CA2921509A1 - Compositions and methods for modulating apolipoprotein (a) expression - Google Patents

Abstract

Provided herein are oligomeric compounds with conjugate groups targeting apoplipoprotein (a) [apo(a)]. In certain embodiments, the apo(a) targeting oligomeric compounds are conjugated to N-Acetylgalactosamine. Also disclosed herein are conjugated oligomeric compounds targeting apo(a) for use in decreasing apo(a) to treat, prevent, or ameliorate diseases, disorders or conditions related to apo(a) and/or Lp(a). Certain diseases, disorders or conditions related to apo(a) and/or Lp(a) include inflammatory, cardiovascular and/or metabolic diseases, disorders or conditions. The conjugated oligomeric compounds disclosed herein can be used to treat such diseases, disorders or conditions in an individual in need thereof.

Description

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COMPOSITIONS AND METHODS FOR MODULATING APOLIPOPROTEIN (a) EXPRESSION
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 BIOL0250WOSEQ_ST25.txt, created on May 1, 2014, which is 432 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
The principle behind antisense technology is that an antisense compound hybridizes to a target nucleic acid and modulates the amount, activity, and/or function of the target nucleic acid. For example in certain instances, antisense compounds result in altered transcription or translation of a target. Such modulation of expression can be achieved by, for example, target mRNA
degradation or occupancy-based inhibition. An example of modulation of RNA target function by degradation is RNase H-based degradation of the target RNA upon hybridization with a DNA-like antisense compound.
Another example of modulation of gene expression by target degradation is RNA interference (RNAi). RNAi refers to antisense-mediated gene silencing through a mechanism that utilizes the RNA-induced siliencing complex (RISC). An additional example of modulation of RNA target function is by an occupancy-based mechanism such as is employed naturally by microRNA. MicroRNAs are small non-coding RNAs that regulate the expression of protein-coding RNAs. The binding of an antisense compound to a microRNA prevents that microRNA from binding to its messenger RNA targets, and thus interferes with the function of the microRNA. MicroRNA mimics can enhance native microRNA function. Certain antisense compounds alter splicing of pre-mRNA.
Regardless of the specific mechanism, sequence-specificity makes antisense compounds attractive as tools for target validation and gene functionalization, as well as therapeutics to selectively modulate the expression of genes involved in the pathogenesis of diseases.
Antisense technology is an effective means for modulating the expression of one or more specific gene products and can therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications. Chemically modified nucleosides may be incorporated into antisense compounds to enhance one or more properties, such as nuclease resistance, pharmacokinetics or affinity for a target nucleic acid. In 1998, the antisense compound, Vitravene0 (fomivirsen; developed by Isis Pharmaceuticals Inc., Carlsbad, CA) was the first antisense drug to achieve marketing clearance from the U.S. Food and Drug Administration (FDA), and is currently a treatment of cytomegalovirus (CMV)-induced retinitis in AIDS
patients.
New chemical modifications have improved the potency and efficacy of antisense compounds, uncovering the potential for oral delivery as well as enhancing subcutaneous administration, decreasing potential for side effects, and leading to improvements in patient convenience. Chemical modifications increasing potency of antisense compounds allow administration of lower doses, which reduces the potential for toxicity, as well as decreasing overall cost of therapy. Modifications increasing the resistance to degradation result in slower clearance from the body, allowing for less frequent dosing. Different types of chemical modifications can be combined in one compound to further optimize the compound's efficacy.
Lipoproteins are globular, micelle-like particles that consist of a non-polar core of acylglycerols and cholesteryl esters surrounded by an amphiphilic coating of protein, phospholipid and cholesterol.
Lipoproteins have been classified into five broad categories on the basis of their functional and physical properties: chylomicrons, very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), low density lipoproteins (LDL), and high density lipoproteins (HDL). Chylomicrons transport dietary lipids from intestine to tissues. VLDLs, IDLs and LDLs all transport triacylglycerols and cholesterol from the liver to tissues. HDLs transport endogenous cholesterol from tissues to the liver Lipoprotein particles undergo continuous metabolic processing and have variable properties and compositions. Lipoprotein densities increase without increasing particle diameter because the density of their outer coatings is less than that of the inner core. The protein components of lipoproteins are known as apolipoproteins. At least nine apolipoproteins are distributed in significant amounts among the various human lipoproteins.
The lipoprotein(a) [Lp(a)] particle was identified nearly 50 years ago and is comprised of a highly unique LDL particle in which one apolipoprotein B (apoB) protein is linked via a disulfide bond to a single apolipoprotein(a) [apo(a)] protein. The apo(a) protein shares a high degree of homology with plasminogen particularly within the kringle IV type 2 repetitive domain. Levels of circulating Lp(a) are inversely proportional to the number of kringle IV type 2 variable repeats present in the molecule and, as both alleles are co-expressed within individuals, can display heterozygous plasma isoform profiles (Kraft et al., Eur J
Hum Genet, 1996; 4(2): 74-87). It is thought that this kringle repeat domain in apo(a) may be responsible for its pro-thrombotic and anti-fibrinolytic properties, potentially enhancing atherosclerotic progression.
Apo(a) is transcriptionally regulated by IL-6 and in studies in rheumatoid arthritis patients treated with an IL-6 inhibitor (tocilizumab), plasma levels were reduced by 30% after 3 month treatment (Schultz et al., PLoS One 2010; 5:e14328).
Apo(a) has been shown to preferentially bind oxidized phospholipids and potentiate vascular inflammation (Bergmark et al., J Lipid Res 2008; 49:2230-2239; Tsimikas et al., Circulation. 2009;
119(13):1711-1719).
Further, studies suggest that the Lp(a) particle may also stimulate endothelial permeability, induce plasminogen activator inhibitor type-1 expression and activate macrophage interleukin-8 secretion (Koschinsky and Marcovina, Curr Opin Lipidol 2004; 15:167-174). Importantly, recent genetic association studies revealed that Lp(a) was an independent risk factor for myocardial infarction, stroke, peripheral vascular disease and abdominal aortic aneurysm (Rifai et al., Clin Chem 2004;
50:1364-71; Erqou et al.,

2 JAMA 2009;302:412-23; Kamstrup et al., Circulation 2008;117:176-84). Further, in the recent Precocious Coronary Artery Disease (PROCARDIS) study, Clarke et al. (Clarke et al., NEJM
(2009)361; 2518-2528) described robust and independent associations between coronary heart disease and plasma Lp(a) concentrations. Additionally, Solfrizzi et al., suggested that increased serum Lp(a) may be linked to an increased risk for Alzheimer's Disease (AD) (Solfrizzi et al., J Neurol Neurosurg Psychiatry 2002, 72:732-736. Currently, in the clinic setting, examples of indirect apo(a) inhibitors for treating cardiovascular disease include aspirin, Niaspan, Mipomersen, Anacetrapib, Epirotirome and Lomitapide which reduce plasma Lp(a) levels by 18%, 39%, 32%, 36%, 43% and 17%, respectively. Additionally, Lp(a) apheresis has been used in the clinic to reduce apo(a) containing Lp(a) particles.
To date, therapeutic strategies to treat cardiovascular disease by directly targeting apo(a) levels have been limited. Ribozyme oligonucleotides (U.S. Patent 5,877,022) and antisense oligonucleotides (WO
2005/000201; WO 2003/014397; W02013/177468; U520040242516; U.S. Patent Nos.
8,138,328, 8,673,632 and 7,259,150; Merki et al., J Am Coll Cardiol 2011; 57:1611-1621; each publication incorporated by reference in its entiretly) have been developed but none have been approved for commercial use.
Thus, there remains a clear unmet medical need for novel agents which can potently and selectively reduce apo(a) levels in patients at enhanced risk for cardiovascular events due to chronically elevated plasma Lp(a) levels.
SUMMARY OF THE INVENTION
Provided herein are compositions and methods for modulating expression of apo(a) mRNA and protein. In certain embodiments, the apo(a) specific inhibitor decreases expression of apo(a) mRNA and protein. Provided herein are compositions and methods for modulating expression of Lp(a) levels.
In certain embodiments, the composition is an apo(a) specific inhibitior. In certain embodiments, the apo(a) specific inhibitor is a nucleic acid, protein, or small molecule. In certain embodiments, the apo(a) specific inhibitor is an antisense oligonucleotide targeting apo(a) with a conjugate. In certain embodiments, the apo(a) specific inhibitor is a modified oligonucleotide and a conjugate, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and comprises a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of nucleobases 3901 to 3920 of SEQ ID NO: 1, wherein the nucleobase sequence of the modified oligonucleotide is at least 80%
complementary to SEQ ID NO: 1. In certain embodiments, the apo(a) specific inhibitor is a modified oligonucleotide and a conjugate, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and has a nucleobase sequence comprising at least 8, least 9, least 10, least 11, at least 12, least 13, at least 14, at least 15, at least 16, least 17, least 18, least 19, or 20 contiguous nucleobases of the nucleobase sequence of SEQ ID NO: 1-130, 133, 134. In certain embodiments, the apo(a) specific inhibitor is a modified oligonucleotide and a conjugate, wherein the modified oligonucleotide consists of 20 linked nucleosides and

3 has a nucleobase sequence comprising at least 8 contiguous nucleobases of any of SEQ ID NO: 58, wherein the modified oligonucleotide comprises: (a) a gap segment consisting of ten linked deoxynucleosides; (b) a 5' wing segment consisting of five linked nucleosides; (c) a 3' wing segment consisting of five linked nucleosides; and wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment, wherein each nucleoside of each wing segment comprises a 2'-0-methoxyethyl sugar, wherein at least one internucleoside linkage is a phosphorothioate linkage and wherein each cytosine residue is a 5-methylcytosine.
Certain embodiments provide a composition comprising a conjugated antisense compound described herein, or a salt thereof, and a pharmaceutically acceptable carrier or diluent.
In certain embodiments, the modulation of apo(a) expression occurs in a cell or tissue. In certain embodiments, the modulations occur in a cell or tissue in an animal. In certain embodiments, the animal is a human. In certain embodiments, the modulation is a reduction in apo(a) mRNA
level. In certain embodiments, the modulation is a reduction in apo(a) protein level. In certain embodiments, both apo(a) mRNA and protein levels are reduced. In certain embodiments, the modulation is a reduction in Lp(a) level.
Such reduction may occur in a time-dependent or in a dose-dependent manner.
Certain embodiments provide conjugated antisense compositions and methods for use in therapy.
Certain embodiments provide compositions and methods for preventing, treating, delaying, slowing the progression and/or ameliorating apo(a) related diseases, disorders, and conditions. Certain embodiments provide compositions and methods for preventing, treating, delaying, slowing the progression and/or ameliorating Lp(a) related diseases, disorders, and conditions. In certain embodiments, such diseases, disorders, and conditions are inflammatory, cardiovascular and/or metabolic diseases, disorders, and conditions. In certain embodiments, the compositions and methods for therapy include administering an apo(a) specific inhibitor to an individual in need thereof In certain embodiments, the apo(a) specific inhibitor is a nucleic acid. In certain embodiments, the nucleic acid is an antisense compound. In certain embodiments, the antisense compound is a modified oligonucleotide. In certain embodiments, the antisense compound is a modified oligonucleotide with a conjugate.
In certain embodiments, the present disclosure provides conjugated antisense compounds. In certain embodiments, the present disclosure provides conjugated antisense compounds comprising an antisense oligonucleotide complementary to a nucleic acid transcript. In certain embodiments, the present disclosure provides methods comprising contacting a cell with a conjugated antisense compound comprising an antisense oligonucleotide complementary to a nucleic acid transcript. In certain embodiments, the present disclosure provides methods comprising contacting a cell with a conjugated antisense compound comprising an antisense oligonucleotide and reducing the amount or activity of a nucleic acid transcript in a cell.
The asialoglycoprotein receptor (ASGP-R) has been described previously. See e.g., Park et al., PNAS vol. 102, No. 47, pp 17125-17129 (2005). Such receptors are expressed on liver cells, particularly hepatocytes. Further, it has been shown that compounds comprising clusters of three N-

4

5 acetylgalactosamine (GalNAc) ligands are capable of binding to the ASGP-R, resulting in uptake of the compound into the cell. See e.g., Khorev et al., Bioorganic and Medicinal Chemistry, 16, 9, pp 5216-5231 (May 2008). Accordingly, conjugates comprising such GalNAc clusters have been used to facilitate uptake of certain compounds into liver cells, specifically hepatocytes. For example it has been shown that certain GalNAc-containing conjugates increase activity of duplex siRNA compounds in liver cells in vivo. In such instances, the GalNAc-containing conjugate is typically attached to the sense strand of the siRNA duplex.
Since the sense strand is discarded before the antisense strand ultimately hybridizes with the target nucleic acid, there is little concern that the conjugate will interfere with activity.
Typically, the conjugate is attached to the 3' end of the sense strand of the siRNA. See e.g., U.S. Patent 8,106,022. Certain conjugate groups described herein are more active and/or easier to synthesize than conjugate groups previously described.
In certain embodiments of the present invention, conjugates are attached to single-stranded antisense compounds, including, but not limited to RNase H based antisense compounds and antisense compounds that alter splicing of a pre-mRNA target nucleic acid. In such embodiments, the conjugate should remain attached to the antisense compound long enough to provide benefit (improved uptake into cells) but then should either be cleaved, or otherwise not interfere with the subsequent steps necessary for activity, such as hybridization to a target nucleic acid and interaction with RNase H or enzymes associated with splicing or splice modulation. This balance of properties is more important in the setting of single-stranded antisense compounds than in siRNA compounds, where the conjugate may simply be attached to the sense strand.
Disclosed herein are conjugated single-stranded antisense compounds having improved potency in liver cells in vivo compared with the same antisense compound lacking the conjugate. Given the required balance of properties for these compounds such improved potency is surprising.
In certain embodiments, conjugate groups herein comprise a cleavable moiety.
As noted, without wishing to be bound by mechanism, it is logical that the conjugate should remain on the compound long enough to provide enhancement in uptake, but after that, it is desirable for some portion or, ideally, all of the conjugate to be cleaved, releasing the parent compound (e.g., antisense compound) in its most active form. In certain embodiments, the cleavable moiety is a cleavable nucleoside. Such embodiments take advantage of endogenous nucleases in the cell by attaching the rest of the conjugate (the cluster) to the antisense oligonucleotide through a nucleoside via one or more cleavable bonds, such as those of a phosphodiester linkage. In certain embodiments, the cluster is bound to the cleavable nucleoside through a phosphodiester linkage. In certain embodiments, the cleavable nucleoside is attached to the antisense oligonucleotide (antisense compound) by a phosphodiester linkage. In certain embodiments, the conjugate group may comprise two or three cleavable nucleosides. In such embodiments, such cleavable nucleosides are linked to one another, to the antisense compound and/or to the cluster via cleavable bonds (such as those of a phosphodiester linkage). Certain conjugates herein do not comprise a cleavable nucleoside and instead comprise a cleavable bond. It is shown that that sufficient cleavage of the conjugate from the oligonucleotide is provided by at least one bond that is vulnerable to cleavage in the cell (a cleavable bond).

In certain embodiments, conjugated antisense compounds are prodrugs. Such prodrugs are administered to an animal and are ultimately metabolized to a more active form. For example, conjugated antisense compounds are cleaved to remove all or part of the conjugate resulting in the active (or more active) form of the antisense compound lacking all or some of the conjugate.
In certain embodiments, conjugates are attached at the 5' end of an oligonucleotide. Certain such 5'-conjugates are cleaved more efficiently than counterparts having a similar conjugate group attached at the 3' end. In certain embodiments, improved activity may correlate with improved cleavage. In certain embodiments, oligonucleotides comprising a conjugate at the 5' end have greater efficacy than oligonucleotides comprising a conjugate at the 3' end (see, for example, Examples 56, 81, 83, and 84).
Further, 5'-attachment allows simpler oligonucleotide synthesis. Typically, oligonucleotides are synthesized on a solid support in the 3' to 5' direction. To make a 3'-conjugated oligonucleotide, typically one attaches a pre-conjugated 3' nucleoside to the solid support and then builds the oligonucleotide as usual. However, attaching that conjugated nucleoside to the solid support adds complication to the synthesis. Further, using that approach, the conjugate is then present throughout the synthesis of the oligonucleotide and can become degraded during subsequent steps or may limit the sorts of reactions and reagents that can be used. Using the structures and techniques described herein for 5'-conjugated oligonucleotides, one can synthesize the oligonucleotide using standard automated techniques and introduce the conjugate with the final (5'-most) nucleoside or after the oligonucleotide has been cleaved from the solid support.
In view of the art and the present disclosure, one of ordinary skill can easily make any of the conjugates and conjugated oligonucleotides herein. Moreover, synthesis of certain such conjugates and conjugated oligonucleotides disclosed herein is easier and/or requires few steps, and is therefore less expensive than that of conjugates previously disclosed, providing advantages in manufacturing. For example, the synthesis of certain conjugate groups consists of fewer synthetic steps, resulting in increased yield, relative to conjugate groups previously described. Conjugate groups such as Ga1NAc3-10 in Example 46 and Ga1NAc3-7 in Example 48 are much simpler than previously described conjugates such as those described in U.S. 8,106,022 or U.S. 7,262,177 that require assembly of more chemical intermediates . Accordingly, these and other conjugates described herein have advantages over previously described compounds for use with any oligonucleotide, including single-stranded oligonucleotides and either strand of double-stranded oligonucleotides (e.g., siRNA).
Similarly, disclosed herein are conjugate groups having only one or two GalNAc ligands. As shown, such conjugates groups improve activity of antisense compounds. Such compounds are much easier to prepare than conjugates comprising three GalNAc ligands. Conjugate groups comprising one or two GalNAc ligands may be attached to any antisense compounds, including single-stranded oligonucleotides and either strand of double-stranded oligonucleotides (e.g., siRNA).
In certain embodiments, the conjugates herein do not substantially alter certain measures of tolerability. For example, it is shown herein that conjugated antisense compounds are not more immunogenic

6 than unconjugated parent compounds. Since potency is improved, embodiments in which tolerability remains the same (or indeed even if tolerability worsens only slightly compared to the gains in potency) have improved properties for therapy.
In certain embodiments, conjugation allows one to alter antisense compounds in ways that have less attractive consequences in the absence of conjugation. For example, in certain embodiments, replacing one or more phosphorothioate linkages of a fully phosphorothioate antisense compound with phosphodiester linkages results in improvement in some measures of tolerability. For example, in certain instances, such antisense compounds having one or more phosphodiester are less immunogenic than the same compound in which each linkage is a phosphorothioate. However, in certain instances, as shown in Example 26, that same replacement of one or more phosphorothioate linkages with phosphodiester linkages also results in reduced cellular uptake and/or loss in potency. In certain embodiments, conjugated antisense compounds described herein tolerate such change in linkages with little or no loss in uptake and potency when compared to the conjugated full-phosphorothioate counterpart. In fact, in certain embodiments, for example, in Examples 44, 57, 59, and 86, oligonucleotides comprising a conjugate and at least one phosphodiester internucleoside linkage actually exhibit increased potency in vivo even relative to a full phosphorothioate counterpart also comprising the same conjugate. Moreover, since conjugation results in substantial increases in uptake/potency a small loss in that substantial gain may be acceptable to achieve improved tolerability.
Accordingly, in certain embodiments, conjugated antisense compounds comprise at least one phosphodiester linkage.
In certain embodiments, conjugation of antisense compounds herein results in increased delivery, uptake and activity in hepatocytes. Thus, more compound is delivered to liver tissue. However, in certain embodiments, that increased delivery alone does not explain the entire increase in activity. In certain such embodiments, more compound enters hepatocytes. In certain embodiments, even that increased hepatocyte uptake does not explain the entire increase in activity. In such embodiments, productive uptake of the conjugated compound is increased. For example, as shown in Example 102, certain embodiments of GalNAc-containing conjugates increase enrichment of antisense oligonucleotides in hepatocytes versus non-parenchymal cells. This enrichment is beneficial for oligonucleotides that target genes that are expressed in hepatocytes.
In certain embodiments, conjugated antisense compounds herein result in reduced kidney exposure.
For example, as shown in Example 20, the concentrations of antisense oligonucleotides comprising certain embodiments of GalNAc-containing conjugates are lower in the kidney than that of antisense oligonucleotides lacking a GalNAc-containing conjugate.
This has several beneficial therapeutic implications. For therapeutic indications where activity in the kidney is not sought, exposure to kidney risks kidney toxicity without corresponding benefit. Moreover, high concentration in kidney typically results in loss of compound to the urine resulting in faster clearance. Accordingly for non-kidney targets, kidney accumulation is undesired.

7 In certain embodiments, the present disclosure provides conjugated antisense compounds represented by the formula:
A¨B¨C¨DiE¨) q wherein A is the antisense oligonucleotide;
B is the cleavable moiety C is the conjugate linker D is the branching group each E is a tether;
each F is a ligand; and q is an integer between 1 and 5.
In the above diagram and in similar diagrams herein, the branching group "D"
branches as many times as is necessary to accommodate the number of (E-F) groups as indicated by "q". Thus, where q = 1, the formula is:
A¨B¨C¨D¨E¨F
where q = 2, the formula is:
E¨F
A¨B¨C¨D/
\ E¨F
where q = 3, the formula is:
E¨F
A¨B¨C¨D/¨ E¨F
\ E¨F
where q = 4, the formula is:

8 E¨F
E¨F
A ¨B ¨C ¨D
E¨F
E¨F
where q = 5, the formula is:
E¨F
/ E¨F E¨F
A ¨ B ¨ C D
N E¨F
E¨F
In certain embodiments, conjugated antisense compounds are provided having the structure:
TargeUng moiety ASO
HO OH
¨ 0=P -OH

N--t1 9H O
ni_li¨Jõ.N
o ?
rq NHAc ____________________________________________________________________ P-0 H
HO

NHAc 0 0 _ 0 Linker C
- Ligand Tether leavable moiety OH
HO HN ----Branching group HO
NHAc 0 =

9 In certain embodiments, conjugated antisense compounds are provided having the structure:
Cell targeting moiety HO OH
HO , 0 -----.2..\.u.õ------.. ,ii:L
Cleavable moiety AcHN 0 1 0¨

OH

_____________________________________________________ , 0--__. 0 O (-1 0 l_p-u H r AcHN OH 0 o _____________________________________________________ , Tether-04=0 Ligand HO OH 9, y P- ASO

HO OH
NHAc Branching group

10 In certain embodiments, conjugated antisense compounds are provided having the structure:
ASO
Cleavable moiety HO¨P=0 I 0 CI\T¨rµ
0¨ rN
N-_-_---, (1 I
HO¨P=0 Cell targeting moiety 1 1 1 HO OH

AcHN u- 0 - _ HO OH _ i ______ , (03 Conjugate linker HO f-NO
µ-/-.,/N P
cCO ,c; \O-13, =0 v- 1 _ AcHN _ O OH
OH
Tether ________________________________________________________ , ' ____ 1 Ligand HO OH 9 y P¨

HO
NHAc Branching group .
In certain embodiments, conjugated antisense compounds are provided having the structure:

11 ASO

_ Ligand Cleavable moiety I
=0 Tether HO¨P
HO OH - -1 _-H _ _ AcHN 0 ( NH
HO OH
H H 01)3 HO-112-\Z N¨K __________________________________ 4 2 (Y N
AcHN 0 ¨ 0 ¨
Conjugate HO OH
/
___.r.Ø....\, H linker 4 oci AcHN 0 Branching group Cell targeting moiety The present disclosure provides the following non-limiting numbered embodiments:
In embodiments having more than one of a particular variable (e.g., more than one "m" or "n"), unless otherwise indicated, each such particular variable is selected independently. Thus, for a structure having more than one n, each n is selected independently, so they may or may not be the same as one another.
In certain embodiments, the present disclosure provides conjugated antisense compounds represented by the following structure. In certain embodiments, the antisense compound comprises modified oligonucleotide ISIS 494372 with a 5'-X, wherein X is a conjugate group comprising GalNAc. In certain embodiments, the antisense compound consists of modified oligonucleotide ISIS
494372 with a 5'-X, wherein X is a conjugate group comprising GalNAc.

12 NI(Ir 0 0 )i 0 NO N1r)1\1H
I'L7/D NIfj.Lr 0 I\10 e ? o,) o ccL'(LI
5-P=0 N 0 0 I 2eLIIH 0 e 1 (:)/ 2 N N NH S-P=0 0 i NH e , NIfjj'r o o -.L
N 0 I\1 0 0 0,) 0 e I
, S-P=0 N
Y'Ll--1 0 1 S-P=0 N2().(NH /0 0 0 o)) 0 0 S-P=0 N) 95-P=O 0 I l le('X 0) NH
0, / N NH0 0 Nsic:0_22 1 N 0 s-=o I illr 0 1\10 i/j-, 0 \

e I
)*
S-P=0 s-=o NH
i ,-1\( N0 00,,) 0 0\1C;L)/ 0 e , o o 0 S-P=0 1 illr 9 ? 0 NH2 9 1 S-P=0 N 0 S-P=0 1 1.;--k-N,H
, 0 C/
0, )tril. , I ,I, \
\
N-<L5/" N NH2 0 CcL) 0,) 0 e 11: S-1'=0 9 1 N = At, S-P.0 2e5; S-P6,0 0 N1(trk O,,Isfi N NH2 -..õ ccLI 0 N 0 Slf:( 0 OH 0) e 1 0 S-P=0 e 1 S-P=0 I
0 _________________________________________________________ I
0 _______________________________

13 In certain embodiments, the present disclosure provides conjugated antisense compounds represented by the following structure. In certain embodiments, the antisense compound comprises the conjugated modified oligonucleotide ISIS 681251. In certain embodiments, the antisense compound consists of the conjugated modified oligonucleotide ISIS 681251.
O
c) HO OH o 0-F' =0 'NC-4C , Nf NH
_____________________________________________________ ()_)/N N NH2 ---,ir NH No csi 0 HO OH 0 0 e ') 0 e0 SO
4 H oI (!µ111"-tr S-F' =0 ILL'i X
)_ (,N 0 HO OH N

---j 2 S-=O /1-' S-F' =0 HO 0-'11---N 0 NH

)_ o a 0 0 0 o') 0 S-F' =0 1 o-----121N 0 S-P=0 Arll'NH

0__ S-P=0 A'41 NH

0 0.,) 0 ,,.-., "'S-p =0 '-'"(LN

1c2i 0 N
, 0 a NH
L
0 0 o,) NH2 S-F' =0 SP SO

-S-=O NTIL'NH
00 ) I
S- r O F'=0 KI-It--:

0 0 (j) 0 e o NH

S-P =0 S-I's '''CjtHNH
0N N __ 0 N ''0 )_04/cy, o a o,1 0 s-=o A---4-1 X e S-P =0 0.--,IN 0 0 N

e N
----) NH2 S-P =0 Irlhi e 0 o N N NH2 S-P =0 A--)."'N
--1_0_)1 0 1 p o S-P =0 ) OH 05) o _________

14 In certain embodiments, the present disclosure provides conjugated antisense compounds represented by the following structure. In certain embodiments, the antisense compound comprises the conjugated modified oligonucleotide ISIS 681257. In certain embodiments, the antisense compound consists of the conjugated modified oligonucleotide ISIS 681257.
O

HO OH 0 O-F'=0 '1)1'1 N4F1 N f NH
I
()_)/N N NH2 --,[r NH 'No cv 0 HO OH 0 N 0 e 0 (:)--) o e 0 SO
HO 021"--N
/ tar S-F' =0 'XLIC,ZIF1 HO OH
9 0-_,-i NH2 0 N
_.....72..v SF'=0 Y r HO 0-'11--N 0 F'=0 N
.-1''''N N NH2 N--.0 0 )_A
0_/cy, e 0 e 0 0) 0 S-F'=0 1 o-----121N 0 0-p=0 NH

0 S-P=0 'fl NH
O 0,) NH2 0 N-,,.-., '-'0-p=0 A-""LN

1c2i 0 O y0 N
(:t 0 0 0 ill' NH
0 0 o,) NH2 S0 ---o 0-F'=0 -O-P =0 NI-11' NH
00 ) 1 S- r O N N NH2 F'=0 (-lt--,, 0 N% N NH2 CLi NHS-P =0 11 I's 'IJICNH

N'O
04/ o., e o,1 0 s-=o 11 X e S-P =0 lUir 0.--.,_)1 0 (:)1/N 0 0 0"
e N lhi S-P =0 O N"' eI
N NH2 S-P =0 'N
---01 o I

op o S-P =0 ) OH 0õ i 0 _____________________________________________________________ In certain embodiments, the present disclosure provides conjugated antisense compounds represented by the following structure. In certain embodiments, the antisense compound comprises a modified oligonucleotide with SEQ ID NO: 58 with a 5'-Ga1NAc with variability in the sugar mods of the wings. In certain embodiments, the antisense compound consists of a modified oligonucleotide with SEQ ID NO: 58 with a 5'-GalNAc with variability in the sugar mods of the wings.
O

HO OH 0 ,0-1?=60 R51-1t.i Nii.F1 N/It NH
__...72...\ HN'< c, 0o N0 1 Iµj N NH2 r NH
HO OH 0 \ 0 e 9 R I 0 ,0 .---____---N H S-P =0 NH

es_i so RVI'IN,H
HO 01-r N
4 H \//NI--IL'N --"i'N H2 O=/
O NH
--- Tr OV-1 _....72..v S-F' =0 4 H Z-F' =0 R5I--k,,,..
I \l ONI0 0 N 1 ' NH2 - Tr NH
' (:1 NH2 0 R51).=,,,,, e 9 RI I
9 o s-1, =0 Z-P =0 R5,se, O NH
N0 o--I-R) 0 R4 , e9 R3 R)-NH
O R" NH2 S-F'=0 i 0 ,R)p/N--'''-0 z-=0 O i¨ 47N110 ' 9 R3 R5 NH2 S-F' =0 Z-=0 'N 0------)NO

0 -_ ZR7!,)p/s R3 <IN Ii):NFI
00 R-, --i0N 0 -F'=0 ol,N111'NNH2 Z 0 R5'NH
_9=
e o R3 R51,[1, S-F' =0 1 NH p Oj_ o N0 o N ---_5/ 0 e 9 R5.,cANH
S-F' =0 1, 9 R5-jt-NH
N,r) 0 N-'-'0 I
)0_)/ 0 o /
eS I?=0 1\1112=

1,r NH2 N N NH2 e 9 0 S-F' =0 N
--1_5 o N''.0 e 9 R0 sezo ) OH R, 0 ______ Wherein either R1 is ¨OCH2CH2OCH3 (M0E)and R2 is H; or R1 and R2 together form a bridge, wherein R1 is ¨0- and R2 is ¨CH2-, -CH(CH3)-, or -CH2CH2-, and R1 and R2 are directly connected such that the resulting bridge is selected from: -0-CH2-, -0-CH(CH3)-, and ¨0-CH2CH2-;

And for each pair of R3 and R4 on the same ring, independently for each ring:
either R3 is selected from H and -OCH2CH2OCH3 and R4 is H; or R3 and R4 together form a bridge, wherein R3 is ¨0-, and R4 is ¨
CH2-, -CH(CH3)-, or -CH2CH2-and R3 and R4 are directly connected such that the resulting bridge is selected from: -0-CH2-, -0-CH(CH3)-, and ¨0-CH2CH2-;
And R5 is selected from H and ¨CH3;
And Z is selected from S- and 0-.
The present disclosure provides the following non-limiting numbered embodiments:
DETAILED DESCRIPTION
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 disclosure. 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, Florida; 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 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, "nucleic acid" refers to molecules composed of monomeric nucleotides. A nucleic acid includes ribonucleic acids (RNA), deoxyribonucleic acids (DNA), single-stranded nucleic acids (ssDNA), double-stranded nucleic acids (dsDNA), small interfering ribonucleic acids (siRNA), and microRNAs (miRNA). A nucleic acid may also comprise any combination of these elements in a single molecule.
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, "nucleobase sequence" means the order of contiguous nucleobases independent of any sugar, linkage, or nucleobase modification.
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)-0-2'bridge.
As used herein, "locked nucleic acid nucleoside" or "LNA" means a nucleoside comprising a bicyclic sugar moiety comprising a 4'-CH2-0-2'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, "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, "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, "linkage" or "linking group" means a group of atoms that link together two or more other groups of atoms.
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, "terminal internucleoside linkage" means the linkage between the last two nucleosides of an oligonucleotide or defined region thereof As used herein, "phosphorus linking group" means a linking group comprising a phosphorus atom.
Phosphorus linking groups include without limitation groups having the formula:
I
Ra Rb=P¨R, I
Rd wherein:
Ra and Rd are each, independently, 0, S, CH2, NH, or NJI wherein J1 is C1-C6 alkyl or substituted CI-C6 alkyl;
Rb is 0 or S;
Re is OH, SH, C1-C6 alkyl, substituted C1-C6 alkyl, Ci-C6 alkoxy, substituted Ci-C6 alkoxy, amino or substituted amino; and J1 is Rb is 0 or S.
Phosphorus linking groups include without limitation, phosphodiester, phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, phosphorothioamidate, thionoalkylphosphonate, phosphotriesters, thionoalkylphosphotriester and boranophosphate.
As used herein, "internucleoside phosphorus linking group" means a phosphorus linking group that directly links two nucleosides.
As used herein, "non-internucleoside phosphorus linking group" means a phosphorus linking group that does not directly link two nucleosides. In certain embodiments, a non-internucleoside phosphorus linking group links a nucleoside to a group other than a nucleoside. In certain embodiments, a non-internucleoside phosphorus linking group links two groups, neither of which is a nucleoside.
As used herein, "neutral linking group" means a linking group that is not charged. Neutral linking groups include without limitation phosphotriesters, methylphosphonates, MMI (-CH2-N(CH3)-0-), amide-3 (-CH2-C(=0)-N(H)-), amide-4 (-CH2-N(H)-C(=0)-), formacetal (-0-CH2-0-), and thioformacetal (-S-CH2-0-).
Further neutral linking groups 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, (pp.
40-65)). Further neutral linking groups include nonionic linkages comprising mixed N, 0, S and CH2 component parts.
As used herein, "internucleoside neutral linking group" means a neutral linking group that directly links two nucleosides.
As used herein, "non-internucleoside neutral linking group" means a neutral linking group that does not directly link two nucleosides. In certain embodiments, a non-internucleoside neutral linking group links a nucleoside to a group other than a nucleoside. In certain embodiments, a non-internucleoside neutral linking group links two groups, neither of which is a nucleoside.
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.
Oligomeric compounds also include naturally occurring nucleic acids. In certain embodiments, an oligomeric compound comprises a backbone of one or more linked monomeric subunits where each linked monomeric subunit is directly or indirectly attached to a heterocyclic base moiety. In certain embodiments, oligomeric compounds may also include monomeric subunits that are not linked to a heterocyclic base moiety, thereby providing abasic sites.
In certain embodiments, the linkages joining the monomeric subunits, the sugar moieties or surrogates and the heterocyclic base moieties can be independently modified. In certain embodiments, the linkage-sugar unit, which may or may not include a heterocyclic base, may be substituted with a mimetic such as the monomers in peptide nucleic acids.
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" or "conjugate group" 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 linker" or "linker" in the context of a conjugate group means a portion of a conjugate group comprising any atom or group of atoms and which covalently link (1) an oligonucleotide to another portion of the conjugate group or (2) two or more portions of the conjugate group.
Conjugate groups are shown herein as radicals, providing a bond for forming covalent attachment to an oligomeric compound such as an antisense oligonucleotide. In certain embodiments, the point of attachment on the oligomeric compound is the 3'-oxygen atom of the 3'-hydroxyl group of the 3' terminal nucleoside of the oligomeric compound. In certain embodiments the point of attachment on the oligomeric compound is the 5'-oxygen atom of the 5'-hydroxyl group of the 5' terminal nucleoside of the oligomeric compound. In certain embodiments, the bond for forming attachment to the oligomeric compound is a cleavable bond. In certain such embodiments, such cleavable bond constitutes all or part of a cleavable moiety.
In certain embodiments, conjugate groups comprise a cleavable moiety (e.g., a cleavable bond or cleavable nucleoside) and a carbohydrate cluster portion, such as a GalNAc cluster portion. Such carbohydrate cluster portion comprises: a targeting moiety and, optionally, a conjugate linker. In certain embodiments, the carbohydrate cluster portion is identified by the number and identity of the ligand. For example, in certain embodiments, the carbohydrate cluster portion comprises 3 GalNAc groups and is designated "Ga1NAc3". In certain embodiments, the carbohydrate cluster portion comprises 4 GalNAc groups and is designated "Ga1NAc4". Specific carbohydrate cluster portions (having specific tether, branching and conjugate linker groups) are described herein and designated by Roman numeral followed by subscript "a". Accordingly "GalNac3-1,7 refers to a specific carbohydrate cluster portion of a conjugate group having 3 GalNac groups and specifically identified tether, branching and linking groups. Such carbohydrate cluster fragment is attached to an oligomeric compound via a cleavable moiety, such as a cleavable bond or cleavable nucleoside.
As used herein, "cleavable moiety" means a bond or group that is capable of being split under physiological conditions. In certain embodiments, a cleavable moiety is cleaved inside a cell or sub-cellular compartments, such as a lysosome. In certain embodiments, a cleavable moiety is cleaved by endogenous enzymes, such as nucleases. In certain embodiments, a cleavable moiety comprises a group of atoms having one, two, three, four, or more than four cleavable bonds.
As used herein, "cleavable bond" means any chemical bond capable of being split. In certain embodiments, a cleavable bond is selected from among: an amide, a polyamide, an ester, an ether, one or both esters of a phosphodiester, a phosphate ester, a carbamate, a di-sulfide, or a peptide.

As used herein, "carbohydrate cluster" means a compound having one or more carbohydrate residues attached to a scaffold or linker group. (see, e.g., Maier et al., "Synthesis of Antisense Oligonucleotides Conjugated to a Multivalent Carbohydrate Cluster for Cellular Targeting,"
Bioconjugate Chemistry, 2003, (14): 18-29, which is incorporated herein by reference in its entirety, or Rensen et al., "Design and Synthesis of Novel N-Acetylgalactosamine-Terminated Glycolipids for Targeting of Lipoproteins to the Hepatic Asiaglycoprotein Receptor," J. Med. Chem. 2004, (47): 5798-5808, for examples of carbohydrate conjugate clusters).
As used herein, "modified carbohydrate" means any carbohydrate having one or more chemical modifications relative to naturally occurring carbohydrates.
As used herein, "carbohydrate derivative" means any compound which may be synthesized using a carbohydrate as a starting material or intermediate.
As used herein, "carbohydrate" means a naturally occurring carbohydrate, a modified carbohydrate, or a carbohydrate derivative.
As used herein "protecting group" means any compound or protecting group known to those having skill in the art. Non-limiting examples of protecting groups may be found in "Protective Groups in Organic Chemistry", T. W. Greene, P. G. M. Wuts, ISBN 0-471-62301-6, John Wiley &
Sons, Inc, New York, which is incorporated herein by reference in its entirety.
As used herein, "single-stranded" means an oligomeric compound that is not hybridized to its complement and which lacks sufficient self-complementarity to form a stable self-duplex.
As used herein, "double stranded" means a pair of oligomeric compounds that are hybridized to one another or a single self-complementary oligomeric compound that forms a hairpin structure. In certain embodiments, a double-stranded oligomeric compound comprises a first and a second 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. In certain embodiments, antisense activity includes modulation of the amount or activity of a target nucleic acid transcript (e.g. mRNA). In certain embodiments, antisense activity includes modulation of the splicing of pre-mRNA.
As used herein, "RNase H based antisense compound" means an antisense compound wherein at least some of the antisense activity of the antisense compound is attributable to hybridization of the antisense compound to a target nucleic acid and subsequent cleavage of the target nucleic acid by RNase H.
As used herein, "RISC based antisense compound" means an antisense compound wherein at least some of the antisense activity of the antisense compound is attributable to the RNA Induced Silencing Complex (RISC).
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 measureable activity" means a statistically significant 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 to result in a desired antisense activity. Antisense oligonucleotides have sufficient complementarity to their target nucleic acids 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.
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.
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, "chemical motif' means a pattern of chemical modifications in an oligonucleotide or a region thereof Motifs may be defined by modifications at certain nucleosides and/or at certain linking groups of an oligonucleotide.

As used herein, "nucleoside motif' means a pattern of nucleoside modifications in an oligonucleotide or a region thereof The linkages of such an oligonucleotide 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 oligonucleotide or a region thereof As used herein, "linkage motif' means a pattern of linkage modifications in an oligonucleotide or region thereof The nucleosides of such an oligonucleotide 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'-0Me modified sugar and an unmodified adenine nucleobase and a nucleoside comprising a 2'-0Me 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 nucleosides 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, "separate regions" means portions of an oligonucleotide wherein the chemical modifications or the motif of chemical modifications of any neighboring portions include at least one difference to allow the separate regions to be distinguished from one another.
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 the term "metabolic disorder" means a disease or condition principally characterized by dysregulation of metabolism ¨ the complex set of chemical reactions associated with breakdown of food to produce energy.
As used herein, the term "cardiovascular disorder" means a disease or condition principally characterized by impaired function of the heart or blood vessels.
As used herein the term "mono or polycyclic ring system" is meant to include all ring systems selected from single or polycyclic radical ring systems wherein the rings are fused or linked and is meant to be inclusive of single and mixed ring systems individually selected from aliphatic, alicyclic, aryl, heteroaryl, aralkyl, arylalkyl, heterocyclic, heteroaryl, heteroaromatic and heteroarylalkyl. Such mono and poly cyclic structures can contain rings that each have the same level of saturation or each, independently, have varying degrees of saturation including fully saturated, partially saturated or fully unsaturated. Each ring can comprise ring atoms selected from C, N, 0 and S to give rise to heterocyclic rings as well as rings comprising only C ring atoms which can be present in a mixed motif such as for example benzimidazole wherein one ring has only carbon ring atoms and the fused ring has two nitrogen atoms. The mono or polycyclic ring system can be further substituted with substituent groups such as for example phthalimide which has two =0 groups attached to one of the rings. Mono or polycyclic ring systems can be attached to parent molecules using various strategies such as directly through a ring atom, fused through multiple ring atoms, through a substituent group or through a bifunctional linking moiety.
As used herein, "prodrug" means an inactive or less active form of a compound which, when administered to a subject, is metabolized to form the active, or more active, compound (e.g., drug).
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'-substuent 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 disclosure 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 that 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(0)Raa), carboxyl (-C(0)0-Raa), aliphatic groups, alicyclic groups, alkoxy, substituted oxy (-O-Raa), aryl, aralkyl, heterocyclic radical, heteroaryl, heteroarylalkyl, amino (-N(Rbb)(Ree)), imino(=NRbb), amido (-C(0)N(Rbb)(Ree) or -N(Rbb)C(0)Ra.), azido (-N3), nitro (-NO2), cyano (-CN), carbamido (-0C(0)N(Rbb)(Ree) or -N(Rbb)C(0)0Raa), ureido (-N(Rbb)C(0)N(Rbb)(Rõ)), thioureido (-N(Rbb)C(S)N(Rbb)-(Rõ)), guanidinyl (-N(Rbb)C(=NRbb)N(Rbb)(Ree)), amidinyl (-C(=NRbb)N(Rbb)(Ree) or -N(Rbb)C(=NRbb)(Raa.)), thiol (-SRbb), sulfinyl (-S(0)Rbb), sulfonyl (-S(0)2Rbb) and sulfonamidyl (-S(0)2N(Rbb)(Ree) or -N(Rbb)S-(0)2Rbb). Wherein each Raa, Rbb and Ree 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 (Ci-C12alkyl) 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-methy1-2-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(0)-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.
As used herein, "conjugate compound" means any atoms, group of atoms, or group of linked atoms suitable for use as a conjugate group. In certain embodiments, conjugate compounds may possess or impart one or more properties, including, but not limited to pharmacodynamic, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, charge and/or clearance properties.
As used herein, unless otherwise indicated or modified, the term "double-stranded" refers to two separate oligomeric compounds that are hybridized to one another. Such double stranded compounds may have one or more or non-hybridizing nucleosides at one or both ends of one or both strands (overhangs) and/or one or more internal non-hybridizing nucleosides (mismatches) provided there is sufficient complementarity to maintain hybridization under physiologically relevant conditions.
As used herein, "5' target site" refers to the nucleotide of a target nucleic acid which is complementary to the 5'-most nucleotide of a particular antisense compound.
As used herein, "About" means within 10% of a value. For example, if it is stated, "a marker may be increased by about 50%", it is implied that the marker may be increased between 45%-55%.
As used herein, "administered concomitantly" refers to the co-administration of two agents in any manner in which the pharmacological effects of both are manifest in the patient at the same time.
Concomitant administration does not require that both agents be administered in a single pharmaceutical composition, in the same dosage form, or by the same route of administration.
The effects of both agents need not manifest themselves at the same time. The effects need only be overlapping for a period of time and need not be coextensive.
As used herein, "administering" or "administration" means providing a pharmaceutical agent to an individual, and includes, but is not limited to, administering by a medical professional and self-administering.
Administration of a pharmaceutical agent to an individual can be continuous, chronic, short or intermittent.
Administration can parenteral or non-parenteral.
As used herein, "agent" means an active substance that can provide a therapeutic benefit when administered to an animal. "First agent" means a therapeutic compound of the invention. For example, a first agent can be an antisense oligonucleotide targeting apo(a). "Second agent"
means a second therapeutic compound of the invention (e.g. a second antisense oligonucleotide targeting apo(a)) and/or a non-apo(a) therapeutic compound.
As used herein, "amelioration" or "ameliorate" or "ameliorating" refers to a lessening of at least one indicator, sign, or symptom of an associated disease, disorder, or condition.
The severity of indicators can be determined by subjective or objective measures, which are known to those skilled in the art.

As used herein, "animal" refers to a human or non-human animal, including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including, but not limited to, monkeys and chimpanzees.
As used herein, "apo(a)" means any nucleic acid or protein sequence encoding apo(a). For example, in certain embodiments, apo(a) includes a DNA sequence encoding apo(a), a RNA
sequence transcribed from DNA encoding apo(a) (including genomic DNA comprising introns and exons), a mRNA sequence encoding apo(a), or a peptide sequence encoding apo(a).
As used herein, "apo(a) nucleic acid" means any nucleic acid encoding apo(a).
For example, in certain embodiments, an apo(a) nucleic acid includes a DNA sequence encoding apo(a), a RNA sequence transcribed from DNA encoding apo(a) (including genomic DNA comprising introns and exons), and a mRNA sequence encoding apo(a).
As used herein, "apo(a) mRNA" means a mRNA encoding an apo(a) protein.
As used herein, "apo(a) protein" means any protein sequence encoding Apo(a).
As used herein, "apo(a) specific inhibitor" refers to any agent capable of specifically inhibiting the expression of an apo(a) nucleic acid and/or apo(a) protein. For example, apo(a) specific inhibitors include nucleic acids (including antisense compounds), peptides, antibodies, small molecules, and other agents capable of inhibiting the expression of apo(a) nucleic acid and/or apo(a) protein. In certain embodiments, by specifically modulating apo(a) nucleic acid expression and/or apo(a) protein expression, apo(a) specific inhibitors can affect other components of the lipid transport system including downstream components.
Similarly, in certain embodiments, apo(a) specific inhibitors can affect other molecular processes in an animal.
As used herein, "atherosclerosis" means a hardening of the arteries affecting large and medium-sized arteries and is characterized by the presence of fatty deposits. The fatty deposits are called "atheromas" or "plaques," which consist mainly of cholesterol and other fats, calcium and scar tissue, and damage the lining of arteries.
As used herein, "coronary heart disease (CHD)" means a narrowing of the small blood vessels that supply blood and oxygen to the heart, which is often a result of atherosclerosis.
As used herein, "diabetes mellitus" or "diabetes" is a syndrome characterized by disordered metabolism and abnormally high blood sugar (hyperglycemia) resulting from insufficient levels of insulin or reduced insulin sensitivity. The characteristic symptoms are excessive urine production (polyuria) due to high blood glucose levels, excessive thirst and increased fluid intake (polydipsia) attempting to compensate for increased urination, blurred vision due to high blood glucose effects on the eye's optics, unexplained weight loss, and lethargy.
As used herein, "diabetic dyslipidemia" or "type 2 diabetes with dyslipidemia"
means a condition characterized by Type 2 diabetes, reduced HDL-C, elevated triglycerides (TG), and elevated small, dense LDL particles.

As used herein, "diluent" means an ingredient in a composition that lacks pharmacological activity, but is pharmaceutically necessary or desirable. For example, the diluent in an injected composition can be a liquid, e.g. saline solution.
As used herein, "dyslipidemia" refers to a disorder of lipid and/or lipoprotein metabolism, including lipid and/or lipoprotein overproduction or deficiency. Dyslipidemias can be manifested by elevation of lipids such as chylomicron, cholesterol and triglycerides as well as lipoproteins such as low-density lipoprotein (LDL) cholesterol.
As used herein, "dosage unit" means a form in which a pharmaceutical agent is provided, e.g. pill, tablet, or other dosage unit known in the art. In certain embodiments, a dosage unit is a vial containing lyophilized antisense oligonucleotide. In certain embodiments, a dosage unit is a vial containing reconstituted antisense oligonucleotide.
As used herein, "dose" means a specified quantity of a pharmaceutical agent provided in a single administration, or in a specified time period. In certain embodiments, a dose can be administered in one, two, or more boluses, tablets, or injections. For example, in certain embodiments where subcutaneous administration is desired, the desired dose requires a volume not easily accommodated by a single injection, therefore, two or more injections can be used to achieve the desired dose. In certain embodiments, the pharmaceutical agent is administered by infusion over an extended period of time or continuously. Doses can be stated as the amount of pharmaceutical agent per hour, day, week, or month.
Doses can also be stated as mg/kg or g/kg.
As used herein, "effective amount" or "therapeutically effective amount" means the amount of active pharmaceutical agent sufficient to effectuate a desired physiological outcome in an individual in need of the agent. The effective amount can vary among individuals depending on the health and physical condition of the individual to be treated, the taxonomic group of the individuals to be treated, the formulation of the composition, assessment of the individual's medical condition, and other relevant factors.
As used herein, "fully complementary" or "100% complementary" means each nucleobase of a nucleobase sequence of a first nucleic acid has a complementary nucleobase in a second nucleobase sequence of a second nucleic acid. In certain embodiments, a first nucleic acid is an antisense compound and a second nucleic acid is a target nucleic acid.
As used herein, "glucose" is a monosaccharide used by cells as a source of energy and inflammatory intermediate. "Plasma glucose" refers to glucose present in the plasma.
As used herein, "high density lipoprotein-C" or "HDL-C" means cholesterol associated with high density lipoprotein particles. Concentration of HDL-C in serum (or plasma) is typically quantified in mg/dL or nmon. "Serum HDL-C" and "plasma HDL-C" mean HDL-C in serum and plasma, respectively.
As used herein, "HMG-CoA reductase inhibitor" means an agent that acts through the inhibition of the enzyme HMG-CoA reductase, such as atorvastatin, rosuvastatin, fluvastatin, lovastatin, pravastatin, and simvastatin.

As used herein, "hypercholesterolemia" means a condition characterized by elevated cholesterol or circulating (plasma) cholesterol, LDL-cholesterol and VLDL-cholesterol, as per the guidelines of the Expert Panel Report of the National Cholesterol Educational Program (NCEP) of Detection, Evaluation of Treatment of high cholesterol in adults (see, Arch. Int. Med. (1988) 148, 36-39).
As used herein, "hyperlipidemia" or "hyperlipemia" is a condition characterized by elevated serum lipids or circulating (plasma) lipids. This condition manifests an abnormally high concentration of fats. The lipid fractions in the circulating blood are cholesterol, low density lipoproteins, very low density lipoproteins, chylomicrons and triglycerides. The Fredrickson classification of hyperlipidemias is based on the pattern of TG and cholesterol-rich lipoprotein particles, as measured by electrophoresis or ultracentrifugation and is commonly used to characterize primary causes of hyperlipidemias such as hypertriglyceridemia (Fredrickson and Lee, Circulation, 1965, 31:321-327; Fredrickson et al., New Eng J Med, 1967, 276 (1): 34-42).
As used herein, "hypertriglyceridemia" means a condition characterized by elevated triglyceride levels. Its etiology includes primary (i.e. genetic causes) and secondary (other underlying causes such as diabetes, metabolic syndrome/insulin resistance, obesity, physical inactivity, cigarette smoking, excess alcohol and a diet very high in carbohydrates) factors or, most often, a combination of both (Yuan et al.
CMAJ, 2007, 176:1113-1120).
As used herein, "identifying" or "selecting an animal with metabolic or cardiovascular disease"
means identifying or selecting a subject prone to or having been diagnosed with a metabolic disease, a cardiovascular disease, or a metabolic syndrome; or, identifying or selecting a subject having any symptom of a metabolic disease, cardiovascular disease, or metabolic syndrome including, but not limited to, hypercholesterolemia, hyperglycemia, hyperlipidemia, hypertriglyceridemia, hypertension increased insulin resistance, decreased insulin sensitivity, above normal body weight, and/or above normal body fat content or any combination thereof Such identification can be accomplished by any method, including but not limited to, standard clinical tests or assessments, such as measuring serum or circulating (plasma) cholesterol, measuring serum or circulating (plasma) blood-glucose, measuring serum or circulating (plasma) triglycerides, measuring blood-pressure, measuring body fat content, measuring body weight, and the like.
As used herein, "improved cardiovascular outcome" means a reduction in the occurrence of adverse cardiovascular events, or the risk thereof Examples of adverse cardiovascular events include, without limitation, death, reinfarction, stroke, cardiogenic shock, pulmonary edema, cardiac arrest, and atrial dysrhythmia.
As used herein, "immediately adjacent" means there are no intervening elements between the immediately adjacent elements, for example, between regions, segments, nucleotides and/or nucleosides.
As used herein, "increasing HDL" or "raising HDL" means increasing the level of HDL in an animal after administration of at least one compound of the invention, compared to the HDL level in an animal not administered any compound.

As used herein, "individual" or "subject" or "animal" means a human or non-human animal selected for treatment or therapy.
As used herein, "individual in need thereof' refers to a human or non-human animal selected for treatment or therapy that is in need of such treatment or therapy.
As used herein, "induce", "inhibit", "potentiate", "elevate", "increase", "decrease", "reduce" or the like denote quantitative differences between two states. For example, "an amount effective to inhibit the activity or expression of apo(a)" means that the level of activity or expression of apo(a) in a treated sample will differ from the level of apo(a) activity or expression in an untreated sample. Such terms are applied to, for example, levels of expression, and levels of activity.
As used herein, "inflammatory condition" refers to a disease, disease state, syndrome, or other condition resulting in inflammation. For example, rheumatoid arthritis and liver fibrosis are inflammatory conditions. Other examples of inflammatory conditions include sepsis, myocardial ischemia/reperfusion injury, adult respiratory distress syndrome, nephritis, graft rejection, inflammatory bowel disease, multiple sclerosis, arteriosclerosis, atherosclerosis and vasculitis.
As used herein, "inhibiting the expression or activity" refers to a reduction or blockade of the expression or activity of a RNA or protein and does not necessarily indicate a total elimination of expression or activity.
As used herein, "insulin resistance" is defined as the condition in which normal amounts of insulin are inadequate to produce a normal insulin response from fat, muscle and liver cells. Insulin resistance in fat cells results in hydrolysis of stored triglycerides, which elevates free fatty acids in the blood plasma. Insulin resistance in muscle reduces glucose uptake whereas insulin resistance in liver reduces glucose storage, with both effects serving to elevate blood glucose. High plasma levels of insulin and glucose due to insulin resistance often leads to metabolic syndrome and type 2 diabetes.
As used herein, "insulin sensitivity" is a measure of how effectively an individual processes glucose.
An individual having high insulin sensitivity effectively processes glucose whereas an individual with low insulin sensitivity does not effectively process glucose.
As used herein, "lipid-lowering" means a reduction in one or more lipids (e.g., LDL, VLDL) in a subject. "Lipid-raising" means an increase in a lipid (e.g., HDL) in a subject. Lipid-lowering or lipid-raising can occur with one or more doses over time.
As used herein, "lipid-lowering therapy" or "lipid lowering agent" means a therapeutic regimen provided to a subject to reduce one or more lipids in a subject. In certain embodiments, a lipid-lowering therapy is provided to reduce one or more of apo(a), CETP, apoB, total cholesterol, LDL-C, VLDL-C, IDL-C, non-HDL-C, triglycerides, small dense LDL particles, and Lp(a) in a subject. Examples of lipid-lowering therapy include, but are not limited to, apoB inhibitors, statins, fibrates and MTP inhibitors.
As used herein, "lipoprotein", such as VLDL, LDL and HDL, refers to a group of proteins found in the serum, plasma and lymph and are important for lipid transport. The chemical composition of each lipoprotein differs, for example, in that the HDL has a higher proportion of protein versus lipid, whereas the VLDL has a lower proportion of protein versus lipid.
As used herein, "Lp(a)" comprises apo(a) and a LDL like particle containing apoB. The apo(a) is linked to the apoB by a disulfide bond.
As used herein, "low density lipoprotein-cholesterol (LDL-C)" means cholesterol carried in low density lipoprotein particles. Concentration of LDL-C in serum (or plasma) is typically quantified in mg/dL
or nmon. "Serum LDL-C" and "plasma LDL-C" mean LDL-C in the serum and plasma, respectively.
As used herein, "major risk factors" refers to factors that contribute to a high risk for a particular disease or condition. In certain embodiments, major risk factors for coronary heart disease include, without limitation, cigarette smoking, hypertension, high LDL, low HDL-C, family history of coronary heart disease, age, and other factors disclosed herein.
As used herein, "metabolic disorder" or "metabolic disease" refers to a condition characterized by an alteration or disturbance in metabolic function. "Metabolic" and "metabolism"
are terms well known in the art and generally include the whole range of biochemical processes that occur within a living organism.
Metabolic disorders include, but are not limited to, hyperglycemia, prediabetes, diabetes (type 1 and type 2), obesity, insulin resistance, metabolic syndrome and dyslipidemia due to type 2 diabetes.
As used herein, "metabolic syndrome" means a condition characterized by a clustering of lipid and non-lipid cardiovascular risk factors of metabolic origin. In certain embodiments, metabolic syndrome is identified by the presence of any 3 of the following factors: waist circumference of greater than 102 cm in men or greater than 88 cm in women; serum triglyceride of at least 150 mg/dL;
HDL-C less than 40 mg/dL in men or less than 50 mg/dL in women; blood pressure of at least 130/85 mmHg;
and fasting glucose of at least 110 mg/dL. These determinants can be readily measured in clinical practice (JAMA, 2001, 285: 2486-2497).
"Parenteral administration" means administration through injection or infusion. Parenteral administration includes subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, or intracranial administration, e.g.
intrathecal or intracerebroventricular administration. Administration can be continuous, chronic, short or intermittent.
As used herein, "peptide" means a molecule formed by linking at least two amino acids by amide bonds. Peptide refers to polypeptides and proteins.
As used herein, "pharmaceutical agent" means a substance that provides a therapeutic benefit when administered to an individual. For example, in certain embodiments, an antisense oligonucleotide targeted to apo(a) is a pharmaceutical agent.
As used herein, "pharmaceutical composition" or "composition" means a mixture of substances suitable for administering to an individual. For example, a pharmaceutical composition can comprise one or more active agents and a pharmaceutical carrier e.g., a sterile aqueous solution.

As used herein, "pharmaceutically acceptable derivative" encompasses derivatives of the compounds described herein such as solvates, hydrates, esters, prodrugs, polymorphs, isomers, isotopically labelled variants, pharmaceutically acceptable salts and other derivatives known in the art.
As used herein, "pharmaceutically acceptable salts" means physiologically and pharmaceutically -- acceptable salts of antisense compounds, i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto. The term "pharmaceutically acceptable salt" or "salt" includes a salt prepared from pharmaceutically acceptable non-toxic acids or bases, including inorganic or organic acids and bases. "Pharmaceutically acceptable salts" of the compounds described herein may be prepared by methods well-known in the art. For a review of pharmaceutically acceptable salts, see -- Stahl and Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection and Use (Wiley-VCH, Weinheim, Germany, 2002). Sodium salts of antisense oligonucleotides are useful and are well accepted for therapeutic administration to humans. Accordingly, in one embodiment the compounds described herein are in the form of a sodium salt.
As used herein, "portion" means a defined number of contiguous (i.e. linked) nucleobases of a -- nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of a target nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of an antisense compound.
As used herein, "prevent"or "preventing" refers to delaying or forestalling the onset or development of a disease, disorder, or condition for a period of time from minutes to indefinitely. Prevent also means -- reducing risk of developing a disease, disorder, or condition.
As used herein, "raise" means to increase in amount. For example, to raise plasma HDL levels means to increase the amount of HDL in the plasma.
As used herein, "reduce" means to bring down to a smaller extent, size, amount, or number. For example, to reduce plasma triglyceride levels means to bring down the amount of triglyceride in the plasma.
As used herein, "region" or "target region" is defined as a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic. For example, a target region may encompass a 3' UTR, a 5' UTR, an exon, an intron, an exon/intron junction, a coding region, a translation initiation region, translation termination region, or other defined nucleic acid region. The structurally defined regions for apo(a) can be obtained by accession number from sequence databases such as NCBI and such information is -- incorporated herein by reference. In certain embodiments, a target region may encompass the sequence from a 5' target site of one target segment within the target region to a 3' target site of another target segment within the target region.
As used herein, "second agent" or "second therapeutic agent" means an agent that can be used in combination with a "first agent". A second therapeutic agent can include, but is not limited to, antisense -- oligonucleotides targeting apo(a) or apoB. A second agent can also include anti- apo(a) antibodies, apo(a) peptide inhibitors, cholesterol lowering agents, lipid lowering agents, glucose lowering agents and anti-inflammatory agents.
As used herein, "segments" are defined as smaller, sub-portions of regions within a nucleic acid. For example, a "target segment" means the sequence of nucleotides of a target nucleic acid to which one or more antisense compounds is targeted. "5' target site" refers to the 5'-most nucleotide of a target segment. "3' target site" refers to the 3'-most nucleotide of a target segment.
Alternatively, a "start site" can refer to the 5'-most nucleotide of a target segment and a "stop site" refers to the 3'-most nucleotide of a target segment. A
target segment can also begin at the "start site" of one sequence and end at the "stop site" of another sequence.
As used herein, "statin" means an agent that inhibits the activity of HMG-CoA
reductase.
As used herein, "subcutaneous administration" means administration just below the skin.
As used herein, "subject" means a human or non-human animal selected for treatment or therapy.
As used herein, "symptom of cardiovascular disease or disorder" means a phenomenon that arises from and accompanies the cardiovascular disease or disorder and serves as an indication of it. For example, angina; chest pain; shortness of breath; palpitations; weakness; dizziness;
nausea; sweating; tachycardia;
bradycardia; arrhythmia; atrial fibrillation; swelling in the lower extremities; cyanosis; fatigue; fainting;
numbness of the face; numbness of the limbs; claudication or cramping of muscles; bloating of the abdomen;
or fever are symptoms of cardiovascular disease or disorder.
As used herein, "targeting" or "targeted" means the process of design and selection of an antisense compound that will specifically hybridize to a target nucleic acid and induce a desired effect.
As used herein, "therapeutically effective amount" means an amount of a pharmaceutical agent that provides a therapeutic benefit to an individual.
As used herein, "therapeutic lifestyle change" means dietary and lifestyle changes intended to lower fat/adipose tissue mass and/or cholesterol. Such change can reduce the risk of developing heart disease, and may includes recommendations for dietary intake of total daily calories, total fat, saturated fat, polyunsaturated fat, monounsaturated fat, carbohydrate, protein, cholesterol, insoluble fiber, as well as recommendations for physical activity.
As used herein, "treat" or "treating" refers to administering a compound described herein to effect an alteration or improvement of a disease, disorder, or condition.
As used herein, "triglyceride" or "TG" means a lipid or neutral fat consisting of glycerol combined with three fatty acid molecules.
As used herein, "type 2 diabetes," (also known as "type 2 diabetes mellitus", "diabetes mellitus, type 2", "non-insulin-dependent diabetes", "NIDDM", "obesity related diabetes", or "adult-onset diabetes") is a metabolic disorder that is primarily characterized by insulin resistance, relative insulin deficiency, and hyperglycemia.

Certain Embodiments In certain embodiments, a compound comprises a siRNA or antisense oligonucleotide targeted to apolipoprotein(a) (apo(a)) known in the art and a conjugate group described herein. Examples of antisense oligonucleotides targeted to apo(a) suitable for conjugation include but are not limited to those disclosed in WO 2013/177468; US 8,673,632; US 7,259,150; and US Patent Application Publication No. US
2004/0242516; which are incorporated by reference in their entireties herein.
In certain embodiments, a compound comprises an antisense oligonucleotide having a nucleobase sequence of any of SEQ ID NOs 12-130, 133, 134 disclosed in WO 2013/177468 and a conjugate group described herein. In certain embodiments, a compound comprises an antisense oligonucleotide having a nucleobase sequence of any of SEQ ID NOs 11-45 and 85-96 disclosed in US 8,673,632 and a conjugate group described herein. In certain embodiments, a compound comprises an antisense oligonucleotide having a nucleobase sequence of any of SEQ ID NOs 11-45 disclosed in US 7,259,150 and a conjugate group described herein. In certain embodiments, a compound comprises an antisense oligonucleotide having a nucleobase sequence of any of SEQ ID NOs 7-41 disclosed in US Patent Application Publication No. US
2004/0242516 and a conjugate group described herein. The nucleobase sequences of all of the aforementioned referenced SEQ ID NOs are incorporated by reference herein.
Certain embodiments provide a compounds and methods for decreasing apo(a) mRNA
and protein expression. In certain embodiments, the compound is an apo(a) specific inhibitor for treating, preventing, or ameliorating an apo(a) associated disease. In certain embodiments, the compound is an antisense oligonucleotide targeting apo(a). In certain embodiments, the compound is an antisense oligonucleotide targeting apo(a) and a conjugate group.
Certain embodiments provide a compounds and methods for decreasing Lp(a) levels. In certain embodiments, the compound is an apo(a) specific inhibitor for treating, preventing, or ameliorating an Lp(a) associated disease. In certain embodiments, the compound is an antisense oligonucleotide targeting apo(a). In certain embodiments, the compound is an antisense oligonucleotide targeting apo(a) and a conjugate group.
Certain embodiments provide a compound comprising a modified oligonucleotide targeting apo(a) and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides. In certain embodiments, the modified oligonucleotide with the conjugate group consists of 15 to 30, 18 to 24, 19 to 22, 13 to 25, 14 to 25, 15 to 25 linked nucleosides. In certain embodiments, the modified oligonucleotide with the conjugate group comprises at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29 or 30 linked nucleosides. In certain embodiments, the modified oligonucleotide with the conjugate group consists of 20 linked nucleosides.
Certain embodiments provide a compound comprising a modified oligonucleotide targeting apo(a) and a conjugate group, wherein the modified oligonucleotide comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 contiguous nucleobases complementary to an equal length portion of any of SEQ
ID NOs: 1-4.
Certain embodiments provide a compound comprising a modified oligonucleotide targeting an apo(a) segment and a conjugate group, wherein the modified oligonucleotide comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 contiguous nucleobases complementary to an equal length portion of any of the target segments shown in, for example, Examples 114 and 117. In the tables, the "Start Site" refers to the 5'-most nucleotide of a target segment and "Stop Site" refers to the 3'-most nucleotide of a target segment.
A target segment can range from the start site to the stop site of each sequence listed in the tables.
Alternatively, the target segment can range from the start site of one sequence and end at the stop site of another sequence. For example, as shown in Table 125, a target segment can range from 3901-3920, the start site to the stop site of SEQ ID NO: 58. In another example, as shown in Table 125, a target segment can range from 3900-3923, the start site of SEQ ID
NO: 57 to the stop site of SEQ ID NO: 61.
Certain embodiments provide a compound comprising a modified oligonucleotide targeting apo(a) and a conjugate group, wherein the nucleobase sequence of the modified oligonucleotide is at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to any of SEQ ID
NOs: 1-4. Certain embodiments provide a compound comprising a modified oligonucleotide targeting apo(a) and a conjugate group, wherein the nucleobase sequence of the modified oligonucleotide is at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to any of the target segments shown in, for example, Examples 114 and 117.
Certain embodiments provide a compound comprising a modified oligonucleotide targeting apo(a) and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and comprises a nucleobase sequence comprising a portion of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 contiguous nucleobases complementary to an equal length portion of nucleobases 3901 to 3920 of SEQ ID NO: 1, wherein the nucleobase sequence of the modified oligonucleotide is at least 80% complementary to SEQ ID
NO: 1.
Certain embodiments provide a compound comprising a modified oligonucleotide targeting apo(a) and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and comprises a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29 or 30 contiguous nucleobases complementary to an equal length portion of nucleobases 3900 to 3923 of SEQ ID
NO: 1, wherein the nucleobase sequence of the modified oligonucleotide is at least 80%
complementary to SEQ ID NO: 1.
Certain embodiments provide a compound comprising a modified oligonucleotide targeting apo(a) and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and has a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 12-130, 133, 134. In certain embodiments, the modified oligonucleotide has a nucleobase sequence comprising at least 8 contiguous nucleobases of any one of the nucleobase sequences of SEQ ID NOs: 12-130, 133, 134. In certain embodiments, the compound consists of any one of SEQ ID NOs: 12-130, 133, 134 and a conjugate group.
Certain embodiments provide a compound comprising a modified oligonucleotide targeting apo(a) and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and has a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 12-20, 22-33, 35-44, 47-50, 51, 53, 57-62, 65-66, 68, 70-79, 81, 85-86, 89-90, 92-94, 97, 105-110, 103-104, 133-134. In certain embodiments, the compound consists of any of the nucleobase sequences of SEQ ID NOs: 12-20, 22-33, 35-44, 47-50, 51, 53, 57-62, 65-66, 68, 70-79, 81, 85-86, 89-90, 92-94, 97, 105-110, 103-104, 133-134 and a conjugate group.
Certain embodiments provide a compound comprising a modified oligonucleotide targeting apo(a) and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and has a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 12-19, 26-30, 32, 35, 38-44, 46-47, 50, 57-58, 61, 64-66, 68, 72-74, 76-77, 92-94, 103-110. In certain embodiments, the compound consists of any of the nucleobase sequences of SEQ ID NOs: 12-19, 26-30, 32, 35, 38-44, 46-47, 50, 57-58, 61, 64-66, 68, 72-74, 76-77, 92-94, 103-110 and a conjugate group.
Certain embodiments provide a compound comprising a modified oligonucleotide targeting apo(a) and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and has a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 111, 114-121, 123-129. In certain embodiments, the compound consists of any of the nucleobase sequences of SEQ ID NOs: 111, 114-121, 123-129 and a conjugate group.
Certain embodiments provide a compound comprising a modified oligonucleotide targeting apo(a) and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and has a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 14, 17, 18, 26-28, 39, 71, 106-107. In certain embodiments, the compound consists of any of the nucleobase sequences of SEQ ID NOs: 14, 17, 18, 26-28, 39, 71, 106-107 and a conjugate group.

Certain embodiments provide a compound comprising a modified oligonucleotide targeting apo(a) and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and has a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 14, 26-29, 39-40, 82. In certain embodiments, the compound consists of any of the nucleobase sequences of SEQ ID NOs: 14, 26-29, 39-40, 82 and a conjugate group.
Certain embodiments provide a compound comprising a modified oligonucleotide targeting apo(a) and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and has a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 14, 16-18. In certain embodiments, the compound consists of any of the nucleobase sequences of SEQ ID NOs: 14, 16-18 and a conjugate group.
Certain embodiments provide a compound comprising a modified oligonucleotide targeting apo(a) and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and has a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 26-27, 107. In certain embodiments, the compound consists of any of the nucleobase sequences of SEQ ID NOs: 26-27, 107 and a conjugate group.
Certain embodiments provide a compound comprising a modified oligonucleotide targeting apo(a) and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and has a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 28-29, 39-40, 47. In certain embodiments, the compound consists of any of the nucleobase sequences of SEQ ID NOs: : 28-29, 39-40, 47 and a conjugate group.
Certain embodiments provide a compound comprising a modified oligonucleotide targeting apo(a) and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and has a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 28, 93, 104, 134. In certain embodiments, the compound consists of any of the nucleobase sequences of SEQ ID NOs: 28, 93, 104, 134 and a conjugate group.
Certain embodiments provide a compound comprising a modified oligonucleotide targeting apo(a) and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and has a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 contiguous nucleobases of the nucleobase sequence of SEQ ID NO: 58. In certain embodiments, the modified oligonucleotide with the conjugate group has a nucleobase sequence comprising at least 8 contiguous nucleobases of the nucleobase sequence of SEQ
ID NO: 58. In certain embodiments, the compound consists of SEQ ID NO: 58 and a conjugate group.
In certain embodiments, the present disclosure provides conjugated antisense compounds represented by the following structure. In certain embodiments, the antisense compound comprises the modified oligonucleotide ISIS 494372 with a 5'-X, wherein X is a conjugate group comprising GalNAc. In certain embodiments, the antisense compound consists of the modified oligonucleotide ISIS 494372 with a 5'-X, wherein X is a conjugate group comprising GalNAc.
o Ill'r o o o NO '1111N1r )2L7/ 'IfjINyH
1\11O
o' 0 01 C)) o coj co_i S-P=0 N 0 0 1 21)(IXo e 1 (:)\,1_4/N N NH2 S-P=0 'IfjINyH 0,S-P=0 '111INNH
o1 o1---V24/N0 o Nvmmy/N-10 o' o 0 0õ) NH2 o' ' o o o,) e 1.0 N 0 1 N o o N
o 1 S-P=0 O ,lcm.(2.32erst-AINH2 9 1 S-P=0 N 0 \1 ol :LAX
o 0 O)0 0 cli 1 e I o' S-P0 S -P==0 N
O 1112.1H o Ir l 0, 2L111-X o,) o 8 ' S-P=0 _0_yN 0 .....IL
O
0 o \ I NI-.0 0 7 0,) NH2 o & 1 S-P=0 1111.IH
S-P=0 o1 /
....r('N
01 I 0) _04zN 0 e 01 0 o o S-P=0 N
o1 A-kr Y o) NH2 9 1 S -P=0 N1--.0 S-P=0 N
O X.112X1 ....r.0 I ,1 \ N NNH2 o' c - -oj c_o_) 0) N 0 e NH2 o 0 o a , o ' N S-P=0 9 1 N S-P,1=0 I 01 N
S-P=0 111:11E.1 0 I
,,Icomm4 N NH2 O I

cON 0 e 1 o S-P=0 S-P=0 O ____________ O ____________________________________ In certain embodiments, the present disclosure provides conjugated antisense compounds represented by the following structure. In certain embodiments, the antisense compound comprises the conjugated modified oligonucleotide ISIS 681251. In certain embodiments, the antisense compound consists of the conjugated modified oligonucleotide ISIS 681251.
O
c) HO OH o 0-F' =0 'NC-4C , Nf NH
_____________________________________________________ ()_)/N N NH2 ---,ir NH No csi 0 HO OH 0 0 e ') 0 e0 SO
4 H oI (!µ111"-tr S-F' =0 ILL'i X
)_ (,N 0 HO OH N

---j 2 S-=O /1-' S-F' =0 HO 0-'11---N 0 NH

)_ o a 0 0 0 o') 0 S-F' =0 1 o-----121N 0 S-P=0 Arll'NH

0__ S-P=0 A'41 NH

0 0.,) 0 ,,.-., "'S-p =0 '-'"(LN

1c2i 0 N
, 0 a NH
L
0 0 o,) NH2 S-F' =0 SP SO

-S-=O NTIL'NH
00 ) I
S- r O F'=0 KI-It--:

0 0 (j) 0 e o NH

S-P =0 S-I's '''CjtHNH
0N N __ 0 N ''0 )_04/cy, o a o,1 0 s-=o A---4-1 X e S-P =0 0.--,IN 0 0 N

e N
----) NH2 S-P =0 Irlhi e 0 o N N NH2 S-P =0 A--)."'N
--1_0_)1 0 1 p o S-P =0 ) OH 05) o _________ In certain embodiments, the present disclosure provides conjugated antisense compounds represented by the following structure. In certain embodiments, the antisense compound comprises the conjugated modified oligonucleotide ISIS 681257. In certain embodiments, the antisense compound consists of the conjugated modified oligonucleotide ISIS 681257.
O

HO OH 0 0-F' =0 '1)1'1 N4F1 Nf NH
I
()_)/N N NH2 --,[r NH 'No cv 0 HO OH 0 N 0 e 0 (:)--) o e 0 SO
HO 01-rN
/ tar S-F' =0 'XLIC,ZIF1 HO OH
9 0-_,-i NH2 0 N
_.....72..v SF' =0 Y 1r HO 0-'11---N 0 F' =0 N
.-1''''N N NH2 N--.0 0 )_A
0_/cy, e 0 e 0 0) 0 S_=0 1 o-----121N 0 0-p=0 NH

0 S-P=0 'fl NH
O 0,) NH2 0 N-,,.-., `-'0-p =0 '1"-LN

1c2i 0 O y0 N
(:t 0 0 0 ill' NH
0 0 CO NH2 S-F' =0 ---o OP SO

-O-P =0 NI-11' NH
00 ) 1 S- r O F'=0 ( 0 Nl N NH2 CLiN N NH2 NHS-P =0 11 I's 'NH

N ''0 04/ o., e s-=o 11 X e S-P =0 lUir 0.,,i_)1 0 (D1_0/N 0 e N lhi S-P =0 O N"' eI
N NH2 S-P =0 'N
---01 o I

op o S-P =0 ) OH 0õ i 0 _____________________________________________________________ In certain embodiments, the present disclosure provides conjugated antisense compounds represented by the following structure. In certain embodiments, the antisense compound comprises a modified oligonucleotide with the nucleobase sequence of SEQ ID NO: 58 with a 5'-GalNAc with variability in the sugar mods of the wings. In certain embodiments, the antisense compound consists of a modified oligonucleotide with the nucleobase seuquence of SEQ ID NO: 58 with a 5'-GalNAc with variability in the sugar mods of the wings.
O

HO OH 0 ,0 -IC2=0 R51-1t- 0 Nil. H
Nlit NH
__...72...\ HN 0o N0I

(r r NH

es_1C2so R5IAINI,H
HO 01-r N

i 9 (L;II-11'N --- _1 --Y-FINH2 0 N¨so - Tr NH OZ

_....72..vS-P =0 (!µif NH

Z-=0 R5I.---1,-,..

