NZ728517B2 - Compositions and methods for modulating ttr expression - Google Patents

Abstract

Disclosed is a compound comprising a modified oligonucleotide and a conjugate group comprising clusters of three N-acetylgalactosamine (GalNAc) ligands, wherein the modified oligonucleotide consists of 16 to 30 linked nucleosides and comprises a nucleobase sequence at least 85% complementary to SEQ ID NO: 2 (TTR). Also disclosed are compositions comprising said compound and the use of said compositions and compounds in the manufacture of a medicament for treating transthyretin amyloidosis. ID NO: 2 (TTR). Also disclosed are compositions comprising said compound and the use of said compositions and compounds in the manufacture of a medicament for treating transthyretin amyloidosis.

Description

ation] Vaughan.Barlow COMPOSITIONS AND METHODS FOR TING TTR 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 BIOL0248WOSEQ_ST25.txt, created on May 1, 2014, which is 16 Kb in size. The information in the electronic format of the sequence g is incorporated herein by reference in its entirety.

BACKGROUND OF THE ION 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 ation or occupancy-based tion. An example of tion of RNA target function by degradation is RNase H-based degradation of the target RNA upon ization with a DNA-like antisense compound. Another example of modulation of gene expression by target degradation is RNA interference (RNAi). RNAi refers to nse-mediated gene silencing through a mechanism that utilizes the RNA-induced siliencing complex (RISC). An additional example of modulation of RNA target fimction is by an occupancy-based mechanism such as is employed lly by microRNA. MicroRNAs are small non-coding RNAs that regulate the expression of protein- coding RNAs. The g of an antisense nd 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 fiJnction. 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 fiJnctionalization, as well as eutics to selectively modulate the expression of genes involved in the pathogenesis of diseases.

Antisense technology is an ive 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 ed 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, Vitravene® (fomivirsen; developed by Isis Pharmaceuticals Inc., Carlsbad, CA) was the first antisense drug to achieve marketing clearance from the US. Food and Drug Administration (FDA), and is currently a treatment of cytomegalovirus (CMV)-induced retinitis in AIDS patients.

New al modifications have improved the y 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 ements in patient convenience. Chemical modifications increasing potency of antisense compounds allow administration of lower doses, which reduces the ial for toxicity, as well as decreasing overall cost of therapy. Modifications increasing the ance to degradation result in slower clearance from the body, allowing for less frequent dosing. Different types of chemical modifications can be combined in one nd to further optimize the compound's efficacy.

SUMMARY OF THE INVENTION In certain ments, the present disclosure provides conjugated antisense compounds. In n embodiments, the present disclosure provides conjugated antisense compounds comprising an antisense oligonucleotide complementary to a nucleic acid transcript. In certain embodiments, the present disclosure es methods comprising contacting a cell with a ated antisense nd comprising an nse oligonucleotide mentary to a nucleic acid transcript. In certain embodiments, the present disclosure es methods comprising ting a cell with a conjugated antisense compound sing an antisense ucleotide 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- 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 .

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 ed to the antisense nd long enough to provide benefit ved 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. sed 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 . 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 ble nucleoside through a phosphodiester e. In certain embodiments, the cleavable nucleoside is ed to the antisense oligonucleotide (antisense compound) by a phosphodiester linkage. In certain embodiments, the conjugate group may comprise two or three ble nucleosides. In such embodiments, such cleavable sides 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 d comprise a cleavable bond. It is shown that that ent 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 nds are d to remove all or part of the conjugate ing in the active (or more active) form of the antisense compound lacking all or some of the conjugate.

In n embodiments, ates are attached at the 5’ end of an oligonucleotide. Certain such 5’- conjugates are d more efficiently than counterparts having a similar ate 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 r efficacy than oligonucleotides comprising a ate 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. r, using that approach, the ate 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 ques described herein for 5’-conjugated oligonucleotides, one can synthesize the ucleotide 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. er, 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 GalNAc3-10 in e 46 and 3-7 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 . ingly, 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 nds. 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, ing single-stranded oligonucleotides and either strand of -stranded oligonucleotides (e.g., siRNA).

In certain ments, the conjugates herein do not ntially alter certain measures of tolerability. For example, it is shown herein that conjugated nse compounds are not more immunogenic than unconjugated parent compounds. Since potency is ed, ments 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 nse 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 es of tolerability. For example, in certain ces, such antisense compounds having one or more phosphodiester are less genic 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, ated antisense compounds described herein tolerate such change in linkages with little or no loss in uptake and potency when compared to the ated full-phosphorothioate counterpart. In fact, in certain ments, for example, in Examples 44, 57, 59, and 86, oligonucleotides comprising a conjugate and at least one phosphodiester intemucleoside linkage ly 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 nse 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 sed 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 n the entire increase in activity. In such ments, productive uptake of the conjugated compound is increased. For example, as shown in Example 102, certain embodiments of GalNAc-containing conjugates se enrichment of antisense oligonucleotides in cytes versus nonparenchymal cells. This ment is beneficial for ucleotides 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 , 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 red.

In certain embodiments, the t disclosure provides conjugated nse compounds represented by the formula: A—B—C—D—éE—F) 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 ; 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: Where q = 3, the formula is: Where q = 4, the a is: Where q = 5, the formula is: E F A—B—C—D In certain embodiments, conjugated antisense compounds are provided having the structure: Targeting moiety / \ HO OH ww 7 O:F"*OH NHz W WW)N NHAc HOOH 0 O N s E: DETAILED DESCRIPTION It is to be understood that both the foregoing general description and the following detailed description are ary 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 ding" 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 se 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 ses, are hereby expressly incorporated by reference in their ty 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 nal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard ques 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 al Society , Washington DC, 1994; "Remington's Pharmaceutical Sciences," Mack Publishing Co., , 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 ," 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, which are hereby incorporated by reference for any purpose. Where permitted, all s, ations, 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 , "nucleoside" means a compound sing 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 intemucleoside 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 ing sugar moiety" means a ribofiJranosyl as found in naturally occurring RNA or a deoxyribofuranosyl as found in lly occurring DNA.

As used herein, "sugar moiety" means a naturally ing 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 " means a fiJranosyl 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 es are bicyclic sugar moieties.

As used herein, "2’-substituted sugar moiety" means a fiJranosyl sing 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 fiJranosyl ring.

As used herein, "MOE" means -OCH2CHZOCH3.

As used herein, "2’-F nucleoside" refers to a nucleoside comprising a sugar comprising fluorine at the 2’ position. Unless otherwise ted, the fluorine in a 2’-F nucleoside is in the ribo position (replacing the OH of a natural ).

As used herein the term "sugar ate" 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 nd which is capable of hybridizing to a complementary oligomeric compound. Such structures include rings comprising a different number of atoms than fiJranosyl (e. g., 4, 6, or 7-membered rings); replacement of the oxygen of a fiJranosyl 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., ered carbocyclic bicyclic sugar surrogates optionally comprising additional substituents). Sugar surrogates also e more complex sugar ements (e. g., the non-ring systems of peptide nucleic acid). Sugar surrogates include without tion linos, cyclohexenyls and exitols.

As used herein, "bicyclic sugar moiety" means a modified sugar moiety comprising a 4 to 7 membered ring (including but not limited to a fiJranosyl) comprising a bridge connecting two atoms of the 4 to 7 ed 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 fiJranosyl.

As used herein, "nucleotide" means a nucleoside fithher sing 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 n those that are ).

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 mentary naturally occurring nucleobase of another oligonucleotide or nucleic acid.

Nucleobases may be naturally occurring or may be modified.

As used herein the terms, "unmodified nucleobase" or ally occurring nucleobase" means the naturally occurring heterocyclic nucleobases of RNA or DNA: the purine bases adenine (A) and e (G), and the pyrimidine bases thymine (T), cytosine (C) (including 5-methyl C), and uracil (U).

As used herein, "modified nucleobase" means any base that is not a naturally occurring nucleobase.

As used , "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 d nucleobase.

As used herein, "bicyclic nucleoside" or "BNA" means a nucleoside comprising a bicyclic sugar moiety.

As used herein, "constrained ethyl nucleoside" or "cEt" means a nucleoside comprising a bicyclic sugar moiety comprising a 4’-CH(CH3)-O-2’bridge.

As used herein, "locked nucleic acid nucleoside" or "LNA" means a nucleoside sing a bicyclic sugar moiety comprising a 4’-CH2-O-2’bridge.

As used herein, "2’-substituted nucleoside" means a side 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 lly occurring deoxyribonucleosides (DNA). In certain embodiments, a 2’-deoxynucleoside may comprise a d nucleobase or may comprise an RNA nucleobase (e. g., uracil).

As used , "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 ibonucleosides (DNA) and/or one or more d 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 ucleotide" 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 ing internucleoside linkage" means a 3' to 5' phosphodiester e.

As used , "modified internucleoside linkage" means any internucleoside linkage other than a naturally occurring internucleoside linkage.

As used herein, nal 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 orus atom.

Phosphorus linking groups include without limitation groups haVing the formula: WO 79627 Rb: ['3‘ Re wherein: Ra and Rd are each, independently, O, S, CH2, NH, or NJ1 wherein J1 is C1-C6 alkyl or substituted C1- C6 alkyl; Rb is O or S; Rc is OH, SH, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, amino or substituted amino; and J1 is Rb is O 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 g group that directly links two nucleosides.

As used herein, "non-internucleoside phosphorus g group" means a phosphorus linking group that does not directly link two nucleosides. In certain embodiments, a non-intemucleoside phosphorus linking group links a side to a group other than a nucleoside. In certain embodiments, a non- cleoside phosphorus g group links two groups, r 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 N(CH3)-O-), amide-3 (- CH2-C(=O)-N(H)-), 4 (-CHZ-N(H)-C(=O)-), formacetal (-O-CH2), and thioformacetal (-S-CH2).

Further neutral linking groups include nonionic linkages comprising siloxane (dialkylsiloxane), ylate ester, carboxamide, sulfide, sulfonate ester and amides (See for example: Carbohydrate Modifications in Antisense Research; Y.S. SanghVi and PD. Cook Eds. ACS Symposium Series 580; Chapters 3 and 4, (pp. 40-65)). Further neutral linking groups include nonionic linkages comprising mixed N, O, 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, nternucleoside 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 n embodiments, a non-intemucleoside neutral linking group links two groups, neither of which is a nucleoside.

As used herein, "oligomeric compound" means a polymeric ure 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 ing c 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 ments, 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 es 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 ed to either, or both, the 3’ end or the 5’ end of an ucleotide. 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 nd. In general, conjugate groups modify one or more properties of the nd to which they are attached, including, but not limited to pharmacodynamic, pharmacokinetic, g, absorption, cellular distribution, ar uptake, charge and/or clearance ties.

As used , "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 ucleotide to r n of the conjugate group or (2) two or more portions of the ate 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 ments 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 ydrate cluster portion, such as a GalNAc cluster portion. Such carbohydrate cluster n ses: 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 ments, the carbohydrate cluster portion ses 3 GalNAc groups and is designated "GalNAc3". In certain embodiments, the carbohydrate cluster portion comprises 4 GalNAc groups and is ated "GalNAc4". Specific carbohydrate cluster portions (having specific tether, branching and conjugate linker groups) are bed herein and designated by Roman numeral followed by subscript "a". Accordingly "GalNac3-1a" 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 nd via a cleavable moiety, such as a cleavable bond or cleavable nucleoside.

As used herein, "cleavable moiety" means a bond or group that is e 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 e.

As used herein, hydrate cluster" means a compound having one or more carbohydrate residues attached to a scaffold or linker group. (see, e.g., Maier et al., esis 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): 5798-5 808, for es of carbohydrate ate clusters).

As used herein, "carbohydrate derivative" means any compound which may be synthesized using a ydrate as a starting material or ediate.

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. miting examples of protecting groups may be found in "Protective Groups in Organic Chemistry", T. W. Greene, P. G. M. Wuts, ISBN 062301-6, John Wiley & Sons, Inc, New York, which is incorporated herein by reference in its entirety.

As used herein, "single-stranded" means an oligomeric nd that is not hybridized to its complement and which lacks sufficient omplementarity to form a stable self-duplex.

As used , "double stranded" means a pair of oligomeric compounds that are hybridized to one another or a single omplementary 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 nse activity.

As used , ense activity" means any detectable and/or measurable change utable 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 tion of the splicing ofpre-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 nse 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 ring" 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 ing or measuring the ty 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 ter, particularly ve to another parameter which changes much more. In certain embodiments, a parameter is essentially unchanged when it changes less than 5%. In certain ments, 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 c acid. In certain such embodiments, the amount of a non-target nucleic acid is essentially unchanged if it s 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 logical 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, e (A) is complementary to uracil (U). In certain embodiments, complementary nucleobase means a nucleobase of an nse compound that is e of base pairing with a nucleobase of its target nucleic acid. For example, if a nucleobase at a n position of an antisense compound is e 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 base 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 nce to nucleobases means a pair of bases that do not form hydrogen bonds with one r.

As used herein, "complementary" in reference to oligomeric compounds (e.g., linked nucleosides, ucleotides, 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 eric 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, mentary oligomeric compounds or regions are 90% complementary. In certain ments, complementary oligomeric compounds or regions are 95% mentary. In certain embodiments, complementary oligomeric compounds or regions are 100% mentary.

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 g 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 ed Hoogsteen en g, between complementary nucleobases.

As used herein, "specifically hybridizes" means the ability of an eric 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 nce to an oligonucleotide or portion thereof means that each nucleobase of the ucleotide 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 mentary to nucleobases at corresponding positions in the target c acid by the total length of the oligomeric compound.

As used , "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, ation" means a change of amount or quality of a molecule, function, or activity when compared to the amount or quality of a molecule, fiJnction, or activity prior to modulation. For e, 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 sing, 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 n of sugar modifications in an ucleotide or a region f.

As used herein, ge motif" means a pattern of linkage modifications in an ucleotide 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 , "nucleobase modification motif’ means a pattern of modifications to nucleobases along an oligonucleotide. Unless ise indicated, a base 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 ations, including no al modifications.

As used , "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 ted, 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 lly-occurring unmodified nucleosides. Nucleosides that are the same but for comprising different nucleobases are not differently modified. For example, a nucleoside comprising a 2’-OMe modified sugar and an unmodified adenine nucleobase and a nucleoside sing a 2’-OMe modified sugar and an unmodified thymine nucleobase are not differently modified.

As used , "the same type of modifications" refers to modifications that are the same as one another, including absence of modifications. Thus, for e, two unmodified DNA nucleosides have "the same type of cation," even though the DNA nucleoside is unmodified. Such nucleosides having the same type modification may se different nucleobases.

As used herein, "separate regions" means ns of an oligonucleotide wherein the chemical modifications or the motif of chemical cations 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 . In certain embodiments, such sterile saline is pharmaceutical grade saline.

As used herein the term "metabolic disorder" means a e 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 fiJnction of the heart or blood vessels.

As used herein the term "mono or polycyclic ring " is meant to include all ring systems selected from single or polycyclic radical ring systems wherein the rings are fiJsed or linked and is meant to be inclusive of single and mixed ring systems individually selected from tic, alicyclic, aryl, heteroaryl, aralkyl, arylalkyl, heterocyclic, heteroaryl, heteroaromatic and heteroarylalkyl. Such mono and poly cyclic structures can n 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, O 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 fiJsed ring has two nitrogen atoms. The mono or polycyclic ring system can be fithher substituted with tuent 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 les using various strategies such as directly through a ring atom, fiJsed through multiple ring atoms, through a substituent group or h a bifiJnctional linking moiety.

As used herein, "prodrug" means an inactive or less active form of a nd which, when administered to a subject, is lized to form the active, or more active, compound (e. g., drug).

As used herein, "substituen " and ituent 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 ition of a nucleoside other than H or OH). Substituent groups can be protected or ected. 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 arbyl group to a parent compound.

Likewise, as used herein, "substituent" in reference to a chemical onal group means an atom or group of atoms that differs from the atom or a group of atoms normally present in the named fiJnctional group. In certain embodiments, a substituent es a hydrogen atom of the fiJnctional group (e.g., in certain embodiments, the substituent of a substituted methyl group is an atom or group other than en which replaces one of the hydrogen atoms of an unsubstituted methyl group). Unless ise indicated, groups amenable for use as substituents include without limitation, n, hydroxyl, alkyl, alkenyl, alkynyl, acyl (-C(O)Raa), carboxyl (-C(O)O-Raa), tic groups, lic groups, alkoxy, substituted oxy (-O-Raa), aryl, aralkyl, heterocyclic radical, heteroaryl, arylalkyl, amino (-N(Rbb)(Rcc)), imino(=NRbb), amido (—C(O)N(Rbb)(Rcc) 0r -N(Rbb)C(O)Raa), azido (-N3), nitro (-NOZ), cyano (-CN), carbamido ('OC(O)N(Rbb)(Rcc) 0r 'N(Rbb)C(O)ORaa)o ureidO ('N(Rbb)C(O)N(Rbb)(Rcc))o thioureido ('N(Rbb)C(S)N(Rbb)' (Rea): guanidinyl ('N(Rbb)c(ZNRbb)N(Rbb)(Rcc))o amidinyl ('C(=NRbb)N(Rbb)(Rcc) or 'N(Rbb)c(=NRbb)(Raa))o thiol (-SRbb), sulfinyl (-S(O)Rbb), sulfonyl (-S(O)2Rbb) and sulfonamidyl (-S(O)2N(Rbb)(Rcc) or -N(Rbb)S- (0)2Rbb). Wherein each Rad, Rbb and Rcc is, independently, H, an optionally linked chemical functional group or a fithher substituent group with a preferred list including without limitation, alkyl, alkenyl, l, aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl, alicyclic, cyclic and heteroarylalkyl. Selected substituents within the compounds described herein are present to a recursive .

As used herein, "alky ," 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, l and the like. Alkyl groups typically include from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms (Cl-Cu alkyl) with from 1 to about 6 carbon atoms being more preferred.

As used herein, "alkenyl," means a straight or branched hydrocarbon chain radical containing up to twenty four carbon atoms and having at least one carbon-carbon double bond. es of alkenyl groups include without tion, ethenyl, propenyl, butenyl, 1-methylbutenyl, 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 ning up to twenty four carbon atoms and haVing at least one carbon-carbon triple bond. Examples of l groups include, t 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 e one or more fithher substituent groups.

As used herein, "acyl," means a radical formed by removal of a hydroxyl group from an organic acid and has the general Formula -C(O)-X where X is typically aliphatic, alicyclic or aromatic. es include aliphatic carbonyls, aromatic yls, aliphatic sulfonyls, aromatic sulf1nyls, aliphatic sulf1nyls, aromatic phosphates, aliphatic phosphates and the like. Acyl groups as used herein may optionally include fiirther 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. lic as used herein may optionally include fiirther 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 ns 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. tic groups as used herein may optionally include fiirther substituent groups.

As used herein, y" 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 tion, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, lerl-butoxy, n- y, neopentoxy, n-hexoxy and the like. Alkoxy groups as used herein may optionally include fiirther substituent groups.

As used herein, "aminoalky " means an amino substituted C1-C12 alkyl radical. The alkyl n of the radical forms a covalent bond with a parent le. 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, "aralky " and "arylalky " 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 nt bond with a parent molecule. Examples include t 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 , "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, , 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 fithher 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 fiJsed ring system wherein at least one of the rings is ic and includes one or more atoms. Heteroaryl is also meant to include fused ring systems including systems where one or more of the fiJsed rings contain no heteroatoms. Heteroaryl groups typically e one ring atom selected from sulfiJr, nitrogen or oxygen. es of heteroaryl groups include without limitation, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, lyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl and the like. Heteroaryl radicals can be attached to a parent le ly 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 nd" means any atoms, group of atoms, or group of linked atoms suitable for use as a conjugate group. In certain embodiments, conjugate nds may possess or impart one or more properties, including, but not limited to pharmacodynamic, cokinetic, 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 ization under physiologically nt conditions.

B. Certain Compounds In n embodiments, the invention provides conjugated antisense nds comprising antisense oligonucleoitdes and a ate. a. n 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 cations) 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 intemucleoside linkages. In certain embodiments, modified sides 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 y with a target nucleic acid relative to an oligonucleotide comprising only nucleosides comprising naturally occurring sugar moieties. In certain embodiments, modified sugar moieties are tued sugar moieties. In n 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, ing but not limited to substituents at the 2’ and/or 5’ positions. es of sugar substituents suitable for the 2’-position, include, but are not d to: 2’-F, 3 ("OMe" or "O-methyl"), and 2'-O(CH2)ZOCH3 ("MOE"). In certain embodiments, sugar substituents at the 2’ position is selected from allyl, amino, azido, thio, O-allyl, O-Cl-Clo alkyl, O-Cl-Clo substituted alkyl, OCF3, 2SCH3, O(CH2)2-O-N(Rm)(Rn), and O-CHz-C(=O)-N(Rm)(Rn), Where each Rm and Rn is, independently, H or tuted or tituted C1-C10 alkyl. Examples of sugar substituents at the 5’- position, include, but are not d to:, 5’-methyl (R or S); 5'—Vinyl, and 5’-methoxy. In certain embodiments, substituted sugars comprise more than one idging sugar substituent, for example, 2'-F- '—methyl sugar moieties (see,e.g., PCT International Application tuted sugar moieties and nucleosides).

Nucleosides comprising 2’-substituted sugar moieties are referred to as 2’-substituted nucleosides. In certain embodiments, a 2’- substituted nucleoside comprises a 2'-substituent group selected from halo, allyl, amino, azido, SH, CN, OCN, CF3, OCF3, O, S, or N(Rm)-alkyl; O, S, or N(Rm)-alkenyl; O, S or N(Rm)- alkynyl, O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH2)2SCH3, O-(CH2)2-O- N(Rm)(Rn) or O-CHZ-C(=O)-N(Rm)(Rn), Where each Rm and RH is, independently, H, an amino protecting group or substituted or unsubstituted C1-C10 alkyl. These 2'-substituent groups can be further tuted with one or more substituent groups independently selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (N02), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.

In n embodiments, a 2’- tuted nucleoside comprises a stituent group selected from F, NH2, N3, OCFg, O-CH3, O(CH2)3NH2, CHz-CH=CH2, O-CHz-CHZCHz, OCHZCHZOCHg, O(CH2)2SCH3, O-(CH2)2-O-N(Rm)(Rn), O(CH2)ZO(CH2)2N(CH3)2, and N—substituted ide (O-CHz-C(=O)-N(Rm)(Rn) where each Rm and RH 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, OCFg, O-CH3, OCHZCHZOCHg, O(CH2)ZSCH3, O-(CH2)2-ON (CH3)2, )ZO(CH2)2N(CH3)2, and O-CHZ-C(=O)-N(H)CH3.

In certain embodiments, a 2’- substituted nucleoside comprises a sugar moiety comprising a 2’- substituent group ed from F, O-CH3, and OCHZCHZOCHg.

Certain d sugar moieties comprise a bridging sugar substituent that forms a second ring resulting in a bicyclic sugar moiety. In n such embodiments, the bicyclic sugar moiety comprises a bridge between the 4' and the 2' fiJranose ring atoms. Examples of such 4’ to 2’ sugar substituents, include, but are not limited to: -[C(Ra)(Rb)]n-, )(Rb)]n-O-, -C(RaRb)-N(R)-O- or, —C(RaRb)-O-N(R)-; -2', 4'-(CH2)2-2', 4'-(CH2)3-2',. 4'-(CH2)-O-2' (LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-O-2' (ENA); 4'-CH(CH3)-O-2' (cEt) and 4'-CH(CH20CH3)-O-2',and analogs thereof (see, eg, U.S. Patent 7,399,845, issued on July 15, 2008); 4'-C(CH3)(CH3)-O-2'and analogs thereof, (see, eg, WO2009/006478, published January 8, 2009); 4'- CHZ-N(OCH3)-2' and analogs thereof (see, eg, WO2008/ 150729, published December 11, 2008); 4'-CH2-O- N(CH3)-2' (see, eg, US2004/0171570, published September 2, 2004 ); 4'-CH2-O-N(R)-2', and 4'-CH2-N(R)- O-2'-, wherein each Ris, ndently, H, a protecting group, or C1-C12 alkyl; 4'-CH2-N(R)-O-2', wherein R is H, C1-C12 alkyl, or a protecting group (see, U.S. Patent 672, issued on September 23, 2008); - C(H)(CH3)-2' (see, eg, Chattopadhyaya, er 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)]n-, =C(Rb)-, -C(Ra)=N-, a)-, -C(=O)-, -C(=S)-, -O-, - Si(Ra)2'o -S(=O)x-, and -N(Ra)-; wherein: x is 0, 1, or 2; nis 1, 2, 3, or 4; each Ra and Rb is, ndently, H, a protecting group, yl, C1-C12 alkyl, tuted 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 cycle radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, tuted C5-C7a1icyclic radical, halogen, 0J1, NJ1J2, SJ 1, N3, C00] 1, acyl (C(=O)- H), substituted acyl, CN, sulfonyl (S(=O)2-J1), or sulfoxyl (S(=O)-J1); and each J1 and J2 is, independently, H, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, acyl - H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C1-C12 aminoalkyl, substituted C1-C12 aminoalkyl, or a protecting group.

Nucleosides comprising ic sugar moieties are referred to as bicyclic nucleosides or BNAs.

Bicyclic sides include, but are not limited to, (A) a-L-Methyleneoxy (4’-CH2-O-2’) BNA , (B) [3-DMethyleneoxy (4’-CH2-O-2’) BNA (also referred to as locked nucleic acid or LNA) , (C) Ethyleneoxy (4’- (CH2)2-O-2’) BNA , (D) Aminooxy (4’-CH2-O-N(R)-2’) BNA, (E) Oxyamino (4’-CH2-N(R)-O-2’) BNA, (F) Methyl(methyleneoxy) (4’-CH(CH3)-O-2’) BNA (also referred to as constrained ethyl or cEt), (G) methylene-thio (4’-CH2-S-2’) BNA, (H) methylene-amino 2-N(R)-2’) BNA, (1) methyl carbocyclic (4’-CH2-CH(CH3)-2’) BNA, and (J) propylene yclic (4’-(CH2)3-2’) BNA as depicted below. 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; n et al., Teirahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci.

U. S. A., 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Leii., 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); i er al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch er al., Chem. Biol, 2001, 8, 1-7; Orum er al., Curr. n Moi. 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; 2005/021570, and US2008/0039618; U.S. Patent Serial Nos. 12/129,154, 60/989,574, 61/026,995, 61/026,998, 61/056,564, WO 79627 2014/036463 61/086,231, 61/097,787, and 61/099,844; and PCT International Applications Nos. , , and .

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 Ot-L configuration or in the [3-D configuration. usly, Ot-L- methyleneoxy (4’-CH2-O-2’) bicyclic nucleosides have been incorporated into antisense oligonucleotides that showed antisense activity (Frieden er al., Nucleic Acids Research, 2003, 21, 6365-6372).

In certain embodiments, substituted sugar moieties comprise one or more idging 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 .

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 en atom. In certain such embodiments, such modified sugar moiety also comprises bridging and/or non-bridging substituents as bed above. For e, certain sugar surrogates comprise a 4’-sulfer atom and a substitution at the 2'-position (see,e.g., published U.S. Patent Application US2005/0130923, published on June 16, 2005) and/or the 5’ on. By way of additional example, yclic bicyclic nucleosides having a 4'-2' bridge have been bed (see, e.g., Freier er al., Nucleic Acids Research, 1997, , 4429-4443 and Albaek er al, J. Org. Chem., 2006, 71 , 740).

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 er al, Biochemistry, 2002, 41, 4503-4510; and U.S. Patents 5,698,685; 5,166,315; 5,185,444; and ,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 lino structure. Such sugar surrogates are refered to herein as "modifed morpholinos." For another e, in certain embodiments, a sugar surrogate comprises a six-membered tetrahydropyran. Such tetrahydropyrans may be further modified or substituted. Nucleosides comprising such modified ydropyrans 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: q1 q2 T3—O cl3 q7 q4 q6 BX / R1 R2 q5 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 ydropyran side 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 minal group; ql, C12, (13, q4, q5, q6 and q7 are each, independently, H, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 l, C2-C6 alkynyl, or substituted C2-C6 alkynyl, and each of R1 and R2 is independently selected from among: hydrogen, halogen, substituted or tituted alkoxy, NJ1J2, SJ 1, N3, J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2, and CN, wherein X is O, S or NJ1, and each J1, J2, and J3 is, independently, H or C1-C6 alkyl.

In certain embodiments, the modified THP sides of Formula VI are ed wherein q1, q2, q3, q4, q5, q6 and q7 are each H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6 and q7 is other than H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6 and q7 is methyl. In certain embodiments, THP nucleosides of Formula VI are provided wherein one of R1 and Rzis 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 nse compounds (see, eg, review article: Leumann, J.

C, Bioorganic & Medicinal try, 2002, 10, 841-854).

Combinations of modifications are also provided without limitation, such as 2'-F-5'-methyl substituted nucleosides (see PCT International Application WO 2008/ 101 157 Published on 8/21/08 for other disclosed 5', 2'-bis substituted nucleosides) and ement of the ribosyl ring oxygen atom with S and fiirther substitution at the 2'-position (see published U.S. Patent Application -0130923, published on June 16, 2005) or alternatively 5'—substitution of a bicyclic nucleic acid (see PCT International Application 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 er al., J. Am. Chem. Soc. 2007, ), 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 t disclosure comprise one or more modifed nucleobases.

In certain ments, modified nucleobases are selected from: universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fiuorinated bases as defined herein. 5-substituted dines, 6-azapyrimidines and N—2, N—6 and 0-6 substituted s, 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 (-CEC- CH3) uracil and cytosine and other alkynyl derivatives of dine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, , 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8- substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifiuoromethyl and other 5-substituted uracils and nes, 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 ated bases as defined herein. r modified nucleobases include tricyclic dines 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 azine cytidine (e.g. minoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indolone), pyridoindole cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3- d]pyrimidinone). d bases 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, 85 8-859; those sed by Englisch er al., Angewandie Chemie, International Edition, 1991, 30, 613; and those disclosed by Sanghvi, Y.S., Chapter 15, Aniisense ch and Applications, Crooke, S.T. and Lebleu, B., Eds., CRC Press, 1993, 8.

Representative United States patents that teach the preparation of n of the above noted modified nucleobases as well as other modified nucleobases include Without tion, U.S. 808; 4,845,205; 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; ,525,711; 5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; 985; 5,681,941; 5,750,692; ,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 ucleoside Linkages In certain ments, the t disclosure provides oligonucleotides comprising linked nucleosides. In such embodiments, nucleosides may be linked together using any intemucleoside e.

The two main classes of intemucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus containing intemucleoside linkages include, but are not limited to, phosphodiesters (PO), phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates (PS). Representative non-phosphorus containing ucleoside g groups include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-O-C(O)-S-), thionocarbamate (-OC (O)(NH)-S-); ne (-O-Si(H)2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Modified linkages, compared to natural phosphodiester linkages, can be used to alter, typically increase, nuclease resistance of the oligonucleotide. In certain embodiments, internucleoside linkages having a chiral atom can be prepared as a racemic mixture, or as separate enantiomers. Representative chiral linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing internucleoside linkages are well known to those skilled in the art.

The oligonucleotides bed herein contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric rations that may be , in terms of absolute stereochemistry, as (R) or (S), 0t or [3 such as for sugar anomers, or as (D) or (L) such as for amino acids etc. ed in the antisense compounds provided herein are all such possible isomers, as well as their racemic and optically pure forms.

Neutral internucleoside linkages include without limitation, phosphotriesters, methylphosphonates, MMI (3'-CH2-N(CH3)-O-5'), amide-3 2-C(=O)-N(H)-5'), amide-4 2-N(H)-C(=O)-5'), formacetal (3'-O-CH2-O-5'), and thioformacetal (3'-S-CH2-O-5'). Further neutral intemucleoside linkages e nonionic linkages sing siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester and amides (See for example: Carbohydrate Modifications in Antisense Research; Y.S. Sanghvi and PD. Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, . Further neutral intemucleoside linkages include nonionic linkages comprising mixed N, O, 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 ndent of one another. a. Certain sugar motifs In certain embodiments, oligonucleotides comprise one or more type of modified sugar es 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 " and a central or internal region or "gap." The three s of a gapmer sugar motif (the g, the gap, and the 3’-wing) form a contiguous sequence of nucleosides wherein at least some of the sugar es 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 ments, 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 n 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 sides. In certain ments, 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 ments, 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 ts of 1 or 2 linked nucleosides. In certain embodiments, the 5’- wing of a gapmer ts of 2 to 4 linked sides. 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 ments, 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 ments, the 5’- wing of a gapmer consists of 5 linked nucleosides. In n embodiments, the ’- 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 ments, the 5’- wing of a gapmer comprises at least three bicyclic nucleosides. In certain embodiments, the 5’- wing of a gapmer ses 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 n embodiments, each side of the 5’- wing of a gapmer is a bicyclic nucleoside. In certain embodiments, each side 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 ments, the 5’- wing of a gapmer comprises at least one 2’-substituted nucleoside. In certain embodiments, the 5’- wing of a gapmer ses at least one 2’-MOE nucleoside. In certain embodiments, the 5’- wing of a gapmer comprises at least one 2’-OMe nucleoside. In certain embodiments, each nucleoside of the 5’- wing of a gapmer is a non-bicyclic modified nucleoside. In certain embodiments, each nucleoside of the 5’- wing of a gapmer is a 2’-substituted nucleoside. In certain embodiments, each nucleoside of the 5’- wing of a gapmer is a 2’-MOE nucleoside. In certain embodiments, each nucleoside of the 5’- wing of a gapmer is a 2’-OMe nucleoside.

In certain embodiments, the 5’- wing of a gapmer comprises at least one 2’-deoxynucleoside. In certain embodiments, each nucleoside of the 5’- wing of a gapmer is a 2’-deoxynucleoside. In a certain embodiments, the 5’- wing of a gapmer comprises at least one cleoside. 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 d nucleoside. In certain ments, the 5’-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2’-substituted nucleoside. In certain embodiments, the 5’-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2’-MOE nucleoside. In certain embodiments, the 5’-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2’-OMe nucleoside. In n 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 ses at least one constrained ethyl nucleoside and at least one 2’-substituted nucleoside. In certain embodiments, the g of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2’-MOE nucleoside. In certain embodiments, the 5’-wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2’-OMe nucleoside. In certain ments, the 5’-wing of a gapmer ses at least one constrained ethyl side and at least one 2’-deoxynucleoside. ii. n 3’-wings In certain embodiments, the 3’- wing of a gapmer consists of 1 to 8 linked nucleosides. In certain ments, the 3’- wing of a gapmer consists of 1 to 7 linked sides. In certain embodiments, the 3’- wing of a gapmer consists of 1 to 6 linked nucleosides. In certain ments, 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 ts 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 n 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 sides. In certain embodiments, the 3’- wing of a gapmer consists of 1 nucleoside. In certain ments, the 3’- wing of a gapmer consists of 2 linked nucleosides. In certain embodiments, the 3’- wing of a gapmer consists of 3linked nucleosides. In certain embodiments, the 3’- wing of a gapmer consists of 4 linked nucleosides. In n 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 side. 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 ic 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 ses at least one 2’-MOE side. In certain embodiments, the 3’- wing of a gapmer comprises at least one 2’-OMe nucleoside. In certain embodiments, each nucleoside of the 3’- wing of a gapmer is a cyclic modified nucleoside. In certain embodiments, each side of the 3’- wing of a gapmer is a 2’-substituted side. In certain embodiments, each side of the 3’- wing of a gapmer is a 2’-MOE side. In certain embodiments, each nucleoside of the 3’- wing of a gapmer is a 2’-OMe nucleoside.

In certain embodiments, the 3’- wing of a gapmer comprises at least one 2’-deoxynucleoside. In certain embodiments, each nucleoside of the 3’- wing of a gapmer is a 2’-deoxynucleoside. In a certain embodiments, the 3’- wing of a gapmer comprises at least one ribonucleoside. In certain embodiments, each nucleoside of the 3’- Wing of a gapmer is a ribonucleoside. In n embodiments, one, more than one, or each of the nucleosides of the 5’- Wing is an RNA-like nucleoside.

In certain embodiments, the g of a gapmer comprises at least one bicyclic side and at least one non-bicyclic d nucleoside. In certain embodiments, the 3’-Wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2’-substituted nucleoside. In certain embodiments, the 3’-wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2’-MOE nucleoside. In certain embodiments, the 3’-Wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2’-OMe nucleoside. In certain embodiments, the 3’-Wing of a gapmer comprises at least one bicyclic nucleoside and at least one 2’-deoxynucleoside.

In certain ments, the g of a gapmer comprises at least one constrained ethyl nucleoside and at least one non-bicyclic modified side. In certain embodiments, the g of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2’-substituted nucleoside. In n embodiments, the 3’-Wing of a gapmer comprises at least one constrained ethyl side and at least one 2’-MOE nucleoside. In n embodiments, the 3’-Wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2’-OMe nucleoside. In certain embodiments, the 3’-Wing of a gapmer comprises at least one constrained ethyl nucleoside and at least one 2’-deoxynucleoside.

In certain embodiments, the 3’-Wing of a gapmer comprises at least one LNA nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 3’-Wing of a gapmer comprises at least one LNA nucleoside and at least one 2’-substituted nucleoside. In n embodiments, the g of a gapmer comprises at least one LNA nucleoside and at least one 2’-MOE nucleoside. In certain ments, the 3’-wing of a gapmer comprises at least one LNA nucleoside and at least one 2’-OMe nucleoside. In certain embodiments, the g of a gapmer comprises at least one LNA nucleoside and at least one 2’- deoxynucleoside.

In certain embodiments, the g 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 ses 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 side, 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 n embodiments, the 3’-Wing of a gapmer comprises at least one bicyclic side, at least one 2’-OMe nucleoside, and at least one 2’-deoxynucleoside. In certain embodiments, the 3’-wing of a gapmer comprises at least one constrained ethyl nucleoside, at least one 2’-OMe nucleoside, and at least one 2’-deoxynucleoside. In certain embodiments, the 3’-wing of a gapmer comprises at least one LNA nucleoside, at least one 2’-OMe nucleoside, and at least one 2’-deoxynucleoside. iii. Certain Central Regions (gaps) In certain embodiments, the gap of a gapmer consists of 6 to 20 linked nucleosides. In n embodiments, the gap of a gapmer consists of 6 to 15 linked sides. In certain embodiments, the gap of a gapmer consists of 6 to 12 linked sides. In certain embodiments, the gap of a gapmer consists of 6 to 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 ts 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 sides. 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 n 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 ses one or more modified nucleosides. In n 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 ting 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 sides).

In n embodiments, gaps comprise a stretch of unmodified 2’-deoxynucleoside interrupted by one or more d 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 s. In certain embodiments, short stretches are achieved by upting a longer gap .

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 n 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 ses 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 ses two or more modified linkages. In certain embodiments, the gap comprises one or more modified es and one or more modified nucleosides. In certain embodiments, the gap comprises one modified linkage and one modified nucleoside. In certain ments, the gap comprises two modified linkages and two or more modified nucleosides. b. Certain Internucleoside Linkage Motifs In certain embodiments, oligonucleotides comprise modified internucleoside es arranged along the ucleotide 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 es. In certain such embodiments, the oligonucleotide comprises a region that is uniformly linked by phosphorothioate internucleoside linkages. In certain ments, 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 ucleoside linkage is phosphorothioate.

In certain embodiments, the oligonucleotide comprises at least 6 phosphorothioate internucleoside linkages. In certain embodiments, the ucleotide comprises at least 7 orothioate internucleoside linkages. In n embodiments, the oligonucleotide comprises at least 8 phosphorothioate internucleoside linkages. In certain ments, the oligonucleotide comprises at least 9 orothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 10 phosphorothioate internucleoside linkages. In certain ments, the oligonucleotide comprises at least 11 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 12 phosphorothioate cleoside linkages. In certain embodiments, the oligonucleotide comprises at least 13 phosphorothioate intemucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 14 phosphorothioate intemucleoside In n embodiments, the oligonucleotide comprises at least one block of at least 6 consecutive phosphorothioate intemucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 7 utive orothioate intemucleoside linkages. In certain ments, the oligonucleotide comprises at least one block of at least 8 consecutive phosphorothioate intemucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 9 consecutive phosphorothioate intemucleoside linkages. In certain embodiments, the ucleotide comprises at least one block of at least 10 utive phosphorothioate cleoside linkages. In certain embodiments, the oligonucleotide comprises at least block of at least one 12 consecutive phosphorothioate intemucleoside 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 intemucleoside linkages. In n embodiments, the oligonucleotide comprises less than 14 phosphoro- thioate intemucleoside linkages. In certain embodiments, the ucleotide comprises less than 13 phosphorothioate cleoside linkages. In certain ments, the oligonucleotide comprises less than 12 phosphorothioate intemucleoside linkages. In certain embodiments, the oligonucleotide ses less than 11 phosphorothioate intemucleoside linkages. In certain embodiments, the oligonucleotide ses less than 10 phosphorothioate intemucleoside linkages. In n embodiments, the oligonucleotide comprises less than 9 phosphorothioate intemucleoside linkages. In certain embodiments, the oligonucleotide comprises less than 8 phosphorothioate intemucleoside linkages. In certain embodiments, the oligonucleotide comprises less than 7 phosphorothioate cleoside linkages. In certain embodiments, the oligonucleotide comprises less than 6 phosphorothioate intemucleoside linkages. In certain embodiments, the oligonucleotide comprises less than 5 phosphorothioate intemucleoside linkages. c. Certain Nucleobase ation 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 ments, each nucleobase is modified. In certain embodiments, none of the nucleobases is chemically d.

In certain embodiments, ucleotides 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 n 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 es in an oligonucleotide are 5- methyl cytosine moieties. Herein, 5-methyl cytosine is not a "modified nucleobase.)9 Accordingly, unless otherwise indicated, unmodified nucleobases e both cytosine residues having a 5-methyl and those lacking a 5 methyl. In certain ments, the methylation state of all or some cytosine nucleobases is specified.

