Huntington (htt) iRNA pharmaceutical compositions and methods of use thereof

Abstract

The present disclosure relates to double stranded ribonucleic acid (dsRNAi) agents and compositions that target a Huntington (HTT) gene (e.g., exon 1 of the HTT gene), as well as methods of inhibiting expression of an HTT gene and methods of treating subjects having an HTT-related disease or disorder (e.g., Huntington's disease) using such dsRNAi agents and compositions.

Description

[0001] This application is a divisional application of the Chinese invention patent application with an application date of October 29, 2020, application number 202080076710.6, and invention name “Huntington (HTT) iRNA pharmaceutical composition and method of use thereof”.

[0002] Related Applications

[0003] This application claims priority to U.S. Provisional Application No. 62 / 929,174, filed on November 1, 2019, the entire contents of which are incorporated herein by reference.

[0004] Sequence Listing

[0005] 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 was created on October 26, 2020, is named 121301_10320_SL.txt, and is 1,468,832 bytes in size. Background Art

[0006] Huntington's disease is a progressive neurodegenerative disease characterized by movement disorders, cognitive loss 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 descent (Harper, PS et al., in Huntington's Disease, WB Saunders, Philadelphia, 1991). The hallmark of Huntington's disease is a unique chorea movement disorder that usually develops insidiously at the age of 4-50 and gradually worsens over 10 to 20 years until death. Occasionally, Huntington's disease manifests itself in adolescents, usually with more severe symptoms, including rigidity and a faster course of the disease. The juvenile onset of Huntington's disease is associated with the paternal genetic advantage of the disease allele. The neuropathology of Huntington's disease also displays a distinctive pattern, with selective neuronal loss being greatest in the caudate and putamen regions of the brain.

[0007] Huntington's disease has been shown to be caused by an expanded glutamine repeat sequence in exon 1 of a gene called IT15 or Huntingtin (HTT). Although the gene is ubiquitously expressed and required for normal development, Huntington's disease pathology is restricted to the brain for reasons that remain poorly understood. In patients with HD, an autosomal linear inherited disease, expansion of the polyglutamine repeat sequence results in wild-type transcripts, full-length mutant transcripts with expanded polyglutamine repeats, and truncated mutant transcripts with expanded polyglutamine repeats. It has been shown that the expansion of the polyglutamine repeat sequence and the presence of full-length mutant transcripts and truncated mutant transcripts cause toxicity despite similar expression levels of the Huntington gene product in patients and controls.

[0008] There is currently no effective treatment for Huntington's disease. Antipsychotic drugs (e.g., chlorpromazine) or reserpine may (usually only partially) suppress chorea and agitated behavior until adverse effects such as drowsiness, hypotension, or parkinsonism occur. Furthermore, despite significant progress in the field of RNAi and Huntington's disease treatment, there is still a need for a drug that can selectively and effectively silence the HD gene using the cell's own RNAi machinery, has high biological activity and in vivo stability, and can effectively inhibit the expression of the target Huntington gene. Summary of the invention

[0009] The present disclosure provides RNAi pharmaceutical compositions that affect RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of the Huntington (HTT) gene. The HTT gene can be in a cell, such as a cell in a subject such as a human. The present disclosure also provides methods of using the RNAi pharmaceutical compositions of the present disclosure to inhibit the expression of the HTT gene or to treat a subject who will benefit from inhibiting or reducing the expression of the HTT gene (e.g., a subject suffering from or prone to suffering from an HTT-related disease).

[0010] In one aspect, the present invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting Huntington's (HTT) expression, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15, 16, 17, 18, 19, 20 or 21 consecutive 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 consecutive 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 on at least one strand.

[0011] 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, or 33.

[0012] 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, 5509-5510 any of the nucleotide sequences of 5531, 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 differs by no more than 0, 1, 2, or 3 nucleotides over at least 15 consecutive nucleotides.

[0013] In some embodiments, the antisense strand comprises at least 15 consecutive nucleotides 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, The nucleotide sequence of any antisense strand of the duplex of D-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 does not differ by more than 0, 1, 2 or 3 nucleotides.

[0014] In some embodiments, the lipophilic moiety is conjugated via a linker or a carrier.

[0015] On the other hand, the present invention provides a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of Huntington (HTT) in a cell, wherein the dsRNA comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a complementary region to an mRNA encoding HTT, and wherein the complementary region comprises at least 15 (e.g., 15, 16, 17, 18, 19, 20 or 21) consecutive nucleotides that differ by no more than 3, 2, 1 or 0 nucleotides from any 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 or 33.

[0016] The sense strand, the antisense strand, or both may be conjugated to one or more lipophilic moieties. In some embodiments, the lipophilic moiety is conjugated to one or more internal positions in the double-stranded region of the dsRNA agent, for example, one or more lipophilic moieties may be conjugated to one or more internal positions on the antisense strand. In some embodiments, one or more lipophilic moieties are conjugated to one or more internal positions on at least one strand via a linker or carrier.

[0017] In some embodiments, the lipophilicity of the lipophilic moiety as measured by logKow is greater than zero.

[0018] 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.

[0019] In some embodiments, the internal position includes all positions except the terminal two positions of each end of the sense strand or antisense strand. In other embodiments, the internal position includes all positions except the terminal three positions of each end of the sense strand or antisense strand.

[0020] In some embodiments, the internal positions do not include the cleavage site region of the sense strand, for example, the internal positions include all positions except positions 9-12 counted from the 5' end of the sense strand, or the internal positions include all positions except positions 11-13 counted from the 3' end of the sense strand.

[0021] In some embodiments, the internal positions do not include the cleavage site region of the antisense strand. In other embodiments, the internal positions include all positions except positions 12-14 counting from the 5' end of the antisense strand. In some embodiments, the internal positions include all positions except positions 11-13 counting from the 3' end on the sense strand and positions 12-14 counting from the 5' end on the antisense strand.

[0022] In some embodiments, one or more lipophilic moieties are conjugated to one or more internal positions selected from 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.

[0023] In some embodiments, one or more lipophilic moieties are conjugated to one or more internal positions selected from positions 5, 6, 7, 15, and 17 on the sense strand and positions 15 and 17 on the antisense strand, counting from the 5' end of each strand.

[0024] In some embodiments, the position in the double-stranded region does not include the cleavage site region of the sense strand.

[0025] 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.

[0026] 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.

[0027] In some embodiments, the lipophilic moiety is an aliphatic, alicyclic, or polyalicyclic compound.

[0028] In some embodiments, the lipophilic moiety is selected from a lipid, cholesterol, retinoic acid, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexanol, hexadecylglycerol, borneol, menthol, 1,3-propylene glycol, heptadecanyl, palmitic acid, myristic acid, (O3)-(oleoyl)lithocholic acid, O3-(oleoyl)cholic acid, dimethoxytrityl, or phenoxazine.

[0029] In some embodiments, the lipophilic moiety comprises a saturated or unsaturated C4-C30 hydrocarbon chain and an optional functional group selected from hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.

[0030] In some embodiments, the lipophilic moiety comprises a saturated or unsaturated C6-C18 hydrocarbon chain.

[0031] In some embodiments, the lipophilic moiety comprises a saturated or unsaturated C16 hydrocarbon chain. In some embodiments, the saturated or unsaturated C16 hydrocarbon chain is conjugated to position 6, counted from the 5' end of the chain.

[0032] In some embodiments, the lipophilic moiety is conjugated by a carrier that replaces one or more nucleotides in an internal position or in a double-stranded region. In some embodiments, the carrier is a cyclic group selected from pyrrolidinyl, pyrazolyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3] dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinone, tetrahydrofuranyl, decahydronaphthyl; or a non-cyclic moiety based on a serinol backbone or a diethanolamine backbone.

[0033] In some embodiments, the lipophilic moiety is conjugated to the dsRNA agent through a linker comprising an ether, thioether, urea, carbonate, amine, amide, maleimide thioether, disulfide, phosphodiester, sulfonamide bond, the product of a click reaction, or a carbamate.

[0034] In some embodiments, the lipophilic moiety is conjugated to a nucleobase, a sugar moiety, or an internucleoside linkage.

[0035] In some embodiments, the dsRNA agent comprises at least one modified nucleotide. In some embodiments, no more than five sense strand nucleotides and no more than five antisense strand nucleotides are unmodified nucleotides. In other embodiments, all nucleotides of the sense strand and all nucleotides of the antisense strand comprise modifications.

[0036] In some embodiments, at least one modified nucleotide is selected from deoxynucleotides, 3'-terminal deoxythymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, unlocked nucleotides, conformationally constrained nucleotides, constrained ethyl nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides, 2'-hydroxy modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing non-natural bases, tetrahydropyranose modified nucleotides, The invention relates to nucleotides comprising: modified nucleotides, 1,5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing 5'-phosphorothioate groups, nucleotides containing 5'-methylphosphonate groups, nucleotides containing 5' phosphate or 5' phosphate mimetics, nucleotides containing vinyl phosphonate, nucleotides containing adenosine-diol nucleic acid (GNA), nucleotides containing thymidine-diol nucleic acid (GNA) S-isomers, nucleotides containing 2-hydroxymethyl-tetrahydrofuranyl-5-phosphate, nucleotides containing 2'-deoxythymidine-3' phosphate, nucleotides containing 2'-deoxyguanosine-3'-phosphate, and terminal nucleotides linked to cholesterol derivatives and combinations thereof.

[0037] In other embodiments, the modified nucleotides are selected from 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, 3'-terminal deoxythymidine nucleotides (dT), locked nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, and nucleotides containing non-natural bases.

[0038] In some embodiments, the at least one modified nucleotide is selected from deoxynucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, diol modified nucleotides (GNA) and vinylphosphonate nucleotides, and combinations thereof.

[0039] In some embodiments, at least one modification on the nucleotide is a thermally unstable nucleotide modification. In some embodiments, the thermally unstable nucleotide modification is selected from abasic modification; mismatch with the opposite nucleotide in the duplex; and destabilizing sugar modification, 2'-deoxy modification, acyclic nucleotide, non-locked nucleic acid (UNA) and glycerol nucleic acid (GNA).

[0040] In some embodiments, the modified nucleotide comprises a short sequence of 3' terminal deoxythymidine nucleotide (dT).

[0041] In some embodiments, the modifications on the nucleotides are 2'-O-methyl, GNA, and 2' fluoro modifications.

[0042] In some embodiments, the dsRNA agent further comprises at least one phosphorothioate internucleotide bond. In some embodiments, the dsRNA agent comprises 6-8 phosphorothioate internucleotide bonds. In one embodiment, the phosphorothioate or methylphosphonate internucleotide bond is located at the 3' end of a strand. Preferably, the strand is an antisense strand. In another embodiment, the strand is an antisense strand. In a related embodiment, the phosphorothioate or methylphosphonate internucleotide bond is located at the 5' end of a strand. Preferably, the strand is an antisense strand. In another embodiment, the strand is an antisense strand. In another embodiment, the phosphorothioate or methylphosphonate internucleotide bond is located at the 5' and 3' ends of a strand. Preferably, the strand is an antisense strand. In another embodiment, the strand is a sense strand.

[0043] In some embodiments, each strand is no more than 30 nucleotides in length.

[0044] In some embodiments, at least one strand comprises a 3' overhang of at least one nucleotide or a 3' overhang of at least 2 nucleotides.

[0045] The double-stranded region can be 15-30 nucleotide pairs in length; 17-23 nucleotide pairs in length; 17-25 nucleotide pairs in length; 23-27 nucleotide pairs in length; 19-21 nucleotide pairs in length; or 21-23 nucleotide pairs in length.

[0046] Each strand may be 19-30 nucleotides; 19-23 nucleotides; or 21-23 nucleotides.

[0047] 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.

[0048] In certain embodiments, the double-stranded RNAi agent further comprises a targeting ligand that targets a receptor that mediates delivery to CNS tissues, such as a hydrophilic ligand.

[0049] In certain embodiments, the targeting ligand is a C16 ligand. In one embodiment, the ligand is

[0050]

[0051] wherein B is a nucleotide base or a nucleotide base analog, optionally wherein B is adenine, guanine, cytosine, thymine or uracil.

[0052] In some embodiments, the hydrophilic moiety or targeting ligand is conjugated via a biocleavable linker selected from the group consisting of DNA, RNA, disulfide, amide, galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

[0053] In some embodiments, the 3' end of the sense strand is protected by a capping agent, wherein the capping agent is a cyclic group having an amine, wherein the cyclic group is selected from pyrrolidinyl, pyrazolyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinone, tetrahydrofuranyl and decahydronaphthyl.

[0054] In some embodiments, the dsRNA agent further comprises a terminal chiral modification at the first internucleotide bond of the 3' end of the antisense strand having an attached phosphorus atom with a Sp configuration, a terminal chiral modification at the first internucleotide bond of the 5' end of the antisense strand having an attached phosphorus atom with a Rp configuration, and a terminal chiral modification at the first internucleotide bond of the 5' end of the sense strand having an attached phosphorus atom with either an Rp configuration or an Sp configuration.

[0055] In some embodiments, the dsRNA agent further comprises a terminal chiral modification at the first and second internucleotide bonds of the 3' end of the antisense strand having an attached phosphorus atom with a Sp configuration, a terminal chiral modification at the first internucleotide bond of the 5' end of the antisense strand having an attached phosphorus atom with a Rp configuration, and a terminal chiral modification at the first internucleotide bond of the 5' end of the sense strand having an attached phosphorus atom with either an Rp configuration or an Sp configuration.

[0056] In some embodiments, the dsRNA agent further comprises a terminal chiral modification at the first, second, and third internucleotide bonds at the 3' end of the antisense strand having an attached phosphorus atom in a Sp configuration, a terminal chiral modification at the first internucleotide bond at the 5' end of the antisense strand having an attached phosphorus atom in an Rp configuration, and a terminal chiral modification at the first internucleotide bond at the 5' end of the sense strand having an attached phosphorus atom in either an Rp configuration or an Sp configuration.

[0057] In some embodiments, the dsRNA agent further comprises a terminal chiral modification at the first and second internucleotide bonds of the 3' end of the antisense strand having an attached phosphorus atom with a Sp configuration, a terminal chiral modification at the third internucleotide bond of the 3' end of the antisense strand having an attached phosphorus atom with a Rp configuration, a terminal chiral modification at the first internucleotide bond of the 5' end of the antisense strand having an attached phosphorus atom with a Rp configuration, and a terminal chiral modification at the first internucleotide bond of the 5' end of the sense strand having an attached phosphorus atom with either an Rp configuration or an Sp configuration.

[0058] In some embodiments, the dsRNA agent further comprises a terminal chiral modification at the first and second internucleotide bonds at the 3' end of the antisense strand having an attached phosphorus atom with a Sp configuration, a terminal chiral modification at the first and second internucleotide bonds at the 5' end of the antisense strand having an attached phosphorus atom with a Rp configuration, and a terminal chiral modification at the first internucleotide bond at the 5' end of the sense strand having an attached phosphorus atom with either an Rp configuration or an Sp configuration.

[0059] In some embodiments, the dsRNA agent further comprises a phosphate or a phosphate mimetic at the 5' end of the antisense strand. In some embodiments, the phosphate mimetic is 5'-vinylphosphonate (VP).

[0060] In some embodiments, the base pair at position 1 of the 5' end of the antisense strand of the duplex is an AU base pair.

[0061] In some embodiments, the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

[0062] Another aspect of the present disclosure provides a double-stranded RNAi agent for inhibiting the expression of Huntington's (HTT) gene, wherein the double-stranded RNAi agent targeting 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) consecutive nucleotides that differ from any one of the nucleotide sequences in SEQ ID NOs: 1-5 by no more than 3 nucleotides (e.g., 3, 2, 1, or 0 nucleotides), and the antisense strand comprises at least 15 (e.g., 15, 16, 17, 18, 19, or 20) consecutive nucleotides that differ from any one of the nucleotide sequences in SEQ ID NOs: 6-10 by no more than 3 nucleotides (e.g., 3, 2, 1, or 0 nucleotides); wherein substitution of any thymine with uracil in the sequences provided in SEQ ID NOs: 1-10 (when comparing the sequences) is not counted as resulting in a difference from SEQ ID NOs: 1-11. Any of the nucleotide sequences provided in NOs: 1-10 differ by no more than 3 nucleotides, wherein 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, wherein the sense strand comprises two phosphorothioate internucleotide bonds at the 5' end, wherein substantially all of the nucleotides of the antisense strand comprise a modification selected from a 2'-O-methyl modification and a 2'-fluoro modification, wherein the antisense strand comprises two phosphorothioate internucleotide bonds at the 5' end and a phosphorothioate internucleotide bond at the 3' end, and wherein the sense strand is conjugated to one or more lipophilic ligands (e.g., C16).

[0063] Another aspect of the present disclosure provides a double-stranded RNAi agent for inhibiting the expression of Huntington's (HTT) gene, wherein the double-stranded RNAi agent targeting 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) consecutive nucleotides that differ from any one of the nucleotide sequences in SEQ ID NOs: 1-5 by no more than 3 nucleotides (e.g., by 3, 2, 1, or 0 nucleotides), and the antisense strand comprises at least 15 (e.g., 15, 16, 17, 18, 19, or 20) consecutive nucleotides that differ from any one of the nucleotide sequences in SEQ ID NOs: 6-10 by no more than 3 nucleotides (e.g., by 3, 2, 1, or 0 nucleotides), wherein substitution of any thymine with uracil in the sequences provided in SEQ ID NOs: 1-10 (when comparing the sequences) is not counted as resulting in a difference from SEQ ID NOs: 1-10. Any of the nucleotide sequences provided in NO: 1-10 differs by no more than 3 nucleotides; wherein the sense strand comprises at least one 3'-terminal deoxythymidine nucleotide (dT), and wherein the antisense strand comprises at least one 3'-terminal deoxythymidine nucleotide (dT).

[0064] Another aspect of the present disclosure provides a double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of Huntington's (HTT) gene, wherein the RNAi agent has a sense strand and an antisense strand, and wherein the antisense strand comprises a complementary region comprising at least 15 consecutive nucleotides, which differ from any antisense strand nucleobase sequence in Tables 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 24, 25, 27-30, 32 and 33 by no more than 3 nucleotides (i.e., by 3, 2, 1 or 0 nucleotides), such as at least 15 nucleotides (i.e., by 3, 2, 1 or 0 nucleotides), such as 19 nucleotides (i.e., by 3, 2, 1 or 0 nucleotides). 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, nucleotides comprising diol nucleic acids (GNA), phosphorothioates (PS), and vinylphosphonates (VP). Optionally, the RNAi agent comprises at least one of the following modifications: 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-C-alkyl-modified nucleotides, nucleotides comprising diol nucleic acids (GNA), phosphorothioates, and vinylphosphonates (VP).

[0065] In another embodiment, the RNAi agent comprises four or more PS modifications, optionally six to ten PS modifications, optionally eight PS modifications.

[0066] In additional embodiments, the sense and antisense strands of the RNAi agent each have a 5' end and a 3' end, and the RNAi agent comprises eight PS modifications located at the penultimate and last internucleotide bonds at the 3' and 5' ends of each sense and antisense strand of the RNAi agent, respectively.

[0067] In another embodiment, the sense strand and antisense strand of the RNAi agent each comprise a 5' end and a 3' end, and the RNAi agent comprises only one nucleotide, including GNA. Optionally, the nucleotide including GNA is located on the antisense strand at the seventh nucleobase residue from the 5' end of the antisense strand.

[0068] In another embodiment, the sense strand and antisense strand of the RNAi agent each comprise 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 nucleotides are 2'-C16 modified nucleotides. Optionally, the RNAi agent comprises a single 2'-C-alkyl, e.g., C16 modified nucleotide. Optionally, a single 2'-C-alkyl, e.g., C16 modified nucleotide is located at the sixth nucleobase on the sense strand from the 5' end of the sense strand.

[0069] In another embodiment, the sense strand and antisense strand of the RNAi agent each comprise a 5' end and a 3' end, and the RNAi agent comprises two or more 2'-fluoro-modified nucleotides. Optionally, the sense strand and antisense strand of the RNAi agent each comprise two or more 2'-fluoro-modified nucleotides. Optionally, the 2'-fluoro-modified nucleotides are located at nucleobase positions 7, 9, 10, and 11 on the sense strand starting from the 5' end of the sense strand and at nucleobase positions 2, 14, and 16 on the antisense strand starting from the 5' end of the antisense strand.

[0070] In additional embodiments, the sense strand and the antisense strand of the RNAi agent each comprise a 5' end and a 3' end, and the RNAi agent comprises one or more VP modifications. Optionally, the RNAi agent comprises a single VP modification at the 5' end of the antisense strand.

[0071] In another embodiment, the sense strand and antisense strand of the RNAi agent each include a 5' end and a 3' end, and the RNAi agent includes two or more 2'-O-methyl modified nucleotides. Optionally, the RNAi agent includes 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, two or more 2'-O-methyl modified nucleotides are located 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 are located 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 on the antisense strand.

[0072] In one embodiment, all nucleotides of the sense strand and all nucleotides of the antisense strand are modified nucleotides.

[0073] In another embodiment, each strand has 19-30 nucleotides.

[0074] In certain embodiments, the antisense strand of the RNAi agent comprises at least one thermally unstable modification of the duplex within the first 9 nucleotide positions of the 5' region or its precursor. Optionally, the thermally unstable modification of the duplex is one or more of the following:

[0075]

[0076]

[0077] Wherein B is a nucleobase.

[0078] The present invention further provides cells comprising any of the dsRNA agents of the present invention and pharmaceutical compositions for inhibiting the expression of a gene encoding HTT, comprising any of the dsRNA agents of the present invention.

[0079] In one embodiment, the double-stranded RNAi agent is in a non-buffered solution. Optionally, the non-buffered solution is saline or water. In another embodiment, the double-stranded RNAi agent is in a buffered solution. Optionally, the buffered solution includes 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 includes lipid nanoparticles (LNP).

[0080] Another aspect of the present disclosure provides a method for inhibiting the expression of the HTT gene in a cell, the method comprising (a) contacting the cell with the double-stranded RNAi agent of the present disclosure or the pharmaceutical composition of the present disclosure; (b) maintaining the cell produced in step (a) for a sufficient time to obtain degradation of the mRNA transcript of the HTT gene, thereby inhibiting the expression of the HTT gene in the cell.

[0081] In one embodiment, the cell is in a subject. Optionally, the subject is a human.

[0082] In certain embodiments, the subject is a rhesus monkey, a cynomolgus monkey, a mouse, or a rat. In certain embodiments, HTT expression is inhibited by at least about 50% by the RNAi agent.

[0083] In certain embodiments, the human subject has been diagnosed with an HTT-related disease, such as Huntington's disease.

[0084] Another aspect of the present disclosure provides a method for treating a subject diagnosed with an HTT-related disease (eg, Huntington's disease), the method 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.

[0085] In one embodiment, treatment includes ameliorating at least one sign or symptom of a disease. In another embodiment, treatment includes preventing the progression of a disease.

[0086] In some embodiments, the dsRNA agent is administered to a subject at a dose of about 0.01 mg / kg to about 50 mg / kg.

[0087] In some embodiments, the dsRNA agent is administered intrathecally to the subject. In one embodiment, the method reduces the expression of the HTT gene in brain (e.g., striatum) or spinal tissue. Optionally, the brain or spinal tissue is striatum, cortex, cerebellum, cervical spine, lumbar spine or thoracic spine.

[0088] In some embodiments, the method further comprises measuring the level of HTT in a sample obtained from the subject.

[0089] Another aspect of the present disclosure provides a method for inhibiting Huntington (HTT) expression in a subject, the method involving: administering a therapeutically effective amount of the double-stranded RNAi agent or the pharmaceutical composition of the present disclosure to the subject, thereby inhibiting the expression of HTT in the subject.

[0090] In some embodiments, the method further comprises administering to the subject an additional agent useful for treating or preventing an HTT-related disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] Figure 1 is a graph showing wild-type human HTT mRNA levels in the liver of mice expressing partial wild-type human HTT (via AAV). On day 14 after AAV administration, these mice were subcutaneously administered a single 3 mg / kg dose of the indicated dsRNA duplexes targeting exon 1 of the human HTT transcript. Human HTT levels shown were normalized to AAV-treated controls 14 days after siRNA administration.

[0092] Figure 2 is a graph showing wild-type human HTT mRNA levels in the liver of mice expressing partial wild-type human HTT (via AAV). On day 14 after AAV administration, these mice were subcutaneously administered a single 3 mg / kg dose of the indicated dsRNA duplexes targeting exon 1 of the human HTT transcript. Human HTT levels shown were normalized to AAV-treated controls 14 days after siRNA administration.

[0093] Figure 3A is a graph showing full-length mutant human HTT mRNA levels in liver of YAC128 mice at day 7 after dosing with a single 10 mg / kg subcutaneous dose of the indicated dsRNA duplexes targeting exon 1 of the human HTT transcript. Human HTT levels shown are normalized to PBS-treated levels.

[0094] Figure 3B is a Western blot showing mutant human HTT protein and wild-type mouse HTT protein levels in the liver of YAC128 mice administered subcutaneously on day 7 after a single 10 mg / kg dose of the indicated dsRNA duplexes targeting exon 1 of the human HTT transcript.

[0095] Figure 3C is a bar graph showing mutant human HTT protein levels in the liver of YAC128 mice administered a single 10 mg / kg dose subcutaneously of the indicated dsRNA duplexes targeting exon 1 of the human HTT transcript on day 7 post-dose. The mutant human HTT levels shown are normalized to PBS-treated levels.

[0096] Figure 4A is a graph showing full-length mutant HTT mRNA levels in the liver of YAC128 mice administered a single 10 mg / kg dose subcutaneously of the indicated dsRNA duplexes targeting exon 1 of the human HTT transcript on day 7 post-dosing. The full-length mutant human HTT levels shown are normalized to PBS-treated levels.

[0097] Figure 4B is a bar graph showing quantification of mutant HTT protein levels in the liver of YAC128 mice administered a single 10 mg / kg dose subcutaneously of the indicated dsRNA duplexes targeting exon 1 of the HTT transcript on day 7 post-dose. The mutant human HTT levels shown are normalized to PBS-treated levels.

[0098] Figure 5 is a graph showing full-length mutant HTT mRNA levels in the liver of YAC128 mice administered a single 10 mg / kg dose subcutaneously of the indicated dsRNA duplexes targeting exon 1 of the HTT transcript on day 7. Human HTT levels shown are normalized to PBS-treated levels.

[0099] Figure 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 following subcutaneous administration of a single 10 mg / kg dose of the indicated dsRNA duplexes on post-dose day 7. The mutant human mRNA and protein HTT levels shown are normalized to PBS-treated levels.

[0100] Figure 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 duplexes subcutaneously on post-dose day 7. The mutant human HTT levels shown are normalized to PBS-treated levels.

