Extrahepatic delivery

Abstract

The invention relates to a method of gene silencing, comprising administering to a cell or a subject in need thereof a therapeutically effective amount of the lipophilic moieties-conjugated double- stranded iRNAs at one or more internal positions on at least one strand, optionally via a linker or carrier.

Description

Extrahepatic Delivery

[0001] This application claims benefit of priority to U.S. Provisional Application No. 62 / 668,072 filed May 7, 2018; U.S. Provisional Application No.62 / 738,747 filed September 28, 2018; and U.S. Provisional Application No.62 / 773,082 filed November 29, 2018, all of which are herein incorporated by reference in their entirety. BACKGROUND

[0002] Efficient delivery of an iRNA agent to cells in vivo requires specific targeting and substantial protection from the extracellular environment, particularly serum proteins. RNAi- based therapeutics show promising clinical data for treatment of liver-associated disorders. However, siRNA delivery into extra-hepatic tissues remains an obstacle, limiting the use of siRNA-based therapies.

[0003] One of the factors that limit the experimental and therapeutic application of iRNA agents in vivo is the ability to deliver intact siRNA efficiently. Particular difficulties have been associated with non-viral gene transfer into the retina in vivo. One of the challenges is to overcome the inner limiting membrane, which impedes the transfection of the retina.Additionally, negatively charged sugars of the vitreous have been shown to interact with positive DNA-transfection reagent complexes, promoting their aggregation, which impedes diffusion and cellular uptake.

[0004] Delivery of oligonucleotides to the central nervous system (CNS) poses particular problems due to the blood brain barrier (BBB) that free oligonucleotides cannot cross. One means to deliver oligonucleotides into the CNS is by intrathecal delivery. However, the oligonucleotides need also to be efficiently internalized into target cells of the CNS to achieve the desired therapeutic effect. Previous work has typically used delivery reagents such as liposomes, cationic lipids, and nanoparticles forming complexes to aid the intracellular internalization of oligonucleotides into cells of neuronal origin.

[0005] Thus, there is a continuing need for new and improved methods for delivering siRNA molecules in vivo, without the use of tissue delivery reagents, to achieve and enhance the therapeutic potential of iRNA agents. SUMMARY

[0006] One aspect of the invention provides a double-stranded iRNA agent comprising:an antisense strand which is complementary to a target gene; a sense strand which is complementary to said antisense strand; and one or more lipophilic moieties conjugated to one or more internal positions on at least one strand, optionally via a linker or carrier.

[0007] In some embodiments, the lipophilicity of the lipophilic moiety, measured by octanol-water partition coefficient, logKow, exceeds 0. The lipophilic moiety may possess a logKow exceeding 1, exceeding 1.5, exceeding 2, exceeding 3, exceeding 4, exceeding 5, or exceeding 10.

[0008] In some embodiments, the hydrophobicity of the double-stranded iRNA agent, measured by the unbound fraction in the plasma protein binding assay of the double-stranded iRNA agent, exceeds 0.2. In one embodiment, the plasma protein binding assay determined is an electrophoretic mobility shift assay (EMSA) using human serum albumin protein. The hydrophobicity of the double-stranded iRNA agent, measured by fraction of unbound siRNA in the binding assay, exceeds 0.15, exceeds 0.2, exceeds 0.25, exceeds 0.3, exceeds 0.35, exceeds 0.4, exceeds 0.45, or exceeds 0.5 for an enhanced in vivo delivery of siRNA.

[0009] In some embodiments, the lipophilic moiety is an aliphatic, cylic such as alicyclic, or polycyclic such as polyalicyclic compound, such as a steroid (e.g., sterol) or a linear or branched aliphatic hydrocarbon. Exemplary lipophilic moieties are lipid, cholesterol, retinoic acid, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis- O(hexadecyl)glycerol, geranyloxyhexyanol, hexadecylglycerol, borneol, menthol, 1,3- propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3- (oleoyl)cholenic acid, ibuprofen, naproxen, dimethoxytrityl, or phenoxazine.

[0010] Suitable lipophilic moieties also include those containing a saturated or unsaturated C4-C30hydrocarbon chain (e.g., C4-C30alkyl or alkenyl), and an optional functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne. The functional groups are useful to attach the lipophilic moiety to the iRNA agent. In some embodiments, the lipophilic moiety contains a saturated or unsaturated C6-C18hydrocarbon chain (e.g., a linear C6-C18alkyl or alkenyl). In one embodiment, the lipophilic moiety contains a saturated or unsaturated C16 hydrocarbon chain (e.g., a linear C16 alkyl or alkenyl).

[0011] In some embodiments, the lipophilic moiety is a C6-C30acid (e.g., hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, dodcanoic acid, tridecanoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, oleic acid, linoleic acid, arachidonic acid, cis-4,7,10,13,16,19-docosahexanoic acid, vitamin A, vitamin E, cholesterol etc.) or a C6-C30alcohol (e.g., hexanol, heptanol, octanol, nonanol, decanol, undecanol, dodcanol, tridecanol, tetradecanol, pentadecanol, hexadecanol, heptadecanol, octadecanol, oleyl alcohol, linoleyl alcohol, arachidonic alcohol, cis-4,7,10,13,16,19-docosahexanol, retinol, vitamin E, cholesterol etc.).

[0012] The lipophilic moiety may be conjugated to the iRNA agent via a direct attachment to the ribosugar of the iRNA agent. Alternatively, the lipophilic moiety may be conjugated to the iRNA agent via a linker or a carrier.

[0013] In certain embodiments, the lipophilic moiety are conjugated to the iRNA agent via one or more linkers (tethers).

[0014] In some embodiments, the lipophilic moiety is conjugated to the double-stranded iRNA agent via a linker a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, a product of a click reaction (e.g., a triazole from the azide-alkyne cycloaddition), or carbamate.

[0015] In some embodiments, at least one of the linkers (tethers) is a redox cleavable linker (such as a reductively cleavable linker; e.g., a disulfide group), an acid cleavable linker (e.g., a hydrazone group, an ester group, an acetal group, or a ketal group), an esterase cleavable linker (e.g., an ester group), a phosphatase cleavable linker (e.g., a phosphate group), or a peptidase cleavable linker (e.g., a peptide bond).

[0016] In other embodiments, at least one of the linkers (tethers) is a bio-clevable linker selected from the group consisting of DNA, RNA, disulfide, amide, functionalized monosaccharides or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

[0017] In certain embodiments, the lipophilic moiety is conjugated to the double-stranded iRNA agent via a carrier that replaces one or more nucleotide(s). The carrier can be a cyclic group or an acyclic group. In one embodiment, the cyclic group is selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, and decalin. In one embodiment, the acyclic group is a moiety based on a serinol backbone or adiethanolamine backbone.

[0018] In some embodiments, the carrier replaces one or more nucleotide(s) in the internal position(s) of the double-stranded iRNA agent.

[0019] In other embodiments, the carrier replaces the nucleotides at the terminal end of the sense strand or antisense strand. In one embodiment, the carrier replaces the terminal nucleotide on the 3’ end of the sense strand, thereby functioning as an end cap protecting the 3’ end of the sense strand. In one embodiment, the carrier is a cyclic group having an amine, for instance, the carrier may be pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, or decalinyl.

[0020] In one embodiment, the lipophilic moiety is conjugated to one or more internal positions on at least one strand, which include all positions except the terminal two positions from each end of the strand. In one embodiment, the lipophilic moiety is conjugated to one or more internal positions on at least one strand, which include all positions except the terminal three positions from each end of the strand.

[0021] In one embodiment, at least one lipophilic moiety is conjugated to one or more positions of at least one end of the duplex region, which include all positions within the duplex region, but not include the overhang region or the carrier that replaces the terminal nucleotide on the 3’ end of the sense strand.

[0022] In one embodiment, at least one lipophilic moiety is conjugated on the sense strand within the first five base pairs at the 5’-end of the antisense strand of the duplex region.

[0023] In one embodiment, at least one lipophilic moiety is conjugated on the sense strand within the first four base pairs at the 5’-end of the antisense strand of the duplex region.

[0024] In one embodiment, at least one lipophilic moiety is conjugated on the sense strand within the first three base pairs at the 5’-end of the antisense strand of the duplex region.

[0025] In one embodiment, at least one lipophilic moiety is conjugated on the sense strand within the first two base pairs at the 5’-end of the antisense strand of the duplex region.

[0026] In one embodiment, at least one lipophilic moiety is conjugated on the sense strand on the first base pair at the 5’-end of the antisense strand of the duplex region.

[0027] In one embodiment, the lipophilic moiety is conjugated to one or more internal positions on at least one strand, which exclude the cleavage site region of the sense strand. For instance, the internal positions exclude positions 9-12 counting from the 5’-end of thesense strand. For example, the internal positions exclude positions 9-11 counting from the 5’-end of the sense strand. Alternatively, the internal positions exclude positions 11-13 counting from the 3’-end of the sense strand.

[0028] In one embodiment, the lipophilic moiety is conjugated to one or more internal positions on at least one strand, which exclude the cleavage site region of the antisense strand. For instance, the internal positions exclude positions 12-14 counting from the 5’-end of the antisense strand.

[0029] In one embodiment, the lipophilic moiety is conjugated to one or more internal positions on at least one strand, which exclude positions 11-13 on the sense strand, counting from the 3’-end, and positions 12-14 on the antisense strand, counting from the 5’-end.

[0030] In one embodiment, one or more lipophilic moieties are conjugated to one or more of the following internal positions: positions 4-8 and 13-18 on the sense strand, and positions 6-10 and 15-18 on the antisense strand, counting from the 5’end of each strand.

[0031] In one embodiment, one or more lipophilic moieties are conjugated to one or more of the following internal positions: positions 5, 6, 7, 15, and 17 on the sense strand, and positions 15 and 17 on the antisense strand, counting from the 5’end of each strand.

[0032] In some embodiments, the sense and antisense strands of the double-stranded iRNA agent are each 15 to 30 nucleotides in length.

[0033] In one embodiment, the sense and antisense strands of a double-stranded iRNA agent are each 19 to 25 nucleotides in length.

[0034] In one embodiment, the sense and antisense strands of the double-stranded iRNA agent are each 21 to 23 nucleotides in length.

[0035] In some embodiments, the double-stranded iRNA agent comprises a single- stranded overhang on at least one of the termini, e.g., 3’ and / or 5’ overhang(s) of 1-10 nucleotides in length, for instance, an overhang of 1, 2, 3, 4, 5, or 6 nucleotides. In some embodiments, both strands have at least one stretch of 1-5 (e.g., 1, 2, 3, 4, or 5) single- stranded nucleotides in the double stranded region. In one embodiment, the single-stranded overhang is 1, 2, or 3 nucleotides in length. In some embodiments, the double-stranded iRNA agent may also have a blunt end, located at the 5’-end of the antisense strand (or the 3’-end of the sense strand), or vice versa. In one embodiment, the double-stranded iRNA agent comprises a 3’ overhang at the 3’-end of the antisense strand, and optionally a blunt end at the 5’-end of the antisense strand. In one embodiment, the double-stranded iRNA agent has a 5’ overhang at the 5’-end of the sense strand, and optionally a blunt end at the 5’-end of the antisense strand. In one embodiment, the double-stranded iRNA agent has two blunt ends at both ends of the iRNA duplex.

[0036] In one embodiment, the sense strand of the double-stranded iRNA agent is 21- nucleotides in length, and the antisense strand is 23-nucleotides in length, wherein the strands form a double-stranded region of 21 consecutive base pairs having a 2-nucleotide long single-stranded overhangs at the 3’-end.

[0037] In some embodiments, the lipophilic moiety is conjugated to a nucleobase, sugar moiety, or internucleosidic linkage of the double-stranded iRNA agent.

[0038] In some embodiments, the double-stranded iRNA agent further comprises a phosphate or phosphate mimic at the 5’-end of the antisense strand. In one embodiment, the phosphate mimic is a 5’-vinyl phosphonate (VP).

[0039] In some embodiments, the 5’-end of the antisense strand of the double-stranded iRNA agent does not contain a 5’-vinyl phosphonate (VP).

[0040] In some embodiments, the double-stranded iRNA agent further comprises at least one terminal, chiral phosphorus atom.

[0041] A site specific, chiral modification to the internucleotide linkage may occur at the 5’ end, 3’ end, or both the 5’ end and 3’ end of a strand. This is being referred to herein as a “terminal” chiral modification. The terminal modification may occur at a 3’ or 5’ terminal position in a terminal region, e.g., at a position on a terminal nucleotide or within the last 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides of a strand. A chiral modification may occur on the sense strand, antisense strand, or both the sense strand and antisense strand. Each of the chiral pure phosphorus atoms may be in either Rp configuration or Sp configuration, and combination thereof. More details regarding chiral modifications and chirally-modified dsRNA agents can be found in PCT / US18 / 67103, entitled“Chirally-Modified Double-Stranded RNA Agents,” filed December 21, 2018, which is incorporated herein by reference in its entirety.

[0042] In some embodiments, the double-stranded iRNA agent further comprises a terminal, chiral modification occuring at the first internucleotide linkage at the 3’ end of the antisense strand, having the linkage phosphorus atom in Sp configuration; a terminal, chiral modification occuring at the first internucleotide linkage at the 5’ end of the antisense strand, having the linkage phosphorus atom in Rp configuration; and a terminal, chiral modification occuring at the first internucleotide linkage at the 5’ end of the sense strand, having the linkage phosphorus atom in either Rp configuration or Sp configuration.

[0043] In one embodiment, the double-stranded iRNA agent further comprises a terminal, chiral modification occuring at the first and second internucleotide linkages at the 3’ end of the antisense strand, having the linkage phosphorus atom in Sp configuration; a terminal, chiral modification occuring at the first internucleotide linkage at the 5’ end of the antisense strand, having the linkage phosphorus atom in Rp configuration; and a terminal, chiral modification occuring at the first internucleotide linkage at the 5’ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.

[0044] In one embodiment, the double-stranded iRNA agent further comprises a terminal, chiral modification occuring at the first, second, and third internucleotide linkages at the 3’ end of the antisense strand, having the linkage phosphorus atom in Sp configuration; a terminal, chiral modification occuring at the first internucleotide linkage at the 5’ end of the antisense strand, having the linkage phosphorus atom in Rp configuration; and a terminal, chiral modification occuring at the first internucleotide linkage at the 5’ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.

[0045] In one embodiment, the double-stranded iRNA agent further comprises a terminal, chiral modification occuring at the first and second internucleotide linkages at the 3’ end of the antisense strand, having the linkage phosphorus atom in Sp configuration; a terminal, chiral modification occuring at the third internucleotide linkages at the 3’ end of the antisense strand, having the linkage phosphorus atom in Rp configuration; a terminal, chiral modification occuring at the first internucleotide linkage at the 5’ end of the antisense strand, having the linkage phosphorus atom in Rp configuration; and a terminal, chiral modification occuring at the first internucleotide linkage at the 5’ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.

[0046] In one embodiment, the double-stranded iRNA agent further comprises a terminal, chiral modification occuring at the first and second internucleotide linkages at the 3’ end of the antisense strand, having the linkage phosphorus atom in Sp configuration; a terminal, chiral modification occuring at the first, and second internucleotide linkages at the 5’ end of the antisense strand, having the linkage phosphorus atom in Rp configuration; and a terminal, chiral modification occuring at the first internucleotide linkage at the 5’ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.

[0047] In some embodiments, the double-stranded iRNA agent has at least two phosphorothioate internucleotide linkages at the first five nucleotides on the antisense strand (counting from the 5’ end).

[0048] In some embodiments, the antisense strand comprises two blocks of one, two, or three phosphorothioate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate internucleotide linkages.

[0049] In some embodiments, the double-stranded iRNA agent further comprises a targeting ligand that targets a receptor which mediates delivery to a specific CNS tissue. In one embodiment, the targeting ligand is selected from the group consisting of Angiopep-2, lipoprotein receptor related protein (LRP) ligand, bEnd.3 cell binding ligand, transferrin receptor (TfR) ligand, manose receptor ligand, glucose transporter protein, and LDL receptor ligand.

[0050] In some embodiments, the double-stranded iRNA agent further comprises a targeting ligand that targets a receptor which mediates delivery to an ocular tissue. In one embodiment, the targeting ligand is selected from the group consisting of trans-retinol, RGD peptide, LDL receptor ligand, and carbohydrate-based ligands. In one embodiment, the targeting ligand is a RGD peptide, such as H-Gly-Arg-Gly-Asp-Ser-Pro-Lys-Cys-OH or Cyclo(-Arg-Gly-Asp-D-Phe-Cys).

[0051] In some embodiments, the double-stranded iRNA agent further comprises a targeting ligand that targets a liver tissue. In some embodiments, the targeting ligand is a carbohydrate-based ligand. In one embodiment, the targeting ligand is a GalNAc conjugate.

[0052] All the above aspects and embodiments would be applicable to an oligonucleotide having one or more lipophilic moieties conjugated to one or more internal positions on the oligonucleotide. In some embodiments, 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35% or 30% of the oligonucleotide is modified. For example, when 50% of the oligonucleotide is modified, 50% of all nucleotides present in the oligonucleotide contain a modification as described herein.

[0053] In one embodiment, the oligonucleotide is a double-stranded dsRNA agent, and at least 50% of the nucleotides of the double-stranded dsRNA agent is independently modified with 2’- O-methyl, 2’-O-allyl, 2’-deoxy, or 2’-fluoro.

[0054] In one embodiment, the oligonucleotide is an antisense, and at least 50% of the nucleotides of the antisense is independently modified with LNA, CeNA, 2’-methoxyethyl, or 2’-deoxy.

[0055] In some embodiments, the double-stranded iRNA agent has less than 12, less than 10, less than 8, less than 6, less than 4, less than 2, or no 2’-F modifications on the sense strand. In some embodiments, the double-stranded iRNA agent has less than 12, less than 10,less than 8, less than 6, less than 4, less than 2, or no 2’-F modifications on the antisense strand.

[0056] In some embodiments, the double-stranded iRNA agent has one or more 2’-F modifications on any position of the sense strand or antisense strand.

[0057] In some embodiments, the double-stranded iRNA agent has less than 20%, less than 15%, less than 10%, less than 5% non-natural nucleotide, or substantially no non-natural nucleotide. Examples of non-natural nucleotide include acyclic nucleotides, LNA, HNA, CeNA, 2’-O-methoxyalkyl (e.g., 2’-O-methoxymethyl, 2’-O-methoxyethyl, or 2’-O-2- methoxypropanyl), 2’-O-allyl, 2’-C-allyl, 2’-fluoro, 2'-O-N-methylacetamido (2'-O-NMA), a 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), 2'-ara-F, L- nucleoside modification (such as 2’-modified L-nucleoside, e.g., 2’-deoxy-L-nucleoside), BNA abasic sugar, abasic cyclic and open-chain alkyl.

[0058] In some embodiments, the double-stranded iRNA agent has greater than 80%, greater than 85%, greater than 90%, greater than 95%, or virtually 100% natural nucleotides. For the purpose of these embodiments, natural nucleotides can include those having 2’-OH, 2’-deoxy, and 2’-OMe.

[0059] In one embodiment, the double-stranded iRNA agent comprises a sense strand and antisense strand each having a length of 15-30 nucleotides; at least two phosphorothioate internucleotide linkages at the first five nucleotides on the antisense strand (counting from the 5’ end); wherein the duplex region is between 19 to 25 base pairs (preferably 19, 20, 21 or 22); wherein the double-stranded iRNA agent has less than 20%, less than 15%, less than 10%, less than 5% non-natural nucleotide, or substantially no non-natural nucleotide.

[0060] In one embodiment, the double-stranded iRNA agent comprises a sense strand and antisense strand each having a length of 15-30 nucleotides; at least two phosphorothioate internucleotide linkages at the first five nucleotides on the antisense strand (counting from the 5’ end); wherein the duplex region is between 19 to 25 base pairs (preferably 19, 20, 21 or 22); wherein the double-stranded iRNA agent has greater than 80%, greater than 85%, greater than 95%, or virtually 100% natural nucleotides, such as those having 2’-OH, 2’-deoxy, or 2’-OMe.

[0061] Another aspect of the invention relates to a method of reducing the expression of a target gene in a cell, comprising contacting said cell with a double-stranded iRNA agent comprising an antisense strand which is complementary to a target gene; a sense strand whichis complementary to said antisense strand; and one or more lipophilic moieties conjugated to one or more internal positions on at least one strand, optionally via a linker or carrier.

[0062] All the above embodiments relating to the lipophilic moieties and theirconjugation to the double-stranded iRNA agent in the first aspect of the invention relating to the double-stranded iRNA agent are suitable in this aspect of the invention relating to a method of reducing the expression of a target gene in a cell.

[0063] In one embodiment, the cell is an extraheptic cell.

[0064] Another aspect of the invention relates to a method of reducing the expression of a target gene in a subject, comprising administering to the subject a double-stranded iRNA agent comprising contacting said cell with a double-stranded iRNA agent comprising an antisense strand which is complementary to a target gene; a sense strand which iscomplementary to said antisense strand; and one or more lipophilic moieties conjugated to one or more internal positions on at least one strand, optionally via a linker or carrier.

[0065] All the above embodiments relating to the lipophilic moieties and theirconjugation to the double-stranded iRNA agent in the first aspect of the invention relating to the double-stranded iRNA agent are suitable in this aspect of the invention relating to a method of reducing the expression of a target gene in a subject.

[0066] In some embodiments, the double-stranded iRNA agent is administered extrahepatically.

[0067] In one embodiment, the double-stranded iRNA agent is administered intrathecally. By intrathecal administration of the double-stranded iRNA agent, the method can reduce the expression of a target gene in a brain or spine tissue, for instance, cortex, cerebellum, cervical spine, lumbar spine, and thoracic spine.

[0068] In some embodiments, exemplary target genes are APP, ATXN2, C9orf72, TARDBP, MAPT(Tau), HTT, SNCA, FUS, ATXN3, ATXN1, SCA1, SCA7, SCA8, MeCP2, PRNP, SOD1, DMPK, and TTR. To reduce the expression of these target genes in the subject, the double-stranded iRNA agent can be administered intravitreally. By intravitreal administration of the double-stranded iRNA agent, the method can reduce the expression of the target gene in an ocular tissue.

[0069] Another aspect of the invention relates to a method of treating a subject having a CNS disorder, comprising administering to the subject a therapeutically effective amount of a double-stranded RNAi agent, thereby treating the subject. The double-stranded RNAi agent comprises an antisense strand which is complementary to a target gene; a sense strand whichis complementary to said antisense strand; and one or more lipophilic moieties conjugated to one or more internal positions on at least one strand, optionally via a linker or carrier.

[0070] All the above embodiments relating to the lipophilic moieties and their conjugation to the double-stranded iRNA agent in the first aspect of the invention relating to the double-stranded iRNA agent are suitable in this aspect of the invention relating to a method of treating a subject having a CNS disorder. Exemplary CNS disorders that can be treated by the method of the invention include alzheimer, amyotrophic lateral schlerosis (ALS), frontotemporal dementia, huntington, Parkinson, spinocerebellar, prion, and lafora. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 is a scheme showing ligands, such as lipophilic moieties, that are conjugated to siRNAs at internal positions of the sense or antisense strand (i.e., somewhere within the siRNA sequence).

[0072] Figure 2 is a scheme showing ligands, such as lipophilic moieties, that are conjugated to siRNAs through linkers or carriers at the 3’- and / or 5’-ends of the sense or antisense strand.

[0073] Figure 3 is a scheme showing ligands, such as lipophilic moieties, that are conjugated to siRNAs via bio-cleavable linkers.

[0074] Figure 4 is a graph showing the results of beta catenin gene (ocular CTNNB1) silencing by an intravitreal injection of various exemplary siRNA conjugates in mice.

[0075] Figure 5 is a graph showing the results of SOD1 mRNA silencing by a single intrathecal injection of various exemplary siRNA conjugates in Cortex of Sprague Dawley Rats.

[0076] Figure 6 is a graph showing the results of SOD1 mRNA silencing by a single intrathecal injection of various exemplary siRNA conjugates in Cerebellum of Sprague Dawley Rats.

[0077] Figure 7 is a graph showing the results of SOD1 mRNA silencing by a single intrathecal injection of various exemplary siRNA conjugates in Cervical Spine of Sprague Dawley Rats.

[0078] Figure 8 is a graph showing the results of SOD1 mRNA silencing by a single intrathecal injection of various exemplary siRNA conjugates in Lumbar Spine of Sprague Dawley Rats.

[0079] Figure 9 is a graph showing the results of SOD1 mRNA silencing by a single intrathecal injection of various exemplary siRNA conjugates in Thoracic Spine of Sprague Dawley Rats.

[0080] Figure 10 shows the results of primay cyno hepatocyte (PCH) free uptake (without transfection agent) for cells incubated with a F12 siRNA, modified by conjugating a lipophilic moiety (C16) at each position of the antisence strand and sense strand, at 2.5 and 250 nM concentrations by measuring F12 mRNA levels after 24 hours using RT-qPCR.

[0081] Figure 11 shows the results of primay cyno hepatocyte (PCH) free uptake (without transfection agent) for cells incubated with a F12 siRNA, modified by conjugating a lipophilic moiety (C16) at each position of the antisence strand and sense strand, at 2.5 and 250 nM concentrations by measuring F12 mRNA levels after 24 hours using RT-qPCR.

[0082] Figure 12 shows the results of relative hydrophobicity for each position of the antisence strand and sense strand of an siRNA duplex, modified by conjugating a lipophilic moiety (C16) at each position of the antisence strand and sense strand, determined by measuring the unbound fraction using an electrophoretic mobility shift assay after each siRNA conjugate was incubated with human serum albumin.

[0083] Figures 13A-13C show that durable SOD1 mRNA silencing is seen in all regions of the brain and spinal cord tested. Figure 13A shows the results of SOD1 mRNA silencing by a single intrathecal injection of various exemplary siRNA conjugates in rats in lumbar spine, thoracic spine, and cervical spine regions, respectively. Figure 13B is a schemetic showing of various tissues tested in the CNS of rats. Figure 13C shows the results of SOD1 mRNA silencing by a single intrathecal injection of various exemplary siRNA conjugates in rats in cerebellum, frontal cortex, and remaining brain regions, respectively.

[0084] Figures 14A-14B show the results of silencing of b-catenin following a single intrathecal dose. Figure 14A shows the results of b-catenin silencing of various exemplary siRNA conjugates in rats in lumbar spine, thoracic spine, and cervical spine regions, respectively. Figure 14B shows the results of b-catenin silencing of various exemplary siRNA conjugates in rats in cerebellum, frontal cortex, and remaining brain regions, respectively.

[0085] Figures 15A-15C show the results of silencing of SOD1 following a single intrathecal dosing of exemplary siRNA duplexes in rats, indicating higher drug levels and robust silencing observed in brain with SOD1 siRNA conjugate. Figure 15A shows the conjugated siRNA levels in CSF, as compared to the unconjugated siRNA levels. Figure 15Bshows the conjugated siRNA levels in brain, as compared to the unconjugated siRNA levels. Figure 15C shows the conjugated siRNA levels in cerebellum, as compared to theunconjugated siRNA levels and control siRNA levels.

[0086] Figures 16A-16B show the results of silencing of SOD1 with different chemistry modifications at various doses. Figure 16A shows the results of SOD1 silencing in rats in lumbar spine, thoracic spine, and cervical spine regions, respectively. Figure 16B shows the results of SOD1 silencing in rats in cerebellum, frontal cortex, and remaining brain regions, respectively.

[0087] Figure 17 shows the results of b-catenin siRNA levels following a single intrathecal (IT) dosing of an exemplary siRNA duplex in various regions of non-human primate (NHP) at Day 31.

[0088] Figure 18 shows the results of robust gene silencing of b-catenin mRNA in various tissues, at Day 31.

[0089] Figure 19 shows pictures illustrating siRNAs distributed throughout the CNS in NHP, following the single IT dosing.

[0090] Figure 20 shows pictures illustrating the siRNA conjugates localized to neurons, following the single IT dosing. MAP2 is a neuronal marker.

[0091] Figure 21 shows pictures illustrating the siRNA conjugates localized to microglia, following the single IT dosing. Iba1 is a microglia marker.

[0092] Figure 22 shows pictures illustrating the siRNA conjugates localized to astrocytes, following the single IT dosing.

[0093] Figure 23 shows a graph comparing the gene silencing activity observed in rats and NHP at compartment scaled dose.

[0094] Figure 24 shows the results of TTR mRNA levels in the eye of mice at Day 14 following administrating various exemplary siRNA duplexes shown in Table 7, at a dosage of 3 mg or 7.5 mg.

[0095] Figure 25 shows the results of TTR mRNA levels in the eye of mice at Day 14, following administrating various exemplary siRNA duplexes shown in Table 7, at a dosage of 7.5 mg.

[0096] Figure 26 shows the results of TTR mRNA levels in the eye of mice at Day 14, following an intravitreal administration of various exemplary siRNA duplexes shown in Table 7, at a dosage of 7.5 mg.DETAILED DESCRIPTION

[0097] The inventors have found, inter alia, that conjugating a lipophilic moiety to one or more internal positions on at least one strand of the double-stranded iRNA agent provides surprisingly good results for in vivo intravitreal delivery and intrathecal delivery of the double-stranded iRNAs, resulting in efficient entry of CNS tissues and ocular tissues and are efficiently internalized into cells of the CNS system and ocular system.

[0098] One aspect of the invention provides a double-stranded iRNA agent comprising: an antisense strand which is complementary to a target gene; a sense strand which is complementary to said antisense strand; and one or more lipophilic moieties conjugated to one or more internal positions on at least one strand, optionally via a linker or carrier.

[0099] The term“lipophile” or“lipophilic moiety” broadly refers to any compound or chemical moiety having an affinity for lipids. One way to characterize the lipophilicity of the lipophilic moiety is by the octanol-water partition coefficient, logKow, where Kow is the ratio of a chemical’s concentration in the octanol-phase to its concentration in the aqueous phase of a two-phase system at equilibrium. The octanol-water partition coefficient is a laboratory- measured property of a substance. However, it may also be predicted by using coefficients attributed to the structural components of a chemical which are calculated using first- principle or empirical methods (see, for example, Tetko et al., J. Chem. Inf. Comput. Sci. 41:1407-21 (2001), which is incorporated herein by reference in its entirety). It provides a thermodynamic measure of the tendency of the substance to prefer a non-aqueous or oily milieu rather than water (i.e. its hydrophilic / lipophilic balance). In principle, a chemical substance is lipophilic in character when its logKow exceeds 0. Typically, the lipophilic moiety possesses a logKowexceeding 1, exceeding 1.5, exceeding 2, exceeding 3, exceeding 4, exceeding 5, or exceeding 10. For instance, the logKowof 6-amino hexanol, for instance, is predicted to be approximately 0.7. Using the same method, the logKow of cholesteryl N- (hexan-6-ol) carbamate is predicted to be 10.7.

[0100] The lipophilicity of a molecule can change with respect to the functional group it carries. For instance, adding a hydroxyl group or amine group to the end of a lipophilic moiety can increase or decrease the partition coefficient (e.g., logKow) value of the lipophilic moiety.

[0101] Alternatively, the hydrophobicity of the double-stranded iRNA agent, conjugated to one or more lipophilic moieties, can be measured by its protein binding characteristics. For instance, the unbound fraction in the plasma protein binding assay of the double-strandediRNA agent can be determined to positively correlate to the relative hydrophobicity of the double-stranded iRNA agent, which can positively correlate to the silencing activity of the double-stranded iRNA agent.

