US20150051389A1 - Selective antisense compounds and uses thereof - Google Patents

Selective antisense compounds and uses thereof Download PDF

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US20150051389A1
US20150051389A1 US14/238,439 US201214238439A US2015051389A1 US 20150051389 A1 US20150051389 A1 US 20150051389A1 US 201214238439 A US201214238439 A US 201214238439A US 2015051389 A1 US2015051389 A1 US 2015051389A1
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nucleoside
oligomeric compound
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Punit P. Seth
Michael Oestergarrd
Eric E. Swayze
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Ionis Pharmaceuticals Inc
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Isis Pharmaceuticals Inc
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Definitions

  • the present invention pertains generally to chemically-modified oligonucleotides for use in research, diagnostics, and/or therapeutics.
  • Antisense compounds have been used to modulate target nucleic acids. Antisense compounds comprising a variety of chemical modifications and motifs have been reported. In certain instances, such compounds are useful as research tools, diagnostic reagents, and as therapeutic agents. In certain instances antisense compounds have been shown to modulate protein expression by binding to a target messenger RNA (mRNA) encoding the protein. In certain instances, such binding of an antisense compound to its target mRNA results in cleavage of the mRNA. Antisense compounds that modulate processing of a pre-mRNA have also been reported. Such antisense compounds alter splicing, interfere with polyadenlyation or prevent formation of the 5′-cap of a pre-mRNA.
  • mRNA target messenger RNA
  • the present invention provides oligomeric compounds comprising oligonucleotides.
  • such oligonucleotides comprise a region having a gapmer motif.
  • such oligonucleotides consist of a region having a gapmer motif.
  • oligomeric compounds including oligonucleotides described herein are capable of modulating expression of a target RNA.
  • the target RNA is associated with a disease or disorder, or encodes a protein that is associated with a disease or disorder.
  • the oligomeric compounds or oligonucleotides provided herein modulate the expression of function of such RNA to alleviate one or more symptom of the disease or disorder.
  • oligomeric compounds including oligonucleotides describe herein are useful in vitro. In certain embodiments such oligomeric compounds are used in diagnostics and/or for target validation experiments.
  • nucleoside means a compound comprising a nucleobase moiety and a sugar moiety. Nucleosides include, but are not limited to, naturally occurring nucleosides (as found in DNA and RNA) and modified nucleosides. Nucleosides may be linked to a phosphate moiety.
  • chemical modification means a chemical difference in a compound when compared to a naturally occurring counterpart.
  • Chemical modifications of oligonucleotides include nucleoside modifications (including sugar moiety modifications and nucleobase modifications) and internucleoside linkage modifications. In reference to an oligonucleotide, chemical modification does not include differences only in nucleobase sequence.
  • furanosyl means a structure comprising a 5-membered ring comprising four carbon atoms and one oxygen atom.
  • naturally occurring sugar moiety means a ribofuranosyl as found in naturally occurring RNA or a deoxyribofuranosyl as found in naturally occurring DNA.
  • sugar moiety means a naturally occurring sugar moiety or a modified sugar moiety of a nucleoside.
  • modified sugar moiety means a substituted sugar moiety or a sugar surrogate.
  • substituted sugar moiety means a furanosyl that is not a naturally occurring sugar moiety.
  • Substituted sugar moieties include, but are not limited to furanosyls comprising substituents at the 2′-position, the 3′-position, the 5′-position and/or the 4′-position.
  • Certain substituted sugar moieties are bicyclic sugar moieties.
  • 2′-substituted sugar moiety means a furanosyl comprising a substituent at the 2′-position other than H or OH. Unless otherwise indicated, a 2′-substituted sugar moiety is not a bicyclic sugar moiety (i.e., the 2′-substituent of a 2′-substituted sugar moiety does not form a bridge to another atom of the furanosyl ring.
  • MOE means —OCH 2 CH 2 OCH 3 .
  • 2′-F nucleoside refers to a nucleoside comprising a sugar comprising fluorine at the 2′ position. Unless otherwise indicated, the fluorine in a 2′-F nucleoside is in the ribo position (replacing the OH of a natural ribose).
  • 2′-(ara)-F refers to a 2′-F substituted nucleoside, wherein the fluoro group is in the arabino position.
  • sugar surrogate means a structure that does not comprise a furanosyl and that is capable of replacing the naturally occurring sugar moiety of a nucleoside, such that the resulting nucleoside sub-units are capable of linking together and/or linking to other nucleosides to form an oligomeric compound which is capable of hybridizing to a complementary oligomeric compound.
  • Such structures include rings comprising a different number of atoms than furanosyl (e.g., 4, 6, or 7-membered rings); replacement of the oxygen of a furanosyl with a non-oxygen atom (e.g., carbon, sulfur, or nitrogen); or both a change in the number of atoms and a replacement of the oxygen.
  • Such structures may also comprise substitutions corresponding to those described for substituted sugar moieties (e.g., 6-membered carbocyclic bicyclic sugar surrogates optionally comprising additional substituents).
  • Sugar surrogates also include more complex sugar replacements (e.g., the non-ring systems of peptide nucleic acid).
  • Sugar surrogates include without limitation morpholinos, cyclohexenyls and cyclohexitols.
  • bicyclic sugar moiety means a modified sugar moiety comprising a 4 to 7 membered ring (including but not limited to a furanosyl) comprising a bridge connecting two atoms of the 4 to 7 membered ring to form a second ring, resulting in a bicyclic structure.
  • the 4 to 7 membered ring is a sugar ring.
  • the 4 to 7 membered ring is a furanosyl.
  • the bridge connects the 2′-carbon and the 4′-carbon of the furanosyl.
  • nucleotide means a nucleoside further comprising a phosphate linking group.
  • linked nucleosides may or may not be linked by phosphate linkages and thus includes, but is not limited to “linked nucleotides.”
  • linked nucleosides are nucleosides that are connected in a continuous sequence (i.e. no additional nucleosides are present between those that are linked).
  • nucleobase means a group of atoms that can be linked to a sugar moiety to create a nucleoside that is capable of incorporation into an oligonucleotide, and wherein the group of atoms is capable of bonding with a complementary naturally occurring nucleobase of another oligonucleotide or nucleic acid. Nucleobases may be naturally occurring or may be modified.
  • unmodified nucleobase or “naturally occurring nucleobase” means the naturally occurring heterocyclic nucleobases of RNA or DNA: the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) (including 5-methyl C), and uracil (U).
  • modified nucleobase means any nucleobase that is not a naturally occurring nucleobase.
  • modified nucleoside means a nucleoside comprising at least one chemical modification compared to naturally occurring RNA or DNA nucleosides. Modified nucleosides comprise a modified sugar moiety and/or a modified nucleobase.
  • bicyclic nucleoside or “BNA” means a nucleoside comprising a bicyclic sugar moiety.
  • constrained ethyl nucleoside or “cEt” means a nucleoside comprising a bicyclic sugar moiety comprising a 4′-CH(CH 3 )—O-2′ bridge.
  • locked nucleic acid nucleoside or “LNA” means a nucleoside comprising a bicyclic sugar moiety comprising a 4′-CH 2 —O-2′ bridge.
  • 2′-substituted nucleoside means a nucleoside comprising a substituent at the 2′-position other than H or OH. Unless otherwise indicated, a 2′-substituted nucleoside is not a bicyclic nucleoside.
  • 2′-deoxynucleoside means a nucleoside comprising 2′-H furanosyl sugar moiety, as found in naturally occurring deoxyribonucleosides (DNA).
  • a 2′-deoxynucleoside may comprise a modified nucleobase or may comprise an RNA nucleobase (e.g., uracil).
  • RNA-like nucleoside means a modified nucleoside that adopts a northern configuration and functions like RNA when incorporated into an oligonucleotide.
  • RNA-like nucleosides include, but are not limited to 3′-endo furanosyl nucleosides and RNA surrogates.
  • 3′-endo-furanosyl nucleoside means an RNA-like nucleoside that comprises a substituted sugar moiety that has a 3′-endo conformation.
  • 3′-endo-furanosyl nucleosides include, but are not limited to: 2′-MOE, 2′-F, 2′-OMe, LNA, ENA, and cEt nucleosides.
  • RNA-surrogate nucleoside means an RNA-like nucleoside that does not comprise a furanosyl. RNA-surrogate nucleosides include, but are not limited to hexitols and cyclopentanes.
  • oligonucleotide means a compound comprising a plurality of linked nucleosides.
  • an oligonucleotide comprises one or more unmodified ribonucleosides (RNA) and/or unmodified deoxyribonucleosides (DNA) and/or one or more modified nucleosides.
  • oligonucleoside means an oligonucleotide in which none of the internucleoside linkages contains a phosphorus atom.
  • oligonucleotides include oligonucleosides.
  • modified oligonucleotide means an oligonucleotide comprising at least one modified nucleoside and/or at least one modified internucleoside linkage.
  • nucleoside linkage means a covalent linkage between adjacent nucleosides in an oligonucleotide.
  • naturally occurring internucleoside linkage means a 3′ to 5′ phosphodiester linkage.
  • modified internucleoside linkage means any internucleoside linkage other than a naturally occurring internucleoside linkage.
  • oligomeric compound means a polymeric structure comprising two or more sub-structures.
  • an oligomeric compound comprises an oligonucleotide.
  • an oligomeric compound comprises one or more conjugate groups and/or terminal groups.
  • an oligomeric compound consists of an oligonucleotide.
  • terminal group means one or more atom attached to either, or both, the 3′ end or the 5′ end of an oligonucleotide. In certain embodiments a terminal group is a conjugate group. In certain embodiments, a terminal group comprises one or more terminal group nucleosides.
  • conjugate means an atom or group of atoms bound to an oligonucleotide or oligomeric compound.
  • conjugate groups modify one or more properties of the compound to which they are attached, including, but not limited to pharmacodynamic, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, charge and/or clearance properties.
  • conjugate linking group means any atom or group of atoms used to attach a conjugate to an oligonucleotide or oligomeric compound.
  • antisense compound means a compound comprising or consisting of an oligonucleotide at least a portion of which is complementary to a target nucleic acid to which it is capable of hybridizing, resulting in at least one antisense activity.
  • antisense activity means any detectable and/or measurable change attributable to the hybridization of an antisense compound to its target nucleic acid.
  • detecting or “measuring” means that a test or assay for detecting or measuring is performed. Such detection and/or measuring may result in a value of zero. Thus, if a test for detection or measuring results in a finding of no activity (activity of zero), the step of detecting or measuring the activity has nevertheless been performed.
  • detectable and/or measurable activity means a measurable activity that is not zero.
  • essentially unchanged means little or no change in a particular parameter, particularly relative to another parameter which changes much more.
  • a parameter is essentially unchanged when it changes less than 5%.
  • a parameter is essentially unchanged if it changes less than two-fold while another parameter changes at least ten-fold.
  • an antisense activity is a change in the amount of a target nucleic acid.
  • the amount of a non-target nucleic acid is essentially unchanged if it changes much less than the target nucleic acid does, but the change need not be zero.
  • expression means the process by which a gene ultimately results in a protein.
  • Expression includes, but is not limited to, transcription, post-transcriptional modification (e.g., splicing, polyadenlyation, addition of 5′-cap), and translation.
  • target nucleic acid means a nucleic acid molecule to which an antisense compound is intended to hybridize.
  • non-target nucleic acid means a nucleic acid molecule to which hybridization of an antisense compound is not intended or desired.
  • antisense compounds do hybridize to a non-target, due to homology between the target (intended) and non-target (un-intended).
  • mRNA means an RNA molecule that encodes a protein.
  • pre-mRNA means an RNA transcript that has not been fully processed into mRNA. Pre-RNA includes one or more intron.
  • object RNA means an RNA molecule other than a target RNA, the amount, activity, splicing, and/or function of which is modulated, either directly or indirectly, by a target nucleic acid.
  • a target nucleic acid modulates splicing of an object RNA.
  • an antisense compound modulates the amount or activity of the target nucleic acid, resulting in a change in the splicing of an object RNA and ultimately resulting in a change in the activity or function of the object RNA.
  • microRNA means a naturally occurring, small, non-coding RNA that represses gene expression of at least one mRNA.
  • a microRNA represses gene expression by binding to a target site within a 3′ untranslated region of an mRNA.
  • a microRNA has a nucleobase sequence as set forth in miRBase, a database of published microRNA sequences found at http://microrna.sanger.ac.uk/sequences/.
  • a microRNA has a nucleobase sequence as set forth in miRBase version 12.0 released September 2008, which is herein incorporated by reference in its entirety.
  • microRNA mimic means an oligomeric compound having a sequence that is at least partially identical to that of a microRNA.
  • a microRNA mimic comprises the microRNA seed region of a microRNA.
  • a microRNA mimic modulates translation of more than one target nucleic acids.
  • a microRNA mimic is double-stranded.
  • “differentiating nucleobase” means a nucleobase that differs between two nucleic acids.
  • a target region of a target nucleic acid differs by 1-4 nucleobases from a non-target nucleic acid. Each of those differences is referred to as a differentiating nucleobase.
  • a differentiating nucleobase is a single-nucleotide polymorphism.
  • target-selective nucleoside means a nucleoside of an antisense compound that corresponds to a differentiating nucleobase of a target nucleic acid.
  • allelic pair means one of a pair of copies of a gene existing at a particular locus or marker on a specific chromosome, or one member of a pair of nucleobases existing at a particular locus or marker on a specific chromosome, or one member of a pair of nucleobase sequences existing at a particular locus or marker on a specific chromosome.
  • each allelic pair will normally occupy corresponding positions (loci) on a pair of homologous chromosomes, one inherited from the mother and one inherited from the father.
  • the organism or cell is said to be “homozygous” for that allele; if they differ, the organism or cell is said to be “heterozygous” for that allele.
  • Wild-type allele refers to the genotype typically not associated with disease or dysfunction of the gene product.
  • Melt allele refers to the genotype associated with disease or dysfunction of the gene product.
  • allelic variant means a particular identity of an allele, where more than one identity occurs.
  • an allelic variant may refer to either the mutant allele or the wild-type allele.
  • single nucleotide polymorphism or “SNP” means a single nucleotide variation between the genomes of individuals of the same species.
  • a SNP may be a single nucleotide deletion or insertion.
  • SNPs occur relatively frequently in genomes and thus contribute to genetic diversity. The location of a SNP is generally flanked by highly conserved sequences. An individual may be homozygous or heterozygous for an allele at each SNP site.
  • single nucleotide polymorphism site or “SNP site” refers to the nucleotides surrounding a SNP contained in a target nucleic acid to which an antisense compound is targeted.
  • targeting means the association of an antisense compound to a particular target nucleic acid molecule or a particular region of a target nucleic acid molecule.
  • An antisense compound targets a target nucleic acid if it is sufficiently complementary to the target nucleic acid to allow hybridization under physiological conditions.
  • nucleobase complementarity or “complementarity” when in reference to nucleobases means a nucleobase that is capable of base pairing with another nucleobase.
  • adenine (A) is complementary to thymine (T).
  • adenine (A) is complementary to uracil (U).
  • complementary nucleobase means a nucleobase of an antisense compound that is capable of base pairing with a nucleobase of its target nucleic acid.
  • nucleobases at a certain position of an antisense compound are capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid
  • the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be complementary at that nucleobase pair.
  • Nucleobases comprising certain modifications may maintain the ability to pair with a counterpart nucleobase and thus, are still capable of nucleobase complementarity.
  • non-complementary in reference to nucleobases means a pair of nucleobases that do not form hydrogen bonds with one another.
  • complementary in reference to oligomeric compounds (e.g., linked nucleosides, oligonucleotides, or nucleic acids) means the capacity of such oligomeric compounds or regions thereof to hybridize to another oligomeric compound or region thereof through nucleobase complementarity under stringent conditions.
  • Complementary oligomeric compounds need not have nucleobase complementarity at each nucleoside. Rather, some mismatches are tolerated.
  • complementary oligomeric compounds or regions are complementary at 70% of the nucleobases (70% complementary).
  • complementary oligomeric compounds or regions are 80% complementary.
  • complementary oligomeric compounds or regions are 90% complementary.
  • complementary oligomeric compounds or regions are 95% complementary.
  • complementary oligomeric compounds or regions are 100% complementary.
  • mismatch means a nucleobase of a first oligomeric compound that is not capable of pairing with a nucleobase at a corresponding position of a second oligomeric compound, when the first and second oligomeric compound are aligned.
  • Either or both of the first and second oligomeric compounds may be oligonucleotides.
  • hybridization means the pairing of complementary oligomeric compounds (e.g., an antisense compound and its target nucleic acid). While not limited to a particular mechanism, the most common mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases.
  • “specifically hybridizes” means the ability of an oligomeric compound to hybridize to one nucleic acid site with greater affinity than it hybridizes to another nucleic acid site.
  • an antisense oligonucleotide specifically hybridizes to more than one target site.
  • oligonucleotide or portion thereof means that each nucleobase of the oligonucleotide or portion thereof is capable of pairing with a nucleobase of a complementary nucleic acid or contiguous portion thereof.
  • a fully complementary region comprises no mismatches or unhybridized nucleobases in either strand.
  • percent complementarity means the percentage of nucleobases of an oligomeric compound that are complementary to an equal-length portion of a target nucleic acid. Percent complementarity is calculated by dividing the number of nucleobases of the oligomeric compound that are complementary to nucleobases at corresponding positions in the target nucleic acid by the total length of the oligomeric compound.
  • percent identity means the number of nucleobases in a first nucleic acid that are the same type (independent of chemical modification) as nucleobases at corresponding positions in a second nucleic acid, divided by the total number of nucleobases in the first nucleic acid.
  • modulation means a change of amount or quality of a molecule, function, or activity when compared to the amount or quality of a molecule, function, or activity prior to modulation.
  • modulation includes the change, either an increase (stimulation or induction) or a decrease (inhibition or reduction) in gene expression.
  • modulation of expression can include a change in splice site selection of pre-mRNA processing, resulting in a change in the absolute or relative amount of a particular splice-variant compared to the amount in the absence of modulation.
  • modification motif means a pattern of chemical modifications in an oligomeric compound or a region thereof. Motifs may be defined by modifications at certain nucleosides and/or at certain linking groups of an oligomeric compound.
  • nucleoside motif means a pattern of nucleoside modifications in an oligomeric compound or a region thereof.
  • the linkages of such an oligomeric compound may be modified or unmodified.
  • motifs herein describing only nucleosides are intended to be nucleoside motifs. Thus, in such instances, the linkages are not limited.
  • sugar motif means a pattern of sugar modifications in an oligomeric compound or a region thereof.
  • linkage motif means a pattern of linkage modifications in an oligomeric compound or region thereof.
  • the nucleosides of such an oligomeric compound may be modified or unmodified.
  • motifs herein describing only linkages are intended to be linkage motifs. Thus, in such instances, the nucleosides are not limited.
  • nucleobase modification motif means a pattern of modifications to nucleobases along an oligonucleotide. Unless otherwise indicated, a nucleobase modification motif is independent of the nucleobase sequence.
  • sequence motif means a pattern of nucleobases arranged along an oligonucleotide or portion thereof. Unless otherwise indicated, a sequence motif is independent of chemical modifications and thus may have any combination of chemical modifications, including no chemical modifications.
  • nucleoside having a modification of a first type may be an unmodified nucleoside.
  • “differently modified” mean chemical modifications or chemical substituents that are different from one another, including absence of modifications.
  • a MOE nucleoside and an unmodified DNA nucleoside are “differently modified,” even though the DNA nucleoside is unmodified.
  • DNA and RNA are “differently modified,” even though both are naturally-occurring unmodified nucleosides. Nucleosides that are the same but for comprising different nucleobases are not differently modified.
  • nucleoside comprising a 2′-OMe modified sugar and an unmodified adenine nucleobase and a nucleoside comprising a 2′-OMe modified sugar and an unmodified thymine nucleobase are not differently modified.
  • the same type of modifications refers to modifications that are the same as one another, including absence of modifications.
  • two unmodified DNA nucleoside have “the same type of modification,” even though the DNA nucleoside is unmodified.
  • Such nucleosides having the same type modification may comprise different nucleobases.
  • pharmaceutically acceptable carrier or diluent means any substance suitable for use in administering to an animal.
  • a pharmaceutically acceptable carrier or diluent is sterile saline.
  • such sterile saline is pharmaceutical grade saline.
  • substituted nucleoside and “substituent group,” means an atom or group that replaces the atom or group of a named parent compound.
  • a substituent of a modified nucleoside is any atom or group that differs from the atom or group found in a naturally occurring nucleoside (e.g., a modified 2′-substituent is any atom or group at the 2′-position of a nucleoside other than H or OH).
  • Substituent groups can be protected or unprotected.
  • compounds of the present invention have substituents at one or at more than one position of the parent compound. Substituents may also be further substituted with other substituent groups and may be attached directly or via a linking group such as an alkyl or hydrocarbyl group to a parent compound.
  • substituted in reference to a chemical functional group means an atom or group of atoms differs from the atom or a group of atoms normally present in the named functional group.
  • a substituent replaces a hydrogen atom of the functional group (e.g., in certain embodiments, the substituent of a substituted methyl group is an atom or group other than hydrogen which replaces one of the hydrogen atoms of an unsubstituted methyl group).
  • groups amenable for use as substituents include without limitation, halogen, hydroxyl, alkyl, alkenyl, alkynyl, acyl (—C(O)R aa ), carboxyl (—C(O)O—R aa ), aliphatic groups, alicyclic groups, alkoxy, substituted oxy (—O—R aa ), aryl, aralkyl, heterocyclic radical, heteroaryl, heteroarylalkyl, amino (—N(R bb )(R cc )), imino( ⁇ NR bb ), amido (—C(O)N(R bb )(R cc ) or —N(R bb )C(O)R aa ), azido (—N 3 ), nitro (—NO 2 ), cyano (—CN), carbamido (—OC(O)N(R bb )(R cc ) or
  • each R aa , R bb and R cc is, independently, H, an optionally linked chemical functional group or a further substituent group with a preferred list including without limitation, alkyl, alkenyl, alkynyl, aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl, alicyclic, heterocyclic and heteroarylalkyl. Selected substituents within the compounds described herein are present to a recursive degree.
  • alkyl means a saturated straight or branched hydrocarbon radical containing up to twenty four carbon atoms.
  • alkyl groups include without limitation, methyl, ethyl, propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl and the like.
  • Alkyl groups typically include from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms (C 1 -C 12 alkyl) with from 1 to about 6 carbon atoms being more preferred.
  • alkenyl means a straight or branched hydrocarbon chain radical containing up to twenty four carbon atoms and having at least one carbon-carbon double bond.
  • alkenyl groups include without limitation, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, dienes such as 1,3-butadiene and the like.
  • Alkenyl groups typically include from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms with from 2 to about 6 carbon atoms being more preferred.
  • Alkenyl groups as used herein may optionally include one or more further substituent groups.
  • alkynyl means a straight or branched hydrocarbon radical containing up to twenty four carbon atoms and having at least one carbon-carbon triple bond.
  • alkynyl groups include, without limitation, ethynyl, 1-propynyl, 1-butynyl, and the like.
  • Alkynyl groups typically include from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms with from 2 to about 6 carbon atoms being more preferred.
  • Alkynyl groups as used herein may optionally include one or more further substituent groups.
  • acyl means a radical formed by removal of a hydroxyl group from an organic acid and has the general Formula —C(O)—X where X is typically aliphatic, alicyclic or aromatic. Examples include aliphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls, aromatic sulfinyls, aliphatic sulfinyls, aromatic phosphates, aliphatic phosphates and the like. Acyl groups as used herein may optionally include further substituent groups.
  • alicyclic means a cyclic ring system wherein the ring is aliphatic.
  • the ring system can comprise one or more rings wherein at least one ring is aliphatic.
  • Preferred alicyclics include rings having from about 5 to about 9 carbon atoms in the ring.
  • Alicyclic as used herein may optionally include further substituent groups.
  • aliphatic means a straight or branched hydrocarbon radical containing up to twenty four carbon atoms wherein the saturation between any two carbon atoms is a single, double or triple bond.
  • An aliphatic group preferably contains from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms with from 1 to about 6 carbon atoms being more preferred.
  • the straight or branched chain of an aliphatic group may be interrupted with one or more heteroatoms that include nitrogen, oxygen, sulfur and phosphorus.
  • Such aliphatic groups interrupted by heteroatoms include without limitation, polyalkoxys, such as polyalkylene glycols, polyamines, and polyimines. Aliphatic groups as used herein may optionally include further substituent groups.
  • alkoxy means a radical formed between an alkyl group and an oxygen atom wherein the oxygen atom is used to attach the alkoxy group to a parent molecule.
  • alkoxy groups include without limitation, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy and the like.
  • Alkoxy groups as used herein may optionally include further substituent groups.
  • aminoalkyl means an amino substituted C 1 -C 12 alkyl radical.
  • the alkyl portion of the radical forms a covalent bond with a parent molecule.
  • the amino group can be located at any position and the aminoalkyl group can be substituted with a further substituent group at the alkyl and/or amino portions.
  • aralkyl and arylalkyl mean an aromatic group that is covalently linked to a C 1 -C 12 alkyl radical.
  • the alkyl radical portion of the resulting aralkyl (or arylalkyl) group forms a covalent bond with a parent molecule. Examples include without limitation, benzyl, phenethyl and the like.
  • Aralkyl groups as used herein may optionally include further substituent groups attached to the alkyl, the aryl or both groups that form the radical group.
  • aryl and “aromatic” mean a mono- or polycyclic carbocyclic ring system radicals having one or more aromatic rings.
  • aryl groups include without limitation, phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyl and the like.
  • Preferred aryl ring systems have from about 5 to about 20 carbon atoms in one or more rings.
  • Aryl groups as used herein may optionally include further substituent groups.
  • halo and “halogen,” mean an atom selected from fluorine, chlorine, bromine and iodine.
  • heteroaryl and “heteroaromatic,” mean a radical comprising a mono- or poly-cyclic aromatic ring, ring system or fused ring system wherein at least one of the rings is aromatic and includes one or more heteroatoms. Heteroaryl is also meant to include fused ring systems including systems where one or more of the fused rings contain no heteroatoms. Heteroaryl groups typically include one ring atom selected from sulfur, nitrogen or oxygen.
  • heteroaryl groups include without limitation, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl and the like.
  • Heteroaryl radicals can be attached to a parent molecule directly or through a linking moiety such as an aliphatic group or hetero atom.
  • Heteroaryl groups as used herein may optionally include further substituent groups.
  • the present invention provides oligomeric compounds.
  • such oligomeric compounds comprise oligonucleotides optionally comprising one or more conjugate and/or terminal groups.
  • an oligomeric compound consists of an oligonucleotide.
  • oligonucleotides comprise one or more chemical modifications. Such chemical modifications include modifications of one or more nucleoside (including modifications to the sugar moiety and/or the nucleobase) and/or modifications to one or more internucleoside linkage.
  • oligomeric compounds comprising or consisting of oligonucleotides comprising at least one modified nucleoside.
  • modified nucleosides comprise a modified sugar moeity, a modified nucleobase, or both a modified sugar moiety and a modified nucleobase.
  • compounds of the invention comprise one or more modified nucleosides comprising a modified sugar moiety.
  • modified nucleosides comprising a modified sugar moiety.
  • Such compounds comprising one or more sugar-modified nucleosides may have desirable properties, such as enhanced nuclease stability or increased binding affinity with a target nucleic acid relative to an oligonucleotide comprising only nucleosides comprising naturally occurring sugar moieties.
  • modified sugar moieties are substituted sugar moieties.
  • modified sugar moieties are sugar surrogates. Such sugar surrogates may comprise one or more substitutions corresponding to those of substituted sugar moieties.
  • modified sugar moieties are substituted sugar moieties comprising one or more non-bridging sugar substituent, including but not limited to substituents at the 2′ and/or 5′ positions.
  • sugar substituents suitable for the 2′-position include, but are not limited to: 2′-F, 2′-OCH 3 (“OMe” or “O-methyl”), and 2′-O(CH 2 ) 2 OCH 3 (“MOE”).
  • sugar substituents at the 2′ position is selected from allyl, amino, azido, thio, O-allyl, O—C 1 -C 10 alkyl, O—C 1 -C 10 substituted alkyl; OCF 3 , O(CH 2 ) 2 SCH 3 , O(CH 2 ) 2 —O—N(Rm)(Rn), and O—CH 2 —C( ⁇ O)—N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted C 1 -C 10 alkyl.
  • sugar substituents at the 5′-position include, but are not limited to: 5′-methyl (R or S); 5′-vinyl, and 5′-methoxy.
  • substituted sugars comprise more than one non-bridging sugar substituent, for example, 2′-F-5′-methyl sugar moieties (see, e.g., PCT International Application WO 2008/101157, for additional 5′,2′-bis substituted sugar moieties and nucleosides).
  • Nucleosides comprising 2′-substituted sugar moieties are referred to as 2′-substituted nucleosides.
  • a 2′-substituted nucleoside comprises a 2′-substituent group selected from halo, allyl, amino, azido, SH, CN, OCN, CF 3 , OCF 3 , O, S, or N(R m )-alkyl; O, S, or N(R m )-alkenyl; O, S or N(R m )-alkynyl; O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH 2 ) 2 SCH 3 , O—(CH 2 ) 2 —O—N(R m )(R n ) or O—CH 2 —C( ⁇ O)—N(R m
  • These 2′-substituent groups can be further substituted with one or more substituent groups independently selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO 2 ), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.
  • a 2′-substituted nucleoside comprises a 2′-substituent group selected from F, NH 2 , N 3 , OCF 3 , O—CH 3 , O(CH 2 ) 3 NH 2 , CH 2 —CH ⁇ CH 2 , O—CH 2 —CH ⁇ CH 2 , OCH 2 CH 2 OCH 3 , O(CH 2 ) 2 SCH 3 , O—(CH 2 ) 2 —O—N(R m )(R n ), O(CH 2 ) 2 —O—(CH 2 ) 2 N(CH 3 ) 2 , and N-substituted acetamide (O—CH 2 —C( ⁇ O)—N(R m )(R n ) where each R m and R n is, independently, H, an amino protecting group or substituted or unsubstituted C 1 -C 10 alkyl.
  • a 2′-substituted nucleoside comprises a sugar moiety comprising a 2′-substituent group selected from F, OCF 3 , O—CH 3 , OCH 2 CH 2 OCH 3 , O(CH 2 ) 2 SCH 3 , O—(CH 2 ) 2 —O—N(CH 3 ) 2 , —O(CH 2 ) 2 O(CH 2 ) 2 N(CH 3 ) 2 , and O—CH 2 —C( ⁇ O)—N(H)CH 3 .
  • a 2′-substituted nucleoside comprises a sugar moiety comprising a 2′-substituent group selected from F, OCF 3 , O—CH 3 , OCH 2 CH 2 OCH 3 , O(CH 2 ) 2 SCH 3 , O—(CH 2 ) 2 —O—N(CH 3 ) 2 , —O(CH 2 ) 2 O(CH 2 ) 2 N(CH 3 ) 2
  • a 2′-substituted nucleoside comprises a sugar moiety comprising a 2′-substituent group selected from F, O—CH 3 , and OCH 2 CH 2 OCH 3 .
  • modified sugar moieties comprise a bridging sugar substituent that forms a second ring resulting in a bicyclic sugar moiety.
  • the bicyclic sugar moiety comprises a bridge between the 4′ and the 2′ furanose ring atoms.
  • 4′ to 2′ sugar substituents include, but are not limited to: —[C(R a )(R b )] n —, —[C(R a )(R b )] n —O—, —C(R a R b )—N(R)—O— or, —C(R a R b )—O—N(R)—; 4′-CH 2 -2′, 4′-(CH 2 ) 2 -2′, 4′-(CH 2 )—O-2′ (LNA); 4′-(CH 2 )—S-2; 4′-(CH 2 ) 2 —O-2′ (ENA); 4′-CH(CH 3 )—O-2′ (cEt) and 4′-CH(CH 2 OCH 3 )—O-2′, and analogs thereof (see, e.g., U.S.
  • such 4′ to 2′ bridges independently comprise from 1 to 4 linked groups independently selected from —[C(R a )(R b )] n —, —C(R a ) ⁇ C(R b )—, —C(R a ) ⁇ N—, —C( ⁇ NR a )—, —C( ⁇ O)—, —C( ⁇ S)—, —O—, —Si(R a ) 2 —, —S( ⁇ O) x —, and —N(R a )—;
  • x 0, 1, or 2;
  • n 1, 2, 3, or 4;
  • each R a and R b is, independently, H, a protecting group, hydroxyl, C 1 -C 12 alkyl, substituted C 1 -C 12 alkyl, C 2 -C 12 alkenyl, substituted C 2 -C 12 alkenyl, C 2 -C 12 alkynyl, substituted C 2 -C 12 alkynyl, C 5 -C 20 aryl, substituted C 5 -C 20 aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C 5 -C 7 alicyclic radical, substituted C 5 -C 7 alicyclic radical, halogen, OJ 1 , NJ 1 J 2 , SJ 1 , N 3 , COOJ 1 , acyl (C( ⁇ O)—H), substituted acyl, CN, sulfonyl (S( ⁇ O) 2 -J 1 ), or sulfoxyl (S( ⁇ O)-J 1 ); and
  • each J 1 and J 2 is, independently, H, C 1 -C 12 alkyl, substituted C 1 -C 12 alkyl, C 2 -C 12 alkenyl, substituted C 2 -C 12 alkenyl, C 2 -C 12 alkynyl, substituted C 2 -C 12 alkynyl, C 5 -C 20 aryl, substituted C 5 -C 20 aryl, acyl (C( ⁇ O)—H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C 1 -C 12 aminoalkyl, substituted C 1 -C 12 aminoalkyl, or a protecting group.
  • Bicyclic nucleosides include, but are not limited to, (A) ⁇ -L-Methyleneoxy (4′-CH 2 —O-2′) BNA, (B) ⁇ -D-Methyleneoxy (4′-CH 2 —O-2′) BNA (also referred to as locked nucleic acid or LNA), (C) Ethyleneoxy (4′-(CH 2 ) 2 —O-2′) BNA, (D) Aminooxy (4′-CH 2 —O—N(R)-2′) BNA, (E) Oxyamino (4′-CH 2 —N(R)—O-2′) BNA, (F) Methyl(methyleneoxy) (4′-CH(CH 3 )—O-2′) BNA (also referred to as constrained ethyl or cEt), (G) methylene-thio (4′-CH 2 —S
  • Bx is a nucleobase moiety and R is, independently, H, a protecting group, or C 1 -C 12 alkyl.
  • bicyclic sugar moieties and nucleosides incorporating such bicyclic sugar moieties are further defined by isomeric configuration.
  • a nucleoside comprising a 4′-2′ methylene-oxy bridge may be in the ⁇ -L configuration or in the ⁇ -D configuration.
  • ⁇ -L-methyleneoxy (4′-CH 2 —O-2′) bicyclic nucleosides have been incorporated into antisense oligonucleotides that showed antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).
  • substituted sugar moieties comprise one or more non-bridging sugar substituent and one or more bridging sugar substituent (e.g., 5′-substituted and 4′-2′ bridged sugars).
  • bridging sugar substituent e.g., 5′-substituted and 4′-2′ bridged sugars.
  • modified sugar moieties are sugar surrogates.
  • the oxygen atom of the naturally occurring sugar is substituted, e.g., with a sulfer, carbon or nitrogen atom.
  • such modified sugar moiety also comprises bridging and/or non-bridging substituents as described above.
  • certain sugar surrogates comprise a 4′-sulfer atom and a substitution at the 2′-position (see, e.g., published U.S. Patent Application US2005/0130923, published on Jun. 16, 2005) and/or the 5′ position.
  • carbocyclic bicyclic nucleosides having a 4′-2′ bridge have been described (see, e.g., Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al., J. Org. Chem., 2006, 71, 7731-7740).
  • sugar surrogates comprise rings having other than 5-atoms.
  • a sugar surrogate comprises a six-membered tetrahydropyran.
  • Such tetrahydropyrans may be further modified or substituted.
  • Nucleosides comprising such modified tetrahydropyrans include, but are not limited to, hexitol nucleic acid (HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) (see Leumann, C J. Bioorg. & Med. Chem . (2002) 10:841-854), fluoro HNA (F-HNA), and those compounds having Formula VII:
  • Bx is a nucleobase moiety
  • T 3 and T 4 are each, independently, an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound or one of T 3 and T 4 is an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound and the other of T 3 and T 4 is H, a hydroxyl protecting group, a linked conjugate group, or a 5′ or 3′-terminal group;
  • q 1 , q 2 , q 3 , q 4 , q 5 , q 6 and q 7 are each, independently, H, C 1 -C 6 alkyl, substituted C 1 -C 6 alkyl, C 2 -C 6 alkenyl, substituted C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, or substituted C 2 -C 6 alkynyl; and
  • each of R 1 and R 2 is independently selected from among: hydrogen, halogen, substituted or unsubstituted alkoxy, NJ 1 J 2 , SJ 1 , N 3 , OC( ⁇ X)J 1 , OC( ⁇ X)NJ 1 J 2 , NJ 3 C( ⁇ X)NJ 1 J 2 , and CN, wherein X is O, S or NJ 1 , and each J 1 , J 2 , and J 3 is, independently, H or C 1 -C 6 alkyl.
  • the modified THP nucleosides of Formula VII are provided wherein q 1 , q 2 , q 3 , q 4 , q 5 , q 6 and q 7 are each H. In certain embodiments, at least one of q 1 , q 2 , q 3 , q 4 , q 5 , q 6 and q 7 is other than H. In certain embodiments, at least one of q 1 , q 2 , q 3 , q 4 , q 5 , q 6 and q 7 is methyl. In certain embodiments, THP nucleosides of Formula VII are provided wherein one of R 1 and R 2 is F. In certain embodiments, R 1 is fluoro and R 2 is H, R 1 is methoxy and R 2 is H, and R 1 is methoxyethoxy and R 2 is H.
  • the present invention provides oligonucleotides comprising modified nucleosides.
  • modified nucleotides may include modified sugars, modified nucleobases, and/or modified linkages. The specific modifications are selected such that the resulting oligonucleotides possess desirable characteristics.
  • oligonucleotides comprise one or more RNA-like nucleosides. In certain embodiments, oligonucleotides comprise one or more DNA-like nucleotides.
  • nucleosides of the present invention comprise one or more unmodified nucleobases. In certain embodiments, nucleosides of the present invention comprise one or more modified nucleobases.
  • modified nucleobases are selected from: universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases as defined herein.
  • nucleobases include tricyclic pyrimidines such as phenoxazine cytidine([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g.
  • nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat.
  • nucleosides may be linked together using any internucleoside linkage to form oligonucleotides.
  • the two main classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom.
  • Representative phosphorus containing internucleoside linkages include, but are not limited to, phosphodiesters (P ⁇ O), phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates (P ⁇ S).
  • Non-phosphorus containing internucleoside linking groups include, but are not limited to, methylenemethylimino (—CH 2 —N(CH 3 )—O—CH 2 —) thiodiester (—O—C(O)—S—), thionocarbamate (—O—C(O)(NH)—S—); siloxane (—O—Si(H) 2 —O—); and N,N′-dimethylhydrazine (—CH 2 —N(CH 3 )—N(CH 3 )—).
  • Modified linkages, compared to natural phosphodiester linkages, can be used to alter, typically increase, nuclease resistance of the oligonucleotide.
  • internucleoside linkages having a chiral atom can be prepared as a racemic mixture, or as separate enantiomers.
  • Representative chiral linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing internucleoside linkages are well known to those skilled in the art.
  • oligonucleotides described herein contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations that may be defined, in terms of absolute stereochemistry, as (R) or (S), ⁇ or ⁇ such as for sugar anomers, or as (D) or (L) such as for amino acids etc. Included in the antisense compounds provided herein are all such possible isomers, as well as their racemic and optically pure forms.
  • Neutral internucleoside linkages include without limitation, phosphotriesters, methylphosphonates, MMI (3′-CH 2 —N(CH 3 )—O-5′), amide-3 (3′-CH 2 —C( ⁇ P)—N(H)-5′), amide-4 (3′-CH 2 —N(H)—C( ⁇ O)-5′), formacetal (3′-O—CH 2 —O-5′), and thioformacetal (3′-S—CH 2 —O-5′).
  • Further neutral internucleoside linkages include nonionic linkages comprising siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester and amides (See for example: Carbohydrate Modifications in Antisense Research ; Y. S. Sanghvi and P. D. Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Further neutral internucleoside linkages include nonionic linkages comprising mixed N, O, S and CH 2 component parts.
  • oligomeric compounds include nucleosides synthetically modified to induce a 3′-endo sugar conformation.
  • a nucleoside can incorporate synthetic modifications of the heterocyclic base moiety, the sugar moiety or both to induce a desired 3′-endo sugar conformation. These modified nucleosides are used to mimic RNA like nucleosides so that particular properties of an oligomeric compound can be enhanced while maintaining the desirable 3′-endo conformational geometry.
  • RNA type duplex A form helix, predominantly 3′-endo
  • duplexes composed of 2′-deoxy-2′-F-nucleosides appear efficient in triggering RNAi response in the C. elegans system.
  • Properties that are enhanced by using more stable 3′-endo nucleosides include but aren't limited to modulation of pharmacokinetic properties through modification of protein binding, protein off-rate, absorption and clearance; modulation of nuclease stability as well as chemical stability; modulation of the binding affinity and specificity of the oligomer (affinity and specificity for enzymes as well as for complementary sequences); and increasing efficacy of RNA cleavage.
  • the present invention provides oligomeric compounds having one or more nucleosides modified in such a way as to favor a C3′-endo type conformation.
  • Nucleoside conformation is influenced by various factors including substitution at the 2′, 3′ or 4′-positions of the pentofuranosyl sugar. Electronegative substituents generally prefer the axial positions, while sterically demanding substituents generally prefer the equatorial positions (Principles of Nucleic Acid Structure, Wolfgang Sanger, 1984, Springer-Verlag.) Modification of the 2′ position to favor the 3′-endo conformation can be achieved while maintaining the 2′-OH as a recognition element, as exemplified in Example 35, below (Gallo et al., Tetrahedron (2001), 57, 5707-5713. Harry-O'kuru et al., J. Org.
  • preference for the 3′-endo conformation can be achieved by deletion of the 2′-OH as exemplified by 2′ deoxy-2′F-nucleosides (Kawasaki et al., J. Med. Chem. (1993), 36, 831-841), which adopts the 3′-endo conformation positioning the electronegative fluorine atom in the axial position.
  • LNA locked nucleic acid
  • ENA ethylene bridged nucleic acids
  • oligomeric compounds comprise or consist of oligonucleotides.
  • such oligonucleotides comprise one or more chemical modification.
  • chemically modified oligonucleotides comprise one or more modified sugars.
  • chemically modified oligonucleotides comprise one or more modified nucleobases.
  • chemically modified oligonucleotides comprise one or more modified internucleoside linkages.
  • the chemical modifications (sugar modifications, nucleobase modifications, and/or linkage modifications) define a pattern or motif.
  • the patterns of chemical modifications of sugar moieties, internucleoside linkages, and nucleobases are each independent of one another.
  • an oligonucleotide may be described by its sugar modification motif, internucleoside linkage motif and/or nucleobase modification motif (as used herein, nucleobase modification motif describes the chemical modifications to the nucleobases independent of the sequence of nucleobases).
  • oligonucleotides comprise one or more type of modified sugar moieties and/or naturally occurring sugar moieties arranged along an oligonucleotide or region thereof in a defined pattern or sugar motif.
  • sugar motifs include but are not limited to any of the sugar modifications discussed herein.
  • the oligonucleotides comprise or consist of a region having a gapmer sugar motif, which comprises two external regions or “wings” and a central or internal region or “gap.”
  • the three regions of a gapmer sugar motif (the 5′-wing, the gap, and the 3′-wing) form a contiguous sequence of nucleosides wherein at least some of the sugar moieties of the nucleosides of each of the wings differ from at least some of the sugar moieties of the nucleosides of the gap.
  • the sugar moieties of the nucleosides of each wing that are closest to the gap differ from the sugar moiety of the neighboring gap nucleosides, thus defining the boundary between the wings and the gap.
  • the sugar moieties within the gap are the same as one another.
  • the gap includes one or more nucleoside having a sugar moiety that differs from the sugar moiety of one or more other nucleosides of the gap.
  • the sugar motifs of the two wings are the same as one another (symmetric sugar gapmer).
  • the sugar motifs of the 5′-wing differs from the sugar motif of the 3′-wing (asymmetric sugar gapmer).
  • oligonucleotides comprise chemical modifications to nucleobases arranged along the oligonucleotide or region thereof in a defined pattern or nucleobases modification motif. In certain embodiments, each nucleobase is modified. In certain embodiments, none of the nucleobases is chemically modified.
  • oligonucleotides comprise a block of modified nucleobases.
  • the block is at the 3′-end of the oligonucleotide.
  • the block is within 3 nucleotides of the 3′-end of the oligonucleotide.
  • the block is at the 5′-end of the oligonucleotide.
  • the block is within 3 nucleotides of the 5′-end of the oligonucleotide.
  • nucleobase modifications are a function of the natural base at a particular position of an oligonucleotide.
  • each purine or each pyrimidine in an oligonucleotide is modified.
  • each adenine is modified.
  • each guanine is modified.
  • each thymine is modified.
  • each cytosine is modified.
  • each uracil is modified.
  • oligonucleotides comprise one or more nucleosides comprising a modified nucleobase.
  • oligonucleotides having a gapmer sugar motif comprise a nucleoside comprising a modified nucleobase.
  • one nucleoside comprising a modified nucleobases is in the central gap of an oligonucleotide having a gapmer sugar motif.
  • the sugar is an unmodified 2′ deoxynucleoside.
  • the modified nucleobase is selected from: a 2-thio pyrimidine and a 5-propyne pyrimidine
  • cytosine moieties in an oligonucleotide are 5-methyl cytosine moieties.
  • 5-methyl cytosine is not a “modified nucleobase.” Accordingly, unless otherwise indicated, unmodified nucleobases include both cytosine residues having a 5-methyl and those lacking a 5 methyl. In certain embodiments, the methylation state of all or some cytosine nucleobases is specified.
  • oligonucleotides comprise nucleosides comprising modified sugar moieties and/or nucleosides comprising modified nucleobases.
  • Such motifs can be described by their sugar motif and their nucleobase motif separately or by their nucleoside motif, which provides positions or patterns of modified nucleosides (whether modified sugar, nucleobase, or both sugar and nucleobase) in an oligonucleotide.
  • the oligonucleotides comprise or consist of a region having a gapmer nucleoside motif, which comprises two external regions or “wings” and a central or internal region or “gap.”
  • the three regions of a gapmer nucleoside motif (the 5′-wing, the gap, and the 3′-wing) form a contiguous sequence of nucleosides wherein at least some of the sugar moieties and/or nucleobases of the nucleosides of each of the wings differ from at least some of the sugar moieties and/or nucleobase of the nucleosides of the gap.
  • the nucleosides of each wing that are closest to the gap differ from the neighboring gap nucleosides, thus defining the boundary between the wings and the gap.
  • the nucleosides within the gap are the same as one another.
  • the gap includes one or more nucleoside that differs from one or more other nucleosides of the gap.
  • the nucleoside motifs of the two wings are the same as one another (symmetric gapmer).
  • the nucleoside motifs of the 5′-wing differs from the nucleoside motif of the 3′-wing (asymmetric gapmer).
  • the 5′-wing of a gapmer consists of 1 to 6 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 1 to 5 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 2 to 5 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 3 to 5 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 4 or 5 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 1 to 4 linked nucleosides.
  • the 5′-wing of a gapmer consists of 1 to 3 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 1 or 2 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 2 to 4 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 2 or 3 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 3 or 4 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 1 nucleoside. In certain embodiments, the 5′-wing of a gapmer consists of 2 linked nucleosides.
  • the 5′-wing of a gapmer consists of 3 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 4 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 5 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 6 linked nucleosides.
  • the 5′-wing of a gapmer comprises at least one bicyclic nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least two bicyclic nucleosides. In certain embodiments, the 5′-wing of a gapmer comprises at least three bicyclic nucleosides. In certain embodiments, the 5′-wing of a gapmer comprises at least four bicyclic nucleosides. In certain embodiments, the 5′-wing of a gapmer comprises at least one constrained ethyl nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one LNA nucleoside.
  • each nucleoside of the 5′-wing of a gapmer is a bicyclic nucleoside. In certain embodiments, each nucleoside of the 5′-wing of a gapmer is a constrained ethyl nucleoside. In certain embodiments, each nucleoside of the 5′-wing of a gapmer is a LNA nucleoside.
  • the 5′-wing of a gapmer comprises at least one non-bicyclic modified nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one 2′-substituted nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one 2′-MOE nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one 2′-OMe nucleoside. In certain embodiments, each nucleoside of the 5′-wing of a gapmer is a non-bicyclic modified nucleoside.
  • each nucleoside of the 5′-wing of a gapmer is a 2′-substituted nucleoside. In certain embodiments, each nucleoside of the 5′-wing of a gapmer is a 2′-MOE nucleoside. In certain embodiments, each nucleoside of the 5′-wing of a gapmer is a 2′-OMe nucleoside.
  • the 5′-wing of a gapmer comprises at least one 2′-deoxynucleoside. In certain embodiments, each nucleoside of the 5′-wing of a gapmer is a 2′-deoxynucleoside. In a certain embodiments, the 5′-wing of a gapmer comprises at least one ribonucleoside. In certain embodiments, each nucleoside of the 5′-wing of a gapmer is a ribonucleoside. In certain embodiments, one, more than one, or each of the nucleosides of the 5′-wing is an RNA-like nucleoside.
  • the 5′-wing of a gapmer comprises at least one bicyclic nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2′-substituted nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2′-MOE nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2′-OMe nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2′-deoxynucleoside.
  • the 5′-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2′-substituted nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2′-MOE nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2′-OMe nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2′-deoxynucleoside.
  • the 5′-wing of a gapmer has a nucleoside motif selected from among the following: ADDA; ABDAA; ABBA; ABB; ABAA; AABAA; AAABAA; AAAABAA; AAAAABAA; AAABAA; AABAA; ABAB; ABADB; ABADDB; AAABB; AAAAA; ABBDC; ABDDC; ABBDCC; ABBDDC; ABBDCC; ABBC; AA; AAA; AAAA; AAAAB; AAAAAAA; AAAAAAAA; ABBB; AB; ABAB; AAAAB; AABBB; AAAAB; and AABBB, wherein each A is a modified nucleoside of a first type, each B is a modified nucleoside of a second type, each C is a modified nucleoside of a third type, and each D is an unmodified deoxynucleoside.
  • the 5′-wing of a gapmer has a nucleoside motif selected from among the following: AB, ABB, AAA, BBB, BBBAA, AAB, BAA, BBAA, AABB, AAAB, ABBW, ABBWW, ABBB, ABBBB, ABAB, ABABAB, ABABBB, ABABAA, AAABB, AAAABB, AABB, AAAAB, AABBB, ABBBB, BBBBB, AAABW, AAAAA, BBBBAA, and AAABW; wherein each A is a modified nucleoside of a first type, each B is a modified nucleoside of a second type, and each W is a modified nucleoside of either the first type, the second type or a third type.
  • the 5′-wing of a gapmer has a nucleoside motif selected from among the following: ABB; ABAA; AABAA; AAABAA; ABAB; ABADB; AAABB; AAAAA; AA; AAA; AAAA; AAAAB; ABBB; AB; and ABAB; wherein each A is a modified nucleoside of a first type, each B is a modified nucleoside of a second type, and each W is a modified nucleoside of either the first type, the second type or a third type.
  • an oligonucleotide comprises any 5′-wing motif provided herein.
  • the oligonucleotide is a 5′-hemimer (does not comprise a 3′-wing).
  • such an oligonucleotide is a gapmer.
  • the 3′-wing of the gapmer may comprise any nucleoside motif.
  • the 5′-wing of a gapmer has a sugar motif selected from among those listed in the following non-limiting tables:
  • each A, each B, and each C located at the 3′-most 5′-wing nucleoside is a modified nucleoside.
  • the 5′-wing motif is selected from among AB B , BB B , and CB B , wherein the underlined nucleoside represents the 3′-most 5′-wing nucleoside and wherein the underlined nucleoside is a modified nucleoside.
  • the 3′-most 5′-wing nucleoside comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, ⁇ -L-LNA, ENA and 2′-thio LNA.
  • the 3′-most 5′-wing nucleoside comprises a bicyclic sugar moiety selected from among cEt and LNA. In certain embodiments, the 3′-most 5′-wing nucleoside comprises cEt. In certain embodiments, the 3′-most 5′-wing nucleoside comprises LNA.
  • each A comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, each A comprises a modified sugar moiety. In certain embodiments, each A comprises a 2′-substituted sugar moiety. In certain embodiments, each A comprises a 2′-substituted sugar moiety selected from among F, ara-F, OCH 3 and O(CH 2 ) 2 —OCH 3 . In certain embodiments, each A comprises a bicyclic sugar moiety. In certain embodiments, each A comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, ⁇ -L-LNA, ENA and 2′-thio LNA.
  • each A comprises a modified nucleobase. In certain embodiments, each A comprises a modified nucleobase selected from among 2-thio-thymidine nucleoside and 5-propyne uridine nucleoside. In certain embodiments, each A comprises an HNA. In certain embodiments, each A comprises a F-HNA. In certain embodiments, each A comprises a 5′-substituted sugar moiety selected from among 5′-Me DNA, and 5′-(R)-Me DNA.
  • each B comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, each B comprises a modified sugar moiety. In certain embodiments, each B comprises a 2′-substituted sugar moiety. In certain embodiments, each B comprises a 2′-substituted sugar moiety selected from among F, (ara)-F, OCH 3 and O(CH 2 ) 2 —OCH 3 . In certain embodiments, each B comprises a bicyclic sugar moiety. In certain embodiments, each B comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, ⁇ -L-LNA, ENA and 2′-thio LNA.
  • each B comprises a modified nucleobase. In certain embodiments, each B comprises a modified nucleobase selected from among 2-thio-thymidine nucleoside and 5-propyne urindine nucleoside. In certain embodiments, each B comprises an HNA. In certain embodiments, each B comprises a F-HNA. In certain embodiments, each B comprises a 5′-substituted sugar moiety selected from among 5′-Me DNA, and 5′-(R)-Me DNA.
  • each A comprises a 2′-substituted sugar moiety selected from among F, ara-F, OCH 3 and O(CH 2 ) 2 —OCH 3 and each B comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, ⁇ -L-LNA, ENA and 2′-thio LNA.
  • each A comprises O(CH 2 ) 2 —OCH 3 and each B comprises cEt.
  • each C comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, each C comprises a modified sugar moiety. In certain embodiments, each C comprises a 2′-substituted sugar moiety. In certain embodiments, each C comprises a 2′-substituted sugar moiety selected from among F, (ara)-F, OCH 3 and O(CH 2 ) 2 —OCH 3 . In certain embodiments, each C comprises a 5′-substituted sugar moiety. In certain embodiments, each C comprises a 5′-substituted sugar moiety selected from among 5′-Me DNA, and 5′-(R)-Me DNA.
  • each C comprises a bicyclic sugar moiety. In certain embodiments, each C comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, ⁇ -L-LNA, ENA and 2′-thio LNA. In certain embodiments, each C comprises a modified nucleobase. In certain embodiments, each C comprises a modified nucleobase selected from among 2-thio-thymidine and 5-propyne uridine. In certain embodiments, each C comprises a 2-thio-thymidine nucleoside. In certain embodiments, each C comprises an HNA. In certain embodiments, each C comprises an F-HNA.
  • the 3′-wing of a gapmer consists of 1 to 6 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 1 to 5 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 2 to 5 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 3 to 5 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 4 or 5 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 1 to 4 linked nucleosides.
  • the 3′-wing of a gapmer consists of 1 to 3 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 1 or 2 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 2 to 4 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 2 or 3 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 3 or 4 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 1 nucleoside. In certain embodiments, the 3′-wing of a gapmer consists of 2 linked nucleosides.
  • the 3′-wing of a gapmer consists of 31 inked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 4 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 5 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 6 linked nucleosides.
  • the 3′-wing of a gapmer comprises at least one bicyclic nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one constrained ethyl nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one LNA nucleoside. In certain embodiments, each nucleoside of the 3′-wing of a gapmer is a bicyclic nucleoside. In certain embodiments, each nucleoside of the 3′-wing of a gapmer is a constrained ethyl nucleoside. In certain embodiments, each nucleoside of the 3′-wing of a gapmer is a LNA nucleoside.
  • the 3′-wing of a gapmer comprises at least one non-bicyclic modified nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least two non-bicyclic modified nucleosides. In certain embodiments, the 3′-wing of a gapmer comprises at least three non-bicyclic modified nucleosides. In certain embodiments, the 3′-wing of a gapmer comprises at least four non-bicyclic modified nucleosides. In certain embodiments, the 3′-wing of a gapmer comprises at least one 2′-substituted nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one 2′-MOE nucleoside.
  • the 3′-wing of a gapmer comprises at least one 2′-OMe nucleoside.
  • each nucleoside of the 3′-wing of a gapmer is a non-bicyclic modified nucleoside.
  • each nucleoside of the 3′-wing of a gapmer is a 2′-substituted nucleoside.
  • each nucleoside of the 3′-wing of a gapmer is a 2′-MOE nucleoside.
  • each nucleoside of the 3′-wing of a gapmer is a 2′-OMe nucleoside.
  • the 3′-wing of a gapmer comprises at least one 2′-deoxynucleoside. In certain embodiments, each nucleoside of the 3′-wing of a gapmer is a 2′-deoxynucleoside. In a certain embodiments, the 3′-wing of a gapmer comprises at least one ribonucleoside. In certain embodiments, each nucleoside of the 3′-wing of a gapmer is a ribonucleoside. In certain embodiments, one, more than one, or each of the nucleosides of the 5′-wing is an RNA-like nucleoside.
  • the 3′-wing of a gapmer comprises at least one bicyclic nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2′-substituted nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2′-MOE nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2′-OMe nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2′-deoxynucleoside.
  • the 3′-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2′-substituted nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2′-MOE nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2′-OMe nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2′-deoxynucleoside.
  • the 3′-wing of a gapmer comprises at least one LNA nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one LNA nucleoside and at least one 2′-substituted nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one LNA nucleoside and at least one 2′-MOE nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one LNA nucleoside and at least one 2′-OMe nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one LNA nucleoside and at least one 2′-deoxynucleoside.
  • the 3′-wing of a gapmer comprises at least one bicyclic nucleoside, at least one non-bicyclic modified nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one constrained ethyl nucleoside, at least one non-bicyclic modified nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one LNA nucleoside, at least one non-bicyclic modified nucleoside, and at least one 2′-deoxynucleoside.
  • the 3′-wing of a gapmer comprises at least one bicyclic nucleoside, at least one 2′-substituted nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one constrained ethyl nucleoside, at least one 2′-substituted nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one LNA nucleoside, at least one 2′-substituted nucleoside, and at least one 2′-deoxynucleoside.
  • the 3′-wing of a gapmer comprises at least one bicyclic nucleoside, at least one 2′-MOE nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one constrained ethyl nucleoside, at least one 2′-MOE nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one LNA nucleoside, at least one 2′-MOE nucleoside, and at least one 2′-deoxynucleoside.
  • the 3′-wing of a gapmer comprises at least one bicyclic nucleoside, at least one 2′-OMe nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one constrained ethyl nucleoside, at least one 2′-OMe nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one LNA nucleoside, at least one 2′-OMe nucleoside, and at least one 2′-deoxynucleoside.
  • the 3′-wing of a gapmer has a nucleoside motif selected from among the following: ABB, ABAA, AAABAA, AAAAABAA, AABAA, AAAABAA, AAABAA, ABAB, AAAAA, AAABB, AAAAAAAA, AAAAAAA, AAAAAA, AAAAB, AAAA, AAA, AA, AB, ABBB, ABAB, AABBB; wherein each A is a modified nucleoside of a first type, each B is a modified nucleoside of a second type.
  • an oligonucleotide comprises any 3′-wing motif provided herein.
  • the oligonucleotide is a 3′-hemimer (does not comprise a 5′-wing). In certain embodiments, such an oligonucleotide is a gapmer. In certain such embodiments, the 5′-wing of the gapmer may comprise any nucleoside motif.
  • the 3′-wing of a gapmer has a nucleoside motif selected from among the following: BBA, AAB, AAA, BBB, BBAA, AABB, WBBA, WAAB, BBBA, BBBBA, BBBB, BBBBBA, ABBBBB, BBAAA, AABBB, BBBAA, BBBBA, BBBBB, BABA, AAAAA, BBAAAA, AABBBB, BAAAA, and ABBBB, wherein each A is a modified nucleoside of a first type, each B is a modified nucleoside of a second type, and each W is a modified nucleoside of either the first type, the second type or a third type.
  • the 3′-wing of a gapmer has a nucleoside motif selected from among the following: ABB; AAABAA; AABAA; AAAABAA; AAAAA; AAABB; AAAAAAAA; AAAAAAA; AAAAAA; AAAAB; AB; ABBB; and ABAB, wherein each A is a modified nucleoside of a first type, each B is a modified nucleoside of a second type, and each W is a modified nucleoside of either the first type, the second type or a third type.
  • the 3′-wing of a gapmer has a sugar motif selected from among those listed in the following non-limiting tables:
  • each A, each B, and each C located at the 5′-most 3′-wing region nucleoside is a modified nucleoside.
  • the 3′-wing motif is selected from among A BB, B BB, and C BB, wherein the underlined nucleoside represents the 5′-most 3′-wing region nucleoside and wherein the underlined nucleoside is a modified nucleoside.
  • each A comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, each A comprises a modified sugar moiety. In certain embodiments, each A comprises a 2′-substituted sugar moiety. In certain embodiments, each A comprises a 2′-substituted sugar moiety selected from among F, ara-F, OCH 3 and O(CH 2 ) 2 —OCH 3 . In certain embodiments, each A comprises a bicyclic sugar moiety. In certain embodiments, each A comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, ⁇ -L-LNA, ENA and 2′-thio LNA.
  • each A comprises a modified nucleobase. In certain embodiments, each A comprises a modified nucleobase selected from among 2-thio-thymidine nucleoside and 5-propyne uridine nucleoside. In certain embodiments, each A comprises a 5′-substituted sugar moiety selected from among 5′-Me DNA, and 5′-(R)-Me DNA.
  • each B comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, each B comprises a modified sugar moiety. In certain embodiments, each B comprises a 2′-substituted sugar moiety. In certain embodiments, each B comprises a 2′-substituted sugar moiety selected from among F, (ara)-F, OCH 3 and O(CH 2 ) 2 —OCH 3 . In certain embodiments, each B comprises a bicyclic sugar moiety. In certain embodiments, each B comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, ⁇ -L-LNA, ENA and 2′-thio LNA.
  • each B comprises a modified nucleobase. In certain embodiments, each B comprises a modified nucleobase selected from among 2-thio-thymidine nucleoside and 5-propyne urindine nucleoside. In certain embodiments, each B comprises an HNA. In certain embodiments, each B comprises an F-HNA. In certain embodiments, each B comprises a 5′-substituted sugar moiety selected from among 5′-Me DNA, and 5′-(R)-Me DNA.
  • each A comprises a 2′-substituted sugar moiety selected from among F, ara-F, OCH 3 and O(CH 2 ) 2 —OCH 3 and each B comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, ⁇ -L-LNA, ENA and 2′-thio LNA.
  • each A comprises O(CH 2 ) 2 —OCH 3 and each B comprises cEt.
  • each C comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, each C comprises a modified sugar moiety. In certain embodiments, each C comprises a 2′-substituted sugar moiety. In certain embodiments, each C comprises a 2′-substituted sugar moiety selected from among F, (ara)-F, OCH 3 and O(CH 2 ) 2 —OCH 3 . In certain embodiments, each C comprises a 5′-substituted sugar moiety. In certain embodiments, each C comprises a 5′-substituted sugar moiety selected from among 5′-Me, and 5′-(R)-Me. In certain embodiments, each C comprises a bicyclic sugar moiety.
  • each C comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, ⁇ -L-LNA, ENA and 2′-thio LNA.
  • each C comprises a modified nucleobase.
  • each C comprises a modified nucleobase selected from among 2-thio-thymidine and 5-propyne uridine.
  • each C comprises a 2-thio-thymidine nucleoside.
  • each C comprises an HNA.
  • each C comprises an F-HNA.
  • the gap of a gapmer consists of 6 to 20 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 to 15 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 to 12 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 to 10 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 to 9 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 to 8 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 or 7 linked nucleosides.
  • the gap of a gapmer consists of 7 to 10 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 7 to 9 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 7 or 8 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 8 to 10 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 8 or 9 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 7 linked nucleosides.
  • the gap of a gapmer consists of 8 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 9 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 10 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 11 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 12 linked nucleosides.
  • each nucleoside of the gap of a gapmer is a 2′-deoxynucleoside.
  • the gap comprises one or more modified nucleosides.
  • each nucleoside of the gap of a gapmer is a 2′-deoxynucleoside or is a modified nucleoside that is “DNA-like.”
  • “DNA-like” means that the nucleoside has similar characteristics to DNA, such that a duplex comprising the gapmer and an RNA molecule is capable of activating RNase H. For example, under certain conditions, 2′-(ara)-F have been shown to support RNase H activation, and thus is DNA-like.
  • one or more nucleosides of the gap of a gapmer is not a 2′-deoxynucleoside and is not DNA-like. In certain such embodiments, the gapmer nonetheless supports RNase H activation (e.g., by virtue of the number or placement of the non-DNA nucleosides).
  • gaps comprise a stretch of unmodified 2′-deoxynucleoside interrupted by one or more modified nucleosides, thus resulting in three sub-regions (two stretches of one or more 2′-deoxynucleosides and a stretch of one or more interrupting modified nucleosides).
  • no stretch of unmodified 2′-deoxynucleosides is longer than 5, 6, or 7 nucleosides.
  • such short stretches is achieved by using short gap regions.
  • short stretches are achieved by interrupting a longer gap region.
  • the gap comprises one or more modified nucleosides. In certain embodiments, the gap comprises one or more modified nucleosides selected from among cEt, FHNA, LNA, and 2-thio-thymidine. In certain embodiments, the gap comprises one modified nucleoside. In certain embodiments, the gap comprises a 5′-substituted sugar moiety selected from among 5′-Me, and 5′-(R)-Me. In certain embodiments, the gap comprises two modified nucleosides. In certain embodiments, the gap comprises three modified nucleosides. In certain embodiments, the gap comprises four modified nucleosides. In certain embodiments, the gap comprises two or more modified nucleosides and each modified nucleoside is the same. In certain embodiments, the gap comprises two or more modified nucleosides and each modified nucleoside is different.
  • the gap comprises one or more modified linkages. In certain embodiments, the gap comprises one or more methyl phosphonate linkages. In certain embodiments the gap comprises two or more modified linkages. In certain embodiments, the gap comprises one or more modified linkages and one or more modified nucleosides. In certain embodiments, the gap comprises one modified linkage and one modified nucleoside. In certain embodiments, the gap comprises two modified linkages and two or more modified nucleosides.
  • the gap comprises a nucleoside motif selected from among the following: DDDDXDDDDD; DDDDDXDDDDD; DDDXDDDDD; DDDDXDDDDDD; DDXDDDDDD; DDDXDDDDDD; DDXDDDDD; DDXDDDDD; DDXDDDXDDD; DDDXDDDXDDD; DDXDDDXDD; DDXDDDDXDD; DDXDDDDXDD; DDXDDDDXDD; DDXDDDDXDD; DDXDDDDXDD; DXDDDDXDDD; DDDDXDDD; DXDDDDXDDD; DDDDXDDD; DDDDDXDDD; DDDDDXDDD; DDDDDXDDDDD; DDDDXDDDDD; DDDDXDDD; DDDXDDDDDDD; DDDDXDDDDD; DDDDXDDD; DDDXDDDDDDD; DDDDXDDD; DDDDXDDD;
  • the gap comprises a nucleoside motif selected from among the following: DDDDDDDDD; DXDDDDD; DDXDDDD; DDDXDDDDD; DDDDXDDDD; DDDDDXDDD; DDDDDDXDDDD; DDDDDDDXD; DDDDDDXDDDD; DDXXXDDDD; DDDDXXDDDDD; DDDDXXDDDDD; DDDDXXDDD; DDDDDXXDD; DDDDDXDD; DXDDDDXDD; DXDDXDDDD; DXDDXDDDD; DDXDDDDXD; DDXDDDDXDD; DDXDDXDD; DDXDDXDD; DDXDDDXDD; DDXDXDDDD; DDXDXDDDD; DDXDXDDDD; DDXDXDDDD; DDXDXDDDD; DDXDXDDDD; DDXDXDDDD; DDXDXDDDD; DDXDXDD
  • the gap comprises a nucleoside motif selected from among the following: DDDDXDDDD, DXDDDDD, DXXDDDDDD, DDXDDDDDD, DDDXDDDDD, DDDDXDDDD, DDDDDXDDD, DDDDDDXDD, and DDDDDDDXD, wherein each D is an unmodified deoxynucleoside; and each X is a modified nucleoside or a substituted sugar moiety.
  • the gap comprises a nucleoside motif selected from among the following: DDDDDDDD, DXDDDD, DDXDDDDD, DDDXDDDD, DDDDXDDD, DDDDDXDD, DDDDDDXD, DXDDDDXD, DXDDDDXDD, DXDDXDDDD, DDXXDDDD, DDXDDXDD, DDXDDXDD, DDXDDXDD, DDXDDXDD, DDXDDXDD, DXDDXD, DDDXXD, DDDXDXDD, DDDXDDXD, DDDDXXDD, DDDDXDXD, and DDDDDXXD, wherein each D is an unmodified deoxynucleoside; and each X is a modified nucleoside or a substituted sugar moiety.
  • the gap comprises a nucleoside motif selected from among the following: DXDDDDD, DDXDDDD, DDDXDDD, DDDDXDD, DDDDDXD, DXDDDXD, DXDDXDD, DXDXDDDD, DDXXDDD, DDXDDXD, DDXDDXD, DDDXXDD, DDDXDXD, and DDDDXXD, wherein each D is an unmodified deoxynucleoside; and each X is a modified nucleoside or a substituted sugar moiety.
  • the gap comprises a nucleoside motif selected from among the following: DXDDDD, DDXDDD, DDDXDD, DDDDXD, DXXDDD, DXDXDD, DXDDXD, DDXXDD, DDXDXD, and DDDXXD, wherein each D is an unmodified deoxynucleoside; and each X is a modified nucleoside or a substituted sugar moiety.
  • the gap comprises a nucleoside motif selected from among the following: DXDDDD, DDXDDD, DDDXDD, DDDDXD, DXDDDDD, DDXDDDD, DDDDDXD, DXDDDDDD, DDXDDDDD, DDDXDDDD, DDDDXDDD, DDDDDXDD, DDDDDDXD, DXDDDDDDD; DDXDDDDDD, DDDXDDDDD, DDDDXDD, DDDDDXDD, DDDDXDD, DDDDDDDXD, DXDDDDDDDD, DDXDDDDD, DDDXDDDDDDDD, DDXDDDDDDD, DDDXDDDDDDDD, DDDDXDDDDDDD, DDDXDDDDDDDD, DDDDXDDDDDDD, DDDDDXDDDDDD, DDDDXDDDDDDD, DDDDDXDDDDDD, DDDDXDDDDDDD, DDDDDXDDDDDD, DDDDXDDDDD
  • each X comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, each X comprises a modified sugar moiety. In certain embodiments, each X comprises a 2′-substituted sugar moiety. In certain embodiments, each X comprises a 2′-substituted sugar moiety selected from among F, (ara)-F, OCH 3 and O(CH 2 ) 2 —OCH 3 . In certain embodiments, each X comprises a 5′-substituted sugar moiety. In certain embodiments, each X comprises a 5′-substituted sugar moiety selected from among 5′-Me, and 5′-(R)-Me.
  • each X comprises a bicyclic sugar moiety. In certain embodiments, each X comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, ⁇ -L-LNA, ENA and 2′-thio LNA. In certain embodiments, each X comprises a modified nucleobase. In certain embodiments, each X comprises a modified nucleobase selected from among 2-thio-thymidine and 5-propyne uridine. In certain embodiments, each X comprises a 2-thio-thymidine nucleoside. In certain embodiments, each X comprises an HNA. In certain embodiments, each C comprises an F-HNA. In certain embodiments, X represents the location of a single differentiating nucleobase.
  • a gapmer comprises a 5′-wing, a gap, and a 3′ wing, wherein the 5′-wing, gap, and 3′ wing are independently selected from among those discussed above.
  • a gapmer has a 5′-wing, a gap, and a 3′-wing having features selected from among any of those listed in the tables above and any 5′-wing may be paired with any gap and any 3′-wing.
  • a 5′-wing may comprise AAABB
  • a 3′-wing may comprise BBA
  • the gap may comprise DDDDDDD.
  • a gapmer has a 5′-wing, a gap, and a 3′-wing having features selected from among those listed in the following non-limiting table, wherein each motif is represented as (5′-wing)-(gap)-(3′-wing), wherein each number represents the number of linked nucleosides in each portion of the motif, for example, a 5-10-5 motif would have a 5′-wing comprising 5 nucleosides, a gap comprising 10 nucleosides, and a 3′-wing comprising 5 nucleosides:
  • a gapmer comprises a 5′-wing, a gap, and a 3′ wing, wherein the 5′-wing, gap, and 3′ wing are independently selected from among those discussed above.
  • a gapmer has a 5′-wing, a gap, and a 3′-wing having features selected from among those listed in the following non-limiting tables:
  • each A is a modified nucleoside of a first type
  • each B is a modified nucleoside of a second type and each W is a modified nucleoside or nucleobase of either the first type, the second type or a third type
  • each D is a nucleoside comprising an unmodified 2′ deoxy sugar moiety and unmodified nucleobase
  • N D is modified nucleoside comprising a modified nucleobase and an unmodified 2′ deoxy sugar moiety.
  • each A comprises a modified sugar moiety. In certain embodiments, each A comprises a 2′-substituted sugar moiety. In certain embodiments, each A comprises a 2′-substituted sugar moiety selected from among F, ara-F, OCH 3 and O(CH 2 ) 2 —OCH 3 . In certain embodiments, each A comprises a bicyclic sugar moiety. In certain embodiments, each A comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, ⁇ -L-LNA, ENA and 2′-thio LNA. In certain embodiments, each A comprises a modified nucleobase.
  • each A comprises a modified nucleobase selected from among 2-thio-thymidine nucleoside and 5-propyne uridine nucleoside.
  • each A comprises an HNA.
  • each A comprises an F-HNA.
  • each A comprises a 5′-substituted sugar moiety selected from among 5′-Me, and 5′-(R)-Me.
  • each B comprises a modified sugar moiety. In certain embodiments, each B comprises a 2′-substituted sugar moiety. In certain embodiments, each B comprises a 2′-substituted sugar moiety selected from among F, (ara)-F, OCH 3 and O(CH 2 ) 2 —OCH 3 . In certain embodiments, each B comprises a bicyclic sugar moiety. In certain embodiments, each B comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, ⁇ -L-LNA, ENA and 2′-thio LNA. In certain embodiments, each B comprises a modified nucleobase.
  • each B comprises a modified nucleobase selected from among 2-thio-thymidine nucleoside and 5-propyne urindine nucleoside.
  • each B comprises an HNA.
  • each B comprises an F-HNA.
  • each B comprises a 5′-substituted sugar moiety selected from among 5′-Me, and 5′-(R)-Me.
  • each C comprises a modified sugar moiety. In certain embodiments, each C comprises a 2′-substituted sugar moiety. In certain embodiments, each C comprises a 2′-substituted sugar moiety selected from among F, (ara)-F, OCH 3 and O(CH 2 ) 2 —OCH 3 . In certain embodiments, each C comprises a 5′-substituted sugar moiety. In certain embodiments, each C comprises a 5′-substituted sugar moiety selected from among 5′-Me, and 5′-(R)-Me. In certain embodiments, each C comprises a bicyclic sugar moiety.
  • each C comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, ⁇ -L-LNA, ENA and 2′-thio LNA.
  • each C comprises a modified nucleobase.
  • each C comprises a modified nucleobase selected from among 2-thio-thymidine and 5-propyne uridine.
  • each C comprises a 2-thio-thymidine nucleoside.
  • each C comprises an HNA.
  • each C comprises an F-HNA.
  • each W comprises a modified sugar moiety. In certain embodiments, each W comprises a 2′-substituted sugar moiety. In certain embodiments, each W comprises a 2′-substituted sugar moiety selected from among F, (ara)-F, OCH 3 and O(CH 2 ) 2 —OCH 3 . In certain embodiments, each W comprises a 5′-substituted sugar moiety. In certain embodiments, each W comprises a 5′-substituted sugar moiety selected from among 5′-Me, and 5′-(R)-Me. In certain embodiments, each W comprises a bicyclic sugar moiety.
  • each W comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, ⁇ -L-LNA, ENA and 2′-thio LNA.
  • each W comprises a sugar surrogate.
  • each W comprises a sugar surrogate selected from among HNA and F—HNA.
  • each W comprises a 2-thio-thymidine nucleoside.
  • At least one of A or B comprises a bicyclic sugar moiety, and the other comprises a 2′-substituted sugar moiety.
  • one of A or B is an LNA nucleoside and the other of A or B comprises a 2′-substituted sugar moiety.
  • one of A or B is a cEt nucleoside and the other of A or B comprises a 2′-substituted sugar moiety.
  • one of A or B is an ⁇ -L-LNA nucleoside and the other of A or B comprises a 2′-substituted sugar moiety.
  • one of A or B is an LNA nucleoside and the other of A or B comprises a 2′-MOE sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises a 2′-MOE sugar moiety. In certain embodiments, one of A or B is an ⁇ -L-LNA nucleoside and the other of A or B comprises a 2′-MOE sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises a 2′-F sugar moiety.
  • one of A or B is a cEt nucleoside and the other of A or B comprises a 2′-F sugar moiety.
  • one of A or B is an ⁇ -L-LNA nucleoside and the other of A or B comprises a 2′-F sugar moiety.
  • one of A or B is an LNA nucleoside and the other of A or B comprises a 2′-(ara)-F sugar moiety.
  • one of A or B is a cEt nucleoside and the other of A or B comprises a 2′-(ara)-F sugar moiety.
  • one of A or B is an ⁇ -L-LNA nucleoside and the other of A or B comprises a 2′-(ara)-F sugar moiety.
  • A comprises a bicyclic sugar moiety, and B comprises a 2′-substituted sugar moiety.
  • A is an LNA nucleoside and B comprises a 2′-substituted sugar moiety.
  • A is a cEt nucleoside and B comprises a 2′-substituted sugar moiety.
  • A is an ⁇ -L-LNA nucleoside and B comprises a 2′-substituted sugar moiety.
  • A comprises a bicyclic sugar moiety, and B comprises a 2′-MOE sugar moiety.
  • A is an LNA nucleoside and B comprises a 2′-MOE sugar moiety.
  • A is a cEt nucleoside and B comprises a 2′-MOE sugar moiety.
  • A is an ⁇ -L-LNA nucleoside and B comprises a 2′-MOE sugar moiety.
  • A comprises a bicyclic sugar moiety, and B comprises a 2′-F sugar moiety.
  • A is an LNA nucleoside and B comprises a 2′-F sugar moiety.
  • A is a cEt nucleoside and B comprises a 2′-F sugar moiety.
  • A is an ⁇ -L-LNA nucleoside and B comprises a 2′-F sugar moiety.
  • A comprises a bicyclic sugar moiety, and B comprises a 2′-(ara)-F sugar moiety.
  • A is an LNA nucleoside and B comprises a 2′-(ara)-F sugar moiety.
  • A is a cEt nucleoside and B comprises a 2′-(ara)-F sugar moiety.
  • A is an ⁇ -L-LNA nucleoside and B comprises a 2′-(ara)-F sugar moiety.
  • B comprises a bicyclic sugar moiety, and A comprises a 2′-MOE sugar moiety.
  • B is an LNA nucleoside and A comprises a 2′-MOE sugar moiety.
  • B is a cEt nucleoside and A comprises a 2′-MOE sugar moiety.
  • B is an ⁇ -L-LNA nucleoside and A comprises a 2′-MOE sugar moiety.
  • B comprises a bicyclic sugar moiety, and A comprises a 2′-F sugar moiety.
  • B is an LNA nucleoside and A comprises a 2′-F sugar moiety.
  • B is a cEt nucleoside and A comprises a 2′-F sugar moiety.
  • B is an ⁇ -L-LNA nucleoside and A comprises a 2′-F sugar moiety.
  • B comprises a bicyclic sugar moiety, and A comprises a 2′-(ara)-F sugar moiety.
  • B is an LNA nucleoside and A comprises a 2′-(ara)-F sugar moiety.
  • B is a cEt nucleoside and A comprises a 2′-(ara)-F sugar moiety.
  • B is an ⁇ -L-LNA nucleoside and A comprises a 2′-(ara)-F sugar moiety.
  • At least one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-substituted sugar moiety and W comprises a modified nucleobase.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-substituted sugar moiety, and W comprises a modified nucleobase.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-substituted sugar moiety, and C comprises a modified nucleobase.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-substituted sugar moiety, and W comprises a modified nucleobase.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a modified nucleobase.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a modified nucleobase.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a modified nucleobase.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a modified nucleobase.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and W comprises a modified nucleobase.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a modified nucleobase.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a modified nucleobase.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a modified nucleobase.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a modified nucleobase.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a modified nucleobase.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a modified nucleobase.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a modified nucleobase.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-substituted sugar moiety, and W comprises a 2-thio-thymidine nucleobase.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-substituted sugar moiety, and W comprises a 2-thio-thymidine nucleobase.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-substituted sugar moiety, and W comprises a 2-thio-thymidine nucleobase.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-substituted sugar moiety, and W comprises a 2-thio-thymidine nucleobase.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 2-thio-thymidine nucleobase.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 2-thio-thymidine nucleobase.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 2-thio-thymidine nucleobase.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 2-thio-thymidine nucleobase.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and W comprises a 2-thio-thymidine nucleobase.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a 2-thio-thymidine nucleobase.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a 2-thio-thymidine nucleobase.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a 2-thio-thymidine nucleobase.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 2-thio-thymidine nucleobase.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 2-thio-thymidine nucleobase.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 2-thio-thymidine nucleobase.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises 2-thio-thymidine nucleobase.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 5-propyne uridine nucleobase.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5-propyne uridine nucleobase.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 5-propyne uridine nucleobase.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5-propyne uridine nucleobase.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and W comprises a 5-propyne uridine nucleobase.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5-propyne uridine nucleobase.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a 5-propyne uridine nucleobase.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a 5-propyne uridine nucleobase.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 5-propyne uridine nucleobase.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 5-propyne uridine nucleobase.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 5-propyne uridine nucleobase.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 5-propyne uridine nucleobase.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a sugar surrogate.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a sugar surrogate.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a sugar surrogate.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a sugar surrogate.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and W comprises a sugar surrogate.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a sugar surrogate.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a sugar surrogate.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a sugar surrogate.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a sugar surrogate.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a sugar surrogate.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a sugar surrogate.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises sugar surrogate.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a HNA sugar surrogate.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a HNA sugar surrogate.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a HNA sugar surrogate.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a HNA sugar surrogate.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and W comprises a HNA sugar surrogate.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a HNA sugar surrogate.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a HNA sugar surrogate.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a sugar HNA surrogate.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a HNA sugar surrogate.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a HNA sugar surrogate.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a HNA sugar surrogate.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a HNA sugar surrogate.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a F-HNA sugar surrogate.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a F-HNA sugar surrogate.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a F-HNA sugar surrogate.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a F-HNA sugar surrogate.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and W comprises a F-HNA sugar surrogate.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a F-HNA sugar surrogate.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a F-HNA sugar surrogate.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a F-HNA sugar surrogate.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a F-HNA sugar surrogate.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a F-HNA sugar surrogate.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a F-HNA sugar surrogate.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a F-HNA sugar surrogate.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 5′-Me DNA sugar moiety.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 5′-Me DNA sugar moiety.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 5′-Me DNA sugar moiety.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 5′-Me DNA sugar moiety.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and W comprises a 5′-Me DNA sugar moiety.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a 5′-Me DNA sugar moiety.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a 5′-Me DNA sugar moiety.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a 5′-Me DNA sugar moiety.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 5′-Me DNA sugar moiety.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 5′-Me DNA sugar moiety.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 5′-Me DNA sugar moiety.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 5′-Me DNA sugar moiety.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety.
  • At least two of A, B or W comprises a 2′-substituted sugar moiety, and the other comprises a bicyclic sugar moiety. In certain embodiments, at least two of A, B or W comprises a bicyclic sugar moiety, and the other comprises a 2′-substituted sugar moiety.
  • a gapmer has a sugar motif other than: E-K-K-(D) 9 -K-K-E; E-E-E-E-K-(D) 9 -E-E-E-E; E-K-K-K-(D) 9 -K-K-E; K-E-E-K-(D) 9 -K-E-E-K; K-D-D-K-(D) 9 -K-D-D-K; K-E-K-E-K-(D) 9 -K-E-K-E-K; K-D-K-D-K-(D) 9 -K-D-K-D-K; E-K-E-K-(D) 9 -K-E-K-E; E-E-E-E-E-K-(D) 8 -E-E-E-E-E; or E-K-E-K-E-(D) 9 -E-K-E-K-E-E-(D) 9
  • a gapmer comprises a A-(D) 4 -A-(D) 4 -A-(D) 4 -AA motif. In certain embodiments a gapmer comprises a B-(D) 4 -A-(D) 4 -A-(D) 4 -AA motif. In certain embodiments a gapmer comprises a A-(D) 4 -B-(D) 4 -A-(D) 4 -AA motif. In certain embodiments a gapmer comprises a A-(D) 4 -A-(D) 4 -B-(D) 4 -AA motif. In certain embodiments a gapmer comprises a A-(D) 4 -A-(D) 4 -A-(D) 4 -BA motif.
  • a gapmer comprises a A-(D) 4 -A-(D) 4 -A-(D) 4 -BB motif. In certain embodiments a gapmer comprises a K-(D) 4 -K-(D) 4 -K-(D) 4 -K-E motif.
  • oligonucleotides comprise modified internucleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or modified internucleoside linkage motif.
  • internucleoside linkages are arranged in a gapped motif, as described above for nucleoside motif.
  • the internucleoside linkages in each of two wing regions are different from the internucleoside linkages in the gap region.
  • the internucleoside linkages in the wings are phosphodiester and the internucleoside linkages in the gap are phosphorothioate.
  • the nucleoside motif is independently selected, so such oligonucleotides having a gapped internucleoside linkage motif may or may not have a gapped nucleoside motif and if it does have a gapped nucleoside motif, the wing and gap lengths may or may not be the same.
  • oligonucleotides comprise a region having an alternating internucleoside linkage motif. In certain embodiments, oligonucleotides of the present invention comprise a region of uniformly modified internucleoside linkages. In certain such embodiments, the oligonucleotide comprises a region that is uniformly linked by phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide is uniformly linked by phosphorothioate. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate and at least one internucleoside linkage is phosphorothioate.
  • the oligonucleotide comprises at least 6 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 8 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 10 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 6 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 8 consecutive phosphorothioate internucleoside linkages.
  • the oligonucleotide comprises at least one block of at least 10 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least block of at least one 12 consecutive phosphorothioate internucleoside linkages. In certain such embodiments, at least one such block is located at the 3′ end of the oligonucleotide. In certain such embodiments, at least one such block is located within 3 nucleosides of the 3′ end of the oligonucleotide.
  • oligonucleotides comprise one or more methylphosphonate linkages.
  • oligonucleotides having a gapmer nucleoside motif comprise a linkage motif comprising all phosphorothioate linkages except for one or two methylphosphonate linkages.
  • one methylphosphonate linkage is in the central gap of an oligonucleotide having a gapmer nucleoside motif.
  • Modification motifs define oligonucleotides by nucleoside motif (sugar motif and nucleobase motif) and linkage motif.
  • nucleoside motif sucrose motif and nucleobase motif
  • linkage motif For example, certain oligonucleotides have the following modification motif:
  • each A is a modified nucleoside comprising a 2′-substituted sugar moiety
  • each D is an unmodified 2′-deoxynucleoside
  • each B is a modified nucleoside comprising a bicyclic sugar moiety
  • N D is a modified nucleoside comprising a modified nucleobase
  • s is a phosphorothioate internucleoside linkage.
  • the sugar motif is a gapmer motif.
  • the nucleobase modification motif is a single modified nucleobase at 8 th nucleoside from the 5′-end.
  • the nucleoside motif is an interrupted gapmer where the gap of the sugar modified gapmer is interrupted by a nucleoside comprising a modified nucleobase.
  • the linkage motif is uniform phosphorothioate.
  • each A and B are nucleosides comprising differently modified sugar moieties
  • each D is a nucleoside comprising an unmodified 2′ deoxy sugar moiety
  • each W is a modified nucleoside of either the first type, the second type or a third type
  • each N D is a modified nucleoside comprising a modified nucleobase
  • s is a phosphorothioate internucleoside linkage
  • z is a non-phosphorothioate internucleoside linkage.
  • each A comprises a modified sugar moiety. In certain embodiments, each A comprises a 2′-substituted sugar moiety. In certain embodiments, each A comprises a 2′-substituted sugar moiety selected from among F, (ara)-F, OCH 3 and O(CH 2 ) 2 —OCH 3 . In certain embodiments, each A comprises a bicyclic sugar moiety. In certain embodiments, each A comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, ⁇ -L-LNA, ENA and 2′-thio LNA. In certain embodiments, each A comprises a modified nucleobase.
  • each A comprises a modified nucleobase selected from among 2-thio-thymidine nucleoside and 5-propyne uridine nucleoside.
  • each B comprises a modified sugar moiety.
  • each B comprises a 2′-substituted sugar moiety.
  • each B comprises a 2′-substituted sugar moiety selected from among F, (ara)-F, OCH 3 and O(CH 2 ) 2 —OCH 3 .
  • each B comprises a bicyclic sugar moiety.
  • each B comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, ⁇ -L-LNA, ENA and 2′-thio LNA.
  • each B comprises a modified nucleobase.
  • each B comprises a modified nucleobase selected from among 2-thio-thymidine nucleoside and 5-propyne urindine nucleoside.
  • each A comprises an HNA.
  • each A comprises an F-HNA.
  • each W comprises a modified sugar moiety. In certain embodiments, each W comprises a 2′-substituted sugar moiety. In certain embodiments, each W comprises a 2′-substituted sugar moiety selected from among F, (ara)-F, OCH 3 and O(CH 2 ) 2 —OCH 3 . In certain embodiments, each W comprises a 5′-substituted sugar moiety. In certain embodiments, each W comprises a 5′-substituted sugar moiety selected from among 5′-Me, and 5′-(R)-Me. In certain embodiments, each W comprises a bicyclic sugar moiety.
  • each W comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, ⁇ -L-LNA, ENA and 2′-thio LNA.
  • each W comprises a sugar surrogate.
  • each W comprises a sugar surrogate selected from among HNA and F—HNA.
  • At least one of A or B comprises a bicyclic sugar moiety, and the other comprises a 2′-substituted sugar moiety.
  • one of A or B is an LNA nucleoside and the other of A or B comprises a 2′-substituted sugar moiety.
  • one of A or B is a cEt nucleoside and the other of A or B comprises a 2′-substituted sugar moiety.
  • one of A or B is an ⁇ -L-LNA nucleoside and the other of A or B comprises a 2′-substituted sugar moiety.
  • one of A or B is an LNA nucleoside and the other of A or B comprises a 2′-MOE sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises a 2′-MOE sugar moiety. In certain embodiments, one of A or B is an ⁇ -L-LNA nucleoside and the other of A or B comprises a 2′-MOE sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises a 2′-F sugar moiety.
  • one of A or B is a cEt nucleoside and the other of A or B comprises a 2′-F sugar moiety.
  • one of A or B is an ⁇ -L-LNA nucleoside and the other of A or B comprises a 2′-F sugar moiety.
  • one of A or B is an LNA nucleoside and the other of A or B comprises a 2′-(ara)-F sugar moiety.
  • one of A or B is a cEt nucleoside and the other of A or B comprises a 2′-(ara)-F sugar moiety.
  • one of A or B is an ⁇ -L-LNA nucleoside and the other of A or B comprises a 2′-(ara)-F sugar moiety.
  • A comprises a bicyclic sugar moiety, and B comprises a 2′-substituted sugar moiety.
  • A is an LNA nucleoside and B comprises a 2′-substituted sugar moiety.
  • A is a cEt nucleoside and B comprises a 2′-substituted sugar moiety.
  • A is an ⁇ -L-LNA nucleoside and B comprises a 2′-substituted sugar moiety.
  • A comprises a bicyclic sugar moiety, and B comprises a 2′-MOE sugar moiety.
  • A is an LNA nucleoside and B comprises a 2′-MOE sugar moiety.
  • A is a cEt nucleoside and B comprises a 2′-MOE sugar moiety.
  • A is an ⁇ -L-LNA nucleoside and B comprises a 2′-MOE sugar moiety.
  • A comprises a bicyclic sugar moiety, and B comprises a 2′-F sugar moiety.
  • A is an LNA nucleoside and B comprises a 2′-F sugar moiety.
  • A is a cEt nucleoside and B comprises a 2′-F sugar moiety.
  • A is an ⁇ -L-LNA nucleoside and B comprises a 2′-F sugar moiety.
  • A comprises a bicyclic sugar moiety, and B comprises a 2′-(ara)-F sugar moiety.
  • A is an LNA nucleoside and B comprises a 2′-(ara)-F sugar moiety.
  • A is a cEt nucleoside and B comprises a 2′-(ara)-F sugar moiety.
  • A is an ⁇ -L-LNA nucleoside and B comprises a 2′-(ara)-F sugar moiety.
  • B comprises a bicyclic sugar moiety, and A comprises a 2′-MOE sugar moiety.
  • B is an LNA nucleoside and A comprises a 2′-MOE sugar moiety.
  • B is a cEt nucleoside and A comprises a 2′-MOE sugar moiety.
  • B is an ⁇ -L-LNA nucleoside and A comprises a 2′-MOE sugar moiety.
  • B comprises a bicyclic sugar moiety, and A comprises a 2′-F sugar moiety.
  • B is an LNA nucleoside and A comprises a 2′-F sugar moiety.
  • B is a cEt nucleoside and A comprises a 2′-F sugar moiety.
  • B is an ⁇ -L-LNA nucleoside and A comprises a 2′-F sugar moiety.
  • B comprises a bicyclic sugar moiety, and A comprises a 2′-(ara)-F sugar moiety.
  • B is an LNA nucleoside and A comprises a 2′-(ara)-F sugar moiety.
  • B is a cEt nucleoside and A comprises a 2′-(ara)-F sugar moiety.
  • B is an ⁇ -L-LNA nucleoside and A comprises a 2′-(ara)-F sugar moiety.
  • At least one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-substituted sugar moiety and W comprises a modified nucleobase.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-substituted sugar moiety, and W comprises a modified nucleobase.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-substituted sugar moiety, and C comprises a modified nucleobase.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-substituted sugar moiety, and W comprises a modified nucleobase.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a modified nucleobase.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a modified nucleobase.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a modified nucleobase.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a modified nucleobase.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and W comprises a modified nucleobase.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a modified nucleobase.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a modified nucleobase.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a modified nucleobase.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a modified nucleobase.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a modified nucleobase.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a modified nucleobase.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a modified nucleobase.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-substituted sugar moiety, and W comprises a 2-thio-thymidine nucleobase.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-substituted sugar moiety, and W comprises a 2-thio-thymidine nucleobase.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-substituted sugar moiety, and W comprises a 2-thio-thymidine nucleobase.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-substituted sugar moiety, and W comprises a 2-thio-thymidine nucleobase.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 2-thio-thymidine nucleobase.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 2-thio-thymidine nucleobase.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 2-thio-thymidine nucleobase.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 2-thio-thymidine nucleobase.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and W comprises a 2-thio-thymidine nucleobase.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a 2-thio-thymidine nucleobase.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a 2-thio-thymidine nucleobase.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a 2-thio-thymidine nucleobase.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 2-thio-thymidine nucleobase.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 2-thio-thymidine nucleobase.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 2-thio-thymidine nucleobase.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises 2-thio-thymidine nucleobase.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 5-propyne uridine nucleobase.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5-propyne uridine nucleobase.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 5-propyne uridine nucleobase.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5-propyne uridine nucleobase.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and W comprises a 5-propyne uridine nucleobase.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5-propyne uridine nucleobase.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a 5-propyne uridine nucleobase.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a 5-propyne uridine nucleobase.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 5-propyne uridine nucleobase.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 5-propyne uridine nucleobase.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 5-propyne uridine nucleobase.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 5-propyne uridine nucleobase.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a sugar surrogate.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a sugar surrogate.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a sugar surrogate.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a sugar surrogate.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and W comprises a sugar surrogate.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a sugar surrogate.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a sugar surrogate.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a sugar surrogate.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a sugar surrogate.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a sugar surrogate.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a sugar surrogate.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises sugar surrogate.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a HNA sugar surrogate.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a HNA sugar surrogate.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a HNA sugar surrogate.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a HNA sugar surrogate.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and W comprises a HNA sugar surrogate.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a HNA sugar surrogate.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a HNA sugar surrogate.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a sugar HNA surrogate.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a HNA sugar surrogate.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a HNA sugar surrogate.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a HNA sugar surrogate.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a HNA sugar surrogate.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a F-HNA sugar surrogate.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a F—HNA sugar surrogate.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a F-HNA sugar surrogate.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a F-HNA sugar surrogate.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and W comprises a F-HNA sugar surrogate.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a F-HNA sugar surrogate.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a F-HNA sugar surrogate.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a F-HNA sugar surrogate.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a F-HNA sugar surrogate.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a F-HNA sugar surrogate.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a F-HNA sugar surrogate.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a F-HNA sugar surrogate.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 5′-Me DNA sugar moiety.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 5′-Me DNA sugar moiety.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 5′-Me DNA sugar moiety.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 5′-Me DNA sugar moiety.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and W comprises a 5′-Me DNA sugar moiety.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a 5′-Me DNA sugar moiety.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a 5′-Me DNA sugar moiety.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a 5′-Me DNA sugar moiety.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 5′-Me DNA sugar moiety.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 5′-Me DNA sugar moiety.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 5′-Me DNA sugar moiety.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 5′-Me DNA sugar moiety.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety.
  • one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety.
  • one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety.
  • one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety.
  • one of A or B is an ⁇ -L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety.
  • At least two of A, B or W comprises a 2′-substituted sugar moiety, and the other comprises a bicyclic sugar moiety. In certain embodiments, at least two of A, B or W comprises a bicyclic sugar moiety, and the other comprises a 2′-substituted sugar moiety.
  • the present invention provides oligomeric compounds including oligonucleotides of any of a variety of ranges of lengths.
  • the invention provides oligomeric compounds or oligonucleotides consisting of X to Y linked nucleosides, where X represents the fewest number of nucleosides in the range and Y represents the largest number of nucleosides in the range.
  • X and Y are each independently selected from 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50; provided that X ⁇ Y.
  • the invention provides oligomeric compounds which comprise oligonucleotides consisting of 8 to 9, 8 to 10, 8 to 11, 8 to 12, 8 to 13, 8 to 14, 8 to 15, 8 to 16, 8 to 17, 8 to 18, 8 to 19, 8 to 20, 8 to 21, 8 to 22, 8 to 23, 8 to 24, 8 to 25, 8 to 26, 8 to 27, 8 to 28, 8 to 29, 8 to 30, 9 to 10, 9 to 11, 9 to 12, 9 to 13, 9 to 14, 9 to 15, 9 to 16, 9 to 17, 9 to 18, 9 to 19, 9 to 20, 9 to 21, 9 to 22, 9 to 23, 9 to 24, 9 to 25, 9 to 26, 9 to 27, 9 to 28, 9 to 29, 9 to 30, 10 to 11, 10 to 12, 10 to 13, 10 to 14, 10 to 15, 10 to 16, 10 to 17, 10 to 18, 10 to 19, 10 to 20, 10 to 21, 10 to 22, 10 to 23, 10 to 24, 10 to 25, 10 to 26, 10 to 27, 10 to 28, 10 to 29, 10 to 30, 11 to 12, 11 to 13, 11 to 14, 11 to 15, 11 to 16, 11 to 17, 11 to 18, 11 to 19, 11 to 20, 11 to 21, 11 to 22, 11 to 23, 11 to 15, 11 to 16, 11 to
  • the oligomeric compound or oligonucleotide may, nonetheless further comprise additional other substituents.
  • an oligonucleotide comprising 8-30 nucleosides excludes oligonucleotides having 31 nucleosides, but, unless otherwise indicated, such an oligonucleotide may further comprise, for example one or more conjugates, terminal groups, or other substituents.
  • a gapmer oligonucleotide has any of the above lengths.
  • an oligonucleotide is described by an overall length range and by regions having specified lengths, and where the sum of specified lengths of the regions is less than the upper limit of the overall length range, the oligonucleotide may have additional nucleosides, beyond those of the specified regions, provided that the total number of nucleosides does not exceed the upper limit of the overall length range.
  • oligonucleotides of the present invention are characterized by their modification motif and overall length. In certain embodiments, such parameters are each independent of one another.
  • each internucleoside linkage of an oligonucleotide having a gapmer sugar motif may be modified or unmodified and may or may not follow the gapmer modification pattern of the sugar modifications.
  • the internucleoside linkages within the wing regions of a sugar-gapmer may be the same or different from one another and may be the same or different from the internucleoside linkages of the gap region.
  • sugar-gapmer oligonucleotides may comprise one or more modified nucleobase independent of the gapmer pattern of the sugar modifications.
  • oligonucleotide motifs may be combined to create a variety of oligonucleotides.
  • oligonucleotide or oligomeric compound is silent with respect to one or more parameter, such parameter is not limited.
  • an oligomeric compound described only as having a gapmer sugar motif without further description may have any length, internucleoside linkage motif, and nucleobase modification motif. Unless otherwise indicated, all chemical modifications are independent of nucleobase sequence.
  • oligomeric compounds are modified by attachment of one or more conjugate groups.
  • conjugate groups modify one or more properties of the attached oligomeric compound including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, cellular distribution, cellular uptake, charge and clearance.
  • Conjugate groups are routinely used in the chemical arts and are linked directly or via an optional conjugate linking moiety or conjugate linking group to a parent compound such as an oligomeric compound, such as an oligonucleotide.
  • Conjugate groups includes without limitation, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins and dyes.
  • Certain conjugate groups have been described previously, for example: cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci.
  • Acids Res., 1990, 18, 3777-3783 a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937).
  • a conjugate group comprises an active drug substance, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fen-bufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indo-methicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.
  • active drug substance for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fen-bufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansyls
  • conjugate groups are directly attached to oligonucleotides in oligomeric compounds.
  • conjugate groups are attached to oligonucleotides by a conjugate linking group.
  • conjugate linking groups including, but not limited to, bifunctional linking moieties such as those known in the art are amenable to the compounds provided herein.
  • Conjugate linking groups are useful for attachment of conjugate groups, such as chemical stabilizing groups, functional groups, reporter groups and other groups to selective sites in a parent compound such as for example an oligomeric compound.
  • a bifunctional linking moiety comprises a hydrocarbyl moiety having two functional groups.
  • One of the functional groups is selected to bind to a parent molecule or compound of interest and the other is selected to bind essentially any selected group such as chemical functional group or a conjugate group.
  • the conjugate linker comprises a chain structure or an oligomer of repeating units such as ethylene glycol or amino acid units.
  • functional groups that are routinely used in a bifunctional linking moiety include, but are not limited to, electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups.
  • bifunctional linking moieties include amino, hydroxyl, carboxylic acid, thiol, unsaturations (e.g., double or triple bonds), and the like.
  • conjugate linking moieties include pyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and 6-aminohexanoic acid (AHEX or AHA).
  • ADO 8-amino-3,6-dioxaoctanoic acid
  • SMCC succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate
  • AHEX or AHA 6-aminohexanoic acid
  • linking groups include, but are not limited to, substituted C 1 -C 10 alkyl, substituted or unsubstituted C 2 -C 10 alkenyl or substituted or unsubstituted C 2 -C 10 alkynyl, wherein a nonlimiting list of preferred substituent groups includes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.
  • Conjugate groups may be attached to either or both ends of an oligonucleotide (terminal conjugate groups) and/or at any internal position.
  • conjugate groups are at the 3′-end of an oligonucleotide of an oligomeric compound. In certain embodiments, conjugate groups are near the 3′-end. In certain embodiments, conjugates are attached at the 3′ end of an oligomeric compound, but before one or more terminal group nucleosides. In certain embodiments, conjugate groups are placed within a terminal group. In certain embodiments, the present invention provides oligomeric compounds. In certain embodiments, oligomeric compounds comprise an oligonucleotide. In certain embodiments, an oligomeric compound comprises an oligonucleotide and one or more conjugate and/or terminal groups.
  • conjugate and/or terminal groups may be added to oligonucleotides having any of the motifs discussed above.
  • an oligomeric compound comprising an oligonucleotide having region of alternating nucleosides may comprise a terminal group.
  • oligomeric compounds provided herein are antisense compounds. Such antisense compounds are capable of hybridizing to a target nucleic acid, resulting in at least one antisense activity. In certain embodiments, antisense compounds specifically hybridize to one or more target nucleic acid.
  • a specifically hybridizing antisense compound has a nucleobase sequence comprising a region having sufficient complementarity to a target nucleic acid to allow hybridization and result in antisense activity and insufficient complementarity to any non-target so as to avoid non-specific hybridization to any non-target nucleic acid sequences under conditions in which specific hybridization is desired (e.g., under physiological conditions for in vivo or therapeutic uses, and under conditions in which assays are performed in the case of in vitro assays).
  • the present invention provides antisense compounds comprising oligonucleotides that are fully complementary to the target nucleic acid over the entire length of the oligonucleotide. In certain embodiments, oligonucleotides are 99% complementary to the target nucleic acid. In certain embodiments, oligonucleotides are 95% complementary to the target nucleic acid. In certain embodiments, such oligonucleotides are 90% complementary to the target nucleic acid.
  • such oligonucleotides are 85% complementary to the target nucleic acid. In certain embodiments, such oligonucleotides are 80% complementary to the target nucleic acid. In certain embodiments, an antisense compound comprises a region that is fully complementary to a target nucleic acid and is at least 80% complementary to the target nucleic acid over the entire length of the oligonucleotide. In certain such embodiments, the region of full complementarity is from 6 to 14 nucleobases in length.
  • hybridization of an antisense compound results in recruitment of a protein that cleaves of the target nucleic acid.
  • certain antisense compounds result in RNase H mediated cleavage of target nucleic acid.
  • RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex.
  • the “DNA” in such an RNA:DNA duplex need not be unmodified DNA.
  • the invention provides antisense compounds that are sufficiently “DNA-like” to elicit RNase H activity.
  • DNA-like antisense compounds include, but are not limited to gapmers having unmodified deoxyfuronose sugar moieties in the nucleosides of the gap and modified sugar moieties in the nucleosides of the wings.
  • Antisense activities may be observed directly or indirectly.
  • observation or detection of an antisense activity involves observation or detection of a change in an amount of a target nucleic acid or protein encoded by such target nucleic acid; a change in the ratio of splice variants of a nucleic acid or protein; and/or a phenotypic change in a cell or animal.
