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

Abstract

Double-stranded ribonucleic acid (dsRNAi) agents targeting exon 1 of the huntingtin (HTT) gene are provided. [Solution] A double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of huntingtin (HTT) is provided, which comprises a sense strand and an antisense strand that form a double-stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides that differ from a specific nucleotide sequence by no more than 3 nucleotides, and the antisense strand comprises at least 15 contiguous nucleotides that differ from another specific nucleotide sequence by no more than 3 nucleotides, and one or more lipophilic moieties are conjugated to one or more internal positions in at least one of the sense strand or the antisense strand.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 929,174, filed November 1, 2019, the entire contents of which are incorporated herein by reference.

[0002] Array List This application contains a Sequence Listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy created on October 36, 2020 is named 121301_10320_SL.txt and is 1,468,832 bytes in size. [Background technology]

[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, PS et al., in Huntington's Disease, WB Saunders, Philadelphia, 1991). The hallmark of Huntington's disease is a distinctive choreiform movement disorder, which typically has subtle, insidious, and insidious symptoms in the fourth or fifth decade of life and gradually worsens over 10 to 20 years before death. Huntington's disease often manifests in younger individuals, typically with more severe symptoms such as rigidity and a more rapid course of the disease. The younger onset of Huntington's disease is associated with paternal dominance of the disease allele. The neuropathology of Huntington's disease also displays 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 unclear. In patients with HD (an autosomal dominant disease), the expansion of polyglutamine repeats 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] Currently, there is no effective treatment for Huntington's disease.Choreic movements 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 paralysis occur.In addition, despite the significant progress in the field of RNAi and Huntington's disease treatment, there is still a need for a drug that can selectively and efficiently silence HD genes using the cell's own RNAi mechanism, has high biological activity and in vivo stability, and can effectively inhibit the expression of the target huntingtin gene. Summary of the Invention [Problem to be solved by 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 huntingtin (HTT) gene.HTT gene can be in a cell, for example, in a cell of 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 HTT gene or to treat the subject who will benefit from inhibiting or reducing the expression of HTT gene, for example, the subject who is suffering from or prone to suffering from HTT-related disease. [Means for solving the problem]

[0007] In one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of huntingtin (HTT), comprising a sense strand and an antisense strand that form a double-stranded region, wherein the sense strand comprises at least 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO: 1 by no more than 3, 2, 1, or 0 nucleotides, and the antisense strand comprises at least 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO: 6 by no more than 3, 2, 1, or 0 nucleotides, and wherein one or more lipophilic moieties are conjugated to one or more internal positions in at least one strand.

[0008] In some embodiments, the nucleotide sequence of the sense strand comprises any one of the sense strand nucleotide sequences in any one of Tables 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 24, 25, 27-30, 32, and 33.

[0009] In some embodiments, the sense strand comprises nucleotides 618-640, 1215-1237, 1248-1270, 1403-1425, 4051-4073, 4393-4415, 4398-4420, 4403-4425, 4441-4463, 4518-4540, 4548-4570, 5105-5127, 5215-5237, 5217-5239, 5221-5243, 5222-5244, 5366-5388, 5372-5394, 5450-5472, 5509-5531, 5548-5552, 5556-5558, 5559-5561, 5562-5563, 5570-5571, 5572-5573, 5580-5581, 5582-5584, 5584-5585, 5586-5588, 5590-5591, 5592-5593, 5594-5595, 5596-5597, 5598-5599, 5599-5600, 5601-5602, 5603-5604, 5605-5606, 5607-5608, 5609-5610, 5611-5612, 5613-5614, 5615-5615, 5616-5616, 5617-5 and at least 15 consecutive nucleotides that differ by 0, 1, 2, or no more than 3 nucleotides from any one of the nucleotide sequences 5883-5905, 6009-6031, 6010-6032, 6011-6033, 6012-6034, 6013-6035, 6014-6036, 6015-6037, 6347-6369, 6512-6534, 7523-7545, 7525-7547, 7526-7548, 9127-9149, 9531-9553, or 9538-9560.

[0010] In some embodiments, the antisense strand is selected from the group consisting of AD-953769.1, AD-953778.1, AD-953784.1, AD-953786.1, AD-953849.1, AD-953854.1, AD-953855.1, AD-953857.1, AD-953862.1, AD-953866.1, AD-953867.1, AD-953880.1, AD-953883.1, AD-953884.1, AD-953885.1, AD-953886.1, AD-953887.1, AD-953888.1, AD-953889.1, AD-953891.1, AD-953896.1 , AD-953898.1, AD-953899.1, AD-953900.1, AD-953901.1, AD-953902.1, AD-953903.1, AD-953904.1, AD-953907.1, AD-953911.1, AD-953921.1, AD-953923.1, AD-953924.1, AD-953932.1, and AD-953933.1, AD-953937.1, and comprising at least 15 contiguous nucleotides that differ by 0, 1, 2, or 3 nucleotides or less from any one of the antisense strand nucleotide sequences of the duplex selected from the group consisting of:

[0011] In some embodiments, the lipophilic moiety is conjugated via a linker or carrier.

[0012] In another aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of huntingtin (HTT) in a cell, the double-stranded ribonucleic acid (dsRNA) comprising a sense strand and an antisense strand that form a duplex region, wherein the antisense strand comprises a region of complementarity to an mRNA encoding HTT, wherein the region of complementarity comprises at least 15, e.g., 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides that differ by no more than 3, 2, 1, or 0 nucleotides from any one of the antisense nucleotide sequences in any one of Tables 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 24, 25, 27-30, 32, and 33.

[0013] Sense strand, antisense strand, or both sense strand and antisense strand can be conjugated to 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 in antisense strand.In some embodiments, one or more lipophilic moieties are conjugated to one or more internal positions in at least one strand via linker or carrier.

[0014] In some embodiments, logK ow The lipophilicity of the lipophilic moiety as measured by is greater than 0.

[0015] In some embodiments, the hydrophobicity of the dsRNA agent, as measured by the unbound fraction in a plasma protein binding assay of the dsRNA agent, is greater than 0.2. In some embodiments, the plasma protein binding assay is an electrophoretic mobility shift assay using human serum albumin protein.

[0016] In some embodiments, internal positions include all but the two most terminal positions from each end of the sense or antisense strand, while in other embodiments, internal positions include all but the three most terminal positions from each end of the sense or antisense strand.

[0017] 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.

[0018] 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, counting from the 3' end of the sense strand, and positions 12-14, counting from the 5' end of the antisense strand.

[0019] In some embodiments, the one or more lipophilic moieties are conjugated to one or more internal positions selected from the group consisting of positions 4-8 and 13-18 in the sense strand and positions 6-10 and 15-18 in the antisense strand, counting from the 5' end of each strand.

[0020] 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 in the sense strand and positions 15 and 17 in the antisense strand, counting from the 5' end of each strand.

[0021] In some embodiments, the positions in the double-stranded region exclude the cleavage site region of the sense strand.

[0022] In some 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, position 15, position 1, position 7, position 6, or position 2 of the sense strand or position 16 of the antisense strand.

[0023] 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, position 20, position 15, position 1, position 7, position 6, or position 2 of the sense strand or position 16 of the antisense strand.

[0024] In some embodiments, the lipophilic moiety is an aliphatic compound, an alicyclic compound, or a polyalicyclic compound.

[0025] In some embodiments, the lipophilic moiety is selected from the group consisting of lipids, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenoic acid, dimethoxytrityl, or phenoxazine.

[0026] 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.

[0027] In some embodiments, the lipophilic moiety contains a saturated or unsaturated C6 to C18 hydrocarbon chain.

[0028] 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 position 6, counting from the 5' end of the chain.

[0029] In some embodiments, lipophilic moiety is conjugated via carrier that replaces one or more nucleotides in internal position or double-stranded region.In some embodiments, carrier is selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3] dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl and decalinyl; or is based on serinol skeleton or diethanolamine skeleton acyclic moiety.

[0030] 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.

[0031] In some embodiments, the lipophilic moiety is conjugated to a nucleobase, a sugar moiety, or an internucleoside linkage.

[0032] In some embodiments, dsRNA agent comprises at least one modified nucleotide.In some embodiments, 5 or less of the nucleotides of sense strand and 5 or less of the nucleotides of antisense strand are unmodified nucleotide.In other embodiments, all of the nucleotides of sense strand and all of the nucleotides of antisense strand comprise modification.

[0033] 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-anhydrohexyl ... The nucleotide is selected from the group consisting of nucleotides containing 5'-phosphate, 5'-phosphate, 5'-phosphate mimics, 5'-phosphate ...

[0034] 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.

[0035] 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.

[0036] 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 opposite 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).

[0037] In some embodiments, the modified nucleotides comprise a short sequence of 3'-terminal deoxy-thymine nucleotides (dT).

[0038] In some embodiments, the modifications in the nucleotides are 2'-O-methyl modifications, GNA modifications, and 2' fluoro modifications.

[0039] 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, the strand may be the antisense strand. In another embodiment, the strand is the sense strand. In a related embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 5'-end of one strand. Optionally, the strand may be the antisense strand. In another embodiment, the 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, the strand may be the antisense strand. In another embodiment, the strand is the sense strand.

[0040] In some embodiments, each strand is 30 nucleotides or less in length.

[0041] 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.

[0042] 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.

[0043] Each strand can be 19 to 30 nucleotides; 19 to 23 nucleotides; or 21 to 23 nucleotides.

[0044] In some embodiments, the dsRNA agent further comprises a targeting ligand that targets liver tissue. In some embodiments, the targeting ligand is a GalNAc conjugate.

[0045] 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.

[0046] In certain embodiments, the targeting ligand is a C16 ligand. In one embodiment, the ligand is:

[0047] [ka] wherein B is a nucleotide base or a nucleotide base analog, and optionally B can be adenine, guanine, cytosine, thymine, or uracil.

[0048] In some embodiments, the lipophilic moiety or targeting ligand is conjugated via a biocleavable 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.

[0049] 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.

[0050] In some embodiments, the dsRNA agent further includes a terminal chiral modification occurring at the first internucleotide linkage at the 3'-end of the antisense strand, the first internucleotide linkage having 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, the first internucleotide linkage having 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, the first internucleotide linkage having the linking phosphorus atom in either the Rp or Sp configuration.

[0051] 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.

[0052] 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, 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.

[0053] 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 third internucleotide linkage at the 3'-end of the antisense strand, with the 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, 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.

[0054] 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.

[0055] 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).

[0056] In some embodiments, the base pair at one position at the 5' end of the antisense strand of the duplex is an AU base pair.

[0057] In some embodiments, the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

[0058] An additional embodiment of the present disclosure provides a double-stranded RNAi agent for inhibiting expression of the huntingtin (HTT) gene, wherein the double-stranded RNAi agent targeted to HTT comprises 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, or 20 contiguous nucleotides that differ by no more than 3 nucleotides (i.e., differs by 3, 2, 1, or 0 nucleotides) from any one of the nucleotide sequences of SEQ ID NOs: 1-5, and the antisense strand comprises at least 15, e.g., 15, 16, 17, 18, 19, or 20 contiguous nucleotides that differ by no more than 3 nucleotides (i.e., differs by 3, 2, 1, or 0 nucleotides) from any one of the nucleotide sequences of SEQ ID NOs: 6-10, and substitution of uracil with any thymine present (when comparing aligned sequences) does not count as a difference within the three nucleotide or less difference from any one of the nucleotide sequences provided in SEQ ID NOs: 1-10; substantially all of the nucleotides of the sense strand comprise a modification that is a 2'-O-methyl modification, a GNA, or a 2'-fluoro modification; the sense strand comprises two phosphorothioate internucleotide linkages at the 5'-terminus; substantially all of the nucleotides of the antisense strand comprise a modification selected from the group consisting of a 2'-O-methyl modification and a 2'-fluoro modification; the antisense strand comprises two phosphorothioate internucleotide linkages at the 5'-terminus and two phosphorothioate internucleotide linkages at the 3'-terminus; and the sense strand is conjugated to one or more lipophilic ligands, e.g., a C16 ligand.

[0059] Another aspect of the present disclosure provides a double-stranded RNAi agent for inhibiting expression of the huntingtin (HTT) gene, wherein the double-stranded RNAi agent targeted to HTT comprises 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, or 20 contiguous nucleotides that differ by no more than 3 nucleotides (i.e., differs by 3, 2, 1, or 0 nucleotides) from any one of the nucleotide sequences of SEQ ID NOs: 1-5, and the antisense strand differs by 3 nucleotides from any one of the nucleotide sequences of SEQ ID NOs: 6-10. the sense strand comprises at least one 3'-terminal deoxythymine nucleotide (dT) and the antisense strand comprises at least one 3'-terminal deoxythymine nucleotide (dT), ...

[0060] Additional aspects of the present disclosure provide double-stranded ribonucleic acid (RNAi) agents for inhibiting expression of the huntingtin (HTT) gene, wherein the RNAi agent has a sense strand and an antisense strand, and the antisense strand comprises a region of complementarity comprising at least 15 contiguous nucleotides, e.g., at least 15 nucleotides (i.e., differs in 3, 2, 1, or 0 nucleotides), at least 19 nucleotides (i.e., differs in 3, 2, 1, or 0 nucleotides), that differ by no more than 3 nucleotides (i.e., differs in 3, 2, 1, or 0 nucleotides) from any one of the antisense strand nucleobase sequences of Tables 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 24, 25, 27-30, 32, and 33. In one embodiment, the RNAi agent comprises one or more of the following modifications: 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-C-alkyl-modified nucleotides, and nucleotides comprising glycol nucleic acids (GNAs), phosphorothioates (PS), and vinyl phosphonates (VPs). Optionally, the RNAi agent may comprise at least one of each of the following modifications: 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-C-alkyl-modified nucleotides, and nucleotides comprising glycol nucleic acids (GNAs), phosphorothioates, and vinyl phosphonates (VPs).

[0061] In another embodiment, the RNAi agent comprises four or more PS modifications, optionally six to ten PS modifications, and optionally eight PS modifications.

[0062] In additional embodiments, each of the sense and antisense strands of the RNAi agent has a 5'-end and a 3'-end, and the RNAi agent includes eight PS modifications located at each of the penultimate and final internucleotide linkages from each of the 3'- and 5'-ends of each of the sense and antisense strands of the RNAi agent.

[0063] In another embodiment, each of the sense strand and antisense strand of the RNAi agent comprises a 5'-end and a 3'-end, and the RNAi agent comprises only one nucleotide that comprises GNA.Optionally, the nucleotide that comprises GNA may be located on the antisense strand at the seventh nucleobase residue from the 5'-end of the antisense strand.

[0064] In a further embodiment, each of the sense strand and antisense strand of the RNAi agent comprises a 5'-end and a 3'-end, and the RNAi agent comprises one to four 2'-C-alkyl-modified nucleotides. Optionally, the 2'-C-alkyl-modified nucleotide may be a 2'-C16-modified nucleotide. Optionally, the RNAi agent may comprise a single 2'-C-alkyl, e.g., C16-modified nucleotide. Optionally, the single 2'-C-alkyl, e.g., C16-modified nucleotide may be located on the sense strand at the sixth nucleobase residue from the 5'-end of the sense strand.

[0065] In another embodiment, each of the sense strand and antisense strand of the RNAi agent comprises a 5'-end and a 3'-end, and the RNAi agent comprises two or more 2'-fluoro modified nucleotides.Optionally, each of the sense strand and antisense strand of the RNAi agent can comprise two or more 2'-fluoro modified nucleotides.Optionally, the 2'-fluoro modified nucleotides can be located on the sense strand at nucleobase positions 7, 9, 10 and 11 from the 5'-end of the sense strand, and on the antisense strand at nucleobase positions 2, 14 and 16 from the 5'-end of the antisense strand.

[0066] In additional embodiments, each of the sense and antisense strands of the RNAi agent comprises a 5'-end and a 3'-end, and the RNAi agent comprises one or more VP modifications. Optionally, the RNAi agent may comprise a single VP modification at the 5'-end of the antisense strand.

[0067] In another embodiment, each of the sense strand and antisense strand of the RNAi agent comprises a 5'-end and a 3'-end, and the RNAi agent comprises two or more 2'-O-methyl modified nucleotides.Optionally, the RNAi agent may comprise 2'-O-methyl modified nucleotides at all nucleobase positions that are not modified by 2'-fluoro, 2'-C-alkyl, or glycol nucleic acid (GNA).Optionally, the two or more 2'-O-methyl modified nucleotides may be located on the sense strand at positions 1, 2, 3, 4, 5, 8, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 from the 5'-end of the sense strand, and on the antisense strand at positions 1, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22, and 23 from the 5'-end of the antisense strand.

[0068] In one embodiment, all of the nucleotides in the sense strand and all of the nucleotides in the antisense strand are modified nucleotides.

[0069] In another embodiment, each strand has between 19 and 30 nucleotides.

[0070] In certain embodiments, the antisense strand of the RNAi agent comprises at least one thermally destabilizing modification of the duplex within the 5' region or the first 9 nucleotide positions of its precursor. Optionally, the thermally destabilizing modification of the duplex is one of the following:

[0071] [ka] where B is a nucleobase.

[0072] 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.

[0073] In one embodiment, the double-stranded RNAi agent is in a non-buffered solution. Optionally, the non-buffered solution may be saline or water. In another embodiment, the double-stranded RNAi agent is in a buffered solution. Optionally, the buffered solution may comprise acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In another embodiment, the buffered solution 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).

[0074] An additional 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 or a pharmaceutical composition of the present disclosure; and (b) maintaining the cell 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 the cell.

[0075] In one embodiment, the cell is in a subject. Optionally, the subject may be a human.

[0076] In certain embodiments, the subject is a rhesus monkey, a cynomolgus monkey, a mouse, or a rat. In certain embodiments, TT expression is inhibited by at least about 50% by the RNAi agent.

[0077] In certain embodiments, the human subject has been diagnosed with an HTT-related disease, such as Huntington's disease.

[0078] Another aspect of the present disclosure provides 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 or a pharmaceutical composition of the present disclosure, thereby treating the subject.

[0079] In one embodiment, treatment comprises ameliorating at least one sign or symptom of the disease, hi another embodiment, treatment comprises preventing the progression of the disease.

[0080] In some embodiments, the dsRNA agent is administered to the subject at a dose of about 0.01 mg / kg to about 50 mg / kg.

[0081] 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 can be striatum, cortex, cerebellum, cervical vertebrae, lumbar vertebrae or thoracic vertebrae.

[0082] In some embodiments, the method further comprises measuring the level of HTT in a sample obtained from the subject.

[0083] 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.