[0102] In one embodiment, the plasma protein binding assay determined is an electrophoretic mobility shift assay (EMSA) using human serum albumin protein. An exemplary protocol of this binding assay is illustrated in detail in Example 14. The hydrophobicity of the double-stranded iRNA agent, measured by fraction of unbound siRNA in the binding assay, exceeds 0.15, exceeds 0.2, exceeds 0.25, exceeds 0.3, exceeds 0.35, exceeds 0.4, exceeds 0.45, or exceeds 0.5 for an enhanced in vivo delivery of siRNA.

[0103] Accordingly, conjugating the lipophilic moieties to the internal position(s) of the double-stranded iRNA agent provides optimal hydrophobicity for the enhanced in vivo delivery of siRNA.

[0104] In certain embodiments, the lipophilic moiety is an aliphatic, cyclic such as alicyclic, or polycyclic such as polyalicyclic compound, such as a steroid (e.g., sterol) or a linear or branched aliphatic hydrocarbon. The lipophilic moiety may generally comprises a hydrocarbon chain, which may be cyclic or acyclic. The hydrocarbon chain may comprise various substituents and / or one or more heteroatoms, such as an oxygen or nitrogen atom. Such lipophilic aliphatic moieties include, without limitation, saturated or unsaturated C4-C30 hydrocarbon (e.g., C6-C18 hydrocarbon), saturated or unsaturated fatty acids, waxes (e.g., monohydric alcohol esters of fatty acids and fatty diamides), terpenes (e.g., C10terpenes, C15sesquiterpenes, C20diterpenes, C30triterpenes, and C40tetraterpenes), and other polyalicyclic hydrocarbons. For instance, the lipophilic moiety may contain a C4-C30 hydrocarbon chain (e.g., C4-C30alkyl or alkenyl). In some embodiment the lipophilic moiety contains a saturated or unsaturated C6-C18hydrocarbon chain (e.g., a linear C6-C18alkyl or alkenyl). In one embodiment, the lipophilic moiety contains a saturated or unsaturated C16 hydrocarbon chain (e.g., a linear C16 alkyl or alkenyl).

[0105] The lipophilic moiety may be attached to the iRNA agent by any method known in the art, including via a functional grouping already present in the lipophilic moiety or introduced into the iRNA agent, such as a hydroxy group (e.g.,—CO—CH2—OH). The functional groups already present in the lipophilic moiety or introduced into the iRNA agent include, but are not limited to, hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.

[0106] Conjugation of the iRNA agent and the lipophilic moiety may occur, for example, through formation of an ether or a carboxylic or carbamoyl ester linkage between the hydroxy and an alkyl group R—, an alkanoyl group RCO— or a substituted carbamoyl groupRNHCO—. The alkyl group R may be cyclic (e.g., cyclohexyl) or acyclic (e.g., straight- chained or branched; and saturated or unsaturated). Alkyl group R may be a butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl or octadecyl group, or the like.

[0107] In some embodiments, the lipophilic moiety is conjugated to the double-stranded iRNA agent via a linker a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, a product of a click reaction (e.g., a triazole from the azide-alkyne cycloaddition), or carbamate.

[0108] In another embodiment, the lipophilic moiety is a steroid, such as sterol. Steroids are polycyclic compounds containing a perhydro-1,2-cyclopentanophenanthrene ring system. Steroids include, without limitation, bile acids (e.g., cholic acid, deoxycholic acid and dehydrocholic acid), cortisone, digoxigenin, testosterone, cholesterol, and cationic steroids, such as cortisone. A“cholesterol derivative” refers to a compound derived from cholesterol, for example by substitution, addition or removal of substituents.

[0109] In another embodiment, the lipophilic moiety is an aromatic moiety. In this context, the term“aromatic” refers broadly to mono- and polyaromatic hydrocarbons.Aromatic groups include, without limitation, C6-C14aryl moieties comprising one to three aromatic rings, which may be optionally substituted;“aralkyl” or“arylalkyl” groups comprising an aryl group covalently linked to an alkyl group, either of which mayindependently be optionally substituted or unsubstituted; and“heteroaryl” groups. As used herein, the term“heteroaryl” refers to groups having 5 to 14 ring atoms, preferably 5, 6, 9, or 10 ring atoms; having 6, 10, or 14p electrons shared in a cyclic array, and having, in addition to carbon atoms, between one and about three heteroatoms selected from the group consisting of nitrogen (N), oxygen (O), and sulfur (S).

[0110] As employed herein, a“substituted” alkyl, cycloalkyl, aryl, heteroaryl, or heterocyclic group is one having between one and about four, preferably between one and about three, more preferably one or two, non-hydrogen substituents. Suitable substituents include, without limitation, halo, hydroxy, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl,alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, and ureido groups.

[0111] In some embodiments, the lipophilic moiety is an aralkyl group, e.g., a 2- arylpropanoyl moiety. The structural features of the aralkyl group are selected so that the lipophilic moiety will bind to at least one protein in vivo. In certain embodiments, the structural features of the aralkyl group are selected so that the lipophilic moiety binds to serum, vascular, or cellular proteins. In certain embodiments, the structural features of the aralkyl group promote binding to albumin, an immunoglobulin, a lipoprotein, a-2- macroglubulin, or a-1-glycoprotein.

[0112] In certain embodiments, the ligand is naproxen or a structural derivative of naproxen. Procedures for the synthesis of naproxen can be found in U.S. Pat. No.3,904,682 and U.S. Pat. No.4,009,197, which are herey incorporated by reference in their entirety. Naproxen has the chemical name (S)-6-Methoxy-a-methyl-2-naphthaleneacetic acid and thestructure is .

[0113] In certain embodiments, the ligand is ibuprofen or a structural derivative of ibuprofen. Procedures for the synthesis of ibuprofen can be found in U.S. Pat. No.3,228,831, which are herey incorporated by reference in their entirety. The structure of ibuprofen is.

[0114] Additional exemplary aralkyl groups are illustrated in U.S. Patent No.7,626,014, which is incorporated herein by reference in its entirety.

[0115] In another embodiment, suitable lipophilic moieties include lipid, cholesterol, retinoic acid, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, ibuprofen, naproxen, dimethoxytrityl, or phenoxazine.

[0116] In some embodiments, the lipophilic moiety is a C6-C30 acid (e.g., hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, dodcanoic acid, tridecanoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid, heptadecanoicacid, octadecanoic acid, oleic acid, linoleic acid, arachidonic acid, cis-4,7,10,13,16,19- docosahexanoic acid, vitamin A, vitamin E, cholesterol etc.) or a C6-C30 alcohol (e.g., hexanol, heptanol, octanol, nonanol, decanol, undecanol, dodcanol, tridecanol, tetradecanol, pentadecanol, hexadecanol, heptadecanol, octadecanol, oleyl alcohol, linoleyl alcohol, arachidonic alcohol, cis-4,7,10,13,16,19-docosahexanol, retinol, vitamin E, cholesterol etc.).

[0117] In certain embodiments, more than one lipophilic moieties can be incorporated into the double-strand iRNA agent, particularly when the lipophilic moiety has a low lipophilicity or hydrophobicity. In one embodiment, two or more lipophilic moieties are incorporated into the same strand of the double-strand iRNA agent. In one embodiment, each strand of the double-strand iRNA agent has one or more lipophilic moieties incorporated. In one embodiment, two or more lipophilic moieties are incorporated into the same position (i.e., the same nucleobase, same sugar moiety, or same internucleosidic linkage) of the double-strand iRNA agent. This can be achieved by, e.g., conjugating the two or more lipophilic moieties via a carrier, and / or conjugating the two or more lipophilic moieties via a branched linker, and / or conjugating the two or more lipophilic moieties via one or more linkers, with one or more linkers linking the lipophilic moieties consecutively.

[0118] The lipophilic moiety may be conjugated to the iRNA agent via a direct attachment to the ribosugar of the iRNA agent. Alternatively, the lipophilic moiety may be conjugated to the double-strand iRNA agent via a linker or a carrier.

[0119] In certain embodiments, the lipophilic moiety may be conjugated to the iRNA agent via one or more linkers (tethers).

[0120] In one embodiment, the lipophilic moiety is conjugated to the double-stranded iRNA agent via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, a product of a click reaction (e.g., a triazole from the azide-alkyne cycloaddition), or carbamate. Some exemplary linkages are illustrated in Figure 1, Examples 2, 3, 5, 6, and 7. Linkers / Tethers

[0121] Linkers / Tethers are connected to the lipophilic moiety at a“tethering attachment point (TAP).” Linkers / Tethers may include any C1-C100carbon-containing moiety, (e.g. C1- C75, C1-C50, C1-C20, C1-C10; C1, C2, C3, C4, C5, C6, C7, C8, C9, or C10), and may have at least one nitrogen atom. In certain embodiments, the nitrogen atom forms part of a terminal amino or amido (NHC(O)-) group on the linker / tether, which may serve as a connection point forthe lipophilic moiety. Non-limited examples of linkers / tethers (underlined) include TAP- (CH2)nNH-; TAP-C(O)(CH2)nNH-; TAP-NR’’’’(CH2)nNH-, TAP-C(O)-(CH2)n-C(O)-; TAP- C(O)-(CH2)n-C(O)O-; TAP-C(O)-O-; TAP-C(O)-(CH2)n-NH-C(O)-; TAP-C(O)-(CH2)n-; TAP-C(O)-NH-; TAP-C(O)-; TAP-(CH2)n-C(O)-; TAP-(CH2)n-C(O)O-; TAP-(CH2)n-; or TAP-(CH2)n-NH-C(O)-; in which n is 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) and R’’’’ is C1-C6 alkyl. Preferably, n is 5, 6, or 11. In other embodiments, the nitrogen may form part of a terminal oxyamino group, e.g., -ONH2, or hydrazino group, -NHNH2. The linker / tether may optionally be substituted, e.g., with hydroxy, alkoxy, perhaloalkyl, and / or optionally inserted with one or more additional heteroatoms, e.g., N, O, or S. Preferred tethered ligands may include, e.g., TAP- (CH2)nNH(LIGAND); TAP-C(O)(CH2)nNH(LIGAND); TAP-NR’’’’(CH2)nNH(LIGAND); TAP-(CH2)nONH(LIGAND); TAP-C(O)(CH2)nONH(LIGAND); TAP- NR’’’’(CH2)nONH(LIGAND); TAP-(CH2)nNHNH2(LIGAND), TAP- C(O)(CH2)nNHNH2(LIGAND); TAP-NR’’’’(CH2)nNHNH2(LIGAND); TAP-C(O)-(CH2)n- C(O)(LIGAND); TAP-C(O)-(CH2)n-C(O)O(LIGAND); TAP-C(O)-O(LIGAND); TAP-C(O)- (CH2)n-NH-C(O)(LIGAND); TAP-C(O)-(CH2)n(LIGAND); TAP-C(O)-NH(LIGAND); TAP- C(O)(LIGAND); TAP-(CH2)n-C(O) (LIGAND); TAP-(CH2)n-C(O)O(LIGAND); TAP- (CH2)n(LIGAND); or TAP-(CH2)n-NH-C(O)(LIGAND). In some embodiments, amino terminated linkers / tethers (e.g., NH2, ONH2, NH2NH2) can form an imino bond (i.e., C=N) with the ligand. In some embodiments, amino terminated linkers / tethers (e.g., NH2, ONH2, NH2NH2) can acylated, e.g., with C(O)CF3.

[0122] In some embodiments, the linker / tether can terminate with a mercapto group (i.e., SH) or an olefin (e.g., CH=CH2). For example, the tether can be TAP-(CH2)n-SH, TAP- C(O)(CH2)nSH, TAP-(CH2)n-(CH=CH2), or TAP-C(O)(CH2)n(CH=CH2), in which n can be as described elsewhere. The tether may optionally be substituted, e.g., with hydroxy, alkoxy, perhaloalkyl, and / or optionally inserted with one or more additional heteroatoms, e.g., N, O, or S. The double bond can be cis or trans or E or Z.

[0123] In other embodiments, the linker / tether may include an electrophilic moiety, preferably at the terminal position of the linker / tether. Exemplary electrophilic moieties include, e.g., an aldehyde, alkyl halide, mesylate, tosylate, nosylate, or brosylate, or an activated carboxylic acid ester, e.g. an NHS ester, or a pentafluorophenyl ester. Preferred linkers / tethers (underlined) include TAP-(CH2)nCHO; TAP-C(O)(CH2)nCHO; or TAP- NR’’’’(CH2)nCHO, in which n is 1-6 and R’’’’ is C1-C6alkyl; or TAP-(CH2)nC(O)ONHS;TAP-C(O)(CH2)nC(O)ONHS; or TAP-NR’’’’(CH2)nC(O)ONHS, in which n is 1-6 and R’’’’ is C1-C6 alkyl; TAP-(CH2)nC(O)OC6F5; TAP-C(O)(CH2) nC(O) OC6F5; or TAP-NR’’’’(CH2) nC(O) OC6F5, in which n is 1-11 and R’’’’ is C1-C6 alkyl; or -(CH2)nCH2LG; TAP- C(O)(CH2)nCH2LG; or TAP-NR’’’’(CH2)nCH2LG, in which n can be as described elsewhere and R’’’’ is C1-C6 alkyl (LG can be a leaving group, e.g., halide, mesylate, tosylate, nosylate, brosylate). Tethering can be carried out by coupling a nucleophilic group of a ligand, e.g., a thiol or amino group with an electrophilic group on the tether.

[0124] In other embodiments, it can be desirable for the monomer to include aphthalimido group (K) at the terminal position of the linker / tether

[0125] In other embodiments, other protected amino groups can be at the terminal position of the linker / tether, e.g., alloc, monomethoxy trityl (MMT), trifluoroacetyl, Fmoc, or aryl sulfonyl (e.g., the aryl portion can be ortho-nitrophenyl or ortho, para-dinitrophenyl).

[0126] Any of the linkers / tethers described herein may further include one or more additional linking groups, e.g., -O-(CH2)n-, -(CH2)n-SS-, -(CH2)n-, or -(CH=CH)-. Cleavable linkers / tethers

[0127] In some embodiments, at least one of the linkers / tethers can be a redox cleavable linker, an acid cleavable linker, an esterase cleavable linker, a phosphatase cleavable linker, or a peptidase cleavable linker.

[0128] In one embodiment, at least one of the linkers / tethers can be a reductively cleavable linker (e.g., a disulfide group).

[0129] In one embodiment, at least one of the linkers / tethers can be an acid cleavable linker (e.g., a hydrazone group, an ester group, an acetal group, or a ketal group).

[0130] In one embodiment, at least one of the linkers / tethers can be an esterase cleavable linker (e.g., an ester group).

[0131] In one embodiment, at least one of the linkers / tethers can be a phosphatase cleavable linker (e.g., a phosphate group).

[0132] In one embodiment, at least one of the linkers / tethers can be a peptidase cleavable linker (e.g., a peptide bond).

[0133] Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential or the presence of degradative molecules. Generally, cleavage agents are moreprevalent or found at higher levels or activities inside cells than in serum or blood. Examples of such degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.

[0134] A cleavable linkage group, such as a disulfide bond can be susceptible to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0. Some tethers will have a linkage group that is cleaved at a preferred pH, thereby releasing the iRNA agent from a ligand (e.g., a targeting or cell- permeable ligand, such as cholesterol) inside the cell, or into the desired compartment of the cell.

[0135] A chemical junction (e.g., a linking group) that links a ligand to an iRNA agent can include a disulfide bond. When the iRNA agent / ligand complex is taken up into the cell by endocytosis, the acidic environment of the endosome will cause the disulfide bond to be cleaved, thereby releasing the iRNA agent from the ligand (Quintana et al., PharmRes.19:1310-1316, 2002; Patri et al., Curr. Opin. Curr. Biol.6:466-471, 2002). The ligand can be a targeting ligand or a second therapeutic agent that may complement the therapeutic effects of the iRNA agent.

[0136] A tether can include a linking group that is cleavable by a particular enzyme. The type of linking group incorporated into a tether can depend on the cell to be targeted by the iRNA agent. For example, an iRNA agent that targets an mRNA in liver cells can be conjugated to a tether that includes an ester group. Liver cells are rich in esterases, and therefore the tether will be cleaved more efficiently in liver cells than in cell types that are not esterase-rich. Cleavage of the tether releases the iRNA agent from a ligand that is attached to the distal end of the tether, thereby potentially enhancing silencing activity of the iRNA agent. Other cell-types rich in esterases include cells of the lung, renal cortex, and testis.

[0137] Tethers that contain peptide bonds can be conjugated to iRNA agents target to cell types rich in peptidases, such as liver cells and synoviocytes. For example, an iRNA agenttargeted to synoviocytes, such as for the treatment of an inflammatory disease (e.g., rheumatoid arthritis), can be conjugated to a tether containing a peptide bond.

[0138] In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linking group. It will also be desirable to also test the candidate cleavable linking group for the ability to resist cleavage in the blood or when in contact with other non-target tissue, e.g., tissue the iRNA agent would be exposed to when administered to a subject. Thus one can determine the relative susceptibility to cleavage between a first and a second condition, where the first is selected to be indicative of cleavage in a target cell and the second is selected to be indicative of cleavage in other tissues or biological fluids, e.g., blood or serum. The evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals. It may be useful to make initial evaluations in cell-free or culture conditions and to confirm by further evaluations in whole animals. In preferred embodiments, useful candidate compounds are cleaved at least 2, 4, 10 or 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions). Redox Cleavable Linking Groups

[0139] One class of cleavable linking groups are redox cleavable linking groups that are cleaved upon reduction or oxidation. An example of reductively cleavable linking group is a disulphide linking group (—S—S—). To determine if a candidate cleavable linking group is a suitable“reductively cleavable linking group,” or for example is suitable for use with a particular iRNA moiety and particular targeting agent one can look to methods described herein. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent using reagents know in the art, which mimic the rate of cleavage which would be observed in a cell, e.g., a target cell. The candidates can also be evaluated under conditions which are selected to mimic blood or serum conditions. In a preferredembodiment, candidate compounds are cleaved by at most 10% in the blood. In preferred embodiments, useful candidate compounds are degraded at least 2, 4, 10 or 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of candidate compounds can be determined using standard enzymekinetics assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media. Phosphate-Based Cleavable Linking Groups

[0140] Phosphate-based linking groups are cleaved by agents that degrade or hydrolyze the phosphate group. An example of an agent that cleaves phosphate groups in cells are enzymes such as phosphatases in cells. Examples of phosphate-based linking groups are— O—P(O)(ORk)-O—,—O—P(S)(ORk)-O—,—O—P(S)(SRk)-O—,—S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—,—S—P(O)(ORk)-S—,—O—P(S)(ORk)-S—,—S—P(S)(ORk)-O— ,—O—P(O)(Rk)-O—,—O—P(S)(Rk)-O—,—S—P(O)(Rk)-O—,—S—P(S)(Rk)-O—,— S—P(O)(Rk)-S—,—O—P(S)(Rk)-S—. Preferred embodiments are—O—P(O)(OH)—O—, —O—P(S)(OH)—O—,—O—P(S)(SH)—O—,—S—P(O)(OH)—O—,—O—P(O)(OH)— S—,—S—P(O)(OH)—S—,—O—P(S)(OH)—S—,—S—P(S)(OH)—O—,—O—P(O)(H)—O—,—O—P(S)(H)—O—,—S—P(O)(H)—O—,—S—P(S)(H)—O—,—S— P(O)(H)—S—,—O—P(S)(H)—S—. A preferred embodiment is—O—P(O)(OH)—O—. These candidates can be evaluated using methods analogous to those described above. Acid Cleavable Linking Groups

[0141] Acid cleavable linking groups are linking groups that are cleaved under acidic conditions. In preferred embodiments acid cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.5, 5.0, or lower), or by agents such as enzymes that can act as a general acid. In a cell, specific low pH organelles, such as endosomes and lysosomes can provide a cleaving environment for acid cleavable linking groups. Examples of acid cleavable linking groups include but are not limited to hydrazones, ketals, acetals, esters, and esters of amino acids. Acid cleavable groups can have the general formula—C═NN—, C(O)O, or—OC(O). A preferred embodiment is when the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl. These candidates can be evaluated using methods analogous to those described above. Ester-Based Linking Groups

[0142] Ester-based linking groups are cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include but are not limited to estersof alkylene, alkenylene and alkynylene groups. Ester cleavable linking groups have the general formula—C(O)O—, or—OC(O)—. These candidates can be evaluated using methods analogous to those described above. Peptide-Based Cleaving Groups

[0143] Peptide-based linking groups are cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides.Peptide-based cleavable groups do not include the amide group (—C(O)NH—). The amide group can be formed between any alkylene, alkenylene or alkynelene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. The peptide based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group. Peptide cleavable linking groups have the general formula— NHCHR1C(O)NHCHR2C(O)—, where R1and R2are the R groups of the two adjacent amino acids. These candidates can be evaluated using methods analogous to those described above. Biocleavable linkers / tethers

[0144] The linkers can also includes biocleavable linkers that are nucleotide and non- nucleotide linkers or combinations thereof that connect two parts of a molecule, for example, one or both strands of two individual siRNA molecule to generate a bis(siRNA). In some embodiments, mere electrostatic or stacking interaction between two individual siRNAs can represent a linker. The non-nucleotide linkers include tethers or linkers derived frommonosaccharides, disaccharides, oligosaccharides, and derivatives thereof, aliphatic, alicyclic, hetercyclic, and combinations thereof.

[0145] In some embodiments, at least one of the linkers (tethers) is a bio-clevable linker selected from the group consisting of DNA, RNA, disulfide, amide, functionalized monosaccharides or oligosaccharides of galactosamine, glucosamine, glucose, galactose, and mannose, and combinations thereof.

[0146] In one embodiment, the bio-cleavable carbohydrate linker may have 1 to 10 saccharide units, which have at least one anomeric linkage capable of connecting two siRNA units. When two or more saccharides are present, these units can be linked via 1-3, 1-4, or 1-6 sugar linkages, or via alkyl chains.

[0147] Exemplary bio-cleavable linkers include:

[0148] Additional exemplary bio-cleavable linkers are illustrated in Schemes 28-30.

[0149] More discussion about the biocleavable linkers may be found in PCT application No. PCT / US18 / 14213, entitled“Endosomal Cleavable Linkers,” filed on January 18, 2018, the content of which is incorporated herein by reference in its entirety. Carriers

[0150] In certain embodiments, the lipophilic moiety is conjugated to the iRNA agent via a carrier that replaces one or more nucleotide(s).

[0151] The carrier can be a cyclic group or an acyclic group. In one embodiment, the cyclic group is selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, and decalin. In one embodiment, the acyclic group is a moiety based on a serinol backbone or a diethanolamine backbone.

[0152] In some embodiments, the carrier replaces one or more nucleotide(s) in the internal position(s) of the double-stranded iRNA agent.

[0153] In other embodiments, the carrier replaces the nucleotides at the terminal end of the sense strand or antisense strand. In one embodiment, the carrier replaces the terminal nucleotide on the 3’ end of the sense strand, thereby functioning as an end cap protecting the 3’ end of the sense strand. In one embodiment, the carrier is a cyclic group having an amine, for instance, the carrier may be pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, or decalinyl.

[0154] A ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is referred to herein as a ribose replacement modification subunit (RRMS). The carrier can be a cyclic or acyclic moiety and include two“backbone attachment points” (e.g., hydroxyl groups) and a ligand (e.g., the lipophilic moiety). The lipophilic moiety can be directly attached to the carrier or indirectly attached to the carrier by an intervening linker / tether, as described above.

[0155] The ligand-conjugated monomer subunit may be the 5’ or 3’ terminal subunit of the iRNA molecule, i.e., one of the two“W” groups may be a hydroxyl group, and the other “W” group may be a chain of two or more unmodified or modified ribonucleotides.Alternatively, the ligand-conjugated monomer subunit may occupy an internal position, and both“W” groups may be one or more unmodified or modified ribonucleotides. More than one ligand-conjugated monomer subunit may be present in an iRNA agent. Sugar Replacement-Based Monomers, e.g., Ligand-Conjugated Monomers (Cyclic)

[0156] Cyclic sugar replacement-based monomers, e.g., sugar replacement-based ligand-conjugated monomers, are also referred to herein as RRMS monomer compounds. The carriers may have the general formula (LCM-2) provided below (In that structure preferred backbone attachment points can be chosen from R1or R2; R3or R4; or R9and R10if Y is CR9R10(two positions are chosen to give two backbone attachment points, e.g., R1and R4, or R4and R9)). Preferred tethering attachment points include R7; R5or R6when X is CH2. The carriers are described below as an entity, which can be incorporated into a strand. Thus, it is understood that the structures also encompass the situations wherein one (in the case of a terminal position) or two (in the case of an internal position) of the attachment points, e.g., R1or R2; R3or R4; or R9or R10(when Y is CR9R10), is connected to the phosphate, or modified phosphate, e.g., sulfur containing, backbone. E.g., one of the above-named R groups can be - CH2-, wherein one bond is connected to the carrier and one to a backbone atom, e.g., a linking oxygen or a central phosphorus atom.(LCM-2)wherein:X is N(CO)R7, NR7or CH2;Y is NR8, O, S, CR9R10;Z is CR11R12or absent;Each of R1, R2, R3, R4, R9, and R10is, independently, H, ORa, or (CH2)nORb, provided that at least two of R1, R2, R3, R4, R9, and R10are ORaand / or (CH2)nORb;Each of R5, R6, R11, and R12is, independently, a ligand, H, C1-C6alkyl optionally substituted with 1-3 R13, or C(O)NHR7; or R5and R11together are C3-C8 cycloalkyl optionally substituted with R14;R7can be a ligand, e.g., R7can be Rd, or R7can be a ligand tethered indirectly to the carrier, e.g., through a tethering moiety, e.g., C1-C20 alkyl substituted with NRcRd; or C1-C20 alkyl substituted with NHC(O)Rd;R8is H or C1-C6alkyl;R13is hydroxy, C1-C4alkoxy, or halo;R14is NRcR7;R15is C1-C6alkyl optionally substituted with cyano, or C2-C6alkenyl;R16is C1-C10alkyl;R17is a liquid or solid phase support reagent;L is -C(O)(CH2)qC(O)-, or -C(O)(CH2)qS-;Rais a protecting group, e.g., CAr3; (e.g., a dimethoxytrityl group) orSi(X5’)(X5”)(X5”’) in which (X5’),(X5”), and (X5”’) are as described elsewhere.Rbis P(O)(O-)H, P(OR15)N(R16)2 or L-R17;Rcis H or C1-C6 alkyl;Rdis H or a ligand;Each Ar is, independently, C6-C10aryl optionally substituted with C1-C4alkoxy; n is 1-4; and q is 0-4.

[0157] Exemplary carriers include those in which, e.g., X is N(CO)R7or NR7, Y is CR9R10, and Z is absent; or X is N(CO)R7or NR7, Y is CR9R10, and Z is CR11R12; or X is N(CO)R7or NR7, Y is NR8, and Z is CR11R12; or X is N(CO)R7or NR7, Y is O, and Z is CR11R12; or X is CH2; Y is CR9R10; Z is CR11R12, and R5and R11together form C6 cycloalkyl (H, z = 2), or the indane ring system, e.g., X is CH2; Y is CR9R10; Z is CR11R12, and R5and R11together form C5 cycloalkyl (H, z = 1).

[0158] In certain embodiments, the carrier may be based on the pyrroline ring system or the 4-hydroxyproline ring system, e.g., X is N(CO)R7or NR7, Y is CR9R10, and Z is absent(D). . OFG1is preferably attached to a primary carbon, e.g., an exocyclic alkylene group, e.g., a methylene group, connected to one of the carbons in the five- membered ring (-CH2OFG1in D). OFG2is preferably attached directly to one of the carbons in the five-membered ring (-OFG2in D). For the pyrroline-based carriers, -CH2OFG1may be attached to C-2 and OFG2may be attached to C-3; or -CH2OFG1may be attached to C-3 and OFG2may be attached to C-4. In certain embodiments, CH2OFG1and OFG2may be geminally substituted to one of the above-referenced carbons. For the 3-hydroxyproline- based carriers, -CH2OFG1may be attached to C-2 and OFG2may be attached to C-4. The pyrroline- and 4-hydroxyproline-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring. Thus, CH2OFG1and OFG2may be cis ortrans with respect to one another in any of the pairings delineated above Accordingly, all cis / trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included (e.g., the centers bearing CH2OFG1and OFG2can both have the R configuration; or both have the S configuration; or one center can have the R configuration and the other center can have the S configuration and vice versa). The tethering attachment point is preferably nitrogen. Preferred examples of carrier D include the following:.

[0159] In certain embodiments, the carrier may be based on the piperidine ring system(E), e.g., X is N(CO)R7or NR7, Y is CR9R10, and Z is CR11R12. .OFG1is preferably attached to a primary carbon, e.g., an exocyclic alkylene group, e.g., a methylene group (n=1) or ethylene group (n=2), connected to one of the carbons in the six- membered ring [-(CH2)nOFG1in E]. OFG2is preferably attached directly to one of the carbons in the six-membered ring (-OFG2in E). -(CH2)nOFG1and OFG2may be disposed in a geminal manner on the ring, i.e., both groups may be attached to the same carbon, e.g., atC-2, C-3, or C-4. Alternatively, -(CH2)nOFG1and OFG2may be disposed in a vicinal manner on the ring, i.e., both groups may be attached to adjacent ring carbon atoms, e.g., - (CH2)nOFG1may be attached to C-2 and OFG2may be attached to C-3; -(CH2)nOFG1may be attached to C-3 and OFG2may be attached to C-2; -(CH2)nOFG1may be attached to C-3 and OFG2may be attached to C-4; or -(CH2)nOFG1may be attached to C-4 and OFG2may be attached to C-3. The piperidine-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring. Thus, -(CH2)nOFG1and OFG2may be cis or trans with respect to one another in any of the pairings delineated above. Accordingly, all cis / trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included (e.g., the centers bearing CH2OFG1and OFG2can both have the R configuration; or both have the S configuration; or one center can have the R configuration and the other center can have the S configuration and vice versa). The tethering attachment point is preferably nitrogen.

[0160] In certain embodiments, the carrier may be based on the piperazine ring system (F), e.g., X is N(CO)R7or NR7, Y is NR8, and Z is CR11R12, or the morpholine ring system(G), e.g., X is N(CO)R7or NR7, Y is O, and Z is CR11R12. . OFG1is preferably attached to a primary carbon, e.g., an exocyclic alkylene group, e.g., a methylene group, connected to one of the carbons in the six-membered ring (-CH2OFG1in F or G). OFG2is preferably attached directly to one of the carbons in the six-membered rings (-OFG2in F or G). For both F and G, -CH2OFG1may be attached to C-2 and OFG2may be attached to C-3; or vice versa. In certain embodiments, CH2OFG1and OFG2may be geminally substituted to one of the above-referenced carbons.The piperazine- andmorpholine-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring. Thus, CH2OFG1and OFG2may be cis or trans with respect to one another in any of the pairings delineated above. Accordingly, all cis / trans isomers areexpressly included. The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included (e.g., the centers bearing CH2OFG1and OFG2can both have the R configuration; or both have the S configuration; or one center can have the R configuration and the other center can have the S configuration and vice versa). R’’’ can be, e.g., C1-C6 alkyl, preferably CH3. The tethering attachment point is preferably nitrogen in both F and G.

