Modified oligonucleotides targeting SNPs

Abstract

Novel nucleotides are provided that enhance silencing of expression of genes containing single nucleotide polymorphisms (SNPs) relative to expression of the corresponding wild-type gene. Methods are provided that use the novel nucleotides to enhance silencing of expression of genes containing SNPs relative to expression of the corresponding wild-type gene.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 717,287, filed August 10, 2018, and U.S. Provisional Patent Application No. 62 / 825,429, filed March 28, 2019, the entire contents of which are incorporated herein by reference.

[0002] Declaration of Federally Sponsored Research This invention was made with Government support under Grant Nos. NS104022 and GM108803 awarded by the National Institutes of Health. The Government has certain rights in this invention. [Background technology]

[0003] RNA interference provides a simple and effective tool for inhibiting gene function. RNA silencing agents are of particular interest as research tools and therapeutic agents due to their ability to knock down the expression of specific proteins with a high degree of sequence specificity. The sequence specificity of RNA silencing agents is particularly useful for treating diseases caused by dominant mutations in heterozygotes with one mutant and one wild-type copy of a specific gene. However, there is still a need for RNA silencing agents that can preferentially silence mutant, disease-causing allele expression while not affecting or only minimally affecting the expression of wild-type allele. Summary of the Invention [Means for solving the problem]

[0004] The present invention is based on the surprising discovery of novel oligonucleotides that enhance the silencing of expression of genes containing single nucleotide polymorphisms (SNPs) (e.g., heterozygous SNPs) relative to the expression of the corresponding wild-type gene in heterozygotes, for example, by more than 100-fold. In certain aspects, oligonucleotides (e.g., dsRNAs) are provided that preferentially target SNP-containing nucleic acids for degradation, and oligonucleotides (e.g., double-stranded RNAs (dsRNAs)) that do not target, or target to a lesser extent, the corresponding wild-type (non-SNP-containing) nucleic acids for degradation. In certain aspects, the oligonucleotides (e.g., dsRNAs) of the present invention are 1) complementary to the SNP position in the target nucleic acid; and 2) contain a mismatch at a specific position of the target nucleic acid relative to the SNP. In certain embodiments, the oligonucleotides (e.g., dsRNAs) of the present invention also contain two mismatches relative to the corresponding wild-type nucleic acid sequence: 1) the wild-type SNP position; and 2) at a specific position of the target nucleic acid sequence relative to the wild-type SNP position. Thus, an exemplary oligonucleotide (e.g., dsRNA) contains one mismatch to the SNP-containing target and two mismatches to the corresponding wild-type sequence, thus resulting in preferential cleavage of the SNP-containing target relative to the corresponding wild-type sequence.

[0005] In one aspect, an oligonucleotide is provided having a 5' end, a 3' end and a seed region, the oligonucleotide comprising a SNP position nucleotide at a position within the seed region that is complementary to a region of a gene comprising an allelic polymorphism, the SNP position nucleotide being complementary to the allelic polymorphism, and a mismatch (MM) position nucleotide located 2-11 nucleotides from the SNP position nucleotide that is mismatched to a nucleotide in the gene. In some cases, the oligonucleotide is complementary to a region of a gene comprising an allelic polymorphism, the oligonucleotide comprising a SNP position nucleotide at any one of positions 2-6 from the 5' end, and a mismatch position nucleotide located 2-11 nucleotides from the SNP position nucleotide that is mismatched to a nucleotide in the gene.

[0006] In certain exemplary embodiments, the oligonucleotide is RNA.

[0007] In certain exemplary embodiments, the RNA has the formula: [ka] The compound further comprises at least one vinyl phosphonate (VP) modification at an intersubunit linkage having the formula:

[0008] In certain exemplary embodiments, a VP motif is inserted next to the SNP position nucleotide or next to the MM position nucleotide.

[0009] In certain exemplary embodiments, the oligonucleotide is selected from the group consisting of siRNA, miRNA, shRNA, CRISPR guide, DNA, antisense oligonucleotide (ASO), gapmer, mixmer, miRNA inhibitor, splice switching oligonucleotide (SSO), phosphorodiamidate morpholino oligomer (PMO) and peptide nucleic acid (PNA).

[0010] In certain exemplary embodiments, the RNA is an antisense oligonucleotide (ASO) or dsRNA.

[0011] In certain exemplary embodiments, the dsRNA comprises a first strand of about 15-35 nucleotides that is complementary to a region of the gene that contains an allelic polymorphism and a second strand of about 15-35 nucleotides that is complementary to at least a portion of the first strand, wherein the first strand comprises a SNP position nucleotide in the seed region (e.g., at positions 2-6 from the 5' end) that is complementary to the allelic polymorphism, and the first strand comprises an MM position nucleotide located 2-6 nucleotides from the SNP position nucleotide that is mismatched to a nucleotide in the gene.

[0012] In certain exemplary embodiments, the SNP position nucleotide is located at position 2, 4 or 6 from the 5' end of the RNA, and the MM position nucleotide is located 2-6 nucleotides from the SNP position nucleotide.

[0013] In certain exemplary embodiments, the MM position nucleotide is located within 2, 3, 4, or 6 nucleotides of the SNP position nucleotide. In certain exemplary embodiments, the MM position nucleotide is located within 5 nucleotides of the SNP position nucleotide.

[0014] In certain exemplary embodiments, dsRNA is blunt-ended.In certain exemplary embodiments, dsRNA comprises at least one single-stranded nucleotide overhang.In certain exemplary embodiments, dsRNA comprises naturally occurring nucleotides.In certain exemplary embodiments, dsRNA comprises at least one modified nucleotide.In certain exemplary embodiments, each nucleotide of dsRNA is modified.

[0015] In certain exemplary embodiments, the at least one modified nucleotide is selected from the group consisting of a 2'-O-methyl modified nucleotide, a nucleotide comprising a 5'-phosphorothioate group, a terminal nucleotide linked to a cholesteryl derivative, and a terminal nucleotide linked to a dodecanoic acid bisdecylamide group.

