Huntintin (HTT)iRNA formulation and method of use thereof

Abstract

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

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 929,174, filed on November 1, 2019, the entirety of which is incorporated herein by reference.

[0002] Array List This application includes an array list submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy created on October 36, 2020, is named 121301_10320_SL.txt and is 1,468,832 bytes in size. [Background technology]

[0003] Huntington's disease is a progressive neurodegenerative disorder characterized by motor impairment, cognitive impairment, and psychiatric manifestations [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 people in most populations of European origin (Harper, PS et al., in Huntington's Disease, WB Saunders, Philadelphia, 1991). A prominent feature of Huntington's disease is its characteristic choreiform movement disorder, which typically presents with subtle, insidious, and persistent manifestations in the 40s and 50s, gradually worsening over 10 to 20 years until death. Often, Huntington's disease presents in younger individuals, typically with more severe symptoms such as rigidity and a more rapid course. The early manifestations of Huntington's disease are associated with paternal dominance of the disease allele. The neuropathology of Huntington's disease also exhibits a distinctive pattern, involving a selective reduction of neurons, most severely affected in the caudate and putamen regions of the brain.

[0004] Huntington's disease is known to be caused by an elongated glutamine repeat in exon 1 of the gene called IT15 or huntingtin (HTT). While this gene is widely expressed and necessary for normal development, the pathology of Huntington's disease is limited to the brain, and the reason for this remains unclear. In patients with HD (autosomal dominant disease), the elongation of the polyglutamine repeat results in wild-type transcripts, full-length mutant transcripts with the elongated polyglutamine repeat, and truncated mutant transcripts with the elongated polyglutamine repeat. The huntingtin gene product is expressed at similar levels in patients and controls, but it is known to be characterized by the elongation of the polyglutamine repeat, the presence of full-length mutant transcripts, and the toxic truncated mutant transcripts.

[0005] Currently, there is no effective treatment for Huntington's disease. Chorea and induced behavior can usually only be partially suppressed with antipsychotics (e.g., chlorpromazine) or reserpine, to the extent that adverse effects such as apathy, hypotension, or tremor paralysis occur. In addition, despite significant advances in the fields of RNAi and Huntington's disease treatment, there is still a need for drugs that possess high bioactivity and in vivo stability, can effectively inhibit the expression of the target huntingtin gene, and can selectively and efficiently silence the HD gene using the cell's own RNAi mechanism. [Overview of the project] [Problems that the invention aims to solve]

[0006] This disclosure provides RNAi agent compositions that perform RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the huntingtin (HTT) gene. The HTT gene may be located within cells, for example, within cells in subjects such as humans. This disclosure also provides methods of using the RNAi agent compositions of this disclosure to inhibit the expression of the HTT gene, or to treat subjects who would benefit from inhibiting or reducing the expression of the HTT gene, for example, subjects suffering from or prone to suffering from HTT-related diseases. [Means for solving the problem]

[0007] In one embodiment, the present invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting huntingtin (HTT) expression, comprising 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 3, 2, 1, or 0 nucleotides or less, 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 3, 2, 1, or 0 nucleotides or less, and one or more lipophilic moieties are conjugated at one or more internal positions on at least one strand.

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

[0009] In some embodiments, the sense strand is nucleotides 618-640, 1215-1237, 1248-1270, 1403-1425, 4051-4073, 4393-4415, 4398-4420, 4403-4425, 4441-4463, 4518-4540, 4548-4570, 5105-5127, 5215-5237, 5217-5239, 5221-5243, 5222-5244, 5366-5388, 5372-5394, 5450-5472, 5509-5531 of SEQ ID NO: 1, It contains at least 15 consecutive nucleotides that differ by 0, 1, 2, or 3 nucleotides or less from any one of the nucleotide sequences 5883-5905, 6009-6031, 6010-6032, 6011-6033, 6012-6034, 6013-6035, 6014-6036, 6015-6037, 6347-6369, 6512-6534, 7523-7545, 7525-7547, 7526-7548, 9127-9149, 9531-9553, or 9538-9560.

[0010] In some embodiments, the antisense chains are AD-953769.1, AD-953778.1, AD-953784.1, AD-953786.1, AD-953849.1, AD-953854.1, AD-953855.1, AD-953857.1, AD-953862.1, AD-953866.1, AD-953867.1, AD-953880.1, AD-953883.1, AD-953884.1, AD-953885.1, AD-953886.1, AD-953887.1, AD-953888.1, AD-953889.1, AD-953891.1, AD-953896.1 It contains at least 15 consecutive nucleotides that differ by 0, 1, 2, or 3 nucleotides or less from any one of the double-stranded antisense nucleotide sequences selected from the group consisting of 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.

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

[0012] In another embodiment, the present invention provides a double-stranded ribonucleic acid (dsRNA) for inhibiting huntingtin (HTT) expression in cells, comprising a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a region complementary to the mRNA encoding HTT, and the complementary region comprises at least 15 consecutive nucleotides, e.g., 15, 16, 17, 18, 19, 20, or 21, which differ by 3, 2, 1, or 0 nucleotides or less from any one of the antisense nucleotide sequences in any one of Tables 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 24, 25, 27-30, 32, and 33.

[0013] The sense strand, antisense strand, or both the sense and antisense strands may be conjugated to one or more lipophilic moieties. In some embodiments, the lipophilic moieties are 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.

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

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

[0016] In some embodiments, the internal position includes all positions except the two terminal positions from each end of the sense or antisense chain. In other embodiments, the internal position includes all positions except the three terminal positions from each end of the sense or antisense chain.

[0017] In some embodiments, the internal positions include all positions except those 9-12, counting from the 5' end of the sense chain, excluding the cleavage region of the sense chain, or the internal positions include all positions except those 11-13, counting from the 3' end of the sense chain.

[0018] In some embodiments, the internal position excludes the cleavage region of the antisense chain. In other embodiments, the internal position includes all positions except positions 12-14, counting from the 5' end of the antisense chain. In some embodiments, the internal position includes all positions except positions 11-13, counting from the 3' end of the sense chain and positions 12-14, counting from the 5' end of the antisense chain.

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

[0020] In some embodiments, one or more lipophilic portions are conjugated to one or more internal positions selected from the group consisting of positions 5, 6, 7, 15, and 17 in the sense chain and positions 15 and 17 in the antisense chain, counting from the 5' end of each chain.

[0021] In some embodiments, the location within the double-stranded region excludes the sense strand cleavage region.

[0022] In some embodiments, the sense strand is 21 nucleotides long, the antisense strand is 23 nucleotides long, and the lipophilic portion is conjugated at positions 20, 15, 1, 7, 6, or 2 on the sense strand, or at position 16 on the antisense strand.

[0023] In other embodiments, the sense chain is 21 nucleotides long, the antisense chain is 23 nucleotides long, and the lipophilic portion is conjugated at positions 21, 20, 15, 1, 7, 6, or 2 on the sense chain or at position 16 on the antisense chain.

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

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

[0026] In some embodiments, the lipophilic portion contains a saturated or unsaturated C4-C30 hydrocarbon chain and a suitable functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.

[0027] In some embodiments, the lipophilic portion contains saturated or unsaturated C6-C18 hydrocarbon chains.

[0028] In some embodiments, the lipophilic portion contains a saturated or unsaturated C16 hydrocarbon chain. In some embodiments, the saturated or unsaturated C16 hydrocarbon chain is conjugated at position 6, counting from the 5' end of the chain.

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

[0030] In some embodiments, the lipophilic moiety is conjugated to a dsRNA agent via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfamide linkage, click reaction product, or carbamate.

[0031] In some embodiments, the lipophilic portion is conjugated to a nucleic acid base, a sugar portion, or an internucleoside linkage.

[0032] In some embodiments, the dsRNA agent contains at least one modified nucleotide. In some embodiments, five or fewer nucleotides in the sense strand and five or fewer nucleotides in the antisense strand are unmodified nucleotides. In other embodiments, all nucleotides in the sense strand and all nucleotides in the antisense strand contain modifications.

[0033] In some embodiments, at least one of the modified nucleotides is a deoxy-nucleotide, a 3'-terminal deoxythymine (dT) nucleotide, a 2'-O-methyl-modified nucleotide, a 2'-fluoro-modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformation-restricted nucleotide, a restricted ethyl nucleotide, a debasalized nucleotide, a 2'-amino-modified nucleotide, a 2'-O-allyl-modified nucleotide, a 2'-C-alkyl-modified nucleotide, a 2'-hydroxy-modified nucleotide, a 2'-methoxyethyl-modified nucleotide, a 2'-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramide, a nucleotide containing a non-natural base, a tetrahydropyran-modified nucleotide, or a 1,5-anhydrohexyl The following are selected from the group consisting of citol-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing a 5'-phosphorothioate group, nucleotides containing a 5'-methylphosphonate group, nucleotides containing a 5'-phosphate or 5'-phosphate mimetic, nucleotides containing vinylphosphonate, nucleotides containing adenosine-glycol nucleic acid (GNA), nucleotides containing thymidine-glycol nucleic acid (GNA) S isomers, nucleotides containing 2-hydroxymethyl-tetrahydrofuran-5-phosphate, nucleotides containing 2'-deoxythymidine-3'-phosphate, nucleotides containing 2'-deoxyguanosine-3'-phosphate, terminal nucleotides linked to cholesteryl derivatives, and dodecanoate bisdecylamide groups, as well as combinations thereof.

[0034] In other embodiments, the modified nucleotide is selected from the group consisting of nucleotides including 2'-deoxy-2'-fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, 3'-terminal deoxy-thymine nucleotides (dT), locked nucleotides, debasalized nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, and non-natural bases.

[0035] In some embodiments, at least one of the modified nucleotides is selected from the group consisting of deoxy-nucleotides, 2'-O-methyl-modified nucleotides, 2'-fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, glycol-modified nucleotides (GNAs), and vinyl-phosphonate nucleotides; and combinations thereof.

[0036] In some embodiments, at least one of the modifications in the nucleotide is a thermally destabilized nucleotide modification. In some embodiments, the thermally destabilized nucleotide modification is selected from the group consisting of debasing modifications; mismatches with opposing nucleotides in the double helix; and destabilized sugar modifications, 2'-deoxy modifications, acyclic nucleotides, unlocked nucleic acids (UNAs), and glycerol nucleic acids (GNAs).

[0037] In some embodiments, the modified nucleotide includes a short sequence of a 3'-terminal deoxythymine nucleotide (dT).

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

[0039] In some embodiments, the dsRNA agent further comprises at least one phosphorothioate nucleotide linkage. In some embodiments, the dsRNA agent comprises 6 to 8 phosphorothioate nucleotide linkages. In one embodiment, the phosphorothioate or methylphosphonate nucleotide linkage is at the 3' end of one strand. Optionally, the strand may be an antisense strand. In another embodiment, the strand is a sense strand. In a related embodiment, the phosphorothioate or methylphosphonate nucleotide linkage is at the 5' end of one strand. Optionally, the strand may be an antisense strand. In another embodiment, the strand is a sense strand. In another embodiment, the phosphorothioate or methylphosphonate nucleotide linkage is at both the 5' and 3' ends of one strand. Optionally, the strand may be an antisense strand. In another embodiment, the strand is a sense strand.

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

[0041] In some embodiments, at least one strand includes a 3' overhang of at least one nucleotide or at least two nucleotides.

[0042] The double-stranded region may be 15–30 nucleotide pairs long, 17–23 nucleotide pairs long, 17–25 nucleotide pairs long, 23–27 nucleotide pairs long, 19–21 nucleotide pairs long, or 21–23 nucleotide pairs long.

[0043] Each chain may consist of 19-30 nucleotides; 19-23 nucleotides; or 21-23 nucleotides.

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

[0045] In certain embodiments, the double-stranded RNAi agent further comprises a targeted ligand, such as a hydrophilic ligand, that targets a receptor that mediates delivery to CNS tissue.

[0046] In a particular embodiment, the targeted ligand is a C16 ligand. In one embodiment, the ligand is as follows:

[0047] [ka] In the formula, B is a nucleotide base or a nucleotide base analog, and B may be adenine, guanine, cytosine, thymine, or uracil, as appropriate.

[0048] In some embodiments, the lipophilic moiety or targeted ligand is conjugated via a biocleavable linker selected from the group consisting of DNA, RNA, disulfides, amides, and functionalized monosaccharides or oligosaccharides of galactosamine, glucosamine, glucose, galactose, and mannose, as well as combinations thereof.

[0049] In some embodiments, the 3' end of the sense chain is protected via an end cap which is a cyclic group having an amine, and the cyclic group is selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanil, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridadinyl, tetrahydrofuranil, and dekalinyl.

[0050] In some embodiments, the dsRNA agent further includes a terminal chiral modification occurring at the first nucleotide linkage at the 3' end of the antisense strand, having a linked phosphorus atom in the Sp configuration; a terminal chiral modification occurring at the first nucleotide linkage at the 5' end of the antisense strand, having a linked phosphorus atom in the Rp configuration; and a terminal chiral modification occurring at the first nucleotide linkage at the 5' end of the sense strand, having a linked phosphorus atom in either the Rp configuration or the Sp configuration.

[0051] In some embodiments, the dsRNA agent further includes terminal chiral modifications occurring at the first and second nucleotide linkages at the 3' end of the antisense strand, having a linked phosphorus atom in the Sp configuration; terminal chiral modifications occurring at the first nucleotide linkage at the 5' end of the antisense strand, having a linked phosphorus atom in the Rp configuration; and terminal chiral modifications occurring at the first nucleotide linkage at the 5' end of the sense strand, having a linked phosphorus atom in either the Rp or Sp configuration.

[0052] In some embodiments, the dsRNA agent further includes terminal chiral modifications occurring at the first, second, and third nucleotide linkages at the 3' end of the antisense strand, having a linked phosphorus atom in the Sp configuration; terminal chiral modifications occurring at the first nucleotide linkage at the 5' end of the antisense strand, having a linked phosphorus atom in the Rp configuration; and terminal chiral modifications occurring at the first nucleotide linkage at the 5' end of the sense strand, having a linked phosphorus atom in either the Rp or Sp configuration.

[0053] In some embodiments, the dsRNA agent further includes terminal chiral modifications occurring at the first and second nucleotide linkages at the 3' end of the antisense strand, having a linked phosphorus atom in the Sp configuration; terminal chiral modifications occurring at the third nucleotide linkage at the 3' end of the antisense strand, having a linked phosphorus atom in the Rp configuration; terminal chiral modifications occurring at the first nucleotide linkage at the 5' end of the antisense strand, having a linked phosphorus atom in the Rp configuration; and terminal chiral modifications occurring at the first nucleotide linkage at the 5' end of the sense strand, having a linked phosphorus atom in either the Rp or Sp configuration.

[0054] In some embodiments, the dsRNA agent further includes terminal chiral modifications occurring at the first and second nucleotide linkages at the 3' end of the antisense strand, having a linked phosphorus atom in the Sp configuration; terminal chiral modifications occurring at the first and second nucleotide linkages at the 5' end of the antisense strand, having a linked phosphorus atom in the Rp configuration; and terminal chiral modifications occurring at the first nucleotide linkage at the 5' end of the sense strand, having a linked phosphorus atom in either the Rp or Sp configuration.

[0055] In some embodiments, the dsRNA agent further comprises a phosphate or phosphate mimetic at the 5' end of the antisense strand. In some embodiments, the phosphate mimetic is a 5'-vinyl phosphonate (VP).

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

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

[0058] An additional aspect of this disclosure provides a double-stranded RNAi agent for inhibiting the expression of the huntingtin (HTT) gene, wherein the HTT-targeted double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, the sense strand comprising at least 15, e.g., 15, 16, 17, 18, 19, or 20 consecutive nucleotides that differ from any one of the nucleotide sequences of SEQ ID NOs. 1 to 5 by 3 nucleotides or less (i.e., by 3, 2, 1, or 0 nucleotides), and the antisense strand comprising at least 15, e.g., 15, 16, 17, 18, 19, or 20 consecutive nucleotides that differ from any one of the nucleotide sequences of SEQ ID NOs. 6 to 10 by 3 nucleotides or less (i.e., by 3, 2, 1, or 0 nucleotides), in the sequence provided in SEQ ID NOs. 1 to 10 Any substitution of uracil with thymine (when comparing aligned sequences) is not counted as a difference of three nucleotides or less with any of the nucleotide sequences provided in SEQ ID NOs. 1-10, substantially all nucleotides of the sense strand contain modifications which are 2'-O-methyl modifications, GNAs, or 2'-fluoro modifications, the sense strand contains two phosphorothioate nucleotide linkages at its 5' end, substantially all nucleotides of the antisense strand contain modifications selected from the group consisting of 2'-O-methyl modifications and 2'-fluoro modifications, the antisense strand contains two phosphorothioate nucleotide linkages at its 5' end and two phosphorothioate nucleotide linkages at its 3' end, and the sense strand is conjugated to one or more lipophilic ligands, e.g., C16 ligand.

[0059] Another aspect of the present disclosure provides a double-stranded RNAi agent for inhibiting the expression of the huntingtin (HTT) gene, wherein the HTT-targeted double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, the sense strand comprising at least 15, e.g., 15, 16, 17, 18, 19, or 20 consecutive nucleotides that differ from any one of the nucleotide sequences of SEQ ID NOs. 1-5 by 3 nucleotides or less (i.e., by 3, 2, 1, or 0 nucleotides), and the antisense strand comprising 3 nucleotides that differ from any one of the nucleotide sequences of SEQ ID NOs. 6-10 The sequence comprises at least 15 consecutive nucleotides that differ in the following order (i.e., differ in 3, 2, 1, or 0 nucleotides), and any substitution of uracil with thymine in the sequences provided in SEQ ID NOs. 1 to 10 (when comparing aligned sequences) is not counted as a difference of 3 nucleotides or less with any one of the nucleotide sequences provided in SEQ ID NOs. 1 to 10, the sense strand comprises at least one 3'-terminal deoxythymine nucleotide (dT), and the antisense strand comprises at least one 3'-terminal deoxythymine nucleotide (dT).

[0060] Additional aspects of the present disclosure provide a double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of the huntingtin (HTT) gene, wherein the RNAi agent has a sense strand and an antisense strand, the antisense strand including a complementary region comprising at least 15 consecutive nucleotides that differ by three or fewer nucleotides (i.e., by 3, 2, 1, or 0 nucleotides) from any one of the antisense strand nucleic acid sequences of Tables 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 24, 25, 27-30, 32, and 33, for example, at least 15 nucleotides (i.e., differing by 3, 2, 1, or 0 nucleotides) and at least 19 nucleotides (i.e., differing by 3, 2, 1, or 0 nucleotides). In one embodiment, the RNAi agent comprises one or more nucleotides from among the following modifications: 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-C-alkyl-modified nucleotides, and glycol nucleic acids (GNA), phosphorothioates (PS), and vinyl phosphonates (VP). Optionally, the RNAi agent may also comprise at least one of each of the following modifications: 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-C-alkyl-modified nucleotides, and glycol nucleic acids (GNA), phosphorothioates, and vinyl phosphonates (VP).

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

[0062] In additional embodiments, the sense and antisense strands of the RNAi agent each have a 5'-terminus and a 3'-terminus, and the RNAi agent includes eight PS modifications located at each of the penalties, and final internucleotide ligations from the 3'- and 5'-terminuses of the sense and antisense strands of the RNAi agent, respectively.

[0063] In another embodiment, the sense and antisense strands of the RNAi agent each include a 5'-terminus and a 3'-terminus, and the RNAi agent contains only one nucleotide containing a GNA. Optionally, the nucleotide containing the GNA may be located on the antisense strand at the seventh nucleic acid base residue from the 5'-terminus of the antisense strand.

[0064] In additional embodiments, the sense strand and antisense strand of the RNAi agent each include a 5'-terminus and a 3'-terminus, and the RNAi agent contains one to four 2'-C-alkyl-modified nucleotides. Optionally, the 2'-C-alkyl-modified nucleotides may be 2'-C16-modified nucleotides. Optionally, the RNAi agent may contain a single 2'-C-alkyl, e.g., a C16-modified nucleotide. Optionally, the single 2'-C-alkyl, e.g., a C16-modified nucleotide, may be located on the sense strand at the 6th nucleic acid base residue from the 5'-terminus of the sense strand.

[0065] In another embodiment, each of the sense and antisense strands of the RNAi agent includes a 5'-terminus and a 3'-terminus, and the RNAi agent contains two or more 2'-fluoromodified nucleotides. Optionally, each of the sense and antisense strands of the RNAi agent may contain two or more 2'-fluoromodified nucleotides. Optionally, the 2'-fluoromodified nucleotides may be located on the sense strand at nucleic acid base positions 7, 9, 10, and 11 from the 5'-terminus of the sense strand, and on the antisense strand at nucleic acid base positions 2, 14, and 16 from the 5'-terminus of the antisense strand.

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

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

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

[0069] In another embodiment, each chain has 19 to 30 nucleotides.

[0070] In certain embodiments, the antisense strand of the RNAi agent includes at least one double-strand thermal destabilization modification within the 5' region or the first nine nucleotides of its precursor. The double-strand thermal destabilization modification may be as follows:

[0071] [ka] It may be one or more of these, where B is a nucleic acid base.

[0072] The present invention further provides cells containing 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.

[0073] In one embodiment, the double-stranded RNAi agent is in a non-buffer. The non-buffer may optionally be saline or water. In another embodiment, the double-stranded RNAi agent is in a buffer. The buffer may optionally contain acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In another embodiment, the buffer is phosphate-buffered saline (PBS). Another aspect of the present disclosure provides a pharmaceutical composition comprising the double-stranded RNAi agent of the present disclosure and a lipid formulation. In one embodiment, the lipid formulation comprises lipid nanoparticles (LNPs).

[0074] Additional aspects of the present disclosure provide a method for inhibiting the expression of the HTT gene in cells, comprising (a) contacting cells with a double-stranded RNAi agent of the present disclosure or a pharmaceutical composition of the present disclosure, and (b) maintaining the cells produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the HTT gene, thereby inhibiting the expression of the HTT gene in the cells.

[0075] In one embodiment, the cells are located within the target. The target may, as appropriate, be a human.

[0076] In certain embodiments, the subjects are rhesus monkeys, cynomolgus monkeys, mice, or rats. In certain embodiments, TT expression is inhibited by at least about 50% by the RNAi agent.

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

[0078] Another aspect of the present disclosure provides a method for treating a subject diagnosed with an HTT-related disease, such as Huntington's disease, comprising administering to the subject a therapeutically effective amount of the double-stranded RNAi agent or the pharmaceutical composition of the present disclosure, thereby treating the subject.

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

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

[0081] In some embodiments, the dsRNA agent is administered to the subject intrathecally. In one embodiment, the method reduces the expression of the HTT gene in brain (e.g., striatum) or spinal tissue. The brain or spinal tissue may be the striatum, cortex, cerebellum, cervical vertebrae, lumbar vertebrae, or thoracic vertebrae, as appropriate.

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

[0083] Another aspect of the present disclosure provides a method for inhibiting huntingtin (HTT) expression in a subject, comprising administering a therapeutically effective amount of the double-stranded RNAi agent or the pharmaceutical composition of the present disclosure to the subject, thereby inhibiting HTT expression in the subject.

[0084] In some embodiments, the method further includes administering additional agents suitable for the treatment or prevention of HTT-related diseases. [Brief explanation of the drawing]

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

[0086] This disclosure provides RNAi compositions that perform RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the huntingtin (HTT) gene. The HTT gene may be present in cells, for example, in cells within a subject such as a human. The use of these iRNAs enables targeted degradation of the mRNA of the corresponding gene (HTT gene) in mammals.

[0087] The iRNAs of the present invention are designed to target the HTT gene, including portions of the gene conserved in HTT orthologs of other mammalian species. The iRNAs of the present invention are also designed to target exon 1, a specific portion of the HTT gene, thereby targeting, for example, full-length wild-type transcripts, full-length mutant transcripts, and truncated mutant transcripts. While not intended to be theoretically limited, the combination or partial combination of the aforementioned properties with specific target sites, such as exon 1, or specific modifications of these iRNAs, is thought to confer improved efficiency, stability, potency, durability, and safety to the iRNAs of the present invention.

[0088] Accordingly, the Disclosure also provides methods for using the RNAi compositions of the Disclosure to inhibit the expression of the HTT gene, or to treat subjects having a disorder, such as an HTT-related disease, such as Huntington's disease (HD), which would benefit from inhibiting or reducing the expression of the HTT gene.

[0089] The RNAi agents disclosed herein are approximately 30 nucleotides or less in length, for example, 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- The RNAi agent of the Disclosure comprises an RNA strand (antisense strand) having a region of 24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, wherein the region is substantially complementary to at least a portion of the mRNA transcript of the HTT gene. In certain embodiments, the RNAi agent of the Disclosure comprises an RNA strand (antisense strand) having a region of about 21-23 nucleotides in length, wherein the region is substantially complementary to at least a portion of the mRNA transcript of the HTT gene.