  • compounds comprising oligonucleotides having a gapmer nucleoside motif described herein have desirable properties compared to non-gapmer oligonucleotides or to gapmers having other motifs. In certain circumstances, it is desirable to identify motifs resulting in a favorable combination of potent antisense activity and relatively low toxicity. In certain embodiments, compounds of the present invention have a favorable therapeutic index (measure of activity divided by measure of toxicity).
  • antisense compounds provided are selective for a target relative to a non-target nucleic acid.
  • the nucleobase sequences of the target and non-target nucleic acids differ by no more than 4 differentiating nucleobases in the targeted region.
  • the nucleobase sequences of the target and non-target nucleic acids differ by no more than 3 differentiating nucleobases in the targeted region.
  • the nucleobase sequences of the target and non-target nucleic acids differ by no more than 2 differentiating nucleobases in the targeted region.
  • the nucleobase sequences of the target and non-target nucleic acids differ by a single differentiating nucleobase in the targeted region.
  • the target and non-target nucleic acids are transcripts from different genes.
  • the target and non-target nucleic acids are different alleles for the same gene.
  • the introduction of a mismatch between an antisense compound and a non-target nucleic acid may alter the RNase H cleavage site of a target nucleic acid compared to a non-target nucleic acid.
  • the target and non-target nucleic acids are not functionally related to one another (e.g., are transcripts from different genes).
  • the target and not-target nucleic acids are allelic variants of one another.
  • the allelic variant contains a single nucleotide polymorphism (SNP).
  • SNP single nucleotide polymorphism
  • a SNP is associated with a mutant allele.
  • a mutant SNP is associated with a disease.
  • a mutant SNP is associated with a disease, but is not causative of the disease.
  • mRNA and protein expression of a mutant allele is associated with disease.
  • antisense compounds are achieved, principally, by nucleobase complementarity. For example, if an antisense compound has no mismatches for a target nucleic acid and one or more mismatches for a non-target nucleic acid, some amount of selectivity for the target nucleic acid will result. In certain embodiments, provided herein are antisense compounds with enhanced selectivity (i.e. the ratio of activity for the target to the activity for non-target is greater).
  • a selective nucleoside comprises a particular feature or combination of features (e.g., chemical modification, motif, placement of selective nucleoside, and/or self-complementary region) that increases selectivity of an antisense compound compared to an antisense compound not having that feature or combination of features.
  • a feature or combination of features increases antisense activity for the target.
  • such feature or combination of features decreases activity for the target, but decreases activity for the non-target by a greater amount, thus resulting in an increase in selectivity.
  • a selective antisense compound comprises a modified nucleoside at that same position as a differentiating nucleobase (i.e., the selective nucleoside is modified). That modification may increase the difference in binding affinity of the antisense compound for the target relative to the non-target.
  • the chemical modification may increase the difference in RNAse H activity for the duplex formed by the antisense compound and its target compared to the RNase activity for the duplex formed by the antisense compound and the non-target.
  • the modification may exaggerate a structure that is less compatible for RNase H to bind, cleave and/or release the non-target.
  • an antisense compound binds its intended target to form a target duplex.
  • RNase H cleaves the target nucleic acid of the target duplex.
  • the same antisense compound hybridizes to a non-target to form a non-target duplex.
  • the non-target differs from the target by a single nucleobase within the target region, and so the antisense compound hybridizes with a single mismatch. Because of the mismatch, in certain embodiments, RNase H cleavage of the non-target may be reduced compared to cleavage of the target, but still occurs. In certain embodiments, though, the primary site of that cleavage of the non-target nucleic acid (primary non-target cleavage site) is different from that of the target. That is; the primary site is shifted due to the mismatch. In such a circumstance, one may use a modification placed in the antisense compound to disrupt RNase H cleavage at the primary non-target cleavage site. Such modification will result in reduced cleavage of the non-target, but will result little or no decrease in cleavage of the target. In certain embodiments, the modification is a modified sugar, nucleobase and/or linkage.
  • the primary non-target cleavage site is towards the 5′-end of the antisense compound, and the 5′-end of an antisense compound may be modified to prevent RNaseH cleavage.
  • the 5′-end of an antisense compound may be modified to prevent RNaseH cleavage.
  • one having skill in the art may modify the 5′-end of an antisense compound, or modify the nucleosides in the gap region of the 5′-end of the antisense compound, or modify the 3′-most 5′-region nucleosides of the antisense compound to selectively inhibit RNaseH cleavage of the non-target nucleic acid duplex while retaining RNase H cleavage of the target nucleic acid duplex.
  • 1-3 of the 3′-most 5′-region nucleosides of the antisense compound comprises a bicyclic sugar moiety.
  • the target nucleic acid may have an allelic variant, e.g. a non-target nucleic acid, containing a single nucleotide polymorphism.
  • An antisense compound may be designed having a single nucleobase mismatch from the non-target nucleic acid, but which has full complementarity to the target nucleic acid. The mismatch between the antisense compound and the non-target nucleic acid may destabilize the antisense compound non-target nucleic acid duplex, and consequently the cleavage site of RNaseH may shift upstream towards the 5′-end of the antisense compound.
  • Modification of the 5′-end of the antisense compound or the gap region near the 5′-end of the antisense compound, or one or more of the 3′-most nucleosides of the 5′-wing region, will then prevent RNaseH cleavage of the non-target nucleic acid. Since the target nucleic acid is fully complementary to the antisense compound, the antisense compound and the target nucleic acid will form a more stabilized antisense compound-target nucleic acid duplex and the cleavage site of RnaseH will be more downstream, towards the 3′ end of the antisense compound.
  • one or more of the 3′-most nucleosides of the 5′-wing region comprises a bicyclic sugar moiety. In certain embodiments, one or more of the 3′-most nucleosides of the 5′-wing region comprises a bicyclic sugar moiety selected from cEt and LNA. In certain embodiments, one or more of the 3′-most nucleosides of the 5′-wing region comprises cEt. In certain embodiments, one or more of the 3′-most nucleosides of the 5′-wing region comprises LNA.
  • the introduction of a mismatch between an antisense compound and a target nucleic acid may alter the RNase H cleavage site of a target nucleic acid compared to a non-target nucleic acid by shifting the RNaseH cleavage site downstream from the mismatch site and towards the 3′-end of the antisense compound.
  • the 3′-end of an antisense compound may be modified to prevent RNaseH cleavage.
  • the target nucleic acid may have an allelic variant, e.g. a non-target nucleic acid, containing a single nucleotide polymorphism.
  • An antisense compound may be designed having a single nucleobase mismatch from the non-target nucleic acid, but which has full complementarity to target nucleic acid. The mismatch between the antisense compound and the non-target nucleic acid may destabilize the antisense compound-non-target nucleic acid duplex, and consequently the cleavage site of RNaseH may shift downstream towards the 3′-end of the antisense compound.
  • Modification of the 3′-end of the antisense compound, or one or more of the 5′-most nucleosides of the 3′-wing region, or the gap region of the antisense compound near the 3′-end will then prevent RNaseH cleavage of the non-target nucleic acid. Since the target nucleic acid is fully complementary to the antisense compound, the antisense compound and the target nucleic acid will form a more stabilized antisense compound-target nucleic acid duplex and the cleavage site of RnaseH will be more upstream, towards the 5′ end of the antisense compound.
  • one or more of the 5′-most nucleosides of the 3′-wing region comprises a bicyclic sugar moiety. In certain embodiments, one or more of the 5′-most nucleosides of the 3′-wing region comprises a bicyclic sugar moiety selected from cEt and LNA. In certain embodiments, one or more of the 5′-most nucleosides of the 3′-wing region comprises cEt. In certain embodiments, one or more of the 5′-most nucleosides of the 3′-wing region comprises LNA.
  • the selectivity of antisense compounds having certain gaps may be improved by the addition of one or more bicyclic nucleosides at the 3′-most 5′-wing nucleoside. In certain embodiments, the selectivity of antisense compounds having certain gaps, e.g. gaps of 7 nucleosides or longer, may be improved by the addition of two or more bicyclic nucleosides at the 3′-most 5′-wing nucleoside. In certain embodiments, the selectivity of antisense compounds having certain gaps, e.g.
  • gaps of 7 nucleosides or longer may be improved by the addition of one bicyclic nucleoside at the 3′-most 5′-wing nucleoside.
  • the selectivity of antisense compounds having certain gaps may be improved by the addition of two bicyclic nucleosides at the 3′-most 5′-wing nucleoside.
  • the selectivity of antisense compounds having certain gaps may be improved by the addition of three bicyclic nucleosides at the 3′-most 5′-wing nucleoside.
  • the selectivity of antisense compounds having certain gaps e.g.
  • gaps of 7 nucleosides or longer may be improved by the addition of four bicyclic nucleosides at the 3′-most 5′-wing nucleoside.
  • the selectivity of antisense compounds having certain gaps e.g. gaps of 7 nucleosides or longer, may be improved by the addition of five bicyclic nucleosides at the 3′-most 5′-wing nucleoside.
  • the bicyclic nucleosides at the 3′-most 5′-wing nucleoside are selected from among cEt, cMOE, LNA, ⁇ -LNA, ENA and 2′-thio LNA.
  • the bicyclic nucleosides at the 3′-most 5′-wing nucleoside comprise cEt. In certain embodiments discussed above, the bicyclic nucleosides at the 3′-most 5′-wing nucleoside comprise LNA.
  • the selectivity of antisense compounds having certain gaps may be improved by the addition of one or more bicyclic nucleosides at the 3′-most 5′-wing nucleoside and the addition of one or more bicyclic nucleosides at the 5′-most 3′-wing nucleoside.
  • the selectivity of antisense compounds having certain gaps may be improved by the addition of two or more bicyclic nucleosides at the 3′-most 5′-wing nucleoside and the addition of one or more bicyclic nucleosides at the 5′-most 3′-wing nucleoside.
  • the selectivity of antisense compounds having certain gaps may be improved by the addition of one bicyclic nucleoside at the 3′-most 5′-wing nucleoside and the addition of one or more bicyclic nucleosides at the 5′-most 3′-wing nucleoside.
  • the selectivity of antisense compounds having certain gaps may be improved by the addition of two bicyclic nucleosides at the 3′-most 5′-wing nucleoside and the addition of one or more bicyclic nucleosides at the 5′-most 3′-wing nucleoside.
  • the selectivity of antisense compounds having certain gaps may be improved by the addition of three bicyclic nucleosides at the 3′-most 5′-wing nucleoside and the addition of one or more bicyclic nucleosides at the 5′-most 3′-wing nucleoside.
  • the selectivity of antisense compounds having certain gaps may be improved by the addition of four bicyclic nucleosides at the 3′-most 5′-wing nucleoside and the addition of one or more bicyclic nucleosides at the 5′-most 3′-wing nucleoside.
  • the selectivity of antisense compounds having certain gaps may be improved by the addition of four bicyclic nucleosides at the 3′-most 5′-wing nucleoside and the addition of one or more bicyclic nucleosides at the 5′-most 3′-wing nucleoside.
  • the selectivity of antisense compounds having certain gaps may be improved by the addition of one or more bicyclic nucleosides at the 3′-most 5′-wing nucleoside. In certain embodiments, the selectivity of antisense compounds having certain gaps, e.g. gaps of 7 nucleosides or shorter, may be improved by the addition of two or more bicyclic nucleosides at the 3′-most 5′-wing nucleoside. In certain embodiments, the selectivity of antisense compounds having certain gaps, e.g.
  • gaps of 7 nucleosides or shorter may be improved by the addition of one bicyclic nucleoside at the 3′-most 5′-wing nucleoside.
  • the selectivity of antisense compounds having certain gaps may be improved by the addition of two bicyclic nucleosides at the 3′-most 5′-wing nucleoside.
  • the selectivity of antisense compounds having certain gaps may be improved by the addition of three bicyclic nucleosides at the 3′-most 5′-wing nucleoside.
  • the selectivity of antisense compounds having certain gaps e.g.
  • gaps of 7 nucleosides or shorter may be improved by the addition of four bicyclic nucleosides at the 3′-most 5′-wing nucleoside.
  • the selectivity of antisense compounds having certain gaps e.g. gaps of 7 nucleosides or shorter, may be improved by the addition of five bicyclic nucleosides at the 3′-most 5′-wing nucleoside.
  • the bicyclic nucleosides at the 3′-most 5′-wing nucleoside are selected from among cEt, cMOE, LNA, ⁇ -LNA, ENA and 2′-thio LNA.
  • the bicyclic nucleosides at the 3′-most 5′-wing nucleoside comprise cEt. In certain embodiments discussed above, the bicyclic nucleosides at the 3′-most 5′-wing nucleoside comprise LNA.
  • Antisense compounds having certain specified motifs have enhanced selectivity, including, but not limited to motifs described above.
  • enhanced selectivity is achieved by oligonucleotides comprising any one or more of:
  • a modification motif comprising a long 5′-wing (longer than 5, 6, or 7 nucleosides);
  • a modification motif comprising a long 3′-wing (longer than 5, 6, or 7 nucleosides);
  • a modification motif comprising a short gap region (shorter than 8, 7, or 6 nucleosides);
  • a modification motif comprising an interrupted gap region (having no uninterrupted stretch of unmodified 2′-deoxynucleosides longer than 7, 6 or 5).
  • selective antisense compounds comprise nucleobase sequence elements.
  • nucleobase sequence elements are independent of modification motifs. Accordingly, oligonucleotides having any of the motifs (modification motifs, nucleoside motifs, sugar motifs, nucleobase modification motifs, and/or linkage motifs) may also comprise one or more of the following nucleobase sequence elements.
  • a target region and a region of a non-target nucleic acid differ by 1-4 differentiating nucleobase.
  • selective antisense compounds have a nucleobase sequence that aligns with the non-target nucleic acid with 1-4 mismatches.
  • a nucleoside of the antisense compound that corresponds to a differentiating nucleobase of the target nucleic acid is referred to herein as a target-selective nucleoside.
  • selective antisense compounds having a gapmer motif align with a non-target nucleic acid, such that a target-selective nucleoside is positioned in the gap.
  • a target-selective nucleoside is the 1 st nucleoside of the gap from the 5′ end. In certain embodiments, a target-selective nucleoside is the 2 nd nucleoside of the gap from the 5′ end. In certain embodiments, a target-selective nucleoside is the 3 rd nucleoside of the gap from the 5′-end. In certain embodiments, a target-selective nucleoside is the 4 th nucleoside of the gap from the 5′-end. In certain embodiments, a target-selective nucleoside is the 5 th nucleoside of the gap from the 5′-end.
  • a target-selective nucleoside is the 6 rd nucleoside of the gap from the 5′-end. In certain embodiments, a target-selective nucleoside is the 8 th nucleoside of the gap from the 3′-end. In certain embodiments, a target-selective nucleoside is the 7 th nucleoside of the gap from the 3′-end. In certain embodiments, a target-selective nucleoside is the 6 th nucleoside of the gap from the 3′-end. In certain embodiments, a target-selective nucleoside is the 5 th nucleoside of the gap from the 3′-end.
  • a target-selective nucleoside is the 4 th nucleoside of the gap from the 3′-end. In certain embodiments, a target-selective nucleoside is the 3 rd nucleoside of the gap from the 3′-end. In certain embodiments, a target-selective nucleoside is the 2 nd nucleoside of the gap from the 3′-end.
  • a target-selective nucleoside comprises a modified nucleoside. In certain embodiments, a target-selective nucleoside comprises a modified sugar. In certain embodiments, a target-selective nucleoside comprises a sugar surrogate. In certain embodiments, a target-selective nucleoside comprises a sugar surrogate selected from among HNA and F-HNA. In certain embodiments, a target-selective nucleoside comprises a 2′-substituted sugar moiety. In certain embodiments, a target-selective nucleoside comprises a 2′-substituted sugar moiety selected from among MOE, F and (ara)-F.
  • a target-selective nucleoside comprises a 5′-substituted sugar moiety. In certain embodiments, a target-selective nucleoside comprises a 5′-substituted sugar moiety selected from 5′-(R)-Me DNA. In certain embodiments, a target-selective nucleoside comprises a bicyclic sugar moiety. In certain embodiments, a target-selective nucleoside comprises a bicyclic sugar moiety selected from among cEt, and ⁇ -L-LNA. In certain embodiments, a target-selective nucleoside comprises a modified nucleobase. In certain embodiments, a target-selective nucleoside comprises a modified nucleobase selected from among 2-thio-thymidine and 5-propyne uridine.
  • selective antisense compounds comprise one or more mismatched nucleobases relative to the target nucleic acid.
  • antisense activity against the target is reduced by such mismatch, but activity against the non-target is reduced by a greater amount.
  • selectivity is improved.
  • Any nucleobase other than the differentiating nucleobase is suitable for a mismatch.
  • the mismatch is specifically positioned within the gap of an oligonucleotide having a gapmer motif.
  • a mismatch relative to the target nucleic acid is at positions 1, 2, 3, 4, 5, 6, 7, or 8 from the 5′-end of the gap region.
  • a mismatch relative to the target nucleic acid is at positions 9, 8, 7, 6, 5, 4, 3, 2, 1 of the antisense compounds from the 3′-end of the gap region. In certain embodiments, a mismatch relative to the target nucleic acid is at positions 1, 2, 3, or 4 of the antisense compounds from the 5′-end of the wing region. In certain embodiments, a mismatch relative to the target nucleic acid is at positions 4, 3, 2, or 1 of the antisense compounds from the 3′-end of the wing region.
  • selective antisense compounds comprise a region that is not complementary to the target. In certain embodiments, such region is complementary to another region of the antisense compound. Such regions are referred to herein as self-complementary regions.
  • an antisense compound has a first region at one end that is complementary to a second region at the other end. In certain embodiments, one of the first and second regions is complementary to the target nucleic acid. Unless the target nucleic acid also includes a self-complementary region, the other of the first and second region of the antisense compound will not be complementary to the target nucleic acid.
  • certain antisense compounds have the following nucleobase motif:
  • such antisense compounds are expected to form self-structure, which is disrupted upon contact with a target nucleic acid.
  • Contact with a non-target nucleic acid is expected to disrupt the self-structure to a lesser degree, thus increasing selectivity compared to the same antisense compound lacking the self-complementary regions.
  • a single antisense compound may include any one, two, three, or more of: self-complementary regions, a mismatch relative to the target nucleic acid, a short nucleoside gap, an interrupted gap, and specific placement of the selective nucleoside.
  • an antisense compound of interest may modulate the expression of a target nucleic acid but possess undesirable properties.
  • an antisense compound of interest may have an undesirably high affinity for one or more non-target nucleic acids.
  • an antisense compound of interest may produce undesirable increases in ALT and/or AST levels when administered to an animal.
  • such an antisense compound of interest may produce undesirable increases in organ weight.
  • an antisense compound of interest effectively modulates the expression of a target nucleic acid, but possess one or more undesirable properties
  • a person having skill in the art may selectively incorporate one or more modifications into the antisense compound of interest that retain some or all of the desired property of effective modulation of expression of a target nucleic acid while reducing one or more of the antisense compound's undesirable properties.
  • the present invention provides methods of altering such an antisense compound of interest to form an improved antisense compound.
  • altering the number of nucleosides in the 5′-region, the 3′-region, and/or the central region of such an antisense compound of interest results in improved properties.
  • an antisense compound having a modification motif of 3-10-3 could be altered to result in an improved antisense compound having a modification motif of 4-9-3 or 5-8-3.
  • the modification state of one or more of nucleosides at or near the 3′-end of the central region may likewise be altered.
  • the modification of one or more of the nucleosides at or near the 5′-end and the 3′-end of the central region may be altered.
  • the central region becomes shorter relative to the central region of the original antisense compound of interest.
  • the modifications to the one or more nucleosides that had been part of the central region are the same as one or more modification that had been present in the 5′-region and/or the 3′-region of the original antisense compound of interest.
  • the improved antisense compound having a shortened central region may retain its ability to effectively modulate the expression of a target nucleic acid, but not possess some or all of the undesirable properties possessed by antisense compound of interest having a longer central region.
  • reducing the length of the central region reduces affinity for off-target nucleic acids.
  • reducing the length of the central region results in reduced cleavage of non-target nucleic acids by RNase H.
  • reducing the length of the central region does not produce undesirable increases in ALT levels.
  • reducing the length of the central region does not produce undesirable increases in AST levels.
  • reducing the length of the central region does not produce undesirable increases organ weights.
  • nucleobase sequence and overall length of an antisense compound it is possible to retain the same nucleobase sequence and overall length of an antisense compound while decreasing the length of the central region. In certain embodiments retaining the same nucleobase sequence and overall length of an antisense compound while decreasing the length of the central region ameliorates one or more undesirable properties of an antisense compound. In certain embodiments retaining the same nucleobase sequence and overall length of an antisense compound while decreasing the length of the central region ameliorates one or more undesirable properties of an antisense compound but does not substantially affect the ability of the antisense compound to modulate expression of a target nucleic acid. In certain such embodiments, two or more antisense compounds would have the same overall length and nucleobase sequence, but would have a different central region length, and different properties.
  • the length of the central region is 9 nucleobases. In certain embodiments, the length of the central region is 8 nucleobases. In certain embodiments, the length of the central region is 7 nucleobases. In certain embodiments, the central region consists of unmodified deoxynucleosides. In certain embodiments, the length of the central region can be decreased by increasing the length of the 5′-region, the 3′-region, or both the 5′-region and the 3′-region.
  • the length of the central region can be decreased by increasing the length of the 5′-region with modified nucleosides. In certain embodiments, the length of the central region can be decreased by increasing the length of the 5′-region with modified nucleosides. In certain embodiments, the length of the central region can be decreased by increasing the length of the 5′-region with modified nucleosides comprising a bicyclic sugar moiety selected from among: cEt, cMOE, LNA, ⁇ -LNA, ENA and 2′-thio LNA. In certain embodiments, the length of the central region can be decreased by increasing the length of the 5′-region with a cEt substituted sugar moiety.
  • the length of the central region can be decreased by increasing the length of the 5′-region with modified nucleosides. In certain embodiments, the length of the central region can be decreased by increasing the length of the 5′-region with modified nucleosides.
  • the length of the central region can be decreased by increasing the length of the 5′-region with modified nucleosides comprising a bicyclic sugar moiety comprising a 2′ substituent selected from among: a halogen, OCH 3 , OCF 3 , OCH 2 CH 3 , OCH 2 CF 3 , OCH 2 —CH ⁇ CH 2 , O(CH 2 ) 2 —OCH 3 (MOE), O(CH 2 ) 2 —O(CH 2 ) 2 —N(CH 3 ) 2 , OCH 2 C( ⁇ O)—N(H)CH 3 , OCH 2 C( ⁇ O)—N(H)—(CH 2 ) 2 —N(CH 3 ) 2 , and OCH 2 —N(H)—C( ⁇ NH)NH 2 .
  • the length of the central region can be decreased by increasing the length of the 5′-region with 2′-O(CH 2 ) 2 —OCH 3 (MOE) substituted sugar moiety
  • the length of the central region can be decreased by increasing the length of the 3′-region with modified nucleosides. In certain embodiments, the length of the central region can be decreased by increasing the length of the 3′-region with modified nucleosides. In certain embodiments, the length of the central region can be decreased by increasing the length of the 3′-region with modified nucleosides comprising a bicyclic sugar moiety selected from among: cEt, cMOE, LNA, ⁇ -LNA, ENA and 2′-thio LNA. In certain embodiments, the length of the central region can be decreased by increasing the length of the 3′-region with a cEt substituted sugar moiety.
  • the length of the central region can be decreased by increasing the length of the 3′-region with modified nucleosides. In certain embodiments, the length of the central region can be decreased by increasing the length of the 3′-region with modified nucleosides.
  • the length of the central region can be decreased by increasing the length of the 3′-region with modified nucleosides comprising a bicyclic sugar moiety comprising a 2′ substituent selected from among: a halogen, OCH 3 , OCF 3 , OCH 2 CH 3 , OCH 2 CF 3 , OCH 2 —CH ⁇ CH 2 , O(CH 2 ) 2 —OCH 3 (MOE), O(CH 2 ) 2 —O(CH 2 ) 2 —N(CH 3 ) 2 , OCH 2 C( ⁇ O)—N(H)CH 3 , OCH 2 C( ⁇ O)—N(H)—(CH 2 ) 2 —N(CH 3 ) 2 , and OCH 2 —N(H)—C( ⁇ NH)NH 2 .
  • the length of the central region can be decreased by increasing the length of the 3′-region with 2′-O(CH 2 ) 2 —OCH 3 (MOE) substituted sugar moiety
  • the length of the central region can be decreased by increasing the length of the 5′-region with modified nucleosides and increasing the length of the 3′-region with modified nucleosides.
  • antisense compounds comprise or consist of an oligonucleotide comprising a region that is complementary to a target nucleic acid.
  • the target nucleic acid is an endogenous RNA molecule.
  • the target nucleic acid is a non-coding RNA.
  • the target non-coding RNA is selected from: a long-non-coding RNA, a short non-coding RNA, an intronic RNA molecule, a snoRNA, a scaRNA, a microRNA (including pre-microRNA and mature microRNA), a ribosomal RNA, and promoter directed RNA.
  • the target nucleic acid encodes a protein.
  • the target nucleic acid is selected from: an mRNA and a pre-mRNA, including intronic, exonic and untranslated regions.
  • oligomeric compounds are at least partially complementary to more than one target nucleic acid.
  • antisense compounds of the present invention may mimic microRNAs, which typically bind to multiple targets.
  • the target nucleic acid is a nucleic acid other than a mature mRNA. In certain embodiments, the target nucleic acid is a nucleic acid other than a mature mRNA or a microRNA. In certain embodiments, the target nucleic acid is a non-coding RNA other than a microRNA. In certain embodiments, the target nucleic acid is a non-coding RNA other than a microRNA or an intronic region of a pre-mRNA. In certain embodiments, the target nucleic acid is a long non-coding RNA. In certain embodiments, the target RNA is an mRNA. In certain embodiments, the target nucleic acid is a pre-mRNA.
  • the target region is entirely within an intron. In certain embodiments, the target region spans an intron/exon junction. In certain embodiments, the target region is at least 50% within an intron. In certain embodiments, the target nucleic acid is selected from among non-coding RNA, including exonic regions of pre-mRNA. In certain embodiments, the target nucleic acid is a ribosomal RNA (rRNA). In certain embodiments, the target nucleic acid is a non-coding RNA associated with splicing of other pre-mRNAs. In certain embodiments, the target nucleic acid is a nuclear-retained non-coding RNA.
  • rRNA ribosomal RNA
  • antisense compounds described herein are complementary to a target nucleic acid comprising a single-nucleotide polymorphism.
  • the antisense compound is capable of modulating expression of one allele of the single-nucleotide polymorphism-containing-target nucleic acid to a greater or lesser extent than it modulates another allele.
  • an antisense compound hybridizes to a single-nucleotide polymorphism-containing-target nucleic acid at the single-nucleotide polymorphism site.
  • the target nucleic acid is a Huntingtin gene transcript.
  • the target nucleic acid is a single-nucleotide polymorphism-containing-target nucleic acid of a Huntingtin gene transcript. In certain embodiments, the target nucleic acid is not a Huntingtin gene transcript. In certain embodiments, the target nucleic acid is a single-nucleotide polymorphism-containing-target nucleic acid of a gene transcript other than Huntingtin. In certain embodiments, the target nucleic acid is any nucleic acid other than a Huntingtin gene transcript.
  • the invention provides selective antisense compounds that have greater activity for a target nucleic acid than for a homologous or partially homologous non-target nucleic acid.
  • the target and non-target nucleic acids are not functionally related to one another (e.g., are transcripts from different genes).
  • the target and not-target nucleic acids are allelic variants of one another.
  • Certain embodiments of the present invention provide methods, compounds, and compositions for selectively inhibiting mRNA and protein expression of an allelic variant of a particular gene or DNA sequence.
  • the allelic variant contains a single nucleotide polymorphism (SNP).
  • SNP single nucleotide polymorphism
  • a mutant SNP is associated with a disease. In certain embodiments a mutant SNP is associated with a disease, but is not causative of the disease. In certain embodiments, mRNA and protein expression of a mutant allele is associated with disease.
  • the expressed gene product of a mutant allele results in aggregation of the mutant proteins causing disease. In certain embodiments, the expressed gene product of a mutant allele results in gain of function causing disease.
  • genes with an autosomal dominant mutation resulting in a toxic gain of function of the protein are the APP gene encoding amyloid precursor protein involved in Alzheimer's disease (Gene, 371: 68, 2006); the PrP gene encoding prion protein involved in Creutzfeldt-Jakob disease and in fatal familial insomnia (Nat. Med. 1997, 3: 1009); GFAP gene encoding glial fibrillary acidic protein involved in Alexander disease (J. Neurosci.
  • alpha-synuclein gene encoding alpha-synuclein protein involved in Parkinson's disease (J. Clin. Invest. 2003, 111: 145); SOD-1 gene encoding the SOD-1 protein involved in amyotrophic lateral sclerosis (Science 1998, 281: 1851); atrophin-1 gene encoding atrophin-1 protein involved in dentato-rubral and pallido-luysian atrophy (DRPA) (Trends Mol. Med. 2001, 7: 479); SCA1 gene encoding ataxin-1 protein involved in spino-cerebellar ataxia-1 (SCA1) (Protein Sci.
  • Ltk gene encoding leukocyte tyrosine kinase protein involved in systemic lupus erythematosus (Hum. Mol. Gen. 2004, 13: 171); PCSK9 gene encoding PCSK9 protein involved in hypercholesterolemia (Hum Mutat. 2009, 30: 520); prolactin receptor gene encoding prolactin receptor protein involved in breast tumors (Proc. Natl. Assoc. Sci. 2008, 105: 4533); CCL5 gene encoding the chemokine CCL5 involved in COPD and asthma (Eur. Respir. J.
  • PTPN22 gene encoding PTPN22 protein involved in Type 1 diabetes, Rheumatoid arthritis, Graves disease, and SLE (Proc. Natl. Assoc. Sci. 2007, 104: 19767); androgen receptor gene encoding the androgen receptor protein involved in spinal and bulbar muscular atrophy or Kennedy's disease (J Steroid Biochem. Mol. Biol. 2008, 108: 245); CHMP4B gene encoding chromatin modifying protein-4B involved in progressive childhood posterior subcapsular cataracts (Am. J. Hum. Genet.
  • AVP gene encoding arginine vasopressin protein in stress-related disorders such as anxiety disorders and comorbid depression (CNS Neurol. Disord. Drug Targets 2006, 5: 167); GNAS gene encoding G proteins involved in congenital visual defects, hypertension, metabolic syndrome (Trends Pharmacol. Sci. 2006, 27: 260); APAF1 gene encoding APAF1 protein involved in a predisposition to major depression (Mol. Psychiatry. 2006, 11: 76); TGF-beta1 gene encoding TGF-beta1 protein involved in breast cancer and prostate cancer (Cancer Epidemiol. Biomarkers Prev.
  • AChR gene encoding acetylcholine receptor involved in congential myasthenic syndrome (Neurology 2004, 62: 1090); P2Y12 gene encoding adenosine diphosphate (ADP) receptor protein involved in risk of peripheral arterial disease (Circulation 2003, 108: 2971); LQT1 gene encoding LQT1 protein involved in atrial fibrillation (Cardiology 2003, 100: 109); RET protooncogene encoding RET protein involved in sporadic pheochromocytoma (J. Clin. Endocrinol. Metab.
  • CA4 gene encoding carbonic anhydrase 4 protein, CRX gene encoding cone-rod homeobox transcription factor protein, FSCN2 gene encoding retinal fascin homolog 2 protein, IMPDH1 gene encoding inosine monophosphate dehydrogenase 1 protein, NR2E3 gene encoding nuclear receptor subfamily 2 group E3 protein, NRL gene encoding neural retina leucine zipper protein, PRPF3 (RP18) gene encoding pre-mRNA splicing factor 3 protein, PRPF8 (RP13) gene encoding pre-mRNA splicing factor 8 protein, PRPF31 (RP11) gene encoding pre-mRNA splicing factor 31 protein, RDS gene encoding peripherin 2 protein, ROM1 gene encoding rod outer membrane protein 1 protein, RHO gene encoding rhodopsin protein, RP1 gene encoding RP1 protein, RPGR gene encoding retinitis pigmentosa GTP
  • the mutant allele is associated with any disease from the group consisting of Alzheimer's disease, Creutzfeldt-Jakob disease, fatal familial insomnia, Alexander disease, Parkinson's disease, amyotrophic lateral sclerosis, dentato-rubral and pallido-luysian atrophy DRPA, spino-cerebellar ataxia, Torsion dystonia, cardiomyopathy, chronic obstructive pulmonary disease (COPD), liver disease, hepatocellular carcinoma, systemic lupus erythematosus, hypercholesterolemia, breast cancer, asthma, Type 1 diabetes, Rheumatoid arthritis, Graves disease, SLE, spinal and bulbar muscular atrophy, Kennedy's disease, progressive childhood posterior subcapsular cataracts, cholesterol gallstone disease, arthrosclerosis, cardiovascular disease, primary hypercalciuria, alpha-thallasemia, obsessive compulsive disorder, Anxiety, comorbid depression, congenital visual defects, hypertension, metabolic syndrome,
  • any disease
  • an allelic variant of huntingtin is selectively reduced.
  • Nucleotide sequences that encode huntingtin include, without limitation, the following: GENBANK Accession No. NT — 006081.18, truncated from nucleotides 1566000 to 1768000 (replaced by GENBANK Accession No. NT — 006051), incorporated herein as SEQ ID NO: 1, and NM — 002111.6, incorporated herein as SEQ ID NO: 2.
  • Table 14 provides SNPs found in the GM04022, GM04281, GM02171, and GM02173B cell lines. Also provided are the allelic variants found at each SNP position, the genotype for each of the cell lines, and the percentage of HD patients having a particular allelic variant. For example, the two allelic variants for SNP rs6446723 are T and C.
  • the GM04022 cell line is heterozygous TC
  • the GM02171 cell line is homozygous CC
  • the GM02173 cell line is heterozygous TC
  • the GM04281 cell line is homozygous TT.
  • Fifty percent of HD patients have a T at SNP position rs6446723.
  • provided herein are methods of treating an animal or individual comprising administering one or more pharmaceutical compositions as described herein.
  • the individual or animal has Huntington's disease.
  • compounds targeted to huntingtin as described herein may be administered to reduce the severity of physiological symptoms of Huntington's disease. In certain embodiments, compounds targeted to huntingtin as described herein may be administered to reduce the rate of degeneration in an individual or an animal having Huntington's disease. In certain embodiments, compounds targeted to huntingtin as described herein may be administered regeneration function in an individual or an animal having Huntington's disease. In certain embodiments, symptoms of Huntingtin's disease may be reversed by treatment with a compound as described herein.
  • compounds targeted to huntingtin as described herein may be administered to ameliorate one or more symptoms of Huntington's disease.
  • administration of compounds targeted to huntingtin as described herein may improve the symptoms of Huntington's disease as measured by any metric known to those having skill in the art.
  • administration of compounds targeted to huntingtin as described herein may improve a rodent's rotaraod assay performance.
  • administration of compounds targeted to huntingtin as described herein may improve a rodent's plus maze assay.
  • administration of compounds targeted to huntingtin as described herein may improve a rodent's open field assay performance.
  • provided herein are methods for ameliorating a symptom associated with Huntington's disease in a subject in need thereof.
  • a method for reducing the rate of onset of a symptom associated with Huntington's disease In certain embodiments, provided is a method for reducing the severity of a symptom associated with Huntington's disease. In certain embodiments, provided is a method for regenerating neurological function as shown by an improvement of a symptom associated with Huntington's disease.
  • the methods comprise administering to an individual or animal in need thereof a therapeutically effective amount of a compound targeted to a huntingtin nucleic acid.
  • Huntington's disease is characterized by numerous physical, neurological, psychiatric, and/or peripheral symptoms. Any symptom known to one of skill in the art to be associated with Huntington's disease can be ameliorated or otherwise modulated as set forth above in the methods described above.
  • the symptom is a physical symptom selected from the group consisting of restlessness, lack of coordination, unintentionally initiated motions, unintentionally uncompleted motions, unsteady gait, chorea, rigidity, writhing motions, abnormal posturing, instability, abnormal facial expressions, difficulty chewing, difficulty swallowing, difficulty speaking, seizure, and sleep disturbances.
  • the symptom is a cognitive symptom selected from the group consisting of impaired planning, impaired flexibility, impaired abstract thinking, impaired rule acquisition, impaired initiation of appropriate actions, impaired inhibition of inappropriate actions, impaired short-term memory, impaired long-term memory, paranoia, disorientation, confusion, hallucination and dementia.
  • the symptom is a psychiatric symptom selected from the group consisting of anxiety, depression, blunted affect, egocentrisms, aggression, compulsive behavior, irritability and suicidal ideation.
  • the symptom is a peripheral symptom selected from the group consisting of reduced brain mass, muscle atrophy, cardiac failure, impaired glucose tolerance, weight loss, osteoporosis, and testicular atrophy.
  • the symptom is restlessness. In certain embodiments, the symptom is lack of coordination. In certain embodiments, the symptom is unintentionally initiated motions. In certain embodiments, the symptom is unintentionally uncompleted motions. In certain embodiments, the symptom is unsteady gait. In certain embodiments, the symptom is chorea. In certain embodiments, the symptom is rigidity. In certain embodiments, the symptom is writhing motions. In certain embodiments, the symptom is abnormal posturing. In certain embodiments, the symptom is instability. In certain embodiments, the symptom is abnormal facial expressions. In certain embodiments, the symptom is difficulty chewing. In certain embodiments, the symptom is difficulty swallowing. In certain embodiments, the symptom is difficulty speaking. In certain embodiments, the symptom is seizures. In certain embodiments, the symptom is sleep disturbances.
  • the symptom is impaired planning. In certain embodiments, the symptom is impaired flexibility. In certain embodiments, the symptom is impaired abstract thinking. In certain embodiments, the symptom is impaired rule acquisition. In certain embodiments, the symptom is impaired initiation of appropriate actions. In certain embodiments, the symptom is impaired inhibition of inappropriate actions. In certain embodiments, the symptom is impaired short-term memory. In certain embodiments, the symptom is impaired long-term memory. In certain embodiments, the symptom is paranoia. In certain embodiments, the symptom is disorientation. In certain embodiments, the symptom is confusion. In certain embodiments, the symptom is hallucination. In certain embodiments, the symptom is dementia.
  • the symptom is anxiety. In certain embodiments, the symptom is depression. In certain embodiments, the symptom is blunted affect. In certain embodiments, the symptom is egocentrism. In certain embodiments, the symptom is aggression. In certain embodiments, the symptom is compulsive behavior. In certain embodiments, the symptom is irritability. In certain embodiments, the symptom is suicidal ideation.
  • the symptom is reduced brain mass. In certain embodiments, the symptom is muscle atrophy. In certain embodiments, the symptom is cardiac failure. In certain embodiments, the symptom is impaired glucose tolerance. In certain embodiments, the symptom is weight loss. In certain embodiments, the symptom is osteoporosis. In certain embodiments, the symptom is testicular atrophy.
  • symptoms of Huntington's disease may be quantifiable.
  • osteoporosis may be measured and quantified by, for example, bone density scans.
  • the symptom may be reduced by about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two of these values.
  • provided are methods of treating an individual comprising administering one or more pharmaceutical compositions as described herein.
  • the individual has Huntington's disease.
  • administering results in reduction of huntingtin expression by at least about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two of these values.
  • compositions comprising an antisense compound targeted to huntingtin are used for the preparation of a medicament for treating a patient suffering or susceptible to Huntington's disease.
  • the present invention provides pharmaceutical compositions comprising one or more antisense compound.
  • such pharmaceutical composition comprises a suitable pharmaceutically acceptable diluent or carrier.
  • a pharmaceutical composition comprises a sterile saline solution and one or more antisense compound.
  • such pharmaceutical composition consists of a sterile saline solution and one or more antisense compound.
  • the sterile saline is pharmaceutical grade saline.
  • a pharmaceutical composition comprises one or more antisense compound and sterile water.
  • a pharmaceutical composition consists of one or more antisense compound and sterile water.
  • the sterile saline is pharmaceutical grade water.
  • a pharmaceutical composition comprises one or more antisense compound and phosphate-buffered saline (PBS). In certain embodiments, a pharmaceutical composition consists of one or more antisense compound and sterile phosphate-buffered saline (PBS). In certain embodiments, the sterile saline is pharmaceutical grade PBS.
  • antisense compounds may be admixed with pharmaceutically acceptable active and/or inert substances for the preparation of pharmaceutical compositions or formulations.
  • Compositions and methods for the formulation of pharmaceutical compositions depend on a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.
  • compositions comprising antisense compounds encompass any pharmaceutically acceptable salts, esters, or salts of such esters.
  • pharmaceutical compositions comprising antisense compounds comprise one or more oligonucleotide which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof.
  • the disclosure is also drawn to pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.
  • Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.
  • a prodrug can include the incorporation of additional nucleosides at one or both ends of an oligomeric compound which are cleaved by endogenous nucleases within the body, to form the active antisense oligomeric compound.
  • Lipid moieties have been used in nucleic acid therapies in a variety of methods.
  • the nucleic acid is introduced into preformed liposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids.
  • DNA complexes with mono- or poly-cationic lipids are formed without the presence of a neutral lipid.
  • a lipid moiety is selected to increase distribution of a pharmaceutical agent to a particular cell or tissue.
  • a lipid moiety is selected to increase distribution of a pharmaceutical agent to fat tissue.
  • a lipid moiety is selected to increase distribution of a pharmaceutical agent to muscle tissue.
  • compositions provided herein comprise one or more modified oligonucleotides and one or more excipients.
  • excipients are selected from water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose and polyvinylpyrrolidone.
  • a pharmaceutical composition provided herein comprises a delivery system.
  • delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful for preparing certain pharmaceutical compositions including those comprising hydrophobic compounds. In certain embodiments, certain organic solvents such as dimethylsulfoxide are used.
  • a pharmaceutical composition provided herein comprises one or more tissue-specific delivery molecules designed to deliver the one or more pharmaceutical agents of the present invention to specific tissues or cell types.
  • pharmaceutical compositions include liposomes coated with a tissue-specific antibody.
  • a pharmaceutical composition provided herein comprises a co-solvent system.
  • co-solvent systems comprise, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase.
  • co-solvent systems are used for hydrophobic compounds.
  • a non-limiting example of such a co-solvent system is the VPD co-solvent system, which is a solution of absolute ethanol comprising 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80TM and 65% w/v polyethylene glycol 300.
  • the proportions of such co-solvent systems may be varied considerably without significantly altering their solubility and toxicity characteristics.
  • co-solvent components may be varied: for example, other surfactants may be used instead of Polysorbate 80TM; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.
  • a pharmaceutical composition provided herein is prepared for oral administration. In certain embodiments, pharmaceutical compositions are prepared for buccal administration.
  • a pharmaceutical composition is prepared for administration by injection (e.g., intravenous, subcutaneous, intramuscular, etc.).
  • a pharmaceutical composition comprises a carrier and is formulated in aqueous solution, such as water or physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
  • other ingredients are included (e.g., ingredients that aid in solubility or serve as preservatives).
  • injectable suspensions are prepared using appropriate liquid carriers, suspending agents and the like.
  • compositions for injection are suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • Certain solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes.
  • Aqueous injection suspensions may contain.
  • the compounds and compositions as described herein are administered parenterally.
  • parenteral administration is by infusion. Infusion can be chronic or continuous or short or intermittent. In certain embodiments, infused pharmaceutical agents are delivered with a pump. In certain embodiments, parenteral administration is by injection.
  • compounds and compositions are delivered to the CNS. In certain embodiments, compounds and compositions are delivered to the cerebrospinal fluid. In certain embodiments, compounds and compositions are administered to the brain parenchyma. In certain embodiments, compounds and compositions are delivered to an animal by intrathecal administration, or intracerebroventricular administration. Broad distribution of compounds and compositions, described herein, within the central nervous system may be achieved with intraparenchymal administration, intrathecal administration, or intracerebroventricular administration.
  • parenteral administration is by injection.
  • the injection may be delivered with a syringe or a pump.
  • the injection is a bolus injection.
  • the injection is administered directly to a tissue, such as striatum, caudate, cortex, hippocampus and cerebellum.
  • delivery of a compound or composition described herein can affect the pharmacokinetic profile of the compound or composition.
  • injection of a compound or composition described herein, to a targeted tissue improves the pharmacokinetic profile of the compound or composition as compared to infusion of the compound or composition.
  • the injection of a compound or composition improves potency compared to broad diffusion, requiring less of the compound or composition to achieve similar pharmacology.
  • similar pharmacology refers to the amount of time that a target mRNA and/or target protein is down-regulated (e.g. duration of action).
  • methods of specifically localizing a pharmaceutical agent decreases median effective concentration (EC50) by a factor of about 50 (e.g. 50 fold less concentration in tissue is required to achieve the same or similar pharmacodynamic effect).
  • methods of specifically localizing a pharmaceutical agent decreases median effective concentration (EC50) by a factor of 20, 25, 30, 35, 40, 45 or 50.
  • the pharmaceutical agent in an antisense compound as further described herein.
  • the targeted tissue is brain tissue.
  • the targeted tissue is striatal tissue.
  • decreasing EC50 is desirable because it reduces the dose required to achieve a pharmacological result in a patient in need thereof.
  • an antisense oligonucleotide is delivered by injection or infusion once every month, every two months, every 90 days, every 3 months, every 6 months, twice a year or once a year.
  • one or more pharmaceutical compositions are co-administered with one or more other pharmaceutical agents.
  • such one or more other pharmaceutical agents are designed to treat the same disease, disorder, or condition as the one or more pharmaceutical compositions described herein.
  • such one or more other pharmaceutical agents are designed to treat a different disease, disorder, or condition as the one or more pharmaceutical compositions described herein.
  • such one or more other pharmaceutical agents are designed to treat an undesired side effect of one or more pharmaceutical compositions as described herein.
  • one or more pharmaceutical compositions are co-administered with another pharmaceutical agent to treat an undesired effect of that other pharmaceutical agent.
  • one or more pharmaceutical compositions are co-administered with another pharmaceutical agent to produce a combinational effect. In certain embodiments, one or more pharmaceutical compositions are co-administered with another pharmaceutical agent to produce a synergistic effect.
  • one or more pharmaceutical compositions and one or more other pharmaceutical agents are administered at the same time. In certain embodiments, one or more pharmaceutical compositions and one or more other pharmaceutical agents are administered at different times. In certain embodiments, one or more pharmaceutical compositions and one or more other pharmaceutical agents are prepared together in a single formulation. In certain embodiments, one or more pharmaceutical compositions and one or more other pharmaceutical agents are prepared separately.
  • pharmaceutical agents that may be co-administered with a pharmaceutical composition of include antipsychotic agents, such as, e.g., haloperidol, chlorpromazine, clozapine, quetiapine, and olanzapine; antidepressant agents, such as, e.g., fluoxetine, sertraline hydrochloride, venlafaxine and nortriptyline; tranquilizing agents such as, e.g., benzodiazepines, clonazepam, paroxetine, venlafaxin, and beta-blockers; mood-stabilizing agents such as, e.g., lithium, valproate, lamotrigine, and carbamazepine; paralytic agents such as, e.g., Botulinum toxin; and/or other experimental agents including, but not limited to, tetrabenazine (Xenazine), creatine, coenzyme Q10, trehalose, docosa
  • RNA nucleoside comprising a 2′-OH sugar moiety and a thymine base
  • RNA methylated uracil
  • nucleic acid sequences provided herein are intended to encompass nucleic acids containing any combination of natural or modified RNA and/or DNA, including, but not limited to such nucleic acids having modified nucleobases.
  • an oligomeric compound having the nucleobase sequence “ATCGATCG” encompasses any oligomeric compounds having such nucleobase sequence, whether modified or unmodified, including, but not limited to, such compounds comprising RNA bases, such as those having sequence “AUCGAUCG” and those having some DNA bases and some RNA bases such as “AUCGATCG” and oligomeric compounds having other modified or naturally occurring bases, such as “AT me CGAUCG,” wherein me C indicates a cytosine base comprising a methyl group at the 5-position.
  • oligomeric compounds are assigned a “Sequence Code.” Oligomeric compounds having the same Sequence Code have the same nucleobase sequence. Oligomeric compounds having different Sequence Codes have different nucleobase sequences.
  • Antisense oligonucleotides were designed targeting a Target-X nucleic acid and were tested for their effects on Target-X mRNA in vitro. ISIS 407939, which was described in an earlier publication (WO 2009/061851) was also tested.
  • the newly designed chimeric antisense oligonucleotides and their motifs are described in Table 15.
  • the internucleoside linkages throughout each gapmer are phosphorothioate linkages (P ⁇ S).
  • Nucleosides followed by “d” indicate 2′-deoxyribonucleosides.
  • Nucleosides followed by “k” indicate 6′-(S)—CH 3 bicyclic nucleoside (e.g cEt) nucleosides.
  • Nucleosides followed by “e” indicate 2′-O-methoxyethyl (2′-MOE) nucleosides.
  • “N” indicates modified or naturally occurring nucleobases (A, T, C, G, U, or 5-methyl C).
  • Each gapmer listed in Table 15 is targeted to the human Target-X genomic sequence.
  • Results are presented as percent inhibition of Target-X, relative to untreated control cells, and indicate that several of the newly designed antisense oligonucleotides are more potent than ISIS 407939. A total of 771 oligonucleotides were tested. Only those oligonucleotides which were selected for further studies are shown in Table 15. Each of the newly designed antisense oligonucleotides provided in Table 1 achieved greater than 80% inhibition and, therefore, are more active than ISIS 407939.
  • Additional antisense oligonucleotides were designed targeting a Target-X nucleic acid and were tested for their effects on Target-X mRNA in vitro. ISIS 407939 was also tested.
  • the chimeric antisense oligonucleotides and their motifs are described in Table 16.
  • the internucleoside linkages throughout each gapmer are phosphorothioate linkages (P ⁇ S).
  • Nucleosides followed by “d” indicate 2′-deoxyribonucleosides.
  • Nucleosides followed by “k” indicate 6′-(S)—CH 3 bicyclic nucleosides (e.g cEt).
  • Nucleosides followed by “e” indicate 2′-O-methoxyethyl (2′-MOE) modified nucleosides.
  • Nucleosides followed by ‘g’ indicate F-HNA modified nucleosides.
  • “N” indicates modified or naturally occurring nucleobases (A, T, C, G, U, or 5-methyl C).
  • Each gapmer listed in Table 16 is targeted to the human Target-X genomic sequence.
  • Results are presented as percent inhibition of Target-X, relative to untreated control cells, and demonstrate that several of the newly designed gapmers are more potent than ISIS 407939.
  • a total of 765 oligonucleotides were tested. Only those oligonucleotides which were selected for further studies are shown in Table 16. All but one of the newly designed antisense oligonucleotides provided in Table 16 achieved greater than 30% inhibition and, therefore, are more active than ISIS 407939.
  • the newly designed chimeric antisense oligonucleotides are 16 nucleotides in length and their motifs are described in Table 17.
  • the chemistry column of Table 17 presents the sugar motif of each oligonucleotide, wherein “e” indicates a 2′-O-methoxyethyl (2′-MOE) nucleoside, “k” indicates a 6′-(S)—CH 3 bicyclic nucleoside (e.g cEt) and “d” indicates a 2′-deoxyribonucleoside.
  • the internucleoside linkages throughout each gapmer are phosphorothioate (P ⁇ S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosines.
  • Each gapmer listed in Table 17 is targeted to the human Target-X genomic sequence.
  • ISIS 403052, ISIS 407594, ISIS 407606, ISIS 407939, and ISIS 416438 Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 2,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS2927 (described hereinabove in Example 1) was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of Target-X, relative to untreated control cells.
  • Additional antisense oligonucleotides were designed targeting a Target-X nucleic acid and were tested for their effects on Target-X mRNA in vitro. Also tested were ISIS 403094, ISIS 407641, ISIS 407643, ISIS 407662, ISIS 407900, ISIS 407910, ISIS 407935, ISIS 407936, ISIS 407939, ISIS 416446, ISIS 416449, ISIS 416455, ISIS 416472, ISIS 416477, ISIS 416507, ISIS 416508, ISIS 422086, ISIS 422087, ISIS 422140, and ISIS 422142, 5-10-5 2′-MOE gapmers targeting human Target-X, which were described in an earlier publication (WO 2009/061851), incorporated herein by reference.
  • the newly designed modified antisense oligonucleotides are 20 nucleotides in length and their motifs are described in Tables 18 and 19.
  • the chemistry column of Tables 18 and 19 present the sugar motif of each oligonucleotide, wherein “e” indicates a 2′-O-methoxyethyl (2′-MOE) nucleoside and “d” indicates a 2′-deoxyribonucleoside.
  • the internucleoside linkages throughout each gapmer are phosphorothioate (P ⁇ S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosines.
  • Each gapmer listed in Table 18 is targeted to the human Target-X genomic sequence.
  • Human primer probe set RTS2927 (described hereinabove in Example 1) was used to measure mRNA levels.
  • Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of Target-X, relative to untreated control cells. A total of 916 oligonucleotides were tested. Only those oligonucleotides which were selected for further studies are shown in Tables 18 and 19.
  • ISIS 407939 which was described in an earlier publication (WO 2009/061851) were also tested.
  • the newly designed chimeric antisense oligonucleotides in Table 20 were designed as 2-10-2 cEt gapmers.
  • the newly designed gapmers are 14 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxyribonucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising five nucleosides each.
  • Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment comprises 6′-(S)—CH 3 bicyclic nucleoside (e.g cEt) modification.
  • the internucleoside linkages throughout each gapmer are phosphorothioate (P ⁇ S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines.
  • Each gapmer listed in Table 20 is targeted to the human Target-X genomic sequence.
  • Human primer probe set RTS2927 (described hereinabove in Example 1) was used to measure mRNA levels.
  • Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of Target-X, relative to untreated control cells.
  • a total of 614 oligonucleotides were tested. Only those oligonucleotides which were selected for further studies are shown in Table 20. Many of the newly designed antisense oligonucleotides provided in Table 20 achieved greater than 72% inhibition and, therefore, are more potent than ISIS 407939.
  • Additional antisense oligonucleotides were designed targeting a Target-X nucleic acid and were tested for their effects on Target-X mRNA in vitro. Also tested was ISIS 407939, a 5-10-5 MOE gapmer targeting human Target-X, which was described in an earlier publication (WO 2009/061851). ISIS 472998 and ISIS 473046, described in the Examples above were also included in the screen.
  • the newly designed chimeric antisense oligonucleotides in Table 21 were designed as 2-10-2 cEt gapmers.
  • the newly designed gapmers are 14 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxyribonucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising five nucleosides each.
  • Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment comprise 6′-(S)—CH 3 bicyclic nucleoside (e.g cEt) modification.
  • the internucleoside linkages throughout each gapmer are phosphorothioate (P ⁇ S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines.
  • Each gapmer listed in Table 21 is targeted to the human Target-X genomic sequence.
  • Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.67 ⁇ M, 2.00 ⁇ M, 1.11 ⁇ M, and 6.00 ⁇ M concentrations of antisense oligonucleotide, as specified in Table 22.
  • RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human Target-X primer probe set RTS2927 was used to measure mRNA levels.
  • Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells.
  • IC 50 half maximal inhibitory concentration of each oligonucleotide is also presented in Table 22. As illustrated in Table 22, Target-X mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells. The data also confirms that several of the newly designed gapmers are more potent than ISIS 407939 of the previous publication.
  • Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells. As illustrated in Table 23, Target-X mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells. The data also confirms that several of the newly designed gapmers are more potent than ISIS 407939.
  • Additional antisense oligonucleotides were designed targeting a Target-X nucleic acid and were tested for their effects on Target-X mRNA in vitro. Also tested were ISIS 403052, ISIS 407939, ISIS 416446, ISIS 416472, ISIS 416507, ISIS 416508, ISIS 422087, ISIS 422096, ISIS 422130, and ISIS 422142 which were described in an earlier publication (WO 2009/061851), incorporated herein by reference. ISIS 490149, ISIS 490197, ISIS 490209, ISIS 490275, ISIS 490277, and ISIS 490424, described in the Examples above, were also included in the screen.
  • the newly designed chimeric antisense oligonucleotides in Table 24 were designed as 3-10-4 2′-MOE gapmers. These gapmers are 17 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxyribonucleosides and is flanked by wing segments on the 5′ direction with three nucleosides and the 3′ direction with four nucleosides. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has 2′-MOE modifications.
  • the internucleoside linkages throughout each gapmer are phosphorothioate (P ⁇ S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines.
  • Each gapmer listed in Table 24 is targeted to the human Target-X genomic sequence.
  • oligonucleotides Activity of the newly designed oligonucleotides was compared to ISIS 403052, ISIS 407939, ISIS 416446, ISIS 416472, ISIS 416507, ISIS 416508, ISIS 422087, ISIS 422096, ISIS 422130, and ISIS 422142.
  • Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 2,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS2927 (described hereinabove in Example 1) was used to measure mRNA levels.
  • Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells. A total of 272 oligonucleotides were tested. Only those oligonucleotides which were selected for further studies are shown in Table 24. Several of the newly designed antisense oligonucleotides provided in Table 24 are more potent than antisense oligonucleotides from the previous publication.
  • IC 50 half maximal inhibitory concentration
  • RNA samples were plated at a density of 20,000 cells per well and transfected using electroporation with 0.3125 ⁇ M, 0.625 ⁇ M, 1.25 ⁇ M, 2.50 ⁇ M, 5.00 ⁇ M and 10.00 ⁇ M concentrations of antisense oligonucleotide, as specified in Table 26.
  • RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR.
  • Human Target-X primer probe set RTS2927 was used to measure mRNA levels.
  • Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells. As illustrated in Table 26, Target-X mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells. The data also confirms that the newly designed gapmers are more potent than gapmers from the previous publication.
  • mice are a multipurpose mice model, frequently utilized for safety and efficacy testing.
  • the mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.
  • mice Groups of male BALB/c mice were injected subcutaneously twice a week for 3 weeks with 50 mg/kg of ISIS 407935, ISIS 416472, ISIS 416549, ISIS 422086, ISIS 422087, ISIS 422096, ISIS 422142, ISIS 490103, ISIS 490149, ISIS 490196, ISIS 490208, ISIS 490209, ISIS 513419, ISIS 513420, ISIS 513421, ISIS 513454, ISIS 513455, ISIS 513456, ISIS 513457, ISIS 513462, ISIS 513463, ISIS 513487, ISIS 513504, ISIS 513508, and ISIS 513642.
  • One group of male BALB/c mice was injected subcutaneously twice a week for 3 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.
  • ISIS oligonucleotides To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, albumin, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.).
  • ISIS oligonucleotides that did not cause any increase in the levels of transaminases, or which caused an increase within three times the upper limit of normal (ULN) were deemed very tolerable.
  • ISIS oligonucleotides that caused an increase in the levels of transaminases between three times and seven times the ULN were deemed tolerable.
  • ISIS 407935, ISIS 416472, ISIS 416549, ISIS 422087, ISIS 422096, ISIS 490103, ISIS 490196, ISIS 490208, ISIS 513454, ISIS 513455, ISIS 513456, ISIS 513457, ISIS 513487, ISIS 513504, and ISIS 513508 were considered very tolerable in terms of liver function.
  • ISIS 422086, ISIS 490209, ISIS 513419, ISIS 513420, and ISIS 513463 were considered tolerable in terms of liver function.
  • Human Target-X primer probe set RTS2927 (described hereinabove in Example 1) was used to measure mRNA levels.
  • Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells.
  • IC 50 half maximal inhibitory concentration of each oligonucleotide is also presented in Table 27. As illustrated in Table 27, Target-X mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells. Many of the newly designed antisense oligonucleotides provided in Table 27 achieved an IC 50 of less than 0.9 ⁇ M and, therefore, are more potent than ISIS 407939.
  • Additional antisense oligonucleotides were designed targeting a Target-X nucleic acid and were tested for their effects on Target-X mRNA in vitro. Also tested was ISIS 407939, a 5-10-5 MOE gapmer targeting human Target-X, which was described in an earlier publication (WO 2009/061851). ISIS 472998, ISIS 492878, and ISIS 493201 and 493182, 2-10-2 cEt gapmers, described in the Examples above were also included in the screen.
  • the newly designed modified antisense oligonucleotides are 16 nucleotides in length and their motifs are described in Table 28.
  • the chemistry column of Table 28 presents the sugar motif of each oligonucleotide, wherein “e” indicates a 2′-O-methoxyethyl (2′-MOE) nucleoside, “k” indicates a 6′-(S)—CH 3 bicyclic nucleoside (e.g cEt) and “d” indicates a 2′-deoxyribonucleoside.
  • the internucleoside linkages throughout each gapmer are phosphorothioate (P ⁇ S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosines.
  • Each gapmer listed in Table 28 is targeted to the human Target-X genomic sequence.
  • mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.
  • the newly designed modified antisense oligonucleotides were also added to this screen.
  • the newly designed chimeric antisense oligonucleotides are 16 nucleotides in length and their motifs are described in Table 29.
  • the chemistry column of Table 29 presents the sugar motif of each oligonucleotide, wherein “e” indicates a 2′-O-methoxyethyl (2′-MOE) nucleoside, “k” indicates a 6′-(S)—CH 3 bicyclic nucleoside (e.g cEt) and “d” indicates a 2′-deoxyribonucleoside.
  • the internucleoside linkages throughout each gapmer are phosphorothioate (P ⁇ S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosines.
  • Each gapmer listed in Table 29 is targeted to the human Target-X genomic sequence.
  • ISIS oligonucleotides To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, albumin, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.).
  • ISIS oligonucleotides that did not cause any increase in the levels of transaminases, or which caused an increase within three times the upper limit of normal (ULN) were deemed very tolerable.
  • ISIS oligonucleotides that caused an increase in the levels of transaminases between three times and seven times the ULN were deemed tolerable.
  • ISIS 457851, ISIS 515635, ISIS 515637, ISIS 515638, ISIS 515643, ISIS 515647, ISIS 515649, ISIS 515650, ISIS 515652, ISIS 515654, ISIS 515656, ISIS 516056, and ISIS 516057 were considered tolerable in terms of liver function.
  • mice were developed at Taconic farms harboring a Target-X genomic DNA fragment. The mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for efficacy.
  • mice Groups of 3-4 male and female transgenic mice were injected subcutaneously twice a week for 3 weeks with 20 mg/kg/week of ISIS 457851, ISIS 515636, ISIS 515639, ISIS 515653, ISIS 516053, ISIS 516065, and ISIS 516066.
  • One group of mice was injected subcutaneously twice a week for 3 weeks with control oligonucleotide, ISIS 141923 (CCTTCCCTGAAGGTTCCTCC, 5-10-5 MOE gapmer with no known murine target, SEQ ID NO: 9).
  • ISIS 141923 control oligonucleotide
  • mice was injected subcutaneously twice a week for 3 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.
  • Plasma protein levels of Target-X were estimated using a Target-X ELISA kit (purchased from Hyphen Bio-Med). Results are presented as percent inhibition of Target-X, relative to control. As shown in Table 31, several antisense oligonucleotides achieved reduction of human Target-X protein expression over the PBS control.
  • mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for efficacy.
  • mice Groups of 2-4 male and female transgenic mice were injected subcutaneously twice a week for 3 weeks with 10 mg/kg/week of ISIS 407935, ISIS 416472, ISIS 416549, ISIS 422087, ISIS 422096, ISIS 473137, ISIS 473244, ISIS 473326, ISIS 473327, ISIS 473359, ISIS 473392, ISIS 473393, ISIS 473547, ISIS 473567, ISIS 473589, ISIS 473630, ISIS 484559, ISIS 484713, ISIS 490103, ISIS 490196, ISIS 490208, ISIS 513419, ISIS 513454, ISIS 513455, ISIS 513456, ISIS 513457, ISIS 513487, ISIS 513508, ISIS 515640, ISIS 515641, ISIS 515642, ISIS 515648, ISIS 515655, ISIS 515657, ISIS 516045, ISIS 516046, ISIS 516047,
  • Plasma protein levels of Target-X were estimated using a Target-X ELISA kit (purchased from Hyphen Bio-Med). Results are presented as percent inhibition of Target-X, relative to control. As shown in Table 32, several antisense oligonucleotides achieved reduction of human Target-X over the PBS control.
  • Antisense oligonucleotides exhibiting in vitro inhibition of Target-X mRNA were selected and tested at various doses in Hep3B cells. Also tested was ISIS 407939, a 5-10-5 MOE gapmer targeting human Target-X, which was described in an earlier publication (WO 2009/061851).
  • Human Target-X primer probe set RTS2927 (described hereinabove in Example 1) was used to measure mRNA levels.
  • Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells.
  • IC 50 half maximal inhibitory concentration of each oligonucleotide is also presented in Table 33. As illustrated in Table 33, Target-X mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells. Many of the newly designed antisense oligonucleotides provided in Table 33 achieved an IC 50 of less than 2.0 ⁇ M and, therefore, are more potent than ISIS 407939.
  • Additional antisense oligonucleotides were designed targeting a Target-X nucleic acid and were tested for their effects on Target-X mRNA in vitro.
  • ISIS 472998, ISIS 515652, ISIS 515653, ISIS 515654, ISIS 515655, ISIS 515656, and ISIS 515657, described in the Examples above were also included in the screen.
  • the newly designed chimeric antisense oligonucleotides are 16 or 17 nucleotides in length and their motifs are described in Table 34.
  • the chemistry column of Table 34 presents the sugar motif of each oligonucleotide, wherein “e” indicates a 2′-O-methoxyethyl (2′-MOE) nucleoside, “k” indicates a 6′-(S)—CH 3 bicyclic nucleoside (e.g cEt) and “d” indicates a 2′-deoxyribonucleoside.
  • the internucleoside linkages throughout each gapmer are phosphorothioate (P ⁇ S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosines.
  • Each gapmer listed in Table 34 is targeted to the human Target-X genomic sequence.
  • Human primer probe set RTS2927 (described hereinabove in Example 1) was used to measure mRNA levels.
  • Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells.
  • additional antisense oligonucleotides were designed targeting a Target-X nucleic acid targeting start positions 1147, 1154 or 12842 of Target-X.
  • the newly designed chimeric antisense oligonucleotides are 16 or 17 nucleotides in length and their motifs are described in Table 35.
  • the chemistry column of Table 35 presents the sugar motif of each oligonucleotide, wherein “e” indicates a 2′-O-methoxyethyl (2′-MOE) nucleoside, “k” indicates a 6′-(S)—CH 3 bicyclic nucleoside (e.g cEt) and “d” indicates a 2′-deoxyribonucleoside.
  • the internucleoside linkages throughout each gapmer are phosphorothioate (P ⁇ S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosine.
  • Each gapmer listed in Table 35 is targeted to the human Target-X genomic sequence.
  • Additional antisense oligonucleotides were designed targeting a Target-X nucleic acid and were tested for their effects on Target-X mRNA in vitro. ISIS 472998 and ISIS 515554, described in the Examples above were also included in the screen.
  • the newly designed chimeric antisense oligonucleotides are 16 nucleotides in length and their motifs are described in Table 36.
  • the chemistry column of Table 36 presents the sugar motif of each oligonucleotide, wherein “e” indicates a 2′-O-methoxyethyl (2′-MOE) nucleoside, “k” indicates a 6′-(S)—CH 3 bicyclic nucleoside (e.g cEt) and “d” indicates a 2′-deoxyribonucleoside.
  • the internucleoside linkages throughout each gapmer are phosphorothioate (P ⁇ S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosine.
  • Each gapmer listed in Table 36 is targeted to the human Target-X genomic sequence.
  • the newly designed chimeric antisense oligonucleotides and their motifs are described in Table 37.
  • the internucleoside linkages throughout each gapmer are phosphorothioate linkages (P ⁇ S) and are designated as “s”.
  • Nucleosides followed by “d” indicate 2′-deoxyribonucleosides.
  • Nucleosides followed by “k” indicate 6′-(S)—CH 3 bicyclic nucleosides (e.g cEt).
  • Nucleosides followed by “e” indicate 2′-O-methoxyethyl (2′-MOE) nucleosides.
  • “N” indicates modified or naturally occurring nucleobases (A, T, C, G, U, or 5-methyl C).
  • Target-X Each gapmer listed in Table 37 is targeted to the intronic region of human Target-X genomic sequence, designated herein as Target-X.
  • mice were treated at a high dose with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.
  • the newly designed antisense oligonucleotides were created with the same sequences as the antisense oligonucleotides from the study described above and were also added to this screen targeting intronic repeat regions of Target-X.
  • the newly designed modified antisense oligonucleotides and their motifs are described in Table 38.
  • the internucleoside linkages throughout each gapmer are phosphorothioate linkages (P ⁇ S).
  • Nucleosides followed by “d” indicate 2′-deoxyribonucleosides.
  • Nucleosides followed by “k” indicate 6′-(S)—CH3 bicyclic nucleoside (e.g cEt) nucleosides.
  • Nucleosides followed by “e” indicate 2′-O-methoxyethyl (2′-MOE) nucleosides.
  • “N” indicates modified or naturally occurring nucleobases (A, T, C, G, U, or 5-methyl C).
  • Target-X Each gapmer listed in Table 38 is targeted to the intronic region of human Target-X genomic sequence, designated herein as Target-X.
  • Start site indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence.
  • Stop site indicates the 3′-most nucleoside to which the gapmer is targeted human gene sequence.
  • mice Male BALB/c mice were injected subcutaneously with a single dose of 200 mg/kg of ISIS 422142, ISIS 457851, ISIS 473294, ISIS 473295, ISIS 473327, ISIS 484714, ISIS 515334, ISIS 515338, ISIS 515354, ISIS 515366, ISIS 515380, ISIS 515381, ISIS 515382, ISIS 515384, ISIS 515386, ISIS 515387, ISIS 515388, ISIS 515406, ISIS 515407, ISIS 515408, ISIS 515422, ISIS 515423, ISIS 515424, ISIS 515532, ISIS 515533, ISIS 515534, ISIS 515538, ISIS 515539, ISIS 515558, ISIS 515656, ISIS 515575, ISIS 515926, ISIS 515944, ISIS 515945, ISIS 515948, ISIS 515949, ISIS 515951, ISIS 515952, ISSI
  • ISIS oligonucleotides To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, albumin, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.).
  • ISIS oligonucleotides that did not cause any increase in the levels of transaminases, or which caused an increase within three times the upper limit of normal (ULN) were deemed very tolerable.
  • ISIS oligonucleotides that caused an increase in the levels of transaminases between three times and seven times the ULN were deemed tolerable.
  • ISIS 529173, ISIS 529854, ISIS 529614, ISIS 515386, ISIS 515388, ISIS 515949, ISIS 544817, and ISIS 545479 were considered tolerable in terms of liver function.
  • Sprague-Dawley rats are a multipurpose model used for safety and efficacy evaluations.
  • the rats were treated with ISIS antisense oligonucleotides from the studies described in the Examples above and evaluated for changes in the levels of various plasma chemistry markers.
  • ALT alanine transaminase
  • AST aspartate transaminase
  • ISIS oligonucleotides that did not cause any increase in the levels of transaminases, or which caused an increase within three times the upper limit of normal (ULN) were deemed very tolerable.
  • ISIS oligonucleotides that caused an increase in the levels of transaminases between three times and seven times the ULN were deemed tolerable.
  • ISIS 473286, ISIS 473547, ISSI 473589, ISIS 473630, ISIS 484559, ISIS 515636, ISIS 515640, ISIS 515655, ISIS 516046, and ISIS 516051 were considered very tolerable in terms of liver function.
  • ISIS 473567, ISIS 515641, ISIS 515657, ISIS 516048, and ISIS 516051 were considered tolerable in terms of liver function.
  • Sprague-Dawley rats were treated with ISIS antisense oligonucleotides from the studies described in the Examples above and evaluated for changes in the levels of various plasma chemistry markers.

Abstract

Disclosed are oligomeric compounds which are useful for hybridizing to a complementary nucleic acid, including but not limited, to nucleic acids in a cell. The hybridization results in modulation of the amount activity or expression of the target nucleic acid in a cell.

Description

    FIELD OF THE INVENTION
  • The present invention pertains generally to chemically-modified oligonucleotides for use in research, diagnostics, and/or therapeutics.
    • SEQUENCE LISTING
  • The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled CORE0099WOSEQ.txt, created Aug. 1, 2012 which is 304 Kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.
    • BACKGROUND OF THE INVENTION
  • Antisense compounds have been used to modulate target nucleic acids. Antisense compounds comprising a variety of chemical modifications and motifs have been reported. In certain instances, such compounds are useful as research tools, diagnostic reagents, and as therapeutic agents. In certain instances antisense compounds have been shown to modulate protein expression by binding to a target messenger RNA (mRNA) encoding the protein. In certain instances, such binding of an antisense compound to its target mRNA results in cleavage of the mRNA. Antisense compounds that modulate processing of a pre-mRNA have also been reported. Such antisense compounds alter splicing, interfere with polyadenlyation or prevent formation of the 5′-cap of a pre-mRNA.
    • SUMMARY OF THE INVENTION
  • In certain embodiments, the present invention provides oligomeric compounds comprising oligonucleotides. In certain embodiments, such oligonucleotides comprise a region having a gapmer motif. In certain embodiments, such oligonucleotides consist of a region having a gapmer motif.
  • The present disclosure provides the following non-limiting numbered embodiments:
    • Embodiment 1
      • A oligomeric compound comprising a modified oligonucleotide consisting of 10 to 30 linked nucleosides, wherein the modified oligonucleotide has a modification motif comprising: a 5′-region consisting of 2-8 linked 5′-region nucleosides, each independently selected from among a modified nucleoside and an unmodified deoxynucleoside, provided that at least one 5′-region nucleoside is a modified nucleoside and wherein the 3′-most 5′-region nucleoside is a modified nucleoside;
      • a 3′-region consisting of 2-8 linked 3′-region nucleosides, each independently selected from among a modified nucleoside and an unmodified deoxynucleoside, provided that at least one 3′-region nucleoside is a modified nucleoside and wherein the 5′-most 3′-region nucleoside is a modified nucleoside; and
      • a central region between the 5′-region and the 3′-region consisting of 6-12 linked central region nucleosides, each independently selected from among: a modified nucleoside and an unmodified deoxynucleoside, wherein the 5′-most central region nucleoside is an unmodified deoxynucleoside and the 3′-most central region nucleoside is an unmodified deoxynucleoside;
      • wherein the modified oligonucleotide has a nucleobase sequence complementary to the nucleobase sequence of a target region of a target nucleic acid.
    Embodiment 2
      • The oligomeric compound of embodiment 1, wherein the nucleobase sequence of the target region of the target nucleic acid differs from the nucleobase sequence of at least one non-target nucleic acid by 1-3 differentiating nucleobases.
    Embodiment 3
      • The oligomeric compound of embodiment 1, the nucleobase sequence of the target region of the target nucleic acid differs from the nucleobase sequence of at least one non-target nucleic acid by a single differentiating nucleobase.
    Embodiment 4
      • The oligomeric compound of embodiment 2 or 3, wherein the target nucleic acid and the non-target nucleic acid are alleles of the same gene.
    Embodiment 5
      • The oligomeric compound of embodiment 4, wherein the single differentiating nucleobase is a single-nucleotide polymorphism.
    Embodiment 6
      • The oligomeric compound of embodiment 5, wherein the single-nucleotide polymorphism is associated with a disease.
    Embodiment 7
      • The oligomeric compound of embodiment 6, wherein the disease is selected from among: Alzheimer's disease, Creutzfeldt-Jakob disease, fatal familial insomnia, Alexander disease, Parkinson's disease, amyotrophic lateral sclerosis, dentato-rubral and pallido-luysian atrophy DRPA, spino-cerebellar ataxia, Torsion dystonia, cardiomyopathy, chronic obstructive pulmonary disease (COPD), liver disease, hepatocellular carcinoma, systemic lupus erythematosus, hypercholesterolemia, breast cancer, asthma, Type 1 diabetes, Rheumatoid arthritis, Graves disease, SLE, spinal and bulbar muscular atrophy, Kennedy's disease, progressive childhood posterior subcapsular cataracts, cholesterol gallstone disease, arthrosclerosis, cardiovascular disease, primary hypercalciuria, alpha-thallasemia, obsessive compulsive disorder, Anxiety, comorbid depression, congenital visual defects, hypertension, metabolic syndrome, prostate cancer, congential myasthenic syndrome, peripheral arterial disease, atrial fibrillation, sporadic pheochromocytoma, congenital malformations, Machado-Joseph disease, Huntington's disease, and Autosomal Dominant Retinitis Pigmentosa disease.
    Embodiment 8
      • The oligomeric compound of embodiment 6, wherein the single-nucleotide polymorphism is selected from among: rs6446723, rs3856973, rs2285086, rs363092, rs916171, rs6844859, rs7691627, rs4690073, rs2024115, rs11731237, rs362296, rs10015979, rs7659144, rs363096, rs362273, rs16843804, rs362271, rs362275, rs3121419, rs362272, rs3775061, rs34315806, rs363099, rs2298967, rs363088, rs363064, rs363102, rs2798235, rs363080, rs363072, rs363125, rs362303, rs362310, rs10488840, rs362325, rs35892913, rs363102, rs363096, rs11731237, rs10015979, rs363080, rs2798235, rs1936032, rs2276881, rs363070, rs35892913, rs12502045, rs6446723, rs7685686, rs3733217, rs6844859, and rs362331.
    Embodiment 9
      • The oligomeric compound of embodiment 8, wherein the single-nucleotide polymorphism is selected from among: rs7685686, rs362303 rs4690072 and rs363088
    Embodiment 10
      • The oligomeric compound of embodiment 2 or 3, wherein the target nucleic acid and the non-target nucleic acid are transcripts from different genes.
    Embodiment 11
      • The oligomeric compound of any of embodiments 1-10, wherein the 3′-most 5′-region nucleoside comprises a bicyclic sugar moiety.
    Embodiment 12
      • The oligomeric compound of embodiment 11, wherein the 3′-most 5′-region nucleoside comprises a cEt sugar moiety.
    Embodiment 13
      • The oligomeric compound of embodiment 11, wherein the 3′-most 5′-region nucleoside comprises an LNA sugar moiety.
    Embodiment 14
      • The oligomeric compound of any of embodiments 1-13, wherein the central region consists of 6-10 linked nucleosides.
    Embodiment 15
      • The oligomeric compound of any of embodiments 1-14, wherein the central region consists of 6-9 linked nucleosides.
    Embodiment 16
      • The oligomeric compound of embodiment 15, wherein the central region consists of 6 linked nucleosides.
    Embodiment 17
      • The oligomeric compound of embodiment 15, wherein the central region consists of 7 linked nucleosides.
    Embodiment 18
      • The oligomeric compound of embodiment 15, wherein the central region consists of 8 linked nucleosides.
    Embodiment 19
      • The oligomeric compound of embodiment 15, wherein the central region consists of 9 linked nucleosides.
    Embodiment 20
      • The oligomeric compound of any of embodiments 1-19, wherein each central region nucleoside is an unmodified deoxynucleoside.
    Embodiment 21
      • The oligomeric compound of any of embodiments 1-19, wherein at least one central region nucleoside is a modified nucleoside.
    Embodiment 22
      • The oligomeric compound of embodiment 21, wherein one central region nucleoside is a modified nucleoside and each of the other central region nucleosides is an unmodified deoxynucleoside.
    Embodiment 23
      • The oligomeric compound of embodiment 21, wherein two central region nucleosides are modified nucleosides and each of the other central region nucleosides is an unmodified deoxynucleoside.
    Embodiment 24
      • The oligomeric compound of any of embodiments 21-23 wherein at least one modified central region nucleoside is an RNA-like nucleoside.
    Embodiment 25
      • The oligomeric compound of any of embodiments 21-23 comprising at least one modified central region nucleoside comprising a modified sugar moiety.
    Embodiment 26
      • The oligomeric compound of any of embodiments 21-25 comprising at least one modified central region nucleoside comprising a 5′-methyl-2′-deoxy sugar moiety.
    Embodiment 27
      • The oligomeric compound of any of embodiments 21-26 comprising at least one modified central region nucleoside comprising a bicyclic sugar moiety.
    Embodiment 28
      • The oligomeric compound of any of embodiments 21-27 comprising at least one modified central region nucleoside comprising a cEt sugar moiety.
    Embodiment 29
      • The oligomeric compound of any of embodiments 21-28 comprising at least one modified central region nucleoside comprising an LNA sugar moiety.
    Embodiment 30
      • The oligomeric compound of any of embodiments 21-29 comprising at least one modified central region nucleoside comprising an α-LNA sugar moiety.
    Embodiment 31
      • The oligomeric compound of any of embodiments 21-29 comprising at least one modified central region nucleoside comprising a 2′-substituted sugar moiety.
    Embodiment 32
      • The oligomeric compound of embodiment 31 wherein at least one modified central region nucleoside comprises a 2′-substituted sugar moiety comprising a 2′ substituent selected from among: halogen, optionally substituted allyl, optionally substituted amino, azido, optionally substituted SH, CN, OCN, CF3, OCF3, O, S, or N(Rm)-alkyl; O, S, or N(Rm)-alkenyl; O, S or N(Rm)-alkynyl; optionally substituted O-alkylenyl-O-alkyl, optionally substituted alkynyl, optionally substituted alkaryl, optionally substituted aralkyl, optionally substituted O-alkaryl, optionally substituted O-aralkyl, O(CH2)2SCH3, O—(CH2)2-O—N(Rm)(Rn) or O—CH2-C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H, an amino protecting group or substituted or unsubstituted C1-C10 alkyl;
      • wherein each optionally substituted group is optionally substituted with a substituent group independently selected from among: hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.
    Embodiment 33
      • The oligomeric compound of embodiment 32 wherein at least one modified central region nucleoside comprises a 2′-substituted sugar moiety comprising a 2′ substituent selected from among: a halogen, OCH3, OCH2F, OCHF2, OCF3, OCH2CH3, O(CH2)2F, OCH2CHF2, OCH2CF3, OCH2—CH═CH2, O(CH2)2—OCH3, O(CH2)2—SCH3, O(CH2)2—OCF3, O(CH2)3—N(R1)(R2), O(CH2)2—ON(R1)(R2), O(CH2)2—O(CH2)2—N(R1)(R2), OCH2C(═O)—N(R1)(R2), OCH2C(═O)—N(R3)—(CH2)2—N(R1)(R2), and O(CH2)2—N(R3)—C(═NR4)[N(R1)(R2)]; wherein R1, R2, R3 and R4 are each, independently, H or C1-C6 alkyl.
    Embodiment 34
      • The oligomeric compound of embodiment 33 wherein the 2′ substituent is selected from among: a halogen, OCH3, OCF3, OCH2CH3, OCH2CF3, OCH2—CH═CH2, O(CH2)2—OCH3 (MOE), O(CH2)2—O(CH2)2—N(CH3)2, OCH2C(═O)—N(H)CH3, OCH2C(═O)—N(H)—(CH2)2—N(CH3)2, and OCH2—N(H)—C(═NH)NH2.
    Embodiment 35
      • The oligomeric compound of any of embodiments 21-34 comprising at least one modified central region nucleoside comprising a 2′-MOE sugar moiety.
    Embodiment 36
      • The oligomeric compound of any of embodiments 21-35 comprising at least one modified central region nucleoside comprising a 2′-OMe sugar moiety.
    Embodiment 37
      • The oligomeric compound of any of embodiments 21-36 comprising at least one modified central region nucleoside comprising a 2′-F sugar moiety.
    Embodiment 38
      • The oligomeric compound of any of embodiments 21-37 comprising at least one modified central region nucleoside comprising a 2′-(ara)-F sugar moiety.
    Embodiment 39
      • The oligomeric compound of any of embodiments 21-38 comprising at least one modified central region nucleoside comprising a sugar surrogate.
    Embodiment 40
      • The oligomeric compound of embodiment 39 comprising at least one modified central region nucleoside comprising an F-HNA sugar moiety.
    Embodiment 41
      • The oligomeric compound of embodiment 39 or 40 comprising at least one modified central region nucleoside comprising an HNA sugar moiety.
    Embodiment 42
      • The oligomeric compound of any of embodiments 21-41 comprising at least one modified central region nucleoside comprising a modified nucleobase.
    Embodiment 43
      • The oligomeric compound of embodiment 42 comprising at least one modified central region nucleoside comprising a modified nucleobase selected from a 2-thio pyrimidine and a 5-propyne pyrimidine.
    Embodiment 44
      • The oligomeric compound of any of embodiments 21-43, wherein the 2nd nucleoside from the 5′-end of the central region is a modified nucleoside.
    Embodiment 45
      • The oligomeric compound of any of embodiments 21-44, wherein the 3rd nucleoside from the 5′-end of the central region is a modified nucleoside.
    Embodiment 46
      • The oligomeric compound of any of embodiments 21-45, wherein the 4th nucleoside from the 5′-end of the central region is a modified nucleoside.
    Embodiment 47
      • The oligomeric compound of any of embodiments 21-46, wherein the 5th nucleoside from the 5′-end of the central region is a modified nucleoside.
    Embodiment 48
      • The oligomeric compound of any of embodiments 21-47, wherein the 6th nucleoside from the 5′-end of the central region is a modified nucleoside.
    Embodiment 49
      • The oligomeric compound of any of embodiments 21-48, wherein the 8th nucleoside from the 3′-end of the central region is a modified nucleoside.
    Embodiment 50
      • The oligomeric compound of any of embodiments 21-49, wherein the 7th nucleoside from the 3′-end of the central region is a modified nucleoside.
    Embodiment 51
      • The oligomeric compound of any of embodiments 21-50, wherein the 6th nucleoside from the 3′-end of the central region is a modified nucleoside.
    Embodiment 52
      • The oligomeric compound of any of embodiments 21-51, wherein the 5th nucleoside from the 3′-end of the central region is a modified nucleoside.
    Embodiment 53
      • The oligomeric compound of any of embodiments 21-52, wherein the 4th nucleoside from the 3′-end of the central region is a modified nucleoside.
    Embodiment 54
      • The oligomeric compound of any of embodiments 21-53, wherein the 3rd nucleoside from the 3′-end of the central region is a modified nucleoside.
    Embodiment 55
      • The oligomeric compound of any of embodiments 21-54, wherein the 2nd nucleoside from the 3′-end of the central region is a modified nucleoside.
    Embodiment 56
      • The oligomeric compound of any of embodiments 21-55, wherein the modified nucleoside is a 5′-methyl-2′-deoxy sugar moiety.
    Embodiment 57
      • The oligomeric compound of any of embodiments 21-55, wherein the modified nucleoside is a 2-thio pyrimidine.
    Embodiment 58
      • The oligomeric compound of any of embodiments 21-55, wherein the central region comprises no region having more than 4 contiguous unmodified deoxynucleosides.
    Embodiment 59
      • The oligomeric compound of any of embodiments 21-55, wherein the central region comprises no region having more than 5 contiguous unmodified deoxynucleosides.
    Embodiment 60
      • The oligomeric compound of any of embodiments 21-55, wherein the central region comprises no region having more than 6 contiguous unmodified deoxynucleosides.
    Embodiment 61
      • The oligomeric compound of any of embodiments 21-55, wherein the central region comprises no region having more than 7 contiguous unmodified deoxynucleosides.
    Embodiment 62
      • The oligomeric compound of any of embodiments 1-14 or 21-59, wherein the central region has a nucleoside motif selected from among: DDDDDDDDDD, DDDDXDDDDD; DDDDDXDDDDD; DDDXDDDDD; DDDDXDDDDDD; DDDDXDDDD; DDXDDDDDD; DDDXDDDDDD; DXDDDDDD; DDXDDDDDDD; DDXDDDDD; DDXDDDXDDD; DDDXDDDXDDD; DXDDDXDDD; DDXDDDXDD; DDXDDDDXDDD; DDXDDDDXDD; DXDDDDXDDD; DDDDXDDD; DDDXDDD; DXDDDDDDD; DDDDXXDDD; and DXXDXXDXX; wherein
      • each D is an unmodified deoxynucleoside; and each X is a modified nucleoside.
    Embodiment 63
      • The oligomeric compound of any of embodiments 1-14 or 21-59, wherein the central region has a nucleoside motif selected from among: DDDDDDDDD; DXDDDDDDD; DDXDDDDDD; DDDXDDDDD; DDDDXDDDD; DDDDDXDDD; DDDDDDXDD; DDDDDDDXD; DXXDDDDDD; DDDDDDXXD; DDXXDDDDD; DDDXXDDDD; DDDDXXDDD; DDDDDXXDD; DXDDDDDXD; DXDDDDXDD; DXDDDXDDD; DXDDXDDDD; DXDXDDDDD; DDXDDDDXD; DDXDDDXDD; DDXDDXDDD; DDXDXDDDD; DDDXDDDXD; DDDXDDXDD; DDDXDXDDD; DDDDXDDXD; DDDDXDXDD; and DDDDDXDXD wherein each D is an unmodified deoxynucleoside; and each X is a modified nucleoside.
    Embodiment 64
      • The oligomeric compound of any of embodiments 1-14 or 21-59, wherein the central region has a nucleoside motif selected from among: DDDDXDDDD, DXDDDDDDD, DXXDDDDDD, DDXDDDDDD, DDDXDDDDD, DDDDXDDDD, DDDDDXDDD, DDDDDDXDD, and DDDDDDDXD.
    Embodiment 65
      • The oligomeric compound of any of embodiments 1-14 or 21-59, wherein the central region has a nucleoside motif selected from among: DDDDDDDD, DXDDDDDD, DDXDDDDD, DDDXDDDD, DDDDXDDD, DDDDDXDD, DDDDDDXD, DXDDDDXD, DXDDDXDD, DXDDXDDD, DXDXDDDD, DXXDDDDD, DDXXDDDD, DDXDXDDD, DDXDDXDD, DXDDDDXD, DDDXXDDD, DDDXDXDD, DDDXDDXD, DDDDXXDD, DDDDXDXD, and DDDDDXXD.
    Embodiment 66
      • The oligomeric compound of any of embodiments 1-14 or 21-59, wherein the central region has a nucleoside motif selected from among: DDDDDDD, DXDDDDD, DDXDDDD, DDDXDDD, DDDDXDD, DDDDDXD, DXDDDXD, DXDDXDD, DXDXDDD, DXXDDDD, DDXXDDD, DDXDXDD, DDXDDXD, DDDXXDD, DDDXDXD, and DDDDXXD.
    Embodiment 67
      • The oligomeric compound of any of embodiments 1-14 or 21-59, wherein the central region has a nucleoside motif selected from among: DDDDDD, DXDDDD, DDXDDD, DDDXDD, DDDDXD, DXXDDD, DXDXDD, DXDDXD, DDXXDD, DDXDXD, and DDDXXD.
    Embodiment 68
      • The oligomeric compound of any of embodiments 1-14 or 21-59, wherein the central region has a nucleoside motif selected from among: DDDDDD, DDDDDDD, DDDDDDDD, DDDDDDDDD, DXDDDD, DDXDDD, DDDXDD, DDDDXD, DXDDDDD, DDXDDDD, DDDXDDD, DDDDXDD, DDDDDXD, DXDDDDDD, DDXDDDDD, DDDXDDDD, DDDDXDDD, DDDDDXDD, DDDDDDXD, DXDDDDDDD; DDXDDDDDD, DDDXDDDDD, DDDDXDDDD, DDDDDXDDD, DDDDDDXDD, DDDDDDDXD, DXDDDDDDDD, DDXDDDDDDD, DDDXDDDDDD, DDDDXDDDDD, DDDDDXDDDD, DDDDDDXDDD, DDDDDDDXDD, and DDDDDDDDXD.
    Embodiment 69
      • The oligomeric compound of embodiments 62-68, wherein each X comprises a modified nucleobase.
    Embodiment 70
      • The oligomeric compound of embodiments 62-68, wherein each X comprises a modified sugar moiety.
    Embodiment 71
      • The oligomeric compound of embodiments 62-68, wherein each X comprises 2-thio-thymidine.
    Embodiment 72
      • The oligomeric compound of embodiments 62-68, wherein each X nucleoside comprises an F-HNA sugar moiety.
    Embodiment 73
      • The oligomeric compound of embodiments 62-68, wherein the nucleobase sequence of the target region of the target nucleic acid differs from the nucleobase sequence of at least one non-target nucleic acid by a single differentiating nucleobase, and wherein the location of the single differentiating nucleobase is represented by X.
    Embodiment 74
      • The oligomeric compound of embodiment 73, wherein the target nucleic acid and the non-target nucleic acid are alleles of the same gene.
    Embodiment 75
      • The oligomeric compound of embodiment 73, wherein the single differentiating nucleobase is a single-nucleotide polymorphism.
    Embodiment 76
      • The oligomeric compound of any of embodiments 1-75, wherein the 5′ region consists of 2 linked 5′-region nucleosides.
    Embodiment 77
      • The oligomeric compound of any of embodiments 1-75, wherein the 5′ region consists of 3 linked 5′-region nucleosides.
    Embodiment 78
      • The oligomeric compound of any of embodiments 1-75, wherein the 5′ region consists of 4 linked 5′-region nucleosides.
    Embodiment 79
      • The oligomeric compound of any of embodiments 1-75, wherein the 5′ region consists of 5 linked 5′-region nucleosides.
    Embodiment 80
      • The oligomeric compound of any of embodiments 1-75, wherein the 5′ region consists of 6 linked 5′-region nucleosides.
    Embodiment 81
      • The oligomeric compound of any of embodiments 1-80, wherein at least one 5′-region nucleoside is an unmodified deoxynucleoside.
    Embodiment 82
      • The oligomeric compound of any of embodiments 1-80, wherein each 5′-region nucleoside is a modified nucleoside.
    Embodiment 83
      • The oligomeric compound of any of embodiments 1-80 wherein at least one 5′-region nucleoside is an RNA-like nucleoside.
    Embodiment 84
      • The oligomeric compound of any of embodiments 1-80 wherein each 5′-region nucleoside is an RNA-like nucleoside.
    Embodiment 85
      • The oligomeric compound of any of embodiments 1-80 comprising at least one modified 5′-region nucleoside comprising a modified sugar.
    Embodiment 86
      • The oligomeric compound of embodiment 80 comprising at least one modified 5′-region nucleoside comprising a bicyclic sugar moiety.
    Embodiment 87
      • The oligomeric compound of embodiment 86 comprising at least one modified 5′-region nucleoside comprising a cEt sugar moiety.
    Embodiment 88
      • The oligomeric compound of embodiment 85 or 86 comprising at least one modified 5′-region nucleoside comprising an LNA sugar moiety.
    Embodiment 89
      • The oligomeric compound of any of embodiments 76-80 comprising of at least one modified 5′-region nucleoside comprising a 2′-substituted sugar moiety.
    Embodiment 90
      • The oligomeric compound of embodiment 89 wherein at least one modified central region nucleoside comprises a 2′-substituted sugar moiety comprising a 2′ substituent selected from among: halogen, optionally substituted allyl, optionally substituted amino, azido, optionally substituted SH, CN, OCN, CF3, OCF3, O, S, or N(Rm)-alkyl; O, S, or N(Rm)-alkenyl; O, S or N(Rm)-alkynyl; optionally substituted O-alkylenyl-O-alkyl, optionally substituted alkynyl, optionally substituted alkaryl, optionally substituted aralkyl, optionally substituted O-alkaryl, optionally substituted O-aralkyl, O(CH2)2SCH3, O—(CH2)2—O—N(Rm)(Rn) or O—CH2—C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H, an amino protecting group or substituted or unsubstituted C1-C10 alkyl;
      • wherein each optionally substituted group is optionally substituted with a substituent group independently selected from among: hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.
    Embodiment 91
      • The oligomeric compound of embodiment 90 wherein at least one modified 5′-region nucleoside comprises a 2′-substituted sugar moiety comprising a 2′-substituent selected from among: a halogen, OCH3, OCH2F, OCHF2, OCF3, OCH2CH3, O(CH2)2F, OCH2CHF2, OCH2CF3, OCH2—CH═CH2, O(CH2)2—OCH3 (MOE), O(CH2)2—SCH3, O(CH2)2—OCF3, O(CH2)3—N(R1)(R2), O(CH2)2—ON(R1)(R2), O(CH2)2—O(CH2)2—N(R1)(R2), OCH2C(═O)—N(R1)(R2), OCH2C(═O)—N(R3)—(CH2)2—N(R1)(R2), and O(CH2)2—N(R3)—C(═NR4)[N(R1)(R2)]; wherein R1, R2, R3 and R4 are each, independently, H or C1-C6 alkyl.
    Embodiment 92
      • The oligomeric compound of embodiment 91, wherein the 2′-substituent is selected from among: a halogen, OCH3, OCF3, OCH2CH3, OCH2CF3, OCH2—CH═CH2, O(CH2)2—OCH3, O(CH2)2—O(CH2)2—N(CH3)2, OCH2C(═O)—N(H)CH3, OCH2C(═O)—N(H)—(CH2)2—N(CH3)2, and OCH2—N(H)—C(═NH)NH2.
    Embodiment 93
      • The oligomeric compound of any of embodiments 89-92 comprising at least one modified 5′-region nucleoside comprising a 2′-MOE sugar moiety.
    Embodiment 94
      • The oligomeric compound of any of embodiments 89-92 comprising at least one modified 5′-region nucleoside comprising a 2′-OMe sugar moiety.
    Embodiment 95
      • The oligomeric compound of any of embodiments 89-92 comprising at least one modified 5′-region nucleoside comprising a 2′-F sugar moiety.
    Embodiment 96
      • The oligomeric compound of any of embodiments 89-92 comprising at least one modified 5′-region nucleoside comprising a 2′-(ara)-F sugar moiety.
    Embodiment 97
      • The oligomeric compound of any of embodiments 82-96 comprising of at least one modified 5′-region nucleoside comprising a sugar surrogate.
    Embodiment 98
      • The oligomeric compound of embodiment 97 comprising at least one modified 5′-region nucleoside comprising an F-HNA sugar moiety.
    Embodiment 99
      • The oligomeric compound of embodiment 97 or 98 comprising at least one modified 5′-region nucleoside comprising an HNA sugar moiety.
    Embodiment 100
      • The oligomeric compound of any of embodiments 1-99 comprising at least one modified 5′-region nucleoside comprising a modified nucleobase.
    Embodiment 101
      • The oligomeric compound of embodiment 100, wherein the modified nucleoside comprises 2-thio-thymidine.
    Embodiment 102
      • The oligomeric compound of any of embodiments 1-101, wherein the 5′-region has a motif selected from among:
      • ADDA; ABDAA; ABBA; ABB; ABAA; AABAA; AAABAA; AAAABAA; AAAAABAA; AAABAA; AABAA; ABAB; ABADB; ABADDB; AAABB; AAAAA; ABBDC; ABDDC; ABBDCC; ABBDDC; ABBDCC; ABBC; AA; AAA; AAAA; AAAAB; AAAAAAA; AAAAAAAA; ABBB; AB; ABAB; AAAAB; AABBB; AAAAB; and AABBB, wherein each A is a modified nucleoside of a first type, each B is a modified nucleoside of a second type, each C is a modified nucleoside of a third type, and each D is an unmodified deoxynucleoside.
    Embodiment 103
      • The oligomeric compound of any of embodiments 1-101, wherein the 5′-region has a motif selected from among:
      • AB, ABB, AAA, BBB, BBBAA, AAB, BAA, BBAA, AABB, AAAB, ABBW, ABBWW, ABBB, ABBBB, ABAB, ABABAB, ABABBB, ABABAA, AAABB, AAAABB, AABB, AAAAB, AABBB, ABBBB, BBBBB, AAABW, AAAAA, BBBBAA, and AAABW wherein each A is a modified nucleoside of a first type, each B is a modified nucleoside of a second type, and each W is a modified nucleoside of a third type.
    Embodiment 104
      • The oligomeric compound of any of embodiments 1-101, wherein the 5′-region has a motif selected from among: ABB; ABAA; AABAA; AAABAA; ABAB; ABADB; AAABB; AAAAA; AA; AAA; AAAA; AAAAB; ABBB; AB; and ABAB, wherein each A is a modified nucleoside of a first type, each B is a modified nucleoside of a second type, and each W is a modified nucleoside of a third type.
    Embodiment 105
      • The oligomeric compound of embodiments 102-104, wherein each A nucleoside comprises a 2′-substituted sugar moiety.
    Embodiment 106
      • The oligomeric compound of embodiment 105 wherein at least one central region nucleoside comprises a 2′-substituted sugar moiety comprising a 2′ substituent selected from among: halogen, optionally substituted allyl, optionally substituted amino, azido, optionally substituted SH, CN, OCN, CF3, OCF3, O, S, or N(Rm)-alkyl; O, S, or N(Rm)-alkenyl; O, S or N(Rm)-alkynyl; optionally substituted O-alkylenyl-O-alkyl, optionally substituted alkynyl, optionally substituted alkaryl, optionally substituted aralkyl, optionally substituted O-alkaryl, optionally substituted O-aralkyl, O(CH2)2SCH3, O—(CH2)2—O—N(Rm)(Rn) or O—CH2—C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H, an amino protecting group or substituted or unsubstituted C1-C10 alkyl; wherein each optionally substituted group is optionally substituted with a substituent group independently selected from among: hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.
    Embodiment 107
      • The oligomeric compound of embodiment 102-106, wherein each A nucleoside comprises a 2′-substituted sugar moiety comprising a 2′-substituent selected from among: a halogen, OCH3, OCF3, OCH2CH3, OCH2CF3, OCH2—CH═CH2, O(CH2)2—OCH3, O(CH2)2—O(CH2)2—N(CH3)2, OCH2C(═O)—N(H)CH3, OCH2C(═O)—N(H)—(CH2)2—N(CH3)2, and OCH2—N(H)—C(═NH)NH2.
    Embodiment 108
      • The oligomeric compound of embodiment 107, wherein each A nucleoside comprises a 2′-substituted sugar moiety comprising a 2′-substituent selected from among: F, OCH3, O(CH2)2—OCH3.
    Embodiment 109
      • The oligomeric compound of embodiments 102-106, wherein each A nucleoside comprises a bicyclic sugar moiety.
    Embodiment 110
      • The oligomeric compound of embodiment 109, wherein each A nucleoside comprises a bicyclic sugar moiety selected from among: cEt, cMOE, LNA, α-LNA, ENA and 2′-thio LNA.
    Embodiment 111
      • The oligomeric compound of any of embodiments 102-110, wherein each A comprises a modified nucleobase.
    Embodiment 112
      • The oligomeric compound of embodiment 111, wherein each A comprises a modified nucleobase selected from among a 2-thio pyrimidine and a 5-propyne pyrimidine.
    Embodiment 113
      • The oligomeric compound of embodiment 112, wherein each A comprises 2-thio-thymidine.
    Embodiment 114
      • The oligomeric compound of embodiment 102-106, wherein each A nucleoside comprises an unmodified 2′-deoxyfuranose sugar moiety.
    Embodiment 115
      • The oligomeric compound of embodiment 102-106, wherein each A nucleoside comprises an F-HNA sugar moiety.
    Embodiment 116
      • The oligomeric compound of any of embodiments 102-115, wherein each B nucleoside comprises a 2′-substituted sugar moiety.
    Embodiment 117
      • The oligomeric compound of embodiment 116, wherein at least one central region nucleoside comprises a 2′-substituted sugar moiety comprising a 2′ substituent selected from among: halogen, optionally substituted allyl, optionally substituted amino, azido, optionally substituted SH, CN, OCN, CF3, OCF3, O, S, or N(Rm)-alkyl; O, S, or N(Rm)-alkenyl; O, S or N(Rm)-alkynyl; optionally substituted O-alkylenyl-O-alkyl, optionally substituted alkynyl, optionally substituted alkaryl, optionally substituted aralkyl, optionally substituted O-alkaryl, optionally substituted O-aralkyl, O(CH2)2SCH3, O—(CH2)2—O—N(Rm)(Rn) or O—CH2—C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H, an amino protecting group or substituted or unsubstituted C1-C10 alkyl; wherein each optionally substituted group is optionally substituted with a substituent group independently selected from among: hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.
    Embodiment 118
      • The oligomeric compound of embodiment 117, wherein each B nucleoside comprises a 2′-substituted sugar moiety comprising a 2′-substituent selected from among: a halogen, OCH3, OCF3, OCH2CH3, OCH2CF3, OCH2—CH═CH2, O(CH2)2—OCH3, O(CH2)2—O(CH2)2—N(CH3)2, OCH2C(═O)—N(H)CH3, OCH2C(═O)—N(H)—(CH2)2—N(CH3)2, and OCH2—N(H)—C(═NH)NH2.
    Embodiment 119
      • The oligomeric compound of embodiment 118, wherein each B nucleoside comprises a 2′-substituted sugar moiety comprising a 2′-substituent selected from among: F, OCH3, O(CH2)2—OCH3.
    Embodiment 120
      • The oligomeric compound of any of embodiments 102-115, wherein each B nucleoside comprises a bicyclic sugar moiety.
    Embodiment 121
      • The oligomeric compound of embodiment 120, wherein each B nucleoside comprises a bicyclic sugar moiety selected from among: cEt, cMOE, LNA, α-LNA, ENA and 2′-thio LNA.
    Embodiment 122
      • The oligomeric compound of any of embodiments 102-115, wherein each B comprises a modified nucleobase.
    Embodiment 123
      • The oligomeric compound of embodiment 122, wherein each B comprises a modified nucleobase selected from among a 2-thio pyrimidine and a 5-propyne pyrimidine.
    Embodiment 124
      • The oligomeric compound of embodiment 123, wherein each B comprises 2-thio-thymidine.
    Embodiment 125
      • The oligomeric compound of embodiment 102-106, wherein each B nucleoside comprises an unmodified 2′-deoxyfuranose sugar moiety.
    Embodiment 126
      • The oligomeric compound of embodiment 102-115, wherein each B nucleoside comprises an F-HNA sugar moiety.
    Embodiment 127
      • The oligomeric compound of any of embodiments 102-126, wherein each C nucleoside comprises a 2′-substituted sugar moiety.
    Embodiment 128
      • The oligomeric compound of embodiment 127, wherein at least one central region nucleoside comprises a 2′-substituted sugar moiety comprising a 2′ substituent selected from among: halogen, optionally substituted allyl, optionally substituted amino, azido, optionally substituted SH, CN, OCN, CF3, OCF3, O, S, or N(Rm)-alkyl; O, S, or N(Rm)-alkenyl; O, S or N(Rm)-alkynyl; optionally substituted O-alkylenyl-O-alkyl, optionally substituted alkynyl, optionally substituted alkaryl, optionally substituted aralkyl, optionally substituted O-alkaryl, optionally substituted O-aralkyl, O(CH2)2SCH3, O—(CH2)2—O—N(Rm)(Rn) or O—CH2—C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H, an amino protecting group or substituted or unsubstituted C1-C10 alkyl; wherein each optionally substituted group is optionally substituted with a substituent group independently selected from among: hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.
    Embodiment 129
      • The oligomeric compound of embodiment 128, wherein each C nucleoside comprises a 2′-substituted sugar moiety comprising a 2′-substituent selected from among: a halogen, OCH3, OCF3, OCH2CH3, OCH2CF3, OCH2—CH═CH2, O(CH2)2—OCH3, O(CH2)2—O(CH2)2—N(CH3)2, OCH2C(═O)—N(H)CH3, OCH2C(═O)—N(H)—(CH2)2—N(CH3)2, and OCH2—N(H)—C(═NH)NH2.
    Embodiment 130
      • The oligomeric compound of embodiment 129, wherein each C nucleoside comprises a 2′-substituted sugar moiety comprising a 2′-substituent selected from among: F, OCH3, O(CH2)2—OCH3.