[0016] In certain exemplary embodiments, the modified nucleotide is selected from the group consisting of 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, and nucleotides containing unnatural bases.

[0017] In certain exemplary embodiments, the dsRNA comprises at least one 2'-O-methyl modified nucleotide and a 5'-phosphorothioate group.

[0018] In certain exemplary embodiments, the first strand comprises at least three 2'-O-methyl modified nucleotides. In certain exemplary embodiments, the first strand comprises 2'-O-methyl modified nucleotides on either side of the SNP position nucleotide.

[0019] In certain exemplary embodiments, the dsRNA comprises a hydrophobic portion.

[0020] In certain exemplary embodiments, the region of the gene that contains the allelic polymorphism comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 10. In certain exemplary embodiments, the first strand comprises UUCUGUAGCAUCAGCUUCUC.

[0021] In certain exemplary embodiments, the RNA described herein (e.g., the first strand of a dsRNA) comprises a combination of SNP position nucleotide (referenced from the 5' end)-MM position nucleotide (referenced from the 5' end) selected from the group consisting of 2-7, 4-7, 4-8, 4-15, 6-5, 6-8, 6-11, 6-14, 6-16, 3-5, 3-7, and 3-8.

[0022] In certain exemplary embodiments, the SNP position nucleotide is complementary to an allelic polymorphism of an htt SNP selected from the group consisting of rs363125, rs362273, rs362307, rs362336, rs362331, rs362272, rs362306, rs362268, rs362267, and rs363099.

[0023] In certain exemplary embodiments, the RNA further comprises a 5' stabilizing moiety selected from the group consisting of phosphate, vinyl phosphonate, C5-methyl (R, or S, or racemic), C5-methyl on vinyl, and reduced vinyl.

[0024] In certain exemplary embodiments, the RNA further comprises a conjugate moiety selected from the group consisting of an alkyl chain, a vitamin, a peptide, a glycosphingolipid, a polyunsaturated fatty acid, a secosteroid, a steroid hormone, and a steroid lipid.

[0025] In one aspect, a dsRNA is provided that comprises a first strand of about 15-35 nucleotides that is complementary to a region of a gene that contains an allelic polymorphism and a second strand of about 15-35 nucleotides that is complementary to at least a portion of the first strand, wherein the first strand comprises a SNP position nucleotide at any one of the positions in the seed region (e.g., one of positions 2-6 from the 5' end) that is complementary to the allelic polymorphism, and the first strand comprises an MM position nucleotide located 2-6 nucleotides from the SNP position nucleotide that is mismatched to a nucleotide in the gene.

[0026] In certain exemplary embodiments, a dsRNA is provided that comprises a first strand of about 15-35 nucleotides that is complementary to a region of a gene that contains an allelic polymorphism and a second strand of about 15-35 nucleotides that is complementary to at least a portion of the first strand, wherein the first strand comprises a SNP position nucleotide at position 2, 4 or 6 from the 5' end that is complementary to the allelic polymorphism, and the first strand comprises an MM position nucleotide located 2-6 nucleotides from the SNP position nucleotide that is mismatched to a nucleotide in the gene.

[0027] In certain exemplary embodiments, the RNA has the formula: [ka] The compound further comprises at least one vinyl phosphonate modification at an intersubunit linkage having the formula:

[0028] In certain exemplary embodiments, a VP motif is inserted next to the SNP position nucleotide or next to the MM position nucleotide.

[0029] In certain exemplary embodiments, the SNP position nucleotide is at position 2 from the 5' end of the first strand, position 4 from the 5' end of the first strand, or position 6 from the 5' end of the first strand. In other exemplary embodiments, the MM position nucleotide is located within 2 nucleotides of the SNP position nucleotide, within 3 nucleotides of the SNP position nucleotide, within 4 nucleotides of the SNP position nucleotide, within 5 nucleotides of the SNP position nucleotide, or within 6 nucleotides of the SNP position nucleotide. In certain exemplary embodiments, the SNP position nucleotide is located 4 nucleotides from the 5' end and the MM position nucleotide is located 7 nucleotides from the 5' end. In other exemplary embodiments, the SNP position nucleotide is located 6 nucleotides from the 5' end and the MM position nucleotide is located 11 nucleotides from the 5' end.

[0030] In another aspect, there is provided a dsRNA comprising a first strand of about 15-35 nucleotides that is complementary to a region of a gene comprising an allelic polymorphism and a second strand of about 15-35 nucleotides that is complementary to at least a portion of the first strand, wherein the first strand comprises a SNP position nucleotide at position 6 from the 5' end that is complementary to the allelic polymorphism, and the first strand comprises a MM position nucleotide located at position 11 from the 5' end that is mismatched to a nucleotide in the gene.

[0031] In certain exemplary embodiments, the RNA has the formula: [ka] In certain exemplary embodiments, the VP motif is inserted next to the SNP position nucleotide or next to the MM position nucleotide.

[0032] In certain exemplary embodiments, the first strand comprises 2'-O-methyl modified nucleotides on either side of the SNP position nucleotide. In certain exemplary embodiments, the first strand comprises at least three 2'-O-methyl modified nucleotides.

[0033] In certain exemplary embodiments, the dsRNA comprises a 5'-phosphorothioate group.

[0034] In certain exemplary embodiments, the gene comprising the allelic polymorphism is the huntingtin (htt) gene.

[0035] In certain exemplary embodiments, the region of the gene that contains the allelic polymorphism comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 10. In certain exemplary embodiments, the first strand comprises UUCUGUAGCAUCAGCUUCUC.

[0036] In certain aspects, a pharmaceutical composition is provided that includes an RNA described herein and a pharma- ceutically acceptable carrier.

[0037] In certain aspects, methods are provided for inhibiting expression of a gene that comprises an allelic variant in a cell, comprising contacting the cell with any of the methods described herein.