[0090] In certain embodiments, the RNAi agents of the present disclosure include an RNA chain (antisense chain) 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, and which may be longer in length, for example, up to 66 nucleotides, for example, 36-66, 26-36, 25-36, 31-60, 22-43, or 27-53 nucleotides. These RNAi agents having longer antisense chains preferably include a second RNA chain (sense chain) of 20-60 nucleotides in length, in which case the sense and antisense chains form a double helix of 18-30 consecutive nucleotides.

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

[0092] The following detailed description discloses methods for preparing and using compositions containing RNAi agents to inhibit the expression of the HTT gene, as well as compositions or methods for treating subjects with disease or disorder who would benefit from the inhibition or reduction of gene expression.

[0093] I. Definition To make this disclosure more easily understandable, certain terms are defined first. In addition, whenever parameter values ​​or ranges of values ​​are listed, intermediate values ​​and ranges of the listed values ​​are also intended to be part of this disclosure.

[0094] The articles "a" and "an" are used herein to mean one or more (i.e., at least one) grammatical objects of the article. For example, "an element" means one or more elements, e.g., multiple elements.

[0095] The term "including" is used herein to mean "including but not limited to" and is interchangeable with the phrase. The term "or" is used herein to mean "and / or" unless explicitly indicated in the context and is interchangeable with the phrase.

[0096] The term “approximately” is used herein to mean within a typical range of crossover in the art. For example, “approximately” can be understood as approximately 2 standard deviations from the mean. In certain embodiments, “approximately” means ±10%. In certain embodiments, “approximately” means ±5%. It will be understood that when “approximately” precedes a series of numbers or ranges, it can modify each of the numbers or ranges in that series.

[0097] The term "at least" preceding a number or a range of numbers is understood, where evident from the context, to include the number adjacent to the term "at least," and all subsequent numbers or integers that may logically be included. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For instance, "at least 18 nucleotides in a 21-nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have the stated characteristic. It will be understood that when the term "at least" precedes a range of numbers or a range, "at least" can modify each of the numbers and ranges in the range.

[0098] Where used herein, “less than” or “less than” is understood to mean from the value adjacent to the phrase and any value or integer logically smaller than that value, down to zero, where logical in context. For example, a double helix with an overhang of “2 nucleotides or less” has an overhang of 2, 1, or 0 nucleotides. Where “less than” precedes a series of numbers or ranges, it will be understood that “less than” can modify each of the numbers or ranges in that series.

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

[0100] If the indicated target site does not match the nucleotide sequence on the sense or antisense strand, the indicated sequence takes precedence.

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

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

[0103] 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 (House mouse HTT, SEQ ID NO. 2; reverse complement, SEQ ID NO. 7); GenBank accession No.: NM_024357.3 (Norway rat HTT, SEQ ID NO. 3, reverse complement, SEQ ID NO. 8); GenBank accession No.: XM_015449989.1 (Cynomolgus monkey HTT, SEQ ID NO. 4, reverse complement, SEQ ID NO. 9); and GenBank accession No.: XM_028848247.1 (Rhesus monkey HTT, SEQ ID NO. 5, reverse complement, SEQ ID NO. 10).

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

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

[0106] The entire contents of the aforementioned GenBank accession numbers and Gene database numbers are incorporated herein by reference as of the filing date of this application.

[0107] When used herein, the term HTT also refers to variations of the HTT gene, including variants provided in SNP databases. Numerous sequence variations within the HTT gene have been identified and can be found, for example, in NCBI dbSNP and UniProt (see, for example, www.ncbi.nlm.nih.gov / snp / ?LinkName=gene_snp&from_uid=3064, whose entire contents are incorporated herein by reference as of the filing date of this application).

[0108] As used herein, “target sequence” means a contiguous portion of a nucleotide sequence in an mRNA molecule formed during the transcription of the HTT gene, such as mRNA which is the product of RNA processing of the primary transcript. In one embodiment, the target portion of the sequence will be at least sufficiently long to function as a substrate for RNAi-dependent cleavage in or near a portion of the nucleotide sequence of the mRNA molecule formed during the transcription of the HTT gene.

[0109] The target sequence is approximately 15-30 nucleotides long. For example, the target sequence is approximately 15-30 nucleotides long, 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, 1 The target sequence may be 9-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides long. In certain embodiments, the target sequence is 19-23 nucleotides long and may optionally be 21-23 nucleotides long. It is conceivable that intermediate ranges and lengths between those listed above are also part of this disclosure.

[0110] As used herein, the term “sequence-containing chain” means an oligonucleotide containing a chain of nucleotides described by a sequence as referred to using the standard nucleotide terminology.

[0111] "G," "C," "A," "T," and "U" generally represent nucleotides containing guanine, cytosine, adenine, thymidine, and uracil as bases, respectively, in relation to modified or unmodified nucleotides. However, it will be understood that the terms "ribonucleotide" or "nucleotide" can also mean modified nucleotides or substituted parts (see, for example, Table 1), as will be described in more detail below. Those skilled in the art are well aware that guanine, cytosine, adenine, thymidine, and uracil can be replaced by other parts without substantially altering the base-pairing properties of oligonucleotides containing such substituted parts. For example, but not limited to, nucleotides containing inosine as a base can base-pair with nucleotides containing adenine, cytosine, or uracil. Thus, nucleotides containing uracil, guanine, or adenine can be substituted, for example, with nucleotides containing inosine in the nucleotide sequences of dsRNAs featured in this disclosure. In another example, adenine and cytosine in either of the oligonucleotides can be substituted with guanine and uracil, respectively, to form G-UWobble base pairs with the target mRNA. Sequences containing such substitutions are suitable for the compositions and methods featured in this disclosure.

[0112] The terms “iRNA,” “RNAi agent,” “iRNA agent,” and “RNA interfering agent,” as used interchangeably herein, mean agents containing RNA as defined herein, which mediate targeted cleavage in RNA transcription via 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 the expression of HTT in cells within a subject, such as a mammalian subject.

[0113] In one embodiment, the RNAi agent of the present disclosure comprises a single-stranded RNAi that interacts with a target RNA sequence, e.g., an HTT target mRNA sequence, to direct the cleavage of the target RNA. While we do not wish to be bound by theory, it is thought that long double-stranded RNA introduced into a cell is degraded into double-stranded small interfering RNA (siRNA) containing sense and antisense strands by a type III endonuclease known as Dicer [Sharp et al. (2001) Genes Dev. 15:485]. Dicer, a ribonuclease III-like enzyme, processes these dsRNAs into 19-23 base pair small interfering RNAs with characteristic two base 3' overhangs [Bernstein, et al., (2001) Nature 409:363]. These siRNAs are then introduced into an RNA-induced silencing complex (RISC), in which one or more helicases unwind the siRNA double helix, allowing the complementary antisense strand to induce target recognition [Nykanen, et al., (2001) Cell 107:309]. Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing [Elbashir, et al., (2001) Genes Dev. 15:188]. Thus, in one embodiment, this disclosure relates to single-stranded RNA (ssRNA) (the antisense strand of the siRNA double helix) that is generated in a cell and facilitates the formation of the RISC complex, thereby silencing a target gene, namely the HTT gene. Accordingly, the term "siRNA" is used herein to also mean the RNAi described above.

[0114] In another embodiment, the RNAi agent may 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 Argonaut 2 and then cleaves the target mRNA. Single-stranded siRNAs are generally 15–30 nucleotides long and are chemically modified. Designs and tests of single-stranded RNAs are described in U.S. Patent No. 8,101,348 and Lima et al., (2012) Cell 150:883–894, the entire contents of which are incorporated herein by reference. Any antisense nucleotide sequences described herein may be used as single-stranded siRNAs described herein, or as single-stranded siRNAs chemically modified by the methods described in Lima et al., (2012) Cell 150:883–894.

[0115] In another embodiment, the “RNAi agent” for use in the compositions and methods of the present disclosure is double-stranded RNA, and is referred to herein as “double-stranded RNAi agent,” “double-stranded RNA (dsRNA) molecule,” “dsRNA agent,” or “dsRNA.” The term “dsRNA” means a complex of ribonucleic acid molecules having a double-stranded structure containing two antiparallel and substantially complementary nucleic acid strands, which are said to have “sense” or “antisense” orientation with respect to the HTT gene. In some embodiments of the present disclosure, double-stranded RNA (dsRNA) induces the degradation of target RNA, e.g., mRNA, by a post-transcriptional gene silencing mechanism referred herein as RNA interference or RNAi.

[0116] Generally, dsRNA molecules may contain ribonucleotides, but as will be described in detail herein, each or both strands may also contain one or more ribonucleotides, such as deoxyribonucleotides, modified nucleotides, etc. In addition, as used herein, “RNAi agent” may include ribonucleotides having chemical modifications; an RNAi agent may include substantial modifications in multiple nucleotides. As used herein, the term “modified nucleotide” means a nucleotide having independently modified sugar moieties, modified nucleotide linkages, or modified nucleic acid bases. Thus, the term modified nucleotide includes substitution, addition, or removal of, for example, functional groups or atoms, to nucleoside linkages, sugar moieties, or nucleic acid bases. Modifications suitable for use in the agents of this disclosure include all types of modifications disclosed herein or known in the art. Any such modifications used in siRNA-type molecules are encompassed by “RNAi agent” for the purposes of this specification and the claims.

[0117] In certain embodiments of this disclosure, the inclusion of a deoxyribonucleotide, when present in an RNAi agent, can be considered to constitute a modified nucleotide.

[0118] The double-stranded region can be of any length that allows for the specific degradation of the desired target RNA by the RISC pathway, as well as lengths of approximately 15–36 base pairs, e.g., approximately 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 The base pair lengths can range from ~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. In certain embodiments, the double-stranded region is 19 to 21 base pairs long, for example, 21 base pairs long. It is conceivable that intermediate ranges and lengths between those listed above are also part of this disclosure.

[0119] The two strands forming a double helix structure may be different parts of one larger RNA molecule, or they may be separate RNA molecules. If the two strands are part of one larger molecule and are therefore connected by an unpaired nucleotide chain between the 3' end of one strand and the 5' end of the other strand forming the double helix structure, then the connecting RNA strands are called a “hairpin loop”. A hairpin loop may contain at least one unpaired nucleotide. In some embodiments, a hairpin loop may contain at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides or nucleotides not targeting the dsRNA site. In some embodiments, a hairpin loop may contain 10 or fewer nucleotides. In some embodiments, a hairpin loop may contain 8 or fewer unpaired nucleotides. In some embodiments, a hairpin loop may contain 4 to 10 unpaired nucleotides. In some embodiments, a hairpin loop may contain 4 to 8 unpaired nucleotides.

[0120] The two substantially complementary strands of dsRNA are contained within separate RNA molecules, which can, though not necessarily, be covalently linked. In certain embodiments, where the two strands are covalently linked between the 3' end of one strand and the 5' end of the other strand forming a double-stranded structure by means other than an uninterrupted chain of nucleotides, the connecting structure is called a “linker” (although certain other structures defined elsewhere in this specification may also be called “linkers”). RNA strands may have the same or different numbers of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of dsRNA minus all the overhangs present in the double-stranded structure. In addition to the double-stranded structure, RNAi may contain one or more nucleotide overhangs. In one embodiment of an RNAi agent, at least one strand contains a 3' overhang of at least one nucleotide. In another embodiment, at least one strand contains a 3' overhang of at least two nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, at least one strand of the RNAi agent includes a 5' overhang of at least one nucleotide. In certain embodiments, at least one strand includes a 5' overhang of at least two nucleotides, for example, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In yet another embodiment, both the 3' and 5' ends of one strand of the RNAi agent include an overhang of at least one nucleotide.

[0121] In one embodiment, the RNAi agent of the present disclosure is a dsRNA, each strand containing 19 to 23 nucleotides that independently interact with a target RNA sequence, such as an HTT target mRNA sequence, to induce cleavage of the target RNA.

[0122] As used herein, the term “nucleotide overhang” means at least one unpaired nucleotide protruding from the double-stranded structure of an RNAi agent, such as a dsRNA. For example, a nucleotide overhang exists if the 3' end of one strand of a dsRNA extends beyond the 5' end of the other strand, or vice versa. A dsRNA may contain an overhang of at least one nucleotide; or the overhang may contain at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. A nucleotide overhang may contain or consist of nucleotide / nucleoside analogs such as deoxynucleotides / nucleosides. The overhang may be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleotides of the overhang may be located at the 5' end, the 3' end, or both of either the antisense strand or the sense strand of the dsRNA.

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

[0124] In one particular embodiment, the antisense strand of the dsRNA has a 1-10 nucleotide overhang at its 3' or 5' end, e.g., 0-3, 1-3, 2-4, 2-5, 4-10, 5-10 nucleotide overhang, e.g., an overhang of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In another embodiment, one or more nucleotides in the overhang are replaced with a nucleoside thiophosphate.

[0125] In certain embodiments, the overhang in the sense or antisense strand may include an extended length longer than 10 nucleotides, for example, 1–30 nucleotides, 2–30 nucleotides, 10–30 nucleotides, or 10–15 nucleotides. In certain embodiments, the extended overhang is located in the sense strand of the double helix. In certain embodiments, the extended overhang is located at the 3' end of the sense strand of the double helix. In certain embodiments, the extended overhang is located at the 5' end of the sense strand of the double helix. In certain embodiments, the extended overhang is located in the antisense strand of the double helix. In certain embodiments, the extended overhang is located at the 3' end of the antisense strand of the double helix. In certain embodiments, the extended overhang is located at the 5' end of the antisense strand of the double helix. In certain embodiments, one or more nucleotides in the overhang are replaced with a nucleoside thiophosphate. In certain embodiments, the overhang includes a self-complementary portion such that the overhang can form a stable hairpin structure under physiological conditions.

[0126] In certain embodiments, at least one end of at least one chain is extended beyond a double-stranded targeting region, such as a structure in which one end of the chain includes a thermodynamically stable tetraloop structure (see, for example, U.S. Patents 8,513,207 and 8,927,705, and W02010033225, whose entire contents are incorporated herein by reference). Such structures may include single-stranded extensions (at one or both ends of the molecule) and double-stranded extensions.

[0127] In certain embodiments, the 3' end of the sense strand and the 5' end of the antisense strand are joined by a polynucleotide sequence containing ribonucleotides, deoxyribonucleotides, or both, and the polynucleotide sequence may optionally include a tetraloop sequence. In certain embodiments, the sense strand is 25 to 35 nucleotides long.

[0128] A tetraloop may include ribonucleotides, deoxyribonucleotides, modified nucleotides, and combinations thereof. Typically, a tetraloop has 4 to 5 nucleotides. In some embodiments, the loop includes a sequence described as GAAA. In some embodiments, at least one of the nucleotides (GAAA) in the loop includes a nucleotide modification. In some embodiments, the modified nucleotide includes a 2' modification. In some embodiments, the 2' modification is a modification selected from the group consisting of 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, 2'-aminodiethoxymethanol, 2'-adem, and 2'-deoxy-2'-filo--d-arabinonucleotides. In some embodiments, all nucleotides in the loop are modified. In some embodiments, G in the GAAA sequence includes a 2'-OH group. In some embodiments, each nucleotide in the GAAA sequence includes a 2'-O-methyl modification. In some embodiments, each A in the GAAA sequence includes a 2'-OH group, and G in the GAAA sequence includes a 2'-O-methyl modification. In preferred embodiments, in some embodiments, each A in the GAAA sequence includes a 2'-O-methoxyethyl (MOE) modification and the G in the GAAA sequence includes a 2'-O-methyl modification; or each A in the GAAA sequence includes a 2'-adem modification and the G in the GAAA sequence includes a 2'-O-methyl modification. See, for example, PCT publication number WO 2020 / 206350, whose entire contents are incorporated herein by reference.

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

[0130] [ka]

[0131] The terms “blunt” or “blunt-ended,” as used herein in relation to dsRNA, mean that there are no unpaired nucleotides or nucleotide analogs at any given end of the dsRNA; that is, there are no nucleotide overhangs. One or both ends of a dsRNA can be blunt. If both ends of a dsRNA are blunt, it is said to be blunt-ended. For clarity, a “blunt-ended” dsRNA is a dsRNA that is blunt at both ends, i.e., a dsRNA in which there are no nucleotide overhangs at either end of the molecule. In most cases, such a molecule will be double-stranded over its entire length.

[0132] The terms "antisense strand" or "guide strand" refer to the strand of an RNAi agent, such as dsRNA, that contains a region substantially complementary to the target sequence, such as HTTmRNA.

[0133] As used herein, the term “complementary region” means a region on the antisense strand that is substantially complementary to a sequence, e.g., a target sequence, e.g., an HTT nucleotide sequence, as defined herein. If the complementary region is not fully complementary to the target sequence, the mismatch may be in an internal or terminal region of the molecule. Generally, the most acceptable mismatches are within 5, 4, 3, or 2 nucleotides in the terminal region, e.g., within the 5' or 3' end of the RNAi agent. In some embodiments, the double-stranded RNA agent of the present invention includes nucleotide mismatches in the antisense strand. In some embodiments, the antisense strand of the double-stranded RNA agent of the present invention includes four or fewer mismatches with the target mRNA, e.g., the antisense strand includes four, three, two, one, or zero mismatches with the target mRNA. In some embodiments, the antisense strand of the double-stranded RNA agent of the present invention includes four or fewer mismatches with the sense strand, e.g., the antisense strand includes four, three, two, one, or zero mismatches with the sense strand. In some embodiments, the double-stranded RNA agent of the present invention contains nucleotide mismatches in the sense strand. In some embodiments, the sense strand of the double-stranded RNA agent of the present invention contains four or fewer mismatches with the antisense strand, for example, the sense strand contains four, three, two, one, or zero mismatches with the antisense strand. In some embodiments, the nucleotide mismatches are, for example, within 5, 4, or 3 nucleotides from the 3' end of the iRNA. In another embodiment, the nucleotide mismatches are, for example, at the 3' terminal nucleotide of the iRNA agent. In some embodiments, there are no mismatches in the seed region.

[0134] Therefore, the RNAi agents described herein may contain one or more mismatches with respect to the target sequence. In one embodiment, the RNAi agents described herein contain three or fewer mismatches (i.e., three, two, one, or zero mismatches). In one embodiment, the RNAi agents described herein contain two or fewer mismatches. In one embodiment, the RNAi agents described herein contain one or fewer mismatches. In one embodiment, the RNAi agents described herein contain zero mismatches. In certain embodiments, if the antisense strand of the RNAi agent contains a mismatch with respect to the target sequence, the mismatch may, as appropriate, be limited to within the last five nucleotides from the 5' or 3' end of the complementary region. For example, in such embodiments, for a 23-nucleotide RNAi agent, the strand complementary to the HTT gene region generally does not contain any mismatches within the central 13 nucleotides. By using the methods described herein or methods known in the art, it is possible to determine whether an RNAi agent containing a mismatch with respect to the target sequence is effective in inhibiting the expression of the HTT gene. In particular, if specific complementary regions in the HTT gene are known to exhibit polymorphic sequence variations within a population, it is important to consider the effectiveness of RNAi agents with mismatches that inhibit HTT expression.

[0135] When used herein, the terms “sense strand” or “passenger strand” mean a strand of an RNAi agent that contains a region substantially complementary to the antisense strand region as defined herein.

[0136] As used herein, the term “cleavage region” means a region located directly adjacent to a cleavage site. A cleavage site is a site on the target where a cleavage occurs. In some embodiments, a cleavage region includes three bases directly adjacent to either end of a cleavage site. In some embodiments, a cleavage region includes two bases directly adjacent to either end of a cleavage site. In some embodiments, in detail, a cleavage site occurs at a site bound by nucleotides 10 and 11 of the antisense strand, and the cleavage region includes nucleotides 11, 12, and 13.

[0137] As used herein, unless otherwise specified, the term “complementary” means, as understood by those skilled in the art, the ability of an oligonucleotide or polynucleotide containing a first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide containing a second nucleotide sequence to form a double helix under certain conditions.

[0138] In RNAi agents, for example, in dsRNA as described herein, complementary sequences include base pairings of an oligonucleotide or polynucleotide containing a first nucleotide sequence with an oligonucleotide or polynucleotide containing a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences may be referred to herein as “fully complementary” with respect to each other. However, where herein the first sequence is considered “substantially complementary” to the second sequence, the two sequences may be fully complementary, or they may form one or more, but generally five, four, three, or two or fewer, mismatched base pairs during hybridization, while maintaining their ability to hybridize under conditions best suited to their final use, e.g., inhibition of gene expression via the RISC pathway, in the case of double helixes of up to 30 base pairs. However, if two oligonucleotides are designed to form one or more single-stranded overhangs during hybridization, such overhangs are not considered mismatches with respect to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide of 21 nucleotides and another oligonucleotide of 23 nucleotides, wherein the longer oligonucleotide contains a 21-nucleotide sequence that is perfectly complementary to the shorter oligonucleotide, can still be considered "perfectly complementary" for the purposes described herein.

[0139] When used herein, "complementary" sequences may include, or may be entirely formed from, non-Watson-Crick base pairs or base pairs formed from non-naturally modified nucleotides, provided that the above requirements regarding their ability to hybridize are met. Examples of such non-Watson-Crick base pairs include, but are not limited to, G:UWobble or Hoogstein base pairings.

[0140] The terms “complementary,” “fully complementary,” and “substantially complementary” can be used herein, as can be understood in relation to their use, in connection with base matching between the sense strand and antisense strand of a dsRNA, or between the antisense strand and target sequence of an RNAi agent.

[0141] As used herein, a polynucleotide that is "substantially complementary to at least a portion of" messenger RNA (mRNA) means a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., the mRNA encoding HTT). For example, a polynucleotide is complementary to at least a portion of HTT mRNA if its sequence is substantially complementary to an uninterrupted portion of the mRNA encoding HTT.

[0142] Accordingly, 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 sequence of nucleotides that is at least 80%, for example, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary over its entire length to any nucleotide sequence or equivalent region of any fragment of any of SEQ ID NOs: 1-5.

[0143] In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to a target HTT sequence and comprise a contiguous nucleotide sequence that is at least 80% over its entire length, for example, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% complementary over its entire length to any one sense strand nucleotide sequence in any one of Tables 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 24, 25, 27–30, 32, and 33.

[0144] In one embodiment, the RNAi agent of the present disclosure comprises a sense strand substantially complementary to an antisense polynucleotide which is identical to the target HTT sequence, wherein the sense strand polynucleotide comprises a sequence of nucleotides which is at least about 80%, e.g., about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% complementary over its entire length to the nucleotide sequences of SEQ ID NOs. 6-10 or an equivalent region of any fragment of SEQ ID NOs. 6-10.

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

[0146] In one embodiment, at least partial repression of HTT gene expression is assessed by a reduction in the amount of HTT mRNA that can be isolated from or detected in the first cells or group of cells that have been treated or are substantially identical to the first group of cells but not treated in the same way (control cells), and in which the HTT gene has been transcribed and HTT gene expression is inhibited. The degree of inhibition is represented by the following formula:

[0147]

number

[0148] The phrase "contacting cells with an RNAi agent," such as dsRNA, as used herein, encompasses contacting cells by any possible means. Contacting cells with an RNAi agent includes contacting cells with an RNAi agent in vitro or in vivo. Contact may be direct or indirect. For example, an RNAi agent may be brought into physical contact with cells by performing the method individually, or an RNAi agent may be placed in a situation that allows or causes subsequent contact with cells.

[0149] Cell contact in vitro can be achieved, for example, by incubating cells with an RNAi agent. Cell contact in vivo can be achieved, for example, by injecting the RNAi agent into or near the tissue in which the cells are located, or by injecting the RNAi agent into another region, such as the central nervous system (CNS), by intra-shelter injection, intravitreous injection, or other injection, as appropriate, or by injecting the RNAi agent into the bloodstream or subcutaneous space so that the agent subsequently reaches the tissue in which the cells to be contacted are located. For example, the RNAi agent may include or be coupled with a ligand that directs or stabilizes the RNAi agent to the desired site, such as the CNS, such as a lipophilic moiety, as described below and further detailed, for example, in PCT / US2019 / 031170, which is incorporated herein by reference. Combinations of in vitro and in vivo methods for contact are also possible. For example, cells may be contacted with an RNAi agent in vitro and then transferred to a target.

[0150] In one embodiment, contacting cells with an RNAi agent includes “introducing” or “delivering the RNAi agent into cells” by promoting or carrying out uptake or absorption into the cells. Absorption or uptake of the RNAi agent may occur by spontaneously diffusive or active cellular processes, or by adjuvants or devices. Introducing the RNAi agent into cells may be in vitro or in vivo. For example, in the case of in vivo introduction, the RNAi agent may 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 approaches are described below in this specification or are known in the art.

