Huntingtin (HTT) iRNA agent compositions and methods of use thereof

Abstract

The present disclosure relates to double-stranded ribonucleic acid (dsRNAi) agents and compositions that target the huntingtin (HTT) gene, and methods of using the dsRNAi agents and compositions to inhibit expression of the HTT gene and to treat subjects with HTT-related diseases or disorders, e.g., Huntington's disease.

Description

[Background technology]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 167,140, ​​filed March 29, 2021, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated by reference in its entirety. The ASCII copy was created on March 22, 2022, is named 121301-_14420_SL.txt, and is 198,616 bytes in size.

[0003] Huntington's disease is a progressive neurodegenerative disorder characterized by movement disorders, cognitive impairment, and psychiatric symptoms (Martin and Gusella (1986) N. Engl. J. Med. 315:1267-1276). It is inherited in an autosomal dominant manner and affects approximately 1 in 10,000 individuals in most populations of European origin (Harper, P.S. et al., in Huntington's Disease, W.B. Saunders, Philadelphia, 1991). The hallmark of Huntington's disease is a distinctive choreiform movement disorder, which typically has a subtle, insidious onset in the fourth or fifth decade of life and gradually worsens over 10 to 20 years before death. Huntington's disease often manifests at an early age, typically manifesting with more severe symptoms, including rigidity and a rapid progression. The early age at onset of Huntington's disease is associated with predominant paternal transmission of the disease allele. The neuropathology of Huntington's disease also shows a distinctive pattern, with selective loss of neurons that is most severe in the caudate and putamen regions of the brain.

[0004] Huntington's disease is known to be caused by an expanded glutamine repeat in exon 1 of a gene called IT15, or huntingtin (HTT). This gene is widely expressed and necessary for normal development, but Huntington's disease pathology is restricted to the brain, for reasons that remain poorly understood. In patients with HD (an autosomal dominant genetic disease), polyglutamine repeat expansion results in wild-type transcripts, full-length mutant transcripts with expanded polyglutamine repeats, and truncated mutant transcripts with expanded polyglutamine repeats. While the huntingtin gene product is expressed at similar levels in patients and controls, it is the presence of expanded polyglutamine repeats and full-length mutant transcripts, as well as truncated mutant transcripts, that induce toxicity.

[0005] No effective treatment for Huntington's disease is currently available. Chorea and agitated behavior can usually only be partially suppressed by antipsychotic drugs (e.g., chlorpromazine) or reserpine, until the adverse effects of lethargy, hypotension, or tremor paralysis occur. In addition, despite significant progress in the fields of RNAi and Huntington's disease treatment, there remains a need for agents that can selectively and efficiently silence the HD gene using the cell's own RNAi mechanism, have high biological activity and in vivo stability, and can effectively inhibit the expression of the target huntingtin gene. Summary of the Invention

[0006] The present disclosure provides an RNAi agent composition that performs RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the huntingtin (HTT) gene.The HTT gene can be present in a cell, for example, in a subject, such as a human.The present disclosure also provides a method for using the RNAi agent composition of the present disclosure to inhibit the expression of the HTT gene or to treat a subject that will benefit from inhibiting or reducing the expression of the HTT gene, for example, a subject that suffers from or is prone to suffer from an HTT-related disease.

[0007] In one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent that inhibits expression of huntingtin (HTT) in a cell, the dsRNA comprising a sense strand and an antisense strand that form a double-stranded region, the antisense strand comprising a region complementary to an mRNA encoding HTT, the complementary region comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides, e.g., no more than 3 nucleotides, no more than 2 nucleotides, no more than 1 nucleotide, or no nucleotide, from any one of the antisense nucleotide sequences in any one of Tables 2-5.

[0008] In another aspect, the present invention provides a double-stranded ribonucleic acid (dsRNA) agent that inhibits expression of huntingtin (HTT) in a cell, the dsRNA comprising a sense strand and an antisense strand that form a double-stranded region, wherein the sense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21, contiguous nucleotides that differ by no more than three nucleotides, e.g., 3 nucleotides, 2 nucleotides, 1 nucleotide, or 0 nucleotides, from any one of the nucleotide sequences of nucleotides 4391-4669, 6500-6540, or 6009-6037 of SEQ ID NO:1, and the antisense strand comprises at least 15 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO:6.

[0009] In one embodiment, the sense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21, contiguous nucleotides that differ by no more than three nucleotides from any one of the nucleotide sequences 4398-4420, 4403-4425, or 6512-6534 of SEQ ID NO:1.

[0010] In one embodiment, the sense strand comprises at least 15, eg, 15, 16, 17, 18, 19, 20, or 21, contiguous nucleotides that differ from nucleotides 4398-4420 of SEQ ID NO:1 by no more than three nucleotides.

[0011] In one embodiment, the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by no more than three nucleotides from any one of the antisense strand nucleotide sequences of a duplex selected from the group consisting of AD-1271085, AD-1271083, or AD-1271084.

[0012] In one embodiment, the antisense strand comprises at least 15, eg, 15, 16, 17, 18, 19, 20, 21, 22, or 23 contiguous nucleotides that differ by no more than three nucleotides from the antisense strand nucleotide sequence of AD-1271085.

[0013] In one embodiment, the sense strand comprises the nucleotide sequence 5'-GCUAUUCAUAAUCACAUUCGA-3' (SEQ ID NO: 15).

[0014] In one embodiment, the sense strand consists of the nucleotide sequence 5'-GCUAUUCAUAAUCACAUUCGA-3' (SEQ ID NO: 15).

[0015] In one embodiment, the antisense strand comprises the nucleotide sequence 5'-UCGAAUGUGAUUAUGAAUAGCAU-3' (SEQ ID NO: 16).

[0016] In one embodiment, the antisense strand consists of the nucleotide sequence 5'-UCGAAUGUGAUUAUGAAUAGCAU-3' (SEQ ID NO: 15).

[0017] In one embodiment, the sense strand comprises the nucleotide sequence 5'-GCUAUUCAUAAUCACAUUCGA-3' (SEQ ID NO: 15) and the antisense strand comprises the nucleotide sequence 5'-UCGAAUGUGAUUAUGAAUAGCAU-3' (SEQ ID NO: 16).

[0018] In one embodiment, the sense strand comprises the nucleotide sequence 5'-GCUAUUCAUAAUCACAUUCGA-3' (SEQ ID NO: 15) and the antisense strand comprises the nucleotide sequence 5'-UCGAAUGUGAUUAUGAAUAGCAU-3' (SEQ ID NO: 16).

[0019] In one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent that inhibits expression of huntingtin (HTT) in a cell, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, the sense strand comprising at least 15 contiguous nucleotides that differ by no more than three nucleotides from nucleotides 26-77 or 142-202 of SEQ ID NO:1, and the antisense strand comprising at least 15 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO:6.

[0020] In one embodiment, the sense strand comprises at least 15 contiguous nucleotides that differ by no more than three nucleotides from the nucleotide sequence of nucleotides 27-49, 55-77, 147-169, or 173-195 of SEQ ID NO:1.

[0021] In one embodiment, the sense strand comprises at least 15 contiguous nucleotides that differ from nucleotides 147-169 of SEQ ID NO:1 by no more than three nucleotides.

[0022] In one embodiment, the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than three nucleotides from the antisense strand nucleotide sequence of a duplex selected from the group consisting of AD-1019448, AD-1498524, AD-1498526, or AD-1498528.

[0023] In one embodiment, the antisense strand comprises at least 15 contiguous nucleotides that differ from the antisense strand nucleotide sequence of AD-1498524 by no more than three nucleotides.

[0024] Sense strand, antisense strand, or both sense strand and antisense strand can be conjugated with one or more lipophilic moieties.In some embodiments, lipophilic moieties are conjugated to one or more internal positions in the double-stranded region of dsRNA agent, for example, one or more lipophilic moieties can be conjugated to one or more internal positions on antisense strand.In some embodiments, one or more lipophilic moieties are conjugated to one or more internal positions on at least one strand via linker or carrier.

[0025] In some embodiments, the lipophilicity of the lipophilic moiety is greater than 0, as measured by log Kow.

[0026] In some embodiments, the hydrophobicity of the dsRNA agent is greater than 0.2 as measured by the unbound fraction in a plasma protein binding assay of the dsRNA agent. In some embodiments, the plasma protein binding assay is an electrophoretic mobility shift assay using human serum albumin protein.

[0027] In some embodiments, internal positions include all but the two most distal positions from each end of the sense or antisense strand, while in other embodiments, internal positions include all but the three most distal positions from each end of the sense or antisense strand.

[0028] In some embodiments, internal positions exclude the cleavage site region of the sense strand, for example, internal positions include all positions except positions 9-12 counting from the 5' end of the sense strand, or internal positions include all positions except positions 11-13 counting from the 3' end of the sense strand.

[0029] In some embodiments, internal positions exclude the cleavage site region of the antisense strand. In other embodiments, internal positions include all positions except positions 12-14 counting from the 5' end of the antisense strand. In some embodiments, internal positions include all positions except 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 some embodiments, the one or more lipophilic moieties are conjugated to one or more of the internal positions selected from the group consisting of 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 some embodiments, the one or more lipophilic moieties are conjugated to one or more internal positions selected from the group consisting of 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 position in the double-stranded region excludes the cleavage site region of the sense strand.

[0033] In other embodiments, the sense strand is 21 nucleotides in length, the antisense strand is 23 nucleotides in length, and the lipophilic moiety is conjugated to position 20, 15, 1, 7, 6, or 2 of the sense strand or position 16 of the antisense strand.

[0034] In other embodiments, the sense strand is 21 nucleotides in length, the antisense strand is 23 nucleotides in length, and the lipophilic moiety is conjugated to position 21, 20, 15, 1, 7, 6, or 2 of the sense strand or position 16 of the antisense strand.

[0035] In some embodiments, the lipophilic moiety is an aliphatic compound, an alicyclic compound, or a polyalicyclic compound.

[0036] In some embodiments, the lipophilic moiety is selected from the group consisting of a lipid, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, a heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.

[0037] In some embodiments, the lipophilic moiety contains a saturated or unsaturated C4-C30 hydrocarbon chain and an optional functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.

[0038] In some embodiments, the lipophilic moiety contains a saturated or unsaturated C6 to C18 hydrocarbon chain.

[0039] In some embodiments, the lipophilic moiety contains a saturated or unsaturated C16 hydrocarbon chain, in some embodiments, the saturated or unsaturated C16 hydrocarbon chain is conjugated to the 6th position, counting from the 5' end of one chain.

[0040] In some embodiments, the lipophilic moiety is conjugated via a carrier that replaces one or more nucleotides in the internal position or double-stranded region. In some embodiments, the carrier is a cyclic group selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl, or an acyclic moiety of a serinol backbone or a diethanolamine backbone system.

[0041] In some embodiments, the lipophilic moiety is conjugated to the dsRNA agent via a linker that contains an ether, a thioether, a urea, a carbonate, an amine, an amide, a maleimide-thioether, a disulfide, a phosphodiester, a sulfonamide linkage, a product of a click reaction, or a carbamate.

[0042] In some embodiments, the lipophilic moiety is conjugated to a nucleobase, a sugar moiety, or an internucleoside linkage.

[0043] In some embodiments, dsRNA agent comprises at least one modified nucleotide.In some embodiments, five or less nucleotides of sense strand and five or less nucleotides of antisense strand are unmodified nucleotides.In other embodiments, all nucleotides of sense strand and all nucleotides of antisense strand comprise modification.

[0044] In some embodiments, at least one of the modified nucleotides is a deoxy-nucleotide, a 3'-terminal deoxy-thymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide, a 2'-O-allyl-modified nucleotide, a 2'-C-alkyl-modified nucleotide, a 2'-hydroxy-modified nucleotide, a 2'-methoxyethyl-modified nucleotide, a 2'-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide containing a non-natural base, a tetrahydropyran-modified nucleotide, a 1,5-anhydrohexitol-modified nucleotide, a cyclohexenyl-modified nucleotide, a nucleotide containing a 5'-phosphorothioate group, a nucleotide containing a 5'-methylphosphonate group, a 5'-phosphoric acid ... The nucleotide is selected from the group consisting of nucleotides containing 5' phosphate or 5' phosphate mimics, nucleotides containing vinyl phosphonates, nucleotides containing adenosine glycol nucleic acid (GNA), nucleotides containing thymidine glycol nucleic acid (GNA) S isomers, nucleotides containing 2-hydroxymethyl-tetrahydrofuran-5-phosphate, nucleotides containing 2'-deoxythymidine-3' phosphate, nucleotides containing 2'-deoxyguanosine-3'-phosphate, and terminal nucleotides linked to cholesteryl derivatives and dodecanoic acid bisdecylamide groups, and combinations thereof.

[0045] In other embodiments, the modified nucleotide is selected from the group consisting of 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, 3'-terminal deoxy-thymine nucleotides (dT), locked nucleotides, abasic nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, and nucleotides containing unnatural bases.

[0046] In some embodiments, at least one of the modified nucleotides is selected from the group consisting of deoxy-nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, glycol-modified nucleotides (GNAs), and vinyl-phosphonate nucleotides; and combinations thereof.

[0047] In some embodiments, at least one of the modifications in the nucleotide is a thermally destabilizing nucleotide modification, in some embodiments, the thermally destabilizing nucleotide modification is selected from the group consisting of an abasic modification, a mismatch with the opposing nucleotide in a duplex, and a destabilizing sugar modification, a 2'-deoxy modification, an acyclic nucleotide, an unlocked nucleic acid (UNA), and a glycerol nucleic acid (GNA).

[0048] In some embodiments, the modified nucleotides include a short sequence of 3'-terminal deoxy-thymine nucleotides (dT).

[0049] In some embodiments, the modifications in the nucleotides are 2'-O-methyl modifications, GNA modifications, and 2' fluoro modifications.

[0050] In some embodiments, the dsRNA agent further comprises at least one phosphorothioate internucleotide linkage. In some embodiments, the dsRNA agent comprises 6 to 8 phosphorothioate internucleotide linkages. In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 3'-end of one strand. Optionally, this strand is the antisense strand. In another embodiment, this strand is the sense strand. In a related embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 5'-end of one strand. Optionally, this strand is the antisense strand. In another embodiment, this strand is the sense strand. In another embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at both the 5'-end and the 3'-end of one strand. Optionally, this strand is the antisense strand. In another embodiment, this strand is the sense strand.

[0051] In some embodiments, each strand is 30 nucleotides or less in length.

[0052] In some embodiments, at least one strand comprises a 3' overhang of at least 1 nucleotide or a 3' overhang of at least 2 nucleotides.

[0053] The double-stranded region can be 15 to 30 nucleotide pairs in length, 17 to 23 nucleotide pairs in length, 17 to 25 nucleotide pairs in length, 23 to 27 nucleotide pairs in length, 19 to 21 nucleotide pairs in length, or 21 to 23 nucleotide pairs in length.

[0054] Each strand can be 19-30 nucleotides, 19-23 nucleotides, or 21-23 nucleotides.

[0055] In some embodiments, dsRNA agent further comprises the targeting ligand that targets liver tissue.In some embodiments, targeting ligand is GalNAc conjugate.In other embodiments, dsRNA agent does not comprise the targeting ligand that targets liver tissue, such as GalNAc conjugate.

[0056] In certain embodiments, the double-stranded RNAi agent further comprises a targeting ligand, eg, a hydrophilic ligand, that targets a receptor that mediates delivery to CNS tissue.

[0057] In certain embodiments, the targeting ligand is a C16 ligand. In one embodiment, the ligand is [ka] , wherein B is a nucleotide base or a nucleotide base analog; optionally, B may be adenine, guanine, cytosine, thymine, or uracil.

[0058] In some embodiments, the dsRNA agent further comprises a targeting ligand that targets a receptor that mediates delivery to CNS tissue, e.g., a hydrophilic ligand such as a C16 ligand, e.g., [ka] , wherein B is a nucleotide base or nucleotide base analog, optionally B is adenine, guanine, cytosine, thymine, or uracil, and does not include a targeting ligand that targets liver tissue, such as a GalNAc conjugate.

[0059] In some embodiments, the lipophilic moiety or targeting ligand is conjugated via a bio-cleavable linker selected from the group consisting of DNA, RNA, disulfide, amide, and functionalized mono- or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

[0060] In some embodiments, the 3' end of the sense strand is protected via an end cap that is an amine-bearing cyclic group selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl.

[0061] In some embodiments, the dsRNA agent further includes a terminal chiral modification occurring at a first internucleotide linkage at the 3'-end of the antisense strand having a linking phosphorus atom in the Sp configuration, a terminal chiral modification occurring at a first internucleotide linkage at the 5'-end of the antisense strand having a linking phosphorus atom in the Rp configuration, and a terminal chiral modification occurring at a first internucleotide linkage at the 5'-end of the sense strand having a linking phosphorus atom in either the Rp or Sp configuration.

[0062] In some embodiments, the dsRNA agent further includes a terminal chiral modification occurring 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 occurring 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 occurring 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.

[0063] In some embodiments, the dsRNA agent further includes a terminal chiral modification occurring at the first, second, and third internucleotide linkages at the 3'-end of the antisense strand having a linking phosphorus atom in the Sp configuration, a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the antisense strand having a linking phosphorus atom in the Rp configuration, and a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the sense strand having a linking phosphorus atom in either the Rp or Sp configuration.

[0064] In some embodiments, the dsRNA agent further includes a terminal chiral modification occurring at the first and second internucleotide linkages at the 3'-end of the antisense strand having a linking phosphorus atom in the Sp configuration, a terminal chiral modification occurring at the third internucleotide linkage at the 3'-end of the antisense strand having a linking phosphorus atom in the Rp configuration, a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the antisense strand having a linking phosphorus atom in the Rp configuration, and a terminal chiral modification occurring at the first internucleotide linkage at the 5'-end of the sense strand having a linking phosphorus atom in either the Rp or Sp configuration.

[0065] In some embodiments, the dsRNA agent further includes a terminal chiral modification occurring 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 occurring at the first and second internucleotide linkages at the 5'-end of the antisense strand, with the linking phosphorus atom in the Rp configuration; and a terminal chiral modification occurring 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.

[0066] In some embodiments, the dsRNA agent further comprises a phosphate or a phosphate mimic at the 5'-end of the antisense strand. In some embodiments, the phosphate mimic is 5'-vinylphosphonate (VP).

[0067] In some embodiments, the base pair at position 1 of the 5'-end of the antisense strand of the duplex is an AU base pair.

[0068] In some embodiments, the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

[0069] In one embodiment, the antisense strand comprises the nucleotide sequence 5'-UAUCAGCUUUUCCAGGGUCGCCG-3' (SEQ ID NO: 53).

[0070] In one embodiment, the sense strand comprises the nucleotide sequence 5'-GCGACCCUGGAAAAGCUGAUA-3' (SEQ ID NO: 40).

[0071] In one embodiment, the sense strand comprises the nucleotide sequence 5'-GCGACCCUGGAAAAGCUGAUA-3' (SEQ ID NO: 40) and the antisense strand comprises the nucleotide sequence 5'-UAUCAGCUUUUCCAGGGUCGCCG-3' (SEQ ID NO: 53).

[0072] In one embodiment, the antisense strand comprises the nucleotide sequence 5'-UAGGACTUGAGGGACUCGAAGGC-3' (SEQ ID NO: 57).

[0073] In one embodiment, the sense strand comprises the nucleotide sequence 5'-CUUCGAGUCCCUCAAGUCCUA-3' (SEQ ID NO: 44).

[0074] In one embodiment, the sense strand comprises the nucleotide sequence 5'-CUUCGAGUCCCUCAAGUCCUA-3' (SEQ ID NO: 44) and the antisense strand comprises the nucleotide sequence 5'-UAGGACTUGAGGGACUCGAAGGC-3' (SEQ ID NO: 57).

[0075] In one embodiment, the antisense strand comprises the nucleotide sequence 5'-UUAAAAGCAGAACCUGACCGGCC-3' (SEQ ID NO: 59).

[0076] In one embodiment, the sense strand comprises the nucleotide sequence 5'-CCGGUCAGGUUCUGCUUUUAA-3' (SEQ ID NO: 46).

[0077] In one embodiment, the sense strand comprises the nucleotide sequence 5'-CCGGUCAGGUUCUGCUUUUAA-3' (SEQ ID NO: 46) and the antisense strand comprises the nucleotide sequence 5'-UUAAAAGCAGAACCUGACCGGCC-3' (SEQ ID NO: 59).

[0078] In one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent that inhibits expression of huntingtin (HTT) in a cell, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, the sense strand comprising at least 15 contiguous nucleotides that differ by no more than 4 nucleotides from the nucleotide sequence 5'-gscsgac(Chd)CfuGfGfAfaaagcugasusa-3' (SEQ ID NO: 65), and the antisense strand comprising the nucleotide sequence 5'-VPusAfsucdAg(C2p)uuu udCcAfgdGgucgcscsg-3' (SEQ ID NO:79), wherein a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; s is a phosphorothioate linkage; Af, Cf, Gf, Uf are 2'-fluoro A, C, G, and U; (Chd) is 2'-O-hexadecyl-cytosine-3'-phosphate; C2p is cytidine-2'-phosphate; and VP is 5'-vinylphosphonate.

[0079] In one embodiment, the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence 5'-gscsgac(Chd)CfuGfGfAfaaagcugasusa-3' (SEQ ID NO: 65), and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence 5'-VPusAfsucdAg(C2p)uuuudCcAfgdGgucgcscsg-3' (SEQ ID NO: 79).

[0080] In one embodiment, the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 2 nucleotides from the nucleotide sequence 5'-gscsgac(Chd)CfuGfGfAfaaagcugasusa-3' (SEQ ID NO: 65), and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 2 nucleotides from the nucleotide sequence 5'-VPusAfsucdAg(C2p)uuuudCcAfgdGgucgcscsg-3' (SEQ ID NO: 79).

[0081] In one embodiment, the sense strand comprises at least 15 contiguous nucleotides that differ by no more than one nucleotide from the nucleotide sequence 5'-gscsgac(Chd)CfuGfGfAfaaagcugasusa-3' (SEQ ID NO: 65), and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than one nucleotide from the nucleotide sequence 5'-VPusAfsucdAg(C2p)uuuudCcAfgdGgucgcscsg-3' (SEQ ID NO: 79).

[0082] In one embodiment, the sense strand comprises the nucleotide sequence 5'-gscsgac(Chd)CfuGfGfAfaaagcugasusa-3' (SEQ ID NO: 65) and the antisense strand comprises the nucleotide sequence 5'-VPusAfsucdAg(C2p)uuuudCcAfgdGgucgcscsg-3' (SEQ ID NO: 79).

[0083] In one embodiment, the sense strand consists of the nucleotide sequence 5'-gscsgac(Chd)CfuGfGfAfaaagcugasusa-3' (SEQ ID NO: 65), and the antisense strand consists of the nucleotide sequence 5'-VPusAfsucdAg(C2p)uuuudCcAfgdGgucgcscsg-3' (SEQ ID NO: 79).

[0084] In another aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent that inhibits expression of huntingtin (HTT) in a cell, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, the sense strand comprising at least 15 contiguous nucleotides that differ by no more than four nucleotides from the nucleotide sequence 5'-csusucg(Ahd)GfuCfCfCfucaaguccsusa-3' (SEQ ID NO: 70), and the antisense strand comprising the nucleotide sequence 5'-VPusAfsggac(Tgn)ugagggAf cUfcgaagsgsc-3' (SEQ ID NO: 83), comprising at least 15 contiguous nucleotides that differ by no more than four nucleotides, wherein a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; s is a phosphorothioate linkage; Af, Cf, Gf, Uf are 2'-fluoro A, C, G, and U; (Ahd) is 2'-O-hexadecyl-adenosine-3'-phosphate; Tgn is a thymidine-glycol nucleic acid (GNA) S isomer; and VP is a 5'-vinylphosphonate.

[0085] In one embodiment, the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence 5'-csusucg(Ahd)GfuCfCfCfucaaguccsusa-3' (SEQ ID NO: 70), and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence 5'-VPusAfsggac(Tgn)ugagggAfcUfcgaagsgsc-3' (SEQ ID NO: 83).

[0086] In one embodiment, the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 2 nucleotides from the nucleotide sequence 5'-csusucg(Ahd)GfuCfCfCfucaaguccsusa-3' (SEQ ID NO: 70), and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 2 nucleotides from the nucleotide sequence 5'-VPusAfsggac(Tgn)ugagggAfcUfcgaagsgsc-3' (SEQ ID NO: 83).

[0087] In one embodiment, the sense strand comprises at least 15 contiguous nucleotides that differ by no more than one nucleotide from the nucleotide sequence 5'-csusucg(Ahd)GfuCfCfCfucaaguccsusa-3' (SEQ ID NO: 70), and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than one nucleotide from the nucleotide sequence 5'-VPusAfsggac(Tgn)ugagggAfcUfcgaagsgsc-3' (SEQ ID NO: 83).

[0088] In one embodiment, the sense strand comprises the nucleotide sequence 5'-csusucg(Ahd)GfuCfCfCfucaaguccsusa-3' (SEQ ID NO: 70) and the antisense strand comprises the nucleotide sequence 5'-VPusAfsggac(Tgn)ugagggAfcUfcgaagsgsc-3' (SEQ ID NO: 83).

[0089] In one embodiment, the sense strand consists of the nucleotide sequence 5'-csusucg(Ahd)GfuCfCfCfucaaguccsusa-3' (SEQ ID NO: 70) and the antisense strand consists of the nucleotide sequence 5'-VPusAfsggac(Tgn)ugagggAfcUfcgaagsgsc-3' (SEQ ID NO: 83).

[0090] In one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent that inhibits expression of huntingtin (HTT) in a cell, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, the sense strand comprising at least 15 contiguous nucleotides that differ by no more than 4 nucleotides from the nucleotide sequence 5'-cscsggu(Chd)AfgGfUfUfcugcuuuusasa-3' (SEQ ID NO: 72), and the antisense strand comprising the nucleotide sequence 5'-VPusUfsa adAadGcagaacCfudGaccggscsc-3' (SEQ ID NO: 85), wherein a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U; s is a phosphorothioate linkage; Af, Cf, Gf, Uf are 2'-fluoro A, C, G, and U; (Chd) is 2'-O-hexadecyl-cytosine-3'-phosphate; and VP is 5'-vinylphosphonate.

[0091] In one embodiment, the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence 5'-cscsggu(Chd)AfgGfUfUfcugcuuuusasa-3' (SEQ ID NO: 72), and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence 5'-VPusUfsaadAadGcagaacCfudGaccggscsc-3' (SEQ ID NO: 85).

[0092] In one embodiment, the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 2 nucleotides from the nucleotide sequence 5'-cscsggu(Chd)AfgGfUfUfcugcuuuusasa-3' (SEQ ID NO: 72), and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 2 nucleotides from the nucleotide sequence 5'-VPusUfsaadAadGcagaacCfudGaccggscsc-3' (SEQ ID NO: 85).

[0093] In one embodiment, the sense strand comprises at least 15 contiguous nucleotides that differ by no more than one nucleotide from the nucleotide sequence 5'-cscsggu(Chd)AfgGfUfUfcugcuuuusasa-3' (SEQ ID NO: 72), and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than one nucleotide from the nucleotide sequence 5'-VPusUfsaadAadGcagaacCfudGaccggscsc-3' (SEQ ID NO: 85).

[0094] In one embodiment, the sense strand comprises the nucleotide sequence 5'-cscsggu(Chd)AfgGfUfUfcugcuuuusasa-3' (SEQ ID NO: 72) and the antisense strand comprises the nucleotide sequence 5'-VPusUfsaadAadGcagaacCfudGaccggscsc-3' (SEQ ID NO: 85).

[0095] In one embodiment, the sense strand consists of the nucleotide sequence 5'-cscsggu(Chd)AfgGfUfUfcugcuuuusasa-3' (SEQ ID NO: 72) and the antisense strand consists of the nucleotide sequence 5'-VPusUfsaadAadGcagaacCfudGaccggscs-3' (SEQ ID NO: 85).

[0096] In another aspect, the invention provides a double-stranded RNAi agent that inhibits expression of huntingtin (HTT) in a cell, the double-stranded RNAi agent comprising a sense strand and an antisense strand that form a double-stranded region, wherein the sense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21, contiguous nucleotides that differ by no more than 3 nucleotides (i.e., differs by 3, 2, 1, or 0 nucleotides) from the nucleotide sequence 5'-GCUAUUCAUAAUCACAUUCGA-3' (SEQ ID NO: 15), and the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21, contiguous nucleotides that differ by no more than 3 nucleotides (i.e., differs by 3, 2, 1, or 0 nucleotides) from the nucleotide sequence 5'-UCGAAUGUGAUUAUGAAUAGCAU-3' (SEQ ID NO: 16). 2'-O-methyl, 2'-fluoro, and 2'-O-hexadecyl modifications, wherein substantially all of the nucleotides in the sense strand, e.g., 18, 19, 20, 21, 22, or 23 contiguous nucleotides, for example, 15, 16, 17, 18, 19, 20, 21, 22, or 23, comprise a nucleotide modification selected from the group consisting of a 2'-O-methyl, a 2'-fluoro, and a 2'-O-hexadecyl modification, and the dsRNA agent comprises six to eight phosphorothioate internucleotide linkages, the sense strand comprises one or more vinylphosphonate (VP) modifications, e.g., a single VP modification at the 5'-end of the antisense strand, and the dsRNA agent does not comprise a ligand that targets liver tissue, e.g., a GalNAc ligand. In one embodiment, the dsRNA agent comprises eight phosphorothioate internucleotide linkages.