- Tr NH 0 ,I ,L1 R2 NO ' (:1 R5 a 0 0 0 S-P=0 RI I .L1 Z-P =0 N
R5,e, O NH
N0 o--I-R) oll R4 o , IR
- R5'C NH
0 R" NH2 S0 i 0 R)p/N --'-'0 Z-F1' =0 R51)..11..

i¨' 47N0 R3 R5'CI NH
a 0 0 R3 IR,,cN 0 NH2 5-F=O
Z-P =0 'N 0------)N ''0 0--__ ZR7DPN)p/s R3 <IN NN H2 NFI
I----i0 R41¨r, 0 S-F'=0 N
ol__/ N1HNH2 R4 , 0 R51) NH
_ p=
o IR- R511 Z'C
S-F' =0 NH 1 ,,.., O
o o N

OP R5e NH
S-F' =0 1 L 0 R5rit.NH
N,o 11'() 0 oi_04/
0S CI 2 = 0 ' I 11 - 1 I - --, -N-11-,H Ri NH2 N N NH2 e R5, -P =0 N

e IR
s-=0 ) OH R, 0 _______ Wherein either R1 is ¨OCH2CH2OCH3 (MOE) and R2 is H; or R1 and R2 together form a bridge, wherein R1 is ¨0- and R2 is ¨CH2-, -CH(CH3)-, or -CH2CH2-, and R1 and R2 are directly connected such that the resulting bridge is selected from: -0-CH2-, -0-CH(CH3)-, and ¨0-CH2CH2-;

And for each pair of R3 and R4 on the same ring, independently for each ring:
either R3 is selected from H and -OCH2CH2OCH3 and R4 is H; or R3 and R4 together form a bridge, wherein R3 is ¨0-, and R4 is ¨
CH2-, -CH(CH3)-, or -CH2CH2-and R3 and R4 are directly connected such that the resulting bridge is selected from: -0-CH2-, -0-CH(CH3)-, and ¨0-CH2CH2-;
And R5 is selected from H and ¨CH3;
And Z is selected from S- and 0-.
Certain embodiments provide a compound comprising a modified oligonucleotide targeting apo(a) and a conjugate group, wherein the modified oligonucleotide is single-stranded.
Certain embodiments provide a compound comprising a modified oligonucleotide targeting apo(a) and a conjugate group, wherein at least one internucleoside linkage is a modified internucleoside linkage. In certain embodiments, the modified internucleoside linkage is a phosphorothioate internucleoside linkage. In certain embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 internucleoside linkages of said modified oligonucleotide are phosphorothioate internucleoside linkages. In certain embodiments, each internucleoside linkage is a phosphorothioate internucleoside linkage. In certain embodiments, the modified oligonucleotide comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 phosphodiester internucleoside linkages. In certain embodiments, each internucleoside linkage of the modified oligonucleotide is selected from a phosphodiester internucleoside linkage and a phosphorothioate internucleoside linkage.
Certain embodiments provide a compound comprising a modified oligonucleotide targeting apo(a) and a conjugate group, wherein at least one nucleoside comprises a modified nucleobase. In certain embodiments, the modified nucleobase is a 5-methylcytosine.
Certain embodiments provide a compound comprising a modified oligonucleotide targeting apo(a) and a conjugate group, wherein the modified oligonucleotide comprises at least one modified sugar. In certain embodiments, the modified sugar is a bicyclic sugar. In certain embodiments, the modified sugar comprises a 2'-0-methoxyethyl, a constrained ethyl, a 3'-fluoro-HNA or a 4'- (CH2)-0-2' bridge, wherein n is 1 or 2.
Certain embodiments provide a compound comprising a modified oligonucleotide targeting apo(a) and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and comprises: (a) a gap segment consisting of linked deoxynucleosides; (b) a 5' wing segment consisting of linked nucleosides; (c) a 3' wing segment consisting of linked nucleosides;
and wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.
Certain embodiments provide a compound comprising a modified oligonucleotide targeting apo(a) and a conjugate group, wherein the modified oligonucleotide consists of 20 linked nucleosides and comprises: (a) a gap segment consisting of ten linked deoxynucleosides; (b) a 5' wing segment consisting of five linked nucleosides; (c) a 3' wing segment consisting of five linked nucleosides; and wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment, wherein each nucleoside of each wing segment comprises a 2'-0-methoxyethyl sugar, wherein at least one internucleoside linkage is a phosphorothioate linkage and wherein each cytosine residue is a 5-methylcytosine.
Certain embodiments provide a compound comprising a modified oligonucleotide targeting apo(a) and a conjugate group, wherein the modified oligonucleotide consists of 20 linked nucleosides and has a nucleobase sequence comprising at least 8 contiguous nucleobases of any of SEQ
ID NOs: 12-130, 133, 134, wherein the modified oligonucleotide comprises: (a) a gap segment consisting of ten linked deoxynucleosides; (b) a 5' wing segment consisting of five linked nucleosides;
(c) a 3' wing segment consisting of five linked nucleosides; and wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment, wherein each nucleoside of each wing segment comprises a 2'-0-methoxyethyl sugar, wherein at least one internucleoside linkage is a phosphorothioate linkage and wherein each cytosine residue is a 5-methylcytosine.
Certain embodiments provide a compound comprising a modified oligonucleotide targeting apo(a) and a conjugate group, wherein the modified oligonucleotide consists of 20 linked nucleosides and has a nucleobase sequence comprising at least 8 contiguous nucleobases of SEQ ID NO:
58, wherein the modified oligonucleotide comprises: (a) a gap segment consisting of ten linked deoxynucleosides; (b) a 5' wing segment consisting of five linked nucleosides; (c) a 3' wing segment consisting of five linked nucleosides;
and wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment, wherein each nucleoside of each wing segment comprises a 2'-0-methoxyethyl sugar, wherein at least one internucleoside linkage is a phosphorothioate linkage and wherein each cytosine residue is a 5-methylcytosine.
Certain embodiments provide a modified oligonucleotide targeting apo(a) and a conjugate group, wherein the modified oligonucleotide consists of 20 linked nucleosides with the nucleobase sequence of SEQ
ID NO: 58, wherein the modified oligonucleotide comprises: (a) a gap segment consisting of ten linked deoxynucleosides; (b) a 5' wing segment consisting of five linked nucleosides;
(c) a 3' wing segment consisting of five linked nucleosides; and wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment, wherein each nucleoside of each wing segment comprises a 2'-0-methoxyethyl sugar, wherein at least one internucleoside linkage is a phosphorothioate linkage and wherein each cytosine residue is a 5-methylcytosine.
In certain embodiments, the conjugate group is linked to the modified oligonucleotide at the 5' end of the modified oligonucleotide. In certain embodiments, the conjugate group is linked to the modified oligonucleotide at the 3' end of the modified oligonucleotide.
In certain embodiments, the conjugate group comprises one or more ligands. In certain embodiments, the conjugate group comprises two or more ligands. In certain embodiments, the conjugate group comprises three or more ligands. In certain embodiments, the conjugate group comprises three ligands. In certain embodiments, each ligand is selected from among: a polysaccharide, modified polysaccharide, mannose, galactose, a mannose derivative, a galactose derivative, D-mannopyranose, L-Mannopyranose, D-Arabinose, L-Galactose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-Galactose, L-Galactose, a-D-Mannofuranose, 13-D-Mannofuranose, a-D-Mannopyranose, 13-D-Mannopyranose, a-D-Glucopyranose, [3-D-Glucopyranose, a-D-Glucofuranose, 13-D-Glucofuranose, a-D-fructofuranose, a-D-fructopyranose, a-D-Galactopyranose, [3 -D-Galactopyranose, a-D-Galactofuranose, [3 -D-Galactofuranose, glucosamine, sialic acid, a-D-galactosamine, N-Acetylgalactosamine, 2-Amino-3-0-[(R)-1-carboxyethy1]-2-deoxy-13-D-glucopyranose, 2-Deoxy-2-methylamino-L-glucopyranose, 4,6-Dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose, 2-Deoxy-2-sulfoamino-D-glucopyranose, N-Glycoloyl-a-neuraminic acid, 5-thio-13-D-glucopyranose, methyl 2,3,4-tri-0-acety1-1-thio-6-0-trityl-a-D-glucopyranoside, 4-Thio-13-D-galactopyranose, ethyl 3,4,6,7-tetra-0-acety1-2-deoxy-1,5-dithio-a-D-g/uco-heptopyranoside, 2,5-Anhydro-D-allononitrile, ribose, D-ribose, D-4-thioribose, L-ribose, L-4-thioribose.
In certain embodiments, each ligand is N-acetyl galactosamine.
In certain embodiments, each ligand is N-acetyl galactosamine.
In certain embodiments, the conjugate group comprises:
HO H
AcHNN-------)r-N H
0 \N
1r o 0 H H ,..., - )LVA,,, fr.su \
N--I----N-,.....,....--NH IN-k,,, ,2)6-0.¨

HO
HNN____j NHAc H
OH

HO\
HOk):\>:"
NHAc In certain embodiments, the conjugate group comprises:

,Ic 0---Tri. hi Ho__T-2-\/-AcHN N
ON
HOOH 0 o 0 0.--iõ...c. =H
AcHN 0Z
HOOH
--CI
0.--Tc=H 0 AcHN
In certain embodiments, the conjugate group comprises:
HOOH
H
HO
---../
AcHN

)L

H
AcHN
---(HO OH
HO_.....r2..\,0,,w¨H 0 \ 14 AcHN
In certain embodiments, the conjugate group comprises:
OH OH

H0.7.2,_ AcHN
OH OH
HO*,_0 HO H (:) 0-......õ---..õ---,,,11, N ....¨.........õ,.........N--K6 e AcHN INI 0 11 0 HO OH
HO 0 ..._./.,,,/ j--NH
.....r?..\/
NHAc In certain embodiments, the conjugate group comprises:

:OH
HO OH
HO
ON

AcHN
0=P¨OH
HOOH
HOOf AcHN
0=P¨OH

HOOH
HO0rNR____0 AcHN
In certain embodiments, the conjugate group comprises at least one phosphorus linking group or neutral linking group.
In certain embodiments, the conjugate group comprises a structure selected from among:
OH OH

OH "3 3 OH OH "3 "3 cssYYLN H)'t cleYLN 6 6 OH =
0 and 0 cs&(,),(HIHN)22- ' cssL(NN 0 OH
m wherein n is from 1 to 12; and wherein m is from 1 to 12.

In certain embodiments, the conjugate group has a tether having a structure selected from among:
0 Zi cssH-1¨HA and csssNI-Se2"
mi mi mi H ml wherein L is either a phosphorus linking group or a neutral linking group;
Z1 is C(=0)0-R2;
Z2 is H, C1-C6 alkyl or substituted C1-C6 alky;
R2 is H, C1-C6 alkyl or substituted C1-C6 alky; and each ml is, independently, from 0 to 20 wherein at least one ml is greater than 0 for each tether.
In certain embodiments, conjugate group has a tether having a structure selected from among:
COOH OH
4*µ
k+0-1g-0 and \. l(,ANO¨. ¨0 m1 mi I mi mi wherein Z2 is H or CH3; and each ml is, independently, from 0 to 20 wherein at least one ml is greater than 0 for each tether.
In certain embodiments, the conjugate group has tether having a structure selected from among:
csss H m HN 0 m OH

=
wherein n is from 1 to 12; and wherein m is from 1 to 12.
In certain embodiments, the conjugate group is covalently attached to the modified oligonucleotide.
In certain embodiments, the compound has a structure represented by the formula:

A¨B¨C¨D¨EE¨F) q wherein A is the modified oligonucleotide;
B is the cleavable moiety C is the conjugate linker D is the branching group each E is a tether;
each F is a ligand; and q is an integer between 1 and 5.
In certain embodiments, the compound has a structure represented by the formula:
A ¨(¨B ) ( C ) ( D ) ( E F) n2 ni n3 q wherein:
A is the modified oligonucleotide;
B is the cleavable moiety C is the conjugate linker D is the branching group each E is a tether;
each F is a ligand;
each n is independently 0 or 1; and q is an integer between 1 and 5.
In certain embodiments, the compound has a structure represented by the formula:
A¨B¨C¨( E¨F) q wherein A is the modified oligonucleotide;
B is the cleavable moiety;
C is the conjugate linker;
each E is a tether;

each F is a ligand; and q is an integer between 1 and 5.
In certain embodiments, the compound has a structure represented by the formula:
A ¨C¨D¨EE¨F) q wherein A is the modified oligonucleotide;
C is the conjugate linker;
D is the branching group;
each E is a tether;
each F is a ligand; and q is an integer between 1 and 5.
In certain embodiments, the compound has a structure represented by the formula:
A ¨C¨( E¨F) q wherein A is the modified oligonucleotide;
C is the conjugate linker;
each E is a tether;
each F is a ligand; and q is an integer between 1 and 5.
In certain embodiments, the compound has a structure represented by the formula:
A ¨B¨D¨EE¨F) q wherein A is the modified oligonucleotide;
B is the cleavable moiety;
D is the branching group;

each E is a tether;
each F is a ligand; and q is an integer between 1 and 5.
In certain embodiments, the compound has a structure represented by the formula:
A¨B¨(E¨F) q wherein A is the modified oligonucleotide;
B is the cleavable moiety;
each E is a tether;
each F is a ligand; and q is an integer between 1 and 5.
In certain embodiments, the compound has a structure represented by the formula:
A¨D¨(¨E¨F) q wherein A is the modified oligonucleotide;
D is the branching group;
each E is a tether;
each F is a ligand; and q is an integer between 1 and 5.

In certain embodiments, the conjugate linker has a structure selected from among:

H
H H N
µ)Le- N I \ I = µ)Yj N N

; n µ) ;
n0 n H , n n H 0 0 , 0 µ)yr .
, ; " n n sk N H , N n 0 ,n H n IN ')iiN'-:,1,/1 iLL? . 1 \ ;
, EN-I =
H õ N , n Clti ' 0 .. H

H H H
H N
1.(01;
n n n n H H H
H
= ci IV -rN.HO CrH n ' n 0 0 OH OH
¨L-,...1.....\-0-,H------0.*õ..y-L .
"n_ n -n _ n _ -n rcYti)LNYL cs.s5 and ssss=L' n N

wherein each L is, independently, a phosphorus linking group or a neutral linking group; and each n is, independently, from 1 to 20.

In certain embodiments, the conjugate linker has a structure selected from among:

H
H N
)NA ' .
, H H 0 , , .

osr / A..
,---11----(õ1-^---N\ ; cly-H-8y\. ;

H
H
csssyK8r N cX0/\/N csss ; H
oss EN1 "s H
N sss ;
QEN-I ,s, s 1,00õ,õss ;

H
csssr 1\k'O//N c,ss ;

OH ''3 3 H

1-0¨P-00 0 0¨A-0-1 ; qsscrHJ(3 N '2'2.6 and 01) - 0-1 H " 60H ' In certain embodiments, the conjugate linker has the followingstructure:
Jsr54 O NO
)227-In certain embodiments, the conjugate linker has a structure selected from among:
ss.Cosss ; ssCoosss ; and In certain embodiments, the conjugate linker has a structure selected from among:
/OH
/OH

I I and 001,A,Oss I
OH 3 '3 OH OH 3 "3 r" =
In certain embodiments, the conjugate linker has a structure selected from among:

; csWN
3 H 6 OH 0 6 and vss'W H

In certain embodiments, the conjugate linker comprises a pyrrolidine. In certain embodiments, the conjugate linker does not comprise a pyrrolidine. In certain embodiments, the conjugate linker comprises PEG. In certain embodiments, the conjugate linker comprises an amide. In certain embodiments, the conjugate linker comprises at least two amides. In certain embodiments, the conjugate linker does not comprise an amide. In certain embodiments, the conjugate linker comprises a polyamide. In certain embodiments, the conjugate linker comprises an amine. In certain embodiments, the conjugate linker comprises one or more disulfide bonds. In certain embodiments, the conjugate linker comprises a protein binding moiety. In certain embodiments, the protein binding moiety comprises a lipid.

In certain embodiments, the protein binding moiety is selected from among:
cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-0(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, 03-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine), a vitamin (e.g., folate, vitamin A, vitamin E, biotin, pyridoxal), a peptide, a carbohydrate (e.g., monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, polysaccharide), an endosomolytic component, a steroid (e.g., uvaol, hecigenin, diosgenin), a terpene (e.g., triterpene, e.g., sarsasapogenin, friedelin, epifriedelanol derivatized lithocholic acid), or a cationic lipid.
In certain embodiments, the protein binding moiety is selected from among: a C16 to C22 long chain saturated or unsaturated fatty acid, cholesterol, cholic acid, vitamin E, adamantane or 1-pentafluoropropyl.
In certain embodiments, the conjugate linker has a structure selected from among:

H H 1¨NH
,22(NN I

0 0).2- II
0 ¨P-0 H
N I
N CNO.,F

H
1¨N 'HI-0 µ,N,Hr<1 ' , H ( L0¨i ( )n N I

4.
r0¨t0H , II
N I 0 1\l'C)cs.s' 1¨NH
I OH .
/ N OH ;
P I
I I
0 04.
0, ,0 I
-.4-0;1' eOH 04.
N (:),ss' O

n 0 Y 'S-SIO
H H N
csss N , riss S' 'KLI 0 N ''Hrsri 191-0 S
H

I
04, -csss mn =

I
'(-4):on JN

I
0 00, ,0 \ , ... 0..._00,; P,9 iOOH
OHJn \ /-- O\ ) S¨S n 0 N
1-1¨ffl -10 H N , and cs's H
N ("ryi N'Hii.L0 v N.Hri.LO H

wherein each n is, independently, is from 1 to 20; and p is from 1 to 6.

In certain embodiments, the conjugate linker has a structure selected from among:
!Pr' \ (:).
(:).
N

_r-r44 \
q, NO
rrsj 0 N \
H H q n =Hi..r= H Nri ;

20;aeL

I H
µ)NyHINN[\11 .
O -^1.
Prc4 0 q.
N )0A
\
q H
csc. ,(,:yS.,sXHiNI ,c) ' N )0A

H
"sy)1LN 1\1)(0 =
in n H ' OA \2.

!Pr' CIN A (')LH, A C
\ NNAA
q xis' n H
\
s o q ), 1 I
N OH ;

vN,.......4 µ)C) , and o H
0 , \1.. ___________________________ SN¨

`55'N N
\ -.)n H

#
HO
wherein each n is, independently, from 1 to 20.

In certain embodiments, the conjugate linker has a structure selected from among:
\
\
E.
s N0)''L
N),OA 0 H
0 µ)........õ...........õ........ir,N.õ,....õ,--.,s,,S.,...õ-----,,,..") , -NH
44\4 0 ,s ZN).,..õ0-P-OH

H H

N
I \ rO--OH
ii J-Pc4 0 =
\
' O. 1-NH
0)2"

H H
I

3 3 \ ..
0 -,L, 0 . ..0 ,,0 1C-- > OH
7 ¨=s o \ ,,===
H N
JIM!

OH
NcYl c) ,1 ,;;,. S 0 H H N )-1 ,S 0 S
/
N(`-rNO ;
H

IO., HHHH H
c ,s.ss /0-) H N ' ; r&S'S'H& =
I

QN \i,...
I 0 0 ,0 O. /OH
IC),s0 1 CY1-0,1,1:)' H

0 _s, \ /-----/-1 L'\,.. ) 0 N)' N
e ; and H
/ .
H

In certain embodiments, the conjugate linker has a structure selected from among:

J-r"rj \
rrr\j 0 and wherein n is from 1 to 20.
In certain embodiments, the conjugate linker has a structure selected from among:

l\)L 0¨P¨OA feYLN

0 and 0 In certain embodiments, the conjugate linker has a structure selected from among:

II µ
n H n OH H n 0 and 0 =
, wherein each n is independently, 0, 1, 2, 3, 4, 5, 6, or 7.
In certain embodiments, the conjugate linker has the following structure:

µ)N101¨i =
In certain embodiments, the branching group has one of the following structures:
I I I

1_ 1 ) A1-1 Nn A1-1 /Al ¨ ri 1 . (/1 )1(fn 1-4 A 10( A1 and F n A1 (*1 n yiIrss sr wherein each Al is independently, 0, S, C=0 or NH; and each n is, independently, from 1 to 20.

In certain embodiments, the branching group has one of the following structures:
,s,s, cgs, )( ) ( fn and s\ss.3 SJ
wherein each Al is independently, 0, S, C=0 or NH; and each n is, independently, from 1 to 20.
In certain embodiments, the branching group has the following structure:
\
0..., /0õ.......õ..--/

/ .
In certain embodiments, the branching group has the following structure:
\
0,, A
/0õ,.......õ--h, / =
In certain embodiments, the branching group has the following structure:
\----...
/
NA\õ----H
avv r.
In certain embodiments, the branching group has the following structure:
isss \
________________ /
µ
\L/
\ss e .

In certain embodiments, the branching group comprises an ether.
In certain embodiments, the branching group has the following structure:
J.J1fll 0 ,111' 0 0 1 \ 0 - 0 HO 0-11=11-0 __ 1 . µ)Li\IN,css =
n H n NH 0 n õL, 3 /ni OH ' 0 I Ld-1 :..

H 0 ( i>n H 0 e-L,Nir-1.,1 rr'sYrnNi e ;
.
( )n 0 , .1-r-`4 r, 0 ( 1.), 0 n JINJ m (/' ______________________________________________ 1 clO NrL . 4 ).r.:..... / n CH3 ..,Fi3 o (4 0 .
, 01 \ ,s:' im H (?¨NH c.r-rr CH3 f,djuk, n 0 jni r 6 n ;and O I
o I o ¨
I
NH H (,NH,s.
?¨ n n 0 I
.e H
0 m each n is, independently, from 1 to 20; and m is from 2 to 6.
In certain embodiments, the branching group has the following structure:

0 ilt 0 ?
0 (:)..21/4 =>- 1 rrss -...,......---..N.ww.--µ
NH H . 0 l<C) I

*
,J1J1J
I / __ NH /0 \
0o 0 NH
41 µ)LN N csss =
0 ___________________________________________________________________ ,0 0 , ..,,, ; AN \ ;and '2ec.¨ NH
rrr' =
H
JIJIlll 0 In certain embodiments, the branching group has the following structure:
\

/
/0...õ...--0 0__.
/
In certain embodiments, the branching group comprises:
o TN H

NH cirA ENINA
H H
csss 4)\-i A
)..:;..... O(/ /1 H
0 / j H . NH

/ / /

o 0 0 0 )rr' 0 V-N*()LN NjjF\JA v¨i-r¨)LN N"N"
H H
n H H

0 ( rre ir NH
v NH
,or o =
, wherein each j is an integer from 1 to 3; and wherein each n is an integer from 1 to 20.
In certain embodiments, the branching group comprises:
µ o HN
) '\/\/\)1.---NH

EN-I NH
N mi s H ,K

HNi H NH

/ / /

0\/. \ / \ )1"---- NH
NH

N ..,....õ-k, NH AF, \-----..."--"--'"--)LN"------ O/
O/
risc,.....Thr NH
NH
or o , .
In certain embodiments, each tether is selected from among:

cKH-L1He'L and cssN I¨H)''' ril 1 m1 r111 H ml wherein L is selected from a phosphorus linking group and a neutral linking group;

Z1 is C(=0)0-R2;
Z2 is H, C1-C6 alkyl or substituted C1-C6 alky;
R2 is H, C1-C6 alkyl or substituted C1-C6 alky; and each ml is, independently, from 0 to 20 wherein at least one ml is greater than 0 for each tether.
In certain embodiments, each tether is selected from among:

k+o-vo and \. f(,ANO-11'-0 m I M2 I I M2 OH 'm2 H 0 wherein Z2 is H or CH3; and each m2 is, independently, from 0 to 20 wherein at least one m2 is greater than 0 for each tether.
In certain embodiments, each tether is selected from among:
H m HN 0 m OH

=
wherein n is from 1 to 12; and wherein m is from 1 to 12.
In certain embodiments, at least one tether comprises ethylene glycol. In certain embodiments, at least one tether comprises an amide. In certain embodiments, at least one tether comprises a polyamide. In certain embodiments, at least one tether comprises an amine. In certain embodiments, at least two tethers are different from one another. In certain embodiments, all of the tethers are the same as one another. In certain embodiments, each tether is selected from among:

N,),(1 C)(:)1,))X . V N .(') . , n =
in Olt-1 1 .
n H n 7 7 H H H
4H1O-C)11L . SM\i'HNH, . r's'N,(,,i,N
n /n .9r\ 7 n P Cl 7 H 7 \H 1¨EN1 ( n rj ¨N N , N 12- =
\
\ n H /2 r 7 in 1 in \ in cs"
; 7 Fri0 ( Jfl , Krri ;and lyNN'n1 " H n wherein each n is, independently, from 1 to 20; and each p is from 1 to about 6.
In certain embodiments, each tether is selected from among:

H
N .C).-0-21'-- ; V- N ;

H
'azz<0,rr' , rrrr0.j'1. ; VI\L.Wcsss ; and rr'sossss .
In certain embodiments, each tether has the following structure:
H H
css5,1(,, N .,N )r''2.z m wherein each n is, independently, from 1 to 20.
In certain embodiments, each tether has the following structure:
H H
cs5r N N .1.µ
0 0 .

In certain embodiments, the tether has a structure selected from among:

Y('r NC YTr NC
4 H n H
or --...,s 'r ; wherein each n is independently, 0, 1, 2, 3, 4, 5, 6, or 7.
In certain embodiments, the tether has a structure selected from among:

Y('r NC

In certain embodiments, the ligand is galactose. In certain embodiments, the ligand is mannose-6-phosphate.
In certain embodiments, each ligand is selected from among:
OH
OH
OH
and HO*
HO .../...C...:)...\
OH
HO 0¨
Ri Ri R1 0 __ .../..!_.).....\____O¨

wherein each R1 is selected from OH and NHCOOH.
In certain embodiments, each ligand is selected from among:

HOOH OH HO HO
OH o\
OH
0 (-1 HO-----.\rOxi . Fis? -0 HO -0 s,r5 =
, NHAc 7 OH r' , 110 ; HO

\Irr' HOOH OH
N. 1-10-1-7\ OH HOOH
HO cs.,T 0 0 FNi HO OH
OH
, HO HO, X OH
rcss ; HO---4--\:) , +40-7110'- ;and OH
OH
HO
OH

\
HO¨

HO OH
OH

HO

0 \s, In certain embodiments, each ligand has the following structure:
HOOH
_...4..).H
N
HO ....\/ Nws NHAc " .
In certain embodiments, each ligand has the following structure:
HOOH
0 (-1 HO-=----\----\V¨ \rxr NHAc 7 =
In certain embodiments, the conjugate group comprises a cell-targeting moiety.
In certain embodiments, the conjugate group comprises a cell-targeting moiety having the following structure:

NOON

.\uNW\
n 0 0 \
AcHN
1)n HO OH OH) O
HO õ
.r, AcHN OH

n HO n OH
NHAc wherein each n is, independently, from 1 to 20.
In certain embodiments, the cell-targeting moiety has the following structure:
HO OH
O
AcHN 0 0 OH

I
AcHN OH 0 HO H 9 y HO OH
NHAc In certain embodiments, the cell-targeting moiety has the following structure:

HO OH

HO n n )n HO OH NHAc H H ) n H I
n n n NHAc 0 OH
HO HN
H

HO_ 0 .\.:....\7) n n NHAc 0 =
wherein each n is, independently, from 1 to 20.
In certain embodiments, the cell-targeting moiety has the following structure:
HO OH

HO

HO OH NHAc 0 ---.....
_.......7Ø....\,___ H H
0,.........õ..õ.õ,,........--N
HO
NHAc 0 OH
HO
H
HO

NHAc =

15 In certain embodiments, the cell-targeting moiety comprises:

HO OH
HO
c 0 AcHN
NH

HO OH
HN
HO \Z( 0 AcHN 0 HO OH
NH
HO
AcHN 0 In certain embodiments, the cell-targeting moiety comprises:

HO-Z/C)HIC
HN
HOOH AcHN0 H
HOCZ/C)1FliN

AcHN
HOOH

AcHN
In certain embodiments, the cell-targeting moiety comprises:
HOOH
O
HO-Z\Z 1)C
AcHN 0 HO-/ N
H
AcHN 0 HO OH

AcHN

In certain embodiments, the cell-targeting moiety comprises:

AcHN

A
N N

AcHN

AcHN
In certain embodiments,the cell-targeting moiety comprises:
pH
HOOH
HO
ON

AcHN
0=P¨OH
HOOH
HO

AcHN
0=P¨OH
HOOH
HO

AcHN
In certain embodiments, the cell-targeting moiety comprises:
HOOH
HO

"4 AcHN

H
H0-CZ7C{Thcill\r AcHN
HOOH
HO_..,72..\,0-r4¨HN
AcHN
In certain embodiments, the cell-targeting moiety comprises:

HO OH
HO 0---&"2-\/
NH
AcHN

HO OH
HO )/.1\r N H o AcHN 0 H
0 , HO OH
HO-&ZO H NH
Z,r--N
AcHN 0 0 HO OH
HO 0 n.i---NH
AcHN
0 =
In certain embodiments, the cell-targeting moiety comprises:
OH OH

AcHN N--NNANH
OH
N
HO 0\.
\-_,---0 ......\_\_x 0 AcHN
N----../----N
H H
H
cr N \(:) _r_ HO,( NH7)....\/
HO
NHAc In certain embodiments, the cell-targeting moiety comprises:

OH OH

AcHN
OH OH
0 crNFi CD
0 N 'Cks?
AcHN H 0 0 r HO
NHAc In certain embodiments, the cell-targeting moiety comprises:

HO "10 H
AcHN 0 HO

AcHN 0 HOdH
HO
NHAc In certain embodiments, the cell-targeting moiety comprises:

HOOH
0 oi_pi -N) AcHN 0 0 0, HO OH r0-kFy0' 0 0' N`
AcHN 0 P, ( H op NHAc In certain embodiments, the cell-targeting moiety comprises:

AcHN H
HOOH 0 11A.
HO---72-=\..L'il 1 AcHN
HOOH 0 ( HO v 04 H 2 H
AcHN .
In certain embodiments, the cell-targeting moiety comprises:

HO
AcHN
H
HOOH 0 N µ.
HOil 0 AcHN

( 0 N ,N 0 HO--r.--\v i-i H
AcHN .
In certain embodiments, the cell-targeting moiety comprises:

AcHN H

_."..!......\D N

N,./
).N\=
H

( AcHN

_....2...v 0 AcHN .
In certain embodiments, the cell-targeting moiety comprises:

pH
HOOH
HO

AcHN
0=P¨OH
HOOH
AcHN
0=P¨OH
HOOH

AcHN
In certain embodiments, the cell-targeting moiety comprises:
oH
HOOH 0 =

AcHN
0=P¨OH

AcHN
0=P¨OH

HOOH H
A
HO
AcHN
In certain embodiments, the cell-targeting moiety comprises:

NOON rNOH

AcHN I
0=P-OH
I

Nk HO--12--\
AcHN I
0=P-OH
I

N
HO__.=,72.013 0 \---0-1 AcHN .
In certain embodiments, the cell-targeting moiety comprises:
H 0 i¨/OH
HO OH
Nj=k)-1---NTh \ --AcHN I
0=P-OH
I

HO OH H O( N)-1--3 N

AcHN
0=P-OH
I

HO OH H 0r' N
AcHN .
In certain embodiments, the cell-targeting moiety comprises:
OH H
01-11........ N 0 0 (:)C)--- 0 7 OH
OH
01-1........\vNHAc H NH

OH .-;.--- 0 NHAc 0i_i NH
OH
NHAc .

In certain embodiments, the cell-targeting moiety comprises:
OH
HOOA
AcHN NH
r.-H OH

HO=-=:).\---01=LN NH
AcHN scss In certain embodiments, the cell-targeting moiety comprises:
OH OH

AcHN
In certain embodiments, the cell-targeting moiety comprises:
OH
H?
.=
AcHN Y
CrY

0¨p/HO , OH Y
HCAcHN =
wherein each Y is selected from 0, S, a substituted or unsubstituted C1-C10 alkyl, amino, substituted amino, azido, alkenyl or alkynyl.
In certain embodiments, the conjugate group comprises:
OH
H?
= \ 0õY
AcHN Y ¨0 0 P\ 0 csss Y
OH Y
HO\r(r1v0 HCAcHN =
wherein each Y is selected from 0, S, a substituted or unsubstituted C1-C10 alkyl, amino, substituted amino, azido, alkenyl or alkynyl.

In certain embodiments, the conjugate group comprises:
OH

HO
AcHN
$/\y =
wherein each Y is selected from 0, S, a substituted or unsubstituted C1-C10 alkyl, amino, substituted amino, azido, alkenyl or alkynyl.
In certain embodiments, the conjugate group comprises:
OH

HO
AcHN
OH
In certain embodiments, the conjugate group comprises:
OH

/.*%%0H
HO
AcHN
In certain embodiments, the conjugate group comprises:
OH
HOµ .õ0A

NN?
AcHN

In certain embodiments, the conjugate group comprises:
OH
HOµ .õOH

AcHN
In certain embodiments, the conjugate group comprises a cleavable moiety selected from among: a phosphodiester, an amide, or an ester.
In certain embodiments, the conjugate group comprises a phosphodiester cleavable moiety.

In certain embodiments,the conjugate group does not comprise a cleavable moiety, and wherein the conjugate group comprises a phosphorothioate linkage between the conjugate group and the oligonucleotide.
In certain embodiments, the conjugate group comprises an amide cleavable moiety. In certain embodiments, the conjugate group comprises an ester cleavable moiety.
In certain embodiments, the compound has the following structure:
HOOH
HO----72.0 0 -K
n 0 1 0 µ
AcHN OH 1))n HOOH

0-, 0 Bx n 0 OH
AcHN OH ,---- 0 Q13 II V
HO-P=0 HO
,070.....\/0.,,L.r....0OH ,P-0-(-- )n I
n A
NHAc wherein each n is, independently, from 1 to 20;
Q13 is H or 0(CH2)2-0CH3;
A is the modified oligonucleotide; and Bx is a heterocyclic base moiety.
In certain embodiments, the compound has the following structure:

HO OH

HO----r52..\v0õ....\ Ag AcHN 0 1Th OH ---HO OH 0,.. 0 II rki(03,,Bx HO--0¨p¨

OH ,.,,, (r, AcHN OH (:) kJ

HO¨P=0 HO H 9 y P, I
A
OH
HO
NHAc wherein each n is, independently, from 1 to 20;
Q13 is H or 0(CH2)2-0CH3;
A is the modified oligonucleotide; and Bx is a heterocyclic base moiety.
In certain embodiments, the compound has the following structure:

A

HO-P=0 I
Bx HO-P=0 O
HO OH On 0 , 0 HO---i......_\L) ,11 , AcHN OH ) --\03\

HO OH n (On 0 0-_,. 0 ___,.õ(.2.\,0*..1,... A , , HO I
0---nie ___________________________________________ NrO-P=0 I
AcHN OH
0 u ,.. _____________________________________________________ OH
H0.12....v F1 0....ci, ,pil...... ......0 ) 010 n HO \ - in OH
NHAc wherein each n is, independently, from 1 to 20;
Q13 is H or 0(CH2)2-0CH3;
A is the modified oligonucleotide;
Z is H or a linked solid support; and Bx is a heterocyclic base moiety.
In certain embodiments, the compound has the following structure:

A
I
HO¨P=0 I
Bx d Q13 I
HO¨P=0 O

o HO----r.C2.,\/' 0 N./\ ,k \O

AcHN \

(03 HO

0 0,_ I
....r.Z\vr) HO =-, AI, -----7()I=C) AcHN OH 0' OH

HO H II
P, HO
/00' l 0 OH
NHAc wherein each n is, independently, from 1 to 20;
Q13 is H or 0(CH2)2-0CH3;
A is the modified oligonucleotide;
Z is H or a linked solid support; and Bx is a heterocyclic base moiety.

In certain embodiments, the compound has the following structure:
OH OH
HO*,0 AcHN N---X-NH
OH \-----\__H
N
H03,.._ \-:_----0 0\.......\___\_. jz 0 )C--N C) AcHN /
N-----Z------N
H H ______________________________________________ ,(1.__(_r0 H 6 HO-P=0 rrN \(:) 0--=õcOrBx _r_i_ -NH
d Q13 I

HO, OH;\..:)....v HO-P=0 HO A
NHAc wherein Q13 is H or 0(CH2)2-0CH3;
A is the modified oligonucleotide;and Bx is a heterocyclic base moiety.
In certain embodiments, the compound has the following structure:
OH OH

H0.7.::2._ -"-----LNH
AcHN
OH OH
H0*.\,.._ 0 ,crH 0 H 0 0-........".....õ..---.,...). N ......õ.õ..-.õ..,Thr-N-i-16 NNN
AcHN 11 0 LII 0 HO-P=0 I
Bx 0 r 0¨cOza HO. 0.,.,/,..õ,z_i¨NH
Cf: Q13 I
HO HO-P=0 NHAc I
A , wherein Q13 is H or 0(CH2)2-0CH3;
A is the modified oligonucleotide;and Bx is a heterocyclic base moiety.
In certain embodiments, the compound has the following structure:

HO
-' loJC
H
AcHN N

HO01`rN)C----C)NNI'hO
io H H H
AcHN N
Z= HO-P=0 Bx HOOH

NHAc loH I
HO-P=0 I
A , wherein Q13 is H or 0(CH2)2-0CH3;
A is the modified oligonucleotide; and Bx is a heterocyclic base moiety.
In certain embodiments, the compound has the following structure:

HOOH
0 (514)i HO-4,0 NrD
AcHN 0 o 0.
HOOH ro-011-Ey'o' ci\
o a" HO-P=0 HO-7"---\,C)1N0 I
AcHN 0Bx P,-HOOH ocN
HO-P=0 0I"' Q13 I

NHAc wherein Q13 is H or 0(CH2)2-0CH3;
A is the modified oligonucleotide; and Bx is a heterocyclic base moiety.
In certain embodiments, the compound has the following structure:

A
HO-P=0 OçOBx HOOH 0 d Q13 HO¨P=0 AcHN
HO OH

HO4H 2 ON AcHN OH
HO OH
HOoN^(¨)N(c) AcHN
wherein Q13 is H or 0(CH2)2-0CH3;
A is the modified oligonucleotide; and Bx is a heterocyclic base moiety.
In certain embodiments, the compound has the following structure:

AcHN 0 0 3 H 0 HO-P=0 AcHN
HOOH
( Bx HO-"(==:?-\VC)3 H 0 AcHN HO¨P=0 A
wherein Q13 is H or 0(CH2)2-0CH3;
A is the modified oligonucleotide; and Bx is a heterocyclic base moiety.
In certain embodiments, the compound has the following structure:

AcHN 0 0 HO -:).-\/ 11A
4 H 0 HO-P=0 AcHN
HOOH 0 0I0z.Bx AcHN HO-P=0 A
wherein Q13 is H or 0(CH2)2-0CH3;
A is the modified oligonucleotide; and Bx is a heterocyclic base moiety.
In certain embodiments, the compound has the following structure:
pH
HOOH
HO

AcHN
0=P-OH
HOOH
HO
OO
AcHN
0=P-OH A
g HO -P = 0 HO OH 0 Bx HO d Q13 AcHN I
P =0 OH
wherein Q13 is H or 0(CH2)2-0CH3;
A is the modified oligonucleotide; and Bx is a heterocyclic base moiety.
In certain embodiments, the compound has the following structure:

pH
HO OH 0 n-N

AcHN
0=P-OH
NOON 0 =
HO-0=72-\/ *cr, N

AcHN
0=P-OH
A
HOOH H 0 _____ HO-p=0 HO-===:)-\/0Thr3 0 N N 0¨cOrBx AcHN

P=0 OH
wherein Q13 is H or 0(CH2)2-0CH3;
A is the modified oligonucleotide; and Bx is a heterocyclic base moiety.
In certain embodiments, the compound has the following structure:
HO OH ("OH
N
HO 3 0 Lo AcHN
0=P-OH

HO--72-\/CYMN
AcHN
0=P-OH A
rO HO-p=0 HO OH HO_ 0¨.(OrBx 0^N

s' AcHN Q13 P=0 OH
wherein Q13 is H or 0(CH2)2-0CH3;
A is the modified oligonucleotide; and Bx is a heterocyclic base moiety.
In certain embodiments, the compound has the following structure:

OH
HO OH H 0 /¨/
H0011-N1--N---\
AcHN I
0=P-OH
I

0.--1,nir N N
HO-112-\--- ---\--_ AcHN I
0=P-OH A
I I
0 HO-P=0 HOOH H
HO -12-=\N (C/JL-1\1 AcHN
P=0 OH
wherein Q13 is H or 0(CH2)2-0CH3;
A is the modified oligonucleotide; and Bx is a heterocyclic base moiety.
In certain embodiments, the conjugate group comprises:
HO OH
HOOH
AcHN N----N----)r-N \--\. NH
0 y------1 0 0 H H 0 0- Kv7k 9 0 Bx N 7N---"N---1-0.----NH
H OH ==

HO
NHAc HN N HO-P=0 ,-.........--.., ____k) H ,-, A
OH 0.-/¨/-%
HO
1-18) NHAc wherein Q13 is H or 0(CH2)2-0CH3;
A is the modified oligonucleotide; and Bx is a heterocyclic base moiety.
In certain embodiments, the conjugate group comprises:

HOOH
)=c AcHN

AcHN O OH 0 HOOH
HO-P=0 AcHN
wherein Q13 is H or 0(CH2)2-0CH3;
A is the modified oligonucleotide; and Bx is a heterocyclic base moiety.
In certain embodiments, the conjugate group comprises:
HOOH

AcHN
HOOH

"4 H

AcHN OH 0 Q13 HOOH
HO-P=0 HO "4 AcHN
wherein Q13 is H or 0(CH2)2-0CH3;
A is the modified oligonucleotide; and Bx is a heterocyclic base moiety.
In certain embodiments, Bx is selected from among from adenine, guanine, thymine, uracil, or cytosine, or 5-methyl cytosine. In certain embodiments, Bx is adenine. In certain embodiments, Bx is thymine. In certain embodiments, Q13 is 0(CH2)2-0CH3. In certain embodiments, Q13 is H.
In certain embodiments, the compound is in a salt form. In further embodiments, the compound further comprises of a pharmaceutically acceptable carrier or diluent. In certain embodiments, the compound comprises a modified oligonucleotide targeting apo(a) and a conjugate group, or a salt thereof, and a pharmaceutically acceptable carrier or diluent.

Certain embodiments provide a composition comprising a conjugated antisense compound as described herein, wherein the viscosity level of the compound is less than 40 centipoise (cP). In certain embodiments, the conjugated antisense compounds as described herein are efficacious by virtue of having a viscosity of less than 40 cP, less than 35 cP, less than 30 cP, less than 25 cP, less than 20 cP or less than 15 cP when measured by the parameters as described in Example 125.
Certain embodiments provide compositions and methods comprising administering to an animal a conjugated antisense compound or composition disclosed herein. In certain embodiments, administering the conjugated antisense compound prevents, treats, ameliorates, or slows progression of a cardiovascular, metabolic and/or inflammatory disease Certain embodiments provide compositions and methods for use in therapy to treat an apo(a) related disease, disorder or condition. Certain embodiments provide compositions and methods for use in therapy to treat an Lp(a) related disease, disorder or condition. In certain embodiments, apo(a) and/or Lp(a) levels are elevated in an animal. In certain embodiments, the composition is a compound comprising an apo(a) specific inhibitor. In certain embodiments, the apo(a) specific inhibitor is a nucleic acid. In certain embodiments, the nucleic acid is an antisense compound. In certain embodiments, the antisense compound is a modified oligonucleotide targeting apo(a). In certain embodiments, the antisense compound is a modified oligonucleotide targeting apo(a) and a conjugate group. In certain embodiments, the modified oligonucleotide targeting apo(a) with the conjugate group, is used in treating, preventing, slowing progression, ameliorating a cardiovascular and/or metabolic disease, disorder or condition. In certain embodiments, the compositions and methods for therapy include administering an apo(a) specific inhibitor to an individual in need thereof Certain embodiments provide compositions and methods for reducing apo(a) levels. Certain embodiments provide compositions and methods for reducing Lp(a) levels. In certain embodiments, reducing apo(a) levels in a tissue, organ or subject improves the ratio of LDL to HDL
or the ratio of TG to HDL.
Certain embodiments provide compositions and methods to reduce apo(a) mRNA or protein expression in an animal comprising administering to the animal a conjugated antisense compound or composition disclosed herein to reduce apo(a) mRNA or protein expression in the animal. Certain embodiments provide compositions and methods to reduce Lp(a) levels in an animal comprising administering to the animal a conjugated antisense compound or composition disclosed herein to reduce apo(a) mRNA or protein expression in the animal.
Certain embodiments provide compositions and methods for preventing, treating, delaying, slowing the progression and/or ameliorating apo(a) related diseases, disorders, and conditions in a subject in need thereof Certain embodiments provide compositions and methods for preventing, treating, delaying, slowing the progression and/or ameliorating Lp(a) related diseases, disorders, and conditions in a subject in need thereof In certain embodiments, such diseases, disorders, and conditions include inflammatory, cardiovascular and/or metabolic diseases, disorders, and conditions. Certain such cardiovascular diseases, disorders or conditions include, but are not limited to, aortic stenosis, aneurysm (e.g., abdominal aortic aneurysm), angina, arrhythmia, atherosclerosis, cerebrovascular disease, coronary artery disease, coronary heart disease, dyslipidemia, hypercholesterolemia, hyperlipidemia, hypertension, hypertriglyceridemia, myocardial infarction, peripheral vascular disease (e.g., peripheral artery disease, peripheral artery occlusive disease), retinal vascular occlusion, or stroke. Certain such metabolic diseases, disorders or conditions include, but are not limited to, hyperglycemia, prediabetes, diabetes (type I
and type II), obesity, insulin resistance, metabolic syndrome and diabetic dyslipidemia. Certain such inflammatory diseases, disorders or conditions include, but are not limited to, aortic stenosis, coronary artey disease (CAD), Alzheimer's Disease and thromboembolic diseases, disorder or conditions. Certain thromboembolic diseases, disorders or conditions include, but are not limited to, stroke, thrombosis (e.g., venous thromboembolism), myocardial infarction and peripheral vascular disease. Certain embodiments provide compositions and methods for preventing, treating, delaying, slowing the progression and/or ameliorating aortic stenosis.
Certain embodiments provide a method of reducing at least one symptom of a cardiovascular disease, disorder or condition. In certain embodiments, the symptoms include, but are not limited to, angina, chest pain, shortness of breath, palpitations, weakness, dizziness, nausea, sweating, tachycardia, bradycardia, arrhythmia, atrial fibrillation, swelling in the lower extremities, cyanosis, fatigue, fainting, numbness of the face, numbness of the limbs, claudication or cramping of muscles, bloating of the abdomen, and fever.
Certain embodiments provide a method of reducing at least one symptom of aortic stenosis.
In certain embodiments, the modulation of apo(a) or Lp(a) expression occurs in a cell, tissue or organ. In certain embodiments, the modulations occur in a cell, tissue or organ in an animal. In certain embodiments, the modulation is a reduction in apo(a) mRNA level. In certain embodiments, the modulation is a reduction in apo(a) protein level. In certain embodiments, both apo(a) mRNA and protein levels are reduced. In certain embodiments, the modulation is a reduction in Lp(a) level.
Such reduction may occur in a time-dependent or in a dose-dependent manner.
In certain embodiments, the subject or animal is human.
In certain embodiments, the conjugated antisense compound is parenterally administered. In further embodiments, the parenteral administration is subcutaneous.
In certain embodiments, the conjugated antisense compound or composition is co-administered with a second agent or therapy. In certain embodiments, the conjugated antisense compound or composition and the second agent are administered concomitantly.
In certain embodiments, the second agent is a glucose-lowering agent. In certain embodiments, the second agent is a LDL, TG or cholesterol lowering agent. In certain embodiments, the second agent is an anti-inflammatory agent. In certain embodiments, the second agent is an Alzheimer Disease drug. In certain embodiments, the second agent can be, but is not limited to, a non-steroidal anti-inflammatory drug (NSAID
e.g., aspirin), niacin (e.g., Niaspan), nicotinic acid, an apoB inhibitor (e.g., Mipomersen), a CETP inhibitor (e.g., Anacetrapib), an apo(a) inhibitor, a thyroid hormone analog (e.g., Eprotirome), a HMG-CoA reductase inhibitor (e.g., a statin), a fibrate (e.g., Gemfibrozil) and an microsomal triglyceride transfer protein inhibitor (e.g., Lomitapide). The therapy can be, but is not limited to, Lp(a) apheresis. Agents or therapies can be co-administered or administered concomitantly. Agents or therapies can be sequentially or subsequently administered.
Certain embodiments provide use of a conjugated antisense compound targeted to apo(a) for decreasing apo(a) levels in an animal. Certain embodiments provide use of a conjugated antisense compound targeted to apo(a) for decreasing Lp(a) levels in an animal. Certain embodiments provide use of a conjugated antisense compounds targeted to apo(a) for the treatment, prevention, or amelioration of a disease, disorder, or condition associated with apo(a). Certain embodiments provide use of a conjugated antisense compounds targeted to apo(a) for the treatment, prevention, or amelioration of a disease, disorder, or condition associated with Lp(a).
Certain embodiments provide use of a conjugated antisense compound targeted to apo(a) in the preparation of a medicament for decreasing apo(a) levels in an animal. Certain embodiments provide use of a conjugated antisense compound targeted to apo(a) in the preparation of a medicament for decreasing Lp(a) levels in an animal. Certain embodiments provide use of a conjugated antisense compound for the preparation of a medicament for the treatment, prevention, or amelioration of a disease, disorder, or condition associated with apo(a). Certain embodiments provide use of a conjugated antisense compound for the preparation of a medicament for the treatment, prevention, or amelioration of a disease, disorder, or condition associated with Lp(a).
Certain embodiments provide the use of a conjugated antisense compound as described herein in the manufacture of a medicament for treating, ameliorating, delaying or preventing one or more of a disease related to apo(a) and/or Lp(a).
Certain embodiments provide a kit for treating, preventing, or ameliorating a disease, disorder or condition as described herein wherein the kit comprises: (i) an apo(a) specific inhibitor as described herein;
and optionally (ii) a second agent or therapy as described herein.
A kit of the present invention can further include instructions for using the kit to treat, prevent, or ameliorate a disease, disorder or condition as described herein by combination therapy as described herein.
B. Certain Compounds In certain embodiments, the invention provides conjugated antisense compounds comprising antisense oligonucleoitdes and a conjugate.
a. Certain Antisense Oligonucleotides In certain embodiments, the invention provides antisense oligonucleotides.
Such antisense oligonucleotides comprise linked nucleosides, each nucleoside comprising a sugar moiety and a nucleobase.
The structure of such antisense oligonucleotides may be considered in terms of chemical features (e.g., modifications and patterns of modifications) and nucleobase sequence (e.g., sequence of antisense oligonucleotide, idenity and sequence of target nucleic acid).
i. Certain Chemistry Features In certain embodiments, antisense oligonucleotide comprise one or more modification. In certain such embodiments, antisense oligonucleotides comprise one or more modified nucleosides and/or modified internucleoside linkages. In certain embodiments, modified nucleosides comprise a modifed sugar moirty and/or modifed nucleobase.
1. Certain Sugar Moieties In certain embodiments, compounds of the disclosure comprise one or more modifed nucleosides comprising a modifed 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 substitued 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 "0-methyl"), and 2'-0(CH2)20CH3("MOE"). In certain embodiments, sugar substituents at the 2' position is selected from allyl, amino, azido, thio, 0-allyl, 0-C1-C10 alkyl, 0-C1-C10 substituted alkyl; OCF3, 0(CH2)2SCH3, 0(CH2)2-0-N(Rm)(R4 and 0-CH2-C(=0)-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, 0, S, or N(Rm)-alkyl; 0, S, or N(Rm)-alkenyl; 0, S or N(Rm)-alkynyl; 0-alkyleny1-0-alkyl, alkynyl, alkaryl, aralkyl, 0-a1karyl, 0-ara1kyl, 0(CH2)25CH3, 0-(CH2)2-0-N(Rm)(Rn) or 0-CH2-C(=0)-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, 0-CH3, 0(CH2)3NH2, CH2-CH=CH2, 0-CH2-CH=CH2, OCH2CH2OCH3, 0(CH2)25CH3, 0-(CH2)2-0-N(Rm)(Ra), 0(CH2)20(CH2)2N(CH3)2, and N-substituted acetamide (0-CH2-C(=0)-N(Rm)(RO
where each Rai and Ra 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, 0-CH3, OCH2CH2OCH3, 0(CH2)2SCH3, 0-(CH2)2-0-N(CH3)2, -0(CH2)20(CH2)2N(CH3)2, and 0-CH2-C(=0)-N(H)CH3.
In certain embodiments, a 2'- substituted nucleoside comprises a sugar moiety comprising a 2'-substituent group selected from F, 0-CH3, and OCH2CH2OCH3.
Certain modifed 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(RO(Rb)].-, -[C(RO(Rb)].-0-, -C(RaRb)-N(R)-0- or, -C(RaRb)-0-N(R)-; 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)3-2',. 4'-(CH2)-0-2' (LNA); 4'4CH2)-S-2'; 4'-(CH2)2-0-2' (ENA); 4'-CH(CH3)-0-2' (cEt) and 4'-CH(CH2OCH3)-0-2',and analogs thereof (see, e.g., U.S. Patent 7,399,845, issued on July 15, 2008); 4'-C(CH3)(CH3)-0-2'and analogs thereof, (see, e.g., W02009/006478, published January 8, 2009); 4'-CH2-N(OCH3)-2' and analogs thereof (see, e.g., W02008/150729, published December 11, 2008); 4'-CH2-0-N(CH3)-2' (see, e.g., U52004/0171570, published September 2, 2004); 4'-CH2-0-N(R)-2', and 4'-CH2-N(R)-0-2'-, wherein each R is, independently, H, a protecting group, or C1-C12 alkyl; 4'-CH2-N(R)-0-2', wherein R
is H, C1-C12 alkyl, or a protecting group (see, U.S. Patent 7,427,672, issued on September 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 December 8, 2008).
In certain embodiments, such 4' to 2' bridges independently comprise from 1 to 4 linked groups independently selected from -[C(Ra)(Rb)]a-, -C(Ra)=C(Rb)-, -C(Ra)=N-, -C(=NRa)-, -C(=0)-, -C(=S)-, -0-, -Si(Ra)2-, -S(=0)x-, and -N(RO-;
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, CI-Cu.
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, OJI, NJ1.1-2, SJI, N3, COOJI, acyl (C(=0)-H), substituted acyl, CN, sulfonyl (S(=0)2-Ji), or sulfoxyl (S(=0)-Ji); and each J1 andJ2 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(=0)-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) a-L-Methyleneoxy (4'-CH2-0-2') BNA, (B) 13-D-Methyleneoxy (4'-CH2-0-2') BNA (also referred to as locked nucleic acid or LNA) , (C) Ethyleneoxy (4'-(CH2)2-0-2') BNA, (D) Aminooxy (4'-CH2-0-N(R)-2') BNA, (E) Oxyamino (4'-CH2-N(R)-0-2') BNA, (F) Methyl(methyleneoxy) (4'-CH(CH3)-0-2') 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, and (J) propylene carbocyclic (4'-(CH2)3-2') BNA as depicted below.
Bx 0 Bx Bx ¨0 ¨ ¨0 (A) (B) (C) ______________ 0 Bx 0 Bx 0 Bx 0¨ ¨N R-N- ¨0 H3C
(D) (E) (F) $0,y,Bx __________________________________________ OyBxBx S (H) ¨N
\
(G) R --- CH3 (I) 0 Bx (J) 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 Mot. Ther., 2001, 3, 239-243; U.S. Patent 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. U52004/0171570, U52007/0287831, and US2008/0039618; U.S. Patent Serial 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/U52008/064591, PCT/U52008/066154, and PCT/U52008/068922.
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 a-L configuration or in the P-D
configuration. Previously, a-L-methyleneoxy (4'-CH2-0-2') 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 11/22/07, 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 occuring 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 U52005/0130923, published on June 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 morphlino. Morpholino compounds and their use in oligomeric compounds has been reported in numerous patents and published articles (see for example:
Braasch et al., Biochemistry, 2002, 41, 4503-4510; and U.S. Patents 5,698,685;
5,166,315; 5,185,444; and 5,034,506). As used here, the term "morpholino" means a sugar surrogate having the following structure:
In certain embodiments, morpholinos may be modified, for example by adding or altering various substituent groups from the above morpholino structure. Such sugar surrogates are refered to herein as "modifed morpholinos."
For another 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, CJ. Bioorg. & Med. Chem.
(2002) 10:841-854), fluoro HNA (F-HNA), and those compounds having Formula VI:
C11 Cl2 CI6 Bx % R1 R2 C15 TvT
wherein independently for each of said at least one tetrahydropyran nucleoside analog of Formula VI:
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;
qi, q2, q3, q4, q5, q6 and (47 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, SJI, N3, OC(=X)Ji, OC(=X)NJ1J2, NJ3C(=X)NJ1J2, and CN, wherein X is 0, S or NJI, and each J1, J2, and J3 is, independently, H or C1-C6 alkyl.
In certain embodiments, the modified THP nucleosides of Formula VI are provided wherein qi, q2, q3, q4, q5, q6and q7 are each H. In certain embodiments, at least one of qi, q2, q3, q4, q5, q6and (47 is other than H. In certain embodiments, at least one of qi, q2, q3, q4, q5, q6and q7 is methyl. In certain embodiments, THP
nucleosides of Formula VI 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, /0, 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 8/21/08 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 June 16, 2005) or alternatively 5'-substitution of a bicyclic nucleic acid (see PCT International Application WO 2007/134181, published on 11/22/07 wherein a 4'-CH2-0-2' 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 disclosure 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 desireable characteristics. In certain embodmiments, oligonucleotides comprise one or more RNA-like nucleosides. In certain embodiments, oligonucleotides comprise one or more DNA-like nucleotides.
2. Certain Nucleobase Modifications In certain embodiments, nucleosides of the present disclosure comprise one or more unmodified nucleobases. In certain embodiments, nucleosides of the present disclosure comprise one or more modifed 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 0-6 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 (-CC-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]indo1-2-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 United States Patent 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. 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.
3. Certain Internucleoside Linkages In certain embodiments, the present disclosure provides oligonucleotides comprising linked nucleosides. In such embodiments, nucleosides may be linked together using any internucleoside linkage.
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 (PO), phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates (PS). Representative non-phosphorus containing internucleoside linking groups include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-0-CH2-), thiodiester (-0-C(0)-S-), thionocarbamate (-0-C(0)(NH)-S-); siloxane (-0-Si(H)2-0-); 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), a or b 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)-0-5'), amide-3 (3'-CH2-C(=0)-N(H)-5'), amide-4 (3'-CH2-N(H)-C(=0)-5'), formacetal (3'-0-CH2-0-5'), and thioformacetal (3'-S-CH2-0-5'). 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, 0, S and CH2 component parts.
4. Certain Motifs In certain embodiments, antisense oligonucleotides comprise one or more modified nucleoside (e.g., nucleoside comprising a modified sugar and/or modified nucleobase) and/or one or more modified internucleoside linkage. The pattern of such modifications on an oligonucleotide is referred to herein as a motif In certain embodiments, sugar, nucleobase, and linkage motifs are independent of one another.
a. 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 modification motif Such motifs may include any of the sugar modifications discussed herein and/or other known sugar modifications.
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).
i. Certain 5'-wings In certain embodiments, the 5'- wing of a gapmer consists of 1 to 8 linked nucleosides. In certain embodiments, the 5'- wing of a gapmer consists of 1 to 7 linked nucleosides.
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'-0Me 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'-0Me 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'-0Me 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'-0Me nucleoside. In certain embodiments, the 5'-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2'-deoxynucleoside.
ii. Certain 3'-wings In certain embodiments, the 3'- wing of a gapmer consists of 1 to 8 linked nucleosides. In certain embodiments, the 3'- wing of a gapmer consists of 1 to 7 linked nucleosides.
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 31inked 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'-0Me 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'-0Me 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'-0Me 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'-0Me 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'-0Me 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'-0Me 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'-0Me 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'-0Me nucleoside, and at least one 2'-deoxynucleoside.
iii. 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.
b. 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, oligonucleotides comprise a region having an alternating internucleoside linkage motif In certain embodiments, oligonucleotides of the present disclosure 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 internucleoside linkages.
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 7 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 8 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 9 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 10 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 11 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 12 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 13 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 14 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 7 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 9 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, the oligonucleotide comprises less than 15 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises less than 14 phosphoro-thioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises less than 13 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises less than 12 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises less than 11 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises less than 10 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises less than 9 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises less than 8 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises less than 7 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises less than 6 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises less than 5 phosphorothioate internucleoside linkages.
c. 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 such embodiments, nucleobase modifications are arranged in a gapped motif In certain embodiments, nucleobase modifications are arranged in an alternating 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, 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.
In certain embodiments, chemical modifications to nucleobases comprise attachment of certain conjugate groups to nucleobases. In certain embodiments, each purine or each pyrimidine in an oligonucleotide may be optionally modified to comprise a conjugate group.
d. Certain Overall Lengths In certain embodiments, the present disclosure provides oligonucleotides of any of a variety of ranges of lengths. In certain embodiments, oligonucleotides consist 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 oligonucleotide may consist 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 oligonucleotide of a compound is limited, whether to a range or to a specific number, the compound 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 conjugate groups, terminal groups, or other substituents.
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.
5. Certain Antisense Oligonucleotide Chemistry Motifs In certain embodiments, the chemical structural features of antisense oligonucleotides are characterized by their sugar motif, internucleoside linkage motif, nucleobase modification motif and overall length. In certain embodiments, such parameters are each independent of one another. Thus, 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. Thus, 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.
In certain embodiments, the selection of internucleoside linkage and nucleoside modification are not independent of one another.
i. Certain Sequences and Targets In certain embodiments, the invention provides antisense oligonucleotides having a sequence complementary to a target nucleic acid. 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 or reduce non-specific hybridization to 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, oligonucleotides are selective between a target and non-target, even though both target and non-target comprise the target sequence. In such embodiments, selectivity may result from relative accessibility of the target region of one nucleic acid molecule compared to the other.
In certain embodiments, the present disclosure 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.
In certain embodiments, oligonucleotides comprise a hybridizing region and a terminal region. In certain such embodiments, the hybridizing region consists of 12-30 linked nucleosides and is fully complementary to the target nucleic acid. In certain embodiments, the hybridizing region includes one mismatch relative to the target nucleic acid. In certain embodiments, the hybridizing region includes two mismatches relative to the target nucleic acid. In certain embodiments, the hybridizing region includes three mismatches relative to the target nucleic acid. In certain embodiments, the terminal region consists of 1-4 terminal nucleosides. In certain embodiments, the terminal nucleosides are at the 3' end. In certain embodiments, one or more of the terminal nucleosides are not complementary to the target nucleic acid.
Antisense mechanisms include any mechanism involving the hybridization of an oligonucleotide with target nucleic acid, wherein the hybridization results in a biological effect.
In certain embodiments, such hybridization results in either target nucleic acid degradation or occupancy with concomitant inhibition or stimulation of the cellular machinery involving, for example, translation, transcription, or splicing of the target nucleic acid.
One type of antisense mechanism involving degradation of target RNA is RNase H
mediated antisense. RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. It is known in the art that single-stranded antisense compounds which are "DNA-like"
elicit RNase H activity in mammalian cells. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of DNA-like oligonucleotide-mediated inhibition of gene expression.
In certain embodiments, a conjugate group comprises a cleavable moiety. In certain embodiments, a conjugate group comprises one or more cleavable bond. In certain embodiments, a conjugate group comprises a linker. In certain embodiments, a linker comprises a protein binding moiety. In certain embodiments, a conjugate group comprises a cell-targeting moiety (also referred to as a cell-targeting group).
In certain embodiments a cell-targeting moiety comprises a branching group. In certain embodiments, a cell-targeting moiety comprises one or more tethers. In certain embodiments, a cell-targeting moiety comprises a carbohydrate or carbohydrate cluster.
ii. Certain Cleavable Moieties In certain embodiments, a cleavable moiety is a cleavable bond. In certain embodiments, a cleavable moiety comprises a cleavable bond. In certain embodiments, the conjugate group comprises a cleavable moiety. In certain such embodiments, the cleavable moiety attaches to the antisense oligonucleotide. In certain such embodiments, the cleavable moiety attaches directly to the cell-targeting moiety. In certain such embodiments, the cleavable moiety attaches to the conjugate linker. In certain embodiments, the cleavable moiety comprises a phosphate or phosphodiester. In certain embodiments, the cleavable moiety is a cleavable nucleoside or nucleoside analog. In certain embodiments, the nucleoside or nucleoside analog comprises an optionally protected heterocyclic base selected from a purine, substituted purine, pyrimidine or substituted pyrimidine. In certain embodiments, the cleavable moiety is a nucleoside comprising an optionally protected heterocyclic base selected from uracil, thymine, cytosine, 4-N-benzoylcytosine, 5-methylcytosine, 4-N-benzoy1-5-methylcytosine, adenine, 6-N-benzoyladenine, guanine and 2-N-isobutyrylguanine. In certain embodiments, the cleavable moiety is 2'-deoxy nucleoside that is attached to the 3' position of the antisense oligonucleotide by a phosphodiester linkage and is attached to the linker by a phosphodiester or phosphorothioate linkage. In certain embodiments, the cleavable moiety is 2'-deoxy adenosine that is attached to the 3' position of the antisense oligonucleotide by a phosphodiester linkage and is attached to the linker by a phosphodiester or phosphorothioate linkage. In certain embodiments, the cleavable moiety is 2'-deoxy adenosine that is attached to the 3' position of the antisense oligonucleotide by a phosphodiester linkage and is attached to the linker by a phosphodiester linkage.
In certain embodiments, the cleavable moiety is attached to the 3' position of the antisense oligonucleotide. In certain embodiments, the cleavable moiety is attached to the 5' position of the antisense oligonucleotide. In certain embodiments, the cleavable moiety is attached to a 2' position of the antisense oligonucleotide. In certain embodiments, the cleavable moiety is attached to the antisense oligonucleotide by a phosphodiester linkage. In certain embodiments, the cleavable moiety is attached to the linker by either a phosphodiester or a phosphorothioate linkage. In certain embodiments, the cleavable moiety is attached to the linker by a phosphodiester linkage. In certain embodiments, the conjugate group does not include a cleavable moiety.

In certain embodiments, the cleavable moiety is cleaved after the complex has been administered to an animal only after being internalized by a targeted cell. Inside the cell the cleavable moiety is cleaved thereby releasing the active antisense oligonucleotide. While not wanting to be bound by theory it is believed that the cleavable moiety is cleaved by one or more nucleases within the cell.
In certain embodiments, the one or more nucleases cleave the phosphodiester linkage between the cleavable moiety and the linker. In certain embodiments, the cleavable moiety has a structure selected from among the following:
0=1:1'-OH

\(),,I3x1 0=P-OH 0=P-OH
LO),13x2 (5_ 0=P-OH
O'-OH 0=1:1'-OH

\(C)Bx c0),13x2 0),13x3 , and 0=P-OH 0=P-OH 0=P-OH
wherein each of Bx, Bxi, Bx2, and Bx3 is independently a heterocyclic base moiety. In certain embodiments, the cleavable moiety has a structure selected from among the following:
0=P-OH NH2 0=P-OH
iii. Certain Linkers In certain embodiments, the conjugate groups comprise a linker. In certain such embodiments, the linker is covalently bound to the cleavable moiety. In certain such embodiments, the linker is covalently bound to the antisense oligonucleotide. In certain embodiments, the linker is covalently bound to a cell-targeting moiety. In certain embodiments, the linker further comprises a covalent attachment to a solid support. In certain embodiments, the linker further comprises a covalent attachment to a protein binding moiety. In certain embodiments, the linker further comprises a covalent attachment to a solid support and further comprises a covalent attachment to a protein binding moiety. In certain embodiments, the linker includes multiple positions for attachment of tethered ligands. In certain embodiments, the linker includes multiple positions for attachment of tethered ligands and is not attached to a branching group. In certain embodiments, the linker further comprises one or more cleavable bond. In certain embodiments, the conjugate group does not include a linker.
In certain embodiments, the linker includes at least a linear group comprising groups selected from alkyl, amide, disulfide, polyethylene glycol, ether, thioether (-S-) and hydroxylamino (-0-N(H)-) groups. In certain embodiments, the linear group comprises groups selected from alkyl, amide and ether groups. In certain embodiments, the linear group comprises groups selected from alkyl and ether groups. In certain embodiments, the linear group comprises at least one phosphorus linking group.
In certain embodiments, the linear group comprises at least one phosphodiester group. In certain embodiments, the linear group includes at least one neutral linking group. In certain embodiments, the linear group is covalently attached to the cell-targeting moiety and the cleavable moiety. In certain embodiments, the linear group is covalently attached to the cell-targeting moiety and the antisense oligonucleotide. In certain embodiments, the linear group is covalently attached to the cell-targeting moiety, the cleavable moiety and a solid support. In certain embodiments, the linear group is covalently attached to the cell-targeting moiety, the cleavable moiety, a solid support and a protein binding moiety. In certain embodiments, the linear group includes one or more cleavable bond.
In certain embodiments, the linker includes the linear group covalently attached to a scaffold group.
In certain embodiments, the scaffold includes a branched aliphatic group comprising groups selected from alkyl, amide, disulfide, polyethylene glycol, ether, thioether and hydroxylamino groups. In certain embodiments, the scaffold includes a branched aliphatic group comprising groups selected from alkyl, amide and ether groups. In certain embodiments, the scaffold includes at least one mono or polycyclic ring system.
In certain embodiments, the scaffold includes at least two mono or polycyclic ring systems. In certain embodiments, the linear group is covalently attached to the scaffold group and the scaffold group is covalently attached to the cleavable moiety and the linker. In certain embodiments, the linear group is covalently attached to the scaffold group and the scaffold group is covalently attached to the cleavable moiety, the linker and a solid support. In certain embodiments, the linear group is covalently attached to the scaffold group and the scaffold group is covalently attached to the cleavable moiety, the linker and a protein binding moiety. In certain embodiments, the linear group is covalently attached to the scaffold group and the scaffold group is covalently attached to the cleavable moiety, the linker, a protein binding moiety and a solid support. In certain embodiments, the scaffold group includes one or more cleavable bond.

In certain embodiments, the linker includes a protein binding moiety. In certain embodiments, the protein binding moiety is a lipid such as for example including but not limited to cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-0(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, 03-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine), a vitamin (e.g., folate, vitamin A, vitamin E, biotin, pyridoxal), a peptide, a carbohydrate (e.g., monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, polysaccharide), an endosomolytic component, a steroid (e.g., uvaol, hecigenin, diosgenin), a terpene (e.g., triterpene, e.g., sarsasapogenin, friedelin, epifriedelanol derivatized lithocholic acid), or a cationic lipid. In certain embodiments, the protein binding moiety is a C16 to C22 long chain saturated or unsaturated fatty acid, cholesterol, cholic acid, vitamin E, adamantane or 1-pentafluoropropyl.
In certain embodiments, a linker has a structure selected from among:

H H ¨NH
\
,N N I

0 )(:)A )0¨P-OH
N (-30 N
N csTs I 0 H
4') , .
N n 0 (,H
NriLO , VV
H
( ) n X , ,, r011-0H , I I

N I 0 CI\ 31 1¨NH
I OH .
)0H = , P jn, I I
0 vwV 0, I , \=10.... 00N, p,p 0,µ
OH CI\ 31 0,1 1 ri,wLii 0 ' rssrS'S' IWL0 N , H n I
0, HHHH H
\., N ,(,),riN ,(,), N ,(,1r1 N ,)-:p õ 0 n N =
0 , /
I

, ..
0 0 \I ON ,0 Fr ________________________________________________________________ OH
OH
ID'a-0- Fr \ __ )\s-1 \ ---(---/ 0 N
and sk H
v N ..(,)riLo H

wherein each n is, independently, from 1 to 20; and p is from 1 to 6.

In certain embodiments, a linker has a structure selected from among:
.rrs' prri \
\ 0.
I:1.
IDA 0 N 0)2-J-rrj \
q OA rrrj 0 N \
0 N 0)'L

I H
4,rtj 0 \ 0 CI.
N 0;zaL , \
q H

NO
n n 0 H
`1.1`)).LNC).40rN 10 =
n H n 0' µ
0 0'µ

0*.
\ n H
o 0*

N I
0 ¨P = 0 N
I
0 ¨P = 0 H OH ;

¨C) , and o H
N,I(CDc16 _______________________ SN-1 n H

HO
wherein each n is, independently, from 1 to 20.

In certain embodiments, a linker has a structure selected from among:

H
N N
n .1.rMjn H n ,, n css' ; css-nS Sk-.0 ; =2t2./(,)ry'l. ;
A, N H
N ; s no H
0 n ck 1 A
cssn µ .
0 n H
0 0 ' H
H N H
; ; H
n c N ., j cs''r N(:)/in cr H
H H
FN; N
YH I css*VNH 10Q0/'H'n csss ; and n O o o H MEI 'F
clY(2 1-ri\I 0 0 ¨ n n wherein n is from 1 to 20.

In certain embodiments, a linker has a structure selected from among:

H
Ad H
H N

Nq. , "L N H n n H n A
H
, 1 \ i .
-..,õ,........---1,..1N ..,.._ss ;
n c' 0 0 ' 0 H
N
cilm NH .(,00/'HNH Y ln NH -Ho'CR:rH'n ; ".(01 i ;

H
H H N
H ci = csssi\j'HO/Hnf ;
, N.(,, 0 0 \ i n n n " n ' 0 0 AOH
-( OH
H .
"n_ n -n _ -n _ -n TcsYtiANYI-csss and &reOL
n NHA
H n H n .

wherein each L is, independently, a phosphorus linking group or a neutral linking group; and each n is, independently, from 1 to 20.
In certain embodiments, a linker has a structure selected from among:

(:), )0A
)0A 0 N

s,S0 , JJ4j\ ¨NH

q N II
O¨P ¨OH

)A I I

H H
3 1¨NH
- H

I
N )0)\ N I
I \ rO¨P ¨OH
II
, (:), 1¨NH
),OA

H
I
)3 EN11.''y MN ),-,k, =
\,,,, 0 ,0 0 =^'^' 0 NO-. -¨s o \,,,.=
N
I H JVN,V
0 \ ..,,, N f,gLo 01,, \ , , , = =
ik N '...-.1`-')-;)=-" NH *--Y6-0 ;
H

I

I 'i HHHH H
CN 31 0 ,ce . csss 0¨) H ' S's '90 , '14 I

0 0 ,0 0, 1 __ i( NO-0/ _________ OH
, 0\
H
0 CN 31 . S¨S 0 N
;
1=L,)0 N NH'(")&
H

,/0 -'4- ,0 N

H VVVI, HH N
IK N
f 31 0, 'LLL)SS'L)&
, and I
O
o \ ".. o 0 õo I
1 10-'6-\
S-S 0 \µ,...
N
H
IKI
H

In certain embodiments, a linker has a structure selected from among:

N N
cs? .

H
µ)- 0 HN
csC N EI\11)"c ; 0 r / \ ;

H
H
cs'vr8,,N (:)c)N 'csss ; H H
N
cs'c N 000/./ csss ;

H H HN =
csc/N.v ; cl.y.iN and oQc)// csss 7 H
H N
ly,.1\1 '0/./ csss =

In certain embodiments, a linker has a structure selected from among:

H H
A
0 0 ' 0 0 so OH

H

0 L222(k...õ. N Irjt...)ss ;
ck N Ell ,,r ; 0 .2k)L'Y'1/4 ;
r `ssLN Thr3 N Si N A .
; ossy.K8yk ;

H
H
H
cs'sr-8.rN cX0/\/N ,sss ; H
`sssrg-r N .0QC)/./ 1 H
isss H
.......c....õ-N.,..)ss ; csssH

/of; '5Co0/; ssC0001 ;

'sss N
yfl.-8.r1-10/\/scs ; 1 1 OH ''3 3 H

1-0¨P-0 0 0 04-0-1 ; O's k 1 (N/y '26 22.
I Ã/' t'r 1 3 .,, and OH 3 '3 OH H

ISWNIO-F1)-O-1 H 60H ' In certain embodiments, a linker has a structure selected from among:

o 0;aaL

and wherein n is from 1 to 20.
In certain embodiments, a linker has a structure selected from among:
ssCecsss ; ssCe.\.7.e.\./isss ; and In certain embodiments, a linker has a structure selected from among:
/OH
/OH
and OH "3 '3 OH OH "3 3 =
In certain embodiments, a linker has a structure selected from among:

YYLNH;\
IWLN

=
0 and 0 In certain embodiments, the conjugate linker has the structure:
o 0)N"
6 0 .
In certain embodiments, the conjugate linker has the structure:

In certain embodiments, a linker has a structure selected from among:

H
0 =
0 and In certain embodiments, a linker has a structure selected from among:

f\e'k)L issy0L, n NKµ
n OH
0 and 0 wherein each n is independently, 0, 1, 2, 3, 4, 5, 6, or 7.
iv. Certain Cell-Targeting Moieties In certain embodiments, conjugate groups comprise cell-targeting moieties.
Certain such cell-targeting moieties increase cellular uptake of antisense compounds. In certain embodiments, cell-targeting moieties comprise a branching group, one or more tether, and one or more ligand. In certain embodiments, cell-targeting moieties comprise a branching group, one or more tether, one or more ligand and one or more cleavable bond.
1. Certain Branching Groups In certain embodiments, the conjugate groups comprise a targeting moiety comprising a branching group and at least two tethered ligands. In certain embodiments, the branching group attaches the conjugate linker. In certain embodiments, the branching group attaches the cleavable moiety. In certain embodiments, the branching group attaches the antisense oligonucleotide. In certain embodiments, the branching group is covalently attached to the linker and each of the tethered ligands. In certain embodiments, the branching group comprises a branched aliphatic group comprising groups selected from alkyl, amide, disulfide, polyethylene glycol, ether, thioether and hydroxylamino groups. In certain embodiments, the branching group comprises groups selected from alkyl, amide and ether groups. In certain embodiments, the branching group comprises groups selected from alkyl and ether groups. In certain embodiments, the branching group comprises a mono or polycyclic ring system. In certain embodiments, the branching group comprises one or more cleavable bond. In certain embodiments, the conjugate group does not include a branching group.
In certain embodiments, a branching group has a structure selected from among:

¨
0 0 ,(27\l(1 0 0 C)\- \ 0 HOj' O¨P-0 n H i n 0 ' n OH /
NH t CH3 /m . (:).) wv /.71/..
0 ( in 0 n n H).L H
0 , ( )n H 8 ( )n H =
, n 1 .pcJj ,Prr 0 ( 0 ' n m ci V22L

CH3 ( , n n 0 ,/<

CH3.4" 01 n \ 03s im ' \
( NH /
\ /).ri rd I
I 0\ __ .(:) NHNH /
J
0 r )n ( /).n , ) NH /
H

INH
NH
= 4sssN.-----r 4ssL N
n 0 / , H
/ j H
0 (/( 0 vNH
0 `2t2.1----NH
n NNA ; and 0 rr"H0 0 rr"H0 µt22(N N N A
n H H 0 di =
(.' i=Fr NH
v NH

wherein each n is, independently, from 1 to 20;
j is from 1 to 3; and m is from 2 to 6.

In certain embodiments, a branching group has a structure selected from among:
WIAM1.1 cn 0 \ NH

HO-\VI-i 1 0 ¨P-0 n H n n OH 1 n x in e I CH3 im 0 >1_ 1 H 0 ("í H 0 o}
.. n n ,222.,,N1NNe-N41....d , 41) .
, ( )n H 8 ( )n H
J4rj rrrr 0 ( 0 n JUL, m (µ . (/(2 .NH f CH3 C7H3 in 1 c'ss n 01 4() , ' A(N
\ vssf im H ' 0 <
).r1 .
, CH3rdPk n Om rd CI) ;and o o I \ , NH q. NH re n n ___________________________________________ 0 I
"tN--1 ,ss ¨NH r H
0 m wherein each n is, independently, from 1 to 20; and m is from 2 to 6.
In certain embodiments, a branching group has a structure selected from among:

0, iIL.

0 /µ11/-csss ;''' ;

H . 0 '/K

NH 0 , / _______ NH crss I

¨
I ,)NH vs-NH
0 0 (:)'\

A . A Nj-\ N sr ) e .
0 , 0,) ; i,N \ .
, ¨ H

µ 0 HN' y\/\)-1------NH
) y\/\)1----- NH
/
H (PI H (F?
I\ N N
, ; .
, ssc N N ,ros ;
H 11\1 \ H

H O/

HN
v NH

NH NH

0 0 H ?
H ,cs N---"NNs ; and L.LHN N
c.