[0161] In certain embodiments, the carrier may be based on the decalin ring system, e.g., X is CH2; Y is CR9R10; Z is CR11R12, and R5and R11together form C6 cycloalkyl (H, z = 2), or the indane ring system, e.g., X is CH2; Y is CR9R10; Z is CR11R12, and R5and R11togetherform C5 cycloalkyl (H, z = 1).. OFG1is preferably attached to a primary carbon, e.g., an exocyclic methylene group (n=1) or ethylene group (n=2) connected to one of C-2, C-3, C-4, or C-5 [-(CH2)nOFG1in H]. OFG2is preferably attached directly to one of C-2, C-3, C-4, or C-5 (-OFG2in H). -(CH2)nOFG1and OFG2may be disposed in a geminal manner on the ring, i.e., both groups may be attached to the same carbon, e.g., at C- 2, C-3, C-4, or C-5. Alternatively, -(CH2)nOFG1and OFG2may be disposed in a vicinal manner on the ring, i.e., both groups may be attached to adjacent ring carbon atoms, e.g., - (CH2)nOFG1may be attached to C-2 and OFG2may be attached to C-3; -(CH2)nOFG1may be attached to C-3 and OFG2may be attached to C-2; -(CH2)nOFG1may be attached to C-3 and OFG2may be attached to C-4; or -(CH2)nOFG1may be attached to C-4 and OFG2may be attached to C-3; -(CH2)nOFG1may be attached to C-4 and OFG2may be attached to C-5; or - (CH2)nOFG1may be attached to C-5 and OFG2may be attached to C-4. The decalin or indane-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring. Thus, -(CH2)nOFG1and OFG2may be cis or trans with respect to one another in any of the pairings delineated above. Accordingly, all cis / trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included (e.g., the centers bearing CH2OFG1and OFG2can both have the R configuration; orboth have the S configuration; or one center can have the R configuration and the other center can have the S configuration and vice versa). In a preferred embodiment, the substituents at C-1 and C-6 are trans with respect to one another. The tethering attachment point is preferably C-6 or C-7.

[0162] Other carriers may include those based on 3-hydroxyproline (J).. Thus, -(CH2)nOFG1and OFG2may be cis or trans with respect to one another. Accordingly, all cis / trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included (e.g., the centers bearing CH2OFG1and OFG2can both have the R configuration; or both have the S configuration; or one center can have the R configuration and the other center can have the S configuration and vice versa). The tethering attachment point is preferably nitrogen.

[0163] Details about more representative cyclic, sugar replacement-based carriers can be found in U.S. Patent Nos.7,745,608 and 8,017,762, which are herein incorporated by reference in their entireties. Sugar Replacement-Based Monomers (Acyclic)

[0164] Acyclic sugar replacement-based monomers, e.g., sugar replacement-based ligand-conjugated monomers, are also referred to herein as ribose replacement monomer subunit (RRMS) monomer compounds. Preferred acyclic carriers can have formula LCM-3 or LCM-4:.

[0165] In some embodiments, each of x, y, and z can be, independently of one another, 0, 1, 2, or 3. In formula LCM-3, when y and z are different, then the tertiary carbon can have either the R or S configuration. In preferred embodiments, x is zero and y and z are each 1 in formula LCM-3 (e.g., based on serinol), and y and z are each 1 in formula LCM-3. Each offormula LCM-3 or LCM-4 below can optionally be substituted, e.g., with hydroxy, alkoxy, perhaloalkyl.

[0166] Details about more representative acyclic, sugar replacement-based carriers can be found in U.S. Patent Nos.7,745,608 and 8,017,762, which are herein incorporated by reference in their entireties.

[0167] In some embodiments, the double stranded iRNA agent comprises one or more lipophilic moieties conjugated to the 5 end of the sense strand or the 5’ end of the antisense strand.

[0168] In certain embodiments, the lipophilic moiety is conjugated to the 5’-end of a strand via a carrier and / or linker. In one embodiment, the lipophilic moiety is conjugated tothe 5’-end of a strand via a carrier of a formula:ligand such as the lipophilic moiety.

[0169] In some embodiments, the double stranded iRNA agent comprises one or more lipophilic moieties conjugated to the 3 end of the sense strand or the 3’ end of the antisense strand.

[0170] In certain embodiments, the lipophilic moiety is conjugated to the 3’-end of a strand via a carrier and / or linker. In one embodiment, the lipophilic moiety is conjugated tothe 3’-end of a strand via a carrier of a formul:ligand such as the lipophilic moiety.

[0171] In some embodiments, the double stranded iRNA agent comprises one or more lipophilic moieties conjugated to both ends of the sense strand.

[0172] In some embodiments, the double stranded iRNA agent comprises one or more lipophilic moieties conjugated to both ends of the antisense strand.

[0173] In some embodiments, the double stranded iRNA agent comprises one or more lipophilic moieties conjugated to the 5 end or 3 end of the sense strand, and one or more lipophilic moieties conjugated to the 5 end or 3 end of the antisense strand,

[0174] In some embodiments, the lipophilic moiety is conjugated to the terminal end of a strand via one or more linkers (tethers) and / or a carrier.

[0175] In one embodiment, the lipophilic moiety is conjugated to the terminal end of a strand via one or more linkers (tethers).

[0176] In one embodiment, the lipophilic moiety is conjugated to the 5’ end of the sense strand or antisense strand via a cyclic carrier, optionally via one or more intervening linkers (tethers).

[0177] In some embodiments, the lipophilic moiety is conjugated to one or more internal positions on at least one strand. Internal positions of a strand refers to the nucleotide on any position of the strand, except the terminal position from the 3’ end and 5’ end of the strand(e.g., excluding 2 positions: position 1 counting from the 3’ end and position 1 counting from the 5’ end).

[0178] In one embodiment, the lipophilic moiety is conjugated to one or more internal positions on at least one strand, which include all positions except the terminal two positions from each end of the strand (e.g., excluding 4 positions: positions 1 and 2 counting from the 3’ end and positions 1 and 2 counting from the 5’ end). In one embodiment, the lipophilic moiety is conjugated to one or more internal positions on at least one strand, which include all positions except the terminal three positions from each end of the strand (e.g., excluding 6 positions: positions 1, 2, and 3 counting from the 3’ end and positions 1, 2, and 3 counting from the 5’ end).

[0179] In one embodiment, the lipophilic moiety is conjugated to one or more internal positions on at least one strand, except the cleavage site region of the sense strand, for instance, the lipophilic moiety is not conjugated to positions 9-12 counting from the 5’-end of the sense strand, for example, the lipophilic moiety is not conjugated to positions 9-11 counting from the 5’-end of the sense strand. Alternatively, the internal positions exclude positions 11-13 counting from the 3’-end of the sense strand.

[0180] In one embodiment, the lipophilic moiety is conjugated to one or more internal positions on at least one strand, which exclude the cleavage site region of the antisense strand. For instance, the internal positions exclude positions 12-14 counting from the 5’-end of the antisense strand.

[0181] In one embodiment, the lipophilic moiety is conjugated to one or more internal positions on at least one strand, which exclude positions 11-13 on the sense strand, counting from the 3’-end, and positions 12-14 on the antisense strand, counting from the 5’-end.

[0182] In one embodiment, one or more lipophilic moieties are conjugated to one or more of the following internal positions: positions 4-8 and 13-18 on the sense strand, and positions 6-10 and 15-18 on the antisense strand, counting from the 5’end of each strand.

[0183] In one embodiment, one or more lipophilic moieties are conjugated to one or more of the following internal positions: positions 5, 6, 7, 15, and 17 on the sense strand, and positions 15 and 17 on the antisense strand, counting from the 5’end of each strand.

[0184] In some embodiments, the lipophilic moiety is conjugated to a nucleobase, sugar moiety, or internucleosidic linkage of the double-stranded iRNA agent.DEFINITIONS

[0185] Unless specific definitions are provided, the nomenclature utilized in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques may be used for chemical synthesis, and chemical analysis. Certain such techniques and procedures may be found for example in “Carbohydrate Modifications in Antisense Research” Edited by Sangvi and Cook, American Chemical Society, Washington D.C., 1994;“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., 18th edition, 1990; and“Antisense Drug Technology, Principles, Strategies, and Applications” Edited by Stanley T. Crooke, CRC Press, Boca Raton, Fla.; and Sambrook et al.,“Molecular Cloning, A laboratory Manual,” 2ndEdition, Cold Spring Harbor Laboratory Press, 1989, which are hereby incorporated by reference for any purpose. Where permitted, all patents, applications, published applications and other publications and other data referred to throughout in the disclosure herein are incorporated by reference in their entirety.

[0186] Unless otherwise indicated, the following terms have the following meanings:

[0187] As used herein, the term“target nucleic acid” refers to any nucleic acid molecule the expression or activity of which is capable of being modulated by an siRNA compound. Target nucleic acids include, but are not limited to, RNA (including, but not limited to pre- mRNA and mRNA or portions thereof) transcribed from DNA encoding a target protein, and also cDNA derived from such RNA, and miRNA. For example, the target nucleic acid can be a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state. In some embodiments, a target nucleic acid can be a nucleic acid molecule from an infectious agent.

[0188] As used herein, the term“iRNA” refers to an agent that mediates the targeted cleavage of an RNA transcript. These agents associate with a cytoplasmic multi-protein complex known as RNAi-induced silencing complex (RISC). Agents that are effective in inducing RNA interference are also referred to as siRNA, RNAi agent, or iRNA agent, herein. Thus, these terms can be used interchangeably herein. As used herein, the term iRNA includes microRNAs and pre-microRNAs. Moreover, the“compound” or“compounds” of the invention as used herein, also refers to the iRNA agent, and can be used interchangeably with the iRNA agent.

[0189] The iRNA agent should include a region of sufficient homology to the target gene, and be of sufficient length in terms of nucleotides, such that the iRNA agent, or a fragment thereof, can mediate downregulation of the target gene. (For ease of exposition the term nucleotide or ribonucleotide is sometimes used herein in reference to one or more monomeric subunits of an iRNA agent. It will be understood herein that the usage of the term“ribonucleotide” or“nucleotide”, herein can, in the case of a modified RNA or nucleotide surrogate, also refer to a modified nucleotide, or surrogate replacement moiety at one or more positions.) Thus, the iRNA agent is or includes a region which is at least partially, and in some embodiments fully, complementary to the target RNA. It is not necessary that there be perfect complementarity between the iRNA agent and the target, but the correspondence must be sufficient to enable the iRNA agent, or a cleavage product thereof, to direct sequence specific silencing, e.g., by RNAi cleavage of the target RNA, e.g., mRNA. Complementarity, or degree of homology with the target strand, is most critical in the antisense strand. While perfect complementarity, particularly in the antisense strand, is often desired someembodiments can include, particularly in the antisense strand, one or more, or for example, 6, 5, 4, 3, 2, or fewer mismatches (with respect to the target RNA). The sense strand need only be sufficiently complementary with the antisense strand to maintain the over all double stranded character of the molecule.

[0190] iRNA agents include: molecules that are long enough to trigger the interferon response (which can be cleaved by Dicer (Bernstein et al.2001. Nature, 409:363-366) and enter a RISC (RNAi-induced silencing complex)); and, molecules which are sufficiently short that they do not trigger the interferon response (which molecules can also be cleaved by Dicer and / or enter a RISC), e.g., molecules which are of a size which allows entry into a RISC, e.g., molecules which resemble Dicer-cleavage products. Molecules that are short enough that they do not trigger an interferon response are termed siRNA agents or shorter iRNA agents herein. “siRNA agent or shorter iRNA agent” as used herein, refers to an iRNA agent, e.g., a double stranded RNA agent or single strand agent, that is sufficiently short that it does not induce a deleterious interferon response in a human cell, e.g., it has a duplexed region of less than 60, 50, 40, or 30 nucleotide pairs. The siRNA agent, or a cleavage product thereof, can down regulate a target gene, e.g., by inducing RNAi with respect to a target RNA, wherein the target may comprise an endogenous or pathogen target RNA.

[0191] A“single strand iRNA agent” as used herein, is an iRNA agent which is made up of a single molecule. It may include a duplexed region, formed by intra-strand pairing, e.g., it may be, or include, a hairpin or pan-handle structure. Single strand iRNA agents may be antisense with regard to the target molecule. A single strand iRNA agent may be sufficiently long that it can enter the RISC and participate in RISC mediated cleavage of a target mRNA. A single strand iRNA agent is at least 14, and in other embodiments at least 15, 20, 25, 29, 35, 40, or 50 nucleotides in length. In certain embodiments, it is less than 200, 100, or 60 nucleotides in length.

[0192] A loop refers to a region of an iRNA strand that is unpaired with the opposing nucleotide in the duplex when a section of the iRNA strand forms base pairs with another strand or with another section of the same strand.

[0193] Hairpin iRNA agents will have a duplex region equal to or at least 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The duplex region will may be equal to or less than 200, 100, or 50, in length. In certain embodiments, ranges for the duplex region are 15-30, 17 to 23, 19 to 23, and 19 to 21 nucleotides pairs in length. The hairpin may have a single strand overhang or terminal unpaired region, in some embodiments at the 3’, and in certain embodiments on the antisense side of the hairpin. In some embodiments, the overhangs are 2-3 nucleotides in length.

[0194] A“double stranded (ds) iRNA agent” as used herein, is an iRNA agent which includes more than one, and in some cases two, strands in which interchain hybridization can form a region of duplex structure.

[0195] As used herein, the terms“siRNA activity” and“RNAi activity” refer to gene silencing by an siRNA.

[0196] As used herein, "gene silencing" by a RNA interference molecule refers to a decrease in the mRNA level in a cell for a target gene by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99% up to and including 100%, and any integer in between of the mRNA level found in the cell without the presence of the miRNA or RNA interference molecule. In one preferred embodiment, the mRNA levels are decreased by at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, up to and including 100% and any integer in between 5% and 100%."

[0197] As used herein the term“modulate gene expression” means that expression of the gene, or level of RNA molecule or equivalent RNA molecules encoding one or more proteins or protein subunits is up regulated or down regulated, such that expression, level, or activity is greater than or less than that observed in the absence of the modulator. For example, the term“modulate” can mean“inhibit,” but the use of the word“modulate” is not limited to this definition.

[0198] As used herein, gene expression modulation happens when the expression of the gene, or level of RNA molecule or equivalent RNA molecules encoding one or more proteins or protein subunits is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 4-fold, 5-fold or more different from that observed in the absence of the siRNA, e.g., RNAi agent. The % and / or fold difference can be calculated relative to the control or the non-control, for example,[expression with siRNA– expression without siRNA]% difference = ------------------------------------------------------------------------------- expression without siRNAor[expression with siRNA– expression without siRNA]% difference = ------------------------------------------------------------------------------- expression without siRNA

[0199] As used herein, the term“inhibit”,“down-regulate”, or“reduce” in relation to gene expresion, means that the expression of the gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits, is reduced below that observed in the absence of modulator. The gene expression is down-regulated when expression of the gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits, is reduced at least 10% lower relative to a corresponding non-modulated control, and preferably at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or most preferably, 100% (i.e., no gene expression).

[0200] As used herein, the term“increase” or“up-regulate” in relation to gene expression means that the expression of the gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or moreproteins or protein subunits, is increased above that observed in the absence of modulator. The gene expression is up-regulated when expression of the gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits, is increased at least 10% relative to a corresponding non-modulated control, and preferably at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 100%, 1.1-fold, 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 3- fold, 4-fold, 5-fold, 10-fold, 50-fold, 100-fold or more.

[0201] The term "increased" or "increase" as used herein generally means an increase by a statically significant amount; for the avoidance of any doubt, "increased" means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.

[0202] The term "reduced" or "reduce" as used herein generally means a decrease by a statistically significant amount. However, for avoidance of doubt, "reduced" means a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (i.e. absent level as compared to a reference sample), or any decrease between 10-100% as compared to a reference level.

[0203] The double-stranded iRNAs comprise two oligonucleotide strands that are sufficiently complementary to hybridize to form a duplex structure. Generally, the duplex structure is between 15 and 30, more generally between 18 and 25, yet more generally between 19 and 24, and most generally between 19 and 21 base pairs in length. In some embodiments, longer double-stranded iRNAs of between 25 and 30 base pairs in length are preferred. In some embodiments, shorter double-stranded iRNAs of between 10 and 15 base pairs in length are preferred. In another embodiment, the double-stranded iRNA is at least 21 nucleotides long.

[0204] In some embodiments, the double-stranded iRNA comprises a sense strand and an antisense strand, wherein the antisense RNA strand has a region of complementarity which iscomplementary to at least a part of a target sequence, and the duplex region is 14-30 nucleotides in length. Similarly, the region of complementarity to the target sequence is between 14 and 30, more generally between 18 and 25, yet more generally between 19 and 24, and most generally between 19 and 21 nucleotides in length.

[0205] The phrase“antisense strand” as used herein, refers to an oligomeric compound that is substantially or 100% complementary to a target sequence of interest. The phrase "antisense strand" includes the antisense region of both oligomeric compounds that are formed from two separate strands, as well as unimolecular oligomeric compounds that are capable of forming hairpin or dumbbell type structures. The terms“antisense strand” and “guide strand” are used interchangeably herein.

[0206] The phrase“sense strand” refers to an oligomeric compound that has the same nucleoside sequence, in whole or in part, as a target sequence such as a messenger RNA or a sequence of DNA. The terms“sense strand” and“passenger strand” are used interchangeably herein.

[0207] By“specifically hybridizable” and "complementary" is meant that a nucleic acid can form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson- Crick or other non- traditional types. In reference to the nucleic molecules of the present invention, the binding free energy for a nucleic acid molecule with its complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., RNAi activity. Determination of binding free energies for nucleic acid molecules is well known in the art (see, e.g., Turner et al, 1987, CSH Symp. Quant. Biol. LII pp.123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA 83:9373-9377; Turner et al., 1987, / . Am. Chem. Soc.109:3783-3785). A percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9,10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Perfectly complementary" or 100% complementarity means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. Less than perfect complementarity refers to the situation in which some, but not all, nucleoside units of two strands can hydrogen bond with each other.“Substantial complementarity” refers to polynucleotide strands exhibiting 90% or greater complementarity, excluding regions of the polynucleotide strands, such as overhangs, that are selected so as to be noncomplementary. Specific binding requires a sufficient degree of complementarity to avoid non-specificbinding of the oligomeric compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, or in the case of in vitro assays, under conditions in which the assays are performed. The non-target sequences typically differ by at least 5 nucleotides.

[0208] In some embodiments, the double-stranded region of a double-stranded iRNA agent is equal to or at least, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotide pairs in length.

[0209] In some embodiments, the antisense strand of a double-stranded iRNA agent is equal to or at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0210] In some embodiments, the sense strand of a double-stranded iRNA agent is equal to or at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0211] In one embodiment, the sense and antisense strands of the double-stranded iRNA agent are each 15 to 30 nucleotides in length.

[0212] In one embodiment, the sense and antisense strands of the double-stranded iRNA agent are each 19 to 25 nucleotides in length.

[0213] In one embodiment, the sense and antisense strands of the double-stranded iRNA agent are each 21 to 23 nucleotides in length.

[0214] In some embodiments, one strand has at least one stretch of 1-5 single-stranded nucleotides in the double-stranded region. By“stretch of single-stranded nucleotides in the double-stranded region” is meant that there is present at least one nucleotide base pair at both ends of the single-stranded stretch. In some embodiments, both strands have at least one stretch of 1-5 (e.g., 1, 2, 3, 4, or 5) single-stranded nucleotides in the double stranded region. When both strands have a stretch of 1-5 (e.g., 1, 2, 3, 4, or 5) single-stranded nucleotides in the double stranded region, such single-stranded nucleotides can be opposite to each other (e.g., a stretch of mismatches) or they can be located such that the second strand has no single-stranded nucleotides opposite to the single-stranded iRNAs of the first strand and vice versa (e.g., a single-stranded loop). In some embodiments, the single-stranded nucleotides are present within 8 nucleotides from either end, for example 8, 7, 6, 5, 4, 3, or 2 nucleotide from either the 5’ or 3’ end of the region of complementarity between the two strands.

[0215] In one embodiment, the double-stranded iRNA agent comprises a single-stranded overhang on at least one of the termini. In one embodiment, the single-stranded overhang is 1, 2, or 3 nucleotides in length.

[0216] In one embodiment, the sense strand of the iRNA agent is 21- nucleotides in length, and the antisense strand is 23-nucleotides in length, wherein the strands form a double-stranded region of 21 consecutive base pairs having a 2-nucleotide longsingle-stranded overhangs at the 3’-end.

[0217] In some embodiments, each strand of the double-stranded iRNA has a ZXY structure, such as is described in PCT Publication No.2004080406, which is hereby incorporated by reference in its entirety.

[0218] In certain embodiment, the two strands of double-stranded oligomeric compound can be linked together. The two strands can be linked to each other at both ends, or at one end only. By linking at one end is meant that 5’-end of first strand is linked to the 3’-end of the second strand or 3’-end of first strand is linked to 5’-end of the second strand. When the two strands are linked to each other at both ends, 5’-end of first strand is linked to 3’-end of second strand and 3’-end of first strand is linked to 5’-end of second strand. The two strands can be linked together by an oligonucleotide linker including, but not limited to, (N)n;wherein N is independently a modified or unmodified nucleotide and n is 3-23. In some embodiemtns, n is 3-10, e.g., 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, theoligonucleotide linker is selected from the group consisting of GNRA, (G)4, (U)4, and (dT)4, wherein N is a modified or unmodified nucleotide and R is a modified or unmodified purine nucleotide. Some of the nucleotides in the linker can be involved in base-pair interactions with other nucleotides in the linker. The two strands can also be linked together by a non- nucleosidic linker, e.g. a linker described herein. It will be appreciated by one of skill in the art that any oligonucleotide chemical modifications or variations describe herein can be used in the oligonucleotide linker.

[0219] Hairpin and dumbbell type oligomeric compounds will have a duplex region equal to or at least 14, 15, 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The duplex region can be equal to or less than 200, 100, or 50, in length. In some embodiments, ranges for the duplex region are 15-30, 17 to 23, 19 to 23, and 19 to 21 nucleotides pairs in length. .

[0220] The hairpin oligomeric compounds can have a single strand overhang or terminal unpaired region, in some embodiments at the 3’, and in some embodiments on the antisense side of the hairpin. In some embodiments, the overhangs are 1-4, more generally 2-3nucleotides in length. The hairpin oligomeric compounds that can induce RNA interference are also referred to as“shRNA” herein.

[0221] In certain embodiments, two oligomeric strands specifically hybridize when there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target nucleic acid sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and under conditions in which assays are performed in the case of in vitro assays.

[0222] As used herein,“stringent hybridization conditions” or“stringent conditions” refers to conditions under which an antisense compound will hybridize to its target sequence, but to a minimal number of other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances, and“stringent conditions” under which antisense compounds hybridize to a target sequence are determined by the nature and composition of the antisense compounds and the assays in which they are being investigated.

[0223] It is understood in the art that incorporation of nucleotide affinity modifications may allow for a greater number of mismatches compared to an unmodified compound.Similarly, certain oligonucleotide sequences may be more tolerant to mismatches than other oligonucleotide sequences. One of ordinary skill in the art is capable of determining an appropriate number of mismatches between oligonucleotides, or between an oligonucleotide and a target nucleic acid, such as by determining melting temperature (Tm). Tm or DTm can be calculated by techniques that are familiar to one of ordinary skill in the art. For example, techniques described in Freier et al. (Nucleic Acids Research, 1997, 25, 22: 4429-4443) allow one of ordinary skill in the art to evaluate nucleotide modifications for their ability to increase the melting temperature of an RNA:DNA duplex. siRNA Design

[0224] In one embodiment, the iRNA agent of the invention is a double ended bluntmer of 19 nt in length, wherein the sense strand contains at least one motif of three 2’-F modifications on three consecutive nucleotides at positions 7,8,9 from the 5’end. The antisense strand contains at least one motif of three 2’-O-methyl modifications on three consecutive nucleotides at positions 11,12,13 from the 5’end.

[0225] In one embodiment, the iRNA agent of the invention is a double ended bluntmer of 20 nt in length, wherein the sense strand contains at least one motif of three 2’-F modifications on three consecutive nucleotides at positions 8,9,10 from the 5’end. Theantisense strand contains at least one motif of three 2’-O-methyl modifications on three consecutive nucleotides at positions 11,12,13 from the 5’end.

[0226] In one embodiment, the iRNA agent of the invention is a double ended bluntmer of 21 nt in length, wherein the sense strand contains at least one motif of three 2’-F modifications on three consecutive nucleotides at positions 9,10,11 from the 5’end. The antisense strand contains at least one motif of three 2’-O-methyl modifications on three consecutive nucleotides at positions 11,12,13 from the 5’end.

[0227] In one embodiment, the iRNA agent of the invention comprises a 21 nucleotides (nt) sense strand and a 23 nucleotides (nt) antisense, wherein the sense strand contains at least one motif of three 2’-F modifications on three consecutive nucleotides at positions 9,10,11 from the 5’end; the antisense strand contains at least one motif of three 2’-O-methyl modifications on three consecutive nucleotides at positions 11,12,13 from the 5’end, wherein one end of the iRNA is blunt, while the other end is comprises a 2 nt overhang. Preferably, the 2 nt overhang is at the 3’-end of the antisense. Optionally, the iRNA agent further comprises a ligand (e.g., GalNAc3).

[0228] In one embodiment, the iRNA agent of the invention comprises a sense and antisense strands, wherein: the sense strand is 25-30 nucleotide residues in length, wherein starting from the 5' terminal nucleotide (position 1) positions 1 to 23 of said first strand comprise at least 8 ribonucleotides; antisense strand is 36-66 nucleotide residues in length and, starting from the 3' terminal nucleotide, comprises at least 8 ribonucleotides in the positions paired with positions 1- 23 of sense strand to form a duplex; wherein at least the 3 ' terminal nucleotide of antisense strand is unpaired with sense strand, and up to 6 consecutive 3' terminal nucleotides are unpaired with sense strand, thereby forming a 3' single stranded overhang of 1-6 nucleotides; wherein the 5' terminus of antisense strand comprises from 10- 30 consecutive nucleotides which are unpaired with sense strand, thereby forming a 10-30 nucleotide single stranded 5' overhang; wherein at least the sense strand 5' terminal and 3' terminal nucleotides are base paired with nucleotides of antisense strand when sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially duplexed region between sense and antisense strands; and antisense strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of antisense strand length to reduce target gene expression when said double stranded nucleic acid is introduced into a mammalian cell; and wherein the sense strand contains at least one motif of three 2’-F modifications on three consecutive nucleotides, where at least one of the motifs occurs at ornear the cleavage site. The antisense strand contains at least one motif of three 2’-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

[0229] In one embodiment, the iRNA agent of the invention comprises a sense and antisense strands, wherein said iRNA agent comprises a first strand having a length which is at least 25 and at most 29 nucleotides and a second strand having a length which is at most 30 nucleotides with at least one motif of three 2’-O-methyl modifications on three consecutive nucleotides at position 11,12,13 from the 5’ end; wherein said 3’ end of said first strand and said 5’ end of said second strand form a blunt end and said second strand is 1-4 nucleotides longer at its 3’ end than the first strand, wherein the duplex region region which is at least 25 nucleotides in length, and said second strand is sufficiently complemenatary to a target mRNA along at least 19 nt of said second strand length to reduce target gene expression when said iRNA agent is introduced into a mammalian cell, and wherein dicer cleavage of said iRNA preferentially results in an siRNA comprising said 3’ end of said second strand, thereby reducing expression of the target gene in the mammal. Optionally, the iRNA agent further comprises a ligand (e.g., GalNAc3).

[0230] In one embodiment, the sense strand of the iRNA agent contains at least one motif of three identical modifications on three consecutive nucleotides, where one of the motifs occurs at the cleavage site in the sense strand. For instance, the sense strand can contain at least one motif of three 2’-F modifications on three consecutive nucleotides within 7-15 positions from the 5’end.

[0231] In one embodiment, the antisense strand of the iRNA agent can also contain at least one motif of three identical modifications on three consecutive nucleotides, where one of the motifs occurs at or near the cleavage site in the antisense strand. For instance, the antisense strand can contain at least one motif of three 2’-O-methyl modifications on three consecutive nucleotides within 9-15 positions from the 5’end.

[0232] For iRNA agent having a duplex region of 17-23 nt in length, the cleavage site of the antisense strand is typically around the 10, 11 and 12 positions from the 5’-end. Thus the motifs of three identical modifications may occur at the 9, 10, 11 positions; 10, 11, 12 positions; 11, 12, 13 positions; 12, 13, 14 positions; or 13, 14, 15 positions of the antisense strand, the count starting from the 1stnucleotide from the 5’-end of the antisense strand, or, the count starting from the 1stpaired nucleotide within the duplex region from the 5’- end of the antisense strand. The cleavage site in the antisense strand may also change according to the length of the duplex region of the iRNA from the 5’-end.

[0233] In some embodiments, the iRNA agent comprises a sense strand and antisense strand each having 14 to 30 nucleotides, wherein the sense strand contains at least two motifs of three identical modifications on three consecutive nucleotides, where at least one of the motifs occurs at or near the cleavage site within the strand and at least one of the motifs occurs at another portion of the strand that is separated from the motif at the cleavage site by at least one nucleotide. In one embodiment, the antisense strand also contains at least one motif of three identical modifications on three consecutive nucleotides, where at least one of the motifs occurs at or near the cleavage site within the strand. The modification in the motif occurring at or near the cleavage site in the sense strand is different than the modification in the motif occurring at or near the cleavage site in the antisense strand.

[0234] In some embodiments, the iRNA agent comprises a sense strand and antisense strand each having 14 to 30 nucleotides, wherein the sense strand contains at least one motif of three 2’-F modifications on three consecutive nucleotides, where at least one of the motifs occurs at or near the cleavage site in the strand. In one embodiment, the antisense strand also contains at least one motif of three 2’-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

[0235] In some embodiments, the iRNA agent comprises a sense strand and antisense strand each having 14 to 30 nucleotides, wherein the sense strand contains at least one motif of three 2’-F modifications on three consecutive nucleotides at positions 9,10,11 from the 5’end, and wherein the antisense strand contains at least one motif of three 2’-O-methyl modifications on three consecutive nucleotides at positions 11,12,13 from the 5’end.

[0236] In one embodiment, the iRNA agent of the invention comprises mismatch(es) with the target, within the duplex, or combinations thereof. The mistmatch can occur in the overhang region or the duplex region. The base pair can be ranked on the basis of their propensity to promote dissociation or melting (e.g., on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on an individual pair basis, though next neighbor or similar analysis can also be used). In terms of promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; and I:C is preferred over G:C (I=inosine). Mismatches, e.g., non-canonical or other than canonical pairings (as described elsewhere herein) are preferred over canonical (A:T, A:U, G:C) pairings; and pairings which include a universal base are preferred over canonical pairings.

[0237] In one embodiment, the iRNA agent of the invention comprises at least one of the first 1, 2, 3, 4, or 5 base pairs within the duplex regions from the 5’- end of the antisensestrand can be chosen independently from the group of: A:U, G:U, I:C, and mismatched pairs, e.g., non-canonical or other than canonical pairings or pairings which include a universal base, to promote the dissociation of the antisense strand at the 5’-end of the duplex.

[0238] In one embodiment, the nucleotide at the 1 position within the duplex region from the 5’-end in the antisense strand is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2 or 3 base pair within the duplex region from the 5’- end of the antisense strand is an AU base pair. For example, the first base pair within the duplex region from the 5’- end of the antisense strand is an AU base pair.

[0239] In one aspect, the invention relates to a double-stranded RNA (dsRNA) agent for inhibiting the expression of a target gene. The dsRNA agent comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides. The dsRNA agent is represented by formula (I):

[0240] In formula (I), B1, B2, B3, B1’, B2’, B3’, and B4’ each are independently a nucleotide containing a modification selected from the group consisting of 2’-O-alkyl, 2’- substituted alkoxy, 2’-substituted alkyl, 2’-halo, ENA, and BNA / LNA. In one embodiment, B1, B2, B3, B1’, B2’, B3’, and B4’ each contain 2’-OMe modifications. In one embodiment, B1, B2, B3, B1’, B2’, B3’, and B4’ each contain 2’-OMe or 2’-F modifications. In one embodiment, at least one of B1, B2, B3, B1’, B2’, B3’, and B4’ contain 2'-O-N- methylacetamido (2'-O-NMA) modification.