    Embodiment 131
      • The oligomeric compound of any of embodiments 102-126, wherein each C nucleoside comprises a bicyclic sugar moiety.
    Embodiment 132
      • The oligomeric compound of embodiment 131, wherein each C nucleoside comprises a bicyclic sugar moiety selected from among: cEt, cMOE, LNA, α-LNA, ENA and 2′-thio LNA.
    Embodiment 133
      • The oligomeric compound of any of embodiments 102-126, wherein each C comprises a modified nucleobase.
    Embodiment 134
      • The oligomeric compound of embodiment 133, wherein each C comprises a modified nucleobase selected from among a 2-thio pyrimidine and a 5-propyne pyrimidine.
    Embodiment 135
      • The oligomeric compound of embodiment 134, wherein each C comprises 2-thio-thymidine.
    Embodiment 136
      • The oligomeric compound of embodiment 102-126, wherein each C comprises an F—HNA sugar moiety.
    Embodiment 137
      • The oligomeric compound of embodiment 102-126, wherein each C nucleoside comprises an unmodified 2′-deoxyfuranose sugar moiety.
    Embodiment 138
      • The oligomeric compound of any of embodiments 102-138, wherein each W nucleoside comprises a 2′-substituted sugar moiety.
    Embodiment 139
      • The oligomeric compound of embodiment 138, wherein at least one central region nucleoside comprises a 2′-substituted sugar moiety comprising a 2′ substituent selected from among: halogen, optionally substituted allyl, optionally substituted amino, azido, optionally substituted SH, CN, OCN, CF3, OCF3, O, S, or N(Rm)-alkyl; O, S, or N(Rm)-alkenyl; O, S or N(Rm)-alkynyl; optionally substituted O-alkylenyl-O-alkyl, optionally substituted alkynyl, optionally substituted alkaryl, optionally substituted aralkyl, optionally substituted O-alkaryl, optionally substituted O-aralkyl, O(CH2)2SCH3, O—(CH2)2—O—N(Rm)(Rn) or O—CH2—C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H, an amino protecting group or substituted or unsubstituted C1-C10 alkyl; wherein each optionally substituted group is optionally substituted with a substituent group independently selected from among: hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.
    Embodiment 140
      • The oligomeric compound of embodiment 139, wherein each W nucleoside comprises a 2′-substituted sugar moiety comprising a 2′-substituent selected from among: a halogen, OCH3, OCF3, OCH2CH3, OCH2CF3, OCH2—CH═CH2, O(CH2)2—OCH3, O(CH2)2—O(CH2)2—N(CH3)2, OCH2C(═O)—N(H)CH3, OCH2C(═O)—N(H)—(CH2)2—N(CH3)2, and OCH2—N(H)—C(═NH)NH2.
    Embodiment 141
      • The oligomeric compound of embodiment 139, wherein each W nucleoside comprises a 2′-substituted sugar moiety comprising a 2′-substituent selected from among: F, OCH3, O(CH2)2—OCH3.
    Embodiment 142
      • The oligomeric compound of any of embodiments 102-137, wherein each W nucleoside comprises a bicyclic sugar moiety.
    Embodiment 143
      • The oligomeric compound of embodiment 142, wherein each W nucleoside comprises a bicyclic sugar moiety selected from among: cEt, cMOE, LNA, α-LNA, ENA and 2′-thio LNA.
    Embodiment 144
      • The oligomeric compound of any of embodiments 102-137, wherein each W comprises a modified nucleobase.
    Embodiment 145
      • The oligomeric compound of embodiment 144, wherein each W comprises a modified nucleobase selected from among a 2-thio pyrimidine and a 5-propyne pyrimidine.
    Embodiment 146
      • The oligomeric compound of embodiment 145, wherein each W comprises 2-thio-thymidine.
    Embodiment 147
      • The oligomeric compound of embodiment 102-137, wherein each W comprises an F—HNA sugar moiety.
    Embodiment 148
      • The oligomeric compound of embodiment 102-137, wherein each W nucleoside comprises an unmodified 2′-deoxyfuranose sugar moiety.
    Embodiment 149
      • The oligomeric compound of any of embodiments 1-148, wherein the 3′ region consists of 2 linked 3′-region nucleosides.
    Embodiment 150
      • The oligomeric compound of any of embodiments 1-148, wherein the 3′ region consists of 3 linked 3′-region nucleosides.
    Embodiment 151
      • The oligomeric compound of any of embodiments 1-148, wherein the 3′ region consists of 4 linked 3′-region nucleosides.
    Embodiment 152
      • The oligomeric compound of any of embodiments 1-148, wherein the 3′ region consists of 5 linked 3′-region nucleosides.
    Embodiment 153
      • The oligomeric compound of any of embodiments 1-148, wherein the 3′ region consists of 6 linked 3′-region nucleosides.
    Embodiment 154
      • The oligomeric compound of any of embodiments 1-153, wherein at least one 3′-region nucleoside is an unmodified deoxynucleoside.
    Embodiment 155
      • The oligomeric compound of any of embodiments 1-154, wherein each 3′-region nucleoside is a modified nucleoside.
    Embodiment 156
      • The oligomeric compound of any of embodiments 1-153, wherein at least one 3′-region nucleoside is an RNA-like nucleoside.
    Embodiment 157
      • The oligomeric compound of any of embodiments 1-154, wherein each 3′-region nucleoside is an RNA-like nucleoside.
    Embodiment 158
      • The oligomeric compound of any of embodiments 1-153, comprising at least one modified 3′-region nucleoside comprising a modified sugar.
    Embodiment 159
      • The oligomeric compound of embodiment 158, comprising at least one modified 3′-region nucleoside comprising a bicyclic sugar moiety.
    Embodiment 160
      • The oligomeric compound of embodiment 159, comprising at least one modified 3′-region nucleoside comprising a cEt sugar moiety.
    Embodiment 161
      • The oligomeric compound of embodiment 159, comprising at least one modified 3′-region nucleoside comprising an LNA sugar moiety.
    Embodiment 162
      • The oligomeric compound of any of embodiments 1-162 comprising of at least one modified 3′-region nucleoside comprising a 2′-substituted sugar moiety.
    Embodiment 163
      • The oligomeric compound of embodiment 162, wherein at least one central region nucleoside comprises a 2′-substituted sugar moiety comprising a 2′ substituent selected from among: halogen, optionally substituted allyl, optionally substituted amino, azido, optionally substituted SH, CN, OCN, CF3, OCF3, O, S, or N(Rm)-alkyl; O, S, or N(Rm)-alkenyl; O, S or N(Rm)-alkynyl; optionally substituted O-alkylenyl-O-alkyl, optionally substituted alkynyl, optionally substituted alkaryl, optionally substituted aralkyl, optionally substituted O-alkaryl, optionally substituted O-aralkyl, O(CH2)2SCH3, O—(CH2)2—O—N(Rm)(Rn) or O—CH2—C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H, an amino protecting group or substituted or unsubstituted C1-C10 alkyl; wherein each optionally substituted group is optionally substituted with a substituent group independently selected from among: hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.
    Embodiment 164
      • The oligomeric compound of embodiment 163 wherein at least one modified 3′-region nucleoside comprises a 2′-substituted sugar moiety comprising a 2′-substituent selected from among: a halogen, OCH3, OCH2F, OCHF2, OCF3, OCH2CH3, O(CH2)2F, OCH2CHF2, OCH2CF3, OCH2—CH═CH2, O(CH2)2—OCH3 (MOE), O(CH2)2—SCH3, O(CH2)2—OCF3, O(CH2)3—N(R1)(R2), O(CH2)2—ON(R1)(R2), O(CH2)2—O(CH2)2—N(R1)(R2), OCH2C(═O)—N(R1)(R2), OCH2C(═O)—N(R3)—(CH2)2—N(R1)(R2), and O(CH2)2—N(R3)—C(═NR4)[N(R1)(R2)]; wherein R1, R2, R3 and R4 are each, independently, H or C1-C6 alkyl.
    Embodiment 165
      • The oligomeric compound of embodiment 164, wherein the 2′-substituent is selected from among: a halogen, OCH3, OCF3, OCH2CH3, OCH2CF3, OCH2—CH═CH2, O(CH2)2—OCH3, O(CH2)2—O(CH2)2—N(CH3)2, OCH2C(═O)—N(H)CH3, OCH2C(═O)—N(H)—(CH2)2—N(CH3)2, and OCH2—N(H)—C(═NH)NH2.
    Embodiment 166
      • The oligomeric compound of any of embodiments 162-165 comprising at least one modified 3′-region nucleoside comprising a 2′-MOE sugar moiety.
    Embodiment 167
      • The oligomeric compound of any of embodiments 162-166 comprising at least one modified 3′-region nucleoside comprising a 2′-OMe sugar moiety.
    Embodiment 168
      • The oligomeric compound of any of embodiments 162-167 comprising at least one modified 3′-region nucleoside comprising a 2′-F sugar moiety.
    Embodiment 169
      • The oligomeric compound of any of embodiments 162-168 comprising at least one modified 3′-region nucleoside comprising a 2′-(ara)-F sugar moiety.
    Embodiment 170
      • The oligomeric compound of any of embodiments 162-169 comprising of at least one modified 3′-region nucleoside comprising a sugar surrogate.
    Embodiment 171
      • The oligomeric compound of embodiment 170 comprising at least one modified 3′-region nucleoside comprising an F-HNA sugar moiety.
    Embodiment 172
      • The oligomeric compound of embodiment 170 comprising at least one modified 3′-region nucleoside comprising an HNA sugar moiety.
    Embodiment 173
      • The oligomeric compound of any of embodiments 1-172 comprising at least one modified 3′-region nucleoside comprising a modified nucleobase.
    Embodiment 174
      • The oligomeric compound of any of embodiments 1-173, wherein each A comprises a 2′-substituted sugar moiety comprising a 2′-substituent selected from among: F, OCH3, O(CH2)2—OCH3, and each B comprises a bicyclic sugar moiety selected from among: LNA and cEt.
    Embodiment 175
      • The oligomeric compound of embodiment 174, wherein each A comprises O(CH2)2—OCH3 and each B comprises cEt.
    Embodiment 176
      • The oligomeric compound of any of embodiments 1-175, wherein the 3′-region has a motif selected from among: ABB, ABAA, AAABAA, AAAAABAA, AABAA, AAAABAA, AAABAA, ABAB, AAAAA, AAABB, AAAAAAAA, AAAAAAA, AAAAAA, AAAAB, AAAA, AAA, AA, AB, ABBB, ABAB, AABBB, wherein each A is a modified nucleoside of a first type, each B is a modified nucleoside of a second type.
    Embodiment 177
      • The oligomeric compound of embodiments 1-175, wherein the 3′-region has a motif selected from among: ABB; AAABAA; AABAA; AAAABAA; AAAAA; AAABB; AAAAAAAA; AAAAAAA; AAAAAA; AAAAB; AB; ABBB; and ABAB, wherein each A is a modified nucleoside of a first type, each B is a modified nucleoside of a second type.
    Embodiment 178
      • The oligomeric compound of embodiments 1-175, wherein the 3′-region has a motif selected from among: BBA, AAB, AAA, BBB, BBAA, AABB, WBBA, WAAB, BBBA, BBBBA, BBBB, BBBBBA, ABBBBB, BBAAA, AABBB, BBBAA, BBBBA, BBBBB, BABA, AAAAA, BBAAAA, AABBBB, BAAAA, and ABBBB, wherein each A is a modified nucleoside of a first type, each B is a modified nucleoside of a second type, and each W is a modified nucleoside of a first type, a second type, or a third type.
    Embodiment 179
      • The oligomeric compound of embodiments 176-178, wherein each A nucleoside comprises a 2′-substituted sugar moiety.
    Embodiment 180
      • The oligomeric compound of embodiments 176-178, wherein at least one central region nucleoside comprises a 2′-substituted sugar moiety comprising a 2′ substituent selected from among: halogen, optionally substituted allyl, optionally substituted amino, azido, optionally substituted SH, CN, OCN, CF3, OCF3, O, S, or N(Rm)-alkyl; O, S, or N(Rm)-alkenyl; O, S or N(Rm)-alkynyl; optionally substituted O-alkylenyl-O-alkyl, optionally substituted alkynyl, optionally substituted alkaryl, optionally substituted aralkyl, optionally substituted O-alkaryl, optionally substituted O-aralkyl, O(CH2)2SCH3, O—(CH2)2—O—N(Rm)(Rn) or O—CH2—C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H, an amino protecting group or substituted or unsubstituted C1-C10 alkyl;
      • wherein each optionally substituted group is optionally substituted with a substituent group independently selected from among: hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.
    Embodiment 181
      • The oligomeric compound of embodiment 180, wherein each A nucleoside comprises a 2′-substituted sugar moiety comprising a 2′-substituent selected from among: a halogen, OCH3, OCF3, OCH2CH3, OCH2CF3, OCH2—CH═CH2, O(CH2)2—OCH3, O(CH2)2—O(CH2)2—N(CH3)2, OCH2C(═O)—N(H)CH3, OCH2C(═O)—N(H)—(CH2)2—N(CH3)2, and OCH2—N(H)—C(═NH)NH2.
    Embodiment 182
      • The oligomeric compound of embodiment 181, wherein each A nucleoside comprises a 2′-substituted sugar moiety comprising a 2′-substituent selected from among: F, OCH3, O(CH2)2—OCH3.
    Embodiment 183
      • The oligomeric compound of embodiments 176-178, wherein each A nucleoside comprises a bicyclic sugar moiety.
    Embodiment 184
      • The oligomeric compound of embodiment 183, wherein each A nucleoside comprises a bicyclic sugar moiety selected from among: cEt, cMOE, LNA, α-LNA, ENA and 2′-thio LNA.
    Embodiment 185
      • The oligomeric compound of any of embodiments 176-178, wherein each B nucleoside comprises a 2′-substituted sugar moiety.
    Embodiment 186
      • The oligomeric compound of embodiment 185, wherein at least one modified central region nucleoside comprises a 2′-substituted sugar moiety comprising a 2′ substituent selected from among: halogen, optionally substituted allyl, optionally substituted amino, azido, optionally substituted SH, CN, OCN, CF3, OCF3, O, S, or N(Rm)-alkyl; O, S, or N(Rm)-alkenyl; O, S or N(Rm)-alkynyl; optionally substituted O-alkylenyl-O-alkyl, optionally substituted alkynyl, optionally substituted alkaryl, optionally substituted aralkyl, optionally substituted O-alkaryl, optionally substituted O-aralkyl, O(CH2)2SCH3, O—(CH2)2—O—N(Rm)(Rn) or O—CH2—C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H, an amino protecting group or substituted or unsubstituted C1-C10 alkyl;
      • wherein each optionally substituted group is optionally substituted with a substituent group independently selected from among: hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.
    Embodiment 187
      • The oligomeric compound of embodiment 185, wherein each B nucleoside comprises a 2′-substituted sugar moiety comprising a 2′-substituent selected from among: a halogen, OCH3, OCF3, OCH2CH3, OCH2CF3, OCH2—CH═CH2, O(CH2)2—OCH3, O(CH2)2—O(CH2)2—N(CH3)2, OCH2C(═O)—N(H)CH3, OCH2C(═O)—N(H)—(CH2)2—N(CH3)2, and OCH2—N(H)—C(═NH)NH2.
    Embodiment 188
      • The oligomeric compound of embodiment 187, wherein each B nucleoside comprises a 2′-substituted sugar moiety comprising a 2′-substituent selected from among: F, OCH3, O(CH2)2—OCH3.
    Embodiment 189
      • The oligomeric compound of any of embodiments 176-178, wherein each B nucleoside comprises a bicyclic sugar moiety.
    Embodiment 190
      • The oligomeric compound of embodiment 189, wherein each B nucleoside comprises a bicyclic sugar moiety selected from among: cEt, cMOE, LNA, α-LNA, ENA and 2′-thio LNA.
    Embodiment 191
      • The oligomeric compound of any of embodiments 176-190, wherein each A comprises a 2′-substituted sugar moiety comprising a 2′-substituent selected from among: F, OCH3, O(CH2)2—OCH3, and each B comprises a bicyclic sugar moiety selected from among: LNA and cEt.
    Embodiment 192
      • The oligomeric compound of embodiment 191, wherein each A comprises O(CH2)2—OCH3 and each B comprises cEt.
    Embodiment 193
      • The oligomeric compound of any of embodiments 176-192, wherein each W nucleoside comprises a 2′-substituted sugar moiety.
    Embodiment 194
      • The oligomeric compound of embodiment 193, wherein at least one central region nucleoside comprises a 2′-substituted sugar moiety comprising a 2′ substituent selected from among: halogen, optionally substituted allyl, optionally substituted amino, azido, optionally substituted SH, CN, OCN, CF3, OCF3, O, S, or N(Rm)-alkyl; O, S, or N(Rm)-alkenyl; O, S or N(Rm)-alkynyl; optionally substituted O-alkylenyl-O-alkyl, optionally substituted alkynyl, optionally substituted alkaryl, optionally substituted aralkyl, optionally substituted O-alkaryl, optionally substituted O-aralkyl, O(CH2)2SCH3, O—(CH2)2—O—N(Rm)(Rn) or O—CH2—C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H, an amino protecting group or substituted or unsubstituted C1-C10 alkyl; wherein each optionally substituted group is optionally substituted with a substituent group independently selected from among: hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.
    Embodiment 195
      • The oligomeric compound of embodiment 193, wherein each W nucleoside comprises a 2′-substituted sugar moiety comprising a 2′-substituent selected from among: a halogen, OCH3, OCF3, OCH2CH3, OCH2CF3, OCH2—CH═CH2, O(CH2)2—OCH3, O(CH2)2—O(CH2)2—N(CH3)2, OCH2C(═O)—N(H)CH3, OCH2C(═O)—N(H)—(CH2)2—N(CH3)2, and OCH2—N(H)—C(═NH)NH2.
    Embodiment 196
      • The oligomeric compound of embodiment 195, wherein each W nucleoside comprises a 2′-substituted sugar moiety comprising a 2′-substituent selected from among: F, OCH3, O(CH2)2—OCH3.
    Embodiment 197
      • The oligomeric compound of any of embodiments 176-192, wherein each W nucleoside comprises a bicyclic sugar moiety.
    Embodiment 198
      • The oligomeric compound of embodiment 197, wherein each W nucleoside comprises a bicyclic sugar moiety selected from among: cEt, cMOE, LNA, α-LNA, ENA and 2′-thio LNA.
    Embodiment 199
      • The oligomeric compound of any of embodiments 176-192, wherein each W comprises a modified nucleobase.
    Embodiment 200
      • The oligomeric compound of embodiment 199, wherein each W comprises a modified nucleobase selected from among a 2-thio pyrimidine and a 5-propyne pyrimidine.
    Embodiment 201
      • The oligomeric compound of embodiment 200, wherein each W comprises 2-thio-thymidine.
    Embodiment 202
      • The oligomeric compound of embodiment 176-192, wherein each W comprises an F-HNA sugar moiety.
    Embodiment 203
      • The oligomeric compound of embodiment 202, wherein each W nucleoside comprises an unmodified 2′-deoxyfuranose sugar moiety.
    Embodiment 204
      • The oligomeric compound of embodiments 1-203, wherein the 5′-region has a motif selected from among: AB, ABB, AAA, BBB, BBBAA, AAB, BAA, BBAA, AABB, AAAB, ABBW, ABBWW, ABBB, ABBBB, ABAB, ABABAB, ABABBB, ABABAA, AAABB, AAAABB, AABB, AAAAB, AABBB, ABBBB, BBBBB, AAABW, AAAAA, and BBBBAA;
        • wherein the 3′-region has a motif selected from among: BBA, AAB, AAA, BBB, BBAA, AABB, WBBA, WAAB, BBBA, BBBBA, BBBB, BBBBBA, ABBBBB, BBAAA, AABBB, BBBAA, BBBBA, BBBBB, BABA, AAAAA, BBAAAA, AABBBB, BAAAA, and ABBBB;
        • wherein the central region has a nucleoside motif selected from among: DDDDDD, DDDDDDD, DDDDDDDD, DDDDDDDDD, DDDDDDDDDD, DXDDDDDDD, DDXDDDDDD, DDDXDDDDD, DDDDXDDDD, DDDDDXDDD, DDDDDDXDD, DDDDDDDXD, DXXDDDDDD, DDDDDDXXD, DDXXDDDDD, DDDXXDDDD, DDDDXXDDD, DDDDDXXDD, DXDDDDDXD, DXDDDDXDD, DXDDDXDDD, DXDDXDDDD, DXDXDDDDD, DDXDDDDXD, DDXDDDXDD, DDXDDXDDD, DDXDXDDDD, DDDXDDDXD, DDDXDDXDD, DDDXDXDDD, DDDDXDDXD, DDDDXDXDD, and DDDDDXDXD, DDDDDDDD, DXDDDDDD, DDXDDDDD, DDDXDDDD, DDDDXDDD, DDDDDXDD, DDDDDDXD, DXDDDDXD, DXDDDXDD, DXDDXDDD, DXDXDDDD, DXXDDDDD, DDXXDDDD, DDXDXDDD, DDXDDXDD, DXDDDDXD, DDDXXDDD, DDDXDXDD, DDDXDDXD, DDDDXXDD, DDDDXDXD, and DDDDDXXD, DXDDDDD, DDXDDDD, DDDXDDD, DDDDXDD, DDDDDXD, DXDDDXD, DXDDXDD, DXDXDDD, DXXDDDD, DDXXDDD, DDXDXDD, DDXDDXD, DDDXXDD, DDDXDXD, and DDDDXXD, DXDDDD, DDXDDD, DDDXDD, DDDDXD, DXXDDD, DXDXDD, DXDDXD, DDXXDD, DDXDXD, and DDDXXD; and
        • wherein each A is a modified nucleoside of a first type, each B is a modified nucleoside of a second type, each W is a modified nucleoside of a first type, a second type, or a third type, each D is an unmodified deoxynucleoside, and each X is a modified nucleoside or a modified nucleobase.
    Embodiment 205
      • The oligomeric compound of embodiment 204, wherein the 5′-region has a motif selected from among:
      • AB, ABB, AAA, BBB, BBBAA, AAB, BAA, BBAA, AABB, ABBW, ABBWW, ABBB, ABBBB, ABAB, ABABAB, ABABBB, ABABAA, AAABB, AAAABB, AABB, AAAAB, AABBB, ABBBB, BBBBB, AAABW, and BBBBAA; and wherein the 3′-region has a BBA motif.
    Embodiment 206
      • The oligomeric compound of embodiment 204 or 205, wherein one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 2-thio-thymidine nucleobase.
    Embodiment 207
      • The oligomeric compound of embodiment 204 or 205, wherein one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and W comprises FHNA.
    Embodiment 208
      • The oligomeric compound of embodiment 204 or 205, wherein one of A or B comprises cEt, another of A or B comprises a 2′-modified sugar moiety, and W comprises a 2-thio-thymidine nucleobase.
    Embodiment 209
      • The oligomeric compound of embodiment 204 or 205, wherein one of A or B comprises cEt, another of A or B comprises a 2′-modified sugar moiety, and W comprises FHNA.
    Embodiment 210
      • The oligomeric compound of embodiment 204 or 205, wherein each A comprises MOE, each B comprises cEt, and each W is selected from among cEt or FHNA.
    Embodiment 211
      • The oligomeric compound of embodiment 204 or 205, wherein each W comprises cEt.
    Embodiment 212
      • The oligomeric compound of embodiment 204 or 205, wherein each W comprises 2-thio-thymidine.
    Embodiment 213
      • The oligomeric compound of embodiment 204 or 205, wherein each W comprises FHNA.
    Embodiment 214
      • The oligomeric compound of any of embodiments 1-213 comprising at least one modified internucleoside linkage.
    Embodiment 215
      • The oligomeric compound of embodiment 214, wherein each internucleoside linkage is a modified internucleoside linkage.
    Embodiment 216
      • The oligomeric compound of embodiment 214 or 215 comprising at least one phosphorothioate internucleoside linkage.
    Embodiment 217
      • The oligomeric compound of any of embodiments 214 or 215 comprising at least one methylphosphonate internucleoside linkage.
    Embodiment 218
      • The oligomeric compound of any of embodiments 214 or 215 comprising one methylphosphonate internucleoside linkage.
    Embodiment 219
      • The oligomeric compound of any of embodiments 214 or 215 comprising two methylphosphonate internucleoside linkages.
    Embodiment 220
      • The oligomeric compound of embodiment 217, wherein at least one of the 3rd, 4th, 5th, 6th and/or, 7th internucleoside from the 5′-end is a methylphosphonate internucleoside linkage.
    Embodiment 221
      • The oligomeric compound of embodiment 217, wherein at least one of the 3rd, 4th, 5th, 6th and/or 7th internucleoside from the 3′-end is a methylphosphonate internucleoside linkage.
    Embodiment 222
      • The oligomeric compound of embodiment 217, wherein at least one of the 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, and/or 12th internucleoside from the 5′-end is a methylphosphonate internucleoside linkage, and wherein at least one of the 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, and/or 12th internucleoside from the 5′-end is a modified nucleoside.
    Embodiment 223
      • The oligomeric compound of embodiment 217, wherein at least one of the 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, and/or 12th internucleoside from the 3′-end is a methylphosphonate internucleoside linkage, and wherein at least one of the 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, and/or 12th internucleoside from the 3′-end is a modified nucleoside.
    Embodiment 224
      • The oligomeric compound of any of embodiments 1-223 comprising at least one conjugate group.
    Embodiment 225
      • The oligomeric compound of embodiment 1-223, wherein the conjugate group consists of a conjugate.
    Embodiment 226
      • The oligomeric compound of embodiment 225, wherein the conjugate group consists of a conjugate and a conjugate linker.
    Embodiment 227
      • The oligomeric compound of any of embodiments 1-226, wherein the nucleobase sequence of the modified oligonucleotide is 100% complementary to the nucleobase sequence of the target region of the target nucleic acid.
    Embodiment 228
      • The oligomeric compound of any of embodiments 1-226, wherein the nucleobase sequence of the modified oligonucleotide contains one mismatch relative to the nucleobase sequence of the target region of the target nucleic acid.
    Embodiment 229
      • The oligomeric compound of any of embodiments 1-226, wherein the nucleobase sequence of the modified oligonucleotide contains two mismatches relative to the nucleobase sequence of the target region of the target nucleic acid.
    Embodiment 230
      • The oligomeric compound of any of embodiments 1-226, wherein the nucleobase sequence of the modified oligonucleotide comprises a hybridizing region and at least one non-targeting region, wherein the nucleobase sequence of the hybridizing region is complementary to the nucleobase sequence of the target region of the target nucleic acid.
    Embodiment 231
      • The oligomeric compound of embodiment 230, wherein the nucleobase sequence of the hybridizing region is 100% complementary to the nucleobase sequence of the target region of the target nucleic acid.
    Embodiment 232
      • The oligomeric compound of embodiment 230, wherein the nucleobase sequence of the hybridizing region contains one mismatched relative to the nucleobase sequence of the target region of the target nucleic acid.
    Embodiment 233
      • The oligomeric compound of any of embodiments 230-232, wherein the nucleobase sequence of at least one non-targeting region is complementary to a portion of the hybridizing region of the modified oligonucleotide.
    Embodiment 234
      • The oligomeric compound of embodiment 233, wherein the nucleobase sequence of at least one non-targeting region is 100% complementary to a portion of the hybridizing region of the modified oligonucleotide.
    Embodiment 235
      • The oligomeric compound of embodiment 1-234 wherein the nucleobase sequence of the modified oligonucleotide comprises two non-targeting regions flanking a central hybridizing region.
    Embodiment 236
      • The oligomeric compound of embodiment 235, wherein the two non-targeting regions are complementary to one another.
    Embodiment 237
      • The oligomeric compound of embodiment 236, wherein the two non-targeting regions are 100% complementary to one another.
    Embodiment 238
      • The oligomeric compound of any of embodiments 2-237, wherein the nucleobase sequence of the modified oligonucleotide aligns with the nucleobase of the target region of the target nucleic acid such that a distinguishing nucleobase of the target region of the target nucleic acid aligns with a target-selective nucleoside within the central region of the modified oligonucleotide.
    Embodiment 239
      • The oligomeric compound of any of embodiments 3-237, wherein the nucleobase sequence of the modified oligonucleotide aligns with the nucleobase of the target region of the target nucleic acid such that the single distinguishing nucleobase of the target region of the target nucleic acid aligns with a target-selective nucleoside within the central region of the modified oligonucleotide.
    Embodiment 240
      • The oligomeric compound of embodiment 238 or 239, wherein the target-selective nucleoside is the 5′-most nucleoside of the central region.
    Embodiment 241
      • The oligomeric compound of embodiment 238 or 239, wherein the target-selective nucleoside is the 2nd nucleoside from the 5′-end of the central region.
    Embodiment 242
      • The oligomeric compound of embodiment 238 or 239, wherein the target-selective nucleoside is at the 3rd nucleoside from the 5′-end of the central region.
    Embodiment 243
      • The oligomeric compound of embodiment 238 or 239, wherein the target-selective nucleoside is at the 5th nucleoside from the 5′-end of the central region.
    Embodiment 244
      • The oligomeric compound of embodiment 238 or 239, wherein the target-selective nucleoside is at the 7th nucleoside from the 5′-end of the central region.
    Embodiment 245
      • The oligomeric compound of embodiment 238 or 239, wherein the target-selective nucleoside is at the 9th nucleoside from the 5′-end of the central region.
    Embodiment 246
      • The oligomeric compound of any of embodiments 238 or 239, or 241-245, wherein the target-selective nucleoside is at the 2nd nucleoside from the 3′-end of the central region.
    Embodiment 247
      • The oligomeric compound of any of embodiments 238 or 239, or 241-245, wherein the target-selective nucleoside is at the 5th nucleoside from the 3′-end of the central region.
    Embodiment 248
      • The oligomeric compound of any of embodiments 1-247, wherein target-selective nucleoside is an unmodified deoxynucleoside.
    Embodiment 249
      • The oligomeric compound of any of embodiments 1-247, wherein target-selective nucleoside is a modified nucleoside.
    Embodiment 250
      • The oligomeric compound of embodiment 249, wherein the target-selective nucleoside is a sugar modified nucleoside.
    Embodiment 251
      • The oligomeric compound of embodiment 250, wherein the target-selective nucleoside comprises a sugar modification selected from among: 2′-MOE, 2′-F, 2′-(ara)-F, HNA, FHNA, cEt, and α-L-LNA.
    Embodiment 252
      • The oligomeric compound of any of embodiments 1-251, wherein the target-selective nucleoside comprises a nucleobase modification.
    Embodiment 253
      • The oligomeric compound of embodiment 252, wherein the modified nucleobase is selected from among: a 2-thio pyrimidine and a 5-propyne pyrimidine.
    Embodiment 254
      • The oligomeric compound of any of embodiments 1-253, wherein the oligomeric compound is an antisense compound.
    Embodiment 255
      • The oligomeric compound of embodiment 254, wherein the oligomeric compound selectively reduces expression of the target relative to the non-target.
    Embodiment 256
      • The oligomeric compound of embodiment 255, wherein the oligomeric compound reduces expression of target at least two-fold more than it reduces expression of the non-target.
    Embodiment 257
      • The oligomeric compound of embodiment 256, having an EC50 for reduction of expression of target that is at least least two-fold lower than its EC50 for reduction of expression of the non-target, when measured in cells.
    Embodiment 258
      • The oligomeric compound of embodiment 256, having an ED50 for reduction of expression of target that is at least least two-fold lower than its ED50 for reduction of expression of the non-target, when measured in an animal.
    Embodiment 259
      • The oligomeric compound of embodiments 1-10, having an E-E-E-K-K-(D)7-E-E-K motif, wherein each E is a 2′-MOE nucleoside and each K is a cEt nucleoside.
    Embodiment 260
      • A method comprising contacting a cell with an oligomeric compound of any of embodiments 1-259.
    Embodiment 261
      • The method of embodiment 260, wherein the cell is in vitro.
    Embodiment 262
      • The method of embodiment 260, wherein the cell is in an animal.
    Embodiment 263
      • The method of embodiment 262, wherein the animal is a human.
    Embodiment 264
      • The method of embodiment 263, wherein the animal is a mouse.
    Embodiment 265
      • A pharmaceutical composition comprising an oligomeric compound of any of embodiments 1-259 and a pharmaceutically acceptable carrier or diluent.
    Embodiment 266
      • A method of administering a pharmaceutical composition of embodiment 265 to an animal.
    Embodiment 267
      • The method of embodiment 266, wherein the animal is a human.
    Embodiment 268
      • The method of embodiment 266, wherein the animal is a mouse.
    Embodiment 269
      • Use of an oligomeric compound of any of embodiments 1-259 for the preparation of a medicament for the treatment or amelioration of Alzheimer's disease, Creutzfeldt-Jakob disease, fatal familial insomnia, Alexander disease, Parkinson's disease, amyotrophic lateral sclerosis, dentato-rubral and pallido-luysian atrophy DRPA, spino-cerebellar ataxia, Torsion dystonia, cardiomyopathy, chronic obstructive pulmonary disease (COPD), liver disease, hepatocellular carcinoma, systemic lupus erythematosus, hypercholesterolemia, breast cancer, asthma, Type 1 diabetes, Rheumatoid arthritis, Graves disease, SLE, spinal and bulbar muscular atrophy, Kennedy's disease, progressive childhood posterior subcapsular cataracts, cholesterol gallstone disease, arthrosclerosis, cardiovascular disease, primary hypercalciuria, alpha-thallasemia, obsessive compulsive disorder, Anxiety, comorbid depression, congenital visual defects, hypertension, metabolic syndrome, prostate cancer, congential myasthenic syndrome, peripheral arterial disease, atrial fibrillation, sporadic pheochromocytoma, congenital malformations, Machado-Joseph disease, Huntington's disease, and Autosomal Dominant Retinitis Pigmentosa disease.
    Embodiment 270
      • A method of ameliorating a symptom of Alzheimer's disease, Creutzfeldt-Jakob disease, fatal familial insomnia, Alexander disease, Parkinson's disease, amyotrophic lateral sclerosis, dentato-rubral and pallido-luysian atrophy DRPA, spino-cerebellar ataxia, Torsion dystonia, cardiomyopathy, chronic obstructive pulmonary disease (COPD), liver disease, hepatocellular carcinoma, systemic lupus erythematosus, hypercholesterolemia, breast cancer, asthma, Type 1 diabetes, Rheumatoid arthritis, Graves disease, SLE, spinal and bulbar muscular atrophy, Kennedy's disease, progressive childhood posterior subcapsular cataracts, cholesterol gallstone disease, arthrosclerosis, cardiovascular disease, primary hypercalciuria, alpha-thallasemia, obsessive compulsive disorder, Anxiety, comorbid depression, congenital visual defects, hypertension, metabolic syndrome, prostate cancer, congential myasthenic syndrome, peripheral arterial disease, atrial fibrillation, sporadic pheochromocytoma, congenital malformations, Machado-Joseph disease, Huntington's disease, and Autosomal Dominant Retinitis Pigmentosa disease, comprising administering an oligomeric compound of any of embodiments 1-259 to an animal in need thereof.
    Embodiment 271
      • The method of embodiment 270, wherein the animal is a human.
    Embodiment 272
      • The method of embodiment 270, wherein the animal is a mouse.
  • In certain embodiments, including but not limited to any of the above numbered embodiments, oligomeric compounds including oligonucleotides described herein are capable of modulating expression of a target RNA. In certain embodiments, the target RNA is associated with a disease or disorder, or encodes a protein that is associated with a disease or disorder. In certain embodiments, the oligomeric compounds or oligonucleotides provided herein modulate the expression of function of such RNA to alleviate one or more symptom of the disease or disorder.
  • In certain embodiments, oligomeric compounds including oligonucleotides describe herein are useful in vitro. In certain embodiments such oligomeric compounds are used in diagnostics and/or for target validation experiments.
    • DETAILED DESCRIPTION OF THE INVENTION
  • It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. Herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “including” as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit, unless specifically stated otherwise.
  • The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose.
    • A. DEFINITIONS
  • Unless specific definitions are provided, the nomenclature used in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for chemical synthesis, and chemical analysis. Certain such techniques and procedures may be found for example in “Carbohydrate Modifications in Antisense Research” Edited by Sangvi and Cook, American Chemical Society, Washington D.C., 1994; “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., 21st edition, 2005; and “Antisense Drug Technology, Principles, Strategies, and Applications” Edited by Stanley T. Crooke, CRC Press, Boca Raton, Fla.; and Sambrook et al., “Molecular Cloning, A laboratory Manual,” 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, which are hereby incorporated by reference for any purpose. Where permitted, all patents, applications, published applications and other publications and other data referred to throughout in the disclosure are incorporated by reference herein in their entirety.
  • Unless otherwise indicated, the following terms have the following meanings:
  • As used herein, “nucleoside” means a compound comprising a nucleobase moiety and a sugar moiety. Nucleosides include, but are not limited to, naturally occurring nucleosides (as found in DNA and RNA) and modified nucleosides. Nucleosides may be linked to a phosphate moiety.
  • As used herein, “chemical modification” means a chemical difference in a compound when compared to a naturally occurring counterpart. Chemical modifications of oligonucleotides include nucleoside modifications (including sugar moiety modifications and nucleobase modifications) and internucleoside linkage modifications. In reference to an oligonucleotide, chemical modification does not include differences only in nucleobase sequence.
  • As used herein, “furanosyl” means a structure comprising a 5-membered ring comprising four carbon atoms and one oxygen atom.
  • As used herein, “naturally occurring sugar moiety” means a ribofuranosyl as found in naturally occurring RNA or a deoxyribofuranosyl as found in naturally occurring DNA.
  • As used herein, “sugar moiety” means a naturally occurring sugar moiety or a modified sugar moiety of a nucleoside.
  • As used herein, “modified sugar moiety” means a substituted sugar moiety or a sugar surrogate.
  • As used herein, “substituted sugar moiety” means a furanosyl that is not a naturally occurring sugar moiety. Substituted sugar moieties include, but are not limited to furanosyls comprising substituents at the 2′-position, the 3′-position, the 5′-position and/or the 4′-position. Certain substituted sugar moieties are bicyclic sugar moieties.
  • As used herein, “2′-substituted sugar moiety” means a furanosyl comprising a substituent at the 2′-position other than H or OH. Unless otherwise indicated, a 2′-substituted sugar moiety is not a bicyclic sugar moiety (i.e., the 2′-substituent of a 2′-substituted sugar moiety does not form a bridge to another atom of the furanosyl ring.
  • As used herein, “MOE” means —OCH2CH2OCH3.
  • As used herein, “2′-F nucleoside” refers to a nucleoside comprising a sugar comprising fluorine at the 2′ position. Unless otherwise indicated, the fluorine in a 2′-F nucleoside is in the ribo position (replacing the OH of a natural ribose).
  • As used herein, “2′-(ara)-F” refers to a 2′-F substituted nucleoside, wherein the fluoro group is in the arabino position.
  • Figure US20150051389A1-20150219-C00001
  • As used herein the term “sugar surrogate” means a structure that does not comprise a furanosyl and that is capable of replacing the naturally occurring sugar moiety of a nucleoside, such that the resulting nucleoside sub-units are capable of linking together and/or linking to other nucleosides to form an oligomeric compound which is capable of hybridizing to a complementary oligomeric compound. Such structures include rings comprising a different number of atoms than furanosyl (e.g., 4, 6, or 7-membered rings); replacement of the oxygen of a furanosyl with a non-oxygen atom (e.g., carbon, sulfur, or nitrogen); or both a change in the number of atoms and a replacement of the oxygen. Such structures may also comprise substitutions corresponding to those described for substituted sugar moieties (e.g., 6-membered carbocyclic bicyclic sugar surrogates optionally comprising additional substituents). Sugar surrogates also include more complex sugar replacements (e.g., the non-ring systems of peptide nucleic acid). Sugar surrogates include without limitation morpholinos, cyclohexenyls and cyclohexitols.
  • As used herein, “bicyclic sugar moiety” means a modified sugar moiety comprising a 4 to 7 membered ring (including but not limited to a furanosyl) comprising a bridge connecting two atoms of the 4 to 7 membered ring to form a second ring, resulting in a bicyclic structure. In certain embodiments, the 4 to 7 membered ring is a sugar ring. In certain embodiments the 4 to 7 membered ring is a furanosyl. In certain such embodiments, the bridge connects the 2′-carbon and the 4′-carbon of the furanosyl.
  • As used herein, “nucleotide” means a nucleoside further comprising a phosphate linking group. As used herein, “linked nucleosides” may or may not be linked by phosphate linkages and thus includes, but is not limited to “linked nucleotides.” As used herein, “linked nucleosides” are nucleosides that are connected in a continuous sequence (i.e. no additional nucleosides are present between those that are linked).
  • As used herein, “nucleobase” means a group of atoms that can be linked to a sugar moiety to create a nucleoside that is capable of incorporation into an oligonucleotide, and wherein the group of atoms is capable of bonding with a complementary naturally occurring nucleobase of another oligonucleotide or nucleic acid. Nucleobases may be naturally occurring or may be modified.
  • As used herein the terms, “unmodified nucleobase” or “naturally occurring nucleobase” means the naturally occurring heterocyclic nucleobases of RNA or DNA: the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) (including 5-methyl C), and uracil (U).
  • As used herein, “modified nucleobase” means any nucleobase that is not a naturally occurring nucleobase.
  • As used herein, “modified nucleoside” means a nucleoside comprising at least one chemical modification compared to naturally occurring RNA or DNA nucleosides. Modified nucleosides comprise a modified sugar moiety and/or a modified nucleobase.
  • As used herein, “bicyclic nucleoside” or “BNA” means a nucleoside comprising a bicyclic sugar moiety.
  • As used herein, “constrained ethyl nucleoside” or “cEt” means a nucleoside comprising a bicyclic sugar moiety comprising a 4′-CH(CH3)—O-2′ bridge.
  • As used herein, “locked nucleic acid nucleoside” or “LNA” means a nucleoside comprising a bicyclic sugar moiety comprising a 4′-CH2—O-2′ bridge.
  • As used herein, “2′-substituted nucleoside” means a nucleoside comprising a substituent at the 2′-position other than H or OH. Unless otherwise indicated, a 2′-substituted nucleoside is not a bicyclic nucleoside.
  • As used herein, “2′-deoxynucleoside” means a nucleoside comprising 2′-H furanosyl sugar moiety, as found in naturally occurring deoxyribonucleosides (DNA). In certain embodiments, a 2′-deoxynucleoside may comprise a modified nucleobase or may comprise an RNA nucleobase (e.g., uracil).
  • As used herein, “RNA-like nucleoside” means a modified nucleoside that adopts a northern configuration and functions like RNA when incorporated into an oligonucleotide. RNA-like nucleosides include, but are not limited to 3′-endo furanosyl nucleosides and RNA surrogates.
  • As used herein, “3′-endo-furanosyl nucleoside” means an RNA-like nucleoside that comprises a substituted sugar moiety that has a 3′-endo conformation. 3′-endo-furanosyl nucleosides include, but are not limited to: 2′-MOE, 2′-F, 2′-OMe, LNA, ENA, and cEt nucleosides.
  • As used herein, “RNA-surrogate nucleoside” means an RNA-like nucleoside that does not comprise a furanosyl. RNA-surrogate nucleosides include, but are not limited to hexitols and cyclopentanes.
  • As used herein, “oligonucleotide” means a compound comprising a plurality of linked nucleosides. In certain embodiments, an oligonucleotide comprises one or more unmodified ribonucleosides (RNA) and/or unmodified deoxyribonucleosides (DNA) and/or one or more modified nucleosides.
  • As used herein “oligonucleoside” means an oligonucleotide in which none of the internucleoside linkages contains a phosphorus atom. As used herein, oligonucleotides include oligonucleosides.
  • As used herein, “modified oligonucleotide” means an oligonucleotide comprising at least one modified nucleoside and/or at least one modified internucleoside linkage.
  • As used herein “internucleoside linkage” means a covalent linkage between adjacent nucleosides in an oligonucleotide.
  • As used herein “naturally occurring internucleoside linkage” means a 3′ to 5′ phosphodiester linkage.
  • As used herein, “modified internucleoside linkage” means any internucleoside linkage other than a naturally occurring internucleoside linkage.
  • As used herein, “oligomeric compound” means a polymeric structure comprising two or more sub-structures. In certain embodiments, an oligomeric compound comprises an oligonucleotide. In certain embodiments, an oligomeric compound comprises one or more conjugate groups and/or terminal groups. In certain embodiments, an oligomeric compound consists of an oligonucleotide.
  • As used herein, “terminal group” means one or more atom attached to either, or both, the 3′ end or the 5′ end of an oligonucleotide. In certain embodiments a terminal group is a conjugate group. In certain embodiments, a terminal group comprises one or more terminal group nucleosides.
  • As used herein, “conjugate” means an atom or group of atoms bound to an oligonucleotide or oligomeric compound. In general, conjugate groups modify one or more properties of the compound to which they are attached, including, but not limited to pharmacodynamic, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, charge and/or clearance properties.
  • As used herein, “conjugate linking group” means any atom or group of atoms used to attach a conjugate to an oligonucleotide or oligomeric compound.
  • As used herein, “antisense compound” means a compound comprising or consisting of an oligonucleotide at least a portion of which is complementary to a target nucleic acid to which it is capable of hybridizing, resulting in at least one antisense activity.
  • As used herein, “antisense activity” means any detectable and/or measurable change attributable to the hybridization of an antisense compound to its target nucleic acid.
  • As used herein, “detecting” or “measuring” means that a test or assay for detecting or measuring is performed. Such detection and/or measuring may result in a value of zero. Thus, if a test for detection or measuring results in a finding of no activity (activity of zero), the step of detecting or measuring the activity has nevertheless been performed.
  • As used herein, “detectable and/or measurable activity” means a measurable activity that is not zero.
  • As used herein, “essentially unchanged” means little or no change in a particular parameter, particularly relative to another parameter which changes much more. In certain embodiments, a parameter is essentially unchanged when it changes less than 5%. In certain embodiments, a parameter is essentially unchanged if it changes less than two-fold while another parameter changes at least ten-fold. For example, in certain embodiments, an antisense activity is a change in the amount of a target nucleic acid. In certain such embodiments, the amount of a non-target nucleic acid is essentially unchanged if it changes much less than the target nucleic acid does, but the change need not be zero.
  • As used herein, “expression” means the process by which a gene ultimately results in a protein. Expression includes, but is not limited to, transcription, post-transcriptional modification (e.g., splicing, polyadenlyation, addition of 5′-cap), and translation.
  • As used herein, “target nucleic acid” means a nucleic acid molecule to which an antisense compound is intended to hybridize.
  • As used herein, “non-target nucleic acid” means a nucleic acid molecule to which hybridization of an antisense compound is not intended or desired. In certain embodiments, antisense compounds do hybridize to a non-target, due to homology between the target (intended) and non-target (un-intended).
  • As used herein, “mRNA” means an RNA molecule that encodes a protein.
  • As used herein, “pre-mRNA” means an RNA transcript that has not been fully processed into mRNA. Pre-RNA includes one or more intron.
  • As used herein, “object RNA” means an RNA molecule other than a target RNA, the amount, activity, splicing, and/or function of which is modulated, either directly or indirectly, by a target nucleic acid. In certain embodiments, a target nucleic acid modulates splicing of an object RNA. In certain such embodiments, an antisense compound modulates the amount or activity of the target nucleic acid, resulting in a change in the splicing of an object RNA and ultimately resulting in a change in the activity or function of the object RNA.
  • As used herein, “microRNA” means a naturally occurring, small, non-coding RNA that represses gene expression of at least one mRNA. In certain embodiments, a microRNA represses gene expression by binding to a target site within a 3′ untranslated region of an mRNA. In certain embodiments, a microRNA has a nucleobase sequence as set forth in miRBase, a database of published microRNA sequences found at http://microrna.sanger.ac.uk/sequences/. In certain embodiments, a microRNA has a nucleobase sequence as set forth in miRBase version 12.0 released September 2008, which is herein incorporated by reference in its entirety.
  • As used herein, “microRNA mimic” means an oligomeric compound having a sequence that is at least partially identical to that of a microRNA. In certain embodiments, a microRNA mimic comprises the microRNA seed region of a microRNA. In certain embodiments, a microRNA mimic modulates translation of more than one target nucleic acids. In certain embodiments, a microRNA mimic is double-stranded.
  • As used herein, “differentiating nucleobase” means a nucleobase that differs between two nucleic acids. In certain instances, a target region of a target nucleic acid differs by 1-4 nucleobases from a non-target nucleic acid. Each of those differences is referred to as a differentiating nucleobase. In certain instances, a differentiating nucleobase is a single-nucleotide polymorphism.
  • As used herein, “target-selective nucleoside” means a nucleoside of an antisense compound that corresponds to a differentiating nucleobase of a target nucleic acid.
  • As used herein, “allele” means one of a pair of copies of a gene existing at a particular locus or marker on a specific chromosome, or one member of a pair of nucleobases existing at a particular locus or marker on a specific chromosome, or one member of a pair of nucleobase sequences existing at a particular locus or marker on a specific chromosome. For a diploid organism or cell or for autosomal chromosomes, each allelic pair will normally occupy corresponding positions (loci) on a pair of homologous chromosomes, one inherited from the mother and one inherited from the father. If these alleles are identical, the organism or cell is said to be “homozygous” for that allele; if they differ, the organism or cell is said to be “heterozygous” for that allele. “Wild-type allele” refers to the genotype typically not associated with disease or dysfunction of the gene product. “Mutant allele” refers to the genotype associated with disease or dysfunction of the gene product.
  • As used herein, “allelic variant” means a particular identity of an allele, where more than one identity occurs. For example, an allelic variant may refer to either the mutant allele or the wild-type allele.
  • As used herein, “single nucleotide polymorphism” or “SNP” means a single nucleotide variation between the genomes of individuals of the same species. In some cases, a SNP may be a single nucleotide deletion or insertion. In general, SNPs occur relatively frequently in genomes and thus contribute to genetic diversity. The location of a SNP is generally flanked by highly conserved sequences. An individual may be homozygous or heterozygous for an allele at each SNP site.
  • As used herein, “single nucleotide polymorphism site” or “SNP site” refers to the nucleotides surrounding a SNP contained in a target nucleic acid to which an antisense compound is targeted.
  • As used herein, “targeting” or “targeted to” means the association of an antisense compound to a particular target nucleic acid molecule or a particular region of a target nucleic acid molecule. An antisense compound targets a target nucleic acid if it is sufficiently complementary to the target nucleic acid to allow hybridization under physiological conditions.
  • As used herein, “nucleobase complementarity” or “complementarity” when in reference to nucleobases means a nucleobase that is capable of base pairing with another nucleobase. For example, in DNA, adenine (A) is complementary to thymine (T). For example, in RNA, adenine (A) is complementary to uracil (U). In certain embodiments, complementary nucleobase means a nucleobase of an antisense compound that is capable of base pairing with a nucleobase of its target nucleic acid. For example, if a nucleobase at a certain position of an antisense compound is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be complementary at that nucleobase pair. Nucleobases comprising certain modifications may maintain the ability to pair with a counterpart nucleobase and thus, are still capable of nucleobase complementarity.
  • As used herein, “non-complementary” in reference to nucleobases means a pair of nucleobases that do not form hydrogen bonds with one another.
  • As used herein, “complementary” in reference to oligomeric compounds (e.g., linked nucleosides, oligonucleotides, or nucleic acids) means the capacity of such oligomeric compounds or regions thereof to hybridize to another oligomeric compound or region thereof through nucleobase complementarity under stringent conditions. Complementary oligomeric compounds need not have nucleobase complementarity at each nucleoside. Rather, some mismatches are tolerated. In certain embodiments, complementary oligomeric compounds or regions are complementary at 70% of the nucleobases (70% complementary). In certain embodiments, complementary oligomeric compounds or regions are 80% complementary. In certain embodiments, complementary oligomeric compounds or regions are 90% complementary. In certain embodiments, complementary oligomeric compounds or regions are 95% complementary. In certain embodiments, complementary oligomeric compounds or regions are 100% complementary.
  • As used herein, “mismatch” means a nucleobase of a first oligomeric compound that is not capable of pairing with a nucleobase at a corresponding position of a second oligomeric compound, when the first and second oligomeric compound are aligned. Either or both of the first and second oligomeric compounds may be oligonucleotides.
  • As used herein, “hybridization” means the pairing of complementary oligomeric compounds (e.g., an antisense compound and its target nucleic acid). While not limited to a particular mechanism, the most common mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases.
  • As used herein, “specifically hybridizes” means the ability of an oligomeric compound to hybridize to one nucleic acid site with greater affinity than it hybridizes to another nucleic acid site. In certain embodiments, an antisense oligonucleotide specifically hybridizes to more than one target site.
  • As used herein, “fully complementary” in reference to an oligonucleotide or portion thereof means that each nucleobase of the oligonucleotide or portion thereof is capable of pairing with a nucleobase of a complementary nucleic acid or contiguous portion thereof. Thus, a fully complementary region comprises no mismatches or unhybridized nucleobases in either strand.
  • As used herein, “percent complementarity” means the percentage of nucleobases of an oligomeric compound that are complementary to an equal-length portion of a target nucleic acid. Percent complementarity is calculated by dividing the number of nucleobases of the oligomeric compound that are complementary to nucleobases at corresponding positions in the target nucleic acid by the total length of the oligomeric compound.
  • As used herein, “percent identity” means the number of nucleobases in a first nucleic acid that are the same type (independent of chemical modification) as nucleobases at corresponding positions in a second nucleic acid, divided by the total number of nucleobases in the first nucleic acid.
  • As used herein, “modulation” means a change of amount or quality of a molecule, function, or activity when compared to the amount or quality of a molecule, function, or activity prior to modulation. For example, modulation includes the change, either an increase (stimulation or induction) or a decrease (inhibition or reduction) in gene expression. As a further example, modulation of expression can include a change in splice site selection of pre-mRNA processing, resulting in a change in the absolute or relative amount of a particular splice-variant compared to the amount in the absence of modulation.
  • As used herein, “modification motif” means a pattern of chemical modifications in an oligomeric compound or a region thereof. Motifs may be defined by modifications at certain nucleosides and/or at certain linking groups of an oligomeric compound.
  • As used herein, “nucleoside motif” means a pattern of nucleoside modifications in an oligomeric compound or a region thereof. The linkages of such an oligomeric compound may be modified or unmodified. Unless otherwise indicated, motifs herein describing only nucleosides are intended to be nucleoside motifs. Thus, in such instances, the linkages are not limited.
  • As used herein, “sugar motif” means a pattern of sugar modifications in an oligomeric compound or a region thereof.
  • As used herein, “linkage motif” means a pattern of linkage modifications in an oligomeric compound or region thereof. The nucleosides of such an oligomeric compound may be modified or unmodified. Unless otherwise indicated, motifs herein describing only linkages are intended to be linkage motifs. Thus, in such instances, the nucleosides are not limited.
  • As used herein, “nucleobase modification motif” means a pattern of modifications to nucleobases along an oligonucleotide. Unless otherwise indicated, a nucleobase modification motif is independent of the nucleobase sequence.
  • As used herein, “sequence motif” means a pattern of nucleobases arranged along an oligonucleotide or portion thereof. Unless otherwise indicated, a sequence motif is independent of chemical modifications and thus may have any combination of chemical modifications, including no chemical modifications.
  • As used herein, “type of modification” in reference to a nucleoside or a nucleoside of a “type” means the chemical modification of a nucleoside and includes modified and unmodified nucleosides. Accordingly, unless otherwise indicated, a “nucleoside having a modification of a first type” may be an unmodified nucleoside.
  • As used herein, “differently modified” mean chemical modifications or chemical substituents that are different from one another, including absence of modifications. Thus, for example, a MOE nucleoside and an unmodified DNA nucleoside are “differently modified,” even though the DNA nucleoside is unmodified. Likewise, DNA and RNA are “differently modified,” even though both are naturally-occurring unmodified nucleosides. Nucleosides that are the same but for comprising different nucleobases are not differently modified. For example, a nucleoside comprising a 2′-OMe modified sugar and an unmodified adenine nucleobase and a nucleoside comprising a 2′-OMe modified sugar and an unmodified thymine nucleobase are not differently modified.
  • As used herein, “the same type of modifications” refers to modifications that are the same as one another, including absence of modifications. Thus, for example, two unmodified DNA nucleoside have “the same type of modification,” even though the DNA nucleoside is unmodified. Such nucleosides having the same type modification may comprise different nucleobases.
  • As used herein, “pharmaceutically acceptable carrier or diluent” means any substance suitable for use in administering to an animal. In certain embodiments, a pharmaceutically acceptable carrier or diluent is sterile saline. In certain embodiments, such sterile saline is pharmaceutical grade saline.
  • As used herein, “substituent” and “substituent group,” means an atom or group that replaces the atom or group of a named parent compound. For example a substituent of a modified nucleoside is any atom or group that differs from the atom or group found in a naturally occurring nucleoside (e.g., a modified 2′-substituent is any atom or group at the 2′-position of a nucleoside other than H or OH). Substituent groups can be protected or unprotected. In certain embodiments, compounds of the present invention have substituents at one or at more than one position of the parent compound. Substituents may also be further substituted with other substituent groups and may be attached directly or via a linking group such as an alkyl or hydrocarbyl group to a parent compound.
  • Likewise, as used herein, “substituent” in reference to a chemical functional group means an atom or group of atoms differs from the atom or a group of atoms normally present in the named functional group. In certain embodiments, a substituent replaces a hydrogen atom of the functional group (e.g., in certain embodiments, the substituent of a substituted methyl group is an atom or group other than hydrogen which replaces one of the hydrogen atoms of an unsubstituted methyl group). Unless otherwise indicated, groups amenable for use as substituents include without limitation, halogen, hydroxyl, alkyl, alkenyl, alkynyl, acyl (—C(O)Raa), carboxyl (—C(O)O—Raa), aliphatic groups, alicyclic groups, alkoxy, substituted oxy (—O—Raa), aryl, aralkyl, heterocyclic radical, heteroaryl, heteroarylalkyl, amino (—N(Rbb)(Rcc)), imino(═NRbb), amido (—C(O)N(Rbb)(Rcc) or —N(Rbb)C(O)Raa), azido (—N3), nitro (—NO2), cyano (—CN), carbamido (—OC(O)N(Rbb)(Rcc) or —N(Rbb)C(O)ORaa), ureido (—N(Rbb)C(O)N(Rbb)(Rcc)), thioureido (—N(Rbb)C(S)N(Rbb)—Rcc)), guanidinyl (—N(Rbb)C(═NRbb)N(Rbb)(Rcc)), amidinyl (—C(═NRbb)N(Rbb)(Rcc) or —N(Rbb)C(═NRbb)(Raa)), thiol (—SRbb), sulfinyl (—S(O)Rbb), sulfonyl (—S(O)2Rbb) and sulfonamidyl (—S(O)2N(Rbb)(Rcc) or —N(Rbb)S—(O)2Rbb). Wherein each Raa, Rbb and Rcc is, independently, H, an optionally linked chemical functional group or a further substituent group with a preferred list including without limitation, alkyl, alkenyl, alkynyl, aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl, alicyclic, heterocyclic and heteroarylalkyl. Selected substituents within the compounds described herein are present to a recursive degree.
  • As used herein, “alkyl,” as used herein, means a saturated straight or branched hydrocarbon radical containing up to twenty four carbon atoms. Examples of alkyl groups include without limitation, methyl, ethyl, propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl and the like. Alkyl groups typically include from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms (C1-C12 alkyl) with from 1 to about 6 carbon atoms being more preferred.
  • As used herein, “alkenyl,” means a straight or branched hydrocarbon chain radical containing up to twenty four carbon atoms and having at least one carbon-carbon double bond. Examples of alkenyl groups include without limitation, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, dienes such as 1,3-butadiene and the like. Alkenyl groups typically include from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms with from 2 to about 6 carbon atoms being more preferred. Alkenyl groups as used herein may optionally include one or more further substituent groups.
  • As used herein, “alkynyl,” means a straight or branched hydrocarbon radical containing up to twenty four carbon atoms and having at least one carbon-carbon triple bond. Examples of alkynyl groups include, without limitation, ethynyl, 1-propynyl, 1-butynyl, and the like. Alkynyl groups typically include from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms with from 2 to about 6 carbon atoms being more preferred. Alkynyl groups as used herein may optionally include one or more further substituent groups.
  • As used herein, “acyl,” means a radical formed by removal of a hydroxyl group from an organic acid and has the general Formula —C(O)—X where X is typically aliphatic, alicyclic or aromatic. Examples include aliphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls, aromatic sulfinyls, aliphatic sulfinyls, aromatic phosphates, aliphatic phosphates and the like. Acyl groups as used herein may optionally include further substituent groups.
  • As used herein, “alicyclic” means a cyclic ring system wherein the ring is aliphatic. The ring system can comprise one or more rings wherein at least one ring is aliphatic. Preferred alicyclics include rings having from about 5 to about 9 carbon atoms in the ring. Alicyclic as used herein may optionally include further substituent groups.
  • As used herein, “aliphatic” means a straight or branched hydrocarbon radical containing up to twenty four carbon atoms wherein the saturation between any two carbon atoms is a single, double or triple bond. An aliphatic group preferably contains from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms with from 1 to about 6 carbon atoms being more preferred. The straight or branched chain of an aliphatic group may be interrupted with one or more heteroatoms that include nitrogen, oxygen, sulfur and phosphorus. Such aliphatic groups interrupted by heteroatoms include without limitation, polyalkoxys, such as polyalkylene glycols, polyamines, and polyimines. Aliphatic groups as used herein may optionally include further substituent groups.
  • As used herein, “alkoxy” means a radical formed between an alkyl group and an oxygen atom wherein the oxygen atom is used to attach the alkoxy group to a parent molecule. Examples of alkoxy groups include without limitation, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy and the like. Alkoxy groups as used herein may optionally include further substituent groups.
  • As used herein, “aminoalkyl” means an amino substituted C1-C12 alkyl radical. The alkyl portion of the radical forms a covalent bond with a parent molecule. The amino group can be located at any position and the aminoalkyl group can be substituted with a further substituent group at the alkyl and/or amino portions.
  • As used herein, “aralkyl” and “arylalkyl” mean an aromatic group that is covalently linked to a C1-C12 alkyl radical. The alkyl radical portion of the resulting aralkyl (or arylalkyl) group forms a covalent bond with a parent molecule. Examples include without limitation, benzyl, phenethyl and the like. Aralkyl groups as used herein may optionally include further substituent groups attached to the alkyl, the aryl or both groups that form the radical group.
  • As used herein, “aryl” and “aromatic” mean a mono- or polycyclic carbocyclic ring system radicals having one or more aromatic rings. Examples of aryl groups include without limitation, phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyl and the like. Preferred aryl ring systems have from about 5 to about 20 carbon atoms in one or more rings. Aryl groups as used herein may optionally include further substituent groups.
  • As used herein, “halo” and “halogen,” mean an atom selected from fluorine, chlorine, bromine and iodine.
  • As used herein, “heteroaryl,” and “heteroaromatic,” mean a radical comprising a mono- or poly-cyclic aromatic ring, ring system or fused ring system wherein at least one of the rings is aromatic and includes one or more heteroatoms. Heteroaryl is also meant to include fused ring systems including systems where one or more of the fused rings contain no heteroatoms. Heteroaryl groups typically include one ring atom selected from sulfur, nitrogen or oxygen. Examples of heteroaryl groups include without limitation, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl and the like. Heteroaryl radicals can be attached to a parent molecule directly or through a linking moiety such as an aliphatic group or hetero atom. Heteroaryl groups as used herein may optionally include further substituent groups.
    • B. OLIGOMERIC COMPOUNDS
  • In certain embodiments, the present invention provides oligomeric compounds. In certain embodiments, such oligomeric compounds comprise oligonucleotides optionally comprising one or more conjugate and/or terminal groups. In certain embodiments, an oligomeric compound consists of an oligonucleotide. In certain embodiments, oligonucleotides comprise one or more chemical modifications. Such chemical modifications include modifications of one or more nucleoside (including modifications to the sugar moiety and/or the nucleobase) and/or modifications to one or more internucleoside linkage.
  • a. Certain Modified Nucleosides
  • In certain embodiments, provided herein are oligomeric compounds comprising or consisting of oligonucleotides comprising at least one modified nucleoside. Such modified nucleosides comprise a modified sugar moeity, a modified nucleobase, or both a modified sugar moiety and a modified nucleobase.
  • i. Certain Modified Sugar Moieties
  • In certain embodiments, compounds of the invention comprise one or more modified nucleosides comprising a modified sugar moiety. Such compounds comprising one or more sugar-modified nucleosides may have desirable properties, such as enhanced nuclease stability or increased binding affinity with a target nucleic acid relative to an oligonucleotide comprising only nucleosides comprising naturally occurring sugar moieties. In certain embodiments, modified sugar moieties are substituted sugar moieties. In certain embodiments, modified sugar moieties are sugar surrogates. Such sugar surrogates may comprise one or more substitutions corresponding to those of substituted sugar moieties.
  • In certain embodiments, modified sugar moieties are substituted sugar moieties comprising one or more non-bridging sugar substituent, including but not limited to substituents at the 2′ and/or 5′ positions. Examples of sugar substituents suitable for the 2′-position, include, but are not limited to: 2′-F, 2′-OCH3 (“OMe” or “O-methyl”), and 2′-O(CH2)2OCH3 (“MOE”). In certain embodiments, sugar substituents at the 2′ position is selected from allyl, amino, azido, thio, O-allyl, O—C1-C10 alkyl, O—C1-C10 substituted alkyl; OCF3, O(CH2)2SCH3, O(CH2)2—O—N(Rm)(Rn), and O—CH2—C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted C1-C10 alkyl. Examples of sugar substituents at the 5′-position, include, but are not limited to: 5′-methyl (R or S); 5′-vinyl, and 5′-methoxy. In certain embodiments, substituted sugars comprise more than one non-bridging sugar substituent, for example, 2′-F-5′-methyl sugar moieties (see, e.g., PCT International Application WO 2008/101157, for additional 5′,2′-bis substituted sugar moieties and nucleosides).
  • Nucleosides comprising 2′-substituted sugar moieties are referred to as 2′-substituted nucleosides. In certain embodiments, a 2′-substituted nucleoside comprises a 2′-substituent group selected from halo, allyl, amino, azido, SH, CN, OCN, CF3, OCF3, O, S, or N(Rm)-alkyl; O, S, or N(Rm)-alkenyl; O, S or N(Rm)-alkynyl; O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH2)2SCH3, O—(CH2)2—O—N(Rm)(Rn) or O—CH2—C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H, an amino protecting group or substituted or unsubstituted C1-C10 alkyl. These 2′-substituent groups can be further substituted with one or more substituent groups independently selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.
  • In certain embodiments, a 2′-substituted nucleoside comprises a 2′-substituent group selected from F, NH2, N3, OCF3, O—CH3, O(CH2)3NH2, CH2—CH═CH2, O—CH2—CH═CH2, OCH2CH2OCH3, O(CH2)2SCH3, O—(CH2)2—O—N(Rm)(Rn), O(CH2)2—O—(CH2)2N(CH3)2, and N-substituted acetamide (O—CH2—C(═O)—N(Rm)(Rn) where each Rm and Rn is, independently, H, an amino protecting group or substituted or unsubstituted C1-C10 alkyl.
  • In certain embodiments, a 2′-substituted nucleoside comprises a sugar moiety comprising a 2′-substituent group selected from F, OCF3, O—CH3, OCH2CH2OCH3, O(CH2)2SCH3, O—(CH2)2—O—N(CH3)2, —O(CH2)2O(CH2)2N(CH3)2, and O—CH2—C(═O)—N(H)CH3.
  • In certain embodiments, a 2′-substituted nucleoside comprises a sugar moiety comprising a 2′-substituent group selected from F, O—CH3, and OCH2CH2OCH3.
  • Certain modified sugar moieties comprise a bridging sugar substituent that forms a second ring resulting in a bicyclic sugar moiety. In certain such embodiments, the bicyclic sugar moiety comprises a bridge between the 4′ and the 2′ furanose ring atoms. Examples of such 4′ to 2′ sugar substituents, include, but are not limited to: —[C(Ra)(Rb)]n—, —[C(Ra)(Rb)]n—O—, —C(RaRb)—N(R)—O— or, —C(RaRb)—O—N(R)—; 4′-CH2-2′, 4′-(CH2)2-2′, 4′-(CH2)—O-2′ (LNA); 4′-(CH2)—S-2; 4′-(CH2)2—O-2′ (ENA); 4′-CH(CH3)—O-2′ (cEt) and 4′-CH(CH2OCH3)—O-2′, and analogs thereof (see, e.g., U.S. Pat. No. 7,399,845, issued on Jul. 15, 2008); 4′-C(CH3)(CH3)—O-2′ and analogs thereof, (see, e.g., WO2009/006478, published Jan. 8, 2009); 4′-CH2—N(OCH3)-2′ and analogs thereof (see, e.g., WO2008/150729, published Dec. 11, 2008); 4′-CH2—O—N(CH3)-2′ (see, e.g., US2004/0171570, published Sep. 2, 2004); 4′-CH2—O—N(R)-2′, and 4′-CH2—N(R)—O-2′-, wherein each R is, independently, H, a protecting group, or C1-C12 alkyl; 4′-CH2—N(R)—O-2′, wherein R is H, C1-C12 alkyl, or a protecting group (see, U.S. Pat. No. 7,427,672, issued on Sep. 23, 2008); 4′-CH2—C(H)(CH3)-2′ (see, e.g., Chattopadhyaya, et al., J. Org. Chem., 2009, 74, 118-134); and 4′-CH2—C(═CH2)-2′ and analogs thereof (see, published PCT International Application WO 2008/154401, published on Dec. 8, 2008).
  • In certain embodiments, such 4′ to 2′ bridges independently comprise from 1 to 4 linked groups independently selected from —[C(Ra)(Rb)]n—, —C(Ra)═C(Rb)—, —C(Ra)═N—, —C(═NRa)—, —C(═O)—, —C(═S)—, —O—, —Si(Ra)2—, —S(═O)x—, and —N(Ra)—;
  • wherein:
  • x is 0, 1, or 2;
  • n is 1, 2, 3, or 4;
  • each Ra and Rb is, independently, H, a protecting group, hydroxyl, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)2-J1), or sulfoxyl (S(═O)-J1); and
  • each J1 and J2 is, independently, H, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, acyl (C(═O)—H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C1-C12 aminoalkyl, substituted C1-C12 aminoalkyl, or a protecting group.
  • Nucleosides comprising bicyclic sugar moieties are referred to as bicyclic nucleosides or BNAs. Bicyclic nucleosides include, but are not limited to, (A) α-L-Methyleneoxy (4′-CH2—O-2′) BNA, (B) β-D-Methyleneoxy (4′-CH2—O-2′) BNA (also referred to as locked nucleic acid or LNA), (C) Ethyleneoxy (4′-(CH2)2—O-2′) BNA, (D) Aminooxy (4′-CH2—O—N(R)-2′) BNA, (E) Oxyamino (4′-CH2—N(R)—O-2′) BNA, (F) Methyl(methyleneoxy) (4′-CH(CH3)—O-2′) BNA (also referred to as constrained ethyl or cEt), (G) methylene-thio (4′-CH2—S-2′) BNA, (H) methylene-amino (4′-CH2—N(R)-2′) BNA, (I) methyl carbocyclic (4′-CH2—CH(CH3)-2′) BNA, (J) propylene carbocyclic (4′-(CH2)3-2′) BNA, and (K) Ethylene(methoxy) (4′-(CH(CH2OMe)-O-2′) BNA (also referred to as constrained MOE or cMOE) as depicted below.
  • Figure US20150051389A1-20150219-C00002
    Figure US20150051389A1-20150219-C00003
  • wherein Bx is a nucleobase moiety and R is, independently, H, a protecting group, or C1-C12 alkyl.
  • Additional bicyclic sugar moieties are known in the art, for example: Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al., J. Am. Chem. Soc., 129(26) 8362-8379 (Jul. 4, 2007); Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8, 1-7; Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; U.S. Pat. Nos. 7,053,207, 6,268,490, 6,770,748, 6,794,499, 7,034,133, 6,525,191, 6,670,461, and 7,399,845; WO 2004/106356, WO 1994/14226, WO 2005/021570, and WO 2007/134181; U.S. Patent Publication Nos. US2004/0171570, US2007/0287831, and US2008/0039618; U.S. patent Ser. Nos. 12/129,154, 60/989,574, 61/026,995, 61/026,998, 61/056,564, 61/086,231, 61/097,787, and 61/099,844; and PCT International Applications Nos. PCT/US2008/064591, PCT/US2008/066154, and PCT/US2008/068922.
  • In certain embodiments, bicyclic sugar moieties and nucleosides incorporating such bicyclic sugar moieties are further defined by isomeric configuration. For example, a nucleoside comprising a 4′-2′ methylene-oxy bridge, may be in the α-L configuration or in the β-D configuration. Previously, α-L-methyleneoxy (4′-CH2—O-2′) bicyclic nucleosides have been incorporated into antisense oligonucleotides that showed antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).
  • In certain embodiments, substituted sugar moieties comprise one or more non-bridging sugar substituent and one or more bridging sugar substituent (e.g., 5′-substituted and 4′-2′ bridged sugars). (see, PCT International Application WO 2007/134181, published on Nov. 22, 2007, wherein LNA is substituted with, for example, a 5′-methyl or a 5′-vinyl group).
  • In certain embodiments, modified sugar moieties are sugar surrogates. In certain such embodiments, the oxygen atom of the naturally occurring sugar is substituted, e.g., with a sulfer, carbon or nitrogen atom. In certain such embodiments, such modified sugar moiety also comprises bridging and/or non-bridging substituents as described above. For example, certain sugar surrogates comprise a 4′-sulfer atom and a substitution at the 2′-position (see, e.g., published U.S. Patent Application US2005/0130923, published on Jun. 16, 2005) and/or the 5′ position. By way of additional example, carbocyclic bicyclic nucleosides having a 4′-2′ bridge have been described (see, e.g., Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al., J. Org. Chem., 2006, 71, 7731-7740).
  • In certain embodiments, sugar surrogates comprise rings having other than 5-atoms. For example, in certain embodiments, a sugar surrogate comprises a six-membered tetrahydropyran. Such tetrahydropyrans may be further modified or substituted. Nucleosides comprising such modified tetrahydropyrans include, but are not limited to, hexitol nucleic acid (HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) (see Leumann, C J. Bioorg. & Med. Chem. (2002) 10:841-854), fluoro HNA (F-HNA), and those compounds having Formula VII:
  • Figure US20150051389A1-20150219-C00004
  • wherein independently for each of said at least one tetrahydropyran nucleoside analog of Formula VII:
  • Bx is a nucleobase moiety;
  • T3 and T4 are each, independently, an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound or one of T3 and T4 is an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound and the other of T3 and T4 is H, a hydroxyl protecting group, a linked conjugate group, or a 5′ or 3′-terminal group;
  • q1, q2, q3, q4, q5, q6 and q7 are each, independently, H, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, or substituted C2-C6 alkynyl; and
  • each of R1 and R2 is independently selected from among: hydrogen, halogen, substituted or unsubstituted alkoxy, NJ1J2, SJ1, N3, OC(═X)J1, OC(═X)NJ1J2, NJ3C(═X)NJ1J2, and CN, wherein X is O, S or NJ1, and each J1, J2, and J3 is, independently, H or C1-C6 alkyl.
  • In certain embodiments, the modified THP nucleosides of Formula VII are provided wherein q1, q2, q3, q4, q5, q6 and q7 are each H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6 and q7 is other than H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6 and q7 is methyl. In certain embodiments, THP nucleosides of Formula VII are provided wherein one of R1 and R2 is F. In certain embodiments, R1 is fluoro and R2 is H, R1 is methoxy and R2 is H, and R1 is methoxyethoxy and R2 is H.
  • Many other bicyclo and tricyclo sugar surrogate ring systems are also known in the art that can be used to modify nucleosides for incorporation into antisense compounds (see, e.g., review article: Leumann, J. C, Bioorganic & Medicinal Chemistry, 2002, 10, 841-854).
  • Combinations of modifications are also provided without limitation, such as 2′-F-5′-methyl substituted nucleosides (see PCT International Application WO 2008/101157 Published on Aug. 21, 2008 for other disclosed 5′,2′-bis substituted nucleosides) and replacement of the ribosyl ring oxygen atom with S and further substitution at the 2′-position (see published U.S. Patent Application US2005-0130923, published on Jun. 16, 2005) or alternatively 5′-substitution of a bicyclic nucleic acid (see PCT International Application WO 2007/134181, published on Nov. 22, 2007 wherein a 4′-CH2—O-2′ bicyclic nucleoside is further substituted at the 5′ position with a 5′-methyl or a 5′-vinyl group). The synthesis and preparation of carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described (see, e.g., Srivastava et al., J. Am. Chem. Soc. 2007, 129(26), 8362-8379).
  • In certain embodiments, the present invention provides oligonucleotides comprising modified nucleosides. Those modified nucleotides may include modified sugars, modified nucleobases, and/or modified linkages. The specific modifications are selected such that the resulting oligonucleotides possess desirable characteristics. In certain embodiments, oligonucleotides comprise one or more RNA-like nucleosides. In certain embodiments, oligonucleotides comprise one or more DNA-like nucleotides.
  • ii. Certain Modified Nucleobases
  • In certain embodiments, nucleosides of the present invention comprise one or more unmodified nucleobases. In certain embodiments, nucleosides of the present invention comprise one or more modified nucleobases.
  • In certain embodiments, modified nucleobases are selected from: universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases as defined herein. 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil; 5-propynylcytosine; 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C≡C—CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, 3-deazaguanine and 3-deazaadenine, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases as defined herein. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, J. I., Ed., John Wiley & Sons, 1990, 858-859; those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, Crooke, S. T. and Lebleu, B., Eds., CRC Press, 1993, 273-288.
  • Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include without limitation, U.S. Pat. Nos. 3,687,808; 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,645,985; 5,681,941; 5,750,692; 5,763,588; 5,830,653 and 6,005,096, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.
  • b. Certain Internucleoside Linkages
  • In certain embodiments, nucleosides may be linked together using any internucleoside linkage to form oligonucleotides. The two main classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus containing internucleoside linkages include, but are not limited to, phosphodiesters (P═O), phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates (P═S). Representative non-phosphorus containing internucleoside linking groups include, but are not limited to, methylenemethylimino (—CH2—N(CH3)—O—CH2—) thiodiester (—O—C(O)—S—), thionocarbamate (—O—C(O)(NH)—S—); siloxane (—O—Si(H)2—O—); and N,N′-dimethylhydrazine (—CH2—N(CH3)—N(CH3)—). Modified linkages, compared to natural phosphodiester linkages, can be used to alter, typically increase, nuclease resistance of the oligonucleotide. In certain embodiments, internucleoside linkages having a chiral atom can be prepared as a racemic mixture, or as separate enantiomers. Representative chiral linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing internucleoside linkages are well known to those skilled in the art.
  • The oligonucleotides described herein contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations that may be defined, in terms of absolute stereochemistry, as (R) or (S), α or β such as for sugar anomers, or as (D) or (L) such as for amino acids etc. Included in the antisense compounds provided herein are all such possible isomers, as well as their racemic and optically pure forms.
  • Neutral internucleoside linkages include without limitation, phosphotriesters, methylphosphonates, MMI (3′-CH2—N(CH3)—O-5′), amide-3 (3′-CH2—C(═P)—N(H)-5′), amide-4 (3′-CH2—N(H)—C(═O)-5′), formacetal (3′-O—CH2—O-5′), and thioformacetal (3′-S—CH2—O-5′). Further neutral internucleoside linkages include nonionic linkages comprising siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester and amides (See for example: Carbohydrate Modifications in Antisense Research; Y. S. Sanghvi and P. D. Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Further neutral internucleoside linkages include nonionic linkages comprising mixed N, O, S and CH2 component parts.
  • i. 3′-Endo Modifications
  • In one aspect of the present disclosure, oligomeric compounds include nucleosides synthetically modified to induce a 3′-endo sugar conformation. A nucleoside can incorporate synthetic modifications of the heterocyclic base moiety, the sugar moiety or both to induce a desired 3′-endo sugar conformation. These modified nucleosides are used to mimic RNA like nucleosides so that particular properties of an oligomeric compound can be enhanced while maintaining the desirable 3′-endo conformational geometry. There is an apparent preference for an RNA type duplex (A form helix, predominantly 3′-endo) as a requirement of RNA interference which is supported in part by the fact that duplexes composed of 2′-deoxy-2′-F-nucleosides appear efficient in triggering RNAi response in the C. elegans system. Properties that are enhanced by using more stable 3′-endo nucleosides include but aren't limited to modulation of pharmacokinetic properties through modification of protein binding, protein off-rate, absorption and clearance; modulation of nuclease stability as well as chemical stability; modulation of the binding affinity and specificity of the oligomer (affinity and specificity for enzymes as well as for complementary sequences); and increasing efficacy of RNA cleavage. The present invention provides oligomeric compounds having one or more nucleosides modified in such a way as to favor a C3′-endo type conformation.
  • Figure US20150051389A1-20150219-C00005
  • Nucleoside conformation is influenced by various factors including substitution at the 2′, 3′ or 4′-positions of the pentofuranosyl sugar. Electronegative substituents generally prefer the axial positions, while sterically demanding substituents generally prefer the equatorial positions (Principles of Nucleic Acid Structure, Wolfgang Sanger, 1984, Springer-Verlag.) Modification of the 2′ position to favor the 3′-endo conformation can be achieved while maintaining the 2′-OH as a recognition element, as exemplified in Example 35, below (Gallo et al., Tetrahedron (2001), 57, 5707-5713. Harry-O'kuru et al., J. Org. Chem., (1997), 62(6), 1754-1759 and Tang et al., J. Org. Chem. (1999), 64, 747-754.) Alternatively, preference for the 3′-endo conformation can be achieved by deletion of the 2′-OH as exemplified by 2′ deoxy-2′F-nucleosides (Kawasaki et al., J. Med. Chem. (1993), 36, 831-841), which adopts the 3′-endo conformation positioning the electronegative fluorine atom in the axial position. Other modifications of the ribose ring, for example substitution at the 4′-position to give 4′-F modified nucleosides (Guillerm et al., Bioorganic and Medicinal Chemistry Letters (1995), 5, 1455-1460 and Owen et al., J. Org. Chem. (1976), 41, 3010-3017), or for example modification to yield methanocarba nucleoside analogs (Jacobson et al., J. Med. Chem. Lett. (2000), 43, 2196-2203 and Lee et al., Bioorganic and Medicinal Chemistry Letters (2001), 11, 1333-1337) also induce preference for the 3′-endo conformation. Some modifications actually lock the conformational geometry by formation of a bicyclic sugar moiety e.g. locked nucleic acid (LNA, Singh et al, Chem. Commun. (1998), 4, 455-456), and ethylene bridged nucleic acids (ENA, Morita et al, Bioorganic & Medicinal Chemistry Letters (2002), 12, 73-76.)
  • c. Certain Motifs
  • In certain embodiments, oligomeric compounds comprise or consist of oligonucleotides. In certain embodiments, such oligonucleotides comprise one or more chemical modification. In certain embodiments, chemically modified oligonucleotides comprise one or more modified sugars. In certain embodiments, chemically modified oligonucleotides comprise one or more modified nucleobases. In certain embodiments, chemically modified oligonucleotides comprise one or more modified internucleoside linkages. In certain embodiments, the chemical modifications (sugar modifications, nucleobase modifications, and/or linkage modifications) define a pattern or motif. In certain embodiments, the patterns of chemical modifications of sugar moieties, internucleoside linkages, and nucleobases are each independent of one another. Thus, an oligonucleotide may be described by its sugar modification motif, internucleoside linkage motif and/or nucleobase modification motif (as used herein, nucleobase modification motif describes the chemical modifications to the nucleobases independent of the sequence of nucleobases).
  • i. Certain Sugar Motifs
  • In certain embodiments, oligonucleotides comprise one or more type of modified sugar moieties and/or naturally occurring sugar moieties arranged along an oligonucleotide or region thereof in a defined pattern or sugar motif. Such sugar motifs include but are not limited to any of the sugar modifications discussed herein.
  • In certain embodiments, the oligonucleotides comprise or consist of a region having a gapmer sugar motif, which comprises two external regions or “wings” and a central or internal region or “gap.” The three regions of a gapmer sugar motif (the 5′-wing, the gap, and the 3′-wing) form a contiguous sequence of nucleosides wherein at least some of the sugar moieties of the nucleosides of each of the wings differ from at least some of the sugar moieties of the nucleosides of the gap. Specifically, at least the sugar moieties of the nucleosides of each wing that are closest to the gap (the 3′-most nucleoside of the 5′-wing and the 5′-most nucleoside of the 3′-wing) differ from the sugar moiety of the neighboring gap nucleosides, thus defining the boundary between the wings and the gap. In certain embodiments, the sugar moieties within the gap are the same as one another. In certain embodiments, the gap includes one or more nucleoside having a sugar moiety that differs from the sugar moiety of one or more other nucleosides of the gap. In certain embodiments, the sugar motifs of the two wings are the same as one another (symmetric sugar gapmer). In certain embodiments, the sugar motifs of the 5′-wing differs from the sugar motif of the 3′-wing (asymmetric sugar gapmer).
  • ii. Certain Nucleobase Modification Motifs
  • In certain embodiments, oligonucleotides comprise chemical modifications to nucleobases arranged along the oligonucleotide or region thereof in a defined pattern or nucleobases modification motif. In certain embodiments, each nucleobase is modified. In certain embodiments, none of the nucleobases is chemically modified.
  • In certain embodiments, oligonucleotides comprise a block of modified nucleobases. In certain such embodiments, the block is at the 3′-end of the oligonucleotide. In certain embodiments the block is within 3 nucleotides of the 3′-end of the oligonucleotide. In certain such embodiments, the block is at the 5′-end of the oligonucleotide. In certain embodiments the block is within 3 nucleotides of the 5′-end of the oligonucleotide.
  • In certain embodiments, nucleobase modifications are a function of the natural base at a particular position of an oligonucleotide. For example, in certain embodiments each purine or each pyrimidine in an oligonucleotide is modified. In certain embodiments, each adenine is modified. In certain embodiments, each guanine is modified. In certain embodiments, each thymine is modified. In certain embodiments, each cytosine is modified. In certain embodiments, each uracil is modified.
  • In certain embodiments, oligonucleotides comprise one or more nucleosides comprising a modified nucleobase. In certain embodiments, oligonucleotides having a gapmer sugar motif comprise a nucleoside comprising a modified nucleobase. In certain such embodiments, one nucleoside comprising a modified nucleobases is in the central gap of an oligonucleotide having a gapmer sugar motif. In certain embodiments, the sugar is an unmodified 2′ deoxynucleoside. In certain embodiments, the modified nucleobase is selected from: a 2-thio pyrimidine and a 5-propyne pyrimidine
  • In certain embodiments, some, all, or none of the cytosine moieties in an oligonucleotide are 5-methyl cytosine moieties. Herein, 5-methyl cytosine is not a “modified nucleobase.” Accordingly, unless otherwise indicated, unmodified nucleobases include both cytosine residues having a 5-methyl and those lacking a 5 methyl. In certain embodiments, the methylation state of all or some cytosine nucleobases is specified.
  • iii. Certain Nucleoside Motifs
  • In certain embodiments, oligonucleotides comprise nucleosides comprising modified sugar moieties and/or nucleosides comprising modified nucleobases. Such motifs can be described by their sugar motif and their nucleobase motif separately or by their nucleoside motif, which provides positions or patterns of modified nucleosides (whether modified sugar, nucleobase, or both sugar and nucleobase) in an oligonucleotide.
  • In certain embodiments, the oligonucleotides comprise or consist of a region having a gapmer nucleoside motif, which comprises two external regions or “wings” and a central or internal region or “gap.” The three regions of a gapmer nucleoside motif (the 5′-wing, the gap, and the 3′-wing) form a contiguous sequence of nucleosides wherein at least some of the sugar moieties and/or nucleobases of the nucleosides of each of the wings differ from at least some of the sugar moieties and/or nucleobase of the nucleosides of the gap. Specifically, at least the nucleosides of each wing that are closest to the gap (the 3′-most nucleoside of the 5′-wing and the 5′-most nucleoside of the 3′-wing) differ from the neighboring gap nucleosides, thus defining the boundary between the wings and the gap. In certain embodiments, the nucleosides within the gap are the same as one another. In certain embodiments, the gap includes one or more nucleoside that differs from one or more other nucleosides of the gap. In certain embodiments, the nucleoside motifs of the two wings are the same as one another (symmetric gapmer). In certain embodiments, the nucleoside motifs of the 5′-wing differs from the nucleoside motif of the 3′-wing (asymmetric gapmer).
  • iv. Certain 5′-Wings
  • In certain embodiments, the 5′-wing of a gapmer consists of 1 to 6 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 1 to 5 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 2 to 5 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 3 to 5 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 4 or 5 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 1 to 4 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 1 to 3 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 1 or 2 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 2 to 4 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 2 or 3 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 3 or 4 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 1 nucleoside. In certain embodiments, the 5′-wing of a gapmer consists of 2 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 3 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 4 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 5 linked nucleosides. In certain embodiments, the 5′-wing of a gapmer consists of 6 linked nucleosides.
  • In certain embodiments, the 5′-wing of a gapmer comprises at least one bicyclic nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least two bicyclic nucleosides. In certain embodiments, the 5′-wing of a gapmer comprises at least three bicyclic nucleosides. In certain embodiments, the 5′-wing of a gapmer comprises at least four bicyclic nucleosides. In certain embodiments, the 5′-wing of a gapmer comprises at least one constrained ethyl nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one LNA nucleoside. In certain embodiments, each nucleoside of the 5′-wing of a gapmer is a bicyclic nucleoside. In certain embodiments, each nucleoside of the 5′-wing of a gapmer is a constrained ethyl nucleoside. In certain embodiments, each nucleoside of the 5′-wing of a gapmer is a LNA nucleoside.
  • In certain embodiments, the 5′-wing of a gapmer comprises at least one non-bicyclic modified nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one 2′-substituted nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one 2′-MOE nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one 2′-OMe nucleoside. In certain embodiments, each nucleoside of the 5′-wing of a gapmer is a non-bicyclic modified nucleoside. In certain embodiments, each nucleoside of the 5′-wing of a gapmer is a 2′-substituted nucleoside. In certain embodiments, each nucleoside of the 5′-wing of a gapmer is a 2′-MOE nucleoside. In certain embodiments, each nucleoside of the 5′-wing of a gapmer is a 2′-OMe nucleoside.
  • In certain embodiments, the 5′-wing of a gapmer comprises at least one 2′-deoxynucleoside. In certain embodiments, each nucleoside of the 5′-wing of a gapmer is a 2′-deoxynucleoside. In a certain embodiments, the 5′-wing of a gapmer comprises at least one ribonucleoside. In certain embodiments, each nucleoside of the 5′-wing of a gapmer is a ribonucleoside. In certain embodiments, one, more than one, or each of the nucleosides of the 5′-wing is an RNA-like nucleoside.
  • In certain embodiments, the 5′-wing of a gapmer comprises at least one bicyclic nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2′-substituted nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2′-MOE nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2′-OMe nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2′-deoxynucleoside.
  • In certain embodiments, the 5′-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2′-substituted nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2′-MOE nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2′-OMe nucleoside. In certain embodiments, the 5′-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2′-deoxynucleoside.
  • In certain embodiments, the 5′-wing of a gapmer has a nucleoside motif selected from among the following: ADDA; ABDAA; ABBA; ABB; ABAA; AABAA; AAABAA; AAAABAA; AAAAABAA; AAABAA; AABAA; ABAB; ABADB; ABADDB; AAABB; AAAAA; ABBDC; ABDDC; ABBDCC; ABBDDC; ABBDCC; ABBC; AA; AAA; AAAA; AAAAB; AAAAAAA; AAAAAAAA; ABBB; AB; ABAB; AAAAB; AABBB; AAAAB; and AABBB, wherein each A is a modified nucleoside of a first type, each B is a modified nucleoside of a second type, each C is a modified nucleoside of a third type, and each D is an unmodified deoxynucleoside.
  • In certain embodiments, the 5′-wing of a gapmer has a nucleoside motif selected from among the following: AB, ABB, AAA, BBB, BBBAA, AAB, BAA, BBAA, AABB, AAAB, ABBW, ABBWW, ABBB, ABBBB, ABAB, ABABAB, ABABBB, ABABAA, AAABB, AAAABB, AABB, AAAAB, AABBB, ABBBB, BBBBB, AAABW, AAAAA, BBBBAA, and AAABW; wherein each A is a modified nucleoside of a first type, each B is a modified nucleoside of a second type, and each W is a modified nucleoside of either the first type, the second type or a third type.
  • In certain embodiments, the 5′-wing of a gapmer has a nucleoside motif selected from among the following: ABB; ABAA; AABAA; AAABAA; ABAB; ABADB; AAABB; AAAAA; AA; AAA; AAAA; AAAAB; ABBB; AB; and ABAB; wherein each A is a modified nucleoside of a first type, each B is a modified nucleoside of a second type, and each W is a modified nucleoside of either the first type, the second type or a third type.
  • In certain embodiments, an oligonucleotide comprises any 5′-wing motif provided herein. In certain such embodiments, the oligonucleotide is a 5′-hemimer (does not comprise a 3′-wing). In certain embodiments, such an oligonucleotide is a gapmer. In certain such embodiments, the 3′-wing of the gapmer may comprise any nucleoside motif.
  • In certain embodiments, the 5′-wing of a gapmer has a sugar motif selected from among those listed in the following non-limiting tables:
  • TABLE 1
    Certain 5′-Wing Sugar Motifs
    Certain 5′-Wing Sugar Motifs
    AAAAA ABCBB BABCC BCBBA CBACC
    AAAAB ABCBC BACAA BCBBB CBBAA
    AAAAC ABCCA BACAB BCBBC CBBAB
    AAABA ABCCB BACAC BCBCA CBBAC
    AAABB ABCCC BACBA BCBCB CBBBA
    AAABC ACAAA BACBB BCBCC CBBBB
    AAACA ACAAB BACBC BCCAA CBBBC
    AAACB ACAAC BACCA BCCAB CBBCA
    AAACC ACABA BACCB BCCAC CBBCB
    AABAA ACABB BACCC BCCBA CBBCC
    AABAB ACABC BBAAA BCCBB CBCAA
    AABAC ACACA BBAAB BCCBC CBCAB
    AABBA ACACB BBAAC BCCCA CBCAC
    AABBB ACACC BBABA BCCCB CBCBA
    AABBC ACBAA BBABB BCCCC CBCBB
    AABCA ACBAB BBABC CAAAA CBCBC
    AABCB ACBAC BBACA CAAAB CBCCA
    AABCC ACBBA BBACB CAAAC CBCCB
    AACAA ACBBB BBACC CAABA CBCCC
    AACAB ACBBC BBBAA CAABB CCAAA
    AACAC ACBCA BBBAB CAABC CCAAB
    AACBA ACBCB BBBAC CAACA CCAAC
    AACBB ACBCC BBBBA CAACB CCABA
    AACBC ACCAA BBBBB CAACC CCABB
    AACCA ACCAB BBBBC CABAA CCABC
    AACCB ACCAC BBBCA CABAB CCACA
    AACCC ACCBA BBBCB CABAC CCACB
    ABAAA ACCBB BBBCC CABBA CCACC
    ABAAB ACCBC BBCAA CABBB CCBAA
    ABAAC ACCCA BBCAB CABBC CCBAB
    ABABA ACCCB BBCAC CABCA CCBAC
    ABABB ACCCC BBCBA CABCB CCBBA
    ABABC BAAAA BBCBB CABCC CCBBB
    ABACA BAAAB BBCBC CACAA CCBBC
    ABACB BAAAC BBCCA CACAB CCBCA
    ABACC BAABA BBCCB CACAC CCBCB
    ABBAA BAABB BBCCC CACBA CCBCC
    ABBAB BAABC BCAAA CACBB CCCAA
    ABBAC BAACA BCAAB CACBC CCCAB
    ABBBA BAACB BCAAC CACCA CCCAC
    ABBBB BAACC BCABA CACCB CCCBA
    ABBBC BABAA BCABB CACCC CCCBB
    ABBCA BABAB BCABC CBAAA CCCBC
    ABBCB BABAC BCACA CBAAB CCCCA
    ABBCC BABBA BCACB CBAAC CCCCB
    ABCAA BABBB BCACC CBABA CCCCC
    ABCAB BABBC BCBAA CBABB
    ABCAC BABCA BCBAB CBABC
    ABCBA BABCB BCBAC CBACA
  • TABLE 2
    Certain 5′-Wing Sugar Motifs
    Certain 5′-Wing Sugar Motifs
    AAAAA BABC CBAB ABBB BAA
    AAAAB BACA CBAC BAAA BAB
    AAABA BACB CBBA BAAB BBA
    AAABB BACC CBBB BABA BBB
    AABAA BBAA CBBC BABB AA
    AABAB BBAB CBCA BBAA AB
    AABBA BBAC CBCB BBAB AC
    AABBB BBBA CBCC BBBA BA
    ABAAA BBBB CCAA BBBB BB
    ABAAB BBBC CCAB AAA BC
    ABABA BBCA CCAC AAB CA
    ABABB BBCB CCBA AAC CB
    ABBAA BBCC CCBB ABA CC
    ABBAB BCAA CCBC ABB AA
    ABBBA BCAB CCCA ABC AB
    ABBBB BCAC CCCB ACA BA
    BAAAA ABCB BCBA ACB
    BAAAB ABCC BCBB ACC
    BAABA ACAA BCBC BAA
    BAABB ACAB BCCA BAB
    BABAA ACAC BCCB BAC
    BABAB ACBA BCCC BBA
    BABBA ACBB CAAA BBB
    BABBB ACBC CAAB BBC
    BBAAA ACCA CAAC BCA
    BBAAB ACCB CABA BCB
    BBABA ACCC CABB BCC
    BBABB BAAA CABC CAA
    BBBAA BAAB CACA CAB
    BBBAB BAAC CACB CAC
    BBBBA BABA CACC CBA
    BBBBB BABB CBAA CBB
    AAAA AACC CCCC CBC
    AAAB ABAA AAAA CCA
    AAAC ABAB AAAB CCB
    AABA ABAC AABA CCC
    AABB ABBA AABB AAA
    AABC ABBB ABAA AAB
    AACA ABBC ABAB ABA
    AACB ABCA ABBA ABB
  • In certain embodiments, each A, each B, and each C located at the 3′-most 5′-wing nucleoside is a modified nucleoside. For example, in certain embodiments the 5′-wing motif is selected from among ABB, BBB, and CBB, wherein the underlined nucleoside represents the 3′-most 5′-wing nucleoside and wherein the underlined nucleoside is a modified nucleoside. In certain embodiments, the 3′-most 5′-wing nucleoside comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, α-L-LNA, ENA and 2′-thio LNA. In certain embodiments, the 3′-most 5′-wing nucleoside comprises a bicyclic sugar moiety selected from among cEt and LNA. In certain embodiments, the 3′-most 5′-wing nucleoside comprises cEt. In certain embodiments, the 3′-most 5′-wing nucleoside comprises LNA.
  • In certain embodiments, each A comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, each A comprises a modified sugar moiety. In certain embodiments, each A comprises a 2′-substituted sugar moiety. In certain embodiments, each A comprises a 2′-substituted sugar moiety selected from among F, ara-F, OCH3 and O(CH2)2—OCH3. In certain embodiments, each A comprises a bicyclic sugar moiety. In certain embodiments, each A comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, α-L-LNA, ENA and 2′-thio LNA. In certain embodiments, each A comprises a modified nucleobase. In certain embodiments, each A comprises a modified nucleobase selected from among 2-thio-thymidine nucleoside and 5-propyne uridine nucleoside. In certain embodiments, each A comprises an HNA. In certain embodiments, each A comprises a F-HNA. In certain embodiments, each A comprises a 5′-substituted sugar moiety selected from among 5′-Me DNA, and 5′-(R)-Me DNA.
  • In certain embodiments, each B comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, each B comprises a modified sugar moiety. In certain embodiments, each B comprises a 2′-substituted sugar moiety. In certain embodiments, each B comprises a 2′-substituted sugar moiety selected from among F, (ara)-F, OCH3 and O(CH2)2—OCH3. In certain embodiments, each B comprises a bicyclic sugar moiety. In certain embodiments, each B comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, α-L-LNA, ENA and 2′-thio LNA. In certain embodiments, each B comprises a modified nucleobase. In certain embodiments, each B comprises a modified nucleobase selected from among 2-thio-thymidine nucleoside and 5-propyne urindine nucleoside. In certain embodiments, each B comprises an HNA. In certain embodiments, each B comprises a F-HNA. In certain embodiments, each B comprises a 5′-substituted sugar moiety selected from among 5′-Me DNA, and 5′-(R)-Me DNA.
  • In certain embodiments, each A comprises a 2′-substituted sugar moiety selected from among F, ara-F, OCH3 and O(CH2)2—OCH3 and each B comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, α-L-LNA, ENA and 2′-thio LNA. In certain embodiments, each A comprises O(CH2)2—OCH3 and each B comprises cEt.
  • In certain embodiments, each C comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, each C comprises a modified sugar moiety. In certain embodiments, each C comprises a 2′-substituted sugar moiety. In certain embodiments, each C comprises a 2′-substituted sugar moiety selected from among F, (ara)-F, OCH3 and O(CH2)2—OCH3. In certain embodiments, each C comprises a 5′-substituted sugar moiety. In certain embodiments, each C comprises a 5′-substituted sugar moiety selected from among 5′-Me DNA, and 5′-(R)-Me DNA. In certain embodiments, each C comprises a bicyclic sugar moiety. In certain embodiments, each C comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, α-L-LNA, ENA and 2′-thio LNA. In certain embodiments, each C comprises a modified nucleobase. In certain embodiments, each C comprises a modified nucleobase selected from among 2-thio-thymidine and 5-propyne uridine. In certain embodiments, each C comprises a 2-thio-thymidine nucleoside. In certain embodiments, each C comprises an HNA. In certain embodiments, each C comprises an F-HNA.
  • v. Certain 3′-Wings
  • In certain embodiments, the 3′-wing of a gapmer consists of 1 to 6 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 1 to 5 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 2 to 5 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 3 to 5 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 4 or 5 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 1 to 4 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 1 to 3 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 1 or 2 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 2 to 4 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 2 or 3 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 3 or 4 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 1 nucleoside. In certain embodiments, the 3′-wing of a gapmer consists of 2 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 31 inked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 4 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 5 linked nucleosides. In certain embodiments, the 3′-wing of a gapmer consists of 6 linked nucleosides.
  • In certain embodiments, the 3′-wing of a gapmer comprises at least one bicyclic nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one constrained ethyl nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one LNA nucleoside. In certain embodiments, each nucleoside of the 3′-wing of a gapmer is a bicyclic nucleoside. In certain embodiments, each nucleoside of the 3′-wing of a gapmer is a constrained ethyl nucleoside. In certain embodiments, each nucleoside of the 3′-wing of a gapmer is a LNA nucleoside.
  • In certain embodiments, the 3′-wing of a gapmer comprises at least one non-bicyclic modified nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least two non-bicyclic modified nucleosides. In certain embodiments, the 3′-wing of a gapmer comprises at least three non-bicyclic modified nucleosides. In certain embodiments, the 3′-wing of a gapmer comprises at least four non-bicyclic modified nucleosides. In certain embodiments, the 3′-wing of a gapmer comprises at least one 2′-substituted nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one 2′-MOE nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one 2′-OMe nucleoside. In certain embodiments, each nucleoside of the 3′-wing of a gapmer is a non-bicyclic modified nucleoside. In certain embodiments, each nucleoside of the 3′-wing of a gapmer is a 2′-substituted nucleoside. In certain embodiments, each nucleoside of the 3′-wing of a gapmer is a 2′-MOE nucleoside. In certain embodiments, each nucleoside of the 3′-wing of a gapmer is a 2′-OMe nucleoside.
  • In certain embodiments, the 3′-wing of a gapmer comprises at least one 2′-deoxynucleoside. In certain embodiments, each nucleoside of the 3′-wing of a gapmer is a 2′-deoxynucleoside. In a certain embodiments, the 3′-wing of a gapmer comprises at least one ribonucleoside. In certain embodiments, each nucleoside of the 3′-wing of a gapmer is a ribonucleoside. In certain embodiments, one, more than one, or each of the nucleosides of the 5′-wing is an RNA-like nucleoside.
  • In certain embodiments, the 3′-wing of a gapmer comprises at least one bicyclic nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2′-substituted nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2′-MOE nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2′-OMe nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2′-deoxynucleoside.
  • In certain embodiments, the 3′-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2′-substituted nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2′-MOE nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2′-OMe nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2′-deoxynucleoside.
  • In certain embodiments, the 3′-wing of a gapmer comprises at least one LNA nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one LNA nucleoside and at least one 2′-substituted nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one LNA nucleoside and at least one 2′-MOE nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one LNA nucleoside and at least one 2′-OMe nucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one LNA nucleoside and at least one 2′-deoxynucleoside.
  • In certain embodiments, the 3′-wing of a gapmer comprises at least one bicyclic nucleoside, at least one non-bicyclic modified nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one constrained ethyl nucleoside, at least one non-bicyclic modified nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one LNA nucleoside, at least one non-bicyclic modified nucleoside, and at least one 2′-deoxynucleoside.
  • In certain embodiments, the 3′-wing of a gapmer comprises at least one bicyclic nucleoside, at least one 2′-substituted nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one constrained ethyl nucleoside, at least one 2′-substituted nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one LNA nucleoside, at least one 2′-substituted nucleoside, and at least one 2′-deoxynucleoside.
  • In certain embodiments, the 3′-wing of a gapmer comprises at least one bicyclic nucleoside, at least one 2′-MOE nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one constrained ethyl nucleoside, at least one 2′-MOE nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one LNA nucleoside, at least one 2′-MOE nucleoside, and at least one 2′-deoxynucleoside.
  • In certain embodiments, the 3′-wing of a gapmer comprises at least one bicyclic nucleoside, at least one 2′-OMe nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one constrained ethyl nucleoside, at least one 2′-OMe nucleoside, and at least one 2′-deoxynucleoside. In certain embodiments, the 3′-wing of a gapmer comprises at least one LNA nucleoside, at least one 2′-OMe nucleoside, and at least one 2′-deoxynucleoside.
  • In certain embodiments, the 3′-wing of a gapmer has a nucleoside motif selected from among the following: ABB, ABAA, AAABAA, AAAAABAA, AABAA, AAAABAA, AAABAA, ABAB, AAAAA, AAABB, AAAAAAAA, AAAAAAA, AAAAAA, AAAAB, AAAA, AAA, AA, AB, ABBB, ABAB, AABBB; wherein each A is a modified nucleoside of a first type, each B is a modified nucleoside of a second type. In certain embodiments, an oligonucleotide comprises any 3′-wing motif provided herein. In certain such embodiments, the oligonucleotide is a 3′-hemimer (does not comprise a 5′-wing). In certain embodiments, such an oligonucleotide is a gapmer. In certain such embodiments, the 5′-wing of the gapmer may comprise any nucleoside motif.