H 0 / =
O/
csss NH
v NH

In certain embodiments, a branching group has a structure selected from among:
\ I
A1, -Lqiõ A1 /
Ail ¨iok.` In ' In Kr Ai / and õImn, wherein each A1 is independently, 0, S, C=0 or NH; and each n is, independently, from 1 to 20.
In certain embodiments, a branching group has a structure selected from among:

Ai I\ )n A1A A1A

Jr) _____________________________________________ ( A n A1 and ¨A1 ) \n wherein each A1 is independently, 0, S, C=0 or NH; and each n is, independently, from 1 to 20.
In certain embodiments, a branching group has a structure selected from among:
sisr S.\
)n )()n n and Ai \ss wherein A1 is 0, S, C=0 or NH; and each n is, independently, from 1 to 20.
In certain embodiments, a branching group has a structure selected from among:
0, /0¨NH
C) =
.rtryt, In certain embodiments, a branching group has a structure selected from among:

\
ON, 4-10k, / .
In certain embodiments, a branching group has a structure selected from among:
/
\
______________________ 22'4' \,5 .5., .
2. Certain Tethers In certain embodiments, conjugate groups comprise one or more tethers covalently attached to the branching group. In certain embodiments, conjugate groups comprise one or more tethers covalently attached to the linking group. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl, ether, thioether, disulfide, amide and polyethylene glycol groups in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl, substituted alkyl, ether, thioether, disulfide, amide, phosphodiester and polyethylene glycol groups in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl, ether and amide groups in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl, substituted alkyl, phosphodiester, ether and amide groups in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl and phosphodiester in any combination.
In certain embodiments, each tether comprises at least one phosphorus linking group or neutral linking group.
In certain embodiments, the tether includes one or more cleavable bond. In certain embodiments, the tether is attached to the branching group through either an amide or an ether group. In certain embodiments, the tether is attached to the branching group through a phosphodiester group. In certain embodiments, the tether is attached to the branching group through a phosphorus linking group or neutral linking group. In certain embodiments, the tether is attached to the branching group through an ether group.
In certain embodiments, the tether is attached to the ligand through either an amide or an ether group. In certain embodiments, the tether is attached to the ligand through an ether group. In certain embodiments, the tether is attached to the ligand through either an amide or an ether group. In certain embodiments, the tether is attached to the ligand through an ether group.
In certain embodiments, each tether comprises from about 8 to about 20 atoms in chain length between the ligand and the branching group. In certain embodiments, each tether group comprises from about 10 to about 18 atoms in chain length between the ligand and the branching group. In certain embodiments, each tether group comprises about 13 atoms in chain length.
In certain embodiments, a tether has a structure selected from among:
N,1'µ . `222. , n ' /11 0 7 \ in n H 7 H H H
H'Iµn rrrr7f\lN
\-n .1=C'H
i pr ;

( 0 4 \ H 1¨NI
¨N----(2n =
O /2 7 r A9' 13 GSS5 1 1 n =

\ n H ' /n I \ n n n - -P
1¨Ell ( 0 0 ' n N
;and H,n1 n H n wherein each n is, independently, from 1 to 20; and each p is from 1 to about 6.
In certain embodiments, a tether has a structure selected from among:

H
N ./Wcss' ;

H
and In certain embodiments, a tether has a structure selected from among:
H H
in H x In wherein each n is, independently, from 1 to 20.

In certain embodiments, a tether has a structure selected from among:
0 Zi "sH-1¨H)22- and cssjLNI¨H)k mi mi miH m 1 wherein L is either a phosphorus linking group or a neutral linking group;
Z1 is C(=0)0-R2;
Z2 is H, C1-C6 alkyl or substituted C1-C6 alkY;
R2 is H, C1-C6 alkyl or substituted C1-C6 alky; and each m1 is, independently, from 0 to 20 wherein at least one m1 is greater than 0 for each tether.
In certain embodiments, a tether has a structure selected from among:
H H
0 0 .
In certain embodiments, a tether has a structure selected from among:
0 I liti.. 0 COOH OH
)-jr0¨P-0 mi -k1 and c&pJ-I, 04-0-WA
4.1/4. mi 6H m NH II

wherein Z2 is H or CH3; and each m1 is, independently, from 0 to 20 wherein at least one m1 is greater than 0 for each tether.
In certain embodiments, a tether has a structure selected from among:

Y('rNC YTrNC
4 H n H
20.rr ''' , or --...,s 'r ; wherein each n is independently, 0, 1, 2, 3, 4, 5, 6, or 7.
In certain embodiments, a tether comprises a phosphorus linking group. In certain embodiments, a tether does not comprise any amide bonds. In certain embodiments, a tether comprises a phosphorus linking group and does not comprise any amide bonds.

3. Certain Ligands In certain embodiments, the present disclosure provides ligands wherein each ligand is covalently attached to a tether. In certain embodiments, each ligand is selected to have an affinity for at least one type of receptor on a target cell. In certain embodiments, ligands are selected that have an affinity for at least one type of receptor on the surface of a mammalian liver cell. In certain embodiments, ligands are selected that have an affinity for the hepatic asialoglycoprotein receptor (ASGP-R). In certain embodiments, each ligand is a carbohydrate. In certain embodiments, each ligand is, independently selected from galactose, N-acetyl galactoseamine, mannose, glucose, glucosamone and fucose. In certain embodiments, each ligand is N-acetyl galactoseamine (GalNAc). In certain embodiments, the targeting moiety comprises 2 to 6 ligands. In certain embodiments, the targeting moiety comprises 3 ligands. In certain embodiments, the targeting moiety comprises 3 N-acetyl galactoseamine ligands.
In certain embodiments, the ligand is a carbohydrate, carbohydrate derivative, modified carbohydrate, multivalent carbohydrate cluster, polysaccharide, modified polysaccharide, or polysaccharide derivative. In certain embodiments, the ligand is an amino sugar or a thio sugar. For example, amino sugars may be selected from any number of compounds known in the art, for example glucosamine, sialic acid, a-D-galactosamine, N-Acetylgalactosamine, 2-acetamido-2-deoxy-D-galactopyranose (GalNAc), 2-Amino-3- 0-[(R)-1- carb oxyethyl] -2- deoxy-13-D- gluc opyranos e (13-muramic acid), 2-D
eoxy-2-methylamino-L-gluc opyranos e, 4,6-Dideoxy-4-formamido-2,3 -di- 0-methyl-D-mannopyranose, 2-D eoxy-2-sulfoamino-D -glucopyranose and N-sulfo-D-glucosamine, and N-Glycoloyl-a-neuraminic acid.
For example, thio sugars may be selected from the group consisting of 5-Thio-13-D-glucopyranose, Methyl 2,3,4-tri-O-acety1-1-thio-6-0-trity1-a-D-g1ucopyranoside, 4-Thio-13-D-ga1actopyranose, and ethyl 3,4,6,7-tetra-0-acety1-2-deoxy-1,5-dithio-a-D-g/uco-heptopyranoside.
In certain embodiments, "GalNac" or "Gal-NAc" refers to 2-(Acetylamino)-2-deoxy-D-galactopyranose, commonly referred to in the literature as N-acetyl galactosamine. In certain embodiments, "N-acetyl galactosamine" refers to 2-(Acetylamino)-2-deoxy-D-galactopyranose.
In certain embodiments, "GalNac" or "Gal-NAc" refers to 2-(Acetylamino)-2-deoxy-D-galactopyranose. In certain embodiments, "GalNac" or "Gal-NAc" refers to 2-(Acetylamino)-2-deoxy-D-galactopyranose, which includes both the [3-form: 2-(Acety1amino)-2-deoxy-13-D-ga1actopyranose and a-form: 2-(Acetylamino)-2-deoxy-D-galactopyranose. In certain embodiments, both the 13-form: 2-(Acety1amino)-2-deoxy-13-D-ga1actopyranose and a-form: 2-(Acetylamino)-2-deoxy-D-galactopyranose may be used interchangeably. Accordingly, in structures in which one form is depicted, these structures are intended to include the other form as well. For example, where the structure for an a-form: 2-(Acetylamino)-2-deoxy-D-galactopyranose is shown, this structure is intended to include the other form as well. In certain embodiments, In certain preferred embodiments, the 13-form 2-(Acetylamino)-2-deoxy-D-galactopyranose is the preferred embodiment.

H044k44 HO ..'"IliN
nr H
OH
2-(Acetylamino)-2-deoxy-D-galactopyranose OH
OH

HO 0¨

NHAc 2-(Acety1amino)-2-deoxy-13-D-ga1actopyranose OH
OH

HO
NHAc 0c, 2-(Acetylamino)-2-deoxy-a-D-galactopyranose In certain embodiments one or more ligand has a structure selected from among:
O
OH H
OH

HOH0-7.2..

and Ri Ri Ri wherein each R1 is selected from OH and NHCOOH.

In certain embodiments one or more ligand has a structure selected from among:
HOOH OH HO HO
OH OH

0 HO Ho 0 -0 HO
HO Ns ; OH ,,s= . 1-1!::?0 X./ ; HO
, NHAc r c' , 11 HOOH OH
N-1 HO---/-1\ OH HOOH
HO \ros ; 0 .
; and ____7(...:)....\ , HO OH
OH \-L,O, OH 0-.71., A
HO HOr Nrris , HO 0 OH OH
OH
HO
OH

HO
0 __________________ HO OH
OH
F-K -\_"?...\

I .
HO

0 \s5j4 In certain embodiments one or more ligand has a structure selected from among:
HOOH

HO N -Nros NHAc =
In certain embodiments one or more ligand has a structure selected from among:
HOOH
HO N., NHAc r =

i. Certain Conjugates In certain embodiments, conjugate groups comprise the structural features above. In certain such embodiments, conjugate groups have the following structure:
HO OH

___.......70...\
HO H c 0 0,1,...y...........õHNI.....r,N--õfli HO
)n NHA
N 0,g 1 \ in H
n n NHAc 0 0 0"--IC in ) n OH
HO
_.,..\.o....\7o,H,.......
n HO n NHAc 0 =
wherein each n is, independently, from 1 to 20.
In certain such embodiments, conjugate groups have the following structure:
HO OH
*...\...,,.._ H 0 HO
NHAc 0 N
/\/-----,....--"---HO
NHAc 0 OH
HO
H
HO

NHAc =
In certain such embodiments, conjugate groups have the following structure:

- K 0=
P-OH
N N
OH
N
in ---fl, 0 HO
NHAc )n 0 LcTOBx n P-0 X=
I
NHAc 0 :4) OH
0 0 n -----H.L:ill\----H HN
N
HO C'Kn 0 n NHAc O
wherein each n is, independently, from 1 to 20;
Z is H or a linked solid support;
Q is an antisense compound;
X is 0 or S; and Bx is a heterocyclic base moiety.
In certain such embodiments, conjugate groups have the following structure:

0=P-OH
OH \V"--....7NN---------(1 O
HO
NHAc LcTONyBx HO
HP-0 X=
I
NHAc OH

O OH
H.:::4:\"-------.
H HN--i:

N'"----.../
HO O
NHAc In certain such embodiments, conjugate groups have the following structure:
HO H
_..,...\..C..)..\.._____ H H 0 0=P-OH

N N

NxJN
HO
NHAc Lco),N N,..) -......_ .
n.,õ\ 6=
O¨P=0 I
NHAc 0 0 OH

HO H
_..,...\...C..)...\,.___ H HN
HO -----ol"r3N-..,&))/ 0 NHAc o In certain such embodiments, conjugate groups have the following structure:
HOOH
OO,H,-\ ,il?
H
n 0 1 0 AcH N 1n HO OH OH) H .._..r.9....\,0 9 0 O 1H-A;`0--hk ] I
AcH N OH n 0 0,1"1-..0-0 )n HO "n OH
N HAc .
In certain such embodiments, conjugate groups have the following structure:

HO OH
o HO---7.2.,'-' 0 -K
AcHN 0 1 0 OH ---HO OH

ii ] I
AcHN OH 0 P... 0 HO OH
NHAc =
In certain such embodiments, conjugate groups have the following structure:
NOON
HOu ,-, 0 -K
AcHN OH) n NH2 HOOH I\I______µ
0 0-, 0 1 N
HO0-P-OnAN¨cm.,:j AcHN OH OH ==
HO H 0 Cr 0 i.i2.\/00,p,0 II ( A-- in 6 HO-P=0 I
HO "n OH
NHAc .
In certain such embodiments, conjugate groups have the following structure:

HO OH

AcHN 0 O-OH
,J\T
HO OH 0 0õ. 0 V
n\I
0 I OC) AcHN OH o OH õ.==
HO-P=0 HO OH 9 jj P, 6 OH
HO
NHAc In certain such embodiments, conjugate groups have the following structure:
HO-P=0 V n\T
0-0)õN
HO-P=0 O
HO OH

HO
AcHN OH 1) 0 HO OH (on HO ' 0-, 0 0 0---7,10, ______________________________________ ,o1=0 AcHN OH o OH

)11 NHAc =
In certain such embodiments, conjugate groups have the following structure:

HO-P=0 I 0 C/N¨rµN
as' HO¨P=0 I

((3 0 , 0 AcHN

(03 HO OH

0-, I
________________________________________________________ z(i) 1) 13 AcHN OH (:) OH
HO OH 9 y P, OH
HO
NHAc .
In certain embodiments, conjugates do not comprise a pyrrolidine.
In certain such embodiments, conjugate groups have the following structure:

¨FI)-0-HOOH

HO-- o2-=\, r--N----N(:) 0=P-0-AcHN 0 I

HOOH
H H õ 0 ji µ ,11 õ..,..,,...õõ.õ,..,õ...- N o____-- __ N-1-48-NN
H \
AcHN 0 0 0' bH
HOOH HN-----kj H____/______/
HO 0_...4)....\,0-------rN

AcHN =
In certain such embodiments, conjugate groups have the following structure:

HO OH

AcHN
HOOH
0 0, 0 HO`-' __....re..\;-% 0 x /X P

,- -.....õ, , 0-V-o^c rB

AcHN (:) 0 HOOH c), P, I
.....12.\/0...,,---_Z-----.70- 69 HO
NHAc .
In certain such embodiments, conjugate groups have the following structure:
HO OH

AcHN N-'---)r-N...--\.

H H 00. )L7).-L I
HO OHo N-(01-i2)6-0-1D¨

H I I
HO/r-V 0 0 0- 0 NHAc I-INrNHN--eo OH /¨/-0 HO\C) HO
NHAc .
In certain such embodiments, conjugate groups have the following structure:

N
r AcHN N

HO 4 H Hji(HN MI :
AcHN OZ
HOOH
--CI
HOOrN__...f.2..\__.

AcHN .
In certain such embodiments, conjugate groups have the following structure:

AcHN N

AcHN 0 HOOH
--CI
HO CY1rN 0 AcHN .
In certain such embodiments, conjugate groups have the following structure:
HOOH H
-'\AcHN

H H
AcHN
HO OH
N--(0 HO__...r.L..v) AcHN .
In certain such embodiments, conjugate groups have the following structure:
HOOH H

HO "4 .....
AcHN

AcHN a HO OH
N--(0 AcHN .
In certain such embodiments, conjugate groups have the following structure:

OH OH

AcHN
OH OH
H0*_ 0 ,crH 0 H 0 Nr"r6 AcHN H 0 0 0 r NH
HV H
HO
NHAc In certain such embodiments, conjugate groups have the following structure:
OH OH

AcHN
OH OH
H0*_ 0 ,crH 0 H 0,0 AcHN 11 0 0 0 r NH
HV.12.%1 HO
NHAc In certain such embodiments, conjugate groups have the following structure:
p H
HO OH

AcHN
0=P-OH
HO OH
HO
AcHN
0=P-OH
HOOH
HO OO
EMI
AcHN
In certain such embodiments, conjugate groups have the following structure:

pH
HOOH

AcHN
0=P¨OH
HO OH

AcHN
0=P¨OH
HOOH

AcHN 0 In certain embodiments, the cell-targeting moiety of the conjugate group has the following structure:
HOOH
HO-0 n AcHN

HOOH
HO
AcHN
HOOH X/
HO
AcHN
wherein X is a substituted or unsubstituted tether of six to eleven consecutively bonded atoms.
In certain embodiments, the cell-targeting moiety of the conjugate group has the following structure:
HOOH
0 n AcHN

HOOH
HO
AcHN
HOOH X/
HO
AcHN
wherein X is a substituted or unsubstituted tether of ten consecutively bonded atoms.

In certain embodiments, the cell-targeting moiety of the conjugate group has the following structure:
HOOH
HO
X, AcHN \

H
AcHN Z
,0 /
HOOH X
__.7.2..\
HO o/
AcHN
wherein X is a substituted or unsubstituted tether of four to eleven consecutively bonded atoms and wherein the tether comprises exactly one amide bond.
In certain embodiments, the cell-targeting moiety of the conjugate group has the following structure:
HOOH

HO YN A , AcHN N z-AJN

HO H
AcHN H H
Z.
,N.....1c ----(7 HOOH ,Y
_......f.e.. I 0 HO
AcHN
wherein Y and Z are independently selected from a C1-C12 substituted or unsubstituted alkyl, alkenyl, or alkynyl group, or a group comprising an ether, a ketone, an amide, an ester, a carbamate, an amine, a piperidine, a phosphate, a phosphodiester, a phosphorothioate, a triazole, a pyrrolidine, a disulfide, or a thioether.
In certain such embodiments, the cell-targeting moiety of the conjugate group has the following structure:

HOOH
N

HO YN
AcHN N z-ON

HO
AcHN H H
HOOH ,Y
HO -O
AcHN
wherein Y and Z are independently selected from a C1-C12 substituted or unsubstituted alkyl group, or a group comprising exactly one ether or exactly two ethers, an amide, an amine, a piperidine, a phosphate, a phosphodiester, or a phosphorothioate.
In certain such embodiments, the cell-targeting moiety of the conjugate group has the following structure:
HOOH
N

HO YN
AcHN N z-ON

HO
AcHN H H
HOOH ,Y

AcHN
wherein Y and Z are independently selected from a C i-C12 substituted or unsubstituted alkyl group.
In certain such embodiments, the cell-targeting moiety of the conjugate group has the following structure:
HOOH
HO
AcHN 0 HOOH
9N)-efFiNA
HO 0 m H
H,..1(4,10Z
AcHN
HOOH c<µ 0 HO ---AcHN
wherein m and n are independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12.
In certain such embodiments, the cell-targeting moiety of the conjugate group has the following structure:

HO
H N
AcHN 0 n HOOH
_......T:.....0 J'2---N)11-i FiNA
o "m H

HO 10, AcHN
N
HOOH_ ... cc\ 0 AcHN
wherein m is 4, 5, 6, 7, or 8, and n is 1, 2, 3, or 4.
In certain embodiments, the cell-targeting moiety of the conjugate group has the following structure:
HOOH

HOOH HO --..'r'...
_...".(2..\___0 AcHN 1/4---HO _________________ X----\
N
AcHN
01-10H r- H

AcHN
wherein X is a substituted or unsubstituted tether of four to thirteen consecutively bonded atoms, and wherein X does not comprise an ether group.
In certain embodiments, the cell-targeting moiety of the conjugate group has the following structure:
HOOH

HOOH HO --..'r'...
_...".(2..\___0 AcHN 1/4---HO _________________ X----\
N
AcHN
01-10H r- H

AcHN
wherein X is a substituted or unsubstituted tether of eight consecutively bonded atoms, and wherein X does not comprise an ether group.
In certain embodiments, the cell-targeting moiety of the conjugate group has the following structure:

HOOH

HOOH HO . X
...".(2..\_AcHN L--...
'N.
HO_ __0 X-----\H\l/
AcHN
01-10H r- H
HO--0¨

AcHN
wherein X is a substituted or unsubstituted tether of four to thirteen consecutively bonded atoms, and wherein the tether comprises exactly one amide bond, and wherein X does not comprise an ether group.
In certain embodiments, the cell-targeting moiety of the conjugate group has the following structure:
HOOH

HOOH HO . X
...".(2..\_AcHN L---..
'N.
HO_ __0 X-----\H\l/
AcHN
01-10H r- H

AcHN
wherein X is a substituted or unsubstituted tether of four to thirteen consecutively bonded atoms and wherein the tether consists of an amide bond and a substituted or unsubstituted C2-Ci1 alkyl group.
In certain embodiments, the cell-targeting moiety of the conjugate group has the following structure:
HOOH H
HO
AcHN

_....72...vONN'''µ.
HO H
H
AcHN
HO OH
HO_..1.(2.vo¨y-----121 u AcHN
wherein Y is selected from a C1-C12 substituted or unsubstituted alkyl, alkenyl, or alkynyl group, or a group comprising an ether, a ketone, an amide, an ester, a carbamate, an amine, a piperidine, a phosphate, a phosphodiester, a phosphorothioate, a triazole, a pyrrolidine, a disulfide, or a thioether.
In certain such embodiments, the cell-targeting moiety of the conjugate group has the following structure:

NOON
HO
AcHN

NA
HO
AcHN
HO OH

HO
OYH
AcHN
wherein Y is selected from a C1-C12 substituted or unsubstituted alkyl group, or a group comprising an ether, an amine, a piperidine, a phosphate, a phosphodiester, or a phosphorothioate.
In certain such embodiments, the cell-targeting moiety of the conjugate group has the following structure:
NOON
HO
AcHN

NA
HO
AcHN
HO OH

HO
OYH
AcHN
wherein Y is selected from a C i-C12 substituted or unsubstituted alkyl group.
In certain such embodiments, the cell-targeting moiety of the conjugate group has the following structure:
HO OH
__7(2.v0.pN 0 HO
AcHN

NA

AcHN
NOON j HO -./O
AcHN
Wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.
In certain such embodiments, the cell-targeting moiety of the conjugate group has the following structure:

HOOH H
_.7.2.vo.pN 0 AcHN

_CZ/0-6.-N)NA
HO nH
H
AcHN
HOOH ( \
HO__..rC...)..\zcil\ i n N--(0 H
AcHN
wherein n is 4, 5, 6, 7, or 8.
b. Certain conjugated antisense compounds In certain embodiments, the conjugates are bound to a nucleoside of the antisense oligonucleotide at the 2', 3', of 5' position of the nucleoside. In certain embodiments, a conjugated antisense compound has the following structure:
A¨B¨C¨D¨EE¨F) q wherein A is the antisense oligonucleotide;
B is the cleavable moiety C is the conjugate linker D is the branching group each E is a tether;
each F is a ligand; and q is an integer between 1 and 5.
In certain embodiments, a conjugated antisense compound has the following structure:
A¨C¨DiE¨F) q wherein A is the antisense oligonucleotide;
C is the conjugate linker D is the branching group each E is a tether;
each F is a ligand; and q is an integer between 1 and 5.
In certain such embodiments, the conjugate linker comprises at least one cleavable bond.
In certain such embodiments, the branching group comprises at least one cleavable bond.
In certain embodiments each tether comprises at least one cleavable bond.
In certain embodiments, the conjugates are bound to a nucleoside of the antisense oligonucleotide at the 2', 3', of 5' position of the nucleoside.
In certain embodiments, a conjugated antisense compound has the following structure:
A¨B¨CiE¨F) q wherein A is the antisense oligonucleotide;
B is the cleavable moiety C is the conjugate linker each E is a tether;
each F is a ligand; and q is an integer between 1 and 5.
In certain embodiments, the conjugates are bound to a nucleoside of the antisense oligonucleotide at the 2', 3', of 5' position of the nucleoside. In certain embodiments, a conjugated antisense compound has the following structure:
A¨CiE¨F) q wherein A is the antisense oligonucleotide;
C is the conjugate linker each E is a tether;
each F is a ligand; and q is an integer between 1 and 5.
In certain embodiments, a conjugated antisense compound has the following structure:
A¨B¨D¨EE¨F) q wherein A is the antisense oligonucleotide;
B is the cleavable moiety D is the branching group each E is a tether;
each F is a ligand; and q is an integer between 1 and 5.
In certain embodiments, a conjugated antisense compound has the following structure:
A¨D¨EE¨F) q wherein A is the antisense oligonucleotide;
D is the branching group each E is a tether;
each F is a ligand; and q is an integer between 1 and 5.
In certain such embodiments, the conjugate linker comprises at least one cleavable bond.
In certain embodiments each tether comprises at least one cleavable bond.
In certain embodiments, a conjugated antisense compound has a structure selected from among the following:

Targeting moiety ASO
HO OH
0 =P -OH

pH O
NHAc 0 1 I 1 s......7..Ø...\_____ H H d N' HO

NHAc g 0 _ 0' Linker Cleavable moiety - Ligand Tether OH
H:.....7........võ.. HN -----0 N Branching group NI-IAe =
In certain embodiments, a conjugated antisense compound has a structure selected from among the following:
Cell targeting moiety HO OH

¨
Cleavable moiety ¨
AcHN 0 1 ic=____ OH
_ -HO OH _________________________________________________ , ir,( cl_liLcrõ,õ.......c ,siC2 --I
HO 0 1 0(Y 6-N----_ AcHN _ _ OH 0"-- 0 Tether _____________ , -04=0 Ligand HO OH 91 y ¨
P¨
ASO
_ 1,2..volp' 1 0 HO OH
NHAc Branching group =
In certain embodiments, a conjugated antisense compound has a structure selected from among the following:

_ ASO
Cleavable moiety _ iNH2 HO¨P=0 I 0 (N¨riN
N-:_---d HO¨P=0 Cell targeting moiety I
¨ ' 0 ' _ u HO-4)...\/' 0 ,k AcHN

_ HO OH _ . _____ , (03 Conjugate 0 0--... 0 I linker _ HO -------------\/N -R. .---,.......---, ,.--0 1 0 0 ------------(31¨(31 _ AcHN _ cy-- - OH
Tether, ' __ 1 Ligand HO H 9 y P, o_ 0 HO
NHAc Branching group In certain embodiments, the conjugated antisense compound has the following structure:

O
Oc) 0 0-P =0 NH <,N Jr 1 I ,, N H
\
N't HNI' cr, 0 N 0 N N

1_0_/
,f NH NO cv 0 HO H O 0 N 0 e 0 o) o e O
..-11-,_õ,-0-.¨NH 5-P=0 = 1 7-1 HO 0N oI (I'lf,N,I,,H S-p 0 NH Oz N N NH2 ' --1_0_)/

0 o N
--i NH2 _....7Ø_v 1 S-p=0 N
HO 01rN
4 H Art'N (fr --õr NH
N,'0 NH2 0 O\ 0W
e S -P =0 o-----loiN 0 S -P=0 ILL:71 O
o 0 NHo S -P =0 O NH2 0 N o es -i.-oolill OZ/No , 8 0 0,) NH2 S-P =0 --c, S -P =0 ' N 0 O a S -P =0 e 0 o1 r S -P =0 O

e 0 0 s -P=0 7,-i s-1;'=0 yi NH

N
_Oj Wcr e 0 0,) o s-p-oNHo ID
S-P =0 All'7H

NH
ID - j NH2 S -P=0 0 ID
ON N NH2 S -P=0 1_ 0 I
N 0 0_/
O

S -P =0 OHO

_____________________________________________________________________________ .
Representative United States patents, United States patent application publications, and international patent application publications that teach the preparation of certain of the above noted conjugates, conjugated antisense compounds, tethers, linkers, branching groups, ligands, cleavable moieties as well as other modifications include without limitation, US 5,994,517, US 6,300,319, US
6,660,720, US 6,906,182, US
7,262,177, US 7,491,805, US 8,106,022, US 7,723,509, US 2006/0148740, US
2011/0123520, WO
2013/033230 and WO 2012/037254, each of which is incorporated by reference herein in its entirety.
Representative publications that teach the preparation of certain of the above noted conjugates, conjugated antisense compounds, tethers, linkers, branching groups, ligands, cleavable moieties as well as other modifications include without limitation, BIESSEN et al., "The Cholesterol Derivative of a Triantennary Galactoside with High Affinity for the Hepatic Asialoglycoprotein Receptor: a Potent Cholesterol Lowering Agent" J. Med. Chem. (1995) 38:1846-1852, BIESSEN et al., "Synthesis of Cluster Galactosides with High Affinity for the Hepatic Asialoglycoprotein Receptor"
J. Med. Chem. (1995) 38:1538-1546, LEE et al., "New and more efficient multivalent glyco-ligands for asialoglycoprotein receptor of mammalian hepatocytes" Bioorganic & Medicinal Chemistry (2011) 19:2494-2500, RENSEN et al., "Determination of the Upper Size Limit for Uptake and Processing of Ligands by the Asialoglycoprotein Receptor on Hepatocytes in Vitro and in Vivo" J. Biol. Chem. (2001) 276(40):37577-37584, RENSEN et al., "Design and Synthesis of Novel N-Acetylgalactosamine-Terminated Glycolipids for Targeting of Lipoproteins to the Hepatic Asialoglycoprotein Receptor" J. Med. Chem. (2004) 47:5798-5808, SLIEDREGT
et al., "Design and Synthesis of Novel Amphiphilic Dendritic Galactosides for Selective Targeting of Liposomes to the Hepatic Asialoglycoprotein Receptor" J. Med. Chem. (1999) 42:609-618, and Valentijn et al., "Solid-phase synthesis of lysine-based cluster galactosides with high affinity for the Asialoglycoprotein Receptor" Tetrahedron, 1997, 53(2), 759-770, each of which is incorporated by reference herein in its entirety.
In certain embodiments, conjugated antisense compounds comprise an RNase H
based oligonucleotide (such as a gapmer) or a splice modulating oligonucleotide (such as a fully modified oligonucleotide) and any conjugate group comprising at least one, two, or three GalNAc groups. In certain embodiments a conjugated antisense compound comprises any conjugate group found in any of the following references: Lee, Carbohydr Res, 1978, 67, 509-514; Connolly et al., J Biol Chem, 1982, 257, 939-945; Pavia et al., Int J Pep Protein Res, 1983, 22, 539-548; Lee et al., Biochem, 1984, 23, 4255-4261; Lee et al., Glycoconjugate J, 1987, 4, 317-328; Toyokuni et al., Tetrahedron Lett, 1990, 31, 2673-2676; Biessen et al., J
Med Chem, 1995, 38, 1538-1546; Valentijn et al., Tetrahedron, 1997, 53, 759-770; Kim et al., Tetrahedron Lett, 1997, 38, 3487-3490; Lee et al., Bioconjug Chem, 1997, 8, 762-765; Kato et al., Glycobiol, 2001, 11, 821-829; Rensen et al., J Biol Chem, 2001, 276, 37577-37584; Lee et al., Methods Enzymol, 2003, 362, 38-43; Westerlind et al., Glycoconj J, 2004, 21, 227-241; Lee et al., Bioorg Med Chem Lett, 2006, 16(19), 5132-5135; Maierhofer et al., Bioorg Med Chem, 2007, 15, 7661-7676; Khorev et al., Bioorg Med Chem, 2008, 16, 5216-5231; Lee et al., Bioorg Med Chem, 2011, 19, 2494-2500; Kornilova et al., Analyt Biochem, 2012, 425, 43-46; Pujol et al., Angew Chemie Int Ed Engl, 2012, 51, 7445-7448; Biessen et al., J Med Chem, 1995, 38, 1846-1852; Sliedregt et al., J Med Chem, 1999, 42, 609-618; Rensen et al., J
Med Chem, 2004, 47, 5798-5808; Rensen et al., Arterioscler Thromb Vasc Biol, 2006, 26, 169-175; van Rossenberg et al., Gene Ther, 2004, 11, 457-464; Sato et al., J Am Chem Soc, 2004, 126, 14013-14022; Lee et al., J Org Chem, 2012, 77, 7564-7571; Biessen et al., FASEB J, 2000, 14, 1784-1792; Rajur et al., Bioconjug Chem, 1997, 8, 935-940;
Duff et al., Methods Enzymol, 2000, 313, 297-321; Maier et al., Bioconjug Chem, 2003, 14, 18-29;
Jayaprakash et al., Org Lett, 2010, 12, 5410-5413; Manoharan, Antisense Nucleic Acid Drug Dev, 2002, 12, 103-128; Merwin et al., Bioconjug Chem, 1994, 5, 612-620; Tomiya et al., Bioorg Med Chem, 2013, 21, 5275-5281; International applications W01998/013381; W02011/038356;
W01997/046098;
W02008/098788; W02004/101619; W02012/037254; W02011/120053; W02011/100131;
W02011/163121; W02012/177947; W02013/033230; W02013/075035; W02012/083185;
W02012/083046; W02009/082607; W02009/134487; W02010/144740; W02010/148013;
W01997/020563; W02010/088537; W02002/043771; W02010/129709; W02012/068187;
W02009/126933; W02004/024757; W02010/054406; W02012/089352; W02012/089602;
W02013/166121; W02013/165816; U.S. Patents 4,751,219; 8,552,163; 6,908,903;
7,262,177; 5,994,517;
6,300,319; 8,106,022; 7,491,805; 7,491,805; 7,582,744; 8,137,695; 6,383,812;
6,525,031; 6,660,720;
7,723,509; 8,541,548; 8,344,125; 8,313,772; 8,349,308; 8,450,467; 8,501,930;
8,158,601; 7,262,177;
6,906,182; 6,620,916; 8,435,491; 8,404,862; 7,851,615; Published U.S. Patent Application Publications US2011/0097264; US2011/0097265; U52013/0004427; U52005/0164235;
U52006/0148740;
U52008/0281044; U52010/0240730; US2003/0119724; U52006/0183886;
U52008/0206869;
US2011/0269814; U52009/0286973; US2011/0207799; U52012/0136042;
U52012/0165393;
U52008/0281041; U52009/0203135; U52012/0035115; U52012/0095075;
U52012/0101148;
U52012/0128760; U52012/0157509; U52012/0230938; U52013/0109817;
US2013/0121954;
U52013/0178512; U52013/0236968; U52011/0123520; U52003/0077829;
U52008/0108801; and U52009/0203132; each of which is incorporated by reference in its entirety.
C. Certain Uses and Features In certain embodiments, conjugated antisense compounds exhibit potent target RNA reduction in vivo. In certain embodiments, unconjugated antisense compounds accumulate in the kidney. In certain embodiments, conjugated antisense compounds accumulate in the liver. In certain embodiments, conjugated antisense compounds are well tolerated. Such properties render conjugated antisense compounds particularly useful for inhibition of many target RNAs, including, but not limited to those involved in metabolic, cardiovascular and other diseases, disorders or conditions. Thus, provided herein are methods of treating such diseases, disorders or conditions by contacting liver tissues with the conjugated antisense compounds targeted to RNAs associated with such diseases, disorders or conditions. Thus, also provided are methods for ameliorating any of a variety of metabolic, cardiovascular and other diseases, disorders or conditions with the conjugated antisense compounds of the present invention.
In certain embodiments, conjugated antisense compounds are more potent than unconjugated counterpart at a particular tissue concentration. Without wishing to be bound by any theory or mechanism, in certain embodiemtns, the conjugate may allow the conjugated antisense compound to enter the cell more efficiently or to enter the cell more productively. For example, in certain embodiments conjugated antisense compounds may exhibit greater target reduction as compared to its unconjugated counterpart wherein both the conjugated antisense compound and its unconjugated counterpart are present in the tissue at the same concentrations. For example, in certain embodiments conjugated antisense compounds may exhibit greater target reduction as compared to its unconjugated counterpart wherein both the conjugated antisense compound and its unconjugated counterpart are present in the liver at the same concentrations.
Productive and non-productive uptake of oligonucleotides has beed discussed previously (See e.g.
Geary, R. S., E. Wancewicz, et al. (2009). "Effect of Dose and Plasma Concentration on Liver Uptake and Pharmacologic Activity of a 2'-Methoxyethyl Modified Chimeric Antisense Oligonucleotide Targeting PTEN." Biochem. Pharmacol. 78(3): 284-91; & Koller, E., T. M. Vincent, et al.
(2011). "Mechanisms of single-stranded phosphorothioate modified antisense oligonucleotide accumulation in hepatocytes." Nucleic Acids Res. 39(11): 4795-807). Conjugate groups described herein may improve productive uptake.
In certain embodiments, the conjugate groups described herein may further improve potency by increasing the affinity of the conjugated antisense compound for a particular type of cell or tissue. In certain embodiments, the conjugate groups described herein may further improve potency by increasing recognition of the conjugated antisense compound by one or more cell-surface receptors. .
In certain embodiments, the conjugate groups described herein may further improve potency by facilitating endocytosis of the conjugated antisense compound.
In certain embodiments, the cleavable moiety may further improve potency by allowing the conjugate to be cleaved from the antisense oligonucleotide after the conjugated antisense compound has entered the cell. Accordingly, in certain embodiments, conjugated antisense compounds can be administed at doses lower than would be necessary for unconjugated antisense oligonucleotides.
Phosphorothioate linkages have been incorporated into antisense oligonucleotides previously. Such phosphorothioate linkages are resistant to nucleases and so improve stability of the oligonucleotide. Further, phosphorothioate linkages also bind certain proteins, which results in accumulation of antisense oligonucleotide in the liver. Oligonucleotides with fewer phosphorothioate linkages accumulate less in the liver and more in the kidney (see, for example, Geary, R., "Pharmacokinetic Properties of 2'4)-(2-Methoxyethyl)-Modified Oligonucleotide Analogs in Rats," Journal of Pharmacology and Experimental Therapeutics, Vol. 296, No. 3, 890-897; & Pharmacological Properties of 2 '-0-Methoxyethyl Modified Oligonucleotides in Antisense a Drug Technology, Chapter 10, Crooke, S.T., ed., 2008) In certain embodiments, oligonucleotides with fewer phosphorothioate internculeoside linkages and more phosphodiester internucleoside linkages accumulate less in the liver and more in the kidney. When treating diseases in the liver, this is undesibable for several reasons (1) less drug is getting to the site of desired action (liver); (2) drug is escaping into the urine; and (3) the kidney is exposed to relatively high concentration of drug which can result in toxicities in the kidney. Thus, for liver diseases, phosphorothioate linkages provide important benefits.

In certain embodiments, however, administration of oligonucleotides uniformly linked by phosphoro-thioate internucleoside linkages induces one or more proinflammatory reactions. (see for example: J Lab Clin Med. 1996 Sep; 128(3):329-38. "Amplification of antibody production by phosphorothioate oligodeoxynucleotides". Branda et al.; and see also for example: Toxicologic Properties in Antisense a Drug Technology, Chapter 12, pages 342-351, Crooke, S.T., ed., 2008). In certain embodiments, administration of oligonucleotides wherein most of the internucleoside linkages comprise phosphorothioate internucleoside linkages induces one or more proinflammatory reactions.
In certain embodiments, the degree of proinflammatory effect may depend on several variables (e.g.
backbone modification, off-target effects, nucleobase modifications, and/or nucleoside modifications) see for example: Toxicologic Properties in Antisense a Drug Technology, Chapter 12, pages 342-351, Crooke, S.T., ed., 2008). In certain embodiments, the degree of proinflammatory effect may be mitigated by adjusting one or more variables. For example the degree of proinflammatory effect of a given oligonucleotide may be mitigated by replacing any number of phosphorothioate internucleoside linkages with phosphodiester internucleoside linkages and thereby reducing the total number of phosphorothioate internucleoside linkages.
In certain embodiments, it would be desirable to reduce the number of phosphorothioate linkages, if doing so could be done without losing stability and without shifting the distribution from liver to kidney. For example, in certain embodiments, the number of phosphorothioate linkages may be reduced by replacing phosphorothioate linkages with phosphodiester linkages. In such an embodiment, the antisense compound having fewer phosphorothioate linkages and more phosphodiester linkages may induce less proinflammatory reactions or no proinflammatory reaction. Although the the antisense compound having fewer phosphoro-thioate linkages and more phosphodiester linkages may induce fewer proinflammatory reactions, the antisense compound having fewer phosphorothioate linkages and more phosphodiester linkages may not accumulate in the liver and may be less efficacious at the same or similar dose as compared to an antisense compound having more phosphorothioate linkages. In certain embodiments, it is therefore desirable to design an antisense compound that has a plurality of phosphodiester bonds and a plurality of phosphorothioate bonds but which also possesses stability and good distribution to the liver.
In certain embodiments, conjugated antisense compounds accumulate more in the liver and less in the kidney than unconjugated counterparts, even when some of the phosporothioate linkages are replaced with less proinflammatory phosphodiester internucleoside linkages. In certain embodiments, conjugated antisense compounds accumulate more in the liver and are not excreted as much in the urine compared to its unonjugated counterparts, even when some of the phosporothioate linkages are replaced with less proinflammatory phosphodiester internucleoside linkages. In certain embodiments, the use of a conjugate allows one to design more potent and better tolerated antisense drugs. Indeed, in certain emobidments, conjugated antisense compounds have larger therapeutic indexes than unconjugated counterparts. This allows the conjugated antisense compound to be administered at a higher absolute dose, because there is less risk of proinflammatory response and less risk of kidney toxicity. This higher dose, allows one to dose less frequently, since the clearance (metabolism) is expected to be similar.
Further, because the compound is more potent, as described above, one can allow the concentration to go lower before the next dose without losing therapeutic activity, allowing for even longer periods between dosing.
In certain embodiments, the inclusion of some phosphorothioate linkages remains desirable. For example, the terminal linkages are vulnerable to exonucleoases and so in certain embodiments, those linkages are phosphorothioate or other modified linkage. Internucleoside linkages linking two deoxynucleosides are vulnerable to endonucleases and so in certain embodiments those those linkages are phosphorothioate or other modified linkage. Internucleoside linkages between a modified nucleoside and a deoxynucleoside where the deoxynucleoside is on the 5' side of the linkage deoxynucleosides are vulnerable to endonucleases and so in certain embodiments those those linkages are phosphorothioate or other modified linkage.
Internucleoside linkages between two modified nucleosides of certain types and between a deoxynucleoside and a modified nucleoside of certain typ where the modified nucleoside is at the 5' side of the linkage are sufficiently resistant to nuclease digestion, that the linkage can be phosphodiester.
In certain embodiments, the antisense oligonucleotide of a conjugated antisense compound comprises fewer than 16 phosphorthioate linkages. In certain embodiments, the antisense oligonucleotide of a conjugated antisense compound comprises fewer than 15 phosphorthioate linkages. In certain embodiments, the antisense oligonucleotide of a conjugated antisense compound comprises fewer than 14 phosphorthioate linkages. In certain embodiments, the antisense oligonucleotide of a conjugated antisense compound comprises fewer than 13 phosphorthioate linkages. In certain embodiments, the antisense oligonucleotide of a conjugated antisense compound comprises fewer than 12 phosphorthioate linkages. In certain embodiments, the antisense oligonucleotide of a conjugated antisense compound comprises fewer than 11 phosphorthioate linkages. In certain embodiments, the antisense oligonucleotide of a conjugated antisense compound comprises fewer than 10 phosphorthioate linkages. In certain embodiments, the antisense oligonucleotide of a conjugated antisense compound comprises fewer than 9 phosphorthioate linkages. In certain embodiments, the antisense oligonucleotide of a conjugated antisense compound comprises fewer than 8 phosphorthioate linkages.
In certain embodiments, antisense compounds comprsing one or more conjugae group described herein has increased activity and/or potency and/or tolerability compared to a parent antisense compound lacking such one or more conjugate group. Accordingly, in certain embodiments, attachment of such conjugate groups to an oligonucleotide is desirable. Such conjugate groups may be attached at the 5'-, and/or 3'- end of an oligonucleotide. In certain instances, attachment at the 5'-end is synthetically desireable.
Typically, oligonucleietides are synthesized by attachment of the 3' terminal nucleoside to a solid support and sequential coupling of nucleosides from 3' to 5' using techniques that are well known in the art.
Accordingly if a conjugate group is desred at the 3'-terminus, one may (1) attach the conjugate group to the 3'-terminal nucleoside and attach that conjugated nucleoside to the solid support for subsequent preparation of the oligonucleotide or (2) attach the conjugate group to the 3'-terminal nucleoside of a completed oligonucleotide after synthesis. Niether of these approaches is very efficient and thus both are costly. In particular, attachment of the conjugated nucleoside to the solid support, while demonstrated in the Examples herein, is an inefficient process. In certain embodiments, attaching a conjugate group to the 5'-terminal nucleoside is synthetically easier than attachment at the 3'-end. One may attach a non-conjugated 3' terminal nucleoside to the solid support and prepare the oligonucleotide using standard and well characterized reastions. One then needs only to attach a 5'nucleoside having a conjugate group at the final coupling step.
In certain embodiments, this is more efficient than attaching a conjugated nucleoside directly to the solid support as is typically done to prepare a 3'-conjugated oligonucleotide. The Examples herein demonstrate attachment at the 5'-end. In addition, certain conjugate groups have synthetic advantages. For Example, certain conjugate groups comprising phosphorus linkage groups are synthetically simpler and more efficiently prepared than other conjugate groups, including conjugate groups reported previously (e.g., WO/2012/037254).
In certain embodiments, conjugated antisense compounds are administered to a subject. In such embodiments, antisense compounds comprsing one or more conjugae group described herein has increased activity and/or potency and/or tolerability compared to a parent antisense compound lacking such one or more conjugate group. Without being bound by mechanism, it is believed that the conjugate group helps with distribution, delivery, and/or uptake into a target cell or tissue. In certain embodiments, once inside the target cell or tissue, it is desirable that all or part of the conjugate group to be cleaved to releas the active oligonucleitde. In certain embodiments, it is not necessary that the entire conjugate group be cleaved from the oligonucleotide. For example, in Example 20 a conjugated oligonucleotide was administered to mice and a number of different chemical species, each comprising a different portion of the conjugate group remaining on the oligonucleotide, were detected (Table 23a). Thisconjugated antisense compound demonstrated good potency (Table 23). Thus, in certain embodiments, such metabolite profile of multiple partial cleavage of the conjugate group does not interfere with activity/potency. Nevertheless, in certain embodiments it is desirable that a prodrug (conjugated oligonucleotide) yield a single active compound. In certain instances, if multiple forms of the active compound are found, it may be necessary to determine relative amounts and activities for each one. In certain embodiments where regulatory review is required (e.g., USFDA or counterpart) it is desirable to have a single (or predominantly single) active species. In certain such embodiments, it is desirable that such single active species be the antisense oligonucleotide lacking any portion of the conjugate group. In certain embodiments, conjugate groups at the 5'-end are more likely to result in complete metabolism of the conjugate group. Without being bound by mechanism it may be that endogenous enzymes responsible for metabolism at the 5' end (e.g., 5' nucleases) are more active/efficient than the 3' counterparts.
In certain embodiments, the specific conjugate groups are more amenable to metabolism to a single active species. In certain embodiments, certain conjugate groups are more amenable to metabolism to the oligonucleotide.
D. Antisense In certain embodiments, oligomeric compounds of the present invention are antisense compounds.
In such embodiments, the oligomeric compound is complementary to a target nucleic acid. In certain embodiments, a target nucleic acid is an RNA. In certain embodiments, a target nucleic acid is a non-coding RNA. In certain embodiments, a target nucleic acid encodes a protein. In certain embodiments, a target nucleic acid is selected from a mRNA, a pre-mRNA, a microRNA, a non-coding RNA, including small non-coding RNA, and a promoter-directed RNA. In certain embodiments, oligomeric compounds are at least partially complementary to more than one target nucleic acid. For example, oligomeric compounds of the present invention may be microRNA mimics, which typically bind to multiple targets.
In certain embodiments, antisense compounds comprise a portion having a nucleobase sequence at least 70% complementary to the nucleobase sequence of a target nucleic acid.
In certain embodiments, antisense compounds comprise a portion having a nucleobase sequence at least 80% complementary to the nucleobase sequence of a target nucleic acid. In certain embodiments, antisense compounds comprise a portion having a nucleobase sequence at least 90% complementary to the nucleobase sequence of a target nucleic acid. In certain embodiments, antisense compounds comprise a portion having a nucleobase sequence at least 95% complementary to the nucleobase sequence of a target nucleic acid. In certain embodiments, antisense compounds comprise a portion having a nucleobase sequence at least 98%
complementary to the nucleobase sequence of a target nucleic acid. In certain embodiments, antisense compounds comprise a portion having a nucleobase sequence that is 100%
complementary to the nucleobase sequence of a target nucleic acid. In certain embodiments, antisense compounds are at least 70%, 80%, 90%, 95%, 98%, or 100% complementary to the nucleobase sequence of a target nucleic acid over the entire length of the antisense compound.
Antisense mechanisms include any mechanism involving the hybridization of an oligomeric compound with target nucleic acid, wherein the hybridization results in a biological effect. In certain embodiments, such hybridization results in either target nucleic acid degradation or occupancy with concomitant inhibition or stimulation of the cellular machinery involving, for example, translation, transcription, or polyadenylation of the target nucleic acid or of a nucleic acid with which the target nucleic acid may otherwise interact.
One type of antisense mechanism involving degradation of target RNA is RNase H
mediated antisense. RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. It is known in the art that single-stranded antisense compounds which are "DNA-like"
elicit RNase H activity in mammalian cells. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of DNA-like oligonucleotide-mediated inhibition of gene expression.
Antisense mechanisms also include, without limitation RNAi mechanisms, which utilize the RISC
pathway. Such RNAi mechanisms include, without limitation siRNA, ssRNA and microRNA mechanisms.
Such mechanisms include creation of a microRNA mimic and/or an anti-microRNA.
Antisense mechanisms also include, without limitation, mechanisms that hybridize or mimic non-coding RNA other than microRNA or mRNA. Such non-coding RNA includes, but is not limited to promoter-directed RNA and short and long RNA that effects transcription or translation of one or more nucleic acids.
In certain embodiments, oligonucleotides comprising conjugates described herein are RNAi compounds. In certain embodiments, oligomeric oligonucleotides comprising conjugates described herein are ssRNA compounds. In certain embodiments, oligonucleotides comprising conjugates described herein are paired with a second oligomeric compound to form an siRNA. In certain such embodiments, the second oligomeric compound also comprises a conjugate. In certain embodiments, the second oligomeric compound is any modified or unmodified nucleic acid. In certain embodiments, the oligonucleotides comprising conjugates described herein is the antisense strand in an siRNA compound. In certain embodiments, the oligonucleotides comprising conjugates described herein is the sense strand in an siRNA compound. In embodiments in which the conjugated oligomeric compound is double-stranded siRnA, the conjugate may be on the sense strand, the antisense strand or both the sense strand and the antisense strand.
C. Apolipoprotein (a) (apo(a)) In certain embodiments, conjugated antisense compounds target any apo(a) nucleic acid. In certain embodiments, the target nucleic acid encodes an apo(a) target protein that is clinically relevant. In such embodiments, modulation of the target nucleic acid results in clinical benefit.
The targeting process usually includes determination of at least one target region, segment, or site within the target nucleic acid for the antisense interaction to occur such that the desired effect will result.
In certain embodiments, a target region is a structurally defined region of the nucleic acid. For example, in certain such embodiments, a target region may encompass a 3' UTR, a 5' UTR, an exon, an intron, a coding region, a translation initiation region, translation termination region, or other defined nucleic acid region or target segment.
In certain embodiments, a target segment is at least about an 8-nucleobase portion of a target region to which a conjugated antisense compound is targeted. Target segments can include DNA or RNA sequences that comprise at least 8 consecutive nucleobases from the 5'-terminus of one of the target segments (the remaining nucleobases being a consecutive stretch of the same DNA or RNA
beginning immediately upstream of the 5'-terminus of the target segment and continuing until the DNA
or RNA comprises about 8 to about 30 nucleobases). Target segments are also represented by DNA or RNA
sequences that comprise at least 8 consecutive nucleobases from the 3'-terminus of one of the target segments (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately downstream of the 3'-terminus of the target segment and continuing until the DNA or RNA
comprises about 8 to about 30 nucleobases). Target segments can also be represented by DNA or RNA sequences that comprise at least 8 consecutive nucleobases from an internal portion of the sequence of a target segment, and may extend in either or both directions until the conjugated antisense compound comprises about 8 to about 30 nucleobases.
In certain embodiments, antisense compounds targeted to an apo(a) nucleic acid can be modified as described herein. In certain embodiments, the antisense compounds can have a modified sugar moiety, an unmodified sugar moiety or a mixture of modified and unmodified sugar moieties as described herein. In certain embodiments, the antisense compounds can have a modified internucleoside linkage, an unmodified internucleoside linkage or a mixture of modified and unmodified internucleoside linkages as described herein. In certain embodiments, the antisense compounds can have a modified nucleobase, an unmodified nucleobase or a mixture of modified and unmodified nucleobases as described herein. In certain embodiments, the antisense compounds can have a motif as described herein.
In certain embodiments, antisense compounds targeted to apo(a) nucleic acids can be conjugated as described herein.
One apo(a) protein is linked via a disulfide bond to a single apolipoprotein B
(apoB) protein to form a lipoprotein(a) (Lp(a)) particle. The apo(a) protein shares a high degree of homology with plasminogen particularly within the kringle IV type 2 repetitive domain. It is thought that the kringle repeat domain in apo(a) may be responsible for its pro-thrombotic and anti-fibrinolytic properties, potentially enhancing atherosclerotic progression. Apo(a) is transcriptionally regulated by IL-6 and in studies in rheumatoid arthritis patients treated with an IL-6 inhibitor (tocilizumab), plasma levels were reduced by 30% after 3 month treatment. Apo(a) has been shown to preferentially bind oxidized phospholipids and potentiate vascular inflammation. Further, studies suggest that the Lp(a) particle may also stimulate endothelial permeability, induce plasminogen activator inhibitor type-1 expression and activate macrophage interleukin-8 secretion. Importantly, recent genetic association studies revealed that Lp(a) was an independent risk factor for myocardial infarction, stroke, peripheral vascular disease and abdominal aortic aneurysm. Further, in the Precocious Coronary Artery Disease (PROCARDIS) study, Clarke et al. described robust and independent associations between coronary heart disease and plasma Lp(a) concentrations.
Additionally, Solfrizzi et al., suggested that increased serum Lp(a) may be linked to an increased risk for Alzheimer's Disease (AD).
Antisense compounds targeting apo(a) have been previously disclosed in W02005/000201 and US2010-0331390, herein incorporated by reference in its entirety. An antisense oligonucleobase targeting Apo(a), ISIS-APOAR,õ was assessed in a Phase I clinical trial to study it's safety profile.
Certain Conjugated Antisense Compounds Targeted to an Apo(a) Nucleic Acid In certain embodiments, conjugated antisense compounds are targeted to an Apo(a) nucleic acid having the sequence of GENBANK Accession No. NM_005577.2, incorporated herein as SEQ ID NO: 1;
GENBANK Accession No. NT_007422.12 truncated from nucleotides 3230000 to 3380000, incorporated herein as SEQ ID NO: 2; GENBANK Accession No. NT_025741.15 truncated from nucleotides 65120000 to 65258000, designated herein as SEQ ID NO: 3; and GENBANK Accession No.
NM_005577.1, incorporated herein as SEQ ID NO: 4. In certain such embodiments, a conjugated antisense compound is at least 90%, at least 95%, or 100% complementary to any of the nucleobase sequences of SEQ ID
NOs: 1-4.
In certain embodiments, a conjugated antisense compound targeted to any of the nucleobase sequences of SEQ ID NOs: 1-4 comprises an at least 8 consecutive nucleobase sequence selected from the nucleobase sequence of any of SEQ ID NOs: 12-130, 133, 134. In certain embodiments, a conjugated antisense compound targeted to any of SEQ ID NOs: 1-4 comprises a nucleobase sequence selected from the nucleobase sequence of any of SEQ ID NOs: 12-130, 133, 134.
Table A: Antisense Compounds targeted to Apo(a) SEQ ID NO: 1 Target Start SEQ ID
ISIS No Sequence (5' -3') Motif Site NO

3901 TGCTCCGTTGGTGCTTGTTC eeeeeddddddddddeeeee 58 494283 TCTTCCTGTGACAGTGGTGG eeeeeddddddddddeeeee 26 494284 TTCTTCCTGTGACAGTGGTG eeeeeddddddddddeeeee 27 GGTTCTTCCTGTGACAGTGG eeeeeddddddddddeeeee 29 CGACTATGCGAGTGTGGTGT eeeeeddddddddddeeeee 38 494302 CCGACTATGCGAGTGTGGTG eeeeeddddddddddeeeee 39 Apo(a) Therapeutic Indications In certain embodiments, the invention provides methods for using a conjugated antisense compound targeted to an apo(a) nucleic acid for modulating the expression of apo(a) in a subject. In certain embodiments, the expression of apo(a) is reduced.
In certain embodiments, provided herein are methods of treating a subject comprising administering one or more pharmaceutical compositions as described herein. In certain embodiments, the invention provides methods for using a conjugated antisense compound targeted to an apo(a) nucleic acid in a pharmaceutical composition for treating a subject. In certain embodiments, the individual has an apo(a) related disease. In certain embodiments, the individual has an Lp(a) related disease. In certain embodiments, the individual has an inflammatory, cardiovascular and/or a metabolic disease, disorder or condition.
In certain embodiments, the subject has an inflammatory, cardiovascular and/or metabolic disease, disorder or condition.
In certain embodiments, the cardiovascular diseases, disorders or conditions include, but are not limited to, aortic stenosis, aneurysm (e.g., abdominal aortic aneurysm), angina, arrhythmia, atherosclerosis, cerebrovascular disease, coronary artery disease, coronary heart disease, dyslipidemia, hypercholesterolemia, hyperlipidemia, hypertension, hypertriglyceridemia, myocardial infarction, peripheral vascular disease (e.g., peripheral artery disease), stroke and the like.
In certain embodiments, the compounds targeted to apo(a) described herein modulate physiological markers or phenotypes of the cardiovascular disease, disorder or condition.
For example, administration of the compounds to animals can decrease LDL and cholesterol levels in those animals compared to untreated animals. In certain embodiments, the modulation of the physiological markers or phenotypes can be associated with inhibition of apo(a) by the compounds.
In certain embodiments, the physiological markers of the cardiovascular disease, disorder or condition can be quantifiable. For example, LDL or cholesterol levels can be measured and quantified by, for example, standard lipid tests. For such markers, in certain embodiments, the marker can be decreased by about 5, 10, 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.
Also, provided herein are methods for preventing, treating or ameliorating a symptom associated with the cardiovascular disease, disorder or condition in a subject in need thereof In certain embodiments, provided is a method for reducing the rate of onset of a symptom associated with the cardiovascular disease, disorder or condition. In certain embodiments, provided is a method for reducing the severity of a symptom associated with the cardiovascular disease, disorder or condition. In such embodiments, the methods comprise administering a therapeutically effective amount of a compound targeted to an apo(a) nucleic acid to an individual in need thereof The cardiovascular disease, disorder or condition can be characterized by numerous physical symptoms. Any symptom known to one of skill in the art to be associated with the cardiovascular disease, disorder or condition can be prevented, treated, ameliorated or otherwise modulated with the compounds and methods described herein. In certain embodiments, the symptom can be any of, but not limited to, angina, chest pain, shortness of breath, palpitations, weakness, dizziness, nausea, sweating, tachycardia, bradycardia, arrhythmia, atrial fibrillation, swelling in the lower extremities, cyanosis, fatigue, fainting, numbness of the face, numbness of the limbs, claudication or cramping of muscles, bloating of the abdomen or fever.
In certain embodiments, the metabolic diseases, disorders or conditions include, but are not limited to, hyperglycemia, prediabetes, diabetes (type I and type II), obesity, insulin resistance, metabolic syndrome and diabetic dyslipidemia.
In certain embodiments, compounds targeted to apo(a) as described herein modulate physiological markers or phenotypes of the metabolic disease, disorder or condition. For example, administrion of the compounds to animals can decrease glucose and insulin resistance levels in those animals compared to untreated animals. In certain embodiments, the modulation of the physiological markers or phenotypes can be associated with inhibition of apo(a) by the compounds.
In certain embodiments, physiological markers of the metabolic disease, disorder or condition can be quantifiable. For example, glucose levels or insulin resistance can be measured and quantified by standard tests known in the art. For such markers, in certain embodiments, the marker can be decreased by about 5, 10, 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 another example, insulin sensitivity can be measured and quantified by standard tests known in the art. For such markers, in certain embodiments, the marker can be increase by about 5, 10, 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.
Also, provided herein are methods for preventing, treating or ameliorating a symptom associated with the metabolic disease, disorder or condition in a subject in need thereof In certain embodiments, provided is a method for reducing the rate of onset of a symptom associated with the metabolic disease, disorder or condition. In certain embodiments, provided is a method for reducing the severity of a symptom associated with the metabolic disease, disorder or condition. In such embodiments, the methods comprise administering a therapeutically effective amount of a compound targeted to an apo(a) nucleic acid to an individual in need thereof The metabolic disease, disorder or condition can be characterized by numerous physical symptoms.
Any symptom known to one of skill in the art to be associated with the metabolic disease, disorder or condition can be prevented, treated, ameliorated or otherwise modulated with the compounds and methods described herein. In certain embodiments, the symptom can be any of, but not limited to, excessive urine production (polyuria), excessive thirst and increased fluid intake (polydipsia), blurred vision, unexplained weight loss and lethargy.
In certain embodiments, the inflammatory diseases, disorders or conditions include, but are not limited to, aortic stenosis, coronary artey disease (CAD), Alzheimer's Disease and thromboembolic diseases, disorder or conditions. Certain thromboembolic diseases, disorders or conditions include, but are not limited to, stroke, thrombosis, myocardial infarction and peripheral vascular disease.
In certain embodiments, the compounds targeted to apo(a) described herein modulate physiological markers or phenotypes of the inflammatory disease, disorder or condition. For example, administration of the compounds to animals can decrease inflammatory cytokine or other inflammatory markers levels in those animals compared to untreated animals. In certain embodiments, the modulation of the physiological markers or phenotypes can be associated with inhibition of apo(a) by the compounds.
In certain embodiments, the physiological markers of the inflammatory disease, disorder or condition can be quantifiable. For example, cytokine levels can be measured and quantified by standard tests known in the art. For such markers, in certain embodiments, the marker can be decreased by at least about 5%, 10%, 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.
Also, provided herein are methods for preventing, treating or ameliorating a symptom associated with the inflammatory disease, disorder or condition in a subject in need thereof In certain embodiments, provided is a method for reducing the rate of onset of a symptom associated with the inflammatory disease, disorder or condition. In certain embodiments, provided is a method for reducing the severity of a symptom associated with the inflammatory disease, disorder or condition. In such embodiments, the methods comprise administering a therapeutically effective amount of a compound targeted to an apo(a) nucleic acid to an individual in need thereof In certain embodiments, provided are methods of treating an individual with an apo(a) related disease, disorder or condition comprising administering a therapeutically effective amount of one or more pharmaceutical compositions as described herein. In certain embodiments, the individual has elevated apo(a) levels. In certain embodiments, provided are methods of treating an individual with an Lp(a) related disease, disorder or condition comprising administering a therapeutically effective amount of one or more pharmaceutical compositions as described herein. In certain embodiments, the individual has elevated Lp(a) levels. In certain embodiments, the individual has an inflammatory, cardiovascular and/or metabolic disease, disorder or condition. In certain embodiments, administration of a therapeutically effective amount of an antisense compound targeted to an apo(a) nucleic acid is accompanied by monitoring of apo(a) or Lp(a) levels. In certain embodiments, administration of a therapeutically effective amount of an antisense compound targeted to an apo(a) nucleic acid is accompanied by monitoring of markers of inflammatory, cardiovascular and/or metabolic disease, or other disease process associated with the expression of apo(a), to determine an individual's response to the antisense compound. An individual's response to administration of the antisense compound targeting apo(a) can be used by a physician to determine the amount and duration of therapeutic intervention with the compound.
In certain embodiments, administration of an antisense compound targeted to an apo(a) nucleic acid results in reduction of apo(a) 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, apo(a) expression is reduced to at least 100 mg/dL, 90 mg/dL, 80 mg/dL, 70 mg/dL, 60 mg/dL, 50 mg/dL, 40 mg/dL, 30 mg/dL, .20 mg/dL or 10 mg/dL.
In certain embodiments, administration of an antisense compound targeted to an apo(a) nucleic acid results in reduction of Lp(a) 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, Lp(a) expression is reduced to at least < 200 mg/dL, <
190 mg/dL, < 180 mg/dL, < 175 mg/dL,'( 170 mg/dL,'( 160 mg/dL, < 150 mg/dL, < 140 mg/dL, < 130 mg/dL, < 120 mg/dL, < 110 mg/dL, <
100 mg/dL, < 90 mg/dL, < 80 mg/dL, < 70 mg/dL, < 60 mg/dL, < 55 mg/dL, < 50 mg/dL, < 45 mg/dL, < 40 mg/dL, < 35 mg/dL, < 30 mg/dL, < 25 mg/dL, < 20 mg/dL, < 15 mg/dL, or < 10 mg/dL.
In certain embodiments, the invention provides methods for using a conjugated antisense compound targeted to an apo(a) nucleic acid in the preparation of a medicament. In certain embodiments, pharmaceutical compositions comprising a conjugated antisense compound targeted to apo(a) are used for the preparation of a medicament for treating a patient suffering or susceptible to an inflammatory, cardiovascular and/or a metabolic disease, disorder or condition.
Apo(a) Treatment Populations Certain subjects with high Lp(a) levels are at a significant risk of various diseases (Lippi et al., Clinica Chimica Acta, 2011, 412:797-801; Solfrizz et al.). In many subjects with high Lp(a) levels, current treatments cannot reduce their Lp(a) levels to safe levels. Apo(a) plays an important role in the formation of Lp(a), hence reducing apo(a) can reduce Lp(a) and prevent, treat or ameliorate a disease associated with Lp(a).
In certain embodiments, treatment with the compounds and methods disclosed herein is indicated for a human animal with elevated apo(a) levels and/or Lp(a) levels. In certain embodiments, the human has apo(a) levels > 10 mg/dL, > 20 mg/dL, > 30 mg/dL, > 40 mg/dL, > 50 mg/dL, > 60 mg/dL, > 70 mg/dL, >
80 mg/dL, > 90 mg/dL or > 100 mg/dL. In certain embodiments, the human has Lp(a) levels > 10 mg/dL, >
15 mg/dL, > 20 mg/dL, > 25 mg/dL, > 30 mg/dL, > 35 mg/dL, > 40 mg/dL, > 50 mg/dL, > 60 mg/dL, > 70 mg/dL, > 80 mg/dL, > 90 mg/dL, > 100 mg/dL, > 110 mg/dL, > 120 mg/dL, > 130 mg/dL, > 140 mg/dL, > 150 mg/dL, > 160 mg/dL, > 170 mg/dL, > 175 mg/dL, > 180 mg/dL, > 190 mg/dL, > 200 mg/dL.
D. Certain Pharmaceutical Compositions In certain embodiments, the present disclosure 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 oligonucleotide which are cleaved by endogenous nucleases within the body, to form the active antisense oligonucleotide.

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 disclosure 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 8OTM 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 8OTM; 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 substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, such suspensions may also contain suitable stabilizers or agents that increase the solubility of the pharmaceutical agents to allow for the preparation of highly concentrated solutions.
In certain embodiments, a pharmaceutical composition is prepared for transmucosal administration.
In certain of such embodiments penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
In certain embodiments, a pharmaceutical composition provided herein comprises an oligonucleotide in a therapeutically effective amount. In certain embodiments, the therapeutically effective amount is sufficient to prevent, alleviate or ameliorate symptoms of a disease or to prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art.
In certain embodiments, one or more modified oligonucleotide provided herein is formulated as a prodrug. In certain embodiments, upon in vivo administration, a prodrug is chemically converted to the biologically, pharmaceutically or therapeutically more active form of an oligonucleotide. In certain embodiments, prodrugs are useful because they are easier to administer than the corresponding active form.
For example, in certain instances, a prodrug may be more bioavailable (e.g., through oral administration) than is the corresponding active form. In certain instances, a prodrug may have improved solubility compared to the corresponding active form. In certain embodiments, prodrugs are less water soluble than the corresponding active form. In certain instances, such prodrugs possess superior transmittal across cell membranes, where water solubility is detrimental to mobility. In certain embodiments, a prodrug is an ester.
In certain such embodiments, the ester is metabolically hydrolyzed to carboxylic acid upon administration. In certain instances the carboxylic acid containing compound is the corresponding active form. In certain embodiments, a prodrug comprises a short peptide (polyaminoacid) bound to an acid group. In certain of such embodiments, the peptide is cleaved upon administration to form the corresponding active form.
In certain embodiments, the present disclosure provides compositions and methods for reducing the amount or activity of a target nucleic acid in a cell. In certain embodiments, the cell is in an animal. In certain embodiments, the animal is a mammal. In certain embodiments, the animal is a rodent. In certain embodiments, the animal is a primate. In certain embodiments, the animal is a non-human primate. In certain embodiments, the animal is a human.
In certain embodiments, the present disclosure provides methods of administering a pharmaceutical composition comprising an oligonucleotide of the present disclosure to an animal. Suitable administration routes include, but are not limited to, oral, rectal, transmucosal, intestinal, enteral, topical, suppository, through inhalation, intrathecal, intracerebroventricular, intraperitoneal, intranasal, intraocular, intratumoral, and parenteral (e.g., intravenous, intramuscular, intramedullary, and subcutaneous). In certain embodiments, pharmaceutical intrathecals are administered to achieve local rather than systemic exposures. For example, pharmaceutical compositions may be injected directly in the area of desired effect (e.g., into the liver).
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 oligonucleotide having the nucleobase sequence "ATCGATCG"
encompasses any oligonucleotides 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 oligonucleotides having other modified bases, such as "ArreCGAUCG," wherein nieC indicates a cytosine base comprising a methyl group at the 5-position.
EXAMPLES
The following examples illustrate certain embodiments of the present disclosure 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.
Example 1: General Method for the Preparation of Phosphoramidites, Compounds 1, la and 2 DMTO \ ___________ /Bx DMTO \ ____ /Bx DMTO
H3NC ON(iPr)2 C
d NCA3-N(iPr)2 NCA3-N(iPr)2 1 la 2 Bx is a heterocyclic base;
Compounds 1, la and 2 were prepared as per the procedures well known in the art as described in the specification herein (see Seth et al., Bioorg. Med. Chem., 2011, 21(4), 1122-1125, J. Org. Chem., 2010, 75(5), 1569-1581, Nucleic Acids Symposium Series, 2008, 52(1), 553-554); and also see published PCT
International Applications (WO 2011/115818, WO 2010/077578, W02010/036698, W02009/143369, WO
2009/006478, and WO 2007/090071), and US patent 7,569,686).
Example 2: Preparation of Compound 7 AcO0Ac Ac0 OAc 0 TMSOTf, 50 C AcO HOCO 5 Ac0 OAc ______________ 1""
AcHN CICH2CH2CI N TMSOTf, DCE
3 (93%) 4 ( 66%) AcO0Ac AcO0Ac H2/Pd Me0H
AcHN 0 AcHN,(95%) Compounds 3 (2-acetamido-1,3,4,6-tetra-O-acety1-2-deoxy-13-Dga1actopyranose or galactosamine pentaacetate) is commercially available. Compound 5 was prepared according to published procedures (Weber et al., J. Med. Chem., 1991, 34, 2692).

Example 3: Preparation of Compound 11 Et0,c, NCT.,...õ._ 0 0, HO-, 0, Et0 _,..CN 9 HCI, Et0H ----NH2 H0,-----NH2 ).-- N0----N--- __ NH2 Is-aq. KOH, Reflux, rt, 0 Et0 0----HO"- 1,4-dioxane, (:) (56%) 8 (40%) NC--__I 10 d----) 11 Compounds 8 and 9 are commercially available.
Example 4: Preparation of Compound 18 ),....___Et0r4).....___ N 00 io Et0 0 0õ benzylchloroformate, Et0 Et0 --I( Li0H, H20 Dioxane, Na2CO3 )------0NH2 ________________________ )...- H Dioxane __ 0.
Et 0" (86%) Et0 0--(91%) ii 12 NH
' 0 )n _....v 9 H 0 9_ 9 HO
0 0, 9 -0 N1 "-'"--NH2 14 __\/-nr __, H N.....7"\----N 0 HOµp------...--00 401 _____________________________ 0 0 0"
H
HBTU, DIEA, DMF
0 HO 0" (69%) 0 +O 15 r,LNN--e 0"--------) 13 H H ¨
AcO0Ac H
H2N ACOrs\r OrOH

17 H 0 0, 1 AcHN 0 ---ir-N.,0õ. N 0 io HBTU, DIEA, HOBt _____________ 1.. H

95 % 0 0' DMF

16 (64%) V\N_____ AcO0Ac Ac0 r Orl\l,.õN,0 AcHN 0 AcO0Ac 0 Ac0_....r.Ø.\r H
0 r0,______ __ N--1(0 io H
AcHN 0 0 0' AcO0AcHN-----kj Ac0_4r0--------r-N

AcHN 18 Compound 11 was prepared as per the procedures illustrated in Example 3.
Compound 14 is commercially available. Compound 17 was prepared using similar procedures reported by Rensen et al., J.
Med. Chem., 2004, 47, 5798-5808.
Example 5: Preparation of Compound 23 1.
0----./0¨b 1. TBDMSCI H
N H3CO)LkOH 21 HBTU, DIEA
) DMF, Imidazode, rt (95 %) DMF, rt (65%) HON) 2. Pd/C, H2, Me0H, rt _ 2.
TEA.3HF, TEA, THF
87% 20 a TBDMS (72%) -OH

1. DMTCI, pyr, rt (75%) --bi)Lq8.LOH
OCH ______________________________________ --b1).L.k.L

2. Li0H, Dioxane (97%) 23 ; 22 H
OH
Compounds 19 and 21 are commercially available.

Example 6: Preparation of Compound 24 AcO0Ac H H
Ac0_,)0,õ----õ..---yN.,,,,-,N.,...,0 AcHN 0 1. H2, Pd/C, Me0H (93%) AcO0Ac Ac0__.

H 0, 2. HBTU, DIEA, DMF
(76%) ......\, ____________________________________________ 0 0 0 ,--OD MT
NNVN.---"N---if---N.---0,--- N -1(ip H
AcHN 0 0 (:)HON :
c 23 AcO0Ac H H N-----j Ac0_...72...\,0--------r AcHN
AcO0Ac Ac0 --AcHN 0 AcO0Ac OD MT
H 0, 0 0 __.....2..\, H
Ac0 C)NNVN.-----N---n---N,-0,.. N --iLqLN
H µ
AcHN 0 0 (:) OH
AcO0Ac H H N-----j __....f.2...\,-____----ri--Ac0 0 24 ArHNI
Compounds 18 and 23 were prepared as per the procedures illustrated in Examples 4 and 5.

Example 7: Preparation of Compound 25 AcO0Ac H H
Ac0 0--12--\' (N--""N `=:) AcHN 0 ODMT
AcO0Ac Ac0 C).r NNõT.-N___--ILH' NQ
H 1. Succinic anhydride, DMAP, DCE
_________________________________________________________________________ -AcHN 0 0 0' OH 2. DMF, HBTU, EtN(iPr)2, PS-SS
AcO0Ac HN-----H....7_____/
Ac00----...--rN

AcHN
AcO0Ac __.....r.CL\r H H
Ac0 0 -......---.....---y N....õ-^õ......-N..õ;...0 AcHN 0 AcO0Ac ODMT

0 p Ac0--72-\r NN7N----N-----rf----i3O------ hi ---ILP q NH\¨
AcHN 0 0 0' 04 AcO0Ac HN-----kj Ac00----.."---r N

AcHN
Compound 24 was prepared as per the procedures illustrated in Example 6.

Example 8: Preparation of Compound 26 AcO0Ac H H
AcO__=.4,0,..NõN,C) AcHN 0 ODMT
AcO0Ac H H 0, 0 0 I.
Phosphitylation H 8 \
AcHN 0 0 0' OH
AcO0AcHN-----kj H__/___/
_....1.1\õ0N
Ac0 0 24 AcHN
AcO0Ac H H
.r N .,,-N,.0 Ac0 0 AcHN 0 AcO0Ac ODMT
H H 0, 0 !
Ac00.....,...õ....NN.7--N..õ-N 0,......--- N-1-1-8.-"Nrs H \
AcHN 0 0 0' 0 I
RN (ip 0 2 H ft¨kJ
AcO0Ac H 0 Ac0 _...!......\,D .õ_..ThrN----7-----/

Compound 24 is prepared as per the procedures illustrated in Example 6.

Example 9: General preparation of conjugated ASOs comprising Ga1NAc3-1 at the 3' terminus, Compound 29 AcO0Ac H H
--f.l\r Ac0 AcHN 0 OD MT
AcO0Ac H H 0, ilt )\¨NH
H \
AcHN 0 0 0' 04 HN 1. DCA, DCM
--j AcO0Ac H 0 2. DCI, NIV1I, ACN
N--,7---/
Ac0 0----(" Phosphoramidite DNA/RNA
building block 1 a.utomated synthesizer AcHN 3. Capping 4. t-BuO0H DMTOr\A,Bx r AcO0Ac --/
H N,-.,,IN1,0 0 Ac0 0=P-0 AcHN 0 O
AcO0Ac NH
AcHN 0 0 0' 04 HN _____ci 1. DCA, DCM 0 2. DCI, NIV1I, ACN
AcO0Ac H 0 Phosphoramidite DNA/RNA ' Ac0 0--building block la a.utomated synthesizer , ---.../\/"---r N
3. Capping 0 27 4. t-BuO0H
AcHN
D M TO ¨Nn,13x a. ________________________________________________________ b_/-0Me 0.FL0CN
\
0 ¨NafrBx AcO0Ac .
_......r..C.). H H
0 N ..,,N..,..,,.,..0 0 I
Ac0 0=P-0-AcHN 0 O
AcO0Ac N
Ac0 ).\¨NH
H µ
AcHN 0 0 0' 04 1. DCA, DCM
¨CI
AcO0Ac H0 2. DCI, NIV1I, ACN
N--,7---/
0----./\/---Tr Phosphoramidite DNA/RNA
Ac0 building blocks a.utomated synthesize AcHN 3. Capping 4. xanthane hydride or t-BuO0H
5. Et3N/CH3CN (1:1) 6. Aaueous NH, (cleavaue) OH
I
, OLIGO ]
I

X=1-0-O-Na,Bx Bx = Heterocyclic base (5.. b-//¨OMe x = 0 or S l 0=P-0-\
0-N(043x HOOH .\ __ /
H H CI
HO_,õ,r,L1,0I
N.,......--N.,,0 0=P-0-AcHN 0 I

H H 0, 0 0 õ,..õ.....õ..õ. N....õ--..........õN
H \
AcHN 0 0 0' OH
HOOH HN----kj HOo 0_____.,----õ--N

AcHN
Wherein the protected Ga1NAc3-1 has the structure:

¨F1)-0-No,,N
- \ __ /
HOOH
H H a 0=P-0-A 0 oI
HOOH cHN
H H

,,..õ....õ.õ.õ... N....õ--..........õN N--111-38-11.**NrsZ
H \
AcHN 0 0 0' OH
HOOH HN----kj HO_FED...o-w---IrN

AcHN
The Ga1NAc3 cluster portion of the conjugate group GalNAc3-1 (Ga1NAc3-1a) can be combined with any cleavable moiety to provide a variety of conjugate groups. Wherein Ga1NAc3-1a has the formula:

HOOH
H H
AcH N 0 I

H H 0, 0O
N ARIL N'..-H \
AcH N 0 0 0' OH
HOOH H I-I N-----Ci _....2.\,0--------Tr N----/----/

AcH N
The solid support bound protected Ga1NAc3-1, Compound 25, was prepared as per the procedures illustrated in Example 7. Oligomeric Compound 29 comprising Ga1NAc3-1 at the 3' terminus was prepared using standard procedures in automated DNA/RNA synthesis (see Dupouy et al., Angew. Chem. Int. Ed., 2006, 45, 3623-3627). Phosphoramidite building blocks, Compounds 1 and la were prepared as per the procedures illustrated in Example 1. The phosphoramidites illustrated are meant to be representative and not intended to be limiting as other phosphoramidite building blocks can be used to prepare oligomeric compounds having a predetermined sequence and composition. The order and quantity of phosphoramidites added to the solid support can be adjusted to prepare gapped oligomeric compounds as described herein.
Such gapped oligomeric compounds can have predetermined composition and base sequence as dictated by any given target.