[0241] C1 is a thermally destabilizing nucleotide placed at a site opposite to the seed region of the antisense strand (i.e., at positions 2-8 of the 5’-end of the antisense strand). For example, C1 is at a position of the sense strand that pairs with a nucleotide at positions 2-8 of the 5’-end of the antisense strand. In one example, C1 is at position 15 from the 5’-end of the sense strand. C1 nucleotide bears the thermally destabilizing modification which can include abasic modification; mismatch with the opposing nucleotide in the duplex; and sugar modification such as 2’-deoxy modification or acyclic nucleotide e.g., unlocked nucleic acids (UNA) or glycerol nucleic acid (GNA). In one embodiment, C1 has thermally destabilizingmodification selected from the group consisting of: i) mismatch with the opposing nucleotide in the antisense strand; ii) abasic modification selected from the group consisting of:iii) sugar modification selected from the group consisting of:, wherein B is a modified or unmodified nucleobase, R1and R2independently are H, halogen, OR3, or alkyl; and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar. In one embodiment, the thermally destabilizing modification in C1 is a mismatch selected from the group consisting of G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, and U:T; and optionally, at least one nucleobase in the mismatch pair is a 2’- deoxy nucleobase. In one example, the thermally destabilizing modification in C1 is GNA or.

[0242] T1, T1’, T2’, and T3’ each independently represent a nucleotide comprising a modification providing the nucleotide a steric bulk that is less or equal to the steric bulk of a 2’-OMe modification. A steric bulk refers to the sum of steric effects of a modification. Methods for determining steric effects of a modification of a nucleotide are known to one skilled in the art. The modification can be at the 2’ position of a ribose sugar of the nucleotide, or a modification to a non-ribose nucleotide, acyclic nucleotide, or the backbone of the nucleotide that is similar or equivalent to the 2’ position of the ribose sugar, and provides the nucleotide a steric bulk that is less than or equal to the steric bulk of a 2’-OMemodification. For example, T1, T1’, T2’, and T3’ are each independently selected from DNA, RNA, LNA, 2’-F, and 2’-F-5’-methyl. In one embodiment, T1 is DNA. In one embodiment, T1’ is DNA, RNA or LNA. In one embodiment, T2’ is DNA or RNA. In one embodiment, T3’ is DNA or RNA.

[0243] n1, n3, and q1are independently 4 to 15 nucleotides in length.

[0244] n5, q3, and q7are independently 1-6 nucleotide(s) in length.

[0245] n4, q2, and q6are independently 1-3 nucleotide(s) in length; alternatively, n4is 0. q5is independently 0-10 nucleotide(s) in length.

[0246] n2and q4are independently 0-3 nucleotide(s) in length.

[0247] Alternatively, n4is 0-3 nucleotide(s) in length.

[0248] In one embodiment, n4can be 0. In one example, n4is 0, and q2and q6are 1. In another example, n4is 0, and q2and q6are 1, with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end of the antisense strand).

[0249] In one embodiment, n4, q2, and q6are each 1.

[0250] In one embodiment, n2, n4, q2, q4, and q6are each 1.

[0251] In one embodiment, C1 is at position 14-17 of the 5’-end of the sense strand, when the sense strand is 19-22 nucleotides in length, and n4is 1. In one embodiment, C1 is at position 15 of the 5’-end of the sense strand

[0252] In one embodiment, T3’ starts at position 2 from the 5’ end of the antisense strand. In one example, T3’ is at position 2 from the 5’ end of the antisense strand and q6is equal to 1.

[0253] In one embodiment, T1’ starts at position 14 from the 5’ end of the antisense strand. In one example, T1’ is at position 14 from the 5’ end of the antisense strand and q2is equal to 1.

[0254] In an exemplary embodiment, T3’ starts from position 2 from the 5’ end of the antisense strand and T1’ starts from position 14 from the 5’ end of the antisense strand. In one example, T3’ starts from position 2 from the 5’ end of the antisense strand and q6is equal to 1 and T1’ starts from position 14 from the 5’ end of the antisense strand and q2is equal to 1.

[0255] In one embodiment, T1’ and T3’ are separated by 11 nucleotides in length (i.e. not counting the T1’ and T3’ nucleotides).

[0256] In one embodiment, T1’ is at position 14 from the 5’ end of the antisense strand. In one example, T1’ is at position 14 from the 5’ end of the antisense strand and q2is equal to 1, and the modification at the 2’ position or positions in a non-ribose, acyclic or backbone that provide less steric bulk than a 2’-OMe ribose.

[0257] In one embodiment, T3’ is at position 2 from the 5’ end of the antisense strand. In one example, T3’ is at position 2 from the 5’ end of the antisense strand and q6is equal to 1, and the modification at the 2’ position or positions in a non-ribose, acyclic or backbone that provide less than or equal to steric bulk than a 2’-OMe ribose.In one embodiment, T1 is at the cleavage site of the sense strand. In one example, T1 is at position 11 from the 5’ end of the sense strand, when the sense strand is 19-22 nucleotides in length, and n2is 1. In an exemplary embodiment, T1 is at the cleavage site of the sense strand at position 11 from the 5’ end of the sense strand, when the sense strand is 19-22 nucleotides in length, and n2is 1,

[0258] In one embodiment, T2’ starts at position 6 from the 5’ end of the antisense strand. In one example, T2’ is at positions 6-10 from the 5’ end of the antisense strand, and q4is 1.In an exemplary embodiment, T1 is at the cleavage site of the sense strand, for instance, at position 11 from the 5’ end of the sense strand, when the sense strand is 19-22 nucleotides in length, and n2is 1; T1’ is at position 14 from the 5’ end of the antisense strand, and q2is equal to 1, and the modification to T1’ is at the 2’ position of a ribose sugar or at positions in a non-ribose, acyclic or backbone that provide less steric bulk than a 2’-OMe ribose; T2’ is at positions 6-10 from the 5’ end of the antisense strand, and q4is 1; and T3’ is at position 2 from the 5’ end of the antisense strand, and q6is equal to 1, and the modification to T3’ is at the 2’ position or at positions in a non-ribose, acyclic or backbone that provide less than or equal to steric bulk than a 2’-OMe ribose.

[0259] In one embodiment, T2’ starts at position 8 from the 5’ end of the antisense strand. In one example, T2’ starts at position 8 from the 5’ end of the antisense strand, and q4is 2.In one embodiment, T2’ starts at position 9 from the 5’ end of the antisense strand. In one example, T2’ is at position 9 from the 5’ end of the antisense strand, and q4is 1.In one embodiment, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’- F, q3is 4, T2’ is 2’-F, q4is 1, B3’ is 2’-OMe or 2’-F, q5is 6, T3’ is 2’-F, q6is 1, B4’ is 2’- OMe, and q7is 1; with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end of the antisense strand).

[0260] In one embodiment, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, T2’ is 2’-F, q4is 1, B3’ is 2’-OMe or 2’-F, q5is 6, T3’ is 2’-F, q6is 1, B4’ is 2’-OMe, and q7is 1; with two phosphorothioateinternucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkagemodifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end of the antisense strand).

[0261] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, T2’ is 2’-F, q4is 2, B3’ is 2’-OMe or 2’-F, q5is 5, T3’ is 2’-F, q6is 1, B4’ is 2’-OMe, and q7is 1.

[0262] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, T2’ is 2’-F, q4is 2, B3’ is 2’-OMe or 2’-F, q5is 5, T3’ is 2’- F, q6is 1, B4’ is 2’-OMe, and q7is 1; with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end of the antisense strand).

[0263] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 6, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 7, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, T2’ is 2’-F, q4is 2, B3’ is 2’-OMe or 2’-F, q5is 5, T3’ is 2’-F, q6is 1, B4’ is 2’-OMe, and q7is 1.

[0264] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 6, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 7, T1’ is 2’-F, q2is1, B2’ is 2’-OMe or 2’-F, q3is 4, T2’ is 2’-F, q4is 2, B3’ is 2’-OMe or 2’-F, q5is 5, T3’ is 2’- F, q6is 1, B4’ is 2’-OMe, and q7is 1; with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end of the antisense strand).

[0265] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, T2’ is 2’-F, q4is 1, B3’ is 2’-OMe or 2’-F, q5is 6, T3’ is 2’-F, q6is 1, B4’ is 2’-OMe, and q7is 1.

[0266] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, T2’ is 2’-F, q4is 1, B3’ is 2’-OMe or 2’-F, q5is 6, T3’ is 2’- F, q6is 1, B4’ is 2’-OMe, and q7is 1; with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end of the antisense strand).

[0267] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 5, T2’ is 2’-F, q4is 1, B3’ is 2’-OMe or 2’-F, q5is 5, T3’ is 2’-F, q6is 1, B4’ is 2’-OMe, and q7is 1; optionally with at least 2 additional TT at the 3’-end of the antisense strand.

[0268] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 5, T2’ is 2’-F, q4is 1, B3’ is 2’-OMe or 2’-F, q5is 5, T3’ is 2’- F, q6is 1, B4’ is 2’-OMe, and q7is 1; optionally with at least 2 additional TT at the 3’-end of the antisense strand; with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end of the antisense strand).

[0269] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, q4is 0, B3’ is 2’-OMe or 2’-F, q5is 7, T3’ is 2’-F, q6is 1, B4’ is 2’-OMe, and q7is 1.

[0270] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, q4is 0, B3’ is 2’-OMe or 2’-F, q5is 7, T3’ is 2’-F, q6is 1, B4’ is 2’-OMe, and q7is 1; with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5’-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end).

[0271] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, T2’ is 2’-F, q4is 2, B3’ is 2’-OMe or 2’-F, q5is 5, T3’ is 2’-F, q6is 1, B4’ is 2’-F, and q7is 1.

[0272] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, T2’ is 2’-F, q4is 2, B3’ is 2’-OMe or 2’-F, q5is 5, T3’ is 2’- F, q6is 1, B4’ is 2’-F, and q7is 1; with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end of the antisense strand).

[0273] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, q4is 0, B3’ is 2’-OMe or 2’-F, q5is 7, T3’ is 2’-F, q6is 1, B4’ is 2’-F, and q7is 1.

[0274] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, q4is 0, B3’ is 2’-OMe or 2’-F, q5is 7, T3’ is 2’-F, q6is 1, B4’ is 2’-F, and q7is 1; with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5’-end of the sense strand), andtwo phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end of the antisense strand).

[0275] The dsRNA agent can comprise a phosphorus-containing group at the 5’-end of the sense strand or antisense strand. The 5’-end phosphorus-containing group can be 5’-end phosphate (5’-P), 5’-end phosphorothioate (5’-PS), 5’-end phosphorodithioate (5’-PS2), 5’-end vinylphosphonate (5’-VP), 5’-end methylphosphonate (MePhos), or 5’-deoxy-5’-C-malonyl ( ). When the 5’-end phosphorus-containing group is 5’-end vinylphosphonate (5’-VP), the 5’-VP can be either 5’-E-VP isomer (i.e., trans-vinylphosphate,isomer (i.e., cis-vinylphosphate,or mixtures thereof.

[0276] In one embodiment, the dsRNA agent comprises a phosphorus-containing group at the 5’-end of the sense strand. In one embodiment, the dsRNA agent comprises a phosphorus-containing group at the 5’-end of the antisense strand.

[0277] In one embodiment, the dsRNA agent comprises a 5’-P. In one embodiment, the dsRNA agent comprises a 5’-P in the antisense strand.In one embodiment, the dsRNA agent comprises a 5’-PS. In one embodiment, the dsRNA agent comprises a 5’-PS in the antisense strand.

[0278] In one embodiment, the dsRNA agent comprises a 5’-VP. In one embodiment, the dsRNA agent comprises a 5’-VP in the antisense strand. In one embodiment, the dsRNA agent comprises a 5’-E-VP in the antisense strand. In one embodiment, the dsRNA agent comprises a 5’-Z-VP in the antisense strand.

[0279] In one embodiment, the dsRNA agent comprises a 5’-PS2. In one embodiment, the dsRNA agent comprises a 5’-PS2 in the antisense strand.

[0280] In one embodiment, the dsRNA agent comprises a 5’-PS2. In one embodiment, the dsRNA agent comprises a 5’-deoxy-5’-C-malonyl in the antisense strand.

[0281] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1,B2’ is 2’-OMe or 2’-F, q3is 4, T2’ is 2’-F, q4is 2, B3’ is 2’-OMe or 2’-F, q5is 5, T3’ is 2’-F, q6is 1, B4’ is 2’-OMe, and q7is 1. The dsRNA agent also comprises a 5’-PS.

[0282] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, T2’ is 2’-F, q4is 2, B3’ is 2’-OMe or 2’-F, q5is 5, T3’ is 2’-F, q6is 1, B4’ is 2’-OMe, and q7is 1. The dsRNA agent also comprises a 5’-P.

[0283] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, T2’ is 2’-F, q4is 2, B3’ is 2’-OMe or 2’-F, q5is 5, T3’ is 2’-F, q6is 1, B4’ is 2’-OMe, and q7is 1. The dsRNA agent also comprises a 5’-VP. The 5’-VP may be 5’-E-VP, 5’-Z-VP, or combination thereof.

[0284] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, T2’ is 2’-F, q4is 2, B3’ is 2’-OMe or 2’-F, q5is 5, T3’ is 2’-F, q6is 1, B4’ is 2’-OMe, and q7is 1. The dsRNA agent also comprises a 5’- PS2.

[0285] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, T2’ is 2’-F, q4is 2, B3’ is 2’-OMe or 2’-F, q5is 5, T3’ is 2’-F, q6is 1, B4’ is 2’-OMe, and q7is 1. The dsRNA agent also comprises a 5’-deoxy-5’-C- malonyl.

[0286] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, T2’ is 2’-F, q4is 2, B3’ is 2’-OMe or 2’-F, q5is 5, T3’ is 2’- F, q6is 1, B4’ is 2’-OMe, and q7is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end of the antisense strand). The dsRNA agent also comprises a 5’-P.

[0287] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, T2’ is 2’-F, q4is 2, B3’ is 2’-OMe or 2’-F, q5is 5, T3’ is 2’- F, q6is 1, B4’ is 2’-OMe, and q7is 1; with two phosphorothioate internucleotide linkagemodifications within position 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end of the antisense strand). The dsRNA agent also comprises a 5’-PS.

[0288] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, T2’ is 2’-F, q4is 2, B3’ is 2’-OMe or 2’-F, q5is 5, T3’ is 2’- F, q6is 1, B4’ is 2’-OMe, and q7is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end of the antisense strand). The dsRNA agent also comprises a 5’-VP. The 5’-VP may be 5’-E-VP, 5’-Z-VP, or combination thereof.

[0289] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, T2’ is 2’-F, q4is 2, B3’ is 2’-OMe or 2’-F, q5is 5, T3’ is 2’- F, q6is 1, B4’ is 2’-OMe, and q7is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end of the antisense strand). The dsRNA agent also comprises a 5’- PS2.

[0290] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, T2’ is 2’-F, q4is 2, B3’ is 2’-OMe or 2’-F, q5is 5, T3’ is 2’- F, q6is 1, B4’ is 2’-OMe, and q7is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end of the antisense strand). The dsRNA agent also comprises a 5’-deoxy-5’-C-malonyl.

[0291] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, q4is 0, B3’ is 2’-OMe or 2’-F, q5is 7, T3’ is 2’-F, q6is 1, B4’ is 2’-OMe, and q7is 1. The dsRNA agent also comprises a 5’-P.

[0292] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, q4is 0, B3’ is 2’-OMe or 2’-F, q5is 7, T3’ is 2’-F, q6is 1, B4’ is 2’-OMe, and q7is 1. The dsRNA agent also comprises a 5’-PS.

[0293] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, q4is 0, B3’ is 2’-OMe or 2’-F, q5is 7, T3’ is 2’-F, q6is 1, B4’ is 2’-OMe, and q7is 1. The dsRNA agent also comprises a 5’-VP. The 5’-VP may be 5’-E-VP, 5’-Z-VP, or combination thereof.

[0294] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, q4is 0, B3’ is 2’-OMe or 2’-F, q5is 7, T3’ is 2’-F, q6is 1, B4’ is 2’-OMe, and q7is 1. The dsRNA agent also comprises a 5’- PS2.

[0295] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, q4is 0, B3’ is 2’-OMe or 2’-F, q5is 7, T3’ is 2’-F, q6is 1, B4’ is 2’-OMe, and q7is 1. The dsRNA agent also comprises a 5’-deoxy-5’-C-malonyl.

[0296] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, q4is 0, B3’ is 2’-OMe or 2’-F, q5is 7, T3’ is 2’-F, q6is 1, B4’ is 2’-OMe, and q7is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end). The dsRNA agent also comprises a 5’-P.

[0297] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, q4is 0, B3’ is 2’-OMe or 2’-F, q5is 7, T3’ is 2’-F, q6is 1, B4’ is 2’-OMe, and q7is 1; with two phosphorothioate internucleotide linkage modificationswithin position 1-5 of the sense strand (counting from the 5’-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end). The dsRNA agent also comprises a 5’-PS.

[0298] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, q4is 0, B3’ is 2’-OMe or 2’-F, q5is 7, T3’ is 2’-F, q6is 1, B4’ is 2’-OMe, and q7is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end). The dsRNA agent also comprises a 5’-VP. The 5’-VP may be 5’-E-VP, 5’- Z-VP, or combination thereof.

[0299] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, q4is 0, B3’ is 2’-OMe or 2’-F, q5is 7, T3’ is 2’-F, q6is 1, B4’ is 2’-OMe, and q7is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end). The dsRNA agent also comprises a 5’- PS2.

[0300] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, q4is 0, B3’ is 2’-OMe or 2’-F, q5is 7, T3’ is 2’-F, q6is 1, B4’ is 2’-OMe, and q7is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end). The dsRNA agent also comprises a 5’-deoxy-5’-C-malonyl.

[0301] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, T2’ is 2’-F, q4is 2, B3’ is 2’-OMe or 2’-F, q5is 5, T3’ is 2’-F, q6is 1, B4’ is 2’-F, and q7is 1. The dsRNA agent also comprises a 5’- P.

[0302] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, T2’ is 2’-F, q4is 2, B3’ is 2’-OMe or 2’-F, q5is 5, T3’ is 2’-F, q6is 1, B4’ is 2’-F, and q7is 1. The dsRNA agent also comprises a 5’- PS.

[0303] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, T2’ is 2’-F, q4is 2, B3’ is 2’-OMe or 2’-F, q5is 5, T3’ is 2’-F, q6is 1, B4’ is 2’-F, and q7is 1. The dsRNA agent also comprises a 5’- VP. The 5’-VP may be 5’-E-VP, 5’-Z-VP, or combination thereof.

[0304] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, T2’ is 2’-F, q4is 2, B3’ is 2’-OMe or 2’-F, q5is 5, T3’ is 2’-F, q6is 1, B4’ is 2’-F, and q7is 1. The dsRNA agent also comprises a 5’- PS2.

[0305] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, T2’ is 2’-F, q4is 2, B3’ is 2’-OMe or 2’-F, q5is 5, T3’ is 2’-F, q6is 1, B4’ is 2’-F, and q7is 1. The dsRNA agent also comprises a 5’-deoxy-5’-C-malonyl.

[0306] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, T2’ is 2’-F, q4is 2, B3’ is 2’-OMe or 2’-F, q5is 5, T3’ is 2’- F, q6is 1, B4’ is 2’-F, and q7is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end of the antisense strand). The dsRNA agent also comprises a 5’- P.

[0307] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, T2’ is 2’-F, q4is 2, B3’ is 2’-OMe or 2’-F, q5is 5, T3’ is 2’- F, q6is 1, B4’ is 2’-F, and q7is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of theantisense strand (counting from the 5’-end of the antisense strand). The dsRNA agent also comprises a 5’- PS.

[0308] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, T2’ is 2’-F, q4is 2, B3’ is 2’-OMe or 2’-F, q5is 5, T3’ is 2’- F, q6is 1, B4’ is 2’-F, and q7is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end of the antisense strand). The dsRNA agent also comprises a 5’- VP. The 5’-VP may be 5’-E-VP, 5’-Z-VP, or combination thereof.

[0309] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, T2’ is 2’-F, q4is 2, B3’ is 2’-OMe or 2’-F, q5is 5, T3’ is 2’- F, q6is 1, B4’ is 2’-F, and q7is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end of the antisense strand). The dsRNA agent also comprises a 5’- PS2.

[0310] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, T2’ is 2’-F, q4is 2, B3’ is 2’-OMe or 2’-F, q5is 5, T3’ is 2’- F, q6is 1, B4’ is 2’-F, and q7is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end of the antisense strand). The dsRNA agent also comprises a 5’-deoxy-5’-C-malonyl.

[0311] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, q4is 0, B3’ is 2’-OMe or 2’-F, q5is 7, T3’ is 2’-F, q6is 1, B4’ is 2’-F, and q7is 1. The dsRNA agent also comprises a 5’- P.

[0312] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, q4is 0, B3’ is 2’-OMe or 2’-F, q5is 7, T3’ is 2’-F, q6is 1, B4’ is 2’-F, and q7is 1. The dsRNA agent also comprises a 5’- PS.

[0313] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, q4is 0, B3’ is 2’-OMe or 2’-F, q5is 7, T3’ is 2’-F, q6is 1, B4’ is 2’-F, and q7is 1. The dsRNA agent also comprises a 5’- VP. The 5’-VP may be 5’-E- VP, 5’-Z-VP, or combination thereof.

[0314] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, q4is 0, B3’ is 2’-OMe or 2’-F, q5is 7, T3’ is 2’-F, q6is 1, B4’ is 2’-F, and q7is 1. The dsRNA agent also comprises a 5’- PS2.

[0315] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, q4is 0, B3’ is 2’-OMe or 2’-F, q5is 7, T3’ is 2’-F, q6is 1, B4’ is 2’-F, and q7is 1. The dsRNA agent also comprises a 5’-deoxy-5’-C-malonyl.

[0316] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, q4is 0, B3’ is 2’-OMe or 2’-F, q5is 7, T3’ is 2’-F, q6is 1, B4’ is 2’-F, and q7is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end of the antisense strand). The dsRNA agent also comprises a 5’- P.

[0317] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, q4is 0, B3’ is 2’-OMe or 2’-F, q5is 7, T3’ is 2’-F, q6is 1, B4’ is 2’-F, and q7is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of theantisense strand (counting from the 5’-end of the antisense strand). The dsRNA agent also comprises a 5’- PS.

[0318] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, q4is 0, B3’ is 2’-OMe or 2’-F, q5is 7, T3’ is 2’-F, q6is 1, B4’ is 2’-F, and q7is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end of the antisense strand). The dsRNA agent also comprises a 5’- VP. The 5’-VP may be 5’-E-VP, 5’-Z-VP, or combination thereof.

[0319] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, q4is 0, B3’ is 2’-OMe or 2’-F, q5is 7, T3’ is 2’-F, q6is 1, B4’ is 2’-F, and q7is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end of the antisense strand). The dsRNA agent also comprises a 5’- PS2.

[0320] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, q4is 0, B3’ is 2’-OMe or 2’-F, q5is 7, T3’ is 2’-F, q6is 1, B4’ is 2’-F, and q7is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end of the antisense strand). The dsRNA agent also comprises a 5’-deoxy-5’-C-malonyl.

[0321] In one embodiment, 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35% or 30% of the dsRNA agent of the invention is modified. For example, when 50% of the dsRNA agent is modified, 50% of all nucleotides present in the dsRNA agent contain a modification as described herein.

[0322] In one embodiment, each of the sense and antisense strands of the dsRNA agent is independently modified with acyclic nucleotides, LNA, HNA, CeNA, 2’-methoxyethyl, 2’- O-methyl, 2’-O-allyl, 2’-C-allyl, 2’-deoxy, 2’-fluoro, 2'-O-N-methylacetamido (2'-O- NMA), a 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), or 2'-ara-F.

[0323] In one embodiment, each of the sense and antisense strands of the dsRNA agent contains at least two different modifications.

[0324] In one embodiment, the dsRNA agent of Formula (I) further comprises 3’ and / or 5’ overhang(s) of 1-10 nucleotides in length. In one example, dsRNA agent of formula (I) comprises a 3’ overhang at the 3’-end of the antisense strand and a blunt end at the 5’-end of the antisense strand. In another example, the dsRNA agent has a 5’ overhang at the 5’-end of the sense strand.

[0325] In one embodiment, the dsRNA agent of the invention does not contain any 2’-F modification.

[0326] In one embodiment, the sense strand and / or antisense strand of the dsRNA agent comprises one or more blocks of phosphorothioate or methylphosphonate internucleotide linkages. In one example, the sense strand comprises one block of two phosphorothioate or methylphosphonate internucleotide linkages. In one example, the antisense strand comprises two blocks of two phosphorothioate or methylphosphonate internucleotide linkages. For example, the two blocks of phosphorothioate or methylphosphonate internucleotide linkages are separated by 16-18 phosphate internucleotide linkages.

[0327] In one embodiment, each of the sense and antisense strands of the dsRNA agent has 15-30 nucleotides. In one example, the sense strand has 19-22 nucleotides, and the antisense strand has 19-25 nucleotides. In another example, the sense strand has 21 nucleotides, and the antisense strand has 23 nucleotides.

[0328] In one embodiment, the nucleotide at position 1 of the 5’-end of the antisense strand in the duplex is selected from the group consisting of A, dA, dU, U, and dT. In one embodiment, at least one of the first, second, and third base pair from the 5’-end of the antisense strand is an AU base pair.

[0329] In one embodiment, the antisense strand of the dsRNA agent of the invention is 100% complementary to a target RNA to hybridize thereto and inhibits its expression through RNA interference. In another embodiment, the antisense strand of the dsRNA agent of theinvention is at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, or at least 50% complementary to a target RNA.

[0330] In one aspect, the invention relates to a dsRNA agent as defined herein capable of inhibiting the expression of a target gene. The dsRNA agent comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides. The sense strand contains at least one thermally destabilizing nucleotide, wherein at least one of said thermally destabilizing nucleotide occurs at or near the site that is opposite to the seed region of the antisense strand (i.e. at position 2-8 of the 5’-end of the antisense strand). Each of the embodiments and aspects described in this specification relating to the dsRNA represented by formula (I) can also apply to the dsRNA containing the thermally destabilizing nucleotide.

[0331] The thermally destabilizing nucleotide can occur, for example, between positions 14-17 of the 5’-end of the sense strand when the sense strand is 21 nucleotides in length. The antisense strand contains at least two modified nucleic acids that are smaller than a sterically demanding 2’-OMe modification. Preferably, the two modified nucleic acids that are smaller than a sterically demanding 2’-OMe are separated by 11 nucleotides in length. For example, the two modified nucleic acids are at positions 2 and 14 of the 5’end of the antisense strand.

[0332] In one embodiment, the dsRNA agent further comprises at least one ASGPR ligand. For example, the ASGPR ligand is one or more GalNAc derivatives attached througha bivalent or trivalent branched linker, such as: . In oneexample, the ASGPR ligand is attached to the 3’ end of the sense strand.

[0333] For example, the dsRNA agent as defined herein can comprise i) a phosphorus- containing group at the 5’-end of the sense strand or antisense strand; ii) with twophosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioateinternucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end of the antisense strand); and iii) a ligand, such as a ASGPR ligand (e.g., one or more GalNAc derivatives) at 5’-end or 3’-end of the sense strand or antisense strand. For instance, the ligand may be at the 3’-end of the sense strand.

[0334] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, T2’ is 2’-F, q4is 2, B3’ is 2’-OMe or 2’-F, q5is 5, T3’ is 2’- F, q6is 1, B4’ is 2’-OMe, and q7is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end of the antisense strand). The dsRNA agent also comprises a 5’-P and a targeting ligand. In one embodiment, the 5’-P is at the 5’-end of the antisense strand, and the targeting ligand is at the 3’-end of the sense strand.

[0335] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, T2’ is 2’-F, q4is 2, B3’ is 2’-OMe or 2’-F, q5is 5, T3’ is 2’- F, q6is 1, B4’ is 2’-OMe, and q7is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end of the antisense strand). The dsRNA agent also comprises a 5’-PS and a targeting ligand. In one embodiment, the 5’-PS is at the 5’-end of the antisense strand, and the targeting ligand is at the 3’-end of the sense strand.

[0336] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, T2’ is 2’-F, q4is 2, B3’ is 2’-OMe or 2’-F, q5is 5, T3’ is 2’- F, q6is 1, B4’ is 2’-OMe, and q7is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end of the antisense strand). The dsRNA agent also comprises a 5’-VP (e.g., a 5’-E-VP, 5’-Z-VP, or combination thereof), and a targeting ligand. In one embodiment, the 5’-VP is at the 5’-end of the antisense strand, and the targeting ligand is at the 3’-end of the sense strand.

[0337] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is1, B2’ is 2’-OMe or 2’-F, q3is 4, T2’ is 2’-F, q4is 2, B3’ is 2’-OMe or 2’-F, q5is 5, T3’ is 2’- F, q6is 1, B4’ is 2’-OMe, and q7is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end of the antisense strand). The dsRNA agent also comprises a 5’- PS2and a targeting ligand. In one embodiment, the 5’-PS2is at the 5’-end of the antisense strand, and the targeting ligand is at the 3’-end of the sense strand.

[0338] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, T2’ is 2’-F, q4is 2, B3’ is 2’-OMe or 2’-F, q5is 5, T3’ is 2’- F, q6is 1, B4’ is 2’-OMe, and q7is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end of the antisense strand). The dsRNA agent also comprises a 5’-deoxy-5’-C-malonyl and a targeting ligand. In one embodiment, the 5’- deoxy-5’-C-malonyl is at the 5’-end of the antisense strand, and the targeting ligand is at the 3’-end of the sense strand.

[0339] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, q4is 0, B3’ is 2’-OMe or 2’-F, q5is 7, T3’ is 2’-F, q6is 1, B4’ is 2’-OMe, and q7is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end). The dsRNA agent also comprises a 5’-P and a targeting ligand. In one embodiment, the 5’-P is at the 5’-end of the antisense strand, and the targeting ligand is at the 3’-end of the sense strand.

[0340] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, q4is 0, B3’ is 2’-OMe or 2’-F, q5is 7, T3’ is 2’-F, q6is 1, B4’ is 2’-OMe, and q7is 1; with two phosphorothioate internucleotide linkage modificationswithin position 1-5 of the sense strand (counting from the 5’-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end). The dsRNA agent also comprises a 5’-PS and a targeting ligand. In one embodiment, the 5’-PS is at the 5’-end of the antisense strand, and the targeting ligand is at the 3’-end of the sense strand.

[0341] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, q4is 0, B3’ is 2’-OMe or 2’-F, q5is 7, T3’ is 2’-F, q6is 1, B4’ is 2’-OMe, and q7is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end). The dsRNA agent also comprises a 5’-VP (e.g., a 5’-E-VP, 5’-Z-VP, or combination thereof) and a targeting ligand. In one embodiment, the 5’-VP is at the 5’-end of the antisense strand, and the targeting ligand is at the 3’-end of the sense strand.

[0342] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, q4is 0, B3’ is 2’-OMe or 2’-F, q5is 7, T3’ is 2’-F, q6is 1, B4’ is 2’-OMe, and q7is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end). The dsRNA agent also comprises a 5’-PS2and a targeting ligand. In one embodiment, the 5’-PS2 is at the 5’-end of the antisense strand, and the targeting ligand is at the 3’-end of the sense strand.