[0084] In some embodiments, the method further comprises administering to the subject an additional agent suitable for the treatment or prevention of an HTT-related disorder. [Brief explanation of the drawings]

[0085] [Figure 1] 1 is a graph showing wild-type human HTT mRNA levels in the livers of mice expressing a portion of wild-type human HTT (via AAV). These mice were subcutaneously administered a single 3 mg / kg dose of the indicated dsRNA duplex targeting exon 1 of the human HTT transcript 14 days after AAV administration. The human HTT levels shown are normalized to AAV-treated controls 14 days after siRNA administration. [Figure 2]2 is a graph showing wild-type human HTT mRNA levels in the livers of mice expressing a portion of wild-type human HTT (via AAV). These mice were subcutaneously administered a single 3 mg / kg dose of the indicated dsRNA duplex targeting exon 1 of the human HTT transcript 14 days after AAV administration. The human HTT levels shown are normalized to AAV-treated controls 14 days after siRNA administration. [Figure 3] Figure 3A is a graph showing full-length mutant human HTT mRNA levels in the livers of YAC128 mice subcutaneously administered a single 10 mg / kg dose of the indicated dsRNA duplex targeting exon 1 of the human HTT transcript at day 7 post-administration. The indicated human HTT levels are normalized to PBS-treated levels. Figure 3B is a Western blot showing mutant and wild-type human HTT protein levels in the livers of YAC128 mice subcutaneously administered a single 10 mg / kg dose of the indicated dsRNA duplex targeting exon 1 of the human HTT transcript at day 7 post-administration. Figure 3C is a bar graph showing mutant human HTT protein levels in the livers of YAC128 mice subcutaneously administered a single 10 mg / kg dose of the indicated dsRNA duplex targeting exon 1 of the human HTT transcript at day 7 post-administration. The indicated mutant human HTT levels are normalized to PBS-treated levels. [Figure 4] Figure 4A is a graph showing full-length mutant HTT mRNA levels in the livers of YAC128 mice subcutaneously administered a single 10 mg / kg dose of the indicated dsRNA duplex targeting exon 1 of the HTT transcript at day 7 post-administration. The indicated full-length mutant human HTT levels are normalized to PBS-treated levels. Figure 4B is a bar graph showing quantification of mutant HTT protein levels in the livers of YAC128 mice subcutaneously administered a single 10 mg / kg dose of the indicated dsRNA duplex targeting exon 1 of the HTT transcript at day 7 post-administration. The indicated mutant human HTT levels are normalized to PBS-treated levels. [Figure 5] 5 is a graph showing full-length mutant human HTT mRNA levels in the liver of YAC128 mice administered a single 10 mg / kg dose of the indicated dsRNA duplexes targeting exon 1 of the HTT transcript subcutaneously on day 7 post-administration. The human HTT levels shown are normalized to PBS-treated levels. [Figure 6] 6 is a graph showing full-length mutant human HTT mRNA levels in YAC128 mice and corresponding full-length mutant human HTT protein levels in the liver after subcutaneous administration of a single 10 mg / kg dose of the indicated dsRNA duplex on day 7. The indicated mutant human mRNA and protein HTT levels are normalized to PBS-treated levels. [Figure 7] 7 is a graph showing mutant full-length human HTT mRNA levels in YAC128 mice administered a single 10 mg / kg dose of the indicated dsRNA duplex subcutaneously on day 7 post-administration. The indicated mutant human HTT levels are normalized to PBS-treated levels. [Figure 8] Figures 8A and 8B are graphs showing full-length human HTT mRNA levels in human fibroblasts transfected with 10 nM or 50 nM of the indicated dsRNA duplexes targeting various exons of human HTT or specifically exon 1. Fibroblasts were obtained from Coriell, an adult healthy control patient ("Control," GM02153), an adult-onset HD patient ("Adult," GM04478), and a young-onset HD patient ("Young," GM09197). The HTT levels shown are normalized to mock-transfected controls. [Figure 9]Figures 9A and 9B are graphs showing full-length human HTT mRNA levels in human fibroblasts transfected with 10 nM or 50 nM of the indicated dsRNA duplexes targeting various exons of human HTT or specifically exon 1. Fibroblasts were obtained from Coriell, an adult healthy control patient ("Control," GM02153), an HD patient with adult disease onset ("Adult," GM04478), and an HD patient with juvenile disease onset ("Juvenile," GM09197). The HTT levels shown are normalized to mock-transfected controls. [Figure 10] Figures 10A-10D are graphs showing full-length human HTT mRNA levels in human fibroblasts transfected with 10 nM or 50 nM of the indicated dsRNA duplexes targeting various exons of human HTT or specifically exon 1. Fibroblasts were obtained from Coriell, an adult healthy control patient ("Control," GM02153), an HD patient with adult disease onset ("Adult," GM04478), and an HD patient with juvenile disease onset ("Juvenile," GM09197). Shown HTT levels are normalized to mock-transfected controls. [Figure 11] Figures 11A-11D are graphs showing full-length HTT mRNA levels in human fibroblasts transfected with 10 nM or 50 nM of the indicated dsRNA duplexes targeting various exons of human HTT or specifically exon 1. Fibroblasts were obtained from Coriell, an adult healthy control patient ("Control," GM02153), an HD patient with adult disease onset ("Adult," GM04478), and an HD patient with juvenile disease onset ("Juvenile," GM09197). HTT levels shown are normalized to mock-transfected controls. [Figure 12]12 is a graph showing full-length mutant human HTT mRNA levels in the livers of YAC 128 or wild-type mice expressing a portion of human wild-type HTT ("AAV") and administered subcutaneously a single dose of the indicated dsRNA duplex targeting the full-length HTT transcript on the indicated days after administration. The HTT levels shown are normalized to PBS-treated levels. [Figure 13] Figures 13A-D are graphs showing full-length human HTT mRNA levels in the livers of mice expressing a portion of human wild-type HTT via AAV. These mice were subcutaneously administered a single 3 mg / kg dose of the indicated dsRNA duplex targeting the full-length HTT transcript 14 days after administration. HTT levels are shown relative to AAV-treated control levels 14 days after the siRNA dose. DETAILED DESCRIPTION OF THE INVENTION

[0086] The present disclosure provides an RNAi composition that carries out RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of huntingtin (HTT) gene.HTT gene can be present in cells, for example, in cells of a subject such as human.The use of these iRNAs allows the targeted degradation of the mRNA of corresponding gene (HTT gene) in mammals.

[0087] The iRNA of the present invention is designed to target HTT gene, including the part of the gene that is conserved in the HTT orthologues of other mammalian species.The iRNA of the present invention is also designed to target a specific part of HTT gene, exon 1, for example, thereby targeting full-length wild-type transcript, full-length mutant transcript, and truncated mutant transcript.Without intending to be limited by theory, it is believed that the combination or partial combination of the above-mentioned characteristics and specific target site, for example, exon 1, or specific modification of these iRNAs will give the iRNA of the present invention improved efficiency, stability, efficacy, durability and safety.

[0088] Thus, the present disclosure also provides methods of using the RNAi compositions of the present disclosure 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, such as Huntington's disease (HD).

[0089] RNAi agents of the present 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, 19-26, 19-27, 19-28, 19-29, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-26, 19-27, 19-28, 19-25, 19-26, 19-25, 19-26, 19-27, 19-28, 19-29, 19-30 ... The RNAi agent of the present disclosure comprises an RNA strand (antisense strand) having a region that is 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, wherein the region 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 about 21-23 nucleotides in length, wherein the region is substantially complementary to at least a portion of an mRNA transcript of the HTT gene.

[0090] In certain embodiments, the RNAi agents of the present disclosure comprise an RNA strand (antisense strand) having 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, and can comprise a longer length, e.g., up to 66 nucleotides, e.g., 36-66, 26-36, 25-36, 31-60, 22-43, 27-53 nucleotides in length. These RNAi agents with longer antisense strand lengths preferably comprise a second RNA strand (sense strand) of 20-60 nucleotides in length, in which case the sense and antisense strands form a duplex of 18-30 contiguous nucleotides.

[0091] The use of these RNAi agents allows the targeted degradation of the mRNA of HTT gene in mammals.Therefore, the methods and compositions comprising these RNAi agents are useful for treating subjects who will benefit from the reduction of HTT protein level or activity, such as subjects with HTT-related diseases, such as Huntington's disease (HD).

[0092] The detailed description below discloses methods for making and using compositions containing RNAi agents to inhibit expression of the HTT gene, as well as compositions or methods for treating subjects with diseases or disorders that would benefit from inhibiting or reducing expression of the gene.

[0093] 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.

[0094] 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 more than one element, e.g., a plurality of elements.

[0095] 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.

[0096] The term "about" is used herein to mean within a range that is typical 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 is present before a series of numbers or ranges, it will be understood that "about" can modify each number or range in the series.

[0097] The term "at least" before a number or a series of numbers, if clear from the context, is understood to include the number adjacent to the term "at least" and all subsequent numbers or integers that may be logically included. 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 the term "at least" is present before a series of numbers or a range, it is understood that "at least" can modify each of the numbers and ranges in the series.

[0098] As used herein, "less than" or "less than" shall be understood as from the value adjacent to the phrase and its logically smaller value or integer, if logical from the context, to zero.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 present before a series of numbers or ranges, it shall be understood that "less than" can modify each of the numbers or ranges in the series.

[0099] As used herein, a method of detection can include determining that the amount of analyte present is below the detection level of the method.

[0100] In the event of a discrepancy between the nucleotide sequence for a given target site and the sense or antisense strand, the given sequence prevails.

[0101] If the chemical structure and chemical name do not agree, the chemical structure takes precedence.

[0102] The term "HTT" or "huntingtin" refers to the well-known gene encoding the protein HTT, also known as "Huntingtin," "Huntington Disease Protein," "IT15," "HD," "HD Protein," or "LOMARS," a gene that is widely expressed and necessary for normal development, and is responsible for Huntington's disease, a neurodegenerative disorder characterized by stretch-induced loss of striatal neurons, a disease gene associated with an unstable trinucleotide (CAG) repeat in the huntingtin gene that is translated as a polyglutamine repeat in the protein product.

[0103] Exemplary 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).

[0104] Further examples of HTT sequences can be found in publicly available databases such as GenBank, OMIM, and UniProt.

[0105] Further information regarding HTT can be found, for example, at www.ncbi.nlm.nih.gov / gene / 3064.

[0106] 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.

[0107] Term HTT as used herein also refers to the variation of HTT gene, including the variant provided in SNP database.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).

[0108] As used herein, " target sequence " refers to a continuous portion of the nucleotide sequence of the mRNA molecule formed during the transcription of HTT gene, such as the mRNA that is the product of RNA processing of the primary transcript.In one embodiment, the target portion of the sequence will be at least long enough to serve as the substrate for RNAi-dependent cleavage at or near a portion of the nucleotide sequence of the mRNA molecule formed during the transcription of HTT gene.

[0109] The target sequence is about 15 to 30 nucleotides in length. For example, the target sequence may be about 15 to 30 nucleotides in length, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 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, 1 The target sequence may be 9-28, 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, where appropriate, may be 21-23 nucleotides in length. Ranges and lengths intermediate to the above-listed ranges and lengths are also contemplated as part of this disclosure.

[0110] As used herein, the term "strand comprising a sequence" means an oligonucleotide comprising a chain of nucleotides described by a sequence referenced using standard nucleotide nomenclature.

[0111] " G ", " C ", " A ", " T " and " U " each generally represent the nucleotide that contains guanine, cytosine, adenine, thymidine and uracil as a base, respectively, in relation to modified or unmodified nucleotide. However, it should be understood that the term "ribonucleotide" or "nucleotide" can also refer to modified nucleotides, as described in more detail below, or substitute replacement moieties (see, for example, Table 1). Those skilled in the art are well aware that guanine, cytosine, adenine, thymidine and uracil can be replaced with other moieties without substantially changing the base pairing properties of the oligonucleotide that contains the nucleotide with such replacement moieties. For example, but not limited to, the nucleotide that contains inosine as its base can form base pairs with the nucleotide that contains adenine, cytosine or uracil. Thus, the nucleotide that contains uracil, guanine or adenine can be replaced with the nucleotide that contains inosine, for example, in the nucleotide sequence of the dsRNA that is featured in the present disclosure. In another example, adenine and cytosine in any of the oligonucleotides can be substituted with guanine and uracil, respectively, to form G-U Wobble base pairs with the target mRNA. Sequences containing such substituted moieties are suitable for the compositions and methods featured in this disclosure.

[0112] The terms " iRNA ", " RNAi agent ", " iRNA agent ", " RNA interference agent ", used interchangeably herein, refer to the agent that contains the RNA as defined herein and mediates the targeted cleavage in RNA transcription via the RNA-induced silencing complex (RISC) pathway.RNA interference (RNAi) is the process that directs the sequence-specific degradation of mRNA.RNAi regulates, for example, inhibits, the expression of HTT in cells, for example, cells in a subject, such as a mammalian subject.

[0113] In one embodiment, the RNAi agent of the present disclosure comprises a single-stranded RNAi that interacts with a target RNA sequence, for example, an HTT target mRNA sequence, to direct the cleavage of the target RNA. Without wishing to be bound by theory, it is believed that long double-stranded RNA introduced into cells is 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 a characteristic two-base 3' overhang [Bernstein, et al., (2001) Nature 409:363]. These siRNAs are then introduced into RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, allowing complementary antisense strands to induce 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 to induce silencing [Elbashir, et al., (2001) Genes Dev. 15:188]. Thus, 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, promotes the formation of RISC complex, and thereby silences the target gene, i.e., HTT gene. Therefore, the term "siRNA" is used herein to also mean the RNAi described above.

[0114] 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 and then cleaves the target mRNA. The single-stranded siRNA is generally 15-30 nucleotides and chemically modified. The design and testing of single-stranded RNA is 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 chemically modified by the method described in Lima et al., (2012) Cell 150:883-894.

[0115] In another embodiment, the "RNAi agent" for use in the compositions and methods of the present disclosure is double-stranded RNA, and is herein referred to 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, said to have "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, by a post-transcriptional gene silencing mechanism, herein referred to as RNA interference or RNAi.

[0116] Generally, dsRNA molecules can comprise ribonucleotides, but as described in detail herein, each or both strands can also comprise one or more ribonucleotides, such as deoxyribonucleotides, modified nucleotides.In addition, as used herein, " RNAi agent " can comprise ribonucleotides with chemical modification; RNAi agent can comprise substantial modifications in multiple nucleotides.As used herein, the term " modified nucleotide " refers to the nucleotide that has independently modified sugar moiety, modified internucleotide linkage, or modified nucleobase.Therefore, the term modified nucleotide encompasses the substitution, addition, or removal of, for example, functional group or atom, etc., in internucleoside linkage, sugar moiety, or nucleobase.The modifications suitable for use in the agent of the present disclosure encompass 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 purpose of this specification and claims.

[0117] In certain embodiments of the present disclosure, the inclusion of deoxy-nucleotides can be considered to constitute modified nucleotides when present within an RNAi agent.

[0118] The duplex region can be any length that allows for specific degradation of the desired target RNA by the RISC pathway, as well as about 15 to 36 base pairs in length, e.g., 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, e.g., about 15 to 30, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 31, 18 to 32, 18 to 33, 18 to 34, 18 to 35, 18 to 36, or 36 base pairs in length. 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 base pairs in length. In certain embodiments, the duplex region is 19-21 base pairs in length, e.g., 21 base pairs in length. Ranges and lengths intermediate to the above-listed ranges and lengths are also contemplated as part of the present disclosure.

[0119] 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 5' end of the other strand forming the duplex structure, the connecting 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 targeted to 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 unpaired nucleotides.

[0120] The two substantially complementary strands of dsRNA are contained in separate RNA molecules, and these molecules can, but do not necessarily, be covalently linked. In certain embodiments, where the two strands are covalently linked between the 3' end of one strand and the 5' end of each other strand forming a duplex structure by means other than an uninterrupted chain of nucleotides, the connecting structure is called a "linker" (although certain other structures defined elsewhere herein may also be 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 all overhangs present in the duplex. In addition to the duplex structure, the RNAi can contain one or more nucleotide overhangs. In one embodiment of an RNAi agent, at least one strand contains a 3' overhang of at least one nucleotide. In another embodiment, at least one strand contains a 3' overhang of at least two 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 one nucleotide. In certain embodiments, at least one strand comprises a 5' overhang of at least two 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' and 5' ends of one strand of the RNAi agent comprise an overhang of at least one nucleotide.

[0121] In one embodiment, an RNAi agent of the present 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.

[0122] 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, when the 3'-end of one strand of dsRNA extends beyond the 5'-end of the other strand, and vice versa, there is a nucleotide overhang.DsRNA can comprise at least one nucleotide overhang; alternatively, the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more nucleotides.The nucleotide overhang can comprise or consist of nucleotide / nucleoside analogs, such as deoxynucleotides / nucleosides.The overhang can be on the sense strand, antisense strand, or any combination thereof.In addition, the nucleotide of the overhang can be present at the 5'-end, 3'-end, or both of the antisense strand or sense strand of dsRNA.

[0123] In one embodiment, the antisense strand of the dsRNA has a 1-10 nucleotide overhang at the 3' or 5' end, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide overhang. In one embodiment, the sense strand of the dsRNA has a 1-10 nucleotide overhang at the 3' or 5' end, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide overhang. In another embodiment, one or more of the nucleotides in the overhang are replaced with nucleoside thiophosphate.

[0124] In certain embodiments, the antisense strand of dsRNA has 1-10 nucleotide overhangs at the 3'-end or 5'-end, for example, 0-3, 1-3, 2-4, 2-5, 4-10, or 5-10 nucleotide overhangs, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide overhangs. In one embodiment, the sense strand of dsRNA has 1-10 nucleotide overhangs at the 3'-end or 5'-end, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide overhangs. In another embodiment, one or more of the nucleotides in the overhang are replaced with nucleoside thiophosphates.

[0125] 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 certain embodiments, the extended overhang is on the sense strand of the duplex. In certain embodiments, the extended overhang is at the 3'-end of the sense strand of the duplex. In certain embodiments, the extended overhang is at 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 at the 3'-end of the antisense strand of the duplex. In certain embodiments, the extended overhang is at the 5'-end of the antisense strand of the duplex. In certain embodiments, one or more of the nucleotides in the overhang are replaced with a nucleoside thiophosphate. In certain embodiments, the overhang comprises a self-complementary portion such that the overhang can form a stable hairpin structure under physiological conditions.

[0126] In certain embodiments, at least one end of at least one chain is extended beyond double-stranded targeting region, such as the structure where one of the chains comprises thermodynamically stable tetraloop structure (see, for example, U.S. Patent Nos. 8,513,207 and 8,927,705, and WO2010033225, each of which is incorporated herein by reference in its entirety).Such structure can comprise single-stranded extension (at one or both ends of molecule) and double-stranded extension.

[0127] 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 may comprise a tetraloop sequence. In certain embodiments, the sense strand is 25-35 nucleotides in length.

[0128] The tetraloop can contain ribonucleotides, deoxyribonucleotides, modified nucleotides, and combinations thereof. Typically, a 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'-phiolo-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 the G in the GAAA sequence comprises a 2'-O-methyl modification. In a preferred embodiment, in some embodiments, each A in the GAAA sequence comprises a 2'-O-methoxyethyl (MOE) modification, and the G in the GAAA sequence comprises a 2'-O-methyl modification; or each A in the GAAA sequence comprises a 2'-adem modification, and the G in the GAAA sequence comprises a 2'-O-methyl modification. For example, see PCT Publication No. WO 2020 / 206350, the entire contents of which are incorporated herein by reference.

[0129] Exemplary 2'adem modified nucleotides are shown below.

[0130] [ka]

[0131] The term "blunt" or "blunt-ended" as used herein in reference to dsRNA means that there are no unpaired nucleotides or nucleotide analogs at a given end of the dsRNA, i.e., there are no nucleotide overhangs. One or both ends of the dsRNA can be blunt. When both ends of the dsRNA are blunt, the dsRNA is said to be blunt-ended. For clarity, a "blunt-ended" dsRNA is a dsRNA that is blunt at both ends, i.e., there are no nucleotide overhangs at either end of the molecule. In most cases, such a molecule will be double-stranded throughout its entire length.

[0132] The term "antisense strand" or "guide strand" refers to the strand of an RNAi agent, eg, a dsRNA, which includes a region that is substantially complementary to a target sequence, eg, HTT mRNA.

[0133] 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 region of complementarity is not completely complementary to the target sequence, the mismatch may be in the internal or 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 contains nucleotide mismatches in the antisense strand. In some embodiments, the antisense strand of the double-stranded RNA agent of the present invention contains four or fewer mismatches with the target mRNA, for example, the antisense strand contains four, three, two, one, or zero mismatches with the target mRNA. In some embodiments, the antisense strand of the double-stranded RNA agent of the present invention contains four or fewer mismatches with the sense strand, for example, the antisense strand contains four, three, two, one, or zero mismatches with the sense strand. In some embodiments, the double-stranded RNA agent of the present invention contains nucleotide mismatches 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 four, three, two, one, or zero 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 another embodiment, the nucleotide mismatch is, for example, at the 3' terminal nucleotide of the iRNA agent. In some embodiments, the mismatch is not in the seed region.

[0134] Thus, the RNAi agents described herein can contain one or more mismatches to the target sequence. In one embodiment, the RNAi agents described herein contain three or fewer mismatches (i.e., three, two, one, or zero mismatches). In one embodiment, the RNAi agents described herein contain two or fewer mismatches. In one embodiment, the RNAi agents described herein contain one or fewer mismatches. In one embodiment, the RNAi agents described herein contain zero mismatches. In certain embodiments, when the antisense strand of an RNAi agent contains a mismatch to the target sequence, the mismatch can also be limited to within the last five nucleotides from the 5' or 3' end of the region of complementarity, as appropriate. For example, in such an embodiment, for a 23-nucleotide RNAi agent, the strand complementary to a region of the HTT gene generally does not contain any mismatches within the central 13 nucleotides. Using the methods described herein or known in the art, it can be determined whether an RNAi agent containing a mismatch to the target sequence is effective in inhibiting expression of the HTT gene. It is important to consider the efficacy of mismatched RNAi agents in inhibiting HTT expression, particularly when the particular region of complementarity in the HTT gene is known to have polymorphic sequence variation within the population.

[0135] 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.

[0136] As used herein, the term "cleavage region" refers to the region located directly adjacent to the cleavage site.Cleavage site is the site on the target where cleavage occurs.In some embodiments, the cleavage region comprises three bases that are directly adjacent to either end of the cleavage site.In some embodiments, the cleavage region comprises two bases that are directly adjacent to either end of the cleavage site.In some embodiments, specifically, the cleavage site occurs at the site that is bound by nucleotides 10 and 11 of the antisense strand, and the cleavage region comprises nucleotides 11, 12, and 13.

[0137] As used herein, unless otherwise specified, 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 with an oligonucleotide or polynucleotide comprising the second nucleotide sequence under certain conditions, as would be understood by one of skill in the art.

[0138] The complementary sequence in an RNAi agent, for example, in the dsRNA described herein, comprises the base pairing of the oligonucleotide or polynucleotide comprising the first nucleotide sequence with the oligonucleotide or polynucleotide comprising the second nucleotide sequence throughout the entire length of one or both nucleotide sequences.Such sequences can be referred to herein as " completely complementary " with respect to each other.However, in the present specification, when a first sequence is considered to be " substantially complementary " with a second sequence, the two sequences can be completely complementary, or they can form one or more, but generally not more than 5, 4, 3, or 2 mismatched base pairs during hybridization, while maintaining the ability to hybridize under the conditions most suitable for their final use, for example, the inhibition of gene expression by the RISC pathway, in the case of a duplex of up to 30 base pairs.However, when two oligonucleotides are designed to form one or more single-stranded overhangs during hybridization, these overhangs are not considered as mismatches when 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, can still be considered "perfectly complementary" for the purposes described herein.