[0101] Fig. 8A and 8BIs a graph showing full-length human HTT mRNA levels in human fibroblasts transfected with 10 nM or 50 nM of the indicated dsRNA duplexes specifically targeting various exons of human HTT or exon 1. Fibroblasts were obtained from Coriell, adult healthy control patients ("Control", GM02153), HD patients with adult disease ("Adult", GM04478), and HD patients with adolescent disease ("Adolescent", GM09197). HTT levels shown are normalized to mock-transfected controls.

[0102] Fig.9A and 9B Is a graph showing full-length human HTT mRNA levels in human fibroblasts transfected with 10 nM or 50 nM of the indicated dsRNA duplexes specifically targeting various exons of human HTT or exon 1. Fibroblasts were obtained from Coriell, adult healthy control patients ("Control", GM02153), HD patients with adult disease ("Adult", GM04478), and HD patients with adolescent disease ("Adolescent", GM09197). HTT levels shown are normalized to mock-transfected controls.

[0103] Figures 10A-10D Is a graph showing full-length human HTT mRNA levels in human fibroblasts transfected with 10 nM or 50 nM of the indicated dsRNA duplexes specifically targeting various exons of human HTT or exon 1. Fibroblasts were obtained from Coriell, adult healthy control patients ("Control", GM02153), HD patients with adult disease ("Adult", GM04478), and HD patients with adolescent disease ("Adolescent", GM09197). HTT levels shown are normalized to mock-transfected controls.

[0104] Figures 11A-11D Is a graph showing full-length human HTT mRNA levels in human fibroblasts transfected with 10 nM or 50 nM of the indicated dsRNA duplexes specifically targeting various exons of human HTT or exon 1. Fibroblasts were obtained from Coriell, adult healthy control patients ("Control", GM02153), HD patients with adult disease ("Adult", GM04478), and HD patients with adolescent disease ("Adolescent", GM09197). HTT levels shown are normalized to mock-transfected controls.

[0105] Fig.12Full-length mutant human HTT mRNA levels are shown in the liver of YAC128 mice or wild-type mice expressing a portion of human wild-type HTT ("AAV") and administered subcutaneously a single dose of the indicated dsRNA duplexes targeting the full-length HTT transcript on the indicated days after dosing. HTT levels shown are normalized to PBS-treated levels.

[0106] Fig.13A -D is a graph showing full-length human HTT mRNA levels in the liver of mice expressing partial human wild-type HTT by AVV. At 14 days post-dose, these mice were subcutaneously administered a single 3 mg / kg dose of the indicated dsRNA duplexes, which target the full-length HTT transcript. At 14 days post-siRNA administration, HTT levels are shown relative to AAV-treated control levels. DETAILED DESCRIPTION

[0107] The present disclosure provides RNAi compositions that affect RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of the Huntington (HTT) gene. The HTT gene can be in a cell, such as a cell in a subject such as a human. Using these iRNAs, the degradation of the mRNA of the corresponding gene (HTT gene) can be targeted in mammals.

[0108] The iRNAs of the present invention have been designed to target the HTT gene, including portions of the gene that are conserved among HTT orthologs of other mammalian species. The iRNAs of the present invention are also designed to target specific portions of the HTT gene, such as exon 1, thereby targeting full-length wild-type transcripts, full-length mutant transcripts, and truncated mutant transcripts. Without being limited by theory, it is believed that the combination or combination of the above properties and specific targets, such as exon 1, or specific modifications in these iRNAs impart improved efficacy, stability, potency, durability, and safety to the iRNAs of the present invention.

[0109] Thus, the disclosure also provides methods of using the RNAi compositions of the disclosure for inhibiting expression of an HTT gene or for treating a subject having a condition that would benefit from inhibiting or reducing expression of an HTT gene (e.g., an HTT-related disease, such as Huntington's disease).

[0110] The RNAi agents of the present disclosure comprise an RNA strand (antisense strand) having a length of about 30 nucleotides 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, 19-24, 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, which region is substantially complementary to at least a portion of the 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 of about 21-23 nucleotides in length, which region is substantially complementary to at least a portion of the mRNA transcript of the HTT gene.

[0111] In certain embodiments, the RNAi agent of the present disclosure comprises an RNA strand (antisense strand) that may comprise a longer length, such as up to 66 nucleotides, such as 36-66, 26-36, 25-36, 31-60, 22-43, 27-53 nucleotides in length, having a region of at least 19 consecutive nucleotides that is substantially complementary to at least a portion of the mRNA transcript of the HTT gene. These RNAi agents with longer antisense strands preferably comprise a second RNA strand (sense strand) of 20-60 nucleotides in length, wherein the sense strand and the antisense strand form a duplex of 18-30 consecutive nucleotides.

[0112] The use of these RNAi agents can target the degradation of mRNA of the HTT gene in mammals. Therefore, the methods and compositions comprising these RNAi agents can be used to treat subjects who will benefit from a reduction in HTT protein levels or activity, such as subjects with HTT-related diseases, such as Huntington's disease (HD).

[0113] The following detailed description discloses how to make and use compositions comprising RNAi agents to inhibit expression of the HTT gene, as well as compositions and methods for treating subjects suffering from diseases and disorders that would benefit from inhibition or reduction of gene expression.

[0114] I. Definitions

[0115] In order that the present disclosure may be more readily understood, certain terms are first defined.In addition, it should be noted that whenever a value or range of values ​​for a parameter is recited, values ​​and ranges intermediate to the recited values ​​are also intended to be part of the present disclosure.

[0116] The articles "a" and "an" are used herein to refer to one or more than one (ie, at least one) of the grammatical object of the article. For example, "an element" refers to one element or a plurality of elements, for example, a plurality of elements.

[0117] The term "including" is used herein to mean the phrase "including but not limited to", and can be used interchangeably with the phrase "including but not limited to". The term "or" is used herein to mean the term "and / or", and can be used interchangeably with the term "and / or", unless the context clearly indicates otherwise.

[0118] The term "about" is used herein to indicate within the typical tolerance range in the art. For example, "about" can be understood to mean about 2 standard deviations from the mean. In some embodiments, about means ±10%. In some embodiments, about means ±5%. When about appears before a series of numbers or ranges, it is understood that "about" can modify each number in the series or range.

[0119] The term "at least" preceding a number or a series of numbers is understood to include the numbers adjacent to the term "at least", as well as all subsequent numbers or integers that can be logically included, as is clear from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleotides of a 21-nucleotide nucleic acid molecule" means that 18, 19, 20 or 21 nucleotides have the specified properties. When at least appears before a series of numbers or a range, it is understood that "at least" can modify each number in the series or range.

[0120] As used herein, "not more than" or "less than" is understood to refer to the value adjacent to the phrase and the logically lower value or integer, logically to zero from the context. For example, a duplex having an overhang of "not more than 2 nucleotides" has 2, 1, or 0 nucleotide overhangs. When "not more than" appears before a series of numbers or a range, it is understood that "not more than" can modify each number in the series or range.

[0121] As used herein, a detection method may include determining that an analyte is present in an amount below the detection level of the method.

[0122] If the designated target site conflicts with the nucleotide sequence of the sense or antisense strand, the designated sequence takes precedence.

[0123] In case of conflict between chemical structure and chemical name, the chemical structure takes precedence.

[0124] The term "HTT" or "huntingtin", also known as "Huntingtin", "Huntington's disease protein", "IT15", "HD", "HD protein" or "LOMARS", refers to the well-known gene encoding the protein HTT, which is ubiquitously expressed and essential for normal development and a disease gene associated with Huntington's disease, a neurodegenerative disease characterized by striatal neuron loss caused by expanded, unstable trinucleotide (CAG) repeat sequences in the huntingtin gene, which are converted to polyglutamine repeat sequences in the protein product.

[0125] Exemplary nucleotide and amino acid sequences of 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 (Mus musculus HTT, SEQ ID NO: 2; reverse complement, SEQ ID NO: 7); GenBank Accession No. NM_024357.3 (Mus musculus HTT, SEQ ID NO: 3, reverse complement, SEQ ID NO: 8); GenBank Accession No. XM_015449989.1 (Cynomolgus macaque HTT, SEQ ID NO: 4, reverse complement, SEQ ID NO: 9); and GenBank Accession No. XM_028848247.1 (Rhesus macaque, SEQ ID NO: 5, reverse complement, SEQ ID NO: 10).

[0126] Other examples of HTT sequences can be found in publicly available databases such as GenBank, OMIM, and UniProt.

[0127] For more information on HTT, visit, for example, www.ncbi.nlm.nih.gov / gene / 3064.

[0128] As of the date of filing this application, the entire contents of each of the aforementioned GenBank Accession Numbers and GenBank Database Numbers are incorporated herein by reference.

[0129] As used herein, the term HTT also refers to variants of the HTT gene, including variants provided in the SNP database. Many sequence variants within the HTT gene have been identified and can be found in, for example, 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 date of filing this application).

[0130] As used herein, "target sequence" refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during transcription of the HTT gene, including mRNA that is an RNA processing product of a depolarized transcription product. In one embodiment, the target portion of the sequence will be at least long enough to serve as a substrate for RNAi-directed cleavage at or near the nucleotide sequence portion of the mRNA molecule formed during transcription of the HTT gene.

[0131] The target sequence is about 15-30 nucleotides in length. For example, the target sequence can be from about 15-30 nucleotides in length, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19 In some embodiments, the target sequence is 19-23 nucleotides in length, optionally 21-23 nucleotides in length. Ranges and lengths intermediate to the above ranges and lengths are also considered to be part of the present disclosure.

[0132] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a chain of nucleotides described by the sequence referred to using standard nucleotide nomenclature.

[0133] "G", "C", "A", "T" and "U" generally represent nucleotides comprising guanine, cytosine, adenine, thymus and uracil as bases, respectively in the case of modified or unmodified nucleotides. However, it should be understood that the term "ribonucleotide" or "nucleotide" may also refer to modified nucleotides, as further described below, or alternative substitution moieties (see, e.g., Table 1). It is well known to those skilled in the art that guanine, cytosine, adenine, thymus and uracil may be replaced by other moieties without significantly changing the base pairing properties of the oligonucleotide comprising the nucleotide with such substitution moieties. For example, but not limited to, a nucleotide comprising inosine as its base may be base paired with a nucleotide comprising adenine, cytosine or uracil. Thus, a nucleotide comprising uracil, guanine or adenine may be replaced by a nucleotide comprising, for example, inosine in the nucleotide sequence of a dsRNA characterized in the present disclosure. In another embodiment, adenine and cytosine anywhere in the oligonucleotide may be replaced with guanine and uracil, respectively, to form a GU wobble base pairing with the target mRNA. Sequences comprising such replacement moieties are suitable for use in the compositions and methods of the present disclosure.

[0134] The terms "iRNA", "RNAi agent", "iRNA agent", "RNA interference agent", as used interchangeably herein, refer to an agent that comprises RNA as that term is defined herein, and which mediates targeted cleavage of RNA transcripts through the RNA-induced silencing complex (RISC) pathway. RNA interference (RNAi) is a process that directs sequence-specific degradation of mRNA. RNAi modulates, for example, inhibits expression of HTT in a cell, for example, a cell within a subject, such as a mammalian subject.

[0135] In one embodiment, the RNAi agent of the present disclosure includes a single-stranded RNAi that interacts with a target RNA sequence (e.g., an HTT target mRNA sequence) to guide the cleavage of the target RNA. Without wishing to be bound by theory, it is believed that the long double-stranded RNA introduced into the cell is broken down by a type III nuclease called Dicer into double-stranded short interfering RNA (siRNA) comprising a sense strand and an antisense strand (Sharp et al. (2001) Genes Dev. 15: 485). Dicer, a ribonuclease III-like enzyme, can process these dsRNAs into 19-23 base pair short interfering RNAs with characteristic two base 3' overhangs (Bernstein et al., (2001) Nature 409: 363). These siRNAs are then integrated into an RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, allowing the complementary antisense strand to guide target recognition (Nykanen et al., (2001) Cell 107: 309). After binding to the appropriate target mRNA, one or more endonucleases within RISC cleave the target to induce silencing (Elbashir et al., (2001) Genes Dev. 15: 188). Therefore, in one aspect, the present disclosure relates to single-stranded RNA (ssRNA) (the antisense strand of the siRNA duplex) that is produced in cells and promotes the formation of a RISC complex to achieve silencing of a target gene, such as the HTT gene. Therefore, the term "siRNA" is also used herein to refer to RNAi as described above.

[0136] In another embodiment, the RNAi agent can be a single-stranded RNA introduced into a cell or organism to inhibit a target mRNA. The single-stranded RNAi agent binds to the RISC endonuclease Argonaute2 and then cuts the target mRNA. Single-stranded siRNA is generally 15-30 nucleotides and is chemically modified. The design and testing of single-stranded RNA are described in U.S. Patent No. 8,101,348 and Lima et al. (2012) Cell 150:883-894, the entire contents of each of which are incorporated herein by reference. Any antisense nucleotide sequence described herein can be used as a single-stranded siRNA as described herein or chemically modified by the method described in Lima et al. (2012) Cell 150:883-894.

[0137] In another embodiment, the "RNAi agent" used in the compositions and methods of the present disclosure is a double-stranded RNA and is referred to herein as a "double-stranded RNAi agent," "double-stranded RNA (dsRNA) molecule," "dsRNA agent," or "dsRNA." The term "dsRNA" refers to a complex of ribonucleic acid molecules having a duplex structure comprising two antiparallel and substantially complementary nucleic acid strands, referred to as having "sense" and "antisense" orientations relative to the target RNA (i.e., HTT gene). In some embodiments of the present disclosure, double-stranded RNA (dsRNA) triggers degradation of a target RNA, such as mRNA, by a post-transcriptional gene silencing mechanism referred to herein as RNA interference or RNAi.

[0138] Typically, dsRNA molecules can include ribonucleotides, but as described in detail herein, each or both chains can also include one or more non-ribonucleotides, such as deoxyribonucleotides, modified nucleotides. In addition, as used in this specification, "RNAi agents" can include chemically modified ribonucleotides; RNAi agents can include substantial modifications on multiple nucleotides. As used herein, the term "modified nucleotides" refers to nucleotides that independently have modified sugar moieties, modified internucleotide bonds, or modified nucleobases. Therefore, the term modified nucleotides includes substitutions, additions, or removals of internucleoside bonds, sugar moieties, or nucleobases, such as functional groups or atoms. Modifications suitable for agents of the present disclosure include all types of modifications disclosed herein or known in the art. For the purposes of this specification and claims, any such modifications as used in siRNA type molecules are included in "RNAi agents".

[0139] In certain embodiments of the present disclosure, the inclusion of deoxynucleotides, if present in an RNAi agent, may be considered to constitute modified nucleotides.

[0140] The duplex region can be any length that allows for specific degradation of the desired target RNA by the RISC pathway, and can be about 15-36 base pairs in length, such as 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, such as about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-29, 18-30, 18-29, 18-31, 18-32, 18-33, 18-34, 18-35, 18-36, 18-37, 18-38, 18-39, 18-40, 18-41, 18-43, 18-44, 18-45, 18-46, 18-47, 18-48, 18-49, 18-50, 18-51, 18-52, 18-53, 18-54, 18-55, 18-56, 18-57, 18-58, 18-59, 18-60, 18-61, 18-62, 18-63, 18-64, 21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-24, 21-23, or 21-22 base pairs. In certain embodiments, the duplex region is 19-21 base pairs in length, for example 21 base pairs in length. Ranges and lengths intermediate to the above ranges and lengths are also considered part of the present disclosure.

[0141] The two chains forming the duplex structure can be different parts of a larger RNA molecule, or they can be separate RNA molecules. If the two chains are part of a larger molecule, the uninterrupted chain connection between the nucleotides between the 3' end of one chain and the 5' end of the other chain forms a duplex structure, and the connecting RNA chain is called a "hairpin loop". The hairpin loop can include at least one unpaired nucleotide. In some embodiments, the hairpin loop can include 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 that do not point to the dsRNA target site. In some embodiments, the hairpin loop can be 10 or less nucleotides. In some embodiments, the hairpin loop can be 8 or less unpaired nucleotides. In some embodiments, the hairpin loop can be 4-10 unpaired nucleotides. In some embodiments, the hairpin loop can be 4-8 nucleotides.

[0142] When the two substantially complementary chains of dsRNA are composed of separate RNA molecules, these molecules do not need to be but can be covalently linked. In certain embodiments, when the two chains are covalently linked by means other than an uninterrupted nucleotide chain between the 3' end of one chain and the 5' end of the other chain, a duplex structure is formed, and the connecting structure is referred to as a "joint" (although it is noted that certain other structures defined elsewhere herein may also be referred to as "joints"). RNA chains may have the same or different numbers of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest chain of the dsRNA minus any overhangs present in the duplex. In addition to the duplex structure, RNAi also comprises one or more nucleotide overhangs. In one embodiment of the RNAi agent, at least one chain comprises a 3' overhang of at least 1 nucleotide. In another embodiment, at least one chain comprises a 3' overhang of at least 2 nucleotides, such as 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14 or 15 nucleotides. In other embodiments, at least one chain of the RNAi agent comprises a 5' overhang of at least 1 nucleotide. In certain embodiments, at least one strand comprises a 5' overhang of at least 2 nucleotides, such as 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, both the 3' and 5' ends of one strand of the RNAi agent comprise an overhang of at least 1 nucleotide.

[0143] In one embodiment, the RNAi agent of the present disclosure is a dsRNA, each strand of which independently comprises 19-23 nucleotides, which interacts with a target RNA sequence, such as an HTT target mRNA sequence, to direct the cleavage of the target RNA.

[0144] As used herein, the term "nucleotide overhang" refers to the protruding unpaired nucleotides from the duplex structure of an RNAi agent, such as a dsRNA. For example, when the 3' end of one strand of a dsRNA extends beyond the 5' end of the other strand, or vice versa, there is a nucleotide overhang. The dsRNA may include an overhang of at least one nucleotide; or, the overhang may include at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more. The nucleotide overhang may include or be composed of nucleotides / nucleotides, including deoxynucleotides / nucleosides. The overhang may be on the sense strand, the antisense strand, or any combination thereof. In addition, the nucleotides of the overhang may be present at the 5' end, the 3' end, or both ends of the sense strand or the antisense strand of the dsRNA.

[0145] In one embodiment, the antisense strand of the dsRNA has an overhang of 1-10 nucleotides at the 3' end or the 5' end, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides. In one embodiment, the sense strand of the dsRNA has an overhang of 1-10 nucleotides at the 3' end or the 5' end, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides. In another embodiment, one or more nucleotides in the overhang are substituted with nucleoside thiophosphates.

[0146] In certain embodiments, the antisense strand of the dsRNA has an overhang of 1-10 nucleotides at the 3' end or the 5' end, such as 0-3, 1-3, 2-4, 2-5, 4-10, 5-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides. In one embodiment, the sense strand of the dsRNA has an overhang of 1-10 nucleotides at the 3' end or the 5' end, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides. In another embodiment, one or more nucleotides in the overhang are substituted with nucleoside thiophosphates.

[0147] In certain embodiments, the overhang of the sense strand or antisense strand may comprise an extension length longer than 10 nucleotides, for example, a length of 1-30 nucleotides, 2-30 nucleotides, 10-30 nucleotides, or 10-15 nucleotides. In certain embodiments, the extended overhang is located on the sense strand of the duplex. In certain embodiments, the extended overhang is located on the 3' end of the sense strand of the duplex. In certain embodiments, the extended overhang is located on the 5' end of the sense strand of the duplex. In certain embodiments, the extended overhang is located on the antisense strand of the duplex. In certain embodiments, the extended overhang is located on the 3' end of the antisense strand of the duplex. In certain embodiments, the extended overhang is located on the 5' end of the antisense strand of the duplex. In certain embodiments, one or more nucleotides in the overhang are substituted by nucleoside thiophosphates. In certain embodiments, the overhang comprises a self-complementary portion so that the overhang can form a hairpin structure that is stable under physiological conditions.

[0148] In certain embodiments, at least one end of at least one strand extends beyond the duplex targeting region, including structures in which one strand includes a thermodynamically stable tetracyclic structure (see, e.g., U.S. Pat. Nos. 8,513,207 and 8,927,705 and WO2010033225, the entire contents of each of which are incorporated herein by reference). Such structures may include single-stranded stretches (on one or both sides of the molecule) as well as double-stranded stretches.

[0149] In certain embodiments, the 3' end of the sense strand and the 5' end of the antisense strand are connected by a polynucleotide sequence comprising ribonucleotides, deoxyribonucleotides, or both, optionally wherein the polynucleotide sequence comprises a tetraloop sequence. In certain embodiments, the sense strand is 25-35 nucleotides in length.

[0150] The four rings can contain ribonucleotides, deoxyribonucleotides, modified nucleotides and combinations thereof. Typically, the four rings have 4 to 5 nucleotides. In some embodiments, the ring contains a GAAA sequence. In some embodiments, at least one nucleotide of the ring (GAAA) contains a nucleotide modification. In some embodiments, the modified nucleotide contains a 2' modification. In some embodiments, the 2' modification is selected from 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, 2'-aminodiethoxymethanol, 2'-adem and 2'-deoxy-2'-phytic acid arabinonucleotides. In some embodiments, all nucleotides of the ring are modified. In some embodiments, the G in the GAAA sequence is 2'-OH. In some embodiments, each nucleotide in the GAAA sequence contains a 2'-O-methyl modification. In some embodiments, each A in the GAAA sequence contains a 2'-OH and the G in the GAAA sequence contains a 2'-O-methyl modification. In preferred embodiments, 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. See, e.g., PCT Publication No. WO 2020 / 206350, the entire contents of which are incorporated herein by reference.

[0151] Exemplary 2'-adem modified nucleotides are shown below:

[0152]

[0153] As used herein, the term "flat end" for dsRNA refers to the absence of unpaired nucleotides or nucleotide analogs, i.e., no nucleotide overhangs, at a given end of the dsRNA. One or both ends of the dsRNA may be flat ends. If both ends of the dsRNA are flat ends, the dsRNA is said to be flat ends. It should be noted that "flat-ended" dsRNA is a dsRNA with both ends being flat, i.e., no nucleotide overhangs at either end of the molecule. In most cases, such molecules are double-stranded over their entire length.

[0154] The term "antisense strand" or "guide strand" refers to the strand of a RNAi agent, such as a dsRNA, that comprises a region that is substantially complementary to a target sequence, such as HTT mRNA.

[0155] As used herein, the term "complementary region" refers to a region on the antisense strand that is substantially complementary to a sequence defined herein, such as a target sequence, such as an HTT nucleotide sequence. In the case where the complementary region is not completely complementary to the target sequence, the mismatch may be in the interior or terminal region of the molecule. Typically, the most tolerated mismatch is within the terminal region, such as 5, 4, 3, or 2 nucleotides at the 5' or 3' end of the RNAi agent. In some embodiments, the double-stranded RNA agent of the present invention comprises nucleotide mismatches in the antisense strand. In some embodiments, the antisense strand of the double-stranded RNA agent of the present invention comprises no more than 4 mismatches with the target mRNA, i.e., the antisense strand comprises 4, 3, 2, 1, or 0 mismatches with the target mRNA. In some embodiments, the antisense strand of the double-stranded RNA agent of the present invention comprises no more than 4 mismatches of the sense strand, i.e., the antisense strand comprises 4, 3, 2, 1, or 0 mismatches with the sense strand. In some embodiments, the double-stranded RNA agent of the present invention comprises nucleotide mismatches in the sense strand. In some embodiments, the sense strand of the double-stranded RNA agent of the invention comprises no more than 4 mismatches with the antisense strand, and the sense strand comprises 4, 3, 2, 1 or 0 mismatches with the antisense strand. In some embodiments, the nucleotide mismatch is, for example, within 5, 4, 3 nucleotides of the 3' end of the iRNA. In another embodiment, the nucleotide mismatch is, for example, in the 3' terminal nucleotide of the iRNA agent. In some embodiments, the mismatch is not in the seed region.

[0156] Therefore, the RNAi agent as described herein may comprise one or more mismatches with the target sequence. In one embodiment, the RNAi agent as described herein comprises no more than 3 mismatches (i.e., 3, 2, 1 or 0 mismatches). In one embodiment, the RNAi agent as described herein comprises no more than 2 mismatches. In one embodiment, the RNAi agent as described herein comprises no more than 1 mismatch. In one embodiment, the RNAi agent as described herein comprises no more than 0 mismatches. In certain embodiments, if the antisense strand of the RNAi agent comprises a mismatch with the target sequence, the mismatch may optionally be limited to the last 5 nucleotides at the 5' or 3' end of the complementary region. For example, in such an embodiment, for a 23-nucleotide RNAi agent, the strand complementary to the HTT gene region generally does not comprise any mismatch within the central 13 nucleotides. Methods as described herein or methods known in the art can be used to determine whether an RNAi agent comprising a mismatch with a target sequence effectively inhibits the expression of the HTT gene. It is important to consider the efficacy of RNAi agents with mismatches in inhibiting HTT gene expression, particularly if a particular complementary region in the HTT gene is known to have polymorphic sequence variation in a population.

[0157] The term "sense strand" or "follower strand" as used herein refers to the strand of the RNAi agent that comprises a region complementary to a region of the antisense strand as defined herein.

[0158] As used in this article, the term "cleavage zone" refers to the area next to the cleavage site. The cleavage site is the site where cleavage occurs on the target. In some embodiments, the cleavage zone comprises three bases at either end of the cleavage site and next to the cleavage site. In some embodiments, the cleavage zone comprises two bases at either end of the cleavage site and next to the cleavage site. In some embodiments, the cleavage site specifically occurs at the site bound by nucleotides 10 and 11 of the antisense strand, and the cleavage site comprises nucleotides 11, 12, and 13.

[0159] As used herein, unless otherwise indicated, the term "complementary" when used to describe a first nucleotide sequence relative to a second nucleotide sequence, as will be understood by the skilled artisan, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide comprising the second nucleotide sequence and form a duplex structure under certain conditions.

[0160] The complementary sequence in the RNAi agent, for example, in the dsRNA as described herein, is included in the full length of one or two nucleotide sequences, and the oligonucleotide or polynucleotide comprising the first nucleotide sequence is paired with the oligonucleotide or polynucleotide comprising the second nucleotide sequence. Such sequences may be referred to as "fully complementary" to each other in this article. However, when the first sequence is referred to as "substantially complementary" relative to the second sequence in this article, the two sequences may be fully complementary, or they may form one or more, but generally no more than 5, 4, 3 or 2 mismatched base pairs, forming up to 30 base pairs of duplexes after hybridization, while retaining the ability to hybridize under the conditions most relevant to its final application, such as inhibition of gene expression by RISC pathways. However, when two oligonucleotides are designed to form one or more single-stranded overhangs when hybridized, such overhangs should not be considered as mismatches with respect to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide of 21 nucleotides in length and another oligonucleotide of 23 nucleotides in length, wherein the longer oligonucleotide comprises a 21 nucleotide sequence that is fully complementary to the shorter oligonucleotide, can still be referred to as "fully complementary" for purposes described herein.

