WO1994023028A2 - Modified oligonucleotides having improved anti-influenza activity - Google Patents
Modified oligonucleotides having improved anti-influenza activity Download PDFInfo
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- WO1994023028A2 WO1994023028A2 PCT/US1994/003454 US9403454W WO9423028A2 WO 1994023028 A2 WO1994023028 A2 WO 1994023028A2 US 9403454 W US9403454 W US 9403454W WO 9423028 A2 WO9423028 A2 WO 9423028A2
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- C—CHEMISTRY; METALLURGY
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
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- C—CHEMISTRY; METALLURGY
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- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
Definitions
- the invention relates to anti-sense oligonucleotides. More particularly, the invention relates to modified oligonucleotides that are capable of inhibiting replication or propagation of influenza virus.
- Influenza A virus is a membrane-enclosed virus whose genome is a segmented minus strand of RNA.
- the ten influenza virus genes are present on eight segments of the single- stranded RNA of strains A and B, and on seven segments of strain C. The segments are of varying sizes (ranging from 890 to 2341 nucleotides in length) and each is a template for synthesis ' for a different mRNA.
- the influenza virus virion contains virus-specific RNA polymerases necessary for mRNA synthesis from these templates and, in the absence of such specific polymerases, the minus strand of influenza virus RNA is not infectious.
- Initiation of transcription of the mRNAs occurs when the influenza virus mRNA polymerase takes 12 to 15 nucleotides from the 5' end of a cellular mRNA or mRNA precursor and uses the borrowed oligonucleotide as a primer.
- the mRNAs made through this process encode only one protein.
- the M RNA and the NS RNA also encode spliced mRNAs, which results in production of two different proteins for each of these two segments.
- Influenza viruses infect humans and animals (e.g., pigs, birds, horses) and may cause acute respiratory disease. There have been numerous attempts to produce vaccines effective against influenza virus. None, however, have been completely successful, particularly on a long-term basis.
- anti-sense oligonucleotides that are capable of inhibiting the replication or propagation of influenza virus.
- anti-sense oligonucleotides hold the promise of providing broader protection against different strains of influenza, because they can be designed to hybridize to conserved regions of the influenza genome that are present in multiple strains of influenza virus.
- Agrawal et al. U.S. Patent No. 5,194,428 the teachings of which are hereby incorporated by reference, discloses oligonucleotides having certain modified internucleotide linkages that are capable of inhibiting influenza replication by hybridizing to the influenza PB1 polymerase gene.
- Cowsert et al., 092/03454 (1992) discloses antisense oligonucleotides that inhibit influenza virus propagation by hybridizing to the influenza polymerase 1, 2 or 3, or the hemagglutinin, nucleoprotein, neuraminidase, matrix protein, or nonstructural protein gene, or to various splice junction sites or packaging sequences.
- anti-sense oligonucleotides show promise as anti- influenza therapeutic agents. As with any such agents, however, there remains a need for improved agents that have even greater efficacy in inhibiting influenza virus replication or propagation. Such improved anti-sense oligonucleotides would be useful for studying which regions of the influenza virus genome are the best candidates for broad cross-strain inhibition as well as for use, and as anti- influenza therapeutic agents.
- the invention provides modified oligonucleotides having greater efficacy in inhibiting the replication or propagation of influenza virus than previously known oligonucleotides.
- modified oligonucleotides are characterized by having a nucleotide sequence sufficiently complementary to an essential nucleic acid of influenza virus origin to hybridize to such influenza virus nucleic acid in a cell.
- the essential nucleic acid is a portion of the PB1, 2 or 3 polymerase gene or to the hemagglutinin, nucleoprotein, neuraminidase, matrix protein, or nonstructural protein gene of influenza or to the various influenza splice junction sites or packaging sequences.
- modified oligonucleotides according to the invention have one or more type of modified internucleotide linkage.
- at least some of the modified internucleotide linkages are phosphorothioate or phosphorodithioate linkages.
- phosphorothioate internucleotide linkages are present in a modified oligonucleotide that also contains other modified, i.e. nonphosphodiester, linkages.
- these other modified internucleotide linkages are alkylphosphonate or alkylphosphonothioate linkages.
- these other modified internucleotide linkages are present at or near one or both ends of the oligonucleotide.
- phosphorothioate or phosphorodithioate internucleotide linkages are present in an oligonucleotide having one or more modified nucleoside.
- such modified nucleoside is a 2'-0 alkyl nucleoside.
- at least some modified nucleosides are present at or near one or both ends of the oligonucleotide.
- phosphorothioate or phosphorodithioate internucleotide linkages are present in an oligonucleotide having an exonuclease resistance-conferring cap structure at one or both ends.
- a cap structure is present at least at the 3 1 end of the molecule.
- Such cap structures may also be present at one or both ends of all embodiments of modified oligonucleotides according to the invention and preferably are present at least at the 3' end of the oligonucleotide.
- phosphorothioate or phosphorodithioate internucleotide linkages are present in an oligonucleotide that is self-stabilized by having a self- complementary region involving nucleotides at or near one or both ends, and preferably at least at or near the 3 1 end, whereby the oligonucleotide forms a hairpin-like structure.
- Each of these modified oligonucleotides according to the invention provides greater efficacy in inhibiting replication or propagation of influenza than any known oligonucleotide or modified oligonucleotide that hybridizes to the same gene.