In certain embodiments, chemical ations to nucleobases comprise ment of certain conjugate groups to nucleobases. In certain embodiments, each purine or each dine in an oligonucleotide may be optionally modified to comprise a conjugate group. d. Certain Overall Lengths In certain embodiments, the present sure es oligonucleotides of any of a variety of ranges of lengths. In certain embodiments, oligonucleotides consist ofX 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 XSY. For example, in certain embodiments, the oligonucleotide may consist of8 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, 9to 11, 9to 12, 9to 13,9to 14, 9t015, 9to 16, 9to 17, 9to 18, 9to 19,9to20, 9to21,9to22, 9to23, 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, 10to 28,10to 29,10to 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 , 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, 12to 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 , 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 , 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 , 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, 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 , 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 nd 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.

. Certain Antisense Oligonucleotide Chemistry Motifs In certain embodiments, the chemical structural features of antisense ucleotides are characterized by their sugar motif, internucleoside linkage motif, nucleobase modification motif and overall length. In certain embodiments, such ters are each ndent of one another. Thus, each internucleoside e 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 base 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 r. i. Certain ces and Targets In certain ments, 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 nse activity. In n 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 logical conditions for in vivo or therapeutic uses, and under conditions in which assays are performed in the case of in vitro ). 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 ed to the other.

In certain ments, the t disclosure provides antisense compounds comprising oligonucleotides that are fiJlly complementary to the target nucleic acid over the entire length of the oligonucleotide. In certain embodiments, ucleotides are 99% mentary to the target nucleic acid.

In n 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 ments, 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 mentary 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 c acid. In certain embodiments, the hybridizing region includes one mismatch ve 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 n 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 c acid, wherein the hybridization results in a ical effect. In certain embodiments, such hybridization results in either target nucleic acid degradation or occupancy with concomitant tion or ation 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 s 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 ses a ble moiety. In n ments, a conjugate group comprises one or more cleavable bond. In certain embodiments, a ate 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 .

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 ble bond. In certain ments, a cleavable moiety ses 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 . In certain embodiments, the ble 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 dine. 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-benzoylmethylcytosine, adenine, nzoyladenine, 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 ucleotide by a phosphodiester linkage and is ed 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 ments, 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 ucleotide. In certain embodiments, the cleavable moiety is attached to a 2' position of the antisense oligonucleotide. In certain embodiments, the cleavable moiety is ed to the nse 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 n embodiments, the cleavable moiety is attached to the linker by a odiester 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 y 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 e between the cleavable moiety and the . In certain embodiments, the cleavable moiety has a structure selected from among the following: o=fi—0H l é O=|I3-OH O=|I3-OH o o 0 Bx1 O Bx2 l o 0‘ O 3 0": — 0=F:)—OH | O=|I3-OH o o O Bx 0 3x2 0 Bx3 ; ;and 9" t? C? 0: -OH O=P-OH O=P-OH wherein each of Bx, Bxl, sz, and Bx3 is independently a heterocyclic base moiety. In certain embodiments, the cleavable moiety has a structure ed from among the following: iii. n s 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- ing moiety. In certain embodiments, the linker fithher comprises a nt 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 fithher comprises a covalent attachment to a protein binding moiety. In certain embodiments, the linker includes multiple ons for ment 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 ments, the linker further ses 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, her (-S—) and hydroxylamino (-O-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 ses 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 ments, 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 ments, the linear group is ntly 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 ble moiety, a solid support and a protein binding moiety. In certain embodiments, the linear group es one or more cleavable bond.

In certain embodiments, the linker includes the linear group covalently attached to a ld group.

In certain embodiments, the scaffold includes a branched tic group comprising groups ed 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 clic ring system.

In certain embodiments, the scaffold includes at least two mono or polycyclic ring systems. In certain ments, 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 ld group and the scaffold group is covalently attached to the ble moiety, the linker and a solid support. In n embodiments, the linear group is covalently attached to the ld 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 es a protein binding moiety. In certain ments, the protein binding moiety is a lipid such as for e including but not limited to 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, ic acid, 03 -(oleoyl)lithocholic acid, 03 -(oleoyl)cholenic acid, oxytrityl, or phenoxazine), a Vitamin (e.g., folate, Vitamin A, Vitamin E, biotin, pyridoxal), a peptide, a carbohydrate (e.g., monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, ccharide), an endosomolytic component, a steroid (e.g., uvaol, hecigenin, diosgenin), a terpene (e.g., triterpene, e.g., apogenin, 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 «W E—NH E/Nwrm/N ('3’, 9 o R0271 &/ N N 0\ O—F|’-OH :kNMO "L" ; W1 "‘9; n L077. (on H n O 0—; vav ( )n m I o é‘s\o I I fi 0, mo—fi—OH , OH . , N ’ H"LL/"\Hfigo \III-Q’O I - \ ’1 0/, N 0"? OH .

O UVN 0‘9 I \ O 0 III- 0 5m NQ’_0\ I O [P OH \ I, O I N 0"? OH O w n H... o 0 Q §_S \....- n O N 'and 1 ;\ H E/Nfl/KOH "W n O wherein each n is, independently, from 1 to 20; and p is from 1 t0 6.

In certain embodiments, a linker has a ure selected from among: H Q EINN NMH O ’ n n fiH "W 0 RCA | H vav 11% WNWNMON ’ 14$, 0 "L, n \O O wherein each n is, independently, from 1 to 20.

WO 79627 In certain embodiments, a linker has a structure selected from among: 0 O O O $673M" 1" , ELANHWM’E EXPIEM? O O n n ’ O O o HNIO :1 H A O (9‘ H X N\ H WW0 0"Mn '3. gmHWOQD/M:\fi . o o 99W"? :and ’ fmnonw/"r n n n n o o o wherein n is from 1 to 20.

In certain embodiments, a 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.

In n embodiments, a linker has a structure selected from among: WO 79627 §—N‘H in’p OH \lll" d WO 79627 OEP’p 0—; O ('3 \|Il|' < j In certain embodiments, a linker has a structure selected from among: 0 O O O EkNMM/EKE;H iJk/NWM/E ;E)J\/N70(\/\)KégH ; O O O O I )k/H o HN o O N;_ Mg, E , EW‘L‘ _ 9»: : ’ WO 79627 In certain embodiments, a linker has a structure selected from among: 0 O O O H H EkNMM/jq/E; EkNWH/E :EJk/NW;H ; O O O O )K/H o HN o O _ , _ In certain ments, a linker has a structure selected from among: Hi" \ Q\’O}i A 0 mo EWO and EW0 wherein n is from 1 to 20.

In certain embodiments, a linker has a ure selected from among: f\O/\/\e;; ,ROMOM; ;and §\O/\/\O/\/\O/\/\é§ .

In certain ments, a linker has a structure selected from among: OH OH g—o—fi—ovokoWo—g—o—gI and OH 3 3 OH E—o—g—ovokofloy-OH 3 In certain embodiments, a linker has a structure selected from among: 0 o swmwg 3H0_||_ _s e; ‘771 and WNW In certain embodiments, the conjugate linker has the structure: In certain embodiments, the conjugate linker has the structure: 0 o EWfiAWO—E In certain embodiments, a linker has a structure selected from among: O 9' f O-P-O—E 5 ‘1 NAM . 2 NM H 5 OH H and o In certain embodiments, a linker has a structure selected from among: 0 o g‘WWW"o—'F'>—o—E :5 ‘91 and WNW. wherein each n is independently, 0, 1, 2, 3, 4, 5, 6, or 7. iv. Certain Cell-Targeting Moieties In certain embodiments, ate groups comprise cell-targeting moieties. Certain such cell-targeting moieties increase cellular uptake of antisense compounds. In certain ments, 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 ble bond. 1. Certain Branching Groups In certain embodiments, the conjugate groups comprise a ing moiety sing a branching group and at least two tethered s. In certain embodiments, the branching group es the conjugate linker. In certain embodiments, the branching group attaches the cleavable moiety. In certain embodiments, the branching group es 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 ments, the branching group comprises groups selected from alkyl, amide and ether . In certain embodiments, the branching group ses 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 n embodiments, a branching group has a structure selected from among: WO 79627 WO 79627 ELL/NH 0 wherein each n is, independently, from 1 to 20; j is from 1 t0 3; and m is fr0m2 t0 6.

In certain embodiments, a ing group has a structure selected from among: wherein each n is, independently, from 1 to 20; and m is from2 to 6.

In certain embodiments, a branching group has a structure selected from among: ’27-?(3 Jé\N '73. E/NH '13 HN/LLL‘ O s‘ £1;ka e HN\; "at/NH ill/NH In certain embodiments, a branching group has a structure selected from among: \ | A‘ 7"" /A1 A1 n AF; ‘15. g—A1 n In n embodiments, a branching group has a structure selected from among: "W" | | in" A1 A1 AA_/Ar§ ; §—A)(’lA)—n(-J"A1—g "()n "APE 1 g—A1 A1 A1 and gr 94‘ 9" wherein each A1 is independently, O, S, C=O or NH; and each n is, independently, from 1 to 20.

In certain embodiments, a branching group has a structure selected from among: "fink" and "‘1..." n ,5" 5:" wherein A1 is O, S, C=O or NH; and each n is, ndently, from 1 to 20.

In certain embodiments, a branching group has a structure selected from among: / ow"m M In certain embodiments, a ing group has a structure selected from among: /O\g—/"m In certain embodiments, a branching group has a structure selected from among: 2. Certain Tethers In certain embodiments, conjugate groups se one or more tethers ntly attached to the branching group. In certain embodiments, conjugate groups comprise one or more tethers covalently ed to the linking group. In n embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl, ether, thioether, de, amide and hylene 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 ments, 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 n embodiments, the tether is attached to the ing group through a phosphorus g 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 ed to the ligand through an ether group. In certain embodiments, the tether is attached to the ligand h 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 WO 79627 2014/036463 about 10 to about 18 atoms in chain length between the ligand and the ing 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: 0 H 1%. awwvtowy ; a/Nenw‘: Emir Encore; ; 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: ii/QLN/Vowo/Vi‘m H ‘44\/\/\n/h91 ; *3/ng ; ; "HMO/Er" ; fimO/Vov‘g ; 1(ng ; and EWOA; _ In certain embodiments, a tether has a structure selected from among: :5 H R ‘2 O 0 wherein each n is, independently, from 1 to 20.

In n embodiments, a tether has a structure selected from among: 0 21 f L "1 {Wk &L "a wherein L is either a phosphorus linking group or a neutral linking group; 21 is C(=O)O-R2; 22 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 r than 0 for each tether.

In certain embodiments, a tether has a structure selected from among: In certain embodiments, a tether has a structure selected from among: o—E—OM: waK(O‘flI‘O'('l/m?0 COOH OH 1% m‘ (5H wherein 22 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: 0 0 "MR "mi , ; wherein each n is independently, 0, l, 2, 3, 4, 5, 6, or 7.

In n ments, a tether comprises a orus 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 n 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 ed that have an y 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 R). In certain embodiments, each ligand is a carbohydrate. In certain embodiments, each ligand is, independently selected from galactose, N—acetyl oseamine, mannose, e, glucosamone and fiJcose. In certain embodiments, each ligand is N—acetyl galactoseamine (GalNAc). In certain embodiments, the targeting moiety comprises 2 to 6 s. In n embodiments, the targeting moiety ses 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, (1-D- galactosamine, N—Acetylgalactosamine, 2-acetamidodeoxy-D-galactopyranose (GalNAc), 2-Amino0- [(R)carboxyethyl]deoxy-B-D-glucopyranose (B-muramic acid), 2-Deoxymethylamino-L- yranose, 4,6-Dideoxyformamido-2,3-dimethyl-D-mannopyranose, 2-Deoxy-2—sulfoamino-D- glucopyranose and o-D-glucosamine, and N—Glycoloyl-a-neuraminic acid. For example, thio sugars may be selected from the group consisting of 5-Thio-B-D-glucopyranose, Methyl triacetylthio yl-a-D-glucopyranoside, 4-Thio-B-D-galactopyranose, and ethyl 3,4,6,7-tetraacetyldeoxy—1,5- dithio-a-D-gluco-heptopyranoside.

In certain embodiments, "GalNac" or "Gal-NAc" refers to 2-(Acetylamino)deoxy-D- galactopyranose, commonly referred to in the literature as N—acetyl galactosamine. In certain embodiments, "N-acetyl galactosamine" refers to 2-(Acetylamino)deoxy-D-galactopyranose. In certain embodiments, "GalNac" or "Gal-NAc" refers to 2-(Acetylamino)deoxy-D-galactopyranose. In n embodiments, "GalNac" or "Gal-NAc" refers to 2-(Acetylamino)deoxy-D-galactopyranose, which includes both the B- form: 2-(Acetylamino)deoxy-B-D-galactopyranose and a-form: 2-(Acetylamino)deoxy-D- galactopyranose. In certain ments, both the B-form: 2-(Acetylamino)deoxy-B-D-galactopyranose and a-form: 2-(Acetylamino)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)deoxy-D-galactopyranose is shown, this structure is intended to include the other form as well. In certain embodiments, In certain preferred embodiments, the B-form tylamino)deoxy-D-galactopyranose is the preferred embodiment.

WO 79627 0 OH HO 0 '0’], k HO 0" 2-(Acety1amino)deoxy-D-galactopyranose HO 0—; 2-(Acetylamino)deoxy-B-D-galactopyranose 2-(Acetylamino)deoxy-a-D-galactopyranose In certain embodiments one or more ligand has a structure selected from among: "0&0 OH HO o HO 0—; Ho OH R and R1 1 R1 0 o wherein each R1 is selected from OH and NHCOOH.

In certain ments one or more ligand has a structure selected from among: OH OH . HO HO \ NHAc‘"I\ HO 0 , "g \ HO , 0H HO ’ #4 ’ "0% VHo N HO OH HOOH ’ Wom" OH - oHO HO \ /1 , HO HO .5 ,and OH OH \%?::7m0 HO 0H0 OH 0 F In certain embodiments one or more ligand has a structure selected from among: In certain embodiments one or more ligand has a structure selected from among: Home», N HAC _ i. Certain Conjugates In certain embodiments, conjugate groups comprise the structural features above. In certain such ments, conjugate groups have the following structure: HO OH HO WHNWNn NHAC o HO OH H H O N N 0 Ho \(v)/\/ NHAC 0 HO HN H o o N HO W n NHAc 0 wherein each n is, independently, from 1 to 20.

In certain such embodiments, conjugate groups have the ing structure: Ho OH O H O o HN\/\/N NHAc o HO OH o O H H H 0 NWN 0 N_| NHAc o Ho H" H o o N HO V\/\n/ In certain such embodiments, conjugate groups have the following structure: NHAc 0 Bx HO OH O N 5 O H H ‘ O N N O (I)— HO \9/ n O—P—X n n " NHAc (IDH o O HO 0" NWN 0 NHAc OW n each n is, independently, from 1 to 20; Z is H or a linked solid support; Q is an antisense compound; X is O or S; and Bx is a heterocyclic base moiety.

In certain such embodiments, conjugate groups have the following structure: HO OH _ O H H 0 o=||=—0H o OH HO \/ o NHAc = HO OH 0 0 KO’BX O N H H 03 O\/\n/ \/N O N I HO H o—F|>=X NHAc 0 OH HO 0H 0 H "N N\/ o NHAc V\n/ In certain such embodiments, conjugate groups have the following structure: %:EQL/ H O M OH I O HO 0H 0 o O H H 0" O N N O 7 Ho WM"W N o F"—o— 3 3 | In certain such embodiments, ate groups have the following structure: AcHN 0H 0 HO 0" ,ii I O O O l O In certain such embodiments, conjugate groups have the following structure: " H O N n 6%er "O 0'1?"O/\(_7’ w AcHN OH OH O Q HO%/ WO O (l3 HO-ll)=0 ' \O’0) (H n O In certain such embodiments, conjugate groups have the ing structure: WO 79627 HO O\v/\v/\v/A\ S? AcHN CYEEO\LW HC}E§::iQL/O\V/"\//\~//\}yfik0/"\/"\O/€E%\/O"EEO/‘\:_}KOo 9 9 () ETZ/< AcHN OH O o In certain such embodiments, conjugate groups have the following structure: AcHN 5H ) HO O" n o 0 o o 0 II HO lvt:\w)l\0’i‘%fo/P '_ o—F—o AcHN 0H HO (9| 0 0 O/l?\0fl)n HO H OH In certain such embodiments, conjugate groups have the following structure: HO—IT=O (<3)3 HOOH o HO /\ ('13? O/|\O §fiOH OH \H (3)3 HOOH O 0 9 oW\/\ o E— =o HO / \ O 1') OMO/a\/ (le AcHN 0H 0 0 o o’Ii\0 In certain embodiments, conjugates do not comprise a pyrrolidine.

In certain such embodiments, conjugate groups have the following structure: 0 H o H o (I) HO \/\/\n/N\/\/ i O=F|)'O ACHN o HOOH o O O o H H HO \/\/\n/ \/\/N\n/\/O "MN AcHN o o o 'bH HOOH HN’i: Ho$xvoO /N In certain such embodiments, conjugate groups have the following structure: O/p\OMO/a\/O—P—O0 0 O 0 II || 0 BX ACH N O O 0:15—0- HO OH le O o\/~V/\/"Rybo In certain such embodiments, conjugate groups have the following structure: HO 0H HO O H AcHN \/A\/:¥’N H HO OH NH N-(CH2)6—o—P—§ \fl/\’0 H H o o o NHAc /\/\ In certain such embodiments, conjugate groups have the ing structure: HoOH o HO 4 "JW\ HOOH fiL/r— o o Hemmer" O "wwfio AcHN O o /£fj O N HO O In certain such embodiments, conjugate groups have the following structure: HOOH o HO 4 NJle HCDOFI O jiV/’__ JOL\/"\/fi)L O Ho 4 N N "MO—Hll AcHN o 0 t HO fl 0 In certain such embodiments, conjugate groups have the following ure: Hoj§:a;L/O/\tz~\HO N O HOOH O o o O "WMWO A4" HQWOWHNo O In certain such embodiments, conjugate groups have the following structure: HOOH H O o N 0 Ho A"? HOOH O o o O o N O HO ’ATZNH "J\’A\/mfi/lfif\o—P—§ O 0 H0 "677" In certain such embodiments, conjugate groups have the following structure: WO 79627 HO O 0\/\/\)OLNH HO 0 H O O OWN N NWN‘9; a AcHN H o H o o o\/A\/\J%~NH In certain such embodiments, conjugate groups have the following structure: H O O O O\/\/\>"NH In certain such embodiments, conjugate groups have the following structure: HoOH ‘ 0 CW" HO 3 o o ACHN o-$ OH HO o"$g7§N o ACHN o—$ OH HO 3 0 -CM In certain such ments, conjugate groups have the following structure: HQOH ‘ 0 CW" HO 3 O O ACHN o—$ OH 0 o"$Y\WN ACHN 0—5 OH Howo N 3 o O 2045—; ACHN ('3 In certain ments, the cell-targeting moiety of the conjugate group has the following structure: HO%Q/0\XO AcHN \\ HOOH O Hog/Oo ___ ACHN 0%M’2 HOOH X/ 0 o/// wherein X is a substituted or unsubstituted tether of six to eleven consecutively bonded atoms.

In certain ments, the cell-targeting moiety of the conjugate group has the following structure: HO%Q/0\Xo AcHN \\ HOOH O o__,X___o HO "A HOOH X/ 0 o/// wherein X is a substituted or unsubstituted tether of ten consecutively bonded atoms.

WO 79627 In certain embodiments, the cell-targeting moiety of the conjugate group has the following structure: 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 ments, the cell-targeting moiety of the conjugate group has the following structure: HO%O\Yo AcHN \NiZ,0 HoOH 0H Hog/OO /3 __ /O Y\N Z M AcHN N z\ HOOH O y’ \W O 0/ 0 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 ate, 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 ing structure: HO O\Y i AcHN \N Z/o HoOH 0H Hog/Oo ,o / __ 3 Y\H Z " AcHN H y’NWz\ HOOH O O 0/ 0 wherein Y and Z are independently selected from a C1-C12 substituted or unsubstituted alkyl group, or a group sing 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 ure: HO O\Y i AcHN \N Z,0 HoOH OH Hog/Oo /is __ /o Y\" Z " AcHN H HOOH y’NW O O 0/ 0 wherein Y and Z are independently selected from a C1-C12 substituted or unsubstituted alkyl group.

In n such embodiments, the cell-targeting moiety of the conjugate group has the following structure: wherein m and n are independently ed 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: HOOH )Lflo 2% 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: HQOH HO \X O X HO NE AcHN H HO O 0//X 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 HOACHN%O\o HOH0\E;;3/o/" /a OHOH X/XEN wherein X is a tuted 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 ate group has the ing structure: 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 ments, the cell-targeting moiety of the conjugate group has the following structure: HQOH HO \X O X HO NEAL AcHN H HOMO/X 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-C11 alkyl group.

In certain embodiments, the cell-targeting moiety of the conjugate group has the following structure: HoOH H O o——Y——N O HoOH 0 0 /AK\ o N is HO H N O o—Y/H O wherein Y is ed from a C1-C12 substituted or unsubstituted alkyl, l, 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 her.

In n such embodiments, the cell-targeting moiety of the conjugate group has the following structure: HOOH H O 0—y——N O Ho§$:f%/OW/AK\ A O O—Y/H 0 wherein Y is selected from a C1-C12 substituted or tituted 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: HoOH H O o——Y~—N O HoOHO W/AK\ 3 OAcHN H C) o——Y"H O wherein Y is selected from a C1-C12 substituted or unsubstituted alkyl group.

In certain such embodiments, the argeting moiety of the conjugate group has the following structure: O o N 0 Ho fl, HoOH O [40%o ofakN A r'H M O CHEN 0 HO H Whereinn is 1, 2, 3, 4, 5, 6, 7, 8, 9,10,11, or 12.

In certain such embodiments, the cell-targeting moiety of the ate group has the following structure: HOomoflh‘o HOOH 0 HOmOWN IZ OACHN wherein n is 4, 5, 6, 7, or 8. b. Certain c0n°u ated antisense com ounds 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: ——C———D——6E-—-a A is the antisense oligonucleotide; B is the cleavable moiety C is the ate 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 nd has the following structure: A—c—D—QE—F) 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 ated antisense compound has the following structure: wherein A is the antisense oligonucleotide; B is the cleavable moiety C is the ate linker each E is a tether; each F is a ligand; and q is an integer between 1 and 5.

In certain ments, the conjugates are bound to a nucleoside of the nse oligonucleotide at the 2’, 3’, of 5’ position of the side. In certain embodiments, a conjugated antisense compound has the following structure: A—c+E—F> 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—o+E—F> wherein A is the antisense oligonucleotide; B is the cleavable moiety D is the ing 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) 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 n ments 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 HO OH 0 o P OH— NH2 H _ WHNWN N (I l \N 0 N NA OH T‘ HO 0 .

N‘ P=O H | Cleavable moiety Ligand Tether HO fl:IHN‘ H O Branching group In certain embodiments, a conjugated antisense compound has a structure selected from among the following: Cell targeting moiety AcHN OH 0 (.3 T the er Ligand O_P=O o l HO OH it ASO NHAC Branching group In n embodiments, a conjugated antisense compound has a structure ed from among the following: WO 79627 Cleavable moiety HO—PZO Cell targeting moiety I Hog/OO\/\/\/\ o O’P\O OH ACHN 00L;— O HO OH O O O Cpnjugate 1.). O-II’_O linker 0/6?0 0 I ACHN O OH Tether '—' Ligand, O O NHAC Branching group 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 nse 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 177, US 7,491,805, US 8,106,022, US 7,723,509, US 148740, US 2011/0123520, WO 2013/033230 and Representative publications that teach the ation of certain of the above noted conjugates, conjugated nse 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 WO 79627 Cholesterol ng 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 & nal Chemistry (2011) 19:2494-2500, RENSEN et al., mination 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):37577-375 84, RENSEN et al., "Design and sis of Novel N—Acetylgalactosamine-Terminated Glycolipids for Targeting of Lipoproteins to the Hepatic glycoprotein 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 -based r 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 se 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., JBiol Chem, 1982, 257, 939-945; Pavia et al., Int J Pep n 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., Bioconjag Chem, 1997, 8, 762-765; Kato et al., Glycobiol, 2001, 11, 821-829; Rensen et al., JBioZ 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 m, 2012, 425, 43-46; Pujol et al., Angew Chemie Int Ed Engl, 2012, 51, 448; Biessen et al., JMed Chem, 1995, 38, 1846-1852; Sliedregt et al., JMed Chem, 1999, 42, 609-618; Rensen et al., JMed Chem, 2004, 47, 5798- 5808; Rensen et al., Arterioscler Thromb Vasc Biol, 2006, 26, 169-175; van Rossenberg et al., Gene Ther, 2004, 11, 4; Sato et al., JAm Chem Soc, 2004, 126, 14013-14022; Lee et al., J Org Chem, 2012, 77, 7564-7571; n et al., FASEB J, 2000, 14, 1784-1792; Rajur et al., Bioconjug Chem, 1997, 8, 935-940; Duff et al., Methods l, 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 WOl998/013381; WO2011/038356; WOl997/046098; /098788; WO2004/101619; WO2012/037254; WO2011/120053; WO2011/100131; WO2011/163121; WO2012/177947; WO2013/033230; WO2013/075035; WO2012/083185; WO2012/083046; /082607; WO2009/134487; WO2010/144740; WO2010/148013; WOl997/020563; WO2010/088537; WO2002/043771; /129709; WO2012/068187; WO2009/126933; WO2004/024757; WO2010/054406; /089352; WO2012/089602; WO2013/166121; WO2013/165816; US. Patents 4,751,219; 8,552,163; 6,908,903; 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; 601; 177; 6,906,182; 6,620,916; 8,435,491; 8,404,862; 7,851,615; hed U.S. Patent Application Publications US2011/0097264; US2011/0097265; US2013/0004427; US2005/0164235; /0148740; US2008/0281044; US2010/0240730; US2003/0119724; US2006/0183886; US2008/0206869; US2011/0269814; US2009/0286973; US2011/0207799; US2012/0136042; /0165393; US2008/0281041; US2009/0203135; US2012/0035115; US2012/0095075; US2012/0101148; US2012/0128760; US2012/0157509; US2012/0230938; US2013/0109817; US2013/0121954; US2013/0178512; US2013/0236968; US2011/0123520; US2003/0077829; /0108801; and US2009/0203132; 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 late in the kidney. In certain ments, 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 ated nse 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 n embodiments conjugated antisense compounds may exhibit greater target reduction as compared to its unconjugated counterpart wherein both the ated antisense compound and its ugated counterpart are present in the tissue at the same concentrations. For example, in certain embodiments ated antisense compounds may exhibit greater target reduction as ed 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. icz, 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 bed herein may fiirther 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 r improve potency by facilitating endocytosis of the conjugated antisense nd.

In certain embodiments, the ble 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, ated 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. ucleotides with fewer phosphorothioate linkages accumulate less in the liver and more in the kidney (see, for example, Geary, R., "Pharmacokinetic ties of 2’-O-(2- yethyl)-Modif1ed Oligonucleotide Analogs in Rats," Journal ofPharmacology and Experimental eutics, Vol. 296, No. 3, 890-897; & Pharmacological Properties of2 ’-0—Metlzoxyetlzyl Modified Oligonucleotides in Antisense a Drug Technology, Chapter 10, Crooke, S.T., ed., 2008) In certain embodiments, ucleotides with fewer phosphorothioate intemculeoside es and more phosphodiester intemucleoside linkages late 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 d action (liver); (2) drug is ng into the urine; and (3) the kidney is exposed to relatively high concentration of drug which can result in ties 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 ammatory reactions. (see for example: J Lab Clin Med. 1996 Sep;128(3):329-38. fication of dy production by phosphorothioate oligodeoxynucleotides". Branda et al.; and see also for example: Toxicologz'c 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 s one or more proinflammatory reactions.

In certain embodiments, the degree of proinflammatory effect may depend on several variables (e. g. backbone modification, rget effects, nucleobase modifications, and/or nucleoside modifications) see for example: logz'c ties in nse 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 ing one or more variables. For e 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 ucleoside linkages.

In certain embodiments, it would be desirable to reduce the number of phosphorothioate linkages, if doing so could be done without losing ity and without shifting the distribution from liver to kidney. For e, 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 ed to an nse 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 n 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 ed as much in the urine compared to its unonjugated counterparts, even when some of the phosporothioate linkages are ed with less proinflammatory phosphodiester internucleoside linkages. In n embodiments, the use of a conjugate allows one to design more potent and better tolerated antisense drugs. Indeed, in n emobidments, conjugated antisense compounds have larger therapeutic indexes than unconjugated counterparts. This allows the conjugated antisense compound to be stered 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 ion of some orothioate linkages remains desirable. For example, the terminal linkages are vulnerable to exonucleoases and so in certain embodiments, those linkages are phosphorothioate or other modified e. Intemucleoside linkages linking two deoxynucleosides are vulnerable to endonucleases and so in certain embodiments those those es are phosphorothioate or other modified linkage. Intemucleoside linkages between a modified nucleoside and a deoxynucleoside where the deoxynucleoside is on the 5’ side of the linkage deoxynucleosides are able to endonucleases and so in certain embodiments those those linkages are phosphorothioate or other modified linkage.

Internucleoside es between two modified nucleosides of n types and between a deoxynucleoside and a modified nucleoside of n typ where the modified side is at the 5’ side of the linkage are sufficiently resistant to nuclease ion, that the linkage can be phosphodiester.

In certain embodiments, the antisense ucleotide 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 ments, the antisense oligonucleotide of a conjugated antisense compound comprises fewer than 14 phosphorthioate linkages. In certain embodiments, the antisense oligonucleotide of a conjugated antisense nd comprises fewer than 13 phosphorthioate linkages. In n 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 ments, 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 es. In certain embodiments, the antisense oligonucleotide of a ated antisense compound comprises fewer than 8 phosphorthioate linkages.

In n 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 nd 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. lly, ucleietides 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 minal nucleoside and attach that conjugated side 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 , is an inefficient process. In certain ments, 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’ al nucleoside to the solid t 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 e a 3 ’-conjugated oligonucleotide. The Examples herein demonstrate attachment at the 5’-end. In addition, certain conjugate groups have synthetic advantages. For e, certain conjugate groups comprising orus linkage groups are synthetically simpler and more efficiently prepared than other conjugate , including conjugate groups ed 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 g such one or more conjugate group. Without being bound by mechanism, it is believed that the ate 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 e 20 a conjugated oligonucleotide was administered to mice and a number of different chemical s, each comprising a different portion of the ate group remaining on the oligonucleotide, were detected (Table 10a). Thisconjugated antisense compound demonstrated good potency (Table 10). 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 n 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 ments where regulatory review is required (e.g., USFDA or counterpart) it is desirable to have a single (or inantly single) active species. In n 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 lism 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 c acid. In certain embodiments, a target nucleic acid is an RNA. In certain embodiments, a target nucleic acid is a non-coding RNA. In n embodiments, a target nucleic acid encodes a protein. In certain embodiments, a target nucleic acid is ed from a mRNA, a pre-mRNA, a microRNA, a non-coding RNA, ing 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 n having a nucleobase sequence at least 70% complementary to the nucleobase sequence of a target nucleic acid. In n embodiments, antisense compounds comprise a portion having a base 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 n having a nucleobase sequence at least 95% complementary to the nucleobase sequence of a target nucleic acid. In certain embodiments, nse compounds comprise a n 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 base sequence of a target nucleic acid. In certain embodiments, antisense compounds are at least 70%, 80%, 90%, 95%, 98%, or 100% complementary to the base sequence of a target nucleic acid over the entire length of the antisense nd.

Antisense mechanisms include any mechanism involving the hybridization of an oligomeric compound with target c acid, wherein the hybridization results in a biological effect. In certain embodiments, such ization results in either target nucleic acid degradation or occupancy with concomitant inhibition or stimulation of the cellular ery 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 ation of target RNA is RNase H mediated nse. RNase H is a cellular clease 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. tion 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 isms.

Such mechanisms include creation of a microRNA mimic and/or an anti-microRNA.

Antisense mechanisms also include, Without limitation, mechanisms that ize 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 ription or ation 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 n embodiments, oligonucleotides comprising ates described herein are paired with a second oligomeric compound to form an siRNA. In n such embodiments, the second oligomeric compound also comprises a conjugate. In certain embodiments, the second eric 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 ments, 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 nse strand.

D. Target c Acids, Regions and Segments In certain embodiments, conjugated antisense compounds target any nucleic acid. In certain embodiments, the target nucleic acid encodes a target n that is clinically relevant. In such embodiments, modulation of the target nucleic acid s in clinical benefit. Certain target nucleic acids include, but are not limited to, the target nucleic acids illustrated in Table 1.

Table 1: Certain Target Nucleic Acids Transthyretin (TTR) NM_000371.3 - The targeting process y 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 ments, a target region is a structurally defined region of the nucleic acid. For example, in certain such embodiments, a target region may ass 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 n 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 utive 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 se at least 8 consecutive nucleobases from the 3'—terminus of one of the target ts (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 bases.

In certain embodiments, antisense compounds targeted to the nucleic acids listed in Table 1 can be d 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 es as described . In certain embodiments, the antisense compounds can have a modified internucleoside linkage, an unmodified internucleoside linkage or a mixture of d and fied internucleoside linkages as described herein. In certain embodiments, the antisense compounds can have a modified nucleobase, an unmodified nucleobase or a mixture ofmodified and unmodified nucleobases as described herein. In certain ments, the antisense compounds can have a motif as bed herein.

In certain embodiments, antisense compounds targeted to the nucleic acids listed in Table 1 can be conjugated as described herein. 1. Hepatitis B (HBVg Hepatitis B is a viral disease itted parenterally by contaminated material such as blood and blood products, contaminated needles, ly and vertically from infected or carrier mothers to their offspring. It is estimated by the World Health Organization that more than 2 billion people have been infected worldwide, with about 4 million acute cases per year, 1 n deaths per year, and 350-400 million chronic carriers (World Health Organization: Geographic Prevalence of Hepatitis B Prevalence, 2004. http://www.who.int/vaccines-surveillance/graphics/htmls/hepbprev.htm).

The virus, HBV, is a -stranded hepatotropic virus which infects only humans and non-human primates. Viral replication takes place predominantly in the liver and, to a lesser extent, in the kidneys, pancreas, bone marrow and spleen (Hepatitis B virus biology. Microbiol Mol Biol Rev. 64: 2000; 51-68.).

Viral and immune markers are detectable in blood and characteristic antigen-antibody patterns evolve over time. The first detectable viral marker is HB sAg, followed by hepatitis B e antigen (HBeAg) and HBV DNA.

Titers may be high during the tion period, but HBV DNA and HBeAg levels begin to fall at the onset of s and may be undetectable at the time of peak clinical illness (Hepatitis B virus infection—natural history and clinical consequences. N Engl J Med.. 350: 2004; 1118-1129). HBeAg is a viral marker detectable in blood and correlates with active viral replication, and therefore high viral load and infectivity (Hepatitis B e antigen—the dangerous end game of tis B. N Engl J Med. 347: 2002; 208-210). The ce of anti-HBsAb and BcAb (IgG) indicates recovery and immunity in a previously ed individual.

Currently the recommended therapies for chronic HBV infection by the American Association for the Study of Liver Diseases (AASLD) and the European Association for the Study of the Liver (EASL) include interferon alpha (INFOL), pegylated interferon alpha-2a (Peg-IFN2a), entecavir, and tenofovir. The nucleoside and nucleobase therapies, entecavir and tenofovir, are successful at reducing viral load, but the rates of HBeAg seroconversion and HBsAg loss are even lower than those obtained using IFNa therapy. Other similar therapies, including lamivudine (3TC), telbivudine (LdT), and adefovir are also used, but for nucleoside/nucleobase therapies in general, the emergence of resistance limits therapeutic efficacy.

Thus, there is a need in the art to discover and develop new anti-viral therapies. Additionally, there is a need for new anti-HBV therapies e of increasing HBeAg and HBsAg seroconversion rates. Recent clinical research has found a correlation between seroconversion and reductions in HBeAg (Fried et al (2008) Hepatology ) and reductions in HBsAg (Moucari et al (2009) Hepatology 49:1151). Reductions in antigen levels may have allowed immunological control of HBV infection because high levels of antigens are thought to induce immunological tolerance. Current side therapies for HBV are e of ic reductions in serum levels of HBV but have little impact on HBeAg and HBsAg levels. nse compounds targeting HBV have been usly disclosed in WO2011/047312, WO2012/145674, and WO2012/145697, each herein incorporated by reference in its entirety. Clinical studies are planned to assess the effect of antisense compounds targeting HBV in patients. However, there is still a need to e patients with onal and more potent ent options.

Certain Conjugated Antisense Compounds Targeted to a HB VNuclet'c Acid In certain embodiments, conjugated antisense compounds are targeted to a HBV nucleic acid having the sequence of GENBANK® Accession No. U95551.1, incorporated herein as SEQ ID NO: 1. In certain such embodiments, a conjugated antisense compound targeted to SEQ ID NO: 1 is at least 90%, at least 95%, or 100% complementary to SEQ ID NO: 1.

In certain embodiments, a conjugated antisense compound targeted to SEQ ID NO: 1 comprises an at least 8 consecutive nucleobase sequence of SEQ ID NO: 3. In certain embodiments, a conjugated antisense compound targeted to SEQ ID NO: 1 comprises a nucleobase sequence of SEQ ID NO: 3.

In certain embodiments, a conjugated nse compound targeted to SEQ ID NO: 1 ses an at least 8 consecutive nucleobase sequence of SEQ ID NO: 4. In certain embodiments, a conjugated nse nd targeted to SEQ ID NO: 1 comprises a nucleobase sequence of SEQ ID NO: 4.

In certain embodiments, a conjugated antisense compound targeted to SEQ ID NO: 1 comprises an at least 8 utive nucleobase sequence of SEQ ID NO: 5. In certain embodiments, a conjugated antisense compound targeted to SEQ ID NO: 1 comprises a nucleobase sequence of SEQ ID NO: 5.

In certain embodiments, a conjugated antisense compound targeted to SEQ ID NO: 1 comprises an at least 8 consecutive base sequence of SEQ ID NO: 6. In certain embodiments, a conjugated antisense compound targeted to SEQ ID NO: 1 comprises a nucleobase sequence of SEQ ID NO: 6.

In certain embodiments, a conjugated antisense compound targeted to SEQ ID NO: 1 comprises an at least 8 consecutive nucleobase sequence of SEQ ID NO: 7. In n embodiments, a conjugated antisense compound targeted to SEQ ID NO: 1 comprises a nucleobase sequence of SEQ ID NO: 7.

In certain embodiments, a conjugated antisense compound targeted to SEQ ID NO: 1 ses an at least 8 consecutive base sequence of SEQ ID NO: 8. In certain embodiments, a conjugated antisense compound targeted to SEQ ID NO: 1 comprises a nucleobase sequence of SEQ ID NO: 8.

In n embodiments, a ated antisense compound targeted to SEQ ID NO: 1 comprises an at least 8 consecutive nucleobase sequence of SEQ ID NO: 9. In certain embodiments, a conjugated antisense compound targeted to SEQ ID NO: 1 ses a nucleobase sequence of SEQ ID NO: 9.

In certain embodiments, a conjugated antisense compound targeted to SEQ ID NO: 1 ses an at least 8 consecutive nucleobase ce of SEQ ID NO: 10. In certain ments, a conjugated antisense compound targeted to SEQ ID NO: 1 comprises a nucleobase sequence of SEQ ID NO: 10.

In certain embodiments, a conjugated antisense compound targeted to SEQ ID NO: 1 comprises an at least 8 consecutive nucleobase sequence of SEQ ID NO: 11. In certain embodiments, a conjugated nse compound targeted to SEQ ID NO: 1 comprises a nucleobase sequence of SEQ ID NO: 11.

Table 2: Antisense Compounds targeted to HBV SEQ ID NO: 1 Target SEQ ID ISIS No Start Sequence (5’-3’) Motif 505358 1583 GCAGAGGTGAAGCGAAGTGC eeeeeddddddddddeeeee 3 509934 1780 CCAATTTATGCCTACAGCCT eeeeeddddddddddeeeee 4 101 00 41 1 GGCATAGCAGCAGGATG eeeddddddddddeeee 5 552023 1266 TCCGCAGTATGGAT eeeeeeddddddddddeeee 6 552024 1577 GTGAAGCGAAGTGCACACGG eeeeeeddddddddddeeee 7 552032 1585 GTGCAGAGGTGAAGCGAAGT ddddddddddeeee 8 552859 1583 AGGTGAAGCGAAGTGC ekkddddddddddkke 9 552925 1264 TCCGCAGTATGGATCG ekddddddddddkeke 10 577119 1780 AATTTATGCCTACAGCCT kdkdkddddddddeeeee 11 In certain embodiments, a compound comprises 0r consists of ISIS 505358 and a conjugate group.

ISIS 505358 is a modified oligonucleotide having the formula: Ges mCes Aes Ges Aes Gds Gds Tds Gds Ads Ads Gds mCds Gds Ads Aes Ges Tes Ges mCe, n, A = an adenine, mC = a 5’-methylcyt0sine G = a guanine, T = a thymine, e = a 2’-O-meth0xyethyl modified nucleoside, d = a xynucle0side, and s = a orothioate internucleoside linkage.

In certain embodiments, a compound comprises 0r consists of ISIS 509934 and a conjugate group.

ISIS 509934 is a modified oligonucleotide having the formula: mCes mCes Aes Aes Tes Tds Tds Ads Tds Gds mCds mCds Tds Ads mCds Aes Ges mCes mCes Te, wherein, A = an adenine, mC = a 5’-methylcytosine G = a e, T = a thymine, e = a 2’-O-methoxyethyl modified nucleoside, d = a 2’-deoxynucleoside, and s = a phosphorothioate intemucleoside linkage.

In certain embodiments, a nd ses or consists of ISIS 510100 and a conjugate group.

ISIS 510100 is a modified oligonucleotide having the formula: Ges Ges mCes Ads Tds Ads Gds mCds Ads Gds mCds Ads Gds Ges Aes Tes Ge, wherein, A = an adenine, mC = a 5’-methylcytosine G = a guanine, T = a thymine, e = a 2’-O-methoxyethyl modified nucleoside, d = a 2’-deoxynucleoside, and s = a phosphorothioate cleoside linkage.

In certain embodiments, a compound ses or consists of ISIS 552023 and a conjugate group.

