Extrahepatic delivery

Abstract

To provide 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.SOLUTION: 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 lipophilic moieties-conjugated double-stranded iRNAs at one or more internal positions on at least one strand, optionally via a linker or carrier.SELECTED DRAWING: None

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 668,072, filed May 7, 2018; U.S. Provisional Patent Application No. 62 / 738,747, filed September 28, 2018; and U.S. Provisional Patent Application No. 62 / 773,082, filed November 29, 2018, the entire contents of each of which are incorporated herein by reference. [Background technology]

[0002] Efficient delivery of iRNA agents to cells in vivo requires specific targeting and substantial protection from extracellular environment, especially serum proteins.RNAi-based therapy has shown promising clinical data for the treatment of liver-related diseases.However, the delivery of siRNA to extrahepatic tissues remains an obstacle, limiting the use of siRNA-based therapy.

[0003] One factor limiting the experimental and therapeutic application of iRNA agents in vivo is the ability to efficiently deliver intact siRNA. Particular difficulties relate to non-viral gene transfer to the retina in vivo. One of the challenges is overcoming the internal limiting membrane, which prevents retinal transfection. Furthermore, negatively charged sugars in the vitreous have been shown to interact with cationic DNA-transfection reagent complexes, promoting their aggregation and hindering diffusion and cellular uptake.

[0004] The delivery of oligonucleotides into the central nervous system (CNS) presents particular challenges due to the blood-brain barrier (BBB), which free oligonucleotides cannot cross. One method for delivering oligonucleotides into the CNS is by intrathecal delivery. However, oligonucleotides also need to be efficiently taken up by target cells in the CNS to achieve the desired therapeutic effect. Previous studies have typically used delivery agents such as liposomes, cationic lipids, and nanoparticle-forming complexes to facilitate the intracellular uptake of oligonucleotides into neuronal-derived cells. Summary of the Invention [Problem to be solved by the invention]

[0005] Thus, there remains a 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. [Means for solving the problem]

[0006] One aspect of the invention provides a double-stranded iRNA agent that includes an antisense strand complementary to a target gene; a sense strand complementary to the 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 octanol-water partition coefficient, log K ow The lipophilic moiety has a log K of greater than 1, greater than 1.5, greater than 2, greater than 3, greater than 4, greater than 5, or greater than 10. ow may have:

[0008] In some embodiments, the hydrophobicity of the double-stranded iRNA agent, as measured by the unbound fraction in a plasma protein binding assay of the double-stranded iRNA agent, is greater than 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, as measured by the fraction of unbound siRNA in a binding assay, is greater than 0.15, greater than 0.2, greater than 0.25, greater than 0.3, greater than 0.35, greater than 0.4, greater than 0.45, or greater than 0.5 for improved in vivo delivery of siRNA.

[0009] In some embodiments, the lipophilic moiety is aliphatic, cyclic, e.g., alicyclic, or polycyclic, e.g., polyalicyclic, such as a steroid (e.g., a sterol) or a straight-chain or branched-chain aliphatic hydrocarbon. Exemplary lipophilic moieties are lipids, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexanol, 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 include saturated or unsaturated C4-C 30 Hydrocarbon chains (e.g., C4 to C 30 Also included are those containing a saturated or unsaturated C6-C8 functional group (e.g., alkyl or alkenyl), and an optional functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne. This functional group is useful for attaching a lipophilic moiety to an iRNA agent. In some embodiments, the lipophilic moiety is a saturated or unsaturated C6-C8 functional group. 18 Hydrocarbon chains (e.g., straight chain C6-C 18 In one embodiment, the lipophilic moiety contains a saturated or unsaturated C 16 Hydrocarbon chains (e.g., linear C 16 alkyl or alkenyl).

[0011] In some embodiments, the lipophilic moiety is a C-C 30 Acids (e.g., hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, dodecanoic 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 C6 to C30 Alcohols (e.g., hexanol, heptanol, octanol, nonanol, decanol, undecanol, dodecanol, tridecanol, tetradecanol, pentadecanol, hexadecanol, heptadecanol, octadecanol, oleyl alcohol, linoleyl alcohol, arachidonic acid alcohol, cis-4,7,10,13,16,19-docosahexanol, retinol, vitamin E, cholesterol, etc.).

[0012] The lipophilic moiety can be conjugated to the iRNA agent via a direct bond to the iRNA agent's ribosugar. Alternatively, the lipophilic moiety can be conjugated to the iRNA agent via a linker or carrier.

[0013] In certain embodiments, the lipophilic moiety is 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 that includes an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide bond, the product of a click reaction (e.g., a triazole from an azide-alkyne cycloaddition), or a carbamate.

[0015] In some embodiments, at least one of the linkers (tethers) is a redox-cleavable linker (a reductively cleavable linker; e.g., a disulfide group), an acidic 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 biocleavable linker selected from the group consisting of DNA, RNA, disulfides, amides, functionalized mono- 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 nucleotides.The carrier can be a cyclic or 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 skeleton or a diethanolamine skeleton.

[0018] In some embodiments, the carrier replaces one or more nucleotides at an internal position of the double-stranded iRNA agent.

[0019] In other embodiments, carrier replaces the nucleotide at the end of sense strand or antisense strand.In one embodiment, carrier replaces the terminal nucleotide at the 3' end of sense strand, thereby functioning as the end cap that protects the 3' end of sense strand.In one embodiment, carrier is a cyclic group that has amine, for example, carrier can 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 moieties are conjugated to one or more internal positions on at least one chain, including all but the two most terminal positions on each chain. In one embodiment, the lipophilic moieties are conjugated to one or more internal positions on at least one chain, including all but the three most terminal positions on each chain.

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

[0022] In one embodiment, at least one lipophilic moiety is conjugated to 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 to 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 to 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 to 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 to the sense strand within 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, excluding the cleavage site region of the sense strand. For example, internal positions exclude positions 9-12, counting from the 5' end of the sense strand. For example, internal positions exclude positions 9-11, counting from the 5' end of the sense strand. Alternatively, 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, excluding the cleavage site region of the antisense strand, e.g., internal positions excluding positions 12-14 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, 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.

[0030] In one embodiment, the 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, the 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 a double-stranded iRNA agent are each 15-30 nucleotides in length.

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

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

[0035] In some embodiments, the double-stranded iRNA agent includes a single-stranded overhang on at least one of its ends, e.g., a 3' and / or 5' overhang 1-10 nucleotides in length, e.g., 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 includes 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, where the strands form a double-stranded region of 21 contiguous base pairs with a 2-nucleotide-long single-stranded overhang at the 3' end.

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

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

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

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

[0041] Site-specific chiral modifications of internucleotide bonds can occur at the 5'-end, 3'-end, or both the 5'-end and 3'-end of the strand. This is referred to herein as "terminal" chiral modification. Terminal modifications can occur at the 3'- or 5'-end position in the terminal region, for example, at the terminal nucleotide or within the last 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of the strand. Chiral modifications can occur in the sense strand, the antisense strand, or both the sense strand and the antisense strand. Each chirally pure phosphorus atom can be in either the Rp or Sp configuration, and combinations thereof. Further details regarding chiral modifications and chiral modified dsRNA agents can be found in PCT / US18 / 67103, filed December 21, 2018, entitled "Chirally Modified Double-Stranded RNA Agents," the entire contents of which are incorporated herein by reference.

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

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

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

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

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

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

[0048] In some embodiments, the antisense strand comprises two blocks of 1, 2, or 3 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 that mediates delivery to a specific central nervous system 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, mannose 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 that mediates delivery to 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 ligand. In one embodiment, the targeting ligand is an 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 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 of the above aspects and embodiments can be applied to the oligonucleotide that has one or more lipophilic moieties conjugated to one or more internal positions on oligonucleotide.In some embodiments, 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35% or 30% of oligonucleotide is modified.For example, when 50% of oligonucleotide is modified, 50% of all nucleotides present in oligonucleotide will comprise the modification described herein.

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

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

[0055] In some embodiments, the double-stranded iRNA agent has fewer than 12, fewer than 10, fewer than 8, fewer than 6, fewer than 4, fewer than 2, or no 2'-F modifications on the sense strand. In some embodiments, the double-stranded iRNA agent has fewer than 12, fewer than 10, fewer than 8, fewer than 6, fewer than 4, fewer 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 anywhere in the sense 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%, or is substantially free of non-natural nucleotides. Examples of non-natural nucleotides 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'-ON-methylacetamide (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), 2'-ara-F, L-nucleoside modifications (2'-modified L-nucleosides, such as 2'-deoxy-L-nucleosides), BNA abasic sugars, abasic cyclic and open-chain alkyls.

[0058] In some embodiments, the double-stranded iRNA agent has more than 80%, more than 85%, more than 90%, more than 95%, or substantially 100% naturally occurring nucleotides. For the purposes of these embodiments, naturally occurring nucleotides can include those with 2'-OH, 2'-deoxy, and 2'-OMe.

[0059] In one embodiment, the double-stranded iRNA agent has sense and antisense strands each having a length of 15-30 nucleotides; includes at least two phosphorothioate internucleotide linkages in the first five nucleotides on the antisense strand (counting from the 5' end); wherein the duplex region is 19-25 base pairs (preferably 19, 20, 21, or 22); and wherein the double-stranded iRNA agent has less than 20%, less than 15%, less than 10%, less than 5% natural nucleotides, or is substantially free of non-natural nucleotides.

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

[0061] Another aspect of the invention relates to a method of reducing expression of a target gene in a cell, the method comprising contacting the cell with a double-stranded iRNA agent comprising: an antisense strand complementary to the target gene; a sense strand complementary to the 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 of the above embodiments relating to lipophilic moieties and their conjugation to double-stranded iRNA agents in the first aspect of the invention relating to double-stranded iRNA agents are suitable for this aspect of the invention relating to methods of reducing expression of a target gene in a cell.

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

[0064] Another aspect of the invention relates to a method of reducing expression of a target gene in a subject comprising administering to a subject a double-stranded iRNA agent comprising contacting said cell with a double-stranded iRNA agent comprising: an antisense strand complementary to the target gene; a sense strand complementary to the 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 of the above embodiments relating to lipophilic moieties and their conjugation to double-stranded iRNA agents in the first aspect of the invention relating to double-stranded iRNA agents are suitable for this aspect of the invention relating to a method of reducing 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 administering the double-stranded iRNA agent intrathecally, the method can reduce the expression of target genes in brain or spinal tissue, for example, the cerebral cortex, cerebellum, cervical vertebrae, lumbar vertebrae, and thoracic vertebrae.

[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 subject, double-stranded iRNA agent can be administered intravitreally.By administering double-stranded iRNA agent intravitreally, this method can reduce the expression of target gene in eye tissue.

[0069] Another aspect of the present invention relates to a method for treating a subject suffering from a central nervous system disease, 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 complementary to a target gene, a sense strand complementary to the 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 of the above embodiments relating to lipophilic moieties and their conjugation to double-stranded iRNA agents in the first aspect of the invention relating to double-stranded iRNA agents are suitable for this aspect of the invention relating to methods of treating a subject suffering from a central nervous system disease. Exemplary central nervous system diseases that can be treated by the methods of the invention include Alzheimer's disease, amyotrophic lateral sclerosis (ALS), frontotemporal dementia, Huntington's disease, Parkinson's disease, spinocerebellar ataxia, prion diseases, and Lafora's disease. [Brief explanation of the drawings]

[0071] [Figure 1] 1 is a scheme showing a ligand, such as a lipophilic moiety, conjugated to an siRNA at an internal position (ie, anywhere within the siRNA sequence) of either the sense or antisense strand. [Figure 2] 1 is a scheme showing a ligand, such as a lipophilic moiety, conjugated to an siRNA via a linker or carrier at the 3' and / or 5' end of the sense or antisense strand. [Figure 3] 1 is a scheme showing a ligand, such as a lipophilic moiety, conjugated to an siRNA via a biocleavable linker. [Figure 4] 1 is a graph showing the results of beta-catenin gene (intraocular CTNNB1) silencing by intravitreal injection of various exemplary siRNA conjugates in mice. [Figure 5]1 is a graph showing the results of SOD1 mRNA silencing in the cerebral cortex of Sprague-Dawley rats by a single intrathecal injection of various exemplary siRNA conjugates. [Figure 6] 1 is a graph showing the results of SOD1 mRNA silencing in the cerebellum of Sprague-Dawley rats by a single intrathecal injection of various exemplary siRNA conjugates. [Figure 7] 1 is a graph showing the results of SOD1 mRNA silencing by a single intrathecal injection of various exemplary siRNA conjugates in the cervical spine of Sprague-Dawley rats. [Figure 8] 1 is a graph showing the results of SOD1 mRNA silencing in the lumbar spine of Sprague-Dawley rats by a single intrathecal injection of various exemplary siRNA conjugates. [Figure 9] 1 is a graph showing the results of SOD1 mRNA silencing by a single intrathecal injection of various exemplary siRNA conjugates in the thoracic spine of Sprague-Dawley rats. [Figure 10] 1 shows the results of primary cynomolgus monkey hepatocyte (PCH) spontaneous uptake (without transfection agent) for cells incubated with F12 siRNA modified by conjugating a lipophilic moiety (C16) at each position of the antisense and sense strands at concentrations of 2.5 and 250 nM, by measuring F12 mRNA levels after 24 hours using RT-qPCR. [Figure 11] 1 shows the results of primary cynomolgus monkey hepatocyte (PCH) spontaneous uptake (without transfection agent) for cells incubated with F12 siRNA modified by conjugating a lipophilic moiety (C16) at each position of the antisense and sense strands at concentrations of 2.5 and 250 nM, by measuring F12 mRNA levels after 24 hours using RT-qPCR. [Figure 12]Figure 1 shows the relative hydrophobicity results for each position of the antisense and sense strands of siRNA duplexes modified by conjugating a lipophilic moiety (C16) at each position in the antisense and sense strands, as determined by measuring the unbound fraction using an electrophoretic mobility shift assay after incubating each siRNA conjugate with human serum albumin. [Figure 13-1] Figure 13A shows that sustained SOD1 mRNA silencing is observed in all brain and spinal cord regions tested. Figure 13A shows the results of SOD1 mRNA silencing by single intrathecal injection of various exemplary siRNA conjugates in rats in each of lumbar, thoracic and cervical spinal regions. Figure 13B shows the various tissues tested in rat central nervous system. [Figure 13-2] Figure 13C shows that sustained SOD1 mRNA silencing is observed in all brain and spinal cord regions tested. Figure 13C shows the results of SOD1 mRNA silencing by single intrathecal injection of various exemplary siRNA conjugates in rats in each of the cerebellum, frontal lobe and remaining brain regions. [Figure 14] Figure 14A shows the beta-catenin silencing results after single intrathecal administration.Figure 14B shows the beta-catenin silencing results of various exemplary siRNA conjugates in rats in lumbar spine, thoracic spine and cervical spine regions.Figure 14B shows the beta-catenin silencing results of various exemplary siRNA conjugates in rats in cerebellum, frontal lobe and remaining brain regions. [Figure 15]Figure 15 shows the results of SOD1 silencing in rats after a single intrathecal administration of exemplary siRNA duplex, showing that higher drug levels and robust silencing are observed in the brain using SOD1 siRNA conjugate. Figure 15A shows the conjugate siRNA level in CSF compared with that of non-conjugated siRNA. Figure 15B shows the conjugated siRNA level in brain compared with that of non-conjugated siRNA. Figure 15C shows the conjugated siRNA level in cerebellum compared with that of non-conjugated siRNA and control siRNA. [Figure 16] Figure 16 shows the results of SOD1 silencing by various chemical modifications at various doses. Figure 16A shows the results of SOD1 silencing in rats in the lumbar spine, thoracic spine, and cervical spine regions, respectively. Figure 16B shows the results of SOD1 silencing in rats in the cerebellum, frontal lobe, and remaining brain regions, respectively. [Figure 17] 1 shows the results of β-catenin siRNA levels following a single intrathecal (IT) administration of exemplary siRNA duplexes in various regions of non-human primates (NHPs) at 31 days. [Figure 18] Results show robust gene silencing of β-catenin mRNA in various tissues at 31 days. [Figure 19] Photographs showing siRNA distribution throughout the central nervous system of NHPs after a single IT administration are shown. [Figure 20] Photographs showing siRNA conjugates localized to neurons after a single IT administration. MAP2 is a neuronal marker. [Figure 21] Photographs showing siRNA conjugates localized to microglia after a single IT administration. Iba1 is a microglial marker. [Figure 22] Photographs showing siRNA conjugates localized to astrocytes after a single IT administration are shown. [Figure 23]Graphs comparing gene silencing activity observed in rats and NHPs at compartment scaled doses are shown. [Figure 24] 1 shows the results of TTR mRNA levels in mouse eyes at day 14 after administration of various exemplary siRNA duplexes shown in Table 7 at doses of 3 μg or 7.5 μg. [Figure 25] 1 shows the results of TTR mRNA levels in mouse eyes at day 14 after administration of various exemplary siRNA duplexes shown in Table 7 at a dose of 7.5 μg. [Figure 26] 1 shows the results of TTR mRNA levels in mouse eyes at day 14 following intravitreal administration of various exemplary siRNA duplexes shown in Table 7 at a dose of 7.5 μg. DETAILED DESCRIPTION OF THE INVENTION

[0072] In particular, the inventors have found that conjugating a lipophilic moiety to one or more internal positions on at least one strand of a double-stranded iRNA agent provides unexpectedly good results for in vivo intravitreal and intrathecal delivery of double-stranded iRNAs, resulting in efficient entry into central nervous system and ocular tissues and efficient uptake by cells of the central nervous system and visual system.

[0073] One aspect of the invention provides a double-stranded iRNA agent that includes an antisense strand complementary to a target gene; a sense strand complementary to the 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.

[0074] The term "lipophilic moiety" or "lipophilic moiety" refers broadly to any compound or chemical moiety that has an affinity for lipids. One method for characterizing the lipophilicity of a lipophilic moiety is to measure the octanol-water partition coefficient, log K ow where K owis 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 can also be predicted by using coefficients attributed to the chemical's components calculated using first-principles or empirical methods (see, e.g., Tetko et al., J. Chem. Inf. Comput. Sci. 41:1407-21 (2001), the entire contents of which are incorporated herein by reference). It provides a thermodynamic measure of a substance's tendency to prefer a non-aqueous or oily environment over water (i.e., its hydrophilic / lipophilic balance). In principle, a chemical can be expressed as its log K ow is greater than 0. Typically, a lipophilic moiety has a log K of greater than 1, greater than 1.5, greater than 2, greater than 3, greater than 4, greater than 5, or greater than 10. ow For example, the log K of 6-aminohexanol ow For example, the log K of cholesteryl N-(hexan-6-ol) carbamate is predicted to be about 0.7. ow is predicted to be 10.7.

[0075] The lipophilicity of a molecule can be altered with respect to the functional groups it possesses. For example, the addition of a hydroxyl or amine group to the terminus of a lipophilic moiety can increase the partition coefficient (e.g., logK ow ) value may be increased or decreased.

[0076] Alternatively, the hydrophobicity of the double-stranded iRNA agent conjugated with one or more lipophilic moieties can be measured by its protein binding properties.For example, the unbound fraction in the plasma protein binding assay of double-stranded iRNA agent can be determined to be correlated with the relative hydrophobicity of double-stranded iRNA agent, which can be positively correlated with the silencing activity of double-stranded iRNA agent.

[0077] In one embodiment, the plasma protein binding assay to be determined is an electrophoretic mobility shift assay (EMSA) using human serum albumin protein. An exemplary protocol for this binding assay is detailed in Example 14. The hydrophobicity of the double-stranded iRNA agent, as measured by the fraction of unbound siRNA in the binding assay, is greater than 0.15, greater than 0.2, greater than 0.25, greater than 0.3, greater than 0.35, greater than 0.4, greater than 0.45, or greater than 0.5 for improved in vivo delivery of siRNA.

[0078] Thus, conjugating a lipophilic moiety to an internal position of a double-stranded iRNA agent provides optimal hydrophobicity for improved in vivo delivery of the siRNA.

[0079] In certain embodiments, the lipophilic moiety is aliphatic, cyclic, e.g., alicyclic, or polycyclic, e.g., polyalicyclic, compounds such as steroids (e.g., sterols) or straight- or branched-chain aliphatic hydrocarbons. The lipophilic moiety generally comprises a hydrocarbon chain, which may be cyclic or acyclic. The hydrocarbon chain may contain various substituents and / or one or more heteroatoms, e.g., oxygen or nitrogen atoms. Such lipophilic aliphatic moieties include, but are not limited to, saturated or unsaturated C4-C6 30 Hydrocarbons (e.g., C6-C 18 hydrocarbons), saturated or unsaturated fatty acids, waxes (e.g., monohydric alcohol esters of fatty acids and fatty diamides), terpenes (e.g., C 10 Terpene, C 15 Sesquiterpene, C 20 Diterpenes, C 30 Triterpenes, and C 40 tetraterpenes), and other polyalicyclic hydrocarbons. For example, the lipophilic moiety is C4-C 30 Hydrocarbon chains (e.g., C4 to C 30 In some embodiments, the lipophilic moiety may contain a saturated or unsaturated C-C 18 Hydrocarbon chains (e.g., straight chain C6-C 18In one embodiment, the lipophilic moiety contains a saturated or unsaturated C 16 Hydrocarbon chains (e.g., linear C 16 alkyl or alkenyl).

[0080] The lipophilic moiety can be attached to the iRNA agent by any method known in the art, such as through a functional group already present in the lipophilic moiety or introduced into the iRNA agent, such as a hydroxy group (e.g., -CO-CH-OH). 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.

[0081] Conjugation of an iRNA agent and a lipophilic moiety can occur, for example, by formation of an ether, carboxyl, or carbamoyl ester bond between a hydroxy and alkyl group R-, an alkanoyl group RCO-, or a substituted carbamoyl group RNHCO-. The alkyl group R can be cyclic (e.g., cyclohexyl) or acyclic (e.g., straight-chain or branched-chain; and saturated or unsaturated). The alkyl group R can be a butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, or octadecyl group, etc.

[0082] In some embodiments, the lipophilic moiety is conjugated to the double-stranded iRNA agent via a linker that includes an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide bond, the product of a click reaction (e.g., a triazole from an azide-alkyne cycloaddition), or a carbamate.

[0083] In another embodiment, the lipophilic moiety is a steroid such as a sterol. Steroids are polycyclic compounds containing a perhydro-1,2-cyclopentanophenanthrene ring system. Steroids include, but are not limited to, bile acids (e.g., cholic acid, deoxycholic acid, and dehydrocholic acid), cortisone, digoxigenin, testosterone, cholesterol, and cationic steroids such as cortisone. "Cholesterol derivative" refers to a compound derived from cholesterol, for example, by substitution, addition, or removal of a substituent.

[0084] In another embodiment, the lipophilic moiety is an aromatic moiety. In this context, the term "aromatic" refers broadly to monocyclic and polycyclic aromatic hydrocarbons. Aromatic groups include, but are not limited to, C6-C8 aromatic groups containing 1-3 aromatic rings, which may be optionally substituted. 14 These include aryl moieties; "aralkyl" or "arylalkyl" groups comprising an aryl group covalently bonded to an alkyl group, either of which may independently 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; 6, 10, or 14 pi-electrons shared in a cyclic arrangement; and, in addition to carbon atoms, 1 to about 3 heteroatoms selected from the group consisting of nitrogen (N), oxygen (O), and sulfur (S).

[0085] As used herein, a "substituted" alkyl, cycloalkyl, aryl, heteroaryl, or heterocyclic group has 1 to about 4, preferably 1 to about 3, and more preferably 1 or 2 non-hydrogen substituents. Suitable substituents include, but are not limited to, 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.