Example 10: General preparation conjugated ASOs comprising Ga1NAc3-1 at the 5' terminus, Compound 34 ODMT 1. Capping (Ac20, NMI, PYr) I
1. DCA, DCM (OLIGO) 2. PADS or t-BuO0H
_____________________________ . 3. DCA, DCM I
0-UNL-ODMT 2. DCI, NMI, ACN 0 ..
I 0, 4. DCI, NMI, ACN
30 Phosphoramidite 0-UNL-0-P-0-'1N Phosphoramidite 1 building blocks , DNA/RNA DNA/RNA
31 ,automated synthesizer, ,.automated synthesizer, DMTO`c rBx 1. Capping (Ac20, NMI, PYr) 2. t-BuO0H 0 3. DCA, DCM NC ' 0-1) - ______________________ 4. DCI, NMI, ACN 0 I
Phosphoramidite 26 (OLIGO) ' DNA/RNA ' I
X = 0, or S ,automated synthesizer 0, I
Bx = Heterocylic base 0-1JNL-Ac0 OAc ?Ac0___.....\, H H
AcHN 0 Ac0 OAc OD MT
H H 0, 0 0 __r.C.)...\,0 Ac0 NN (:),....,-- NA(4.LN
H \
AcHN 0 0 C) 0 I
NC c), 1=',00,.Bx H N------Cj AcO0Ac H 0 /

___.2,----- NC 0 4 =0 Ac0 ,0__ 0 0 AcHN I
(OLIGO) I
1. Capping (Ac20, NMI, PYr) 0 2. t-BuO0H I
3. Et3N:CH3CN (1:1 \Iv) X
4. DCA, DCM
5. NH4, rt (cleavage) 33 HOOH
H H

r'N' AcHN

HOOH OH
H H 0, 0 0 _______________________________________________ )1 HO_....,o.._.---.._...----..õ--NN--N
H \
AcHN 0 0 0' CI) -0--;p,_,....,(OrBx HOOH HN----CI 0/ u 0 :

AcHN 34 0 I
Willi()) I
()T T
The UnylinkerTM 30 is commercially available. Oligomeric Compound 34 comprising a Ga1NAc3-1 cluster at the 5' terminus is prepared using standard procedures in automated DNA/RNA synthesis (see Dupouy et al., Angew. Chem. Int. Ed., 2006, 45, 3623-3627). Phosphoramidite building blocks, Compounds 1 and la were prepared as per the procedures illustrated in Example 1. The phosphoramidites illustrated are meant to be representative and not intended to be limiting as other phosphoramidite building blocks can be used to prepare an oligomeric compound having a predetermined sequence and composition. The order and quantity of phosphoramidites added to the solid support can be adjusted to prepare gapped oligomeric compounds as described herein. Such gapped oligomeric compounds can have predetermined composition and base sequence as dictated by any given target.
Example 11: Preparation of Compound 39 AcO0Ac 1. HO, N)L0. AcO0Ac Ac0_...7Ø.\0 ... H
35 TMSOTf, DCE
______________________________________________ ..- 8 N--__H----- 2. H2/Pd, Me0H AcHN 36 Ac0 OAc HBTU, DMF, EtN(iP02 Ac0 o\
1. H2, Pd/C, Me0H
____________________ ),.. ----NO__N__kli _______________________ ..-Compound 13 AcHN 8 2. HBTU, DIEA, DMF
OAc H 0 0 Compound Ac0 8 0 NHAc 0 0 0 ,----) C) Ac0OAc Ö0 0 NH
AcOr**--V

AcHN

Ac0 OAc Ac0-...7,0 F /0DMT
N---1+N__NI
Phosphitylation AcHN 8 0 -OAc0\ )¨N
OH
Ac0\0..Ø....\/0......,".r.,..rN
y.õ-0 ¨ NH
Ac0 8 NHAc 0 0 0 OAc Ac0 )\---) 38 NH
AcHN
Ac0 OAc AcOD0 /
ODMT
N---NWN__FNI
AcHN 8 0 =
OAc0\ )¨N
Ac0 H 0 ...,0., ) __ 8 Ac0 8 P
NC /,0' N UP 02 NHAc 0 0 0 OAc )\----) Ac0 AcO,C) NH
AcHN
Compounds 4, 13 and 23 were prepared as per the procedures illustrated in Examples 2, 4, and 5.
Compound 35 is prepared using similar procedures published in Rouchaud et al., Eur. J. Org. Chem., 2011, 12, 2346-2353.

Example 12: Preparation of Compound 40 Ac0 OAc Ac00 zODMT

AcHN 8 0 -OAc 0\ N
H 0 ..,0, > __ 8 OH
Ac00.......,..r.,....õNy_õ.0 NH
Ac0 8 NHAc 0 0 0 1. Succinic anhydride, DMAP, DCE
OAc )\----) __________________________________________________________________________ ,.-AcOr.o....\, NH
0 2. DMF, HBTU, EtN(iPr)2, PS-SS
Ac0 .õ,,,,,,,f_r.õõ
,8 38 AcHN
Ac0 OAc Ac00 zODMT
AcHN 8 0 -00 le OAc Ac0 Ac0 0 0 8 0----C}---1 i.(...).\./0õ),8\rENl0NH

NHAc (:) 0 0 OAc )\----) Ac0 Ac0=1"1 . NH
AcHN
Compound 38 is prepared as per the procedures illustrated in Example 11.
5 Example 13: Preparation of Compound 44 AcO0Ac HBTU, DMF, EtN(iPr)2 Ac0 AcHN 36 H01.1.70--,. )-0 *
¨N

HO\ /-0". 41 ii Ac0 OAc Ac0-A
....,0 AcHN X\W\)1\1 O o 1. H2, Pd/C, Me0H
]¨E N1 _________________________________________________________ ).
0 0 2. HBTU, DIEA, DMF
0)\___) Compound 23 OAc 0 AcOrc..)..\, 0..õ...õ,....f_r-...õ_,..NH
ft Ac0 AcHN
Ac0 OAc Ac0 u ¨ _ N ODMT
=
AcHN 8 0 0 - Phosphitylation , 0 0 ) (-8 NaOH
IN -H

0\\ ) 43 OAc Ac00.70,...\, N7----C) J.-.H
Ac0 AcHN
Ac0 OAc Ac0 ¨u N ODMT
_ AcHN-0 0 , (FN
OAc IN 8 ?
H NC01p ,N(iPr)2 0)\__ j AcO4, 44 ONH
Ac0 AcHN
Compounds 23 and 36 are prepared as per the procedures illustrated in Examples 5 and 11.
Compound 41 is prepared using similar procedures published in WO 2009082607.

Example 14: Preparation of Compound 45 Ac0 OAc Ac0-1....õ0 ODMT
H

H

OAc Ac0 1. Succinic anhydride, DMAP, DCE

_______________________________________________________________________________ I.
AcHN 2. DMF, HBTU, EtN(iPr)2, PS-SS
Ac0 OAc Ac00 ,ODMT
H
AcHN 8 0 =

OAc \----) 45 Ac0 NH
Ac0 AcHN
Compound 43 is prepared as per the procedures illustrated in Example 13.
Example 15: Preparation of Compound 47 HO o 11 b DMTO l 1. DMTCI, pyr --b1H
_____________________________________ ,..
2. Pd/C, H2, Me0H

Hd Compound 46 is commercially available.

Example 16: Preparation of Compound 53 HBTU, EtN(iPr)2, DMF 0 _____________________________________ )..
z Boc H3C0---/Ic FNi N

, Boc \CBz 50 CBz,-NH
HN,CBz H3C0 ,CBz 1. TFA N NH H 1. Li0H, Me0H
_________________ , 0 H ________________________________________ x 2. HBTU, EtN(iPr)2, DMF 2. HBTU, EtN(iPr)2, DMF

HN' Compound 47 HN
NH HN,CBz 0 \CBz OH

DMTO HN-CBz 1. H2, Pd/C

2. HBTU, EtN(iPr)2 DMF -N- C'..____7"-L------7--.N ,CBz Compound 17 ' HO' NH H
0 ' H

HN-CBz OAc OAc...._\______\_ 0 Ac0 0 ,z_l_____ NHAc OAc 0 Ac0 OAc____.\....___\_ 0 i1------..µ10H

HN-- HN
7-1...r.
r NHAc 0 OAc Ac 0 ODMT
?.....\___ 7)1 NH 53 Ac0 0 NHAc Compounds 48 and 49 are commercially available. Compounds 17 and 47 are prepared as per the procedures illustrated in Examples 4 and 15.

Example 17: Preparation of Compound 54 OAc OAc....,...._.\_ Ac0 0 .1.____ NHAc OAc 0 OAc.....___\_ 0 ,........m,y_ 7-------.00H
Ac0 0 NHAc 0 .------OAc ODMT
OAT( 0 Ac0----\------\---0 NHAc Phosphitylation y OAc OAc O
Ac0 0 ,1_____, NHAc \
(iPr)2N, OAc ----- 0 P `-'\______\
OAc.......\____\ CN
0 1.1_,...,,ri,...... 7----..,,6 Ac0 0 0 HN HN -------N/" 1\1_______ NHAc 0 ( OAc ODMT
0A?( 0 0 I _______________________________ NH 54 Ac0----\------\--0 NHAc Compound 53 is prepared as per the procedures illustrated in Example 16.

Example 18: Preparation of Compound 55 OAc OAc 0 .....\.......\_ 0 )__1____ Ac0 NHAc OAc 0 OAc.....\.......\_ .%%%0H
0 illiN
Ac0 0 NHAc HN--.7-1....(L.-- s-' -, p OAcC)Ac 0 ODMT
.....4.:?....\___ol NH 53 Ac0 NHAc 1. Succinic anhydride, DMAP, DCE
2. DMF, HBTU, EtN(iPr)2, PS-SS
-OAc OAc.....\.......\_ 0 Ac0 0 ,...i_ NHAc OAc 0 o\J' Ac0 NHAc ft OAc.....\.......\_ .,%%0 0 IlLid)LNp H

HN HN--.../s-y----, 0 s-' OAcC)Ac 0 ODMT
Ac0.....4.:?....\___(:) ______________ NH 55 NHAc Compound 53 is prepared as per the procedures illustrated in Example 16.
Example 19: General method for the preparation of conjugated ASOs comprising GaINAc3-1 at the 3' position via solid phase techniques (preparation of ISIS 647535, 647536 and 651900) Unless otherwise stated, all reagents and solutions used for the synthesis of oligomeric compounds are purchased from commercial sources. Standard phosphoramidite building blocks and solid support are used for incorporation nucleoside residues which include for example T, A, G, and InC residues. A 0.1 M
solution of phosphoramidite in anhydrous acetonitrile was used for 13-D-2'-deoxyribonucleoside and 2'-MOE.
The ASO syntheses were performed on ABI 394 synthesizer (1-2 [mot scale) or on GE Healthcare Bioscience AKTA oligopilot synthesizer (40-200 [mot scale) by the phosphoramidite coupling method on an Ga1NAc3-1 loaded VIMAD solid support (110 i.tmol/g, Guzaev et al., 2003) packed in the column. For the coupling step, the phosphoramidites were delivered 4 fold excess over the loading on the solid support and phosphoramidite condensation was carried out for 10 min. All other steps followed standard protocols supplied by the manufacturer. A solution of 6% dichloroacetic acid in toluene was used for removing dimethoxytrityl (DMT) group from 5'-hydroxyl group of the nucleotide. 4,5-Dicyanoimidazole (0.7 M) in anhydrous CH3CN was used as activator during coupling step. Phosphorothioate linkages were introduced by sulfurization with 0.1 M solution of xanthane hydride in 1:1 pyridine/CH3CN
for a contact time of 3 minutes.
A solution of 20% tert-butylhydroperoxide in CH3CN containing 6% water was used as an oxidizing agent to provide phosphodiester internucleoside linkages with a contact time of 12 minutes.
After the desired sequence was assembled, the cyanoethyl phosphate protecting groups were deprotected using a 1:1 (v/v) mixture of triethylamine and acetonitrile with a contact time of 45 minutes. The solid-support bound ASOs were suspended in aqueous ammonia (28-30 wt %) and heated at 55 C for 6 h.
The unbound ASOs were then filtered and the ammonia was boiled off The residue was purified by high pressure liquid chromatography on a strong anion exchange column (GE
Healthcare Bioscience, Source 30Q, 30 [tin, 2.54 x 8 cm, A = 100 mM ammonium acetate in 30% aqueous CH3CN, B
= 1.5 M NaBr in A, 0-40% of B in 60 min, flow 14 mL min-1, k = 260 nm). The residue was desalted by HPLC on a reverse phase column to yield the desired ASOs in an isolated yield of 15-30% based on the initial loading on the solid support. The ASOs were characterized by ion-pair-HPLC coupled MS analysis with Agilent 1100 MSD
system.
Antisense oligonucleotides not comprising a conjugate were synthesized using standard oligonucleotide synthesis procedures well known in the art.
Using these methods, three separate antisense compounds targeting ApoC III
were prepared. As summarized in Table 17, below, each of the three antisense compounds targeting ApoC III had the same nucleobase sequence; ISIS 304801 is a 5-10-5 MOE gapmer having all phosphorothioate linkages; ISIS
647535 is the same as ISIS 304801, except that it had a Ga1NAc3-1 conjugated at its 3'end; and ISIS 647536 is the same as ISIS 647535 except that certain internucleoside linkages of that compound are phosphodiester linkages. As further summarized in Table 17, two separate antisense compounds targeting SRB-1 were synthesized. ISIS 440762 was a 2-10-2 cEt gapmer with all phosphorothioate internucleoside linkages; ISIS
651900 is the same as ISIS 440762, except that it included a Ga1NAc3-1 at its 3'-end.
Table 17 Modified ASO targeting ApoC III and SRB-1 SEQ
CalCd Observed ASO Sequence (5 to 3') Target ID
Mass Mass No.
ISIS

3 1 s s ANC
AesGesniCesTesTesmCd Td Td Gd Td mCd mCd Ad Gd mCd TesTesTesAesTe 7165.4 7164.4 135 ISIS AesGesmCesTesTesmCdsTdsTdsGdsTdsmCdsmCdsAdsGdsmCdsTesTesTesAesTeoAdo,-ApoC
9239.5 9237.8 136 647535 Ga1NAc3-1. III
ISIS AesGeomCeeTeeTeemCdsTdsTdsGdsTdsmCdsmCdsAdsGdsmCdsTeoTeoTesAesTeoAdo,-ApoC
9142.9 9140.8 136 647536 Ga1NAc3-1. III

ISISSRB-TksmCksAdsGdsTdsmCdsAdsTdsGdsAdsmCdsTdsTksmCk 4647.0 4646.4 137 ISIS

TksmCksAdsGdsTdsmCdsAdsTdsGdsAdsmCdsTdsTksmCkoAdo,-GaINAC3-1a 6721.1 6719.4 138 Subscripts: "e" indicates 2'-MOE modified nucleoside; "d" indicates [3-D-2'-deoxyribonuc1eoside; "k"
indicates 6'-(S)-CH3 bicyclic nucleoside (e.g. cEt); "s" indicates phosphorothioate internucleoside linkages (PS); "o" indicates phosphodiester internucleoside linkages (PO); and "o¨
indicates -0-P(=0)(OH)-.
Superscript "m" indicates 5-methylcytosines. "Ga1NAc3-1" indicates a conjugate group having the structure shown previously in Example 9. Note that Ga1NAc3-1 comprises a cleavable adenosine which links the ASO
to remainder of the conjugate, which is designated "Ga1NAc3-1.." This nomenclature is used in the above table to show the full nucleobase sequence, including the adenosine, which is part of the conjugate. Thus, in the above table, the sequences could also be listed as ending with "Ga1NAc3-1"
with the "Ado" omitted. This convention of using the subscript "a" to indicate the portion of a conjugate group lacking a cleavable nucleoside or cleavable moiety is used throughout these Examples. This portion of a conjugate group lacking the cleavable moiety is referred to herein as a "cluster" or "conjugate cluster" or "Ga1NAc3 cluster." In certain instances it is convenient to describe a conjugate group by separately providing its cluster and its cleavable moiety.
Example 20: Dose-dependent antisense inhibition of human ApoC III in huApoC
III transgenic mice ISIS 304801 and ISIS 647535, each targeting human ApoC III and described above, were separately tested and evaluated in a dose-dependent study for their ability to inhibit human ApoC III in human ApoC III
transgenic mice.
Treatment Human ApoCIII transgenic mice were maintained on a 12-hour light/dark cycle and fed ad libitum Teklad lab chow. Animals were acclimated for at least 7 days in the research facility before initiation of the experiment. ASOs were prepared in PBS and sterilized by filtering through a 0.2 micron filter. ASOs were dissolved in 0.9% PBS for injection.
Human ApoC III transgenic mice were injected intraperitoneally once a week for two weeks with ISIS 304801 or 647535 at 0.08, 0.25. 0.75, 2.25 or 6.75 [tmol/kg or with PBS
as a control. Each treatment group consisted of 4 animals. Forty-eight hours after the administration of the last dose, blood was drawn from each mouse and the mice were sacrificed and tissues were collected.
ApoC III mRNA Analysis ApoC III mRNA levels in the mice's livers were determined using real-time PCR
and RIBOGREENO RNA quantification reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols. ApoC III mRNA levels were determined relative to total RNA (using Ribogreen), prior to normalization to PBS-treated control. The results below are presented as the average percent of ApoC III

mRNA levels for each treatment group, normalized to PBS-treated control and are denoted as "% PBS". The half maximal effective dosage (ED50) of each ASO is also presented in Table

18, below.
As illustrated, both antisense compounds reduced ApoC III RNA relative to the PBS control.
Further, the antisense compound conjugated to Ga1NAc3-1 (ISIS 647535) was substantially more potent than the antisense compound lacking the Ga1NAc3-1 conjugate (ISIS 304801).
Table 18 Effect of ASO treatment on ApoC III mRNA levels in human ApoC III transgenic mice Dose ED50Internucleoside SEQ ID
ASO 3' Conjugate Olmol/kg) PBS Onnol/kg) linkage/Length No.

0.08 95 ISIS 0.75 42 0.77 None PS/20 304801 2.25 32 6.75 19 0.08 50 SIS 0.75 15 I
647535 2.25 17 0.074 Ga1NAc3-1 PS/20 6.75 8 ApoC III Protein Analysis (Turbidometric Assay) Plasma ApoC III protein analysis was determined using procedures reported by Graham et al, Circulation Research, published online before print March 29, 2013.
Approximately 100 1 of plasma isolated from mice was analyzed without dilution using an Olympus Clinical Analyzer and a commercially available turbidometric ApoC III assay (Kamiya, Cat# KAI-006, Kamiya Biomedical, Seattle, WA). The assay protocol was performed as described by the vendor.
As shown in the Table 19 below, both antisense compounds reduced ApoC III
protein relative to the PBS control. Further, the antisense compound conjugated to Ga1NAc3-1 (ISIS
647535) was substantially more potent than the antisense compound lacking the Ga1NAc3-1 conjugate (ISIS
304801).
Table 19 Effect of ASO treatment on ApoC III plasma protein levels in human ApoC III
transgenic mice Dose ED50Internucleoside SEQ ID
ASO 3' Conjugate (jlmol/kg) PBS (jnnol/kg) Linkage/Length No.

ISIS 0.08 86 0.73 None PS/20 304801 0.75 51 2.25 23 6.75 13 0.08 72 ISIS 0.75 14 647535 2.25 12 0.19 Ga1NAc3-1 PS/20 136 6.75 11 Plasma triglycerides and cholesterol were extracted by the method of Bligh and Dyer (Bligh, E.G.
and Dyer, W.J. Can. J. Biochem. Physiol. 37: 911-917, 1959)(Bligh, E and Dyer, W, Can J Biochem Physiol, 37, 911-917, 1959)(Bligh, E and Dyer, W, Can J Biochem Physiol, 37, 911-917, 1959) and measured by using a Beckmann Coulter clinical analyzer and commercially available reagents.
The triglyceride levels were measured relative to PBS injected mice and are denoted as "%
PBS". Results are presented in Table 20. As illustrated, both antisense compounds lowered triglyceride levels. Further, the antisense compound conjugated to Ga1NAc3-1 (ISIS 647535) was substantially more potent than the antisense compound lacking the Ga1NAc3-1 conjugate (ISIS
304801).
Table 20 Effect of ASO treatment on triglyceride levels in transgenic mice ASO Dose % ED50 3' Internucleoside SEQ ID
(jlmol/kg) PBS (jlmol/kg) Conjugate Linkage/Length No.

0.08 87 ISIS 0.75 46 0.63 None PS/20 135 304801 2.25 21 6.75 12 0.08 65 ISIS 0.75 9 0.13 Ga1NAc3-1 PS/20 136 647535 2.25 8 6.75 9 Plasma samples were analyzed by HPLC to determine the amount of total cholesterol and of different fractions of cholesterol (HDL and LDL). Results are presented in Tables 21 and 22. As illustrated, both antisense compounds lowered total cholesterol levels; both lowered LDL; and both raised HDL. Further, the antisense compound conjugated to Ga1NAc3-1 (ISIS 647535) was substantially more potent than the antisense compound lacking the Ga1NAc3-1 conjugate (ISIS 304801). An increase in HDL and a decrease in LDL levels is a cardiovascular beneficial effect of antisense inhibition of ApoC III.

Table 21 Effect of ASO treatment on total cholesterol levels in transgenic mice ASO Dose Total Cholesterol 3' Internucleoside SEQ
(jlmol/kg) (mg/dL) Conjugate Linkage/Length ID
No.

0.08 226 ISIS 0.75 164 None PS/20 304801 2.25 110 6.75 82 0.08 230 ISIS 0.75 82 647535 2.25 86 Ga1NAc3-1 PS/20 6.75 99 Table 22 Effect of ASO treatment on HDL and LDL cholesterol levels in transgenic mice ASO Dose HDL LDL 3' Internucleoside SEQ
(jlmol/kg) (mg/dL) (mg/dL) Conjugate Linkage/Length ID No.

0.08 17 23 ISIS 0.75 27 12 None PS/20 304801 2.25 50 4 6.75 45 2 0.08 21 21 ISIS 0.75 44 2 Ga1NAc3-1 PS/20 647535 2.25 50 2 6.75 58 2 Pharmacokinetics Analysis (PK) The PK of the ASOs was also evaluated. Liver and kidney samples were minced and extracted using standard protocols. Samples were analyzed on MSD1 utilizing IP-HPLC-MS. The tissue level (lg/g) of full-length ISIS 304801 and 647535 was measured and the results are provided in Table 23. As illustrated, liver concentrations of total full-length antisense compounds were similar for the two antisense compounds.
Thus, even though the Ga1NAc3-1 -conjugated antisense compound is more active in the liver (as demonstrated by the RNA and protein data above), it is not present at substantially higher concentration in the liver. Indeed, the calculated EC50 (provided in Table 23) confirms that the observed increase in potency of the conjugated compound cannot be entirely attributed to increased accumulation. This result suggests that the conjugate improved potency by a mechanism other than liver accumulation alone, possibly by improving the productive uptake of the antisense compound into cells.
The results also show that the concentration of Ga1NAc3-1 conjugated antisense compound in the kidney is lower than that of antisense compound lacking the GalNAc conjugate.
This has several beneficial therapeutic implications. For therapeutic indications where activity in the kidney is not sought, exposure to kidney risks kidney toxicity without corresponding benefit. Moreover, high concentration in kidney typically results in loss of compound to the urine resulting in faster clearance.
Accordingly, for non-kidney targets, kidney accumulation is undesired. These data suggest that Ga1NAc3-1 conjugation reduces kidney accumulation.
Table 23 PK analysis of ASO treatment in transgenic mice Internucleoside Dose Liver Kidney Liver EC50 3'SEQ
ASO Linkage/Length (jlmol/kg) (m/g) (pg/g) (11,g/g) Conjugate ID No.
0.1 5.2 2.1 ISIS 0.8 62.8 119.6 53 None PS/20 304801 2.3 142.3 191.5 6.8 202.3 337.7 0.1 3.8 0.7 ISIS 0.8 72.7 34.3 3.8 Ga1NAc3-1 PS/20 647535 2.3 106.8 111.4 6.8 237.2 179.3 Metabolites of ISIS 647535 were also identified and their masses were confirmed by high resolution mass spectrometry analysis. The cleavage sites and structures of the observed metabolites are shown below.
The relative % of full length ASO was calculated using standard procedures and the results are presented in Table 23a. The major metabolite of ISIS 647535 was full-length ASO lacking the entire conjugate (i.e. ISIS
304801), which results from cleavage at cleavage site A, shown below. Further, additional metabolites resulting from other cleavage sites were also observed. These results suggest that introducing other cleabable bonds such as esters, peptides, disulfides, phosphoramidates or acyl-hydrazones between the Ga1NAc3-1 sugar and the ASO, which can be cleaved by enzymes inside the cell, or which may cleave in the reductive environment of the cytosol, or which are labile to the acidic pH inside endosomes and lyzosomes, can also be useful.
Table 23a Observed full length metabolites of ISIS 647535 Metabolite ASO Cleavage site Relative %
1 ISIS 304801 A 36.1 2 ISIS 304801 + dA B 10.5 3 ISIS 647535 minus [3 GalNAc] C 16.1 ISIS 647535 minus 4 D 17.6 [3 GalNAc + 1 5-hydroxy-pentanoic acid tether]
ISIS 647535 minus D 9.9 [2 GalNAc + 2 5-hydroxy-pentanoic acid tether]
ISIS 647535 minus D
6 [3 GalNAc + 3 5-hydroxy-pentanoic acid tether] 9.8 Cleavage Sites ,, Cleavage site A
HO OH Cleavage site C 0=P-OH

Cleavage site D O

HO
(3 N N-_-_-]
Ni NHAc 0 0---__ N 1 0 Cleavage site C -Cleavage site B
H H I
HO \-0 \ N N 0 0 __ P=0 NHAc Cleavage site D 0 OH

OH
HO HN

\ 0 \ N
HO-Z
NHAc Cleavage site C 0 Cleavage site D

() 0=P-OH NH2 Metabolite 1 Metabolite 2Ki4_1.--1õ.N
,C).,N N-, OH

O=P¨OH NH2 H 0Nx-J.k, OH
HO , ON N7 N js I

C2.,.\
H H
N N
HO -----_----- H 0 __ 17=0 0-'-2 Metabolite 3 H

O=P¨OH NH2 O
H
0,Nxik, N
H2N N--____( OH

N

N N
H 0 ____ 17=0 O_=_=
Metabolite 4 H

0=P¨OH NH2 O Ny.õ

N---__t OH

C),,,...<N N.-) ___________________________________________________________________ /

R
H
H2N NH 0 ____ P=0 Metabolite 5 H

O=P¨OH

Nx-J.k, H O

N
OH
N.) _____________________________________________________________________ /
0---___ 0 H
H
H2N N 0 __ 17=0 0 ..------Metabolite 6 HN

Example 21: Antisense inhibition of human ApoC III in human ApoC III
transgenic mice in single administration study ISIS 304801, 647535 and 647536 each targeting human ApoC III and described in Table 17, were further evaluated in a single administration study for their ability to inhibit human ApoC III in human ApoC
III transgenic mice.
Treatment Human ApoCIII transgenic mice were maintained on a 12-hour light/dark cycle and fed ad libitum Teklad lab chow. Animals were acclimated for at least 7 days in the research facility before initiation of the experiment. ASOs were prepared in PBS and sterilized by filtering through a 0.2 micron filter. ASOs were dissolved in 0.9% PBS for injection.
Human ApoC III transgenic mice were injected intraperitoneally once at the dosage shown below with ISIS 304801, 647535 or 647536 (described above) or with PBS treated control. The treatment group consisted of 3 animals and the control group consisted of 4 animals. Prior to the treatment as well as after the last dose, blood was drawn from each mouse and plasma samples were analyzed.
The mice were sacrificed 72 hours following the last administration.
Samples were collected and analyzed to determine the ApoC III mRNA and protein levels in the liver; plasma triglycerides; and cholesterol, including HDL and LDL fractions were assessed as described above (Example 20). Data from those analyses are presented in Tables 24-28, below. Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols. The ALT and AST
levels showed that the antisense compounds were well tolerated at all administered doses.
These results show improvement in potency for antisense compounds comprising a Ga1NAc3-1 conjugate at the 3' terminus (ISIS 647535 and 647536) compared to the antisense compound lacking a Ga1NAc3-1 conjugate (ISIS 304801). Further, ISIS 647536, which comprises a Ga1NAc3-1 conjugate and some phosphodiester linkages was as potent as ISIS 647535, which comprises the same conjugate and all internucleoside linkages within the ASO are phosphorothioate.
Table 24 Effect of ASO treatment on ApoC III mRNA levels in human ApoC III transgenic mice PBS
ASO
Dose ED50 3' Internucleoside SEQ ID

(mg/kg) (mg/kg) Conjugate linkage/Length No.

304801 10 71 13.2 None PS/20 0.3 98 ISIS
647535 1 70 1.9 Ga1NAc3-1 PS/20 0.3 103 1.7 Ga1NAc3-1 PS/PO/20 136 Table 25 Effect of ASO treatment on ApoC III plasma protein levels in human ApoC III
transgenic mice Dose ASO % PBS ED5o 3' Internucleoside SEQ ID
(mg/kg) (mg/kg) Conjugate Linkage/Length No.

1 104 23.2 None PS/20 135 0.3 98 2.1 Ga1NAc3-1 PS/20 136 0.3 103 1.8 Ga1NAc3-1 PS/PO/20 136 Table 26 Effect of ASO treatment on triglyceride levels in transgenic mice Dose ED5o Internucleoside SEQ ID
ASO % PBS 3' Conjugate (mg/kg) (mg/kg) Linkage/Length No.

29.1 None PS/20 135 0.3 100 2.2 Ga1NAc3-1 PS/20 136 0.3 95 1.9 Ga1NAc3-1 PS/PO/20 136 Table 27 Effect of ASO treatment on total cholesterol levels in transgenic mice Dose Internucleoside ASO % PBS 3' Conjugate SEQ ID No.
(mg/kg) Linkage/Length 304801 10 86 None PS/20 135 0.3 93 GaINAc3-1 PS/20 136 0.3 115 GaINAc3-1 PS/PO/20 136 Table 28 Effect of ASO treatment on HDL and LDL cholesterol levels in transgenic mice ASO Dose HDL LDL 3' Internucleoside SEQ
ID
(mg/kg) % PBS % PBS Conjugate Linkage/Length No.

304801 10 226 63 None PS/20 0.3 98 86 647535 3 212 39 Ga1NAc3-1 PS/20 0.3 143 89 647536 3 213 33 Ga1NAc3-1 PS/PO/20 5 These results confirm that the Ga1NAc3-1 conjugate improves potency of an antisense compound.
The results also show equal potency of a Ga1NAc3-1 conjugated antisense compounds where the antisense oligonucleotides have mixed linkages (ISIS 647536 which has six phosphodiester linkages) and a full phosphorothioate version of the same antisense compound (ISIS 647535).
Phosphorothioate linkages provide several properties to antisense compounds.
For example, they 10 resist nuclease digestion and they bind proteins resulting in accumulation of compound in the liver, rather than in the kidney/urine. These are desirable properties, particularly when treating an indication in the liver.
However, phosphorothioate linkages have also been associated with an inflammatory response. Accordingly, reducing the number of phosphorothioate linkages in a compound is expected to reduce the risk of inflammation, but also lower concentration of the compound in liver, increase concentration in the kidney and urine, decrease stability in the presence of nucleases, and lower overall potency. The present results show that a Ga1NAc3-1 conjugated antisense compound where certain phosphorothioate linkages have been replaced with phosphodiester linkages is as potent against a target in the liver as a counterpart having full phosphorothioate linkages. Such compounds are expected to be less proinflammatory (See Example 24 describing an experiment showing reduction of PS results in reduced inflammatory effect).
Example 22: Effect of GaINAc3-1 conjugated modified ASO targeting SRB-1 in vivo ISIS 440762 and 651900, each targeting SRB-1 and described in Table 17, were evaluated in a dose-dependent study for their ability to inhibit SRB-1 in Balb/c mice.
Treatment Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, ME) were injected subcutaneously once at the dosage shown below with ISIS 440762, 651900 or with PBS treated control. Each treatment group consisted of 4 animals. The mice were sacrificed 48 hours following the final administration to determine the SRB-1 mRNA levels in liver using real-time PCR and RIBOGREENO
RNA quantification reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols.
SRB-1 mRNA levels were determined relative to total RNA (using Ribogreen), prior to normalization to PBS-treated control. The results below are presented as the average percent of SRB-1 mRNA levels for each treatment group, normalized to PBS-treated control and is denoted as "% PBS".
As illustrated in Table 29, both antisense compounds lowered SRB-1 mRNA
levels. Further, the antisense compound comprising the Ga1NAc3-1 conjugate (ISIS 651900) was substantially more potent than the antisense compound lacking the Ga1NAc3-1 conjugate (ISIS 440762). These results demonstrate that the potency benefit of Ga1NAc3-1 conjugates are observed using antisense oligonucleotides complementary to a different target and having different chemically modified nucleosides, in this instance modified nucleosides comprise constrained ethyl sugar moieties (a bicyclic sugar moiety).
Table 29 Effect of ASO treatment on SRB-1 mRNA levels in Balb/c mice Internucleosid ASO
Dose Liver ED50 Conugate e SEQ ID
3' j (mg/kg) % PBS (mg/kg) linkage/Lengt No.

0.7 85 440762 7 12 2.2 None PS/14 0.07 98 ISIS 0.2 63 651900 0.7 20 0.3 Ga1NAc3-1 PS/14 Example 23: Human Peripheral Blood Mononuclear Cells (hPBMC) Assay Protocol The hPBMC assay was performed using BD Vautainer 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 x 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 1 sample was pipette into a sample vial (Beckman Coulter) with 600 1 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 Vice11 XR cell viability analyzer (Beckman Coulter) using PBMC cell type (dilution factor of 1:11 was stored with other parameters). The live cell/m1 and viability were recorded. The cell suspension was diluted to 1 x 107 live PBMC/ml in RPMI+ 10%
FBS+pen/strep.
The cells were plated at 5 x 105 in 50 [tl/well of 96-well tissue culture plate (Falcon Microtest). 50 [d/well of 2x concentration oligos/controls diluted in RPMI+10% FBS+pen/strep.
was added according to experiment template (100 [Ll/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 x 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 24: Evaluation of Proinflammatory Effects in hPBMC Assay for GaINAc3-1 conjugated ASOs The antisense oligonucleotides (AS0s) listed in Table 30 were evaluated for proinflammatory effect in hPBMC assay using the protocol described in Example 23. ISIS 353512 is an internal standard known to be a high responder for IL-6 release in the assay. The hPBMCs were isolated from fresh, volunteered donors and were treated with ASOs at 0, 0.0128, 0.064, 0.32, 1.6, 8, 40 and 200 [LM
concentrations. After a 24 hr treatment, the cytokine levels were measured.
The levels of IL-6 were used as the primary readout. The EC50 and Emax was calculated using standard procedures. Results are expressed as the average ratio of Emax/EC50 from two donors and is denoted as "En./EC50." The lower ratio indicates a relative decrease in the proinflammatory response and the higher ratio indicates a relative increase in the proinflammatory response.
With regard to the test compounds, the least proinflammatory compound was the PS/P0 linked ASO
(ISIS 616468). The Ga1NAc3-1 conjugated ASO, ISIS 647535 was slightly less proinflammatory than its non-conjugated counterpart ISIS 304801. These results indicate that incorporation of some PO linkages reduces proinflammatory reaction and addition of a Ga1NAc3-1 conjugate does not make a compound more proinflammatory and may reduce proinflammatory response. Accordingly, one would expect that an antisense compound comprising both mixed PS/P0 linkages and a Ga1NAc3-1 conjugate would produce lower proinflammatory responses relative to full PS linked antisense compound with or without a Ga1NAc3-1 conjugate. These results show that Ga1NAc3_1 conjugated antisense compounds, particularly those having reduced PS content are less proinflammatory.
Together, these results suggest that a Ga1NAc3-1 conjugated compound, particularly one with reduced PS content, can be administered at a higher dose than a counterpart full PS antisense compound lacking a Ga1NAc3-1 conjugate. Since half-life is not expected to be substantially different for these compounds, such higher administration would result in less frequent dosing.
Indeed such administration could be even less frequent, because the Ga1NAc3-1 conjugated compounds are more potent (See Examples 20-22) and re-dosing is necessary once the concentration of a compound has dropped below a desired level, where such desired level is based on potency.
Table 30 Modified ASOs ASO Sequence (5' to 3') Target SEQ ID
No.
ISIS GesmCesTesGesAesTdsTdsAdsGdsAdsGds TNFa 139 104838 AdsGdsAdsGdsGesTesmCesmCesmCe ISIS TesmCesmCesmCdsAdsTdsrrdsTdsmCdsAdsGds 353512 GdsAdsGdsAdsmCdsmCdsTesGesGe ISIS ikesGesmCesTesTesmCdsTdsTdsGdsTds Ap OC III 135 304801 mCdsmCdsAdsGdsmCds TesTesTesAesTe ISIS ikesGesmCesTesTesmCdsTdsTdsGdsTds ApOC III 136 647535 mCdsmCdsAdsGdsmCdsTesTesTesAesTeoAdo,-GaINAC3-la ISIS ikesGeomCeoTeoTeomCdsTdsTdsGdsTds Ap OC III 135 616468 mCdsmCdsAdsGasmCdsTeoTeoTesAesTe Subscripts: "e" indicates 2'-MOE modified nucleoside; "d" indicates 13-D-2'-deoxyribonucleoside; "k" indicates 6'-(S)-CH3 bicyclic nucleoside (e.g. cEt);
"s" indicates phosphorothioate internucleoside linkages (PS); "o" indicates phosphodiester internucleoside linkages (PO); and "o¨ indicates -0-P(=0)(OH)-. Superscript "m" indicates 5-methylcytosines. "Ado¨Ga1NAc3-1."
indicates a conjugate having the structure Ga1NAc3-1 shown in Example 9 attached to the 3'-end of the antisense oligonucleotide, as indicated.
Table 31 Proinflammatory Effect of ASOs targeting ApoC III in hPBMC assay ASO
EC50 Emax E /EC 3' Internucleoside SEQ ID
(11M) (11M) max 5 0 Conjugate Linkage/Length No.

0.01 265.9 26,590 None PS/20 140 (high responder) ISIS 304801 0.07 106.55 1,522 None PS/20 135 ISIS 647535 0.12 138 1,150 Ga1NAc3-1 PS/20 136 ISIS 616468 0.32 71.52 224 None PS/PO/20 135 Example 25: Effect of Ga1NAc3-1 conjugated modified ASO targeting human ApoC
III in vitro ISIS 304801 and 647535 described above were tested in vitro. Primary hepatocyte cells from transgenic mice at a density of 25,000 cells per well were treated with 0.03,0.08, 0.24, 0.74, 2.22, 6.67 and 20 [LM 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 and the hApoC
III mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN.
The IC50 was calculated using the standard methods and the results are presented in Table 32. As illustrated, comparable potency was observed in cells treated with ISIS 647535 as compared to the control, ISIS 304801.
Table 32 Modified ASO targeting human ApoC III in primary hepatocytes Internucleoside SEQ
ASO ICso (P,M) 3 Conjugate linkage/Length ID No.
ISIS
304801 0.44 None PS/20 135 ISIS
0.31 Ga1NAc3-1 PS/20 136 In this experiment, the large potency benefits of Ga1NAc3-1 conjugation that are observed in vivo were not observed in vitro. Subsequent free uptake experiments in primary hepatocytes in vitro did show increased potency of oligonucleotides comprising various GalNAc conjugates relative to oligonucleotides that lacking the GalNAc conjugate.(see Examples 60, 82, and 92) Example 26: Effect of PO/PS linkages on ApoC III ASO Activity Human ApoC III transgenic mice were injected intraperitoneally once at 25 mg/kg of ISIS 304801, or ISIS 616468 (both described above) or with PBS treated control once per week for two weeks. The treatment group consisted of 3 animals and the control group consisted of 4 animals. Prior to the treatment as well as after the last dose, blood was drawn from each mouse and plasma samples were analyzed. The mice were sacrificed 72 hours following the last administration.
Samples were collected and analyzed to determine the ApoC III protein levels in the liver as described above (Example 20). Data from those analyses are presented in Table 33, below.
These results show reduction in potency for antisense compounds with PO/PS
(ISIS 616468) in the wings relative to full PS (ISIS 304801).
Table 33 Effect of ASO treatment on ApoC III protein levels in human ApoC III
transgenic mice PBS
ASO
Dose 3' Internucleoside SEQ ID

(mg/kg) Conjugate linkage/Length No.

304801 mg/kg/wk 24 None Full PS 135 for 2 wks 616468 mg/kg/wk 40 None 14 PS/6 PO 135 for 2 wks Example 27: Compound 56 N(iPr)2 DMTO---....õ..-----......_-- 1 DMTO
V
DMTO () Compound 56 is commercially available from Glen Research or may be prepared according to published procedures reported by Shchepinov et al., Nucleic Acids Research, 1997, 25(22), 4447-4454.

Example 28: Preparation of Compound 60 Ac0 OAc Ac0 OAc _.....1:2\ HO L) Bn 57 H2/Pd Ac0 3..._ Ac0 __........\,o\
Me0H
ci TMSOTf, DCE
( 71%) 1\1"---H- AcHN 58 (quant.) CNEtO(N(iPr)2)PC1, Ac0 OAc Ac0 OAc ED1P
N(iPr)2 __,.........._\,0 CN

_........2..\,0 0.1".0/\/
Ac0 Ac0 OH CH2C12 AcHN 59 (80%) AcHN 60 Compound 4 was prepared as per the procedures illustrated in Example 2.
Compound 57 is commercially available. Compound 60 was confirmed by structural analysis.
Compound 57 is meant to be representative and not intended to be limiting as other monoprotected substituted or unsubstituted alkyl diols including but not limited to those presented in the specification herein can be used to prepare phosphoramidites having a predetermined composition.
Example 29: Preparation of Compound 63 CN
1. BnC1 õ..OH 1. DMTC1, pyr H
,..ODMT

HO 2. KOH, DMSO , ,..,õ 2. Pd/C, H2 __ 0 õ ---..---ODMT
\ ________ (:)..)¨ ¨CH -"1" .-----------OH
3. HC1, Me0H 3.
Phosphitylation I
0 '¨'01-1 'N Pr ---ODMT
4. NaHCO3 Compounds 61 and 62 are prepared using procedures similar to those reported by Tober et al., Eur. J.
Org. Chem., 2013, 3, 566-577; and Jiang et al., Tetrahedron, 2007, 63(19), 3982-3988.
Alternatively, Compound 63 is prepared using procedures similar to those reported in scientific and patent literature by Kim et al., Synlett, 2003, 12, 1838-1840; and Kim et al., published PCT International Application, WO 2004063208.Example 30: Preparation of Compound 63b OH ODMT
,c ij CN
1. DMTC1, pyr H
2. TBAF 0, (:)............--ODMT
).- P
0 3. Phosphitylation I(i NPr)2 \--\
63a OH 63b ODMT
Compound 63a is prepared using procedures similar to those reported by Hanessian et al., Canadian Journal of Chemistry, 1996, 74(9), 1731-1737.
Example 31: Preparation of Compound 63d HO¨ \ DMT0¨\
\ \
0, N(iPr)2 0, 1. DMTC1, pyr I
HO 0 \/00Bn 2. Pd/C, H2 1.---- 3. Phosphitylation 0.---/ 63c / 63d HO¨/ DMT0¨/
Compound 63c is prepared using procedures similar to those reported by Chen et al., Chinese Chemical Letters, 1998, 9(5), 451-453.
Example 32: Preparation of Compound 67 CO2Bn Ac0 OAc 0 Ac0 oOH H2N )0TBDMS Ac0 OAc 0 CO2Bn > Ac0 AcHN 64 HBTU, DIEA AcHN 66 H
R
R = H or CH3 Ac0 OAc 1. TEA.3HF, THF 0 CO2Bn 2. Phosphi ____________ I.-Ac0_,,,,;,4,,i) 0........,.....-...õ.õ.......-jt,N..-Ii0,p,O........õ.....--...,CN
tylation H I
AcHN R N(iPr)2 Compound 64 was prepared as per the procedures illustrated in Example 2.
Compound 65 is prepared using procedures similar to those reported by Or et al., published PCT International Application, WO 2009003009. The protecting groups used for Compound 65 are meant to be representative and not 10 intended to be limiting as other protecting groups including but not limited to those presented in the specification herein can be used.
Example 33: Preparation of Compound 70 H2NOBn Ac0 OAc 68 0 CH3 Ac0 OAc 0 Ac0 _...r..?4,0,L, HBTU, DIEA
OH ____________________________________________ 1.-- Ac0 AcHN 64 MT H
AcHN 69 Ac0 OAc 1. Pd/C, H2 0 ______________________ 1.-_.....Ø,...\/0 CN
2. Phosphitylation H I I
AcHN 70 CH3 N(iPr)2 15 Compound 64 was prepared as per the procedures illustrated in Example 2.
Compound 68 is commercially available. The protecting group used for Compound 68 is meant to be representative and not intended to be limiting as other protecting groups including but not limited to those presented in the specification herein can be used.

Example 34: Preparation of Compound 75a 1. TBDMSC1, pyr 2. Pd/C, H2 HN
N(iP02 --....õ........õ--0--..._ 1 NC 3. CF3CO2Et, Me0H H
NC ' .'"-----1 OH ______________________ x= F3C
NC (:)...-' 4. TEA.314F, THF
5. Phosphitylation 0 CF 3 75a Compound 75 is prepared according to published procedures reported by Shchepinov et al., Nucleic 5 Acids Research, 1997, 25(22), 4447-4454.

Example 35: Preparation of Compound 79 DMTO 0 HO,..õ.....7"...õ..0,.
-....,,...õ..-....õ...,õ
DCI, NMI, ACN
1. BnCl, NaH
Phosphoramidite 60 _____________________________________________________________________________ ).-,..-...,..õ7- HO
-.Ø--- 2. DCA, CH2C12 õ,..-..,...7----..Ø---DMTO

Ac0 OAc NC m _____...C..)..\
Ac0 0 0 AcHN
NC 1. H2/Pd, Me0H
---.1 Ac0 OAc ___________________________________________________________________ ,...
L 0 ,.., O 2. Phosphitylation \ 13n AcHN (:) NC----\
\--._ )----j Ac0 OAc 2....\/00-11)0 Ac0 NHAc 78 Ac0 OAc NC-...1 Ac0 0 0 \---------õ,..--N i!, AcHN
NC--_\
Ac0 Ac \,....-0-.... ,-- '-....------"Th AcHN (:) 1 NC---\NOT)02 Ac0 OAc Ac0 NHAc Compound 76 was prepared according to published procedures reported by Shchepinov et al., Nucleic Acids Research, 1997, 25(22), 4447-4454.
Example 36: Preparation of Compound 79a HO-.......7-....õ701...., 1. FmocC1, pyr H0_,,=-,....0,,....,1 OBn 2. Pd/C, H2 Fmoc00 AD CN

___________________________________________ 0.-.7. .7.
HO co 3. Phosphitylation Fmoc0 co 77 79a Compound 77 is prepared as per the procedures illustrated in Example 35.
Example 37: General method for the preparation of conjugated oligomeric compound 82 comprising a phosphodiester linked Ga1NAc3-2 conjugate at 5' terminus via solid support (Method I) Ç,....._0 --/--/ODMT
nr-----7--ODMT
DMT0((i5Bx " ru t-,i-- ------/---ODMT

NC0¨P=0 1. DCA, DCM NC (:)coOr Bx 0 2. DCI, NMI, ACN 0õ
I
OLIGO Phosphoramidite 56 NC0¨P=0 , ______________________________________________ s. ____________________ ., DNA/RNA 0 I
0 ,autom I
, ated synthesizer , , I OLIGO
0¨VIMAD_o_p_..0CN ,. _______ 31( 0 79b I
_0_130CN
0¨VIMAD
X = S- or 0- X
Bx = Heterocylic base 1. Capping (Ac20, NMI, pyr) 80 2. t-BuO0H
3. DCA, DCM
4. DCI, NMI, ACN
Ac0 OAc NC ---,\
, Phosphoramidite 60 Ac0--4\00 AcHN 0 sC,--.
CN
----\
Ac0 OAc NC of _...r2...\vr\
__________________________________________________ 04-0( Or Bx AcHN Cr 0 NC-----\ j 0 j NC Th¨P = 0 L
Ac0 Ac 0 1 1200' KO , OLIGO , Ac0 ,.
NHAc 01 O¨VIMAD
X
1. Capping (Ac20, NMI, pyr) 81 2. t-BuO0H
3. 20% Et2NH inToluene (v/v) 4. NH4, 55 C, , HO OH

HO--4,_ AcHN 0 HO OH
0 0, 0 0 Bx HO -==="TZ,C) (31'Pl `030' l"3/ --0 AcHN 0' O.
O=P-O-OH) HO 9 y OLIGO ______________________________________________________________ ' NHAc 82 wherein Ga1NAc3-2 has the structure:
HOOH

AcHN

HOOH
_________________________________________ 0-P-0 r o-AcHN -6 ==
O _____________________________________ o HO H 9, P, I
HO
NHAc The Ga1NAc3 cluster portion of the conjugate group Ga1NAc3-2 (Ga1NAc3-2a) can be combined with any cleavable moiety to provide a variety of conjugate groups. Wherein Ga1NAc3-2a has the formula:
HO OH
H0.7.,2..\,0 0 ..----,,, AcHN

HO OH
0 0, _......i2..\,,-, o , o o-AcHN (:) HO H 9, P, NHAc The VIMAD-bound oligomeric compound 79b was prepared using standard procedures for automated DNA/RNA synthesis (see Dupouy et al., Angew. Chem. Int. Ed, 2006, 45, 3623-3627). The phosphoramidite Compounds 56 and 60 were prepared as per the procedures illustrated in Examples 27 and 28, respectively. The phosphoramidites illustrated are meant to be representative and not intended to be limiting as other phosphoramidite building blocks including but not limited those presented in the specification herein can be used to prepare an oligomeric compound having a phosphodiester linked conjugate group at the 5' terminus. The order and quantity of phosphoramidites added to the solid support can be adjusted to prepare the oligomeric compounds as described herein having any predetermined sequence and composition.

Example 38: Alternative method for the preparation of oligomeric compound 82 comprising a phosphodiester linked Ga1NAc3-2 conjugate at 5' terminus (Method II) DMT0(3)Bx"*.
1. DCA, DCM
NC
0_0 2. DCI, NMI, ACN
O Phosphoramidite 79 OLIGO DNA/RNA
µautomated synthesizer X = S- or 0-Bx ¨ Heterocyclic base 79b Ac0 OAc NC
AcO

0 ¨
AcH N
NC CN
Ac0 OAc 0 0 jrN 0 ()(0)"Bx Ac0 0 00 AcHN
NC NC
0_0 Ac0 Ac =
Ac0 OLIGO
N HAc 0 1. Capping 2. t-BuO0H
3. Et3N:CH3CN (1:1 v/v) 83 4. NH4, 55 C
Oligomeric Compound 82 The VIMAD-bound oligomeric compound 79b was prepared using standard procedures for automated DNA/RNA synthesis (see Dupouy et al., Angew. Chem. Int. Ed, 2006, 45, 3623-3627). The Ga1NAc3-2 cluster phosphoramidite, Compound 79 was prepared as per the procedures illustrated in Example 35. This alternative method allows a one-step installation of the phosphodiester linked Ga1NAc3-2 conjugate to the oligomeric compound at the final step of the synthesis. The phosphoramidites illustrated are meant to be representative and not intended to be limiting, as other phosphoramidite building blocks including but not limited to those presented in the specification herein can be used to prepare oligomeric compounds having a phosphodiester conjugate at the 5' terminus. The order and quantity of phosphoramidites added to the solid support can be adjusted to prepare the oligomeric compounds as described herein having any predetermined sequence and composition.

Example 39: General method for the preparation of oligomeric compound 83h comprising a Ga1NAc3-3 Conjugate at the 5' Terminus (Ga1NAc3-1 modified for 5' end attachment) via Solid Support Ac0 OAc Ac0-0 H
N-------)r--N H 1. H2, Pd/C, Me0H (93%) AcHN
Fi H0 0 0,, 2. BnO0H
83a N
OAc Ac0) 0....../---õ/".1 N7N--- N')---N--0.---NA 0 0 Ac0 HBTU, DIEA, DMF, 76%
________________________________________________________________ i.-NHAc HNVN/NN____ 3. H2,Pd/C,Me0H

OAc _/¨/--0 Ac0/) Ac0 OAc Ac0 Ac0-.,0 NHAc H
N"--N----),iN H
AcHN Nõ,.....õ..

F
H 8 L )c OH

83b N
COCF3 Ac0 F
F Ac0 F NHAc HNN____,Cj , 83c Pyridine, DMF
OAc __/¨/- H 0 C\>) Ac0 OAc AcO\ P
Ac0 NHAc Ac0 H 5 0 0 83e 3' N--------)r--N H '1 I I
AcHN
F
0 0 F í.F
OLIGO 0-P-0-(CH2)6-NH2 Fi H 0 0-, ./,1 F I
OH
a Ac0 0Borate buffer, DMSO, pH 8.5, rt Ac0 NHAc HN F FN____ H 0 83d OAc j¨r1/40 Ac0\(:\>) i Ac0 NHAc Ac0 OAc Ac0 0 H
N--N----)_-N H
AcHN

H H 0 0_. /=Lv I
N-(CH2)6-0-P-0¨[ OLIGO j Ac0 NHAc HN---N..---NN _____________ kJ 83f OAc j¨r1/40 AcO\C\.>/) Ac0 NHAc Aqueous ammonia HO----74:0 H
N"---------¨N H
).1 AcHN \----NN 1 0 0, /õ..................--õ)IN
H H0....- _____ NH N-(CH2)6-0-P-0¨ [
OLIGO ) HO OH 0,... H I I
HO 83h OH 0,....../---/-1 83h HN N ,-, NHAc H L) /
OH __/ 0 HO'1-11;
NHAc Compound 18 was prepared as per the procedures illustrated in Example 4.
Compounds 83a and 83b are commercially available. Oligomeric Compound 83e comprising a phosphodiester linked hexylamine was prepared using standard oligonucleotide synthesis procedures. Treatment of the protected oligomeric compound with aqueous ammonia provided the 5'-Ga1NAc3-3 conjugated oligomeric compound (83h).
Wherein Ga1NAc3-3 has the structure:
HO OH
HO---74:,..\ H
AcHN H
Ni.--N
0 \N
1r o 0 OH
H H 0 0- )L7).- I
N---ir--N-0----NH

HO H H
HO
NHAc HNrNHN-0 0Hr, 0.-7-7-HO
NHAc .
The Ga1NAc3 cluster portion of the conjugate group Ga1NAc3-3 (Ga1NAc3-3a) can be combined with any cleavable moiety to provide a variety of conjugate groups. Wherein Ga1NAc3-3a has the formula:

HO OH

AcHN N---N---)---N H
O
H H 0 0...
7X---N-ii-N--0..---NH N¨(CH2)6-0¨

H
0 0"
HO
NHAc HN N
OH
HO\
HO
NHAc .

Example 40: General method for the preparation of oligomeric compound 89 comprising a phosphodiester linked Ga1NAc3-4 conjugate at the 3' terminus via solid support r0--7---/ODMT

1. DCA NL-ODMT . ________________________ 0 0 v-----.7--OFmoc 2. DCI, NMI, ACN I
Fmoc0 -0 N(/Pr)2 0-UNL-0-0CN
--,. I 0 DMT0,--..0,7"---0-1:

3. Capping ODMTr CN
4. t-BuO0H r0,/----/ r OFmoc Co 0/ /-0Fmoc 1. 2% Piperidine, 2% DBU, 96% DMF or\-----ov--7-- "1'-o-,OFmoc _________________ .. I
3. DCI, NMI, ACN cyum_70_p_oCN 86 0-/
Phosphoramidite 79a 6 ' DNA/RNA ' 1. Capping ,automated synthesizer 2. t-BuO0H, 3. 2% Piperidine, Ac0 OAc 2% DBU, 96% DMF
Ac0 c......_ 4. DCI, NMI, ACN
0 Phosphoramidite 60 , DNA/RNA , AcHN o___\NC ,.automated synthesizer, 5. Capping Ac0 OAc (0 0-P, AcOo \
NC) oN-----\-0 j--CN
0---PN0,/¨/ o 0----joN
P=0 NC --\ yi \-0 N---"\----0 AcO1/4...\/Ac o 0 _....../....._/".---DMTO"N----No-j¨\

I ,CN
Ac0 0-UNL-0-NHAc 1. t-BuO0H
6' 2. DCA
3. Oligo synthesis (DNA/RNA automated synthesizer) 4. Capping 5. Oxidation 6. Et3N:CH3CN (1:1, v/v) Ac0 OAc Ac0*\01_ AcHN 0 0-P' AcOo / \
-o o_______\__o AcHN
\--"\""\---\ P P 88 0-P\ 0--9------r---/ 0 ,--, ,.
Ac0C)Ac Ac0 0- -o o-------N }---\
o o DMT-( OLIGO 1)------2Pt/ o_uNL_O-p-c , r NHAc 5' 3' (13 HO OH NH4, 55 C
HO\\0L
AcHN 0 O-P' HO:Lo /\
o-AcHN
\--"\--"\---\ 89 0-PN P=0 =P--0 ()--"\--0 HO OH
3-----\OH
....--.1--0 HO
NHAc [ OLIGO ) __ [CMY
5' 3' Wherein Ga1NAc3-4 has the structure:

HO*\01_ AcHN 0 HO OH \--\--\--\ p HO / \
0\--\--\------\ 9 0- o------\,o AcHN
0 ,...-j,0, /0 -p. .., / 0,7----/ P=0 q ()0 ,13-0 H0 O 0 \
L 0,//- 0-j------\OH

NHAc 1 Car Wherein CM is a cleavable moiety. In certain embodiments, cleavable moiety is:
I
0=P-OH N NH2 0-Ncy N____:j (5 I
0=P-OH
I
The Ga1NAc3 cluster portion of the conjugate group Ga1NAc3-4 (Ga1NAc3-4a) can be combined with any cleavable moiety to provide a variety of conjugate groups. Wherein Ga1NAc3-4a has the formula:

HON
HO oFi AcHN 0 HO OH p HO o-po ' AcHN
0-1\
P=0 9P¨(1-1 ()0 \¨
HO H
/L.0 0 0-HO ______________ N\
NHAc The protected Unylinker functionalized solid support Compound 30 is commercially available.
Compound 84 is prepared using procedures similar to those reported in the literature (see Shchepinov et al., Nucleic Acids Research, 1997, 25(22), 4447-4454; Shchepinov et al., Nucleic Acids Research, 1999, 27, 3035-3041; and Hornet et al., Nucleic Acids Research, 1997, 25, 4842-4849).
The phosphoramidite building blocks, Compounds 60 and 79a are prepared as per the procedures illustrated in Examples 28 and 36. The phosphoramidites illustrated are meant to be representative and not intended to be limiting as other phosphoramidite building blocks can be used to prepare an oligomeric compound having a phosphodiester linked conjugate at the 3' terminus with a predetermined sequence and composition. The order and quantity of phosphoramidites added to the solid support can be adjusted to prepare the oligomeric compounds as described herein having any predetermined sequence and composition.
Example 41: General method for the preparation of ASOs comprising a phosphodiester linked Ga1NAc3-2 (see Example 37, Bx is adenine) conjugate at the 5' position via solid phase techniques (preparation of ISIS 661134) Unless otherwise stated, all reagents and solutions used for the synthesis of oligomeric compounds are purchased from commercial sources. Standard phosphoramidite building blocks and solid support are used for incorporation nucleoside residues which include for example T, A, G, and mC residues.
Phosphoramidite compounds 56 and 60 were used to synthesize the phosphodiester linked Ga1NAc3-2 conjugate at the 5' terminus. A 0.1 M solution of phosphoramidite in anhydrous acetonitrile was used for 13-D-2'-deoxyribonucleoside and 2'-M0E.
The ASO syntheses were performed on ABI 394 synthesizer (1-2 [mot scale) or on GE Healthcare Bioscience AKTA oligopilot synthesizer (40-200 [mot scale) by the phosphoramidite coupling method on VIMAD solid support (110 [tmol/g, Guzaev et al., 2003) packed in the column.
For the coupling step, the phosphoramidites were delivered at a 4 fold excess over the initial loading of the solid support and phosphoramidite coupling was carried out for 10 min. All other steps followed standard protocols supplied by the manufacturer. A solution of 6% dichloroacetic acid in toluene was used for removing the dimethoxytrityl (DMT) groups from 5'-hydroxyl groups of the nucleotide. 4,5-Dicyanoimidazole (0.7 M) in anhydrous CH3CN was used as activator during the coupling step.
Phosphorothioate linkages were introduced by sulfurization with 0.1 M solution of xanthane hydride in 1:1 pyridine/CH3CN for a contact time of 3 minutes. A solution of 20% tert-butylhydroperoxide in CH3CN containing 6%
water was used as an oxidizing agent to provide phosphodiester internucleoside linkages with a contact time of 12 minutes.
After the desired sequence was assembled, the cyanoethyl phosphate protecting groups were deprotected using a 20% diethylamine in toluene (v/v) with a contact time of 45 minutes. The solid-support bound ASOs were suspended in aqueous ammonia (28-30 wt %) and heated at 55 C
for 6 h.
The unbound ASOs were then filtered and the ammonia was boiled off The residue was purified by high pressure liquid chromatography on a strong anion exchange column (GE
Healthcare Bioscience, Source 30Q, 30 [tin, 2.54 x 8 cm, A = 100 mM ammonium acetate in 30% aqueous CH3CN, B =
1.5 M NaBr in A, 0-40%
of B in 60 min, flow 14 mL min-1, k = 260 nm). The residue was desalted by HPLC on a reverse phase column to yield the desired ASOs in an isolated yield of 15-30% based on the initial loading on the solid support. The ASOs were characterized by ion-pair-HPLC coupled MS analysis with Agilent 1100 MSD
system.
Table 34 ASO comprising a phosphodiester linked Ga1NAc3-2 conjugate at the 5' position targeting SRB-1 Observed SEQ
ID
ISIS No. Sequence (5 to 3') CalCd Mass Mass No.
GalNAC3-2a-otAdoTksmCksAdsGdsTdsmCdsAdsrrds 661134 6482.2 6481.6 141 Gds AdsmCdsTdsTksmCk Subscripts: "e" indicates 2'-MOE modified nucleoside;
"d" indicates 13-D-2' -deoxyribonucleoside; "k" indicates 6'-(S)-CH3 bicyclic nucleoside (e.g. cEt);
"s" indicates phosphorothioate internucleoside linkages (PS); "o" indicates phosphodiester internucleoside linkages (PO); and "o¨ indicates -0-P(=0)(OH)-. Superscript "m" indicates 5-methylcytosines. The structure of Ga1NAc3-2a is shown in Example 37.

Example 42: General method for the preparation of ASOs comprising a Ga1NAc3-3 conjugate at the 5' position via solid phase techniques (preparation of ISIS 661166) The synthesis for ISIS 661166 was performed using similar procedures as illustrated in Examples 39 and 41.
ISIS 661166 is a 5-10-5 MOE gapmer, wherein the 5' position comprises a Ga1NAc3-3 conjugate.
The ASO was characterized by ion-pair-HPLC coupled MS analysis with Agilent 1100 MSD system.
Table 34a ASO comprising a Ga1NAc3-3 conjugate at the 5' position via a hexylamino phosphodiester linkage targeting Malat-1 ISIS,Conjugate Calcd Observed No. Mass Mass Sequence (5 to 3') SEQ ID No.
5'-Ga1NAC3-3,-0,mCesGesGesTesGes 661166 mCdsAdsAdsGdsGdsmCdsTdsTdsAdsGds 5'-Ga1NAc3-3 8992.16 8990.51 GesAesAes TesTe Subscripts: "e" indicates 2'-MOE modified nucleoside; "d" indicates [3-D-2'-deoxyribonuc1eoside;
"s" indicates phosphorothioate internucleoside linkages (PS); "o" indicates phosphodiester internucleoside linkages (PO); and "o" indicates -0-P(=0)(OH)-. Superscript "m" indicates 5-methylcytosines. The structure of "5'-GalNAc3-3a" is shown in Example 39.
Example 43: Dose-dependent study of phosphodiester linked Ga1NAc3-2 (see examples 37 and 41, Bx is adenine) at the 5' terminus targeting SRB-1 in vivo ISIS 661134 (see Example 41) comprising a phosphodiester linked Ga1NAc3-2 conjugate at the 5' terminus was tested in a dose-dependent study for antisense inhibition of SRB-1 in mice. Unconjugated ISIS
440762 and 651900 (Ga1NAc3-1 conjugate at 3' terminus, see Example 9) were included in the study for comparison and are described previously in Table 17.
Treatment Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, ME) were injected subcutaneously once at the dosage shown below with ISIS 440762, 651900, 661134 or with PBS
treated control. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration to determine the liver SRB-1 mRNA levels using real-time PCR and RIBOGREENO
RNA quantification reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols.
SRB-1 mRNA levels were determined relative to total RNA (using Ribogreen), prior to normalization to PBS-treated control. The results below are presented as the average percent of SRB-1 mRNA levels for each treatment group, normalized to PBS-treated control and is denoted as "% PBS". The ED50s were measured using similar methods as described previously and are presented below.

As illustrated in Table 35, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner. Indeed, the antisense oligonucleotides comprising the phosphodiester linked Ga1NAc3-2 conjugate at the 5' terminus (ISIS 661134) or the Ga1NAc3-1 conjugate linked at the 3' terminus (ISIS 651900) showed substantial improvement in potency compared to the unconjugated antisense oligonucleotide (ISIS 440762). Further, ISIS 661134, which comprises the phosphodiester linked Ga1NAc3-2 conjugate at the 5' terminus was equipotent compared to ISIS 651900, which comprises the Ga1NAc3-1 conjugate at the 3' terminus.
Table 35 ASOs containing Ga1NAc3-1 or Ga1NAc3-2 targeting SRB-1 ISIS Dosage SRB-1 mRNA ED50 Conjugate SE Q ID No.
No. (mg/kg) levels (% PBS) (mg/kg) 0.2 116 0.7 91 440762 2 69 2.58 No conjugate 137 0.07 95 0.2 77 651900 0.7 28 0.26 3' Ga1NAc3-1 138 0.07 107 0.2 86 661134 0.7 28 0.25 5' GalNAc3-2 141 Structures for 3' Ga1NAc3-1 and 5' Ga1NAc3-2 were described previously in Examples 9 and 37.
Pharmacokinetics Analysis (PK) The PK of the ASOs from the high dose group (7 mg/kg) was examined and evaluated in the same 15 manner as illustrated in Example 20. Liver sample was minced and extracted using standard protocols. The full length metabolites of 661134 (5' Ga1NAc3-2) and ISIS 651900 (3' Ga1NAc3-1) were identified and their masses were confirmed by high resolution mass spectrometry analysis. The results showed that the major metabolite detected for the ASO comprising a phosphodiester linked Ga1NAc3-2 conjugate at the 5' terminus (ISIS 661134) was ISIS 440762 (data not shown). No additional metabolites, at a detectable level, were 20 observed. Unlike its counterpart, additional metabolites similar to those reported previously in Table 23a were observed for the ASO having the Ga1NAc3-1 conjugate at the 3' terminus (ISIS 651900). These results suggest that having the phosphodiester linked GalNAc3-1 or Ga1NAc3-2 conjugate may improve the PK
profile of ASOs without compromising their potency.

Example 44: Effect of PO/PS linkages on antisense inhibition of ASOs comprising GaINAc3-1 conjugate (see Example 9) at the 3' terminus targeting SRB-1 ISIS 655861 and 655862 comprising a Ga1NAc3-1 conjugate at the 3' terminus each targeting SRB-1 were tested in a single administration study for their ability to inhibit SRB-1 in mice. The parent unconjugated compound, ISIS 353382 was included in the study for comparison.
The ASOs are 5-10-5 MOE gapmers, wherein the gap region comprises ten 2'-deoxyribonucleosides and each wing region comprises five 2'-MOE modified nucleosides. The ASOs were prepared using similar methods as illustrated previously in Example 19 and are described Table 36, below.
Table 36 Modified ASOs comprising GaINAc3-1 conjugate at the 3' terminus targeting SRB-Chemistry SEQ
ISIS No. Sequence (5' to 3') ID
No.
353382 GesmCesTesTesmCesAdsGdsTdsmCdsAdsTdsGdsAds Full PS no conjugate 143 (parent) mCdsTdsTesmCesmCesTesTe GesmCesTesTesmCesAdsGdsTdsmCdsAdsTdsGdsAds Full PS with 655861 mCdsTdsTesmCesmCesTesTeoAdo,-Ga1NAC3-1 a Ga1NAc3-1 conjugate 655862 GesmCeoTeoTeomCeoAdsGdsTdsmCdsAdsTdsGdsAds Mixed PS/P0 with 144 m CdsTdsrreom Ceom CesTesTe0Ada,¨GaINAC3¨la Ga1NAc3-1 conjugate Subscripts: "e" indicates 2'-MOE modified nucleoside; "d" indicates [3-D-2'-deoxyribonuc1eoside;
"s" indicates phosphorothioate internucleoside linkages (PS); "o" indicates phosphodiester internucleoside linkages (PO); and "o" indicates -0-P(=0)(OH)-. Superscript "m" indicates 5-methylcytosines. The structure of "GalNAc3-1" is shown in Example 9.
Treatment Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, ME) were injected subcutaneously once at the dosage shown below with ISIS 353382, 655861, 655862 or with PBS
treated control. Each treatment group consisted of 4 animals. Prior to the treatment as well as after the last dose, blood was drawn from each mouse and plasma samples were analyzed. The mice were sacrificed 72 hours following the final administration to determine the liver SRB-1 mRNA levels using real-time PCR
and RIBOGREENO RNA
quantification reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols. SRB-1 mRNA
levels were determined relative to total RNA (using Ribogreen), prior to normalization to PBS-treated control. The results below are presented as the average percent of SRB-1 mRNA
levels for each treatment group, normalized to PBS-treated control and is denoted as "% PBS". The ED50s were measured using similar methods as described previously and are reported below.
As illustrated in Table 37, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner compared to PBS treated control. Indeed, the antisense oligonucleotides comprising the Ga1NAc3-1 conjugate at the 3' terminus (ISIS 655861 and 655862) showed substantial improvement in potency comparing to the unconjugated antisense oligonucleotide (ISIS 353382). Further, ISIS 655862 with mixed PS/P0 linkages showed an improvement in potency relative to full PS (ISIS
655861).
Table 37 Effect of PO/PS linkages on antisense inhibition of ASOs comprising Ga1NAc3-1 conjugate at 3' terminus targeting SRB-1 ISIS Dosage SRB-1 mRNA ED50 Chemistry SEQ ID
No.
No. (mg/kg) levels (% PBS) (mg/kg) 3 76.65 10 52.40 10.4 Full PS without conjugate 143 (parent) 30 24.95 0.5 81.22 Full PS with GalNAc3-1 1.5 63.51 655861 2.2 conjugate 144 5 24.61 15 14.80 0.5 69.57 1.5 45.78 Mixed PS/P0 with 655862 1.3 144 5 19.70 Ga1NAc3-1 conjugate 15 12.90 Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols.
Organ weights were also evaluated. The results demonstrated that no elevation in transaminase levels (Table 38) or organ weights (data not shown) were observed in mice treated with ASOs compared to PBS
control. Further, the ASO with mixed PS/P0 linkages (ISIS 655862) showed similar transaminase levels compared to full PS (ISIS 655861).
Table 38 Effect of PO/PS linkages on transaminase levels of ASOs comprising Ga1NAc3-1 conjugate at 3' terminus targeting SRB-1 ISIS Dosage ALT AST
Chemistry SEQ ID No.
No. (mg/kg) (U/L) (U/L) PBS 0 28.5 65 3 50.25 89 353382 Full PS without 10 27.5 79.3 143 (parent) conjugate 30 27.3 97 0.5 28 55.7 1.5 30 78 Full PS with 5 29 63.5 Ga1NAc3-1 15 28.8 67.8 0.5 50 75.5 Mixed PS/P0 with 655862 1.5 21.7 58.5 144 GalNAc3-1 5 29.3 69 Example 45: Preparation of PFP Ester, Compound 110a OAc OAc Pd/C, H2 " n OAc OAc Et0Ac, Me0H
103a; n=1 ..,0...) , ----.,..õ.....õ,N3 _______________________________________________________________________________ ).
Ac0* 103b; n=7 Ac0 n 0.-- AcHN
N 104a; n=1 104b; n= 7 4 OAc AcONC:Ac AcHN 0/,) OAc OAc OAc OAc n N
H
__T!.C...D...\
Ac0--C-D-_\ -----NH2 PFPTFA
______________________________________ I.- Ac0 0 n NHIr.______NO2 AcHN DMF, Pyr AcHN n 105a; n=1 Compound 90 0 r OAc OAc 105b; n=7 Ac0 n AcHN
106a; n=1 106b; n=7 OAc AcONC:Ac AcHN 0 O
OAc OAc n Ra-Ni, H2 Ac0 Me0H, Et0Ac AcHN
...C..:)..\. HBTU, DIEA, DMF
_____________ ,- 0 ___________________________________________ )..-0 r "t Bn OAc OAc HO2C" ri '0-.r!C...:)..Ø----- jrHN ---.0 Ac0 AcHN 99 107a; n=1 107b; n= 7 OAc AcONC:Ac AcHN 0 OAc OAc µ 11 N¨C
H
__(!.C...:)...
Ac0 0 )r'.NH
AcHN Ir\--- NH

0 r OAc OAc , µ
Ac0 0.--N ---.0 'n AcHN
108a; n=1 0 108b; n= 7 1 Bn OAc Ac0 OAc AcHN 0 Pd/C H2, 108a; n=1 Et0Ac,, Me0H OAc OAc 108b; n= 7 Ac0 0 NH
AcHN \ n O

OAc OAc Ac0 'n AcHN
109a; n=1 HO
109b; n= 7 OAc Ac0 OAc AcHN 0 0 rAc OAc AcHN NH

PFPTFA, DMF, 0 pyr OAc OAc 109a Ac0 OHNO
AcHN
O
110a O F
F F
F F
Compound 4 (9.5g, 28.8 mmoles) was treated with compound 103a or 103b (38 mmoles), individually, and TMSOTf (0.5 eq.) and molecular sieves in dichloromethane (200 mL), and stirred for 16 hours at room temperature. At that time, the organic layer was filtered thru celite, then washed with sodium bicarbonate, water and brine. The organic layer was then separated and dried over sodium sulfate, filtered and reduced under reduced pressure. The resultant oil was purified by silica gel chromatography (2%-->10%
methanadichloromethane) to give compounds 104a and 104b in >80% yield. LCMS
and proton NMR was consistent with the structure.
Compounds 104a and 104b were treated to the same conditions as for compounds 100a-d (Example 47), to give compounds 105a and 105b in >90% yield. LCMS and proton NMR was consistent with the structure.
Compounds 105a and 105b were treated, individually, with compound 90 under the same conditions as for compounds 901a-d, to give compounds 106a (80%) and 106b (20%). LCMS and proton NMR was consistent with the structure.

Compounds 106a and 106b were treated to the same conditions as for compounds 96a-d (Example 47), to give 107a (60%) and 107b (20%). LCMS and proton NMR was consistent with the structure.
Compounds 107a and 107b were treated to the same conditions as for compounds 97a-d (Example 47), to give compounds 108a and 108b in 40-60% yield. LCMS and proton NMR was consistent with the structure.
Compounds 108a (60%) and 108b (40%) were treated to the same conditions as for compounds 100a-d (Example 47), to give compounds 109a and 109b in >80% yields. LCMS and proton NMR was consistent with the structure.
Compound 109a was treated to the same conditions as for compounds 101a-d (Example 47), to give Compound 110a in 30-60% yield. LCMS and proton NMR was consistent with the structure. Alternatively, Compound 110b can be prepared in a similar manner starting with Compound 109b.
Example 46: General Procedure for Conjugation with PFP Esters (Oligonucleotide 111); Preparation of ISIS 666881 (Ga1NAc3-10) A 5'-hexylamino modified oligonucleotide was synthesized and purified using standard solid-phase oligonucleotide procedures. The 5'-hexylamino modified oligonucleotide was dissolved in 0.1 M sodium tetraborate, pH 8.5 (200 [LI-) and 3 equivalents of a selected PFP esterified Ga1NAc3 cluster dissolved in DMSO (50 [LI-) was added. If the PFP ester precipitated upon addition to the ASO solution DMSO was added until all PFP ester was in solution. The reaction was complete after about 16 h of mixing at room temperature. The resulting solution was diluted with water to 12 mL and then spun down at 3000 rpm in a spin filter with a mass cut off of 3000 Da. This process was repeated twice to remove small molecule impurities. The solution was then lyophilized to dryness and redissolved in concentrated aqueous ammonia and mixed at room temperature for 2.5 h followed by concentration in vacuo to remove most of the ammonia.
The conjugated oligonucleotide was purified and desalted by RP-HPLC and lyophilized to provide the Ga1NAc3 conjugated oligonucleotide.

OH
HONC:H
83e 3' ) I I AcHN
[ OLIGO O-P-0-(CH2)6-NH2 01-1 r OH
H
110a OH
HOONH 1 Borate buffer, DMSO, pH 8.5, rt AcHN NH

2 NH3 (aq), rt 0 OH OH
HOOH

AcHN

Oligonucleotide 111 is conjugated with Ga1NAc3-10. The Ga1NAc3 cluster portion of the conjugate group Ga1NAc3-10 (Ga1NAc3-10a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is -P(=0)(OH)-Ad-P(=0)(OH)- as shown in 5 the oligonucleotide (ISIS 666881) synthesized with Ga1NAc3-10 below. The structure of Ga1NAc3-10 (Ga1NAc3-10a-CM-) is shown below:
HO OH

HO "4 AcHN

HO El AcHN
HO OH

HO
AcHN
Following this general procedure ISIS 666881 was prepared. 5'-hexylamino modified oligonucleotide, ISIS 660254, was synthesized and purified using standard solid-phase oligonucleotide procedures. ISIS 660254 (40 mg, 5.2 [mot) was dissolved in 0.1 M sodium tetraborate, pH 8.5 (200 I.LL) and 3 equivalents PFP ester (Compound 110a) dissolved in DMSO (50 I.LL) was added.
The PFP ester precipitated upon addition to the ASO solution requiring additional DMSO (600 I.LL) to fully dissolve the PFP
ester. The reaction was complete after 16 h of mixing at room temperature. The solution was diluted with water to 12 mL total volume and spun down at 3000 rpm in a spin filter with a mass cut off of 3000 Da. This process was repeated twice to remove small molecule impurities. The solution was lyophilized to dryness and redissolved in concentrated aqueous ammonia with mixing at room temperature for 2.5 h followed by concentration in vacuo to remove most of the ammonia. The conjugated oligonucleotide was purified and desalted by RP-HPLC and lyophilized to give ISIS 666881 in 90% yield by weight (42 mg, 4.7 [mot).
Ga1NAc3-10 conjugated oligonucleotide SEQ
ASO Sequence (5' to 3') 5' group ID No.
NH2(CH2)6-0AdoGesmCesTesTesmCesAdsGdsTds ISIS 660254 Hexylamine 145 mCdsAdsTdsGdsAdsmCdsTdsTesmCesmCesTesTe GallNAC3-10,-0'AdoGesmCesTesTesmCesAdsGdsTds ISIS 666881 GalNAc3-10 145 mCdsAdsTdsGdsAdsmCdsTdsTesniCesniCesrfesrfe Capital letters indicate the nucleobase for each nucleoside and mC
indicates a 5-methyl cytosine.
Subscripts: "e" indicates a 2'-MOE modified nucleoside; "d" indicates a [3-D-2'-deoxyribonuc1eoside; "s"
indicates a phosphorothioate internucleoside linkage (PS); "o" indicates a phosphodiester internucleoside linkage (PO); and "o" indicates -0-P(=0)(OH)-. Conjugate groups are in bold.