ISIS 552023 is a modified oligonucleotide having the a: Aes Ges Ges Aes Ges Tes Tds mCds mCds Gds mCds Ads Gds Tds Ads Tds Ges Ges Aes Te, wherein, A = an adenine, mC = a 5’-methylcytosine G = a guanine, T = a thymine, e = a 2’-O-methoxyethyl modified nucleoside, d = a 2’-deoxynucleoside, and s = a phosphorothioate intemucleoside linkage.

In certain embodiments, a compound comprises or consists of ISIS 552024 and a conjugate group.

ISIS 552024 is a modified oligonucleotide having the formula: Ges Tes Ges Aes Aes Ges mCds Gds Ads Ads Gds Tds Gds mCds Ads mCds Aes mCes Ges Ge, wherein, A = an adenine, mC = a 5’-methylcytosine G = a guanine, T = a thymine, e = a 2’-O-methoxyethyl modified nucleoside, d = a 2’-deoxynucleoside, and s = a phosphorothioate cleoside linkage.

In certain embodiments, a compound comprises or consists of ISIS 552032 and a ate group.

ISIS 552032 is a modified oligonucleotide having the a: Ges Tes Ges mCes Aes Ges Ads Gds Gds Tds Gds Ads Ads Gds mCds Gds Aes Aes Ges Te, wherein, A = an adenine, mC = a 5’-methylcytosine G = a e, T = a thymine, e = a 2’-O-methoxyethyl modified nucleoside, d = a 2’-deoxynucleoside, and s = a phosphorothioate intemucleoside linkage.

In certain embodiments, a compound comprises or consists of ISIS 552859 and a conjugate group.

ISIS 552859 is a modified oligonucleotide having the formula: Aes Gks Gks Tds Gds Ads Ads Gds mCds Gds Ads Ads Gds Tks Gks mCe, wherein, A = an adenine, mC = a hylcytosine G = a guanine, T = a thymine, e = a 2’-O-methoxyethyl modified nucleoside, k = a cEt modified nucleoside, d = a 2’-deoxynucleoside, and s = a phosphorothioate intemucleoside linkage.

In certain embodiments, a compound comprises or consists of ISIS 552925 and a conjugate group.

ISIS 552925 is a modified ucleotide having the a: Tes kas mCds Gds mCds Ads Gds Tds Ads Tds Gds Gds Aks Tes kas Ge, wherein, A = an adenine, mC = a 5’-methylcytosine G = a guanine, T = a thymine, e = a 2’-O-methoxyethyl modified nucleoside, k = a cEt modified nucleoside, d = a 2’-deoxynucleoside, and s = a phosphorothioate internucleoside linkage. s = a phosphorothioate internucleoside linkage.

In certain embodiments, a compound ses or consists of ISIS 577119 and a conjugate group.

ISIS 577119 is a d oligonucleotide haVing the formula: Aks Ads Tks Tds Tks Ads Tds Gds mCds mCds Tds Ads mCds Aes Ges mCes mCes Te, wherein, A = an e, mC = a 5’-methylcytosine G = a guanine, T = a thymine, e = a 2’-O-methoxyethyl modified nucleoside, k = a cEt modified nucleoside, d = a 2’-deoxynucleoside, and s = a phosphorothioate internucleoside linkage.

In n embodiments, a compound haVing the following chemical structure comprises or consists of ISIS 505358 with a 5’-X, wherein X is a conjugate group as described herein: WO 79627 In certain embodiments, a compound comprises or consists of ISIS 712408 having the following chemical structure: In certain embodiments, a nd comprises or consists of ISIS 695324 having the following chemical structure: In certain embodiments, a compound comprises or consists of SEQ ID NO: 3, 5’-GalNAc, and chemical modifications as represented by the following chemical structure: wherein either R1 is —OCH2CH20CH3 (MOE)and R2 is H; or R1 and R2 together form a bridge, wherein R1 is —O- and R2 is —CH2-, -CH(CH3)-, or -CH2CH2-, and R1 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 -OCH2CH20CH3 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 ing bridge is selected from: - O-CH2-, CH3)-, and CH2-; and R5 is selected from H and —CH3; and Z is selected from S' and O'.

In certain embodiments, a compound comprises an antisense oligonucleotide disclosed in WC 2012/ 145697, which is incorporated by reference in its entirety herein, and a conjugate group described herein. In certain ments, a nd comprises an antisense oligonucleotide having a nucleobase sequence of any of SEQ ID NOs 5-310, 2, 804-1272, 1288-1350, 372, 1375, 1376, and 1379 disclosed in WC 2012/ 145697 and a conjugate group described herein. In certain ments, a compound comprises an nse oligonucleotide sed in WC 2011/ 047312, which is incorporated by reference in its entirety herein, 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 14-22 disclosed in WO 2011/ 047312 and a conjugate group described herein. In certain embodiments, a compound comprises an antisense oligonucleotide disclosed in herein, and a conjugate group described . In certain embodiments, a nd comprises an antisense oligonucleotide having a nucleobase sequence of any of SEQ ID NOs 18-35 disclosed in WC 2012/ 145674.

In certain embodiments, a compound comprises a double-stranded oligonucleotide disclosed in WC 201 3/ 159109, which is orated by reference in its ty herein, and a conjugate group described herein. In certain embodiments, a compound comprises a double-stranded oligonucleotide in which one strand has a nucleobase sequence of any of SEQ ID NOs 30-125 disclosed in WC 2013/ 159109. The base sequences of all of the aforementioned referenced SEQ ID NOs are incorporated by reference HBV Therapeutic Indications In certain embodiments, the invention provides methods for using a conjugated antisense compound targeted to a HBV nucleic acid for modulating the expression of HBV in a subject. In certain embodiments, the expression ofHBV is reduced.

In certain embodiments, the invention provides methods for using a conjugated antisense compound targeted to a HBV nucleic acid in a pharmaceutical composition for treating a subject. In certain embodiments, the subject has a HBV-related condition. In certain ments, the HBV-related condition includes, but is not limited to, chronic HBV infection, inflammation, fibrosis, cirrhosis, liver cancer, serum hepatitis, ce, liver cancer, liver inflammation, liver fibrosis, liver cirrhosis, liver failure, e cellular inflammatory disease, hemophagocytic syndrome, serum hepatitis, and HBV viremia. In certain embodiments, the lated condition may have symptoms which may include any or all of the following: flu-like illness, weakness, aches, headache, fever, loss of appetite, diarrhea, jaundice, nausea and vomiting, pain over the liver area of the body, clay- or grey-colored stool, itching all over, and dark-colored urine, when d With a positive test for presence of a tis B virus, a hepatitis B viral antigen, or a positive test for the presence of an antibody specific for a hepatitis B viral antigen. In certain embodiments, the subject is at risk for an HBV-related condition. This includes subjects having one or more risk factors for developing an HBV-related condition, including sexual exposure to an individual infected with Hepatitis B virus, living in the same house as an individual with a lifelong hepatitis B virus infection, re to human blood infected with the hepatitis B virus, injection of illicit drugs, being a person Who has hemophilia, and visiting an area Where hepatitis B is common. In n embodiments, the subject has been identified as in need of treatment for an HBV-related condition.

Certain embodiments provide a method of reducing HBV DNA and/or HBV n levels in a animal infected with HBV comprising administering to the animal a conjugated antisense compound targeted to a HBV nucleic acid. In certain embodiments, the antigen is HBsAG or HBeAG. In certain embodiments, the amount ofHBV antigen may be iently d to result in seroconversion.

In certain ments, the invention provides methods for using a conjugated antisense compound ed to a HBV nucleic acid in the preparation of a medicament.

In n embodiments, the invention provides a conjugated antisense compound targeted to a HBV nucleic acid, or a pharmaceutically acceptable salt thereof, for use in therapy.

Certain embodiments provide a ated antisense compound targeted to a HBV nucleic acid for use in the treatment of a HBV-related condition. The HBV-related condition includes, but is not limited to, c HBV infection, inflammation, fibrosis, cirrhosis, liver cancer, serum hepatitis, jaundice, liver cancer, liver inflammation, liver fibrosis, liver cirrhosis, liver e, diffuse hepatocellular inflammatory disease, hemophagocytic syndrome, serum hepatitis, and HBV viremia.

Certain embodiments provide a conjugated antisense compound targeted to a HBV nucleic acid for use in reducing HBV DNA and/or HBV antigen levels in a animal infected with HBV comprising administering to the animal a conjugated antisense compound ed to a HBV nucleic acid. In certain embodiments, the antigen is HBsAG or HBeAG. In certain embodiments, the amount of HBV antigen may be sufficiently d to result in seroconversion.

It will be understood that any of the compounds described herein can be used in the aforementioned methods and uses. For example, in certain embodiments a conjugated antisense compound targeted to a HBV nucleic acid in the aforementioned methods and uses can include, but is not limited to, a conjugated antisense compound targeted to SEQ ID NO: 1 sing an at least 8 consecutive nucleobase sequence of any of SEQ ID NOs: 3-11; a conjugated antisense nd targeted to SEQ ID NO: 1 sing a nucleobase sequence of any of SEQ ID NOs: 3-11; a compound comprising or consisting of ISIS 505358, ISIS 509934, ISIS 510100, ISIS , ISIS 552024, ISIS 552032, ISIS 552859, ISIS 552925, or ISIS 577119 and a conjugate group; a nd comprising an antisense oligonucleotide disclosed in WC 2012/ 145697, which is incorporated by reference in its ty herein, and a conjugate group; a compound comprising an antisense oligonucleotide having a nucleobase sequence of any of SEQ ID NOs 5-310, 321-802, 72, 1288-1350, 1364-1372, 1375, 1376, and 1379 disclosed in herein; a compound sing an antisense oligonucleotide having a nucleobase ce of any of SEQ ID NOs 14-22 disclosed in WC 2011/ 047312 and a conjugate group described herein; a compound comprising an antisense oligonucleotide having a nucleobase sequence of any of SEQ ID NOs 18-35 disclosed in WC 2012/ ; or a compound comprising a double-stranded oligonucleotide in which one strand has a nucleobase sequence of any of SEQ ID NOs 30-125 disclosed in 2. Transthfletin g TTR! TTR (also known as prealbumin, hyperthytoxinemia, dysprealbuminemic, ine; senile systemic amyloidosis, amyloid polyneuropathy, amyloidosis I, PALB; dystransthyretinemic, 1; TBPA; dysprealbuminemic euthyroidal hyperthyroxinemia) is a serum/plasma and cerebrospinal fluid protein responsible for the transport of thyroxine and retinol (Sakaki et al, Mol Biol Med. 1989, 6: 161-8).

Structurally, TTR is a homotetramer; point mutations and misfolding of the n leads to deposition of amyloid fibrils and is associated with disorders, such as senile systemic amyloidosis (SSA), familial amyloid polyneuropathy (FAP), and familial amyloid cardiopathy (FAC).

TTR is synthesized primarily by the liver and the choroid plexus of the brain and, to a lesser degree, by the retina in humans (Palha, Clin Chem Lab Med, 2002, 40, 300). hyretin that is synthesized in the liver is ed into the blood, whereas transthyretin originating in the choroid plexus is destined for the CSF. In the choroid plexus, transthyretin synthesis represents about 20% of total local protein synthesis and as much as 25% of the total CSF protein (Dickson et al., JBz'oZ Chem, 1986, 261, 3475-3478).

With the availability of genetic and immunohistochemical stic tests, patients with TTR amyloidosis have been found in many nations worldwide. Recent studies indicate that TTR amyloidosis is not a rare endemic disease as previously thought, and may affect as much as 25% of the elderly population (Tanskanen et al, Ann Med. 2008;40(3):232-9).

At the biochemical level, TTR was identified as the major protein component in the amyloid deposits of PAP patients (Costa et al, Proc. Natl. Acad. Sci. USA 1978, 75:4499—4503) and later, a substitution of methionine for valine at position 30 of the protein was found to be the most common molecular defect causing the e (Saraiva et al, J. Clin. Invest. 1984, 74: 104—119). In FAP, widespread systemic extracellular deposition of TTR aggregates and amyloid fibrils occurs throughout the connective tissue, particularly in the peripheral nervous system (Sousa and Saraiva, Prog. Neurobiol. 2003, 71: 385—400).

Following TTR deposition, axonal degeneration occurs, starting in the inated and myelinated fibers of low diameter, and ultimately leading to neuronal loss at ganglionic sites.

Antisense compounds targeting TTR have been previously disclosed in /0244869, W020 1 09, and WO201 1/139917, each herein incorporated by reference in its entirety. An antisense oligonucleobase targeting TTR, ISIS-TTRRX, is currently in Phase 2/3 clinical trials to study its effectiveness in treating subjects with Familial Amyloid Polyneuropathy. However, there is still a need to provide patients with additional and more potent treatment options.

Certain Conjugated Antisense Compounds ed to a TTR c Acid In n embodiments, conjugated nse compounds are targeted to a TTR nucleic acid having the ce of GENBANK® Accession No. 371.3, incorporated herein as SEQ ID NO: 2. In certain such embodiments, a conjugated antisense compound targeted to SEQ ID NO: 2 is at least 90%, at least 95%, or 100% complementary to SEQ ID NO: 2.

In certain ments, a conjugated antisense compound targeted to SEQ ID NO: 2 comprises an at least 8 consecutive nucleobase sequence of any one of SEQ ID NOs: 12-19. In certain embodiments, a conjugated antisense compound targeted to SEQ ID NO: 2 comprises a nucleobase sequence of any one of SEQ ID NO:12-19.

In certain embodiments, a conjugated antisense compound targeted to SEQ ID NO: 2 ses an at least 8 consecutive nucleobase sequence of SEQ ID NO: 12. In certain embodiments, a conjugated antisense compound targeted to SEQ ID NO: 2 comprises a nucleobase sequence of SEQ ID NO: 12.

In certain embodiments, a conjugated antisense compound targeted to SEQ ID NO: 2 comprises an at least 8 consecutive nucleobase sequence of SEQ ID NO: 13. In certain embodiments, a conjugated antisense compound targeted to SEQ ID NO: 2 comprises a nucleobase sequence of SEQ ID NO: 13.

In certain ments, a conjugated antisense compound targeted to SEQ ID NO: 2 comprises an at least 8 consecutive nucleobase sequence of SEQ ID NO: 14. In certain embodiments, a conjugated antisense compound targeted to SEQ ID NO: 2 comprises a base sequence of SEQ ID NO: 14.

In n embodiments, a conjugated antisense compound targeted to SEQ ID NO: 2 comprises an at least 8 consecutive nucleobase sequence of SEQ ID NO: 15. In certain embodiments, a conjugated nse compound targeted to SEQ ID NO: 2 comprises a nucleobase sequence of SEQ ID NO: 15.

In certain embodiments, a conjugated nse compound targeted to SEQ ID NO: 16 comprises an at least 8 consecutive nucleobase sequence of SEQ ID NO: 78. In certain embodiments, a conjugated antisense compound targeted to SEQ ID NO: 16 comprises a nucleobase sequence of SEQ ID NO: 78.

In certain embodiments, a conjugated antisense compound targeted to SEQ ID NO: 2 comprises an at least 8 consecutive nucleobase sequence of SEQ ID NO: 17. In certain embodiments, a conjugated antisense nd targeted to SEQ ID NO: 2 comprises a nucleobase ce of SEQ ID NO: 17.

In certain embodiments, a conjugated antisense compound targeted to SEQ ID NO: 2 comprises an at least 8 consecutive nucleobase sequence of SEQ ID NO: 18. In certain ments, a conjugated antisense compound targeted to SEQ ID NO: 2 comprises a nucleobase ce of SEQ ID NO: 18.

In n embodiments, a conjugated antisense compound targeted to SEQ ID NO: 2 comprises an at least 8 consecutive nucleobase sequence of SEQ ID NO: 19. In certain embodiments, a conjugated antisense compound targeted to SEQ ID NO: 2 comprises a nucleobase sequence of SEQ ID NO: 19.

Table 3: nse Compounds targeted to TTR SEQ ID NO: 2 ISIS N0 Targsittftart Sequence (5’-3’) SEQ ID NO 08 eeeeeddddddddddeeeee 12 07 eeeeeddddddddddeeeee 13 eeeeeddddddddddeeeee 14 eeeeeddddddddddeeeee 15 80 dddddddddeeeee 16 85 eeeeeddddddddddeeeee 17 87 eeeeeddddddddddeeeee 18 89 eeeeeddddddddddeeeee 19 In certain embodiments, a compound comprises or consists of ISIS 420915 and a conjugate group.

ISIS 420915 is a modified ucleotide having the formula: Tes mCes Tes Tes Ges Gds Tds Tds Ads mCds Ads Tds Gds Ads Ads Aes Tes mCes mCes mCe, wherein A = an adenine, mC = a 5’-methylcytosine G = a guanine, T = a thymine, e = a 2’-O-methoxyethyl d nucleoside, d = a 2’-deoxynucleoside, and s = a phosphorothioate internucleoside linkage.

In certain embodiments, a compound comprises or consists of ISIS 304299 and a conjugate group.

ISIS 304299 is a modified oligonucleotide having the formula: mCes Tes Tes Ges Ges Tds Tds Ads mCds Ads Tds Gds Ads Ads Ads Tes mCes mCes mCes Ae, wherein A = an adenine, mC = a 5’-methylcytosine G = a guanine, T = a thymine, e = a 2’-O-methoxyethyl modified nucleoside, d = a 2’-deoxynucleoside, and s = a phosphorothioate ucleoside linkage.

In certain embodiments, a compound comprises or consists of ISIS 420921 and a conjugate group.

ISIS 420921 is a modified oligonucleotide haVing the formula: Ges Ges Aes Aes Tes Ads mCds Tds mCds Tds Tds Gds Gds Tds Tds Aes mCes Aes Tes Ge, wherein A = an adenine, mC = a hylcytosine G = a guanine, T = a thymine, e = a 2’-O-methoxyethyl d nucleoside, d = a 2’-deoxynucleoside, and s = a phosphorothioate ucleoside linkage.

In certain embodiments, a compound comprises or consists of ISIS 420922 and a conjugate group.

ISIS 420922 is a modified oligonucleotide haVing the formula: Tes Ges Ges Aes Aes Tds Ads mCds Tds mCds Tds Tds Gds Gds Tds Tes Aes mCes Aes Te, n A = an adenine, mC = a 5’-methylcytosine G = a e, T = a thymine, e = a 2’-O-methoxyethyl modified nucleoside, d = a 2’-deoxynucleoside, and s = a phosphorothioate internucleoside linkage.

In certain embodiments, a compound comprises or consists of ISIS 420950 and a conjugate group.

ISIS 420950 is a modified oligonucleotide having the formula: Tes Tes Tes Tes Aes Tds Tds Gds Tds mCds Tds mCds Tds Gds mCds mCes Tes Ges Ges Ae, wherein A = an adenine, mC = a 5’-methylcytosine G = a guanine, T = a e, e = a 2’-O-methoxyethyl modified nucleoside, d = a 2’-deoxynucleoside, and s = a phosphorothioate internucleoside linkage.

In certain embodiments, a compound comprises or consists of ISIS 420955 and a conjugate group.

ISIS 420955 is a d oligonucleotide haVing the formula: Ges Aes Aes Tes Ges Tds Tds Tds Tds Ads Tds Tds Gds Tds mCds Tes mCes Tes Ges mCe, wherein A = an e, mC = a 5’-methylcytosine G = a guanine, T = a thymine, e = a 2’-O-methoxyethyl modified nucleoside, d = a 2’-deoxynucleoside, and s = a orothioate internucleoside linkage.

In certain ments, a compound comprises or consists of ISIS 420957 and a conjugate group.

ISIS 420957 is a modified oligonucleotide haVing the formula: Aes Ges Ges Aes Aes Tds Gds Tds Tds Tds Tds Ads Tds Tds Gds Tes mCes Tes mCes Te, n A = an adenine, mC = a 5’-methylcytosine G = a guanine, T = a thymine, e = a 2’-O-methoxyethyl modified nucleoside, d = a xynucleoside, and s = a phosphorothioate intemucleoside linkage.

In certain embodiments, a compound ses or ts of ISIS 420959 and a conjugate group.

ISIS 420959 is a modified oligonucleotide having the formula: Aes mCes Aes Ges Ges Ads Ads Tds Gds Tds Tds Tds Tds Ads Tds Tes Ges Tes mCes Te, wherein A = an adenine, mC = a 5’-methylcytosine G = a guanine, T = a thymine, e = a 2’-O-methoxyethyl modified nucleoside, d = a 2’-deoxynucleoside, and s = a phosphorothioate intemucleoside e.

In certain embodiments, a compound having the following chemical structure comprises or consists of ISIS 420915 with a 5’-X, wherein X is a conjugate group as described herein: In certain embodiments, a compound comprises or consists of ISIS 682877 having the following chemical structure: In n embodiments, a compound comprises or consists of ISIS 682884 having the following chemical structure: In certain embodiments, a compound comprises or consists of SEQ ID NO: 12, 5’-GalNAc, and chemical modifications as represented by the following chemical structure: wherein either R1 is —OCH2CH20CH3 nd R2 is H; or R1 and R2 together form a bridge, wherein R1 is —O- and R2 is —CH2-, -CH(CH3)-, or -CH2CH2-, and R1 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, ndently for each ring: either R3 is selected from H and -OCH2CH20CH3 and R4 is H; or R3 and R4 together form a bridge, n 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'.

In certain embodiments, a compound ses an antisense oligonucleotide disclosed in WC 201 1/ 139917 or US 8,101,743, which are incorporated by reference in their entireties herein, and a conjugate group. In n embodiments, a nd comprises an antisense oligonucleotide having a nucleobase ce of any of SEQ ID NOs 8-160, 170-177 disclosed in described herein. In certain embodiments, a compound comprises an antisense oligonucleotide having a nucleobase sequence of any of SEQ ID NOs 12-89 disclosed in US 8,101,743 and a conjugate group described herein. In n embodiments, a compound comprises an antisense oligonucleotide having a nucleobase ce complementary to a red target segment of any of SEQ ID NOs 90-133 disclosed in US 8,101,743 and a conjugate group described herein. The nucleobase sequences of all of the aforementioned referenced SEQ ID NOs are incorporated by reference herein.

TTR Therapeutic Indications In n embodiments, the invention provides methods for using a conjugated antisense compound targeted to a TTR nucleic acid for modulating the expression of TTR in a subject. In certain embodiments, the expression of TTR is reduced.

In certain embodiments, the invention provides methods for using a conjugated antisense compound targeted to a TTR nucleic acid in a pharmaceutical composition for treating a subject. In certain embodiments, the subject has a transthyretin related e, disorder or condition, or symptom thereof. In certain embodiments, the transthyretin d disease, disorder or condition is transthyretin amyloidosis.

"Transthyretin-related amyloidosis" or "transthyretin amyloidosis" or "Transthyretin amyloid disease", as used herein, is any pathology or disease associated With dysfiinction or dysregulation of transthyretin that result in formation of hyretin-containing amyloid fibrils. Transthyretin amyloidosis includes, but is not d to, hereditary TTR amyloidosis, leptomeningeal amyloidosis, familial amyloid uropathy (FAP), al amyloid cardiomyopathy, familial oculoleptomeningeal amyloidosis, senile cardiac amyloidosis, or senile systemic amyloidosis.

In certain embodiments, the invention provides methods for using a conjugated antisense nd targeted to a TTR nucleic acid in the preparation of a medicament.

In n embodiments, the invention es a conjugated antisense compound targeted to a TTR nucleic acid, or a pharmaceutically acceptable salt thereof, for use in therapy.

Certain embodiments provide a conjugated antisense compound targeted to a TTR nucleic acid for use in the treatment of a transthyretin related disease, disorder or ion, or symptom thereof. In n embodiments, the transthyretin related disease, disorder or condition is transthyretin amyloidosis.

It will be understood that any of the compounds described herein can be used in the aforementioned methods and uses. For e, in certain embodiments a conjugated antisense compound targeted to a TTR nucleic acid in the aforementioned methods and uses can include, but is not limited to, a conjugated antisense compound targeted to SEQ ID NO: 2 sing an at least 8 utive nucleobase sequence of any one of SEQ ID NOs: 12-19; a conjugated antisense compound targeted to SEQ ID NO: 2 comprising a nucleobase sequence of any one of SEQ ID NO: 12-19; a compound comprising or consisting of ISIS 420915, ISIS 304299, ISIS 420921, ISIS 420922, ISIS 420950, ISIS 420955, ISIS 420957, or ISIS 420959 and a conjugate group; a compound comprising an nse oligonucleotide disclosed in WC 201 1/ 139917 or US 8,101,743, which are incorporated by reference in their entireties herein, and a conjugate group; a compound comprising an antisense oligonucleotide having a nucleobase sequence of any of SEQ ID NOs 8-160, 170-177 sed in WC 201 1/ 139917 and a conjugate group described herein; an antisense oligonucleotide having a nucleobase sequence of any of SEQ ID NOs 12-89 disclosed in US 8,101,743 and a conjugate group bed herein; or a compound comprising an antisense oligonucleotide having a base sequence complementary to a red target segment of any of SEQ ID NOs 90-133 disclosed in US 8,101,743 and a conjugate group bed herein. The nucleobase sequences of all of the aforementioned nced SEQ ID NOs are orated by reference herein.

E. Certain Pharmaceutical Compositions In certain embodiments, the present disclosure provides pharmaceutical compositions comprising one or more antisense nd. 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 nse compound. In n 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 ments, a pharmaceutical composition comprises one or more antisense compound and sterile water. In n embodiments, a pharmaceutical composition consists of one or more antisense nd and e water.

In certain embodiments, the sterile saline is pharmaceutical grade water. In certain embodiments, a pharmaceutical composition comprises one or more nse 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 e, 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 ically active metabolite or residue f. 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 s, DNA complexes with mono- or poly-cationic lipids are formed without the ce of a l lipid. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to a particular cell or tissue. In n embodiments, a lipid moiety is selected to increase distribution of a ceutical agent to fat tissue. In n embodiments, a lipid moiety is selected to increase distribution of a ceutical 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, ymethylcellulose and polyvinylpyrrolidone.

In certain embodiments, a pharmaceutical ition provided herein comprises a delivery system.

Examples of delivery systems include, but are not limited to, liposomes and emulsions. n delivery systems are usefiJl for preparing certain pharmaceutical compositions including those comprising hydrophobic compounds. In certain embodiments, certain c solvents such as dimethylsulfoxide are used.

In certain embodiments, a pharmaceutical composition provided herein comprises one or more - specific delivery molecules designed to deliver the one or more pharmaceutical agents of the t disclosure to specific tissues or cell types. For example, in certain embodiments, pharmaceutical compositions include liposomes coated with a tissue-specific antibody.

In certain ments, a pharmaceutical composition ed herein comprises a co-solvent system. Certain of such co-solvent systems comprise, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic r, and an aqueous phase. In certain embodiments, such co-solvent systems are used for hydrophobic compounds. A non-limiting e of such a co-solvent system is the VPD co-solvent system, which is a solution of absolute ethanol sing 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80"" and 65% w/v polyethylene glycol 300. The tions of such co-solvent systems may be varied considerably without significantly altering their solubility and toxicity characteristics.

Furthermore, the ty of co-solvent components may be varied: for example, other surfactants may be used instead of Polysorbate 80""; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e. g., polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.

In certain embodiments, a pharmaceutical composition provided herein is prepared for oral administration. In certain embodiments, pharmaceutical itions 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 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 sions are prepared using appropriate liquid carriers, suspending agents and the like. Certain ceutical 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, ons or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing . Certain solvents le for use in ceutical compositions for injection e, but are not d 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. ally, such sions may also contain suitable izers or agents that increase the solubility of the pharmaceutical agents to allow for the preparation of highly concentrated solutions.

In certain embodiments, a ceutical 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 lly known in the art.

In certain embodiments, a pharmaceutical composition provided herein comprises an oligonucleotide in a therapeutically effective . In certain embodiments, the eutically 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 g. In certain embodiments, upon in viva 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 ces, a g may be more bioavailable (e. g., through oral administration) than is the corresponding active form. In certain ces, a prodrug may have improved solubility compared to the corresponding active form. In n embodiments, prodrugs are less water soluble than the corresponding active form. In certain instances, such gs 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 ylic 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 n 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 . In certain ments, 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 s of stering a pharmaceutical composition comprising an oligonucleotide of the present sure to an animal. Suitable administration routes e, but are not limited to, oral, rectal, transmucosal, inal, enteral, topical, suppository, through inhalation, intrathecal, intracerebroventricular, eritoneal, intranasal, intraocular, intratumoral, and parenteral (e.g., intravenous, uscular, intramedullary, and subcutaneous). In certain embodiments, ceutical 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, k accession s, and the like recited in the present application is incorporated herein by reference in its entirety.

Certain compounds, compositions, and methods herein are described as "comprising exactly" or "comprises exactly" a particular number of a particular t or feature. Such descriptions are used to indicate that While the compound, composition, or method may comprise additional other elements, the number of the particular element or feature is the identified number. For example, "a conjugate comprising exactly one GalNAc" is a conjugate that contains one and only one , though it may contain other elements in addition to the one GalNAc.

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 al modifications. One of skill in the art Will readily appreciate that such designation as "RNA" or "DNA" to describe d 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 bed 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 ed herein, including, but not limited to those in the sequence g, are intended to encompass nucleic acids ning any combination of natural or modified RNA and/or DNA, including, but not limited to such nucleic acids having modified nucleobases. By way of fithher example and without limitation, an oligonucleotide having the nucleobase sequence "ATCGATCG" encompasses any oligonucleotides having such base sequence, r modified or unmodified, including, but not limited to, such compounds sing RNA bases, such as those having ce UCG" and those having some DNA bases and some RNA bases such as "AUCGATCG" and oligonucleotides having other modified bases, such as "ATmeCGAUCG," wherein meC indicates a cytosine base comprising a methyl group at the 5-position.

EXAMPLES The following examples illustrate certain embodiments of the t 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. e 1: General Method for the ation of Phosphoramidites, Compounds 1, 1a and 2 O O BX B Bx : s '9 /\/OMe H3C SK 5 o‘ o O o o ' 1'3 NC 1" NC\/\o/P‘N(ipr)2 NC\/\o/ NM» "\0/ NUPF>2 1 la 2 Bx is a heterocyclic base; Compounds 1, 1a 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, 2009/006478, and Example 2: Preparation of Compound 7 AcOOAc AcOOAc O O o TMSOTf, 50 00 HOWO/b 5 A00 OAC —> N\o AcHN CICHzConCI TMSOTf, DCE 3 (93 Au) 4 (66%) AcOOAc AcOOAc Acofi/ \/\/\n/ \/© O O O —>AcO MeOH V\/\"/ A HNC 0 AcHN o (95%) 6 7 Compounds 3 (2-acetamid0-1,3,4,6-tetra-O-acetyl-Z-deoxy-B-Dgalactopyranose 0r galactosamine pentaacetate) is cially available. Compound 5 was prepared according to published procedures (Weber er al., J. Med. Chem, 1991, 34, 2692).

Example 3: Preparation of Compound 11 \H/\\ NC/\\ O 0 NH —>2 NC/\/O NH —’2 aq. KOH, Reflux, rt, 0 ED 0 HO 0xane, O (56%) 8 (40%) Nod 10 ON 11 Compounds 8 and 9 are commercially available.

Example 4: Preparation of Compound 18 "fig EC benzylchloroformate, 311 o BOW/V0 NH e: NazCO3 NAO fl, 2 T» /\© e 0 EC ( 0) EtOZI/\/OOEtO (91%) ON 11 ON 12 m 0 0 N’\/‘NH2 HOW/:09»H 14 NA H %O\Cf)l/H 0/\© HBTU DIEA DMF 3N 13 +11%"Mfir" AcOOAc H2N H O m 17 ACHN W CF3COOH 2 \/\/N\n/\O/O§~ka OAQ HBTU, DIEA, HOBt 95 % DMF < 16 (64%) H2NMN O AcOOAc O H H ACO St $5 /O\/\/\n/N\/\/N\;O ACHN O AcOOAc AcowwwwwwbflimHAcHN O o o AcOOAc HN’C H 0 A00 OM AcHN 1 8 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 er al., J.

Med. Chem, 2004, 47, 5798-5808.

Example 5: Preparation of Compound 23 1. chOWOH oj/O Z > 1. TBDMSCI TBDMSO HBTU, DIEA DMF, ImIdazode, "(95 %) DMF, rt (65%) HO ’ 2- Pd/Cy H2: 'V'eOHv rt 2. TEA.3HF TEA THF 87% 0TBD'V'S "0H (72%) HO 0 o 1. DMTCI pyr n(75%) NMOH N OCH3 —> 2. LiOH, Dioxane (97%) a 22 nds 19 and 21 are cially available.

Example 6: Preparation of Compound 24 AcOOAc ACO \/\/ AcHN W K 1- H2, Pd/C. MeOH (93%) AcOOAc 2. HBTU DIEA DMF (76%) O H H i ACHN O o o HOMN AcOOAc HN OH 0 N\/\/H ACO OM O 18 AcOOAc 0 H o H 0 A00 \/\/\n/N\/\/ K ACHN O AcOOA O O 1 o H H AcO V\/\n/ \/\/NTVO€>HNWa N: AcHN o o o AcOOAc H HN’EO o N\/\/ A00 O\/\/\n/ 0 24 Compounds 18 and 23 were prepared as per the procedures rated in Examples 4 and 5.

Example 7: Preparation of Compound 25 AcOOAc O H o H 0 A00 WNW ACHN O AcOOAc ACOmOWNWHwog—HWN:H o o [ODMT 1. ic anhydride, DMAP, DCE ACHN O o 0 2. DMF, HBTU, EtN(iPr)2, PS-SS AcOOAc H HN’CO Acog/O\/\/\n/O 2" AcOOAc O H o H 0 A00 \/\/\n/N\/\/ K ACHN O AcOOAc o o ACO WY \/\/N\n/\/O N 8 NQ NH ACHN O O O AcOOAc "Nf Acomom 25 Compound 24 was prepared as per the procedures illustrated in Example 6.

Example 8: Preparation of Compound 26 ACOOAC ACO$§/O\/\/\n/N\/\/NxiOO H H ACHN O ACOOAC o /ODMT O O o H H 2' ACO W\n/ WNWI/Vog5HWNQ Phosphitylation AcHN o o 0 ACOOAC H HN/ ACO%Q/ \/\/\n/N\/\/N\;OH 0 O=F|"O/\/| l. DCA, DCM O 2. DCl, NMl, ACN AcOOAc HN H o Phosphoramidite DNA/RNA $0 O\/\/\n/N\/\/ building blockla A00 3. Capping 27 4. t-BuOOH DMTOWBX (If. beMe O:P_O/\/CN AcOOAc O H o H o (l) ACO WYNW x; O=F|"O_ ACHN O AcOOAc o /O s ACO&H/W \/\/N NH ACHN O \(q/VOgiNWNQO l. DCA, DCM AcOOAc HN o 2. DCI, NMI, ACN o OWNH\/\/ Phosphoramidite DNA/RNA 0 ng bIOCkS automated synthesize ACHN 3. Capping 4. xanthane hydride or t-BuOOH . Et3N/CH3CN (1:1) 6. Aaueous NHQ (cleavage) OLIGO X=F<-O O o BX Bx = Heterocycllc base. VfOMe0 X=OorS l O=F<—O o H H o (l) HO \/\/\n/ W K O=||3_O ACHN O o /O O 0 o H H H0 W \/\/N\[I/\/ %H0 NW8 N: ACHN O O O o H H \/\/\n/ \/\/ Ko HQ 0=|ID—O ACHN O o P O O o H H HO \/\/\n/ g’" 8 N: ACHN O O O HN’CO OH HOOH H 0 NW HO 0M The GalNAc3 r portion of the conjugate group GalNAc3-1 (GalNAc3-1a) can be combined with any cleavable moiety to provide a variety of conjugate groups. Wherein GalNAc3-1a has the formula: O H H HOOH MNWNf o ,0 O O O H o H HO \/\/\n/ WNYVog>MMLNQ ACHN O O O HOWOM The solid support bound ted GalNAcg-l, Compound 25, was prepared as per the procedures illustrated in Example 7. Oligomeric Compound 29 comprising GalNAc3-1 at the 3’ terminus was prepared using standard procedures in automated DNA/RNA synthesis (see Dupouy er al., Angew. Chem. Int. Ed, 2006, 45, 3623-3627). oramidite building blocks, Compounds 1 and 1a 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 e oligomeric compounds haVing a predetermined ce 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 ermined composition and base sequence as dictated by any given target.

Example 10: General preparation conjugated ASOs comprising GalNAc3-1 at the 5’ terminus, nd 34 ODMT 1. Capping (A020, NMI, pyr) \_ 3. DCA, DCM Q UNL—ODMT 2. DCI, NMI, ACN 0 PhOSphoramidite Q UNL—O-llk /\/CNO MIfdéCIIV building blocks 05p orami 16 DNA/RNA DNA/RNA 3 1 automated s nthe51zer 1. Cappmg (A020, NMI, pyr). DMTO/\<:7’BX 2. t-BuOOH N \/\O-PC (I) 3. DCA, DCM 4. DCI, NMI, ACN Phosphoramidite 26 DNA/RNA X = O, or S automated s zer BX = Heterocylic base AcOOAc o H H 0 A00 W\n/ W K AcHN 0 AcOOAc o H O o [ODMT O H =.

AcO \/\/\H/ \/\/N\"/\/O\E~MJ\HSJLNQ AcHN o o o NC\/\ O BX AcOOAc HN/CO O W ACO%Q/O\/\/\g/O N O NC\/\O—l:’=0 OLlGO 1. Capping (A020, NMl, pyr) O 2. t-BuOOH , 3. Et3NzCH3CN (1:1 v/v) 4. DCA, DCM . N114, rt (cleavage) 33 WO 79627 O H o H 0 HO \/\/\n/N\/\/ x: AcHN O HO \/\/\n/ \/\/N\n/V \E’HO N AcHN O o O ACH N 34 The UnylinkerTM 30 is cially available. Oligomeric Compound 34 comprising a GalNAc3-1 cluster at the 5’ terminus is prepared using standard procedures in ted DNA/RNA synthesis (see Dupouy er al., Angew. Chem. Int. Ed, 2006, 45, 3623-3627). Phosphoramidite building blocks, Compounds 1 and 1a were prepared as per the procedures illustrated in Example 1. The phosphoramidites illustrated are meant to be representative and not intended to be ng as other phosphoramidite building blocks can be used to prepare an oligomeric compound having a ermined ce 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 ition and base sequence as dictated by any given target.

Example 11: Preparation of Compound 39 AcOOAc 1. HoWfi/ko/D AcOOAc A00 35 0 TMSOTf, DCE o AGO$¢0MNH28 Ni‘ 2. H2/Pd, MeOH ACHN 35 AcO OAc HBTU, DMF, EtN(iPr)2 "Cg/0M9").o 1- H2, Pd/C, MeOH Compound 13 AcHN 8 \n/\\O 2 HBTU DIEA DMF AcO Compound 23 AcO O\/\H/\/N AcO \n/\O/OO o NH AcO WW AcO OAc Ammo /ODMT A mi phitylation mkw‘wwwjrNHAC AcO w o 38 A00 OWNH ACO OAC AcO o /ODMT NHAc O o AcO W O 39 AcO OWNH 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 er al., Eur. J. Org. Chem, 2011, 12, 2346-2353.

Example 12: Preparation of Compound 40 A00 OAC Afmo /ODMT Ac0 Co "Nil/WOff—3?;Mic...H NHAC 0):)O 1. Succinic anhydride, DMAP, DCE O . 0 NH 2. DMF, HBTU, EtN(IPr)2, PS—ss ACO W ACO OAc A00 o /ODMT ACHN \/\(\98/\/N O : N HA0 0 0 A00 p 40 0 NH ACO w ACH N nd 38 is prepared as per the procedures rated in Example 11.

Example 13: Preparation of Compound 44 AGOOAC HBTU, DMF, EtN(iPr)2 ACQfl/OVW/NHZ o AcHN HO 0 36 /—© IV byH 0 ACO OAc ACO O AcHN %H8 O 0}H 1. H2, Pd/C, MeOH 2. HBTU, DIEA, DMF o >10 Compound 23 OAc 0 0 NH ACO W AcO OAC 0%? /ODMT AcHN 8 j/ZI NMNiOH Phosphitylation O 43 O W ACO O\/H\/\/NH ACO OAc AcHN 8 jZI N08\/\O//P\NN(iPI')2 ACQk/OwéggA/pNHOAcA00 44 0 W},0\oH Compounds 23 and 36 are prepared as per the procedures illustrated in Examples 5 and 11.

Compound 41 is prepared using similar ures published in WO 2009082607.

Example 14: Preparation of Compound 45 ACO OAC MHo {ODMT AcHN 8 ? 0 1. Succinic anhydride, DMAP, DCE AcO O\/H\/\/NH 8 —> AcHN 2. DMF, HBTU, EtN(iPr)2, Ps-ss ACO OAC AcHN 8 3 o W AcO OVHWNH nd 43 is prepared as per the procedures illustrated in Example 13.

Example 15: Preparation of Compound 47 HO DMTO >\_ / o < > N 1. DMTCI, pyr 2. Pd/C, H2, MeOH HO: 46 Compound 46 is commercially available.

Example 16: Preparation of Compound 53 HBTU, EtN(iPr)21DMF H300WNHZ O / H3CO\n/\fi/\NO 0 \CB2 49 Bz/NH H300 mN/CBZ . 1 TFA WHO 1.LIOH,MeOH 2. HBTU EtN(iPr)z DMF 2. HBTU, EtN(iPr)2,DMF /CBz Compound 47 DMTO HN 1. H2, Pd/C o .

,CBZ 2. HBTU, r)21 DMF HOI'" NW" NH I2 Compound 17 HN‘CBz OACOAc CO 0 NH A::§§¥OAC/\/\)I\N O HN N NHAC O OACO O ODMT O _NH 53 AcO O 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 CO 0 NH A:&::AC/\/\)OJ\HN O HN "M ""0" N NHACO O NHACO Phosphitylation OACOAc CO /\/\)J\ 0 NH (iPr)2N‘ SQOC/A\/\)OJ\N o P \/\ ""0 CN HN 7 NHAC O O NE: OACO O ODMT O /\/\/u‘NH 54 A00 0 nd 53 is prepared as per the procedures illustrated in Example 16.