[0343] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, q4is 0, B3’ is 2’-OMe or 2’-F, q5is 7, T3’ is 2’-F, q6is 1, B4’ is 2’-OMe, and q7is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (countingfrom the 5’-end). The dsRNA agent also comprises a 5’-deoxy-5’-C-malonyl and a targeting ligand. In one embodiment, the 5’-deoxy-5’-C-malonyl is at the 5’-end of the antisense strand, and the targeting ligand is at the 3’-end of the sense strand.

[0344] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, T2’ is 2’-F, q4is 2, B3’ is 2’-OMe or 2’-F, q5is 5, T3’ is 2’- F, q6is 1, B4’ is 2’-F, and q7is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end of the antisense strand). The dsRNA agent also comprises a 5’-P and a targeting ligand. In one embodiment, the 5’-P is at the 5’-end of the antisense strand, and the targeting ligand is at the 3’-end of the sense strand.

[0345] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, T2’ is 2’-F, q4is 2, B3’ is 2’-OMe or 2’-F, q5is 5, T3’ is 2’- F, q6is 1, B4’ is 2’-F, and q7is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end of the antisense strand). The dsRNA agent also comprises a 5’-PS and a targeting ligand. In one embodiment, the 5’-PS is at the 5’-end of the antisense strand, and the targeting ligand is at the 3’-end of the sense strand.

[0346] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, T2’ is 2’-F, q4is 2, B3’ is 2’-OMe or 2’-F, q5is 5, T3’ is 2’- F, q6is 1, B4’ is 2’-F, and q7is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end of the antisense strand). The dsRNA agent also comprises a 5’-VP (e.g., a 5’-E-VP, 5’-Z-VP, or combination thereof) and a targeting ligand.In one embodiment, the 5’-VP is at the 5’-end of the antisense strand, and the targeting ligand is at the 3’-end of the sense strand.

[0347] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, T2’ is 2’-F, q4is 2, B3’ is 2’-OMe or 2’-F, q5is 5, T3’ is 2’- F, q6is 1, B4’ is 2’-F, and q7is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end of the antisense strand). The dsRNA agent also comprises a 5’-PS2and a targeting ligand. In one embodiment, the 5’-PS2is at the 5’-end of the antisense strand, and the targeting ligand is at the 3’-end of the sense strand.

[0348] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, T2’ is 2’-F, q4is 2, B3’ is 2’-OMe or 2’-F, q5is 5, T3’ is 2’- F, q6is 1, B4’ is 2’-F, and q7is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end of the antisense strand). The dsRNA agent also comprises a 5’-deoxy-5’-C-malonyl and a targeting ligand. In one embodiment, the 5’- deoxy-5’-C-malonyl is at the 5’-end of the antisense strand, and the targeting ligand is at the 3’-end of the sense strand.

[0349] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, q4is 0, B3’ is 2’-OMe or 2’-F, q5is 7, T3’ is 2’-F, q6is 1, B4’ is 2’-F, and q7is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end of the antisense strand). The dsRNA agent also comprises a 5’-P and a targeting ligand. In one embodiment, the 5’-P is at the 5’-end of the antisense strand, and the targeting ligand is at the 3’-end of the sense strand.

[0350] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, q4is 0, B3’ is 2’-OMe or 2’-F, q5is 7, T3’ is 2’-F, q6is 1, B4’ is 2’-F, and q7is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end of the antisense strand). The dsRNA agent also comprises a 5’- PS and a targeting ligand. In one embodiment, the 5’-PS is at the 5’-end of the antisense strand, and the targeting ligand is at the 3’-end of the sense strand.

[0351] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, q4is 0, B3’ is 2’-OMe or 2’-F, q5is 7, T3’ is 2’-F, q6is 1, B4’ is 2’-F, and q7is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end of the antisense strand). The dsRNA agent also comprises a 5’- VP (e.g., a 5’-E-VP, 5’-Z-VP, or combination thereof) and a targeting ligand. In one embodiment, the 5’-VP is at the 5’-end of the antisense strand, and the targeting ligand is at the 3’-end of the sense strand.

[0352] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is 1, B2’ is 2’-OMe or 2’-F, q3is 4, q4is 0, B3’ is 2’-OMe or 2’-F, q5is 7, T3’ is 2’-F, q6is 1, B4’ is 2’-F, and q7is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end of the antisense strand). The dsRNA agent also comprises a 5’- PS2and a targeting ligand. In one embodiment, the 5’-PS2is at the 5’-end of the antisense strand, and the targeting ligand is at the 3’-end of the sense strand.

[0353] In one embodiment, B1 is 2’-OMe or 2’-F, n1is 8, T1 is 2’F, n2is 3, B2 is 2’- OMe, n3is 7, n4is 0, B3 is 2’-OMe, n5is 3, B1’ is 2’-OMe or 2’-F, q1is 9, T1’ is 2’-F, q2is1, B2’ is 2’-OMe or 2’-F, q3is 4, q4is 0, B3’ is 2’-OMe or 2’-F, q5is 7, T3’ is 2’-F, q6is 1, B4’ is 2’-F, and q7is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5’-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5’-end of the antisense strand). The dsRNA agent also comprises a 5’-deoxy-5’-C-malonyl and a targeting ligand. In one embodiment, the 5’- deoxy-5’-C-malonyl is at the 5’-end of the antisense strand, and the targeting ligand is at the 3’-end of the sense strand.

[0354] In a particular embodiment, the dsRNA agents of the present invention comprise: (a) a sense strand having:(i) a length of 21 nucleotides;(ii) optionally an ASGPR ligand attached to the 3’-end, wherein said ASGPR ligand comprises three GalNAc derivatives attached through a trivalent branched linker; and(iii) 2’-F modifications at positions 1, 3, 5, 7, 9 to 11, 13, 17, 19, and 21, and 2’-OMe modifications at positions 2, 4, 6, 8, 12, 14 to 16, 18, and 20 (counting from the 5’ end);and(b) an antisense strand having:(i) a length of 23 nucleotides;(ii) 2’-OMe modifications at positions 1, 3, 5, 9, 11 to 13, 15, 17, 19, 21, and 23, and 2’F modifications at positions 2, 4, 6 to 8, 10, 14, 16, 18, 20, and 22 (counting from the 5’ end); and(iii) phosphorothioate internucleotide linkages between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5’ end);wherein the dsRNA agents have a two nucleotide overhang at the 3’-end of the antisense strand, and a blunt end at the 5’-end of the antisense strand.

[0355] In another particular embodiment, the dsRNA agents of the present invention comprise:(a) a sense strand having:(i) a length of 21 nucleotides;(ii) optionally an ASGPR ligand attached to the 3’-end, wherein said ASGPR ligand comprises three GalNAc derivatives attached through a trivalent branched linker; (iii) 2’-F modifications at positions 1, 3, 5, 7, 9 to 11, 13, 15, 17, 19, and 21, and 2’- OMe modifications at positions 2, 4, 6, 8, 12, 14, 16, 18, and 20 (counting from the 5’ end); and(iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5’ end); and(b) an antisense strand having:(i) a length of 23 nucleotides;(ii) 2’-OMe modifications at positions 1, 3, 5, 7, 9, 11 to 13, 15, 17, 19, and 21 to 23, and 2’F modifications at positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 (counting from the 5’ end); and(iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5’ end); wherein the dsRNA agents have a two nucleotide overhang at the 3’-end of the antisense strand, and a blunt end at the 5’-end of the antisense strand.

[0356] In another particular embodiment, the dsRNA agents of the present invention comprise:(a) a sense strand having:(i) a length of 21 nucleotides;(ii) optionally an ASGPR ligand attached to the 3’-end, wherein said ASGPR ligand comprises three GalNAc derivatives attached through a trivalent branched linker; (iii) 2’-OMe modifications at positions 1 to 6, 8, 10, and 12 to 21, 2’-F modifications at positions 7, and 9, and a desoxy-nucleotide (e.g. dT) at position 11 (counting from the 5’ end); and(iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5’ end); and(b) an antisense strand having:(i) a length of 23 nucleotides;(ii) 2’-OMe modifications at positions 1, 3, 7, 9, 11, 13, 15, 17, and 19 to 23, and 2’- F modifications at positions 2, 4 to 6, 8, 10, 12, 14, 16, and 18 (counting from the 5’ end); and(iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5’ end); wherein the dsRNA agents have a two nucleotide overhang at the 3’-end of the antisense strand, and a blunt end at the 5’-end of the antisense strand.

[0357] In another particular embodiment, the dsRNA agents of the present invention comprise:(a) a sense strand having:(i) a length of 21 nucleotides;(ii) optionally an ASGPR ligand attached to the 3’-end, wherein said ASGPR ligand comprises three GalNAc derivatives attached through a trivalent branched linker; (iii) 2’-OMe modifications at positions 1 to 6, 8, 10, 12, 14, and 16 to 21, and 2’-F modifications at positions 7, 9, 11, 13, and 15; and(iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5’ end); and(b) an antisense strand having:(i) a length of 23 nucleotides;(ii) 2’-OMe modifications at positions 1, 5, 7, 9, 11, 13, 15, 17, 19, and 21 to 23, and 2’-F modifications at positions 2 to 4, 6, 8, 10, 12, 14, 16, 18, and 20 (counting from the 5’ end); and(iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5’ end); wherein the dsRNA agents have a two nucleotide overhang at the 3’-end of the antisense strand, and a blunt end at the 5’-end of the antisense strand.

[0358] In another particular embodiment, the dsRNA agents of the present invention comprise:(a) a sense strand having:(i) a length of 21 nucleotides;(ii) optionally an ASGPR ligand attached to the 3’-end, wherein said ASGPR ligand comprises three GalNAc derivatives attached through a trivalent branched linker; (iii) 2’-OMe modifications at positions 1 to 9, and 12 to 21, and 2’-F modifications at positions 10, and 11; and(iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5’ end); and(b) an antisense strand having:(i) a length of 23 nucleotides;(ii) 2’-OMe modifications at positions 1, 3, 5, 7, 9, 11 to 13, 15, 17, 19, and 21 to 23, and 2’-F modifications at positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 (counting from the 5’ end); and(iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5’ end); wherein the dsRNA agents have a two nucleotide overhang at the 3’-end of the antisense strand, and a blunt end at the 5’-end of the antisense strand.

[0359] In another particular embodiment, the dsRNA agents of the present invention comprise:(a) a sense strand having:(i) a length of 21 nucleotides;(ii) optionally an ASGPR ligand attached to the 3’-end, wherein said ASGPR ligand comprises three GalNAc derivatives attached through a trivalent branched linker; (iii) 2’-F modifications at positions 1, 3, 5, 7, 9 to 11, and 13, and 2’-OMemodifications at positions 2, 4, 6, 8, 12, and 14 to 21; and(iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5’ end); and(b) an antisense strand having:(i) a length of 23 nucleotides;(ii) 2’-OMe modifications at positions 1, 3, 5 to 7, 9, 11 to 13, 15, 17 to 19, and 21 to 23, and 2’-F modifications at positions 2, 4, 8, 10, 14, 16, and 20 (counting from the 5’ end); and(iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5’ end); wherein the dsRNA agents have a two nucleotide overhang at the 3’-end of the antisense strand, and a blunt end at the 5’-end of the antisense strand.

[0360] In another particular embodiment, the dsRNA agents of the present invention comprise:(a) a sense strand having:(i) a length of 21 nucleotides;(ii) optionally an ASGPR ligand attached to the 3’-end, wherein said ASGPR ligand comprises three GalNAc derivatives attached through a trivalent branched linker; (iii) 2’-OMe modifications at positions 1, 2, 4, 6, 8, 12, 14, 15, 17, and 19 to 21, and 2’-F modifications at positions 3, 5, 7, 9 to 11, 13, 16, and 18; and(iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5’ end); and(b) an antisense strand having:(i) a length of 25 nucleotides;(ii) 2’-OMe modifications at positions 1, 4, 6, 7, 9, 11 to 13, 15, 17, and 19 to 23, 2’- F modifications at positions 2, 3, 5, 8, 10, 14, 16, and 18, and desoxy-nucleotides (e.g. dT) at positions 24 and 25 (counting from the 5’ end); and(iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5’ end); wherein the dsRNA agents have a four nucleotide overhang at the 3’-end of the antisense strand, and a blunt end at the 5’-end of the antisense strand.

[0361] In another particular embodiment, the dsRNA agents of the present invention comprise:(a) a sense strand having:(i) a length of 21 nucleotides;(ii) optionally an ASGPR ligand attached to the 3’-end, wherein said ASGPR ligand comprises three GalNAc derivatives attached through a trivalent branched linker;(iii) 2’-OMe modifications at positions 1 to 6, 8, and 12 to 21, and 2’-F modifications at positions 7, and 9 to 11; and(iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5’ end); and(b) an antisense strand having:(i) a length of 23 nucleotides;(ii) 2’-OMe modifications at positions 1, 3 to 5, 7, 8, 10 to 13, 15, and 17 to 23, and 2’-F modifications at positions 2, 6, 9, 14, and 16 (counting from the 5’ end); and (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5’ end); wherein the dsRNA agents have a two nucleotide overhang at the 3’-end of the antisense strand, and a blunt end at the 5’-end of the antisense strand.

[0362] In another particular embodiment, the dsRNA agents of the present invention comprise:(a) a sense strand having:(i) a length of 21 nucleotides;(ii) optionally an ASGPR ligand attached to the 3’-end, wherein said ASGPR ligand comprises three GalNAc derivatives attached through a trivalent branched linker; (iii) 2’-OMe modifications at positions 1 to 6, 8, and 12 to 21, and 2’-F modifications at positions 7, and 9 to 11; and(iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5’ end); and(b) an antisense strand having:(i) a length of 23 nucleotides;(ii) 2’-OMe modifications at positions 1, 3 to 5, 7, 10 to 13, 15, and 17 to 23, and 2’- F modifications at positions 2, 6, 8, 9, 14, and 16 (counting from the 5’ end); and (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23 (counting from the 5’ end);wherein the dsRNA agents have a two nucleotide overhang at the 3’-end of the antisense strand, and a blunt end at the 5’-end of the antisense strand.

[0363] In another particular embodiment, the dsRNA agents of the present invention comprise:(a) a sense strand having:(i) a length of 19 nucleotides;(ii) optionally an ASGPR ligand attached to the 3’-end, wherein said ASGPR ligand comprises three GalNAc derivatives attached through a trivalent branched linker; (iii) 2’-OMe modifications at positions 1 to 4, 6, and 10 to 19, and 2’-F modifications at positions 5, and 7 to 9; and(iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3 (counting from the 5’ end); and(b) an antisense strand having:(i) a length of 21 nucleotides;(ii) 2’-OMe modifications at positions 1, 3 to 5, 7, 10 to 13, 15, and 17 to 21, and 2’- F modifications at positions 2, 6, 8, 9, 14, and 16 (counting from the 5’ end); and (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21 (counting from the 5’ end); wherein the dsRNA agents have a two nucleotide overhang at the 3’-end of the antisense strand, and a blunt end at the 5’-end of the antisense strand.

[0364] In one embodiment, the dsRNA agents of the present invention comprise:(a) a sense strand having:(i) a length of 18-23 nucleotides;(ii) three consecutive 2’-F modifications at positions 7-15; and(b) an antisense strand having:(i) a length of 18-23 nucleotides;(ii) at least 2’-F modifications anywhere on the strand; and(iii) at least two phosphorothioate internucleotide linkages at the first five nucleotides (counting from the 5’ end);wherein the dsRNA agents have one or more lipophilic moieties conjugated to one or more positions on at least one strand; and either have two nucleotides overhang at the 3’-end of theantisense strand, and a blunt end at the 5’-end of the antisense strand; or blunt end both ends of the duplex.

[0365] In one embodiment, the dsRNA agents of the present invention comprise:(a) a sense strand having:(i) a length of 18-23 nucleotides;(ii) less than four 2’-F modifications;(b) an antisense strand having:(i) a length of 18-23 nucleotides;(ii) at less than twelve 2’-F modfication; and(iii) at least two phosphorothioate internucleotide linkages at the first five nucleotides (counting from the 5’ end);wherein the dsRNA agents have one or more lipophilic moieties conjugated to one or more positions on at least one strand; and either have two nucleotides overhang at the 3’-end of the antisense strand, and a blunt end at the 5’-end of the antisense strand; or blunt end both ends of the duplex.

[0366] In one embodiment, the dsRNA agents of the present invention comprise:(a) a sense strand having:(i) a length of 19-35 nucleotides;(ii) less than four 2’-F modifications;(b) an antisense strand having:(i) a length of 19-35 nucleotides;(ii) at less than twelve 2’-F modfication; and(iii) at least two phosphorothioate internucleotide linkages at the first five nucleotides (counting from the 5’ end);wherein the duplex region is between 19 to 25 base pairs (preferably 19, 20, 21 or 22); and wherein the dsRNA agents have one or more lipophilic moieties conjugated to one or more positions on at least one strand; and either have two nucleotides overhang at the 3’-end of the antisense strand, and a blunt end at the 5’-end of the antisense strand; or blunt end both ends of the duplex.

[0367] In one embodiment, the dsRNA agents of the present invention comprise a sense strand and antisense strands having a length of 15-30 nucleotides; at least twophosphorothioate internucleotide linkages at the first five nucleotides on the antisense strand (counting from the 5’ end); wherein the duplex region is between 19 to 25 base pairs(preferably 19, 20, 21 or 22); wherein the dsRNA agents have one or more lipophilic moieties conjugated to one or more positions on at least one strand; and wherein the dsRNA agents have less than 20% , less than 15% and less than 10% non-natural nucleotide.

[0368] Examples of non-natural nucleotide includes acyclic nucleotides, LNA, HNA, CeNA, 2’-methoxyethyl, , 2’-O-allyl, 2’-C-allyl, 2’-deoxy, 2’-fluoro, 2'-O-N- methylacetamido (2'-O-NMA), a 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O- aminopropyl (2'-O-AP), or 2'-ara-F, and others.

[0369] In one embodiment, the dsRNA agents of the present invention comprise a sense strand and antisense strands having a length of 15-30 nucleotides; at least twophosphorothioate internucleotide linkages at the first five nucleotides on the antisense strand (counting from the 5’ end); wherein the duplex region is between 19 to 25 base pairs(preferably 19, 20, 21 or 22); wherein the dsRNA agents have one or more lipophilic moieties conjugated to one or more positions on at least one strand; and wherein the dsRNA agents have greater than 80% , greater than 85% and greater than 90% natural nucleotide, such as 2’- OH, 2’-deoxy and 2’-OMe are natural nucleotides.

[0370] In one embodiment, the dsRNA agents of the present invention comprise a sense strand and antisense strands having a length of 15-30 nucleotides; at least twophosphorothioate internucleotide linkages at the first five nucleotides on the antisense strand (counting from the 5’ end); wherein the duplex region is between 19 to 25 base pairs(preferably 19, 20, 21 or 22); wherein the dsRNA agents have one or more lipophilic moieties conjugated to one or more positions on at least one strand; and wherein the dsRNA agents have 100% natural nucleotide, such as 2’-OH, 2’-deoxy and 2’-OMe are natural nucleotides.

[0371] Examples of lipophilic moieties include, but not limted to, lipid (a saturated or unsaturated C4-C30 hydrocarbon chain, and an optional functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne), cholesterol, retinoic acid, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3- (oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.

[0372] In some embodiments, the lipophilic moiety is a C6-C30acid (e.g., hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, dodcanoic acid, tridecanoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, oleic acid, linoleic acid, arachidonic acid, cis-4,7,10,13,16,19-docosahexanoic acid, vitamin A, vitamin E, cholesterol etc.) or a C6-C30alcohol (e.g., hexanol, heptanol, octanol, nonanol, decanol, undecanol, dodcanol, tridecanol, tetradecanol, pentadecanol, hexadecanol, heptadecanol, octadecanol, oleyl alcohol, linoleyl alcohol, arachidonic alcohol, cis-4,7,10,13,16,19-docosahexanol, retinol, vitamin E, cholesterol etc.).

[0373] In one example, the lipophilic moiety is a saturated or unsaturated C6-C18 hydrocarbon chain.

[0374] In one example, the lipohilic moiety is docosahexaenoic acid.

[0375] In one embodiment, the dsRNA agents of the present invention a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, wherein the sense strand sequence is represented by formula (I): 5' np-Na-(X X X )i-Nb-Y Y Y -Nb-(Z Z Z )j-Na-nq 3'(I) wherein:i and j are each independently 0 or 1;p and q are each independently 0-6;each Na independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modifiednucleotides;each Nbindependently represents an oligonucleotide sequence comprising 1, 2, 3, 4, 5, or 6 modified nucleotides;each npand nqindependently represent an overhang nucleotide;wherein Nband Y do not have the same modification;wherein XXX, YYY and ZZZ each independently represent one motif of three identical modifications on three consecutive nucleotides;wherein wherein the dsRNA agents have one or more lipophilic moieties conjugated to one or more positions on at least one strand; andwherein the antisense strand of the dsRNA comprises two blocks of one, two pr three phosphorothioate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate internucleotide linkages.

[0376] Various publications described multimeric siRNA and can all be used with the iRNA of the invention. Such publications include WO2007 / 091269, US Patent No.7858769,WO2010 / 141511, WO2007 / 117686, WO2009 / 014887 and WO2011 / 031520, which are hereby incorporated by reference in their entirety.

[0377] In some embodiments, 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35% or 30% of the iRNA agent of the invention is modified.

[0378] In some embodiments, each of the sense and antisense strands of the iRNA agent is independently modified with acyclic nucleotides, LNA, HNA, CeNA, 2’-methoxyethyl, 2’- O-methyl, 2’-O-allyl, 2’-C-allyl, 2’-deoxy, 2’-fluoro, 2'-O-N-methylacetamido (2'-O- NMA), a 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), or 2'-ara-F.

[0379] In some embodiments, each of the sense and antisense strands of the iRNA agent contains at least two different modifications.

[0380] In some embodiments, the double-stranded iRNA agent of the invention of the invention does not contain any 2’-F modification.

[0381] In some embodiments, the double-stranded iRNA agent of the invention contains one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve 2’-F modification(s). In one example, double-stranded iRNA agent of the invention contains nine or ten 2’-F modifications.

[0382] The iRNA agent of the invention may further comprise at least onephosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate internucleotide linkage modification may occur on any nucleotide of the sense strand or antisense strand or both in any position of the strand. For instance, the internucleotide linkage modification may occur on every nucleotide on the sense strand or antisense strand; each internucleotide linkage modification may occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand may contain both internucleotide linkage modifications in an alternating pattern. The alternating pattern of the internucleotide linkage modification on the sense strand may be the same or different from the antisense strand, and the alternating pattern of the internucleotide linkage modification on the sense strand may have a shift relative to the alternating pattern of the internucleotide linkage modification on the antisense strand.

[0383] In one embodiment, the iRNA comprises the phosphorothioate ormethylphosphonate internucleotide linkage modification in the overhang region. For example, the overhang region may contain two nucleotides having a phosphorothioate or methylphosphonate internucleotide linkage between the two nucleotides. Internucleotidelinkage modifications also may be made to link the overhang nucleotides with the terminal paired nucleotides within duplex region. For example, at least 2, 3, 4, or all the overhang nucleotides may be linked through phosphorothioate or methylphosphonate internucleotide linkage, and optionally, there may be additional phosphorothioate or methylphosphonate internucleotide linkages linking the overhang nucleotide with a paired nucleotide that is next to the overhang nucleotide. For instance, there may be at least two phosphorothioate internucleotide linkages between the terminal three nucleotides, in which two of the three nucleotides are overhang nucleotides, and the third is a paried nucleotide next to the overhang nucleotide. Preferably, these terminal three nucleotides may be at the 3’-end of the antisense strand.

[0384] In some embodiments, the sense strand and / or antisense strand of the iRNA agent comprises one or more blocks of phosphorothioate or methylphosphonate internucleotide linkages. In one example, the sense strand comprises one block of two phosphorothioate or methylphosphonate internucleotide linkages. In one example, the antisense strand comprises two blocks of two phosphorothioate or methylphosphonate internucleotide linkages. For example, the two blocks of phosphorothioate or methylphosphonate internucleotide linkages are separated by 16-18 phosphate internucleotide linkages.

[0385] In some embodiments, the antisense strand of the iRNA agent of the invention is 100% complementary to a target RNA to hybridize thereto and inhibits its expression through RNA interference. In another embodiment, the antisense strand of the iRNA agent of the invention is at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, or at least 50% complementary to a target RNA.

[0386] In one aspect, the invention relates to a iRNA agent capable of inhibiting the expression of a target gene. The iRNA agent comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides. The sense strand contains at least one thermally destabilizing nucleotide, wherein at at least one said thermally destabilizing nucleotide occurs at or near the site that is opposite to the seed region of the antisense strand (i.e .at position 2-8 of the 5’-end of the antisense strand), For example, the thermally destabilizing nucleotide occurs between positions 14-17 of the 5’-end of the sense strand when the sense strand is 21 nucleotides in length. The antisense strand contains at least two modified nucleic acids that are smaller than a sterically demanding 2’-OMe modification. Preferably, the two modified nucleic acids that is smaller than a sterically demanding 2’-OMeare separated by 11 nucleotides in length. For example, the two modified nucleic acids are at positions 2 and 14 of the 5’end of the antisense strand.

[0387] In some embodiments, the compound of the invention disclosed herein is a miRNA mimic. In one design, miRNA mimics are double stranded molecules (e.g., with a duplex region of between about 16 and about 31 nucleotides in length) and contain one or more sequences that have identity with the mature strand of a given miRNA. Double- stranded miRNA mimics have designs similar to as described above for double-stranded iRNAs. In some embodiments, a miRNA mimic comprises a duplex region of between 16 and 31 nucleotides and one or more of the following chemical modification patterns: the sense strand contains 2'-O-methyl modifications of nucleotides 1 and 2 (counting from the 5' end of the sense oligonucleotide), and all of the Cs and Us; the antisense strand modifications can comprise 2' F modification of all of the Cs and Us, phosphorylation of the 5' end of the oligonucleotide, and stabilized internucleotide linkages associated with a 2 nucleotide 3 ' overhang.

[0388] In some embodiments, the compound of the invention disclosed herein is an antimir. In some embodiments, compound of the invention comprises at least two antimirs covalently linked to each other via a nucleotide-based or non-nucleotide-based linker, for example a linker described in the disclosure, or non-covlantly linked to each other. The terms“antimir” "microRNA inhibitor" or "miR inhibitor" are synonymous and refer to oligonucleotides or modified oligonucleotides that interfere with the activity of specific miRNAs. Inhibitors can adopt a variety of configurations including single stranded, double stranded (RNA / RNA or RNA / DNA duplexes), and hairpin designs, in general, microRNA inhibitors comprise one or more sequences or portions of sequences that are complementary or partially complementary with the mature strand (or strands) of the miRNA to be targeted, in addition, the miRNA inhibitor can also comprise additional sequences located 5' and 3' to the sequence that is the reverse complement of the mature miRNA. The additional sequences can be the reverse complements of the sequences that are adjacent to the mature miRNA in the pri-miRNA from which the mature miRNA is derived, or the additional sequences can be arbitrary sequences (having a mixture of A, G, C, U, or dT). In some embodiments, one or both of the additional sequences are arbitrary sequences capable of forming hairpins. Thus, in some embodiments, the sequence that is the reverse complement of the miRNA is flanked on the 5' side and on the 3' side by hairpin structures. MicroRNA inhibitors, when double stranded, can include mismatches between nucleotides on opposite strands. Furthermore,microRNA inhibitors can be linked to conjugate moieties in order to facilitate uptake of the inhibitor into a cell.

[0389] MicroRNA inhibitors, including hairpin miRNA inhibitors, are described in detail in Vermeulen et al., "Double-Stranded Regions Are Essential Design Components Of Potent Inhibitors of RISC Function," RNA 13: 723-730 (2007) and in WO2007 / 095387 and WO 2008 / 036825 each of which is incorporated herein by reference in its entirety. A person of ordinary skill in the art can select a sequence from the database for a desired miRNA and design an inhibitor useful for the methods disclosed herein.

[0390] In some embodiments, compound of the invention disclosed herein is an antagomir. In some embodiments, the compound of the invention comprises at least two antagomirs covalently linked to each other via a nucleotide-based or non-nucleotide-based linker, for example a linker described in the disclosure, or non-covlantly linked to each other. Antagomirs are RNA-like oligonucleotides that harbor various modifications for RNAse protection and pharmacologic properties, such as enhanced tissue and cellular uptake. They differ from normal RNA by, for example, complete 2'-O-methylation of sugar,phosphorothioate intersugar linkage and, for example, a cholesterol-moiety at 3'-end. In a preferred embodiment, antagomir comprises a 2’-O-methyl modification at all nucleotides, a cholesterol moiety at 3’-end, two phsophorothioate intersugar linkages at the first two positions at the 5’-end and four phosphorothioate linkages at the 3’-end of the molecule. Antagomirs can be used to efficiently silence endogenous miRNAs by forming duplexes comprising the antagomir and endogenous miRNA, thereby preventing miRNA-induced gene silencing. An example of antagomir-mediated miRNA silencing is the silencing of miR-122, described in Krutzfeldt et al, Nature, 2005, 438: 685-689, which is expressly incorporated by reference herein in its entirety.

[0391] Recent studies have found that dsRNA can also activate gene expression, a mechanism that has been termed "small RNA-induced gene activation" or RNAa (activating RNA). See for example Li, L.C. et al. Proc Natl Acad Sci U S A. (2006), 103(46):17337-42 and Li L.C. (2008). "Small RNA-Mediated Gene Activation". RNA and the Regulation of Gene Expression: A Hidden Layer of Complexity. Caister Academic Press. ISBN 978-1- 904455-25-7. It has been shown that dsRNAs targeting gene promoters induce potent transcriptional activation of associated genes. Endogenous miRNA that cause RNAa has also been found in humans. Check E. Nature (2007).448 (7156): 855–858.

[0392] Another surprising observation is that gene activation by RNAa is long-lasting. Induction of gene expression has been seen to last for over ten days. The prolonged effect of RNAa could be attributed to epigenetic changes at dsRNA target sites. In someembodiments, the RNA activator can increase the expression of a gene. In someembodiments, increased gene expression inhibits viability, growth development, and / or reproduction.

[0393] Accordingly, in some embodiments compound of the invention disclosed herein is activating RNA. In some embodiments, the compound of the invention comprises at least two activating RNAs scovalently linked to each other via a nucleotide-based or non- nucleotide-based linker, for example a linker described in the disclosure, or non-covlantly linked to each other.

[0394] Accordingly, in some embodiments, compound of the invention disclosed herein is a triplex forming oligonucotide (TFO). In some embodiments, the compound of the invention comprises at least two TFOs covalently linked to each other via a nucleotide-based or non-nucleotide-based linker, for example a linker described in the disclosure, or non- covlantly linked to each other. Recent studies have shown that triplex formingoligonucleotides can be designed which can recognize and bind to polypurine / polypyrimidine regions in double-stranded helical DNA in a sequence-specific manner. These recognition rules are outline by Maher III, L.J., et al., Science (1989) vol.245, pp 725-730; Moser, H. E., et al., Science (1987) vol.238, pp 645-630; Beal, P.A., et al., Science (1992) vol.251, pp 1360-1363; Conney, M., et al., Science (1988) vol.241, pp 456-459 and Hogan, M.E., et al., EP Publication 375408. Modification of the oligonucleotides, such as the introduction of intercalators and intersugar linkage substitutions, and optimization of binding conditions (pH and cation concentration) have aided in overcoming inherent obstacles to TFO activity such as charge repulsion and instability, and it was recently shown that synthetic oligonucleotides can be targeted to specific sequences (for a recent review see Seidman and Glazer, J Clin Invest 2003;l 12:487-94). In general, the triplex-forming oligonucleotide has the sequence correspondence:oligo 3'-A G G Tduplex 5'-A G C Tduplex 3'-T C G A

[0395] However, it has been shown that the A-AT and G-GC triplets have the greatest triple helical stability (Reither and Jeltsch, BMC Biochem, 2002, Sertl2, Epub). The sameauthors have demonstrated that TFOs designed according to the A-AT and G-GC rule do not form non-specific triplexes, indicating that the triplex formation is indeed sequence specific.