[0161] As used herein, "complementary" sequences may also include or consist entirely of non-Watson-Crick base pairs or base pairs formed by non-natural and modified nucleotides, as long as the above requirements for their hybridization ability are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble or Hoogstein base pairs.

[0162] The terms "complementary", "fully complementary" and "substantially complementary" herein may be used for base pairing between the sense and antisense strands of a dsRNA, or the antisense strand between an RNAi agent and a target sequence, as will be understood from the context of their use.

[0163] As used herein, a polynucleotide that is substantially complementary to at least a portion of a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of an mRNA of interest (e.g., an mRNA encoding HTT). For example, if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding HTT, then the polynucleotide is complementary to at least a portion of an HTT mRNA.

[0164] Therefore, 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 continuous nucleotide sequence that is at least 80% complementary to the equivalent region of any nucleotide sequence in SEQ ID NO: 1-5, or any fragment in SEQ ID NO: 1-5, for example, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% complementary over its entire length.

[0165] In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target HTT sequence and comprise a contiguous nucleotide sequence that is at least 80% complementary to any of the sense strand nucleotide sequences in any of Tables 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 24, 25, 27-30, 32, and 33, or to a fragment of any of the sense strand nucleotide sequences in any of Tables 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 24, 25, 27-30, 32, and 33, 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.

[0166] In one embodiment, the RNAi agent of the present disclosure comprises a sense strand that is substantially complementary to an antisense polynucleotide, which in turn is identical to the target HTT sequence, and wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80% complementary to an equivalent region of a nucleotide sequence of SEQ ID NOs: 6-10, or any fragment of SEQ ID NOs: 6-10, over its entire length, 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.

[0167] In some embodiments, the iRNA of the invention comprises a sense strand that is substantially complementary to an antisense polynucleotide, which in turn is complementary to a target HTT sequence, and wherein the sense strand polynucleotide comprises an antisense strand nucleotide sequence that is substantially complementary to 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 over its entire length, or a sense strand nucleotide sequence that is substantially complementary to 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. A continuous nucleotide sequence that is at least about 80% complementary to a fragment of any antisense strand nucleotide sequence of any one of , 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 24, 25, 27-30, 32 and 33, 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.

[0168] In one embodiment, at least partial inhibition of HTT gene expression can be assessed by a reduction in the amount of HTT mRNA that can be isolated or detected from a first cell or group of cells in which the HTT gene is transcribed and which has been or has been treated such that expression of the HTT gene is inhibited, compared to a second cell or group of cells that is substantially identical to the first cell or group of cells but has or has not been so treated (control cells). The degree of inhibition can be expressed as:

[0169]

[0170] As used herein, the phrase "contacting a cell with an RNAi agent" includes contacting the cell by any possible means. Contacting a cell with an RNAi agent includes contacting the cell with an RNAi agent in vitro or contacting the cell with an RNAi agent in vivo. Contact can be made directly or indirectly. Thus, for example, the RNAi agent can be brought into physical contact with the cell by an individual practicing the method, or the RNAi agent can be placed in an environment that allows or causes it to subsequently contact the cell.

[0171] For example, cells can be contacted in vitro by incubating cells with RNAi agents. For example, RNAi agents can be injected into or near the tissue where the cells are located, or by injecting RNAi agents into another area (such as the central nervous system (CNS)), optionally by intrathecal injection, intravitreal injection or other injection, or injection into the bloodstream or subcutaneous space, so that the agent then reaches the tissue where the cells are to be contacted. For example, RNAi agents can include or be coupled to a ligand, such as a lipophilic part or a part as described below and further described in detail, such as PCT / US2019 / 031170, which is incorporated herein by reference, and it guides or otherwise stabilizes RNAi agents at sites of interest, such as CNS. A combination of in vitro and in vivo contact methods is also possible. For example, cells can also be contacted with RNAi agents in vitro and then transplanted into a subject.

[0172] In one embodiment, contacting a cell with an RNAi agent includes "introducing" or "delivering an RNAi agent to a cell" by promoting or influencing uptake or absorption by the cell. Absorption or uptake of the RNAi agent can occur by independent diffusion or active cellular processes, or by auxiliary agents or devices. The introduction of the RNAi agent into the cell can be performed in vitro or in vivo. For example, for in vivo introduction, the RNAi agent can be injected into a tissue site or administered systemically. In vitro introduction into cells includes methods known in the art, such as electroporation and lipofection. Further methods are described below or are known in the art.

[0173] The term "lipophilic" or "lipophilic moiety" refers broadly to any compound or chemical moiety that has an affinity for lipids. One method of characterizing the lipophilicity of a lipophilic moiety is by octanol-water partitioning of the dilute log K ow , where K ow is the ratio of the concentration of a chemical in the octanol phase to its concentration in the aqueous phase of a two-phase system at equilibrium. The octanol-water partition coefficient is a laboratory-measured property of a substance. However, it can also be predicted by using coefficients attributed to chemical structural components, which are calculated using first principles or empirical methods (see, e.g., Tetko et al., J. Chem. Inf. Comput. Sci. 41: 1407-21 (2001), which is incorporated herein by reference in its entirety). It provides a thermodynamic measure of the tendency of a substance to prefer a non-aqueous or oily environment rather than water (i.e., its hydrophile / lipophile balance). In principle, when logK ow When it is greater than 0, the chemical is lipophilic. Typically, the lipophilic moiety has a logK greater than 1, greater than 1.5, greater than 2, greater than 3, greater than 4, greater than 5, or greater than 10. ow For example, to predict the logK of 6-aminohexanol owUsing the same method, we can predict the logK of cholesterol carbamate. ow It is 10.7.

[0174] The lipophilicity of a molecule can be altered depending on the oligomeric groups it carries. For example, adding a hydroxyl or amine group to the end of a lipophilic moiety can increase or decrease the partition coefficient (i.e., logK ow )value.

[0175] Alternatively, the hydrophobicity of a double-stranded RNAi agent conjugated to one or more lipophilic moieties can be measured by its protein binding properties. For example, in certain embodiments, the unbound fraction in a plasma protein binding assay of a double-stranded RNAi agent can be determined to be positively correlated with the relative hydrophobicity of the double-stranded RNAi agent, which may then be positively correlated with the silencing activity of the double-stranded RNAi agent.

[0176] In one embodiment, the plasma protein binding assay determined is an electrophoretic mobility shift assay (EMSA) using human serum albumin. Exemplary protocols for such binding assays are described in detail, for example, in PCT / US2019 / 031170. The hydrophobicity of the double-stranded RNAi agent, as measured by the fraction of unbound siRNA in the binding assay, is greater than 0.15, greater than 0.2, greater than 0.25, greater than 0.3, greater than 0.35, greater than 0.4, greater than 0.45, or greater than 0.5 for enhanced siRNA body delivery.

[0177] Therefore, conjugating a lipophilic moiety to an internal position of a double-stranded RNAi agent provides optimal hydrophobicity for enhanced siRNA delivery in vivo.

[0178] The term "lipid nanoparticle" or "LNP" is 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 an RNAi agent is transcribed. LNP is described in, for example, 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.

[0179] As used herein, a "subject" is an animal, such as a mammal, including a primate (e.g., a human, a non-human primate, such as a monkey and a chimpanzee), or a non-primate (e.g., a mouse or a rat). In a preferred embodiment, the subject is a human, such as a human being treated or evaluated for a disease, disorder, or condition that would benefit from reduced HTT expression; a human being who has a disease, disorder, or condition that would benefit from reduced HTT expression; a human being suffering from a disease, disorder, or condition that would benefit from reduced HTT expression; or a human being being treated for a disease, disorder, or condition that would benefit from reduced HTT expression as described herein. In some embodiments, the subject is a female human. In other embodiments, the subject is a male human. In one embodiment, the subject is an adult subject. In one embodiment, the subject is a pediatric subject. In another embodiment, the subject is a juvenile subject, such as a subject under 20 years old.

[0180] As used herein, the term "treat" or "treatment" refers to beneficial or desired results, including but not limited to alleviating or ameliorating one or more signs or symptoms associated with HTT gene expression or HTT protein production, such as HTT-related diseases such as Huntington's disease. "Treatment" may also refer to prolonging survival compared to expected survival in the absence of treatment.

[0181] With respect to the HTT level of a subject or a disease marker or symptom, the term "lower" refers to a statistically significant decrease in the 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 disease marker, such as protein or gene expression level, is reduced by at least 50%. With respect to the HTT level in a subject, "lower" preferably decreases to a level that is accepted within the normal range for individuals without such a condition. In certain embodiments, "lower" is a reduction in the difference between the level of a marker or symptom of a subject with a disease and an acceptable level within the normal range for an individual, such as the level of weight loss between an obese individual and an individual whose weight is acceptable within the normal range.

[0182] As used herein, "prevention" or "prevention" when used in reference to a disease, disorder, or condition that would benefit from a reduction in HTT gene or HTT protein production, refers to a reduction in the likelihood that a subject will develop symptoms associated with such a disease, disorder, or condition, e.g., symptoms of an HTT-related disease. Failure to develop a disease, disorder, or condition, or a reduction in the development of symptoms associated with such a disease, disorder, or condition (e.g., a reduction of at least about 10% on a clinically acceptable scale for the disease or condition), or a delay in the manifestation of symptoms (e.g., days, weeks, months, or years) is considered effective prevention.

[0183] As used herein, the term "HTT-associated disease" or "HTT-associated disorder" is understood as any disease or disorder that would benefit from a reduction in the expression and / or activity of HTT. Exemplary HTT-associated diseases include Huntington's disease.

[0184] Huntington's disease, also known as HD, Huntington's chorea, chorea, chronic progressive chorea, and hereditary chorea, is an autosomal dominant disorder characterized by chorea-like movements and progressive intellectual deterioration, usually beginning in middle age (35 to 50 years old). The disease affects both sexes equally. The caudate nucleus atrophies, small cell groups degenerate, and levels of the neurotransmitters gamma-aminobutyric acid (GABA) and substance P decrease. This degeneration results in the characteristic "carriage-like ventricles" seen on CT scans.

[0185] Signs and symptoms of HD develop insidiously. The most obvious symptom of HD is abnormal body movements called chorea and lack of coordination, but it can also affect many mental abilities and certain aspects of personality. These physical symptoms usually become noticeable when a person is in their 40s, but can occur at any age. If the age of onset is before the age of 20, it is called juvenile HD.

[0186] Dementia or psychotic disorders, ranging from apathy and irritability to full-blown bipolar or schizophrenia, may precede or develop during movement disorders. Anhedonia or unsociable behavior may be the first behavioral manifestation. Motor manifestations include flicking movements of the limbs, a brisk gait, motor insuspensions (inability to sustain motor behaviors, such as tongue protrusion), facial grimaces, ataxia, and dystonia.

[0187] HD is caused by a trinucleotide repeat expansion in the Huntington (HTT) gene and is one of several polyglutamine expansion (or PolyQ expansion) diseases. This produces an expanded form of mutant huntingtin protein (mHtt), which causes cell death in selective areas of the brain.

[0188] As used herein, "therapeutically effective amount" is intended to include an amount sufficient to achieve disease treatment (e.g., by reducing, ameliorating, or maintaining existing disease or one or more disease symptoms) when administered to a subject suffering from an HTT-related disease. A "therapeutically effective amount" may vary depending on the RNAi agent, the mode of administration, the disease and its severity, as well as medical history, age, weight, family history, genetic makeup, type of prior or concomitant treatment (if any), and other individual characteristics of the subject to be treated.

[0189] As used herein, a "prophylactically effective amount" is intended to include an amount of an RNAi agent sufficient to prevent or ameliorate the disease or one or more disease symptoms when administered to a subject suffering from an HTT-related disease. Amelioration of the disease includes slowing the progression of the disease or reducing the severity of the disease at a later stage of development. A "prophylactically effective amount" may vary depending on the RNAi agent, the route of administration, the degree of risk of the disease, and the medical history, age, weight, family history, genetic makeup, type of prior or concomitant treatment (if any), and other individual characteristics of the patient to be treated.

[0190] "Therapeutically effective amount" or "prophylactically effective amount" also includes the amount of 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 method of the present disclosure can be administered in sufficient amount to produce a reasonable benefit / risk ratio applicable to such treatment.

[0191] As used herein, the phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions or agents which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human and animal subjects without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0192] As used herein, the phrase "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or carrier, 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 carrying or transporting a subject compound from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable", i.e., compatible with the other ingredients of the formulation and not harmful to the subject to be treated. Some examples of materials that can be used 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) tragacanth powder; (5) malt; (6) gelatin; (7) lubricants, such as magnesium salts, sodium lauryl sulfate, talc, etc.; (8) excipients, such as cocoa butter, suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol. ; (11) polyols, such as glycerol, sorbitol, mannitol, polyethylene glycol, etc.; (12) esters, such as ethyl oleate, ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide, aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) pH buffer solution; (21) polyesters, polycarbonates or polyanhydrides; (22) fillers, such as polypeptides and amino acids; (23) serum components, such as serum albumin, HDL and LDL; and (22) other non-toxic compatible substances used in pharmaceutical preparations.

[0193] The term "sample" as used in this article includes a collection of similar fluids, cells or tissues separated from a subject, and fluids, cells or tissues present in a subject. The example of biological fluids includes blood, serum and serous fluid, plasma, cerebrospinal fluid, ocular fluid, lymphatic fluid, urine, saliva, etc. Tissue samples can include samples from tissues, organs or local areas. For example, the sample can be from the fluid or cells in a specific organ, the part of an organ or these organs. In some embodiments, the sample can be from the brain (for example, some fragments of the whole brain or brain, such as cells of certain types in the striatum or brain, such as neurons and glial cells (astroglial cells, oligodendrocytes, microglia)). In some embodiments, "sample derived from a subject" refers to blood taken from a subject or plasma or serum derived from it. In a further embodiment, "sample derived from a subject" refers to brain tissue (or its subcomponents) or retinal tissue (or its subcomponents) taken from a subject.

[0194] II. RNAi Agents of the Disclosure

[0195] RNAi agents that inhibit HTT gene expression are described herein. In one embodiment, RNAi agents include double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of HTT genes in cells, such as cells in a subject, such as mammals, such as people with HTT-related diseases (such as Huntington's disease). dsRNA includes an antisense strand with a complementary region, which is complementary to at least a portion of the mRNA formed in the expression of the HTT gene, and the length of the complementary region is about 15-30 nucleotides or shorter. After contacting cells expressing the HTT gene, RNAi agents inhibit the expression of HTT genes (such as human genes, primate genes, non-primate genes) by at least 50%, which is determined by, for example, PCR-based or branched DNA (bDNA) methods, or by protein-based methods, such as by immunofluorescence analysis, using, for example, Western blotting or flow cytometry. On the one hand, the knockout level in Cos7 cells is determined using a dual luciferase assay.

[0196] dsRNA comprises two complementary chains that hybridize to form a duplex structure under the conditions of using dsRNA. One chain (antisense chain) of dsRNA comprises a complementary region that is substantially complementary to the target sequence and is usually completely complementary. The target sequence can be derived from the mRNA sequence formed during the expression of the HTT gene. The other chain (sense chain) comprises a region complementary to the antisense chain, so that the two chains hybridize and form a duplex structure when combined under appropriate conditions. As described elsewhere herein and known in the art, the complementary sequence of dsRNA can also be included as a self-complementary region of a single nucleic acid molecule, rather than on a separate oligonucleotide.

[0197] Typically, the length of the duplex structure is 15 to 30 base pairs, for example, 15-29, 15-29, 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, 1 21-29, 21-28, 21-27, 21-6, 21-25, 21-4, 21-23, or 21-22 base pairs. In certain preferred embodiments, the length of the duplex structure is 18 to 25 base pairs, such as 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, 21-4, 21-23, 21-22, 22-25, 22-24, 22-23, 23-25, 23-24 or 24-25 base pairs, such as 19-20 base pairs in length. Ranges and lengths intermediate to the above ranges and lengths are also considered part of the present disclosure.

[0198] Similarly, the region complementary to the target sequence is 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, for example, 19-23 nucleotides in length or 21-23 nucleotides in length. Ranges and lengths intermediate to the above ranges and lengths are also considered part of the disclosure.

[0199] In some embodiments, the length of the duplex structure is 19 to 30 base pairs. Similarly, the region complementary to the target sequence is 19 to 30 nucleotides in length.

[0200] In some embodiments, the dsRNA is 15 to 23 nucleotides in length, 19 to 23 nucleotides in length, or 25 to 30 nucleotides in length. Typically, the length of the dsRNA is sufficient to serve as a substrate for the Dicer enzyme. For example, it is well known in the art that dsRNAs longer than about 21-23 nucleotides can be used as Dicer substrates. As will be appreciated by those of ordinary skill, the RNA region targeted for cutting is typically a portion of a larger RNA molecule, typically an mRNA molecule. In related cases, a "portion" of an mRNA target is a continuous sequence of an mRNA target that is long enough to serve as a substrate for RNAi directed cutting (i.e., cutting by the RISC pathway).

[0201] One skilled in the art will also recognize that the duplex region is the major functional portion of the dsRNA, such as a duplex region of about 15 to 36 base pairs, such as 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, 18-26 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, for example 19-21 base pairs. Thus, in one embodiment, an RNA molecule or RNA molecule complex having a duplex region greater than 30 base pairs is a dsRNA insofar as it is processed into a functional duplex of, for example, 15-30 base pairs, which targets the desired RNA for cleavage. Thus, one of ordinary skill will recognize that in one embodiment, the miRNA is a dsRNA. In another embodiment, the dsRNA is not a naturally occurring miRNA. In another embodiment, the RNAi agent useful for target HTT expression is not produced in the target cell by cleaving a larger dsRNA.

[0202] As described herein, dsRNA can further comprise one or more single-stranded nucleotide overhangs, such as 1, 2, 3 or 4 nucleotides. The nucleotide overhangs can comprise or be similarly composed of nucleotides / nucleotides, including deoxynucleotides / nucleosides. The overhangs can be on the sense strand, the antisense strand or any combination thereof. In addition, the nucleotides of the overhangs can be present at the 5' end, 3' end or both ends of the sense strand or antisense strand of the dsRNA.

[0203] 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 chain of the double-stranded RNA molecule is prepared separately. Then, the component chains are annealed. The single chain of the siRNA compound can be prepared using solution phase or solid phase organic synthesis or both. The advantage provided by organic synthesis is that oligonucleotide chains containing non-natural or modified nucleotides can be easily prepared. Similarly, single-stranded oligonucleotide antigens of the present invention are prepared using solution phase or solid phase organic synthesis or both.

[0204] In one aspect, the dsRNA of the present disclosure comprises at least two nucleotide sequences, a sense sequence and an antisense sequence. The sense strand sequence of HTT can be selected from the sequence groups 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 nucleotide sequence of the antisense strand of the sense strand can be selected from the sequence groups 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 regard, one of the two sequences is complementary to the other of the two sequences, wherein one of the sequences is substantially complementary to the mRNA sequence produced in the expression of the HTT gene. Thus, in this regard, the dsRNA will comprise two oligonucleotides, one of which is described as the sense strand (follower 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) of 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.

[0205] In one embodiment, the substantially complementary sequence to the dsRNA is contained on a separate oligonucleotide. In another embodiment, the substantially complementary sequence to the dsRNA is contained on a single oligonucleotide.

[0206] It should be understood that 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, the RNA of the RNAi agent of the present disclosure, such as the dsRNA of the present disclosure, can comprise any of the sequences listed in any of Tables 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 24, 25, 27-30, 32 and 33, which are unmodified, unconjugated, or modified or conjugated different from those described herein. For example, although the sense strands of the agents of the invention shown in Tables 3, 9, 12, 15, 17, 27, 29 and 32 are conjugated to a GalNAc ligand, these agents may be conjugated to a moiety (e.g., a C16 ligand) that directs delivery to the CNS as described herein. The lipophilic ligand may be included in any position provided in this application.

[0207] It is well known to those skilled in the art that dsRNAs having a duplex structure of about 20 to 23 base pairs (e.g., 21 base pairs) are 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 may also be effective (Chu and Rana (2007) RNA 14: 1714-1719; Kim et al. (2005) Nat Biotech 23: 222-226). In the above embodiments, due to the nature of the oligonucleotide sequences provided herein, the dsRNA described herein may comprise at least one chain having a minimum length of 21 nucleotides. It is reasonable to expect that, compared to the above dsRNA, a shorter duplex having only a few nucleotides removed at one or both ends may be equally effective. Thus, dsRNAs having a sequence of at least 15, 16, 17, 18, 19, 20 or more consecutive nucleotides derived from one of the sequences provided herein, and their ability to inhibit HTT gene expression does not exceed 10, 15, 20, 25 or 30% inhibition, as provided in the Examples herein, in vitro assays using Cos7 and 10 nM concentrations of the RNA agent and PCR assays to extract dsRNAs containing the complete sequence from dsRNAs containing the complete sequence are considered to be within the scope of the present disclosure.

[0208] In addition, the RNA described herein recognizes a site in the HTT transcript that is susceptible to RISC-mediated cleavage. Therefore, the present disclosure further describes an RNAi agent that targets the site. As used herein, if the RNAi agent promotes cleavage of the transcript at any position within the specific site, the RNAi agent is said to be targeted within a specific site of the RNA transcript. Such an RNAi agent typically comprises at least about 15 consecutive nucleotides, preferably at least 19 nucleotides, which are derived from one of the sequences provided herein and coupled to an additional nucleotide sequence taken from a neighboring region of a selected sequence in the HTT gene.

[0209] III. Modified RNAi Agents of the Disclosure

[0210] In one embodiment, the RNA of the RNAi agent of the present disclosure, such as dsRNA, is unmodified and does not include chemical modifications or conjugations such as those known in the art and described herein. In a preferred embodiment, the RNA of the RNAi agent of the present disclosure, such as dsRNA, is chemically modified to enhance stability or other beneficial properties. In certain embodiments of the present disclosure, substantially all nucleotides of the RNAi agent of the present disclosure are modified. In other embodiments of the present disclosure, all nucleotides of the RNAi agent of the present disclosure are modified. Most but not all of the RNAi agents of the present disclosure where "substantially all nucleotides are modified" are modified and may not exceed 5, 4, 3, 2 or unmodified nucleotides. In other embodiments of the present disclosure, the RNAi agent of the present disclosure may include no more than 5, 4, 3, 2 or 1 modified nucleotides.

[0211] The nucleic acids in the present disclosure may be synthesized or modified by methods well established in the art, such as those described in "Current Protocols in Nucleic Acid Chemistry", Beaucage, SL et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. Modifications include, for example, terminal modifications, such as 5' terminal modifications (phosphorylation, conjugation, reverse inter-bonds) or 3' terminal modifications (conjugation, DNA nucleotides, reverse inter-bonds, etc.); base modifications, such as replacement with stable bases, destabilizing bases, or bases paired with an extended partner library, removal of bases (abasic nucleotides) or conjugated bases; sugar modifications (e.g., at the 2' or 4' position) or replacement of sugars; or backbone modifications, including phosphodiester bond modifications or replacements. Specific examples of RNAi agents that can be used in the embodiments described herein include, but are not limited to, RNAs comprising modified backbones or non-natural internucleoside bonds. RNAs having modified backbones particularly include those without phosphorus atoms in the backbone. For the purpose of this specification, and as sometimes mentioned in the art, a modified RNA that does not have a phosphorus atom in its internucleoside backbone can also be considered an oligonucleoside. In some embodiments, the modified RNAi agent will have a phosphorus atom in the internucleoside backbone.

[0212] The RNA backbone of modification includes for example thiophosphate, chiral thiophosphate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl and other alkylphosphonates, including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates include 3'-aminophosphoramidate and aminoalkylphosphoramidate, thiophosphoramidate, thioalkylphosphonates, thioalkylphosphotriester and boric acid phosphate with normal 3'-5' key, the analogs of their 2'-5' connection, and wherein adjacent nucleoside units are connected to 5'-3' or 2'-5' to 5'-2' with 3'-5'. Various salts, mixed salts and free acid forms are also included. In some embodiments of the present invention, dsRNA agent of the present invention is free acid form. In other embodiments of the present invention, dsRNA agent of the present invention is salt form. In one embodiment, dsRNA agent of the present invention is sodium salt form. In certain embodiments, when the dsRNA agent of the present invention is in the form of a sodium salt, sodium ions are present in the agent as counterions for substantially all phosphodiester and / or phosphorothioate groups present in the agent. Agents in which substantially all phosphodiester and / or phosphorothioate bonds have sodium counterions include no more than 5, 4, 3, 2, or 1 phosphodiester and / or phosphorothioate bonds without sodium counterions. In some embodiments, when the dsRNA agent of the present invention is in the form of a sodium salt, sodium ions are present as counterions for all phosphodiester and / or phosphorothioate groups present in the agent.

[0213] Exemplary U.S. patents that teach the above phosphorus-containing interlinkages 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; 5,276,0195; 278,302; 5,286,717; 5,32 1,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,58 7,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,53 7,321,029; and U.S. Patent RE39464, the entire contents of each of which are hereby incorporated by reference herein.

[0214] The modified RNA backbones that do not contain phosphorus atoms have backbones formed by short-chain alkyl or cycloalkyl nucleoside bonds, mixed heteroatoms and alkyl or cycloalkyl nucleoside bonds, or one or more short-chain heteroatoms or heterocyclic nucleoside bonds. These include those with morpholine bonds (partially formed by the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; methyl and thiomethyl backbones; methylenemethyl and thiomethyl backbones; olefin-containing backbones; aminosulfonic acid ester backbones; methyleneimido and methylenehydrazine backbones; sulfonic acid ester and sulfonamide backbones; amide backbones; and those with other mixed N, O, S and CH2 components.

[0215] Exemplary U.S. patents that teach the preparation of the above oligonucleotides 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; 5, 5,677,437 and 5,677,439, the entire contents of each of which are hereby incorporated by reference.

[0216] In other embodiments, suitable RNA mimics are contemplated for use in RNAi agents, wherein the sugar and internucleoside bonds of the nucleotide units, i.e., the backbone, are replaced by new groups. The base units are retained for hybridization with suitable nucleic acid target compounds. One such oligomeric compound, RNA mimics, has been shown to have excellent hybridization properties and is referred to as peptide nucleic acids (PNAs). In PNA compounds, the sugar backbone of RNAi is replaced by a backbone comprising an amide, particularly an aminoethylglycine backbone. The nucleobase is retained and directly or indirectly bound to the aza nitrogen atom of the backbone amide portion. Exemplary U.S. patents teaching the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082; 5,714,331 and 5,719,262, the entire contents of each of which are incorporated herein by reference. Additional PNA compounds suitable for RNAi agents of the present disclosure are described in, for example, Nielsen et al., Science, 1991, 254, 1497-1500.