[0086] In some embodiments, the lipophilic moiety is an aralkyl group, for example, a 2-arylpropanoyl moiety. The structural features of the aralkyl group are selected so that the lipophilic moiety binds 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, immunoglobulins, lipoproteins, α-2-macroglobulin, or α-1-glycoprotein.

[0087] 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. Patent Nos. 3,904,682 and 4,009,197, the entire contents of which are incorporated herein by reference. Naproxen has the chemical name (S)-6-methoxy-α-methyl-2-naphthaleneacetic acid and the structure: [ka] is.

[0088] In certain embodiments, the ligand is ibuprofen or an ibuprofen structural derivative. Procedures for the synthesis of ibuprofen can be found in U.S. Pat. No. 3,228,831, the entire contents of which are incorporated herein by reference. The structure of ibuprofen is: [ka] is.

[0089] Further exemplary aralkyl groups are set forth in US Pat. No. 7,626,014, the entire contents of which are incorporated herein by reference.

[0090] In another embodiment, suitable lipophilic moieties include lipids, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl groups, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, ibuprofen, naproxen, dimethoxytrityl, or phenoxazine.

[0091] In some embodiments, the lipophilic moiety is a C-C 30 Acids (e.g., hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, dodecanoic 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 C6 to C 30Alcohols (e.g., hexanol, heptanol, octanol, nonanol, decanol, undecanol, dodecanol, tridecanol, tetradecanol, pentadecanol, hexadecanol, heptadecanol, octadecanol, oleyl alcohol, linoleyl alcohol, arachidonic acid alcohol, cis-4,7,10,13,16,19-docosahexanol, retinol, vitamin E, cholesterol, etc.).

[0092] In certain embodiments, two or more lipophilic moieties can be incorporated into a double-stranded iRNA agent, particularly if the lipophilic moieties have low lipophilicity or hydrophobicity. In one embodiment, two or more lipophilic moieties are incorporated into the same strand of a double-stranded iRNA agent. In one embodiment, one or more lipophilic moieties are incorporated into each strand of a double-stranded iRNA agent. In one embodiment, two or more lipophilic moieties are incorporated into the same position of a double-stranded iRNA agent (i.e., the same nucleobase, the same sugar moiety, or the same internucleoside linkage). This can be achieved, for example, by conjugating two or more lipophilic moieties via a carrier, and / or by conjugating two or more lipophilic moieties via a branched linker, and / or by conjugating two or more lipophilic moieties via one or more linkers, with one or more linkers connecting the lipophilic moieties consecutively.

[0093] The lipophilic moiety can be conjugated to the iRNA agent via a direct bond to the iRNA agent's ribosugar. Alternatively, the lipophilic moiety can be conjugated to the double-stranded iRNA agent via a linker or carrier.

[0094] In certain embodiments, the lipophilic moiety can be conjugated to the iRNA agent via one or more linkers (tethers).

[0095] 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 bond, the product of a click reaction (e.g., a triazole from an azide-alkyne cycloaddition), or a carbamate. Some exemplary linkages are shown in Figure 1 and Examples 2, 3, 5, 6, and 7.

[0096] Linker / Tether The linker / tether is linked to the lipophilic moiety at a "tethering attachment point (TAP)." The linker / tether can be any C1-C 100 Carbon-containing moieties (e.g., C1-C 75 , C1~C 50 , C1~C 20 , C1~C 10 ;C1, C2, C3, C4, C5, C6, C7, C8, C9, or C 10 ) 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 in the linker / tether that can serve as a point of attachment for a lipophilic moiety. Non-limiting examples of linkers / tethers (underlined) include: [ka] wherein 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 a 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 a hydrazino group, -NHNH2. The linker / tether may be optionally substituted, e.g., with hydroxy, alkoxy, perhaloalkyl, and / or may optionally contain one or more additional heteroatoms, e.g., N, O, or S. Preferred linking ligands include, for example, [ka] In some embodiments, the amino-terminated linker / tether (e.g., NH, ONH, NHNH) can form an imino bond (i.e., C=N) with the ligand. In some embodiments, the amino-terminated linker / tether (e.g., NH, ONH, NHNH) can be acylated, for example, with C(O)CF.

[0097] In some embodiments, the linker / tether may terminate in a mercapto group (i.e., SH) or an olefin (e.g., CH=CH). For example, the tether may be: [ka] where n can be as described elsewhere. The tether can be optionally substituted, e.g., with hydroxy, alkoxy, perhaloalkyl, and / or can be 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.

[0098] In other embodiments, the linker / tether may include an electrophilic moiety, preferably at the terminal position of the linker / tether. Exemplary electrophilic moieties include, for example, an aldehyde, an alkyl halide, a mesylate, a tosylate, a nosylate, or a brosylate, or an activated carboxylic acid ester, such as an NHS ester, or a pentafluorophenyl ester. Preferred linkers / tethers (underlined) include: [ka] where n is 1 to 6 and R"" is C1 to C6 alkyl; or [ka] where n is 1 to 6 and R'''' is C1 to C6 alkyl; [ka] where n is 1 to 11 and R"" is C1 to C6 alkyl; or [ka] where n can be as described elsewhere and R"" is a C1-C6 alkyl (LG can be a leaving group, e.g., halide, mesylate, tosylate, nosylate, brosylate). Tethering can be performed by coupling a nucleophilic group, e.g., a thiol or amino group, of the ligand with an electrophilic group on the tether.

[0099] In other embodiments, it may be desirable for the monomer to contain a phthalimide group (K) at the terminal position of the linker / tether. [ka]

[0100] In other embodiments, other protected amino groups may be at the terminal positions of the linker / tether, such as alloc, monomethoxytrityl (MMT), trifluoroacetyl, Fmoc, or arylsulfonyl (e.g., the aryl moiety may be ortho-nitrophenyl or ortho, para-dinitrophenyl).

[0101] Any of the linkers / tethers described herein may contain one or more additional linking groups, such as -O-(CH) n -, -(CH2) n -SS-, -(CH2) n It may further comprise - or (CH=CH)-.

[0102] Cleavable Linker / Tether In some embodiments, at least one of the linkers / tethers can be a redox-cleavable linker, an acidic-cleavable linker, an esterase-cleavable linker, a phosphatase-cleavable linker, or a peptidase-cleavable linker.

[0103] In one embodiment, at least one of the linkers / tethers can be a reductively cleavable linker (eg, a disulfide group).

[0104] In one embodiment, at least one of the linkers / tethers can be an acid-cleavable linker (eg, a hydrazone group, an ester group, an acetal group, or a ketal group).

[0105] In one embodiment, at least one of the linkers / tethers can be an esterase-cleavable linker (eg, an ester group).

[0106] In one embodiment, at least one of the linkers / tethers can be a phosphatase-cleavable linker (eg, a phosphate group).

[0107] In one embodiment, at least one of the linkers / tethers can be a peptidase-cleavable linker (eg, a peptide bond).

[0108] Cleavable linking groups are susceptible to cleaving agents, such as pH, redox potential, or the presence of degradable molecules.Generally, cleaving agents are more prevalent or present at higher levels or activity in cells than in serum or blood.Examples of such degrading agents include, for example, oxidizing enzymes or reductases or reducing agents present in cells, which can degrade redox-cleavable linking groups by reduction, such as mercaptans, which are selective for specific substrates or do not have substrate specificity; esterases; endosomes or agents that can create an acidic environment, for example, a pH of 5 or less; enzymes that can hydrolyze or degrade acidic cleavable linking groups by acting as general acids, peptidases (which may be substrate specific), and phosphatases.

[0109] Cleavable linking groups, e.g., disulfide bonds, are sensitive to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1 to 7.3. Endosomes have a more acidic pH, ranging from 5.5 to 6.0, and lysosomes have an even more acidic pH of about 5.0. Some tethers have linking groups that cleave at a preferred pH, thereby releasing the iRNA agent from the ligand (e.g., a targeting or cell-permeable ligand, e.g., cholesterol) into the cell or into a desired compartment of the cell.

[0110] The chemical bond (e.g., linking group) that links the ligand to the iRNA agent can include a disulfide bond. When the iRNA agent / ligand complex is taken up into a cell by endocytosis, the acidic environment of the endosome cleaves the disulfide bond, thereby releasing the iRNA agent from the ligand (Quintana et al., Pharm Res. 19:1310-1316, 2002; Patri et al., Curr. Opin. Curr. Biol. 6:466-471, 2002). The ligand can be a secondary therapeutic agent that can complement the therapeutic effect of the targeting ligand or iRNA agent.

[0111] The tether may contain a linking group that can be cleaved by a specific enzyme. The type of linking group incorporated into the tether may vary depending on the cell targeted by the iRNA agent. For example, an iRNA agent targeting mRNA in liver cells may be conjugated to a tether containing an ester group. Liver cells are rich in esterases, and therefore, the tether is cleaved more efficiently in liver cells than in cell types that are not rich in esterases. Cleavage of the tether releases the iRNA agent from the ligand attached to the distal end of the tether, thereby potentially increasing the silencing activity of the iRNA agent. Other cell types rich in esterases include lung, renal cortex, and testicular cells.

[0112] Tethers containing peptide bonds can be conjugated to iRNA agents that target cell types rich in peptidases, such as hepatocytes and synovial cells. For example, for the treatment of inflammatory diseases (e.g., rheumatoid arthritis), iRNA agents targeted to synovial cells can be conjugated to tethers containing peptide bonds.

[0113] Generally, the suitability of a candidate cleavable linking group can be evaluated by testing the ability (or conditions) of a degrading agent to cleave the candidate linking group. It is also desirable to test the candidate cleavable linking group for its ability to resist cleavage in blood or when in contact with other non-target tissues, such as tissues to which the iRNA agent is exposed when administered to a subject. Thus, the relative ease of cleavage between a first condition and a second condition can be determined, with the first condition being selected to demonstrate cleavage within target cells, and the second condition being selected to demonstrate cleavage in other tissues or body fluids, such as blood or serum. Evaluation can be performed in a cell-free system, cells, cell cultures, organ or tissue cultures, or whole animals. It can be useful to perform initial evaluation in a cell-free or cultured condition and confirm with further evaluation in whole animals. In preferred embodiments, useful candidate compounds are cleaved at least 2-fold, 4-fold, 10-fold, or 100-fold faster inside cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0114] Redox-cleavable linking groups One class of cleavable linkers is redox-cleavable linkers, which are cleaved upon reduction or oxidation. One example of a reductively cleavable linker is a disulfide linker (-SS-). The methods described herein can be used to determine whether a candidate cleavable linker is a suitable "reductively cleavable linker," or whether it is suitable for use with, for example, a particular iRNA moiety and a particular targeting agent. For example, candidates can be evaluated by incubation with dithiothreitol (DTT) or other reducing agents using reagents known in the art that mimic cleavage rates observed in cells, e.g., target cells. Candidates can also be evaluated under conditions selected to mimic blood or serum conditions. In preferred embodiments, candidate compounds are cleaved at most 10% in blood. In preferred embodiments, useful candidate compounds are degraded at least 2-fold, 4-fold, 10-fold, or 100-fold faster in cells (or under in vitro conditions selected to mimic intracellular conditions) than in blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of a candidate compound can be determined using standard enzyme kinetic assays under conditions selected to mimic the intracellular medium and compared to conditions selected to mimic the extracellular medium.

[0115] Phosphate-based cleavable linkers Phosphate-based linking groups are cleaved by agents that decompose or hydrolyze phosphate groups. An example of an agent that cleaves phosphate groups within a cell is an intracellular enzyme, such as a phosphatase. Examples of phosphate-based linking groups are -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, and -OP(S)(Rk)-S-. Preferred embodiments are -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O-, -SP(S)(H)-O-, -SP(O)(H)-S-, -OP(S)(H)-S-. A preferred embodiment is -OP(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

[0116] acidic cleavable linking group An acidic cleavable tether is a tether that is cleaved under acidic conditions. In a preferred embodiment, the acidic cleavable tether is cleaved in an acidic environment with a pH of about 6.5 or less (e.g., about 6.0, 5.5, 5.0, or less) or by an agent that can function as a general acid, such as an enzymatic agent. In cells, certain organelles with low pH, such as endosomes and lysosomes, can provide a cleavage environment for the acidic cleavable tether. Examples of acidic cleavable tethers include, but are not limited to, hydrazones, ketals, acetals, esters, and esters of amino acids. Acidic cleavable groups can have the general formula -C=NN-, C(O)O, or OC(O). A preferred embodiment is when the carbon bonded to the oxygen of the ester (alkoxy group) is an aryl group, a substituted alkyl group, or a tertiary alkyl group, such as dimethylpentyl or t-butyl. These candidates can be evaluated using methods similar to those described above.

[0117] Ester-based linking group Ester-based linking groups are cleaved by intracellular enzymes, such as esterases and amidases. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene groups, alkenylene groups, and alkynylene groups. Ester-cleavable linking groups have the general formula -C(O)O- or -C(O)-. These candidates can be evaluated using methods similar to those described above.

[0118] Peptide-based cleavable linkers Peptide-based linking groups are cleaved by intracellular enzymes, such as peptidases and proteases. Peptide-based cleavable linking groups are peptide bonds formed between amino acids, resulting in oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable linking groups do not include amide groups (-C(O)NH-). Amide groups can be formed between any alkylene, alkenylene, or alkynylene. A peptide bond is a special type of amide bond formed between amino acids, resulting in peptides and proteins. Peptide-based cleaving groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids, resulting in peptides and proteins, and do not include the entire amide functionality. Peptide cleavable linking groups have the general formula -NHCHR 1 C(O)NHCHR 2 C(O)—, where R 1 and R 2 are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

[0119] Biocleavable linkers / tethers Linker can also include biocleavable linker, which is nucleotide and non-nucleotide linker or their combination, which connects two parts of molecule, for example, one or both strands of two individual siRNA molecules to generate bis(siRNA).In some embodiments, the simple electrostatic or stacking interaction between two individual siRNA can represent linker.Non-nucleotide linker includes tether or linker derived from monosaccharide, disaccharide, oligosaccharide, and their derivatives, aliphatic, alicyclic, heterocyclic, and their combinations.

[0120] In some embodiments, at least one of the linkers (tethers) is a biocleavable linker selected from the group consisting of DNA, RNA, disulfide, amide, galactosamine, glucosamine, glucose, galactose, and mannose functionalized mono- or oligosaccharides, and combinations thereof.

[0121] In one embodiment, the biocleavable carbohydrate linker can have 1-10 sugar units with at least one aromatic linkage capable of linking two siRNA units. When more than one sugar is present, the units can be linked via 1-3, 1-4, or 1-6 sugar linkages or via alkyl chains.

[0122] Exemplary biocleavable linkers include: [ka] [ka] [ka] Examples include:

[0123] Further exemplary biocleavable linkers are shown in Schemes 28-30.

[0124] Further description of biocleavable linkers can be found in PCT Application No. PCT / US18 / 14213, entitled "Endosomal Cleavable Linkers," filed January 18, 2018, the entire contents of which are incorporated herein by reference.

[0125] Carrier In certain embodiments, a lipophilic moiety is conjugated to an iRNA agent via a carrier that replaces one or more nucleotides.

[0126] The carrier can be a cyclic or 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 skeleton or a diethanolamine skeleton.

[0127] In some embodiments, the carrier replaces one or more nucleotides at an internal position of the double-stranded iRNA agent.

[0128] In other embodiments, carrier replaces the nucleotide at the end of sense strand or antisense strand.In one embodiment, carrier replaces the terminal nucleotide at the 3' end of sense strand, thereby functioning as the end cap that protects the 3' end of sense strand.In one embodiment, carrier is a cyclic group that has amine, for example, carrier can be pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3] dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl or decalinyl.

[0129] Ribonucleotide subunits in which the ribose sugar of the subunit has been replaced in this way are called ribose-replacement modified subunits (RRMS). The carrier may be a cyclic or acyclic moiety and comprises two "backbone attachment points" (e.g., hydroxyl groups) and a ligand (e.g., a lipophilic moiety). The lipophilic moiety may be directly attached to the carrier or indirectly attached to the carrier by an intervening linker / tether, as described above. [ka]

[0130] The ligand-conjugated monomer subunit can be the 5' or 3' terminal subunit of an iRNA molecule, i.e., one of the two "W" groups can be a hydroxyl group and the other "W" group can be a chain of two or more unmodified or modified ribonucleotides. Alternatively, the ligand-conjugated monomer subunit can occupy an internal position, and both "W" groups can be one or more unmodified or modified ribonucleotides. Two or more ligand-conjugated monomer subunits can be present in an iRNA agent.

[0131] Monomers based on sugar substitutions, e.g., ligand-conjugated monomers (cyclic) Monomers based on cyclic sugar substitutions, e.g., ligand-conjugate monomers based on the substitutions, are also referred to herein as RRMS monomer compounds. The carriers may have the general formula (LCM-2) shown below, in which the preferred backbone attachment points are R 1 or R 2 ;R 3 or R 4 ; or Y is CR 9 R 10 If R 9 and R 10 (two positions may be selected from two backbone attachment points, e.g., R 1 and R 4 , or R 4 and R 9 The preferred tether attachment point is R 7 ; When X is CH2, R 5 or R 6 Carriers are described below as substances that can be incorporated into the chain. Thus, the structure may have one (for terminal positions) or two (for internal positions) attachment points, e.g., R 1 or R 2 ;R 3 or R 4 ; or R 9 or R 10 (Y is CR 9 R 10It is understood that the term "R" also encompasses cases where the R group is linked to a phosphate or modified phosphate, e.g., a sulfur-containing backbone. For example, one of the R groups listed above can be -CH2-, where one bond is linked to the carrier and one is linked to a backbone atom, e.g., the linking oxygen or central phosphorus atom. [ka] During the ceremony: X is N(CO)R 7 , N.R. 7 or CH2; Y is NR 8 ,O,S,CR 9 R 10 and; Z is CR 11 R 12 is or does not exist; R 1 , R 2 , R 3 , R 4 , R 9 , and R 10 each of which is independently H, OR a , or (CH2) n OR b where R 1 , R 2 , R 3 , R 4 , R 9 , and R 10 At least two of the a and / or (CH2) n OR b and; R 5 , R 6 , R 11 , and R 12 each independently comprising a ligand, H, one to three R 13 optionally substituted C1-C6 alkyl, or C(O)NHR 7 or R 5 and R 11 Together, R 14 and optionally substituted C3-C8 cycloalkyl; R 7can be a ligand, e.g., R 7 is R d or R 7 is a ligand, e.g., NR, that is indirectly linked to a carrier, e.g., via a tether moiety. c R d C1~C substituted with 20 alkyl; or NHC(O)R d C1~C substituted with 20 may be alkyl; R 8 is H or C1-C6 alkyl; R 13 is hydroxy, C1-C4 alkoxy, or halo; R 14 is NR c R 7 and; R 15 is cyano, optionally substituted C1-C6 alkyl, or C2-C6 alkenyl; R 16 is C1~C 10 is alkyl; R 17 is a liquid or solid phase carrier reagent; L is -C(O)(CH2) q C(O)- or C(O)(CH2) q S- and; R a is a protecting group, such as CAr; (e.g., dimethoxytrityl group) or Si(X 5’ )(X 5” )(X 5”’ ), where (X 5’ ),(X 5” ), and (X 5”’ ) is as described above. R b is P(O)(O - )H, P(OR 15 )N(R 16 )2 or LR 17 and; R c is H or C1-C6 alkyl; R dis H or a ligand; Each Ar is independently selected from C1-C4 alkoxy, C6-C 10 is aryl; n is 1 to 4; q is 0 to 4.

[0132] Exemplary carriers include, for example, those in which X is N(CO)R 7 or NR 7 and Y is CR 9 R 10 and Z is absent; or X is N(CO)R 7 or NR 7 and Y is CR 9 R 10 and Z is CR 11 R 12 or X is N(CO)R 7 or NR 7 and Y is NR 8 and Z is CR 11 R 12 or X is N(CO)R 7 or NR 7 and Y is O and Z is CR 11 R 12 or X is CH; Y is CR 9 R 10 and Z is CR 11 R 12 and R 5 and R 11 together form a C6 cycloalkyl (H, z=2), or indane ring system, for example, X is CH2; Y is CR 9 R 10 and Z is CR 11 R 12 and R 5 and R 11 taken together to form a C5 cycloalkyl (H, z=1).

[0133] In certain embodiments, the carrier may be based on a pyrroline ring system or a 4-hydroxyproline ring system, e.g., X is N(CO)R 7 or NR7 and Y is CR 9 R 10 and Z does not exist (D). [ka] OFG 1 is preferably a carbon atom in the five-membered ring (-CH2OFG in D) 1 ) is attached to a primary carbon, e.g., an exocyclic alkylene group, e.g., a methylene group, which is linked to one of the OFG 2 is preferably a carbon atom in the five-membered ring (-OFG in D) 2 ) for pyrroline-based carriers, -CH2OFG 1 may be attached to C-2, OFG 2 may be attached to C-3; or CH2OFG 1 may be attached to C-3, OFG 2 may be attached to C-4. In certain embodiments, CH2OFG 1 and OFG 2 may be geminally substituted at one of the above carbons. For 3-hydroxyproline-based carriers, -CHOFG 1 may be attached to C-2, OFG 2 may be attached to C-4. Thus, pyrroline-based and 4-hydroxyproline-based monomers may contain bonds (e.g., carbon-carbon bonds) where bond rotation is restricted around that particular bond (e.g., due to the presence of a ring). Thus, CHOFG 1 and OFG 2 can be cis or trans relative to each other in any of the pairs described above. Thus, all cis / trans isomers are expressly included. Monomers may also contain one or more asymmetric centers and thus may exist as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomers of the monomers are expressly included (e.g., CHOFG 1 and OFG 2(The centers having the formula (I) and (II) 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, or vice versa). The tether attachment point is preferably nitrogen. Preferred examples of carrier D include the following: [ka] Examples include:

[0134] In certain embodiments, the carrier may be based on a piperidine ring system (E), e.g., X is N(CO)R 7 or NR 7 and Y is CR 9 R 10 and Z is CR 11 R 12 is. [ka] OFG 1 is preferably a carbon atom in the six-membered ring [—(CH) in E] n OFG 1

[0023] OFG is bonded to a primary carbon, for example, an exocyclic alkylene group, for example, a methylene group (n=1) or an ethylene group (n=2), which is linked to one of the following: 2 is preferably a carbon atom in the six-membered ring (-OFG in E) 2 ) is directly bonded to one of the -(CH2) n OFG 1 and OFG 2 may be geminally positioned on the ring, i.e., both groups may be attached to the same carbon, e.g., at C-2, C-3, or C-4. Alternatively, -(CH) n OFG 1 and OFG 2 may be vicinal on the ring, i.e., both groups may be attached to adjacent ring carbon atoms, for example, -(CH) n OFG 1 may be attached to C-2, OFG 2 may be attached to C-3; -(CH2) n OFG 1may be attached to C-3, OFG 2 may be attached to C-2; -(CH2) n OFG 1 may be attached to C-3, OFG 2 may be attached to C-4; or (CH) n OFG 1 may be attached to C-4, OFG 2 may be attached to C-3. Thus, a piperidine-based monomer may contain a bond (e.g., a carbon-carbon bond) where bond rotation is restricted around that particular bond (e.g., due to the presence of a ring). Thus, -(CH2) n OFG 1 and OFG 2 can be cis or trans relative to each other in any of the pairs described above. Thus, all cis / trans isomers are expressly included. Monomers may also contain one or more asymmetric centers and thus may exist as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomers of the monomers are expressly included (e.g., CHOFG 1 and OFG 2 (The centers having 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, or vice versa.) The tether attachment point is preferably nitrogen.