[0151] The term "lipophilic" or "lipophilic moiety" broadly refers to any compound or chemical moiety that has an affinity for lipids. One way to characterize the lipophilicity of a lipophilic moiety is by the octanol-water partition coefficient logK. ow This is by which, in this case, K ow The octanol-water partition coefficient is the ratio of the concentration of a chemical in the octanol phase to the concentration of a chemical in the aqueous phase in a two-phase system at equilibrium. The octanol-water partition coefficient is a laboratory-measured property of a substance. However, it can also be predicted by using a coefficient derived from the structural components of the chemical, calculated using first-principles or empirical methods [see, for example, Tetko et al., J. Chem. Inf. Comput. Sci. 41:1407-21 (2001), whose entirety is incorporated herein by reference]. It provides a thermodynamic measure of a substance's tendency to prefer non-aqueous or oily environments rather than water (i.e., the hydrophilic / lipophilic balance). In principle, a chemical is logK ow If logK is greater than 0, it is lipophilic. Typically, the lipophilic portion is greater than 1, greater than 1.5, greater than 2, greater than 3, greater than 4, greater than 5, or greater than 10. ow It has, for example, the logK of 6-aminohexanol. owIt is expected to be approximately 0.7. Using the same method, the logK of cholesteryl N-(hexane-6-ol) carbamate can be obtained. ow It is expected to be 10.7.

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

[0153] 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 of a plasma protein-binding assay for a double-stranded RNAi agent can be determined to be positively correlated with the relative hydrophobicity of the double-stranded RNAi agent, which may be positively correlated with the silencing activity of the double-stranded RNAi agent.

[0154] In one embodiment, the plasma protein binding assay to be determined is an electrophoretic mobility shift assay (EMSA) using human serum albumin protein. An exemplary protocol for this binding assay is described in detail, for example, 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 in the case of enhanced in vivo delivery of siRNA.

[0155] Therefore, by conjugating the lipophilic portion to the internal position of the double-stranded RNAi agent, optimal hydrophobicity for enhanced in vivo delivery in siRNA is provided.

[0156] The term “lipid nanoparticle” or “LNP” refers to a vesicle containing 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. LNPs are described, for example, in U.S. Patents 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are incorporated herein by reference.

[0157] As used herein, “Subject” means an animal, e.g., a mammal, e.g., a primate (e.g., human, non-human primates, e.g., monkeys and chimpanzees), or a non-primate (e.g., a rat or mouse). In preferred embodiments, the subject is a human, e.g., a human being treated or evaluated for a disease, disorder, or condition that would benefit from reduced HTT expression; a human being at risk for a disease, disorder, or condition that would benefit from reduced HTT expression; a human having a disease, disorder, or condition that would benefit from reduced HTT expression; or a human being treated for a disease, disorder, or condition that would benefit from reduced HTT expression as described herein. In some embodiments, the subject is a human female. In other embodiments, the subject is a human male. In one embodiment, the subject is a human adult. In one embodiment, the subject is a human child. In another embodiment, the subject is a human minor, i.e., a subject under 20 years of age.

[0158] As used herein, the terms “to treat” or “treatment” mean the relief or improvement of one or more signs or symptoms associated with HTT gene expression or HTT protein production, such as, but not limited to, HTT-related diseases such as Huntington's disease. “Treatment” may also mean extending survival compared to the survival expected if no treatment is performed.

[0159] The term “lower” in relation to the level of HTT or disease markers or symptoms in a subject means a statistically significant decrease in such level. The decrease may be, for example, at least 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. In certain embodiments, the decrease is at least 20%. In certain embodiments, the decrease is at least 50% of a disease marker, e.g., a protein level or a gene expression level. When relating to the level of HTT in a subject, “lower” preferably means reducing it to a level that is acceptable as within the normal range in an individual without such disorder. In certain embodiments, “lower” means reducing the difference between the level of a marker or symptom in a subject suffering from the disease and a level that is acceptable within the normal range for the individual, e.g., the level of weight reduction between an obese individual and an individual with a weight that is acceptable within the normal range.

[0160] Where used herein, “prevention” or “prevention” means, when used in relation to a disease, disorder, or condition that would benefit from reduced expression of the HTT gene or reduced production of the HTT protein, a reduction in the likelihood of the subject developing symptoms associated with such disease, disorder, or condition, e.g., symptoms of an HTT-related disease. The absence of developing a disease, disorder, or condition, or a reduction in the development of symptoms associated with such disease, disorder, or condition (e.g., a reduction of at least about 10% of a clinically acceptable scale for that disease or disorder), or a delay in the onset of delayed symptoms (e.g., a delay of days, weeks, months, or years) is considered effective prevention.

[0161] As used herein, the terms “HTT-related disease” or “HTT-related disorder” are understood to mean any disease or disorder that would benefit from reduced expression and / or activity of HTT. An exemplary HTT-related disease is Huntington’s disease.

[0162] Huntington's disease, also known as HD, Huntington's chorea, major chorea, chronic progressive chorea, and hereditary chorea, is an autosomal dominant genetic disorder that typically develops in middle age (35-50 years) and is characterized by choreiform movements and progressive intellectual regression. The disease affects both sexes. The caudate dermal nucleus degenerates, the small cell population deteriorates, and levels of the neurotransmitters gamma-aminobutyric acid (GABA) and substance P decrease. This deterioration results in a characteristic "boxcar ventricle" visible on CT scans.

[0163] The symptoms and signs of HD often develop without the person being aware of it. The most obvious symptoms of HD are abnormal body movements called chorea and incoordination, but it also affects many aspects of intelligence and personality. These physical symptoms generally become more pronounced in the 40s, but can occur at any age. If the onset occurs before the age of 20, it is known as Juvenile HD.

[0164] Dementia or psychiatric disorders, ranging from apathy and excitability to terminal bipolar disorder or schizophrenic disorder, may precede or develop during motor impairment. Anhenamic or antisocial behavior may be the first behavioral signs. Motor signs include limb fluttering, light footing, difficulty maintaining movement (inability to maintain motor activities, e.g., tongue protrusion), grimacing, ataxia, and dystonia.

[0165] HD is caused by trinucleotide repeat elongation in the huntingtin (HTT) gene and is one of several polyglutamine elongation (or PolyQ elongation) disorders. This results in an elongated form of mutant huntingtin protein (mHtt) that causes cell death in select areas of the brain.

[0166] When used herein, “therapeutic dose” is intended to include an amount of RNAi agent sufficient to treat the disease (for example, by reducing, improving, or maintaining one or more symptoms of the existing disease or disease) when administered to a subject with HTT-related disease. “Therapeutic dose” may vary depending on the RNAi agent, how the drug is administered, the disease and its severity, as well as the patient’s medical history, age, weight, family history, genetic makeup, type of prior or concurrent treatment, and any other individual characteristics of the subject being treated.

[0167] When used herein, “Prophylactic effective dose” is intended to include an amount of RNAi agent sufficient to prevent or improve the disease or one or more symptoms of the disease when administered to a subject having an HTT-related disease. Improvement of the disease includes slowing the course of the disease or reducing the severity of the disease if it develops later. The “Prophylactic effective dose” may vary depending on the RNAi agent, how the drug is administered, the degree of the disease risk, and the patient’s medical history, age, weight, family history, genetic makeup, type of prior or concurrent treatment, and any other individual characteristics of the treated patient.

[0168] The “therapeutic dose” or “preventive dose” also includes the amount of RNAi agent that produces several desired local or systemic effects in a reasonable benefit / risk ratio applicable to any treatment. The RNAi agent used in the method of this disclosure may be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0169] The term "pharmaceutically acceptable" is used herein to mean a compound, material, composition, or dosage form that is suitable for use in contact with the tissues of human and animal subjects within the bounds of sound medical judgment, at a reasonable benefit / risk ratio, without excessive toxicity, irritation, allergic reaction, or other problems or complications.

[0170] When used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, magnesium talc, calcium or zinc stearate, or stearic acid), or solvent encapsulating material, that is involved in the transport or delivery of the compound of interest from one organ or part of the body to another organ, e.g., another part of the body. Each carrier must be “acceptable” in the sense that it is compatible with the other raw materials of the formulation and must not be harmful to the subject being treated. Some examples of materials that can function as pharmaceutically acceptable carriers include: (1) sugars, e.g., lactose, glucose, and sucrose; (2) starches, e.g., corn starch and potato starch; (3) cellulose and its derivatives, e.g., sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) tragacanth powder; (5) malt; (6) gelatin; (7) lubricants, e.g., magnesium stearate, sodium lauryl sulfate, and talc; (8) excipients, e.g., cocoa butter and suppository waxes; (9) oils, e.g., peanut oil, cottonseed oil, safflower oil, sesame oil. (10) Glycols, e.g., propylene glycol; (11) Polyols, e.g., glycerin, sorbitol, mannitol, and polyethylene glycol; (12) Esters, e.g., ethyl oleate and ethyl laurate; (13) Agar; (14) Buffers, e.g., magnesium hydroxide and aluminum hydroxide; (15) Alginic acid; (16) Phenothermally hydrated; (17) Isotonic saline; (18) Ringer's solution; (19) Ethyl alcohol; (20) pH buffer solution; (21) Polyesters, polycarbonates, or polyanhydrides; (22) Bulking agents, e.g., polypeptides and amino acids; (23) Serum components, e.g., serum albumin, HDL, and LDL; and (22) other non-toxic affinity substances used in pharmaceutical formulations.

[0171] The term “sample,” as used herein, encompasses similar bodily fluids, cells, or tissues isolated from a subject, as well as collections of bodily fluids, cells, or tissues present within the subject. Examples of bodily fluids include blood, serum, and serous fluid, plasma, cerebrospinal fluid, ocular fluid, lymph, urine, saliva, and the like. Tissue samples may include samples from tissues, organs, or local areas. For example, a sample may be obtained from a specific organ, a part of an organ, or bodily fluids or cells within those organs. In certain embodiments, a sample may be obtained from the brain (e.g., the whole brain or a segment of the brain, e.g., striatum, or a certain type of cell in the brain, e.g., neurons and glial cells (astrocytes, oligodendrocytes, microglial cells)). In some embodiments, “sample obtained from subject” means blood obtained from the subject or plasma or serum obtained therefrom. In further embodiments, “sample obtained from subject” means brain tissue (or its minor components) obtained from the subject. ) or retinal tissue (or its minor components).

[0172] II. RNAi agents of the present disclosure This specification describes RNAi agents that inhibit the expression of the HTT gene. In one embodiment, the RNAi agent comprises a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting the expression of HTT in cells, e.g., cells within a subject, e.g., mammals, e.g., humans with HTT-related diseases such as Huntington's disease. The dsRNA comprises an antisense strand having a complementary region that is complementary to at least a portion of the mRNA formed during HTT gene expression. The complementary region is approximately 15 to 30 nucleotides long or less. In contact with cells expressing the HTT gene, the RNAi agent inhibits the expression of the HTT gene (e.g., human gene, primate gene, non-primate gene) by at least 50%, as evaluated by, for example, PCR or a branched DNA (bDNA)-based method, or a protein-based method, e.g., immunofluorescence analysis using Western blotting or flow cytometry techniques. In one embodiment, the level of knockdown is evaluated in Cos7 cells using a dual-luciferase assay method.

[0173] dsRNA comprises two RNA strands, which are complementary and hybridize to form a double-stranded structure under the conditions in which the dsRNA is used. One strand of the dsRNA (the antisense strand) contains a complementary region that is substantially complementary and generally perfectly complementary to the target sequence. The target sequence can be obtained from the sequence of mRNA formed during the expression of the HTT gene. The other strand (the sense strand) contains a region complementary to the antisense strand, thereby the two strands hybridize to form a double-stranded structure when combined under suitable conditions. As described elsewhere in this specification and known in the art, the complementary sequence of the dsRNA can also be included as a self-complementary region of a single nucleic acid molecule, so as to be relative on separate oligonucleotides.

[0174] Generally, double-stranded structures are 15 to 30 base pairs long, for example, 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, 19-2 The lengths are 9, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. In certain preferred embodiments, the double-stranded structure is 18 to 25 base pairs long, e.g., 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-25, 20-24, 20-23, 20-22, 20-21, 21-25, 21-24, 21-23, 21-22, 22-25, 22-24, 22-23, 23-25, 23-24, or 24-25 base pairs long, e.g., 19-21 base pairs long. It is conceivable that intermediate ranges and lengths between those listed above are also part of this disclosure.

[0175] Similarly, the complementary region to the target sequence is 15 to 30 nucleotides long, 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 nucleotide lengths, for example, 19-23 nucleotide lengths or 21-23 nucleotide lengths. It is conceivable that intermediate ranges and lengths between the above-listed ranges and lengths are also part of this disclosure.

[0176] In some embodiments, the double-stranded structure is 19 to 30 base pairs long. Similarly, the complementary region to the target sequence is 19 to 30 nucleotides long.

[0177] In some embodiments, dsRNAs are 15 to 23 nucleotides long, 19 to 23 nucleotides long, or 25 to 30 nucleotides long. Generally, dsRNAs are long enough to function as substrates for Dicer enzymes. For example, it is well known in the art that dsRNAs longer than about 21–23 nucleotides can function as substrates for Dicer. As those skilled in the art will also recognize, the RNA region targeted for cleavage is in most cases a longer RNA molecule, often a portion of an mRNA molecule. Where applicable, the “portion” of the mRNA target is a sequence of mRNA targets long enough to allow it to be a substrate for RNAi-dependent cleavage (i.e., cleavage via the RISC pathway).

[0178] Those skilled in the art will know that the double-stranded region is the primary functional portion of dsRNA, for example, 15 to 36 base pairs, for example, 15-36, 15-35, 15-34, 15-33, 15-32, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 1 You will also recognize that these are 8-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs, for example, a double-stranded region of 19-21 base pairs. Therefore, in one embodiment, an RNA molecule or complex of RNA molecules having a double-stranded region of more than 30 base pairs is a dsRNA, insofar as it is processed into a functional double helix of, for example, 15-30 base pairs, which targets the desired RNA for cleavage. Thus, those skilled in the art will recognize that in one embodiment, miRNA is a dsRNA. In another embodiment, dsRNA is not a naturally occurring miRNA. In another embodiment, RNAi agents useful for targeting HTT expression are not generated in target cells by cleavage of larger dsRNAs.

[0179] The dsRNAs described herein may further include one or more single-stranded nucleotide overhangs, for example, 1, 2, 3, or 4 nucleotides. The nucleotide overhangs may include or consist of nucleotide / nucleoside analogs such as deoxynucleotides / nucleosides. The overhangs may be on the sense strand, on the antisense strand, or in any combination thereof. Furthermore, the nucleotides of the overhangs may be located on the 5' end, the 3' end, or both of either the antisense strand or the sense strand of the dsRNA.

[0180] dsRNA can be synthesized by standard methods known in the art. The double-stranded RNAi compounds of the present invention can be prepared using a two-step method. First, the individual strands of the double-stranded RNA molecule are prepared separately. Then, the strands of the component are annealed. The individual strands of the siRNA compound can be prepared using solution-phase or solid-phase organic synthesis or both. Organic synthesis offers the advantage that oligonucleotide chains containing non-natural or modified nucleotides can be easily prepared. Similarly, the single-stranded oligonucleotides of the present invention can be prepared using solution-phase or solid-phase organic synthesis or both.

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

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

[0183] 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. However, it will be understood that the RNA of the RNAi agent of this disclosure, for example, the dsRNA of this disclosure, may contain any one of the sequences described in any one of Tables 2, 3, 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 21, 24, 25, 27-30, 32, and 33, which may be unmodified, unconjugated, or modified or conjugated in a way different from those described. For example, the sense strands of the agents of the present invention, as shown in Tables 3, 9, 12, 15, 17, 27, 29, and 32, are conjugated to a GalNAc ligand, but these agents may also be conjugated to a portion that induces delivery to a CNS, such as a C16 ligand, as described herein. Lipophilic ligands may be included at any of the positions provided herein.

[0184] Those skilled in the art are well aware that dsRNAs having double-stranded structures of about 20 to 23 base pairs, for example, 21 base pairs, have been welcomed as particularly effective in introducing RNA interference [Elbashir et al., (2001) EMBO J., 20:6877-6888]. However, others have found that shorter or longer RNA double-stranded structures may also be effective [Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226]. In the embodiments described above, due to the nature of the oligonucleotide sequences provided herein, the dsRNAs described herein may include at least one strand of a minimum length of 21 nucleotides. It can be reasonably expected that shorter double-stranded structures, with some nucleotides subtracted from one or both ends, may be equally effective compared to the dsRNAs described above. Accordingly, dsRNAs having a sequence of at least 15, 16, 17, 18, 19, 20, or more consecutive nucleotides obtained from one of the sequences provided herein, and which differ in their ability to inhibit the expression of the HTT gene by 10, 15, 20, 25, or 30% or less inhibition from dsRNAs containing the complete sequence, using in vitro assays with Cos7 and 10 nM concentration RNA agents and PCR assays provided in the examples herein, are conceivable to be within the scope of this disclosure.

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

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

[0187] The nucleic acids featured in this disclosure can be synthesized or modified by methods well established in the art, for example, 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'-end modifications (phosphorylation, conjugation, reverse linking) or 3'-end modifications (conjugation, DNA nucleotides, reverse linking, etc.), base modifications, such as replacement of stabilizing bases, destabilizing bases or bases that form base pairs with partners in an expanded repertoire, base removal (debasing nucleotides) or conjugated bases, sugar modifications (e.g., at the 2' or 4' position) or sugar replacement, or skeletal modifications including modification or replacement of phosphodiester bonds. Specific examples of RNAi agents useful in the embodiments described herein, but not limited to, RNA containing a modified skeleton or lacking natural nucleoside linkages, include RNA containing a modified skeleton or lacking natural nucleoside linkages. Among RNAs having a modified skeleton, those lacking a phosphorus atom in their skeleton are particularly noteworthy. For the purposes of this specification, as sometimes mentioned in the art, modified RNAs lacking a phosphorus atom in their internucleoside skeleton can also be considered oligonucleosides. In some embodiments, the modified RNAi agent has a phosphorus atom in its internucleoside skeleton.

[0188] Examples of modified RNA backbones include phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotryesters, aminoalkyl phosphotryesters, methylphosphonates, and other alkylphosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotryesters, and boranophosphates having the usual 3'-5' linkage, their analogues with 2'-5' linkages, and those having reverse polarity where adjacent pairs of nucleoside units are linked from 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included. In some embodiments of the present invention, the dsRNA agent of the present invention is in free acid form. In other embodiments of the present invention, the dsRNA agent of the present invention is in salt form. In one embodiment, the dsRNA agent of the present invention is in sodium salt form. In certain embodiments, when the dsRNA agent of the present invention is in sodium salt form, sodium ions are present in the agent as counterions to substantially all of the phosphodiester and / or phosphorothioate groups present in the agent. Agents in which substantially all of the phosphodiester and / or phosphorothioate linkages have sodium counterions include 5, 4, 3, 2, or 1 or fewer phosphodiester and / or phosphorothioate linkages that do not have sodium counterions. In some embodiments, when the dsRNA agent of the present invention is in sodium salt form, sodium ions are present as counterions to all of the phosphodiester and / or phosphorothioate groups present in the agent.

[0189] Representative U.S. patents teaching the preparation of the phosphorus-containing linkages described above include, but are not limited to, U.S. Patents 3,687,808, 4,469,863, 4,476,301, 5,023,243, 5,177,195, 5,188,897, 5,264,423, 5,276,019, 5,278,302, and 5,286,71 No. 7, No. 5,321,131, No. 5,399,676, No. 5,405,939, No. 5,453,496, No. 5,455,233, No. 5,466,677, No. 5,476 , No. 925, No. 5,519,126, No. 5,536,821, No. 5,541,316, No. 5,550,111, No. 5,563,253, No. 5,571,799, No. 5,5 87,361, 5,625,050, 6,028,188, 6,124,445, 6,160,109, 6,169,170, 6,172,209, No. 6,239,265, No. 6,277,603, No. 6,326,199, No. 6,346,614, No. 6,444,423, No. 6,531,590, No. 6,534,639 Examples include U.S. Patent Nos. 6,608,035, 6,683,167, 6,858,715, 6,867,294, 6,878,805, 7,015,315, 7,041,816, 7,273,933, 7,321,029 and U.S. Reissue Patent No. RE39464, the entirety of each of these is incorporated herein by reference.

[0190] Modified RNA skeletons that do not contain phosphorus atoms have skeletons formed by short alkyl or cycloalkyl nucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl nucleoside linkages, or one or more short heteroatoms or heterocyclic nucleoside linkages. These include morpholino linkages (some formed from the sugar moiety of nucleosides), siloxane skeletons, sulfide, sulfoxide and sulfone skeletons, formacetyl and thioformacetyl skeletons, methyleneformacetyl and thioformacetyl skeletons, alkene-containing skeletons, sulfamate skeletons, methyleneimino and methylenehydrazino skeletons, sulfonate and sulfonamide skeletons, amide skeletons, and others having mixed N, O, S and CH2 component moieties.

[0191] Representative U.S. patents teaching the preparation of the above-mentioned oligonucleotides include, but are not limited to, U.S. Patents 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, and the same. Nos. 5,489,677, 5,541,307, 5,561,225, 5,596,086, 5,602,240, 5,608,046, 5,610,289, 5,618,704, 5,623,070, 5,663,312, 5,633,360, 5,677,437, and 5,677,439 are examples, the entire contents of each of these are incorporated herein by reference.

[0192] In other embodiments, RNA mimetics suitable for use in RNAi agents are envisioned in which both sugar and nucleoside linkages, i.e., the nucleotide unit backbone, are replaced with novel groups. The base units are maintained for hybridization with suitable nucleic acid target compounds. One such oligomeric compound, an RNA mimetic known to have excellent hybridization properties, is called a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, in particular an aminoethylglycine backbone. The nucleic acid bases are retained and directly or indirectly bonded to the aza nitrogen atom of the amide portion of the backbone. Representative U.S. patents teaching the preparation of PNA compounds, but not limited to, U.S. Patents 5,539,082, 5,714,331, and 5,719,262, the entire contents of each of which are incorporated herein by reference. Further PNA compounds suitable for use in the RNAi agents of this disclosure are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0193] Some embodiments featured in this disclosure include RNAs and heteroatom skeletons having a phosphorothioate backbone, in particular the --CH2--NH--CH2-, --CH2--N(CH3)--O--CH2-- [known as the methylene (methylimino) or MMI backbone], --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2-- and --N(CH3)--CH2--CH2-- [the natural phosphodiester backbone is represented as --O--P--O--CH2--] and oligonucleosides having an amide backbone as referenced above in U.S. Patent No. 5,602,240. In some embodiments, the RNAs featured herein have a morpholino backbone structure as referenced above in U.S. Patent No. 5,034,506.

[0194] The modified RNA may also contain one or more substituted sugar moieties. The RNAi agents characterized herein, e.g., dsRNA, may contain at the 2'-position one of the following: OH; F; O-, S- or N-alkyl; O-, S- or N-alkenyl; O-, S- or N-alkynyl or O-alkyl-O-alkyl, where alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1-C 10 alkyl or C2-C 10 alkenyl and alkynyl. 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 CH3)]2, where n and m are from 1 to about 10. In other embodiments, the dsRNA has at the 2'-position the following: C1-C 10The modifications include one of the following: 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, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, interfering substance, group for improving the pharmacokinetic properties of RNAi agents or group for improving the pharmacokinetic properties of RNAi agents, and other substituents having similar properties. In some embodiments, the modifications include 2'-methoxyethoxy (2'-O-(2-methoxyethyl) or 2'-MOE, also known as 2'-O--CH2CH2OCH3) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., an alkoxy-alkoxy group. Other exemplary modifications include 2'-dimethylaminooxyethoxy, i.e., the O(CH2)2ON(CH3)2 group also known as 2'-DMAOE, as described below in the examples herein, 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 nucleotide, 5'-Me-2'-OMe nucleotide, 5'-Me-2'-deoxynucleotide (both R and S isomers in these three families), 2'-alkoxyalkyl, and 2'-NMA (N-methylacetamide).

[0195] 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 on the RNA of the RNAi agent, particularly on the 3' terminal nucleotide or at the 3' position and 5' position of the sugar in the 2'-5' ligated dsRNA. The RNAi agent may also have sugar mimetic moieties, such as a cyclobutyl moiety instead of a pentofuranosyl sugar. Representative U.S. patents teaching the preparation of such modified sugar structures include, but are not limited to, U.S. Patents 4,981,957, 5,118,800, 5,319,080, 5,359,044, 5,393,878, 5,446,137, 5,466,786, 5,514,785, 5,519,134, and 5,56 Examples include patents 7,811, 5,576,427, 5,591,722, 5,597,909, 5,610,300, 5,627,053, 5,639,873, 5,646,265, 5,658,873, 5,670,633, and 5,700,920, some of which are jointly owned with this application. The entire content of each of the aforementioned is incorporated herein by reference.

[0196] The RNAi agents of this disclosure may also include modifications or substitutions of nucleic acid bases (often simply referred to as “bases” in the art). As used herein, “unmodified” or “natural” nucleic acid bases include the purine bases adenine (A) and guanine (G), the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleic acid bases include other synthetic and natural nucleic acid bases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, and 5-uracil. This includes (pseudracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and (anal) 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-deazaadenine (daazaadenine), as well as 3-deazaguanine and 3-deazaadenine.Further nucleic acid bases include those disclosed in U.S. Patent No. 3,687,808, Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008, The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., (1991) Angewandte Chemie, International Edition, 30:613, and Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleic acid bases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in this 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 substitution has been shown to increase nucleic acid double-strand 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 more specifically, is an exemplary base substitution when combined with 2'-O-methoxyethyl sugar modification.