[0139] "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 the above requirements regarding their ability to hybridize are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogstein base pairing.

[0140] The terms "complementary," "fully complementary," and "substantially complementary" can be used herein 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 understood in connection with their use.

[0141] 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 continuous 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.

[0142] 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%, 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 to the nucleotide sequence of any of SEQ ID NOs: 1-5 or the equivalent region of a fragment of any of SEQ ID NOs: 1-5 over its entire length.

[0143] 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 80%, 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 over its entire length to any one of the sense strand nucleotide sequences in any one of Tables 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 24, 25, 27-30, 32, and 33, or a fragment of any one of the sense strand nucleotide sequences in any one of Tables 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 24, 25, 27-30, 32, and 33.

[0144] In one embodiment, an RNAi agent of the present disclosure comprises a sense strand that is substantially complementary to an antisense polynucleotide that is identical to a target HTT sequence, and the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80%, 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 to the nucleotide sequence of SEQ ID NOs:6-10 or an equivalent region of a fragment of any of SEQ ID NOs:6-10 throughout its entire length.

[0145] In some embodiments, the iRNA of the invention comprises a sense strand that is substantially complementary to an antisense polynucleotide that is complementary to a target HTT sequence, wherein the sense strand polynucleotide is any one of the antisense strand nucleotide sequences in any one of Tables 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 24, 25, 27-30, 32, and 33, or any one of the antisense strand nucleotide sequences in Tables 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 24, 25, 27-30, 32, and 33. The antisense strand nucleotide sequence of any one of the sequences 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 24, 25, 27-30, 32, and 33 is at least 80%, for example, 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 over its entire length to a fragment of any one of the antisense strand nucleotide sequences in any one of the sequences 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 24, 25, 27-30, 32, and 33.

[0146] 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 has been treated or has been treated to inhibit expression of the HTT gene compared to a second cell or group of cells (control cells) that is substantially identical to the first cell or group of cells but has not been treated as the first. The degree of inhibition is expressed in terms of the following formula:

[0147]

number

[0148] As used herein, the phrase " contacting cell with RNAi agent " such as dsRNA includes contacting cell by any possible means.Contacting cell with RNAi agent includes contacting cell with RNAi agent in vitro or contacting cell with RNAi agent in vivo.Contacting can be carried out directly or indirectly.Therefore, for example, RNAi agent can be physically contacted with cell by carrying out a method separately, or RNAi agent can be placed in a situation that can allow or cause it to contact cell afterwards.

[0149] Contacting cells in vitro can be achieved, for example, by incubating cells with an RNAi agent. Contacting cells in vivo can be achieved, 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), by intrathecal injection, intravitreal injection, or other injection, as appropriate, 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 stabilizes the RNAi agent to the desired site, for example, 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.

[0150] 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 the 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 includes methods known in the art, such as electroporation and lipofection. Further approaches are described herein below or known in the art.

[0151] The terms "lipophilic" 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 in 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., hydrophilic / lipophilic balance). In principle, a chemical can be expressed as a function of the log K ow is greater than 0, it is lipophilic in nature. Typically, a lipophilic moiety has a log K ow For example, the log K of 6-aminohexanol owis expected to be approximately 0.7. Using the same method, the log K ow is expected to be 10.7.

[0152] The lipophilicity of a molecule can be modified with respect to the functional groups it carries. For example, adding a hydroxyl or amine group to the end of the lipophilic moiety can increase the partition coefficient (e.g., logK ow ) value can be increased or decreased.

[0153] 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 properties.For example, in certain embodiments, the unbound fraction of the plasma protein binding assay of double-stranded RNAi agent can be determined to be positively correlated with the relative hydrophobicity of double-stranded RNAi agent, which can be positively correlated with the silencing activity of double-stranded RNAi agent.

[0154] In one embodiment, the plasma protein binding assay that is determined is electrophoretic mobility shift assay (EMSA) using 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 fraction of unbound 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 for enhanced in vivo delivery of siRNA.

[0155] 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.

[0156] 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, such as an RNAi agent or a plasmid from which the 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.

[0157] As used herein, a "subject" refers to an animal, for example, a mammal, such as 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, for example, a human being who is being treated or evaluated for a disease, disorder, or condition that would benefit from reduced HTT expression; a human being who is at risk for a disease, disorder, or condition that would benefit from reduced HTT expression; a human being who has a disease, disorder, or condition that would benefit from reduced HTT expression; or a human being who is 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 human female. In other embodiments, the subject is a human male. In one embodiment, the subject is a human adult. In one embodiment, the subject is a human child. In another embodiment, the subject is a human juvenile, i.e., a subject under the age of 20.

[0158] As used herein, the terms "treating" or "treatment" refer to a beneficial or desired result, 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, e.g., an HTT-related disease such as Huntington's disease. "Treatment" can also mean prolonging survival as compared to expected survival if no treatment is administered.

[0159] The term "lower" in relation to 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% of the disease marker, e.g., protein level or gene expression level. When referring to the level of HTT in a subject, "lower" preferably refers to a reduction to a level that is accepted as being within the normal range in individuals without such a disorder. In certain embodiments, "lower" refers to a reduction in the difference between the level of the 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 reduction between an obese individual and an individual with a weight that is accepted to be within the normal range.

[0160] 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 scale clinically acceptable 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.

[0161] 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.

[0162] 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 genders. The caudate nucleus degenerates, small cell populations deteriorate, and levels of the neurotransmitters gamma-aminobutyric acid (GABA) and substance P decrease. This deterioration results in the characteristic "boxcar ventricles" seen on CT scans.

[0163] 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 typically become noticeable in the fourth decade of life but can occur at any age. When the age of onset is before 20 years of age, it is known as Juvenile HD.

[0164] Dementia or psychiatric disorders, ranging from apathy and irritability to end-stage bipolar or schizophreniform disorders, may precede movement disorders or develop during their course. Anhedonia or antisocial behavior may be the first behavioral manifestation. Motor signs include throbbing movements of the limbs, stomping, motor impermanence (inability to maintain a motor act, e.g., tongue protrusion), facial grimacing, ataxia, and dystonia.

[0165] 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) that causes cell death in selected areas of the brain.

[0166] 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, improving, or maintaining the existing disease or one or more symptoms of the disease). A "therapeutically effective amount" may vary depending on the RNAi agent, how the agent is administered, the disease and its severity, as well as medical history, age, weight, family history, genetic makeup, type of previous or concurrent treatment, if any, and other individual characteristics of the subject being treated.

[0167] As used herein, the term "prophylactically effective amount" is intended to include the amount of an RNAi agent that is sufficient to prevent or ameliorate the disease or one or more symptoms of the disease when administered to a subject with an HTT-related disease. Amelioration of the disease includes slowing the progression of the disease or reducing the severity of the disease that subsequently develops. The "prophylactically effective amount" may vary depending on the RNAi agent, how the agent is administered, the degree of risk of the disease, as well as the patient's medical history, age, weight, family history, genetic makeup, type of previous or concurrent treatment, if any, and other individual characteristics of the patient being treated.

[0168] 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.

[0169] The phrase "pharmaceutically acceptable" is used herein to mean compounds, materials, compositions, or dosage forms that are suitable for use in contact with the tissues of human and animal subjects, within the scope of sound medical judgment, without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0170] The phrase "pharmaceutically acceptable carrier," as used herein, refers to 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, involved in the transport or transportation of a compound of interest from one organ or part of the body to another organ, e.g., part of the body. 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 function 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 stearate, sodium lauryl sulfate, and talc; (8) excipients, such as cocoa butter and suppository wax; and (9) oils, such as peanut oil, cottonseed oil, safflower oil, and 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) buffers, 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 affinity substances used in pharmaceutical formulations.

[0171] 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 obtained 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 entire brain or a segment in the brain, e.g., the striatum, or a type of cell in the brain, e.g., 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) obtained from a subject. ) or retinal tissue (or a subcomponent thereof).

[0172] 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 for inhibiting expression of HTT in a cell, e.g., a cell in a subject, e.g., a mammal, e.g., a human with an HTT-related disorder such as 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 about 15-30 nucleotides in length or less. Upon contact 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 assessed, for example, by PCR or branched DNA (bDNA)-based methods, or protein-based methods, such as immunofluorescence analysis using Western blotting or flow cytometry techniques. In one embodiment, the level of knockdown is assessed in Cos7 cells using a dual-luciferase assay method.

[0173] dsRNA comprises two RNA strands, which are complementary, and hybridize to form a duplex structure under the conditions that dsRNA is used.One strand (antisense strand) of dsRNA comprises a complementary region that is substantially complementary, and generally completely complementary to target sequence.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 antisense strand, so that when the two strands are combined under suitable conditions, they hybridize to form a duplex structure.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 as to be opposite on separate oligonucleotides.

[0174] Generally, the duplex structure is 15 to 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, 19-31, 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- 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, e.g., 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-25, 20-24, 20-23, 20-22, 20-21, 21-25, 21-24, 21-23, 21-22, 22-25, 22-24, 22-23, 23-25, 23-24, or 24-25 base pairs in length, e.g., 19-21 base pairs in length. Ranges and lengths intermediate to the above-listed ranges and lengths are also contemplated as part of the present disclosure.

[0175] Similarly, the region of complementarity to the target sequence may be 15 to 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 intermediate to the above-listed ranges and lengths are also contemplated as part of this disclosure.

[0176] In some embodiments, the duplex structure is 19 to 30 base pairs in length. Similarly, the region of complementarity to the target sequence is 19 to 30 nucleotides in length.

[0177] 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 function as a substrate for the Dicer enzyme. For example, it is well known in the art that dsRNA longer than about 21 to 23 nucleotides can function as a substrate for Dicer. As those skilled in the art will recognize, the region of RNA targeted for cleavage is most often a portion of a longer RNA molecule, often an mRNA molecule. In relevant cases, a "portion" of the mRNA target is a continuous sequence of the mRNA target long enough to be a substrate for RNAi-dependent cleavage (i.e., cleavage by the RISC pathway).

[0178] Those skilled in the art will appreciate that the duplex region may be a primary functional portion of a dsRNA, e.g., 15 to 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, 18-21, 18-22, 18-23, 18-24, 18-25, 18-24, 18-23, 18-22, 18-21, 18-25, 18-26, 18-27, 18-28, 18-29, 18-30, 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- It will also be appreciated that the duplex region may be 8-20, 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, e.g., 19-21 base pairs. Therefore, in one embodiment, the RNA molecule or complex of RNA molecules with a double-stranded region of more than 30 base pairs is dsRNA, as long as it is processed into a functional double-stranded region of, for example, 15-30 base pairs, which targets the desired RNA for cleavage.Therefore, those skilled in the art will recognize that in one embodiment, miRNA is dsRNA.In another embodiment, dsRNA is not naturally occurring miRNA.In another embodiment, the RNAi agent useful for targeting HTT expression is not produced in target cells by cleavage of larger dsRNA.

[0179] dsRNA described herein can further comprise one or more single-stranded nucleotide overhangs, for example, 1, 2, 3 or 4 nucleotides.Nucleotide overhangs can comprise or consist of nucleotide / nucleoside analogs such as deoxynucleotide / nucleoside.Overhangs can be on sense strand, antisense strand, or any combination thereof.In addition, the nucleotide of overhang can be on the 5' end, 3' end, or both of the antisense strand or sense strand of dsRNA.

[0180] 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 the 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 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.

[0181] In one embodiment, the dsRNA of the present disclosure comprises at least two nucleotide sequences, that is, sense strand and antisense strand.The sense strand sequence of HTT can be selected from the group of sequences provided in any one of Tables 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 24, 25, 27-30, 32 and 33, and the corresponding nucleotides of the sense strand and the antisense strand can be selected from the group of sequences provided in any one of Tables 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 24, 25, 27-30, 32 and 33.In this embodiment, one of the two sequences is complementary to the other of the two sequences, and in this case, one of the sequences is substantially complementary to the sequence of mRNA generated during the expression of HTT gene. Thus, in this embodiment, the dsRNA will comprise two oligonucleotides, where one oligonucleotide is described as the sense strand (passenger strand) in any one of Tables 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 24, 25, 27-30, 32, and 33, and the second oligonucleotide is described as the corresponding antisense strand (guide strand) to the sense strand in any one of Tables 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 24, 25, 27-30, 32, and 33.

[0182] In one embodiment, the sequences substantially complementary to the dsRNA are contained in separate oligonucleotides, hi another embodiment, the sequences substantially complementary to the dsRNA are contained in a single oligonucleotide.

[0183] Although the sequences in Tables 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 24, 25, 27-30, 32, and 33 are described as modified or conjugated sequences, it will be understood that an RNA of an RNAi agent of the disclosure, e.g., a dsRNA of the disclosure, can comprise any one of the sequences described in any one of Tables 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 24, 25, 27-30, 32, and 33 that is unmodified, unconjugated, or modified or conjugated differently than described. For example, the sense strands of the agents of the invention shown in Tables 3, 9, 12, 15, 17, 27, 29, and 32 are conjugated to GalNAc ligands, although these agents may also be conjugated to moieties that direct delivery to the CNS, e.g., C16 ligands, as described herein. Lipophilic ligands can be included at any of the positions provided herein.

[0184] Those skilled in the art are well aware that dsRNAs with duplex structures of about 20 to 23 base pairs, for example, 21 base pairs, are hailed as being particularly effective in inducing RNA interference [Elbashir et al., (2001) EMBO J., 20:6877-6888]. However, others have found 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 embodiment, due to the nature of the oligonucleotide sequences provided herein, the dsRNAs described herein can comprise at least one strand with a minimum length of 21 nucleotides. It can be reasonably expected that shorter duplexes, minus 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 that 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 complete sequence using in vitro assays with Cos7 and 10 nM concentrations of the RNA agent and the PCR assays provided in the Examples herein are contemplated to be within the scope of this disclosure.

[0185] In addition, the RNA described herein specifies the site of HTT transcript that is susceptible to RISC-mediated cleavage.Therefore, the present disclosure further features the RNAi agent that targets within this site.As used herein, if an RNAi agent promotes the cleavage of the transcript at any of specific sites, it is said to target within specific site of RNA transcript.Such RNAi agent will generally comprise at least about 15 nucleotides, preferably at least 19 nucleotides, from one of the sequences provided herein, which is coupled with additional nucleotide sequence taken from the region adjacent to the selected sequence in HTT gene.

[0186] III. Modified RNAi Agents of the Present Disclosure In one embodiment, the RNA of the RNAi agent of the present disclosure, for example, dsRNA, is unmodified, and does not include, for example, chemical modifications or conjugations known in the art and described herein.In a preferred embodiment, the RNA of the RNAi agent of the present disclosure, for example, 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.The RNAi agent of the present disclosure in which "substantially all of the nucleotides are modified" is mostly, but not entirely, modified, and may include 5, 4, 3, 2, or unmodified nucleotides.In yet other embodiments of the present disclosure, the RNAi agent of the present disclosure may include 5, 4, 3, 2, or 1 modified nucleotide.

[0187] 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, S.L. et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, incorporated herein by reference. Modifications include, for example, terminal modifications, such as 5'-end modifications (phosphorylation, conjugation, reverse ligation) or 3'-end modifications (conjugation, DNA nucleotides, reverse ligation, etc.), base modifications, such as replacement with a stabilizing base, a destabilizing base, or a base that base-pairs with an expanded repertoire partner, removal of a base (abasic nucleotide) or a conjugated base, sugar modifications (e.g., at the 2' or 4' position) or sugar replacement, or backbone modifications, including modification or replacement of a phosphodiester bond. Specific examples of RNAi agents useful in the embodiments described herein include, but are not limited to, RNAs containing modified backbones or that do not contain natural internucleoside linkages. Among the RNAs with modified backbone, those that do not have phosphorus atom in backbone include.For the purpose of this specification, as sometimes referred to in the art, the modified RNA that does not have phosphorus atom in its internucleoside backbone can also be considered as oligonucleoside.In some embodiments, modified RNAi agent has phosphorus atom in its internucleoside backbone.

[0188] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methylphosphonates, 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 analogs linked in 2'-5', and those with reverse polarity, where adjacent pairs of nucleoside units are linked in 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 agent of the present invention is in free acid form. In other embodiments of the present invention, the dsRNA agent of the present invention is 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 the counterion for 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 the counterion for all of the phosphodiester and / or phosphorothioate groups present in the agent.

[0189] 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. 5,476 , No. 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. 5,5 87,361, 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. 6,534,639 , 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. Reissue Patent No. RE39464, the entire contents of each of which are incorporated herein by reference.

[0190] Modified RNA backbones that do not contain phosphorus atoms 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. These include 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.

[0191] 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.

[0192] In other embodiments, RNA mimetics suitable for use in RNAi agents are contemplated, in which both sugar and internucleoside linkage, i.e., the backbone of nucleotide unit, are replaced with novel groups. Base unit is maintained for hybridization with appropriate nucleic acid target compound. One such oligomeric compound, an RNA mimetic that has been found 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. Nucleic acid bases are retained and are directly or indirectly linked to the aza nitrogen atom of the amide part of the backbone. Representative US patents that teach the preparation of PNA compounds include, but are not limited to, US 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.

[0193] Some embodiments featured in this disclosure include RNAs 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-- (the natural phosphodiester backbone is represented as --O--P--O--CH2--) of the above-referenced U.S. Patent No. 5,489,677, and oligonucleosides with amide backbones of the above-referenced U.S. Patent No. 5,602,240. In some embodiments, the RNAs featured herein have the morpholino backbone structure of the above-referenced U.S. Patent No. 5,034,506.

[0194] Modified RNAs may also contain one or more substituted sugar moieties. The RNAi agents, e.g., dsRNAs, featured herein may contain 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 and 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 contains at the 2' position: C1 to C 10The modification may include one of the following: lower 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 group, reporter group, interfering substance, group for improving the pharmacokinetic properties of RNAi agents or group 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 in these three families), 2'-alkoxyalkyl, and 2'-NMA (N-methylacetamide).

[0195] 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 at the 3'-position of the sugar on the 3'-terminal nucleotide or in a 2'-5'-linked dsRNA and at the 5'-position of the 5'-terminal nucleotide. RNAi agents can also have sugar mimetics, such as cyclobutyl moieties instead of pentofuranosyl sugars. 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, certain of which are commonly owned with the present application, the entire contents of each of which are incorporated herein by reference.

[0196] The RNAi agents of the present disclosure may also include nucleobase (often simply referred to in the art as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic 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, 5-uracil ... These include 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and adenine, 8-azaguanine and adenine, 7-deazaguanine and adenine, and 3-deazaguanine and adenine.Additional nucleobases include those disclosed in U.S. 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, J. L, ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., (1991) Angewandte Chemie, International Edition, 30:613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., Ed., CRC Press, 1993. Certain 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 O-6 substituted purines, including 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, YS, Crooke, ST, and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), and are exemplary base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications.

[0197] Representative United States patents that teach the preparation of certain of the above-mentioned modified nucleobases, as well as other modified nucleobases, include, but are not limited to, the above-mentioned 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.

[0198] The RNAi agent of the present disclosure can also be modified to include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide with a modified ribose moiety, which includes an additional bridge connecting the 2' and 4' carbons of the ribose moiety. This structure effectively "locks" the ribose in a 3'-endo conformation. The addition of locked nucleic acids 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 anc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193].

[0199] The RNAi agents of the present disclosure can also be modified to include one or more bicyclic sugar moieties. A "bicyclic sugar" is a furanosyl ring modified by bridging two atoms. A "bicyclic nucleoside" ("BNA") is a nucleoside having a sugar moiety containing a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring structure. In certain embodiments, the bridge connects the 4'-carbon and 2'-carbon of the sugar ring. Thus, in some embodiments, the agents of the present disclosure can 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 contains an additional bridge connecting the 2' and 4' carbons. In other words, an LNA is a nucleotide containing a bicyclic sugar moiety containing a 4'-CH2-O-2' bridge. This structure effectively "locks" the ribose into a 3'-endo conformation. The addition of a locked 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 disclosure include, but are not limited to, nucleosides comprising a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, the antisense polynucleotide agent of the present disclosure includes one or more bicyclic nucleosides comprising a 4' to 2' bridge.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 known as "constrained ethyl" or "cEt") and 4'-CH(CHOCH3)-O-2' (and analogs, see e.g., U.S. Pat. No. 7,399,845), 4'-C(CH3)(CH3)-O-2' (and analogs, see e.g., U.S. Pat. No. 8,399,845). 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 Publication No. 2004 / 0171570), 4'-CH2-N(R)-O-2' (wherein R is H, C1-C12 alkyl, or a 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 which are incorporated herein by reference.

[0200] Additional representative U.S. patents and publications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: 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, Nos. 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, US2008 / 0039618 and US2009 / 0012281, the entire contents of each of which are incorporated herein by reference.

[0201] For example, any of the above bicyclic nucleosides can be prepared with one or more stereochemical sugar configurations including α-L-ribofuranose and β-D-ribofuranose (see WO 99 / 14226).

[0202] RNAi agents of the present disclosure can also be modified to include 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)-0 to 2' bridge. In one embodiment, the constrained ethyl nucleotide is in the S conformation and is referred to herein as an "S-cEt."