[0038] In another aspect, there is provided a method of treating a disease or disorder characterized or caused by a gene comprising an allelic polymorphism in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of RNA having a 5' end, a 3' end and a seed region that is complementary to a region of the gene comprising an allelic polymorphism, the RNA comprising a SNP position nucleotide at a position in the seed region that is complementary to the allelic polymorphism and a MM position nucleotide located 2-11 nucleotides from the SNP position nucleotide that is mismatched to a nucleotide in the gene. In certain exemplary embodiments, the RNA has the formula: [ka] The compound further comprises at least one vinyl phosphonate modification at an intersubunit linkage having the formula:

[0039] In certain exemplary embodiments, a VP motif is inserted next to the SNP position nucleotide or next to the MM position nucleotide.

[0040] In certain exemplary embodiments, the RNA is an ASO or a dsRNA.

[0041] In certain exemplary embodiments, the dsRNA comprises a first strand of about 15-35 nucleotides that is complementary to a region of the gene that contains an allelic polymorphism and a second strand of about 15-35 nucleotides that is complementary to at least a portion of the first strand, wherein the first strand comprises a SNP position nucleotide within the seed region (e.g., at any one of positions 2-6 from the 5' end, e.g., at position 2, 4 or 6 from the 5' end) that is complementary to the allelic polymorphism, and the first strand comprises an MM position nucleotide located 2-6 nucleotides from the SNP position nucleotide that is mismatched to a nucleotide in the gene.

[0042] In some exemplary embodiments, the dsRNA is administered to the brain of the subject.In some exemplary embodiments, the dsRNA is administered by intrastriatal injection.In some exemplary embodiments, the expression of gene is reduced in the striatum.In some exemplary embodiments, the expression of gene is reduced in the cortex.

[0043] In certain exemplary embodiments, the gene comprising the allelic polymorphism is the huntingtin (htt) gene, hi certain exemplary embodiments, the disease is Huntington's disease.

[0044] In certain exemplary embodiments, the SNP position nucleotide is located at position 2, 4 or 6 from the 5' end of the RNA, and the MM position nucleotide is located 2-6 nucleotides from the SNP position nucleotide.

[0045] In another aspect, a bifurcated oligonucleotide compound is provided that comprises two RNAs, the RNAs being linked to one another by one or more moieties selected from a linker, a spacer, and a branch point, each RNA having a 5' end, a 3' end, and a seed region, each RNA being complementary to a region of a gene comprising an allelic polymorphism, each RNA comprising a SNP position nucleotide at a position within the seed region, the SNP position nucleotide being complementary to the allelic polymorphism and an MM position nucleotide located 2 to 11 nucleotides from the SNP position nucleotide that is mismatched to the SNP position nucleotide complementary to the allelic polymorphism and a nucleotide in the gene.

[0046] In certain exemplary embodiments, the RNA has the formula: [ka] The compound further comprises at least one vinyl phosphonate modification at an intersubunit linkage having the formula:

[0047] In certain exemplary embodiments, a VP motif is inserted next to the SNP position nucleotide or next to the MM position nucleotide.

[0048] In certain exemplary embodiments, the biantennary oligonucleotide compound has an hsi-RNA structure.

[0049] In certain exemplary embodiments, the SNP position nucleotide is complementary to an allelic polymorphism of an htt SNP selected from the group consisting of rs363125, rs362273, rs362307, rs362336, rs362331, rs362272, rs362306, rs362268, rs362267, and rs363099.

[0050] In another aspect, there is provided a bifurcated oligonucleotide compound comprising two or more nucleic acid sequences, the nucleic acid sequences (N) being linked to each other by one or more moieties selected from a linker (L), a spacer (S) and optionally a branch point (B), each nucleic acid sequence being double-stranded and comprising a sense strand and an antisense strand, the sense strand and the antisense strand having a 5' end and a 3' end, respectively, the sense strand and the antisense strand each comprising one or more chemically modified nucleotides, each antisense strand having a seed region, each antisense strand being complementary to a region of a gene comprising an allelic polymorphism, each antisense strand comprising a SNP position nucleotide at a position within the seed region, the SNP position nucleotide being complementary to the allelic polymorphism and a mismatch (MM) position nucleotide located 2 to 11 nucleotides from the SNP position nucleotide that is mismatched with the SNP position nucleotide complementary to the allelic polymorphism and the nucleotide in the gene.

[0051] In certain exemplary embodiments, the RNA has the formula: [ka] The compound further comprises at least one vinyl phosphonate modification at an intersubunit linkage having the formula:

[0052] In certain exemplary embodiments, a VP motif is inserted next to the SNP position nucleotide or next to the MM position nucleotide.

[0053] In certain exemplary embodiments, the sense and antisense strands each contain >80% chemically modified nucleotides.

[0054] In certain exemplary embodiments, the nucleotides at positions 1 and 2 from the 5' end of the sense and antisense strands are linked to adjacent nucleotides via phosphorothioate bonds.

[0055] In certain exemplary embodiments, each antisense strand comprises at least 15 contiguous nucleotides and each sense strand comprises at least 15 contiguous nucleotides and is complementary to the antisense strand.

[0056] In certain exemplary embodiments, the compound further comprises a hydrophobic moiety attached to the terminal 5' position of the branched oligonucleotide compound.

[0057] In certain exemplary embodiments, each duplex nucleic acid sequence is independently linked to a linker, spacer, or branch point at the 3' or 5' end of the sense or antisense strand.

[0058] In certain exemplary embodiments, the SNP position nucleotide is complementary to an allelic polymorphism of an htt SNP selected from the group consisting of rs363125, rs362273, rs362307, rs362336, rs362331, rs362272, rs362306, rs362268, rs362267, and rs363099.

[0059] In another aspect, a nucleic acid is provided having a 5' end, a 3' end and a seed region that is complementary to a region of a gene comprising an allelic polymorphism, the nucleic acid comprising a SNP position nucleotide at a position within the seed region, the SNP position nucleotide that is complementary to the allelic polymorphism, an MM position that is mismatched with a nucleotide in the gene and at least one modified nucleotide (X) on either side of the SNP position nucleotide, wherein each X is located within 4, 3 or 2 nucleotides of the SNP position nucleotide.