[0097] In another aspect, the present invention provides a double-stranded RNAi agent that inhibits expression of the huntingtin (HTT) gene in a cell, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21, contiguous nucleotides that differ from the nucleotide sequence 5'-GCUAUUCAUAAUCACAUUCGA-3' (SEQ ID NO: 15) by no more than 3 nucleotides (i.e., differs by 3, 2, 1, or 0 nucleotides), and the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, 21, 22, or 23, contiguous nucleotides that differ from the nucleotide sequence 5'-UCGAAUGUGAUUAUGAAUAGCAU-3' (SEQ ID NO: 16) by no more than 3 nucleotides (i.e., differs by 3, 2, 1, or 0 nucleotides), Substantially all of the nucleotides of the strand, e.g., 18, 19, 20, or 21 or more, and substantially all of the nucleotides of the antisense strand, e.g., 20, 21, 22, or 23 or more, comprise a nucleotide modification selected from the group consisting of a 2'-O-methyl modification, a 2'-fluoro modification, and a 2'-O-hexadecyl modification; the sense strand comprises four phosphorothioate internucleotide linkages, e.g., two phosphorothioate internucleotide linkages at the 5'-terminus and two phosphorothioate internucleotide linkages at the 3'-terminus; and the antisense strand comprises four phosphorothioate internucleotide linkages, e.g., two phosphorothioate internucleotide linkages at the 5'-terminus and two phosphorothioate internucleotide linkages at the 3'-terminus; and the sense strand comprises one or more vinyl phosphonate (VP) modifications, e.g., a single VP modification at the 5'-terminus of the antisense strand.

[0098] In another embodiment, the sense strand comprises the nucleotide sequence 5'-gscsuau(Uhd)CfaUfAfAfucacauucsgsa-3' (SEQ ID NO: 17) and the antisense strand comprises the nucleotide sequence 5'-VPusCfsgaaUfgUfGfauuaUfgAfauagcsasu-3' (SEQ ID NO: 18), where a, c, g, and u are 2'-O-methyl (2'-OMe) modified A, C, G, and U, respectively; Af, Cf, Gf, and Uf are 2'-fluoro modified A, C, G, and U, respectively; s is a phosphorothioate linkage; and VP is vinylphosphonate. In one embodiment, the dsRNA agent further comprises a ligand, such as N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol.

[0099] In one embodiment, the sense strand consists of the nucleotide sequence 5'-gscsuau(Uhd)CfaUfAfAfucacauucsgsa-3' (SEQ ID NO: 17) and the antisense strand consists of the nucleotide sequence 5'-VPusCfsgaaUfgUfGfauuaUfgAfauagcsasu-3' (SEQ ID NO: 18), where a, c, g, and u are 2'-O-methyl (2'-OMe) modified A, C, G, and U, respectively; Af, Cf, Gf, and Uf are 2'-fluoro modified A, C, G, and U, respectively; s is a phosphorothioate linkage; and VP is vinylphosphonate. In one embodiment, the dsRNA agent further comprises a ligand, such as, for example, N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol.

[0100] In another embodiment, the sense strand comprises the nucleotide sequence 5'-gscsuau(Uhd)CfaUfAfAfucacauucsgsa-3' (SEQ ID NO: 17) and the antisense strand comprises the nucleotide sequence 5'-VPusCfsgaaUfgUfGfauuaUfgAfauagcsasu-3' (SEQ ID NO: 18), wherein a, c, g, and u are 2'-O-methyl (2'-OMe) modified A, C, G, and U, respectively; Af, Cf, Gf, and Uf are 2'-fluoro modified A, C, G, and U, respectively; s is a phosphorothioate linkage; and VP is vinyl phosphonate; and the ligand is conjugated to the 3' end of the sense strand as shown in the diagram below: [ka] wherein X is O.

[0101] In one embodiment, the sense strand consists of the nucleotide sequence 5'-gscsuau(Uhd)CfaUfAfAfucacauucsgsa-3' (SEQ ID NO: 17) and the antisense strand consists of the nucleotide sequence 5'-VPusCfsgaaUfgUfGfauuaUfgAfauagcsasu-3' (SEQ ID NO: 18), wherein a, c, g, and u are 2'-O-methyl (2'-OMe) modified A, C, G, and U, respectively; Af, Cf, Gf, and Uf are 2'-fluoro modified A, C, G, and U, respectively; s is a phosphorothioate linkage; and VP is vinyl phosphonate; and the ligand is conjugated to the 3' end of the sense strand as shown in the diagram below: [ka] wherein X is O.

[0102] In one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent that inhibits expression of huntingtin (HTT) in a cell, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, the sense strand comprising a modified nucleotide sequence 5'-uscsaua(Ahd)UfcAfCfAfuucguuugsusa-3' (SEQ ID NO: 19) and the antisense strand comprising a modified nucleotide sequence 5'-VPusAfs caaAfcGfAfauguGfaUfuaugasasu-3' (SEQ ID NO: 20), wherein a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U, s is a phosphorothioate linkage, Af, Cf, Gf, Uf are 2'-fluoro A, C, G, and U, (Ahd) is 2'-O-hexadecyl-adenosine-3'-phosphate, and VP is 5'-vinylphosphonate.

[0103] In one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent that inhibits expression of huntingtin (HTT) in a cell, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, the sense strand comprising a modified nucleotide sequence 5'-gscsugg(Uhd)GfaAfUfCfggauuccusgsa-3' (SEQ ID NO: 21) and the antisense strand comprising a modified nucleotide sequence 5'-VPusCfsaggA faUfCfcgauUfcAfccagcsusc-3' (SEQ ID NO: 22), wherein a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U, respectively; s is a phosphorothioate linkage; Af, Cf, Gf, Uf are 2'-fluoro A, C, G, and U, respectively; (Uhd) is 2'-O-hexadecyl-uridine-3'-phosphate; and VP is 5'-vinylphosphonate.

[0104] In one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent that inhibits expression of huntingtin (HTT) in a cell, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, the sense strand comprising a modified nucleotide sequence 5'-gscsuau(Uhd)CfaUfAfAfucacauucsgsa-3' (SEQ ID NO: 17) and the antisense strand comprising a modified nucleotide sequence 5'-VPusCfsgaaUfgUfGfauuaUf gAfauagcsasu-3' (SEQ ID NO: 18), wherein a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U, respectively; s is a phosphorothioate linkage; Af, Cf, Uf are 2'-fluoro A, C, and U, respectively; dG and dT are 2'-deoxy G and T, respectively; (Uhd) is 2'-O-hexadecyl-uridine-3'-phosphate; and VP is 5'-vinylphosphonate.

[0105] In one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent that inhibits expression of huntingtin (HTT) in a cell, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, the sense strand comprising a modified nucleotide sequence 5'-usgsgaa(Ahd)AfgCfUfGfaugaaggcscsa-3' (SEQ ID NO: 23) and the antisense strand comprising a modified nucleotide sequence 5'-VPusGfsgccu(Tgn)caucagCfuUf uuccasgsg-3' (SEQ ID NO: 24), where a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U, respectively; s is a phosphorothioate linkage; Af, Cf, Gf, Uf are 2'-fluoro A, C, G, and U, respectively; (Ahd) is 2'-O-hexadecyl-adenosine-3'-phosphate; (Tgn) is thymidine-glycol nucleic acid (GNA) S isomer; and VP is 5'-vinylphosphonate.

[0106] In one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent that inhibits expression of huntingtin (HTT) in a cell, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, the sense strand consisting of a modified nucleotide sequence 5'-uscsaua(Ahd)UfcAfCfAfuucguuugsusa-3' (SEQ ID NO: 19) and the antisense strand consisting of a modified nucleotide sequence 5'-VPusAfscaaA fcGfAfauguGfaUfuaugasasu-3' (SEQ ID NO: 20), wherein a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U, respectively; s is a phosphorothioate linkage; Af, Cf, Gf, Uf are 2'-fluoro A, C, G, and U, respectively; (Ahd) is 2'-O-hexadecyl-adenosine-3'-phosphate; and VP is 5'-vinylphosphonate.

[0107] In one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent that inhibits expression of huntingtin (HTT) in a cell, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, the sense strand consisting of a modified nucleotide sequence 5'-gscsugg(Uhd)GfaAfUfCfggauuccusgsa-3' (SEQ ID NO: 21) and the antisense strand consisting of a modified nucleotide sequence 5'-VPusCfsaggA faUfCfcgauUfcAfccagcsusc-3' (SEQ ID NO: 22), where a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U, respectively; s is a phosphorothioate linkage; Af, Cf, Gf, Uf are 2'-fluoro A, C, G, and U, respectively; (Uhd) is 2'-O-hexadecyl-uridine-3'-phosphate; and VP is 5'-vinylphosphonate.

[0108] In one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent that inhibits expression of huntingtin (HTT) in a cell, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, the sense strand consisting of a modified nucleotide sequence 5'-gscsuau(Uhd)CfaUfAfAfucacauucsgsa-3' (SEQ ID NO: 17) and the antisense strand consisting of a modified nucleotide sequence 5'-VPusCfsgaaUfgUfGfauuaUf gAfauagcsasu-3' (SEQ ID NO: 18), where a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U, respectively; s is a phosphorothioate linkage; Af, Cf, Uf are 2'-fluoro A, C, and U, respectively; dG and dT are 2'-deoxy G and T, respectively; (Uhd) is 2'-O-hexadecyl-uridine-3'-phosphate; and VP is 5'-vinylphosphonate.

[0109] In one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent that inhibits expression of huntingtin (HTT) in a cell, the dsRNA agent comprising a sense strand and an antisense strand that form a double-stranded region, the sense strand consisting of the modified nucleotide sequence 5'-usgsgaa(Ahd)AfgCfUfGfaugaaggcscsa-3' (SEQ ID NO: 23) and the antisense strand consisting of the modified nucleotide sequence 5'-VPusGfsgccu(Tgn)caucagCfu Ufuuccasgsg-3' (SEQ ID NO: 24), where a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U, respectively; s is a phosphorothioate linkage; Af, Cf, Gf, Uf are 2'-fluoro A, C, G, and U; (Ahd) is 2'-O-hexadecyl-adenosine-3'-phosphate; (Tgn) is thymidine-glycol nucleic acid (GNA) S isomer; and VP is 5'-vinylphosphonate.

[0110] The invention further provides cells containing any of the dsRNA agents of the invention, and pharmaceutical compositions for inhibiting expression of the gene encoding HTT, comprising any of the dsRNA agents of the invention.

[0111] In one embodiment, the double-stranded RNAi agent is in a non-buffered solution. Optionally, the non-buffered solution is saline or water. In another embodiment, the double-stranded RNAi agent is in a buffered solution. Optionally, the buffer comprises acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In another embodiment, the buffer is phosphate-buffered saline (PBS). Another aspect of the present disclosure provides a pharmaceutical composition comprising a double-stranded RNAi agent of the present disclosure and a lipid formulation. In one embodiment, the lipid formulation comprises a lipid nanoparticle (LNP).

[0112] The present invention provides compositions comprising two or more dsRNA agents that inhibit expression of huntingtin (HTT) in a cell, such as a first dsRNA agent that targets exon 1 of human HTT and a second dsRNA agent that targets full-length human HTT.

[0113] In one aspect, the invention provides a composition comprising two or more dsRNA agents that inhibit expression of huntingtin (HTT) in a cell, each of the dsRNA agents independently comprising a sense strand and an antisense strand that form a double-stranded region, each of the antisense strands independently comprising a region complementary to an mRNA encoding HTT, each of the complementary regions independently comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the antisense nucleotide sequences in any one of Tables 2-5.

[0114] In another aspect, the invention provides a composition comprising two or more dsRNA agents that inhibit expression of huntingtin (HTT) in a cell, each of the dsRNA agents independently comprising a sense strand and an antisense strand that form a double-stranded region, each of the sense strands independently comprising a nucleotide sequence comprising at least 15 contiguous nucleotides that differ by no more than three nucleotides from any one of the nucleotide sequences of nucleotides 4391-4669, 6500-6540, or 6009-6037 of SEQ ID NO:1, and each of the antisense strands independently comprising a nucleotide sequence comprising at least 15 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO:6.

[0115] In one embodiment, the sense strands each independently comprise at least 15 contiguous nucleotides that differ by no more than three nucleotides from any one of the nucleotide sequences of nucleotides 4398-4420, 4403-4425, or 6512-6534 of SEQ ID NO:1.

[0116] In one embodiment, one of the sense strands comprises at least 15 contiguous nucleotides that differ from nucleotides 4398-4420 of SEQ ID NO:1 by no more than three nucleotides.

[0117] In one embodiment, each antisense strand independently comprises at least 15 contiguous nucleotides that differ by no more than three nucleotides from any one of the antisense strand nucleotide sequences of a duplex selected from the group consisting of AD-1271085, AD-1271083, or AD-1271084.

[0118] In one embodiment, one of the antisense strands comprises at least 15 contiguous nucleotides that differ from the antisense strand nucleotide sequence of AD-1271085 by no more than three nucleotides.

[0119] In one aspect, the invention provides a composition comprising two or more dsRNA agents that inhibit expression of huntingtin (HTT) in a cell, each of the dsRNA agents independently comprising a sense strand and an antisense strand that form a double-stranded region, each of the sense strands independently comprising a nucleotide sequence comprising at least 15 contiguous nucleotides that differs by no more than three nucleotides from any one of the nucleotide sequences of nucleotides 26-77 or 142-202 of SEQ ID NO:1, and each of the antisense strands independently comprising a nucleotide sequence comprising at least 15 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO:6.

[0120] In one embodiment, the sense strands each independently comprise at least 15 contiguous nucleotides that differ by no more than three nucleotides from any one of the nucleotide sequences of nucleotides 27-49, 55-77, 147-169, or 173-195 of SEQ ID NO:1.

[0121] In one embodiment, one of the sense strands comprises at least 15 contiguous nucleotides that differ from nucleotides 147-169 of SEQ ID NO:1 by no more than three nucleotides.

[0122] In one embodiment, each antisense strand comprises at least 15 contiguous nucleotides that differ by no more than three nucleotides from any one of the antisense strand nucleotide sequences of the duplex independently selected from the group consisting of AD-1019448, AD-1498524, AD-1498526, or AD-1498528.

[0123] In one embodiment, one of the antisense strands comprises at least 15 contiguous nucleotides that differ from the antisense strand nucleotide sequence of AD-1498524 by no more than three nucleotides.

[0124] In one embodiment, at least one of the sense strands or at least one of the antisense strands is independently conjugated to one or more lipophilic moieties.

[0125] In one embodiment, all of the sense strands or all of the antisense strands of each dsRNA agent are independently conjugated to one or more lipophilic moieties.

[0126] In one embodiment, each lipophilic moiety is independently conjugated to one or more positions within the double-stranded region of the dsRNA agent.

[0127] In one embodiment, each lipophilic moiety is independently conjugated via a linker or carrier.

[0128] In one embodiment, the lipophilicity of each lipophilic moiety, as measured in log Kow, is independently greater than 0.

[0129] In one embodiment, the hydrophobicity of each double-stranded RNAi agent is independently greater than 0.2, as measured by the unbound fraction in a plasma protein binding assay of the double-stranded RNAi agent.

[0130] In one embodiment, the plasma protein binding assay is an electrophoretic mobility shift assay using human serum albumin protein.

[0131] In one embodiment, the dsRNA agents each independently include at least one modified nucleotide.

[0132] In one embodiment, each sense strand and each antisense strand of each dsRNA agent independently includes five or fewer unmodified nucleotides.

[0133] In one embodiment, all of the nucleotides in each sense strand and all of the nucleotides in each antisense strand independently include a modification.

[0134] In one embodiment, at least one of the modified nucleotides is a deoxy-nucleotide, a 3'-terminal deoxy-thymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl-modified nucleotide, a 2'-C-alkyl-modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide containing a non-natural base, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing a 5'-phosphorothioate group, nucleotides containing a 5'-methylphosphonate group, nucleotides containing a 5' phosphate or a 5' phosphate mimic, nucleotides containing vinylphosphonate, nucleotides containing adenosine-glycol nucleic acid (GNA), nucleotides containing thymidine-glycol nucleic acid (GNA) S-isomer, nucleotides containing 2-hydroxymethyl-tetrahydrofuran-5-phosphate, nucleotides containing 2'-deoxythymidine-3' phosphate, nucleotides containing 2'-deoxyguanosine-3'-phosphate, 2'-O-hexadecyl nucleotides, nucleotides containing 2'-phosphate, cytidine-2'-phosphate nucleotides, guanosine-2'-phosphate nucleotides 2'-O-hexadecyl-cytidine-3'-phosphate nucleotide, 2'-O-hexadecyl-adenosine-3'-phosphate nucleotide, 2'-O-hexadecyl-guanosine-3'-phosphate nucleotide, 2'-O-hexadecyl-uridine-3'-phosphate nucleotide, 5'-vinylphosphonate (VP), 2'-deoxyadenosine-3'-phosphate nucleotide, 2'-deoxycytidine-3'-phosphate nucleotide, 2'-deoxyguanosine-3'-phosphate nucleotide, 2'-deoxythymidine-3'-phosphate nucleotide, 2'-deoxyuridine nucleotide, and terminal nucleotides linked to cholesteryl derivatives and dodecanoic acid bisdecylamide groups, and combinations thereof.

[0135] In one embodiment, the modified nucleotides are independently selected from the group consisting of 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, 3'-terminal deoxy-thymine nucleotides (dT), locked nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, and nucleotides containing unnatural bases.

[0136] In another embodiment, the modified nucleotides comprise a short sequence of 3'-terminal deoxy-thymine nucleotides (dT).

[0137] In one embodiment, the modifications on the nucleotide are each independently selected from the group consisting of a 2'-O-methyl modification, a 2'-deoxy-modification, or a 2'-fluoro modification.

[0138] In one embodiment, at least one of the dsRNA agents further comprises at least one phosphorothioate internucleotide linkage.

[0139] In one embodiment, at least one of the dsRNA agents includes 6 to 8 phosphorothioate internucleotide linkages.

[0140] In one embodiment, each strand of each dsRNA agent is independently 30 nucleotides or less in length.

[0141] In one embodiment, at least one strand of at least one dsRNA agent independently includes a 3' overhang of at least 1 nucleotide.

[0142] In another embodiment, at least one strand of at least one dsRNA agent independently comprises a 3' overhang of at least 2 nucleotides.

[0143] The double-stranded region of each dsRNA agent can independently be 15 to 30 nucleotide pairs in length, 17 to 23 nucleotide pairs in length, 17 to 25 nucleotide pairs in length, 23 to 27 nucleotide pairs in length, 19 to 21 nucleotide pairs in length, or 21 to 23 nucleotide pairs in length.

[0144] Each strand of each dsRNA agent can independently be 19-30 nucleotides, 19-23 nucleotides, or 21-23 nucleotides in length.

[0145] In one embodiment, each dsRNA agent includes one or more lipophilic moieties independently conjugated to one or more interior positions on at least one strand.

[0146] In one embodiment, one or more lipophilic moieties are each independently conjugated via a linker or carrier to one or more interior positions on at least one chain.

[0147] In one embodiment, each interior position independently includes all positions except the two most distal positions from each end of at least one strand.

[0148] In one embodiment, each interior position independently includes all but the three most distal positions on each end of at least one strand.

[0149] In one embodiment, each internal position independently excludes the cleavage site region of the sense strand.

[0150] In one embodiment, each internal position independently includes all positions except positions 9-12 counting from the 5' end of the sense strand.

[0151] In one embodiment, each internal position independently includes all positions except positions 11-13 counting from the 3' end of the sense strand.

[0152] In one embodiment, each internal position independently excludes the cleavage site region of the antisense strand.

[0153] In one embodiment, each internal position independently includes all positions except positions 12-14 counting from the 5' end of the antisense strand.

[0154] In one embodiment, the internal positions each independently include all positions except positions 11-13 on the sense strand counting from the 3' end, and all positions except positions 12-14 on the antisense strand counting from the 5' end.

[0155] In one embodiment, the one or more lipophilic moieties are each independently conjugated to one or more of the internal positions selected from the group consisting of 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.

[0156] In one embodiment, one or more lipophilic moieties are each independently conjugated to one or more internal positions selected from the group consisting of 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.

[0157] In one embodiment, each position in the double-stranded region independently excludes the cleavage site region of the sense strand.

[0158] In one embodiment, each sense strand is independently 21 nucleotides in length, each antisense strand is independently 23 nucleotides in length, and each lipophilic moiety is independently conjugated to position 21, 20, 15, 1, 7, 6, or 2 of the sense strand or position 16 of the antisense strand.

[0159] In one embodiment, each of the lipophilic moieties is independently conjugated to position 21, 20, 15, 1, or 7 of the sense strand.

[0160] In one embodiment, each of the lipophilic moieties is independently conjugated to position 21, 20, or 15 of the sense strand.

[0161] In one embodiment, each of the lipophilic moieties is independently conjugated to position 20 or 15 of the sense strand.

[0162] In one embodiment, each of the lipophilic moieties is independently conjugated to position 16 of the antisense strand.

[0163] In one embodiment, each lipophilic moiety is independently an aliphatic compound, an alicyclic compound, or a polyalicyclic compound.

[0164] In one embodiment, each lipophilic moiety is independently selected from the group consisting of a lipid, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, a heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.

[0165] In one embodiment, each lipophilic moiety independently contains a saturated or unsaturated C4-C30 hydrocarbon chain and an optional functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.

[0166] In one embodiment, each lipophilic moiety independently contains a saturated or unsaturated C6 to C18 hydrocarbon chain.

[0167] In one embodiment, each lipophilic moiety independently contains a saturated or unsaturated C16 hydrocarbon chain.

[0168] In one embodiment, each saturated or unsaturated C16 hydrocarbon chain is conjugated to the 6th position counting from the 5' end of one chain.

[0169] In one embodiment, each lipophilic moiety is conjugated via a carrier that independently replaces one or more nucleotides in an internal position or in the double-stranded region.

[0170] In one embodiment, each carrier is independently a cyclic group selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl, or an acyclic portion of a serinol or diethanolamine backbone system.

[0171] In one embodiment, each lipophilic moiety is independently conjugated to the double-stranded iRNA agent via a linker that contains an ether, a thioether, a urea, a carbonate, an amine, an amide, a maleimide-thioether, a disulfide, a phosphodiester, a sulfonamide linkage, a product of a click reaction, or a carbamate.

[0172] In one embodiment, each lipophilic moiety is independently conjugated to a nucleobase, a sugar moiety, or an internucleoside linkage.

[0173] In one embodiment, each of the lipophilic moieties or one or more targeting ligands is independently conjugated via a bio-cleavable linker selected from the group consisting of DNA, RNA, disulfide, amide, galactosamine, glucosamine, glucose, galactose, mannose functionalized mono- or oligosaccharides, and combinations thereof.

[0174] In one embodiment, at least one 3' end of the sense strand is independently protected via an end cap that is an amine-bearing cyclic group selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl.

[0175] In one embodiment, at least one of the dsRNA agents further comprises a phosphate or a phosphate mimetic at the 5'-end of the antisense strand.

[0176] In one embodiment, each phosphate mimic is independently a 5'-vinylphosphonate (VP).

[0177] In one embodiment, each sense strand independently has a total of 21 nucleotides and each antisense strand independently has a total of 23 nucleotides.

[0178] In another embodiment, the RNAi agent is a pharmaceutically acceptable salt thereof. The "pharmaceutically acceptable salt" of each RNAi agent herein includes, but is not limited to, sodium, calcium, lithium, potassium, ammonium, magnesium salts, and mixtures thereof. Those skilled in the art will understand that when an RNAi agent is provided as a polycationic salt, it will have one cation per free acid group of the optionally modified phosphodiester backbone and / or any other acidic modifications (e.g., a phosphonate group at the 5' end). For example, an oligonucleotide "n" nucleotides in length contains n-1 optionally modified phosphodiesters, such that an oligonucleotide 21 nt in length can be provided as a salt with up to 20 cations (e.g., 20 sodium cations). Similarly, an RNAi agent having a 21 nt sense strand and a 23 nt antisense strand can be provided as a salt with up to 42 cations (e.g., 42 sodium cations). In the foregoing examples, if the RNAi agent also includes a 5'-terminal phosphate group or a 5'-terminal vinylphosphonate group, the RNAi agent can be provided as a salt with up to 44 cations (e.g., 44 sodium cations).

[0179] The invention also provides cells and pharmaceutical compositions comprising the compositions of the invention, including, for example, lipid formulations.

[0180] A further aspect of the present disclosure provides a method for inhibiting expression of the HTT gene in a cell, the method comprising: (a) contacting the cell with a double-stranded RNAi agent of the present disclosure, a composition of the present disclosure, or a pharmaceutical composition of the present disclosure; and (b) maintaining the cell resulting from step (a) for a time sufficient to obtain degradation of mRNA transcripts of the HTT gene, thereby inhibiting expression of the HTT gene in the cell.

[0181] In one embodiment, the cell is contacted with two or more, e.g., 2, 3, or 4, dsRNA agents of the invention (or compositions of the invention), such as a first dsRNA agent that targets exon 1 of human HTT and a second dsRNA agent that targets full-length human HTT.

[0182] In one embodiment, the cell is in a subject. Optionally, the subject is a human.

[0183] In certain embodiments, the subject is a rhesus monkey, a cynomolgous monkey, a mouse, or a rat. In certain embodiments, HTT expression is inhibited by at least about 50% by the RNAi agent.

[0184] In certain embodiments, the human subject has been diagnosed with an HTT-related disorder, such as Huntington's disease.

[0185] In another aspect of the present disclosure, there is provided a method of treating a subject diagnosed with an HTT-related disease, e.g., Huntington's disease, comprising administering to the subject a therapeutically effective amount of a double-stranded RNAi agent of the present disclosure, a composition of the invention, or a pharmaceutical composition of the present disclosure, thereby treating the subject.

[0186] In one embodiment, a subject is administered two or more, e.g., two, three, or four, dsRNA agents of the invention (or compositions of the invention), such as a first dsRNA agent that targets exon 1 of human HTT and a second dsRNA agent that targets full-length human HTT.

[0187] The first dsRNA agent and the second dsRNA agent can be present in the same composition or in separate compositions.

[0188] The first dsRNA agent and the second dsRNA agent can be administered to the subject at the same time or at different times.

[0189] In one embodiment, treating includes ameliorating at least one sign or symptom of the disease, hi another embodiment, treating includes preventing the progression of the disease.

[0190] In some embodiments, a dsRNA agent is administered to a subject at a dose of about 0.01 mg / kg to about 50 mg / kg.

[0191] In some embodiments, dsRNA agent is administered to subject intrathecally.In one embodiment, method reduces the expression of HTT gene in brain (for example, striatum) or spinal tissue.Optionally, brain or spinal tissue is striatum, cortex, cerebellum, cervical vertebrae, lumbar vertebrae or thoracic vertebrae.

[0192] In some embodiments, the method further comprises measuring the level of HTT in a sample obtained from the subject.

[0193] Another aspect of the present disclosure provides a method for inhibiting expression of huntingtin (HTT) in a subject, the method involving administering to the subject a therapeutically effective amount of a double-stranded RNAi agent of the present disclosure or a pharmaceutical composition of the present disclosure, thereby inhibiting expression of HTT in the subject.