[0203] The RNAi agents of the present disclosure may also contain one or more "conformation-restricting 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 and increases hybridization affinity to mRNA. The linker is of sufficient length to position the oxygen in an optimal position for stability and affinity, resulting in less ribose ring puckering.

[0204] Representative publications that teach the preparation of certain of the above CRNs include, but are not limited to, US2013 / 0190383 and WO2013 / 036868, the entire contents of each of which are incorporated herein by reference.

[0205] In some embodiments, the RNAi agent of the present disclosure comprises one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNA is an unlocked acyclic nucleic acid, and any of the sugar bonds have been removed to form an unlocked "sugar" residue. In one example, UNA also encompasses a monomer in which the bond between C1'-C4' (i.e., the covalent carbon-oxygen-carbon bond between the C1' and C4' carbons) has been removed. In another example, the C2'-C3' bond of the sugar (i.e., the 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].

[0206] Representative U.S. publications that teach the preparation of UNAs include, but are not limited to, U.S. Pat. Nos. 8,314,227 and U.S. Patent Publication Nos. 2013 / 0096289, 2013 / 0011922, and 2011 / 0313020, the entire contents of each of which are incorporated herein by reference.

[0207] Potentially stabilizing modifications to the ends of RNA molecules 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 base dT (idT), and others. Disclosure of this modification can be found in WO2011 / 005861.

[0208] Other modifications of the RNAi agent of the present disclosure include 5' phosphate or 5' phosphate mimic, for example, the 5' terminal phosphate or phosphate mimic on the antisense strand of RNAi agent.Suitable phosphate mimic is disclosed in, for example, US2012 / 0157511, the entire content of which is incorporated herein by reference.

[0209] A. Modified RNAi Agents Containing 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, for example, as disclosed in WO2013 / 075035, the entire contents of which are incorporated herein by reference.As shown herein and in WO2013 / 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 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 disrupts the modification pattern of the sense or antisense strand, if present.The RNAi agent can also be conjugated with a lipophilic ligand, for example, a C16 ligand on the sense strand.The RNAi agent can also be modified, for example, with (S)-glycol nucleic acid (GNA) modification at one or more residues of the antisense strand.The resulting RNAi agent exhibits excellent gene silencing activity.

[0210] Thus, the present disclosure provides double-stranded RNAi agents capable of inhibiting the 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. For example, each strand can be 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.

[0211] 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 19-21 nucleotide pairs in length.

[0212] In one embodiment, the RNAi agent may contain one or more overhang regions or capping groups at the 3', 5', or both ends of one or both strands. The overhangs may be 1 to 6 nucleotides in length, e.g., 2 to 6 nucleotides, 1 to 5 nucleotides, 2 to 5 nucleotides, 1 to 4 nucleotides, 2 to 4 nucleotides, 1 to 3 nucleotides, 2 to 3 nucleotides, 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 staggered. 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 joined by additional bases, for example, to form a hairpin, or by other non-basic linkers.

[0213] In one embodiment, the nucleotides in the overhang region of an RNAi agent can each independently be a modified or unmodified nucleotide, including, but not limited to, a 2'-sugar modified, e.g., 2-F, 2'-O-methyl, thymidine (T), and any combination thereof.

[0214] For example, TT can be an overhang sequence at either end on either strand. The overhang can form a mismatch with the target mRNA, or can be complementary to the targeted gene sequence, or can be another sequence.

[0215] 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(s) contain two nucleotides with 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.

[0216] 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 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 the 5' end is blunt.Without wishing to be bound by theory, the blunt end at the 5' end of the asymmetric antisense strand and the 3' end overhang of the antisense strand are favorable for the guide strand loading into the RISC process.

[0217] In one embodiment, the RNAi agent is a 19-nucleotide double-ended bluntmer, where the sense strand contains 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 contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0218] In another embodiment, the RNAi agent is a 20-nucleotide double-ended bluntmer, and the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 8, 9, and 10 from the 5' end. The antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0219] In yet another embodiment, the RNAi agent is a 21-nucleotide double-ended bluntmer, and the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 11 from the 5' end. The antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0220] In one embodiment, the RNAi agent comprises a 21-nucleotide sense strand and a 23-nucleotide antisense strand, wherein the sense strand contains 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 contains 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 contains 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 the three nucleotides being overhanging nucleotides, and the third nucleotide being the next paired nucleotide after 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, in the alternating motif.The RNAi agent may further comprise a ligand (for example, a lipophilic ligand, optionally a C16 ligand).

[0221] In one embodiment, the RNAi agent comprises a sense and an antisense strand, wherein the sense strand is 25-30 nucleotide residues in length, and starting from the 5'-most nucleotide (position 1), positions 1-23 of the first strand comprise at least 8 ribonucleotides; and the antisense strand is 36-66 nucleotide residues in length, and starting from the 3'-most nucleotide, comprises at least 8 ribonucleotides at positions that pair with positions 1-23 of the sense strand to form a duplex, wherein at least the 3'-most nucleotide of the antisense strand is not paired with the sense strand, and up to six consecutive 3'-most nucleotides are not paired with the sense strand, thereby forming a 3' single-stranded overhang of 1-6 nucleotides. The double-stranded nucleic acid comprises at least 19 ribonucleotides of the sense strand, forming a 10-30 nucleotide single-stranded 5' overhang, at least the 5'- and 3'-terminal nucleotides of the sense strand being base-paired with nucleotides of the antisense strand when the sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially double-stranded region between the sense and antisense strands, the antisense strand being sufficiently complementary to the target RNA along at least 19 ribonucleotides of the length of the antisense strand, and reducing target gene expression when the double-stranded nucleic acid is introduced into a mammalian cell, the sense strand containing 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, and the antisense strand containing at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

[0222] In one embodiment, the RNAi agent comprises a sense and an antisense strand, the RNAi agent comprising a first strand having a length of at least 25 and at most 29 nucleotides, and a second strand having a length of at most 30 nucleotides and having 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 1-4 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 second strand length, the RNAi agent reducing target gene expression when introduced into a mammalian cell, and Dicer cleavage of the RNAi agent preferentially yielding siRNAs comprising the 3' end of the second strand, thereby reducing target gene expression in the mammal. Optionally, the RNAi agent may further comprise a ligand.

[0223] 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 in the sense strand.

[0224] 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 the motifs occurring at or near the cleavage site in the antisense strand.

[0225] For RNAi agents with a duplex region 17-23 nucleotides long, the cleavage sites in the antisense strand are typically approximately positions 10, 11, and 12 from the 5' end. Thus, three identical modification motifs may occur in the antisense strand at positions 9, 10, and 11; positions 10, 11, and 12; positions 11, 12, and 13; positions 12, 13, and 14; or positions 13, 14, and 15, with the numbers starting from the first nucleotide from the 5' end of the antisense strand, or the numbers starting from the first paired nucleotide in the duplex region from the 5' end of the antisense strand. The cleavage site in the antisense strand may also vary depending on the length of the duplex region of the RNAi agent from the 5' end.

[0226] The sense strand of RNAi agent can contain at least one motif of three identical modifications on three consecutive nucleotides at the site of strand breakage, and antisense strand can have at least one motif of three identical modifications on three consecutive nucleotides at or near the site of strand breakage.When sense strand and antisense strand form dsRNA duplex, sense strand and antisense strand can be arranged 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 pairs.Alternatively, at least two nucleotides can overlap, or all three nucleotides can overlap.

[0227] In one embodiment, the sense strand of an RNAi agent may contain two or more motifs of three identical modifications on three consecutive nucleotides. The first motif may occur at or near the cleavage site of the strand, and the other motifs may be wing modifications. As used herein, the term "wing modification" refers to a motif occurring in another part of the strand, away from a motif at or near the cleavage site 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 chemistry of the motifs is distinct from each other, and when the motifs are separated by one or more nucleotides, the chemistry 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.

[0228] Similar to the sense strand, the antisense strand of an RNAi agent may contain two or more motifs of three identical modifications on three consecutive nucleotides, with at least one of the motifs occurring at or near the site of strand cleavage.The antisense strand may also contain one or more wing modifications in the same sequence as the wing modifications that may be present on the sense strand.

[0229] 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.

[0230] 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 in the duplex region at the 3' end, 5' end, or both ends of the strand.

[0231] When the sense and antisense strands of an RNAi agent each contain at least one wing modification, the wing modifications can fall at the same end of the duplex region and have an overlap of 1, 2, or 3 nucleotides.

[0232] When the sense or antisense strand of an RNAi agent each contains at least two wing modifications, the sense and antisense strands can be arranged such that two modifications from one strand each occupy one end of the duplex region, with an overlap of 1, 2, or 3 nucleotides; two modifications from one strand each occupy the other end of the duplex region, with an overlap of 1, 2, or 3 nucleotides; and two modifications from one strand occupy either side of the lead motif, with an overlap of 1, 2, or 3 nucleotides in the duplex region.

[0233] In one embodiment, the RNAi agent contains mismatch(es) or combinations thereof within the double strand with the target. Mismatches can occur in overhang regions or double-stranded regions. Base pairs can be ranked based on their tendency to promote dissociation or melting (e.g., the free energy of association or dissociation of a particular pairing; the simplest approach is to examine pairs on an individual basis, but next-neighbor analysis or similar analysis can also be used). In terms of promoting dissociation, A:U is preferred over G:C, G:U is preferred over G:C, and I:C is preferred over G:C (I=inosine). Mismatches, such as non-canonical pairings or those other than canonical pairings (as described elsewhere herein), are preferred over canonical (A:T, A:U, G:C) pairings, and pairings involving universal bases are preferred over canonical pairings.

[0234] In one embodiment, the RNAi agent comprises at least one of the first 1, 2, 3, 4, or 5 base pairs within the duplex region from the 5' end of the antisense strand independently selected from the group of A:U, G:U, I:C, and mismatch pairs, e.g., non-canonical pairings or other than canonical pairings or pairings including universal bases, to promote dissociation of the antisense strand at the 5' end of the duplex.

[0235] In one embodiment, the nucleotide at position 1 in the double-stranded region from the 5' end of the antisense strand is selected from the group consisting of A, dA, dU, U and dT.Alternatively, at least one of the first 1, 2 or 3 base pairs in the double-stranded region from the 5' end of the antisense strand is an AU base pair.For example, the first base pair in the double-stranded region from the 5' end of the antisense strand is an AU base pair.

[0236] 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 at the 3' end of the sense or antisense strand.

[0237] In one embodiment, the sense strand sequence has formula (I): 5'n p -N a -(XXX) i -N b -YYY -N b -(ZZZ) j -N a -n q 3' (I) [In the formula, i and j each independently represent 0 or 1; p and q each independently represent 0 to 6; each N a independently represent oligonucleotide sequences comprising 0 to 25 modified nucleotides, each sequence comprising at least two differently modified nucleotides; each N bindependently represent an oligonucleotide sequence comprising 0 to 10 modified nucleotides; each n p and n q independently represent an overhanging nucleotide; 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, all of YYY are 2'-F modified nucleotides.

[0238] In one embodiment, N a or N b includes alternating pattern modifications.

[0239] In one embodiment, the YYY motif occurs at or near the cleavage site of the sense strand. For example, if the RNAi agent has a duplex region 17-23 nucleotides in length, the YYY motif can occur at or near the cleavage site of the sense strand (e.g., can occur at positions 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12, or 11, 12, 13), with the number starting from the first nucleotide from the 5' end, or, optionally, the number starting from the first paired nucleotide within the duplex region from the 5' end.

[0240] In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or i and j are both 1. Thus, the sense strand has the following formula: 5' n p -N a -YYY-N b -ZZZ-N a -n q 3' (Ib), 5' n p -N a -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:

[0241] 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.

[0242] each N a can independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0243] 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 may independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0244] 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 may independently represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0245] Each of X, Y and Z may be the same as or different from one another.

[0246] In other embodiments, i is 0, 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:

[0247] When the sense strand is represented by formula (Ia), each N a may independently comprise an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0248] In one embodiment, the antisense strand sequence of the RNAi has the 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) [In the formula, 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 comprising 0 to 25 modified nucleotides, each sequence comprising 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, X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides. It can be expressed as:

[0249] In one embodiment, N a ' or N b ' includes alternating pattern modifications.

[0250] The Y'Y'Y' motif occurs at or near the cleavage site of the sense strand. For example, if the RNAi agent has a duplex region 17-23 nucleotides long, the Y'Y'Y' motif can occur at positions 9, 10, 11, 10, 11, 12, 11, 12, 13, 12, 13, 14, or 13, 14, 15 of the antisense strand, the numbers starting from the first nucleotide from the 5' end, or, where appropriate, the numbers starting from the first paired nucleotide in the duplex region from the 5' end. Preferably, the Y'Y'Y' motif occurs at positions 11, 12, 13.

[0251] In one embodiment, the Y'Y'Y' motif is all 2'-OMe modified nucleotides.

[0252] 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.

[0253] 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:

[0254] 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 represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0255] When the antisense strand is represented by formula (IIc), N b ' represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. a ' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0256] 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.

[0257] In other embodiments, k is 0, l is 0, and the antisense strand has the formula: 5' n p’ -N a’ -Y'Y'Y'- N a’ -n q’ 3' (Ia) It can be expressed as:

[0258] When the antisense strand is represented by formula (IIa), each N a ' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0259] Each of X', Y' and Z' may be the same as or different from one another.

[0260] 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.

[0261] In one embodiment, the sense strand of the RNAi agent may 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, where appropriate, the numbers may start from the 5' end with the first paired nucleotide in the duplex region, and Y represents a 2'-F modification. The sense strand may further contain a XXX motif or a ZZZ motif as a wing modification at the opposite end of the duplex region, where XXX and ZZZ each independently represent a 2'-OMe modification or a 2'-F modification.

[0262] In one embodiment, the antisense strand may contain a Y'Y'Y' motif occurring at positions 11, 12, 13 of the strand, where the number starts from the first nucleotide from the 5' end, or, where appropriate, the number may start 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, where X'X'X' and Z'Z'Z' each independently represent a 2'-OMe modification or a 2'-F modification.

[0263] 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 formulas (IIa), (IIb), (IIc) and (IId), respectively.

[0264] Thus, an RNAi agent for use in the methods of the disclosure can include a sense strand and an antisense strand, each strand having 14-30 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) [In the formula, 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 ’ independently represent oligonucleotide sequences comprising 0 to 25 modified nucleotides, each sequence comprising at least two differently modified nucleotides; each N b and N b ’ independently represent an oligonucleotide sequence comprising 0 to 10 modified nucleotides; each n p ',n p , n q ' and n q independently represent overhanging nucleotides, each of which may or may not be present; 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. is expressed by

[0265] 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.

[0266] An exemplary combination of sense and antisense strands that form an RNAi duplex has the following 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) Includes.

[0267] 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.

[0268] 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.

[0269] 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.

[0270] 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 comprises an alternating pattern of modifications.

[0271] In one embodiment, when the RNAi agent is represented by formula (IIId), N a The modification is a 2'-O-methyl or a 2'-fluoro modification. In another embodiment, when the RNAi agent is represented by formula (IIId), N a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p In yet another embodiment, when the RNAi agent is represented by formula (IIId), N' is linked to the adjacent nucleotide via a phosphorothioate linkage. a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n pIn another embodiment, when the RNAi agent is represented by formula (IIId), N' is linked to adjacent nucleotides via phosphorothioate linkages, and the sense strand is conjugated to one or more C16 (or related) moieties attached by a bivalent or trivalent branched linker (described below). a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p ' are linked to adjacent nucleotides via phosphorothioate linkages, and the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more lipophilic, e.g., C16 (or related) moieties, which may be attached by a divalent or trivalent branched linker.

[0272] In one embodiment, when the RNAi agent is represented by formula (IIIa), N a The modifications are 2'-O-methyl or 2'-fluoro modifications, n p '>0 and at least one n p ' are linked to adjacent nucleotides via phosphorothioate linkages, and the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more lipophilic, e.g., C16 (or related) moieties attached by a divalent or trivalent branched linker.

[0273] In one embodiment, the RNAi agent is a multimer that contains at least two 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. The multimer may further comprise a ligand. Each double strand may target the same gene, or may target two different genes, or each double strand may target the same gene at two different target sites.

[0274] In one embodiment, the RNAi agent is a multimer that contains 3, 4, 5, 6 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. The multimer may further comprise a ligand. Each double strand may target the same gene, or may target two different genes, or each double strand may target the same gene at two different target sites.

[0275] In one embodiment, two RNAi agents represented by formula (III), (IIIa), (IIIb), (IIIc) and (IIId) are linked to each other at the 5' end, and one or both of the 3' ends may be conjugated to a ligand. Each of the agents may target the same gene, or may target two different genes, or each of the agents may target the same gene at two different target sites.

[0276] Various publications describe the multimeric RNAi agent that can be used in the method of the present disclosure.Such publications include WO2007 / 091269, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887 and WO2011 / 031520 and US7858769, each of whose entire contents is incorporated herein by reference.

[0277] 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 vinyl phosphonates of the present disclosure have the following structure:

[0278] [ka] It has.

[0279] The vinyl phosphonate of the present disclosure can be attached to either the antisense or sense strand of the dsRNA of the present disclosure. In certain preferred embodiments, the vinyl phosphonate of the present disclosure is attached to the antisense strand of the dsRNA at the 5' end of the antisense strand of the dsRNA, as appropriate.

[0280] Vinyl phosphate modifications are also contemplated for the compositions and methods of the present disclosure. Exemplary vinyl phosphate structures include:

[0281] [ka] There is.

[0282] i.Thermal destabilization modification In certain embodiments, dsRNA molecules can be optimized for RNA interference by incorporating a thermally destabilizing modification into the seed region of the antisense strand (i.e., positions 2-9 at 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 thermally destabilizing duplex modification 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., 1, 2, 3, 4, 5, or more) thermally destabilizing duplex modification within the first 9 nucleotide positions of the 5' region of the antisense strand. In some embodiments, one or more thermally destabilizing duplex modifications are located in positions 2-9, or preferably positions 4-8, from the 5' end of the antisense strand. In some further embodiments, the thermally destabilizing duplex modification(s) are located at positions 6, 7, or 8 from the 5' end of the antisense strand. In still 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(s)" includes modification(s) that will result in a dsRNA having a lower overall melting temperature (Tm) (preferably 1, 2, 3, or 4 degrees lower than the Tm of a dsRNA without such modification(s). 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.

[0283] 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 or acyclic nucleotides, such as unlocked nucleic acids (UNAs) or glycol nucleic acids (GNAs).

[0284] Exemplary abasic modifications include, but are not limited to, the following:

[0285] [ka] wherein R=H, Me, Et, or OMe; R′=H, Me, Et, or OMe; and R″=H, Me, Et, or OMe.

[0286] [ka] wherein B is a modified or unmodified nucleobase. Examples include:

[0287] Exemplary sugar modifications include, but are not limited to, the following:

[0288] [ka] wherein B is a modified or unmodified nucleobase. Examples include:

[0289] In some embodiments, the thermally destabilizing modification of the duplex is one of the following:

[0290] [ka] where B is a modified or unmodified nucleobase and the asterisk on each structure represents either R, S or racemic. is selected from the group consisting of:

[0291] The term "acyclic nucleotide" refers to any nucleotide having an acyclic ribose sugar, e.g., in which any of the bonds between the ribose carbons (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

[0292] [ka] wherein B is a modified or unmodified nucleobase and R 1 and R 2 are independently H, halogen, OR3, or alkyl; and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar. The term "UNA" refers to an unlocked acyclic nucleic acid in which one of the sugar bonds has been removed to form an unlocked "sugar" residue. In one example, UNA also encompasses a monomer in which the C1'-C4' bond has been removed (i.e., a covalent carbon-oxygen-carbon bond between the C1' and C4' carbons). In another example, the C2'-C3' bond of the sugar (i.e., a covalent carbon-carbon 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.

[0293] The term "GNA" refers to glycol nucleic acid, a polymer similar to DNA or RNA, but differing in the composition of its "backbone" in that it is made up of repeating glycerol units linked by phosphodiester bonds:

[0294] [ka]

[0295] The thermally destabilizing modification of the double strand 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 combinations thereof.Other mismatch base pairings known in the art are also suitable for the present invention.Mismatches can occur between nucleotides that are either naturally occurring or modified nucleotides, that is, mismatch base pairing can occur between the nucleobases derived from each nucleotide independently of the modification on the ribose sugar of the nucleotide.In certain embodiments, the dsRNA molecule contains at least one nucleobase in mismatch pairing that is a 2'-deoxynucleobase, for example, the 2'-deoxynucleobase is in the sense strand.

[0296] In some embodiments, the duplex thermally destabilizing modification in the seed region of the antisense strand is a nucleotide that has impaired WCH bonding with the complementary base on the target mRNA, such as:

[0297] [ka] Includes.

[0298] Many more examples of abasic nucleotides, acyclic nucleotide modifications (including UNA and GNA) and mismatch modifications are described in detail in WO2011 / 133876, which is incorporated herein by reference in its entirety.

[0299] Thermally destabilizing modifications can also include universal base and phosphate modifications that have reduced or eliminated ability to form hydrogen bonds with opposing bases.