[0217] Some embodiments of the present disclosure include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones of the aforementioned U.S. Pat. No. 5,489,677, particularly --CH2--NH--CH2-, --CH2--N(CH3)--O--CH2-- (referred to as a methylene(methylimino) or MMI backbone), --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2--, and --N(CH3)--CH2--CH 2- - (wherein the natural phosphodiester backbone is represented as --O--P--O--CH2-), and the amide backbone of the aforementioned US Pat. No. 5,602,240. In some embodiments, the RNA characterized herein has the morpholine backbone structure of the aforementioned US Pat. No. 5,034,506.

[0218] The modified RNA may also comprise one or more substituted sugar moieties. The RNAi agents, e.g., dsRNAs, featured herein may comprise 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, wherein alkyl, alkenyl, and alkynyl groups may be substituted or unsubstituted C1 to C 10 Alkyl or C2 to C 10 Exemplary suitable modifications include O[(CH2) 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 C3)]2, wherein n and m are 1 to about 10. In other embodiments, the dsRNA comprises one of the following at the 2' position: C1 to C 10 Lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkylaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, 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, modification includes 2'methoxyethoxy (2'-O--CH2CH2OCH3, also referred to as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv.Chim.Acta, 1995, 78:486-504), i.e., alkoxy-alkoxy groups. Another exemplary modification is 2'-dimethylaminoethoxy, i.e., O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described in the Examples below, 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 are in these three families); 2'-alkoxyalkyl; and 2'-NMA (N-methylacetamide).

[0219] Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2), 2'-O-hexadecyl and 2'-fluoro (2'-F). Similar modifications can also be made at other positions of the RNA of the RNAi agent, particularly the 3' position of the sugar on the 3' terminal nucleotide or the 5' position of the 2'-5' linked dsRNA and the 5' terminal nucleotide. The RNAi agent can also have a sugar mimetic, such as a cyclobutyl moiety to replace the pentofuranosyl sugar. Exemplary U.S. 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; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633 and 5,700,920, some of which are commonly owned by the instant application. The entire contents of each of the foregoing are hereby incorporated herein by reference.

[0220] RNAi agents of the present disclosure may also include modifications or substitutions of nucleobases (commonly referred to in the art as "bases"). As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). 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 thymidine. adenine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halo, in particular 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Other nucleobases include those disclosed in U.S. Pat. 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, pp. 858-859, Kroschwitz, JL, ed. John Wiley & Sons, 1990; those disclosed in Englisch et al. (1991) Angewandte Chemie, International Edition, 30:613, and those disclosed in Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pp. 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 0-6 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.

[0221] Exemplary U.S. patents that teach the preparation of some of the above-mentioned modified nucleobases as well as other modified nucleobases include, but are not limited to, 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; 5,59 7,045,610; 7,427,672 and 7,495,088, the entire contents of each of which are hereby incorporated by reference.

[0222] The RNAi agents disclosed herein can also be modified to include one or more locked nucleic acids (LNA). Locked nucleic acids are nucleotides with modified ribose moieties, wherein the ribose moiety includes an additional bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose in a 3'-endo structural conformation. It has been shown that adding locked nucleic acids to siRNA can improve the stability of siRNA in serum and reduce off-target effects (Elmen, J. et al. (2005) Nucleic Acids Research 33 (1); Mook, OR. et al. (2007) Mol Canc Ther 6 (3): 833-843; Grunweller, A. et al. (2003) Nucleic Acids Research 31 (12): 3185-3193).

[0223] RNAi agents of the present disclosure can also be modified to include one or more bicyclic sugar moieties." Bicyclic sugar " is a furanosyl ring modified by two atom bridges. " Bicyclic nucleotides " (" BNA ") are nucleosides with sugar moieties, which include bridges connecting two carbon atoms of sugar rings, thereby forming a bicyclic system. In certain embodiments, the bridge connects 4' carbon and 2' carbon of sugar rings. Therefore, in some embodiments, agents of the present disclosure can include one or more locked nucleic acids (LNA). Locked nucleic acids are nucleotides with modified ribose moieties, wherein the ribose moiety includes an extra bridge connecting 2' and 4' carbon. In other words, LNA is a nucleotide comprising a bicyclic sugar moiety comprising a 4'-CH2-O-2' bridge. This structure effectively "locks" ribose in a 3'-endo structural conformation. It has been shown that adding locked nucleic acid to siRNA can improve the stability of siRNA in serum and reduce off-target effects (Elmen, J. et al. (2005) Nucleic Acids Research 33 (1); 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 nucleotides 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 polynucleotides of the present disclosure comprise 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(CH2OCH3)-O-2' (and analogs thereof; see, e.g., U.S. Pat. No. 7,399,845); 4'-C(CH3)(CH3)-O-2' (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,283); 4'-CH2-N(OCH3)-2' (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,283); No. 8,278,425); 4'-CH2-O-N(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. Patent 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 its analogs, see, e.g., U.S. Patent No. 8,278,426).The entire contents of each of the foregoing are hereby incorporated herein by reference.

[0224] Other exemplary US patents and US patent publications that teach locked nucleic acid nucleotide preparation include, but are not limited to, the following: US Patent Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133; 7,084,125; 7,399,845; 7,427,672; 7,569,686; 7,741,457; 8,022,193; 8,030,467; 8,278,425; 8,278,426; 8,278,283; US 2008 / 0039618 and US 2009 / 0012281, the entire contents of each of which are incorporated herein by reference.

[0225] Any of the foregoing bicyclic nucleosides may be prepared with one or more stereochemical sugar configurations, including, for example, α-L-ribofuranose and β-D-ribofuranose (see WO 99 / 14226).

[0226] RNAi medicament of the present disclosure can be modified to comprise one or more restricted ethyl nucleotides.As used in this article, " restricted ethyl nucleotide " or " cEt " is the locked nucleic acid comprising the bicyclic sugar moiety comprising 4 '-CH (CH 3 ) -0-2 ' bridge.In one embodiment, the restricted ethyl nucleotide is that the S conformation is referred to as " S-cEt " in this article.

[0227] The RNAi agents of the present disclosure may also include one or more "conformationally restricted nucleotides" ("CRNs"). CRNs are nucleotide analogs that have a linker connecting the C2' and C4' carbons of ribose or the C3 and C5' carbons of ribose. CRNs lock the ribose ring into a stable conformation and increase hybridization affinity with mRNA. The length of the linker is sufficient to place the oxygen in the optimal position for stability and affinity, thereby reducing ribose ring folding.

[0228] Exemplary publications that teach some of the above-described CRN preparations include, but are not limited to, US 2013 / 0190383; and WO 2013 / 036868, the entire contents of each of which are hereby incorporated by reference herein.

[0229] In some embodiments, the RNAi agent of the present disclosure comprises one or more monomers, which are UNA (unlocked nucleic acid) nucleotides. UNA is a non-locked nucleotide in which any bond of a sugar has been removed to form a non-locked "sugar" residue. In one example, UNA also comprises a monomer in which the bond between C1'-C4' has been removed (i.e., a covalent carbon-oxygen-carbon bond between C1' and C4' carbons). In another example, the C2'-C3' bond of the sugar (i.e., a covalent carbon-carbon bond between 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, incorporated herein by reference).

[0230] Exemplary U.S. patent publications that teach UNA preparation include, but are not limited to, US 8,314,227; and U.S. Patent Publication Nos. 2013 / 0096289; 2013 / 0011922; and 2011 / 0313020, each of which is hereby incorporated by reference in its entirety.

[0231] Potential stabilizing modifications to the ends of RNA molecules may include N-(acetylaminohexanoyl)-4-hydroxyproline (Hyp-C6-NHAc), N-(hexanoyl-4-hydroxyproline (Hyp-C6), N-(acetyl-4-hydroxyproline) (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminohexyl)-4-hydroxyproline (Hyp-C6-amino), 2-docosahexyluridine-3"-phosphate, inverted base dT (idT) and others. Disclosure of such modifications may be found in WO 2011 / 005861.

[0232] Other modifications of the RNAi agents of the present disclosure include a 5' phosphate or a 5' phosphate mimetic, such as a 5' terminal phosphate or a phosphate mimetic on the antisense strand of the RNAi agent. Suitable phosphate mimetics are disclosed, for example, in US2012 / 0157511, the entire contents of which are incorporated herein by reference.

[0233] A. Modified RNAi Agents Comprising Motifs of the Disclosure

[0234] In certain aspects of the present disclosure, the double-stranded RNAi agent of the present disclosure includes an agent with chemical modification, such as disclosed in WO 2013 / 075035, the entire contents of which are incorporated herein by reference. As shown in this article and WO 2013 / 075035, by introducing one or more motifs of three identical modifications on three consecutive nucleotides into the sense strand or antisense strand of the RNAi agent, particularly at or near the cleavage site, better results can be obtained. In some embodiments, the sense strand and antisense strand of the RNAi agent can be completely modified in addition. The introduction of these motifs interrupts the modification pattern of the sense or antisense strand (if present). The RNAi agent can optionally be conjugated with a lipophilic ligand, such as a C16 ligand, such as on the sense strand. The RNAi agent can optionally be modified with (S)-diol nucleic acid (GNA), such as on one or more residues of the antisense strand. The RNAi agent produced has excellent gene silencing activity.

[0235] Therefore, the present disclosure provides a double-stranded RNAi agent that can inhibit the expression of a target gene (e.g., HTT gene) in vivo. The RNAi agent comprises a sense strand and an antisense strand. Each strand of the RNAi agent is 15-30 nucleotides in length. For example, each strand is 16-30 nucleotides in length, 17-30 nucleotides in length, 25-30 nucleotides in length, 27-30 nucleotides in length, 17-23 nucleosides in length, 17-21 nucleotides in length, 17-19 nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides in length, 19-21 nucleotides in length, 21-25 nucleotides in length, or 21-23 nucleotides in length. In certain embodiments, each strand is 19-23 nucleotides in length.

[0236] The sense strand and the antisense strand typically form a duplex double-stranded RNA ("dsRNA"), also referred to herein as an "RNAi agent". The duplex region of an RNAi agent is 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 nucleotides in length.

[0237] In one embodiment, the RNAi agent can include one or more overhang regions or capping groups at the 3' end, 5' end or both ends of one or both chains. The overhang length is 1-6 nucleotides, for example, 2-6 nucleotides in length, 1-5 nucleotides in length, 2-5 nucleotides in length, 1-4 nucleotides in length, 2-4 nucleotides in length, 1-3 nucleotides in length, 2-3 nucleotides in length or 1-2 nucleotides in length. In a preferred embodiment, the nucleotide overhang region length is 2 nucleotides. The overhang may be the result that one chain is longer than the other chain, or the result of two chains of the same length interlacing. The overhang may form a mismatch with the target mRNA, or it may be complementary to the targeted gene sequence, or it may be another sequence. The first and second chains may also be connected, for example, by additional bases to form a hairpin, or by other base-free joints.

[0238] In one embodiment, the nucleotides in the overhang region of the RNAi agent can each independently be a modified or unmodified nucleotide, including but not limited to 2'-sugar modifications such as 2-F, 2'-O-methyl, thymidine (T), and any combination thereof.

[0239] 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 it can be complementary to the targeted gene sequence, or it can be another sequence.

[0240] The 5' or 3' overhang on the sense strand, antisense strand, or both strands of the RNAi agent may be phosphorylated. In some embodiments, the overhang region comprises two nucleotides with a thiophosphate between the two nucleotides, wherein the two nucleotides may 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 on the antisense strand. In one embodiment, the 3' overhang is present on the sense strand.

[0241] RNAi agents may contain only one overhang, which can enhance the interference activity of RNAi without affecting its overall stability. For example, a single-stranded overhang can be located at the 3' end of the sense strand, or 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), and vice versa. Typically, the antisense strand of RNAi has a nucleotide overhang at the 3' end, and the 5' end is a blunt end. Although not wishing to be bound by theory, the asymmetric blunt end at the 5' end of the antisense strand and the 3' terminal overhang of the antisense strand are conducive to the loading of the guide strand into the RISC process.

[0242] In one embodiment, the RNAi agent is a double-terminal flat polymer of 19 nucleotides in length, wherein the sense strand comprises at least one three 2'-F modified motifs at three consecutive nucleotides at positions 7, 8, and 9 from the 5' end. The antisense strand comprises at least one three 3'-O-methyl modified motifs at three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0243] In another embodiment, the RNAi agent is a double-terminal flat polymer of 20 nucleotides in length, wherein the sense strand comprises at least one three 2'-F modified motif at three consecutive nucleotides at positions 8, 9, and 10 from the 5' end. The antisense strand comprises at least one three 3'-O-methyl modified motif at three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0244] In another embodiment, the RNAi agent is a double-terminal flat polymer of 21 nucleotides in length, wherein the sense strand comprises at least one three 2'-F modified motif at three consecutive nucleotides at positions 9, 10, and 11 from the 5' end. The antisense strand comprises at least one three 3'-O-methyl modified motif at three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0245] In one embodiment, the RNAi agent comprises a sense strand of 21 nucleotides and an antisense strand of 23 nucleotides, wherein the sense strand comprises three consecutive nucleotides at positions 9, 10, and 11 from the 5' end comprising at least one three 2'-F modified motifs; the antisense strand comprises three consecutive nucleotides at positions 11, 12, and 13 from the 5' end comprising at least one three 3'-O-methyl modified motifs, wherein one end of the RNAi agent is a blunt end, and the other end comprises two nucleotide overhangs. Preferably, the two nucleotide overhangs are at the 3' end of the antisense strand. When the two nucleotide overhangs are at the 3' end of the antisense strand, the terminal three nucleotides may be separated by two phosphorothioate internucleoside bonds, wherein two of the three nucleotides are overhang nucleotides, and the third nucleotide is a paired nucleotide adjacent to the overhang nucleotide. In one embodiment, the RNAi agent additionally has two phosphorothioate internucleotide bonds between the terminal three nucleotides at the 5' end of the sense strand and the 5' end of the antisense strand. In one embodiment, each nucleotide on the sense strand and antisense strand of the RNAi agent, including nucleotides as part of the motif, is a modified nucleotide. In one embodiment, each residue is independently modified by 2'-methyl or 3'-fluoro, such as in an alternating motif. Optionally, the RNAi agent also comprises a ligand (e.g., a lipophilic ligand, optionally a C16 ligand).

[0246] In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, wherein the sense strand is 25-30 nucleotide residues in length, wherein starting from the 5' terminal nucleotide (position 1), positions 1 to 23 comprise at least 8 ribonucleotides; the antisense strand is 36-66 nucleotide residues in length, starting from the 3' terminal nucleotide, comprising at least 8 ribonucleotides at positions paired with positions 1-23 of the sense strand to form a duplex; wherein at least the 3' terminal nucleotide of the antisense strand is unpaired with the sense strand, and up to 6 consecutive 3' terminal nucleotides are unpaired with the sense strand, thereby forming a 3' single-stranded overhang of 1-6 nucleotides; wherein the 5' end of the antisense strand comprises 10-30 consecutive nucleotides that are not paired with the sense strand, thereby forming a 10-30 nucleotide single-stranded 5' overhang; wherein when the sense strand and the antisense strand are aligned for maximum complementarity, at least the 5' terminal and 3' terminal nucleotides of the sense strand are base paired with nucleotides of the antisense strand, thereby forming a substantially double-stranded region between the sense strand and the antisense strand; and the antisense strand is sufficiently complementary to the target RNA of at least 19 ribonucleotides of the antisense strand to reduce target gene expression when the double-stranded nucleic acid is introduced into a mammalian cell; and wherein the sense strand comprises at least one three 2'-F modified motif on three consecutive nucleotides, wherein at least one motif occurs at or near the cleavage site. The antisense strand comprises at least one three 2'-O-methyl modified motif on three consecutive nucleotides at or near the cleavage site.

[0247] In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, wherein the RNAi agent comprises a first strand of at least 25 and at most 29 nucleotides in length and a second strand of at most 30 nucleotides in length and at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end; wherein the 3' end of the first strand and the 5' end of the second strand form a blunt end, and the 3' end of the second strand is 1-4 nucleotides longer than the first strand, wherein the duplex region of at least 25 nucleotides in length and the second strand along at least 19 nucleotides of the length of the second strand are sufficiently complementary to the target mRNA to reduce target gene expression when the RNAi agent is introduced into a mammalian cell, and wherein dimer cleavage of the RNAi agent preferentially results in siRNA comprising the 3' end of the second strand, thereby reducing target gene expression in a mammal. Optionally, the RNAi agent further comprises a ligand.

[0248] In one embodiment, the sense strand of the RNAi agent comprises at least one motif of three identical modifications on three consecutive nucleotides, wherein one motif occurs at the cleavage site of the sense strand.

[0249] In one embodiment, the antisense strand of the RNAi agent may also comprise at least one motif of three identical modifications on three consecutive nucleotides, wherein one of the motifs occurs at or near the cleavage site of the antisense strand.

[0250] For RNAi agents with a duplex region of 17-23 nucleotides, the cleavage site of the antisense strand is usually near position 10, 11, or 12 of the 5' end. Thus, three identical modified motifs may occur at positions 9, 10, or 11 of the antisense strand; positions 11, 12, or 13, 14, or positions 13, 14, or 15 of the antisense strand, counting from the first nucleotide of the 5' end of the antisense strand, or from the first pair of nucleotides in the duplex region of the 5' end of the antisense strand. The cleavage site of the antisense strand may also vary depending on the length of the duplex region at the 5' end of the RNAi.

[0251] The sense strand of the RNAi agent may include at least one motif of three identical modifications on three consecutive nucleotides at the cleavage site of the strand; and the antisense strand may have at least one motif of three identical modifications on three consecutive nucleic acids at or near the cleavage site of the strand. When the sense strand and the antisense strand form a dsRNA duplex, the sense strand and the antisense strand may be arranged so that a motif of three nucleotides on the sense strand and a motif of three nucleotides on the antisense strand have at least one nucleotide overlap, that is, at least one of the nucleotides of the motif in the sense strand forms a base pair with at least one of the three nucleotides of the motif in the antisense strand. Alternatively, at least two nucleotides overlap, or all three nucleotides overlap.

[0252] In one embodiment, the sense strand of an RNAi agent can include one or more three identically modified motifs on three continuous nucleotides. The first motif can appear at or near the cleavage site of the chain and another motif can be a wing modification. The term "wing modification" herein refers to a motif that appears in another part of the chain, and the remaining motifs at or near the cleavage site of the same chain are separated. The wing modification is adjacent to the first motif or is separated by at least one or more nucleotides. When the motifs are directly adjacent to each other, the chemical properties of the motifs are different from each other, and when the motifs are separated by one or more nucleotides, the chemical form can be the same or different. There are two or more wing modifications. For example, when there are two wing modifications, each wing modification can occur at one end of the first motif located at or near the cleavage site, or on either side of the leading motif.

[0253] As with the sense strand, the antisense strand of the RNAi agent may contain one or more identical modified motifs on three consecutive nucleotides, wherein a motif appears at or near the cleavage site of the strand. The antisense strand may also contain one or more wing modifications arranged in a manner similar to the wing modifications that may be present in the sense strand.

[0254] In one embodiment, wing modifications on the sense or antisense strand of the RNAi agent typically exclude the first or two terminal nucleotides at the 3' end, the 5' end, or both ends of the strand.

[0255] In another embodiment, the wing modifications of the sense or antisense strand of the RNAi agent generally exclude the first one or two paired nucleotides at the 3' end, the 5' end, or both ends of the strand.

[0256] When the sense and antisense strands of the RNAi agent each comprise at least one wing modification, the wing modifications can fall at the same end of the duplex region and have an overlap of 1, two, or three nucleotides.

[0257] When the sense strand and the antisense strand of the RNAi agent each comprise at least two wing modifications, the sense strand and the antisense strand can be arranged such that the two modifications from one strand fall at one end of the duplex region with an overlap of one, two, or three nucleotides; the two modifications from one strand fall at the other end of the duplex region with an overlap of one, two, or three nucleotides; the two modifications of one strand fall on each side of the leader motif with an overlap of one, two, or three nucleotides in the duplex region.

[0258] In one embodiment, the RNAi agent comprises mismatches with the target surface, within the duplex, or a combination thereof. Mismatches may occur in the overhang region or the duplex region. Base pairs can be ranked according to their tendency to promote dissociation or melting (e.g., based on the binding or dissociation free energy of a particular pairing, the simplest method is to examine the pairing on the basis of a single pairing, although next adjacent or similar analysis may also be used). In terms of promoting dissociation: A:U is better than G:C; G:U is better than G:C; and I:C is better than G:C. Mismatches, such as non-standard or non-standard pairings (as described elsewhere herein) are better than standard (A:T, A:U, G:C) pairings; and pairs containing universal bases are better than standard pairings.

[0259] In one embodiment, the RNAi agent comprises at least one of the first 1, 2, 3, 4 or 5 base pairs in the duplex region at the 5' end of the antisense strand independently selected from the following groups: A:U, G:U, I:C, and mismatched pairs, such as non-standard or different from standard pairs or pairs containing universal bases, to promote dissociation of the antisense strand at the 5' end of the duplex.

[0260] In one embodiment, the nucleotide at position 1 in the duplex region starting from the 5' end of the antisense strand is selected from A, dA, dU, U and dT. Alternatively, at least one of the 1st, 2nd or 3rd base pairs in the duplex region starting from the 5' end of the antisense strand is an AU base pair. For example, the first base pair in the duplex region starting from the 5' end of the antisense strand is an AU base pair.

[0261] 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, such as two dT nucleotides at the 3' end of the sense strand or the antisense strand.

[0262] In one embodiment, the sense strand sequence can be represented by formula (I):

[0263] 5'n p -N a -(XXX) i -N b -YY YN b -(ZZZ) j -N a -n q 3'(I)

[0264] in:

[0265] i and j are each independently 0 or 1;

[0266] p and q are each independently 0-6;

[0267] Each N a Independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides.

[0268] Each N b independently represent an oligonucleotide sequence comprising 0-10 modified nucleotides;

[0269] Each n p and n q independently represent the overhang nucleotides;

[0270] wherein Nb and Y do not have the same modifications; and

[0271] XXX, YYY and ZZZ each independently represent a motif of three identical modifications on three consecutive nucleotides. Preferably YYY is all 2'-F modified nucleotides.

[0272] In one embodiment, N a or N b Contains alternating patterns of modifications.

[0273] In one embodiment, the YYY motif occurs at or near the cleavage site of the sense strand. For example, when the RNAi agent has a duplex region of 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), counting from the first nucleotide at the 5' end; or alternatively, counting from the first paired nucleotide in the duplex region, starting from the 5' end.

[0274] In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. Thus, the sense strand can be represented by the following formula:

[0275] 5'n p -N a -YYY-N b -ZZZ-N a -n q 3'(Ib);

[0276] 5'n p -N a -XXX-N b -YYY-N a -n q 3'(Ic); or

[0277] 5'n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3'(Id).

[0278] When the sense strand is represented by formula (Ib), N b represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides.

[0279] Each N a Independently represents an oligonucleotide sequence comprising 2-20, 2-15 or 2-10 modified nucleotides.

[0280] When the sense strand is represented by formula (Ic), N b represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each N a Oligonucleotide sequences comprising 2-20, 2-15 or 2-10 modified nucleotides may independently be represented.

[0281] When the sense strand is represented by formula (Id), each N b N independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. b is 0, 1, 2, 3, 4, 5, or 6. Each N a Oligonucleotide sequences comprising 2-20, 2-15 or 2-10 modified nucleotides may independently be represented.

[0282] Each of X, Y, and Z may be the same as or different from each other.

[0283] In other embodiments, i is 0 and j is 0, and the sense strand can be represented by the formula:

[0284] 5'n p -N a -YYY-N a -n q 3'(Ia).

[0285] When the sense strand is represented by formula (Ia), each N a An oligonucleotide sequence comprising 2-20, 2-15 or 2-10 modified nucleotides may independently be represented.

[0286] In one embodiment, the antisense strand of RNAi can be represented by formula (II):

[0287] 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)

[0288] in:

[0289] k and l are each independently 0 or 1;

[0290] p' and q' are each independently 0-6;

[0291] Each N a ' independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

[0292] Each N b ' independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;

[0293] Each n p′ and n q ' independently represent overhang nucleotides;

[0294] Where N b ' and Y' do not have the same modification;

[0295] and

[0296] X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent a motif of three identical modifications on three consecutive nucleotides.

[0297] In one embodiment, N a ' or N b 'Contains alternating pattern modifications.

[0298] The Y'Y'Y' motif occurs at or near the cleavage site of the antisense strand. For example, when the RNAi agent has a duplex region of 17-23 nucleotides in length, 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, counting from the first nucleotide from the 5' end; or alternatively, counting from the first paired nucleotide from the 5' end within the duplex region. Preferably, the Y'Y'Y' motif occurs at positions 11, 12, 13.

[0299] In one embodiment, the Y'Y'Y' motifs are all 2'-OMe modified nucleotides.

[0300] 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.

[0301] Therefore, the antisense strand can be expressed by the following formula:

[0302] 5'n q’ -N a ′-Z′Z′Z′-N b ′-Y′Y′Y′-N a ′-n p’ 3' (IIb);

[0303] 5'n q '-N a ′-Y′Y′Y′-N b′ -X′X′X′-n p’ 3'(IIc); or

[0304] 5'n q’ -N a ′-Z′Z′Z′-N b ′-Y′Y′Y′-N b ′-X′X′X′-Na ′-n p '3'(IId).

[0305] When the antisense strand is represented by formula (IIb), N b ' represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N a ' independently represents an oligonucleotide sequence comprising 2-20, 2-15 or 2-10 modified nucleotides.

[0306] When the antisense strand is represented by formula (IIc), N b ' represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N a ' independently represents an oligonucleotide sequence comprising 2-20, 2-15 or 2-10 modified nucleotides.

[0307] When the antisense strand is represented by formula (IId), each N b ' independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N a ' independently represents an oligonucleotide sequence comprising 2-20, 2-15 or 2-10 modified nucleotides. Preferably, N b Is 0, 1, 2, 3, 4, 5, or 6.

[0308] In other embodiments, k is 0 and l is 0, and the antisense strand can be represented by the formula:

[0309] 5'n p’ -N a '-Y'Y'Y'-N a’ -n q’ 3'(Ia).

[0310] When the antisense strand is represented by formula (IIa), each N a ' independently represents an oligonucleotide sequence comprising 2-20, 2-15 or 2-10 modified nucleotides.

[0311] Each of X', Y' and Z' may be the same as or different from each other.

[0312] Each nucleotide of the sense strand and the 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'-fluorine. For example, each nucleotide of the sense strand and the antisense strand is independently modified with 2'-O-methyl or 2'-fluorine. In particular, each of X, Y, Z, X', Y' and Z' can represent a 2'-O-methyl modification or a 2'-fluorine modification.