[0194] In some embodiments, the method further comprises administering to the subject an additional agent suitable for treating or preventing an HTT-related disorder. [Brief explanation of the drawings]

[0195] [Figure 1] FIG. 1 is a graph showing knockdown of HTT in the indicated regions of the brain and spine of non-human primates with greater than 1000 ng / mL of siRNA in the CSF 24 hours after intrathecal administration of a single 60 mg dose of the indicated duplex. [Figure 2] FIG. 2 is a table showing inhibition of HTT mRNA expression in the indicated regions of the brain and spine of non-human primates (n=5 per group) 24 hours after intrathecal administration of a single 60 mg dose of the indicated duplex. [Figure 3] Figure 3A is a graph showing the concentration of siRNA in the CSF of the illustrated non-human primate (see Figure 2) 24 hours after intrathecal administration of a single 60 mg dose of duplex. Figure 3B is a graph showing the concentration of siRNA in the prefrontal cortex of the illustrated non-human primate (see Figure 2) 45 days after intrathecal administration of a single 60 mg dose of duplex. Figure 3C is a graph showing the concentration of siRNA in the striatum (caudate nucleus) of the illustrated non-human primate (see Figure 2) 45 days after intrathecal administration of a single 60 mg dose of duplex. Figure 3D is a graph showing the concentration of siRNA in the striatum (putamen) of the illustrated non-human primate (see Figure 2) 45 days after intrathecal administration of a single 60 mg dose of duplex. FIG. 3E is a graph showing the concentration of siRNA in the thoracic spine of the depicted non-human primate (see FIG. 2) 45 days after intrathecal administration of a single 60 mg dose of the duplex. [Figure 4]Figure 4A is a graph showing the correlation of siRNA concentration in brain and spinal cord tissues with HTT knockdown levels in non-human primates administered a single 60 mg dose of all tested duplexes intrathecally. Figure 4B is a graph showing the correlation of siRNA concentration in brain and spinal cord tissues with HTT knockdown levels in non-human primates administered a single 60 mg dose of duplex AD-1019465 intrathecally. Figure 4C is a graph showing the correlation of siRNA concentration in brain and spinal cord tissues with HTT knockdown levels in non-human primates administered a single 60 mg dose of duplex AD-1271082 intrathecally. Figure 4D is a graph showing the correlation of siRNA concentration in brain and spinal cord tissues with HTT knockdown levels in non-human primates administered a single 60 mg dose of duplex AD-1271085 intrathecally. [Figure 5] Figure 5A is a graph showing the concentration of siRNA in plasma samples over time after a single 60 mg intrathecal dose of duplexes. Figure 5B is a graph showing the concentration of siRNA in CSF and plasma at the indicated time points after a single 60 mg intrathecal dose of all duplexes tested (see Figure 2). [Figure 6]Figure 6A is a graph showing knockdown levels of mutant human HTT mRNA in the frontal cortex of YAC128 mice administered a single 300 μg / kg dose of the indicated duplex or an artificial CSF (aCSF) control by intracerebroventricular injection (ICV). For comparison, the effects of the antisense oligonucleotide Tominersen (Roche, also known as IONIS-HTTRx and RG6042) are also shown. Figure 6B is a Western blot and graph showing knockdown levels of mutant human HTT protein and mouse wild-type protein in the frontal cortex of YAC128 mice administered a single 300 μg / kg dose of the indicated duplex or an artificial CSF (aCSF) control by intracerebroventricular injection (ICV). Human protein levels are quantified in the graph below. For comparison, the effects of the antisense oligonucleotide Tominersen (Roche, also known as IONIS-HTTRx and RG6042) are also shown. [Figure 7] Figure 7 shows a schematic representation of the portion of Htt transcripts assessed by each assay in the QuantiGene panel from Papadopoulou, et al. (2019) Scientific Reports volume 9, Article number 16137. [Figure 8A] Figure 8A shows the mean fluorescence intensity (MFI) of Htt (endogenous mouse) intron 1 expression with polyA site 1 (680 bp variant) in the spinal cord and striatum of Q175 KI mice after a single 300 μg intracerebroventricular administration of the indicated agent or aCSF control, as well as the expression levels of Htt (endogenous mouse) intron 1 with polyA site 1 (680 bp variant) in the spinal cord and striatum of wild-type mice. Mean ± SD, ***p<0.001 (one-way analysis of variance (ANOVA) with Dunnett's multiple comparison test), and ###p<0.001 (unpaired t-test with Si control vs. WT). Normalized to the geomeans of three reference genes. [Figure 8B]Figure 8B shows the mean fluorescence intensity (MFI) of Htt (endogenous mouse) intron 1 expression with polyA site 2 (1145 bp variant) in the spinal cord and striatum of Q175 KI mice after a single 300 μg intracerebroventricular injection of the indicated agent or aCSF control, as well as the level of Htt (endogenous mouse) intron 1 expression with polyA site 1 (680 bp variant) in the spinal cord and striatum of wild-type mice. Mean ± SD, *p<0.05 (one-way analysis of variance (ANOVA) with Dunnett's multiple comparison test), and ##p<0.01, ###p<0.001 (unpaired t-test with WT vs. Si control). Normalized to the geomean of three reference genes. [Figure 8C] Figure 8C shows the mean fluorescence intensity (MFI) of huntingtin, full-length (total), expression in the spinal cord and striatum of Q175 KI mice after a single 300 μg intracerebroventricular injection of the indicated agent or aCSF control, as well as the level of huntingtin, full-length (total), expression in the spinal cord and striatum of wild-type mice. Data are presented as means with SD, n = 3–6 per group. Pairwise comparisons are as follows: *p<0.05; **p<0.01; ****p<0.0001 (one-way analysis of variance (ANOVA) with Dunnett's multiple comparisons test) and ###p<0.001; ####p<0.0001 (unpaired t-test with WT vs. Si control). Comparisons between Group 1 and Group 6 serve as controls for model (genotype) effects. Data were normalized to the geomeans of the three reference genes. [Figure 8D] Figure 8D is a graph showing the mean fluorescence intensity (MFI) of expression of intron 1 at its 3' end of Htt (endogenous to the mouse) (as a negative control for detection of retained intronic region expression) in the spinal cord and striatum of Q175 KI mice after a single 300 μg intracerebroventricular injection of the indicated agent or aCSF control, as well as the level of huntingtin, full-length (overall), expression in the spinal cord and striatum of wild-type mice, normalized to the geomeans of three reference genes. [Figure 8E] Figure 8E is a graph showing the mean fluorescence intensity (MFI) of Htt (endogenous to the mouse) intron 3 expression (as a negative control for detection of retained intronic region expression) in the spinal cord and striatum of Q175 KI mice after a single 300 μg intracerebroventricular injection of the indicated agent or aCSF control, as well as the level of huntingtin, full-length (overall) expression in the spinal cord and striatum of wild-type mice, normalized to the geomeans of three reference genes. [Figure 8F] Figure 8F is a graph showing the concentration of mutant huntingtin protein in the spinal cord and striatum of heterozygous Q175 KI mice after intracerebroventricular injection of a single dose of 300 μg of the indicated agent, a non-targeting control agent, or an aCSF control. The concentration of mutant huntingtin protein is shown as femtomoles of mutant huntingtin protein per milligram of total protein. [Figure 8G] Figure 8G is a graph showing wild-type huntingtin protein concentrations in the spinal cord and striatum of heterozygous Q175 KI mice after intracerebroventricular injection of a single dose of 300 μg of the indicated agent, a non-targeting control agent, or an aCSF control. Wild-type huntingtin protein concentrations are shown as femtomoles of wild-type huntingtin protein per milligram of total protein. [Figure 9] 9 shows the percent HTT protein remaining in the prefrontal cortex, hippocampus, striatum caudate, lumbar spine, and thoracic spine tissues of non-human primates administered a single 60 mg dose of AD-1019465 (n=5), AD-1271082 (n=5), or AD-1271085 (n=5) intrathecally at 45 days post-administration. The percent HTT protein remaining is compared to the level of HTT protein in the corresponding tissues of non-human primates administered artificial CSF (aCSF). [Figure 10A]Figure 10A is a graph showing the level of mutant human HTT mRNA knockdown in the frontal cortex of YAC128 mice administered a single dose of 300 μg or 600 μg of the indicated duplex or an artificial CSF (aCSF) control via intracerebroventricular injection (ICV). For comparison, the effect of the antisense oligonucleotide A-1800326 (Tominersen, Roche, also known as IONIS-HTTRx and RG6042) is also shown. [Figure 10B] Figure 10B is a graph showing the level of wild-type mouse HTT mRNA knockdown in the frontal cortex of YAC128 mice administered a single dose of 300 μg or 600 μg of the indicated duplex or an artificial CSF (aCSF) control via intracerebroventricular injection (ICV). For comparison, the effect of the antisense oligonucleotide A-1800326 (Tominersen, Roche, also known as IONIS-HTTRx and RG6042) is also shown. [Figure 10C] Figure 10C is a Western blot showing knockdown levels of mutant human HTT protein and wild-type mouse protein in the frontal cortex of YAC128 mice administered a single dose of 300 μg or 600 μg of the indicated duplex or an artificial CSF (aCSF) control via intracerebroventricular injection (ICV). "a-CSF" refers to the artificial CSF, "476-600" refers to the 600 μg / kg dose of AD-1019476, and "476-300" refers to the 300 μg / kg dose of AD-1019476. MAB2166 (EMD Millipore) was used for protein detection. [Figure 10D]Figure 10D is a Western blot showing knockdown levels of mutant human HTT protein and wild-type mouse protein in the frontal cortex of YAC128 mice administered a single dose of 300 μg or 600 μg of the indicated duplex or an artificial CSF (aCSF) control via intracerebroventricular injection (ICV). "a-CSF" refers to artificial CSF, "079-600" refers to a 600 μg / kg dose of AD-1443079, "079-300" refers to a 300 μg / kg dose of AD-1443079, "080-600" refers to a 600 μg / kg dose of AD-1443080, and "080-300" refers to a 300 μg / kg dose of AD-1443080. MAB2166 (EMD Millipore) was used for protein detection. [Figure 10E] Figure 10E shows Western blots and graphs showing knockdown levels of mutant human HTT protein and mouse wild-type protein in the frontal cortex of YAC128 mice administered a single dose of 300 μg or 600 μg of the indicated duplex or an artificial CSF (aCSF) control via intracerebroventricular injection (ICV). Lysates from Figure 10C were reanalyzed with the D7F7 antibody to confirm the observed HTT protein knockdown. Mutant human HTT protein and mouse wild-type protein levels are quantified in the graphs below. "a-CSF" refers to the artificial CSF, "476-600" refers to the 600 μg / kg dose of AD-1019476, and "476-300" refers to the 300 μg / kg dose of AD-1019476. [Figure 10F]Figure 10F is a Western blot and graph showing knockdown levels of mutant human HTT protein and mouse wild-type protein in the frontal cortex of YAC128 mice administered a single 300 μg or 600 μg dose of the indicated duplex or an artificial CSF (aCSF) control via intracerebroventricular injection (ICV). Lysates from Figure 10D were reanalyzed with the D7F7 antibody to confirm the observed HTT protein knockdown. Mutant human HTT protein and mouse wild-type protein levels are quantified in the graph below. Quantitations from both blots (Figure 10E and Figure 10F) are shown side-by-side for comparison. "a-CSF" refers to artificial CSF, "079-600" refers to a 600 μg / kg dose of AD-1443079, "079-300" refers to a 300 μg / kg dose of AD-1443079, "080-600" refers to a 600 μg / kg dose of AD-1443080, and "080-300" refers to a 300 μg / kg dose of AD-1443080. [Figure 11] Figure 11A is a graph showing mutant human HTT protein levels in the frontal cortex of YAC128 mice administered a single dose of 37.5 μg, 75 μg, 150 μg, or 300 μg of AD-1019476 or an artificial CSF (aCSF) control by intracerebroventricular injection (ICV). Two different antibodies, MAB2166 (EMD Millipore) and D7F7 (Cell Signaling), were used, and results from both antibodies showed similar HTT protein knockdown. Figure 11B is a graph showing mouse wild-type protein levels in the frontal cortex of YAC128 mice administered a single dose of 37.5 μg, 75 μg, 150 μg, or 300 μg of AD-1019476 or an artificial CSF (aCSF) control by intracerebroventricular injection (ICV). Two different antibodies, MAB2166 (EMD Millipore) and D7F7 (Cell Signaling), were used, and results from both antibodies showed similar HTT protein knockdown. [Figure 12A]Figure 12A is a graph showing the mean fluorescence intensity (MFI) of Htt (endogenous mouse) intron 1 expression with polyA site 1 (680 bp variant) in the spinal cord of Q175 WT and Q175 HET mice at 10 and 12 months after a single intracerebroventricular injection of 300 μg of the indicated agent or aCSF control (Si-Control). Data are expressed as mean with SD, n = 3-10 per group. Statistically significant: ****p<0.0001, Q175 HET Si-Control 10 months vs. Q175 WT Si-Control 10 months; *p<0.05, ***p<0.001, Q175 WT Si-Control 12 months and Q175 HET AD-1498524 12 months vs. Q175 HET Si-Control 12 months (Welch's one-way ANOVA test with Welch's correction, unpaired t-test with Dunnett's T3 multiple comparison test). Normalized to the geomeans of the three reference genes. [Figure 12B] Figure 12B is a graph showing the mean fluorescence intensity (MFI) of Htt (endogenous mouse) intron 1 expression with polyA site 2 (1145 bp variant) in the spinal cord of Q175 WT and Q175 HET mice at 10 and 12 months after a single intracerebroventricular injection of 300 μg of the indicated agent or aCSF control (Si-Control). Data are expressed as mean with SD, n = 3-10 per group. Statistically significant: **p<0.01, Q175 HET Si-Control 10 months vs. Q175 WT Si-Control 10 months; *p<0.05, **p<0.001, Q175 WT Si-Control 12 months, Q175 HET AD-1498524 12 months and Q175 HET AD-1271085 12 months vs. Q175 HET Si-Control 12 months (Welch's correction / Ordinary one-way ANOVA, unpaired t-test with Dunnett's multiple comparison test). Normalized to the geomeans of the three reference genes. [Figure 12C]Figure 12C is a graph showing the mean fluorescence intensity (MFI) of expression of intron 1 at its 3' end of Htt (endogenous to the mouse) (as a negative control for detecting retained intronic region expression) in the spinal cord of Q175 WT and Q175 HET mice at 10 and 12 months after a single intracerebroventricular injection of 300 μg of the indicated agent or aCSF control (Si-Control). Data are presented as mean with SD, n = 3-10 per group. Statistically significant: *p<0.05, Q175 HET AD-1019448 10 months vs. Q175 HET Si-Control 10 months (ordinary one-way ANOVA, Dunnett's multiple comparison test). Normalized to the geomeans of the three reference genes. [Figure 12D] Figure 12D is a graph showing the mean fluorescence intensity (MFI) of Htt (endogenous to the mouse) intron 3 expression (as a negative control for detecting retained intronic region expression) in the spinal cord of Q175 WT and Q175 HET mice at 10 and 12 months after a single intracerebroventricular injection of 300 μg of the indicated agent or aCSF control (Si-control). Data are expressed as mean with SD, n = 3-10 per group. Statistically not significant: p > 0.05 (unpaired t-test with Welch's correction / ordinary one-way ANOVA). Normalized to the geomeans of three reference genes. [Figure 12E]Figure 12E is a graph showing the mean fluorescence intensity (MFI) of huntingtin, full-length (global) expression in the spinal cord of Q175 WT and Q175 HET mice at 10 and 12 months after a single intracerebroventricular injection of 300 μg of the indicated agent or aCSF control (Si-Control). Data are expressed as mean with SD, n = 3-10 per group. Statistically significant: **p<0.01, ***p<0.001, ****p<0.0001, Q175 WT Si-Control 10 months, Q175 HET AD-1271085 10 months, Q175 HET AD-1498524 10 months, Q175 HET AD-1019448 10 months vs. Q175 HET Si-Control 10 months; ***p<0.001, ****p<0.0001, Q175 WT Si-Control 12 months, Q175 HET AD-1271085 12 months, and Q175 HET AD-1498524 12 months vs. Q175 HET Si-Control 12 months (Ordinary one-way ANOVA with Welch's correction, unpaired t-test with Dunnett's multiple comparison test). Normalized to the geomeans of three reference genes. [Figure 12F] Figure 12F is a graph showing the mean fluorescence intensity (MFI) of Htt (endogenous to mouse) intron 1 expression with polyA site 1 (680 bp variant) in the striatum of Q175 WT and Q175 HET mice at 10 and 12 months after a single intracerebroventricular injection of 300 μg of the indicated agent or aCSF control (Si-Control). Data are expressed as mean with SD, n = 3-10 per group. Statistically significant: ****p<0.0001, Q175 HET Si-Control 10 months vs. Q175 WT Si-Control 10 months; ****p<0.0001, Q175 WT Si-Control 12 months and Q175 HET AD-1498524 12 months vs. Q175 HET Si-Control 12 months (Welch's ANOVA test with Welch's correction, unpaired t-test with Dunnett's T3 multiple comparison test). Normalized to the geomeans of three reference genes. [Figure 12G]Figure 12G is a graph showing the mean fluorescence intensity (MFI) of Htt (endogenous mouse) intron 1 expression with polyA site 2 (1145 bp variant) in the striatum of Q175 WT and Q175 HET mice at 10 and 12 months after a single 300 μL intracerebroventricular injection of the indicated agent or aCSF control (Si-Control). Data are expressed as mean with SD, n = 3-10 per group. Statistically significant: ***p<0.001, Q175 HET Si-Control 10 months vs. Q175 WT Si-Control 10 months; *p<0.05, ***p<0.001, Q175 WT Si-Control 12 months and Q175 HET AD-1498524 12 months vs. Q175 HET Si-Control 12 months (Welch's correction / Ordinary one-way ANOVA, unpaired t-test with Dunnett's multiple comparison test). Normalized to the geomeans of the three reference genes. [Figure 12H] Figure 12H is a graph showing the mean fluorescence intensity (MFI) of expression of intron 1 at its 3' end of Htt (endogenous to the mouse) (as a negative control for detecting retained intronic region expression) in the striatum of Q175 WT and Q175 HET mice at 10 and 12 months after a single intracerebroventricular injection of 300 μg of the indicated agent or aCSF control (Si-Control). Data are presented as means with SD, n = 3-10 per group. Statistically significant: *p<0.05, Q175 HET AD-1271085 10 months and Q175 HET AD-1019448 10 months vs. Q175 HET Si-Control 10 months (One-way ANOVA, Dunnett's multiple comparison test). Normalized to the geomeans of the three reference genes. [Figure 12I]Figure 12I shows the mean fluorescence intensity (MFI) of intron 3 expression of Htt (endogenous to the mouse) in the striatum of Q175 WT and Q175 HET mice at 10 and 12 months after a single 300 μL intracerebroventricular injection of the indicated agent or aCSF control (Si-Control). Data are presented as mean with SD, n = 3-10 per group. Statistically significant: p > 0.05 (unpaired t-test with Welch's correction / ordinary one-way ANOVA). Normalized to the geomeans of the three reference genes. [Figure 12J] Figure 12J is a graph showing the mean fluorescence intensity (MFI) of huntingtin, full-length (total) expression in the striatum of Q175 WT and Q175 HET mice at 10 and 12 months after a single intracerebroventricular injection of 300 μg of the indicated agent or aCSF control (Si-Control). Data are expressed as mean with SD, n = 3-10 per group. Statistically significant: **p<0.01, ***p<0.001, Q175 WT Si-Control 10 months, Q175 HET AD-1271085 10 months, Q175 HET AD-1498524 10 months, Q175 HET AD-1019448 10 months vs. Q175 HET Si-Control 10 months, ****p<0.0001, Q175 WT Si-Control 12 months, Q175 HET AD-1271085 12 months, and Q175 HET AD-1498524 12 months vs. Q175 HET Si-Control 12 months (Ordinary one-way ANOVA with Welch's correction, unpaired t-test with Dunnett's multiple comparison test). Normalized to the geomeans of the three reference genes. [Figure 12K]Figure 12K is a graph showing mutant huntingtin protein concentrations (top panel) and total HTT protein concentrations (bottom panel) in the striatum of WT and Q175 HET mice after a single 300 μg intracerebroventricular injection of the indicated agent or aCSF control (Si-Control). Mutant and total huntingtin protein concentrations are shown as femtomoles of mutant huntingtin protein per milligram of total protein. Statistically significant: Q175 Het Si-Control vs. treatment: one-way ANOVA followed by Dunnett's multiple comparison test compared at each time point. *p≦0.05, ***p<0.001, **p<0.01, ****p<0.0001. [Figure 12L] Figure 12L is a graph showing mutant huntingtin protein concentrations (top panel) and total HTT protein concentrations (bottom panel) in the spinal cord of WT and Q175 HET mice after a single intracerebroventricular injection of 300 μg of the indicated agent or aCSF control (Si-Control). Mutant and total huntingtin protein concentrations are shown as femtomoles of mutant huntingtin protein per milligram of total protein. Statistically significant: Q175 Het Si-Control vs. treatment: One-way ANOVA followed by Dunnett's multiple comparison test compared at each time point. ***p<0.001, **p<0.01, ****p<0.0001. [Figure 13A] Figure 13A is a graph showing the persistence of HTT transcripts in the prefrontal cortex, hippocampus, striatopamen, striatopaudate, and lumbar spine (lumbar vertebrae L1-L3) tissues of non-human primates administered a single 3 mg or 10 mg dose of AD-1271085 (n=8) intrathecally at day 33. The persistence of HTT transcripts is compared to the levels of HTT transcripts in the corresponding tissues of non-human primates administered artificial CSF (aCSF). [Figure 13B]Figure 13B is a graph showing HTT transcript persistence in prefrontal cortex, hippocampus, striatopumen, and striatopaudate tissues from non-human primates administered a single 3 mg or 10 mg dose of AD-1271085 (n=8) intrathecally at day 33. HTT transcript persistence is compared to HTT transcript levels in corresponding tissues from non-human primates administered artificial CSF (aCSF). [Figure 13C] Figure 13C is a graph showing HTT protein retention in tissues of the prefrontal cortex, hippocampus, striatopamen, striatopaudate, and lumbar spine (lumbar vertebrae L1-L3) of non-human primates administered a single 3 mg or 10 mg dose of AD-1271085 (n=8) intrathecally 33 days after administration. HTT protein retention is compared to HTT protein levels in the corresponding tissues of non-human primates administered artificial CSF (aCSF). [Figure 13D] Figure 13D is a graph showing HTT protein persistence in prefrontal cortex, hippocampus, striatopumen, and striatopaudate tissues from non-human primates administered a single 3 mg or 10 mg dose of AD-1271085 (n=8) intrathecally at day 33. HTT protein persistence is compared to HTT protein levels in corresponding tissues from non-human primates administered artificial CSF (aCSF). [Figure 13E] Figure 13E is a graph showing the correlation of the effect of a single 3 mg or 10 mg intrathecal dose of AD-1271085 (n=8) on HTT transcript and protein persistence in the prefrontal cortex, hippocampus, striatopumen, striatopaudate, and lumbar spine (lumbar vertebrae L1-L3) tissues of non-human primates at day 33 post-administration. HTT protein persistence is compared to HTT protein and HTT transcript levels in the corresponding tissues of non-human primates administered artificial CSF (aCSF). [Figure 14A]14A is a graph showing HTT protein persistence in striatopamen, striatopaudate, hippocampus, prefrontal cortex, and lumbar tissues from non-human primates administered a single 60 mg intrathecal dose of AD-1498524, AD-1498526, or AD-1498528 (n=5) at 59 days post-administration. HTT protein persistence is compared to HTT protein levels in corresponding tissues from non-human primates administered artificial CSF (aCSF). [Figure 14B] Figure 14B is a graph showing the persistence of HTT transcripts in striatopamen, striatopamen, hippocampus, prefrontal cortex, and lumbar tissues from non-human primates administered a single 60 mg intrathecal dose of AD-1498524, AD-1498526, or AD-1498528 (n=5) at 59 days post-administration. HTT transcript persistence is compared to HTT transcript levels in the corresponding tissues from non-human primates administered artificial CSF (aCSF). [Figure 14C] Figure 14C is a graph showing the persistence of HTT transcripts in prefrontal cortex, hippocampus, striatopumen, and lumbar tissues from non-human primates administered a single 60 mg intrathecal dose of AD-1498524, AD-1498526, or AD-1498528 (n=5) at 59 days post-administration. The persistence of HTT transcripts is compared to the levels of HTT transcripts in the corresponding tissues from non-human primates administered artificial CSF (aCSF). [Figure 14D] Figure 14D is a graph showing the correlation of the effect of a single 60 mg intrathecal dose of AD-1498524, AD-1498526, or AD-1498528 (n=5) on HTT transcript and HTT protein persistence in tissues of non-human primates at day 59 post-administration. HTT protein and HTT transcript persistence are compared to HTT protein and HTT transcript levels in corresponding tissues of non-human primates administered artificial CSF (aCSF). [Figure 15] FIG. 15 is a schematic diagram of the structure of the duplex shown. DETAILED DESCRIPTION OF THE INVENTION

[0196] The present disclosure provides RNAi compositions that perform RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the huntingtin (HTT) gene. The HTT gene can be present in a cell, for example, in a subject, such as a human. The use of these iRNAs allows for the targeted degradation of the mRNA of the corresponding gene (HTT gene) in a mammal.

[0197] The iRNAs of the invention are designed to target the full-length HTT gene, including portions of the gene that are conserved among HTT orthologs in other mammalian species, e.g., thereby targeting the full-length wild-type transcript and the full-length mutant transcript. Without intending to be bound by theory, it is believed that the combination or subcombination of the above features with specific target sites or specific modifications in these iRNAs improves the efficacy, stability, potency, durability, and safety of the iRNAs of the invention.

[0198] Accordingly, the present disclosure also provides methods of using the RNAi compositions of the present disclosure, including compositions comprising one or more, e.g., two, three, or four, dsRNA agents of the invention, to inhibit expression of the HTT gene or to treat a subject having a disorder that would benefit from inhibiting or reducing expression of the HTT gene, such as an HTT-associated disease, e.g., Huntington's disease (HD).

[0199] RNAi agents of the disclosure can be about 30 nucleotides in length or less, e.g., 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-2 In certain embodiments, the RNAi agent of the present disclosure comprises an RNA strand (antisense strand) having a region that is approximately 4, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, which is substantially complementary to at least a portion of an mRNA transcript of the HTT gene. In certain embodiments, the RNAi agent of the present disclosure comprises an RNA strand (antisense strand) having a region that is approximately 21-23 nucleotides in length, which is substantially complementary to at least a portion of an mRNA transcript of the HTT gene.

[0200] In certain embodiments, RNAi agents of the present disclosure comprise an RNA strand (antisense strand) that can comprise a longer length, e.g., up to 66 nucleotides in length, e.g., 36-66, 26-36, 25-36, 31-60, 22-43, 27-53 nucleotides, with a region of at least 19 contiguous nucleotides that is substantially complementary to at least a portion of an mRNA transcript of the HTT gene. These RNAi agents with longer antisense strand lengths preferably comprise a second RNA strand (sense strand) 20-60 nucleotides in length, where the sense and antisense strands form a duplex of 18-30 contiguous nucleotides.

[0201] The use of these RNAi agents allows for the targeted degradation of the mRNA of the HTT gene in mammals. Thus, methods and compositions comprising these RNAi agents are useful for treating subjects who would benefit from a reduction in the level or activity of HTT protein, such as those with HTT-related diseases, such as Huntington's disease (HD).

[0202] The following detailed description discloses methods for making and using compositions containing RNAi agents that inhibit expression of the HTT gene, as well as compositions and methods for treating subjects with diseases and disorders that would benefit from inhibiting or reducing expression of the gene.

[0203] I. Definition In order that this disclosure may be more readily understood, certain terms are first defined. Additionally, whenever a value or range of values ​​for a parameter is listed, it is intended that values ​​and ranges intermediate to the listed values ​​are also intended to be part of this disclosure.

[0204] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. For example, "an element" means one element or to more than one element, e.g., a plurality of elements.

[0205] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to." The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless context clearly indicates otherwise.

[0206] The term "about" is used herein to mean within a typical tolerance in the art. For example, "about" can be understood as about 2 standard deviations from the mean. In certain embodiments, about means ±10%. In certain embodiments, about means ±5%. When about precedes a series of numbers or ranges, it is understood that "about" can modify each of the consecutive numbers or ranges.

[0207] The terms "at least," "more than," or "or more" preceding a number or series of numbers are understood to include the number adjacent to the term "at least," and all subsequent numbers or integers that may be logically included, if this is clear from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleotides of a 21-nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have the specified property. When at least precedes a series of numbers or ranges, it is understood that "at least" can modify each of the series of numbers or ranges.

[0208] As used herein, "less than" or "less than" refers to the value adjacent to the term and any logically smaller value or integer up to zero, if logical from the context. For example, a duplex with an overhang of "2 nucleotides or less" has an overhang of 2, 1, or 0 nucleotides. When "less than" is used before a series of numbers or ranges, it is understood that "less than" can modify each of the series of numbers or ranges.

[0209] As used herein, a method of detection can include determining that the amount of analyte present is below the detection level of the method.

[0210] In the event of a conflict between a given target site and the nucleotide sequence for either the sense or antisense strand, the given sequence controls.

[0211] If the chemical structure and chemical name do not match, the chemical structure takes precedence.

[0212] The term "HTT" or "Huntingtin," also known as "Huntingtin," "Huntington disease protein," "IT15," "HD," "HD protein," or "LOMARS," refers to the well-known gene encoding the protein HTT, which is widely expressed and required for normal development, and the disease gene involved in Huntington's disease, a neurodegenerative disorder characterized by loss of striatal neurons caused by an expanded unstable trinucleotide (CAG) repeat in the huntingtin gene, which is translated as a polyglutamine repeat in the protein product.

[0213] Examples of nucleotide and amino acid sequences for HTT can be found, for example, in GenBank Accession No. NM_002111.8 (Homo sapiens HTT, SEQ ID NO: 1, reverse complement, SEQ ID NO: 6), GenBank Accession No. NM_010414.3 (House mouse HTT, SEQ ID NO: 2, reverse complement, SEQ ID NO: 7), GenBank Accession No. NM_024357.3 (Rattus norvegicus HTT, SEQ ID NO: 3, reverse complement, SEQ ID NO: 8); GenBank Accession No. XM_015449989.1 (Cynomolgus monkey HTT, SEQ ID NO: 4, reverse complement, SEQ ID NO: 9); and GenBank Accession No. XM_028848247.1 (Rhesus monkey HTT, SEQ ID NO: 5, reverse complement, SEQ ID NO: 10).

[0214] Further examples of HTT sequences can be found in publicly available databases such as GenBank, OMIM, and UniProt.

[0215] Further information regarding HTT can be found, for example, at www.ncbi.nlm.nih.gov / gene / 3064.

[0216] The entire contents of each of the foregoing GenBank Accession Numbers and Gene Database Numbers are incorporated herein by reference as of the filing date of this application.

[0217] As used herein, the term HTT also refers to the variation of HTT gene, including the variants provided in SNP databases.A large number of sequence variations in HTT gene have been identified and can be found, for example, in NCBI dbSNP and UniProt (see, for example, www.ncbi.nlm.nih.gov / snp / ?LinkName=gene_snp&from_uid=3064, the entire contents of which are incorporated herein by reference as of the filing date of this application).

[0218] As used herein, "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of the HTT gene, such as an mRNA that is the product of RNA processing of a primary transcript. In one embodiment, the target portion of the sequence will be at least long enough to serve as a substrate for RNAi-dependent cleavage at or near a portion of the nucleotide sequence of an mRNA molecule formed during transcription of the HTT gene.

[0219] The target sequence is about 15-30 nucleotides in length. For example, the target sequence can be about 15-30 nucleotides in length, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, The target sequence may be 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. In certain embodiments, the target sequence is 19-23 nucleotides in length, and optionally 21-23 nucleotides in length. Ranges and lengths between the ranges and lengths listed above are also intended to be part of this disclosure.

[0220] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a chain of nucleotides described by a sequence referenced using standard nucleotide nomenclature.

[0221] "G," "C," "A," "T," and "U" generally refer to nucleotides containing guanine, cytosine, adenine, thymidine, and uracil as bases, respectively, in the context of modified or unmodified nucleotides. However, it will be understood that the term "ribonucleotide" or "nucleotide" can also refer to modified nucleotides, as described in more detail below, or alternative replacement moieties (see, for example, Table 1). Those skilled in the art are well aware that guanine, cytosine, adenine, thymidine, and uracil can be substituted with other moieties without substantially altering the base pairing properties of oligonucleotides containing nucleotides with such replacement moieties. For example, but not limited to, a nucleotide containing inosine as its base can base pair with a nucleotide containing adenine, cytosine, or uracil. Thus, a nucleotide containing uracil, guanine, or adenine can be substituted with a nucleotide containing, for example, inosine, in the nucleotide sequence of a dsRNA featured in the present disclosure. In another example, adenine and cytosine anywhere within an oligonucleotide can be substituted with guanine and uracil, respectively, to form GU wobble bases that pair with the target mRNA. Sequences containing such substitutions are suitable for the compositions and methods featured in this disclosure.