[0135] In certain embodiments, the carrier may be based on a piperazine ring system (F), e.g., X is N(CO)R 7 or NR 7 and Y is NR 8 and Z is CR 11 R 12 or a morpholine ring system (G), e.g., X is N(CO)R 7 or NR 7 and Y is O and Z is CR 11 R 12 is. [ka] OFG 1 is preferably a carbon atom in a six-membered ring (-CH2OFG in F or G) 1 ) is attached to a primary carbon, e.g., an exocyclic alkylene group, e.g., a methylene group, which is linked to one of the OFG 2 is preferably a carbon atom in a six-membered ring (-OFG in F or G) 2 ) for both F and G. 1 may be attached to C-2, OFG 2 may be attached to C-3; or vice versa. In certain embodiments, CH2OFG 1 and OFG 2 can be geminally substituted on one of the carbons. Thus, piperazine- and morpholine-based monomers can contain bonds (e.g., carbon-carbon bonds) where bond rotation is restricted around that particular bond (e.g., due to the presence of a ring). Thus, CHOFG 1 and OFG 2 can be cis or trans relative to each other in any of the pairs described above. Thus, all cis / trans isomers are expressly included. Monomers may also contain one or more asymmetric centers and thus may exist as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomers of the monomers are expressly included (e.g., CHOFG 1 and OFG 2 (The centers having 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, or vice versa). R''' can be, for example, a C1-C6 alkyl, preferably CH3. The tether attachment points are preferably nitrogen in both F and G.

[0136] In certain embodiments, the carrier may be based on a decalin ring system, e.g., X is CH; Y is CR 9 R 10 and Z is CR11 R 12 and R 5 and R 11 together form a C6 cycloalkyl (H, z=2), or indane ring system, e.g., X is CH2; Y is CR 9 R 10 and Z is CR 11 R 12 and R 5 and R 11 together form a C5 cycloalkyl (H, z=1). [ka] OFG 1 is preferably —(CH) at C-2, C-3, C-4, or C-5 [H] n OFG 1 ], for example, an exocyclic methylene group (n=1) or an ethylene group (n=2). OFG 2 is preferably at C-2, C-3, C-4, or C-5 (H in -OFG 2 ) is directly bonded to one of the -(CH2) n OFG 1 and OFG 2 may be geminally positioned 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, -(CH) n OFG 1 and OFG 2 may be vicinal on the ring, i.e., both groups may be attached to adjacent ring carbon atoms, for example, -(CH) n OFG 1 may be attached to C-2, OFG 2 may be attached to C-3; -(CH2) n OFG 1 may be attached to C-3, OFG 2 may be attached to C-2; -(CH2) n OFG 1 may be attached to C-3, OFG 2 may be attached to C-4; or (CH)n OFG 1 may be attached to C-4, OFG 2 may be attached to C-3; -(CH2) n OFG 1 may be attached to C-4, OFG 2 may be attached to C-5; or (CH) n OFG 1 may be attached to C-5, OFG 2 may be attached to C-4. Thus, decalin or indane-based monomers may contain bonds (e.g., carbon-carbon bonds) where bond rotation is restricted around that particular bond (e.g., due to the presence of a ring). Thus, -(CH2) n OFG 1 and OFG 2 can be cis or trans relative to each other in any of the pairs described above. Thus, all cis / trans isomers are expressly included. Monomers may also contain one or more asymmetric centers and thus may exist as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomers of the monomers are expressly included (e.g., CHOFG 1 and OFG 2 (The centers having the R and S configurations may both have the R and S configurations, or one center may have the R and S configurations, or vice versa.) In preferred embodiments, the substituents at C-1 and C-6 are trans relative to each other. The tether attachment point is preferably at C-6 or C-7.

[0137] Other carriers may include those based on 3-hydroxyproline (J). [ka] Therefore, -(CH2) n OFG 1 and OFG 2can be cis or trans relative to each other. Thus, all cis / trans isomers are expressly included. Monomers may also contain one or more asymmetric centers and thus may exist as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomers of the monomers are expressly included (e.g., CHOFG 1 and OFG 2 (The centers having 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, or vice versa.) The tether attachment point is preferably nitrogen.

[0138] Details regarding more representative cyclic sugar-based carriers can be found in US Pat. Nos. 7,745,608 and 8,017,762, the entire contents of which are incorporated herein by reference.

[0139] Monomers based on sugar substitution (acyclic) Acyclic sugar-substituted based monomers, e.g., sugar-substituted based ligand-conjugate monomers, are also referred to herein as ribose-substituted monomer subunit (RRMS) monomer compounds. Preferred acyclic carriers are represented by the formula LCM-3 or LCM-4: [ka] may have:

[0140] In some embodiments, each of x, y, and z can be, independently of one another, 0, 1, 2, or 3. When y and z are different in formula LCM-3, the tertiary carbon can have either the R or S configuration. In a preferred embodiment, in formula LCM-3 (e.g., based on serinol), x is 0, y and z are each 1, and in formula LCM-3, y and z are each 1. Each of formulas LCM-3 or LCM-4 below can be optionally substituted, for example, with hydroxy, alkoxy, or perhaloalkyl.

[0141] Details regarding more representative acyclic sugar-based carriers can be found in US Pat. Nos. 7,745,608 and 8,017,762, the entire contents of which are incorporated herein by reference.

[0142] In some embodiments, the double-stranded iRNA agent includes one or more lipophilic moieties conjugated to the 5' end of the sense strand or the 5' end of the antisense strand.

[0143] In certain embodiments, the lipophilic moiety is conjugated to the 5' end of the chain via a carrier and / or linker. In one embodiment, the lipophilic moiety has the formula: [ka] R is a ligand, such as a lipophilic moiety.

[0144] In some embodiments, the double-stranded iRNA agent includes one or more lipophilic moieties conjugated to the 3' end of the sense strand or the 3' end of the antisense strand.

[0145] In certain embodiments, the lipophilic moiety is conjugated to the 3' end of the chain via a carrier and / or a linker. In one embodiment, the lipophilic moiety has the formula: [ka] R is a ligand, such as a lipophilic moiety.

[0146] In some embodiments, the double-stranded iRNA agent includes one or more lipophilic moieties conjugated to both ends of the sense strand.

[0147] In some embodiments, the double-stranded iRNA agent includes one or more lipophilic moieties conjugated to both ends of the antisense strand.

[0148] In some embodiments, the double-stranded iRNA agent includes one or more lipophilic moieties conjugated to the 5' or 3' end of the sense strand and one or more lipophilic moieties conjugated to the 5' or 3' end of the antisense strand.

[0149] In some embodiments, the lipophilic moiety is conjugated to the end of the chain via one or more linkers (tethers) and / or carriers.

[0150] In one embodiment, the lipophilic moiety is conjugated to the end of the chain via one or more linkers (tethers).

[0151] In one embodiment, the lipophilic moiety is conjugated to the 5' end of the sense strand or the antisense strand via a cyclic carrier, optionally via one or more intervening linkers (tethers).

[0152] In some embodiments, the lipophilic moiety is conjugated to one or more internal positions on at least one strand. Internal positions on a strand refer to nucleotides at any position on the strand, excluding the positions from the 3' and 5' ends of the strand (e.g., excluding two positions: position 1 counting from the 3' end and position 1 counting from the 5' end).

[0153] In one embodiment, the lipophilic moiety is conjugated to one or more internal positions on at least one chain, including all but the two most terminal positions on each end of the chain (e.g., four positions: excluding 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 chain, including all but the three most terminal positions on each end of the chain (e.g., six positions: excluding positions 1, 2, and 3 counting from the 3' end and positions 1, 2, and 3 counting from the 5' end).

[0154] In one embodiment, the lipophilic moiety is conjugated to one or more internal positions on at least one strand excluding the cleavage site region of the sense strand, e.g., the lipophilic moiety is not conjugated to positions 9-12 counting from the 5' end of the sense strand, e.g., 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.

[0155] In one embodiment, the lipophilic moiety is conjugated to one or more internal positions on at least one strand, excluding the cleavage site region of the antisense strand, e.g., internal positions excluding positions 12-14 from the 5' end of the antisense strand.

[0156] In one embodiment, the lipophilic moiety is conjugated to one or more internal positions on at least one strand, 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.

[0157] In one embodiment, the 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.

[0158] In one embodiment, the 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.

[0159] In some embodiments, the lipophilic moiety is conjugated to a nucleobase, sugar moiety, or internucleoside linkage of a double-stranded iRNA agent.

[0160] definition Unless specific definitions are provided, the nomenclature used in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for chemical synthesis and chemical analysis. Some such techniques and procedures are described, for example, in "Carbohydrate Modifications in Antisense Research," Edited by Sangvi and Cook, American Chemical Society, Washington, DC, 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," 2002. nd Edition, Cold Spring Harbor Laboratory Press, 1989, which are incorporated herein by reference for all purposes. Where permitted, all patents, applications, published applications and other publications and other data referred to throughout this disclosure are incorporated herein by reference in their entirety.

[0161] Unless otherwise indicated, the following terms have the following meanings:

[0162] The term "target nucleic acid" as used herein refers to any nucleic acid molecule whose expression or activity can be regulated by siRNA compounds.Target nucleic acid includes but is not limited to RNA transcribed from DNA encoding target protein (including but not limited to pre-mRNA and mRNA or its part), and also cDNA and miRNA obtained from such RNA.For example, target nucleic acid can be a cellular gene (or mRNA transcribed from the gene) whose expression is related to a specific disorder or pathology.In some embodiments, target nucleic acid can be a nucleic acid molecule derived from an infectious agent.

[0163] As used herein, the term "iRNA" refers to an agent that mediates targeted cleavage of RNA transcripts. These agents associate with a cytoplasmic multiprotein complex known as the RNAi-induced silencing complex (RISC). Agents that are effective in inducing RNA interference are also referred to herein as siRNAs, RNAi agents, or iRNA agents. Thus, these terms can be used interchangeably herein. As used herein, the term iRNA includes microRNAs and pre-microRNAs. Furthermore, as used herein, the terms "compound" or "compounds" of the present invention also refer to iRNA agents and can be used interchangeably with iRNA agents.

[0164] An iRNA agent should contain a region sufficiently homologous to a target gene and be of sufficient length in terms of nucleotides so that the iRNA agent, or a fragment thereof, can mediate downregulation of the target gene. (For ease of explanation, the terms nucleotide or ribonucleotide may be used herein in reference to one or more monomeric subunits of an iRNA agent. It will be understood herein that the use of the terms "ribonucleotide" or "nucleotide" herein, in the case of a modified RNA or nucleotide surrogate, can also refer to the modified nucleotide or surrogate replacement moiety at one or more positions.) Thus, an iRNA agent is or contains a region that is at least partially, and in some embodiments, completely, complementary to the target RNA. While perfect complementarity between an iRNA agent and a target is not required, the match must be sufficient to enable the iRNA agent, or its cleavage product, to direct sequence-specific silencing, e.g., by RNAi cleavage of the target RNA, e.g., mRNA. The degree of complementarity, or homology, with the target strand is most important in the antisense strand. While perfect complementarity is often desirable, particularly in the antisense strand, some embodiments may include one or more, or for example, 6, 5, 4, 3, 2, or fewer, mismatches (with respect to the target RNA), particularly in the antisense strand. The sense strand need only be sufficiently complementary to the antisense strand to maintain the overall double-stranded nature of the molecule.

[0165] iRNA agents include molecules that are long enough to trigger an interferon response (which can be cleaved by Dicer (Bernstein et al. 2001. Nature, 409:363-366) and enter RISC (RNAi-induced silencing complex)); and molecules that are short enough not to trigger an interferon response (which can also be cleaved by Dicer and / or enter RISC), e.g., molecules of a size that allows entry into RISC, e.g., molecules similar to Dicer cleavage products. Molecules that are short enough not to trigger an interferon response are referred to herein as siRNA agents or short iRNA agents. As used herein, "siRNA agent or short iRNA agent" refers to an iRNA agent, e.g., a double-stranded RNA agent or a single-stranded agent, that is short enough not to induce a deleterious interferon response in human cells, e.g., which has a duplex region of less than 60, 50, 40, or 30 nucleotide pairs. The siRNA agent, or its cleavage product, can downregulate a target gene, for example, by inducing RNAi against the target RNA, where the target can include an endogenous or pathogen target RNA.

[0166] As used herein, a "single-stranded iRNA agent" is an iRNA agent that is composed of a single molecule. It may contain a double-stranded region formed by intrastrand pairing, for example, it may be or include a hairpin or panhandle structure. A single-stranded iRNA agent may be antisense to a target molecule. A single-stranded iRNA agent may be long enough to enter RISC and participate in RISC-mediated cleavage of a target mRNA. A single-stranded 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.

[0167] A loop refers to a region of an iRNA strand that, when base-pairing with another strand or another portion of the same strand, does not pair with the opposite nucleotide in the duplex.

[0168] Hairpin iRNA agents have a duplex region of at least or equal to 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The duplex region can be 200, 100, or 50 or less in length. In certain embodiments, the duplex region ranges from 15 to 30, 17 to 23, 19 to 23, and 19 to 21 nucleotide pairs in length. The hairpin has a single-stranded overhang or terminal unpaired region at the 3' end in some embodiments, and in certain embodiments, on the antisense side of the hairpin. In some embodiments, the overhang is 2 to 3 nucleotides in length.

[0169] As used herein, a "double-stranded (ds) iRNA agent" is an iRNA agent that includes two or more, and in some cases two, strands that can form a region of duplex structure by hybridization between the strands.

[0170] As used herein, the terms "siRNA activity" and "RNAi activity" refer to gene silencing by siRNA.

[0171] As used herein, "gene silencing" by an RNA interference molecule refers to a reduction in the mRNA level of a target gene in a cell 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%, including up to 100%, and any integer percentage between these. In a preferred embodiment, the mRNA level is reduced by at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, including up to 100%, and any integer percentage between 5% and 100%.

[0172] As used herein, the term "modulate gene expression" means up-regulating or down-regulating the expression of a gene encoding one or more proteins or protein subunits, or the level of an RNA molecule or equivalent RNA molecule, such that the expression, level, or activity is higher or lower than that observed in the absence of the modulator. For example, the term "modulate" can mean "inhibit," although the use of the word "modulate" is not limited to this definition.

[0173] As used herein, gene expression modulation occurs when the expression of a gene encoding one or more proteins or protein subunits, or the level of an RNA molecule or equivalent RNA molecule, differs by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, or more from that observed in the absence of an siRNA, e.g., an RNAi agent. The percentage and / or fold difference can be calculated relative to a control or non-control, for example, as follows:

number

[0174] As used herein, the terms "inhibit," "down-regulate," or "reduce," with respect to gene expression, mean that the expression of a gene encoding one or more proteins or protein subunits, or the level of an RNA molecule or equivalent RNA molecule, or the activity of one or more proteins or protein subunits, is reduced below that observed in the absence of a modulator. Gene expression is down-regulated when the expression of a gene encoding one or more proteins or protein subunits, or the level of an RNA molecule or equivalent RNA molecule, or the activity of one or more proteins or protein subunits, is reduced by at least 10% compared to a corresponding unmodulated control, preferably by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or most preferably, 100% (i.e., no gene expression).

[0175] As used herein, the terms "increase" or "up-regulate," with respect to gene expression, mean that the expression of a gene encoding one or more proteins or protein subunits, or the level of an RNA molecule or equivalent RNA molecule, or the activity of one or more proteins or protein subunits, is increased above that observed in the absence of a modulator. Gene expression is up-regulated when the expression of a gene encoding one or more proteins or protein subunits, or the level of an RNA molecule or equivalent RNA molecule, or the activity of one or more proteins or protein subunits, is increased by at least 10%, 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, compared to a corresponding unmodulated control.

[0176] As used herein, the terms "increased" or "increasing" generally refer to an increase by a statistically significant amount; for the avoidance of doubt, "increased" means an increase of at least 10% 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% compared to a reference sample, or an increase of up to 100%, or any increase between 10-100%, or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold or more increase compared to a reference level.

[0177] The term "reduced" or "reducing" as used herein generally refers to a statistically significant reduction. However, for the avoidance of doubt, "reduced" refers to a reduction of at least 10% compared to the reference level, for example, 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% reduction, or a reduction of 100% or less (i.e., zero level compared to the reference sample), or any reduction between 10% and 100% compared to the reference level.

[0178] A double-stranded iRNA comprises two oligonucleotide strands sufficiently complementary to hybridize to form a duplex structure. Typically, the duplex structure is 15-30, more commonly 18-25, even more commonly 19-24, and most commonly 19-21 base pairs in length. In some embodiments, longer double-stranded iRNAs, 25-30 base pairs in length, are preferred. In some embodiments, shorter double-stranded iRNAs, 10-15 base pairs in length, are preferred. In another embodiment, the double-stranded iRNA is at least 21 nucleotides in length.

[0179] 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 complementary to at least a portion of a target sequence, and the duplex region is 14-30 nucleotides in length. Similarly, the region of complementarity to the target sequence is 14-30, more commonly 18-25, even more commonly 19-24, and most commonly 19-21 nucleotides in length.

[0180] The phrase " antisense strand " used herein refers to the oligomeric compound that is substantially or 100% complementary to the target sequence of interest.The phrase " antisense strand " includes the antisense region of both oligomeric compounds that are formed from two separate strands, and the unimolecular oligomeric compound that can form hairpin or dumbbell structure.The terms " antisense strand " and " guide strand " are used interchangeably.

[0181] The phrase "sense strand" refers to an oligomeric compound having a nucleoside sequence that is wholly or partially identical to a target sequence, such as a sequence of messenger RNA or DNA. The terms "sense strand" and "passenger strand" are used interchangeably.

[0182] " Specifically hybridizable " and " complementary " mean that nucleic acid can form hydrogen bonds with another nucleic acid sequence, either by Watson-Crick or other non-traditional type. In relation to the nucleic acid molecule of the present invention, the binding free energy between nucleic acid molecule and its complementary sequence is sufficient to allow the relevant function of nucleic acid to proceed, such as RNAi activity. The determination of the binding free energy of nucleic acid molecules is well known in the art (see, for example, 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). The percentage of complementarity indicates the percentage of consecutive 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 are 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Perfectly complementary" or 100% complementarity means that all consecutive residues of a nucleic acid sequence will hydrogen bond with the same number of consecutive residues in a second nucleic acid sequence. Less than perfect complementarity refers to a situation in which some (but not all) nucleoside units of two strands can hydrogen bond with each other. "Substantial complementarity" refers to polynucleotide strands that exhibit 90% or greater complementarity, excluding regions of the polynucleotide strands, such as overhangs, that are selected to be non-complementary. Specific binding requires a sufficient degree of complementarity to avoid non-specific binding of the oligomeric compound to non-target sequences under the conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatments, or under the conditions in which the assay is performed in the case of in vitro assays. Non-target sequences typically differ by at least 5 nucleotides.

[0183] In some embodiments, the double-stranded region of a double-stranded iRNA agent is at least or equal to 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.

[0184] In some embodiments, the antisense strand of a double-stranded iRNA agent is at least equal to or is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0185] In some embodiments, the sense strand of a double-stranded iRNA agent is at least equal to or is 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.

[0186] In one embodiment, the sense and antisense strands of the double-stranded iRNA agent are each 15-30 nucleotides in length.

[0187] In one embodiment, the sense and antisense strands of the double-stranded iRNA agent are each 19-25 nucleotides in length.

[0188] In one embodiment, the sense and antisense strands of the double-stranded iRNA agent are each 21-23 nucleotides in length.

[0189] In some embodiments, one strand has at least one stretch of 1 to 5 single-stranded nucleotides within the double-stranded region. A "single-stranded nucleotide stretch within a double-stranded region" means that there is at least one nucleotide base pair on both ends of the single-stranded stretch. In some embodiments, both strands have at least one stretch of 1 to 5 (e.g., 1, 2, 3, 4, or 5) single-stranded nucleotides within the double-stranded region. When both strands have a stretch of 1 to 5 (e.g., 1, 2, 3, 4, or 5) single-stranded nucleotides within the double-stranded region, such single-stranded nucleotides can be highly opposite (e.g., a mismatched stretch), or they can be positioned such that the second strand does not have a single-stranded nucleotide opposite the single-stranded iRNA of the first strand, or 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 nucleotides from either the 5' or 3' end of the region of complementarity between the two strands.

[0190] In one embodiment, the double-stranded iRNA agent includes a single-stranded overhang on at least one of its ends. In one embodiment, the single-stranded overhang is 1, 2, or 3 nucleotides in length.

[0191] In one embodiment, the sense strand of the iRNA agent is 21 nucleotides in length and the antisense strand is 23 nucleotides in length, where the strands form a double-stranded region of 21 contiguous base pairs with a 2-nucleotide-long single-stranded overhang at the 3' end.

[0192] In some embodiments, each strand of the double-stranded iRNA has a ZXY structure, such as that described in PCT Publication No. WO2004080406, the entire contents of which are incorporated herein by reference.

[0193] In certain embodiments, the two strands of a double-stranded oligomeric compound may be linked to each other. The two strands may be linked to each other at both ends or only one end. Linked at one end means that the 5' end of the first strand is linked to the 3' end of the second strand, or the 3' end of the first strand is linked to the 5' end of the second strand. When the two strands are linked to each other at both ends, the 5' end of the first strand is linked to the 3' end of the second strand, and the 3' end of the first strand is linked to the 5' end of the second strand. The two strands may be, but are not limited to, (N) n (where N is independently a modified or unmodified nucleotide, and n is 3 to 23). In some embodiments, n is 3 to 10, e.g., 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the oligonucleotide linker is selected from the group consisting of GNRA, (G)4, (U)4, and (dT)4, where N is a modified or unmodified nucleotide and R is a modified or unmodified purine nucleotide. Some of the nucleotides in the linker may participate in base-pairing interactions with other nucleotides in the linker. The two strands may also be linked to each other by a non-nucleoside linker, such as the linkers described herein. It will be understood by those skilled in the art that any of the oligonucleotide chemical modifications or alterations described herein may be used in the oligonucleotide linker.

[0194] Hairpin and dumbbell-shaped oligomeric compounds have duplex regions of at least or equal to 14, 15, 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The duplex region can be 200, 100, or 50 or less in length. In some embodiments, the duplex region ranges from 15-30, 17-23, 19-23, and 19-21 nucleotide pairs in length.

[0195] Hairpin oligomeric compounds may have a single-stranded overhang or terminal unpaired region at the 3' end, and in some embodiments, at the antisense end of the hairpin. In some embodiments, the overhang is 1 to 4, more typically 2 to 3, nucleotides in length. Hairpin oligomeric compounds capable of inducing RNA interference are also referred to herein as "shRNAs."

[0196] In certain embodiments, two oligomer strands specifically hybridize when there is a sufficient degree of complementarity to avoid nonspecific binding of the antisense compound to non-target nucleic acid sequences under conditions where specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatments, and under the conditions under which the assay is performed in the case of in vitro assays.

[0197] As used herein, " stringent hybridization conditions " or " stringent conditions " refers to the conditions under which an antisense compound hybridizes to its target sequence but hybridizes to a minimum number of other sequences. Stringent conditions are sequence-dependent and vary in various circumstances, and the " stringent conditions " under which an antisense compound hybridizes to a target sequence are determined by the nature and composition of the antisense compound and the assay they are tested.

[0198] It is understood in the art that incorporating nucleotide affinity modification can allow a greater number of mismatches compared to unmodified compounds.Similarly, certain oligonucleotide sequences can be more tolerant to mismatches than other oligonucleotide sequences.Those skilled in the art can determine the appropriate number of mismatches between oligonucleotides or between oligonucleotides and target nucleic acid, for example, by determining melting temperature (Tm).Tm or ΔTm can be calculated by techniques well known to those skilled in the art.For example, those skilled in the art can evaluate nucleotide modification for its ability to increase the melting temperature of RNA:DNA duplex by the technique described in Freier et al. (Nucleic Acids Research, 1997,25,22:4429-4443).