[0300] In some embodiments, the thermal destabilizing modification of duplex comprises the nucleotide with non-canonical base, for example but not limited to, the nucleobase modification that the ability to form hydrogen bond with the base in opposite strand is impaired or completely lost.These nucleobase modifications have been evaluated for the destabilization of the central region of dsRNA duplex, as described in WO2010 / 0011895, the entirety of which is incorporated herein by reference.Exemplary nucleobase modifications include:

[0301] [ka] There is.

[0302] In some embodiments, the duplex thermally destabilizing modifications 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 the following:

[0303] [ka] wherein R is H, OH, OCH, F, NH, NHMe, NMe, or O-alkyl. Includes:

[0304] Exemplary phosphate modifications known to reduce the thermal stability of dsRNA duplexes compared to native phosphodiester linkages include:

[0305] [ka] There is.

[0306] The alkyl R group can be a C1-C6 alkyl. Particular alkyl R groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, pentyl, and hexyl.

[0307] 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, for introducing destabilizing modifications into the RNAi agent of the present disclosure, but the range of modifications that can be used and generally exist on the RNAi agent of the present disclosure tends to be greater for non-nucleobase modifications, for example, modifications to the sugar group or phosphate backbone of polyribonucleotide.Such modifications are described in more detail in other sections of this 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.

[0308] In addition to the antisense strand that contains thermal destabilizing modifications, dsRNA can also contain one or more stabilizing modifications.For example, dsRNA can contain at least two (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) stabilizing modifications.Without being limited, all stabilizing modifications can be present in one strand.In some embodiments, both sense and antisense strands contain at least two stabilizing modifications.Stabilizing modifications can occur at any nucleotide of sense strand or antisense strand.For example, stabilizing modifications can occur at any nucleotide on sense strand or antisense strand, and each stabilizing modification can occur in an alternating pattern on sense strand or antisense strand, or both sense strand and antisense strand contain stabilizing modifications in an alternating pattern.The alternating pattern of stabilizing modifications on sense strand can be the same or different from that of antisense strand, and the alternating pattern of stabilizing modifications on sense strand can have a shift compared to the alternating pattern of stabilizing modifications on antisense strand.

[0309] In some embodiments, the antisense strand comprises at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) stabilizing modifications. Without limitation, the stabilizing modifications in 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 some other embodiments, the antisense strand comprises stabilizing modifications at positions 2, 14, and 16 from the 5' end.

[0310] In some embodiments, antisense strand comprises at least one stabilizing modification adjacent to destabilizing modification.For example, stabilizing modification can be at the 5'-end or 3'-end of destabilizing modification, that is, at the nucleotide of position -1 or +1 from the position of destabilizing modification.In some embodiments, antisense strand comprises stabilizing modification at each of the 5'-end and 3'-end of destabilizing modification, that is, at the nucleotide of position -1 and +1 from the position of destabilizing modification.

[0311] 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.

[0312] In some embodiments, the sense strand comprises at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) stabilizing modifications. Without limitation, the stabilizing modifications in 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 blocks of two, three, or four stabilizing modifications.

[0313] 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.

[0314] Exemplary thermally stabilizing modifications include, but are not limited to, 2'-fluoro modifications. Other thermally stabilizing modifications include, but are not limited to, LNA.

[0315] In some embodiments, the dsRNA of the present disclosure comprises at least four (for example, 4, 5, 6, 7, 8, 9, 10 or more) 2'-fluoro nucleotides. Without being limited thereto, all 2'-fluoro nucleotides may be present in one strand. In some embodiments, both sense and antisense strands comprise at least two 2'-fluoro nucleotides. 2'-fluoro modification can occur at any nucleotide of sense strand or antisense strand. For example, 2'-fluoro modification can occur at any nucleotide on sense strand or antisense strand, and each 2'-fluoro modification can occur in an alternating pattern on sense strand or antisense strand, or both sense strand and antisense strand contain 2'-fluoro modification in an alternating pattern. The alternating pattern of 2'-fluoro modification on sense strand can be the same or different from that of antisense strand, and the alternating pattern of 2'-fluoro modification on sense strand can have a shift compared to the alternating pattern of 2'-fluoro modification on antisense strand.

[0316] In some embodiments, the antisense strand comprises at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-fluoro nucleotides. Without limitation, the 2'-fluoro modifications in the antisense strand can be present at any position. In some embodiments, the antisense strand comprises 2'-fluoro nucleotides at positions 2, 6, 8, 9, 14 and 16 from the 5' end. In some other embodiments, the antisense strand comprises 2'-fluoro nucleotides at positions 2, 6, 14 and 16 from the 5' end. In yet some other embodiments, the antisense strand comprises 2'-fluoro nucleotides at positions 2, 14 and 16 from the 5' end.

[0317] In some embodiments, antisense strand comprises at least one 2'-fluoro nucleotide adjacent to destabilizing modification.For example, 2'-fluoro nucleotide can be at the 5' end or 3' end of destabilizing modification, that is, at the nucleotide of position -1 or +1 from the position of destabilizing modification.In some embodiments, antisense strand comprises 2'-fluoro nucleotide at each of the 5' end and 3' end of destabilizing modification, that is, at the position -1 and +1 from the position of destabilizing modification.

[0318] 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.

[0319] In some embodiments, the sense strand comprises at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-fluoro nucleotides. Without limitation, the 2'-fluoro modifications in the sense strand can be present at any position. In some embodiments, the antisense strand comprises 2'-fluoro nucleotides at positions 7, 10 and 11 from the 5' end. In some other embodiments, the sense strand comprises 2'-fluoro nucleotides at positions 7, 9, 10 and 11 from the 5' end. In some embodiments, the sense strand comprises 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 comprises 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 comprises 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 comprises 2, 3 or 4 blocks of 2'-fluoro nucleotides.

[0320] In some embodiments, the sense strand does not contain 2'-fluoro nucleotides at positions opposite or complementary to thermally destabilizing modifications of the duplex in the antisense strand.

[0321] In some embodiments, a dsRNA molecule of the present disclosure comprises a 21-nucleotide (nt) sense strand and a 23-nucleotide (nt) antisense strand, wherein the antisense strand contains at least one thermally destabilized nucleotide, wherein the at least one thermally destabilized nucleotide occurs in the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), one end of the dsRNA is blunt and the other end comprises a 2-nt overhang, and the dsRNA further has at least one (e.g., 1, 2, 3, 4, 5, 6, or all 7) of the following features: The dsRNA may comprise: (i) the antisense strand comprises 2, 3, 4, 5, or 6 2'-fluoro modifications, (ii) the antisense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages, (iii) the sense strand is conjugated with 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, and (vii) the dsRNA comprises 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.

[0322] In some embodiments, a dsRNA molecule of the present disclosure comprises a sense 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), positions 1 to 23 of the sense strand comprise at least 8 ribonucleotides; the antisense strand is 36 to 66 nucleotide residues in length, and starting from the 3'-terminal nucleotide, at least 8 ribonucleotides in positions paired with positions 1 to 23 of the sense strand 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 3' single-stranded overhang of 1 to 6 nucleotides; and the 5'-end of the antisense strand is 10 to 30 consecutive nucleotides that are not paired with the sense strand. the antisense strand comprises a nucleotide sequence of at least 19 ribonucleotides along its length, thereby forming a single-stranded 5' overhang of 10 to 30 nucleotides, wherein at least the 5'- and 3'-terminal nucleotides of the sense strand are base-paired with nucleotides in the antisense strand when the sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially double-stranded region between the sense and antisense strands, the antisense strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides along its length, thereby reducing target gene expression when the double-stranded nucleic acid is introduced into a mammalian cell, and the antisense strand contains at least one thermally destabilizing nucleotide, the at least one thermally destabilizing nucleotide being in 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 thermally destabilizing nucleotide occurs between positions 14-17 at the 5' end of the sense strand and the positions opposite or complementary to them, and the dsRNA may further have at least one (e.g., one, two, three, four, five, six, or all seven) of the following features: (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; (vi) the dsRNA contains at least four 2'-fluoro modifications; and (vii) the dsRNA contains a duplex region 12-30 nucleotide pairs in length.

[0323] In some embodiments, a dsRNA molecule of the present disclosure comprises a sense and an antisense strand, wherein the dsRNA molecule comprises a sense strand having a length of at least 25 and at most 29 nucleotides and an antisense strand having a length of at most 30 nucleotides, wherein the sense strand comprises a modified nucleotide at position 11 from its 5' end that is susceptible to enzymatic degradation, wherein the 3' end of the sense strand and the 5' end of the antisense strand form a blunt end, wherein the antisense strand is 1-4 nucleotides longer at its 3' end than the sense strand, wherein the duplex region is at least 25 nucleotides in length, and wherein the antisense strand is sufficiently complementary to a target mRNA along at least 19 nucleotides of the length of the antisense strand, wherein the dsRNA molecule reduces target gene expression when introduced into a mammalian cell, wherein Dicer cleavage of the dsRNA preferentially yields siRNA comprising the 3' end of the antisense strand, thereby reducing target gene expression in the mammal, and wherein the antisense strand comprises at least one The dsRNA contains thermally destabilized nucleotides, with at least one thermally destabilized nucleotide in the seed region of the antisense strand (i.e., positions 2-9 at the 5' end of the antisense strand), and may further have at least one (e.g., one, two, three, four, five, six, or all seven) of the following features: (i) the antisense contains two, three, four, five, or six 2'-fluoro modifications; (ii) the antisense contains two, three, four, five, or six 2'-fluoro modifications; (i) the antisense strand contains 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand contains 2, 3, 4, or 5 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 has a duplex region 12 to 29 nucleotide pairs in length.

[0324] 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, which can include one or more of non-linked phosphate oxygen or one or more of linking phosphate oxygen, one or both of the modifications of ribose sugar components, for example, 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.

[0325] 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 non-linked Os at 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' or 5' terminal position, or only in the terminal region, for example, at the terminal nucleotide position of the chain, or at the last 2, 3, 4, 5, or 10 nucleotides. 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 non-linked O positions can occur only at one or both ends, or only in the terminal region, for example, at the terminal nucleotide position of the chain, or at the last 2, 3, 4, 5, or 10 nucleotides, or in double-stranded and single-stranded regions, especially at the ends. The 5' end or both ends can be phosphorylated.

[0326] For example, it may be possible 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' or 3' overhang, or both.For example, it may be desirable to include purine nucleotides in the overhang.In some embodiments, all or part of the bases in the 3' 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 nucleic acid base, and modifications at the phosphate group, for example, phosphorothioate modifications.The overhang does not need to be homologous to the target sequence.

[0327] In some embodiments, each residue of sense strand and 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.Strands can contain two or more modifications.In some embodiments, each residue of sense strand and 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 thermally destabilizing modification of the double strand that exists in the antisense strand.

[0328] At least two different modifications are usually present on the sense strand and the antisense strand. These two 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 of the sense strand and the antisense strand is independently modified with 2'-O-methyl nucleotides, 2'-deoxy nucleotides, 2'-deoxy-2'-fluoro nucleotides, 2'-ON-methylacetamide (2'-O-NMA) nucleotides, 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) nucleotides, 2'-O-aminopropyl (2'-O-AP) nucleotides, or 2'-ara-F nucleotides. It should be understood that these modifications are in addition to at least one thermally destabilizing modification of the duplex present in the antisense strand.

[0329] In some embodiments, the dsRNA molecules of the present disclosure comprise alternating patterns 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 with one or more modifications, where each modification occurs at alternating nucleotides in a single strand. Alternating nucleotides may refer to one every other nucleotide or one every third nucleotide, or similar patterns. For example, if A, B, and C each represent one type of modification to a nucleotide, the alternating motif may be "ABABABABABAB...", "AABBAABBAABB...", "AABAABAABAAB...", "AAABAAABAAAB...", "AABBBAAABBB...", or "ABCABCABCABC...", etc.

[0330] The types of modifications contained within the alternating motifs can be the same or different. For example, if A, B, C, and D each represent one type of modification on a nucleotide, the alternating turns, 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 possibilities for modifications within the alternating motif, such as "ABABAB...", "ACACAC...", "BDBDBD..." or "CDCDCD...".

[0331] In some embodiments, the dsRNA molecules of the present disclosure comprise 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 the modified groups of the nucleotides of the sense strand correspond to the differently modified groups of the nucleotides of 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 may begin with "ABABAB" from the 5'-3' end of the strand, and the alternating motif in the antisense strand may begin with "BABABA" from the 3'-5' end of the strand within the duplex region. As another example, the alternating motif in the sense strand may begin with "AABBAABB" from the 5'-3' end of the strand, and the alternating motif in the antisense strand may begin with "BBAABBAA" from the 3'-5' end of the strand within the duplex region, resulting in a complete or partial shift in the modification pattern between the sense strand and the antisense strand.

[0332] 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 at any nucleotide of sense strand or antisense strand or both at any position of chain.For example, internucleotide linkage modification can occur at any nucleotide on sense strand or antisense strand, and 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 have a shift with respect to the alternating pattern of internucleotide linkage modification on antisense strand.

[0333] In some embodiments, dsRNA molecule comprises phosphorothioate or methylphosphonate internucleotide linkage modification in overhang region.For example, overhang region comprises two nucleotides with phosphorothioate or methylphosphonate internucleotide linkage between the two nucleotides.Internucleotide linkage modification can also be performed to connect overhang nucleotide with the terminal pairing nucleotide in duplex region.For example, at least 2, 3, 4 or all overhang nucleotides can be linked by phosphorothioate or methylphosphonate internucleotide linkage, and there can be additional phosphorothioate or methylphosphonate internucleotide linkage that connects overhang nucleotide with the nucleotide that is adjacent to the overhang nucleotide.For example, there can be at least two phosphorothioate internucleotide linkages between the terminal three nucleotides, two of which are overhang nucleotides, and the third is the nucleotide that is adjacent to the overhang nucleotide that is adjacent to the overhang nucleotide.Preferably, these terminal three nucleotides can be the 3' end of antisense strand.

[0334] In some embodiments, the sense strand of the dsRNA molecule comprises 1 to 10 blocks of 2 to 10 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, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is positioned at any position in 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.

[0335] In some embodiments, the antisense strand of the 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 located at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages and an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0336] In some embodiments, the antisense strand of the 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 the phosphorothioate or methylphosphonate internucleotide linkages being located at any position in 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.

[0337] In some embodiments, the antisense strand of the 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 the phosphorothioate or methylphosphonate internucleotide linkages being located at any position in 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.

[0338] In some embodiments, the antisense strand of the 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 the phosphorothioate or methylphosphonate internucleotide linkages being located at any position in 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.

[0339] In some embodiments, the antisense strand of the 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 the phosphorothioate or methylphosphonate internucleotide linkages being located at any position in 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.

[0340] In some embodiments, the antisense strand of the 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 the phosphorothioate or methylphosphonate internucleotide linkages being located at any position in 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.

[0341] In some embodiments, the antisense strand of the 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 the phosphorothioate or methylphosphonate internucleotide linkages being located at any position in 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.

[0342] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of nine phosphorothioate or methylphosphonate internucleotide linkages separated by one, two, three, or four phosphate internucleotide linkages, one of the phosphorothioate or methylphosphonate internucleotide linkages being located at any position in 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.

[0343] In some embodiments, the dsRNA molecules of this disclosure further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within the 1-10 terminal positions of the sense or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides can be linked by phosphorothioate or methylphosphonate internucleotide linkages at one or both termini of the sense or antisense strand.

[0344] In some embodiments, dsRNA molecules of the present disclosure further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 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 by phosphorothioate methylphosphonate internucleotide linkages at positions 8-16 of the duplex region, counting from the 5' end of the sense strand. The dsRNA molecule may further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 1-10 of the terminal ends.

[0345] In some embodiments, dsRNA molecules of this disclosure further comprise one to five phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 1-5 of the sense strand and one to five phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) and one to five phosphorothioate or methylphosphonate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and one to five within positions 18-23 (counting from the 5' end).

[0346] In some embodiments, dsRNA molecules of this disclosure further comprise one phosphorothioate internucleotide linkage modification within positions 1-5 and one phosphorothioate or methylphosphonate internucleotide linkage modification within positions 18-23 (counting from the 5' end) of the sense strand and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the antisense strand.

[0347] In some embodiments, dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications within positions 1-5 and one phosphorothioate internucleotide linkage modification within positions 18-23 (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 positions 18-23 (counting from the 5' end) of the antisense strand.

[0348] In some embodiments, dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications within positions 1-5 and two phosphorothioate internucleotide linkage modifications within positions 18-23 (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 positions 18-23 (counting from the 5' end) of the antisense strand.

[0349] In some embodiments, dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications within positions 1-5 and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the sense strand and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 (counting from the 5' end) of the antisense strand.

[0350] In some embodiments, dsRNA molecules of this disclosure further comprise one phosphorothioate internucleotide linkage modification within positions 1-5 and one phosphorothioate internucleotide linkage modification within positions 18-23 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the antisense strand.

[0351] In some embodiments, dsRNA molecules of this disclosure further comprise one phosphorothioate internucleotide linkage modification within positions 1-5 and one within positions 18-23 (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 positions 18-23 (counting from the 5' end) of the antisense strand.

[0352] In some embodiments, dsRNA molecules of this disclosure further comprise one phosphorothioate internucleotide linkage modification within positions 1-5 of the sense strand (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 of the antisense strand and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5' end).

[0353] In some embodiments, dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications (counting from the 5' end) within positions 1-5 of the sense strand and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications (counting from the 5' end) within positions 18-23 of the antisense strand.

[0354] In some embodiments, dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications within positions 1-5 and one within positions 18-23 (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 positions 18-23 (counting from the 5' end) of the antisense strand.

[0355] In some embodiments, dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications within positions 1-5 and one phosphorothioate internucleotide linkage modification within positions 18-23 (counting from the 5' end) of the sense strand and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 5' end) of the antisense strand.

[0356] In some embodiments, dsRNA molecules of this disclosure further comprise two phosphorothioate internucleotide linkage modifications within positions 1-5 and one phosphorothioate internucleotide linkage modification within positions 18-23 (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 positions 18-23 (counting from the 5' end) of the antisense strand.

[0357] In some embodiments, dsRNA molecules of the present disclosure further comprise 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 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.

[0358] In some embodiments, dsRNA molecules of the present 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.

[0359] In some embodiments, dsRNA molecules of the present disclosure further comprise 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 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.

[0360] In some embodiments, dsRNA molecules of the present 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.

[0361] In some embodiments, dsRNA molecules of the present disclosure further comprise two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 22 and 23 (counting from the 5' end) 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.

[0362] In some embodiments, dsRNA molecules of the present 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.

[0363] In some embodiments, the compounds of the present disclosure comprise a pattern of backbone chiral centers. In some embodiments, the general pattern of backbone chiral centers comprises at least 5 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 6 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 7 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 8 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 9 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 10 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 11 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 12 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 13 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 14 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 15 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 16 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 17 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 18 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises at least 19 internucleotide linkages in the Sp configuration. In some embodiments, the general pattern of backbone chiral centers comprises no more than 8 internucleotide linkages in the Rp configuration.In some embodiments, the general pattern of backbone chiral centers comprises seven or fewer internucleotide linkages in the Rp configuration. In some embodiments, the general pattern of backbone chiral centers comprises six or fewer internucleotide linkages in the Rp configuration. In some embodiments, the general pattern of backbone chiral centers comprises five or fewer internucleotide linkages in the Rp configuration. In some embodiments, the general pattern of backbone chiral centers comprises four or fewer internucleotide linkages in the Rp configuration. In some embodiments, the general pattern of backbone chiral centers comprises three or fewer internucleotide linkages in the Rp configuration. In some embodiments, the general pattern of backbone chiral centers comprises two or fewer internucleotide linkages in the Rp configuration. In some embodiments, the general pattern of backbone chiral centers comprises one or fewer internucleotide linkages in the Rp configuration. In some embodiments, the general pattern of backbone chiral centers comprises eight or fewer non-chiral internucleotide linkages (phosphodiesters as a non-limiting example). In some embodiments, the general pattern of backbone chiral centers comprises seven or fewer non-chiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises six or fewer non-chiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises five or fewer non-chiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises four or fewer non-chiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises three or fewer non-chiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises two or fewer non-chiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises one or fewer non-chiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises at least 10 internucleotide linkages in the Sp configuration and eight or fewer non-chiral internucleotide linkages.In some embodiments, the general pattern of backbone chiral centers comprises at least 11 internucleotide linkages in the Sp configuration and no more than 7 nonchiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises at least 12 internucleotide linkages in the Sp configuration and no more than 6 nonchiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises at least 13 internucleotide linkages in the Sp configuration and no more than 6 nonchiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises at least 14 internucleotide linkages in the Sp configuration and no more than 5 nonchiral internucleotide linkages. In some embodiments, the general pattern of backbone chiral centers comprises at least 15 internucleotide linkages in the Sp configuration and no more than 4 nonchiral internucleotide linkages. In some embodiments, the internucleotide linkages in the Sp configuration may be contiguous or noncontiguous. In some embodiments, the internucleotide linkages in the Rp configuration may be contiguous or noncontiguous. In some embodiments, the nonchiral internucleotide linkages may be contiguous or noncontiguous.