[0222] As used herein interchangeably, the terms "iRNA", "RNAi agent", "iRNA agent", "RNA interference agent" refer to the agent that contains the RNA as defined herein and mediates the targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway.RNA interference (RNAi) is the process that controls the sequence-specific degradation of mRNA.RNAi regulates, for example, inhibits, the expression of HTT in cells, for example, in cells of a subject, such as a mammalian subject.

[0223] In one embodiment, the RNAi agent of the present disclosure comprises a single-stranded RNAi that interacts with a target RNA sequence, such as an HTT target mRNA sequence, to mediate cleavage of the target RNA. Without wishing to be bound by theory, it is believed that long double-stranded RNAs introduced into cells are degraded into double-stranded small interfering RNAs (siRNAs) comprising a sense strand and an antisense strand by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, an RNase III-like enzyme, processes these dsRNAs into 19-23 base pair small interfering RNAs with characteristic two-base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). These siRNAs are then incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, thereby allowing complementary antisense strands to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases in RISC cleave the target, inducing silencing (Elbashir, et al., (2001) Genes Dev. 15:188). That is, in one aspect, the present disclosure relates to a single-stranded RNA (ssRNA) (the antisense strand of the siRNA duplex) that is produced in cells and promotes the formation of a RISC complex to silence the target gene, i.e., the HTT gene. Therefore, the term "siRNA" is used herein to also refer to the RNAi described above.

[0224] In another embodiment, the RNAi agent can be a single-stranded RNA introduced into a cell or organism to inhibit target mRNA. The single-stranded RNAi agent binds to the RISC endonuclease Argonaute 2, which then cleaves the target mRNA. Single-stranded siRNAs are generally 15-30 nucleotides and chemically modified. The design and testing of single-stranded RNAs are described in U.S. Patent No. 8,101,348 and Lima et al., (2012) Cell 150:883-894, the entire contents of each of which are incorporated herein by reference. Any of the antisense nucleotide sequences described herein can be used as the single-stranded siRNA described herein or as the single-stranded siRNA that is chemically modified by the method described in Lima et al., (2012) Cell 150:883-894.

[0225] In another embodiment, the "RNAi agent" used in the compositions and methods of the present disclosure is double-stranded RNA, and is also referred to herein as "double-stranded RNAi agent", "double-stranded RNA (dsRNA) molecule", "dsRNA agent" or "dsRNA". The term "dsRNA" refers to a complex of ribonucleic acid molecules with a duplex structure, comprising two antiparallel, substantially complementary nucleic acid strands, which are referred to as having "sense" or "antisense" orientation with respect to target RNA, i.e., HTT gene. In some embodiments of the present disclosure, double-stranded RNA (dsRNA) induces the degradation of target RNA, for example, mRNA, through a post-transcriptional gene silencing mechanism, referred to herein as RNA interference or RNAi.

[0226] Generally, dsRNA molecules can contain ribonucleotides, but as described in detail herein, each or both strands can also contain one or more non-ribonucleotides, such as deoxyribonucleotides or modified nucleotides. In addition, as used herein, "RNAi agent" can include ribonucleotides with chemical modifications, and RNAi agents can contain substantial modifications in multiple nucleotides. As used herein, the term "modified nucleotide" refers to a nucleotide that independently has a modified sugar moiety, a modified internucleotide linkage, or a modified nucleobase. Thus, the term modified nucleotide encompasses the substitution, addition, or removal of, for example, functional groups or atoms, to the internucleoside linkage, sugar moiety, or nucleobase. Modifications suitable for use in the agents of the present disclosure include all types of modifications disclosed herein or known in the art. Any such modifications used in siRNA-type molecules are encompassed by "RNAi agent" for the purposes of this specification and claims.

[0227] In certain embodiments of the present disclosure, the inclusion of deoxy-nucleotides, if present within an RNAi agent, can be considered to constitute modified nucleotides.

[0228] The duplex region can be of any length that allows for specific degradation of the desired target RNA via the RISC pathway, and can range from about 15 to 36 base pairs in length, for example, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, for example, about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-31, 18-32, 18-33, 18-34, 18-35, 18-36, 18-37, 18-38, 18-39, 18-40, 18-41, 18-42, 18-43, 18-44, 18-45, 18-46, 18-47, 18-48, 18-49, 18-50, 18-51, 18-52, 18-53, 18-54, 18-55, 18-56, 18-57, 18-58, 18-59, 18-60, 18-61, 18-62, 18-63, 18-64, 18-65, 18-66, 18 The length may be, for example, 8 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or about 21 to 22 base pairs. In certain embodiments, the duplex region is 19-21 base pairs in length, e.g., 21 base pairs in length. Ranges and lengths between the ranges and lengths listed above are also intended to be part of this disclosure.

[0229] The two strands forming the duplex structure can be different portions of a larger RNA molecule, or they can be separate RNA molecules. When the two strands are part of a larger molecule and are therefore connected by an uninterrupted chain of nucleotides between the 3' end of one strand and the corresponding 5' end of the other strand, the connected RNA strands are called "hairpin loops." A hairpin loop can contain at least one unpaired nucleotide. In some embodiments, a hairpin loop can contain at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23, or more unpaired nucleotides or nucleotides not directed toward the target site of the dsRNA. In some embodiments, a hairpin loop can be 10 or fewer nucleotides. In some embodiments, a hairpin loop can be 8 or fewer unpaired nucleotides. In some embodiments, a hairpin loop can be 4 to 10 unpaired nucleotides. In some embodiments, a hairpin loop can be 4 to 8 nucleotides.

[0230] When the two substantially complementary strands of dsRNA are composed of separate RNA molecules, these molecules can, but do not necessarily, be covalently linked. In certain embodiments, when the two strands are covalently linked by means other than an uninterrupted chain of nucleotides between the 3'-end of one strand and the corresponding 5'-end of the other strand that form a duplex structure, this connecting structure is called a "linker" (note, however, that other specific structures as defined elsewhere in this specification are also called "linkers"). The RNA strands can have the same or different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs present in the duplex. In addition to the duplex structure, RNAi can include one or more nucleotide overhangs. In one embodiment of an RNAi agent, at least one strand includes a 3'-overhang of at least one nucleotide. In another embodiment, at least one strand comprises a 3' overhang of at least 2 nucleotides, for example, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, at least one strand of the RNAi agent comprises a 5' overhang of at least 1 nucleotide. In certain embodiments, at least one strand comprises a 5' overhang of at least 2 nucleotides, for example, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In still other embodiments, both the 3' end and the 5' end of one strand of the RNAi agent comprise an overhang of at least 1 nucleotide.

[0231] In one embodiment, an RNAi agent of the disclosure is a dsRNA, each strand of which independently comprises 19-23 nucleotides that interact with a target RNA sequence, e.g., an HTT target mRNA sequence, to induce cleavage of the target RNA.

[0232] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the double-stranded structure of an RNAi agent, such as dsRNA.For example, a nucleotide overhang exists when the 3'-end of one strand of dsRNA extends beyond the 5'-end of the other strand, or vice versa.A dsRNA can comprise an overhang of at least one nucleotide, or the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more.A nucleotide overhang can comprise or consist of a nucleotide / nucleoside analog, such as deoxynucleotide / nucleoside.An overhang can be on the sense strand, on the antisense strand, or any combination thereof.Furthermore, a certain overhanging nucleotide can be present on the 5'-end, 3'-end, or both ends of either the antisense strand or the sense strand of dsRNA.

[0233] In one embodiment, the antisense strand of the dsRNA has an overhang of 1 to 10 nucleotides at the 3' or 5' end, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In one embodiment, the sense strand of the dsRNA has an overhang of 1 to 10 nucleotides at the 3' or 5' end, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In another embodiment, one or more of the nucleotides in the overhang are substituted with nucleoside thiophosphates.

[0234] In certain embodiments, the antisense strand of the dsRNA has an overhang of 1 to 10 nucleotides at the 3' or 5' end, for example, 0 to 3, 1 to 3, 2 to 4, 2 to 5, 4 to 10, 5 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In one embodiment, the sense strand of the dsRNA has an overhang of 1 to 10 nucleotides at the 3' or 5' end, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In another embodiment, one or more of the nucleotides in the overhang are substituted with nucleoside thiophosphates.

[0235] In certain embodiments, the overhang on the sense strand or the antisense strand can comprise an extended length of more than 10 nucleotides, e.g., 1 to 30 nucleotides, 2 to 30 nucleotides, 10 to 30 nucleotides, or 10 to 15 nucleotides in length. In certain embodiments, the extended overhang is on the sense strand of the duplex. In certain embodiments, the extended overhang is on the 3'-end of the sense strand of the duplex. In certain embodiments, the extended overhang is on the 5'-end of the sense strand of the duplex. In certain embodiments, the extended overhang is on the antisense strand of the duplex. In certain embodiments, the extended overhang is on the 3'-end of the antisense strand of the duplex. In certain embodiments, the extended overhang is on the 5'-end of the antisense strand of the duplex. In certain embodiments, one or more of the nucleotides in the overhang are substituted with a nucleoside thiophosphate. In certain embodiments, the overhang comprises a self-complementary portion that enables the overhang to form a stable hairpin structure under physiological conditions.

[0236] In certain embodiments, at least one end of at least one strand extends beyond the double-stranded targeting region, including structures in which one of the strands contains a thermodynamically stable tetraloop structure (see, e.g., U.S. Pat. Nos. 8,513,207 and 8,927,705 and International Publication No. WO 2010033225, the contents of each of which are incorporated herein by reference in their entirety). Such structures can include single-stranded extensions (on one or both sides of the molecule) as well as double-stranded extensions.

[0237] In certain embodiments, the 3'-end of the sense strand and the 5'-end of the antisense strand are joined by a polynucleotide sequence comprising ribonucleotides, deoxyribonucleotides, or both, and optionally the polynucleotide sequence comprises a tetraloop sequence. In certain embodiments, the sense strand is 25-35 nucleotides in length.

[0238] The tetraloop can comprise ribonucleotides, deoxyribonucleotides, modified nucleotides, and combinations thereof. Typically, the tetraloop has 4 to 5 nucleotides. In some embodiments, the loop comprises a sequence described as GAAA. In some embodiments, at least one of the nucleotides (GAAA) of the loop comprises a nucleotide modification. In some embodiments, the modified nucleotide comprises a 2' modification. In some embodiments, the 2' modification is a modification selected from the group consisting of 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, 2'-aminodiethoxymethanol, 2'-adem, and 2'-deoxy-2'-fioro-d-arabinonucleic acid. In some embodiments, all nucleotides of the loop are modified. In some embodiments, the G in the GAAA sequence comprises a 2'-OH. In some embodiments, each nucleotide in the GAAA sequence comprises a 2'-O-methyl modification. In some embodiments, each A in the GAAA sequence comprises a 2'-OH, and each G in the GAAA sequence comprises a 2'-O-methyl modification. In preferred embodiments, each A in the GAAA sequence comprises a 2'-O-methoxyethyl (MOE) modification, and each G in the GAAA sequence comprises a 2'-O-methyl modification, or each A in the GAAA sequence comprises a 2'-adem modification, and each G in the GAAA sequence comprises a 2'-O-methyl modification. See, for example, PCT Publication WO 2020 / 206350, the entire contents of which are incorporated herein by reference.

[0239] Exemplary 2' adem modified nucleotides are shown below. [ka]

[0240] The term "blunt" or "blunt-end" when used herein in relation to dsRNA means that there is no unpaired nucleotide or nucleotide analogue at the predetermined end of dsRNA, that is, there is no nucleotide overhang.One end or both ends of dsRNA can be blunt.When both ends of dsRNA are blunt, dsRNA is said to be blunt-ended.For clarity, "blunt-end" dsRNA is the dsRNA that has both ends blunt, that is, there is no nucleotide overhang at either end of the molecule.In most cases, this molecule will be double-stranded throughout its entire length.

[0241] The term "antisense strand" or "guide strand" refers to the strand of an RNAi agent, e.g., dsRNA, that includes a region that is substantially complementary to a target sequence, e.g., HTT mRNA.

[0242] As used herein, the term "region of complementarity," as defined herein, refers to a region on the antisense strand that is substantially complementary to a sequence, for example, a target sequence, for example, an HTT nucleotide sequence. If the complementary region is not completely complementary to the target sequence, the mismatch can be in the internal region or in the terminal region of the molecule. Generally, the most tolerable mismatch is in the terminal region, for example, within 5, 4, 3, or 2 nucleotides of the 5' or 3' end of the RNAi agent. In some embodiments, the double-stranded RNA agent of the present invention comprises a nucleotide mismatch in the antisense strand. In some embodiments, the antisense strand of the double-stranded RNA agent of the present invention comprises 4 or less mismatches with the target mRNA, for example, the antisense strand comprises 4, 3, 2, 1, or 0 mismatches with the target mRNA. In some embodiments, the antisense strand of the double-stranded RNA agent of the present invention comprises 4 or less mismatches with the sense strand, for example, the antisense strand comprises 4, 3, 2, 1, or 0 mismatches with the sense strand. In some embodiments, the double-stranded RNA agent of the present invention contains a nucleotide mismatch in the sense strand. In some embodiments, the sense strand of the double-stranded RNA agent of the present invention contains four or fewer mismatches with the antisense strand, for example, the sense strand contains 4, 3, 2, 1, or 0 mismatches with the antisense strand. In some embodiments, the nucleotide mismatch is, for example, within 5, 4, or 3 nucleotides from the 3' end of the iRNA. In other embodiments, the nucleotide mismatch is, for example, in the 3' terminal nucleotide of the iRNA agent. In some embodiments, the mismatch is not in the seed region.

[0243] Therefore, the RNAi agent described herein may contain one or more mismatches with respect to the target sequence. In one embodiment, the RNAi agent described herein contains 3 or less mismatches (i.e., 3, 2, 1, or 0 mismatches). In one embodiment, the RNAi agent described herein contains 2 or less mismatches. In one embodiment, the RNAi agent described herein contains 1 or less mismatches. In one embodiment, the RNAi agent described herein contains 0 mismatches. In certain embodiments, when the antisense strand of an RNAi agent contains a mismatch with respect to the target sequence, the mismatch can be optionally limited to be within the last 5 nucleotides from either the 5'-end or 3'-end of the complementary region. For example, in such an embodiment, for a 23-nucleotide RNAi agent, the strand complementary to the region of the HTT gene generally does not contain any mismatches within the central 13 nucleotides. By using the methods described herein or known in the art, it can be determined whether an RNAi agent containing a mismatch with respect to the target sequence is effective in inhibiting the expression of the HTT gene. It is important to consider the efficacy of mismatched RNAi agents to inhibit expression of HTT, particularly when specific regions of complementarity in the HTT gene are known to have polymorphic sequence variation within the population.

[0244] The term "sense strand" or "passenger strand," as used herein, refers to the strand of an RNAi agent that includes a region that is substantially complementary to a region of the antisense strand, as those terms are defined herein.

[0245] As used herein, the term "cleavage region" refers to the region located directly adjacent to the cleavage site. The cleavage site is the site on the target where cleavage occurs. In some embodiments, the cleavage region comprises three bases directly adjacent to either end of the cleavage site. In some embodiments, the cleavage region comprises two bases directly adjacent to either end of the cleavage site. In some embodiments, the cleavage site specifically occurs at the site bound by nucleotides 10 and 11 of the antisense strand, and this cleavage region comprises nucleotides 11, 12, and 13.

[0246] As used herein, unless specifically indicated otherwise, the term "complementary," when used to describe a first nucleotide sequence in the context of a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize to form a duplex structure under specified conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as would be understood by one of skill in the art.

[0247] A complementary sequence within an RNAi agent, such as within a dsRNA described herein, involves base pairing of an oligonucleotide or polynucleotide comprising a first nucleotide sequence with an oligonucleotide or polynucleotide comprising a second nucleotide sequence throughout the entire length of one or both nucleotide sequences. Such sequences may be referred to herein as being "fully complementary" to each other. However, when a first sequence is referred to herein as being "substantially complementary" to a second sequence, the two sequences may be perfectly complementary, or they may form one or more, but generally no more than five, four, three, or two, mismatched base pairs upon hybridization of a duplex of up to 30 base pairs, while maintaining the ability to hybridize under conditions most relevant to its end use, such as inhibiting gene expression via the RISC pathway. However, if two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs shall not be considered mismatches in determining complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, where the longer oligonucleotide comprises a 21-nucleotide sequence that is perfectly complementary to the shorter oligonucleotide, may still be referred to as "fully complementary" for purposes described herein.

[0248] "Complementary" sequences, as used herein, may also include or be formed entirely of non-Watson-Crick base pairs, or base pairs formed from non-naturally occurring modified nucleotides, so long as they satisfy the above requirements regarding their ability to hybridize. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble or Hoogsteen base pairing.

[0249] The terms "complementary," "fully complementary," and "substantially complementary" herein may be used in reference to base matching between the sense and antisense strands of a dsRNA, or between the antisense strand of an RNAi agent and a target sequence, as will be understood from the context of their use.

[0250] As used herein, a polynucleotide that is "substantially complementary to at least a portion of" a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of an mRNA of interest (e.g., an mRNA encoding HTT). For example, a polynucleotide is complementary to at least a portion of an HTT mRNA if the sequence is substantially complementary to an uninterrupted portion of the mRNA encoding HTT.

[0251] Thus, in some embodiments, the antisense polynucleotides disclosed herein are fully complementary to the target HTT sequence. In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target complement component HTT sequence and comprise a contiguous nucleotide sequence that is at least 80% complementary over its entire length to the equivalent region of the nucleotide sequence of any one of SEQ ID NOS: 1-5 or a fragment of any one of SEQ ID NOS: 1-5, e.g., about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

[0252] In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a target HTT sequence and comprise a contiguous nucleotide sequence that is at least about 80% complementary over its entire length to any one of the sense strand nucleotide sequences in any one of Tables 2-5 or a fragment of any one of the sense strand nucleotide sequences in Tables 2-5, e.g., about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% complementary.

[0253] In one embodiment, an RNAi agent of the present disclosure comprises a sense strand that is substantially complementary to an antisense polynucleotide that is also identical to a target HTT sequence, and the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80% complementary over its entire length to the nucleotide sequence of SEQ ID NOs: 6-10 or an equivalent region of a fragment of any one of SEQ ID NOs: 6-10, e.g., about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% complementary.

[0254] In some embodiments, the iRNAs of the invention comprise a sense strand that is substantially complementary to an antisense polynucleotide that is also complementary to a target HTT sequence, and the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80% complementary over its entire length to any one of the antisense strand nucleotide sequences in any one of Tables 2-5, or a fragment of any one of the antisense strand nucleotide sequences in Tables 2-5, e.g., about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% complementary.

[0255] In one embodiment, at least partial suppression of HTT gene expression is assessed by a decrease in the amount of HTT mRNA that can be isolated from or detected in a first cell or group of cells in which the HTT gene is transcribed and that have been treated to inhibit expression of the HTT gene, compared to a second cell or group of cells (control cells) that are substantially identical to the first cell or group of cells but have not been treated like the first cell or group of cells. The degree of inhibition can be expressed as follows:

number

[0256] The phrase "contacting a cell with an RNAi agent" such as dsRNA as used herein includes contacting a cell by any possible means.Contacting a cell with an RNAi agent includes contacting a cell with an RNAi agent in vitro or contacting a cell with an RNAi agent in vivo.Contacting can be performed directly or indirectly.Therefore, for example, the RNAi agent can be physically contacted with a cell by separately performing a method, or the RNAi agent can be placed in a situation that can allow or cause it to subsequently contact a cell.

[0257] Contacting cells in vitro can be carried out, for example, by incubating cells with an RNAi agent. Contacting cells in vivo can be carried out, for example, by injecting an RNAi agent into or near the tissue where the cells are located, or by injecting the RNAi agent into another region, for example, the central nervous system (CNS), optionally by intrathecal injection, intravitreal injection, or other injection, or by injecting the RNAi agent into the bloodstream or subcutaneous space so that the agent subsequently reaches the tissue where the cells to be contacted are located. For example, the RNAi agent can contain or be coupled to a ligand that directs or otherwise stabilizes the RNAi agent to the desired site, for example, in the CNS, for example, a lipophilic moiety, as described below and further detailed in, for example, PCT / US2019 / 031170, which is incorporated herein by reference. A combination of in vitro and in vivo contacting methods is also possible. For example, cells can be contacted with an RNAi agent in vitro and then transferred to a subject.

[0258] In one embodiment, contacting a cell with an RNAi agent includes "introducing" or "delivering an RNAi agent into a cell" by promoting or performing uptake or absorption into the cell. The absorption or uptake of an RNAi agent can occur by spontaneous diffusive or active cellular processes, or by auxiliary agents or devices. The introduction of an RNAi agent into a cell can be in vitro or in vivo. For example, in the case of in vivo introduction, the RNAi agent can be injected into a tissue site or administered systemically. In vitro introduction into a cell can include methods known in the art, such as electroporation and lipofection. Further approaches are described herein below or known in the art.

[0259] The terms "lipid-soluble" or "lipophilic moiety" refer broadly to any compound or chemical moiety that has an affinity for lipids. One way to characterize the lipophilicity of a lipophilic moiety is by the octanol-water partition coefficient, log K ow In this case, K ow is the ratio of the concentration of a chemical in the octanol phase to the concentration of the chemical 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 attributable to the structural components of a chemical calculated using first principles or empirical methods (see, e.g., Tetko et al., J. Chem. Inf. Comput. Sci. 41:1407-21 (2001), incorporated herein by reference in its entirety). It provides a thermodynamic measure of a substance's tendency to prefer a non-aqueous or oily environment rather than water (i.e., its hydrophilic / lipophilic balance). In principle, a chemical can be expressed as its logK ow is greater than 0, the molecule is lipophilic in nature. Typically, a lipophilic moiety has a logK 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 approximately 0.7. ow is predicted to be 10.7.

[0260] The lipophilicity of a molecule can be changed depending on the functional groups it contains. For example, adding a hydroxyl or amine group to the end of the lipophilic moiety can increase the partition coefficient (e.g., logK ow ) may increase or decrease in value.

[0261] Alternatively, the hydrophobicity of the double-stranded RNAi agent that is conjugated with one or more lipophilic moieties can be measured by its protein binding property.For example, in certain embodiments, if the unbound fraction of the plasma protein binding assay of double-stranded RNAi agent is determined to be positively correlated with the relative hydrophobicity of double-stranded RNAi agent, then it will be positively correlated with the silencing activity of double-stranded RNAi agent.

[0262] In one embodiment, the plasma protein binding assay that is determined is electrophoretic mobility shift assay (EMSA) that uses human serum albumin protein.The exemplary protocol of this binding assay is described in detail in, for example, PCT / US2019 / 031170.The hydrophobicity of double-stranded RNAi agent measured by the unbound fraction of siRNA in 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 in the case of enhanced siRNA in vivo delivery.

[0263] Thus, conjugating a lipophilic moiety to an internal position of a double-stranded RNAi agent provides optimal hydrophobicity in siRNA for enhanced in vivo delivery.

[0264] The term "lipid nanoparticle" or "LNP" refers to a vesicle comprising a lipid layer that encapsulates a pharmaceutically active molecule, such as a nucleic acid molecule, e.g., an RNAi agent or a plasmid from which an RNAi agent is transcribed. LNPs are described, for example, in U.S. Patent Nos. 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are incorporated herein by reference.

[0265] As used herein, a "subject" refers to an animal, such as a mammal, including a primate (e.g., a human, a non-human primate, such as a monkey or chimpanzee) or a non-primate (e.g., a rat or a mouse). In a preferred embodiment, the subject is a human, such as a human being treated or evaluated for a disease, disorder, or condition that would benefit from reduced HTT expression, a human being at risk for a disease, disorder, or condition that would benefit from reduced HTT expression, a human having a disease, disorder, or condition that would benefit from reduced HTT expression, or a human being treated for a disease, disorder, or condition that would benefit from reduced HTT expression as described herein. In some embodiments, the subject is a female human. In other embodiments, the subject is a male human. In one embodiment, the subject is an adult subject. In one embodiment, the subject is a pediatric subject. In another embodiment, the subject is a juvenile subject, i.e., a subject under the age of 20.

[0266] As used herein, the terms "treating" or "treatment" refer to a beneficial or desired outcome, such as, but not limited to, the alleviation or amelioration of one or more signs or symptoms associated with HTT gene expression or HTT protein production, for example, of an HTT-related disease, such as Huntington's disease. "Treatment" can also mean increasing survival time compared to expected survival time in the absence of treatment.

[0267] The term "lower" in the context of the level of HTT or a disease marker or symptom in a subject refers to a statistically significant decrease in such level. The decrease can be, for example, at least 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. In certain embodiments, the decrease is at least 20%. In certain embodiments, the decrease is at least 50% in a disease marker, for example, in the protein level or gene expression level. "Lowering" in the context of the level of HTT in a subject preferably refers to lowering to a level that is accepted as being within the normal range for individuals without such a disorder. In certain embodiments, "lowering" refers to a decrease in the difference between the level of a marker or symptom in a subject suffering from a disease and the level that an individual would consider to be within the normal range, for example, the level of weight loss between an obese individual and an individual whose weight is considered to be within the normal range.

[0268] As used herein, "prevention" or "preventing", when used in reference to a disease, disorder, or condition that would benefit from a reduction in HTT gene expression or HTT protein production, refers to a reduction in the likelihood that a subject will develop symptoms associated with such disease, disorder, or condition, such as symptoms of an HTT-related disease.Not developing a disease, disorder, or condition, or a reduction in the onset of symptoms associated with such disease, disorder, or condition (e.g., a reduction of at least about 10% on a clinically acceptable scale for the disease or disorder), or a delay in the onset of symptoms (e.g., a delay of several days, weeks, months, or years) is considered effective prevention.

[0269] As used herein, the term "HTT-related disease" or "HTT-related disorder" refers to any disease or disorder that would benefit from a reduction in the expression and / or activity of HTT. Exemplary HTT-related diseases include Huntington's disease.

[0270] Huntington's disease, also known as HD, Huntington's disease, grand mal chorea, chronic progressive chorea, and hereditary chorea, is an autosomal dominant disorder characterized by choreiform movements and progressive intellectual deterioration, usually occurring in middle age (35-50 years of age). The disease affects both sexes equally. The caudate nucleus atrophies, its small cell clusters degenerate, and levels of the neurotransmitters gamma-aminobutyric acid (GABA) and substance P decrease. This degeneration results in the characteristic "boxcar-shaped ventricles" seen on CT scans.

[0271] The symptoms and signs of HD develop insidiously. The most obvious symptoms of HD are abnormal body movements called chorea and dyscoordination, but it also affects many aspects of intelligence and personality. These physical symptoms usually become noticeable in the fourth decade of life but can occur at any age. When the age of onset is before 20 years, it is known as Juvenile HD.

[0272] Dementia or psychiatric disorders, ranging from apathy and irritability to end-stage bipolar or schizophreniform disorders, may precede the movement disorder or develop during its course. Anhedonia or antisocial behavior may be the first behavioral manifestation. Motor signs include quick, flicking movements of the limbs, a footstep, impermanence (inability to sustain a motor act, e.g., tongue protrusion), facial grimacing, ataxia, and dystonia.

[0273] HD is caused by a trinucleotide repeat expansion in the huntingtin (HTT) gene and is one of several polyglutamine expansion (or PolyQ expansion) diseases, which results in an expanded form of the mutant huntingtin protein (mHtt), which causes cell death in select regions of the brain.

[0274] As used herein, a "therapeutically effective amount" is intended to include an amount of an RNAi agent that, when administered to a subject with an HTT-related disease, is sufficient to treat the disease (e.g., by reducing, ameliorating, or maintaining an existing disease or one or more symptoms of the disease). A "therapeutically effective amount" can vary depending on the RNAi agent, the method of administration of the agent, the disease and its severity and medical history, age, weight, family history, genetic makeup, type of prior or concurrent treatment, if any, and other personal characteristics of the subject to be treated.

[0275] As used herein, a "prophylactically effective amount" is intended to include an amount of an RNAi agent that, when administered to a subject with an HTT-related disease, is sufficient to prevent or ameliorate the disease or one or more symptoms of the disease. Amelioration of the disease includes slowing the progression of the disease or reducing the severity of any future disease. A "prophylactically effective amount" may vary depending on the RNAi agent, the method of administration of the agent, the degree of risk of the disease, and the patient's medical history, age, weight, family history, genetic makeup, type of previous or concurrent treatment, if any, and other personal characteristics of the patient to be treated.

[0276] A "therapeutically effective amount" or a "prophylactically effective amount" also encompasses the amount of an RNAi agent that produces some desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. The RNAi agent used in the methods of the present disclosure can be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0277] The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, materials (including salts), compositions or dosage forms that are suitable for use in contact with the tissues of human and animal subjects without excessive toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio.

[0278] The phrase "pharmaceutically acceptable carrier," as used herein, means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, magnesium talc, calcium or zinc stearate, or stearic acid), or solvent encapsulating material, that is involved in the transport or transportation of a subject compound from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the subject being treated. Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants, such as magnesium state, sodium lauryl sulfate, and talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar. (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide, (15) alginic acid, (16) pyrogen-free water, (17) isotonic saline, (18) Ringer's solution, (19) ethyl alcohol, (20) pH buffer solutions, (21) polyesters, polycarbonates, or polyanhydrides, (22) bulking agents, such as polypeptides and amino acids, (23) serum components, such as serum albumin, HDL, and LDL, and (22) other non-toxic, compatible substances employed in pharmaceutical formulations.