[0199] siRNA design In one embodiment, an iRNA agent of the invention is a 19-nt long double-ended bluntmer, in which the sense strand contains at least one motif of three 2'-F modifications in three consecutive nucleotides at positions 7, 8, and 9 from the 5' end, and the antisense strand contains at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0200] In one embodiment, an iRNA agent of the invention is a 20 nt long blunt-ended duplex, in which the sense strand contains at least one motif of three 2'-F modifications in three consecutive nucleotides at positions 8, 9, and 10 from the 5' end, and the antisense strand contains at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0201] In one embodiment, an iRNA agent of the invention is a 21 nt long blunt-ended duplex, in which the sense strand contains at least one motif of three 2'-F modifications in three consecutive nucleotides at positions 9, 10, and 11 from the 5' end, and the antisense strand contains at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0202] In one embodiment, the iRNA agent of the present invention comprises a 21-nucleotide (nt) sense strand and a 23-nucleotide (nt) antisense strand, wherein the sense strand comprises at least one motif of three 2'-F modifications in three consecutive nucleotides at positions 9, 10, and 11 from the 5' end; and the antisense strand comprises at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, wherein one end of the iRNA is blunt, while the other end comprises a 2-nt overhang. Preferably, the 2-nt overhang is at the 3' end of the antisense strand. Optionally, the iRNA agent further comprises a ligand (e.g., GalNAc3).

[0203] In one embodiment, the iRNA agent of the invention comprises a sense strand and an antisense strand, wherein the sense strand is 25 to 30 nucleotide residues long and, starting from the 5'-terminal nucleotide (position 1), positions 1 to 23 of the first strand comprise at least 8 ribonucleotides; the antisense strand is 36 to 66 nucleotide residues long and, starting from the 3'-terminal nucleotide, comprises at least 8 ribonucleotides at positions paired with positions 1 to 23 of the sense strand to form a duplex; wherein at least the 3'-terminal nucleotide of the antisense strand is not paired with the sense strand, and up to 6 consecutive 3'-terminal nucleotides are not paired with the sense strand, thereby forming a 3' single-stranded overhang of 1 to 6 nucleotides; wherein the 5' end of the antisense strand comprises 10 to 30 consecutive ribonucleotides that are not paired with the sense strand. the sense strand comprises consecutive nucleotides, thereby forming a 10-30 nucleotide single-stranded 5' overhang; wherein at least the 5'- and 3'-terminal nucleotides of the sense strand are base-paired with nucleotides of the antisense strand when the sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially duplexed region between the sense and antisense strands; the antisense strand is sufficiently complementary to the target RNA along at least 19 ribonucleotides of the antisense strand length so as to reduce target gene expression when the double-stranded nucleic acid is introduced into a mammalian cell; wherein the sense strand comprises at least one motif of three 2'-F modifications in three consecutive nucleotides, at least one of the motifs being at or near the cleavage site; and the antisense strand comprises at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides at or near the cleavage site.

[0204] In one embodiment, an iRNA agent of the invention includes a sense strand and an antisense strand, wherein the iRNA agent includes a first strand having a length of at least 25 and no more than 29 nucleotides, and a second strand having a length of no more than 30 nucleotides, the second strand including at least one motif of three 2'-O-methyl modifications at three consecutive nucleotides at positions 11, 12, and 13 from the 5' end; wherein the 3' end of the first strand and the 5' end of the second strand form a blunt end, the second strand is 1-4 nucleotides longer than the first strand at its 3' end, the duplex region is at least 25 nucleotides long, and the second strand is sufficiently complementary to a target mRNA along at least 19 nt of the length of the second strand such that the iRNA agent reduces target gene expression when introduced into a mammalian cell, and wherein Dicer cleavage of the iRNA preferentially results in an siRNA including the 3' end of the second strand, thereby reducing target gene expression in a mammal. Optionally, the iRNA agent further includes a ligand (eg, GalNAc3).

[0205] In one embodiment, the sense strand of the iRNA agent contains at least one motif of three identical modifications in three consecutive nucleotides, where one of the motifs is at the cleavage site of the sense strand. For example, the sense strand can contain at least one motif of three 2'-F modifications in three consecutive nucleotides within positions 7-15 from the 5' end.

[0206] In one embodiment, the antisense strand of an iRNA agent can also contain at least one motif of three identical modifications in three consecutive nucleotides, where one of the motifs is at or near the cleavage site of the antisense strand. For example, the antisense strand can contain at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides within positions 9-15 from the 5' end.

[0207] For iRNA agents having a duplex region 17-23 nt in length, the cleavage sites in the antisense strand are typically near positions 10, 11, and 12 from the 5' end. Thus, the three identical modification motifs can be located at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, counting from the first nucleotide from the 5' end of the antisense strand, or from the first paired nucleotide in the duplex region from the 5' end of the antisense strand. The cleavage site in the antisense strand can also vary depending on the length of the duplex region of the iRNA from the 5' end.

[0208] In some embodiments, the iRNA agent includes a sense strand and an antisense strand, each having 14 to 30 nucleotides, wherein the sense strand includes at least two motifs of three identical modifications in three consecutive nucleotides, where at least one of the motifs is at or near the cleavage site within the strand, and at least one of the motifs is in another portion of the strand separated from the motif at the cleavage site by at least one nucleotide. In one embodiment, the antisense strand can also include at least one motif of three identical modifications in three consecutive nucleotides, where at least one of the motifs is at or near the cleavage site within the strand. The modifications in the motifs at or near the cleavage site in the sense strand are different from the modifications in the motifs at or near the cleavage site in the antisense strand.

[0209] In some embodiments, the iRNA agent includes a sense strand and an antisense strand, each having 14 to 30 nucleotides, wherein the sense strand includes at least one motif of three 2'-F modifications in three consecutive nucleotides, where at least one of the motifs is at or near the site of cleavage in the strand. In one embodiment, the antisense strand also includes at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides at or near the site of cleavage.

[0210] In some embodiments, the iRNA agent includes a sense strand and an antisense strand, each having 14 to 30 nucleotides, wherein the sense strand includes at least one motif of three 2'-F modifications in three consecutive nucleotides at positions 9, 10, and 11 from the 5' end, and the antisense strand includes at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0211] In one embodiment, the iRNA agent of the present invention contains mismatches with the target, mismatches within the duplex, or a combination thereof. Mismatches can occur in overhang regions or duplex regions. Base pairs can be evaluated based on their tendency to promote dissociation or melting (e.g., relative to the free energy of binding or dissociation of a particular pairing; the simplest approach is to examine each pair individually, but similar or equivalent analyses can also be used). With regard to promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; I:C is preferred over G:C (I = inosine). Mismatches, such as non-canonical or non-canonical pairings (described elsewhere herein), are preferred over canonical (A:T, A:U, G:C) pairings; pairings involving universal bases are preferred over canonical pairings.

[0212] In one embodiment, an iRNA agent of the invention includes at least one of the first 1, 2, 3, 4, or 5 base pairs within the duplex region from the 5' end of the antisense strand, which may be independently selected from the group A:U, G:U, I:C, and a mismatch pair, e.g., a non-canonical or non-canonical pairing or a pairing containing a universal base, to promote dissociation of the antisense strand at the 5' end of the duplex.

[0213] In one embodiment, the nucleotide at position 1 in the double-stranded region from the 5' end of 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 pairs in the double-stranded region from the 5' end of the antisense strand is an AU base pair. For example, the first base pair in the double-stranded region from the 5' end of the antisense strand is an AU base pair.

[0214] In one aspect, the present invention relates to a double-stranded RNA (dsRNA) agent for inhibiting expression of a target gene. The dsRNA agent includes a sense strand and an antisense strand, each strand having 14 to 40 nucleotides. The dsRNA agent is represented by formula (I): [ka]

[0215] In Formula (I), B1, B2, B3, B1', B2', B3', and B4' are each 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 a 2'-OMe modification. In one embodiment, B1, B2, B3, B1', B2', B3', and B4' each contain a 2'-OMe modification or a 2'-F modification. In one embodiment, at least one of B1, B2, B3, B1', B2', B3', and B4' contains a 2'-ON-methylacetamide (2'-O-NMA) modification.

[0216] C1 is a thermolabile nucleotide located at the site opposite the seed region of the antisense strand (i.e., positions 2-8 of the 5' end of the antisense strand). For example, C1 is located in the sense strand at a position that pairs with nucleotides 2-8 of the 5' end of the antisense strand. In one example, C1 is located at position 15 of the 5' end of the sense strand. The C1 nucleotide has a thermolabile modification that can include an abasic modification; a mismatch with the opposing nucleotide of the duplex; and a sugar modification, such as a 2'-deoxy modification, or an acyclic nucleotide, such as an unlocked nucleic acid (UNA) or a glycerol nucleic acid (GNA). In one embodiment, C1 (i) is a mismatch with the opposing nucleotide of the antisense strand; or (ii) an abasic modification selected from the group consisting of: [ka] and (iii) a sugar modification selected from the group consisting of: [ka] wherein B is a modified or unmodified nucleobase and R 1 and R 2 are independently H, halogen, OR3, or alkyl; and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar. In one embodiment, the thermolabilizing modification of 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; optionally, at least one nucleobase in the mismatch pair is a 2'-deoxy-nucleobase. In one example, the thermolabilizing modification in C1 is GNA or [ka] is.

[0217] T1, T1', T2', and T3' each independently represent a nucleotide containing a modification that provides the nucleotide with steric bulk equal to or less than that of a 2'-OMe modification. Steric bulk refers to the total steric effect of the modification. Methods for determining the steric effect of a nucleotide modification are known to those skilled in the art. The modification may be a modification at the 2' position of the ribose sugar of the nucleotide, or a modification of the backbone of a non-ribose nucleotide, an acyclic nucleotide, or a similar or equivalent modification at the 2' position of the ribose sugar, and provides the nucleotide with steric bulk equal to or less than that of a 2'-OMe modification. 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.

[0218] n 1 , n 3 , and q 1 are independently 4 to 15 nucleotides in length.

[0219] n 5 , q 3 , and q 7 are independently 1 to 6 nucleotides in length.

[0220] n 4 , q 2 , and q 6 are independently 1 to 3 nucleotides in length; or 4 is 0.

[0221] q 5 are independently 0 to 10 nucleotides in length.

[0222] n 2 and q 4 are independently 0 to 3 nucleotides in length.

[0223] Or, n 4is 0 to 3 nucleotides in length.

[0224] In one embodiment, n 4 can be 0. In one example, n 4 is 0 and q 2 and q 6 is 1. In another example, n 4 is 0 and q 2 and q 6 is 1, and has two phosphorothioate internucleotide linkage modifications within positions 1 to 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 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand.

[0225] In one embodiment, n 4 , q 2 , and q 6 are each 1.

[0226] In one embodiment, n 2 , n 4 , q 2 , q 4 , and q 6 are each 1.

[0227] In one embodiment, when the sense strand is 19 to 22 nucleotides in length, C1 is at positions 14 to 17 of the 5' end of the sense strand, and n 4 is 1. In one embodiment, C1 is at position 15 of the 5' end of the sense strand.

[0228] In one embodiment, T3' begins at position 2 of the 5' end of the antisense strand. In one example, T3' is at position 2 of the 5' end of the antisense strand, and 6 is equal to 1.

[0229] In one embodiment, T1' begins at position 14 of the 5' end of the antisense strand. In one example, T1' is at position 14 of the 5' end of the antisense strand and 2is equal to 1.

[0230] In an exemplary embodiment, T3' begins at position 2 of the 5' end of the antisense strand, and T1' begins at position 14 of the 5' end of the antisense strand. In one example, T3' begins at position 2 of the 5' end of the antisense strand, and q 6 is equal to 1, T1' starts from position 14 of the 5' end of the antisense strand, and q 2 is equal to 1.

[0231] In one embodiment, T1' and T3' are separated by 11 nucleotides (ie, not counting the T1' and T3' nucleotides).

[0232] In one embodiment, T1' is at position 14 of the 5' end of the antisense strand. 2 is equal to 1 and the modification is at the 2' position or at a non-ribose, acyclic, or backbone position that provides less steric bulk than 2'-OMe ribose.

[0233] In one embodiment, T3' is at position 2 of the 5' end of the antisense strand. In one example, T3' is at position 2 of the 5' end of the antisense strand, and 6 is equal to 1 and the modification is at the 2' position or at a non-ribose, acyclic, or backbone position that provides steric bulk less than or equal to that of 2'-OMe ribose.

[0234] In one embodiment, T1 is at the cleavage site of the sense strand. In one example, when the sense strand is 19-22 nucleotides long, T1 is at position 11 of the 5' end of the sense strand, n 2 is 1. In an exemplary embodiment, when the sense strand is 19-22 nucleotides in length, T1 is at the cleavage site of the sense strand at position 11 at the 5' end of the sense strand, and n 2 is 1.

[0235] In one embodiment, T2' begins at position 6 of the 5' end of the antisense strand. In one example, T2' is from position 6 to position 10 of the 5' end of the antisense strand, and 4 is 1.

[0236] In an exemplary embodiment, T1 is at the cleavage site of the sense strand, e.g., at position 11 of the 5' end of the sense strand, when the sense strand is 19-22 nucleotides in length, and n 2 is 1; T1' is at position 14 of the 5' end of the antisense strand, and q 2 is equal to 1, and the T1' modification is at the 2' position of the ribose sugar or at a non-ribose, acyclic, or backbone position that provides less steric bulk than 2'-OMe ribose; T2' is located at positions 6-10 of the 5' end of the antisense strand, and q 4 is 1; and T3' is at position 2 of the 5' end of the antisense strand, and q 6 is equal to 1, and the T3' modification is at the 2' position or at a non-ribose, acyclic, or backbone position that provides steric bulk less than or equal to that of 2'-OMe ribose.

[0237] In one embodiment, T2' begins at position 8 of the 5' end of the antisense strand. 4 is 2.

[0238] In one embodiment, T2' begins at position 9 of the 5' end of the antisense strand. 4 is 1.

[0239] In one embodiment, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5is 6, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 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 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand.

[0240] In one embodiment, n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5 is 6, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 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 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand.

[0241] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1.

[0242] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 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 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand.

[0243] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 6, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 7, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1.

[0244] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 6, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 7, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 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 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand.

[0245] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5 is 6, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1.

[0246] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5 is 6, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 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 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand.

[0247] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 5, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; optionally with at least two additional TTs at the 3' end of the antisense strand.

[0248] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 5, T2' is 2'-F, and q 4 is 1, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7is 1; optionally has at least two additional TTs at the 3' end of the antisense strand; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5' end of the sense strand), as well as two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand.

[0249] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1.

[0250] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 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 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand.

[0251] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1.

[0252] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 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 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand.

[0253] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1.

[0254] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 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 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand.

[0255] The dsRNA agent can include a phosphorus-containing group at the 5'-end of either the sense or antisense strand. The 5'-terminal phosphorus-containing group can be 5'-terminal phosphate (5'-P), 5'-terminal phosphorothioate (5'-PS), 5'-terminal phosphorodithioate (5'-PS2), 5'-terminal vinylphosphonate (5'-VP), 5'-terminal methylphosphonate (MePhos), or 5'-deoxy 5'-C-malonyl. [ka] When the 5'-terminal phosphorus-containing group is a 5'-terminal vinyl phosphonate (5-VP), the 5'-VP may be a 5'-E-VP isomer (i.e., trans-vinyl phosphate, [ka] ), or the 5'-Z-VP isomer (i.e., cis-vinyl phosphate, [ka] ) or a mixture thereof.

[0256] In one embodiment, the dsRNA agent includes a phosphorus-containing group at the 5'-end of the sense strand.In one embodiment, the dsRNA agent includes a phosphorus-containing group at the 5'-end of the antisense strand.

[0257] In one embodiment, the dsRNA agent includes a 5'-P. In one embodiment, the dsRNA agent includes a 5'-P in the antisense strand.

[0258] In one embodiment, the dsRNA agent comprises a 5'-PS.In one embodiment, the dsRNA agent comprises a 5'-PS on the antisense strand.

[0259] 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.

[0260] In one embodiment, the dsRNA agent comprises a 5'-PS2.In one embodiment, the dsRNA agent comprises a 5'-PS2 in the antisense strand.

[0261] In one embodiment, the dsRNA agent comprises a 5'-PS2. In one embodiment, the dsRNA agent comprises a 5'deoxy5'-C-malonyl in the antisense strand.

[0262] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The dsRNA agent also includes a 5'-PS.

[0263] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The dsRNA agent also includes a 5'-P.

[0264] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The dsRNA agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0265] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The dsRNA agent also includes a 5'-PS2.

[0266] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The dsRNA agent also includes a 5'-deoxy-5'-C-malonyl.

[0267] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 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 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes a 5'-P.

[0268] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 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 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes a 5'-PS.

[0269] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 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 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0270] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 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 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes a 5'-PS2.

[0271] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 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 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes 5'-deoxy-5'-C-malonyl.

[0272] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The dsRNA agent also includes a 5'-P.

[0273] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The dsRNA agent also includes a 5'-PS.

[0274] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The dsRNA agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0275] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The dsRNA agent also includes a 5'-PS2.

[0276] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1. The dsRNA agent also includes a 5'-deoxy-5'-C-malonyl.

[0277] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 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 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes a 5'-P.

[0278] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 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 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes a 5'-PS.

[0279] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 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 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0280] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 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 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes a 5'-PS2.

[0281] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 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 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes 5'-deoxy-5'-C-malonyl.

[0282] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The dsRNA agent also includes a 5'-P.

[0283] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The dsRNA agent also includes a 5'-PS.

[0284] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The dsRNA agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0285] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The dsRNA agent also includes a 5'-PS2.

[0286] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The dsRNA agent also includes a 5'-deoxy-5'-C-malonyl.

[0287] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 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 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes a 5'-P.

[0288] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 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 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes a 5'-PS.

[0289] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 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 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0290] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 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 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes a 5'-PS2.

[0291] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 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 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes 5'-deoxy-5'-C-malonyl.

[0292] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The dsRNA agent also includes a 5'-P.

[0293] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The dsRNA agent also includes a 5'-PS.

[0294] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The dsRNA agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0295] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The dsRNA agent also includes a 5'-PS2.

[0296] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1. The dsRNA agent also includes a 5'-deoxy-5'-C-malonyl.

[0297] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 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 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes a 5'-P.

[0298] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 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 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes a 5'-PS.

[0299] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 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 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes a 5'-VP. The 5'-VP can be a 5'-E-VP, a 5'-Z-VP, or a combination thereof.

[0300] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 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 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes a 5'-PS2.

[0301] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 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 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes 5'-deoxy-5'-C-malonyl.

[0302] 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 present invention is modified.For example, when 50% of the dsRNA agent is modified, 50% of all the nucleotides present in the dsRNA agent comprise modification as described herein.

[0303] In one embodiment, the sense and antisense strands of a dsRNA agent are each independently modified with an acyclic nucleotide, LNA, HNA, CeNA, 2'-methoxyethyl, 2'O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-fluoro, 2'-ON-methylacetamide (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), or 2'-ara-F.

[0304] In one embodiment, the sense and antisense strands of the dsRNA agent each contain at least two different modifications.

[0305] In one embodiment, the dsRNA agent of Formula (I) further comprises a 3' and / or 5' overhang of 1 to 10 nucleotides in length. In one example, the 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.

[0306] In one embodiment, a dsRNA agent of the invention does not include any 2'-F modifications.

[0307] In one embodiment, the sense strand and / or antisense strand of a 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 to 18 phosphate internucleotide linkages.

[0308] In one embodiment, the sense and antisense strands of the dsRNA agent each have 15 to 30 nucleotides. In one example, the sense strand has 19 to 22 nucleotides and the antisense strand has 19 to 25 nucleotides. In another example, the sense strand has 21 nucleotides and the antisense strand has 23 nucleotides.

[0309] 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 pairs from the 5' end of the antisense strand is an AU base pair.

[0310] In one embodiment, the antisense strand of the dsRNA agent of the present invention is 100% complementary to target RNA, so as to hybridize with target RNA and inhibit its expression by RNA interference.In another embodiment, the antisense strand of the dsRNA agent of the present 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 target RNA.

[0311] In one aspect, the present invention relates to a dsRNA agent, as defined herein, capable of inhibiting 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 comprises at least one thermolabile nucleotide, at least one of which is located opposite or near the seed region of the antisense strand (i.e., positions 2 to 8 of the 5' end of the antisense strand). Each of the embodiments and aspects described herein relating to a dsRNA represented by Formula (I) can also be applied to a dsRNA comprising a thermolabile nucleotide.

[0312] For example, if the sense strand is 21 nucleotides long, the thermally destabilizing nucleotide may be located between positions 14 and 17 at the 5' end of the sense strand. The antisense strand comprises at least two modified nucleic acids that are smaller than sterically demanding 2'-OMe modifications. Preferably, the two modified nucleic acids that are smaller than sterically demanding 2'-OMe are spaced 11 nucleotides apart. For example, the two modified nucleic acids are located at positions 2 and 14 at the 5' end of the antisense strand.

[0313] In one embodiment, the dsRNA agent further comprises at least one ASGPR ligand. For example, the ASGPR ligand may be a bivalent or trivalent branched linker, such as: [ka] In one example, the ASGPR ligand is added to the 3' end of the sense strand.

[0314] For example, the dsRNA agent described herein can comprise: (i) a phosphorus-containing group at the 5'-end of the sense strand or antisense strand; (ii) two phosphorothioate internucleotide bond modifications within the 1st to 5th positions (counting from the 5'-end of the sense strand) of the sense strand, and two phosphorothioate internucleotide bond modifications at the 1st and 2nd positions (counting from the 5'-end of the antisense strand) of the antisense strand, and two phosphorothioate internucleotide bond modifications within the 18th to 23rd positions; and (iii) a ligand at the 5'-end or 3'-end of the sense strand or antisense strand, for example, an ASGPR ligand (for example, one or more GalNAc derivatives).For example, this ligand can be present at the 3'-end of the sense strand.

[0315] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 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 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand. The dsRNA agent also includes 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.

[0316] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 5 (counting from the 5' end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand. The dsRNA agent also includes 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.

[0317] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 5 (counting from the 5' end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand. The dsRNA agent also includes a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a 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.

[0318] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 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 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand. The dsRNA agent also includes a 5'-PS2 and 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.

[0319] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 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 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes 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.

[0320] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 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 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand. The dsRNA agent also includes 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.

[0321] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 5 (counting from the 5' end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand. The dsRNA agent also includes 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.

[0322] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 5 (counting from the 5' end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand. The dsRNA agent also includes a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a 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.

[0323] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 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 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand. The dsRNA agent also includes a 5'-PS2 and 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.

[0324] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-OMe, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 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 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes 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.

[0325] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 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 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand. The dsRNA agent also includes 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.

[0326] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 5 (counting from the 5' end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand. The dsRNA agent also includes 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.

[0327] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 5 (counting from the 5' end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand. The dsRNA agent also includes a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a 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.

[0328] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 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 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand. The dsRNA agent also includes a 5'-PS2 and 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.

[0329] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4, T2' is 2'-F, and q 4 is 2, B3' is 2'-OMe or 2'-F, and q 5 is 5, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 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 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes 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.

[0330] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 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 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand. The dsRNA agent also includes 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.

[0331] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 5 (counting from the 5' end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand. The dsRNA agent also includes 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.

[0332] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 5 (counting from the 5' end of the sense strand) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end of the antisense strand) of the antisense strand, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand. The dsRNA agent also includes a 5'-VP (e.g., 5'-E-VP, 5'-Z-VP, or a 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.