[0313] In one embodiment, when the duplex region is 21 nt, the sense strand of the RNAi agent may include a YYY motif occurring at positions 9, 10, and 11 of the sense strand, counting from the first nucleotide at the 5' end, or optionally, counting from the first paired nucleotide in the duplex region, starting from the 5' end; and Y represents a 2'-F modification. The sense strand may additionally include a XXX motif or a ZZZ motif as a wing modification at the other end of the duplex region; XXX and ZZZ each independently represent a 2'-OMe modification or a '-F modification.

[0314] In one embodiment, the antisense strand may comprise a Y'Y"Y' motif appearing at positions 11, 12, 13 of the strand, counting from the first nucleotide at the 5' end, or optionally, counting from the first paired nucleotide in the duplex region, starting from the 5' end; and Y represents a 2'-O-methyl modification. The antisense strand may additionally comprise a X'X'X' motif or a Z'Z'Z' motif as a wing modification at the other end of the duplex region; and X'X'X' and Z'Z'Z' each independently represent a 2'-OMe modification or a 2'-F modification.

[0315] The sense strand represented by any one of the above formulae (Ia), (Ib), (Ic) and (Id) forms a duplex with the antisense strand represented by any one of the formulae (IIa), (IIb), (IIb), (IIc) and (IId).

[0316] Therefore, the RNAi agent used in the method of the present invention may comprise a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, and the RNAi duplex is represented by formula (III):

[0317] Meaning: 5'n p -N a -(XXX) i -N b -YY YN b -(ZZZ) j -N a -n q 3'

[0318] Antonym: 3'n p '-Na '-(X'X′X′) k -N b '-Y′Y′Y′-N b '-(Z′Z′Z′) l -N a '-n q '5'

[0319] (III)

[0320] in:

[0321] i, j, k and l are each independently 0 or 1.

[0322] p, p', q and q' are each independently 0-6.

[0323] Each N a and N a 'Independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

[0324] Each N b and N b ' independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;

[0325] in

[0326] Each n p '、n p 、n q ' and n q , each of which may or may not be present and independently represents an overhang nucleotide; and

[0327] XXX, YYY, ZZZ, X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent a motif of three identical modifications on three consecutive nucleotides.

[0328] 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; k is 0 and l is 1; or k and l are both 0; or k and l are both 1.

[0329] Exemplary combinations of sense and antisense strands that form RNAi duplexes include the following:

[0330] 5'n p -N a -YY YN a -n q 3'

[0331] 3's p '-N a '-Y′Y′Y′-N a the q '5'

[0332] (IIIa)

[0333] 5's p -N a -YY THE b -ZZ ZN a -n q 3'

[0334] 3's p '-N a '-Y′Y′Y′-N b '-Z′Z′Z′-N a the q '5'

[0335] (IIIb)

[0336] 5's p -N a -XX XN b -YY THE a -n q 3'

[0337] 3's p '-N a '-X′X′X′-N b '-Y′Y′Y′-N a '-n q '5'

[0338] (IIIc)

[0339] 5's p -N a -XX XN b -YY THE b -ZZ ZN a -n q 3'

[0340] 3's p '-N a '-X′X′X′-N b '-Y′Y′Y′-N b '-Z′Z′Z′-N a -n q '5'

[0341] (IIId)

[0342] When the RNAi agent is represented by formula (IIIa), each Na independently represents an oligonucleotide sequence comprising 2-20, 2-15 or 2-10 modified nucleotides.

[0343] When the RNAi agent is represented by formula (IIIb), each Nb independently represents an oligonucleotide sequence comprising 1-10, 1-7, 1-5 or 1-4 modified nucleotides. Each Na independently represents an oligonucleotide sequence comprising 2-20, 2-15 or 2-10 modified nucleotides.

[0344] When the RNAi agent is represented by formula (IIIc), each N b 、N b ' independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N a Independently refers to an oligonucleotide sequence comprising 2-20, 2-15 or 2-10 modified nucleotides.

[0345] When the RNAi agent is represented by formula (IIId), each N b 、N b ' independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides.

[0346] Each N a 、N a ' independently represents an oligonucleotide sequence comprising 2-20, 2-15 or 2-10 modified nucleotides. N a 、N a '、N b and N b 'Each independently contains alternating patterns of modifications.

[0347] In one embodiment, when the RNAi agent is represented by formula (IIId), N a In another embodiment, when the RNAi agent is represented by formula (IIId), N a The modification is 2'-O-methyl or 2'-fluoro modification and n p ′>0 and at least one n p ' is connected to the adjacent nucleotide via a phosphorothioate bond. In yet another embodiment, when the RNAi agent is represented by formula (IIId), N a The modification is 2'-O-methyl or 2'-fluoro, n p ′>0 and at least one n p' is linked to adjacent nucleotides via a phosphorothioate bond, and the sense is conjugated to one or more C16 (or related) moieties via a divalent or trivalent branched linker (as described below). In another embodiment, when the RNAi agent is represented by formula (IIId), N a The modification is 2'-O-methyl or 2'-fluoro, n p ′>0 and at least one n p ' is linked to an adjacent nucleotide via a phosphorothioate bond, the sense strand comprises at least one phosphorothioate bond, and the sense strand is conjugated to one or more lipophilic moieties (e.g., a C16 (or related) moiety), optionally linked via a divalent or trivalent linker.

[0348] In one embodiment, when the RNAi agent is represented by formula (IIIa), N a The modification is 2'-O-methyl or 2'-fluoro, n p '>0 and at least one n p ' is linked to an adjacent nucleotide via a phosphorothioate bond, the sense strand comprises at least one phosphorothioate bond, and the sense strand is conjugated to one or more lipophilic moieties (eg, a C16 (or related) moiety) via a divalent or trivalent branched linker.

[0349] In one embodiment, the RNAi agent is a polymer comprising at least two duplexes represented by formula (III), (IIIa), (IIIb), (IIIc) and (IIId), wherein the duplexes are connected by a linker. The linker can be cleavable or non-cleavable. Optionally, the polymer further comprises a ligand. Each polymer can target the same gene or two different genes; or each polymer can target the same gene at two different target sites.

[0350] In one embodiment, the RNAi agent is a polymer comprising three, four, five, six or more duplexes represented by formula (III), (IIIa), (IIIb), (IIIc) and (IIId), wherein the duplexes are connected by a linker. The linker can be cleavable or non-cleavable. Optionally, the polymer further comprises a ligand. Each polymer can target the same gene or two different genes; or each polymer can target the same gene at two different target sites.

[0351] 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 3' ends, and are optionally conjugated to a ligand. Each agent can target the same gene or two different genes; or each agent can target the same gene at two different target sites.

[0352] Various publications describe multimeric RNAi agents that can be used in the disclosed methods. Such publications include WO2007 / 091269, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887, and WO2011 / 031520; and US7858769, the entire contents of each of which are incorporated herein by reference.

[0353] In certain embodiments, the compositions and methods of the present disclosure include vinyl phosphonate (VP) modifications of RNAi agents as described herein. In an exemplary embodiment, the vinyl phosphonate of the present disclosure has the following structure:

[0354]

[0355] The vinyl phosphonates of the present disclosure may be linked to the antisense strand or the sense strand of the dsRNA of the present disclosure. In certain preferred embodiments, the vinyl phosphonates of the present disclosure are linked to the antisense strand of the dsRNA, optionally at the 5' end of the antisense strand of the dsRNA.

[0356] The compositions and methods of the present disclosure also contemplate vinyl phosphonate modifications. An exemplary vinyl phosphonate structure is:

[0357]

[0358] i. Thermally unstable modifications

[0359] In certain embodiments, dsRNA molecules can be optimized for RNA interference by incorporating thermolabile modifications in 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 found that dsRNAs with antisense strands contain thermolabile modifications of at least one duplex within the first 9 nucleotide positions of the antisense strand counting from the 5' end, with reduced off-target gene silencing activity. Therefore, in some embodiments, the antisense strand contains at least one (e.g., one, two, three, four, five or more) thermolabile modified duplex within the first 9 nucleotide positions of the 5' region of the antisense strand. In some embodiments, one or more thermolabile modified duplexes are located at positions 2-9, or preferably positions 4-8, at the 5' end of the antisense strand. In some embodiments, the thermolabile modifications of the doublet are located at positions 6, 7 or 8 at the translated 5' end. In some further embodiments, the thermolabile modification of the duplex is located at position 7 at the 5' end of the antisense strand. The term "thermostable modification" includes modifications that result in a dsRNA with a lower overall melting temperature (Tm) (preferably, the Tm is one, two, three, or four degrees lower than the Tm of the dsRNA without such modification). In some embodiments, the thermolabile modification of the duplex is located 2, 3, 4, 5, or 9 of the 5' end of the antisense strand.

[0360] Thermally labile modifications may include, but are not limited to, abasic modifications; mismatches with the opposite nucleotide in the opposite strand; and sugar modifications, such as 2'-deoxy modifications or acyclic nucleotides, such as unlocked nucleic acids (UNA) and glycol nucleic acids (GNA).

[0361] Exemplary abasic modifications include, but are not limited to, the following:

[0362]

[0363] wherein R=H, Me, Et or OMe; R'=H, Me, Et or OMe; R"=H, Me, Et or OMe.

[0364]

[0365] wherein B is a modified or unmodified nucleobase.

[0366] Exemplary sugar modifications include, but are not limited to, the following:

[0367]

[0368]

[0369] wherein B is a modified or unmodified nucleobase.

[0370] In some embodiments, the thermolabile modification of the duplex is selected from:

[0371]

[0372] where B is a modified or unmodified nucleobase and the asterisk on each structure represents R, S or the racemate.

[0373] The term "acyclic nucleotide" refers to any nucleotide having an acyclic ribose sugar, for example, wherein any bond between the ribose carbons (e.g., C1'-C2', C2'-C3', C3'-C4', C4'-O4', or C1'-O4') is absent or at least one ribose carbon or oxygen (e.g., C1', C2', C3', C4', or O4') is absent, independently or in combination, in the nucleotide. In some embodiments, an acyclic nucleotide is Where B is a modified or unmodified nucleobase, R 1 and R 2R is independently H, halogen, OR3 or alkyl; R3 is H, cycloalkyl, aryl, aralkyl, heteroaryl or sugar). The term "UNA" refers to a non-locked nucleotide in which any bond to the sugar has been removed to form a non-locked "sugar" residue. In one example, UNA also refers to a monomer in which the bond between C1'-C4' is 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) is removed (see Mikhailov et al. Tetrahedron Letters, 26(17):2059(1985); and Fluiter et al., Mol. Biosyst., 10:1039(2009), the entire contents of which are incorporated herein by reference. Acyclic derivatives provide greater backbone flexibility without affecting Watson-Crick pairing. Acyclic nucleotides can be connected by 2'-5' or 3'-5' bonds.

[0374] The term "GNA" refers to diol nucleic acid, a polymer similar to DNA or RNA, but with a different "backbone" consisting of repeating glycerol units linked by phosphodiester bonds:

[0375]

[0376] The heat unstable modification of the duplex can be a mismatch (i.e., non-complementary base pair) between a heat unstable nucleotide and an opposite nucleotide in the opposite strand in the dsRNA duplex. Exemplary mismatch base pairs include G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, U:T, or a combination thereof. Other mismatch base pairs known in the art are also applicable to the present invention. Mismatches can occur between nucleotides of naturally occurring nucleotides or modified nucleotides, i.e., mismatch base pairs can occur between nucleobases from each nucleotide, regardless of the modification on the ribose of the nucleotide. In certain embodiments, the dsRNA molecule comprises at least one nucleobase, i.e., a 2'-deoxynucleobase, in the mismatch pair; for example, a 2'-deoxynucleobase is located in the sense strand.

[0377] In some embodiments, the thermolabile modification of the duplex in the seed region of the antisense strand includes nucleotides that impair WCH-bonding to complementary bases on the target mRNA, for example:

[0378]

[0379] Further examples of abasic nucleotides, acyclic nucleotide modifications (including UNA and GNA), and mismatch modifications have been described in detail in WO2011 / 133876, which is incorporated herein by reference in its entirety.

[0380] Thermally labile modifications may also include universal bases whose ability to form hydrogen bonds with opposing bases is reduced or eliminated, and phosphate modifications.

[0381] In some embodiments, the thermally unstable modification of the duplex includes nucleotides with non-standard bases, such as, but not limited to, nucleobase modifications with impaired or completely eliminated ability to form hydrogen bonds with the oppositely connected base. These nucleobase modifications have been evaluated for destabilization of the central region of the dsRNA duplex as described in WO 2010 / 0011895, which is incorporated herein by reference in its entirety. Exemplary nucleobase modifications are:

[0382]

[0383]

[0384] In some embodiments, the thermolabile modification of the duplex in the seed region during translation includes one or more α-nucleotides complementary to bases on the target mRNA, for example:

[0385]

[0386] wherein R is H, OH, OCH3, F, NH2, NHMe, NMe2 or O-alkyl.

[0387] Exemplary phosphate modifications known to reduce the thermal stability of dsRNA duplexes compared to native phosphodiester bonds are:

[0388]

[0389] R=alkyl

[0390] The alkyl group of the R group can be a C1-C6 alkyl group. Specific alkyl groups of the R group include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, pentyl, and hexyl.

[0391] As will be appreciated by those skilled in the art, in view of the functional role of the core base is to define the specificity of the RNAi agent of the present disclosure, although the core base modification can be carried out in various ways as described herein, such as to introduce destabilizing modifications into the RNAi agent of the present disclosure, such as to enhance the on-target effect relative to the off-target effect, for non-core base modifications, such as modifications to the sugar group or phosphate backbone of a polyribonucleotide, the range of modifications available and generally present on the RNAi agent of the present disclosure is often greater. Such modifications are described in more detail in other parts of the present disclosure, and RNAi agents of the present disclosure are explicitly contemplated, which have natural core bases or modified core bases as described above or elsewhere herein.

[0392] In addition to the antisense strand containing a thermally unstable modification, the dsRNA may also contain one or more stable modifications. For example, the dsRNA may contain at least two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) stable modifications. Without limitation, stable modifications may be present in one strand. In some embodiments, both the sense strand and the antisense strand contain at least two stable modifications. Stable modifications may occur on any nucleotide of the sense strand or the antisense strand. For example, a stable modification may occur on each nucleotide on the sense strand or the antisense strand; each stable modification may occur on the sense strand or the antisense strand in an alternating pattern; or both the sense strand and the antisense strand contain a stable modification of an alternating pattern. The alternating pattern of the stable modification on the sense strand may be the same or different from the alternating pattern of the stable modification on the antisense strand, and the alternating pattern of the stable modification on the sense strand may be offset relative to the alternating pattern of the stable modification on the antisense strand.

[0393] In some embodiments, the antisense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) stabilizing modifications. Without limitation, the stabilizing modifications in the antisense strand can be present at any position. In some embodiments, the antisense comprises stabilizing modifications at positions 2, 6, 8, 9, 14, and 16 starting from the 5' end. In some other embodiments, the antisense comprises stabilizing modifications at positions 2, 6, 14, and 16 starting from the 5' end. In yet other embodiments, the antisense comprises stabilizing modifications at positions 2, 14, and 16 starting from the 5' end.

[0394] In some embodiments, the antisense strand comprises at least one stabilizing modification adjacent to a destabilizing modification. For example, the stabilizing modification can be a nucleotide at the 5' or 3' end of the destabilizing modification, i.e., a nucleotide at -1 or +1 from the position of the destabilizing modification. In some embodiments, the antisense strand comprises a stabilizing modification at each of the 5' and 3' ends of the destabilizing modification, i.e., -1 and +1 from the position of the destabilizing modification.

[0395] In some embodiments, the antisense strand comprises at least two stabilizing modifications 3' to the destabilizing modification, ie, at positions +1 and +2 to the position of the destabilizing modification.

[0396] In some embodiments, the sense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) stabilizing modifications. Without limitation, the stabilizing modifications in the sense strand may be present at any position. In some embodiments, the sense strand comprises stabilizing modifications at positions 7, 10, and 11 starting from the 5' end. In some other embodiments, the sense strand comprises stabilizing modifications at positions 7, 9, 10, and 11 starting from the 5' end. In some embodiments, the sense strand comprises stabilizing modifications at positions opposite or complementary to the 11th, 12th, and 15th positions 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 the 11th, 12th, 13th, and 15th positions on the antisense strand counting from the 5' end of the antisense strand. In some embodiments, the sense strand comprises a set of two, three, or four stabilizing modifications.

[0397] In some embodiments, the sense strand does not comprise a stabilizing modification at a position opposite or complementary to a thermally labile modification of the duplex in the antisense strand.

[0398] Exemplary thermostable modifications include, but are not limited to, 2'-fluoro modifications. Other thermostable modifications include, but are not limited to, LNA.

[0399] In some embodiments, the dsRNA of the present disclosure comprises at least four (e.g., four, five, six, seven, eight, nine, ten or more) 2'-fluoro nucleotides. Without limitation, 2'-fluoro nucleotides may be present in one strand. In some embodiments, both the sense strand and the antisense strand comprise at least two 2'-fluoro nucleotides. The 2'-fluoro modification may occur on any nucleotide of the sense strand or the antisense strand. For example, the 2'-fluoro modification may occur on each nucleotide on the sense strand or the antisense strand; each 2'-fluoro modification may occur on the sense strand or the antisense strand in an alternating pattern; or both the sense strand and the antisense strand comprise an alternating pattern of 2'-fluoro modifications. The alternating pattern of 2'-fluoro modifications on the sense strand may be the same or different from the alternating pattern of stable modifications on the antisense strand, and the alternating pattern of 2'-fluoro modifications on the sense strand may be offset relative to the alternating pattern of 2'-fluoro modifications on the antisense strand.

[0400] In some embodiments, the antisense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) 2'-fluoro modifications. Without limitation, the 2'-fluoro modifications in the antisense strand can be present at any position. In some embodiments, the antisense comprises 2'-fluoro modifications at positions 2, 6, 8, 9, 14, and 16 starting from the 5' end. In some embodiments, the antisense comprises 2'-fluoro modifications at positions 2, 6, 14, and 16 starting from the 5' end. In yet other embodiments, the antisense comprises 2'-fluoro modifications at positions 2, 14, and 16 starting from the 5' end.

[0401] In some embodiments, the antisense strand comprises at least one 2'-fluoro modification adjacent to the destabilizing modification. For example, the 2'-fluoro modified nucleotide can be a nucleotide at the 5' or 3' end of the destabilizing modification, i.e., a nucleotide at -1 or +1 from the position of the destabilizing modification. In some embodiments, the antisense strand comprises a 2'-fluoro nucleotide at each of the 5' and 3' ends of the destabilizing modification, i.e., -1 and +1 from the position of the destabilizing modification.

[0402] In some embodiments, the antisense strand comprises at least two 2'-fluoro nucleotides 3' to the destabilizing modification, ie, at positions +1 and +2 of the destabilizing modification.

[0403] In some embodiments, the sense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) 2'-fluoro nucleotides. Without limitation, the 2'-fluoro modification in the sense strand may be present at any position. In some embodiments, the antisense comprises 2'-fluoro nucleotides at positions 7, 10 and 11 starting from the 5' end. In other embodiments, the sense strand comprises 2'-fluoro nucleotides at positions 7, 9, 10 and 11 starting from the 5' end. In some embodiments, the sense strand comprises 2'-fluoro nucleotides at positions opposite or complementary to the 11th, 12th and 15th positions 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 the 11th, 12th, 13th and 15th positions of the antisense strand counting from the 5' end of the antisense strand. In some embodiments, the sense strand comprises a group of two, three or four 2'-fluoro nucleotides.

[0404] In some embodiments, the sense strand does not comprise a 2'-fluoro nucleotide at a position opposite or complementary to the thermolabile modification of the duplex in the antisense strand.

[0405] In other embodiments, the dsRNA molecules of the present disclosure comprise a sense strand of 21 nucleotides (nt) and an antisense strand of 23 nucleotides (nt), wherein the antisense strand comprises at least one thermolabile nucleotide, wherein at least one thermolabile nucleotide occurs in the seed region of the antisense strand (i.e., positions 2-9 of the 5' end of the antisense strand), wherein one end of the dsRNA is blunt-ended and the other end comprises a 2 nt overhang, and wherein the dsRNA optionally further has at least one of the following features (e.g., one, two, three, four , five, six or all seven): (i) the antisense comprises 2, 3, 4, 5 or 6 2'-fluoro modifications; (ii) the antisense comprises 1, 2, 3, 4 or 5 phosphorothioate internucleotide bonds; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand comprises 2, 3, 4 or 5 2'-fluoro modifications; (v) the sense strand comprises 1, 2, 3, 4 or 5 phosphorothioate internucleotide bonds; (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, the 2nt overhang is at the 3' end of the antisense.

[0406] In some embodiments, the dsRNA molecules of the present disclosure comprise a sense strand and an antisense strand, wherein: the sense strand is 25-30 nucleotide residues in length, wherein starting from the 5' terminal nucleotide (position 1), positions 1 to 23 of the sense strand comprise at least 8 ribonucleotides; the antisense strand is 36-66 nucleotide residues in length, and starting from the 3' terminal nucleotide, at least 8 ribonucleotides form a duplex at positions paired with positions 1-23 of the sense strand; wherein at least the 3' terminal nucleotide of the antisense strand is unpaired with the sense strand, and up to 6 consecutive 3' terminal nucleotides are unpaired with the sense strand, thereby forming a 3' single-stranded overhang of 1-6 nucleotides; wherein the 5' end of the antisense strand comprises The invention relates to a method for producing a double-stranded RNA comprising 10-30 consecutive nucleotides that are not paired with the sense strand, thereby forming a single-stranded 5' overhang of 10-30 nucleotides; wherein when the sense strand and the antisense strand are aligned for maximum complementarity, at least the 5' and 3' terminal nucleotides of the sense strand are base paired with nucleotides of the antisense strand, thereby forming a substantially double-stranded region between the sense strand and the antisense strand; and the antisense strand is sufficiently complementary to a target RNA of at least 19 ribonucleotides of the antisense strand to reduce target gene expression when the double-stranded nucleic acid is introduced into a mammalian cell; and wherein the antisense strand comprises at least one thermolabile nucleotide, wherein the at least one thermolabile nucleotide is in the seed region of the antisense strand (i.e., at positions 2-9 at the 5' end of the antisense strand). For example, the thermolabile nucleotide occurs between positions opposite or complementary to positions 14-17 of the 5' end of the sense strand, and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six, or all seven) of the following features: (i) the antisense comprises 2, 3, 4, 5, or 6 2'-fluoro modifications; (ii) the antisense comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand comprises 2, 3, 4, or 5 2'-fluoro modifications; (v) the sense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; and (vi) the dsRNA comprises at least four 2'-fluoro modifications; and (vii) the dsRNA comprises a duplex region of 12-30 nucleotide pairs in length.

[0407] In some embodiments, the dsRNA of the present disclosure comprises a sense strand and an antisense strand, wherein the dsRNA molecule comprises a sense strand of at least 25 and at most 29 nucleotides in length and an antisense strand of at most 30 nucleotides in length, wherein the sense strand comprises a modified nucleotide at position 11 (from the 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, and the antisense strand is 1-4 nucleotides longer at its 3' end than the sense strand, wherein there is a duplex region of at least 25 nucleotides in length, and the antisense strand is sufficiently complementary to a target mRNA along at least 19 nt of the length of the antisense strand to reduce target gene expression when the dsRNA molecule is introduced into a mammalian cell, and wherein dimer cleavage of the dsRNA preferentially produces an siRNA comprising the 3' end of the antisense strand, thereby reducing target mRNA expression in a mammalian cell. The invention relates to a method for producing a dsRNA, wherein the antisense strand comprises at least one thermolabile nucleotide, wherein the at least one thermolabile nucleotide is in the seed region of the antisense strand (i.e., at positions 2-9 of the 5' end of the antisense strand), and wherein the dsRNA optionally further has at least one of the following characteristics (e.g., one, two, three, four, five, six, or all seven): (i) the antisense comprises 2, 3, 4, 5, or 6 2'-fluoro modifications; (ii) the antisense comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide bonds; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand comprises 2, 3, 4, or 5 2'-fluoro modifications; (v) the sense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide bonds; and (vi) the dsRNA comprises at least four 2'-fluoro modifications; and (vii) the dsRNA has a duplex region of 12-29 nucleotide pairs in length.

[0408] In some embodiments, each nucleotide on the sense and antisense strands of the dsRNA molecule can be modified. Each nucleotide can be modified with the same or different modifications, which can include one or more changes in one or both of the non-linked phosphate oxygens or one or more of the linked phosphate oxygens; changes in the composition of the phosphate group, such as the 2' hydroxyl group on the ribose; large replacement of the phosphate moiety with a "dephosphorylation" linker; modification or replacement of the natural bases; and replacement or modification of the ribose-phosphate backbone.

[0409] Since nucleic acid is a polymer of subunits, many modifications occur at repeated positions within nucleic acid, such as modifications of bases or phosphate moieties, or non-connected O of phosphate moieties. In some cases, the modification will occur at all positions in the nucleic acid, but in many cases it will not. For example, the modification may occur only at the 3' or 5' terminal position, may occur only at the terminal region, such as the position on the terminal nucleotide of a chain or the last 2, 3, 4, 5 or 10 nucleotides. The modification may occur in a double-stranded region, a single-stranded region or both. The modification may occur only in the double-stranded region of RNA, or may occur in the single-stranded region of RNA. For example, the phosphorothioate modification at the non-connected O position may occur only at one or both ends, may occur only at the terminal region, such as the terminal nucleotide position of a chain or the last 2, 3, 4, 5 or 10 nucleotides, or may occur in double-stranded and single-stranded regions, particularly at the end. The 5' end or the end may be phosphorylated.

[0410] For example, stability can be improved by including specific bases at the overhangs, or by including modified nucleotides or nucleotide substitutes at the single-stranded overhangs, such as at the 5' or 3' overhangs or both. For example, it may be necessary to include purine nucleotides at the overhangs. In some embodiments, all or some bases at the 3' or 5' overhangs can be modified, such as with modifications as described herein. Modifications can include, for example, using modifications known in the art at the 2' position of ribose, such as using deoxyribonucleotides, 2'-deoxy-2'-fluoro (2'-F) or 2'-O-methyl modifications instead of the ribose of the nucleobase, and modifications of phosphate groups, such as phosphorothioate modifications. The overhangs do not need to be homologous to the target sequence.

[0411] In some embodiments, each residue of the sense strand and the antisense strand is independently modified by LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy or 2'-fluoro. The strand may contain more than one modification. In some embodiments, each residue of the sense strand and the antisense strand is independently modified by 2'-O-methyl or 2'-fluoro. It should be understood that these modifications are in addition to at least one thermally unstable modification of the duplex present in the antisense strand.