Example 47: Preparation of Oligonucleotide 102 Comprising GaINAc3-8 HO--/(_____ H2N" 'n1 'NHBoc BocHNN---(_:).___ n H
91a; n=1 BocHN NH TFA, DCM
91b, n=2 n 1.-------NO2 _______ D.-________________________________ )0-0 r 0 PFPTFA, DIPEA, DMF
H0c) BocHN ,(:/HN --(0 in 92a; n=1 92b, n=2 H2Ni Iji OAcr- OAc H2N-- NH NO2 ; TMSOTf, DCM
Ac0 0 AcHN 3 H2N eHN 0 /n 93a; n=1 93b, n=2 94a; m=1 94b, m=2 0 OAc OAc OAc OAc 0,Bn Ac0 __.:)..\ m --P...\----(:): OH
Ac0 HO __________ ... AcHN

N0 TMSOTf 7; m=1 Pd/C. H2 64, m=2 OAc AcONC:Ac 0 AcHN ,m 0 93a (93b) OAc OAc 0 NH Ra-Ni, H2 HBTU, DIPEA, DMF Ac0 _____________ i..-% / m N n y\--- NO2 H
AcHN
OAc OAc 0 r H
__/L:_.:)...\o....,õ...,,,i.õ."Thii i Ac0 r\IN,hHN '0 AcHN n 96a; n=1, m=1 96b; n=1, m=2 96c; n=2, m=1 96d; n=2, m=2 OAc AcONC:Ac 0 0 õ.........c.......}...
AcHN 0 , / HBTU, DIEA, DMF
\ i n OAc OAc 0 H ___________________________ a-Ac0 0 , / m N n NHN H2 A H 40 ODMTr AcHN
OAc OAc 0 r H
...;..:)..\. ' N
Ac0 0 HO r N N,i/HN---0 % m -e-P Nb., AcHN n 0 0 'OH
97a; n=1, m=1 97b; n=1, m=2 97c; n=2, m=1 97d; n=2, m=2 OAc AcONC_)Ac 0 AcHN
OAc OAc 0 H
0)),( 7,hNH H 0 ODMTr Ac0 , / m N n Ir\--- N
p N
AcHN H
OAc OAc 0 r cb H
Ac0 0iNN_rr HN--.0 0 ''OH
, m AcHN n 98a; n=1, m=1 98b; n=1, m=2 98c; n=2, m=1 98d; n=2, m=2 OAc AcON(:)Ac 0 0 0 ,hAcHN mA [Ii HBTU, DIEA, DMF k 7 n [1 97a, n=1, m=1 OAc OAc 0 97b, n=1, m=2 a Ac0- CY Nr$'hNH ...----0, 97c, n=2, m=1 0 -)-ii LH n 1r .\, ---- [1 97d, n=2, m=2 AcHN Bn HO2C,,qL,0,Bn OAc OAc 0 r Ac0 HNo 99 m AcHN n 100a, n=1, m=1 100b, n=1, m=2 100c, n=2, m=1 OAc 100d, n=2, m=2 AcON(:)Ac 0 AcHN 0').',..),,, LN0 Pd(OH)2/C, OAc OAc 0 MpC)H
_ 0 0 H2, Et0Ac, PFPTFA, DMF, , ..:..:).\N/$,i"NH ..-----OH
Ac0 n 1õr \,.---- [1 pyr _ m H
AcHN
OAc OAc 0 r H
HN ---o 101a, n=1, m=1 AcHN m 0 n 101b, n=1, m=2 101c, n=2, m=1 101d, n=2, m=2 OAc Ac0(:)Ac 0 0 F

AcHN 01'h)LN 0 F
OAc OAc m H.-If\I [1--(.....
0 0 io F
Ac0c)\)LNZ'HNH
F
AcHN H
OAc OAc 0 r F
H
N HN---%
Ac0--).._\171 1 102a, n=1, m=1 AcHN 102b, n=1, m=2 102c, n=2, m=1 102d, n=2, m=2 The triacid 90 (4 g, 14.43 mmol) was dissolved in DMF (120 mL) and N,N-Diisopropylethylamine (12.35 mL, 72 mmoles). Pentafluorophenyl trifluoroacetate (8.9 mL, 52 mmoles) was added dropwise, under argon, and the reaction was allowed to stir at room temperature for 30 minutes. Boc-diamine 91a or 91b (68.87 mmol) was added, along with N,N-Diisopropylethylamine (12.35 mL, 72 mmoles), and the reaction was allowed to stir at room temperature for 16 hours. At that time, the DMF
was reduced by >75% under reduced pressure, and then the mixture was dissolved in dichloromethane. The organic layer was washed with sodium bicarbonate, water and brine. The organic layer was then separated and dried over sodium sulfate, filtered and reduced to an oil under reduced pressure. The resultant oil was purified by silica gel chromatography (2%-->10% methanol/dichloromethane) to give compounds 92a and 92b in an approximate 80% yield. LCMS and proton NMR were consistent with the structure.
Compound 92a or 92b (6.7 mmoles) was treated with 20 mL of dichloromethane and 20 mL of trifluoroacetic acid at room temperature for 16 hours. The resultant solution was evaporated and then dissolved in methanol and treated with DOWEX-OH resin for 30 minutes. The resultant solution was filtered and reduced to an oil under reduced pressure to give 85-90% yield of compounds 93a and 93b.
Compounds 7 or 64 (9.6 mmoles) were treated with HBTU (3.7g, 9.6 mmoles) and N,N-Diisopropylethylamine (5 mL) in DMF (20 mL) for 15 minutes. To this was added either compounds 93a or 93b (3 mmoles), and allowed to stir at room temperature for 16 hours. At that time, the DMF was reduced by >75% under reduced pressure, and then the mixture was dissolved in dichloromethane. The organic layer was washed with sodium bicarbonate, water and brine. The organic layer was then separated and dried over sodium sulfate, filtered and reduced to an oil under reduced pressure. The resultant oil was purified by silica gel chromatography (5%-->20% methanol/dichloromethane) to give compounds 96a-d in 20-40% yield.
LCMS and proton NMR was consistent with the structure.
Compounds 96a-d (0.75 mmoles), individually, were hydrogenated over Raney Nickel for 3 hours in Ethanol (75 mL). At that time, the catalyst was removed by filtration thru celite, and the ethanol removed under reduced pressure to give compounds 97a-d in 80-90% yield. LCMS and proton NMR were consistent with the structure.
Compound 23 (0.32g, 0.53 mmoles) was treated with HBTU (0.2g, 0.53 mmoles) and N,N-Diisopropylethylamine (0.19 mL, 1.14 mmoles) in DMF (30mL) for 15 minutes. To this was added compounds 97a-d (0.38 mmoles), individually, and allowed to stir at room temperature for 16 hours. At that time, the DMF was reduced by >75% under reduced pressure, and then the mixture was dissolved in dichloromethane. The organic layer was washed with sodium bicarbonate, water and brine. The organic layer was then separated and dried over sodium sulfate, filtered and reduced to an oil under reduced pressure.
The resultant oil was purified by silica gel chromatography (2%-->20%
methanol/dichloromethane) to give compounds 98a-d in 30-40% yield. LCMS and proton NMR was consistent with the structure.
Compound 99 (0.17g, 0.76 mmoles) was treated with HBTU (0.29 g, 0.76 mmoles) and N,N-Diisopropylethylamine (0.35 mL, 2.0 mmoles) in DMF (50mL) for 15 minutes. To this was added compounds 97a-d (0.51 mmoles), individually, and allowed to stir at room temperature for 16 hours. At that time, the DMF was reduced by >75% under reduced pressure, and then the mixture was dissolved in dichloromethane. The organic layer was washed with sodium bicarbonate, water and brine. The organic layer was then separated and dried over sodium sulfate, filtered and reduced to an oil under reduced pressure.
The resultant oil was purified by silica gel chromatography (5%-->20%
methanol/ dichloromethane) to give compounds 100a-d in 40-60% yield. LCMS and proton NMR was consistent with the structure.
Compounds 100a-d (0.16 mmoles), individually, were hydrogenated over 10%
Pd(OH)2/C for 3 hours in methanoVethyl acetate (1:1, 50 mL). At that time, the catalyst was removed by filtration thru celite, and the organics removed under reduced pressure to give compounds 101a-d in 80-90% yield. LCMS and proton NMR was consistent with the structure.
Compounds 101a-d (0.15 mmoles), individually, were dissolved in DMF (15 mL) and pyridine (0.016 mL, 0.2 mmoles). Pentafluorophenyl trifluoroacetate (0.034 mL, 0.2 mmoles) was added dropwise, under argon, and the reaction was allowed to stir at room temperature for 30 minutes. At that time, the DMF
was reduced by >75% under reduced pressure, and then the mixture was dissolved in dichloromethane. The organic layer was washed with sodium bicarbonate, water and brine. The organic layer was then separated and dried over sodium sulfate, filtered and reduced to an oil under reduced pressure. The resultant oil was purified by silica gel chromatography (2%-->5% methanadichloromethane) to give compounds 102a-d in an approximate 80% yield. LCMS and proton NMR were consistent with the structure.
83e 3'5', 11 ou t Go O-P-0-(CH2)6NH2 OH
Borate buffer, DMSO, pH 8.5, rt 102d 2. aq. ammonia, rt AcHN 0 0 AcHN

AcHN
Oligomeric Compound 102, comprising a Ga1NAc3-8 conjugate group, was prepared using the general procedures illustrated in Example 46. The Ga1NAc3 cluster portion of the conjugate group Ga1NAc3-8 (Ga1NAc3-8a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In a preferred embodiment, the cleavable moiety is -P(=0)(OH)-Ad-P(=0)(OH)-.
The structure of Ga1NAc3-8 (Ga1NAc3-8a-CM-) is shown below:

AcHN 0 0 N)N,i 0 El ' AcHN

HO

AcHN =

Example 48: Preparation of Oligonucleotide 119 Comprising GaINAc3-7 AcO0Ac Ac0 OAc Ac0-.4-\0 TMSOTf, DCE Ac0rONHCBz Pd(OH)2/C
> 4 N--1 HO ..-rNHCBz AcHN H2, Me0H, Et0Ac 4 i 35b 112 HO---in HBTU, DIEA
Ac0 OAc 0 .(:),._ DMF
NHCBZ _______________________________________________ .
Ac04,r(:)NH2 + HO 0 AcHN 0 C)L) 105a HO

Ac0 OAc , Ac0 ,r H
-4 \)"k/N----t;

Ac0 OAc AcHN
Ac0 0(--N
HCBZ

AcHN

Ac0 OAc Ac0 r0_ 0(-)NH

AcHN

Ac0 OAc H , Ac0.....).,..\,0N----ts.;

AcHN
Ac0 OAc Pd/C, H2,2 0 AcHN 0 0 Ac0 OAc .......2...\õONH
Ac0 AcHN

Ac0 OAc H , Ac047 N------t;

HBTU, DIEA, DMF AcHN 0 0 Ac0 OAc _________________________ ).-Ac0 4 )..n....بNH
0 AcHN 0 0 HOO
Ac0 OAc Ac0 ........2...\õONH

83a AcHN

Compound 112 was synthesized following the procedure described in the literature (J. Med. Chem.
2004, 47, 5798-5808).
Compound 112 (5 g, 8.6 mmol) was dissolved in 1:1 methanol/ethyl acetate (22 mL/22 mL).
Palladium hydroxide on carbon (0.5 g) was added. The reaction mixture was stirred at room temperature under hydrogen for 12 h. The reaction mixture was filtered through a pad of celite and washed the pad with 1:1 methanol/ethyl acetate. The filtrate and the washings were combined and concentrated to dryness to yield Compound 105a (quantitative). The structure was confirmed by LCMS.
Compound 113 (1.25 g, 2.7 mmol), HBTU (3.2 g, 8.4 mmol) and DIEA (2.8 mL, 16.2 mmol) were dissolved in anhydrous DMF (17 mL) and the reaction mixture was stirred at room temperature for 5 min. To this a solution of Compound 105a (3.77 g, 8.4 mmol) in anhydrous DMF (20 mL) was added. The reaction was stirred at room temperature for 6 h. Solvent was removed under reduced pressure to get an oil. The residue was dissolved in CH2C12 (100 mL) and washed with aqueous saturated NaHCO3 solution (100 mL) and brine (100 mL). The organic phase was separated, dried (Na2SO4), filtered and evaporated. The residue was purified by silica gel column chromatography and eluted with 10 to 20 %
Me0H in dichloromethane to yield Compound 114 (1.45 g, 30%). The structure was confirmed by LCMS and 1H
NMR analysis.
Compound 114 (1.43 g, 0.8 mmol) was dissolved in 1:1 methanol/ethyl acetate (4 mL/4 mL).
Palladium on carbon (wet, 0.14 g) was added. The reaction mixture was flushed with hydrogen and stirred at room temperature under hydrogen for 12 h. The reaction mixture was filtered through a pad of celite. The celite pad was washed with methanol/ethyl acetate (1:1). The filtrate and the washings were combined together and evaporated under reduced pressure to yield Compound 115 (quantitative). The structure was confirmed by LCMS and 1H NMR analysis.
Compound 83a (0.17 g, 0.75 mmol), HBTU (0.31 g, 0.83 mmol) and DIEA (0.26 mL, 1.5 mmol) were dissolved in anhydrous DMF (5 mL) and the reaction mixture was stirred at room temperature for 5 min. To this a solution of Compound 115 (1.22 g, 0.75 mmol) in anhydrous DMF
was added and the reaction was stirred at room temperature for 6 h. The solvent was removed under reduced pressure and the residue was dissolved in CH2C12. The organic layer was washed aqueous saturated NaHCO3 solution and brine and dried over anhydrous Na2SO4 and filtered. The organic layer was concentrated to dryness and the residue obtained was purified by silica gel column chromatography and eluted with 3 to 15 % Me0H in dichloromethane to yield Compound 116 (0.84 g, 61%). The structure was confirmed by LC MS and 1H
NMR analysis.
Ac0 OAc H , Ac0.....1, N----t...\-' AcHN
Pd/C, H2, Ac0 OAc 0 0 Et0Ac, Me0H
_ AcHN 0 0 Ac0 OAc .....?....\,0(¨)NH
Ac0 AcHN
Ac0 OAc H , F
F
F
AcHN 0 -----t....\' PFPTFA, DMF, Pyr Ac0 OAc )c) 0 .......2...\r0(--)NH F
F

AcHN 0 0 Ac0 OAc )j .......2...\,,ONH 118 Ac0 AcHN

Compound 116 (0.74 g, 0.4 mmol) was dissolved in 1:1 methanol/ethyl acetate (5 mL/5 mL).
Palladium on carbon (wet, 0.074 g) was added. The reaction mixture was flushed with hydrogen and stirred at room temperature under hydrogen for 12 h. The reaction mixture was filtered through a pad of celite. The celite pad was washed with methanoVethyl acetate (1:1). The filtrate and the washings were combined together and evaporated under reduced pressure to yield compound 117 (0.73 g, 98%). The structure was confirmed by LCMS and 1H NMR analysis.
Compound 117 (0.63 g, 0.36 mmol) was dissolved in anhydrous DMF (3 mL). To this solution N,N-Diisopropylethylamine (70 [LL, 0.4 mmol) and pentafluorophenyl trifluoroacetate (72 [tt, 0.42 mmol) were added. The reaction mixture was stirred at room temperature for 12 h and poured into a aqueous saturated NaHCO3 solution. The mixture was extracted with dichloromethane, washed with brine and dried over anhydrous Na2SO4. The dichloromethane solution was concentrated to dryness and purified with silica gel column chromatography and eluted with 5 to 10 % Me0H in dichloromethane to yield compound 118 (0.51 g, 79%). The structure was confirmed by LCMS and 1H and 1H and 19F NMR.
83e 3'5') 11 j [ OLIGO ¨0¨P-0¨(CH2)6-NH2 I
OH
1. Borate buffer, DMSO, pH 8.5, rt 118 __________________________ )1.-2. aq. ammonia, rt AcHN N

OLIGO

AcHN 07 HO OH

AcHN
Oligomeric Compound 119, comprising a Ga1NAc3-7 conjugate group, was prepared using the general procedures illustrated in Example 46. The Ga1NAc3 cluster portion of the conjugate group Ga1NAc3-7 (Ga1NAc3-7a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is -P(=0)(OH)-Ad-P(=0)(OH)-.
The structure of Ga1NAc3-7 (Ga1NAc3-7a-CM-) is shown below:

HOOH

AcHN No AcHN 0/
HOOH

AcHN
=
Example 49: Preparation of Oligonucleotide 132 Comprising GaINAc3-5 ,Boc HN
,Boc HN,Boc HN
H HN,Boc H2NC) Bocj-Lo 'NThrN
Boc,Ny NJ-LOH

Boc,NOH

0Li0H, H20 HBTU, TEA
,Boo Me0H, THF
DMF HN HN,Boc 78% 123 Compound 120 (14.01 g, 40 mmol) and HBTU (14.06 g, 37 mmol) were dissolved in anhydrous DMF (80 mL). Triethylamine (11.2 mL, 80.35 mmol) was added and stirred for 5 min. The reaction mixture was cooled in an ice bath and a solution of compound 121 (10 g, mmol) in anhydrous DMF (20 mL) was added. Additional triethylamine (4.5 mL, 32.28 mmol) was added and the reaction mixture was stirred for 18 h under an argon atmosphere. The reaction was monitored by TLC (ethyl acetate:hexane; 1:1; Rf = 0.47).
The solvent was removed under reduced pressure. The residue was taken up in Et0Ac (300 mL) and washed with 1M NaHSO4 ( 3 x 150 mL), aqueous saturated NaHCO3 solution (3 x 150 mL) and brine (2 x 100 mL).
Organic layer was dried with Na2SO4. Drying agent was removed by filtration and organic layer was concentrated by rotary evaporation. Crude mixture was purified by silica gel column chromatography and eluted by using 35 ¨ 50% Et0Ac in hexane to yield a compound 122 (15.50 g, 78.13%). The structure was confirmed by LCMS and 1H NMR analysis. Mass m/z 589.3 [M + H] .
A solution of LiOH (92.15 mmol) in water (20 mL) and THF (10 mL) was added to a cooled solution of Compound 122 (7.75 g,13.16 mmol) dissolved in methanol (15 mL). The reaction mixture was stirred at room temperature for 45 min. and monitored by TLC (Et0Ac:hexane; 1:1). The reaction mixture was concentrated to half the volume under reduced pressure. The remaining solution was cooled an ice bath and neutralized by adding concentrated HC1. The reaction mixture was diluted, extracted with Et0Ac (120 mL) and washed with brine (100 mL). An emulsion formed and cleared upon standing overnight. The organic layer was separated dried (Na2SO4), filtered and evaporated to yield Compound 123 (8.42 g). Residual salt is the likely cause of excess mass. LCMS is consistent with structure. Product was used without any further purification. M.W.cal:574.36; M.W.fd:575.3 [M + H] .

4. ii ¨OH
0 S = H20 F-13N-----L
li H2N-L,OH + HO 11O
0 0¨g Toluene, Reflux 0li 996%
Compound 126 was synthesized following the procedure described in the literature (J. Am. Chem.
Soc. 2011, /33, 958-963).

H N, Boc 126 Boc, N j.LN ...."...p, 0 CF3COOH
123 Vo- NThr 0 _____________________________________________________________________ )v-HOBt, DIEA, 0 8 CH2Cl2 PyBop, Bop, DMF
r HN,Boc 127 CF3C00- 1\13 Ac0 OAc H

AcO OH
H3N N 1-4 AcHN 7 0 ( CF3C00- a 0 ________________________________________________ - 7..-HATU, HOAt, DIEA, DMF
r cF3coo- 0 NH3 128 Ac0 OAc _....7.2_\7 Ac0 CI0 AcHN
NH

Ac0 OAc N j'L r3-(c) 0 HN----r N
*Ø...\, H
Ac0 Oz--i 0 0 AcHN 0 /
Ac0 OAc Ac0---4r, H
-\,¨

AcHN 0 Ac0 OAc AcO
AcHN
NH
H
Pd/C, H2, Me0H 0 Ac0 OAc OF
HN
i\jj.LHr Ac0 0 0 AcHN
Ac0 OAc AcO 0 r, NH
Ac0 OAc AcHN 0 130 Ac0 ______________________ AcHN
NH
L
PFPTFA, DMF, Pyr H
Ac0 OAc HN
"3 H
Ac0 0 0 AcHN
Ac0 OAc 0 r, NH
AcHN

Compound 123 (7.419 g, 12.91 mmol), HOBt (3.49 g, 25.82 mmol) and compound 126 (6.33 g, 16.14 mmol) were dissolved in and DMF (40 mL) and the resulting reaction mixture was cooled in an ice bath. To this N,N-Diisopropylethylamine (4.42 mL, 25.82 mmol), PyBop (8.7 g, 16.7 mmol) followed by Bop coupling reagent (1.17 g, 2.66 mmol) were added under an argon atmosphere.
The ice bath was removed and the solution was allowed to warm to room temperature. The reaction was completed after 1 h as determined by TLC (DCM:MeOH:AA; 89:10:1). The reaction mixture was concentrated under reduced pressure. The residue was dissolved in Et0Ac (200 mL) and washed with 1 M
NaHSO4 (3x100 mL), aqueous saturated NaHCO3 (3x100 mL) and brine (2x100 mL). The organic phase separated dried (Na2SO4), filtered and concentrated. The residue was purified by silica gel column chromatography with a gradient of 50% hexanes/EtOAC to 100% Et0Ac to yield Compound 127 (9.4 g) as a white foam.
LCMS and 1H NMR

were consistent with structure. Mass m/z 778.4 [M + H] +.
Trifluoroacetic acid (12 mL) was added to a solution of compound 127 (1.57 g, 2.02 mmol) in dichloromethane (12 mL) and stirred at room temperature for 1 h. The reaction mixture was co-evaporated with toluene (30 mL) under reduced pressure to dryness. The residue obtained was co-evaporated twice with acetonitrile (30 mL) and toluene (40 mL) to yield Compound 128 (1.67 g) as trifluoro acetate salt and used for next step without further purification. LCMS and 1H NMR were consistent with structure. Mass m/z 478.2 [M + H] +.
Compound 7 (0.43 g, 0.963 mmol), HATU (0.35 g, 0.91 mmol), and HOAt (0.035 g, 0.26 mmol) were combined together and dried for 4 h over P205 under reduced pressure in a round bottom flask and then dissolved in anhydrous DMF (1 mL) and stirred for 5 min. To this a solution of compound 128 (0.20 g, 0.26 mmol) in anhydrous DMF (0.2 mL) and N,N-Diisopropylethylamine (0.2 mL) was added. The reaction mixture was stirred at room temperature under an argon atmosphere. The reaction was complete after 30 min as determined by LCMS and TLC (7% Me0H/DCM). The reaction mixture was concentrated under reduced pressure. The residue was dissolved in DCM (30 mL) and washed with 1 M NaHSO4 (3x20 mL), aqueous saturated NaHCO3 (3 x 20 mL) and brine (3x20 mL). The organic phase was separated, dried over Na2504, filtered and concentrated. The residue was purified by silica gel column chromatography using 5-15%
Me0H in dichloromethane to yield Compound 129 (96.6 mg). LC MS and 1H NMR are consistent with structure. Mass m/z 883.4 [M + 2H] .
Compound 129 (0.09 g, 0.051 mmol) was dissolved in methanol (5 mL) in 20 mL
scintillation vial.
To this was added a small amount of 10% Pd/C (0.015 mg) and the reaction vessel was flushed with H2 gas.
The reaction mixture was stirred at room temperature under H2 atmosphere for 18 h. The reaction mixture was filtered through a pad of Celite and the Celite pad was washed with methanol. The filtrate washings were pooled together and concentrated under reduced pressure to yield Compound 130 (0.08 g). LCMS and 1H NMR were consistent with structure. The product was used without further purification. Mass m/z 838.3 [M + 2H] .
To a 10 mL pointed round bottom flask were added compound 130 (75.8 mg, 0.046 mmol), 0.37 M
pyridine/DMF (200 [LI-) and a stir bar. To this solution was added 0.7 M
pentafluorophenyl trifluoroacetate/DMF (100 [LL) drop wise with stirring. The reaction was completed after 1 h as determined by LC MS. The solvent was removed under reduced pressure and the residue was dissolved in CHC13 (¨ 10 mL). The organic layer was partitioned against NaHSO4 (1 M, 10 mL) , aqueous saturated NaHCO3 (10 mL) and brine (10 mL) three times each. The organic phase separated and dried over Na2504, filtered and concentrated to yield Compound 131 (77.7 mg). LCMS is consistent with structure. Used without further purification. Mass m/z 921.3 [M + 2H] .

HO OH
O 83e HO---4-v0 N...õ.....õ--Nr0 3' J¨ I I AcHN
[ OLIGO O¨P-0¨(CH2)6-NH2 NH
OH
1. Borate buffer, DMSO, pH 8.5, rt 131 ______________________ ).-2. aq. ammonia, rt HO OH NA
NH
HN----....ir ___&....D..\, HOOZ"-i 0 .., AcHN 0 /
HO OH
0 n NH
HO--rs-Wy 0 N0 co OLIGO

AcHN 0 Oligomeric Compound 132, comprising a Ga1NAc3-5 conjugate group, was prepared using the general procedures illustrated in Example 46. The Ga1NAc3 cluster portion of the conjugate group Ga1NAc3-5 (Ga1NAc3-5a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is -P(=0)(01-1)-Ad-P(=0)(OH)-.
The structure of Ga1NAc3-5 (Ga1NAc3-5a-CM-) is shown below:
HO OH
*,,Ø.
HO 0r0 AcHN
NH

HO OH
HN=ri\INH
*...\zoz...i HO 0 2_ AcHN 0 /
HO OH
0 r, NH
HO--4-V- 0 NO¨(CIVO-AcHN 0 =

Example 50: Preparation of Oligonucleotide 144 Comprising Ga1NAc4-11 DMTO Fmoc 1. TBTU, DIEA DMTO Fmoc La ACN, VIMAD Resin La pip:DBU:DMF
________________________________ OP- ___________________________ VP
. 2. Ac 20 Capping - 0 0 (2:2:96) O --- )-OH Kaiser: Negetive HN-Fmoc DMTO H Fmoc,N OH
LO
H /
0 DMTF-...

L. al b 135 HBTU, DIEA, DMF

b NH-Fmoc DMTr ) ib 1. pip:DBU:DMF 0 / H N'j 0 1. 2%
hydrazine/DMF
Kaiser: Positive Kaiser: Positive _________________ 10 ___ ....iN)L(CH2)5'N
Y ________________________________________________________________________ VP

2. Dde-Lys(Fmoc)-OH (138) 0 H 2. Fmoc-Lys(Fmoc)-OH
(140) HATU, DIEA, DMF d 0 HATU, DIEA, DMF
Kaiser: Negative 0 Kaiser: Negative ,Fmoc HN
) /
HNOyizi,Fmoc DMTr O

N)L(cH2)5-1-N1 NNFmoc ... .1 HN,Fmoc Ac0 OAc AcHN
=

Ac0 OAc AcOol\rH 0 AcHN
1. pip:DBU:DMF 0 H

141 Kaiser: Positive HN2. 7, HATU, DIEA, Ac0 OAc DMF DMTO
Kaiser: Negative NH
AcHN 0 0 Ac0 OAc AcOOC
AcHN 0 Synthesis of Compound 134. To a Merrifield flask was added aminomethyl VIMAD
resin (2.5 g, 450 [tmol/g) that was washed with acetonitrile, dimethylformamide, dichloromethane and acetonitrile. The resin was swelled in acetonitrile (4 mL). Compound 133 was pre-activated in a 100 mL round bottom flask by adding 20 (1.0 mmol, 0.747 g), TBTU (1.0 mmol, 0.321 g), acetonitrile (5 mL) and DIEA (3.0 mmol, 0.5 mL). This solution was allowed to stir for 5 min and was then added to the Merrifield flask with shaking.
The suspension was allowed to shake for 3 h. The reaction mixture was drained and the resin was washed with acetonitrile, DMF and DCM. New resin loading was quantitated by measuring the absorbance of the DMT cation at 500 nm (extinction coefficient = 76000) in DCM and determined to be 238 [tmol/g. The resin was capped by suspending in an acetic anhydride solution for ten minutes three times.
The solid support bound compound 141 was synthesized using iterative Fmoc-based solid phase peptide synthesis methods. A small amount of solid support was withdrawn and suspended in aqueous ammonia (28-30 wt%) for 6 h. The cleaved compound was analyzed by LC-MS and the observed mass was consistent with structure. Mass m/z 1063.8 [M + 2I-1] .
The solid support bound compound 142 was synthesized using solid phase peptide synthesis methods.

Ac0 OAc AcO,0 AcHN -NH

Ac0 OAc Ac0 N Ni AcHN 0 H g DNA syntesizer 0 H Ny 142 ________________ Ac0 OAc 0 __&.(2.\,0 H NH C' Ac0 ___________________________________________________________________________ , AcHN CM .) _____ , ASO , Ac0 OAc Ac0 )i---NH
AcHN 0 HO OH
HO__0 /----NH
AcHN 0 HO OH
HO NI
AcHN
aqueous NH3 0 H )Ny ___________________ ).-I
HO
AcHN )0ri\r)¨\<0 ( CM _____ ASO
) HO OH

AcHN 0 The solid support bound compound 143 was synthesized using standard solid phase synthesis on a DNA synthesizer.
The solid support bound compound 143 was suspended in aqueous ammonia (28-30 wt%) and heated at 55 C for 16 h. The solution was cooled and the solid support was filtered.
The filtrate was concentrated and the residue dissolved in water and purified by HPLC on a strong anion exchange column. The fractions containing full length compound 144 were pooled together and desalted. The resulting Ga1NAc4-11 conjugated oligomeric compound was analyzed by LC-MS and the observed mass was consistent with structure.
The Ga1NAc4 cluster portion of the conjugate group Ga1NAc4-11 (Ga1NAc4-11a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is -P(=0)(OH)-Ad-P(=0)(OH)-.
The structure of GalNAc4-11 (Ga1NAc4-11a-CM) is shown below:
HO OH

AcHN

HO OH

AcHN 0 H pH

NH
HO 1))r¨r_r_r<oN IE2 AcHN
HO OH
HO
AcHN

Example 51: Preparation of Oligonucleotide 155 Comprising GaINAc3-6 OH

I I OH
j-LOH
N

2M NaOH 0 OH

Compound 146 was synthesized as described in the literature (Analytical Biochemistry 1995, 229, 54-60).

HO .,...õ--,..õ-.,-.....N I Ao 0 Ac0 OAc 35b &i.......\,0 0.,.. A
4 ____________________________________ v.- Ac0 N 0 40 TMS-0Tf, 4 A molecular sieves, CH2Cl2, rt H
AcHN

Ac0 OAc 0 0 NÄ0 II
H2, Pd(OH)2 /C
Z ,r-1 0 147 Et0Ac/Me0H AcHN 105a 2 HBTU, DIEA, DMF, rt Ac0 OAc o ___________________________________________________________________________ 0 H ...C.,:)..\, H2, Pd(OH)2/C, Et0Ac/Me0H
0..,, }..,.....N 0 AcHN H

Ac0 OAc Ac0--&"-:)--\,0---,./\/\/\N}..,...NH2 AcHN H

Compound 4 (15 g, 45.55 mmol) and compound 35b (14.3 grams, 57 mmol) were dissolved in CH2C12 (200 m1). Activated molecular sieves (4 A. 2 g, powdered) were added, and the reaction was allowed to stir for 30 minutes under nitrogen atmosphere. TMS-0Tf was added (4.1 ml, 22.77 mmol) and the reaction was allowed to stir at room temp overnight. Upon completion, the reaction was quenched by pouring into solution of saturated aqueous NaHCO3 (500 ml) and crushed ice (¨
150 g). The organic layer was separated, washed with brine, dried over MgSO4, filtered, and was concentrated to an orange oil under reduced pressure. The crude material was purified by silica gel column chromatography and eluted with 2-10 % Me0H in CH2C12to yield Compound 112 (16.53 g, 63 %). LCMS and 1H NMR were consistent with the expected compound.
Compound 112 (4.27 g, 7.35 mmol) was dissolved in 1:1 Me0H/Et0Ac (40 m1). The reaction mixture was purged by bubbling a stream of argon through the solution for 15 minutes. Pearlman's catalyst (palladium hydroxide on carbon, 400 mg) was added, and hydrogen gas was bubbled through the solution for 30 minutes. Upon completion (TLC 10% Me0H in CH2C12, and LCMS), the catalyst was removed by filtration through a pad of celite. The filtrate was concentrated by rotary evaporation, and was dried briefly under high vacuum to yield Compound 105a (3.28 g). LCMS and 1H NMR were consistent with desired product.
Compound 147 (2.31 g, 11 mmol) was dissolved in anhydrous DMF (100 mL). N,N-Diisopropylethylamine (DIEA, 3.9 mL, 22 mmol) was added, followed by HBTU (4 g, 10.5 mmol). The reaction mixture was allowed to stir for ¨ 15 minutes under nitrogen. To this a solution of compound 105a (3.3 g, 7.4 mmol) in dry DMF was added and stirred for 2 h under nitrogen atmosphere. The reaction was diluted with Et0Ac and washed with saturated aqueous NaHCO3 and brine. The organics phase was separated, dried (MgSO4), filtered, and concentrated to an orange syrup. The crude material was purified by column chromatography 2-5 % Me0H in CH2C12 to yield Compound 148 (3.44 g, 73 %). LCMS and 1H
NMR were consistent with the expected product.
Compound 148 (3.3 g, 5.2 mmol) was dissolved in 1:1 Me0H/Et0Ac (75 m1). The reaction mixture was purged by bubbling a stream of argon through the solution for 15 minutes.
Pearlman's catalyst (palladium hydroxide on carbon) was added (350 mg). Hydrogen gas was bubbled through the solution for 30 minutes. Upon completion (TLC 10% Me0H in DCM, and LCMS), the catalyst was removed by filtration through a pad of celite. The filtrate was concentrated by rotary evaporation, and was dried briefly under high vacuum to yield Compound 149 (2.6 g). LCMS was consistent with desired product. The residue was dissolved in dry DMF (10 ml) was used immediately in the next step.
Ac0 OAc Ac0- iv PrN)EF1 0 0 Ac0 OAc AcHN 3 H

N
AcOoN --)L-----N--r---N )L0 0 H
AcHN 3 H 0 146 ________________ )..= Ac0 OAc 0 HBTU, DIEA, DMF )..........NH
,a,L.:).....\/0.........."¨...1..õ.)----.., N
Ac0 3 H
NHAc Ac0 OAc Ac0 OAcAc0oN)1 0 Pd(OH)2/C, H2 AcHN 3 H

Me0H, Et0Ac AcHN 3 H 0 AcO\ tOAc 0 Ac0 NH
0 0........."....¨is.r, N)1.-NHAc Compound 146 (0.68 g, 1.73 mmol) was dissolved in dry DMF (20 m1). To this DIEA (450 [tt, 2.6 mmol, 1.5 eq.) and HBTU (1.96 g, 0.5.2 mmol) were added. The reaction mixture was allowed to stir for 15 minutes at room temperature under nitrogen. A solution of compound 149 (2.6 g) in anhydrous DMF (10 mL) was added. The pH of the reaction was adjusted to pH = 9-10 by addition of DIEA (if necessary). The reaction was allowed to stir at room temperature under nitrogen for 2 h. Upon completion the reaction was diluted with Et0Ac (100 mL), and washed with aqueous saturated aqueous NaHCO3, followed by brine. The organic phase was separated, dried over MgSO4, filtered, and concentrated. The residue was purified by silica gel column chromatography and eluted with 2-10 % Me0H in CH2C12to yield Compound 150 (0.62 g, 20 %). LCMS and 1H NMR were consistent with the desired product.
Compound 150 (0.62 g) was dissolved in 1:1 Me0H/ Et0Ac (5 L). The reaction mixture was purged by bubbling a stream of argon through the solution for 15 minutes. Pearlman's catalyst (palladium hydroxide on carbon) was added (60 mg). Hydrogen gas was bubbled through the solution for 30 minutes. Upon completion (TLC 10% Me0H in DCM, and LCMS), the catalyst was removed by filtration (syringe-tip Teflon filter, 0.45 [Lin). The filtrate was concentrated by rotary evaporation, and was dried briefly under high vacuum to yield Compound 151 (0.57 g). The LCMS was consistent with the desired product. The product was dissolved in 4 mL dry DMF and was used immediately in the next step.

Ac0 OAc Ac0---4/ N)N
Ac0 OAc 0 0 AcHN 3 H Z(:_r,) 0 0 0 H ) Bn001-1 AC0-4r()NN----inN N
3 H OBn 83a 0 151 ),,.. AcHN 3 H
'-----_-.:---0 PFP-TFA, DIEA, DMF
Ac0 OAc 0 N)./NH
Ac0 3 H
NHAc Ac0 OAc Ac0(:)NN
Ac0 OAc AcHN 3 H Z,),,C) 0 0 Pd(OH)2/C, H2 ..\.r 0 Ho N N)COH
Ac0 )1N---Tr---N

Me0H, Et0Ac AcHN 3 H 0 ----r.----0 Ac0 OAc 0 ..i..,0....\/0.,_____õ...-õ=(....r, N)./NH
Ac0 NHAc Ac0 OAc Ac0 N)N F
Ac0 OAc AcHN 3 H ,C) F
0 0 .

PFP-TFA, Dl EA
o N) --..,,, }..,,...N---r----NZ,), F
o ____________ ). Ac0 N 3 H F
DMF AcHN 3 H 0 -----r.---0 F
Ac0 OAc 0 NH N)-----/
Ac0 NHAc Compound 83a (0.11 g, 0.33 mmol) was dissolved in anhydrous DMF (5 mL) and N,N-Diisopropylethylamine (75 I.LL, 1 mmol) and PFP-TFA (90 I.LL, 0.76 mmol) were added. The reaction mixture turned magenta upon contact, and gradually turned orange over the next 30 minutes. Progress of reaction was monitored by TLC and LCMS. Upon completion (formation of the PFP
ester), a solution of compound 151 (0.57 g, 0.33 mmol) in DMF was added. The pH of the reaction was adjusted to pH = 9-10 by addition of N,N-Diisopropylethylamine (if necessary). The reaction mixture was stirred under nitrogen for ¨
30 min. Upon completion, the majority of the solvent was removed under reduced pressure. The residue was diluted with CH2C12 and washed with aqueous saturated NaHCO3, followed by brine. The organic phase separated, dried over MgSO4, filtered, and concentrated to an orange syrup.
The residue was purified by silica gel column chromatography (2-10 % Me0H in CH2C12) to yield Compound 152 (0.35 g, 55 %). LCMS
and 1H NMR were consistent with the desired product.
Compound 152 (0.35 g, 0.182 mmol) was dissolved in 1:1 Me0H/Et0Ac (10 mL). The reaction mixture was purged by bubbling a stream of argon thru the solution for 15 minutes. Pearlman's catalyst (palladium hydroxide on carbon) was added (35 mg). Hydrogen gas was bubbled thru the solution for 30 minutes. Upon completion (TLC 10% Me0H in DCM, and LCMS), the catalyst was removed by filtration (syringe-tip Teflon filter, 0.45 [tin). The filtrate was concentrated by rotary evaporation, and was dried briefly under high vacuum to yield Compound 153 (0.33 g, quantitative). The LCMS was consistent with desired product.
Compound 153 (0.33 g, 0.18 mmol) was dissolved in anhydrous DMF (5 mL) with stirring under nitrogen. To this N,N-Diisopropylethylamine (65 [tt, 0.37 mmol) and PFP-TFA
(35 [tt, 0.28 mmol) were added. The reaction mixture was stirred under nitrogen for ¨ 30 min. The reaction mixture turned magenta upon contact, and gradually turned orange. The pH of the reaction mixture was maintained at pH = 9-10 by adding more N,-Diisopropylethylamine. The progress of the reaction was monitored by TLC and LCMS.
Upon completion, the majority of the solvent was removed under reduced pressure. The residue was diluted with CH2C12 (50 mL), and washed with saturated aqueous NaHCO3, followed by brine. The organic layer was dried over Mg504, filtered, and concentrated to an orange syrup. The residue was purified by column chromatography and eluted with 2-10 % Me0H in CH2C12 to yield Compound 154 (0.29 g, 79 %). LCMS
and 1H NMR were consistent with the desired product.
83e 3 5', II HOOH 0 p 1. OLIGO 0-1-0-(CH2)6 NH2 O HO- 0r=C=?-\1'rA NC H 4 H
HN\r0 H
154 1 Borate buffer, DMSO, .. HOOH AcHN

pH 8.5, rt 0 H
ON)C1\11.r ,_ _ cm i¨ OLIGO

______________________ 2 aq AcHN
ammonia, rt 0 0 0 HO

AcHN
Oligomeric Compound 155, comprising a Ga1NAc3-6 conjugate group, was prepared using the general procedures illustrated in Example 46. The Ga1NAc3 cluster portion of the conjugate group Ga1NAc3-6 (Ga1NAc3-6a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is -P(=0)(OH)-Ad-P(=0)(OH)-.
The structure of Ga1NAc3-6 (Ga1NAc3-6a-CM-) is shown below:

HoOH 0 HO-----\---01`-r'N

AcHN HN

H
N)C'1\11(cyH

HO N
---r-\--"
4 H l''r50 El I
0 o 0 AcHN
ri---µ
HooH , 0 AcHN .
Example 52: Preparation of Oligonucleotide 160 Comprising GaINAc3-9 AcO0Ac 0 AcO\ rc ___.....7..\\O 0 HO('')---100 Ac0 ,, TMSOTf, 50 C Ac0 ' IS
-.......).\OAc __ ).- _______________________________ ).-AcHN CICH2CH2CI, rt, 93% N"------ ---__:_( TMSOTf, DCE, 66%

Ac0 OAc Ac0 OAc H2, Pd/C
Ac0 0, 01 µ '10 Me0H, 95 k 100 0 AcHN 0 OH
Ac0 OAc HBTU, DMF, EtN(iP02 Phosphitylation __________________________________________________________________________ ).-Ac0 'D'H7 81%
DMTO '10 AcHN 0 NCR. ODMT
b1H

Hd 47 NC

/
p¨P
Ac0 OAc N(iP02 ....Ø0.4, Ac0 IIIR
C)tiVµ10 AcHN 0 ODMT

Compound 156 was synthesized following the procedure described in the literature (J. Med. Chem.
2004, 47, 5798-5808).
Compound 156, (18.60 g, 29.28 mmol) was dissolved in methanol (200 mL).
Palladium on carbon (6.15 g, 10 wt%, loading (dry basis), matrix carbon powder, wet) was added.
The reaction mixture was stirred at room temperature under hydrogen for 18 h. The reaction mixture was filtered through a pad of celite and the celite pad was washed thoroughly with methanol. The combined filtrate was washed and concentrated to dryness. The residue was purified by silica gel column chromatography and eluted with 5-10 % methanol in dichloromethane to yield Compound 157 (14.26 g, 89%). Mass m/z 544.1 [M-H].
Compound 157 (5 g, 9.17 mmol) was dissolved in anhydrous DMF (30 mL). HBTU
(3.65 g, 9.61 mmol) and N,N-Diisopropylethylamine (13.73 mL, 78.81 mmol) were added and the reaction mixture was stirred at room temperature for 5 minutes. To this a solution of compound 47 (2.96 g, 7.04 mmol) was added.
The reaction was stirred at room temperature for 8 h. The reaction mixture was poured into a saturated NaHCO3 aqueous solution. The mixture was extracted with ethyl acetate and the organic layer was washed with brine and dried (Na2SO4), filtered and evaporated. The residue obtained was purified by silica gel column chromatography and eluted with 50% ethyl acetate in hexane to yield compound 158 (8.25g, 73.3%).
The structure was confirmed by MS and 1H NMR analysis.
Compound 158 (7.2 g, 7.61 mmol) was dried over P205 under reduced pressure.
The dried compound was dissolved in anhydrous DMF (50 mL). To this 1H-tetrazole (0.43 g, 6.09 mmol) and N-methylimidazole (0.3 mL, 3.81 mmol) and 2-cyanoethyl-N,N,/VV\P-tetraisopropyl phosphorodiamidite (3.65 mL, 11.50 mmol) were added. The reaction mixture was stirred t under an argon atmosphere for 4 h. The reaction mixture was diluted with ethyl acetate (200 mL). The reaction mixture was washed with saturated NaHCO3 and brine. The organic phase was separated, dried (Na2SO4), filtered and evaporated. The residue was purified by silica gel column chromatography and eluted with 50-90 % ethyl acetate in hexane to yield Compound 159 (7.82 g, 80.5%). The structure was confirmed by LCMS and 31P NMR
analysis.
pH
HOOH

AcHN
0=P¨OH
O
1 DNA synthesizer HOOH
159 _______________ 2 aq NH4OH Ho00 AcHN
0=P¨OH

HOOH
_______________________________________________________ OLIGO
AcHN

Oligomeric Compound 160, comprising a Ga1NAc3-9 conjugate group, was prepared using standard oligonucleotide synthesis procedures. Three units of compound 159 were coupled to the solid support, followed by nucleotide phosphoramidites. Treatment of the protected oligomeric compound with aqueous ammonia yielded compound 160. The Ga1NAc3 cluster portion of the conjugate group Ga1NAc3-9 (Ga1NAc3-9a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is -P(=0)(OH)-Ad-P(=0)(OH)-. The structure of Ga1NAc3-9 (Ga1NAc3-9a-CM) is shown below:
pH
HOOH
NR.
HO

AcHN
0=P¨OH
HO OH
HO0or AcHN
0=P¨OH

HOOH
HO
AcHN
=

Example 53: Alternate procedure for preparation of Compound 18 (Ga1NAc3-la and Ga1NAc3-3a) A0 H2NNHR H TMSOTf ________________________________ ,... HO NNHR _________________ ,...
\) R = H or Cbz OAc 0 OAc 161 I¨ R = H 162a ...7...C.pl..
CbzCI, Et3N i_x_ R = Cb' z , 162b Ac0 4 Ny) PFPO
OAc)1--------\
H
0 ..õ.v¨ ¨0,--Ac0 ,,n ,.....r.N NHR + PFPOC) NHCBZ
NHAc 0 0 0' R = Cbz, 163a K) Pd/C, H2 I¨ PFPO
= H, 163b OAc Ogv...... 0 Ac0 0 J.L
H
NHAc " IIINN)7.-------1 OAc OAc 0 0-, H
Ac0---/)-- Ji ___________________ NNH
I0õ,..¨NHCBZ
zi , NHAc (:) ?I 10 OAc OAc HNN)4---7 Ac0 NHAc Lactone 161 was reacted with diamino propane (3-5 eq) or Mono-Boc protected diamino propane (1 eq) to provide alcohol 162a or 162b. When unprotected propanediamine was used for the above reaction, the excess diamine was removed by evaporation under high vacuum and the free amino group in 162a was protected using CbzCl to provide 162b as a white solid after purification by column chromatography.
Alcohol 162b was further reacted with compound 4 in the presence of TMSOTf to provide 163a which was converted to 163b by removal of the Cbz group using catalytic hydrogenation.
The pentafluorophenyl (PFP) ester 164 was prepared by reacting triacid 113 (see Example 48) with PFPTFA
(3.5 eq) and pyridine (3.5 eq) in DMF (0.1 to 0.5 M). The triester 164 was directly reacted with the amine 163b (3-4 eq) and DIPEA (3-4 eq) to provide Compound 18. The above method greatly facilitates purification of intermediates and minimizes the formation of byproducts which are formed using the procedure described in Example 4.

Example 54: Alternate procedure for preparation of Compound 18 (GaINAc3-la and Ga1NAc3-3a) HO2C7Th PFPTFA PFPO
0, DMF, pyr 0 C), ,-........7v ________________ NHCBZ ___________ ,. PFP0-..{r0......... NHCBZ
-, r Or 0 Ott 0 HO2C) PFPO

BocHN N

BocHN NH2 H 1. HCI or TFA
____________________ ,-DIPEA BocHN N y---,r --,.V __ NHCBZ ______________ ,..
,r 2.

0.. ingc.7....... 0 0 o BocHNN)/ Ac0 -4.LI OPFF
H
165 NHAc OAc 166 0.. ing_v_____. 0 0 0, ,,,I.,L 1. 1,6-hexanediol Ac0 H or 1,5-pentane-diol NHAc '-) IIINN),r-----,\ TMSOTf + compound 4 OAc 2. TEMPO
0.. ing 0 0 0, 3. PFPTFA, pyr H H
_______________________ NNI.r--......, -----..V __ NHCBZ
M4 r NHAc o 0 0 OAc K
HNN) OAc Ac0 NHAc The triPFP ester 164 was prepared from acid 113 using the procedure outlined in example 53 above and reacted with mono-Boc protected diamine to provide 165 in essentially quantitative yield. The Boc groups were removed with hydrochloric acid or trifluoroacetic acid to provide the triamine which was reacted with the PFP activated acid 166 in the presence of a suitable base such as DIPEA to provide Compound 18.
The PFP protected Gal-NAc acid 166 was prepared from the corresponding acid by treatment with PFPTFA (1-1.2 eq) and pyridine (1-1.2 eq) in DMF. The precursor acid in turn was prepared from the corresponding alcohol by oxidation using TEMPO (0.2 eq) and BAIB in acetonitrile and water. The precursor alcohol was prepared from sugar intermediate 4 by reaction with 1,6-hexanediol (or 1,5-pentanediol or other diol for other n values) (2-4 eq) and TMSOTf using conditions described previously in example 47.

Example 55: Dose-dependent study of oligonucleotides comprising either a 3' or 5'-conjugate group (comparison of Ga1NAc3-1, 3, 8 and 9) targeting SRB-1 in vivo The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice. Unconjugated ISIS 353382 was included as a standard. Each of the various Ga1NAc3 conjugate groups was attached at either the 3' or 5' terminus of the respective oligonucleotide by a phosphodiester linked 2'-deoxyadenosine nucleoside (cleavable moiety).
Table 39 Modified ASO targeting SRB-1 SEQ
ASO Sequence (5 to 3') Motif Conjugate ID No.
ISIS 353382 GesmCesTesTesmCesAdsGdsTdsmCdsAdsTdsGdsAds 5/10/5 none (parent) mCdaTdaTeamCeamCeaTeaTe G mC T T mC Ad Gd IT,' Ad I'd Gd ISIS 655861 es eseses essss sssss 5/10/5 Ga1NAc3-1 mCds'r ds'f esmCesmCesT esTeoAdo¨GaINAC3¨la Ges es es es es s s mC T T mC Ad Gd s IT,'s s Ad I'ds Gds s ISIS 664078 5/10/5 Ga1NAc3-9 mC m daTdsTesC m esCesTesTeoAdo¨es-9a Ga1NAc3-3a-o'Ado ISIS 661161 GeamCeaTeaTeamCesAdaGasTasmCdsAdsTasGasAds 5/10/5 Ga1NAc3-3 145 mCdaTdaTeamCesmCesTesTe Ga1NAC3-8a¨o'Ado ISIS 665001 GeamCeaTeaTeamCesAdaGdaTasmCdsAdsTasGasAds 5/10/5 Ga1NAc3-8 145 mCdaTdaTeamCesmCesTesTe Capital letters indicate the nucleobase for each nucleoside and mC indicates a 5-methyl cytosine.
Subscripts: "e" indicates a 2'-MOE modified nucleoside; "d" indicates a [3-D-2'-deoxyribonuc1eoside; "s"
indicates a phosphorothioate internucleoside linkage (PS); "o" indicates a phosphodiester internucleoside linkage (PO); and "o" indicates -0-P(=0)(OH)-. Conjugate groups are in bold.
The structure of Ga1NAc3-1a was shown previously in Example 9. The structure of Ga1NAc3-9 was shown previously in Example 52. The structure of Ga1NAc3-3 was shown previously in Example 39. The structure of Ga1NAc3-8 was shown previously in Example 47.
Treatment Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, ME) were injected subcutaneously once at the dosage shown below with ISIS 353382, 655861, 664078, 661161, 665001 or with saline. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration to determine the liver SRB-1 mRNA levels using real-time PCR and RIBOGREENO
RNA quantification reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols.
The results below are presented as the average percent of SRB-1 mRNA levels for each treatment group, normalized to the saline control.

As illustrated in Table 40, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner. Indeed, the antisense oligonucleotides comprising the phosphodiester linked Ga1NAc3-1 and Ga1NAc3-9 conjugates at the 3' terminus (ISIS 655861 and ISIS
664078) and the Ga1NAc3-3 and Ga1NAc3-8 conjugates linked at the 5' terminus (ISIS 661161 and ISIS
665001) showed substantial improvement in potency compared to the unconjugated antisense oligonucleotide (ISIS 353382).
Furthermore, ISIS 664078, comprising a Ga1NAc3-9 conjugate at the 3' terminus was essentially equipotent compared to ISIS 655861, which comprises a Ga1NAc3-1 conjugate at the 3' terminus. The 5' conjugated antisense oligonucleotides, ISIS 661161 and ISIS 665001, comprising a Ga1NAc3-3 or Ga1NAc3-9, respectively, had increased potency compared to the 3' conjugated antisense oligonucleotides (ISIS 655861 and ISIS 664078).
Table 40 ASOs containing Ga1NAc3-1, 3, 8 or 9 targeting SRB-1 Dosage SRB-1 mRNA
ISIS No.Conj ug ate (mg/kg) (`)/0 Saline) Saline n/a 100 353382 10 68 none 0.5 98 1.5 76 655861 GalNac3 -1 (3') 0.5 88 1.5 85 664078 GalNac3-9 (3') 0.5 92 1.5 59 661161 GalNac3-3 (5') 0.5 100 1.5 73 665001 GalNac3-8 (5') Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in 15 serum were measured relative to saline injected mice using standard protocols. Total bilirubin and BUN were also evaluated. The change in body weights was evaluated with no significant change from the saline group.
ALTs, ASTs, total bilirubin and BUN values are shown in the table below.

Table 41 Dosage Total ISIS No. ALT AST BUN Conjugate mg/kg Bilirubin Saline 24 59 0.1 37.52 3 21 66 0.2 34.65 353382 10 22 54 0.2 34.2 none 30 22 49 0.2 33.72 0.5 25 62 0.2 30.65 1.5 23 48 0.2 30.97 655861 GalNac3-1 (3') 28 49 0.1 32.92 40 97 0.1 31.62 0.5 40 74 0.1 35.3 1.5 47 104 0.1 32.75 664078 GalNac3-9 (3') 5 20 43 0.1 30.62 15 38 92 0.1 26.2 0.5 101 162 0.1 34.17 1.5 g 42 100 0.1 33.37 661161 GalNac3-3 (5') 5 g 23 99 0.1 34.97 15 53 83 0.1 34.8 0.5 28 54 0.1 31.32 1.5 42 75 0.1 32.32 665001 GalNac3-8 (5') 5 24 42 0.1 31.85 15 32 67 0.1 31.
Example 56: Dose-dependent study of oligonucleotides comprising either a 3' or 5'-conjugate group 5 (comparison of Ga1NAc3-1, 2, 3, 5, 6, 7 and 10) targeting SRB-1 in vivo The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice. Unconjugated ISIS 353382 was included as a standard. Each of the various Ga1NAc3 conjugate groups was attached at the 5' terminus of the respective oligonucleotide by a phosphodiester linked 2'-deoxyadenosine nucleoside (cleavable moiety) except for ISIS 655861 which had the Ga1NAc3 conjugate 10 group attached at the 3' terminus.
Table 42 Modified ASO targeting SRB-1 SEQ
ASO Sequence (5' to 3') Motif Conjugate ID No.

GesmCesTesTesmCesAdsGasTasmCdsAdsTasGasAds 5/10/5 no conjugate 143 (parent) mCdsTdsTesmCesmCesTesTe G mC T T mC Ad Gd Td mCd Ad I'd Gd Ad ISIS 655861 es eseses essss sssss 5/10/5 Ga1NAc3-1 mCdsTdsrresmCesmCesTesTecAdo,-GaINAC3-1a GalNAC3-2a-0,AdoGesmCesTesTesmCesAdsGdsTas 5/10/5 ISIS 664507 Ga1NAc3-2 145 mCdsAdsTdsGdsAdsmCdsTdsTesmCesmCesTesTe Ga1NAc3-3a-o'Ado GesmCesTesTesmCesAdsGdsTdsmCdsAdsTdsGdsAds 5/10/5 Ga1NAc3-3 145 mCdsTdsTesmCesmCesTesTe ISIS 666224 Ga1NAc3-5a-0,AdoGesmCesTesTesmCesAdsGdsTds 5/10/5 Ga1NAc3-5 145 mCdsAdsTdsGdsAdsmCdsTasTesmCesmCesTesTe GalNAc3-6.-0,AdoGesmCesTesTesmCesAdsGasTas ISIS 666961 5/10/5 Ga1NAc3-6 145 mCdaAdaTdaGdaAdamCdaTasTesmCesmCesTesTe GalNAc3-7.-0,AdoGesmCesTesTesmCesAdsGasTas 5/10/5 ISIS 666981 Ga1NAc3-7 145 mCdaAdaTdaGdaAdamCdaTasTesmCesmCesTesTe GalNAc3-10.-0,AdoGesmCesTesTesmCesAdsGasTas 5/10/5 ISIS 666881 Ga1NAc3-10 145 mCdaAdaTdaGdaAdamCdaTasTesmCesmCesTesTe Capital letters indicate the nucleobase for each nucleoside and mC indicates a 5-methyl cytosine.
Subscripts: "e" indicates a 2'-MOE modified nucleoside; "d" indicates a [3-D-2'-deoxyribonuc1eoside; "s"
indicates a phosphorothioate internucleoside linkage (PS); "o" indicates a phosphodiester internucleoside linkage (PO); and "o" indicates -0-P(=0)(OH)-. Conjugate groups are in bold.
The structure of Ga1NAc3-1a was shown previously in Example 9. The structure of Ga1NAc3-2a was shown previously in Example 37. The structure of Ga1NAc3-3a was shown previously in Example 39. The structure of Ga1NAc3-5a was shown previously in Example 49. The structure of Ga1NAc3-6a was shown previously in Example 51. The structure of Ga1NAc3-7a was shown previously in Example 48. The structure of GalNAc3-10a was shown previously in Example 46.
Treatment Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, ME) were injected subcutaneously once at the dosage shown below with ISIS 353382, 655861, 664507, 661161, 666224, 666961, 666981, 666881 or with saline. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration to determine the liver SRB-1 mRNA levels using real-time PCR and RIBOGREENO RNA quantification reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols. The results below are presented as the average percent of SRB-1 mRNA levels for each treatment group, normalized to the saline control.
As illustrated in Table 43, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner.
Indeed, the conjugated antisense oligonucleotides showed substantial improvement in potency compared to the unconjugated antisense oligonucleotide (ISIS 353382). The 5' conjugated antisense oligonucleotides showed a slight increase in potency compared to the 3' conjugated antisense oligonucleotide.
Table 43 Dosage SRB-1 mRNA
ISIS No.Conjugate (mg/kg) (% Saline) Saline n/a 100.0 3 96.0 353382 10 73.1 none 36.1 655861 0.5 99.4 GalNac3-1 (3') 1.5 81.2 33.9 15.2 0.5 102.0 1.5 73.2 664507 GalNac3-2 (5') 5 31.3 15 10.8 0.5 90.7 1.5 67.6 661161 GalNac3-3 (5') 5 24.3 15 11.5 0.5 96.1 1.5 61.6 666224 GalNac3-5 (5') 5 25.6 15 11.7 0.5 85.5 1.5 56.3 666961 Ga1NAc3-6 (5') 5 34.2 15 13.1 0.5 84.7 1.5 59.9 666981 Ga1NAc3-7 (5') 5 24.9 15 8.5 0.5 100.0 1.5 65.8 666881 Ga1NAc3-10 (5') 5 26.0 15 13.0 Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols.
Total bilirubin and BUN were also evaluated. The change in body weights was evaluated with no significant change from the saline group.
5 ALTs, ASTs, total bilirubin and BUN values are shown in Table 44 below.
Table 44 Dosage Total ISIS No. ALT ASTBUN Conjugate mg/kg Bilirubin Saline 26 57 0.2 27 3 25 92 0.2 27 353382 10 23 40 0.2 25 none 30 29 54 0.1 28 0.5 25 71 0.2 34 1.5 28 60 0.2 26 655861 GalNac3-1 (3') 5 26 63 0.2 28 15 25 61 0.2 28 0.5 25 62 0.2 25 1.5 24 49 0.2 26 664507 GalNac3-2 (5') 5 21 50 0.2 26 15 59 84 0.1 22 0.5 20 42 0.2 29 1.5 g 37 74 0.2 25 661161 GalNac3-3 (5') g 28 61 0.2 29 21 41 0.2 25 0.5 34 48 0.2 21 1.5 23 46 0.2 26 666224 GalNac3-5 (5') 5 24 47 0.2 23 15 32 49 0.1 26 0.5 17 63 0.2 26 1.5 23 68 0.2 26 666961 Ga1NAc3-6 (5') 5 25 66 0.2 26 15 29 107 0.2 28 0.5 24 48 0.2 26 1.5 30 55 0.2 24 666981 Ga1NAc3-7 (5') 5 46 74 0.1 24 15 29 58 0.1 26 0.5 20 65 0.2 27 1.5 23 59 0.2 24 666881 Ga1NAc3-1 0 (5') 5 45 70 0.2 26 15 21 57 0.2 24 Example 57: Duration of action study of oligonucleotides comprising a 3'-conjugate group targeting ApoC III in vivo Mice were injected once with the doses indicated below and monitored over the course of 42 days for ApoC-III and plasma triglycerides (Plasma TG) levels. The study was performed using 3 transgenic mice that express human APOC-III in each group.
Table 45 Modified ASO targeting ApoC III
ASO Sequence (5' to 3') Linkages SEQ ID
No.
ISIS AesGesmCesTesTesmCdsTdsTdsGdsTds PS 135 304801 mCdsmCdsAdsGdsmCdsTeaTeaTesAes're ISIS
ikesGesmCesTesTesmCdsTdsTdsGdsTdsmCdsmCds PS 136 647535 AdsGdsmCdsTesTesTesAesTeoAdo,-GaINAC3-la ISIS AesGeomCeoTeoTeomCdsTdsTdsGdsTdsmCdsmCds PO/PS

647536 AdaGdamCdaTeoTeorresAesTeoAdo-GalNAc3-la Capital letters indicate the nucleobase for each nucleoside and mC indicates a 5-methyl cytosine.
Subscripts: "e" indicates a 2'-MOE modified nucleoside; "d" indicates a [3-D-2'-deoxyribonuc1eoside; "s"
indicates a phosphorothioate internucleoside linkage (PS); "o" indicates a phosphodiester internucleoside linkage (PO); and "o" indicates -0-P(=0)(OH)-. Conjugate groups are in bold.
The structure of Ga1NAc3-11 was shown previously in Example 9.

Table 46 ApoC III mRNA ( /0 Saline on Day 1) and Plasma TG Levels ( /0 Saline on Day 1) ASO Dose Target Day 3 Day 7 Day 14 Day 35 Day 42 Saline 0 mg/kg ApoC-III 98 100 100 95 ISIS 304801 30 mg/kg ApoC-III 28 30 41 65 74 ISIS 647535 10 mg/kg ApoC-III 16 19 25 74 94 ISIS 647536 10 mg/kg ApoC-III 18 16 17 35 51 Saline 0 mg/kg Plasma TG 121 130 123 105 ISIS 304801 30 mg/kg Plasma TG 34 37 50 69 69 ISIS 647535 10 mg/kg Plasma TG 18 14 24 18 71 ISIS 647536 10 mg/kg Plasma TG 21 19 15 32 35 As can be seen in the table above the duration of action increased with addition of the 3'-conjugate group compared to the unconjugated oligonucleotide. There was a further increase in the duration of action for the conjugated mixed PO/PS oligonucleotide 647536 as compared to the conjugated full PS
oligonucleotide 647535.
Example 58: Dose-dependent study of oligonucleotides comprising a 3'-conjugate group (comparison of Ga1NAc3-1 and Ga1NAc4-11) targeting SRB-1 in vivo The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice. Unconjugated ISIS 440762 was included as an unconjugated standard. Each of the conjugate groups were attached at the 3' terminus of the respective oligonucleotide by a phosphodiester linked 2'-deoxyadenosine nucleoside cleavable moiety.
The structure of Ga1NAc3-la was shown previously in Example 9. The structure of Ga1NAc3-11a was shown previously in Example 50.
Treatment Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, ME) were injected subcutaneously once at the dosage shown below with ISIS 440762, 651900, 663748 or with saline. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration to determine the liver SRB-1 mRNA levels using real-time PCR and RIBOGREENO RNA quantification reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols. The results below are presented as the average percent of SRB-1 mRNA levels for each treatment group, normalized to the saline control.
As illustrated in Table 47, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner. The antisense oligonucleotides comprising the phosphodiester linked GalNAc3-1 and Ga1NAc4-11 conjugates at the 3' terminus (ISIS 651900 and ISIS 663748) showed substantial improvement in potency compared to the unconjugated antisense oligonucleotide (ISIS 440762). The two conjugated oligonucleotides, GalNAc3-1 and Ga1NAc4-11, were equipotent.
Table 47 Modified ASO targeting SRB-1 % Saline SEQ ID
ASO Sequence (5 to 3') Dose mg/kg control No.
Saline 100 0.6 73.45 IlsmCksAdsGdsTdsmCdsAdsrrdsGdsAds 2 59.66 ISIS 440762 mCdsTdsrllsmCk 6 23.50 0.2 62.75 TksmCksAdsGasTasmCdSAdSrrdSGdSAdS 0.6 29.14 mCdsTdsrllsmCkoAdo,-GalNAC3-19 2 8.61 6 5.62 0.2 63.99 TksmCksAdsGdsrrdsmCdsAdsrrdsGdsAds 0.6 33.53 mCdsTdsTIsmCkoAdo-GallsTAC4-1 1 a 2 7.58 6 5.52 Capital letters indicate the nucleobase for each nucleoside and mC indicates a 5-methyl cytosine.
Subscripts: "e" indicates a 2'-MOE modified nucleoside; "k" indicates 6'-(S)-CH3 bicyclic nucleoside; "d"
indicates a 13-D-2'-deoxyribonuc1eoside; "s" indicates a phosphorothioate internucleoside linkage (PS); "o"
indicates a phosphodiester internucleoside linkage (PO); and "o- indicates -0-P(=0)(OH)-. Conjugate groups are in bold.
Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols.
Total bilirubin and BUN were also evaluated. The change in body weights was evaluated with no significant change from the saline group.
ALTs, ASTs, total bilirubin and BUN values are shown in Table 48 below.
Table 48 Dosage Total ISIS No. ALT AST BUN Conjugate mg/kg Bilirubin Saline 30 76 0.2 40 0.60 32 70 0.1 35 440762 2 26 57 0.1 35 none 6 31 48 0.1 39 0.2 32 115 0.2 39 0.6 33 61 0.1 35 651900 Ga1Nac3-1 (3') 2 30 50 0.1 37 6 34 52 0.1 36 0.2 28 56 0.2 36 663748 0.6 34 60 0.1 35 GalNac4-11 (3') 2 44 62 0.1 36 6 38 71 0.1 33 Example 59: Effects of GaINAc3-1 conjugated ASOs targeting FXI in vivo The oligonucleotides listed below were tested in a multiple dose study for antisense inhibition of FXI
in mice. ISIS 404071 was included as an unconjugated standard. Each of the conjugate groups was attached at the 3' terminus of the respective oligonucleotide by a phosphodiester linked 2'-deoxyadenosine nucleoside cleavable moiety.
Table 49 Modified ASOs targeting FXI
SEQ ID
ASO Sequence (5' to 3') Linkages No.
ISIS TeaGesGesTesAesAdsTdamCdamCdaAdamCds 404071 TdsTdsTdsmCdsAesGesAesGesGe ISIS TesGesGesTesAesAdsTasmCdsmCdsAdsmCds PS 147 656172 TdsTdsTdsmCdsAesGesAesGesGeoAdo,-Ga1NAc3-1a ISIS TesGeoGeorreoAeoAdsTdsmCdsmCdsAdsmCds 656173 TdsTdsTdsmCdsAeoGeoAesGesGeoAdo,-Ga1NAC3-1a Capital letters indicate the nucleobase for each nucleoside and mC indicates a 5-methyl cytosine.
Subscripts: "e" indicates a 2'-MOE modified nucleoside; "d" indicates a [3-D-2'-deoxyribonuc1eoside; "s"
indicates a phosphorothioate internucleoside linkage (PS); "o" indicates a phosphodiester internucleoside linkage (PO); and "o" indicates -0-P(=0)(OH)-. Conjugate groups are in bold.
The structure of Ga1NAc3-1a was shown previously in Example 9.
Treatment Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, ME) were injected subcutaneously twice a week for 3 weeks at the dosage shown below with ISIS 404071, 656172, 656173 or with PBS treated control. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration to determine the liver FXI mRNA levels using real-time PCR and RIBOGREENO RNA
quantification reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols. Plasma FXI
protein levels were also measured using ELISA. FXI mRNA levels were determined relative to total RNA
(using RIBOGREENO), prior to normalization to PBS-treated control. The results below are presented as the average percent of FXI mRNA levels for each treatment group. The data was normalized to PBS-treated control and is denoted as "% PBS". The ED50s were measured using similar methods as described previously and are presented below.
Table 50 Factor XI mRNA (% Saline) Dose ASO % Control Conjugate Linkages mg/kg Saline 100 none 404071 10 40 none PS

SIS 0.7 74 I
656172 2 33 GaINAc3-1 PS

SIS 0.7 49 I
656173 2 22 GaINAc3-1 PO/PS

As illustrated in Table 50, treatment with antisense oligonucleotides lowered FXI mRNA levels in a dose-dependent manner.
The oligonucleotides comprising a 3'-Ga1NAc3-1 conjugate group showed substantial improvement in potency compared to the unconjugated antisense oligonucleotide (ISIS 404071).
Between the two conjugated oligonucleotides an improvement in potency was further provided by substituting some of the PS linkages with PO (ISIS 656173).
As illustrated in Table 50a, treatment with antisense oligonucleotides lowered FXI protein levels in a dose-dependent manner.
The oligonucleotides comprising a 3'-Ga1NAc3-1 conjugate group showed substantial improvement in potency compared to the unconjugated antisense oligonucleotide (ISIS 404071).
Between the two conjugated oligonucleotides an improvement in potency was further provided by substituting some of the PS linkages with PO (ISIS 656173).
Table 50a Factor XI protein (% Saline) Dose Protein (%
ASO Conjugate Linkages mg/kg Control) Saline 100 none 404071 10 32 none PS

0.7 ISIS
656172 2 23 Ga1NAc3-1 PS

0.7 ISIS
656173 2 6 Ga1NAc3-1 PO/PS

Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols.
Total bilirubin, total albumin, CRE and BUN were also evaluated. The change in body weights was evaluated with no significant change from the saline group. ALTs, ASTs, total bilirubin and BUN values are shown in the table below.

Table 51 Dosage Total Total ISIS No. ALT AST CRE BUN
Conjugate mg/kg Albumin Bilirubin Saline 71.8 84.0 3.1 0.2 0.2 22.9 3 152.8 176.0 3.1 0.3 0.2 23.0 404071 10 73.3 121.5 3.0 0.2 0.2 21.4 none 30 82.5 92.3 3.0 0.2 0.2 23.0 0.7 62.5 111.5 3.1 0.2 0.2 23.8 656172 2 33.0 51.8 2.9 0.2 0.2 22.0 GalNac3-1 (3') 6 65.0 71.5 3.2 0.2 0.2 23.9 0.7 54.8 90.5 3.0 0.2 0.2 24.9 656173 2 85.8 71.5 3.2 0.2 0.2 21.0 GalNac3-1 (3') 6 114.0 101.8 3.3 0.2 0.2 22.7 Example 60: Effects of conjugated ASOs targeting SRB-1 in vitro The oligonucleotides listed below were tested in a multiple dose study for antisense inhibition of SRB-1 in primary mouse hepatocytes. ISIS 353382 was included as an unconjugated standard. Each of the conjugate groups were attached at the 3' or 5' terminus of the respective oligonucleotide by a phosphodiester linked 2'-deoxyadenosine nucleoside cleavable moiety.
Table 52 Modified ASO targeting SRB-1 SEQ
ASO Sequence (5' to 3') Motif Conjugate ID No.
GesdmsCdessTeessTe,smsCesAesd eGsd eTd mCd Ad Ta Gd Ad s 5/10/5 none 143ISIS 353382m,rrm,mCTT
GesmCesTesTesmCesAdsGdsTdsmCdsAdsTdsGdsAds ISIS 655861 5/10/5 GalNAc3-1 144 mCdsTdsTesmCesmCesTesTeoAGalNAC3-1 a GesdmsCdesoTeeooTeoemoCeoeAsde sGde oTdd mo,C-d Ad Td G3-d Ad s 5/10/5 Ga1NAc3-1 144ISIS 655862mCTTmCmCTTAGa1NAC1 a GalNAc3-3a-0,AdoGesmCesTesTesmCesAdsGds ISIS 661161 5/10/5 Ga1NAc3-3 145 TdsmCdsAdsTdsGdsAdsmCdsTdsTesmCesmCesTesTe GalNAC3-8a-o'AdoGesmCesTesTesmCesAdsGds ISIS 665001 5/10/5 Ga1NAc3-8 145 TdsmCdsAdsTdsGdsAdsmCdsTdsTesmCesmCesTesTe GesmCesTesTesmCesAdsGdsTdsmCdsAdsTdsGdsAds ISIS 664078 5/10/5 GalNAc3-9 144 mCdsTdsTesmCesmCesTesTeoAGalNAC3-9a GalNAC3-6a-o'AdoGesmCesTesTesmCesAdsGds ISIS 666961 5/10/5 Ga1NAc3-6 TdsmCdsAdsTdsGdsAdsmCdsTdsTesmCesmCesTesTe Gad1 sNAdsC3d-s2a-ds0,AddsoGedssmCdsesTesesTeessmCeessAde sGed Td s 5/10/5 Ga1NAc3-2 145ISIS 664507mCATGAmCTTmCmCTT
Ga1NAC3-10a-0,AdoGesmCesTesTesmCesAd Gd Td s 5/10/5 Ga1NAc3-10 145ISIS 666881mCdsAdsTdsGdsAdsmCdsTdsTesmCesmCesTesTe Gad1 sNAdsC3d-s5a-ds0,AddsoGedssmCdsesTesesTeessmCeessAde sGed Td s 5/10/5 Ga1NAc3-5 145ISIS 666224mCATGAmCTTmCmCTT
Gad1 sNAdsC3d-s7a-ds0,AddsoGedssmCdsesTesesTeessmCeessAde sGed Td s 5/10/5 Ga1NAc3-7 145ISIS 666981mCATGAmCTTmCmCTT

Capital letters indicate the nucleobase for each nucleoside and niC indicates a 5-methyl cytosine.
Subscripts: "e" indicates a 2'-MOE modified nucleoside; "d" indicates a [3-D-2'-deoxyribonuc1eoside; "s"
indicates a phosphorothioate internucleoside linkage (PS); "o" indicates a phosphodiester internucleoside linkage (PO); and "o" indicates -0-P(=0)(OH)-. Conjugate groups are in bold.
The structure of Ga1NAc3-la was shown previously in Example 9. The structure of Ga1NAc3-3a was shown previously in Example 39. The structure of Ga1NAc3-8a was shown previously in Example 47. The structure of Ga1NAc3-9a was shown previously in Example 52. The structure of Ga1NAc3-6a was shown previously in Example 51. The structure of GalNAc3-2a was shown previously in Example 37. The structure of Ga1NAc3-10a was shown previously in Example 46. The structure of Ga1NAc3-5a was shown previously in Example 49. The structure of Ga1NAc3-7a was shown previously in Example 48.
Treatment The oligonucleotides listed above were tested in vitro in primary mouse hepatocyte cells plated at a density of 25,000 cells per well and treated with 0.03, 0.08, 0.24, 0.74, 2.22, 6.67 or 20 nM modified oligonucleotide. After a treatment period of approximately 16 hours, RNA was isolated from the cells and mRNA levels were measured by quantitative real-time PCR and the SRB-1 mRNA
levels were adjusted according to total RNA content, as measured by RIBOGREENO.
The 1050 was calculated using standard methods and the results are presented in Table 53. The results show that, under free uptake conditions in which no reagents or electroporation techniques are used to artificially promote entry of the oligonucleotides into cells, the oligonucleotides comprising a GalNAc conjugate were significantly more potent in hepatocytes than the parent oligonucleotide (ISIS 353382) that does not comprise a GalNAc conjugate.
Table 53 Internucleoside SEQ ID
ASO IC50 (nM) Conjugate linkages No.
ISIS 353382 190a PS none 143 ISIS 655861 1 la PS Ga1NAc3-1 ISIS 655862 3 PO/PS Ga1NAc3-1 144 ISIS 661161 15a PS Ga1NAc3-3 ISIS 665001 20 PS Ga1NAc3-8 145 ISIS 664078 55 PS Ga1NAc3-9 144 ISIS 666961 22a PS Ga1NAc3-6 145 ISIS 664507 30 PS Ga1NAc3-2 145 ISIS 666881 30 PS Ga1NAc3-10 145 ISIS 666224 30a PS Ga1NAc3-5 145 ISIS 666981 40 PS Ga1NAc3-7 145 aAverage of multiple runs.
Example 61: Preparation of oligomeric compound 175 comprising GaINAc3-12 Ac0 0 OAc Bo c , N N H2 Ac0NOAc HN
,)-C) /...C240Ac 91a ________ B oc , --lcõ..--__NO 0 Pfp0 N N
NAc H H
OAc HN , Ac HOOC
H ) N
0 OAc Ac0 CBz,N \¨COOH
COOH

OAc ________________________________________________________________ H
DC M HN
"Ac HBTU DIEA DMF

Ac0 OAc )r HN ¨Ac HN

}¨ N ---/---j 101 Ac0 0 y ENI N 1N \ C) 0 oAc 1.
.......--..,N ).-----0 ,_,,,=\ N "Ac OAc H H H
HN
0 u ----\----A 0 HN Ac0 :)Ac OAc "Ac Ac0 OAc 0 o j21___OAc )1' __ HN Ac HN
Pd(OH)2/C, H2 0 H
}¨N --7-----/
Me0H/Et0Ac _)=._ Ac0 m 0 0 Nz..7.0Ac k ,\ N N OAc u HN H H
HN
,-, "Ac----\-----\ 0 HN Ac0 \z_ii)Ac OAc "Ac F
F

F
benzyl (perfluorophenyl) glutarate _______________________________ Om-DMF
A,c0.1õ.1)Ac )0 OAc HN HN....Ac ¨N¨/--/
O
I
Ac0 0 EN- I N \ ?I 0 OAc \NN)0 0 0 0 r, \ OAc µ-' HN--1; 0 H
HN Ac HN AcOil OAc \/
OAc HN
"Ac Ac0 OAc K0 0,..2-0Ac ,,Ac HN

Pd(OH)2 i C , H2 },.- N--/-----/
172 __________ >
H m 0 0 Ac0 )Ac T
Me0H / Et0Ac HO N
0 0 ,-,\ \ N N
OAc H
HN'Ac HN Ac0 1:1:)Ac 0\ /0 OAc 173 HN, Ac Ac0 OAc PFP-TFA
0 0,21.__OAc DIEA DMF
HN)LVy HN --Ac OH
},¨N --.7----/
F F
HH Ac0 OAc 0 Ni_i F ii 0 N,,,N, ;:).