[0333] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4 is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 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 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand. The dsRNA agent also includes a 5'-PS2 and 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.

[0334] In one embodiment, B1 is 2'-OMe or 2'-F, and n 1 is 8, T1 is 2'F, and n 2 is 3, B2 is 2'-OMe, and n 3 is 7, and n 4 is 0, B3 is 2'-OMe, and n 5 is 3, B1' is 2'-OMe or 2'-F, and q 1 is 9, T1' is 2'-F, and q 2 is 1, B2' is 2'-OMe or 2'-F, and q 3 is 4 and q 4is 0, B3' is 2'-OMe or 2'-F, and q 5 is 7, T3' is 2'-F, and q 6 is 1, B4' is 2'-F, and q 7 is 1; and has two phosphorothioate internucleotide linkage modifications within positions 1 to 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 of the antisense strand (counting from the 5' end of the antisense strand) and two phosphorothioate internucleotide linkage modifications within positions 18 to 23. The dsRNA agent also includes 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.

[0335] In one particular embodiment, a dsRNA agent of the invention: (a) a sense strand comprising: (i) 21 nucleotides long; (ii) an ASGPR ligand attached to its 3' terminus, optionally comprising three GalNAc derivatives attached by a trivalent branched linker; and (iii) a sense strand having 2'-F modifications at positions 1, 3, 5, 7, 9-11, 13, 17, 19, and 21 (counting from the 5' end) and 2'-OMe modifications at positions 2, 4, 6, 8, 12, 14-16, 18, and 20; and (b) an antisense strand: (i) 23 nucleotides long; (ii) 2'-OMe modifications at positions 1, 3, 5, 9, 11-13, 15, 17, 19, 21, and 23 (counting from the 5' end) and 2'F modifications at positions 2, 4, 6-8, 10, 14, 16, 18, 20, and 22; and (iii) an antisense strand having phosphorothioate internucleotide linkages between nucleotide positions 21 and 22 (counting from the 5' end) and between nucleotide positions 22 and 23; The dsRNA agent has an antisense strand with a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0336] In another specific embodiment, the dsRNA agent of the invention is: (a) a sense strand comprising: (i) 21 nucleotides long; (ii) an ASGPR ligand attached to the 3' end, comprising three GalNAc derivatives optionally attached by a trivalent branched linker; (iii) 2'-F modifications at positions 1, 3, 5, 7, 9-11, 13, 15, 17, 19, and 21 (counting from the 5' end) and 2'-OMe modifications at positions 2, 4, 6, 8, 12, 14, 16, 18, and 20; and (iv) a sense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end) and between nucleotide positions 2 and 3; and (b) an antisense strand: (i) 23 nucleotides long; (ii) 2'-OMe modifications at positions 1, 3, 5, 7, 9, 11-13, 15, 17, 19, and 21-23 (counting from the 5' end) and 2'-F modifications at positions 2, 4, 6, 8, 10, 14, 16, 18, and 20; and (iii) an antisense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end), between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23; The dsRNA agent comprises an antisense strand with a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0337] In another specific embodiment, the dsRNA agent of the invention is: (a) a sense strand comprising: (i) 21 nucleotides long; (ii) an ASGPR ligand attached to the 3' end, comprising three GalNAc derivatives optionally attached by a trivalent branched linker; (iii) 2'-OMe modifications at positions 1 to 6, 8, 10, and 12 to 21 (counting from the 5' end), 2'-F modifications at positions 7 and 9, and a deoxy-nucleotide (e.g., dT) at position 11; and (iv) a sense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end) and between nucleotide positions 2 and 3; and (b) an antisense strand comprising: (i) 23 nucleotides long; (ii) 2'-OMe modifications at positions 1, 3, 7, 9, 11, 13, 15, 17, and 19-23 (counting from the 5' end), and 2'-F modifications at positions 2, 4-6, 8, 10, 12, 14, 16, and 18; and (iii) an antisense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end), between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23; The dsRNA agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0338] In another specific embodiment, the dsRNA agent of the invention is: (a) a sense strand comprising: (i) 21 nucleotides long; (ii) an ASGPR ligand attached to the 3' end, comprising three GalNAc derivatives optionally attached by 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) a sense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end) and between nucleotide positions 2 and 3; and (b) an antisense strand: (i) 23 nucleotides long; (ii) 2'-OMe modifications at positions 1, 5, 7, 9, 11, 13, 15, 17, 19, and 21-23 (counting from the 5' end), and 2'-F modifications at positions 2-4, 6, 8, 10, 12, 14, 16, 18, and 20; and (iii) an antisense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end), between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23; The dsRNA agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0339] In another specific embodiment, the dsRNA agent of the invention is: (a) a sense strand comprising: (i) 21 nucleotides long; (ii) an ASGPR ligand attached to the 3' end, comprising three GalNAc derivatives optionally attached by 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) a sense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end) and between nucleotide positions 2 and 3; and (b) an antisense strand: (i) 23 nucleotides long; (ii) 2'-OMe modifications at positions 1, 5, 7, 9, 11-13, 15, 17, 19, and 21-23 (counting from the 5' end) and 2'-F modifications at positions 2, 4, 6, 8, 10, 14, 16, 18, and 20; and (iii) an antisense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end), between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23; The dsRNA agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0340] In another specific embodiment, the dsRNA agent of the invention is: (a) a sense strand comprising: (i) 21 nucleotides long; (ii) an ASGPR ligand attached to the 3' end, comprising three GalNAc derivatives optionally attached by a trivalent branched linker; (iii) 2'-F modifications at positions 1, 3, 5, 7, 9-11, and 13, and 2'-OMe modifications at positions 2, 4, 6, 8, 12, and 14-21; and (iv) a sense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end) and between nucleotide positions 2 and 3; and (b) an antisense strand: (i) 23 nucleotides long; (ii) 2'-OMe modifications at positions 1, 3, 5-7, 9, 11-13, 15, 17-19, and 21-23 (counting from the 5' end), and 2'-F modifications at positions 2, 4, 8, 10, 14, 16, and 20; and (iii) an antisense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end), between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23; The dsRNA agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0341] In another specific embodiment, the dsRNA agent of the invention is: (a) a sense strand comprising: (i) 21 nucleotides long; (ii) an ASGPR ligand attached to the 3' end, comprising three GalNAc derivatives optionally attached by a trivalent branched linker; (iii) 2'-OMe modifications at positions 1, 2, 4, 6, 8, 12, 14, 15, 17, and 19-21, and 2'-F modifications at positions 3, 5, 7, 9-11, 13, 16, and 18; and (iv) a sense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end) and between nucleotide positions 2 and 3; and (b) an antisense strand: (i) 25 nucleotides long; (ii) 2'-OMe modifications at positions 1, 4, 6, 7, 9, 11-13, 15, 17, and 19-23 (counting from the 5' end), 2'-F modifications at positions 2, 3, 5, 8, 10, 14, 16, and 18, and deoxy-nucleotides (e.g., dT) at positions 24 and 25; and (iii) an antisense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end), between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23; The dsRNA agent has a four nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0342] In another specific embodiment, the dsRNA agent of the invention is: (a) a sense strand comprising: (i) 21 nucleotides long; (ii) an ASGPR ligand attached to the 3' end, comprising three GalNAc derivatives optionally attached by 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) a sense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end) and between nucleotide positions 2 and 3; and (b) an antisense strand comprising: (i) 23 nucleotides long; (ii) 2'-OMe modifications at positions 1, 3-5, 7, 8, 10-13, 15, and 17-23 (counting from the 5' end), and 2'-F modifications at positions 2, 6, 8, 9, 14, and 16; and (iii) an antisense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end), between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23; The dsRNA agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0343] In another specific embodiment, the dsRNA agent of the invention is: (a) a sense strand comprising: (i) 21 nucleotides long; (ii) an ASGPR ligand attached to the 3' end, comprising three GalNAc derivatives optionally attached by 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) a sense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end) and between nucleotide positions 2 and 3; and (b) an antisense strand comprising: (i) 23 nucleotides long; (ii) 2'-OMe modifications at positions 1, 3-5, 7, 10-13, 15, and 17-23 (counting from the 5' end), and 2'-F modifications at positions 2, 6, 8, 9, 14, and 16; and (iii) an antisense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end), between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23; The dsRNA agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0344] In another specific embodiment, the dsRNA agent of the invention is: (a) a sense strand comprising: (i) 19 nucleotides long; (ii) an ASGPR ligand attached to the 3' end, comprising three GalNAc derivatives optionally attached by 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) a sense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end) and between nucleotide positions 2 and 3; and (b) an antisense strand: (i) 21 nucleotides long; (ii) 2'-OMe modifications at positions 1, 3-5, 7, 10-13, 15, and 17-21 (counting from the 5' end), and 2'-F modifications at positions 2, 6, 8, 9, 14, and 16; and (iii) an antisense strand having phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5' end), between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21; The dsRNA agent has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0345] In one embodiment, a dsRNA agent of the invention comprises: (a) a sense strand, (i) 18–23 nucleotides long; (ii) a sense strand having three consecutive 2'-F modifications at positions 7 to 15; and (b) an antisense strand, (i) 18–23 nucleotides long; (ii) at least a 2'-F modification anywhere on the strand; and (iii) comprises an antisense strand having at least two phosphorothioate internucleotide linkages in the first five nucleotides (counting from the 5' end); The dsRNA agent has either one or more lipophilic moieties conjugated to one or more positions on at least one strand; a two-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand; or blunt ends at both ends of the duplex.

[0346] In one embodiment, a dsRNA agent of the invention comprises: (a) a sense strand, (i) 18–23 nucleotides long; (ii) the sense strand, having fewer than four 2'-F modifications; (b) an antisense strand, (i) 18–23 nucleotides long; (ii) fewer than 12 2'-F modifications; and (iii) comprises an antisense strand having at least two phosphorothioate internucleotide linkages in the first five nucleotides (counting from the 5' end); The dsRNA agent has either one or more lipophilic moieties conjugated to one or more positions on at least one strand; a two-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand; or blunt ends at both ends of the duplex.

[0347] In one embodiment, a dsRNA agent of the invention comprises: (a) a sense strand, (i) 19–35 nucleotides long; (ii) the sense strand, having fewer than four 2'-F modifications; (b) an antisense strand, (i) 19–35 nucleotides long; (ii) fewer than 12 2'-F modifications; and (iii) comprises an antisense strand having at least two phosphorothioate internucleotide linkages in the first five nucleotides (counting from the 5' end); wherein the duplex region is 19 to 25 base pairs (preferably 19, 20, 21, or 22); the dsRNA agent has one or more lipophilic moieties conjugated to one or more positions in at least one strand; a two-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand; or blunt ends at both ends of the duplex.

[0348] In one embodiment, a dsRNA agent of the invention has sense and antisense strands having lengths of 15-30 nucleotides; at least two phosphorothioate internucleotide linkages in the first five nucleotides on the antisense strand (counting from the 5' end); wherein the duplexed region is 19-25 base pairs (preferably 19, 20, 21, or 22); the dsRNA agent has one or more lipophilic moieties conjugated to one or more positions in at least one strand; and the dsRNA agent has less than 20%, less than 15%, and less than 10% non-natural nucleotides.

[0349] Examples of non-natural nucleotides include acyclic nucleotides, LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-fluoro, 2'-ON-methylacetamide (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), or 2'-ara-F, and others.

[0350] In one embodiment, a dsRNA agent of the invention has sense and antisense strands having lengths of 15-30 nucleotides; at least two phosphorothioate internucleotide linkages in the first five nucleotides on the antisense strand (counting from the 5' end); wherein the duplexed region is 19-25 base pairs (preferably 19, 20, 21, or 22); the dsRNA agent has one or more lipophilic moieties conjugated to one or more positions in at least one strand; and the dsRNA agent has greater than 80%, greater than 85%, and greater than 90% naturally occurring nucleotides, e.g., 2'-OH, 2'-deoxy, and 2'-OMe are naturally occurring nucleotides.

[0351] In one embodiment, a dsRNA agent of the invention has sense and antisense strands having lengths of 15-30 nucleotides; at least two phosphorothioate internucleotide linkages in the first five nucleotides on the antisense strand (counting from the 5' end); wherein the duplexed region is 19-25 base pairs (preferably 19, 20, 21, or 22); the dsRNA agent has one or more lipophilic moieties conjugated to one or more positions in at least one strand; and the dsRNA agent has 100% naturally occurring nucleotides, e.g., 2'-OH, 2'-deoxy, and 2'-OMe are naturally occurring nucleotides.

[0352] Examples of lipophilic moieties include, but are not limited to, lipids (saturated or unsaturated C4-C30 hydrocarbon chains and optional functional groups selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne), cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl groups, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenoic acid, dimethoxytrityl, or phenoxazine.

[0353] In some embodiments, the lipophilic moiety is a C-C 30 Acids (e.g., hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid, dodecanoic 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 C6 to C 30Alcohols (e.g., hexanol, heptanol, octanol, nonanol, decanol, undecanol, dodecanol, tridecanol, tetradecanol, pentadecanol, hexadecanol, heptadecanol, octadecanol, oleyl alcohol, linoleyl alcohol, arachidonic acid alcohol, cis-4,7,10,13,16,19-docosahexanol, retinol, vitamin E, cholesterol, etc.).

[0354] In one example, the lipophilic moiety is a saturated or unsaturated C6 to C18 hydrocarbon chain.

[0355] In one example, the lipophilic moiety is docosahexaenoic acid.

[0356] In one embodiment, a dsRNA agent of the invention includes a sense strand and an antisense strand, each having 14 to 30 nucleotides, wherein the sense strand sequence is represented by formula (I): 5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' (I) During the ceremony: i and j are each independently 0 or 1; p and q are each independently 0 to 6; each N a independently represent oligonucleotide sequences containing 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each N b represents an oligonucleotide sequence containing, independently, 1, 2, 3, 4, 5, or 6 modified nucleotides; each n p and n q independently represent overhanging nucleotides; N b and Y do not have the same modification; XXX, YYY and ZZZ each independently represent one motif of three identical modifications in three consecutive nucleotides; the dsRNA agent has one or more lipophilic moieties conjugated to one or more positions in at least one strand; The antisense strand of the dsRNA contains two blocks of 1, 2, or 3 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.

[0357] Various publications describe multimeric siRNA, and all of them can be used with the iRNA of the present invention.Such publications include WO2007 / 091269, US Patent No. 7,858,769, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887 and WO2011 / 031520, the entire contents of which are incorporated herein by reference.

[0358] In some embodiments, 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, or 30% of the iRNA agents of the invention are modified.

[0359] In some embodiments, each of the sense and antisense strands of an iRNA agent is independently modified with an acyclic nucleotide, LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-fluoro, 2'-ON-methylacetamido (2'-O-NMA), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-aminopropyl (2'-O-AP), or 2'-ara-F.

[0360] In some embodiments, the sense and antisense strands of an iRNA agent each contain at least two different modifications.

[0361] In some embodiments, the double-stranded iRNA agents of the invention do not include any 2'-F.

[0362] In some embodiments, a double-stranded iRNA agent of the invention includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 2'-F modifications. In one example, a double-stranded iRNA agent of the invention includes 9 or 10 2'-F modifications.

[0363] The iRNA agent of the present invention can further comprise at least one phosphorothioate or methylphosphonate internucleotide bond.Phosphorothioate or methylphosphonate internucleotide bond modification can be present at any nucleotide in the sense strand or antisense strand or both at any position of the strand.For example, internucleotide bond modification can be present at all nucleotides in the sense strand or antisense strand; each internucleotide bond modification can be present in an alternating pattern in the sense strand or antisense strand; or the sense strand or antisense strand can contain both internucleotide bond modifications in an alternating pattern.The alternating pattern of internucleotide bond modification in the sense strand can be the same or different from that of the antisense strand, and the alternating pattern of internucleotide bond modification in the sense strand can have a shift relative to the alternating pattern of internucleotide bond modification in the antisense strand.

[0364] In one embodiment, the iRNA agent comprises a phosphorothioate or methylphosphonate internucleotide bond modification in the overhang region. For example, the overhang region can contain two nucleotides with a phosphorothioate or methylphosphonate internucleotide bond between the two nucleotides. The internucleotide bond modification can also be formed to link the overhang nucleotide to the terminal base-pairing nucleotide in the duplex region. For example, at least two, three, four, or all of the overhang nucleotides can be linked by phosphorothioate or methylphosphonate internucleotide bonds, and optionally, an additional phosphorothioate or methylphosphonate internucleotide bond can be present to link the overhang nucleotide to the next base-pairing nucleotide. For example, at least two phosphothioate internucleotide bonds can be present between the terminal three nucleotides, two of which are overhanging nucleotides, and the third nucleotide is the next base-pairing nucleotide to the overhanging nucleotide. Preferably, these terminal three nucleotides can be present at the 3' end of the antisense strand.

[0365] In some embodiments, the sense strand and / or antisense strand of an iRNA agent contains one or more blocks of phosphorothioate or methylphosphonate internucleotide linkages. In one example, the sense strand contains one block of two phosphorothioate or methylphosphonate internucleotide linkages. In one example, the antisense strand contains two blocks of two phosphorothioate or methylphosphonate internucleotide linkages. For example, the two blocks of phosphorothioate or methylphosphonate internucleotide linkages are separated by 16 to 18 phosphate internucleotide linkages.

[0366] In some embodiments, the antisense strand of an iRNA agent of the invention is 100% complementary to the target RNA in order to hybridize to the target RNA and inhibit its expression by RNA interference. In other embodiments, the antisense strand of an 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 the target RNA.

[0367] In one aspect, the present invention relates to an iRNA agent capable of inhibiting the expression of a target gene. The iRNA agent includes a sense strand and an antisense strand, each strand having 14 to 40 nucleotides. The sense strand includes at least one thermolabile nucleotide, where the at least one thermolabile nucleotide is located at or near the opposite site from the seed region of the antisense strand (i.e., positions 2 to 8 of the 5' end of the antisense strand). For example, if the sense strand is 21 nucleotides long, the thermolabile nucleotide is located at positions 14 to 17 of the 5' end of the sense strand. The antisense strand includes at least two modified nucleic acids that are smaller than sterically bulky 2'-OMe modifications. Preferably, the two modified nucleic acids that are smaller than sterically bulky 2'-OMe are separated by a length of 11 nucleotides. For example, the two modified nucleic acids are located at positions 2 and 14 of the 5' end of the antisense strand.

[0368] In some embodiments, the compounds of the present invention disclosed herein are miRNA mimics. In one design, miRNA mimics are double-stranded molecules (e.g., having a duplex region of about 16 to about 31 nucleotides in length) and contain one or more sequences that share identity with the mature strand of a given miRNA. Double-stranded miRNA mimics have designs similar to those described above for double-stranded iRNAs. In some embodiments, miRNA mimics contain a 16-31 nucleotide duplex region 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 Cs and Us; the antisense strand modifications may include 2'F modifications of all Cs and Us, phosphorylation of the 5' end of the oligonucleotide, and a stable internucleotide linkage coupled with a two-nucleotide 3' overhang.

[0369] In some embodiments, the compounds of the present invention disclosed herein are antimirs. In some embodiments, the compounds of the present invention comprise at least two antimirs covalently or non-covalently linked to each other via a nucleotide-based or non-nucleotide-based linker, such as those described in this disclosure. The terms "antimer," "microRNA inhibitor," or "miR inhibitor" are synonymous and refer to oligonucleotides or modified oligonucleotides that inhibit the activity of specific miRNAs. Inhibitors can take various forms, including single-stranded, double-stranded (RNA / RNA or RNA / DNA duplexes), and hairpin designs. Generally, microRNA inhibitors contain one or more sequences or portions of sequences that are complementary or partially complementary to the mature strand (or strands) of the targeted miRNA. In addition, miRNA inhibitors may also contain additional sequences located 5' and 3' to the sequence that is the reverse complement of the mature miRNA. The additional sequence may be the reverse complement of the sequence adjacent to the mature miRNA in the pri-miRNA from which the mature miRNA is derived, or the additional sequence may be any sequence (having a mixture of A, G, C, U, or dT). In some embodiments, one or both of the additional sequences is any sequence capable of forming a hairpin. Thus, in some embodiments, the sequence that is the reverse complement of the miRNA is flanked on the 5' and 3' sides by hairpin structures. When the microRNA inhibitor is double-stranded, it may contain mismatches between nucleotides in the opposite strands. Furthermore, the microRNA inhibitor may be linked to a conjugate moiety to facilitate uptake of the inhibitor into a cell.

[0370] 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 WO 2007 / 095387 and WO 2008 / 036825, the entire contents of each of which are incorporated herein by reference. One skilled in the art can select a sequence from a database for a desired miRNA and design an inhibitor useful in the methods disclosed herein.

[0371] In some embodiments, the compounds of the present invention disclosed herein are antagomirs. In some embodiments, the compounds of the present invention comprise at least two antagomirs covalently or non-covalently linked to each other via a nucleotide-based or non-nucleotide-based linker, such as those described in this disclosure. Antagomirs are RNA-like oligonucleotides with various modifications for RNAse protection and pharmacological properties, such as improved tissue and cellular uptake. They differ from normal RNAs by, for example, complete 2'-O-methylation of the sugars, phosphorothioate intersugar linkages, and, for example, a cholesterol moiety at the 3' end. In a preferred embodiment, the antagomir contains 2'-O-methyl modifications at all nucleotides, a cholesterol moiety at the 3' end, two phosphorothioate 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 a duplex containing the antagomir and the endogenous miRNA, thereby preventing miRNA-induced gene silencing. An example of antagomir-mediated miRNA silencing is the silencing of miR-122, as described in Krutzfeldt et al., Nature, 2005, 438:685-689, the entire contents of which are expressly incorporated herein by reference.

[0372] Recent research has found that dsRNA can also activate gene expression, a mechanism known as "small RNA-induced gene activation" or RNAa (activating RNA).See, for example, Li, LC et al. Proc Natl Acad Sci US A. (2006), 103 (46): 17337-42 and Li LC (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 dsRNA targeting gene promoters induces strong transcriptional activation of related genes.Endogenous miRNAs that cause RNAa are also found in humans.Check E. Nature (2007). 448 (7156): 855-858.

[0373] Another surprising observation is that RNAa-induced gene activation is long-lasting.The induction of gene expression has been observed to continue for more than 10 days.The sustained effect of RNAa may be due to epigenetic changes at dsRNA target sites.In some embodiments, RNA activators can increase gene expression.In some embodiments, increased gene expression inhibits survival, growth and development, and / or reproduction.

[0374] Thus, in some embodiments, the compounds of the present invention disclosed herein are activator-RNAs. In some embodiments, the compounds of the present invention comprise at least two activator-RNAs that are covalently or non-covalently linked to each other via a nucleotide-based or non-nucleotide-based linker, such as a linker described in this disclosure.

[0375] Therefore, in some embodiments, the compound of the present invention disclosed herein is triplex-forming oligonucleotide (TFO).In some embodiments, the compound of the present invention comprises at least two TFOs that are covalently linked to each other through nucleotide-based or non-nucleotide-based linkers, for example, the linkers described in the present disclosure, or are non-covalently linked to each other.Recent research has shown that triplex-forming oligonucleotides can be designed that can recognize and bind to polypurine / polypyrimidine regions in double-stranded helical DNA in a sequence-specific manner. These recognition rules are outlined by Maher III, LJ, et al., Science (1989) vol. 245, pp. 725-730; Moser, HE, et al., Science (1987) vol. 238, pp. 645-630; Beal, PA, et al., Science (1992) vol. 251, pp. 1360-1363; Conney, M., et al., Science (1988) vol. 241, pp. 456-459, and Hogan, ME et al., EP Publication No. 375408. Modification of oligonucleotides, such as the introduction of intercalating agents and intersugar bond replacement, and optimization of binding conditions (pH and cation concentration) have helped overcome the inherent obstacles to TFO activity, such as charge repulsion and instability, and it has recently been shown that synthetic oligonucleotides can be targeted to specific sequences (for a recent review, see Seidman and Glazer, J Clin Invest 2003;1 12:487-94). Generally, triplex-forming oligonucleotides have the sequence match: Oligo 3'-AGGT Double-stranded 5'-AGCT Double-stranded 3'-TCGA

[0376] However, A-AT and G-GC triplets have been shown to have the highest triple helix stability (Reither and Jeltsch, BMC Biochem, 2002, Sept. 12, Epub). The same authors demonstrated that TFOs designed according to the A-AT and G-GC rules did not form nonspecific triplexes, indicating that triplex formation is indeed sequence-specific.