Example 18: Preparation of Compound 55 ACO§$7O0Ac O OACOAC O 0 HM ...\0H 0 W N N NHAC 0 GAO O ODMT 0 /\/\/u—NH 53 A00 0 1. Succinic anhydride, DMAP, DCE 2. DMF, HBTU, EtN(iPr)2, PS-SS 0Ac$03 WK ACO O NH N HAC O 0 OAc o W 0 H . I \ \O 0 Wk N HN N ACOOA& 7 O HN NHAC 0 OAc o ODMT 0 N—NH 55 AcO O nd 53 is prepared as per the procedures illustrated in Example 16.

Example 19: General method for the preparation of conjugated ASOs comprising 3-1 at the 3’ position via solid phase ques (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 oramidite building blocks and solid support are used for incorporation nucleoside residues which e for example T, A, G, and mC residues. A 0.1 M solution of phosphoramidite in anhydrous acetonitrile was used for B-D-Z’-deoxyribonucleoside and 2’- The ASO syntheses were performed on ABI 394 synthesizer (1-2 umol scale) or on GE Healthcare Bioscience AKTA oligopilot synthesizer (40-200 umol scale) by the phosphoramidite ng method on an GalNAc3-1 loaded VIMAD solid support (110 umol/g, GuzaeV er 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. orothioate linkages were introduced by sulfurization with 0.1 M on 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 intemucleoside linkages with a contact time of 12 minutes.

After the desired sequence was assembled, the thyl phosphate ting 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 um, 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, )t = 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 rd oligonucleotide synthesis procedures well known in the art.

Using these methods, three separate antisense nds ing ApoC III were prepared. As summarized in Table 4, below, each of the three antisense nds targeting ApoC III had the same nucleobase sequence; ISIS 304801 is a 55 MOE gapmer having all phosphorothioate linkages; ISIS 647535 is the same as ISIS 304801, except that it had a GalNAc3-1 conjugated at its 3’end; and ISIS 647536 is the same as ISIS 647535 except that certain intemucleoside linkages of that compound are odiester linkages. As fithher summarized in Table 4, two separate nse compounds targeting SRB-l were synthesized. ISIS 440762 was a 22 cEt gapmer with all phosphorothioate intemucleoside linkages; ISIS 651900 is the same as ISIS 440762, except that it included a GalNAc3-l at its 3’-end.

Table 4 ed ASO targeting ApoC III and SRB-l CalCd Observed , , 353-1831 Aes(}esmcesTesTesmcdsTdsTds(}dsTdsmcdsmcdsAds(}dsmcds TesAesTe Aflgc 71 65 .4 71 64.4 ISIS Aes(}esmcesTesTesmcdsTdsTds(}dsTdsmcdsmcdsAds(}dsmcdsTesTesTesAesTeoAdo’' -9239.5APOC 9237.8 21 647535 GalNAc3-la ISIS omceor-l—‘eorl—‘eomcdsTdsTds(}dsTdsmCdsmCdsAds(}dsmcdsTeoTeoTesAesTeoAdo" APOC 647536 GalNAc3-la -9142.9 9140.8 WO 79627 45.317862 TksmcksAdsGdsTdsmcdsAdsTdsGdsAdsmCdsTdsTmmck SRIB' 4647-0 46464 £19?" AdsGdsTdsmCdsAdsTdsGdsAdsmcdsTdsTkskaoAdowGalNAcg-la SRIB' 67211 67194 Subscripts: "e" indicates 2’-MOE modified nucleoside; "d" indicates B-D-2’-deoxyribonucleoside; "k" indicates 6’-(S)-CH3 bicyclic nucleoside (e.g. cEt); "s" indicates phosphorothioate intemucleoside linkages (PS); "0" tes odiester intemucleoside linkages (PO); and "0’" indicates -O-P(=O)(OH)-.

Superscript "m" indicates 5-methylcytosines. "GalNAc3-1" indicates a conjugate group having the structure shown previously in Example 9. Note that GalNAc3-l comprises a ble ine which links the ASO to remainder of the conjugate, which is designated "GalNAc3-la." This nomenclature is used in the above table to show the filll 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 "GalNAc3-1" 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 "GalNAc3 cluster.’ 9 In certain instances it is convenient to describe a conjugate group by separately providing its cluster and its cleavable moiety. e 20: Dose-dependent nse 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.

Trealmenl Human I transgenic mice were maintained on a 12-hour dark cycle and fed ad libilum Teklad lab chow. Animals were ated 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 umol/kg or with PBS as a control. Each treatment group consisted of 4 animals. 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 [H mRNA Analysis ApoC III mRNA levels in the mice’s livers were determined using real-time PCR and RIBOGREEN® RNA quantification t (Molecular Probes, Inc. Eugene, OR) according to rd protocols. ApoC III mRNA levels were determined relative to total RNA (using Ribogreen), prior to ization to PBS-treated control. The results below are presented as the average percent of ApoC III mRNA levels for each treatment group, normalized to PB S-treated control and are denoted as "% PBS". The half maximal effective dosage (ED50) of each ASO is also presented in Table 5, below.

As illustrated, both nse compounds reduced ApoC III RNA relative to the PBS control.

Further, the antisense compound conjugated to GalNAc3-1 (ISIS 647535) was substantially more potent than the antisense compound lacking the GalNAc3-1 conjugate (ISIS 304801).

Table 5 Effect ofASO treatment on ApoC III mRNA levels in human ApoC III transgenic mice Internucleoside 3’ ate linkage/Length 304801 ISIS - 0-074 GalNAc3-1 PS/20 21 647535 .

ApoC [[1 Protein is (Turbidomeiric Assay) Plasma ApoC III n analysis was ined using procedures reported by Graham er al, Circulation Research, published online before print March 29, 2013.

Approximately 100 pl 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 .

As shown in the Table 6 below, both antisense compounds reduced ApoC III protein relative to the PBS control. Further, the nse compound conjugated to GalNAc3-1 (ISIS 647535) was substantially more potent than the antisense compound lacking the GalNAc3-1 conjugate (ISIS 304801).

Table 6 Effect of ASO treatment on ApoC III plasma protein levels in human ApoC III enic mice Dose % ED50 ucleoside SEQ ID AS0 3, C0n'J gu ate (umovkg) PBS (umol/kg) Linkage/Length No. "n———— ISIS 0-08 ISIS —- 019‘ GalNAc3'1 PS/20 21 647535 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 JBl'ochem Physiol, 37, 911-917, 1959) and measured by using a Beckmann Coulter clinical analyzer and commercially available reagents.

The ceride levels were measured ve to PBS injected mice and are denoted as "% PBS". Results are presented in Table 7. As illustrated, both antisense compounds lowered triglyceride levels. Further, the antisense compound conjugated to GalNAc3-1 (ISIS 647535) was substantially more potent than the antisense compound lacking the GalNAc3-1 conjugate (ISIS 304801).

Table 7 Effect ofASO treatment on triglyceride levels in transgenic mice Dose % 3 ’ Internucleoside (HumOI/kg) PBS Conjugate Linkage/Length PBS 100 ISIS 075 647535 2-2— . 3-l 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 8 and 9. As illustrated, both antisense compounds lowered total cholesterol levels; both lowered LDL; and both raised HDL. Further, the antisense nd conjugated to GalNAc3-1 (ISIS ) was substantially more potent than the antisense nd lacking the 3-1 conjugate (ISIS 304801). An increase in HDL and a decrease in LDL levels is a cardiovascular cial effect of nse inhibition of ApoC III.

Table 8 Effect ofASO treatment on total cholesterol levels in transgenic mice Dose Total Cholesterol 3’ Intemucleoside SEQ (umol/kg) (mg/dL) Conjugate Linkage/Length ID No.

PBS 0 257 _- __ 0.08 226 ISIS 0.75 164 None PS/20 20 304801 —2.25110 6.75 82 0.08 230 ISIS 0.75 82 GalNAc3-1 PS/20 21 647535 2.25 86 6.75 99 Table 9 Effect ofASO treatment on HDL and LDL terol levels in transgenic mice Dose (mg/7dIL) C_onj3ugateL—inkage/LengthIntemucleoside SEQ (umol/kg) (mg/dL) ID NO PBS 0 ISIS 0.75 None PS/20 304801 2.25 ISIS 0.75 al-NAc31PS/20 647535 2.25 Pharmacokz'netics 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 MSDl utilizing IP-HPLC-MS. The tissue level (pg/g) of fiJll-length ISIS 304801 and 647535 was measured and the results are ed in Table 10. As illustrated, liver concentrations of total full-length antisense compounds were r for the two nse compounds.

Thus, even though the GalNAc3-1 -conjugated antisense compound is more active in the liver (as demonstrated by the RNA and protein data above), it is not t at substantially higher concentration in the liver. Indeed, the calculated EC50 (provided in Table 10) confirms that the observed increase in potency of the conjugated compound cannot be entirely uted to increased accumulation. This result suggests that the conjugate improved potency by a mechanism other than liver accumulation alone, possibly by ing the productive uptake of the antisense compound into cells.

The s also show that the concentration of GalNAc3-l 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 GalNAc3-1 conjugation reduces kidney lation.

Table 10 PK analysis ofASO ent in transgenic mice Intemucleoside L'1ver EC ’ 50 3 e/Length (Hg/g) Conjugate ISIS 0.8 62.8 304801 2.3 142.3 6. 8 202.3 0.1 3.8 ISIS 0.8 72.7 647535 2.3 106.8 6.8 237.2 Metabolites of ISIS 647535 were also identified and their masses were confirmed by high resolution mass ometry analysis. The cleavage sites and structures of the observed metabolites are shown below.

The relative % of fill length ASO was calculated using standard procedures and the results are presented in Table 10a. The major metabolite of ISIS 647535 was fiJll-length ASO lacking the entire conjugate (i.e. ISIS 304801), which results from cleavage at ge site A, shown below. Further, additional metabolites resulting from other cleavage sites were also observed. These results suggest that introducing other ble bonds such as , peptides, disulfides, phosphoramidates or acyl-hydrazones between the GalNAc3-l 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 1021 Observed full length metabolites of ISIS 647535 ISIS 304801 2 ISIS 304801 + dA 3 ISIS 647535 minus [3 GalNAc] ISIS 647535 minus [3 GalNAc -- 1 0xy—pentan0ic acid tether] ISIS 647535 minus [2 GalNAc -- 2 5-hydr0xy—pentan0ic acid tether] ISIS 647535 minus 6 [3 GalNAc -- 3 5-hydr0xy—pentan0ic acid tether] ASO 304801 Cleavage Sites_ Cleavage site A HO OH Cleavage site C O=?_OH NH2 Howe Cleavage site D N WNW" 0 9H I ;‘N / E (W N HO = NHAc WT :- age site C —Cleavage site B H\]c])/\/O " O _-u_=0 NHAc \O\/\/\H/\Cl:a\/\/vagesite D HO \/O\/\/\]THCleavage site D NHAc Cleavage site C A50 304801 O=If—0H NH2 A30 304801 N Metabolite 1 | Metabolite 2 OH Wm WO 79627 lesoso4sn1 O=P-OH NH2 0 N H o ’ N QH I o 5 O o o‘ H H I NW\n/\/O N o I?2o HO Metabolite 3 HN H QH (/N l 2W No)"o N/j," g Metabolite 4 HN/: l|\50 304801 O N H o ’ N H2N\/\/N EH 0 (N [NJ 0 0 o n | H N2W O N 0 H Fl’=0 Metabolite 5.

HN\/\/ o T80 304301 HOW/Y o o l O=P-OH NH2 H o /NlAN HZNWN 9H WM[NJ 0 0 o H | H N2W O N O = o H F; Metabolite 6 /: Example 21: nse inhibition of human ApoC III in human ApoC III transgenic mice in single administration study ISIS 304801, 647535 and 647536 each ing human ApoC III and bed in Table 4, were fithher evaluated in a single administration study for their ability to inhibit human ApoC III in human ApoC III transgenic mice.

Trealmenl Human ApoCIII transgenic mice were maintained on a 12-hour dark cycle and fed ad libilum Teklad lab chow. Animals were acclimated for at least 7 days in the research facility before initiation of the experiment. ASOs were ed 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 eritoneally 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 ted 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 stration .

Samples were collected and analyzed to determine the ApoC III mRNA and n 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 11-15, 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 GalNAc3-1 conjugate at the 3’ terminus (ISIS 647535 and 647536) compared to the antisense compound lacking a GalNAc3-1 conjugate (ISIS 304801). Further, ISIS 647536, which comprises a GalNAc3-1 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 11 Effect ofASO treatment on ApoC III mRNA levels in human ApoC III transgenic mice SENQID 304801 ISIS 0.3 98 1.9 GalNAc3-1 PS/20 WO 79627 647535 70:3 ISIS == GalNAc3-1 PS/PO/20 647536 Table 12 Effect of ASO treatment on ApoC III plasma protein levels in human ApoC III enic mice E1350 3’ Intemucleoside SEQ ID ASO . (mg/kg) Conjugate Linkage/Length. N0. 104 23 2 ISIS 92 None PS/20 20 304801 71 98 2.1 ISIS 70 GalNAc3-1 PS/20 21 647535 33 103 1.8 ISIS 60 GalNAc3-1 PS/PO/20 21 647536 31 Table 13 Effect ofASO treatment on triglyceride levels in transgenic mice Dose Intemucleoside SEQ ID ASO (V PBS0 3’ Conjuga et (mg/kg) (mg/kg) Linkage/Length N0.

PBS 0 98 — -- __ 1 80 ISIS 3 92 47 0.3 100 ISIS 1 70 2-149GalNAc3-1GalNAc3-1 PS/20 21 647535 3 34 23 ISIS 0.3 95 PS/PO/ZO 21 647536 1 66 Table 14 Effect ofASO treatment on total cholesterol levels in transgenic mice ASO (111391;) % PBS 3’ Conjugate 3:335:33: SEQ ID No.

PBS 0 96 -- -- 1 104 ISIS 3 96 None PS/20 20 304801 10 86 72 0.3 93 ISIS 1 85 GalNAc3-1 PS/20 21 647535 3 61 53 0.3 1 15 ISIS 1 79 GalNAc3-1 PS/PO/20 21 647536 3 51 54 Table 15 Effect ofASO treatment on HDL and LDL cholesterol levels in transgenic mice Dose HDL LDL 3’ Internucleoside SEQ ID (mg/kg) % PBS % PBS Conjugate e/Length No. 1 130 ISIS 3 186 304801 10 226 240 0.3 98 ISIS 1 214 3-1 21 647535 3 212 218 0.3 143 ISIS 1 187 GalNAc3-1 PS/PO/20 21 647536 3 213 221 WO 79627 These results confirm that the GalNAc3-l conjugate improves y of an nse compound.

The results also show equal y of a GalNAc3-1 conjugated antisense compounds where the antisense oligonucleotides have mixed linkages (ISIS 647536 which has six phosphodiester linkages) and a full orothioate version of the same antisense compound (ISIS 647535).

Phosphorothioate linkages e several properties to antisense compounds. For example, they 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 es in a compound is expected to reduce the risk of inflammation, but also lower tration of the compound in liver, se tration in the kidney and urine, decrease stability in the presence of nucleases, and lower overall potency. The present results show that a GalNAc3-l 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 orothioate 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 GalNAc3-l conjugated modified ASO targeting SRB-l in vivo ISIS 440762 and 651900, each targeting SRB-1 and described in Table 4, were evaluated in a dose- dependent study for their ability to inhibit SRB-1 in Balb/c mice.

Trealmenl Six week old male Balb/c mice (Jackson Laboratory, Bar , ME) were injected aneously 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 RIBOGREEN® RNA quantification reagent ular Probes, Inc. Eugene, OR) according to standard protocols. SRB-l mRNA levels were determined relative to total RNA (using Ribogreen), prior to normalization to PBS-treated control. The results below are ted as the average percent of SRB-1 mRNA levels for each treatment group, normalized to PB S-treated control and is denoted as "% PBS".

As rated in Table 16, both antisense compounds lowered SRB-l mRNA levels. Further, the antisense compound comprising the GalNAc3-1 conjugate (ISIS ) was substantially more potent than the antisense compound lacking the GalNAc3-l conjugate (ISIS 440762). These results trate that the potency benefit of GalNAc3-1 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 16 Effect of ASO treatment on SRB-1 mRNA levels in Balb/c mice Intemucleosid 440762 651900 e 23: Human eral 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 WWR 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 ing tubes 8-10 times. CPT tubes were centrifiJged at rt (18-25 0C) in a horizontal (swing-out) rotor for 30 min. at 1500-1800 RCF with brake off (2700 RPM Beckman a 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 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 centrifiJged 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 (N1 ml / 10 ml ng whole blood volume). A 60 ul sample was pipette into a sample vial (Beckman Coulter) with 600 pl VersaLyse reagent (Beckman Coulter Cat# A09777) and was gently vortexed for 10-15 sec. The sample was allowed to incubate for 10 min. at rt and being mixed again before counting. The cell suspension was counted on Vicell XR cell viability analyzer (Beckman Coulter) using PBMC cell type ion factor of 1:11 was stored with other ters). The live cell/ml 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 ul/well of 96-well tissue culture plate (Falcon Microtest). 50 ul/well of 2x concentration oligos/controls diluted in RPMI+10% FBS+pen/strep. was added according to experiment te (100 ul/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% C02, the plates were centrifuged at 400 x g for 10 minutes before removing the supernatant for MSD ne assay (i.e. human IL-6, IL-10, IL-8 and MCP-1). e 24: Evaluation of Proinflammatory Effects in hPBMC Assay for GalNAc3-1 conjugated ASOs The antisense ucleotides (ASOs) listed in Table 17 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 der 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 uM concentrations. After a 24 hr treatment, the cytokine levels were ed.

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/ECSO from two donors and is denoted as 50." The lower ratio indicates a relative se in the proinflammatory response and the higher ratio indicates a relative increase in the proinflammatory response.

With regard to the test compounds, the least ammatory compound was the PS/PO linked ASO (ISIS 616468). The GalNAc3-1 conjugated ASO, ISIS 647535 was slightly less ammatory than its non-conjugated counterpart ISIS 304801. These results te that incorporation of some PO linkages reduces proinflammatory reaction and addition of a GalNAc3-1 conjugate does not make a compound more proinflammatory and may reduce proinflammatory response. ingly, one would expect that an antisense compound comprising both mixed PS/PO linkages and a GalNAc3-1 conjugate would produce lower proinflammatory responses relative to fill PS linked antisense compound with or without a 3-l conjugate. These results show that GalNAc3_1 conjugated antisense compounds, particularly those having reduced PS content are less proinflammatory.

Together, these results suggest that a GalNAc3-l conjugated compound, particularly one with reduced PS content, can be stered at a higher dose than a counterpart fiJll PS antisense compound lacking a GalNAc3-l conjugate. Since half-life is not expected to be substantially different for these compounds, such higher administration would result in less nt dosing. Indeed such administration could be even less frequent, because the GalNAc3-1 conjugated nds are more potent (See Examples -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 17 d ASOs ASO Sequence (5’ to 3’) Target SE13 ID0 ISIS sTesGesAesTdsTdsAdsGdsAdsGds 10483 8 AdsGdsAdsGdsGesTesmCesmCesmCe ISIS TesmcesmCesmCdsAdsTdsTdsTdsmCdsAdsGds CRP 25 353512 GdsAdSGdSAdsmCdsmCdsTesGmGe ISIS AesGesmCeSTesTesmCdsTdsTdsGdsTds ApoC III 20 304801 IncdsmCdsAdsGdsmcds TesTeSTeSAmTe ISIS A,SGesmC,STesTesmCdsTdsTdSGdsTdS ApOC III 21 647535 mCLisInC:dslAL1s(IdsmcdsTesTesTes[AesTeolAdo"(;alNAAC3-1a ISIS AesGmmCmTe(,Te(,mCdsTdSTdsGdSTds 616468 mCdsmCdsAdsGdsmcdsTeoTeoTfiAeSTe ApoC III 20 Subscripts: a; 99 e indicates 2’-MOE modified nucleoside; "(1" indicates B-D-2’- deoxyribonucleoside; "k" indicates 6’-(S)-CH3 bicyclic nucleoside (e. g. cEt); "s" tes phosphorothioate intemucleoside linkages (PS); "0" indicates phosphodiester internucleoside linkages (PO); and "0’" indicates -O-P(=O)(OH)-. Superscript "m" indicates 5-methylcytosines. "AdowGalNAc3-la" indicates a conjugate having the structure GalNAc3-1 shown in Example 9 ed to the 3’-end of the antisense oligonucleotide, as indicated.

Table 18 Proinfiammatory Effect of ASOs targeting ApoC III in hPBMC assay EC50 Emax 3’ Internucleoside SEQ ID ASO me/ECSO (HM) (HM) Conjugate e/Length No.

ISIS 35 3512 0.01 265.9 None PS/20 25 . 26,590 (high responder) ISIS 304801 0.07 106.55 1,522 None PS/20 20 ISIS 647535 0.12 1,150 GalNAc3-1 PS/20 21 ISIS 616468 0.32 224 None PS/PO/20 20 Example 25: Effect of GalNAc3-l 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 003,008, 0.24, 0.74, 2.22, 6.67 and 20 uM 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 s and the results are presented in Table 19. As illustrated, able potency was observed in cells treated with ISIS 647535 as compared to the control, ISIS 304801.

Table 19 Modified ASO targeting human ApoC III in primary hepatocytes cleoside SEQ IC50 (uM) Conjugate. linkage/Length 304801 647535 In this experiment, the large potency benefits of GalNAc3-l conjugation that are observed in vivo were not ed in vilro. Subsequent free uptake experiments in primary hepatocytes in vitro did show increased potency of oligonucleotides comprising various GalNAc ates 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 ed. The mice were ced 72 hours following the last administration.

Samples were collected and analyzed to determine the ApoC III protein levels in the liver as bed above (Example 20). Data from those analyses are presented in Table 20, below.

These results show reduction in potency for antisense compounds with PO/PS (ISIS ) in the wings relative to full PS (ISIS 304801).

Table 20 Effect ofASO treatment on ApoC III protein levels in human ApoC III transgenic mice Dose 3’ Intemucleoside 0 SEQ ID ASO A) PBS (mg/kg) Conjugate linkage/Length No.

PBS 0 99 - -- WO 79627 Wk 24 None Full PS 20 304801 for 2 Wks wk 40 None 14 PS/6 P0 20 616468 for 2 Wks Example 27: Compound 56 Compound 56 is commercially available from Glen Research or may be prepared according to published procedures reported by Shchepinov er al., Nucleic Acids Research, 1997, 25(22), 4447-4454.

Example 28: Preparation of Compound 60 AcO OAC AcO OAC O /\/\/\/OBIl 5 7 AGO HO O Hz/Pd O\/\/\/\ AcO , —> OBn MeOH \O , DCE AcHN 58 Nfl ( uant )q i (71%) AcO OAC CNEtOgégrhWCL A00 0A0 O 1101,02 CN 0 —> H AcO O\/\/\/\O/P\O/\/ ACO 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 1. BnCl OH 1. DMTCl,pyr 0 H HO 2. KOH,DMSO 2. Pd/C, 112 BnO ODMT 09—013 on _ O\P’O 3. HCl,MeOH 3. ltylatlon . 4. NaHCO; N(iPr)2 61 ~ 62 63 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 etaZ., Tetrahedron, 2007, , 988.

Alternatively, nd 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 O a O TPDBSOonA/OH l. DMTCl, pyr 2. TBAF —O\P/O\/E\o/\/ODMT O 3. Phosphitylation ' 63a OH ODMT nd 63a is prepared using procedures similar to those reported by Hanessian et al., Canadian Journal ofChemiStry, 1996, 74(9), 1731-1737.

Example 31: Preparation of Compound 63d HO—\_\ DMTO—\_\ HOWOgAOMOBn 1' DMTCl’pyr DMTO 0 /\/\ / \1'3 2' Pd/C,H2 W O /\/CN O O O Phosphitylation o _/_/ 63c _/__/ 63d Compound 63c is prepared using ures similar to those reported by Chen et al., Chinese Chemical Letters, 1998, 9(5), 451-453.

Example 32: Preparation of Compound 67 COZBn AcOOAc $¢O Ovij HZNKKOTBDMS ACOOAC 0 COZBH 65 O AcO OH R "0%0M ACHN 64 BDMSH HBTU,D1EA ACHN 66 R R = H or CH3 AGO 0Ac 1. TEA.3HF, THF O COZBn O OWL O\ /O\/\ 2. Phosphitylation c E 1|) CN AcHN R N(1Pr)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 WO 79627 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 AcOOAc 68 0 AcOOAc O CH3 0 A00 O\»/\\/"\/JLI&1 0 HBTUJMEA AC0 0\V/~\/»\,JL DMF ENOB" A 00AC C 1. 2 0 O OWL 111%O\ /O\/\ A00 2. Phosphitylation 1" CN AcHN CH3 N(iPr)2 nd 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: ation of Compound 75a O CF3 l. TBDMSCI, pyr Y N(iPr) /\/O 2. Pd/C, H2 HNWQ 2 NC 3. CF3COZEt,MeOH H ,P\ /\/CN NC/\/O OH F3C\[rN\/\/O 0 0 NC\/\O 4. TEA.3ldF, THF . Phosphltylatlon 0 HN/\/\O 75 A 75a 0 CF3 Compound 75 is prepared according to published procedures reported by Shchepinov et al., Nucleic Acids Research, 1997, 25(22), 4447-4454.

Example 35: ation of Compound 79 DMTOWO HOWO 1. BnCl NaH DOLNMLACN DMTO\/\/O OH —, HO\/\/O OBn Phosphoramidite 60 DMTOMO 2. DCA,CH2C12 HO/\/\O 76 77 A00 OAC NC 0 1 A00 O\\//\\/’\V//«\ 9 O/P\0 NC\\\ \H 1. Hz/Pd, MeOH AcO OAC ACHN O ACO 1‘) O O 0’ \0 A00 OAc NC 0 1 A00 O\/\/\/\ z \I? O 0 ACO OAC \\\ 0 o 9 AcHN 0 NC N(z’Pr)2 A00 [K O O\/\/\/\O/ 0 Compound 76 was prepared according to published procedures reported by Shchepinov er al., Nucleic Acids Research, 1997, 25(22), 4447-4454.

Example 36: ation of Compound 79a HOWO 1. , pyr FmOCOWO I?K1Pr)2 HO\/\/O 2.

OBn Pd/C, H2 —FmocOWO\%/\O/P\O/\/CN HOMO 3. Phosphitylation FmocOMO 77 79a Compound 77 is prepared as per the procedures illustrated in Example 35.

Example 37: General method for the preparation of conjugated oligomeric nd 82 comprising a odiester linked GalNAc3-2 conjugate at 5’ terminus via solid support (Method 1) o\/\/ O O/\/\ODMT DMTO/m’BX O/\/\ODMT \ O 0‘ NC /ll)\ 0 BX NC\/\o—1'>=o 1. DCA, DCM \/\O 0W 0 2. DCI, NMI, ACN 0" . . NC Phosphoramidite 56 \/\ OLIGO 1|) 0 A O O automated synthesizer ® VIMAD'O‘E’ro/VCN X = S' or O' X BX = Heterocylic base 80 1' Capping (A020, NM], pyr) 2. t-BuOOH 3. DCA, DCM 4. DCI NMI ACN AC0 OAC NC 9 9 O \ Phosphoramidite 60 ACHN 0 NC 7} 0 (I) I O BX O\/\/\/\O/P\O/\/\O O_(.P.) W"O ACHN O O 0 ('3 A00 OAC II) NHAC (I) Q VIMAD—O-fi~O/\/CN l. Capping (ACZO, NM], pyr) 81 2. t-BuOOH 3. 20% EtZNH inToluene (V/V) 4. NH4, 55 0C, NHAC 82 wherein GalNAc3-2 has the structure: O || 0 BX O\/\/\/\ H _ ACHN O O o=e—o- HO OH le O Q\/~V/\/Afiij The GalNAc3 cluster portion of the conjugate group GalNAc3-2 (GalNAc3-2a) can be combined With any cleavable moiety to provide a y of conjugate . Wherein GalNAc3-2a has the formula: The VIMAD-bound oligomeric compound 79b was prepared using standard procedures for ted DNA/RNA synthesis (see Dupouy er 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 e an eric compound haVing a odiester 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 GalNAc3-2 conjugate at 5’ terminus d 11) 0‘ 1. DCA, DCM . 2. DCI, NMI, ACN Phosphoramidite 79 OLIGO DNA/RNA ted s nthesizer X = S' or O' BX = Heterocyclic base 1. Capping 2. t-BuOOH 3. Et3N2CH3CN(1:1V/V) 83 4. NH4, 55 0C Oligomeric Compound 82 The VIMAD-bound oligomeric compound 79b was prepared using standard procedures for automated DNA/RNA synthesis (see Dupouy er al., Angew. Chem. Int. Ed, 2006, 45, 3623-3627). The g-Z cluster phosphoramidite, Compound 79 was prepared as per the procedures illustrated in Example . This alternative method allows a ep installation of the phosphodiester linked GalNAc3-2 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 ng blocks including but not limited to those ted 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 sing a GalNAc3- 3 Conjugate at the 5’ Terminus (GalNAc3-1 modified for 5' end attachment) via Solid Support ACO OAC ACO O H AcHN WNW/N H2, Pd/C, MeOH (93%) o h o BnO OH N a meowA \/\/ rah figO N O 0 o NHAC HN/\/\N 3. H2,Pd/C,MeOH A00 OAC A"$76 A00 AcO N HAc H AcH N DOWN O O 0 \/\/\n/\\N F H H OO F /U\/\/U\ o N \/\/NwogiN \COCF3 OACO OH H OH F NHAC NN/\/\ 830 Pyridine, DMF ACO 0Ac A00 83e 3| 5' H fi AcHN (DOWNWNW F OLIGO (CH2)6—NH2 HN0 H M; F —>OH \/\/N\n/EI/jovjiNH Borate buffer, DMSO, pH 8.5, rt AcORog/O0M F F N HAc NN/\/\ ACO\&/OAcO N HAc AcO o H N H ACHN W \/\/N HO Em M H ' 3' N\/\/N ) OLIGO OAC OM NH N—(CH26 —O— A00 H O woo O:'U—O NHAc /\/\ HN N H 0 0Ac #0 Agog?OAcO N HAC Aqueous ammonia HO OH AcHN COW/firWN\n/\‘ N— Wherein GalNAc3-3 has the ure: HO OH HOfi/O H AcHN WN H H H O 0 0H NWN 77/"0 NHMN "0 0M INF—(CH2)6—-O-F|’—§II NHAc HNMN "0305H0 The GalNAc3 cluster portion of the conjugate group GalNAc3-3 (GalNAc3-3a) can be combined with any cleavable moiety to provide a variety of conjugate groups. Wherein GalNAc3-3a has the formula: \/\/N 0 NH N—(CH2)6—O—§ HO 0" Y" H NHAC M Example 40: General method for the preparation of oligomeric compound 89 comprising a odiester linked GalNAc3-4 conjugate at the 3’ terminus via solid support WODMT 1. DCA Q 0&:\//\/\0Fmoc UNL—ODMT 2. DCI NMI ACN N(iPr)2 .—UNL—0—P\O/\/CN FmocOWO /P\ /\/CN 85 DMTOWO 0 0 3 Capping ODMT CN 4. t-BuOOH O\/\/ /_/_OFmoc O O OFmoc 1. r1dine,' ' ' ’1') /—/— 2% DBU, 96% DMF 0 0M0 \0 o OFmoc 3. DCl, NMl, ACN 86 Phosphoramidite 79a A l. Capping automated synthesizer 2- t-BUOOH 3. 2% Piperidine, AcO OAC 2% DBU, 96% DMF 4. DCl, NMI, ACN O Phosphoramidite 60 DNA/RNA ACHN O automated synthesizer Z 5. g ACO OAC O AcO O-P’O AcHN OO\/\/\/\NCOI 0W0 NHAc QP‘QWF’H. t-BuOOHDCAOligo synthesis (DNA/RNA automated synthesizer)CappingOxidationEt3N.CH3CN (1:1, V/V) A00 OAc ACHN O 98138 \/\/o 0 [0' 88 ,P\ \ fi"0_ OI 0\/\/ 0 0’ 5'0 DMT -OLIGO O\P/O/\/\O \0\ Q:x . 3, UNL—o—lgl— NH4, 55°C 0' 89 OO/P\0\/\/, / o \PI’=O Q r \/\/O P\\_0 jjOH OLIGO ' 3' WO 79627 Wherein GalNAc3-4 has the structure: HO OH AcHN 0 HO OH O AcHN \/\/\/\ o O- O O OH \/\/\/\ 0 HO 0’ \ NHAc é Wherein CM is a cleavable moiety. In certain embodiments, cleavable moiety is: I NH2 O=P-OHIva/1N(/N O=||3-OH The GalNAc3 cluster portion of the conjugate group GalNAc3-4 (GalNAc3-4a) can be combined with any cleavable moiety to e a y of conjugate groups. Wherein GalNAc3-4a has the formula: WO 79627 HO OH AcHN O HO OH ’0 HO o 07K AcHN \ o o- 0’15’\ 0 O\ /_ /_ O\/\/ IIJ—O The protected ker fimctionalized solid support Compound 30 is commercially available.

Compound 84 is prepared using procedures similar to those reported in the literature (see Shchepinov er al., Nucleic Acids Research, 1997, 25(22), 4447-4454; inov er al., Nucleic Acids Research, 1999, 27, 041; and Hornet er 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 ition. The order and quantity of phosphoramidites added to the solid support can be adjusted to prepare the oligomeric compounds as bed herein having any predetermined sequence and composition.

Example 41: General method for the preparation of ASOs comprising a phosphodiester linked GalNAc3-2 (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 sis 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 odiester linked GalNAc3-2 conjugate at the 5’ terminus. A 0.1 M solution of phosphoramidite in anhydrous acetonitrile was used for B- D-2’-deoxyribonucleoside and 2’-MOE.

The ASO syntheses were performed on ABI 394 synthesizer (1-2 umol scale) or on GE Healthcare Bioscience AKTA oligopilot synthesizer (40-200 umol scale) by the phosphoramidite coupling method on VIMAD solid support (110 umol/g, Guzaev er 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 t and phosphoramidite ng 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 sulfiJrization with 0.1 M solution of ne hydride in 1:1 ne/CH3CN for a contact time of 3 s. A on of 20% tert-butylhydroperoxide in CH3CN containing 6% water was used as an oxidizing agent to provide phosphodiester intemucleoside linkages with a contact time of 12 minutes.

After the desired sequence was led, 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 ed and the a was boiled off. The residue was ed by high pressure liquid chromatography on a strong anion ge column (GE Healthcare Bioscience, Source 30Q, um, 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, )t = 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 g on the solid support. The ASOs were characterized by ion-pair-HPLC coupled MS analysis with Agilent 1100 MSD system.

Table 21 ASO comprising a phosphodiester linked GalNAc3-2 conjugate at the 5’ position targeting SRB-l Observed , , SEQ ID ISIS No. Sequence (5 to 3 ) CalCd Mass GalNAc3-2a'o'AdoTkskasAdsGdsTdsmCdsAdsTds 661134 6482.2 6481.6 26 Gds AdsmcdsTdsTkska Subscripts: a; 99 e indicates 2’-MOE modified nucleoside; "d" indicates B-D-2’- deoxyribonucleoside; "k" indicates 6’-(S)-CH3 bicyclic nucleoside (e. g. cEt); "s" indicates phosphorothioate intemucleoside linkages (PS); "0" indicates odiester internucleoside linkages (PO); and "0’" indicates -O-P(=O)(OH)-. Superscript "m" indicates 5-methylcytosines. The structure of GalNAc3-2a is shown in Example 37.

WO 79627 Example 42: General method for the preparation of ASOs comprising a GalNAc3-3 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 55 MOE gapmer, wherein the 5’ position comprises a GalNAc3-3 conjugate.

The ASO was characterized by ion-pair-HPLC coupled MS analysis with Agilent 1100 MSD system.

Table 21a ASO comprising a GalNAc3-3 conjugate at the 5’ position Via a hexylamino phosphodiester linkage targeting l ISIS , , Conjugate Calcd Observed "GalNAc33’a-omcesC}GesTesGes 661166 mmCdsAdsAdsGdsGdsCdSTdsTdsAdsGds 5’-GalNAc3-3 6 8990.51 27 GeseASAS TseT ipts: "e" indicates 2’-MOE modified nucleoside; "(1" indicates B-D-Z’-deoxyribonucleoside; "s99 indicates phosphorothioate internucleoside linkages (PS); "099 indicates phosphodiester internucleoside’ linkages (PO); and "o"’ indicates -O-P(=O)(OH)-. Superscript "m" indicates 5-methylcytosines. The structure of "5’-GalNAcg-3a" is shown in e 39.

Example 43: Dose-dependent study of phosphodiester linked 3-2 (see es 37 and 41, Bx is adenine) at the 5’ terminus targeting SRB-l in vivo ISIS 661134 (see Example 41) sing a phosphodiester linked GalNAc3-2 conjugate at the 5’ terminus was tested in a dose-dependent study for nse tion of SRB-l in mice. Unconjugated ISIS 440762 and 651900 (GalNAc3-1 conjugate at 3’ us, see Example 9) were included in the study for ison and are described previously in Table 4.

Trealmenl 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-l mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. , OR) according to standard protocols. SRB-l mRNA levels were determined ve to total RNA (using Ribogreen), prior to normalization to PBS-treated control. The results below are presented as the average percent of SRB-l mRNA levels for each treatment group, normalized to PBS-treated control and is denoted as "% PBS". The EDsos were measured using similar methods as described previously and are presented below.

As illustrated in Table 22, treatment with antisense oligonucleotides lowered SRB-l mRNA levels in a dose-dependent manner. Indeed, the antisense oligonucleotides sing the odiester linked GalNAc3-2 conjugate at the 5’ terminus (ISIS 661134) or the GalNAc3-1 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 ses the phosphodiester linked GalNAc3-2 conjugate at the 5’ terminus was equipotent compared to ISIS 651900, which comprises the GalNAc3-1 conjugate at the 3’ terminus.

Table 22 A805 containing GalNAc3-l 0r 3-2 targeting SRB-l ISIS Dosage SRB-l mRNA ED50 ate SEQ ID No.

No (mg/kg) levels (% PB S) (mg/kg) 0 100 __ 440762 . No conjugate 651900 . . 3’ GalNAc3-1 661134 . . 5’ GalNAc3-2 Structures for 3’ GalNAc3-1 and 5’ GalNAc3-2 were described previously in es 9 and 37.

Pharmacokz'netics Analysis (PK) The PK of the ASOs from the high dose group (7 mg/kg) was examined and evaluated in the same manner as illustrated in Example 20. Liver sample was minced and extracted using standard protocols. The fill length metabolites of 661134 (5’ GalNAc3-2) and ISIS 651900 (3’ GalNAc3-1) were identified and their masses were confirmed by high resolution mass ometry analysis. The s showed that the major metabolite detected for the ASO sing a phosphodiester linked GalNAc3-2 conjugate at the 5’ terminus (ISIS 661134) was ISIS 440762 (data not shown). No additional metabolites, at a able level, were observed. Unlike its counterpart, additional metabolites similar to those reported previously in Table 10a were observed for the ASO having the GalNAc3-1 conjugate at the 3’ terminus (ISIS 651900). These results suggest that having the phosphodiester linked GalNAc3-1 or GalNAc3-2 ate may improve the PK profile of ASOs without compromising their potency.

WO 79627 Example 44: Effect of PO/PS linkages on antisense inhibition of ASOs comprising 3-1 conjugate (see Example 9) at the 3’ terminus targeting SRB-l ISIS 655861 and 655862 comprising a GalNAcg-l conjugate at the 3’ us 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 55 MOE gapmers, wherein the gap region ses 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 23, below.

Table 23 Modified ASOs comprising GalNAc3-1 conjugate at the 3’ terminus targeting SRB-l Chemistry SEQ ISIS No. Sequence (5’ to 3’) ID 353382 GeSmCesTesTeSmCeSAdSGdsTdsmCdsAdSTdsGdsAds Full PS no ate 28 t) mCdsTdsTesmCesmcesTesTe GesmCeSTeSTeSmCesAdsGdsTdsmCdsAdsTdsGdsAds Full PS Wlth mCdsFl-‘dsFl-‘esn’lcesmCeSTesTeolAdowc;alN14c3'1a GalNAc3-1 conjugate GesmCmTeoTeomCmAdsGdSTdsmCdSAdSTdsGdsAds Mixed PS/PO with mCdsTdsTeon’lceon’lcesTesTeo‘Ado"(;alN‘Ac3'1a GalNAC3-1 conjUgate Subscripts: "e" indicates 2’-MOE modified side; "d" indicates B-D-2’-deoxyribonucleoside; "s" indicates phosphorothioate ucleoside linkages (PS); "0" indicates phosphodiester internucleoside linkages (PO); and "0’" indicates -O-P(=O)(OH)-. Superscript "m" indicates 5-methylcytosines. The structure of "GalNAcg-l" is shown in Example 9.

Trealmenl Six week old male Balb/c mice (Jackson Laboratory, Bar , 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 ced 72 hours following the final administration to determine the liver SRB-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. , OR) ing 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 EDsos were measured using r methods as described previously and are reported below.

As illustrated in Table 24, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner compared to PBS treated control. Indeed, the antisense oligonucleotides sing the GalNAc3-1 conjugate at the 3’ terminus (ISIS 655861 and 655862) showed substantial ement in potency comparing to the unconjugated antisense oligonucleotide (ISIS ). Further, ISIS 655862 with mixed PS/PO linkages showed an improvement in potency relative to fill PS (ISIS 655861).

Table 24 Effect of PO/PS linkages on antisense inhibition of ASOs comprising GalNAc3-1 conjugate at 3’ terminus targeting SRB-l Dosage SRB-l mRNA ED50 Chemlstry. SEQ ID No, (mg/kg) levels (% PBS) (mg/kg) Full PS without conjugate Full PS with GalNAc3-l Mixed PS/PO with GalNAc3-l conjugate Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols. Organ s were also evaluated. The results demonstrated that no elevation in transaminase levels (Table 25) or organ weights (data not shown) were observed in mice treated with ASOs compared to PBS control. Further, the ASO with mixed PS/PO linkages (ISIS 655862) showed r transaminase levels compared to filll PS (ISIS 655861).