[0060] In certain exemplary embodiments, the RNA has the formula: [ka] The compound further comprises at least one vinyl phosphonate modification at an intersubunit linkage having the formula:

[0061] In certain exemplary embodiments, a VP motif is inserted next to the SNP position nucleotide or next to the MM position nucleotide.

[0062] In certain exemplary embodiments, X comprises a sugar modification selected from the group consisting of 2'-O-methyl (2'-OMe), 2'-fluoro (2'-F), 2'-ribo, 2'-deoxyribo, 2'-F-4'-thioarabino (2'-F-ANA), 2-O-(2-methoxyethyl) (2'-MOE), 4'-S-RNA, locked nucleic acid (LNA), 4'-SF-ANA, 2'-O-allyl, 2'-O-ethylamine, 2-cyanoethyl-RNA (CNet-RNA), tricyclo-DNA, cyclohexyl nucleic acid (CeNA), arabino nucleic acid (ANA), and hexitol nucleic acid (HNA).

[0063] In certain exemplary embodiments, X is located immediately 5' to the SNP position nucleotide or immediately 3' to the SNP position nucleotide. In certain exemplary embodiments, X is located immediately 5' to the SNP position nucleotide and immediately 3' to the SNP position nucleotide.

[0064] In certain exemplary embodiments, the SNP position nucleotide is located 2-6 from the 5' end. In certain exemplary embodiments, the MM position nucleotide is located 2-11 nucleotides from the SNP position nucleotide. In certain exemplary embodiments, the MM position nucleotide is located 2-6 nucleotides from the SNP position nucleotide.

[0065] In another aspect, a nucleic acid is provided having a 5' end, a 3' end and a seed region that is complementary to a region of a gene comprising an allelic polymorphism, the nucleic acid comprising a SNP position nucleotide at a position within the seed region, the SNP position nucleotide being complementary to the allelic polymorphism, an MM position that is mismatched with a nucleotide in the gene and at least one modified nucleotide (Y) on either side of the MM position nucleotide, wherein each Y is located within 4, 3 or 2 nucleotides of the MM position nucleotide.

[0066] In certain exemplary embodiments, the RNA has the formula: [ka] The compound further comprises at least one vinyl phosphonate modification at an intersubunit linkage having the formula:

[0067] In certain exemplary embodiments, a VP motif is inserted next to the SNP position nucleotide or next to the MM position nucleotide.

[0068] In certain exemplary embodiments, Y is located immediately 5' to the nucleotide at the MM position or immediately 3' to the nucleotide at the MM position. In certain exemplary embodiments, Y is located immediately 5' to the nucleotide at the MM position and immediately 3' to the nucleotide at the MM position.

[0069] In certain exemplary embodiments, the SNP position nucleotide is located 2-6 from the 5' end. In certain exemplary embodiments, the MM position nucleotide is located 2-11 nucleotides from the SNP position nucleotide. In certain exemplary embodiments, the MM position nucleotide is located 2-6 nucleotides from the SNP position nucleotide.

[0070] In another aspect, a nucleic acid is provided having a 5' end, a 3' end and a seed region that is complementary to a region of a gene comprising an allelic polymorphism, the nucleic acid comprising a SNP position nucleotide at a position within the seed region, the SNP position nucleotide being complementary to the allelic polymorphism, an MM position that is mismatched with a nucleotide in the gene, and at least one modified nucleotide (X) on either side of the SNP position nucleotide, where each X is located within 4, 3 or 2 nucleotides of the SNP position nucleotide, and at least one modified nucleotide (Y) on either side of the MM position nucleotide, where each Y is located within 4, 3 or 2 nucleotides of the MM position nucleotide.

[0071] In certain exemplary embodiments, the RNA has the formula: [ka] The compound further comprises at least one vinyl phosphonate modification at an intersubunit linkage having the formula:

[0072] In certain exemplary embodiments, a VP motif is inserted next to the SNP position nucleotide or next to the MM position nucleotide.

[0073] In certain exemplary embodiments, X comprises a sugar modification selected from the group consisting of 2'-OMe, 2'-F, 2'-ribo, 2'-deoxyribo, 2'-F-ANA, 2'-MOE, 4'-S-RNA, LNA, 4'-SF-ANA, 2'-O-allyl, 2'-O-ethylamine, CNet-RNA, tricyclo-DNA, CeNA, ANA, and HNA. In certain exemplary embodiments, Y comprises a sugar modification selected from the group consisting of 2'-OMe, 2'-F, 2'-ribo, 2'-deoxyribo, 2'-F-ANA, 2'-MOE, 4'-S-RNA, LNA, 4'-SF-ANA, 2'-O-allyl, 2'-O-ethylamine, CNet-RNA, tricyclo-DNA, CeNA, ANA, and HNA.

[0074] In certain exemplary embodiments, X is located immediately 5' to the SNP position nucleotide or immediately 3' to the SNP position nucleotide. In certain exemplary embodiments, X is located immediately 5' to the SNP position nucleotide and immediately 3' to the SNP position nucleotide.

[0075] In certain exemplary embodiments, Y is located immediately 5' to the nucleotide at the MM position or immediately 3' to the nucleotide at the MM position. In certain exemplary embodiments, Y is located immediately 5' to the nucleotide at the MM position and immediately 3' to the nucleotide at the MM position.

[0076] In certain exemplary embodiments, the SNP position nucleotide is located 2-6 from the 5' end. In certain exemplary embodiments, the MM position nucleotide is located 2-11 nucleotides from the SNP position nucleotide. In certain exemplary embodiments, the MM position nucleotide is located 2-6 nucleotides from the SNP position nucleotide.

[0077] In certain exemplary embodiments, X and Y are the same.