N
F F ' 0 ,_, u ,\ N
OAc HN--1-1 0 HN'Ac HN Ac0 1:1:)Ac \ /0 OAc 174 HN, Ac 83e 3'5') 11 F
( OLIGO O¨P-0¨(CH2)e¨NH2 OH
174 1. Borate buffer, DMSO, pH 8.5, rt ________________________________ yo.
2. aq. ammonia, rt OH OH
HO.r.C.:)..o 0 AcHN
NH

\-------\¨ENI\.,_-_¨__o HO..)\...,o 0 AcHN
--____Z------N)C--NN ___________________________ /,(7(Ei 0 =¨= ¨
N H H N--K cm __ OLIGO
N

ri 0 j--NH

HO
NHAc Compound 169 is commercially available. Compound 172 was prepared by addition of benzyl (perfluorophenyl) glutarate to compound 171. The benzyl (perfluorophenyl) glutarate was prepared by adding PFP-TFA and DIEA to 5-(benzyloxy)-5-oxopentanoic acid in DMF. Oligomeric compound 175, comprising a Ga1NAc3-12 conjugate group, was prepared from compound 174 using the general procedures illustrated in Example 46. The Ga1NAc3 cluster portion of the conjugate group Ga1NAc3-12 (Ga1NAc3-12a) can be combined with any cleavable moiety to provide a variety of conjugate groups.
In a certain embodiments, the cleavable moiety is -P(=0)(OH)-Ad-P(=0)(OH)-. The structure of GalNAc3-12 (Ga1NAc3-12a-CM-) is shown below:

OH OH
HO*,o 0 AcHN N-------NANH
01-0H \------\__H
HOo\__N__\_ N N
AcHN /0 H H ri __ (,.,.....,r ENi0 En N
ri 0 0 _r_ri\--NH

HO,(7)....\./
HO
NHAc Example 62: Preparation of oligomeric compound 180 comprising Ga1NAc3-13 OAc OAc 0 \ , 0 Ac0----P__\_-0(OH + H 1.1 HATU, HOAt AcHN _A
__________________________________________________________________________ )IN-176 H2N- y N \V\V\v(:) H DIEA, DMF
O___¨ 0 V

OAc OAc Ac0--C--)-.\--ONc AcHN NH
OAc OAc H 0 H2, Pd/C
Ac0----!---1.--ON7NNO 401 AcHN
H H
0 y 0 /
OAc OAc HN

Ac0-----\:) .-0 AcHN 0 OAcr- OAc Ac0-....).1_-0.),c AcHN NH
OAcr- OAc O.

PFPTFA, TEA
Ac0---)...\---0,,,NThri\iNrOH _________________________________________ AcHN Ito-H H DMF

OAcc- OAc r 178 HN
Ac0-..:)...\_-0 AcHN 0 r-Ac akc AcOO
AcHN NH
r-Ac OAc j=L
AcHN NrC) F
r-Ac OAc AcOO
AcHN
83e 3' OLIGO FO-P-0-(CH2)6-NH2 OH
1. Borate buffer, DMSO, pH 8.5, rt _______________________________ Jo-2. aq. ammonia, rt HOO

AcHN NH
r..-H OH

AcHN cm ¨
OLIGO

011-1 r OH 180 HOO
HN
AcHN 0 Compound 176 was prepared using the general procedure shown in Example 2.
Oligomeric compound 180, comprising a Ga1NAc3-13 conjugate group, was prepared from compound 177 using the general procedures illustrated in Example 49. The Ga1NAc3 cluster portion of the conjugate group Ga1NAc3-13 (Ga1NAc3-130 can be combined with any cleavable moiety to provide a variety of conjugate groups. In a certainembodiments, the cleavable moiety is -P(=0)(OH)-Ad-P(=0)(OH)-. The structure of Ga1NAc3-13 (Ga1NAc3-13a-CM-) is shown below:
OH OH

H0*.\..._ -----LNH
AcHN
OH OH
HOr.C..D 0 (HO H 0 0-........---...õ.."..,õA N k AcHN H0 LLil 0 O( ..,.y.......z_ HOH
-10.\/0 HO
NHAc Example 63: Preparation of oligomeric compound 188 comprising Ga1NAc3-14 H OAc HOIn HON)-NH2 HO 6 N-N Ac01r) 0 0 6 H 0 OTh Ac0 HO 0.¨NHCBz 181 HO)'61\11 H¨NHCBz N \ 0 4 r 0 0 HBTU, DIEA 0 0 HOHO.( OAc OAc Ac0\_( _ AcO\ ( H H
N
Ac0 111110N-6NICI Ac0 Villikk. -0N-6 lin OAc NHAc 0 0 OAc NHAc 0 0 Ac0 , H AcO\ ( _ oNE11..r 0N--N 0NHCBz Pd/C, H2 Ac0 '6 Ac0 .Ø..4-N
H21111./ ' / 6 NHAc NHAc 0 0 0 OAc OAc HN4 Ac0 ,( __ 16 0 Ac00{)\---) 0 Ac0 6H
Ac0 NHAc NHAc 183 OAc Ac0 H
N
Ac0 06 In 0 H01..i0 el OAc NHAc 0 0 H 1. Pd/C, H2 2. PFP.TFA, pyr, Ac0 -N--6-N1c,--0,4-NI DMF

HBTU, NHAc 0 OAc DIEA, Ac000N)\----) 4.
DMF
Ac0 6H
NHAc OAc Ac0 H F
Ac0 ON-6N1n o F 0 F
\.,:,/0Ac Ac0 h NHAc µ o o 0 .---IL
0-1 N y(:)/}h 0 F
Ac0 / 6 F
NHAc 0 0 0 OAc AcOor.N)----) Ac0 % /6H
NHAc 83e H 0OH 1 11 4.v...A
( OLIG0J-0-P-0-(CH2)6-NH2 0 0 01H HO NI-1c vr_0-N I
1.r.--0 H
187 1. Borate buffer, DMSO, pH 8.5, rt HOZ-- ----1----V \ 76 H 6 CM
______ OLIGO ., ________________________ 0.- NHAc 0 0 0 )---) 2. aq. ammonia, rt HO OH\_( 0/ AN

NHAc Compounds 181 and 185 are commercially available. Oligomeric compound 188, comprising a Ga1NAc3-14 conjugate group, was prepared from compound 187 using the general procedures illustrated in Example 46.
The Ga1NAc3 cluster portion of the conjugate group Ga1NAc3-14 (Ga1NAc3-14a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is -P(=0)(OH)-Ad-P(=0)(OH)-. The structure of GalNAc3-14 (Ga1NAc3-14a-CM-) is shown below:

io H
AcHN N

Z
AcHN 0 HO OH
H0_01-r N--c---:
io H
AcHN
Example 64: Preparation of oligomeric compound 197 comprising GaINAc3-15 Ac0 OA OTBS OTBS
AcO__..r!..:)..\0/---,/---1OH
)\ Ac0 OA

Ac0_..i.Ø...vo._z=-----.../'-""
AcHN N 0 H

HBTU, DIEA AcHN

7 N NH3/Me0H OTBS
___________ ..-0 _________________________________________________________ 1 HO Bz20, DMAP
-112--\ ----/-----/'""1 AcHN

OH
OTBS
Bz0 Bz Bz0 OBz_* 0 0 Et3N.HF Bz0 ___T.O.s\z 0 Bz0 0 AcHN
AcHN 193 -----( (0-F,\,'r Phosphitylation Bz0 Bz __________ i.
___..Ø....\.,0õ./-----.../N 5 Bz0 1\i AcHN

DMTO
N(iPr)2 DMTO /
DMTO
O
DMT0/\7---0 5' 3' 195 CM j Oligo =
DMT0/\7----0 SS, DNA synthesizer 196 OH
,---OH
HO NI_ 1. 194, DNA synthesizer AcHN

2. Aq NH3 55 C, 18 h OH o 0-17N/7) __________________________________________________________________ Oligo NHAc 0 0¨P¨OH

OH
H0/0\...>/
HO NHAc Compound 189 is commercially available. Compound 195 was prepared using the general procedure shown in Example 31. Oligomeric compound 197, comprising a Ga1NAc3-15 conjugate group, was prepared from compounds 194 and 195 using standard oligonucleotide synthesis procedures. The Ga1NAc3 cluster portion of the conjugate group Ga1NAc3-15 (Ga1NAc3-15a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is -P(=0)(OH)-Ad-P(=0)(OH)-.
The structure of GalNAc3-15 (Ga1NAc3-15a-CM-) is shown below:

HOOH -PH, 0 oi_pi Nr-D
AcHN 0 0 0, HOOH
o HO =rN
AcHN 0 P, HO OH oov0 NHAc Example 65: Dose-dependent study of oligonucleotides comprising a 5'-conjugate group (comparison of Ga1NAc3-3, 12, 13, 14, and 15) targeting SRB-1 in vivo The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice. Unconjugated ISIS 353382 was included as a standard. Each of the Ga1NAc3 conjugate groups was attached at the 5' terminus of the respective oligonucleotide by a phosphodiester linked 2'-deoxyadenosine nucleoside (cleavable moiety).
Table 54 Modified ASOs targeting SRB-1 ISIS Sequences (5' to 3') Conjugate SEQ
No.
ID
No.
353382 none GesmCesTesTesmCesAdsGdsTdsmCdsAdsTdsGdsAdsmCdsTdsTesmCesmCesTesTe 661161 c_ INT A 2 A r.;_ c A T A A
varilrvC3--,a-0,,vdco=-.esmsesesesmses, .-ass=-=ds dsms-,ds, .clsnisds ds Ga1NAc3-3 145 m m Tes Ces CesTesTe 671144 GalNAc3-12a-0,AdoGesmCesTesTesmCesAdsGdsTdsmCdsAdsTdsGdsAdsmCdsTds Ga1NAc3-12 145 m m Tes Ces CesTesTe 670061 GalNAc3-13a-0,AdoGesmCesTesTesmCesAdsGdsTdsmCdsAdsTdsGdsAdsmCdsTds Ga1NAc3-13 145 m m Tes Ces CesTesTe 671261 GalNAC3-14a-0,AdoGesmCesTesTesmCesAdsGdsTdsmCdsAdsTdsGdsAdsmCdsTds Ga1NAc3-14 145 m m Tes Ces CesTesTe 671262 GalNAc3-15a-0,AdoGesmCesTesTesmCesAdsGdsrrdsmCdsAdsrrdsGdsAdsmCdsrrds Ga1NAc3-15 145 m m Tes Ces CesTesTe Capital letters indicate the nucleobase for each nucleoside and mC indicates a 5-methyl cytosine. Subscripts:
"e" indicates a 2'-MOE modified nucleoside; "d" indicates a [3-D-2'-deoxyribonuc1eoside; "s" indicates a phosphorothioate internucleoside linkage (PS); "o" indicates a phosphodiester internucleoside linkage (PO);
and "o" indicates -0-P(=0)(OH)-. Conjugate groups are in bold.

The structure of Ga1NAc3-3a was shown previously in Example 39. The structure of Ga1NAc3-12a was shown previously in Example 61. The structure of Ga1NAc3-13a was shown previously in Example 62.
The structure of Ga1NAc3-14a was shown previously in Example 63. The structure of Ga1NAc3-15a was shown previously in Example 64.
Treatment Six to eight week old C57b16 mice (Jackson Laboratory, Bar Harbor, ME) were injected subcutaneously once or twice at the dosage shown below with ISIS 353382, 661161, 671144, 670061, 671261, 671262, or with saline. Mice that were dosed twice received the second dose three days after the first dose. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration to determine the liver SRB-1 mRNA levels using real-time PCR and RIBOGREENO
RNA quantification reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols. The results below are presented as the average percent of SRB-1 mRNA levels for each treatment group, normalized to the saline control.
As illustrated in Table 55, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner. No significant differences in target knockdown were observed between animals that received a single dose and animals that received two doses (see ISIS
353382 dosages 30 and 2 x 15 mg/kg; and ISIS 661161 dosages 5 and 2 x 2.5 mg/kg). The antisense oligonucleotides comprising the phosphodiester linked Ga1NAc3-3, 12, 13, 14, and 15 conjugates showed substantial improvement in potency compared to the unconjugated antisense oligonucleotide (ISIS 335382).
Table 55 SRB-1 mRNA (% Saline) ISIS No. Dosage (mg/kg) SRB-1 mRNA (% ED50 (mg/kg) Conjugate Saline) Saline n/a 100.0 n/a n/a 3 85.0 10 69.2 353382 30 34.2 22.4 none 2 x 15 36.0 0.5 87.4 1.5 59.0 661161 5 25.6 2.2 Ga1NAc3-3 2 x 2.5 27.5 15 17.4 0.5 101.2 1.5 76.
671144 3.4 Ga1NAc3-12 5 32.0 15 17.6 0.5 94.8 670061 1.5 57.8 2.1 Ga1NAc3-13 5 20.7 15 13.3 0.5 110.7 1.5 81.9 671261 4.1 Ga1NAc3-14 39.8 15 14.1 0.5 109.4 1.5 99.5 671262 9.8 Ga1NAc3-15 5 69.2 15 36.1 Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols.
Total bilirubin and BUN were also evaluated. The changes in body weights were evaluated with no significant differences from the saline 5 group (data not shown). ALTs, ASTs, total bilirubin and BUN values are shown in Table 56 below.
Table 56 Total Dosage ALT BUN
ISIS No. AST (U/L) Bilirubin Conjugate (mg/kg) (U/L) (mg/d1-) (mg/d1-) Saline n/a 28 60 0.1 39 n/a 3 30 77 0.2 36 10 25 78 0.2 36 353382 none 30 28 62 0.2 35 2 x 15 22 59 0.2 33 0.5 39 72 0.2 34 1.5 26 50 0.2 33 661161 5 41 80 0.2 32 Ga1NAc3-3 2 x 2.5 24 72 0.2 28 15 32 69 0.2 36 0.5 25 39 0.2 34 1.5 26 55 0.2 28 671144 Ga1NAc3-5 48 82 0.2 34 15 23 46 0.2 32 0.5 27 53 0.2 33 1.5 24 45 0.2 35 670061 Ga1NAc3-5 23 58 0.1 34 15 24 72 0.1 31 0.5 69 99 0.1 33 1.5 34 62 0.1 33 671261 Ga1NAc3-5 43 73 0.1 32 15 32 53 0.2 30 0.5 24 51 0.2 29 1.5 32 62 0.1 31 671262 Ga1NAc3-5 30 76 0.2 32 15 31 64 0.1 32 Example 66: Effect of various cleavable moieties on antisense inhibition in vivo by oligonucleotides targeting SRB-1 comprising a 5'-GaINAc3 cluster The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice. Each of the Ga1NAc3 conjugate groups was attached at the 5' terminus of the respective oligonucleotide by a phosphodiester linked nucleoside (cleavable moiety (CM)).
Table 57 Modified ASOs targeting SRB-1 ISIS Sequences (5' to 3') Ga1NAc3 CM SEQ
No. Cluster ID
No.
661161 Ga1NAc1-3 - Ad mC T T mC A G T mC A T
- a ()' ¨ Ga1NAc3-3 a Ad es es es es es ds ds ds ds ds ds m m GdsAds CdsT ds es es Ces CesTesTe 670699 Ga1NAc3-3a-0 -0 ,Td mC T T mC A G T mC A T Ga1NAc3-3 a Td es eo eo eo eo ds ds ds ds ds ds m m GdsAds CdsTdsTeo Ceo C esT esTe 670700 Ga1NAc1-3 - A G mC T T mC A G T mC A T
- a ()' a Ga1NAc3-3 a Ae es eo eo eo eo ds ds ds ds ds ds m m GdsAds CdsTdsTeo Ceo C esT es 670701 Ga1NAc3-3a - ,T mC T T mC A G T mC A T
a Ga1NAc3-3 a Te 148 es eo eo eo eo ds ds ds ds ds ds m m GdsAds CdsTdsTeo Ceo C esT esTe 671165 Ga1NAc3-13 - mC T T mC A G T m o,A
a doC A T Ga1NAc3-13 a Ad es eo eo eo eo ds ds ds ds ds ds m m GdsAds CdsTdsTeo Ceo C esT es Capital letters indicate the nucleobase for each nucleoside and mC indicates a 5-methyl cytosine. Subscripts:
"e" indicates a 2'-MOE modified nucleoside; "d" indicates a [3-D-2'-deoxyribonuc1eoside; "s" indicates a phosphorothioate internucleoside linkage (PS); "o" indicates a phosphodiester internucleoside linkage (PO);
and "o" indicates -0-P(=0)(OH)-. Conjugate groups are in bold.
The structure of Ga1NAc3-3a was shown previously in Example 39. The structure of Ga1NAc3-13a was shown previously in Example 62.
Treatment Six to eight week old C57b16 mice (Jackson Laboratory, Bar Harbor, ME) were injected subcutaneously once at the dosage shown below with ISIS 661161, 670699, 670700, 670701, 671165, or with saline. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration to determine the liver SRB-1 mRNA levels using real-time PCR
and RIBOGREENO RNA
quantification reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols. The results below are presented as the average percent of SRB-1 mRNA levels for each treatment group, normalized to the saline control.
As illustrated in Table 58, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner. The antisense oligonucleotides comprising various cleavable moieties all showed similar potencies.
Table 58 SRB-1 mRNA (% Saline) ISIS No. Dosage (mg/kg) SRB-1 mRNA Ga1NAc3 CM
(% Saline) Cluster Saline n/a 100.0 n/a n/a 0.5 87.8 1.5 61.3 661161 Ga1NAc3-3a Ad 33.8 14.0 0.5 89.4 1.5 59.4 670699 Ga1NAc3-3a Td 5 31.3 15 17.1 0.5 79.0 1.5 63.3 670700 Ga1NAc3-3a Ae 5 32.8 15 17.9 0.5 79.1 1.5 59.2 670701 Ga1NAc3-3a I', 5 35.8 15 17.7 0.5 76.4 1.5 43.2 671165 Ga1NAc3-13a Ad 5 22.6 15 10.0 Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols.
Total bilirubin and BUN were 5 also evaluated. The changes in body weights were evaluated with no significant differences from the saline group (data not shown). ALTs, ASTs, total bilirubin and BUN values are shown in Table 56 below.
Table 59 Dosage ALT AST Total BUN Ga1NAc3 CM
ISIS No. Bilirubin (mg/kg) (U/L) (U/L) (mg/d1-) (mg/d1-) Cluster Saline n/a 24 64 0.2 31 n/a n/a 0.5 25 64 0.2 31 1.5 24 50 0.2 32 Ga1NAc3-3a Ad 5 26 55 0.2 28 15 27 52 0.2 31 0.5 42 83 0.2 31 1.5 33 58 0.2 32 Ga1NAc3-3a Td 5 26 70 0.2 29 15 25 67 0.2 29 0.5 40 74 0.2 27 1.5 23 62 0.2 27 Ga1NAc3-3a A, 5 24 49 0.2 29 15 25 87 0.1 25 0.5 30 77 0.2 27 Ga1NAc3-3a Te 1.5 22 55 0.2 30 81 101 0.2 25 31 82 0.2 24 0.5 44 84 0.2 26 1.5 47 71 0.1 24 671165 Ga1NAc3-13a Ad 5 33 91 0.2 26 15 33 56 0.2 29 Example 67: Preparation of oligomeric compound 199 comprising GaINAc3-16 OAc AcONC:Ac 0 AcHN ON
2 F:\(\r\j___ OAc OAc 0 H
Ac0.1\r\/IL ,,-, NH H __ ( /0DMTr õ 2 hl \ i 41. Succinic anhydride, AcHN DMAP, DCE

OAc OAc 0 Ac0 0 r -17 NaOH 2. DMF, HBTU, DIEA, ' H .
PS-SS
,1µ,-.1\INN----0 AcHN / 2 98d Ac0 OAc ____......:......\,0 0 H H N N 0 N
Ac0 AcHN 0 ODMT
Ac0 OAc H
_.....C...D..\ H /-- 1. DNA Synthesizer Ac0_ , 1 2. aq. NH3 AcHN 0 0 0 HN
Ac0 OAc 0 NI -:_2(/

' i 2 Ac0--/r,' 2 AcHN 198 t HO OH
H H
____.......2...\,0/ NN 0 HO , __ 1 = ____ HO OH AcHN 0 0-- , CM , ______ , oligo , H H
AcHN 0 0 OH
HO OH HN

H , HO__ 0 2 _..!.:).\ _.........1(N --../r/

AcHN

Oligomeric compound 199, comprising a Ga1NAc3-16 conjugate group, is prepared using the general procedures illustrated in Examples 7 and 9. The Ga1NAc3 cluster portion of the conjugate group Ga1NAc3-16 (Ga1NAc3-16a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is -P(=0)(OH)-Ad-P(=0)(OH)-.The structure of Ga1NAc3-16 (Ga1NAc3-16a-CM-) is shown below:

4 H 2 H [ cm D
/
AcHN H 0 0 _-0 Ny,-.N.,,,...-.N.11..N.....
HO--12-CY1))).L4 FiN12 0 H
AcHN("OH

HOr...?.....\õ0 .---AcHN
Example 68: Preparation of oligomeric compound 200 comprising GaINAc3-17 OAc 83e Ac00Ac 0 3' 0 5, II
AcHN 0" ' ' .N1-\ 0 F (OLIG0J-0-P-0-(CH2)6-NH2 1. Borate buffer, DMSO, pH 8.5, rt OAc OAc H _______________________________________________ 7 H 0 r F 2. aq. ammonia, rt Ac0--r----07,,,7--IfNHN-N
AcHN 0 102a HO_..7.2..\01-ENIN
H
AcHN 0 0 (OLIGO]

AcHN

( HOv-ii;11-./E1,--..õ..---.,N 0 H
AcHN

Oligomeric compound 200, comprising a Ga1NAc3-17 conjugate group, was prepared using the general procedures illustrated in Example 46. The Ga1NAc3 cluster portion of the conjugate group Ga1NAc3-17 (Ga1NAc3-17a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is -P(=0)(OH)-Ad-P(=0)(OH)-. The structure of Ga1NAc3-17 (Ga1NAc3-17a-CM-) is shown below:

_ii,!.....\,D .õ0.---ie.N.--...,......--..,Nõ.11.........\

AcHN H 0 0 HO__,.,E)._\vON 'K-NNH HO EMI
µ 3 H 0 AcHN

N.--...,s.õ----õN(c) AcHN
Example 69: Preparation of oligomeric compound 201 comprising GaINAc3-18 OAc Ac0,0Ac 0 0 83e AcHN O'H'2).LN1---1\1_40 F 3' 5,' H 0 0 F 1,6 F ( OLIG0)-0-17-0-(CH2)6-OAc OAc NNH ..---- OH
AcHN H

OAc H ir\----11 0 F 1. Borate buffer, DMSO, pH 8.5, rt H 0 r F ____________________ >
Ac0--r---%.-- 0HN--0 2. aq. ammonia, rt AcHN 2 0 102b H
AcHN 0 0 ,....u......_ " "4 0¨ ____________________________________________________________ CM __ OLIGO
HO--).-\--C4L111\1.--------'7 H H õ _______ H
AcHN
(/HOOH 0 __.....f.C.2..\.õ..
HOOv().4$1N O
H
AcHN 201 Oligomeric compound 201, comprising a Ga1NAc3-18 conjugate group, was prepared using the general procedures illustrated in Example 46. The Ga1NAc3 cluster portion of the conjugate group Ga1NAc3-18 (Ga1NAc3-18a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is -P(=0)(OH)-Ad-P(=0)(OH)-. The structure of Ga1NAc3-18 (Ga1NAc3-18a-CM-) is shown below:
HOOH o 0 ---..."-----'N'IL--- \

AcHN H 0 0 11)Fr\jr0¨[cm H
HO--72-\FNI 0 AcHN

HO 4 0.--eN---....,.....---.NO
H H
AcHN

Example 70: Preparation of oligomeric compound 204 comprising GaINAc3-19 AcO0Ac AcO0Ac Ac0- r 0H HBTU, DMF, DIEA
AcHN DMTO AcHN

DMTO

HO
AcO0Ac Phosphitylation Ac0 1C
NC 1. DNA
synthesizer AcHN
2. aq. NH3 203 DMTO (iPr)2N
.gH
HO OH
HO

AcHN
0=P¨OH
HO OH
HO

AcHN
0=P¨OH
,0 HO OH
HO
0 0 __ Cm __ OLIGO
AcHN

Oligomeric compound 204, comprising a Ga1NAc3-19 conjugate group, was prepared from compound 64 using the general procedures illustrated in Example 52. The Ga1NAc3 cluster portion of the conjugate group Ga1NAc3-19 (Ga1NAc3-19a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is -P(=0)(OH)-Ad-P(=0)(OH)-. The structure of Ga1NAc3-19 (Ga1NAc3-19a-CM-) is shown below:

pH
HOOH
HO

AcHN
0=P¨OH
HOOH
HO--2--\Aro 0 AcHN
0=P¨OH
HOOH
HO
AcHN

Example 71: Preparation of oligomeric compound 210 comprising GaINAc3-20 F 0 F EtN(iPr)2, CH3CN
o FF H ___________________________________ 0 __ F -N ..iii0H
FNO F

-b1h1 HO
AcO0Ac 0 Ac0C)kopfp K2CO3/Methanol 3 N -110H AcHN 166 ACN

AcO0Ac 0 Phosphitylation -(Z,()NO)N ""i0H )..-Ac0 AcHN
DMTO

I
AcO0Ac NO 1. DNA synthesizer NC ___________________________________________________________ .
)....0 Ac0 ...\,0 p \põ..0) 2. aq. NH3 AcHN I
209 DMTO (iPr)2N
pH
HO OH

___7õ.....\, H
0 02,),..---N1---NR, AcHN I
0=P¨OH

OH 0 .;
HO_.....i......\, AcHN
I
0=P¨OH
I
p OH 0 ..'' HO
HO ,__....r.....\
0 0.y\2------110LNIZ

0 0 __ avl __ OLIGO
AcHN 210 Compound 205 was prepared by adding PFP-TFA and DIEA to 6-(2,2,2-trifluoroacetamido)hexanoic acid in acetonitrile ,which was prepared by adding triflic anhydride to 6-aminohexanoic acid. The reaction mixture was heated to 80 C, then lowered to rt. Oligomeric compound 210, comprising a Ga1NAc3-20 conjugate group, was prepared from compound 208 using the general procedures illustrated in Example 52. The Ga1NAc3 cluster portion of the conjugate group Ga1NAc3-20 (Ga1NAc3-20a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is -P(=0)(OH)-Ad-P(=0)(OH)-. The structure of Ga1NAc3-20 (Ga1NAc3-20a-CM-) is shown below:
pH

AcHN 0 0 I
0=P ¨OH
I
OH p 0 0,--...(r.,,,-AcHN 0 0 I
O=P¨OH
I
sp OH

HO (-,,,)------NRA___ OfrrN

AcHN 0 0 Ell Example 72: Preparation of oligomeric compound 215 comprising Ga1NAc3-21 HO----L AcO0Ac 0 OH
NH
H O AcO0Ac Ac0--4.,kOH
AcHN 176 _______________________________________ ).- 0 Ac0 11---rti-j BOP, EtN(iPr)2, 1,2-dichloroethane AcHN OH

ODMT
AcO0Ac 0 DMICI, Pyridine,rt Phosphitylation ______________ )1.- Ac0---12--\,(:).----Ifl AcHN
OH

NC
0¨) /
0P\ 1. DNA synthesizer AcO0Ac 0N(iPr)2 ___________________________ .
2. aq. NH3 Ac0---, 11-----rti-j AcHN
---I¨ODMT

OH
OH
rj HO___72..\
HO N--.____\._____.

AcHN
I
0=P¨OH
I

OH
r----/
HO
HO___4.
0 (:) I N
"3 I
O
AcHN
I
0-=P¨OH
I

OH
nj HO*,,,,(2..\
0' HO N
0 -----10 ____ cm OLIGO
AcHN

Compound 211 is commercially available. Oligomeric compound 215, comprising a Ga1NAc3-21 conjugate group, was prepared from compound 213 using the general procedures illustrated in Example 52. The Ga1NAc3 cluster portion of the conjugate group GalNAc3-21 (GalNAc3-21 a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is -P(=0)(OH)-Ad-P(=0)(OH)-. The structure of GalNAc3-21 (Ga1NAc3-21a-CM-) is shown below:
OH
HO OH
HO
r---1 ___72.
Ofr'rN
3 ------1_, AcHN
I
0=P¨OH
I

OH
r---i O N

0 ---.
AcHN

I
0=P¨OH
I

OH

0 -----0 El AcHN

Example 73: Preparation of oligomeric compound 221 comprising Ga1NAc3-22 , H
F3C ., H N OH

II 0 F3C N )-L,NxOH
II

H
OH
.-DIEA ACN
F

H
DMT-CI F3C NN x-x0DMTr _________________________ , II
pyridine 0 H Me0H / H20 H2N Nx0DMTr Ac0 /0Ac F

OH 1.1 F
NHAc F F

F
_________________________________ 0 _________________________ 0 OAc Ac011 )-.N1 ODMTr L CO\70 Phosphitylation Ac0 ________________ 0 H

NHAc OAc AcO
Ac0 \ EN11)-LN ,ODMTr 0 0-r \/
H
NHAc 220 NC c), PI NN(i p 02 \Z

H
NHAc 1. DNA Synthesizer 0 l ,0 OH2. Aq. NH3 õ..---...õ,.--,...õ,.----.1r, 11 ..,._,,..õ...--,..,..)1.., 0\z0 N

NHAc H

OH 0 1,0 , OH
HO ____________________ \Z
CO 0.r NHAc H

_________________________________________________________________ Oligo ' 221 , ., Compound 220 was prepared from compound 219 using diisopropylammonium tetrazolide. Oligomeric compound 221, comprising a Ga1NAc3-21 conjugate group, is prepared from compound 220 using the general procedure illustrated in Example 52. The Ga1NAc3 cluster portion of the conjugate group Ga1NAc3-22 (Ga1NAc3-22a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is -P(=0)(OH)-Ad-P(=0)(OH)-. The structure of Ga1NAc3-22 (Ga1NAc3-22a-CM-) is shown below:

0 F-& 0 z=. r NI N z z OH
\/

H
NHAc OH H 0 1,0 OF& orN=LNvz0 OH
\/

NHAc H

OH 0 1,0 ,P( OH
HO ______________________ 0 H
NHAc 0( __ Example 74: Effect of various cleavable moieties on antisense inhibition in vivo by oligonucleotides targeting SRB-1 comprising a 5'-GaINAc3 conjugate The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice. Each of the Ga1NAc3 conjugate groups was attached at the 5' terminus of the respective oligonucleotide.

Table 60 Modified ASOs targeting SRB-1 ISISGalNAc3 SEQ
Sequences (5' to 3') CM
No. Cluster ID No.
m m m m G CTT CAGT CA TGA CTT
353382 es es es es es ds ds ds ds ds ds ds ds ds ds es m m n/a n/a 143 Ces CesTes mTe m GaINAC3-3am m 'Ad G CTT CAGT CAT
661161 0 es es es es es ds ds ds ds ds ds m m m Ga1NAc3-3 a Ad GdsAds CdsTdsTes Ces CesTesTe o GalNAc3-3a - ,G m m m CTT CAGT CAT
666904 es es es es es ds ds ds ds ds ds m m m Ga1NAc3-3 a PO

GdsAds CdsTdsTes Ce CesTesTe Ga1NAc3-17a-0,AdoG m m m CT T CAGT CAT
675441 m es es em es m es ds ds ds ds ds ds Ga1NAc3-17a Ad 145 GdsAds CdsTdsTes Ce CesTesTe GaINAC3-18a-0,AdoG m m m CTT CAGT CAT
675442 m es es em es m es ds ds ds ds ds ds Ga1NAc3-18 a Ad 145 GdsAds CdsTdsTes Ces CesTesTe In all tables, capital letters indicate the nucleobase for each nucleoside and mC indicates a 5-methyl cytosine.
Subscripts: "e" indicates a 2'-MOE modified nucleoside; "d" indicates a [3-D-2'-deoxyribonuc1eoside; "s"
indicates a phosphorothioate internucleoside linkage (PS); "o" indicates a phosphodiester internucleoside linkage (PO); and "o" indicates -0-P(=0)(OH)-. Conjugate groups are in bold.
The structure of Ga1NAc3-3a was shown previously in Example 39. The structure of Ga1NAc3-17a was shown previously in Example 68, and the structure of GalNAc3-18a was shown in Example 69.
Treatment Six to eight week old C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME) were injected subcutaneously once at the dosage shown below with an oligonucleotide listed in Table 60 or with saline.
Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration to determine the SRB-1 mRNA levels using real-time PCR and RIBOGREENO RNA
quantification reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols. The results below are presented as the average percent of SRB-1 mRNA levels for each treatment group, normalized to the saline control.
As illustrated in Table 61, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner. The antisense oligonucleotides comprising a GalNAc conjugate showed similar potencies and were significantly more potent than the parent oligonucleotide lacking a GalNAc conjugate.
Table 61 SRB-1 mRNA (% Saline) ISIS No. Dosage (mg/kg) SRB-1 mRNA
Ga1NAc3 CM
(% Saline) Cluster Saline n/a 100.0 n/a n/a 3 79.38 353382 10 68.67 n/a n/a 30 40.70 0.5 79.18 1.5 75.96 661161 Ga1NAc3-3a Ad 30.53 12.52 0.5 91.30 1.5 57.88 666904 Ga1NAc3-3a PO
5 21.22 15 16.49 0.5 76.71 1.5 63.63 675441 Ga1NAc3-17a Ad 5 29.57 15 13.49 0.5 95.03 1.5 60.06 675442 Ga1NAc3-18a Ad 5 31.04 15 19.40 Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols.
Total bilirubin and BUN were also evaluated. The change in body weights was evaluated with no significant change from the saline group 5 (data not shown). ALTs, ASTs, total bilirubin and BUN values are shown in Table 62 below.
Table 62 Dosage ALT AST Total BUN Ga1NAc3 CM
ISIS No.
(mg/kg) (U/L) (U/L) Bilirubin(Ing/d1-) Cluster (mg/d1-) Saline n/a 26 59 0.16 42 n/a n/a 3 23 58 0.18 39 353382 10 28 58 0.16 43 n/a n/a 30 20 48 0.12 34 0.5 30 47 0.13 35 1.5 23 53 0.14 37 Ga1NAc3-3a Ad 5 26 48 0.15 39 15 32 57 0.15 42 0.5 24 73 0.13 36 1.5 21 48 0.12 32 Ga1NAc3-3a PO
5 19 49 0.14 33 15 20 52 0.15 26 0.5 42 148 0.21 36 1.5 60 95 0.16 34 Ga1NAc3-17a Ad 5 27 75 0.14 37 15 24 61 0.14 36 0.5 26 65 0.15 37 1.5 25 64 0.15 43 Ga1NAc3-18a Ad 5 27 69 0.15 37 15 30 84 0.14 37 Example 75: Pharmacokinetic analysis of oligonucleotides comprising a 5'-conjugate group The PK of the ASOs in Tables 54, 57 and 60 above was evaluated using liver samples that were obtained following the treatment procedures described in Examples 65, 66, and 74. The liver samples were minced and extracted using standard protocols and analyzed by IP-HPLC-MS
alongside an internal standard.
The combined tissue level (Kg/g) of all metabolites was measured by integrating the appropriate UV peaks, and the tissue level of the full-length ASO missing the conjugate ("parent,"
which is Isis No. 353382 in this case) was measured using the appropriate extracted ion chromatograms (EIC).
Table 63 PK Analysis in Liver ISIS No. Dosage Total Tissue Level Parent ASO Tissue Ga1NAc3 CM
(mg/kg) by UV (Kg/g) Level by EIC (pg/g) Cluster 353382 3 8.9 8.6 10 22.4 21.0 n/a n/a 30 54.2 44.2 661161 5 32.4 20.7 Ga1NAc3-3a Ad 63.2 44.1 671144 5 20.5 19.2 Ga1NAc3-12a Ad 15 48.6 41.5 670061 5 31.6 28.0 Ga1NAc3-13a Ad 15 67.6 55.5 671261 5 19.8 16.8 Ga1NAc3-14a Ad 15 64.7 49.1 671262 5 18.5 7.4 Ga1NAc3-15a Ad 15 52.3 24.2 670699 5 16.4 10.4 Ga1NAc3-3a Td 15 31.5 22.5 670700 5 19.3 10.9 Ga1NAc3-3a A, 15 38.1 20.0 670701 5 21.8 8.8 Ga1NAc3-3a Te 15 35.2 16.1 671165 5 27.1 26.5 Ga1NAc3-13a Ad 15 48.3 44.3 666904 5 30.8 24.0 Ga1NAc3-3a PO
15 52.6 37.6 675441 5 25.4 19.0 Ga1NAc3-17a Ad 15 54.2 42.1 675442 5 22.2 20.7 Ga1NAc3-18a Ad 15 39.6 29.0 The results in Table 63 above show that there were greater liver tissue levels of the oligonucleotides comprising a Ga1NAc3 conjugate group than of the parent oligonucleotide that does not comprise a Ga1NAc3 conjugate group (ISIS 353382) 72 hours following oligonucleotide administration, particularly when taking 15 into consideration the differences in dosing between the oligonucleotides with and without a Ga1NAc3 conjugate group. Furthermore, by 72 hours, 40-98% of each oligonucleotide comprising a Ga1NAc3 conjugate group was metabolized to the parent compound, indicating that the Ga1NAc3 conjugate groups were cleaved from the oligonucleotides.
Example 76: Preparation of oligomeric compound 230 comprising GaINAc3-23 L, ToSCI NaN3 HO00,---...õ.0 11 ¨).- H0000Ts Pyr 4, TMSOTf OAc 0 N3 OAc...T.......

OAc 224 NHAc OAc Pd(01-1)2 OAc...T....... ACN
_____________ ).-H2, Et0Ac, Me0H OAc 7 F F \
NHAc \ F 0¨/( )/3 C¨N 02 OAc OAc NH 0 OAc OAc OAc NHAc H NO2 1) Reduce 0 C)---ON 2) Couple Diacid 0 3) Pd/C
OAc 0 O 4) PFPTFA
NHAc oAcOAc NH
(:)C) OAc NHAc 228 OAc OAc...T....... H

OAc F
OAc OAc NHAc H NH
1C) 0 F
OAc ____________ 0 0 0 NHAc OAc OAc ---0 OAc F
NH
NHAc 229 83e 3' 5' 11 ( OLIGO )-O-P-0-(CH2)6-NH2 OH
1. Borate buffer, DMSO, pH 8.5, rt ____________________________ ii-2. aq. ammonia, rt OH H
OH___\.._....\, N 0 OH
OH H
N Ir......... \11-11(.....õ,..........r.N ........4*cõ.0 Eli _______________________ 0 =-=-=0 ¨i oligo, __________________________ ., OH \r0 NHAc 01_1 OH 1.._ 0...... -.NH
NHAc 230 Compound 222 is commercially available. 44.48 ml (0.33 mol) of compound 222 was treated with tosyl chloride (25.39 g, 0.13 mol) in pyridine (500mL) for 16 hours. The reaction was then evaporated to an oil, dissolved in Et0Ac and washed with water, sat. NaHCO3, brine, and dried over Na2SO4. The ethyl acetate was concentrated to dryness and purified by column chromatography, eluted with Et0Ac/hexanes (1:1) followed by 10% methanol in CH2C12 to give compound 223 as a colorless oil. LCMS and NMR were consistent with the structure. 10 g (32.86 mmol) of 1-Tosyltriethylene glycol (compound 223) was treated with sodium azide (10.68 g, 164.28 mmol) in DMSO (100mL) at room temperature for 17 hours. The reaction mixture was then poured onto water, and extracted with Et0Ac. The organic layer was washed with water three times and dried over Na2504. The organic layer was concentrated to dryness to give 5.3g of compound 224 (92%). LCMS and NMR were consistent with the structure. 1-Azidotriethylene glycol (compound 224, 5.53 g, 23.69 mmol) and compound 4 (6 g, 18.22 mmol) were treated with 4A molecular sieves (5g), and TMSOTf (1.65 ml, 9.11 mmol) in dichloromethane (100mL) under an inert atmosphere.
After 14 hours, the reaction was filtered to remove the sieves, and the organic layer was washed with sat.
NaHCO3, water, brine, and dried over Na2504. The organic layer was concentrated to dryness and purified by column chromatography, eluted with a gradient of 2 to 4% methanol in dichloromethane to give compound 225. LCMS and NMR were consistent with the structure. Compound 225 (11.9 g, 23.59 mmol) was hydrogenated in Et0Ac/Methanol (4:1, 250mL) over Pearlman's catalyst.
After 8 hours, the catalyst was removed by filtration and the solvents removed to dryness to give compound 226. LCMS and NMR were consistent with the structure.
In order to generate compound 227, a solution of nitromethanetrispropionic acid (4.17 g, 15.04 mmol) and Hunig's base (10.3 ml, 60.17 mmol) in DMF (100mL) were treated dropwise with pentaflourotrifluoro acetate (9.05 ml, 52.65 mmol). After 30 minutes, the reaction was poured onto ice water and extracted with Et0Ac. The organic layer was washed with water, brine, and dried over Na2SO4. The organic layer was concentrated to dryness and then recrystallized from heptane to give compound 227 as a white solid. LCMS and NMR were consistent with the structure. Compound 227 (1.5 g, 1.93 mmol) and compound 226 (3.7 g, 7.74 mmol) were stirred at room temperature in acetonitrile (15 mL) for 2 hours. The reaction was then evaporated to dryness and purified by column chromatography, eluting with a gradient of 2 tol 0% methanol in dichloromethane to give compound 228. LCMS and NMR were consistent with the structure. Compound 228 (1.7 g, 1.02 mmol) was treated with Raney Nickel (about 2g wet) in ethanol (100mL) in an atmosphere of hydrogen. After 12 hours, the catalyst was removed by filtration and the organic layer was evaporated to a solid that was used directly in the next step. LCMS and NMR were consistent with the structure. This solid (0.87 g, 0.53 mmol) was treated with benzylglutaric acid (0.18 g, 0.8 mmol), HBTU (0.3 g, 0.8 mmol) and DIEA (273.7 [tl, 1.6 mmol) in DMF (5mL).
After 16 hours, the DMF
was removed under reduced pressure at 65 C to an oil, and the oil was dissolved in dichloromethane. The organic layer was washed with sat. NaHCO3, brine, and dried over Na2504. After evaporation of the organic layer, the compound was purified by column chromatography and eluted with a gradient of 2 to 20%
methanol in dichloromethane to give the coupled product. LCMS and NMR were consistent with the structure. The benzyl ester was deprotected with Pearlman's catalyst under a hydrogen atmosphere for 1 hour. The catalyst was them removed by filtration and the solvents removed to dryness to give the acid.
LCMS and NMR were consistent with the structure. The acid (486 mg, 0.27 mmol) was dissolved in dry DMF (3 mL). Pyridine (53.61 [tl, 0.66 mmol) was added and the reaction was purged with argon.
Pentaflourotriflouro acetate (46.39 [tl, 0.4 mmol) was slowly added to the reaction mixture. The color of the reaction changed from pale yellow to burgundy, and gave off a light smoke which was blown away with a stream of argon. The reaction was allowed to stir at room temperature for one hour (completion of reaction was confirmed by LCMS). The solvent was removed under reduced pressure (rotovap) at 70 C. The residue was diluted with DCM and washed with 1N NaHSO4, brine, saturated sodium bicarbonate and brine again. The organics were dried over Na2504, filtered, and were concentrated to dryness to give 225 mg of compound 229 as a brittle yellow foam. LCMS and NMR were consistent with the structure.
Oligomeric compound 230, comprising a Ga1NAc3-23 conjugate group, was prepared from compound 229 using the general procedure illustrated in Example 46. The Ga1NAc3 cluster portion of the Ga1NAc3-23 conjugate group (Ga1NAc3-23a) can be combined with any cleavable moiety to provide a variety of conjugate groups. The structure of GalNAc3-23 (Ga1NAc3-23a-CM) is shown below:

OH
01-1.v..... H
OH N ,0 OH H
01-1....\vNHAcam H
N ir.............xl,\I 0 N.,,..õ...,+
0 C)=-=-=0 IH

OH 0\r0 NHAc 01._ OH
NHAc Example 77: Antisense inhibition in vivo by oligonucleotides targeting SRB-1 comprising a GaINAc3 conjugate The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice.
Table 64 Modified ASOs targeting SRB-1 ISISGalNAc3 SEQ
Sequences (5' to 3') CM
No. Cluster ID
No.
m m m GalNAc3-3a-0,AdoGes CesTesTes CesAdsGdsTds CdsAdsTds 661161 m m m Ga1NAc3-3a Ad 145 GdsAds CdsTdsTes Ces CesTesTe m m m Ga1NAc3-3 - ,G CTT CAGT CAT
666904 a es es es es es ds ds ds ds ds ds m m m Ga1NAc3-3a PO 143 GdsAds CdsTdsTes Ces CesTesTe m m m Ga1NAC3-10a-0,AdoG CTT CAGT CAT
673502m es eo eom eo m eo ds ds ds ds ds ds Ga1NAc3-10a Ad GdsAds CdsTdsTeo Ceo CesTesTe m m m GaINAC3-9am'AdoGes CesTesTes CesAdsGdsTds CdsAdsTds 677844 m m m Ga1NAc3-9a Ad 145 GdsAds CdsTdsTes Ces CesTesTe m m m GaINAC3-23a-0,AdoG CTT CAGT CAT
es es es es es ds ds ds ds ds ds 677843 m m m Ga1NAc3-23a Ad 145 GdsAds CdsTdsTes Ces CesTesTe m m m m m G CTT CAGT CATGA CTT C
655861 es es es es es ds ds ds ds ds ds ds ds ds ds es es m Ga1NAc3-la Ad 144 CesTesTeeAdo,-Ga1NAc3-1a m m m m m G CTT CAGT CATGA CTT C
677841 es es es es mes ds ds ds ds ds ds ds ds ds ds es es Ga1NAc3-19a Ad 144 CesTesTeeAdo,-Ga1NAc3-19a m m m m m G CTT CAGT CAT GA CTT C
677842 es es es es mes ds ds ds ds ds ds ds ds ds ds es es Ga1NAc3-20a Ad 144 CesTesTeeAdo,-Ga1NAc3-20, The structure of Ga1NAc3-1a was shown previously in Example 9, Ga1NAc3-3a was shown in Example 39, Ga1NAc3-9a was shown in Example 52, Ga1NAc3-10a was shown in Example 46, Ga1NAc3-19a was shown in Example 70, Ga1NAc3-20a was shown in Example 71, and Ga1NAc3-23a was shown in Example 76.

Treatment Six to eight week old C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME) were each injected subcutaneously once at a dosage shown below with an oligonucleotide listed in Table 64 or with saline. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration to determine the SRB-1 mRNA levels using real-time PCR and RIBOGREENO RNA
quantification reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols. The results below are presented as the average percent of SRB-1 mRNA levels for each treatment group, normalized to the saline control.
As illustrated in Table 65, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner.
Table 65 SRB-1 mRNA (% Saline) ISIS No. Dosage (mg/kg) SRB-1 mRNA Ga1NAc3 CM
(% Saline) Cluster Saline n/a 100.0 n/a n/a 0.5 89.18 1. 77.02 661161 Ga1NAc3-3a Ad 5 29.10 12.64 0.5 93.11 1. 55.85 666904 Ga1NAc3-3a PO
5 21.29 15 13.43 0.5 77.75 1. 41.05 673502 Ga1NAc3-10a Ad 5 19.27 15 14.41 0.5 87.65 1. 93.04 677844 Ga1NAc3-9a Ad 5 40.77 15 16.95 0.5 102.28 1. 70.51 677843 Ga1NAc3-23a Ad 5 30.68 15 13.26 0.5 79.72 1. 55.48 655861 Ga1NAc3-la Ad 5 26.99 15 17.58 0.5 67.43 1. 45.13 677841 Ga1NAc3-19a Ad 5 27.02 15 12.41 0.5 64.13 1. 53.56 677842 Ga1NAc3-20a Ad 5 20.47 15 10.23 Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were also measured using standard protocols. Total bilirubin and BUN
were also evaluated. Changes in body weights were evaluated, with no significant change from the saline group (data not shown). ALTs, ASTs, total bilirubin and BUN values are shown in Table 66 below.
Table 66 Total CM
Dosage ALT AST BUN Ga1NAc3 ISIS No. Bilirubin (mg/kg) (U/L) (U/L) (mg/d1-) Cluster (mg/d1-) Saline n/a 21 45 0.13 34 n/a n/a 0.5 28 51 0.14 39 1.5 23 42 0.13 39 Ga1NAc3-3a Ad 5 22 59 0.13 37 21 56 0.15 35 0.5 24 56 0.14 37 1.5 26 68 0.15 35 Ga1NAc3-3a PO
5 23 77 0.14 34 15 24 60 0.13 35 0.5 24 59 0.16 34 1.5 20 46 0.17 32 Ga1NAc3-10 a Ad 5 24 45 0.12 31 15 24 47 0.13 34 0.5 25 61 0.14 37 1.5 23 64 0.17 33 Ga1NAc3-9a Ad 5 25 58 0.13 35 15 22 65 0.14 34 0.5 53 53 0.13 35 1.5 25 54 0.13 34 Ga1NAc3-23a Ad 5 21 60 0.15 34 15 22 43 0.12 38 0.5 21 48 0.15 33 1.5 28 54 0.12 35 Ga1NAc3-la Ad 5 22 60 0.13 36 15 21 55 0.17 30 0.5 32 54 0.13 34 1.5 24 56 0.14 34 Ga1NAc3-19a Ad 5 23 92 0.18 31 15 24 58 0.15 31 0.5 23 61 0.15 35 1.5 24 57 0.14 34 Ga1NAc3-20a Ad 5 41 62 0.15 35 15 24 37 0.14 32 Example 78: Antisense inhibition in vivo by oligonucleotides targeting Angiotensinogen comprising a Ga1NAc3 conjugate 10 The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of Angiotensinogen (AGT) in normotensive Sprague Dawley rats.

Table 67 Modified ASOs targeting AGT
ISISGalNAc3 SEQ
Sequences (5' to 3') CM
No. Cluster ID
No.
mCesAesmCesTesGesikdsTdsTdsTasTasTdsGdsmCdsmCdsmCdsAesGes 552668 A n/a n/a 149 e mCesAesmCesTesGesAdsTdsTdsTdsTdsTdsGdsmCdsmCdsmCdsAesGes GalNAc3-la Ad 150 GesAesTeoAdo'¨Ga1NAc3-1a The structure of Ga1NAc3-1a was shown previously in Example 9.
Treatment Six week old, male Sprague Dawley rats were each injected subcutaneously once per week at a dosage shown below, for a total of three doses, with an oligonucleotide listed in Table 67 or with PBS. Each treatment group consisted of 4 animals. The rats were sacrificed 72 hours following the final dose. AGT liver mRNA levels were measured using real-time PCR and RIBOGREENO RNA
quantification reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols. AGT
plasma protein levels were measured using the Total Angiotensinogen ELISA (Catalog # JP27412, IBL
International, Toronto, ON) with plasma diluted 1:20,000. The results below are presented as the average percent of AGT mRNA levels in liver or AGT protein levels in plasma for each treatment group, normalized to the PBS control.
As illustrated in Table 68, treatment with antisense oligonucleotides lowered AGT liver mRNA and plasma protein levels in a dose-dependent manner, and the oligonucleotide comprising a GalNAc conjugate was significantly more potent than the parent oligonucleotide lacking a GalNAc conjugate.
Table 68 AGT liver mRNA and plasma protein levels ISIS Dosage (mg/kg) AGT liver AGT plasma Ga1NAc3 Cluster CM
No. mRNA (% PBS) protein (% PBS) PBS n/a 100 100 n/a n/a 552668 n/a n/a 0.3 95 70 669509 Ga1NAc3-la Ad Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in plasma and body weights were also measured at time of sacrifice using standard protocols. The results are shown in Table 69 below.
Table 69 Liver transaminase levels and rat body weights Body CM
Dosage GalNAc3 ISIS No. ALT (U/L) AST (U/L) Weight (%
(mg/kg) Cluster of baseline) PBS n/a 51 81 186 n/a n/a 552668 n/a n/a 0.3 53 90 190 GalNAc3-la Ad Example 79: Duration of action in vivo of oligonucleotides targeting APOC-III
comprising a GaINAc3 conjugate The oligonucleotides listed in Table 70 below were tested in a single dose study for duration of action 10 in mice.
Table 70 Modified ASOs targeting APOC-III
ISISGalNAc3 SEQ
Sequences (5' to 3') CM
No. Cluster ID No.
AesGesmCesTesTesmCdsTdsTdsGdsTdsmCdsmCdsAdsGdsmCdsTesTes 304801 n/a n/a TesAesTe AesGesmCesTesTesmCdsTdsTdsGdsTdsmCdsmCdsAdsGdsmCdsTesTes 647535 Ga1NAc3-la Ad 136 TesAesTeoAdo'¨GalNAc3¨la GalNAC3-3,-0'AdoAesGesmCesTesTesmCdsTdsTdsGdsTdsmCds 663083 Ga1NAc3-3a Ad 151 mCdsAdsGdsmCdsTesTes TesAesTe GalNAC3-7,-0'AdoAesGesmCesTesTesmCdsTdsTdsGdsTdsmCds 674449 Ga1NAc3-7a Ad 151 mCdsAdsGdsmCdsTesTes TesAesTe GalNAc3-10am'AdoAesGesmCesTesTesmCdsrrdsrrdsGdsTasmCds GalNAc3-10 a Ad 151 mCdsAdsGdsmCdsTesTes TesAesTe GalNAc3-13 am'AdoAesGesmCesTesTesmCdsrrdsrrdsGdsTasmCds GalNAc3-13a Ad 151 mCdsAdsGdsmCdsTesTes TesAesTe The structure of Ga1NAc3-1 a was shown previously in Example 9, Ga1NAc3-3a was shown in Example 39, Ga1NAc3-7a was shown in Example 48, Ga1NAc3-10a was shown in Example 46, and Ga1NAc3-13a was shown in Example 62.

Treatment Six to eight week old transgenic mice that express human APOC-III were each injected subcutaneously once with an oligonucleotide listed in Table 70 or with PBS.
Each treatment group consisted of 3 animals. Blood was drawn before dosing to determine baseline and at 72 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, and 6 weeks following the dose. Plasma triglyceride and APOC-III protein levels were measured as described in Example 20. The results below are presented as the average percent of plasma triglyceride and APOC-III levels for each treatment group, normalized to baseline levels, showing that the oligonucleotides comprising a GalNAc conjugate group exhibited a longer duration of action than the parent oligonucleotide without a conjugate group (ISIS 304801) even though the dosage of the parent was three times the dosage of the oligonucleotides comprising a GalNAc conjugate group.
Table 71 Plasma triglyceride and APOC-III protein levels in transgenic mice Time pointAPOC-III
ISIS Dosage TriglyceridesGalNAc3 CM
(days post- ne protein ( /0 No. (mg/kg) (% baseline) Cluster dose) baseli) PBS n/a 21 107 107 n/a n/a 304801 30 21 50 50 n/a n/a 647535 10 21 41 41 Ga1NAc3-la Ad 663083 10 21 28 28 Ga1NAc3-3a Ad 674449 10 Ga1NAc3-7a Ad 674450 10 21 44 44 Ga1NAc3-10 a Ad 674451 10 21 48 48 Ga1NAc3-13a Ad Example 80: Antisense inhibition in vivo by oligonucleotides targeting Alpha-1 Antitrypsin (AlAT) comprising a Ga1NAc3 Conjugate The oligonucleotides listed in Table 72 below were tested in a study for dose-dependent inhibition of Al AT in mice.
Table 72 Modified ASOs targeting AlAT
ISIS Ga1NAc3 SEQ ID
Sequences (5' to 3') CM
No. Cluster No.
A mC mC 'V A Ad Td Td mCd Ad Gd Ad Ad Gd Gd A A
476366 es es es es es sss s s s s s s ses es n/a n/a GesGesAe AesmCesmCesmCesAesAdsTdsTdsmCdsAdsGdsAdsAdsGdsGdsAesAes GalNAc3-la Ad 153 GesGesAeoAdo'¨Ga1NAc3-1a GalNAc3-39-0'AdoAesmCesmCesmCesAesAdsTdsTdsmCdsAdsGdsAds GalNAc3-3a Ad 154 AdsGdsGdsAesAes GesGesAe GalNAC3-7a¨o'AdoAesmCesmCesmCesAesAdsTdsTdsmCdsAdsGdsAds GalNAc3-7a Ad 154 AdsGdsGdsAesAes GesGesAe GalNAc3-10a-0,AdoAesmCesmCesmCesAesAdsTasTasmCdsAdsGds GalNAc3-10a Ad 154 AdsAdsGdsGdsAesAes GesGesAe GalNAc3-13am'AdoAesmCesniCesniCesAesAdsTdsTdsmCdsAdsGds GalNAc3-13a Ad 154 AdsAdsGdsGdsAesAes GesGesAe The structure of Ga1NAc3-la was shown previously in Example 9, Ga1NAc3-3a was shown in Example 39, Ga1NAc3-7a was shown in Example 48, Ga1NAc3-10a was shown in Example 46, and Ga1NAc3-13a was shown in Example 62.

Treatment Six week old, male C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME) were each injected subcutaneously once per week at a dosage shown below, for a total of three doses, with an oligonucleotide listed in Table 72 or with PBS. Each treatment group consisted of 4 animals.
The mice were sacrificed 72 hours following the final administration. Al AT liver mRNA levels were determined using real-time PCR and RIBOGREENO RNA quantification reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols. AlAT plasma protein levels were determined using the Mouse Alpha 1-Antitrypsin ELISA
(catalog # 41-A1AMS-E01, Alpco, Salem, NH). The results below are presented as the average percent of Al AT liver mRNA and plasma protein levels for each treatment group, normalized to the PBS control.
As illustrated in Table 73, treatment with antisense oligonucleotides lowered Al AT liver mRNA and Al AT plasma protein levels in a dose-dependent manner. The oligonucleotides comprising a GalNAc conjugate were significantly more potent than the parent (ISIS 476366).
Table 73 AlAT liver mRNA and plasma protein levels ISIS Dosage (mg/kg) Al AT liver Al AT plasma Ga1NAc3 Cluster CM
No. mRNA (% PBS) protein (% PBS) PBS n/a 100 100 n/a n/a 15 73 61 n/a n/a 0.6 99 90 656326 Ga1NAc3-la Ad 0.6 105 90 6 16 20 Ga1NAc3-3a Ad 0.6 90 79 678382 Ga1NAc3-7a Ad 0.6 94 84 678383 Ga1NAc3-10 a Ad 0.6 106 91 678384 Ga1NAc3-13a Ad Liver transaminase and BUN levels in plasma were measured at time of sacrifice using standard protocols. Body weights and organ weights were also measured. The results are shown in Table 74 below.

Body weight is shown as % relative to baseline. Organ weights are shown as %
of body weight relative to the PBS control group.
Table 74 Body Liver Kidney Spleen ISIS Dosage ALT AST BUN
weight (% weight (Rel weight (Rel weight (Rel No. (mg/kg) (U/L) (U/L) (mg/dL) baseline) % BW) % BW) % BW) PBS n/a 25 51 37 119 100 100 0.6 29 57 40 123 100 103 0.6 26 57 32 117 93 109 0.6 26 42 35 114 100 103 0.6 30 67 38 121 91 100 0.6 36 63 31 118 98 103 Example 81: Duration of action in vivo of oligonucleotides targeting AlAT
comprising a GaINAc3 cluster The oligonucleotides listed in Table 72 were tested in a single dose study for duration of action in mice.
Treatment Six week old, male C57BL/6 mice were each injected subcutaneously once with an oligonucleotide listed in Table 72 or with PBS. Each treatment group consisted of 4 animals.
Blood was drawn the day before dosing to determine baseline and at 5, 12, 19, and 25 days following the dose. Plasma Al AT protein levels were measured via ELISA (see Example 80). The results below are presented as the average percent of plasma Al AT protein levels for each treatment group, normalized to baseline levels. The results show that the oligonucleotides comprising a GalNAc conjugate were more potent and had longer duration of action than the parent lacking a GalNAc conjugate (ISIS 476366). Furthermore, the oligonucleotides comprising a 5'-GalNAc conjugate (ISIS 678381, 678382, 678383, and 678384) were generally even more potent with even longer duration of action than the oligonucleotide comprising a 3'-GalNAc conjugate (ISIS 656326).
Table 75 Plasma AlAT protein levels in mice ISIS Dosage Time point Al AT (% Ga1NAc3 CM
No. (mg/kg) (days post- baseline) Cluster dose) PBS n/a n/a n/a

19 90 476366 100 n/a n/a 656326 18 Ga1NAc3-la Ad 678381 18 Ga1NAc3-3a Ad 678382 18 Ga1NAc3-7a Ad 678383 18 Ga1NAc3-10 a Ad 678384 18 Ga1NAc3-13a Ad Example 82: Antisense inhibition in vitro by oligonucleotides targeting SRB-1 comprising a GaINAc3 conjugate Primary mouse liver hepatocytes were seeded in 96 well plates at 15,000 cells/well 2 hours prior to treatment. The oligonucleotides listed in Table 76 were added at 2, 10, 50, or 250 nM in Williams E medium and cells were incubated overnight at 37 C in 5% CO2. Cells were lysed 16 hours following oligonucleotide addition, and total RNA was purified using RNease 3000 BioRobot (Qiagen). SRB-1 mRNA levels were determined using real-time PCR and RIBOGREENO RNA quantification reagent (Molecular Probes, Inc.
Eugene, OR) according to standard protocols. IC50 values were determined using Prism 4 software (GraphPad). The results show that oligonucleotides comprising a variety of different GalNAc conjugate groups and a variety of different cleavable moieties are significantly more potent in an in vitro free uptake experiment than the parent oligonucleotides lacking a GalNAc conjugate group (ISIS 353382 and 666841).
Table 76 Inhibition of SRB-1 expression in vitro ISISGalNAc IC50 SEQ
Sequence (5' to 3') Linkages CM
No. cluster (nM) ID No.
m m m Ges CesTesTes CesAd Gd Tds Cd Ad Td Gd Ad s353382 m m m PS n/a n/a 250 143 CdsTdsTes Ces CesTesTe GesmCesTesTesmCesAd Gd I'd mCd Ad I'd Gd Ad GalNAc3 655861 sss sssss PS
Ad 40 144 mCdsTdsTesmCesmCesTesTeoAdo,-GalNAC3-1 a -1a m m GalNAC3-3a-0,AdoGes CesTesTes CesAdsGdsTds GalNAc3 661161 m m m m PS Ad 40 CdsAdsTdsGdsAds CdsTds Tes Ces CesTesTe -3a m m GalNAc3-3a-0,AGes CeeTeeTee Ce0AdsGd Ta GalNAc3 661162 m m m mdo s s PO/PS Ad 8 CdsAdsTdsGdsAds CdsTds Teo Ceo CesTesTe -3a GesmCesTesTesmCesAdsGdsTdsmCdsAdsTdsGasAds Ga1NAc3 Ad 20 144 mCdsTdsTesmCesmCesTesTeoAdo¨GalNAc3-9a -9a GalNAC3-8a-o'AdoGesmCesTesTesmCesAdsGdsTds GalNAc3 665001 PS Ad 70 mCdsAdsTdsGdsAdsmCdsTdsTesmCesmCesTesTe -8a GalNAC3-5a-0,AdoGesmCesTesTesmCesAdsGdsTds Ga1NAc3 Ad 80 145 mCdsAdsTdsGdsAdsmCdsTdsTesmCesmCesTesTe -5a m m m Ges CeoTeoTeo CesAdsGd I'd Cd Ad Td Gd Ad 666841 ss sssss PO/PS
m m m n/a n/a >250 143 CdsTds Teo Ceo CesTesTe GalNAc3-10a-0,AdoGesmCesTesTesmCesAdsGdsTds Ga1NAc3 666881 PS Ad 30 mCdsAdsTdsGdsAdsmCdsTdsTesmCesmCesTesTe -10a m m m GalNAC3-3a-0,Ges CesTesTes CesAdsGdsTd Cd GalNAc3 666904 m m m s s ps PO 9 AdsTdsGdsAds CdsTds Tes Ces CesTesTe -3a m m GalNAC3-3a-0,TdoGes CesTesTes CesAdsGdsTds GalNAc3 666924 m m m m PS Td 15 CdsAdsTdsGdsAds CdsTds Tes Ces CesTesTe -3a GalNAC3-6a-0,AdoGesmCesTesTesmCesAdsGdsTds GalNAc3 Ad 150 145 mCdsAdsTdsGdsAdsmCdsTdsTesmCesmCesTesTe -6a GalNAC3-7a-0,AdoGesmCesTesTesmCesAdsGdsTds Ga1NAc3 666981 PS Ad 20 mCdsAdsTdsGdsAdsmCdsTdsTesmCesmCesTesTe -7a m m GalNAC3-13a-0,AdoGes CesTesTes CesAd ssGd 'fas GalNAc3 670061 m m m m ps Ad 30 CdsAdsTdsGdsAds CdsTds Tes Ces CesTesTe -13a m m Ga1NAC3-3a-0,TdoG C T T C A G T Ga1NAc3 670699 m es eo eo eo eo ds ds ds m m m PO/PS
Td 15 148 CdsAdsTdsGdsAds CdsTdsTeo Ceo CesTesTe -3a m m Ga1NAC3-3a-0,AeoG C T T C A G T GalNAc3 670700 m es eo eo eo eo ds ds ds m m m PO/PS
Ae 30 145 CdsAdsTdsGdsAds CdsTdsTeo Ceo CesT 3a esT -m m Ga1NAC3-3a-0,Te0G CT T CAGT GalNAc3 T
670701 m es eo eo eo eo ds ds ds m m m PO/PS
e 25 148 dsdsdsdsds dsdseo eo esesTe -3 CATGA CTT C CT a m m GalNAC3-12a-0,AdoGes CesTesTes CesAdsGds I'ds GalNAc3 671144 m m m m ps Ad 40 CdsAdsTdsGdsAds CdsTds Tes Ces CesTesTe -12a m m Ga1NAc3-139-0,AdoG CTT CAGT Ga1NAc3 Ad 8 145 671165 m m es m eo eo eo eo ds ds ds m PO/PS
CdsAdsTdsGdsAds CdsTdsTeo Ceo C es es 13a esT -m m GalNAc3-14a-0,AdoGes CesTesTes CesAdsGdsTds GalNAc3 671261 m m m m PS
Ad >250 145 CdsAdsTdsGdsAds CdsTds Tes Ces CesTesTe -14a m m GalNAc3-15a-0,AdoGes CesTesTes CesAdsGdsTds GalNAc3 671262 m m m m PS
Ad >250 145 CdsAdsTdsGdsAds CdsTds Tes Ces CesTesTe -15a m m GalNAc3-7a-0,AGes CeoTeoTeo CeoAdsGdsTds GalNAc3 do673501 m m m m PO/PS Ad 30 145 CdsAdsTdsGdsAds CdsTdsTeo Ceo CesTesTe -7a m m GalNAC3-10a-0,AGes CeoTeoTeo CeoAdsGdsTds GalNAc3 do673502 m m m m PO/PS Ad 8 145 CdsAdsTdsGdsAds CdsTds Teo Ceo CesTesTe - 1 Oa m m GalNAC3-17a-0,AGes CesTesTes CesAdsGdsTds GalNAc3 do675441 m m m m PS Ad 30 145 CdsAdsTdsGdsAds CdsTds Tes Ces CesTesTe -17a m m GalNAc3-18a-0,AGes CesTesTes CesAdsGdsTds GalNAc3 do675442 m m m m PS Ad 20 145 CdsAdsTdsGdsAds CdsTds Tes Ces CesTesTe -18a GeamCeaTeaTeamCesAdaGdsTdsmCdsAdsTdsGdsAds GalNAc3 677841 PS Ad 40 mCdaTdaTeamCesmCeaTearreoAdo¨GalNAc3-19a -19a GeamCeaTeaTeamCesAdaGdsTdsmCdsAdsTdsGdsAds GalNAc3 Ad 30 144 mCdaTdaTeamCesmCearrearreoAdo¨GalNAc3-20a -20a m m GalNAc3-23a-0,AGes CesTesTes CesAdsGdsTds GalNAc3 do677843 m m m m PS Ad 40 145 CdsAdsTdsGdsAds CdsTds Tes Ces CesTesTe 23a-The structure of Ga1NAc3-1 a was shown previously in Example 9, Ga1NAc3-3a was shown in Example 39, Ga1NAc3-5a was shown in Example 49, Ga1NAc3-6a was shown in Example 51, Ga1NAc3-7a was shown in Example 48, Ga1NAc3-8a was shown in Example 47, Ga1NAc3-9a was shown in Example 52, Ga1NAc3-10a was shown in Example 46, Ga1NAc3-12a was shown in Example 61, Ga1NAc3-13a was shown in Example 62, Ga1NAc3-14a was shown in Example 63, Ga1NAc3-15a was shown in Example 64, Ga1NAc3-17a was shown in Example 68, Ga1NAc3-18a was shown in Example 69, Ga1NAc3-19a was shown in Example 70, Ga1NAc3-20a was shown in Example 71, and Ga1NAc3-23a was shown in Example 76.
Example 83: Antisense inhibition in vivo by oligonucleotides targeting Factor XI comprising a GaINAc3 cluster The oligonucleotides listed in Table 77 below were tested in a study for dose-dependent inhibition of Factor XI in mice.
Table 77 Modified oligonucleotides targeting Factor XI
ISIS GalNAc SEQ
Sequence (5' to 3') CM
No. cluster ID No.
TesGesGesTesAesAdsTdsmCdsmCdsAdsmCdsTdsTdsTdsmCdsAesGes 404071 n/a n/a AesGesGe TesGeoGeoTecAeoAdsTdsmCdsmCdsAdsmCdsTdsTdsTdsmCdsAeoGeo 656173 Ga1NAc3-1 a Ad 147 AesGesGeoAdo¨GalNAc3-1 a 663086 Ga1NAc3-3a-0,AdoTesGeoGeoTeoAeoAdsTdsmCdsmCdsAdsmCdsTds Ga1NAc3-3 a Ad 155 TasTasmCdsAeoGeoAesGesGe GalNAc3-7a-0,AdoTesGeoGeoTeoAeoAdsTdsmCdsmCdsAdsmCdsTds GalNAc3-7a Ad 155 678347 TdsTdsmCdsAeoGeoAesGesGe GalNAc3-10a-0,AdoTesGeoGeoTeoAeoAdsTdsmCdsmCdsAdsmCds GalNAc3-10a Ad 155 678348 TdsTdsTdsmCdsAeoGeoAesGesGe GalNAc3-13a-0,AdoTesGeoGeoTeoAeoAdsTdsmCdsmCdsAdsmCds GalNAc3-13a Ad 155 678349 TdsTdsTdsmCdsAeoGeoAesGesGe The structure of Ga1NAc3-la was shown previously in Example 9, Ga1NAc3-3a was shown in Example 39, Ga1NAc3-7a was shown in Example 48, Ga1NAc3-10a was shown in Example 46, and Ga1NAc3-13a was shown in Example 62.
Treatment Six to eight week old mice were each injected subcutaneously once per week at a dosage shown below, for a total of three doses, with an oligonucleotide listed below or with PBS. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final dose. Factor XI liver mRNA
levels were measured using real-time PCR and normalized to cyclophilin according to standard protocols.
Liver transaminases, BUN, and bilirubin were also measured. The results below are presented as the average percent for each treatment group, normalized to the PBS control.
As illustrated in Table 78, treatment with antisense oligonucleotides lowered Factor XI liver mRNA
in a dose-dependent manner. The results show that the oligonucleotides comprising a GalNAc conjugate were more potent than the parent lacking a GalNAc conjugate (ISIS 404071).
Furthermore, the oligonucleotides comprising a 5'-GalNAc conjugate (ISIS 663086, 678347, 678348, and 678349) were even more potent than the oligonucleotide comprising a 3'-GalNAc conjugate (ISIS 656173).
Table 78 Factor XI liver mRNA, liver transaminase, BUN, and bilirubin levels ISIS Dosage Factor XI ALT AST BUN Bilirubin Ga1NAc3 SEQ
No. (mg/kg) mRNA (% PBS) (U/L) (U/L) (mg/dL) (mg/dL) Cluster ID No.
PBS n/a 100 63 70 21 0.18 n/a n/a 3 65 41 58 21 0.15 10 33 49 53 23 0.15 n/a 30 17 43 57 22 0.14 0.7 43 90 89 21 0.16 656173 2 9 36 58 26 0.17 Ga1NAc3-la 6 3 50 63 25 0.15 0.7 33 91 169 25 0.16 2 7 38 55 21 0.16 Ga1NAc3-3a 6 1 34 40 23 0.14 0.7 35 28 49 20 0.14 678347 2 10 180 149 21 0.18 Ga1NAc3-7a 6 1 44 76 19 0.15 0. 39 43 54 21 0.16 678348 Ga1NAc3-10 a 2 5 38 55 22 0.17 6 2 25 38 20 0.14 0.7 34 39 46 20 0.16 678349 2 8 43 63 21 0.14 Ga1NAc3-13 a 6 2 28 41 20 0.14 Example 84: Duration of action in vivo of oligonucleotides targeting Factor XI
comprising a GaINAc3 Conj ug ate The oligonucleotides listed in Table 77 were tested in a single dose study for duration of action in mice.
Treatment Six to eight week old mice were each injected subcutaneously once with an oligonucleotide listed in Table 77 or with PBS. Each treatment group consisted of 4 animals. Blood was drawn by tail bleeds the day before dosing to determine baseline and at 3, 10, and 17 days following the dose. Plasma Factor XI protein levels were measured by ELISA using Factor XI capture and biotinylated detection antibodies from R & D
Systems, Minneapolis, MN (catalog # AF2460 and # BAF2460, respectively) and the OptEIA Reagent Set B
(Catalog # 550534, BD Biosciences, San Jose, CA). The results below are presented as the average percent of plasma Factor XI protein levels for each treatment group, normalized to baseline levels. The results show that the oligonucleotides comprising a GalNAc conjugate were more potent with longer duration of action than the parent lacking a GalNAc conjugate (ISIS 404071). Furthermore, the oligonucleotides comprising a 5'-GalNAc conjugate (ISIS 663086, 678347, 678348, and 678349) were even more potent with an even longer duration of action than the oligonucleotide comprising a 3'-GalNAc conjugate (ISIS 656173).
Table 79 Plasma Factor XI protein levels in mice ISIS Dosage Time point (days Factor XI (% CM SEQ ID
GalNAc3 Cluster No. (mg/kg) post-dose) baseline) No.