[0377] Thus, for any given sequence, triplex-forming sequences can be devised. The triplex-forming oligonucleotides are preferably at least 15, more preferably 25, even more preferably 30 or more nucleotides in length, up to a maximum of 50 or 100 nucleotides.

[0378] The formation of triple helix structures with target DNA induces steric and functional changes, blocking transcription initiation and elongation, allowing the introduction of desired sequence changes in endogenous DNA, resulting in specific downregulation of gene expression. Examples of such suppression of gene expression in cells treated with TFO include the 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), as well as the sequence-specific and target-specific downregulation of the expression of Ets2 transcription factor, which is important in the pathogenesis of prostate cancer (Carbone, et al., Nucl Acids Res. 2003; 31: 833-43), and the inflammatory ICAM-I gene (Besch et al., J Biol Chem, 2002; 277: 32473-79). Furthermore, Vuyisich and Beal recently showed that sequence-specific TFOs can bind to dsRNA and inhibit the activity of dsRNA-dependent enzymes such as RNA-dependent kinases (Vuyisich and Beal, Nuc. Acids Res 2000;28:2369-74).

[0379] Furthermore, TFOs designed according to the above principles can induce directed mutagenesis capable of DNA repair, thereby providing both down-regulation and up-regulation of endogenous gene expression (Seidman and Glazer, J Clin Invest 2003;112:487-94).Detailed descriptions of the design, synthesis and administration of effective TFOs can be found in U.S. Patent Application Publication Nos. 2003-017068 and 2003-0096980 to Froehler et al., U.S. Patent Application Publication Nos. 2002-0128218 and 2002-0123476 to Emanuele et al., and U.S. Patent No. 5,721,138 to Lawn, the entire contents of which are incorporated herein by reference.

[0380] Nucleic acid modification In some embodiments, the double-stranded iRNA agent of the present invention comprises at least one nucleic acid modification as described herein.For example, at least one modification is selected from the group consisting of modified internucleoside linkage, modified nucleobase, modified sugar, and any combination thereof.Without being limited, such modification can be present anywhere in the double-stranded iRNA agent of the present invention.For example, modification can be present in one of RNA molecules.

[0381] Nucleic acid modifications (nucleobases) The naturally occurring base moiety of a nucleoside is typically a heterocyclic base. The two most common types of such heterocyclic bases are purines and pyrimidines. In nucleosides containing a pentofuranosyl sugar, the phosphate group can be linked to the 2', 3', or 5' hydroxyl moiety of the sugar. When forming oligonucleotides, these phosphate groups covalently link adjacent nucleosides to form a linear polymeric compound. Within oligonucleotides, the phosphate groups are generally considered to form the internucleoside backbone of the oligonucleotide. The naturally occurring linkage or backbone of RNA and DNA is the 3'-5' phosphodiester bond.

[0382] 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 mimics known to those skilled in the art can be used in the compounds described herein. Unmodified or natural nucleobases can be modified or substituted to provide iRNAs with improved properties. For example, nuclease-resistant oligonucleotides can be prepared using these bases or synthetic and natural nucleobases (e.g., inosine, xanthine, hypoxanthine, nubularine, isoguanisine, or tubercidin) 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 used. When a natural base is replaced with an unnatural and / or universal base, the nucleotide is said herein to contain a modified nucleobase and / or nucleobase modification. Modified nucleobases and / or nucleobase modifications also include natural, unnatural, and universal bases, including conjugate moieties, such as the ligands described herein. Preferred conjugate moieties for conjugation with nucleobases include cationic amino groups, which can be conjugated to the nucleobases via linkers having suitable alkyl, alkenyl, or amide bonds.

[0383] The oligomeric compounds described herein may also contain nucleobase (often simply referred to in the art 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, tubercidin, 2-(halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2-(amino)adenine, 2-(aminoalkyl)adenine, 2-(aminopropyl)adenine, 2-(methylthio)-N 6-(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, N 6 -(Isopentyl)adenine, N 6 -(methyl)adenine, N 6 , N 6 -(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 cytosine, 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-(trifluoromethyl)cytosine, 6-(azo)cytosine, N 4-(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-(methoxycarbonylmethyl)uracil, 5-(propynyl)uracil, 5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 6-(azo)uracil, dihydrouracil, N 3-(methyl)uracil, 5-uracil (i.e., pseudouracil), 2-(thio)pseudouracil, 4-(thio)pseudouracil, 2,4-(dithio)pseudouracil, 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 Uracil, 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 Uracil, 1-(aminoalkylaminocarbonylethylenyl)-pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-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)-phenoxazin-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)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl (hydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(guanidiniumalkyl-hydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-(guanidiniumalkyl-hydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 1,3,5-(triaza)-2,6-(dioxa-naphthalene, inosine, xanthine, hypoxanthine, nubularine, tubercidin, isoguanisine, inosinyl, 2-aza-inosinyl, 7-deazaino Indolyl, 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, pyrrolopyridinyl, isocarbostyrilyl, 7-(propynyl)isocarbostyrilyl, propynyl-7-(aza)indolyl allyl, 2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl, phenyl, naphthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stivenyl, 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, N, 2 -substituted purines, N6 -substituted purines, O 6 -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 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-pyridopyrimidin-3-yl, or any O- or N-alkylated derivative thereof. Alternatively, substituted or modified analogs of any of the above bases and "universal bases" can be used.

[0384] As used herein, a universal nucleobase is any nucleobase that can base pair with all four naturally occurring nucleobases without substantially affecting the melting behavior, recognition by intracellular enzymes, or activity of an iRNA duplex. Some exemplary universal nucleobases include, but are not limited to, 2,4-difluorotoluene, nitropyrrolyl, nitroindolyl, 8-aza-7-deazaadenine, 4-fluoro-6-methylbenzimidazole, 4-methylbenzimidazole, 3-methylisocarbostyrilyl, 5-methylisocarbostyrilyl, 3-methyl-7-propynylisocarbostyrilyl, 7-azaindolyl, 6-methyl-7-azaindolyl, and imidizopyridinyl. In some embodiments, the aryl groups include phenyl, 9-methyl-imidizopyridinyl, pyrrolopyridinyl, isocarbostyryl, 7-propynylisocarbostyryl, propynyl-7-azaindolyl, 2,4,5-trimethylphenyl, 4-methylinolyl, 4,6-dimethylindolyl, phenyl, naphthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stibenyl, tetracenyl, pentacenyl, and structural derivatives thereof (see, e.g., Loakes, 2001, Nucleic Acids Research, 29, 2437-2447).

[0385] 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, JI, 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, YS, Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., Eds., CRC Press, 1993, all of which are incorporated herein by reference.

[0386] In certain embodiments, modified nucleobase is the nucleobase that has a structure that is substantially similar to that of parent nucleobase, such as 7-deazapurine, 5-methylcytosine or G-clamp.In certain embodiments, nucleobase mimics include more complex structures, such as tricyclic phenoxazine nucleobase mimics.The preparation method of the above-mentioned modified nucleobase is well known to those skilled in the art.

[0387] Nucleic acid modification (sugar) The double-stranded iRNA agents of the invention provided herein can contain one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) monomers, including nucleosides or nucleotides, having modified sugar moieties. For example, the furanosyl sugar ring of a nucleoside can be modified in several ways, including, but not limited to, the addition of a substituent or bridging of two non-geminal ring atoms to form a locked nucleic acid or bicyclic nucleic acid. In certain embodiments, an oligomeric compound contains 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 LNAs.

[0388] In some embodiments of the locked nucleic acid, the 2' position of the furanosyl is -[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 -ON(R1)-, -C(R1)=C(R2)-O-, -C(R1)=N-, -C(R1)=NO-, -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)-; During the ceremony: 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, heterocyclic radical, substituted heterocyclic radical, heteroaryl, substituted heteroaryl, C5-C7 cycloaliphatic radical, substituted C5-C7 cycloaliphatic radical, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(=O)-H), substituted acyl, CN, sulfonyl (S(=O)2-J1), or sulfoxyl (S(=O)-J1); Each J1 and J2 is independently H, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, acyl (C(=O)-H), substituted acyl, heterocyclic radical, substituted heterocyclic radical, C1-C12 aminoalkyl, substituted C1-C12 aminoalkyl, or a protecting group.

[0389] In some embodiments, each of the linkers in the LNA compound is independently -[C(R1)(R2)]n-, -[C(R1)(R2)]nO-, -C(R1R2)-N(R1)-O-, or C(R1R2)-ON(R1)-. In other embodiments, each of the 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-ON(R1)-2', and 4'-CH2-N(R1)-O-2'-, where each R1 is independently H, a protecting group, or a C1-C12 alkyl.

[0390] Several LNAs have been prepared and disclosed in the patent and 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. USA, 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; WO 94 / 14226; WO 2005 / 021570; Singh et al. al., J. Org. Chem., 1998, 63, 10035-10039; examples of issued U.S. patents and published applications disclosing LNAs 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. Patent Application Publication Nos. 2004-0171570; 2004-0219565; 2004-0014959; 2003-0207841; 2004-0143114; and 20030082807.

[0391] 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') bond to form a bicyclic sugar moiety (reviewed in Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8, 1-7; and Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; see also U.S. Patent 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, and the term methyleneoxy(4'-CH2-O-2')LNA is used for the bicyclic moiety; if there is an ethylene group at 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 exhibit very high duplex thermal stability with complementary DNA and RNA (Tm = +3 to +10°C), stability against 3'-exonuclease degradation, and good solubility properties. Potent and non-toxic antisense oligonucleotides, including BNAs, have been described (Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 5633-5638).

[0392] A similarly described isomer of methyleneoxy(4'-CH2-O-2')LNA is α-L-methyleneoxy(4'-CH2-O-2')LNA, which has been shown to have excellent stability against 3'-exonucleases. α-L-methyleneoxy(4'-CH2-O-2')LNA has been incorporated into antisense gapmers and chimeras, which have shown potent antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

[0393] The synthesis and preparation of methyleneoxy (4'-CH2-O-2') LNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine, and uracil have been described, along with their oligomerization and nucleic acid recognition properties (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). BNAs and their preparation are also described in WO 98 / 39352 and WO 99 / 14226.

[0394] Analogs of methyleneoxy (4'-CH2-O-2') LNA, phosphorothioate-methyleneoxy (4'-CH2-O-2') LNA and 2'-thio-LNA have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). The preparation of locked nucleoside analogs containing oligodeoxyribonucleotide duplexes as substrates for nucleic acid polymerases has also been described (Wengel et al., WO 99 / 14226). Furthermore, the synthesis of a novel conformationally restricted, high-affinity oligonucleotide analog, 2'-amino-LNA, has been described in the art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). Furthermore, 2'-amino- and 2'-methylamino-LNAs have been prepared and the thermal stability of their duplexes with complementary RNA and DNA strands has been reported.

[0395] Modified sugar moieties are well known and can be used to alter, typically increase, the affinity of an 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'-bridged) ENA; substituted sugars, particularly 2'-substituted sugars with 2'-F, 2'-OCH3, or 2'-O(CH2)2-OCH3 substituents; and 4'-thio modified sugars. Sugars can also be substituted with, among other things, sugar mimetic groups. Methods for preparing 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; and 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; and 6,600,032; and WO 2005 / 121371.

[0396] Examples of "oxy"-2' hydroxyl group modifications include alkoxy or aryloxy (OR, e.g., R = H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar); polyethylene glycol (PEG), O(CH2CHO) nCH2CH2OR, n=1-50; "locked" nucleic acids (LNA) in which the furanose portion of the nucleoside contains a bridge connecting two carbon atoms on the furanose ring, thereby forming a bicyclic ring system; O-AMINE or O-(CH2) n AMINE (n=1-10, AMINE=NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, ethylenediamine or polyamino); and O-CH2CH2(NCH2CH2NMe2)2.

[0397] "Deoxy" modifications include hydrogen (i.e., deoxyribose sugars particularly associated with single-stranded overhangs); halo (e.g., fluoro); amino (e.g., NH; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH(CHCHNH) n Included are CH2CH2-AMINE (AMINE = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino); -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 may be optionally substituted, for example, with an amino functional group.

[0398] Other suitable 2'-modifications, such as modified MOEs, are described in US Patent Application Publication No. 20130130378, the contents of which are incorporated herein by reference.

[0399] Modifications at the 2' position can be in the arabinose configuration. The term "arabinose configuration" refers to the placement of the substituent on C2' of the ribose in the same configuration as the 2'-OH in arabinose.

[0400] A sugar may contain two different modifications, such as gem modifications, at the same carbon of the sugar. The sugar group may also contain one or more carbons with the opposite stereochemical configuration to that of the corresponding carbon in ribose. Thus, an oligomeric compound may contain, for example, one or more monomers containing arabinose as the sugar. The monomer may have an α-linkage at the 1'-position of the sugar, such as an α-nucleoside. The monomer may also have the opposite configuration at the 4'-position, for example, C5' and H4' or the substituents replacing them are interchanged. When C5' and H4' or the substituents replacing them are interchanged, the sugar is said to be modified at the 4'-position.

[0401] The double-stranded iRNA agents of the invention disclosed herein can also contain abasic sugars, i.e., sugars lacking a nucleobase at C-1' or having another chemical group at C1' in place of a nucleobase. See, for example, U.S. Pat. No. 5,998,203, the entire contents of which are incorporated herein. These abasic sugars can also further contain modifications to one or more of the constituent sugar atoms. The double-stranded iRNA agents of the invention can also contain one or more sugars that are L-isomers, e.g., L-nucleosides. Modifications to the sugar group can also include replacing the 4'-O with sulfur, an optionally substituted nitrogen, or a CH2 group. In some embodiments, the linkage between C1' and the nucleobase is in the α-configuration.

[0402] Sugar modifications can also include acyclic nucleotides in which a C-C bond between ribose carbons (e.g., C1'-C2', C2'-C3', C3'-C4', C4'-O4', C1'-O4') is absent and / or at least one of the ribose carbons or oxygens (e.g., C1', C2', C3', C4', or O4'), independently or in combination, is absent from the nucleotide. In some embodiments, an acyclic nucleotide is [ka] wherein B is a modified or unmodified nucleobase, R1 and R2 are independently H, halogen, OR3, or alkyl; and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar.

[0403] In some embodiments, the sugar modification is 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 gem2'-OMe / 2'F with 2'-O-Me in the arabinose configuration.

[0404] It should be understood that when a particular nucleotide is linked to the next nucleotide through its 2' position, the sugar modifications described herein can be located at the 3' position of the sugar for that particular nucleotide, e.g., the nucleotide linked through its 2' position. The modification at the 3' position can be in the xylose configuration. The term "xylose configuration" refers to the placement of the substituent on the C3' of the ribose in the same configuration as the 3'-OH in a xylose sugar.

[0405] The hydrogen attached to C4' and / or C1' can be replaced with a straight or branched chain, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, wherein the 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'), a phosphorus-containing bond, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclic, or optionally substituted cycloalkyl, wherein R' is hydrogen, acyl, or optionally substituted aliphatic, and Z' is OR 11 , C.O.R. 11 , CO2R 11 , [ka] , N.R. 21 R 31 ,CONR 21 R 31 , CON(H)NR 21 R 31 , ONR 21 R 31 , CON(H)N=CR 41 R 51 , N(R 21 )C(=NR 31 )NR 21 R 31 , N(R 21 )C(O)NR 21 R 31 , N(R 21 )C(S)NR 21 R 31 , OC(O)NR 21 R 31 , SC(O)NR 21 R 31 , N(R 21 )C(S)OR 11 , N(R 21 )C(O)OR 11 , N(R 21 )C(O)SR 11 , N(R 21 )N=CR 41 R 51 , ON=CR 41 R51 , SO2R 11 , SOR 11 , S.R. 11 and substituted or unsubstituted heterocyclic; R 21 and R 31 is independently for each occurrence hydrogen, acyl, unsubstituted or substituted aliphatic, aryl, heteroaryl, heterocyclic, OR 11 , C.O.R. 11 , CO2R 11 , or NR 11 R 11 ' or R 21 and R 31 together with the atoms to which they are attached form a heterocyclic ring; R 41 and R 51 is independently for each occurrence hydrogen, acyl, unsubstituted or substituted aliphatic, aryl, heteroaryl, heterocyclic, OR 11 , C.O.R. 11 , or CO2R 11 , or NR 11 R 11 ' and ;R 11 and R 11 is independently hydrogen, aliphatic, substituted aliphatic, aryl, heteroaryl, or heterocyclic. In some embodiments, the hydrogen attached to C4' of the 5'-terminal nucleotide is substituted.

[0406] In some embodiments, C4' and C5' together preferably form an optionally substituted heterocycle containing at least one -PX(Y)-, where 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 alkylmetal or transition metal having a total charge of +1; and Y is O, S, or NR', where R' is hydrogen or an optionally substituted aliphatic. Preferably, this modification is at the 5' end of the iRNA.

[0407] In certain embodiments, the LNA has the formula: [ka] and a bicyclic nucleoside having the formula: During the ceremony: Bx is a heterocyclic base moiety; T1 is H or a hydroxyl protecting group; T2 is H, a hydroxyl protecting group or a reactive phosphorus group; Z 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 amido.

[0408] In some embodiments, each of the substituted groups is independently mono- or polysubstituted with optionally protected substituents independently selected from halogen, oxo, hydroxyl, OJ1, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2 and CN, where each J1, J2 and J3 is independently H or C1-C6 alkyl, and X is O, S or NJ1.

[0409] In certain such embodiments, each of the substituted groups is independently mono- or polysubstituted with substituents independently selected from halogen, oxo, hydroxyl, OJ1, NJ1J2, SJ1, N3, OC(=X)J1, and NJ3C(=X)NJ1J2, where each J1, J2, and J3 is independently H, C1-C6 alkyl, or substituted C1-C6 alkyl, and X is O or NJ1.

[0410] In certain embodiments, the Z group is C-C alkyl substituted with one or more X, where each X is independently OJ, NJJ, SJ, N, OC(=X)J, OC(=X)NJJ, NJC(=X)NJJ, or CN; where each J, J, and J is independently H or C-C alkyl, and X is O, S, or NJ. In another embodiment, the Z group is C-C alkyl substituted with one or more X, where each X is independently halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CHO—), substituted alkoxy, or azido.

[0411] In certain embodiments, the Z group is -CH2Xx, where Xx is OJ1, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2, or CN; where 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, where Xx is halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CHO-), or azido.

[0412] In certain such embodiments, the Z group is in the (R)-configuration: [ka] is located.

[0413] In certain such embodiments, the Z group is in the (S)-configuration: [ka] is located.

[0414] 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-phenylxanthin-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthin-9-yl (MOX). In certain embodiments, T1 is a hydroxyl protecting group selected from acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl and dimethoxytrityl, where the more preferred hydroxyl protecting group is T1 which is 4,4'-dimethoxytrityl.

[0415] In certain embodiments, T2 is a reactive phosphorus group, and preferred reactive phosphorus groups include diisopropylcyanoethoxyphosphoramidite and H-phosphonate. In certain embodiments, T1 is 4,4'-dimethoxytrityl and T2 is diisopropylcyanoethoxyphosphoramidite.

[0416] In certain embodiments, the compounds of the present invention have the formula: [ka] or the expression: [ka] or the expression: [ka] and at least one monomer of During the ceremony, Bx 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, nucleotide, oligonucleoside, oligonucleotide, monomeric subunit, or oligomeric compound; T4 is H, a hydroxyl protecting group, a linked conjugate group, or an internucleoside linking group attached to a nucleoside, nucleotide, oligonucleoside, oligonucleotide, monomeric subunit, or oligomeric compound; wherein at least one of T3 and T4 is an internucleoside linking group attached to a nucleoside, nucleotide, oligonucleoside, oligonucleotide, monomeric subunit, or oligomeric compound; Z 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 amido.

[0417] In some embodiments, each of the substituted groups is independently mono- or polysubstituted with optionally protected substituents independently selected from halogen, oxo, hydroxyl, OJ1, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2 and CN, where each J1, J2 and J3 is independently H or C1-C6 alkyl, and X is O, S or NJ1.

[0418] In some embodiments, each of the substituted groups is independently mono- or polysubstituted with substituents independently selected from halogen, oxo, hydroxyl, OJ1, NJ1J2, SJ1, N3, OC(=X)J1, and NJ3C(=X)NJ1J2, where each J1, J2, and J3 is independently H or C1-C6 alkyl, and X is O or NJ1.

[0419] In certain such embodiments, at least one Z is C1-C6 alkyl or substituted C1-C6 alkyl. In certain embodiments, each Z is independently C1-C6 alkyl or substituted C1-C6 alkyl. In certain embodiments, at least one Z is C1-C6 alkyl. In certain embodiments, each Z is independently C1-C6 alkyl. 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-C6 alkyl. 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.

[0420] In certain embodiments, at least one substituent is C1-C6 alkoxy (e.g., at least one Z is C1-C6 alkyl substituted with one or more C1-C6 alkoxy). In other embodiments, each substituent is independently C1-C6 alkoxy (e.g., each Z is independently C1-C6 alkyl substituted with one or more C1-C6 alkoxy).

[0421] In certain embodiments, at least one C1-C6 alkoxy substituent is CHO- (e.g., at least one Z is CHOCH-). In other embodiments, each C1-C6 alkoxy substituent is CHO- (e.g., each Z is CHOCH-).

[0422] In certain embodiments, at least one substituent is halogen (e.g., at least one Z is C-C alkyl substituted with one or more halogens). In certain embodiments, each substituent is independently halogen (e.g., each Z is independently C-C alkyl substituted with one or more halogens). In certain embodiments, at least one halogen substituent is fluoro (e.g., at least one Z is CHFCH-, CHFCH-, or CFCH-). In certain embodiments, each halo substituent is fluoro (e.g., each Z is independently CHFCH-, CHFCH-, or CFCH-).

[0423] In certain embodiments, at least one substituent is hydroxyl (e.g., at least one Z is C1-C6 alkyl substituted with one or more hydroxyl). In certain embodiments, each substituent is independently hydroxyl (e.g., each Z is independently C1-C6 alkyl substituted with one or more hydroxyl). In certain embodiments, at least one Z is HOCH2-. In other embodiments, each Z is HOCH2-.

[0424] In certain embodiments, at least one Z is CH3-, CH3CH2-, CHOCH3-, CH2F-, or HOCH2-. In certain embodiments, each Z is independently CH3-, CH3CH2-, CHOCH3-, CH2F-, or HOCH2-.

[0425] In certain embodiments, at least one Z group is C-C alkyl substituted with one or more X, where each X is independently OJ, NJJ, SJ, N, OC(=X)J, OC(=X)NJJ, NJC(=X)NJJ, or CN; where each J, J, and J is independently H or C-C alkyl, and X is O, S, or NJ. In another embodiment, at least one Z group is C-C alkyl substituted with one or more X, where each X is independently halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CHO—), or azido.