  • In certain embodiments, the 3′-wing of a gapmer has a nucleoside motif selected from among the following: BBA, AAB, AAA, BBB, BBAA, AABB, WBBA, WAAB, BBBA, BBBBA, BBBB, BBBBBA, ABBBBB, BBAAA, AABBB, BBBAA, BBBBA, BBBBB, BABA, AAAAA, BBAAAA, AABBBB, BAAAA, and ABBBB, wherein each A is a modified nucleoside of a first type, each B is a modified nucleoside of a second type, and each W is a modified nucleoside of either the first type, the second type or a third type.
  • In certain embodiments, the 3′-wing of a gapmer has a nucleoside motif selected from among the following: ABB; AAABAA; AABAA; AAAABAA; AAAAA; AAABB; AAAAAAAA; AAAAAAA; AAAAAA; AAAAB; AB; ABBB; and ABAB, wherein each A is a modified nucleoside of a first type, each B is a modified nucleoside of a second type, and each W is a modified nucleoside of either the first type, the second type or a third type.
  • In certain embodiments, the 3′-wing of a gapmer has a sugar motif selected from among those listed in the following non-limiting tables:
  • TABLE 3
    Certain 3′-Wing Sugar Motifs
    Certain 3′-Wing Sugar Motifs
    AAAAA ABCBB BABCC BCBBA CBACC
    AAAAB ABCBC BACAA BCBBB CBBAA
    AAAAC ABCCA BACAB BCBBC CBBAB
    AAABA ABCCB BACAC BCBCA CBBAC
    AAABB ABCCC BACBA BCBCB CBBBA
    AAABC ACAAA BACBB BCBCC CBBBB
    AAACA ACAAB BACBC BCCAA CBBBC
    AAACB ACAAC BACCA BCCAB CBBCA
    AAACC ACABA BACCB BCCAC CBBCB
    AABAA ACABB BACCC BCCBA CBBCC
    AABAB ACABC BBAAA BCCBB CBCAA
    AABAC ACACA BBAAB BCCBC CBCAB
    AABBA ACACB BBAAC BCCCA CBCAC
    AABBB ACACC BBABA BCCCB CBCBA
    AABBC ACBAA BBABB BCCCC CBCBB
    AABCA ACBAB BBABC CAAAA CBCBC
    AABCB ACBAC BBACA CAAAB CBCCA
    AABCC ACBBA BBACB CAAAC CBCCB
    AACAA ACBBB BBACC CAABA CBCCC
    AACAB ACBBC BBBAA CAABB CCAAA
    AACAC ACBCA BBBAB CAABC CCAAB
    AACBA ACBCB BBBAC CAACA CCAAC
    AACBB ACBCC BBBBA CAACB CCABA
    AACBC ACCAA BBBBB CAACC CCABB
    AACCA ACCAB BBBBC CABAA CCABC
    AACCB ACCAC BBBCA CABAB CCACA
    AACCC ACCBA BBBCB CABAC CCACB
    ABAAA ACCBB BBBCC CABBA CCACC
    ABAAB ACCBC BBCAA CABBB CCBAA
    ABAAC ACCCA BBCAB CABBC CCBAB
    ABABA ACCCB BBCAC CABCA CCBAC
    ABABB ACCCC BBCBA CABCB CCBBA
    ABABC BAAAA BBCBB CABCC CCBBB
    ABACA BAAAB BBCBC CACAA CCBBC
    ABACB BAAAC BBCCA CACAB CCBCA
    ABACC BAABA BBCCB CACAC CCBCB
    ABBAA BAABB BBCCC CACBA CCBCC
    ABBAB BAABC BCAAA CACBB CCCAA
    ABBAC BAACA BCAAB CACBC CCCAB
    ABBBA BAACB BCAAC CACCA CCCAC
    ABBBB BAACC BCABA CACCB CCCBA
    ABBBC BABAA BCABB CACCC CCCBB
    ABBCA BABAB BCABC CBAAA CCCBC
    ABBCB BABAC BCACA CBAAB CCCCA
    ABBCC BABBA BCACB CBAAC CCCCB
    ABCAA BABBB BCACC CBABA CCCCC
    ABCAB BABBC BCBAA CBABB
    ABCAC BABCA BCBAB CBABC
    ABCBA BABCB BCBAC CBACA
  • TABLE 4
    Certain 3′-Wing Sugar Motifs
    Certain 3′-Wing Sugar Motifs
    AAAAA BABC CBAB ABBB BAA
    AAAAB BACA CBAC BAAA BAB
    AAABA BACB CBBA BAAB BBA
    AAABB BACC CBBB BABA BBB
    AABAA BBAA CBBC BABB AA
    AABAB BBAB CBCA BBAA AB
    AABBA BBAC CBCB BBAB AC
    AABBB BBBA CBCC BBBA BA
    ABAAA BBBB CCAA BBBB BB
    ABAAB BBBC CCAB  AAA BC
    ABABA BBCA CCAC  AAB CA
    ABABB BBCB CCBA  AAC CB
    ABBAA BBCC CCBB  ABA CC
    ABBAB BCAA CCBC  ABB AA
    ABBBA BCAB CCCA  ABC AB
    ABBBB BCAC CCCB  ACA BA
    BAAAA ABCB BCBA ACB
    BAAAB ABCC BCBB ACC
    BAABA ACAA BCBC BAA
    BAABB ACAB BCCA BAB
    BABAA ACAC BCCB BAC
    BABAB ACBA BCCC BBA
    BABBA ACBB CAAA BBB
    BABBB ACBC CAAB BBC
    BBAAA ACCA CAAC BCA
    BBAAB ACCB CABA BCB
    BBABA ACCC CABB BCC
    BBABB BAAA CABC CAA
    BBBAA BAAB CACA CAB
    BBBAB BAAC CACB CAC
    BBBBA BABA CACC CBA
    BBBBB BABB CBAA CBB
    AAAA AACC CCCC CBC
    AAAB ABAA AAAA CCA
    AAAC ABAB AAAB CCB
    AABA ABAC AABA CCC
    AABB ABBA AABB AAA
    AABC ABBB ABAA AAB
    AACA ABBC ABAB ABA
    AACB ABCA ABBA ABB
  • In certain embodiments, each A, each B, and each C located at the 5′-most 3′-wing region nucleoside is a modified nucleoside. For example, in certain embodiments the 3′-wing motif is selected from among ABB, BBB, and CBB, wherein the underlined nucleoside represents the 5′-most 3′-wing region nucleoside and wherein the underlined nucleoside is a modified nucleoside.
  • In certain embodiments, each A comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, each A comprises a modified sugar moiety. In certain embodiments, each A comprises a 2′-substituted sugar moiety. In certain embodiments, each A comprises a 2′-substituted sugar moiety selected from among F, ara-F, OCH3 and O(CH2)2—OCH3. In certain embodiments, each A comprises a bicyclic sugar moiety. In certain embodiments, each A comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, α-L-LNA, ENA and 2′-thio LNA. In certain embodiments, each A comprises a modified nucleobase. In certain embodiments, each A comprises a modified nucleobase selected from among 2-thio-thymidine nucleoside and 5-propyne uridine nucleoside. In certain embodiments, each A comprises a 5′-substituted sugar moiety selected from among 5′-Me DNA, and 5′-(R)-Me DNA.
  • In certain embodiments, each B comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, each B comprises a modified sugar moiety. In certain embodiments, each B comprises a 2′-substituted sugar moiety. In certain embodiments, each B comprises a 2′-substituted sugar moiety selected from among F, (ara)-F, OCH3 and O(CH2)2—OCH3. In certain embodiments, each B comprises a bicyclic sugar moiety. In certain embodiments, each B comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, α-L-LNA, ENA and 2′-thio LNA. In certain embodiments, each B comprises a modified nucleobase. In certain embodiments, each B comprises a modified nucleobase selected from among 2-thio-thymidine nucleoside and 5-propyne urindine nucleoside. In certain embodiments, each B comprises an HNA. In certain embodiments, each B comprises an F-HNA. In certain embodiments, each B comprises a 5′-substituted sugar moiety selected from among 5′-Me DNA, and 5′-(R)-Me DNA.
  • In certain embodiments, each A comprises a 2′-substituted sugar moiety selected from among F, ara-F, OCH3 and O(CH2)2—OCH3 and each B comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, α-L-LNA, ENA and 2′-thio LNA. In certain embodiments, each A comprises O(CH2)2—OCH3 and each B comprises cEt.
  • In certain embodiments, each C comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, each C comprises a modified sugar moiety. In certain embodiments, each C comprises a 2′-substituted sugar moiety. In certain embodiments, each C comprises a 2′-substituted sugar moiety selected from among F, (ara)-F, OCH3 and O(CH2)2—OCH3. In certain embodiments, each C comprises a 5′-substituted sugar moiety. In certain embodiments, each C comprises a 5′-substituted sugar moiety selected from among 5′-Me, and 5′-(R)-Me. In certain embodiments, each C comprises a bicyclic sugar moiety. In certain embodiments, each C comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, α-L-LNA, ENA and 2′-thio LNA. In certain embodiments, each C comprises a modified nucleobase. In certain embodiments, each C comprises a modified nucleobase selected from among 2-thio-thymidine and 5-propyne uridine. In certain embodiments, each C comprises a 2-thio-thymidine nucleoside. In certain embodiments, each C comprises an HNA. In certain embodiments, each C comprises an F-HNA.
  • vi. Certain Central Regions (Gaps)
  • In certain embodiments, the gap of a gapmer consists of 6 to 20 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 to 15 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 to 12 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 to 10 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 to 9 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 to 8 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 or 7 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 7 to 10 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 7 to 9 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 7 or 8 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 8 to 10 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 8 or 9 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 6 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 7 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 8 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 9 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 10 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 11 linked nucleosides. In certain embodiments, the gap of a gapmer consists of 12 linked nucleosides.
  • In certain embodiments, each nucleoside of the gap of a gapmer is a 2′-deoxynucleoside. In certain embodiments, the gap comprises one or more modified nucleosides. In certain embodiments, each nucleoside of the gap of a gapmer is a 2′-deoxynucleoside or is a modified nucleoside that is “DNA-like.” In such embodiments, “DNA-like” means that the nucleoside has similar characteristics to DNA, such that a duplex comprising the gapmer and an RNA molecule is capable of activating RNase H. For example, under certain conditions, 2′-(ara)-F have been shown to support RNase H activation, and thus is DNA-like. In certain embodiments, one or more nucleosides of the gap of a gapmer is not a 2′-deoxynucleoside and is not DNA-like. In certain such embodiments, the gapmer nonetheless supports RNase H activation (e.g., by virtue of the number or placement of the non-DNA nucleosides).
  • In certain embodiments, gaps comprise a stretch of unmodified 2′-deoxynucleoside interrupted by one or more modified nucleosides, thus resulting in three sub-regions (two stretches of one or more 2′-deoxynucleosides and a stretch of one or more interrupting modified nucleosides). In certain embodiments, no stretch of unmodified 2′-deoxynucleosides is longer than 5, 6, or 7 nucleosides. In certain embodiments, such short stretches is achieved by using short gap regions. In certain embodiments, short stretches are achieved by interrupting a longer gap region.
  • In certain embodiments, the gap comprises one or more modified nucleosides. In certain embodiments, the gap comprises one or more modified nucleosides selected from among cEt, FHNA, LNA, and 2-thio-thymidine. In certain embodiments, the gap comprises one modified nucleoside. In certain embodiments, the gap comprises a 5′-substituted sugar moiety selected from among 5′-Me, and 5′-(R)-Me. In certain embodiments, the gap comprises two modified nucleosides. In certain embodiments, the gap comprises three modified nucleosides. In certain embodiments, the gap comprises four modified nucleosides. In certain embodiments, the gap comprises two or more modified nucleosides and each modified nucleoside is the same. In certain embodiments, the gap comprises two or more modified nucleosides and each modified nucleoside is different.
  • In certain embodiments, the gap comprises one or more modified linkages. In certain embodiments, the gap comprises one or more methyl phosphonate linkages. In certain embodiments the gap comprises two or more modified linkages. In certain embodiments, the gap comprises one or more modified linkages and one or more modified nucleosides. In certain embodiments, the gap comprises one modified linkage and one modified nucleoside. In certain embodiments, the gap comprises two modified linkages and two or more modified nucleosides.
  • In certain embodiments, the gap comprises a nucleoside motif selected from among the following: DDDDXDDDDD; DDDDDXDDDDD; DDDXDDDDD; DDDDXDDDDDD; DDDDXDDDD; DDXDDDDDD; DDDXDDDDDD; DXDDDDDD; DDXDDDDDDD; DDXDDDDD; DDXDDDXDDD; DDDXDDDXDDD; DXDDDXDDD; DDXDDDXDD; DDXDDDDXDDD; DDXDDDDXDD; DXDDDDXDDD; DDDDXDDD; DDDXDDD; DXDDDDDDD; DDDDXXDDD; and DXXDXXDXX; wherein each D is an unmodified deoxynucleoside; and each X is a modified nucleoside or a substituted sugar moiety.
  • In certain embodiments, the gap comprises a nucleoside motif selected from among the following: DDDDDDDDD; DXDDDDDDD; DDXDDDDDD; DDDXDDDDD; DDDDXDDDD; DDDDDXDDD; DDDDDDXDD; DDDDDDDXD; DXXDDDDDD; DDDDDDXXD; DDXXDDDDD; DDDXXDDDD; DDDDXXDDD; DDDDDXXDD; DXDDDDDXD; DXDDDDXDD; DXDDDXDDD; DXDDXDDDD; DXDXDDDDD; DDXDDDDXD; DDXDDDXDD; DDXDDXDDD; DDXDXDDDD; DDDXDDDXD; DDDXDDXDD; DDDXDXDDD; DDDDXDDXD; DDDDXDXDD; and DDDDDXDXD, wherein each D is an unmodified deoxynucleoside; and each X is a modified nucleoside or a substituted sugar moiety.
  • In certain embodiments, the gap comprises a nucleoside motif selected from among the following: DDDDXDDDD, DXDDDDDDD, DXXDDDDDD, DDXDDDDDD, DDDXDDDDD, DDDDXDDDD, DDDDDXDDD, DDDDDDXDD, and DDDDDDDXD, wherein each D is an unmodified deoxynucleoside; and each X is a modified nucleoside or a substituted sugar moiety.
  • In certain embodiments, the gap comprises a nucleoside motif selected from among the following: DDDDDDDD, DXDDDDDD, DDXDDDDD, DDDXDDDD, DDDDXDDD, DDDDDXDD, DDDDDDXD, DXDDDDXD, DXDDDXDD, DXDDXDDD, DXDXDDDD, DXXDDDDD, DDXXDDDD, DDXDXDDD, DDXDDXDD, DXDDDDXD, DDDXXDDD, DDDXDXDD, DDDXDDXD, DDDDXXDD, DDDDXDXD, and DDDDDXXD, wherein each D is an unmodified deoxynucleoside; and each X is a modified nucleoside or a substituted sugar moiety.
  • In certain embodiments, the gap comprises a nucleoside motif selected from among the following: DXDDDDD, DDXDDDD, DDDXDDD, DDDDXDD, DDDDDXD, DXDDDXD, DXDDXDD, DXDXDDD, DXXDDDD, DDXXDDD, DDXDXDD, DDXDDXD, DDDXXDD, DDDXDXD, and DDDDXXD, wherein each D is an unmodified deoxynucleoside; and each X is a modified nucleoside or a substituted sugar moiety.
  • In certain embodiments, the gap comprises a nucleoside motif selected from among the following: DXDDDD, DDXDDD, DDDXDD, DDDDXD, DXXDDD, DXDXDD, DXDDXD, DDXXDD, DDXDXD, and DDDXXD, wherein each D is an unmodified deoxynucleoside; and each X is a modified nucleoside or a substituted sugar moiety.
  • In certain embodiments, the gap comprises a nucleoside motif selected from among the following: DXDDDD, DDXDDD, DDDXDD, DDDDXD, DXDDDDD, DDXDDDD, DDDXDDD, DDDDXDD, DDDDDXD, DXDDDDDD, DDXDDDDD, DDDXDDDD, DDDDXDDD, DDDDDXDD, DDDDDDXD, DXDDDDDDD; DDXDDDDDD, DDDXDDDDD, DDDDXDDDD, DDDDDXDDD, DDDDDDXDD, DDDDDDDXD, DXDDDDDDDD, DDXDDDDDDD, DDDXDDDDDD, DDDDXDDDDD, DDDDDXDDDD, DDDDDDXDDD, DDDDDDDXDD, and DDDDDDDDXD, wherein each D is an unmodified deoxynucleoside; and each X is a modified nucleoside or a substituted sugar moiety.
  • In certain embodiments, each X comprises an unmodified 2′-deoxyfuranose sugar moiety. In certain embodiments, each X comprises a modified sugar moiety. In certain embodiments, each X comprises a 2′-substituted sugar moiety. In certain embodiments, each X comprises a 2′-substituted sugar moiety selected from among F, (ara)-F, OCH3 and O(CH2)2—OCH3. In certain embodiments, each X comprises a 5′-substituted sugar moiety. In certain embodiments, each X comprises a 5′-substituted sugar moiety selected from among 5′-Me, and 5′-(R)-Me. In certain embodiments, each X comprises a bicyclic sugar moiety. In certain embodiments, each X comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, α-L-LNA, ENA and 2′-thio LNA. In certain embodiments, each X comprises a modified nucleobase. In certain embodiments, each X comprises a modified nucleobase selected from among 2-thio-thymidine and 5-propyne uridine. In certain embodiments, each X comprises a 2-thio-thymidine nucleoside. In certain embodiments, each X comprises an HNA. In certain embodiments, each C comprises an F-HNA. In certain embodiments, X represents the location of a single differentiating nucleobase.
  • vii. Certain Gapmer Motifs
  • In certain embodiments, a gapmer comprises a 5′-wing, a gap, and a 3′ wing, wherein the 5′-wing, gap, and 3′ wing are independently selected from among those discussed above. For example, in certain embodiments, a gapmer has a 5′-wing, a gap, and a 3′-wing having features selected from among any of those listed in the tables above and any 5′-wing may be paired with any gap and any 3′-wing. For example, in certain embodiments, a 5′-wing may comprise AAABB, a 3′-wing may comprise BBA, and the gap may comprise DDDDDDD. For example, in certain embodiments, a gapmer has a 5′-wing, a gap, and a 3′-wing having features selected from among those listed in the following non-limiting table, wherein each motif is represented as (5′-wing)-(gap)-(3′-wing), wherein each number represents the number of linked nucleosides in each portion of the motif, for example, a 5-10-5 motif would have a 5′-wing comprising 5 nucleosides, a gap comprising 10 nucleosides, and a 3′-wing comprising 5 nucleosides:
  • TABLE 5
    Certain Gapmer Sugar Motifs
    Certain Gapmer Sugar Motifs
    2-10-2 3-10-2 4-10-2 5-10-2
    2-10-3 3-10-3 4-10-3 5-10-3
    2-10-4 3-10-4 4-10-4 5-10-4
    2-10-5 3-10-5 4-10-5 5-10-5
    2-9-2 3-9-2 4-9-2 5-9-2
    2-9-3 3-9-3 4-9-3 5-9-3
    2-9-4 3-9-4 4-9-4 5-9-4
    2-9-5 3-9-5 4-9-5 5-9-5
    2-11-2 3-11-2 4-11-2 5-11-2
    2-11-3 3-11-3 4-11-3 5-11-3
    2-11-4 3-11-4 4-11-4 5-11-4
    2-11-5 3-11-5 4-11-5 5-11-5
    2-8-2 3-8-2 4-8-2 5-8-2
    2-8-3 3-8-3 4-8-3 5-8-3
    2-8-4 3-8-4 4-8-4 5-8-4
    2-8-5 3-8-5 4-8-5 5-8-5
  • In certain embodiments, a gapmer comprises a 5′-wing, a gap, and a 3′ wing, wherein the 5′-wing, gap, and 3′ wing are independently selected from among those discussed above. For example, in certain embodiments, a gapmer has a 5′-wing, a gap, and a 3′-wing having features selected from among those listed in the following non-limiting tables:
  • TABLE 6
    Certain Gapmer Nucleoside Motifs
    5′-wing Central gap 3′-wing
    region region region
    ADDA DDDDDD ABB
    ABBA DDDADDDD ABAA
    AAAAAAA DDDDDDDDDDD AAA
    AAAAABB DDDDDDDD BBAAAAA
    ABB DDDDADDDD ABB
    ABB DDDDBDDDD BBA
    ABB DDDDDDDDD BBA
    AABAA DDDDDDDDD AABAA
    ABB DDDDDD AABAA
    AAABAA DDDDDDDDD AAABAA
    AAABAA DDDDDDDDD AAB
    ABAB DDDDDDDDD ABAB
    AAABB DDDDDDD BBA
    ABADB DDDDDDD BBA
    ABA DBDDDDDDD BBA
    ABA DADDDDDDD BBA
    ABAB DDDDDDDD BBA
    AA DDDDDDDD BBBBBBBB
    ABB DDDDDD ABADB
    AAAAB DDDDDDD BAAAA
    ABBB DDDDDDDDD AB
    AB DDDDDDDDD BBBA
    ABBB DDDDDDDDD BBBA
    AB DDDDDDDD ABA
    ABB DDDDWDDDD BBA
    AAABB DDDWDDD BBAAA
    ABB DDDDWWDDD BBA
    ABADB DDDDDDD BBA
    ABBDC DDDDDDD BBA
    ABBDDC DDDDDD BBA
    ABBDCC DDDDDD BBA
    ABB DWWDWWDWW BBA
    ABB DWDDDDDDD BBA
    ABB DDWDDDDDD BBA
    ABB DWWDDDDDD BBA
    AAABB DDWDDDDDD AA
    BB DDWDWDDDD BBABBBB
    ABB DDDD(ND)DDDD BBA
    AAABB DDD(ND)DDD BBAAA
    ABB DDDD(ND)(ND)DDD BBA
    ABB D(ND)(ND)D(ND)(ND)D(ND)(ND) BBA
    ABB D(ND)DDDDDDD BBA
    ABB DD(ND)DDDDDD BBA
    ABB D(ND)(ND)DDDDDD BBA
    AAABB DD(ND)DDDDDD AA
    BB DD(ND)D(ND)DDDD BBABBBB
    ABAB DDDDDDDDD BABA
  • TABLE 7
    Certain Gapmer Nucleoside Motifs
    5′-wing Central gap 3′-wing
    region region region
    ABBW DDDDDDDD BBA
    ABB DWDDDDDDD BBA
    ABB DDWDDDDDD BBA
    ABB DDDWDDDDD BBA
    ABB DDDDWDDDD BBA
    ABB DDDDDWDDD BBA
    ABB DDDDDDWDD BBA
    ABB DDDDDDDWD BBA
    ABB DDDDDDDD WBBA
    ABBWW DDDDDDD BBA
    ABB DWWDDDDDD BBA
    ABB DDWWDDDDD BBA
    ABB DDDWWDDDD BBA
    ABB DDDDWWDDD BBA
    ABB DDDDDWWDD BBA
    ABB DDDDDDWWD BBA
    ABB DDDDDDD WWBBA
    ABBW DDDDDDD WBBA
    ABBW DDDDDDWD BBA
    ABBW DDDDDWDD BBA
    ABBW DDDDWDDD BBA
    ABBW DDDWDDDD BBA
    ABBW DDWDDDDD BBA
    ABBW DWDDDDDD BBA
    ABB DWDDDDDD WBBA
    ABB DWDDDDDWD BBA
    ABB DWDDDDWDD BBA
    ABB DWDDDWDDD BBA
    ABB DWDDWDDDD BBA
    ABB DWDWDDDDD BBA
    ABB DDWDDDDD WBBA
    ABB DDWDDDDWD BBA
    ABB DDWDDDWDD BBA
    ABB DDWDDWDDD BBA
    ABB DDWDWDDDD BBA
    ABB DDWWDDDDD BBA
    ABB DDDWDDDD WBBA
    ABB DDDWDDDWD BBA
    ABB DDDWDDWDD BBA
    ABB DDDWDWDDD BBA
    ABB DDDWWDDDD BBA
    ABB DDDDWDDD WBBA
    ABB DDDDWDDWD BBA
    ABB DDDDWDWDD BBA
    ABB DDDDWWDDD BBA
    ABB DDDDDWDD WBBA
    ABB DDDDDWDWD BBA
    ABB DDDDDWWDD BBA
    ABB DDDDDDWD WBBA
  • TABLE 8
    Certain Gapmer Nucleoside Motifs
    5′-wing Central gap 3′-wing
    region region region
    ABBB DDDDDDDD BBA
    ABB DBDDDDDDD BBA
    ABB DDBDDDDDD BBA
    ABB DDDBDDDDD BBA
    ABB DDDDBDDDD BBA
    ABB DDDDDBDDD BBA
    ABB DDDDDDBDD BBA
    ABB DDDDDDDBD BBA
    ABB DDDDDDDD BBBA
    ABBBB DDDDDDD BBA
    ABB DBBDDDDDD BBA
    ABB DDBBDDDDD BBA
    ABB DDDBBDDDD BBA
    ABB DDDDBBDDD BBA
    ABB DDDDDBBDD BBA
    ABB DDDDDDBBD BBA
    ABB DDDDDDD BBBBA
    ABBB DDDDDDD BBBA
    ABB DDDDDDBD BBA
    ABBB DDDDDBDD BBA
    ABBB DDDDBDDD BBA
    ABBB DDDBDDDD BBA
    ABBB DDBDDDDD BBA
    ABBB DBDDDDDD BBA
    ABB DBDDDDDD BBBA
    ABB DBDDDDDBD BBA
    ABB DBDDDDBDD BBA
    ABB DBDDDBDDD BBA
    ABB DBDDBDDDD BBA
    ABB DBDBDDDDD BBA
    ABB DDBDDDDD BBBA
    ABB DDBDDDDBD BBA
    ABB DDBDDDBDD BBA
    ABB DDBDDBDDD BBA
    ABB DDBDBDDDD BBA
    ABB DDBBDDDDD BBA
    ABB DDDBDDDD BBBA
    ABB DDDBDDDBD BBA
    ABB DDDBDDBDD BBA
    ABB DDDBDBDDD BBA
    ABB DDDBBDDDD BBA
    ABB DDDDBDDD BBBA
    ABB DDDDBDDBD BBA
    ABB DDDDBDBDD BBA
    ABB DDDDBBDDD BBA
    ABB DDDDDBDD BBBA
    ABB DDDDDBDBD BBA
    ABB DDDDDBBDD BBA
    ABB DDDDDDBD BBBA
  • TABLE 9
    Certain Gapmer Nucleoside Motifs
    5′-wing Central gap 3′-wing
    region region region
    ABB DDDDDDDDD BBA
    AB DBDDDDDDDD BBA
    AB DDBDDDDDDD BBA
    AB DDDBDDDDDD BBA
    AB DDDDBDDDDD BBA
    AB DDDDDBDDDD BBA
    AB DDDDDDBDDD BBA
    AB DDDDDDDBDD BBA
    AB DDDDDDDDBD BBA
    AB DDDDDDDDD BBBA
    ABBB DDDDDDDD BBA
    AB DBBDDDDDDD BBA
    AB DDBBDDDDDD BBA
    AB DDDBBDDDDD BBA
    AB DDDDBBDDDD BBA
    AB DDDDDBBDDD BBA
    AB DDDDDDBBDD BBA
    AB DDDDDDDBBD BBA
    AB DDDDDDDD BBBBA
    ABBBB DDDDDDD BBA
    AB DBBBDDDDDD BBA
    AB DDBBBDDDDD BBA
    AB DDDBBBDDDD BBA
    AB DDDDBBBDDD BBA
    AB DDDDDBBBDD BBA
    AB DDDDDDBBBD BBA
    AB DDDDDDD BBBBBA
    AB DDDDDDDDD BBBA
    AB DDDDDDDBD BBBA
    AB DDDDDBDD BBBA
    AB DDDDBDDD BBBA
    AB DDDBDDDD BBBA
    AB DDBDDDDD BBBA
    AB DBDDDDDD BBBA
    AB DDDDDBD BBBBA
    AB DDDDBDD BBBBA
    AB DDDBDDD BBBBA
    AB DDBDDDD BBBBA
    AB DBDDDDD BBBBA
    AB DDDDBD BBBBBA
    AB DDDBDD BBBBBA
    AB DDBDDD BBBBBA
    AB DBDDDD BBBBBA
  • TABLE 10
    Certain Gapmer Nucleoside Motifs
    5′-wing Central gap 3′-wing
    region region region
    AAAAAA DDDDDDD BABA
    AAAAAB DDDDDDD BABA
    AAAABA DDDDDDD BABA
    AAABAA DDDDDDD BABA
    AABAAA DDDDDDD BABA
    ABAAAA DDDDDDD BABA
    BAAAAA DDDDDDD BABA
    ABAAAB DDDDDDD BABA
    ABAABA DDDDDDD BABA
    ABABAA DDDDDDD BABA
    ABBAAA DDDDDDD BABA
    AABAAB DDDDDDD BABA
    AABABA DDDDDDD BABA
    AABBAA DDDDDDD BABA
    AAABAB DDDDDDD BABA
    AAABBA DDDDDDD BABA
    AAAABB DDDDDDD BABA
    BAAAAB DDDDDDD BABA
    BAAABA DDDDDDD BABA
    BAABAA DDDDDDD BABA
    BABAAA DDDDDDD BABA
    BBAAAA DDDDDDD BABA
    BBBAAA DDDDDDD BABA
    BBABAA DDDDDDD BABA
    BBAABA DDDDDDD BABA
    BBAAAB DDDDDDD BABA
    ABABAB DDDDDDD BABA
    BBBBAA DDDDDDD BABA
    BBBABA DDDDDDD BABA
    BBBAAB DDDDDDD BABA
    BBBBBA DDDDDDD BABA
    BBBBAB DDDDDDD BABA
    AAABBB DDDDDDD BABA
    AABABB DDDDDDD BABA
    ABAABB DDDDDDD BABA
    BAAABB DDDDDDD BABA
    AABBBB DDDDDDD BABA
    ABABBB DDDDDDD BABA
    BAABBB DDDDDDD BABA
    ABBBBB DDDDDDD BABA
    BABBBB DDDDDDD BABA
    BBBBBB DDDDDDD BABA
  • TABLE 11
    Certain Gapmer Nucleoside Motifs
    5′-wing Central gap 3′-wing
    region region region
    AAAAA DDDDDDD AAAAA
    AAAAB DDDDDDD AAAAA
    AAABA DDDDDDD AAAAA
    AAABB DDDDDDD AAAAA
    AABAA DDDDDDD AAAAA
    AABAB DDDDDDD AAAAA
    AABBA DDDDDDD AAAAA
    AABBB DDDDDDD AAAAA
    ABAAA DDDDDDD AAAAA
    ABAAB DDDDDDD AAAAA
    ABABA DDDDDDD AAAAA
    ABABB DDDDDDD AAAAA
    ABBAA DDDDDDD AAAAA
    ABBAB DDDDDDD AAAAA
    ABBBA DDDDDDD AAAAA
    ABBBB DDDDDDD AAAAA
    BAAAA DDDDDDD AAAAA
    BAAAB DDDDDDD AAAAA
    BAABA DDDDDDD AAAAA
    BAABB DDDDDDD AAAAA
    BABAA DDDDDDD AAAAA
    BABAB DDDDDDD AAAAA
    BABBA DDDDDDD AAAAA
    BABBB DDDDDDD AAAAA
    BBAAA DDDDDDD AAAAA
    BBAAB DDDDDDD AAAAA
    BBABA DDDDDDD AAAAA
    BBABB DDDDDDD AAAAA
    BBBAA DDDDDDD AAAAA
    BBBAB DDDDDDD AAAAA
    BBBBA DDDDDDD AAAAA
    BBBBB DDDDDDD AAAAA
    AAAAA DDDDDDD BAAAA
    AAAAB DDDDDDD BAAAA
    AAABA DDDDDDD BAAAA
    AAABB DDDDDDD BAAAA
    AABAA DDDDDDD BAAAA
    AABAB DDDDDDD BAAAA
    AABBA DDDDDDD BAAAA
    AABBB DDDDDDD BAAAA
    ABAAA DDDDDDD BAAAA
    ABAAB DDDDDDD BAAAA
    ABABA DDDDDDD BAAAA
    ABABB DDDDDDD BAAAA
    ABBAA DDDDDDD BAAAA
    ABBAB DDDDDDD BAAAA
    ABBBA DDDDDDD BAAAA
    ABBBB DDDDDDD BAAAA
    BAAAA DDDDDDD BAAAA
    BAAAB DDDDDDD BAAAA
    BAABA DDDDDDD BAAAA
    BAABB DDDDDDD BAAAA
    BABAA DDDDDDD BAAAA
    BABAB DDDDDDD BAAAA
    BABBA DDDDDDD BAAAA
    BABBB DDDDDDD BAAAA
    BBAAA DDDDDDD BAAAA
    BBAAB DDDDDDD BAAAA
    BBABA DDDDDDD BAAAA
    BBABB DDDDDDD BAAAA
    BBBAA DDDDDDD BAAAA
    BBBAB DDDDDDD BAAAA
    BBBBA DDDDDDD BAAAA
    BBBBB DDDDDDD BAAAA
    AAAAA DDDDDDD BBAAA
    AAAAB DDDDDDD BBAAA
    AAABA DDDDDDD BBAAA
    AAABB DDDDDDD BBAAA
    AABAA DDDDDDD BBAAA
    AABAB DDDDDDD BBAAA
    AABBA DDDDDDD BBAAA
    AABBB DDDDDDD BBAAA
    ABAAA DDDDDDD BBAAA
    ABAAB DDDDDDD BBAAA
    ABABA DDDDDDD BBAAA
    ABABB DDDDDDD BBAAA
    ABBAA DDDDDDD BBAAA
    ABBAB DDDDDDD BBAAA
    ABBBA DDDDDDD BBAAA
    ABBBB DDDDDDD BBAAA
    BAAAA DDDDDDD BBAAA
    BAAAB DDDDDDD BBAAA
    BAABA DDDDDDD BBAAA
    BAABB DDDDDDD BBAAA
    BABAA DDDDDDD BBAAA
    BABAB DDDDDDD BBAAA
    BABBA DDDDDDD BBAAA
    BABBB DDDDDDD BBAAA
    BBAAA DDDDDDD BBAAA
    BBAAB DDDDDDD BBAAA
    BBABA DDDDDDD BBAAA
    BBABB DDDDDDD BBAAA
    BBBAA DDDDDDD BBAAA
    BBBAB DDDDDDD BBAAA
    BBBBA DDDDDDD BBAAA
    BBBBB DDDDDDD BBAAA
    AAAAA DDDDDDD BBBAA
    AAAAB DDDDDDD BBBAA
    AAABA DDDDDDD BBBAA
    AAABB DDDDDDD BBBAA
    AABAA DDDDDDD BBBAA
    AABAB DDDDDDD BBBAA
    AABBA DDDDDDD BBBAA
    AABBB DDDDDDD BBBAA
    ABAAA DDDDDDD BBBAA
    ABAAB DDDDDDD BBBAA
    ABABA DDDDDDD BBBAA
    ABABB DDDDDDD BBBAA
    ABBAA DDDDDDD BBBAA
    ABBAB DDDDDDD BBBAA
    ABBBA DDDDDDD BBBAA
    ABBBB DDDDDDD BBBAA
    BAAAA DDDDDDD BBBAA
    BAAAB DDDDDDD BBBAA
    BAABA DDDDDDD BBBAA
    BAABB DDDDDDD BBBAA
    BABAA DDDDDDD BBBAA
    BABAB DDDDDDD BBBAA
    BABBA DDDDDDD BBBAA
    BABBB DDDDDDD BBBAA
    BBAAA DDDDDDD BBBAA
    BBAAB DDDDDDD BBBAA
    BBABA DDDDDDD BBBAA
    BBABB DDDDDDD BBBAA
    BBBAA DDDDDDD BBBAA
    BBBAB DDDDDDD BBBAA
    BBBBA DDDDDDD BBBAA
    BBBBB DDDDDDD BBBAA
    AAAAA DDDDDDD BBBBA
    AAAAB DDDDDDD BBBBA
    AAABA DDDDDDD BBBBA
    AAABB DDDDDDD BBBBA
    AABAA DDDDDDD BBBBA
    AABAB DDDDDDD BBBBA
    AABBA DDDDDDD BBBBA
    AABBB DDDDDDD BBBBA
    ABAAA DDDDDDD BBBBA
    ABAAB DDDDDDD BBBBA
    ABABA DDDDDDD BBBBA
    ABABB DDDDDDD BBBBA
    ABBAA DDDDDDD BBBBA
    ABBAB DDDDDDD BBBBA
    ABBBA DDDDDDD BBBBA
    ABBBB DDDDDDD BBBBA
    BAAAA DDDDDDD BBBBA
    BAAAB DDDDDDD BBBBA
    BAABA DDDDDDD BBBBA
    BAABB DDDDDDD BBBBA
    BABAA DDDDDDD BBBBA
    BABAB DDDDDDD BBBBA
    BABBA DDDDDDD BBBBA
    BABBB DDDDDDD BBBBA
    BBAAA DDDDDDD BBBBA
    BBAAB DDDDDDD BBBBA
    BBABA DDDDDDD BBBBA
    BBABB DDDDDDD BBBBA
    BBBAA DDDDDDD BBBBA
    BBBAB DDDDDDD BBBBA
    BBBBA DDDDDDD BBBBA
    BBBBB DDDDDDD BBBBA
    AAAAA DDDDDDD BBBBB
    AAAAB DDDDDDD BBBBB
    AAABA DDDDDDD BBBBB
    AAABB DDDDDDD BBBBB
    AABAA DDDDDDD BBBBB
    AABAB DDDDDDD BBBBB
    AABBA DDDDDDD BBBBB
    AABBB DDDDDDD BBBBB
    ABAAA DDDDDDD BBBBB
    ABAAB DDDDDDD BBBBB
    ABABA DDDDDDD BBBBB
    ABABB DDDDDDD BBBBB
    ABBAA DDDDDDD BBBBB
    ABBAB DDDDDDD BBBBB
    ABBBA DDDDDDD BBBBB
    ABBBB DDDDDDD BBBBB
    BAAAA DDDDDDD BBBBB
    BAAAB DDDDDDD BBBBB
    BAABA DDDDDDD BBBBB
    BAABB DDDDDDD BBBBB
    BABAA DDDDDDD BBBBB
    BABAB DDDDDDD BBBBB
    BABBA DDDDDDD BBBBB
    BABBB DDDDDDD BBBBB
    BBAAA DDDDDDD BBBBB
    BBAAB DDDDDDD BBBBB
    BBABA DDDDDDD BBBBB
    BBABB DDDDDDD BBBBB
    BBBAA DDDDDDD BBBBB
    BBBAB DDDDDDD BBBBB
    BBBBA DDDDDDD BBBBB
    BBBBB DDDDDDD BBBBB
  • TABLE 12
    Certain Gapmer Nucleoside Motifs
    5′-wing Central gap 3′-wing
    region region region
    AAAW DDDDDDDD BBA
    AABW DDDDDDDD BBA
    ABAW DDDDDDDD BBA
    ABBW DDDDDDDD BBA
    BAAW DDDDDDDD BBA
    BABW DDDDDDDD BBA
    BBAW DDDDDDDD BBA
    BBBW DDDDDDDD BBA
    ABB DDDDDDDD WAAA
    ABB DDDDDDDD WAAB
    ABB DDDDDDDD WABA
    ABB DDDDDDDD WABB
    ABB DDDDDDDD WBAA
    ABB DDDDDDDD WBAB
    ABB DDDDDDDD WBBA
    ABB DDDDDDDD WBBB
    AAAWW DDDDDDD BBA
    AABWW DDDDDDD BBA
    ABAWW DDDDDDD BBA
    ABBWW DDDDDDD BBA
    BAAWW DDDDDDD BBA
    BABWW DDDDDDD BBA
    BBAWW DDDDDDD BBA
    BBBWW DDDDDDD BBA
    ABB DDDDDDD WWAAA
    ABB DDDDDDD WWAAB
    ABB DDDDDDD WWABA
    ABB DDDDDDD WWABB
    ABB DDDDDDD WWBAA
    ABB DDDDDDD WWBAB
    ABB DDDDDDD WWBBA
    ABB DDDDDDD WWBBB
    AAAAW DDDDDDD BBA
    AAABW DDDDDDD BBA
    AABAW DDDDDDD BBA
    AABBW DDDDDDD BBA
    ABAAW DDDDDDD BBA
    ABABW DDDDDDD BBA
    ABBAW DDDDDDD BBA
    ABBBW DDDDDDD BBA
    BAAAW DDDDDDD BBA
    BAABW DDDDDDD BBA
    BABAW DDDDDDD BBA
    BABBW DDDDDDD BBA
    BBAAW DDDDDDD BBA
    BBABW DDDDDDD BBA
    BBBAW DDDDDDD BBA
    BBBBW DDDDDDD WAAAA
    ABB DDDDDDD WAAAB
    ABB DDDDDDD WAABA
    ABB DDDDDDD WAABB
    ABB DDDDDDD WABAA
    ABB DDDDDDD WABAB
    ABB DDDDDDD WABBA
    ABB DDDDDDD WABBB
    ABB DDDDDDD WBAAA
    ABB DDDDDDD WBAAB
    ABB DDDDDDD WBABA
    ABB DDDDDDD WBABB
    ABB DDDDDDD WBBAA
    ABB DDDDDDD WBBAB
    ABB DDDDDDD WBBBA
    ABB DDDDDDD WBBBB
  • wherein each A is a modified nucleoside of a first type, each B is a modified nucleoside of a second type and each W is a modified nucleoside or nucleobase of either the first type, the second type or a third type, each D is a nucleoside comprising an unmodified 2′ deoxy sugar moiety and unmodified nucleobase, and ND is modified nucleoside comprising a modified nucleobase and an unmodified 2′ deoxy sugar moiety.
  • In certain embodiments, each A comprises a modified sugar moiety. In certain embodiments, each A comprises a 2′-substituted sugar moiety. In certain embodiments, each A comprises a 2′-substituted sugar moiety selected from among F, ara-F, OCH3 and O(CH2)2—OCH3. In certain embodiments, each A comprises a bicyclic sugar moiety. In certain embodiments, each A comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, α-L-LNA, ENA and 2′-thio LNA. In certain embodiments, each A comprises a modified nucleobase. In certain embodiments, each A comprises a modified nucleobase selected from among 2-thio-thymidine nucleoside and 5-propyne uridine nucleoside. In certain embodiments, each A comprises an HNA. In certain embodiments, each A comprises an F-HNA. In certain embodiments, each A comprises a 5′-substituted sugar moiety selected from among 5′-Me, and 5′-(R)-Me.
  • In certain embodiments, each B comprises a modified sugar moiety. In certain embodiments, each B comprises a 2′-substituted sugar moiety. In certain embodiments, each B comprises a 2′-substituted sugar moiety selected from among F, (ara)-F, OCH3 and O(CH2)2—OCH3. In certain embodiments, each B comprises a bicyclic sugar moiety. In certain embodiments, each B comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, α-L-LNA, ENA and 2′-thio LNA. In certain embodiments, each B comprises a modified nucleobase. In certain embodiments, each B comprises a modified nucleobase selected from among 2-thio-thymidine nucleoside and 5-propyne urindine nucleoside. In certain embodiments, each B comprises an HNA. In certain embodiments, each B comprises an F-HNA. In certain embodiments, each B comprises a 5′-substituted sugar moiety selected from among 5′-Me, and 5′-(R)-Me.
  • In certain embodiments, each C comprises a modified sugar moiety. In certain embodiments, each C comprises a 2′-substituted sugar moiety. In certain embodiments, each C comprises a 2′-substituted sugar moiety selected from among F, (ara)-F, OCH3 and O(CH2)2—OCH3. In certain embodiments, each C comprises a 5′-substituted sugar moiety. In certain embodiments, each C comprises a 5′-substituted sugar moiety selected from among 5′-Me, and 5′-(R)-Me. In certain embodiments, each C comprises a bicyclic sugar moiety. In certain embodiments, each C comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, α-L-LNA, ENA and 2′-thio LNA. In certain embodiments, each C comprises a modified nucleobase. In certain embodiments, each C comprises a modified nucleobase selected from among 2-thio-thymidine and 5-propyne uridine. In certain embodiments, each C comprises a 2-thio-thymidine nucleoside. In certain embodiments, each C comprises an HNA. In certain embodiments, each C comprises an F-HNA.
  • In certain embodiments, each W comprises a modified sugar moiety. In certain embodiments, each W comprises a 2′-substituted sugar moiety. In certain embodiments, each W comprises a 2′-substituted sugar moiety selected from among F, (ara)-F, OCH3 and O(CH2)2—OCH3. In certain embodiments, each W comprises a 5′-substituted sugar moiety. In certain embodiments, each W comprises a 5′-substituted sugar moiety selected from among 5′-Me, and 5′-(R)-Me. In certain embodiments, each W comprises a bicyclic sugar moiety. In certain embodiments, each W comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, α-L-LNA, ENA and 2′-thio LNA. In certain embodiments, each W comprises a sugar surrogate. In certain embodiments, each W comprises a sugar surrogate selected from among HNA and F—HNA. In certain embodiments, each W comprises a 2-thio-thymidine nucleoside.
  • In certain embodiments, at least one of A or B comprises a bicyclic sugar moiety, and the other comprises a 2′-substituted sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises a 2′-substituted sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises a 2′-substituted sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside and the other of A or B comprises a 2′-substituted sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises a 2′-MOE sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises a 2′-MOE sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside and the other of A or B comprises a 2′-MOE sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises a 2′-F sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises a 2′-F sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside and the other of A or B comprises a 2′-F sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises a 2′-(ara)-F sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises a 2′-(ara)-F sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside and the other of A or B comprises a 2′-(ara)-F sugar moiety.
  • In certain embodiments, A comprises a bicyclic sugar moiety, and B comprises a 2′-substituted sugar moiety. In certain embodiments, A is an LNA nucleoside and B comprises a 2′-substituted sugar moiety. In certain embodiments, A is a cEt nucleoside and B comprises a 2′-substituted sugar moiety. In certain embodiments, A is an α-L-LNA nucleoside and B comprises a 2′-substituted sugar moiety.
  • In certain embodiments, A comprises a bicyclic sugar moiety, and B comprises a 2′-MOE sugar moiety. In certain embodiments, A is an LNA nucleoside and B comprises a 2′-MOE sugar moiety. In certain embodiments, A is a cEt nucleoside and B comprises a 2′-MOE sugar moiety. In certain embodiments, A is an α-L-LNA nucleoside and B comprises a 2′-MOE sugar moiety.
  • In certain embodiments, A comprises a bicyclic sugar moiety, and B comprises a 2′-F sugar moiety. In certain embodiments, A is an LNA nucleoside and B comprises a 2′-F sugar moiety. In certain embodiments, A is a cEt nucleoside and B comprises a 2′-F sugar moiety. In certain embodiments, A is an α-L-LNA nucleoside and B comprises a 2′-F sugar moiety.
  • In certain embodiments, A comprises a bicyclic sugar moiety, and B comprises a 2′-(ara)-F sugar moiety. In certain embodiments, A is an LNA nucleoside and B comprises a 2′-(ara)-F sugar moiety. In certain embodiments, A is a cEt nucleoside and B comprises a 2′-(ara)-F sugar moiety. In certain embodiments, A is an α-L-LNA nucleoside and B comprises a 2′-(ara)-F sugar moiety.
  • In certain embodiments, B comprises a bicyclic sugar moiety, and A comprises a 2′-MOE sugar moiety. In certain embodiments, B is an LNA nucleoside and A comprises a 2′-MOE sugar moiety. In certain embodiments, B is a cEt nucleoside and A comprises a 2′-MOE sugar moiety. In certain embodiments, B is an α-L-LNA nucleoside and A comprises a 2′-MOE sugar moiety.
  • In certain embodiments, B comprises a bicyclic sugar moiety, and A comprises a 2′-F sugar moiety. In certain embodiments, B is an LNA nucleoside and A comprises a 2′-F sugar moiety. In certain embodiments, B is a cEt nucleoside and A comprises a 2′-F sugar moiety. In certain embodiments, B is an α-L-LNA nucleoside and A comprises a 2′-F sugar moiety.
  • In certain embodiments, B comprises a bicyclic sugar moiety, and A comprises a 2′-(ara)-F sugar moiety. In certain embodiments, B is an LNA nucleoside and A comprises a 2′-(ara)-F sugar moiety. In certain embodiments, B is a cEt nucleoside and A comprises a 2′-(ara)-F sugar moiety. In certain embodiments, B is an α-L-LNA nucleoside and A comprises a 2′-(ara)-F sugar moiety.
  • In certain embodiments, at least one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-substituted sugar moiety and W comprises a modified nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-substituted sugar moiety, and W comprises a modified nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-substituted sugar moiety, and C comprises a modified nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-substituted sugar moiety, and W comprises a modified nucleobase.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a modified nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a modified nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a modified nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a modified nucleobase.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and W comprises a modified nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a modified nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a modified nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a modified nucleobase.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a modified nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a modified nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a modified nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a modified nucleobase.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-substituted sugar moiety, and W comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-substituted sugar moiety, and W comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-substituted sugar moiety, and W comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-substituted sugar moiety, and W comprises a 2-thio-thymidine nucleobase.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 2-thio-thymidine nucleobase.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and W comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a 2-thio-thymidine nucleobase.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises 2-thio-thymidine nucleobase.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5-propyne uridine nucleobase.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and W comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a 5-propyne uridine nucleobase.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 5-propyne uridine nucleobase.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a sugar surrogate.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and W comprises a sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a sugar surrogate.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises sugar surrogate.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a HNA sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a HNA sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a HNA sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a HNA sugar surrogate.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and W comprises a HNA sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a HNA sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a HNA sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a sugar HNA surrogate.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a HNA sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a HNA sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a HNA sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a HNA sugar surrogate.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a F-HNA sugar surrogate.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and W comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a F-HNA sugar surrogate.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a F-HNA sugar surrogate.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 5′-Me DNA sugar moiety.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and W comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a 5′-Me DNA sugar moiety.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 5′-Me DNA sugar moiety.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety.
  • In certain embodiments, at least two of A, B or W comprises a 2′-substituted sugar moiety, and the other comprises a bicyclic sugar moiety. In certain embodiments, at least two of A, B or W comprises a bicyclic sugar moiety, and the other comprises a 2′-substituted sugar moiety. In certain embodiments, a gapmer has a sugar motif other than: E-K-K-(D)9-K-K-E; E-E-E-E-K-(D)9-E-E-E-E-E; E-K-K-K-(D)9-K-K-K-E; K-E-E-K-(D)9-K-E-E-K; K-D-D-K-(D)9-K-D-D-K; K-E-K-E-K-(D)9-K-E-K-E-K; K-D-K-D-K-(D)9-K-D-K-D-K; E-K-E-K-(D)9-K-E-K-E; E-E-E-E-E-K-(D)8-E-E-E-E-E; or E-K-E-K-E-(D)9-E-K-E-K-E, E-E-E-K-K-(D)7-E-E-K, E-K-E-K-K-K-(D)7-K-E-K-E, E-K-E-K-E-K-(D)7-K-E-K-E, wherein K is a nucleoside comprising a cEt sugar moiety and E is a nucleoside comprising a 2′-MOE sugar moiety.
  • In certain embodiments a gapmer comprises a A-(D)4-A-(D)4-A-(D)4-AA motif. In certain embodiments a gapmer comprises a B-(D)4-A-(D)4-A-(D)4-AA motif. In certain embodiments a gapmer comprises a A-(D)4-B-(D)4-A-(D)4-AA motif. In certain embodiments a gapmer comprises a A-(D)4-A-(D)4-B-(D)4-AA motif. In certain embodiments a gapmer comprises a A-(D)4-A-(D)4-A-(D)4-BA motif. In certain embodiments a gapmer comprises a A-(D)4-A-(D)4-A-(D)4-BB motif. In certain embodiments a gapmer comprises a K-(D)4-K-(D)4-K-(D)4-K-E motif.
  • viii. Certain Internucleoside Linkage Motifs
  • In certain embodiments, oligonucleotides comprise modified internucleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or modified internucleoside linkage motif. In certain embodiments, internucleoside linkages are arranged in a gapped motif, as described above for nucleoside motif. In such embodiments, the internucleoside linkages in each of two wing regions are different from the internucleoside linkages in the gap region. In certain embodiments the internucleoside linkages in the wings are phosphodiester and the internucleoside linkages in the gap are phosphorothioate. The nucleoside motif is independently selected, so such oligonucleotides having a gapped internucleoside linkage motif may or may not have a gapped nucleoside motif and if it does have a gapped nucleoside motif, the wing and gap lengths may or may not be the same.
  • In certain embodiments, oligonucleotides comprise a region having an alternating internucleoside linkage motif. In certain embodiments, oligonucleotides of the present invention comprise a region of uniformly modified internucleoside linkages. In certain such embodiments, the oligonucleotide comprises a region that is uniformly linked by phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide is uniformly linked by phosphorothioate. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate and at least one internucleoside linkage is phosphorothioate.
  • In certain embodiments, the oligonucleotide comprises at least 6 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 8 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 10 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 6 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 8 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 10 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least block of at least one 12 consecutive phosphorothioate internucleoside linkages. In certain such embodiments, at least one such block is located at the 3′ end of the oligonucleotide. In certain such embodiments, at least one such block is located within 3 nucleosides of the 3′ end of the oligonucleotide.
  • In certain embodiments, oligonucleotides comprise one or more methylphosphonate linkages. In certain embodiments, oligonucleotides having a gapmer nucleoside motif comprise a linkage motif comprising all phosphorothioate linkages except for one or two methylphosphonate linkages. In certain embodiments, one methylphosphonate linkage is in the central gap of an oligonucleotide having a gapmer nucleoside motif.
  • ix. Certain Modification Motifs
  • Modification motifs define oligonucleotides by nucleoside motif (sugar motif and nucleobase motif) and linkage motif. For example, certain oligonucleotides have the following modification motif:
  • AsAsAsDsDsDsDs(ND)sDsDsDsDsBsBsB;
  • wherein each A is a modified nucleoside comprising a 2′-substituted sugar moiety; each D is an unmodified 2′-deoxynucleoside; each B is a modified nucleoside comprising a bicyclic sugar moiety; ND is a modified nucleoside comprising a modified nucleobase; and s is a phosphorothioate internucleoside linkage. Thus, the sugar motif is a gapmer motif. The nucleobase modification motif is a single modified nucleobase at 8th nucleoside from the 5′-end. Combining the sugar motif and the nucleobase modification motif, the nucleoside motif is an interrupted gapmer where the gap of the sugar modified gapmer is interrupted by a nucleoside comprising a modified nucleobase. The linkage motif is uniform phosphorothioate. The following non-limiting Table further illustrates certain modification motifs:
  • TABLE 13
    Certain Modification Motifs
    5′-wing Central gap 3′-wing
    region region region
    BsBs sDsDsDsDsDsDsDsDsDs AsAsAsAsAsAsAsA
    AsBsBs DsDsDsDsDsDsDsDsDs BsBsA
    AsBsBs DsDsDsDs(ND)sDsDsDsDs BsBsA
    AsBsBs DsDsDsDsAsDsDsDsDs BsBsA
    AsBsBs DsDsDsDsBsDsDsDsDs BsBsA
    AsBsBs DsDsDsDsWsDsDsDsDs BsBsA
    AsBsBsBs DsDsDsDsDsDsDsDsDs BsBsAsBsB
    AsBsBs DsDsDsDsDsDsDsDsDs BsBsAsBsB
    BsBsAsBsBs DsDsDsDsDsDsDsDsDs BsBsA
    AsBsBs DsDsDsDsDsDsDsDsDs BsBsAsBsBsBsB
    AsAsBsAsAs DsDsDsDsDsDsDsDsDs BsBsA
    AsAsAsBsAsAs DsDsDsDsDsDsDsDsDs BsBsA
    AsAsBsAsAs DsDsDsDsDsDsDsDsDs AsAsBsAsA
    AsAsAsBsAsAs DsDsDsDsDsDsDsDsDs AsAsBsAsAsA
    AsAsAsAsBsAsAs DsDsDsDsDsDsDsDsDs BsBsA
    AsBsAsBs DsDsDsDsDsDsDsDsDs BsAsBsA
    AsBsAsBs DsDsDsDsDsDsDsDsDs AsAsBsAsAs
    AsBsBs DsDsDsDsDsDsDsDsDs BsAsBsA
    BsBsAsBsBsBsB DsDsDsDsDsDsDsDsDs BsAsBsA
    AsAsAsAsAs DsDsDsDsDsDsDsDsDs AsAsAsAsA
    AsAsAsAsAs DsDsDsDsDsDsDs AsAsAsAsA
    AsAsAsAsAs DsDsDsDsDsDsDsDsDs BsBsAsBsBsBsB
    AsAsAsBsBs DsDsDsDsDsDsDs BsBsA
    AsBsAsBs DsDsDsDsDsDsDsDs BsBsA
    AsBsAsBs DsDsDsDsDsDsDs AsAsAsBsBs
    AsAsAsAsBs DsDsDsDsDsDsDs BsAsAsAsA
    BsBs DsDsDsDsDsDsDsDs AsA
    AsAs DsDsDsDsDsDsDs AsAsAsAsAsAsAsA
    AsAsAs DsDsDsDsDsDsDs AsAsAsAsAsAsA
    AsAsAs DsDsDsDsDsDsDs AsAsAsAsAsA
    AsBs DsDsDsDsDsDsDs BsBsBsA
    AsBsBsBs DsDsDsDsDsDsDsDsDs BsA
    AsBs DsDsDsDsDsDsDsDsDs BsBsBsA
    AsAsAsBsBs DsDsDs(ND)sDsDsDs BsBsAsAsA
    AsAsAsBsBs DsDsDsAsDsDsDs BsBsAsAsA
    AsAsAsBsBs DsDsDsBsDsDsDs BsBsAsAsA
    AsAsAsAsBs DsDsDsDsDsDsDs BsAsAsAsA
    AsAsBsBsBs DsDsDsDsDsDsDs BsBsBsAsA
    AsAsAsAsBs DsDsDsDsDsDsDs AsAsAsAsAs
    AsAsAsBsBs DsDsDsDsDsDsDs AsAsAsAsAs
    AsAsBsBsBs DsDsDsDsDsDsDs AsAsAsAsAs
    AsAsAsAsAs DsDsDsDsDsDsDs BsAsAsAsAs
    AsAsAsAsAs DsDsDsDsDsDsDs BsBsAsAsAs
    AsAsAsAsAs DsDsDsDsDsDsDs BsBsBsAsAs
    AsBsBs DsDsDsDs(ND)s(ND)sDsDsDs BsBsA
    AsBsBs Ds(ND)s(ND)sDs(ND)s(ND)sDs(ND)s(ND)s BsBsA
    AsBsBs Ds(ND)sDsDsDsDsDsDsDs BsBsA
    AsBsBs DsDs(ND)sDsDsDsDsDsDs BsBsA
    AsBsBs Ds(ND)s(ND)sDsDsDsDsDsDs BsBsA
    AsBsBs DsDs(D)zDsDsDsDsDsDs BsBsA
    AsBsBs Ds(D)zDsDsDsDsDsDsDs BsBsA
    AsBsBs (D)zDsDsDsDsDsDsDsDs BsBsA
    AsBsBs DsDsAsDsDsDsDsDsDs BsBsA
    AsBsBs DsDsBsDsDsDsDsDsDs BsBsA
    AsBsBs AsDsDsDsDsDsDsDsDs BsBsA
    AsBsBs BsDsDsDsDsDsDsDsDs BsBsA
    AsBsAsBs DsDs(D)zDsDsDsDsDsDs BsBsBsAsAs
    AsAsAsBsBs DsDs(ND)sDsDsDsDsDsDs AsA
    AsBsBsBs Ds(D)zDsDsDsDsDsDsDs AsAsAsBsBs
    AsBsBs DsDsDsDsDsDsDsDs(D)z BsBsA
    AsAsBsBsBs DsDsDsAsDsDsDs BsBsBsAsA
    AsAsBsBsBs DsDsDsBsDsDsDs BsBsBsAsA
    AsBsAsBs DsDsDsAsDsDsDs BsBsAsBsBsBsB
    AsBsBsBs DsDsDsDs(D)zDsDsDsDs BsA
    AsAsBsBsBs DsDsAsAsDsDsDs BsBsA
    AsBsBs DsDsDsDs(D)zDsDsDsDs BsBsBsA
    BsBs DsDs(ND)sDs(ND)sDsDsDsDs BsBsAsBsBsBsB
  • wherein each A and B are nucleosides comprising differently modified sugar moieties, each D is a nucleoside comprising an unmodified 2′ deoxy sugar moiety, each W is a modified nucleoside of either the first type, the second type or a third type, each ND is a modified nucleoside comprising a modified nucleobase, s is a phosphorothioate internucleoside linkage, and z is a non-phosphorothioate internucleoside linkage.
  • In certain embodiments, each A comprises a modified sugar moiety. In certain embodiments, each A comprises a 2′-substituted sugar moiety. In certain embodiments, each A comprises a 2′-substituted sugar moiety selected from among F, (ara)-F, OCH3 and O(CH2)2—OCH3. In certain embodiments, each A comprises a bicyclic sugar moiety. In certain embodiments, each A comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, α-L-LNA, ENA and 2′-thio LNA. In certain embodiments, each A comprises a modified nucleobase. In certain embodiments, each A comprises a modified nucleobase selected from among 2-thio-thymidine nucleoside and 5-propyne uridine nucleoside. In certain embodiments, each B comprises a modified sugar moiety. In certain embodiments, each B comprises a 2′-substituted sugar moiety. In certain embodiments, each B comprises a 2′-substituted sugar moiety selected from among F, (ara)-F, OCH3 and O(CH2)2—OCH3. In certain embodiments, each B comprises a bicyclic sugar moiety. In certain embodiments, each B comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, α-L-LNA, ENA and 2′-thio LNA. In certain embodiments, each B comprises a modified nucleobase. In certain embodiments, each B comprises a modified nucleobase selected from among 2-thio-thymidine nucleoside and 5-propyne urindine nucleoside. In certain embodiments, each A comprises an HNA. In certain embodiments, each A comprises an F-HNA.
  • In certain embodiments, each W comprises a modified sugar moiety. In certain embodiments, each W comprises a 2′-substituted sugar moiety. In certain embodiments, each W comprises a 2′-substituted sugar moiety selected from among F, (ara)-F, OCH3 and O(CH2)2—OCH3. In certain embodiments, each W comprises a 5′-substituted sugar moiety. In certain embodiments, each W comprises a 5′-substituted sugar moiety selected from among 5′-Me, and 5′-(R)-Me. In certain embodiments, each W comprises a bicyclic sugar moiety. In certain embodiments, each W comprises a bicyclic sugar moiety selected from among cEt, cMOE, LNA, α-L-LNA, ENA and 2′-thio LNA. In certain embodiments, each W comprises a sugar surrogate. In certain embodiments, each W comprises a sugar surrogate selected from among HNA and F—HNA.
  • In certain embodiments, at least one of A or B comprises a bicyclic sugar moiety, and the other comprises a 2′-substituted sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises a 2′-substituted sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises a 2′-substituted sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside and the other of A or B comprises a 2′-substituted sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises a 2′-MOE sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises a 2′-MOE sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside and the other of A or B comprises a 2′-MOE sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises a 2′-F sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises a 2′-F sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside and the other of A or B comprises a 2′-F sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside and the other of A or B comprises a 2′-(ara)-F sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside and the other of A or B comprises a 2′-(ara)-F sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside and the other of A or B comprises a 2′-(ara)-F sugar moiety.
  • In certain embodiments, A comprises a bicyclic sugar moiety, and B comprises a 2′-substituted sugar moiety. In certain embodiments, A is an LNA nucleoside and B comprises a 2′-substituted sugar moiety. In certain embodiments, A is a cEt nucleoside and B comprises a 2′-substituted sugar moiety. In certain embodiments, A is an α-L-LNA nucleoside and B comprises a 2′-substituted sugar moiety.
  • In certain embodiments, A comprises a bicyclic sugar moiety, and B comprises a 2′-MOE sugar moiety. In certain embodiments, A is an LNA nucleoside and B comprises a 2′-MOE sugar moiety. In certain embodiments, A is a cEt nucleoside and B comprises a 2′-MOE sugar moiety. In certain embodiments, A is an α-L-LNA nucleoside and B comprises a 2′-MOE sugar moiety.
  • In certain embodiments, A comprises a bicyclic sugar moiety, and B comprises a 2′-F sugar moiety. In certain embodiments, A is an LNA nucleoside and B comprises a 2′-F sugar moiety. In certain embodiments, A is a cEt nucleoside and B comprises a 2′-F sugar moiety. In certain embodiments, A is an α-L-LNA nucleoside and B comprises a 2′-F sugar moiety.
  • In certain embodiments, A comprises a bicyclic sugar moiety, and B comprises a 2′-(ara)-F sugar moiety. In certain embodiments, A is an LNA nucleoside and B comprises a 2′-(ara)-F sugar moiety. In certain embodiments, A is a cEt nucleoside and B comprises a 2′-(ara)-F sugar moiety. In certain embodiments, A is an α-L-LNA nucleoside and B comprises a 2′-(ara)-F sugar moiety.
  • In certain embodiments, B comprises a bicyclic sugar moiety, and A comprises a 2′-MOE sugar moiety. In certain embodiments, B is an LNA nucleoside and A comprises a 2′-MOE sugar moiety. In certain embodiments, B is a cEt nucleoside and A comprises a 2′-MOE sugar moiety. In certain embodiments, B is an α-L-LNA nucleoside and A comprises a 2′-MOE sugar moiety.
  • In certain embodiments, B comprises a bicyclic sugar moiety, and A comprises a 2′-F sugar moiety. In certain embodiments, B is an LNA nucleoside and A comprises a 2′-F sugar moiety. In certain embodiments, B is a cEt nucleoside and A comprises a 2′-F sugar moiety. In certain embodiments, B is an α-L-LNA nucleoside and A comprises a 2′-F sugar moiety.
  • In certain embodiments, B comprises a bicyclic sugar moiety, and A comprises a 2′-(ara)-F sugar moiety. In certain embodiments, B is an LNA nucleoside and A comprises a 2′-(ara)-F sugar moiety. In certain embodiments, B is a cEt nucleoside and A comprises a 2′-(ara)-F sugar moiety. In certain embodiments, B is an α-L-LNA nucleoside and A comprises a 2′-(ara)-F sugar moiety.
  • In certain embodiments, at least one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-substituted sugar moiety and W comprises a modified nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-substituted sugar moiety, and W comprises a modified nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-substituted sugar moiety, and C comprises a modified nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-substituted sugar moiety, and W comprises a modified nucleobase.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a modified nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a modified nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a modified nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a modified nucleobase.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and W comprises a modified nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a modified nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a modified nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a modified nucleobase.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a modified nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a modified nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a modified nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a modified nucleobase.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-substituted sugar moiety, and W comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-substituted sugar moiety, and W comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-substituted sugar moiety, and W comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-substituted sugar moiety, and W comprises a 2-thio-thymidine nucleobase.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 2-thio-thymidine nucleobase.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and W comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a 2-thio-thymidine nucleobase.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 2-thio-thymidine nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises 2-thio-thymidine nucleobase.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and C comprises a 5-propyne uridine nucleobase.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and W comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and C comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a 5-propyne uridine nucleobase.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 5-propyne uridine nucleobase. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 5-propyne uridine nucleobase.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a sugar surrogate.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and W comprises a sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a sugar surrogate.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises sugar surrogate.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a HNA sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a HNA sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a HNA sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a HNA sugar surrogate.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and W comprises a HNA sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a HNA sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a HNA sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a sugar HNA surrogate.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a HNA sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a HNA sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a HNA sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a HNA sugar surrogate.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a F—HNA sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a F-HNA sugar surrogate.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and W comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a F-HNA sugar surrogate.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a F-HNA sugar surrogate. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a F-HNA sugar surrogate.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 5′-Me DNA sugar moiety.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and W comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a 5′-Me DNA sugar moiety.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 5′-Me DNA sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 5′-Me DNA sugar moiety.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-F sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-F sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety.
  • In certain embodiments, one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is an LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is a cEt nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety. In certain embodiments, one of A or B is an α-L-LNA nucleoside, another of A or B comprises a 2′-(ara)-F sugar moiety, and W comprises a 5′-(R)-Me DNA sugar moiety.
  • In certain embodiments, at least two of A, B or W comprises a 2′-substituted sugar moiety, and the other comprises a bicyclic sugar moiety. In certain embodiments, at least two of A, B or W comprises a bicyclic sugar moiety, and the other comprises a 2′-substituted sugar moiety.
  • d. Certain Overall Lengths
  • In certain embodiments, the present invention provides oligomeric compounds including oligonucleotides of any of a variety of ranges of lengths. In certain embodiments, the invention provides oligomeric compounds or oligonucleotides consisting of X to Y linked nucleosides, where X represents the fewest number of nucleosides in the range and Y represents the largest number of nucleosides in the range. In certain such embodiments, X and Y are each independently selected from 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50; provided that X≦Y. For example, in certain embodiments, the invention provides oligomeric compounds which comprise oligonucleotides consisting of 8 to 9, 8 to 10, 8 to 11, 8 to 12, 8 to 13, 8 to 14, 8 to 15, 8 to 16, 8 to 17, 8 to 18, 8 to 19, 8 to 20, 8 to 21, 8 to 22, 8 to 23, 8 to 24, 8 to 25, 8 to 26, 8 to 27, 8 to 28, 8 to 29, 8 to 30, 9 to 10, 9 to 11, 9 to 12, 9 to 13, 9 to 14, 9 to 15, 9 to 16, 9 to 17, 9 to 18, 9 to 19, 9 to 20, 9 to 21, 9 to 22, 9 to 23, 9 to 24, 9 to 25, 9 to 26, 9 to 27, 9 to 28, 9 to 29, 9 to 30, 10 to 11, 10 to 12, 10 to 13, 10 to 14, 10 to 15, 10 to 16, 10 to 17, 10 to 18, 10 to 19, 10 to 20, 10 to 21, 10 to 22, 10 to 23, 10 to 24, 10 to 25, 10 to 26, 10 to 27, 10 to 28, 10 to 29, 10 to 30, 11 to 12, 11 to 13, 11 to 14, 11 to 15, 11 to 16, 11 to 17, 11 to 18, 11 to 19, 11 to 20, 11 to 21, 11 to 22, 11 to 23, 11 to 24, 11 to 25, 11 to 26, 11 to 27, 11 to 28, 11 to 29, 11 to 30, 12 to 13, 12 to 14, 12 to 15, 12 to 16, 12 to 17, 12 to 18, 12 to 19, 12 to 20, 12 to 21, 12 to 22, 12 to 23, 12 to 24, 12 to 25, 12 to 26, 12 to 27, 12 to 28, 12 to 29, 12 to 30, 13 to 14, 13 to 15, 13 to 16, 13 to 17, 13 to 18, 13 to 19, 13 to 20, 13 to 21, 13 to 22, 13 to 23, 13 to 24, 13 to 25, 13 to 26, 13 to 27, 13 to 28, 13 to 29, 13 to 30, 14 to 15, 14 to 16, 14 to 17, 14 to 18, 14 to 19, 14 to 20, 14 to 21, 14 to 22, 14 to 23, 14 to 24, 14 to 25, 14 to 26, 14 to 27, 14 to 28, 14 to 29, 14 to 30, 15 to 16, 15 to 17, 15 to 18, 15 to 19, 15 to 20, 15 to 21, 15 to 22, 15 to 23, 15 to 24, 15 to 25, 15 to 26, 15 to 27, 15 to 28, 15 to 29, 15 to 30, 16 to 17, 16 to 18, 16 to 19, 16 to 20, 16 to 21, 16 to 22, 16 to 23, 16 to 24, 16 to 25, 16 to 26, 16 to 27, 16 to 28, 16 to 29, 16 to 30, 17 to 18, 17 to 19, 17 to 20, 17 to 21, 17 to 22, 17 to 23, 17 to 24, 17 to 25, 17 to 26, 17 to 27, 17 to 28, 17 to 29, 17 to 30, 18 to 19, 18 to 20, 18 to 21, 18 to 22, 18 to 23, 18 to 24, 18 to 25, 18 to 26, 18 to 27, 18 to 28, 18 to 29, 18 to 30, 19 to 20, 19 to 21, 19 to 22, 19 to 23, 19 to 24, 19 to 25, 19 to 26, 19 to 29, 19 to 28, 19 to 29, 19 to 30, 20 to 21, 20 to 22, 20 to 23, 20 to 24, 20 to 25, 20 to 26, 20 to 27, 20 to 28, 20 to 29, 20 to 30, 21 to 22, 21 to 23, 21 to 24, 21 to 25, 21 to 26, 21 to 27, 21 to 28, 21 to 29, 21 to 30, 22 to 23, 22 to 24, 22 to 25, 22 to 26, 22 to 27, 22 to 28, 22 to 29, 22 to 30, 23 to 24, 23 to 25, 23 to 26, 23 to 27, 23 to 28, 23 to 29, 23 to 30, 24 to 25, 24 to 26, 24 to 27, 24 to 28, 24 to 29, 24 to 30, 25 to 26, 25 to 27, 25 to 28, 25 to 29, 25 to 30, 26 to 27, 26 to 28, 26 to 29, 26 to 30, 27 to 28, 27 to 29, 27 to 30, 28 to 29, 28 to 30, or 29 to 30 linked nucleosides. In embodiments where the number of nucleosides of an oligomeric compound or oligonucleotide is limited, whether to a range or to a specific number, the oligomeric compound or oligonucleotide may, nonetheless further comprise additional other substituents. For example, an oligonucleotide comprising 8-30 nucleosides excludes oligonucleotides having 31 nucleosides, but, unless otherwise indicated, such an oligonucleotide may further comprise, for example one or more conjugates, terminal groups, or other substituents. In certain embodiments, a gapmer oligonucleotide has any of the above lengths.
  • Further, where an oligonucleotide is described by an overall length range and by regions having specified lengths, and where the sum of specified lengths of the regions is less than the upper limit of the overall length range, the oligonucleotide may have additional nucleosides, beyond those of the specified regions, provided that the total number of nucleosides does not exceed the upper limit of the overall length range.
  • e. Certain Oligonucleotides
  • In certain embodiments, oligonucleotides of the present invention are characterized by their modification motif and overall length. In certain embodiments, such parameters are each independent of one another. Thus, unless otherwise indicated, each internucleoside linkage of an oligonucleotide having a gapmer sugar motif may be modified or unmodified and may or may not follow the gapmer modification pattern of the sugar modifications. For example, the internucleoside linkages within the wing regions of a sugar-gapmer may be the same or different from one another and may be the same or different from the internucleoside linkages of the gap region. Likewise, such sugar-gapmer oligonucleotides may comprise one or more modified nucleobase independent of the gapmer pattern of the sugar modifications. One of skill in the art will appreciate that such motifs may be combined to create a variety of oligonucleotides. Herein if a description of an oligonucleotide or oligomeric compound is silent with respect to one or more parameter, such parameter is not limited. Thus, an oligomeric compound described only as having a gapmer sugar motif without further description may have any length, internucleoside linkage motif, and nucleobase modification motif. Unless otherwise indicated, all chemical modifications are independent of nucleobase sequence.
  • f. Certain Conjugate Groups
  • In certain embodiments, oligomeric compounds are modified by attachment of one or more conjugate groups. In general, conjugate groups modify one or more properties of the attached oligomeric compound including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, cellular distribution, cellular uptake, charge and clearance. Conjugate groups are routinely used in the chemical arts and are linked directly or via an optional conjugate linking moiety or conjugate linking group to a parent compound such as an oligomeric compound, such as an oligonucleotide. Conjugate groups includes without limitation, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins and dyes. Certain conjugate groups have been described previously, for example: cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-5-tritylthiol (Manoharan et al., Ann N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., do-decan-diol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937).
  • In certain embodiments, a conjugate group comprises an active drug substance, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fen-bufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indo-methicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.
  • In certain embodiments, conjugate groups are directly attached to oligonucleotides in oligomeric compounds. In certain embodiments, conjugate groups are attached to oligonucleotides by a conjugate linking group. In certain such embodiments, conjugate linking groups, including, but not limited to, bifunctional linking moieties such as those known in the art are amenable to the compounds provided herein. Conjugate linking groups are useful for attachment of conjugate groups, such as chemical stabilizing groups, functional groups, reporter groups and other groups to selective sites in a parent compound such as for example an oligomeric compound. In general a bifunctional linking moiety comprises a hydrocarbyl moiety having two functional groups. One of the functional groups is selected to bind to a parent molecule or compound of interest and the other is selected to bind essentially any selected group such as chemical functional group or a conjugate group. In some embodiments, the conjugate linker comprises a chain structure or an oligomer of repeating units such as ethylene glycol or amino acid units. Examples of functional groups that are routinely used in a bifunctional linking moiety include, but are not limited to, electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups. In some embodiments, bifunctional linking moieties include amino, hydroxyl, carboxylic acid, thiol, unsaturations (e.g., double or triple bonds), and the like.
  • Some nonlimiting examples of conjugate linking moieties include pyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and 6-aminohexanoic acid (AHEX or AHA). Other linking groups include, but are not limited to, substituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl or substituted or unsubstituted C2-C10 alkynyl, wherein a nonlimiting list of preferred substituent groups includes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.
  • Conjugate groups may be attached to either or both ends of an oligonucleotide (terminal conjugate groups) and/or at any internal position.
  • In certain embodiments, conjugate groups are at the 3′-end of an oligonucleotide of an oligomeric compound. In certain embodiments, conjugate groups are near the 3′-end. In certain embodiments, conjugates are attached at the 3′ end of an oligomeric compound, but before one or more terminal group nucleosides. In certain embodiments, conjugate groups are placed within a terminal group. In certain embodiments, the present invention provides oligomeric compounds. In certain embodiments, oligomeric compounds comprise an oligonucleotide. In certain embodiments, an oligomeric compound comprises an oligonucleotide and one or more conjugate and/or terminal groups. Such conjugate and/or terminal groups may be added to oligonucleotides having any of the motifs discussed above. Thus, for example, an oligomeric compound comprising an oligonucleotide having region of alternating nucleosides may comprise a terminal group.
    • C. ANTISENSE COMPOUNDS
  • In certain embodiments, oligomeric compounds provided herein are antisense compounds. Such antisense compounds are capable of hybridizing to a target nucleic acid, resulting in at least one antisense activity. In certain embodiments, antisense compounds specifically hybridize to one or more target nucleic acid. In certain embodiments, a specifically hybridizing antisense compound has a nucleobase sequence comprising a region having sufficient complementarity to a target nucleic acid to allow hybridization and result in antisense activity and insufficient complementarity to any non-target so as to avoid non-specific hybridization to any non-target nucleic acid sequences under conditions in which specific hybridization is desired (e.g., under physiological conditions for in vivo or therapeutic uses, and under conditions in which assays are performed in the case of in vitro assays).
  • In certain embodiments, the present invention provides antisense compounds comprising oligonucleotides that are fully complementary to the target nucleic acid over the entire length of the oligonucleotide. In certain embodiments, oligonucleotides are 99% complementary to the target nucleic acid. In certain embodiments, oligonucleotides are 95% complementary to the target nucleic acid. In certain embodiments, such oligonucleotides are 90% complementary to the target nucleic acid.
  • In certain embodiments, such oligonucleotides are 85% complementary to the target nucleic acid. In certain embodiments, such oligonucleotides are 80% complementary to the target nucleic acid. In certain embodiments, an antisense compound comprises a region that is fully complementary to a target nucleic acid and is at least 80% complementary to the target nucleic acid over the entire length of the oligonucleotide. In certain such embodiments, the region of full complementarity is from 6 to 14 nucleobases in length.
  • a. Certain Antisense Activities and Mechanisms
  • In certain antisense activities, hybridization of an antisense compound results in recruitment of a protein that cleaves of the target nucleic acid. For example, certain antisense compounds result in RNase H mediated cleavage of target nucleic acid. RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. The “DNA” in such an RNA:DNA duplex, need not be unmodified DNA. In certain embodiments, the invention provides antisense compounds that are sufficiently “DNA-like” to elicit RNase H activity. Such DNA-like antisense compounds include, but are not limited to gapmers having unmodified deoxyfuronose sugar moieties in the nucleosides of the gap and modified sugar moieties in the nucleosides of the wings.
  • Antisense activities may be observed directly or indirectly. In certain embodiments, observation or detection of an antisense activity involves observation or detection of a change in an amount of a target nucleic acid or protein encoded by such target nucleic acid; a change in the ratio of splice variants of a nucleic acid or protein; and/or a phenotypic change in a cell or animal.
  • In certain embodiments, compounds comprising oligonucleotides having a gapmer nucleoside motif described herein have desirable properties compared to non-gapmer oligonucleotides or to gapmers having other motifs. In certain circumstances, it is desirable to identify motifs resulting in a favorable combination of potent antisense activity and relatively low toxicity. In certain embodiments, compounds of the present invention have a favorable therapeutic index (measure of activity divided by measure of toxicity).
  • b. Certain Selective Antisense Compounds
  • In certain embodiments, antisense compounds provided are selective for a target relative to a non-target nucleic acid. In certain embodiments, the nucleobase sequences of the target and non-target nucleic acids differ by no more than 4 differentiating nucleobases in the targeted region. In certain embodiments, the nucleobase sequences of the target and non-target nucleic acids differ by no more than 3 differentiating nucleobases in the targeted region. In certain embodiments, the nucleobase sequences of the target and non-target nucleic acids differ by no more than 2 differentiating nucleobases in the targeted region. In certain embodiments, the nucleobase sequences of the target and non-target nucleic acids differ by a single differentiating nucleobase in the targeted region. In certain embodiments, the target and non-target nucleic acids are transcripts from different genes. In certain embodiments, the target and non-target nucleic acids are different alleles for the same gene. In certain embodiments, the introduction of a mismatch between an antisense compound and a non-target nucleic acid may alter the RNase H cleavage site of a target nucleic acid compared to a non-target nucleic acid. In certain embodiments, the target and non-target nucleic acids are not functionally related to one another (e.g., are transcripts from different genes). In certain embodiments, the target and not-target nucleic acids are allelic variants of one another. In certain embodiments, the allelic variant contains a single nucleotide polymorphism (SNP). In certain embodiments, a SNP is associated with a mutant allele. In certain embodiments, a mutant SNP is associated with a disease. In certain embodiments a mutant SNP is associated with a disease, but is not causative of the disease. In certain embodiments, mRNA and protein expression of a mutant allele is associated with disease.
  • Selectivity of antisense compounds is achieved, principally, by nucleobase complementarity. For example, if an antisense compound has no mismatches for a target nucleic acid and one or more mismatches for a non-target nucleic acid, some amount of selectivity for the target nucleic acid will result. In certain embodiments, provided herein are antisense compounds with enhanced selectivity (i.e. the ratio of activity for the target to the activity for non-target is greater). For example, in certain embodiments, a selective nucleoside comprises a particular feature or combination of features (e.g., chemical modification, motif, placement of selective nucleoside, and/or self-complementary region) that increases selectivity of an antisense compound compared to an antisense compound not having that feature or combination of features. In certain embodiments, such feature or combination of features increases antisense activity for the target. In certain embodiments, such feature or combination of features decreases activity for the target, but decreases activity for the non-target by a greater amount, thus resulting in an increase in selectivity.
  • Without being limited by mechanism, enhanced selectivity may result from a larger difference in the affinity of an antisense compound for its target compared to its affinity for the non-target and/or a larger difference in RNase H activity for the resulting duplexes. For example, in certain embodiments, a selective antisense compound comprises a modified nucleoside at that same position as a differentiating nucleobase (i.e., the selective nucleoside is modified). That modification may increase the difference in binding affinity of the antisense compound for the target relative to the non-target. In addition or in the alternative, the chemical modification may increase the difference in RNAse H activity for the duplex formed by the antisense compound and its target compared to the RNase activity for the duplex formed by the antisense compound and the non-target. For example, the modification may exaggerate a structure that is less compatible for RNase H to bind, cleave and/or release the non-target.
  • In certain embodiments, an antisense compound binds its intended target to form a target duplex. In certain embodiments, RNase H cleaves the target nucleic acid of the target duplex. In certain such embodiments, there is a primary cleavage site between two particular nucleosides of the target nucleic acid (the primary target cleavage site), which accounts for the largest amount of cleavage of the target nucleic acid. In certain embodiments, there are one or more secondary target cleavage sites. In certain embodiments, the same antisense compound hybridizes to a non-target to form a non-target duplex. In certain such embodiments, the non-target differs from the target by a single nucleobase within the target region, and so the antisense compound hybridizes with a single mismatch. Because of the mismatch, in certain embodiments, RNase H cleavage of the non-target may be reduced compared to cleavage of the target, but still occurs. In certain embodiments, though, the primary site of that cleavage of the non-target nucleic acid (primary non-target cleavage site) is different from that of the target. That is; the primary site is shifted due to the mismatch. In such a circumstance, one may use a modification placed in the antisense compound to disrupt RNase H cleavage at the primary non-target cleavage site. Such modification will result in reduced cleavage of the non-target, but will result little or no decrease in cleavage of the target. In certain embodiments, the modification is a modified sugar, nucleobase and/or linkage.
  • In certain embodiments, the primary non-target cleavage site is towards the 5′-end of the antisense compound, and the 5′-end of an antisense compound may be modified to prevent RNaseH cleavage. In this manner, it is thought that one having skill in the art may modify the 5′-end of an antisense compound, or modify the nucleosides in the gap region of the 5′-end of the antisense compound, or modify the 3′-most 5′-region nucleosides of the antisense compound to selectively inhibit RNaseH cleavage of the non-target nucleic acid duplex while retaining RNase H cleavage of the target nucleic acid duplex. In certain embodiments, 1-3 of the 3′-most 5′-region nucleosides of the antisense compound comprises a bicyclic sugar moiety.
  • For example, in certain embodiments the target nucleic acid may have an allelic variant, e.g. a non-target nucleic acid, containing a single nucleotide polymorphism. An antisense compound may be designed having a single nucleobase mismatch from the non-target nucleic acid, but which has full complementarity to the target nucleic acid. The mismatch between the antisense compound and the non-target nucleic acid may destabilize the antisense compound non-target nucleic acid duplex, and consequently the cleavage site of RNaseH may shift upstream towards the 5′-end of the antisense compound. Modification of the 5′-end of the antisense compound or the gap region near the 5′-end of the antisense compound, or one or more of the 3′-most nucleosides of the 5′-wing region, will then prevent RNaseH cleavage of the non-target nucleic acid. Since the target nucleic acid is fully complementary to the antisense compound, the antisense compound and the target nucleic acid will form a more stabilized antisense compound-target nucleic acid duplex and the cleavage site of RnaseH will be more downstream, towards the 3′ end of the antisense compound. Accordingly, modifications at the 5′-end of the antisense compound will prevent RNaseH cleavage of the non-target nucleic acid, but will not substantially effect RNaseH cleavage of the target nucleic acid, and selectivity between a target nucleic acid and its allelic variant may be achieved. In certain embodiments, one or more of the 3′-most nucleosides of the 5′-wing region comprises a bicyclic sugar moiety. In certain embodiments, one or more of the 3′-most nucleosides of the 5′-wing region comprises a bicyclic sugar moiety selected from cEt and LNA. In certain embodiments, one or more of the 3′-most nucleosides of the 5′-wing region comprises cEt. In certain embodiments, one or more of the 3′-most nucleosides of the 5′-wing region comprises LNA.
  • In certain embodiments, the introduction of a mismatch between an antisense compound and a target nucleic acid may alter the RNase H cleavage site of a target nucleic acid compared to a non-target nucleic acid by shifting the RNaseH cleavage site downstream from the mismatch site and towards the 3′-end of the antisense compound. In certain embodiments where the cleavage site of a target nucleic acid compared to a non-target nucleic acid has shifted downstream towards the 3′-end of the antisense compound, the 3′-end of an antisense compound may be modified to prevent RNaseH cleavage. In this manner, it is thought that one having skill in the art may modify the 3′-end of an antisense compound, or modify the nucleosides in the gap region near the 3′-end of antisense compound, to selectively inhibit RNaseH cleavage of the non-target nucleic acid while retaining RNase H cleavage of the target nucleic acid.
  • For example, in certain embodiments the target nucleic acid may have an allelic variant, e.g. a non-target nucleic acid, containing a single nucleotide polymorphism. An antisense compound may be designed having a single nucleobase mismatch from the non-target nucleic acid, but which has full complementarity to target nucleic acid. The mismatch between the antisense compound and the non-target nucleic acid may destabilize the antisense compound-non-target nucleic acid duplex, and consequently the cleavage site of RNaseH may shift downstream towards the 3′-end of the antisense compound. Modification of the 3′-end of the antisense compound, or one or more of the 5′-most nucleosides of the 3′-wing region, or the gap region of the antisense compound near the 3′-end will then prevent RNaseH cleavage of the non-target nucleic acid. Since the target nucleic acid is fully complementary to the antisense compound, the antisense compound and the target nucleic acid will form a more stabilized antisense compound-target nucleic acid duplex and the cleavage site of RnaseH will be more upstream, towards the 5′ end of the antisense compound. Accordingly, modifications at the 3′-end of the antisense compound will prevent RNaseH cleavage of the non-target nucleic acid, but will not substantially effect RNaseH cleavage of the target nucleic acid, and selectivity between a target nucleic acid and its allelic variant may be achieved. In certain embodiments, one or more of the 5′-most nucleosides of the 3′-wing region comprises a bicyclic sugar moiety. In certain embodiments, one or more of the 5′-most nucleosides of the 3′-wing region comprises a bicyclic sugar moiety selected from cEt and LNA. In certain embodiments, one or more of the 5′-most nucleosides of the 3′-wing region comprises cEt. In certain embodiments, one or more of the 5′-most nucleosides of the 3′-wing region comprises LNA.
  • In certain embodiments, the selectivity of antisense compounds having certain gaps, e.g. gaps of 7 nucleosides or longer, may be improved by the addition of one or more bicyclic nucleosides at the 3′-most 5′-wing nucleoside. In certain embodiments, the selectivity of antisense compounds having certain gaps, e.g. gaps of 7 nucleosides or longer, may be improved by the addition of two or more bicyclic nucleosides at the 3′-most 5′-wing nucleoside. In certain embodiments, the selectivity of antisense compounds having certain gaps, e.g. gaps of 7 nucleosides or longer, may be improved by the addition of one bicyclic nucleoside at the 3′-most 5′-wing nucleoside. In certain embodiments, the selectivity of antisense compounds having certain gaps, e.g. gaps of 7 nucleosides or longer, may be improved by the addition of two bicyclic nucleosides at the 3′-most 5′-wing nucleoside. In certain embodiments, the selectivity of antisense compounds having certain gaps, e.g. gaps of 7 nucleosides or longer, may be improved by the addition of three bicyclic nucleosides at the 3′-most 5′-wing nucleoside. In certain embodiments, the selectivity of antisense compounds having certain gaps, e.g. gaps of 7 nucleosides or longer, may be improved by the addition of four bicyclic nucleosides at the 3′-most 5′-wing nucleoside. In certain embodiments, the selectivity of antisense compounds having certain gaps, e.g. gaps of 7 nucleosides or longer, may be improved by the addition of five bicyclic nucleosides at the 3′-most 5′-wing nucleoside. In certain embodiments discussed above, the bicyclic nucleosides at the 3′-most 5′-wing nucleoside are selected from among cEt, cMOE, LNA, α-LNA, ENA and 2′-thio LNA. In certain embodiments discussed above, the bicyclic nucleosides at the 3′-most 5′-wing nucleoside comprise cEt. In certain embodiments discussed above, the bicyclic nucleosides at the 3′-most 5′-wing nucleoside comprise LNA.
  • In certain embodiments, the selectivity of antisense compounds having certain gaps, e.g. gaps of 7 nucleosides or longer, may be improved by the addition of one or more bicyclic nucleosides at the 3′-most 5′-wing nucleoside and the addition of one or more bicyclic nucleosides at the 5′-most 3′-wing nucleoside. In certain embodiments, the selectivity of antisense compounds having certain gaps, e.g. gaps of 7 nucleosides or longer, may be improved by the addition of two or more bicyclic nucleosides at the 3′-most 5′-wing nucleoside and the addition of one or more bicyclic nucleosides at the 5′-most 3′-wing nucleoside. In certain embodiments, the selectivity of antisense compounds having certain gaps, e.g. gaps of 7 nucleosides or longer, may be improved by the addition of one bicyclic nucleoside at the 3′-most 5′-wing nucleoside and the addition of one or more bicyclic nucleosides at the 5′-most 3′-wing nucleoside. In certain embodiments, the selectivity of antisense compounds having certain gaps, e.g. gaps of 7 nucleosides or longer, may be improved by the addition of two bicyclic nucleosides at the 3′-most 5′-wing nucleoside and the addition of one or more bicyclic nucleosides at the 5′-most 3′-wing nucleoside. In certain embodiments, the selectivity of antisense compounds having certain gaps, e.g. gaps of 7 nucleosides or longer, may be improved by the addition of three bicyclic nucleosides at the 3′-most 5′-wing nucleoside and the addition of one or more bicyclic nucleosides at the 5′-most 3′-wing nucleoside. In certain embodiments, the selectivity of antisense compounds having certain gaps, e.g. gaps of 7 nucleosides or longer, may be improved by the addition of four bicyclic nucleosides at the 3′-most 5′-wing nucleoside and the addition of one or more bicyclic nucleosides at the 5′-most 3′-wing nucleoside. In certain embodiments, the selectivity of antisense compounds having certain gaps, e.g. gaps of 7 nucleosides or longer, may be improved by the addition of four bicyclic nucleosides at the 3′-most 5′-wing nucleoside and the addition of one or more bicyclic nucleosides at the 5′-most 3′-wing nucleoside.
  • In certain embodiments, the selectivity of antisense compounds having certain gaps, e.g. gaps of 7 nucleosides or shorter, may be improved by the addition of one or more bicyclic nucleosides at the 3′-most 5′-wing nucleoside. In certain embodiments, the selectivity of antisense compounds having certain gaps, e.g. gaps of 7 nucleosides or shorter, may be improved by the addition of two or more bicyclic nucleosides at the 3′-most 5′-wing nucleoside. In certain embodiments, the selectivity of antisense compounds having certain gaps, e.g. gaps of 7 nucleosides or shorter, may be improved by the addition of one bicyclic nucleoside at the 3′-most 5′-wing nucleoside. In certain embodiments, the selectivity of antisense compounds having certain gaps, e.g. gaps of 7 nucleosides or shorter, may be improved by the addition of two bicyclic nucleosides at the 3′-most 5′-wing nucleoside. In certain embodiments, the selectivity of antisense compounds having certain gaps, e.g. gaps of 7 nucleosides or shorter, may be improved by the addition of three bicyclic nucleosides at the 3′-most 5′-wing nucleoside. In certain embodiments, the selectivity of antisense compounds having certain gaps, e.g. gaps of 7 nucleosides or shorter, may be improved by the addition of four bicyclic nucleosides at the 3′-most 5′-wing nucleoside. In certain embodiments, the selectivity of antisense compounds having certain gaps, e.g. gaps of 7 nucleosides or shorter, may be improved by the addition of five bicyclic nucleosides at the 3′-most 5′-wing nucleoside. In certain embodiments discussed above, the bicyclic nucleosides at the 3′-most 5′-wing nucleoside are selected from among cEt, cMOE, LNA, α-LNA, ENA and 2′-thio LNA. In certain embodiments discussed above, the bicyclic nucleosides at the 3′-most 5′-wing nucleoside comprise cEt. In certain embodiments discussed above, the bicyclic nucleosides at the 3′-most 5′-wing nucleoside comprise LNA.
  • Antisense compounds having certain specified motifs have enhanced selectivity, including, but not limited to motifs described above. In certain embodiments, enhanced selectivity is achieved by oligonucleotides comprising any one or more of:
  • a modification motif comprising a long 5′-wing (longer than 5, 6, or 7 nucleosides);
  • a modification motif comprising a long 3′-wing (longer than 5, 6, or 7 nucleosides);
  • a modification motif comprising a short gap region (shorter than 8, 7, or 6 nucleosides); and
  • a modification motif comprising an interrupted gap region (having no uninterrupted stretch of unmodified 2′-deoxynucleosides longer than 7, 6 or 5).
  • i. Certain Selective Nucleobase Sequence Elements
  • In certain embodiments, selective antisense compounds comprise nucleobase sequence elements. Such nucleobase sequence elements are independent of modification motifs. Accordingly, oligonucleotides having any of the motifs (modification motifs, nucleoside motifs, sugar motifs, nucleobase modification motifs, and/or linkage motifs) may also comprise one or more of the following nucleobase sequence elements.
  • ii. Alignment of Differentiating Nucleobase/Target-Selective Nucleoside
  • In certain embodiments, a target region and a region of a non-target nucleic acid differ by 1-4 differentiating nucleobase. In such embodiments, selective antisense compounds have a nucleobase sequence that aligns with the non-target nucleic acid with 1-4 mismatches. A nucleoside of the antisense compound that corresponds to a differentiating nucleobase of the target nucleic acid is referred to herein as a target-selective nucleoside. In certain embodiments, selective antisense compounds having a gapmer motif align with a non-target nucleic acid, such that a target-selective nucleoside is positioned in the gap. In certain embodiments, a target-selective nucleoside is the 1st nucleoside of the gap from the 5′ end. In certain embodiments, a target-selective nucleoside is the 2nd nucleoside of the gap from the 5′ end. In certain embodiments, a target-selective nucleoside is the 3rd nucleoside of the gap from the 5′-end. In certain embodiments, a target-selective nucleoside is the 4th nucleoside of the gap from the 5′-end. In certain embodiments, a target-selective nucleoside is the 5th nucleoside of the gap from the 5′-end. In certain embodiments, a target-selective nucleoside is the 6rd nucleoside of the gap from the 5′-end. In certain embodiments, a target-selective nucleoside is the 8th nucleoside of the gap from the 3′-end. In certain embodiments, a target-selective nucleoside is the 7th nucleoside of the gap from the 3′-end. In certain embodiments, a target-selective nucleoside is the 6th nucleoside of the gap from the 3′-end. In certain embodiments, a target-selective nucleoside is the 5th nucleoside of the gap from the 3′-end. In certain embodiments, a target-selective nucleoside is the 4th nucleoside of the gap from the 3′-end. In certain embodiments, a target-selective nucleoside is the 3rd nucleoside of the gap from the 3′-end. In certain embodiments, a target-selective nucleoside is the 2nd nucleoside of the gap from the 3′-end.
  • In certain embodiments, a target-selective nucleoside comprises a modified nucleoside. In certain embodiments, a target-selective nucleoside comprises a modified sugar. In certain embodiments, a target-selective nucleoside comprises a sugar surrogate. In certain embodiments, a target-selective nucleoside comprises a sugar surrogate selected from among HNA and F-HNA. In certain embodiments, a target-selective nucleoside comprises a 2′-substituted sugar moiety. In certain embodiments, a target-selective nucleoside comprises a 2′-substituted sugar moiety selected from among MOE, F and (ara)-F. In certain embodiments, a target-selective nucleoside comprises a 5′-substituted sugar moiety. In certain embodiments, a target-selective nucleoside comprises a 5′-substituted sugar moiety selected from 5′-(R)-Me DNA. In certain embodiments, a target-selective nucleoside comprises a bicyclic sugar moiety. In certain embodiments, a target-selective nucleoside comprises a bicyclic sugar moiety selected from among cEt, and α-L-LNA. In certain embodiments, a target-selective nucleoside comprises a modified nucleobase. In certain embodiments, a target-selective nucleoside comprises a modified nucleobase selected from among 2-thio-thymidine and 5-propyne uridine.
  • iii. Mismatches to the Target Nucleic Acid
  • In certain embodiments, selective antisense compounds comprise one or more mismatched nucleobases relative to the target nucleic acid. In certain such embodiments, antisense activity against the target is reduced by such mismatch, but activity against the non-target is reduced by a greater amount. Thus, in certain embodiments selectivity is improved. Any nucleobase other than the differentiating nucleobase is suitable for a mismatch. In certain embodiments, however, the mismatch is specifically positioned within the gap of an oligonucleotide having a gapmer motif. In certain embodiments, a mismatch relative to the target nucleic acid is at positions 1, 2, 3, 4, 5, 6, 7, or 8 from the 5′-end of the gap region. In certain embodiments, a mismatch relative to the target nucleic acid is at positions 9, 8, 7, 6, 5, 4, 3, 2, 1 of the antisense compounds from the 3′-end of the gap region. In certain embodiments, a mismatch relative to the target nucleic acid is at positions 1, 2, 3, or 4 of the antisense compounds from the 5′-end of the wing region. In certain embodiments, a mismatch relative to the target nucleic acid is at positions 4, 3, 2, or 1 of the antisense compounds from the 3′-end of the wing region.
  • iv. Self Complementary Regions
  • In certain embodiments, selective antisense compounds comprise a region that is not complementary to the target. In certain embodiments, such region is complementary to another region of the antisense compound. Such regions are referred to herein as self-complementary regions. For example, in certain embodiments, an antisense compound has a first region at one end that is complementary to a second region at the other end. In certain embodiments, one of the first and second regions is complementary to the target nucleic acid. Unless the target nucleic acid also includes a self-complementary region, the other of the first and second region of the antisense compound will not be complementary to the target nucleic acid. For illustrative purposes, certain antisense compounds have the following nucleobase motif:
  • ABCXXXXXXXXXC′B′A′;
    ABCXXXXXXX(X/C′)(X/B′)(X/A′);
    (X/A)(X/B)(X/C)XXXXXXXXXC′B′A′

    where each of A, B, and C are any nucleobase; A′, B′, and C′ are the complementary bases to A, B, and C, respectively; each X is a nucleobase complementary to the target nucleic acid; and two letters in parentheses (e.g., (X/C′)) indicates that the nucleobase is complementary to the target nucleic acid and to the designated nucleoside within the antisense oligonucleotide.
  • Without being bound to any mechanism, in certain embodiments, such antisense compounds are expected to form self-structure, which is disrupted upon contact with a target nucleic acid. Contact with a non-target nucleic acid is expected to disrupt the self-structure to a lesser degree, thus increasing selectivity compared to the same antisense compound lacking the self-complementary regions.
  • v. Combinations of Features
  • Though it is clear to one of skill in the art, the above motifs and other elements for increasing selectivity may be used alone or in combination. For example, a single antisense compound may include any one, two, three, or more of: self-complementary regions, a mismatch relative to the target nucleic acid, a short nucleoside gap, an interrupted gap, and specific placement of the selective nucleoside.
    • D. CERTAIN SHORT GAP ANTISENSE COMPOUNDS
  • In certain embodiments, an antisense compound of interest may modulate the expression of a target nucleic acid but possess undesirable properties. In certain embodiments, for example, an antisense compound of interest may have an undesirably high affinity for one or more non-target nucleic acids. In certain embodiments, whether as a result of such affinity for one or more non-target nucleic acid or by some other mechanism, an antisense compound of interest may produce undesirable increases in ALT and/or AST levels when administered to an animal. In certain embodiments, such an antisense compound of interest may produce undesirable increases in organ weight.
  • In certain such embodiments wherein an antisense compound of interest effectively modulates the expression of a target nucleic acid, but possess one or more undesirable properties, a person having skill in the art may selectively incorporate one or more modifications into the antisense compound of interest that retain some or all of the desired property of effective modulation of expression of a target nucleic acid while reducing one or more of the antisense compound's undesirable properties. In certain embodiments, the present invention provides methods of altering such an antisense compound of interest to form an improved antisense compound. In certain embodiments, altering the number of nucleosides in the 5′-region, the 3′-region, and/or the central region of such an antisense compound of interest results in improved properties. For example, in certain embodiments, one may alter the modification state of one or more nucleosides at or near the 5′-end of the central region. Having been altered, those nucleosides may then be characterized as being part of the 5′-region. Thus, in such embodiments, the overall number of nucleosides of the 5′-region is increased and the number of nucleosides in the central region is decreased. For example, an antisense compound having a modification motif of 3-10-3 could be altered to result in an improved antisense compound having a modification motif of 4-9-3 or 5-8-3. In certain embodiments, the modification state of one or more of nucleosides at or near the 3′-end of the central region may likewise be altered. In certain embodiments, the modification of one or more of the nucleosides at or near the 5′-end and the 3′-end of the central region may be altered. In such embodiments in which one or more nucleosides at or near the 5′-end and the 3′-end of the central region is altered the central region becomes shorter relative to the central region of the original antisense compound of interest. In such embodiments, the modifications to the one or more nucleosides that had been part of the central region are the same as one or more modification that had been present in the 5′-region and/or the 3′-region of the original antisense compound of interest. In certain embodiments, the improved antisense compound having a shortened central region may retain its ability to effectively modulate the expression of a target nucleic acid, but not possess some or all of the undesirable properties possessed by antisense compound of interest having a longer central region. In certain embodiments, reducing the length of the central region reduces affinity for off-target nucleic acids. In certain embodiments, reducing the length of the central region results in reduced cleavage of non-target nucleic acids by RNase H. In certain embodiments, reducing the length of the central region does not produce undesirable increases in ALT levels. In certain embodiments, reducing the length of the central region does not produce undesirable increases in AST levels. In certain embodiments, reducing the length of the central region does not produce undesirable increases organ weights.
  • In certain embodiments it is possible to retain the same nucleobase sequence and overall length of an antisense compound while decreasing the length of the central region. In certain embodiments retaining the same nucleobase sequence and overall length of an antisense compound while decreasing the length of the central region ameliorates one or more undesirable properties of an antisense compound. In certain embodiments retaining the same nucleobase sequence and overall length of an antisense compound while decreasing the length of the central region ameliorates one or more undesirable properties of an antisense compound but does not substantially affect the ability of the antisense compound to modulate expression of a target nucleic acid. In certain such embodiments, two or more antisense compounds would have the same overall length and nucleobase sequence, but would have a different central region length, and different properties. In certain embodiments, the length of the central region is 9 nucleobases. In certain embodiments, the length of the central region is 8 nucleobases. In certain embodiments, the length of the central region is 7 nucleobases. In certain embodiments, the central region consists of unmodified deoxynucleosides. In certain embodiments, the length of the central region can be decreased by increasing the length of the 5′-region, the 3′-region, or both the 5′-region and the 3′-region.
  • In certain embodiments, the length of the central region can be decreased by increasing the length of the 5′-region with modified nucleosides. In certain embodiments, the length of the central region can be decreased by increasing the length of the 5′-region with modified nucleosides. In certain embodiments, the length of the central region can be decreased by increasing the length of the 5′-region with modified nucleosides comprising a bicyclic sugar moiety selected from among: cEt, cMOE, LNA, α-LNA, ENA and 2′-thio LNA. In certain embodiments, the length of the central region can be decreased by increasing the length of the 5′-region with a cEt substituted sugar moiety.
  • In certain embodiments, the length of the central region can be decreased by increasing the length of the 5′-region with modified nucleosides. In certain embodiments, the length of the central region can be decreased by increasing the length of the 5′-region with modified nucleosides. In certain embodiments, the length of the central region can be decreased by increasing the length of the 5′-region with modified nucleosides comprising a bicyclic sugar moiety comprising a 2′ substituent selected from among: a halogen, OCH3, OCF3, OCH2CH3, OCH2CF3, OCH2—CH═CH2, O(CH2)2—OCH3 (MOE), O(CH2)2—O(CH2)2—N(CH3)2, OCH2C(═O)—N(H)CH3, OCH2C(═O)—N(H)—(CH2)2—N(CH3)2, and OCH2—N(H)—C(═NH)NH2. In certain embodiments, the length of the central region can be decreased by increasing the length of the 5′-region with 2′-O(CH2)2—OCH3 (MOE) substituted sugar moiety.