[0197] Representative U.S. patents teaching the preparation of the above-mentioned modified nucleic acid bases and certain other modified nucleic acid bases include, but are not limited to, U.S. Patents 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, and 5,587,469, Reference numbers 5,594,121, 5,596,091, 5,614,617, 5,681,941, 5,750,692, 6,015,886, 6,147,200, 6,166,197, 6,222,025, 6,235,887, 6,380,368, 6,528,640, 6,639,062, 6,617,438, 7,045,610, 7,427,672, and 7,495,088 are examples, the entire contents of each of these are incorporated herein by reference.

[0198] The RNAi agents of this disclosure can also be modified to include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety includes an additional crosslink connecting the 2' and 4' carbons. This structure efficiently "locks" the ribose into a 3'-end conformation. The addition of locked nucleic acids to siRNA has been shown to increase siRNA stability in serum and reduce off-target effects [Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447, Mook, OR. et al., (2007) Mol anc Ther 6(3):833-843, Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193].

[0199] The RNAi agents of this disclosure can also be modified to include one or more bicyclic sugar moieties. A “bicyclic sugar” is a furanosyl ring modified by bridging two atoms. A “bicyclic nucleoside” (“BNA”) is a nucleoside having a sugar moiety that includes a bridge connecting two carbon atoms of a sugar ring, thereby forming a bicyclic ring structure. In certain embodiments, the bridge connects the 4'-carbon and 2'-carbon of the sugar ring. Thus, in some embodiments, the agents of this disclosure may include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety includes an additional bridge connecting the 2' and 4' carbons. In other words, an LNA is a nucleotide having a bicyclic sugar moiety that includes a 4'-CH2-O-2' bridge. This structure efficiently “locks” the ribose into a 3'-end conformation. The addition of locked nucleic acids to siRNA has been shown to increase siRNA stability in serum and reduce off-target effects [Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447, Mook, OR. et al., (2007) Mol Canc Ther 6(3):833-843, Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193]. Examples of bicyclic nucleosides for use in the polynucleotides of this disclosure include, but are not limited to, nucleosides containing a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, one or more bicyclic nucleosides containing a 4'-to-2' bridge may be used as antisense polynucleotide agents of this disclosure.Examples of such 4'-to-2' cross-linked 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 "restricted ethyl" or "cEt") and 4'-CH(CH2OCH3)-O-2' (and its analogues, see, for example, U.S. Patent No. 7,399,845), 4'-C(CH3)(CH3)-O-2' (and its analogues, see, for example, U.S. Patent No. 8, See Patent Nos. 278,283), 4'-CH2-N(OCH3)-2' (and its analogues, e.g., see U.S. Patent No. 8,278,425), 4'-CH2-ON(CH3)-2' (e.g., see U.S. Patent Publication No. 2004 / 0171570), 4'-CH2-N(R)-O-2' (wherein R is H, C1-C12 alkyl or protecting group) (e.g., see U.S. Patent No. 7,427,672), 4'-CH2-C(H)(CH3)-2' (e.g., Chattopadhyaya Examples include 4'-CH2-C(-CH2)-2' (and its analogues, see, for example, U.S. Patent No. 8,278,426), and 4'-CH2-C(-CH2)-2' (see, et al., J. Org. Chem., 2009, 74, 118-134). The entire contents of each of the foregoing are incorporated herein by reference.

[0200] Further representative U.S. patents and U.S. patent publications teaching the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Patent Nos. 6,268,490, 6,525,191, 6,670,461, 6,770,748, 6,794,499, 6,998,484, 7,053,207, 7,034,133, 7,084,125, and the same. Examples include patent numbers 7,399,845, 7,427,672, 7,569,686, 7,741,457, 8,022,193, 8,030,467, 8,278,425, 8,278,426, 8,278,283, US2008 / 0039618, and US2009 / 0012281, the entire contents of each of these are incorporated herein by reference.

[0201] For example, any of the aforementioned bicyclic nucleosides having one or more stereochemical sugar configurations, including α-L-ribofuranose and β-D-ribofuranose, can be prepared (see WO99 / 14226).

[0202] The RNAi agents of this disclosure can also be modified to include one or more restricted ethyl nucleotides. As used herein, “restricted ethyl nucleotide” or “cEt” is a locked nucleic acid comprising a bicyclic sugar moiety including a 4'-CH(CH3)-0~2' bridge. In one embodiment, the restricted ethyl nucleotide is in the S conformation and is referred to herein as “S-cEt”.

[0203] The RNAi agents of this disclosure may also comprise one or more “conformation-restricted nucleotides” (“CRNs”). CRNs are nucleotide analogs having 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 to mRNA. The linker is long enough to position the oxygen optimally for stability and affinity, resulting in less ribose ring puckering.

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

[0205] In some embodiments, the RNAi agents of the present disclosure include one or more monomers that are unlocked nucleic acid (UNA) nucleotides. UNA is an unlocked acyclic nucleic acid in which any sugar linkages have been removed to form an unlocked “sugar” residue. In one example, UNA also includes monomers in which the bond between C1’-C4’ (i.e., the carbon-oxygen-carbon bond of the covalent bond between C1’ and C4’ carbons) has been removed. In another example, the C2’-C3’ bond of the sugar (i.e., the carbon-carbon bond of the covalent 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, which are incorporated herein by reference].

[0206] Representative U.S. publications teaching the preparation of UNA include, but are not limited to, US8,314,227 and U.S. Patent Publications 2013 / 0096289, 2013 / 0011922, and 2011 / 0313020, the entire contents of each of which are incorporated herein by reference.

[0207] Potentially stabilizing modifications to the ends of RNA molecules include, but are not limited to, N-(acetylaminocaproyl)-4-hydroxyproline (Hyp-C6-NHAc), N-(caproyl-4-hydroxyproline (Hyp-C​​6), N-(acetyl-4-hydroxyproline (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyproline (Hyp-C6-amino), 2-docosanoyl-uridine-3”-phosphate, reverse base dT (idT), and others. The disclosure of this modification can be found in WO2011 / 005861.

[0208] Other modifications of the RNAi agents of this disclosure include 5' phosphates or 5' phosphate mimics, for example, a 5' terminal phosphate or phosphate mimic on the antisense strand of the RNAi agent. Suitable phosphate mimics are disclosed, for example, in US2012 / 0157511, the entirety of which is incorporated herein by reference.

[0209] A. Modified RNAi agents containing motifs of the present disclosure In certain embodiments of this disclosure, the double-stranded RNAi agents of this disclosure include agents having chemical modifications such as those disclosed in WO2013 / 075035, the entirety of which is incorporated herein by reference. Excellent results can be obtained by introducing one or more motifs of three identical modifications on a triple nucleotide into the sense or antisense strand of the RNAi agent at or near the cleavage site, as shown herein and in WO2013 / 075035. In some embodiments, the sense and antisense strands of the RNAi agent may otherwise be fully modified. The introduction of these motifs disrupts the modification pattern of the sense or antisense strand, if present. The RNAi agent may be conjugated with a lipophilic ligand, for example, a C16 ligand on the sense strand. The RNAi agent may be modified, for example, with (S)-glycol nucleic acid (GNA) modification at one or more residues on the antisense strand. The resulting RNAi agent exhibits excellent gene silencing activity.

[0210] Accordingly, this disclosure provides a double-stranded RNAi agent capable of inhibiting the expression of a target gene (i.e., the HTT gene) in vivo. The RNAi agent comprises a sense strand and an antisense strand. Each strand of the RNAi agent may be 15 to 30 nucleotides long. For example, each strand may be 16 to 30 nucleotides long, 17 to 30 nucleotides long, 25 to 30 nucleotides long, 27 to 30 nucleotides long, 17 to 23 nucleotides long, 17 to 21 nucleotides long, 17 to 19 nucleotides long, 19 to 25 nucleotides long, 19 to 23 nucleotides long, 19 to 21 nucleotides long, 21 to 25 nucleotides long, or 21 to 23 nucleotides long. In a particular embodiment, each strand is 19 to 23 nucleotides long.

[0211] The sense strand and antisense strand typically form a double-stranded RNA ("dsRNA"), also referred herein as the "RNAi agent." The double-stranded region of the RNAi agent may be 15–30 nucleotide pairs long. For example, the double-stranded region may be 16–30 nucleotide pairs long, 17–30 nucleotide pairs long, 27–30 nucleotide pairs long, 17–23 nucleotide pairs long, 17–21 nucleotide pairs long, 17–19 nucleotide pairs long, 19–25 nucleotide pairs long, 19–23 nucleotide pairs long, 19–21 nucleotide pairs long, 21–25 nucleotide pairs long, or 21–23 nucleotide pairs long. In another example, the double-stranded region is selected from lengths of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides. In a preferred embodiment, the double-stranded region is 19–21 nucleotide pairs long.

[0212] In one embodiment, the RNAi agent may contain one or more overhang regions or capping groups at the 3' end, 5' end, or both ends of one or both strands. The overhangs may be 1 to 6 nucleotides long, for example, 2 to 6 nucleotides, 1 to 5 nucleotides, 2 to 5 nucleotides, 1 to 4 nucleotides, 2 to 4 nucleotides, 1 to 3 nucleotides, 2 to 3 nucleotides, or 1 to 2 nucleotides. In a preferred embodiment, the nucleotide overhang region is 2 nucleotides long. The overhangs may result from one strand being longer than the other, or from two strands of equal length being twisted. The overhangs may form a mismatch with the target mRNA, or may be complementary to the targeted gene sequence, or may be a different sequence. The first and second strands may also be joined, for example, by additional bases forming a hairpin, or by other non-base linkers.

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

[0214] For example, TT could be an overhang sequence at any end of either strand. The overhang could form a mismatch with the target mRNA, or it could be complementary to the targeted gene sequence, or it could be a different sequence altogether.

[0215] The sense strand, antisense strand, or 5'- or 3'-overhangs of both strands of an RNAi agent can be phosphorylated. In some embodiments, the overhang region(s) contains two nucleotides with a phosphorothioate between them, and the two nucleotides may be identical or different. In one embodiment, the overhang is located at the 3' end of the sense strand, antisense strand, or both strands. In one embodiment, this 3'-overhang is located in the antisense strand. In one embodiment, this 3'-overhang is located in the sense strand.

[0216] RNAi agents may contain only a single overhang that can enhance RNAi interference activity without affecting their overall stability. For example, a single-stranded overhang may be located at the 3' end of the sense strand or the 3' end of the antisense strand. RNAi may also have a blunt end located at the 5' end of the antisense strand (or the 3' end of the sense strand), or vice versa. Generally, the antisense strand of RNAi has a nucleotide overhang at the 3' end and a blunt end at the 5' end. Without getting bogged down in theory, the blunt end at the 5' end of an asymmetric antisense strand and the 3' end overhang of the antisense strand are advantageous for guide strand loading into RISC processes.

[0217] In one embodiment, the RNAi agent is a 19-nucleotide-long double bluntmer, where the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 7, 8, and 9 from the 5' end. The antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

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

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

[0220] In one embodiment, the RNAi agent comprises a 21-nucleotide sense strand and a 23-nucleotide antisense strand, wherein the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 11 from the 5' end, and the antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, with one end of the RNAi agent being blunt and the other end containing a 2-nucleotide overhang. Preferably, the 2-nucleotide overhang is at the 3' end of the antisense strand. If the 2-nucleotide overhang is at the 3' end of the antisense strand, there may be two phosphorothioate nucleotide linkages between the three terminal nucleotides, where two of the three nucleotides are the overhang nucleotides and the third nucleotide is the nucleotide that follows the overhang nucleotide. In one embodiment, the RNAi agent further has two phosphorothioate nucleotide linkages between the three terminal nucleotides at both the 5' end of the sense strand and the 5' end of the antisense strand. In one embodiment, any nucleotide in the sense strand and antisense strand of the RNAi agent, including a nucleotide that is part of a motif, is a modified nucleotide. In one embodiment, each residue is independently modified, for example, with 2'-O-methyl or 3'-fluoro in an alternating motif. The RNAi agent may further contain a ligand (e.g., a lipophilic ligand, optionally a C16 ligand).

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

[0222] In one embodiment, the RNAi agent comprises sense and antisense strands, the RNAi agent comprising a first strand having a length of at least 25 and at most 29 nucleotides, and a second strand having a length of at most 30 nucleotides and having at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, the 3' end of the first strand and the 5' end of the second strand form blunt ends, the second strand is 1 to 4 nucleotides longer at its 3' end than the first strand, the double-stranded region is a region of at least 25 nucleotides, the second strand is sufficiently complementary to the target mRNA along the length of the second strand of at least 19 nucleotides, and the RNAi agent reduces the expression of the target gene when introduced into mammalian cells, the dicer cleavage of the RNAi agent preferentially yields the siRNA including the 3' end of the second strand, thereby reducing the expression of the target gene in mammals. Optionally, the RNAi agent may further comprise a ligand.

[0223] In one embodiment, the sense strand of the RNAi agent contains at least one motif of three identical modifications on a triple nucleotide sequence, one of which occurs at a cleavage site in the sense strand.

[0224] In one embodiment, the antisense strand of the RNAi agent may also contain at least one motif of three identical modifications on a triple nucleotide, one of which occurs at or near a cleavage site in the antisense strand.

[0225] For RNAi agents having a double-stranded region of 17–23 nucleotides in length, the cleavage sites on the antisense strand are typically at positions 10, 11, and 12 from the 5' end. Therefore, three identical modification motifs may occur at positions 9, 10, 11, 10, 11, 12, 11, 12, 13, 12, 13, 12, 13, 14, or 13, 14, 15 of the antisense strand, with the number starting from the first nucleotide from the 5' end of the antisense strand, or the number starting from the first pair-formed nucleotide within the double-stranded region from the 5' end of the antisense strand. The cleavage sites in the antisense strand may also vary depending on the length of the double-stranded region of the RNAi from the 5' end.

[0226] The sense strand of an RNAi agent may contain at least one motif of three identical modifications on a triple nucleotide at the cleavage site of the strand, and the antisense strand may have at least one motif of three identical modifications on a triple nucleotide at or near the cleavage site of the strand. When the sense strand and antisense strand form a dsRNA double helix, the sense strand and antisense strand can be sequenced such that one motif of three nucleotides on the sense strand and one motif of three nucleotides on the antisense strand have at least one nucleotide duplication, i.e., at least one of the three 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 may be duplicated, or all three nucleotides may be duplicated.

[0227] In one embodiment, the sense strand of an RNAi agent may contain two or more motifs of three identical modifications on a triple nucleotide sequence. The first motif may occur at or near a cleavage site on the strand, and the other motifs may be wing modifications. In this specification, the term “wing modification” refers to a motif occurring on a different part of the strand, away from the motif at or near the cleavage site on the same strand. Wing modifications are either adjacent to the first motif or at least one or more nucleotides away. If the motifs are immediately adjacent to each other, their chemistry is distinct from each other; if the motifs are one or more nucleotides away, their chemistry may be identical or different. There may be two or more wing modifications. For example, if there are two wing modifications, each wing modification may occur at one end relative to the first motif at or near the cleavage site, or on either side of the read motif.

[0228] Similar to the sense strand, the antisense strand of an RNAi agent may contain two or more motifs of three identical modifications on a triple nucleotide sequence, with at least one motif occurring at or near a cleavage site on the strand. This antisense strand may also contain one or more wing modifications in a sequence similar to those present on the sense strand.

[0229] In one embodiment, wing modifications on the sense or antisense strand of an RNAi agent typically do not include the first one or two terminal nucleotides at the 3' end, 5' end, or both ends of the strand.

[0230] In another embodiment, wing modifications on the sense or antisense strand of an RNAi agent typically do not include the first one or two pairs of nucleotides in the double-stranded region at the 3' end, 5' end, or both ends of the strand.

[0231] If the sense strand and antisense strand of an RNAi agent each contain at least one wing modification, the wing modification may be located at the same end of the double-stranded region and may have one, two, or three nucleotide duplicates.

[0232] If the sense strand or antisense strand of the RNAi agent each contains at least two wing modifications, the sense strand and antisense strand can be arranged such that two modifications from one strand each enter one end of the double-stranded region with 1, 2, or 3 nucleotide duplicates, and two modifications from one strand each enter the other end of the double-stranded region with 1, 2, or 3 nucleotide duplicates, and one strand of the two modifications enters each side of the read motif with 1, 2, or 3 nucleotide duplicates in the double-stranded region.

[0233] In one embodiment, the RNAi agent includes a double-strand mismatch(s) or combination thereof with the target. Mismatches may occur in overhang regions or double-strand regions. Base pairs can be ranked based on their tendency to promote dissociation or dissolution (e.g., by the free energy of association or dissociation of a particular pairing, the simplest approach being to examine pairs on a basis of individual pairs, although the following adjacent analysis or similar analysis can also be used). In terms of promoting dissociation: A:U is preferred over G:C, G:U is preferred over G:C, and I:C is preferred over G:C (I = inosine). Mismatches, e.g., non-canonical pairing or non-canonical pairing (as described elsewhere herein) are preferred over canonical (A:T, A:U, G:C) pairing, and pairing involving universal bases is preferred over canonical pairing.

[0234] In one embodiment, the RNAi agent includes at least one of the first 1, 2, 3, 4, or 5 base pairs in the double-stranded region from the 5' end of the antisense strand, independently selected from the group of A:U, G:U, I:C, and mismatch pairs, e.g., non-canonical pairing, non-canonical pairing, or pairings involving universal bases, in order to facilitate the dissociation of the antisense strand at the 5' end of the double helix.

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

[0236] In another embodiment, the nucleotide at the 3' end of the sense strand is deoxythymine (dT). In yet 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, e.g., two dT nucleotides at the 3' ends of the sense or antisense strand.

[0237] In one embodiment, the sense strand sequence is given by formula (I): 5'n p -N a -(XXX) i -N b -YYY -N b -(ZZZ) j -N a -n q 3' (I) [In the formula, i and j are independently either 0 or 1. p and q are each independently between 0 and 6. each N a Each independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, where each sequence contains at least two differently modified nucleotides. each N bEach independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides. each n p and n q These independently represent overhang nucleotides, Nb and Y do not have the same modifications, and XXX, YYY, and ZZZ each independently represent one motif of three identical modifications on a sequence of three nucleotides. It can be represented by the following. Preferably, all YYY nucleotides are 2'-F modified nucleotides.

[0238] In one embodiment, N a or N b This includes alternating modification patterns.

[0239] In one embodiment, the YYY motif occurs at or near the sense strand cleavage site. For example, if the RNAi agent has a double-stranded region of 17-23 nucleotides in length, the YYY motif may occur at or near the sense strand cleavage site (e.g., at positions 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12 or 11, 12, 13), the number may start from the first nucleotide from the 5' end, or, as appropriate, the number may start from the first pair-formed nucleotide in the double-stranded region from the 5' end.

[0240] In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. Therefore, the sense chain is given by the following equation: 5' n p -N a -YYY-N b -ZZZ-N a -n q 3' (Ib), 5' n p -N a -XXX-N b -YYY-N a -n q 3' (Ic), or 5' n p -N a-XXX-N b -YYY-N b -ZZZ-N a -n q 3' (Id) It can be represented by [this].

[0241] If the sense chain is represented by formula (Ib), then N b This represents an oligonucleotide sequence containing modified nucleotides of 0-10, 0-7, 0-5, 0-4, 0-2, or 0.

[0242] each N a These can independently represent oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides.

[0243] If the sense chain is expressed as equation (Ic), then N b This represents an oligonucleotide sequence containing modified nucleotides of 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0. a These may also independently represent oligonucleotide sequences containing 2–20, 2–15, or 2–10 modified nucleotides.

[0244] When the sense chain is expressed as formula (Id), each N b Each independently represents an oligonucleotide sequence containing modified nucleotides of 0-10, 0-7, 0-5, 0-4, 0-2, or 0. Preferably, N b is 0, 1, 2, 3, 4, 5, or 6. a These can also independently represent oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides.

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

[0246] In other embodiments, i is 0, j is 0, and the sense chain is given by: 5' n p -N a-YYY-N a -n q 3' (Ia) It can be represented by [this].

[0247] If the sense chain is represented by equation (Ia), then each N a These may independently contain oligonucleotide sequences comprising 2-20, 2-15, or 2-10 modified nucleotides.

[0248] In one embodiment, the antisense strand sequence of RNAi is given by formula (II): 5' n q’ -N a '-(Z'Z'Z') k -N b '-Y'Y'Y'-N b '-(X'X'X') l -N' a -n p ' 3' (II) [In the formula, k and l are independently either 0 or 1. p' and q' are each independently between 0 and 6. each N a ' independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, where each sequence contains at least two differently modified nucleotides. each N b 'Independently, this represents an oligonucleotide sequence containing 0 to 10 modified nucleotides, each n p 'and n q ' independently represents an overhang nucleotide, N b 'and Y' do not have the same modifier, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent a single motif of three identical modifications on a triple nucleotide chain. It can be represented by [this].

[0249] In one embodiment, N a 'or N b ' includes alternating modification patterns.

[0250] The Y’Y’Y’ motif occurs at or near the cleavage site of the sense strand. For example, when the RNAi agent has a double-stranded region 17 to 23 nucleotides in length, the Y’Y’Y’ motif may occur at positions 9, 10, 11, positions 10, 11, 12, positions 11, 12, 13, positions 12, 13, 14 or positions 13, 14, 15 of the antisense strand, the numbers starting from the first nucleotide from the 5’ end, or, as appropriate, the numbers may start from the first paired nucleotide within the double-stranded region from the 5’ end. Preferably, the Y’Y’Y’ motif occurs at positions 11, 12, 13.

[0251] In one embodiment, the Y’Y’Y’ motif consists of nucleotides that are all 2’-OMe modified.

[0252] In one embodiment, k is 1 and l is 0, or k is 0 and l is 1, or both k and l are 1.

[0253] Therefore, the antisense strand has the following formula: 5’ n q’ -N<了 a ’-Z’Z’Z’-N b ’-Y’Y’Y’-N a ’-n p’ 3’ (IIb), 5’ n q’ -N a ’-Y’Y’Y’-N b ’-X’X’X’-n p’ 3’ (IIc), or 5’ n q’ -N a ’- Z’Z’Z’-N b ’-Y’Y’Y’-N b ’- X’X’X’-N a ’-n p’ 3’ (IId) and can be represented by.

[0254] When the antisense strand is represented by formula (IIb), N b’ represents an oligonucleotide sequence containing modified nucleotides of 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2 or 0. Each N a ’ independently represents an oligonucleotide sequence containing modified nucleotides of 2 to 20, 2 to 15 or 2 to 10.

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

[0256] When the antisense strand is represented by formula (IId), each N b ’ independently represents an oligonucleotide sequence containing modified nucleotides of 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2 or 0. Each N a ’ independently represents an oligonucleotide sequence containing modified nucleotides of 2 to 20, 2 to 15 or 2 to 10. Preferably, N b is 0, 1, 2, 3, 4, 5 or 6.

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

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

[0259] Each of X', Y', and Z' may be identical or different from the others.

[0260] Each nucleotide in the sense and antisense strands can be independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-hydroxyl, or 2'-fluoro. For example, each nucleotide in the sense and antisense strands can be independently modified with 2'-O-methyl or 2'-fluoro. Each X, Y, Z, X', Y', and Z' may, in particular, represent a 2'-O-methyl modification or a 2'-fluoro modification.

[0261] In one embodiment, the sense strand of the RNAi agent may contain a YYY motif occurring at positions 9, 10, and 11 of the strand when the double-stranded region is 21nt, the number starting from the first nucleotide from the 5' end, or optionally starting from the first pair-formed nucleotide in the double-stranded region from the 5' end, where Y represents a 2'-F modification. The sense strand may further contain an XXX motif or a ZZZ motif as a wing modification at the opposite end of the double-stranded region, where XXX and ZZZ independently represent a 2'-OMe modification or a 2'-F modification.

[0262] In one embodiment, the antisense strand may contain a Y'Y'Y' motif occurring at positions 11, 12, and 13 of the strand, the number starting from the first nucleotide from the 5' end, or optionally starting from the first pair-formed nucleotide in the double-stranded region from the 5' end, where Y' represents a 2'-O-methyl modification. The antisense strand may further contain an X'X'X' motif or a Z'Z'Z' motif as a wing modification at the opposite end of the double-stranded region, where X'X'X' and Z'Z'Z' independently represent a 2'-OMe modification or a 2'-F modification.

[0263] A sense strand represented by any one of the above equations (Ia), (Ib), (Ic), and (Id) forms a double helix with an antisense strand represented by any one of the above equations (IIa), (IIb), (IIc), and (IId).