Table 25 Effect of PO/PS linkages on transaminase levels of ASOs comprising GalNAc3-1 conjugate at 3’ terminus targeting SRB-l ISIS Dosage ALT AST Chemlstry SEQ ID No, No. (mg/kg) (U/L) (U/L) PBS 0 28.5 65 __ 3 50.25 89 353.233) . 28 p 18 27.3 97 0.5 28 55.7 1-5 30 78 Full PS with 655861 29 29 63.5 3-1 28.8 67.8 0.5 50 75.5 Mlxéiflsfglwh. . 655862 1.5 21.7 58.5 29 29.3 69 WO 79627 ____—— Example 45: Preparation of PFP Ester, Compound 110a WWW Pd/C H n 0A0 , 2 OAc OAc OOAC EtOAC, MGOH 103a; n=1 0 OWN 103b; n= 7 A00 3 —> AGO n ’ ACHN N 104a; n=1 7/0 104b; n= 7 4 GAO 0:00%ACCAcHN o OAC OAC O WNH PFPTFA OOAC ow" ACO$0 2 n —>ACO AcHN WNH DMF’ pyr AcHN N02 105a; n=1 Compound 90 0 105b; n: 7 OOACWHN A00 0 106a; n=1 106b; n= 7 OAACO\%,OACCAcHN OW o OOAC N Ra-Ni, H2 HBTU, DIEA, DMF AcO WW —> MeOH, EtOAc ACHNACOfi/On 2 ACHN 99 107a; n 1 107b; n 7 AcoivovwGAO NH ACHN NH ACOfi/On 0 OAc OAC 108a; n=1 O 108b;n=7 I Pd/C, H2, OACACHS‘A 108a; n=1 EtOAc, MeOH ACO%W 108b; n= 7 AcO ACHNfi/CWWnWNH WOACW 109a; n= 1 109b; n= 7 PFPTFA,DMF, O OAC OAc —’ O O/V\/\/HN 0 109a AcO 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 d 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% methanol/dichloromethane) to give compounds 104a and 104b in >80% yield. LCMS and proton NMR was tent with the structure.

Compounds 104a and 104b were treated to the same conditions as for compounds 100a-d (Example 47), to give nds 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. nds 106a and 106b were d 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 d 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 le 47), to give compounds 109a and 10% in >80% yields. LCMS and proton NMR was consistent with the structure.

Compound 109a was d to the same conditions as for compounds 101a-d (Example 47), to give Compound 110a in 30-60% yield. LCMS and proton NMR was tent with the structure. Alternatively, nd 110b can be prepared in a similar manner starting with nd 10%.

Example 46: General Procedure for Conjugation with PFP Esters (Oligonucleotide 111); Preparation of ISIS 666881 (GalNAc3-10) A 5’-hexylamino d 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 uL) and 3 equivalents of a selected PFP esterified GalNAc3 cluster dissolved in DMSO (50 uL) was added. If the PFP ester precipitated upon addition to the A80 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 GalNAc3 conjugated oligonucleotide.

HO OH o 839 3. 5, H AcHN574$,O\/\/\/\ O OLIGO O—Fl’-O-(CH2)6-NH2 OH OH 110a —>OH HOiVO\/\/\/\NH N 0 1. Borate buffer, DMSO, pH 8.5, rt ACHN 2. NH 3 (aq) rt , O OH OH HOfi/OO mHN O O OLIGO o/WNH Oligonucleotide 111 is conjugated with GalNAc3-10. The GalNAc3 cluster portion of the conjugate group GalNAc3-10 (GalNAc3-10a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain ments, the cleavable moiety is (OH)—Ad-P(=O)(OH)- as shown in the oligonucleotide (ISIS 666881) synthesized with GalNAc3-10 below. The structure of GalNAc3-10 (GalNAc3-10a-CM-) is shown below: O N 0 Ho "W HOOH O o o Hog/OW"O "WMWO E O 0 Ho All" Following this l procedure ISIS 666881 was prepared. ylamino modified oligonucleotide, ISIS 660254, was synthesized and purified using standard phase ucleotide procedures. ISIS 660254 (40 mg, 5.2 umol) was dissolved in 0.1 M sodium tetraborate, pH 8.5 (200 uL) and 3 equivalents PFP ester (Compound 110a) ved in DMSO (50 uL) was added. The PFP ester precipitated upon addition to the ASO solution requiring additional DMSO (600 uL) 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 olved in concentrated aqueous a with mixing at room ature 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 umol).

GalNAc3-10 conjugated oligonucleotide v v . SEQ sequence(5 m3) 5 gm" ID No.

NH2(CH2)6'oAdoGesmCeSTesTesmCesAdsGdsTds ISIS 660254 HeXYIamlne~ mCdslAdsTdsC}dslAdsn’lcdsTdsTmmcesmcesTesTe GalNAc3'10-A'o’AdoGesmCesTesTesmCesAdsGdsTds ISIS 6668 81 GalNAC3-10 mCdslAdsTdsGdsAdsmCdsTdsTfimCeSmCeSTeSTe 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 B-D-2’-deoxyribonucleoside; "s" indicates a phosphorothioate internucleoside e (PS); "099 indicates a phosphodiester internucleoside linkage (PO); and "o "’ indicates O)(OH)-. Conjugate groups are in bold.

Example 47: Preparation of Oligonucleotide 102 Comprising GalNAc3-8 AWNHBOCn BocHN/flA O 91a; n= 1 91b n—2 BocHNMNH N02 fl, PFPTFA DIPEA DMF BocHNWHN o 92a; n=1 92b,n=2 HZN/HjéANl-I OAC OAC N02 ; O TMSOTf,DCM AcO OAc —> HZNVQVHN o 933;n=1 93b,n=2 94a; m=1 94b, m=2 0 GAO OAc \/J\O/Bn OAC "O OOAC 0 m Aco AcHN 0M0"m N o ?/0 TMSOTf 7;m=1 Pd/C.H2 64m=2 —>93a(93b) $0AWN"MAN: Ra-Ni, H2 A00 —> HBTU, DIPEA, DMF N02 AcHNO Acofi/OOAcOmefi/HN O AcHN n 963; n=1, m=1 96b; n=1, m=2 96c; n=2, m=1 96d: n=2. m=2 ACOWNN O HBTU DIEA DMF Acofi/OOAcWN NH2 AcHNO o ODMTr OAC HO 0 0MWWH 0 >7 ACHN n N 0 ,, 97a; n=1, m=1 97b; n=1, m=2 97c; n=2, m=1 97d; n=2, m=2 A00%AcHN OAc 0 o N 0 OAc 0 N OOACOWN"W ODMTr H O AcO n N O ) OAc OAC 7 H N 0 OMNWHN A00 O "OH ACHN n 98a; n=1, m=1 98b; n=1, m=2 98c; n=2, m=1 98d; n=2, m=2 ACO\(\7r 0Ac O O OMNWHN AC0 0 101a n=1,m=1 AcHN n 101b m=1, m=2 0 1010 n=2, m=1 101d m=2, m=2 ACO\%OAC O AcHN o m "WM 0 F 0Ac 0Ac O H O O F F Acofi/OW"O /\H/\ MNHn " o F 0Ac O A00 OMNWHN O =1,m=1 AcHN n o 102b; n=1, m=2 1020; m=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 moles). Pentafluorophenyl trifluoroacetate (8.9 mL, 52 moles) was added dropwise, under argon, and the reaction was d 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 onate, water and brine. The c layer was then separated and dried over sodium sulfate, filtered and reduced to an oil under d 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 d 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 , 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 romethane. The organic layer was washed with sodium bicarbonate, water and brine. The c 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 20% methanol/dichloromethane) to give compounds 96a-d in 20-40% yield.

LCMS and proton NMR was tent with the structure.

Compounds 96a-d (0.75 mmoles), individually, were enated 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 ) 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 d with HBTU (0.29 g, 0.76 mmoles) and N,N— Diisopropylethylamine (0.35 mL, 2.0 mmoles) in DMF (50mL) for 15 s. To this was added nds 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 romethane. The organic layer was washed with sodium bicarbonate, water and brine. The c 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 methanol/ethyl acetate (1:1, 50 mL). At that time, the catalyst was removed by filtration thru celite, and the organics d 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 ). Pentafluorophenyl roacetate (0.034 mL, 0.2 mmoles) was added dropwise, under argon, and the on 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 c 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% methanol/dichloromethane) to give compounds 102a-d in an approximate 80% yield. LCMS and proton NMR were consistent with the structure. 3‘ 5‘ || -O-ll3-O-(CH2)5 NHz Borate , DMSO, pH 8.5, rt 102d —> 2. aq. ammonia, r1 HoOH o o HO%0 0/\(\’)JLN 4 H/W" AcHN o O H0OH 0 0 g. N O Own/W" N W"H H 4 O HoOH o HOEE:%¢/o O/d7kN O 4 HATE\H 1M Oligomeric Compound 102, comprising a GalNAc3-8 conjugate group, was prepared using the general procedures illustrated in Example 46. The 3 cluster n of the conjugate group GalNAc3- 8 (GalNAc3-8a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In a preferred embodiment, the cleavable moiety is (OH)-Ad-P(=O)(OH)-.

The structure of GalNAc3-8 (GalNAc3-8a-CM-) is shown below: HOOH O O Ho:E%4/O0 WA4"/+:\N ACHN O O HOOH O 0 OMANAHAN MWNWO 3 HO 4 H 2 H O HOOH O 0 AWN 0 Example 48: Preparation of Oligonucleotide 119 Comprising GalNAc3-7 ACO OAC ACO OAC O 0 A00 , DCE AC0 OWNHCBZ Pd(OH)2/C —> 4 —’ Nic‘) HOWNHCBZ ACHN H2 EtOAc , MeOH, 4 35b 112 HO\n/\\ HBTU, DIEA A00 OAC 0 0 DMF 3% O O NHCBZ A00 WNH HO 2 + \3‘ 1053 p AcO OAc o H 0 A00 W \C‘ A HNC ACO OAC o H 0 ACHN WNYVog'NHCBz o o AcO OAc H ACO%Q/O\X1\/NHO AcO OAc o H 0 A00 \k‘k/ A HNC AcO 0A0 Pd/C, H2, O 114 CH30H ACO ACHN OWNHY/0\%NH2 O 0 A00 0Ac p A00 OAc ACO$Q/O\M\/NHO O HBTU, DIEA, DMF A HNC O O ACO OAc AcO$Q/o\/H4\/Nl'\n/\/O\3—NHoo OBn \/© ACHN o 0 HOM0 o AcO OAc O O 0 )L) A00 0\/H4\/NH Compound 112 was synthesized following the procedure described in the literature (J. Med. Chem. 2004, 47, 5798-5808). nd 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 d 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 itative). 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 d at room ature 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 (NaZSO4), filtered and evaporated. The residue WO 79627 was purified by silica gel column tography and eluted with 10 to 20 % MeOH 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 d 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 re to yield Compound 115 (quantitative). The ure 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 e 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 s saturated NaHC03 solution and brine and dried over anhydrous NaZSO4 and filtered. The organic layer was concentrated to dryness and the residue obtained was d by silica gel column chromatography and eluted with 3 to 15 % MeOH in dichloromethane to yield Compound 116 (0.84 g, 61%). The structure was confirmed by LC MS and 1H NMR analysis.

AcO 0A0 0 OWHQ A00 l 4 AcHN 0 Pd/C, H2, AcO ON: 115 —>EtOAc, MeOH O WOH OVH‘F/NH ACHN \U/\/0%NH AcO OAc H AcO OAc ACO%Q/O\/H\/N\/£O\HO F 4 F PFPTFA, DMF, Pyr AC0 OAc — gemwwfiw AcO OAC H nd 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 methanol/ethyl acetate (1:1). The e 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 uL, 0.4 mmol) and pentafluorophenyl trifluoroacetate (72 uL, 0.42 mmol) were added. The on mixture was stirred at room ature for 12 h and poured into a aqueous saturated NaHCO3 on. The mixture was extracted with dichloromethane, washed with brine and dried over anhydrous NaZSO4. The romethane solution was concentrated to dryness and d with silica gel column chromatography and eluted with 5 to 10 % MeOH in dichloromethane to yield compound 118 (0.51 g, 79%). The structure was confirmed by LCMS and 1H and 1H and 19F NMR. 3‘ 5' -O-H—O-(CH2)6-NH2 1. Borate buffer, DMSO, pH 8.5, rt 1 18 —> 2. aq. ammonia, rt H%WM%MH0M HO 0W" 0 119 eric Compound 119, comprising a GalNAc3-7 conjugate group, was prepared using the general procedures illustrated in Example 46. The GalNAc3 cluster portion of the conjugate group GalNAc3- 7 (GalNAc3-7a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is -P(=O)(OH)-Ad-P(=O)(OH)-.

The structure of 3-7 (GalNAc3-7a-CM-) is shown below: HoOH o HO 4 "AI o L— mo O : HO 4" N "W0 i AcHN o 0 t HO 4 fl 0 Example 49: Preparation of Oligonucleotide 132 Comprising GalNAc3-5 .2; R in?» O Boc\N OH HBTU TEA L'OH H20 DMF MeOH, THF HN\ 120 122 78% 123 Compound 120 (14.01 g, 40 mmol) and HBTU (14.06 g, 37 mmol) were dissolved in ous 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 nd 121 (10 g, mol) 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 here. The reaction was monitored by TLC (ethyl acetate:hexane; 1:1; Rf = 0.47).

The solvent was removed under reduced pressure. The e was taken up in EtOAc (300 mL) and washed with 1M NaHSO4 ( 3 x 150 mL), aqueous ted NaHC03 solution (3 x 150 mL) and brine (2 x 100 mL).

Organic layer was dried with NaZSO4. 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% EtOAc 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 (EtOAc:hexane; 1:1). The reaction mixture was concentrated to half the volume under reduced pressure. The ing solution was cooled an ice bath and neutralized by adding trated HCl. The reaction mixture was diluted, extracted with EtOAc (120 mL) and washed with brine (100 mL). An emulsion formed and cleared upon ng overnight. The organic layer was separated dried (NaZSO4), filtered and evaporated to yield Compound 123 (8.42 g). Residual salt is the likely cause of excess mass. LCMS is consistent with structure. t was used without any further purification. M.W.cal:574.36; M.W.fd:575.3 [M + H]+.

(P. e O o @fi-OH - H20 HsNWJxo HZNMOH + Ho/\© O . ('3' /\© Toluene, Reflux " 124 125 126 99.6% Compound 126 was synthesized following the procedure described in the literature (J. Am. Chem.

Soc. 2011, 133, 958-963). 123 —126, BORN ONAWOOp C—>F3COOH HOBt DIEA C"'20'2 PyBop, Bop, DMF HN\ 127 CF3000 AcO OAC 3 kWO OH H3N 3W0 AcHN 7 o CFgCOO'ea —> HATU, HOAt, DIEA, DMF CF3000' @NH3 128 A00 OAc AcHN W0 AcOOAcOO:EjW/HfifiW0oi) Aco$w MO O AcO OAC Acok WNHo o AcHN o AcO OAc Aco%wOO o ACHN W Pd/C, H2, MeOH o A 0 OAcC H O} HN N AcO OAC AcokowO NH AC0 OAc ACHN o 130 AcO 0 ACHN W0 PFPTFA, DMF, Pyr AcO OAC Acog/0O:H(No NW: AcO OAC AcO OWY ACHN O 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 ved 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 t (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 on was completed after 1 h as determined by TLC eOH:AA; 89:10:1). The reaction mixture was concentrated under reduced pressure. The residue was dissolved in EtOAc (200 mL) and washed with 1 M NaHSO4 (3x100 mL), aqueous saturated NaHC03 (3x100 mL) and brine (2x100 mL). The organic phase separated dried (NaZSO4), filtered and concentrated. The residue was purified by silica gel column chromatography with a gradient of 50% hexanes/EtOAC to 100% EtOAc to yield Compound 127 (9.4 g) as a white foam. LCMS and 1H NMR were consistent with ure. Mass m/z 778.4 [M + H] L Trifluoroacetic acid (12 mL) was added to a on of nd 127 (1.57 g, 2.02 mmol) in romethane (12 mL) and stirred at room ature 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 tent 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 ous 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% MeOH/DCM). The on mixture was concentrated under reduced pressure. The residue was dissolved in DCM (30 mL) and washed with 1 M NaHSO4 (3x20 mL), aqueous saturated NaHC03 (3 x 20 mL) and brine (3x20 mL). The organic phase was separated, dried over , filtered and concentrated. The residue was purified by silica gel column chromatography using 5-15% MeOH 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 here 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 ure. 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 uL) and a stir bar. To this solution was added 0.7 M pentafluorophenyl trifiuoroacetate/DMF (100 uL) 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 (N 10 mL). The organic layer was partitioned against NaHSO4 (1 M, 10 mL) saturated NaHCO3 (10 mL) , aqueous and brine (10 mL) three times each. The organic phase separated and dried over NaZSO4, filtered and concentrated to yield Compound 131 (77.7 mg). LCMS is consistent with structure. Used t fiirther purification. Mass m/z 921.3 [M + 2H]+.

WO 79627 2014/036463 HO OH O HO 3. 5. fi 83e A HNC W0 OLIGO O—IID—O—(CH2)6'NH2 1. Borate buffer, DMSO, pH 8.5, rt 131—> 2. aq. ammonia, [1 Hog/HOOHR HOJfi/HOOH NW0" Oligomeric nd 132, comprising a GalNAc3-5 conjugate group, was prepared using the general procedures illustrated in Example 46. The GalNAc3 cluster portion of the conjugate group GalNAc3- (GalNAc3-5a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is -P(=O)(OH)-Ad-P(=O)(OH)-.

The structure of GalNAc3-5 (GalNAcg-Sa-CM-) is shown below: HO OH HO&/O\/\/\fOAcHN HO OH N Ho§¢ovflHN NH HO OH HO$WOWYNH NAMAo—u—é O H 4 e 50: Preparation of Oligonucleotide 144 Comprising GalNAc4-11 KEN!) ACN, VIMAD Resin K67 pip:DBU:DMF —> —> O O 2 A020 Capping (2:2:96) b‘UOH Kaiser: Negetive H Fmoc\NW\n/OHH L, N o HBTU, DIEA, DMF KL? NH-Fmoc 1. pip:DBU:DMF_ 2% hydrazine/DMF : Positive Kaiser: Positive —> MCHZ 2. Dde-Lys(Fmoc)-OH (138) 2 Fmoc-Lys(Fmoc)-O—H (140) HATU, DIEA,IDMF 0‘ HATU, DIEA, DMF Kaiser: Negative Kaiser. Negative O /Fmoc H H N \Fmoc HN\Fmoc ACO OAc AcHN OW>\NH A00 OAc AcO O\/\/>\ H N N 1. pip:DBU:DMF 0 H Kaiser: Posmve 2. 7, HATU, DIEA, AcO OAc K ' alser:Nega Ivet' H AcO OWN O O AcO OAC AcHN OWNH Synthesis of Compound 134. To a Merrifield flask was added aminomethyl VIMAD resin (2.5 g, 450 umol/g) that was washed with acetonitrile, dimethylformamide, dichloromethane and acetonitrile. The resin was d in acetonitrile (4 mL). Compound 133 was tivated 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 . The resin was capped by suspending in an acetic anhydride solution for ten minutes three times.

The solid support bound compound 141 was sized 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 + 2H]+.

The solid support bound compound 142 was sized using solid phase peptide synthesis methods.

AcO OAC AcO OAC ACO "WHOZI DNA izer 142—> Aim."AcAcOOAC WACHNAcO OAC HO OH ACHN00% HO OH aqueous NH3 mi HO OH OW» NH | HO OH 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 0C 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 ge column. The fractions containing full length compound 144 were pooled together and desalted. The resulting GalNAc4-11 conjugated eric compound was analyzed by LC-MS and the observed mass was consistent with structure.

The GalNAc4 cluster portion of the conjugate group GalNAc4-11 (GalNAc4-11a) can be combined with any cleavable moiety to provide a y of conjugate groups. In certain embodiments, the cleavable moiety is -P(=O)(OH)-Ad-P(=O)(OH)-.

The structure of GalNAc4-11 (GalNAc4-11a-CM) is shown below: HO OH ACHN OWNH HO OH H0$§/OW>\ H o N N ACHN OH HO OH 0 j f O O O HO OH HO&/ WNHO O Example 51: Preparation of Oligonucleotide 155 Comprising GalNAc3-6 QVOTN O B o o NH2 erOH Q/O H _ NQLOH O \n/ \/\/1 O OH 0 2M NaOH 0 OH Compound 146 was synthesized as described in the ture (Analytical Biochemistry 1995, 229, 54- 35b O o i TMS-OTf, 4 A molecular sieves, CH2C|2, rt H Q O OTNQLOHH AcO OAC H2, Pd(OH)2 /C O O 147 —>ACO VW\NH2 EtOAc/MeOH ACHN rt 105a HBTU, DIEA, DMF, AcO OAC Aco\%/o\/W\NJJ\/ \H/OVQ —>O H 0 H2, Pd(OH)2 lC, EtOAC/MeOH 148 0 A00 OAC ACO OWNJK/ 2 Compound 4 (15 g, 45.55 mmol) and compound 35b (14.3 grams, 57 mmol) were dissolved in CH2C12 (200 ml). Activated molecular sieves (4 A. 2 g, powdered) were added, and the reaction was allowed to stir for 30 minutes under en atmosphere. TMS-OTf was added (4.1 ml, 22.77 mmol) and the reaction was allowed to stir at room temp overnight. Upon completion, the reaction was ed by pouring into solution of saturated aqueous NaHC03 (500 ml) and crushed ice (N 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 tography and eluted with 2-10 % MeOH in CH2C12 to yield Compound 112 (16.53 g, 63 %). LCMS and 1H NMR were consistent with the ed compound.

Compound 112 (4.27 g, 7.35 mmol) was dissolved in 1:1 MeOH/EtOAc (40 ml). The reaction mixture was purged by bubbling a stream of argon through the solution for 15 minutes. Pearlman’s catalyst dium hydroxide on carbon, 400 mg) was added, and hydrogen gas was bubbled through the solution for minutes. Upon completion (TLC 10% MeOH in CH2C12, and LCMS), the catalyst was removed by filtration through a pad of . 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 ous 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 N 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 EtOAc and washed with saturated aqueous NaHC03 and brine. The organics phase was separated, dried ), filtered, and concentrated to an orange syrup. The crude material was d by column chromatography 2-5 % MeOH 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 tOAc (75 ml). 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 minutes. Upon completion (TLC 10% MeOH in DCM, and LCMS), the catalyst was removed by filtration through a pad of . The e was concentrated by rotary evaporation, and was dried briefly under high vacuum to yield Compound 149 (2.6 g). LCMS was consistent with desired t. The residue was dissolved in dry DMF (10 ml) was used immediately in the next step.

ACO OAC o o AC0 OWN/lK/H 0 A00 OAC AcHN 3 H O i O WNJK/ \‘OKNH N 0 ~20 H AcHN 3 H 146 —> AC0 OAc 0 HBTU,D|EA,DMF NH o OWN/[K/ ACO OAC o o AC0 OAC Pd(OH)2/C, H2 AcHN 3 ’ o I? O N EMNH Compound 146 (0.68 g, 1.73 mmol) was dissolved in dry DMF (20 ml). To this DIEA (450 uL, 2.6 mmol, 1.5 eq.) and HBTU (1.96 g, 0.5.2 mmol) were added. The on 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 on of DIEA (if necessary). The reaction was d to stir at room temperature under nitrogen for 2 h. Upon completion the reaction was diluted with EtOAc (100 mL), and washed with aqueous ted s NaHC03, followed by brine. The organic phase was separated, dried over MgSO4, filtered, and concentrated. The e was purified by silica gel column chromatography and eluted with 2-10 % MeOH in CH2C12 to yield Compound 150 (0.62 g, %). LCMS and 1H NMR were consistent with the desired product.

Compound 150 (0.62 g) was dissolved in 1:1 MeOH/ EtOAc (5 L). The reaction mixture was purged by bubbling a stream of argon through the solution for 15 minutes. Pearlman’s catalyst dium hydroxide on carbon) was added (60 mg). Hydrogen gas was bubbled through the solution for 30 minutes. Upon completion (TLC 10% MeOH in DCM, and LCMS), the catalyst was removed by filtration (syringe-tip Teflon filter, 0.45 um). 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.

ACO OAC o 0 mo OWNJK/H A00 OAC o 0 AcHN 3 H O O W o H BnO OH AcO OWNJK/Nf,"K:M3 M OBn 151 —> 3 H O AcHN O PFP—TFA, DIEA, DMF Aco OAc O \/\(/)’\N A00 3 H ACO OAC o 0 A00 OVW k/HN O AcO OAC AcHN 3 H O O Pd(OH)/C,H o H 4» ka/Nf'"o M 3 H Lfo OH MeOH, EtOAc AcHN 3 H O AcO OAC o ACO OAC O O 0 H A00 /U\/N A00 0A0 AcHN W" o o F PFP TFA, DIEA- M A o $ 2 OWNJK/NfN H O —>C 3 DMF AcHN 3 H O Lro F AcO OAC 0 0 GM )\/ AcO N nd 83a (0.11 g, 0.33 mmol) was dissolved in anhydrous DMF (5 mL) and N,N- Diisopropylethylamine (75 uL, 1 mmol) and A (90 uL, 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 on of N,N-Diisopropylethylamine (if necessary). The reaction mixture was stirred under nitrogen for N min. Upon completion, the majority of the solvent was removed under reduced pressure. The residue was diluted with CH2C12 and washed with aqueous saturated NaHC03, followed by brine. The organic phase ted, dried over MgSO4, filtered, and concentrated to an orange syrup. The residue was purified by silica gel column chromatography (2-10 % MeOH in CH2C12) to yield Compound 152 (0.35 g, 55 %). LCMS and 1H NMR were consistent with the desired product. nd 152 (0.35 g, 0.182 mmol) was dissolved in 1:1 MeOH/EtOAc (10 mL). The on mixture was purged by ng 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% MeOH in DCM, and LCMS), the catalyst was removed by filtration ge-tip Teflon filter, 0.45 um). The filtrate was trated 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 uL, 0.37 mmol) and PFP-TFA (35 uL, 0.28 mmol) were added. The on mixture was stirred under nitrogen for N 30 min. The reaction mixture turned magenta upon contact, and gradually turned orange. The pH of the reaction mixture was ined 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 e was diluted with CH2C12 (50 mL), and washed with saturated aqueous NaHC03, followed by brine. The organic layer was dried over MgSO4, filtered, and concentrated to an orange syrup. The residue was purified by column chromatography and eluted with 2-10 % MeOH in CH2C12 to yield Compound 154 (0.29 g, 79 %). LCMS and 1H NMR were consistent with the desired product. 3' 5' II HOOH O OLIGO O-P-O- CH< 2).; NH O OH HokowNflH ACHN HN 1. Borate buffer, DMSO, HOOH O 154 ' pH85 rt 0 JV" H H Ho 0mm VN "MNWO 2. aq. ammonia, rt 0 4 5 ACHN O O HOOH LN o HO OAWHN O Oligomeric Compound 155, comprising a GalNAc3-6 conjugate group, was prepared using the general procedures illustrated in e 46. The GalNAc3 cluster portion of the conjugate group GalNAc3- 6 (GalNAc3-6a) can be ed with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is -P(=O)(OH)-Ad-P(=O)(OH)-.

The structure of GalNAc3-6 (GalNAc3-6a-CM-) is shown below: HO O Example 52: Preparation of Oligonucleotide 160 Comprising GalNAc3-9 AcO0Ac AcOOAcO TMSOTf, 50 C0 41/1130 ACHN CICHZCHZCI TMSOTf DOE 66% rt 93% 3 4\1 AcO OAC AcO OAc "3%WV© —’ Ai$wW0"0H2, OH, 95 A) ACHN AcHN 156 157 HBTU, DMF, EtN(iPr)2 ’ A00OAC Phosphitylation DMTO 81% ACHN ODMT H5 47 NC \N(iPr)2 ACOWWI"AcOOAc ACHN 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 ol (200 mL). Palladium on carbon (6.15 g, 10 wt%, loading (dry basis), matrix carbon powder, wet) was added. The reaction mixture was d at room temperature under hydrogen for 18 h. The reaction e was filtered through a pad of celite and the celite pad was washed thoroughly with methanol. The combined filtrate was washed and concentrated to s. 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 d at room ature for 8 h. The reaction mixture was poured into a saturated NaHC03 aqueous solution. The mixture was extracted with ethyl acetate and the organic layer was washed with brine and dried (NaZSO4), filtered and evaporated. The residue ed 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 re. The dried compound was ved 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,N’,N’-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 NaHC03 and brine. The organic phase was separated, dried (NaZSO4), filtered and evaporated. The residue was purified by silica gel column tography and eluted with 50-90 % ethyl e in hexane to yield Compound 159 (7.82 g, 80.5%). The structure was confirmed by LCMS and 31P NMR is.

HOOH ‘ 0 ow"? HO 9 O O ACHN I O=F|’-OH H00" ' 1.DNAsynthesizer 159 0 MN H0 9 2. aq. NH4OH o o AcHN I O=F|’—OH HowOWN/{OHoOH CM OLIGO Oligomeric Compound 160, sing a GalNAc3-9 conjugate group, was prepared using standard oligonucleotide sis 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 GalNAc3 cluster portion of the ate group GalNAc3-9 (GalNA03- 9a) can be combined With any cleavable moiety to e a variety of conjugate groups. In certain embodiments, the cleavable moiety is -P(=O)(OH)-Ad-P(=O)(OH)-. The structure of GalNAc3-9 (GalNAcg- 9a-CM) is shown below: HoOH ‘ 0 ow"? HO 9 o o AcHN I OZT-OH HO o/dfi"firN9 o o ACHN o-$ OH o N HO 0% o 2 Example 53: Alternate procedure for preparation of Compound 18 (GalNAcg-la and GalNAc3-3a) /\/\ H R = H or Cbz 0Ac 0 GAO 161 = 0 CszI, Et3N 1:, 223526212" A00 4 NYC OAc PFPO7I/\\ 0Ac O O AcO o\/\/\n/N\/\/NHR + NHAc PFPOWOgVNHCBZ ' ) o 0 O O R = Cbz' 163a Pd/C. H2 E PFPOM R=H,163b ACO 0% H 0Ac HNWNh 0Ac o o O IL_H H o NHCBZ A00 0 N\/\/NW 0Ac HN/\/\ O N A60 OM14 Lactone 161 was reacted with diamino propane (3-5 eq) 0r Mono-B00 ted diamino propane (1 eq) to provide alcohol 162a 0r 162b. When unprotected ediamine 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 Csz1 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 ed 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 nd 18. The above method greatly facilitates purification of intermediates and zes the formation of byproducts which are formed using the procedure described in Example 4.

Example 54: Alternate procedure for preparation of nd 18 (GalNAcg-la and GalNAc3-3a) HOZC/\\ PFPTFA PFPO 0 DMF,pyr O /\/O NHCBZ O NHCBZ H020 \n/V o O 0 H020\) PFPOM 113 H 164 BocHN\/\/Ngi/\\O 1 HCI or TFA —’ NHCBZ —> BOCHNW \‘fo/0%N DIPEA BOCHN H ACOQHO OPFF 165 NHAC a: 166 O 1. 1 6-hexanediol ACO Dix/9k H ' 4 HN N or1,5-pentane-d|o| NHAC W h TMSOTf + compound 4 OAc 2. TEMPO OAc O O O 3. PFPTFA, pyr O H H NHCBZ AC0 orE jJJ—4NWNWO O o O The triPFP ester 164 was prepared from acid 113 using the procedure outlined in example 53 above and reacted with mono-Boo protected diamine to provide 165 in essentially quantitative yield. The Boc groups were removed with hydrochloric acid or roacetic 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-NAG acid 166 was prepared from the ponding 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 l by oxidation using TEMPO (0.2 eq) and BAIB in acetonitrile and water. The precursor alcohol was ed 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 sing either a 3' or 5'-conjugate group (comparison of GalNAc3-1, 3, 8 and 9) targeting SRB-l in vivo The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-l in mice. Unconjugated ISIS 353382 was included as a standard. Each of the various GalNA03 conjugate groups was attached at either the 3' or 5' us of the respective oligonucleotide by a phosphodiester linked 2'-deoxyadenosine side (cleavable moiety).

Table 26 Modified ASO targeting SRB-l ASO Sequence (5’ to 3’) Motif Conjugate ID No.

ISIS 353382 GCSmCGSTGSTGSmCCSACSGCSTCSmCCSACSTCSGCSACS / 10/5 "one 28 (parent) mccsTcsTesmcesmcesTesT.

Gesm CSTGSTGSmCCSACSGCSTCSmCCSACSTCSGCSACS ISIS 655861 5/ 10/5 3-1 29 mCCSTcsTeSmCesmCesTeSTe d0’-GalNAC3- a GGSmCCSTCSTGSmCGSACSGCSTCSmCCSACSTCSGCSACS ISIS 664078 5/ 1 0/5 GalNAc3-9 29 sTesmCesmCesTesTeoAdo"GalNAc3' a 3-3a'o’Ado ISIS 661 161 GesmcesTesTesmCCSAcSGCSTCSmCCSACSTCSGCSACS 5/ 1 0/5 GalNAc3-3 30 IncCSTCSTGSmCGSmCCSTGSTe GalNAC30’Ad0 ISIS 665001 GesmCesTesTesmCesAcSGCSTCSmCCSACSTCSGCSACS 5/ 10/5 GalNAc3-8 30 IncCSTCSTGSmCGSmCCSTGSTe Capital s indicate the base for each nucleoside and mC indicates a 5-methyl cytosine.

Subscripts: "e" indicates a 2’-MOE modified nucleoside; "(1" indicates a B-D-2’-deoxyribonucleoside; "s" indicates a orothioate internucleoside linkage (PS); "0" indicates a phosphodiester intemucleoside linkage (PO); and "0’" indicates -O-P(=O)(OH)-. Conjugate groups are in bold.

The structure of GalNA03-1a was shown previously in Example 9. The structure of GalNAcg-9 was shown previously in Example 52. The structure of GalNA03-3 was shown usly in Example 39. The structure of GalNAcg-8 was shown previously in Example 47.

Trealmenl 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 ent group consisted of 4 animals. The mice were sacrificed 72 hours ing the final administration to determine the liver SRB-l mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols. The results below are presented as the average percent of SRB-l mRNA levels for each treatment group, normalized to the saline control.

As illustrated in Table 27, treatment with antisense oligonucleotides lowered SRB-l mRNA levels in a ependent manner. Indeed, the antisense oligonucleotides comprising the phosphodiester linked GalNAc3-1 and GalNAc3-9 ates at the 3’ terminus (ISIS 655861 and ISIS 664078) and the GalNAcg-3 and GalNAc3-8 conjugates linked at the 5’ terminus (ISIS 661161 and ISIS 665001) showed substantial improvement in potency compared to the ugated antisense oligonucleotide (ISIS 353382). rmore, ISIS , comprising a GalNAc3-9 conjugate at the 3' terminus was essentially equipotent compared to ISIS 655861, which comprises a GalNAc3-1 conjugate at the 3’ terminus. The 5' conjugated antisense oligonucleotides, ISIS 661161 and ISIS 665001, comprising a 3-3 or 3-9, respectively, had increased potency compared to the 3' conjugated antisense oligonucleotides (ISIS 655861 and ISIS ).

Table 27 A805 containing GalNAc3-1, 3, 8 0r 9 targeting SRB-l SRB-l mRNA ISIS N0. Conjugate % Saline Saline n/a 353382 655861 GalNac3 —1 (3') 664078 GalNac3-9 (3') 661 161 GalNac3-3 (5') 665001 GalNac3-8 (5') 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 the table below.

Table 28 Dosage Total ISIS No. ALT AST BUN ConJugate. m/k Bilirubin Saline 24 59 0.1 37.52 3 21 66 0.2 34.65 353382 10 22 54 0.2 34.2 none 22 49 0.2 33.72 0.5 25 62 0.2 30.65 1.5 23 48 0.2 30.97 655861 3-1 (3), 28 49 01 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 ), 20 43 01 30.62 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), g 23 99 0.1 34.97 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 ), 24 42 01 31.85 32 67 0.1 31.

Example 56: Dose-dependent study of oligonucleotides comprising either a 3' or 5'-conjugate group (comparison of GalNAc3-1, 2, 3, 5, 6, 7 and 10) ing SRB-l in vivo The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-l in mice. Unconjugated ISIS 353382 was ed as a standard. Each of the various GalNA03 conjugate groups was attached at the 5' terminus of the respective oligonucleotide by a phosphodiester linked xyadenosine nucleoside (cleavable moiety) except for ISIS 655 861 which had the GalNA03 conjugate group attached at the 3’ terminus.

Table 29 Modified ASO targeting SRB-l ASO Sequence (5’ to 3’) Motif. Conjugate ID No.

ISIS 353382 GesmcesTesTesmCesAdsGdsTdsmCdsAdsTdsGdsAds /10/5 ’ no conjugate 28 (parent) mCdsTdsTesmCesmCesTesTe GesmcesTesTesmCesAdsGdsTdsmCdsAdsTdsGdsAds ISIS 655861 5/10/5 3-1 29 mCdsTdsTesmCesmcesTesTeoAdo"GalNAc3'1a GalNAc3'2a'o’AdoGesmCesTfiTesmCesAdsGdsTds ISIS 664507 5/10/5 GalNAc3-2 30 mCdsAdsTdsGdsAdsmcdsTdsTesmCesmCesTesTe GalNAC3-3a-0’Ad0 ISIS 661161 GesmCesTesTesmCesAdsGdsTdSmCdSAdSTdSGdsAds 5/ 10/5 GalNAc3-3 30 mCdsTdsTesmCesmcesTesTe ISIS 666224 GalNAc3-Sa-o,AdoGeSmCesTmTesmCesAdsGdsTds 5/10/5 GalNAc3-5 30 —"‘CasAdsTasGasAdsmCdsTdsTesmCesmCesTesTe _—_ GalNAc3'6a'0’AdoGesmCesTfiTesmCesAdsGdsTds ISIS 666961 5/ 10/5 GalNAc3-6 mCdsAdsTdsGdsAdsmcdsTdsTesmCesmCesTesTe 3'7a'0’AdoGesmCesTfiTesmCesAdsGdsTds ISIS 666981 5/ 10/5 GalNAc3-7 mCdsAdsTdsGdsAdsmcdsTdsTesmCesmCesTesTe GalNAc3'10-A'o’AdoGesmCesTesTesmCesAdsGdsTds ISIS 666881 5/ 10/5 GalNAc3-10 mCdsAdsTdsGdsAdsmcdsTdsTesmCesmCesTesTe Capital letters indicate the nucleobase for each nucleoside and mC tes a 5-methyl ne.

Subscripts: "e" indicates a 2’-MOE modified nucleoside; "d" indicates a B-D-2’-deoxyribonucleoside; a;s" indicates a phosphorothioate internucleoside linkage (PS); "099 indicates a phosphodiester intemucleoside e (PO); and "o "’ indicates -O-P(=O)(OH)-. Conjugate groups are in bold.

The structure of GalNAc3-1a was shown previously in Example 9. The structure of GalNAc3-2a was shown previously in Example 37. The structure of GalNAc3-3a was shown previously in Example 39. The structure of GalNAc3-5a was shown previously in e 49. The structure of GalNAc3-6a was shown previously in Example 51. The ure of GalNAc3-7a was shown previously in e 48. The structure of GalNAc3-10a was shown previously in Example 46.

Trealmenl 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, , 666224, 666961, 666981, 666881 or with saline. Each treatment group consisted of 4 s. The mice were sacrificed 72 hours following the final administration to determine the liver SRB-l mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. , OR) according to rd protocols. The results below are presented as the average percent of SRB-l mRNA levels for each treatment group, normalized to the saline control.

As illustrated in Table 30, treatment with antisense oligonucleotides lowered SRB-l 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 se in potency compared to the 3' ated antisense oligonucleotide.

Table 30 Dosage SRB-l mRNA . 353382 none 655861 GalNac3-1(3') WO 79627 1.5 81.2 33.9 15.2 0.5 102.0 1.5 73.2 664507 —5 GalNac3—2(5), 10.8 0.5 90.7 1.5 67.6 661161 —5 GalNac3-3(5), 11.5 0.5 96.1 1.5 61.6 666224 GalNac3-5 (5). 256 11.7 0.5 85.5 1.5 56.3 666961 —5 GalNAc3-6(5), 13.1 0.5 84.7 1.5 59.9 666981 —5 GalNAc3-7(5), 8.5 0.5 100.0 1.5 65.8 666881 —5 GalNAc3-10(5), 13.0 Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline ed 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 31 below.

Table 31 Total ISIS N0. AST BUN ConJugate.

Bilirubin 26 57 0-2 27 92 0.2 27 353382 655861 664507 661161 ' ' GalNac3-3 (5') 666224 ' ' GalNac3-5 (5) 666961 ' ' GalNAc3-6 (5') 666981 ' ' 3-7 (5') 666881 ' ' GalNAc3-10 (5 ') 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 cerides (Plasma TG) levels. The study was performed using 3 transgenic mice that s human APOC-III in each group.

Table 32 Modified ASO targeting ApoC III ASO Sequence (5’ to 3’) ISIS AesGesmCeSTesTesmCdsTdsTdsGdsTds PS 20 304801 mCdsmcdslAdsC}dsmcdsTesTesTesAesTe ISIS mCeSTeSTeSmCdsTdsTdsGdsTdsmCdsmCds PS 21 6475 35 AdSGdsmCdsTesTesTesAesTeoAdo,-GalNAc3-1 a ISIS AesGeomCeoTeoTeomCdsTdsTdsGdsTdsmCdsmCds PO/PS 21 6475 36 AdsGdsmCdsTeoTeoTfiAesTeoAdoy-GalNAc3-1 a l letters indicate the nucleobase for each nucleoside and mC indicates a 5-methyl cytosine.

Subscripts: "6" indicates a 2’-MOE modified nucleoside; "(1" indicates a B-D-2’-deoxyribonucleoside; "5" indicates a phosphorothioate internucleoside linkage (PS); "0" indicates a phosphodiester intemucleoside e (PO); and "0’" indicates -O-P(=O)(OH)-. Conjugate groups are in bold.

The structure of GalNA03-1a was shown previously in Example 9.