  • In certain embodiments, the length of the central region can be decreased by increasing the length of the 3′-region with modified nucleosides. In certain embodiments, the length of the central region can be decreased by increasing the length of the 3′-region with modified nucleosides. In certain embodiments, the length of the central region can be decreased by increasing the length of the 3′-region with modified nucleosides comprising a bicyclic sugar moiety selected from among: cEt, cMOE, LNA, α-LNA, ENA and 2′-thio LNA. In certain embodiments, the length of the central region can be decreased by increasing the length of the 3′-region with a cEt substituted sugar moiety.
  • In certain embodiments, the length of the central region can be decreased by increasing the length of the 3′-region with modified nucleosides. In certain embodiments, the length of the central region can be decreased by increasing the length of the 3′-region with modified nucleosides. In certain embodiments, the length of the central region can be decreased by increasing the length of the 3′-region with modified nucleosides comprising a bicyclic sugar moiety comprising a 2′ substituent selected from among: a halogen, OCH3, OCF3, OCH2CH3, OCH2CF3, OCH2—CH═CH2, O(CH2)2—OCH3 (MOE), O(CH2)2—O(CH2)2—N(CH3)2, OCH2C(═O)—N(H)CH3, OCH2C(═O)—N(H)—(CH2)2—N(CH3)2, and OCH2—N(H)—C(═NH)NH2. In certain embodiments, the length of the central region can be decreased by increasing the length of the 3′-region with 2′-O(CH2)2—OCH3 (MOE) substituted sugar moiety.
  • In certain embodiments, the length of the central region can be decreased by increasing the length of the 5′-region with modified nucleosides and increasing the length of the 3′-region with modified nucleosides.
    • E. CERTAIN TARGET NUCLEIC ACIDS
  • In certain embodiments, antisense compounds comprise or consist of an oligonucleotide comprising a region that is complementary to a target nucleic acid. In certain embodiments, the target nucleic acid is an endogenous RNA molecule. In certain embodiments, the target nucleic acid is a non-coding RNA. In certain such embodiments, the target non-coding RNA is selected from: a long-non-coding RNA, a short non-coding RNA, an intronic RNA molecule, a snoRNA, a scaRNA, a microRNA (including pre-microRNA and mature microRNA), a ribosomal RNA, and promoter directed RNA. In certain embodiments, the target nucleic acid encodes a protein. In certain such embodiments, the target nucleic acid is selected from: an mRNA and a pre-mRNA, including intronic, exonic and untranslated regions. In certain embodiments, oligomeric compounds are at least partially complementary to more than one target nucleic acid. For example, antisense compounds of the present invention may mimic microRNAs, which typically bind to multiple targets.
  • In certain embodiments, the target nucleic acid is a nucleic acid other than a mature mRNA. In certain embodiments, the target nucleic acid is a nucleic acid other than a mature mRNA or a microRNA. In certain embodiments, the target nucleic acid is a non-coding RNA other than a microRNA. In certain embodiments, the target nucleic acid is a non-coding RNA other than a microRNA or an intronic region of a pre-mRNA. In certain embodiments, the target nucleic acid is a long non-coding RNA. In certain embodiments, the target RNA is an mRNA. In certain embodiments, the target nucleic acid is a pre-mRNA. In certain such embodiments, the target region is entirely within an intron. In certain embodiments, the target region spans an intron/exon junction. In certain embodiments, the target region is at least 50% within an intron. In certain embodiments, the target nucleic acid is selected from among non-coding RNA, including exonic regions of pre-mRNA. In certain embodiments, the target nucleic acid is a ribosomal RNA (rRNA). In certain embodiments, the target nucleic acid is a non-coding RNA associated with splicing of other pre-mRNAs. In certain embodiments, the target nucleic acid is a nuclear-retained non-coding RNA.
  • In certain embodiments, antisense compounds described herein are complementary to a target nucleic acid comprising a single-nucleotide polymorphism. In certain such embodiments, the antisense compound is capable of modulating expression of one allele of the single-nucleotide polymorphism-containing-target nucleic acid to a greater or lesser extent than it modulates another allele. In certain embodiments an antisense compound hybridizes to a single-nucleotide polymorphism-containing-target nucleic acid at the single-nucleotide polymorphism site. In certain embodiments, the target nucleic acid is a Huntingtin gene transcript. In certain embodiments, the target nucleic acid is a single-nucleotide polymorphism-containing-target nucleic acid of a Huntingtin gene transcript. In certain embodiments, the target nucleic acid is not a Huntingtin gene transcript. In certain embodiments, the target nucleic acid is a single-nucleotide polymorphism-containing-target nucleic acid of a gene transcript other than Huntingtin. In certain embodiments, the target nucleic acid is any nucleic acid other than a Huntingtin gene transcript.
  • a. Single-Nucleotide Polymorphism
  • In certain embodiments, the invention provides selective antisense compounds that have greater activity for a target nucleic acid than for a homologous or partially homologous non-target nucleic acid. In certain such embodiments, the target and non-target nucleic acids are not functionally related to one another (e.g., are transcripts from different genes). In certain embodiments, the target and not-target nucleic acids are allelic variants of one another. Certain embodiments of the present invention provide methods, compounds, and compositions for selectively inhibiting mRNA and protein expression of an allelic variant of a particular gene or DNA sequence. In certain embodiments, the allelic variant contains a single nucleotide polymorphism (SNP). In certain embodiments, a SNP is associated with a mutant allele. In certain embodiments, a mutant SNP is associated with a disease. In certain embodiments a mutant SNP is associated with a disease, but is not causative of the disease. In certain embodiments, mRNA and protein expression of a mutant allele is associated with disease.
  • In certain embodiments, the expressed gene product of a mutant allele results in aggregation of the mutant proteins causing disease. In certain embodiments, the expressed gene product of a mutant allele results in gain of function causing disease. In certain embodiments, genes with an autosomal dominant mutation resulting in a toxic gain of function of the protein are the APP gene encoding amyloid precursor protein involved in Alzheimer's disease (Gene, 371: 68, 2006); the PrP gene encoding prion protein involved in Creutzfeldt-Jakob disease and in fatal familial insomnia (Nat. Med. 1997, 3: 1009); GFAP gene encoding glial fibrillary acidic protein involved in Alexander disease (J. Neurosci. 2006, 26:111623); alpha-synuclein gene encoding alpha-synuclein protein involved in Parkinson's disease (J. Clin. Invest. 2003, 111: 145); SOD-1 gene encoding the SOD-1 protein involved in amyotrophic lateral sclerosis (Science 1998, 281: 1851); atrophin-1 gene encoding atrophin-1 protein involved in dentato-rubral and pallido-luysian atrophy (DRPA) (Trends Mol. Med. 2001, 7: 479); SCA1 gene encoding ataxin-1 protein involved in spino-cerebellar ataxia-1 (SCA1) (Protein Sci. 2003, 12: 953); PLP gene encoding proteolipid protein involved in Pelizaeus-Merzbacher disease (NeuroMol. Med. 2007, 4: 73); DYT1 gene encoding torsinA protein involved in Torsion dystonia (Brain Res. 2000, 877: 379); and alpha-B crystalline gene encoding alpha-B crystalline protein involved in protein aggregation diseases, including cardiomyopathy (Cell 2007, 130: 427); alpha1-antitrypsin gene encoding alpha1-antitrypsin protein involved in chronic obstructive pulmonary disease (COPD), liver disease and hepatocellular carcinoma (New Engl J. Med. 2002, 346: 45); Ltk gene encoding leukocyte tyrosine kinase protein involved in systemic lupus erythematosus (Hum. Mol. Gen. 2004, 13: 171); PCSK9 gene encoding PCSK9 protein involved in hypercholesterolemia (Hum Mutat. 2009, 30: 520); prolactin receptor gene encoding prolactin receptor protein involved in breast tumors (Proc. Natl. Assoc. Sci. 2008, 105: 4533); CCL5 gene encoding the chemokine CCL5 involved in COPD and asthma (Eur. Respir. J. 2008, 32: 327); PTPN22 gene encoding PTPN22 protein involved in Type 1 diabetes, Rheumatoid arthritis, Graves disease, and SLE (Proc. Natl. Assoc. Sci. 2007, 104: 19767); androgen receptor gene encoding the androgen receptor protein involved in spinal and bulbar muscular atrophy or Kennedy's disease (J Steroid Biochem. Mol. Biol. 2008, 108: 245); CHMP4B gene encoding chromatin modifying protein-4B involved in progressive childhood posterior subcapsular cataracts (Am. J. Hum. Genet. 2007, 81: 596); FXR/NR1H4 gene encoding Farnesoid X receptor protein involved in cholesterol gallstone disease, arthrosclerosis and diabetes (Mol. Endocrinol. 2007, 21: 1769); ABCA1 gene encoding ABCA1 protein involved in cardiovascular disease (Transl. Res. 2007, 149: 205); CaSR gene encoding the calcium sensing receptor protein involved in primary hypercalciuria (Kidney Int. 2007, 71: 1155); alpha-globin gene encoding alpha-globin protein involved in alpha-thallasemia (Science 2006, 312: 1215); httlpr gene encoding HTTLPR protein involved in obsessive compulsive disorder (Am. J. Hum. Genet. 2006, 78: 815); AVP gene encoding arginine vasopressin protein in stress-related disorders such as anxiety disorders and comorbid depression (CNS Neurol. Disord. Drug Targets 2006, 5: 167); GNAS gene encoding G proteins involved in congenital visual defects, hypertension, metabolic syndrome (Trends Pharmacol. Sci. 2006, 27: 260); APAF1 gene encoding APAF1 protein involved in a predisposition to major depression (Mol. Psychiatry. 2006, 11: 76); TGF-beta1 gene encoding TGF-beta1 protein involved in breast cancer and prostate cancer (Cancer Epidemiol. Biomarkers Prev. 2004, 13: 759); AChR gene encoding acetylcholine receptor involved in congential myasthenic syndrome (Neurology 2004, 62: 1090); P2Y12 gene encoding adenosine diphosphate (ADP) receptor protein involved in risk of peripheral arterial disease (Circulation 2003, 108: 2971); LQT1 gene encoding LQT1 protein involved in atrial fibrillation (Cardiology 2003, 100: 109); RET protooncogene encoding RET protein involved in sporadic pheochromocytoma (J. Clin. Endocrinol. Metab. 2003, 88: 4911); filamin A gene encoding filamin A protein involved in various congenital malformations (Nat. Genet. 2003, 33: 487); TARDBP gene encoding TDP-43 protein involved in amyotrophic lateral sclerosis (Hum. Mol. Gene.t 2010, 19: 671); SCA3 gene encoding ataxin-3 protein involved in Machado-Joseph disease (PLoS One 2008, 3: e3341); SCAT gene encoding ataxin-7 protein involved in spino-cerebellar ataxia-7 (PLoS One 2009, 4: e7232); and HTT gene encoding huntingtin protein involved in Huntington's disease (Neurobiol Dis. 1996, 3:183); and the CA4 gene encoding carbonic anhydrase 4 protein, CRX gene encoding cone-rod homeobox transcription factor protein, FSCN2 gene encoding retinal fascin homolog 2 protein, IMPDH1 gene encoding inosine monophosphate dehydrogenase 1 protein, NR2E3 gene encoding nuclear receptor subfamily 2 group E3 protein, NRL gene encoding neural retina leucine zipper protein, PRPF3 (RP18) gene encoding pre-mRNA splicing factor 3 protein, PRPF8 (RP13) gene encoding pre-mRNA splicing factor 8 protein, PRPF31 (RP11) gene encoding pre-mRNA splicing factor 31 protein, RDS gene encoding peripherin 2 protein, ROM1 gene encoding rod outer membrane protein 1 protein, RHO gene encoding rhodopsin protein, RP1 gene encoding RP1 protein, RPGR gene encoding retinitis pigmentosa GTPase regulator protein, all of which are involved in Autosomal Dominant Retinitis Pigmentosa disease (Adv Exp Med. Biol. 2008, 613:203)
  • In certain embodiments, the mutant allele is associated with any disease from the group consisting of Alzheimer's disease, Creutzfeldt-Jakob disease, fatal familial insomnia, Alexander disease, Parkinson's disease, amyotrophic lateral sclerosis, dentato-rubral and pallido-luysian atrophy DRPA, spino-cerebellar ataxia, Torsion dystonia, cardiomyopathy, chronic obstructive pulmonary disease (COPD), liver disease, hepatocellular carcinoma, systemic lupus erythematosus, hypercholesterolemia, breast cancer, asthma, Type 1 diabetes, Rheumatoid arthritis, Graves disease, SLE, spinal and bulbar muscular atrophy, Kennedy's disease, progressive childhood posterior subcapsular cataracts, cholesterol gallstone disease, arthrosclerosis, cardiovascular disease, primary hypercalciuria, alpha-thallasemia, obsessive compulsive disorder, Anxiety, comorbid depression, congenital visual defects, hypertension, metabolic syndrome, prostate cancer, congential myasthenic syndrome, peripheral arterial disease, atrial fibrillation, sporadic pheochromocytoma, congenital malformations, Machado-Joseph disease, Huntington's disease, and Autosomal Dominant Retinitis Pigmentosa disease.
  • i. Certain Huntingtin Targets
  • In certain embodiments, an allelic variant of huntingtin is selectively reduced. Nucleotide sequences that encode huntingtin include, without limitation, the following: GENBANK Accession No. NT—006081.18, truncated from nucleotides 1566000 to 1768000 (replaced by GENBANK Accession No. NT—006051), incorporated herein as SEQ ID NO: 1, and NM—002111.6, incorporated herein as SEQ ID NO: 2.
  • Table 14 provides SNPs found in the GM04022, GM04281, GM02171, and GM02173B cell lines. Also provided are the allelic variants found at each SNP position, the genotype for each of the cell lines, and the percentage of HD patients having a particular allelic variant. For example, the two allelic variants for SNP rs6446723 are T and C. The GM04022 cell line is heterozygous TC, the GM02171 cell line is homozygous CC, the GM02173 cell line is heterozygous TC, and the GM04281 cell line is homozygous TT. Fifty percent of HD patients have a T at SNP position rs6446723.
  • TABLE 14
    Allelic Variations for SNPs Associated with HD
    SNP Variation GM04022 GM02171 GM02173 GM04281 TargetPOP allele
    rs6446723 T/C TC CC TC TT 0.50 T
    rs3856973 A/G AG AA AG GG 0.50 G
    rs2285086 A/G AG GG AG AA 0.50 A
    rs363092 A/C AC AA AC CC 0.49 C
    rs916171 C/G GC GG GC CC 0.49 C
    rs6844859 T/C TC CC TC TT 0.49 T
    rs7691627 A/G AG AA AG GG 0.49 G
    rs4690073 A/G AG AA AG GG 0.49 G
    rs2024115 A/G AG GG AG AA 0.48 A
    rs11731237 T/C CC CC TC TT 0.43 T
    rs362296 A/C CC AC AC AC 0.42 C
    rs10015979 A/G AA AA AG GG 0.42 G
    rs7659144 C/G CG CG CG CC 0.41 C
    rs363096 T/C CC CC TC TT 0.40 T
    rs362273 A/G AA AG AG AA 0.39 A
    rs16843804 T/C CC TC TC CC 0.38 C
    rs362271 A/G GG AG AG GG 0.38 G
    rs362275 T/C CC TC TC CC 0.38 C
    rs3121419 T/C CC TC TC CC 0.38 C
    rs362272 A/G GG — AG GG 0.38 G
    rs3775061 A/G AA AG AG AA 0.38 A
    rs34315806 T/C CC TC TC CC 0.38 C
    rs363099 T/C CC TC TC CC 0.38 C
    rs2298967 T/C TT TC TC TT 0.38 T
    rs363088 A/T AA TA TA AA 0.38 A
    rs363064 T/C CC TC TC CC 0.35 C
    rs363102 A/G AG AA AA AA 0.23 G
    rs2798235 A/G AG GG GG GG 0.21 A
    rs363080 T/C TC CC CC CC 0.21 T
    rs363072 A/T TA TA AA AA 0.13 A
    rs363125 A/C AC AC CC CC 0.12 C
    rs362303 T/C TC TC CC CC 0.12 C
    rs362310 T/C TC TC CC CC 0.12 C
    rs10488840 A/G AG AG GG GG 0.12 G
    rs362325 T/C TC TC TT TT 0.11 T
    rs35892913 A/G GG GG GG GG 0.10 A
    rs363102 A/G AG AA AA AA 0.09 A
    rs363096 T/C CC CC TC TT 0.09 C
    rs11731237 T/C CC CC TC TT 0.09 C
    rs10015979 A/G AA AA AG GG 0.08 A
    rs363080 T/C TC CC CC CC 0.07 C
    rs2798235 A/G AG GG GG GG 0.07 G
    rs1936032 C/G GC CC CC CC 0.06 C
    rs2276881 A/G GG GG GG GG 0.06 G
    rs363070 A/G AA AA AA AA 0.06 A
    rs35892913 A/G GG GG GG GG 0.04 G
    rs12502045 T/C CC CC CC CC 0.04 C
    rs6446723 T/C TC CC TC TT 0.04 C
    rs7685686 A/G AG GG AG AA 0.04 G
    rs3733217 T/C CC CC CC CC 0.03 C
    rs6844859 T/C TC CC TC TT 0.03 C
    rs362331 T/C TC CC TC TT 0.03 C
    • F. CERTAIN INDICATIONS
  • In certain embodiments, provided herein are methods of treating an animal or individual comprising administering one or more pharmaceutical compositions as described herein. In certain embodiments, the individual or animal has Huntington's disease.
  • In certain embodiments, compounds targeted to huntingtin as described herein may be administered to reduce the severity of physiological symptoms of Huntington's disease. In certain embodiments, compounds targeted to huntingtin as described herein may be administered to reduce the rate of degeneration in an individual or an animal having Huntington's disease. In certain embodiments, compounds targeted to huntingtin as described herein may be administered regeneration function in an individual or an animal having Huntington's disease. In certain embodiments, symptoms of Huntingtin's disease may be reversed by treatment with a compound as described herein.
  • In certain embodiments, compounds targeted to huntingtin as described herein may be administered to ameliorate one or more symptoms of Huntington's disease. In certain embodiments administration of compounds targeted to huntingtin as described herein may improve the symptoms of Huntington's disease as measured by any metric known to those having skill in the art. In certain embodiments, administration of compounds targeted to huntingtin as described herein may improve a rodent's rotaraod assay performance. In certain embodiments, administration of compounds targeted to huntingtin as described herein may improve a rodent's plus maze assay. In certain embodiments, administration of compounds targeted to huntingtin as described herein may improve a rodent's open field assay performance.
  • Accordingly, provided herein are methods for ameliorating a symptom associated with Huntington's disease in a subject in need thereof. In certain embodiments, provided is a method for reducing the rate of onset of a symptom associated with Huntington's disease. In certain embodiments, provided is a method for reducing the severity of a symptom associated with Huntington's disease. In certain embodiments, provided is a method for regenerating neurological function as shown by an improvement of a symptom associated with Huntington's disease. In such embodiments, the methods comprise administering to an individual or animal in need thereof a therapeutically effective amount of a compound targeted to a huntingtin nucleic acid.
  • Huntington's disease is characterized by numerous physical, neurological, psychiatric, and/or peripheral symptoms. Any symptom known to one of skill in the art to be associated with Huntington's disease can be ameliorated or otherwise modulated as set forth above in the methods described above. In certain embodiments, the symptom is a physical symptom selected from the group consisting of restlessness, lack of coordination, unintentionally initiated motions, unintentionally uncompleted motions, unsteady gait, chorea, rigidity, writhing motions, abnormal posturing, instability, abnormal facial expressions, difficulty chewing, difficulty swallowing, difficulty speaking, seizure, and sleep disturbances. In certain embodiments, the symptom is a cognitive symptom selected from the group consisting of impaired planning, impaired flexibility, impaired abstract thinking, impaired rule acquisition, impaired initiation of appropriate actions, impaired inhibition of inappropriate actions, impaired short-term memory, impaired long-term memory, paranoia, disorientation, confusion, hallucination and dementia. In certain embodiments, the symptom is a psychiatric symptom selected from the group consisting of anxiety, depression, blunted affect, egocentrisms, aggression, compulsive behavior, irritability and suicidal ideation. In certain embodiments, the symptom is a peripheral symptom selected from the group consisting of reduced brain mass, muscle atrophy, cardiac failure, impaired glucose tolerance, weight loss, osteoporosis, and testicular atrophy.
  • In certain embodiments, the symptom is restlessness. In certain embodiments, the symptom is lack of coordination. In certain embodiments, the symptom is unintentionally initiated motions. In certain embodiments, the symptom is unintentionally uncompleted motions. In certain embodiments, the symptom is unsteady gait. In certain embodiments, the symptom is chorea. In certain embodiments, the symptom is rigidity. In certain embodiments, the symptom is writhing motions. In certain embodiments, the symptom is abnormal posturing. In certain embodiments, the symptom is instability. In certain embodiments, the symptom is abnormal facial expressions. In certain embodiments, the symptom is difficulty chewing. In certain embodiments, the symptom is difficulty swallowing. In certain embodiments, the symptom is difficulty speaking. In certain embodiments, the symptom is seizures. In certain embodiments, the symptom is sleep disturbances.
  • In certain embodiments, the symptom is impaired planning. In certain embodiments, the symptom is impaired flexibility. In certain embodiments, the symptom is impaired abstract thinking. In certain embodiments, the symptom is impaired rule acquisition. In certain embodiments, the symptom is impaired initiation of appropriate actions. In certain embodiments, the symptom is impaired inhibition of inappropriate actions. In certain embodiments, the symptom is impaired short-term memory. In certain embodiments, the symptom is impaired long-term memory. In certain embodiments, the symptom is paranoia. In certain embodiments, the symptom is disorientation. In certain embodiments, the symptom is confusion. In certain embodiments, the symptom is hallucination. In certain embodiments, the symptom is dementia.
  • In certain embodiments, the symptom is anxiety. In certain embodiments, the symptom is depression. In certain embodiments, the symptom is blunted affect. In certain embodiments, the symptom is egocentrism. In certain embodiments, the symptom is aggression. In certain embodiments, the symptom is compulsive behavior. In certain embodiments, the symptom is irritability. In certain embodiments, the symptom is suicidal ideation.
  • In certain embodiments, the symptom is reduced brain mass. In certain embodiments, the symptom is muscle atrophy. In certain embodiments, the symptom is cardiac failure. In certain embodiments, the symptom is impaired glucose tolerance. In certain embodiments, the symptom is weight loss. In certain embodiments, the symptom is osteoporosis. In certain embodiments, the symptom is testicular atrophy.
  • In certain embodiments, symptoms of Huntington's disease may be quantifiable. For example, osteoporosis may be measured and quantified by, for example, bone density scans. For such symptoms, in certain embodiments, the symptom may be reduced by about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two of these values.
  • In certain embodiments, provided are methods of treating an individual comprising administering one or more pharmaceutical compositions as described herein. In certain embodiments, the individual has Huntington's disease.
  • In certain embodiments, administration of an antisense compound targeted to a huntingtin nucleic acid results in reduction of huntingtin expression by at least about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two of these values.
  • In certain embodiments, pharmaceutical compositions comprising an antisense compound targeted to huntingtin are used for the preparation of a medicament for treating a patient suffering or susceptible to Huntington's disease.
    • G. CERTAIN PHARMACEUTICAL COMPOSITIONS
  • In certain embodiments, the present invention provides pharmaceutical compositions comprising one or more antisense compound. In certain embodiments, such pharmaceutical composition comprises a suitable pharmaceutically acceptable diluent or carrier. In certain embodiments, a pharmaceutical composition comprises a sterile saline solution and one or more antisense compound. In certain embodiments, such pharmaceutical composition consists of a sterile saline solution and one or more antisense compound. In certain embodiments, the sterile saline is pharmaceutical grade saline. In certain embodiments, a pharmaceutical composition comprises one or more antisense compound and sterile water. In certain embodiments, a pharmaceutical composition consists of one or more antisense compound and sterile water. In certain embodiments, the sterile saline is pharmaceutical grade water. In certain embodiments, a pharmaceutical composition comprises one or more antisense compound and phosphate-buffered saline (PBS). In certain embodiments, a pharmaceutical composition consists of one or more antisense compound and sterile phosphate-buffered saline (PBS). In certain embodiments, the sterile saline is pharmaceutical grade PBS.
  • In certain embodiments, antisense compounds may be admixed with pharmaceutically acceptable active and/or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions depend on a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.
  • Pharmaceutical compositions comprising antisense compounds encompass any pharmaceutically acceptable salts, esters, or salts of such esters. In certain embodiments, pharmaceutical compositions comprising antisense compounds comprise one or more oligonucleotide which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.
  • A prodrug can include the incorporation of additional nucleosides at one or both ends of an oligomeric compound which are cleaved by endogenous nucleases within the body, to form the active antisense oligomeric compound.
  • Lipid moieties have been used in nucleic acid therapies in a variety of methods. In certain such methods, the nucleic acid is introduced into preformed liposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids. In certain methods, DNA complexes with mono- or poly-cationic lipids are formed without the presence of a neutral lipid. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to a particular cell or tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to fat tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to muscle tissue.
  • In certain embodiments, pharmaceutical compositions provided herein comprise one or more modified oligonucleotides and one or more excipients. In certain such embodiments, excipients are selected from water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose and polyvinylpyrrolidone.
  • In certain embodiments, a pharmaceutical composition provided herein comprises a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful for preparing certain pharmaceutical compositions including those comprising hydrophobic compounds. In certain embodiments, certain organic solvents such as dimethylsulfoxide are used.
  • In certain embodiments, a pharmaceutical composition provided herein comprises one or more tissue-specific delivery molecules designed to deliver the one or more pharmaceutical agents of the present invention to specific tissues or cell types. For example, in certain embodiments, pharmaceutical compositions include liposomes coated with a tissue-specific antibody.
  • In certain embodiments, a pharmaceutical composition provided herein comprises a co-solvent system. Certain of such co-solvent systems comprise, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. In certain embodiments, such co-solvent systems are used for hydrophobic compounds. A non-limiting example of such a co-solvent system is the VPD co-solvent system, which is a solution of absolute ethanol comprising 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80™ and 65% w/v polyethylene glycol 300. The proportions of such co-solvent systems may be varied considerably without significantly altering their solubility and toxicity characteristics. Furthermore, the identity of co-solvent components may be varied: for example, other surfactants may be used instead of Polysorbate 80™; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.
  • In certain embodiments, a pharmaceutical composition provided herein is prepared for oral administration. In certain embodiments, pharmaceutical compositions are prepared for buccal administration.
  • In certain embodiments, a pharmaceutical composition is prepared for administration by injection (e.g., intravenous, subcutaneous, intramuscular, etc.). In certain of such embodiments, a pharmaceutical composition comprises a carrier and is formulated in aqueous solution, such as water or physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. In certain embodiments, other ingredients are included (e.g., ingredients that aid in solubility or serve as preservatives). In certain embodiments, injectable suspensions are prepared using appropriate liquid carriers, suspending agents and the like. Certain pharmaceutical compositions for injection are presented in unit dosage form, e.g., in ampoules or in multi-dose containers. Certain pharmaceutical compositions for injection are suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Certain solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes. Aqueous injection suspensions may contain.
    • H. ADMINISTRATION
  • In certain embodiments, the compounds and compositions as described herein are administered parenterally.
  • In certain embodiments, parenteral administration is by infusion. Infusion can be chronic or continuous or short or intermittent. In certain embodiments, infused pharmaceutical agents are delivered with a pump. In certain embodiments, parenteral administration is by injection.
  • In certain embodiments, compounds and compositions are delivered to the CNS. In certain embodiments, compounds and compositions are delivered to the cerebrospinal fluid. In certain embodiments, compounds and compositions are administered to the brain parenchyma. In certain embodiments, compounds and compositions are delivered to an animal by intrathecal administration, or intracerebroventricular administration. Broad distribution of compounds and compositions, described herein, within the central nervous system may be achieved with intraparenchymal administration, intrathecal administration, or intracerebroventricular administration.
  • In certain embodiments, parenteral administration is by injection. The injection may be delivered with a syringe or a pump. In certain embodiments, the injection is a bolus injection. In certain embodiments, the injection is administered directly to a tissue, such as striatum, caudate, cortex, hippocampus and cerebellum.
  • Therefore, in certain embodiments, delivery of a compound or composition described herein can affect the pharmacokinetic profile of the compound or composition. In certain embodiments, injection of a compound or composition described herein, to a targeted tissue improves the pharmacokinetic profile of the compound or composition as compared to infusion of the compound or composition. In a certain embodiment, the injection of a compound or composition improves potency compared to broad diffusion, requiring less of the compound or composition to achieve similar pharmacology. In certain embodiments, similar pharmacology refers to the amount of time that a target mRNA and/or target protein is down-regulated (e.g. duration of action). In certain embodiments, methods of specifically localizing a pharmaceutical agent, such as by bolus injection, decreases median effective concentration (EC50) by a factor of about 50 (e.g. 50 fold less concentration in tissue is required to achieve the same or similar pharmacodynamic effect). In certain embodiments, methods of specifically localizing a pharmaceutical agent, such as by bolus injection, decreases median effective concentration (EC50) by a factor of 20, 25, 30, 35, 40, 45 or 50. In certain embodiments the pharmaceutical agent in an antisense compound as further described herein. In certain embodiments, the targeted tissue is brain tissue. In certain embodiments the targeted tissue is striatal tissue. In certain embodiments, decreasing EC50 is desirable because it reduces the dose required to achieve a pharmacological result in a patient in need thereof.
  • In certain embodiments, an antisense oligonucleotide is delivered by injection or infusion once every month, every two months, every 90 days, every 3 months, every 6 months, twice a year or once a year.
    • I. CERTAIN COMBINATION THERAPIES
  • In certain embodiments, one or more pharmaceutical compositions are co-administered with one or more other pharmaceutical agents. In certain embodiments, such one or more other pharmaceutical agents are designed to treat the same disease, disorder, or condition as the one or more pharmaceutical compositions described herein. In certain embodiments, such one or more other pharmaceutical agents are designed to treat a different disease, disorder, or condition as the one or more pharmaceutical compositions described herein. In certain embodiments, such one or more other pharmaceutical agents are designed to treat an undesired side effect of one or more pharmaceutical compositions as described herein. In certain embodiments, one or more pharmaceutical compositions are co-administered with another pharmaceutical agent to treat an undesired effect of that other pharmaceutical agent. In certain embodiments, one or more pharmaceutical compositions are co-administered with another pharmaceutical agent to produce a combinational effect. In certain embodiments, one or more pharmaceutical compositions are co-administered with another pharmaceutical agent to produce a synergistic effect.
  • In certain embodiments, one or more pharmaceutical compositions and one or more other pharmaceutical agents are administered at the same time. In certain embodiments, one or more pharmaceutical compositions and one or more other pharmaceutical agents are administered at different times. In certain embodiments, one or more pharmaceutical compositions and one or more other pharmaceutical agents are prepared together in a single formulation. In certain embodiments, one or more pharmaceutical compositions and one or more other pharmaceutical agents are prepared separately.
  • In certain embodiments, pharmaceutical agents that may be co-administered with a pharmaceutical composition of include antipsychotic agents, such as, e.g., haloperidol, chlorpromazine, clozapine, quetiapine, and olanzapine; antidepressant agents, such as, e.g., fluoxetine, sertraline hydrochloride, venlafaxine and nortriptyline; tranquilizing agents such as, e.g., benzodiazepines, clonazepam, paroxetine, venlafaxin, and beta-blockers; mood-stabilizing agents such as, e.g., lithium, valproate, lamotrigine, and carbamazepine; paralytic agents such as, e.g., Botulinum toxin; and/or other experimental agents including, but not limited to, tetrabenazine (Xenazine), creatine, coenzyme Q10, trehalose, docosahexanoic acids, ACR16, ethyl-EPA, atomoxetine, citalopram, dimebon, memantine, sodium phenylbutyrate, ramelteon, ursodiol, zyprexa, xenasine, tiapride, riluzole, amantadine, [123I]MNI-420, atomoxetine, tetrabenazine, digoxin, detromethorphan, warfarin, alprozam, ketoconazole, omeprazole, and minocycline.
    • Nonlimiting Disclosure and Incorporation by Reference
  • While certain compounds, compositions and methods described herein have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds described herein and are not intended to limit the same. Each of the references, GenBank accession numbers, and the like recited in the present application is incorporated herein by reference in its entirety.
  • Although the sequence listing accompanying this filing identifies each sequence as either “RNA” or “DNA” as required, in reality, those sequences may be modified with any combination of chemical modifications. One of skill in the art will readily appreciate that such designation as “RNA” or “DNA” to describe modified oligonucleotides is, in certain instances, arbitrary. For example, an oligonucleotide comprising a nucleoside comprising a 2′-OH sugar moiety and a thymine base could be described as a DNA having a modified sugar (2′-OH for the natural 2′-H of DNA) or as an RNA having a modified base (thymine (methylated uracil) for natural uracil of RNA).
  • Accordingly, nucleic acid sequences provided herein, including, but not limited to those in the sequence listing, are intended to encompass nucleic acids containing any combination of natural or modified RNA and/or DNA, including, but not limited to such nucleic acids having modified nucleobases. By way of further example and without limitation, an oligomeric compound having the nucleobase sequence “ATCGATCG” encompasses any oligomeric compounds having such nucleobase sequence, whether modified or unmodified, including, but not limited to, such compounds comprising RNA bases, such as those having sequence “AUCGAUCG” and those having some DNA bases and some RNA bases such as “AUCGATCG” and oligomeric compounds having other modified or naturally occurring bases, such as “ATmeCGAUCG,” wherein meC indicates a cytosine base comprising a methyl group at the 5-position.
    • EXAMPLES
  • The following examples illustrate certain embodiments of the present invention and are not limiting. Moreover, where specific embodiments are provided, the inventors have contemplated generic application of those specific embodiments. For example, disclosure of an oligonucleotide having a particular motif provides reasonable support for additional oligonucleotides having the same or similar motif. And, for example, where a particular high-affinity modification appears at a particular position, other high-affinity modifications at the same position are considered suitable, unless otherwise indicated.
  • To allow assessment of the relative effects of nucleobase sequence and chemical modification, throughout the examples, oligomeric compounds are assigned a “Sequence Code.” Oligomeric compounds having the same Sequence Code have the same nucleobase sequence. Oligomeric compounds having different Sequence Codes have different nucleobase sequences.
    • Example 1 Modified Antisense Oligonucleotides Targeting Human Target-X
  • Antisense oligonucleotides were designed targeting a Target-X nucleic acid and were tested for their effects on Target-X mRNA in vitro. ISIS 407939, which was described in an earlier publication (WO 2009/061851) was also tested.
  • The newly designed chimeric antisense oligonucleotides and their motifs are described in Table 15. The internucleoside linkages throughout each gapmer are phosphorothioate linkages (P═S). Nucleosides followed by “d” indicate 2′-deoxyribonucleosides. Nucleosides followed by “k” indicate 6′-(S)—CH3 bicyclic nucleoside (e.g cEt) nucleosides. Nucleosides followed by “e” indicate 2′-O-methoxyethyl (2′-MOE) nucleosides. “N” indicates modified or naturally occurring nucleobases (A, T, C, G, U, or 5-methyl C).
  • Each gapmer listed in Table 15 is targeted to the human Target-X genomic sequence.
  • Activity of the newly designed gapmers was compared to a 5-10-5 2′-MOE gapmer, ISIS 407939 targeting human Target-X and is further described in USPN XXX, incorporated herein by reference. Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 2,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS2927 was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells, and indicate that several of the newly designed antisense oligonucleotides are more potent than ISIS 407939. A total of 771 oligonucleotides were tested. Only those oligonucleotides which were selected for further studies are shown in Table 15. Each of the newly designed antisense oligonucleotides provided in Table 1 achieved greater than 80% inhibition and, therefore, are more active than ISIS 407939.
  • TABLE 15
    Inhibition of human Target-X mRNA levels by chimeric antisense
    oligonucleotides targeted to Target-X
    Wing SEQ
    ISIS % Gap Chemistry SEQ ID
    Sequence (5′ to 3′) NO inhibition Motif Chemistry 5′ 3′ CODE NO
    NkNkNkNdNdNdNdNkNd 473359  92 3-10-3 Deoxy/ kkk eee 21 6
    NdNdNdNdNeNeNe cEt
    NkNkNkNdNdNdNdNkNd 473360  96 3-10-3 Deoxy/ kkk eee 22 6
    NdNdNdNdNeNeNe cEt
    NkNkNkNdNdNdNdNdNd 473168  94 3-10-3 Full kkk kkk 23 6
    NdNdNdNdNkNkNk deoxy
    NkNkNkNdNdNdNdNdNd 473317  95 3-10-3 Full kkk eee 23 6
    NdNdNdNdNeNeNe deoxy
    NkNkNkNdNdNdNdNkNd 473471  90 3-10-3 Deoxy/ kkk eee 23 6
    NdNdNdNdNeNeNe cEt
    NkNdNkNdNkNdNdNdNd 473620  94 5-9-2 Full kdkdk ee 23 6
    NdNdNdNdNdNeNe deoxy
    NkNkNdNdNdNdNdNdNd 473019  88 2-10-2 Full kk kk 24 7
    NdNdNdNkNk deoxy
    NkNkNdNdNdNdNdNdNd 473020  93 2-10-2 Full kk kk 25 7
    NdNdNdNkNk deoxy
    NkNkNkNdNdNdNdNdNd 473321  93 3-10-3 Full kkk eee 26 6
    NdNdNdNdNeNeNe deoxy
    NkNkNkNdNdNdNdNdNd 473322  94 3-10-3 Full kkk eee 27 6
    NdNdNdNdNeNeNe deoxy
    NkNkNkNdNdNdNdNdNd 473323  96 3-10-3 Full kkk eee 28 6
    NdNdNdNdNeNeNe deoxy
    NkNkNkNdNdNdNdNdNd 473326  94 3-10-3 Full kkk eee 29 6
    NdNdNdNdNeNeNe deoxy
    NkNkNkNdNdNdNdNkNd 473480  92 3-10-3 Deoxy/ kkk eee 29 6
    NdNdNdNdNeNeNe cEt
    NkNkNkNdNdNdNdNdNd 473178  96 3-10-3 Full kkk kkk 30 6
    NdNdNdNdNkNkNk deoxy
    NkNkNkNdNdNdNdNdNd 473327  96 3-10-3 Full kkk eee 30 6
    NdNdNdNdNeNeNe deoxy
    NkNkNkNdNdNdNdNkNd 473481  93 3-10-3 Deoxy/ kkk eee 30 6
    NdNdNdNdNeNeNe cEt
    NkNdNkNdNkNdNdNdNd 473630  89 5-9-2 Full kdkdk ee 30 6
    NdNdNdNdNdNeNe deoxy
    NkNkNdNdNdNdNdNdNd 473029  96 2-10-2 Full kk kk 31 7
    NdNdNdNkNk deoxy
    NkNkNdNdNdNdNdNdNd 472925  93 2-10-2 Full kk kk 32 7
    NdNdNdNkNk deoxy
    NkNkNdNdNdNdNdNdNd 472926  85 2-10-2 Full kk kk 33 7
    NdNdNdNkNk deoxy
    NkNkNkNdNdNdNdNdNd 473195  97 3-10-3 Full kkk kkk 34 6
    NdNdNdNdNkNkNk deoxy
    NkNkNdNdNdNdNdNdNd 473046  90 2-10-2 Full kk kk 35 7
    NdNdNdNkNk deoxy
    NkNkNdNdNdNdNdNdNd 472935  92 2-10-2 Full kk kk 36 7
    NdNdNdNkNk deoxy
    NkNkNkNdNdNdNdNdNd 473089  95 3-10-3 Full kkk kkk 37 6
    NdNdNdNdNkNkNk deoxy
    NkNkNkNdNdNdNdNdNd 473350  93 3-10-3 Full kkk eee 38 6
    NdNdNdNdNeNeNe deoxy
    NkNkNkNdNdNdNdNdNd 473353  93 3-10-3 Full kkk eee 39 6
    NdNdNdNdNeNeNe deoxy
    NkNkNdNdNdNdNdNdNd 473055  91 2-10-2 Full kk kk 40 7
    NdNdNdNkNk deoxy
    NkNkNkNdNdNdNdNkNd 473392  95 3-10-3 Deoxy/ kkk eee 41 6
    NdNdNdNdNeNeNe cEt
    NkNkNkNdNdNdNdNdNd 473095 100 3-10-3 Full kkk kkk 42 6
    NdNdNdNdNkNkNk deoxy
    NkNkNkNdNdNdNdNdNd 473244  99 3-10-3 Full kkk eee 42 6
    NdNdNdNdNeNeNe deoxy
    NkNkNkNdNdNdNdNkNd 473393  99 3-10-3 Deoxy/ kkk eee 42 6
    NdNdNdNdNeNeNe cEt
    NkNdNkNdNkNdNdNdNd 473547  98 5-9-2 Full kdkdk ee 42 6
    NdNdNdNdNdNeNe deoxy
    NkNkNdNdNdNdNdNdNd 472942  87 2-10-2 Full kk kk 43 7
    NdNdNdNkNk deoxy
    NkNkNkNdNdNdNdNdNd 473098  97 3-10-3 Full kkk kkk 44 6
    NdNdNdNdNkNkNk deoxy
    NkNkNkNdNdNdNdNkNd 473408  92 3-10-3 Deoxy/ kkk eee 45 6
    NdNdNdNdNeNeNe cEt
    NkNkNdNdNdNdNdNdNd 472958  89 2-10-2 Full kk kk 46 7
    NdNdNdNkNk deoxy
    NkNkNdNdNdNdNdNdNd 472959  90 2-10-2 Full kk kk 47 7
    NdNdNdNkNk deoxy
    NkNdNkNdNkNdNdNdNd 473566  94 5-9-2 Full kdkdk ee 48 6
    NdNdNdNdNdNeNe deoxy
    NkNdNkNdNkNdNdNdNd 473567  95 5-9-2 Full kdkdk ee 49 6
    NdNdNdNdNdNeNe deoxy
    NkNdNkNdNkNdNdNdNd 473569  92 5-9-2 Full kdkdk ee 50 6
    NdNdNdNdNdNeNe deoxy
    NkNkNdNdNdNdNdNdNd 457851  90 2-10-2 Full kk kk 51 7
    NdNdNdNkNk deoxy
    NkNkNdNdNdNdNdNdNd 472970  91 2-10-2 Full kk kk 32 7
    NdNdNdNkNk deoxy
    NkNkNkNdNdNdNdNdNd 473125  90 3-10-3 Full kkk kkk 53 6
    NdNdNdNdNkNkNk deoxy
    NkNkNkNdNdNdNdNdNd 473274  98 3-10-3 Full kkk eee 53 6
    NdNdNdNdNeNeNe deoxy
    NkNkNkNdNdNdNdNkNd 473428  90 3-10-3 Deoxy/ kkk eee 53 6
    NdNdNdNdNeNeNe cEt
    NkNdNkNdNkNdNdNdNd 473577  93 5-9-2 Full kdkdk ee 53 6
    NdNdNdNdNdNeNe deoxy
    NkNkNdNdNdNdNdNdNd 472976  97 2-10-2 Full kk kk 54 7
    NdNdNdNkNk deoxy
    NkNkNdNdNdNdNdNd 472983  94 2-10-2 Full kk kk 55 7
    NdNdNdNdNkNk deoxy
    NkNkNdNdNdNdNdNd 472984  90 2-10-2 Full kk kk 56 7
    NdNdNdNdNkNk deoxy
    NkNkNkNdNdNdNdNd 473135  97 3-10-3 Full kkk kkk 57 6
    NdNdNdNdNdNkNkNk deoxy
    NkNkNdNdNdNdNdNd 472986  95 2-10-2 Full kk kk 58 7
    NdNdNdNdNkNk deoxy
    NkNkNkNdNdNdNdNd 473137  95 3-10-3 Full kkk kkk 59 6
    NdNdNdNdNdNkNkNk deoxy
    NkNkNkNdNdNdNdNd 473286  95 3-10-3 Full kkk eee 59 6
    NdNdNdNdNdNeNeNe deoxy
    NkNkNkNdNdNdNdNkNd 473440  88 3-10-3 Deoxy/ kkk eee 59 6
    NdNdNdNdNeNeNe cEt
    NkNdNkNdNkNdNdNd 473589  97 5-9-2 Full kdkdk ee 59 6
    NdNdNdNdNdNdNeNe deoxy
    NkNkNdNdNdNdNdNd 472988  85 2-10-2 Full kk kk 60 7
    NdNdNdNdNkNk deoxy
    NkNkNkNdNdNdNdNd 473140  96 3-10-3 Full kkk kkk 61 6
    NdNdNdNdNdNkNkNk deoxy
    NkNkNdNdNdNdNdNd 472991  90 2-10-2 Full kk kk 62 7
    NdNdNdNdNkNk deoxy
    NkNkNkNdNdNdNdNkNd 473444  94 3-10-3 Deoxy/ kkk eee 63 6
    NdNdNdNdNeNeNe cEt
    NkNkNkNdNdNdNdNd 473142  96 3-10-3 Full kkk kkk 64 6
    NdNdNdNdNdNkNkNk deoxy
    NkNkNkNdNdNdNdNd 473291  95 3-10-3 Full kkk eee 64 6
    NdNdNdNdNdNeNeNe deoxy
    NkNdNkNdNkNdNdNd 473594  95 5-9-2 Full kdkdk ee 64 6
    NdNdNdNdNdNdNeNe deoxy
    NkNkNkNdNdNdNdNdNd 473143  97 3-10-3 Full kkk kkk 65 6
    NdNdNdNdNkNkNk deoxy
    NkNkNkNdNdNdNdNd 473292  96 3-10-3 Full kkk eee 65 6
    NdNdNdNdNdNeNeNe deoxy
    NkNkNkNdNdNdNdNkNd 473446  96 3-10-3 Deoxy/ kkk eee 65 6
    NdNdNdNdNeNeNe cEt
    NkNdNkNdNkNdNdNdNd 473595  84 5-9-2 Full kdkdk ee 65 6
    NdNdNdNdNdNeNe deoxy
    NkNkNdNdNdNdNdNdNd 472994  96 2-10-2 Full kk kk 66 7
    NdNdNdNkNk deoxy
    NkNkNkNdNdNdNdNdNd 473144  98 3-10-3 Full kkk kkk 67 6
    NdNdNdNdNkNkNk deoxy
    NkNkNkNdNdNdNdNdNd 473293  96 3-10-3 Full kkk eee 67 6
    NdNdNdNdNeNeNe deoxy
    NkNkNdNdNdNdNdNdNd 472995  96 2-10-2 Full kk kk 68 7
    NdNdNdNkNk deoxy
    NkNkNkNdNdNdNdNd 473294  91 3-10-3 Full kkk eee 69 6
    NdNdNdNdNdNeNeNe deoxy
    NkNdNkNdNkNdNdNdNd 473597  94 5-9-2 Full kdkdk ee 69 6
    NdNdNdNdNdNeNe deoxy
    NkNkNdNdNdNdNdNdNd 472996  94 2-10-2 Full kk kk 70 7
    NdNdNdNkNk deoxy
    NkNkNkNdNdNdNdNd 473295  92 3-10-3 Full kkk eee 71 6
    NdNdNdNdNdNeNeNe deoxy
    NeNeNeNeNeNdNdNdNdNd 407939  80 5-10-5 Full eeeee eeee 72 8
    NdNdNdNdNdNeNeNeNeNe deoxy e
    NkNkNkNdNdNdNdNdNd 473296  98 3-10-3 Full kkk eee 73 6
    NdNdNdNdNeNeNe deoxy
    NkNkNkNdNdNdNdNkNd 473450  95 3-10-3 Deoxy/ kkk eee 73 6
    NdNdNdNdNeNeNe cEt
    NkNkNdNdNdNdNdNdNd 472998  97 2-10-2 Full kk kk 74 7
    NdNdNdNkNk deoxy
    e = 2′MOE, k = cEt, d = 2′-deoxyribonucleoside
    • Example 2 Modified Antisense Oligonucleotides Comprising 6′-(S)—CH3 Bicyclic Nucleoside (cEt) and F-HNA Modifications Targeting Human Target-X
  • Additional antisense oligonucleotides were designed targeting a Target-X nucleic acid and were tested for their effects on Target-X mRNA in vitro. ISIS 407939 was also tested.
  • The chimeric antisense oligonucleotides and their motifs are described in Table 16. The internucleoside linkages throughout each gapmer are phosphorothioate linkages (P═S). Nucleosides followed by “d” indicate 2′-deoxyribonucleosides. Nucleosides followed by “k” indicate 6′-(S)—CH3 bicyclic nucleosides (e.g cEt). Nucleosides followed by “e” indicate 2′-O-methoxyethyl (2′-MOE) modified nucleosides. Nucleosides followed by ‘g’ indicate F-HNA modified nucleosides. “N” indicates modified or naturally occurring nucleobases (A, T, C, G, U, or 5-methyl C).
  • Each gapmer listed in Table 16 is targeted to the human Target-X genomic sequence.
  • Activity of the newly designed gapmers was compared to a 5-10-5 2′-MOE gapmer, ISIS 407939 targeting human Target-X. Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 2,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS2927 was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells, and demonstrate that several of the newly designed gapmers are more potent than ISIS 407939. A total of 765 oligonucleotides were tested. Only those oligonucleotides which were selected for further studies are shown in Table 16. All but one of the newly designed antisense oligonucleotides provided in Table 16 achieved greater than 30% inhibition and, therefore, are more active than ISIS 407939.
  • TABLE 16
    Inhibition of human Target-X mRNA levels by chimeric antisense
    oligonucleotides targeted to Target-X
    Wing SEQ
    ISIS % Gap Chemistry SEQ ID
    Sequence (5′ to 3′) No inhibition Motif Chemistry 5′ 3′ CODE NO
    NgNgNdNdNdNdNdNdNd 482838 81 2-10-2 Full gg gg 25 7
    NdNdNdNgNg deoxy
    NgNgNgNdNdNdNdNdNd 482992 93 3-10-3 Full ggg ggg 28 6
    NdNdNdNdNgNgNg deoxy
    NgNgNgNdNdNdNdNdNd 482996 97 3-10-3 Full ggg ggg 30 6
    NdNdNdNdNgNgNg deoxy
    NgNdNgNdNgNdNdNdNd 483284 82 5-9-2 Full gdgdg ee 23 6
    NdNdNdNdNdNeNe deoxy
    NgNdNgNdNgNdNdNdNd 483289 70 5-9-2 Full gdgdg ee 27 6
    NdNdNdNdNdNeNe deoxy
    NgNdNgNdNgNdNdNdNd 483290 80 5-9-2 Full gdgdg ee 28 6
    NdNdNdNdNdNeNe deoxy
    NgNdNgNdNgNdNdNdNd 483294 69 5-9-2 Full gdgdg ee 30 6
    NdNdNdNdNdNeNe deoxy
    NgNgNdNdNdNdNdNdNd 483438 81 2-10-4 Full gg eeee 23 6
    NdNdNdNeNeNeNe deoxy
    NgNgNdNdNdNdNdNdNd 483444 84 2-10-4 Full gg eeee 28 6
    NdNdNdNeNeNeNe deoxy
    NgNgNdNdNdNdNdNdNd 483448 77 2-10-4 Full gg eeee 30 6
    NdNdNdNeNeNeNe deoxy
    NgNgNdNdNdNdNdNdNd 482847 79 2-10-2 Full gg gg 31 7
    NdNdNdNgNg deoxy
    NgNgNdNdNdNdNdNdNd 482747 85 2-10-2 Full gg gg 32 7
    NdNdNdNgNg deoxy
    NgNgNdNdNdNdNdNdNd 482873 81 2-10-2 Full gg gg 40 7
    NdNdNdNgNg deoxy
    NgNgNdNdNdNdNdNdNdNd 482874 82 2-10-2 Full gg gg 75 7
    NdNdNgNg deoxy
    NgNgNdNdNdNdNdNd 482875 82 2-10-2 Full gg gg 76 7
    NdNdNdNdNgNg deoxy
    NgNgNgNdNdNdNdNd 482896 95 3-10-3 Full ggg ggg 77 6
    NdNdNdNdNdNgNgNg deoxy
    NgNgNgNdNdNdNdNdNd 483019 89 3-10-3 Full ggg ggg 38 6
    NdNdNdNdNgNgNg deoxy
    NgNdNgNdNdNdNdNdNd 483045 92 3-10-3 Full gdg gdg 77 6
    NdNdNdNdNgNdNg deoxy
    NgNdNgNdNgNdNdNdNd 483194 64 3-10-3 Full gdg gdg 77 6
    NdNdNdNdNdNeNe deoxy
    NgNdNgNdNgNdNdNdNd 483317 79 5-9-2 Full gdgdg ee 38 6
    NdNdNdNdNdNeNe deoxy
    NgNgNdNdNdNdNdNdNd 483343 75 2-10-4 Full gg eeee 57 6
    NdNdNdNeNeNeNe deoxy
    NgNgNdNdNdNdNdNdNdNdN 483471 76 2-10-4 Full gg eeee 38 6
    dNdNeNeNeNe deoxy
    NgNgNdNdNdNdNdNdNd 483478 20 2-10-4 Full gg eeee 78 6
    NdNdNdNeNeNeNe deoxy
    NeNeNeNeNeNdNdNdNdNd 407939 30 5-10-5 Full eeeee eeeee 72 8
    NdNdNdNdNdNeNeNeNeNe deoxy
    NgNgNdNdNdNdNdNd 482784 83 2-10-2 Full gg gg 79 7
    NdNdNdNdNgNg deoxy
    NgNgNdNdNdNdNdNd 482794 91 2-10-2 Full gg gg 54 7
    NdNdNdNdNgNg deoxy
    NgNgNdNdNdNdNdNd 482804 80 2-10-2 Full gg gg 58 7
    NdNdNdNdNgNg deoxy
    NgNgNdNdNdNdNdNd 482812 81 2-10-2 Full gg gg 66 7
    NdNdNdNdNgNg deoxy
    NgNgNdNdNdNdNdNd 482813 92 2-10-2 Full gg gg 68 7
    NdNdNdNdNgNg deoxy
    NgNgNdNdNdNdNdNd 482814 94 2-10-2 Full gg gg 70 7
    NdNdNdNdNgNg deoxy
    NgNgNdNdNdNdNdNd 482815 81 2-10-2 Full gg gg 80 7
    NdNdNdNdNgNg deoxy
    NgNgNdNdNdNdNdNd 482816 71 2-10-2 Full gg gg 74 7
    NdNdNdNdNgNg deoxy
    NgNgNgNdNdNdNdNd 482916 90 3-10-3 Full ggg ggg 44 6
    NdNdNdNdNdNgNgNg deoxy
    NgNgNgNdNdNdNdNd 482932 89 3-10-3 Full ggg ggg 48 6
    NdNdNdNdNdNgNgNg deoxy
    NgNgNgNdNdNdNdNd 482953 93 3-10-3 Full ggg ggg 57 6
    NdNdNdNdNdNgNgNg deoxy
    NgNgNgNdNdNdNdNd 482962 97 3-10-3 Full ggg ggg 67 6
    NdNdNdNdNdNgNgNg deoxy
    NgNgNgNdNdNdNdNd 482963 96 3-10-3 Full ggg ggg 69 6
    NdNdNdNdNdNgNgNg deoxy
    NgNgNgNdNdNdNdNd 482965 89 3-10-3 Full ggg ggg 73 6
    NdNdNdNdNdNgNgNg deoxy
    NgNdNgNdNdNdNdNd 483065 69 3-10-3 Full ggg ggg 44 6
    NdNdNdNdNdNgNdNg deoxy
    NgNdNgNdNdNdNdNd 483092 89 3-10-3 Full gdg gdg 53 6
    NdNdNdNdNdNgNdNg deoxy
    NgNdNgNdNgNdNdNd 483241 79 5-9-2 Full gdgdg ee 53 6
    NdNdNdNdNdNdNeNe deoxy
    NgNdNgNdNgNdNdNd 483253 76 5-9-2 Full gdgdg ee 59 6
    NdNdNdNdNdNdNeNe deoxy
    NgNdNgNdNgNdNdNd 483258 70 5-9-2 Full gdgdg ee 64 6
    NdNdNdNdNdNdNeNe deoxy
    NgNdNgNdNgNdNdNd 483260 62 5-9-2 Full gdgdg ee 67 6
    NdNdNdNdNdNdNeNe deoxy
    NgNdNgNdNgNdNdNd 483261 76 5-9-2 Full gdgdg ee 69 6
    NdNdNdNdNdNdNeNe deoxy
    NgNdNgNdNgNdNdNd 483262 75 5-9-2 Full gdgdg ee 71 6
    NdNdNdNdNdNdNeNe deoxy
    NgNdNgNdNgNdNdNd 483263 73 5-9-2 Full gdgdg ee 73 6
    NdNdNdNdNdNdNeNe deoxy
    NgNgNdNdNdNdNdNd 483364 78 2-10-4 Full gg eeee 81 6
    NdNdNdNdNeNeNeNe deoxy
    NgNgNdNdNdNdNdNd 483395 86 2-10-4 Full gg eeee 53 6
    NdNdNdNdNeNeNeNe deoxy
    NgNgNdNdNdNdNdNd 483413 83 2-10-4 Full gg eeee 65 6
    NdNdNdNdNeNeNeNe deoxy
    NgNgNdNdNdNdNdNd 483414 76 2-10-4 Full gg eeee 67 6
    NdNdNdNdNeNeNeNe deoxy
    NgNgNdNdNdNdNdNd 483415 85 2-10-4 Full gg eeee 69 6
    NdNdNdNdNeNeNeNe deoxy
    NgNgNdNdNdNdNdNd 483416 77 2-10-4 Full gg eeee 71 6
    NdNdNdNdNeNeNeNe deoxy
    NgNgNdNdNdNdNdNd 483417 83 2-10-4 Full gg eeee 73 6
    NdNdNdNdNeNeNeNe deoxy
    e = 2′-MOE, d = 2′-deoxyribonucleoside, g = F-HNA
    • Example 3 Modified Antisense Oligonucleotides Comprising 2′-MOE and 6′-(S)—CH3 Bicyclic Nucleoside (e.g cEt) Modifications Targeting Human Target-X
  • Additional antisense oligonucleotides were designed targeting a Target-X nucleic acid and were tested for their effects on Target-X mRNA in vitro. ISIS 403052, ISIS 407594, ISIS 407606, ISIS 407939, and ISIS 416438, which were described in an earlier publication (WO 2009/061851) were also tested.
  • The newly designed chimeric antisense oligonucleotides are 16 nucleotides in length and their motifs are described in Table 17. The chemistry column of Table 17 presents the sugar motif of each oligonucleotide, wherein “e” indicates a 2′-O-methoxyethyl (2′-MOE) nucleoside, “k” indicates a 6′-(S)—CH3 bicyclic nucleoside (e.g cEt) and “d” indicates a 2′-deoxyribonucleoside. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosines.
  • Each gapmer listed in Table 17 is targeted to the human Target-X genomic sequence.
  • Activity of the newly designed gapmers was compared to ISIS 403052, ISIS 407594, ISIS 407606, ISIS 407939, and ISIS 416438. Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 2,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS2927 (described hereinabove in Example 1) was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of Target-X, relative to untreated control cells. A total of 380 oligonucleotides were tested. Only those oligonucleotides which were selected for further studies are shown in Table 17. Each of the newly designed antisense oligonucleotides provided in Table 17 achieved greater than 64% inhibition and, therefore, are more potent than each of ISIS 403052, ISIS 407594, ISIS 407606, ISIS 407939, and ISIS 416438.
  • TABLE 17
    Inhibition of human Target-X mRNA levels by chimeric
    antisense oligonucleotides targeted to Target-X
    ISIS No Chemistry Motif % inhibition SEQ CODE
    403052 eeeee-(d10)-eeeee 5-10-5 64 82
    407594 eeeee-(d10)-eeeee 5-10-5 40 83
    407606 eeeee-(d10)-eeeee 5-10-5 39 84
    407939 eeeee-(d10)-eeeee 5-10-5 57 72
    416438 eeeee-(d10)-eeeee 5-10-5 62 85
    484487 kdk-(d10)-dkdk 3-10-3 91 77
    484539 kdk-d(10)-kdk 3-10-3 92 53
    484546 kdk-d(10)-kdk 3-10-3 92 86
    484547 kdk-d(10)-kdk 3-10-3 89 87
    484549 kdk-d(10)-kdk 3-10-3 91 57
    484557 kdk-d(10)-kdk 3-10-3 92 65
    484558 kdk-d(10)-kdk 3-10-3 94 67
    484559 kdk-d(10)-kdk 3-10-3 90 69
    484582 kdk-d(10)-kdk 3-10-3 88 23
    484632 kk-d(10)-eeee 2-10-4 90 88
    484641 kk-d(10)-eeee 2-10-4 91 77
    484679 kk-d(10)-eeee 2-10-4 90 49
    484693 kk-d(10)-eeee 2-10-4 93 53
    484711 kk-d(10)-eeee 2-10-4 92 65
    484712 kk-d(10)-eeee 2-10-4 92 67
    484713 kk-d(10)-eeee 2-10-4 85 69
    484714 kk-d(10)-eeee 2-10-4 83 71
    484715 kk-d(10)-eeee 2-10-4 93 73
    484736 kk-d(10)-eeee 2-10-4 89 23
    484742 kk-d(10)-eeee 2-10-4 93 28
    484746 kk-d(10)-eeee 2-10-4 88 30
    484771 kk-d(10)-eeee 2-10-4 89 89
    e = 2′-MOE, k = cEt, d = 2′-deoxyribonucleoside
    • Example 4 Antisense Inhibition of Human Target-X with 5-10-5 2′-MOE Gapmers
  • Additional antisense oligonucleotides were designed targeting a Target-X nucleic acid and were tested for their effects on Target-X mRNA in vitro. Also tested were ISIS 403094, ISIS 407641, ISIS 407643, ISIS 407662, ISIS 407900, ISIS 407910, ISIS 407935, ISIS 407936, ISIS 407939, ISIS 416446, ISIS 416449, ISIS 416455, ISIS 416472, ISIS 416477, ISIS 416507, ISIS 416508, ISIS 422086, ISIS 422087, ISIS 422140, and ISIS 422142, 5-10-5 2′-MOE gapmers targeting human Target-X, which were described in an earlier publication (WO 2009/061851), incorporated herein by reference.
  • The newly designed modified antisense oligonucleotides are 20 nucleotides in length and their motifs are described in Tables 18 and 19. The chemistry column of Tables 18 and 19 present the sugar motif of each oligonucleotide, wherein “e” indicates a 2′-O-methoxyethyl (2′-MOE) nucleoside and “d” indicates a 2′-deoxyribonucleoside. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosines.
  • Each gapmer listed in Table 18 is targeted to the human Target-X genomic sequence.
  • Activity of the newly designed gapmers was compared to ISIS 403094, ISIS 407641, ISIS 407643, ISIS 407662, ISIS 407900, ISIS 407910, ISIS 407935, ISIS 407936, ISIS 407939, ISIS 416446, ISIS 416449, ISIS 416455, ISIS 416472, ISIS 416477, ISIS 416507, ISIS 416508, ISIS 422086, ISIS 422087, ISIS 422140, and ISIS 422142. Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 2,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS2927 (described hereinabove in Example 1) was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of Target-X, relative to untreated control cells. A total of 916 oligonucleotides were tested. Only those oligonucleotides which were selected for further studies are shown in Tables 18 and 19.
  • TABLE 18
    Inhibition of human Target-X mRNA levels by chimeric
    antisense oligonucleotides targeted to Target-X
    ISIS No Chemistry % inhibition SEQ CODE
    490275 e5-d(10)-e5 35 90
    490277 e5-d(10)-e5 73 91
    490278 e5-d(10)-e5 78 92
    490279 e5-d(10)-e5 66 93
    490323 e5-d(10)-e5 65 94
    490368 e5-d(10)-e5 78 95
    490396 e5-d(10)-e5 76 96
    416507 e5-d(10)-e5 73 97
    422140 e5-d(10)-e5 59 98
    422142 e5-d(10)-e5 73 99
    416508 e5-d(10)-e5 75 100
    490424 e5-d(10)-e5 57 101
    490803 e5-d(10)-e5 70 102
    416446 e5-d(10)-e5 73 103
    416449 e5-d(10)-e5 33 104
    407900 e5-d(10)-e5 66 105
    490103 e5-d(10)-e5 87 106
    416455 e5-d(10)-e5 42 107
    407910 e5-d(10)-e5 25 108
    490149 e5-d(10)-e5 82 109
    403094 e5-d(10)-e5 60 110
    416472 e5-d(10)-e5 78 111
    407641 e5-d(10)-e5 64 112
    416477 e5-d(10)-e5 25 113
    407643 e5-d(10)-e5 78 114
    490196 e5-d(10)-e5 81 115
    490197 e5-d(10)-e5 85 116
    490208 e5-d(10)-e5 89 117
    490209 e5-d(10)-e5 81 118
    422086 e5-d(10)-e5 90 119
    407935 e5-d(10)-e5 91 120
    422087 e5-d(10)-e5 89 121
    407936 e5-d(10)-e5 80 122
    407939 e5-d(10)-e5 67 72
    e = 2′-MOE, d = 2′-deoxynucleoside
  • TABLE 19
    Inhibition of human Target-X mRNA levels
    by chimeric antisense oligonucleotides
    ISIS No Motif % inhibition SEQ CODE
    407662 e5-d(10)-e5 76 123
    416446 e5-d(10)-e5 73 103
    e = 2′-MOE, d = 2′-deoxynucleoside
    • Example 5 Modified Chimeric Antisense Oligonucleotides Comprising 6′-(S)—CH3 Bicyclic Nucleoside (e.g cEt) Modifications at 5′ and 3′ Wing Regions Targeting Human Target-X
  • Additional antisense oligonucleotides were designed targeting a Target-X nucleic acid and were tested for their effects on Target-X mRNA in vitro. ISIS 407939, which was described in an earlier publication (WO 2009/061851) were also tested. ISIS 457851, ISIS 472925, ISIS 472926, ISIS 472935, ISIS 472942, ISIS 472958, ISIS 472959, ISIS 472970, ISIS 472976, ISIS 472983, ISIS 472984, ISIS 472988, ISIS 472991, ISIS 472994, ISIS 472995, ISIS 472996, ISIS 472998, and ISIS 473020, described in the Examples above were also included in the screen.
  • The newly designed chimeric antisense oligonucleotides in Table 20 were designed as 2-10-2 cEt gapmers. The newly designed gapmers are 14 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxyribonucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising five nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment comprises 6′-(S)—CH3 bicyclic nucleoside (e.g cEt) modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines.
  • Each gapmer listed in Table 20 is targeted to the human Target-X genomic sequence.
  • Activity of the newly designed oligonucleotides was compared to ISIS 407939. Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 2,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS2927 (described hereinabove in Example 1) was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of Target-X, relative to untreated control cells. A total of 614 oligonucleotides were tested. Only those oligonucleotides which were selected for further studies are shown in Table 20. Many of the newly designed antisense oligonucleotides provided in Table 20 achieved greater than 72% inhibition and, therefore, are more potent than ISIS 407939.
  • TABLE 20
    Inhibition of human Target-X mRNA levels by chimeric
    antisense oligonucleotides targeted to Target-X
    ISIS No % inhibition Motif Wing Chemistry SEQ CODE
    407939 72 5-10-5 cEt 72
    473020 90 2-10-2 cEt 25
    492465 83 2-10-2 cEt 124
    492467 74 2-10-2 cEt 125
    492492 84 2-10-2 cEt 126
    492494 91 2-10-2 cEt 127
    492503 89 2-10-2 cEt 128
    492530 91 2-10-2 cEt 129
    492534 91 2-10-2 cEt 130
    492536 90 2-10-2 cEt 131
    492541 84 2-10-2 cEt 132
    492545 89 2-10-2 cEt 133
    492566 90 2-10-2 cEt 134
    492571 82 2-10-2 cEt 135
    492572 89 2-10-2 cEt 136
    492573 90 2-10-2 cEt 137
    492574 92 2-10-2 cEt 138
    492575 88 2-10-2 cEt 139
    492593 83 2-10-2 cEt 140
    492617 91 2-10-2 cEt 141
    492618 92 2-10-2 cEt 142
    492619 90 2-10-2 cEt 143
    492621 75 2-10-2 cEt 144
    492104 89 2-10-2 cEt 145
    492105 86 2-10-2 cEt 146
    492189 88 2-10-2 cEt 147
    492194 92 2-10-2 cEt 148
    492195 90 2-10-2 cEt 149
    472925 87 2-10-2 cEt 32
    492196 91 2-10-2 cEt 150
    472926 88 2-10-2 cEt 33
    492205 92 2-10-2 cEt 151
    492215 77 2-10-2 cEt 152
    492221 79 2-10-2 cEt 153
    472935 82 2-10-2 cEt 36
    492234 86 2-10-2 cEt 154
    472942 85 2-10-2 cEt 43
    492276 75 2-10-2 cEt 155
    492277 75 2-10-2 cEt 156
    492306 85 2-10-2 cEt 157
    492317 93 2-10-2 cEt 158
    472958 92 2-10-2 cEt 46
    472959 88 2-10-2 cEt 47
    492329 88 2-10-2 cEt 159
    492331 95 2-10-2 cEt 160
    492333 85 2-10-2 cEt 161
    492334 88 2-10-2 cEt 162
    457851 89 2-10-2 cEt 51
    472970 92 2-10-2 cEt 52
    492365 69 2-10-2 cEt 163
    472976 94 2-10-2 cEt 54
    472983 76 2-10-2 cEt 55
    472984 72 2-10-2 cEt 56
    492377 70 2-10-2 cEt 164
    492380 80 2-10-2 cEt 165
    492384 61 2-10-2 cEt 166
    472988 59 2-10-2 cEt 60
    492388 70 2-10-2 cEt 167
    492389 70 2-10-2 cEt 168
    492390 89 2-10-2 cEt 169
    492391 80 2-10-2 cEt 170
    472991 84 2-10-2 cEt 62
    492398 88 2-10-2 cEt 171
    492399 94 2-10-2 cEt 172
    492401 91 2-10-2 cEt 173
    492403 78 2-10-2 cEt 174
    472994 95 2-10-2 cEt 66
    472995 91 2-10-2 cEt 68
    492404 84 2-10-2 cEt 175
    492405 87 2-10-2 cEt 176
    472996 85 2-10-2 cEt 70
    492406 43 2-10-2 cEt 177
    472998 92 2-10-2 cEt 74
    492440 89 2-10-2 cEt 178
    • Example 6 Modified Chimeric Antisense Oligonucleotides Comprising 6′-(S)—CH3 Bicyclic Nucleoside (e.g cEt) Modifications at 5′ and 3′ Wing Regions Targeting Human Target-X
  • Additional antisense oligonucleotides were designed targeting a Target-X nucleic acid and were tested for their effects on Target-X mRNA in vitro. Also tested was ISIS 407939, a 5-10-5 MOE gapmer targeting human Target-X, which was described in an earlier publication (WO 2009/061851). ISIS 472998 and ISIS 473046, described in the Examples above were also included in the screen.
  • The newly designed chimeric antisense oligonucleotides in Table 21 were designed as 2-10-2 cEt gapmers. The newly designed gapmers are 14 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxyribonucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising five nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment comprise 6′-(S)—CH3 bicyclic nucleoside (e.g cEt) modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines.
  • Each gapmer listed in Table 21 is targeted to the human Target-X genomic sequence.
  • Activity of the newly designed gapmers was compared to ISIS 407939. Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 2,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS2927 (described hereinabove in Example 1) was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of Target-X, relative to untreated control cells. A total of 757 oligonucleotides were tested. Only those oligonucleotides which were selected for further studies are shown in Table 21. Each of the newly designed antisense oligonucleotides provided in Table 21 achieved greater than 67% inhibition and, therefore, are more potent than 407939.
  • TABLE 21
    Inhibition of human Target-X mRNA levels by chimeric
    antisense oligonucleotides targeted to Target-X
    ISIS No % inhibition Motif Wing chemistry SEQ CODE
    407939 67 5-10-5 cEt 72
    492651 77 2-10-2 cEt 179
    492652 84 2-10-2 cEt 180
    492658 87 2-10-2 cEt 181
    492725 74 2-10-2 cEt 182
    492730 78 2-10-2 cEt 183
    492731 72 2-10-2 cEt 184
    492784 72 2-10-2 cEt 185
    492816 70 2-10-2 cEt 186
    492818 73 2-10-2 cEt 187
    492877 83 2-10-2 cEt 188
    492878 79 2-10-2 cEt 189
    492913 73 2-10-2 cEt 190
    492914 82 2-10-2 cEt 191
    492928 76 5-10-5 cEt 192
    492938 80 2-10-2 cEt 193
    492991 91 2-10-2 cEt 194
    492992 73 2-10-2 cEt 195
    493087 81 2-10-2 cEt 196
    493114 80 2-10-2 cEt 197
    493178 86 2-10-2 cEt 198
    493179 69 2-10-2 cEt 199
    493182 79 2-10-2 cEt 200
    493195 71 2-10-2 cEt 201
    473046 79 2-10-2 cEt 35
    493201 86 2-10-2 cEt 202
    493202 76 2-10-2 cEt 203
    493255 80 2-10-2 cEt 204
    493291 84 2-10-2 cEt 205
    493292 90 2-10-2 cEt 206
    493296 82 2-10-2 cEt 207
    493298 77 2-10-2 cEt 208
    493299 76 5-10-5 cEt 209
    493304 77 2-10-2 cEt 210
    493312 75 2-10-2 cEt 211
    493333 76 2-10-2 cEt 212
    472998 85 2-10-2 cEt 74
    • Example 7 Dose-Dependent Antisense Inhibition of Human Target-X in Hep3B Cells
  • Antisense oligonucleotides from the studies above, exhibiting in vitro inhibition of Target-X mRNA, were selected and tested at various doses in Hep3B cells. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.67 μM, 2.00 μM, 1.11 μM, and 6.00 μM concentrations of antisense oligonucleotide, as specified in Table 22. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human Target-X primer probe set RTS2927 was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells.
  • The half maximal inhibitory concentration (IC50) of each oligonucleotide is also presented in Table 22. As illustrated in Table 22, Target-X mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells. The data also confirms that several of the newly designed gapmers are more potent than ISIS 407939 of the previous publication.
  • TABLE 22
    Dose-dependent antisense inhibition of human
    Target-X in Hep3B cells using electroporation
    666.6667 2000.0 6000.0 IC50
    ISIS No nM nM nM (μM)
    407939 47 68 85 0.7
    457851 60 80 93 <0.6
    472916 53 80 87 <0.6
    472925 62 86 95 <0.6
    472926 66 77 85 <0.6
    472935 54 84 94 <0.6
    472958 66 82 88 <0.6
    472959 64 81 93 <0.6
    472970 72 87 86 <0.6
    472976 78 92 97 <0.6
    472994 79 92 96 <0.6
    472995 61 82 93 <0.6
    472996 73 91 95 <0.6
    472998 63 90 95 <0.6
    473019 55 80 86 <0.6
    473020 61 76 85 <0.6
    473046 61 80 94 <0.6
    473055 55 84 94 <0.6
    492104 53 76 88 <0.6
    492105 62 80 90 <0.6
    492189 57 80 92 <0.6
    492194 57 83 91 <0.6
    492195 58 81 95 <0.6
    492196 62 86 95 <0.6
    492205 62 87 95 <0.6
    492215 60 78 89 <0.6
    492221 63 76 92 <0.6
    492234 51 74 91 0.5
    492276 50 56 95 0.8
    492277 58 73 81 <0.6
    492306 61 75 84 <0.6
    492317 59 80 93 <0.6
    492329 59 70 89 <0.6
    492331 69 87 95 <0.6
    492333 47 70 85 0.7
    492334 57 77 90 <0.6
    492390 72 88 95 <0.6
    492399 68 91 96 <0.6
    492401 68 89 95 <0.6
    492404 65 87 94 <0.6
    492405 44 81 90 0.7
    492406 65 82 92 <0.6
    492440 50 70 89 0.6
    492465 16 80 79 1.4
    492467 58 77 92 <0.6
    492492 45 80 94 0.7
    492494 63 82 93 <0.6
    492503 55 81 93 <0.6
    492530 70 86 90 <0.6
    492534 67 85 91 <0.6
    492536 54 81 89 <0.6
    492541 54 71 85 <0.6
    492545 59 78 89 <0.6
    492566 59 84 85 <0.6
    492571 52 81 89 <0.6
    492572 67 83 90 <0.6
    492573 69 83 92 <0.6
    492574 65 82 91 <0.6
    492575 72 83 91 <0.6
    492593 61 78 90 <0.6
    492617 62 80 93 <0.6
    492618 47 79 94 0.6
    492619 54 82 95 <0.6
    492621 44 85 92 0.6
    492651 53 66 91 0.6
    492652 61 78 88 <0.6
    492658 59 79 88 <0.6
    492725 43 84 89 0.6
    492730 51 87 93 0.4
    492731 46 82 90 0.6
    492784 56 88 96 <0.6
    492816 68 89 97 <0.6
    492818 64 84 96 <0.6
    492877 67 91 93 <0.6
    492878 80 89 93 <0.6
    492913 53 87 92 <0.6
    492914 75 89 96 <0.6
    492928 60 83 94 <0.6
    492938 70 90 92 <0.6
    492991 67 93 99 <0.6
    492992 0 82 95 2.1
    493087 54 81 90 <0.6
    493114 50 73 90 0.6
    493178 71 88 96 <0.6
    493179 47 82 95 0.6
    493182 79 87 91 <0.6
    493195 55 78 90 <0.6
    493201 87 93 96 <0.6
    493202 68 89 94 <0.6
    493255 57 79 93 <0.6
    493291 57 87 93 <0.6
    493292 70 89 93 <0.6
    493296 35 84 91 0.9
    493298 57 84 92 <0.6
    493299 65 84 93 <0.6
    493304 68 86 94 <0.6
    493312 53 82 91 <0.6
    493333 66 84 87 <0.6
    • Example 8 Dose-Dependent Antisense Inhibition of Human Target-X in Hep3B Cells
  • Additional antisense oligonucleotides from the studies described above, exhibiting in vitro inhibition of Target-X mRNA, were selected and tested at various doses in Hep3B cells. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.67 μM, 2.00 μM, 1.11 μM, and 6.00 μM concentrations of antisense oligonucleotide, as specified in Table 23. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human Target-X primer probe set RTS2927 was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells. As illustrated in Table 23, Target-X mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells. The data also confirms that several of the newly designed gapmers are more potent than ISIS 407939.
  • TABLE 23
    Dose-dependent antisense inhibition of human
    Target-X in Hep3B cells using electroporation
    0.67 2.00 6.00 IC50
    ISIS No μM μM μM (μM)
    407939 52 71 86 0.6
    472983 49 83 97 0.5
    472984 51 82 95 0.5
    472991 49 82 95 0.5
    472998 59 88 96 <0.6
    492365 74 91 96 <0.6
    492377 56 76 91 <0.6
    492380 63 79 95 <0.6
    492384 67 84 94 <0.6
    492388 69 87 97 <0.6
    492389 62 90 96 <0.6
    492391 56 84 94 <0.6
    492398 63 80 95 <0.6
    492403 58 81 91 <0.6
    • Example 9 Modified Chimeric Antisense Oligonucleotides Comprising 2′-Methoxyethyl (2′-MOE) Modifications at 5′ and 3′ Wing Regions Targeting Human Target-X
  • Additional antisense oligonucleotides were designed targeting a Target-X nucleic acid and were tested for their effects on Target-X mRNA in vitro. Also tested were ISIS 403052, ISIS 407939, ISIS 416446, ISIS 416472, ISIS 416507, ISIS 416508, ISIS 422087, ISIS 422096, ISIS 422130, and ISIS 422142 which were described in an earlier publication (WO 2009/061851), incorporated herein by reference. ISIS 490149, ISIS 490197, ISIS 490209, ISIS 490275, ISIS 490277, and ISIS 490424, described in the Examples above, were also included in the screen.
  • The newly designed chimeric antisense oligonucleotides in Table 24 were designed as 3-10-4 2′-MOE gapmers. These gapmers are 17 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxyribonucleosides and is flanked by wing segments on the 5′ direction with three nucleosides and the 3′ direction with four nucleosides. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has 2′-MOE modifications. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines.
  • Each gapmer listed in Table 24 is targeted to the human Target-X genomic sequence.
  • Activity of the newly designed oligonucleotides was compared to ISIS 403052, ISIS 407939, ISIS 416446, ISIS 416472, ISIS 416507, ISIS 416508, ISIS 422087, ISIS 422096, ISIS 422130, and ISIS 422142. Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 2,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS2927 (described hereinabove in Example 1) was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells. A total of 272 oligonucleotides were tested. Only those oligonucleotides which were selected for further studies are shown in Table 24. Several of the newly designed antisense oligonucleotides provided in Table 24 are more potent than antisense oligonucleotides from the previous publication.
  • TABLE 24
    Inhibition of human Target-X mRNA levels by chimeric
    antisense oligonucleotides targeted to Target-X
    ISIS No % inhibition Motif Wing Chemistry SEQ CODE
    403052 51 5-10-5 2′-MOE 82
    407939 78 5-10-5 2′-MOE 72
    416446 70 5-10-5 2′-MOE 103
    416472 79 5-10-5 2′-MOE 111
    416507 84 5-10-5 2′-MOE 97
    416508 80 5-10-5 2′-MOE 100
    422087 89 5-10-5 2′-MOE 121
    422096 78 5-10-5 2′-MOE 219
    422130 81 5-10-5 2′-MOE 225
    422142 84 5-10-5 2′-MOE 99
    490275 77 5-10-5 2′-MOE 90
    513462 79 3-10-4 2′-MOE 213
    513463 81 3-10-4 2′-MOE 214
    490277 74 5-10-5 2′-MOE 91
    513487 83 3-10-4 2′-MOE 215
    513504 81 3-10-4 2′-MOE 216
    513507 86 3-10-4 2′-MOE 217
    513508 85 3-10-4 2′-MOE 218
    490424 69 5-10-5 2′-MOE 101
    491122 87 5-10-5 2′-MOE 220
    513642 79 3-10-4 2′-MOE 221
    490149 71 5-10-5 2′-MOE 109
    513419 90 3-10-4 2′-MOE 222
    513420 89 3-10-4 2′-MOE 223
    513421 88 3-10-4 2′-MOE 224
    490197 77 5-10-5 2′-MOE 116
    513446 89 3-10-4 2′-MOE 226
    513447 83 3-10-4 2′-MOE 227
    490209 79 5-10-5 2′-MOE 118
    513454 84 3-10-4 2′-MOE 228
    513455 92 3-10-4 2′-MOE 229
    513456 89 3-10-4 2′-MOE 230
    513457 83 3-10-4 2′-MOE 231
    • Example 10 Dose-Dependent Antisense Inhibition of Human Target-X in Hep3B Cells
  • Antisense oligonucleotides from the studies above, exhibiting in vitro inhibition of Target-X mRNA, were selected and tested at various doses in Hep3B cells. ISIS 403052, ISIS 407643, ISIS 407935, ISIS 407936, ISIS 407939, ISIS 416446, ISIS 416459, ISIS 416472, ISIS 416507, ISIS 416508, ISIS 416549, ISIS 422086, ISIS 422087, ISIS 422130, ISIS and 422142, 5-10-5 MOE gapmers targeting human Target-X, which were described in an earlier publication (WO 2009/061851).
  • Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.625 μM, 1.25 μM, 2.50 μM, 5.00 μM and 10.00 μM concentrations of antisense oligonucleotide, as specified in Table 25. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human Target-X primer probe set RTS2927 was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells.
  • The half maximal inhibitory concentration (IC50) of each oligonucleotide is also presented in Table 25. As illustrated in Table 25, Target-X mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells. The data also confirms that the newly designed gapmers are potent than gapmers from the previous publication.
  • TABLE 25
    Dose-dependent antisense inhibition of human
    Target-X in Hep3B cells using electroporation
    0.625 1.25 2.50 5.00 10.00 IC50
    ISIS No μM μM μM μM μM (μM)
    403052 21 35 63 82 89 1.9
    407643 29 46 67 83 90 1.4
    407935 52 68 80 89 91 <0.6
    407936 31 51 62 78 84 1.4
    407939 30 61 74 83 88 1.0
    416446 37 53 64 76 83 1.2
    416459 51 76 83 90 92 <0.6
    416472 37 52 66 78 85 1.2
    416507 45 68 82 87 90 0.7
    416508 33 56 74 84 89 1.1
    416549 57 71 78 82 85 <0.6
    422086 46 67 77 89 92 0.7
    422087 50 69 74 86 91 0.6
    422130 32 65 78 92 93 0.9
    422142 59 73 84 86 88 <0.6
    490103 52 57 66 83 88 0.9
    490149 34 58 71 85 91 1.0
    490196 26 59 66 79 84 1.3
    490197 39 63 74 81 90 0.8
    490208 44 70 76 83 88 0.6
    490275 36 58 76 85 89 1.0
    490277 37 63 73 87 87 0.8
    490279 40 54 72 83 89 1.0
    490323 49 68 79 86 90 <0.6
    490368 39 62 76 86 91 0.8
    490396 36 53 69 80 87 1.1
    490424 45 65 69 76 82 0.6
    490803 57 74 85 89 92 <0.6
    513419 60 71 85 95 96 <0.6
    513420 37 69 79 94 96 0.7
    513421 46 64 84 95 97 0.6
    513446 47 81 88 95 96 <0.6
    513447 56 74 81 92 96 <0.6
    513454 50 77 82 93 95 <0.6
    513455 74 82 91 96 96 <0.6
    513456 66 80 88 94 95 <0.6
    513457 54 67 80 87 89 <0.6
    513462 49 72 84 87 89 <0.6
    513463 36 62 76 85 89 0.9
    513487 42 56 73 87 93 0.9
    513504 47 65 81 90 91 0.6
    513505 39 50 78 85 92 1.0
    513507 52 73 83 89 93 <0.6
    513508 56 78 85 91 94 <0.6
    • Example 11 Dose-Dependent Antisense Inhibition of Human Target-X in Hep3B Cells
  • Additional antisense oligonucleotides from the studies above, exhibiting in vitro inhibition of Target-X mRNA, were tested at various doses in Hep3B cells. ISIS 407935, ISIS 407939, ISIS 416446, ISIS 416472, ISIS 416507, ISIS 416549, ISIS 422086, ISIS 422087, ISIS 422096, and ISIS 422142 5-10-5 MOE gapmers targeting human Target-X, which were described in an earlier publication (WO 2009/061851).
  • Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.3125 μM, 0.625 μM, 1.25 μM, 2.50 μM, 5.00 μM and 10.00 μM concentrations of antisense oligonucleotide, as specified in Table 26. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human Target-X primer probe set RTS2927 was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells. As illustrated in Table 26, Target-X mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells. The data also confirms that the newly designed gapmers are more potent than gapmers from the previous publication.
  • TABLE 26
    Dose-dependent antisense inhibition of human
    Target-X in Hep3B cells using electroporation
    0.3125 0.625 1.250 2.500 5.000 10.000 IC50
    ISIS No μM μM μM μM μM μM (μM)
    407935 30 49 75 86 91 94 0.6
    407939 30 48 61 78 85 90 0.8
    416446 27 52 63 75 85 90 0.7
    416472 38 51 72 83 88 94 0.5
    416507 58 81 76 84 89 92 <0.3
    416549 52 67 75 81 88 89 0.3
    422086 48 49 68 78 86 91 0.5
    422087 30 56 66 83 72 92 0.6
    422096 47 63 70 77 83 85 <0.3
    422142 69 85 87 85 89 91 <0.3
    490103 52 57 68 78 87 93 0.4
    490149 33 64 62 77 86 93 0.5
    490197 38 46 60 75 87 93 0.7
    490208 46 62 73 83 88 91 0.4
    490209 40 54 72 79 85 94 0.5
    490275 52 61 67 78 85 91 0.3
    490277 33 59 77 79 91 94 0.5
    490323 43 61 72 69 84 87 0.4
    490368 50 64 78 83 90 92 <0.3
    490396 46 64 68 84 84 90 0.3
    490424 24 47 58 72 76 82 1.0
    490803 45 60 70 84 88 89 0.3
    513419 32 53 76 88 93 95 0.5
    513420 35 59 72 82 94 97 0.5
    513421 46 67 78 86 94 96 <0.3
    513446 26 61 77 89 91 97 0.5
    513447 22 48 60 82 91 95 0.8
    513454 25 59 76 86 94 96 0.5
    513455 60 73 85 89 95 96 <0.3
    513456 49 60 81 88 94 95 <0.3
    513457 43 50 72 77 87 92 0.5
    513462 25 48 58 76 83 88 0.8
    513463 22 45 66 73 85 88 0.9
    513487 41 56 65 79 86 90 0.4
    513504 19 48 63 76 87 92 0.9
    513505 11 21 54 73 85 90 1.4
    513507 47 55 72 82 90 91 0.3
    513508 31 59 74 85 92 93 0.5
    513642 43 55 67 80 88 92 0.4
    • Example 12 Tolerability of 2′-MOE Gapmers Targeting Human Target-X in BALB/c Mice
  • BALB/c mice are a multipurpose mice model, frequently utilized for safety and efficacy testing. The mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.
    • Treatment
  • Groups of male BALB/c mice were injected subcutaneously twice a week for 3 weeks with 50 mg/kg of ISIS 407935, ISIS 416472, ISIS 416549, ISIS 422086, ISIS 422087, ISIS 422096, ISIS 422142, ISIS 490103, ISIS 490149, ISIS 490196, ISIS 490208, ISIS 490209, ISIS 513419, ISIS 513420, ISIS 513421, ISIS 513454, ISIS 513455, ISIS 513456, ISIS 513457, ISIS 513462, ISIS 513463, ISIS 513487, ISIS 513504, ISIS 513508, and ISIS 513642. One group of male BALB/c mice was injected subcutaneously twice a week for 3 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.
    • Plasma Chemistry Markers
  • To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, albumin, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.).
  • ISIS oligonucleotides that did not cause any increase in the levels of transaminases, or which caused an increase within three times the upper limit of normal (ULN) were deemed very tolerable. ISIS oligonucleotides that caused an increase in the levels of transaminases between three times and seven times the ULN were deemed tolerable. Based on these criteria, ISIS 407935, ISIS 416472, ISIS 416549, ISIS 422087, ISIS 422096, ISIS 490103, ISIS 490196, ISIS 490208, ISIS 513454, ISIS 513455, ISIS 513456, ISIS 513457, ISIS 513487, ISIS 513504, and ISIS 513508 were considered very tolerable in terms of liver function. Based on these criteria, ISIS 422086, ISIS 490209, ISIS 513419, ISIS 513420, and ISIS 513463 were considered tolerable in terms of liver function.
    • Example 13 Dose-Dependent Antisense Inhibition of Human Target-X in Hep3B cells
  • Additional antisense oligonucleotides from the studies above, exhibiting in vitro inhibition of Target-X mRNA were selected and tested at various doses in Hep3B cells. Also tested was ISIS 407939, a 5-10-5 MOE gapmer, which was described in an earlier publication (WO 2009/061851).
  • Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.074 μM, 0.222 μM, 0.667 μM, 2.000 μM, and 6.000 μM concentrations of antisense oligonucleotide, as specified in Table 27. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human Target-X primer probe set RTS2927 (described hereinabove in Example 1) was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells.
  • The half maximal inhibitory concentration (IC50) of each oligonucleotide is also presented in Table 27. As illustrated in Table 27, Target-X mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells. Many of the newly designed antisense oligonucleotides provided in Table 27 achieved an IC50 of less than 0.9 μM and, therefore, are more potent than ISIS 407939.
  • TABLE 27
    Dose-dependent antisense inhibition of human
    Target-X in Hep3B cells using electroporation
    0.074 0.222 0.667 2.000 6.000 IC50
    ISIS No μM μM μM μM μM (μM)
    407939 2 17 53 70 87 0.9
    472970 17 47 75 92 95 0.3
    472988 0 8 21 54 92 1.4
    472996 18 59 74 93 95 0.2
    473244 91 95 97 99 99 <0.07
    473286 6 53 85 92 98 0.3
    473359 2 3 20 47 67 2.6
    473392 71 85 88 92 96 <0.07
    473393 91 96 97 98 99 <0.07
    473547 85 88 93 97 98 <0.07
    473567 0 25 66 88 95 0.7
    473589 8 47 79 94 99 0.3
    482814 23 68 86 93 96 0.1
    482815 6 48 65 90 96 0.4
    482963 3 68 85 94 96 0.2
    483241 14 33 44 76 93 0.6
    483261 14 21 41 72 88 0.7
    483290 0 1 41 69 92 1.0
    483414 8 1 36 76 91 0.9
    483415 0 40 52 84 94 0.6
    484559 26 51 78 87 97 0.2
    484713 6 5 53 64 88 0.9
    • Example 14 Modified Antisense Oligonucleotides Comprising 2′-O-Methoxyethyl (2′-MOE) and 6′-(S)—CH3 Bicyclic Nucleoside (e.g cEt) Modifications Targeting Human Target-X
  • Additional antisense oligonucleotides were designed targeting a Target-X nucleic acid and were tested for their effects on Target-X mRNA in vitro. Also tested was ISIS 407939, a 5-10-5 MOE gapmer targeting human Target-X, which was described in an earlier publication (WO 2009/061851). ISIS 472998, ISIS 492878, and ISIS 493201 and 493182, 2-10-2 cEt gapmers, described in the Examples above were also included in the screen.
  • The newly designed modified antisense oligonucleotides are 16 nucleotides in length and their motifs are described in Table 28. The chemistry column of Table 28 presents the sugar motif of each oligonucleotide, wherein “e” indicates a 2′-O-methoxyethyl (2′-MOE) nucleoside, “k” indicates a 6′-(S)—CH3 bicyclic nucleoside (e.g cEt) and “d” indicates a 2′-deoxyribonucleoside. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosines.
  • Each gapmer listed in Table 28 is targeted to the human Target-X genomic sequence.
  • Activity of newly designed gapmers was compared to ISIS 407939. Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 2,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS2927 was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells and demonstrate that several of the newly designed gapmers are more potent than ISIS 407939. A total of 685 oligonucleotides were tested. Only those oligonucleotides which were selected for further studies are shown in Table 28.
  • TABLE 28
    Inhibition of human Target-X mRNA levels by chimeric
    antisense oligonucleotides targeted to Target-X
    ISIS No % inhibition Chemistry SEQ CODE
    407939 68 eeeee-d(10)-eeeee 72
    492878 73 kk-d(10)-kk
    493182 80 kk-d(10)-kk
    493201 84 kk-d(10)-kk
    472998 91 kk-d(10)-kk
    515640 75 eee-d(10)-kkk 23
    515637 77 eee-d(10)-kkk 232
    515554 72 eee-d(10)-kkk 233
    515406 80 kkk-d(10)-eee 234
    515558 81 eee-d(10)-kkk 234
    515407 88 kkk-d(10)-eee 235
    515408 85 kkk-d(10)-eee 236
    515422 86 kkk-d(10)-eee 237
    515423 90 kkk-d(10)-eee 238
    515575 84 eee-d(10)-kkk 238
    515424 87 kkk-d(10)-eee 239
    515432 78 kkk-d(10)-eee 240
    515433 71 kkk-d(10)-eee 241
    515434 76 kkk-d(10)-eee 242
    515334 85 kkk-d(10)-eee 243
    515649 61 eee-d(10)-kkk 88
    515338 86 kkk-d(10)-eee 244
    515438 76 kkk-d(10)-eee 245
    515439 75 kkk-d(10)-eee 246
    516003 87 eee-d(10)-kkk 247
    515647 60 eee-d(10)-kkk 77
    515639 78 eee-d(10)-kkk 34
    493201 84 eee-d(10)-kkk 202
    515648 36 kkk-d(10)-eee 248
    515641 69 kk-d(10)-eeee 39
    515650 76 kkk-d(10)-eee 44
    515354 87 eee-d(10)-kkk 249
    515926 87 eee-d(10)-kkk 250
    515366 87 kk-d(10)-eeee 251
    515642 58 kkk-d(10)-eee 252
    515643 81 eee-d(10)-kkk 53
    515944 84 kk-d(10)-eeee 253
    515380 90 kkk-d(10)-eee 254
    515532 83 kkk-d(10)-eee 254
    515945 85 kk-d(10)-eeee 254
    515381 82 kk-d(10)-eeee 255
    515382 95 kkk-d(10)-eee 256
    515948 94 eee-d(10)-kkk 256
    515949 87 eee-d(10)-kkk 257
    515384 89 kkk-d(10)-eee 258
    515635 82 kk-d(10)-eeee 65
    515638 90 kkk-d(10)-eee 67
    515386 92 kk-d(10)-eeee 259
    515951 84 eee-d(10)-kkk 259
    515387 78 kkk-d(10)-eee 260
    515952 89 kkk-d(10)-eee 260
    515636 90 kkk-d(10)-eee 69
    515388 84 eee-d(10)-kkk 261
    e = 2′-MOE, k = cEt, d = 2′-deoxyribonucleoside
    • Example 15 Tolerability of Modified Oligonucleotides Comprising 2′-O-Methoxyethyl (2′-MOE) and 6′-(S)—CH3 Bicyclic Nucleoside (e.g cEt) Modifications Targeting Human Target-X in BALB/c Mice
  • BALB/c mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.
  • Additionally, the newly designed modified antisense oligonucleotides were also added to this screen. The newly designed chimeric antisense oligonucleotides are 16 nucleotides in length and their motifs are described in Table 29. The chemistry column of Table 29 presents the sugar motif of each oligonucleotide, wherein “e” indicates a 2′-O-methoxyethyl (2′-MOE) nucleoside, “k” indicates a 6′-(S)—CH3 bicyclic nucleoside (e.g cEt) and “d” indicates a 2′-deoxyribonucleoside. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosines.
  • Each gapmer listed in Table 29 is targeted to the human Target-X genomic sequence.
  • TABLE 29
    Modified chimeric antisense oligonucleotides targeted to Target-X
    ISIS No Chemistry SEQ CODE
    516044 eee-d(10)-kkk 21
    516045 eee-d(10)-kkk 22
    516058 eee-d(10)-kkk 26
    516059 eee-d(10)-kkk 27
    516060 eee-d(10)-kkk 28
    516061 eee-d(10)-kkk 29
    516062 eee-d(10)-kkk 30
    516046 eee-d(10)-kkk 37
    516063 eee-d(10)-kkk 38
    516064 eee-d(10)-kkk 89
    516065 eee-d(10)-kkk 262
    516066 eee-d(10)-kkk 263
    516047 eee-d(10)-kkk 41
    516048 eee-d(10)-kkk 42
    516049 eee-d(10)-kkk 81
    516050 eee-d(10)-kkk 45
    516051 eee-d(10)-kkk 48
    516052 eee-d(10)-kkk 49
    515652 eee-d(10)-kkk 50
    508039 eee-d(10)-kkk 264
    516053 eee-d(10)-kkk 265
    515654 eee-d(10)-kkk 76
    515656 eee-d(10)-kkk 77
    516054 eee-d(10)-kkk 57
    516055 eee-d(10)-kkk 59
    515655 eee-d(10)-kkk 61
    516056 eee-d(10)-kkk 63
    516057 eee-d(10)-kkk 64
    515653 eee-d(10)-kkk 71
    515657 eee-d(10)-kkk 73
    e = 2′-MOE, k = cEt, d = 2′-deoxyribonucleoside
    • Treatment
  • Groups of 4-6-week old male BALB/c mice were injected subcutaneously twice a week for 3 weeks with 50 mg/kg/week of ISIS 457851, ISIS 515635, ISIS 515636, ISIS 515637, ISIS 515638, ISIS 515639, ISIS 515640, ISIS 515641, ISIS 515642, ISIS 515643, ISIS 515647, ISIS 515648, ISIS 515649, ISSI 515650, ISIS 515652, ISIS 515653, ISIS 515654, ISIS 515655, ISIS 515656, ISIS 515657, ISIS 516044, ISIS 516045, ISIS 516046, ISIS 516047, ISIS 516048, ISIS 516049, ISIS 516050, ISIS 516051, ISIS 516052, ISIS 516053, ISIS 516054, ISIS 516055, ISIS 516056, ISIS 516057, ISIS 516058, ISIS 516059, ISIS 516060, ISIS 516061, ISIS 516062, ISIS 516063, ISIS 516064, ISIS 516065, and ISIS 516066. One group of 4-6-week old male BALB/c mice was injected subcutaneously twice a week for 3 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.
    • Plasma Chemistry Markers
  • To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, albumin, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.).
  • ISIS oligonucleotides that did not cause any increase in the levels of transaminases, or which caused an increase within three times the upper limit of normal (ULN) were deemed very tolerable. ISIS oligonucleotides that caused an increase in the levels of transaminases between three times and seven times the ULN were deemed tolerable. Based on these criteria, ISIS 515636, ISIS 515639, ISIS 515641, ISIS 515642, ISIS 515648, ISIS 515650, ISIS 515652, ISIS 515653, ISIS 515655, ISIS 515657, ISIS 516044, ISIS 516045, ISIS 516047, ISIS 516048, ISIS 516051, ISIS 516052, ISIS 516053, ISIS 516055, ISIS 516056, ISIS 516058, ISIS 516059, ISIS 516060, ISIS 516061, ISIS 516062, ISIS 516063, ISIS 516064, ISIS 516065, and ISIS 516066 were considered very tolerable in terms of liver function. Based on these criteria, ISIS 457851, ISIS 515635, ISIS 515637, ISIS 515638, ISIS 515643, ISIS 515647, ISIS 515649, ISIS 515650, ISIS 515652, ISIS 515654, ISIS 515656, ISIS 516056, and ISIS 516057 were considered tolerable in terms of liver function.
    • Example 16 Efficacy of Modified Oligonucleotides Comprising 2′-O-Methoxyethyl (2′-MOE) and 6′-(S)—CH3 Bicyclic Nucleoside (e.g cEt) Modifications Targeting Human Target-X in Transgenic Mice
  • Transgenic mice were developed at Taconic farms harboring a Target-X genomic DNA fragment. The mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for efficacy.
    • Treatment
  • Groups of 3-4 male and female transgenic mice were injected subcutaneously twice a week for 3 weeks with 20 mg/kg/week of ISIS 457851, ISIS 515636, ISIS 515639, ISIS 515653, ISIS 516053, ISIS 516065, and ISIS 516066. One group of mice was injected subcutaneously twice a week for 3 weeks with control oligonucleotide, ISIS 141923 (CCTTCCCTGAAGGTTCCTCC, 5-10-5 MOE gapmer with no known murine target, SEQ ID NO: 9). One group of mice was injected subcutaneously twice a week for 3 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.
    • RNA Analysis
  • RNA was extracted from plasma for real-time PCR analysis of Target-X, using primer probe set RTS2927. The mRNA levels were normalized using RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to control. As shown in Table 30, each of the antisense oligonucleotides achieved reduction of human Target-X mRNA expression over the PBS control. Treatment with the control oligonucleotide did not achieve reduction in Target-X levels, as expected.
  • TABLE 30
    Percent inhibition of Target-X mRNA in transgenic mice
    ISIS No % inhibition
    141923 0
    457851 76
    515636 66
    515639 49
    515653 78
    516053 72
    516065 59
    516066 39
    • Protein Analysis
  • Plasma protein levels of Target-X were estimated using a Target-X ELISA kit (purchased from Hyphen Bio-Med). Results are presented as percent inhibition of Target-X, relative to control. As shown in Table 31, several antisense oligonucleotides achieved reduction of human Target-X protein expression over the PBS control.
  • TABLE 31
    Percent inhibition of Target-X protein levels in transgenic mice
    ISIS No % inhibition
    141923 0
    457851 64
    515636 68
    515639 46
    515653 0
    516053 19
    516065 0
    516066 7
    • Example 17 Efficacy of Modified Oligonucleotides Comprising 2′-O-Methoxyethyl (2′-MOE) and 6′-(S)—CH3 Bicyclic Nucleoside (e.g cEt) Modifications Targeting Human Target-X in Transgenic Mice
  • Transgenic mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for efficacy.
    • Treatment
  • Groups of 2-4 male and female transgenic mice were injected subcutaneously twice a week for 3 weeks with 10 mg/kg/week of ISIS 407935, ISIS 416472, ISIS 416549, ISIS 422087, ISIS 422096, ISIS 473137, ISIS 473244, ISIS 473326, ISIS 473327, ISIS 473359, ISIS 473392, ISIS 473393, ISIS 473547, ISIS 473567, ISIS 473589, ISIS 473630, ISIS 484559, ISIS 484713, ISIS 490103, ISIS 490196, ISIS 490208, ISIS 513419, ISIS 513454, ISIS 513455, ISIS 513456, ISIS 513457, ISIS 513487, ISIS 513508, ISIS 515640, ISIS 515641, ISIS 515642, ISIS 515648, ISIS 515655, ISIS 515657, ISIS 516045, ISIS 516046, ISIS 516047, ISIS 516048, ISIS 516051, ISIS 516052, ISIS 516055, ISIS 516056, ISIS 516059, ISIS 516061, ISIS 516062, and ISIS 516063. One group of mice was injected subcutaneously twice a week for 3 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.
    • Protein Analysis
  • Plasma protein levels of Target-X were estimated using a Target-X ELISA kit (purchased from Hyphen Bio-Med). Results are presented as percent inhibition of Target-X, relative to control. As shown in Table 32, several antisense oligonucleotides achieved reduction of human Target-X over the PBS control.
  • TABLE 32
    Percent inhibition of Target-X plasma
    protein levels in transgenic mice
    ISIS No % inhibition
    407935 80
    416472 49
    416549 29
    422087 12
    422096 21
    473137 57
    473244 67
    473326 42
    473327 100
    473359 0
    473392 22
    473393 32
    473547 73
    473567 77
    473589 96
    473630 75
    484559 75
    484713 56
    490103 0
    490196 74
    490208 90
    513419 90
    513454 83
    513455 91
    513456 81
    513457 12
    513487 74
    513508 77
    515640 83
    515641 87
    515642 23
    515648 32
    515655 79
    515657 81
    516045 52
    516046 79
    516047 65
    516048 79
    516051 84
    516052 72
    516055 70
    516056 0
    516059 39
    516061 64
    516062 96
    516063 24
    • Example 18 Dose-Dependent Antisense Inhibition of Human Target-X in Hep3B Cells
  • Antisense oligonucleotides exhibiting in vitro inhibition of Target-X mRNA were selected and tested at various doses in Hep3B cells. Also tested was ISIS 407939, a 5-10-5 MOE gapmer targeting human Target-X, which was described in an earlier publication (WO 2009/061851).
  • Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.074 μM, 0.222 μM, 0.667 μM, 2.000 μM, and 6.000 μM concentrations of antisense oligonucleotide, as specified in Table 33. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human Target-X primer probe set RTS2927 (described hereinabove in Example 1) was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells.
  • The half maximal inhibitory concentration (IC50) of each oligonucleotide is also presented in Table 33. As illustrated in Table 33, Target-X mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells. Many of the newly designed antisense oligonucleotides provided in Table 33 achieved an IC50 of less than 2.0 μM and, therefore, are more potent than ISIS 407939.