[0078] The foregoing and other features and advantages of the present invention will become more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0079] [Figure 1] FIG. 1 shows the psiCHECK reporter plasmids containing either the wild-type region of htt or the same region of htt with the SNP, rs362273. [Diagram 2]Figure 2 shows a bar graph depicting luciferase activity following psiCHECK reporter plasmid assay in HeLa cells transfected with hsiRNAs carrying SNP nucleotides at various positions. This primary screening yielded multiple effective hydrophobically modified siRNA (hsiRNA) sequences. [Diagram 3] FIG. 3 shows dose-response curves demonstrating the silencing effect of three exemplary hsiRNAs of the invention on the psiCHECK reporter plasmid. [Figure 4] FIG. 4 shows dose-response curves demonstrating the potency of two hsiRNAs for silencing htt mRNA. [Diagram 5] FIG. 5 shows a bar graph depicting luciferase activity following psiCHECK reporter plasmid assay in HeLa cells transfected with hsiRNAs containing a second mismatch at various positions. [Figure 6] FIG. 6 shows dose-response curves comparing the silencing efficacy of SNP2 hsiRNA with (mm2-7) or without (mm2-0) additional mismatches. [Figure 7] FIG. 7 shows dose-response curves comparing the silencing efficacy of SNP4 hsiRNAs with or without additional mismatches. [Figure 8] FIG. 8 shows dose-response curves comparing the silencing efficacy of SNP6 hsiRNAs with or without additional mismatches. [Figure 9] Figure 9 shows dose-response curves comparing the silencing effect of SNP4 or SNP6 hsiRNAs with additional mismatches (SNP4-7 and SNP6-11, respectively) compared to the same hsiRNAs without additional mismatches (SNP4-0 and SNP4-11). HeLa cells transfected with one of the two reporter plasmids were reverse transfected with hsiRNAs by passive uptake and treated for 72 hours. Reporter expression was measured by dual luciferase assay. [Figure 10]FIG. 10 shows dose-response curves of htt mRNA expression measuring the silencing potency of hsiRNAs with additional mismatches. [Figure 11] FIG. 11 shows a schematic of hsiRNAs and exemplary modifications according to certain embodiments of the invention. [Figure 12A] 12A-12C show the SNP2, SNP4 and SNP6 hsiRNA libraries, respectively. The antisense strand is shown 5' to 3' with the SNP site in red and the mismatch in blue. [Figure 12B] 12A-12C show the SNP2, SNP4 and SNP6 hsiRNA libraries, respectively. The antisense strand is shown 5' to 3' with the SNP site in red and the mismatch in blue. [Figure 12C] 12A-12C show the SNP2, SNP4 and SNP6 hsiRNA libraries, respectively. The antisense strand is shown 5' to 3' with the SNP site in red and the mismatch in blue. [Figure 13] 13 shows the antisense and sense strand sequences and modification patterns for various hsiRNA constructs according to certain embodiments. mm4-7 and mm6-11 demonstrated excellent SNP discrimination and were selected for further screening. [Figure 14] FIG. 14 shows exemplary SNP-selective compounds designed as di-siRNAs. [Figure 15] Figure 15 shows backbone linkages according to certain exemplary embodiments. The oligonucleotide backbone may include one or any combination of phosphate, phosphorothioate (racemic or stereospecific), diphosphorothioate, phosphoramidate, peptide nucleic acid (PNA), boranophosphate, 2'-5'-phosphodiester, amide, phosphonoacetate, morpholino, and the like. [Figure 16]16 illustrates sugar modifications according to certain exemplary embodiments, including one or any combination of 2'-O-methyl, 2'-fluoro, 2'-ribo, 2'-deoxyribo, 2'-F-ANA, MOE, 4'-S-RNA, LNA, 4'-SF-ANA, 2'-O-allyl, 2'-O-ethylamine, CNet-RNA, tricyclo-DNA, CeNA, ANA, HNA, and the like. [Figure 17] Figure 17 shows internucleotide linkages according to certain exemplary embodiments. The possible internucleotide linkages can be stabilized by any of the moieties shown between the first two nucleotides at the 5' or 3' end of any given oligonucleotide strand. [Figure 18] Figure 18 shows 5' stabilizing modifications according to certain exemplary embodiments. Suitable 5' stabilizing modifications can be phosphate, non-phosphate, vinyl phosphonate, C5-methyl (R or S or racemic), C5-methyl on vinyl, reduced vinyl (e.g., three carbon alkyl), and the like. [Figure 19] 19 shows a conjugate moiety according to certain exemplary embodiments. Suitable conjugate moieties can be alkyl chains of any length, vitamins, ligands, peptides or bioactive conjugates such as glycosphingolipids, polyunsaturated fatty acids, secosteroids, steroid hormones, steroid lipids, and the like. [Figure 20] FIG. 20 graphically illustrates that the activity of a SNP discrimination scaffold containing a SNP position nucleotide at position 6 from the 5' end and a mismatch position nucleotide located at position 11 from the 5' end is sequence dependent. [Figure 21] FIG. 21 shows a representative synthesis of a vinylphosphonate (VP) modified intersubunit linkage described herein. [Figure 22] FIG. 22 shows a method for preparing oligonucleotides having VP modified intersubunit linkages. [Figure 23] FIG. 23 is a diagrammatic illustration of a VP-modified RNA according to certain exemplary embodiments. [Figure 24]FIG. 24 shows the sequences of VP-modified oligonucleotides synthesized according to certain exemplary embodiments. [Diagram 25] FIG. 25 summarizes the comprehensive study of siRNA efficacy. [Figure 26] FIG. 26 is a schematic of the hsiRNA antisense scaffold aligned to the HTT sequence surrounding the SNP site rs362273, with the green box indicating the location of the SNP site. [Figure 27A] 27A-27B show the effect of adding mismatches in the siRNA sequence to improve allele discrimination without compromising silencing of the mutant allele. [Figure 27B] 27A-27B show the effect of adding mismatches in the siRNA sequence to improve allele discrimination without compromising silencing of the mutant allele. [Figure 28A] 28A-28B show VP modified sequences prepared by the synthesizer. [Figure 28B] 28A-28B show VP modified sequences prepared by the synthesizer. [Figure 29] FIG. 29 demonstrates another method for preparing a VP-modified oligonucleotide provided herein. [Figure 30-1] FIG. 30 demonstrates the effect that VP modified binding has on the target / non-target discrimination of SNP-selective siRNAs. [Figure 30-2] FIG. 30 demonstrates the effect that VP modified binding has on the target / non-target discrimination of SNP-selective siRNAs. [Diagram 31] FIG. 31 shows examples of biantennary siRNA chemical scaffolds. [Figure 32A] FIG. 32A is a Western blot performed to measure HTT protein levels. [Figure 32B] FIG. 32B shows protein levels normalized to vinculin. [Diagram 33] FIG. 33 shows dose-response curves comparing the silencing efficacy for oligonucleotides targeting a G at the SNP site instead of an A. [Diagram 34] FIG. 34 shows examples of sequences that introduce a single mismatch in a sequence previously selected for dose response. [Diagram 35] FIG. 35 shows some exemplary oligonucleotide backbone modifications. [Diagram 36] 36 shows oligonucleotide branching motifs according to certain exemplary embodiments. The double helix represents the oligonucleotide. The combination of different linkers, spacers and branching points allows for the generation of a variety of branched hsiRNA structures. [Figure 37] FIG. 37 shows branched oligonucleotides of the invention having conjugated biologically active moieties. [Figure 38] FIG. 38 shows exemplary amidite linkers, spacers and branching moieties. [Figure 39] FIG. 39 is a schematic diagram of the hsiRNA antisense scaffold aligned to the HTT sequence surrounding the alternative SNP site rs362273. [Diagram 40] Figure 40 shows a bar graph depicting luciferase activity following a psiCHECK reporter plasmid assay in HeLa cells transfected with the hsiRNAs of Figure 39. The number following "SNP" represents the position of the SNP in the siRNA. [Diagram 41] FIG. 41 shows dose-response curves comparing the silencing effect for the oligonucleotides of FIG. 39 targeting C or T at the SNP3 site. [Diagram 42] FIG. 42 shows a bar graph depicting luciferase activity following psiCHECK reporter plasmid assay in HeLa cells transfected with the hsiRNAs of FIG. 39 modified at various positions to provide a second mismatch. [Diagram 43] FIG. 43 shows examples of modified intersubunit linkers. [Figure 44A] FIG. 44A shows a representative example for preparing monomers for modified phosphinate-containing oligonucleotides provided herein. [Figure 44B]FIG. 44B shows a representative example for preparing another monomer for the modified phosphinate-containing oligonucleotides provided herein. [Figure 44C] FIG. 44C shows a representative example for preparing modified phosphinate-containing oligonucleotides provided herein. [Diagram 45] FIG. 45 shows exemplary SNPs within the HTT gene (SEQ ID NOs: 1-10, numbered from top to bottom). [Figure 46-1] FIG. 46 is a flow chart showing the methodology for generating and selecting SNP-discriminating siRNAs. [Figure 46-2] FIG. 46 is a flow chart showing the methodology for generating and selecting SNP-discriminating siRNAs. [Figure 47] FIG. 47 shows the nomenclature convention that denotes the location of a SNP within an siRNA. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0080] The present disclosure relates to compositions comprising oligonucleotides, e.g., RNA, silencing agents, e.g., RNA, such as double-stranded RNA ("dsRNA"), antisense oligonucleotides ("ASO"), etc., that are useful for silencing allelic polymorphisms located in genes encoding mutant proteins. In certain embodiments, the oligonucleotides, e.g., RNA, silencing agents, are dsRNA agents provided herein that destroy corresponding mutant mRNAs (e.g., SNP-containing mRNAs) with nucleotide specificity and selectivity. The oligonucleotides, e.g., RNA, silencing agents, e.g., dsRNA agents disclosed herein, target the mRNAs corresponding to the polymorphic regions of mutant genes, resulting in the cleavage of the mutant mRNAs, and preventing the synthesis of the corresponding mutant proteins, e.g., the acquisition of functional mutant proteins, such as huntingtin proteins.