[0426] In certain embodiments, each Z group is independently C-C alkyl substituted with one or more X, where each X is independently OJ, NJJ, SJ, N, OC(=X)J, OC(=X)NJJ, NJC(=X)NJJ, or CN; where each J, J, and J is independently H or C-C alkyl, and X is O, S, or NJ. In another embodiment, each Z group is independently C-C alkyl substituted with one or more X, where each X is independently halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CHO—), or azido.

[0427] In certain embodiments, at least one Z group is -CH2Xx, where Xx is OJ1, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2, or CN; where each J1, J2, and J3 is independently H or C1-C6 alkyl and X is O, S, or NJ1. In certain embodiments, at least one Z group is -CH2Xx, where Xx is halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CHO-), or azido.

[0428] In certain embodiments, each Z group is independently -CH2Xx, where each Xx is independently OJ1, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2, or CN; where 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, where each Xx is independently halo (e.g., fluoro), hydroxyl, alkoxy (e.g., CHO-), or azido.

[0429] In certain embodiments, at least one Z is CH3-. In other embodiments, each Z is CH3-.

[0430] In certain embodiments, the Z group of at least one monomer has the formula: [ka] or the expression: [ka] or the expression: [ka] The compound is in the (R)-configuration represented by

[0431] In certain embodiments, the Z group of each monomer of the formula is in the (R)-configuration.

[0432] In certain embodiments, the Z group of at least one monomer has the formula: [ka] or the expression: [ka] or the expression: [ka] The ion beam is in the (S)-configuration represented by

[0433] In certain embodiments, the Z group of each monomer of the formula is in the (S)-configuration.

[0434] In certain embodiments, T3 is H or a hydroxyl protecting group. In certain embodiments, T4 is H or a hydroxyl protecting group. In further embodiments, T3 is an internucleoside linking group attached to a nucleoside, nucleotide, or monomer subunit. In certain embodiments, T4 is an internucleoside linking group attached to a nucleoside, nucleotide, or monomer subunit. In certain embodiments, T3 is an internucleoside linking group attached to an oligonucleoside or oligonucleotide. In certain embodiments, T4 is an internucleoside linking group attached to an oligonucleoside or 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 oligomeric compound. In certain embodiments, at least one of T3 and T4 comprises an internucleoside linking group selected from phosphodiester or phosphorothioate.

[0435] In certain embodiments, the double-stranded iRNA agent of the invention has the formula: [ka] or the expression: [ka] or the expression: [ka] and at least one region of at least two consecutive monomers of

[0436] In certain such embodiments, LNAs include, but are not limited to, (A) α-L-methyleneoxy (4'-CH2-O-2') LNA, (B) β-D-methyleneoxy (4'-CH2-O-2') LNA, (C) ethyleneoxy (4'-(CH2)2-O-2') LNA, (D) aminooxy (4'-CH2-ON(R)-2') LNA, and (E) oxyamino (4'-CH2-N(R)-O-2') LNA, as shown below. [ka]

[0437] In certain embodiments, a double-stranded iRNA agent of the invention comprises at least two regions of at least two consecutive monomers of the above formula. In certain embodiments, a double-stranded iRNA agent of the invention comprises a gap motif. In certain embodiments, a double-stranded iRNA agent of the invention comprises at least one region of about 8 to about 14 consecutive β-D-2'-deoxyribofuranosyl nucleosides. In certain embodiments, a double-stranded iRNA agent of the invention comprises at least one region of about 9 to about 12 consecutive β-D-2'-deoxyribofuranosyl nucleosides.

[0438] In certain embodiments, the double-stranded iRNA agent of the invention has the formula: [ka] wherein Bx is a heterocyclic base moiety. and at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more) (S)-cEt monomer.

[0439] In certain embodiments, the monomer comprises a sugar mimetic. In certain such embodiments, the mimetic is used in place of the sugar or sugar-internucleoside linkage combination, while the nucleobase is maintained for hybridization with a selected target. Representative examples of sugar mimetics include, but are not limited to, cyclohexenyl or morpholino. Representative examples of mimetics of sugar-internucleoside linkage combinations include, but are not limited to, peptide nucleic acids (PNAs) and morpholino groups linked by uncharged achiral bonds. In some examples, the 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 for synthesizing sugar, nucleoside, and nucleobase mimetics are well known to those skilled in the art.

[0440] Nucleic acid modification (sugar bond) Described herein are linking groups that link monomers (including, but not limited to, modified and unmodified nucleosides and nucleotides) together to form oligomeric compounds, e.g., oligonucleotides. Such linking groups are also referred to as intersugar linkages. Two major 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, phosphodiester (P=O), phosphotriester, methylphosphonate, phosphoramidate, and phosphorothioate (P=S). Representative non-phosphorus-containing linking groups include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-OC(O)-S-), thionocarbamate (-OC(O)(NH)-S-); siloxane (-O-Si(H)2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Compared with natural phosphodiester bond, modified bond can be used to modify, typically increase, the nuclease resistance of oligonucleotide.In certain embodiments, bond with chiral atom can be prepared as separate enantiomers, as racemic mixture.Representative chiral bond includes, but is not limited to, alkyl phosphonate and phosphorothioate.The preparation method of phosphorus-containing and non-phosphorus-containing bond is well known to those skilled in the art.

[0441] The phosphate group in the linking group can be modified by replacing one of the oxygen atoms with a different substituent. One result of this modification can be increased resistance of the oligonucleotide to nucleolytic degradation. Examples of modified phosphate groups include phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, alkyl or aryl phosphonates, and phosphotriesters. In some embodiments, one of the non-bridging phosphate oxygen atoms in the linking group can be replaced with any of the following: S, Se, BR3 (R is hydrogen, alkyl, aryl), C (i.e., alkyl group, aryl group, etc.), H, NR2 (R is hydrogen, optionally substituted alkyl, aryl), or OR (R is optionally substituted alkyl or aryl). The phosphorus atom in an unmodified phosphate group is achiral. However, replacing one of the non-bridging oxygen atoms with one of the above atoms or atomic groups makes the phosphorus atom chiral; in other words, the phosphorus atom in such a modified phosphate group is a stereocenter. The stereogenic phosphorus atom can have either the "R" configuration (herein Rp) or the "S" configuration (herein Sp).

[0442] Phosphorodithioates have both non-bridging oxygens replaced by sulfur. The phosphorus center in phosphorodithioates is chiral, which prevents the formation of oligonucleotide diastereomers. Therefore, without wishing to be bound by theory, modification of both non-bridging oxygens may be desirable because it eliminates asymmetric centers, such as phosphorodithioate formation, and they cannot produce diastereomeric mixtures. Therefore, the non-bridging oxygens can independently be any one of O, S, Se, B, C, H, N, or OR (R is alkyl or aryl).

[0443] Phosphate linkers can also be modified by replacing the bridging oxygen (i.e., the oxygen linking the phosphate to the sugar of the monomer) with nitrogen (bridging phosphoramidates), sulfur (bridging phosphorothioates), and carbon (bridging methylene phosphonates). This replacement can occur at either or both of the linking oxygens. When the bridging oxygen is the 3'-oxygen of the nucleoside, substitution with carbon is preferred. When the bridging oxygen is the 5'-oxygen of the nucleoside, substitution with nitrogen is preferred.

[0444] Modified phosphate linkages in which at least one of the oxygens linked to the phosphate is replaced or the phosphate group is replaced by a non-phosphorus group are also called "non-phosphodiester intersugar linkages" or "non-phosphodiester linkers."

[0445] In certain embodiments, the phosphate group can be replaced by a non-phosphorus-containing connector, such as a dephosphoryl linker. Dephosphoryl linkers are herein referred to as non-phosphodiester linkers. Without wishing to be bound by theory, it is believed that because the charged phosphodiester group is the reactive center of nucleic acid degradation, its replacement with a neutral structural mimic should confer improved nuclease stability. Again, without wishing to be bound by theory, in some embodiments, it may be desirable to introduce a modification in which the charged phosphate group is replaced by a neutral moiety.

[0446] Examples of moieties that can replace the phosphate group include, but are not limited to, amide (e.g., amide-3 (3'-CH2-C(=O)-N(H)-5') and amide-4 (3'-CH2-N(H)-C(=O)-5')), hydroxylamino, siloxane (dialkylsiloxane), carboxamide, carbonate, carboxymethyl, carbamate, carboxylic acid ester, thioether, ethylene oxide linker, sulfide, sulfonate, sulfonamide, sulfonate ester, thioformacetal (3'-S-CH2-O-5'), formacetal (3'-O-CH2-O-5'), oxime, methyleneimino, methylene These include carbonylamino, methylenemethylimino (MMI, 3'-CH2-N(CH3)-O-5'), methylenehydrazo, methylenedimethylhydrazo, methyleneoxymethylimino, ether (C3'-O-C5'), thioether (C3'-S-C5'), thioacetamide (C3'-N(H)-C(=O)-CH2-S-C5', C3'-O-P(O)-O-S-S-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 moieties. For example, Carbohydrate See Modifications in Antisense Research; YS Sanghvi and PDCook Eds. ACS Symposium Series 580; Chapters 3 and 4, (pp. 40-65). Preferred embodiments include methylenemethylimino (MMI), methylenecarbonylamino, amide, carbamate, and ethylene oxide linkers.

[0447] Those skilled in the art will appreciate that, in certain instances, substitution of a non-bridging oxygen can result in enhanced cleavage of the intersugar bond by the adjacent 2'-OH, and therefore, in many cases, modification of the non-bridging oxygen may require modification of the 2'-OH, e.g., a modification that does not involve cleavage of the adjacent intersugar bond, e.g., arabinose sugars, 2'-O-alkyl, 2'-F, LNA, and ENA.

[0448] Preferred non-phosphodiester intersugar linkages include phosphorothioates, phosphorothioates containing an enantiomeric excess of the Sp isomer of at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, phosphorothioates containing an enantiomeric excess of the Rp isomer of at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, alkyl-phosphonates (e.g., methyl-phosphonates), selenophosphates, phosphoramidates (e.g., N-alkylphosphoramidates), and boranophosphonates.

[0449] In some embodiments, a double-stranded iRNA agent of the invention includes at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more and up to all) modified or non-phosphodiester linkage. In some embodiments, a double-stranded iRNA agent of the invention includes at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more and up to all) phosphorothioate linkage.

[0450] The double-stranded iRNA agent of the present invention can also be constructed in which the phosphate linker and sugar are replaced by a nuclease-resistant nucleoside or nucleotide surrogate. Without wishing to be bound by theory, it is believed that the absence of a repeatedly charged backbone weakens binding to proteins that recognize polyanions (e.g., nucleases). Again, without wishing to be bound by theory, in some embodiments, it may be desirable to introduce a modification in which the bases are connected by a neutral surrogate backbone. Examples include morpholino, cyclobutyl, pyrrolidine, peptide nucleic acid (PNA), aminoethylglycyl PNA (aegPNA), and backbone-extended pyrrolidine PNA (bepPNA) nucleoside surrogates. A preferred surrogate is a PNA surrogate.

[0451] The double-stranded iRNA agents of the invention described herein can contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations that can be defined with respect to absolute stereochemistry as (R) or (S), such as in the case of sugar anomers, or as (D) or (L), such as in the case of amino acids. The double-stranded iRNA agents of the invention provided herein include all such possible isomers, as well as their racemic and optically pure forms.

[0452] Nucleic acid modification (end modification) In some embodiments, the double-stranded iRNA agent further comprises a phosphate or a phosphate mimic at the 5'-end of the antisense strand. In one embodiment, the phosphate mimic is 5'-vinylphosphonate (VP).

[0453] In some embodiments, the 5' end of the antisense strand of the double-stranded iRNA agent does not contain a 5'-vinylphosphonate (VP).

[0454] The termini of the iRNA agent of the present invention may be modified. Such modifications may occur at one or both termini. For example, the 3' and / or 5' termini of the iRNA may be conjugated to a labeling moiety, such as a fluorophore (e.g., pyrene, TAMRA, fluorescein, Cy3, or Cy5 dye) or other functional molecular entity, such as a protecting group (e.g., sulfur, silicon, boron, or ester-based). The functional molecular entity may be attached to the sugar via a phosphate group and / or a linker. The terminal atom of the linker may be linked 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 may be linked to or replace the terminal atom of a nucleotide surrogate (e.g., PNA).

[0455] When a linker / phosphate-functional molecular entity-linker / phosphate array is placed between the two strands of a double-stranded oligomeric compound, the array can take the place of a hairpin loop in a hairpin-shaped oligomeric compound.

[0456] Terminal modifications useful for modulating activity include modification of the 5' end of an iRNA with phosphate or a phosphate analog. In certain embodiments, the 5' end of an iRNA is phosphorylated or contains a phosphoryl analog. Exemplary 5'-phosphate modifications include modifications compatible with RISC-mediated gene silencing. Modifications at the 5' end may also be useful for stimulating or inhibiting the immune system of a subject. In some embodiments, the 5' end of an oligomeric compound is modified [ka] wherein W, X, and Y are each independently O, OR (wherein R is hydrogen, alkyl, or aryl), S, Se, BR3 (wherein R is hydrogen, alkyl, or aryl), BH3 - , C (i.e., alkyl, aryl, etc.), H, NR (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, CH, NR (R is hydrogen, alkyl, aryl), or optionally substituted alkylene, where the alkylene backbone can include internally and / or terminally one or more of O, S, SS, and NR (R is hydrogen, alkyl, aryl); and n is 0-2. In some embodiments, n is 1 or 2. It is understood that A replaces the oxygen linked to the 5' carbon of the 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 heterocycle, where W and Y are each independently O, S, NR', or alkylene. Preferably, the heterocycle is substituted with aryl or heteroaryl. In some embodiments, one or both hydrogens on the C5' of the 5' terminal nucleotide are replaced with a halogen, eg, F.

[0457] Exemplary 5'-modifications include, but are not limited to, 5'-monophosphate ((HO)2(O)PO-5'); 5'-diphosphate ((HO)2(O)POP(HO)(O)-O-5'); 5'-triphosphate ((HO)2(O)PO-(HO)(O)POP(HO)(O)-O-5'); 5'-monothiophosphate (phosphorothioate; (HO)2(S)PO-5'); 5'-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)PO-5'), 5'-phosphorothiolate ((HO)2(O)PS-5'); 5'-alpha-thiotriphosphate; 5'-beta-thiotriphosphate; 5'-gamma-thiotriphosphate; 5'-phosphoramidate ((HO)2(O)P-NH-5', (HO)(NH2)(O)PO-5'). Other 5'-modifications include 5'-alkyl phosphonates (R(OH)(O)PO-5', R = alkyl, e.g., methyl, ethyl, isopropyl, propyl, etc.), 5'-alkyl ether phosphonates (R(OH)(O)PO-5', R = alkyl ether, e.g., methoxymethyl (CHOMe), ethoxymethyl, etc.). Other exemplary 5'-modifications include those in which Z is alkyl, optionally substituted at least once, e.g., ((HO)(X)PO[-(CH) a -OP(X)(OH)-O] b -5', ((HO)2(X)PO[-(CH2) a -P(X)(OH)-O] b -5', ((HO)2(X)P-[-(CH2) a -OP(X)(OH)-O] b -5'; Dialkyl-terminated phosphates and phosphate mimetics: HO[-(CH2) a -OP(X)(OH)-O] b -5', H2N[-(CH2) a -OP(X)(OH)-O] b -5', H[-(CH2) a -OP(X)(OH)-O] b -5', Me2N[-(CH2) a -OP(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', where a and b are each independently 1 to 10. Other embodiments include BH3, BH3 - and / or containing substitution of oxygen and / or sulfur by Se.

[0458] Terminal modifications can also be useful for monitoring distribution; in such cases, preferred groups to be added include fluorophores, such as fluorescein, or Alexa dyes, such as Alexa 488. Terminal modifications can also be useful for promoting uptake; useful modifications for this purpose include targeting ligands. Terminal modifications can also be useful for crosslinking the oligonucleotide to another moiety; useful modifications for this purpose include mitomycin C, psoralens, and their derivatives.

[0459] Thermal destabilization modification Compounds of the invention, such as iRNA or dsRNA agents, can be optimized for RNA interference by introducing a thermostabilizing modification in the sense strand opposite the antisense strand's seed region (i.e., positions 2-8 at the 5' end of the antisense strand) to enhance the dissociation or melting propensity of the iRNA duplex (reducing the free energy of duplex binding). This modification can enhance the dissociation or melting propensity of the duplex at the antisense strand's seed region.

[0460] Thermally destabilizing modifications can include abasic modifications; mismatches with opposing nucleotides in the opposing strand; and sugar modifications, such as 2'-deoxy modifications or acyclic nucleotides, such as unlocked nucleic acids (UNAs) or glycerol nucleic acids (GNAs).

[0461] Exemplary abasic modifications are as follows: [ka]

[0462] Exemplary sugar modifications are as follows: [ka]

[0463] The term "acyclic nucleotide" refers to any nucleotide having an acyclic ribose sugar in which, for example, there is no bond between the ribose carbons (e.g., C1'-C2', C2'-C3', C3'-C4', C4'-O4', or C1'-O4') and / or at least one of the ribose carbons or oxygens (e.g., C1', C2', C3', C4', or O4'), alone or in combination, is absent from the nucleotide. In some embodiments, an acyclic nucleotide is: [ka] wherein B is a modified or unmodified nucleobase and R 1 and R 2are independently H, halogen, OR3, or alkyl; R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar. The term "UNA" refers to an unlocked acyclic nucleic acid in which all bonds of the sugar have been removed to form an unlocked "sugar" residue. In one example, a UNA also includes a monomer in which the C1'-C4' bond (i.e., the carbon-oxygen-carbon covalent bond between the C1' and C4' carbons) has been removed. In another example, the C2'-C3' bond of the sugar (i.e., the carbon-carbon covalent bond between the C2' and C3' carbons) has been removed (see Mikhailov et al., Tetrahedron Letters, 26 (17):2059 (1985); and Fluiter et al., Mol. Biosyst., 10:1039 (2009), the entire contents of each of which are incorporated herein by reference). Acyclic derivatives offer greater backbone flexibility without affecting Watson-Crick base pairing. Acyclic nucleotides can be linked by 2'-5' or 3'-5' linkages.

[0464] The term "GNA" refers to glycol nucleic acid, a polymer similar to DNA or RNA, but differing in the composition of its "backbone" as it consists of repeating glycerol units linked by phosphodiester bonds: [ka]

[0465] The heat destabilizing modification can be a mismatch (i.e., non-complementary base pair) between the heat destabilizing nucleotide and the opposite nucleotide in the opposite strand in the dsRNA duplex.Exemplary mismatch base pairs 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 combinations thereof.Other mismatch base pairings known in the art are also suitable for the present invention.Mismatches can occur between nucleotides that are naturally occurring nucleotides or modified nucleotides, that is, mismatch base pairing can occur between the nucleobases from each nucleotide regardless of the modification in the ribose sugar of the nucleotide.In certain embodiments, the compound of the present invention, such as siRNA or iRNA agent, comprises at least one nucleobase that is a 2'-deoxynucleobase in mismatch pairing; for example, the 2'-deoxynucleobase is in the sense strand.

[0466] Further examples of abasic nucleotides, acyclic nucleotide modifications (including UNA and GNA), and mismatch modifications are described in detail in WO 2011 / 133876, the entire contents of which are incorporated herein by reference.

[0467] Thermodestabilizing modifications can include universal bases that have reduced or lost the ability to form hydrogen bonds with opposing bases, and phosphate modifications.

[0468] Nucleobase modifications that reduce or completely eliminate the ability to form hydrogen bonds with bases in the opposing strand were evaluated for destabilizing the central region of the dsRNA agent duplex, as described in International Publication No. 2010 / 0011895, the entire contents of which are incorporated herein by reference. Exemplary nucleobase modifications are as follows: [ka]

[0469] Exemplary phosphate modifications known to reduce the thermal stability of dsRNA duplexes compared to natural phosphodiester linkages are: [ka]

[0470] In some embodiments, compounds of the invention may contain 2'-5' linkages (with 2'-H, 2'-OH, and 2'-OMe, and with P=O or P=S). For example, 2'-5' linkage modifications may be used to increase nuclease resistance or to inhibit binding of the sense strand to the antisense strand, or may be used at the 5' end of the sense strand to avoid activation of the sense strand by RISC.

[0471] In another embodiment, the compounds of the present invention may contain L-sugars (e.g., L-ribose, L-arabinose, including 2'-H, 2'-OH, and 2'-OMe). For example, these L-sugar modifications may be used to increase nuclease resistance or to inhibit binding of the sense strand to the antisense strand, or may be used at the 5' end of the sense strand to prevent activation of the sense strand by RISC.

[0472] In one embodiment, an iRNA agent of the invention is conjugated to a ligand via a carrier, which can be a cyclic 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 decalin; preferably, the acyclic group is selected from a serinol backbone or a diethanolamine backbone.

[0473] In some embodiments, at least one strand of an iRNA agent of the invention disclosed herein is 5' phosphorylated or contains a phosphoryl analog at the 5' end. The 5'-phosphate modification includes modifications that are compatible with RISC-mediated gene silencing. Suitable modifications include: 5'-monophosphate ((HO)2(O)PO-5'); 5'-diphosphate ((HO)2(O)POP(HO)(O)-O-5'); 5'-triphosphate ((HO)2(O)PO-(HO)(O)POP(HO)(O)-O-5'); 5'-guanosine cap (7-methylated or unmethylated) (7m-GO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5'); 5'-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (NO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5'); 5'-monothiophosphate (phosphorothioate; (HO)2(S)PO-5'); 5'-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P O-5'), 5'-phosphorothiolate ((HO)2(O)PS-5'); oxygen / sulfur substituted monophosphates, diphosphates, and triphosphates (e.g., 5'-α-thiotriphosphate, 5'-γ-thiotriphosphate, etc.), 5'-phosphoramidates ((HO)2(O)P-NH-5', (HO)(NH2)(O)PO-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'-alkyl ether phosphonates (R = alkyl ether = methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g., RP(OH)(O)-O-5'-).

[0474] Target gene Non-limiting examples of target genes for siRNA include genes that promote unwanted cell proliferation, growth factor genes, growth factor receptor genes, gene expression kinases, adaptor protein genes, genes encoding G-protein superfamily molecules, genes encoding transcription factors, genes that mediate angiogenesis, viral genes, genes required for viral replication, cellular genes that mediate viral function, genes of bacterial pathogens, genes of amoebic pathogens, genes of parasitic pathogens, genes of fungal pathogens, genes that mediate unwanted immune responses, genes that mediate pain processing, genes that mediate neurological disorders, alleles found in cells characterized by loss of heterozygosity, or one allele of a polymorphic gene.