[0412] There are usually at least two different modifications on the sense strand and the antisense strand. These two modifications can be 2'-deoxy, 2'-O-methyl or 2'-fluoro modifications, acyclic nucleotides or other. In some embodiments, the sense strand and the antisense strand each contain two different modified nucleotides selected from 2'-O-methyl or 2'-deoxy. In some embodiments, the sense strand and the antisense strand are each independently 2'-O-methyl nucleotides, 2'-deoxy nucleotides, 2'-deoxy-2'-fluoro nucleotides, 2'-ON-methylacetamido (2'-O-NMA) nucleotides, 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) nucleotides, 2'-O-aminopropyl (2'-O-AP) nucleotides or 2'-ara-F nucleotides. Similarly, it should be understood that these modifications are supplementary to at least one thermally unstable modification of the duplex present in the antisense strand.

[0413] In some embodiments, the dsRNA of the present disclosure comprises an alternating pattern of modifications, particularly in B1, B2, B3, B1', B2', B3', B4' regions. As used herein, the term "alternating motif" or "alternating pattern" refers to a motif having one or more modifications, each modification occurring on alternating nucleotides of a chain. Alternating nucleotides may refer to one for each other nucleotide or one for every three nucleotides, or a similar pattern. For example, if A, B, and C represent a modification to a nucleotide, respectively, the alternating motif may be "ABABABABABAB...", "AABBAABBAABB...", "AABAABAABAAB...", "AAABAAABAAAB...", "AAABBBAAABBB...", or "ABCABCABCABC...", etc.

[0414] The types of modifications contained in the alternating motif can be the same or different. For example, if A, B, C, and D each represent a modification on a nucleotide, the alternating pattern, i.e., the modification on every other nucleotide may be the same, but each sense strand or antisense strand can choose from several modification possibilities within the alternating motif, such as "ABABAB...", "ACACAC...", "BDBDBD...", or "CDCDCD...", etc.

[0415] In some embodiments, the dsRNA molecules of the present disclosure include a modification pattern of an alternating motif on the sense strand that is displaced relative to the modification pattern of an alternating motif on the antisense strand. The displacement can cause the modified nucleotide group of the sense strand to correspond to the differently modified nucleotide group of the antisense strand, and vice versa. For example, when the sense strand is paired with the antisense strand in the dsRNA duplex, the alternating motif in the sense strand can start with "ABABAB" from the 5'-3' of the strand and the alternating motif in the antisense strand can start with "BABABA" from the 3'-5' in the duplex region. As another example, the alternating motif in the sense strand can start with "AABBAABB" from the 5'-3' of the strand and the alternating motif in the antisense strand can start with "BBAABBAA" from the 3'-5' in the duplex region, so that the modification pattern between the sense strand and the antisense strand is completely or partially displaced.

[0416] The dsRNA of the present disclosure also comprises at least one phosphorothioate or methylphosphonate internucleotide bond. The phosphorothioate or methylphosphonate internucleotide bond modification can occur on any nucleotide of the sense strand or antisense strand or at any position of the strand. For example, the internucleotide bond modification can occur on each nucleotide of the sense strand or antisense strand; each internucleotide bond modification can occur on the sense strand or antisense strand in an alternating pattern; or the sense strand or antisense strand comprises two internucleotide bond modifications in an alternating pattern. The alternating pattern of the internucleotide bond modification on the sense strand can be the same or different from the antisense strand, and the alternating pattern of the internucleotide bond modification on the sense strand can be shifted relative to the alternating pattern of the internucleotide bond modification on the antisense strand.

[0417] In some embodiments, the dsRNA molecule comprises a phosphorothioate or methylphosphonate internucleotide bond modification on the overhang region. For example, the overhang region comprises two nucleotides with a phosphorothioate or methylphosphonate internucleotide bond. It is also possible to carry out internucleotide bond modification to connect the overhang nucleotide to the terminal pairing nucleotide in the duplex region. For example, at least 2,3,4 or all overhang nucleotides can be connected by a phosphorothioate or methylphosphonate internucleotide bond, and optionally, there may be an additional phosphorothioate or methylphosphonate internucleotide bond to connect the overhang nucleotide to the pairing nucleotide next to the overhang nucleotide. For example, there may be at least two phosphorothioate internucleotide bonds between the three nucleotides at the end, wherein two of the three nucleotides are overhang nucleotides, and the third is the pairing nucleotide next to the overhang nucleotide. Preferably, these three nucleotides at the end can be at the 3' end of the antisense strand.

[0418] In some embodiments, the sense strand of the dsRNA molecule comprises 1-10 blocks of two to ten phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located 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 an antisense strand comprising phosphorothioate or methylphosphonate or phosphate linkages.

[0419] 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 sense strand is paired with an antisense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or an antisense strand comprising phosphorothioate or methylphosphonate or phosphate linkages.

[0420] 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, 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 an antisense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or an antisense strand comprising phosphorothioate or methylphosphonate or phosphate linkages.

[0421] 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, 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 an antisense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or an antisense strand comprising phosphorothioate or methylphosphonate or phosphate linkages.

[0422] 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, 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 an antisense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or an antisense strand comprising phosphorothioate or methylphosphonate or phosphate linkages.

[0423] 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, or 10 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 an antisense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or an antisense strand comprising phosphorothioate or methylphosphonate or phosphate linkages.

[0424] 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, 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 an antisense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or an antisense strand comprising phosphorothioate or methylphosphonate or phosphate linkages.

[0425] 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, 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 an antisense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or an antisense strand comprising phosphorothioate or methylphosphonate or phosphate linkages.

[0426] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of nine phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, or 4 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 an antisense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages or an antisense strand comprising phosphorothioate or methylphosphonate or phosphate linkages.

[0427] In some embodiments, the dsRNA of the present disclosure further comprises one or more phosphorothioate or methylphosphonate internucleotide linkage modifications at 1-10 of the terminal positions of the sense strand or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides may be linked by phosphorothioate or methylphosphonate internucleotide linkages at one or both ends of the sense strand or antisense strand.

[0428] In some embodiments, the dsRNA molecules of the present disclosure further comprise one or more phosphorothioate or methylphosphonate internucleotide bond modifications within positions 1-10 of the internal region of the duplex of each sense strand or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides may be linked by phosphorothioate methylphosphonate internucleotide bonds at positions 8-16 of the duplex region counting from the 5′ end of the sense strand; the dsRNA molecules may optionally further comprise one or more phosphorothioate or methylphosphonate internucleotide bond modifications at terminal positions 1-10.

[0429] In some embodiments, the dsRNA molecules of the present disclosure further comprise one to five phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 1-5 of the sense strand counting from the 5' end and one to five phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 18-23 of the sense strand counting from the 5' end, and one to five phosphorothioate or methylphosphonate internucleotide linkage modifications at positions 1 and 2 and 18-23 of the antisense strand counting from the 5' end.

[0430] In some embodiments, the dsRNA molecules of the present disclosure further comprise one phosphorothioate internucleotide linkage modification within positions 1-5 and one phosphorothioate or methylphosphonate internucleotide linkage modification within positions 18-23 of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 18-23 of the antisense strand, counting from the 5' end.

[0431] In some embodiments, the dsRNA molecules of the present disclosure further comprise two phosphorothioate internucleotide linkage modifications within positions 1-5 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the sense strand, as counted from the 5' end, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand, as counted from the 5' end.

[0432] In some embodiments, the dsRNA molecules of the present disclosure further comprise two phosphorothioate internucleotide linkage modifications within positions 1-5 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the sense strand, counted from the 5' end, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand, counted from the 5' end.

[0433] In some embodiments, the dsRNA molecules of the present disclosure further comprise two phosphorothioate internucleotide linkage modifications within positions 1-5 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the sense strand, counted from the 5' end, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand, counted from the 5' end.

[0434] In some embodiments, the dsRNA molecules of the present disclosure further comprise one phosphorothioate internucleotide linkage modification within positions 1-5 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the sense strand, counted from the 5' end, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand, counted from the 5' end.

[0435] In some embodiments, the dsRNA molecules of the present disclosure further comprise one phosphorothioate internucleotide linkage modification within positions 1-5 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the sense strand, counted from the 5' end, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand, counted from the 5' end.

[0436] In some embodiments, the dsRNA molecules of the present disclosure further comprise one phosphorothioate internucleotide linkage modification within positions 1-5 counting from the 5' end of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand counting from the 5' end.

[0437] In some embodiments, the dsRNA molecules of the present disclosure further comprise two phosphorothioate internucleotide linkage modifications within positions 1-5 counting from the 5' end of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand counting from the 5' end.

[0438] In some embodiments, the dsRNA molecules of the present disclosure further comprise two phosphorothioate internucleotide linkage modifications within positions 1-5 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the sense strand, as counted from the 5' end, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand, as counted from the 5' end.

[0439] In some embodiments, the dsRNA molecules of the present disclosure further comprise two phosphorothioate internucleotide linkage modifications within positions 1-5 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the sense strand, as counted from the 5' end, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand, as counted from the 5' end.

[0440] In some embodiments, the dsRNA molecules of the present disclosure further comprise two phosphorothioate internucleotide linkage modifications within positions 1-5 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the sense strand, as counted from the 5' end, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand, as counted from the 5' end.

[0441] In some embodiments, the 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 of the sense strand, and one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 of the antisense strand, counting from the 5' end.

[0442] In some embodiments, the 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 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 of the antisense strand, counting from the 5' end.

[0443] In some embodiments, the 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 of the sense strand, and one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 of the antisense strand, counting from the 5' end.

[0444] In some embodiments, the 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 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 of the antisense strand, counting from the 5' end.

[0445] In some embodiments, the 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 of the sense strand, and one phosphorothioate internucleotide linkage modification at position 1 and one phosphorothioate internucleotide linkage modification at position 21 of the antisense strand, counting from the 5' end.

[0446] In some embodiments, the 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 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 of the antisense strand, counting from the 5' end.

[0447] In some embodiments, the compounds of the present disclosure comprise a pattern of backbone chiral centers. In some embodiments, the common pattern of backbone chiral centers comprises at least 5 internucleotide bonds in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 6 internucleotide bonds in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 7 internucleotide bonds in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 8 internucleotide bonds in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 9 internucleotide bonds in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 10 internucleotide bonds in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 11 internucleotide bonds in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 12 internucleotide bonds in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 13 internucleotide bonds in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 14 internucleotide bonds in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 15 internucleotide bonds in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 16 internucleotide bonds in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 17 internucleotide bonds in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 18 internucleotide bonds in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises at least 19 internucleotide bonds in the Sp configuration. In some embodiments, the common pattern of backbone chiral centers comprises no more than 8 internucleotide bonds in the Rp configuration. In some embodiments, the common pattern of backbone chiral centers comprises no more than 7 internucleotide bonds in the Rp configuration. In some embodiments, the common pattern of backbone chiral centers comprises no more than 6 internucleotide bonds in the Rp configuration. In some embodiments, the common pattern of backbone chiral centers comprises no more than 5 internucleotide bonds in the Rp configuration. In some embodiments, the common pattern of backbone chiral centers comprises no more than 4 internucleotide bonds in the Rp configuration. In some embodiments, the common pattern of backbone chiral centers comprises no more than 3 internucleotide bonds in the Rp configuration. In some embodiments, the common pattern of backbone chiral centers comprises no more than 2 internucleotide bonds in the Rp configuration. In some embodiments, the common pattern of backbone chiral centers comprises no more than 1 internucleotide bond in the Rp configuration. In some embodiments, the common pattern of backbone chiral centers comprises no more than 8 achiral internucleotide bonds (as a non-limiting example, phosphodiester).In some embodiments, the common pattern of backbone chiral centers comprises no more than 7 achiral internucleotide bonds. In some embodiments, the common pattern of backbone chiral centers comprises no more than 6 achiral internucleotide bonds. In some embodiments, the common pattern of backbone chiral centers comprises no more than 5 achiral internucleotide bonds. In some embodiments, the common pattern of backbone chiral centers comprises no more than 4 achiral internucleotide bonds. In some embodiments, the common pattern of backbone chiral centers comprises no more than 3 achiral internucleotide bonds. In some embodiments, the common pattern of backbone chiral centers comprises no more than 2 achiral internucleotide bonds. In some embodiments, the common pattern of backbone chiral centers comprises no more than 1 achiral internucleotide bond. In some embodiments, the common pattern of backbone chiral centers comprises at least 10 internucleotide bonds in the Sp configuration, and no more than 8 achiral internucleotide bonds. In some embodiments, the common pattern of backbone chiral centers comprises at least 11 internucleotide bonds in the Sp configuration, and no more than 7 achiral internucleotide bonds. In some embodiments, the common pattern of backbone chiral centers comprises at least 12 internucleotide bonds of Sp configuration, and no more than 6 achiral internucleotide bonds. In some embodiments, the common pattern of backbone chiral centers comprises at least 13 internucleotide bonds of Sp configuration, and no more than 6 achiral internucleotide bonds. In some embodiments, the common pattern of backbone chiral centers comprises at least 14 internucleotide bonds of Sp configuration, and no more than 5 achiral internucleotide bonds. In some embodiments, the common pattern of backbone chiral centers comprises at least 15 internucleotide bonds of Sp configuration, and no more than 4 achiral internucleotide bonds. In some embodiments, internucleotide bonds in Sp configuration are optionally continuous or discontinuous. In some embodiments, internucleotide bonds in Rp configuration are optionally continuous or discontinuous. In some embodiments, achiral internucleotide bonds are optionally continuous or discontinuous.

[0448] In some embodiments, the compounds of the present disclosure include blocks that are stereochemical blocks. In some embodiments, the block is an Rp block because each internucleotide bond 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 block is an Sp block because each internucleotide bond 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, the oligonucleotides provided include Rp and Sp blocks. In some embodiments, the oligonucleotides provided include one or more Rp but do not include Sp blocks. In some embodiments, the oligonucleotides provided include one or more Sp but do not include Rp blocks. In some embodiments, the oligonucleotides provided include one or more PO blocks, wherein each internucleotide bond is a natural phosphate bond.

[0449] In some embodiments, the compounds of the present disclosure include 5' blocks that are Sp blocks, wherein each sugar moiety includes 2'-F modifications. In some embodiments, 5' blocks are Sp blocks, wherein each internucleotide bond is a modified internucleotide bond and each sugar moiety includes 2'-F modifications. In some embodiments, 5' blocks are Sp blocks, wherein each internucleotide bond is a phosphorothioate bond and each sugar moiety includes 2'-F modifications. In some embodiments, 5' blocks include 4 or more nucleoside units. In some embodiments, 5' blocks include 5 or more nucleoside units. In some embodiments, 5' blocks include 6 or more nucleoside units. In some embodiments, 5' blocks include 7 or more nucleoside units. In some embodiments, 3' blocks are Sp blocks, wherein each sugar moiety includes 2'-F modifications. In some embodiments, 3' blocks are Sp blocks, wherein each internucleotide bond is a modified nucleotide and each sugar moiety includes 2'-F modifications. In some embodiments, the 3' block is an Sp block, wherein each internucleotide bond is a phosphorothioate bond and each sugar moiety comprises a 2'-F modification. In some embodiments, the 3' block comprises 4 or more nucleoside units. In some embodiments, the 3' block comprises 5 or more nucleoside units. In some embodiments, the 3' block comprises 6 or more nucleoside units. In some embodiments, the 3' block comprises 7 or more nucleoside units.

[0450] In some embodiments, the compounds of the present disclosure contain a type of nucleotide or oligonucleotide followed by a specific type of internucleotide bond in a region, such as a natural phosphate bond, a modified internucleotide bond, an Rp chiral internucleotide bond, an Sp chiral internucleotide bond, 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 bond (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 bond (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 bond (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 bond (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 bond (PO). In some embodiments, A and G are followed by Sp. In some embodiments, A and G are followed by Rp.

[0451] In some embodiments, the antisense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 21 and 22 and between nucleotide positions 22 and 23, wherein the antisense strand comprises at least one thermolabile modification of the duplex located in the seed region of the antisense strand (i.e., positions 2-9 at the 5' end of the antisense strand), and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six, seven, or all eight) of the following features: (i) the antisense strand comprises 2, 3, 4, 5, or 6 2'-fluoro modification; (ii) the antisense comprises 3, 4 or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand comprises 2, 3, 4 or 5 2'-fluoro modifications; (v) the sense strand comprises 1, 2, 3, 4 or 5 phosphorothioate internucleotide linkages; (vi) the dsRNA comprises at least four 2'-fluoro modifications; (vii) the dsRNA comprises a duplex region of 12-40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at the 5' end of the antisense strand.

[0452] In some embodiments, the antisense strand comprises phosphorothioate internucleotide bonds 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, wherein the antisense strand comprises at least one thermolabile modification of the duplex located in the seed region of the antisense strand (i.e., positions 2-9 at the 5' end of the antisense strand), and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six, seven, or all eight) of the following features: (i) the antisense strand comprises 2, 3, 4 , 5 or 6 2'-fluoro modifications; (ii) the sense strand is conjugated to a ligand; (iii) the sense strand comprises 2, 3, 4 or 5 2'-fluoro modifications; (iv) the sense strand comprises 1, 2, 3, 4 or 5 phosphorothioate internucleotide linkages; (v) the dsRNA comprises at least four 2'-fluoro modifications; (vi) the dsRNA comprises a duplex region of 12-40 nucleotide pairs in length; (vii) the dsRNA comprises a duplex region of 12-40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at the 5' end of the antisense strand.

[0453] In some embodiments, the sense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotides 2 and 3, wherein the antisense strand comprises at least one thermolabile modification of the duplex located in the seed region of the antisense strand (i.e., positions 2-9 at the 5′ end of the antisense strand), and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six, seven, or all eight) of the following characteristics: (i) the antisense comprises 2, 3, 4, 5, or 6 2′-fluoro modifications; (ii) the antisense comprises 1, 2, 3, 4 or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated to a ligand; (iv) the sense strand comprises 2, 3, 4 or 5 2'-fluoro modifications; (v) the sense strand comprises 3, 4 or 5 phosphorothioate internucleotide linkages; (vi) the dsRNA comprises at least four 2'-fluoro modifications; (vii) the dsRNA comprises a duplex region of 12-40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at the 5' end of the antisense strand.

[0454] In some embodiments, the sense strand comprises a phosphorothioate internucleotide bond between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3, the antisense strand comprises a phosphorothioate internucleotide bond 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, wherein the antisense strand comprises at least one thermolabile modification of the duplex located in the seed region of the antisense strand (i.e., positions 2-9 at the 5' end of the antisense strand), and wherein the dsRNA optionally further has at least one of the following features (e.g. one, two, three, four, five, six or all seven): (i) the antisense comprises 2, 3, 4, 5 or 6 2'-fluoro modifications; (ii) the sense strand is conjugated to a ligand; (iii) the sense strand comprises 2, 3, 4 or 5 2'-fluoro modifications; (iv) the sense strand comprises 3, 4 or 5 phosphorothioate internucleotide linkages; (v) the dsRNA comprises at least four 2'-fluoro modifications; (vi) the dsRNA comprises a duplex region of 12-40 nucleotide pairs in length; and (vii) the dsRNA has a blunt end at the 5' end of the antisense strand.

[0455] In some embodiments, the dsRNA molecules of the present disclosure comprise mismatches with the target, within the duplex, or a combination thereof. Mismatches may occur in the overhang region or in the duplex region. Base pairs may be ranked according to their propensity to promote dissociation or melting (e.g., based on the binding or dissociation free energy of a particular pairing, the simplest approach being to examine the pairing on the basis of a single pairing, although next adjacent or similar analysis may also be used). In terms of promoting dissociation: A:U is better than G:C; G:U is better than G:C; and I:C is better than G:C. Mismatches, such as non-standard or non-standard pairings (as described elsewhere herein) are better than standard (A:T, A:U, G:C) pairings; and pairings comprising universal bases are better than standard pairings.

[0456] 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 at the 5' end of the antisense strand independently selected from the following groups: A:U, G:U, I:C, and mismatched pairs, such as non-standard or different from standard pairs or pairs comprising universal bases, to promote dissociation of the antisense strand at the 5' end of the duplex.

[0457] In some embodiments, the nucleotide at position 1 in the duplex region starting from the 5' end of the antisense strand is selected from A, dA, dU, U and dT. Alternatively, at least one of the 1st, 2nd or 3rd base pairs in the duplex region starting from the 5' end of the antisense strand is an AU base pair. For example, the first base pair in the duplex region starting from the 5' end of the antisense strand is an AU base pair.

[0458] It was found that the introduction of 4'-modified or 5'-modified nucleotides at the 3' end of a phosphodiester (PO), phosphorothioate (PS) or phosphorodithioate (PS2) linkage of a dinucleotide at any position in a single-stranded or double-stranded oligonucleotide can produce a steric effect on the internucleotide bond, thereby protecting or stabilizing it from nucleases.

[0459] In some embodiments, a 5' modified nucleoside is introduced into the 3' end of a dinucleotide at any position of a single-stranded or double-stranded siRNA. For example, a 5'-alkylated nucleoside can be introduced into the 3' end of a dinucleotide at any position of a single-stranded or double-stranded siRNA. The alkyl group at the 5' position of the ribose can be a racemic or chirally pure R or S isomer. An exemplary 5'-alkylated nucleoside is a 5'-methyl nucleoside. The 5'-methyl group can be a racemic or a chirally pure R or S isomer.

[0460] In some embodiments, a 4' modified nucleoside is introduced into the 3' end of a dinucleotide at any position of a single-stranded or double-stranded siRNA. For example, a 4'-alkylated nucleoside can be introduced into the 3' end of a dinucleotide at any position of a single-stranded or double-stranded siRNA. The alkyl group at the 4' position of the ribose can be a racemic or chirally pure R or S isomer. Exemplary 4'-alkylated nucleosides are 4'-methyl nucleosides. The 4'-methyl group can be a racemic or chirally pure R or S isomer. Alternatively, a 4'-O-alkylated nucleoside can be introduced into the 3' end of a dinucleotide at any position of a single-stranded or double-stranded siRNA. The 4'-O-methyl group of the ribose can be a racemic or chirally pure R or S isomer. Exemplary 4'-O-alkylated nucleosides are 4'-O-methyl nucleosides. The 4'-O-methyl group can be a racemic or chirally pure R or S isomer.

[0461] In some embodiments, 5'-alkylated nucleosides are introduced into any position of the sense strand or antisense strand of a dsRNA, and such modifications maintain or improve the efficacy of the dsRNA. The 5'-alkyl can be a racemic or chirally pure R or S isomer. Exemplary 5'-alkylated nucleosides are 5'-methyl nucleosides. The 5'-methyl can be a racemic or chirally pure R or S isomer.

[0462] In some embodiments, 4'-alkylated nucleosides are introduced into any position of the sense strand or antisense strand of a dsRNA, and such modifications maintain or improve the efficacy of the dsRNA. The 4'-alkyl can be a racemic or chirally pure R or S isomer. Exemplary 4'-alkylated nucleosides are 4'-methyl nucleosides. The 4'-methyl can be a racemic or chirally pure R or S isomer.

[0463] In some embodiments, 4'-O-alkylated nucleosides are introduced into any position of the sense strand or antisense strand of a dsRNA, and such modifications maintain or improve the efficacy of the dsRNA. The 5'-alkyl group can be a racemic or chirally pure R or S isomer. Exemplary 4'-O-alkylated nucleosides are 4'-O-methyl nucleosides. The 4'-O-methyl group can be a racemic or chirally pure R or S isomer.

[0464] In some embodiments, the dsRNA molecules of the present disclosure may include a 2'-5' bond (having 2'-H, 2'-OH and 2'-OMe and having P=O or P=S). For example, the 2'-5' bond modification can be used to promote nuclease resistance or inhibit the binding of the sense strand to the antisense strand, or can be used at the 5' end of the sense strand to avoid RISC activation of the sense strand.

[0465] In another embodiment, the dsRNA molecules of the present disclosure can include 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 the binding of the sense strand to the antisense strand, or can be used at the 5' end of the sense strand to avoid RISC activation of the sense strand.

[0466] Various publications describe multimeric siRNAs that can all be used with the dsRNA of the present disclosure. Such publications include WO2007 / 091269, US 7858769, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887, and WO2011 / 031520, all of which are incorporated herein in their entirety.

[0467] As described in more detail below, the RNAi agent comprising the conjugation of one or more carbohydrate moieties with RNAi agents can optimize one or more properties of RNAi agents. In many cases, the carbohydrate moiety will be connected to the modified subunit of the RNAi agent. For example, the ribose of one or more ribonucleotide subunits of a dsRNA agent can be replaced by another part, such as a non-carbohydrate (preferably cyclic) carrier connected to a carbohydrate ligand. The ribonucleotide subunits of the subunits so replaced are referred to herein as ribose replacement modified subunits (RRMS). The cyclic carrier can be a carbocyclic system, i.e., all ring atoms are carbon atoms, or a heterocyclic system, i.e., one or more ring atoms can be heteroatoms, such as nitrogen, oxygen, sulfur. The cyclic carrier can be a monocyclic system or can include two or more rings, such as a fused ring. The cyclic carrier can be a fully saturated ring system, or it can include one or more double bonds.

[0468] The ligand can be connected to the polynucleotide by a carrier. The carrier includes (i) at least one "main chain connection point", preferably two "main chain connection points" and (ii) at least one "tethering connection point". "Main chain connection point" as used in this article refers to a functional group, such as a hydroxyl group, or is generally a key that can be used and applied to incorporate the carrier into the main chain, such as a phosphate ester of ribonucleic acid or a modified phosphate ester, such as a sulfur-containing main chain. "Tethering connection point" (TAP) refers to the composition of the circular carrier connecting the selected part in some embodiments, such as a carbon atom or a heteroatom (different from the atom providing the main chain connection point). The part can be, for example, a carbohydrate, such as a monosaccharide, a disaccharide, a trisaccharide, a tetrasaccharide, an oligosaccharide and a polysaccharide. Optionally, the selected part is connected to the circular carrier by inserting a tether. Therefore, the circular carrier will generally include a functional group, such as an amino group, or generally provide another chemical entity suitable for binding or tethering, such as a ligand to a key constituting the ring.

[0469] The RNAi agent can be conjugated to the ligand through a carrier, wherein the carrier can be a cyclic group or a non-cyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinone, tetrahydrofuranyl and decahydronaphthyl; preferably, the acyclic group is selected from a serinol backbone or a diethanolamine backbone.

[0470] In certain embodiments, the RNAi agent used in the disclosed methods 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. These agents may further comprise a ligand.