[0364] In some embodiments, compounds of the present disclosure include blocks that are stereochemical blocks. In some embodiments, the blocks are Rp blocks, 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, the blocks are Sp blocks, 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 Rp and Sp blocks. In some embodiments, provided oligonucleotides include one or more Rp blocks but do not include Sp blocks. In some embodiments, provided oligonucleotides include one or more Sp blocks but do not include Rp blocks. In some embodiments, provided oligonucleotides include one or more PO blocks, in which each internucleotide linkage is a natural phosphate linkage.

[0365] 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 internucleotide linkage 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 includes a 2'-F modification. In some embodiments, the 3'-block includes 4 or more nucleoside units. In some embodiments, the 3'-block includes 5 or more nucleoside units. In some embodiments, the 3'-block includes 6 or more nucleoside units. In some embodiments, the 3'-block includes 7 or more nucleoside units.

[0366] In some embodiments, compounds of the disclosure include a certain type of nucleoside in a region, or an oligonucleotide is followed by a particular type of internucleotide linkage, e.g., 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.

[0367] In some embodiments, the antisense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 21 and 22 and between nucleotide positions 22 and 23, the antisense strand contains at least one thermally destabilizing modification of the duplex located in the seed region of the antisense strand (i.e., at positions 2-9 at the 5' end of the antisense strand), and the dsRNA may further have 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; (ii) the antisense strand comprises two, three, four, five, or six 2'-fluoro modifications; (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 one, two, 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.

[0368] 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 thermally destabilizing modification of the duplex located in the seed region of the antisense strand (i.e., at positions 2-9 at the 5' end of the antisense strand); and the dsRNA may further have at least one (e.g., 1, 2, 3, 4, 5, 6, 7, or all 8) of the following features: (i) the antisense strand comprises at least two, three, four, five, or six 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. (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 one, two, three, four, or five phosphorothioate internucleotide linkages; (v) the dsRNA comprises at least four 2'-fluoro modifications; (vi) the dsRNA comprises a duplex region between 12 and 40 nucleotide pairs in length; (vii) the dsRNA comprises a duplex region between 12 and 40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at the 5' end of the antisense strand.

[0369] In some embodiments, the sense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3, the antisense strand contains at least one thermally destabilizing modification of the duplex located in the seed region of the antisense strand (i.e., at positions 2-9 at the 5' end of the antisense strand), and the dsRNA may further have 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; (ii) the antisense strand comprises two, three, four, five, or six 2'-fluoro modifications; (iii) the sense strand contains 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (iv) the sense strand contains 2, 3, 4, or 5 2'-fluoro modifications; (v) the sense strand contains 3, 4, or 5 phosphorothioate internucleotide linkages; (vi) the dsRNA contains at least four 2'-fluoro modifications; (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.

[0370] In some embodiments, the sense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3, 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 thermally destabilizing modification of the duplex located in the seed region of the antisense strand (i.e., at positions 2-9 at the 5' end of the antisense strand), and the dsRNA has at least one of the following characteristics (e.g., 1, 2): (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 four 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.

[0371] In some embodiments, the dsRNA molecule of the present disclosure comprises a mismatch (or mismatches) or combinations thereof within the double strand with the target. Mismatches can occur in the overhang region or the double-stranded region. Base pairs can be ranked based on their tendency to promote dissociation or melting (for example, the free energy of association or dissociation of a particular pairing; the simplest approach is to examine pairs on an individual basis, but next-neighbor analysis or similar analysis can also be used). In terms of promoting dissociation, A:U is preferred over G:C, G:U is preferred over G:C, and I:C is preferred over G:C (I=inosine). Mismatches, such as non-canonical pairings or those other than canonical pairings (as described elsewhere herein), are preferred over canonical (A:T, A:U, G:C) pairings, and pairings involving universal bases are preferred over canonical pairings.

[0372] In some embodiments, the dsRNA molecules of the present disclosure comprise at least one of the first 1, 2, 3, 4, or 5 base pairs within the duplex region from the 5' end of the antisense strand, which can be independently selected from the group of A:U, G:U, I:C, and mismatch pairs, e.g., non-canonical or other than canonical pairings or pairings including universal bases, to promote dissociation of the antisense strand at the 5' end of the duplex.

[0373] In some embodiments, the nucleotide at position 1 in the double-stranded region from the 5' end of the antisense strand is selected from the group consisting of A, dA, dU, U and dT.Alternatively, at least one of the first 1, 2 or 3 base pairs in the double-stranded region from the 5' end of the antisense strand is an AU base pair.For example, the first base pair in the double-stranded region from the 5' end of the antisense strand is an AU base pair.

[0374] 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, thus protecting it from nucleases and stabilizing it.

[0375] 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 is 5'-methyl nucleoside.5'-methyl can be racemic or chirally pure R or S isomer.

[0376] 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 5' 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 group at 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.

[0377] 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 is 5'-methyl nucleoside.5'-methyl can be either racemic or chirally pure R or S isomer.

[0378] 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 chirally pure R or S isomer.Exemplary 4'-alkylated nucleoside is 4'-methyl nucleoside.4'-methyl can be either racemic or chirally pure R or S isomer.

[0379] In some embodiments, 4'-O-alkylated nucleosides are introduced at any position of the sense strand or antisense strand of dsRNA, and such modifications maintain or improve the efficacy of dsRNA.5'-Alkyl can be either racemic or chirally pure R or S isomer.Exemplary 4'-O-alkylated nucleosides include 4'-O-methyl nucleosides.4'-O-methyl can be either racemic or chirally pure R or S isomer.

[0380] In some embodiments, the dsRNA molecules of the present disclosure may contain 2'-5' linkages (having 2'-H, 2'-OH, and 2'-OMe, with P=O or P=S). For example, 2'-5' linkage modifications can be used to promote nuclease resistance or inhibit binding of the sense strand to the antisense strand, or can be used at the 5' end of the sense strand to prevent sense strand activation by RISC.

[0381] 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 inhibit binding of the sense strand to the antisense strand, or can be used at the 5' end of the sense strand to prevent sense strand activation by RISC.

[0382] Various publications have described multimeric siRNA, and all of them can be used with the dsRNA of the present disclosure.Such publications include WO2007 / 091269, US7858769, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887 and WO2011 / 031520, all of which are incorporated herein in their entirety.

[0383] 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. In many cases, the carbohydrate moiety is 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 carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring structure, i.e., one or more ring atoms can be heteroatoms, such as nitrogen, oxygen, or sulfur. The cyclic carrier can be a monocyclic ring structure or can contain two or more rings, such as fused rings. The cyclic carrier can be a fully saturated ring structure or can contain one or more double bonds.

[0384] Ligands can be attached to polynucleotides via carriers. The carriers include (i) at least one "backbone attachment point," preferably two "backbone attachment points," and (ii) at least one "tethering attachment point." As used herein, "backbone attachment point" refers to a functional group, e.g., a hydroxyl group, or generally a bond available and suitable for incorporation of the carrier into a backbone, e.g., a phosphate or modified phosphate, e.g., sulfur-containing backbone, of a ribonucleic acid. "Tethering attachment point" (TAP) refers, in some embodiments, to a constituent ring atom, e.g., a carbon atom or heteroatom (separate from the atom providing the backbone attachment point), of a cyclic carrier that connects the selected moiety. The moiety can be, for example, a carbohydrate, e.g., a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. The selected moiety may be connected to the cyclic carrier by an intervening tether. Thus, cyclic carriers often contain a functional group, e.g., an amino group, or generally provide a bond suitable for incorporation or tethering another chemical entity, e.g., a ligand, to the constituent ring.

[0385] The RNAi agent may be conjugated to the ligand via a carrier, which can be a cyclic or acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, and decalin; preferably, the acyclic group is selected from a serinol backbone or a diethanolamine backbone.

[0386] In certain specific embodiments, the RNAi agent for use in the methods of the disclosure is an agent selected from the group of agents listed in any one of Tables 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 24, 25, 27-30, 32 and 33.

[0387] IV. Ligand-Conjugated iRNA Another modification of the iRNA of the invention includes chemically linking the iRNA to one or more ligands, moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the iRNA, for example, into cells. Such moieties include, but are not limited to, lipid moieties such as cholesterol moieties (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), thioethers, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), and the like. 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 di-hexadecyl-rac-glycerol or triethyl-ammonium 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 moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0388] In certain embodiments, the ligand alters the distribution, targeting, or life span of the iRNA agent into which it is incorporated. In some embodiments, the ligand provides enhanced affinity for a selected target, for example, a molecule, a cell or cell type, a compartment, for example, a cellular or organ compartment, a tissue, an organ, or a region of the body, for example, compared to a species in which such a ligand is not present. Conventional ligands do not participate in duplex pairing in double-stranded nucleic acids.

[0389] 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-glycolized) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylamide), 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 alpha-helical peptides.

[0390] Ligands can also include targeting groups, such as cell or tissue targeting agents, such as lectins, glycoproteins, lipids, or proteins, for example, antibodies that bind to specific cell types, such as kidney cells.Targeting groups can be thyroid-stimulating hormone, melanocyte-stimulating hormone, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, multivalent fucose, glycosylated polyamino acids, multivalent galactose, transferrin, bisphosphonates, polyglutamic acid, polyaspartic acid, lipids, cholesterol steroids, bile acid, folic acid, vitamin B12, biotin, or RGD peptide or RGD peptide mimetics.In certain embodiments, the ligand is multivalent galactose, such as N-acetyl-galactosamine.

[0391] 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-pyrenebutyric 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 cleavage inhibitors include cleavage inhibitors (e.g., oleic acid, O3-(oleoyl)cholenoic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bis-imidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

[0392] The ligand can be a protein, such as a glycoprotein or peptide, a molecule with specific affinity for the co-ligand, or an antibody, such as an antibody that binds to a specific cell type, 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.

[0393] The ligand can be, for example, a substance, e.g., a drug, that can increase cellular uptake of an iRNA agent by, for example, disrupting the cytoskeleton of a cell, e.g., by disrupting cellular microtubules, microfilaments, or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, jasplakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0394] In some embodiments, the ligand attached to the iRNA described herein acts as a pharmacokinetic modulator (PK modulator). 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 are also known to bind to serum proteins. Therefore, short oligonucleotides, e.g., oligonucleotides of about 5, 10, 15, or 20 bases containing multiple phosphorothioate linkages in the backbone, are also suitable as ligands (e.g., as PK-modulating ligands) for the present invention. Furthermore, aptamers that bind to serum components (e.g., serum proteins) are also suitable for use as PK-modulating ligands in the embodiments described herein.

[0395] Ligand-conjugated iRNAs of the invention can be synthesized by using oligonucleotides bearing pendant reactive functionalities, e.g., derived from the attachment of a linking molecule onto an oligonucleotide (described below). This reactive oligonucleotide can be reacted directly with commercially available ligands, ligands that have been synthesized with any of a variety of protecting groups, or ligands that have a linking moiety attached to them.

[0396] The oligonucleotides used in the conjugates of the present invention can be conveniently and routinely produced by known techniques of solid phase synthesis.The 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 can also or alternatively be used.It is also known to use similar techniques to prepare other oligonucleotides, for example, phosphorothioates and alkylated derivatives.

[0397] For the ligand-conjugated oligonucleotides and molecules having ligand-sequence-specific linked nucleosides of the present invention, the oligonucleotides and oligonucleosides can be assembled on a suitable DNA synthesizer using standard nucleotide or nucleoside precursors or nucleotide or nucleoside conjugate precursors that already have a linking moiety, ligand-nucleotide or nucleoside conjugate precursors that already have a ligand molecule, or non-nucleoside ligand-containing building blocks.

[0398] When using a nucleotide-conjugate precursor that already has a linking moiety, the synthesis of the sequence-specific linked nucleoside is usually completed, and then a ligand molecule is reacted with the linking moiety to form a ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the present invention are synthesized by automated synthesizers using phosphoramidites derived from ligand-nucleoside conjugates, in addition to standard and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis.

[0399] A. Lipid Conjugates In certain embodiments, the ligand or conjugate is lipid or lipid-based molecule.This lipid or lipid-based molecule can usually bind to serum protein, for example, human serum albumin (HSA).HSA-binding ligand allows the distribution of conjugate to target tissue in the body, for example, non-renal target tissue.For example, the target tissue can be the liver, including liver parenchymal cells.Other molecules that can bind to 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) adjust the binding to serum protein, for example, HSA.

[0400] The lipid-based ligand can be used to modulate, e.g., manage (e.g., inhibit), the binding of the conjugate to the target tissue. For example, a lipid or lipid-based ligand that binds more strongly to HSA is less likely to be targeted to the kidney and therefore less likely to be eliminated from the body. A lipid or lipid-based ligand that binds less strongly to HSA can be used to target the conjugate to the kidney.

[0401] In certain embodiments, lipid-based ligand binds to HSA.For example, the ligand can bind to HSA with sufficient affinity, so that the distribution of conjugate to non-renal tissue is enhanced.However, the affinity is usually not so strong that HSA-ligand binding cannot be reversed.

[0402] 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.

[0403] In another embodiment, the ligand is a moiety, such as a vitamin, that is taken up by target cells, for example, proliferating cells. These are particularly useful for treating disorders characterized by unwanted cell proliferation, for example, malignant or non-malignant types, such as cancer cells. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include B vitamins, such as folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients taken up by cancer cells. Also included are HSA and low-density lipoprotein (LDL).

[0404] B. Cell-penetrating agents In another aspect, the ligand is a cell-penetrating agent, for example, a helical cell-penetrating agent. In certain embodiments, these cell-penetrating agents are amphipathic. Exemplary cell-penetrating agents include peptides, such as tat or antennopedia. When the agent is a peptide, it may be modified, including peptidyl mimetics, invertomers, non-peptide or pseudopeptide linkages, and the use of D-amino acids. Helical agents are usually α-helical agents and may have a lipophilic and lipophobic phase.

[0405] The ligand can be a peptide or peptidomimetic. Peptidomimetics (also referred to herein as oligopeptidomimetics) are molecules capable of folding into defined three-dimensional structures similar to natural peptides. Attachment of peptides and peptidomimetics to iRNA agents can affect the pharmacokinetic distribution of iRNAs, such as by enhancing cellular recognition and uptake. The peptide or peptidomimetic moiety can be about 5 to 50 amino acids in length, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0406] The peptide or peptidomimetic can be, for example, a cell-penetrating peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide (e.g., composed primarily of Tyr, Trp, or Phe). The peptide moiety can be a dendrimeric peptide, a constrained peptide, or a cross-linked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF, which has the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 11). An RFGF analog containing a hydrophobic MTS (e.g., the amino acid sequence AALLPVLLAAP (SEQ ID NO: 12)) can also be a targeting moiety. The peptide moiety can be a "delivery" peptide, capable of transporting large polar molecules, including peptides, oligonucleotides, and proteins, across cell membranes. For example, the sequence derived from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 13)) and the sequence derived from the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 14)) have been found to function as delivery peptides. Peptides or peptidomimetics can be encoded by random sequences of DNA, such as peptides identified from phage display libraries or one-bead-one-compound (OBOC) combinatorial libraries (Lam et al., Nature, 354:82-84, 1991). Typically, peptides or peptidomimetics tethered to dsRNA agents via incorporated monomer units include cell-targeting peptides, such as arginine-glycine-aspartic acid (RGD) peptides or RGD mimics. The peptide portion can range in length from about 5 amino acids to about 40 amino acids. The peptide portion can have structural modifications, for example, to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.

[0407] The RGD peptide for use in the compositions and methods of the present invention can be linear or cyclic, and can be modified, for example, glycosylated or methylated, to facilitate targeting to specific tissue(s).RGD-containing peptides and peptidomimetics can contain D-amino acids and synthetic RGD mimics.In addition to RGD, other moieties can be used to target integrin ligands.Preferred conjugates of this ligand target PECAM-1 or VEGF.

[0408] RGD peptide moieties can be used to target specific cell types, such as tumor cells, e.g., endothelial tumor cells or breast cancer tumor cells (Zitzmann et al., Cancer Res., 62:5139-43, 2002). RGD peptides can facilitate targeting of dsRNA agents to tumors in various other tissues, including the lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001). Typically, RGD peptides facilitate targeting of iRNA agents to the kidney. RGD peptides can be linear or cyclic, and can be modified, e.g., glycosylated or methylated, to facilitate targeting to a specific tissue(s). For example, glycosylated RGD peptides can facilitate targeting of iRNA agents to α- V It can be delivered to tumor cells that express β3 (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001).

[0409] A "cell-penetrating peptide" is capable of penetrating cells, such as microbial cells, e.g., bacterial or fungal cells, or mammalian cells, e.g., human cells. Microbial cell-penetrating peptides can be, for example, α-helical linear peptides (e.g., LL-37 or seropin P1), disulfide bond-containing peptides (e.g., α-defensins, β-defensins, or bactenecins), or peptides containing only one or two dominant amino acids (e.g., PR-39 or indolicidin). Cell-penetrating peptides can also contain a nuclear localization signal (NLS). For example, a cell-penetrating peptide can be a bisected amphipathic peptide such as MPG, derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

[0410] C. Carbohydrate conjugates In some embodiments of the compositions and methods of the present invention, the iRNA further comprises a carbohydrate. Carbohydrate-conjugated iRNA is advantageous for in vivo delivery of nucleic acids and compositions suitable for in vivo therapeutic use, as described herein. As used herein, "carbohydrate" refers to a compound that is either a carbohydrate itself, composed of one or more monosaccharide units having at least six carbon atoms (which may be linear, branched, or cyclic), with an oxygen, nitrogen, or sulfur atom bonded to each carbon atom, or a compound that has as part thereof a carbohydrate moiety composed of one or more monosaccharide units, each having at least six carbon atoms (which may be linear, branched, or cyclic), with an oxygen, nitrogen, or sulfur atom bonded to each carbon atom. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units) and polysaccharides, such as starch, glycogen, cellulose, and polysaccharide gums. Particular monosaccharides include C5, and above (e.g., C5, C6, C7, or C8) sugars, disaccharides, and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).

[0411] In certain embodiments, the carbohydrate conjugate comprises a monosaccharide.

[0412] In certain embodiments, the monosaccharide is N-acetylgalactosamine (GalNAc). GalNAc conjugates containing one or more N-acetylgalactosamine (GalNAc) derivatives are described, for example, in US 8,106,022, the entire contents of which are incorporated herein by reference. In some embodiments, the GalNAc conjugate serves as a ligand that targets iRNA to specific cells. In some embodiments, the GalNAc conjugate targets iRNA to liver cells, for example, by serving as a ligand for the asialoglycoprotein receptor of liver cells (e.g., hepatocytes).

[0413] In some embodiments, the carbohydrate conjugate comprises one or more GalNAc derivatives. The GalNAc derivatives can be attached via a linker, e.g., a bivalent or trivalent branched linker. In some embodiments, the GalNAc conjugate is conjugated to the 3' end of the sense strand. In some embodiments, the GalNAc conjugate is conjugated to the iRNA agent (e.g., to the 3' end of the sense strand) via a linker, e.g., a linker as described herein. In some embodiments, the GalNAc conjugate is conjugated to the 5' end of the sense strand. In some embodiments, the GalNAc conjugate is conjugated to the iRNA agent (e.g., to the 5' end of the sense strand) via a linker, e.g., a linker as described herein.

[0414] In certain embodiments of the invention, GalNAc or GalNAc derivatives are attached to iRNA agents of the invention via a monovalent linker. In some embodiments, GalNAc or GalNAc derivatives are attached to iRNA agents of the invention via a bivalent linker. In yet other embodiments of the invention, GalNAc or GalNAc derivatives are attached to iRNA agents of the invention via a trivalent linker. In other embodiments of the invention, GalNAc or GalNAc derivatives are attached to iRNA agents of the invention via a tetravalent linker.

[0415] In certain embodiments, double-stranded RNAi agents of the invention comprise one GalNAc or GalNAc derivative attached to the iRNA agent. In certain embodiments, double-stranded RNAi agents of the invention comprise multiple (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each independently attached to multiple nucleotides of the double-stranded RNAi agent via multiple monovalent linkers.

[0416] In some embodiments, for example, when the two strands of an iRNA agent of the invention are part of a single larger molecule connected by an uninterrupted chain of nucleotides between the 3' end of one strand and the 5' end of the other strand, forming a hairpin loop containing multiple unpaired nucleotides, each unpaired nucleotide in the hairpin loop can independently comprise a GalNAc or GalNAc derivative attached via a monovalent linker. Hairpin loops can also be formed by an extended overhang on one strand of the duplex.

[0417] In some embodiments, for example, when the two strands of an iRNA agent of the invention are part of a single larger molecule connected by an uninterrupted chain of nucleotides between the 3' end of one strand and the 5' end of the other strand, forming a hairpin loop containing multiple unpaired nucleotides, each unpaired nucleotide in the hairpin loop can independently comprise a GalNAc or GalNAc derivative attached via a monovalent linker. Hairpin loops can also be formed by an extended overhang on one strand of the duplex.

[0418] In some embodiments, the GalNAc conjugate is

[0419] [ka] is.

[0420] In some embodiments, the RNAi agent is attached to the carbohydrate conjugate via a linker as shown in the following schematic diagram, where X is O or S:

[0421] [ka]

[0422] In some embodiments, the RNAi agent is conjugated to L96 as defined in Table 1 and shown below:

[0423] [ka]

[0424] In certain embodiments, the carbohydrate conjugate for use in the compositions and methods of the invention is selected from the group consisting of:

[0425] [ka] [ka] [ka] [ka] [ka]

[0426] [ka]

[0427] [ka]

[0428] [ka] [ka]

[0429] In certain embodiments, the carbohydrate conjugate for use in the compositions and methods of the present invention is a monosaccharide. In certain embodiments, the monosaccharide is N-acetylgalactosamine,

[0430] [ka] is.