[0081] definition Unless otherwise defined herein, scientific and technical terms used herein shall have the meanings commonly understood by those of ordinary skill in the art. In case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or extrinsic definitions. Unless otherwise required by context, singular terms shall include plurals and plural terms shall include the singular. The use of "or" means "and / or" unless otherwise indicated. The use of the term "comprising" and other forms such as "including" and "included" is not limiting.

[0082] As used herein in connection with oligonucleotide sequences, "A" represents a nucleoside that contains the base adenine (e.g., adenosine or a chemically modified derivative thereof), "G" represents a nucleoside that contains the base guanine (e.g., guanosine or a chemically modified derivative thereof), "U" represents a nucleoside that contains the base uracil (e.g., uridine or a chemically modified derivative thereof), and "C" represents a nucleoside that contains the base adenine (e.g., cytidine or a chemically modified derivative thereof).

[0083] As used herein, the term "capping group" refers to a chemical moiety that replaces a hydrogen atom in a functional group such as an alcohol (ROH), a carboxylic acid (RCOH), or an amine (RNH). Non-limiting examples of capping groups include alkyl (e.g., methyl, tertiary butyl); alkenyl (e.g., vinyl, alkyl); carboxyl (e.g., acetyl, benzoyl); carbamoyl; phosphate; and phosphonate (e.g., vinyl phosphonate). Other suitable capping groups are known to those of skill in the art.

[0084] The term "nucleotide analog" or "altered nucleotide" or "modified nucleotide" refers to a non-standard nucleotide, including non-naturally occurring ribonucleotides or deoxyribonucleotides. Exemplary nucleotide analogs are modified at any position to alter certain chemical properties of the nucleotide while retaining the ability of the nucleotide analog to perform its intended function. Examples of nucleotide positions that can be derivatized include the 5-position, such as 5-(2-amino)propyluridine, 5-bromouridine, 5-propyneuridine, 5-propenyluridine, etc.; the 6-position, such as 6-(2-amino)propyluridine; the 8-position relative to adenosine and / or guanosine, such as 8-bromoguanosine, 8-chloroguanosine, 8-fluoroguanosine, etc. Nucleotide analogs also include deazanucleotides, such as 7-deaza-adenosine; O- and N-modified (e.g., alkylated, such as N6-methyladenosine or others known in the art) nucleotides; and other heterocyclic modified nucleotide analogs such as those described in Herdewijn, Antisense Nucleic Acid Drug Dev., 2000 Aug. 10(4):297-310.