  • TABLE 33
    Dose-dependent antisense inhibition of human
    Target-X in Hep3B cells using electroporation
    0.074 0.222 0.667 2.000 6.000 IC50
    ISIS No μM μM μM μM μM (μM)
    407939 0 9 21 58 76 2.0
    515636 14 32 50 62 81 0.7
    515639 10 24 41 61 67 1.3
    515640 4 16 35 52 63 2.0
    515641 0 21 27 55 66 1.9
    515642 3 13 36 44 66 2.2
    515648 8 10 10 5 16 >6.0
    515653 9 35 26 55 71 1.5
    515655 0 0 6 13 42 >6.0
    515657 0 13 17 38 51 6.0
    516045 0 6 15 19 40 >6.0
    516046 0 7 32 48 69 2.1
    516047 12 27 41 50 63 1.8
    516051 9 8 34 52 66 2.0
    516052 17 42 27 53 75 1.2
    516053 9 7 28 63 77 1.3
    516055 0 3 27 54 75 2.0
    516056 0 4 14 52 66 2.6
    516057 0 34 33 51 70 1.6
    516058 13 12 25 47 74 2.0
    516059 4 15 36 47 68 1.9
    516060 0 1 39 29 63 3.2
    516061 0 0 24 0 3 <6.0
    516062 0 20 43 65 78 1.0
    516063 0 8 10 37 61 3.8
    516064 0 3 13 45 69 2.7
    516065 0 14 38 63 76 1.3
    516066 0 3 30 55 75 1.7
    • Example 19 Modified Oligonucleotides Comprising 2′-O-Methoxyethyl (2′-MOE) and 6′-(S)—CH3 Bicyclic Nucleoside (e.g cEt) Modifications Targeting Human Target-X
  • Additional antisense oligonucleotides were designed targeting a Target-X nucleic acid and were tested for their effects on Target-X mRNA in vitro. ISIS 472998, ISIS 515652, ISIS 515653, ISIS 515654, ISIS 515655, ISIS 515656, and ISIS 515657, described in the Examples above were also included in the screen.
  • The newly designed chimeric antisense oligonucleotides are 16 or 17 nucleotides in length and their motifs are described in Table 34. The chemistry column of Table 34 presents the sugar motif of each oligonucleotide, wherein “e” indicates a 2′-O-methoxyethyl (2′-MOE) nucleoside, “k” indicates a 6′-(S)—CH3 bicyclic nucleoside (e.g cEt) and “d” indicates a 2′-deoxyribonucleoside. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosines.
  • Each gapmer listed in Table 34 is targeted to the human Target-X genomic sequence.
  • Activity of newly designed gapmers was compared to ISIS 407939. Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 2,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS2927 (described hereinabove in Example 1) was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells.
  • TABLE 34
    Inhibition of human Target-X mRNA levels by chimeric
    antisense oligonucleotides targeted to Target-X
    ISIS No % inhibition Chemistry SEQ CODE
    472998 85 kk-d(10)-kk 74
    515652 63 eee-d(10)-kkk 50
    515653 67 eee-d(10)-kkk 71
    515654 78 eee-d(10)-kkk 86
    515655 41 eee-d(10)-kkk 61
    515656 74 eee-d(10)-kkk 87
    515657 49 eee-d(10)-kkk 73
    529265 52 eek-d(10)-keke 267
    529332 82 eek-d(10)-keke 268
    529334 78 eek-d(10)-keke 269
    529186 85 eek-d(10)-keke 213
    529223 81 eek-d(10)-kkke 213
    529129 75 eee-d(10)-kkk 270
    529149 82 kkk-d(10)-eee 270
    529177 77 eek-d(10)-keke 214
    529214 78 eek-d(10)-kkke 214
    529178 79 eek-d(10)-keke 271
    529215 82 eek-d(10)-kkke 271
    529179 71 eek-d(10)-keke 272
    529216 77 eek-d(10)-kkke 272
    529193 69 eek-d(10)-keke 273
    529230 70 eek-d(10)-kkke 273
    529136 48 eee-d(10)-kkk 274
    529156 68 kkk-d(10)-eee 274
    529194 44 eek-d(10)-keke 275
    529231 56 eek-d(10)-kkke 275
    529137 34 eee-d(10)-kkk 276
    529157 79 kkk-d(10)-eee 276
    529336 57 eek-d(10)-keke 277
    529338 73 eek-d(10)-keke 278
    529195 55 eek-d(10)-keke 279
    529232 68 eek-d(10)-kkke 279
    529340 65 eek-d(10)-keke 280
    529342 69 eek-d(10)-keke 281
    529812 69 k-d(10)-kekee 282
    529831 62 k-d(10)-kdkee 282
    529733 64 ke-d(10)-keke 283
    529753 52 ek-d(10)-keke 283
    529773 57 ke-d(10)-kdke 283
    529793 36 ek-d(10)-kdke 283
    529862 48 kde-d(10)-kdke 284
    529882 35 edk-d(10)-kdke 284
    529902 44 k-(d4)-k-(d4)-k-(d4)-ke 284
    529559 71 eek-d(10)-kke 26
    529584 57 kee-d(10)-kke 26
    529609 58 edk-d(10)-kke 26
    529634 49 kde-d(10)-kke 26
    529659 52 kddk-d(9)-kke 26
    529684 48 kdde-d(9)-kke 26
    529709 61 eddk-d(9)-kke 26
    529922 52 eeee-d(9)-kke 26
    529344 50 eek-d(10)-keke 285
    529138 32 eee-d(10)-kkk 286
    529158 75 kkk-d(10)-eee 286
    529184 75 eek-d(10)-keke 215
    529221 78 eek-d(10)-kkke 215
    529127 67 eee-d(10)-kkk 287
    529147 79 kkk-d(10)-eee 287
    529346 58 eek-d(10)-keke 288
    529348 65 eek-d(10)-keke 289
    529350 77 eek-d(10)-keke 290
    529813 20 k-d(10)-kekee 291
    529832 47 k-d(10)-kdkee 291
    529734 63 ke-d(10)-keke 292
    529754 58 ek-d(10)-keke 292
    529774 49 ke-d(10)-kdke 292
    529794 51 ek-d(10)-kdke 292
    529863 64 kde-d(10)-kdke 293
    529883 78 edk-d(10)-kdke 293
    529903 36 k-d(4)-k-d(4)-k-d(4)-ke 293
    529560 71 eek-d(10)-kke 27
    529585 70 kee-d(10)-kke 27
    529610 66 edk-d(10)-kke 27
    529635 45 kde-d(10)-kke 27
    529660 53 kddk-d(9)-kke 27
    529685 42 kdde-d(9)-kke 27
    529710 60 eddk-d(9)-kke 27
    529923 63 eeee-d(9)-kke 27
    529196 74 eek-d(10)-keke 294
    529233 80 eek-d(10)-kkke 294
    529139 75 eee-d(10)-kkk 295
    529159 62 kkk-d(10)-eee 295
    529352 74 eek-d(10)-keke 296
    529354 67 eek-d(10)-keke 297
    529197 43 eek-d(10)-keke 298
    529234 58 eek-d(10)-kkke 298
    529140 29 eee-d(10)-kkk 299
    529160 59 kkk-d(10)-eee 299
    529180 80 eek-d(10)-keke 216
    529217 79 eek-d(10)-kkke 216
    529814 51 k-d(10)-kekee 300
    529833 52 k-d(10)-kdkee 300
    529735 43 ke-d(10)-keke 301
    529755 60 ek-d(10)-keke 301
    529775 38 ke-d(10)-kdke 301
    529795 58 ek-d(10)-kdke 301
    529864 41 kde-d(10)-kdke 302
    529884 48 edk-d(10)-kdke 302
    529904 44 k-d(4)-k-(d4)-k-d(4)-ke 302
    529934 61 eek-d(10)-keke 302
    529356 71 eek-d(10)-keke 303
    529561 75 eek-d(10)-kke 28
    529586 65 kee-d(10)-kke 28
    529611 54 edk-d(10)-kke 28
    529636 39 kde-d(10)-kke 28
    529661 67 kddk-d(9)-kke 28
    529686 66 kdde-d(9)-kke 28
    529711 60 eddk-d(9)-kke 28
    529924 62 eeee-d(9)-kke 28
    529358 82 eek-d(10)-keke 304
    529181 79 eek-d(10)-keke 217
    529218 73 eek-d(10)-kkke 217
    529182 85 eek-d(10)-keke 218
    529219 84 eek-d(10)-kkke 218
    529360 84 eek-d(10)-keke 305
    529362 87 eek-d(10)-keke 306
    529364 81 eek-d(10)-keke 307
    529366 77 eek-d(10)-keke 308
    529198 28 eek-d(10)-keke 309
    529235 8 eek-d(10)-kkke 309
    529141 34 eee-d(10)-kkk 310
    529161 66 kkk-d(10)-eee 310
    529368 27 eek-d(10)-keke 311
    529370 44 eek-d(10)-keke 312
    529372 61 eek-d(10)-keke 313
    529374 71 eek-d(10)-keke 314
    529376 63 eek-d(10)-keke 315
    529378 68 eek-d(10)-keke 316
    529380 79 eek-d(10)-keke 317
    529382 77 eek-d(10)-keke 318
    529384 75 eek-d(10)-keke 319
    529386 40 eek-d(10)-keke 320
    529240 73 eek-d(10)-keke 321
    529241 67 eek-d(10)-keke 322
    529242 42 eek-d(10)-keke 323
    529243 60 eek-d(10)-keke 324
    529388 65 eek-d(10)-keke 325
    529815 37 k-d(10)-kekee 326
    529834 44 k-d(10)-kdkee 326
    529736 47 ke-d(10)-keke 327
    529756 78 ek-d(10)-keke 327
    529776 37 ke-d(10)-kdke 327
    529796 71 ek-d(10)-kdke 327
    529865 70 kde-d(10)-kdke 328
    529885 59 edk-d(10)-kdke 328
    529905 54 k-(d4)-k-(d4)-k-(d4)-ke 328
    529935 70 eek-d(10)-keke 328
    529562 87 eek-d(10)-kke 29
    529587 68 kee-d(10)-kke 29
    529612 67 edk-d(10)-kke 29
    529637 64 kde-d(10)-kke 29
    529662 62 kddk-d(9)-kke 29
    529687 63 kdde-d(9)-kke 29
    529712 61 eddk-d(9)-kke 29
    529925 61 eeee-d(9)-kke 29
    529816 77 k-d(10)-kekee 329
    529835 80 k-d(10)-kdkee 329
    529737 82 ke-d(10)-keke 330
    529757 83 ek-d(10)-keke 330
    529777 68 ke-d(10)-kdke 330
    529797 77 ek-d(10)-kdke 330
    529866 15 kde-d(10)-kdke 331
    529886 71 edk-d(10)-kdke 331
    529906 63 k-(d4)-k-(d4)-k-(d4)-ke 331
    529936 78 eek-d(10)-keke 331
    529563 89 eek-d(10)-kke 30
    529588 84 kee-d(10)-kke 30
    529613 80 edk-d(10)-kke 30
    529638 48 kde-d(10)-kke 30
    529663 85 kddk-d(9)-kke 30
    529688 42 kdde-d(9)-kke 30
    529713 81 eddk-d(9)-kke 30
    529926 67 eeee-d(9)-kke 30
    529390 53 eek-d(10)-keke 332
    529392 63 eek-d(10)-keke 333
    529394 58 eek-d(10)-keke 334
    529396 56 eek-d(10)-keke 335
    529398 62 eek-d(10)-keke 336
    529400 44 eek-d(10)-keke 337
    529402 39 eek-d(10)-keke 338
    529404 46 eek-d(10)-keke 339
    529406 63 eek-d(10)-keke 340
    529244 58 eek-d(10)-keke 341
    529245 68 eek-d(10)-keke 342
    529246 60 eek-d(10)-keke 343
    529247 36 eek-d(10)-keke 344
    529248 43 eek-d(10)-keke 345
    529249 23 eek-d(10)-keke 346
    529250 69 eek-d(10)-keke 347
    529251 15 eek-d(10)-keke 348
    529252 44 eek-d(10)-keke 349
    529253 42 eek-d(10)-keke 350
    529408 67 eek-d(10)-keke 351
    529410 19 eek-d(10)-keke 352
    529412 57 eek-d(10)-keke 353
    529414 80 eek-d(10)-keke 354
    529416 85 eek-d(10)-keke 355
    529418 70 eek-d(10)-keke 356
    529420 78 eek-d(10)-keke 357
    529422 19 eek-d(10)-keke 358
    529424 48 eek-d(10)-keke 359
    529426 66 eek-d(10)-keke 360
    529428 59 eek-d(10)-keke 361
    529430 83 eek-d(10)-keke 362
    529432 84 eek-d(10)-keke 363
    529199 71 eek-d(10)-keke 364
    529236 76 eek-d(10)-kkke 364
    529142 64 eee-d(10)-kkk 365
    529162 60 kkk-d(10)-eee 365
    529254 46 eek-d(10)-keke 366
    529255 52 eek-d(10)-keke 367
    529256 57 eek-d(10)-keke 368
    529257 55 eek-d(10)-keke 369
    529258 3 eek-d(10)-keke 370
    529259 71 eek-d(10)-keke 371
    529260 72 eek-d(10)-keke 372
    529261 56 eek-d(10)-keke 373
    529262 56 eek-d(10)-keke 374
    529263 59 eek-d(10)-keke 375
    529264 49 eek-d(10)-keke 376
    529434 83 eek-d(10)-keke 377
    529436 80 eek-d(10)-keke 378
    529438 79 eek-d(10)-keke 379
    529440 87 eek-d(10)-keke 380
    529442 68 eek-d(10)-keke 381
    529443 72 eek-d(10)-keke 382
    529444 68 eek-d(10)-keke 383
    529445 85 eek-d(10)-keke 384
    529446 72 eek-d(10)-keke 385
    529447 60 eek-d(10)-keke 386
    529448 77 eek-d(10)-keke 387
    529807 78 k-d(10)-kekee 388
    529826 61 k-d(10)-kdkee 388
    529449 81 eek-d(10)-keke 389
    529728 75 ke-d(10)-keke 390
    529748 80 ek-d(10)-keke 390
    529768 68 ke-d(10)-kdke 390
    529788 74 ek-d(10)-kdke 390
    529857 67 kde-d(10)-kdke 389
    529877 77 edk-d(10)-kdke 389
    529897 26 k-(d4)-k-(d4)-k-(d4)-ke 389
    529200 78 eek-d(10)-keke 391
    529237 84 eek-d(10)-kkke 391
    529564 90 eek-d(10)-kke 34
    529589 86 kee-d(10)-kke 34
    529614 82 edk-d(10)-kke 34
    529639 80 kde-d(10)-kke 34
    529664 69 kddk-d(9)-kke 34
    529689 71 kdde-d(9)-kke 34
    529714 73 eddk-d(9)-kke 34
    529917 73 eeee-d(9)-kke 34
    529143 68 eee-d(10)-kkk 392
    529163 50 kkk-d(10)-eee 392
    529201 76 eek-d(10)-keke 393
    529238 72 eek-d(10)-kkke 393
    529144 57 eee-d(10)-kkk 394
    529164 71 kkk-d(10)-eee 394
    529450 91 eek-d(10)-keke 395
    529451 85 eek-d(10)-keke 396
    529266 63 eek-d(10)-keke 397
    529806 52 k-d(10)-kekee 398
    529825 44 k-d(10)-kdkee 398
    529267 56 eek-d(10)-keke 399
    529727 67 ke-d(10)-keke 400
    529747 63 ek-d(10)-keke 400
    529767 67 ke-d(10)-kdke 400
    529787 68 ek-d(10)-kdke 400
    529856 42 kde-d(10)-kdke 399
    529876 36 edk-d(10)-kdke 399
    529896 56 k-(d4)-k-(d4)-k-(d4)-ke 399
    529546 65 eek-d(10)-kke 248
    529571 80 kee-d(10)-kke 248
    529596 43 edk-d(10)-kke 248
    529621 38 kde-d(10)-kke 248
    529646 68 kddk-d(9)-kke 248
    529671 50 kdde-d(9)-kke 248
    529696 53 eddk-d(9)-kke 248
    529916 22 eeee-d(9)-kke 248
    529547 86 eek-d(10)-kke 37
    529572 75 kee-d(10)-kke 37
    529597 58 edk-d(10)-kke 37
    529622 58 kde-d(10)-kke 37
    529647 18 kddk-d(9)-kke 37
    529672 23 kdde-d(9)-kke 37
    529697 28 eddk-d(9)-kke 37
    529928 36 eeee-d(9)-kke 37
    529452 63 eek-d(10)-keke 401
    529453 73 eek-d(10)-keke 402
    529454 82 eek-d(10)-keke 403
    529455 84 eek-d(10)-keke 404
    529202 61 eek-d(10)-keke 405
    529239 59 eek-d(10)-kkke 405
    529145 54 eee-d(10)-kkk 406
    529165 77 kkk-d(10)-eee 406
    529456 69 eek-d(10)-keke 407
    529457 81 eek-d(10)-keke 408
    529458 72 eek-d(10)-keke 409
    529459 86 eek-d(10)-keke 410
    529460 88 eek-d(10)-keke 411
    529817 46 k-d(10)-kekee 412
    529836 49 k-d(10)-kdkee 412
    529738 51 ke-d(10)-keke 413
    529758 53 ek-d(10)-keke 413
    529778 39 ke-d(10)-kdke 413
    529798 52 ek-d(10)-kdke 413
    529867 56 kde-d(10)-kdke 414
    529887 68 edk-d(10)-kdke 414
    529907 28 k-(d4)-k-(d4)-k-(d4)-ke 414
    529938 64 eek-d(10)-keke 414
    529565 81 eek-d(10)-kke 38
    529590 49 kee-d(10)-kke 38
    529615 65 edk-d(10)-kke 38
    529640 54 kde-d(10)-kke 38
    529665 77 kddk-d(9)-kke 38
    529690 77 kdde-d(9)-kke 38
    529715 63 eddk-d(9)-kke 38
    529927 62 eeee-d(9)-kke 38
    529185 66 eek-d(10)-keke 221
    529222 62 eek-d(10)-kkke 221
    529808 75 k-d(10)-kekee 89
    529827 67 k-d(10)-kdkee 89
    529128 64 eee-d(10)-kkk 415
    529148 78 kkk-d(10)-eee 415
    529461 87 eek-d(10)-keke 416
    529729 71 ke-d(10)-keke 415
    529749 83 ek-d(10)-keke 415
    529769 63 ke-d(10)-kdke 415
    529789 10 ek-d(10)-kdke 415
    529800 69 k-d(10)-kekee 415
    529819 78 k-d(10)-kdkee 415
    529858 60 kde-d(10)-kdke 416
    529878 75 edk-d(10)-kdke 416
    529898 34 k-(d4)-k-(d4)-k-(d4)-ke 416
    529566 61 eek-d(10)-kke 39
    529591 71 kee-d(10)-kke 39
    529616 71 edk-d(10)-kke 39
    529641 65 kde-d(10)-kke 39
    529666 70 kddk-d(9)-kke 39
    529691 67 kdde-d(9)-kke 39
    529716 75 eddk-d(9)-kke 39
    529721 71 ke-d(10)-keke 39
    529741 81 ek-d(10)-keke 39
    529761 66 ke-d(10)-kdke 39
    529781 65 ek-d(10)-kdke 39
    529801 71 k-d(10)-kekee 39
    529820 74 k-d(10)-kdkee 39
    529850 63 kde-d(10)-kdke 417
    529870 72 edk-d(10)-kdke 417
    529890 23 k-(d4)-k-(d4)-k-(d4)-ke 417
    529918 54 eeee-d(9)-kke 39
    529567 75 eek-d(10)-kke 262
    529592 80 kee-d(10)-kke 262
    529617 65 edk-d(10)-kke 262
    529642 62 kde-d(10)-kke 262
    529667 75 kddk-d(9)-kke 262
    529692 53 kdde-d(9)-kke 262
    529717 69 eddk-d(9)-kke 262
    529722 74 ke-d(10)-keke 262
    529742 81 ek-d(10)-keke 262
    529762 66 ke-d(10)-kdke 262
    529782 68 ek-d(10)-kdke 262
    529851 68 kde-d(10)-kdke 418
    529871 77 edk-d(10)-kdke 418
    529891 36 k-(d4)-k-(d4)-k-(d4)-ke 418
    529910 60 eeee-d(9)-kke 262
    529568 79 eek-d(10)-kke 263
    529593 70 kee-d(10)-kke 263
    529618 77 edk-d(10)-kke 263
    529643 72 kde-d(10)-kke 263
    529668 73 kddk-d(9)-kke 263
    529693 62 kdde-d(9)-kke 263
    529718 69 eddk-d(9)-kke 263
    529911 66 eeee-d(9)-kke 263
    529462 76 eek-d(10)-keke 419
    529268 18 eek-d(10)-keke 420
    529187 46 eek-d(10)-keke 421
    529224 48 eek-d(10)-kkke 421
    529130 34 eee-d(10)-kkk 422
    529150 51 kkk-d(10)-eee 422
    529549 85 eek-d(10)-kke 42
    529574 81 kee-d(10)-kke 42
    529599 64 edk-d(10)-kke 42
    529624 68 kde-d(10)-kke 42
    529649 77 kddk-d(9)-kke 42
    529674 65 kdde-d(9)-kke 42
    529699 63 eddk-d(9)-kke 42
    529931 59 eeee-d(9)-kke 42
    529810 80 k-d(10)-kekee 423
    529829 67 k-d(10)-kdkee 423
    529269 65 eek-d(10)-keke 424
    529731 66 ke-d(10)-keke 425
    529751 76 ek-d(10)-keke 425
    529771 73 ke-d(10)-kdke 425
    529791 65 ek-d(10)-kdke 425
    529860 73 kde-d(10)-kdke 424
    529880 74 edk-d(10)-kdke 424
    529900 62 k-(d4)-k-(d4)-k-(d4)-ke 424
    529270 69 eek-d(10)-keke 480
    529550 81 eek-d(10)-kke 44
    529575 88 kee-d(10)-kke 44
    529600 78 edk-d(10)-kke 44
    529625 74 kde-d(10)-kke 44
    529650 81 kddk-d(9)-kke 44
    529675 76 kdde-d(9)-kke 44
    529700 73 eddk-d(9)-kke 44
    529920 67 eeee-d(9)-kke 44
    529271 43 eek-d(10)-keke 427
    529272 0 eek-d(10)-keke 428
    529273 62 eek-d(10)-keke 429
    529274 78 eek-d(10)-keke 430
    529275 70 eek-d(10)-keke 431
    529276 73 eek-d(10)-keke 432
    529277 71 eek-d(10)-keke 433
    529278 72 eek-d(10)-keke 434
    529279 10 eek-d(10)-keke 435
    529280 11 eek-d(10)-keke 436
    529281 82 eek-d(10)-keke 437
    529282 87 eek-d(10)-keke 438
    529803 71 k-d(10)-kekee 250
    529822 72 k-d(10)-kdkee 250
    529724 76 ke-d(10)-keke 439
    529744 81 ek-d(10)-keke 439
    529764 65 ke-d(10)-kdke 439
    529784 68 ek-d(10)-kdke 439
    529853 64 kde-d(10)-kdke 440
    529873 69 edk-d(10)-kdke 440
    529893 45 k-(d4)-k-(d4)-k-(d4)-ke 440
    529937 81 eek-d(10)-keke 440
    529551 88 eek-d(10)-kke 48
    529576 71 kee-d(10)-kke 48
    529601 74 edk-d(10)-kke 48
    529626 72 kde-d(10)-kke 48
    529651 85 kddk-d(9)-kke 48
    529676 67 kdde-d(9)-kke 48
    529701 82 eddk-d(9)-kke 48
    529913 76 eeee-d(9)-kke 48
    529811 56 k-d(10)-kekee 441
    529830 46 k-d(10)-kdkee 441
    529732 63 ke-d(10)-keke 442
    529752 72 ek-d(10)-keke 442
    529772 61 ke-d(10)-kdke 442
    529792 68 ek-d(10)-kdke 442
    529861 54 kde-d(10)-kdke 443
    529881 78 edk-d(10)-kdke 443
    529901 29 k-(d4)-k-(d4)-k-(d4)-ke 443
    529939 67 eek-d(10)-keke 443
    529283 70 eek-d(10)-keke 444
    529552 72 eek-d(10)-kke 49
    529577 80 kee-d(10)-kke 49
    529602 64 edk-d(10)-kke 49
    529627 56 kde-d(10)-kke 49
    529652 57 kddk-d(9)-kke 49
    529677 43 kdde-d(9)-kke 49
    529702 54 eddk-d(9)-kke 49
    529921 42 eeee-d(9)-kke 49
    529284 76 eek-d(10)-keke 445
    529285 77 eek-d(10)-keke 446
    529286 68 eek-d(10)-keke 447
    529287 65 eek-d(10)-keke 448
    529719 73 ke-d(10)-keke 264
    529739 83 ek-d(10)-keke 264
    529759 63 ke-d(10)-kdke 264
    529779 70 ek-d(10)-kdke 244
    529848 60 kde-d(10)-kdke 449
    529868 63 edk-d(10)-kdke 449
    529888 53 k-(d4)-k-(d4)-k-(d4)-ke 449
    529553 81 eek-d(10)-kke 265
    529578 65 kee-d(10)-kke 265
    529603 60 edk-d(10)-kke 265
    529628 59 kde-d(10)-kke 265
    529653 76 kddk-d(9)-kke 265
    529678 56 kdde-d(9)-kke 265
    529703 68 eddk-d(9)-kke 265
    529908 69 eeee-d(9)-kke 265
    529168 64 eek-d(10)-keke 450
    529205 62 eek-d(10)-kkke 450
    529290 53 eek-d(10)-keke 451
    529802 57 k-d(10)-kekee 452
    529821 61 k-d(10)-kdkee 452
    529292 74 eek-d(10)-keke 453
    529723 68 ke-d(10)-keke 454
    529743 84 ek-d(10)-keke 454
    529763 64 ke-d(10)-kdke 454
    529783 72 ek-d(10)-kdke 454
    529852 66 kde-d(10)-kdke 453
    529872 62 edk-d(10)-kdke 453
    529892 43 k-(d4)-k-(d4)-k-(d4)-ke 453
    529554 80 eek-d(10)-kke 252
    529579 83 kee-d(10)-kke 252
    529604 73 edk-d(10)-kke 252
    529629 64 kde-d(10)-kke 252
    529654 69 kddk-d(9)-kke 252
    529679 52 kdde-d(9)-kke 252
    529704 63 eddk-d(9)-kke 252
    529912 64 eeee-d(9)-kke 252
    529294 74 eek-d(10)-keke 455
    529296 52 eek-d(10)-keke 456
    529298 60 eek-d(10)-keke 457
    529300 71 eek-d(10)-keke 458
    529188 79 eek-d(10)-keke 459
    529225 78 eek-d(10)-kkke 459
    529131 58 eee-d(10)-kkk 460
    529151 71 kkk-d(10)-eee 460
    529302 74 eek-d(10)-keke 461
    529189 64 eek-d(10)-keke 222
    529226 50 eek-d(10)-kkke 222
    529132 78 eee-d(10)-kkk 462
    529152 62 kkk-d(10)-eee 462
    529190 76 eek-d(10)-keke 223
    529227 88 eek-d(10)-kkke 250
    529133 81 eee-d(10)-kkk 463
    529153 68 kkk-d(10)-eee 463
    529191 78 eek-d(10)-keke 224
    529228 85 eek-d(10)-kkke 224
    529134 75 eee-d(10)-kkk 464
    529154 61 kkk-d(10)-eee 464
    529304 89 eek-d(10)-keke 465
    529306 84 eek-d(10)-keke 466
    529308 68 eek-d(10)-keke 467
    529310 59 eek-d(10)-keke 468
    529169 79 eek-d(10)-keke 469
    529206 82 eek-d(10)-kkke 469
    529312 68 eek-d(10)-keke 470
    529314 61 eek-d(10)-keke 471
    529316 62 eek-d(10)-keke 472
    529555 78 eek-d(10)-kke 59
    529580 73 kee-d(10)-kke 59
    529605 71 edk-d(10)-kke 59
    529630 64 kde-d(10)-kke 59
    529655 63 kddk-d(9)-kke 59
    529680 43 kdde-d(9)-kke 59
    529705 63 eddk-d(9)-kke 59
    529932 60 eeee-d(9)-kke 59
    529318 82 eek-d(10)-keke 473
    529170 85 eek-d(10)-keke 474
    529207 88 eek-d(10)-kkke 474
    529171 81 eek-d(10)-keke 475
    529208 84 eek-d(10)-kkke 475
    529805 40 k-d(10)-kekee 476
    529824 32 k-d(10)-kdkee 476
    529320 74 eek-d(10)-keke 477
    529726 80 ke-d(10)-keke 478
    529746 82 ek-d(10)-keke 478
    529766 63 ke-d(10)-kdke 478
    529786 69 ek-d(10)-kdke 478
    529855 39 kde-d(10)-kdke 477
    529875 40 edk-d(10)-kdke 477
    529895 27 k-(d4)-k-(d4)-k-(d4)-ke 477
    529556 72 eek-d(10)-kke 61
    529581 68 kee-d(10)-kke 61
    529606 54 edk-d(10)-kke 61
    529631 29 kde-d(10)-kke 61
    529656 74 kddk-d(9)-kke 61
    529681 32 kdde-d(9)-kke 61
    529706 41 eddk-d(9)-kke 61
    529915 51 eeee-d(9)-kke 61
    529172 88 eek-d(10)-keke 226
    529209 87 eek-d(10)-kkke 226
    529173 92 eek-d(10)-keke 227
    529210 89 eek-d(10)-kkke 227
    529183 85 eek-d(10)-keke 479
    529220 92 eek-d(10)-kkke 479
    529126 83 eee-d(10)-kkk 257
    529146 84 kkk-d(10)-eee 257
    529174 85 eek-d(10)-keke 480
    529211 86 eek-d(10)-kkke 480
    529322 71 eek-d(10)-keke 481
    529324 79 eek-d(10)-keke 482
    529326 85 eek-d(10)-keke 483
    529175 92 eek-d(10)-keke 228
    529212 92 eek-d(10)-kkke 228
    529176 89 eek-d(10)-keke 229
    529213 90 eek-d(10)-kkke 229
    529804 89 k-d(10)-kekee 259
    529823 89 k-d(10)-kdkee 259
    529166 83 eek-d(10)-keke 230
    529203 86 eek-d(10)-kkke 230
    529725 92 ke-d(10)-keke 260
    529745 91 ek-d(10)-keke 260
    529765 88 ke-d(10)-kdke 260
    529785 91 ek-d(10)-kdke 260
    529799 89 k-d(10)-kekee 260
    529818 88 k-d(10)-kdkee 260
    529854 90 kde-d(10)-kdke 230
    529874 81 edk-d(10)-kdke 230
    529894 60 k-(d4)-k-(d4)-k-(d4)-ke 230
    529167 71 eek-d(10)-keke 231
    529204 70 eek-d(10)-kkke 231
    529557 86 eek-d(10)-kke 69
    529582 86 kee-d(10)-kke 69
    529607 84 edk-d(10)-kke 69
    529632 81 kde-d(10)-kke 69
    529657 85 kddk-d(9)-kke 69
    529682 78 kdde-d(9)-kke 69
    529707 79 eddk-d(9)-kke 69
    529720 75 ke-d(10)-keke 69
    529740 70 ek-d(10)-keke 69
    529760 78 ke-d(10)-kdke 69
    529780 83 ek-d(10)-kdke 69
    529849 80 kde-d(10)-kdke 231
    529869 72 edk-d(10)-kdke 231
    529889 49 k-(d4)-k-(d4)-k-(d4)-ke 231
    529914 69 eeee-d(9)-kke 69
    529328 68 eek-d(10)-keke 484
    529558 71 eek-d(10)-kke 71
    529583 81 kee-d(10)-kke 71
    529608 68 edk-d(10)-kke 71
    529633 73 kde-d(10)-kke 71
    529658 63 kddk-d(9)-kke 71
    529683 74 kdde-d(9)-kke 71
    529708 70 eddk-d(9)-kke 71
    529909 59 eeee-d(9)-kke 71
    529192 51 eek-d(10)-keke 485
    529229 69 eek-d(10)-kkke 485
    529135 54 eee-d(10)-kkk 486
    529155 56 kkk-d(10)-eee 486
    529330 37 eek-d(10)-keke 487
    e = 2′ -MOE, k = cEt, d = 2′-deoxyribonucleoside
    • Example 20 Design of Modified Oligonucleotides Comprising 2′-O-Methoxyethyl (2′-MOE) or 6′-(S)—CH3 Bicyclic Nucleoside (e.g cEt) Modifications
  • Based on the activity of the antisense oligonucleotides listed above, additional antisense oligonucleotides were designed targeting a Target-X nucleic acid targeting start positions 1147, 1154 or 12842 of Target-X.
  • The newly designed chimeric antisense oligonucleotides are 16 or 17 nucleotides in length and their motifs are described in Table 35. The chemistry column of Table 35 presents the sugar motif of each oligonucleotide, wherein “e” indicates a 2′-O-methoxyethyl (2′-MOE) nucleoside, “k” indicates a 6′-(S)—CH3 bicyclic nucleoside (e.g cEt) and “d” indicates a 2′-deoxyribonucleoside. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosine.
  • Each gapmer listed in Table 35 is targeted to the human Target-X genomic sequence.
  • TABLE 35
    Chimeric antisense oligonucleotides targeted to Target-X
    ISIS No Chemistry SEQ CODE
    529544 eek-d(10)-kke 21
    529569 kee-d(10)-kke 21
    529594 edk-d(10)-kke 21
    529619 kde-d(10)-kke 21
    529644 kddk-d(9)-kke 21
    529669 kdde-d(9)-kke 21
    529694 eddk-d(9)-kke 21
    529929 eeee-d(9)-kke 21
    529809 k-d(10)-kekee 488
    529828 k-d(10)-kdkee 488
    529730 ke-d(10)-keke 489
    529750 ek-d(10)-keke 489
    529770 ke-d(10)-kdke 489
    529790 ek-d(10)-kdke 489
    529859 kde-d(10)-kdke 490
    529879 edk-d(10)-kdke 490
    529899 k-d(4)-k-d(4)-k-d(4)-ke 490
    529545 eek-d(10)-kke 22
    529570 kee-d(10)-kke 22
    529595 edk-d(10)-kke 22
    529620 kde-d(10)-kke 22
    529645 kddk-d(9)-kke 22
    529670 kdde-d(9)-kke 22
    529695 eddk-d(9)-kke 22
    529919 eeee-d(9)-kke 22
    529548 eek-d(10)-kke 41
    529573 kee-d(10)-kke 41
    529598 edk-d(10)-kke 41
    529623 kde-d(10)-kke 41
    529648 kddk-d(9)-kke 41
    529673 kdde-d(9)-kke 41
    529698 eddk-d(9)-kke 41
    529930 eeee-d(9)-kke 41
    e = 2′-MOE, k = cEt, d = 2′-deoxyribonucleoside
    • Example 21 Modified Oligonucleotides Comprising 2′-O-Methoxyethyl (2′-MOE) and 6′-(S)—CH3 Bicyclic Nucleoside (e.g cEt) Modifications Targeting Human Target-X
  • Additional antisense oligonucleotides were designed targeting a Target-X nucleic acid and were tested for their effects on Target-X mRNA in vitro. ISIS 472998 and ISIS 515554, described in the Examples above were also included in the screen.
  • The newly designed chimeric antisense oligonucleotides are 16 nucleotides in length and their motifs are described in Table 36. The chemistry column of Table 36 presents the sugar motif of each oligonucleotide, wherein “e” indicates a 2′-O-methoxyethyl (2′-MOE) nucleoside, “k” indicates a 6′-(S)—CH3 bicyclic nucleoside (e.g cEt) and “d” indicates a 2′-deoxyribonucleoside. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosine.
  • Each gapmer listed in Table 36 is targeted to the human Target-X genomic sequence.
  • Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 2,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS2927 was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells.
  • TABLE 36
    Inhibition of human Target-X mRNA levels by chimeric
    antisense oligonucleotides targeted to Target-X
    ISIS No % inhibition Chemistry SEQ CODE
    472998 88 kk-d(10)-kk 74
    515554 75 eee-d(10)-kkk 493
    534530 92 keke-d(9)-kek 491
    534563 92 kek-d(9)-ekek 491
    534596 88 ekee-d(9)-kke 491
    534629 89 eke-d(9)-ekke 491
    534662 87 eekk-d(9)-eke 491
    534695 92 eek-d(9)-keke 491
    534732 90 ekek-d(8)-keke 491
    534767 92 keek-d(8)-keek 491
    534802 93 ekk-d(10)-kke 491
    534832 83 edk-d(10)-kke 491
    534862 72 kde-d(10)-kke 491
    534892 82 eek-d(10)-kke 491
    534922 80 kddk-d(9)-kke 491
    534952 72 kdde-d(9)-kke 491
    534982 77 eddk-d(9)-kke 491
    535012 70 eeee-d(9)-kke 491
    535045 84 eeee-d(9)-kkk 491
    535078 87 eeek-d(9)-kke 491
    535111 63 eeeee-d(8)-kke 491
    535144 69 ededk-d(8)-kke 491
    535177 68 edkde-d(8)-kke 491
    534531 61 keke-d(9)-kek 492
    534564 30 kek-d(9)-ekek 492
    534597 67 ekee-d(9)-kke 492
    534630 54 eke-d(9)-ekke 492
    534663 94 eekk-d(9)-eke 492
    534696 68 eek-d(9)-keke 492
    534733 44 ekek-d(8)-keke 492
    534768 55 keek-d(8)-keek 492
    534803 73 ekk-d(10)-kke 492
    534833 65 edk-d(10)-kke 492
    534863 53 kde-d(10)-kke 492
    534893 61 eek-d(10)-kke 492
    534923 70 kddk-d(9)-kke 492
    534953 54 kdde-d(9)-kke 492
    534983 58 eddk-d(9)-kke 492
    535013 52 eeee-d(9)-kke 492
    535046 67 eeee-d(9)-kkk 492
    535079 57 eeek-d(9)-kke 492
    535112 42 eeeee-d(8)-kke 492
    535145 41 ededk-d(8)-kke 492
    535178 35 edkde-d(8)-kke 492
    534565 87 kek-d(9)-ekek 493
    534598 72 ekee-d(9)-kke 493
    534631 70 eke-d(9)-ekke 493
    534664 94 eekk-d(9)-eke 493
    534697 90 eek-d(9)-keke 493
    534734 74 ekek-d(8)-keke 493
    534769 80 keek-d(8)-keek 493
    534804 87 ekk-d(10)-kke 493
    534834 76 edk-d(10)-kke 493
    534864 56 kde-d(10)-kke 493
    534894 67 eek-d(10)-kke 493
    534924 71 kddk-d(9)-kke 493
    534954 54 kdde-d(9)-kke 493
    534984 48 eddk-d(9)-kke 493
    535014 43 eeee-d(9)-kke 493
    535047 60 eeee-d(9)-kkk 493
    535080 64 eeek-d(9)-kke 493
    535113 32 eeeee-d(8)-kke 493
    535146 31 ededk-d(8)-kke 493
    535179 28 edkde-d(8)-kke 493
    534533 82 keke-d(9)-kek 494
    534566 88 kek-d(9)-ekek 494
    534599 65 ekee-d(9)-kke 494
    534632 69 eke-d(9)-ekke 494
    534665 87 eekk-d(9)-eke 494
    534698 64 eek-d(9)-keke 494
    534735 63 ekek-d(8)-keke 494
    534770 66 keek-d(8)-keek 494
    534805 87 ekk-d(10)-kke 494
    534835 68 edk-d(10)-kke 494
    534865 66 kde-d(10)-kke 494
    534895 57 eek-d(10)-kke 494
    534925 82 kddk-d(9)-kke 494
    534955 76 kdde-d(9)-kke 494
    534985 71 eddk-d(9)-kke 494
    535015 59 eeee-d(9)-kke 494
    535048 69 eeee-d(9)-kkk 494
    535081 67 eeek-d(9)-kke 494
    535114 37 eeeee-d(8)-kke 494
    535147 32 ededk-d(8)-kke 494
    535180 31 edkde-d(8)-kke 494
    534534 94 keke-d(9)-kek 234
    534567 92 kek-d(9)-ekek 234
    534600 92 ekee-d(9)-kke 234
    534633 91 eke-d(9)-ekke 234
    534666 89 eekk-d(9)-eke 234
    534699 91 eek-d(9)-keke 234
    534736 83 ekek-d(8)-keke 234
    534771 80 keek-d(8)-keek 234
    534806 96 ekk-d(10)-kke 234
    534836 86 edk-d(10)-kke 234
    534866 82 kde-d(10)-kke 234
    534896 82 eek-d(10)-kke 234
    534926 89 kddk-d(9)-kke 234
    534956 91 kdde-d(9)-kke 234
    534986 87 eddk-d(9)-kke 234
    535016 83 eeee-d(9)-kke 234
    535049 87 eeee-d(9)-kkk 234
    535082 87 eeek-d(9)-kke 234
    535115 77 eeeee-d(8)-kke 234
    535148 73 ededk-d(8)-kke 234
    535181 68 edkde-d(8)-kke 234
    534535 66 keke-d(9)-kek 236
    534568 85 kek-d(9)-ekek 236
    534601 51 ekee-d(9)-kke 236
    534634 80 eke-d(9)-ekke 236
    534667 90 eekk-d(9)-eke 236
    534700 88 eek-d(9)-keke 236
    534737 65 ekek-d(8)-keke 236
    534772 77 keek-d(8)-keek 236
    534807 84 ekk-d(10)-kke 236
    534837 78 edk-d(10)-kke 236
    534867 44 kde-d(10)-kke 236
    534897 82 eek-d(10)-kke 236
    534927 61 kddk-d(9)-kke 236
    534957 58 kdde-d(9)-kke 236
    534987 49 eddk-d(9)-kke 236
    535017 38 eeee-d(9)-kke 236
    535050 32 eeee-d(9)-kkk 236
    535083 43 eeek-d(9)-kke 236
    535116 9 eeeee-d(8)-kke 236
    535149 23 ededk-d(8)-kke 236
    535182 18 edkde-d(8)-kke 236
    534536 89 keke-d(9)-kek 238
    534569 90 kek-d(9)-ekek 238
    534602 85 ekee-d(9)-kke 238
    534635 87 eke-d(9)-ekke 238
    534668 90 eekk-d(9)-eke 238
    534701 92 eek-d(9)-keke 238
    534738 81 ekek-d(8)-keke 238
    534773 79 keek-d(8)-keek 238
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    534692 91 eek-d(9)-keke 258
    534729 91 ekek-d(8)-keke 258
    534764 91 keek-d(8)-keek 258
    534799 96 ekk-d(10)-kke 258
    534829 91 edk-d(10)-kke 258
    534859 87 kde-d(10)-kke 258
    534889 81 eek-d(10)-kke 258
    534919 92 kddk-d(9)-kke 258
    534949 91 kdde-d(9)-kke 258
    534979 84 eddk-d(9)-kke 258
    535009 78 eeee-d(9)-kke 258
    535042 76 eeee-d(9)-kkk 258
    535075 83 eeek-d(9)-kke 258
    535108 64 eeeee-d(8)-kke 258
    535141 69 ededk-d(8)-kke 258
    535174 65 edkde-d(8)-kke 258
    534528 94 keke-d(9)-kek 260
    534561 0 kek-d(9)-ekek 260
    534594 92 ekee-d(9)-kke 260
    534627 90 eke-d(9)-ekke 260
    534660 92 eekk-d(9)-eke 260
    534693 95 eek-d(9)-keke 260
    534730 93 ekek-d(8)-keke 260
    534765 92 keek-d(8)-keek 260
    534800 93 ekk-d(10)-kke 260
    534830 93 edk-d(10)-kke 260
    534860 85 kde-d(10)-kke 260
    534890 91 eek-d(10)-kke 260
    534920 93 kddk-d(9)-kke 260
    534950 90 kdde-d(9)-kke 260
    534980 88 eddk-d(9)-kke 260
    535010 88 eeee-d(9)-kke 260
    535043 89 eeee-d(9)-kkk 260
    535076 88 eeek-d(9)-kke 260
    535109 76 eeeee-d(8)-kke 260
    535142 86 ededk-d(8)-kke 260
    535175 71 edkde-d(8)-kke 260
    534529 70 keke-d(9)-kek 261
    534562 86 kek-d(9)-ekek 261
    534595 56 ekee-d(9)-kke 261
    534628 73 eke-d(9)-ekke 261
    534661 64 eekk-d(9)-eke 261
    534694 75 eek-d(9)-keke 261
    534731 47 ekek-d(8)-keke 261
    534766 30 keek-d(8)-keek 261
    534801 83 ekk-d(10)-kke 261
    534831 84 edk-d(10)-kke 261
    534861 71 kde-d(10)-kke 261
    534891 73 eek-d(10)-kke 261
    534921 55 kddk-d(9)-kke 261
    534951 61 kdde-d(9)-kke 261
    534981 48 eddk-d(9)-kke 261
    535011 54 eeee-d(9)-kke 261
    535044 46 eeee-d(9)-kkk 261
    535077 29 eeek-d(9)-kke 261
    535110 19 eeeee-d(8)-kke 261
    535143 15 ededk-d(8)-kke 261
    535176 37 edkde-d(8)-kke 261
    e = 2′-MOE, k = cEt, d = 2′-deoxynucleoside
    • Example 22 Modified Antisense Oligonucleotides Comprising 2′-O-Methoxyethyl (2′-MOE) and 6′-(S)—CH3Bicyclic Nucleoside (e.g cEt) Modifications Targeting Human Target-X Targeting Intronic Repeats
  • Additional antisense oligonucleotides were designed targeting the intronic repeat regions of Target-X.
  • The newly designed chimeric antisense oligonucleotides and their motifs are described in Table 37. The internucleoside linkages throughout each gapmer are phosphorothioate linkages (P═S) and are designated as “s”. Nucleosides followed by “d” indicate 2′-deoxyribonucleosides. Nucleosides followed by “k” indicate 6′-(S)—CH3 bicyclic nucleosides (e.g cEt). Nucleosides followed by “e” indicate 2′-O-methoxyethyl (2′-MOE) nucleosides. “N” indicates modified or naturally occurring nucleobases (A, T, C, G, U, or 5-methyl C).
  • Each gapmer listed in Table 37 is targeted to the intronic region of human Target-X genomic sequence, designated herein as Target-X.
  • Cultured Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 2,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human primer probe set was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells.
  • TABLE 37
    Inhibition of human Target-X mRNA levels by chimeric antisense
    oligonucleotides targeted to Target-X
    SEQ
    ISIS % SEQ ID
    Sequence (5′ to 3′) No inhibition CODE NO
    Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds 472998 90 508 7
    Nds Nks Nk
    Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds 473327 88  30 6
    Nds Nds Nes Nes Ne
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537024 74 509 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537025 79 510 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537026 76 511 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537028 37 512 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537029 45 513 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537030 67 514 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537031 59 515 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537032  9 516 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537033 65 517 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537034 71 518 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537035 68 519 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537036 74 520 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537038 69 521 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537039 67 522 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537040 68 523 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537041 76 524 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537042 77 525 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537043 70 526 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537044 82 527 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537045 69 528 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537047 35 529 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537049 62 530 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537051 62 531 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537055 16 532 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537056 25 533 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537057 49 534 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537058 49 535 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537059 53 536 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537060 73 537 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537061 70 538 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537062 69 539 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537063 68 540 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537064 71 541 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537065 67 542 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537066 68 543 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537067 71 544 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537068 86 545 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537069 82 546 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537070 87 547 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537792 36 548 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537793 35 549 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537794 35 550 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537795 33 551 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537796 49 552 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537797 54 553 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537798 68 554 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537799 72 555 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537800 69 556 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537801 82 557 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537802 72 558 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537803 72 559 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537804 67 560 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537805 74 561 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537806 70 562 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537809 60 563 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537810 71 564 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537811 69 565 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537812 80 566 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537813 74 567 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537814 54 568 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537837 70 569 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537838 76 570 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537839 76 571 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537840 80 572 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537841 81 573 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537842 75 574 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537843 70 575 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537844 73 576 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537845 59 577 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537846 51 578 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537847 52 579 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537848 41 580 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 537849 44 581 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538160 69 582 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538172 24 583 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538173 23 584 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538185 68 585 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538187 69 585 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538189 81 587 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538191 66 588 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538192 59 589 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538193 16 590 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538194 10 591 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538195 15 592 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538196  3 593 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538197 36 594 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538198 49 595 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538199 47 596 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538200 57 597 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538201 71 598 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538202 60 599 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538203 55 600 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538204 62 601 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538205 68 602 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538228 63 603 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538229 26 604 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538230 75 605 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538231 75 606 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538233 52 607 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538235 26 608 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538237 28 609 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538239 54 610 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538241 73 611 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538242 68 612 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538243 61 613 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538245 75 614 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538253 37 615 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538254 45 616 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538361 56 617 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538378 70 618 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538380 68 619 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 538381 57 620 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540361 71 621 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540362 73 622 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540363 78 623 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540364 89 624 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540365 83 625 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540366 84 626 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540367 65 627 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540368 55 628 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540369 82 629 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540370 86 630 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540371 74 631 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540372 82 632 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540373 81 633 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540374 87 634 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540375 78 635 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540376 69 636 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540377 88 637 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540378 85 638 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540379 77 639 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540380 84 640 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540381 85 641 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540382 69 642 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540383 85 643 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540384 88 644 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540385 87 645 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540386 86 646 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540387 77 647 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540388 86 648 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540389 86 649 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540390 85 650 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540391 83 651 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540392 43 652 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540393 88 653 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540394 68 654 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540395 87 655 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540396 87 656 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540397 59 657 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540398 36 658 6
    Nds Nds Nks Nks Nk
    Nes Nes Nes Nds Nds Nds Nds Nds Nds Nds Nds 540399 81 659 6
    Nds Nds Nks Nks Nk
    • Example 23 High Dose Tolerability of Modified Oligonucleotides Comprising 2′-O-Methoxyethyl (2′-MOE) and 6′-(S)—CH3 Bicyclic Nucleoside (e.g cEt) Modifications Targeting Human Target-X in BALB/c Mice
  • BALB/c mice were treated at a high dose with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.
  • Additionally, the newly designed antisense oligonucleotides were created with the same sequences as the antisense oligonucleotides from the study described above and were also added to this screen targeting intronic repeat regions of Target-X.
  • The newly designed modified antisense oligonucleotides and their motifs are described in Table 38. The internucleoside linkages throughout each gapmer are phosphorothioate linkages (P═S). Nucleosides followed by “d” indicate 2′-deoxyribonucleosides. Nucleosides followed by “k” indicate 6′-(S)—CH3 bicyclic nucleoside (e.g cEt) nucleosides. Nucleosides followed by “e” indicate 2′-O-methoxyethyl (2′-MOE) nucleosides. “N” indicates modified or naturally occurring nucleobases (A, T, C, G, U, or 5-methyl C).
  • Each gapmer listed in Table 38 is targeted to the intronic region of human Target-X genomic sequence, designated herein as Target-X. “Start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence. “Stop site” indicates the 3′-most nucleoside to which the gapmer is targeted human gene sequence.
  • TABLE 38
    Modified antisense oligonucleotides targeted to Target-X
    SEQ
    ISIS SEQ ID
    Sequence (5′ to 3′) No CODE NO
    Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 537721 509 6
    Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 537738 524 6
    Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 537759 539 6
    Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 537761 541 6
    Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 537763 543 6
    Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 537850 548 6
    Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 537858 556 6
    Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 537864 562 6
    Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 537869 565 6
    Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 537872 568 6
    Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 537897 571 6
    Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 540118 582 6
    Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 540138 602 6
    Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 540139 603 6
    Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 540148 612 6
    Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 540153 617 6
    Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 540155 619 6
    Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540162 624 6
    Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540164 626 6
    Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540168 630 6
    Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540172 634 6
    Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540175 637 6
    Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540176 638 6
    Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540178 640 6
    Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540179 641 6
    Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540181 643 6
    Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540182 644 6
    Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540183 645 6
    Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540184 646 6
    Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540186 648 6
    Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540187 649 6
    Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540188 650 6
    Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540191 653 6
    Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540193 655 6
    Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 540194 656 6
    Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544811 547 6
    Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544812 545 6
    Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544813 527 6
    Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544814 557 6
    Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544815 546 6
    Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544816 573 6
    Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544817 572 6
    Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544818 566 6
    Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544819 510 6
    Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544820 525 6
    Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544821 567 6
    Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544826 537 6
    Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544827 538 6
    Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544828 539 6
    Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544829 540 6
    Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 544830 541 6
    Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 545471 542 6
    Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 545472 543 6
    Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 545473 544 6
    Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 545474 558 6
    Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 545475 559 6
    Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 545476 560 6
    Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 545477 561 6
    Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 545478 562 6
    Nes Nes Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nks Nks Ne 545479 556 6
    Nks Nks Nks Nds Nds Nds Nds Nds Nds Nds Nds Nds Nds Nes Nes Ne 537727 514 6
    • Treatment
  • Male BALB/c mice were injected subcutaneously with a single dose of 200 mg/kg of ISIS 422142, ISIS 457851, ISIS 473294, ISIS 473295, ISIS 473327, ISIS 484714, ISIS 515334, ISIS 515338, ISIS 515354, ISIS 515366, ISIS 515380, ISIS 515381, ISIS 515382, ISIS 515384, ISIS 515386, ISIS 515387, ISIS 515388, ISIS 515406, ISIS 515407, ISIS 515408, ISIS 515422, ISIS 515423, ISIS 515424, ISIS 515532, ISIS 515533, ISIS 515534, ISIS 515538, ISIS 515539, ISIS 515558, ISIS 515656, ISIS 515575, ISIS 515926, ISIS 515944, ISIS 515945, ISIS 515948, ISIS 515949, ISIS 515951, ISIS 515952, ISSI 516003, ISIS 516055, ISIS 516057, ISIS 516060, ISIS 516062, ISIS 529126, ISIS 529146, ISIS 529166, ISIS 529170, ISIS 529172, ISIS 529173, ISIS 529174, ISIS 529175, ISSI 529176, ISIS 529182, ISIS 529183, ISIS 529186, ISIS 529282, ISIS 529304, ISIS 529306, ISIS 529360, ISIS 529450, ISIS 529459, ISIS 529460, ISIS 529461, ISIS 529547, ISIS 529550, ISIS 529551, ISIS 529553, ISIS 529557, ISIS 529562, ISIS 529563, ISIS 529564, ISIS 529565, ISIS 529575, ISIS 529582, ISIS 529589, ISIS 529607, ISIS 529614, ISIS 529632, ISIS 529650, ISIS 529651, ISIS 529657, ISIS 529663, ISIS 529725, ISIS 529745, ISIS 529765, ISIS 529785, ISIS 529804, ISIS 529818, ISIS 529823, ISIS 529854, ISIS 534528, ISIS 534534, ISIS 534594, ISIS 534660, ISIS 534663, ISIS 534664, ISIS 534676, ISIS 534677, ISIS 537679, ISIS 537683, ISIS 534693, ISIS 534701, ISIS 534716, ISIS 534730, ISIS 534765, ISIS 534795, ISIS 534796, ISIS 534797, ISIS 534798, ISIS 534799, ISIS 534800, ISIS 534802, ISIS 534806, ISSI 534830, ISIS 534838, ISIS 534888, ISIS 534890, ISIS 534898, ISIS 534911, ISIS 534920, ISIS 534926, ISIS 534937, ISIS 534950, ISSI 534956, ISIS 534980, ISIS 534986, ISIS 535010, ISIS 535043, ISIS 535049, ISIS 535076, ISIS 535082, ISSI 535142, ISIS 537024, ISIS 537030, ISIS 537041, ISIS 537062, ISIS 537064, ISIS 537066, ISIS 537721, ISIS 537727, ISIS 537738, ISIS 537759, ISIS 537761, ISIS 537763, ISIS 537792, ISIS 537800, ISIS 537806, ISIS 537811, ISIS 537814, ISIS 537839, ISIS 537850, ISSI 537858, ISIS 537864, ISIS 537869, ISIS 537872, ISIS 537897, ISIS 538160, ISIS 538196, ISIS 538205, ISIS 538228, ISIS 538242, ISIS 538361, ISIS 538380, ISIS 540118, ISIS 540138, ISIS 540139, ISIS 540148, ISIS 540153, ISIS 540155, ISIS 540162, ISIS 540164, ISIS 540168, ISIS 540172, ISIS 540175, ISIS 540176, ISIS 540178, ISIS 540179, ISIS 540181, ISIS 540182, ISIS 540183, ISIS 540184, ISIS 540186, ISIS 540187, ISIS 540188, ISIS 540191, ISIS 540193, ISIS 540194, ISIS 544811, ISIS 544812, ISIS 544813, ISIS 544814, ISIS 544815, ISIS 544816, ISIS 544817, ISIS 544818, ISIS 544819, ISIS 544820, ISIS 544821, ISIS 544826, ISIS 544827, ISIS 544828, ISIS 544829, ISIS 544830, ISIS 545471, ISIS 545472, ISIS 545473, ISIS 545474, ISIS 545475, ISIS 545476, ISIS 545477, ISIS 545478, and ISIS 545479. One set of male BALB/c mice was injected with a single dose of PBS. Mice were euthanized 96 hours later, and organs and plasma were harvested for further analysis.
    • Plasma Chemistry Markers
  • To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, albumin, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.).
  • ISIS oligonucleotides that did not cause any increase in the levels of transaminases, or which caused an increase within three times the upper limit of normal (ULN) were deemed very tolerable. ISIS oligonucleotides that caused an increase in the levels of transaminases between three times and seven times the ULN were deemed tolerable. Based on these criteria, ISIS 529166, ISIS 529170, ISIS 529175, ISIS 529176, ISIS 529186, ISIS 529282, ISIS 529360, ISIS 529450, ISIS 529459, ISIS 529460, ISIS 529547, ISIS 529549, ISIS 529551, ISIS 529553, ISIS 529557, ISIS 529562, ISIS 529575, ISIS 529582, ISIS 529607, ISIS 529589, ISIS 529632, ISIS 529657, ISIS 529725, ISIS 529745, ISIS 529785, ISIS 529799, ISIS 529804, ISIS 529818, ISIS 529823, ISIS 534950, ISIS 534980, ISIS 535010, ISIS 537030, ISIS 537041, ISIS 537062, ISIS 537064, ISIS 537066, ISIS 537759, ISIS 537792, ISIS 537800, ISIS 537839, ISIS 538228, ISIS 473294, ISIS 473295, ISIS 484714, ISIS 515338, ISIS 515366, ISIS 515380, ISIS 515381, ISIS 515387, ISIS 515408, ISIS 515423, ISIS 515424, ISIS 515532, ISIS 515534, ISIS 515538, ISIS 515539, ISIS 515558, ISIS 515575, ISIS 515926, ISIS 515944, ISIS 515945, ISIS 515951, ISIS 515952, ISIS 529126, ISIS 529765, ISIS 534528, ISIS 534534, ISIS 534594, ISIS 534663, ISIS 534676, ISIS 534677, ISIS 534679, ISIS 534683, ISIS 534693, ISIS 534701, ISIS 534716, ISIS 534730, ISIS 534806, ISIS 534830, ISIS 534838, ISIS 534890, ISIS 534898, ISIS 534911, ISIS 534937, ISIS 534956, ISIS 534986, ISIS 535043, ISIS 535049, ISIS 535076, ISIS 535082, ISIS 535142, ISIS 538160, ISIS 538242, ISIS 538361, ISIS 538380, ISIS 534795, ISIS 534796, ISIS 534797, ISIS 540162, ISIS 540164, ISIS 540168, ISIS 540172, ISIS 540175, ISIS 540176, ISIS 540178, ISIS 540179, ISIS 540181, ISIS 540182, ISIS 540183, ISIS 540184, ISIS 540186, ISIS 540187, ISIS 540188, ISIS 540191, ISIS 540193, ISIS 540194, ISIS 544813, ISIS 544814, ISIS 544816, ISIS 544826, ISIS 544827, ISIS 544828, ISIS 544829, ISIS 545473, and ISIS 545474 were considered very tolerable in terms of liver function. Based on these criteria, ISIS 529173, ISIS 529854, ISIS 529614, ISIS 515386, ISIS 515388, ISIS 515949, ISIS 544817, and ISIS 545479 were considered tolerable in terms of liver function.
    • Example 24 Tolerability of Antisense Oligonucleotides Targeting Human Target-X in Sprague-Dawley Rats
  • Sprague-Dawley rats are a multipurpose model used for safety and efficacy evaluations. The rats were treated with ISIS antisense oligonucleotides from the studies described in the Examples above and evaluated for changes in the levels of various plasma chemistry markers.
    • Treatment
  • Six-eight week old male Sprague-Dawley rats were maintained on a 12-hour light/dark cycle and fed ad libitum with Teklad normal rat chow. Groups of four Sprague-Dawley rats each were injected subcutaneously twice a week for 6 weeks with 25 mg/kg of ISIS 473286, ISIS 473547, ISIS 473567, ISIS 473589, ISIS 473630, ISIS 484559, ISIS 515636, ISIS 515640, ISIS 515641, ISIS 515655, ISIS 515657, ISIS 516046, ISIS 516048, ISIS 516051, ISIS 516052, and ISIS 516062. A group of four Sprague-Dawley rats was injected subcutaneously twice a week for 6 weeks with PBS. Forty eight hours after the last dose, rats were euthanized and organs and plasma were harvested for further analysis.
    • Liver Function
  • To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma levels of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma levels of ALT (alanine transaminase) and AST (aspartate transaminase) were measured. Plasma levels of Bilirubin and BUN were also measured using the same clinical chemistry analyzer.
  • ISIS oligonucleotides that did not cause any increase in the levels of transaminases, or which caused an increase within three times the upper limit of normal (ULN) were deemed very tolerable. ISIS oligonucleotides that caused an increase in the levels of transaminases between three times and seven times the ULN were deemed tolerable. Based on these criteria, ISIS 473286, ISIS 473547, ISSI 473589, ISIS 473630, ISIS 484559, ISIS 515636, ISIS 515640, ISIS 515655, ISIS 516046, and ISIS 516051 were considered very tolerable in terms of liver function. Based on these criteria, ISIS 473567, ISIS 515641, ISIS 515657, ISIS 516048, and ISIS 516051 were considered tolerable in terms of liver function.
    • Example 25 Tolerability of Chimeric Antisense Oligonucleotides Comprising 2′-O-Methoxyethyl (2′-MOE) Modifications Targeting Human Target-X in Sprague-Dawley Rats
  • Sprague-Dawley rats were treated with ISIS antisense oligonucleotides from the studies described in the Examples above and evaluated for changes in the levels of various plasma chemistry markers.
    • Treatment
  • Six-eight week old male Sprague-Dawley rats were maintained on a 12-hour light/dark cycle and fed ad libitum with Purina normal rat chow. Groups of four Sprague-Dawley rats each were injected subcutaneously twice a week for 6 weeks with 50 mg/kg of ISIS 407936, ISIS 416507, ISIS 416508, ISIS 490208, ISIS 490279, ISIS 490323, ISIS 490368, ISIS 490396, ISIS 490803, ISIS 491122, ISIS 513419, ISIS 513446, ISIS 513454, ISIS 513455, ISIS 513456, ISIS 513504, ISIS 513507, and ISIS 513508. A group of four Sprague-Dawley rats was injected subcutaneously twice a week for 6 weeks with PBS. Forty eight hours after the last dose, rats were euthanized and organs and plasma were harvested for further analysis.
    • Liver Function
  • To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma levels of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma levels of Bilirubin and BUN were also measured using the same clinical chemistry analyzer.
  • ISIS oligonucleotides that did not cause any increase in the levels of transaminases, or which caused an increase within three times the upper limit of normal (ULN) were deemed very tolerable. ISIS oligonucleotides that caused an increase in the levels of transaminases between three times and seven times the ULN were deemed tolerable. Based on these criteria, ISIS 416507, ISIS 490208, ISIS 490368, ISIS 490396, ISIS 490803, ISIS 491122, ISIS 513446, ISIS 513454, ISIS 513455, ISIS 513456, ISIS 513504, and ISIS 513508 were considered very tolerable in terms of liver function. Based on these criteria, ISIS 407936, ISIS 416508, ISIS 490279, and ISIS 513507 were considered tolerable in terms of liver function.
    • Example 26 Tolerability of Chimeric Antisense Oligonucleotides Comprising 2′-O-Methoxyethyl (2′-MOE) Modifications Targeting Human Target-X in CD-1 Mice
  • CD-1 mice are a multipurpose mice model, frequently utilized for safety and efficacy testing. The mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.
    • Treatment
  • Groups of 3 male CD-1 mice each were injected subcutaneously twice a week for 6 weeks with 50 mg/kg of ISIS 473244, ISIS 473295, ISIS 484714, ISIS 515386, ISIS 515424, ISIS 515534, ISIS 515558, ISIS 515926, ISIS 515949, ISIS 515951, ISIS 515952, ISIS 529126, ISIS 529166, ISIS 529173, ISIS 529186, ISIS 529360, ISIS 529461, ISIS 529553, ISIS 529564, ISIS 529582, ISIS 529614, ISIS 529725, ISIS 529745, ISIS 529765, ISIS 529785, ISIS 529799, ISIS 529818, ISIS 529823, ISIS 534528, ISIS 534594, and ISIS 534664. One group of male CD-1 mice was injected subcutaneously twice a week for 6 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.
    • Plasma Chemistry Markers
  • To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, albumin, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.).
  • ISIS oligonucleotides that did not cause any increase in the levels of transaminases, or which caused an increase within three times the upper limit of normal (ULN) were deemed very tolerable. ISIS oligonucleotides that caused an increase in the levels of transaminases between three times and seven times the ULN were deemed tolerable. Based on these criteria, ISIS 473295, ISIS 473714, ISIS 515558, ISIS 515926, 515951, ISIS 515952, ISIS 529126, ISIS 529166, 529564, ISIS 529582, ISIS 529614, ISIS 529725, ISIS 529765, ISIS 529799, ISIS 529823, and ISIS 534594 were considered very tolerable in terms of liver function. Based on these criteria, ISIS 515424, ISIS 515534, ISIS 515926, ISIS 529785, and ISIS 534664 were considered tolerable in terms of liver function.
    • Example 27 Tolerability of Chimeric Antisense Oligonucleotides Comprising 2′-O-Methoxyethyl (2′-MOE) Modifications Targeting Human Target-X in CD-1 Mice
  • CD-1 mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.
    • Treatment
  • Groups of 3 male CD-1 mice each were injected subcutaneously twice a week for 6 weeks with 100 mg/kg of ISIS 490208, ISIS 490279, ISIS 490323, ISIS 490368, ISIS 490396, ISIS 490803, ISIS 491122, ISIS 513419, ISIS 513446, ISIS 513454, ISIS 513455, ISIS 513456, ISIS 513504, ISIS 513507, and ISIS 513508. Groups of 3 male CD-1 mice each were injected subcutaneously twice a week for 6 weeks with 100 mg/kg of ISIS 407936, ISIS 416507, and ISIS 416508, which are gapmers described in a previous publication. One group of male CD-1 mice was injected subcutaneously twice a week for 6 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.
    • Plasma Chemistry Markers
  • To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.).
  • ISIS oligonucleotides that did not cause any increase in the levels of transaminases, or which caused an increase within three times the upper limit of normal (ULN) were deemed very tolerable. ISIS oligonucleotides that caused an increase in the levels of transaminases between three times and seven times the ULN were deemed tolerable. Based on these criteria, ISIS 407936, ISIS 416507, ISIS 490279, ISIS 490368, ISIS 490396, ISIS 490803, ISIS 491122, ISIS 513446, ISIS 513454, ISIS 513456, and ISIS 513504 were considered very tolerable in terms of liver function. Based on these criteria, ISIS 490208, ISIS 513455, ISIS 513507, and ISIS 513508 were considered tolerable in terms of liver function.
    • Example 28 Efficacy of Modified Oligonucleotides Comprising 2′-O-Methoxyethyl (2′-MOE) and 6′-(S)—CH3 Bicyclic Nucleoside (e.g cEt) Modifications Targeting Human Target-X in Transgenic Mice
  • Transgenic mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for efficacy.
    • Treatment
  • Groups of 2-3 male and female transgenic mice were injected subcutaneously twice a week for 3 weeks with 5 mg/kg/week of ISIS 473244, ISIS 473295, ISIS 484714, ISIS 515926, ISIS 515951, ISIS 515952, ISIS 516062, ISIS 529126, ISIS 529553, ISIS 529745, ISIS 529799, ISIS 534664, ISIS 534826, ISIS 540168, ISIS 540175, ISIS 544826, ISIS 544827, ISIS 544828, and ISIS 544829. One group of mice was injected subcutaneously twice a week for 3 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.
    • Protein Analysis
  • Plasma protein levels of Target-X were estimated using a Target-X ELISA kit (purchased from Hyphen Bio-Med). Results are presented as percent inhibition of Target-X, relative to control. As shown in Table 39, several antisense oligonucleotides achieved reduction of human Target-X over the PBS control. ‘n.d.’ indicates that the value for that particular oligonucleotide was not measured.
  • TABLE 39
    Percent inhibition of Target-X plasma
    protein levels in transgenic mice
    ISIS No % inhibition
    473244 2
    473295 13
    484714 19
    515926 11
    515951 13
    515952 0
    516062 62
    529126 0
    529553 0
    529745 22
    529799 26
    534664 32
    534826 n.d.
    540168 94
    540175 98
    544813 0
    544826 23
    544827 60
    544828 33
    544829 53
    • Example 29 Efficacy of Modified Oligonucleotides Comprising 2′-Methoxyethyl (2′-MOE) and 6′-(S)—CH3 Bicyclic Nucleoside (e.g cEt) Modifications Targeting Human Target-X in Transgenic Mice
  • Transgenic mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for efficacy.
    • Treatment
  • Groups of 2-3 male and female transgenic mice were injected subcutaneously twice a week for 3 weeks with 1 mg/kg/week of ISIS 407936, ISIS 490197, ISIS 490275, ISIS 490278, ISIS 490279, ISIS 490323, ISIS 490368, ISIS 490396, ISIS 490803, ISIS 491122, ISIS 513446, ISIS 513447, ISIS 513504, ISIS 516062, ISIS 529166, ISIS 529173, ISIS 529360, ISIS 529725, ISIS 534557, ISIS 534594, ISIS 534664, ISIS 534688, ISIS 534689, ISIS 534915, ISIS 534916, ISIS 534917, and ISIS 534980. One group of mice was injected subcutaneously twice a week for 3 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.
    • Protein Analysis
  • Plasma protein levels of Target-X were estimated using a Target-X ELISA kit (purchased from Hyphen Bio-Med). Results are presented as percent inhibition of Target-X, relative to control. As shown in Table 40, several antisense oligonucleotides achieved reduction of human Target-X over the PBS control.
  • TABLE 40
    Percent inhibition of Target-X plasm
    protein levels in transgenic mice
    ISIS No % inhibition
    407936 28
    490197 50
    490275 21
    490278 20
    490279 59
    490323 54
    490368 22
    490396 31
    490803 30
    491122 51
    513446 29
    513447 44
    513504 45
    516062 75
    529166 37
    529173 64
    529360 43
    529725 53
    534557 76
    534594 40
    534664 14
    534687 12
    534688 48
    534689 25
    534915 40
    534916 45
    534917 66
    534980 62
    • Example 30 Tolerability of Antisense Oligonucleotides Targeting Human Target-X in Sprague-Dawley Rats
  • Sprague-Dawley rats were treated with ISIS antisense oligonucleotides from the studies described in the Examples above and evaluated for changes in the levels of various plasma chemistry markers.
    • Treatment
  • Six-eight week old male Sprague-Dawley rats were maintained on a 12-hour light/dark cycle and fed ad libitum with Teklad normal rat chow. Groups of four Sprague-Dawley rats each were injected subcutaneously twice a week for 4 weeks with ISIS 515380, ISIS 515381, ISIS 515387, ISIS 529175, ISIS 529176, ISIS 529575, ISIS 529804, and ISIS 537064. Doses 1, 5, 6, 7, and 8 were 25 mg/kg; dose 2 was 75 mg/kg; doses 3 and 4 were 50 mg/kg. One group of four Sprague-Dawley rats was injected subcutaneously twice a week for 4 weeks with PBS. Forty eight hours after the last dose, rats were euthanized and organs and plasma were harvested for further analysis.
    • Liver Function
  • To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma levels of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma levels of ALT (alanine transaminase) and AST (aspartate transaminase) were measured. Plasma levels of Bilirubin and BUN were also measured using the same clinical chemistry analyzer.
  • ISIS oligonucleotides that did not cause any increase in the levels of transaminases, or which caused increase in the levels within three times the upper limit of normal levels of transaminases were deemed very tolerable. ISIS oligonucleotides that caused increase in the levels of transaminases between three times and seven times the upper limit of normal levels were deemed tolerable. Based on these criteria, ISIS 515380, ISIS 515387, ISIS 529175, ISIS 529176, ISIS 529804, and ISIS 537064 were considered very tolerable in terms of liver function. Based on these criteria, ISIS 515381 was considered tolerable in terms of liver function.
    • Example 31 Efficacy of Antisense Oligonucleotides Targeting Human Target-X in Transgenic Mice
  • Transgenic mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for efficacy.
    • Treatment
  • Two groups of 3 male and female transgenic mice were injected subcutaneously twice a week for 2 weeks with 0.5 mg/kg/week or 1.5 mg/kg/week of ISIS 407935 and ISIS 513455. Another group of mice was subcutaneously twice a week for 2 weeks with 0.6 mg/kg/week or 2.0 mg/kg/week of ISIS 473286. Another 16 groups of mice were subcutaneously twice a week for 2 weeks with 0.1 mg/kg/week or 0.3 mg/kg/week of ISIS 473589, ISIS 515380, ISIS 515423, ISIS 529804, ISIS 534676, ISIS 534796, ISIS 540162, ISIS 540164, ISIS 540175, ISIS 540179, ISIS 540181, ISIS 540182, ISIS 540186, ISIS 540191, ISIS 540193, ISIS 544827, or ISIS 545474. Another 3 groups of mice were injected subcutaneously twice a week for 2 weeks with 0.3 mg/kg/week of ISIS 516062, ISIS 534528 or ISIS 534693. One group of mice was injected subcutaneously twice a week for 2 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.
    • Protein Analysis
  • Plasma protein levels of Target-X were estimated using a Target-X ELISA kit (purchased from Hyphen Bio-Med). Results are presented as percent inhibition of Target-X, relative to control. As shown in Table 41, several antisense oligonucleotides achieved reduction of human Target-X over the PBS control.
  • TABLE 41
    Percent inhibition of Target-X plasma
    protein levels in transgenic mice
    Dose %
    ISIS No (mg/kg/wk) inhibition
    407935 1.5 65
    0.5 31
    513455 1.5 64
    0.5 52
    473286 2 67
    0.6 11
    473589 0.3 42
    0.1 12
    515380 0.3 64
    0.1 32
    515423 0.3 72
    0.1 37
    529804 0.3 36
    0.1 24
    534676 0.3 31
    0.1 18
    534796 0.3 54
    0.1 43
    540162 0.3 84
    0.1 42
    540164 0.3 25
    0.1 17
    540175 0.3 90
    0.1 55
    540179 0.3 29
    0.1 24
    540181 0.3 53
    0.1 0
    540182 0.3 78
    0.1 21
    540186 0.3 72
    0.1 46
    540191 0.3 62
    0.1 35
    540193 0.3 74
    0.1 46
    544827 0.3 28
    0.1 19
    545474 0.3 59
    0.1 0
    516062 0.3 33
    534528 0.3 41
    534693 0.3 34
    • Example 32 Tolerability of Antisense Oligonucleotides Targeting Human Target-X in Sprague-Dawley Rats
  • Sprague-Dawley rats were treated with ISIS antisense oligonucleotides from the studies described in the Examples above and evaluated for changes in the levels of various plasma chemistry markers.
    • Treatment
  • Five-six week old male Sprague-Dawley rats were maintained on a 12-hour light/dark cycle and fed ad libitum with Teklad normal rat chow. Groups of four Sprague-Dawley rats each were injected subcutaneously twice a week for 4 weeks with 50 mg/kg of ISIS 515423, ISIS 515424, ISIS 515640, ISIS 534676, ISIS 534796, ISIS 534797, ISIS 540162, ISIS 540164, ISIS 540172, ISIS 540175, ISIS 540179, ISIS 540181, ISIS 540182, ISIS 540183, ISIS 540186, ISIS 540191, and ISIS 545474. A group of four Sprague-Dawley rats was injected subcutaneously twice a week for 4 weeks with PBS. Forty eight hours after the last dose, rats were euthanized and organs and plasma were harvested for further analysis.
    • Liver Function
  • To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma levels of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma levels of ALT (alanine transaminase) and AST (aspartate transaminase) were measured. Plasma levels of Bilirubin and BUN were also measured using the same clinical chemistry analyzer.
  • ISIS oligonucleotides that did not cause any increase in the levels of transaminases, or which caused an increase within three times the upper limit of normal (ULN) were deemed very tolerable. ISIS oligonucleotides that caused an increase in the levels of transaminases between three times and seven times the ULN were deemed tolerable. Based on these criteria, ISIS 540164, ISIS 540172, and ISIS 540175 were considered very tolerable in terms of liver function. Based on these criteria, ISIS 534676, ISIS 534796, ISIS 534797, ISIS 540162, and ISIS 540179 were considered tolerable in terms of liver function.
    • Example 33 Dose-Dependent Antisense Inhibition of Human Target-X in Hep3B Cells
  • Antisense oligonucleotides selected from the studies described above were tested at various doses in Hep3B cells. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.05 μM, 0.15 μM, 0.44 μM, 1.33 μM, and 4.00 μM concentrations of antisense oligonucleotide, as specified in Table 42. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Human Target-X primer probe set RTS2927 was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells.
  • The half maximal inhibitory concentration (IC50) of each oligonucleotide is also presented in Table 42. As illustrated in Table 42, Target-X mRNA levels were reduced in a dose-dependent manner in several of the antisense oligonucleotide treated cells.
  • TABLE 42
    Dose-dependent antisense inhibition of human
    Target-X in Hep3B cells using electroporation
    0.05 0.15 0.44 1.33 4.00 IC50
    ISIS No μM μM μM μM μM (μM)
    473286 0 1 13 12 15 >4.0
    457851 23 32 57 80 93 0.3
    473286 3 20 43 71 88 0.5
    473286 15 26 24 28 36 >4.0
    473286 6 3 10 26 29 >4.0
    473327 14 28 35 67 90 0.5
    473589 29 53 76 89 95 0.1
    515380 44 72 85 93 95 <0.05
    515423 43 64 87 95 98 <0.05
    515424 38 55 85 92 97 0.1
    515636 21 33 74 82 93 0.2
    516046 29 23 29 48 78 0.9
    516048 35 24 41 67 87 0.4
    516052 18 6 48 63 80 0.6
    516062 24 14 21 47 68 1.6
    529166 16 47 75 87 94 0.2
    529173 14 49 77 91 96 0.2
    529175 30 69 88 93 96 0.1
    529176 34 63 85 93 96 0.1
    529360 35 53 74 91 93 0.1
    529725 53 69 85 92 95 <0.05
    529804 37 41 71 90 94 0.1
    534528 50 68 78 93 97 <0.05
    534557 48 78 90 94 95 <0.05
    534594 39 47 76 87 94 0.1
    534676 29 20 40 64 87 0.5
    534687 41 37 56 80 93 0.2
    534688 16 56 88 94 96 0.1
    534689 21 59 82 94 95 0.1
    534693 18 58 81 93 95 0.1
    534795 19 43 68 90 94 0.2
    534796 25 59 80 93 96 0.1
    534890 31 55 77 90 96 0.1
    534898 22 61 80 94 97 0.1
    534915 19 26 51 77 94 0.3
    534916 20 36 66 86 93 0.2
    534917 34 53 82 89 94 0.1
    540162 40 64 84 90 92 <0.05
    540164 34 60 83 91 92 0.1
    540168 51 79 90 92 94 <0.05
    540172 40 66 80 88 92 <0.05
    540175 30 61 80 88 91 0.1
    540176 7 17 50 75 85 0.5
    540179 11 22 25 16 19 >4.0
    540181 19 46 72 86 91 0.2
    540182 16 66 83 86 92 0.1
    540183 39 74 87 92 93 <0.05
    540186 31 69 85 91 94 0.1
    540191 38 54 80 88 91 0.1
    540193 57 67 84 94 97 <0.05
    540194 30 45 62 77 91 0.2
    544827 37 42 67 82 96 0.1
    544829 26 41 42 71 93 0.3
    545473 28 27 49 80 97 0.3
    545474 23 27 55 84 96 0.3
    • Example 34 Tolerability of Antisense Oligonucleotides Targeting Human Target-X in CD-1 Mice
  • CD-1 mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.
    • Treatment
  • Two groups of 4 male 6-8 week old CD-1 mice each were injected subcutaneously twice a week for 6 weeks with 50 mg/kg of ISIS 407935 and ISIS 490279. Another seven groups of 4 male 6-8 week old CD-1 mice each were injected subcutaneously twice a week for 6 weeks with 25 mg/kg of ISIS 473589, ISIS 529804, ISIS 534796, ISIS 540162, ISIS 540175, ISIS 540182, and ISIS 540191. One group of male CD-1 mice was injected subcutaneously twice a week for 6 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.
    • Plasma Chemistry Markers
  • To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, albumin, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in Table 43. Treatment with the newly designed antisense oligonucleotides were more tolerable compared to treatment with ISIS 407935 (disclosed in an earlier publication), which caused elevation of ALT levels greater than seven times the upper limit of normal (ULN).
  • TABLE 43
    Effect of antisense oligonucleotide treatment on liver function in CD-1 mice
    Dose AST BUN Bilirubin
    Motif (mg/kg/wk) ALT(IU/L) (IU/L) (mg/dL) (mg/dL)
    PBS — — 37 47 28 0.2
    407935 e5-d(10)-e5 100 373 217 24 0.2
    490279 kdkdk-d(9)-ee 100 96 82 24 0.2
    473589 e5-d(10)-e5 50 93 116 22 0.2
    529804 k-d(10)-kekee 50 54 74 27 0.2
    534796 ekk-d(10)-kke 50 60 63 27 0.2
    540162 eek-d(10)-kke 50 43 55 29 0.2
    540175 eek-d(10)-kke 50 113 78 24 0.3
    540182 eek-d(10)-kke 50 147 95 26 0.1
    540191 eek-d(10)-kke 50 79 88 28 0.2
    e = 2′-MOE, k = cEt, d = 2′-deoxynucleoside
    • Body and Organ Weights
  • Body weights, as well as liver, heart, lungs, spleen and kidney weights were measured at the end of the study, and are presented in Table 44. Several of the ISIS oligonucleotides did not cause any changes in organ weights outside the expected range and were therefore deemed tolerable in terms of organ weights.
  • TABLE 44
    Body and organ weights (grams) of CD-1 mice
    Dose
    (mg/ Body Liv- Kid-
    Motif kg/wk) weight er Spleen ney
    PBS — — 42 2.2 0.12 0.64
    407935 e5-d(10)-e5 100 40 2.6 0.20 0.62
    490279 kdkdk-d(9)-ee 100 42 2.8 0.17 0.61
    473589 e5-d(10)-e5 50 41 2.5 0.16 0.67
    529804 k-d(10)-kekee 50 40 2.3 0.14 0.62
    534796 ekk-d(10)-kke 50 37 2.6 0.15 0.51
    540162 eek-d(10)-kke 50 42 2.4 0.15 0.60
    540175 eek-d(10)-kke 50 39 2.2 0.11 0.62
    540182 eek-d(10)-kke 50 41 2.6 0.16 0.61
    540191 eek-d(10)-kke 50 40 2.4 0.13 0.60
    e = 2′-MOE, k = cEt, d = 2′-deoxynucleoside
    • Example 35 Tolerability of Antisense Oligonucleotides Targeting Human Target-X in Sprague-Dawley Rats
  • Sprague-Dawley rats were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.
    • Treatment
  • Two groups of 4 male 7-8 week old Sprague-Dawley rats each were injected subcutaneously twice a week for 6 weeks with 50 mg/kg of ISIS 407935 and ISIS 490279. Another seven groups of 4 male 6-8 week old Sprague-Dawley rats each were injected subcutaneously twice a week for 6 weeks with 25 mg/kg of ISIS 473589, ISIS 529804, ISIS 534796, ISIS 540162, ISIS 540175, ISIS 540182, and ISIS 540191. One group of male Sprague-Dawley rats was injected subcutaneously twice a week for 6 weeks with PBS. The rats were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.
    • Plasma Chemistry Markers
  • To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, albumin, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in Table 45. Treatment with the all antisense oligonucleotides was tolerable in terms of plasma chemistry markers in this model.
  • TABLE 45
    Effect of antisense oligonucleotide treatment
    on liver function in Sprague-Dawley rats
    Dose AST BUN Bilirubin
    Motif (mg/kg/wk) ALT(IU/L) (IU/L) (mg/dL) (mg/dL)
    PBS — — 71 83 19 0.2
    407935 e5-d(10)-e5 100 74 96 22 0.2
    490279 kdkdk-d(9)-ee 100 96 181 22 0.4
    473589 e5-d(10)-e5 50 57 73 21 0.2
    529804 k-d(10)-kekee 50 54 78 21 0.2
    534796 ekk-d(10)-kke 50 68 98 22 0.2
    540162 eek-d(10)-kke 50 96 82 21 0.1
    540175 eek-d(10)-kke 50 55 73 18 0.2
    540182 eek-d(10)-kke 50 45 87 21 0.2
    540191 eek-d(10)-kke 50 77 104 21 0.2
    e = 2′-MOE, k = cEt, d = 2′-deoxynucleoside
    • Body and Organ Weights
  • Body weights, as well as liver, heart, lungs, spleen and kidney weights were measured at the end of the study, and are presented in Table 46. Treatment with all the antisense oligonucleotides was tolerable in terms of body and organ weights in this model.
  • TABLE 46
    Body and organ weights (grams) of Sprague-Dawley rats
    Dose
    (mg/ Body Liv- Kid-
    Motif kg/wk) weight er Spleen ney
    PBS — — 443 16 0.8 3.5
    ISIS 407935 e5-d(10)-e5 100 337 14 1.8 3.2
    ISIS 490279 kdkdk-d(9)-ee 100 365 18 2.2 2.9
    ISIS 473589 e5-d(10)-e5 50 432 18 1.3 3.3
    ISIS 529804 k-d(10)-kekee 50 429 18 2.2 3.4
    ISIS 534796 ekk-d(10)-kke 50 434 15 1.4 3.3
    ISIS 540162 eek-d(10)-kke 50 446 18 1.1 3.3
    ISIS 540175 eek-d(10)-kke 50 467 16 1.0 3.5
    ISIS 540182 eek-d(10)-kke 50 447 22 2.5 4.5
    ISIS 540191 eek-d(10)-kke 50 471 21 1.4 3.9
    e = 2′-MOE, k = cEt, d = 2′-deoxynucleoside
    • Example 36 Dose-Dependent Antisense Inhibition of Human Target-X in Cynomolgos Monkey Primary Hepatocytes
  • Antisense oligonucleotides selected from the studies described above were tested at various doses in cynomolgus monkey primary hepatocytes. Cells were plated at a density of 35,000 cells per well and transfected using electroporation with 0.009 μM, 0.03 μM, 0.08 μM, 0.25 μM, 0.74 μM, 2.22 μM, 6.67 μM, and 20.00 μM concentrations of antisense oligonucleotide, as specified in Table 47. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Target-X primer probe set RTS2927 was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells. As illustrated in Table 47, Target-X mRNA levels were reduced in a dose-dependent manner with some of the antisense oligonucleotides that are cross-reactive with the rhesus monkey genomic sequence.
  • TABLE 47
    Dose-dependent antisense inhibition of Target-X in cynomolgous
    monkey primary hepatocytes using electroporation
    0.009 0.03 0.08 0.25 0.74 2.22 6.67 20.00
    ISIS No μM μM μM μM μM μM μM μM
    407935 10 18 15 29 56 73 82 88
    490279 19 12 13 0 6 18 27 22
    473589 5 10 19 42 64 76 88 92
    529804 10 3 23 25 57 80 86 91
    534796 0 28 23 49 71 81 87 90
    540162 9 14 9 6 13 13 11 31
    540175 0 4 12 9 10 16 12 22
    540182 0 7 0 6 36 12 10 0
    540191 6 7 0 0 0 0 21 42
    • Example 37 Dose-Dependent Antisense Inhibition of Human Target-X in Hep3B Cells
  • Antisense oligonucleotides from the study described above were also tested at various doses in Hep3B cells. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.009 μM, 0.03 μM, 0.08 μM, 0.25 μM, 0.74 μM, 2.22 μM, 6.67 μM, and 20.00 μM concentrations of antisense oligonucleotide, as specified in Table 48. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Target-X mRNA levels were measured by quantitative real-time PCR. Target-X primer probe set RTS2927 was used to measure mRNA levels. Target-X mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Target-X, relative to untreated control cells. As illustrated in Table 48, Target-X mRNA levels were reduced in a dose-dependent manner with several of the antisense oligonucleotides.
  • TABLE 48
    Dose-dependent antisense inhibition of Target-X
    in Hep3B cells using electroporation
    0.009 0.03 0.08 0.25 0.74 2.22 6.67 20.00 IC50
    ISIS No μM μM μM μM μM μM μM μM (μM)
    407935 3 9 11 35 64 83 87 93 4.5
    473244 20 33 50 69 77 89 7 14 0.9
    473589 0 14 23 44 74 88 90 94 2.7
    490279 0 5 7 15 25 61 76 78 11.6
    515533 0 12 21 36 63 78 88 94 3.6
    515952 0 12 27 57 76 89 93 94 2.2
    516066 6 0 12 26 52 70 81 86 6.0
    529459 0 4 24 40 61 78 88 94 3.5
    529553 9 7 17 40 58 74 87 93 4.6
    529804 0 3 34 64 83 89 93 95 2.0
    534796 8 18 43 67 82 89 95 96 1.4
    537806 6 11 5 20 37 69 79 86 7.1
    540162 18 33 63 75 87 91 91 92 0.7
    540175 10 25 55 76 86 89 89 93 1.0
    540182 13 36 61 75 84 88 90 93 0.7
    540191 3 12 28 61 79 80 88 94 2.2
    • Example 38 Efficacy of Antisense Oligonucleotides Targeting Human Target-X in Transgenic Mice
  • Transgenic mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for efficacy.
    • Treatment
  • Eight groups of 3 transgenic mice each were injected subcutaneously twice a week for 3 weeks with 20 mg/kg/week, 10 mg/kg/week, 5 mg/kg/week, or 2.5 mg/kg/week of ISIS 407935 or ISIS 490279. Another 24 groups of 3 transgenic mice each were subcutaneously twice a week for 3 weeks with 5 mg/kg/week, 2.5 mg/kg/week, 1.25 mg/kg/week, or 0.625 mg/kg/week of ISIS 473589, ISIS 529804, ISIS 534796, ISIS 540162, ISIS 540175, or ISIS 540191. One group of mice was injected subcutaneously twice a week for 3 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.
    • RNA Analysis
  • RNA was extracted from plasma for real-time PCR analysis of Target-X, using primer probe set RTS2927. The mRNA levels were normalized using RIBOGREEN®. As shown in Table 49, several antisense oligonucleotides achieved reduction of human Target-X over the PBS control. Results are presented as percent inhibition of Target-X, relative to control. Treatment with newly designed 2′-MOE gapmer, ISIS 490279, caused greater reduction in human Target-X mRNA levels than treatment with ISIS 407935, the 2′-MOE gapmer from the earlier publication. Treatment with several of the newly designed oligonucleotides also caused greater reduction in human Target-X mRNA levels than treatment with ISIS 407935.
  • TABLE 49
    Percent inhibition of Target-X mRNA in transgenic mice
    Dose %
    ISIS No Motif (mg/kg/wk) inhibition
    407935 e5-d(10)-e5 20.0 85
    10.0 57
    5.0 45
    2.5 28
    490279 kdkdk-d(9)-ee 20.0 88
    10.0 70
    5.0 51
    2.5 33
    473589 e5-d(10)-e5 5.00 80
    2.50 62
    1.25 44
    0.625 25
    529804 k-d(10)-kekee 5.00 55
    2.50 41
    1.25 0
    0.625 1
    534796 ekk-d(10)-kke 5.00 56
    2.50 41
    1.25 5
    0.625 0
    540162 eek-d(10)-kke 5.00 97
    2.50 92
    1.25 69
    0.625 78
    540175 eek-d(10)-kke 5.00 95
    2.50 85
    1.25 65
    0.625 55
    540182 eek-d(10)-kke 5.00 97
    2.50 83
    1.25 54
    0.625 10
    540191 eek-d(10)-kke 5.00 91
    2.50 74
    1.25 58
    0.625 34
    e = 2′-MOE, k = cEt, d = 2′-deoxynucleoside
    • Protein Analysis
  • Plasma protein levels of Target-X were estimated using a Target-X ELISA kit (purchased from Hyphen Bio-Med). As shown in Table 50, several antisense oligonucleotides achieved reduction of human Target-X over the PBS control. Results are presented as percent inhibition of Target-X, relative to control.
  • TABLE 50
    Percent inhibition of Target-X plasm
    protein levels in transgenic mice
    Dose %
    ISIS No Motif (mg/kg/wk) inhibition
    407935 e5-d(10)-e5 20 65
    10 47
    5 0
    2.5 3
    490279 kdkdk-d(9)-ee 20 91
    10 75
    5 31
    2.5 23
    473589 e5-d(10)-e5 5 78
    2.5 40
    1.25 6
    0.625 0
    529804 k-d(10)-kekee 5 50
    2.5 36
    1.25 0
    0.625 8
    534796 ekk-d(10)-kke 5 45
    2.5 26
    1.25 0
    0.625 8
    540162 eek-d(10)-kke 5 98
    2.5 96
    1.25 78
    0.625 74
    540175 eek-d(10)-kke 5 93
    2.5 83
    1.25 49
    0.625 24
    540182 eek-d(10)-kke 5 97
    2.5 71
    1.25 50
    0.625 0
    540191 eek-d(10)-kke 5 97
    2.5 74
    1.25 46
    0.625 25
    e = 2′-MOE, k = cEt, d = 2′-deoxynucleoside
    • Example 39 Effect of ISIS Antisense Oligonucleotides Targeting Human Target-X in Cynomolgus Monkeys
  • Cynomolgus monkeys were treated with ISIS antisense oligonucleotides selected from studies described above, including ISIS 407935, ISIS 490279, ISIS 473589, ISIS 529804, ISIS 534796, ISIS 540162, ISIS 540175, ISIS 540182, and ISIS 540191. Antisense oligonucleotide efficacy was evaluated. ISIS 407935, from the earlier publication, was included in the study for comparison.
    • Treatment
  • Prior to the study, the monkeys were kept in quarantine for at least a 30-day period, during which the animals were observed daily for general health. Standard panels of serum chemistry and hematology, examination of fecal samples for ova and parasites, and a tuberculosis test were conducted immediately after the animals' arrival to the quarantine area. The monkeys were 2-4 years old at the start of treatment and weighed between 2 and 4 kg. Ten groups of four randomly assigned male cynomolgus monkeys each were injected subcutaneously with ISIS oligonucleotide or PBS using a stainless steel dosing needle and syringe of appropriate size into one of 4 sites on the back of the monkeys; each site used in clock-wise rotation per dose administered. Nine groups of monkeys were dosed four times a week for the first week (days 1, 3, 5, and 7) as loading doses, and subsequently once a week for weeks 2-12, with 35 mg/kg of ISIS 407935, ISIS 490279, ISIS 473589, ISIS 529804, ISIS 534796, ISIS 540162, ISIS 540175, ISIS 540182, or ISIS 540191. A control group of cynomolgus monkeys was injected with PBS subcutaneously thrice four times a week for the first week (days 1, 3, 5, and 7), and subsequently once a week for weeks 2-12. The protocols described in the Example were approved by the Institutional Animal Care and Use Committee (IACUC).
    • Hepatic Target Reduction RNA Analysis
  • On day 86, RNA was extracted from liver tissue for real-time PCR analysis of Target-X using primer probe set RTS2927. Results are presented as percent inhibition of Target-X mRNA, relative to PBS control, normalized to RIBOGREEN® or to the house keeping gene, GAPDH. As shown in Table 52, treatment with ISIS antisense oligonucleotides resulted in reduction of Target-X mRNA in comparison to the PBS control.
  • TABLE 52
    Percent Inhibition of cynomolgous monkey Target-X mRNA in
    the cynomolgus monkey liver relative to the PBS control
    ISIS No Motif RTS2927/Ribogreen RTS2927/GAPDH
    407935 e5-d(10)-e5 90 90
    490279 kdkdk-d(9)-ee 72 66
    473589 e5-d(10)-e5 96 96
    529804 k-d(10)-kekee 90 87
    534796 ekk-d(10)-kke 80 78
    540162 eek-d(10)-kke 66 58
    540175 eek-d(10)-kke 68 66
    540182 eek-d(10)-kke 0 0
    540191 eek-d(10)-kke 34 14
    e = 2′-MOE, k = cEt, d = 2′-deoxynucleoside
    • Protein Levels and Activity Analysis
  • Plasma Target-X levels were measured prior to dosing, and on day 3, day 5, day 7, day 16, day 30, day 44, day 65, and day 86 of treatment. Target-X activity was measured using Target-X deficient plasma. Approximately 1.5 mL of blood was collected from all available study animals into tubes containing 3.2% sodium citrate. The samples were placed on ice immediately after collection. Collected blood samples were processed to platelet poor plasma and the tubes were centrifuged at 3,000 rpm for 10 min at 4° C. to obtain plasma.
  • Protein levels of Target-X were measured by a Target-X elisa kit (purchased from Hyphen BioMed). The results are presented in Table 53.
  • TABLE 53
    Plasma Target-X protein levels (% reduction compared
    to the baseline) in the cynomolgus monkey plasma
    Day Day Day Day Day Day Day Day
    ISIS No 3 5 7 16 30 44 65 86
    407935 21 62 69 82 84 85 84 90
    490279 0 29 35 30 38 45 51 58
    473589 12 67 85 97 98 98 98 98
    529804 19 65 76 87 88 89 90 90
    534796 1 46 54 64 64 67 66 70
    540162 0 24 26 37 45 49 49 50
    540175 0 28 36 38 47 52 55 55
    540182 0 17 8 0 0 0 5 0
    540191 0 12 4 0 0 4 9 10
    • Example 40 Single Nucleotide Polymorphisms (SNPs) in the Huntingtin (HTT) Gene Sequence
  • SNP positions (identified by Hayden et al, WO/2009/135322) associated with the HTT gene were mapped to the HTT genomic sequence, designated herein as SEQ ID NO: 1 (NT—006081.18 truncated from nucleotides 1566000 to 1768000). Table 56 provides SNP positions associated with the HTT gene. Table 56 provides a reference SNP ID number from the Entrez SNP database at the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/sites/entrez?db=snp), incorporated herein by reference. Table 56 furnishes further details on each SNP. The ‘Reference SNP ID number’ or ‘RS number’ is the number designated to each SNP from the Entrez SNP database at NCBI, incorporated herein by reference. ‘SNP position’ refers to the nucleotide position of the SNP on SEQ ID NO: 1. ‘Polymorphism’ indicates the nucleotide variants at that SNP position. ‘Major allele’ indicates the nucleotide associated with the major allele, or the nucleotide present in a statistically significant proportion of individuals in the human population. ‘Minor allele’ indicates the nucleotide associated with the minor allele, or the nucleotide present in a relatively small proportion of individuals in the human population.
  • TABLE 56
    Single Nuclear Polymorphisms (SNPs)
    and their positions on SEQ ID NO: 1
    SNP Major Minor
    RS No. position Polymorphism allele allele
    rs2857936 1963 C/T C T
    rs12506200 3707 A/G G A
    rs762855 14449 A/G G A
    rs3856973 19826 G/A G A
    rs2285086 28912 G/A A G
    rs7659144 37974 C/G C G
    rs16843804 44043 C/T C T
    rs2024115 44221 G/A A G
    rs10015979 49095 A/G A G
    rs7691627 51063 A/G G A
    rs2798235 54485 G/A G A
    rs4690072 62160 G/T T G
    rs6446723 66466 C/T T C
    rs363081 73280 G/A G A
    rs363080 73564 T/C C T
    rs363075 77327 G/A G A
    rs363064 81063 T/C C T
    rs3025849 83420 A/G A G
    rs6855981 87929 A/G G A
    rs363102 88669 G/A A G
    rs11731237 91466 C/T C T
    rs4690073 99803 A/G G A
    rs363144 100948 T/G T G
    rs3025838 101099 C/T C T
    rs34315806 101687 A/G G A
    rs363099 101709 T/C C T
    rs363096 119674 T/C T C
    rs2298967 125400 C/T T C
    rs2298969 125897 A/G G A
    rs6844859 130139 C/T T C
    rs363092 135682 C/A C A
    rs7685686 146795 A/G A G
    rs363088 149983 A/T A T
    rs362331 155488 C/T T C
    rs916171 156468 G/C c G
    rs362322 161018 A/G A G
    rs362275 164255 T/C C T
    rs362273 167080 A/G A G
    rs2276881 171314 G/A G A
    rs3121419 171910 T/C C T
    rs362272 174633 G/A G A
    rs362271 175171 G/A G A
    rs3775061 178407 C/T C T
    rs362310 179429 A/G G A
    rs362307 181498 T/C C T
    rs362306 181753 G/A G A
    rs362303 181960 T/C C T
    rs362296 186660 C/A C A
    rs1006798 198026 A/G A G
    • Example 41 Modified Oligonucleotides Targeting Huntingtin (HTT) Single Nucleotide Polymorphism (SNP)
  • A series of modified oligonucleotides were designed based on the parent gapmer, ISIS 460209 wherein the central gap region contains nine 2′-deoxyribonucleosides. These modified oligonucleotides were designed by introducing various chemical modifications in the central gap region and were tested for their ability to selectively inhibit mutant (mut) HTT mRNA expression levels targeting rs7685686 while leaving the expression of the wild-type (wt) intact. The activity and selectivity of the modified oligonucleotides were evaluated and compared to the parent gapmer, ISIS 460209.
  • The modified oligonucleotides were created with a 3-9-3 motif and are described in Table 57. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine nucleobases throughout each gapmer are 5-methyl cytosines. Nucleosides without a subscript are β-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e”, “k”, “y”, or “z” are sugar modified nucleosides. A subscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside, a subscript “k” indicates a 6′-(S)—CH3 bicyclic nucleoside (e.g. cEt), a subscript “y” indicates an α-L-LNA bicyclic nucleoside and a subscript “z” indicates a F-HNA modified nucleoside. pU indicates a 5-propyne uridine nucleoside and xT indicates a 2-thio-thymidine nucleoside.
  • The number in parentheses indicates the position on the modified oligonucleotide opposite to the SNP position, as counted from the 5′-terminus.
    • Cell Culture and Transfection
  • The modified oligonucleotides were tested in vitro. Heterozygous fibroblast GM04022 cell line was used (from Coriell Institute). Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with 0.12, 0.37, 1.1, 3.3 and 10 μM concentrations of modified oligonucleotides. After a treatment period of approximately 24 hours, cells were washed with DPBS buffer and lysed. RNA was extracted using Qiagen RNeasy purification and mRNA levels were measured by quantitative real-time PCR using ABI assay C—2229297—10 which measures at dbSNP rs362303. RT-PCR method in short; A mixture was made using 2020 uL 2×PCR buffer, 101 uL primers (300 uM from ABI), 1000 uL water and 40.4 uL RT MIX. To each well was added 15 uL of this mixture and 5 uL of purified RNA. The mutant and wild-type HTT mRNA levels were measured simultaneously by using two different fluorophores, FAM for mutant allele and VIC for wild-type allele. The HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN and the results are presented below.
    • Analysis of IC50's
  • The half maximal inhibitory concentration (IC50) of each oligonucleotide is presented in Table 58 and was calculated by plotting the concentrations of oligonucleotides used versus the percent inhibition of HTT mRNA expression achieved at each concentration, and noting the concentration of oligonucleotide at which 50% inhibition of HTT mRNA expression was achieved compared to the control. The IC50 at which each oligonucleotide inhibits the mutant HTT mRNA expression is denoted as ‘mut IC50’. The IC50 at which each oligonucleotide inhibits the wild-type HTT mRNA expression is denoted as ‘wt IC50’. Selectivity was calculated by dividing the IC50 for inhibition of the wild-type HTT versus the IC50 for inhibiting expression of the mutant HTT mRNA.
  • The parent gapmer, ISIS 460209 is marked with an asterisk (*) in the table and was included in the study as a benchmark oligonucleotide against which the activity and selectivity of the modified oligonucleotides targeting nucleotides overlapping the SNP position could be compared.
  • As illustrated in Table 58, modified oligonucleotides having chemical modifications in the central gap region at the SNP position exhibited similar activity with an increase in selectivity comparing to the parent gapmer, wherein the central gap region contains full deoxyribonucleosides.
  • TABLE 57
    Modified oligonucleotides targeting HTT rs7685686
    Wing SEQ
    ISIS Gap chemistry ID
    NO Sequence (5′ to 3′) chemistry 5′ 3′ NO
    460209* (8) TeAkAkATTGTCATCAkCkCe Full Deoxy ekk kke 10
    539560 (8) TeAkAkATTGpUCATCAkCkCe Deoxy/5-Propyne ekk kke 11
    539563 (8) TeAkAkATTGxTCATCAkCkCe Deoxy/2-Thio ekk kke 10
    539554 (8) TeAkAkATTGUyCATCAkCkCe Deoxy/α-L-LNA ekk kke 11
    542686 (8) TeAkAkATTGTzCATCAkCkCe Deoxy/F-HNA ekk kke 10
    e = 2′-MOE, k = cEt
  • TABLE 58
    Comparison of inhibition of HTT mRNA levels and selectivity of modified
    oligonucleotides with ISIS 460209 targeted to rs7685686 in GM04022 cells
    ISIS Mut IC50 Wt IC50 Selectivity Wing chemistry
    NO (μM) (μM) (mut vs wt) Gap chemistry 5′ 3′
     460209* (8) 0.41 2.0 4.9 Full Deoxy ekk kke
    539560 (8) 0.29 1.1 3.8 Deoxy/5-Propyne ekk kke
    539563 (8) 0.45 3.1 6.9 Deoxy/2-Thio ekk kke
    539554 (8) 3.5 >10 >3 Deoxy/α-L-LNA ekk kke
    542686 (8) 0.5 3.1 6.0 Deoxy/F-HNA ekk kke
    • Example 42 Modified Oligonucleotides Comprising Chemical Modifications in the Gap Region Targeting Huntingtin (HTT) Single Nucleotide Polymorphism (SNP)
  • Additional modified oligonucleotides were designed in a similar manner as the antisense oligonucleotides described in Table 57. Various chemical modifications were introduced in the central gap region at the SNP position in an effort to improve selectivity while maintaining activity in reducing mutant HTT mRNA levels.
  • The modified oligonucleotides were created with a 3-9-3 motif and are described in Table 59. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine nucleobases throughout each gapmer are 5-methyl cytosines. Nucleosides without a subscript are β-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “a”, “e”, “f”, “h”, “k”, “1”, “R”, “w” are sugar modified nucleosides. A subscript “a” indicates a 2′-(ara)-F modified nucleoside, a subscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside, a subscript “f” indicates a 2′-F modified nucleoside, a subscript “h” indicates a HNA modified nucleoside, a subscript “k” indicates a 6′-(S)—CH3 bicyclic nucleoside (e.g. cEt), a subscript “1” indicates a LNA modified nucleoside, a subscript “R” indicates a 5′-(R)-Me DNA, a subscript “w” indicates an unlocked nucleic acid (UNA) modified nucleoside. nT indicates an N3-ethylcyano thymidine nucleoside and bN indicates an abasic nucleoside (e.g. 2′-deoxyribonucleoside comprising a H in place of a nucleobase). Underlined nucleoside or the number in parentheses indicates the position on the modified oligonucleotide opposite to the SNP position, as counted from the 5′-terminus.