[0471] IV. iRNA conjugated to ligand

[0472] Another modification of the RNA of the iRNAs of the invention involves chemically linking to the iRNA one or more ligands, moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the iRNA, e.g., entry into cells. Such moieties include, but are not limited to, lipid moieties, such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86:6553-6556), bile acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), thioethers, such as beryl-S-triphenylthiol (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, 20:533-538), aliphatic 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 hexadecyl-racemic glycerol or 1,2-di-O-hexadecyl-racemic-glycero-3-phosphonic acid triethylammonium (Manoharan et al., Tetrahedron Lett., 1995, 36: 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18: 3777-3783), polyamines 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; Shea et al., Nucl. Acids Res., 1990, 18: 3777-3783), Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0473] In certain embodiments, the ligand alters the distribution, targeting or lifespan of the iRNA agent into which it is incorporated. In some embodiments, the ligand provides enhanced affinity for a selected target, such as a molecule, cell or cell type, compartment, such as a cell or organ compartment, tissue, organ or body region, such as compared to a species without such a ligand. Typical ligands do not participate in duplex pairing in double-stranded nucleic acids.

[0474] The ligand may 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. The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, such as a synthetic polyamino acid. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolic acid) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or poly(L-lactide-co-glycolic acid) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphazene. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines or alpha helical peptides.

[0475] The ligand can also include a targeting group that binds to a specific cell type such as a nephrocyte, such as a cell or tissue targeting agent, such as a lectin, a glycoprotein, a lipid or an albumen, such as an antibody. The targeting group can be thyrotropin, melanocyte stimulating hormone, a lectin, a glycoprotein, a surfactant protein A, a mucin carbohydrate, a multivalent lactose, a multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, a multivalent fucose, a glycosylated polyamino acid, a multivalent galactose, transferrin, a bisphosphonate, a polyglutamate, a polyaspartate, a lipid, a cholesterol, a steroid, a bile acid, a folic acid, a vitamin B12, a biotin or an RGD peptide or an RGD peptide mimetic. In some embodiments, the ligand is a multivalent galactose, such as N-acetyl-galactosamine.

[0476] Other ions of ligands include dyes, intercalators (e.g., acridine), crosslinkers (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaporphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules such as cholesterol, monosuccinic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexadecyl, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecanyl, palmitic acid, myristic acid, O3-(oleoyl)glycerol bile acid, dimethoxytrityl or phenoxazine) and peptide conjugates (e.g., antennary peptides, Tat peptides), alkylating agents, phosphates, amino groups, sulfhydryl groups, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled labels, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP or AP.

[0477] The ligand can be a protein, such as a glycoprotein, or a peptide, such as a molecule with a specific affinity for a co-ligand, or an antibody, such as an antibody that binds to a specific cell type, such as a cancer cell, an endothelial cell, or an osteocyte. The ligand can also include a hormone or a hormone receptor. They can also include non-peptide substances, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB.

[0478] The ligand can be a substance, such as a drug, that can increase the uptake of an iRNA agent into a cell, such as by disrupting the cytoskeleton of the cell, such as by disrupting the microtubules, microfilaments or intermediate filaments of the cell. The drug can be, for example, a taxon, vincristine, vinblastine, cytochalasin, nocodazole, amphetamine, gibberellin A, phalloidin, cytokine A, indanoxine or myoglobin.

[0479] In some embodiments, the ligand connected to the iRNA as described herein serves as a pharmacokinetic regulator (pK regulator). PK regulators include lipophilic substances, bile acid, steroids, phospholipid analogs, polypeptides, protein binders, PEG, vitamins, etc. Exemplary PK regulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkyl glycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, etc. It is also known that the oligonucleotides comprising multiple thiophosphate inter-bonds are combined with albumin, so the short oligonucleotides comprising multiple thiophosphate inter-bonds in the main chain, such as oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases are also suitable for the present invention as a ligand (e.g., PK regulating ligand). In addition, the aptamers in conjunction with serum moieties (e.g., serum proteins) are also suitable for use as PK regulators in embodiments described herein.

[0480] The ligand-conjugated iRNA of the present invention can be synthesized by using oligonucleotides with pendant reactive functional groups, such as those derived from the attachment of linker molecules to the oligonucleotides (described below). Such reactive oligonucleotides can be reacted directly with commercially available ligands, synthetic ligands with various protecting groups, or ligands having linker moieties attached thereto.

[0481] Oligonucleotides for use in the conjugates of the invention can be conveniently and routinely prepared by well-known solid phase synthesis techniques. Equipment for such synthesis is sold by a number of suppliers, including, for example, Applied (Foster City, Calif.). Any other means for such synthesis known in the art may be used in addition or alternatively. It is also known to use similar techniques to prepare other oligonucleotides, such as phosphorothioate and alkylated derivatives.

[0482] In the ligand-conjugated oligonucleotides and nucleosides with sequence-specifically linked ligand molecules of the present invention, the oligonucleotides and oligonucleosides can be assembled on a suitable DNA synthesizer using standard nucleotide or nucleoside precursors, or nucleotides or nucleoside conjugate precursors that already have a linking portion, ligand-nucleotides or ligand-conjugate precursors that already have a ligand molecule, or building blocks with non-nucleoside ligands.

[0483] When using a nucleotide-conjugate precursor that already carries a linking moiety, the synthesis of the sequence-specific nucleoside is completed in the same city, and then the ligand molecule reacts with the linking moiety to form a ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides of the present invention or the linked nucleosides are synthesized by an automated synthesizer using phosphoramidites derived from ligand-nucleoside conjugates and standard phosphoramidites or non-standard phosphoramidites that are commercially available and routinely used for oligonucleotide synthesis.

[0484] A. Lipid Conjugates

[0485] In certain embodiments, the ligand or conjugate is a lipid or lipid-based molecule. Such lipids or lipid-based molecules can generally bind to serum proteins, such as human serum albumin (HSA). HSA binding ligands allow the conjugate to be distributed to target tissues, such as non-renal target tissues of the body. For example, the target tissue can be the liver, including parenchymal cells of the liver. Other molecules that can bind to HSA can also be used as ligands. For example, naproxen or aspirin can be used. Lipids or lipid-based ligands can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport to target cells or cell membranes, or (c) can be used to regulate binding to albumin, such as HSA.

[0486] Lipid-based ligands can be used to modulate, e.g., control (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 will be less likely to be targeted to the kidneys and, therefore, less likely to be cleared from the body. A lipid or lipid-based ligand that binds less strongly to HSA can be used to target the conjugate to the kidneys.

[0487] In certain embodiments, the lipid-based ligand binds to HSA. For example, the ligand may bind to HSA with sufficient affinity so as to enhance the distribution of the conjugate in non-renal tissues. However, the affinity is generally not so strong that it cannot reverse HSA ligand binding.

[0488] In certain embodiments, the lipid-based ligand binds weakly or not at all to HSA, resulting in enhanced distribution of the conjugate in the kidney.Other moieties that target renal cells may also be used to replace or supplement the lipid-based ligand.

[0489] On the other hand, ligands are moieties taken up by targeted cells, such as proliferating cells, such as vitamins. These are particularly useful for treating diseases characterized by undesirable cell proliferation, such as malignant or non-malignant types of diseases such as cancer cells. Exemplary vitamins include vitamins A, E and K. Other exemplary vitamins include B family microorganisms, such as folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients absorbed by the animal. HSA and low-density lipoprotein (LDL) are also included.

[0490] B. Cell Permeabilization Agents

[0491] In another aspect, the ligand is a cell permeating agent, such as a helical cell permeating agent. In certain embodiments, the agent is amphipathic. Exemplary agents are peptides, such as tat or antennopedia. If the agent is a peptide, it can be modified, including peptide mimetics, transformants, non-peptide or pseudopeptide inter-bonds, and the use of D amino acids. Helical agents are typically alpha-helical agents and can have lipophilic and lipophobic phases.

[0492] The ligand can be a peptide or a peptide mimetic. Peptide mimics (also referred to herein as oligopeptide mimics) are molecules that can be folded into a definite three-dimensional structure similar to a natural peptide. The connection of peptides and peptide mimics to iRNA medicaments can affect the pharmacokinetic distribution of iRNA, for example, by enhancing cell recognition and absorption. Peptide or peptide mimics can be about 5-50 amino acids long, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids long.

[0493] The peptide or peptide mimetic can be, for example, a cell permeable peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide (e.g., mainly composed of Tyr, Trp, or Phe). The peptide portion can be a dendritic peptide, a restricted peptide, or a cross-linked peptide. In another alternative, the platform portion can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic peptide comprising MTS is RFGF with an amino acid sequence of AAVALLPAVLLALLAP (SEQ ID NO: 11). An RFGF analog comprising a hydrophobic MTS (e.g., an amino acid sequence of AALLPVLLAAP (SEQ ID NO: 12)) can also be a targeting portion. The peptide portion can be a "delivery" peptide, which can carry large polar molecules including peptides, oligonucleotides, and proteins through the cell membrane. For example, it has been found that sequences from HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 13)) and Drosophila antenna protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 14)) can be used as delivery peptides. The peptide or peptide mimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage display library or a single strain one compound (OBOC) combinatorial library (Lam et al., Nature, 354: 82-84, 1991). Typically, the peptide or peptide mimetic connected to the dsRNA agent by the incorporated monomer unit is a cell targeting peptide, such as an arginine-glycine-aspartic acid (RGD)-peptide or RGD mimetic. The length of the peptide portion can range from about 5 amino acids to about 40 amino acids. The peptide portion can have structural modifications, such as to increase stability or direct conformational properties. Any structural modification described below can be used.

[0494] The RGD peptides used in the compositions and methods of the invention can be linear or cyclic and can be modified, such as glycosylated or methylated, to facilitate targeting to specific tissues. RGD-containing peptides and peptide mimetics can include D-amino acids as well as synthetic RGD mimetics. In addition to RGD, other moieties that target integrin ligands can also be used. Preferred conjugates of the ligand target PECAM-1 or VEGF.

[0495] The RGD peptide portion can be used to target specific cell types, such as cancer cells, such as endothelial tumor cells or breast cancer tumor cells (Zitzmann et al., Cancer Res., 62: 5139-43, 2002). RGD peptides can promote dsRNA agents to target tumors of various other tissues, including lungs, kidneys, spleens or livers (Aoki et al., Cancer Gene Therapy 8: 783-787, 2001). Typically, RGD peptides will promote iRNA agents to target the kidneys. RGD peptides can be linear or cyclic and can be modified, such as glycosylated or methylated, to facilitate targeting specific tissues. For example, glycosylated RGD peptides can deliver iRNA agents to tumor cells expressing αVα3 (Haubner et al., Jour. Nucl. Med., 42: 326-336, 2001).

[0496] "Cell penetrating peptides" can penetrate cells, such as microbial cells, such as bacteria or fungal cells, or mammalian cells, such as human cells. Microbial cell penetrating peptides can be, for example, α-helical linear peptides (e.g., IL-37 or CeropinP1), disulfide bond-containing peptides (e.g., α-defensins, β-defensins or bacteriocins), or peptides containing only one or two major amino acids (e.g., PR-39 or indolicidin). Cell penetrating peptides can also include a nuclear localization signal (NLS). For example, a cell penetrating peptide can be a dichotomous amphipathic peptide, such as MPG, which is derived from the fusion platform domain of HIV-1gp41 and the NLS of the large T antigen of SV40 (Simeoni et al., Nucl.Acids Res.31:2717-2724, 2003).

[0497] C. Carbohydrate Conjugates

[0498] In some embodiments of the compositions and methods of the present invention, the iRNA also comprises a carbohydrate. Carbohydrate-conjugated iRNA is conducive to the in vivo delivery of nucleic acids, and compositions suitable for in vivo therapeutic uses, as described herein. As used herein, "carbohydrate" refers to a compound that is a carbohydrate itself, which is composed of one or more monosaccharide units with at least 6 carbon atoms (which can be straight, branched or cyclic), and each carbon atom has an oxygen, nitrogen or sulfur atom; or a compound whose carbohydrate portion is composed of one or more monosaccharide units, each monosaccharide unit has at least six carbon atoms (which can be straight, branched or cyclic), and each carbon atom has oxygen, nitrogen or sulfur atoms. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides or oligosaccharides containing about 4, 5, 6, 7, 8 or 9 monosaccharide units) and polysaccharides, such as starch, glycogen, cellulose and polysaccharide gum. Specific monosaccharides include C5 and above (e.g., C5, C6, C7 or C8) sugars; disaccharides and trisaccharides include having two or three monosaccharide units (e.g., C5, C6, C7 or C8).

[0499] In certain embodiments, the carbohydrate conjugate comprises a monosaccharide.

[0500] In certain embodiments, the monosaccharide is N-acetylgalactosamine (GalNAc). GalNAc conjugates comprising one or more N-acetylgalactosamine (GalNAc) derivatives are described in, for example, US 8,106,022, the entire contents of which are incorporated herein by reference. In some embodiments, the GalNAc conjugate is used as a ligand for targeting iRNA to specific cells. In some embodiments, the GalNAc conjugate targets iRNA to stem cells, for example, by acting as a ligand for the asialoglycoprotein receptor of a stem cell (e.g., a stem cell).

[0501] In some embodiments, the carbohydrate conjugate comprises one or more GalNAc derivatives. The GalNAc derivatives can be connected by a linker, such as a divalent 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 an iRNA agent (e.g., to the 3' end of the sense strand) through 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 an iRNA agent (e.g., to the 5' end of the sense strand) through a linker (e.g., a linker as described herein).

[0502] In certain embodiments of the invention, GalNAc or a GalNAc derivative is connected to the iRNA agent of the invention via a monovalent linker. In some embodiments, GalNAc or a GalNAc derivative is connected to the iRNA agent of the invention via a divalent linker. In other embodiments of the invention, GalNAc or a GalNAc derivative is connected to the iRNA agent of the invention via a trivalent linker. In other embodiments of the invention, GalNAc or a GalNAc derivative is connected to the iRNA agent of the invention via a tetravalent linker.

[0503] In certain embodiments, the double-stranded RNAi agent of the present invention comprises a GalNAc or GalNAc derivative connected to the iRNA agent. In certain embodiments, the double-stranded RNAi agent of the present invention comprises a plurality of (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each of which is independently connected to a plurality of nucleotides of the double-stranded RNAi agent via a plurality of monovalent linkers.

[0504] In some embodiments, for example, when the two chains of the iRNA agent of the present invention are part of a larger molecule, the 3' end of one chain and the 5' end of the corresponding other chain are connected by an uninterrupted nucleotide chain to form a hairpin loop, which contains a plurality of unpaired nucleotides, and each unpaired nucleotide in the hairpin loop can independently contain GalNAc or GalNAc derivatives connected by a monovalent linker. The hairpin loop can also be formed by an extended overhang in one chain of the double strand.

[0505] In some embodiments, for example, when the two chains of the iRNA agent of the present invention are part of a larger molecule, the 3' end of one chain and the 5' end of the corresponding other chain are connected by an uninterrupted nucleotide chain to form a hairpin loop, which contains a plurality of unpaired nucleotides, and each unpaired nucleotide in the hairpin loop can independently contain GalNAc or GalNAc derivatives connected by a monovalent linker. The hairpin loop can also be formed by an extended overhang in one chain of the double strand.

[0506] In some embodiments, the GalNAc conjugate is

[0507]

[0508] In some embodiments, the RNAi agent is linked to the carbohydrate conjugate via a linker as shown in the following schematic diagram, wherein X is O or S

[0509]

[0510] In some embodiments, the RNAi agent is conjugated to L96 as defined in Table 1 and shown below:

[0511]

[0512] In certain embodiments, the carbohydrate conjugates used in the compositions and methods of the invention are selected from the group consisting of:

[0513]

[0514]

[0515]

[0516]

[0517]

[0518]

[0519]

[0520]

[0521] In certain embodiments, the carbohydrate conjugate used in the compositions and methods of the invention is a monosaccharide. In certain embodiments, the monosaccharide is N-acetylgalactosamine, such as

[0522]

[0523] Another exemplary carbohydrate conjugate for use in the embodiments described herein includes, but is not limited to,

[0524]

[0525] (Formula XXXVI),

[0526] When one of X or Y is an oligonucleotide, the other is hydrogen.

[0527] In some embodiments, suitable ligands are ligands disclosed in WO 2019 / 055633, the entire contents of which are incorporated herein by reference. In one embodiment, the ligand comprises the following structure:

[0528]

[0529] In certain embodiments, the RNAi agents of the present disclosure may comprise a GalNAc ligand, even though such GalNAc ligands are presently expected to be of limited value for the preferred intrathecal / CNS delivery routes of the present disclosure.

[0530] In certain embodiments of the invention, GalNAc or a GalNAc derivative is connected to the iRNA agent of the invention via a monovalent linker. In some embodiments, GalNAc or a GalNAc derivative is connected to the iRNA agent of the invention via a divalent linker. In other embodiments of the invention, GalNAc or a GalNAc derivative is connected to the iRNA agent of the invention via a trivalent linker.

[0531] In one embodiment, the double-stranded RNAi agent of the present invention comprises one or more GalNAc or GalNAc derivatives connected to the iRNA agent. GalNAc can be connected to any nucleotide via a linker on the sense strand or antisense strand. GalNAc can be connected 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 connected to the 3' end of the sense strand, for example, via a trivalent linker.

[0532] In other embodiments, the double-stranded RNAi agent of the present invention comprises a plurality (eg, 2, 3, 4, 5 or 6) of GalNAc or GalNAc derivatives, each of which is independently linked to a plurality of nucleotides of the double-stranded RNAi agent via a plurality of monovalent linkers.

[0533] In some embodiments, for example, when the two chains of the iRNA agent of the present invention are part of a larger molecule, the 3' end of one chain and the 5' end of the corresponding other chain are connected by an uninterrupted nucleotide chain to form a hairpin loop, which contains a plurality of unpaired nucleotides, and each unpaired nucleotide in the hairpin loop can independently contain GalNAc or a GalNAc derivative connected by a monovalent linker.

[0534] In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, a PK modifier or a cell penetrating peptide.

[0535] Other carbohydrate conjugates and linkers suitable for use in the present invention include those described in WO 2014 / 179620 and WO 2014 / 179627, the entire contents of each of which are incorporated herein by reference.

[0536] D.Connector

[0537] In some embodiments, the conjugates or ligands described herein can be linked to iRNA oligonucleotides with various cleavable or non-cleavable linkers.

[0538] The term "linker" or "linking group" refers to an organic moiety that connects two parts of a compound, such as covalently connecting two parts of a compound. The linker typically comprises a direct bond or an atom such as oxygen, sulfur, a unit such as NR8, C(O), C(O)NH, SO, SO2, SO2NH or a chain of atoms such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkyl, alkylarylalkynyl, alkenylarylalkyl, alkenylaryl, alkenylarylalkynyl, alkynylalkyl, alkynylalkenyl, alkynylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkyl, alkylheteroarylalkyl, alkylheteroaryl wherein the one or more methylene radicals are alkyl, alkyl, alkylheteroaryl, alkylheteroarylalkyl, alkylheteroarylalkyl, alkylheteroarylalkynyl, alkylheterocycloalkyl, alkylheterocycloalkyl, alkylheterocycloalkyl, alkylheterocycloalkyl, alkylheterocycloalkyl, alkynylheterocycloalkyl, alkynylheterocycloalkenyl, alkynylheterocycloalkynyl, alkylaryl, alkenylaryl, alkynyl, alkylheteroaryl, alkenylheteroaryl, alkynyl, wherein the one or more methylene radicals may be interrupted or terminated by O, S, S(O), SO2, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle; wherein R8 is hydrogen, acyl, aliphatic or substituted aliphatic. In certain embodiments, the linker is between 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.

[0539] A cleavable linking group is a linking group that is sufficiently stable outside the cell, but it is cleaved upon entry into the target cell to release the two parts of the linker that are held together. In a preferred embodiment, the cleavable linking group is cleaved at least about 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or more, or at least about 100 times, in the target cell or under a first reference condition (e.g., one that can be selected to simulate or represent intracellular conditions) than in the subject's blood or under a second reference condition (e.g., one that can be selected to simulate or represent conditions in blood or serum) faster.

[0540] The cleavable linking group is susceptible to the influence of cleavage agents, such as pH, redox potential or the presence of degradation molecules. Typically, cleavage agents are more prevalent or present at higher levels or activities in cells than in serum or blood. Examples of such degradation agents include: redox agents selected for specific substrates or without substrate specificity, including, for example, oxidative or reductases or reductants such as thiols present in cells, which can degrade redox cleavable linking groups by reduction; esterases; endosomes or agents that can produce an acidic environment, such as those that result in a pH of 5 or less; enzymes, peptidases (which can be substrate specific) and phosphatases that can hydrolyze or degrade acid cleavable linking groups by acting as general acids.

[0541] Cleavable linking groups, such as disulfide bonds, may be sensitive to pH. The pH of human serum is 7.4, while the average pH inside cells is slightly lower, ranging from about 7.1-7.3. Endosomes have a more acidic pH, ranging from 5.5-6.0, and lysosomes have an even more acidic pH of about 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, or into a desired cellular compartment.

[0542] The linker can include a cleavable linker group that can be cleaved by a specific enzyme. The type of cleavable linker group incorporated into the linker may depend on the cell to be targeted. For example, a liver targeting ligand can be connected to a cationic lipid by a linker that includes an ester group. Hepatocytes are rich in esterases, and therefore the linker in stem cells will be more effectively cleaved than cell types that are not rich in esterases. Other cell types rich in esterases include lung cells, renal cortical cells, and testicular cells.

[0543] When targeting cell types rich in peptidases (e.g., stem cells and chemokines), linkers containing peptide bonds can be used.

[0544] Generally, the suitability of a candidate cleavable linker group can be evaluated by testing the ability of a degradation agent (or condition) to cut a candidate linking group. It is also necessary to test the ability of a candidate cleavable linker group to resist cutting in blood or when in contact with other non-target tissues. Therefore, the relative susceptibility to cutting between the first or second conditions can be determined, wherein the first is selected to indicate the cutting in the target cell, and the second is selected to indicate the cutting in other tissues or fluids, such as blood or serum. The assessment can be carried out in a cell-free system, in a cell, in a cell culture, in an organ or tissue culture, or in a whole animal. It may be useful to conduct a preliminary assessment under cell-free or culture conditions and confirm by further assessment of the whole animal. In a preferred embodiment, the cleavage rate of a useful candidate compound in a cell (or under in vitro conditions of simulating extracellular conditions) is at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90 or about 100 times faster than in blood or serum (or under in vitro conditions of simulating intracellular conditions).

[0545] i. Redox-cleavable linker

[0546] In certain embodiments, the cleavable linking group is a redox cleavable linking group that is cleaved when reduced or oxidized. The example of a linking group that can be reductively cleaved is a disulfide bond linking group (-SS-). In order to determine whether a candidate cleavable linking group is a suitable "reducible cleavable linking group" or, for example, whether it is suitable for use with a specific iRNA portion and a specific targeting agent, reference can be made to the methods described herein. For example, candidates can be evaluated by incubating with dithiothreitol (DTT) or other reducing agents, using agents known in the art to simulate the cleavage rate observed in cells such as target cells. Candidates can also be evaluated under the conditions of selecting simulated blood or serum. One method is that the candidate compound is cut at most by about 10% in blood. In other embodiments, the degradation of useful candidate compounds in cells (or under in vitro conditions simulating intracellular conditions) is at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90 or about 100 times faster than in blood or serum (or under in vitro conditions simulating extracellular conditions). The rate of cleavage of a candidate compound can be determined using standard enzyme kinetic assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media.

[0547] ii. Phosphate-based cleavable linking groups

[0548] In certain embodiments, the cleavable linker comprises a phosphate-based cleavable linker group. The phosphate-based cleavable linker group is cleaved by an agent that degrades or hydrolyzes the phosphate group. Examples of agents that cleave phosphate groups in cells are enzymes, such as phosphatases in cells. Examples of phosphate-based linkers 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)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, -OP(S)(Rk)-S-. Preferred embodiments are -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O, -SP(S)(H)-O-, -SP(O)(H)-S-, -OP(S)(H)-S-. Preferred embodiments are -OP(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

[0549] iii. Acid cleavable linking group

[0550] 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 in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0 or lower), or is cleaved by agents such as enzymes that can act as general acids. In cells, specific low pH organelles, such as endosomes and lysosomes, can provide a cleavage environment for acid cleavable linking groups. Examples of acid cleavable linking groups include, but are not limited to, hydrazones, esters, and amino acid esters. The acid cleavable group can have the general formula -C=NN-, C(O)O, or -OC(O). A preferred embodiment is when the carbon connected to the oxygen (alkoxy) of the ester is an aryl, substituted alkyl, or tertiary alkyl group such as dimethylpentyl or tert-butyl. These candidates can be evaluated using methods similar to those described above.

[0551] iv. Ester-based cleavable linking groups

[0552] In certain embodiments, the cleavable linker comprises an ester-based cleavable linking group. The ester-based cleavable linking group is cleaved by enzymes in the cell such as esterases and amidases. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene. The ester-cleavable linking group has the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0553] v. Peptide-based cleavable linking groups

[0554] In another embodiment, the cleavable linker comprises a peptide-based cleavable linking group. The peptide-based cleavable linking group is cleaved by enzymes in the cell, such as peptidases and proteases. The peptide-based cleavable linking group is a peptide bond formed between amino acids to produce oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. The peptide-based cleavable group does not include an amide group (-C(O)NH-). The amide group can be formed between any alkylene, alkenylene or alkynylene. The peptide bond is a special type of amide bond formed between amino acids that can produce peptides and proteins. The peptide-based cleavable group is generally limited to the peptide bond (i.e., amide bond) formed between the amino acids that produce peptides and proteins, and does not include the entire amide functional group. The peptide-based cleavable group has the general formula -NHCHRAC(O)NHCHRBC(O)-, where RA and RB are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to the above method.

[0555] In some embodiments, the iRNA of the 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 invention include, but are not limited to

[0556]

[0557]

[0558]

[0559] (Formula XLIV), when one of X or Y is an oligonucleotide, the other is hydrogen.

[0560] In certain embodiments of the compositions and methods of the invention, the ligand is one or more "GalNAc" (N-acetylgalactosamine) derivatives, linked via a divalent or trivalent branched linker.

[0561] In certain embodiments, the dsRNA of the invention is conjugated to a divalent or trivalent branched linker selected from the group represented by any of the structures of Formula (XLV) or Formula (XLVI).

[0562]

[0563]

[0564] in:

[0565] q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C each occurrence independently represent 0-20 and wherein the repeating units may be the same or different;

[0566] 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 Each occurrence is independently CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH, or CH2O;

[0567] Q 2A , Q 2B , Q 3A , Q 3B , Q 4A , Q 4B , Q 5A , Q 5B , Q 5C In each occurrence, independently absent, alkylene, substituted alkylene, wherein one or more methylene groups may be replaced by O, S, S(O), SO2, N(R N ), C(R')=C(R"),C≡C or C(O);

[0568] R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , R 5C Each occurrence is independently 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, or a heterocyclic group;

[0569] L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B and L 5C represents a ligand; that is, each occurrence is independently a monosaccharide (eg, GalNAc), a disaccharide, a trisaccharide, a tetrasaccharide, an oligosaccharide, or a polysaccharide; and R a Is H or an amino acid side chain. Trivalent conjugated GalNAc derivatives are particularly suitable for use with RNAi agents to inhibit the expression of target genes, such as those of formula (XLIX):

[0570]

[0571] Where L 5A , L 5B and L 5C represents a monosaccharide, such as a GalNAc derivative.