[0279] The term "sample," as used herein, encompasses similar fluids, cells, or tissues isolated from a subject, as well as collections of fluids, cells, or tissues present within a subject. Examples of biological fluids include blood, serum, and serous fluid, plasma, cerebrospinal fluid, ocular fluid, lymphatic fluid, urine, saliva, and the like. Tissue samples can include samples from tissues, organs, or localized regions. For example, samples can be derived from specific organs, parts of organs, or fluids or cells within those organs. In certain embodiments, samples can be obtained from the brain (e.g., the whole brain or specific segments of the brain, such as the striatum, or specific types of cells within the brain, such as neurons and glial cells (astrocytes, oligodendrocytes, microglia)). In some embodiments, "a sample obtained from a subject" refers to blood obtained from a subject or plasma or serum obtained therefrom. In further embodiments, "a sample obtained from a subject" refers to brain tissue (or a subcomponent thereof) or retinal tissue (or a subcomponent thereof) obtained from a subject.

[0280] II. RNAi Agents of the Disclosure Described herein are RNAi agents that inhibit expression of the HTT gene. In one embodiment, the RNAi agent comprises a double-stranded ribonucleic acid (dsRNA) molecule that inhibits expression of the HTT gene in a cell, e.g., a cell in a subject, e.g., a mammal, e.g., a human with an HTT-related disorder, e.g., Huntington's disease. The dsRNA comprises an antisense strand having a region of complementarity that is complementary to at least a portion of the mRNA formed upon expression of the HTT gene. The region of complementarity is no more than about 15-30 nucleotides in length. When contacted with a cell expressing the HTT gene, the RNAi agent inhibits expression of the HTT gene (e.g., human, primate, or non-primate gene) by at least 50%, as assayed, e.g., by PCR or branched DNA (bDNA)-based methods, or by protein-based methods, e.g., immunofluorescence, e.g., using Western blot or flow cytometry techniques. In one embodiment, the level of knockdown is assayed in Cos7 cells using a Dual-Luciferase assay.

[0281] dsRNA comprises two RNA strands, which are complementary and hybridize to form a duplex structure under the conditions in which dsRNA is used.One strand of dsRNA (antisense strand) comprises a complementary region that is substantially complementary to the target sequence, and generally completely complementary.The target sequence can be obtained from the sequence of mRNA formed during the expression of HTT gene.The other strand (sense strand) comprises a region that is complementary to the antisense strand, so that the two strands hybridize to form a duplex structure when combined under suitable conditions.As described elsewhere herein and known in the art, the complementary sequence of dsRNA can also be contained as a self-complementary region of a single nucleic acid molecule, so that it is opposite on separate oligonucleotides.

[0282] Generally, the duplex structure is 15-30 base pairs in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-22, 19-23, 19-24, 19-25, 19-26, 19-27, 19-28, 19-29, 19-30, 19-32, 19-33, 19-34, 19-35, 19-36, 19-37, 19-38, 19-39, 19-40, 19-41, 19-42, 19-43, 19-44, 19-45, 19-46, 19-47, 19-48, 19-49, 19-50, 19-51, 19-52, 19-53, 19-54, 19-55, 19-56, 19-57, 19-58, 19-59, 19-60, 19-61, 19-62, 19-63, 19-64, 19-65, 19-66, 19-6 9, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. In certain preferred embodiments, the duplex structure is 18 to 25 base pairs in length, such as 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 25, 21 to 24, 21 to 23, 21 to 22, 22 to 25, 22 to 24, 22 to 23, 23 to 25, 23 to 24, or 24 to 25 base pairs in length, e.g., 19 to 21 base pairs in length. Ranges and lengths between the ranges and lengths listed above are also intended to be part of this disclosure.

[0283] Similarly, the region complementary to the target sequence may be 15-30 nucleotides in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19- 27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, e.g., 19-23 nucleotides in length, or 21-23 nucleotides in length. Ranges and lengths that lie between the ranges and lengths listed above are also intended to be part of this disclosure.

[0284] In some embodiments, the duplex structure is 19 to 30 base pairs in length. Similarly, the region complementary to the target sequence is 19 to 30 nucleotides in length.

[0285] In some embodiments, the dsRNA is 15 to 23 nucleotides long, 19 to 23 nucleotides long, or 25 to 30 nucleotides long. Generally, the dsRNA is long enough to serve as a substrate for the Dicer enzyme. For example, it is well known in the art that dsRNA longer than about 21-23 nucleotides can function as a substrate for Dicer. As those skilled in the art will recognize, the region of RNA targeted for cleavage will most often be a portion of a longer RNA molecule, often an mRNA molecule. In relevant cases, a "portion" of an mRNA target is a contiguous sequence of the mRNA target long enough to allow it to serve as a substrate for RNAi-dependent cleavage (i.e., cleavage via the RISC pathway).

[0286] Those skilled in the art will appreciate that the double-stranded region is the primary functional portion of the dsRNA, and may be, for example, about 15-36 base pairs, e.g., 15-36, 15-35, 15-34, 15-33, 15-32, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-23, 18-24, 18-25, 18-26, 18-24, 18-23, 18-22, 18-25, 18-26, 18-25, 18-24, 18-23, 18-22, 18-26, 18-26, 18-25, 18-26 ... It will also be appreciated that the duplex region may be 21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23 or 21-22 base pairs, for example 19-21 base pairs. That is, in one embodiment, an RNA molecule or a complex of RNA molecules having a duplex region of more than 30 base pairs is dsRNA, as long as it is processed into a functional duplex of, for example, 15-30 base pairs that targets the desired RNA for cleavage.Therefore, those skilled in the art will recognize that in one embodiment, miRNA is dsRNA.In another embodiment, the dsRNA is not a naturally occurring miRNA.In another embodiment, the RNAi agent useful for targeting HTT expression is not generated in target cells by cleavage of a larger dsRNA.

[0287] The dsRNA described herein can further comprise one or more single-stranded nucleotide overhangs, for example, 1, 2, 3, or 4 nucleotides. The nucleotide overhangs can comprise or consist of nucleotide / nucleoside analogs, such as deoxynucleotides / nucleosides. The overhangs can be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleotides of an overhang can be present at the 5'-end, 3'-end, or both ends of either the antisense strand or the sense strand of the dsRNA.

[0288] dsRNA can be synthesized by standard methods known in the art.Double-stranded RNAi compounds of the present invention can be prepared using a two-step method.First, each strand of double-stranded RNA molecules is prepared separately.Then, the strands of these components are annealed.The individual strands of siRNA compounds can be prepared using solution phase or solid phase organic synthesis or both.Organic synthesis has the advantage that it can easily prepare the oligonucleotide strands that contain unnatural nucleotides or modified nucleotides.Similarly, single-stranded oligonucleotides of the present invention can be prepared using solution phase or solid phase organic synthesis or both.

[0289] In one embodiment, the dsRNA of the present disclosure comprises at least two nucleotide sequences, a sense strand and an antisense strand. The sense strand sequence for HTT can be selected from the sequences provided in any one of Tables 2-5, and the corresponding nucleotide sequence of the antisense strand of the sense strand can be selected from the sequences provided in any one of Tables 2-5. In this embodiment, one of the two sequences is complementary to the other of the two sequences, where one of the sequences is substantially complementary to the sequence of mRNA produced upon expression of the HTT gene. Thus, in this embodiment, the dsRNA comprises two oligonucleotides, one of which is designated in any one of Tables 2-5 as the sense strand (passenger strand) and the second of which is designated in any one of Tables 2-5 as the corresponding antisense strand (guide strand).

[0290] In one embodiment, the substantially complementary sequences of the dsRNA are contained on separate oligonucleotides, hi another embodiment, the substantially complementary sequences of the dsRNA are contained on a single oligonucleotide.

[0291] Although some of the sequences in Tables 2-5 are described as modified or conjugated sequences, it will be understood that the RNA of the RNAi agent of the present disclosure, e.g., the dsRNA of the present disclosure, can comprise any one of the sequences set forth in any one of Tables 2-5, unmodified, unconjugated, or modified or conjugated differently from those set forth therein. For example, while the sense strand of the agent of the present invention shown in Table 3 is conjugated to a C16 ligand, these agents can also be conjugated to a moiety responsible for delivery to the liver, e.g., a GalNAc ligand, as described herein. The lipophilic ligand can be included at any of the positions provided herein.

[0292] Those skilled in the art are well aware that dsRNAs having duplex structures of approximately 20-23 base pairs, e.g., 21 base pairs, have been hailed as particularly effective in inducing RNA interference (Elbashir et al., (2001) EMBO J., 20:6877-6888). However, others have discovered that shorter or longer RNA duplex structures can also be effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In the above-described embodiments, due to the nature of the oligonucleotide sequences provided herein, the dsRNAs described herein can comprise at least one strand that is a minimum of 21 nucleotides in length. It can be reasonably predicted that shorter duplexes, minus only a few nucleotides at one or both ends, can be similarly effective compared to the dsRNAs described above. Thus, dsRNAs having a sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides derived from one of the sequences provided herein and which differ in their ability to inhibit expression of the HTT gene by no more than 10, 15, 20, 25, or 30% inhibition from dsRNAs containing the entire sequence using in vitro assays with Cos7 and an RNA agent at a concentration of 10 nM and the PCR assays provided in the Examples herein are intended to be within the scope of this disclosure.

[0293] In addition, the RNA described herein specifies the site in HTT transcript that is susceptible to RISC-mediated cleavage.Therefore, the present disclosure also features the RNAi agent that targets within this site.As used herein, if the RNAi agent promotes the cleavage of the transcript at any of specific sites, the RNAi agent is said to target within the specific site of RNA transcript.This RNAi agent generally comprises at least about 15 consecutive nucleotides, preferably at least 19 nucleotides, from one of the sequences provided herein, which is combined with additional nucleotide sequences taken from the region adjacent to the selected sequence in HTT gene.

[0294] III. Modified RNAi Agents of the Present Disclosure In one embodiment, the RNA of the RNAi agent of the present disclosure, e.g., dsRNA, is unmodified and does not contain any chemical modifications or conjugations, for example, those known in the art and described herein. In a preferred embodiment, the RNA of the RNAi agent of the present disclosure, e.g., dsRNA, is chemically modified to enhance stability or other beneficial characteristics. In certain embodiments of the present disclosure, substantially all of the nucleotides of the RNAi agent of the present disclosure are modified. In other embodiments of the present disclosure, all of the nucleotides of the RNAi agent of the present disclosure are modified. An RNAi agent of the present disclosure in which "substantially all of the nucleotides are modified" may be widely modified but not entirely modified, and may contain no more than 5, 4, 3, 2, or unmodified nucleotides. In yet other embodiments of the present disclosure, an RNAi agent of the present disclosure may contain no more than 5, 4, 3, 2, or 1 modified nucleotide.

[0295] Nucleic acids featured in this disclosure can be synthesized or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, SLet et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. Modifications include, for example, terminal modifications, such as 5'-end modifications (phosphorylation, conjugation, inverted ligation) or 3'-end modifications (conjugation, DNA nucleotides, inverted ligation, etc.), base modifications, such as substitution with stabilizing bases, destabilizing bases, or bases that base pair with partners in an extended repertoire, base removal (abasic nucleotides) or conjugated bases, sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions, or backbone modifications, including modification or substitution of phosphodiester linkages. Specific examples of RNAi agents useful in the embodiments described herein include, but are not limited to, RNAs containing modified backbones or lacking natural internucleoside linkages. Among the RNAs with modified backbones, those that do not have phosphorus atom in backbones include those that do not have phosphorus atom.For the purpose of this specification, and as sometimes referred to in the art, the modified RNAs that do not have phosphorus atom in their internucleoside backbones can also be considered as oligonucleosides.In some embodiments, modified RNAi agent will have phosphorus atom in its internucleoside backbones.

[0296] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates, including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates with normal 3'-5' linkages, their 2'-5' linked analogs, and those with reversed polarity, where adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included. In some embodiments of the present invention, the dsRNA agents of the present invention are in free acid form. In other embodiments of the present invention, the dsRNA agents of the present invention are in salt form. In one embodiment, the dsRNA agent of the present invention is in sodium salt form.In certain embodiments, when the dsRNA agent of the present invention is in sodium salt form, sodium ion exists in the agent as counterion to substantially all of the phosphodiester and / or phosphorothioate groups present in the agent.The agent in which substantially all of the phosphodiester and / or phosphorothioate linkages have sodium counterion comprises 5, 4, 3, 2 or 1 or less phosphodiester and / or phosphorothioate linkages that do not have sodium counterion.In some embodiments, when the dsRNA agent of the present invention is in sodium salt form, sodium ion exists as counterion to all of the phosphodiester and / or phosphorothioate groups present in the agent.

[0297] Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Patent Nos. 3,687,808, 4,469,863, 4,476,301, 5,023,243, 5,177,195, 5,188,897, 5,264,423, 5,276,019, 5,278,302, 5,286,711, and 5,286,712. No. 7, No. 5,321,131, No. 5,399,676, No. 5,405,939, No. 5,453,496, No. 5,455,233, No. 5,466,677, No. 5,47 No. 6,925, No. 5,519,126, No. 5,536,821, No. 5,541,316, No. 5,550,111, No. 5,563,253, No. 5,571,799, No. 5 , 587,361, 5,625,050, 6,028,188, 6,124,445, 6,160,109, 6,169,170, 6,172,209 No. 6,239,265, No. 6,277,603, No. 6,326,199, No. 6,346,614, No. 6,444,423, No. 6,531,590, No. 6,534, Nos. 6,639, 6,608,035, 6,683,167, 6,858,715, 6,867,294, 6,878,805, 7,015,315, 7,041,816, 7,273,933, 7,321,029, and U.S. Patent No. RE39464, the contents of each of which are incorporated herein by reference in their entirety.

[0298] Modified RNA backbones that do not contain a phosphorus atom in the backbone have backbones formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages, including those with morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and others with mixed N, O, S, and CH2 constituent moieties.

[0299] Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Patent Nos. 5,034,506, 5,166,315, 5,185,444, 5,214,134, 5,216,141, 5,235,033, 5,64,562, 5,264,564, 5,405,938, 5,434,257, 5,466,677, 5,470,967, Nos. 5,489,677, 5,541,307, 5,561,225, 5,596,086, 5,602,240, 5,608,046, 5,610,289, 5,618,704, 5,623,070, 5,663,312, 5,633,360, 5,677,437, and 5,677,439, the entire contents of each of which are incorporated herein by reference.

[0300] In another embodiment, RNA mimics suitable for use in RNAi agents are considered, in which both the sugar and internucleoside linkages, i.e., the backbone, of nucleotide units are replaced with novel groups. The base units are maintained for hybridization with appropriate nucleic acid target compounds. One such oligomeric compound, an RNA mimic known to have excellent hybridization properties, is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobases are retained and are directly or indirectly linked to the aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents teaching the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082, 5,714,331, and 5,719,262, the entire contents of each of which are incorporated herein by reference. Additional PNA compounds suitable for use in the RNAi agents of the present disclosure are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0301] Some embodiments featured in this disclosure include RNA with phosphorothioate backbones and oligonucleosides with heteroatom backbones, particularly --CH2--NH--CH2-, --CH2--N(CH3)--O--CH2-- (known as the methylene (methylimino) or MMI backbone), --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2--, and --N(CH3)--CH2--CH2-- (representing the natural phosphodiester backbone as --O--P--O--CH2--) of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNA featured herein has the morpholino backbone structure of the above-referenced US Pat. No. 5,034,506.

[0302] Modified RNAs can also contain one or more substituted sugar moieties. The RNAi agents, e.g., dsRNAs featured herein, can include one of the following at the 2' position: OH, F, O-, S-, or N-alkyl, O-, S-, or N-alkenyl, O-, S-, or N-alkynyl, or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C6. 10 Alkyl or C2-C 10 It can be alkenyl or alkynyl. Exemplary suitable modifications include O[(CH) n O] m CH3, O(CH2). n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2, and O(CH2) n ON[(CH2) n CH3)]2, where n and m are from 1 to about 10. In other embodiments, the dsRNA includes one of the following at the 2' position: C1 to C 10lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH, OCN, Cl, Br, CN, CF, OCF, SOCH, SOCH, ONO, NO, N, NH, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving groups, reporter groups, interfering substances, groups for improving the pharmacokinetic properties of RNAi agents or groups for improving the pharmacodynamic properties of RNAi agents, and other substituents with similar properties. In some embodiments, the modification includes 2'-methoxyethoxy (2'-O-CHCHOCH, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., an alkoxy-alkoxy group. Another exemplary modification is the 2'-dimethylaminooxyethoxy, i.e., O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described herein below in the Examples, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-CH2-O-CH2-N(CH2)2. Further exemplary modifications include 5'-Me-2'-F nucleotides, 5'-Me-2'-OMe nucleotides, 5'-Me-2'-deoxynucleotides (both R and S isomers within these three families), 2'-alkoxyalkyl, and 2'-NMA (N-methylacetamide).

[0303] Other modifications include 2'-methoxy (2'-OCH), 2'-aminopropoxy (2'-OCHCHNH), 2'-O-hexadecyl, and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of an RNAi agent, particularly the 3' position of the sugar on the 3'-terminal nucleotide or in a 2'-5'-linked dsRNA and the 5' position of the 5'-terminal nucleotide. RNAi agents can also have sugar mimetics, such as a cyclobutyl moiety in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Patent 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, and 5,56 Nos. 7,811, 5,576,427, 5,591,722, 5,597,909, 5,610,300, 5,627,053, 5,639,873, 5,646,265, 5,658,873, 5,670,633, and 5,700,920, some of which are commonly owned with this application, the entire contents of each of the foregoing being incorporated herein by reference.

[0304] The RNAi agent of the present disclosure can also comprise modification or substitution of nucleobase (often simply referred to in the art as "base").As used herein, "unmodified" or "natural" nucleobase includes purine bases adenine (A) and guanine (G), and pyrimidine bases thymine (T), cytosine (C) and uracil (U).Modified nucleobases include other synthetic nucleobases and natural nucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, These include 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine.Additional nucleobases include those disclosed in United States Patent No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P.ed.Wiley-VCH, 2008, those disclosed in The Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, Kroschwitz, JL, ed.John Wiley & Sons, 1990, those disclosed in Englisch et al., (1991) Angewandte Chemie, International Edition, 30:613, and those disclosed in Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., Ed., CRC Press, 1993.Some of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the present disclosure. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and 0-6 substituted purines, such as 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, Y.S., Crooke, S.T., and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), and are exemplary base substitutions, especially when combined with 2'-O-methoxyethyl sugar modifications.

[0305] Representative United States patents that teach the preparation of certain of the above modified nucleobases, as well as other modified nucleobases, include, but are not limited to, the above-referenced U.S. Patent Nos. 3,687,808, 4,845,205, 5,130,30, 5,134,066, 5,175,273, 5,367,066, 5,432,272, 5,457,187, 5,459,255, 5,484,908, 5,502,177, 5,525,711, 5,552,540, 5,587,469, Nos. 5,594,121, 5,596,091, 5,614,617, 5,681,941, 5,750,692, 6,015,886, 6,147,200, 6,166,197, 6,222,025, 6,235,887, 6,380,368, 6,528,640, 6,639,062, 6,617,438, 7,045,610, 7,427,672, and 7,495,088, the entire contents of each of which are incorporated herein by reference.

[0306] In some embodiments, the RNAi agents of the present disclosure may also be modified to include one or more bicyclic sugar moieties. A "bicyclic sugar" is a furanosyl ring modified by a ring formed by a bridge between two carbon atoms, whether adjacent or non-adjacent. A "bicyclic nucleoside" ("BNA") is a nucleoside having a sugar moiety that includes a ring formed by bridging two carbon atoms of the sugar ring, whether adjacent or non-adjacent, thereby forming a bicyclic ring system. In certain embodiments, a bridge connects the 4'-carbon and 2'-carbon of the sugar ring, optionally via a 2'-acyclic oxygen atom. Thus, in some embodiments, the agents of the present invention may include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety includes an additional bridge connecting the 2' and 4' carbons. In other words, an LNA is a nucleotide containing a bicyclic sugar moiety that includes a 4'-CH2-O-2' bridge. This structure effectively "locks" the ribose in the 3'-endo conformation. The addition of a locking nucleic acid to siRNA has been shown to increase siRNA stability in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, OR. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Examples of bicyclic nucleosides for use in the polynucleotides of the present invention include, but are not limited to, nucleosides containing a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, the antisense polynucleotide agent of the present invention includes one or more bicyclic nucleosides containing a 4' to 2' bridge.

[0307] A locked nucleoside can be represented by this structure (stereochemistry omitted): TIFF2024512635000007.tif3844, where B is a nucleobase or modified nucleobase, and L is a linking group connecting the 2'-carbon and 4'-carbon of the ribose ring. Examples of such 4' to 2' bridged bicyclic nucleosides include, but are not limited to, 4'-(CH2)-O-2' (LNA), 4'-(CH2)-S-2', 4'-(CH2)2-O-2' (ENA), 4'-CH(CH3)-O-2' (also referred to as "constrained ethyl" or "cEt"), and 4'-CH(CHOCH3)-O-2' (and analogs thereof, see e.g., U.S. Pat. No. 7,399,845), 4'-C(CH3)(CH3)-O-2' (and analogs thereof, e.g., U.S. Pat. No. 7,399,845). No. 8,278,283), 4'-CH2-N(OCH3)-2' (and analogs thereof, see, e.g., U.S. Pat. No. 8,278,425), 4'-CH2-ON(CH3)-2' (see, e.g., U.S. Patent Application Publication No. 2004 / 0171570), 4'-CH2-N(R)-O-2', where R is H, C1-C12 alkyl, or a nitrogen protecting group (see, e.g., U.S. Pat. No. 7,427,672), 4'-CH2-C(H)(CH3)-2' (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134), and 4'-CH2-C(=CH2)-2' (and analogs thereof, see, e.g., U.S. Pat. No. 8,278,426). The entire contents of each of the foregoing are incorporated herein by reference.

[0308] Additional representative U.S. patents and U.S. patent application publications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, U.S. Patent Nos. 6,268,490, 6,525,191, 6,670,461, 6,770,748, 6,794,499, 6,998,484, 7,053,207, 7,034,133, 7,084,125, 7,399, ,845, 7,427,672, 7,569,686, 7,741,457, 8,022,193, 8,030,467, 8,278,425, 8,278,426, 8,278,283, U.S. Patent Application Publication No. 2008 / 0039618 and U.S. Patent Application Publication No. 2009 / 0012281, the entire contents of each of which are incorporated herein by reference.

[0309] Any of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations, including, for example, α-L-ribofuranose and β-D-ribofuranose (see WO 99 / 14226).

[0310] The RNA of an iRNA can also be modified to contain one or more constrained ethyl nucleotides. As used herein, a "constrained ethyl nucleotide" or "cEt" is a locked nucleic acid containing a bicyclic sugar moiety containing a 4'-CH(CH3)-O-2' bridge (L in the preceding structure). In one embodiment, the constrained ethyl nucleotide is in the S conformation and is referred to herein as an "S-cEt."

[0311] The iRNA of the present invention may also contain one or more "conformationally restricted nucleotides" ("CRNs"). A CRN is a nucleotide analogue with a linker connecting the C2' and C4' carbons of ribose or the C3 and C5' carbons of ribose. The CRN locks the ribose ring into a stable conformation, increasing hybridization affinity to mRNA. The linker is long enough to position the oxygen in an optimal position for stability and affinity, thereby reducing puckering of the ribose ring.

[0312] Representative publications that teach the preparation of certain of the above CRNs include, but are not limited to, U.S. Patent Application Publication No. 2013 / 0190383 and PCT Publication No. WO 2013 / 036868, the entire contents of each of which are incorporated herein by reference.

[0313] In some embodiments, the iRNA of the invention includes one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNAs are unlocked acyclic nucleic acids in which any of their sugar linkages have been removed to form an unlocked "sugar" residue. In one example, UNAs also include monomers in which the C1'-C4' linkage has been removed (i.e., a covalent carbon-oxygen-carbon bond between the C1' and C4' carbons). In another example, the C2'-C3' linkage of the sugar (i.e., a covalent carbon-carbon bond between the C2' and C3' carbons) has been removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039, which are incorporated herein by reference).

[0314] Representative U.S. publications that teach the preparation of UNAs include, but are not limited to, U.S. Patent No. 8,314,227, and U.S. Patent Application Publication Nos. 2013 / 0096289, 2013 / 0011922, and 2011 / 0313020, the entire contents of each of which are incorporated herein by reference.

[0315] Potential stabilizing modifications to the ends of RNA molecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-0-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3'-phosphate, inverted 2'-deoxy-modified ribonucleotides such as inverted dT (idT) and inverted dA (idA), and inverted abasic 2'-deoxyribonucleotides (iAb), among others. Disclosure of these modifications can be found in WO 2011 / 005861.

[0316] In one embodiment, the 3' or 5' end of the oligonucleotide is linked to an inverted 2'-deoxy modified ribonucleotide, such as an inverted dT (idT), an inverted dA (idA), or an inverted abasic 2'-deoxyribonucleotide (iAb). In one particular embodiment, the inverted 2'-deoxy-modified ribonucleotide is linked to the 3' end of the oligonucleotide, such as the 3' end of the sense strand described herein, where the linkage is via a 3'-3' phosphodiester linkage or a 3'-3'-phosphorothioate linkage.

[0317] In another embodiment, the 3' end of the sense strand is linked to an inverted abasic ribonucleotide (iAb) via a 3'-3'-phosphorothioate linkage. In another embodiment, the 3' end of the sense strand is linked to an inverted dA (idA) via a 3'-3'-phosphorothioate linkage.

[0318] In one particular example, the inverted 2'-deoxy-modified ribonucleotide is linked to the 3' end of an oligonucleotide, such as the 3' end of the sense strand described herein, wherein the linkage is via a 3'-3' phosphodiester linkage or a 3'-3'-phosphorothioate linkage.

[0319] In another example, the 3'-terminal nucleotide of the sense strand is an inverted dA (idA) and is joined to the preceding nucleotide via a 3'-3'-linkage (eg, a 3'-3'-phosphorothioate linkage).

[0320] Other modifications of the nucleotides of the iRNA of the present invention include 5' phosphates or 5' phosphate mimics, such as 5' terminal phosphates or phosphate mimics on the antisense strand of the iRNA. Suitable phosphate mimics are disclosed, for example, in U.S. Patent Application Publication No. 2012 / 0157511, the entire contents of which are incorporated herein by reference.

[0321] A. Modified RNAi Agents Comprising Motifs of the Disclosure In certain aspects of the present disclosure, the double-stranded RNAi agent of the present disclosure includes an agent having chemical modifications, such as those disclosed in International Publication No. 2013 / 075035, the entire contents of which are incorporated herein by reference. As shown herein and in International Publication No. 2013 / 075035, excellent results can be obtained by introducing one or more motifs of three identical modifications on three consecutive nucleotides into the sense or antisense strand of the RNAi agent, particularly at or near the cleavage site. In some embodiments, the sense and antisense strands of the RNAi agent can be otherwise completely modified. The introduction of these motifs interrupts the modification pattern of the sense or antisense strand, if present. The RNAi agent can optionally be conjugated with a lipophilic ligand, for example, a C16 ligand, for example, on the sense strand. The RNAi agent can optionally be modified with an (S)-glycol nucleic acid (GNA) modification, for example, at one or more residues of the antisense strand. The resulting RNAi agent exhibits excellent gene silencing activity.

[0322] Thus, the present disclosure provides a double-stranded RNAi agent capable of inhibiting expression of a target gene (i.e., the HTT gene) in vivo. The RNAi agent comprises a sense strand and an antisense strand. Each strand of the RNAi agent can be 15 to 30 nucleotides in length. Each strand can be, for example, 16 to 30 nucleotides in length, 17 to 30 nucleotides in length, 25 to 30 nucleotides in length, 27 to 30 nucleotides in length, 17 to 23 nucleotides in length, 17 to 21 nucleotides in length, 17 to 19 nucleotides in length, 19 to 25 nucleotides in length, 19 to 23 nucleotides in length, 19 to 21 nucleotides in length, 21 to 25 nucleotides in length, or 21 to 23 nucleotides in length. In certain embodiments, each strand is 19 to 23 nucleotides in length.

[0323] The sense and antisense strands typically form a duplex, double-stranded RNA ("dsRNA"), also referred to herein as an "RNAi agent." The duplex region of an RNAi agent can be 15-30 nucleotide pairs in length. For example, the duplex region can be 16-30 nucleotide pairs in length, 17-30 nucleotide pairs in length, 27-30 nucleotide pairs in length, 17-23 nucleotide pairs in length, 17-21 nucleotide pairs in length, 17-19 nucleotide pairs in length, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs in length, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs in length. In another example, the duplex region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides in length. In a preferred embodiment, the duplex region is 19 to 21 nucleotide pairs in length.

[0324] In one embodiment, the RNAi agent may contain one or more overhang regions or capping groups at the 3'-end, 5'-end, or both ends of one or both strands. The overhangs may be 1 to 6 nucleotides in length, e.g., 2 to 6 nucleotides in length, 1 to 5 nucleotides in length, 2 to 5 nucleotides in length, 1 to 4 nucleotides in length, 2 to 4 nucleotides in length, 1 to 3 nucleotides in length, 2 to 3 nucleotides in length, or 1 to 2 nucleotides in length. In a preferred embodiment, the nucleotide overhang region is 2 nucleotides in length. The overhang may be the result of one strand being longer than the other, or the result of two strands of the same length being twisted. The overhang may form a mismatch with the target mRNA, be complementary to the targeted gene sequence, or be a different sequence. The first and second strands may also be linked by additional bases, for example, to form a hairpin, or by other non-basic linkers.

[0325] In one embodiment, the nucleotides in the overhang region of an RNAi agent can each independently be modified or unmodified nucleotides, including but not limited to 2'-sugar modifications, such as 2-F, 2'-O-methyl, thymidine (T), and any combination thereof.

[0326] For example, TT can be an overhang sequence for either end on either strand, which can form a mismatch with the target mRNA, or can be complementary to the targeted gene sequence, or can be another sequence.

[0327] The 5'- or 3'-overhang of the sense strand, antisense strand, or both strands of the RNAi agent can be phosphorylated. In some embodiments, the overhang region contains two nucleotides with a phosphorothioate between them, and the two nucleotides can be the same or different. In one embodiment, the overhang is present at the 3'-end of the sense strand, antisense strand, or both strands. In one embodiment, the 3'-overhang is present in the antisense strand. In one embodiment, the 3'-overhang is present in the sense strand.