[0085] The term "oligonucleotide" refers to a short polymer of nucleotides and / or nucleotide analogs. The term "RNA analog" refers to a polynucleotide (e.g., a chemically synthesized polynucleotide) that has at least one altered or modified nucleotide compared to the corresponding unaltered or unmodified RNA, but retains the same or similar properties or functions as the corresponding unaltered or unmodified RNA. As described above, oligonucleotides can be linked with linkages that result in a slower rate of hydrolysis of the RNA analog compared to RNA molecules with phosphodiester linkages. For example, the nucleotides of the analog can include methylenediol, ethylenediol, oxymethylthio, oxyethylthio, oxycarbonyloxy, phosphorodiamidate, phosphoroamidate, and / or phosphorothioate linkages. In certain embodiments, the RNA analog includes sugar and / or backbone modified ribonucleotides and / or deoxyribonucleotides. Such alterations or modifications can further include the addition of non-nucleotide material, such as at the end of the RNA or internally (at one or more nucleotides of the RNA). The RNA analog can be sufficiently similar to natural RNA to have the ability to mediate RNA interference.

[0086] As used herein, exemplary oligonucleotides include, but are not limited to, siRNA, miRNA, shRNA, CRISPR guides, DNA oligonucleotides, antisense oligonucleotides, AAV oligonucleotides, gapmers, mixmers, miRNA inhibitors, SSOs, PMOs, PNAs, and the like.

[0087] As used herein, the term "RNA interference" ("RNAi") refers to the selective intracellular degradation of RNA. RNAi occurs naturally in cells to remove foreign RNA (e.g., viral RNA). Natural RNAi proceeds through fragments cleaved from free dsRNA that induce the degradation mechanism to other similar RNA sequences. Alternatively, RNAi can be artificially initiated, for example, to silence the expression of a target gene.

[0088] As used herein, the term "hsiRNA" refers to an embodiment of a double-stranded RNA provided herein, wherein the RNA molecule is fully chemically modified and contains one or more hydrophobic modifications as described herein.

[0089] An RNAi agent, e.g., an RNA silencing agent, having a strand that is "sufficiently complementary to a target mRNA sequence to induce target-specific RNA interference (RNAi)" means that the strand has sufficient sequence to cause destruction of the target mRNA by the RNAi machinery or process.

[0090] As used herein, the term "isolated RNA" (e.g., "isolated siRNA" or "isolated siRNA precursor") refers to an RNA molecule that is substantially free of other cellular material or culture when produced by recombinant techniques, or that is substantially free of chemical precursors or other chemicals when chemically synthesized.

[0091] As used herein, the term "RNA silencing" refers to a group of sequence-specific regulatory mechanisms mediated by RNA molecules (e.g., RNA interference (RNAi), transcriptional gene silencing (TGS), post-transcriptional gene silencing (PTGS), quelling, co-suppression, translational repression, etc.) that result in the inhibition or "silencing" of expression of genes that code for corresponding proteins. RNA silencing has been observed in many types of organisms, including plants, animals, and fungi.

[0092] The term "discriminatory RNA silencing" refers to the ability of an RNA molecule to substantially inhibit the expression of a "first" or "target" polynucleotide sequence while not substantially inhibiting the expression of a "second" or "non-target" polynucleotide sequence (e.g., when both polynucleotide sequences are present in the same cell). In certain embodiments, the target polynucleotide sequence corresponds to a target gene, while the non-target polynucleotide sequence corresponds to a non-target gene. In other embodiments, the target polynucleotide sequence corresponds to a target allele, while the non-target polynucleotide sequence corresponds to a non-target allele. In certain embodiments, the target polynucleotide sequence is a DNA sequence that encodes a regulatory region (e.g., a promoter or enhancer element) of a target gene. In other embodiments, the target polynucleotide sequence is a target mRNA encoded by a target gene.

[0093] A gene "involved in" a disease or disorder includes a gene whose normal or abnormal expression or function results in or causes the disease or disorder or at least one symptom of said disease or disorder.

[0094] As used herein, the term "target gene" (e.g., a mutant allele of a heterozygous polymorphism, e.g., a heterozygous SNP) is a gene whose expression is to be substantially inhibited or "silenced". This silencing can be achieved by RNA silencing, e.g., by cleaving the mRNA of the target gene or by translational repression of the target gene. The term "non-target gene" (e.g., a wild-type allele) is a gene whose expression is not substantially silenced. In one embodiment, the polynucleotide sequences of the target and non-target genes (e.g., the mRNAs encoded by the target and non-target genes) can differ by one or more nucleotides. In another embodiment, the target and non-target genes can differ by one or more polymorphisms (e.g., single nucleotide polymorphisms or SNPs). In another embodiment, the target and non-target genes can share less than 100% sequence identity. In another embodiment, the non-target gene can be a homolog (e.g., an ortholog or a paralog) of the target gene.

[0095] A "target allele" is an allele (e.g., an SNP allele) whose expression is to be selectively inhibited or "silenced". This silencing can be achieved by RNA silencing, for example, by cleaving the mRNA of the target gene or target allele with an siRNA. The term "non-target allele" is an allele (e.g., a corresponding wild-type allele) whose expression is not substantially silenced. In certain embodiments, the target and non-target alleles may correspond to the same target gene. In other embodiments, the target allele corresponds to or is associated with a target gene, and the non-target allele corresponds to or is associated with a non-target gene. In one embodiment, the polynucleotide sequences of the target and non-target alleles may differ by one or more nucleotides. In another embodiment, the target and non-target alleles may differ by one or more allelic polymorphisms (e.g., one or more SNPs). In another embodiment, the target and non-target alleles may share less than 100% sequence identity.