    • Thermal Stability Assay
  • The modified oligonucleotides were evaluated in thermal stability (Tm) assay. The Tm's were measured using the method described herein. A Cary 100 Bio spectrophotometer with the Cary Win UV Thermal program was used to measure absorbance vs. temperature. For the Tm experiments, oligonucleotides were prepared at a concentration of 8 μM in a buffer of 100 mM Na+, 10 mM phosphate, 0.1 mM EDTA, pH 7. Concentration of oligonucleotides were determined at 85° C. The oligonucleotide concentration was 4 μM with mixing of equal volumes of test oligonucleotide and mutant or wild-type RNA strand. Oligonucleotides were hybridized with the mutant or wild-type RNA strand by heating duplex to 90° C. for 5 min and allowed to cool at room temperature. Using the spectrophotometer, Tm measurements were taken by heating duplex solution at a rate of 0.5 C/min in cuvette starting @ 15° C. and heating to 85° C. Tm values were determined using Vant Hoff calculations (A260 vs temperature curve) using non self-complementary sequences where the minimum absorbance which relates to the duplex and the maximum absorbance which relates to the non-duplex single strand are manually integrated into the program.
  • Presented in Table 60 is the Tm for the modified oligonucleotides when duplexed to mutant or wild-type RNA complement. The Tm of the modified oligonucleotides duplexed with mutant RNA complement is denoted as “Tm (° C.) mut”. The Tm of the modified oligonucleotides duplexed with wild-type RNA complement is denoted as “Tm (° C.) wt”.
    • Cell Culture, Transfection and Selectivity Analysis
  • The modified oligonucleotides were also tested in vitro. Heterozygous fibroblast GM04022 cell line was used. Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with a single dose at 2 μM concentration of the modified oligonucleotide. After a treatment period of approximately 24 hours, cells were washed with DPBS buffer and lysed. RNA was extracted using Qiagen RNeasy purification and mRNA levels were measured by quantitative real-time PCR using ABI assay C—2229297—10 which measures at dbSNP rs362303. RT-PCR method in short; A mixture was made using 2020 uL 2×PCR buffer, 101 uL primers (300 uM from ABI), 1000 uL water and 40.4 uL RT MIX. To each well was added 15 uL of this mixture and 5 uL of purified RNA. The mutant and wild-type HTT mRNA levels were measured simultaneously by using two different fluorophores, FAM for mutant allele and VIC for wild-type allele. The HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. The results in Table 60 are presented as percent of HTT mRNA expression, relative to untreated control levels and is denoted as “% UTC”. Selectivity as was also evaluated and measured by dividing the percent of wild-type HTT mRNA levels vs. the percent of mutant HTT mRNA levels.
  • The parent gapmer, ISIS 460209 is marked with an asterisk (*) in the table and was included in the study as a benchmark oligonucleotide against which the selectivity of the modified oligonucleotides targeting nucleotides overlapping the SNP position could be compared.
  • As illustrated in Table 60, improvement in selectivity was observed for antisense oligonucleotides comprising chemical modifications in the central gap region at the SNP site such as 5′-(R)-Me (ISIS 539558), HNA (ISIS 539559), and 2′-(ara)-F (ISIS 539565) in comparison to the parent full deoxy gapmer, ISIS 460209. Modified oligonucleotides comprising LNA (ISIS 539553) or 2′-F (ISIS 539570) showed comparable selectivity while UNA modification (ISIS 539556 or 543909) showed no selectivity. Modified oligonucleotides comprising modified nucleobase, N3-ethylcyano (ISIS 539564) or abasic nucleobase (ISIS 543525) showed little to no improvement in selectivity.
  • TABLE 59
    Modified oligonucleotides comprising chemical modifications
    in the central gap region
    Wing SEQ
    ISIS chemistry ID
    NO Sequence (5′ to 3′) Gap chemistry 5′ 3′ NO.
    460209* (8) TeAkAkATTGTCATCAkCkCe Full Deoxy ekk kke 10
    539553 (8) TeAkAkATTGTl CATCAkCkCe Deoxy/LNA ekk kke 10
    539556 (8) TeAkAkATTGUw CATCAkCkCe Deoxy/UNA ekk kke 11
    539558 (8) TeAkAkATTGTR CATCAkCkCe Deoxy/5′-(R)-Me DNA ekk kke 10
    539559 (8) TeAkAkATTGTh CATCAkCkCe Deoxy/HNA ekk kke 10
    539564 (8) TeAkAkATTG nTCATCAkCkCe Deoxy/deoxy with N3- ekk kke 10
    Ethylcyano nucleobase
    539565 (8) TeAkAkATTGTa CATCAkCkCe Deoxy/2′-(ara)-F ekk kke 10
    539570 (8) TeAkAkATTGTf CATCAkCkCe Deoxy/2′-F ekk kke 10
    543525 (8) TeAkAkATTG bNCATCAkCkCe Deoxy/Deoxy-Abasic ekk kke 12
    543909 (5) TeAkAkAUw TGTCATCAkCkCe Deoxy/UNA ekk kke 13
    e = 2′-MOE, k = cEt, d = 2′-deoxyribonucleoside
  • TABLE 60
    Comparison of selectivity in inhibition of HTT mRNA levels and Tm of modified
    oligonucleotides with ISIS 460209 targeted to rs7685686 in GM04022 cells
    Wing
    ISIS Tm (° C.) % UTC Selectivity chemistry
    NO mutant wt mutant wt (wt vs mut) Gap chemistry 5′ 3′
     460209* (8) 53.7 52.2 23 57 2.4 Full Deoxy ekk kke
    539553 (8) 57.7 55.3 54 102 1.9 Deoxy/LNA ekk kke
    539556 (8) 43.7 44.1 90 105 1.2 Deoxy/UNA ekk kke
    539558 (8) 51.2 49.7 25 83 3.3 Deoxy/5′-(R)-Me DNA ekk kke
    539559 (8) 55.4 50.5 18 62 3.5 Deoxy/HNA ekk kke
    539564 (8) 42.8 43.1 86 135 1.6 Deoxy/Deoxy N3- ekk kke
    ethylcyano nucleobase
    539565 (8) 53.8 52.5 14 46 3.4 Deoxy/2′-(ara)-F ekk kke
    539570 (8) 54.4 51.8 25 50 2.0 Deoxy/2′-F ekk kke
    543525 (8) 43.1 43.8 87 97 1.1 Deoxy/Deoxy Abasic ekk kke
    543909 (5) 44.7 42.1 68 79 1.2 Deoxy/UNA ekk kke
    e = 2′-MOE, k = cEt, d = 2′-deoxyribonucleoside
    • Example 43 Chimeric Oligonucleotides Comprising Self-Complementary Regions Targeting Huntingtin (HTT) Single Nucleotide Polymorphism (SNP)
  • Chimeric oligonucleotides were designed based on the parent gapmer, ISIS 460209. These gapmers comprise self-complementary regions flanking the central gap region, wherein the central gap region contains nine deoxyribonucleosides and the self-complementary regions are complementary to one another. The underlined nucleosides indicate the portion of the 5′-end that is self-complement to the portion of the 3′-end.
  • The gapmers and their motifs are described in Table 61. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine nucleobases throughout each gapmer are 5-methyl cytosines. Nucleosides without a subscript are β-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e” or “k” are sugar modified nucleosides. A subscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside and a subscript “k” indicates a 6′-(S)—CH3 bicyclic nucleoside (e.g. cEt).
  • The modified oligonucleotides were tested in vitro. Heterozygous fibroblast GM04022 cell line was used. Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with a single dose at 2 μM concentration of the modified oligonucleotide. After a treatment period of approximately 24 hours, cells were washed with DPBS buffer and lysed. RNA was extracted using Qiagen RNeasy purification and mRNA levels were measured by quantitative real-time PCR using ABI assay C—2229297—10 which measures at dbSNP rs362303. RT-PCR method in short; A mixture was made using 2020 uL 2×PCR buffer, 101 uL primers (300 uM from ABI), 1000 uL water and 40.4 uL RT MIX. To each well was added 15 uL of this mixture and 5 uL of purified RNA. The mutant and wild-type HTT mRNA levels were measured simultaneously by using two different fluorophores, FAM for mutant allele and VIC for wild-type allele. HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. The results in Table 62 are presented as percent of HTT mRNA expression, relative to untreated control levels and is denoted as “% UTC”. Selectivity was also evaluated and measured by dividing the percent of wild-type HTT mRNA levels vs. the percent of the mutant HTT mRNA levels.
  • The parent gapmer, ISIS 460209 is marked with an asterisk (*) in the table and was included in the study as a benchmark oligonucleotide against which the selectivity of the modified oligonucleotides targeting nucleotides overlapping the SNP position could be compared.
  • As illustrated in Table 62, improvement in selectivity was observed for chimeric oligonucleotides comprising 5-9-5 (ISIS 550913), 6-9-6 (ISIS 550912), 6-9-3 (ISIS 550907) or 3-9-7 (ISIS 550904) in comparison to the parent gapmer motif, 3-9-3 (ISIS 460209). The remaining gapmers showed moderate to little improvement in selectivity.
  • TABLE 61
    Chimeric oligonucleotides comprising various wing motifs 
    targeted to HTT rs7685686
    Wing SEQ
    ISIS chemistry ID
    NO Sequence (5′ to 3′) Motif 5′ 3′ NO.
    460209* TeAkAkATTGTCATCAkCkCe 3-9-3 ekk kke 10
    544838 Te AkAkATTGTCATCAkCkCe Ak 3-9-4 ekk kkek 14
    544840 TeAkAk ATTGTCATCAkCkCe TkTkAk 3-9-6 ekk kkekkk 15
    544842 TeAkAkATTGTCATCAkCkCe AkTkTkTkAk 3-9-8 ekk kkekkkkk 16
    550903 TeAk AkATTGTCATCAkCkCe TkAk 3-9-5 ekk kkekk 17
    550904 TeAkAkATTGTCATCAkCkCe TkTkTkAk 3-9-7 ekk kkekkkk 18
    550905 Gk TeAkAkATTGTCATCAkCk Ce 4-9-3 kekk kke 19
    550906 GkGk TeAkAkATTGTCATCAk CkCe 5-9-3 kkekk kke 20
    550907 GkGkT kTeAkAkATTGTCATCAkGkCe 6-9-3 kkkekk kke 21
    550908 GkGkTkGk TeAkAkATTGTCATCAkCkCe 7-9-3 kkkkekk kke 22
    550909 GkGkTkGkAk TeAkAkATTGTCATCAkCkCe 8-9-3  kkkkkekk kke 23
    550910 GkGkCk TeAkAkATTGTCATCAkCkCe GkCkCk 6-9-6 kkkekk kkekkk 24
    550911 GkCk TeAkAkATTGTCATCAkCkCe GkCk 5-9-5 kkekk kkekk 25
    550912 TkAkAk TeAkAkATTGTCATCAkCkCe TkTkAk 6-9-6 kkkekk kkekkk 26
    550913 AkAk TeAkAkATTGTCATCAkCkCe TkTk 5-9-5 kkekk kkekk 27
    550914 TkCkTk TeAkAkATTGTCATCAkCkCe AkGkAk 6-9-6 kkkekk kkekkk 28
    550915 CkTk TeAkAkATTGTCATCAkCkCe AkGk 5-9-5 kkekk kkekk 29
    e = 2′-MOE, k = cEt
  • TABLE 62
    Comparison of selectivity in inhibition of HTT mRNA
    levels of chimeric oligonucleotides with ISIS 460209
    targeted to rs7685686 in GM04022 cells
    ISIS % UTC Selectivity wing chemistry
    NO mut wt (wt vs. mut) Motif 5′ 3′
     460209* 23 57 2.4 3-9-3 ekk kke
    544838 13 25 2.0 3-9-4 ekk kkek
    544840 17 31 1.8 3-9-6 ekk kkekkk
    544842 55 102 1.9 3-9-8 ekk kkekkkkk
    550903 13 36 2.7 3-9-5 ekk kkekk
    550904 23 67 3.0 3-9-7 ekk kkekkkk
    550905 21 51 2.4 4-9-3 kekk kke
    550906 23 67 2.9 5-9-3 kkekk kke
    550907 30 93 3.1 6-9-3 kkkekk kke
    550908 60 80 2.4 7-9-3 kkkkekk kke
    550909 42 101 2.4 8-9-3 kkkkkekk kke
    550910 57 102 1.8 6-9-6 kkkekk kkekkk
    550911 18 40 2.2 5-9-5 kkekk kkekk
    550912 14 51 3.6 6-9-6 kkkekk kkekkk
    550913 8 36 4.5 5-9-5 kkekk kkekk
    550914 29 45 1.5 6-9-6 kkkekk kkekkk
    550915 13 28 2.1 5-9-5 kkekk kkekk
    e = 2′-MOE, k = cEt
    • Example 44 Chimeric Antisense Oligonucleotides Comprising Non-Self-Complementary Regions Targeting Huntingtin (HTT) Single Nucleotide Polymorphism (SNP)
  • Additional gapmers are designed based on the most selective gapmers from studies described in Tables 61 and 62 (ISIS 550912 and 550913). These gapmers are created such that they cannot form self-structure in the effort to evaluate if the increased activity simply is due to higher binding affinity. Gapmers are designed by deleting two or three nucleotides at the 3′-terminus and are created with 6-9-3 or 5-9-3 motif.
  • The chimeric oligonucleotides and their motifs are described in Table 63. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine nucleobases throughout each gapmer are 5-methyl cytosines. Nucleosides without a subscript are β-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e” or “k” are sugar modified nucleosides. A subscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside and a subscript “k” indicates a 6′-(S)—CH3 bicyclic nucleoside (e.g. cEt).
  • The gapmers, ISIS 550912 and ISIS 550913, from which the newly designed gapmers are derived from, are marked with an asterisk (*) in the table.
  • TABLE 63
    Non-self-complementary chimeric oligonucleotides  
    targeting HTT SNP
    Wing SEQ
    ISIS chemistry ID
    NO Sequence (5′ to 3′) Motif 5′ 3′ NO.
    550912* TkAkAkTeAkAkATTGTCATCAkCkCeTkTkAk 6-9-6 kkkekk kkekkk 26
    550913* AkAkTeAkAkATTGTCATCAkCkCeTkTk 5-9-5 kkekk kkekk 27
    556879 TkAkAkTeAkAkATTGTCATCAkCkCe 6-9-3 kkkekk kke 30
    556880 AkAkTeAkAkATTGTCATCAkCkCe 5-9-3 kkekk kke 31
    e = 2′-MOE, k = cEt
    • Example 45 Chimeric Oligonucleotides Containing Mismatches Targeting Huntingtin (HTT) Single Nucleotide Polymorphism (SNP)
  • A series of chimeric antisense oligonucleotides were designed based on the parent gapmer, ISIS 460209, wherein the central gap region contains nine 2′-deoxyribonucleosides. These gapmers were designed by introducing modified nucleosides at both 5′ and 3′ termini. Gapmers were also created with a single mismatch shifted slightly upstream and downstream (i.e. “microwalk”) within the central gap region and with the SNP position opposite position 5 of the parent gapmer, as counted from the 5′-gap terminus.
  • The gapmers and their motifs are described in Table 64. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine nucleobases throughout each gapmer are 5-methyl cytosines. Nucleosides without a subscript are β-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e” or “k” are sugar modified nucleosides. A subscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside and a subscript “k” indicates a 6′-(S)—CH3 bicyclic nucleoside (e.g. cEt). Underlined nucleosides indicate the mismatch position, as counted from the 5′-gap terminus.
  • These gapmers were evaluated for thermal stability (Tm) using methods described in Example 42. Presented in Table 65 are the Tm measurements for chimeric antisense oligonucleotides when duplexed to mutant or wild-type RNA complement. The Tm of chimeric antisense oligonucleotides duplexed with mutant RNA complement is denoted as “Tm (° C.) mut”. The Tm of chimeric antisense oligonucleotides duplexed with wild-type RNA complement is denoted as “Tm (° C.) wt”.
  • These gapmers were also tested in vitro. Heterozygous fibroblast GM04022 cell line was used. Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with a single dose at 2 μM concentration of the modified oligonucleotide. After a treatment period of approximately 24 hours, cells were washed with DPBS buffer and lysed. RNA was extracted using Qiagen RNeasy purification and mRNA levels were measured by quantitative real-time PCR using ABI assay C—2229297—10 which measures at dbSNP rs362303. RT-PCR method in short; A mixture was made using 2020 uL 2×PCR buffer, 101 uL primers (300 uM from ABI), 1000 uL water and 40.4 uL RT MIX. To each well was added 15 uL of this mixture and 5 uL of purified RNA. The mutant and wild-type HTT mRNA levels were measured simultaneously by using two different fluorophores, FAM for mutant allele and VIC for wild-type allele. HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. The results in Table 65 are presented as percent of HTT mRNA expression, relative to untreated control levels and is denoted as “% UTC”. Selectivity was also evaluated and measured by dividing the percent of wild-type HTT mRNA levels vs. the percent of mutant HTT mRNA levels.
  • The parent gapmer, ISIS 460209 is marked with an asterisk (*) in the table and was included in the study as a benchmark oligonucleotide against which the selectivity of the modified oligonucleotides targeting nucleotides overlapping the SNP position could be compared.
  • As illustrated in Table 65, improvement in selectivity was observed for gapmers comprising a 4-9-4 motif with a central deoxy gap region (ISIS 476333) or a single mismatch at position 8 within the gap region (ISIS 543531) in comparison to the parent gapmer. The remaining gapmers showed moderate to little improvement in selectivity.
  • TABLE 64
    Chimeric oligonucleotides containing a single 
    mismatch targeting mutant HTT SNP
    Wing SEQ
    ISIS Mismatch chemistry ID
    NO Sequence (5′ to 3′) position Motif 5′ 3′ NO.
    460209* TeAkAkATTGTCATCAkCkCe — 3-9-3 ekk kke 10
    476333 AeTkAeAkATTGTCATCAkCeCkAe — 4-9-4 ekek keke 32
    543526 AeTkAeAkATTCTCATCAkCeCkAe 4 4-9-4 ekek keke 33
    543527 AeTkAeAkATAGTCATCAkCeCkAe 3 4-9-4 ekek keke 34
    543529 AeTkAeAkATTGTGATCAkCeCkAe 6 4-9-4 ekek keke 35
    543530 AeTkAeAkATTGTCTTCAkCeCkAe 7 4-9-4 ekek keke 36
    543531 AeTkAeAkATTGTCAACAkCeCkAe 8 4-9-4 ekk keke 37
    543532 TeAkAkATTCTCATCAkCkCe 4 3-9-3 ekk kke 38
    543534 TeAkAkAATGTCATCAkCkCe 2 3-9-3 ekk kke 39
    543535 TeAkAkATTGTGATCAkCkCe 6 3-9-3 ekk kke 40
    543536 TeAkAkATTGTCTTCAkCkCe 7 3-9-3 ekk kke 41
    543537 TeAkAkATTGTCAACAkCkCe 8 3-9-3 ekk kke 42
    e = 2′-MOE, k = cEt
  • TABLE 65
    Comparison of selectivity and Tm of chimeric oligonucleotides
    with ISIS 460209 targeted to rs7685686 in GM04022 cells
    ISIS Tm (° C.) % UTC Selectivity Mismatch Wing chemistry
    NO mut wt mut wt (wt vs mut) position Motif 5′ 3′
     460209* 53.7 52.2 23 57 2.4 — 3-9-3 ekk kke
    476333 60.2 58.4 10 37 3.6 — 4-9-4 ekek keke
    543526 47.9 46.6 70 86 1.2 4 4-9-4 ekek keke
    543527 52.6 49.9 40 103 2.6 3 4-9-4 ekek keke
    543529 50.3 49.0 66 102 1.5 6 4-9-4 ekek keke
    543530 52.9 50.9 67 110 1.6 7 4-9-4 ekek keke
    543531 53.3 50.3 46 136 3.0 8 4-9-4 ekk keke
    543532 43.6 42.8 127 151 1.2 4 3-9-3 ekk kke
    543534 45.9 43.8 67 95 1.4 2 3-9-3 ekk kke
    543535 44.0 43.3 96 113 1.2 6 3-9-3 ekk kke
    543536 46.8 44.6 106 104 1.0 7 3-9-3 ekk kke
    543537 45.9 44.3 77 81 1.1 8 3-9-3 ekk kke
    e = 2′-MOE, k = cEt
    • Example 46 Chimeric Oligonucleotides Comprising Mismatches Targeting Huntingtin (HTT) Single Nucleotide Polymorphism (SNP)
  • Additional chimeric antisense oligonucleotides are designed based on two gapmers selected from studies described in Tables 64 and 65 (ISIS 476333 and ISIS 460209) wherein the central gap region contains nine 2′-deoxyribonucleosides. These gapmers are designed by introducing a single mismatch, wherein the mismatch will be shifted throughout the antisense oligonucleotide (i.e. “microwalk”). Gapmers are also created with 4-9-4 or 3-9-3 motifs and with the SNP position opposite position 8 of the original gapmers, as counted from the 5′-terminus.
  • The gapmers and their motifs are described in Table 66. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine nucleobases throughout each gapmer are 5-methyl cytosines. Nucleosides without a subscript are β-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e” or “k” are sugar modified nucleosides. A subscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside and a subscript “k” indicates a 6′-(S)—CH3 bicyclic nucleoside (e.g. cEt). Underlined nucleosides indicate the mismatch position, as counted from the 5′-terminus.
  • The gapmers, ISIS 476333 and ISIS 460209, in which the newly designed antisense oligonucleotides are derived from, are marked with an asterisk (*) in the table.
  • TABLE 66
    Chimeric oligonucleotides comprising mismatches 
    targeting HTT SNP
    Wing SEQ
    ISIS Mismatch chemistry ID
    NO Sequence (5′ to 3′) position Motif 5′ 3′ NO
    476333* AeTkAeAkATTGTCATCAkCeCkAe — 4-9-4 ekek keke 32
    554209 Te TkAeAkATTGTCATCAkCeCkAe  1 4-9-4 ekek keke 43
    554210 Ae Ak AeAkATTGTCATCAkCeCkAe  2 4-9-4 ekek keke 44
    554211 AeTk Te AkATTGTCATCAkCeCkAe  3 4-9-4 ekek keke 45
    554212 AeTkAe Tk ATTGTCATCAkCeCkAe  4 4-9-4 ekek keke 46
    554213 AeTkAeAk TTTGTCATCAkCeCkAe  5 4-9-4 ekek keke 47
    554214 AeTkAeAkATTGTCATGAkCeCkAe 13 4-9-4 ekek keke 48
    554215 AeTkAeAkATTGTCATCT kCeCkAe 14 4-9-4 ekek keke 49
    554216 AeTkAeAkATTGTCATCAk Ge CkAe 15 4-9-4 ekek keke 50
    554217 AeTkAeAkATTGTCATCAkCe Gk Ae 16 4-9-4 ekek keke 51
    554218 AeTkAeAkATTGTCATCAkCeCk Te 17 4-9-4 ekek keke 52
    460209* TeAkAkATTGTCATCAkCkCe — 3-9-3 ekk kke 10
    562481 TeAkAk GTTGTCATCAkCkCe  4 3-9-3 ekk kke 53
    554482 TeAkAkAGTGTCATCAkCkCe  5 3-9-3 ekk kke 54
    554283 TeAkAkATGGTCATCAkCkCe  6 3-9-3 ekk kke 55
    e = 2′-MOE, k = cEt
    • Example 47 Short-Gap Chimeric Oligonucleotides Targeting Huntingtin (HTT) Single Nucleotide Polymorphism (SNP)
  • Chimeric antisense oligonucleotides were designed based on the parent gapmer, ISIS 460209, wherein the central gap region contains nine 2′-deoxyribonucleosides. These gapmers were designed by shortening the central gap region to seven 2′-deoxyribonucleosides. Gapmers were also created with 5-7-5 motif and with the SNP position opposite position 8 or 9 of the parent gapmer, as counted from the 5′-terminus.
  • The gapmers and their motifs are described in Table 67. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine nucleobases throughout each gapmer are 5-methyl cytosines. Nucleosides without a subscript are β-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e” or “k” are sugar modified nucleosides. A subscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside and a subscript “k” indicates a 6′-(S)—CH3 bicyclic nucleoside (e.g. cEt). Underlined nucleoside or the number in parentheses indicates the position on the modified oligonucleotide opposite to the SNP position, as counted from the 5′-terminus.
  • The chimeric antisense oligonucleotides were tested in vitro. ISIS 141923 was included in the study as a negative control and is denoted as “neg control”. A non-allele specific antisense oligonucleotide, ISIS 387916 was used as a positive control and is denoted as “pos control”. ISIS 460209 was included in the study for comparison. Heterozygous fibroblast GM04022 cell line was used. Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with 0.12, 0.37, 1.1, 3.3, and 10 μM concentration of the modified oligonucleotide. After a treatment period of approximately 24 hours, cells were washed with DPBS buffer and lysed. RNA was extracted using Qiagen RNeasy purification and mRNA levels were measured by quantitative real-time PCR using ABI assay C—2229297—10 which measures at dbSNP rs362303. RT-PCR method in short; A mixture was made using 2020 uL 2×PCR buffer, 101 uL primers (300 uM from ABI), 1000 uL water and 40.4 uL RT MIX. To each well was added 15 uL of this mixture and 5 uL of purified RNA. The mutant and wild-type HTT mRNA levels were measured simultaneously by using two different fluorophores, FAM for mutant allele and VIC for wild-type allele. HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN and the results are presented in Table 68.
  • The IC50 and selectivity were calculated using methods described previously in Example 41. As illustrated in Table 68, no improvement in potency and selectivity was observed for the chimeric antisense oligonucleotides as compared to ISIS 460209.
  • TABLE 67
    Chimeric antisense oligonucleotides targeting HTT rs7685686
    Wing SEQ
    ISIS Chemistry ID
    NO Sequence (5′ to 3′) Motif 5′ 3′ NO.
    460209* (8) TeAkAkATTGTCATCAkCkCe 3-9-3 ekk kke 10
    460085 (9) AeTeAeAeAeTTGTCATCeAeCeCeAe 5-7-5 eeeee eeeee 32
    540108 (9) AeTeAeAkAkTTGTCATCkAkCeCeAe 5-7-5 eeekk kkeee 32
    387916 TeCeTeCeTeATTGCACATTCeCeAeAeGe 5-10-5 eeeee eeeee 56
    (pos control)
    141923 CeCeTeTeCeCCTGAAGGTTCeCeTeCeCe 5-10-5 eeeee eeeee 57
    (neg control)
    e = 2′-MOE, k = cEt
  • TABLE 68
    Comparison of inhibition of HTT mRNA levels and selectivity
    of chimeric antisense oligonucleotides with ISIS 460209
    targeted to rs7685686 in GM04022 cells
    Wing
    Mut IC50 Wt IC50 Selectivity chemistry
    ISIS NO (μM) (μM) (mut vs wt) Motif 5′ 3′
    460209* (8) 0.41 2.0 4.9 3-9-3 ekk kke
    460085 (9) 3.5 >10 >3 5-7-5 eeeee eeeee
    540108 (9) 0.41 — — 5-7-5 eeekk kkeee
    387916 0.39 0.34 1.0 5-10-5 eeeee eeeee
    (pos control)
    141923 >10 >10 — 5-10-5 eeeee eeeee
    (neg control)
    e = 2′-MOE,
    k = cEt
    • Example 48 Short-Gap Chimeric Oligonucleotides Targeting Huntingtin (HTT) Single Nucleotide Polymorphism (SNP)
  • Additional chimeric antisense oligonucleotides were designed based on the parent gapmer, ISIS 460209, wherein the central gap region contains nine 2′-deoxyribonucleosides. These gapmers were designed with the central gap region shortened or interrupted by introducing various modifications either within the gap or by adding one or more modified nucleosides to the 3′-most 5′-region or to the 5′-most 3′-region. Gapmers were created with the SNP position opposite position 8 of the parent gapmer, as counted from the 5′-terminus.
  • The gapmers and their motifs are described in Table 69. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine nucleobases throughout each gapmer are 5-methyl cytosines. Nucleosides without a subscript are β-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e” or “k” are sugar modified nucleosides. A subscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside and a subscript “k” indicates a 6′-(S)—CH3 bicyclic nucleoside (e.g. cEt).
  • The chimeric antisense oligonucleotides were tested in vitro. Heterozygous fibroblast GM04022 cell line was used. Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with 2 μM concentration of the modified oligonucleotide. After a treatment period of approximately 24 hours, cells were washed with DPBS buffer and lysed. RNA was extracted using Qiagen RNeasy purification and mRNA levels were measured by quantitative real-time PCR using ABI assay C—2229297—10 which measures at dbSNP rs362303. RT-PCR method in short; A mixture was made using 2020 uL 2×PCR buffer, 101 uL primers (300 uM from ABI), 1000 uL water and 40.4 uL RT MIX. To each well was added 15 uL of this mixture and 5 uL of purified RNA. The mutant and wild-type HTT mRNA levels were measured simultaneously by using two different fluorophores, FAM for mutant allele and VIC for wild-type allele. HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. The results in Table 70 are presented as percent of HTT mRNA expression, relative to untreated control levels and is denoted as “% UTC”. Selectivity was also evaluated and measured by dividing the percent of wild-type HTT mRNA levels vs. the percent of mutant HTT mRNA levels. ISIS 460209 marked with an asterisk (*) in the table was included in the study for comparison.
  • As illustrated in Table 70, modifications to the 3′-most 5′-region nucleosides that shorten the gap from 9 to 7 or 8 nucleotides (ISIS 551429 and ISIS 551426) improved selectivity and potency comparing to the parent gapmer (ISIS 460209). The remaining chimeric antisense oligonucleotides showed moderate to little improvement in selectivity.
  • TABLE 69
    Short-gap antisense oligonucleotides targeting 
    HTT rs7685686
    Wing SEQ
    ISIS Chemistry ID
    NO Sequence (5′ to 3′) Motif 5′ 3′ NO.
    460209* TeAkAkATTGTCATCAkCkCe 3-9-3 ekk kke 10
    551426 TeAkAeAkTTGTCATCAkCkCe 4-8-3 ekek kke 10
    551427 TeAkAeATkTGTCATCAkCkCe 3-9-3 or eke or kke 10
    5-7-3 ekedk
    551428 TeAkAeATTkGTCATCAkCkCe 3-9-3 or eke or kke 10
    6-6-3 ekeddk
    551429 TeAeAeAkTkTGTCATCAkCkCe 5-7-3 eeekk kke 10
    e = 2′-MOE, k = cEt, d = 2′-deoxyribonucleoside
  • TABLE 70
    Comparison of selectivity in inhition of HTT mRNA
    levels of antisense oligonucleotides with ISIS 460209
    targeted to rs7685686 in GM4022 cells
    % UTC Selectivity Wing chemistry
    ISIS NO mut wt (wt vs. mut) Motif 5′ 3′
    460209* 23 57 2.4 3-9-3 ekk kke
    551426 14 66 4.8 4-8-3 ekek kke
    551427 35 97 2.8 3-9-3 or eke or kke
    5-7-3 ekedk
    551428 61 110 1.8 3-9-3 or eke or kke
    6-6-3 ekeddk
    551429 19 94 5.0 5-7-3 eeekk kke
    e = 2′-MOE,
    k = cEt,
    d = 2′-deoxyribonucleoside
    • Example 49 Modified Oligonucleotides Targeting HTT SNP
  • A series of modified antisense oligonucleotides are designed based on the parent gapmer, ISIS 460209, wherein the central gap region contains nine 2′-deoxynucleosides and is marked with an asterisk (*) in the table. These modified oligonucleotides are designed by shortening or interrupting the gap with a single mismatch or various chemical modifications within the central gap region. The modified oligonucleotides are created with the SNP position opposite position 8 of the parent gapmer, as counted from the 5′-terminus.
  • The gapmers and their motifs are described in Table 71. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages, except for the internucleoside linkage with a subscript “p”, “pz” or “pw”. Subscript “p” indicates methyl phosphonate internucleoside linkage. Subscript “pz” indicates (R)-methyl phosphonate internucleoside linkage. Subscript “pw” indicates (S)-methyl phosphonate internucleoside linkage. All cytosine nucleobases throughout each gapmer are 5-methyl cytosines. xT indicates a 2-thio thymidine nucleoside. Nucleosides without a subscript are β-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e”, “k” or “b” are sugar modified nucleosides. A subscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside, a subscript “k” indicates a 6′-(S)—CH3 bicyclic nucleoside (e.g. cEt) and a subscript “b” indicates a 5′-Me DNA modified nucleoside. Underlined nucleosides indicate the position of modification. Bold and underlined nucleosides indicate the mismatch position.
  • TABLE 71
    Short-gap chimeric oligonucleotides targeting 
    HTT SNP
    Wing SEQ
    ISIS Sequence Gap Chemistry ID
    NO (5′ to 3′) Motif Chemistry 5′ 3′ NO.
    460209* TeAkAkATTGTC 3-9-3 — ekk kke 10
    ATCAkCkCe
    XXXX16 TeAkAkA xTTGT 3-9-3 Deoxy/2-thio ekk kke 10
    CATCAkCkCe
    XXXX17 TeAkAkAT xTGT 3-9-3 Deoxy/2-thio ekk kke 10
    CATCAkCkCe
    XXXX18 TeAkAkA xTxTGT 3-9-3 Deoxy/2-thio ekk kke 10
    CATCAkCkCe
    XXXX19 TeAkAkATTp GT 3-9-3 Deoxy/Methyl ekk kke 10
    (558257) CATCAkCkCe phosphonate
    XXXX20 TeAkAkATp TGT 3-9-3 Deoxy/Methyl ekk kke 10
    (558256) CATCAkCkCe phosphonate
    XXXX20a TeAkAkATpz TGT 3-9-3 Deoxy/(R)- ekk kke 10
    CATCAkCkCe Methyl
    phosphonate
    XXXX20b TeAkAkATpw TG 3-9-3 Deoxy/(S)- ekk kke 10
    TCATCAkCkCe Methyl
    phosphonate
    XXXX21 TeAkAk Ap TTGT 3-9-3 Methyl ekk kke 10
    (558255) CATCAkCkCe phosphonate
    XXXX22 TeAkAkATTb GT 3-9-3 5′-Me-DNA ekk kke 10
    CATCAkCkCe
    XXXX23 TeAkAkATb TGT 3-9-3 5′-Me-DNA ekk kke 10
    CATCAkCkCe
    XXXX24 TeAkAk Ab TTGT 3-9-3 5′-Me-DNA ekk kke 10
    CATCAkCkCe
    XXXX25 TeAkAk G TTGTC 4-8-3 Mismatch at ekk kke 53
    ATCAkCkCe position 4
    XXXX26 TeAkAkA G TGT 5-7-3 Mismatch at ekk kke 54
    CATCAkCkCe position 5
    XXXX27 TeAkAkAT G GT 6-6-3 Mismatch at ekk kke 55
    CATCAkCkCe position 6
    e = 2′-MOE, k = cEt
    • Example 50 Short-Gap Chimeric Oligonucleotides Comprising Modifications at the Wing Regions Targeting Huntingtin (HTT) Single Nucleotide Polymorphism (SNP)
  • Additional chimeric antisense oligonucleotides were designed based on the parent gapmer, ISIS 460209, wherein the central gap region contains nine 2′-deoxynucleosides. These gapmers were designed by shortening the central gap region to seven 2′-deoxynucleosides and introducing various modifications at the wing regions.
  • The gapmers and their motifs are described in Table 72. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine nucleobases throughout each gapmer are 5-methyl cytosines. Nucleosides without a subscript are β-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e” or “k” are sugar modified nucleosides. A subscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside and a subscript “k” indicates a 6′-(S)—CH3 bicyclic nucleoside (e.g. cEt).
  • The number in parentheses indicates the position on the chimeric oligonucleotide opposite to the SNP position, as counted from the 5′-terminus.
  • These gapmers were evaluated for thermal stability (Tm) using methods described in Example 42. Presented in Table 73 is the Tm measurements for chimeric antisense oligonucleotides when duplexed to mutant or wild-type RNA complement. The Tm of chimeric antisense oligonucleotides duplexed with mutant RNA complement is denoted as “Tm (° C.) mut”. The Tm of chimeric antisense oligonucleotides duplexed with wild-type RNA complement is denoted as “Tm (° C.) wt”.
  • These gapmers were also tested in vitro. Heterozygous fibroblast GM04022 cell line was used. Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with a single dose at 2 μM concentration of the modified oligonucleotide. After a treatment period of approximately 24 hours, cells were washed with DPBS buffer and lysed. RNA was extracted using Qiagen RNeasy purification and mRNA levels were measured by quantitative real-time PCR using ABI assay C—2229297—10 which measures at dbSNP rs362303. RT-PCR method in short; A mixture was made using 2020 uL 2×PCR buffer, 101 uL primers (300 uM from ABI), 1000 uL water and 40.4 uL RT MIX. To each well was added 15 uL of this mixture and 5 uL of purified RNA. The mutant and wild-type HTT mRNA levels were measured simultaneously by using two different fluorophores, FAM for mutant allele and VIC for wild-type allele. HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. The results in Table 73 are presented as percent of HTT mRNA expression, relative to untreated control levels and is denoted as “% UTC”. Selectivity was also evaluated and measured by dividing the percent of wild-type HTT mRNA levels vs. the percent of mutant HTT mRNA levels. ISIS 460209 marked with an asterisk (*) in the table was included in the study for comparison.
  • As illustrated in Table 73, improvement in selectivity was observed for gapmers comprising 2-7-8 or 5-7-5 motifs having cEt subunits at the wing regions in comparison to the parent gapmer, ISIS 460209. The remaining gapmers showed moderate to little improvement in selectivity.
  • TABLE 72
    Short-gap chimeric oligonucleotides comprising  
    wing modifications
    wing SEQ
    ISIS chemistry ID
    NO Sequence (5′ to 3′) Motif 5′ 3′ NO.
    460209* (8) TeAkAkATTGTCATCAkCkCe 3-9-3 ekk kke 10
    540103 (6) AkAkTTGTCATCeAeCeCeAeGeAeAe 2-7-8 kk e8 58
    540104(6)  AeAeTTGTCATCeAeCeCeAeGeAeAe 2-7-8 ee e8 59
    540105 (7) AeAeAeTTGTCATCeAeCeCeAeGeAe 3-7-7 eee e7 60
    540106 (8) TeAeAeAeTTGTCATCeAeCeCeAeGe 4-7-6 eeee e6 61
    540107 (9) AeTeAeAeAkTTGTCATCkAeCeCeAe 5-7-5 eeeek keeee 32
    540109 (10) AeAeTeAeAeAeTTGTCATCeAeCeCe 6-7-4 e6 e4 62
    540110 (11) TeAeAeTeAeAeAeTTGTCATCeAeCe 7-7-3 e7 eee 63
    540111 (12) TeTeAeAeTeAeAeAeTTGTCATCeAe 8-7-2 e8 ee 64
    540112 (12) TeTeAeAeTeAeAeAeTTGTCATCkAk 8-7-2 e8 kk 64
    e = 2′-MOE (e.g. e6 = eeeeee), and k = cEt
  • TABLE 73
    Comparison of selectivity in inhibition of HTT mRNA levels of
    antisense oligonucleotides with ISIS 460209 targeted to
    RS7685686 in GM04022 cells
    Selec-
    Tm tivity wing
    (° C.) % UTC (wt vs chemistry
    ISIS NO mut wt mut wt mut) Motif 5′ 3′
    460209* (8) 53.7 52.2 23 57 2.4 3-9-3 ekk kke
    540103 (6) 57.6 56.4 23 74 3.3 2-7-8 kk e8
    540104 (6) 54.8 52.8 36 91 2.5 2-7-8 ee e8
    540105 (7) 54.2 52.2 53 135 2.6 3-7-7 eee e7
    540106 (8) 52.4 50.8 30 77 2.6 4-7-6 eeee e6
    540107 (9) 56.6 54.7 19 62 3.3 5-7-5 eeeek keeee
    540109 (10) 49.1 47.3 78 127 1.6 6-7-4 e6 e4
    540110 (11) 42.8 41.2 89 112 1.3 7-7-3 e7 eee
    540111 (12) 39.0 36.9 111 128 1.1 8-7-2 e8 ee
    540112 (12) 44.2 42.4 86 102 1.2 8-7-2 e8 kk
    • Example 51 Chimeric Oligonucleotides with SNP Site Shifting within the Central Gap Region
  • Chimeric antisense oligonucleotides were designed based on the parent gapmer, ISIS 460209 wherein the SNP site aligns with position 5 of the parent gapmer, as counted from the 5′-gap terminus. These gapmers were designed by shifting the SNP site upstream or downstream (i.e. microwalk) within the central gap region of the parent gapmer.
  • The gapmers and their motifs are described in Table 74. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine nucleobases throughout each gapmer are 5-methyl cytosines. Nucleosides without a subscript are β-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e” or “k” are sugar modified nucleosides. A subscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside and a subscript “k” indicates a 6′-(S)—CH3 bicyclic nucleoside (e.g. cEt). Underline nucleosides indicate the position on the chimeric oligonucleotide aligns with the SNP site.
  • The SNP site indicates the position on the chimeric antisense oligonucleotide opposite to the SNP position, as counted from the 5′-gap terminus and is denoted as “SNP site”.
  • The chimeric oligonucleotides were tested in vitro. Heterozygous fibroblast GM04022 cell line was used. Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with 0.12, 0.37, 1.1, 3.3 and 10 μM concentrations of modified oligonucleotides. After a treatment period of approximately 16 hours, RNA was isolated from the cells and mRNA levels were measured by quantitative real-time PCR using ABI assay C—2229297—10 which measures at dbSNP rs362303. The HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. ISIS 460209 marked with an asterisk (*) in the table was included in the study for comparison.
  • The IC50 and selectivity were calculated using the methods previously described in Example 41. As illustrated in Table 75, chimeric oligonucleotides comprising 4-9-2 (ISIS 540082) or 2-9-4 (ISIS 540095) motif with the SNP site at position 1 or 3 showed comparable activity and 2.5 fold selectivity as compared to their counterparts.
  • TABLE 74
    Chimeric oligonucleotides designed by microwalk
    wing SEQ
    ISIS SNP chemistry ID
    NO Sequence (5′ to 3′) Motif site 5′ 3′ NO.
    460209* TeAkAkATTGTCATCAkCkCe 3-9-3 5 ekk kke 10
    540082 AeTkTkGk TCATCACCAGkAe 4-9-2 1 ekkk ke 65
    540089 TeTkAkAkTAAATTGTCAkTe 4-9-2 8 ekkk ke 66
    540095 AeTkTGTCATCACCkAkGkAe 2-9-4 3 ek kkke 65
    e = 2′-MOE, and k = cEt
  • TABLE 75
    Comparison of inhibition of HTT mRNA levels and
    selectivity of chimeric oligonucleotides with ISIS
    460209 targeted to HTT SNP
    Mut Wing
    IC50 Wt IC50 Selectivity SNP chemistry
    ISIS NO (μM) (μM) (wt vs mut) Motif site 5′ 3′
    460209 0.41 2.0 4.9 3-9-3 5 ekk kke
    540082 0.45 5.6 12 4-9-2 1 ekkk ke
    540089 >10 >10 — 4-9-2 8 ekkk ke
    540095 0.69 8.4 12 2-9-4 3 ek kkke
    e = 2′-MOE, and
    k = cEt
    • Example 52 Chimeric Oligonucleotides with SNP Site Shifting at Various Positions
  • Chimeric antisense oligonucleotides were designed based on the parent gapmer, ISIS 460209 wherein the SNP site aligns with position 8 of the parent gapmer, as counted from the 5′-terminus. These gapmers were designed by shifting the SNP site upstream or downstream (i.e. microwalk) of the original oligonucleotide.
  • The gapmers and their motifs are described in Table 76. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine nucleobases throughout each gapmer are 5-methyl cytosines. Nucleosides without a subscript are β-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e” or “k” are sugar modified nucleosides. A subscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside and a subscript “k” indicates a 6′-(S)—CH3 bicyclic nucleoside (e.g. cEt). Underline nucleosides indicate the SNP site.
  • The SNP site indicates the position on the chimeric antisense oligonucleotide opposite to the SNP position, as counted from the 5′-terminus and is denoted as “SNP site”.
  • The chimeric oligonucleotides were tested in vitro. Heterozygous fibroblast GM04022 cell line was used. Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with 0.12, 0.37, 1.1, 3.3 and 10 μM concentrations of modified oligonucleotides. After a treatment period of approximately 16 hours, cells were washed with DPBS buffer and lysed. RNA was extracted using Qiagen RNeasy purification and mRNA levels were measured by quantitative real-time PCR using ABI assay C—2229297—10 which measures at dbSNP rs362303. RT-PCR method in short; A mixture was made using 2020 uL 2×PCR buffer, 101 uL primers (300 uM from ABI), 1000 uL water and 40.4 uL RT MIX. To each well was added 15 uL of this mixture and 5 uL of purified RNA. The mutant and wild-type HTT mRNA levels were measured simultaneously by using two different fluorophores, FAM for mutant allele and VIC for wild-type allele. HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. The results in Table 77 are presented as percent of HTT mRNA expression, relative to untreated control levels and is denoted as “% UTC”. Selectivity was also evaluated and measured by dividing the percent of wild-type HTT mRNA levels vs. the percent of mutant HTT mRNA levels.
  • The parent gapmer, ISIS 460209 is marked with an asterisk (*) in the table and was included in the study as a benchmark oligonucleotide against which the selectivity of the modified oligonucleotides targeting nucleotides overlapping the SNP position could be compared.
  • As illustrated in Table 77, improvement in potency and selectivity was observed for chimeric oligonucleotides comprising 4-9-2 or 2-9-4 motif having the target SNP site at positions 3, 4, 6, 7 and 8 (ISIS540083, ISIS540084, ISIS 540085, ISIS 540094, ISIS 540096, ISIS 540097 and ISIS 540098) in comparison to position 8 of the parent gapmer (ISIS 460209). The remaining gapmers showed little to no improvement in potency or selectivity.
  • TABLE 76
    Chimeric oligonucleotides designed by
    microwalk
    SEQ
    ISIS SNP ID
    NO Sequence (5′ to 3′) site Motif NO.
    460209* TeAkAkATTGTCATCAkCkCe  8 3-9-3 10
    (ekk-
    d9-
    kke)
    543887 TeTkGk Tk CATCACCAGAkAe  4 4-9-2 67
    (ekkk-
    d9-ke)
    540083 AeAkTkTkGTCATCACCAkGe  6 4-9-2 68
    (ekkk-
    d9-ke)
    540084 AeAkAkTkTGTCATCACCkAe  7 4-9-2 69
    (ekkk-
    d9-ke)
    540085 TeAkAkAkTTGTCATCACkCe  8 4-9-2 10
    (ekkk-
    d9-ke)
    540087 AeAkTkAkAATTGTCATCkAe 10 4-9-2 70
    (ekkk-
    d9-ke)
    540090 AeTkTkAkATAAATTGTCkAe 13 4-9-2 71
    (ekkk-
    d9-ke)
    540091 TeAkTkTkAATAAATTGTk Ce 14 4-9-2 72
    (ekkk-
    d9-ke)
    540092 Ge Tk CATCACCAGAkAkAkAe  2 2-9-4 73
    (ek-
    d9-
    kkke)
    540093 TeGk TCATCACCAGkAkAkAe  3 2-9-4 74
    (ek-
    d9-
    kkke)
    540094 TeTkGTCATCACCAkGkAkAe  4 2-9-4 67
    (ek-
    d9-
    kkke)
    540096 AeAkTTGTCATCACkCkAkGe  6 2-9-4 68
    (ek-
    d9-
    kkke)
    540097 AeAkATTGTCATCAkCkCkAe  8 2-9-4 69
    (ek-
    d9-
    kkke)
    540098 TeAkAATTGTCATCkAkCkCe  8 2-9-4 10
    (ek-
    d9-
    kkke)
    540099 AeTkAAATTGTCATkCkAkCe  9 2-9-4 75
    (ek-
    d9-
    kkke)
    540100 AeAkTAAATTGTCAkTkCkAe 10 2-9-4 70
    (ek-
    d9-
    kkke)
    540101 TeAkATAAATTGTCkAkTkCe 11 2-9-4 76
    (ek-
    d9-
    kkke)
    540102 TeTkAATAAATTGTk CkAkTe 12 2-9-4 66
    (ek-
    d9-
    kkke)
    e = 2′-MOE; k = cEt; d = 2′-deoxyribonucleoside
  • TABLE 77
    Comparison of selectivity in HTT SNP inhibition
    of chimeric oligonucleotides with ISIS 460209
    % UTC Selectivity SNP
    ISIS NO mut wt (wt vs. mut) site Motif
     460209* 23 57 2.4 8 3-9-3
    (ekk-d9-kke)
    543887 18 43 2.3 4 4-9-2
    (ekkk-d9-ke)
    540083 18 67 3.7 6 4-9-2
    (ekkk-d9-ke)
    540084 10 49 4.9 7 4-9-2
    (ekkk-d9-ke)
    540085 21 86 4.1 8 4-9-2
    (ekkk-d9-ke)
    540087 60 98 1.6 10 4-9-2
    (ekkk-d9-ke)
    540090 129 137 1.1 13 4-9-2
    (ekkk-d9-ke)
    540091 93 105 1.1 14 4-9-2
    (ekkk-d9-ke)
    540092 28 55 2.0 2 2-9-4
    (ek-d9-kkke)
    540093 18 62 3.4 3 2-9-4
    (ek-d9-kkke)
    540094 13 45 3.4 4 2-9-4
    (ek-d9-kkke)
    540096 17 68 4.0 6 2-9-4
    (ek-d9-kkke)
    540097 8 35 4.2 8 2-9-4
    (ek-d9-kkke)
    540098 12 45 3.9 8 2-9-4
    (ek-d9-kkke)
    540099 62 91 1.5 9 2-9-4
    (ek-d9-kkke)
    540100 80 106 1.3 10 2-9-4
    (ek-d9-kkke)
    540101 154 152 1.0 11 2-9-4
    (ek-d9-kkke)
    540102 102 106 1.0 12 2-9-4
    (ek-d9-kkke)
    e = 2′-MOE; k = cEt; d = 2′-deoxyribonucleoside
    • Example 53 Selectivity in Inhibition of HTT mRNA Levels Targeting SNP by Chimeric Oligonucleotides Designed by Microwalk
  • A series of modified oligonucleotides were designed based on the parent gapmer, ISIS 460209, wherein the central gap region comprises nine 2′-deoxyribonucleosides. These gapmers were created with various motifs and modifications at the wings and/or the central gap region.
  • The modified oligonucleotides and their motifs are described in Table 78. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine nucleobases throughout each gapmer are 5-methyl cytosines. Nucleosides without a subscript are β-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e”, “k”, “y”, or “z” are sugar modified nucleosides. A subscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside, a subscript “k” indicates a 6′-(S)—CH3 bicyclic nucleoside (e.g. cEt), a subscript “y” indicates an α-L-LNA modified nucleoside, and a subscript “z” indicates a F-HNA modified nucleoside. pU indicates a 5-propyne uridine nucleoside and xT indicates a 2-thio-thymidine nucleoside. Underlined nucleosides indicate the mismatch position.
  • These gapmers were evaluated for thermal stability (Tm) using methods described in Example 42. Presented in Table 79 are the Tm measurements for chimeric antisense oligonucleotides when duplexed to mutant or wild-type RNA complement. The Tm of chimeric antisense oligonucleotides duplexed with mutant RNA complement is denoted as “Tm (° C.) mut”. The Tm of chimeric antisense oligonucleotides duplexed with wild-type RNA complement is denoted as “Tm (° C.) wt”.
  • These gapmers were also tested in vitro. ISIS 141923 was included in the study as a negative control and is denoted as “neg control”. The non-allele specific antisense oligonucleotides, ISIS 387916 was used as a positive control and is denoted as “pos control”. Heterozygous fibroblast GM04022 cell line was used. Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with a single dose at 2 μM concentration of the modified oligonucleotide. After a treatment period of approximately 24 hours, cells were washed with DPBS buffer and lysed. RNA was extracted using Qiagen RNeasy purification and mRNA levels were measured by quantitative real-time PCR using ABI assay C—2229297—10 which measures at dbSNP rs362303. RT-PCR method in short; A mixture was made using 2020 uL 2×PCR buffer, 101 uL primers (300 uM from ABI), 1000 uL water and 40.4 uL RT MIX. To each well was added 15 uL of this mixture and 5 uL of purified RNA. The mutant and wild-type HTT mRNA levels were measured simultaneously by using two different fluorophores, FAM for mutant allele and VIC for wild-type allele. HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. ISIS 460209 marked with an asterisk (*) in the table was included in the study for comparison. The results in Table 79 are presented as percent of HTT mRNA expression, relative to untreated control levels and is denoted as “% UTC”. Selectivity was also evaluated and measured by dividing the percent of wild-type HTT mRNA levels vs. the percent of mutant HTT mRNA levels.
  • As illustrated, several of the newly designed antisense oligonucleotides showed improvement in potency and/or selectivity in inhibiting mut HTT mRNA levels comparing to ISIS 460209.
  • TABLE 78
    Modified oligonucleotides comprising various modifications targeting 
    HTT SNP
    Wing SEQ
    ISIS Chemistry ID
    NO Sequence (5′ to 3′) Modification 5′ 3′ NO.
    460209* TeAkAkATTGTCATCAkCkCe 3-9-3 ekk kke 10
    (ekk-d9-kke)
    539560 TeAkAkATTGpUCATCAkCkCe 5-propyne in ekk kke 11
    gap
    539563 TeAkAkATTGxTCATCAkCkCe 2-thio in gap ekk kke 10
    539554 TeAkAkATTGUyCATCAkCkCe α-L-LNA in gap ekk kke 11
    542686 TeAkAkATTGTzCATCAkCkCe F-HNA in gap ekk kke 10
    540108 AeTeAeAkAkTTGTCATCkAkCeCeAe 5-7-5 eeekk kkeee 23
    (eeekk-d7-kkeee)
    544840 TeAkAkATTGTCATCAkCkCeTkTkAk 3-9-6 ekk kkekkk 15
    (ekk-d9-kkekkk)
    550904 TeAkAkATTGTCATCAkCkCeTkTkTkAk 3-9-7 ekk kkekkkk 18
    (ekk-d9-kkekkkk)
    540082 AeTkTkGkTCATCACCAGkAe 4-9-2 ekkk ke 65
    (ekkk-d9-ke)
    540089 TeTkAkAkTAAATTGTCAkTe 4-9-2 ekkk ke 66
    (ekkk-d9-ke)
    540095 AeTkTGTCATCACCkAkGkAe 2-9-4 ek kkke 67
    (ek-d9-kkke)
    543528 AeTkAeAkAATGTCATCAkCeCkAe Mismatch at ekek keke 77
    position 2 
    counting
    from 5′ gap
    543533 TeAkAkATAGTCATCAkCkCe Mismatch at ekk kke 78
    position 3 
    counting
    from 5′ gap
    387916 TeCeTeCeTeATTGCACATTCeCeAeAeGe 5-10-5 eeeee eeeee 56
    (pos control)
    141923 CeCeTeTeCeCCTGAAGGTTCeCeTeCeCe 5-10-5 eeeee eeeee 57
    (neg control)
    e = 2′-MOE; k = cEt; d = 2′-deoxyribonucleoside
  • TABLE 79
    Comparison of selectivity in inhibition of HTT mRNA levels, and Tm of modified
    oligonucleotides with ISIS 460209 targeted tors7685686 in GM04022 cells
    Tm (° C.) % UTC Selectivity Wing Chemistry
    ISIS NO mutant wt mut wt (wt vs mut) Modification 5′ 3′
    460209* 53.7 52.2 23 57 2.7 3-9-3 ekk kke
    (ekk-d9-kke)
    539560 54.1 50.8 13 32 2.4 5-propyne in gap ekk kke
    539563 53.8 49.1 13 40 3.2 2-thio in gap ekk kke
    539554 56.5 54.5 54 89 1.7 α-L-LNA in gap ekk kke
    542686 56.1 50.4 26 62 2.4 F-HNA in gap ekk kke
    540108 60.0 57.9 27 63 2.3 5-7-5 eeekk kkeee
    (eeekk-d7-kkeee)
    544840 — — 19 40 2.1 3-9-6 ekk kkekkk
    (ekk-d9-kkekkk)
    550904 — — 39 65 1.7 3-9-7 ekk kkekkkk
    (ekk-d9-
    kkekkkk)
    540082 — — 21 62 3.0 4-9-2 ekkk ke
    (ekkk-d9-ke)
    540089 — — 78 86 1.1 4-9-2 ekkk ke
    (ekkk-d9-ke)
    540095 — — 22 66 3.1 2-9-4 ek kkke
    (ek-d9-kkke)
    543528 50.5 49.1 44 90 2.1 Mismatch at ekek keke
    position 2
    counting from 5′
    gap
    543533 47.0 44.8 83 97 1.2 Mismatch at ekk kke
    position 3
    counting from 5′
    gap
    387916 — — 21 19 0.9 5-10-5 eeeee eeeee
    (pos control)
    141923 — — 95 99 1.0 5-10-5 eeeee eeeee
    (neg control)
    e = 2′-MOE;
    k = cEt;
    d = 2′-deoxyribonucleoside
    • Example 54 Chimeric Oligonucleotides Comprising Modifications at the SNP Site of HTT Gene
  • Additional gapmers are designed based on the gapmer selected from studies described in Tables 73 and 74 (ISIS 540108) and is marked with an asterisk (*). These gapmers are designed by introducing modifications at the SNP site at position 9 of the oligonucleotides, as counted from the 5′-terminus and are created with a 5-7-5 motif.
  • The gapmers are described in Table 80. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine nucleobases throughout each gapmer are 5-methyl cytosines. Nucleosides without a subscript are β-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “a”, “b”, “e”, or “k” are sugar modified nucleosides. A subscript “a” indicates 2′-(ara)-F modified nucleoside, a subscript “b” indicates a 5′-Me DNA modified nucleoside, a subscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside, and a subscript “k” indicates a 6′-(S)—CH3 bicyclic nucleoside (e.g. cEt). xT indicates a 2-thio-thymidine nucleoside. Underline nucleoside or the number in parentheses indicates the position on the oligonucleotides opposite to the SNP position, as counted from the 5′-terminus.
  • TABLE 80
    Modified oligonucleotides targeting HTT SNP
    Wing SEQ
    ISIS Gap chemistry ID
    NO Sequence (5′ to 3′) Chemistry 5′ 3′ NO.
    540108* (9) AeTeAeAkAkTTGTCATCkAkCeCeAe Deoxy eeekk kkeee 32
    XXXX28 (9) AeTeAeAkAkTTG xTCATCkAkCeCeAe Deoxy/2- eeekk kkeee 32
    thio
    XXXX29 (9) AeTeAeAkAkTTGTa CATCkAkCeCeAe Deoxy/2′- eeekk kkeee 32
    (ara)-F
    XXXX30 (9) AeTeAeAkAkTTGTb CATCkAkCeCeAe Deoxy/5′- eeekk kkeee 32
    Me-DNA
    e = 2′-MOE, k = cEt
    • Example 55 Chimeric Oligonucleotides Comprising Modifications at the Wing Regions Targeting HTT SNP
  • Additional gapmers are designed based on the gapmer selected from studies described in Tables 89 and 21 (ISIS 540107) and is marked with an asterisk (*). These gapmers are designed by introducing bicyclic modified nucleosides at the 3′ or 5′ terminus and are tested to evaluate if the addition of bicyclic modified nucleosides at the wing regions improves the activity and selectivity in inhibition of mutant HTT SNP.
  • The gapmers comprise a 5-7-5 motif and are described in Table 81. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine nucleobases throughout each gapmer are 5-methyl cytosines. Nucleosides without a subscript are β-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e”, or “k” are sugar modified nucleosides. A subscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside, and a subscript “k” indicates a 6′-(S)—CH3 bicyclic nucleoside (e.g. cEt).
  • TABLE 81
    Modified oligonucleotides targeting HTT SNP
    wing SEQ
    ISIS chemistry ID
    NO Sequence (5′ to 3′) Motif 5′ 3′ NO.
    540107* AeTeAeAeAkTTGTCATCkAeCeCeAe 5-7-5 eeeek keeee 32
    (eeeek-d7-keeee)
    XXXX31 AeTeAkAkAkTTGTCATCkAkCkCeAe 5-7-5 eekkk kkkee 32
    (eekkk-d7-kkkee)
    XXXX32 AeTeAeAeAkTTGTCATCeAeCeCeAe 5-7-5 eeeek eeeee 32
    (eeeek-d7-eeeee)
    XXXX33 AeTeAeAkAkTTGTCATCeAeCeCeAe 5-7-5 eeekk eeeee 32
    (eeekk-d7-eeeee)
    XXXX34 AeTeAkAkAkTTGTCATCeAeCeCeAe 5-7-5 eekkk eeeee 32
    (eekkk-d7-eeeee)
    XXXX35 AeTeAeAeAeTTGTCATCkAeCeCeAe 5-7-5 eeeee keeee 32
    (eeeee-d7-keeee)
    XXXX36 AeTeAeAeAeTTGTCATCkAkCeCeAe 5-7-5 eeeee kkeee 32
    (eeeee-d7-kkeee)
    XXXX37 AeTeAeAeAeTTGTCATCkAkCkCeAe 5-7-5 eeeee kkkee 32
    (eeeee-d7-kkkee)
    e = 2′-MOE; k = cEt; d = 2′-deoxyribonucleoside
    • Example 56 Chimeric Oligonucleotides Comprising Wing and Central Gap Modifications Targeting HTT SNP
  • Additional gapmers are designed based on the parent gapmer, ISIS 460209, wherein the central gap region comprises nine 2′-deoxyribonucleosides and is marked with an asterisk (*) in the table. These gapmers were designed by introducing modifications at the wings or the central gap region and are created with a 3-9-3 motif.
  • The gapmers are described in Table 82. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine nucleobases throughout each gapmer are 5-methyl cytosines. Nucleosides without a subscript are β-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e”, or “k” are sugar modified nucleosides. A subscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside, and a subscript “k” indicates a 6′-(S)—CH3 bicyclic nucleoside (e.g. cEt). PT indicates a 5-propyne thymidine nucleoside. PC indicates a 5-propyne cytosine nucleoside. Underline nucleoside or the number in parentheses indicates the position on the oligonucleotides opposite to the SNP position, as counted from the 5′-terminus.
  • TABLE 82
    Modified oligonucleotides targeting HTT SNP
    wing SEQ
    ISIS chemistry ID
    NO Sequence (5′ to 3′) Modification 5′ 3′ NO
    460209* (8) TeAkAkATTGTCATCAkCkCe Deoxy gap ekk kke 10
    (3-9-3)
    552103 (8) TeAeAeATTGTCATCAkCkCk Deoxy gap eee kkk 10
    (3-9-3)
    552104 (8) TkAkAkATTGTCATCAeCeCe Deoxy gap kkk eee 10
    (3-9-3)
    552105 (8) TeAkAkATTG PT PCATCAkCkCe Deoxy/5- ekk kke 10
    Propyne
    552106 (8) TeAkAkAPTPTG PT PCAPTPCAkCkCe Deoxy/5- ekk kke 10
    Propyne
    e = 2′-MOE; k = cEt
    • Example 57 Modified Oligonucleotides Comprising F-HNA Modification at the Central Gap or Wing Region Targeting HTT SNP
  • A series of modified oligonucleotides were designed based on ISIS 460209, wherein the central gap region contains nine 2′-deoxyribonucleosides. These modified oligonucleotides were designed by incorporating one or more F-HNA(s) modification within the central gap region or on the wing regions. The F-HNA containing oligonucleotides were tested for their ability to selectively inhibit mutant (mut) HTT mRNA expression levels targeting rs7685686 while leaving the expression of the wild-type (wt) intact. The activity and selectivity of the modified oligonucleotides were evaluated and compared to ISIS 460209.
  • The modified oligonucleotides and their motifs are described in Table 83. The internucleoside linkages throughout each modified oligonucleotide are phosphorothioate linkages (P═S). Nucleosides without a subscript are β-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e” indicate 2′-O-methoxyethyl (MOE) modified nucleosides. Nucleosides followed by a subscript “k” indicate 6′-(S)—CH3 bicyclic nucleosides (e.g. cEt). Nucleosides followed by a subscript “z” indicate F-HNA modified nucleosides. mC indicates a 5-methyl cytosine nucleoside. Underlined nucleoside indicates the position on the oligonucleotides opposite to the SNP position, which is position 8 as counted from the 5′-terminus.
  • The gap-interrupted antisense oligonucleotides were tested in vitro. Heterozygous fibroblast GM04022 cell line was used. Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with 0.12, 0.37, 1.1, 3.3 and 10 μM concentrations of modified oligonucleotides. After a treatment period of approximately 16 hours, RNA was isolated from the cells and mRNA levels were measured by quantitative real-time PCR using ABI assay C—2229297—10 which measures at dbSNP rs362303. The HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN and the results are presented in Table 84.
  • The IC50 and selectivity were calculated using methods previously described in Example 41. The IC50 at which each oligonucleotide inhibits the mutant HTT mRNA expression is denoted as ‘mut IC50’. The IC50 at which each oligonucleotide inhibits the wild-type HTT mRNA expression is denoted as ‘wt IC50’. Selectivity was calculated by dividing the IC50 for inhibition of the wild-type HTT versus the IC50 for inhibiting expression of the mutant HTT mRNA.
  • The parent gapmer, 460209 is marked with an asterisk (*) in the table and was included in the study as a benchmark oligonucleotide against which the activity and selectivity of antisense oligonucleotides targeting nucleotides overlapping the SNP position could be compared.
  • As illustrated in Table 84, oligonucleotides comprising F-HNA modification(s) showed improvement in selectivity while maintaining activity as compared to the parent gapmer, ISIS 460209.
  • TABLE 83
    Gap-interrupted antisense oligonucleotides targeting
    HTT SNP
    Wing SEQ
    ISIS Sequence Gap chemistry ID
    NO. (5′ to 3′) Motif chemistry 5′ 3′ NO.
    460209* TeAkAkATTGT 3-9-3 Full deoxy ekk kke 10
    mCATmCAk mCk mCe
    566266 TeAkAkAzTTGT 3-9-3 or Deoxy/F- ekk or kke 10
    mCATmCAk mCk mCe 4-8-3 HNA ekkz
    566267 TeAkAkATzTGT 3-9-3 or Deoxy/F- ekk or kke 10
    mCATmCAk mCk mCe 5-7-3 HNA ekkdz
    566268 TeAkAkATTzGT 3-9-3 or Deoxy/F- ekk or kke 10
    mCATmCAk mCk mCe 6-6-3 HNA ekkddz
    566269 TeAkAkATTGz T 3-9-3 or Deoxy/F- ekk or kke 10
    mCATmCAk mCk mCe 7-5-3 HNA ekkdddz
    567369 TeAkAkAzTzTGT 3-9-3 or Deoxy/F- ekk or kke 10
    mCATmCAk mCk mCe 5-7-3 HNA ekkzz
    e = 2′-MOE, k = cEt, d = 2′-β-deoxyribonucleoside, z = F-HNA
  • TABLE 84
    Comparison of inhibition of HTT mRNA levels and selectivity
    of gap-interrupted antisense oligonucleotides with
    ISIS 460209 targeting HTT SNP
    IC50 (μM) Selectivity Gap Wing Chemistry
    ISIS NO Mut Wt (wt vs mut) Motif chemistry 5′ 3′
    460209* 0.28 3.1 11 3-9-3 Full deoxy ekk kke
    566266 0.20 >10 >50 3-9-3 or Deoxy/F- ekk or ekkz kke
    4-8-3 HNA
    566267 0.90 >9.9 >11 3-9-3 or Deoxy/F- ekk or ekkdz kke
    5-7-3 HNA
    566268 1.0 >10 >10 3-9-3 or Deoxy/F- ekk or ekkddz kke
    6-6-3 HNA
    566269 1.7 >10.2 >6 3-9-3 or Deoxy/F- ekk or kke
    7-5-3 HNA ekkdddz
    567369 0.82 >9.8 >12 3-9-3 or Deoxy/F- ekk or ekkzz kke
    5-7-3 HNA
    e = 2′-MOE,
    k = cEt,
    d = 2′-β-deoxyribonucleoside,
    z = F-HNA
    • Example 58 Modified Oligonucleotides Comprising cEt Modification(s) at the Central Gap Region Targeting HTT SNP
  • A series of modified oligonucleotides were designed in the same manner as described in Example 57.
  • These modified oligonucleotides were designed by replacing F-HNA(s) with cEt modification(s) in the central gap region while maintaining the wing configuration. The modified oligonucleotides were tested for their ability to selectively inhibit mutant (mut) HTT mRNA expression levels targeting rs7685686 while leaving the expression of the wild-type (wt) intact. The activity and selectivity of the modified oligonucleotides were evaluated and compared to ISIS 460209.
  • The modified oligonucleotides and their motifs are described in Table 85. The internucleoside linkages throughout each modified oligonucleotide are phosphorothioate linkages (P═S). Nucleosides without a subscript are β-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e” indicate 2′-β-methoxyethyl (MOE) modified nucleosides. Nucleosides followed by a subscript “k” indicate 6′-(S)—CH3 bicyclic nucleosides (e.g. cEt). mC indicates a 5-methyl cytosine nucleoside. Underlined nucleoside indicates the position on the oligonucleotides opposite to the SNP position, which is position 8 as counted from the 5′-terminus.
  • The gap-interrupted antisense oligonucleotides were tested in vitro. Heterozygous fibroblast GM04022 cell line was used. Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with 0.12, 0.37, 1.1, 3.3 and 10 μM concentrations of modified oligonucleotides. After a treatment period of approximately 16 hours, RNA was isolated from the cells and mRNA levels were measured by quantitative real-time PCR using ABI assay C—2229297—10 which measures at dbSNP rs362303. The HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN and the results are presented below.
  • The IC50 and selectivity were calculated using methods previously described in Example 41. The IC50 at which each oligonucleotide inhibits the mutant HTT mRNA expression is denoted as ‘mut IC50’. The IC50 at which each oligonucleotide inhibits the wild-type HTT mRNA expression is denoted as ‘wt IC50’. Selectivity was calculated by dividing the IC50 for inhibition of the wild-type HTT versus the IC50 for inhibiting expression of the mutant HTT mRNA.
  • As illustrated in Table 86, some of the newly designed antisense oligonucleotides (ISIS 575006, 575007, and 575008) showed improvement in potency and/or selectivity in inhibiting mut HTT mRNA levels comparing to ISIS 460209.
  • TABLE 85
    Gap-interrupted antisense oligonucleotides targeting
    HTT SNP
    Wing SEQ
    ISIS Sequence Gap chemistry ID
    NO. (5′ to 3′) Motif chemistry 5′ 3′ NO.
    460209* TeAkAkATTGT 3-9-3 Full deoxy ekk kke 10
    mCATmCAk mCk mCe
    575006 TeAkAkAkTTGT 4-8-3 Full deoxy ekkk kke 10
    mCATmCAk mCk mCe
    575007 TeAkAkATkTGT 3-9-3 or Full deoxy or ekk or kke 10
    mCATmCAk mCk mCe 5-7-3 Deoxy/cEt ekkdk
    575133 TeAkAkATTkGT 3-9-3 or Full deoxy or ekk or kke 10
    mCATmCAk mCk mCe 6-6-3 Deoxy/cEt ekkddk
    575134 TeAkAkATTGk T 3-9-3 or Full deoxy or ekk or kke 10
    mCATmCAk mCk mCe 7-5-3 Deoxy/cEt ekkdddk
    575008 TeAkAkAkTkTGT 5-7-3 Deoxy ekkkk kke 10
    mCATmCAk mCk mCe
    e = 2′-MOE, k = cEt, d = 2′-β-deoxyribonucleoside
  • TABLE 86
    Comparison of inhibition of HTT mRNA levels and selectivity of gap-interrupted
    antisense oligonucleotides with ISIS 460209 targeting HTT SNP
    IC50 (μM) Selectivity Gap Wing Chemistry
    ISIS NO Mut Wt (wt vs mut) Motif chemistry 5′ 3′
    460209* 0.28 3.1 11 3-9-3 Full deoxy ekk kke
    575006 0.27 3.8 14 4-8-3 Full deoxy ekkk kke
    575007 0.67 >10.1 >15 3-9-3 or Full deoxy or ekk or kke
    5-7-3 Deoxy/cEt ekkdk
    575133 3.0 >9 >3 3-9-3 or Full deoxy or ekk or kke
    6-6-3 Deoxy/cEt ekkddk
    575134 2.6 >10.4 >4 3-9-3 or Full deoxy or ekk or kke
    7-5-3 Deoxy/cEt ekkdddk
    575008 0.18 >9.9 >55 5-7-3 Full deoxy ekkkk kke
    e = 2′-MOE,
    k = cEt,
    d = 2′-β-deoxyribonucleoside
    • Example 59 Modified Oligonucleotides Comprising F-HNA Modification at the 3′-End of Central Gap Region Targeting HTT SNP
  • A series of modified oligonucleotides were designed based on ISIS 460209, wherein the central gap region contains nine 2′-deoxyribonucleosides. These modified oligonucleotides were designed by incorporating one F-HNA modification at the 3′-end of the central gap region. The F-HNA containing oligonucleotides were tested for their ability to selectively inhibit mutant (mut) HTT mRNA expression levels targeting HTT SNP while leaving the expression of the wild-type (wt) intact. The activity and selectivity of the modified oligonucleotides were evaluated and compared to ISIS 460209.
  • The modified oligonucleotides and their motifs are described in Table 87. The internucleoside linkages throughout each modified oligonucleotide are phosphorothioate linkages (P═S). Nucleosides without a subscript are β-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e” indicate 2′-β-methoxyethyl (MOE) modified nucleosides. Nucleosides followed by a subscript “k” indicate 6′-(S)—CH3 bicyclic nucleosides (e.g. cEt). Nucleosides followed by a subscript “z” indicate F-HNA modified nucleosides. mC indicates a 5-methyl cytosine nucleoside. Underlined nucleoside indicates the position on the oligonucleotides opposite to the SNP position, which is position 8 as counted from the 5′-terminus.
  • The modified oligonucleotides were tested in vitro. Heterozygous fibroblast GM04022 cell line was used. Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with 0.12, 0.37, 1.1, 3.3 and 10 μM concentrations of modified oligonucleotides. After a treatment period of approximately 16 hours, RNA was isolated from the cells and mRNA levels were measured by quantitative real-time PCR using ABI assay C—2229297—10 which measures at dbSNP rs362303. The HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN and the results are presented in Table 88.
  • The IC50 and selectivity were calculated using methods previously described in Example 41. The IC50 at which each oligonucleotide inhibits the mutant HTT mRNA expression is denoted as ‘mut IC50’. The IC50 at which each oligonucleotide inhibits the wild-type HTT mRNA expression is denoted as ‘wt IC50’. Selectivity was calculated by dividing the IC50 for inhibition of the wild-type HTT versus the IC50 for inhibiting expression of the mutant HTT mRNA.
  • As illustrated in Table 88, a couple of the newly designed antisense oligonucleotides (ISIS 575833 and 575834) showed improvement in selectivity while maintaining potency as compared to ISIS 460209. ISIS 575836 showed an increase in potency without improvement in selectivity while ISIS 575835 showed comparable selectivity without improvement in potency.
  • TABLE 87
    Modified oligonucleotides targeting HTT SNP
    Gap Wing SEQ
    ISIS Sequence  chem- chemistry ID
    NO. (5′ to 3′) Motif istry 5′ 3′ NO.
    460209* TeAkAkATTGT 3-9-3 Full  ekk kke 10
    mCATmCAk mCk mCe deoxy
    575833 TeAkAkATTGT 3-9-3  Deoxy/ ekk kke or 10
    mCzATmCAk mCk mCe or F-HNA zdddkke
    3-5-7
    575834 TeAkAkATTGT 3-9-3  Deoxy/ ekk kke or 10
    mCAzTmCAk mCk mCe or F-HNA zddkke
    3-6-6
    575835 TeAkAkATTGT 3-9-3  Deoxy/ ekk kke or 10
    mCATz mCAk mCk mCe or F-HNA zdkke
    3-7-5
    575836 TeAkAkATTGT 3-9-3  Deoxy/ ekk  kke or 10
    mCATmCzAk mCk mCe or F-HNA zkke
    3-8-4
    e = 2′-MOE, k = cEt, d = 2′-β-deoxyribonucleoside, z = F-HNA
  • TABLE 88
    Comparison of inhibition of HTT mRNA levels and selectivity
    of modified oligonucleotides with ISIS 460209 targeting HTT SNP
    IC50 (μM) Selectivity Wing Chemistry
    ISIS NO Mut Wt (wt vs mut) Motif Gap chemistry 5′ 3′
    460209* 0.28 3.1 11 3-9-3 Full deoxy ekk kke
    575833 0.22 4.2 19 3-9-3 or Deoxy/F-HNA ekk kke or
    3-5-7 zdddkke
    575834 0.30 6.3 21 3-9-3 or Deoxy/F-HNA ekk kke or
    3-6-6 zddkke
    575835 0.89 9.8 11 3-9-3 or Deoxy/F-HNA ekk kke or
    3-7-5 zdkke
    575836 0.09 0.4 4.6 3-9-3 or Deoxy/F-HNA ekk kke or zkke
    3-8-4
    e = 2′-MOE,
    k = cEt,
    d = 2′-β-deoxyribonucleoside,
    z = F-HNA
    • Example 60 Short-Gap Chimeric Oligonucleotides Targeting Huntingtin (HTT) Single Nucleotide Polymorphism (SNP)
  • Additional chimeric antisense oligonucleotides were designed based on ISIS 460209 and ISIS 540094 wherein the central gap region contains nine 2′-deoxynucleosides. These gapmers were designed with the central gap region shortened by introducing cEt modifications to the wing regions, or interrupted by introducing cEt modifications at the 3′-end of the central gap region. The modified oligonucleotides were tested for their ability to selectively inhibit mutant (mut) HTT mRNA expression levels targeting HTT SNP while leaving the expression of the wild-type (wt) intact. The activity and selectivity of the modified oligonucleotides were evaluated and compared to ISIS 460209 and 540094.
  • The gapmers and their motifs are described in Table 89. The internucleoside linkages throughout each modified oligonucleotide are phosphorothioate linkages (P═S). Nucleosides without a subscript are (3-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e” indicate 2′-O-methoxyethyl (MOE) modified nucleosides. Nucleosides followed by a subscript “k” indicate 6′-(S)—CH3 bicyclic nucleosides (e.g. cEt). mC indicates a 5-methyl cytosine nucleoside. Underlined nucleoside indicates the position on the oligonucleotides opposite to the SNP position, which is position 4 or 8 as counted from the 5′-terminus.
  • The modified oligonucleotides were tested in vitro. Heterozygous fibroblast GM04022 cell line was used. Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with 0.12, 0.37, 1.1, 3.3 and 10 μM concentrations of modified oligonucleotides. After a treatment period of approximately 16 hours, RNA was isolated from the cells and mRNA levels were measured by quantitative real-time PCR using ABI assay C—2229297—10 which measures at dbSNP rs362303. The HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN and the results are presented in Table 90.
  • The IC50 and selectivity were calculated using methods previously described in Example 41. The IC50 at which each oligonucleotide inhibits the mutant HTT mRNA expression is denoted as ‘mut IC50’. The IC50 at which each oligonucleotide inhibits the wild-type HTT mRNA expression is denoted as ‘wt IC50’. Selectivity was calculated by dividing the IC50 for inhibition of the wild-type HTT versus the IC50 for inhibiting expression of the mutant HTT mRNA.
  • As illustrated in Table 90, the newly designed antisense oligonucleotides (ISIS 575003) showed improvement in selectivity while maintaining potency as compared to ISIS 460209.
  • TABLE 89
    Short-gap antisense oligonucleotides 
    targeting HTT SNP
    Wing SEQ
    ISIS Sequence Gap chemistry ID
    NO. (5′ to 3′) Motif chemistry 5′ 3′ NO.
    460209* TeAkAkATTGT 3-9-3 Full  ekk kke 10
    mCATmCAk mCk mCe deoxy
    540094* TeTkGT mCATmCA 2-9-4 Full  ek kkke 67
    mCmCAkGkAkAe deoxy
    575003 TeTkGT mCATmCA 2-8-5 Full  ek kkkke 67
    mCmCkAkGkAkAe deoxy
    575004 TeTkGT mCATmCA 2-9-4  Full   ek kkke  67
    mCk mCAkGkAkAe or deoxy or
    2-7-6 or kdkkke
    Deoxy/cEt
    575005 TeTkGT mCATmCA 2-7-6 Full  ek kkkkke 67
    mCk mCkAkGkAkAe deoxy
    e = 2′-MOE, k = cEt, d = 2′-deoxyribonucleoside
  • TABLE 90
    Comparison of inhibition of HTT mRNA levels and selectivity
    of modified oligonucleotides with ISIS 460209 targeting HTT SNP
    IC50 (μM) Selectivity Wing Chemistry
    ISIS NO Mut Wt (wt vs mut) Motif Gap chemistry 5′ 3′
    460209* 0.34 3.3 9.7 3-9-3 Full deoxy ekk kke
    540094* 0.17 2.4 14 2-9-4 Full deoxy ek kkke
    575003 0.40 10 25 2-8-5 Full deoxy ek kkkke
    575004 1.2 >9.6 >8 2-9-4 or Full deoxy or ek kkke or
    2-7-6 Deoxy/cEt kdkkke
    575005 >10 >100 >10 2-7-6 Full deoxy ek kkkkke
    e = 2′-MOE,
    k = cEt,
    d = 2′-deoxyribonucleoside
    • Example 61 Short-Gap Chimeric Oligonucleotides Targeting Huntingtin (HTT) Single Nucleotide Polymorphism (SNP)
  • Additional chimeric antisense oligonucleotides were designed based on 15-mer, ISIS 460209 and 17-mer, ISIS 476333 wherein the central gap region contains nine 2′-deoxynucleosides. These gapmers were designed with the central gap region shortened at the 5′-end of the central gap region. The gapmers were tested for their ability to selectively inhibit mutant (mut) HTT mRNA expression levels targeting HTT SNP while leaving the expression of the wild-type (wt) intact. The activity and selectivity of the gapmers were evaluated and compared to ISIS 460209 and ISIS 476333.
  • The gapmers and their motifs are described in Table 91. The internucleoside linkages throughout each modified oligonucleotide are phosphorothioate linkages (P═S). Nucleosides without a subscript are (3-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e” indicate 2′-O-methoxyethyl (MOE) modified nucleosides. Nucleosides followed by a subscript “k” indicate 6′-(S)—CH3 bicyclic nucleosides (e.g. cEt). mC indicates a 5-methyl cytosine nucleoside. Underlined nucleoside indicates the position on the oligonucleotides opposite to the SNP position, which is position 8 or 9 as counted from the 5′-terminus.
  • The modified oligonucleotides were tested in vitro. Heterozygous fibroblast GM04022 cell line was used. Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with 0.12, 0.37, 1.1, 3.3 and 10 μM concentrations of modified oligonucleotides. After a treatment period of approximately 16 hours, RNA was isolated from the cells and mRNA levels were measured by quantitative real-time PCR using ABI assay C—2229297—10 which measures at dbSNP rs362303. The HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN and the results are presented in Table 92.
  • The IC50 and selectivity were calculated using methods previously described in Example 41. The IC50 at which each oligonucleotide inhibits the mutant HTT mRNA expression is denoted as ‘mut IC50’. The IC50 at which each oligonucleotide inhibits the wild-type HTT mRNA expression is denoted as ‘wt IC50’. Selectivity was calculated by dividing the IC50 for inhibition of the wild-type HTT versus the IC50 for inhibiting expression of the mutant HTT mRNA.
  • As illustrated in Table 92, a couple of the newly designed antisense oligonucleotides (ISIS 571036 and 571037) showed improvement in potency and selectivity in inhibiting mut HTT mRNA levels as compared to ISIS 460209 and 476333.
  • TABLE 91
    Short-gap antisense oligonucleotides 
    targeting HTT SNP
    Gap Wing SEQ
    ISIS Sequence chem- chemistry ID
    NO. (5′ to 3′) Motif istry 5′ 3′ NO.
    460209* TeAkAkATTGT 3-9-3 Full  ekk kke 10
    mCATmCAk mCk mCe deoxy
    476333* AeTkAeAkATTGT 4-9-4 Full  ekek keke 32
    mCATmCAk mCe mCkAe deoxy
    571036 AeTkAeAkAeTkTGT 6-7-4 Full  ekekek keke 32
    mCATmCAk mCe mCkAe deoxy
    571037 AeTeAeAeAkTkTGT 6-7-4 Full  eeeekk keke 32
    mCATmCAk mCe mCkAe deoxy
    571038 AeTkAeAkAeTeTGT 6-7-4 Full  ekekee keke 32
    mCATmCAk mCe mCkAe deoxy
    e = 2′-MOE, k = cEt, d = 2′-deoxyribonucleoside
  • TABLE 92
    Comparison of inhibition of HTT mRNA levels and selectivity of
    modified oligonucleotides with ISIS 460209 targeting HTT SNP
    Selectivity Wing
    IC50 (μM) (wt vs Gap Chemistry
    ISIS NO Mut Wt mut) Motif chemistry 5′ 3′
    460209* 0.34 3.3 9.7 3-9-3 Full deoxy ekk kke
    476333* 0.32 1.5 4.7 4-9-4 Full deoxy ekek keke
    571036 0.17 >10.0 >59 6-7-4 Full deoxy ekekek keke
    571037 0.11 >9.9 >90 6-7-4 Full deoxy eeeekk keke
    571038 1.5 >10.5 >7 6-7-4 Full deoxy ekekee keke
    e = 2′-MOE,
    k = cEt,
    d = 2′-deoxyribonucleoside
    • Example 62 Short-Gap Chimeric Oligonucleotides Targeting Huntingtin (HTT) Single Nucleotide Polymorphism (SNP)
  • Additional chimeric antisense oligonucleotides were designed based on 15-mer, ISIS 460209 wherein the central gap region contains nine 2′-deoxynucleosides. These gapmers were designed by having the central gap region shortened to seven 2′-deoxynucleosides. The gapmers were tested for their ability to selectively inhibit mutant (mut) HTT mRNA expression levels targeting HTT SNP while leaving the expression of the wild-type (wt) intact. The activity and selectivity of the gapmers were evaluated and compared to ISIS 460209.
  • The gapmers and their motifs are described in Table 93. The internucleoside linkages throughout each modified oligonucleotide are phosphorothioate linkages (P═S). Nucleosides without a subscript are 13-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e” indicate 2′-O-methoxyethyl (MOE) modified nucleosides. Nucleosides followed by a subscript “k” indicate 6′-(S)—CH3 bicyclic nucleosides (e.g. cEt). mC indicates a 5-methyl cytosine nucleoside. Underlined nucleoside indicates the position on the oligonucleotides opposite to the SNP position, which is position 8 or 9 as counted from the 5′-terminus.
  • The modified oligonucleotides were tested in vitro. Heterozygous fibroblast GM04022 cell line was used. Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with 0.12, 0.37, 1.1, 3.3 and 10 μM concentrations of modified oligonucleotides. After a treatment period of approximately 16 hours, RNA was isolated from the cells and mRNA levels were measured by quantitative real-time PCR using ABI assay C—2229297—10 which measures at dbSNP rs362303. The HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN and the results are presented in Table 94.
  • The IC50 and selectivity were calculated using methods previously described in Example 41. The IC50 at which each oligonucleotide inhibits the mutant HTT mRNA expression is denoted as ‘mut IC50’. The IC50 at which each oligonucleotide inhibits the wild-type HTT mRNA expression is denoted as ‘wt IC50’. Selectivity was calculated by dividing the IC50 for inhibition of the wild-type HTT versus the IC50 for inhibiting expression of the mutant HTT mRNA.
  • As illustrated in Table 94, each of the newly designed antisense oligonucleotides (ISIS 540108 and 571069) showed improvement in potency and/or selectivity in inhibiting mut HTT mRNA levels as compared to ISIS 460209.
  • TABLE 93
    Short-gap antisense oligonucleotides 
    targeting HTT SNP
    Gap Wing SEQ
    ISIS Sequence Mo- chem- chemistry ID
    NO. (5′ to 3′) tif istry 5′ 3′ NO.
    460209 TeAkAkATTGT 3-9- Full  ekk kke 10
    mCATmCAk mCk mCe 3 deoxy
    540108 AeTeAeAkAkTTGT 5-7- Full  eeekk kkeee 32
    mCATmCkAk mCe mCeAe 5 deoxy
    571069 AeTeAeAeAkTkTGT 6-7- Full  eeeekk kkee 32
    mCATmCAk mCk mCeAe 4 deoxy
    571173 AeTeAkAkATTGT 4-7- Full  eekk kkeeee 32
    mCATk mCkAe mCe mCeAe 6 deoxy
    572773 TeAeAkAkTTGT 4-7- Full  eekk kkee 10
    mCATmCkAk mCe mCe 4 deoxy
    e = 2′-MOE, k = cEt, d = 2′-deoxyribonucleoside
  • TABLE 94
    Comparison of inhibition of HTT mRNA levels and selectivity
    of modified oligonucleotides with ISIS 460209 targeting HTT SNP
    Selectivity
    ISIS IC50 (μM) (wt vs Gap Wing Chemistry
    NO Mut Wt mut) Motif chemistry 5′ 3′
    460209 0.34 3.3 9.7 3-9-3 Full deoxy ekk kke
    540108 0.20 >10 >50 5-7-5 Full deoxy eeekk kkeee
    571069 0.29 >9.9 >34 6-7-4 Full deoxy eeeekk kkee
    571173 1.0 >10 >10 4-7-6 Full deoxy eekk kkeeee
    572773 0.71 >7.8 11 4-7-4 Full deoxy eekk kkee
    e = 2′-MOE,
    k = cEt,
    d = 2′-deoxyribonucleoside
    • Example 63 Short-Gap Chimeric Oligonucleotides Targeting Huntingtin (HTT) Single Nucleotide Polymorphism (SNP)
  • Additional chimeric antisense oligonucleotides were designed based on 15-mer, ISIS 460209 and 17-mer, ISIS 540108 wherein the central gap region contains nine and seven 2′-deoxynucleosides, respectively. These gapmers were designed by introducing one or more cEt modification(s) at the 5′-end of the central gap region. The gapmers were tested for their ability to selectively inhibit mutant (mut) HTT mRNA expression levels targeting HTT SNP while leaving the expression of the wild-type (wt) intact. The activity and selectivity of the gapmers were evaluated and compared to ISIS 460209 and ISIS 540108.
  • The gapmers and their motifs are described in Table 95. The internucleoside linkages throughout each modified oligonucleotide are phosphorothioate linkages (P═S). Nucleosides without a subscript are β-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e” indicate 2′-O-methoxyethyl (MOE) modified nucleosides. Nucleosides followed by a subscript “k” indicate 6′-(S)—CH3 bicyclic nucleosides (e.g. cEt). mC indicates a 5-methyl cytosine nucleoside. Underlined nucleoside indicates the position on the oligonucleotides opposite to the SNP position, which is position 8 or 9 as counted from the 5′-terminus.
  • The modified oligonucleotides were tested in vitro. Heterozygous fibroblast GM04022 cell line was used. Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with 0.12, 0.37, 1.1, 3.3 and 10 μM concentrations of modified oligonucleotides. After a treatment period of approximately 16 hours, RNA was isolated from the cells and mRNA levels were measured by quantitative real-time PCR using ABI assay C—2229297—10 which measures at dbSNP rs362303. The HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN and the results are presented in Table 96.
  • The IC50 and selectivity were calculated using methods previously described in Example 41. The IC50 at which each oligonucleotide inhibits the mutant HTT mRNA expression is denoted as ‘mut IC50’. The IC50 at which each oligonucleotide inhibits the wild-type HTT mRNA expression is denoted as ‘wt IC50’. Selectivity was calculated by dividing the IC50 for inhibition of the wild-type HTT versus the IC50 for inhibiting expression of the mutant HTT mRNA.
  • As illustrated in Table 96, most of the newly designed oligonucleotides showed improvement in selectivity while maintaining potency as compared to 460209.
  • TABLE 95
    Short-gap antisense oligonucleotides 
    targeting HTT SNP
    Gap Wing SEQ
    ISIS Sequence chem- chemistry ID
    NO. (5′ to 3′) Motif istry 5′ 3′ NO.
    460209 TeAkAkATTGT 3-9-3 Full  ekk kke 10
    mCATmCAk mCk mCe deoxy
    540108 AeTeAeAkAkTTGT 5-7-5 Full  eeekk kkeee 32
    mCATmCkAk mCe mCeAe deoxy
    556872 AeTeAeAeAkTTGT 5-7-5 Full  eeeek eeeee 32
    mCATmCeAe mCe mCeAe deoxy
    556873 AeTeAeAkAkTTGT 5-7-5 Full  eeekk eeeee 32
    mCATmCeAe mCe mCeAe deoxy
    556874 AeTeAkAkAkTTGT 5-7-5 Full  eekkk eeeee 32
    mCATmCeAe mCe mCeAe deoxy
    568877 AeTkAkAkAkTTGT 5-7-5 Full  ekkkk eeeee 32
    mCATmCeAe mCe mCeAe deoxy
    568878 AkTkAkAkAkTTGT 5-7-5 Full  kkkkk eeeee 32
    mCATmCeAe mCe mCeAe deoxy
    e = 2′-MOE, k = cEt, d = 2′-deoxyribonucleoside
  • TABLE 96
    Comparison of inhibition of HTT mRNA levels and selectivity
    of modified oligonucleotides with ISIS 460209 targeting HTT SNP
    Wing
    IC50 (μM) Selectivity Gap Chemistry
    ISIS NO Mut Wt (wt vs mut) Motif chemistry 5′ 3′
    460209 0.45 2.3 5.1 3-9-3 Full deoxy ekk kke
    540108 0.25 9.5 38 5-7-5 Full deoxy eeekk kkeee
    556872 1.0 9.9 9.9 5-7-5 Full deoxy eeeek eeeee
    556873 0.67 3.4 5.1 5-7-5 Full deoxy eeekk eeeee
    556874 0.38 1.9 5.0 5-7-5 Full deoxy eekkk eeeee
    568877 0.44 6.2 14 5-7-5 Full deoxy ekkkk eeeee
    568878 0.41 8.6 21 5-7-5 Full deoxy kkkkk eeeee
    e = 2′-MOE,
    k = cEt,
    d = 2′-deoxyribonucleoside
    • Example 64 Short-Gap Chimeric Oligonucleotides Targeting Huntingtin (HTT) Single Nucleotide Polymorphism (SNP)
  • Additional chimeric antisense oligonucleotides were designed based on 15-mer, ISIS 460209 and 17-mer, ISIS 540108 wherein the central gap region contains nine and seven 2′-deoxynucleosides, respectively. These gapmers were designed by introducing one or more cEt modification(s) at the 3′-end of the central gap region. The gapmers were tested for their ability to selectively inhibit mutant (mut) HTT mRNA expression levels targeting HTT SNP while leaving the expression of the wild-type (wt) intact. The activity and selectivity of the gapmers were evaluated and compared to ISIS 460209 and ISIS 540108.
  • The gapmers and their motifs are described in Table 97. The internucleoside linkages throughout each modified oligonucleotide are phosphorothioate linkages (P═S). Nucleosides without a subscript are (3-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e” indicate 2′-O-methoxyethyl (MOE) modified nucleosides. Nucleosides followed by a subscript “k” indicate 6′-(S)—CH3 bicyclic nucleosides (e.g. cEt). mC indicates a 5-methyl cytosine nucleoside. Underlined nucleoside indicates the position on the oligonucleotides opposite to the SNP position, which is position 8 or 9 as counted from the 5′-terminus.
  • The modified oligonucleotides were tested in vitro. Heterozygous fibroblast GM04022 cell line was used. Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with 0.12, 0.37, 1.1, 3.3 and 10 μM concentrations of modified oligonucleotides. After a treatment period of approximately 16 hours, RNA was isolated from the cells and mRNA levels were measured by quantitative real-time PCR using ABI assay C—2229297—10 which measures at dbSNP rs362303. The HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN and the results are presented in Table 98.
  • The IC50 and selectivity were calculated using methods previously described in Example 41. The IC50 at which each oligonucleotide inhibits the mutant HTT mRNA expression is denoted as ‘mut IC50’. The IC50 at which each oligonucleotide inhibits the wild-type HTT mRNA expression is denoted as ‘wt IC50’. Selectivity was calculated by dividing the IC50 for inhibition of the wild-type HTT versus the IC50 for inhibiting expression of the mutant HTT mRNA.
  • As illustrated in Table 98, each of the newly designed oligonucleotides showed improvement in selective inhibition of mutant HTT mRNA levels compared to ISIS 460209. Comparable potency was observed for ISIS 568879 and 568880 while a slight loss in potency was observed for ISIS 556875, 556876 and 556877.
  • TABLE 97
    Short-gap antisense oligonucleotides 
    targeting HTT SNP
    Gap Wing SEQ
    ISIS Sequence chem- chemistry ID
    NO. (5′ to 3′) Motif istry 5′ 3′ NO.
    460209 TeAkAkATTGT 3-9-3 Full  ekk kke 10
    mCATmCAk mCk mCe deoxy
    540108 AeTeAeAkAkTTGT 5-7-5 Full  eeekk kkeee 32
    mCATmCkAk mCe mCeAe deoxy
    556875 AeTeAeAeAeTTGT 5-7-5 Full  eeeee keeee 32
    mCATmCkAe mCe mCeAe deoxy
    556876 AeTeAeAeAeTTGT 5-7-5 Full  eeeee kkeee 32
    mCATmCkAk mCe mCeAe deoxy
    556877 AeTeAeAeAeTTGT 5-7-5 Full  eeeee kkkee 32
    mCATmCkAk mCk mCeAe deoxy
    568879 AeTeAeAeAeTTGT 5-7-5 Full  eeeee kkkke 32
    mCATmCkAk mCk mCkAe deoxy
    568880 AeTeAeAeAeTTGT 5-7-5 Full  eeeee kkkkk 32
    mCATmCkAk mCk mCkAk deoxy
    e = 2′-MOE, k = cEt, d = 2′-deoxyribonucleoside
  • TABLE 98
    Comparison of inhibition of HTT mRNA levels and selectivity of
    modified oligonucleotides with ISIS 460209 targeting HTT SNP
    Selectivity Wing
    IC50 (μM) (wt vs Gap Chemistry
    ISIS NO Mut Wt mut) Motif chemistry 5′ 3′
    460209 0.45 2.3 5.1 3-9-3 Full deoxy ekk kke
    540108 0.25 9.5 38 5-7-5 Full deoxy eeekk kkeee
    556875 1.9 >9.5 >5 5-7-5 Full deoxy eeeee keeee
    556876 0.99 >9.9 >10 5-7-5 Full deoxy eeeee kkeee
    556877 1.0 >10 >10 5-7-5 Full deoxy eeeee kkkee
    568879 0.44 >10.1 >23 5-7-5 Full deoxy eeeee kkkke
    568880 0.59 >10 >17 5-7-5 Full deoxy eeeee kkkkk
    e = 2′-MOE,
    k = cEt,
    d = 2′-deoxyribonucleoside
    • Example 65 Modified Oligonucleotides Targeting Huntingtin (HTT) Single Nucleotide Polymorphism (SNP)
  • A series of modified oligonucleotides were designed based on the parent gapmer, ISIS 460209 wherein the central gap region contains nine 2′-deoxyribonucleosides. These modified oligonucleotides were designed by introducing various chemical modifications in the central gap region and were tested for their ability to selectively inhibit mutant (mut) HTT mRNA expression levels targeting SNP while leaving the expression of the wild-type (wt) intact. The activity and selectivity of the modified oligonucleotides were evaluated and compared to the parent gapmer, ISIS 460209.
  • The modified oligonucleotides were created with a 3-9-3 motif and are described in Table 99. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages, except for the internucleoside linkage having a subscript “p” which indicates a methyl phosphonate internucleoside linkage (—O—P(CH3)(═O)—O—). Nucleosides without a subscript are β-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside. Nucleosides followed by a subscript “k” indicates a 6′-(S)—CH3 bicyclic nucleoside (e.g. cEt). mC indicates a 5-methyl cytosine nucleoside. xT indicates a 2-thio-thymidine nucleoside. Underlined nucleoside indicates the position on the oligonucleotides opposite to the SNP position, which is position 8 as counted from the 5′-terminus.
  • The modified oligonucleotides were tested in vitro. Heterozygous fibroblast GM04022 cell line was used (from Coriell Institute). Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with 0.12, 0.37, 1.1, 3.3 and 10 μM concentrations of modified oligonucleotides. After a treatment period of approximately 24 hours, cells were washed with DPBS buffer and lysed. RNA was extracted using Qiagen RNeasy purification and mRNA levels were measured by quantitative real-time PCR using ABI assay C—2229297—10 which measures at dbSNP rs362303. RT-PCR method in short; A mixture was made using 2020 uL 2×PCR buffer, 101 uL primers (300 uM from ABI), 1000 uL water and 40.4 uL RT MIX. To each well was added 15 uL of this mixture and 5 uL of purified RNA. The mutant and wild-type HTT mRNA levels were measured simultaneously by using two different fluorophores, FAM for mutant allele and VIC for wild-type allele. The HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN and the results are presented in Table 100.
  • The IC50 and selectivity were calculated using methods previously described in Example 41. The IC50 at which each oligonucleotide inhibits the mutant HTT mRNA expression is denoted as ‘mut IC50’. The IC50 at which each oligonucleotide inhibits the wild-type HTT mRNA expression is denoted as ‘wt IC50’. Selectivity was calculated by dividing the IC50 for inhibition of the wild-type HTT versus the IC50 for inhibiting expression of the mutant HTT mRNA.
  • As illustrated in Table 100, improvement in selectivity with a slight decrease in potency was observed for the newly designed oligonucleotides as compared to ISIS 460209.
  • TABLE 99
    Short-gap antisense oligonucleotides 
    targeting HTT SNP
    Wing SEQ
    ISIS Sequence  Gap chemistry ID
    NO. (5′ to 3′) chemistry 5′ 3′ NO.
    460209 TeAkAkATTGT Full deoxy ekk kke 10
    mCATmCAk mCk mCe
    556845 TeAkAkAxTTGT Deoxy/2-Thio ekk kke 10
    mCATmCAk mCk mCe
    556847 TeAkAkAxTxTGT Deoxy/2-Thio ekk kke 10
    mCATmCAk mCk mCe
    558257 TeAkAkATTpGT Deoxy/Methyl ekk kke 10
    mCATmCAk mCk mC Phosphonate
    571125 TeAkAkAxTTpGT Deoxy/2-Thio/ ekk kke 10
    mCATmCAk mCk mCe Methyl
    Phosphonate
    e = 2′-MOE, k = cEt, d = 2′-deoxyribonucleoside
  • TABLE 100
    Comparison of inhibition of HTT mRNA levels and selectivity
    of modified oligonucleotides with ISIS 460209 targeting HTT SNP
    Wing
    IC50 (μM) Selectivity Chemistry
    ISIS NO Mut Wt (wt vs mut) Gap chemistry 5′ 3′
    460209 0.56 3.8 6.8 Full deoxy ekk kke
    556845 0.98 >9.8 >10 Deoxy/2-Thio ekk kke
    556847 1.3 >10.4 >8 Deoxy/2-Thio ekk kke
    558257 1.7 >10.2 >6 Deoxy/Methyl ekk kke
    Phosphonate
    571125 1.8 >10.8 >6 Deoxy/2- ekk kke
    Thio/Methyl
    Phosphonate
    e = 2′-MOE,
    k = cEt,
    d = 2′-deoxyribonucleoside
    • Example 66 Modified Oligonucleotides Comprising Chemical Modifications in the Central Gap Region Targeting Huntingtin (HTT) Single Nucleotide Polymorphism (SNP)
  • Additional chimeric antisense oligonucleotides were designed in the same manner as the antisense oligonucleotides described in Example 65. These gapmers were designed by introducing various modifications in the central gap region and were tested for their ability to selectively inhibit mutant (mut) HTT mRNA expression levels targeting SNP while leaving the expression of the wild-type (wt) intact. The activity and selectivity of the modified oligonucleotides were evaluated and compared to the parent gapmer, ISIS 460209.
  • The modified oligonucleotides and their motifs are described in Table 101. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages, except for the internucleoside linkage having a subscript “p” which indicates a methyl phosphonate internucleoside linkage (—O—P(CH3)(═O)—O—). Nucleosides without a subscript are β-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside. Nucleosides followed by a subscript “k” indicates a 6′-(S)—CH3 bicyclic nucleoside (e.g. cEt). mC indicates a 5-methyl cytosine nucleoside. xT indicates a 2-thio-thymidine nucleoside. Underlined nucleoside indicates the position on the oligonucleotides opposite to the SNP position, which is position 8 as counted from the 5′-terminus.
  • The modified oligonucleotides were tested in vitro. Heterozygous fibroblast GM04022 cell line was used (from Coriell Institute). Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with 0.12, 0.37, 1.1, 3.3 and 10 μM concentrations of modified oligonucleotides. After a treatment period of approximately 24 hours, cells were washed with DPBS buffer and lysed. RNA was extracted using Qiagen RNeasy purification and mRNA levels were measured by quantitative real-time PCR using ABI assay C—2229297—10 which measures at dbSNP rs362303. RT-PCR method in short; A mixture was made using 2020 uL 2×PCR buffer, 101 uL primers (300 uM from ABI), 1000 uL water and 40.4 uL RT MIX. To each well was added 15 uL of this mixture and 5 uL of purified RNA. The mutant and wild-type HTT mRNA levels were measured simultaneously by using two different fluorophores, FAM for mutant allele and VIC for wild-type allele. The HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN and the results are presented in Table 102.
  • The IC50 and selectivity were calculated using methods previously described in Example 41. The IC50 at which each oligonucleotide inhibits the mutant HTT mRNA expression is denoted as ‘mut IC50’. The IC50 at which each oligonucleotide inhibits the wild-type HTT mRNA expression is denoted as ‘wt IC50’. Selectivity was calculated by dividing the IC50 for inhibition of the wild-type HTT versus the IC50 for inhibiting expression of the mutant HTT mRNA.
  • As illustrated in Table 102, some of the newly designed oligonucleotides showed improvement in selectivity while maintaining potency as compared to 460209.
  • TABLE 101
    Short-gap antisense oligonucleotides targeting HTT SNP
    Wing SEQ
    ISIS Sequence Gap chemistry ID
    NO. (5′ to 3′) Motif chemistry 5′ 3′ NO.