Table 33 ApoC III mRNA (% Saline on Day 1) and Plasma TG Levels (% Saline on Day 1) ASO Dose Saline 0 mg/kg ISIS 304801 30 mg/kg ISIS 647535 10 mg/kg ApoC-III ISIS 647536 10 mg/kg ApoC-III Saline 0 mg/kg Plasma TG ISIS 304801 30 mg/kg Plasma TG ISIS 647535 10 m_ . Plasma TG ISIS 647536 10 mg/kg Plasma TG As can be seen in the table above the duration of action increased with addition of the 3'-c0njugate group compared to the unconjugated oligonucleotide. There was a r 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: ependent study of oligonucleotides comprising a 3'-conjugate group (comparison of GalNAc3-l and 4-11) ing SRB-l in vivo The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-l 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'-de0xyadenosine nucleoside cleavable moiety.

The structure of GalNAc3-1a was shown previously in Example 9. The structure of GalNAc3-11a was shown previously in Example 50.

Trealmenl 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 s. The mice were sacrificed 72 hours following the final administration to determine the liver SRB-l mRNA levels using real-time PCR and RIBOGREEN® RNA quantification t (Molecular Probes, Inc. Eugene, OR) according to standard protocols. The results below are presented as the average t of SRB-l mRNA levels for each treatment group, normalized to the saline control.

As illustrated in Table 34, ent with antisense oligonucleotides lowered SRB-l mRNA levels in a dose-dependent manner. The antisense ucleotides comprising the phosphodiester linked 3-1 and GalNAc4-11 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 ucleotides, GalNAc3-1 and GalNAc4-11, were equipotent.

Table 34 Modified ASO ing SRB-l 0 . ce (5’ t0 3’) Dose mg/kg 331183236 11 de and Is1s44o762 8 s 22 mc TST inc TksmcksAdsGdsTdsmcdsAdsTdsGdsAds_— 1518651900 23 33651111336040.Gamma-1.

TksmcksAdsGdsTdsmCdsAdsTdsGdsAds_— ISIS 663748 23 mCdsTdsTksmckoAdo.—Ga1NAc4-11.

Capital letters indicate the nucleobase for each nucleoside and mC indicates a 5-methyl cytosine.

Subscripts: "e" indicates a 2’-MOE modified nucleoside; "k" tes 6’-(S)-CH3 bicyclic nucleoside; "d" indicates a B-D-2’-deoxyribonucleoside; "s" indicates a phosphorothioate internucleoside linkage (PS); "0" indicates a phosphodiester internucleoside linkage (PO); and "0’" indicates -O-P(=O)(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 rd 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 35 below.

Table 35 Total ST .

. . . BUN Saline —_76 0-2 40 0.60 —70 0.1 35 440762 2 57 0.1 35 none 6 48 0.1 39 0.2 0.2 39 0.6 61 0.1 35 651900 GalNac3-1 (3') 56 0 2 36 663748 . 60 0.1 35 GalNac4-11 (3') 62 0.1 36 6 38 71 0.1 33 Example 59: Effects of GalNAc3-1 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 36 Modified ASOs targeting FXI ISIS TesGesGesTesAesAdsTdsmCdsmCdsAdsmCds PS 3 1 404071 TdsTdsTdsmCdsAesGesAesGesGe ISIS TesGesGesTesAesAdsTdsmCdsmCdsAdsmCds PS 32 656172 TdsTdSTdsmCdsAesGesAesGesGeoAdO, -GalNAc3-1 a ISIS TesGeoGeoTeeroAdsTdsmCdsmCdsAdsmCds 656173 TdsTdSTdsmCdsAeoGeersGesGeoAdo,-GalNAc3-1 a Capital letters indicate the nucleobase for each nucleoside and mC indicates a 5-methyl cytosine.

Subscripts: "6" indicates a 2’-MOE modified nucleoside; "d" indicates a B-D-Z’-deoxyribonucleoside; "5" indicates a phosphorothioate intemucleoside linkage (PS); "0" indicates a phosphodiester cleoside linkage (PO); and "0’" tes -O-P(=O)(OH)-. ate groups are in bold.

The structure of GalNAcg-la was shown previously in Example 9.

Trealmenl 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 , 656172, 656173 or with PBS d l. Each treatment group consisted of 4 animals. The mice were ced 72 hours following the final administration to determine the liver FXI mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, OR) according to rd protocols. Plasma FXI protein levels were also ed using ELISA. FXI mRNA levels were determined relative to total RNA (using RIBOGREEN®), prior to normalization to PB S-treated control. The results below are presented as the average t of FXI mRNA levels for each treatment group. The data was normalized to PBS-treated control and is denoted as "% PB S". The ED50s were measured using similar methods as described usly and are presented below.

Table 37 Factor XI mRNA (% Saline) ASO % Control Conjugate Linkages trig/kg Saline 404071 GalNAc3-1 656172 GalNAc3-1 656173 As illustrated in Table 37, treatment with antisense oligonucleotides lowered FXI mRNA levels in a dose-dependent manner. The oligonucleotides comprising a 3'-GalNAc3-1 ate group showed substantial improvement in potency compared to the unconjugated antisense oligonucleotide (ISIS 404071).

Between the two conjugated oligonucleotides an ement in potency was further provided by substituting some of the PS linkages with P0 (ISIS 656173).

As illustrated in Table 37a, treatment with antisense oligonucleotides lowered FXI protein levels in a dose-dependent manner. The oligonucleotides comprising a 3'-GalNAc3-1 conjugate group showed substantial improvement in potency compared to the unconjugated nse oligonucleotide (ISIS 404071).

Between the two conjugated oligonucleotides an improvement in potency was further provided by substituting some of the PS linkages with P0 (ISIS 656173).

Table 37a Factor XI protein (% Saline) Dose Protein (A)0 Conjugate Linkages trig/kg l) 404071 GalNAc3-1 Liver transaminase levels, alanine ransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using rd protocols. Total bilirubin, total n, 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 38 Dosage Total Total ISIS No. CRE BUN ConJugate. mg/kg Albumin Bilirubin Saline 0.2 22.9 _152 8 1760 404071 none 0.2 23.0 656172 2 330 518 29 02 22.0 3-1(3') 656173 GalNaCs-l (3') e 60: Effects of conjugated ASOs targeting SRB-l in vitro The oligonucleotides listed below were tested in a multiple dose study for antisense inhibition of SRB-l 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 tive oligonucleotide by a phosphodiester linked 2'-deoxyaden0sine nucleoside cleavable .

Table 39 Modified ASO targeting SRB-l ASO Sequence (5’ to 3’) Motif Conjugate ID No.

ISIS 353382 SES:%§::E§:EECS:T‘1:E}T‘ETdSmCdSAdSTdSGdSAdS 5/10/5 none 28 ISIS 655861 mgCATdfidsoTfidmCég‘iskgdiAds 5/10/5 GalNAc3-l 29 ISIS 655862 SC;ggcmfcfidfididzmcégfiffhs 5/10/5 GalNAc3-1 29 ISIS 661161 gillgAXdidsadAimmcchiTT:51?ng 5/10/5 GalNAc3-3 30 ISIS 665001 $2L§A§d§fdsadA€mtncchiTT$1113ng 5/10/5 GalNAc3-8 30 ISIS 664078 EC;igcmgcA;%&kdmC&$%1§cf§Ae 5/10/5 GalNAc3-9 29 ISIS 666961 gjiiAfdidsG‘jdAfmnéfoggm??? 5/10/5 GalNAc3-6 30 ISIS 664507 SgilifiaédsgmgfliTTigmgédsgdflds 5/10/5 GalNAc3-2 30 ISIS 666881 Sgilifi3dgdsA§§CiT1CTECTmZCTAdTSGdsTd 5/10/5 GalNAc3-10 30 ISIS 666224 SgilifiazdsgmggigiggédsgdsTds 5/10/5 GalNAc3-5 30 ISIS 666981 Sggfidédsgmgdflifggmgédsgdflds 5/10/5 GalNAc3-7 30 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 B-D-2’-deoxyribonucleoside; "s" indicates a phosphorothioate intemucleoside linkage (PS); "0" indicates a phosphodiester cleoside linkage (PO); and "0’" indicates -O-P(=O)(OH)-. Conjugate groups are in bold.

The structure of GalNAc3-1a was shown previously in e 9. The structure of GalNAc3-3a was shown previously in Example 39. The structure of GalNAc3-8a was shown previously in Example 47. The structure of GalNAc3-9a was shown previously in Example 52. The structure of 3-6a was shown previously in Example 51. The structure of GalNAc3-2a was shown previously in e 37. The structure of GalNAc3-10a was shown previously in Example 46. The ure of GalNAc3-5a was shown previously in Example 49. The ure of GalNAc3-7a was shown previously in Example 48.

Trealmenl The oligonucleotides listed above were tested in vitro in y 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 ed according to total RNA content, as measured by RIBOGREEN®.

The IC50 was calculated using standard methods and the results are presented in Table 40. The s show that, under free uptake conditions in which no reagents or electroporation ques 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 40 Internucleoside SEQ ID ASO IC50 (nM) Conjugate linkages N0. none 28 ISIS 655861 11a PS GalNAc3-l 29 GalNAcs'l 29 GalNAc-3 30 GalNAc3-8 30 ISIS 664078 55 PS GalNAc3-9 29 ISIS 666961 22a PS GalNAc3-6 30 ISIS 664507 30 ISIS 666881 30 ISIS 666224 30 ISIS 666981 40 PS GalNAc3-7 30 21Average of multiple runs.

Example 61: Preparation of oligomeric nd 175 comprising GalNAc3-12 0 GAO \/\NH2 prOJKA/VO 0 o A Oc 0A0 91 a OAc HN\ \HMH O 0 166 HN DOM HN\AC HBTU DIEA DMF A00 OAc WC0 OAc HN HN o H \AC Q/OTN HLNW 0 AOC N o 0Ac L /\/\ W0 0 O N N OAc o HN H H HN AcO 17o HN Ac AcO OAc WC0 OAc Pd(OH)2/C,H2 HN o H HN\AC MeOH/EtOAc —> HLNJV O A0C H2N N O OAc L /\/\ W0 0 N N OAc o HN H H HN AcO 171 HN\ benzyl (perfluorophenyl) glutarate AcO 0Ac 3W00 GAO HN HN\ O A N\/\/H Q/OMN O ACO N O OAc A o o o HMHWO OAc WO 79627 PCT/USZOl4/036463 AcO OAC W0O OAC HN HN\ O Ac N\/\/H Pd(OH)2 / C , H2 MeOH / EtOAc HO ’P-TFA iA DMF 3' 5' || OLIGO O—T—O—(CH2)6—NH2 1. Borate buffer, DMSO, pH 8.5, rt 2. aq. ammonia, rt HO O ACHN \/\/\/U\ fiNH 175 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)oxopentanoic acid in DMF. Oligomeric compound 175, sing a GalNAc3-12 conjugate group, was prepared from compound 174 using the l procedures illustrated in e 46. The GalNAc3 cluster n of the conjugate group GalNAc3-12 (GalNAc3-12a) can be ed with any cleavable moiety to provide a variety of conjugate groups. In a certain embodiments, the cleavable moiety is -P(=O)(OH)-Ad-P(=O)(OH)-. The structure of GalNAc3-12 (GalNAc3-12a-CM-) is shown below: OH OH "0570»o o AcHN WNH OHDH in "0&0 o >0Iz i: 8: Izéz ZIA12 ’5:ZI(A;O ig Example 62: Preparation of eric compound 180 comprising GalNAc3-13 WO 79627 AcHN OWLOH OHWYO HATU, HOAt DIEA, DMF 0A0 OAc AcO 0M O0Ac H2, Pd/C AcO OWN —> AcHN (DH/MO 0Ac 0Ac O 177 AC0 OW AcHN 0 OAc 0Ac AcO O\/\/\/U\ "3&00AcOWN PFPTFA TEA AcHN ()wa 0Ac OAc 178 AcO 0W AcHN o WO 79627 OAC OAc ACHN NH OAc 0Ac AcO OW\/U\ GAO OAc AcO OW AcHN Mo 3' 5' || -o-F|’-o-(CH2>e-NH2 1. Borate buffer, DMSO, pH 8.5, rt 2. aq. ammonia, rt OH OH HO OM OH OH HO OW ACHN W0 Compound 176 was prepared using the general procedure shown in Example 2. Oligomeric compound 180, comprising a GalNAc3-13 conjugate group, was prepared from compound 177 using the l procedures illustrated in Example 49. The GalNAc3 cluster portion of the conjugate group GalNAc3-13 (GalNAc3-13a) can be ed with any cleavable moiety to provide a variety of conjugate groups. In a WO 79627 certainembodiments, the cleavable moiety is -P(=O)(OH)-Ad-P(=O)(OH)-. The structure of GalNAc3-13 (GalNAc3-13a-CM-) is shown below: HO O WJL 0 NH HO 0 H O O 0\WL N NWN\92 § N a AcHN H O H O OH M—NH Example 63: Preparation of oligomeric compound 188 comprising GalNAc3-14 N NHCBz \ How00?;NHCB 181 2 HO NWo O O 1400):; HBTU,D|EA o WWW Acofl/ONBNWH NHAC O AC NHAcO o OAc O WNW AGO H Pd/C H N o NHCBz ’ 2 NH2 —* 0W \n/\/O A00 6 0 O 0 NHAc O A00 p ACO&/04/)N6H ACO‘21 "fr: ,ON HO o\/© 1. Pd/C H2 NHACW" M 0A0 2. PFP.TFA AcO Dyr 0 O NH 185 0N —> AcO HBTU, NHggc DIEA, AcO DMF o AcO 6H>X§2 ACO H F NHAC 0 O W Ace H CNN 0 N o F A00 6 \H/\/ F NHAc 0 0 A00 p AcO OWN6H 3' 5' Hog; NH -'O'||:ILO'(CH2)6_NH2 HOH8H NHA:D 6 p0 MH 187 1. Borate buffeOri-IDMSO, pH 8.5, rt /ZN’6Ng/:O O N "fl 2. aq. ammonia, rt HO p Ho&/0 6" 188 Compounds 181 and 185 are commercially available. Oligomeric compound 188, comprising a GalNAc3-14 conjugate group, was prepared from compound 187 using the general procedures illustrated in Example 46.

The GalNAc3 cluster n of the conjugate group GalNAc3-14 c3-14a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is -P(=O)(OH)-Ad-P(=O)(OH)-. The structure of GalNAc3-14 (GalNAc3-14a-CM-) is shown below: HOOH O 0 o N H0 10 HJI AcHN o HOOH O O O O O NJJ\/—O JJ\/\/U\ E H0 10 H M "WC 2 ACHN O 0 b o N O H0 10 H e 64: Preparation of oligomeric compound 197 comprising GalNAc3-15 WO 79627 AcO 0A0 OTBS OTBS o (5 @N ACHN N A00 0 HBTU, DIEA AcHN 7 N NH3/MeOH 0TBS HO OH 0/ 8220, DMAP HO 0 820 082 (jN 320 032 N Et3N.HF 0 0M 0 OM B20 0 B20 0 ACHN 193 Phosphitylation 820 082 d 0 B20 0 NC \/\/o MTO /N(iPr)2 \/\/O O‘P \o DMTO DMTOMO ‘LCN \/\/o 5' DMTO 3' 195 WO$O m @ —’ DMTON\ 88, DNA synthesizer 196 1. 194, DNA synthesizer AcHN —> o /O N \P/ 2.Aq NH3 55 °C, 18h |\ NHAC O OH 0M0 HO NHAc Compound 189 is cially ble. Compound 195 was prepared using the l procedure shown in Example 31. Oligomeric compound 197, comprising a GalNAc3-15 conjugate group, was prepared from compounds 194 and 195 using standard ucleotide synthesis procedures. The GalNAc3 cluster portion of the conjugate group GalNAc3-15 (GalNAc3-15a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is -P(=O)(OH)-Ad-P(=O)(OH)-.

The structure of GalNAc3-15 (GalNAc3-15a-CM-) is shown below: HOOH oi HO 0 NCf (Wk AcHN W Q 0 OR o o CV0mm0 HO /N AcHN O ('133 Example 65: Dose-dependent study of oligonucleotides comprising a jugate group (comparison of GalNAc3-3, 12, 13, 14, and 15) targeting SRB-l in vivo The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-l in mice. Unconjugated ISIS 353382 was included as a standard. Each of the GalNAc3 conjugate groups was ed at the 5' terminus of the respective oligonucleotide by a phosphodiester linked 2'- deoxyadenosine nucleoside (cleavable moiety).

Table 41 Modified ASOs targeting SRB-l ISIS Sequences (5’ to 3’) Conjugate SEQ N0. ID 353 3 82 GesmcesTesTesmCesAdsGdsTdsmCdsAdsTdsGdsAdsmEdsTdsTesmCesmCesTesTe none 28 661161 GalNAc3-3a-oAdoGesmCesTTHCesAdsGdsTdsCdsAdSTdSGdSAdSmCdsTds s-3 30 Te:ncesmeCesTesT 671144 GalNAc3oAdoGesmCesTesTesmCesAdsGdsTdSmCdSAdsTdsGdsAdsmCdsTds Ga1\ACs-12 30 Te:ncesmeCesTesT 670061 GalNAc3-13a-oAdoGCSmCCSTCSTesmCesAdsGdsTdSmCdsAdsTdSGdsAdsmCdSTds Ga1\ACs-13 30 Te,mcesmeCesTesT 671261 GalNAc3-14a-oAdoGesmCESTesTesmCesAdsGdsTdSmCdSAdsTdsGdsAdSmCdSTds s-14 30 Te,mcesmeCesTesT 671262 GalNAc30,AdoGesmCesTesT,smCesAdsGdsTdSmCdSAdsTdSGdSAdSmCdSTdS Ga1\ACs-15 30 Tes Ces CesTesTe Capital letters indicate the nucleobase for each nucleoside and mC indicates a 5-methyl cytosine. Subscripts: "e99 indicates a 2’-MOE modified nucleoside; "(1" indicates a B-D-2’ -deoxyrib0nucle0side; "s99 indicates’ a phosphorothioate internucleoside linkage (PS); "099’indicates a phosphodiester ucleoside linkage (PO); and "0’" tes -O-P(=O)(OH)-. Conjugate groups are in bold.

WO 79627 The structure of GalNAc3-3a was shown previously in Example 39. The structure of GalNAc3-12a was shown previously in Example 61. The structure of GalNAc3-13a was shown previously in Example 62.

The ure of GalNAc3-14a was shown previously in Example 63. The structure of GalNAc3-15a was shown previously in Example 64.

Trealmenl Six to eight week old C57bl6 mice (Jackson Laboratory, Bar , ME) were injected subcutaneously once or twice at the dosage shown below with ISIS 353382, 661161, 671144, , 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-l mRNA levels using ime PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols. The s below are ted as the average percent of SRB-l mRNA levels for each treatment group, normalized to the saline control.

As illustrated in Table 42, treatment with antisense oligonucleotides d SRB-l mRNA levels in a ependent manner. No significant differences in target knockdown were observed between animals that ed 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 GalNAc3-3, 12, 13, 14, and 15 conjugates showed substantial improvement in potency compared to the unconjugated antisense oligonucleotide (ISIS 335382).

Table 42 SRB-l mRNA (% Saline) ISIS No. Dosage (mg/kg) SRB-l mRNA (% ED50 (mg/kg) Conjugate Saline n/a 100.0 n/a 3 85.0 69.2 353382 —30 22.4 none 2 x 15 36.0 0.5 87.4 1.5 59.0 661161 5 25.6 2.2 GalNAc3-3 2 x 2.5 27.5 17.4 0.5 101.2 671144 —;'533(1) 3.4 GalNAc3-12 17.6 0.5 94.8 670061 1.5 57.8 2.1 20.7 WO 79627 671261 . 3-14 671262 GalNAc3-15 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 group (data not shown). ALTs, ASTs, total bin and BU\I values are shown in Table 43 below.

Table 43 Total ALT . . . BUN AST (U/L) Bilirubin Conjugate.

(U/L) ) (m dL) 28 60 0.1 39 n/a 77 0.2 36 353382 661161 . GalNAc3-3 671144 ' ' GalNAc3-12 670061 ' ' GalNAc3-13 671261 ' ' GalNAc3-14 671262 ' ' GalNAc3-15 Example 66: Effect of various cleavable moieties on antisense inhibition in vivo by oligonucleotides targeting SRB-l comprising a 5’-GalNAc3 cluster The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-l in mice. Each of the GalNAc3 conjugate groups was attached at the 5' terminus of the respective ucleotide by a phosphodiester linked nucleoside able moiety (CM)).

Table 44 Modified ASOs targeting SRB-l ISIS Sequences (5’ to 3’) Gal\Ac3 SEQ No. Cluster ID No. 661161 GalNAC30:AdoGesmCeSTeSTesmCesAdsGdsTdSmCdSAdSTds s-3a IAd 30 qSACSmCCSTCSTesmcesmceSTesre 670699 GalNAC30:TdoGesmCeoTCOTCOmCmAdSGdSTdSmCdSAdSTdS Ga1\ACs-3a Ta 33 670700 GalNAC30,A60GesmCeoTeoTeomCeoAdsGdsTdSmCdSAdsTdS Ga1\ACs-3a Ac 30 670701 GalNAc30,Te,GesmccormrmmcmAdsGdsTdsmcdsAdsTds Ga1\ACs-3a Te 33 671165 3-13WAd,GesmcmTWTmmcmAdsGdsTdSmCdSAdSTds Gal\ACs-13a Ad 30 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 -deoxyribonucleoside; "s" indicates a phosphorothioate internucleoside linkage (PS); "0" indicates a phosphodiester internucleoside e (PO); and "0’" indicates -O-P(=O)(OH)-. Conjugate groups are in bold.

The structure of GalNAc3-3a was shown previously in Example 39. The structure of 3-13a was shown previously in Example 62.

Trealmenl Six to eight week old C57bl6 mice (Jackson Laboratory, Bar , 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 s. The mice were sacrificed 72 hours following the final administration to determine the liver SRB-l mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. , OR) according to standard protocols. The results below are presented as the average percent of SRB-l mRNA levels for each treatment group, normalized to the saline control.

As illustrated in Table 45, treatment with antisense oligonucleotides lowered SRB-l mRNA levels in a dose-dependent manner. The antisense oligonucleotides comprising s cleavable moieties all showed similar potencies.

Table 45 SRB-l mRNA (% Saline) ISIS No. Dosage (mg/kg) SRB-l mRNA 3 (% Saline) Cluster Saline Es: 100.0 n/a 661161 GalNAc3-3a 0101‘" 670699 GalNAc3-3a 0101‘" 670700 GalNAc3-3a 0101‘" 670701 GalNAc3-3a 0101‘" 671165 GalNAc3 - 1 3a Liver transaminase , alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were ed relative to saline injected mice using standard protocols. Total bilirubin and BUN were also ted. 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 46 below.

Table 46 Total ALT AST GalNAc3 ISIS No. Bilirubin (U/L) (U/L) Cluster (mg/dL) Saline 24 64 0.2 n/a 661161 GalNAc3-3a 670699 GalNAc3-3a 670700 GalNAc3-3a 670701 GalNAc3-3a WO 79627 -15 81 101 02 ___—_ 671165 _____ GalNAc3-13a Ad _____ Example 67:0APreparation of oligomeric compound 199 comprising GalNAc3-16 OOAC MAM—l /ODMTF AcO OWN 1. Succinic anhydride, AGONgg/OMNWHNO0 OAc 2. DMF, HBTU, DIEA, OHS": PS—SS AcHN 2 AcOOAc OWAcHN ODMT AcOOAc AcO OWNWNO NW ynthesizer H 8 N, Z 2.aq.NH3 AcOOAc O OMH Mc 2 AcHN 198 H H HOOH AcHN O - o /0 AcHN Z H O Oligomeric compound 199, comprising a GalNAcg-16 conjugate group, is prepared using the general procedures illustrated in Examples 7 and 9. The GalNA03 r portion of the conjugate group GalNA03-16 (GalNAcg-16a) can be combined with any cleavable moiety to provide a variety of ate groups. In certain embodiments, the cleavable moiety is (OH)-Ad-P(=O)(OH)-.The structure of GalNA03-16 (GalNAc3-16a-CM-) is shown below: VV()2014/179627 2014/036463 HoOH o o HO%o oAmJLN 4 Hm" H o o ,0 HOOH 0 N 5 4 H 2 o AcHN OH HoOH o o N o Example 68: ation of oligomeric compound 200 comprising 3-17 AcO OOAc 3' IO|| AcHN OW F OLIGO o—P-o—(CH2)6-NH2 0‘" OA° OH 0 OWN/\H/\NHH 1. Borate buffer DMSO pH 8.5 it A00 NMgm: AcHNOAC OAC 2. aq. ammonia rt A00 0MN\/A\/HN o HoOH o o [40%O N 3 H N HoOH o o [—10%O W /\/\O N N 3 H H HoOH o [40%O N O 3 H N Oligomeric compound 200, comprising a GalNA03-17 conjugate group, was prepared using the general procedures illustrated in Example 46. The GalNA03 cluster portion of the conjugate group GalNA03-17 (GalNAc3-17a) can be combined with any cleavable moiety to provide a variety of conjugate . certain embodiments, the cleavable moiety is -P(=O)(OH)-Ad-P(=O)(OH)-. The structure of GalNA03-17 (GalNAc3-17a-CM-) is shown below: HoOH o o Ho$¢O O N/\/\N 3 H H HO N O /\/ N 0 N H NWO_.—§H 3 H O HOOH o O N/\/\ O HO 3 H H Example 69: Preparation of oligomeric compound 201 comprising 3-18 AcO OOAC 836 AcHN 0W 3- 5- H OAc OOAcOO’\$i§VJ:N NM; OLIGO o—P-o—(CH2)6-NH2 NHH I /\/\ OH AcHN 1. Borate buffer DMSO pH 8.5 rt OAc OAC AcO OmOH\/\/HN 2. aq. ammonia rt HoOH o o O N/\/\ 4 H H ACHN O O HOOH O O Ho§¢ofifimmm JJ\/\/U\ o N N"$¥\4 0 OLIGO H H HOOH O o N"\/\ 0 4 H H Oligomeric compound 201, comprising a GalNAcg-18 conjugate group, was prepared using the general procedures rated in Example 46. The GalNA03 cluster portion of the conjugate group GalNA03-18 (GalNAcg-18a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the ble moiety is -P(=O)(OH)-Ad-P(=O)(OH)-. The structure of GalNAcg-l8 (GalNAc3-18a-CM-) is shown below: HoOH o o Hopjo§¢o N 4 HH H HACHN OO/lfiN/v m N NAM" 4 O .CM H H OAcHN HoOHO Example 70: Preparation of oligomeric compound 204 comprising GalNAc3-19 AcOOAC A00OAc 0 O 0 ON O HBTU’DMF’DIEA O\/\/\)J\ ACO AGO OH —> 64 bNH 202 puma—l PhosphityIation 0M i AcO$¢ N "mo NC 1.DNA synthesizer AcHN \ /o\) —» I 2. aq. NH3 DMTO (/Pr)2N HO OH HO 3 o o AcHN | CIT—OH HO OH Homo/W0 N o AcHN | O=P—OH HO OH o 0 - -CM OLIGO Oligomeric nd 204, comprising a GalNA03-19 conjugate group, was prepared from compound 64 using the general procedures rated in Example 52. The 3 cluster portion of the conjugate group GalNAc3-19 (GalNAc3-19a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is -P(=O)(OH)-Ad-P(=O)(OH)-. The structure of GalNAc3-19 (GalNAc3-19a-CM-) is shown below: WO 79627 O=P-OH AcHN | O=P—OH Example 71: Preparation of oligomeric compound 210 comprising 3-20 NWL: F H EtN(iPr)2, CHgCN F><fl/ MNN F)<fl/N ---IIOH DMTO 0 206 DMTO AcOOAcO O o Aco%wO\/\/\)J\ "ZN/"52kb, Opr AcHN 166 KzCO3/Methanol ""OH ACOOAC O Phosphitylation 0 0M 3 N .uIIOH I AcO NH 1. DNA syntheSIzer. AcOOAc 0 0M 3 N ""0 NC — AcO NH \P/OV 2. aq. NH3 AcHN I 209 DMTO (iPr)2N Ho H"; . O N HO 3 O o AcHN | O=|l3—OH OH : HO HVé‘L/OK o N HO 3 AcHN ? O—T—OH OH 0 ¢ 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 GalNAc3-20 conjugate group, was prepared from compound 208 using the general procedures rated in Example 52. The GalNAc3 cluster portion of the conjugate group GalNAc3-20 (GalNAc3-20a) can be ed with any ble moiety to provide a variety of ate groups. In certain embodiments, the cleavable moiety is -P(=O)(OH)-Ad-P(=O)(OH)-. The structure of GalNAc3-20 (GalNAc3-20a-CM-) is shown below: HO o s Hog/OWHO NMN AcHN (I) CIT—OH O ‘30 O NMN HO OW 3 AcHN (I) O=Il3—OH 0 39 Example 72: Preparation of oligomeric compound 215 comprising 3-21 INH ACOOAc H Rog/OMO AcOOAc OH o AcHN 176 OW\)J\Nr/ ACO BOP, EtN(iPr)2, 1,2-dichloroethane AcHN \\\ 212 OH AcOOAC DMTCI Pyridine rt ' ' 0 Phosphltylatlon A00 $0: WkN AcHN \\\ 1. DNA synthesizer ACOOAC Acog/O o (1 N(iF>r)2 —> O \/\/\)J\ 2' 8 'NHq 3 AcHN \\\ Ho H o N AcHN Cl) o=Fl>—0H HO H o N Ho 0% L AcHN Cl) O‘T—OH Compound 211 is cially available. Oligomeric compound 215, comprising a GalNAc3-21 conjugate group, was ed from compound 213 using the general procedures illustrated in Example 52. The 3 cluster portion of the conjugate group GalNAc3-21 (GalNAc3-21a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is -P(=O)(OH)-Ad-P(=O)(OH)-. The structure of GalNAc3-21 (GalNAc3-21a-CM-) is shown below: HO H ACHN 0| OZIT—OH HO f/ O N Ho 0% L HO H O N Example 73: Preparation of eric compound 221 comprising GalNAc3-22 O O F3c3\n/N\/\/\)J\OH H a F3C\n/N\/\/\)LN/\,OHH o F F 211 o OH % 205 F F 216 F DIEA ACN O K2003 DMT'C' FacTNWLNNODMTr —>MeOH / H20 pyridine O R 217 OH HZNMNNODMTr 0A0 F O/\/\/\n/o F % o 218 NHAc F F OH 166 A OC goe/o/VWHMN/\/ODMTr Phosphitylation AcO o —’ OAc H Ac goe/O/WWN0 MN/\/ODMTr A00 0 NC\/\o,P\ 220 N(IPr)2.

OHQrvwH\¢/\v/\vjiN HO O 1. DNASynthesizer OH H F|,/,o OH NWL O, \OH 2. Aq. NH3 goe/O/Ml/ N/\/ HO O OH "W1 O/KgH QWOWY N/V HO O 221 W Compound 220 was prepared from compound 219 using diisopropylammonium tetrazolide. Oligomeric compound 221, comprising a GalNAc3-21 conjugate group, is prepared from compound 220 using the general ure illustrated in Example 52. The GalNAc3 cluster portion of the conjugate group GalNAc3-22 (GalNAc3-22a) can be combined with any cleavable moiety to e a variety of conjugate groups. In certain embodiments, the ble moiety is (OH)-Ad-P(=O)(OH)-. The structure of GalNAc3-22 (GalNAc3-22a-CM-) is shown below: OH%WH\/\/\)OJ\M HO O OH M ’ \ HO O NHAc S OH90mm 0’ w OH HO O NHAc S O E Example 74: Effect of various cleavable moieties on antisense inhibition in vivo by oligonucleotides targeting SRB-l comprising a 5’-GalNAc3 conjugate The oligonucleotides listed below were tested in a dose-dependent study for nse tion of SRB-l in mice. Each of the GalNAc3 conjugate groups was attached at the 5' terminus of the respective oligonucleotide.

Table 47 Modified ASOs targeting SRB-l ISIS GalNA03 SEQ sequences (5 , to 3 , ) CM No. Cluster ID No.

GmCTTmCAGTmCA TGAmCTT 353382 es es es es es dsm ds mds ds ds ds ds ds ds ds es n/a n/a 28 Ces CesTesTe GalNAc,A GmC T T mc A G T mc A T3 a 0 661161 domes es esmes mes ds ds ds ds ds ds GalNA03-3a Ad 30 GdsAds CdsTdsTes Ces CesTesTe GalNAcsG mc T T mc A G T mc A T3 3° :3, cs 65 i; i; ‘15 d5 d5 d5 d5 ‘15 666904 GalNAc3-3a P0 28 GdsAds CdsTdsTes Ces CesTesTe GalNAc-l7-3A GmC TT mc A G T mc A T3 a 0 din es es ersn es es (15 ds d5 d5 (15 ds 675441 m GalNAC3'17a Ad 30 GdsAds CdsTdsTes Ces CesTesTe GalNAc-l8-aA GmC TT mc A G T mc A T3 a 0 675442 din es es ersn es es (15 ds d5 d5 (15 ds m GalNA03-l8a Ad 30 GdsAds Tes Ces CesTesTe In all , l letters indicate the nucleobase for each nucleoside and mC indicates a 5-methyl ne.

Subscripts: "e" indicates a 2’-MOE modified nucleoside; "(1" indicates a B-D-Z’-deoxyribonucleoside; "s" indicates a phosphorothioate intemucleoside linkage (PS); "0" indicates a phosphodiester intemucleoside linkage (PO); and "0’" indicates -O-P(=O)(OH)-. ate groups are in bold.

The structure of GalNAc3-3a was shown previously in Example 39. The structure of GalNAc3-17a was shown previously in Example 68, and the structure of GalNAc3-18a was shown in Example 69.

Trealmenl 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 47 or with saline.

Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours ing the final administration to determine the SRB-l mRNA levels using ime PCR and RIBOGREEN® RNA quantification t (Molecular Probes, Inc. , OR) according to standard protocols. The results below are presented as the average percent of SRB-l mRNA levels for each treatment group, normalized to the saline control.

As illustrated in Table 48, treatment with antisense oligonucleotides lowered SRB-l 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 48 SRB-l mRNA (% Saline) ISIS No. Dosage (mg/kg) SRB-l mRNA GalNAc3 CM (% Saline) Cluster 100.0 353382 661161 ' 3-3a 666904 ' GalNAc3-3a 675441 ' GalNAc3-l7a 675442 ' GalNAc3-18a Liver transaminase levels, alanine aminotransferase (ALT) and aspartate ransferase (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 ted with no significant change from the saline group (data not shown). ALTs, ASTs, total bilirubin and BUN values are shown in Table 49 below.

Table 49 Total Dosage ALT AST .. . GalNAc3 ISIS No. Bilirubin (mg/kg) (U/L) (U/L) Cluster (mg/(1L) Saline n/a 26 59 3 23 58 353382 28 58 48 47 23 53 661161 GalNAc3-3a 26 48 32 57 21 48 666904 GalNAc3-3a 19 49 52 6O 95 675441 GalNAc3-17a 27 75 24 61 26 65 64 675442 GalNAc3-18a 27 69 84 Example 75: Pharmacokinetic analysis of oligonucleotides comprising a 5’-conjugate group The PK of the ASOs in Tables 41, 44 and 47 above was evaluated using liver samples that were obtained following the ent procedures described in Examples 65, 66, and 74. The liver samples were minced and extracted using rd protocols and analyzed by IP-HPLC-MS alongside an internal standard.

The combined tissue level (ug/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 50 PK Analysis in Liver ISIS No. Dosage Total Tissue Level Parent ASO Tissue GalNAc3 CM (mg/kg) by UV (ug/g) Level by EIC (ug/g) Cluster 353382 8.9 8.6 22.4 21.0 n/a n/a 54.2 44.2 661161 _5 32.4 20.7 Gal\A03-3a Ad 63 2 44 1 671144 _5 20.5 19.2 Gal\AC3-12a Ad 48 6 41 5 670061 _5 31.6 28.0 Gal\A03-13a Ad 67 6 55 5 g3: 33 A. 671262 5 18.5 7.4 Gal\A03-15a Ad 52 3 24 2 670699 _5 16.4 10.4 Gal\Ac3-3a Td 31 5 22 5 670700 _5 19.3 10.9 Gal\A03-3a Ae 38 1 20 0 670701 _5 21.8 8.8 Gal\AC3-3a Te 2 16 1 671165 _5 27.1 26.5 Gal\A03-13a Ad 48 3 44 3 666904 E :32 3:2 g-3a PO 675441 33-: 33(1) Ga1\AC,-17a A. 675442 22.2 20.7 Gal\Ac3-18a Ad 39.6 29-0 The results in Table 50 above show that there were greater liver tissue levels of the ucleotides comprising a GalNA03 ate group than of the parent oligonucleotide that does not comprise a GalNA03 conjugate group (ISIS 353382) 72 hours following oligonucleotide administration, particularly when taking into consideration the differences in dosing between the oligonucleotides with and without a 3 conjugate group. Furthermore, by 72 hours, 40-98% of each oligonucleotide comprising a 3 conjugate group was metabolized to the parent compound, indicating that the GalNAc3 conjugate groups were cleaved from the oligonucleotides.

Example 76: ation of oligomeric compound 230 comprising GalNAc3-23 ToSCI NaN3 HO/\/O\/\O/\/OH —> HO/\/O\/\O/\/OTS 222 223 4, TMSOTf O N OAc Pd(OH)2 OACOAC ACN O O NH —,2 H2, MeOH o/\/ \/\o/\/ F F F F F O C_NOE OAC H OAC N O O O/\/O\/\O/\/ OACOAC NHAc H No2 1)Reduce O /\/O\/\O/\/N 2) Couple Diacid OAC 3) Pd/C o o OAc 4) PFPTFA NHAc OAC OAC H N O O O/\/O\/\O/\/ OACOAC NHAC H NHMO F O o/\/O\/\O/\/ OAC O O O O F F NHAC OAC F 0 O/\/O\/\O/\/ 3' 5' II -O-F|’(CH2)6-NH2 1. Borate buffer, DMSO, pH 8.5, rt 2. aq. ammonia, rt 0H0" NHAc H NH N M"m0 OO/\/O\/\o/\/ 4 -m OH O O O 0 NHAc OH o/\/O\/\O/\/NH 0 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 on was then ated to an oil, ved in EtOAc and washed with water, sat. NaHCO3, brine, and dried over NaZSO4. The ethyl acetate was concentrated to dryness and purified by column chromatography, eluted with EtOAc/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 e was then poured onto water, and extracted with EtOAc. The organic layer was washed with water three times and dried over NaZSO4. 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 lar sieves (5g), and TMSOTf (1.65 ml, 9.11 mmol) in dichloromethane (100mL) under an inert atmosphere.

After 14 hours, the reaction was d to remove the sieves, and the c layer was washed with sat.

NaHC03, water, brine, and dried over NaZSO4. 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 EtOAc/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 nd 226. LCMS and NMR were tent 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 d dropwise with pentaflourotrifiuoro acetate (9.05 ml, 52.65 mmol). After 30 minutes, the on was poured onto ice water and extracted with EtOAc. The organic layer was washed with water, brine, and dried over NaZSO4. 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 d at room temperature in acetonitrile (15 mL) for 2 hours. The reaction was then evaporated to s and purified by column chromatography, eluting with a gradient of 2 t010% 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 en. After 12 hours, the st was removed by filtration and the c layer was evaporated to a solid that was used ly 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 ul, 1.6 mmol) in DMF (5mL). After 16 hours, the DMF was removed under reduced re at 65°C to an oil, and the oil was dissolved in dichloromethane. The organic layer was washed with sat. NaHC03, brine, and dried over NaZSO4. 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 ul, 0.66 mmol) was added and the reaction was purged with argon.

Pentafiourotriflouro acetate (46.39 ul, 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 ed by LCMS). The solvent was removed under d pressure (rotovap) at 70 °C. The residue was diluted with DCM and washed with 1N NaHSO4, brine, ted sodium bicarbonate and brine again. The organics were dried over NaZSO4, 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, sing a GalNAc3-23 conjugate group, was prepared from compound 229 using the general procedure rated in Example 46. The GalNAc3 cluster portion of the GalNAc3-23 conjugate group (GalNAc3-23a) can be combined with any cleavable moiety to provide a variety of conjugate groups. The structure of GalNAc3-23 (GalNAc3-23a-CM) is shown below: O O/\/O\/\O/\/ OHOH NHAC H NH "W0 o o/\/O\/\o/\/N M OH OO 0 NHAc OHOH e 77: Antisense inhibition in vivo by oligonucleotides targeting SRB-l comprising a GalNAc3 conjugate The ucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-l in mice.

Table 51 Modified ASOs targeting SRB-l ISIS GalNA03 SEQ Sequences (5 9 t0 3 , ) N0. r ID No.

GalNAC30,AdoGesmCesTesTesmcesAdsGdsTdSmCdSAdsTdS 661161 m m GalNAcg-3a 30 TCSTGS CGS CGSTGSTG mCTTmCAGTmCAT 666904 S 65 i; i; ‘15 d5 d5 d3 d5 ‘13 GalNAcg-3a 28 sTes Ces CesTesTe 673502 m m GalNAc3-10a 30 677844 S S S m m GalNA03-9a 30 esTesTes CesAdsGdsTds CdSAdsTds 677843 3-23a 30 STCS CC C T T S 6S 6S e csGdsTds C A TdsdsdsGdsAds CdsTdsTfi Ce 655861 S GalNAcg-la 29 TesTeoAdowGalNAc3-la csGdsTds C A TdsdsdsGdsAds CdSTdSTeS C 677841 SS GalNAc3-19a 29 esTeoAdo’'GalNAC3-19a GTmCAT GAmCTTmC 677842 cs ds ds ds ds ds ds ds ds ds cs 65 GalNAC3'203 29 TesTeoAdo"GalNAc3'203 The structure of Gal‘\IAc3-1a was shown previously in Example 9, GalNAc3-3a was shown in e 39, GalNAc3-9a was shown in Example 52, 3-10a was shown in Example 46, GalNAc3-19a was shown in Example 70, GalNA03-20a was shown in Example 71, and GalNA03-23a was shown in Example Trealmenl Six to eight week old 6 mice (Jackson Laboratory, Bar Harbor, ME) were each injected subcutaneously once at a dosage shown below with an oligonucleotide listed in Table 51 or with saline. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration to determine the SRB-l mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent ular , Inc. Eugene, OR) according to standard protocols. The results below are presented as the average percent of SRB-l mRNA levels for each treatment group, normalized to the saline control.