- Each of these modified oligonucleotides according to the invention may optionally also contain other modifications to the sugars or bases of the oligonucleotide and may also optionally have additional internucleotide linkages other than phosphorothioate, phosphorodithioate, alkylphosphonate or alkylphosphonothioate linkages.
- Figure 1 shows the nucleotide sequence of an influenza virus PBl (polymerase 1) gene, against which complementary antisense oligonucleotides can be prepared.
- Figure 2 shows a self-stabilized anti-influenza modified oligonucleotide according to the invention.
- Figure 3 shows an alternative form of a self-stabilized anti-influenza modified oligonucleotide according to the invention.
- Figure 4 shows certain preferred cap structures that confer exonuclease resistance upon oligonucleotides.
- Figure 5 shows in vivo nucleolytic degradation patterns for 5'-capped, 3'-capped and uncapped oligonucleotides.
- Figure 6 shows DNA Polymerase I 3•-exonuclease degradation patterns for self-stabilized and non-self- stabilized oligodeoxynucleotide phosphodiesters.
- Figure 7 shows DNA Polymerase I 3'-exonuclease degradation patterns for self-stabilized and non-self- stabilized oligodeoxynucleotide phosphorothioates.
- the invention relates to anti-sense oligonucleotides having anti-influenza activity.
- Modified oligonuclestides having in vivo activity against influenza are referred to herein as anti-influenza modified oligonucleotides.
- the invention provides anti-influenza modified oligonucleotides that have greater efficacy in inhibiting replication or propagation of influenza virus than known oligonucleotides or modified oligonucleotides that hybridize to the same gene.
- Modified oligonucleotides according to the invention have specific preferred characteristics that are discussed in greater detail for each preferred embodiment below.
- modified oligonucleotides according to the invention may optionally have additional ribonucleotide, 2'-substituted ribonucleotide, and/or deoxyribonucleotide monomers, any of which are connected together via 5* to 3' linkages which may include any of the internucleotide linkages known in the art.
- such modified oligonucleotides may optionally contain phosphodiester, phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphorothioate and/or sulfone internucleotide linkages.
- modified oligonucleotides should contain from about 6 to about 100 monomers in total.
- modified oligonucleotides may also optionally contain modified nucleic acid bases and/or sugars, as well as added substituents, such as diamines, cholesteryl or other lipophilic groups.
- modified oligonucleotides according to the invention are illustrated in Table I, below. Although these embodiments all have a nucleotide sequence from the same region of the influenza PBl polymerase gene, those skilled in the art will recognize that the anti-influenza efficacy of oligonucleotides having nucleotide sequences complementary to other essential nucleic acid sequences of influenza virus can also be enhanced by incorporating into such oligonucleotides the structural features of preferred embodiments of modified oligonucleotides according to the invention.
- complementary means having a sequence that hybridizes to the essential nucleic acid sequence under physiological conditions.
- An essential nucleic acid sequence of influenza virus means a nucleic acid sequence that is required for replication or propagation of influenza virus.
- Such essential nucleic acid sequences can be from any known strain of influenza virus.
- oligonucleotides can have other sequences from the influenza PBl polymerase gene
- polymerase 1 gene [SEQ. ID. No. 1] (see Figure 1) should serve as the basis for modified oligonucleotides according to the invention.
- sequences from other influenza sequences or genes can be used (see Table II) .
- the structural features of preferred embodiments of modified oligonucleotides according to the invention should enhance the anti-influenza activity of any antisense oligonucleotide having a nucleotide sequence that hybridizes in a cell with any essential nucleic acid sequence of influenza virus.
- CMPD B CAGAGCAAAATCATCAGAAGA 2 [SEQ. ID NO.
- AAA ACT ACC TTG TTT CTA CT packaging [SEQ. ID. No.36] sequence segment 7
- anti-influenza modified oligonucleotides according to the invention are in the form of a mixed backbone chimeric oligonucleotide having one or more regions of nucleotides connected by phosphorothioate or phosphorodithioateinternucleotide linkages ("phosphorothioate or phosphorodithioate region") as well as one or more regions of nucleotides connected by alkylphosphonate internucleotide linkages (“alkylphosphonate region”) .
- at least one alkylphosphonate region preferably includes nucleotides at or near the 5' end and/or the 3' end of the oligonucleotide.
- the alkylphosphonate region comprises from about 2 to about 10 contiguous nucleotides connected by alkylphosphonate linkages.
- the phosphorothioate or phosphorodithioate region comprises at least 3, and up to about 100 contiguous nucleotides connected by phosphorothioate or phosphorodithioate linkages.
- Table I An example of an anti-influenza modified oligonucleotide according to this embodiment of the invention is shown in Table I as CMPD A.
- Anti-influenza modified oligonucleotides according to this embodiment of the invention are synthesized by solid phase methods, alternating H-phosphonate chemistry and sulfur oxidation for phosphorothioate regions, and alkylphosphonamidate chemistry for alkylphosphonate regions.
- a preferred H-phosphonate approach is taught by Agrawal et al., U.S. Patent No. 5,149,798, the teachings of which are hereby incorporated by reference.
- Alkylphosphonamidite chemistry is well known in the art, as illustrated by Agrawal and Goodchild, Tetrahedron Lett. Klein:3539-3542 (1987).
- anti-influenza modified oligonucleotides according to the invention are in the form of a mixed backbone chimeric oligonucleotide having one or more region of nucleotides connected by phosphorothioate or phosphorodithioate internucleotidelinkages ("phosphorothioate or phosphorodithioate region”) , as well as one or more region of nucleotides connected by alkylphosphonothioate or arylphosphonothioate internucleotide linkages (“alkylphosphonothioate region”) .