[0328] RNAi agent can contain only a single overhang, which can enhance the interference activity of RNAi without affecting its overall stability.For example, the single-stranded overhang can be located at the 3'-end of the sense strand or at the 3'-end of the antisense strand.RNAi can also have a blunt end, which is located at the 5'-end of the antisense strand (or the 3'-end of the sense strand), or vice versa.Generally, the antisense strand of RNAi has a nucleotide overhang at the 3'-end, and its 5'-end is blunt.Without wishing to be bound by theory, the asymmetry between the blunt end at the 5'-end of the antisense strand and the 3'-end overhang of the antisense strand is favorable for the insertion of guide strand into RISC process.

[0329] In one embodiment, the RNAi agent is a double-ended bluntmer 19 nucleotides in length, the sense strand containing at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 7, 8, and 9 from the 5' end, and the antisense strand containing at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0330] In another embodiment, the RNAi agent is a double-ended bluntmer 20 nucleotides in length, the sense strand containing at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 8, 9, and 10 from the 5' end, and the antisense strand containing at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0331] In yet another embodiment, the RNAi agent is a double-ended bluntmer 21 nucleotides in length, the sense strand containing at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 11 from the 5' end, and the antisense strand containing at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0332] In one embodiment, the RNAi agent comprises a 21-nucleotide sense strand and a 23-nucleotide antisense strand, wherein the sense strand comprises at least one motif of three 2'-F modifications on 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 on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, and one end of the RNAi agent is blunt, and the other end comprises a two-nucleotide overhang. Preferably, the two-nucleotide overhang is at the 3' end of the antisense strand. When the two-nucleotide overhang is at the 3' end of the antisense strand, there may be two phosphorothioate internucleotide linkages between the three terminal nucleotides, two of which are overhanging nucleotides, and the third nucleotide is a paired nucleotide adjacent to the overhanging nucleotide. In one embodiment, the RNAi agent further comprises two phosphorothioate internucleotide linkages between the three terminal nucleotides at both the 5'-end of the sense strand and the 5'-end of the antisense strand.In one embodiment, every nucleotide in the sense strand and the antisense strand of the RNAi agent, including the nucleotide that is part of a motif, is a modified nucleotide.In one embodiment, each residue is independently modified with 2'-O-methyl or 3'-fluoro, for example, within an alternating motif.Optionally, the RNAi agent further comprises a ligand (for example, a lipophilic ligand, optionally a C16 ligand).

[0333] In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, wherein the sense strand is 25 to 30 nucleotide residues in length and, starting from the 5'-terminal nucleotide (position 1), comprises at least 8 ribonucleotides at positions 1 to 23 of the first strand; and the antisense strand is 36 to 66 nucleotide residues in length and, starting from the 3'-terminal nucleotide, comprises at least 8 ribonucleotides at positions 1 to 23 of the sense strand to form a duplex; wherein at least the 3'-terminal nucleotide of the antisense strand is unpaired with the sense strand, and up to 6 consecutive 3'-terminal nucleotides are unpaired with the sense strand, thereby forming a 3' single-stranded overhang of 1 to 6 nucleotides; and wherein the 5'-end of the antisense strand comprises 10 to 30 ribonucleotides that are not paired with the sense strand. The double-stranded nucleic acid comprises consecutive nucleotides, thereby forming a single-stranded 5' overhang of 10 to 30 nucleotides, at least the 5'- and 3'-terminal nucleotides of the sense strand base-pair with nucleotides of the antisense strand when the sense strand and the antisense strand are aligned for maximum complementarity, thereby forming a substantially double-stranded region between the sense strand and the antisense strand, and the antisense strand is sufficiently complementary to the target RNA along at least 19 ribonucleotides of the antisense strand length, so that when the double-stranded nucleic acid is introduced into a mammalian cell, target gene expression is reduced, and the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides, at least one of the motifs occurring at or near the cleavage site. The antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

[0334] In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, the RNAi agent comprising a first strand having a length of at least 25 nucleotides and at most 29 nucleotides, and a second strand having a length of at most 30 nucleotides with at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, the 3' end of the first strand and the 5' end of the second strand forming a blunt end, the second strand being 1 to 4 nucleotides longer at its 3' end than the first strand, the duplex region being at least 25 nucleotides in length, the second strand being sufficiently complementary to a target mRNA along at least 19 nucleotides of the length of the second strand, the RNAi agent reducing target gene expression when introduced into a mammalian cell, and Dicer cleavage of the RNAi agent preferentially resulting in siRNA comprising the 3' end of the second strand, thereby reducing target gene expression in the mammalian cell. Optionally, the RNAi agent further comprises a ligand.

[0335] In one embodiment, the sense strand of the RNAi agent contains at least one motif of three identical modifications on three consecutive nucleotides, one of the motifs occurring at the cleavage site within the sense strand.

[0336] In one embodiment, the antisense strand of the RNAi agent can also contain at least one motif of three identical modifications on three consecutive nucleotides, one of which motifs occurs at or near the cleavage site in the antisense strand.

[0337] For RNAi agents with a duplex region 17-23 nucleotides in length, the cleavage sites in the antisense strand are typically approximately 10, 11, and 12 from the 5' end. Thus, three identical modification motifs can occur at positions 9, 10, 11, 10, 11, 12, 11, 12, 13, 12, 13, 14, or 13, 14, 15 of the antisense strand, starting from the first nucleotide from the 5' end of the antisense strand, or starting from the first paired nucleotide within 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 RNAi agent from the 5' end.

[0338] The sense strand of RNAi agent can contain at least one motif of three identical modifications on three consecutive nucleotides at the cleavage site of its strand, and antisense strand can have at least one motif of three identical modifications on three consecutive nucleotides at or near the cleavage site of strand.When sense strand and antisense strand form dsRNA duplex, sense strand and antisense strand can be aligned so that one motif of three nucleotides on sense strand and one motif of three nucleotides on antisense strand have at least one nucleotide overlap, that is, at least one of the three nucleotides of the motif in sense strand and at least one of the three nucleotides of the motif in antisense strand form base pairing.Alternatively, at least two nucleotides can overlap, or all three nucleotides can overlap.

[0339] In one embodiment, the sense strand of an RNAi agent may contain multiple motifs of three identical modifications on three consecutive nucleotides. The first motif may occur at or near the site of strand cleavage, and the other motifs may be wing modifications. As used herein, the term "wing modification" refers to a motif that occurs in another portion of the strand separated from a motif at or near the site of cleavage of the same strand. The wing modifications may be adjacent to the first motif or separated by at least one or more nucleotides. When the motifs are immediately adjacent to each other, the chemical nature of the motifs is distinct from each other, and when the motifs are separated by one or more nucleotides, the chemical nature may be the same or different. Two or more wing modifications may be present. For example, when two wing modifications are present, each wing modification may occur at one end of the first motif at or near the cleavage site, or on either side of the lead motif.

[0340] Like the sense strand, the antisense strand of an RNAi agent may contain multiple motifs of three identical modifications on three consecutive nucleotides, at least one of which occurs at or near the site of cleavage on that strand. The antisense strand may also contain one or more wing modifications in the same alignment as the wing modifications that may be present on the sense strand.

[0341] In one embodiment, wing modifications on the sense or antisense strand of an RNAi agent typically do not include the first one or two terminal nucleotides at the 3' end, 5' end, or both ends of the strand.

[0342] In another embodiment, wing modifications on the sense or antisense strand of an RNAi agent typically do not include the first one or two paired nucleotides within the duplex region at the 3' end, 5' end, or both ends of the strand.

[0343] When the sense and antisense strands of an RNAi agent each contain at least one wing modification, the wing modifications can be at the same end of the duplex region and have an overlap of one, two, or three nucleotides.

[0344] When the sense and antisense strands of an RNAi agent each contain at least two wing modifications, the sense and antisense strands can be aligned such that the two modifications from each single strand are at one end of a duplex region with an overlap of one, two, or three nucleotides, the two modifications from each single strand are at the other end of the duplex region with an overlap of one, two, or three nucleotides, and the two modifications from the single strand are on either side of a lead motif within the duplex region with an overlap of one, two, or three nucleotides.

[0345] In one embodiment, the RNAi agent comprises mismatches or combinations thereof in the duplex with the target. Mismatches can occur in the overhang region or the duplex region. Base pairs can be ranked based on their tendency to promote dissociation or melting (for example, based on the free energy of association or dissociation of a particular pairing; the simplest approach is to examine each pair individually, but adjacent or similar analysis can also be used). In terms of promoting dissociation, A:U is more preferable than G:C, G:U is more preferable than G:C, and I:C is more preferable than G:C (I=inosine). Mismatches, such as non-standard pairings or non-standard pairings (described elsewhere herein), are more preferable than standard pairings (A:T, A:U, G:C), and pairings containing universal bases are more preferable than standard pairings.

[0346] In one embodiment, the RNAi agent comprises the first 1, 2, 3, 4, or 5 base pairs within the duplex region from the 5' end of the antisense strand independently selected from the group of A:U, G:U, I:C, and at least one mismatch pair, e.g., a non-canonical pairing or a non-canonical pairing or a pairing containing a universal base, to promote dissociation of the antisense strand at the 5' end of the duplex.

[0347] 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 one, two, or three 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.

[0348] In another embodiment, the nucleotide at the 3' end of the sense strand is deoxythymine (dT). In another embodiment, the nucleotide at the 3' end of the antisense strand is deoxythymine (dT). In one embodiment, there is a short sequence of deoxythymine nucleotides, for example, two dT nucleotides on the 3' end of the sense strand or the antisense strand.

[0349] In one embodiment, 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) It can be expressed as During the ceremony, i and j each independently represent 0 or 1; p and q each independently represent 0 to 6; each N aindependently represent oligonucleotide sequences containing 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each N b independently represent an oligonucleotide sequence comprising 0 to 10 modified nucleotides; each n p and n q independently represent overhanging nucleotides, wherein Nb and Y do not have the same modification, and XXX, YYY and ZZZ each independently represent one motif of three identical modifications on three consecutive nucleotides. Preferably, YYY are all 2'-F modified nucleotides.

[0350] In one embodiment, N a or N b includes alternating pattern modifications.

[0351] In one embodiment, the YYY motif occurs at or near the cleavage site of the sense strand.For example, when the RNAi agent has a double-stranded region of 17-23 nucleotides in length, the YYY motif can occur at or near the cleavage site of the sense strand (for example, can occur at positions 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12, or 11, 12, 13), and this number starts from the first nucleotide from the 5' end, or optionally this number starts from the first paired nucleotide in the double-stranded region from the 5' end.

[0352] In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. Thus, the sense strand has the formula: 5'n p -N a -YYY-N b -ZZZ-N a -n q 3'(Ib), 5'n p -Na -XXX-N b -YYY-N a -n q 3'(Ic), or 5'n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3'(Id) It can be expressed as:

[0353] When the sense strand is represented by formula (Ib), N b represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides.

[0354] each N a can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0355] When the sense strand is represented by formula (Ic), N b represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0356] When the sense strand is represented by formula (Id), each N b independently represent an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. b is 0, 1, 2, 3, 4, 5 or 6. Each N a can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0357] Each of X, Y and Z may be the same or different from one another.

[0358] In other embodiments, i is 0 and j is 0, and the sense strand has the formula 5'n p -N a -YYY-N a -n q 3' (Ia) It can be expressed as:

[0359] When the sense strand is represented by formula (Ia), each N a can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0360] In one embodiment, the antisense strand sequence of the RNAi is represented by formula (II): 5'n q’ -N a '-(Z'Z'Z') k -N b '-Y'Y'Y'-N b '-(X'X'X') l -N' a -n p '3' (II) It can be expressed as During the ceremony, k and l each independently represent 0 or 1; p' and q' each independently represent 0 to 6; each N a each independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each N b ' independently represents an oligonucleotide sequence comprising 0 to 10 modified nucleotides; each n p ' and n q ' independently represent an overhanging nucleotide; N b ' and Y' do not have the same modification, and X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides.

[0361] In one embodiment, N a ' or N b ' includes alternating pattern modifications.

[0362] Y'Y'Y' motif occurs at or near the cleavage site of antisense strand.For example, when RNAi agent has a double-stranded region of 17-23 nucleotides in length, Y'Y'Y' motif can occur at position 9, 10, 11, 10, 11, 12, 11, 12, 13, 12, 13, 14, or 13, 14, 15 of antisense strand, this number starting from the first nucleotide from the 5' end, or optionally this number starting from the first paired nucleotide in the double-stranded region from the 5' end.Preferably, Y'Y'Y' motif occurs at position 11, 12, 13.

[0363] In one embodiment, the Y'Y'Y' motif is all 2'-OMe modified nucleotides.

[0364] In one embodiment, k is 1 and l is 0, or k is 0 and l is 1, or k and l are both 1.

[0365] Thus, the antisense strand has the formula: 5'n q’ -N a '-Z'Z'Z'-N b '-Y'Y'Y'-N a '-n p’ 3'(IIb), 5'n q’ -N a '-Y'Y'Y'-N b '-X'X'X'-n p’ 3'(IIc), or 5'n q’ -N a '-Z'Z'Z'-N b'-Y'Y'Y'-N b '-X'X'X'-N a '-n p’ 3' (IId) It can be expressed as:

[0366] When the antisense strand is represented by formula (IIb), N b ’ represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a ' independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0367] When the antisense strand is represented by formula (IIc), N b ' represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a ' independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0368] When the antisense strand is represented by formula (IId), each N b Each N' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a ' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. b is 0, 1, 2, 3, 4, 5 or 6.

[0369] In other embodiments, k is 0 and l is 0, and the antisense strand has the formula

[0370] 5'n p’ -N a’ -Y'Y'Y'-N a’ -n q’ 3'(Ia) It can be expressed as:

[0371] When the antisense strand is represented by formula (IIa), each N a ' independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0372] Each of X', Y' and Z' may be the same as or different from one another.

[0373] Each nucleotide of sense strand and antisense strand can be independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-hydroxyl or 2'-fluoro.For example, each nucleotide of sense strand and antisense strand is independently modified with 2'-O-methyl or 2'-fluoro.Each X, Y, Z, X', Y' and Z' can specifically represent 2'-O-methyl modification or 2'-fluoro modification.

[0374] In one embodiment, the sense strand of the RNAi agent can contain a YYY motif occurring at positions 9, 10, and 11 of the strand when the duplex region is 21 nt, the numbers starting from the first nucleotide from the 5' end, or optionally, the numbers starting from the 5' end with the first paired nucleotide in the duplex region, and Y represents a 2'-F modification. The sense strand can further contain a XXX motif or a ZZZ motif as a wing modification at the opposite end of the duplex region, and XXX and ZZZ each independently represent a 2'-OMe modification or a 2'-F modification.

[0375] In one embodiment, the antisense strand may contain a Y'Y'Y' motif occurring at positions 11, 12, or 13 of the strand, where the numbering starts from the first nucleotide from the 5' end, or optionally, the numbering starts from the 5' end with the first paired nucleotide in the duplex region, and Y' represents a 2'-O-methyl modification. The antisense strand may further contain an X'X'X' motif or a Z'Z'Z' motif as a wing modification at the opposite end of the duplex region, and X'X'X' and Z'Z'Z' each independently represent a 2'-OMe modification or a 2'-F modification.

[0376] The sense strand represented by any one of the above formulas (Ia), (Ib), (Ic), and (Id) forms a duplex with the antisense strand represented by any one of the above formulas (IIa), (IIb), (IIc), and (IId), respectively.

[0377] Thus, the RNAi agent used in the methods of the present disclosure can include a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, and the RNAi duplex can have the formula (III): Sense:5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' Antisense: 3'n p ’ -N a ’ -(X'X'X') k -N b ’ -Y'Y'Y'-N b ’ -(Z'Z'Z') l -N a ’ -n q ’ 5' (III) is expressed as During the ceremony, i, j, k, and l each independently represent 0 or 1; p, p', q and q' each independently represent 0 to 6; each N a and N a each independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, each sequence containing at least two differently modified nucleotides; each N b and N b ’ independently represent an oligonucleotide sequence comprising 0 to 10 modified nucleotides; During the ceremony, each n p ',n p , n q ' and n q each of which may or may not be present independently represents an overhanging nucleotide; XXX, YYY, ZZZ, X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides.

[0378] In one embodiment, i is 0 and j is 0, or i is 1 and j is 0, or i is 0 and j is 1, or i and j are both 0, or i and j are both 1. In another embodiment, k is 0 and l is 0, or k is 1 and l is 0, or k is 0 and l is 1, or k and l are both 0, or k and l are both 1.

[0379] An exemplary combination of sense and antisense strands that form an RNAi duplex is represented by the formula: 5'n p -N a -YYY-N a -n q 3' 3'n p ’ -N a ’ -Y'Y'Y'-N a ’ n q’ 5’ (IIIa) 5’n p -N a -YYY-N b -ZZZ-N a -n q 3’ 3’n p ’ -N a ’ -Y’Y’Y’-N b ’ -Z’Z’Z’-N a ’ n q ’ 5’ (IIIb) 5’n p -N a -XXX-N b -YYY-N a -n q 3’ 3’n p ’ -N a ’ -X’X’X’-N b ’ -Y’Y’Y’-N a ’ -n q ’ 5’ (IIIc) 5’n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3’ 3’n p ’ -N a ’ -X’X’X’-N b ’ -Y’Y’Y’-N b ’ -Z’Z’Z’-N a -n q ’ 5’ (IIId) including

[0380] When the RNAi agent is represented by formula (IIIa), each N a independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0381] When the RNAi agent is represented by formula (IIIb), each N b independently represent an oligonucleotide sequence containing 1 to 10, 1 to 7, 1 to 5, or 1 to 4 modified nucleotides. a ' independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0382] When the RNAi agent is represented by formula (IIIc), each N b , N b Each N' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a ' independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0383] When the RNAi agent is represented by formula (IIId), each N b , N b Each N' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. a , N a ’ independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. a , N a ', N b , and N b ' each independently includes an alternating pattern of modifications.

[0384] In one embodiment, when the RNAi agent has formula (IIId), N a The modification is a 2'-O-methyl modification or a 2'-fluoro modification. In another embodiment, when the RNAi agent has formula (IIId), N aThe modifications are 2'-O-methyl or 2'-fluoro modifications, and p '>0 and at least one n p In yet another embodiment, when the RNAi agent has formula (IIId), N' is linked to the adjacent nucleotide via a phosphorothioate linkage. a The modification is 2'-O-methyl or 2'-fluoro modification, p '>0 and at least one n p In another embodiment, when the RNAi agent has formula (IIId), N' is linked to adjacent nucleotides via phosphorothioate linkages, and the sense strand is conjugated to one or more C16 (or related thereto) moieties attached via a divalent or trivalent branched linker (described below). a The modification is 2'-O-methyl or 2'-fluoro modification, p '>0 and at least one n p ' are linked to adjacent nucleotides via phosphorothioate linkages, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more lipophilic moieties, such as C16 (or related) moieties, which may be attached via a bivalent or trivalent branched linker.

[0385] In one embodiment, when the RNAi agent has formula (IIIa), N a The modification is 2'-O-methyl or 2'-fluoro modification, p '>0 and at least one n p ' are linked to adjacent nucleotides via phosphorothioate linkages, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more lipophilic moieties, such as C16 (or related) moieties, which may be attached via a bivalent or trivalent branched linker.

[0386] In one embodiment, the RNAi agent is a multimer comprising at least two duplexes represented by formula (III), (IIIa), (IIIb), (IIIc) and (IIId), and these duplexes are connected by a linker. The linker may be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each duplex may target the same gene or two different genes, or each duplex may target the same gene at two different target sites.

[0387] In one embodiment, the RNAi agent is a multimer that contains three, four, five, six or more double strands represented by formula (III), (IIIa), (IIIb), (IIIc) and (IIId), and the double strands are connected by a linker. The linker may be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each double strand can target the same gene or two different genes, or each double strand can target the same gene at two different target sites.

[0388] In one embodiment, two RNAi agents represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId) are linked to each other at their 5' ends, and one or both of their 3' ends are optionally conjugated to a ligand. The agents can each target the same gene or two different genes, or the agents can each target the same gene at two different target sites.

[0389] Various publications describe the multimeric RNAi agent that can be used in the method of the present disclosure.These publications include International Publication No. 2007 / 091269, International Publication No. 2010 / 141511, International Publication No. 2007 / 117686, International Publication No. 2009 / 014887 and International Publication No. 2011 / 031520 and United States Patent No. 7858769, each of whose entire contents is incorporated herein by reference.

[0390] In certain embodiments, the compositions and methods of the present disclosure include vinyl phosphonate (VP) modifications of RNAi agents as described herein. In exemplary embodiments, the 5'-vinyl phosphonate modified nucleotides of the present disclosure have the following structure: [ka] wherein X is O or S; R is hydrogen, hydroxy, fluoro, or C 1~20 alkoxy (e.g., methoxy or n-hexadecyloxy); R 5’ is =C(H)-P(O)(OH)2, and the C5' carbon and R 5’ and the double bond between B is a nucleobase or modified nucleobase, optionally B is adenine, guanine, cytosine, thymine, or uracil.

[0391] In one embodiment, R 5’ is ═C(H)—P(O)(OH) 2 and the double bond between the C5′ carbon and R5′ is in the E configuration. In another embodiment, R is methoxy and R 5’ is ═C(H)—P(O)(OH) and the double bond between the C5′ carbon and R5′ is in the E configuration. In another embodiment, X is S, R is methoxy and R 5’ is =C(H)-P(O)(OH)2, and the double bond between the C5' carbon and R5' is in the E configuration.

[0392] The vinyl phosphonates of the present disclosure can be attached to either the antisense or sense strand of the dsRNA of the present disclosure. In certain embodiments, the vinyl phosphonates of the present disclosure are attached to the antisense strand of the dsRNA, optionally at the 5' end of the antisense strand of the dsRNA.

[0393] Vinyl phosphate modifications are also contemplated for the compositions and methods of the present disclosure. Exemplary vinyl phosphate structures include those described above, where R5' is =C(H)-OP(O)(OH)2, and the double bond between the C5' carbon and R5' is in the E or Z configuration (e.g., E configuration).

[0394] i. Thermal destabilization modification In certain embodiments, dsRNA molecules can be optimized for RNA interference by incorporating a thermodestabilizing modification into the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand) to reduce or inhibit off-target gene silencing. It has been discovered that dsRNAs having an antisense strand containing at least one thermodestabilizing modification of the duplex within the first 9 nucleotide positions counting from the 5' end of the antisense strand have reduced off-target gene silencing activity. Thus, in some embodiments, the antisense strand contains at least one (e.g., one, two, three, four, five, or more) thermodestabilizing modification of the duplex within the first 9 nucleotide positions of the 5' region of the antisense strand. In some embodiments, the thermodestabilizing modification of one or more duplexes is located at positions 2-9, or preferably 4-8, from the 5' end of the antisense strand. In some further embodiments, the thermodestabilizing modification of the duplex is located at positions 6, 7, or 8 from the 5' end of the antisense strand. In some further embodiments, the thermally destabilizing modification of the duplex is located at position 7 from the 5' end of the antisense strand. The term "thermally destabilizing modification" includes modifications that result in a dsRNA having a lower overall melting temperature (Tm), preferably one, two, three or four degrees lower than the melting temperature (Tm) of a dsRNA that does not have such a modification. In some embodiments, the thermally destabilizing modification of the duplex is located at position 2, 3, 4, 5 or 9 from the 5' end of the antisense strand.

[0395] Thermally destabilizing modifications can include, but are not limited to, abasic modifications, mismatches with the opposing nucleotide in the opposing strand, and sugar modifications such as 2'-deoxy modifications, acyclic nucleotides such as unlocked nucleic acids (UNAs) or glycol nucleic acids (GNAs), and 2'-5'-linked ribonucleotides ("3'-RNAs").

[0396] Exemplary abasic modifications include, but are not limited to, the following: [ka] In the formula, R=H, Me, Et or OMe, R'=H, Me, Et or OMe, R''=H, Me, Et or OMe [ka] wherein B is a modified or unmodified nucleobase.

[0397] Exemplary sugar modifications include, but are not limited to: [ka] wherein B is a modified or unmodified nucleobase.

[0398] In some embodiments, the thermally destabilizing modification of the duplex is selected from the group consisting of: [ka] where B is a modified or unmodified nucleobase, and the asterisk on each structure represents either R, S, or racemic.

[0399] In some embodiments, the thermally destabilizing modification of the duplex is selected from the group consisting of: [ka] wherein B is a modified or unmodified nucleobase and the asterisk represents either R, S or racemic (e.g., S).

[0400] The term "acyclic nucleotide" refers to any nucleotide having an acyclic ribose sugar, for example, one in which any of the bonds of the ribose carbon ring (e.g., C1'-C2', C2'-C3', C3'-C4', C4'-O4', or C1'-O4') are absent, 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] B is a modified or unmodified nucleobase; R 1 and R 2 are independently H, halogen, OR3, or alkyl, and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar. The term "UNA" refers to an acyclic unlocked nucleic acid in which any of the sugar bonds has been removed to form an unlocked "sugar" residue. In one embodiment, UNA also encompasses monomers in which the C1'-C4' bond has been removed (i.e., a carbon-oxygen-carbon covalent bond between the C1' and C4' carbons). In another embodiment, the C2'-C3' bond of the sugar (i.e., a 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), which are incorporated herein by reference in their entireties). Acyclic derivatives offer greater backbone flexibility without affecting Watson-Crick pairing. Acyclic nucleotides can be linked via 2'-5' or 3'-5' linkages.

[0401] The term "GNA" refers to glycol nucleic acid, which is a polymer similar to DNA or RNA, but differs in the composition of its "backbone" in that it is made up of repeating glycerol units linked by phosphodiester bonds. [ka]

[0402] The thermally destabilizing modification of the duplex can be a mismatch (i.e., non-complementary base pair) between the thermally destabilizing nucleotide and the opposite nucleotide in the opposite strand of 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 a combination thereof.Other mismatch base pairings known in the art are also contemplated by the present invention.Mismatches can occur between nucleotides that are either naturally occurring or modified nucleotides, i.e., mismatch base pairing can occur between the nucleobases derived from each nucleotide, regardless of the modification on the ribose sugar of the nucleotide.In certain embodiments, the dsRNA molecule contains at least one nucleobase in the mismatch pairing that is a 2'-deoxynucleobase, for example, the 2'-deoxynucleobase is in the sense strand.

[0403] In some embodiments, the thermodestabilizing modifications of the duplex in the seed region of the antisense strand include nucleotides that impair WCH bonding with complementary bases on the target mRNA, such as: [ka]

[0404] Many examples of abasic nucleotides, acyclic nucleotide modifications (including UNA and GNA) and mismatch modifications are described in detail in WO 2011 / 133876, which is incorporated herein by reference in its entirety.

[0405] Thermodestabilizing modifications can also include universal base and phosphate modifications that have reduced or eliminated ability to form hydrogen bonds with opposing bases.

[0406] In some embodiments, the thermal destabilizing modification of duplex comprises the nucleotide with non-standard base, for example but not limited to, the nucleobase modification that the ability to form hydrogen bond with the base in the opposite strand is impaired or completely lost.These nucleobase modifications have been evaluated for destabilizing the central region of dsRNA duplex, as described in International Publication No. 2010 / 0011895, the entire contents of which are incorporated herein by reference.Exemplary nucleobase modifications include: [ka]

[0407] In some embodiments, the thermodestabilizing modifications of the duplex in the seed region of the antisense strand include one or more α-nucleotides that are complementary to bases on the target mRNA, such as: [ka] wherein R is H, OH, OCH3, F, NH2, NHMe, NMe2 or O-alkyl.

[0408] Exemplary phosphate modifications that have been shown to decrease the thermal stability of dsRNA duplexes, relative to natural phosphodiester linkages, include the following: [ka]

[0409] The alkyl R group can be a C1-C6 alkyl. Specific alkyl R groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, pentyl, and hexyl.

[0410] As those skilled in the art will recognize, considering that the functional role of nucleobase defines the specificity of the RNAi agent of the present disclosure, nucleobase modification can be carried out in various ways as described herein, for example, for the purpose of enhancing on-target effect against off-target effect, for example, introducing destabilizing modification into the RNAi agent of the present disclosure, and generally the range of modifications present on the RNAi agent of the present disclosure tends to be much greater than the non-nucleobase modifications, for example, the modification of the sugar group or phosphate backbone of polyribonucleotide.Such modifications will be described in more detail in other sections of the present disclosure, and are expressly intended for the RNAi agent of the present disclosure that has either natural nucleobase or modified nucleobase as described above or elsewhere herein.

[0411] In addition to the antisense strand containing a thermally destabilizing modification, the dsRNA may also contain one or more stabilizing modifications. For example, the dsRNA may contain at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) stabilizing modifications. Without limitation, all of the stabilizing modifications may be present in one strand. In some embodiments, both the sense and antisense strands contain at least two stabilizing modifications. The stabilizing modifications may occur at any nucleotide in either the sense or antisense strand. For example, the stabilizing modifications may occur at any nucleotide in the sense or antisense strand, and each stabilizing modification may occur in an alternating pattern on the sense or antisense strand, or the sense or antisense strand may contain both stabilizing modifications in an alternating pattern. The alternating pattern of stabilizing modifications on the sense strand may be the same as or different from that of the antisense strand, and the alternating pattern of stabilizing modifications on the sense strand may be shifted relative to the alternating pattern of stabilizing modifications on the antisense strand.

[0412] In some embodiments, the antisense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) stabilizing modifications. Without limitation, the stabilizing modifications within the antisense strand can be located at any position. In some embodiments, the antisense strand comprises stabilizing modifications at positions 2, 6, 8, 9, 14, and 16 from the 5' end. In some other embodiments, the antisense strand comprises stabilizing modifications at positions 2, 6, 14, and 16 from the 5' end. In yet other embodiments, the antisense strand comprises stabilizing modifications at positions 2, 14, and 16 from the 5' end.