[0396] Thus for any given sequence a triplex forming sequence can be devised. Triplex- forming oligonucleotides preferably are at least 15, more preferably 25, still more preferably 30 or more nucleotides in length, up to 50 or 100 nucleotides.

[0397] Formation of the triple helical structure with the target DNA induces steric and functional changes, blocking transcription initiation and elongation, allowing the introduction of desired sequence changes in the endogenous DNA and resulting in the specific down- regulation of gene expression. Examples of such suppression of gene expression in cells treated with TFOs include knockout of episomal supFGl and endogenous HPRT genes in mammalian cells (Vasquez et al., Nucl Acids Res.1999;27: 1176-81, and Puri, et al, J Biol Chem, 2001;276:28991-98), and the sequence- and target specific downregulation of expression of the Ets2 transcription factor, important in prostate cancer etiology (Carbone, et al, Nucl Acid Res.2003 ;31:833-43), and the pro-inflammatory ICAM-I gene (Besch et al, J Biol Chem, 2002;277:32473-79). In addition, Vuyisich and Beal have recently shown that sequence specific TFOs can bind to dsRNA, inhibiting activity of dsRNA-dependent enzymes such as RNA- dependent kinases (Vuyisich and Beal, Nuc. Acids Res2000;28:2369-74).

[0398] Additionally, TFOs designed according to the abovementioned principles can induce directed mutagenesis capable of effecting DNA repair, thus providing both down- regulation and up-regulation of expression of endogenous genes (Seidman and Glazer, J Clin Invest 2003; 112:487-94). Detailed description of the design, synthesis and administration of effective TFOs can be found in U.S. Pat. App. Nos.2003017068 and 20030096980 to Froehler et al, and 20020128218 and 20020123476 to Emanuele et al, and U.S. Pat. No. 5,721,138 to Lawn, contents of which are herein incorporated in their entireties. Nucleic acid modifications

[0399] In some embodiments, the double-stranded iRNA agent of the invention comprises at least one nucleic acid modification described herein. For example, at least one modification selected from the group consisting of modified internucleoside linkage, modified nucleobase, modified sugar, and any combinations thereof. Without limitations, such a modification can be present anywhere in the double-stranded iRNA agent of the invention. For example, the modification can be present in one of the RNA molecules.Nucleic acid modifications (Nucleobases)

[0400] The naturally occurring base portion of a nucleoside is typically a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. For those nucleosides that include a pentofuranosyl sugar, a phosphate group can be linked to the 2 , 3 or 5 hydroxyl moiety of the sugar. In forming oligonucleotides, those phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. Within oligonucleotides, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The naturally occurring linkage or backbone of RNA and of DNA is a 3 to 5 phosphodiester linkage.

[0401] In addition to“unmodified” or“natural” nucleobases such as the purine nucleobases adenine (A) and guanine (G), and the pyrimidine nucleobases thymine (T), cytosine (C) and uracil (U), many modified nucleobases or nucleobase mimetics known to those skilled in the art are amenable with the compounds described herein. The unmodified or natural nucleobases can be modified or replaced to provide iRNAs having improved properties. For example, nuclease resistant oligonucleotides can be prepared with these bases or with synthetic and natural nucleobases (e.g., inosine, xanthine, hypoxanthine, nubularine, isoguanisine, or tubercidine) and any one of the oligomer modifications described herein. Alternatively, substituted or modified analogs of any of the above bases and“universal bases” can be employed. When a natural base is replaced by a non-natural and / or universal base, the nucleotide is said to comprise a modified nucleobase and / or a nucleobase modification herein. Modified nucleobase and / or nucleobase modifications also include natural, non-natural and universal bases, which comprise conjugated moieties, e.g. a ligand described herein. Preferred conjugate moieties for conjugation with nucleobases include cationic amino groups which can be conjugated to the nucleobase via an appropriate alkyl, alkenyl or a linker with an amide linkage.

[0402] An oligomeric compound described herein can also include nucleobase (often referred to in the art simply as“base”) modifications or substitutions. As used herein, “unmodified” or“natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Exemplary modified nucleobases include, but are not limited to, other synthetic and natural nucleobases such as inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidine, 2-(halo)adenine, 2- (alkyl)adenine, 2-(propyl)adenine, 2-(amino)adenine, 2-(aminoalkyll)adenine,2-(aminopropyl)adenine, 2-(methylthio)-N6-(isopentenyl)adenine, 6-(alkyl)adenine,6-(methyl)adenine, 7-(deaza)adenine, 8-(alkenyl)adenine, 8-(alkyl)adenine,8-(alkynyl)adenine, 8-(amino)adenine, 8-(halo)adenine, 8-(hydroxyl)adenine,8-(thioalkyl)adenine, 8-(thiol)adenine, N6-(isopentyl)adenine, N6-(methyl)adenine,N6, N6-(dimethyl)adenine, 2-(alkyl)guanine,2-(propyl)guanine, 6-(alkyl)guanine,6-(methyl)guanine, 7-(alkyl)guanine, 7-(methyl)guanine, 7-(deaza)guanine, 8-(alkyl)guanine, 8-(alkenyl)guanine, 8-(alkynyl)guanine, 8-(amino)guanine, 8-(halo)guanine, 8- (hydroxyl)guanine, 8-(thioalkyl)guanine, 8-(thiol)guanine, N-(methyl)guanine, 2- (thio)cytosine, 3-(deaza)-5-(aza)cytosine, 3-(alkyl)cytosine, 3-(methyl)cytosine, 5- (alkyl)cytosine, 5-(alkynyl)cytosine, 5-(halo)cytosine, 5-(methyl)cytosine,5-(propynyl)cytosine, 5-(propynyl)cytosine, 5-(trifluoromethyl)cytosine, 6-(azo)cytosine, N4-(acetyl)cytosine, 3-(3-amino-3-carboxypropyl)uracil, 2-(thio)uracil,5-(methyl)-2-(thio)uracil, 5-(methylaminomethyl)-2-(thio)uracil, 4-(thio)uracil,5-(methyl)-4-(thio)uracil, 5-(methylaminomethyl)-4-(thio)uracil,5-(methyl)-2,4-(dithio)uracil, 5-(methylaminomethyl)-2,4-(dithio)uracil, 5-(2- aminopropyl)uracil, 5-(alkyl)uracil, 5-(alkynyl)uracil, 5-(allylamino)uracil,5-(aminoallyl)uracil, 5-(aminoalkyl)uracil, 5-(guanidiniumalkyl)uracil, 5-(1,3-diazole-1- alkyl)uracil, 5-(cyanoalkyl)uracil, 5-(dialkylaminoalkyl)uracil, 5-(dimethylaminoalkyl)uracil, 5-(halo)uracil, 5-(methoxy)uracil, uracil-5-oxyacetic acid, 5-(methoxycarbonylmethyl)-2- (thio)uracil, 5-(methoxycarbonyl-methyl)uracil, 5-(propynyl)uracil, 5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 6-(azo)uracil, dihydrouracil, N3-(methyl)uracil, 5-uracil (i.e., pseudouracil), 2-(thio)pseudouracil,4-(thio)pseudouracil,2,4-(dithio)psuedouracil,5- (alkyl)pseudouracil, 5-(methyl)pseudouracil, 5-(alkyl)-2-(thio)pseudouracil, 5-(methyl)-2- (thio)pseudouracil, 5-(alkyl)-4-(thio)pseudouracil, 5-(methyl)-4-(thio)pseudouracil, 5-(alkyl)- 2,4-(dithio)pseudouracil, 5-(methyl)-2,4-(dithio)pseudouracil, 1-substituted pseudouracil, 1-substituted 2(thio)-pseudouracil, 1-substituted 4-(thio)pseudouracil, 1-substituted 2,4- (dithio)pseudouracil, 1-(aminocarbonylethylenyl)-pseudouracil, 1-(aminocarbonylethylenyl)- 2(thio)-pseudouracil, 1-(aminocarbonylethylenyl)-4-(thio)pseudouracil,1-(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil,1-(aminoalkylaminocarbonylethylenyl)-pseudouracil, 1-(aminoalkylamino- carbonylethylenyl)-2(thio)-pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)- 4-(thio)pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1,3- (diaza)-2-(oxo)-phenoxazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-substituted 1,3-(diaza)-2- (oxo)-phenoxazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)- 2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1- yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7- (guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7- (guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(guanidiniumalkyl- hydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-(guanidiniumalkylhydroxy)-1-(aza)-2- (thio)-3-(aza)-phenthiazin-1-yl, 1,3,5-(triaza)-2,6-(dioxa)-naphthalene, inosine, xanthine, hypoxanthine, nubularine, tubercidine, isoguanisine, inosinyl, 2-aza-inosinyl, 7-deaza- inosinyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl, 3-(methyl)isocarbostyrilyl, 5-(methyl)isocarbostyrilyl, 3-(methyl)-7- (propynyl)isocarbostyrilyl, 7-(aza)indolyl, 6-(methyl)-7-(aza)indolyl, imidizopyridinyl, 9- (methyl)-imidizopyridinyl, pyrrolopyrizinyl, isocarbostyrilyl, 7-(propynyl)isocarbostyrilyl, propynyl-7-(aza)indolyl, 2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl, phenyl, napthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl, difluorotolyl, 4-(fluoro)-6-(methyl)benzimidazole, 4-(methyl)benzimidazole, 6-(azo)thymine, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 6-(aza)pyrimidine, 2-(amino)purine, 2,6- (diamino)purine, 5-substituted pyrimidines, N2-substituted purines, N6-substituted purines, O6-substituted purines, substituted 1,2,4-triazoles, pyrrolo-pyrimidin-2-on-3-yl, 6-phenyl- pyrrolo-pyrimidin-2-on-3-yl, para-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, ortho- substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, bis-ortho-substituted-6-phenyl-pyrrolo- pyrimidin-2-on-3-yl, para-(aminoalkylhydroxy)- 6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, ortho-(aminoalkylhydroxy)- 6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, bis-ortho-- (aminoalkylhydroxy)- 6-phenyl-pyrrolo-pyrimidin-2-on-3-yl, pyridopyrimidin-3-yl, 2-oxo-7- amino-pyridopyrimidin-3-yl, 2-oxo-pyridopyrimidine-3-yl, or any O-alkylated or N-alkylated derivatives thereof. Alternatively, substituted or modified analogs of any of the above bases and“universal bases” can be employed.

[0403] As used herein, a universal nucleobase is any nucleobase that can base pair with all of the four naturally occurring nucleobases without substantially affecting the melting behavior, recognition by intracellular enzymes or activity of the iRNA duplex. Some exemplary universal nucleobases include, but are not limited to, 2,4-difluorotoluene,nitropyrrolyl, nitroindolyl, 8-aza-7-deazaadenine, 4-fluoro-6-methylbenzimidazle, 4- methylbenzimidazle, 3-methyl isocarbostyrilyl, 5- methyl isocarbostyrilyl, 3-methyl-7- propynyl isocarbostyrilyl, 7-azaindolyl, 6-methyl-7-azaindolyl, imidizopyridinyl, 9-methyl- imidizopyridinyl, pyrrolopyrizinyl, isocarbostyrilyl, 7-propynyl isocarbostyrilyl, propynyl-7- azaindolyl, 2,4,5-trimethylphenyl, 4-methylinolyl, 4,6-dimethylindolyl, phenyl, napthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl, and structural derivatives thereof (see for example, Loakes, 2001, Nucleic Acids Research, 29, 2437-2447).

[0404] Further nucleobases include those disclosed in U.S. Pat. No.3,687,808; those disclosed in International Application No. PCT / US09 / 038425, filed March 26, 2009; those disclosed in the Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990; those disclosed by English et al.,Angewandte Chemie, International Edition, 1991, 30, 613; those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijin, P.Ed. Wiley-VCH, 2008; and those disclosed by Sanghvi, Y.S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S.T. and Lebleu, B., Eds., CRC Press, 1993. Contents of all of the above are herein incorporated by reference.

[0405] In certain embodiments, a modified nucleobase is a nucleobase that is fairly similar in structure to the parent nucleobase, such as for example a 7-deaza purine, a 5- methyl cytosine, or a G-clamp. In certain embodiments, nucleobase mimetic include more complicated structures, such as for example a tricyclic phenoxazine nucleobase mimetic. Methods for preparation of the above noted modified nucleobases are well known to those skilled in the art. Nucleic acid modifications (sugar)

[0406] Double-stranded iRNA agent of the inventions provided herein can comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) monomer, including a nucleoside or nucleotide, having a modified sugar moiety. For example, the furanosyl sugar ring of a nucleoside can be modified in a number of ways including, but not limited to, addition of a substituent group, bridging of two non-geminal ring atoms to form a locked nucleic acid or bicyclic nucleic acid. In certain embodiments, oligomeric compounds comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) monomers that are LNA.

[0407] In some embodiments of a locked nucleic acid, the 2 ^ position of furnaosyl is connected to the 4’ position by a linker selected independently from–[C(R1)(R2)]n–,–[C(R1)(R2)]n–O–, –[C(R1)(R2)]n-N(R1)–,–[C(R1)(R2)]n-N(R1)–O-,—[C(R1R2)]n-O- N(R1)—,–C(R1)=C(R2)–O–,–C(R1)=N–,–C(R1)=N–O-,—C(═NR1)-,—C(═NR1)-O-,— C(═O)—,—C(═O)O—,—C(═S)—,—C(═S)O—,—C(═S)S—,—O—,—Si(R1)2-,— S(═O)x- and—N(R1)-;wherein:x is 0, 1, or 2;n is 1, 2, 3, or 4;each R1 and R2 is, independently, H, a protecting group, hydroxyl, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(═O)— H), substituted acyl, CN, sulfonyl (S(═O)2-J1), or sulfoxyl (S(═O)-J1); andeach J1 and J2 is, independently, H, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, acyl (C(═O)—H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C1-C12 aminoalkyl, substituted C1-C12 aminoalkyl or a protecting group.

[0408] In some embodiments, each of the linkers of the LNA compounds is,independently,—[C(R1)(R2)]n-,—[C(R1)(R2)]n-O—,—C(R1R2)-N(R1)-O— or—C(R1R2)-O—N(R1)-. In another embodiment, each of said linkers is, independently, 4 -CH2- 2 , 4 -(CH2)2-2 , 4 -(CH2)3-2 , 4 -CH2-O-2 , 4 -(CH2)2-O-2, 4 -CH2-O—N(R1)-2 and 4 -CH2- N(R1)-O-2 - wherein each R1 is, independently, H, a protecting group or C1-C12 alkyl.

[0409] Certain LNA's have been prepared and disclosed in the patent literature as well as in scientific literature (Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; WO 94 / 14226; WO 2005 / 021570; Singh et al., J. Org. Chem., 1998, 63, 10035-10039; Examples of issued US patents and published applications that disclose LNA s include, for example, U.S. Pat. Nos. 7,053,207; 6,268,490; 6,770,748; 6,794,499; 7,034,133; and 6,525,191; and U.S. Pre-GrantPublication Nos.2004-0171570; 2004-0219565; 2004-0014959; 2003-0207841; 2004- 0143114; and 20030082807.

[0410] Also provided herein are LNAs in which the 2 -hydroxyl group of the ribosyl sugar ring is linked to the 4 carbon atom of the sugar ring thereby forming a methyleneoxy (4 -CH2-O-2 ) linkage to form the bicyclic sugar moiety (reviewed in Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 81-7; and Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; see also U.S. Pat. Nos.6,268,490 and 6,670,461). The linkage can be a methylene (—CH2-) group bridging the 2 oxygen atom and the 4 carbon atom, for which the term methyleneoxy (4 -CH2-O-2 ) LNA is used for the bicyclic moiety; in the case of an ethylene group in this position, the term ethyleneoxy (4 - CH2CH2-O-2 ) LNA is used (Singh et al., Chem. Commun., 1998, 4, 455-456: Morita et al., Bioorganic Medicinal Chemistry, 2003, 11, 2211-2226). Methyleneoxy (4 -CH2-O-2) LNA and other bicyclic sugar analogs display very high duplex thermal stabilities withcomplementary DNA and RNA (Tm=+3 to +10° C.), stability towards 3 -exonucleolytic degradation and good solubility properties. Potent and nontoxic antisense oligonucleotides comprising BNAs have been described (Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638).

[0411] An isomer of methyleneoxy (4 -CH2-O-2 ) LNA that has also been discussed is alpha-L-methyleneoxy (4 -CH2-O-2 ) LNA which has been shown to have superior stability against a 3 -exonuclease. The alpha-L-methyleneoxy (4 -CH2-O-2 ) LNA's were incorporated into antisense gapmers and chimeras that showed potent antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

[0412] The synthesis and preparation of the methyleneoxy (4 -CH2-O-2 ) LNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil, along with their oligomerization, and nucleic acid recognition properties have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). BNAs and preparation thereof are also described in WO 98 / 39352 and WO 99 / 14226.

[0413] Analogs of methyleneoxy (4 -CH2-O-2 ) LNA, phosphorothioate-methyleneoxy (4 -CH2-O-2 ) LNA and 2 -thio-LNAs, have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). Preparation of locked nucleoside analogs comprising oligodeoxyribonucleotide duplexes as substrates for nucleic acid polymerases has also been described (Wengel et al., WO 99 / 14226). Furthermore, synthesis of 2 -amino-LNA, a novel comformationally restricted high-affinity oligonucleotide analog has been described in the art(Singh et al., J. Org. Chem., 1998, 63, 10035-10039). In addition, 2 -Amino- and 2 - methylamino-LNA's have been prepared and the thermal stability of their duplexes with complementary RNA and DNA strands has been previously reported.

[0414] Modified sugar moieties are well known and can be used to alter, typically increase, the affinity of the antisense compound for its target and / or increase nuclease resistance. A representative list of preferred modified sugars includes but is not limited to bicyclic modified sugars, including methyleneoxy (4 -CH2-O-2 ) LNA and ethyleneoxy (4 - (CH2)2-O-2 bridge) ENA; substituted sugars, especially 2 -substituted sugars having a 2 -F, 2 -OCH3 or a 2 -O(CH2)2-OCH3 substituent group; and 4 -thio modified sugars. Sugars can also be replaced with sugar mimetic groups among others. Methods for the preparations of modified sugars are well known to those skilled in the art. Some representative patents and publications that teach the preparation of such modified sugars include, but are not limited to, U.S. Pat. Nos.4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; 5,700,920; 6,531,584; and6,600,032; and WO 2005 / 121371.

[0415] Examples of“oxy”-2 ^ hydroxyl group modifications include alkoxy or aryloxy (OR, e.g., R = H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar); polyethyleneglycols (PEG), O(CH2CH2O)nCH2CH2OR, n =1-50;“locked” nucleic acids (LNA) in which the furanose portion of the nucleoside includes a bridge connecting two carbon atoms on the furanose ring, thereby forming a bicyclic ring system; O-AMINE or O-(CH2)nAMINE (n = 1- 10, AMINE = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, ethylene diamine or polyamino); and O- CH2CH2(NCH2CH2NMe2)2.

[0416] “Deoxy” modifications include hydrogen (i.e. deoxyribose sugars, which are of particular relevance to the single-strand overhangs); halo (e.g., fluoro); amino (e.g. NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); NH(CH2CH2NH)nCH2CH2-AMINE (AMINE = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino); -NHC(O)R (R = alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar); cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; thioalkyl; alkyl; cycloalkyl; aryl; alkenyl and alkynyl, which can be optionally substituted with e.g., an amino functionality.

[0417] Other suitable 2’-modifications, e.g., modified MOE, are described in U.S. Patent Application PublicationNo.20130130378, contents of which are herein incorporated by reference.

[0418] A modification at the 2’ position can be present in the arabinose configuration The term“arabinose configuration” refers to the placement of a substituent on the C2’ of ribose in the same configuration as the 2’-OH is in the arabinose.

[0419] The sugar can comprise two different modifications at the same carbon in the sugar, e.g., gem modification. The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, an oligomeric compound can include one or more monomers containing e.g., arabinose, as the sugar. The monomer can have an alpha linkage at the 1’ position on the sugar, e.g., alpha-nucleosides. The monomer can also have the opposite configuration at the 4’-position, e.g., C5’ and H4’ or substituents replacing them are interchanged with each other. When the C5’ and H4’ or substituents replacing them are interchanged with each other, the sugar is said to be modified at the 4’ position.

[0420] Double-stranded iRNA agent of the inventions disclosed herein can also include abasic sugars, i.e., a sugar which lack a nucleobase at C-1 ^ or has other chemical groups in place of a nucleobase at C1’. See for example U.S. Pat. No.5,998,203, content of which is herein incorporated in its entirety. These abasic sugars can also be further containing modifications at one or more of the constituent sugar atoms. Double-stranded iRNA agent of the inventions can also contain one or more sugars that are the L isomer, e.g. L-nucleosides. Modification to the sugar group can also include replacement of the 4’-O with a sulfur, optionally substituted nitrogen or CH2 group. In some embodiments, linkage between C1’ and nucleobase is in a configuration.

[0421] Sugar modifications can also include acyclic nucleotides, wherein a C-C bonds between ribose carbons (e.g., C1’-C2’, C2’-C3’, C3’-C4’, C4’-O4’, C1’-O4’) is absent and / or at least one of ribose carbons or oxygen (e.g., C1’, C2’, C3’, C4’ or O4’) are independently or in combination absent from the nucleotide. In some embodiments, acyclic nucleotide iswherein B is amodified or unmodified nucleobase, R1and R2independently are H, halogen, OR3, or alkyl; and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar).

[0422] In some embodiments, sugar modifications are selected from the group consisting of 2’-H, 2 -O-Me (2 -O-methyl), 2 -O-MOE (2 -O-methoxyethyl), 2’-F, 2 -O-[2- (methylamino)-2-oxoethyl] (2 -O-NMA), 2’-S-methyl, 2’-O-CH2-(4’-C) (LNA), 2’-O- CH2CH2-(4’-C) (ENA), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O- DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'- O-DMAEOE) and gem 2’-OMe / 2’F with 2’-O-Me in the arabinose configuration.

[0423] It is to be understood that when a particular nucleotide is linked through its 2’- position to the next nucleotide, the sugar modifications described herein can be placed at the 3’-position of the sugar for that particular nucleotide, e.g., the nucleotide that is linked through its 2’ -position. A modification at the 3’ position can be present in the xylose configuration The term“xylose configuration” refers to the placement of a substituent on the C3’ of ribose in the same configuration as the 3’-OH is in the xylose sugar.

[0424] The hydrogen attached to C4’ and / or C1’ can be replaced by a straight- or branched- optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, wherein backbone of the alkyl, alkenyl and alkynyl can contain one or more of O, S, S(O), SO2, N(R’), C(O), N(R’)C(O)O, OC(O)N(R’), CH(Z’), phosphorous containing linkage, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclic or optionally substituted cycloalkyl, where R’ is hydrogen, acyl or optionally substituted aliphatic, Z’ is selected from the group consisting of OR11, COR11, CO2R11,CON(H)N=CR41R51, N(R21)C(=NR31)NR21R31, N(R21)C(O)NR21R31, N(R21)C(S)NR21R31, OC(O)NR21R31, SC(O)NR21R31, N(R21)C(S)OR11, N(R21)C(O)OR11, N(R21)C(O)SR11, N(R21)N=CR41R51, ON=CR41R51, SO2R11, SOR11, SR11, and substituted or unsubstituted heterocyclic; R21 and R31 for each occurrence are independently hydrogen, acyl, unsubstituted or substituted aliphatic, aryl, heteroaryl, heterocyclic, OR11, COR11, CO2R11, or NR11R11’; or R21and R31, taken together with the atoms to which they are attached, form a heterocyclic ring; R41and R51for each occurrence are independently hydrogen, acyl, unsubstituted or substituted aliphatic, aryl, heteroaryl, heterocyclic, OR11, COR11, or CO2R11, or NR11R11’; and R11and R11’ are independently hydrogen, aliphatic, substituted aliphatic, aryl, heteroaryl,or heterocyclic. In some embodiments, the hydrogen attached to the C4’ of the 5’ terminal nucleotide is replaced.

[0425] In some embodiments, C4’ and C5’ together form an optionally substituted heterocyclic, preferably comprising at least one -PX(Y)-, wherein X is H, OH, OM, SH, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkylthio, optionally substituted alkylamino or optionally substituted dialkylamino, where M is independently for each occurrence an alki metal or transition metal with an overall charge of +1; and Y is O, S, or NR’, where R’ is hydrogen, optionally substituted aliphatic. Preferably this modification is at the 5 terminal of the iRNA.

[0426] In certain embodiments, LNA's include bicyclic nucleoside having the formula:wherein:Bx is a heterocyclic base moiety;T1is H or a hydroxyl protecting group;T2 is H, a hydroxyl protecting group or a reactive phosphorus group;Z is C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, substituted C1-C6alkyl, substituted C2- C6alkenyl, substituted C2-C6alkynyl, acyl, substituted acyl, or substituted amide.

[0427] In some embodiments, each of the substituted groups, is, independently, mono or poly substituted with optionally protected substituent groups independently selected from halogen, oxo, hydroxyl, OJ1, NJ1J2, SJ1, N3, OC(═X)J1, OC(═X)NJ1J2, NJ3C(═X)NJ1J2 and CN, wherein each J1, J2 and J3 is, independently, H or C1-C6 alkyl, and X is O, S or NJ1.

[0428] In certain such embodiments, each of the substituted groups, is, independently, mono or poly substituted with substituent groups independently selected from halogen, oxo, hydroxyl, OJ1, NJ1J2, SJ1, N3, OC(═X)J1, and NJ3C(═X)NJ1J2, wherein each J1, J2 and J3 is, independently, H, C1-C6 alkyl, or substituted C1-C6 alkyl and X is O or NJ1.

[0429] In certain embodiments, the Z group is C1-C6alkyl substituted with one or more Xx, wherein each Xx is independently OJ1, NJ1J2, SJ1, N3, OC(═X)J1, OC(═X)NJ1J2, NJ3C(═X)NJ1J2 or CN; wherein each J1, J2 and J3 is, independently, H or C1-C6 alkyl, and X is O, S or NJ1. In another embodiment, the Z group is C1-C6 alkyl substituted with one ormore Xx, wherein each Xx is independently halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH3O—), substituted alkoxy or azido.

[0430] In certain embodiments, the Z group is—CH2Xx, wherein Xx is OJ1, NJ1J2, SJ1, N3, OC(═X)J1, OC(═X)NJ1J2, NJ3C(═X)NJ1J2 or CN; wherein each J1, J2 and J3 is, independently, H or C1-C6 alkyl, and X is O, S or NJ1. In another embodiment, the Z group is —CH2Xx, wherein Xx is halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH3O—) or azido.

[0431] In certain such embodiments, the Z group is in the (R)-configuration:.

[0432] In certain such embodiments, the Z group is in the (S)-configuration:.

[0433] In certain embodiments, each T1 and T2 is a hydroxyl protecting group. A preferred list of hydroxyl protecting groups includes benzyl, benzoyl, 2,6-dichlorobenzyl, t- butyldimethylsilyl, t-butyldiphenylsilyl, mesylate, tosylate, dimethoxytrityl (DMT), 9- phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl (MOX). In certain embodiments, T1is a hydroxyl protecting group selected from acetyl, benzyl, t- butyldimethylsilyl, t-butyldiphenylsilyl and dimethoxytrityl wherein a more preferred hydroxyl protecting group is T1 is 4,4 -dimethoxytrityl.

[0434] In certain embodiments, T2 is a reactive phosphorus group wherein preferred reactive phosphorus groups include diisopropylcyanoethoxy phosphoramidite and H- phosphonate. In certain embodiments T1 is 4,4 -dimethoxytrityl and T2 isdiisopropylcyanoethoxy phosphoramidite.

[0435] In certain embodiments, the compounds of the invention comprise at least one monomer of the formula:or of the formula:or of the formula:whereinBx is a heterocyclic base moiety;T3 is H, a hydroxyl protecting group, a linked conjugate group or an internucleoside linking group attached to a nucleoside, a nucleotide, an oligonucleoside, an oligonucleotide, a monomeric subunit or an oligomeric compound;T4 is H, a hydroxyl protecting group, a linked conjugate group or an internucleoside linking group attached to a nucleoside, a nucleotide, an oligonucleoside, an oligonucleotide, a monomeric subunit or an oligomeric compound;wherein at least one of T3 and T4 is an internucleoside linking group attached to a nucleoside, a nucleotide, an oligonucleoside, an oligonucleotide, a monomeric subunit or an oligomeric compound; andZ is C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted C1-C6 alkyl, substituted C2- C6 alkenyl, substituted C2-C6 alkynyl, acyl, substituted acyl, or substituted amide.

[0436] In some embodiments, each of the substituted groups, is, independently, mono or poly substituted with optionally protected substituent groups independently selected from halogen, oxo, hydroxyl, OJ1, NJ1J2, SJ1, N3, OC(═X)J1, OC(═X)NJ1J2, NJ3C(═X)NJ1J2 and CN, wherein each J1, J2 and J3 is, independently, H or C1-C6 alkyl, and X is O, S or NJ1.

[0437] In some embodiments, each of the substituted groups, is, independently, mono or poly substituted with substituent groups independently selected from halogen, oxo, hydroxyl, OJ1, NJ1J2, SJ1, N3, OC(═X)J1, and NJ3C(═X)NJ1J2, wherein each J1, J2 and J3 is, independently, H or C1-C6 alkyl, and X is O or NJ1.

[0438] In certain such embodiments, at least one Z is C1-C6alkyl or substituted C1-C6alkyl. In certain embodiments, each Z is, independently, C1-C6alkyl or substituted C1-C6alkyl. In certain embodiments, at least one Z is C1-C6 alkyl. In certain embodiments, each Zis, independently, C1-C6alkyl. In certain embodiments, at least one Z is methyl. In certain embodiments, each Z is methyl. In certain embodiments, at least one Z is ethyl. In certain embodiments, each Z is ethyl. In certain embodiments, at least one Z is substituted C1-C6 alkyl. In certain embodiments, each Z is, independently, substituted C1-C6alkyl. In certain embodiments, at least one Z is substituted methyl. In certain embodiments, each Z is substituted methyl. In certain embodiments, at least one Z is substituted ethyl. In certain embodiments, each Z is substituted ethyl.

[0439] In certain embodiments, at least one substituent group is C1-C6alkoxy (e.g., at least one Z is C1-C6 alkyl substituted with one or more C1-C6 alkoxy). In anotherembodiment, each substituent group is, independently, C1-C6alkoxy (e.g., each Z is, independently, C1-C6alkyl substituted with one or more C1-C6alkoxy).

[0440] In certain embodiments, at least one C1-C6 alkoxy substituent group is CH3O— (e.g., at least one Z is CH3OCH2-). In another embodiment, each C1-C6 alkoxy substituent group is CH3O— (e.g., each Z is CH3OCH2-).

[0441] In certain embodiments, at least one substituent group is halogen (e.g., at least one Z is C1-C6 alkyl substituted with one or more halogen). In certain embodiments, each substituent group is, independently, halogen (e.g., each Z is, independently, C1-C6alkyl substituted with one or more halogen). In certain embodiments, at least one halogen substituent group is fluoro (e.g., at least one Z is CH2FCH2-, CHF2CH2- or CF3CH2-). In certain embodiments, each halo substituent group is fluoro (e.g., each Z is, independently, CH2FCH2-, CHF2CH2- or CF3CH2-).