[0264] Therefore, the RNAi agent for use in the method of this disclosure may include a sense strand and an antisense strand, each having 14 to 30 nucleotides, and the RNAi double helix is ​​given by formula (III): Sense: 5' n p -N a -(XXX) i -N b - YYY -N b -(ZZZ) j -N a -n q 3' Antisense: 3' n p ’ -N a ’ -(X'X'X') k -N b ’ -Y'Y'Y'-N b ’ -(Z'Z'Z') l -N a ’ -n q ’ 5' (III) [In the formula, i, j, k, and l are each independently either 0 or 1. p, p', q, and q' are each independently between 0 and 6. each N a and N a ’ Each independently represents an oligonucleotide sequence containing 0 to 25 modified nucleotides, where each sequence contains at least two differently modified nucleotides. each N b and N b ’ Each independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides. each n p ',n p , n q 'and n q Each of these may or may not be present, but they independently represent an overhang nucleotide. XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent a single motif of three identical modifications on three consecutive nucleotides. It is represented by [this].

[0265] In one embodiment, i is 0 and j is 0, or i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 0, or both i and j are 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 both k and l are 0, or both k and l are 1.

[0266] An exemplary combination of sense and antisense strands that form an RNAi double helix is ​​given by the following formula: 5' n p - N a -YYY -N a -n q 3' 3' n p ’ -N a ’ -Y'Y'Y' -N a ’ n q ’ 5' (IIIa) 5' n p -N a -YYY -N b -ZZZ -N a -n q 3' 3' n p ’ -N a ’ -Y'Y'Y'-N b ’ -Z'Z'Z'-N a ’ n q ’ 5' (IIIb) 5' n p -N a- XXX -N b -YYY - N a -n q 3' 3' n p ’ -N a ’ -X'X'X'-N b ’ -Y'Y'Y'-N a ’ -n q ’ 5' (IIIc) 5' n p -N a -XXX -N b -YYY -N b - ZZZ -N a -n q 3' 3' n p ’ -N a ’ -X'X'X'-N b ’ -Y'Y'Y'-N b ’ -Z'Z'Z'-N a -n q ’ 5' (IIId) Includes.

[0267] When an RNAi agent is represented by formula (IIIa), each N a Each of these independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0268] When an RNAi agent is represented by formula (IIIb), each N b Each N independently represents an oligonucleotide sequence containing modified nucleotides 1-10, 1-7, 1-5, or 1-4. a Each of these independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0269] When an RNAi agent is represented by formula (IIIc), each N b , N b ' independently represents an oligonucleotide sequence containing modified nucleotides of 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0. Each N a Each of these independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0270] When an RNAi agent is represented by formula (IIId), each N b , N b ' independently represents an oligonucleotide sequence containing modified nucleotides of 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0. Each N a , N a ’ N independently represents oligonucleotide sequences containing 2-20, 2-15, or 2-10 modified nucleotides. a , N a ', N b and N b ’ Each of these independently includes alternating modification patterns.

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

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

[0273] In one embodiment, the RNAi agent is a multimer containing at least two double helixes represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), the double helixes being linked by a linker. The linker may or may not be cleavable. The multimer may further contain ligands. Each double helix may target the same gene, or two different genes, or each double helix may target the same gene at two different target sites.

[0274] In one embodiment, the RNAi agent is a multimer containing 3, 4, 5, 6 or more double helixes represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), where the double helixes are linked by linkers. The linkers may or may not be cleavable. The multimer may further contain ligands. Each double helix may target the same gene, or two different genes, or each double helix may target the same gene at two different target sites.

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

[0276] Various publications describe multimeric RNAi agents that may be used in the methods of this disclosure. Such publications include WO2007 / 091269, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887, and WO2011 / 031520, as well as US7858769, the entire contents of each of these publications being incorporated herein by reference.

[0277] In certain embodiments, the compositions and methods of the Disclosure include vinyl phosphonate (VP) modification of an RNAi agent as described herein. In exemplary embodiments, the vinyl phosphonate of the Disclosure has the following structure:

[0278] [ka] It has.

[0279] The vinyl phosphonates of the Disclosure may be attached to either the antisense or sense strand of the dsRNA of the Disclosure. In certain preferred embodiments, the vinyl phosphonates of the Disclosure may be attached to the antisense strand of the dsRNA at the 5' end, as appropriate.

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

[0281] [ka] There is.

[0282] i.Thermal destabilization modification In certain embodiments, a dsRNA molecule can be optimized for RNA interference by incorporating a thermal destabilization modification within 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 having an antisense strand containing at least one double-strand thermal destabilization modification within the first nine nucleotide positions counting from the 5' end of the antisense strand exhibit reduced off-target gene silencing activity. Therefore, in some embodiments, the antisense strand contains at least one (e.g., 1, 2, 3, 4, 5 or more) double-strand thermal destabilization modification within the first nine nucleotide positions of the 5' region of the antisense strand. In some embodiments, one or more double-strand thermal destabilization modifications are located within positions 2–9, or preferably 4–8, from the 5' end of the antisense strand. In some further embodiments, the double-strand thermal destabilization modification(s) are located within positions 6, 7, or 8 from the 5' end of the antisense strand. In some further embodiments, the double-strand thermal destabilization modification is located at position 7 from the 5' end of the antisense strand. The term “thermal destabilization modification” includes modifications(s) that would result in a dsRNA having a lower overall melting temperature (Tm) (preferably 1, 2, 3, or 4 degrees lower than the Tm of a dsRNA without such modifications(s). In some embodiments, the double-strand thermal destabilization modification is located at positions 2, 3, 4, 5, or 9 from the 5' end of the antisense strand.

[0283] Examples of thermal destabilization modifications, though not limited to these, include debasing modifications, mismatches with opposing nucleotides on opposing chains, and sugar modifications, such as 2'-deoxy modifications or acyclic nucleotides, such as unlocked nucleic acids (UNAs) or glycol nucleic acids (GNAs).

[0284] Examples of debase modification include, but are not limited to, the following:

[0285] [ka] [In the formula, R = H, Me, Et or OMe; R' = H, Me, Et or OMe; R” = H, Me, Et or OMe]

[0286] [ka] [In the formula, B is a modified or unmodified nucleic acid base.] These are some examples.

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

[0288] [ka] [In the formula, B is a modified or unmodified nucleic acid base.] These are some examples.

[0289] In some embodiments, the thermal destabilization modification of the double chain is as follows:

[0290] [ka] [In the formula, B is a modified or unmodified nucleic acid base, and each asterisk in the structure represents either R, S, or racemic.] It is selected from the group consisting of the following.

[0291] The term "acyclic nucleotide" refers to any nucleotide having an acyclic ribose sugar in which, for example, one of the bonds between ribose carbons (e.g., C1'-C2', C2'-C3', C3'-C4', C4'-O4', or C1'-O4') is absent, or at least one of the ribose carbons or oxygen atoms (e.g., C1', C2', C3', C4', or O4') is absent independently or in combination in the nucleotide. In some embodiments, an acyclic nucleotide is,

[0292] [ka] [In the formula, B is a modified or unmodified nucleic acid base, and R 1 and R 2 R3 is independently H, halogen, OR3, or alkyl; and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar. The term "UNA" refers to an unlocked acyclic nucleic acid in which one of the sugar bonds has been removed to form an unlocked "sugar" residue. In one example, UNA also encompasses monomers in which the bond between C1'-C4' has been removed (i.e., the carbon-oxygen-carbon bond of the covalent bond between the C1' and C4' carbons). In another example, the C2'-C3' bond of the sugar (i.e., the carbon-carbon bond of the covalent bond between the C2' and C3' carbons) has been removed [see Mikhailov et al., Tetrahedron Letters, 26 (17): 2059 (1985) and Fluiter et al., Mol. Biosyst., 10: 1039 (2009), whose entirety is incorporated herein by reference]. Acyclic derivatives offer greater skeletal flexibility without affecting Watson-Crick pair formation. Acyclic nucleotides can be linked via 2'-5' or 3'-5' ligatures.

[0293] The term "GNA" refers to glycol nucleic acids, which are polymers similar to DNA or RNA, but differ in the composition of their "backbone" in that it consists of repeating glycerol units linked by phosphodiester bonds.

[0294] [ka]

[0295] Double-strand thermal destabilization modifications can be a mismatch (i.e., a non-complementary base pair) between a thermally destabilized nucleotide and an opposing nucleotide in the opposing strand within the dsRNA double-strand. Exemplary mismatch base pairs include G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, U:T, or combinations thereof. Other mismatch base pair formations known in the art are also suitable for the present invention. Mismatches can occur between nucleotides that are either naturally occurring or modified nucleotides; that is, mismatch base pair formation can occur between nucleic acid bases derived from each nucleotide independently of modifications on the ribose sugar of the nucleotides. In certain embodiments, the dsRNA molecule contains at least one nucleic acid base in mismatch pair formation, for example, a 2'-deoxynucleotide, which is located in the sense strand.

[0296] In some embodiments, thermal destabilization modification of the double helix in the seed region of the antisense strand results in a nucleotide whose WHC bond with the complementary base on the target mRNA is impaired, for example:

[0297] [ka] Includes.

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

[0299] Thermal destabilization modifications may also include universal base and phosphate modifications in which the ability to form hydrogen bonds with opposing bases is reduced or lost.

[0300] In some embodiments, thermal destabilization modifications of the double helix include nucleotides with non-canonical bases, for example, but not limited to, nucleic acid base modifications in which the ability to form hydrogen bonds with bases in the opposing strand is impaired or completely lost. These nucleic acid base modifications have been evaluated for destabilization of the central region of the dsRNA double helix, as described in WO2010 / 0011895, which is incorporated herein by reference in its entirety. Exemplary nucleic acid base modifications include:

[0301] [ka] There is.

[0302] In some embodiments, the thermal destabilization modification of the double helix in the seed region of the antisense strand involves one or more α-nucleotides complementary to the base on the target mRNA, for example:

[0303] [ka] [In the formula, R is H, OH, OCH3, F, NH2, NHMe, NMe2, or O-alkyl] It includes.

[0304] As an example of phosphate modifications known to reduce the thermal stability of dsRNA double helix compared to natural phosphodiester bonds:

[0305] [ka] There is.

[0306] The alkyl group of the R group can be C1-C6 alkyl. Specific examples of alkyl groups of the R group, though not limited to these, include methyl, ethyl, propyl, isopropyl, butyl, pentyl, and hexyl.

[0307] As those skilled in the art will recognize, given that the functional roles of nucleic acid bases define the specificity of the RNAi agents of this disclosure, nucleic acid base modifications can be carried out in various ways as described herein, for example, to enhance on-target effects against off-target effects, or to introduce destabilizing modifications into the RNAi agents of this disclosure. However, the range of modifications available and generally present on the RNAi agents of this disclosure tends to be greater with respect to non-nucleonucleotide modifications, such as modifications to the sugar groups or phosphate backbone of polyribonucleotides. Such modifications are described in more detail in other sections of this disclosure and are explicitly intended for the RNAi agents of this disclosure having either natural nucleic acid bases or modified nucleic acid bases, as described above or elsewhere herein.

[0308] In addition to the antisense strand containing thermal destabilization modifications, the dsRNA may also contain one or more stabilization modifications. For example, the dsRNA may contain at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) stabilization modifications. While not limiting, all stabilization modifications may be present on one of the strands. In some embodiments, both the sense and antisense strands contain at least two stabilization modifications. Stabilization modifications can occur on any nucleotide of the sense or antisense strand. For example, a stabilization modification may occur on any nucleotide on the sense or antisense strand, each stabilization modification may occur in an alternating pattern on the sense or antisense strand, or both the sense or antisense strand may contain stabilization modifications in an alternating pattern. The alternating pattern of stabilization modifications on the sense strand may be identical or different to that on the antisense strand, and the alternating pattern of stabilization modifications on the sense strand may have a shift compared to the alternating pattern of stabilization modifications on the antisense strand.

[0309] In some embodiments, the antisense chain includes at least two stabilization modifications (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more). Stabilization modifications in the antisense chain may be located at any position, but are not limited. In some embodiments, the antisense includes stabilization modifications at positions 2, 6, 8, 9, 14, and 16 from the 5' end. In some other embodiments, the antisense includes stabilization modifications at positions 2, 6, 14, and 16 from the 5' end. In yet another embodiment, the antisense includes stabilization modifications at positions 2, 14, and 16 from the 5' end.

[0310] In some embodiments, the antisense strand includes at least one stabilizing modification adjacent to the destabilizing modification. For example, the stabilizing modification may be a nucleotide at the 5' or 3' end of the destabilizing modification, i.e., at position -1 or +1 from the position of the destabilizing modification. In some embodiments, the antisense strand includes stabilizing modifications at each of the 5' and 3' ends of the destabilizing modification, i.e., at positions -1 and +1 from the position of the destabilizing modification.

[0311] In some embodiments, the antisense chain includes at least two stabilizing modifications at the 3' end of the destabilizing modification, i.e., at positions +1 and +2 from the position of the destabilizing modification.

[0312] In some embodiments, the sense chain includes at least two stabilization modifications (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more). Stabilization modifications in the sense chain may be located at any position, but are not limited to these. In some embodiments, the sense chain includes stabilization modifications at positions 7, 10, and 11 from the 5' end. In some other embodiments, the sense chain includes stabilization modifications at positions 7, 9, 10, and 11 from the 5' end. In some embodiments, the sense chain includes stabilization modifications at positions opposite or complementary to positions 11, 12, and 15 of the antisense chain, counting from the 5' end of the antisense chain. In some other embodiments, the sense chain includes stabilization modifications at positions opposite or complementary to positions 11, 12, 13, and 15 of the antisense chain, counting from the 5' end of the antisense chain. In some embodiments, the sense chain includes blocks of two, three, or four stabilization modifications.

[0313] In some embodiments, the sense chain does not contain stabilizing modifications in positions that counteract or complement the thermal destabilizing modifications of the double chain in the antisense chain.

[0314] Examples of thermal stabilization modifications include, but are not limited to, 2'-fluoro modifications. Other examples of thermal stabilization modifications include, but are not limited to, LNA.

[0315] In some embodiments, the dsRNA of this disclosure contains at least four (e.g., 4, 5, 6, 7, 8, 9, 10 or more) 2'-fluoronucleotides. Not limited to, all 2'-fluoronucleotides may be present in one of the strands. In some embodiments, both the sense and antisense strands contain at least two 2'-fluoronucleotides. 2'-fluoro modifications may occur on any nucleotide of the sense or antisense strand. For example, a 2'-fluoro modification may occur on any nucleotide on the sense or antisense strand, each 2'-fluoro modification may occur in an alternating pattern on the sense or antisense strand, or both the sense or antisense strand may contain 2'-fluoro modifications in an alternating pattern. The alternating pattern of 2'-fluoro modifications on the sense strand may be identical or different to that on the antisense strand, and the alternating pattern of 2'-fluoro modifications on the sense strand may have a shift compared to the alternating pattern of 2'-fluoro modifications on the antisense strand.

[0316] In some embodiments, the antisense chain contains at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-fluoronucleotides. While not limiting, 2'-fluoro modifications in the antisense chain can be located at any position. In some embodiments, the antisense contains 2'-fluoronucleotides at positions 2, 6, 8, 9, 14, and 16 from the 5' end. In some other embodiments, the antisense contains 2'-fluoronucleotides at positions 2, 6, 14, and 16 from the 5' end. In yet another embodiment, the antisense contains 2'-fluoronucleotides at positions 2, 14, and 16 from the 5' end.

[0317] In some embodiments, the antisense strand includes at least one 2'-fluoronucleotide adjacent to the destabilization modification. For example, the 2'-fluoronucleotide may be at the 5' or 3' end of the destabilization modification, i.e., at position -1 or +1 from the position of the destabilization modification. In some embodiments, the antisense strand includes 2'-fluoronucleotides at each of the 5' and 3' ends of the destabilization modification, i.e., at positions -1 and +1 from the position of the destabilization modification.

[0318] In some embodiments, the antisense strand includes at least two 2'-fluoronucleotides at the 3' end of the destabilization modification, i.e., at positions +1 and +2 from the position of the destabilization modification.

[0319] In some embodiments, the sense strand contains at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-fluoronucleotides. While not limiting, 2'-fluoro modifications in the sense strand can be present at any position. In some embodiments, the antisense strand contains 2'-fluoronucleotides at positions 7, 10, and 11 from the 5' end. In some other embodiments, the sense strand contains 2'-fluoronucleotides at positions 7, 9, 10, and 11 from the 5' end. In some embodiments, the sense strand contains 2'-fluoronucleotides at positions opposite or complementary to positions 11, 12, and 15 of the antisense strand, counting from the 5' end of the antisense strand. In some other embodiments, the sense strand contains 2'-fluoronucleotides at positions opposite or complementary to positions 11, 12, 13, and 15 of the antisense strand, counting from the 5' end of the antisense strand. In some embodiments, the sense strand contains blocks of 2, 3, or 4 2'-fluoronucleotides.

[0320] In some embodiments, the sense strand does not contain a 2'-fluoronucleotide in a position that counteracts or complements the thermal destabilization modification of the double helix in the antisense strand.

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

[0322] In some embodiments, the dsRNA molecule of the present disclosure comprises a sense and an antisense strand, the sense strand being 25–30 nucleotides long and starting from the 5' terminal nucleotide (position 1), positions 1–23 of the sense strand containing at least 8 ribonucleotides; the antisense strand being 36–66 nucleotides long and starting from the 3' terminal nucleotide, at least 8 ribonucleotides in positions that pair with positions 1–23 of the sense strand form a double helix; at least 3' terminal nucleotides of the antisense strand do not pair with the sense strand, up to 6 consecutive 3' terminal nucleotides do not pair with the sense strand, thereby forming a 3' single-stranded overhang of 1–6 nucleotides; and the 5' end of the antisense strand contains 10–30 consecutive nucleotides that do not pair with the sense strand. The sense strand contains, thereby forming a single-stranded 5' overhang of 10–30 nucleotides, and at least the 5' and 3' terminal nucleotides of the sense strand are bases that pair with the nucleotides of the antisense strand when the sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially double-stranded region between the sense and antisense strands, the antisense strand being sufficiently complementary to the target RNA along at least 19 ribonucleotides of the length of the antisense strand, reducing target gene expression when the double-stranded nucleic acid is introduced into mammalian cells, the antisense strand containing at least one thermally destabilized nucleotide, the at least one thermally destabilized nucleotide located in the seed region of the antisense strand (i.e., at positions 2–9 at the 5' end of the antisense strand).For example, thermally destabilized nucleotides occur between positions 14-17 at the 5' end of the sense strand and complementary positions, and the dsRNA may further have at least one of the following features (e.g., 1, 2, 3, 4, 5, 6, or all 7): (i) the antisense strand contains 2, 3, 4, 5, or 6 2'-fluoro modifications; (ii) the antisense strand contains 1, 2, 3, 4, or 5 phosphorothioate nucleotide linkages; (iii) the sense strand is conjugated with a ligand; (iv) the sense strand contains 2, 3, 4, or 5 2'-fluoro modifications; (v) the sense strand contains 1, 2, 3, 4, or 5 phosphorothioate nucleotide linkages; (vi) the dsRNA contains at least 4 2'-fluoro modifications; and (vii) the dsRNA contains a double-stranded region 12-30 nucleotide pairs long.

[0323] In some embodiments, the dsRNA molecule of the present disclosure comprises a sense strand and an antisense strand, the dsRNA molecule comprising a sense strand having a length of at least 25 and at most 29 nucleotides, and an antisense strand having a length of at most 30 nucleotides, wherein the sense strand comprises a modified nucleotide sensitive to enzymatic degradation from its 5' end to position 11, the 3' end of the sense strand and the 5' end of the antisense strand form a blunt end, the antisense strand is 1 to 4 nucleotides longer at its 3' end than the sense strand, the double-stranded region is at least 25 nucleotides long, the antisense strand is sufficiently complementary to the target mRNA along the length of the antisense strand by at least 19 nucleotides, the dsRNA molecule reduces target gene expression when introduced into mammalian cells, the dicer cleavage of the dsRNA preferentially yields siRNA including the 3' end of the antisense strand, thereby reducing target gene expression in mammals, and the antisense strand comprises at least one The dsRNA contains thermally destabilized nucleotides, at least one of which is located in the seed region of the antisense strand (i.e., at positions 2-9 at the 5' end of the antisense strand), and the dsRNA may further have at least one of the following features (e.g., all of 1, 2, 3, 4, 5, 6, or 7): (i) the antisense strand contains 2, 3, 4, 5, or 6 2'-fluoro modifications, (ii) the antisense strand contains 2, 3, 4, 5, or 6 2'-fluoro modifications, (i i) the antisense strand contains 1, 2, 3, 4, or 5 phosphorothioate nucleotide interlinks, (iii) the sense strand is conjugated with a ligand, (iv) the sense strand contains 2, 3, 4, or 5 2'-fluoro modifications, (v) the sense strand contains 1, 2, 3, 4, or 5 phosphorothioate nucleotide interlinks, (vi) the dsRNA contains at least 4 2'-fluoro modifications, and (vii) the dsRNA has a double-stranded region of 12–29 nucleotide pairs in length.

[0324] In some embodiments, any nucleotide in the sense and antisense strands of a dsRNA molecule may be modified. Each nucleotide may be modified with the same or different modifications, which may include alterations of one or more unbound phosphate oxygens, or one or more bound phosphate oxygens, alterations of the 2' hydroxyl group on the ribose sugar components, large-scale substitution of the phosphate moiety with a "dephospho" linker, modifications or substitutions of naturally occurring bases, and substitutions or modifications of the ribose-phosphate backbone.

[0325] Since nucleic acids are polymers of subunits, many modifications occur at repeating positions within the nucleic acid, for example, modifications of bases or phosphate moieties or unbound oxygen atoms of phosphate moieties. In some cases, modifications occur at all target positions in the nucleic acid, but often they do not. For example, modifications may occur only at the 3' or 5' end, or only in the terminal region, for example, at the terminal nucleotides of the strand, or at the last 2, 3, 4, 5, or 10 nucleotides. Modifications may occur in double-stranded regions, single-stranded regions, or both. Modifications may occur only in the double-stranded regions of RNA, or only in the single-stranded regions of RNA. For example, phosphorothioate modifications at unbound oxygen atoms may occur only at one or both ends, or only in the terminal region, for example, at the terminal nucleotides of the strand, or at the last 2, 3, 4, 5, or 10 nucleotides, or in both double-stranded and single-stranded regions, especially at the ends. The 5' end or both ends may be phosphorylated.

[0326] For example, it may be possible to enhance stability, include specific bases in the overhang, or include modified nucleotides or nucleotide substitutes in single-stranded overhangs, e.g., in the 5' or 3' overhang, or both. For example, it may be desirable to include purine nucleotides in the overhang. In some embodiments, all or some of the bases in the 3' or 5' overhang may be modified, for example, with modifications described herein. Modifications may include, for example, the use of 2'-position modification of ribose sugar in modifications known in the art, e.g., the use of deoxyribonucleotides, 2'-deoxy-2'-fluoro(2'-F) or 2'-O-methyl modified in place of ribosaccharides in nucleic acid bases, and modifications at phosphate groups, e.g., phosphorothioate modifications. The overhang does not need to be homologous to the target sequence.

[0327] In some embodiments, each residue in the sense and antisense chains is independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, or 2'-fluoro. The chains may contain two or more modifications. In some embodiments, each residue in the sense and antisense chains is independently modified with 2'-O-methyl or 2'-fluoro. It should be understood that these modifications are in addition to at least one thermal destabilization modification of the double helix present in the antisense chain.

[0328] At least two distinct modifications are typically present on the sense and antisense strands. These two modifications may include 2'-deoxy, 2'-O-methyl, or 2'-fluoro modifications, acyclic nucleotides, etc. In some embodiments, the sense and antisense strands each contain two distinctly modified nucleotides selected from 2'-O-methyl or 2'-deoxy. In some embodiments, each residue in the sense and antisense strands is independently modified with 2'-O-methyl nucleotide, 2'-deoxy nucleotide, 2'-deoxy-2'-fluoro nucleotide, 2'-ON-methylacetamide (2'-O-NMA) nucleotide, 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) nucleotide, 2'-O-aminopropyl (2'-O-AP) nucleotide, or 2'-ala-F nucleotide. Again, it should be understood that these modifications are in addition to at least one thermal destabilization modification of the double helix present in the antisense strand.

[0329] In some embodiments, the dsRNA molecules of this disclosure include alternating pattern modifications, particularly in the B1, B2, B3, B1', B2', B3', and B4' regions. The terms “alternating motif” or “alternating pattern,” as used herein, refer to a motif having one or more modifications, each modification occurring in alternating nucleotides on a single strand. Alternating nucleotides may refer to one every other nucleotide, one every three nucleotides, or a similar pattern. For example, if A, B, and C each represent one type of modification to a nucleotide, the alternating motif may be “ABABABABABAB…”, “AABBAABBAABB…”, “AABAABAABAAB…”, “AAABAAABAAAB…”, “AABBBAAABBB…”, or “ABCABCABCABC…”.