    460209 TeAkAkATTGT 3-9-3 Full deoxy ekk kke 10
    mCATmCAk mCk mCe
    551429 TeAeAeAkTkTGT 5-7-3 Full deoxy eeekk kke 10
    mCATmCAk mCk mCe
    571122 TeAeAeAk xTTGT 4-8-3 Deoxy/2-Thio eeek kke 10
    mCATmCAk mCk mCe
    571123 TeAeAeAkTkTpGT 5-7-3 Deoxy/Methyl eeekk kke 10
    mCATmCAk mCk mCe Phosphonate
    571124 TeAeAeAk xTTpGT 4-8-3 Deoxy/2- eeek kke 10
    mCATmCAk mCk mCe Thio/Methyl
    Phosphonate
    579854 TeAeAeAkTTpGT 4-8-3 Deoxy/Methyl eeek kke 10
    mCATmCAk mCk mCe Phosphonate
    566282 TeAkAkAdxTdxTdGdTd mCd 3-9-3 Deoxy/Methyl ekk kke 10
    AdTd mCdAk mCk mCe Phosphonate
    e = 2′-MOE, k = cEt, d = 2′-deoxyribonucleoside
  • TABLE 102
    Comparison of inhibition of HTT mRNA levels and selectivity
    of modified oligonucleotides with ISIS 460209 targeting HTT SNP
    Selectivity Wing
    ISIS IC50 (μM) (wt vs Chemistry
    NO Mut Wt mut) Motif Gap chemistry 5′ 3′
    460209 0.56 3.8 6.8 3-9-3 Full deoxy ekk kke
    551429 0.50 >10 >20 5-7-3 Full deoxy eeekk kke
    571122 1.8 >10.8 >6 4-8-3 Deoxy/2-Thio eeek kke
    571123 0.96 >9.6 >10 5-7-3 Deoxy/Methyl eeekk kke
    Phosphonate
    571124 2.3 >9.2 >4 4-8-3 Deoxy/2- eeek kke
    Thio/Methyl
    Phosphonate
    579854 0.63 >10.1 >16 4-8-3 Deoxy/Methyl eeek kke
    Phosphonate
    566282 0.51 6.3 12.4 3-9-3 Deoxy/Methyl ekk kke
    Phosphonate
    e = 2′-MOE,
    k = cEt
    • Example 67 Modified Oligonucleotides Comprising Chemical Modifications in the Central Gap Region Targeting Huntingtin (HTT) Single Nucleotide Polymorphism (SNP)
  • Additional chimeric antisense oligonucleotides were designed in the same manner as the antisense oligonucleotides described in Example 65. These gapmers were designed by introducing various modifications in the central gap region and were tested for their ability to selectively inhibit mutant (mut) HTT mRNA expression levels targeting SNP while leaving the expression of the wild-type (wt) intact. The activity and selectivity of the modified oligonucleotides were evaluated and compared to the parent gapmer, ISIS 460209.
  • The modified oligonucleotides and their motifs are described in Table 103. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages, except for the internucleoside linkage having a subscript “p” which indicates a methyl phosphonate internucleoside linkage (—O—P(CH3)(═O)—O—). Nucleosides without a subscript are β-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside. Nucleosides followed by a subscript “k” indicates a 6′-(S)—CH3 bicyclic nucleoside (e.g. cEt). mC indicates a 5-methyl cytosine nucleoside. xT indicates a 2-thio-thymidine nucleoside. Underlined nucleoside indicates the position on the oligonucleotides opposite to the SNP position, which is position 8 or 9 as counted from the 5′-terminus.
  • The modified oligonucleotides were tested in vitro. Heterozygous fibroblast GM04022 cell line was used (from Coriell Institute). Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with 0.12, 0.37, 1.1, 3.3 and 10 μM concentrations of modified oligonucleotides. After a treatment period of approximately 24 hours, cells were washed with DPBS buffer and lysed. RNA was extracted using Qiagen RNeasy purification and mRNA levels were measured by quantitative real-time PCR using ABI assay C—2229297—10 which measures at dbSNP rs362303. RT-PCR method in short; A mixture was made using 2020 uL 2×PCR buffer, 101 uL primers (300 uM from ABI), 1000 uL water and 40.4 uL RT MIX. To each well was added 15 uL of this mixture and 5 uL of purified RNA. The mutant and wild-type HTT mRNA levels were measured simultaneously by using two different fluorophores, FAM for mutant allele and VIC for wild-type allele. The HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN and the results are presented in Table 104.
  • The IC50 and selectivity were calculated using methods previously described in Example 41. The IC50 at which each oligonucleotide inhibits the mutant HTT mRNA expression is denoted as ‘mut IC50’. The IC50 at which each oligonucleotide inhibits the wild-type HTT mRNA expression is denoted as ‘wt IC50’. Selectivity was calculated by dividing the IC50 for inhibition of the wild-type HTT versus the IC50 for inhibiting expression of the mutant HTT mRNA.
  • As illustrated in Table 104, all but one of the newly designed oligonucleotides showed improvement in selectivity while maintaining potency as compared to ISIS 460209.
  • TABLE 103
    Short-gap antisense oligonucleotides targeting 
    HTT SNP
    Wing SEQ
    ISIS Sequence Gap chemistry ID
    NO. (5′ to 3′) Motif chemistry 5′ 3′ NO.
    460209 TeAkAkATTGT 3-9-3 Full  ekk kke 10
    mCATmCAk mCk mCe deoxy
    476333 AeTkAeAkATTGT 4-9-4 Full  ekek keke 32
    mCATmCAk mCe mCkAe deoxy
    571039 AeTkAeAkAxTTGT 4-9-4 Deoxy/ ekek keke 32
    mCATmCAk mCe mCkAe 2-Thio
    571171 AeTkAeAkATTpGT 4-9-4 Deoxy/ ekek keke 32
    mCATmCAk mCe mCkAe Methyl
    Phospho-
    nate
    571041 AeTkAeAkAxTTpGT 4-9-4 Deoxy/2- ekek keke 32
    mCATmCAk mCe mCkAe Thio/
    Methyl
    Phospho-
    nate
    e = 2′-MOE, k = cEt, d = 2′-deoxyribonucleoside
  • TABLE 104
    Comparison of inhibition of HTT mRNA levels and selectivity of
    modified oligonucleotides with ISIS 460209 targeting HTT SNP
    ISIS IC50 (μM) Selectivity Gap Wing Chemistry
    NO Mut Wt (wt vs mut) chemistry 5′ 3′
    460209 0.56 3.8 6.8 Full deoxy ekk kke
    476333 0.56 3.4 6.1 Full deoxy ekek keke
    571039 0.34 >9.9 >29 Deoxy/2-Thio ekek keke
    571171 0.54 >10.3 >19 Deoxy/Methyl ekek keke
    Phosphonate
    571041 0.75 >9.8 >13 Deoxy/2- ekek keke
    Thio/Methyl
    Phosphonate
    e = 2′-MOE, k = cEt, d = 2′-deoxyribonucleoside
    • Example 68 Selectivity in Inhibition of HTT mRNA Levels Targeting SNP by Gap-Interrupted Modified Oligonucleotides
  • Additional modified oligonucleotides were designed based on the parent gapmer, ISIS 460209 wherein the central gap region contains nine 2′-deoxyribonucleosides. These modified oligonucleotides were designed by introducing one or more modified nucleobase(s) in the central gap region and were tested for their ability to selectively inhibit mutant (mut) HTT mRNA expression levels targeting SNP while leaving the expression of the wild-type (wt) intact. The activity and selectivity of the modified oligonucleotides were evaluated and compared to ISIS 460209.
  • The modified oligonucleotides were created with a 3-9-3 motif and are described in Table 105. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. Nucleosides without a subscript are β-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside. Nucleosides followed by a subscript “k” indicates a 6′-(S)—CH3 bicyclic nucleoside (e.g. cEt). mC indicates a 5-methyl cytosine nucleoside. xT indicates a 2-thio-thymidine nucleoside. Underlined nucleoside indicates the position on the oligonucleotides opposite to the SNP position, which is position 8 as counted from the 5′-terminus.
  • The modified oligonucleotides were tested in vitro. Heterozygous fibroblast GM04022 cell line was used (from Coriell Institute). Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with 0.12, 0.37, 1.1, 3.3 and 10 μM concentrations of modified oligonucleotides. After a treatment period of approximately 24 hours, cells were washed with DPBS buffer and lysed. RNA was extracted using Qiagen RNeasy purification and mRNA levels were measured by quantitative real-time PCR using ABI assay C—2229297—10 which measures at dbSNP rs362303. RT-PCR method in short; A mixture was made using 2020 uL 2×PCR buffer, 101 uL primers (300 uM from ABI), 1000 uL water and 40.4 uL RT MIX. To each well was added 15 uL of this mixture and 5 uL of purified RNA. The mutant and wild-type HTT mRNA levels were measured simultaneously by using two different fluorophores, FAM for mutant allele and VIC for wild-type allele. The HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN.
  • The IC50 and selectivity were calculated using methods previously described in Example 41. The IC50 at which each oligonucleotide inhibits the mutant HTT mRNA expression is denoted as ‘mut IC50’. The IC50 at which each oligonucleotide inhibits the wild-type HTT mRNA expression is denoted as ‘wt IC50’. Selectivity was calculated by dividing the IC50 for inhibition of the wild-type HTT versus the IC50 for inhibiting expression of the mutant HTT mRNA.
  • As illustrated in Table 106, ISIS 556845 showed improvement in selectivity and potency as compared to ISIS 460209. ISIS 556847 showed improvement in selectivity with comparable potency while ISIS 556846 showed improvement in potency with comparable selectivity.
  • TABLE 105
    Gap-interrupted modified oligonucleotides 
    targeting HTT SNP
    Wing SEQ
    ISIS Sequence Gap chemistry ID
    NO. (5′ to 3′) chemistry 5′ 3′ NO.
    460209 TeAkAkATTGT Full  ekk kke 10
    mCATmCAk mCk mCe deoxy
    556845 TeAkAkAxTTGT Deoxy/ ekk kke 10
    mCATmCAk mCk mCe 2-Thio
    556846 TeAkAkATxTGT Deoxy/ ekk kke 10
    mCATmCAk mCk mCe 2-Thio
    556847 TeAkAkAxTxTGT Deoxy/ ekk kke 10
    mCATmCAk mCk mCe 2-Thio
    e = 2′-MOE, k = cEt, d = 2′-deoxyribonucleoside
  • TABLE 106
    Comparison of inhibition of HTT mRNA levels and
    selectivity of gap-interrupted modified oligonucleotides
    with ISIS 460209 targeting HTT SNP
    ISIS IC50 (μM) Selectivity Gap Wing Chemistry
    NO Mut Wt (wt vs mut) chemistry 5′ 3′
    460209 0.30 0.99 3.3 Full deoxy ekk kke
    556845 0.13 10.01 >77 Deoxy/2-Thio ekk kke
    556846 0.19 0.48 2.5 Deoxy/2-Thio ekk kke
    556847 0.45 9.9 >22 Deoxy/2-Thio ekk kke
    e = 2′-MOE, k = cEt, d = 2′-deoxyribonucleoside
    • Example 69 Evaluation of Modified Oligonucleotides Targeting HTT SNP—In Vivo Study
  • Additional modified oligonucleotides were selected and tested for their effects on mutant and wild type HTT protein levels in vivo targeting various SNP sites as illustrated below.
  • The gapmers and their motifs are described in Table 107. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine nucleobases throughout each gapmer are 5-methyl cytosines. Nucleosides without a subscript are β-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e” or “k” are sugar modified nucleosides. A subscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside and a subscript “k” indicates a 6′-(S)—CH3 bicyclic nucleoside (e.g. cEt).
  • The gapmer, ISIS 460209 was included in the study as a benchmark oligonucleotide against which the potency and selectivity of the modified oligonucleotides could be compared. A non-allele specific oligonucleotide, ISIS 387898, was used as a positive control.
  • Hu97/18 mice, the first murine model of HD that fully genetically recapitulates human HD were used in the study. They were generated in Hayden's lab by cross bred BACHD, YAC 18 and Hdh (−/−) mice.
  • Hu97/18 mice were treated with 300 μg of modified oligonucleotides by a single unilateral intracerebroventricular (ICV) bolus injection. This treatment group consisted of 4 animals/oligonucleotide. The control group received a 10 μl bolus injection of sterile PBS and consisted of 4 animals.
  • Animals were sacrificed at 4 weeks post-injection. The second most anterior 2 mm coronal slab for each brain hemisphere was collected using a 2 mm rodent brain matrix. The remaining portion of the brain was post-fixed in 4% paraformaldehyde, cryoprotected in 30% sucrose and sectioned into 25 μm coronal sections for immunohistochemical analysis.
  • The HTT protein levels were analyzed by high molecular weight western blot (modified from Invitrogen's NuPAGE Bis-Tris System Protocol). The tissue was homogenized in ice cold SDP lysis buffer. 40 μg of total protein lysate was resolved on 10% low-BIS acrylamide gels (200:1 acrylamide:BIS) with tris-glycine running buffer (25 mM Tris, 190 mM Glycince, 0.1% SDS) containing 10.7 mM β-mercaptoethanol added fresh. Gels were run at 90V for 40 min through the stack, then 190V for 2.5 h, or until the 75 kDa molecular weight marker band was at the bottom of the gel. Proteins were transferred to nitrocellulose at 24V for 2 h with NuPage transfer buffer (Invitrogen: 25 mM Bicine, 25 mM Bis-Tris, 1.025 mM EDTA, 5% MeOH, pH 7.2). Membranes were blocked with 5% milk in PBS, and then blotted for HTT with MAB2166 (1:1000, millipore). Anti-calnexin (Sigma C4731) immunoblotting was used as loading control. Proteins were detected with IR dye 800CW goat anti-mouse (Rockland 610-131-007) and AlexaFluor 680 goat anti-rabbit (Molecular Probes A21076)-labeled secondary antibodies, and the LiCor Odyssey Infrared Imaging system.
  • The results in Table 108 are presented as the average percent of HTT protein levels for each treatment group, normalized to PBS-treated control and is denoted as “% UTC”. The percent of mutant HTT protein levels is denoted as “mut”. The percent of wild-type HTT protein levels is denoted as “wt”. Selectivity was also evaluated and measured by dividing the percent of wild-type HTT protein levels vs. the percent of the mutant HTT protein levels.
  • As illustrated in Table 108, treatment with the newly designed oligonucleotides, ISIS 476333 and 460085 showed improvement in potency and selectivity in inhibiting mutant HTT protein levels as compared to the parent gapmer, 460209. Comparable or a slight loss in potency and/or selectivity was observed for the remaining oligonucleotides.
  • TABLE 107
    Modified oligonucleotides targeting HTT rs7685686,
    rs4690072 and rs363088 in Hu97/18 mice
    Wing SEQ
    ISIS Chemistry ID
    NO Sequence (5′ to 3′) Motif 5′ 3′ NO.
    387898 CeTeCeGeAeCTAAAGCAGGAeTeTeTeCe 5-10-5 e5 e5 79
    460209 TeAkAkATTGTCATCAkCkCe 3-9-3 ekk kke 10
    435879 AeAeTeAeAeATTGTCATCAeCeCeAeGe 5-9-5 e5 e5 80
    476333 AeTkAeAkATTGTCATCAkCeCkAe 4-9-4 ekek keke 32
    435874 CeAeCeAeGeTGCTACCCAAeCeCeTeTe 5-9-5 e5 e5 81
    435871 TeCeAeCeAeGCTATCTTCTeCeAeTeCe 5-9-5 e5 e5 82
    460085 AeTeAeAeAeTTGTCATCeAeCeCeAe 5-7-5 e5 e5 32
    e = 2′-MOE (e.g. e5 = eeeee), k = cEt
  • TABLE 108
    Effects of modified oligonucleotides on mutant and
    wild type HTT protein levels in Hu97/18 mice
    Dosage % UTC Selectivity
    ISIS NO SNP site (μg) mut wt (wt vs mut)
    PBS — 300 100 100 1
    387898 — 300 23.76 25.66 1
    460209 rs7685686 300 18.16 48.99 2.7
    435879 rs7685686 300 41.48 73.11 1.8
    476333 rs7685686 300 6.35 22.05 3.5
    460085 rs7685686 300 2.9 40.1 13.8
    435874 rs4690072 300 44.18 76.63 1.7
    435871 rs363088 300 33.07 89.30 2.7
    • Example 70 Evaluation of ISIS 435871 in Central Nervous System (CNS) Targeting HTT Rs363088—In Vivo Study
  • A modified oligonucleotide from Example 68, ISIS 435871 was selected and tested for its effects on mutant and wild type HTT protein levels in the CNS in vivo targeting rs363088.
  • Hu97/18 mouse was treated with 300 μg of ISIS 435871 by a single unilateral intracerebroventricular (ICV) bolus injection. The animal was sacrificed at 4 weeks post-injection. Regional CNS structures were then micro-dissected including bilateral samples from the most anterior portion of cortex (Cortex 1), an intermediate section of cortex (Cortex 2), the most posterior section of cortex (Cortex 3), the striatum, the hippocampus, the cerebellum, and a 1 cm section of spinal cord directly below the brain stem. Tissue was homogenized and assessed for mutant and wild-type HTT levels by Western blotting using the procedures as described in Example 69. The results are presented below. As no untreated or vehicle treated control is shown, HTT intensity of each allele is expressed as a ratio of calnexin loading control intensity. The ratio of the mutant HTT to the wt HTT in the treated animal was determined and is denoted as “wt/mut”. Having a ratio higher than 1 is indicative of allele-specific silencing.
  • As illustrated in Table 109, a single unilateral ICV bolus injection of the modified antisense oligonucleotide showed selective HTT silencing throughout the CNS except in the cerebellum, where the antisense oligonucleotide did not distribute evenly.
  • TABLE 109
    Effects of ISIS 435871 on mutant and wild type HTT protein
    levels in CNS targeting rs363088 in Hu97/18 mice
    HTT intensity/calnexin intensity
    Tissue wt mut wt/mut
    Cortex 1 0.032 0.014 2.29
    Cortex 2 0.027 0.009 3
    Cortex 3 0.023 0.007 3.29
    Striatum 0.030 0.012 2.5
    Hippocampus 0.016 0.006 2.67
    Cerebellum 0.023 0.019 1.21
    Spinal Cord 0.014 0.007 2
    • Example 71 Evaluation of Modified Oligonucleotides Targeting HTT Rs7685686—In Vivo Study
  • Several modified oligonucleotides from Examples 43, 51, 52, 53 and 66 were selected and tested for their effects on mutant and wild type HTT protein levels in vivo targeting HTT rs7685686.
  • The gapmer, ISIS 460209 was included in the study as a benchmark oligonucleotide against which the potency and selectivity of the modified oligonucleotides could be compared.
  • Hu97/18 mice were treated with 300 μg of modified oligonucleotides by a single unilateral intracerebroventricular (ICV) bolus injection. This treatment group consisted of 4 animals/oligonucleotide. The control group received a 10 μl bolus injection of sterile PBS and consisted of 4 animals.
  • Animals were sacrificed at 4 weeks post-injection. The second most anterior 2 mm coronal slab for each brain hemisphere was collected using a 2 mm rodent brain matrix. The HTT protein levels were analyzed in the same manner as described in Example 69 and the results are presented below.
  • The results in Table 110 are presented as the average percent of HTT protein levels for each allele and treatment group, normalized to PBS-treated control and is denoted as “% UTC”. The percent of mutant HTT protein levels is denoted as “mut”. The percent of wild-type HTT protein levels is denoted as “wt”.
  • As shown in Table 110, each of the newly designed oligonucleotides showed improvement in selective inhibition of mutant HTT protein levels as compared to ISIS 460209. ISIS 550913 and 540095 showed improvement in potency while the remaining modified oligonucleotides showed comparable or a slight decrease in potency as compared to the parent gapmer.
  • TABLE 110
    Effects of modified oligonucleotides on mutant and wild type
    HTT protein levels targeting rs7685686 in Hu97/18 mice
    Wing SEQ
    ISIS % UTC chemistry Gap ID
    NO mut wt Motif 5′ 3′ chemistry NO
    PBS 100 100 — — — — —
    460209 18.16 48.99 3-9-3 ekk kke Full deoxy 10
    550913 9.31 34.26 5-9-5 kkekk kkekk Full deoxy 27
    540095 12.75 106.05 2-9-4 ek kkke Full deoxy 65
    551429 19.07 108.31 5-7-3 eeekk kke Full deoxy 10
    540094 24.68 87.56 2-9-4 ek kkke Full deoxy 67
    540096 24.89 98.26 2-9-4 ek kkke Full deoxy 68
    540108 28.34 85.62 5-7-5 eeekk kkeee Full deoxy 23
    e = 2′-MOE, k = cEt
    • Example 72 Evaluation of Modified Oligonucleotides Targeting HTT Rs7685686—In Vivo Study
  • Several modified oligonucleotides selected from Examples 57, 58, 61 and 62 were tested and evaluated for their effects on mutant and wild type HTT protein levels in vivo targeting HTT rs7685686.
  • Hu97/18 mice were treated with 300 μg of modified oligonucleotides by a single unilateral intracerebroventricular (ICV) bolus injection and the control group received a 10 μl bolus injection of sterile PBS. Each treatment group consisted of 4 animals.
  • Animals were sacrificed at 4 weeks post-injection. The second most anterior 2 mm coronal slab for each brain hemisphere was collected using a 2 mm rodent brain matrix. The HTT protein levels were analyzed in the same manner as described in Example 69. The in vivo study for ISIS 575008 and 571069 marked with an asterisk (*) was performed independently and the results are presented below.
  • The results in Table 111 are presented as the average percent of HTT protein levels for each allele and treatment group, normalized to PBS-treated control and is denoted as “% UTC”. The percent of mutant HTT protein levels is denoted as “mut”. The percent of wild-type HTT protein levels is denoted as “wt”.
  • As illustrated in Table 111, selective inhibition of mut HTT protein levels was achieved with the newly designed oligonucleotide treatment as compared to PBS treated control.
  • TABLE 111
    Effects of modified oligonucleotides on mutant and wild type
    HTT protein levels targeting rs7685686 in Hu97/18 mice
    Wing SEQ
    ISIS % UTC chemistry Gap ID
    NO mut wt Motif 5′ 3′ chemistry NO
    PBS 100 100 — — — — —
    575007 26.9 104.5 3-9-3 ekk kke Deoxy/cEt 10
     575008* 21.7 105.9 5-7-3 ekkkk kke Deoxy/cEt 10
    566267 32.8 109.3 3-9-3 ekk kke Deoxy/F- 10
    HNA
    571036 30.3 103.3 6-7-4 ekekek keke Full deoxy 32
    571037 32.8 111.9 6-7-4 eeeekk keke Full deoxy 32
     571069* 29.4 109.8 6-7-4 eeeekk kkee Full deoxy 32
    e = 2′-MOE, k = cEt
    • Example 73 Evaluation of Modified Oligonucleotides Targeting HTT Rs7685686—In Vivo Dose Response Study
  • ISIS 476333, 435871, 540108, 575007 and 551429 from previous examples were selected and evaluated at various doses for their effect on mutant and wild type HTT protein levels in vivo targeting HTT rs7685686.
  • Hu97/18 mice were treated with various doses of modified oligonucleotides as presented in Table 112 by a single unilateral intracerebroventricular (ICV) bolus injection. This treatment group consisted of 4 animals/oligonucleotide. The control group received a 10 μl bolus injection of sterile PBS and consisted of 4 animals.
  • Animals were sacrificed at 4 weeks post-injection. The second most anterior 2 mm coronal slab for each brain hemisphere was collected using a 2 mm rodent brain matrix. The HTT protein levels were analyzed in the same manner as described in Example 69. The dose response study was performed independently for each modified oligonucleotide and the results are presented below.
  • The results in Table 112 are presented as the average percent of HTT protein levels for each allele and treatment group, normalized to PBS-treated control and is denoted as “% UTC”. The percent of mutant HTT protein levels is denoted as “mut”. The percent of wild-type HTT protein levels is denoted as “wt”.
  • As illustrated in Table 112, selective inhibition of mut HTT protein levels was achieved in a dose-dependent manner for the newly designed oligonucleotides.
  • TABLE 112
    Dose-dependent effect of modified oligonucleotides on mutant and
    wild type HTT protein levels targeting rs7685686 in Hu97/18 mice
    Dosage % UTC SEQ
    ISIS NO (μg) mut wt Motif ID NO.
    PBS 0 100 100 —
    476333 50 48.7 115 4-9-4 32
    150 23.1 53.3 (ekek-d9-keke)
    300 8.8 36.7
    435871 75 114 118 5-9-5 82
    150 47.3 80.3 (e5-d9-e5)
    300 33 89.3
    500 36 97.5
    540108 75 30.5 71.7 5-7-5 32
    150 22 81 (eeekk-d7-kkeee)
    300 8.6 59.6
    575007 150 41.5 110.7 3-9-3 10
    300 29 119.4 (ekk-d-k-d7-kke)
    (deoxy gap
    interrupted with cEt)
    551429 75 58 101.3 5-7-3 10
    150 36.2 110.4 (eeekk-d7-kke)
    300 19.7 107.8
    e = 2′-MOE (e.g. e5 = eeeee), k = cEt, d = 2′-deoxyribonucleoside
    • Example 74 Modified Oligonucleotides Targeting Huntingtin (HTT) Single Nucleotide Polymorphism (SNP)
  • A series of modified oligonucleotides was designed based on a parent gapmer, ISIS 460209, wherein the central gap region contains nine β-D-2′-deoxyribonucleosides. The modified oligonucleotides were designed by introducing a 5′-(R)-Me DNA modification within the central gap region. The 5′-(R)-Me DNA containing oligonucleotides were tested for their ability to selectively inhibit mutant (mut) HTT mRNA expression levels targeting rs7685686 while leaving the expression of the wild-type (wt) intact. The potency and selectivity of the modified oligonucleotides were evaluated and compared to ISIS 460209.
  • The position on the oligonucleotides opposite to the SNP position, as counted from the 5′-terminus is position 8.
  • The modified oligonucleotides were created with a 3-9-3 motif and are described in Table 113. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. Nucleosides followed by a subscript “d” are β-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside. Nucleosides followed by a subscript “k” indicates a 6′-(S)—CH3 bicyclic nucleoside (e.g. cEt). Nucleosides followed by a subscript “z” indicates a 5′-(R)-Me DNA. “mC” indicates a 5-methyl cytosine nucleoside.
  • The modified oligonucleotides were tested in vitro. Heterozygous fibroblast GM04022 cell line was used. Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with a single dose at 2 μM concentration of the modified oligonucleotide. After a treatment period of approximately 24 hours, cells were washed with DPBS buffer and lysed. RNA was extracted using Qiagen RNeasy purification and mRNA levels were measured by quantitative real-time PCR using ABI assay C—2229297—10 which measures at dbSNP rs362303. RT-PCR method in short; A mixture was made using 2020 uL 2×PCR buffer, 101 uL primers (300 uM from ABI), 1000 uL water and 40.4 uL RT MIX. To each well was added 15 uL of this mixture and 5 uL of purified RNA. The mutant and wild-type HTT mRNA levels were measured simultaneously by using two different fluorophores, FAM for mutant allele and VIC for wild-type allele. The HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN.
  • The IC50s and selectivities as expressed in “fold” were measured and calculated using methods described previously in Example 41. As illustrated in Table 114, treatment with the newly designed oligonucleotides showed comparable or a slight increase in potency and/or selectivity as compared to ISIS 460209.
  • TABLE 113
    Gap-interrupted oligonucleotides comprising 5′-(R)-Me DNA
    targeting HTT SNP
    Wing SEQ
    ISIS chemistry ID
    NO. Sequence (5′ to 3′) Gap chemistry 5′ 3′ NO.
    460209 TeAkAkAdTdTdGdTd mCdAdTd mCdAk mCk mCe Full deoxy ekk kke 10
    556848 TeAkAkAzTdTdGdTd mCdAdTd mCdAk mCk mCe Deoxy/5′-(R)-Me DNA ekk kke 10
    556849 TeAkAkAdTzTdGdTd mCdAdTd mCdAk mCk mCe Deoxy/5′-(R)-Me DNA ekk kke 10
    556850 TeAkAkAdTdTzGdTd mCdAdTd mCdAk mCk mCe Deoxy/5′-(R)-Me DNA ekk kke 10
    e = 2′-MOE, k = cEt
  • TABLE 114
    Comparison of inhibition of HTT mRNA levels and selectivity of gap-
    interrupted oligonucleotides with ISIS 460209 targeting HTT SNP
    IC50 Wing
    ISIS (μM) Selectivity Gap chemistry
    NO. Mut Wt (wt vs mut) chemistry 5′ 3′
    460209 0.30 0.99 3.3 Full deoxy ekk kke
    556848 0.15 0.6 4.0 Deoxy/5′-(R)- ekk kke
    Me DNA
    556849 0.16 0.46 2.9 Deoxy/5′-(R)- ekk kke
    Me DNA
    556850 0.33 0.96 2.9 Deoxy/5′-(R)- ekk kke
    Me DNA
    e = 2′-MOE, k = cEt
    • Example 75 Modified Oligonucleotides Comprising 5′-(R)- or 5′-(S)-Me DNA Modification Targeting HTT SNP
  • A series of modified oligonucleotides was designed based on a parent gapmer, ISIS 460209, wherein the central gap region contains nine β-D-2′-deoxyribonucleosides. The modified oligonucleotides were designed by introducing 5′-(S)- or 5′-(R)-Me DNA modification slightly upstream or downstream (i.e. “microwalk”) within the central gap region. The gapmers were created with a 3-9-3 motif and were tested for their ability to selectively inhibit mutant (mut) HTT mRNA expression. The potency and selectivity of the modified oligonucleotides were evaluated and compared to ISIS 460209.
  • The position on the oligonucleotides opposite to the SNP position, as counted from the 5′-terminus is position 8.
  • The modified oligonucleotides and their motifs are described in Table 115. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. Nucleosides followed by a subscript “d” are β-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside. Nucleosides followed by a subscript “k” indicates a 6′-(S)—CH3 bicyclic nucleoside (e.g. cEt). Nucleosides followed by a subscript “v” indicates a 5′-(S)-Me DNA. Nucleosides followed by a subscript “z” indicates a 5′-(R)-Me DNA. “mC” indicates a 5-methyl cytosine nucleoside.
  • The modified oligonucleotides were tested in vitro. Heterozygous fibroblast GM04022 cell line was used. Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with 0.1, 0.4, 1.1, 3.3 and 10 μM concentrations of modified oligonucleotides. After a treatment period of approximately 24 hours, cells were washed with DPBS buffer and lysed. RNA was extracted using Qiagen RNeasy purification and mRNA levels were measured by quantitative real-time PCR using ABI assay C—2229297—10 which measures at dbSNP rs362303. RT-PCR method in short; A mixture was made using 2020 uL 2×PCR buffer, 101 uL primers (300 uM from ABI), 1000 uL water and 40.4 uL RT MIX. To each well was added 15 uL of this mixture and 5 uL of purified RNA. The mutant and wild-type HTT mRNA levels were measured simultaneously by using two different fluorophores, FAM for mutant allele and VIC for wild-type allele. The HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN and the results are presented below.
  • The IC50s and selectivities as expressed in “fold” were measured and calculated using methods described previously in Example 41. The results in Table 116 demonstrated that each of the newly designed oligonucleotides comprising 5′-(S)- or 5′-(R)-Me DNA within the central gap region achieved improvement in potency and selectivity as compared to the parent gapmer, ISIS 460209.
  • TABLE 115
    Gap-interrupted oligonucleotides comprising 5′-(S)- or
    5′-(R)-Me DNA targeting HTT SNP
    Wing SEQ
    ISIS Gap Chemistry ID
    NO Sequence (5′ to 3′) Motif Chemistry 5′ 3′ NO
    460209 TeAkAkAdTdTdGdTd mCdAdTd mCdAk mCk mCe 3-9-3 Full deoxy ekk kke 10
    589429 TeAkAkAdTvTdGdTd mCdAdTd mCdAk mCk mCe 3-9-3 Deoxy/5′-(S)- ekk kke 10
    Me DNA
    589430 TeAkAkAdTdTvGdTd mCdAdTd mCdAk mCk mCe 3-9-3 Deoxy/5′-(S)- ekk kke 10
    Me DNA
    589431 TeAkAkAdTdTdGdTv mCdAdTd mCdAk mCk mCe 3-9-3 Deoxy/5′-(S)- ekk kke 10
    Me DNA
    589432 TeAkAkAdTdTdGdTd mCdAdTv mCdAk mCk mCe 3-9-3 Deoxy/5′-(S)- ekk kke 10
    Me DNA
    594588 TeAkAkAdTvTvGdTd mCdAdTd mCdAk mCk mCe 3-9-3 Deoxy/5′-(S)- ekk kke 10
    Me DNA
    556848 TeAkAkAzTdTdGdTd mCdAdTd mCdAk mCk mCe 3-9-3 Deoxy/5′-(R)- ekk kke 10
    Me DNA
    556849 TeAkAkAdTzTdGdTd mCdAdTd mCdAk mCk mCe 3-9-3 Deoxy/5′-(R)- ekk kke 10
    Me DNA
    556850 TeAkAkAdTdTzGdTd mCdAdTd mCdAk mCk mCe 3-9-3 Deoxy/5′-(R)- ekk kke 10
    Me DNA
    539558 TeAkAkAdTdTdGdTz mCdAdTd mCdAk mCk mCe 3-9-3 Deoxy/5′-(R)- ekk kke 10
    Me DNA
    594160 TeAkAkAdTdTdGdTd mCzAdTd mCdAk mCk mCe 3-9-3 Deoxy/5′-(R)- ekk kke 10
    Me DNA
    594161 TeAkAkAdTdTdGdTd mCdAzTd mCdAk mCk mCe 3-9-3 Deoxy/5′-(R)- ekk kke 10
    Me DNA
    589433 TeAkAkAdTdTdGdTd mCdAdTz mCdAk mCk mCe 3-9-3 Deoxy/5′-(R)- ekk kke 10
    Me DNA
    594162 TeAkAkAdTdTdGdTd mCdAdTd mCzAk mCk mCe 3-9-3 Deoxy/5′-(R)- ekk kke 10
    Me DNA
    594589 TeAkAkAdTzTzGdTd mCdAdTd mCdAk mCk mCe 3-9-3 Deoxy/5′-(R)- ekk kke 10
    Me DNA
    e = 2′-MOE; k = cEt
  • TABLE 116
    Comparison of inhibition of HTT mRNA levels and selectivity of gap-
    interrupted oligonucleotides with ISIS 460209 targeting HTT SNP
    ISIS IC50 (μM) Selectivity Wing Chemistry
    NO. Mut Wt (wt vs. mut) Motif Gap Chemistry 5′ 3′
    460209 1.2 1.4 1.2 3-9-3 Full deoxy ekk kke
    589429 0.22 3.3 15 3-9-3 Deoxy/5′-(S)-Me DNA ekk kke
    589430 0.22 >10 >45.5 3-9-3 Deoxy/5′-(S)-Me DNA ekk kke
    589431 0.16 1.9 11.9 3-9-3 Deoxy/5′-(S)-Me DNA ekk kke
    589432 0.23 >10 >43.5 3-9-3 Deoxy/5′-(S)-Me DNA ekk kke
    594588 0.81 >10 >12.3 3-9-3 Deoxy/5′-(S)-Me DNA ekk kke
    556848 0.16 1.8 11.3 3-9-3 Deoxy/5′-(R)-Me DNA ekk kke
    556849 0.14 1.1 7.9 3-9-3 Deoxy/5′-(R)-Me DNA ekk kke
    556850 0.22 1.7 7.7 3-9-3 Deoxy/5′-(R)-Me DNA ekk kke
    539558 0.38 3.8 10 3-9-3 Deoxy/5′-(R)-Me DNA ekk kke
    594160 0.28 3.3 11.8 3-9-3 Deoxy/5′-(R)-Me DNA ekk kke
    594161 0.28 >10 >35.7 3-9-3 Deoxy/5′-(R)-Me DNA ekk kke
    589433 0.27 4.4 16.3 3-9-3 Deoxy/5′-(R)-Me DNA ekk kke
    594162 0.27 3.5 13.0 3-9-3 Deoxy/5′-(R)-Me DNA ekk kke
    594589 0.48 4.4 9.2 3-9-3 Deoxy/5′-(R)-Me DNA ekk kke
    e = 2′-MOE; k = cEt
    • Example 76 Inhibition of HTT mRNA Levels Targeting SNP by Modified Oligonucleotides
  • Additional modified oligonucleotides were designed in a similar manner as the antisense oligonucleotides described in Example 75. Various chemical modifications were introduced slightly upstream or downstream (i.e. “microwalk”) within the central gap region. The gapmers were created with a 3-9-3 motif and were tested for their ability to selectively inhibit mutant (mut) HTT mRNA expression. The position on the oligonucleotides opposite to the SNP position, as counted from the 5′-terminus is position 8. The potency and selectivity of the modified oligonucleotides were evaluated and compared to ISIS 460209.
  • The modified oligonucleotides and their motifs are described in Table 117. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. Nucleosides followed by a subscript “d” are β-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e” indicates a 2′-β-methoxyethyl (MOE) modified nucleoside. Nucleosides followed by a subscript “k” indicates a 6′-(S)—CH3 bicyclic nucleoside (e.g. cEt). Nucleosides followed by a subscript “b” indicates a 5′-(R)-allyl DNA. Nucleosides followed by a subscript “c” indicates a 5′-(S)-allyl DNA. Nucleosides followed by a subscript “g” indicates a 5′-(R)-hydroxyethyl DNA. Nucleosides followed by a subscript “i” indicates a 5′-(S)-hydroxyethyl DNA. “mC” indicates a 5-methyl cytosine nucleoside.
  • The modified oligonucleotides were tested in vitro using heterozygous fibroblast GM04022 cell line. The transfection method and analysis of HTT mRNA levels adjusted according to total RNA content, as measured by RIBOGREEN were performed in the same manner as described in Example 76. The IC50s and selectivities as expressed in “fold” were measured and calculated using methods described previously and the results are shown below. As presented in Table 118, several modified oligonucleotides achieved greater than 4.5 fold selectivity in inhibiting mutant HTT mRNA levels and, therefore, are more selective than ISIS 460209.
  • TABLE 117
    Gap-interrupted oligonucleotides comprising 5′-substituted DNA
    targeting HTT SNP
    Wing SEQ
    ISIS Gap Chemistry Chemisty ID
    NO Sequence (5′ to 3′) Motif (mod position) 5′ 3′ NO
    460209 TeAkAkAdTdTdGdTd mCdAdTd mCdAk mCk mCe 3-9-3 Full deoxy ekk kke 10
    589414 TeAkAkAdTbTdGdTd mCdAdTd mCdAk mCk mCe 3-9-3 Deoxy/5′-(R)- ekk kke 10
    allyl DNA
    (pos 5)
    589415 TeAkAkAdTdTbGdTd mCdAdTd mCdAk mCk mCe 3-9-3 Deoxy/5′-(R)- ekk kke 10
    allyl DNA
    (pos 6)
    589416 TeAkAkAdTdTdGdTb mCdAdTd mCdAk mCk mCe 3-9-3 Deoxy/5′-(R)- ekk kke 10
    allyl DNA
    (pos 8)
    589417 TeAkAkAdTdTdGdTd mCdAdTb mCdAk mCk mCe 3-9-3 Deoxy/5′-(R)- ekk kke 10
    allyl DNA
    (pos 11)
    589418 TeAkAkAdTcTdGdTd mCdAdTd mCdAk mCk mCe 3-9-3 Deoxy/5′-(S)- ekk kke 10
    allyl DNA
    (pos 5)
    589419 TeAkAkAdTdTcGdTd mCdAdTd mCdAk mCk mCe 3-9-3 Deoxy/5′-(S)- ekk kke 10
    allyl DNA
    (pos 6)
    589420 TeAkAkAdTdTdGdTc mCdAdTd mCdAk mCk mCe 3-9-3 Deoxy/5′-(S)- ekk kke 10
    allyl DNA
    (pos 8)
    589421 TeAkAkAdTdTdGdTd mCdAdTc mCdAk mCk mCe 3-9-3 Deoxy/5′-(S)- ekk kke 10
    allyl DNA
    (pos 11)
    589422 TeAkAkAdTgTdGdTd mCdAdTd mCdAk mCk mCe 3-9-3 Deoxy/5′-(R)- ekk kke 10
    hydroxyethyl
    DNA (pos 5)
    589423 TeAkAkAdTdTgGdTd mCdAdTd mCdAk mCk mCe 3-9-3 Deoxy/5′-(R)- ekk kke 10
    hydroxyethyl
    DNA (pos 6)
    589424 TeAkAkAdTdTdGdTg mCdAdTd mCdAk mCk mCe 3-9-3 Deoxy/5′-(R)- ekk kke 10
    hydroxyethyl
    DNA (pos 8)
    589437 TeAkAkAdTdTdGdTd mCdAdTg mCdAk mCk mCe 3-9-3 Deoxy/5′-(R)- ekk kke 10
    hydroxyethyl
    DNA (pos 11)
    589426 TeAkAkAdTiTdGdTd mCdAdTd mCdAk mCk mCe 3-9-3 Deoxy/5′-(S)- ekk kke 10
    hydroxyethyl
    DNA (pos 5)
    589427 TeAkAkAdTdTiGdTd mCdAdTd mCdAk mCk mCe 3-9-3 Deoxy/5′-(S)- ekk kke 10
    hydroxyethyl
    DNA (pos 6)
    589428 TeAkAkAdTdTdGdTi mCdAdTd mCdAk mCk mCe 3-9-3 Deoxy/5′-(S)- ekk kke 10
    hydroxyethyl
    DNA (pos 8)
    589425 TeAkAkAdTdTdGdTd mCdAdTi mCdAk mCk mCe 3-9-3 Deoxy/5′-(S)- ekk kke 10
    hydroxyethyl
    DNA (pos 11)
    e = 2′-MOE; k = cEt
  • TABLE 118
    Comparison of inhibition of HTT mRNA levels and selectivity of gap-
    interrupted oligonucleotides with ISIS 460209 targeting HTT SNP
    ISIS IC50 (μM) Selectivity Gap Chemistry Wing Chemistry
    NO Mut Wt (wt vs. mut) (mod position) Motif 5′ 3′
    460209 0.47 2.1 4.5 Full deoxy 3-9-3 ekk kke
    589414 1.0 7.6 7.6 Deoxy/5′-(R)-Allyl DNA 3-9-3 ekk kke
    (pos 5)
    589415 1.4 >10 >7.1 Deoxy/5′-(R)-Allyl DNA 3-9-3 ekk kke
    (pos 6)
    589416 2.7 >10 >3.7 Deoxy/5′-(R)-Allyl DNA 3-9-3 ekk kke
    (pos 8)
    589417 5.4 >10 >1.9 Deoxy/5′-(R)-Allyl DNA 3-9-3 ekk kke
    (pos 11)
    589418 1.2 >10 >8.3 Deoxy/5′-(S)-Allyl DNA 3-9-3 ekk kke
    (pos 5)
    589419 1.1 >10 >9.1 Deoxy/5′-(S)-Allyl DNA 3-9-3 ekk kke
    (pos 6)
    589420 3.2 >10 >3.1 Deoxy/5′-(S)-Allyl DNA 3-9-3 ekk kke
    (pos 8)
    589421 2.0 >10 >5.0 Deoxy/5′-(S)-Allyl DNA 3-9-3 ekk kke
    (pos 11)
    589422 0.73 3.2 4.4 Deoxy/5′-(R)- 3-9-3 ekk kke
    Hydroxyethyl DNA
    (pos 5)
    589423 0.92 9.2 10 Deoxy/5′-(R)- 3-9-3 ekk kke
    Hydroxyethyl DNA
    (pos 6)
    589424 0.21 4.4 21 Deoxy/5′-(R)- 3-9-3 ekk kke
    Hydroxyethyl DNA
    (pos 8)
    589437 0.73 >10.2 >14 Deoxy/5′-(R)- 3-9-3 ekk kke
    Hydroxyethyl DNA
    (pos 11)
    589426 0.91 5.1 5.6 Deoxy/5′-(5> 3-9-3 ekk kke
    Hydroxyethyl DNA
    (pos 5)
    589427 0.91 >10 >11 Deoxy/5′-(S)- 3-9-3 ekk kke
    Hydroxyethyl DNA
    (pos 6)
    589428 1.1 >11 >10 Deoxy/5′-(S)- 3-9-3 ekk kke
    Hydroxyethyl DNA
    (pos 8)
    589425 1.5 >10.5 >7 Deoxy/5′-(S)- 3-9-3 ekk kke
    Hydroxyethyl DNA
    (pos 11)
    e = 2′-MOE; k = cEt
    • Example 77 Modified Oligonucleotides Comprising 5′-(R)-Me DNA(s) Targeting Human C-Reactive Protein (hCRP)
  • A series of modified oligonucleotides were designed based on ISIS 353512, wherein the central gap region contains fourteen β-D-2′-deoxyribonucleoside. These modified oligonucleotides were designed by replacement of two or three β-D-2′-deoxyribonucleoside in the 14 nucleoside gap region with 5′-(R)-Me DNA(s). The thermal stability (Tm) and potency of these modified oligonucleotides targeting hCRP was evaluated. The 3-14-3 MOE gapmer, ISIS 353512 and 5-10-5 MOE gapmer, ISIS 330012 were included in the study for comparison.
  • The modified oligonucleotides and their motifs are described in Table 119. Each internucleoside linkage is a phosphorothioate (P═S) except for nucleosides followed by a subscript “o”which are phosphodiester internucleoside linkages (P═O). Nucleosides followed by a subscript “d” indicates a β-D-2′-deoxyribonucleoside. Nucleosides followed by a subscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside. Nucleosides followed by a subscript “z” indicates a 5′-(R)-Me DNA. “mC” indicates a 5-methyl cytosine modified nucleoside. Underlined nucleosides indicate a region comprising 5′-(R)-Me DNA modification.
    • Thermal Stability Assay
  • The modified oligonucleotides were evaluated in thermal stability (Tm) assay. The Tm's were measured using the method described herein. A Cary 100 Bio spectrophotometer with the Cary Win UV Thermal program was used to measure absorbance vs. temperature. For the Tm experiments, oligonucleotides were prepared at a concentration of 8 μM in a buffer of 100 mM Na+, 10 mM phosphate, 0.1 mM EDTA, pH 7. Concentration of oligonucleotides were determined at 85° C. The oligonucleotide concentration was 4 μM with mixing of equal volumes of test oligonucleotide and complimentary RNA strand. Oligonucleotides were hybridized with the complimentary RNA strand by heating duplex to 90° C. for 5 min and allowed to cool at room temperature. Using the spectrophotometer, Tm measurements were taken by heating duplex solution at a rate of 0.5 C/min in cuvette starting @ 15° C. and heating to 85° C. Tm values were determined using Vant Hoff calculations (A260 vs temperature curve) using non self-complementary sequences where the minimum absorbance which relates to the duplex and the maximum absorbance which relates to the non-duplex single strand are manually integrated into the program. The results are presented below.
    • Cell Culture and Transfection
  • The modified oligonucleotides were tested in vitro. Hep3B cells were plated at a density of 40,000 cells per well and transfected using electroporation with 0.009 μM, 0.027 μM, 0.082 μM, 0.25 μM, 0.74 μM, 2.2 μM, 6.7 μM and 20 μM concentrations of antisense oligonucleotides. After a treatment period of approximately 16 hours, RNA was isolated from the cells and hCRP mRNA levels were measured by quantitative real-time PCR. Human CRP primer probe set RTS1887 was used to measure mRNA levels. hCRP mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®.
    • Analysis of IC50's
  • The half maximal inhibitory concentration (IC50) of each oligonucleotide is presented below and was calculated by plotting the concentrations of oligonucleotides used versus the percent inhibition of hCRP mRNA expression achieved at each concentration, and noting the concentration of oligonucleotide at which 50% inhibition of hCRP mRNA expression was achieved compared to the control.
  • As illustrated in Table 120, treatment with the newly designed oligonucleotides showed no improvement in potency as compared to the controls, ISIS 353512 and 330012.
  • TABLE 119
    Gap-interrupted oligonucleotides comprising 5′-(R)-Me DNA 
    targeting hCRP
    Wing SEQ
    ISIS Gap Chemistry Linkage ID
    NO Sequence (5′ to 3′) Motif Chemistry 5′ 3′ backbone NO
    353512 Te mCe mCe mCdAdTdTdTd mCdAd 3-14-3 Full deoxy eee eee Full PS 83
    GdGdAdGdAd mCd mCdTeGeGe
    546127 Te mCe mCe mCdAdTdTdTd mCdo Azo 3-14-3 Deoxy/5′-(R)- eee eee Mixed 83
    Gz GdAdGdAd mCd mCdTeGeGe Me DNA PS/PO
    544810 Te mCe mCe mCdAdTdTdTd mCdAd 3-14-3 Deoxy/5′-(R)- eee eee Mixed 83
    GdGdAdGdAdo mCzo mCz TeGeGe Me DNA PS/PO
    544806 Te mCe mCeo mCzoAzoTz TdTd mCdAd 3-14-3 Deoxy/5′-(R)- eee eee Mixed 83
    GdGdAdGdAd mCd mCdTeGeGe Me DNA PS/PO
    544807 Te mCe mCe mCdAdTdo TzoTzo mCz Ad 3-14-3 Deoxy/5′-(R)- eee eee Mixed 83
    GdGdAdGdAd mCd mCdTeGeGe Me DNA PS/PO
    544809 Te mCe mCe mCdAdTdTdTd mCdAd 3-14-3 Deoxy/5′-(R)- eee eee Mixed 83
    GdGdo AzoGzoAz mCd mCdTeGeGe Me DNA PS/PO
    330012 Te mCe mCe mCeAeTdTdTd mCdAd 5-10-5 Full deoxy e5 e5 Full PS 83
    GdGdAdGdAd mCe mCeTeGeGe
    e = 2′-MOE (e.g. e5 = eeeee)
  • TABLE 120
    Effect of gap-interrupted oligonucleotide
    treatment on Tm and hCRP inhibition
    Wing
    ISIS Tm IC50 Gap Chemistry Linkage
    NO (° C.) (μM) Motif Chemistry 5′ 3′ backbone
    353512 66.7 1.1 3-14-3 Full deoxy eee eee Full PS
    546127 65.9 2.5 3-14-3 Deoxy/5′-(R)- eee eee Mixed
    Me DNA PS/PO
    544810 64.3 2.4 3-14-3 Deoxy/5′-(R)- eee eee Mixed
    Me DNA PS/PO
    544806 62.8 2.8 3-14-3 Deoxy/5′-(R)- eee eee Mixed
    Me DNA PS/PO
    544807 65.1 2.7 3-14-3 Deoxy/5′-(R)- eee eee Mixed
    Me DNA PS/PO
    544809 64.2 5.0 3-14-3 Deoxy/5′-(R)- eee eee Mixed
    Me DNA PS/PO
    330012 71.7 0.6 5-10-5 Full deoxy e5 e5 Full PS
    e = 2′-MOE (e.g. e5 = eeeee), PS/PO = phosphorothioate/phosphodiester internucleoside linkage
    • Example 78 Human Peripheral Blood Mononuclear Cells (hPBMC) Assay Protocol—In Vitro
  • The hPBMC assay was performed using BD Vacutainer CPT tube method. A sample of whole blood from volunteered donors with informed consent at US HealthWorks clinic (Faraday & El Camino Real, Carlsbad) was obtained and collected in 4-15 BD Vacutainer CPT 8 ml tubes (VWR Cat.# BD362753). The approximate starting total whole blood volume in the CPT tubes for each donor was recorded using the PBMC assay data sheet.
  • The blood sample was remixed immediately prior to centrifugation by gently inverting tubes 8-10 times. CPT tubes were centrifuged at rt (18-25° C.) in a horizontal (swing-out) rotor for 30 min. at 1500-1800 RCF with brake off (2700 RPM Beckman Allegra 6R). The cells were retrieved from the buffy coat interface (between Ficoll and polymer gel layers); transferred to a sterile 50 ml conical tube and pooled up to 5 CPT tubes/50 ml conical tube/donor. The cells were then washed twice with PBS (Ca++, Mg++ free; GIBCO). The tubes were topped up to 50 ml and mixed by inverting several times. The sample was then centrifuged at 330×g for 15 minutes at rt (1215 RPM in Beckman Allegra 6R) and aspirated as much supernatant as possible without disturbing pellet. The cell pellet was dislodged by gently swirling tube and resuspended cells in RPMI+10% FBS+pen/strep (˜1 ml/10 ml starting whole blood volume). A 60 μl sample was pipette into a sample vial (Beckman Coulter) with 600 μl VersaLyse reagent (Beckman Coulter Cat# A09777) and was gently vortexed for 10-15 sec. The sample was allowed to incubate for 10 min. at rt and being mixed again before counting. The cell suspension was counted on Vicell XR cell viability analyzer (Beckman Coulter) using PBMC cell type (dilution factor of 1:11 was stored with other parameters). The live cell/ml and viability were recorded. The cell suspension was diluted to 1×107 live PBMC/ml in RPMI+10% FBS+pen/strep.
  • The cells were plated at 5×105 in 50 μl/well of 96-well tissue culture plate (Falcon Microtest). 50 μl/well of 2× concentration oligos/controls diluted in RPMI+10% FBS+pen/strep. was added according to experiment template (100 μl/well total). Plates were placed on the shaker and allowed to mix for approx. 1 min. After being incubated for 24 hrs at 37° C.; 5% CO2, the plates were centrifuged at 400×g for 10 minutes before removing the supernatant for MSD cytokine assay (i.e. human IL-6, IL-10, IL-8 and MCP-1).
    • Example 79 Evaluation of the Proinflammatory Effects in hPBMC Assay for 5′-(R)-Me DNA Containing Modified Oligonucleotides—In Vitro Study
  • The modified oligonucleotides targeting hCRP from Example 77 were tested and evaluated for the proinflammatory response in hPBMC assay using methods described previously in Example 78. The hPBMCs were isolated from fresh, volunteered donors and were treated with modified oligonucleotides at 0, 0.0128, 0.064, 0.32, 1.6, 8, 40 and 200 μM concentrations using the hPBMC assay protocol described herein. After a 24 hr treatment, the cytokine levels were measured.
  • IL-6 was used as the primary readout. The resulting IL-6 level was compared to the positive control, ISIS 353512 and negative control, ISIS 104838. The results are presented in Table 121. As illustrated, reduction in proinflammatory response was achieved with the newly designed oligonucleotides at doses evaluated as compared to the positive control, ISIS 353512.
  • ISIS 104838 designated herein as SEQ ID NO: 84, is a 5-10-5 MOE gapmer with the following sequence, Ge mCeTeGeAeTdTdAdGdAdGdAdGdAdGdGeTe mCe mCe mCe. Each internucleoside linkage is a phosphorothioate (P═S). Each nucleoside followed by a subscript “d” is a β-D-2′-deoxyribonucleoside. Each “mC” is a 5-methyl cytosine modified nucleoside and each nucleoside followed by a subscript “e” is a 2′-O-methoxyethyl(MOE) modified nucleoside.
  • TABLE 121
    Effect of gap-interrupted oligonucleotide treatment
    on proinflammatory response in hPBMC
    Wing
    ISIS Conc. IL-6 Gap Chemistry Linkage
    NO (uM) (pg/mL) Motif Chemistry 5′ 3′ backbone
    353512 0 26.9 3-14-3 Full deoxy eee eee Full PS
    (pos 0.0128 10.6
    control) 0.064 73.3
    0.32 219.8
    1.6 200.1
    8 287.8
    40 376.9
    200 181.5
    546127 0 11.5 3-14-3 Deoxy/5′-(R)- eee eee Mixed
    0.0128 15.1 Me DNA PS/PO
    0.064 19.0
    0.32 37.3
    1.6 67.5
    8 86.3
    40 111.2
    200 83.1
    544810 0 11.5 3-14-3 Deoxy/5′-(R)- eee eee Mixed
    0.0128 13.9 Me DNA PS/PO
    0.064 15.1
    0.32 24.9
    1.6 34.0
    8 66.2
    40 96.8
    200 76.5
    06/544806 0 11.3 3-14-3 Deoxy/5′-(R)- eee eee Mixed
    0.0128 10.8 Me DNA PS/PO
    0.064 25.8
    0.32 15.6
    1.6 25.4
    8 52.3
    40 69.3
    200 341.7
    06/544807 0 13.3 3-14-3 Deoxy/5′-(R)- eee eee Mixed
    0.0128 13.7 Me DNA PS/PO
    0.064 18.4
    0.32 53.3
    1.6 18.4
    8 164.9
    40 202.7
    200 606.5
    06/544809 0 10.8 3-14-3 Deoxy/5′-(R)- eee eee Mixed
    0.0128 13.3 Me DNA PS/PO
    0.064 14.3
    0.32 34.8
    1.6 62.3
    8 100.9
    40 213.1
    200 225.0
    06/330012 0 10.9 5-10-5 Full deoxy e5 e5 Full PS
    0.0128 12.9
    0.064 10.8
    0.32 25.3
    1.6 44.2
    8 87.5
    40 80.2
    200 82.3
    07/104838 0 9.3 5-10-5 Full deoxy e5 e5 Full PS
    (neg 0.0128 10.4
    control) 0.064 17.6
    0.32 30.1
    1.6 53.9
    8 124.8
    40 94.5
    200 89.3
    e = 2′-MOE (e.g. e5 = eeeee)
    • Example 80 Evaluation of the Proinflammatory Effects in hPBMC Assay for a Modified Oligonucleotide Comprising Methyl Thiophosphonate Internucleoside Linkages—In Vitro Study
  • A modified oligonucleotide was designed based on the 3/14/3 MOE gapmer, ISIS 353512. This modified oligonucleotide was created by having alternating methyl thiophosphonate (—P(CH3)(═S)—) internucleoside linkages throughout the gap region. The proinflammatory effect of the modified oligonucleotide targeting hCRP was evaluated in hPBMC assay using the protocol described in Example 78.
  • The modified oligonucleotide and its motif are described in Table 122. Each internucleoside linkage is a phosphorothioate (P═S) except for nucleosides followed by a subscript “w”. Each nucleoside followed by a subscript “w” indicates a methyl thiophosphonate internucleoside linkage (—P(CH3)(═S)—). Nucleosides followed by a subscript “d” is a β-D-2′-deoxyribonucleoside. Nucleosides followed by a subscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside. “mC” indicates a 5-methyl cytosine modified nucleoside.
  • The hPBMCs were isolated from fresh, volunteered donors and were treated with modified oligonucleotides at 0, 0.0128, 0.064, 0.32, 1.6, 8, 40 and 200 μM concentrations. After a 24 hr treatment, the cytokine levels were measured.
  • IL-6 was used as the primary readout. The resulting IL-6 level was compared to the positive control oligonucleotide, ISIS 353512 and negative control, ISIS 104838. The results from two donors denoted as “Donor 1” and “Donor 2” are presented in Table 123. As illustrated, reduction in proinflammatory response was achieved with the newly designed oligonucleotide at doses evaluated as compared to the positive control, ISIS 353512.
  • TABLE 122
    Modified oligonucleotide comprising alternating methyl 
    thiophosphonate internucleoside linkages throughout 
    the gap region
    Wing SEQ
    ISIS Gap Chemistry ID
    NO Sequence (5′ to 3′) Motif Chemistry 5′ 3′ NO
    353512 Te mCe mCe mCdAdTdTdTd mCdAdGd 3-14-3 Full deoxy eee eee 83
    GdAdGdAd mCd mCdTeGeGe
    560221 Te mCe mCeCdwAdTdwTdTdw mCdAdwGdGdw 3-14-3 Deoxy/methyl eee eee 83
    AdGdwAdCdw mCdTeGeGe thiophosphonate
    104838 Ge mCeTeGeAeTdTdAdGdAdGdAd 5-10-5 Full deoxy e5 e5 84
    GdAdGdGeTe mCe mCe mCe
    e = 2′-MOE (e.g. e5 = eeeee)
  • TABLE 123
    Effect of modified oligonucleotide treatment
    on proinflammatory response in hPBMC assay
    Wing
    ISIS Conc. IL-6 (Donor 1) IL-6 (Donor 2) Gap Chemistry
    NO (μM) (pg/mL) (pg/mL) Motif Chemistry 5′ 3′
    353512 0 6.3 7.8 3-14-3 Full deoxy eee eee
    0.0128 8.3 10.2
    0.064 77.2 118.2
    0.32 151.9 394.3
    1.6 152.4 395.3
    8 147.6 337.2
    40 122.5 228.4
    200 119.7 193.5
    560221 0 5.6 7.6 3-14-3 Deoxy/methyl eee eee
    0.0128 6.4 6.9 thiophosphonate
    0.064 6.7 7.6
    0.32 7.6 8.9
    1.6 9.1 11.8
    8 17.5 24.3
    40 65.8 50.2
    200 60.0 100.4
    104838 0 5.8 7.3 5-10-5 Full deoxy e5 e5
    0.0128 7.7 7.9
    0.064 7.5 11.6
    0.32 15.1 22.0
    1.6 73.1 112.8
    8 29.6 51.5
    40 41.6 69.5
    200 55.4 4018
    e = 2′-MOE (e.g. e5 = eeeee)
    • Example 81 Modified Oligonucleotides Comprising Methyl Phosphonate Internucleoside Linkage Targeting HTT SNP—In Vitro Study
  • ISIS 558255 and 558256 from Example 49 were selected and evaluated for their effect on mutant and wild type HTT mRNA expression levels targeting rs7685686. ISIS 46020 was included in the study for comparison. The position on the oligonucleotides opposite to the SNP position, as counted from the 5′-terminus is position 8.
  • Heterozygous fibroblast GM04022 cell line was used for the in vitro assay (from Coriell Institute). Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with 0.12, 0.37, 1.1, 3.3 and 10 μM concentrations of modified oligonucleotides. After a treatment period of approximately 24 hours, cells were washed with DPBS buffer and lysed. RNA was extracted using Qiagen RNeasy purification and mRNA levels were measured by quantitative real-time PCR using ABI assay C—2229297—10 which measures at dbSNP rs362303. RT-PCR method in short; A mixture was made using 2020 μL 2×PCR buffer, 101 μL primers (300 μM from ABI), 1000 μL water and 40.4 μL RT MIX. To each well was added 15 μL of this mixture and 5 μL of purified RNA. The mutant and wild-type HTT mRNA levels were measured simultaneously by using two different fluorophores, FAM for mutant allele and VIC for wild-type allele. The HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN.
  • The IC50s and selectivities as expressed in “fold” were measured and calculated using methods described previously in Example 41. As illustrated in Table 124, improvement in selectivity and potency was achieved with modified oligonucleotides comprising methyl phosphonate internucleoside linkage as compared to ISIS 460209.
  • TABLE 124
    Comparison of selectivity in inhition of HTT mRNA levels of antisense
    oligonucleotides with ISIS 460209 targeted to rs7685686 in GM4022 cells
    ISIS IC50 (μM) Selectivity Wing Chemistry SEQ
    NO Mut Wt (wt vs mut) Motif Gap Chemistry 5′ 3′ ID NO
    460209 0.30 0.99 3.3 3-9-3 Full deoxy ekk kke 10
    558255 0.19 1.3 6.8 3-9-3 Deoxy/Methyl ekk kke 10
    phosphonate
    558256 0.20 1.3 6.5 3-9-3 Deoxy/Methyl ekk kke 10
    phosphonate
    e = 2′-MOE (e.g. e5 = eeeee), k = cEt
    • Example 82 Modified Oligonucleotides Comprising Methyl Phosphonate or Phosphonoacetate Internucleoside Linkage(s) Targeting HTT SNP
  • A series of modified oligonucleotides were designed based on ISIS 460209 wherein the gap region contains nine β-D-2′-deoxyribonucleosides. The modified oligonucleotides were synthesized to include one or more methyl phosphonate or phosphonoacetate internucleoside linkage modifications within the gap region. The oligonucleotides with modified phosphorus containing backbone were tested for their ability to selectively inhibit mutant (mut) HTT mRNA expression levels targeting rs7685686 while leaving the expression of the wild-type (wt) intact. The potency and selectivity of the modified oligonucleotides were evaluated and compared to ISIS 460209.
  • The position on the oligonucleotides opposite to the SNP position, as counted from the 5′-terminus is position 8.
  • The modified oligonucleotides and their motifs are described in Table 125. Each internucleoside linkage is a phosphorothioate (P═S) except for the internucleoside linkage having a subscript “x” or “y”. Each nucleoside followed by a subscript “x” indicates a methyl phosphonate internucleoside linkage (—P(CH3)(═O)—). Each nucleoside followed by a subscript “y” indicates a phosphonoacetate internucleoside linkage (—P(CH2CO2 −)(═O)—). Nucleosides followed by a subscript “d” is a β-D-2′-deoxyribonucleoside. Nucleosides followed by a subscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside. Nucleosides followed by a subscript “k” indicates a 6′-(S)—CH3 bicyclic nucleoside (e.g. cEt). “mC” indicates a 5-methyl cytosine modified nucleoside.
  • The modified oligonucleotides were tested in vitro. Heterozygous fibroblast GM04022 cell line was used (from Coriell Institute). Cultured GM04022 cells at a density of 25,000 cells per well were transfected using electroporation with 0.12, 0.37, 1.1, 3.3 and 10 μM concentrations of modified oligonucleotides. After a treatment period of approximately 24 hours, cells were washed with DPBS buffer and lysed. RNA was extracted using Qiagen RNeasy purification and mRNA levels were measured by quantitative real-time PCR using ABI assay C—2229297—10 which measures at dbSNP rs362303. RT-PCR method in short; A mixture was made using 2020 μL 2×PCR buffer, 101 μL primers (300 μM from ABI), 1000 uL water and 40.4 μL RT MIX. To each well was added 15 μL of this mixture and 5 μL of purified RNA. The mutant and wild-type HTT mRNA levels were measured simultaneously by using two different fluorophores, FAM for mutant allele and VIC for wild-type allele. The HTT mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN.
  • The IC50s and selectivities as expressed in “fold” were measured and calculated using methods described previously in Example 41. As illustrated in Table 126, most of the newly design oligonucleotides achieved improvement in selectivity while maintaining potency as compared to ISIS 460209.
  • TABLE 125
    Modified oligonucleotides comprising methyl phosphonate or
    phosphonoacetate internucleoside linkage(s) targeting HTT SNP
    Wing SEQ
    ISIS Chemistry ID
    NO Sequence (5′ to 3′) Motif Gap Chemistry 5′ 3′ NO
    460209 TeAkAkAdTdTdGdTd mCdAdTd mCdAk mCk mCe 3-9-3 Full deoxy ekk kke 10
    566276 TeAkAkAdTdTdGdxTd mCdAdTd mCdAk mCk mCe 3-9-3 Deoxy/Methyl  ekk kke 10
    phosphonate
    566277 TeAkAkAdTdTdGdTdx mCdAdTd mCdAk mCk mCe 3-9-3 Deoxy/Methyl  ekk kke 10
    phosphonate
    566278 TeAkAkAdTdTdGdTd mCdxAdTd mCdAk mCk mCe 3-9-3 Deoxy/Methyl  ekk kke 10
    phosphonate
    566279 TeAkAkAdTdTdGdTd mCdAdxTd mCdAk mCk mCe 3-9-3 Deoxy/Methyl  ekk kke 10
    phosphonate
    566280 TeAkAkAdTdTdGdTd mCdAdTdx mCdAk mCk mCe 3-9-3 Deoxy/Methyl  ekk kke 10
    phosphonate
    566283 TeAkAkAdTdxTdxGdTd mCdAdTd mCdAk mCk mCe 3-9-3 Deoxy/Methyl  ekk kke 10
    phosphonate
    573815 TeAkAkAdTdyTdGdTd mCdAdTd mCdAk mCk mCe 3-9-3 Deoxy/ ekk kke 10
    Phosphonoacetate
    573816 TeAkAkAdTdTdyGdTd mCdAdTd mCdAk mCk mCe 3-9-3 Deoxy/ ekk kke 10
    Phosphonoacetate
    573817 TeAkAkAdTdTdGdTdy mCdAdTd mCdAk mCk mCe 3-9-3 Deoxy/ ekk kke 10
    Phosphonoacetate
    573818 TeAkAkAdTdTdGdTd mCdAdTdy mCdAk mCk mCe 3-9-3 Deoxy/ ekk kke 10
    Phosphonoacetate
    e = 2′-MOE, k = cEt
  • TABLE 126
    Comparison of selectivity in inhition of HTT mRNA levels of antisense
    oligonucleotides with ISIS 460209 targeted to rs7685686 in GM4022 cells
    ISIS Mut IC50 Selectivity Wing Chemistry SEQ
    NO (μM)) (wt vs mut) Motif Gap Chemistry 5′ 3′ ID NO
    460209 0.15 9.4 3-9-3 Full deoxy ekk kke 10
    566276 0.76 12.8 3-9-3 Deoxy/Methyl phosphonate ekk kke 10
    566277 0.20 17 3-9-3 Deoxy/Methyl phosphonate ekk kke 10
    566278 0.25 8.9 3-9-3 Deoxy/Methyl phosphonate ekk kke 10
    566279 0.38 — 3-9-3 Deoxy/Methyl phosphonate ekk kke 10
    566280 0.27 47 3-9-3 Deoxy/Methyl phosphonate ekk kke 10
    566283 0.8 >100 3-9-3 Deoxy/Methyl phosphonate ekk kke 10
    573815 0.16 18.8 3-9-3 Deoxy/Phosphonoacetate ekk kke 10
    573816 0.55 18.1 3-9-3 Deoxy/Phosphonoacetate ekk kke 10
    573817 0.17 22.5 3-9-3 Deoxy/Phosphonoacetate ekk kke 10
    573818 0.24 13.5 3-9-3 Deoxy/Phosphonoacetate ekk kke 10
    e = 2′-MOE, k = cEt
    • Example 83 Modified Oligonucleotides Comprising Methyl Phosphonate Internucleoside Linkages Targeting PTEN and SRB-1—In Vivo Study
  • Additional modified oligonucleotides were designed based on ISIS 482050 and 449093 wherein the gap region contains ten β-D-2′-deoxyribonucleosides. The modified oligonucleotides were designed by introducing two methyl phosphonate internucleoside linkages at the 5′-end of the gap region with a 3/10/3 motif. The oligonucleotides were evaluated for reduction in PTEN and SRB-1 mRNA expression levels in vivo. The parent gapmers, ISIS 482050 and 449093 were included in the study for comparison.
  • The modified oligonucleotides and their motifs are described in Table 127. Each internucleoside linkage is a phosphorothioate (P═S) except for the internucleoside linkage having a subscript “x”. Each nucleoside followed by a subscript “x” indicates a methyl phosphonate internucleoside linkage (—P(CH3)(═O)—). Nucleosides followed by a subscript “d” is a β-D-2′-deoxyribonucleoside. Nucleosides followed by a subscript “k” indicates a 6′-(S)—CH3 bicyclic nucleoside (e.g. cEt). “mC” indicates a 5-methyl cytosine modified nucleoside.
    • Treatment
  • Six week old BALB/C mice (purchased from Charles River) were injected subcutaneously twice a week for three weeks at dosage 10 mg/kg or 20 mg/kg with the modified oligonucleotides shown below or with saline control. Each treatment group consisted of 3 animals. The mice were sacrificed 48 hrs following last administration, and organs and plasma were harvested for further analysis.
  • mRNA Analysis
  • Liver tissues were homogenized and mRNA levels were quantitated using real-time PCR and normalized to RIBOGREEN as described herein. The results in Table 128 are listed as PTEN or SRB-1 mRNA expression for each treatment group relative to saline-treated control (% UTC). As illustrated, reduction in PTEN or SRB-1 mRNA expression levels was achieved with the oligonucleotides comprising two methyl phosphonate internucleoside linkages at the 5′-end of the gap region, ISIS 582073 and 582074.
    • Plasma Chemistry Markers
  • Plasma chemistry markers such as liver transaminase levels, alanine aminotranferase (ALT) in serum were measured relative to saline injected mice and the results are presented in Table 128. Treatment with the oligonucleotides resulted in reduction in ALT level compared to treatment with the parent gapmer, ISIS 482050 or 449093. The results suggest that introduction of methyl phosphonate internucleoside linkage(s) can be useful for reduction of hepatoxicity profile of otherwise unmodified parent gapmers.
    • Body and Organ Weights
  • Body weights, as well as liver, kidney and spleen weights were measured at the end of the study. The results below are presented as the average percent of body and organ weights for each treatment group relative to saline-treated control. As illustrated in Table 129, treatment with ISIS 582073 resulted in a reduction in liver and spleen weights compared to treatment with the parent gapmer, ISIS 482050. The remaining oligonucleotide, ISIS 582074 did not cause any changes in body and organ weights outside the expected range as compared to ISIS 449093.
  • TABLE 127
    Modified oligonucleotides comprising methyl phosphonate internu-
    cleoside linkages
    Wing SEQ
    ISIS Gap Chemistry ID
    NO Sequence (5′ to 3′) Motif Chemistry 5′ 3′ NO.
    482050 AkTk mCkAdTdGdGd mCdTdGd mCdAdGd mCkTkTk 3-10-3 Full deoxy kkk kkk 85
    582073 AkTk mCkAdxTdxGdGd mCdTdGd mCdAdGd mCkTkTk 3-10-3 Deoxy/Methyl kkk kkk 85
    phosphonate
    449093 TkTk mCkAdGdTd mCdAdTdGdAd mCdTdTk mCk mCk 3-10-3 Full deoxy kkk kkk 86
    582074 TkTk mCkAdxGdxTd mCdAdTdGdAd mCdTdTk mCk mCk 3-10-3 Deoxy/Methyl kkk kkk 86
    phosphonate
    k = cEt
  • TABLE 128
    Effect of modified oligonucleotide treatment on target
    reduction and liver function in BALB/C mice
    ISIS Dosage % ALT Gap Wing Chemistry SEQ
    NO. Target (mg/kg/wk) UTC (IU/L) Motif Chemistry 5′ 3′ ID NO.
    Saline — 0 100 30 — — — — —
    482050 PTEN 10 50 228 3-10-3 Full deoxy kkk kkk 85
    482050 20 36.1 505
    582073 10 72.2 47.7 Deoxy/Methyl kkk kkk 85
    582073 20 57.4 46 phosphonate
    449093 SRB-1 10 48 543 3-10-3 Full deoxy kkk kkk 86
    449093 20 18.5 1090
    582074 10 51.3 58.3 Deoxy/Methyl kkk kkk 86
    582074 20 30.3 126.3 phosphonate
    k = cEt
  • TABLE 129
    Effect of modified oligonucleotide treatment on body and organ weights in BALB/C mice
    ISIS Dosage Body wt rel to Liver/Body Spleen/Body Kidney/Body SEQ
    NO. Target (mg/kg/wk) predose (%) Wt (%) Wt (%) Wt (%) ID NO.
    Saline — 0 108.4 100 100 100
    482050 PTEN 10 107.4 154.9 141.8 115.7 85
    482050 20 111.3 176.7 142.3 112.5
    582073 10 108.9 122.9 111.7 100.0 85
    582073 20 107.9 133.8 114.6 102.9
    449093 SRB-1 10 101.3 105.9 117.9 89.3 86
    449093 20 95.3 118.6 129.6 93.0
    582074 10 107.1 92.2 116.4 89.2 86
    582074 20 103.8 95.5 128.8 91.9
    • Example 84 Modified Oligonucleotides Comprising Methyl Phosphonate Internucleoside Linkages Targeting Target-Y—In Vivo Study
  • Additional modified oligonucleotides were designed in the same manner as the antisense oligonucleotides described in Example 24, wherein two methyl phosphonate internucleoside linkages are introduced at the 5′-end of the gap region. The modified oligonucleotides were designed based on ISIS 464917, 465178, 465984 and 466456 with a 3/10/3 motif. The oligonucleotides were evaluated for reduction in Target-Y mRNA expression levels in vivo. The parent gapmers, ISIS 464917, 465178, 465984 and 466456 were included in the study for comparison.
  • The modified oligonucleotides and their motifs are presented in Table 130. Each internucleoside linkage is a phosphorothioate (P═S) except for the internucleoside linkage having a subscript “x”. Each nucleoside followed by a subscript “x” indicates a methyl phosphonate internucleoside linkage (—P(CH3)(═O)—). Each nucleoside followed by a subscript “d” is a β-D-2′-deoxyribonucleoside. Nucleosides followed by a subscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside. Nucleosides followed by a subscript “k” indicates a 6′-(S)—CH3 bicyclic nucleoside (e.g. cEt). “N” indicates modified or naturally occurring nucleobases (A, T, C, G, U, or 5-methyl C).
    • Treatment
  • Six week old BALB/C mice (purchased from Charles River) were injected subcutaneously twice a week for three weeks at dosage 10 mg/kg or 20 mg/kg with the modified oligonucleotides shown below or with saline control. Each treatment group consisted of 3 animals. The mice were sacrificed 48 hrs following last administration, and organs and plasma were harvested for further analysis.
  • mRNA Analysis
  • Liver tissues were homogenized and mRNA levels were quantitated using real-time PCR and normalized to RIBOGREEN as described herein. The results below are listed as Target-Y mRNA expression for each treatment group relative to saline-treated control (% UTC). As illustrated in Table 131, reduction in Target-Y mRNA expression levels was achieved with the oligonucleotides comprising two methyl phosphonate internucleoside linkages at the 5′-end of the gap region, ISIS 582071, 582072, 582069 and 582070.
    • Plasma Chemistry Markers
  • Plasma chemistry markers such as liver transaminase levels, alanine aminotranferase (ALT) in serum were measured relative to saline treated mice and the results are presented in Table 131. Treatment with the oligonucleotides resulted in reduction in ALT level compared to treatment with the parent gapmer, ISIS 464917, 465178, 465984 or 466456. The results suggest that introduction of methyl phosphonate internucleoside linkage(s) can be useful for reduction of hepatoxicity profile of otherwise unmodified parent gapmers.
    • Body and Organ Weights
  • Body weights, as well as liver, kidney and spleen weights were measured at the end of the study. The results in Table 132 are presented as the average percent of body and organ weights for each treatment group relative to saline-treated control. As illustrated, treatment with ISIS 582070 resulted in a reduction in liver and spleen weights compared to treatment with the parent gapmer, ISIS 466456. An increase in body and organ weights was observed for ISIS 582071 as compared to ISIS 464917. The remaining oligonucleotides, ISIS 582072 and 582069 did not cause any changes in body and organ weights outside the expected range as compared to ISIS 465178 and 465984.
  • TABLE 130
    Modified oligonucleotides comprising methyl phosphonate internu-
    cleoside linkages
    Wing SEQ
    ISIS Gap Chemistry ID
    NO Sequence (5′ to 3′) Motif Chemistry 5′ 3′ NO.
    464917 NkNkNkNdNdNdNdNdNdNdNdNdNdNkNkNk 3-10-3 Full deoxy kkk kkk 6
    582071 NkNkNkNdxNdxNdNdNdNdNdNdNdNdNkNkNk 3-10-3 Deoxy/Methyl kkk kkk
    phosphonate
    465178 NkNkNkNdNdNdNdNdNdNdNdNdNdNkNkNk 3-10-3 Full deoxy kkk kkk 6
    582072 NkNkNkNdxNdxNdNdNdNdNdNdNdNdNkNkNk 3-10-3 Deoxy/Methyl kkk kkk
    phosphonate
    465984 NkNkNkNdNdNdNdNdNdNdNdNdNdNeNeNe 3-10-3 Full deoxy kkk eee 6
    582069 NkNkNkNdxNdxNdNdNdNdNdNdNdNdNkNkNk 3-10-3 Deoxy/Methyl kkk kkk
    phosphonate
    466456 NkNdNkNdNkNdNdNdNdNdNdNdNdNdNeNe 5-9-2 or Full deoxy or kdkdk ee 6
    3-11-2 deoxy/cEt or kdk
    582070 NkNdNkNdxNdxNdNdNdNdNdNdNdNdNdNeNe 3-11-2 Deoxy/Methyl kdk ee
    phosphonate
    e = 2′-MOE, k = cEt, d = 2′-deoxyribonucleoside
  • TABLE 131
    Effect of modified oligonucleotide treatment on Target-Y
    reduction and liver function in BALB/C mice
    ISIS Dosage % ALT Gap Wing Chemistry
    NO. (mg/kg/wk) UTC (IU/L) Motif Chemistry 5′ 3′
    Saline 0 100 30 — — — —
    464917 10 29 1244 3-10-3 Full deoxy kkk kkk
    464917 20 30.1 2335
    582071 20 10.2 274 3-10-3 Deoxy/Methyl kkk kkk
    phosphonate
    465178 10 4.9 1231 3-10-3 Full deoxy kkk kkk
    465178 20 10.6 6731
    582072 10 36.7 44.7 3-10-3 Deoxy/Methyl kkk kkk
    582072 20 23.6 43.7 phosphonate
    465984 10 4.7 61 3-10-3 Full deoxy kkk eee
    465984 20 0.9 57
    582069 10 11.1 39.7 3-10-3 Deoxy/Methyl kkk kkk
    582069 20 3.3 27.7 phosphonate
    466456 10 9.5 692 5-9-2 or Full deoxy or kdkdk ee
    466456 20 10.5 2209 3-11-2 deoxy/cEt or kdk
    582070 10 73.9 24 3-11-2 Deoxy/Methyl kdk ee
    582070 20 51.3 36.7 phosphonate
    e = 2′-MOE, k = cEt, d = 2′-deoxyribonucleoside
  • TABLE 132
    Effect of modified oligonucleotide treatment
    on body and organ weights in BALB/C mice
    Body Liver/ Spleen/ Kidney/
    Dosage wt rel to Body Body Body
    ISIS NO. (mg/kg/wk) predose (%) Wt (%) Wt (%) Wt (%)
    Saline 0 108 100 100 100
    464917 10 92.9 125 106.2 102.3
    464917 20 71.1 110.9 67.2 107.3
    582071 20 104.6 135.2 142.8 89.8
    465178 10 94.9 131.3 108.1 85.3
    465178 20 79.5 147.5 112 95.3
    582072 10 109.2 117.3 111.7 104.8
    582072 20 107.1 130.1 107.2 99.8
    465984 10 111.4 117.6 110.1 98.8
    465984 20 111.3 122.6 134.5 96.1
    582069 10 107.8 106.2 97 100.6
    582069 20 105.4 115.8 106.2 100.4
    466456 10 109.7 148.6 198.7 105.9
    466456 20 101.2 182.3 213.7 101.9
    582070 10 111.2 100.3 116.7 100.8
    582070 20 111.1 108.9 115.6 95.7
    • Example 85 Short-Gap Chimeric Oligonucleotides Targeting Target-Y
  • A series of chimeric antisense oligonucleotides was designed based on ISIS 464917 or 465178, wherein the central gap region contains ten 2′-deoxyribonucleosides. These gapmers were designed by introducing 2′-MOE modified nucleoside(s) at the wing(s) and/or shortening the central gap region to nine, eight, or seven 2′-deoxyribonucleosides.
  • The gapmers and their motifs are described in Table 133. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine nucleobases throughout each gapmer are 5-methyl cytosines. Nucleosides without a subscript are β-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e” or “k” are sugar modified nucleosides. A subscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside and a subscript “k” indicates a 6′-(S)—CH3 bicyclic nucleoside (e.g. cEt).
  • TABLE 133
    Short-gap antisense oligonucleotides
    targeting Target-Y
    SEQ
    ISIS ID
    NO Sequence (5′ to 3′) Motif NO.
    464917 NkNkNkNNNNNNNNNNNkNkNk 3-10-3 6
    (kkk-d10-kkk)
    465977 NkNkNkNNNNNNNNNNNeNeNe 3-10-3 6
    (kkk-d10-eee)
    573331 NeNkNkNNNNNNNNNNNkNkNe 3-10-3 6
    (ekk-d10-kke)
    573332 NeNeNkNkNNNNNNNNNNkNkNe 4-9-3 6
    (eekk-d9-kke)
    573333 NeNeNeNkNkNNNNNNNNNkNkNe 5-8-3 6
    (eeekk-d8-kke)
    573334 NeNeNeNeNkNkNNNNNNNNkNkNe 6-7-3 6
    (eeeekk-d7-kke)
    573335 NeNkNkNNNNNNNNNNkNkNeNe 3-9-4 6
    (ekk-d9-kkee)
    573336 NeNkNkNNNNNNNNNkNkNeNeNe 3-8-5 6
    (ekk-d8-kkeee)
    573361 NeNkNkNNNNNNNNkNkNeNeNeNe 3-7-6 6
    (ekk-d7-kkeeee)
    573338 NeNeNkNkNNNNNNNNNkNkNeNe 4-8-4 6
    (eekk-d8-kkee)
    573339 NeNeNeNkNkNNNNNNNNkNkNeNe 5-7-4 6
    (eeekk-d7-kkee)
    573340 NeNeNkNkNNNNNNNNkNkNeNeNe 4-7-5 6
    (eekk-d7-kkeee)
    573779 NkNkNkNNNNNNNNNkNeNeNeNe 3-8-5 6
    (kkk-d8-keeee)
    573780 NkNkNkNNNNNNNNNkNeNeNeNe 3-8-5 6
    (kkk-d8-keeee)
    573806 NkNkNkNNNNNNNNNkNeNeNeNe 3-8-5 6
    (kkk-d8-keeee)
    573782 NkNkNkNNNNNNNNNkNeNeNeNe 3-8-5 6
    (kkk-d8-keeee)
    573783 NkNkNkNNNNNNNNNkNeNeNeNe 3-8-5 6
    (kkk-d8-keeee)
    573784 NkNkNkNNNNNNNNNkNeNeNeNe 3-8-5 6
    (kkk-d8-keeee)
    573785 NkNkNkNNNNNNNNNkNeNeNeNe 3-8-5 6
    (kkk-d8-keeee)
    573786 NkNkNkNNNNNNNNNkNeNeNeNe 3-8-5 6
    (kkk-d8-keeee)
    573787 NkNkNkNNNNNNNNNkNeNeNeNe 3-8-5 6
    (kkk-d8-keeee)
    465178 NkNkNkNNNNNNNNNNNkNkNk 3-10-3 6
    (kkk-d10-kkk)
    466140 NkNkNkNNNNNNNNNNNeNeNe 3-10-3 6
    (kkk-d10-eee)
    573341 NeNkNkNNNNNNNNNNNkNkNe 3-10-3 6
    (ekk-d10-kke)
    573342 NeNeNkNkNNNNNNNNNNkNkNe 4-9-3 6
    (eekk-d9-kke)
    573343 NeNeNeNkNkNNNNNNNNNkNkNe 5-8-3 6
    (eeekk-d8-kke)
    573344 NeNeNeNeNkNkNNNNNNNNkNkNe 6-7-3 6
    (eeeekk-d7-kke)
    573345 NeNkNkNNNNNNNNNNkNkNeNe 3-9-4 6
    (ekk-d9-kkee)
    573346 NeNkNkNNNNNNNNNkNkNeNeNe 3-8-5 6
    (ekk-d8-kkeee)
    573347 NeNkNkNNNNNNNNkNkNeNeNeNe 3-7-6 6
    (ekk-d7-kkeeee)
    573348 NeNeNkNkNNNNNNNNNkNkNeNe 4-8-4 6
    (eekk-d8-kkee)
    573349 NeNeNeNkNkNNNNNNNNkNkNeNe 5-7-4 6
    (eeekk-d7-kkee)
    573350 NeNeNkNkNNNNNNNNkNkNeNeNe 4-7-5 6
    (eekk-d7-kkeee)
    573788 NkNkNkNNNNNNNNNkNeNeNeNe 3-8-5 6
    (kkk-d8-keeee)
    573789 NkNkNkNNNNNNNNNkNeNeNeNe 3-8-5 6
    (kkk-d8-keeee)
    573790 NkNkNkNNNNNNNNNkNeNeNeNe 3-8-5 6
    (kkk-d8-keeee)
    573791 NkNkNkNNNNNNNNNkNeNeNeNe 3-8-5 6
    (kkk-d8-keeee)
    573792 NkNkNkNNNNNNNNNkNeNeNeNe 3-8-5 6
    (kkk-d8-keeee)
    573793 NkNkNkNNNNNNNNNkNeNeNeNe 3-8-5 6
    (kkk-d8-keeee)
    573794 NkNkNkNNNNNNNNNkNeNeNeNe 3-8-5 6
    (kkk-d8-keeee)
    573795 NkNkNkNNNNNNNNNkNeNeNeNe 3-8-5 6
    (kkk-d8-keeee)
    573796 NkNkNkNNNNNNNNNkNeNeNeNe 3-8-5 6
    (kkk-d8-keeee)
    141923 CeCeTeTeCeCCTGAAGGTTCeCeTeCeCe 5-10-5 9
    (neg control) (e5-d10-e5)
    e = 2′-MOE (e.g. e5 = eeeee), k = cEt, d = 2′-deoxyribonucleoside
    • Example 86 Short-Gap Chimeric Oligonucleotides Targeting Target-Y—In Vitro Study
  • Several short-gap chimeric oligonucleotides from Table 133 were selected and evaluated for their effects on Target-Y mRNA in vitro. The parent gapmer, ISIS 464917 and 465178 were included in the study for comparison. ISIS 141923 was used as a negative control.
  • The newly designed gapmers were tested in vitro. Primary mouse hepatocytes at a density of 35,000 cells per well were transfected using electroporation with 0.0625, 0.25, 1, 4 and 16 μM concentrations of chimeric oligonucleotides. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Target-Y mRNA levels were measured by quantitative real-time PCR. Primer probe set RTSXXXX was used to measure mRNA levels. Target-Y mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN.
  • The half maximal inhibitory concentration (IC50) of each oligonucleotide is presented in Table 134 and was calculated by plotting the concentrations of oligonucleotides used versus the percent inhibition of Target-Y mRNA expression achieved at each concentration, and noting the concentration of oligonucleotide at which 50% inhibition of Target-Y mRNA expression was achieved compared to the control. As illustrated in Table 134 and 135, several short-gap oligonucleotides showed comparable inhibition of Target-Y mRNA levels as compared to the parent gapmers, ISIS 464917 or 465178.
  • TABLE 134
    Comparison of inhibition of Target-Y mRNA levels
    of short-gap oligonucleotides with ISIS 464917
    IC50 SEQ ID
    ISIS NO Motif (μM) NO.
    464917 3-10-3 0.5 6
    (kkk-d10-kkk)
    573331 3-10-3 0.5 6
    (ekk-d10-kke)
    573332 4-9-3 0.6 6
    (eekk-d9-kke)
    573333 5-8-3 0.5 6
    (eeekk-d8-kke)
    573335 3-9-4 0.4 6
    (ekk-d9-kkee)
    573336 3-8-5 0.5 6
    (ekk-d8-kkeee)
    573361 3-7-6 0.6 6
    (ekk-d7-kkeeee)
    573340 4-7-5 2.3 6
    (eekk-d7-kkeee)
    141923 5-10-5 >16 9
    (neg control) (e5-d10-e5)
    e = 2′-MOE (e.g. e5 = eeeee), k = cEt, d = 2′-deoxyribonucleoside
  • TABLE 135
    Comparison of inhibition of Target-Y mRNA levels
    of short-gap oligonucleotides with ISIS 465178
    IC50 SEQ
    ISIS NO Motif (μM) ID NO.
    465178 3-10-3 0.2 6
    (kkk-d10-kkk)
    573341 3-10-3 0.2 6
    (ekk-d10-kke)
    573342 4-9-3 0.4 6
    (eekk-d9-kke)
    573345 3-9-4 0.2 6
    (ekk-d9-kkee)
    573346 3-8-5 0.4 6
    573348 (ekk-d8-kkeee) 0.5 6
    573350 4-8-4 0.9 6
    (eekk-d8-kkee)
    573806 4-7-5 0.8 6
    (eekk-d7-kkeee)
    573783 3-8-5 1.0 6
    (kkk-d8-keeee)
    573784 3-8-5 1.3 6
    (kkk-d8-keeee)
    573785 3-8-5 1.0 6
    (kkk-8-keeee)
    573792 3-8-5 0.5 6
    (kkk-8-keeee)
    573794 3-8-5 0.4 6
    (kkk-d8-keeee)
    573795 3-8-5 0.5 6
    (kkk-d8-keeee)
    573796 3-8-5 0.8 6
    (kkk-d8-keeee)
    141923 5-10-5 >16 6
    (neg control) (e5-d10-e5)
    e = 2′-MOE (e.g. e5 = eeeee), k = cEt, d = 2′-deoxyribonucleoside
    • Example 87 Short-Gap Chimeric Oligonucleotides Targeting Target-Y—In Vivo Study
  • Several short-gap oligonucleotides described in Example 85 were selected and evaluated for efficacy in vivo and for changes in the levels of various plasma chemistry markers targeting Target-Y. The parent gapmer, ISIS 464917 was included in the study for comparison.
    • Treatment
  • Six week male BALB/C mice (purchased from Charles River) were injected subcutaneously with a single dose of antisense oligonucleotide at 10 mg/kg or 20 mg/kg or with saline control. Each treatment group consisted of 4 animals. The mice were sacrificed 96 hrs following last administration, and organs and plasma were harvested for further analysis.
  • mRNA Analysis
  • Liver tissues were homogenized and mRNA levels were quantitated using real-time PCR and normalized to Cyclophilin A as described herein. The results below are listed as Target-Y mRNA expression for each treatment group relative to saline-injected control (% UTC). As illustrated in Table 136, Target-Y mRNA expression levels were reduced in a dose-dependent manner with the newly designed oligonucleotides.
    • Plasma Chemistry Markers
  • Plasma chemistry markers such as liver transaminase levels, alanine aminotranferase (ALT) in serum were measured relative to saline treated mice and the results are presented in Table 136. Treatment with the newly designed oligonucleotides resulted in reduction in ALT levels compared to treatment with the parent gapmer, ISIS 464917. The results suggest that shortening the central gap region and introducing 2′-MOE modified nucleoside(s) at the wing(s) can be useful for the reduction of hepatoxicity profile of ISIS 464917.
    • Body and Organ Weights
  • Body weights, as well as liver, kidney and spleen weights were also measured at the end of the study. The results showed that treatment with the newly designed oligonucleotides did not cause any changes in body and organ weights outside the expected range as compared to ISIS 464917 (data not shown).
  • TABLE 136
    Effect of short-gap antisense oligonucleotide treatment
    on Target-Y reduction and liver function in BALB/C mice
    Dosage % ALT SEQ
    ISIS NO (mg/kg/wk) UTC (IU/L) Motif ID NO.
    Saline 0 99 23 —
    464917 10 11.5 1834 3-10-3 6
    20 5.1 8670 (kkk-d10-kkk)
    573333 10 32.8 79 5-8-3 6
    20 21.2 370 (eeekk-d8-kke)
    573334 10 79.5 26 6-7-3 6
    20 69.4 29 (eeeekk-d7-kke)
    573336 10 23.2 179 3-8-5 6
    20 12.0 322 (ekk-d8-kkeee)
    573339 10 47.9 35 5-7-4 6
    20 32.8 199 (eeekk-d7-kkee)
    573340 10 81.3 63 4-7-5 6
    20 66.2 33 (eekk-d7-kkeee)
    573361 10 33.6 150 3-7-6 6
    20 19.2 722 (ekk-d7-kkeeee)
    573783 10 16.5 734 3-8-5 6
    20 6.3 1774 (kkk-d8-keeee)
    573785 10 20.2 61 3-8-5 6
    20 14.2 40 (kkk-d8-keeee)
    573806 10 19.3 346 3-8-5 6
    20 15.4 1389 (kkk-d8-keeee)
    e = 2′-MOE, k = cEt, d = 2′-deoxyribonucleoside
    • Example 88 Short-Gap Chimeric Oligonucleotides Targeting PTEN
  • A series of chimeric antisense oligonucleotides was designed based on ISIS 482050, wherein the central gap region contains ten 2′-deoxyribonucleosides. These gapmers were designed by introducing 2′-MOE modified nucleoside(s) at the wing(s) and/or shortening the central gap region to nine, or eight 2′-deoxyribonucleosides.
  • The gapmers and their motifs are described in Table 137. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine nucleobases throughout each gapmer are 5-methyl cytosines. Nucleosides without a subscript are β-D-2′-deoxyribonucleosides. Nucleosides followed by a subscript “e” or “k” are sugar modified nucleosides. A subscript “e” indicates a 2′-O-methoxyethyl (MOE) modified nucleoside and a subscript “k” indicates a 6′-(S)—CH3 bicyclic nucleoside (e.g. cEt).
  • TABLE 137
    Short-gap antisense oligonucleotides 
    targeting PTEN
    SEQ
    ISIS ID
    NO. Sequence (5′ to 3′) Motif NO.
    482050 AkTkCkATGGCTGCAGCkTkTk 3-10-3 85
    (kkk-d10-kkk)
    508033 AkTkCkATGGCTGCAGCeTeTe 3-10-3 85
    (kkk-d10-eee)
    573351 AeTkCkATGGCTGCAGCkTkTe 3-10-3 85
    (ekk-d10-kke)
    573352 AeTeCkAkTGGCTGCAGCkTkTe 4-9-3 85
    (eekk-d9-kke)
    573353 AeTeCeAkTkGGCTGCAGCkTkTe 5-8-3 85
    (eeekk-d8-kke)
    573354 AeTeCeAeTkGkGCTGCAGCkTkTe 6-7-3 85
    (eeeekk-d7-kke)
    573355 AeTkCkATGGCTGCAGkCkTeTe 3-9-4 85
    (ekk-d9-kkee)
    573356 AeTkCkATGGCTGCAkGkCeTeTe 3-8-5 85
    (ekk-d8-kkeee)
    573357 AkTkCkATGGCTGCkAkGeCeTeTe 3-7-6 85
    (ekk-d7-kkeeee)
    573358 AeTeCkAkTGGCTGCAGkCkTeTe 4-8-4 85
    (eekk-d8-kkee)
    573359 AeTeCeAkTkGGCTGCAGkCkTeTe 5-7-4 85
    (eeekk-d7-kkee)
    573360 AeTeCkAkTGGCTGCAkGkCeTeTe 4-7-5 85
    (eekk-d7-kkeee)
    573797 TkGkGkCTGCAGCTTkCeCeGeAe 3-8-5 87
    (kkk-d8-keeee)
    573798 AkTkGkGCTGCAGCTkTeCeCeGe 3-8-5 88
    (kkk-d8-keeee)
    573799 CkAkTkGGCTGCAGCkTeTeCeCe 3-8-5 89
    (kkk-d8-keeee)
    573800 TkCkAkTGGCTGCAGkCeTeTeCe 3-8-5 90
    (kkk-d8-keeee)
    573801 AkTkCkATGGCTGCAkGeCeTeTe 3-8-5 85
    (kkk-d8-keeee)
    573802 CkAkTkCATGGCTGCkAeGeCeTe 3-8-5 91
    (kkk-d8-keeee)
    573803 CkCkAkTCATGGCTGkCeAeGeCe 3-8-5 92
    (kkk-d8-keeee)
    573804 TkCkCkATCATGGCTkGeCeAeGe 3-8-5 93
    (kkk-d8-keeee)
    573805 TkTkCkCATCATGGCkTeGeCeAe 3-8-5 94
    (kkk-d8-keeee)
    e = 2′-MOE, k = cEt, d = 2′-deoxyribonucleoside
    • Example 89 Short-Gap Chimeric Oligonucleotides Targeting PTEN—In Vitro Study
  • Several short-gap chimeric oligonucleotides from Table 137 were selected and evaluated for their effects on PTEN mRNA in vitro. The parent gapmer, ISIS 482050 were included in the study for comparison. ISIS 141923 was used as a negative control.
  • The newly designed gapmers were tested in vitro. Primary mouse hepatocytes at a density of 35,000 cells per well were transfected using electroporation with 0.0625, 0.25, 1, 4 and 16 μM concentrations of chimeric oligonucleotides. After a treatment period of approximately 24 hours, RNA was isolated from the cells and PTEN mRNA levels were measured by quantitative real-time PCR. Primer probe set RTS186 was used to measure mRNA levels. PTEN mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN.
  • The half maximal inhibitory concentration (IC50) of each oligonucleotide was calculated in the same manner as described previously and the results are presented in Table 138. As illustrated, most short-gap oligonucleotides showed comparable inhibition of PTEN mRNA levels as compared to ISIS 482050.
  • TABLE 138
    Comparison of inhibition of PTEN mRNA levels of
    short-gap oligonucleotides with ISIS 482050
    IC50 SEQ
    ISIS NO Motif (μM) ID NO.
    482050 3-10-3 1.9 85
    (kkk-d10-kkk)
    573351 3-10-3 2.8 85
    573353 (ekk-d10-kke) 6.1 85
    573355 3-9-4 2.6 85
    (ekk-d9-kkee)
    573798 3-8-5 1.6 88
    (kkk-d8-keeee)
    573799 3-8-5 1.9 89
    (kkk-d8-keeee)
    573803 3-8-5 1.4 92
    (kkk-d8-keeee)
    141923 5-10-5 >16 9
    (neg control) (e5-d10-e5)
    e = 2′-MOE (e.g. e5 = eeeee), k = cEt, d = 2′-deoxyribonucleoside

Claims (21)

1.-272. (canceled)
273. A oligomeric compound comprising a modified oligonucleotide consisting of 10 to 30 linked nucleosides, wherein the modified oligonucleotide has a modification motif comprising:
a 5′-region consisting of 2-8 linked 5′-region nucleosides, each independently selected from among a modified nucleoside and an unmodified deoxynucleoside, provided that at least one 5′-region nucleoside is a modified nucleoside and wherein the 3′-most 5′-region nucleoside is a modified nucleoside;
a 3′-region consisting of 2-8 linked 3′-region nucleosides, each independently selected from among a modified nucleoside and an unmodified deoxynucleoside, provided that at least one 3′-region nucleoside is a modified nucleoside and wherein the 5′-most 3′-region nucleoside is a modified nucleoside; and
a central region between the 5′-region and the 3′-region consisting of 6-12 linked central region nucleosides, each independently selected from among: a modified nucleoside and an unmodified deoxynucleoside, wherein the 5′-most central region nucleoside is an unmodified deoxynucleoside and the 3′-most central region nucleoside is an unmodified deoxynucleoside;
wherein the modified oligonucleotide has a nucleobase sequence complementary to the nucleobase sequence of a target region of a target nucleic acid.
274. The oligomeric compound of claim 273, wherein the 5′-region has a motif selected from among: AB, ABB, AAA, BBB, BBBAA, AAB, BAA, BBAA, AABB, AAAB, ABBW, ABBWW, ABBB, ABBBB, ABAB, ABABAB, ABABBB, ABABAA, AAABB, AAAABB, AABB, AAAAB, AABBB, ABBBB, BBBBB, AAABW, AAAAA, and BBBBAA;
wherein the 3′-region has a motif selected from among: BBA, AAB, AAA, BBB, BBAA, AABB, WBBA, WAAB, BBBA, BBBBA, BBBB, BBBBBA, ABBBBB, BBAAA, AABBB, BBBAA, BBBBA, BBBBB, BABA, AAAAA, BBAAAA, AABBBB, BAAAA, and ABBBB;
wherein the central region has a nucleoside motif selected from among: DDDDDD, DDDDDDD, DDDDDDDD, DDDDDDDDD, DDDDDDDDDD, DDDDDDDDD, DXDDDDDDD, DDXDDDDDD, DDDXDDDDD, DDDDXDDDD, DDDDDXDDD, DDDDDDXDD, DDDDDDDXD, DXXDDDDDD, DDDDDDXXD, DDXXDDDDD, DDDXXDDDD, DDDDXXDDD, DDDDDXXDD, DXDDDDDXD, DXDDDDXDD, DXDDDXDDD, DXDDXDDDD, DXDXDDDDD, DDXDDDDXD, DDXDDDXDD, DDXDDXDDD, DDXDXDDDD, DDDXDDDXD, DDDXDDXDD, DDDXDXDDD, DDDDXDDXD, DDDDXDXDD, and DDDDDXDXD, DDDDDDDD, DXDDDDDD, DDXDDDDD, DDDXDDDD, DDDDXDDD, DDDDDXDD, DDDDDDXD, DXDDDDXD, DXDDDXDD, DXDDXDDD, DXDXDDDD, DXXDDDDD, DDXXDDDD, DDXDXDDD, DDXDDXDD, DXDDDDXD, DDDXXDDD, DDDXDXDD, DDDXDDXD, DDDDXXDD, DDDDXDXD, and DDDDDXXD, DXDDDDD, DDXDDDD, DDDXDDD, DDDDXDD, DDDDDXD, DXDDDXD, DXDDXDD, DXDXDDD, DXXDDDD, DDXXDDD, DDXDXDD, DDXDDXD, DDDXXDD, DDDXDXD, and DDDDXXD, DXDDDD, DDXDDD, DDDXDD, DDDDXD, DXXDDD, DXDXDD, DXDDXD, DDXXDD, DDXDXD, and DDDXXD; and
wherein each A is a modified nucleoside of a first type, each B is a modified nucleoside of a second type, each W is a modified nucleoside of a first type, a second type, or a third type, each D is an unmodified deoxynucleoside, and each X is a modified nucleoside or a modified nucleobase.
275. The oligomeric compound of claim 274, wherein the 5′-region has a motif selected from among:
AB, ABB, AAA, BBB, BBBAA, AAB, BAA, BBAA, AABB, ABBW, ABBWW, ABBB, ABBBB, ABAB, ABABAB, ABABBB, ABABAA, AAABB, AAAABB, AABB, AAAAB, AABBB, ABBBB, BBBBB, AAABW, and BBBBAA; and wherein the 3′-region has a BBA motif.
276. The oligomeric compound of claim 274, wherein each A nucleoside comprises a bicyclic sugar moiety selected from among: cEt, cMOE, LNA, α-LNA, ENA and 2′-thio LNA.
277. The oligomeric compound of claim 274, wherein each A nucleoside comprises a cEt.
278. The oligomeric compound of claim 276, wherein each B nucleoside comprises a 2′-substituted sugar moiety comprising a 2′-substituent selected from among: a halogen, OCH3, OCF3, OCH2CH3, OCH2CF3, OCH2—CH═CH2, O(CH2)2—OCH3, O(CH2)2—O(CH2)2—N(CH3)2, OCH2C(═O)—N(H)CH3, OCH2C(═O)—N(H)—(CH2)2—N(CH3)2, and OCH2—N(H)—C(═NH)NH2.
279. The oligomeric compound of claim 278, wherein each B nucleoside comprises a 2′-substituted sugar moiety comprising a 2′-substituent selected from among: F, OCH3, O(CH2)2—OCH3.
280. The oligomeric compound of claim 274, wherein one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and W comprises a 2-thio-thymidine nucleobase.
281. The oligomeric compound of claim 274, wherein one of A or B comprises a bicyclic sugar moiety, another of A or B comprises a 2′-MOE sugar moiety, and W comprises FHNA.
282. The oligomeric compound of claim 274, wherein one of A or B comprises cEt, another of A or B comprises a 2′-modified sugar moiety, and W comprises a 2-thio-thymidine nucleobase.
283. The oligomeric compound of claim 274, wherein one of A or B comprises cEt, another of A or B comprises a 2′-modified sugar moiety, and W comprises FHNA.
284. The oligomeric compound of claim 274, wherein each A comprises MOE, each B comprises cEt, and each W is selected from among cEt or FHNA.
285. The oligomeric compound of claim 284, wherein each W comprises cEt.
286. The oligomeric compound of claim 284, wherein each W comprises 2-thio-thymidine.
287. The oligomeric compound of claim 284, wherein each W comprises FHNA.
288. The oligomeric compound of claim 274 comprising at least one modified internucleoside linkage.
289. The oligomeric compound of claim 288, wherein each internucleoside linkage is a modified internucleoside linkage.
290. The oligomeric compound of claim 289 comprising at least one phosphorothioate internucleoside linkage.
291. The oligomeric compound of claim 288 comprising at least one methylphosphonate internucleoside linkage.
292. The oligomeric compound of claim 275, wherein each A nucleoside comprises a bicyclic sugar moiety selected from among: cEt and LNA and each B nucleoside comprises a 2′-MOE.
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