[0413] In some embodiments, the antisense strand comprises at least one stabilizing modification adjacent to the destabilizing modification.For example, the stabilizing modification can be at the 5'-end or 3'-end of the destabilizing modification, i.e., at the -1 or +1 position from the position of the destabilizing modification, nucleotide.In some embodiments, the antisense strand comprises a stabilizing modification at each of the 5'-end and 3'-end of the destabilizing modification, i.e., at the -1 and +1 positions from the position of the destabilizing modification.

[0414] In some embodiments, the antisense strand comprises at least two stabilizing modifications at the 3' end of the destabilizing modification, ie, at positions +1 and +2 from the position of the destabilizing modification.

[0415] In some embodiments, the sense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) stabilizing modifications. Without limitation, the stabilizing modifications within the sense strand can be located at any position. In some embodiments, the sense strand comprises stabilizing modifications at positions 7, 10, and 11 from the 5' end. In some other embodiments, the sense strand comprises stabilizing modifications at positions 7, 9, 10, and 11 from the 5' end. In some embodiments, the sense strand comprises stabilizing modifications at positions opposite or complementary to positions 11, 12, and 15 of the antisense strand, counting from the 5' end of the antisense strand. In some other embodiments, the sense strand comprises stabilizing modifications at positions opposite or complementary to positions 11, 12, 13, and 15 of the antisense strand, counting from the 5' end of the antisense strand. In some embodiments, the sense strand comprises two, three, or four blocks of stabilizing modifications.

[0416] In some embodiments, the sense strand does not contain a stabilizing modification at a position opposite or complementary to a thermally destabilizing modification of the duplex in the antisense strand.

[0417] Exemplary thermostabilizing modifications include, but are not limited to, 2'-fluoro modifications. Other thermostabilizing modifications include, but are not limited to, LNA.

[0418] In some embodiments, the dsRNA of the present disclosure contains at least four (e.g., four, five, six, seven, eight, nine, ten or more) 2'-fluoro nucleotides. Without limitation, all of the 2'-fluoro nucleotides can be present in one strand. In some embodiments, both the sense and antisense strands contain at least two 2'-fluoro nucleotides. The 2'-fluoro modification can occur on either the sense strand or the antisense strand's nucleotide. For example, the 2'-fluoro modification can occur on any nucleotide on the sense strand or the antisense strand, and each 2'-fluoro modification can occur in an alternating pattern on the sense strand or the antisense strand, or both the sense strand and the antisense strand contain 2'-fluoro modifications in an alternating pattern. The alternating pattern of 2'-fluoro modifications on the sense strand can be the same as or different from that of the antisense strand, and the alternating pattern of 2'-fluoro modifications on the sense strand can be shifted relative to the alternating pattern of 2'-fluoro modifications on the antisense strand.

[0419] In some embodiments, the antisense strand contains at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) 2'-fluoro nucleotides. Without limitation, the 2'-fluoro modifications in the antisense strand can be located at any position. In some embodiments, the antisense strand contains 2'-fluoro nucleotides at positions 2, 6, 8, 9, 14, and 16 from the 5' end. In some other embodiments, the antisense strand contains 2'-fluoro nucleotides at positions 2, 6, 14, and 16 from the 5' end. In yet other embodiments, the antisense strand contains 2'-fluoro nucleotides at positions 2, 14, and 16 from the 5' end.

[0420] In some embodiments, the antisense strand comprises at least one 2'-fluoronucleotide adjacent to the destabilizing modification. For example, the 2'-fluoronucleotide can be at the 5' or 3' end of the destabilizing modification, i.e., at the -1 or +1 position from the position of the destabilizing modification. In some embodiments, the antisense strand comprises a 2'-fluoronucleotide at each of the 5' and 3' ends of the destabilizing modification, i.e., at the -1 and +1 positions from the position of the destabilizing modification.

[0421] In some embodiments, the antisense strand comprises at least two 2'-fluoro nucleotides at the 3' end of the destabilizing modification, ie, at positions +1 and +2 from the position of the destabilizing modification.

[0422] In some embodiments, the sense strand contains at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) 2'-fluoro nucleotides. Without limitation, the 2'-fluoro modifications within the sense strand can be present at any position. In some embodiments, the antisense strand contains 2'-fluoro nucleotides at positions 7, 10, and 11 from the 5'-end. In some other embodiments, the sense strand contains 2'-fluoro nucleotides at positions 7, 9, 10, and 11 from the 5'-end. In some embodiments, the sense strand contains 2'-fluoro nucleotides at positions opposite or complementary to positions 11, 12, and 15 of the antisense strand, counting from the 5'-end of the antisense strand. In some other embodiments, the sense strand contains 2'-fluoro nucleotides at positions opposite or complementary to positions 11, 12, 13, and 15 of the antisense strand, counting from the 5'-end of the antisense strand. In some embodiments, the sense strand contains two, three, or four blocks of 2'-fluoro nucleotides.

[0423] In some embodiments, the sense strand does not contain a 2'-fluoro nucleotide at a position opposite or complementary to a thermally destabilizing modification of the duplex in the antisense strand.

[0424] In some embodiments, a dsRNA molecule of the disclosure comprises a 21-nucleotide (nt) sense strand and a 23-nucleotide (nt) antisense strand, wherein the antisense strand contains at least one thermolabile nucleotide, the at least one thermolabile nucleotide occurring within the seed region of the antisense strand (positions 2-9 of the 5' end of the antisense strand), one end of the dsRNA is blunt while the other end comprises a 2-nt overhang, and the dsRNA optionally further comprises at least one (e.g., one, two, three, four, five, six, or all seven) of the following features: (i) the antisense strand comprises two, three, four, five, or six 2'-fluoro modifications; (ii) the antisense strand comprises one, two, three, four, or five phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated to a ligand; or (iv) the sense strand comprises two, three, four, or five 2'-fluoro modifications. (v) the sense strand contains 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages, (vi) the dsRNA contains at least four 2'-fluoro modifications, and (vii) the dsRNA contains a blunt end at the 5' end of the antisense strand. Preferably, a 2-nt overhang is at the 3' end of the antisense strand.

[0425] In some embodiments, a dsRNA molecule of the present disclosure comprises a sense strand and an antisense strand, wherein the sense strand is 25 to 30 nucleotide residues in length and, starting from the 5'-terminal nucleotide (position 1), comprises at least 8 ribonucleotides at positions 1 to 23 of the sense strand; the antisense strand is 36 to 66 nucleotide residues in length and, starting from the 3'-terminal nucleotide, at least 8 ribonucleotides at these positions are paired with positions 1 to 23 of the sense strand to form a duplex; 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 1-6 nucleotide 3' single-stranded overhang; the 5'-end of the sense strand is composed of 10 to 30 contiguous nucleotides that are not paired with the sense strand, thereby forming a 10 to 30 nucleotide single-stranded 5' overhang; at least the 5'- and 3'-terminal nucleotides of the sense strand base-pair with nucleotides of the antisense strand when the sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially duplex region between the sense and antisense strands; the antisense strand is sufficiently complementary to the target RNA along at least 19 ribonucleotides of the length of the antisense strand to reduce target gene expression when the double-stranded nucleic acid is introduced into a mammalian cell; and the antisense strand contains at least one thermolabile nucleotide, the at least one thermolabile nucleotide being within the seed region of the antisense strand (i.e., at positions 2 to 9 of the 5'-end of the antisense strand).For example, the heat-destabilizing nucleotide occurs between positions 14-17 opposite or complementary to positions 14-17 of the 5' end of the sense strand, and the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six, or all seven) of the following characteristics: (i) the antisense strand contains two, three, four, five, or six 2'-fluoro modifications; (ii) the antisense strand contains one, two, three, four, or five phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand contains two, three, four, or five 2'-fluoro modifications; (v) the sense strand contains one, two, three, four, or five phosphorothioate internucleotide linkages; and (vi) the dsRNA contains at least four 2'-fluoro modifications; and (vii) the dsRNA contains a duplex region 12-30 nucleotide pairs in length.

[0426] In some embodiments, a dsRNA molecule of the present disclosure comprises a sense strand and an antisense strand, the dsRNA molecule comprising a sense strand having a length of at least 25 nucleotides and at most 29 nucleotides, and an antisense strand having a length of at most 30 nucleotides, the sense strand comprising a modified nucleotide at position 11 from the 5' end that is susceptible to enzymatic degradation, the 3' end of the sense strand and the 5' end of the antisense strand forming a blunt end, and the antisense strand is 1 to 4 nucleotides longer at its 3' end than the sense strand, the duplex region being at least 25 nucleotides in length, and the antisense strand being sufficiently complementary to a target mRNA along at least 19 nt of the antisense strand, such that the dsRNA molecule reduces target gene expression when introduced into mammalian cells, and Dicer cleavage of the dsRNA preferentially results in an siRNA comprising the 3' end of the antisense strand, thereby enabling expression of the dsRNA molecule in mammalian cells. wherein the antisense strand contains at least one thermolabile nucleotide, the at least one thermolabile nucleotide being located within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), and the dsRNA optionally further has at least one of the following features (e.g., one, two, three, four, five, six, or all seven): (i) the antisense strand contains two, three, four, five, or six 2'-fluoro modifications; (ii) the antisense strand contains at least one thermolabile nucleotide; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand contains two, three, four, or five 2'-fluoro modifications; (v) the sense strand contains one, two, three, four, or five phosphorothioate internucleotide linkages; and (vi) the dsRNA contains at least four 2'-fluoro modifications; and (vii) the dsRNA has a duplex region 12 to 29 nucleotide pairs in length.

[0427] In some embodiments, any nucleotide in the sense strand and antisense strand of dsRNA molecule can be modified.Each nucleotide can be modified with the same or different modifications, and these modifications can include one or more of the non-linked phosphate oxygen or both, or one or more of the linked phosphate oxygen, the modification of ribose sugar components, for example, the modification of the 2' hydroxyl on ribose sugar, the large-scale replacement of phosphate moiety with " dephosphorylation " linker, the modification or replacement of naturally occurring base, and the replacement or modification of ribose phosphate backbone.

[0428] Because nucleic acids are polymers of subunits, many modifications occur at positions that are repeated within nucleic acids, such as modifications of bases or phosphate moieties, or unlinked Os in phosphate moieties.In some cases, modifications occur at all target positions in nucleic acids, but in many cases, they do not occur.For example, modifications can occur only at the 3'-end or 5'-end positions, or only in terminal regions, such as at the terminal nucleotide position of the chain, or at the last 2, 3, 4, 5, or 10 nucleotides of the chain.Modifications can occur in double-stranded regions, single-stranded regions, or both.Modifications can occur only in the double-stranded region of RNA, or only in the single-stranded region of RNA.For example, phosphorothioate modifications at unlinked O positions can occur only at one or both ends, or only in terminal regions, such as at the terminal nucleotide position of the chain or within the last 2, 3, 4, 5, or 10 nucleotides, or in double-stranded and single-stranded regions, especially at the ends. The 5' end can be phosphorylated.

[0429] This may allow, for example, to enhance stability, include specific bases in the overhang, or include modified nucleotides or nucleotide substitutes in the single-stranded overhang, for example, in the 5' overhang or 3' overhang, or in both overhangs. For example, it may be desirable to include purine nucleotides in the overhang. In some embodiments, all or some of the bases in the 3' overhang or 5' overhang can be modified, for example, with the modifications described herein. Modifications can include, for example, the use of modifications at the 2' position of the ribose sugar, with modifications known in the art, for example, the use of modified deoxyribonucleotides, 2'-deoxy-2'-fluoro (2'-F) or 2'-O-methyl, instead of the ribosugar of the nucleobase, and modifications at the phosphate group, for example, phosphorothioate modifications. The overhang does not need to be homologous to the target sequence.

[0430] In some embodiments, each residue of the sense strand and the antisense strand is independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, or 2'-fluoro. These strands may contain two or more modifications. In some embodiments, each residue of the sense strand and the antisense strand is independently modified with 2'-O-methyl or 2'-fluoro. It should be understood that these modifications are in addition to at least one thermostabilizing modification of the duplex present in the antisense strand.

[0431] At least two different modifications are typically present on the sense strand and the antisense strand. These modifications can be 2'-deoxy, 2'-O-methyl, or 2'-fluoro modifications, acyclic nucleotides, etc. In some embodiments, the sense strand and the antisense strand each contain two differently modified nucleotides selected from 2'-O-methyl or 2'-deoxy. In some embodiments, each residue in the sense strand and the antisense strand is independently modified with a 2'-O-methyl nucleotide, a 2'-deoxy nucleotide, a 2'-deoxy-2'-fluoro nucleotide, a 2'-ON-methylacetamide (2'-O-NMA) nucleotide, a 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) nucleotide, a 2'-O-aminopropyl (2'-O-AP) nucleotide, or a 2'-ara-F nucleotide. Again, it should be understood that these modifications are in addition to at least one thermostabilizing modification of the duplex present in the antisense strand.

[0432] In some embodiments, the dsRNA molecules of the present disclosure contain an alternating pattern of modifications, particularly in the B1, B2, B3, B1', B2', B3', and B4' regions. The term "alternating motif" or "alternating pattern," as used herein, refers to a motif having one or more modifications, each occurring on alternating nucleotides in a single strand. The alternating nucleotides may refer to one every other nucleotide, one every three nucleotides, or similar patterns. For example, if A, B, and C each represent a type of modification to a nucleotide, the alternating motif may be "ABABABABABAB...," "AABBAABBAABB...," "AABAABAABAAB...," "AAABAAABAAAB...," "AAABBBAAABBB...," or "ABCABCABCABC...," etc.

[0433] The types of modifications contained within an alternating motif can be the same or different. For example, if A, B, C, and D each represent one type of modification on a nucleotide, the alternation pattern, i.e., the modifications on every other nucleotide, can be the same, but each of the sense or antisense strands can be selected from several possible modifications within the alternating motif, such as "ABABAB...", "ACACAC...", "BDBDBD...", or "CDCDCD...".

[0434] In some embodiments, dsRNA molecules of the present disclosure include an alternating motif modification pattern on the sense strand that is shifted relative to the alternating motif modification pattern on the antisense strand. The shift can be such that modified groups on nucleotides in the sense strand correspond to differently modified groups on nucleotides in the antisense strand, or vice versa. For example, when the sense strand is paired with the antisense strand in a dsRNA duplex, the alternating motif in the sense strand can begin with "ABABAB" from the 5'-3' end of the strand, and the alternating motif in the antisense strand can begin with "BABABA" from the 3'-5' end of the strand in the duplex region. As another example, the alternating motif in the sense strand can begin with "AABBAABB" from the 5'-3' end of the strand, and the alternating motif in the antisense strand can begin with "BBAABBAA" from the 3'-5' end of the strand in the duplex region, resulting in a complete or partial shift in the modification pattern between the sense and antisense strands.

[0435] In one particular embodiment, the alternating motif in the sense strand is "ABABAB" from 5'-3' of the strand, where each A is an unmodified ribonucleotide and each B is a 2'-O methyl modified nucleotide.

[0436] In one particular embodiment, the alternating motif in the sense strand is "ABABAB" from 5'-3' of the strand, where each A is a 2'-deoxy-2'-fluoro modified nucleotide and each B is a 2'-O methyl modified nucleotide.

[0437] In another specific embodiment, the alternating motif in the antisense strand is "BABABA" from 3'-5' of the strand, where each A is a 2'-deoxy-2'-fluoro modified nucleotide and each B is a 2'-O methyl modified nucleotide.

[0438] In one particular embodiment, the alternating motif in the sense strand is "ABABAB" from 5'-3' of that strand and the alternating motif in the antisense strand is "BABABA" from 3'-5' of that strand, where each A is an unmodified ribonucleotide and each B is a 2'-O methyl modified nucleotide.

[0439] In one particular embodiment, the alternating motif in the sense strand is "ABABAB" from 5'-3' of the strand and the alternating motif in the antisense strand is "BABABA" from 3'-5' of the strand, where each A is a 2'-deoxy-2'-fluoro modified nucleotide and each B is a 2'-O methyl modified nucleotide.

[0440] The dsRNA molecule of the present disclosure can further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage.Phosphorothioate or methylphosphonate internucleotide linkage modification can occur on the nucleotide of either sense strand or antisense strand or both strands at any position of the strand.For example, internucleotide linkage modification can occur on any nucleotide on sense strand or antisense strand, each internucleotide linkage modification can occur in an alternating pattern on sense strand or antisense strand, or sense strand or antisense strand contains both internucleotide linkage modifications in an alternating pattern.The alternating pattern of internucleotide linkage modification on sense strand can be the same or different from that of antisense strand, and the alternating pattern of internucleotide linkage modification on sense strand can be shifted relative to the alternating pattern of internucleotide linkage modification on antisense strand.

[0441] In some embodiments, the dsRNA molecule comprises phosphorothioate or methylphosphonate internucleotide linkage modification in the overhang region.For example, the overhang region comprises two nucleotides, and the two nucleotides comprise two nucleotides with phosphorothioate or methylphosphonate internucleotide linkage between the two nucleotides.Internucleotide linkage modification can also be made to link the overhang nucleotide to the terminal paired nucleotide in the double-stranded region.For example, at least 2, 3, 4 or all of the overhang nucleotides can be linked by phosphorothioate or methylphosphonate internucleotide linkage, and optionally, there can be an additional phosphorothioate or methylphosphonate internucleotide linkage connecting the overhang nucleotide to the paired nucleotide adjacent to the overhang nucleotide.For example, there can be at least two phosphorothioate internucleotide linkages between the terminal three nucleotides, such that two of the three nucleotides are overhang nucleotides and the third is the paired nucleotide adjacent to the overhang nucleotide.Preferably, these terminal three nucleotides can be at the 3'-end of the antisense strand.

[0442] In some embodiments, the sense strand of a dsRNA molecule comprises 1 to 10 blocks of two to ten phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate internucleotide linkages, one of which is positioned at any position within the oligonucleotide sequence, and the sense strand is paired with an antisense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0443] In some embodiments, the antisense strand of a dsRNA molecule comprises two blocks of two phosphorothioate or methylphosphonate 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, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is positioned at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0444] In some embodiments, the antisense strand of a dsRNA molecule comprises two blocks of three phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate internucleotide linkages, one of which is positioned at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0445] In some embodiments, the antisense strand of a dsRNA molecule comprises two blocks of four phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 phosphate internucleotide linkages, one of which is positioned at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0446] In some embodiments, the antisense strand of a dsRNA molecule comprises two blocks of five phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 phosphate internucleotide linkages, one of which is positioned at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0447] In some embodiments, the antisense strand of a dsRNA molecule comprises two blocks of six phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphate internucleotide linkages, one of which is positioned at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0448] In some embodiments, the antisense strand of a dsRNA molecule comprises two blocks of seven phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, or 8 phosphate internucleotide linkages, one of which is positioned at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0449] In some embodiments, the antisense strand of a dsRNA molecule comprises two blocks of eight phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, or 6 phosphate internucleotide linkages, one of which is positioned at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or with an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0450] In some embodiments, the antisense strand of a dsRNA molecule comprises two blocks of nine phosphorothioate or methylphosphonate internucleotide linkages separated by one, two, three, or four phosphate internucleotide linkages, one of which is positioned at any position within the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or with an antisense strand comprising either phosphorothioate, methylphosphonate, or phosphate linkages.

[0451] In some embodiments, dsRNA molecules of this disclosure further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 1 to 10 of the termini of the sense or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides can be linked via phosphorothioate or methylphosphonate internucleotide linkages at one or both termini of the sense or antisense strand.

[0452] In some embodiments, the dsRNA molecules of the present disclosure further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within 1-10 of the internal region of the duplex of each of the sense or antisense strands. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides can be linked via phosphorothioate methylphosphonate internucleotide linkage modifications at positions 8-16 of the duplex region, counting from the 5' end of the sense strand, and the dsRNA molecule can optionally further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 1-10 of that end.

[0453] In some embodiments, dsRNA molecules of the disclosure comprise one to five phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) of the sense strand and one to five phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 18-23 of the sense strand, and one to five phosphorothioate or methylphosphonate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) of the antisense strand and one to five within positions 18-23 of the antisense strand.

[0454] In some embodiments, dsRNA molecules of the disclosure further comprise one phosphorothioate internucleotide linkage modification within positions 1-5 (counting from the 5' end) and one phosphorothioate or methylphosphonate internucleotide linkage modification within positions 18-23 of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 (counting from the 5' end) and two phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 18-23 of the antisense strand.

[0455] In some embodiments, dsRNA molecules of the disclosure further comprise two phosphorothioate internucleotide linkage modifications within the range of positions 1-5 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within the range of positions 18-23 of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within the range of positions 18-23 of the antisense strand.

[0456] In some embodiments, dsRNA molecules of the disclosure further comprise two phosphorothioate internucleotide linkage modifications within the range of positions 1 to 5 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within the range of positions 18 to 23 of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within the range of positions 18 to 23 of the antisense strand.

[0457] In some embodiments, dsRNA molecules of the disclosure further comprise two phosphorothioate internucleotide linkage modifications within the range of positions 1 to 5 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within the range of positions 18 to 23 of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within the range of positions 18 to 23 of the antisense strand.

[0458] In some embodiments, dsRNA molecules of the disclosure further comprise one phosphorothioate internucleotide linkage modification within the range of positions 1-5 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within the range of positions 18-23 of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within the range of positions 18-23 of the antisense strand (counting from the 5' end).

[0459] In some embodiments, dsRNA molecules of the disclosure further comprise one phosphorothioate internucleotide linkage modification within the range of positions 1-5 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within the range of positions 18-23 of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within the range of positions 18-23 of the antisense strand.

[0460] In some embodiments, dsRNA molecules of the present disclosure further comprise one phosphorothioate internucleotide linkage modification within the range of positions 1 to 5 (counting from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within the range of positions 18 to 23 (counting from the 5' end) of the antisense strand.

[0461] In some embodiments, dsRNA molecules of the disclosure further comprise two phosphorothioate internucleotide linkage modifications within the range of positions 1 to 5 (counting from the 5' end) of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within the range of positions 18 to 23 (counting from the 5' end) of the antisense strand.

[0462] In some embodiments, dsRNA molecules of the disclosure further comprise two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) and one within positions 18-23 of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand.

[0463] In some embodiments, dsRNA molecules of the disclosure further comprise two phosphorothioate internucleotide linkage modifications within the range of positions 1 to 5 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within the range of positions 18 to 23 of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within the range of positions 18 to 23 of the antisense strand.

[0464] In some embodiments, dsRNA molecules of the disclosure further comprise two phosphorothioate internucleotide linkage modifications within the range of positions 1-5 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within the range of positions 18-23 of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within the range of positions 18-23 of the antisense strand.

[0465] In some embodiments, the dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications at positions 20 and 21 of the sense strand, and one phosphorothioate internucleotide linkage modification at position 1 and one at position 21 (counting from the 5' end) of the antisense strand.

[0466] In some embodiments, dsRNA molecules of the disclosure further comprise one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 20 and 21 (counting from the 5' end) of the antisense strand.

[0467] In some embodiments, dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications at positions 21 and 22 of the sense strand, and one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the antisense strand.

[0468] In some embodiments, dsRNA molecules of the disclosure further comprise one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 21 and 22 (counting from the 5' end) of the antisense strand.

[0469] In some embodiments, dsRNA molecules of the disclosure further comprise two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications at positions 22 and 23 of the sense strand, and one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the antisense strand.

[0470] In some embodiments, dsRNA molecules of the disclosure further comprise one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 (counting from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 23 and 23 (counting from the 5' end) of the antisense strand.

[0471] In some embodiments, the compounds of the present disclosure comprise a pattern of backbone chiral centers. In some embodiments, the regular pattern of backbone chiral centers comprises at least 5 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 6 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 7 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 8 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 9 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 10 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 11 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 12 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 13 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 14 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 15 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 16 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 17 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 18 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises at least 19 internucleotide linkages in the Sp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises 8 or fewer internucleotide linkages in the Rp configuration.In some embodiments, the regular pattern of backbone chiral centers comprises 7 or fewer internucleotide linkages in the Rp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises 6 or fewer internucleotide linkages in the Rp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises 5 or fewer internucleotide linkages in the Rp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises 4 or fewer internucleotide linkages in the Rp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises 3 or fewer internucleotide linkages in the Rp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises 2 or fewer internucleotide linkages in the Rp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises 1 or fewer internucleotide linkages in the Rp configuration. In some embodiments, the regular pattern of backbone chiral centers comprises 8 or fewer non-chiral internucleotide linkages (phosphodiesters as a non-limiting example). In some embodiments, the regular pattern of backbone chiral centers comprises 7 or fewer non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises 6 or fewer non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises 5 or fewer non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises 4 or fewer non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises 3 or fewer non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises 2 or fewer non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises 1 or fewer non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises at least 10 internucleotide linkages in the Sp configuration and 8 or fewer non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises at least 11 internucleotide linkages in the Sp configuration and 7 or fewer non-chiral internucleotide linkages.In some embodiments, the regular pattern of backbone chiral centers comprises at least 12 internucleotide linkages in the Sp configuration and no more than 6 non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises at least 13 internucleotide linkages in the Sp configuration and no more than 6 non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises at least 14 internucleotide linkages in the Sp configuration and no more than 5 non-chiral internucleotide linkages. In some embodiments, the regular pattern of backbone chiral centers comprises at least 15 internucleotide linkages in the Sp configuration and no more than 4 non-chiral internucleotide linkages. In some embodiments, the internucleotide linkages in the Sp configuration may be contiguous or non-contiguous. In some embodiments, the internucleotide linkages in the Rp configuration may be contiguous or non-contiguous. In some embodiments, the non-chiral internucleotide linkages may be contiguous or non-contiguous.

[0472] In some embodiments, compounds of the present disclosure include blocks that are stereochemical blocks. In some embodiments, a block is an Rp block, in that each internucleotide linkage of the block is Rp. In some embodiments, the 5'-block is an Rp block. In some embodiments, the 3'-block is an Rp block. In some embodiments, a block is an Sp block, in that each internucleotide linkage of the block is Sp. In some embodiments, the 5'-block is an Sp block. In some embodiments, the 3'-block is an Sp block. In some embodiments, provided oligonucleotides include both an Rp block and an Sp block. In some embodiments, provided oligonucleotides include one or more Rp blocks but no Sp blocks. In some embodiments, provided oligonucleotides include one or more Sp blocks but no Rp blocks. In some embodiments, provided oligonucleotides include one or more PO blocks, where each internucleotide linkage is a natural phosphate linkage.

[0473] In some embodiments, compounds of the present disclosure include a 5'-block that is an Sp block in which each sugar moiety comprises a 2'-F modification. In some embodiments, the 5'-block is an Sp block in which each internucleotide linkage is a modified internucleotide linkage and each sugar moiety comprises a 2'-F modification. In some embodiments, the 5'-block is an Sp block in which each internucleotide linkage is a phosphorothioate linkage and each sugar moiety comprises a 2'-F modification. In some embodiments, the 5'-block comprises four or more nucleoside units. In some embodiments, the 5'-block comprises five or more nucleoside units. In some embodiments, the 5'-block comprises six or more nucleoside units. In some embodiments, the 5'-block comprises seven or more nucleoside units. In some embodiments, the 3'-block is an Sp block in which each sugar moiety comprises a 2'-F modification. In some embodiments, the 3'-block is an Sp block, in which each of the internucleotide linkages is a modified internucleotide linkage and each sugar moiety comprises a 2'-F modification. In some embodiments, the 3'-block is an Sp block, in which each of the internucleotide linkages is a phosphorothioate linkage and each sugar moiety comprises a 2'-F modification. In some embodiments, the 3'-block comprises 4 or more nucleoside units. In some embodiments, the 3'-block comprises 5 or more nucleoside units. In some embodiments, the 3'-block comprises 6 or more nucleoside units. In some embodiments, the 3'-block comprises 7 or more nucleoside units.

[0474] In some embodiments, compounds of the disclosure include a region of nucleosides or oligonucleotides followed by a particular type of internucleotide linkage, such as a natural phosphate linkage, a modified internucleotide linkage, an Rp chiral internucleotide linkage, an Sp chiral internucleotide linkage, etc. In some embodiments, A is followed by Sp. In some embodiments, A is followed by Rp. In some embodiments, A is followed by a natural phosphate linkage (PO). In some embodiments, U is followed by Sp. In some embodiments, U is followed by Rp. In some embodiments, U is followed by a natural phosphate linkage (PO). In some embodiments, C is followed by Sp. In some embodiments, C is followed by Rp. In some embodiments, C is followed by a natural phosphate linkage (PO). In some embodiments, G is followed by Sp. In some embodiments, G is followed by Rp. In some embodiments, G is followed by a natural phosphate linkage (PO). In some embodiments, C and U are followed by Sp. In some embodiments, C and U are followed by Rp. In some embodiments, C and U are followed by a natural phosphate linkage (PO). In some embodiments, A and G are followed by Sp. In some embodiments, A and G are followed by Rp.

[0475] In some embodiments, the antisense strand comprises phosphorothioate internucleotide linkages between nucleotides 21 and 22 and between nucleotides 22 and 23, the antisense strand contains at least one thermodestabilizing modification of the duplex located within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), and the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six, seven, or all eight) of the following features: (i) the antisense strand contains two, three, four, five, or six 2'-fluoro modifications; (ii) the antisense strand comprises 3, 4, or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand comprises 2, 3, 4, or 5 2'-fluoro modifications; (v) the sense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (vi) the dsRNA comprises at least four 2'-fluoro modifications; (vii) the dsRNA comprises a duplex region 12 to 40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at the 5' end of the antisense strand.

[0476] In some embodiments, the antisense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23, the antisense strand contains at least one thermodestabilizing modification of the duplex located within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), and the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six, seven, or all eight) of the following features: (i) the antisense strand comprises two, three, (ii) the sense strand is conjugated to a ligand; (iii) the sense strand contains two, three, four, or five 2'-fluoro modifications; (iv) the sense strand contains one, two, three, four, or five phosphorothioate internucleotide linkages; (v) the dsRNA contains at least four 2'-fluoro modifications; (vi) the dsRNA contains a duplex region 12 to 40 nucleotide pairs in length; (vii) the dsRNA contains a duplex region 12 to 40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at the 5' end of the antisense strand.