PBS n/a 10 56 n/a n/a n/a 404071 30 10 47 n/a n/a 656173 6 10 3 Ga1NAc3-la Ad 663086 6 10 2 Ga1NAc3-3 a Ad 678347 6 10 1 Ga1NAc3-7a Ad 678348 6 10 1 Ga1NAc3-10 a Ad 678349 6 10 1 Ga1NAc3-13a Ad Example 85: Antisense inhibition in vivo by oligonucleotides targeting SRB-1 comprising a GaINAc3 Conjugate Oligonucleotides listed in Table 76 were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice.
Treatment Six to eight week old C57BL/6 mice were each injected subcutaneously once per week at a dosage shown below, for a total of three doses, with an oligonucleotide listed in Table 76 or with saline. Each treatment group consisted of 4 animals. The mice were sacrificed 48 hours following the final administration to determine the SRB-1 mRNA levels using real-time PCR and RIBOGREENO RNA
quantification reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols. The results below are presented as the average percent of liver SRB-1 mRNA levels for each treatment group, normalized to the saline control.
As illustrated in Tables 80 and 81, treatment with antisense oligonucleotides lowered SRB-1 mRNA
levels in a dose-dependent manner.
Table 80 SRB-1 mRNA in liver ISIS No. Dosage (mg/kg) SRB-1 mRNA (% Ga1NAc3 Cluster CM
Saline) Saline n/a 100 n/a n/a 0.1 94 0.3 119 655861 Ga1NAc3-la Ad 0.1 120 0.3 107 661161 Ga1NAc3-3a Ad 0.1 107 0.3 107 666881 Ga1NAc3-10 a Ad 0.1 120 0. 103 666981 Ga1NAc3-7a Ad 0.
670061 Ga1NAc3-13a Ad 0.3 89 0.1 119 0.3 96 677842 Ga1NAc3-20a Ad Table 81 SRB-1 mRNA in liver ISIS No. Dosage (mg/kg) SRB-1 mRNA (% Ga1NAc3 Cluster CM
Saline) 0.1 107 0.3 95 661161 Ga1NAc3-3a Ad 0.1 110 0.3 88 677841 Ga1NAc3-19a Ad Liver transaminase levels, total bilirubin, BUN, and body weights were also measured using standard protocols. Average values for each treatment group are shown in Table 82 below.
Table 82 ISIS Dosage ALT AST Bilirubin BUN Body Weight Ga1NAc3 CM
No. (mg/kg) (U/L) (U/L) (mg/dL) (mg/dL) (% baseline) Cluster Saline n/a 19 39 0.17 26 118 n/a n/a 0.1 25 47 0.17 27 114 0.3 29 56 0.15 27 118 655861 Ga1NAc3-la Ad 1 20 32 0.14 24 112 3 27 54 0.14 24 115 0.1 35 83 0.13 24 113 0.3 42 61 0.15 23 117 661161 Ga1NAc3-3a Ad 1 34 60 0.18 22 116 3 29 52 0.13 25 117 0.1 30 51 0.15 23 118 0.3 49 82 0.16 25 119 666881 Ga1NAc3-10 a Ad 1 23 45 0.14 24 117 3 20 38 0.15 21 112 0.1 21 41 0.14 22 113 0.3 29 49 0.16 24 112 666981 Ga1NAc3-7a Ad 1 19 34 0.15 22 111 3 77 78 0.18 25 115 0.1 20 63 0.18 24 111 0.3 20 57 0.15 21 115 670061 Ga1NAc3-13a Ad 1 20 35 0.14 20 115 3 27 42 0.12 20 116 0.1 20 38 0.17 24 114 677842 0.3 31 46 0.17 21 117 Ga1NAc3-20a Ad 1 22 34 0.15 21 119 3 41 57 0.14 23 118 Example 86: Antisense inhibition in vivo by oligonucleotides targeting TTR
comprising a Ga1NAc3 cluster Oligonucleotides listed in Table 83 below were tested in a dose-dependent study for antisense inhibition of human transthyretin (TTR) in transgenic mice that express the human TTR gene.
Treatment Eight week old TTR transgenic mice were each injected subcutaneously once per week for three weeks, for a total of three doses, with an oligonucleotide and dosage listed in the tables below or with PBS.
Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration. Tail bleeds were performed at various time points throughout the experiment, and plasma TTR protein, ALT, and AST levels were measured and reported in Tables 85-87.
After the animals were sacrificed, plasma ALT, AST, and human TTR levels were measured, as were body weights, organ weights, and liver human TTR mRNA levels. TTR protein levels were measured using a clinical analyzer (AU480, Beckman Coulter, CA). Real-time PCR and RIBOGREENO RNA quantification reagent (Molecular Probes, Inc. Eugene, OR) were used according to standard protocols to determine liver human TTR mRNA levels.
The results presented in Tables 84-87 are the average values for each treatment group. The mRNA levels are the average values relative to the average for the PBS group. Plasma protein levels are the average values relative to the average value for the PBS group at baseline. Body weights are the average percent weight change from baseline until sacrifice for each individual treatment group.
Organ weights shown are normalized to the animal's body weight, and the average normalized organ weight for each treatment group is then presented relative to the average normalized organ weight for the PBS
group.
In Tables 84-87, "BL" indicates baseline, measurements that were taken just prior to the first dose.
As illustrated in Tables 84 and 85, treatment with antisense oligonucleotides lowered TTR expression levels in a dose-dependent manner. The oligonucleotides comprising a GalNAc conjugate were more potent than the parent lacking a GalNAc conjugate (ISIS 420915). Furthermore, the oligonucleotides comprising a GalNAc conjugate and mixed PS/P0 internucleoside linkages were even more potent than the oligonucleotide comprising a GalNAc conjugate and full PS linkages.
Table 83 Oligonucleotides targeting human TTR
GalNAc SEQ
Isis No. Sequence 5 to 3' Linkages CM
cluster ID No.
TesmCesT esT es es GdsT dsT dsAdsmC dsAdsTdsGdsAdsAds 420915 PS n/a n/a 156 AesTesmCesmCesmCe TesmCesTesTesGesGdsTdsTdsAdsmCdsAdsTdsGdsAdsAds 660261 PS GalNAc3-1 a Ad 157 AesTesmCesmCesmCeoAdo'-GalNAc3-1, 682883 Ga1NAc3-3a_0,TesmCeoTeoTeoGeoGdsTdsTdsAdsmC dsAds PS/PO
Ga1NAc3-3 a PO 156 TdsGasAdsAdsAeeTeemCesmCesmCe GalNAc3-7a_0,TesmCeorreorreoGeoGasTasTasAdsmCdsAd 682884 s PS/P0 Ga1NAc3-7a PO 156 TdsGdsAdsAdsAeeTeemCesmCesmCe GalNAc3-10a_0,TesmCeoTeoTeoGeoGdsTdsTdsAdsmCd 682885 s PS/P0 Ga1NAc3-10a PO 156 AdsTdsGdsAdsAdsAeoTeomCesmCesmCe GalNAc3-13a_0,TesmCeoTeoTeoGeoGdsTdsTdsAdsmCd 682886 s PS/P0 Ga1NAc3-13a PO 156 AdsTdsGdsAdsAdsAeoTeomCesmCesmCe TesmCeoTeoTer;i3GeoinGdsTindsTdsAdsmCdsAdsTdsGdsAdsAds 684057 PS/P0 Ga1NAc3-19a Ad 157 AeoTeo Ces Ces CeoAdo'-GalNAc3-19a The legend for Table 85 can be found in Example 74. The structure of Ga1NAc3-1 was shown in Example 9.
The structure of Ga1NAc3-3a was shown in Example 39. The structure of Ga1NAc3-7a was shown in Example 48. The structure of GalNAc3-10a was shown in Example 46. The structure of GalNAc3-13a was shown in Example 62. The structure of GalNAc3-19a was shown in Example 70.
Table 84 Antisense inhibition of human TTR in vivo Dosage TTR mRNA (% Plasma TTR protein SEQ
Isis No. GalNAc cluster CM
(mg/kg) PBS) (% PBS) ID No.
PBS n/a 100 100 n/a n/a 420915 20 48 65 n/a n/a 156 0.6 113 87 660261 Ga1NAc3-la Ad 157

20 9 11 Table 85 Antisense inhibition of human TTR in vivo TTR Plasma TTR protein (% PBS at BL) SEQ
Dosage GalNAc Isis No. mRNA Day 17 CM ID
(mg/kg) (% PBS) BL Day 3 Day 10 (After sac) cluster No.
PBS n/a 100 100 96 90 114 n/a n/a 58 n/a n/a 156 0.6 60 88 73 63 68 Ga1NAc3- PO

3a 0.6 56 88 78 63 67 GalNAc3- PO

7a 0.6 60 92 77 68 76 GalNAc3- PO

10a 682886 0.6 57 91 70 64 69 Ga1NAc3- PO 156 2 21 89 50 31 30 13a 0.6 53 80 69 56 62 684057 2 21 92 55 34 30 Ga1NAc3-Ad 157 6 11 82 50 18 13 19a Table 86 Transaminase levels, body weight changes, and relative organ weights Dos ALT (U/L) AST (U/L) Body Liver Spleen Kidne SEQ
age Isis No. ( BL BL Day Day Day Day Day Day (% (%
(% Y (% ID nig /kg) 3 10 17 3 10 17 BL) PBS) PBS) PBS) No.
PBS n/a 33 34 33 24 58 62 67 52 105 100 100 100 n/a 0.6 33 38 28 26 70 71 63 59 111 96 99 92 Table 87 Transaminase levels, body weight changes, and relative organ weights Dos ALT (U/L) AST (U/L) Body Liver Spleen Kidne SEQ
age Isis No. (mg BL Day Day Day BL Day Day Day (% (% (% Y (% ID
/kg) 3 10 17 3 10 17 BL) PBS) PBS) PBS) No.
PBS n/a 32 34 37 41 62 78 76 77 104 100 100 100 n/a 0.6 32 35 38 40 53 81 74 76 104 101 112 95 0.6 33 32 35 34 70 74 75 67 101 100 130 99 0.6 39 26 37 35 63 63 77 59 100 109 109 112 0.6 30 40 34 36 58 87 54 61 104 99 120 101 0.6 35 26 33 39 56 51 51 69 104 99 110 102 Example 87: Duration of action in vivo by single doses of oligonucleotides targeting TTR comprising a Ga1NAc3 cluster ISIS numbers 420915 and 660261 (see Table 83) were tested in a single dose study for duration of action in mice. ISIS numbers 420915, 682883, and 682885 (see Table 83) were also tested in a single dose study for duration of action in mice.
Treatment Eight week old, male transgenic mice that express human TTR were each injected subcutaneously once with 100 mg/kg ISIS No. 420915 or 13.5 mg/kg ISIS No. 660261. Each treatment group consisted of 4 animals. Tail bleeds were performed before dosing to determine baseline and at days 3, 7, 10, 17, 24, and 39 following the dose. Plasma TTR protein levels were measured as described in Example 86. The results below are presented as the average percent of plasma TTR levels for each treatment group, normalized to baseline levels.
Table 88 Plasma TTR protein levels ISIS Dosage Time pointGalNAc3 CM
TTR (% baseline) SEQ
ID No.
No. (mg/kg) (days post-dose) Cluster 420915 100 n/a n/a 660261 13.5 Ga1NAc3-la Ad Treatment Female transgenic mice that express human TTR were each injected subcutaneously once with 100 mg/kg ISIS No. 420915, 10.0 mg/kg ISIS No. 682883, or 10.0 mg/kg 682885. Each treatment group consisted of 4 animals. Tail bleeds were performed before dosing to determine baseline and at days 3, 7, 10, 17, 24, and 39 following the dose. Plasma TTR protein levels were measured as described in Example 86.
The results below are presented as the average percent of plasma TTR levels for each treatment group, normalized to baseline levels.

Table 89 Plasma TTR protein levels ISIS Dosage Time pointGalNAc3 CM
TTR (% baseline) SEQ ID No.
No. (mg/kg) (days post-dose) Cluster 420915 100 10 48 n/a n/a 682883 10.0 10 38 Ga1NAc3-3a PO 156 682885 10.0 10 34 Ga1NAc3-10 a PO

The results in Tables 88 and 89 show that the oligonucleotides comprising a GalNAc conjugate are more potent with a longer duration of action than the parent oligonucleotide lacking a conjugate (ISIS 420915).
Example 88: Splicing modulation in vivo by oligonucleotides targeting SMN
comprising a GaINAc3 conjugate The oligonucleotides listed in Table 90 were tested for splicing modulation of human survival of motor neuron (SMN) in mice.
Table 90 Modified ASOs targeting SMN
ISIS, SEQ
Sequences (5' to 3') CM
No. Cluster ID No.
ATTmCAmCTTTmCATAATGmCTG
387954 es es es es es es es es es es es es es es es es es es es n/a n/a 158 Ge C A. C C
Ga1NAc3-7a PO 158 ,sTes ¨T esm es ,sm es ¨T es ¨T es ¨T esm es ,sTes ,s 699819 GaINAc3-7 TesGesmCesTesGesGe 699821 GaINAc3-7a-0 AesTeoTeomCeoAeomCeoTeoTeoTeomCeoAeoTeoAe Ga1NAc3-7a PO 158 AeoTeoGeomCeoTesGesGe 700000 A T T mC A mC T T T mC A T A A T G mC T G
Ga1NAc3-1a Ad 157 GeoAdo=¨GaINAc3-1a 703421 X-ATTmCAmCTTTmCATAATGmCTGG
n/a n/a 158 703422 GaINAc3-7b-X-ATTmCAmCTTTmCATAATGmCTGG Ga1NAc3-76 n/a 158 The structure of Ga1NAc3-7a was shown previously in Example 48. "X" indicates a 5' primary amine generated by Gene Tools (Philomath, OR), and Ga1NAc3-7b indicates the structure of Ga1NAc3-7a lacking the ¨NH-C6-0 portion of the linker as shown below:

AcHN N

Z
AcHN 0 HOOH

AcHN .
ISIS numbers 703421 and 703422 are morphlino oligonucleotides, wherein each nucleotide of the two oligonucleotides is a morpholino nucleotide.
Treatment Six week old transgenic mice that express human SMN were injected subcutaneously once with an oligonucleotide listed in Table 91 or with saline. Each treatment group consisted of 2 males and 2 females.
The mice were sacrificed 3 days following the dose to determine the liver human SMN mRNA levels both with and without exon 7 using real-time PCR according to standard protocols.
Total RNA was measured using Ribogreen reagent. The SMN mRNA levels were normalized to total mRNA, and further normalized to the averages for the saline treatment group. The resulting average ratios of SMN mRNA including exon 7 to SMN mRNA missing exon 7 are shown in Table 91. The results show that fully modified oligonucleotides that modulate splicing and comprise a GalNAc conjugate are significantly more potent in altering splicing in the liver than the parent oligonucleotides lacking a GlaNAc conjugate.
Furthermore, this trend is maintained for multiple modification chemistries, including 2'-MOE and morpholino modified oligonucleotides.
Table 91 Effect of oligonucleotides targeting human SMN in vivo ISIS
Ga1NAc3 CM SEQ
Dose (mg/kg) +Exon 7 / -Exon 7 No. Cluster ID
No.
Saline n/a 1.00 n/a n/a n/a 387954 32 1.65 n/a n/a 387954 288 5.00 n/a n/a 699819 32 7.84 Ga1NAc3-7 a PO 158 699821 32 7.22 Ga1NAc3-7 a PO 158 700000 32 6.91 Ga1NAc3-la Ad 159 703421 32 1.27 n/a n/a 703422 32 4.12 Ga1NAc3-76 n/a 158 Example 89: Antisense inhibition in vivo by oligonucleotides targeting Apolipoprotein A (Apo(a)) comprising a Ga1NAc3 conjugate The oligonucleotides listed in Table 92 below were tested in a study for dose-dependent inhibition of Apo(a) in transgenic mice.
Table 92 Modified ASOs targeting Apo(a) ISISGalNAc3 SEQ ID
Sequences (5' to 3') CM
No. Cluster No.
es "-esmCes mCdsGdsTasTdsGdsGdsTasGasmC
Tes es ds 494372 n/a n/a 58 TasTesGesTesTesmCe GalNAc3-7a-0,TesGeomCeoTeomCeomCdsGdsTdsTdsGdsGds 681257 Ga1NAc3-7a PO 58 TdsGdsmCds TdsTeoGeoTesTesmCe The structure of GalNAc3-7a was shown in Example 48.
Treatment Eight week old, female C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME) were each injected subcutaneously once per week at a dosage shown below, for a total of six doses, with an oligonucleotide listed in Table 92 or with PBS. Each treatment group consisted of 3-4 animals.
Tail bleeds were performed the day before the first dose and weekly following each dose to determine plasma Apo(a) protein levels. The mice were sacrificed two days following the final administration. Apo(a) liver mRNA levels were determined using real-time PCR and RIBOGREENO RNA quantification reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols. Apo(a) plasma protein levels were determined using ELISA, and liver transaminase levels were determined. The mRNA and plasma protein results in Table 93 are presented as the treatment group average percent relative to the PBS treated group. Plasma protein levels were further normalized to the baseline (BL) value for the PBS group. Average absolute transaminase levels and body weights (% relative to baseline averages) are reported in Table 94.
As illustrated in Table 93, treatment with the oligonucleotides lowered Apo(a) liver mRNA and plasma protein levels in a dose-dependent manner. Furthermore, the oligonucleotide comprising the GalNAc conjugate was significantly more potent with a longer duration of action than the parent oligonucleotide lacking a GalNAc conjugate. As illustrated in Table 94, transaminase levels and body weights were unaffected by the oligonucleotides, indicating that the oligonucleotides were well tolerated.
Table 93 Apo(a) liver mRNA and plasma protein levels ISIS Dosage Apo(a) mRNA Apo(a) plasma protein (% PBS) No. (mg/kg) (% PBS) BL Week 1 Week 2 Week 3 Week 4 Week 5 Week PBS n/a 100 100 120 119 113 88 121 0.3 75 79 76 89 98 71 94 Table 94 ISIS No. Dosage (mg/kg) ALT (U/L) AST (U/L) Body weight (%
baseline) PBS n/a 37 54 103 0.3 30 80 104 Example 90: Antisense inhibition in vivo by oligonucleotides targeting TTR
comprising a Ga1NAc3 5 cluster Oligonucleotides listed in Table 95 below were tested in a dose-dependent study for antisense inhibition of human transthyretin (TTR) in transgenic mice that express the human TTR gene.
Treatment 10 TTR transgenic mice were each injected subcutaneously once per week for three weeks, for a total of three doses, with an oligonucleotide and dosage listed in Table 96 or with PBS. Each treatment group consisted of 4 animals. Prior to the first dose, a tail bleed was performed to determine plasma TTR protein levels at baseline (BL). The mice were sacrificed 72 hours following the final administration. TTR protein levels were measured using a clinical analyzer (AU480, Beckman Coulter, CA).
Real-time PCR and RIBOGREENO RNA quantification reagent (Molecular Probes, Inc. Eugene, OR) were used according to standard protocols to determine liver human TTR mRNA levels. The results presented in Table 96 are the average values for each treatment group. The mRNA levels are the average values relative to the average for the PBS group. Plasma protein levels are the average values relative to the average value for the PBS group at baseline. "BL" indicates baseline, measurements that were taken just prior to the first dose. As illustrated in Table 96, treatment with antisense oligonucleotides lowered TTR expression levels in a dose-dependent manner. The oligonucleotides comprising a GalNAc conjugate were more potent than the parent lacking a GalNAc conjugate (ISIS 420915), and oligonucleotides comprising a phosphodiester or deoxyadenosine cleavable moiety showed significant improvements in potency compared to the parent lacking a conjugate (see ISIS numbers 682883 and 666943 vs 420915 and see Examples 86 and 87).

Table 95 Oligonucleotides targeting human TTR
GalNAc SEQ
Isis No. Sequence 5' to 3' Linkages CM
cluster ID No.
T mC T T G Gd I'd I'd Ad mCd Ad Td Gd Ad Ad 420915 es esesesesssss ssssss PS n/a n/a 156 AesTesmCesmCesmCe GalNAc3-3.,,,TesmCeorreorreoGeoGdsTdsTdsAdsmCdsAd s PS/P0 Ga1NAc3-3a PO 156 TdaGdaAdaAdsAeoTeomCeamCesmCe GalNAc3-3.,,,AdoTesmCeoTeoTeoGeoGdsTdsTdsAd s PS/P0 GalNAc3-3a Ad 160 mCdaAdaTdaGdaAdaAda AeoTeomCesmCesmCe GalNAC3-7,-0,AdoTesmCeoTeoTeoGeoGdsTdsTdsAd s PS/P0 GalNAc3-7a Ad 160 mCdaAdaTdsGdsAdsAdsAeoTeomCesmCesmCe GalNAc3-10._0,AdorresmCeorreorreoGeoGdsTdsTdsAd 682888 s PS/PO
GalNAc3-10 a Ad 160 mCdaAdaTdsGdsAdsAdsAeoTeomCesmCesmCe GalNAc3-13._0,AdoTesmCeorreorreoGeoGdsTdsTdsAd s PS/P0 GalNAc3-13a Ad 160 mCdaAdaTdsGdsAdsAdsAeoTeomCesmCesmCe The legend for Table 95 can be found in Example 74. The structure of Ga1NAc3-3a was shown in Example 39. The structure of GalNAc3-7a was shown in Example 48. The structure of GalNAc3-10a was shown in Example 46. The structure of GalNAc3-13a was shown in Example 62.
Table 96 Antisense inhibition of human TTR in vivo Isis No. Dosage (mg/kg) TTR mRNA (% PBS) TTR protein (% BL) GalNAc cluster CM
PBS n/a 100 124 n/a n/a 420915 20 71 86 n/a n/a 0.6 61 73 Ga1NAc3-3a PO

0.6 74 93 Ga1NAc3-3a Ad 0.6 60 97 Ga1NAc3-7a Ad 0.6 65 92 Ga1NAc3-10 a Ad 0.6 72 74 Ga1NAc3-13a Ad Example 91: Antisense inhibition in vivo by oligonucleotides targeting Factor VII comprising a Ga1NAc3 conjugate in non-human primates Oligonucleotides listed in Table 97 below were tested in a non-terminal, dose escalation study for antisense inhibition of Factor VII in monkeys.
Treatment Non-naïve monkeys were each injected subcutaneously on days 0, 15, and 29 with escalating doses of an oligonucleotide listed in Table 97 or with PBS. Each treatment group consisted of 4 males and 1 female. Prior to the first dose and at various time points thereafter, blood draws were performed to determine plasma Factor VII protein levels. Factor VII protein levels were measured by ELISA. The results presented in Table 98 are the average values for each treatment group relative to the average value for the PBS group at baseline (BL), the measurements taken just prior to the first dose. As illustrated in Table 98, treatment with antisense oligonucleotides lowered Factor VII expression levels in a dose-dependent manner, and the oligonucleotide comprising the GalNAc conjugate was significantly more potent in monkeys compared to the oligonucleotide lacking a GalNAc conjugate.
Table 97 Oligonucleotides targeting Factor VII
GalNAc SEQ
Isis No. Sequence 5' to 3' Linkages CM
cluster ID No.
AesTesGesmCesAesTdsGdsGdsTdsGdsAdsTdsGdsmCdsTds 407935 PS n/a n/a TesmCesTesGesAe GalNAc3-10.-0,AesTesGesmCesAesTdsGdsGdsrrdsGds 686892 PS GalNAc3-10a PO 161 ikdsTdsGdsmCdsTds TeamCesTesGesAe The legend for Table 97 can be found in Example 74. The structure of Ga1NAc3-10a was shown in Example 46.
Table 98 Factor VII plasma protein levels ISIS No. Day Dose (mg/kg) Factor VII (% BL) 0 n/a 100 22 n/a 92 36 n/a 46 43 n/a 43 22 n/a 29 36 n/a 15 43 n/a 11 Example 92: Antisense inhibition in primary hepatocytes by antisense oligonucleotides targeting Apo-CIII comprising a Ga1NAc3 conjugate Primary mouse hepatocytes were seeded in 96-well plates at 15,000 cells per well, and the oligonucleotides listed in Table 99, targeting mouse ApoC-III, were added at 0.46, 1.37, 4.12, or 12.35, 37.04, 111.11, or 333.33 nM or 1.00 [LM. After incubation with the oligonucleotides for 24 hours, the cells were lysed and total RNA was purified using RNeasy (Qiagen). ApoC-III mRNA
levels were determined using real-time PCR and RIBOGREENO RNA quantification reagent (Molecular Probes, Inc.) according to standard protocols. IC50 values were determined using Prism 4 software (GraphPad). The results show that regardless of whether the cleavable moiety was a phosphodiester or a phosphodiester-linked deoxyadensoine, the oligonucleotides comprising a GalNAc conjugate were significantly more potent than the parent oligonucleotide lacking a conjugate.
Table 99 Inhibition of mouse APOC-III expression in mouse primary hepatocytes ISIS,IC50 SEQ
Sequence (5 to 3') CM
No. (nM) ID
No.
440670 mCesAesGesmCesTesTdsTdsAdsTdsTdsAdsGdsGdsGdsAdsmCesAesGesmCesAe n/a 13.20 162 niCesAesGesniCesTesTdsrf dsAdsrf dsrf dsAdsGdsGdsGdsAdsmCes 661180 Ad 1.40 163 AesGesmCesAeo Ado' -GalNAc3-la GallsTAc3-3.,InCesAesGesmCesTesTasTasAdsTasTdsAdsGdsGdsGdsAdsmC
680771 es PO 0.70 162 /kesGesmCesAe GallsTAc3-7.,mCesAesGesmCesrresTdsrrdsAdsrrdsrrdsAdsGdsGdsGdsAdsinC
680772 es PO 1.70 162 /kesGesmCesAe GallsTAc3-10.,mCesAesGesmCesTesTdsrf dsAdsrrdsrrdsAdsGdsGdsGdsAdsmC
680773 es PO 2.00 162 /kesGesmCesAe GallsTAc3-13.,mCesAesGesmCesTesTdsrf dsAdsrrdsrrdsAdsGdsGdsGdsAdsmC
680774 es PO 1.50 162 /kesGesmCesAe GallsTAc3-3.,mCesAeoGeomCeorreorrdsrrdsAdsTdsrrdsAdsGdsGdsGdsAdsinCe PO < 0.46 162 AeoGesmCesAe GalNAC3-3a-0,AdomCesAesGesmCesTesTdsr-f dsAdsrf dsrrdsAdsGdsGdsGdsAdsAd mCesAesGesmCesAe mCesAesGesmCesT es rr dS rr dAdS dS ds Ads Gds Gds Gds Ads mC es 683733 Ad 2.50 163 AesGesmCesAeoAdo' -GalNAc3-19a The structure of Ga1NAc3-11 was shown previously in Example 9, Ga1NAc3-31 was shown in Example 39, Ga1NAc3-71 was shown in Example 48, Ga1NAc3-101 was shown in Example 46, Ga1NAc3-131 was shown in Example 62, and Ga1NAc3-191 was shown in Example 70.
Example 93: Antisense inhibition in vivo by oligonucleotides targeting SRB-1 comprising mixed wings and a 5'-Ga1NAc3 conjugate The oligonucleotides listed in Table 100 were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice.

Table 100 Modified ASOs targeting SRB-1 ISIS Sequences (5' to 3') Ga1NAc3 CM
SEQ
No. Cluster ID
No.
449093 TkaTkamCkaAdaGdaTdamCds AdsTas Gds AdsmCdsTasTIsmCksmCk n/a n/a 165 699806 GaINAc3-3a-0,TI,TIsmCksAdsGdsTdsmCds AdsTds GdsAdsmCds Ga1NAc3-3 a PO

m-TdsTIsksmCk 699807 GaINAc3-7a-0,TI,TIsmCksAdsGdsTdsmCds AdsTds GdsAdsmCds Ga1NAc3-7a PO

m-TdsTIsksmCk 699809 GaINAc3-7a-o, TiOlsmCksAdsGasTasmCds AdsTds Gds AdsmCds Ga1NAc3-7a PO

m-TdaTeaesmCe 699811 GaINAc3-7 = a-0,TesTesmCesAdsGasTasmCds AdsTas GasAdsmCds Ga1NAc3-7a PO

m TdsTIsksmCk 699813 GalNAc3-7 = a-o'rrksTasmCksAdsGasTasmCds AdsTas GasAdsmCds Ga1NAc3-7a PO

m TdsTIsCdsmCk 699815 Ga1NAc3-7a-0,TesTIsmCksAdsGdsTdsmCds AdsTds GdsAdsmCds Ga1NAc3-7a PO

m TdsTIsCksinCe The structure of Ga1NAc3-3a was shown previously in Example 39, and the structure of Ga1NAc3-7a was shown previously in Example 48. Subscripts: "e" indicates 2'-MOE modified nucleoside; "d" indicates [3-D-2'-deoxyribonucleoside; "k" indicates 6'-(S)-CH3 bicyclic nucleoside (cEt);
"s" indicates phosphorothioate internucleoside linkages (PS); "o" indicates phosphodiester internucleoside linkages (PO). Supersript "m"
indicates 5-methylcytosines.
Treatment Six to eight week old C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME) were injected subcutaneously once at the dosage shown below with an oligonucleotide listed in Table 100 or with saline.
Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration. Liver SRB-1 mRNA levels were measured using real-time PCR. SRB-1 mRNA levels were normalized to cyclophilin mRNA levels according to standard protocols. The results are presented as the average percent of SRB-1 mRNA levels for each treatment group relative to the saline control group. As illustrated in Table 101, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner, and the gapmer oligonucleotides comprising a GalNAc conjugate and having wings that were either full cEt or mixed sugar modifications were significantly more potent than the parent oligonucleotide lacking a conjugate and comprising full cEt modified wings.
Body weights, liver transaminases, total bilirubin, and BUN were also measured, and the average values for each treatment group are shown in Table 101. Body weight is shown as the average percent body weight relative to the baseline body weight (% BL) measured just prior to the oligonucleotide dose.

Table 101 SRB-1 mRNA, ALT, AST, BUN, and total bilirubin levels and body weights ISIS Dosage SRB-1 mRNA ALT ASTBil BUN
Body weight No. (mg/kg) (% PBS) (U/L) (U/L) (%
BL) PBS n/a 100 31 84 0.15 28 1 111 18 48 0.17 31 449093 3 94 20 43 0.15 26 36 19 50 0.12 29 104 0.1 114 23 58 0.13 26 699806 0.3 59 21 45 0.12 27 1 25 30 61 0.12 30 0.1 121 19 41 0.14 25 699807 0.3 73 23 56 0.13 26 1 24 22 69 0.14 25 0.1 125 23 57 0.14 26 699809 0.3 70 20 49 0.10 25 1 33 34 62 0.17 25 0.1 123 48 77 0.14 24 699811 0.3 94 20 45 0.13 25 1 66 57 104 0.14 24 0.1 95 20 58 0.13 28 699813 0.3 98 22 61 0.17 28 1 49 19 47 0.11 27 0.1 93 30 79 0.17 25 699815 0.3 64 30 61 0.12 26 1 24 18 41 0.14 25 Example 94: Antisense inhibition in vivo by oligonucleotides targeting SRB-1 comprising 2'-sugar 5 modifications and a 5'-GaINAc3 conjugate The oligonucleotides listed in Table 102 were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice.
Table 102 Modified ASOs targeting SRB-1 ISIS Sequences (5' to 3') Ga1NAc3 CM
SEQ
No. Cluster ID No.
353382 GesmCesTesTesmCesikasGasTasmCdsAds'r dsGdsAdsmCdsTdsTesmCesmCes n/a n/a TesTe 700989 GmsCmsUmsUmsCmsAdsGasTasmCdsAdsTdsGdsAdsmCdsTdsUmsCmsCms 1" n/a UmsUm 666904 Ga1NAc3-3a-o, GesmCesTesTesmCesAdsGdsTdsmCdsAdsTdsGdsAds Ga1NAc3-3a PO

mCdsTdsTesmCesmCesTesTe 700991 Ga1NAc3-7.70,GmsCinsUinsUmsCinsAdsGdsTdsmCdsAdsTdsGds Ga1NAc3-7a PO

AdsmCdsTdsUmsCmsCmsUmsUm 10 Subscript "m" indicates a 2'-0-methyl modified nucleoside. See Example 74 for complete table legend. The structure of Ga1NAc3-31 was shown previously in Example 39, and the structure of Ga1NAc3-7a was shown previously in Example 48.

Treatment The study was completed using the protocol described in Example 93. Results are shown in Table 103 below and show that both the 2'-MOE and 2'-0Me modified oligonucleotides comprising a GalNAc conjugate were significantly more potent than the respective parent oligonucleotides lacking a conjugate. The results of the body weights, liver transaminases, total bilirubin, and BUN
measurements indicated that the compounds were all well tolerated.
Table 103 SRB-1 mRNA
ISIS No. Dosage (mg/kg) SRB-1 mRNA (% PBS) PBS n/a 100 Example 95: Antisense inhibition in vivo by oligonucleotides targeting SRB-1 comprising bicyclic nucleosides and a 5'-Ga1NAc3 conjugate The oligonucleotides listed in Table 104 were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice.
Table 104 Modified ASOs targeting SRB-1 ISIS, SEQ
Sequences (5' to 3') CM
No. Cluster ID No 440762 TkamCkaAdaGdaTdamCdaAdaTdaGdaAdsmCdsTasTksmCk n/a n/a 137 666905 Ga1NAc3-3.-0,TIsmCksAdsGdsTdsmCdsAdsTdsGdsAdsmCdsTdsTIsmCk Ga1NAc3-3a 699782 Ga1NAc3-7.-0,TIsmCksAdsGdsTdsmCdsAdsTdsGdsAdsmCdsTdsTIsmCk Ga1NAc3-7a 699783 Ga1NAc3-39-0,TismCisAdsGasTasmCdsAdsTdsGdsAdsmCdsTdsTismCI Ga1NAc3-3a 653621 TiamCisAdsGasTasmCdsAdsTdsGdsAdsmCdsTdsrrlsmCloAdo,-GalNAC3-19 Ga1NAc3-1a Ad 138 439879 TgamCgaAdaGdaTdamCdaAdaTd GdaAdamCdaTdaTgamCg n/a n/a 137 699789 Ga1NAc3-39-0,TgsmC
gsAdsGdsTdsmCdsAdsTa GdsAdsmCdsTdsTgsmC g Ga1NAc3-3a PO 137 Subscript "g" indicates a fluoro-HNA nucleoside, subscript "1" indicates a locked nucleoside comprising a 2'-0-CH2-4' bridge. See the Example 74 table legend for other abbreviations. The structure of Ga1NAc3-1a was shown previously in Example 9, the structure of Ga1NAc3-3a was shown previously in Example 39, and the structure of Ga1NAc3-7a was shown previously in Example 48.
Treatment The study was completed using the protocol described in Example 93. Results are shown in Table 105 below and show that oligonucleotides comprising a GalNAc conjugate and various bicyclic nucleoside modifications were significantly more potent than the parent oligonucleotide lacking a conjugate and comprising bicyclic nucleoside modifications. Furthermore, the oligonucleotide comprising a GalNAc conjugate and fluoro-HNA modifications was significantly more potent than the parent lacking a conjugate and comprising fluoro-HNA modifications. The results of the body weights, liver transaminases, total bilirubin, and BUN measurements indicated that the compounds were all well tolerated.
Table 105 SRB-1 mRNA, ALT, AST, BUN, and total bilirubin levels and body weights ISIS No. Dosage (mg/kg) SRB-1 mRNA (% PBS) PBS n/a 100 0.1 105 666905 0.3 56 0.1 93 699782 0.3 63 0.1 105 699783 0.3 53 0.1 109 653621 0.3 82 0.1 82 699789 0.3 69 Example 96: Plasma protein binding of antisense oligonucleotides comprising a GaINAc3 conjugate group Oligonucleotides listed in Table 70 targeting ApoC-III and oligonucleotides in Table 106 targeting Apo(a) were tested in an ultra-filtration assay in order to assess plasma protein binding.

Table 106 Modified oligonucleotides targeting Apo(a) ISISGalNAc3 SEQ
Sequences (5' to 3') CM
No. Cluster ID No TesGesmCesTesmCesmCdsGdsTdsTdsGdsGdsTdsGdsmCdsTdsTesGesTes 494372 n/a n/a 5 8 TesmCe TesGeomCeoTeomCeomCdsGdsTdsTdsGdsGdsTdsGdsmCdsTdsTeoGeoTes 693401 n/a n/a 58 TesmCe GalNAC3-7a-o'TesGesmCesTesmCesmCdsGdsTdsTdsGdsGdsTdsGdsmCds GalNAc3-7a PO

TdsTesGesTesTesmCe GalNAC3-7a-o'TesGeomCeoTeomCeomCdsGdsTdsTdsGdsGdsTdsGdsmCds GalNAc3-7a PO

TdsTeoGeoTesTesmCe See the Example 74 for table legend. The structure of Ga1NAc3-7a was shown previously in Example 48.
Ultrafree-MC ultrafiltration units (30,000 NMWL, low-binding regenerated cellulose membrane, Millipore, Bedford, MA) were pre-conditioned with 300 [tt of 0.5% Tween 80 and centrifuged at 2000 g for minutes, then with 300 L of a 300 [tg/mL solution of a control oligonucleotide in H20 and centrifuged at 2000 g for 16 minutes. In order to assess non-specific binding to the filters of each test oligonucleotide from Tables 70 and 106 to be used in the studies, 300 [tt of a 250 ng/mL solution of oligonucleotide in H20 at pH
7.4 was placed in the pre-conditioned filters and centrifuged at 2000 g for 16 minutes. The unfiltered and 10 filtered samples were analyzed by an ELISA assay to determine the oligonucleotide concentrations. Three replicates were used to obtain an average concentration for each sample. The average concentration of the filtered sample relative to the unfiltered sample is used to determine the percent of oligonucleotide that is recovered through the filter in the absence of plasma (% recovery).
Frozen whole plasma samples collected in K3-EDTA from normal, drug-free human volunteers, cynomolgus monkeys, and CD-1 mice, were purchased from Bioreclamation LLC
(Westbury, NY). The test oligonucleotides were added to 1.2 mL aliquots of plasma at two concentrations (5 and 150 [tg/mL). An aliquot (300 [tt) of each spiked plasma sample was placed in a pre-conditioned filter unit and incubated at 37 C for 30 minutes, immediately followed by centrifugation at 2000 g for 16 minutes. Aliquots of filtered and unfiltered spiked plasma samples were analyzed by an ELISA to determine the oligonucleotide concentration in each sample. Three replicates per concentration were used to determine the average percentage of bound and unbound oligonucleotide in each sample. The average concentration of the filtered sample relative to the concentration of the unfiltered sample is used to determine the percent of oligonucleotide in the plasma that is not bound to plasma proteins (%
unbound). The final unbound oligonucleotide values are corrected for non-specific binding by dividing the % unbound by the % recovery for each oligonucleotide. The final % bound oligonucleotide values are determined by subtracting the final %
unbound values from 100. The results are shown in Table 107 for the two concentrations of oligonucleotide tested (5 and 150 [tg/mL) in each species of plasma. The results show that GalNAc conjugate groups do not have a significant impact on plasma protein binding. Furthermore, oligonucleotides with full PS

internucleoside linkages and mixed PO/PS linkages both bind plasma proteins, and those with full PS
linkages bind plasma proteins to a somewhat greater extent than those with mixed PO/PS linkages.
Table 107 Percent of modified oligonucleotide bound to plasma proteins ISIS Human plasma Monkey plasma Mouse plasma No. 5 ug/mL 150 ug/mL 5 ug/mL 150 ug/mL 5 ug/mL 150 ug/mL
304801 99.2 98.0 99.8 99.5 98.1 97.2 663083 97.8 90.9 99.3 99.3 96.5 93.0 674450 96.2 97.0 98.6 94.4 94.6 89.3 494372 94.1 89.3 98.9 97.5 97.2 93.6 693401 93.6 89.9 96.7 92.0 94.6 90.2 681251 95.4 93.9 99.1 98.2 97.8 96.1 681257 93.4 90.5 97.6 93.7 95.6 92.7 Example 97: Modified oligonucleotides targeting TTR comprising a GaINAc3 conjugate group The oligonucleotides shown in Table 108 comprising a GalNAc conjugate were designed to target TTR.
Table 108 Modified oligonucleotides targeting TTR
Ga1NAc3 SEQ ID
ISIS No. Sequences (5' to 3') CM
Cluster No GalNAc3-3a_0,Ado Tes mCes Tes Tes Ges Gds Tds Tds Ads mCds GalNAc3-3 Ad Ads Tds Gds Ads Ads Aes Tes mCes mCes mCe Tes mCeo Teo Teo Gmeo Gnsid Tds TdsAds mCds Ads Tds Gds Ads Ads GalNAc3-1 Ad 157 Aeo Teo Ces Ces Ceo Ado'-Ga1NAc3-3a Ga1NAc3-3a_0,Tes mCes Tes Tes Ges Gds Tds Tds Ads mCds Ads Tds GalNAc3-3 Gds Ads Ads Aes Tes mCes mCes mCe Ga1NAc3-7a_0,Tes mCes Tes Tes Ges Gds Tds Tds Ads mCds Ads Tds GalNAc3-7 Gds Ads Ads Aes T es mCes mCes mCe GalNAC3-10a_0,Tes mCes Tes Tes Ges Gds Tds Tds Ads mCds Ad 682878 s Ga1NAc3-10 PO 156 Tds Gds Ads Ads Aes Tes mCes mCes mCe GalNAC3-13a_0,Tes mCes Tes Tes Ges Gds Tds Tds Ads mCds Ad.682879 Ga1NAc3-13 PO 156 Tds Gds Ads Ads Aes Tes mCes mCes mCe Ga1NAC3-7a-0,Ado Tes mCes Tes Tes Ges Gds Tds Tds Ads mCdsGalNAc3-7 Ad Ads Tds Gds Ads Ads Aes Tes mCes mCes mCe Ga1NAC3-10a_0,Ado Tes mCes Tes Tes Ges Gds Tds Tds Ads mCdsGalNAc3-10 Ad Ads Tds Gds Ads Ads Aes Tes mCes mCes mCe Ga1NAC3-13a_0,Ado Tes mCes Tes Tes Ges Gds Tds Tds Ads mCdsGalNAc3-13 Ad Ads Tds Gds Ads Ads Aes Tes mCes mCes mCe Tes mCes Tes Tes Ges Gmds Tds TdsAds mCds Ads Tds Gds Ads Ads GalNAc3-19 Ad 157 Aes Tes Ces Ces Ceo Ado'-Ga1NAc3-19 a The legend for Table 108 can be found in Example 74. The structure of Ga1NAc3-1 was shown in Example 9.
The structure of Ga1NAc3-3a was shown in Example 39. The structure of Ga1NAc3-7a was shown in Example 48. The structure of Ga1NAc3-10a was shown in Example 46. The structure of Ga1NAc3-13a was shown in Example 62. The structure of GalNAc3-19a was shown in Example 70.

Example 98: Evaluation of pro-inflammatory effects of oligonucleotides comprising a GaINAc conjugate in hPMBC assay The oligonucleotides listed in Table 109 and were tested for pro-inflammatory effects in an hPMBC
assay as described in Examples 23 and 24. (See Tables 30, 83, 95, and 108 for descriptions of the oligonucleotides.) ISIS 353512 is a high responder used as a positive control, and the other oligonucleotides are described in Tables 83, 95, and 108. The results shown in Table 109 were obtained using blood from one volunteer donor. The results show that the oligonucleotides comprising mixed PO/PS internucleoside linkages produced significantly lower pro-inflammatory responses compared to the same oligonucleotides having full PS linkages. Furthermore, the GalNAc conjugate group did not have a significant effect in this assay.
Table 109 ISIS No. Einax/EC50 Ga1NAc3 cluster Linkages CM
353512 3630 n/a PS n/a 420915 802 n/a PS n/a 682881 1311 Ga1NAc3-10 PS Ad 682888 0.26 Ga1NAc3-10 PO/PS Ad 684057 1.03 Ga1NAc3-19 PO/PS Ad Example 99: Binding affinities of oligonucleotides comprising a GaINAc conjugate for the asialoglycoprotein receptor The binding affinities of the oligonucleotides listed in Table 110 (see Table 76 for descriptions of the oligonucleotides) for the asialoglycoprotein receptor were tested in a competitive receptor binding assay. The competitor ligand, al-acid glycoprotein (AGP), was incubated in 50 mM sodium acetate buffer (pH 5) with 1 U neuraminidase-agarose for 16 hours at 37 C, and > 90% desialylation was confirmed by either sialic acid assay or size exclusion chromatography (SEC). Iodine monochloride was used to iodinate the AGP according to the procedure by Atsma et al. (see J Lipid Res. 1991 Jan; 32(1):173-81.) In this method, desialylated al -acid glycoprotein (de-AGP) was added to 10 mM iodine chloride, Na1251, and 1 M
glycine in 0.25 M NaOH.
After incubation for 10 minutes at room temperature, 1251 -labeled de-AGP was separated from free 1251 by concentrating the mixture twice utilizing a 3 KDMWCO spin column. The protein was tested for labeling efficiency and purity on a HPLC system equipped with an Agilent SEC-3 column (7.8x300mm) and a 13-RAM 1251 counter. Competition experiments utilizing -labeled de-AGP and various GalNAc-cluster containing ASOs were performed as follows. Human HepG2 cells (106 cells/m1) were plated on 6-well plates in 2 ml of appropriate growth media. MEM media supplemented with 10% fetal bovine serum (FBS), 2 mM
L-Glutamine and 10mM HEPES was used. Cells were incubated 16-20 hours @ 37 C
with 5% and 10% CO2 respectively. Cells were washed with media without FBS prior to the experiment. Cells were incubated for 30 min @37 C with lml competition mix containing appropriate growth media with 2%
FBS, 10-8 M 1251 _ labeled de-AGP and GalNAc-cluster containing ASOs at concentrations ranging from 10-11 to 10-5 M. Non-specific binding was determined in the presence of 10-2 M GalNAc sugar. Cells were washed twice with media without FBS to remove unbound 1251 -labeled de-AGP and competitor GalNAc ASO. Cells were lysed using Qiagen's RLT buffer containing 1% 13-mercaptoethanol. Lysates were transferred to round bottom assay tubes after a brief 10 min freeze/thaw cycle and assayed on a y-counter.
Non-specific binding was subtracted before dividing 1251 protein counts by the value of the lowest GalNAc-ASO concentration counts.
The inhibition curves were fitted according to a single site competition binding equation using a nonlinear regression algorithm to calculate the binding affinities (KD's).
The results in Table 110 were obtained from experiments performed on five different days. Results for oligonucleotides marked with superscript "a" are the average of experiments run on two different days.
The results show that the oligonucleotides comprising a GalNAc conjugate group on the 5'-end bound the asialoglycoprotein receptor on human HepG2 cells with 1.5 to 16-fold greater affinity than the oligonucleotides comprising a GalNAc conjugate group on the 3'-end.
Table 110 Asialoglycoprotein receptor binding assay results Oligonucleotide end to ISIS No. GalNAc conjugate which GalNAc conjugate KD (nM) is attached 661161' Ga1NAc3-3 5' 3.7 666881' Ga1NAc3-10 5' 7.6 666981 Ga1NAc3-7 5' 6.0 670061 Ga1NAc3-13 5' 7.4 655861' Ga1NAc3-1 3' 11.6 677841' Ga1NAc3-19 3' 60.8 Example 100: Antisense inhibition in vivo by oligonucleotides comprising a GaINAc conjugate group targeting Apo(a) in vivo The oligonucleotides listed in Table 111a below were tested in a single dose study for duration of action in mice.
Table 111a Modified ASOs targeting APO(a) ISISGalNAc3 SEQ
Sequences (5' to 3') CM
No. Cluster ID
No.
GalNAc3-7a-0,TeaGeamCeaTeamCeamCdsGasTasTasGasGas 681251 Ga1NAc3-7a PO 58 TdsGdsmCdsTdsTesGes TesTesmCe GalNAC3-7a-o'TesGeomCeoTeomCeomCdsGdsTdsTdsGdsGds 681257 Ga1NAc3-7a PO 58 TdsGdsmCdsTdsTeoGeo TesTesmCe The structure of GalNAc3-7a was shown in Example 48.

Treatment Female transgenic mice that express human Apo(a) were each injected subcutaneously once per week, for a total of 6 doses, with an oligonucleotide and dosage listed in Table 111b or with PBS. Each treatment group consisted of 3 animals. Blood was drawn the day before dosing to determine baseline levels of Apo(a) protein in plasma and at 72 hours, 1 week, and 2 weeks following the first dose. Additional blood draws will occur at 3 weeks, 4 weeks, 5 weeks, and 6 weeks following the first dose. Plasma Apo(a) protein levels were measured using an ELISA. The results in Table 111b are presented as the average percent of plasma Apo(a) protein levels for each treatment group, normalized to baseline levels (% BL), The results show that the oligonucleotides comprising a GalNAc conjugate group exhibited potent reduction in Apo(a) expression. This potent effect was observed for the oligonucleotide that comprises full PS internucleoside linkages and the oligonucleotide that comprises mixed PO and PS linkages.
Table 111b Apo(a) plasma protein levels Apo(a) at 72 hours Apo(a) at 1 week Apo(a) at 3 weeks ISIS No. Dosage (mg/kg) (% BL) (% BL) (% BL) PBS n/a 116 104 0.3 97 108 93 681251 1.0 85 77 57 3.0 54 49 11 10.0 23 15 4 0.3 114 138 6812 1.0 91 98 54 3.0 69 40 6 10.0 30 21 4 Example 101: Antisense inhibition by oligonucleotides comprising a GaINAc cluster linked via a stable moiety The oligonucleotides listed in Table 112 were tested for inhibition of mouse APOC-III expression in vivo. C57B1/6 mice were each injected subcutaneously once with an oligonucleotide listed in Table 112 or with PBS. Each treatment group consisted of 4 animals. Each mouse treated with ISIS 440670 received a dose of 2, 6, 20, or 60 mg/kg. Each mouse treated with ISIS 680772 or 696847 received 0.6, 2, 6, or 20 mg/kg. The GalNAc conjugate group of ISIS 696847 is linked via a stable moiety, a phosphorothioate linkage instead of a readily cleavable phosphodiester containing linkage. The animals were sacrificed 72 hours after the dose. Liver APOC-III mRNA levels were measured using real-time PCR. APOC-III mRNA
levels were normalized to cyclophilin mRNA levels according to standard protocols. The results are presented in Table 112 as the average percent of APOC-III mRNA levels for each treatment group relative to the saline control group. The results show that the oligonucleotides comprising a GalNAc conjugate group were significantly more potent than the oligonucleotide lacking a conjugate group. Furthermore, the oligonucleotide comprising a GalNAc conjugate group linked to the oligonucleotide via a cleavable moiety (ISIS 680772) was even more potent than the oligonucleotide comprising a GalNAc conjugate group linked to the oligonucleotide via a stable moiety (ISIS 696847).
Table 112 Modified oligonucleotides targeting mouse APOC-III
Dosage APOC-III
ISIS
SEQ
N Sequences (5' to 3') CM (mg/kg) mRNA (%
No.
ID
o.
PBS) mCesAesGesmCesTesTdsrrdsAdsrrdsrrdsAds 6 86 440670 n/a GdsGdsGdsAdsmCes AesGes mCesAe 20 59 0.6 79 GalNAc3-7._0,mCesAesGesmCesTesTdsrrdsAds 2 58 TdsTdsAdsGds GdsGdsAdsmCes ikesGesmCesAe 6 31 0.6 83 Ga1NAc3-7._s,mCesAesGesmCesTesTdsrrdsAdsrrds n/a (PS 2 7 ) TdsAdsGdsGdsGdsAdsmCesikesGesmCesAe 6 40 The structure of GalNAc3-7a was shown in Example 48.
Example 102: Distribution in liver of antisense oligonucleotides comprising a GaINAc conjugate The liver distribution of ISIS 353382 (see Table 36) that does not comprise a GalNAc conjugate and ISIS 655861 (see Table 36) that does comprise a GalNAc conjugate was evaluated. Male balb/c mice were subcutaneously injected once with ISIS 353382 or 655861 at a dosage listed in Table 113. Each treatment group consisted of 3 animals except for the 18 mg/kg group for ISIS 655861, which consisted of 2 animals.
The animals were sacrificed 48 hours following the dose to determine the liver distribution of the oligonucleotides. In order to measure the number of antisense oligonucleotide molecules per cell, a Ruthenium (II) tris-bipyridine tag (MSD TAG, Meso Scale Discovery) was conjugated to an oligonucleotide probe used to detect the antisense oligonucleotides. The results presented in Table 113 are the average concentrations of oligonucleotide for each treatment group in units of millions of oligonucleotide molecules per cell. The results show that at equivalent doses, the oligonucleotide comprising a GalNAc conjugate was present at higher concentrations in the total liver and in hepatocytes than the oligonucleotide that does not comprise a GalNAc conjugate. Furthermore, the oligonucleotide comprising a GalNAc conjugate was present at lower concentrations in non-parenchymal liver cells than the oligonucleotide that does not comprise a GalNAc conjugate. And while the concentrations of ISIS 655861 in hepatocytes and non-parenchymal liver cells were similar per cell, the liver is approximately 80% hepatocytes by volume. Thus, the majority of the ISIS 655861 oligonucleotide that was present in the liver was found in hepatocytes, whereas the majority of the ISIS 353382 oligonucleotide that was present in the liver was found in non-parenchymal liver cells.

Table 113 Concentration in whole Concentration in Concentration in non-ISIS Dosage liver (molecules*10^
N 6 hepatocytes parenchymal liver cells o. mg/kg) ( per cell) (molecules*10^6 per cell) (molecules*10^6 per cell) 3 9.7 1.2 37.2 17.3 4.5 34.0 23.6 6.6 65.6 29.1 11.7 80.0 60 73.4 14.8 98.0 90 89.6 18.5 119.9 0.5 2.6 2.9 3.2 1 6.2 7.0 8.8 655861 3 19.1 25.1 28.5 6 44.1 48.7 55.0 18 76.6 82.3 77.1 Example 103: Duration of action in vivo of oligonucleotides targeting APOC-III
comprising a GaINAc3 5 conjugate The oligonucleotides listed in Table 114 below were tested in a single dose study for duration of action in mice.
Table 114 Modified ASOs targeting APOC-III
ISIS Sequences (5' to 3') Ga1NAc3 CM
SEQ
No. Cluster ID No.
304801 AesGesmCesTesTesmCdsTdsTdsGdsTdsmCdsmCdsAdsGdsmCdsTesTes n/a n/a 135 TesAesTe 663084 Ga1NAc3-3.-0,AdoAesGeomCeoTeoTeomCdsTdsTdsGdsTdsmCds Ga1NAc3-3a Ad 151 mCdsAdsGdsmCdsTeoTeo TesAesTe 679241 AesGeomCeoTeoTeomCdsTdsTdsGdsTdsmCdsmCdsAdsGdsmCdsTeoTeo Ga1NAc3-19a Ad TesAesTeoAdo'-GaINAc3-19.
The structure of GalNAc3-3a was shown in Example 39, and Ga1NAc3-19a was shown in Example 70.
Treatment Female transgenic mice that express human APOC-III were each injected subcutaneously once with an oligonucleotide listed in Table 114 or with PBS. Each treatment group consisted of 3 animals. Blood was drawn before dosing to determine baseline and at 3, 7, 14, 21, 28, 35, and 42 days following the dose. Plasma triglyceride and APOC-III protein levels were measured as described in Example 20. The results in Table 115 are presented as the average percent of plasma triglyceride and APOC-III
levels for each treatment group, normalized to baseline levels. A comparison of the results in Table 71 of example 79 with the results in Table 115 below show that oligonucleotides comprising a mixture of phosphodiester and phosphorothioate internucleoside linkages exhibited increased duration of action than equivalent oligonucleotides comprising only phosphorothioate internucleoside linkages.
Table 115 Plasma triglyceride and APOC-III protein levels in transgenic mice Time point APOC-III
ISIS Dosage Triglycerides Ga1NAc3 CM
(days post- protein (%
No. (mg/kg) (% baseline) Cluster dose) baseline) PBS n/a 21 69 92 n/a n/a 304801 30 21 67 81 n/a n/a 663084 10 21 23 29 Ga1NAc3-3a Ad GalNAc3-Ad 19a Example 104: Synthesis of oligonucleotides comprising a 5'-Ga1NAc2 conjugate HN,Boc HN-Bc)c Boc\.N OH H2No 0 ..
H HBTU, HOBt DIEA, DMF 0 ___________________________________________ )10"- Boc.N EN11.....õ..".õ.õ--,..õ.11..o 0 H TFA

DCM

120 126 85% 231 HN Ed 0 +

AcO-L-:-D-\--0......õko 411) F DIEA
-70.-0 101 AcHN F DMF

OAc/..- OAc 0 OAc OAc ----Z-0, Q
AcHN NH ..24, AcHN NH
1 H2, Pd/C, Me0H
_____________________________________________________________________________ )11.-F
OAc 11.-- OAc 2 PFPTFA , DMF OAc OAc 0 H 0 lk-----4-.0 0\
F F
0r H 0 AcHN AcHN
F

F

0 83e OHOH
3' 5', 11 --0 0 ( OLIGO j-0-P-0-(CH2)6-NH2 __ HO
01H AcHN
1 Borate buffer, DMSO, pH 8.5, rt OHOH
HO /
2 aq ammonia, ...A. N
rt AcHN N

Compound 120 is commercially available, and the synthesis of compound 126 is described in Example 49. Compound 120 (1 g, 2.89 mmol), HBTU (0.39 g, 2.89 mmol), and HOBt (1.64 g, 4.33 mmol) were dissolved in DMF (10 mL. and N,N-diisopropylethylamine (1.75 mL, 10.1 mmol) were added. After about 5 min, aminohexanoic acid benzyl ester (1.36 g, 3.46 mmol) was added to the reaction. After 3h, the reaction mixture was poured into 100 mL of 1 M NaHSO4 and extracted with 2 x 50 mL ethyl acetate.
Organic layers were combined and washed with 3 x 40 mL sat NaHCO3 and 2 x brine, dried with Na2SO4, filtered and concentrated. The product was purified by silica gel column chromatography (DCM:EA:Hex , 1:1:1) to yield compound 231. LCMS and NMR were consistent with the structure.
Compounds 231 (1.34 g, 2.438 mmol) was dissolved in dichloromethane (10 mL) and trifluoracetic acid (10 mL) was added. After stirring at room temperature for 2h, the reaction mixture was concentrated under reduced pressure and co-evaporated with toluene ( 3 x 10 mL). The residue was dried under reduced pressure to yield compound 232 as the trifuloracetate salt. The synthesis of compound 166 is described in Example 54. Compound 166 (3.39 g, 5.40 mmol) was dissolved in DMF (3 mL). A solution of compound 232 (1.3 g, 2.25 mmol) was dissolved in DMF (3 mL) and N,N-diisopropylethylamine (1.55 mL) was added. The reaction was stirred at room temperature for 30 minutes, then poured into water (80 mL) and the aqueous layer was extracted with Et0Ac (2x100 mL). The organic phase was separated and washed with sat. aqueous NaHCO3 (3 x 80 mL), 1 M NaHSO4 (3 x 80 mL) and brine (2 x 80 mL), then dried (Na2SO4), filtered, and concentrated. The residue was purified by silica gel column chromatography to yield compound 233. LCMS
and NMR were consistent with the structure. Compound 233 (0.59 g, 0.48 mmol) was dissolved in methanol (2.2 mL) and ethyl acetate (2.2 mL). Palladium on carbon (10 wt% Pd/C, wet, 0.07 g) was added, and the reaction mixture was stirred under hydrogen atmosphere for 3 h. The reaction mixture was filtered through a pad of Celite and concentrated to yield the carboxylic acid. The carboxylic acid (1.32 g, 1.15 mmol, cluster free acid) was dissolved in DMF (3.2 mL). To this N,N-diisopropylehtylamine (0.3 mL, 1.73 mmol) and PFPTFA (0.30 mL, 1.73 mmol) were added. After 30 min stirring at room temperature the reaction mixture was poured into water (40 mL) and extracted with Et0Ac (2 x 50 mL). A standard work-up was completed as described above to yield compound 234. LCMS and NMR were consistent with the structure.
Oligonucleotide 235 was prepared using the general procedure described in Example 46. The Ga1NAc2 cluster portion (Ga1NAc2-24a) of the conjugate group Ga1NAc2-24 can be combined with any cleavable moiety present on the oligonucleotide to provide a variety of conjugate groups. The structure of Ga1NAc2-24 (Ga1NAc2-24a-CM) is shown below:
r_H OH

AcHN NH
r.-H OH

NrEN1NO
AcHN

Example 105: Synthesis of oligonucleotides comprising a Ga1NAc1-25 conjugate 0 83e 3'5', 11 OAc OAc F
OLIGO O¨P-0¨(CH2)e-NFI2 Ac0 F = F *,_ 0 OH
1. Borate buffer, DMSO, pH 8.5, rt AcHN
166 2. aq. ammonia, rt OH OH

CM OLIGO

AcHN H 6 The synthesis of compound 166 is described in Example 54. Oligonucleotide 236 was prepared using the general procedure described in Example 46.
Alternatively, oligonucleotide 236 was synthesized using the scheme shown below, and compound 238 was used to form the oligonucleotide 236 using procedures described in Example 10.

HNOH OAc 7,5 2 ._...\......\,0Ac 0:5____\......\õ

, Ac0 N.---N.,....N.),( -F PFPTFA 239 Ac0 ,...õ..N.,õN}......
NHAc OH NHAc N...--..,............õ.........,......, TEA, Acetonitrile H

OAc 0:5....\......\õ
tetrazole, 1-Methylimidazole, DMF

_______________________________ ..- Ac0 0 11 2-cyanoethyltetraisopropyl phosphorod iam id ite NHAcNP-NI
o1 LCN
Oligonucleotide OH OH
synthesis H0*._ 0 ___________________________________________________________ , AcHN CM )¨ OLIGO
N " 0 H 6 _______________________________________________________ , The GalNAci cluster portion (GalNAci-25,) of the conjugate group GalNAc1-25 can be combined with any cleavable moiety present on the oligonucleotide to provide a variety of conjugate groups. The structure of Ga1NAc1-25 (GalNAci-25a-CM) is shown below:
OH OH
4)No1133 AcHN H 6 Example 106: Antisense inhibition in vivo by oligonucleotides targeting SRB-1 comprising a 5'-Ga1NAc2or a 5'-Ga1NAc3 conjugate Oligonucleotides listed in Tables 116 and 117 were tested in dose-dependent studies for antisense inhibition of SRB-1 in mice.
Treatment Six to week old, male C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME) were injected subcutaneously once with 2, 7, or 20 mg/kg of ISIS No. 440762; or with 0.2, 0.6, 2, 6, or 20 mg/kg of ISIS
No. 686221, 686222, or 708561; or with saline. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration. Liver SRB-1 mRNA
levels were measured using real-time PCR. SRB-1 mRNA levels were normalized to cyclophilin mRNA levels according to standard protocols. The antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner, and the ED50 results are presented in Tables 116 and 117. Although previous studies showed that trivalent GalNAc-conjugated oligonucleotides were significantly more potent than divalent GalNAc-conjugated oligonucleotides, which were in turn significantly more potent than monovalent GalNAc conjugated oligonucleotides (see, e.g., Khorev et al., Bioorg. & Med. Chem., Vol. 16, 5216-5231 (2008)), treatment with antisense oligonucleotides comprising monovalent, divalent, and trivalent GalNAc clusters lowered SRB-1 mRNA levels with similar potencies as shown in Tables 116 and 117.
Table 116 Modified oligonucleotides targeting SRB-1 ISIS,ED5o SEQ
Sequences (5 to 3') GalNAc Cluster No.
(mg/kg) ID No 440762 TkamCkaAdaGdaTdsmCdsAdsr-f dsGdsAdsmCdsTdsTksmCk n/a 4.7 137 GalNAc2-24a-0,AdoTkamCksAdsGasTasmCdsAdsrrdsGasAds 686221 Ga1NAc2-24a 0.39 141 mCdsTdsTksmCk GalNAc3-13a-0,AdoTIcsmCksAdsGdsTdsmCdsAdsTdsGdsAds 686222 Ga1NAc3-13a 0.41 141 mCdsTdsTksmCk See Example 93 for table legend. The structure of Ga1NAc3-13a was shown in Example 62, and the structure of GalNAc2-24a was shown in Example 104.
Table 117 Modified oligonucleotides targeting SRB-1 ISIS,ED5o SEQ
Sequences (5 to 3') GalNAc Cluster No.
(mg/kg) ID No 440762 TkamCkaAdaGdaTdsmCdsAdsr-f dsGdsAdsmCdsTdsTksmCk n/a 5 137 GalNAci-25a-0,TksmCksAdsGasTasmCdsAdsTasGasAds 708561 Ga1NAc1-25a 0.4 137 mCm daTdaTkaCk See Example 93 for table legend. The structure of GalNAci-25a was shown in Example 105.
The concentrations of the oligonucleotides in Tables 116 and 117 in liver were also assessed, using procedures described in Example 75. The results shown in Tables 117a and 117b below are the average total antisense oligonucleotide tissues levels for each treatment group, as measured by UV in units of [tg oligonucleotide per gram of liver tissue. The results show that the oligonucleotides comprising a GalNAc conjugate group accumulated in the liver at significantly higher levels than the same dose of the oligonucleotide lacking a GalNAc conjugate group. Furthermore, the antisense oligonucleotides comprising one, two, or three GalNAc ligands in their respective conjugate groups all accumulated in the liver at similar levels. This result is surprising in view of the Khorev et al. literature reference cited above and is consistent with the activity data shown in Tables 116 and 117 above.
Table 117a Liver concentrations of oligonucleotides comprising a Ga1NAc2 or Ga1NAc3 conjugate group Dosage ISIS No. [Antisense oligonucleotide] (m/g) GalNAc cluster CM
(mg/kg) 2 2.1 440762 7 13.1 n/a n/a 20 31.1 0.2 0.9 2.7 686221 0.6 Ga1NAc2-24a Ad 2 12.0 6 26.5 0.2 0.5 0.6 1.6 686222 Ga1NAc3-13a Ad 2 11.6 6 19.8 Table 117b Liver concentrations of oligonucleotides comprising a GalNAci conjugate group Dosage ISIS No. [Antisense oligonucleotide] (pg/g) GalNAc cluster CM
(mg/kg) 2 2.3 440762 7 8.9 n/a n/a 20 23.7 0.2 0.4 0.6 1.1 708561 2 5.9 Ga1NAc1-25a PO
6 23.7 20 53.9 Example 107: Synthesis of oligonucleotides comprising a Ga1NAc1-26 or Ga1NAc1-27 conjugate OH A.

HO CM ago ' ___.r.....\, ______________________ HO W---...Nr----AcHN

...----OH
Oligonucleotide 239 is synthesized via coupling of compound 47 (see Example 15) to acid 64 (see Example 32) using HBTU and DIEA in DMF. The resulting amide containing compound is phosphitylated, then added to the 5'-end of an oligonucleotide using procedures described in Example 10. The GalNAci cluster portion (GalNAci-26a) of the conjugate group GalNAc1-26 can be combined with any cleavable moiety present on the oligonucleotide to provide a variety of conjugate groups. The structure of Ga1NAci-26 (GalNAci-26a-CM) is shown below:
HCO MI
H__............\, 0 HO C)/\/\/\ N17'"µC) AcHN...----OH .
In order to add the GalNAci conjugate group to the 3'-end of an oligonucleotide, the amide formed from the reaction of compounds 47 and 64 is added to a solid support using procedures described in Example 7. The oligonucleotide synthesis is then completed using procedures described in Example 9 in order to form oligonucleotide 240.
HO OH
O
HO
ON ,OH
AcHN
3' 04 CM Oligo The GalNAci cluster portion (GalNAci-27a) of the conjugate group Ga1NAc1-27 can be combined with any cleavable moiety present on the oligonucleotide to provide a variety of conjugate groups. The structure of Ga1NAc1-27 (GalNAci-27a-CM) is shown below:

AcHN

Example 108: Antisense inhibition in vivo by oligonucleotides comprising a GaINAc conjugate group targeting Apo(a) in vivo The oligonucleotides listed in Table 118 below were tested in a single dose study in mice.
Table 118 Modified ASOs targeting APO(a) ISISGalNAc3 SEQ
Sequences (5' to 3') CM
No. Cluster ID No.
TesGesmCesTesmCesmCdsGdsTdsTdsGdsGdsTdsGdsmCds 494372 n/a n/a 58 TdsTesGesTesTesmCe GalNAC3-7a-o'TesGesmCesTesmCesmCdsGdsTdsTdsGdsGds Ga1NAc3-7a PO 58 TdsGdsmCdsTdsTesGes TesTesmCe GalNAc3-3a-0,TesGeomCeoTeomCeomCdsGdsTdsTdsGdsGds Ga1NAc3-3a PO 58 TdsGdsmCdsTdsTeoGeo TesTesmCe GalNAc3-10a-0,TesGeomCeoTeomCeomCdsGdsTdsTdsGdsGds 681256 Ga1NAc3-10 a PO

TdsGdsmCdsTdsTeoGeo TesTesmCe GalNAC3-7a-o'TesGeomCeoTeomCeomCdsGdsTdsTdsGdsGds Ga1NAc3-7a PO 58 TdsGdsmCdsTdsTeoGeo TesTesmCe GalNAc3-13a-0,TesGeomCeoTeomCeomCdsGdsTdsTdsGdsGds Ga1NAc3-13a PO 58 TdsGdsmCdsTdsTeoGeo TesTesmCe TesGeomCeoTeomCeomCdsGdsTdsTdsGdsGds TdsGdsmCdsTdsTeoGeo Ga1NAc3-19a Ad 167 TesTesmCeoAdo,-Ga1NAc3-19 The structure of GalNAc3-7a was shown in Example 48.

Treatment Male transgenic mice that express human Apo(a) were each injected subcutaneously once with an oligonucleotide and dosage listed in Table 119 or with PBS. Each treatment group consisted of 4 animals.
Blood was drawn the day before dosing to determine baseline levels of Apo(a) protein in plasma and at 1 week following the first dose. Additional blood draws will occur weekly for approximately 8 weeks. Plasma Apo(a) protein levels were measured using an ELISA. The results in Table 119 are presented as the average percent of plasma Apo(a) protein levels for each treatment group, normalized to baseline levels (% BL), The results show that the antisense oligonucleotides reduced Apo(a) protein expression. Furthermore, the oligonucleotides comprising a GalNAc conjugate group exhibited even more potent reduction in Apo(a) expression than the oligonucleotide that does not comprise a conjugate group.
Table 119 Apo(a) plasma protein levels ISIS No. Dosage (mg/kg) Apo(a) at 1 week PBS n/a 143 Example 109: Synthesis of oligonucleotides comprising a Ga1NAc1-28 or Ga1NAc1-29 conjugate OH 5 __________________________________________________ 3 HOµ
= =
0 =ss CM ¨ Olga HO )C, AcHN

Oligonucleotide 241 is synthesized using procedures similar to those described in Example 71 to form the phosphoramidite intermediate, followed by procedures described in Example 10 to synthesize the oligonucleotide. The GalNAci cluster portion (GalNAci-28a) of the conjugate group Ga1NAc1-28 can be combined with any cleavable moiety present on the oligonucleotide to provide a variety of conjugate groups.
The structure of Ga1NAc1-28 (GalNAci-28a-CM) is shown below:

OH
HO....r 0 .õ0 ___ cm __ H

HO (:).)c .........iN?
N
AcHN H

In order to add the GalNAci conjugate group to the 3'-end of an oligonucleotide, procedures similar to those described in Example 71 are used to form the hydroxyl intermediate, which is then added to the solid support using procedures described in Example 7. The oligonucleotide synthesis is then completed using procedures described in Example 9 in order to form oligonucleotide 242.
HOOF1c):)c .õOH
HO N---",..--N?
AcHN H _______________________ , 3' 5' 0 = ___ N
242 0¨ CM ¨ Oligo _________________________________________________ õ ..
The GalNAci cluster portion (GalNAci-29a) of the conjugate group Ga1NAc1-29 can be combined with any cleavable moiety present on the oligonucleotide to provide a variety of conjugate groups. The structure of Ga1NAc1-29 (GalNAci-29a-CM) is shown below:
OH
HOµ OH
0 .0 HOOw....).c N.-----...,.......,.....õ..-õ...õõN?
¨ l AcHN H

.
Example 110: Synthesis of oligonucleotides comprising a Ga1NAc1-30 conjugate OAc OAc AIL Ac0...7......
W

Ac0-t--/---.1 HO OTBDPS Ac0 OOTBDPS
TMSOTf AcHN
yO 243 1. NH3/Me0H ODMTr 2. DMTrCI Ac0...7......
1. TBAF
3. Ac20, PYr 0 Ac0 O 2.
Phosphitilation ________________ 0- OTBDPS _____________ r AcHN 244 ODMTr Ac0.7......\, 1. Couple to 5'-end of ASO

Ac0 00õOCE ____________________________________________________ AcHN i 245 NOP02 2. Deprotect and purify ASO using DMT-on purification methods OH
HO
HO 00õ0 = _________________ =
P, Oligo AcHN
Y

Oligonucleotide 246 comprising a Ga1NAc1-30 conjugate group, wherein Y is selected from 0, S, a substituted or unsubstituted CI-CI alkyl, amino, substituted amino, azido, alkenyl or alkynyl, is synthesized as shown above. The GalNAci cluster portion (GalNAci-30a) of the conjugate group Ga1NAc1-30 can be combined with any cleavable moiety to provide a variety of conjugate groups.
In certain embodiments, Y is part of the cleavable moiety. In certain embodiments, Y is part of a stable moiety, and the cleavable moiety is present on the oligonucleotide. The structure of GalNAci-30a is shown below:
O
HO H\
0 r, HO
AcHN
Example 111: Synthesis of oligonucleotides comprising a Ga1NAc2-31 or Ga1NAc2-32 conjugate HO 1. DMTrCI DMTrO OCE Couple to 5'-end of ASO
2. Phosphitilation ¨OH ¨0-F' N(iPr)2 HO DMTrO

Bx 1. Remove DMTr groups DMTrO
2. Couple amidite 245 ¨0õ0 F' 3. Deprotect and purify ASO using DMTrO Y 601igo DMT-on purification methods -OH

HO 00õ0 e ID\
AcHN ¨ _______ 3:
(:)'1D\ Oligo Y
y HOO

H AcHN
Oligonucleotide 250 comprising a GalNAc2-31 conjugate group, wherein Y is selected from 0, S, a substituted or unsubstituted C1-C10 alkyl, amino, substituted amino, azido, alkenyl or alkynyl, is synthesized as shown above. The Ga1NAc2 cluster portion (Ga1NAc2-31a) of the conjugate group GalNAc2-31 can be combined with any cleavable moiety to provide a variety of conjugate groups.
In certain embodiments, the Y-containing group directly adjacent to the 5'-end of the oligonucleotide is part of the cleavable moiety. In certain embodiments, the Y-containing group directly adjacent to the 5'-end of the oligonucleotide is part of a stable moiety, and the cleavable moiety is present on the oligonucleotide. The structure of Ga1NAc2-31a is shown below:
OH
HO.../.......

HO 00,1:v0 AcHN 0; \ y _R
/ sjsj HOricl.\/C) HO
AcHN

The synthesis of an oligonucleotide comprising a Ga1NAc2-32 conjugate is shown below.
1. DMTrCI
2. Allyl Br 3. 0s04, Na104 1. Couple to 5'-end of ASO
HO 4. NaBH4 DMTrO 2. Remove DMTr groups 5. Phosphitilation3. Couple amidite 245 ¨OH _____________________ , ¨0¨\_ ________________________________ , 0, 4. Deprotect and purify ASO using HO DMTrO
P-N(iPr)2 DMT-on purification methods 247 CEO, OH
HOr.,......\

HO0.õ....õ....,...õ..--..,.....õ0õ0,, e P\ 0 Y 5' 3:
AcHN o' Y ¨0õ0 µp õ A Oligo P 0- '0 , ii \
O___ O Y
0-p/, OH ___7--/----/ O Y
HO\O

HO NHAc Oligonucleotide 252 comprising a Ga1NAc2-32 conjugate group, wherein Y is selected from 0, S, a substituted or unsubstituted CI-CI alkyl, amino, substituted amino, azido, alkenyl or alkynyl, is synthesized as shown above. The Ga1NAc2 cluster portion (Ga1NAc2-32a) of the conjugate group Ga1NAc2-32 can be combined with any cleavable moiety to provide a variety of conjugate groups.
In certain embodiments, the Y-containing group directly adjacent to the 5'-end of the oligonucleotide is part of the cleavable moiety. In certain embodiments, the Y-containing group directly adjacent to the 5'-end of the oligonucleotide is part of a stable moiety, and the cleavable moiety is present on the oligonucleotide. The structure of Ga1NAc2-32a is shown below:
OH
HO/_____\.

HO0.õ....,..--,,.....,...---õ,õõ0õ0õ, , P\
AcHN 0' y ,/ \
e 0 Y
0-p, HO\O
HO NHAc Example 112: Modified oligonucleotides comprising a GalNAci conjugate The oligonucleotides in Table 120 targeting SRB-1 were synthesized with a GalNAci conjugate group in order to further test the potency of oligonucleotides comprising conjugate groups that contain one GalNAc ligand.

Table 120 GalNAc SEQ
ISIS No. Sequence (5' to 3') CM
cluster ID NO.
711461 Ga1NAC1-25a-0'Ado Ges mCes Tes Tes mCes Ads Gds Tds mCds Ads Tds GalNAci-25a Ad 145 Gds Ads mCds Tds Tes mCes mCes Tes Te 711462 Ga1NAC1-25a-o'Ges mCes Tes Tes mCes Ads Gds Tds mCds Ads Tds Gds GalNAci-25a PO 143 Ads mCds Tds Tes mces mCes Tes Te 711463 Ga1NAC1-25a-o'Ges mCeo Teo Teo mCeo Ads Gds Tds mCds Ads Tds GalNAci-25a PO 143 Gds Ads mCds Tds Teo mCeo mCes Tes Te 711465 Ga1NAC1-26a-o'Ado Ges mCes Tes Tes mCes Ads Gds Tds mCds Ads Tds GalNAci-26a Ad 145 Gds Ads mCds Tds Tes mCes mCes Tes Te 711466 Ga1NAC1-26a-o'Ges mCes Tes Tes mCes Ads Gds Tds mCds Ads Tds Gds GalNAci-26a PO 143 Ads mCds Tds Tes mces mCes Tes Te 711467 Ga1NAC1-26a-o'Ges mCeo Teo Teo mCeo Ads Gds Tds mCds Ads Tds GalNAci-26a PO 143 Gds Ads mCds Tds Teo mCeo mCes Tes Te 711468 GalNAC1-28a-o'Ado Ges mCes Tes Tes mCes Ads Gds Tds mCds Ads Tds GalNAci-28a Ad 145 Gds Ads mCds Tds Tes mCes mCes Tes Te 711469 GalNAci-28a_0,Ges mCes Tes Tes mCes Ads Gds Tds mCds Ads Tds Gds GalNAci-28a PO 143 Ads mCds Tds Tes mCes mCes Tes Te 711470 GalNAci-28a_0,Ges mCeo Teo Teo mCeo Ads Gds Tds mCds Ads Tds GalNAci-28a PO 143 Gds Ads mCds Tds Teo mCeo mCes Tes Te 713844 Ges mCes Tes Tes mCes Ads Gds Tds mCds Ads Tds Gds Ads mCds Tds GalNAci-27a PO 143 Tes mCes mCes Tes Te0,_GalNAel-27a 713845 Ges mCeo Teo Teo mCeo Ads Gds Tds mCds Ads Tds Gds Ads mCds Tds GalNAci-27a PO 143 Teo mCee mCes Tes Te0,_GalNAel-27a 713846 Ges mCeo Teo Teo mCeo Ads Gds Tds mCds Ads Tds Gds Ads mCds Tds GalNAci-27a Ad 144 Teo mCee mCes Tes Teo Ado,_GalNAei-27a 713847 Ges mCes Tes Tes mCes Ads Gds Tds mCds Ads Tds Gds Ads mCds Tds GalNAci-29a PO 143 Tes mCes mCes Tes Te0,_GalNAel-29a 713848 Ges mCeo Teo Teo mCeo Ads Gds Tds mCds Ads Tds Gds Ads mCds Tds GalNAci-29a PO 143 Teo mCee mCes Tes Te0,_GalNAel-29a 713849 Ges mCes Tes Tes mCes Ads Gds Tds mCds Ads Tds Gds Ads mCds Tds GalNAci-29a Ad 144 Tes mCes mCes Tes Teo Ado,_GalNAei-29a 713850 Ges mCeo Teo Teo mCeo Ads Gds Tds mCds Ads Tds Gds Ads mCds Tds GalNAci-29a Ad 144 Teo mCee mCes Tes Teo Ado,_GalNAei-29a Example 113: Dose-dependent antisense inhibition of human apolipoprotein (a) (apo(a)) in human primary hepatocytes Selected gapmer antisense oligonucleotides from a previous publication (W02005/000201, the content of which is incorporated by reference in its entirety herein) were tested in a single dose assay in human primary hepatocytes. Cells were obtained from Tissue Transformation Technologies (BD Biosciences, Franklin Lakes, NJ) and treated with 150 nM of antisense oligonucleotide.
After a treatment period of approximately 16 hours, RNA was isolated from the cells and apo(a) mRNA levels were measured by quantitative real-time PCR. Human apo(a) primer probe set hAPO(a)3' (forward sequence ACAGCAATCAAACGAAGACACTG, designated herein as SEQ ID NO: 5; reverse sequence AGCTTATACACAAAAATACCAAAAATGC, designated herein as SEQ ID NO: 6; probe sequence TCCCAGCTACCAGCTATGCCAAACCTT, designated herein as SEQ ID NO: 7) was used to measure mRNA levels. Additionally, mRNA levels were also measured using human apo(a) primer probe set hAPO(a)12kB (forward sequence CCACAGTGGCCCCGGT, designated herein as SEQ ID
NO: 8; reverse sequence ACAGGGCTTTTCTCAGGTGGT, designated herein as SEQ ID NO: 9; probe sequence CCAAGCACAGAGGCTCCTTCTGAACAAG, designated herein as SEQ ID NO: 10). Apo(a) mRNA
levels were normalized to GAPDH mRNA expression. Results are presented in the table below as percent inhibition of apo(a), relative to untreated control cells.
Table 121 Antisense inhibition of human apo(a) in human primary hepatocytes % inhibition % inhibition ISIS No (hAPO(a)3' (hAPO(a)12kB
PPset) PPset) Several antisense oligonucleotides were selected for further testing in a dose response assay.
The selected antisense oligonucleotides were tested in human primary hepatocytes with 25 nM, 50 nM, 150 nM, or 300 nM concentrations of antisense oligonucleotide, as specified in the table below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and apo(a) mRNA levels were measured by quantitative real-time PCR. Human apo(a) primer probe set hAPO(a)3' was used to measure mRNA levels. Apo(a) mRNA levels were normalized to GAPDH mRNA expression.
Results are presented as percent inhibition of apo(a), relative to untreated control cells.
Table 122 Dose-dependent antisense inhibition of human apo(a) in human primary hepatocytes, as measured with hAPO(a)3' ISIS No 25 nM 50 nM 150 nM 300 nM

ISIS 144367 demonstrated better efficacy and dose-dependency than the other antisense oligonucleotides. Hence, ISIS 144367 was considered the benchmark antisense oligonucleotide to compare the potency of newly designed antisense oligonucleotides disclosed herein.
Example 114: Antisense inhibition of human apo(a) in transgenic mouse primary hepatocytes Antisense oligonucleotides were newly designed targeting an apo(a) nucleic acid and were tested for their effects on apo(a) mRNA in vitro. The antisense oligonucleotides were tested for potency in a series of parallel experiments that had similar culture conditions. Primary hepatocytes from human apo(a) transgenic mice (Frazer, K.A. et al., Nat. Genet. 1995. 9: 424-431) were used in this study. Hepatocytes at a density of 35,000 cells per well were transfected using electroporation with 1,000 nM
antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and apo(a) mRNA levels were measured by quantitative real-time PCR. Human primer probe set hAPO(a)12kB was used to measure mRNA levels. Apo(a) mRNA levels were adjusted according to total RNA content, as measured by RIBOGREENO. The results for each experiment are presented in separate tables shown below. ISIS 144367 from was used as a benchmark for the new antisense oligonucleotides and also included in the studies.
Results are presented as percent inhibition of apo(a), relative to untreated control cells. A total of 1,511 gapmers were tested under these culture conditions. Only those antisense oligonucleotides that were selected for further study are presented in the table below with each table representing a separate experiment.
The newly designed chimeric antisense oligonucleotides were designed as 5-10-5 MOE gapmers.
The gapmers are 20 nucleosides in length, wherein the central gap segment comprises of ten 2'-deoxynucleosides 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 has a 2'-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P=S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines.
The apo(a) target sequence contains multiple Kringle repeat sequences, therefore, an antisense oligonucleotide may target one or more regions of apo(a) depending whether on the oligonucleotide targets a Kringle sequence or not. "Start site" indicates the 5'-most nucleoside to which the gapmer is targeted in the human sequence. "Stop site" indicates the 3'-most nucleoside to which the gapmer is targeted human sequence. An apo(a) antisense oligonucleotide may have more than one "Start site" or "Stop site" depending on whether or not it targets a Kringle repeat.
Most gapmers listed in the tables are targeted with 100% complementarity to one or more regions of either the human apo(a) mRNA, designated herein as SEQ ID NO: 1 (GENBANK
Accession No.