- At least one alkylphosphonothioate region preferably includes nucleotides at or near the 5' end and/or the 3 ' end of the oligonucleotide.
- the alkylphosphonothioate region comprises from about 2 to about 10 contiguous nucleotides connected by alkylphosphonothioate linkages.
- the phosphorothioate or phosphorodithioate region comprises at least 3, and up to about 100 contiguous nucleotides connected by phosphorothioate or phosphorodithioate linkages. Examples of anti-influenza modified oligonucleotides according to this embodiment of the invention are shown in Table I as CMPD B, CMPD E and CMPD F.
- Anti-influenza modified oligonucleotides according to this embodiment of the invention are synthesized by solid phase methods, alternating chemistries for each region to be synthesized.
- Phosphorothioate or phosphorodithioate regions are synthesized as described for the first embodiment.
- Alkylphosphonothioate regions are synthesized by coupling together two or more nucleosides via alkylphosphite linkages, then oxidatively thiolating the alkylphosphite linkages to produce alkylphosphonothioate linkages. This synthesis procedure is set forth in detail in Example 1.
- anti-influenza modified oligonucleotides according to the invention are in the form of a hybrid oligonucleotide having regions of deoxyribonucleotides ("deoxyribonucleotide regions") and regions of ribonucleotides or 2'-substituted ribonucleotides ("ribonucleotide regions”) .
- deoxyribonucleotide regions regions of deoxyribonucleotides
- ribonucleotide regions regions of ribonucleotides or 2'-substituted ribonucleotides
- from about one to about all of the internucleotide linkages are phosphorothioate or phosphorodithioate linkages.
- Preferred 2'-substituted ribonucleotides are halo, amino, alkyl, aryl or lower alkyl (1-6 carbon atoms) substituted ribonucleotides, especially 2'- OMe-ribonucleotides.
- at least some of the ribonucleotide regions include nucleotides present at or near the 5 1 end and/or the 3' end of the oligonucleotide.
- the ribonucleotide regions each comprise from about 2 and preferably from about 4 to about 100 contiguous ribonucleotides and/or 2•-substitute oligonucleotides.
- deoxyribonucleotide regions are optional, and when present may contain from about 1 to about 100 contiguous deoxyribo ⁇ nucleotides.
- Examples of anti-influenza modified oligonucleotides according to this embodiment of the invention are shown in Table I as CMPD C, CMPD D, CMPD G, CMPD H, CMPD K, CMPD M and CMPD N.
- Anti-influenza modified oligonucleotides according to this embodiment of the invention are typically synthesized by solid phase methods, preferably by the H-phosphonate approach, using deoxynucleotide H-phosphonates for deoxyribonucleotide regions, and ribonucleotide or 2'-substituted ribonucleotide H-phosphonates for ribonucleotide regions.
- anti-influenza modified oligonucleotides according to the invention are in the form of an oligonucleotide having at its 5' and/or 3' end a cap structure that confers exonuclease resistance to the oligonucleotide.
- modified oligonucleotides preferably also have from 1 to about all modified (non-phosphodiester) internucleotide linkage.
- Preferred cap structures include those shown in Figure 4, as well as lower alkyl (C1-C12) or alcohol groups.
- Preferred modified internucleotide linkages include phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphorothioate, sulfone, phosphorothioate and phosphorodithioate linkages.
- Anti-influenza modified oligonucleotides according to this embodiment of the invention are synthesized according to procedures well known in the art (see e.g.. Uhlmann and Peyman, Chemical Reviews 9_0:543-584 (1990); Schneider and Banner, Tetrahedron Lett. 3_1:335 (1990)).
- the cap structure is reversibly attached to the solid support and is then coupled to the first nucleotide monomer in the synthesis scheme.
- the cap structure is coupled to the end of the oligonucleotide after addition of the last nucleotide monomer in the synthesis scheme.
- anti-influenza modified oligonucleotides are self-stabilized by having a self- complementary region that hybridizes intra olecularly with the oligonucleotide to form an exonuclease resistant hairpin-like structure.
- Anti-influenza modified oligonucleotides according to this embodiment of the invention are generally characterized by having two regions: an influenza hybridizing region and a self-complementary region.
- the influenza hybridizing region has a nucleotide sequence that is complementary to an essential nucleic acid sequence of influenza virus. Preferably, this region has from about 6 to about 100 nucleotides.
- Figure 2 One form of this embodiment of the invention is shown in Figure 2.
- influenza hybridizing region is shown as connected rectangular squares, and the self-complementary region is shown as connected circles.
- the complementary nucleic acid sequence in a target influenza messenger RNA molecule is represented by connected diamonds. Hydrogen bonding between nucleotides is indicated by dots.
- the oligonucleotide is stabilized, i.e.. rendered resistant to exonucleolytic degradation by base-pairing between the target hybridizing region and the self- complementary region and/or by base-pairing between complementary sequences within the self-complementary region.
- a second form of an oligonucleotide according to this embodiment of the invention operates in a similar way as the first form, but forms a different structure upon self- complementary base-pairing.
- This alternative form forms a hammer-like structure as shown in Figure 3.
- the self-complementary region contains oligonucleotide sequences that can base pair with other oligonucleotide sequences within the self-complementary region.