[0330] The types of modifications contained within an alternating motif may be identical or different. For example, if A, B, C, and D each represent one type of modification on a nucleotide, then the alternating turns, i.e., the modifications on every other nucleotide, may be identical, but each of the sense or antisense strands may be selected from several possible modifications within the alternating motif, such as "ABABAB…", "ACACAC…", "BDBDBD…", or "CDCDCD…".

[0331] In some embodiments, the dsRNA molecules of this disclosure include a modification pattern of alternating motifs on the sense strand that is shifted relative to the modification pattern of alternating motifs on the antisense strand. The shift may be such that modified groups of nucleotides on the sense strand correspond to differently modified groups of nucleotides on the antisense strand, and vice versa. For example, when the sense strand is paired with the antisense strand in a dsRNA double helix, the alternating motifs on the sense strand may begin with "ABABAB" from 5'-3' of the strand, and the alternating motifs on the antisense strand may begin with "BABABA" from 3'-5' of the strand in the double helix region. As another example, the alternating motifs on the sense strand may begin with "AABBAABB" from 5'-3' of the strand, and the alternating motifs on the antisense strand may begin with "BBAABBAA" at 3'-5' of the strand in the double helix region, resulting in a complete or partial shift of the modification patterns between the sense and antisense strands.

[0332] The dsRNA molecules of this disclosure may further include at least one phosphorothioate or methylphosphonate internucleotide ligation. Phosphothioate or methylphosphonate internucleotide ligation modifications may occur at any position on the chain, on the sense strand, the antisense strand, or on any nucleotide of both. For example, an internucleotide ligation modification may occur on any nucleotide on the sense strand or the antisense strand, each internucleotide ligation modification may occur in an alternating pattern on the sense strand or the antisense strand, or the sense strand or the antisense strand may contain both internucleotide ligation modifications in an alternating pattern. The alternating pattern of internucleotide ligation modifications on the sense strand may be identical or different to that on the antisense strand, and the alternating pattern of internucleotide ligation modifications on the sense strand may have a shift relative to the alternating pattern of internucleotide ligation modifications on the antisense strand.

[0333] In some embodiments, the dsRNA molecule includes phosphorothioate or methylphosphonate internucleotide linkage modifications within the overhang region. For example, the overhang region includes two nucleotides having a phosphorothioate or methylphosphonate internucleotide linkage between the two nucleotides. The internucleotide linkage modification may also be performed to link the overhang nucleotides to the terminal pair-forming nucleotides in the double-stranded region. For example, at least two, three, four, or all of the overhang nucleotides may be linked by phosphorothioate or methylphosphonate internucleotide linkages, and there may be further phosphorothioate or methylphosphonate internucleotide linkages linking the overhang nucleotides to the pair-forming nucleotides adjacent to the overhang nucleotides. For example, there may be at least two phosphorothioate internucleotide linkages between three terminal nucleotides, where two of the three nucleotides are overhang nucleotides and the third is the pair-forming nucleotide adjacent to the overhang nucleotide. Preferably, these terminal 3 nucleotides may be the 3' end of the antisense strand.

[0334] In some embodiments, the sense strand of a dsRNA molecule comprises 1 to 10 blocks of 2 to 10 phosphorothioate or methylphosphonate nucleotide links, separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate nucleotide links, one of which is positioned at any position in the oligonucleotide sequence, and the sense strand is paired with an antisense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate nucleotide links, or with an antisense strand comprising either phosphorothioate, methylphosphonate, or phosphate links.

[0335] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of two phosphorothioate or methylphosphonate nucleotide links, separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate nucleotide links, one of which is positioned at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate nucleotide links, and an antisense strand comprising either phosphorothioate, methylphosphonate, or phosphate links.

[0336] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of three phosphorothioate or methylphosphonate internucleotide links, separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate nucleotide interlinks, one of which is positioned at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate nucleotide interlinks, or with an antisense strand comprising either phosphorothioate, methylphosphonate, or phosphate linkage.

[0337] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of four phosphorothioate or methylphosphonate internucleotide links, separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 phosphate nucleotide interlinks, one of which is positioned at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate nucleotide interlinks, or with an antisense strand comprising either phosphorothioate, methylphosphonate, or phosphate linkage.

[0338] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of five phosphorothioate or methylphosphonate internucleotide links, separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 phosphate nucleotide interlinks, one of which is positioned at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate nucleotide interlinks, or with an antisense strand comprising either phosphorothioate, methylphosphonate, or phosphate linkage.

[0339] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of six phosphorothioate or methylphosphonate internucleotide links, separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphate nucleotide interlinks, one of which is positioned at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate nucleotide interlinks, or with an antisense strand comprising either phosphorothioate, methylphosphonate, or phosphate linkage.

[0340] In some embodiments, the antisense strand of a dsRNA molecule comprises two blocks of seven phosphorothioate or methylphosphonate internucleotide links, separated by one, two, three, four, five, six, seven, or eight phosphate nucleotide interlinks, one of which is positioned at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate nucleotide interlinks, or with an antisense strand comprising either phosphorothioate, methylphosphonate, or phosphate links.

[0341] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of eight phosphorothioate or methylphosphonate internucleotide links, separated by one, two, three, four, five, or six phosphate nucleotide interlinks, one of which is positioned at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate nucleotide interlinks, or with an antisense strand comprising either phosphorothioate, methylphosphonate, or phosphate links.

[0342] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of nine phosphorothioate or methylphosphonate nucleotide links, separated by one, two, three, or four phosphate nucleotide links, one of which is positioned at any position in the oligonucleotide sequence, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate nucleotide links, or with an antisense strand comprising either phosphorothioate, methylphosphonate, or phosphate links.

[0343] In some embodiments, the dsRNA molecules of this disclosure further include one or more phosphorothioate or methylphosphonate internucleotide ligation modifications within 1 to 10 terminal positions of the sense or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides may be ligated by phosphorothioate or methylphosphonate internucleotide ligations at one or both ends of the sense or antisense strand.

[0344] In some embodiments, the dsRNA molecules of this disclosure further include one or more phosphorothioate or methylphosphonate nucleotide ligation modifications within 1 to 10 of the internal regions of each duplex of the sense or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides may be ligated by phosphorothioate-methylphosphonate ligations at positions 8 to 16 of the duplex region, counting from the 5' end of the sense strand. The dsRNA molecules may further include one or more phosphorothioate or methylphosphonate nucleotide ligation modifications within terminal positions 1 to 10.

[0345] In some embodiments, the dsRNA molecule of the present disclosure further comprises 1 to 5 phosphorothioate or methylphosphonate nucleotide ligation modifications within positions 1 to 5 of the sense strand and 1 to 5 phosphorothioate or methylphosphonate nucleotide ligation modifications within positions 18 to 23 (counting from the 5' end), as well as 1 to 5 phosphorothioate or methylphosphonate nucleotide ligation modifications within positions 1 and 2 of the antisense strand and 1 to 5 within positions 18 to 23 (counting from the 5' end).

[0346] In some embodiments, the dsRNA molecule of the present disclosure further comprises one phosphorothioate nucleotide ligation modification within positions 1-5 of the sense strand and one phosphorothioate or methylphosphonate nucleotide ligation modification within positions 18-23 (counting from the 5' end), as well as one phosphorothioate nucleotide ligation modification at positions 1 and 2 of the antisense strand and two phosphorothioate or methylphosphonate nucleotide ligation modifications within positions 18-23 (counting from the 5' end).

[0347] In some embodiments, the dsRNA molecule of the present disclosure further comprises two phosphorothioate nucleotide ligation modifications within positions 1–5 of the sense strand and one phosphorothioate nucleotide ligation modification within positions 18–23 (counting from the 5' end), as well as one phosphorothioate nucleotide ligation modification at positions 1 and 2 of the antisense strand and two phosphorothioate nucleotide ligation modifications within positions 18–23 (counting from the 5' end).

[0348] In some embodiments, the dsRNA molecule of the present disclosure further comprises two phosphorothioate nucleotide ligation modifications within positions 1-5 of the sense strand and two phosphorothioate nucleotide ligation modifications within positions 18-23 (counting from the 5' end), as well as one phosphorothioate nucleotide ligation modification at positions 1 and 2 of the antisense strand and two phosphorothioate nucleotide ligation modifications within positions 18-23 (counting from the 5' end).

[0349] In some embodiments, the dsRNA molecule of the present disclosure further comprises two phosphorothioate nucleotide ligation modifications within positions 1–5 of the sense strand and two phosphorothioate nucleotide ligation modifications within positions 18–23 (counting from the 5' end), as well as one phosphorothioate nucleotide ligation modification at positions 1 and 2 of the antisense strand and one phosphorothioate nucleotide ligation modification within positions 18–23 (counting from the 5' end).

[0350] In some embodiments, the dsRNA molecule of the present disclosure further comprises one phosphorothioate nucleotide ligation modification within positions 1-5 of the sense strand and one phosphorothioate nucleotide ligation modification within positions 18-23 (counting from the 5' end), as well as two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand and two phosphorothioate nucleotide ligation modifications within positions 18-23 (counting from the 5' end).

[0351] In some embodiments, the dsRNA molecule of the present disclosure further comprises one phosphorothioate nucleotide ligation modification within positions 1–5 of the sense strand and one within positions 18–23 (counting from the 5' end), as well as two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand and one phosphorothioate nucleotide ligation modification within positions 18–23 (counting from the 5' end).

[0352] In some embodiments, the dsRNA molecule of the present disclosure further includes one phosphorothioate nucleotide ligation modification (counting from the 5' end) within positions 1–5 of the sense strand, and two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand, and one phosphorothioate nucleotide ligation modification (counting from the 5' end) within positions 18–23.

[0353] In some embodiments, the dsRNA molecule of the present disclosure further comprises two phosphorothioate nucleotide ligation modifications (counting from the 5' end) within positions 1–5 of the sense strand, one phosphorothioate nucleotide ligation modification at positions 1 and 2 of the antisense strand, and two phosphorothioate nucleotide ligation modifications (counting from the 5' end) within positions 18–23.

[0354] In some embodiments, the dsRNA molecule of the present disclosure further comprises two phosphorothioate nucleotide ligation modifications within positions 1–5 of the sense strand and one within positions 18–23 (counting from the 5' end), as well as two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand and one within positions 18–23 (counting from the 5' end).

[0355] In some embodiments, the dsRNA molecule of the present disclosure further comprises two phosphorothioate nucleotide ligation modifications within positions 1-5 of the sense strand and one phosphorothioate nucleotide ligation modification within positions 18-23 (counting from the 5' end), as well as two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand and two phosphorothioate nucleotide ligation modifications within positions 18-23 (counting from the 5' end).

[0356] In some embodiments, the dsRNA molecule of the present disclosure further comprises two phosphorothioate nucleotide ligation modifications within positions 1–5 of the sense strand and one phosphorothioate nucleotide ligation modification within positions 18–23 (counting from the 5' end), as well as one phosphorothioate nucleotide ligation modification at positions 1 and 2 of the antisense strand and two phosphorothioate nucleotide ligation modifications within positions 18–23 (counting from the 5' end).

[0357] In some embodiments, the dsRNA molecule of the present disclosure further includes two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the sense strand and two phosphorothioate nucleotide ligation modifications at positions 20 and 21 (counting from the 5' end), as well as one phosphorothioate nucleotide ligation modification at position 1 of the antisense strand and one at position 21 (counting from the 5' end).

[0358] In some embodiments, the dsRNA molecule of the present disclosure further comprises one phosphorothioate nucleotide ligation modification at position 1 of the sense strand and one phosphorothioate nucleotide ligation modification at position 21 (counting from the 5' end), and two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand and two phosphorothioate nucleotide ligation modifications at positions 20 and 21 (counting from the 5' end).

[0359] In some embodiments, the dsRNA molecule of the present disclosure further comprises two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the sense strand and two phosphorothioate nucleotide ligation modifications at positions 21 and 22 (counting from the 5' end), as well as one phosphorothioate nucleotide ligation modification at position 1 of the antisense strand and one phosphorothioate nucleotide ligation modification at position 21 (counting from the 5' end).

[0360] In some embodiments, the dsRNA molecule of the present disclosure further comprises one phosphorothioate nucleotide ligation modification at position 1 of the sense strand and one phosphorothioate nucleotide ligation modification at position 21 (counting from the 5' end), and two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand and two phosphorothioate nucleotide ligation modifications at positions 21 and 22 (counting from the 5' end).

[0361] In some embodiments, the dsRNA molecule of the present disclosure further includes two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the sense strand and two phosphorothioate nucleotide ligation modifications at positions 22 and 23 (counting from the 5' end), as well as one phosphorothioate nucleotide ligation modification at position 1 of the antisense strand and one phosphorothioate nucleotide ligation modification at position 21 (counting from the 5' end).

[0362] In some embodiments, the dsRNA molecule of the present disclosure further includes one phosphorothioate nucleotide ligation modification at position 1 of the sense strand and one phosphorothioate nucleotide ligation modification at position 21 (counting from the 5' end), and two phosphorothioate nucleotide ligation modifications at positions 1 and 2 of the antisense strand and two phosphorothioate nucleotide ligation modifications at positions 23 and 23 (counting from the 5' end).

[0363] In some embodiments, the compounds of the present disclosure include a pattern of skeletal chiral centers. In some embodiments, the general pattern of skeletal chiral centers includes at least five nucleotide linkages in the Sp configuration. In some embodiments, the general pattern of skeletal chiral centers includes at least six nucleotide linkages in the Sp configuration. In some embodiments, the general pattern of skeletal chiral centers includes at least seven nucleotide linkages in the Sp configuration. In some embodiments, the general pattern of skeletal chiral centers includes at least eight nucleotide linkages in the Sp configuration. In some embodiments, the general pattern of skeletal chiral centers includes at least nine nucleotide linkages in the Sp configuration. In some embodiments, the general pattern of skeletal chiral centers includes at least ten nucleotide linkages in the Sp configuration. In some embodiments, the general pattern of skeletal chiral centers includes at least eleven nucleotide linkages in the Sp configuration. In some embodiments, the general pattern of skeletal chiral centers includes at least twelve nucleotide linkages in the Sp configuration. In some embodiments, the general pattern of skeletal chiral centers includes at least thirteen nucleotide linkages in the Sp configuration. In some embodiments, the general pattern of the skeletal chiral center includes at least 14 nucleotide linkages in the Sp configuration. In some embodiments, the general pattern of the skeletal chiral center includes at least 15 nucleotide linkages in the Sp configuration. In some embodiments, the general pattern of the skeletal chiral center includes at least 16 nucleotide linkages in the Sp configuration. In some embodiments, the general pattern of the skeletal chiral center includes at least 17 nucleotide linkages in the Sp configuration. In some embodiments, the general pattern of the skeletal chiral center includes at least 18 nucleotide linkages in the Sp configuration. In some embodiments, the general pattern of the skeletal chiral center includes at least 19 nucleotide linkages in the Sp configuration. In some embodiments, the general pattern of the skeletal chiral center includes 8 or fewer nucleotide linkages in the Rp configuration.In some embodiments, the general pattern of the skeletal chiral center includes seven or fewer nucleotide linkages in the Rp configuration. In some embodiments, the general pattern of the skeletal chiral center includes six or fewer nucleotide linkages in the Rp configuration. In some embodiments, the general pattern of the skeletal chiral center includes five or fewer nucleotide linkages in the Rp configuration. In some embodiments, the general pattern of the skeletal chiral center includes four or fewer nucleotide linkages in the Rp configuration. In some embodiments, the general pattern of the skeletal chiral center includes three or fewer nucleotide linkages in the Rp configuration. In some embodiments, the general pattern of the skeletal chiral center includes two or fewer nucleotide linkages in the Rp configuration. In some embodiments, the general pattern of the skeletal chiral center includes one or fewer nucleotide linkages in the Rp configuration. In some embodiments, the general pattern of the skeletal chiral center includes eight or fewer non-chiral nucleotide linkages (phosphodiesters are an example, not limited to this). In some embodiments, the general pattern of the skeletal chiral center includes seven or fewer non-chiral nucleotide linkages. In some embodiments, the general pattern of the skeletal chiral center includes six or fewer non-chiral nucleotide linkages. In some embodiments, the general pattern of the skeletal chiral center includes five or fewer non-chiral nucleotide linkages. In some embodiments, the general pattern of the skeletal chiral center includes four or fewer non-chiral nucleotide linkages. In some embodiments, the general pattern of the skeletal chiral center includes three or fewer non-chiral nucleotide linkages. In some embodiments, the general pattern of the skeletal chiral center includes two or fewer non-chiral nucleotide linkages. In some embodiments, the general pattern of the skeletal chiral center includes one or fewer non-chiral nucleotide linkages. In some embodiments, the general pattern of the skeletal chiral center includes at least 10 nucleotide linkages and eight or fewer non-chiral nucleotide linkages in the Sp configuration.In some embodiments, the general pattern of the skeletal chiral center includes at least 11 internucleotide links and 7 or fewer non-chiral internucleotide links in the Sp configuration. In some embodiments, the general pattern of the skeletal chiral center includes at least 12 internucleotide links and 6 or fewer non-chiral internucleotide links in the Sp configuration. In some embodiments, the general pattern of the skeletal chiral center includes at least 13 internucleotide links and 6 or fewer non-chiral internucleotide links in the Sp configuration. In some embodiments, the general pattern of the skeletal chiral center includes at least 14 internucleotide links and 5 or fewer non-chiral internucleotide links in the Sp configuration. In some embodiments, the general pattern of the skeletal chiral center includes at least 15 internucleotide links and 4 or fewer non-chiral internucleotide links in the Sp configuration. In some embodiments, the internucleotide links in the Sp configuration may or may not be continuous. In some embodiments, the internucleotide links in the Rp configuration may or may not be continuous. In some embodiments, the non-chiral internucleotide links may or may not be continuous.

[0364] In some embodiments, the compounds of the Disclosure include blocks that are stereochemical blocks. In some embodiments, a block is an Rp block in that each nucleotide linkage in the block is Rp. In some embodiments, a 5'-block is an Rp block. In some embodiments, a 3'-block is an Rp block. In some embodiments, a block is an Sp block in that each nucleotide linkage in the block is Sp. In some embodiments, a 5'-block is an Sp block. In some embodiments, a 3'-block is an Sp block. In some embodiments, the oligonucleotides provided include both 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 in which each nucleotide linkage is a native phosphate linkage.

[0365] In some embodiments, the compounds of the present disclosure include a 5'-block in which each sugar moiety is an Sp block containing a 2'-F modification. In some embodiments, the 5'-block is an Sp block in which each nucleotide linkage is a modified nucleotide linkage and each sugar moiety is an Sp block containing a 2'-F modification. In some embodiments, the 5'-block is an Sp block in which each nucleotide linkage is a phosphorothioate linkage and each sugar moiety is an Sp block containing a 2'-F modification. In some embodiments, the 5'-block contains four or more nucleoside units. In some embodiments, the 5'-block contains five or more nucleoside units. In some embodiments, the 5'-block contains six or more nucleoside units. In some embodiments, the 5'-block contains seven or more nucleoside units. In some embodiments, the 3'-block is an Sp block in which each sugar moiety is an Sp block containing a 2'-F modification. In some embodiments, the 3'-block is an Sp block in which each nucleotide linkage is a modified nucleotide linkage and each sugar moiety is an Sp block containing a 2'-F modification. In some embodiments, the 3'-block is an Sp block in which each nucleotide linkage is a phosphorothioate linkage and each sugar moiety contains a 2'-F modification. In some embodiments, the 3'-block contains four or more nucleoside units. In some embodiments, the 3'-block contains five or more nucleoside units. In some embodiments, the 3'-block contains six or more nucleoside units. In some embodiments, the 3'-block contains seven or more nucleoside units.

[0366] In some embodiments, the compounds of the Disclosure comprise a nucleoside of a certain type in the region, or an oligonucleotide followed by a specific type of internucleotide linkage, such as a native phosphate linkage, a modified internucleotide linkage, an Rp chiral internucleotide linkage, an Sp chiral internucleotide linkage, and the like. In some embodiments, A is followed by Sp. In some embodiments, A is followed by Rp. In some embodiments, A is followed by a native phosphate linkage (PO). In some embodiments, U is followed by Sp. In some embodiments, U is followed by Rp. In some embodiments, U is followed by a native phosphate linkage (PO). In some embodiments, C is followed by Sp. In some embodiments, C is followed by Rp. In some embodiments, C is followed by a native phosphate linkage (PO). In some embodiments, G is followed by Sp. In some embodiments, G is followed by Rp. In some embodiments, G is followed by a native phosphate linkage (PO). In some embodiments, C and U are followed by Sp. In some embodiments, C and U are followed by Rp. In some embodiments, C and U are followed by a natural phosphate linkage (PO). In some embodiments, A and G are followed by Sp. In some embodiments, A and G are followed by Rp.

[0367] In some embodiments, the antisense strand includes phosphorothioate internucleotide linkages between nucleotide positions 21 and 22 and between nucleotide positions 22 and 23, the antisense strand contains at least one double-stranded thermal destabilization modification located in the seed region of the antisense strand (i.e., at positions 2-9 at the 5' end of the antisense strand), and the dsRNA may further have at least one of the following features (e.g., all of 1, 2, 3, 4, 5, 6, 7, or 8): (i) the antisense strand includes 2, 3, 4, 5, or 6 2'-fluoro modifications, (ii) (iii) the antisense strand contains 3, 4, or 5 phosphorothioate nucleotide interlinks, (iii) the sense strand is conjugated with a ligand, (iv) the sense strand contains 2, 3, 4, or 5 2'-fluoro modifications, (v) the sense strand contains 1, 2, 3, 4, or 5 phosphorothioate nucleotide interlinks, (vi) the dsRNA contains at least 4 2'-fluoro modifications, (vii) the dsRNA contains a double-stranded region of 12–40 nucleotide pairs in length, and (viii) the dsRNA has a blunt end at the 5' end of the antisense strand.

[0368] In some embodiments, the antisense strand includes phosphorothioate internucleotide links 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, and the antisense strand contains at least one double-stranded thermal destabilization modification located in the seed region of the antisense strand (i.e., at positions 2-9 at the 5' end of the antisense strand), and the dsRNA may further have at least one of the following features (e.g., all of 1, 2, 3, 4, 5, 6, 7, or 8): (i) the antisense strand has 2, 3, 4, 5, or 6 (ii) the sense strand contains one 2'-fluoro modification, is conjugated with a ligand, (iii) the sense strand contains two, three, four or five 2'-fluoro modifications, (iv) the sense strand contains one, two, three, four or five phosphorothioate nucleotide interlinks, (v) the dsRNA contains at least four 2'-fluoro modifications, (vi) the dsRNA contains a double-stranded region of 12 to 40 nucleotide pairs in length, (vii) the dsRNA contains a double-stranded region of 12 to 40 nucleotide pairs in length, and (viii) the dsRNA has a blunt end at the 5' end of the antisense strand.

[0369] In some embodiments, the sense strand includes phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3, the antisense strand includes at least one double-stranded thermal destabilization modification located in the seed region of the antisense strand (i.e., at positions 2-9 at the 5' end of the antisense strand), and the dsRNA may further have at least one of the following features (e.g., all of 1, 2, 3, 4, 5, 6, 7, or 8): (i) the antisense includes 2, 3, 4, 5, or 6 2'-fluoro modifications, (ii) the anti (iii) the sense strand contains 1, 2, 3, 4 or 5 phosphorothioate nucleotide interlinks, (iv) the sense strand contains 2, 3, 4 or 5 2'-fluoro modifications, (v) the sense strand contains 3, 4 or 5 phosphorothioate nucleotide interlinks, (vi) the dsRNA contains at least 4 2'-fluoro modifications, (vii) the dsRNA contains a double-stranded region of 12 to 40 nucleotide pairs in length, and (viii) the dsRNA has a blunt end at the 5' end of the antisense strand.

[0370] In some embodiments, the sense strand includes phosphorothioate internucleotide links between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3, the antisense strand includes phosphorothioate internucleotide links between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22 and between nucleotide positions 22 and 23, the antisense strand contains at least one double-stranded thermal destabilization modification located in the seed region of the antisense strand (i.e., at positions 2-9 at the 5' end of the antisense strand), and the dsRNA has at least one of the following features (e.g., 1, 2) The dsRNA may further have (3, 4, 5, 6 or 7) all of the following: (i) the antisense strand contains 2, 3, 4, 5 or 6 2'-fluoro modifications; (ii) the sense strand is conjugated with a ligand; (iii) the sense strand contains 2, 3, 4 or 5 2'-fluoro modifications; (iv) the sense strand contains 3, 4 or 5 phosphorothioate internucleotide linkages; (v) the dsRNA contains at least 4 2'-fluoro modifications; (vi) the dsRNA contains a double-stranded region 12–40 nucleotide pairs long; and (vii) the dsRNA has a blunt end at the 5' end of the antisense strand.