[0442] In certain embodiments, at least one substituent group is hydroxyl (e.g., at least one Z is C1-C6 alkyl substituted with one or more hydroxyl). In certain embodiments, each substituent group is, independently, hydroxyl (e.g., each Z is, independently, C1-C6alkyl substituted with one or more hydroxyl). In certain embodiments, at least one Z is HOCH2-. In another embodiment, each Z is HOCH2-.

[0443] In certain embodiments, at least one Z is CH3-, CH3CH2-, CH2OCH3-, CH2F— or HOCH2-. In certain embodiments, each Z is, independently, CH3-, CH3CH2-, CH2OCH3-, CH2F— or HOCH2-.

[0444] In certain embodiments, at least one Z group is C1-C6alkyl substituted with one or more Xx, wherein each Xx is, independently, OJ1, NJ1J2, SJ1, N3, OC(═X)J1,OC(═X)NJ1J2, NJ3C(═X)NJ1J2 or CN; wherein each J1, J2 and J3 is, independently, H or C1-C6alkyl, and X is O, S or NJ1. In another embodiment, at least one Z group is C1-C6alkylsubstituted with one or more Xx, wherein each Xx is, independently, halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH3O—) or azido.

[0445] In certain embodiments, each Z group is, independently, C1-C6 alkyl substituted with one or more Xx, wherein each Xx is independently OJ1, NJ1J2, SJ1, N3, OC(═X)J1, OC(═X)NJ1J2, NJ3C(═X)NJ1J2 or CN; wherein each J1, J2 and J3 is, independently, H or C1-C6 alkyl, and X is O, S or NJ1. In another embodiment, each Z group is, independently, C1-C6alkyl substituted with one or more Xx, wherein each Xx is independently halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH3O—) or azido.

[0446] In certain embodiments, at least one Z group is—CH2Xx, wherein Xx is OJ1, NJ1J2, SJ1, N3, OC(═X)J1, OC(═X)NJ1J2, NJ3C(═X)NJ1J2 or CN; wherein each J1, J2 and J3 is, independently, H or C1-C6alkyl, and X is O, S or NJ1 In certain embodiments, at least one Z group is—CH2Xx, wherein Xx is halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH3O—) or azido.

[0447] In certain embodiments, each Z group is, independently,—CH2Xx, wherein each Xx is, independently, OJ1, NJ1J2, SJ1, N3, OC(═X)J1, OC(═X)NJ1J2, NJ3C(═X)NJ1J2 or CN; wherein each J1, J2 and J3 is, independently, H or C1-C6 alkyl, and X is O, S or NJ1. In another embodiment, each Z group is, independently,—CH2Xx, wherein each Xx is, independently, halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CH3O—) or azido.

[0448] In certain embodiments, at least one Z is CH3-. In another embodiment, each Z is, CH3-.

[0449] In certain embodiments, the Z group of at least one monomer is in the (R)— configuration represented by the formula:or the formula:or the formula:.

[0450] IN certain embodiments, the Z group of each monomer of the formula is in the (R)—configuration.

[0451] In certain embodiments, the Z group of at least one monomer is in the (S)— configuration represented by the formula:or the formula:or the formula:

[0452] In certain embodiments, the Z group of each monomer of the formula is in the (S)— configuration.

[0453] In certain embodiments, T3 is H or a hydroxyl protecting group. In certain embodiments, T4 is H or a hydroxyl protecting group. In a further embodiment T3 is an internucleoside linking group attached to a nucleoside, a nucleotide or a monomeric subunit. In certain embodiments, T4 is an internucleoside linking group attached to a nucleoside, a nucleotide or a monomeric subunit. In certain embodiments, T3 is an internucleoside linking group attached to an oligonucleoside or an oligonucleotide. In certain embodiments, T4is an internucleoside linking group attached to an oligonucleoside or an oligonucleotide. In certain embodiments, T3 is an internucleoside linking group attached to an oligomeric compound. In certain embodiments, T4 is an internucleoside linking group attached to an oligomericcompound. In certain embodiments, at least one of T3and T4comprises an internucleoside linking group selected from phosphodiester or phosphorothioate.

[0454] In certain embodiments, double-stranded iRNA agent of the invention comprise at least one region of at least two contiguous monomers of the formula:or of the formula:or of the formula:.

[0455] In certain such embodiments, LNAs include, but are not limited to, (A) a-L- Methyleneoxy (4 -CH2-O-2 ) LNA, (B) b-D-Methyleneoxy (4 -CH2-O-2 ) LNA, (C)Ethyleneoxy (4 -(CH2)2-O-2 ) LNA, (D) Aminooxy (4 -CH2-O—N(R)-2 ) LNA and (E) Oxyamino (4 -CH2-N(R)—O-2 ) LNA, as depicted below:

[0456] In certain embodiments, the double-stranded iRNA agent of the invention comprises at least two regions of at least two contiguous monomers of the above formula. In certain embodiments, the double-stranded iRNA agent of the invention comprises a gapped motif. In certain embodiments, the double-stranded iRNA agent of the invention comprises at least one region of from about 8 to about 14 contiguous b-D-2 -deoxyribofuranosyl nucleosides. In certain embodiments, the Double-stranded iRNA agent of the invention comprises at least one region of from about 9 to about 12 contiguous b-D-2 - deoxyribofuranosyl nucleosides.

[0457] In certain embodiments, the double-stranded iRNA agent of the invention comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) comprises at least one (S)-cEt monomer of the formula:,wherein Bx is heterocyclic base moiety.

[0458] In certain embodiments, monomers include sugar mimetics. In certain such embodiments, a mimetic is used in place of the sugar or sugar-internucleoside linkage combination, and the nucleobase is maintained for hybridization to a selected target.Representative examples of a sugar mimetics include, but are not limited to, cyclohexenyl or morpholino. Representative examples of a mimetic for a sugar-internucleoside linkage combination include, but are not limited to, peptide nucleic acids (PNA) and morpholino groups linked by uncharged achiral linkages. In some instances a mimetic is used in place of the nucleobase. Representative nucleobase mimetics are well known in the art and include, but are not limited to, tricyclic phenoxazine analogs and universal bases (Berger et al., Nuc Acid Res.2000, 28:2911-14, incorporated herein by reference). Methods of synthesis of sugar, nucleoside and nucleobase mimetics are well known to those skilled in the art. Nucleic acid modifications (intersugar linkage)

[0459] Described herein are linking groups that link monomers (including, but not limited to, modified and unmodified nucleosides and nucleotides) together, thereby forming an oligomeric compound, e.g., an oligonucleotide. Such linking groups are also referred to as intersugar linkage. The two main classes of linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus containing linkages include, but are not limited to, phosphodiesters (P═O), phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates (P═S). Representative non-phosphorus containing linking groups include, but are not limited to, methylenemethylimino (—CH2-N(CH3)-O— CH2-), thiodiester (—O—C(O)—S—), thionocarbamate (—O—C(O)(NH)—S—); siloxane (—O—Si(H)2-O—); and N,N -dimethylhydrazine (—CH2-N(CH3)-N(CH3)-). Modified linkages, compared to natural phosphodiester linkages, can be used to alter, typically increase, nuclease resistance of the oligonucleotides. In certain embodiments, linkages having a chiral atom can be prepared as racemic mixtures, as separate enantomers. Representative chiral linkages include, but are not limited to, alkylphosphonates and phosphorothioates.Methods of preparation of phosphorous-containing and non-phosphorous-containing linkages are well known to those skilled in the art.

[0460] The phosphate group in the linking group can be modified by replacing one of the oxygens with a different substituent. One result of this modification can be increased resistance of the oligonucleotide to nucleolytic breakdown. Examples of modified phosphate groups include phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters. In some embodiments, one of the non-bridging phosphate oxygen atoms in the linkage can be replaced by any of the following: S, Se, BR3 (R is hydrogen, alkyl, aryl), C (i.e. an alkyl group, an aryl group, etc…), H, NR2(R is hydrogen, optionally substituted alkyl, aryl), or OR (R is optionally substituted alkyl or aryl). The phosphorous atom in an unmodified phosphate group is achiral. However, replacement of one of the non-bridging oxygens with one of the above atoms or groups of atoms renders the phosphorous atom chiral; in other words a phosphorous atom in a phosphate group modified in this way is a stereogenic center. The stereogenic phosphorous atom can possess either the“R”configuration (herein Rp) or the“S” configuration (herein Sp).

[0461] Phosphorodithioates have both non-bridging oxygens replaced by sulfur. The phosphorus center in the phosphorodithioates is achiral which precludes the formation of oligonucleotides diastereomers. Thus, while not wishing to be bound by theory,modifications to both non-bridging oxygens, which eliminate the chiral center, e.g.phosphorodithioate formation, can be desirable in that they cannot produce diastereomer mixtures. Thus, the non-bridging oxygens can be independently any one of O, S, Se, B, C, H, N, or OR (R is alkyl or aryl).

[0462] The phosphate linker can also be modified by replacement of bridging oxygen, (i.e. oxygen that links the phosphate to the sugar of the monomer), with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridgedmethylenephosphonates). The replacement can occur at the either one of the linking oxygens or at both linking oxygens. When the bridging oxygen is the 3’-oxygen of a nucleoside, replacement with carbon is preferred. When the bridging oxygen is the 5’-oxygen of a nucleoside, replacement with nitrogen is preferred.

[0463] Modified phosphate linkages where at least one of the oxygen linked to the phosphate has been replaced or the phosphate group has been replaced by a non-phosphorousgroup, are also referred to as“non-phosphodiester intersugar linkage” or“non-phosphodiester linker.”

[0464] In certain embodiments, the phosphate group can be replaced by non-phosphorus containing connectors, e.g. dephospho linkers. Dephospho linkers are also referred to as non- phosphodiester linkers herein. While not wishing to be bound by theory, it is believed that since the charged phosphodiester group is the reaction center in nucleolytic degradation, its replacement with neutral structural mimics should impart enhanced nuclease stability. Again, while not wishing to be bound by theory, it can be desirable, in some embodiment, to introduce alterations in which the charged phosphate group is replaced by a neutral moiety.

[0465] Examples of moieties which can replace the phosphate group include, but are not limited to, amides (for example amide-3 (3'-CH2-C(=O)-N(H)-5') and amide-4 (3'-CH2-N(H)- C(=O)-5')), hydroxylamino, siloxane (dialkylsiloxxane), carboxamide, carbonate,carboxymethyl, carbamate, carboxylate ester, thioether, ethylene oxide linker,sulfide,sulfonate, sulfonamide, sulfonate ester, thioformacetal (3'-S-CH2-O-5'), formacetal (3 '-O-CH2-O-5'), oxime, methyleneimino, methykenecarbonylamino, methylenemethylimino (MMI, 3'-CH2-N(CH3)-O-5'), methylenehydrazo, methylenedimethylhydrazo,methyleneoxymethylimino, ethers (C3’-O-C5’), thioethers (C3’-S-C5’), thioacetamido (C3’- N(H)-C(=O)-CH2-S-C5’, C3’-O-P(O)-O-SS-C5’, C3’-CH2-NH-NH-C5’, 3'-NHP(O)(OCH3)- O-5' and 3'-NHP(O)(OCH3)-O-5’ and nonionic linkages containing mixed N, O, S and CH2 component parts. See for example, Carbohydrate Modifications in Antisense Research; Y.S. Sanghvi and P.D. Cook Eds. ACS Symposium Series 580; Chapters 3 and 4, (pp.40-65). Preferred embodiments include methylenemethylimino (MMI),methylenecarbonylamino, amides,carbamate and ethylene oxide linker.

[0466] One skilled in the art is well aware that in certain instances replacement of a non- bridging oxygen can lead to enhanced cleavage of the intersugar linkage by the neighboring 2’-OH, thus in many instances, a modification of a non-bridging oxygen can necessitate modification of 2’-OH, e.g., a modification that does not participate in cleavage of the neighboring intersugar linkage, e.g., arabinose sugar, 2’-O-alkyl, 2’-F, LNA and ENA.

[0467] Preferred non-phosphodiester intersugar linkages include phosphorothioates, phosphorothioates with an at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% , 90% 95% or more enantiomeric excess of Sp isomer, phosphorothioates with an at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% , 90% 95% or more enantiomeric excess of Rp isomer, phosphorodithioates, phsophotriesters, aminoalkylphosphotrioesters, alkyl-phosphonaters (e.g., methyl-phosphonate), selenophosphates, phosphoramidates (e.g., N- alkylphosphoramidate), and boranophosphonates.

[0468] In some embodiments, the double-stranded iRNA agent of the invention comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more and upto including all) modified or nonphosphodiester linkages. In some embodiments, the double- stranded iRNA agent of the invention comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more and upto including all) phosphorothioate linkages.

[0469] The double-stranded iRNA agent of the inventions can also be constructed wherein the phosphate linker and the sugar are replaced by nuclease resistant nucleoside or nucleotide surrogates. While not wishing to be bound by theory, it is believed that the absence of a repetitively charged backbone diminishes binding to proteins that recognize polyanions (e.g. nucleases). Again, while not wishing to be bound by theory, it can be desirable in some embodiment, to introduce alterations in which the bases are tethered by a neutral surrogate backbone. Examples include the morpholino, cyclobutyl, pyrrolidine, peptide nucleic acid (PNA), aminoethylglycyl PNA (aegPNA) and backnone-extended pyrrolidine PNA (bepPNA) nucleoside surrogates. A preferred surrogate is a PNA surrogate.

[0470] The double-stranded iRNA agent of the inventions described herein can contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations that may be defined, in terms of absolute stereochemistry, as (R) or (S), such as for sugar anomers, or as (D) or (L) such as for amino acids et al. Included in the double-stranded iRNA agent of the inventions provided herein are all such possible isomers, as well as their racemic and optically pure forms. Nucleic acid modifications (terminal modifications

[0471] In some embodiments, the double-stranded iRNA agent further comprises a phosphate or phosphate mimic at the 5’-end of the antisense strand. In one embodiment, the phosphate mimic is a 5’-vinyl phosphonate (VP).

[0472] In some embodiments, the 5’-end of the antisense strand of the double-stranded iRNA agent does not contain a 5’-vinyl phosphonate (VP).

[0473] Ends of the iRNA agent of the invention can be modified. Such modifications can be at one end or both ends. For example, the 3 ^ and / or 5 ^ ends of an iRNA can be conjugated to other functional molecular entities such as labeling moieties, e.g., fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) or protecting groups (based e.g., on sulfur,silicon, boron or ester). The functional molecular entities can be attached to the sugar through a phosphate group and / or a linker. The terminal atom of the linker can connect to or replace the linking atom of the phosphate group or the C-3 ^ or C-5 ^ O, N, S or C group of the sugar. Alternatively, the linker can connect to or replace the terminal atom of a nucleotide surrogate (e.g., PNAs).

[0474] When a linker / phosphate-functional molecular entity-linker / phosphate array is interposed between two strands of a double stranded oligomeric compound, this array can substitute for a hairpin loop in a hairpin-type oligomeric compound.

[0475] Terminal modifications useful for modulating activity include modification of the 5’ end of iRNAs with phosphate or phosphate analogs. In certain embodiments, the 5’end of an iRNA is phosphorylated or includes a phosphoryl analog. Exemplary 5'-phosphate modifications include those which are compatible with RISC mediated gene silencing.Modifications at the 5’-terminal end can also be useful in stimulating or inhibiting the immune system of a subject. In some embodiments, the 5’-end of the oligomeric compoundcomprises the modification, wherein W, X and Y are eachindependently selected from the group consisting of O, OR (R is hydrogen, alkyl, aryl), S, Se, BR3(R is hydrogen, alkyl, aryl), BH - 3 , C (i.e. an alkyl group, an aryl group, etc…), H, NR2(R is hydrogen, alkyl, aryl), or OR (R is hydrogen, alkyl or aryl); A and Z are each independently for each occurrence absent, O, S, CH2, NR (R is hydrogen, alkyl, aryl), or optionally substituted alkylene, wherein backbone of the alkylene can comprise one or more of O, S, SS and NR (R is hydrogen, alkyl, aryl) internally and / or at the end; and n is 0-2. In some embodiments, n is 1 or 2. It is understood that A is replacing the oxygen linked to 5’ carbon of sugar. When n is 0, W and Y together with the P to which they are attached can form an optionally substituted 5-8 membered heterocyclic, wherein W an Y are each independently O, S, NR’ or alkylene. Preferably the heterocyclic is substituted with an aryl or heteroaryl. In some embodiments, one or both hydrogen on C5’ of the 5’- terminal nucleotides are replaced with a halogen, e.g., F.

[0476] Exemplary 5’-modifications include, but are not limited to, 5'-monophosphate ((HO)2(O)P-O-5'); 5'-diphosphate ((HO)2(O)P-O-P(HO)(O)-O-5'); 5'-triphosphate((HO)2(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-monothiophosphate (phosphorothioate;(HO)2(S)P-O-5'); 5'-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P-O-5'), 5'- phosphorothiolate ((HO)2(O)P-S-5'); 5'-alpha-thiotriphosphate; 5’-beta-thiotriphosphate; 5'- gamma-thiotriphosphate; 5'-phosphoramidates ((HO)2(O)P-NH-5', (HO)(NH2)(O)P-O-5'). Other 5’-modification include 5'-alkylphosphonates (R(OH)(O)P-O-5', R=alkyl, e.g., methyl, ethyl, isopropyl, propyl, etc…), 5'-alkyletherphosphonates (R(OH)(O)P-O-5', R=alkylether, e.g., methoxymethyl (CH2OMe), ethoxymethyl, etc…). Other exemplary 5’-modifications include where Z is optionally substituted alkyl at least once, e.g., ((HO)2(X)P-O[-(CH2)a-O- P(X)(OH)-O]b- 5', ((HO)2(X)P-O[-(CH2)a-P(X)(OH)-O]b- 5', ((HO)2(X)P-[-(CH2)a-O- P(X)(OH)-O]b- 5'; dialkyl terminal phosphates and phosphate mimics: HO[-(CH2)a-O- P(X)(OH)-O]b- 5' , H2N[-(CH2)a-O-P(X)(OH)-O]b- 5', H[-(CH2)a-O-P(X)(OH)-O]b- 5', Me2N[-(CH2)a-O-P(X)(OH)-O]b- 5', HO[-(CH2)a-P(X)(OH)-O]b- 5' , H2N[-(CH2)a-P(X)(OH)- O]b- 5', H[-(CH2)a-P(X)(OH)-O]b- 5', Me2N[-(CH2)a-P(X)(OH)-O]b- 5', wherein a and b are each independently 1-10. Other embodiments, include replacement of oxygen and / or sulfur with BH3, BH - 3 and / or Se.

[0477] Terminal modifications can also be useful for monitoring distribution, and in such cases the preferred groups to be added include fluorophores, e.g., fluorescein or an Alexa dye, e.g., Alexa 488. Terminal modifications can also be useful for enhancing uptake, useful modifications for this include targeting ligands. Terminal modifications can also be useful for cross-linking an oligonucleotide to another moiety; modifications useful for this include mitomycin C, psoralen, and derivatives thereof. Thermally Destabilizing Modifications

[0478] The compounds of the invention, such as iRNAs or dsRNA agents, can be optimized for RNA interference by increasing the propensity of the iRNA duplex to disassociate or melt (decreasing the free energy of duplex association) by introducing a thermally destabilizing modification in the sense strand at a site opposite to the seed region of the antisense strand (i.e., at positions 2-8 of the 5’-end of the antisense strand). This modification can increase the propensity of the duplex to disassociate or melt in the seed region of the antisense strand.

[0479] The thermally destabilizing modifications can include abasic modification;mismatch with the opposing nucleotide in the opposing strand; and sugar modification such as 2’-deoxy modification or acyclic nucleotide, e.g., unlocked nucleic acids (UNA) or glycerol nuceltic acid (GNA).

[0480] Exemplified abasic modifications are:.

[0481] Exemplified sugar modifications are:

[0482] The term "acyclic nucleotide" refers to any nucleotide having an acyclic ribose sugar, for example, where any of bonds between the ribose carbons (e.g., C1’-C2’, C2’-C3’, C3’-C4’, C4’-O4’, or C1’-O4’) is absent and / or at least one of ribose carbons or oxygen (e.g., C1’, C2’, C3’, C4’ or O4’) are independently or in combination absent from the nucleotide.In some embodiments, acyclic nucleotidewherein B is a modified or unmodified nucleobase, R1and R2independently are H, halogen, OR3, or alkyl; and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar). The term“UNA” refers to unlocked acyclic nucleic acid, wherein any of the bonds of the sugar has been removed, forming an unlocked "sugar" residue. In one example, UNA also encompasses monomers with bonds between C1'-C4' being removed (i.e. the covalent carbon-oxygen-carbon bond between the C1' and C4' carbons). In another example, the C2'-C3' bond (i.e. the covalent carbon-carbon bond between the C2' and C3' carbons) of the sugar is removed (see Mikhailov et. al., Tetrahedron Letters, 26 (17): 2059 (1985); and Fluiter et al., Mol. Biosyst., 10: 1039 (2009), which arehereby incorporated by reference in their entirety). The acyclic derivative provides greater backbone flexibility without affecting the Watson-Crick pairings. The acyclic nucleotide can be linked via 2’-5’ or 3’-5’ linkage.

[0483] The term‘GNA’ refers to glycol nucleic acid which is a polymer similar to DNA or RNA but differing in the composition of its“backbone” in that is composed of repeating glycerol units linked by phosphodiester bonds:.

[0484] The thermally destabilizing modification can be mismatches (i.e.,noncomplementary base pairs) between the thermally destabilizing nucleotide and the opposing nucleotide in the opposite strand within the dsRNA duplex. Exemplary mismatch basepairs include G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, U:T, or a combination thereof. Other mismatch base pairings known in the art are also amenable to the present invention. A mismatch can occur between nucleotides that are either naturally occurring nucleotides or modified nucleotides, i.e., the mismatch base pairing can occur between the nucleobases from respective nucleotides independent of the modifications on the ribose sugars of the nucleotides. In certain embodiments, the compounds of the invention, such as siRNA or iRNA agent, contains at least one nucleobase in the mismatch pairing that is a 2’-deoxy nucleobase; e.g., the 2’-deoxy nucleobase is in the sense strand.

[0485] More examples of abasic nucleotide, acyclic nucleotide modifications (including UNA and GNA), and mismatch modifications have been described in detail in WO2011 / 133876, which is herein incorporated by reference in its entirety.

[0486] The thermally destabilizing modifications may also include universal base with reduced or abolished capability to form hydrogen bonds with the opposing bases, and phosphate modifications.

[0487] Nucleobase modifications with impaired or completely abolished capability to form hydrogen bonds with bases in the opposite strand have been evaluated fordestabilization of the central region of the dsRNA duplex as described in WO 2010 / 0011895, which is herein incorporated by reference in its entirety. Exemplary nucleobasemodifications are:.

[0488] Exemplary phosphate modifications known to decrease the thermal stability of dsRNA duplexes compared to natural phosphodiester linkages are:.

[0489] In some embodiments, compounds of the invention can comprise 2’-5’ linkages (with 2’-H, 2’-OH and 2’-OMe and with P=O or P=S). For example, the 2’-5’ linkages modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5’ end of the sense strand to avoid sense strand activation by RISC.

[0490] In another embodiment, compounds of the invention can comprise L sugars (e.g., L ribose, L-arabinose with 2’-H, 2’-OH and 2’-OMe). For example, these Lsugar modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5’ end of the sense strand to avoid sense strand activation by RISC.

[0491] In one embodimennt the iRNA agent of the invention is conjugated to a ligand via a carrier, wherein the carrier can be cyclic group or acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl,thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and and decalin; preferably, the acyclic group is selected from serinol backbone or diethanolamine backbone.

[0492] In some embodoments, at least one strand of the iRNA agent of the invention disclosed herein is 5’ phosphorylated or includes a phosphoryl analog at the 5’ prime terminus. 5'-phosphate modifications include those which are compatible with RISC mediated gene silencing. Suitable modifications include: 5'-monophosphate ((HO)2(O)P-O- 5'); 5'-diphosphate ((HO)2(O)P-O-P(HO)(O)-O-5'); 5'-triphosphate ((HO)2(O)P-O-(HO)(O)P- O-P(HO)(O)-O-5'); 5'-guanosine cap (7-methylated or non-methylated) (7m-G-O-5'- (HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N-O-5'-(HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'- monothiophosphate (phosphorothioate; (HO)2(S)P-O-5'); 5'-monodithiophosphate(phosphorodithioate; (HO)(HS)(S)P-O-5'), 5'-phosphorothiolate ((HO)2(O)P-S-5'); any additional combination of oxygen / sulfur replaced monophosphate, diphosphate and triphosphates (e.g.5'-alpha-thiotriphosphate, 5'-gamma-thiotriphosphate, etc.), 5'- phosphoramidates ((HO)2(O)P-NH-5', (HO)(NH2)(O)P-O-5'), 5'-alkylphosphonates(R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g. RP(OH)(O)-O-5'-, 5'- alkenylphosphonates (i.e. vinyl, substituted vinyl), (OH)2(O)P-5'-CH2-), 5'- alkyletherphosphonates (R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g. RP(OH)(O)-O-5'-). Target genes

[0493] Without limitations, target genes for siRNAs include, but are not limited to genes promoting unwanted cell proliferation, growth factor gene, growth factor receptor gene, genes expressing kinases, an adaptor protein gene, a gene encoding a G protein super family molecule, a gene encoding a transcription factor, a gene which mediates angiogenesis, a viral gene, a gene required for viral replication, a cellular gene which mediates viral function, a gene of a bacterial pathogen, a gene of an amoebic pathogen, a gene of a parasitic pathogen, a gene of a fungal pathogen, a gene which mediates an unwanted immune response, a gene which mediates the processing of pain, a gene which mediates a neurological disease, an allene gene found in cells characterized by loss of heterozygosity, or one allege gene of a polymorphic gene.

[0494] Specific exemplary target genes for the siRNAs include, but are not limited to, PCSK-9, ApoC3, AT3, AGT, ALAS1, TMPR, HAO1, AGT, C5, CCR-5, PDGF beta gene;Erb-B gene, Src gene; CRK gene; GRB2 gene; RAS gene; MEKK gene; JNK gene; RAF gene; Erk1 / 2 gene; PCNA(p21) gene; MYB gene; c-MYC gene; JUN gene; FOS gene; BCL- 2 gene; Cyclin D gene; VEGF gene; EGFR gene; Cyclin A gene; Cyclin E gene; WNT-1 gene; beta-catenin gene; c-MET gene; PKC gene; NFKB gene; STAT3 gene; survivin gene; Her2 / Neu gene; topoisomerase I gene; topoisomerase II alpha gene; p73 gene;p21(WAF1 / CIP1) gene, p27(KIP1) gene; PPM1D gene; caveolin I gene; MIB I gene; MTAI gene; M68 gene; tumor suppressor genes; p53 gene; DN-p63 gene; pRb tumor suppressor gene; APC1 tumor suppressor gene; BRCA1 tumor suppressor gene; PTEN tumor suppressor gene; MLL fusion genes, e.g., MLL-AF9, BCR / ABL fusion gene; TEL / AML1 fusion gene; EWS / FLI1 fusion gene; TLS / FUS1 fusion gene; PAX3 / FKHR fusion gene; AML1 / ETO fusion gene; alpha v-integrin gene; Flt-1 receptor gene; tubulin gene; Human Papilloma Virus gene, a gene required for Human Papilloma Virus replication, Human Immunodeficiency Virus gene, a gene required for Human Immunodeficiency Virus replication, Hepatitis A Virus gene, a gene required for Hepatitis A Virus replication, Hepatitis B Virus gene, a gene required for Hepatitis B Virus replication, Hepatitis C Virus gene, a gene required for Hepatitis C Virus replication, Hepatitis D Virus gene, a gene required for Hepatitis D Virus replication, Hepatitis E Virus gene, a gene required for Hepatitis E Virus replication, Hepatitis F Virus gene, a gene required for Hepatitis F Virus replication, Hepatitis G Virus gene, a gene required for Hepatitis G Virus replication, Hepatitis H Virus gene, a gene required for Hepatitis H Virus replication, Respiratory Syncytial Virus gene, a gene that is required for Respiratory Syncytial Virus replication, Herpes Simplex Virus gene, a gene that is required for Herpes Simplex Virus replication, herpes Cytomegalovirus gene, a gene that is required for herpes Cytomegalovirus replication, herpes Epstein Barr Virus gene, a gene that is required for herpes Epstein Barr Virus replication, Kaposi’s Sarcoma-associated Herpes Virus gene, a gene that is required for Kaposi’s Sarcoma-associated Herpes Virus replication, JC Virus gene, human gene that is required for JC Virus replication, myxovirus gene, a gene that is required for myxovirus gene replication, rhinovirus gene, a gene that is required for rhinovirus replication, coronavirus gene, a gene that is required for coronavirus replication, West Nile Virus gene, a gene that is required for West Nile Virus replication, St. Louis Encephalitis gene, a gene that is required for St. Louis Encephalitis replication, Tick-borne encephalitis virus gene, a gene that is required for Tick-borne encephalitis virus replication, Murray Valley encephalitis virus gene, a gene that is required for Murray Valley encephalitis virus replication, dengue virus gene, a gene that is required for dengue virus gene replication,Simian Virus 40 gene, a gene that is required for Simian Virus 40 replication, Human T Cell Lymphotropic Virus gene, a gene that is required for Human T Cell Lymphotropic Virus replication, Moloney-Murine Leukemia Virus gene, a gene that is required for Moloney- Murine Leukemia Virus replication, encephalomyocarditis virus gene, a gene that is required for encephalomyocarditis virus replication, measles virus gene, a gene that is required for measles virus replication, Vericella zoster virus gene, a gene that is required for Vericella zoster virus replication, adenovirus gene, a gene that is required for adenovirus replication, yellow fever virus gene, a gene that is required for yellow fever virus replication, poliovirus gene, a gene that is required for poliovirus replication, poxvirus gene, a gene that is required for poxvirus replication, plasmodium gene, a gene that is required for plasmodium gene replication, Mycobacterium ulcerans gene, a gene that is required for Mycobacterium ulcerans replication, Mycobacterium tuberculosis gene, a gene that is required forMycobacterium tuberculosis replication, Mycobacterium leprae gene, a gene that is required for Mycobacterium leprae replication, Staphylococcus aureus gene, a gene that is required for Staphylococcus aureus replication, Streptococcus pneumoniae gene, a gene that is required for Streptococcus pneumoniae replication, Streptococcus pyogenes gene, a gene that is required for Streptococcus pyogenes replication, Chlamydia pneumoniae gene, a gene that is required for Chlamydia pneumoniae replication, Mycoplasma pneumoniae gene, a gene that is required for Mycoplasma pneumoniae replication, an integrin gene, a selectin gene, complement system gene, chemokine gene, chemokine receptor gene, GCSF gene, Gro1 gene, Gro2 gene, Gro3 gene, PF4 gene, MIG gene, Pro-Platelet Basic Protein gene, MIP-1I gene, MIP-1J gene, RANTES gene, MCP-1 gene, MCP-2 gene, MCP-3 gene, CMBKR1 gene, CMBKR2 gene, CMBKR3 gene, CMBKR5v, AIF-1 gene, I-309 gene, a gene to a component of an ion channel, a gene to a neurotransmitter receptor, a gene to aneurotransmitter ligand, amyloid-family gene, presenilin gene, HD gene, DRPLA gene, SCA1 gene, SCA2 gene, MJD1 gene, CACNL1A4 gene, SCA7 gene, SCA8 gene, allele gene found in loss of heterozygosity (LOH) cells, one allele gene of a polymorphic gene and combinations thereof.