- the self-complementary region may also contain oligonucleotide sequences that are complementary to the influenza hybridizing region.
- the second significant region of self-stabilized oligonucleotides according to the invention is the self- complementary region.
- the self-complementary region contains oligonucleotide sequences that are complementary to other oligonucleotide sequences within the oligonucleotide. These other oligonucleotide sequences may be within the influenza hybridizing region or within the self-complementary region, or they may span both regions.
- the complementary sequences form base pairs, resulting in the formation of a hairpin structure, as shown in Figure 2, or a hammer-like structure, as shown in Figure 3.
- Either the hairpin structure or the hammer-like structure can have loops resulting from non-base-paired nucleotides, as shown in Figure 2 for the hairpin structure, or can be devoid of such loops, as shown in Figure 3 for the hammer-like structure.
- the number of base-pairs to be formed by intra-molecular hybridization involving the self- complementary region may vary, but should be adequate to maintain a double-stranded structure so that the 3' end is not accessible to endonucleases. Generally, about 4 or more base- pairs will be necessary to maintain such a double-stranded structure.
- intramolecular base-pairs formed in the self-stabilized oligonucleotide, with the 10 base pairs being consecutive and involving the 3'-most nucleotides.
- the intra ⁇ molecular base-pairing can be so extensive as to involve every nucleotide of the oligonucleotide. Preferably, this will involve a self-complementary region of about 50 nucleotides or less.
- Oligonucleotides according to this embodiment may have from 1 to about all modified internucleotide linkages, as described for the fourth embodiment.
- at least either the influenza hybridizing region or the self- complementary region, and most preferably both, will contain from about 2 to about all nucleotides being coupled by phosphorothioate and/or phosphorodithioate linkages.
- CMPD O An example of an anti-influenza modified oligonucleotide according to this embodiment of the invention is shown in Table I as CMPD O.
- the invention contemplates anti-influenza modified oligonucleotides having every possible combination of chimeric features, hybrid features, cap structures and self-stabilizing character, all as described herein.
- Each of the preferred embodiments of the present invention has greater ability to inhibit influenza replication or propagation than anti-sense oligonucleotides of the prior art that hybridize to the same influenza mRNA.
- U.S. Patent No. 5,194,428, teaches modified oligonucleotides that inhibit influenza virus replication.
- modified oligonucleotides are oligonucleotide phosphorothioates that hybridize to the influenza PBl
- polymerase 1 polymerase 1 gene.
- an oligonucleotide phosphorothioate that hybridizes to the influenza PBl polymerase gene was tested for its ability to inhibit influenza virus replication in comparison with various preferred embodiments of the present invention.
- Each embodiment of the present invention tested demonstrated surprisingly improved efficacy in anti-influenza activity, relative to the oligonucleotide phosphorothioate that binds to the same site on the same gene.
- Table III below, anti-influenza modified oligonucleotides according to the invention gave a reduction in the 50 per cent inhibitory concentration (IC 50 ) ranging from 2 to nearly fifteen fold.
- oligonucleotides are useful for a variety of purposes. First, they can be used in studies to determine which influenza genes and sites within such genes provide the best ba ⁇ is for anti-sense oligonucleotides having broad effectiveness against multiple strains of influenza virus. Second, they can be used to determine what structural features or combination of structural features provide the greatest effectiveness against influenza in vitro and m vivo. Finally, such oligonucleotides are useful as therapeutic agents for treating influenza infections. For such treatment, oligonucleotides may be administered intraperitoneally, intranasally, orally or anally. Preferably, such oligonucleotides will be administered at a concentration of from about 1 to about 50 mg/kg body weight.
- Chimeric oligonucleotides having methylphosphonate and phosphorothioate regions were synthesized using methylphosphonamindites for methylphosphonate regions and nucleoside H-phosphonates for phosphorothioate regions.
- Methylphosphonamidite synthesis was as follows:
- Methylchloro-N, N-diisopropylaminosphophine was prepared by reaction of methyldichlorophosphine (51 mmol) with diisopropylamine (102 mmol) in ether at 15° under nitrogen. After removal of salt by filtration and evaporation of solvent, methylchloro- ⁇ N-diisopropylaminophosphine was obtained as an oil (48 mmol, 95% of theory) that was characterized by J H and 31 P NMR and was stable at -20° for at least eight weeks. The product was reacted with the usual protected nucleosides in dichloromethane containing N, N- diisopropylethylamine at room temperature for 10-20 min.
- Coupling efficiency was followed by the dmethoxytrityl assay and was found to be the same as for control syntheses run in parallel using phosphoramidites.
- a modified oligonucleotide having regions of four contiguous nucleotide methylphosphonothioates at either end and a region of 12 contiguous oligonucleotide phosphorothioates in the middle, the following procedure on 8 micromole scale was used.
- a first monomer was pre-bound to a control pore glass (CPG) solid support.
- a second monomer which was a deoxynucleotide methylphosphonamidite, was coupled to the first monomer using a standard amidite coupling cycle (see e.g.. Agrawal and Goodchild, Tetrahedron Lett. 28:3539- 3542 (1987)).
- the H- phosphonates were converted to phosphorothioates by oxidation with 0.2 M sulfur in carbon disulfide/pyridine/ triethylamine (9:9:1 vol/vol) .
- the resulting oligonucleotide was deprotected at room temperature for 30 minutes in 0.5 ml ethylene diamine, then kept at room temperature for 6 hours with occasional stirring. Finally, the mixture was filtered and evaporated in vacuo to obtain a solid mass and the mass was dissolved in water and desalted on Sep Pak C 18 .