[0431] Further exemplary carbohydrate conjugates for use in the embodiments described herein include, but are not limited to:

[0432] [ka] wherein one of X or Y is an oligonucleotide and the other is hydrogen. Examples include:

[0433] In some embodiments, suitable ligands are those disclosed in WO2019 / 055633, the entire contents of which are incorporated herein by reference. In one embodiment, the ligand has the following structure:

[0434] [ka] Includes.

[0435] In certain embodiments, RNAi agents of the present disclosure may include GalNAc ligands, even though such GalNAc ligands are currently predicted to be of limited value for the preferred intrathecal / CNS delivery route(s) of the present disclosure.

[0436] In certain embodiments of the invention, GalNAc or GalNAc derivatives are attached to iRNA agents of the invention via a monovalent linker. In some embodiments, GalNAc or GalNAc derivatives are attached to iRNA agents of the invention via a bivalent linker. In yet other embodiments of the invention, GalNAc or GalNAc derivatives are attached to iRNA agents of the invention via a trivalent linker.

[0437] In one embodiment, the double-stranded RNAi agent of the present invention comprises one or more GalNAc or GalNAc derivatives attached to the iRNA agent. GalNAc can be attached to any nucleotide via a linker on the sense strand or antisense strand. GalNAc can be attached to the 5' end of the sense strand, the 3' end of the sense strand, the 5' end of the antisense strand, or the 3' end of the antisense strand. In one embodiment, GalNAc is attached to the 3' end of the sense strand, for example, via a trivalent linker.

[0438] In other embodiments, a double-stranded RNAi agent of the invention comprises multiple (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives independently attached to each of multiple nucleotides of the double-stranded RNAi agent via multiple linkers, e.g., monovalent linkers.

[0439] In some embodiments, for example, when the two strands of an iRNA agent of the invention are part of one larger molecule connected by an uninterrupted chain of nucleotides between the 3' end of one strand and the 5' end of each of the other strands, forming a hairpin loop containing multiple unpaired nucleotides, each unpaired nucleotide in the hairpin loop can independently comprise a GalNAc or GalNAc derivative attached via a monovalent linker.

[0440] In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, a PK modulator or a cell-penetrating peptide.

[0441] Further carbohydrate conjugates and linkers suitable for use in the present invention include those described in WO2014 / 179620 and WO2014 / 179627, the contents of each of which are incorporated herein by reference in their entirety.

[0442] D. Linker In some embodiments, the conjugates or ligands described herein can be attached to the iRNA oligonucleotide using a variety of linkers, which may or may not be cleavable.

[0443] The term "linker" or "linking group" means an organic moiety that connects two parts of a compound, e.g., attaches two parts of a compound by a covalent bond.A linker is typically a direct bond or an atom such as oxygen or sulfur, a unit such as NR, C(O), C(O)NH, SO, SO, SONH, or a substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, or heteroarylalkynyl, where one or more methylenes may be interrupted or terminated by O, S, S(O), SO, N(R), C(O). ynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkynyl

[0044] In some embodiments, R8 is substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, etc. In some embodiments, R8 is substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, etc. In some embodiments, R8 is substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, etc. In some embodiments, R8 is substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, etc. In some embodiments, R8 is substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, etc. In some embodiments, R8 is substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, etc. In some embodiments, R8 is substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, etc.In certain embodiments, the linker is about 1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17, 8-17, 6-16, 7-16, or 8-16 atoms.

[0444] A cleavable linking group is one that is sufficiently stable outside a cell, but that, once inside a target cell, is cleaved to release the two moieties held together by the linker. In preferred embodiments, the cleavable linking group is cleaved at least about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or more, or at least about 100-fold faster in the target cell or under a first reference condition (which may, for example, be selected to mimic or represent intracellular conditions) than in the subject's blood or under a second reference condition (which may be selected to mimic or represent conditions found in blood or serum).

[0445] Cleavable linking groups are susceptible to the influence of cleaving agents, such as pH, redox potential, or the presence of degradable molecules.Generally, cleaving agents are more common or found at higher levels or activity inside cells than in serum or blood.Examples of such degrading agents include redox agents that are selected for specific substrates or do not have substrate specificity, such as oxidizing enzymes or reductases or reducing agents present in cells, such as mercaptans, which can degrade redox-cleavable linking groups by reduction, esterases, endosomes, or agents that can create an acidic environment, such as those that produce a pH of 5 or less, general acids, peptidases (can be substrate-specific), and enzymes that can hydrolyze or degrade acid-cleavable linking groups by acting as phosphatases.

[0446] Cleavable linking groups, such as disulfide bonds, can be pH-sensitive. While the pH of human serum is 7.4, the average intracellular pH is slightly lower, ranging from approximately 7.1 to 7.3. Endosomes have a more acidic pH, ranging from 5.5 to 6.0, and lysosomes have an even more acidic pH of approximately 5.0. Some linkers will have a cleavable linking group that is cleaved at a preferred pH, thereby releasing the cationic lipid from the ligand inside the cell into the desired compartment of the cell.

[0447] Linker can comprise a cleavable linking group that can be cleaved by specific enzyme.The type of cleavable linking group incorporated into linker can vary depending on the cell to be targeted.For example, liver targeting ligand can be linked to cationic lipid via a linker that comprises ester group.Liver cell is rich in esterase, therefore, linker is cleaved more efficiently in liver cell than in cell types that are not rich in esterase.Other cell types that are rich in esterase include lung, renal cortex and testis cell.

[0448] When targeting cell types that are rich in peptidases, such as liver cells and synovial cells, linkers containing peptide bonds can be used.

[0449] Generally, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degrading agent (or condition) to cleave the candidate linking group. It may also be desirable to test the candidate cleavable linking group for its ability to resist cleavage when in blood or in contact with other non-target tissues. Thus, the relative susceptibility to cleavage between a first and a second condition can be determined, with the first being selected to exhibit cleavage in target cells, and the second being selected to exhibit cleavage in other tissues or biological fluids, such as blood or serum. Evaluation can be carried out in a cell-free system, in cells, in cell culture, in organs or tissue culture, or in whole animals. It may be useful to perform initial evaluation in cell-free or culture conditions and confirm by further evaluation in whole animals. In preferred embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0450] i. Redox-cleavable linking group In certain embodiments, the cleavable linking group is a redox-cleavable linking group that is cleaved upon reduction or oxidation. An example of a reductively cleavable linking group is a disulfide linking group (-SS-). To determine whether a candidate cleavable linking group is a suitable "reductively cleavable linking group" or is suitable for use with, for example, a particular iRNA moiety and a particular targeting agent, one can refer to the methods described herein. For example, candidates can be evaluated by incubating with dithiothreitol (DTT) or other reducing agents using reagents known in the art that mimic the cleavage rate that would be observed in cells, e.g., target cells. Candidates can also be evaluated under conditions selected to mimic blood or serum conditions. In some cases, candidate compounds are cleaved at a maximum of about 10% in blood. In other embodiments, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of the candidate compound can be determined using standard enzyme kinetic assays under conditions selected to mimic intracellular media compared to conditions selected to mimic extracellular media.

[0451] ii. Phosphate-based cleavable linking groups In certain embodiments, the cleavable linker comprises a phosphate-based cleavable linking group. The phosphate-based cleavable linking group is cleaved by an agent that degrades or hydrolyzes the phosphate group. An example of an agent that cleaves phosphate groups in cells is an enzyme such as a phosphatase in the cell. Examples of phosphate-based linking groups are -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, and -OP(S)(Rk)-S-. Preferred embodiments include -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O, -SP(S)(H)-O-, -SP(O)(H)-S-, and -OP(S)(H)-S-. Preferred embodiments include -OP(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

[0452] iii. Acid-cleavable linking group In certain embodiments, the cleavable linker comprises an acid-cleavable linking group. An acid-cleavable linking group is a linking group that is cleaved under acidic conditions. In a preferred embodiment, the acid-cleavable linking group is cleaved in an acidic environment having a pH of about 6.5 or less (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower) or by an agent, such as an enzyme, that can act as a general acid. In cells, certain low-pH organelles, such as endosomes and lysosomes, can provide a cleavage environment for the acid-cleavable linking group. Examples of acid-cleavable linking groups include, but are not limited to, hydrazones, esters, and esters of amino acids. Acid-cleavable groups can have the general formula -C=NN-, C(O)O, or -OC(O). In a preferred embodiment, the carbon bonded to the oxygen of the ester (alkoxy group) is an aryl group, a substituted alkyl group, or a tertiary alkyl group, such as dimethylpentyl or t-butyl. These candidates can be evaluated using methods similar to those described above.

[0453] iv. Ester-based cleavable linking groups In certain embodiments, the cleavable linker comprises an ester-based cleavable linking group. Ester-based cleavable linking groups are cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. Ester-cleavable linking groups have the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0454] v. Peptide-based cleavable linking groups In yet another embodiment, the cleavable linker comprises a peptide-based cleavable linking group. Peptide-based cleavable linking groups are cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids, resulting in oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable groups do not include amide groups (—C(O)NH—). Amide groups can be formed between any alkylene, alkenylene, or alkynylene. A peptide bond is a special type of amide bond formed between amino acids, resulting in peptides and proteins. Peptide-based cleaving groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids, resulting in peptides and proteins, and do not include the entire amide functionality. Peptide-based cleavable linking groups have the general formula —NHCHRAC(O)NHCHRBC(O)—, where R and R are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

[0455] In some embodiments, the iRNA of the present invention is conjugated to a carbohydrate via a linker. Non-limiting examples of iRNA carbohydrate conjugates with linkers of the compositions and methods of the present invention include, but are not limited to:

[0456] [ka] [ka] [When one of X or Y is an oligonucleotide, the other is hydrogen] Examples include:

[0457] In certain embodiments of the compositions and methods of the present invention, the ligand is one or more "GalNAc" (N-acetylgalactosamine) derivatives attached via a bivalent or trivalent branched linker.

[0458] In certain embodiments, the dsRNA of the invention is conjugated to a bivalent or trivalent branched linker selected from the group of structures shown in any of formulas (XLV)-(XLVI):

[0459] [ka] [In the formula, q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B, and q5C independently for each occurrence represent 0 to 20, and the repeat units may be the same or different; P 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , P 5C , T 2A , T 2B , T 3A , T 3B , T 4A , T 4B , T 4A , T 5B , T 5C is, independently for each occurrence, absent, CO, NH, O, S, OC(O), NHC(O), CH, CHNH, or CHO; Q 2A , Q 2B , Q 3A , Q 3B , Q 4A , Q 4B , Q 5A , Q 5B , Q 5C is, independently for each occurrence, absent, alkylene, substituted alkylene, and one or more methylenes are O, S, S(O), SO, N(R N), C(R')=C(R''), C≡C or C(O), R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , R 5C is independently for each occurrence absent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R a )C(O), -C(O)-CH(R a )-NH-, CO, CH=NO,

[0460] [ka] or heterocyclyl, L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B and L 5C represents a ligand, i.e., each independently for each occurrence, a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; R a is H or an amino acid side chain]. The trivalent conjugating GalNAc derivatives are used to conjugate target genes, for example, those of formula (XLIX):

[0461] [ka] [In the formula, L 5A , L 5B and L 5C represents a monosaccharide, e.g., a GalNAc derivative] It is particularly useful for use with RNAi agents to inhibit expression of

[0462] Examples of suitable divalent and trivalent branched linker groups for conjugating GalNAc derivatives include, but are not limited to, the structures listed above, such as Formulas II, VII, XI, X, and XIII.

[0463] Representative United States patents that teach the preparation of RNA conjugates include, but are not limited to, U.S. Patent Nos. 4,828,979, 4,948,882, 5,218,105, 5,525,465, 5,541,313, 5,545,730, 5,552,538, 5,578,717, 5,580,731, 5,591,584, 5,109,124, 5,118,802, 5,138,045, 5,414,07 ... 5,486,603, 5,512,439, 5,578,718, 5,608,046, 4,587,044, 4,605,735, 4,667,025, 4,762,779, 4,789,737, 4,824,941, 4,835,263, 4,876,335, 4,904,582, 4,958,013, 5,082,830, 5,112,963, 5,214,136, 5,082,830, No. 5,112,963, No. 5,214,136, No. 5,245,022, No. 5,254,469, No. 5,258,506, No. 5,262,536, No. 5,272,250, No. 5,292,873, No. 5,317,098, No. No. 5,371,241, No. 5,391,723, No. 5,416,203, No. 5,451,463, No. 5,510,475, No. 5,512,667, No. 5,514,785, No. 5,565,552, No. 5,567,810, No. Nos. 5,574,142, 5,585,481, 5,587,371, 5,595,726, 5,597,696, 5,599,923, 5,599,928, 5,688,941, 6,294,664, 6,320,017, 6,576,752, 6,783,931, 6,900,297, 7,037,646 and 8,106,022, the entire contents of each of which are incorporated herein by reference.

[0464] Not all positions in a given compound need be uniformly modified, and in fact more than one of the above modifications can be incorporated in a single compound, or even at a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds.

[0465] In the context of the present invention, a "chimeric" iRNA compound or "chimera" refers to an iRNA compound, preferably a dsRNA agent, that contains two or more chemically distinct regions, each composed of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs typically contain at least one region in which the RNA has been modified to confer increased resistance to nuclease degradation, increased cellular uptake, or increased binding affinity for the target nucleic acid. An additional region of the iRNA can serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. For example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. Thus, activation of RNase H results in cleavage of the RNA target, thereby greatly enhancing the efficiency of iRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter iRNAs when chimeric dsRNAs are used compared to phosphorothioate deoxydsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis, optionally followed by associated nucleic acid hybridization techniques known in the art.

[0466] In certain instances, the RNA of an iRNA can be modified with a non-ligand group. To enhance the activity, cellular distribution, or cellular uptake of an iRNA, several non-ligand molecules have been conjugated to the iRNA, and procedures for performing such conjugation are available in the scientific literature. Such non-ligand moieties include lipid moieties such as cholesterol [Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553], cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), thioethers, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3:2765), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), fatty chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969) or adamantaneacetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), palmityl moieties (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative U.S. patents teaching the preparation of such RNA conjugates are listed above. A typical conjugation protocol involves the synthesis of RNA bearing an amino linker at one or more positions in the sequence. The amino group is then reacted with the molecule being conjugated using an appropriate coupling or activating reagent. The conjugation reaction can be carried out while the RNA is still attached to the solid support or after cleavage of the RNA in solution phase. Purification of the RNA conjugate by HPLC usually yields a pure conjugate.

[0467] V. Delivery of RNAi Agents of the Present Disclosure Delivery of the RNAi agent of the present disclosure to a cell, for example, a cell in a subject, for example, a cell in a human subject (e.g., a subject in need thereof, such as a subject with an HTT-related disease, e.g., Huntington's disease), can be achieved in several different ways. For example, delivery can be performed by contacting a cell with the RNAi agent of the present disclosure either in vitro or in vivo. In vivo delivery can be performed directly by administering a composition containing an RNAi agent, for example, a dsRNA, to a subject. Alternatively, in vivo delivery can be performed indirectly by administering one or more vectors that encode and induce the expression of the RNAi agent. These alternatives are further described below.

[0468] Generally, any method of delivering nucleic acid molecules (in vitro or in vivo) can be adapted for use with the RNAi agents of the present disclosure (see, for example, Akhtar S. and Julian RL., (1992) Trends Cell. Biol. 2(5):139-144 and WO94 / 02595, which are incorporated herein by reference in their entirety). For in vivo delivery, factors to consider when delivering RNAi agents include, for example, the biological stability of the delivered agent, prevention of non-specific effects, and accumulation of the delivered agent in the target tissue. Non-specific effects of RNAi agents can be minimized by local administration, such as direct injection or implantation into tissue or local administration of a preparation. Local administration at the treatment site maximizes the local concentration of the agent, limits exposure of the agent to systemic tissues that may be harmed by or degrade the agent, and allows for a smaller total dose of the administered RNAi agent. Several studies have shown the success of knocking down gene products when RNAi agents are administered locally. For example, intraocular delivery of VEGF dsRNA by intravitreal injection in cynomolgus monkeys [Tolentino, MJ. et al., (2004) Retina 24:132-138] and subretinal injection in mice [Reich, SJ. et al. (2003) Mol. Vis. 9:210-216] have both been shown to prevent neovascularization in experimental models of age-related macular degeneration. In addition, direct intratumoral injection of dsRNA into mice can reduce tumor volume [Pille, J. et al. (2005) Mol. Ther. 11:267-274] and prolong the survival of tumor-bearing mice [Kim, WJ. et al., (2006) Mol. Ther. 14:343-350; Li, S. et al., (2007) Mol. Ther. 15:515-523].RNA interference can be delivered to the CNS by direct injection [Dorn, G. et al., (2004) Nucleic Acids 32:e49; Tan, PH. et al. (2005) Gene Ther. 12:59-66; Makimura, H. et al. (2002) BMC Neurosci. 3:18; Shishkina, GT., et al. (2004) Neuroscience 129:521-528; Thakker, ER., et al. (2004) Proc. Natl. Acad. Sci. USA 101:17270-17275; Akaneya,Y., et al. (2005) J. Neurophysiol. 93 :594-602] and to the lungs by intranasal administration [Howard, KA. et al., (2006) Mol. Ther. 14:476-484; Zhang, X. et al., (2004) J. Biol. Chem. 279:10677-10684; Bitko, V. et al., (2005) Nat. Med. 11:50-55], and local delivery has also shown success. When administering RNAi agents systemically to treat disease, the RNA can be modified or, alternatively, delivered using a drug delivery system; both methods function to prevent the rapid degradation of dsRNA by endonucleases and exonucleases in vivo. Modification of RNA or pharmaceutical carrier can also enable targeting of RNAi agent to target tissue and avoid undesirable off-target effects (for example, without wishing to be bound by theory, it has been identified that the use of GNA described herein destabilizes the seed region of dsRNA, and such off-target effects are significantly weakened by destabilizing such seed region, thereby increasing the priority of such dsRNA for on-target effectiveness compared to off-target effects).RNAi agent can be modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation.For example, systemic injection of ApoB-directed RNAi agents conjugated to lipophilic cholesterol moieties into mice resulted in knockdown of apoB mRNA in both the liver and jejunum [Soutschek, J. et al., (2004) Nature 432:173-178]. Conjugation of RNAi agents to aptamers has been shown to inhibit tumor growth and mediate tumor regression in a mouse model of prostate cancer [McNamara, JO. et al., (2006) Nat. Biotechnol. 24:1005-1015]. In alternative embodiments, RNAi agents can be delivered using drug delivery systems, such as nanoparticles, dendrimers, polymers, liposomes, or cationic delivery systems. Positively charged cationic delivery systems promote the binding of molecular RNAi agents (negatively charged) and also enhance their interaction with the negatively charged cell membrane, thereby enabling efficient uptake of RNAi agents by cells. Cationic lipids, dendrimers, or polymers can be bound to RNAi agents or can be induced to form vesicles or micelles that encapsulate RNAi agents (see, for example, Kim SH. et al., (2008) Journal of Controlled Release 129(2):107-116). The formation of vesicles or micelles also prevents the degradation of RNAi agents when administered systemically. The method of creating and administering cationic RNAi agent complexes is well within the capabilities of those skilled in the art (see, for example, Sorensen, DR., et al. (2003) J. Mol. Biol 327:761-766; Verma, UN. et al., (2003) Clin. Cancer Res. 9:1291-1300; Arnold, AS et al. (2007) J. Hypertens. 25:197-205, the entire contents of which are incorporated herein by reference).Some non-limiting examples of drug delivery systems useful for systemic delivery of RNAi agents include DOTAP [Sorensen, DR., et al (2003), supra; Verma, UN. et al., (2003), supra], Oligofectamine, "solid nucleic acid lipid particles" [Zimmermann, TS. et al., (2006) Nature 441:111-114], cardiolipin [Chien, PY. et al., (2005) Cancer Gene Ther. 12:321-328; Pal, A. et al., (2005) Int J. Oncol. 26:1087-1091], polyethyleneamine [Bonnet ME. et al., (2008) Pharm. Res. Aug 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659], Arg-Gly-Asp (RGD) peptide [Liu, S. (2006) Mol. Pharm. 3:472-487], and polyamidoamine [Tomalia, DA. et al., (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H. et al., (1999) Pharm. Res. 16:1799-1804]. In some embodiments, the RNAi agent is complexed with cyclodextrin for systemic administration. Methods of administration and pharmaceutical compositions of RNAi agents and cyclodextrins can be found in U.S. Patent No. 7,427,605, the entire contents of which are incorporated herein by reference.

[0469] Certain aspects of the present disclosure relate to a method for reducing expression of an HTT target gene in a cell, the method comprising contacting the cell with a double-stranded RNAi agent of the present disclosure. In one embodiment, the cell is an extrahepatic cell, optionally a CNS cell.