[0371] In some embodiments, the dsRNA molecules of this disclosure include double-stranded mismatches(s) or combinations thereof with respect to the target. Mismatches may occur in overhang regions or double-stranded regions. Base pairs can be ranked based on their tendency to promote dissociation or fusion (e.g., by the free energy of association or dissociation of a particular pairing, the simplest approach being to examine pairs on a basis of individual pairs, although the following adjacency analysis or similar analysis may also be used). In terms of promoting dissociation: A:U is preferred over G:C, G:U is preferred over G:C, and I:C is preferred over G:C (I = inosine). Mismatches, e.g., non-canonical pairing or non-canonical pairing (as described elsewhere herein) are preferred over canonical (A:T, A:U, G:C) pairing, and pairing involving universal bases is preferred over canonical pairing.

[0372] In some embodiments, the dsRNA molecule of the present disclosure includes at least one of the first 1, 2, 3, 4, or 5 base pairs in the double-stranded region from the 5' end of the antisense strand, which can be independently selected from the group of A:U, G:U, I:C, and mismatch pairs, e.g., non-canonical pairing or pairing other than canonical pairing or pairing including universal bases, in order to facilitate the dissociation of the antisense strand at the 5' end of the double helix.

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

[0374] It has been found that introducing a 4'-modified or 5'-modified nucleotide to the 3' end of a phosphodiester (PO), phosphorothioate (PS), or phosphorodithioate (PS2) linkage of a dinucleotide at any position on a single-stranded or double-stranded oligonucleotide exerts a steric effect on the nucleotide linkage, thereby protecting and stabilizing it from nucleases.

[0375] In some embodiments, a 5'-modified nucleoside is introduced at the 3' end of a dinucleotide at any position in a single-stranded or double-stranded siRNA. For example, a 5'-alkylated nucleoside can be introduced at the 3' end of a dinucleotide at any position in a single-stranded or double-stranded siRNA. The alkyl group at the 5' position of the ribose sugar can be a racemic mixture or a chiralally pure R or S isomer. An exemplary 5'-alkylated nucleoside is the 5'-methyl nucleoside. The 5'-methyl can be either a racemic mixture or a chirally pure R or S isomer.

[0376] In some embodiments, a 4'-modified nucleoside is introduced at the 3' end of a dinucleotide at any position in a single-stranded or double-stranded siRNA. For example, a 4'-alkylated nucleoside can be introduced at the 3' end of a dinucleotide at any position in a single-stranded or double-stranded siRNA. The alkyl group at the 5' position of the ribose sugar can be racemic or a chiralally pure R or S isomer. An exemplary 4'-alkylated nucleoside is the 4'-methyl nucleoside, which can be either racemic or a chirally pure R or S isomer. Alternatively, a 4'-O-alkylated nucleoside can be introduced at the 3' end of a dinucleotide at any position in a single-stranded or double-stranded siRNA. The 4'-O-alkyl of the ribose sugar can be racemic or a chirally pure R or S isomer. An exemplary 4'-O-alkylated nucleoside is the 4'-O-methyl nucleoside. The 4'-O-methyl nucleoside can be either a racemic mixture or a chiralally pure R or S isomer.

[0377] In some embodiments, a 5'-alkylated nucleoside is introduced at any position on the sense or antisense strand of the dsRNA, and such modification maintains or improves the potency of the dsRNA. The 5'-alkyl can be either a racemic mixture or a chiralally pure R or S isomer. An exemplary 5'-alkylated nucleoside is the 5'-methyl nucleoside. The 5'-methyl can be either a racemic mixture or a chirally pure R or S isomer.

[0378] In some embodiments, a 4'-alkylated nucleoside is introduced at any position on the sense or antisense strand of the dsRNA, and such modification maintains or improves the potency of the dsRNA. The 4'-alkyl can be either a racemic or a chiralally pure R or S isomer. An exemplary 4'-alkylated nucleoside is the 4'-methyl nucleoside. The 4'-methyl can be either a racemic or a chirally pure R or S isomer.

[0379] In some embodiments, the 4'-O-alkylated nucleoside is introduced at any position on the sense or antisense strand of the dsRNA, and such modification maintains or improves the potency of the dsRNA. The 5'-alkyl can be either racemic or a chiralally pure R or S isomer. An exemplary 4'-O-alkylated nucleoside is the 4'-O-methyl nucleoside. The 4'-O-methyl can be either racemic or a chirally pure R or S isomer.

[0380] In some embodiments, the dsRNA molecules of this disclosure may include a 2'-5' ligation (having 2'-H, 2'-OH, and 2'-OMe, and being P=O or P=S). For example, the 2'-5' ligation modification can be used to promote nuclease resistance, to inhibit the binding of sense to the antisense strand, or to avoid sense strand activation by RISC at the 5' end of the sense strand.

[0381] In another embodiment, the dsRNA molecule of this disclosure may contain L-sugars (e.g., L-ribose, L-arabinose having 2'-H, 2'-OH, and 2'-OMe). For example, these L-sugar modifications can be used to promote nuclease resistance, to inhibit the binding of sense to the antisense strand, or to avoid sense strand activation by RISC at the 5' end of the sense strand.

[0382] Multimeric siRNAs have been described in various publications, all of which can be used in conjunction with the dsRNAs of this disclosure. Such publications include WO2007 / 091269, US7858769, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887, and WO2011 / 031520, which are incorporated in their entirety herein.

[0383] As described in more detail below, RNAi agents containing the conjugation of one or more carbohydrate moieties can optimize one or more properties of the RNAi agent. Often, the carbohydrate moiety is attached to a modified subunit of the RNAi agent. For example, the ribose sugar of one or more ribonucleotide subunits of a dsRNA agent can be replaced with another moiety, e.g., a non-carbohydrate (preferably cyclic) carrier to which a carbohydrate ligand is attached. A ribonucleotide subunit in which the ribose sugar of the subunit is thus replaced is referred herein to as a ribose-replaced modified subunit (RRMS). The cyclic carrier may be a carbocyclic system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring structure, i.e., one or more ring atoms are heteroatoms, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be a monocyclic ring structure, or may contain two or more rings, e.g., a fused ring. The cyclic carrier may be a fully saturated ring structure, or may contain one or more double bonds.

[0384] Ligands can be attached to polynucleotides via a carrier. The carrier comprises (i) at least one “skeleton attachment site,” preferably two “skeleton attachment sites,” and (ii) at least one “tethering attachment site.” “Skeleton attachment site,” as used herein, refers to a functional group, e.g., a hydroxyl group, or generally, a bond available and suitable for the incorporation of the carrier into a skeleton, e.g., a phosphate or modified phosphate of ribonucleic acid, e.g., a sulfur-containing skeleton. “Tethering attachment site” (TAP) refers, in some embodiments, to a constituent ring atom of the cyclic carrier connecting a selected moiety, e.g., a carbon atom or heteroatom (separate from the atom providing the skeleton attachment sites). The moiety may be, for example, a carbohydrate, e.g., monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, and polysaccharides. The selected moiety may be connected to the cyclic carrier by an intervening tether. Thus, the cyclic carrier will often provide a bond suitable for the incorporation or tethering of another chemical entity, e.g., a ligand, into a constituent ring, e.g., containing a functional group, e.g., an amino group.

[0385] RNAi agents may be conjugated to ligands via a carrier, which may be a cyclic or acyclic group. Preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridadinyl, tetrahydrofuryl, and decalin. Preferably, the acyclic group is selected from a selinol skeleton or a diethanolamine skeleton.

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

[0387] IV. iRNA conjugated to a ligand Another modification of the iRNA of the present invention involves chemically linking the iRNA with one or more ligands, a moiety or conjugate that enhances the activity, cell distribution, or, for example, cellular uptake into cells of the iRNA. These include, but are not limited to, lipid parts such as cholesterol (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), thioethers, for example, beryl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), fatty acid chains, e.g., 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, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654, Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), polyamine or polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetate (Manoharan et al., Tetrahedron Lett.Examples include the palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237) or the octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0388] In certain embodiments, ligands alter the distribution, targeting, or lifespan of the iRNA agent into which they are incorporated. In some embodiments, ligands provide enhanced affinity to selected targets, such as molecules, cells or cell types, compartments, such as cellular or organ compartments, tissues, organs, or regions of the body, compared to species in which such ligands are absent. Conventional ligands do not participate in double-strand pairing in double-stranded nucleic acids.

[0389] Ligands can be naturally occurring substances, such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin), carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid), or lipids. Ligands can also be recombinant or synthetic molecules, such as synthetic polymers, such as synthetic polyamino acids. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymer, or polyphosphatidine. 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 α-helix peptides.

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

[0391] Other examples of ligands include dyes, intercalating agents (e.g., acridine), crosslinking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyllin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules (e.g., cholesterol, cholic acid, adamantane acetate, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl) lithoglycerol Examples include oenic acid, O3-(oleoyl)colenic acid, dimethoxytrityl or phenoxazine, and peptide conjugates (e.g., Antennapedia peptide, Tat peptide), alkylating agents, phosphoric acid, amino acids, mercaptos, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino acids, alkyls, substituted alkyls, radiolabeled markers, 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 tetraaza macrocyclic molecules), dinitrophenyl, HRP, or AP.

[0392] Ligands can be proteins, such as glycoproteins or peptides, molecules or antibodies that have a specific affinity for a co-ligand, such as antibodies that bind to specific cell types, such as cancer cells, endothelial cells, or osteocytes. Ligands can also include hormones and hormone receptors. They can also include non-peptide species, such as lipids, lectins, carbohydrates, vitamins, cofactors, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, or polyvalent fucose. Ligands can also be, for example, lipopolysaccharides, p38 MAP kinase activators, or NF-κB activators.

[0393] A ligand can be a substance, such as a drug, that can increase the uptake of an iRNA agent into a cell by, for example, disrupting the cytoskeleton of a cell, for example, by disrupting the microtubules, microfibrils, or intermediate fibers of a cell. A drug may be, for example, taxone, vincristine, vinblastine, cytochalasin, nocodazole, jasplakinolide, latruncrine A, phalloidin, swinford A, indanosine, or myoserbine.

[0394] In some embodiments, ligands attached to iRNAs, as described herein, act as pharmacokinetic modulators (PK modulators). PK modulators include lipophilic substances, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEGs, vitamins, and the like. Exemplary PK modulators, but not limited to, include cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, and biotin. Oligonucleotides containing several phosphorothioate linkages are also known to bind to serum proteins; therefore, short oligonucleotides, e.g., oligonucleotides of about 5, 10, 15, or 20 bases containing multiple phosphorothioate linkages in their backbone, are also suitable as ligands (e.g., as PK-modulating ligands) in the present invention. Furthermore, aptamers that bind to serum components (e.g., serum proteins) are also suitable for use as PK-modulating ligands in embodiments described herein.

[0395] iRNAs conjugated with the ligand of the present invention can be synthesized using oligonucleotides having pendant-reactive functionality, for example, those derived from the attachment of a linking molecule to an oligonucleotide (as described below). These reactive oligonucleotides can be directly reacted with commercially available ligands, synthetic ligands having any of the various protecting groups, or ligands having a linking portion attached thereto.

[0396] The oligonucleotides used in the conjugates of the present invention can be conveniently and routinely prepared by known solid-phase synthesis techniques. Equipment for such synthesis is available from several vendors, including, for example, Applied Biosystems® (Foster City, California). Any other means for such synthesis known in the art may be used further or instead. It is also known that similar techniques can be used to prepare other oligonucleotides, such as phosphorothioates and alkylated derivatives.

[0397] In the ligand-conjugated oligonucleotide and ligand-sequence-specific linked nucleosides of the present invention, the oligonucleotide and oligonucleosides can be assembled in a suitable DNA synthesizer using a standard nucleotide or nucleoside precursor, a nucleotide or nucleoside conjugate precursor already having a linking portion, a ligand-nucleotide or nucleoside conjugate precursor already having a ligand molecule, or a non-nucleoside ligand having a building block.

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

[0399] A. Lipid conjugates In certain embodiments, the ligand or conjugate is a lipid or lipid-based molecule. Such lipid or lipid-based molecules can typically bind to serum proteins, such as human serum albumin (HSA). HSA-binding ligands enable the distribution of the conjugate to target tissues in the body, such as non-renal target tissues. For example, the target tissue could be the liver, including the 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. Lipid or lipid-based ligands can (a) increase the resistance of the conjugate to degradation, (b) increase the targeting or transport into target cells or cell membranes, or (c) modulate binding to serum proteins, such as HSA.

[0400] Lipid-based ligands can be used to modulate, for example, control (e.g., inhibit) the binding of conjugates to target tissues. For instance, lipids or lipid-based ligands that bind more strongly to HSA are less likely to be targeted to the kidneys and therefore less likely to be eliminated from the body. Lipids or lipid-based ligands that do not bind less strongly to HSA can be used to target conjugates to the kidneys.

[0401] In certain embodiments, lipid-based ligands bind to HSA. For example, the ligand can bind to HSA with sufficient affinity, resulting in enhanced distribution of the conjugate to non-renal tissue. However, the affinity is usually not strong enough to reverse the HSA-ligand binding.

[0402] In certain embodiments, the lipid-based ligand may bind weakly to HSA or not bind at all, resulting in enhanced distribution of the conjugate to the kidney. Other moieties that target kidney cells can be used instead of, or in addition to, the lipid-based ligand.

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

[0404] B. Cell permeability agents In another embodiment, the ligand is a cell permeabilizer, such as a helix cell permeabilizer. In certain embodiments, these cell permeabilizers are amphiphilic. Exemplary cell permeabilizers include peptides, such as tat or antennopedia. If the agent is a peptide, it may be modified, including peptidyl mimetic, inverted isomers, non-peptide or pseudopeptide linkages, and the use of D-amino acids. Helix agents are typically α-helix agents and may have lipophilic and oleophobic phases.

[0405] Ligands can be peptides or peptidomimetic molecules. Peptidomimetic molecules (also referred to herein as oligopeptidomimetic molecules) are molecules that can fold into a defined three-dimensional structure similar to that of natural peptides. The attachment of peptides and peptidomimetic molecules to iRNA agents can affect the pharmacokinetic distribution of the iRNA, for example, by enhancing cell recognition and absorption. The peptide or peptidomimetic moiety may be about 5 to 50 amino acids long, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

[0406] Peptides or peptidomimetic molecules can be, for example, cell-penetrating peptides, cationic peptides, amphiphilic peptides, or hydrophobic peptides (e.g., mainly composed of Tyr, Trp, or Phe). The peptide moiety can be a dendrimer peptide, a restrictive peptide, or a cross-linked peptide. Alternatively, the peptide moiety may contain a hydrophobic membrane-transfer sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF with the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 11). RFGF analogues containing a hydrophobic MTS (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO: 12)) can also be targeting moieties. The peptide moiety can be a "delivery" peptide capable of carrying large polar molecules, including peptides, oligonucleotides, and proteins, across the cell membrane. For example, sequences derived from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 13)) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 14)) have been found to be functional as delivery peptides. Peptides or peptidomimetic molecules can be encoded by random sequences of DNA, such as peptides identified from phage display libraries or 1-bead-1-compound (OBOC) combinatorial libraries (Lam et al., Nature, 354:82-84, 1991). Typically, peptides or peptidomimetic molecules tethered to dsRNA agents via integrated monomer units include cell-targeting peptides, such as arginine-glycine-aspartate (RGD) peptides or RGD mimics. The peptide moiety can range in length from approximately 5 to 40 amino acids. The peptide moiety may have structural modifications that increase stability or direct conformational properties, for example. Any of the structural modifications described below are available.

[0407] The RGD peptides for use in the compositions and methods of the present invention may be linear or cyclic, and may be modified to facilitate targeting of specific tissues, for example, by glycosylation or methylation. RGD-containing peptides and peptidiomimemtics may include D-amino acids and synthetic RGD mimics. In addition to RGD, other parts that target integrin ligands may be used. Preferred conjugates of these ligands target PECAM-1 or VEGF.

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

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

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

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

[0412] In certain embodiments, the monosaccharide is N-acetylgalactosamine (GalNAc). GalNAc conjugates comprising one or more N-acetylgalactosamine (GalNAc) derivatives are described, for example, in US8,106,022, the entire contents of which are incorporated herein by reference. In some embodiments, the GalNAc conjugate acts as a ligand that targets iRNA to specific cells. In some embodiments, the GalNAc conjugate targets iRNA to liver cells, for example, by acting as a ligand for the asialocrycoprotein receptor in liver cells (e.g., hepatocytes).

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

[0414] In certain embodiments of the present invention, GalNAc or a GalNAc derivative is attached to the iRNA agent of the present invention via a monovalent linker. In some embodiments, GalNAc or a GalNAc derivative is attached to the iRNA agent of the present invention via a divalent linker. In yet another embodiment of the present invention, GalNAc or a GalNAc derivative is attached to the iRNA agent of the present invention via a trivalent linker. In yet another embodiment of the present invention, GalNAc or a GalNAc derivative is attached to the iRNA agent of the present invention via a tetravalent linker.

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

[0416] In some embodiments, for example, if the two strands of the iRNA agent of the present invention are part of one larger molecule connected by an uninterrupted chain of nucleotides between the 3' end of one strand and the 5' end of each of the other strands, forming a hairpin loop containing a plurality of unpaired nucleotides, each unpaired nucleotide in the hairpin loop may independently contain GalNAc or a GalNAc derivative attached via a monovalent linker. The hairpin loop may also be formed by an extended overhang in one of the two strands.

[0417] In some embodiments, for example, if the two strands of the iRNA agent of the present invention are part of one larger molecule connected by an uninterrupted chain of nucleotides between the 3' end of one strand and the 5' end of each of the other strands, forming a hairpin loop containing a plurality of unpaired nucleotides, each unpaired nucleotide in the hairpin loop may independently contain GalNAc or a GalNAc derivative attached via a monovalent linker. The hairpin loop may also be formed by an extended overhang in one of the two strands.

[0418] In some embodiments, the GalNAc conjugate is

[0419] [ka] That is the case.

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

[0421] [ka]

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

[0423] [ka]

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

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

[0426] [ka]

[0427] [ka]

[0428] [ka] [ka]

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

[0430] [ka] That is the case.

[0431] Other representative carbohydrate conjugates for use in the embodiments described herein, but not limited to,

[0432] [ka] [In the formula, one of X or Y is an oligonucleotide, and the other is hydrogen.] These are some examples.

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

[0434] [ka] Includes.

[0435] In certain embodiments, the RNAi agents of the Disclosure may include a GalNAc ligand, even if such a GalNAc ligand is currently expected to have limited value for the preferred subarachnoid / CNS delivery pathway(s) of the Disclosure.

[0436] In certain embodiments of the present invention, GalNAc or a GalNAc derivative is attached to the iRNA agent of the present invention via a monovalent linker. In some embodiments, GalNAc or a GalNAc derivative is attached to the iRNA agent of the present invention via a divalent linker. In yet another embodiment of the present invention, GalNAc or a GalNAc derivative is attached to the iRNA agent of the present invention via a trivalent linker.

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

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

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

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

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

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

[0443] The term "linker" or "linking group" refers to an organic part that connects two parts of a compound, for example, by covalent bonding.Linkers are typically directly bonded or composed of atoms such as oxygen or sulfur, units such as NR8, C(O), C(O)NH, SO, SO2, SO2NH, or not, but one or more methylene groups may be interrupted or terminated by O, S, S(O), SO2, N(R8), C(O), substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylal Quinnyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylal Kenyl, alkyl heteroarylalkynyl, alkenyl heteroarylalkyl, alkenyl heteroarylalkenyl, alkenyl heteroarylalkynyl, alkenyl heteroarylalkynyl, alkynyl heteroarylalkyl, alkynyl heteroarylalkenyl, alkynyl heteroarylalkynyl, alkyl heterocyclylalkyl, alkyl heterocyclylalkenyl, alkyl heterocyclylalkynyl (alkylhererocyclylalkynyl), alkenyl heterocyclylalkyl, alkenyl heterocyclylalkenyl, alkenyl heterocyclylalkynyl, alkynyl heterocyclylalkynyl, alkynyl heterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkyl heteroaryl, alkenyl heteroaryl, alkynyl heteroaryl (alkynylhereroaryl), R8 is hydrogen, acyl, aliphatic or substituted aliphatic, and includes a chain of atoms such as substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, etc.In certain embodiments, the linker has approximately 1 to 24 atoms, 2 to 24, 3 to 24, 4 to 24, 5 to 24, 6 to 24, 6 to 18, 7 to 18, 8 to 18 atoms, 7 to 17, 8 to 17, 6 to 16, 7 to 16, or 8 to 16 atoms.

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

[0445] Cleavable linking groups are susceptible to the influence of cleavage agents, such as pH, redox potential, or the presence of degradable molecules. Generally, cleavage agents are more common or found at higher levels or activity inside cells than in serum or blood. Examples of such degrading agents include redox agents selected for specific substrates or those without substrate specificity, including reducing agents such as mercaptans present in cells that can degrade redox-cleavable linking groups by oxidase or reductase or reduction, esterases, endosomes, or agents that can create an acidic environment, such as those that result in a pH of 5 or less, general acids, peptidases (which may be substrate-specific), and enzymes that can hydrolyze or degrade acid-cleavable linking groups by acting as phosphatases.

[0446] Cleavable linking groups, such as disulfide bonds, can be susceptible to pH changes. While human serum has a pH of 7.4, the average intracellular pH is slightly lower, ranging from approximately 7.1 to 7.3. Endosomes have a more acidic pH in the range of 5.5 to 6.0, and lysosomes have an even more acidic pH of approximately 5.0. Some linkers may have cleavable linking groups that are cleaved at a favorable pH, thereby releasing cationic lipids from ligands into desired compartments within the cell.

[0447] Linkers may contain cleavable linking groups that can be cleaved by specific enzymes. The type of cleavable linking group incorporated into the linker may vary depending on the cell to be targeted. For example, a liver-targeting ligand can be linked to a cationic lipid via a linker containing an ester group. Liver cells are rich in esterases, and therefore, linkers are cleaved more efficiently in liver cells than in cell types that are not rich in esterases. Other cell types rich in esterases include lung, renal cortex, and testicular cells.

[0448] When targeting peptidase-rich cell types such as liver cells and synovial cells, linkers containing peptide bonds can be used.

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

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

[0451] ii. Phosphate-based cleavable linking groups In certain embodiments, the cleavable linker includes a phosphate-based cleavable linking group. The phosphate-based cleavable linking group is cleaved by agents that decompose or hydrolyze the phosphate group. Examples of agents that cleave phosphate groups in cells include enzymes such as phosphatases in cells. Examples of phosphate-based linking groups include -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, and -OP(S)(Rk)-S-. Preferred embodiments include -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O-, -SP(S)(H)-O-, -SP(O)(H)-S-, and -OP(S)(H)-S-. A preferred embodiment is -OP(O)(OH)-O-. These candidates can be evaluated using a method similar to that described above.

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

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

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

[0455] In some embodiments, the iRNA of the present invention is conjugated to a carbohydrate via a linker. Examples of iRNA carbohydrate conjugates having linkers of the compositions and methods of the present invention, but not limited to these, include:

[0456] [ka] [ka] [If either X or Y is an oligonucleotide, then the other is hydrogen.] These are some examples.

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

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

[0459] [ka] [In the formula, q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B, and q5C each independently represent a number from 0 to 20 for each occurrence, and the repeating units may be the same or different. P 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , P 5C , T 2A , T 2B , T 3A , T 3B , T 4A , T 4B , T 4A , T 5B , T 5C Each occurrence is independently of the following: non-existent, CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH, or CH2O. Q 2A Q 2B Q 3A Q 3B Q 4A Q 4B Q 5A Q 5B Q 5C Each occurrence is independently of whether it is a non-existent alkylene, a substituted alkylene, and one or more methylenes, O, S, S(O), SO2, N(R) N), C(R')=C(R''), C≡C or C(O) may interrupt or terminate by one or more of these. R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , R 5C For each occurrence, independently, the non-existent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R) a )C(O), -C(O)-CH(R a )-NH-, CO, CH=NO,

[0460] [ka] or heterocycline, L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B and L 5C R represents a ligand, that is, each instance independently represents a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. a [H is an amino acid side chain]. Trivalent conjugate GalNAc derivatives target genes, e.g., those of formula (XLIX):

[0461] [ka] [In the formula, L 5A , L 5B and L 5C This represents a monosaccharide, for example, a GalNAc derivative. It is particularly useful for use in conjunction with RNAi agents to inhibit the expression of [specific gene / substance].

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

[0463] Representative U.S. patents teaching the preparation of RNA conjugates include, but are not limited to, U.S. Patents 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, and 5,486,603, 5,512,439, 5,578,718, 5,608,046, 4,587,044, 4,605,735, 4,667,025, 4,762,779, 4,789,737, 4,824,941, 4,835,263, 4,876,335, 4,904,582, 4,958,013, 5,082,830, 5,112,963, 5,214,136, 5,082,830, No. 5,112,963, No. 5,214,136, No. 5,245,022, No. 5,254,469, No. 5,258,506, No. 5,262,536, No. 5,272,250, No. 5,292,873, No. 5,317,098, No. No. 5,371,241, No. 5,391,723, No. 5,416,203, No. 5,451,463, No. 5,510,475, No. 5,512,667, No. 5,514,785, No. 5,565,552, No. 5,567,810, No. Examples include Nos. 5,574,142, 5,585,481, 5,587,371, 5,595,726, 5,597,696, 5,599,923, 5,599,928, 5,688,941, 6,294,664, 6,320,017, 6,576,752, 6,783,931, 6,900,297, 7,037,646, and 8,106,022, the entire contents of each of these are incorporated herein by reference.