- Oligonucleotides having 2-4 contiguous deoxynucleotide methylphosphonates at 3' ends and otherwise having all deoxynucleotide phosphodiesters were tested for their relative resistance to 3 • exonucleolytic degradation compared with an oligonucleotide phosphodiester. For each oligonucleotide, 0.4
- a jj ⁇ Q units of oligonucleotide was lyophilized, dissolved in 0.5 ml buffer (10 mM Tris, 10 mM MgCl 2 , pH 8.5) and mixed with 5 ⁇ l (1.5 milliunits) of snake venom phosphodiesterase. The mixture was incubated at 37°C in a thermally regulated cell and A jg o was plotted against time. Increase in hyperchromicity was used as the indicator for oligonucleotide degradation. The results are shown in Table IV, below.
- Hybrid oligonucleotide phosphorothioates were synthesized on CPG on a 5-6 ⁇ mole scale on an automated synthesizer (model
- Deoxyribonucleoside H-phosphonates were obtained from Millipore. 2'-OMe ribonucleotide H-phosphonates were synthesized by standard procedures. Segments of oligonucleotides containing 2'-OMe nucleoside were assembled by using 2'-OMe ribonucleoside H-phosphonates for the desired cycles. Similarly, segments of oligonucleotides containing deoxyribonucleosides were assembled by using deoxynucleoside H-phosphonates for the desired cycles. After assembly, CPG bound oligonucleotide H-phosphonate was oxidized with sulfur as described in Example 2 to generate the phosphorothioate linkage. Oligonucleotides were then deprotected in concentrated NH 4 0H at 40°C for 48 hours.
- oligonucleotides were treated with snake venom phosphodiesterase (SVPD) .
- SVPD snake venom phosphodiesterase
- oligonucleotide having all 2•-OMe-ribonucleotides was very resistant to SVPD, whereas the oligonucleotide having no 2'- OMe-ribonucleotides was digested almost to completion and the oligonucleotide having 5' and 3' terminal 2'-0Me-RNA regions was digested to 50%.
- An oligonucleotide phosphodiester was digested to about 80% in one minute using one tenth of the concentration of SVPD.
- Oligonucleoside phosphorothioates were synthesized on a
- 5'-capped oligonucleoside phosphorothioates were prepared by carrying out the last coupling, after the assembly of the required sequence, with N-Fmoc-0'-DMTr-3-amino-l,2- propanediol-H-phosphonate. The 5'-capped oligonucleoside H- phosphonate was then oxidized with sulfur. 3'-capped oligonucleoside phosphorothioates were assembled on N-Fmoc-0'- DMTr-3-amino-l,2-propanediol-CPG, followed by sulfur oxidation. Combination of these procedures was used to produce 3',5'-capped oligonucleoside phosphorothioates.
- oligonucleoside phosphorothioates having other 3' or 5' cap structures are prepared by substituting the phosphonate or CPG-derivatized cap structures forthe N-Fmoc-0'-DMTr-3-amino-l,2-propanediol- H phosphonate or CPG in the capping procedure.
- capped modified oligonucleotides other than oligonucleotide phosphorothioates are prepared in an analogous manner by appending the capping procedure to the appropriate synthetic procedure.
- mice Male CDC2F1 mice (average weight 20 grams) were treated by intravenous or intraperitoneal injection with a 30 mg/kg dose of radiolabelled oligonucleotides dissolved in 200 microlitres physiological saline. Each capped or uncapped oligonucleotide was administered to three mice. Urine was collected separately from each animal up to 24 hours post- dosing, then extracted with proteinase K. (2 mg/ml, final concentration) in 0.5% SDS, 10 mM 20 M Tris Cl (pH 7.6), 10 mM EDTA for one hour at 37°C, followed by phenol-chloroform extraction and ethanol precipitation.
- oligonucleoside phosphorothioates were then analyzed by PAGE (20% polyacrylamide/7 M urea) followed by autoradiography. Radioactivity was also measure from cage rinse to account for urine spill. Twenty-four hours after dosing, about 30% of oligonucleoside phosphorothioates were excreted, whether capped or uncapped. Excreted uncapped and 5'-capped oligonucleoside phosphorothioates were extensively degraded, as shown in Figure 5. Excreted 3•-capped and 3• ,5'-capped oligonucleoside phosphorothioates, in contrast, demonstrated virtually no degradation.
- control oligonucleotide used for this study was an oligodeoxynucleotide phosphodiester without a self- complementary region.
- the test compound was identical, except that it had a 3' self complementary region of 10 nucleotides.
- another control oligodeoxynucleotide phosphodiester was used that was identical to the first control oligonucleotide except for having at its 3' end 10 mismatched nucleotides (T 10 ) .
- oligonucleotides were tested for their relative resistance to 3' exonucleolytic degradation.
- oligonucleotide 0.4 A ⁇ units of oligonucleotide was lyophilized, dissolved in 0.5 ml buffer (10 mM Tris, 10 mM MgCl 2 , pH 8.5) and mixed with 5 ⁇ l (1.5 milliunits) of snake venom phosphodiesterase (SVPD) . The mixture was incubated at 37° C in a thermally regulated cell and A ⁇ was plotted against time. Oligonucleotide degradation was measured as function of increase in hyperchromicity.