[0470] Another aspect of the present disclosure relates to a method of reducing expression of an HTT target gene in a subject, the method comprising administering to the subject a double-stranded RNAi agent of the present disclosure.

[0471] Another aspect of the present disclosure relates to a method of treating a subject with a CNS disorder, comprising administering to the subject a therapeutically effective amount of a double-stranded HTT-targeting RNAi agent of the present disclosure, thereby treating the subject. Exemplary CNS disorders that can be treated by the methods of the present disclosure include Huntington's disease.

[0472] In one embodiment, double-stranded RNAi agent is administered intrathecally.By administering double-stranded RNAi agent intrathecally, this method can reduce the expression of HTT target gene in brain (for example, striatum) or spinal tissue, for example, cortex, cerebellum, cervical vertebrae, lumbar vertebrae and thoracic vertebrae.

[0473] For ease of explanation, the formulation, composition and method in this section are mainly described with respect to modified siRNA compounds.However, it can be understood that these formulations, compositions and methods can be practiced with other siRNA compounds, for example, unmodified siRNA compounds, and such practice is within the scope of the present disclosure.The composition comprising RNAi agent can be delivered to subject by various routes.Exemplary routes include intrathecal, intravenous, topical, rectal, anal, vaginal, nasal, pulmonary and ocular.

[0474] The RNAi agent of the present disclosure can be incorporated into pharmaceutical compositions suitable for administration.Such compositions usually comprise one or more RNAi agents and pharmaceutically acceptable carriers.As used herein, the phrase " pharmaceutically acceptable carriers " is intended to include any solvent, dispersion medium, coating agent, antifungal and antifungal agent, isotonic and absorption retardant agent, and the like, that are compatible with pharmaceutical administration.The use of such media and agents for pharmaceutically active substances is well known in the art.Unless any conventional media or agent is incompatible with active ingredient, its use in composition is contemplated.A supplementary active ingredient can also be incorporated into composition.

[0475] The pharmaceutical compositions of the present disclosure can be administered in several ways, depending on whether local or systemic treatment is desired and the area to be treated. Administration can be topical (e.g., ophthalmic, vaginal, rectal, intranasal, transdermal, etc.), oral, or parenteral. Parenteral administration includes infusion, subcutaneous, intraperitoneal, or intramuscular injection, or intrathecal or intraventricular administration.

[0476] The route and site of administration can be selected to enhance targeting.For example, for targeting muscle cells, intramuscular injection into the target muscle would be a logical choice.Lung cells can be targeted by administering RNAi agent in aerosol form.Vascular endothelial cells can be targeted by coating balloon catheter with RNAi agent and mechanically introducing RNA.

[0477] Preparations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, solutions, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners, and the like may also be necessary or desirable. Coated condoms, gloves, and the like may also be useful.

[0478] Oral administration compositions include powders or granules, suspensions or solutions in water, syrups, elixirs or non-aqueous media, tablets, capsules, drops, or lozenges.For tablets, carriers that can be used include lactose, sodium citrate, and salts of phosphoric acid.Various disintegrants, such as starch, and lubricants, such as magnesium stearate, sodium lauryl sulfate, and talc, are commonly used in tablets.For oral administration in capsule form, useful diluents are lactose and high molecular weight polyethylene glycol.When aqueous suspensions are required for oral administration, nucleic acid compositions can be combined with emulsifiers and suspending agents.If desired, certain sweeteners or flavorings can be added.

[0479] Compositions for intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.

[0480] Preparations for parenteral administration may include sterile aqueous solutions, which may also contain buffers, diluents, and other suitable additives. Intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir. For intravenous use, the total concentration of solutes may be controlled to make the preparation isotonic.

[0481] In one embodiment, the administration of siRNA compounds, for example, double-stranded siRNA compounds or ssiRNA compounds, compositions is parenteral, for example, intravenous (for example, as a bolus or as a diffuse infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary, intranasal, urethral, ​​or ocular.Administration can be provided by the patient or by another person, such as a healthcare provider.Medicinal products can be provided in measured doses or in dispensers that deliver metered doses.Selected delivery modes are described in more detail below.

[0482] A. Intrathecal administration In one embodiment, double-stranded RNAi agent is delivered by intrathecal injection (i.e., injection into the cerebrospinal fluid that bathes brain and spinal cord tissue).The intrathecal injection of RNAi agent into cerebrospinal fluid can be carried out as a bolus injection or by a minipump that can be injected subcutaneously, thereby providing regular and constant delivery of siRNA into cerebrospinal fluid.The circulation of cerebrospinal fluid from the choroid plexus where cerebrospinal fluid is produced descends around the spinal cord and dorsal root ganglion, then passes through the cerebellum and crosses the cortex to the arachnoid granulation, where the fluid can exit the CNS, and depending on the size, stability and solubility of the compound that is injected, the molecule that is delivered by intrathecal can attack targets throughout the CNS.

[0483] In some embodiments, intrathecal administration is via a pump. The pump may be a surgically implanted osmotic pump. In one embodiment, the osmotic pump is implanted in the subarachnoid space of the spinal canal to facilitate intrathecal administration.

[0484] In some embodiments, intrathecal administration is via an intrathecal pharmaceutical delivery system that includes a reservoir containing a quantity of the pharmaceutical agent and a pump configured to deliver a portion of the pharmaceutical agent contained in the reservoir. Further details about this intrathecal delivery system can be found in WO2015 / 116658, which is incorporated herein by reference in its entirety.

[0485] The amount of RNAi agent injected intrathecally may vary from one target gene to another, and the appropriate amount to be applied may have to be determined individually for each target gene. Typically, this amount is in the range of 10 μg to 2 mg, preferably 50 μg to 1500 μg, more preferably 100 μg to 1000 μg.

[0486] B. Vector-encoded RNAi agents of the present disclosure RNAi agents targeting HTT gene can be expressed from transcription units inserted into DNA or RNA vectors (see, for example, Couture, A, et al., TIG. (1996), 12:5-10; WO 00 / 22113, WO 00 / 22114, and US 6,054,299).Expression preferably continues (for several months or longer) depending on the specific construct used and the target tissue or cell type.These transgenes can be introduced as linear constructs, circular plasmids, or viral vectors, and they can be integrated or non-integrated vectors.Transgenes can also be constructed to allow them to be propagated as extrachromosomal plasmids (Gassmann, et al., (1995) Proc. Natl. Acad. Sci. USA 92:1292).

[0487] Each strand of RNAi agent can be transcribed from the promoter on expression vector.When two separate strands are expressed, for example, to produce dsRNA, two separate expression vectors can be co-introduced into target cells (for example, by transfection or infection).Alternatively, each separate strand of dsRNA can be transcribed by the promoter located on the same expression plasmid.In one embodiment, dsRNA is expressed as an inverted repeat polynucleotide that is connected by linker polynucleotide sequence, so that dsRNA has a stem-and-loop structure.

[0488] RNAi agent expression vector is generally DNA plasmid or virus vector.By using the expression vector that is compatible with eukaryotic cells, preferably the expression vector that is compatible with vertebrate cells, the recombinant construct for expressing the RNAi agent described herein can be produced.The delivery of RNAi agent expression vector can be systemic, for example, by intravenous or intramuscular administration, by administration to the target cell that is explanted from the patient and then reintroduced into the patient, or by any other means that can be introduced into desired target cell.

[0489] Viral vector systems that can be used with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, such as, but not limited to, lentivirus vectors and Moloney murine leukemia virus; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyomavirus vectors; (g) papillomavirus vectors; (h) picornavirus vectors; (i) vesicular virus vectors, such as orthopox, e.g., vaccinia virus vectors, or avipox, e.g., canarypox or fowl diphtheria; and (j) helper-dependent or gutless adenoviruses. Replication-deficient viruses may also be advantageous. Different vectors may or may not integrate into the cellular genome. The construct can contain viral sequences for transfection, if desired. Alternatively, the constructs can be incorporated into vectors capable of episomal replication, such as EPV and EBV vectors. Constructs for recombinant expression of RNAi agents will generally require regulatory elements, such as promoters, enhancers, etc., to ensure expression of the RNAi agent in target cells. Other aspects of vector and construct considerations are known in the art.

[0490] VI. Pharmaceutical Compositions of the Present Invention The present disclosure also includes pharmaceutical compositions and formulations comprising the RNAi agents of the present disclosure. In one embodiment, a pharmaceutical composition comprising an RNAi agent described herein and a pharmaceutically acceptable carrier is provided. Provided herein are pharmaceutical compositions comprising: Pharmaceutical compositions comprising an RNAi agent are useful for treating diseases or disorders associated with HTT expression or activity, such as Huntington's disease.

[0491] In some embodiments, the pharmaceutical compositions of the present invention are sterile. In other embodiments, the pharmaceutical compositions of the present invention are pyrogen-free or non-pyrogenic.

[0492] Such pharmaceutical compositions are formulated based on the mode of delivery. One example is a composition formulated for systemic administration by parenteral delivery, for example, intravenous (IV), intramuscular (IM), or subcutaneous (subQ) delivery. Another example is a composition formulated for direct delivery into the CNS, for example, by intrathecal or intravitreal route of injection, optionally by injection into the brain (e.g., corpus striatum), for example, by continuous pump infusion.

[0493] The pharmaceutical composition of the present disclosure can be administered in a dosage that is sufficient to inhibit the expression of HTT gene.Generally, the suitable dosage of the RNAi agent of the present disclosure will be in the range of about 0.001 milligrams to about 200.0 milligrams per kilogram of recipient body weight per day, generally in the range of about 1 mg to 50 mg per kilogram of recipient body weight per day.

[0494] A repeat dosing regimen can involve administering a therapeutic amount of an RNAi agent on a regular basis, for example, from once a month to once every six months, etc. In certain embodiments, the RNAi agent is administered from about once a quarter (i.e., about once every three months) to about twice a year.

[0495] After an initial treatment regimen (eg, a loading dose), treatment can be administered less frequently.

[0496] In other embodiments, the single administration of the pharmaceutical composition can be continued, whereby subsequent doses are administered at intervals of one month or less, two months or less, three months or less, or four months or less, or more. In some embodiments of the present disclosure, the single administration of the pharmaceutical composition of the present disclosure is administered monthly. In other embodiments of the present disclosure, the single administration of the pharmaceutical composition of the present disclosure is administered quarterly to twice a year.

[0497] Those skilled in the art will understand that certain factors, such as, but not limited to, the severity of the disease or disorder, previous treatments, the general health or age of the subject, and other existing diseases, can affect the dosage and timing of dosages required to effectively treat a subject. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments.

[0498] Advances in mouse genetics have produced many mouse models for the study of various human diseases, such as HD, which may benefit from the reduction of HTT expression.Such models can be used for the in vivo testing of RNAi agents and for determining therapeutically effective doses.Suitable rodent models are known in the art, and include, for example, those described in Cepeda, et al. (ASN Neuro (2010) 2(2):e00033) and Pouladi, et al. (Nat Reviews (2013) 14:708).

[0499] The pharmaceutical composition of the present disclosure can be administered in several ways, depending on whether local or systemic treatment is desired and the area to be treated.Administration can be topical (for example, by transdermal patch), pulmonary (for example, by inhalation or insufflation of powder or aerosol, such as by nebulizer); intratracheal, intranasal, epidermal and transdermal, oral or parenteral.Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subcutaneous (for example, by implanted device) or intracerebral (for example, by intraparenchymal, intrathecal or intraventricular administration).

[0500] RNAi agents can be delivered in a manner that targets specific tissues, such as the CNS (eg, neurons, glial cells, or vascular tissue of the brain).

[0501] Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powdery, or oily bases, thickeners, etc. may be necessary or desirable. Coated condoms, gloves, etc. may also be useful. Suitable topical formulations include those in which the RNAi agent featured in the present disclosure is mixed with a topical delivery agent, such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents, and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidylethanolamine DOPE, dimyristoylphosphatidylcholine DMPC, distearoylphosphatidylcholine), cationic (e.g., dimyristoylphosphatidylglycerol DMPG), and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). The RNAi agent featured in the present disclosure can be encapsulated in liposomes or can be complexed with liposomes, particularly cationic liposomes.Alternatively, the RNAi agent can be complexed with lipids, particularly cationic lipids.Suitable fatty acids and esters include, but are not limited to, arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaproate, tricaproate, monoolein, dilaurin, glyceryl 1-monocaproate, 1-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine, or C 1~20Examples of suitable topical formulations include alkyl esters (e.g., isopropyl myristate IPM), monoglycerides, diglycerides, or pharmaceutically acceptable salts thereof. Topical formulations are described in detail in US 6,747,014, which is incorporated herein by reference.

[0502] A. RNAi agent formulations containing membrane molecular assemblies RNAi agents for use in the compositions and methods of the present disclosure can be formulated for delivery in membrane molecular assemblies, such as liposomes or micelles. As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one or more bilayers. Liposomes include unilamellar and multilamellar vesicles with a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the RNAi agent composition. The lipophilic material separates the aqueous interior from the aqueous exterior, which typically does not contain (but may in some instances contain) the RNAi agent composition. Liposomes are useful for transporting and delivering activ...

Claims

1. A double-stranded ribonucleic acid (dsRNA) agent or a salt thereof for inhibiting huntingtin (HTT) expression, dsRNA or a salt thereof comprises a sense strand and an antisense strand that form a double-stranded region, The antisense strand contains at least 17 consecutive nucleotides from the nucleotide sequence 5'-UCGAAUGUGAAUUAUGAAUAGCAU-3' of SEQ ID NO: 2009, All nucleotides in the sense strand and all nucleotides in the antisense strand contain nucleotide modifications, and, A sense chain, an antisense chain, or both a sense chain and an antisense chain are conjugated to one or more lipophilic moieties. dsRNA preparation or a salt thereof.

2. The dsRNA agent or a salt thereof according to claim 1, wherein the antisense strand comprises at least 19 consecutive nucleotides from the nucleotide sequence 5'-UCGAAUGUGAAUAUGAAUAGCAU-3' of SEQ ID NO: 2009.

3. A dsRNA agent or salt thereof according to claim 1 or 2, wherein the antisense strand comprises the nucleotide sequence 5'-UCGAAUGUGAAUUAUGAAUAGCAU-3' of SEQ ID NO: 2009.

4. The dsRNA agent or a salt thereof according to claim 3, wherein the antisense strand consists of the nucleotide sequence 5'-UCGAAUGUGAAUUAUGAAUAGCAU-3' of SEQ ID NO: 2009.

5. A dsRNA agent or a salt thereof according to any one of claims 1 to 4, wherein the sense strand comprises at least 17 consecutive nucleotides from the nucleotide sequence 5'-GCUAUUCAUAAUCACAUUCGA-3' of SEQ ID NO: 1846.

6. A dsRNA agent or a salt thereof according to any one of claims 1 to 5, wherein the sense strand comprises at least 19 consecutive nucleotides from the nucleotide sequence 5'-GCUAUUCAUAAUCACAUUCGA-3' of SEQ ID NO: 1846.

7. A dsRNA agent or a salt thereof according to any one of claims 1 to 6, wherein the sense strand comprises the nucleotide sequence 5'-GCUAUUCAUAAUCACAUUCGA-3' of SEQ ID NO: 1846.

8. The dsRNA agent or a salt thereof according to claim 7, wherein the sense strand consists of the nucleotide sequence 5'-GCUAUUCAUAAUCACAUUCGA-3' of SEQ ID NO: 1846.

9. A dsRNA agent or a salt thereof according to any one of claims 1 to 8, wherein the sense strand comprises the nucleotide sequence 5'-GCUAUUCAUAAUCACAUUCGA-3' of SEQ ID NO: 1846, and the antisense strand comprises the nucleotide sequence 5'-UCGAAUGUGAAUUAUGAAUAGCAU-3' of SEQ ID NO: 2009.

10. A dsRNA agent or salt thereof according to any one of claims 1 to 9, wherein one or more lipophilic moieties are conjugated to one or more internal positions in at least one strand via a linker or carrier.

11. A dsRNA agent or salt thereof according to any one of claims 1 to 10, wherein one or more lipophilic moieties are conjugated to one or more 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.

12. A dsRNA agent or a salt thereof according to any one of claims 1 to 11, wherein one or more lipophilic portions contain saturated or unsaturated C4-C30 hydrocarbon chains.

13. The dsRNA agent or a salt thereof according to claim 12, wherein one or more lipophilic moieties contain a saturated or unsaturated C4-C30 hydrocarbon chain and a functional group selected from the group consisting of amines, carboxylic acids, sulfonates, phosphates, thiols, azides, and alkynes.

14. A dsRNA agent or a salt thereof according to any one of claims 1 to 13, wherein one or more lipophilic moieties are saturated or unsaturated C16 hydrocarbon chains and are conjugated at position 6 counting from the 5' end of the chain.

15. A dsRNA agent or salt thereof according to any one of claims 1 to 14, wherein one or more saturated or unsaturated C6-C18 hydrocarbon chains are conjugated to a dsRNA agent or salt thereof via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfamide linkage, click reaction product, or carbamate.

16. A dsRNA agent or a salt thereof according to any one of claims 1 to 14, wherein one or more lipophilic moieties are conjugated to nucleic acid bases, sugar moieties, or internucleoside links.

17. At least one of the nucleotide modifications is a deoxy-nucleotide modification, a 3'-terminal deoxythymine (dT) nucleotide modification, a 2'-O-methyl nucleotide modification, a 2'-fluoro nucleotide modification, a 2'-deoxy-nucleotide modification, a locked nucleotide modification, an unlocked nucleotide modification, a conformation-restricted nucleotide modification, a restricted ethyl nucleotide modification, a debasalized nucleotide modification, a 2'-amino-nucleotide modification, a 2'-O-allyl-nucleotide modification, a 2'-C-alkyl-nucleotide modification, a 2'-hydroxyl-nucleotide modification, a 2'-methoxyethyl nucleotide modification, a 2'-O-alkyl-nucleotide modification, a morpholino nucleotide modification, a phosphoramide 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 adenosine glycol nucleic acid (GNA), a thymid Nucleotide modifications including n-glycolic 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, dodecanoic acid bisdecylamide group modifications, cytidine-2'-phosphate modifications, guanosine-2'-phosphate A dsRNA agent or a salt thereof according to any one of claims 1 to 16, selected from the group consisting of phosphate modification, uridine-2'-phosphate modification, adenosine-2'-phosphate modification, 2'-O-hexadecyl-adenosine-3'-phosphate modification, 2'-O-hexadecyl-cytidine-3'-phosphate modification, 2'-O-hexadecyl-guanosine-3'-phosphate modification, and 2'-O-hexadecyl-uridine-3'-phosphate modification, and combinations thereof.

18. A dsRNA agent or a salt thereof according to any one of claims 1 to 17, further comprising at least one phosphorothioate nucleotide linkage.

19. The dsRNA agent or salt thereof according to claim 18, wherein the dsRNA agent or salt thereof comprises 6 to 8 phosphorothioate nucleotide linkages.

20. A dsRNA agent or a salt thereof according to any one of claims 1 to 19, wherein each chain is 30 nucleotides or less in length.

21. A dsRNA agent or salt thereof according to any one of claims 1 to 20, wherein at least one strand comprises a 3' overhang of at least one nucleotide.

22. A dsRNA agent or a salt thereof according to any one of claims 1 to 21, wherein the double-stranded region is 15 to 30 nucleotide pairs long.

23. A dsRNA agent or a salt thereof according to any one of claims 1 to 22, wherein the 3' end of the sense strand is protected via an end cap which is a cyclic group having an amine, and the cyclic group is selected from the group consisting of pyrrolidinyl, pyrazolinil, pyrazolidinyl, imidazolinil, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanil, oxazolidinyl, isoxazolidinyl, morpholinil, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridadinyl, tetrahydrofuranil, and dekalinil.

24. A dsRNA agent or salt thereof according to any one of claims 1 to 23, further comprising a phosphate or phosphate mimetic at the 5' end of the antisense strand.

25. The dsRNA agent or a salt thereof according to claim 24, wherein the phosphate mimetic is 5'-vinylphosphonate (VP).

26. Cells containing the dsRNA agent or a salt thereof according to any one of claims 1 to 25.

27. A pharmaceutical composition for inhibiting the expression of a gene encoding HTT, comprising a dsRNA agent or a salt thereof as described in any one of claims 1 to 25.

28. An in vitro method for inhibiting the expression of the huntingtin (HTT) gene in cells, (a) Contacting cells with a dsRNA agent or a salt thereof according to any one of claims 1 to 25 or the pharmaceutical composition according to claim 27, and (b) Maintain the cells generated in step (a) for a sufficient amount of time to obtain degradation of the mRNA transcript of the HTT gene, thereby inhibiting the expression of the HTT gene in the cells. In vitro methods including those mentioned above.

29. A pharmaceutical composition for treating a subject diagnosed with an HTT-related disease, comprising a therapeutically effective amount of a dsRNA agent or a salt thereof according to any one of claims 1 to 25 or the pharmaceutical composition according to claim 27.

30. The pharmaceutical composition according to claim 29, wherein the HTT-related disease is Huntington's disease.

31. The pharmaceutical composition according to claim 29 or 30, wherein the dsRNA agent or a salt thereof is for intrathecal administration.

32. The pharmaceutical composition according to any one of claims 29 to 31, further comprising additional agents suitable for the treatment or prevention of HTT-related diseases.

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