NM_005577.2) or the human apo(a) genomic sequence, designated herein as SEQ ID
NO: 2 (GENBANK
Accession No. NT_007422.12 truncated from nucleotides 3230000 to 3380000), or both. `n/a' indicates that the antisense oligonucleotide does not target that particular sequence with 100% complementarity.
Table 123 SEQ ID SEQ SEQ
ISIS NO: 1 SEQ ID %
ID NO: ID NO: SEQ
NO: 1 Sequence NO Start inhibition 2 Start 2 Stop ID
Stop SiteNO
Site Site Site Table 124 SEQ ID SEQ
SEQ ID SEQ ID
NO: 1 ID NO: %
SEQ
ISIS NO Sequence O. N :
2 NO. 2 Start 1 Stop inhibition = .
ID NO
Start Site Stop Site Site Site 494307 CTGCTCAGTCGGTGCTTGTT 91 n/a n/a 494310 CCTCTGCTCAGTCGGTGCTT 90 n/a n/a Table 125 SEQ ID SEQ
SEQ ID SEQ ID
ISIS % NO: 2 ID NO: SEQ
NO: 1 NO: 1 Sequence NO inhibition Start 2 Stop ID NO
Start Site Stop Site Site Site 494371 3900 3919 GCTCCGTTGGTGCTTGTTCA 93 n/a n/a 57 494372 3901 3920 TGCTCCGTTGGTGCTTGTTC 93 n/a n/a 58 494373 3902 3921 TTGCTCCGTTGGTGCTTGTT 83 n/a n/a 59 494374 3903 3922 TTTGCTCCGTTGGTGCTTGT 89 n/a n/a 60 494375 3904 3923 CTTTGCTCCGTTGGTGCTTG 85 n/a n/a 61 Table 126 SEQ ID SEQ ID SEQ ID SEQ
ID
%
SEQ
ISIS NO NO: 1 NO: 1 Sequence NO: 2 NO. 2 inhibition = .
ID NO
Start Site Stop Site Start Site Stop Site Table 127 SEQ ID SEQ ID SEQ
ID SEQ ID
ISIS %
SEQ
NO: 1 NO: 1 Sequence NO: 2 NO. 2 NO inhibition = .
ID NO
Start Site Stop Site Start Site Stop Site GCTTGTTCAGAAGGAGCCTC 98 n/a n/a 78 TGCTTGTTCAGAAGGAGCCT 94 n/a n/a 79 GTGCTTGTTCAGAAGGAGCC 96 n/a n/a 80 498243 GGTGCTTGTTCAGAAGGAGC 97 n/a n/a 81 TGGTGCTTGTTCAGAAGGAG 92 n/a n/a 82 498251 2898 2917 GCTCAGTTGGTGCTTGTTCA 90 n/a n/a 83 498252 2899 2918 TGCTCAGTTGGTGCTTGTTC 90 n/a n/a 84 Table 128 SEQ ID SEQ ID
SEQ ID SEQ ID SEQ
%
ISIS NO NO: 1 NO: 1 Sequence NO: 2 NO:

inhibition Start Site Stop Site Start Site Stop Site NO

Table 129 SEQ ID SEQ ID
ISIS NO: 1 NO: 1 SEQ ID
SEQ ID SEQ
%
Sequence NO: 2 NO: 2 ID
NO Start Stop inhibition Start Site Stop Site NO
Site Site 498859 4926 4945 GCTCAGTTGGTGCTGCTTCA 92 n/a n/a 87 Table 130 SEQ ID SEQ ID
ISIS NO: 1 NO: 1 % SEQ ID
SEQ ID SEQ
Sequence NO: 2 NO: 2 inhibition NO Start Stop ID
NO
Start Site Stop Site Site Site Table 131 SEQ ID SEQ ID SEQ
ID SEQ ID
ISIS % SEQ
NO: 1 NO: 1 Sequence NO: 2 NO: 2 NO
inhibitionID NO
Start Site Stop Site Start Site Stop Site 498550 3582 3601 GCTCCGTTGGTGCTTCTTCA 90 n/a n/a 97 498553 3585 3604 TTTGCTCCGTTGGTGCTTCT 87 n/a n/a 98 498555 GCTTTGCTCCGTTGGTGCTT 90 n/a n/a 99 Table 132 SEQ ID SEQ ID SEQ
ID SEQ ID
ISIS % SEQ ID
NO: 1 NO: 1 SequenceNO: 2 NO: 2 NO inhibition NO
Start Site Stop Site Start Site Stop Site 494243 1520 1539 TGCTTGTTCGGAAGGAGCCT 93 n/a n/a 106 494244 1521 1540 GTGCTTGTTCGGAAGGAGCC 95 n/a n/a 107 Table 133 SEQ ID SEQ ID SEQ ID SEQ ID
SEQ
ISIS NO NO: 1 NO: 1 Sequence NO: 2 inhibition = NO. 2 Site ID Site Stop Site Start Site Stop Sfte ID NO

Example 115: Dose-dependent antisense inhibition of apo(a) in transgenic mouse primary hepatocytes Gapmers from the studies described above exhibiting significant in vitro inhibition of apo(a) mRNA
were selected and tested at various doses in transgenic mouse primary hepatocytes in a series of parallel studies with similar culture conditions. Cells were plated at a density of 35,000 per well and transfected using electroporation with 0.0625 tiM, 0.125 tiM, 0.25 ttM, 0.500 [tM, or 1.000 tiM concentrations of antisense oligonucleotide. After a treatment period of approximately 16 hours, RNA was isolated from the cells and apo(a) mRNA levels were measured by quantitative real-time PCR.
Apo(a) primer probe set hAPO(a)12kB was used to measured mRNA levels. Apo(a) mRNA levels were adjusted according to total RNA content, as measured by RIBOGREENO. Results are presented as percent inhibition of apo(a), relative to untreated control cells.

The results of each of the studies are depicted in the tables presented below with each table representing a separate experiment. The half maximal inhibitory concentration (IC50) of each oligonucleotide is also presented in the tables. Apo(a) mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide treated cells. The potency of the newly designed oligos was compared with the benchmark oligonucleotide ISIS 144367.
Table 134 ISIS No 0'0625 0.125 0.250 0.500 1.000 1050 P.M P.M P.M P.M P.M (PM) 144367 11 27 46 62 80 0.31 494157 11 47 53 76 87 0.23 494158 19 57 75 84 88 0.13 494159 41 65 77 84 92 0.07 494160 44 69 76 85 91 0.06 494161 40 64 74 85 91 0.08 494162 36 63 76 87 88 0.09 494163 20 59 75 85 92 0.13 494164 3 45 62 74 90 0.21 494165 25 39 57 71 75 0.19 494166 17 30 47 59 76 0.31 494167 30 43 55 72 80 0.18 494168 25 36 44 59 75 0.28 494169 19 39 51 61 81 0.25 Table 135 ISIS No 0'0625 0.125 0.250 0.500 1.000 P.M P.M P.M P.M P.M (PM) 144367 23 40 58 76 88 0.19 494170 38 34 60 76 84 0.13 494230 55 71 89 95 97 0.03 494243 47 73 87 92 97 0.05 494244 58 73 86 92 96 0.03 494283 54 70 84 93 94 0.05 494284 45 62 83 92 95 0.07 494285 56 70 84 92 95 0.04 494286 51 70 87 93 95 0.05 494287 32 60 67 87 91 0.11 494288 26 41 61 79 88 0.17 494290 30 43 64 81 87 0.15 494291 29 40 56 75 85 0.18 Table 136 ISIS No 0'0625 0.125 0.250 0.500 1.000 1050 PA4 PA4 PA4 PA4 PA4 (PM) 144367 10 38 62 68 84 0.23 494292 17 36 74 85 90 0.17 494294 10 34 53 80 91 0.22 494299 32 29 56 77 88 0.16 494300 34 46 76 86 90 0.12 494301 44 56 72 86 89 0.09 494302 42 59 78 88 89 0.08 494303 37 58 70 86 89 0.10 494304 46 71 78 89 90 0.05 494305 39 58 62 85 87 0.10 494306 31 52 65 79 88 0.13 494307 23 23 39 65 78 0.34 494310 14 29 62 70 88 0.25 Table 137 ISIS No 0'0625 0.125 0.250 0.500 1.000 IC50 PA4 PA4 PA4 PA4 PA4 (PM) 144367 0 29 45 73 92 0.27 494311 28 53 65 85 95 0.13 494334 20 44 66 86 96 0.16 494336 15 38 54 84 97 0.20 494337 28 50 77 90 98 0.12 494338 21 40 68 91 98 0.15 494371 19 0 71 89 97 0.15 494372 33 44 77 91 97 0.12 494373 15 36 65 83 95 0.19 494374 3 17 51 83 90 0.24 494375 1 34 56 80 93 0.23 494386 13 26 46 73 91 0.25 494387 17 27 45 67 88 0.28 Table 138 ISIS No 0'0625 0.125 0.250 0.500 1.000 1050 PA4 ILLM PA4 PA4 PA4 (PM) 144367 35 42 62 70 91 0.15 494537 19 34 54 79 90 0.21 494544 10 38 73 86 94 0.17 498229 36 58 80 92 97 0.10 498238 41 57 75 91 97 0.09 498239 56 71 79 90 94 0.03 498240 91 94 98 99 100 <0.06 498241 75 84 91 96 98 <0.06 498242 11 27 42 47 63 0.49 498243 91 93 96 98 99 <0.06 498244 4 0 0 13 43 >1.00 498251 30 30 42 73 89 0.26 498252 37 33 58 80 92 0.20 498369 22 22 10 22 34 >1.00 Table 139 ISIS No 0'0625 0.125 0.250 0.500 1.000 1050 P,M P,M P,M P,M P,M (PM) 144367 15 32 54 75 90 0.22 498379 29 48 71 80 95 0.13 498408 38 57 77 88 96 0.09 498433 29 36 70 88 96 0.15 498434 49 43 50 78 90 0.19 498435 27 39 57 78 93 0.18 498517 64 72 82 93 98 <0.06 498721 77 84 88 96 97 <0.06 498833 73 78 91 95 99 <0.06 498859 7 24 37 62 75 0.36 498868 7 14 39 63 81 0.36 498875 16 21 33 55 81 0.39 499020 7 24 23 55 78 0.36 499041 6 16 33 64 83 0.35 Table 140 ISIS No 0'0625 0.125 0.250 0.500 1.000 1050 P,M P,M P,M P,M P,M (PM) 144367 14 47 64 79 91 0.14 498523 36 50 80 87 95 0.11 498524 43 79 87 93 97 0.01 498525 32 49 75 86 96 0.12 498529 21 49 57 78 90 0.17 498535 20 34 55 76 86 0.21 498550 12 50 69 84 96 0.11 498553 8 43 55 77 91 0.21 498555 13 35 68 86 94 0.19 498556 27 37 71 85 91 0.15 498557 18 42 75 89 95 0.16 498579 16 38 67 89 95 0.16 498580 36 57 81 91 96 0.10 498581 34 64 75 93 97 0.05 Table 141 0.0625 0.125 0.250 0.500 1.000 1050 ISIS No ILLM ILLM IINI IINI IINI (PM) 144367 0 9 26 49 77 0.47 494388 0 0 21 33 55 0.89 494389 0 15 22 50 79 0.46 494390 5 20 37 68 81 0.33 494391 7 20 32 54 68 0.46 494392 18 24 40 57 76 0.35 494466 33 45 58 69 82 0.16 494470 45 58 68 79 87 0.08 494472 37 50 60 69 83 0.13 494521 0 0 0 15 54 0.17 494525 0 0 2 28 65 0.85 494530 0 6 27 51 80 0.46 494535 0 7 24 53 74 0.49 494536 0 2 15 42 67 0.63 Table 142 ISIS No 0'0625 0.125 0.250 0.500 1.000 1050 IINI IINI IINI IINI IINI (PM) 144367 0 4 16 26 77 0.65 498379 12 18 27 32 63 0.81 498408 0 11 46 50 77 0.41 498433 22 30 46 60 83 0.27 498434 39 29 25 47 78 0.40 498435 21 28 26 43 73 0.50 498517 44 48 63 70 84 0.11 498721 54 54 66 75 89 <0.06 498833 44 51 58 67 83 0.11 498859 0 29 14 35 66 0.69 498868 0 12 9 26 60 1.07 498875 0 30 31 53 78 0.40 499020 0 27 19 45 74 0.51 499041 0 12 10 37 65 0.77 As presented in the tables above, ISIS 494157 (SEQ ID NO: 12), ISIS 494158 (SEQ ID NO:13), ISIS 494159 (SEQ ID NO:14), ISIS 494160 (SEQ ID NO: 15), ISIS 494161 (SEQ ID NO:16), ISIS
494162 (SEQ ID NO:
17), ISIS 494163 (SEQ ID NO: 18), ISIS 494164 (SEQ ID NO: 19), ISIS 494165 (SEQ ID NO: 20), ISIS
494167 (SEQ ID NO: 22), ISIS 494168 (SEQ ID NO: 23), ISIS 494169 (SEQ ID NO:
24), ISIS 494170 (SEQ ID NO: 25), ISIS 494230 (SEQ ID NO: 105), ISIS 494243 (SEQ ID NO: 106), ISIS 494244 (SEQ ID
NO: 107), ISIS 494283 (SEQ ID NO: 26), ISIS 494284 (SEQ ID NO: 27), ISIS
494285 (SEQ ID NO: 28), ISIS 494286 (SEQ ID NO: 29), ISIS 494287 (SEQ ID NO: 30), ISIS 494288 (SEQ ID
NO: 31), ISIS 494290 (SEQ ID NO: 32), ISIS 494291 (SEQ ID NO: 33), ISIS 494292 (SEQ ID NO: 35), ISIS 494294 (SEQ ID
NO: 36), ISIS 494299 (SEQ ID NO: 37), ISIS 494300 (SEQ ID NO: 38), ISIS 494301 (SEQ ID NO: 39), ISIS 494302 (SEQ ID NO: 40), ISIS 494303 (SEQ ID NO: 41), ISIS 494304 (SEQ ID
NO: 42), ISIS 494305 (SEQ ID NO:43), ISIS 494306 (SEQ ID NO: 44), ISIS 494311 (SEQ ID NO: 47), ISIS
494334 (SEQ ID NO:
48), ISIS 494336 (SEQ ID NO: 49), ISIS 494337 (SEQ ID NO: 50), ISIS 494338 (SEQ ID NO: 133), ISIS
494371 (SEQ ID NO: 57), ISIS 494372 (SEQ ID NO: 58), ISIS 494373 (SEQ ID NO:
59), ISIS 494374 (SEQ ID NO: 60), ISIS 494375 (SEQ ID NO: 61), ISIS 494386 (SEQ ID NO: 62), ISIS 494389 (SEQ ID
NO: 65), ISIS 494390 (SEQ ID NO: 66), ISIS 494392 (SEQ ID NO: 68), ISIS 494466 (SEQ ID NO: 108), ISIS 494470 (SEQ ID NO: 109), ISIS 494472 (SEQ ID NO: 110), ISIS 494521 (SEQ
ID NO: 51), ISIS
494530 (SEQ ID NO: 53), ISIS 498229 (SEQ ID NO: 75), ISIS 498238 (SEQ ID NO:
76), ISIS 498239 (SEQ ID NO: 77), ISIS 498240 (SEQ ID NO: 78), ISIS 498241 (SEQ ID NO: 79), ISIS 498243 (SEQ ID
NO: 81), ISIS 498379 (SEQ ID NO: 70), ISIS 498408 (SEQ ID NO: 71), ISIS 498433 (SEQ ID NO: 72), ISIS 498434 (SEQ ID NO: 73), ISIS 498435 (SEQ ID NO: 74), ISIS 498517 (SEQ ID
NO: 85), ISIS 498523 (SEQ ID NO: 92), ISIS 498524 (SEQ ID NO: 93), ISIS 498525 (SEQ ID NO: 94), ISIS 498550 (SEQ ID
NO: 97), ISIS 498580 (SEQ ID NO: 103), ISIS 498581 (SEQ ID NO: 104), ISIS

(ATGCCTCGATAACTCCGTCC; SEQ ID NO: 134), ISIS 498833 (SEQ ID NO: 86), ISIS
498875 (SEQ ID
NO: 89), and ISIS 499020 (SEQ ID NO: 90) were more potent than ISIS 144367 (SEQ ID NO: 11).
Example 116: Dose-dependent antisense inhibition of apo(a) in transgenic mouse primary hepatocytes Potent gapmers from the studies described above were further selected and tested at various doses in transgenic mouse primary hepatocytes in a series of studies with similar culture conditions. Cells were plated at a density of 35,000 per well and transfected using electroporation with 0.049 [tIVI, 0.148 [tIVI, 0.444 [LM, 1.333 [tIVI, or 4.000 [LNI concentrations of antisense oligonucleotide, as specified in tables below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and apo(a) mRNA levels were measured by quantitative real-time PCR. Apo(a) primer probe set hAPO(a)12kB
was used to measured mRNA levels. Apo(a) mRNA levels were adjusted according to total RNA content, as measured by RIBOGREENO. Results are presented as percent inhibition of apo(a), relative to untreated control cells.
The results of each of the studies are depicted in the tables presented below with each table representing a separate experiment. The half maximal inhibitory concentration (IC50) of each oligonucleotide is also presented in the tables. Apo(a) mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide treated cells. The potency of the newly designed oligos was compared with the benchmark oligonucleotide, ISIS 144367. As presented in the tables below, ISIS
494157 (SEQ ID NO: 12), ISIS 494158 (SEQ ID NO:13), ISIS 494159 (SEQ ID NO:14), ISIS 494160 (SEQ ID
NO: 15), ISIS 494161 (SEQ ID NO:16), ISIS 494162 (SEQ ID NO: 17), ISIS 494163 (SEQ ID NO: 18), ISIS
494164 (SEQ ID NO:
19), ISIS 494230 (SEQ ID NO: 105), ISIS 494243 (SEQ ID NO: 106), ISIS 494244 (SEQ ID NO: 107), ISIS
494283 (SEQ ID NO: 26), ISIS 494284 (SEQ ID NO: 27), ISIS 494285 (SEQ ID NO:
28), ISIS 494286 (SEQ ID NO: 29), ISIS 494287 (SEQ ID NO: 30), ISIS 494290 (SEQ ID NO: 32), ISIS 494292 (SEQ ID
NO: 35), ISIS 494300 (SEQ ID NO: 38), ISIS 494301 (SEQ ID NO: 39), ISIS 494302 (SEQ ID NO: 40), ISIS 494303 (SEQ ID NO: 41), ISIS 494304 (SEQ ID NO: 42), ISIS 494305 (SEQ ID
NO: 43), ISIS 494306 (SEQ ID NO: 44), ISIS 494310 (SEQ ID NO: 46), ISIS 494311 (SEQ ID NO: 47), ISIS 494337 (SEQ ID
NO: 50), ISIS 494371 (SEQ ID NO: 57), ISIS 494372 (SEQ ID NO: 58), ISIS 494375 (SEQ ID NO: 61), ISIS 494388 (SEQ ID NO: 64), ISIS 494389 (SEQ ID NO: 65), ISIS 494390 (SEQ ID
NO: 66), ISIS 494392 (SEQ ID NO: 68), ISIS 494466 (SEQ ID NO: 108), ISIS 494470 (SEQ ID NO: 109), ISIS 494472 (SEQ ID
NO: 110), ISIS 498238 (SEQ ID NO: 76), ISIS 498239 (SEQ ID NO: 77), ISIS
498433 (SEQ ID NO: 72), ISIS 498434 (SEQ ID NO: 73), ISIS 498435 (SEQ ID NO: 74), ISIS 498523 (SEQ ID
NO: 92), ISIS 498524 (SEQ ID NO: 93), ISIS 498525 (SEQ ID NO: 94), ISIS 498580 (SEQ ID NO: 103), and ISIS 498581 (SEQ
ID NO: 104) were more potent than ISIS 144367 (SEQ ID NO: 11).
Table 143 ISIS No 0'049 0.148 0.444 1.333 4.000 P.M P.M P.M P.M P.M (PM) 144367 0 26 67 89 92 0.32 494157 23 50 83 96 96 0.15 494158 26 62 85 96 96 0.11 494159 42 65 87 95 94 0.07 494160 51 70 88 94 94 <0.05 494161 36 67 87 95 96 0.08 494162 40 69 89 94 95 0.07 494163 41 57 87 95 94 0.08 494164 15 43 75 93 96 0.20 494230 39 77 94 99 99 0.05 494243 39 76 92 98 99 0.06 494244 58 79 91 97 99 0.02 494283 18 45 80 93 91 0.18 494284 9 53 80 95 94 0.18 Table 144 ISIS No 0'049 0.148 0.444 1.333 4.000 1050 P,M P,M P,M P,M P,M (PM) 144367 21 40 79 94 93 0.18 494285 53 68 90 97 97 <0.05 494286 46 69 89 96 97 0.05 494287 31 38 79 94 95 0.15 494290 22 53 74 93 94 0.16 494292 37 51 81 93 95 0.11 494294 22 40 72 91 94 0.19 494299 15 43 75 93 95 0.20 494300 25 38 79 95 95 0.17 494301 23 48 82 92 95 0.15 494302 26 59 86 93 94 0.12 494303 10 58 84 92 91 0.16 494304 25 62 83 93 93 0.12 Table 145 ISIS No 0'049 0.148 0.444 1.333 4.000 IC50 P,M P,M P,M P,M P,M (PM) 144367 23 40 70 90 94 0.19 494305 20 48 82 93 95 0.16 494306 26 53 78 91 92 0.14 494310 36 50 79 88 92 0.12 494311 38 50 74 93 95 0.12 494334 20 42 73 90 94 0.19 494336 5 39 74 92 95 0.23 494337 23 51 87 96 96 0.14 494338 12 42 82 93 95 0.19 494371 28 49 82 94 94 0.14 494372 28 54 81 93 88 0.13 494373 21 28 67 86 92 0.25 494375 26 40 77 85 92 0.18 Table 146 ISIS N 0.049 0.148 0.444 1.333 4.000 1050 o PA4 PA4 PA4 PA4 PA4 (PM) 144367 5 33 65 78 81 0.32 494388 30 32 60 82 86 0.25 494389 30 45 69 84 84 0.17 494390 32 47 67 83 87 0.16 494392 23 38 54 79 82 0.31 494466 48 67 86 91 95 0.04 494470 74 87 92 96 98 <0.05 494472 69 84 92 96 97 <0.05 494544 5 18 49 74 79 0.48 498238 25 51 76 92 96 0.15 498239 25 62 83 93 97 0.12 498379 5 21 53 71 81 0.55 498408 1 38 63 79 80 0.32 498433 23 43 70 77 79 0.21 Table 147 ISIS No 0'049 0.148 0.444 1.333 4.000 IC50 ILLM PA4 PA4 PA4 PA4 (P,M) 144367 0 40 76 90 93 0.26 498434 32 44 64 78 84 0.20 498435 24 42 64 77 79 0.23 498517 28 23 53 81 85 0.45 498523 50 64 81 90 93 <0.05 498524 53 70 84 93 96 <0.05 498525 38 55 80 92 96 0.09 498550 12 18 62 81 83 0.33 498557 13 33 67 79 83 0.33 498579 6 42 69 80 85 0.31 498580 6 46 76 82 83 0.23 498581 5 40 78 81 84 0.25 498721 40 31 58 78 83 0.35 498833 21 20 58 80 90 0.44 Example 117: Antisense inhibition of human apo(a) in transgenic mouse primary hepatocytes Additional antisense oligonucleotides were newly designed targeting an apo(a) nucleic acid and were tested for their effects on apo(a) mRNA in vitro. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. Primary hepatocytes from human apo(a) transgenic mice were used in this study. Hepatocytes at a density of 35,000 cells per well were transfected using electroporation with 1,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and apo(a) mRNA levels were measured by quantitative real-time PCR. Human primer probe set hAPO(a)12kB was used to measure mRNA levels.
Apo(a) mRNA levels were adjusted according to total RNA content, as measured by RIBOGREENO. The results for each experiment are presented in separate tables shown below. ISIS 144367 was also included in the studies for comparison.
Results are presented as percent inhibition of apo(a), relative to untreated control cells. A total of 231 antisense oligonucleotides were tested under these culture conditions. Only those antisense oligonucleotides that were selected for further studies are presented below.
The newly designed chimeric antisense oligonucleotides were designed as 3-10-4 MOE gapmers.
The gapmers are 17 nucleosides in length, wherein the central gap segment comprises of ten 2'-deoxynucleosides and is flanked by wing segments on the 5' direction and the 3' direction comprising three nucleosides and four nucleosides respectively. Each nucleoside in the 5' wing segment and each nucleoside in the 3' wing segment has a 2'-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P=S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines.
The apo(a) target sequence contains multiple Kringle repeat sequences, therefore, an antisense oligonucleotide may target one or more regions of apo(a) depending whether on the oligonucleotide targets a Kringle sequence or not. "Start site" indicates the 5'-most nucleoside to which the gapmer is targeted in the human sequence. "Stop site" indicates the 3'-most nucleoside to which the gapmer is targeted human sequence. An apo(a) antisense oligonucleotide may have more than one "Start site" or "Stop site" depending on whether or not it targets a Kringle repeat.
Most gapmers listed in the tables are targeted with 100% complementarity to multiple regions of either the human apo(a) mRNA, designated herein as SEQ ID NO: 1 (GENBANK
Accession No.
NM_005577.2) or the human apo(a) genomic sequence, designated herein as SEQ ID
NO: 2 (GENBANK
Accession No. NT_007422.12 truncated from nucleotides 3230000 to 3380000), or both. `n/a' indicates that the antisense oligonucleotide does not target that particular sequence with 100% complementarity.
Table 148 SEQ ID SEQ ID SEQ ID SEQ ID
ISIS
SEQ ID
NO: 1 NO: 1 Sequence NO: 2 NO: 2 NOinhibition NO
Start Site Stop Site Start Site Stop Site Table 149 ISIS
SEQ ID SEQ ID SEQ ID SEQ ID SEQ

NO: 1 NO: 1 Sequence NO: 2 NO: 2 ID
NO inhibition Start Site Stop Site Start Site Stop Site NO

512959 3585 3601 GCTCCGTTGGTGCTTCT 77 n/a n/a 126 Table 150 SEQ ID SEQ ID SEQ ID SEQ ID
% SEQ
ID
ISIS NO NO: 1 NO: 1 Sequence NO: 2 NO: 2 inhibitionNO
Start Site Stop Site Start Site Stop Site n/a n/a 130 Example 118: Dose-dependent antisense inhibition of apo(a) in transgenic mouse primary hepatocytes Potent gapmers from the studies described above were further selected and tested at various doses in transgenic mouse primary hepatocytes in a series of studies with similar culture conditions. Cells were plated at a density of 35,000 per well and transfected using electroporation with 0.156 j.tM, 0.313 1\4, 0.625 1\4, 1.250 uM, 2.500 [tM, or 5.000 i.LM concentrations of antisense oligonucleotide, as specified in the tables below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and apo(a) mRNA levels were measured by quantitative real-time PCR. Apo(a) primer probe set hAPO(a)12kB was used to measured mRNA levels. Apo(a) mRNA levels were adjusted according to total RNA content, as measured by RIBOGREENO. Results are presented as percent inhibition of apo(a), relative to untreated control cells.
The results of each of the studies are depicted in the tables presented below with each study represented in a separate table. The half maximal inhibitory concentration (IC50) of each oligonucleotide is also presented in the tables.
Table 151 ISIS No 0"156 0.312 0.625 1.250 2.500 5.000 !1M 1-11\4 1-1M !1M !1M 1-1M (11M) 144367 28 55 70 83 90 92 0.31 510542 33 58 75 87 89 90 0.27 510543 33 45 68 78 89 89 0.34 510544 33 50 65 78 88 90 0.33 510545 33 58 76 87 91 90 0.26 510546 39 62 76 87 89 91 0.22 510547 36 66 82 84 86 91 0.22 510548 50 70 82 91 88 90 0.13 510549 32 59 73 85 86 90 0.27 510595 26 57 78 88 90 90 0.29 510597 30 53 76 85 89 89 0.30 Table 152 ISIS No 0'156 0.312 0.625 1.250 2.500 5.000 1050 PA4 PA4 ILLM ILLM PA4 PA4 (PM) 144367 36 52 78 87 93 94 0.26 510598 48 58 81 88 93 92 0.18 510701 45 59 78 87 95 95 0.18 510702 49 63 75 90 94 95 0.15 510704 55 67 80 93 94 95 <0.16 510757 34 48 68 79 90 93 0.33 510783 21 32 51 58 78 84 0.69 512944 57 72 81 91 96 97 <0.16 512947 64 74 86 92 96 97 <0.16 512958 48 69 83 91 96 97 0.13 512959 39 59 76 84 93 93 0.22 Table 153 ISIS No 0'156 0.312 0.625 1.250 2.500 5.000 IC50 PA4 PA4 PA4 PA4 PA4 PA4 (PM) 144367 41 58 75 81 88 87 0.22 510542 38 54 69 74 85 83 0.27 510545 21 43 73 77 80 78 0.39 510546 37 58 73 81 83 81 0.24 510547 38 58 72 79 84 86 0.24 510548 40 63 77 79 81 84 0.21 510549 37 47 67 77 81 83 0.31 510595 34 66 73 81 80 75 0.23 510597 39 59 74 83 76 77 0.23 Table 154 ISIS No 0.156 0.312 0.625 1.250 2.500 5.000 1050 P,M P,M P,M P,M P,M P,M (PM) 144367 33 60 72 83 81 81 0.26 510598 47 62 75 75 76 76 0.18 510701 41 67 80 87 92 91 0.19 510702 51 64 77 80 80 83 0.13 510704 54 61 77 84 89 80 0.12 512944 71 74 81 88 92 94 0.02 512947 65 77 86 90 93 95 0.03 512958 63 73 84 92 93 96 0.06 512959 39 62 80 82 86 82 0.22 Apo(a) mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide-treated cells. The potency of the newly designed oligonucleotides was compared with the benchmark oligonucleotide, ISIS 144367. As presented in the tables above, ISIS
510542 (SEQ ID NO: 111), ISIS 510545 (SEQ ID NO: 114), ISIS 510546 (SEQ ID NO: 115), ISIS 510547 (SEQ
ID NO: 116), ISIS
510548 (SEQ ID NO: 117), ISIS 510549 (SEQ ID NO: 118), ISIS 510595 (SEQ ID NO:
119), ISIS 510597 (SEQ ID NO: 120), ISIS 510598 (SEQ ID NO: 121), ISIS 510701 (SEQ ID NO: 127), ISIS 510702 (SEQ ID
NO: 128), ISIS 510704 (SEQ ID NO: 129), ISIS 512944 (SEQ ID NO: 123), ISIS
512947 (SEQ ID NO:
124), ISIS 512958 (SEQ ID NO: 125), and ISIS 512959 (SEQ ID NO: 126) were more potent than ISIS
144367 (SEQ ID NO: 11).
Example 119: Effect of in vivo antisense inhibition of human apo(a) in human apo(a) transgenic mice Transgenic mice with the human apo(a) gene (Frazer, K.A. et al., Nat. Genet.
1995. 9: 424-431) were utilized in the studies described below. ISIS antisense oligonucleotides that demonstrated statistically significant inhibition of apo(a) mRNA in vitro as described above were evaluated further in this model.
Study 1 Female human apo(a) transgenic mice were maintained on a 12-hour light/dark cycle and fed ad libitum normal lab chow. The mice were divided into treatment groups consisting of 4 mice each. The groups received intraperitoneal injections of ISIS 494159, ISIS 494160, ISIS 494161, ISIS 494162, ISIS 494163, ISIS 494230, ISIS 494243, ISIS 494244, ISIS 494283, ISIS 494284, ISIS 494285, ISIS 494286, ISIS 494301, ISIS 494302, ISIS 494304, ISIS 494466, ISIS 494470, ISIS 494472, ISIS 498239, ISIS 498408, ISIS 498517, ISIS 494158, ISIS 494311, ISIS 494337, ISIS 494372, ISIS 498238, ISIS 498523, ISIS 498525, ISIS 510548, ISIS 512944, ISIS 512947, or ISIS 512958 at a dose of 25 mg/kg twice a week for 2 weeks. One group of mice received intraperitoneal injections of PBS twice a week for 2 weeks. The PBS group served as the control group. Two days following the final dose, the mice were euthanized, organs harvested and analyses done.
Inhibition of human apo(a) mRNA
Total RNA was extracted from the livers of some of the treatment groups, and human apo(a) mRNA
was quantitated by RT-PCR. The results are presented in the table below, expressed as percent inhibition of apo(a) mRNA compared to the PBS control.
Table 155 Percent inhibition of human apo(a) mRNA in transgenic mice %
ISIS No . . . .
inhibition The data demonstrates significant inhibition of apo(a) mRNA by several ISIS
oligonucleotides. ISIS
494159 (SEQ ID NO: 14), ISIS 494162 (SEQ ID NO: 17), ISIS 494163 (SEQ ID NO:
18), ISIS 494243 (SEQ ID NO: 106), ISIS 494244 (SEQ ID NO: 107), ISIS 494283 (SEQ ID NO: 26), ISIS 494284 (SEQ ID
NO: 27), ISIS 494285 (SEQ ID NO: 28), ISIS 494301 (SEQ ID NO: 39), and ISIS
498408 (SEQ ID NO: 71) were more potent than the benchmark ISIS 144367 (SEQ ID NO: 11).

Inhibition of human apo(a) protein Plasma human apo(a) protein was measured from all treatment groups using an Apo(a) ELISA kit (Mercodia 10-1106-01, Uppsala, Sweden). The results are presented in the table below, expressed as percent inhibition of apo(a) mRNA compared to the PBS control.
Table 156 Percent inhibition of human apo(a) protein in transgenic mice ISIS %
No inhibition The data demonstrates significant inhibition of apo(a) mRNA by several ISIS
oligonucleotides.
ISIS 494159 (SEQ ID NO: 14), ISIS 494244 (SEQ ID NO: 82), ISIS 494283 (SEQ ID
NO: 26), ISIS 494284 (SEQ ID NO: 27), ISIS 494285 (SEQ ID NO: 28), ISIS 494286 (SEQ ID NO: 29), ISIS 494301 (SEQ ID
NO: 39), and ISIS 494302 (SEQ ID NO: 40) were as potent as or more potent than the benchmark ISIS
144367 (SEQ ID NO: 11)..
Study 2 ISIS 494159, ISIS 494161, ISIS 494162, ISIS 494163, and ISIS 494243 were further evaluated in this transgenic model. ISIS 144367 was included for comparison.
Treatment Female human apo(a) transgenic mice were divided into treatment groups consisting of 4 mice each. The groups received intraperitoneal injections of ISIS 144367, ISIS
494159, ISIS 494161, ISIS 494162, ISIS 494163, or ISIS 494243 at doses of 1.5 mg/kg, 5 mg/kg, 15 mg/kg, or 50 mg/kg twice a week for 2 weeks. One group of mice received intraperitoneal injections of PBS twice a week for 2 weeks. The PBS
group served as the control group. Two days following the final dose, the mice were euthanized, organs harvested and analyses done.
Inhibition of human apo(a) mRNA
Total RNA was extracted from the livers of the treatment groups, and human apo(a) mRNA was quantitated by RT-PCR. The results are presented in the table below, expressed as percent inhibition of apo(a) mRNA compared to the PBS control.
Table 157 Dose-dependent inhibition of human apo(a) mRNA in transgenic mice Dose %
ISIS No ED50 (mg/kg/wk) inhibition The data demonstrates significant inhibition of apo(a) mRNA by several ISIS
oligonucleotides. ISIS
494159 (SEQ ID NO: 14), ISIS 494161 (SEQ ID NO: 16), 494162 (SEQ ID NO:17), and ISIS 94163 (SEQ
ID NO: 18) were more efficacious than the benchmark ISIS 144367 (SEQ ID NO:
11).Reduction of human 5 apo(a) protein levels Blood was collected from the treatment groups, and human apo(a) protein levels were quantitated by an Apo(a) ELISA kit (Mercodia 10-1106-01, Uppsala, Sweden). The results are presented in the table below, expressed as percent reduction of apo(a) protein levels compared to the PBS
control.
Table 158 10 Dose-dependent inhibition of human apo(a) protein in transgenic mice Dose %
ISIS No ED5o (mg/kg/wk) inhibition The data demonstrates significant reduction of apo(a) plasma protein levels by several ISIS
oligonucleotides. ISIS 494159 (SEQ ID NO: 14), ISIS 494161 (SEQ ID NO: 16), ISIS 494162 (SEQ ID NO:
17), and ISIS 494163 (SEQ ID NO: 18) were more efficacious than the benchmark ISIS 144367 (SEQ ID
NO: 11).
5 Study 3 ISIS 494244, ISIS 494283, and ISIS 494284 were further evaluated in this model. ISIS 144367 was included for comparison.
Treatment Female human apo(a) transgenic mice were divided into treatment groups consisting of 4 mice 10 each. The groups received intraperitoneal injections of ISIS 144367, ISIS 494244, ISIS 494283, or ISIS
494284 at doses of 0.75 mg/kg, 2.5 mg/kg, 7.5 mg/kg, or 25 mg/kg twice a week for 2 weeks. One group of mice received intraperitoneal injections of PBS twice a week for 2 weeks. The PBS group served as the control group. Two days following the final dose, the mice were euthanized, organs harvested and analyses done.
Inhibition of human apo(a) mRNA
Total RNA was extracted from the livers of the treatment groups, and human apo(a) mRNA was quantitated by RT-PCR. The results are presented in the table below, expressed as percent inhibition of apo(a) mRNA compared to the PBS control.

Table 159 Dose-dependent inhibition of human apo(a) mRNA in transgenic mice Dose %
ISIS No ED5o (mg/kg/wk) inhibition 1.5 0 1.5 0 1.5 0 1.5 2 The data demonstrates significant inhibition of apo(a) mRNA by several ISIS
oligonucleotides. ISIS
494244 (SEQ ID NO: 107), ISIS 494283 (SEQ ID NO: 26), and ISIS 494284 (SEQ ID
NO: 27) were more 5 efficacious than the benchmark, ISIS 144367 (SEQ ID NO: 11).
Reduction of human apo(a) protein levels Blood was collected from the treatment groups, and human apo(a) protein levels were quantitated by an Apo(a) ELISA kit (Mercodia 10-1106-01, Uppsala, Sweden). The results are presented in the table below, expressed as percent reduction of apo(a) protein levels compared to the PBS
control.
Table 160 Dose-dependent inhibition of human apo(a) plasma protein in transgenic mice Dose %
ISIS No ED5o (mg/kg/wk) inhibition 1.5 0 1.5 0 1.5 69 1.5 54 The data demonstrates significant reduction of apo(a) plasma protein levels by several ISIS
oligonucleotides. ISIS 494244 (SEQ ID NO: 107), ISIS 494283 (SEQ ID NO: 26), and ISIS 494284 (SEQ ID
NO: 27) were more efficacious than the benchmark, ISIS 144367 (SEQ ID NO: 11).
5 Study 4 ISIS 494285, ISIS 494286, ISIS 494301, ISIS 494302, and ISIS 494311 were further evaluated in this model.
Treatment Male human apo(a) transgenic mice were divided into treatment groups consisting of 4 mice each.
Each such group received intraperitoneal injections of ISIS 494285, ISIS
494286, ISIS 494301, ISIS 494302, or ISIS 494311 at doses of 5 mg/kg, 15 mg/kg, or 50 mg/kg once a week for 2 weeks. One group of 3 mice received intraperitoneal injections of PBS once a week for 2 weeks. The PBS
group served as the control group. Two days following the final dose, the mice were euthanized, organs harvested and analyses done.
Inhibition of human apo(a) mRNA
Total RNA was extracted from the livers of the treatment groups, and human apo(a) mRNA was quantitated by RT-PCR. The results are presented in the table below, expressed as percent inhibition of apo(a) mRNA compared to the PBS control. The data demonstrates significant inhibition of apo(a) mRNA by ISIS 494285 (SEQ ID NO: 28), ISIS 494286 (SEQ ID NO: 29), ISIS 494301 (SEQ ID
NO: 39), ISIS 494302 (SEQ ID NO: 40) and ISIS 494311 (SEQ ID NO: 47).
Table 161 Dose-dependent inhibition of human Apo(a) mRNA in transgenic mice Dose %
ISIS No ED50 (mg/kg/wk) inhibition Reduction of human apo(a) protein levels Blood was collected from the treatment groups, and human apo(a) protein levels were quantitated by an Apo(a) ELISA kit (Mercodia 10-1106-01, Uppsala, Sweden). The results are presented in the table below, expressed as percent reduction of apo(a) protein levels compared to the PBS
control. The data demonstrates 5 significant reduction of apo(a) plasma protein levels by ISIS 494285, ISIS 494286, ISIS 494301, ISIS 494302 and ISIS 494311.
Table 162 Dose-dependent inhibition of human apo(a) protein in transgenic mice Dose %
ISIS No ED5o (mg/kg/wk) inhibition Study 5 ISIS 494372, ISIS 498524, ISIS 498581, ISIS 498721, and ISIS 498833 were further evaluated in this model.
Treatment Female human apo(a) transgenic mice were divided into treatment groups consisting of 4 mice each. The groups received intraperitoneal injections of ISIS 494372, ISIS
498524, ISIS 498581, ISIS 498721, or ISIS 498833 at doses of 5 mg/kg, 15 mg/kg, or 50 mg/kg once a week for 2 weeks. One group of 3 mice received intraperitoneal injections of PBS once a week for 2 weeks. The PBS
group served as the control group. Two days following the final dose, the mice were euthanized, organs harvested and analyses done.
Inhibition of human apo(a) mRNA
Total RNA was extracted from the livers of the treatment groups, and human apo(a) mRNA was quantitated by RT-PCR. The results are presented in the table below, expressed as percent inhibition of apo(a) mRNA compared to the PBS control. The data demonstrates significant inhibition of apo(a) mRNA by ISIS 494372 (SEQ ID NO: 28), ISIS 498524 (SEQ ID NO: 93), ISIS 498581 (SEQ ID
NO: 104), and ISIS
498721 (ATGCCTCGATAACTCCGTCC; SEQ ID NO: 134).
Table 163 Dose-dependent inhibition of human Apo(a) mRNA in transgenic mice Dose %
ISIS No ED50 (mg/kg/wk) inhibition Reduction of human apo(a) protein levels Blood was collected from the treatment groups, and human apo(a) protein levels were quantitated by an Apo(a) ELISA kit (Mercodia 10-1106-01, Uppsala, Sweden). The results are presented in the table below, expressed as percent reduction of apo(a) protein levels compared to the PBS
control. The data demonstrates significant reduction of apo(a) plasma protein levels by ISIS 494372 (SEQ ID
NO: 28), ISIS 498581 (SEQ
ID NO: 104), and ISIS 498721 (ATGCCTCGATAACTCCGTCC; SEQ ID NO: 134).
Table 164 Dose-dependent inhibition of human apo(a) protein in transgenic mice Dose %
ISIS No ED50 (mg/kg/wk) inhibition Example 120: Tolerability of antisense oligonucleotides targeting human apo(a) in rodent models Gapmer antisense oligonucleotides targeting human apo(a) were selected from the studies described above for tolerability studies in CD1 mice and in Sprague Dawley rats. Rodents do not express endogenous apo(a), hence these studies tested the tolerability of each human antisense oligonucleotide in an animal rather than any phenotypic changes that may be caused by inhibiting apo(a) in the animal.
Tolerability in CD1 mice: Study 1 CD1 0 mice (Charles River, MA) 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 CD1 mice were injected subcutaneously twice a week for 6 weeks with 50 mg/kg of ISIS 494159, ISIS 494161, ISIS 494162, ISIS 494244, ISIS 494283, ISIS 494284, ISIS 494285, ISIS 494286, ISIS 494301, ISIS 494302, ISIS 494311, ISIS 494337, ISIS 494372, and ISIS
510548. One group of six-week old male CD1 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, creatinine, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, NY). The results are presented in the table below. ISIS
oligonucleotides that caused changes in the levels of any of the liver or kidney function markers outside the expected range for antisense oligonucleotides were excluded in further studies.
Table 165 Plasma chemistry markers of CD1 mice ALT AST Albumin BUN Creatinine Bilirubin (IU/L (IU/L) (g/dL) (mg/dL) (mg/dL) (mg/dL) PBS 38 71 2.9 25.2 0.16 0.15 ISIS 494159 615 525 2.7 23.9 0.11 0.20 ISIS 494161 961 670 2.6 23.7 0.15 0.14 ISIS 494162 1373 1213 2.7 23.7 0.14 0.18 ISIS 494283 237 242 2.5 26.2 0.14 0.13 ISIS 494284 192 307 2.3 27.1 0.14 0.10 ISIS 494285 582 436 2.3 25.4 0.16 0.11 ISIS 494286 191 227 2.5 21.1 0.12 0.15 ISIS 494301 119 130 2.7 26.4 0.15 0.12 ISIS 494302 74 96 2.8 24.8 0.14 0.15 ISIS 494311 817 799 2.7 28.7 0.12 0.17 ISIS 494337 722 397 2.5 20.0 0.13 0.11 ISIS 494372 73 164 2.6 28.5 0.16 0.11 ISIS 510548 2819 2245 3.1 26.0 0.15 0.15 Organ weights Liver, spleen and kidney weights were measured at the end of the study, and are presented in the table below. ISIS oligonucleotides that caused any changes in organ weights outside the expected range for antisense oligonucleotides were excluded from further studies.

Table 166 Organ weights of CD1 mice (g) Kidney Liver Spleen PBS 0.68 2.0 0.13 ISIS 494159 0.68 3.0 0.21 ISIS 494161 0.62 3.5 0.20 ISIS 494162 0.60 3.3 0.20 ISIS 494283 0.65 2.8 0.24 ISIS 494284 0.69 2.7 0.29 ISIS 494285 0.59 3.2 0.21 ISIS 494286 0.64 2.8 0.25 ISIS 494301 0.72 3.0 0.43 ISIS 494302 0.63 2.3 0.23 ISIS 494311 0.61 3.2 0.19 ISIS 494337 0.56 2.3 0.17 ISIS 494372 0.60 2.5 0.27 ISIS 510548 0.55 3.7 0.20 Tolerability 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 selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.
Treatment Groups of male Sprague Dawley rats were injected subcutaneously twice a week for 8 weeks with 30 mg/kg of ISIS 494159, ISIS 494161, ISIS 494162, ISIS 494244, ISIS 494283, ISIS
494284, ISIS 494285, ISIS 494286, ISIS 494301, ISIS 494302, ISIS 494311, ISIS 494337, ISIS 494372, and ISIS 510548. One group of six male Sprague Dawley rats was injected subcutaneously twice a week for 8 weeks with PBS. 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, creatinine, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, NY). The results are presented in the table below. ISIS

oligonucleotides that caused changes in the levels of any of the liver or kidney function markers outside the expected range for antisense oligonucleotides were excluded in further studies.
Table 167 Plasma chemistry markers of Sprague Dawley rats ALT AST Bilirubin Albumin BUN
Creatinine (IU/L) (IU/L) (mg/dL) (mg/dL) (mg/dL) (mg/dL) PBS 30 82 0.09 3.2 19 0.28 ISIS 494159 182 208 0.14 3.4 22 0.35 ISIS 494161 36 86 0.13 3.4 23 0.35 ISIS 494162 102 158 0.17 2.6 28 0.32 ISIS 494283 53 156 0.13 2.9 24 0.32 ISIS 494284 34 113 0.08 2.0 28 0.32 ISIS 494285 110 294 0.10 1.4 110 0.52 ISIS 494286 40 83 0.07 1.6 48 0.44 ISIS 494301 38 132 0.08 3.0 18 0.33 ISIS 494302 47 105 0.09 3.2 19 0.34 ISIS 494311 93 185 0.51 2.7 23 0.30 ISIS 494372 54 119 0.12 3.0 19 0.33 ISIS 510548 116 181 0.11 1.7 65 0.66 Kidney function To evaluate the effect of ISIS oligonucleotides on kidney function, urine levels of total protein and creatinine were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, NY). Results are presented in the table below, expressed in mg/dL.
Table 168 Kidney function markers (mg/dL) in Sprague-Dawley rats .
Total Creatinme protein Organ weights Liver, spleen and kidney weights were measured at the end of the study, and are presented in the table below. ISIS oligonucleotides that caused any changes in organ weights outside the expected range for antisense oligonucleotides were excluded from further studies.
Table 169 Organ weights of Sprague Dawley rats (g) Kidney liver Spleen PBS 3.5 13.1 0.9 ISIS 494159 3.1 11.7 1.6 ISIS 494161 2.8 12.5 2 ISIS 494162 3.1 14.2 1.6 ISIS 494283 3.3 12.9 2.3 ISIS 494284 4.1 15.8 2.7 ISIS 494285 3.8 13.4 0.8 ISIS 494286 4.2 16.7 2.5 ISIS 494301 3.2 12.1 2.3 ISIS 494302 3.4 13.3 2.4 ISIS 494311 3.5 17.4 3.2 ISIS 494372 3.6 12.9 3.2 ISIS 510548 6.4 21.2 1.5 The finding from the rodent tolerability studies showed that in general, taking into consideration all the tolerability markers screened, ISIS 494372 was the best tolerated antisense compound in both the CD1 mouse model and the Sprague Dawley rat model.
Example 121: Pharmacokinetics of antisense oligonucleotide in CD1 mice CD1 mice were treated with ISIS oligonucleotides and the oligonucleotide concentrations in the liver and kidney were evaluated.
Treatment Groups of four CD1 mice each were injected subcutaneously twice per week for 6 weeks with 50 mg/kg of ISIS 494283, ISIS 494284, ISIS 494286, ISIS 494301, ISIS 494302, or ISIS 494372. The mice were sacrificed 2 days following the final dose. Livers were harvested for analysis.
Measurement of oligonucleotide concentration The concentration of the total oligonucleotide concentration was measured. The method used is a modification of previously published methods (Leeds et al., 1996; Geary et al., 1999) which consist of a phenol-chloroform (liquid-liquid) extraction followed by a solid phase extraction. An internal standard (ISIS
355868, a 27-mer 2'-0-methoxyethyl modified phosphorothioate oligonucleotide, GCGTTTGCTCTTCTTCTTGCGTTTTTT, designated herein as SEQ ID NO: 131) was added prior to extraction. Tissue sample concentrations were calculated using calibration curves, with a lower limit of quantitation (LLOQ) of approximately 1.14 [tg/g. Half-lives were then calculated using WinNonlin software (PHARSIGHT).
The results are presented in the table below, expressed as [tg/g liver or kidney tissue. The data indicates that ISIS 494372 was at an acceptable concentration in the liver and kidneys.
Table 170 Oligonucleotide concentration ( g/g tissue) of ISIS oligonucleotides in CD1 mice ISIS No Liver Kidney DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.

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THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME

NOTE: For additional volumes, please contact the Canadian Patent Office NOM DU FICHIER / FILE NAME:
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Claims (193)

CLAIMS:

1. A compound comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and comprises a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of nucleobases 3901 to 3920 of SEQ ID NO: 1, wherein the nucleobase sequence of the modified oligonucleotide is at least 80%
complementary to SEQ ID NO: 1.

2. The compound of claim 1, wherein the modified oligonucleotide comprises a nucleobase sequence comprising a portion of at least 10, at least 12, at least 14, at least 16, at least 18, at least 19, or at least 20 contiguous nucleobases complementary to an equal length portion of SEQ ID NO:

3. A compound comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and comprises a nucleobase sequence comprising a portion of at least 8, at least 10, at least 12, at least 14, at least 15, or at least 16 contiguous nucleobases complementary to an equal length portion of nucleobases 3900 to 3923 of SEQ ID
NO: 1, wherein the nucleobase sequence of the modified oligonucleotide is at least 80%
complementary to SEQ ID NO: 1.

4. The compound of any preceding claim, wherein the nucleobase sequence of the modified oligonucleotide is at least 85%, at least 90%, at least 95%, or 100%
complementary to SEQ ID NO: 1.

5. A compound comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and has a nucleobase sequence comprising at least 8, least 9, least 10, least 11, at least 12, least 13, at least 14, at least 15, at least 16, least 17, least 18, least 19, or 20 contiguous nucleobases of the nucleobase sequence of SEQ ID NO: 58.

6. A compound comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and has a nucleobase sequence comprising at least 8, least 9, least 10, least 11, at least 12, least 13, at least 14, at least 15, at least 16, least 17, least 18, least 19, or 20 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NO: 12-130, 133, 134.

7. The compound of any preceding claim, wherein the modified oligonucleotide is single-stranded.

8. The compound of any of claims 1-6, wherein the modified oligonucleotide is double stranded.

9. The compound of any preceding claim, wherein the modified oligonucleotide comprises at least one modified internucleoside linkage.

10. The compound of claim 9, wherein the modified internucleoside linkage is a phosphorothioate internucleoside linkage.

11. The compound of claim 10, wherein the modified oligonucleotide comprises at least one phosphodiester internucleoside linkage.

12. The compound of claim 10, wherein the modified oligonucleotide comprises at least 2 phosphodiester internucleoside linkages.

13. The compound of claim 10, wherein the modified oligonucleotide comprises at least 3 phosphodiester internucleoside linkages.

14. The compound of claim 10, wherein the modified oligonucleotide comprises at least 4 phosphodiester internucleoside linkages.

15. The compound of claim 10, wherein the modified oligonucleotide comprises at least 5 phosphodiester internucleoside linkages.

16. The compound of claim 10, wherein the modified oligonucleotide comprises at least 6 phosphodiester internucleoside linkages.

17. The compound of claim 10, wherein the modified oligonucleotide comprises at least 7 phosphodiester internucleoside linkages.

18. The compound of any of claims 11 to 17, wherein each internucleoside linkage of the modified oligonucleotide is selected from a phosphodiester internucleoside linkage and a phosphorothioate internucleoside linkage.

19. The compound of any of claims 1 to 8, wherein each internucleoside linkage of the modified oligonucleotide comprises is a phosphorothioate internucleoside linkage.

20. A compound consisting of ISIS 494372 and a conjugate group.

21. The compound of any preceding claim, wherein the modified oligonucleotide comprises at least one modified sugar.

22. The compound of claim 21, wherein at least one modified sugar is a bicyclic sugar.

23. The compound of claim 21, wherein at least one modified sugar comprises a 2'-O-methoxyethyl, a constrained ethyl, a 3'-fluoro-HNA or a 4'-(CH2)n-O-2' bridge, wherein n is 1 or 2.

24. The compound of any preceding claim, wherein at least one nucleoside comprises a modified nucleobase.

25. The compound of claim 24, wherein the modified nucleobase is a 5-methylcytosine.

26. The compound of any preceding claim, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides and comprises:
a gap segment consisting of linked deoxynucleosides;
a 5' wing segment consisting of linked nucleosides;
a 3' wing segment consisting of linked nucleosides;
wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.

27. The compound of any preceding claim, wherein the modified oligonucleotide consists of 15 to 30, 18 to 24, 19 to 22, 13 to 25, 14 to 25, 15 to 25, 16 or 20 linked nucleosides.

28. A compound comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 20 linked nucleosides and has a nucleobase sequence comprising at least 8 contiguous nucleobases complementary to an equal length portion of any of SEQ
ID NO: 58, wherein the modified oligonucleotide comprises:
a gap segment consisting of ten linked deoxynucleosides;
a 5' wing segment consisting of five linked nucleosides;
a 3' wing segment consisting of five linked nucleosides;
wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment, wherein each nucleoside of each wing segment comprises a 2'-O-methoxyethyl sugar, wherein each internucleoside linkage is a phosphorothioate linkage and wherein each cytosine residue is a 5-methylcytosine.

29. The compound of any of claims 1 to 28, wherein the conjugate group is linked to the modified oligonucleotide at the 5' end of the modified oligonucleotide.

30. The compound of any of claims 1 to 28, wherein the conjugate group is linked to the modified oligonucleotide at the 3' end of the modified oligonucleotide.

31. The compound of any of claims 1-30, wherein the conjugate group comprises exactly one ligand.

32. The compound of any of claims 1-30, wherein the conjugate group comprises exactly two ligands.

33. The compound of any of claims 1-30, wherein the conjugate group comprises three or more ligands.

34. The compound of any of claims 1-30, wherein the conjugate group comprises exactly three ligands.

35. The compound of any of claims 31-34, wherein each ligand is selected from among: a polysaccharide, modified polysaccharide, mannose, galactose, a mannose derivative, a galactose derivative, D-mannopyranose, L-Mannopyranose, D-Arabinose, L-Galactose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-Galactose, L-Galactose, .alpha.-D-Mannofuranose,.beta.-D-Mannofuranose, .alpha.-D-Mannopyranose,.beta.-D-Mannopyranose, .alpha.-D-Glucopyranose, [3-D-Glucopyranose, .alpha.-D-Glucofuranose, [3-D-Glucofuranose, .alpha.-D-fructofuranose, .alpha.-D-fructopyranose, .alpha.-D-Galactopyranose, [3 -D-Galactopyranose, .alpha.-D-Galactofuranose, .beta. -D-Galactofuranose, glucosamine, sialic acid, .alpha.-D-galactosamine, N-Acetylgalactosamine, 2-Amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-.beta.-D-glucopyranose, 2-Deoxy-2-methylamino-L-glucopyranose, 4,6-Dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose, 2-Deoxy-2-sulfoamino-D-glucopyranose, N-Glycoloyl-.alpha.-neuraminic acid, 5-thio-.beta.-D-glucopyranose, methyl 2,3,4-tri-O-acetyl-1-thio-6-O-trityl-.alpha.-D-glucopyranoside, 4-Thio-.beta.-D-galactopyranose, ethyl 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-.alpha.-D-gluco-heptopyranoside, 2,5-Anhydro-D-allononitrile, ribose, D-ribose, D-4-thioribose, L-ribose, L-4-thioribose.

36. The compound of claim 35, wherein each ligand is N-acetyl galactosamine.

37. The compound of any of claims 1 to 30, wherein the conjugate group comprises:

38. The compound of any of claims 1 to 30, wherein the conjugate group comprises:

39. The compound of any of claims 1 to 30, wherein the conjugate group comprises:

40. The compound of any of claims 1 to 30, wherein the conjugate group comprises:

41. The compound of any of claims 1 to 30, wherein the conjugate group comprises:

42. The compound of any of claims 30 to 36, wherein the conjugate group comprises at least one phosphorus linking group or neutral linking group.

43. The compound of any of claims 1 to 42, wherein the conjugate group comprises a structure selected from among:

wherein n is from 1 to 12; and wherein m is from 1 to 12.

44. The compound of any of claims 30 to 36, wherein the conjugate group has a tether having a structure selected from among:
wherein L is either a phosphorus linking group or a neutral linking group;
Z1 is C(=O)O-R2;
Z2 is H, C1-C6 alkyl or substituted C1-C6 alky;
R2 is H, C1-C6 alkyl or substituted C1-C6 alky; and each m1 is, independently, from 0 to 20 wherein at least one m1 is greater than 0 for each tether.

45. The compound of claim 44, wherein conjugate group has a tether having a structure selected from among:
wherein Z2 is H or CH3; and each m1 is, independently, from 0 to 20 wherein at least one m1 is greater than 0 for each tether.

46. The compound of any of claims 30 to 36, wherein the conjugate group has tether having a structure selected from among:
wherein n is from 1 to 12; and wherein m is from 1 to 12.

47. The compound of any of claims 1 to 46, wherein the conjugate group is covalently attached to the modified oligonucleotide.

48. The compound of any of claims 1 to 47, wherein the compound has a structure represented by the formula:
wherein A is the modified oligonucleotide;
B is the cleavable moiety C is the conjugate linker D is the branching group each E is a tether;
each F is a ligand; and q is an integer between 1 and 5.

49. The compound of any of claims 1 to 47, wherein the compound has a structure represented by the formula:
wherein:
A is the modified oligonucleotide;
B is the cleavable moiety C is the conjugate linker D is the branching group each E is a tether;
each F is a ligand;
each n is independently 0 or 1; and q is an integer between 1 and 5.

50. The compound of any of claims 1 to 47, wherein the compound has a structure represented by the formula:
wherein A is the modified oligonucleotide;
B is the cleavable moiety;
C is the conjugate linker;
each E is a tether;
each F is a ligand; and q is an integer between 1 and 5.

51. The compound of any of claims 1 to 47, wherein the compound has a structure represented by the formula:
wherein A is the modified oligonucleotide;
C is the conjugate linker;
D is the branching group;
each E is a tether;
each F is a ligand; and q is an integer between 1 and 5.

52. The compound of any of claims 1 to 47, wherein the compound has a structure represented by the formula:
wherein A is the modified oligonucleotide;
C is the conjugate linker;
each E is a tether;
each F is a ligand; and q is an integer between 1 and 5.

53. The compound of any of claims 1 to 47, wherein the compound has a structure represented by the formula:
wherein A is the modified oligonucleotide;
B is the cleavable moiety;
D is the branching group;
each E is a tether;
each F is a ligand; and q is an integer between 1 and 5.

54. The compound of any of claims 1 to 47, wherein the compound has a structure represented by the formula:
wherein A is the modified oligonucleotide;
B is the cleavable moiety;
each E is a tether;
each F is a ligand; and q is an integer between 1 and 5.

55. The compound of any of claims 1 to 47, wherein the compound has a structure represented by the formula:
wherein A is the modified oligonucleotide;
D is the branching group;
each E is a tether;
each F is a ligand; and q is an integer between 1 and 5.

56. The compound of any of claims 48 to 55, wherein the conjugate linker has a structure selected from among:

wherein each L is, independently, a phosphorus linking group or a neutral linking group; and each n is, independently, from 1 to 20.

57. The compound of any of claims 48 to 55, wherein the conjugate linker has a structure selected from among:

58. The compound of any of claims 48 to 55, wherein the conjugate linker has the followingstructure:

59. The compound of any of claims 48 to 55, wherein the conjugate linker has a structure selected from among:

60. The compound of any of claims 48 to 55, wherein the conjugate linker has a structure selected from among:

61. The compound of any of claims 48 to 55, wherein the conjugate linker has a structure selected from among:

62. The compound of any of claims 48 to 61, wherein the conjugate linker comprises a pyrrolidine.

63. The compound of any of claims 48 to 61, wherein the conjugate linker does not comprise a pyrrolidine.

64. The compound of any of claims 48 to 63, wherein the conjugate linker comprises PEG.

65. The compound of any of claims 48 to 64, wherein the conjugate linker comprises an amide.

66. The compound of any of claims 48 to 64, wherein the conjugate linker comprises at least two amides.

67. The compound of any of claims 48 to 64, wherein the conjugate linker does not comprise an amide.

68. The compound of any of claims 48 to 67, wherein the conjugate linker comprises a polyamide.

69. The compound of any of claims 48 to 68, wherein the conjugate linker comprises an amine.

70. The compound of any of claims 48 to 69, wherein the conjugate linker comprises one or more disulfide bonds.

71. The compound of any of claims 48 to 70, wherein the conjugate linker comprises a protein binding moiety.

72. The compound of claim 71, wherein the protein binding moiety comprises a lipid.

73. The compound of claim 71, wherein the protein binding moiety is selected from among: cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine), a vitamin (e.g., folate, vitamin A, vitamin E, biotin, pyridoxal), a peptide, a carbohydrate (e.g., monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, polysaccharide), an endosomolytic component, a steroid (e.g., uvaol, hecigenin, diosgenin), a terpene (e.g., triterpene, e.g., sarsasapogenin, friedelin, epifriedelanol derivatized lithocholic acid), or a cationic lipid.

74. The compound of claim 71, wherein the protein binding moiety is selected from among: a C16 to C22 long chain saturated or unsaturated fatty acid, cholesterol, cholic acid, vitamin E, adamantane or 1-pentafluoropropyl.

75. The compound of any of claims 48 to 74, wherein the conjugate linker has a structure selected from among:
wherein each n is, independently, is from 1 to 20; and p is from 1 to 6.

76. The compound of any of claims 48 to 75, wherein the conjugate linker has a structure selected from among:

wherein each n is, independently, from 1 to 20.

77. The compound of any of claims 48 to 75, wherein the conjugate linker has a structure selected from among:

78. The compound of any of claims 48 to 75, wherein the conjugate linker has a structure selected from among:
wherein n is from 1 to 20.

79. The compound of any of claims 48 to 75, wherein the conjugate linker has a structure selected from among:

80. The compound of any of claims 48 to 75, wherein the conjugate linker has a structure selected from among:
wherein each n is independently, 0, 1, 2, 3, 4, 5, 6, or 7.

81. The compound of any of claims 48 to 75, wherein the conjugate linker has the following structure:

82. The compound of any of claims 48 to 81, wherein the branching group has one of the following structures:

wherein each A1 is independently, O, S, C=O or NH; and each n is, independently, from 1 to 20.

83. The compound of any of claims 48 to 81, wherein the branching group has one of the following structures:
wherein each A1 is independently, O, S, C=O or NH; and each n is, independently, from 1 to 20.

84. The compound of any of claims 48 to 81, wherein the branching group has the following structure:

85. The compound of any of claims 48 to 81, wherein the branching group has the following structure:

86. The compound of any of claims 48 to 81, wherein the branching group has the following structure:

87. The compound of any of claims 48 to 81, wherein the branching group has the following structure:

88. The compound of any of claims 48 to 81, wherein the branching group comprises an ether.

89. The compound of any of claims 48 to 81, wherein the branching group has the following structure:
each n is, independently, from 1 to 20; and m is from 2 to 6.

90. The compound of any of claims 48 to 81, wherein the branching group has the following structure:

91. The compound of any of claims 48 to 81, wherein the branching group has the following structure:

92. The compound of any of claims 48 to 81, wherein the branching group comprises:
wherein each j is an integer from 1 to 3; and wherein each n is an integer from 1 to 20.

93. The compound of any of claims 48 to 81, wherein the branching group comprises:

94. The compound of any of claims 48 to 93, wherein each tether is selected from among:
wherein L is selected from a phosphorus linking group and a neutral linking group;
Z1 is C(=O)O-R2;
Z2 is H, C1-C6 alkyl or substituted C1-C6 alky;
R2 is H, C1-C6 alkyl or substituted C1-C6 alky; and each m1 is, independently, from 0 to 20 wherein at least one m1 is greater than 0 for each tether.

95. The compound of any of claims 48 to 93, wherein each tether is selected from among:
wherein Z2 is H or CH3; and each m2 is, independently, from 0 to 20 wherein at least one m2 is greater than 0 for each tether.

96. The compound of any of claims 48 to 93, wherein each tether is selected from among:
wherein n is from 1 to 12; and wherein m is from 1 to 12.

97. The compound of any of claims 48 to 93, wherein at least one tether comprises ethylene glycol.

98. The compound of any of claims 48 to 93 or 95, wherein at least one tether comprises an amide.

99. The compound of any of claims 48 to 93 or 95, wherein at least one tether comprises a polyamide.

100. The compound of any of claims 48 to 93 or 95, wherein at least one tether comprises an amine.

101. The compound of any of claims 48 to 93 or 95, wherein at least two tethers are different from one another.

102. The compound of any of claims 48 to 93 or 95, wherein all of the tethers are the same as one another.

103. The compound of any of claims 48 to 93, wherein each tether is selected from among:
wherein each n is, independently, from 1 to 20; and each p is from 1 to about 6.

104. The compound of any of claims 48 to 93, wherein each tether is selected from among:

105. The compound of any of claims 48 to 93, wherein each tether has the following structure:

wherein each n is, independently, from 1 to 20.

106. The compound of any of claims 48 to 93, wherein each tether has the following structure:

107. The compound of any of claims 48 to 93, wherein the tether has a structure selected from among:
; wherein each n is independently, 0, 1, 2, 3, 4, 5, 6, or 7.

108. The compound of any of claims 48 to 93, wherein the tether has a structure selected from among:

109. The compound of any of claims 47 to 108, wherein the ligand is galactose.

110. The compound of any of claims 47 to 108, wherein the ligand is mannose-6-phosphate.

111. The compound of any of claims 47 to 108, wherein each ligand is selected from among:
wherein each R1 is selected from OH and NHCOOH.

112. The compound of any of claims 47 to 108, wherein each ligand is selected from among:

113. The compound of any of claims 47 to 108, wherein each ligand has the following structure:

114. The conjugated antisense compound of any of claims 47 to 108, wherein each ligand has the following structure:

115. The compound of any of claims 1 to 30 or 56 to 81, wherein the conjugate group comprises a cell-targeting moiety.

116. The compound of claim 116, wherein the conjugate group comprises a cell-targeting moiety having the following structure:
wherein each n is, independently, from 1 to 20.

117. The compound of any of claims 116, wherein the cell-targeting moiety has the following structure:

118. The compound of claim 116, wherein the cell-targeting moiety has the following structure:
wherein each n is, independently, from 1 to 20.

119. The compound of claim 116, wherein the cell-targeting moiety has the following structure:

120. The compound of claim 116, wherein the cell-targeting moiety comprises:

121. The compound of claim 116, wherein the cell-targeting moiety comprises:

122. The compound of claim 116, wherein the cell-targeting moiety comprises:

123. The compound of claim 116, wherein the cell-targeting moiety comprises:

124. The compound of claim 116, wherein the cell-targeting moiety comprises:

125. The compound of claim 116, wherein the cell-targeting moiety comprises:

126. The compound of claim 116, wherein the cell-targeting moiety comprises:

127. The compound of claim 116, wherein the cell-targeting moiety comprises:

128. The compound of claim 116, wherein the cell-targeting moiety comprises:

129. The compound of claim 116, wherein the cell-targeting moiety comprises:

130. The compound of claim 116, wherein the cell-targeting moiety comprises:

131. The compound of claim 116, wherein the cell-targeting moiety comprises:

132. The compound of claim 116, wherein the cell-targeting moiety comprises:

133. The compound of claim 116, wherein the cell-targeting moiety comprises:

134. The compound of claim 116, wherein the cell-targeting moiety comprises:

135. The compound of claim 116, wherein the cell-targeting moiety comprises:

136. The compound of claim 116, wherein the cell-targeting moiety comprises:

137. The compound of claim 116, wherein the cell-targeting moiety comprises:

138. The compound of claim 116, wherein the cell-targeting moiety comprises:

139. The compound of claim 116, wherein the cell-targeting moiety comprises:

140. The compound of claim 116, wherein the cell-targeting moiety comprises:

141. The compound of claim 116, wherein the cell-targeting moiety comprises:
wherein each Y is selected from O, S, a substituted or unsubstituted C1-C10 alkyl, amino, substituted amino, azido, alkenyl or alkynyl.

142. The compound of any of claims 1 to 30, wherein the conjugate group comprises:
wherein each Y is selected from O, S, a substituted or unsubstituted C1-C10 alkyl, amino, substituted amino, azido, alkenyl or alkynyl.

143. The compound of any of claims 1 to 30, wherein the conjugate group comprises:
wherein each Y is selected from O, S, a substituted or unsubstituted C1-C10 alkyl, amino, substituted amino, azido, alkenyl or alkynyl.

144. The compound of any of claims 1 to 30, wherein the conjugate group comprises:

145. The compound of any of claims 1 to 30, wherein the conjugate group comprises:

146. The compound of any of claims 1 to 30, wherein the conjugate group comprises:

147. The compound of claim 117, wherein the conjugate group comprises:

148. The compound of any of claims 1 to 147, wherein the conjugate group comprises a cleavable moiety selected from among: a phosphodiester, an amide, or an ester.

149. The compound of any of claims 1 to 147, wherein the conjugate group comprises a phosphodiester cleavable moiety.

150. The compound of any of claims 1 to 147, wherein the conjugate group does not comprise a cleavable moiety, and wherein the conjugate group comprises a phosphorothioate linkage between the conjugate group and the oligonucleotide.

151. The compound of any of claims 1 to 150, wherein the conjugate group comprises an amide cleavable moiety.

152. The compound of any of claims 1 to 150, wherein the conjugate group comprises an ester cleavable moiety.

153. The compound of any of claims 1 to 30, wherein the compound has the following structure:
wherein each n is, independently, from 1 to 20;
Q13 is H or O(CH2)2-OCH3;
A is the modified oligonucleotide; and Bx is a heterocyclic base moiety.

154. The compound of any of claims 1 to 30, wherein the compound has the following structure:

wherein each n is, independently, from 1 to 20;
Q13 is H or O(CH2)2-OCH3;
A is the modified oligonucleotide; and Bx is a heterocyclic base moiety.

155. The compound of any of claims 1 to 30, wherein the compound has the following structure:
wherein each n is, independently, from 1 to 20;
Q13 is H or O(CH2)2-OCH3;
A is the modified oligonucleotide;
Z is H or a linked solid support; and Bx is a heterocyclic base moiety.

156. The compound of any of claims 1 to 30, wherein the compound has the following structure:
wherein each n is, independently, from 1 to 20;
Q13 is H or O(CH2)2-OCH3;
A is the modified oligonucleotide;
Z is H or a linked solid support; and Bx is a heterocyclic base moiety.

157. The compound of any of claims 1 to 30, wherein the compound has the following structure:
wherein Q13 is H or O(CH2)2-OCH3;
A is the modified oligonucleotide;and Bx is a heterocyclic base moiety.

158. The compound of any of claims 1 to 30, wherein the compound has the following structure:
wherein Q13 is H or O(CH2)2-OCH3;
A is the modified oligonucleotide;and Bx is a heterocyclic base moiety.

159. The compound of any of claims 1 to 30, wherein the compound has the following structure:
wherein Q13 is H or O(CH2)2-OCH3;
A is the modified oligonucleotide; and Bx is a heterocyclic base moiety.

160. The compound of any of claims 1 to 30, wherein the compound has the following structure:
wherein Q13 is H or O(CH2)2-OCH3;
A is the modified oligonucleotide; and Bx is a heterocyclic base moiety.

161. The compound of any of claims 1 to 30, wherein the compound has the following structure:
wherein Q13 is H or O(CH2)2-OCH3;
A is the modified oligonucleotide; and Bx is a heterocyclic base moiety.

162. The compound of any of claims 1 to 30, wherein the compound has the following structure:
wherein Q13 is H or O(CH2)2-OCH3;
A is the modified oligonucleotide; and Bx is a heterocyclic base moiety.

163. The compound of any of claims 1 to 30, wherein the compound has the following structure:
wherein Q13 is H or O(CH2)2-OCH3;
A is the modified oligonucleotide; and Bx is a heterocyclic base moiety.

164. The compound of any of claims 1 to 30, wherein the compound has the following structure:
wherein Q13 is H or O(CH2)2-OCH3;
A is the modified oligonucleotide; and Bx is a heterocyclic base moiety.

165. The compound of any of claims 1 to 30, wherein the compound has the following structure:
wherein Q13 is H or O(CH2)2-OCH3;
A is the modified oligonucleotide; and Bx is a heterocyclic base moiety.

166. The compound of any of claims 1 to 30, wherein the compound has the following structure:
wherein Q13 is H or O(CH2)2-OCH3;
A is the modified oligonucleotide; and Bx is a heterocyclic base moiety.

167. The compound of any of claims 1 to 30, wherein the compound has the following structure:
wherein Q13 is H or O(CH2)2-OCH3;
A is the modified oligonucleotide; and Bx is a heterocyclic base moiety.

168. The compound of any of claims 1 to 30, wherein the conjugate group comprises:
wherein Q13 is H or O(CH2)2-OCH3;
A is the modified oligonucleotide; and Bx is a heterocyclic base moiety.

169. The compound of any of claims 1 to 30, wherein the conjugate group comprises:
wherein Q13 is H or O(CH2)2-OCH3;
A is the modified oligonucleotide; and Bx is a heterocyclic base moiety.

170. The compound of any of claims 1 to 30, wherein the conjugate group comprises:
wherein Q13 is H or O(CH2)2-OCH3;
A is the modified oligonucleotide; and Bx is a heterocyclic base moiety.

171. The compound of any of claims 153 to 170, wherein B x is selected from among from adenine, guanine, thymine, uracil, or cytosine, or 5-methyl cytosine.

172. The compound of any of claims 153 to 170, wherein B x is adenine.

173. The compound of any of claims 153 to 170, wherein B x is thymine.

174. The compound of any of claims 153 to 170, wherein Q13 is O(CH2)2-OCH3.

175. The compound of any of claims 153 to 170, wherein Q13 is H.

176. A compound having the formula:
wherein x is a conjugate group comprising GalNAc.

177. A compound having the formula:

178. A compound having the formula:

179. A compound haying the formula:
Wherein either R' is ¨OCH2CH2OCH3 (MOE)and R2 is H; or R' and R2 together form a bridge, wherein R' is ¨O- and R2 is ¨CH2-, -CH(CH3)-, or -CH2CH2-, and R' and R2 are directly connected such that the resulting bridge is selected from: -O-CH2-, -O-CH(CH3)-, and ¨O-CH2CH2-;
And for each pair of R3 and R4 on the same ring, independently for each ring:
either R3 is selected from H
and -OCH2CH2OCH3 and R4 is H; or R3 and R4 together form a bridge, wherein R3 is ¨O-, and R4 is ¨CH2-, -CH(CH3)-, or -CH2CH2-and R3 and R4 are directly connected such that the resulting bridge is selected from: -O-CH2-, -O-CH(CH3)-, and ¨O-CH2CH2-;

And R5 is selected from H and ¨CH3;
And Z is selected from S- and O-.

180. A composition comprising the compound of any of claims 1-179 or salt thereof and at least one of a pharmaceutically acceptable carrier or diluent.

181. A prodrug comprising the compound of any of claims 1-180.

182. A method comprising administering to an animal the compound or composition of any of claims 1-181.

183. The method of claim 181, wherein the animal is a human.

184. The method of claim 181, wherein administering the compound prevents, treats, ameliorates, or slows progression of a cardiovascular, metabolic and/or inflammatory disease.

185. The method of claim 182, comprising co-administering the compound or composition and a second agent.

186. The method of claim 185, wherein the compound or composition and the second agent are administered concomitantly.

187. The method of claim 176, wherein the administering is parenteral.

188. The method of claim 176, wherein the administering is subcutaneous.

189. A method to reduce apo(a) mRNA or protein expression in an animal comprising administering to the animal the compound or composition of any of claims 1-181 to reduce apo(a) mRNA or protein expression in the animal.

190. A method to reduce Lp(a) levels in an animal comprising administering to the animal the compound or composition of any of claims 1-181 to reduce apo(a) mRNA or protein expression in the animal.

191. A composition comprising the compound according to any preceding claim, for use in therapy.

192. The compound of claim 191, for use in treating, preventing, or slowing progression of a disease related to elevated apo(a) and/or elevated Lp(a).

193. The compound of claim 191, wherein the disease is an inflammatory, cardiovascular or metabolic disease, disorder or condition.