- SVPD snake venom phosphodiesterase
- the oligonucleotides were also subjected to DNA Polymerase I 3'- exonuclease digestion. As shown in Figure 6 the non-self- stabilized oligonucleotides were digested to completion in 30 minutes, whereas the self-stabilized oligonucleotide with a 10 nucleotide self-complementary region was only partly digested over 30 minutes. TABLE V SVPD Half-Life Of Oligonucleotides
- Example 8 for oligonucleotide phosphodiesters were digested to almost 50% within 4 hours.
- the oligonucleotide phosphorothioate having a 10 nucleotide self-complementary region was undegraded after 4 hours.
- Oligonucleotide phosphorothioates having 6 or 4 nucleotide self-complementary regions were also found to be stable.
- MDCK canine kidney cells were seeded in Minimum Essential Medium (MEM) with non-essential amino acids (GIBCO BRL, Grand Island, New York) with 5% fetal bovine serum (Hyclone Laboratories, Logen, Utah), 0.1% NaHC0 3 , at a concentration of 4 x 10 ⁇ cells/ml in 96 well tissue culture plates (Corning, Corning, New York) at 0.2 ml/well. The cells were incubated overnight to establish monolayers of cells. Growth medium was then removed and 0.1 ml of oligonucleotide at pre-selected concentrations in serum-free MEM containing 0.18% NaHC0 3 and 50 ⁇ g/ml gentamycin.
- MEM Minimum Essential Medium
- GEBCO BRL Grand Island, New York
- fetal bovine serum Hyclone Laboratories, Logen, Utah
- NaHC0 3 fetal bovine serum
- Virus CPE was graded on a scale of 0-4, with 4 being 95-100% CPE.
- the effective dose, 50% endpoint (ED 60 ) was calculated by regression analysis of the mean CPE grade at each concentration of the compound where activity was seen to bracket the CPE grade that was 50% of that seen in the virus controls.
- Control is oligodeoxynucleotide phosphorothioate having the sequence 5» CAGAGCAAAATCATCAGAAGA 3 ' .
- influenza strains were the HlNl strains A/NWS/33 and A/PR/8/34, the H3N2 strains A/Washington/897/80, A/Victoria/3/75 andA/Port Chalmers/1/73, and the H2N2 strain A2/Japan/305/57.
- the results were calculated as described in Example 10, and are shown in Table VII below.
- compound M showed great efficacy in inhibiting three of the six strains of influenza tested and had some efficacy against a fourth strain.
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- ANTI-SENSE YES
- SEQUENCE DESCRIPTION SEQ ID NO:2:
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- ANTI-SENSE YES
- SEQUENCE DESCRIPTION SEQ ID NO:4:
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- ANTI-SENSE YES
- SEQUENCE DESCRIPTION SEQ ID NO:3:
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- ANTI-SENSE YES
- SEQUENCE DESCRIPTION SEQ ID NO:4:
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- ANTI-SENSE YES
- SEQUENCE DESCRIPTION SEQ ID NO:5:
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- ANTI-SENSE YES
- SEQUENCE DESCRIPTION SEQ ID NO:6:
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- ANTI-SENSE YES
- SEQUENCE DESCRIPTION SEQ ID NO:7:
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- ANTI-SENSE YES
- SEQUENCE DESCRIPTION SEQ ID NO:8:
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- ANTI-SENSE YES
- SEQUENCE DESCRIPTION SEQ ID NO:9:
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- ANTI-SENSE YES
- SEQUENCE DESCRIPTION SEQ ID NO:10:
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- ANTI-SENSE YES
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- ANTI-SENSE YES
- SEQUENCE DESCRIPTION SEQ ID NO:12:
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- ANTI-SENSE YES
- SEQUENCE DESCRIPTION SEQ ID NO:13:
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- ANTI-SENSE YES
- SEQUENCE DESCRIPTION SEQ ID NO:14:
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- ANTI-SENSE YES
- SEQUENCE DESCRIPTION SEQ ID NO:15:
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- ANTI-SENSE YES
- SEQUENCE DESCRIPTION SEQ ID NO:16:
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- ANTI-SENSE YES
- SEQUENCE DESCRIPTION SEQ ID NO:17:
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- ANTI-SENSE YES
- SEQUENCE DESCRIPTION SEQ ID NO:18:
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- ANTI-SENSE YES
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- ANTI-SENSE YES
- SEQUENCE DESCRIPTION SEQ ID NO:21:
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- ANTI-SENSE YES
- SEQUENCE DESCRIPTION SEQ ID NO:22:
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- ANTI-SENSE YES
- SEQUENCE DESCRIPTION SEQ ID NO:23:
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- ANTI-SENSE YES
- SEQUENCE DESCRIPTION SEQ ID NO:24:
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- ANTI-SENSE YES
- SEQUENCE DESCRIPTION SEQ ID NO:25:
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- ANTI-SENSE YES
- SEQUENCE DESCRIPTION SEQ ID NO:26:
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- ANTI-SENSE YES
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- ANTI-SENSE YES
- SEQUENCE DESCRIPTION SEQ ID NO:28:
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- ANTI-SENSE YES
- SEQUENCE DESCRIPTION SEQ ID NO:29:
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- ANTI-SENSE YES
- SEQUENCE DESCRIPTION SEQ ID NO:30:
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- ANTI-SENSE YES
- SEQUENCE DESCRIPTION SEQ ID NO:31:
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- ANTI-SENSE YES
- SEQUENCE DESCRIPTION SEQ ID NO:32:
- AAAAGTACCT TGTTTCTACT 20 INFORMATION FOR SEQ ID NO:33:
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- ANTI-SENSE YES
- SEQUENCE DESCRIPTION SEQ ID NO:33:
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- ANTI-SENSE YES
- SEQUENCE DESCRIPTION SEQ ID NO:34:
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- ANTI-SENSE YES
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- ANTI-SENSE YES
- SEQUENCE DESCRIPTION SEQ ID NO:36:
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- ANTI-SENSE YES
- SEQUENCE DESCRIPTION SEQ ID NO:37:
Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU65270/94A AU6527094A (en) | 1993-03-31 | 1994-03-30 | Modified oligonucleotides having improved anti-influenza activity |
JP6522317A JPH08510900A (en) | 1993-03-31 | 1994-03-30 | Modified oligonucleotides with improved anti-influenza activity |
EP94912901A EP0693123A1 (en) | 1993-03-31 | 1994-03-30 | Modified oligonucleotides having improved anti-influenza activity |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US4075293A | 1993-03-31 | 1993-03-31 | |
US08/040,752 | 1993-03-31 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO1994023028A2 true WO1994023028A2 (en) | 1994-10-13 |
WO1994023028A3 WO1994023028A3 (en) | 1995-02-16 |
Family
ID=21912738
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1994/003454 WO1994023028A2 (en) | 1993-03-31 | 1994-03-30 | Modified oligonucleotides having improved anti-influenza activity |
Country Status (7)
Country | Link |
---|---|
EP (1) | EP0693123A1 (en) |
JP (1) | JPH08510900A (en) |
CN (1) | CN1124980A (en) |
AU (1) | AU6527094A (en) |
CA (1) | CA2159350A1 (en) |
NZ (1) | NZ263985A (en) |
WO (1) | WO1994023028A2 (en) |
Cited By (33)
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WO1996031600A1 (en) * | 1995-04-03 | 1996-10-10 | Hybridon, Inc. | Method of modulating gene expression without depleting complement |
US5565350A (en) * | 1993-12-09 | 1996-10-15 | Thomas Jefferson University | Compounds and methods for site directed mutations in eukaryotic cells |
WO1996032474A1 (en) * | 1995-04-12 | 1996-10-17 | Hybridon, Inc. | Cooperative oligonucleotides |
WO1996041812A1 (en) * | 1995-06-09 | 1996-12-27 | Isis Innovation Limited | Oligonucleotide phosphorylation method and products |
WO1997006662A2 (en) * | 1995-08-17 | 1997-02-27 | Hybridon, Inc. | Inverted chimeric and hybrid oligonucleotides |
WO1997011171A1 (en) * | 1995-09-22 | 1997-03-27 | Hybridon, Inc. | Modified protein kinase a-specific oligonucleotides and methods of their use |
WO1997038097A1 (en) * | 1995-04-12 | 1997-10-16 | Hybridon, Inc. | Cooperative oligonucleotides |
WO1998040479A1 (en) * | 1997-03-12 | 1998-09-17 | Hybridon, Inc. | Modified protein kinase a-specific oligonucleotides and methods of their use |
WO1998053073A1 (en) * | 1997-05-16 | 1998-11-26 | Plant Bioscience Limited | A plant disease resistance signalling gene, materials and methods relating thereto |
US5886165A (en) * | 1996-09-24 | 1999-03-23 | Hybridon, Inc. | Mixed backbone antisense oligonucleotides containing 2'-5'-ribonucleotide- and 3'-5'-deoxyribonucleotides segments |
WO2002008420A2 (en) * | 2000-06-06 | 2002-01-31 | Hybridon, Inc. | A method of down-regulating gene expression |
US6624293B1 (en) | 1995-08-17 | 2003-09-23 | Hybridon, Inc. | Modified protein kinase A-specific oligonucleotides and methods of their use |
US6645943B1 (en) | 1994-10-25 | 2003-11-11 | Hybridon, Inc. | Method of down-regulating gene expression |
US7074768B2 (en) | 1995-08-17 | 2006-07-11 | Idera Pharmaceuticals, Inc. | Modified protein kinase A-specific oligonucleotides and methods of their use |
WO2007053696A3 (en) * | 2005-11-01 | 2008-01-17 | Alnylam Pharmaceuticals Inc | Rnai inhibition of influenza virus replication |
EP2128248A1 (en) | 2002-02-01 | 2009-12-02 | Life Technologies Corporation | Oligonucleotide compositions with enhanced efficiency |
US7691996B2 (en) | 1996-09-26 | 2010-04-06 | Lakewood-Amedex | Three component chimeric antisense oligonucleotides |
WO2010090762A1 (en) | 2009-02-04 | 2010-08-12 | Rxi Pharmaceuticals Corporation | Rna duplexes with single stranded phosphorothioate nucleotide regions for additional functionality |