[0572] Examples of suitable divalent and trivalent branched linker groups for conjugating GalNAc derivatives include, but are not limited to, the structures described above as Formulae II, VII, XI, X, and XIII.

[0573] Exemplary U.S. 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; 5,414,077; 5,486,6 03; 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; 5, 112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241; 5,391,723; 5,416,203; 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567, 7,037,646 and 8,106,022, the entire contents of each of which are hereby incorporated by reference.

[0574] It is not necessary that all positions in a given compound be uniformly modified, and in fact more than one of the above modifications may be incorporated into a single compound, or even into a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds.

[0575] In the context of the present invention, a "chimeric" iRNA compound or "chimera" is an iRNA compound, preferably a dsRNA agent, comprising two or more regions of different chemical formulae, each consisting of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs typically comprise at least one region in which the RNA is modified to confer upon the iRNA increased resistance to nuclease degradation, increased cellular uptake, or increased binding affinity to a target nucleotide. Another region of the iRNA may serve as a substrate for an enzyme capable of cleaving RNA:DNA or RNA:RNA hybrids. For example, RNase H is an endonucleolytic cleavage that cleaves the RNA strand of an RNA:DNA duplex. Thus, activation of RNase H results in cleavage of the RNA target, thereby greatly increasing the efficiency of the iRNA in inhibiting gene expression. Thus, when using chimeric dsRNAs, comparable results can generally be obtained using shorter iRNAs than with thiophosphate deoxy dsRNAs hybridized to the same target region. The cleavage of the RNA target may be routinely detected by gel electrophoresis and, if desired, by related nucleic acid hybridization techniques known in the art.

[0576] In some cases, the RNA of an iRNA can be modified with non-ligand groups. Many non-ligand molecules have been conjugated to iRNAs to enhance the activity, cellular distribution or cellular uptake of the iRNAs, and procedures for performing such conjugations 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), bile acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), thioethers such as hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), fatty chains, such as dodecandiol or undecyl residues (Aison-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 hexadecyl-racemic glycerol or, 2-di-O-hexadecyl-racemic-glycero-3-H-phosphonic acid triethylammonium (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18: 3777), polyamine or polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14: 969) or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36: 3651), palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264: 229), or octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277: 923). Representative U.S. patents that teach the preparation of such RNA conjugates are listed above. A typical conjugation scheme involves synthesizing RNA with an amino linker at one or more positions in the sequence. The amino group is then reacted with the molecule to be conjugated using a suitable coupling agent or activator.The conjugation reaction can be performed while the RNA is still bound to a solid support or in solution phase after RNA cleavage. Purification of the RNA conjugate by HPLC will generally yield a pure conjugate.

[0577] V. Delivery of RNAi Agents of the Disclosure

[0578] Delivery of RNAi agents of the present invention to cells, such as cells in a subject, such as a human subject (e.g., a subject in need, such as a subject suffering from an HTT-related disease, such as Huntington's disease), can be achieved in a variety of different ways. For example, delivery can be performed by contacting cells with RNAi agents of the present invention in vitro or in vivo. In vivo delivery can also be performed directly by administering a composition comprising an RNAi agent, such as dsRNA, to the subject. Alternatively, in vivo delivery can be performed indirectly by administering one or more vectors encoding and directing the expression of the RNAi agent. These alternatives will be discussed further below.

[0579] In general, any method of delivering nucleic acid molecules (in vivo or in vitro) may be suitable for use with the RNAi agents of the present disclosure (see, e.g., Akhtar S. and Julian RL., (1992) Trends Cell. Biol. 2(5): 139-144 and WO94 / 02595, the entire contents of which are incorporated herein by reference). For in vivo delivery, factors to be considered in delivering RNAi agents include, for example, the biological stability of the delivered agent, the prevention of nonspecific effects, and the accumulation of the delivered agent in the target tissue. The nonspecific effects of RNAi agents can be minimized by local administration, such as by direct injection or implantation of tissues or local administration of formulations. Local administration to the treatment site maximizes the local concentration of the agent, limits exposure of the agent to systemic tissues that would otherwise be damaged by the agent or degradable agent, and allows the administration of a lower total dose of the RNAi agent. Several studies have shown successful knockdown of gene products when RNAi agents are administered locally. For example, in experimental models of age-related macular degeneration, intravitreal delivery of VEGF dsRNA in cynomolgus monkeys (Tolentino, MJ. et al., (2004) Retina 24: 132-138) and mice (Reich, SJ. et al., (2003) Mol. Vis. 9: 210-216) was shown to prevent angiogenesis. In addition, direct intratumoral injection of dsRNA in mice reduced tumor volume (Pille, J. et al., (2005) Mol. Ther. 11: 267-274) and prolonged 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 has also been shown to be successfully delivered locally 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 by intranasal administration to the lungs (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). In order to administer RNAi agents systemically to treat disease, RNA can be modified or delivered using a drug delivery system; both methods can prevent rapid degradation of dsRNA by endonucleases and exonucleases in vivo. Modification of RNA or drug carriers can also allow RNAi agents to be targeted to target tissues and avoid undesirable off-target effects (e.g., without wishing to be bound by theory, the use of GNA described herein has been identified as destabilizing the seed region of dsRNA, thereby resulting in an enhanced preference for targeting effectiveness of such dsRNA relative to off-target effects, because the instability of such seed region significantly weakens such off-target effects). RNAi agents can be modified by chemical conjugation with lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation. For example, RNAi agents for ApoB conjugated to lipophilic cholesterol moieties were systemically injected into mice, resulting in apoB mRNA being knocked down in the liver and jejunum (Soutschek, J. et al., (2004) Nature 432: 173-178). In a mouse model of prostate cancer, RNAi agents conjugated to aptamers have been shown to inhibit tumor growth and mediate tumor regression (McNamara, JO. et al., (2006) Nat. Biotechnol. 24: 1005-1015). In an alternative embodiment, 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 interactions on negatively charged cell membranes to allow cells to effectively take up RNAi agents.Cationic lipids, dendrimers or polymers can bind to RNAi agents or induce the formation of vesicles or micelles that encase RNAi agents (see, e.g., Kim SH. et al., (2008) Journal of Controlled Release 129(2):107-116). et al., (2008) Journal of Controlled Release 129(2):107-116) that encases an RNAiagent. When administered systemically, the formation of vesicles or micelles further prevents the degradation of the RNAi agent. Methods of preparing and administering cationic-RNAi agent complexes are within the capabilities of those skilled in the art (see, e.g., 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, which are incorporated herein by reference in their entireties). Some non-limiting examples of drug delivery systems that can be used 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), polyethyleneimine (Bonnet ME. et al., (2008) Pharm. Res. Aug 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm. 3: 472-487), and polyamidoamines (Tomalia, DA. et al., (2007) Biochem. Soc. Trans. 35: 61-67; Yoo, H. et al., (1999) Pharm. Res. 16: 1799-1804). In some embodiments, RNAi agents are complexed with cyclodextrins for systemic administration. Pharmaceutical compositions and methods of administration of RNAi agents and cyclodextrins can be found in U.S. Pat. No. 7,427,605, which is incorporated herein by reference in its entirety.

[0580] Certain aspects of the present disclosure relate to methods of reducing HTT target gene expression in a cell, 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.

[0581] Another aspect of the present disclosure relates to a method of reducing HTT target gene expression in a subject, comprising administering a double-stranded RNAi agent of the present disclosure to the subject.

[0582] Another aspect of the present disclosure relates to a method of treating a subject suffering from 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.

[0583] In one embodiment, the double-stranded RNAi agent is administered intrathecally. By administering the double-stranded RNAi agent intrathecally, the method can reduce the expression of HTT target genes in the brain (eg, striatum) or spinal tissues such as the cortex, cerebellum, cervical spine, lumbar spine, and thoracic spine.

[0584] For ease of explanation, the preparations, compositions and methods in this section are mainly discussed for modified siRNA compounds. However, it is understood that these preparations, compositions and methods can be practiced with other siRNA compounds, such as unmodified siRNA compounds, and this practice is within the present disclosure. The composition comprising the RNAi medicament can be delivered to the subject by a variety of approaches. Exemplary approaches include: intrathecal, intravenous, local, rectal, anal, vaginal, nasal, lung and eye.

[0585] The RNAi agents disclosed herein can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically include one or more RNAi agents and a pharmaceutically acceptable carrier. As used herein, the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are compatible with drug administration. It is well known in the art that such media and agents are used for pharmaceutically active substances. Unless any conventional media or agents are incompatible with the active compound, their use in the composition is considered. Supplementary active compounds can also be incorporated into the composition.

[0586] Pharmaceutical compositions of the present disclosure can be administered in a variety of ways, depending on whether local or systemic treatment is needed and on the area to be treated. Administration can be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral or parenteral. Parenteral administration includes intravenous infusion, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or intraventricular administration.

[0587] The route and site of administration can be selected to enhance targeting. For example, to target muscle cells, intramuscular injection into the muscle of interest would be a logical choice. Lung cells can be targeted by using an RNAi agent in aerosol form. Vascular endothelial cells can be targeted by coating a balloon catheter with an RNAi agent and mechanically introducing the RNA.

[0588] Preparations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids or powders. Conventional pharmaceutical carriers, liquids, powders or oily bases, thickeners, etc. may be necessary or desirable. Coated condoms, gloves, etc. may also be useful.

[0589] Compositions for oral administration include powders or granules, suspensions or aqueous solutions, syrups, elixirs or non-aqueous media, tablets, capsules, lozenges or pastilles. In the case of tablets, carriers that can be used include lactose, sodium citrate and phosphate esters. Commonly used disintegrants in tablets include various disintegrants such as starch, lubricants such as magnesium stearate, sodium lauryl sulfate, talcum powder. For oral administration in capsule form, useful diluents are lactose and high molecular weight polyethylene glycol. When oral use requires an aqueous suspension, the nucleic acid composition can be combined with an emulsifier and a suspending agent. If necessary, certain sweeteners or flavorings can be added.

[0590] Compositions for intrathecal or intraventricular administration may include sterile aqueous solutions, which may also contain buffers, diluents or other suitable additives.

[0591] Preparations for parenteral administration may include sterile aqueous solutions, which may also include buffers, diluents or other suitable additives. Intraventricular injection may be facilitated by intraventricular catheters, such as connected to a reservoir. For intravenous use, the total concentration of solutes may be controlled to make the preparation isotonic.

[0592] In one embodiment, the administration of the siRNA compound (e.g., double-stranded siRNA compound or ssiRNA compound) composition is parenteral, such as intravenous (e.g., bolus or as a diffusible infusion), intradermal intraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, subcutaneous, transmucosal, oral, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary, intranasal, urethral or ocular. Administration can be provided by the subject or other persons (e.g., healthcare providers). The drug can be provided in a metered dose or in a dispenser that distributes a metered dose. The selected delivery mode will be discussed in more detail below.

[0593] A. Intrathecal administration

[0594] In one embodiment, double-stranded RNAi agents are delivered by intrathecal injection (i.e., injection into the cerebrospinal fluid that submerges the brain and spinal cord tissue). Intrathecal injection of RNAi agents into the cerebrospinal fluid can be performed as a rapid bolus or by a subcutaneous micropump, thereby regularly and continuously delivering siRNA to the cerebrospinal fluid. The cerebrospinal fluid circulates around the spinal cord and dorsal root ganglia from the choroid plexus that produces it, and then passes upward through the cerebellum and cortex to the arachnoid granules, where the liquid can leave the central nervous system, that is, depending on the size, stability, and solubility of the injected compound, the molecules delivered intrathecally can hit targets throughout the CNS.

[0595] In some embodiments, intrathecal administration is performed by a pump. The pump can be an osmotic pump that is surgically implanted. In one embodiment, the osmotic pump is implanted in the subarachnoid space of the spinal canal to facilitate intrathecal administration.

[0596] In some embodiments, intrathecal administration is by an intrathecal delivery system for a drug, the system comprising a reservoir containing a volume of medicament, and a pump configured to deliver a portion of the medicament contained in the reservoir. More details about this intrathecal delivery system can be found in WO 2015 / 116658, which is incorporated by reference in its entirety.

[0597] The amount of RNAi agent injected intrathecally may vary from one target gene to another, and the appropriate amount that must be applied may have to be determined individually for each target gene. Typically, the amount ranges from 10 μg to 2 mg, preferably 50 μg to 1500 μg, and more preferably 100 μg to 1000 μg.

[0598] B. Vector-Encoded RNAi Agents of the Disclosure

[0599] RNAi agents targeting the HTT gene can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, d et al., TIG. (1996), 12: 5-10; WO 00 / 22113, WO 00 / 22114 and US 6,054,299). Expression is preferably sustained (several months or longer) depending on the specific construct and target tissue or cell type used. These transgenes can be introduced as linear constructs, circular plasmids or viral vectors, which can be integrating or non-integrating vectors. Transgenes can also be constructed so that they are inherited as extrachromosomal plasmids (Gassmann, et al., (1995) Proc. Natl. Acad. Sci. USA 92: 1292).

[0600] A single or multiple chains of RNAi medicaments can be transcribed from a promoter on an expression vector. In the case where two separate chains are to be expressed to produce, for example, dsRNA, two separate expression vectors can be introduced (e.g., by transfection or infection) into a target cell. Alternatively, each separate chain of dsRNA can be transcribed by a promoter located on a corresponding expression plasmid. In one embodiment, dsRNA is expressed as an inverted repeat polynucleotide connected by a joint polynucleotide sequence so that the dsRNA has a stem and loop structure.

[0601] RNAi agent expression vectors are typically DNA plasmids or viral vectors. Expression vectors compatible with eukaryotic cells, preferably those compatible with vertebrate cells, can be used to produce recombinant constructs for expressing RNAi agents as described herein. Delivery of RNAi agent expression vectors can be systemic, for example, by intravenous or intramuscular administration, by administration to target cells explanted from a patient and then reintroduced into the patient, or by any other means that allows introduction into the desired target cells.

[0602] Viral vector systems that can be used for the methods and compositions described herein include, but are not limited to, (a) adenoviral vectors; (b) retroviral vectors, including, but not limited to, lentiviral vectors, moloney mouse leukemia virus, and the like; (c) adeno-associated viral vectors; (d) herpes simplex virus vectors; (e) SV 40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) poxvirus vectors, such as orthopoxviruses, such as vaccinia virus vectors or avian poxviruses such as canary pox or fowl pox; (j) helper-dependent or gutless adenoviruses. Replication-deficient viruses may also be advantageous. Different vectors will or will not be incorporated into the cell genome. If desired, the construct may include viral sequences for transfection. Alternatively, the construct may be incorporated into a vector capable of repeated replication, such as EPV and EBV vectors. Constructs for recombinant expression of RNAi agents typically require regulatory elements, such as promoters, enhancers, and the like, to ensure expression of the RNAi agent in the target cell. Other aspects for considering vectors and constructs are known in the art.

[0603] VI. Pharmaceutical Compositions of the Invention

[0604] The present disclosure also includes pharmaceutical compositions and formulations comprising the RNAi agents of the present disclosure. In one embodiment, provided herein are pharmaceutical compositions comprising RNAi agents, as described herein, and a pharmaceutically acceptable carrier. Pharmaceutical compositions comprising RNAi agents can be used to treat diseases or conditions associated with the expression or activity of HTT, such as Huntington's disease.

[0605] In some embodiments, the pharmaceutical compositions of the invention are sterile. In another embodiment, the pharmaceutical compositions of the invention are pyrogenic or non-pyrogenic.

[0606] Such pharmaceutical compositions are formulated based on the mode of delivery. One example is a composition prepared for systemic delivery by parenteral delivery, such as intravenous (IV), intramuscular (IM) or subcutaneous (subQ) delivery. Another example is a composition for direct delivery to the CNS, such as by intrathecal or intravitreal injection routes, optionally infused into the brain (e.g., striatum), such as by continuous pump infusion.

[0607] The pharmaceutical compositions of the present disclosure can be administered at a dose sufficient to inhibit HTT gene expression. Typically, a suitable dose of the RNAi agent of the present disclosure will be about 0.001 to about 200.0 mg per kilogram of body weight per day, typically within the range of about 1 to 50 mg per kilogram of body weight per day.

[0608] Repeated dosage regimens can include regular administration of therapeutic amounts of RNAi agents, such as once a month to once every six months. In certain embodiments, RNAi agents are administered once a quarter (ie, about once every three months) to about twice a year.

[0609] Following an initial treatment regimen (eg, a loading dose), treatment may be administered on a less frequent basis.

[0610] In other embodiments, a single dose of the pharmaceutical composition can be long-lasting, so that subsequent doses are administered at intervals of no more than 1, 2, 3, or 4 or more months. In some embodiments of the present disclosure, the administration of the pharmaceutical composition of the present disclosure is administered once a month. In other embodiments of the present disclosure, a single dose of the pharmaceutical composition of the present disclosure is administered once a quarter or twice a year.

[0611] The skilled artisan will appreciate that certain factors may influence the dosage and application required to effectively treat a subject, including but not limited to the severity of the disease or condition, previous treatment, the general health or age of the subject, and other diseases present. In addition, treatment of a subject with a therapeutically effective amount of the composition may include a single treatment or a series of treatments.

[0612] Advances in mouse genetics have generated a number of mouse models for the study of various human diseases, such as HD, which benefit from reduced HTT expression. These models can be used for in vivo testing of RNAi agents, as well as determining therapeutically effective amounts. 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).

[0613] The pharmaceutical compositions of the present disclosure can be applied in a variety of ways as needed for local or systemic treatment and on the area to be treated. Administration can be topical (e.g., by transdermal patch), pulmonary, such as by inhalation or insufflation of powder or aerosol, including a nebulizer; intratumoral, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; by implant device, such as by implant device; or intracranial, such as intracranial, intraorgan or intraventricular, administration.

[0614] RNAi agents can be delivered in a manner that targets specific tissues, such as the CNS (eg, neurons of the brain, neurons, or vascular tissue).

[0615] Pharmaceutical compositions and preparations for topical administration can include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. It can be necessary or desired conventional drug carriers, aqueous, powdered or oily bases, thickeners, etc. Coated condoms, gloves, etc. can also be useful. Suitable topical preparations include those local delivery agents of the RNAi medicaments in the present disclosure such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants mixed. Suitable lipids and liposomes include neutral (e.g., dioleyl phosphatidyl DOPE ethanolamine, dipyrimidine sulfonyl phosphatidylcholine DMPC, distearoyl phosphatidylcholine), negative (e.g., dimethyl styryl phosphatidylglycerol DMPG) and cations (dioleyl tetramethylaminopropyl DOTAP and dihexyl phosphatidylethanolamine DOTMA). The RNAi medicaments in the present disclosure can be encapsulated in liposomes or can form complexes with them, particularly cationic liposomes. Alternatively, the RNAi medicaments can be compounded to lipids, particularly cationic lipids. Suitable fatty acids and esters include, but are not limited to, arachidic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, triphosphate, monooleate, dilaurin, 1-monoformylglycerol, 1-dodecylazacycloheptane-2-one, acylguanine, acylcholine or C1-20 alkyl esters (e.g., isopropyl myristate IPM), monoglycerides, aminoglycerides or pharmaceutically acceptable salts thereof. Topical preparations are described in detail in US 6,747,014, which is incorporated herein by reference.

[0616] A. RNAi Agent Formulations Containing Membranous Molecular Components

[0617] RNAi agents for compositions and methods of the present disclosure can be formulated for delivery in a membranous assembly, such as a liposome or micelle. As used herein, the term "liposome" refers to a vesicle composed of at least one bilayer, such as a bilayer or multiple bilayers, of amphiphilic lipids arranged in at least one bilayer or multiple bilayers. Liposomes include single-layer and multi-layer vesicles having a membrane formed by 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 generally does not contain the RNAi agent composition, although in some examples, it can. Liposomes can be used to transfer or deliver active ingredients to the site of action. Because the liposome membrane is structurally similar to a biological membrane, when the liposome is applied to a tissue, the liposome bilayer fuses with the bilayer of the cell membrane. As the merging of liposomes and cells progresses, the internal water content containing the RNAi agent is delivered to the cell, wherein the RNAi agent can specifically bind to the target RNA and can mediate RNAi. In some cases, liposomes are also particularly targeted, for example, to direct the RNAi agent to a specific cell type.

[0618] Liposomes containing RNAi agents can be prepared by various methods. In one example, the lipid component of the liposome is dissolved in a detergent so that micelles are formed with the lipid component. For example, the lipid component can be an amphipathic cationic lipid or a lipid conjugate. The detergent can have a high critical micelle concentration and can be non-ionic. Exemplary detergents include cholic acid lipids, CHAPS, octyl glucoside, deoxycholic acid lipids and lauroyl sarcosine. The RNAi agent preparation is then added to the micelle containing the liposome component. The cationic groups on the lipid interact with the RNAi agent and condense around the RNAi agent to form liposomes. After condensation, the detergent is removed, for example, by dialysis to produce a liposome preparation of the RNAi agent.

[0619] If desired, a carrier compound that aids condensation can be added during the condensation reaction, for example by controlled addition. For example, the carrier compound can be a polymer other than nucleic acid (e.g., spermine or spermidine). The pH can also be adjusted to promote condensation.

[0620] Methods for producing stable polynucleotide delivery vectors, which contain polynucleotide / cationic lipid complexes as structural components of the delivery vector, are described, for example, in WO 96 / 37194, the entire contents of which are incorporated herein by reference. Liposome formation can also include the methods described in Felgner, PL et al., (1987) Proc. Natl. Acad. Sci. USA 8:7413-7417; U.S. Pat. No. 4,897,355; U.S. Pat. No. 5,171,678; Bangham et al., (1965) M. Mol. Biol. 23:238; Olson et al., (1979) Biochim. Biophys. Acta 557:9; Szoka et al., (1978) Proc. Natl. Acad. Sci. 75:4194; Mayhew et al., (1984) Biochim. Biophys. Acta 775:169; Kim et al., (1983) Biochim. Biophys. Acta 728:339; and Fukunaga et al., (1984) Endocrinol. 115:757 exemplary methods of one or more aspects. Common techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw plus extrusion molding (see, for example, Mayer et al., (1986) Biochim. Biophys. Acta 858:161). When consistently small (50 to 200 nm) and relatively uniform aggregates are required, microfluidization can be used (Mayhew et al., (1984) Biochim. Biophys. Acta 775:169). These methods are easily adapted to packaging RNAi agent formulations into liposomes.

[0621] Liposomes are divided into two major categories. Cationic liposomes are positively charged liposomes that interact with negatively charged nucleic acid molecules to form stable complexes. Positively charged nucleic acid / liposome complexes bind to the negatively charged cell surface and are internalized. Due to the acidic pH in the endosome, the liposomes rupture and release their contents into the cytoplasm (Wang et al. (1987) Biochem. Biophys. Res. Commun., 147: 980-985).

[0622] pH-sensitive or negatively charged liposomes embed nucleic acids rather than complex with them. Since nucleic acids and lipids are similarly charged, repulsion rather than complex formation occurs. However, some nucleic acids are trapped in the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver nucleic acids encoding thymidine kinase genes to cell monolayers in culture. The expression of exogenous genes was detected in target cells (Zhou et al. (1992) Journal of Controlled Release, 19: 269-274).

[0623] One major type of liposome composition includes phospholipids other than naturally derived phosphatidylcholine. For example, neutral liposome compositions can be formed by dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC). Anionic liposome compositions are usually formed by dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are mainly formed by dioleoylphosphatidylethanolamine (DOPE). Another type of liposome composition is formed by phosphatidylcholine (PC) such as soybean PC and egg PC. Another type is formed by a mixture of phospholipids or phosphatidylcholine or cholesterol.

[0624] Examples of other methods for introducing liposomes into cells in vitro and in vivo include U.S. Pat. No. 5,283,185; U.S. Pat. No. 5,171,678; WO 94 / 00569; WO 93 / 24640; WO 91 / 16024; Felgner, (1994) J. Biol. Chem. 269:2550; Nabel, (1993) Proc. Natl. Acad. Sci. 90:11307; Nabel, (1992) Human Gene Ther. 3:649; Gershon, (1993) Biochem. 32:7143; and Strauss, (1992) EMBO J. 11:417.

[0625] Nonionic liposomal systems have also been examined to determine their utility in delivering drugs to the skin, particularly systems containing nonionic surfactants and cholesterol. TM I (Dilaurin / Cholesterol / Polyoxyethylene-10-stearyl ether) and Novasome TM A nonionic liposome formulation of II (distearin / cholesterol / polyoxyethylene-10-stearyl ether) delivered cyclosporin A into the dermis of mouse skin. The results showed that this nonionic liposome system can effectively promote the deposition of cyclosporin A into different layers of the skin (Hu et al., (1994) STP Pharma. Sci., 4(6):466).

[0626] Liposomes also include "sterically stabilized" liposomes, as used herein, which are liposomes that contain one or more special lipids that, when incorporated into liposomes, result in an increased circulation life relative to liposomes lacking such special lipids....

Claims

1. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting Huntington's (HTT) expression, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 1, and the antisense strand comprises at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 6, and wherein one or more lipophilic moieties are conjugated to one or more internal positions on at least one of the sense strand or the antisense strand.

2. The dsRNA agent of claim 1, wherein 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 or 33.

3. The dsRNA agent according to claim 1 or 2, wherein the lipophilic moiety is conjugated via a linker or a carrier.

4. A double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of Huntington (HTT) in a cell, wherein the dsRNA comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a complementary region to an mRNA encoding HTT, and wherein the complementary region comprises at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from any 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 or 33.

5. The dsRNA agent of claim 4, wherein the sense strand, the antisense strand, or both the sense strand and the antisense strand are conjugated to one or more lipophilic moieties.

6. The dsRNA agent of claim 5, wherein the lipophilic moiety is conjugated to one or more internal positions in the double-stranded region of the dsRNA agent.

7. The dsRNA agent of claim 5 or 6, wherein the one or more lipophilic moieties are conjugated to one or more internal positions on the antisense strand.

8. The dsRNA agent of claim 5 or 6, wherein the one or more lipophilic moieties are conjugated to one or more internal positions on at least one chain via a linker or carrier.

9. The dsRNA agent of any one of claims 1-3 and 5-8, wherein the lipophilicity of the lipophilic portion measured by logKow exceeds 0.

10. The dsRNA agent according to any one of claims 1-3 and 5-9, wherein the hydrophobicity of the dsRNA agent as measured by the unbound fraction in a plasma protein binding assay of the dsRNA agent exceeds 0.2.

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