[0464] It is not necessary for all positions in a given compound to be uniformly modified; in fact, two or more of the modifications can 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.

[0465] In relation to the present invention, "chimeric" iRNA compound or "chimeric" refers to an iRNA compound, preferably a dsRNA agent, that contains two or more chemically distinct regions, each consisting of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs typically contain at least one region in which the RNA is modified to confer increased resistance to nuclease degradation, increased cellular uptake, or increased binding affinity to a target nucleic acid. Further regions of the iRNA can act as substrates for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. For example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA double-stranded DNA. Therefore, activation of RNase H results in cleavage of the RNA target, thereby greatly enhancing the efficiency of iRNA inhibition of gene expression. Consequently, compared to phosphorothioate deoxy dsRNAs that hybridize to the same target region, it is often possible to obtain results that can be compared with shorter iRNAs when chimeric dsRNAs are used. Cleavage of RNA targets can be routinely detected by gel electrophoresis and, if necessary, by relevant nucleic acid hybridization techniques known in the art.

[0466] In certain cases, the RNA of an iRNA can be modified with a non-ligand group. Several non-ligand molecules have been conjugated to iRNAs to enhance their activity, cell distribution, or cell uptake, and procedures for carrying out such conjugations are available in the scientific literature. These non-ligand portions include lipid portions such as cholesterol [Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61, Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553], cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), thioethers, for example, 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 acid chains, e.g., dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111, Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), phospholipids, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651, Shea et al., Nucl. Acids Res., 1990, 18:3777), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969) or adamantane acetate (Manoharan et al.These included (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 teaching the preparation of such RNA conjugates are listed above. A typical conjugation protocol involves the synthesis of RNA having aminolinkers at one or more positions in its sequence. The amino group then reacts with the conjugated molecule using an appropriate coupling or activating reagent. The conjugation reaction can be performed while the RNA is still bound to a solid support or after the RNA has been cleaved in solution. Purification of the RNA conjugate by HPLC usually yields a pure conjugate.

[0467] V. Delivery of RNAi agents of this disclosure The delivery of the RNAi agents of this disclosure to cells, for example, cells within a subject, for example, cells within a human subject (for example, a subject that requires it, for example, a subject with HTT-related disease, for example, Huntington's disease), can be achieved in several different ways. For example, delivery may be carried out by contacting cells with the RNAi agents of this disclosure either in vitro or in vivo. In vivo delivery may be carried out directly by administering the RNAi agent, for example, a composition comprising dsRNA, to the subject. Alternatively, in vivo delivery may be carried out indirectly by administering one or more vectors that encode the RNAi agent and induce its expression. These alternatives are described further below.

[0468] In general, any method of delivering nucleic acid molecules (in vitro or in vivo) can be adapted to the use of RNAi agents in this disclosure [see, for example, Akhtar S. and Julian RL., (1992) Trends Cell. Biol. 2(5):139-144 and WO94 / 02595, which are incorporated herein in their entirety by reference]. In the case of in vivo delivery, factors to consider for delivering RNAi agents include, for example, the biological stability of the delivered agent, prevention of nonspecific effects, and accumulation of the delivered agent in the target tissue. Nonspecific effects of RNAi agents can be minimized by local administration, e.g., direct injection or transplantation into tissue or local administration of preparations. Local administration to the treatment site maximizes the local concentration of the agent, limits exposure of the agent to systemic tissues that may be harmed or degraded by the agent, and allows for a lower total dose of RNAi agent administered. Several studies have demonstrated successful knockdown of gene products when RNAi agents are administered locally. For example, intraocular delivery of VEGF dsRNA by intravitreous injection in cynomolgus monkeys [Tolentino, MJ. et al., (2004) Retina 24:132-138] and subretinal injection in mice [Reich, SJ. et al. (2003) Mol. Vis. 9:210-216] have both been shown to prevent neovascularization in experimental models of age-related macular degeneration. In addition, direct intratumoral injection of dsRNA into mice can reduce tumor volume [Pille, J. et al. (2005) Mol. Ther. 11:267-274] and prolong the survival of mice with tumors [Kim, WJ. et al., (2006) Mol. Ther. 14:343-350; Li, S. et al., (2007) Mol. Ther. 15:515-523].RNA interference can be administered to the CNS by direct injection [Dorn, G. et al., (2004) Nucleic Acids 32:e49; Tan, PH. et al. (2005) Gene Ther. 12:59-66; Makimura, H. et al. (2002) BMC Neurosci. 3:18; Shishkina, GT., et al. (2004) Neuroscience 129:521-528; Thakker, ER., et al. (2004) Proc. Natl. Acad. Sci. USA 101:17270-17275; Akaneya, Y., et al. (2005) J. Neurophysiol. 93:594-602] and to the lungs by intranasal administration [Howard, KA. et al., (2006) Mol. Ther. [14:476-484; Zhang, X. et al., (2004) J. Biol. Chem. 279:10677-10684; Bitko, V. et al., (2005) Nat. Med. 11:50-55], success has also been demonstrated by local delivery. When RNAi agents are administered systemically to treat a disease, the RNA can be modified, or instead, delivered using a drug delivery system; both methods function to prevent the rapid degradation of dsRNA by endonucleases and exonucleases in vivo. Modification of RNA or drug carriers can also enable the targeting of RNAi agents to target tissues and avoid undesirable off-target effects (for example, although we do not wish to be bound by theory, the use of GNAs described herein has been identified to destabilize the seed region of dsRNAs, and such off-target effects are significantly attenuated by such seed region destabilization, thereby increasing the preference of such dsRNAs for on-target efficacy compared to off-target effects). RNAi agents can be modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation.For example, when RNAi agents derived to ApoB conjugated to a lipophilic cholesterol portion were systemically injected into mice, it resulted in knockdown of apoB mRNA in both the liver and jejunum [Soutschek, J. et al., (2004) Nature 432:173-178]. Conjugation of RNAi agents to aptamers has been shown to inhibit tumor growth and mediate tumor reduction in a mouse model of prostate cancer [McNamara, JO. et al., (2006) Nat. Biotechnol. 24:1005-1015]. In alternative embodiments, RNAi agents can be delivered using drug delivery systems, such as nanoparticles, dendrimers, polymers, liposomes, or cationic delivery systems. Positively charged cationic delivery systems promote the binding of molecular RNAi agents (negatively charged) and enhance interactions with negatively charged cell membranes, thereby enabling efficient uptake of RNAi agents by cells. Cationic lipids, dendrimers, or polymers can bind to RNAi agents or be induced to form vesicles or micelles that encapsulate RNAi agents [see, for example, Kim SH. et al., (2008) Journal of Controlled Release 129(2):107-116]. Vesicle or micelle formation further prevents the degradation of RNAi agents when administered systemically. Methods for preparing and administering cationic RNAi agent conjugates are well within the capabilities of those skilled in the art [see, for example, Sorensen, DR., et al. (2003) J. Mol. Biol 327:761-766; Verma, UN. et al., (2003) Clin. Cancer Res. 9:1291-1300; Arnold, AS et al. (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entirety].Some non-limiting examples of drug delivery systems useful for systemic delivery of RNAi agents include DOTAP [Sorensen, DR., et al (2003), supra; Verma, UN. et al., (2003), supra], oligofectamine, "solid nucleic acid lipid particles" [Zimmermann, TS. et al., (2006) Nature 441:111-114], cardiolipin [Chien, PY. et al., (2005) Cancer Gene Ther. 12:321-328; Pal, A. et al., (2005) Int J. Oncol. 26:1087-1091], and polyethylenemine [Bonnet ME. et al., (2008) Pharm. Res. Aug 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol.]. Examples include

[71659] , Arg-Gly-Asp(RGD) peptide [Liu, S. (2006) Mol. Pharm. 3:472-487], and polyamidoamine [Tomalia, DA. et al., (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H. et al., (1999) Pharm. Res. 16:1799-1804]. In some embodiments, the RNAi agent forms a complex with cyclodextrin for systemic administration. Methods of administration and pharmaceutical compositions of RNAi agents and cyclodextrin can be found in U.S. Patent No. 7,427,605, which is incorporated herein by reference in whole.

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

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

[0471] Another aspect of the present disclosure relates to a method for treating a subject having a CNS disorder, comprising administering to the subject a therapeutically effective dose of the double-stranded HTT-targeted RNAi agent of the present disclosure, thereby treating the subject. An exemplary CNS disorder that can be treated by the method of the present disclosure is Huntington's disease.

[0472] In one embodiment, the double-stranded RNAi agent is administered intrathecally. By intrathecal administration of the double-stranded RNAi agent, the method can reduce the expression of HTT target genes in brain (e.g., striatum) or spinal tissue, such as the cortex, cerebellum, cervical vertebrae, lumbar vertebrae, and thoracic vertebrae.

[0473] For the sake of clarity, the formulations, compositions, and methods described in this section will primarily relate to modified siRNA compounds. However, it should be understood that these formulations, compositions, and methods can be implemented with other siRNA compounds, such as unmodified siRNA compounds, and that such implementations are also covered in this disclosure. Compositions containing RNAi agents can be delivered to the target by various routes. Exemplary routes include intrathecal, intravenous, topical, transrectal, transanal, transvaginal, transnasal, transpulmonary, and transocular delivery.

[0474] The RNAi agents of this disclosure can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise one or more RNAi agents and a pharmaceutically acceptable carrier. As used herein, the phrase “pharmaceutically acceptable carrier” is intended to include any solvent, dispersion medium, coating agent, antifungal and antifungal agent, isotonic and absorption retardant, and similar, that is suitable for pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agent is incompatible with the active ingredient, their use in a composition is conceived. Supplementary active ingredients may also be incorporated into a composition.

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

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

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

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

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

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

[0481] In one embodiment, the administration of an siRNA compound, such as a double-stranded siRNA compound or ssiRNA compound, composition, is parenteral, for example, intravenous (e.g., as a bolus or diffuse infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracerebral, subcutaneous, transmucosal, buccal, sublingual, endoscopic, transrectal, oral, transvaginal, topical, transpulmonary, intranasal, transurethral, ​​or transocular. The administration may be given by the patient or by another person, such as a healthcare provider. The drug may be provided in measured doses or in a dispenser that delivers weighed doses. The selected mode of delivery is described in more detail below.

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

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

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

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

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

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

[0488] RNAi agent expression vectors are generally DNA plasmids or viral vectors. By using expression vectors compatible with eukaryotic cells, preferably expression vectors compatible with vertebrate cells, recombinant constructs for the expression of RNAi agents described herein can be generated. The delivery of RNAi agent expression vectors may be systemic, for example, by intravenous or intramuscular administration, by administration to target cells explanted from a patient and subsequent reintroduction into the patient, or by any other means that enable introduction into desired target cells.

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

[0490] VI. Pharmaceutical composition of the present invention This disclosure also includes pharmaceutical compositions and formulations comprising the RNAi agents of this disclosure. In one embodiment, the RNAi agent described herein and a pharmaceutically acceptable carrier A pharmaceutical composition comprising the RNAi agent is provided herein. The pharmaceutical composition comprising the RNAi agent is useful for the treatment of diseases or disorders related to the expression or activity of HTT, such as Huntington's disease.

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

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

[0493] The pharmaceutical compositions of this disclosure may be administered in doses sufficient to inhibit the expression of the HTT gene. Generally, preferred doses of the RNAi agents of this disclosure will range from about 0.001 milligrams to about 200.0 milligrams per kilogram of body weight of the recipient per day, generally ranging from about 1 mg to 50 mg per kilogram of body weight per day.

[0494] A recurrent dosing regimen may involve regular, therapeutic doses of the RNAi agent, for example, once a month to once every six months. In certain embodiments, the RNAi agent is administered approximately once a quarter (i.e., approximately once every three months) to approximately twice a year.

[0495] After the initial treatment plan (e.g., initial loading dose), treatment can be administered at a low frequency.

[0496] In other embodiments, a single dose of the pharmaceutical composition may be continued, thereby administering subsequent doses at intervals of one month or less, two months or less, three months or less, four months or less, or longer. In some embodiments of the Disclosure, a single dose of the pharmaceutical composition of the Disclosure is administered once a month. In other embodiments of the Disclosure, a single dose of the pharmaceutical composition of the Disclosure is administered quarterly to twice a year.

[0497] Those skilled in the art will understand that certain factors, such as, but not limited to, the severity of the disease or disability, previous treatments, the subject's overall health or age, and other pre-existing conditions, may influence the dosage and timing of administration required to effectively treat the subject. Furthermore, treatment of the subject with a therapeutically effective amount of the composition may consist of a single treatment or a series of treatments.

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

[0499] The pharmaceutical compositions of this disclosure can be administered in several ways, depending on whether a topical or systemic treatment is desired and the area to be treated. Administration may be topical (e.g., by transdermal patch), transpulmonary (e.g., by inhalation or blowing of powder or aerosol formulations, such as by a nebulizer); intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration may include intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subcutaneous (e.g., by an implanted device), or intracerebral (e.g., by intraparenchymal, intrathecal, or intraventricular administration).

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

[0501] Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, solutions, and powders. Conventional pharmaceutical carriers, aqueous, powdery, or oily bases, thickeners, etc., may be necessary or desirable. Coated condoms, gloves, etc., may also be useful. Suitable topical formulations include those in which the RNAi agent characterized in this disclosure is a mixture with a topical delivery agent, such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents, and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidylethanolamine DOPE, dimyristoylphosphatidylcholine DMPC, distearoylphosphatidylcholine), negative (e.g., dimyristoylphosphatidylglycerol DMPG), and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). The RNAi agents featured in this disclosure can be encapsulated within liposomes or can form complexes with liposomes, particularly cationic liposomes. Alternatively, the RNAi agents can be complexed with lipids, particularly cationic lipids. Suitable fatty acids and esters, but not limited to these, include arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaproate, tricaproate, monoolein, dilaurin, glyceryl 1-monocaproate, 1-dodecyl azacycloheptana-2-one, acylcarnitine, acylcholine, or C 1~20Examples include alkyl esters (e.g., isopropyl myristate IPM), monoglycerides, deglycerides, or pharmaceutically acceptable salts thereof. Topical formulations are described in detail in US6,747,014, which is incorporated herein by reference.

[0502] A. RNAi preparations containing membrane molecular assemblies RNAi agents for use in the compositions and methods of this disclosure can be formulated for delivery in membrane molecular assemblies, e.g., liposomes or micelles. As used herein, the term “liposome” means a vesicle composed of amphiphilic lipids aligned in at least one bilayer, e.g., one or more bilayers. Liposomes include monolayer and multilayer lamellar vesicles having a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the RNAi agent composition. The lipophilic material isolates the aqueous interior from the aqueous exterior, which typically does not contain the RNAi agent composition (although in some examples it may). Liposomes are useful for the transport and delivery of active ingredients to the site of action. Because the liposome membrane is structurally similar to that of a biological membrane, when a liposome is applied to a tissue, the liposome bilayer fuses with the bilayer of the cell membrane. As the liposome-cell fusion proceeds, the internal aqueous contents containing the RNAi agent are delivered into the cell, where the RNAi agent can specifically bind to the target RNA and mediate RNAi. In some cases, liposomes can also be specifically targeted, for example, to induce RNAi agents into specific cell types.

[0503] Liposomes containing RNAi agents can be prepared by various methods. In one example, the lipid component of the liposome is dissolved in a surfactant, thereby forming micelles. For example, the lipid component may be an amphiphilic cationic lipid or a lipid conjugate. The surfactant may have a high critical micelle concentration and may be nonionic. Exemplary surfactants include cholates, CHAPS, octyl glucoside, deoxycholate, and lauroyl sarcosine. The RNAi agent is then added to the micelles containing the lipid component. The cationic groups on the lipid interact with the RNAi agent and condense around it to form liposomes. After condensation, the surfactant is removed, for example, by dialysis, to obtain a liposome preparation of the RNAi agent.

[0504] If necessary, a support compound can be added during the aggregation reaction, for example, by controlled addition, to aid aggregation. For example, the support compound may be a polymer other than nucleic acid (e.g., spermine or spermidine). The pH can also be adjusted to support aggregation.

[0505] A method for generating a stable polynucleotide delivery vehicle by incorporating polynucleotide / cationic lipid complexes as structural elements of the delivery vehicle is described in more detail, for example, in WO96 / 37194, the entire content of which is incorporated herein by reference. Formation of liposomes is described in Felgner, PL et al., (1987) Proc. Natl. Acad. Sci. USA 8:7413-7417; US Patent No. 4,897,355; National Patent No. 5,171,678; 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) This may also include one or more embodiments of the exemplary methods described in Endocrinol. 115:757. Commonly used methods for preparing lipid assemblies of appropriate size for use as delivery vehicles include sonication, freeze-thaw, and extrusion [see, e.g., Mayer et al., (1986) Biochim. Biophys. Acta 858:161]. Micro-solution preparation can be used when consistently small (50 nm to 2...

Claims

1. A double-stranded ribonucleic acid (dsRNA) agent or a salt thereof for inhibiting huntingtin (HTT) expression, The dsRNA agent or a salt thereof comprises a sense strand and an antisense strand that form a double-stranded region, The sense strand is 19-21 nucleotides long, and the antisense strand is 21-23 nucleotides long. The antisense strand contains at least the first 19 consecutive nucleotides from the nucleotide sequence 5'-UAUCAGCCUUUUUCCAGGGUGCCG-3' of SEQ ID NO: 4200, counting from the 5' end. All nucleotides of the sense strand and all nucleotides of the antisense strand independently include nucleotide modifications selected from the group consisting of 2'-O-methyl nucleotide modifications, 2'-fluoro nucleotide modifications, debasing modifications, mismatches with opposing nucleotides on the opposing strand, acyclic nucleotides, 2'-deoxy-nucleotide modifications, unlocked nucleic acids (UNAs), glycol nucleic acids (GNAs), cytidine-2'-phosphate, guanosine-2'-phosphate, uridine-2'-phosphate, and adenosine-2'-phosphate, and thermal destabilization modifications selected from the group consisting of these. When thermal destabilization modifications are present, only the antisense strand of the dsRNA agent or its salt contains the thermal destabilization modifications. The dsRNA agent or a salt thereof further comprises 6 to 8 phosphorothioate nucleotide linkages, and One or more lipophilic moieties containing one or more saturated or unsaturated C6-C18 hydrocarbon chains are conjugated to one or more internal positions selected from the group consisting of positions 4-8 and 13-18 on the sense chain, counting from the 5' end of the sense chain. dsRNA preparation or a salt thereof.

2. The dsRNA agent or a salt thereof according to claim 1, wherein the thermal destabilization modification is an unlocked nucleic acid (UNA) or a glycol nucleic acid (GNA).

3. The dsRNA agent or a salt thereof according to claim 1, wherein the thermal destabilization modification is cytidine-2'-phosphate.

4. The dsRNA agent or salt thereof according to claim 1, wherein the dsRNA agent or salt thereof comprises a 2'-5' nucleotide ligation modification.

5. A dsRNA agent or salt thereof according to any one of claims 1 to 4, wherein one or more saturated or unsaturated C6-C18 hydrocarbon chains are conjugated at one or more internal positions in the sense chain via a linker or carrier.

6. A dsRNA agent or a salt thereof according to any one of claims 1 to 5, wherein the saturated or unsaturated C6-C18 hydrocarbon chain further comprises a functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.

7. A dsRNA agent or a salt thereof according to any one of claims 1 to 5, wherein one or more saturated or unsaturated C6-C18 hydrocarbon chains are saturated or unsaturated C16 hydrocarbon chains and are conjugated at position 6 counting from the 5' end of the sense chain.

8. A dsRNA agent or salt thereof according to any one of claims 1 to 7, wherein one or more saturated or unsaturated C6-C18 hydrocarbon chains are conjugated via a carrier that replaces one or more nucleotides in an internal position or double-stranded region.

9. The dsRNA agent or a salt thereof according to claim 8, wherein the carrier is a cyclic group selected from the group consisting of pyrrolidinil, pyrazolinil, pyrazolidinil, imidazolinil, imidazolidinil, piperidinil, piperazinil, [1,3]dioxolanil, oxazolidinil, isoxazolidinil, morpholinil, thiazolidinil, isothiazolidinil, quinoxalinil, pyridadinil, tetrahydrofuranil, and dekalinil.

10. A dsRNA agent or salt thereof according to any one of claims 1 to 7, wherein one or more saturated or unsaturated C6-C18 hydrocarbon chains are conjugated to a dsRNA agent or salt thereof via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfamide linkage, or carbamate.

11. A dsRNA agent or a salt thereof according to any one of claims 1 to 10, wherein one or more saturated or unsaturated C6-C18 hydrocarbon chains are conjugated to nucleic acid bases, sugar moieties, or internucleoside links.

12. A dsRNA agent or a salt thereof according to any one of claims 1 to 11, wherein two 5' terminal nucleotides and two 3' terminal nucleotides of the antisense strand contain phosphorothioate nucleotide linkages, and two 5' terminal nucleotides and two 3' terminal nucleotides of the sense strand contain phosphorothioate nucleotide linkages.

13. A dsRNA agent or a salt thereof according to any one of claims 1 to 12, wherein at least one strand comprises a 3' overhang of at least one nucleotide.

14. A dsRNA agent or a salt thereof according to any one of claims 1 to 13, wherein the 3' end of the sense strand is protected via an end cap which is a cyclic group having an amine, and the cyclic group is selected from the group consisting of pyrrolidinyl, pyrazolinil, pyrazolidinyl, imidazolinil, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanil, oxazolidinyl, isoxazolidinyl, morpholinil, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridadinyl, tetrahydrofuranil, and dekalinil.

15. A dsRNA agent or salt thereof according to any one of claims 1 to 14, further comprising a phosphate or phosphate mimetic at the 5' end of an antisense strand, wherein the phosphate mimetic is a 5'-vinylphosphonate (VP).

16. A dsRNA agent or a salt thereof according to any one of claims 1 to 15, wherein the sense strand comprises at least 19 consecutive nucleotides from the nucleotide sequence 5'-GCGAACCCUGGGAAAAAGCUGAAUA-3' of SEQ ID NO: 3814.

17. A dsRNA agent or a salt thereof according to any one of claims 1 to 16, wherein the sense strand comprises at least 19 consecutive nucleotides from the nucleotide sequence 5'-GCGAACCCUGGGAAAAAGCUGAAUA-3' of SEQ ID NO: 3814, and the antisense strand comprises at least 19 consecutive nucleotides from the nucleotide sequence 5'-UAUCAGCCUUUUCCAGGGUGCCG-3' of SEQ ID NO: 4200.

18. A dsRNA agent or salt thereof according to any one of claims 1 to 16, wherein the sense strand comprises the nucleotide sequence 5'-GCGAACCCUGGGAAAAAGCUGAAUA-3' of SEQ ID NO: 3814, and the antisense strand comprises the nucleotide sequence 5'-UAUCAGCCUUUUCCAGGGUGCCG-3' of SEQ ID NO: 4200.

19. Cells containing the dsRNA agent or a salt thereof according to any one of claims 1 to 18.

20. A pharmaceutical composition for inhibiting the expression of a gene encoding HTT, comprising a dsRNA agent or a salt thereof according to any one of claims 1 to 18.

21. A pharmaceutical composition comprising a dsRNA agent or a salt thereof and a lipid preparation according to any one of claims 1 to 18.

22. An in vitro method for inhibiting the expression of the huntingtin (HTT) gene in cells, (a) Contacting cells with a dsRNA agent or a salt thereof according to any one of claims 1 to 18 or a pharmaceutical composition according to claim 20 or 21, and (b) Maintain the cells generated in step (a) for a sufficient amount of time to obtain degradation of the mRNA transcript of the HTT gene, thereby inhibiting the expression of the HTT gene in the cells. In vitro methods including those mentioned above.

23. A pharmaceutical composition for treating subjects diagnosed with HTT-related disease, comprising a therapeutically effective amount of a dsRNA agent or a salt thereof according to any one of claims 1 to 18 or the pharmaceutical composition according to claim 20 or 21.

24. The pharmaceutical composition according to claim 23, wherein the HTT-related disease is Huntington's disease.

25. The pharmaceutical composition according to claim 23 or 24, wherein the dsRNA agent or a salt thereof is to be administered to a subject in a dose of 0.01 mg / kg to 50 mg / kg.

26. The pharmaceutical composition according to any one of claims 23 to 25, wherein the dsRNA agent or a salt thereof is for intrathecal administration.

27. ​​The pharmaceutical composition according to any one of claims 23 to 26, further comprising an additional agent suitable for the treatment or prevention of HTT-related disorders, selected from the group consisting of monoamine inhibitors, reserpine, anticonvulsants, antipsychotics, and antidepressants, and combinations thereof.

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