[0475] Specific exemplary target genes for siRNA include, but are not limited to, PCSK-9, ApoC3, AT3, AGT, ALAS1, TMPR, HAO1, AGT, C5, CCR-5, PDGF bet 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 gene; cyclin E gene; WNT-1 gene; β-catenin gene; c-MET gene; PKC gene; NFKB gene; STAT3 gene; survivin gene; Her2 / Neu gene; topoisomerase I gene; and topoisomerase II gene. α 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; α v-integrin gene; Flt-1 receptor gene; tubulin gene; human papillomavirus gene, gene required for human papillomavirus replication, human immunodeficiency virus gene, gene required for human immunodeficiency virus replication, hepatitis A virus gene, gene required for hepatitis A virus replication, hepatitis B virus gene, gene required for hepatitis B virus replication, hepatitis C virus gene, gene required for hepatitis C virus replication, hepatitis D virus gene, gene required for hepatitis D virus replication, hepatitis E virus gene, gene required for hepatitis E virus replication, hepatitis F virus gene, gene required for hepatitis F virus replication, hepatitis G virus gene, gene required for hepatitis G virus replication, hepatitis H virus gene, gene required for hepatitis H virus replication, respiratory syncytial virus geneGenes required for respiratory syncytial virus replication, Herpes simplex virus genes, Genes required for herpes simplex virus replication, Herpes cytomegalovirus genes, Genes required for herpes cytomegalovirus replication, Herpes Epstein-Barr virus genes, Genes required for herpes Epstein-Barr virus replication, Kaposi's sarcoma-associated herpesvirus genes, Genes required for Kaposi's sarcoma-associated herpesvirus replication, JC virus genes, Human genes required for JC virus replication, Myxovirus genes, Genes required for myxovirus gene replication, Rhinovirus genes, Genes required for rhinovirus replication, Coronavirus genes, Genes required for coronavirus replication, West Nile virus genes, Genes required for West Nile virus replication, St. Louis encephalitis genes, Genes required for St. Louis encephalitis replication, Tick-borne encephalitis virus genes, Genes required for tick-borne encephalitis virus replication, Murray Valley encephalitis virus genes, Genes required for Murray Valley encephalitis virus replication Dengue virus genes, genes required for dengue virus replication, simian virus 40 genes, genes required for simian virus 40 replication, human T-cell lymphotropic virus genes, genes required for human T-cell lymphotropic virus replication, Moloney murine leukemia virus genes, genes required for Moloney murine leukemia virus replication, encephalomyocarditis virus genes, genes required for encephalomyocarditis virus replication, measles virus genes, genes required for measles virus replication, varicella-zoster virus genes, genes required for varicella-zoster virus replication, adenovirus genes, genes required for adenovirus replication, yellow fever virus genes, genes required for yellow fever virus replication, poliovirus genes, genes required for poliovirus replication, poxvirus genes, genes required for poxvirus replication, plasmodium genes, genes required for plasmodium replication, Mycobacterium ulcerans ulcerans genes, genes required for Mycobacterium ulcerans replication, Mycobacterium tuberculosis genes,Genes required for Mycobacterium tuberculosis replication, Mycobacterium leprae genes, Genes required for Mycobacterium leprae replication, Staphylococcus aureus genes, Genes required for Staphylococcus aureus replication, Streptococcus pneumoniae genes, Genes required for Streptococcus pneumoniae replication, Streptococcus pyogenes genes, Genes required for Streptococcus pyogenes replication, Chlamydia pneumoniae genes, Genes required for Chlamydia pneumoniae replication, Mycoplasma pneumoniae genes, Mycoplasma pneumoniae) replication-required genes, integrin genes, selectin genes, complement system genes, chemokine genes, chemokine receptor genes, GCSF gene, Gro1 gene, Gro2 gene, Gro3 gene, PF4 gene, MIG gene, proplatelet 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, CM Examples of genes that may be involved in the pathogenesis of rheumatoid arthritis include BKR5v, AIF-1 gene, I-309 gene, genes for components of ion channels, genes for neurotransmitter receptors, genes for neurotransmitter ligands, amyloid family genes, presenilin genes, HD gene, DRPL gene, SCA1 gene, SCA2 gene, MJD1 gene, CACNL1A4 gene, SCA7 gene, SCA8 gene, alleles found in loss of heterozygosity (LOH) cells, single alleles of polymorphic genes, and combinations thereof.

[0476] Loss of heterozygosity (LOH) can result in hemizygous sequences, such as genes, in the region of LOH. This can result in significant genetic differences between normal cells and diseased cells, such as cancer cells, and provide useful differentiation between normal cells and diseased cells, such as cancer cells. This difference can occur because genes or other sequences are heterozygous in diploid cells but hemizygous in cells with LOH. Regions of LOH often contain genes whose loss promotes unnecessary proliferation, such as tumor suppressor genes, and other sequences, including other genes, in some cases, genes essential for normal function, such as growth. The methods of the present invention partially rely on the specific regulation of one allele of essential genes by the compositions of the present invention.

[0477] In certain embodiments, the invention provides double-stranded iRNA agents of the invention that regulate microRNAs.

[0478] Central Nervous System Targeting In some embodiments, the invention provides double-stranded iRNA agents that target APP for early-onset familial Alzheimer's disease, ATXN2 for spinocerebellar ataxia type 2 and ALS, and C9orf72 for amyotrophic lateral sclerosis and frontotemporal dementia.

[0479] In some embodiments, the invention provides double-stranded iRNA agents that target TARDBP for ALS, MAPT (tau) for frontotemporal dementia, and HTT for Huntington's disease.

[0480] In some embodiments, the invention provides double-stranded iRNA agents that target SNCA for Parkinson's disease, FUS for ALS, ATXN3 for spinocerebellar ataxia type 3, ATXN1 for SCA1, genes for SCA7 and SCA8, ATN1 for DRPLA, MeCP2 for XLMR, PRNP for prion diseases, recessive central nervous system disorders: Lafora disease, DMPK for DM1 (central nervous system and skeletal muscle), and TTR for hATTR (central nervous system, intraocular and systemic).

[0481] Spinocerebellar ataxia is a genetic brain dysfunction.Dominantly inherited spinocerebellar ataxia, such as SCA1-8, is a serious disease that does not have disease-modifying treatment.Exemplary targets include SCA2, SCA3 and SCA1.

[0482] Targeting ATXN2 for SCA2 Spinocerebellar ataxia type 2 (SCA2), a progressive ataxia, is the second most common form of SCA. Another disease related to this target is amyotrophic lateral sclerosis (ALS). These diseases are debilitating and ultimately fatal, with no disease-modifying treatment available. The prevalence of SCA is 2–6 per 100,000; ATXN2 causes 15% of the SCA population worldwide and a much higher SCA population in some countries, particularly Cuba (40 per 100,000). Targeting ATXN2 through human molecular genetics may be promising. For example, a coding CAG repeat expansion in ATXN2 has been found in tissues such as the spinal cord, brainstem, or cerebellum in familial and sporadic SCA and ALS. The mechanism of this targeting may be that the autosomal dominant coding CAG expansion in ATXN2 causes the expression of harmful misfolded proteins and Purkinje cell and neuronal cell death. Efficacy was demonstrated by 70% knockdown (KD) of ATXN2 mRNA; mATXN2 mouse KD proof of concept was demonstrated. Regarding safety, mATXN2 knockout (KO) mice were reported to be healthy. Possible diagnoses include family history; genetic testing; or early symptoms. Biomarkers that can be used include, for example, CSF CAG mRNA and peptide repeat protein.

[0483] Targeting ATXN3 for SCA3 Spinocerebellar ataxia type 3 (SCA3), a progressive ataxia, is the most common form of SCA worldwide. It is a debilitating, ultimately fatal disease with no disease-modifying treatment. It is the most common cause of SCA, with a prevalence of 2–6 per 100,000 individuals; ATXN3 causes 21% of SCA cases in the United States and is much more prevalent in Europe, particularly Portugal. Targeting ATXN3 through human molecular genetics may be promising. For example, a coding CAG repeat expansion in ATXN3 has been found in tissues such as the spinal cord, brainstem, or cerebellum in familial and sporadic SCA. The mechanism of this targeting may be due to the autosomal dominant coding CAG expansion in ATXN3, which leads to the expression of a deleterious misfolded protein, resulting in Purkinje cell and neuronal death. Efficacy was demonstrated by a 70% knockdown of ATXN3 mRNA; proof-of-concept in ATXN3 knockdown mice was demonstrated. Regarding safety, mATXN3 KO mice were reported to be healthy. Possible diagnoses include family history, genetic testing, or early symptoms. Biomarkers that can be used include, for example, CSF CAG mRNA and peptide repeat protein.

[0484] Targeting ATXN1 for SCA1 Spinocerebellar ataxia type 1 (SCA1), a progressive ataxia, was the first SCA gene discovered in 1993. This disease is debilitating and ultimately fatal, with no disease-modifying treatment available. The prevalence of SCA is 2–6 per 100,000 people; ATXN1 causes 6% of the SCA population in the United States and worldwide, with much higher prevalence in some countries (25% in Japan), particularly Poland (64%) and Siberia (100%). Targeting ATXN1 through human molecular genetics may be promising; for example, a coding CAG repeat expansion in ATXN1 has been found in tissues such as the spinal cord, brainstem, or cerebellum in familial and sporadic SCA. The mechanism of this targeting may be due to the autosomal dominant coding CAG expansion in ATXN1, which leads to the expression of a deleterious misfolded protein, Purkinje cell death, and neuronal death. Efficacy was demonstrated by a 70% reduction in ATXN1 mRNA; mATXN1 mouse POC was demonstrated. Regarding safety, mATXN1 KO mice were reported to be healthy. Possible diagnoses include family history; genetic testing; or early symptoms. Biomarkers that can be used include, for example, CSF CAG mRNA and peptide repeat proteins.

[0485] Targeting ATXN7 for SCA7 Spinocerebellar ataxia type 7 (SCA7) causes progressive ataxia and retinal degeneration. This disease is a debilitating, ultimately fatal retinal and cerebellar disorder with no disease-modifying treatment. The prevalence of SCA is 2-6 per 100,000 people; ATXN7 causes 5% of the global SCA population, with a much higher prevalence in some countries, particularly South Africa. Targeting ATXN7 through human molecular genetics may be promising. For example, a coding CAG repeat expansion in ATXN7 has been found in tissues such as the spinal cord, brainstem, cerebellum, or retina in familial and sporadic SCA. The mechanism of this targeting may be through the autosomal dominant coding CAG expansion in ATXN1, which causes the expression of a deleterious misfolded protein, leading to cone-rod degeneration, Purkinje cell death, and neuronal death. Efficacy was demonstrated by a 70% reduction in ATXN1 mRNA expression following intrathecal (IT) and intravitreal (IVT) administration. Possible diagnoses include family history, genetic testing, or early symptoms. Biomarkers that can be used include, for example, CSF CAG mRNA and peptide repeat protein.

[0486] Targeting ATXN8 for SCA8 Spinocerebellar ataxia type 8 (SCA8), a progressive neurodegenerative disorder, is caused by a CTG repeat expansion in ATXN8. This disease is debilitating and ultimately fatal, with no disease-modifying treatment available. Prevalence: SCA is 2-6 per 100,000 individuals; ATXN8 causes 3% of the global SCA population, with a much higher incidence in some countries, particularly Finland. Targeting ATXN8 through human molecular genetics may be promising. For example, coding CTG repeat expansions in ATXN8 have been found in tissues such as the spinal cord, brainstem, and cerebellum in familial and sporadic SCA. The mechanism of this targeting may be that the autosomal dominant coding CTG expansion in ATXN8 leads to the expression of a deleterious misfolded protein, causing Purkinje cell and neuronal death. Efficacy was demonstrated by a 70% reduction in ATXN8 mRNA expression. Possible diagnoses include family history; genetic testing; or early symptoms. Biomarkers that can be used include, for example, CSF CTG mRNA and peptide repeat proteins.

[0487] Targeting CACNA1A for SCA6 Spinocerebellar ataxia 6 (SCA6) is a progressive ataxia. It is a debilitating, ultimately fatal disease with no disease-modifying treatment. The prevalence of SCA is 2-6 per 100,000 people; CACNA1A causes 15% of the SCA population worldwide. Targeting CACNA1A through human molecular genetics may be promising. For example, a coding CAG repeat expansion in CACNA1A has been found in tissues such as the spinal cord, brainstem, or cerebellum in familial and sporadic SCA. The mechanism of this targeting may be that the autosomal dominant coding CAG expansion in CACNA1A causes the expression of a deleterious misfolded protein and Purkinje cell and neuronal cell death. Efficacy was demonstrated by a 70% KD of the CACNA1A CAG expansion. Possible diagnoses include family history; genetic testing; or early symptoms. Biomarkers that can be used include, for example, CSF CAG mRNA and peptide repeat proteins.

[0488] Exemplary targets of inherited polyglutamine disorders include Huntington's disease (HD).

[0489] Targeting HTT for Huntington's disease Huntington's disease mutations cause HD, a progressive central nervous system degenerative disorder. It is a debilitating, ultimately fatal disease with no disease-modifying treatment. The prevalence of HD is 5-10 per 100,000 people worldwide, with much higher rates in some countries, particularly Venezuela. Targeting HTT through human molecular genetics may be promising. For example, a coding CAG repeat expansion in HTT has been found in tissues such as the striatum or cerebral cortex in familial and sporadic HD. The mechanism of this targeting may be that the autosomal dominant coding CAG expansion in HTT leads to the expression of a deleterious misfolded protein and neuronal death. Efficacy was demonstrated by a 70% knockdown of the HTT CAG expansion alone; proof-of-concept in mice has been demonstrated. Regarding safety, knockdown of HTT in mice can be lethal; knockdown in humans has been demonstrated. Potential diagnostic criteria include family history, genetic testing, and early symptoms. Biomarkers that can be used include, for example, CSF mRNA and peptide repeat proteins.

[0490] Targeting ATN1 for DRPLA Atrophin 1 mutations cause dentatorubral-pallidoluysian atrophy (DRPLA), a progressive spinocerebellar disease similar to HD. This disease is debilitating and ultimately fatal, with no disease-modifying treatment available. The prevalence of DRPLA is 2-7 per 1,000,000 people in Japan. Targeting ATN1 through human molecular genetics may be promising. For example, a coding CAG repeat expansion in ATN1 has been found in familial and sporadic SCA in tissues such as the spinal cord, brainstem, cerebellum, or cerebral cortex. The mechanism of this targeting may be due to the autosomal dominant coding CAG expansion in ATN1 causing the expression of harmful misfolded proteins and neuronal death. Efficacy was demonstrated by a 70% knockout of ATN1. Regarding safety, ATN1 knockout mice were reported to be healthy. Possible diagnoses include family history, genetic testing, or early symptoms. Biomarkers that can be used include, for example, CSF CAG mRNA and peptide repeat proteins.

[0491] Targeting AR for spinal and bulbar muscular atrophy Androgen receptor mutations cause spinal and bulbar muscular atrophy (SBMA, Kennedy's disease), a progressive muscle-wasting disorder, and other diseases. This disease is debilitating and ultimately fatal, with no disease-modifying treatment. The prevalence of SBMA is 2 per 100,000 men; women have a milder phenotype. Targeting the AR through human molecular genetics may be successful; for example, a coding CAG repeat expansion in the AR has been discovered in tissues such as the spinal cord or brainstem in familial SBMA. The mechanism of this targeting may be that the X-linked coding CAG expansion in the AR causes deleterious gain of function and motor neuron death. Efficacy has been demonstrated by a 70% reduction in AR KD. Possible diagnoses include family history, genetic testing, or early symptoms. Biomarkers that can be used include, for example, CSF CAG mRNA and peptide repeat protein.

[0492] Targeting FXN for Friedreich's ataxia A recessive loss-of-function GAA expansion in FXN causes Friedreich's ataxia (FA), a progressive degenerative ataxia. It is a debilitating, ultimately fatal disease with no disease-modifying treatment. The prevalence of FA is 2 per 100,000 people worldwide. Targeting FXN through human molecular genetics may be promising; for example, an intronic GAA repeat expansion in FXN has been found in familial FA in tissues such as the spinal cord, cerebellum, and possibly the retina and heart. The mechanism of this targeting may be that the autosomal recessive noncoding FAA expansion in FXN reduces the expression of FXN, an important mitochondrial protein. Efficacy was demonstrated by a 70% knockdown of the FXN intronic GAS expansion. Regarding safety, knockdown of the intronic GAA expansion was safe and effective in mice. Potential diagnostics include family history, genetic testing, or early symptoms. Biomarkers that can be used include, for example, CSF mRNA and peptide repeat proteins.

[0493] Targeting FMR1 for FXTAS Fragile X-associated tremor / ataxia syndrome (FXTAS), a progressive disorder of adult ataxia and cognitive impairment, is caused by FMR1 overexpression. This disease is severe and has no disease-modifying treatment. The prevalence of FMR1 premutations is 1 in 500 males. Targeting FMR1 through human molecular genetics may be promising; for example, a coding CCG repeat expansion premutation in FMR1 has been found in FXTAS in tissues such as the spinal cord, cerebellum, or cerebral cortex. The mechanism of this targeting may be that the X-linked coding CCG expansion of FMR1 causes toxic mRNA. Efficacy was demonstrated by a 70% reduction in toxic mRNA. Possible diagnoses include family history, genetic testing, or early symptoms. Biomarkers that can be used include, for example, CSF mRNA and peptide repeat proteins.

[0494] Targeting FMR1 upstream for fragile X syndrome Fragile X syndrome (FRAXA), a progressive mental retardation disorder, can be treated by targeting the upstream mRNA of FMR1. This disease is debilitating and has no disease-modifying treatment. The prevalence of FRAXA is 1 in 4,000 men and 1 in 8,000 women. Targeting FMR1 through human molecular genetics may be promising; for example, a coding CCG repeat expansion in FMR1 has been discovered in FRAXA in tissues such as the central nervous system. The mechanism of this targeting may be that the X-linked coding CCG expansion in FMR1 causes loss of function; normal FMR1 functions to export specific mRNAs from the nucleus. Efficacy was demonstrated by a 70% reduction in toxic mRNA levels. Possible diagnoses include family history, genetic testing, or early symptoms. Biomarkers that can be used include, for example, CSF mRNA and peptide repeat proteins.

[0495] Dominantly inherited amyotrophic lateral sclerosis is a severe disease with no disease-...

Claims

1. 1. A composition for reducing expression of a target gene in a cell of the central nervous system (CNS) or in the central nervous system of a subject, the composition comprising a double-stranded iRNA agent, the double-stranded iRNA agent comprising: an antisense strand complementary to the target mRNA; a sense strand complementary to the antisense strand; and one lipophilic moiety attached directly to the 2'-O of a sugar moiety at an internal position of the sense strand; Including, the sense strand and the antisense strand are each independently 19 to 25 nucleotides in length, forming a double-stranded region having a length of 19 to 21 nucleotide pairs and a blunt end at the 5' end of the antisense strand; The lipophilic moiety is a saturated or unsaturated C ４ ~C ３０ is a hydrocarbon chain, the internal positions are positions on the sense strand excluding the three terminal positions from each of the 5'-end and 3'-end of the sense strand and positions 11 to 13 counting from the 3'-end of the sense strand; A composition in which the double-stranded iRNA agent is contacted with the cell or administered to the subject.

2. The composition of claim 1, which reduces expression of a target gene in brain or spinal cord tissue.

3. 3. The composition of claim 2, wherein the brain or spinal tissue is selected from the group consisting of cerebral cortex, cerebellum, cervical vertebrae, lumbar vertebrae, and thoracic vertebrae.

4. 2. The composition of claim 1, wherein the target gene is selected from the group consisting of APP, ATXN2, C9orf72, TARDBP, MAPT (tau), HTT, SNCA, FUS, ATXN3, ATXN1, SCA1, ATXN7, SCA7, ATXN8, SCA8, ATN1, MeCP2, PRNP, SOD1, DMPK, CACNA1A, AR, FXN, FMR1, LRRK2, GARS, seipin, PSEN1, PSEN2, Apo E, ITM2B, CST3, SPAST, KIF5A, ATL1, NIPA1, ZNF9, glycogen synthase, and TTR.

5. 1. A composition for reducing expression of a target gene in a subject, the composition comprising a double-stranded iRNA agent, the double-stranded iRNA agent comprising: an antisense strand complementary to the target mRNA; a sense strand complementary to the antisense strand; and one lipophilic moiety attached directly to the 2'-O of a sugar moiety at an internal position of the sense strand; Including, the sense strand and the antisense strand are each independently 19 to 25 nucleotides in length, forming a double-stranded region having a length of 19 to 21 nucleotide pairs and a blunt end at the 5' end of the antisense strand; The lipophilic moiety is a saturated or unsaturated C ４ ~C ３０ is a hydrocarbon chain, the internal positions are positions on the sense strand excluding the three terminal positions from each of the 5'-end and 3'-end of the sense strand and positions 11 to 13 counting from the 3'-end of the sense strand, The composition is administered intrathecally to the subject.

6. 6. The composition of claim 5 for treating a subject having a central nervous system (CNS) disorder.

7. 7. The composition of claim 6, wherein the central nervous system (CNS) disorder is selected from the group consisting of spinocerebellar ataxia (SCA), X-linked mental retardation (XLMR), myotonic dystrophy (DM), dentatorubral-pallidoluysian atrophy (DRPLA), spinal-bulbar muscular atrophy (SBMA), spastic paraplegia, cerebral amyloid angiopathy (CAA), spinal muscular atrophy (SMA), distal hereditary motor neuropathy, progressive supranuclear palsy (PSP), fragile X syndrome (FRAXA), fragile X-associated tremor / ataxia syndrome (FXTAS), Friedreich's ataxia (FA), Alzheimer's disease, amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Huntington's disease, Parkinson's disease, spinocerebellar disorders, prion diseases, and Lafora's disease.

8. The lipophilic moiety is a saturated or unsaturated C ６ ~C １８ The composition of any one of claims 1 to 7, comprising a hydrocarbon chain.

9. The lipophilic moiety is a linear C １６ The composition of claim 8 which is an alkyl chain.

10. The composition of any one of claims 1-9, wherein the double-stranded iRNA agent does not include an N-acetyl-D-galactosamine (GalNAc) conjugate.

11. The composition of any one of claims 1 to 10, wherein the internal position excludes positions 9 to 12 counting from the 5' end of the sense strand.

12. 11. The composition of any one of claims 1 to 10, wherein the sense strand and the antisense strand are each independently 21 to 23 nucleotides in length, and the internal position is any of positions 4 to 8 and 13 to 18 counting from the 5' end of the sense strand.

13. The composition of any one of claims 1 to 10, wherein the internal position is position 5, 6, or 7 counting from the 5' end of the sense strand.

14. The composition of any one of claims 1-13, wherein the double-stranded iRNA agent further comprises an overhang region at the 3' end of the antisense strand comprising two nucleotides, and has a phosphorothioate or methylphosphonate internucleotide linkage between the two overhanging nucleotides.

15. The composition of any one of claims 1-13, wherein the double-stranded iRNA agent further comprises an overhang region at the 3' end of the antisense strand, comprising two phosphorothioate internucleotide linkages between the terminal three nucleotides of the 3' end of the antisense strand, two of the three nucleotides being overhang nucleotides and the third being the next paired nucleotide to the overhang nucleotide.

16. The composition of any one of claims 1 to 15, wherein the double-stranded iRNA agent further comprises a phosphate or a phosphate mimetic at the 5'-end of the antisense strand.

17. The composition of claim 16, wherein the phosphate mimic is 5'-vinylphosphonic acid (VP).

18. the double stranded iRNA agent (a) a sense strand, (i) 21 nucleotides in length; (ii) 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 (iii) phosphorothioate internucleotide bonds between the 1st and 2nd nucleotides and between the 2nd and 3rd nucleotides, counting from the 5' end of the sense strand; a sense strand comprising: (b) an antisense strand, (i) 23 nucleotides in length; (ii)(1) 2'-OMe modifications at positions 1, 3-5, 7, 8, 10-13, 15, and 17-23, and 2'-F modifications at positions 2, 6, 9, 14, and 16, counting from the 5' end of the antisense strand; or (2) 2'-OMe modifications at positions 1, 3-5, 7, 10-13, 15, and 17-23, and 2'-F modifications at positions 2, 6, 8, 9, 14, and 16, counting from the 5' end of the antisense strand; and (iii) phosphorothioate internucleotide bonds between nucleotides at positions 1 and 2, between nucleotides at positions 2 and 3, between nucleotides at positions 21 and 22, and between nucleotides at positions 22 and 23, counting from the 5′ end of the antisense strand; The antisense strand comprises Including, The composition according to any one of claims 1 to 10, which has a 2-nucleotide overhang at the 3'-end of the antisense strand and a blunt end at the 5'-end of the antisense strand.

Images