As rated in Table 52, treatment with antisense oligonucleotides lowered SRB-l mRNA levels in a dose-dependent manner.

Table 52 SRB-l mRNA (% Saline) ISIS No. Dosage (mg/kg) SRB-l mRNA GalNAc3 0Z (% Saline) Cluster Saline —n/a 100.0 n/a 661161 GalNAc3-3a 666904 3-3a 673502 GalNAc3-10a 677844 GalNAc3-9a 677843 GalNAc3-23a 655 861 GalNAc3 - 1 a 677841 GalNAc3 - 1 9a 677842 GalNAc3-20a IIIIIIIIiI Liver transaminase levels, alanine aminotransferase (ALT) and ate 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 53 below.

Table 53 Dosage ALT AST . . . GalNAc ISIS NO' 3 (mg/kg) (U/L) (U/L) $321131; Cluster Saline 21 45 0.13 n/a 28 51 g; g; N. 21 56 24 56 666904 - —3: g: GalNAc3-3a 24 60 24 59 46 673502 GalNAc3-10a 24 45 24 47 61 677844 3: g: GalNAc3-9a 22 65 _ 53 53 677843 - —3? 23 GalNAc3-23a 22 43 21 48 655861 - —33 23 GalNAc3-la 21 55 _ 32 54 677841 3: 33 GalNAc3-l9a 24 58 23 61 677842 3: 2; 3-20a 24 37 Example 78: Antisense tion in vivo by oligonucleotides targeting Angiotensinogen comprising a GalNAc; ate The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of Angiotensinogen (AGT) in normotensive Sprague Dawley rats.

WO 79627 Table 54 Modified ASOs targeting AGT ISIS , , GalNAc3 SEQ 552668 2C21AzincesTesGesAdsTdsTdsTdsTdesGdSmCdSmCdsmCdsAfiG63 CGSAES CesTesGesAdsTdsTdsTdsTdsTdsGds Cds Cds CdsAfiGes 669509 GalNAC3—1a Ad 35 GesAesTeoAdo"GalNAc3'la The ure of GalNAc3-1a was shown usly in Example 9.

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 54 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 RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. , 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 120,000. The results below are presented as the e 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 55, treatment with antisense ucleotides 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 55 AGT liver mRNA and plasma protein levels ISIS Dosage (mg/kg) AGT liver AGT plasma GalNAc3 Cluster CM No. mRNA (% PBS) protein (% PB S) PBS ma 100 ma 3 95 122 97 552668 n/a n/a 46 669509 ; GalNAcg-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 56 below.

Table 56 Liver transaminase levels and rat body weights GalNAc3 ISIS No. ALT (U/L) AST (U/L) Weight (% Cluster of baseline) PBS 51 81 186 n/a 552668 669509 GalNAc3- 1 a Example 79: Duration of action in vivo of oligonucleotides targeting APOC-III comprising a GalNAc3 conjugate The ucleotides listed in Table 57 below were tested in a single dose study for duration of action in mice.

Table 57 Modified ASOs targeting APOC-III ISIS GalNAc3 SEQ Sequences (5’ to 3’) CM No. Cluster ID No.

AesGesmcesTesTesmCdsTdsTdsGdsTdsmCdsmCdsAdsGdsmcdsTfiTfi 304801 n/a n/a 20 TesAesTe mcesTesTesmCdsTdsTdsGdsTdsmCdsmCdsAdsGdsmcdsTfiTfi 647535 GalNAc3- 1 a 21 TesAfiTeoAd03-GalNAc3-la GalNAc3-32l-0,AderSGesmCmTeSTesmCdSTdsTdsGdsTdsmCdS 663083 GalNAc3-3a 36 mCdsAdSGdsmCdSTesTes TmAesTe GalNAc3-7a-0,AderSGesmCmTeSTesmCdSTdsTdsGdsTdSmCdS 674449 GalNAc3-7a 36 mCdSAdSGdSmCdSTCSTes TmAeSTe ,AdoAesGesmCeSTESTesmCdSTdsTdSGdsTdSmCdS 674450 3- 1 0a 36 mCdsAdSGdsmCdSTesTes TmAesTe GalNAc30,AdoAesGesmCeSTESTesmCdSTdsTdSGdsTdSmCdS 674451 GalNAc3- 1 3a 36 SGdsmCdSTesTes TmAesTe The structure of GalNAc3-1a was shown previously in Example 9, GalNAc3-3a was shown in Example 39, GalNAc3-7a was shown in e 48, GalNAc3-10a was shown in Example 46, and GalNAc3-13a was shown in Example 62.

Trealmenl Six to eight week old transgenic mice that express human APOC-HI were each injected subcutaneously once with an oligonucleotide listed in Table 57 or with PBS. Each treatment group consisted of 3 s. 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 bed in Example 20. The results below are presented as the average t of plasma triglyceride and APOC-HI 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 58 Plasma tri_l ceride and APOC-III rotein levels in trans ' enic mice ISIS (2:1: p315? cerides $3513} GalNAc3 y p p 0 No. (% baseline) Cluster dose) baseline). \o \1 5‘N PBS «35‘i—q n/a 304801 mmmggoooogguoo~o~o<>4>qoo ##kawq Jk-lkwouummww mquowoqq-b 647535 wwooom 41 GalNAc3-1a NN~5\IUJOON 663083 LAUJNNNNOO LII\]OOUIOO-I>UI GalNAc3-3a 674449 camNo 6452qu woooqg GalNAc3-7a DJ 00 1—K 4;4; 44 LII DJ 63 UJUJUJQ OO 30 674450 4;4; 44 GalNAc3-10a LII O\ 1— U300 LIIUJ 95 N4; 32 OLA-54> 000 34 674451 48 GalNAc3-13a \1 4; 97 Example 80: Antisense inhibition in vivo by oligonucleotides targeting Alpha-1 Antitrypsin (AlAT) comprising a GalNAc3 Conjugate The oligonucleotides listed in Table 59 below were tested in a study for ependent tion of A1AT in mice.

Table 59 Modified ASOs targeting AlAT Sequences (5 , 3 SEQ ID t0 3 , ) CM N0. Cluster No.

AesmcesmcesmcesAesAdsTdsTdsmCdsAdsGdsAdsAdsGdsGdsAesAes 476366 11/a 11/a 37 GesGesAe AesmcesmcesmcesAesAdsTdsTdsmCdsAdsGdsAdsAdsGdsGdsAesAes 656326 G lNAa 03'1 AC 38 GesGesAeoAdowGalNAcg-la GalNAc3'3a'o’Aders Ces Ces CesAesAdsTdsTds CdsAdsGdsAds 678381 GalNA03-3a Ac 39 AdsGdsGdsAesAes GesGesAe GalNAc3'7a'o’AdersmCesmCesmCesAesAdsTdsTdsmCdsAdsGdsAds 678382 GalNAC37a AC 39 AdsGdsGdsAesAes GesGesAe GalNAc3'1Oa'o’AdersmCeSmCesmCesAesAdsTdsTdsmCdsAdsGds 678383 G lNAa C310a AC 39 AdsAdsGdsGdsAesAes GesGesAe GalNAc3'13a'o’AdersmCeSmCesmCesAesAdsTdsTdsmCdsAdsGds 678384 GalNAC3 1 3a AC 39 AdsAdsGdsGdsAesAes GesGesAe The structure of GalNAc3-1a was shown usly in Example 9, GalNAc3-3a was shownin Example 39, GalNAc3-7a was shown in Example 48, GalNAc3-10a was shown in Example 46, and Gal\IAc3-13a was shown in Example 62.

Trealmenl Six week old, male C57BL/6 mice on Laboratory, Bar Harbor, ME) were each ed subcutaneously once per week at a dosage shown below, for a total of three doses, with an oligonucleotide listed in Table 59 or with PBS. Each treatment group consisted of 4 animals. The mice were ced 72 hours following the final stration. AlAT liver mRNA levels were determined using real-time PCR and RIBOGREEN® 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 # MS-E01, Alpco, Salem, NH). The results below are presented as the average percent of AlAT liver mRNA and plasma protein levels for each treatment group, normalized to the PBS control.

As illustrated in Table 60, treatment with antisense oligonucleotides lowered AlAT liver mRNA and AlAT plasma protein levels in a dose-dependent manner. The oligonucleotides comprising a GalNAc conjugate were significantly more potent than the parent (ISIS 4763 66).

Table 60 AlAT liver mRNA and plasma protein levels ISIS Dosage (mg/kg) AlAT liver AlAT plasma GalNAc3 Cluster CM No. mRNA (% PBS) protein (% PB S) PBS n/a 100 100 n/a n/a 86 476366 73 ma n/a 45 30 0-6 99 _ 2 61 656326 GalNAc3-1a Ad 6 15 18 6 0.6 105 _ 678381 2 53 _ 991N906" 6 16 l8 7 13 0.6 90 79 2 49 678382 GalNAc3-7a 6 21 18 8 0-6 94 2 44 678383 3-10a Ad 6 13 18 6 0-6 106 2 65 678384 3-13a Ad 6 26 18 11 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 61 below.

Body weight is shown as % relative to baseline. Organ weights are shown as % of body weight ve to the PBS control group.

Table 61 Body Liver Kidney Spleen If}?' 39?:g (1:55) (135) (Egg) weight (% weight (Rel weight (Rel weight (Rel baseline) % BW) % BW) % BW) PBS n/a 25 51 37 119 100 100 100 34 68 35 116 91 98 106 476366 15 37 74 30 122 92 101 128 45 30 47 31 118 99 108 123 0.6 29 57 40 123 100 103 119 2 36 75 39 114 98 111 106 656326 6 32 67 39 125 99 97 122 18 46 77 36 116 102 109 101 0.6 26 57 32 117 93 109 110 2 26 52 33 121 96 106 125 678381 6 40 78 32 124 92 106 126 18 31 54 28 118 94 103 120 0.6 26 42 35 114 100 103 103 2 25 50 31 117 91 104 117 678382 6 30 79 29 117 89 102 107 18 65 112 31 120 89 104 113 0.6 30 67 38 121 91 100 123 2 33 53 33 118 98 102 121 678383 6 32 63 32 117 97 105 105 18 36 68 31 118 99 103 108 0.6 36 63 31 118 98 103 98 2 32 61 32 119 93 102 114 678384 6 34 69 34 122 100 100 96 18 28 54 30 117 98 101 104 Example 81: Duration of action in vivo of oligonucleotides targeting AlAT comprising a GalNAc3 cluster The oligonucleotides listed in Table 59 were tested in a single dose study for duration of action in mice.

Trealmenl Six week old, male C57BL/6 mice were each injected subcutaneously once with an oligonucleotide listed in Table 59 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 AlAT protein levels were measured via ELISA (see Example 80). The results below are presented as the e percent of plasma AlAT protein levels for each treatment group, normalized to baseline levels. The results show that the ucleotides comprising a GalNAc conjugate were more potent and had longer duration of action than the parent g a GalNAc conjugate (ISIS ). 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 62 Plasma AlAT protein levels in mice ISIS Dosage Time point AlAT (% GalNAc3 CM No. (mg/kg) (days post- baseline) Cluster dose) m m 476366 100 n/a n/a ——12 36 656326 18 GalNAc3-1a Ad ——12 21 678381 18 GalNAc3-3a Ad ——12 21 678382 18 GalNAc3-7a Ad ——12 21 678383 18 GalNAc3-10a Ad 678384 18 GalNAc3-13a Ad Example 82: Antisense inhibition in vitro by oligonucleotides targeting SRB-l comprising a 3 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 63 were added at 2, 10, 50, or 250 nM in Williams E medium and cells were incubated overnight at 37 0C in 5% C02. 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 ime PCR and RIBOGREEN® RNA fication reagent (Molecular , 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 63 Inhibition of SRB-l expression in vitro , , . GalNAc IC50 SEQ Sequence (5 to 3 ) Linkages CM No. r (nM) ID No. 353382 CGSTGSEGS CESAddeTndS CdSAdSTdSGdSAdS PS n/a n/a 250 28 Tes Ces CesTesTe GesmCeSTesTesmCesAdsGdsTdsmCdsAdsTdsGdsAds Gal\AC3 633861 PS A" 40 29 mcdsTdsTesmcesmcesTesTmAdo,-Ga1NAc3-1a -1a Gal\Ac3 661161 GglNAC30’Adognes TIES CtgsAdsGdsTds PS Ac 40 30 CdsAdsTdsGdsAds Tes Ces CesTesTe '33.

GglNAc3-3a-03Ad0CifS CeOTeOTIEO CtgoAdsGdsTds Gal\Ac3 661162 PO/PS Ac 8 30 TdsGdsAds CdsTds Teo Ceo CesTesTe '33.

GesmCeSTesTesmCesAdsGdsTdsmCdsAdsTdsGdsAds 3 664078 PS 33° 30 39 mcdsTdsTesmcesmcesTesTmAdo,-Ga1NAc3-9a -9a GalNAC30’Ad0GesmCeSTesTesmCesAdsGdsTds Gal\Ac3 665001 PS AC 70 30 IncdslAdsTdsGdsAdsmCdsTdsTfimCeSmCesTesTe '83.

GalNAC3-Sa-0’AdoGesmCeSTesTesmCesAdsGdsTds Gal\Ac3 666224 PS AC 80 30 mCdslAdsTdsCils[AdsmcdsTdsTmmcesmcesTesTe '53. 666841 C6°T6°nTC6°TC6f°ini°°TnEE CTdslffdsTdsGdsAds PO/PS n/a n/a >250 28 ds ds e0 e0 es es e GalNAc3-102l-0,AdoGESmCesTesTESmCesAdsGdsTdS Gal\Ac3 666881 PS Ad 30 30 mCdslAdsTds(his[AdsmcdsTdsTmmcesmcesTesTe "103. 666904 3-3a-0:GGin CesTesTesmCestlgdsGdSTds Cds Gal\Ac3 PS PO 9 28 AdsTdsGdsAds CdsTds Tes Ces CesTesTe '33. 666924 GrillNAC3-3a-0’TdoCifs CesTfiTtgs CrisAdsGdsTds Gal\Ac3 PS Td 15 33 CdsAdsTcsGdsAds CdsTds Tes Ces CesTesTe '33.

GalNAC3-6a-0’Ad0GesmCeSTesTesmCesAdsGdsTds Gal\Ac3 666961 PS Ad 15 0 30 mCdslAdsTcsGdsAdsmCdSTdsTfimCesmCesTesTe '63.

GalNAC3'7a'0’Ad0GesmCeSTeSTeSmCeSAdSGdSTdS Gal\Ac3 666981 PS Ad 20 30 mCdslAdsTcsCils[AdsmcdsTdsTmmcesmcesTesTe '73. 670061 ngNAc3'13-A'O’Adoges CesTesTnes (tilesAdsGdsTds Gal\AC3 PS Ad 30 30 CdsAdsTcsGdsAds CdsTdsTes Ces CesTesTe "133.

GalNAc -3a-.,,T 0G mc T T3 ‘3 Inc A G T 670699 5 6° 6° G31\AC3 3° $1" 66 6° ‘6 m PO/PS Td 15 33 CdsAdsTcs GdsAds Teo Ceo CesTesTe '33.

GalNAc -3a-.,,AeoG mc T T3 Inc A G T 670700 6° 6° G31\AC3 6° 66 6° m 6; 6;: 6;; PO/PS AC 30 30 CdslAdsTds GdsAds CdsTdsTeo Ceo CesTesT '33.

GalNAc -3a-0,Te.,G mc T T3 Inc A G T 670701 6; 6° 6° 61° 61° 66 6° ‘6 G31\AC3 m PO/PS Te 25 33 CdsAdsTds GdsAds CdsTdsTeo Ceo CesTesTe '33. ngNAc3'12-A'O’Adoges Eles (tilesAdsGdsTds Gal\AC3 671144 PS Ad 40 30 CdsAdsTdsGdsAds CdsTdsTes Ces CesTesTe _123' WO 79627 GalNAc -13a-O’AOGesmCe0Te0TCOmCe0A G T3 d ‘13 d ‘13 Gal\AC3 671165 PO/PS A, 8 30 InCdslAcsTcs GdsAdsmCdsTdsTeomCEneomCesTesT "133. ngNAc3'14a'o’AdogesmCesTesTnesm(tilesAdsGdsTds Gal\A03 671261 PS Ac >250 30 CdsAcsTcchsAcs CdsTds Tes Ces CesTesTe "143.

Gal\A03 671262 ngNAc3'15a'o’Adoges CesTesTnes (tilesAdsGdsTds PS Ac >250 30 CdsAcsTcchsAcs CdsTds Tes Ces Te "153. 673501 NAC3-7a-0’AdoCigs CeOTeOTIEO CfleoAdsGdsTds Gal\A03 PO/PS Ac 30 30 CdsAcsTcchsAcs CdsTdsTeo Ceo CesTfiTe '73. 673502 GanllNAchOa'o’Adoges CeoFl-‘eo'gleo geoAdsGdsTds Gal\A03 PO/PS Ac 8 30 CdslAcsTcchsAcs CdsTds Teo Ceo CesTesTe "103. 675441 ngNAc3'17a'o’Adoges CesTesTnes (tilesAdsGdsTds 3 PS Ac 30 30 CdsAcsTcchsAcs CdsTds Tes Ces CesTesTe "173.

GillNAc3-182l-(,,Ad(,Ges CesTeSTes CeSAdSGdSTdS Gal\A03 675442 PS Ac 20 30 mMdscssTcchssAcmCdsTds TesmCesmeCesTesT _18a C}esmCesTesTesmCeslAdsChsTdsmCdslAdsTdsCidslAds Gal\A03 677841 PS AC 40 29 mcdsTdSTesmcesmcesTeSTmAdo-Ga1NAc3-19 -19a GesmCeSTesTesmCesAdsGdsTdsmCdsAdsTdsGdsAds Gal\A03 677842 PS A" 30 29 mcdsTdsTesmcesmcesTesTmAdo,GalNAc-20.1 -20a ngNAc3'23a'o’Adoges CesTesTnes (tilesAdsGdsTds Gal\A03 677843 PS Ac 40 30 CdslAdsTdsChslAds CdsTds Tes Ces CesTesTe '233.

The structure of GalNAc3-1a was shown previously in e 9, GalNAc3-3a was shown in Example 39, GalNAc3-5a was shown in Example 49, GalNAc3-6a was shown in Example 51, GalNAc3-7a was shown in Example 48, GalNAc3-8a was shown in Example 47, GalNAc3-9a was shown in Example 52, GalNAc3-10a was shown in Example 46, GalNAc3-12a was shown in Example 61, GalNAc3-13a was shown in Example 62, GalNAc3-14a was shown in Example 63, GalNAc3-15a was shown in Example 64, 3-17a was shown in Example 68, GalNAc3-18a was shown in e 69, GalNAc3-19a was shown in Example 70, GalNAc3-20a was shown in Example 71, and GalNAc3-23a was shown in Example 76.

Example 83: Antisense inhibition in vivo by ucleotides targeting Factor XI comprising a GalNAc3 cluster The oligonucleotides listed in Table 64 below were tested in a study for dose-dependent inhibition of Factor XI in mice.

Table 64 Modified oligonucleotides targeting Factor XI ISIS GalNAc Sequence (5 ’ t0 3 ’ ) No. cluster TesGdsG68TGSAGSAdSTdSmCZmE}dS%dSmCdSTdSTdSTdSmCdsAesGes 40407 1 TesC}eoC}eoTeol/XeolikdsTdsmcdsmcdsllkdsmcdsTdsTdsTdsmCdsAeoC}eo 656173 31 AssG65G60AwGalNAc3-1, 663086 GalNAC3-3a-0’AdoTeSGeoGeoTeeroAdsTdsmCdsmCdsAdsmCdsTds GalNAc3-3 678347 GalNAc3-7a GalNAC3—103-0:AdoTesGeoGeoTeeroAds dsmCdsmCdsAdSmCds 678348 GalNAC3_10a TdsTdsTdsmCdsAeoGekoGes e GalNAC30:AdoTesGeoGeoTeeroAdsTdsmCdsmCdsAdsmCds 678349 GalNA03-13, TdsTdsTdsmCdsAeoGeoAasGesGe The structure of GalNAc3-la was shown previously in Example 9, GalNAc3-3a was shown in Example 39, GalNAc3-7a was shown in Example 48, GalNAc3-10a was shown in Example 46, and GalNAc3-13a was shown in Example 62.

Trealmenl 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 ted of 4 animals. The mice were ced 72 hours following the final dose. Factor XI liver mRNA levels were measured using ime PCR and normalized to cyclophilin ing to standard protocols.

Liver transaminases, BUN, and bilirubin were also measured. The results below are presented as the average percent for each ent group, normalized to the PBS control.

As illustrated in Table 65, treatment with antisense oligonucleotides lowered Factor XI liver mRNA in a dose-dependent . 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 65 Factor XI liver mRNA, liver transaminase, BUN, and bilirubin levels ISIS Factor x1 ALT AST BUN Bilirubin No. mRNA (% PBS) (U/L) (U/L) (m_ dL) (m_ dL) Cluster ID No.

PBS 63 41 —_- 404071 49 Na - 90 89 21 656173 36 58 26 GalNAcg-la 32 50 63 25 . 91 169 25 663086 38 55 21 GalNAc3-3a 40 34 40 23 - 28 49 20 678347 180 149 21 GalNAc3-7a 44 76 19 - 43 54 21 6 2 25 38 20 0.14 0.7 34 39 46 20 0.16 "WGalNACs-13a 40 _—--__ Example 84: Duration of action in vivo of oligonucleotides targeting Factor XI comprising a GalNAc3 Conjugate The oligonucleotides listed in Table 64 were tested in a single dose study for duration of action in mice.

Trealmenl Six to eight week old mice were each injected aneously once with an oligonucleotide listed in Table 64 or with PBS. Each treatment group consisted of 4 animals. Blood was drawn by tail bleeds the day before dosing to ine 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 dies from R & D s, 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 ’-GalNAc conjugate (ISIS 663086, 678347, 678348, and 678349) were even more potent with an even longer duration of action than the oligonucleotide comprising a NAc conjugate (ISIS 656173).

Table 66 Plasma Factor XI protein levels in mice Time point (days Factor XI (% SEQ ID GalNAC3 Cluster DOSt'dOSG) baseline) No. 656173 GalNAcg-la 32 663086 GalNAcg-3a 40 678347 1 GalNA03-7a 40 678349-== GalNACs-13a Example 85: Antisense inhibition in vivo by ucleotides targeting SRB-l comprising a GalNAc3 Oligonucleotides listed in Table 63 were tested in a ependent study for antisense inhibition of SRB-l in mice.

Trealmenl 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 63 or with saline. Each treatment group consisted of 4 animals. The mice were sacrificed 48 hours following the final stration to determine the SRB-l mRNA levels using real-time PCR and RIBOGREEN® RNA quantification t (Molecular Probes, Inc. Eugene, OR) according to standard protocols. The results below are presented as the average percent of liver SRB-l mRNA levels for each treatment group, normalized to the saline control.

As illustrated in Tables 67 and 68, treatment with antisense oligonucleotides lowered SRB-l mRNA levels in a dose-dependent manner.

Table 67 SRB-l mRNA in liver ISIS No. Dosage (mg/kg) SRB-l mRNA (% GalNAc3 Cluster 655861 Game?" 66"" GalNA03'3a 666881 GalNAC3'1Oa 666981 WAC?" 670061 GalNAC3'13a Ad WO 79627 1 52 3 18 677842 GalNAC3'ZOa Ad Table 68 SRB-l mRNA in liver ISIS No. Dosage (mg/kg) SRB-l mRNA (% GalNA03 Cluster CM Saline) -—661161 GalNA03-3a Ad 677841 _ GalNA03-19a Ad Liver minase levels, total bilirubin, BUN, and body weights were also measured using standard ols. Average values for each treatment group are shown in Table 69 below.

Table 69 ISIS Dosage ALT AST Bilirubin BUN Body Weight GalNAc3 N6. (mg/kg) (U/L) (U/L) (mg/dL) (mg/dL) (% baseline) Cluster n/a 19 39 0.17 26 118 n/a 0.1 25 47 0.17 —0.3 29 56 0 15 655861 . GalNAc3-1a 3 0-14 3 27 5 0.14 3 0-13 0.3 4 6 0.15 661161 (""0333 3 6 0.18 —3 29 52 0.13 01 30 51 0.15 666881 GalNAc3-1 0a 666981 81:- 6611016346 670061 GalNA03-13a 1 20 35 0.14 3 27 42 0.12 677842 0.17 GalNAc3-20a A 0.15 .nnnnna. 3 41 57 0.14 23 118 Example 86: nse inhibition in vivo by oligonucleotides targeting TTR comprising a GalNAc3 cluster Oligonucleotides listed in Table 70 below were tested in a dose-dependent study for antisense inhibition of human transthyretin (TTR) in transgenic mice that express the human TTR gene.

Trealmenl 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 s. The mice were sacrificed 72 hours following the final administration. Tail bleeds were performed at s time points throughout the experiment, and plasma TTR protein, ALT, and AST levels were measured and reported in Tables 72-74. 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 er (AU480, Beckman Coulter, CA). Real-time PCR and RIBOGREEN® 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 71-74 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 e percent weight change from baseline until sacrifice for each individual treatment group. Organ weights shown are normalized to the ’s body weight, and the average normalized organ weight for each treatment group is then ted relative to the e normalized organ weight for the PBS group.

In Tables 71-74, "BL" indicates baseline, measurements that were taken just prior to the first dose.

As illustrated in Tables 71 and 72, 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 ). Furthermore, the oligonucleotides sing a GalNAc conjugate and mixed PS/PO internucleoside linkages were even more potent than the ucleotide comprising a GalNAc conjugate and fill PS linkages.

Table 70 Oligonucleotides targeting human TTR . Sequence 5 , . GalNAc SEQ ISIS No. to 3 , Linkages cluster ID No.

TesmCesTesTesGesGdsTdsTdsAdsmCdsAdsTdsGdsAdsAds 420915 AesTfimcesmcesmce TesmcesTesTesGesGdsTdsTdsAdsmCdsAdsTdsGdsAdsAds 682883 GalNAc3-3a_0,TesmCeoTeoTeoGeoGdsTdSTdsAdSmCdsAds PS/PO GalNA03-3a In- TdsGdsAdsAdsAeoTeomCeSmCeSmCe GalNAc3'7a-0’Tesmce0TeoTeoGeoGdsTdsTdsAdsmCdsAds 682884 GalNAc3-7a TdsGdsAdsAdsAeoTeomCeSmCeSmCe GalNAc3'10-a-0’TesmceoTeoTeoGeoGdsTdsTdsAdsmCds 682885 AdsTdsGdsAdsAdsAeoTeomCesmCesmCe GalNAc3'13a-0’TesmceoTeoTeoGeoGdsTdsTdsAdsmCds 682886 AdsTdsGdsAdsAdSAGOTeomCCSmCesmce TesmceoTeoTeoGeoGdsTdsTdsAdsmCdsAdsTdsGdsAdsAds 684057 PS/PO GalNAC3'19a AeoTeomcesmcesmcmAdo,—Ga1NAc3-19a The legend for Table 72 can be found in Example 74. The structure of GalNAc3-1 was shown in Example 9.

The structure of GalNAc3-3a was shown in Example 39. The ure of 3-7a 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 e 70.

Table 71 Antisense inhibition of human TTR in vivo . Dosage TTR mRNA (% Plasma TTR protein SEQ Is1s No. GalNAc cluster CM (mg/kg) PBS) (% PBS) ID No.

PBS ma 100 Na Na 6 m 95 420915 20 65 n/a n/a 41 60 28 0-6 87 2 56 660261 GalNAc3-la Ad 42 6 27 n 11 Table 72 Antisense inhibition of human TTR in vivo TTR Plasma TTR prote1n (A) PBS at BL)0 . Dosage GalNAc ISIS NO' mRNA Day 17 (mg/kg) Da 3 Da 10 cluster (% PB S) y y (After sac) PBS n/a 100 _L" 114 n/a 420915 026 60 682883 —l§-_ 0-6 56 682884 0-6 60 682885 682886 0-6 57 21 89 50 31 mfimmz—18 102 41 GalNAc _ 684057 _____—19813 n————— Table 73 Transaminase levels, body weight changes, and relative organ weights ALT (U/L) AST (U/L) Da Da Da Da 58 62 67 52 21 64 59 73 47 -m-m 420915 19 64 54 56 42m41 . 26 70 71 63 111 96 99 92 34 61 6o 68 61 ---m 660261 34 58 59 70 90mm 33 64 54 68 95 Table 74 Transaminase , body weight changes, and relative organ weights ALT (U/L) Isis N0. (Tie D3ay Dlay Day (% 8 BL 17 BL) 1313s27-176 77 100 102 103 102 420915 54 106 107 135 6 106 105 104 682883 16 143 682884 31 321227"" 682885 30 26 _-El111 —-m113 —-m120 682886 55 69 105 40 61 69 109 100 102 1—-m110 684057 112 2""121 Example 87: Duration of action in vivo by single doses of oligonucleotides targeting TTR comprising a GalNAc; cluster ISIS numbers 420915 and 660261 (see Table 70) were tested in a single dose study for duration of action in mice. ISIS numbers 420915, 682883, and 682885 (see Table 70) 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 ed 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 ne 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 t of plasma TTR levels for each treatment group, normalized to baseline levels.

Table 75 Plasma TTR protein levels (mg/15g)Dosa e Time oint . GalNAc3 CM TTR(% baseline) SEQ ID No. (days p01;dose) Cluster 420915 13 2: n/a n/a 41 24 75 39 100 660261 . GalNAc3-1a Ad 42 Treatment Female transgenic mice that express human TTR were each injected subcutaneously once with 100 mg/kg ISIS No. , 10.0 mg/kg ISIS No. 682883, or 10.0 mg/kg 682885. Each treatment group consisted of 4 animals. Tail bleeds were med before dosing to determine ne 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 76 Plasma TTR protein levels ISIS Dosage Time point TTR (% baseline) N0. (mg/kg) (days post-dose) (2111:2233 SEQ ID No. 7 48 420915 100 10 48 n/a n/a 41 17 66 31 80 682883 10.0 38 3-3a P0 41 682885 10.0 34 3- 1 0a P0 41 The results in Tables 75 and 76 show that the oligonucleotides comprising a GalNAc conjugate are more potent with a longer on of action than the parent ucleotide lacking a conjugate (ISIS 420915).

Example 88: Splicing modulation in vivo by oligonucleotides targeting SMN comprising a GalNAc3 conjugate The oligonucleotides listed in Table 77 were tested for splicing modulation of human survival of motor neuron (SMN) in mice.

Table 77 Modified ASOs targeting SMN ISIS GalNAc3 SEQ Sequences (5’ to 3’) CM No. Cluster ID No.

ABSTGSTGSmCGSAGSmCESTESTGSTGSmCGSAGSTGSAGSAGSTCSGGSmCGSTGSGGS 387954 n/a n/a 43 GalNAc30’A68TESTGSmCGSAGSmCGSTGSTGSTGSmCGSABSTGSAGSAGS 699819 3-7a P0 43 TGSGGSmCGSTGSGGSGe GalNAc3'7a'o’AesTeoTeomCeeromCeoTeoTeoTeomceeroTeero 699821 GalNAc3-7a P0 43 ACOTCOGCOmCGOTCSGCSGC ASSTBSTeSmCBSABSmC6STCSTBSTCSmCCSABSTBSABSABSTfiGeSmCBSTCSGCS 700000 GalNAc3- 1 a Ad 44 GeoAdowGalNAc3-la 703421 X-ATTmCAmCTTTmCATAATGmCTGG n/a n/a 43 703422 GalNAc3-7b-X-ATTmCAmCTTTmCATAATGmCTGG GalNAc3-7b n/a 43 The structure of GalNAc3-7a was shown previously in Example 48. "X" indicates a 5’ primary amine generated by Gene Tools (Philomath, OR), and GalNAc3-7b indicates the structure of GalNAc3-7a g the —NH-C6-O portion of the linker as shown below: HoOH o HO 4 "AI HoOH O o o 0 NM0 NJK/\/U\fl HO 'v 4 H H AcHN o 0 t HO 4 M 0 ISIS numbers 703421 and 703422 are morphlino oligonucleotides, wherein each nucleotide of the two ucleotides is a morpholino nucleotide.

Trealmenl Six week old transgenic mice that express human SMN were ed subcutaneously once with an oligonucleotide listed in Table 78 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 rd 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 78. The results show that fiJlly modified oligonucleotides that modulate splicing and comprise a GalNAc conjugate are significantly more potent in ng splicing in the liver than the parent oligonucleotides lacking a GlaNAc conjugate. Furthermore, this trend is maintained for multiple ation chemistries, including 2’-MOE and morpholino modified oligonucleotides.

Table 78 Effect of oligonucleotides targeting human SMN in vivo 113:8 Dose (mg/kg) +Exon 7 / -Exon 7 (2:112:133 ISBN?) Saline n/a 1.00 n/a n/a 387954 32 1.65 n/a 43 387954 288 5.00 n/a 43 699819 32 7.84 GalNAc3-7a IE 43 699821 32 7.22 GalNAc3-7a IE 43 700000 32 6.91 GalNAcg-la 44 703421 32 1.27 n/a 43 703422 32 4.12 GalNAc3-7b 43 Example 89: Antisense inhibition in vivo by oligonucleotides targeting Apolipoprotein A (Apo(a)) sing a GalNAc3 conjugate The oligonucleotides listed in Table 79 below were tested in a study for dose-dependent inhibition of Apo(a) in transgenic mice.

Table 79 Modified ASOs targeting Apo(a) , , GalNA03 SEQ ID TesGesmCesTesmCesmCdsGdsTdsTdsGdsGdsTdsGdsmCds GalNAc3'7a'O’TesCIeomCeoTeommCeoCdsGdsTdsTdsGdsGds 681257 GalNAc3-7a 53 TdsCIdsmCds TdsTeoGeoTeSTmmeC The structure of g-7 was n e 48.

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 79 or with PBS. Each treatment group consisted of 3-4 animals. Tail bleeds were performed the day before the first dose and weekly ing 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 RIBOGREEN® 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 80 are presented as the treatment group average percent relative to the PBS treated group. Plasma protein levels were fithher 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 81.

As rated in Table 80, treatment with the ucleotides lowered Apo(a) liver mRNA and plasma protein levels in a dose-dependent manner. Furthermore, the ucleotide 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 81, transaminase levels and body weights were unaffected by the oligonucleotides, indicating that the oligonucleotides were well tolerated.

Table 80 Apo(a) liver mRNA and plasma protein levels Apo(a) mRNA Apo(a) plasma protein (% PB S) (% PBS) Week 2 Week 3 Week 4 Week 5 —-1m-m——"—7 494372_—-______ 681257 Table 81 ISIS No. Dosae (m k) Bod weiht (% baseline) ——m_— 494372 ——-§_— 681257 Example 90: Antisense inhibition in vivo by ucleotides targeting TTR comprising a GalNAc3 cluster Oligonucleotides listed in Table 82 below were tested in a ependent study for antisense inhibition of human transthyretin (TTR) in transgenic mice that express the human TTR gene.

Trealmenl 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 83 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 ing the final administration. TTR protein levels were measured using a clinical analyzer (AU480, Beckman Coulter, CA). Real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. , OR) were used according to standard protocols to determine liver human TTR mRNA levels. The results presented in Table 83 are the average values for each treatment group. The mRNA levels are the e values relative to the average for the PBS group. Plasma n levels are the average values relative to the average value for the PBS group at baseline. "BL" indicates baseline, ements that were taken just prior to the first dose. As illustrated in Table 83, ent 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 sing 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 82 Oligonucleotides targeting human TTR Isis No. Sequence 5’ to 3’ Linkages (22111111120 CM ISBN?) 420915 T"mc"T"TesGegffggcffigggfdfldSGdSAdSAdS PS n/a n/a 41 682883 GalNAc3glgfsinfgkfifggjgcggfdSmCdsAds PS/PO GalNAc3-3a P0 41 666943 3:gjgxgggfidgggw PS/PO GalNAc3-3a Ad 45 682887 fzzsafl‘éidsnfds:gmGCgéTEngdsAd PS/PO GalNAc3-7a Ad 45 682888 Galli‘égfiOTdéigdSZfsATTnggéTgchSAdS PS/PO GalNAc3-10a Ad 45 682889 Galli‘égfi‘Q’TdéigdSZfsATTnggéTgchSAdS PS/PO GalNAc3-13a Ad 45 The legend for Table 82 can be found in Example 74. The structure of 3-3a 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 83 Antisense inhibition of human TTR in vivo Isis No. Dosage (mg/kg) TTR mRNA (% PB S) TTR protein (% BL) GalNAc cluster CM PBS ma 100 ma n/a 6 69 420915 20 71 —_ Na n/a 60 21 0.6 61 682883 2 23 GalNA03-3a PO 6 18 0-6 74 666943 2 33 GalNAc3-3a 6 17 0.6 60 97 682887 2 36 49 3-7a 6 12 0-6 65 682888 2 32 GalNACs-IOa 6 17 0.6 72 682889 2 38 GalNACs-13a 6 16 WO 79627 Example 91: Antisense inhibition in vivo by oligonucleotides targeting Factor VII sing a GalNAc3 conjugate in non-human primates Oligonucleotides listed in Table 84 below were tested in a non-terminal, dose escalation study for antisense inhibition of Factor VII in monkeys.

Trealmenl Non-naive s were each injected subcutaneously on days 0, 15, and 29 with escalating doses of an oligonucleotide listed in Table 84 or with PBS. Each treatment group ted 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 85 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 85, treatment with antisense oligonucleotides lowered Factor VII expression levels in a ependent manner, and the ucleotide comprising the GalNAc conjugate was significantly more potent in monkeys compared to the oligonucleotide lacking a GalNAc conjugate.

Table 84 Oligonucleotides targeting Factor VII . , , . GalNAc SEQ AesTesGesmcesAesTdsGdsGdsTdsGdsAdsTdsGdsmCdsTds GalNAc3'10a-o’AesTesGesmCesAesTdsGdsGdsTdsGds 686892 GalNAC3_10a "- AdsTdsGdsmCdsTds TesmCeSTesGesAe The legend for Table 84 can be found in Example 74. The structure of GalNAc3-10a was shown in Example Table 85 Factor VII plasma n levels ISIS N0. Day Dose (mg/kg) Factor VII (% BL) 0 Na 10 407935 686892 Example 92: Antisense inhibition in primary hepatocytes by antisense oligonucleotides targeting Apo- CIII comprising a GalNAc3 conjugate Primary mouse hepatocytes were seeded in 96-well plates at 15,000 cells per well, and the oligonucleotides listed in Table 86, 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 "M. 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 RIBOGREEN® RNA quantification t (Molecular Probes, Inc.) ing to standard protocols. IC50 values were determined using Prism 4 re (GraphPad). The results show that regardless of whether the cleavable moiety was a phosphodiester or a phosphodiester-linked deoxyadensoine, the ucleotides comprising a GalNAc conjugate were significantly more potent than the parent ucleotide g a conjugate.

Table 86 Inhibition of mouse APOC-III expression in mouse primary hepatocytes Sequence (5 , IC50 SEQ t0 3 , ) CM N0. (nM) ID No. 440670 mCesAesGesmCesTesTdsTdsAdsTdSTdsAdsGdsGdsGdsAdSmCesAesGesmCesAe n/a 13.20 47 mCeslAesCiesmcesTesTdsTdslAdsTdsTdslAdsCidsCidsCilsAdsmCes 661180 Ad 1.40 48 AesGesmcesA60 lNAc3-la GalNAc3'3a-o’ CesAesGes Ces 680771 CesTesTgdesAdsTdsTdsAdsGdsGdsGdsAds PO 070 47 AesCles CesAe GalNAc3'7a-o’ CesAesGes 680772 ngTdsAdsTdsTdsAdsGdsGdsGdsAds Ces PO 170 47 AesCles CesAe GalNAc3'10a-o’ CesAesGes Ces 680773 CesTesTIgsTdsAdsTdsTdsAdsGdsGdsGdsAds PO 200 47 AesCles CesAe GalNAc3'13a-o’ CesAesGes 680774 CesTesTIgsTdsAdsTdsTdsAdsGdsGdsGdsAds Ces PO 150 47 AesCles CesAe GalNAc3'3a-o’ CesAeoGeo 681272 CeoTeoTIdlsTdsAdsTdsTdsAdsGdsGdsGdsAds Ceo PO < 046 47 AeoCles CesAe GalNAc3'3a'o’AdomCesAesGesmcesTesTdsTdsAdsTdsTdsAdsGdsGdsGdsAds 681273 Ad 1.10 49 mCesAesGesmCesAe mCeslAesCiesmcesTesTdsTdslAdsTdsTdslAdsCidsCidsCils[Adsmces 683733 Ad 2'50 48 AesGesmCCSAeoAdo,-GalNAc3-19a The structure of GalNA03-1a was shown previously in Example 9, GalNA03-3a was shown in Example 39, 3-7a was shown in Example 48, GalNA03-10a was shown in e 46, GalNAcg-13a was shown in Example 62, and GalNA03-19a was shown in Example 70.

Example 93: Antisense inhibition in vivo by oligonucleotides targeting SRB-l comprising mixed wings and a 5’-GalNAc3 conjugate The oligonucleotides listed in Table 87 were tested in a dose-dependent study for antisense inhibition of SRB-l in mice.

WO 79627 Table 87 Modified ASOs ing SRB-l ISIS ces (5’ to 3’) Gal\Ac3 SEQ No. Cluster ID No. 449093 TkST (SkaSAdSGdSTdSmCdS AdSTdS Gds AdsmCdsTdsTkskaska n/a 50 699806 GalNAc3-3a-kasTkskasAdsGdsTdSmCds AdsTds GdsAdsmCds 3-3a w TCST (smoksmck 699807 GalNAC3-7a-0’TksTkskasAdsGdsTdsmCds AdsTds GdsAdsmCds Gal\AC3'7a w TEST (smoksmck 699809 GalNAc3-7a-o, TkSTkskasAdsGdsTdsmCds AdsTds Gds Adsmcds Ga1\A03-7a w Tcs,TesmCesmCe 699811 GalNAc3-7a-0:TesTesmcesAdsGdSTdsmCds AdSTds GdsAdsmCds Ga1\A03-7a w TCST (smcksmck 699813 GalNAC3-7a-0’TksTdskasAdsGdsTdsmCds AdsTds GdsAdsmCds Gal\AC3'7a w TEST (sincdsmck 699815 GalNAc3-7a-0:TesTkskasAdsGdsTdsmCds AdSTds GdsAdsmCds Ga1\A03-7a w TCST (smcksmce The structure of GalNAc3-3a was shown previously in Example 39, and the structure of GalNAc3-7a was shown previously in Example 48. Subscripts: "6" indicates 2’-MOE modified side; "(1" indicates B-D- 2’-deoxyribonucleoside; "k" tes 6’-(S)-CH3 bicyclic nucleoside (cEt); "s" indicates orothioate intemucleoside linkages (PS); "0" indicates phosphodiester intemucleoside linkages (PO). Supersript "m" tes 5-methylcytosines.