WO2011119887A1 (en) | 2010-03-24 | 2011-09-29 | Rxi Pharmaceuticals Corporation | Rna interference in dermal and fibrotic indications |
US8664189B2 (en) | 2008-09-22 | 2014-03-04 | Rxi Pharmaceuticals Corporation | RNA interference in skin indications |
WO2015085113A1 (en) | 2013-12-04 | 2015-06-11 | Rxi Pharmaceuticals Corporation | Methods for treatment of wound healing utilizing chemically modified oligonucleotides |
US9074211B2 (en) | 2008-11-19 | 2015-07-07 | Rxi Pharmaceuticals Corporation | Inhibition of MAP4K4 through RNAI |
US9080171B2 (en) | 2010-03-24 | 2015-07-14 | RXi Parmaceuticals Corporation | Reduced size self-delivering RNAi compounds |
WO2016037071A2 (en) | 2014-09-05 | 2016-03-10 | Rxi Pharmaceuticals Corporation | Methods for treating aging and skin disorders using nucleic acids targeting tyr or mmp1 |
US9493774B2 (en) | 2009-01-05 | 2016-11-15 | Rxi Pharmaceuticals Corporation | Inhibition of PCSK9 through RNAi |
WO2017070151A1 (en) | 2015-10-19 | 2017-04-27 | Rxi Pharmaceuticals Corporation | Reduced size self-delivering nucleic acid compounds targeting long non-coding rna |
US10106793B2 (en) | 2002-02-01 | 2018-10-23 | Life Technologies Corporation | Double-stranded oligonucleotides |
US10131904B2 (en) | 2008-02-11 | 2018-11-20 | Rxi Pharmaceuticals Corporation | Modified RNAi polynucleotides and uses thereof |
US10184124B2 (en) | 2010-03-24 | 2019-01-22 | Phio Pharmaceuticals Corp. | RNA interference in ocular indications |
US10808247B2 (en) | 2015-07-06 | 2020-10-20 | Phio Pharmaceuticals Corp. | Methods for treating neurological disorders using a synergistic small molecule and nucleic acids therapeutic approach |
US10934550B2 (en) | 2013-12-02 | 2021-03-02 | Phio Pharmaceuticals Corp. | Immunotherapy of cancer |
US11001845B2 (en) | 2015-07-06 | 2021-05-11 | Phio Pharmaceuticals Corp. | Nucleic acid molecules targeting superoxide dismutase 1 (SOD1) |
US11279934B2 (en) | 2014-04-28 | 2022-03-22 | Phio Pharmaceuticals Corp. | Methods for treating cancer using nucleic acids targeting MDM2 or MYCN |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0386563A1 (en) * | 1989-03-09 | 1990-09-12 | Bayer Ag | Antisense-oligonucleotides for inhibiting the transactivator target sequence (TAR) and the synthesis of the transactivator protein (Tat) of HIV-I, and their use |
WO1991012323A1 (en) * | 1990-02-15 | 1991-08-22 | Worcester Foundation For Experimental Biology | Method of site-specific alteration of rna and production of encoded polypeptides |
WO1991016902A1 (en) * | 1990-04-30 | 1991-11-14 | Hybridon, Inc. | Inhibition of influenza virus replication by oligonucleotides |
WO1992003454A1 (en) * | 1990-08-14 | 1992-03-05 | Isis Pharmaceuticals, Inc. | Inhibition of influenza virus type a, ann arbor strain h2n2 by antisense oligonucleotides |
-
1994
- 1994-03-30 NZ NZ263985A patent/NZ263985A/en unknown
- 1994-03-30 AU AU65270/94A patent/AU6527094A/en not_active Abandoned
- 1994-03-30 WO PCT/US1994/003454 patent/WO1994023028A2/en not_active Application Discontinuation
- 1994-03-30 CA CA002159350A patent/CA2159350A1/en not_active Abandoned
- 1994-03-30 CN CN94192300A patent/CN1124980A/en active Pending
- 1994-03-30 EP EP94912901A patent/EP0693123A1/en not_active Withdrawn
- 1994-03-30 JP JP6522317A patent/JPH08510900A/en active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0386563A1 (en) * | 1989-03-09 | 1990-09-12 | Bayer Ag | Antisense-oligonucleotides for inhibiting the transactivator target sequence (TAR) and the synthesis of the transactivator protein (Tat) of HIV-I, and their use |
WO1991012323A1 (en) * | 1990-02-15 | 1991-08-22 | Worcester Foundation For Experimental Biology | Method of site-specific alteration of rna and production of encoded polypeptides |
WO1991016902A1 (en) * | 1990-04-30 | 1991-11-14 | Hybridon, Inc. | Inhibition of influenza virus replication by oligonucleotides |
WO1992003454A1 (en) * | 1990-08-14 | 1992-03-05 | Isis Pharmaceuticals, Inc. | Inhibition of influenza virus type a, ann arbor strain h2n2 by antisense oligonucleotides |
Non-Patent Citations (2)
Title |
---|
FEBS LETTERS, vol.232, no.1, May 1988, AMSTERDAM NL pages 96 - 98 ATABEKOV, K. ET AL. 'Site-specific enzymatic cleavage of TMV RNA directed by deoxyribo- and chimeric (deoxyribo-ribo)oligonucleotides' * |
NUCLEIC ACIDS RESEARCH, vol.22, no.3, 11 February 1994, ARLINGTON, VIRGINIA US pages 453 - 456 ZHOU, L. ET AL. 'Synthesis of phosphorothioate-methylphosphonate oligonucleotide co-polymers' * |
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Also Published As
Publication number | Publication date |
---|---|
WO1994023028A3 (en) | 1995-02-16 |
EP0693123A1 (en) | 1996-01-24 |
AU6527094A (en) | 1994-10-24 |
CN1124980A (en) | 1996-06-19 |
JPH08510900A (en) | 1996-11-19 |
CA2159350A1 (en) | 1994-10-13 |
NZ263985A (en) | 1997-07-27 |
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