[0495] The loss of heterozygosity (LOH) can result in hemizygosity for sequence, e.g., genes, in the area of LOH. This can result in a significant genetic difference between normal and disease-state cells, e.g., cancer cells, and provides a useful difference between normal and disease-state cells, e.g., cancer cells. This difference can arise because a gene or other sequence is heterozygous in duploid cells but is hemizygous in cells having LOH. Theregions of LOH will often include a gene, the loss of which promotes unwanted proliferation, e.g., a tumor suppressor gene, and other sequences including, e.g., other genes, in some cases a gene which is essential for normal function, e.g., growth. Methods of the invention rely, in part, on the specific modulation of one allele of an essential gene with a composition of the invention.

[0496] In certain embodiments, the invention provides a double-stranded iRNA agent of the invention that modulates a micro-RNA. Targeting CNS

[0497] In some embodiments, the invention provides a double-stranded iRNA agent that targets APP for Early Onset Familial Alzheimer Disease, ATXN2 for Spinocerebellar Ataxia 2 and ALS, and C9orf72 for Amyotrophic Lateral Sclerosis and Frontotemporal Dementia.

[0498] In some embodiments, the invention provides a double-stranded iRNA agent that targets TARDBP for ALS, MAPT (Tau) for Frontotemporal Dementia, and HTT forHuntington Disease.

[0499] In some embodiments, the invention provides a double-stranded iRNA agent that targets SNCA for Parkinson Disease, FUS for ALS, ATXN3 for Spinocerebellar Ataxia 3, ATXN1 for SCA1, genes for SCA7 and SCA8, ATN1 for DRPLA, MeCP2 for XLMR, PRNP for Prion Diseases, recessive CNS disorders: Lafora Disease, DMPK for DM1 (CNS and Skeletal Muscle), and TTR for hATTR (CNS, ocular and systemic).

[0500] Spinocerebellar ataxia is an inherited brain-function disorder. Dominantly inherited forms of spinocerebellar ataxias, such as SCA1-8, are devastating disorders with no disease-modifying therapy. Exemplary targets include SCA2, SCA3, and SCA1. Targeting ATXN2 for SCA2

[0501] Spinocerebellar Ataxia 2 (SCA2), a progressive ataxia, is the second most common SCA. Another disease associated with this target is amyotrophic lateral sclerosis (ALS). These diseases are debilitating and ultimately lethal diseases with no disease- modifying therapy. The prevalence of SCA is 2-6 per 100,000 people; ATXN2 causes 15% of SCA population worldwide and much more SCA populations in some countries, especially in Cuba (40 per 100,000 people). Targeting ATXN2 can be excellent via human molecular genetics, e.g., coding CAG repeat expansion in ATXN2 was discovered in familial and sporadic SCA and ALS, in tissues such as spinal cord, brainstem, or cerebellum. Themechanism of this targeting may be because autosomal dominant coding CAG expansion of ATXN2 causes expression of toxic, misfolded protein and Purkinje cell and neuronal death. The efficacy has been shown by 70% knockdown (KD) of ATXN2 mRNA; and mATXN2 mice KD POC has been demonstrated. With respect to safety, mATXN2 knockout (KO) mice have been reported healthy. Possible diagnosis includes family history; genetic testing; or early symptoms. Biomarkers that can be used include, e.g., CSF CAG mRNA and peptide repeat proteins Targeting ATXN3 for SCA3

[0502] Spinocerebellar Ataxia 3 (SCA3), a progressive ataxia, is the most common SCA worldwide. This disease is debilitating and ultimately lethal disease with no disease- modifying therapy. It is the most common cause of SCA and the prevalence of SCA is 2-6 per 100,000 people; ATXN3 causes 21% of SCA population in US and much more in Europe, especially in Portugal. Targeting ATXN3 can be excellent via human molecular genetics, e.g., coding CAG repeat expansion in ATXN3 was discovered in familial and sporadic SCA, in tissues such as spinal cord, brainstem, or cerebellum. The mechanism of this targeting may be because autosomal dominant coding CAG expansion of ATXN3 causes expression of toxic, misfolded protein, Purkinje cell and neuron death. The efficacy has been shown by 70% KD of ATXN3 mRNA; and mATXN3 KD mice POC has been demonstrated. With respect to safety, mATXN3 KO mice have been reported healthy. Possible diagnosis includes family history; genetic testing; or early symptoms. Biomarkers that can be used include, e.g., CSF CAG mRNA and peptide repeat proteins. Targeting ATXN1 for SCA1

[0503] Spinocerebellar Ataxia 1 (SCA1), a progressive ataxia, is the first SCA gene discovered in 1993. This disease is debilitating and ultimately lethal disease with no disease- modifying therapy. The prevalence of SCA is 2-6 per 100,000 people; ATXN1 causes 6% of SCA population in US and worldwide, and much more in some countries (25% in Japan), especially in Poland (64%) and Siberia (100%). Targeting ATXN1 can be excellent via human molecular genetics, e.g., coding CAG repeat expansion in ATXN1 was discovered in familial and sporadic SCA, in tissues such as spinal cord, brainstem, or cerebellum. The mechanism of this targeting may be because autosomal dominant coding CAG expansion of ATXN1 causes expression of toxic, misfolded protein, Purkinje cell and neuronal death. Theefficacy has been shown by 70% KD of ATXN1 mRNA; and mATXN1 mice POC has been demonstrated. With respect to safety, mATXN1 KO mice have been reported healthy.Possible diagnosis includes family history; genetic testing; or early symptoms. Biomarkers that can be used include, e.g., CSF CAG mRNA and peptide repeat proteins. Targeting ATXN7 for SCA7

[0504] Spinocerebellar Ataxia 7 (SCA7) causes progressive ataxia and retinal degeneration. This disease is debilitating and ultimately lethal retinal and cerebellar disorder with no disease-modifying therapy. The prevalence of SCA is 2-6 per 100,000 people;ATXN7 causes 5% of SCA population worldwide, and much more in some countries, especially in South Africa. Targeting ATXN7 can be excellent via human molecular genetics, e.g., coding CAG repeat expansion in ATXN7 discovered in familial and sporadic SCA, in tissues such as spinal cord , brainstem, cerebellum, or retina. The mechanism of this targeting may be because autosomal dominant coding CAG expansion of ATXN1 causes expression of toxic, misfolded protein, inciting cone and rod dystrophy, Purkinje cell and neuronal lethality. The efficacy has been shown by 70% KD of ATXN1 mRNA, via intrathecal (IT) and intravitreal (IVT) administrations. Possible diagnosis includes family history; genetic testing; or early symptoms. Biomarkers that can be used include, e.g., CSF CAG mRNA and peptide repeat proteins. Targeting ATXN8 for SCA8

[0505] Spinocerebellar Ataxia 8 (SCA8), a progressive neurodegenerative ataxia is caused by CTG repeat expansion in ATXN8. This disease is debilitating and ultimately lethal disease with no disease-modifying therapy. The prevalence: SCA is 2-6 per 100,000 people; ATXN8 causes 3% of SCA population worldwide, and much more in some countries, especially in Finland. Targeting ATXN8 can be excellent via human molecular genetics, e.g., coding CTG repeat expansion in ATXN8 was discovered in familial and sporadic SCA, in tissues such as spinal cord , brainstem, or cerebellum. The mechanism of this targeting may be because autosomal dominant coding CTG expansion of ATXN8 causes expression of toxic, misfolded protein, inciting Purkinje cell and neuronal lethality. The efficacy has been shown by 70% KD of ATXN8 mRNA. Possible diagnosis includes family history; genetic testing; or early symptoms. Biomarkers that can be used include, e.g., CSF CTG mRNA and peptide repeat proteins.Targeting CACNA1A for SCA6

[0506] Spinocerebellar ataxia 6 (SCA6) is a progressive ataxia. This disease is debilitating and ultimately lethal disease with no disease-modifying therapy. The prevalence of SCA is 2-6 per 100,000 people; and CACNA1A causes 15% of SCA population worldwide. Targeting CACNA1A can be excellent via human molecular genetics, e.g., coding CAG repeat expansion in CACNA1A was discovered in familial and sporadic SCA, in tissues such as spinal cord, brainstem, or cerebellum. The mechanism of this targeting may be because autosomal dominant coding CAG expansion of CACNA1A causes expression of toxic, misfolded protein and Purkinje cell and neuronal death. The efficacy has been shown by 70% KD of CACNA1A CAG expansion. Possible diagnosis includes family history; genetic testing; or early symptoms. Biomarkers that can be used include, e.g., CSF CAG mRNA and peptide repeat proteins.

[0507] Exemplary target for inherited polyglutamine disorders includes huntington disease (HD). Targeting HTT for Huntington Disease

[0508] Huntington mutations causes HD, a progressive CNS degenerative disease. This disease is debilitating and ultimately lethal disease with no disease-modifying therapy. The prevalence of HD is 5-10 per 100,000 people worldwide, and much more common in certain countries, especially in Venezuela. Targeting HTT can be excellent via human molecular genetics, e.g., coding CAG repeat expansion in HTT discovered in familial and sporadic HD, in tissues such as striatum, or cortex. The mechanism of this targeting may be because autosomal dominant coding CAG expansion of HTT causes expression of toxic, misfolded protein and neuronal death. The efficacy has been shown by 70% KD of HTT CAG expansion only; and murine POC has been demonstrated. With respect to safety, KO of HTT in mice can be lethal; KD in humans has been demonstrated. Possible diagnosis includes family history; genetic testing; early symptoms. Biomarkers that can be used include, e.g., CSF mRNA and peptide repeat proteins. Targeting ATN1 for DRPLA

[0509] Atrophin 1 mutations causes dentatorubral-pallidoluysian atrophy (DRPLA), which is a progressive spinocerebellar disorder similar to HD. This disease is debilitating and ultimately lethal disease with no disease-modifying therapy. The prevalence of DRPLAis 2-7 per 1,000,000 people in Japan. Targeting ATN1 can be excellent via human molecular genetics, e.g., coding CAG repeat expansion in ATN1 was discovered in familial and sporadic SCA, in tissues such as spinal cord, brainstem, cerebellum, or cortex. The mechanism of this targeting may be because autosomal dominant coding CAG expansion of ATN1 causes expression of toxic, misfolded protein and neuronal death. The efficacy has been shown by 70% KD of ATN1. With respect to safety, ATN1 KO mice have been reported healthy. Possible diagnosis includes family history; genetic testing; or early symptoms. Biomarkers that can be used include, e.g., CSF CAG mRNA and peptide repeat proteins. Targeting AR for Spinal and Bulbar Muscular Atrophy

[0510] Androgen receptor mutations causes spinal and bulbar muscular atrophy (SBMA, Kennedy disease), a progressive muscle wasting disease, and other diseases. This disease is debilitating and ultimately lethal disease with no disease-modifying therapy. The prevalence of SBMA is 2 per 100,000 males; females have a mild phenotype. Targeting AR can be excellent via human molecular genetics, e.g., coding CAG repeat expansion in AR discovered in familial SBMA, in tissues such as spinal cord, or brainstem. The mechanism of this targeting may be because X-linked coding CAG expansion of AR causes toxic gain-or- function and motor neuron lethality. The efficacy has been shown by 70% KD of AR.Possible diagnosis includes family history; genetic testing; or early symptoms. Biomarkers that can be used include, e.g., CSF CAG mRNA and peptide repeat proteins. Targeting FXN for Friedrich Ataxia

[0511] Recessive loss of function GAA expansion of FXN causes friedrich ataxia (FA), a progressive degenerative ataxia. This disease is debilitating and ultimately lethal disease with no disease-modifying therapy. The prevalence of FA is 2 per 100,000 people worldwide. Targeting FXN can be excellent via human molecular genetics, e.g., intron GAA repeat expansion in FXN was discovered in familial FA, in tissues such as spinal cord, cerebellum, or perhaps retina and heart. The mechanism of this targeting may be because autosomal recessive non-coding FAA expansion of FXN causes deceased expression of FXN, an important mitochondrial protein. The efficacy has been shown by 70% KD of FXN intron GAS expansion. With respect to safety, KD of intron GAA is safe and effective in mice.Possible diagnosis includes family history; genetic testing; or early symptoms. Biomarkers that can be used include, e.g., CSF mRNA and peptide repeat proteins. Targeting FMR1 for FXTAS

[0512] Fragile X-associated tremor / ataxia syndrome (FXTAS), a progressive disorder of ataxia and cognitive loss in adults caused by FMR1 overexpression. This disease is debilitating disease with no disease-modifying therapy. The prevalence of FMR1permutation is 1 in 500 males. Targeting FMR1 can be excellent via human molecular genetics, e.g., coding CCG repeat expansion pre-mutations in FMR1 was discovered in FXTAS, in tissues such as spinal cord, cerebellum, or cortex. The mechanism of this targeting may be because X-linked coding CCG expansion of FMR1 causes toxic mRNA. The efficacy has been shown by 70% KD of toxic mRNA. Possible diagnosis includes family history; genetic testing; or early symptoms. Biomarkers that can be used include, e.g., CSF mRNA and peptide repeat proteins. Targeting upstream of FMR1 for Fragile X Syndrome

[0513] Fragile X syndrome (FRAXA), a progressive disorder of mental retardation, may be treated by targeting upstream mRNA of FMR1. This disease is debilitating disease with no disease-modifying therapy. The prevalence of FRAXA is 1 per 4,000 males and 1 per 8,000 females. Targeting FMR1 can be excellent via human molecular genetics, e.g., coding CCG repeat expansion in FMR1 was discovered in FRAXA, in tissues such as CNS. The mechanism of this targeting may be because X-linked coding CCG expansion of FMR1 causes LOF; and normal FMR1 functions to transport specific mRNAs from nucleus. The efficacy has been shown by 70% KD of toxic mRNA. Possible diagnosis includes family history; genetic testing; or early symptoms. Biomarkers that can be used include, e.g., CSF mRNA and peptide repeat proteins.

[0514] Dominant Inherited Amyotrophic Lateral Sclerosis is a devastating disorders with no disease-modifying therapy. Exemplary targets include C9orf72, ATXN2 (also causes SCA2), and MAPT. Targeting C9orf72 for ALS

[0515] C9orf72 is the most common cause of Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD). These diseases are lethal disorders of motor neurons withno disease-modifying therapy. The prevalence of ALS is 2-5 per 100,000 people (10% is familial); C9orf72 causes 39% of familial ALS in US and Europe and 7% of sporadic ALS. Targeting C9orf72 can be excellent via human molecular genetics, e.g., hexa-nucleotide expansion was discovered in familial and sporadic ALS, in tissues such as upper and lower motor neurons (for ALS); or cortex (for FTD). The mechanism of this targeting may be because autosomal dominant hexa-nucleotide expansion causes repeat-associated non-AUG- dependent translation of toxic dipeptide repeat proteins and neuron lethality. The efficacy has been shown by 70% KD of C9orf72. With respect to safety, heterozygous LOF mutations of C9orf72 appear to be safe in humans and mice. Possible diagnosis includes family history; genetic testing; or early symptoms. Biomarkers that can be used include, e.g., CSF hexa-nucleotide repeat mRNAs and dipeptide repeat proteins. Targeting TARDBP for ALS

[0516] TARDBP mutations causes ALS and Frontotemporal Dementia (FTD). These deseases are lethal disorders of motor neurons with no disease-modifying therapy. The prevalence of ALS is 2-5 per 100,000 people (10% is familial); TARDBP causes 5% of familial ALS and 1.5% of sporadic ALS. Targeting TARDBP can be excellent via human molecular genetics, e.g., mutations were discovered in familial and sporadic ALS, in tissues such as upper and lower motor neurons (for ALS); or cortex (for FTD). The mechanism of this targeting may be because autosomal dominant TRDBP mutations cause toxic TRDBP protein and neuron lethality. The efficacy has been shown by 70% KD of TARDBP mutant alleles. Possible diagnosis includes family history; genetic testing; or early symptoms.Biomarkers that can be used include, e.g., CSF proteins. Targeting FUS for ALS

[0517] FUS mutations causes ALS and FTD. These diseases are lethal disorder of motor neurons with no disease-modifying therapy. The prevalence of ALS is 2-5 per 100,000 people (10% is familial); FUS causes 5% of familial ALS; FUS inclusions are often found in sporadic ALS. Targeting FUS can be excellent via human molecular genetics, e.g., mutations were discovered in familial ALS, in tissues such as upper and lower motor neurons for ALS. The mechanism of this targeting may be because autosomal dominant FUS mutations cause abnormal protein folding and neuron lethality. The efficacy has been shown by 70% KD of FUS mutant alleles. With respect to safety, KO mice struggle but survive and have anADHD phenotype. Possible diagnosis includes family history; genetic testing; or early symptoms. Biomarkers that can be used include, e.g., CSF proteins. Targeting SOD1 for ALS

[0518] Dominant and recessive mutations of SOD1 cause ALS. This disease is lethal disorder of motor neurons with no disease-modifying therapy. The prevalence of ALS is 2-5 per 100,000 people (10% is familial); SOD1 causes5-20% of familial ALS. Target SOD1 can be excellent via human molecular genetics, e.g., many SOD1 mutations associate with AD and AR ALS in families, in tissues such as upper and lower motor neurons for ALS. The efficacy of this targeting may need mutation-specific KD. Possible diagnosis includes family history; genetic testing; or early symptoms. Biomarkers may be mutation-specific.

[0519] Dominant Inherited Frontotemporal Dementia and Progressive Supra-nuclear Palsy. The targets include MAPT because it may be important for AD, or C9orf72. Targeting Microtubule-associated protein Tau for FTD-17 and PSP

[0520] Familial Frontotemporal Dementia 17 (FTD-17), a familial form of FTD lined to chromosome 17, and Familial Progressive Supra-nuclear Palsy may be caused by MAPT mutations, which may also cause rare forms of Progressive Supra-nuclear Palsy, Corticobasal Degeneration, Tauopathy with Respiratory Failure, Dementia with Seizures. These diseases are lethal neurodegenerative disorders with no disease-modifying therapy. The prevalence of FTD is 15-22 per 100,000 people; the prevalence of FTD-17 in Netherlands is 1 in 1,000,000 population. Targeting MAPT can be excellent via human molecular genetics, e.g., GOF point and splice site mutations of MAPT were discovered in familial and sporadic FTD, in tissues such as frontal or temporal cortex. The mechanism of this targeting may be because autosomal dominant GOF mutations of MAPT lead to toxic Tau peptides and neuronal death. The efficacy has been shown by 70% KD of MAPT. With respect to safety, MAPT KO mice have been reported healthy. Possible diagnosis includes family history; genetic testing; early symptoms. Biomarkers that can be used include, e.g., CSF Tau mRNAs and proteins. Targeting Sequestosome 1 for FTD and ALS

[0521] Sporadic FTD / ALS associate with dominant SQSTM1 mutations. This disease is lethal neurodegenerative disorder with no disease-modifying therapy. This is a very rare disease. Targeting Sequestosome 1 is reasonable via human molecular genetic association insporadic cases, in tissues such as frontal and temporal cortex, or cerebellum and spinal cord. Possible diagnosis includes genetic testing; early symptoms.

[0522] Dominant Inherited Parkinson Disease is a devastating disorders with no disease- modifying therapy. The targets include SNCA. Targeting SNCA for Parkinson Disease

[0523] Alpha Synuclein mutations causes familial Parkinson disease (PD) and Lewy body dementia. These diseases are lethal neurodegenerative disorders with no disease- modifying therapy. The prevalence of PD is 4 million worldwide; 1 / 3 of PD is familial; 1% of fPD is caused by SNCA. Targeting SNCA can be excellent via human molecular genetics, e.g., SNCA point mutations and duplications cause familial PD, in tissues such as medulla oblongata; or substantia nigra of the midbrain. The mechanism of this targeting may be because overexpression or expression of abnormal SNCA protein leads to toxic peptides and neuronal death. The efficacy has been shown by 70% KD of SNCA. With respect to safety, SNCA KO mice are healthy. Possible diagnosis includes family history; genetic testing; or early symptoms. Biomarkers that can be used include, e.g., CSF SNCA mRNAs and proteins. Targeting LRRK2 for Parkinson Disease

[0524] Leucine-rich repeat kinase 2 mutations causes familial Parkinson disease. This disease is lethal neurodegenerative disorder with no disease-modifying therapy. The prevalence of PD is 4 million worldwide; 1 / 3 of PD is familial; 3-7% of fPD is caused by LRRK2. Targeting LRRK2 can be excellent via human molecular genetics, e.g., LRRK2 point mutations cause familial PD, in tissues such as medulla oblongata; or substantia nigra of the midbrain. Possible diagnosis includes family history; genetic testing; early symptoms. Biomarkers that can be used include, e.g.,CSF mRNAs and proteins. Targeting GARS for Spinal Muscular Atrophy V

[0525] Autosomal dominant Glycyl-tRNA Synthetase mutations causes spinal muscular atrophy V (SMAV) or distal hereditary motor neuropathy Va. These diseases areneurodegenerative disorders with no disease-modifying therapy. These are very rare diseases. Targeting GARs can be good via human molecular genetics, e.g., GARS pointmutations cause familial SMA, in tissues such as spinal cord. Possible diagnosis includes family history; genetic testing; early symptoms. Targeting Seipin for spinal Muscular Atrophy

[0526] Autosomal dominant Seipin mutations causes spinal muscular atrophy (SMA) or distal hereditary motor neuropathy. These diseases are neurodegenerative disorders with no disease-modifying therapy. These are very rare diseases. Targeting Seipin can be good via human molecular genetics, e.g., Seipin point mutations cause familial SMA, in tissues such as spinal cord. The mechanism of this targeting is probably GOF and toxic peptides. The efficacy has been shown by 50% KD. With respect to safety, recessive LOF mutations cause progressive encephalopathy with or without lipodystrophy. Possible diagnosis includes family history; genetic testing; or early symptoms.

[0527] Dominant Inherited Alzheimer Disease is a devastating disorders with no disease- modifying therapy. The targets include APP because of central mechanistic role in familial disease and possible role in common AD. Targeting APP for Alzheimer Disease

[0528] Amyloid precursor protein mutations causes early onset familial Alzheimer disease (EOFAD); AD in down syndrome; or AD. These diseases are lethalneurodegenerative disorders with no disease-modifying therapy. The prevalence of EOFAD- APP is 1% AD; the prevalence of Trisomy 21 is 1% AD; and the prevalence of AD is about 2.5-5 million in US. Targeting APP can be excellent via human molecular genetics, e.g., APP duplications and point mutations cause EOFAD, in tissues such as cerebral cortex or hippocampus. The mechanism of this targeting may be because APP overexpression or expression of toxic metabolites cause progressive neuronal death. The efficacy has been shown by 70% KD of APP. With respect to safety, KD mice have been reported healthy with some behaviora...

Claims

We claim:

1. A double-stranded iRNA agent comprising:an antisense strand which is complementary to a target gene; a sense strand which is complementary to said antisense strand; and one or more lipophilic moieties conjugated to one or more internal positions on at least one strand, optionally via a linker or carrier.

2. The double-stranded iRNA agent of claim 1, wherein the lipophilicity of the lipophilic moiety, measured by logKow, exceeds 0.

3. The double-stranded iRNA agent of claim 1, wherein the hydrophobicity of the double-stranded iRNA agent, measured by the unbound fraction in the plasma protein binding assay of the double-stranded iRNA agent, exceeds 0.

2.

4. The double-stranded iRNA agent of claim 3, wherein the plasma protein binding assay is an electrophoretic mobility shift assay using human serum albumin protein.

5. The double-stranded iRNA agent of claim 1, wherein the internal positions include all positions except terminal two positions from each end of the strand.

6. The double-stranded iRNA agent of claim 5, wherein the internal positions include all positions except terminal three positions from each end of the strand.

7. The double-stranded iRNA agent of claim 5 or 6, wherein the internal positions exclude the cleavage site region of the sense strand.

8. The double-stranded iRNA agent of claim 7, wherein the internal positions exclude positions 9-12, counting from the 5’-end of the sense strand.

9. The double-stranded iRNA agent of claim 7, wherein the internal positions exclude positions 11-13, counting from the 3’-end of the sense strand.

10. The double-stranded iRNA agent of claim 5 or 6, wherein the internal positions exclude the cleavage site region of the antisense strand.

11. The double-stranded iRNA agent of claim 10, wherein the internal positions exclude positions 12-14, counting from the 5’-end of the antisense strand.

12. The double-stranded iRNA agent of claim 5 or 6, wherein the internal positions excluding positions 11-13 on the sense strand, counting from the 3’-end, and positions 12-14 on the antisense strand, counting from the 5’-end.

13. The double-stranded iRNA agent of claim 1, wherein one or more lipophilic moieties are conjugated to one or more of the following internal positions: positions 4-8 and 13-18 on the sense strand, and positions 6-10 and 15-18 on the antisense strand, counting from the 5’end of each strand.

14. The double-stranded iRNA agent of claim 13, wherein one or more lipophilic moieties are conjugated to one or more of the following internal positions: positions 5, 6, 7, 15, and 17 on the sense strand, and positions 15 and 17 on the antisense strand, counting from the 5’-end of each strand.

15. The double-stranded iRNA agent of any one of claims 1-14, wherein said sense and antisense strands are each 15 to 30 nucleotides in length.

16. The double-stranded iRNA agent of any one of claims 1-14, wherein said sense and antisense strands are each 19 to 25 nucleotides in length.

17. The double-stranded iRNA agent of any one of claims 1-14, wherein said sense and antisense strands are each 21 to 23 nucleotides in length.

18. The double-stranded iRNA agent of claim 17, wherein the sense strand is 21- nucleotides in length, and the antisense strands are 23-nucleotides in length, wherein the strands form a double-stranded region of 21 consecutive base pairs having a 2-nucleotide long single-stranded overhangs at the 3’-end.

19. The double-stranded iRNA agent of claim 1, wherein the lipophilic moiety is an aliphatic, alicyclic, or polyalicyclic compound.

20. The double-stranded iRNA agent of claim 19, wherein the lipophilic moiety is lipid, cholesterol, retinoic acid, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol,geranyloxyhexyanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3- (oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.

21. The double-stranded iRNA agent of claim 19, wherein the lipophilic moietycontains a saturated or unsaturated C4-C30 hydrocarbon chain, and an optional functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.

22. The double-stranded iRNA agent of claim 21, wherein the lipophilic moietycontains a saturated or unsaturated C6-C18hydrocarbon chain.

23. The double-stranded iRNA agent of claim 22, wherein the lipophilic moietycontains a saturated or unsaturated C16hydrocarbon chain.

24. The double-stranded iRNA agent of any one of claims 1-23, wherein the lipophilic moiety is conjugated via a carrier that replaces one or more nucleotide(s) in the internal position(s).

25. The double-stranded iRNA agent of claim 24, wherein the carrier is a cyclic group selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl; or is an acyclic moiety based on a serinol backbone or a diethanolamine backbone.

26. The double-stranded iRNA agent of any one of claims 1-25, wherein the lipophilic moiety is conjugated to the double-stranded iRNA agent via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, a product of a click reaction, or carbamate.

27. The double-stranded iRNA agent of any one of claims 1-26, wherein said iRNA agent comprises a single-stranded overhang on at least one of the termini.

28. The double-stranded iRNA agent of claim 27, wherein said single-strandedoverhang is 1, 2 or 3 nucleotides in length.

29. The double-stranded iRNA agent of any one of claims 1-28, wherein the lipophilic moiety is conjugated to a nucleobase, sugar moiety, or internucleosidic linkage.

30. The double-stranded iRNA agent of any one of claims 1-29, further comprising a phosphate or phosphate mimic at the 5’-end of the antisense strand.

31. The double-stranded iRNA agent of claim 30, wherein the phosphate mimic is a 5’-vinyl phosphonate (VP).

32. The double-stranded iRNA agent of any one of claims 1-31, further comprising a targeting ligand that targets a receptor which mediates delivery to a CNS tissue.

33. The double-stranded iRNA agent of claim 32, wherein the targeting ligand is selected from the group consisting of Angiopep-2, lipoprotein receptor related protein (LRP) ligand, bEnd.3 cell binding ligand, transferrin receptor (TfR) ligand, manose receptor ligand, glucose transporter protein, and LDL receptor ligand.

34. The double-stranded iRNA agent of any one of claims 1-31, further comprising a targeting ligand that targets a receptor which mediates delivery to an ocular tissue.

35. The double-stranded iRNA agent of claim 34, wherein the targeting ligand is selected from the group consisting of trans-retinol, RGD peptide, LDL receptor ligand, and carbohydrate based ligands.

36. The double-stranded iRNA agent of claim 35, wherein the RGD peptide is H-Gly- Arg-Gly-Asp-Ser-Pro-Lys-Cys-OH or Cyclo(-Arg-Gly-Asp-D-Phe-Cys).

37. The double-stranded iRNA agent of any one of claims 1-36, further comprising a targeting ligand that targets a liver tissue.

38. The double-stranded iRNA agent of claim 37, wherein the targeting ligand is a GalNAc conjugate.

39. The double-stranded iRNA agent of any one of claims 1-38, wherein the lipophilic moeity or targeting ligand is conjugated via a bio-clevable linker selected from the group consisting of DNA, RNA, disulfide, amide, funtionalized monosaccharides or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

40. The double stranded iRNA agent of any one of claims 1-39, wherein the 3’ end of the sense strand is protected via an end cap which is a cyclic group having an amine, said cyclic group being selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl.

41. A method of reducing the expression of a target gene in a cell, comprisingcontacting said cell with a double-stranded iRNA agent comprising:an antisense strand which is complementary to a target gene;a sense strand which is complementary to said antisense strand; and one or more lipophilic moieties conjugated to one or more internal positions on at least one strand, optionally via a linker or carrier.

42. The method of claim 41, wherein the cell is an extraheptic cell.

43. The method of claim 41, wherein the lipophilicity of the lipophilic moiety,measured by logKow, exceeds 0.

44. The method of claim 41, wherein the hydrophobicity of the double-stranded iRNA agent, measured by the unbound fraction in the plasma protein binding assay of the double-stranded iRNA agent, exceeds 0.

2.

45. The method of claim 44, wherein the plasma protein binding assay is anelectrophoretic mobility shift assay using human serum albumin protein.

46. The method of claim 41, wherein the lipophilic moiety contains a saturated or unsaturated C16 hydrocarbon chain.

47. A method of reducing the expression of a target gene in a subject, comprising administering to the subject a double-stranded iRNA agent comprising:an antisense strand which is complementary to a target gene;a sense strand which is complementary to said antisense strand; and one or more lipophilic moieties conjugated to one or more internal positions on at least one strand, optionally via a linker or carrier.

48. The method of claim 47, wherein the double-stranded iRNA agent is administered extrahepatically.

49. The method of claim 48, wherein the double-stranded iRNA agent is administered intrathecally.

50. The method of claim 49, wherein the method reduces the expression of a target gene in a brain or spine tissue.

51. The method of claim 50, wherein the brain or spine tissue is selected from the group consisting of cortex, cerebellum, cervical spine, lumbar spine, and thoracic spine.

52. The method of claim 48, wherein the target gene is selected from the groupconsisiting of APP, ATXN2, C9orf72, TARDBP, MAPT(Tau), HTT, SNCA, FUS, ATXN3, ATXN1, SCA1, SCA7, SCA8, MeCP2, PRNP, SOD1, DMPK, and TTR.

53. The method of claim 52, wherein the double-stranded iRNA agent is administered intravitreally.

54. The method of claim 53, wherein the method reduces the expression of a target gene in an ocular tissue.

55. A method of treating a subject having a CNS disorder, comprising:administering to the subject a therapeutically effective amount of the double- stranded RNAi agent of any one of claims 1-40, thereby treating the subject.

56. The method of claim 55, wherein the CNS disorder is selected from the group of alzheimer, amyotrophic lateral schlerosis (ALS), frontotemporal dementia, huntington, Parkinson, spinocerebellar, prion, and lafora.

Images