Trealmenl 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 87 or with saline.

Each treatment group ted of 4 s. The mice were sacrificed 72 hours following the final administration. Liver SRB-l mRNA levels were measured using real-time PCR. SRB-l mRNA levels were normalized to cyclophilin mRNA levels according to standard protocols. The results are presented as the average percent of SRB-l mRNA levels for each treatment group relative to the saline control group. As illustrated in Table 88, treatment with antisense oligonucleotides lowered SRB-l mRNA levels in a dose- dependent manner, and the gapmer oligonucleotides comprising a GalNAc conjugate and having wings that were either fiJll cEt or mixed sugar modifications were significantly more potent than the parent oligonucleotide lacking a conjugate and sing full cEt modified wings.

Body weights, liver transaminases, total bilirubin, and BUN were also measured, and the average values for each ent group are shown in Table 88. 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 88 SRB-l mRNA, ALT, AST, BUN, and total bilirubin levels and body weights ISIS Dosage SRB-l mRNA ALT AST Bil Body weight N0. (mg/kg) (% PBS) (U/L) (U/L) (% BL) PBS n/a 100 31 84 0.15 102 1 111 18 48 0.17 104 449093 3 94 20 43 0.15 26 103 104 0.1 114 23 58 0.13 107 699806 0.3 108 1 104 0.1 121 19 41 0.14 100 699807 0.3 105 1 102 0.1 104 699809 0.3 105 1 33 34 62 0.17 107 0.1 123 48 77 0.14 24 106 699811 0.3 94 20 45 0.13 25 101 1 66 57 104 0.14 107 0.1 104 699813 0.3 105 1 106 0.1 105 699815 0.3 64 30 61 0.12 105 1 24 18 41 0.14 106 Example 94: Antisense inhibition in vivo by oligonucleotides targeting SRB-l comprising 2’-sugar modifications and a NAc3 conjugate The oligonucleotides listed in Table 89 were tested in a dose-dependent study for antisense inhibition of SRB-l in mice.

Table 89 Modified ASOs targeting SRB-l ISIS ces (5’ t0 3’) GalNA03 CM SEQ N0. Cluster ID No. 353382 GesmcesTesTesmcesAdsGdsTdsmcdsAdsTdsGdsAdsmcdsTdsTesmcesmces ma TESTG 700989 GmsCmUmUmcmAdsGdsTdsmcdsAdsTdsGdsAdsmcdsTdsUmcmcm Na UmSUm 666904 GalNAC3-3 a'o’ sTesTesmCesAdsGdsTdsmCdsAdsTdsGdsAds 3—3a P0 mCdSTdSTesmCesmCesTesTe 700991 GalNAC3-7a-0’GmsCmsUmsUmsCmsAdsGdsTdsmCdsAdsTdsGds GalNAC3'7a P0 AdSmCdSTdSUmsCmsCmsUmsUm Subscript "m" indicates a 2’-O-methyl modified nucleoside. See Example 74 for complete table legend. The structure of GalNAcg-3a was shown previously in Example 39, and the structure of GalNAcg-7a was shown previously in Example 48.

Trealmenl The study was completed using the protocol described in Example 93. Results are shown in Table 90 below and show that both the 2’-MOE and 2’-OMe modified ucleotides 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 90 SRB-l mRNA ISIS No. Dosage (mg/kg) SRB-1 mRNA (% PBS) PBS n/a 100 116 353382 15 58 45 27 120 700989 15 92 45 46 1 98 666904 3 45 17 1 118 700991 3 63 14 Example 95: Antisense tion in vivo by oligonucleotides targeting SRB-l comprising bicyclic nucleosides and a 5’-GalNAc3 conjugate The oligonucleotides listed in Table 91 were tested in a dose-dependent study for antisense tion of SRB-1 in mice.

Table 91 Modified ASOs targeting SRB-l Sequences (5’ to 3’) (3111:2233 CM No. IIS)E1\C120 440762 TksmcksAdSGdSTdSmCdsAdsTdsGdsAdsmCdsTdsTkska n/a n/a 22 666905 GalNAc3-3a'o’TkskasAdsGdsTdsmCdSAdsTdsGdsAdsmCdSTdsTksmCk GalNAc3-3 a P0 22 699782 GalNAc3-7a-0,TkskasAdsGdsTdsmCdSAdsTdsGdsAdsmCdSTdsTksmCk GalNAc3-7a P0 22 699783 GalNAc30,TlsmclsAdsGdsTdSmCdSAdsTdsGdsAdSmCdSTdSTlsmC1 GalNAc3-3a P0 22 653621 TlsmClsAdsGdsTdSmCdSAdSTdsGdSAdSmCdSTdSTISmeAdO,-GalNAc3-la GalNA03-1a Ad 23 439879 T_ InC_ AdSGdsTdsmCdSAdSTd GdSAdsmCdsTdST_ InC_ n/a n/a 22 699789 GalNAc3-3a-0,T_ mC_sAdsGdsTdsmCdsAdsTd GdsAdsmCdSTdST_ mC_ GalNAc3-321 P0 22 Subscript "g" tes a fluoro-HNA side, subscript "1" indicates a locked nucleoside comprising a 2’- O-CH2-4’ bridge. See the Example 74 table legend for other iations. The structure of GalNAc3-1a was shown previously in Example 9, the structure of GalNAc3-3a was shown previously in Example 39, and the ure of GalNAc3-7a was shown usly in Example 48.

Trealmenl The study was completed using the protocol described in Example 93. Results are shown in Table 92 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 ic side modifications. Furthermore, the oligonucleotide comprising a GalNAc conjugate and fiuoro-HNA modifications was significantly more potent than the parent lacking a ate and comprising fiuoro-HNA ations. The results of the body weights, liver transaminases, total bilirubin, and BUN measurements indicated that the compounds were all well tolerated.

Table 92 SRB-l mRNA, ALT, AST, BUN, and total bilirubin levels and body weights ISIS No. Dosage (mg/kg) SRB-1 mRNA (% PB S) PBS n/a 100 1 104 440762 3 65 35 0.1 105 666905 0.3 56 1 18 0.1 93 699782 0.3 63 1 15 0.1 105 699783 0.3 53 1 12 0.1 109 653621 0.3 82 1 27 1 96 439879 3 77 37 0.1 82 699789 0.3 69 1 26 Example 96: Plasma protein binding of antisense ucleotides comprising a GalNAc; conjugate group Oligonucleotides listed in Table 57 targeting ApoC-IH and oligonucleotides in Table 93 targeting Apo(a) were tested in an ultra-filtration assay in order to assess plasma protein binding.

Table 93 Modified oli_0nucleotides tar__etin A0 a , , GalNA03 SEQ sequences (5 to 3 ) CM No. Cluster ID No TesGes CesTes Ces 494372 TL'lls-‘TgsgdsGdsTdsGds CdsTdsTesGesTes n/a n/a 53 TsesC}seo CeoTeo Ceo T$S&GdsT&Gds CdsTdsTeoGeoTes 693401 n/a n/a 53 GalNAc3'7a'0’TesGesmcesTesmCesmCdSGdsTdsTdsGdsGdsTdsGdsmCds 68 125 1 G lNAa C3-7a P0 53 TdsTesGesTesTesmCe GalNAc3'7a'o’TeSGeo CeoTeo Ceo 68125 7 CdsgdsTdsTdsGdsGdsTdsGds Cds GalNAc3-7a P0 53 TdsTeoGeoTeSTeS Ce See the Example 74 for table legend. The structure of GalNAc3-7a was shown previously in Example 48.

Ultrafree-MC ultrafiltration units (30,000 NMWL, low-binding regenerated ose membrane, Millipore, Bedford, MA) were nditioned with 300 uL of 0.5% Tween 80 and fiJged at 2000 g for minutes, then with 300uL of a 300 ug/mL solution of a control oligonucleotide in H20 and fuged at 2000 g for 16 minutes. In order to assess ecific binding to the filters of each test oligonucleotide from Tables 57 and 93 to be used in the s, 300 uL 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 d samples were analyzed by an ELISA assay to determine the oligonucleotide concentrations. Three replicates were used to obtain an e concentration for each . 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 ucleotides were added to 1.2 mL aliquots of plasma at two concentrations (5 and 150 ug/mL). An aliquot (300 uL) of each spiked plasma sample was placed in a pre-conditioned filter unit and incubated at 37°C for 30 minutes, immediately followed by centrifiJgation 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 ucleotide 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 94 for the two concentrations of oligonucleotide tested (5 and 150 ug/mL) in each species of plasma. The results show that GalNAc conjugate groups do not have a significant impact on plasma n binding. Furthermore, oligonucleotides with full PS intemucleoside 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 es.

Table 94 Percent of modified oli_0nucle0tide bound to lasma roteins ISIS Human plasma Monkey plasma Mouse plasma No- 150 ug/mL 304301 27.2 003033 230 074430 32.3 434372 230 693401 90-2 030231 20.1 030237 22.7 Example 97: Modified ucleotides ing TTR comprising a GalNAc3 conjugate group The oligonucleotides shown in Table 95 comprising a GalNAc conjugate were designed to target Table 95 Modified oligonucleotides targeting TTR ISIS No. Sequences (5’to 3’) CM SEISOID AiGAAAdSAds A3 42 Ga‘NA"3'3"'°’é::Zii:i:AW P0 41 Ga‘NA"3'7"'°’é::Zi"AZ:S§:AAds "3 P0 41 Ads "3 The legend for Table 95 can be found in Example 74. The structure of GalNAc3-1 was shown in Example 9.

The structure of GalNAc3-3a was shown in Example 39. The structure of GalNAc3-7a was shown in Example 48. The structure of GalNAc3-10a was shown in e 46. The structure of 3-13a 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 GalNAc conjugate in hPMBC assay The oligonucleotides listed in Table 96 and were tested for pro-inflammatory effects in an hPMBC assay as described in Examples 23 and 24. (See Tables 17, 70, 82, and 95 for descriptions of the oligonucleotides.) ISIS 353512 is a high der used as a positive control, and the other oligonucleotides are described in Tables 70, 82, and 95. The results shown in Table 96 were obtained using blood from one volunteer donor. The results show that the oligonucleotides comprising mixed PO/PS ucleoside linkages produced significantly lower pro-inflammatory responses compared to the same oligonucleotides having fiJll PS linkages. Furthermore, the GalNAc conjugate group did not have a significant effect in this assay.

Table 96 ISIS No. Em/ECSO GalNAc3 cluster 353512 3630 420915 682881 1311 GalNACs—IO 682888 GalNAc3-10 PO/Ps 684057 GalNAc3-19 PO/Ps Example 99: Binding affinities of oligonucleotides comprising a GalNAc conjugate for the asialoglycoprotein or The binding affinities of the oligonucleotides listed in Table 97 (see Table 63 for descriptions of the oligonucleotides) for the asialoglycoprotein receptor were tested in a competitive receptor binding assay. The itor ligand, d 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 ure by Atsma et al. (see J Lipid Res. 1991 Jan; 32(1):173-81.) In this method, desialylated a1- acid glycoprotein (de-AGP) was added to 10 mM iodine chloride, Na1251, and 1 M e 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 ed with an Agilent SEC-3 column 00mm) and a 13- RAM counter. ition experiments utilizing 1251 ed de-AGP and various GalNAc-cluster containing ASOs were performed as s. Human HepG2 cells (106 cells/ml) were plated on 6-well plates in 2 ml of appropriate grth 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% C02 respectively. Cells were washed with media without FBS prior to the experiment. Cells were incubated for 30 min @37°C with 1ml competition mix ning 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 125 I ed de-AGP and competitor GalNAc ASO. Cells were lysed using Qiagen’s RLT buffer containing 1% B-mercaptoethanol. Lysates were transferred to round bottom assay tubes after a brief 10 min freeze/thaw cycle and assayed on a v-counter. Non-specific binding was subtracted before dividing 1251 n counts by the value of the lowest GalNAc-ASO tration 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 97 were ed from experiments performed on five different days. Results for oligonucleotides marked with superscript "a" are the average of ments 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 97 Asialol corotein recetor bindin_ assa results Oligonucleotide end to ISIS No. GalNAc conjugate which GalNAc conjugate KD (nM) is attached a GalNAc3-3 66688" GalNA03-10 ———-x_ Example 100: Antisense inhibition in vivo by oligonucleotides comprising a GalNAc conjugate group targeting Ap0(a) in vivo The ucleotides listed in Table 98a below were tested in a single dose study for duration of action in mice.

Table 98a , , 3 SEQ I GalNAc3'7a'0’TesGesmCesTesmCesmCdsGdsTdsTdsGdsGds 681251 -m-G lNA-7 53 GalNAc3'7a'o’TesGeomCeoTeomCeOmCdSGdsTdsTdsGdsGds 68 l 25 7 lNA - The structure of GalNAc3-7a was shown in Example 48.

Trealmenl Female transgenic mice that express human Apo(a) were each injected subcutaneously once per week, for a total of 6 doses, with an ucleotide and dosage listed in Table 98b or with PBS. Each treatment group consisted of 3 s. Blood was drawn the day before dosing to determine ne 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 98b 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 filll PS ucleoside linkages and the oligonucleotide that comprises mixed PO and PS es.

Table 98b A0 a lasma rotein levels A oap ( 2%at72hours A oa at1weekp ((02) BL) A oa at3weeksp ISIS No. Dosage ) BL) ((2%) BL) 03 97 77 57 13000 23 —— 03 114 681257 Example 101: Antisense inhibition by oligonucleotides comprising a GalNAc cluster linked via a stable moiety The oligonucleotides listed in Table 99 were tested for inhibition of mouse II expression in viva. C57Bl/6 mice were each injected subcutaneously once with an oligonucleotide listed in Table 99 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 ed 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 99 as the average t 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 ucleotide lacking a conjugate group. Furthermore, the oligonucleotide comprising a GalNAc WO 79627 conjugate group linked to the oligonucleotide via a cleavable moiety (ISIS 680772) was even more potent than the ucleotide comprising a GalNAc conjugate group linked to the ucleotide via a stable moiety (ISIS 696847).

Table 99 Modified oligonucleotides targeting mouse APOC-III Dosage APOC-III Sequences (5’ to 3’) (mgkg) mRNA (% mCeslAesGesmCeSTesTdsTdsAdsTdsTdsAds 440670 GdsGdsGdsAdSmCes AeSGes mCCSAC 3'7a-o’mcesAesGesmCesTesTdsTdsAds 680772 TdsTdsAdsGds GdsGdsAdsmCes AesGesmCesAe GalNAc3'7a-s’mCesAesGesmCesTesTdsTdsAdsTds 696847 TdsAdsGdsGdsGdsAdsmCes AesGesmCeSAe The structure of GalNAc3-7a was shown in Example 48.

Example 102: Distribution in liver of antisense oligonucleotides comprising a GalNAc conjugate The liver distribution of ISIS 3533 82 (see Table 23) that does not comprise a GalNAc conjugate and ISIS 655861 (see Table 23) 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 100. Each treatment group consisted of 3 animals except for the 18 mg/kg group for ISIS 655861, which ted of 2 animals.

The animals were sacrificed 48 hours following the dose to ine 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 100 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 cytes than the oligonucleotide that does not comprise a GalNAc conjugate. rmore, the oligonucleotide comprising a GalNAc ate was t 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 ucleotide that was present in the liver was found in non-parenchymal liver cells.

Table 100 ISIS Dosage Concentration in whole Concentration in Concentration in non- liver (molecules*10A6 cytes parenchymal liver cells No' (mg/kg) per cell) (molecules*10A6 per cell) (molecules*10A6 per cell) 3 9.7 1.2 37.2 17.3 4.5 34.0 23 .6 6.6 65.6 353382 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 GalNAc3 conjugate The oligonucleotides listed in Table 101 below were tested in a single dose study for duration of action in mice.

Table 101 Modified ASOs targeting APOC-III ISIS Sequences (5 ’ to 3 ’) GalNAc3 CM SEQ No. Cluster ID No. 304801 AesGesmCesTesTesmCdsTdsTdsGdsTdsmCdsmCdsAdsGdsmcdsTesTes TesAesTe mCdsAdsGdsmCdsTeoTeo TesAesTe AesGmmceonTmmcdsTdsTdsGdsTdsmcismcdsAdsGdsmcdsTme TesAfiTeoAdowGalNAc3-l9a The structure of 3-3a was shown in Example 39, and GalNAc3-19a was shown in Example 70.

Trealmenl Female transgenic mice that express human APOC-III were each injected subcutaneously once with an oligonucleotide listed in Table 101 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 ceride and APOC-HI protein levels were measured as described in e 20. The s in Table 102 are presented as the average percent of plasma ceride and APOC-III levels for each treatment group, normalized to baseline levels. A comparison of the results in Table 58 of example 79 with the results in Table 102 below show that oligonucleotides comprising a mixture of phosphodiester and phosphorothioate WO 79627 internucleoside linkages exhibited increased duration of action than equivalent oligonucleotides comprising only phosphorothioate internucleoside linkages.

Table 102 Plasma triglyceride and APOC-III protein levels in transgenic mice ISIS (2:1: p318? cerides GalNAc3 CM y p p£3133}0 No. (% baseline) . Cluster dose) baseline) 304801 663084 GalNAc3-3a- 679241 Example 104: Synthesis of ucleotides comprising a 5’-GalNAc2 conjugate HN , Boc o O HBTU,HOBt + H TFA Boce OH —> N H2N\/\/\)J\O —>BOC\ N\/\/\)J\ DIEA,DMF N 0 H H DCM o o 120 126 85% 231 H2N O + AcO ON: DMF 0 AcHN 166 F 0A6 0A6 ACO&/OWN-l0 0A0 0A6 ACHN Acofi/OW 1. H2 Pd/C MeOH 0A0 0A6 2. PFPTFA DMF OAcOOAc Acofi/OWKo ONVWLO Acofi/ o AcHN N\/\/\j\: o/\© AcHN 233 2034 O 83e OH OH 3' 5' II 0 0_ _F1 0_(CH2)e NH2' HOgo O\/\/\/"\ AcHN NH 1. Borate buffer, DMSO, pH 8.5, rt —>HOE/H 2. aq. ammonia, n HAcHN OWN HM '-NAM/\o O_IGO 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 NaHC03 and 2 x brine, dried with NaZSO4, 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 romethane (10 mL) and trifluoracetic acid (10 mL) was added. After ng at room temperature for 2h, the on e 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 d at room temperature for 30 minutes, then poured into water (80 mL) and the aqueous layer was ted with EtOAc (2x100 mL). The organic phase was separated and washed with sat. aqueous NaHC03 (3 x 80 mL), 1 M NaHSO4 (3 x 80 mL) and brine (2 x 80 mL), then dried (NaZSO4), 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 e was stirred under hydrogen atmosphere for 3 h. The reaction mixture was filtered through a pad of Celite and trated to yield the ylic acid. The carboxylic acid (1.32 g, 1.15 mmol, r 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 ted with EtOAc (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. ucleotide 235 was prepared using the general procedure described in Example 46. The GalNAcZ cluster portion (GalNAc2-24a) of the conjugate group GalNAc2-24 can be combined with any cleavable moiety present on the oligonucleotide to provide a variety of conjugate groups. The structure of GalNAc2-24 (GalNAc2-24a-CM) is shown below: OH OH Hoflowm0ACHN OHOHO Example 105: Synthesis of oligonucleotides comprising a GalNAcl-ZS conjugate o 83e 3' 5' H O__-IPI T o— 166 2. aq. ammonia rt OH OH ACHN DOWN/HEM The synthesis of compound 166 is described in Example 54. ucleotide 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 ures described in Example 10. 0A0 /\/\/\/OH OAc ACOé‘Q’OWOA H2N OA A00 OW + PFPTFA /\/\/\/OH NHAc NHAc OH N TEA, Acetonltrlle_ _ H tetrazole, ylimidazole, DMF 2-cyanoethyltetraisopropyl ACOgQ/OWO O \NK phosphorodiamidite NHAc N/\/\/\/ ‘P’ \r H l 238 01 OH OH Oligonucleotide synthesis HO 0 ’ O\Wk /H\ OLIGO N o ACHN H 6 The GalNAc1 cluster portion (GalNAc1-25a) 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 GalNAc1-25 (GalNAc1-25a-CM) is shown below: OH OH OMNfiO/I—E ACHN H 6 Example 106: Antisense inhibition in vivo by oligonucleotides targeting SRB-l comprising a 5’- GalNAc; or a 5’-GalNAc3 conjugate ucleotides listed in Tables 103 and 104 were tested in dose-dependent studies for antisense inhibition of SRB-l in mice.

Trealmenl Six to week old, male C57BL/6 mice (Jackson Laboratory, Bar , ME) were injected aneously 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. , 686222, or ; or with saline. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours ing the final administration. Liver SRB-l mRNA levels were measured using real- time PCR. SRB-l mRNA levels were normalized to cyclophilin mRNA levels according to standard protocols. The antisense oligonucleotides d SRB-l mRNA levels in a dose-dependent manner, and the ED50 results are presented in Tables 103 and 104. 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-l mRNA levels with similar potencies as shown in Tables 103 and 104.

Table 103 Modified oligonucleotides ing SRB-l , , ED50 SEQ Sequences (5 to 3 ) GalNAc Cluster (mg/kg) 440762 sAdsGdsTdsmCdsAdsTdsGdsAdsmCdsTdsTkska 686221 GalNAc2-24a-0’Adogks CkSAdsgdsTds CdSAdsTdsGdsAds GalNACZ-24a 039 26 CdsTdsTks Ck 686222 3'13a'o’Adogks CksAdsgdsTds CdsAdsTdsGdsAds GalNA03-1 3a 0.41 26 CdsTdsTks Ck See Example 93 for table legend. The structure of 3-13a was shown in Example 62, and the ure of GalNAc2-24a was shown in Example 104.

Table 104 Modified oli onucleotides tar ' etin SRB-l ISIS , , ED50 SEQ Sequences (5 to 3 ) GalNAc Cluster (mg/kg) 440762 TksmcksAdsGdsTdsmCdsAdsTdsGdsAdsmCdsTdsTkska 708561 GalNAc1'25"'°’Tk; CkSAdSijTdS CdSAdSTdSGdSAdS GalNAc1-25a 0.4 22 CdsTdsTks ck See e 93 for table legend. The structure of GalNAc1-25a was shown in Example 105.

The concentrations of the oligonucleotides in Tables 103 and 104 in liver were also assessed, using procedures described in Example 75. The results shown in Tables 104a and 104b below are the average total antisense oligonucleotide tissues levels for each ent group, as measured by UV in units of ug oligonucleotide per gram of liver . The s 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 ate 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 103 and 104 above.

Table 104a Liver concentrations of oligonucleotides comprising a GalNAc; or GalNAc; conjugate group - Dosage —_-ISIS No. [Antisense oligonucleotide] ("g/g) GalNAc cluster CM (mg/kg) 440762 ma 686221 GalNAcZ-24a 0.6 1.6 686222 GalNAC3'13a Ad Table 104b Liver concentrations of oligonucleotides comprising a GalNAcl conjugate group 5I 3' 0 W 0 ..\\O 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 e 10. The GalNAc1 cluster portion (GalNAc1-26a) 0f the conjugate group GalNAc1-26 can be combined with any cleavable moiety present on the ucleotide to provide a variety of conjugate groups. The structure of GalNAc1-26 (GalNAc1-26a-CM) is shown below: O ..|\O In order to add the GalNAc1 conjugate group to the 3’-end of an oligonucleotide, the amide formed from the on of compounds 47 and 64 is added to a solid t using procedures described in Example 7. The oligonucleotide synthesis is then completed using procedures described in Example 9 in order to form oligonucleotide 240.

HOk/OHO O "I OH‘ 240 3' 5' The GalNAc1 cluster portion (GalNAc1-27a) of the conjugate group 1-27 can be combined with any cleavable moiety present on the oligonucleotide to provide a variety of ate groups. The structure of GalNAc1-27 (GalNAc1-27a-CM) is shown below: O '"OH' o E Example 108: Antisense inhibition in vivo by ucleotides comprising a GalNAc conjugate group targeting Ap0(a) in vivo The oligonucleotides listed in Table 105 below were tested in a single dose study in mice.

Table 105 Modified ASOs targeting APO(a) , , GalNAc3 SEQ ces (5 to 3 ) CM No. Cluster ID No.

TesC}esmCesTesmCesmCdsC}dsTdsTdsC}dsC}dsTdsC}dsmCds 4 49 372 n/a n a/ 53 TdsTesGesTesTesmCe GalNAc3'7a'0’TesGesmCesTesmCesmCdsGdsTdsTdsGdsGds 681251 G lNAa C3-7a P0 53 TdsC}dsmCdsTdsTesC}es TesTesmCe 681255 GalNAc3'3a'o’Tesgeo CeoTeo Ceo CdsgdsTdsTdsGdsGds 3-3a P0 53 TdsCids TeoC}eo TesTes Ce GalNAc3'1Oa'o’TesGeomCeoTeomCeomCdsGdsTdsTdsGdsGds 681256 G lNAa C3-10a P0 53 Tds(LlsmcdsTdsTeoC}eo TesTesmCe GalNAc3'7a'o’TesGeomCeoTeomCeomCdsGdsTdsTdsGdsGds 681257 G lNAa C3-7a P0 53 Tds(LlsmcdsTdsTeoC}eo TesTesmCe GalNAc3'13a'o’TesGeomCeoTeomCeomCdsGdsTdsTdsGdsGds 681258 G lNAa C3-13a P0 53 Tds(LlsmcdsTdsTeoC}eo TesTesmCe TesC}eomceoTeomCeomcdsC}dsTdsTdsC}dsC}ds TdsC}dsn’lcdsTdsTeoC}eo 68 1260 G lNAa 03 - l 9a Ad 52 TesTesmCeoAdo,-GalNAc3-19 The structure of GalNAc3-7a was shown in Example 48.

Trealmenl Male transgenic mice that s human Apo(a) were each injected subcutaneously once with an oligonucleotide and dosage listed in Table 106 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. onal blood draws will occur weekly for approximately 8 weeks. Plasma Apo(a) protein levels were measured using an ELISA. The results in Table 106 are presented as the average percent of plasma Apo(a) protein levels for each treatment group, normalized to ne levels (% BL), The results show that the antisense oligonucleotides d Apo(a) protein expression. Furthermore, the ucleotides comprising a GalNAc conjugate group ted even more potent reduction in Apo(a) expression than the oligonucleotide that does not comprise a conjugate group.

Table 106 Apo(a) plasma protein levels ISIS No. Dosage (mg/kg) Ap0(&a1;114)week PBS Na 494372 50 681251 10 IIIIIIIIIEIIIIIIIII 681257 10 24 Example 109: Synthesis of oligonucleotides comprising a GalNAc1-28 or GalNAc1-29 conjugate OH 5' 3' HogowjkHO ..\O\- -mCM "WYN ACHN 241 OH Oligonucleotide 241 is synthesized using procedures similar to those described in Example 71 to form the phosphoramidite intermediate, followed by procedures bed in Example 10 to synthesize the oligonucleotide. The GalNAc1 cluster portion (GalNAc1-28a) of the conjugate group GalNAc1-28 can be combined with any cleavable moiety present on the ucleotide to provide a variety of conjugate groups.

The structure of GalNAc1-28 (GalNAc1-28a-CM) is shown below: HO .\O AcHN NW In order to add the 1 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 e 7. The oligonucleotide synthesis is then completed using procedures described in Example 9 in order to form oligonucleotide 242.

O .\\OH HO OM N ACHN "W 3| 5| 0 0—- w The GalNAc1 cluster portion (GalNAc1-29a) of the conjugate group GalNAc1-29 can be combined with any cleavable moiety present on the oligonucleotide to provide a variety of conjugate groups. The structure of GalNAc1-29 (GalNAc1-29a-CM) is shown below: O "OH HO O\/\/\)J\ N ACHN "W o 0—1; Example 110: Synthesis of ucleotides comprising a GalNAc1-30 conjugate OAC OAC AcO AcO 0 HO OTBDPS 0 A00 W AcO O\/\/\/OTBDPS N ACHN 7/0 243 1. NH /MeOH 0DMTr 2. DM§Fr0| A00 1. TBAF 3_ Ac20, pyr O 2. itilation AcO /OTBDPS —’ ODMTr 1. Couple to 5'-end of A80 ACO O\/\/\/O\P/OCE 245 "("302 2. Deprotect and purify ASO using DMT-on purification methods HO&/O 5' 3' HO O\/\/\/O\ /O\ ACHN I/P\ Oligonucleotide 246 comprising a GalNAc1-30 conjugate group, wherein Y is ed from O, S, a substituted or unsubstituted C1-C10 alkyl, amino, substituted amino, azido, alkenyl or alkynyl, is synthesized as shown above. The GalNAc1 cluster portion (GalNAc1-30a) of the conjugate group GalNAc1-30 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 GalNAc1-30a is shown below: Ho$womoxfeHO 0 Example 111: Synthesis of oligonucleotides comprising a 2-31 0r GalNAc2-32 conjugate DMTrO HO 1- DMTrCI IOCE CoupIe to 5‘-end of A80 2. Phosphitilation Bx 1. Remove DMTr groups DMTFO 2. Couple amidite 245 O O "'X —’ ‘p’ 3. Deprotect and purlfy ASO usmg HCAcHN Oligonucleotide 250 comprising a GalNAc2-31 conjugate group, wherein Y is selected from O, S, a substituted or unsubstituted C1-C10 alkyl, amino, substituted amino, azido, alkenyl or l, is synthesized as shown above. The GalNAcz r portion (GalNAc2-3la) of the ate group GalNAc2-31 can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the Y- OH fl0" Y 305 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 GalNAc2-3la is shown below: The synthesis of an oligonucleotide comprising a GalNAc2-32 conjugate is shown below. 1. DMTrCI 2. Allyl Br 3. 0304, NaIO4 1. Couple to 5'-end of A80 HO 4- NaBH4 DMTrO 2. Remove DMTr groups - ltllatlon__ _ 3. Couple amidite 245 OH O—\_ O 4. Deprotect and purify ASO using DMTrO HO :—PN(|Pi')2 DMT--on purification methods 247 CEO HO /O\ /O ' ' AcHN 0"P‘v 0" ’Y 07,F’\/O\/\O’ P‘o o O Y OH 0" Y HO O OJf/ Oligonucleotide 252 comprising a GalNAc2-32 conjugate group, wherein Y is selected from O, S, a substituted or unsubstituted C1-C10 alkyl, amino, substituted amino, azido, alkenyl or alkynyl, is synthesized as shown above. The GalNAcZ r portion (GalNAc2-32a) of the conjugate group GalNAc2-32 can be combined with any cleavable moiety to proVide a variety of ate groups. In n embodiments, the Y- ning 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 GalNAc2-32a is shown below: HO§&/O\/\/\/O\P\/OHO AcHN 0" Y }07,PCO\/\O}‘ o O Y OH fl0" Y HO O O Example 112: d oligonucleotides comprising a GalNAc1 conjugate The oligonucleotides in Table 107 targeting SRB-l were synthesized with a GalNAc1 conjugate group in order to further test the potency of oligonucleotides comprising conjugate groups that contain one GalNAc ligand.

Table 107 , , GalNAc SEQ ISIS No. ce (5 to 3 ) CM cluster ID NO. 711461 GalNAcl'ZSa-O’Ado Ges mCes Tes Tes mCes Ads Gds Tds mCds Ads Tds Ga1\AC '253, Ad 30 Gcs Ads mCds Tds Tes mCes mCes Tes Te 711462 GalNAcl'ZSa-O’Ges mCes Tes Tes mCes Ads Gds Tds mCds Ads Tds Gds Ga1\AC '253, P0 28 Acs mCds Tds Tes mCes mCes Tes Te 711463 GalNAcl-ZSM:Ges InCco T60 T60 InCco Ads Gds Tds mCds Ads Tds Gal\Ac -25a P0 28 Gcs Ads mCds Tds Teo mCeo mCes Tes Te 711465 GalNAc1'26a-O’Ado Ges mCes Tes Tes mCes Ads Gds Tds mCds Ads Tds Gal\AC '263, Ad 30 Gcs Ads mCds Tds Tes mCes mCes Tes Te 711466 GalNAc1'26a-O’Ges mCes Tes Tes mCes Ads Gds Tds mCds Ads Tds Gds Gal\AC -26a P0 28 Acs mCds Tds Tes mCes mCes Tes Te 711467 GalNAc1-26a_0,Ges mC60 T60 T60 mC60 Ads Gds Tds mCds Ads Tds Gal\Ac -26a P0 28 Gcs Ads mCds Tds Teo mCeo mCes Tes Te 711468 GalNAcl'Zsa-O’Ado Ges mCes Tes Tes mCes Ads Gds Tds mCds Ads Tds Ga1\AC '283, Ad 30 Gcs Ads mCds Tds Tes mCes mCes Tes Te 711469 GalNAcl'Zsa-O’Ges mCes Tes Tes mCes Ads Gds Tds mCds Ads Tds Gds Ga1\AC '283. P0 28 Acs mCds Tds Tes mCes mCes Tes Te 711470 GalNAcl'Zsa-O’Ges mCeo Teo Teo mCeo Ads Gds Tds mCds Ads Tds Ga1\AC '283, P0 28 Gcs Ads mCds Tds Teo mCeo mCes Tes Te 713844 Ges mCes Tee Tes mCes Ads Gds Tds mCds Ads Tds Gds Ads mCds Tds Ga1\AC '273. PO 2'8 Tes InCes InCes Tes Teo:_GalNAc1-27a 713845 Ges mCeo Teo Teo mCeo Ads Gds Tds mCds Ads Tds Gds Ads mCds Tds Gal\AC '273, PO 28 Tco InCco InCes Tes T60,_GalNAc1-27a 713846 Ges mCeo Teo Teo mCeo Ads Gds Tds mCds Ads Tds Gds Ads mCds Tds Ga1\AC '273, Ad 29 Teo mCeo mCes Tes Teo Ad0’-GalNAc1'27a 713847 Ges mCes Tee Tes mCes Ads Gds Tds mCds Ads Tds Gds Ads mCds Tds Ga1\AC '293. P0 28 Tes mCes mCes Tes Teo:_GalNAc1-29a 713848 Ges mCeo Teo Teo mCeo Ads Gds Tds mCds Ads Tds Gds Ads mCds Tds Ga1\AC '293, P0 28 T60 InCCO InCes Tes Te(,,_GalNAc1-29a 713849 Ges mCes Tee Tes mCes Ads Gds Tds mCds Ads Tds Gds Ads mCds Tds Ga1\AC '293, Ad 2'9 Tes mCes mCes Tes Teo Ad0’-GalNAc1'29a 713850 Ges mCeo Teo Teo mCeo Ads Gds Tds mCds Ads Tds Gds Ads mCds Tds Gal\AC '293, Ad 29 Teo mCeo mCes Tes Teo alNAc1'29a Example 113: Modified ucleotides comprising a GalNAc conjugate group targeting Hepatits B Virus (HBV) The oligonucleotides listed in Table 108 below were designed to target HBV. In n embodiments, the cleavable moiety is a phosphodiester linkage.

Table 108 Sequences (5’ t0 3’) SEQ ID No.

GalNAc3GesmCesAesGmAes dsGdsTdsGdsAdsAdsGdsmCdsGdsAdsAesGesTesGesmce GalNAc3GesmCeoAmGmACOQQQQdsGdsTdsGdsAdsAdsGdsmcdsGdsAdsAeoGeoTesGesmCe GalNAc3GesmCesAesGfiAes dsGdsTdsGdsAdsAdsGdsmCdsGdSAdsAesGesTesGesmCe GalNAc3GesmCeeroGeero dSGdsTdsGdsAdsAdsGdSmCdSGdSAdsAeoGeoTfiGesmCe GalNAcgGesmCesAesGCSAesGdsGdSTdSGdSAdsAdsGdsmCdsGdSAdsAesGesTesGesmCe GalNAc3GesmCeeroGeeroGdsGdsTdsGdsAdsAdsGdSmCdsGdsAdSAmeTeSGeSmCe GalNAc3GesmCesAesGESAesGdsGdSTdsGdSAdsAdsGdsmCdsGdsAdsAesGesTesGesmCe GalNAc3GesmCeeroGeeroGdsGdsTdsGdsAdsAdsGdSmCdsGdsAdSAmeTeSGeSmCe GCSmCeSAeSGeSAfiGdsGdsTdsGdsAdSAdsGdSmCdsGdsAdsAesGesTfiGesmCe-GalNAc3-19 eroGeeroGdSGdSTdSGdSAdSAdSGdSmCdsGdsAdsAeoGeoTesGesmCe-GalNAc3-l9 GalNAc3'24'GesmCesAesGesAesGdsGdsTdsGdsAdsAdsGdsmCdsGdsAdsAesGesTesGesmCe GalNAc3'24'GesmCeOAeoGeeroGdsGdsTdsGdsAdsAdsGdsmcdsGdsAdsAeoGeoTesGesmCe 3-2S'GeSmCeSAeSGesAesGdsGdsTdsGdsAdsAdsGdsmCdsGdsAdsAesGesTesGesmCe GalNAc3'2S'GesmCeOAeoGeeroGdsGdsTdsGdsAdsAdsGdsmcdsGdsAdsAeoGeoTesGesmCe

Claims (33)

CLAIMS :

1. A compound comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 16 to 30 linked nucleosides and comprises a base sequence at least 85% complementary to SEQ ID NO: 2, n the conjugate group comprises:

2. The compound of claim 1, wherein the modified oligonucleotide ses at least one modified sugar.

3. The compound of claim 2, wherein at least one modified sugar is a bicyclic sugar.

4. The compound of either one of claims 2 or 3, 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.

5. The compound of claim 2, wherein at least one modified sugar is 2’-O-methoxyethyl.

6. The compound of any one of claims 1-5, wherein at least one nucleoside comprises a ed nucleobase.

7. The compound of claim 6, wherein the modified nucleobase is a 5-methylcytosine.

8. The compound of any one of claims 1-7, wherein the conjugate group is linked to the modified oligonucleotide at the 5’ end of the modified oligonucleotide.

9. The compound of any one of claims 1-7, wherein the conjugate group is linked to the modified oligonucleotide at the 3’ end of the modified ucleotide.

10. The compound of any one of claims 1-9, wherein each internucleoside linkage of the modified oligonucleotide is selected from a phosphodiester internucleoside linkage and a phosphorothioate internucleoside linkage.

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

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

13. The compound of any one of claims 1-12, wherein the modified oligonucleotide is single- stranded.

14. The compound of any one of claims 1-12, wherein the modified oligonucleotide is double stranded.

15. The compound of any one of claims 1-14, wherein the ed oligonucleotide 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 oned between the 5’ wing segment and the 3’ wing segment and wherein each nucleoside of each wing segment ses a modified sugar.

16. The compound of claim 15, wherein each internucleoside linkage in the gap t of the modified oligonucleotide is a phosphorothioate linkage.

17. The compound of claim 16, wherein the modified oligonucleotide further comprises at least one phosphorothioate internucleoside linkage in each wing segment.

18. The compound of any one of claims 1-17, wherein the base sequence of the modified ucleotide is at least 90% complementary to SEQ ID NO: 2.

19. The compound of any one of claims 1-17, wherein the base ce of the modified oligonucleotide is at least 95% complementary to SEQ ID NO: 2.

20. The compound of any one of claims 1-17, wherein the nucleobase sequence of the modified oligonucleotide is 100% complementary to SEQ ID NO: 2.

21. The compound of any one of claims 1-20, wherein the modified oligonucleotide comprises the nucleobase sequence of any of SEQ ID NOs: 12-19.

22. The compound of any one of claims 1-20, wherein the modified oligonucleotide ts of linked nucleosides consisting of the nucleobase sequence of any of SEQ ID NOs: 12-

23. The compound of claim 1, wherein the modified ucleotide consists of 20 linked nucleosides having a nucleobase sequence consisting of SEQ ID NO: 12, and wherein the modified oligonucleotide comprises: a gap t 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 n the 5’ wing segment and the 3’ wing segment, wherein each nucleoside of each wing segment comprises a 2’-O-methoxyethyl sugar, and wherein each cytosine residue is a 5-methylcytosine.

24. The nd of claim 1, wherein the modified oligonucleotide consists of 20 linked nucleosides having a nucleobase sequence consisting of any one of SEQ ID NOs: 13-19, and wherein the modified oligonucleotide comprises: a gap segment ting 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 t, wherein each nucleoside of each wing t ses a 2’-O-methoxyethyl sugar, and wherein each cytosine residue is a 5-methylcytosine.

25. The compound of either one of claims 23 or 24, wherein each internucleoside linkage in the gap segment of the modified oligonucleotide is a phosphorothioate linkage.

26. The nd of claim 25, wherein the modified oligonucleotide further comprises at least one phosphorothioate internucleoside linkage in each wing segment.

27. The compound of either one of claims 23 or 24, wherein each internucleoside linkage of the modified oligonucleotide is a phosphorothioate linkage.

28. The compound of any one of claims 23-27, wherein the wherein the modified oligonucleotide is -stranded.

29. The compound of claim 1, wherein the compound consists of the formula: , or a pharmaceutically acceptable salt thereof.

30. The compound of claim 1, n the compound consists of the formula:

31. A composition comprising the compound of any one of claims 1-30 and a pharmaceutically acceptable carrier or diluent.

32. Use of the compound of any one of claims 1-30, or the composition of claim 31, in the manufacture of a ment for treating transthyretin amyloidosis.

33. The use according to claim 32, n the transthyretin amyloidosis is senile systemic amyloidosis (SSA), familial amyloid polyneuropathy (FAP), or familial amyloid cardiopathy (FAC).