[0477] In some embodiments, the sense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3, and the antisense strand contains at least one thermodestabilizing modification of the duplex located within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), and the dsRNA optionally further comprises at least one (e.g., one, two, three, four, five, six, seven, or all eight) of the following features: (i) the antisense strand comprises two, three, four, five, or six 2'-fluoro modifications; (i) the antisense strand comprises one, two, three, four, or five phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand comprises two, three, four, or five 2'-fluoro modifications; (v) the sense strand comprises three, four, or five phosphorothioate internucleotide linkages; (vi) the dsRNA comprises at least four 2'-fluoro modifications; (vii) the dsRNA comprises a duplex region 12 to 40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at the 5' end of the antisense strand.

[0478] In some embodiments, the sense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3, and the antisense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23, the antisense strand contains at least one thermodestabilizing modification of the duplex located within the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), and the dsRNA optionally has at least one of the following characteristics: (i) the antisense strand comprises two, three, four, five, six, or all seven of the following: (i) the antisense strand comprises two, three, four, five, six, or seven 2'-fluoro modifications; (ii) the sense strand is conjugated to a ligand; (iii) the sense strand comprises two, three, four, or five 2'-fluoro modifications; (iv) the sense strand comprises three, four, or five phosphorothioate internucleotide linkages; (v) the dsRNA comprises at least two 2'-fluoro modifications; (vi) the dsRNA comprises a duplex region 12 to 40 nucleotide pairs in length; and (vii) the dsRNA has a blunt end at the 5' end of the antisense strand.

[0479] In some embodiments, the dsRNA molecule of the present disclosure comprises mismatches in the duplex with the target, or a combination thereof. Mismatches can occur in the overhang region or the duplex region. Base pairs can be ranked based on their tendency to promote dissociation or melting (for example, based on the free energy of association or dissociation of a particular pairing; the simplest approach is to examine each pair individually, but then adjacent or similar analysis can also be used). In terms of promoting dissociation, A:U is more preferable than G:C, G:U is more preferable than G:C, and I:C is more preferable than G:C (I=inosine). Mismatches, such as non-standard pairings or non-standard pairings (described elsewhere herein), are more preferable than standard pairings (A:T, A:U, G:C), and pairings containing universal bases are more preferable than standard pairings.

[0480] In some embodiments, the dsRNA molecules of the present disclosure comprise at least one mismatch pair within the duplex region from the 5' end of the antisense strand, which may be independently selected from the group of A:U, G:U, I:C, and a non-canonical or non-standard or universal base pair, to promote dissociation of the antisense strand at the 5' end of the duplex.

[0481] In some embodiments, the nucleotide at position 1 from the 5' end of the antisense strand into the duplex region is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, at least one of the first one, two, or three base pairs from the 5' end of the antisense strand into the duplex region is an AU base pair. For example, the first base pair from the 5' end of the antisense strand into the duplex region is an AU base pair.

[0482] It has been found that the introduction of a 4'- or 5'-modified nucleotide at the 3' end of a dinucleotide phosphodiester (PO), phosphorothioate (PS), or phosphorodithioate (PS2) linkage at any position in a single- or double-stranded oligonucleotide can exert a steric effect on the internucleotide linkage, thereby protecting or stabilizing it against nucleases.

[0483] In some embodiments, 5'-modified nucleoside is introduced at the 3' end of dinucleotide at any position in single-stranded or double-stranded siRNA.For example, 5'-alkylated nucleoside can be introduced at the 3' end of dinucleotide at any position in single-stranded or double-stranded siRNA.The alkyl group at the 5' position of ribose sugar can be racemic or chirally pure R or S isomer.Exemplary 5'-alkylated nucleoside includes 5'-methyl nucleoside.5'-methyl can be racemic or chirally pure R or S isomer.

[0484] In some embodiments, 4'-modified nucleosides are introduced at the 3' end of dinucleotides at any position in single-stranded or double-stranded siRNA. For example, 4'-alkylated nucleosides can be introduced at the 3' end of dinucleotides at any position in single-stranded or double-stranded siRNA. The alkyl group at the 4' position of the ribose sugar can be racemic or chirally pure R or S isomer. An exemplary 4'-alkylated nucleoside is 4'-methyl nucleoside. 4'-methyl can be racemic or chirally pure R or S isomer. Alternatively, 4'-O-alkylated nucleosides can be introduced at the 3' end of dinucleotides at any position in single-stranded or double-stranded siRNA. The 4'-O-alkyl of the ribose sugar can be racemic or chirally pure R or S isomer. Exemplary 4'-O-alkylated nucleosides include 4'-O-methyl nucleosides, which can be either racemic or chirally pure R or S isomers.

[0485] In some embodiments, 5'-alkylated nucleoside is introduced at any position of the sense strand or antisense strand of dsRNA, and this modification maintains or improves the efficacy of dsRNA.5'-alkyl can be either racemic or chirally pure R or S isomer.Exemplary 5'-alkylated nucleoside includes 5'-methyl nucleoside.5'-methyl can be either racemic or chirally pure R or S isomer.

[0486] In some embodiments, 4'-alkylated nucleoside is introduced at any position of the sense strand or antisense strand of dsRNA, and this modification maintains or improves the efficacy of dsRNA.4'-alkyl can be either racemic or chiral pure R or S isomer.Exemplary 4'-alkylated nucleoside includes 4'-methyl nucleoside.4'-methyl can be either racemic or chiral pure R or S isomer.

[0487] In some embodiments, 4'-O-alkylated nucleoside is introduced at any position of the sense strand or antisense strand of dsRNA, and this modification maintains or improves the efficacy of dsRNA.5'-Alkyl can be either racemic or chirally pure R or S isomer.Exemplary 4'-O-alkylated nucleoside includes 4'-O-methyl nucleoside.4'-O-methyl can be either racemic or chirally pure R or S isomer.

[0488] In some embodiments, dsRNA molecules of the present disclosure may contain 2'-5' linkages (having 2'-H, 2'-OH, and 2'-OMe, and having P=O or P=S). For example, 2'-5' linkage modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5' end of the sense strand to prevent activation of the sense strand by RISC.

[0489] In another embodiment, the dsRNA molecules of the present disclosure can contain L sugars (e.g., L-ribose, L-arabinose, with 2'-H, 2'-OH, and 2'-OMe). For example, these L sugar modifications can be used to promote nuclease resistance, or to inhibit binding of the sense strand to the antisense strand, or can be used at the 5' end of the sense strand to prevent activation of the sense strand by RISC.

[0490] Multimeric siRNA has been described in various publications, and all of them can be used with the dsRNA of the present disclosure.These publications include WO2007 / 091269, US Patent No. 7858769, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887 and WO2011 / 031520, and each of them is incorporated herein by reference in its entirety.

[0491] As described in more detail below, RNAi agents containing one or more carbohydrate moieties conjugated to the RNAi agent can optimize one or more properties of the RNAi agent. Often, the carbohydrate moiety will be attached to a modified subunit of the RNAi agent. For example, the ribose sugar of one or more ribonucleotide subunits of a dsRNA agent can be replaced with another moiety, such as a non-carbohydrate (preferably cyclic) carrier to which a carbohydrate ligand is attached. A ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is referred to herein as a ribose-replacement modified subunit (RRMS). The cyclic carrier can be a carbon-cyclic system, i.e., all ring atoms are carbon atoms, or a heterocyclic system, i.e., one or more ring atoms can be heteroatoms, such as nitrogen, oxygen, or sulfur. The cyclic carrier can be a monocyclic ring system or can contain two or more rings, e.g., fused rings. The cyclic carrier can be a fully saturated ring system or can contain one or more double bonds.

[0492] Ligands can be attached to polynucleotides via carriers. The carriers contain (i) at least one "backbone attachment point," preferably two "backbone attachment points," and (ii) at least one "tethering attachment point." As used herein, a "backbone attachment point" refers to a functional group, e.g., a hydroxyl group, or generally a bond, available and suitable for incorporating a carrier into the backbone of a ribonucleic acid, e.g., a phosphate or modified phosphate backbone, e.g., sulfur-containing. In some embodiments, a "tethering attachment point" (TAP) refers to a ring atom, e.g., a carbon atom or heteroatom (other than the atom providing the backbone attachment point), of a cyclic carrier to which a selected moiety is attached. This moiety can be, for example, a carbohydrate, e.g., a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. Optionally, the selected moiety is connected to the cyclic carrier by an intervening tether. Thus, cyclic carriers often contain a functional group, e.g., an amino group, or generally a bond, that provides a bond suitable for incorporating or tethering another chemical entity, e.g., a ligand, to the ring.

[0493] The RNAi agent may be conjugated to the ligand via a carrier, which may 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.

[0494] In certain embodiments, the RNAi agent used in the methods of the present disclosure is an agent selected from the group of agents listed in any one of Tables 2 through 5. These agents may further comprise a ligand.

[0495] IV. iRNA conjugated to a ligand Another modification of the iRNA of the invention involves chemically linking the iRNA to one or more ligands, moieties, or conjugates that enhance the activity, cellular distribution, or cellular uptake, for example, of the iRNA. Such moieties include, but are not limited to, lipid moieties such as cholesterol moieties (Letsinger et al., Proc. Natl. Acids Sci. USA, 1989, 86:6553-6556), cholic acid (Manoharan et al., Bior. Med. Chem. Let., 1994, 4:1053-1060), thioethers such as beryl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306-309; Manoharan et al., Bior. Med. Chem. Let., 1993, 3:2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), fatty chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), phospholipids such as dihexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), palmityl moieties (Mishra et al., Biochim.Biophys. Acta, 1995, 1264:229-237), or octadecylamine or hexylamino-carbonyloxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0496] In certain embodiments, the ligand changes the distribution, targeting, or life span of the iRNA agent into which it is incorporated.In some embodiments, the ligand enhances the affinity of a selected target, for example, a molecule, a cell or cell type, a compartment, such as a cell or organ compartment, tissue, organ, or region of the body, for example, when compared with a species in which such a ligand is not present.Normal ligands do not participate in double-stranded pairing in double-stranded nucleic acids.

[0497] Ligands can include naturally occurring substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL) or globulins), carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid), or lipids. Ligands can also be recombinant or synthetic molecules, such as synthetic polymers, e.g., synthetic polyamino acids. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryl acid), N-isopropylacrylamide polymer, or polyphosphazine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimeric polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or α-helical peptides.

[0498] The ligand can also include a targeting group, such as a cell or tissue targeting agent, such as a lectin, a glycoprotein, lipid, or protein, such as an antibody, that binds to a specific cell type, such as a kidney cell. The targeting group can be thyroid-stimulating hormone, melanocyte-stimulating hormone, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, polyvalent fucose, glycosylated polyamino acid, polyvalent galactose, transferrin, bisphosphonate, polyglutamic acid, polyaspartic acid, lipid, cholesterol, steroid, bile acid, folic acid, vitamin B12, biotin, or an RGD peptide or RGD peptide mimic. In certain embodiments, the ligand is a polyvalent galactose, such as N-acetyl-galactosamine.

[0499] Other examples of ligands include dyes, intercalating agents (e.g., acridine), crosslinkers (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules such as cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutanoic acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithium, and the like. Examples of suitable nucleotides include hydroxybenzoates (e.g., hydroxybenzoates ...

[0500] Ligands can be proteins, such as glycoproteins or peptides, e.g., molecules with specific affinity for co-ligands, or antibodies, e.g., antibodies that bind to specific cell types, such as cancer cells, endothelial cells, or bone cells. Ligands can also include hormones and hormone receptors. They can also include non-peptide species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose, or multivalent fucose. Ligands can be, for example, lipopolysaccharides, p38 MAP kinase activators, or NF-κB activators.

[0501] The ligand can be a substance, e.g., a drug, that can increase the uptake of an iRNA agent into a cell, e.g., by disrupting the cytoskeleton of the cell, e.g., by disrupting the cell's microtubules, microfilaments, or intermediate filaments. The drug can be, e.g., taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0502] In some embodiments, the ligands that bind to the iRNAs described herein act as pharmacokinetic modulators (PK modulators). PK modulators include lipophilic substances, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, and the like. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, and the like. Oligonucleotides containing several phosphorothioate linkages have also been shown to bind to serum proteins, and therefore short oligonucleotides containing multiple phosphorothioate linkages in the backbone, e.g., oligonucleotides of about 5, 10, 15, or 20 bases, are also suitable as ligands (e.g., as PK-modulating ligands) for the present invention. Furthermore, aptamers that bind serum components (e.g., serum proteins) are also suitable for use as PK-modulating ligands in the embodiments described herein.

[0503] The ligand-conjugated iRNAs of the invention can be synthesized by using oligonucleotides bearing reactive functional pendant side chains, such as those derived from the attachment of a linking molecule onto the oligonucleotide (described below). The reactive oligonucleotides can be reacted directly with commercially available ligands, synthesized ligands bearing any of a variety of protecting groups, or ligands bearing a linking moiety attached thereto.

[0504] The oligonucleotides used in the conjugates of the present invention can be conveniently and routinely prepared by the well-known technique of solid-phase synthesis. Equipment for such synthesis is sold by several vendors, including, for example, Applied Biosystems® (Foster City, California). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is also known to use similar techniques to prepare other oligonucleotides, such as phosphorothioates and alkylated derivatives.

[0505] In the ligand-conjugated oligonucleotides and sequence-specific linked nucleosides bearing ligand molecules of the present invention, the oligonucleotides and oligonucleosides can be assembled on a suitable DNA synthesizer utilizing standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors already bearing a linking moiety, ligand-nucleotide or ligand-nucleoside conjugate precursors already bearing a ligand molecule, or non-nucleoside ligand-bearing building blocks.

[0506] When using a nucleotide-conjugate precursor that already possesses a linking moiety, synthesis of the sequence-specifically linked nucleoside is typically completed, and then a ligand molecule is reacted with the linking moiety to form the ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the invention are synthesized by automated synthesizers using phosphoramidites derived from ligand-nucleoside conjugates, in addition to commercially available standard and non-standard phosphoramidites routinely used in oligonucleotide synthesis.

[0507] A. Lipid Conjugates In certain embodiments, the ligand or conjugate is a lipid or lipid-based molecule. Such lipid or lipid-based molecule can usually bind serum protein, for example, human serum albumin (HSA). HSA-binding ligand allows the distribution of conjugate to target tissue in the body, for example, to target tissue other than the kidney. For example, the target tissue can be the liver, including the liver parenchymal cells. Other molecules that can bind HSA can also be used as ligand. For example, naproxen or aspirin can be used. Lipid or lipid-based ligand can (a) increase the resistance of conjugate to degradation, (b) increase targeting or transport into target cell or cell membrane, or (c) be used to adjust the binding to serum protein, for example, HSA.

[0508] Lipid-based ligands can be used to regulate, for example, control (for example, inhibit) the binding of conjugate to target tissue.For example, lipids or lipid-based ligands that bind more strongly to HSA are less likely to target the kidney, and therefore are less likely to be eliminated from the body.Lipids or lipid-based ligands that bind less strongly to HSA can be used so that the conjugate targets the kidney.

[0509] In certain embodiments, the lipid-based ligand binds HSA. For example, the ligand can bind to HSA with sufficient affinity to enhance distribution of the conjugate to non-renal tissues. However, this affinity is usually not so strong that the HSA-ligand binding is irreversible.

[0510] In certain embodiments, the lipid-based ligand binds weakly or not at all to HSA, thereby enhancing distribution of the conjugate to the kidney. Other moieties that target kidney cells can be used instead of or in addition to the lipid-based ligand.

[0511] In another embodiment, the ligand is a moiety, such as a vitamin, that is taken up by target cells, such as proliferating cells.These are particularly useful for treating disorders characterized by unwanted cell proliferation, such as malignant or non-malignant types, such as cancer cells.Exemplary vitamins include vitamins A, E and K.Other exemplary vitamins include vitamin B, such as folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients that are taken up by cancer cells.HSA and low-density lipoprotein (LDL) are also included.

[0512] B. Cell-penetrating agents In another aspect, the ligand is a cell-penetrating agent, such as a helical cell-penetrating agent. In certain embodiments, the cell-penetrating agent is amphipathic. Exemplary cell-penetrating agents include peptides, such as tat or antennopedia. When the cell-penetrating agent is a peptide, it ca...

Claims

1. A double-stranded ribonucleic acid (dsRNA) agent or a pharma- ceutical acceptable salt thereof that inhibits expression of huntingtin (HTT) in a cell, the dsRNA agent or the pharma-ceutical acceptable salt thereof comprising a sense strand and an antisense strand that form a double-stranded region; a) the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 4 nucleotides, modified or unmodified, from the nucleotide sequence 5'-gscsgac(Chd)CfuGfGfAfaaagcugasusa-3' of SEQ ID NO:65, and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 4 nucleotides, modified or unmodified, from the nucleotide sequence 5'-VPusAfsucdAg(C2p)uuuudCcAfgdGgucgcscsg-3' of SEQ ID NO:79; wherein a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U, respectively, s is a phosphorothioate linkage, Af, Gf, and Cf are 2'-fluoro A, G, and C, respectively, dA is 2'-deoxyadenosine-3'-phosphate, dC is 2'-deoxycytidine-3'-phosphate, dG is 2'-deoxyguanosine-3'-phosphate, (Chd) is 2'-O-hexadecyl-cytosine-3'-phosphate, C2p is cytidine-2'-phosphate, and VP is 5'-vinylphosphonate; b) the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides, modified or unmodified, from the nucleotide sequence 5'-gscsgac(Chd)CfuGfGfAfaaagcugasusa-3' of SEQ ID NO:65, and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides, modified or unmodified, from the nucleotide sequence 5'-VPusAfsucdAg(C2p)uuuudCcAfgdGgucgcscsg-3' of SEQ ID NO:79; wherein a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U, respectively, s is a phosphorothioate linkage, Af, Gf, and Cf are 2'-fluoro A, G, and C, respectively, dA is 2'-deoxyadenosine-3'-phosphate, dC is 2'-deoxycytidine-3'-phosphate, dG is 2'-deoxyguanosine-3'-phosphate, (Chd) is 2'-O-hexadecyl-cytosine-3'-phosphate, C2p is cytidine-2'-phosphate, and VP is 5'-vinylphosphonate; c) the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 2 nucleotides, modified or unmodified, from the nucleotide sequence 5'-gscsgac(Chd)CfuGfGfAfaaagcugasusa-3' of SEQ ID NO:65, and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 2 nucleotides, modified or unmodified, from the nucleotide sequence 5'-VPusAfsucdAg(C2p)uuuudCcAfgdGgucgcscsg-3' of SEQ ID NO:79; wherein a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U, respectively, s is a phosphorothioate linkage, Af, Gf, and Cf are 2'-fluoro A, G, and C, respectively, dA is 2'-deoxyadenosine-3'-phosphate, dC is 2'-deoxycytidine-3'-phosphate, dG is 2'-deoxyguanosine-3'-phosphate, (Chd) is 2'-O-hexadecyl-cytosine-3'-phosphate, C2p is cytidine-2'-phosphate, and VP is 5'-vinylphosphonate; d) the sense strand comprises at least 15 contiguous nucleotides that differ by no more than one nucleotide, modified or unmodified, from the nucleotide sequence 5'-gscsgac(Chd)CfuGfGfAfaaagcugasusa-3' of SEQ ID NO:65, and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than one nucleotide, modified or unmodified, from the nucleotide sequence 5'-VPusAfsucdAg(C2p)uuuudCcAfgdGgucgcscsg-3' of SEQ ID NO:79; wherein a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U, respectively, s is a phosphorothioate linkage, Af, Gf, and Cf are 2'-fluoro A, G, and C, respectively, dA is 2'-deoxyadenosine-3'-phosphate, dC is 2'-deoxycytidine-3'-phosphate, dG is 2'-deoxyguanosine-3'-phosphate, (Chd) is 2'-O-hexadecyl-cytosine-3'-phosphate, C2p is cytidine-2'-phosphate, and VP is 5'-vinylphosphonate; e) the sense strand comprises the nucleotide sequence of SEQ ID NO:65: 5'-gscsgac(Chd)CfuGfGfAfaaagcugasusa-3' and the antisense strand comprises the nucleotide sequence of SEQ ID NO:79: 5'-VPusAfsucdAg(C2p)uuuudCcAfgdGgucgcscsg-3'; wherein a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U, respectively, s is a phosphorothioate linkage, Af, Gf, and Cf are 2'-fluoro A, G, and C, respectively, dA is 2'-deoxyadenosine-3'-phosphate, dC is 2'-deoxycytidine-3'-phosphate, dG is 2'-deoxyguanosine-3'-phosphate, (Chd) is 2'-O-hexadecyl-cytosine-3'-phosphate, C2p is cytidine-2'-phosphate, and VP is 5'-vinylphosphonate; f) the sense strand consists of the nucleotide sequence of SEQ ID NO: 65: 5'-gscsgac(Chd)CfuGfGfAfaaagcugasusa-3' and the antisense strand consists of the nucleotide sequence of SEQ ID NO: 79: 5'-VPusAfsucdAg(C2p)uuuudCcAfgdGgucgcscsg-3'; wherein a, g, c, and u are 2'-O-methyl (2'-OMe) A, G, C, and U, respectively, s is a phosphorothioate linkage, Af, Gf, and Cf are 2'-fluoro A, G, and C, respectively, dA is 2'-deoxyadenosine-3'-phosphate, dC is 2'-deoxycytidine-3'-phosphate, dG is 2'-deoxyguanosine-3'-phosphate, (Chd) is 2'-O-hexadecyl-cytosine-3'-phosphate, C2p is cytidine-2'-phosphate, and VP is 5'-vinylphosphonate; g) the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides, modified or unmodified, from any one of the nucleotide sequences of nucleotides 26-77, 142-202, 27-49, 147-169, or 173-195 of SEQ ID NO:1, and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides, modified or unmodified, from the corresponding nucleotide sequence of SEQ ID NO:6; h) the antisense strand comprises a region complementary to an mRNA encoding HTT, the complementary region comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides, modified or unmodified, from any one of the antisense nucleotide sequences in any one of Tables 2-5, excluding duplexes AD-1019465 and AD-1019476; or i) the sense strand comprises at least 15 contiguous nucleotides that differ by no more than three nucleotides, modified or unmodified, from any one of the nucleotide sequences of nucleotides 4391-4669, 6009-6037, 4398-4420, 4403-4425, or 6512-6534 of SEQ ID NO:1, and the antisense strand comprises at least 15 contiguous nucleotides that differ by three nucleotides, modified or unmodified, from the corresponding nucleotide sequence of SEQ ID NO:6; , a double-stranded ribonucleic acid (dsRNA) agent, or a pharma- ceutically acceptable salt thereof.

2. The dsRNA agent of claim 1, wherein the sense strand, the antisense strand, or both the sense strand and the antisense strand are conjugated to one or more lipophilic moieties, or a pharma- ceutically acceptable salt thereof.

3. The dsRNA agent, or a pharma- ceutically acceptable salt thereof, of claim 2, wherein the lipophilic moiety is conjugated to one or more interior positions in the double-stranded region of the dsRNA agent, or a pharma- ceutically acceptable salt thereof.

4. 4. The dsRNA agent of claim 2 or 3, or a pharma- ceutically acceptable salt thereof, wherein the one or more lipophilic moieties are conjugated to one or more of the internal positions selected from the group consisting of 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.

5. 5. The dsRNA agent of any one of claims 2-4, or a pharma- ceutically acceptable salt thereof, wherein said lipophilic moiety is an aliphatic, alicyclic, or polyalicyclic compound.

6. 6. The dsRNA agent of claim 5, or a pharma- ceutically acceptable salt thereof, wherein said lipophilic moiety contains a saturated or unsaturated C6-C18 hydrocarbon chain.

7. 7. The dsRNA agent of any one of claims 1g)-1i), and 2-6, or a pharma- ceutically acceptable salt thereof, wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a nucleotide modification.

8. At least one of the nucleotide modifications is a deoxy-nucleotide modification, a 3'-terminal deoxy-thymine (dT) nucleotide modification, a 2'-O-methyl nucleotide modification, a 2'-fluoro nucleotide modification, a 2'-deoxy-nucleotide modification, a 2'-5'-linked ribonucleotide (3'-RNA) modification, a locked nucleotide modification, an unlocked nucleotide modification, a conformationally restricted nucleotide modification, a constrained ethyl nucleotide modification, an abasic nucleotide modification, a 2'-amino nucleotide modification, a 2'-O-allyl nucleotide modification, a 2'-C-alkyl nucleotide modification, a 2'-hydroxy-nucleotide modification, a 2'-methoxyethyl nucleotide modification, a 2'-O-alkyl nucleotide modification, a morpholino nucleotide modification, a phosphoramidate modification, a nucleotide modification containing a non-natural base, a tetrahydropyran nucleotide modification, a 1,5-anhydrohexitol nucleotide modification, a cyclohexenyl nucleotide modification, a nucleotide modification containing a 5'-phosphorothioate group, a nucleotide modification containing a 5'-methylphosphonate group, a 5'-phosphate nucleotide modifications including 5' phosphate mimetics, nucleotide modifications including vinyl phosphonates, nucleotide modifications including adenosine-glycol nucleic acid (GNA), nucleotide modifications including thymidine-glycol nucleic acid (GNA) S-isomers, nucleotide modifications including 2-hydroxymethyl-tetrahydrofuran-5-phosphate, nucleotide modifications including 2'-deoxythymidine-3' phosphate, nucleotide modifications including 2'-deoxyguanosine-3'-phosphate, and terminal nucleotide modifications linked to cholesteryl derivatives.

8. The dsRNA agent of claim 7, or a pharma- ceutically acceptable salt thereof, selected from the group consisting of a dodecanoic acid bisdecylamide group modification, a cytidine-2'-phosphate modification, a guanosine-2'-phosphate modification, a uridine-2'-phosphate modification, an adenosine-2'-phosphate modification, a 2'-O-hexadecyl-adenosine-3'-phosphate modification, a 2'-O-hexadecyl-cytidine-3'-phosphate modification, a 2'-O-hexadecyl-guanosine-3'-phosphate modification, and a 2'-O-hexadecyl-uridine-3'-phosphate modification, and combinations thereof.

9. 9. The dsRNA agent of any one of claims 1g)-1i), and 2-8, or a pharma- ceutically acceptable salt thereof, further comprising at least one phosphorothioate internucleotide linkage.

10. 10. The dsRNA agent of any one of claims 1 to 9, or a pharma- ceutically acceptable salt thereof, wherein each strand is 30 nucleotides or less in length.

11. 11. The dsRNA agent of any one of claims 1 to 10, or a pharma- ceutically acceptable salt thereof, wherein at least one strand comprises a 3' overhang of at least one nucleotide.

12. 12. The dsRNA agent of any one of claims 1 to 11, or a pharma- ceutically acceptable salt thereof, wherein said double-stranded region is 15 to 30 nucleotide pairs in length.

13. 13. The dsRNA agent of any one of claims 1g)-i), and 2-12, or a pharma- ceutically acceptable salt thereof, further comprising a phosphate or a phosphate mimetic at the 5'-end of the antisense strand.

14. A cell comprising the dsRNA agent of any one of claims 1 to 13, or a pharma- ceutically acceptable salt thereof.

15. A pharmaceutical composition for inhibiting expression of a gene encoding HTT comprising the dsRNA agent of any one of claims 1 to 13 or a pharma- ceutically acceptable salt thereof.

16. A composition comprising two or more dsRNA agents or pharma- ceutically acceptable salts thereof that inhibit expression of huntingtin (HTT) in a cell, each of said dsRNA agents or pharma- ceutically acceptable salts thereof independently comprising a sense strand and an antisense strand that form a double-stranded region; each of said antisense strands independently comprises a region of complementarity to an mRNA encoding HTT, and each of said regions of complementarity independently comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides, modified or unmodified, from any one of said antisense nucleotide sequences in any one of Tables 2-5.

17. A method for inhibiting expression of the huntingtin (HTT) gene in a cell, comprising: (a) contacting said cell with a dsRNA agent according to any one of claims 1 to 13 or a pharma- ceutically acceptable salt thereof, or with a composition according to claim 16 or a pharmaceutical composition according to claim 15; (b) maintaining the cells produced in step (a) for a time sufficient to obtain degradation of mRNA transcripts of the HTT gene, thereby inhibiting expression of the HTT gene in said cells.

18. 17. A dsRNA agent or a pharma- ceutically acceptable salt thereof or pharmaceutical composition according to any one of claims 1 to 13, or the pharmaceutical composition according to claim 15, or the composition according to claim 16, for use in treating a subject diagnosed with an HTT-related disease, said use comprising administering to said subject a therapeutically effective amount of the dsRNA agent or the composition according to claim 16, or the pharmaceutical composition according to claim 15.

19. 19. The dsRNA agent or a pharma- ceutically acceptable salt thereof or composition or pharmaceutical composition for use according to claim 18, wherein the HTT-related disease is Huntington's disease.

20. 20. The dsRNA agent or pharma- ceutically acceptable salt thereof or composition or pharmaceutical composition for use according to claim 18 or 19, wherein said dsRNA agent or pharma- ceutically acceptable salt thereof is administered at a dose of about 0.01 mg / kg to about 50 mg / kg.

21. 21. The dsRNA agent or pharma- ceutically acceptable salt thereof or composition or pharmaceutical composition for use according to any one of claims 18 to 20, wherein the dsRNA agent or pharma- ceutically acceptable salt thereof is administered intrathecally.

22. The dsRNA agent or a pharma- ceutically acceptable salt thereof or composition or pharmaceutical composition for use according to any one of claims 17 to 21, wherein the use further comprises administering to the subject an additional agent suitable for treating or preventing an HTT-related disease.

23. A kit, vial or syringe comprising a dsRNA agent or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 13, or a composition according to claim 16, or a pharmaceutical composition according to claim 15.

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