[0096] The term "polymorphism" as used herein refers to a variant (e.g., one or more deletions, insertions, or substitutions) in a gene sequence that is identified or detected when the same sequences from different sources or subjects (but the same organism) are compared. For example, polymorphisms can be identified when the same gene sequences from different subjects are compared. Identification of such polymorphisms is routine in the art, and the methodology is similar to that used to detect, for example, breast cancer point mutations. Identification can be done, for example, from DNA extracted from lymphocytes of a subject, and then the polymorphic region is amplified using specific primers for said polymorphic region. Alternatively, polymorphisms can be identified when two alleles of the same gene are compared.

[0097] In certain embodiments, the polymorphism is a single nucleotide polymorphism (SNP). Variations in sequence between two alleles of the same gene in an organism are referred to herein as "allelic polymorphisms." In certain embodiments, an allelic polymorphism corresponds to an SNP allele. For example, an allelic polymorphism may include a single nucleotide variation between two alleles of a SNP, also referred to herein as a heterozygous SNP. A polymorphism may occur in a nucleotide within a coding region, but does not code for a change in amino acid sequence due to the degeneracy of the genetic code. Instead, a polymorphic sequence may code for a different amino acid at a particular position, but the change in amino acid does not affect protein function. Polymorphic regions may also be found in non-coding regions of a gene. In certain embodiments, a polymorphism is found in a coding region of a gene or in a non-translated region of a gene (e.g., 5'UTR or 3'UTR).

[0098] As used herein, the term "allele frequency" is a measure (e.g., a ratio or percentage) of the relative frequency of an allele (e.g., a SNP allele) at a single locus in a population of individuals. For example, if a population of individuals has n loci of a particular chromosomal locus (and the genes occupying the locus) in each of their somatic cells, the allele frequency of an allele is the proportion or percentage of loci that the allele occupies in the population. In certain embodiments, the allele frequency of an allele (e.g., a SNP allele) is at least 10% (e.g., at least 15%, 20%, 25%, 30%, 35%, 40% or more) in a sample population.

[0099] The term "gain of function mutation", as used herein, refers to any mutation in a gene in which the protein encoded by said gene (i.e., mutant protein) gains a function not normally associated with the protein (i.e., wild-type protein) that causes or contributes to a disease or disorder. A gain of function mutation can be a deletion, addition, or substitution of one or more nucleotides in a gene that causes a change in the function of the encoded protein. In one embodiment, a gain of function mutation alters the function of the mutant protein or results in its interaction with other proteins. In another embodiment, a gain of function mutation results in the reduction or elimination of the normal wild-type protein, for example, by interaction of the altered mutant protein with said normal wild-type protein.

[0100] As used herein, the term "gain of function disorder" refers to a disorder characterized by a gain of function mutation. In one embodiment, the gain of function disorder is a neurodegenerative disease caused by a gain of function mutation, such as a polyglutamine disorder and / or a trinucleotide repeat disease, such as Huntington's disease. In another embodiment, the gain of function disorder is caused by a gain of function in an oncogene, and the mutated gene product is a cancer caused by a gain of function mutation, such as a mutation in the ret oncogene (e.g., ret-1), such as an endocrine tumor, medullary thyroid tumor, parathyroid hormone tumor, multiple endocrine neoplasm type 2, etc. Additional exemplary gain of function disorders include, but are not limited to, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), human immunodeficiency disorder (HIV), and slow channel congenital myasthenia gravis (SCCMS).

[0101] The term "trinucleotide repeat disease" as used herein refers to any disease or disorder characterized by an expanded trinucleotide repeat region located in a gene, and the expanded trinucleotide repeat region is the cause of the disease or disorder. Examples of trinucleotide repeat diseases include, but are not limited to, spinocerebellar ataxia type 12, spinocerebellar ataxia type 8, fragile X syndrome, fragile XE mental retardation, Friedreich's ataxia, and myotonic dystrophy. The preferred trinucleotide repeat disease for treatment according to the present invention is characterized or caused by an expanded trinucleotide repeat region at the 5' end of the coding region of the gene that codes for the mutant protein that causes or is responsible for the disease or disorder. Certain trinucleotide diseases in which the mutation is not associated with the coding region, such as fragile X syndrome, may not be suitable for treatment according to the methodology of the present invention, since there is no suitable mRNA to be targeted by RNAi. In contrast, diseases such as Friedreich's ataxia may be suitable for treatment according to the methodology of the invention because the causative mutation is not within a coding region (i.e., within an intron), but the mutation may be, for example, within a pre-mRNA (e.g., a pre-mRNA before it is spliced).

[0102] The term "polyglutamine disorder" as used herein refers to a (CAG) n Polyglutamine disorders refer to any disease or disorder characterized by the expansion of repeats (thus encoding an expanded polyglutamine region in the encoded protein). In one embodiment, the polyglutamine disorder is characterized by progressive degeneration of nerve cells. Examples of polyglutamine disorders include, but are not limited to, Huntington's disease, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type 3 (also known as Machado-Joseph disease), spinocerebellar ataxia type 6, spinocerebellar ataxia type 7, dentatoiubral-pallidoluysian atrophy, and the like.

[0103] The term "single nucleotide polymorphism disorder" or "SNP disorder" refers to a disorder characterized by the presence of a SNP, such as a heterozygous SNP. SNP disorders include, but are not limited to, phenylketonuria, cystic fibrosis, sickle cell anemia, albinism, Huntington's disease, myotonic dystrophy type 1, hypercholesterolemia (autosomal dominant, type B), neurofibromatosis (type 1), polycystic kidney disease (1 and 2), hemophilia A, Duchenne muscular dystrophy, X-linked hypophosphatemic rickets, Rett syndrome, nonobstructive spermatogenesis failure, and the like. An exemplary heterozygous SNP disorder is Huntington's disease.

[0104] In certain aspects, a double-stranded RNA (dsRNA) is provided that includes a first strand of about 15-35 nucleotides that is complementary to a region of a gene that includes an allelic polymorphism and a second strand of about 15-35 nucleotides that is complementary to at least a portion of the first strand, the first strand including a single nucleotide polymorphism (SNP) position nucleotide at positions 2-7 from the 5' end that is complementary to the allelic polymorphism; and a mismatch (MM) position nucleotide located 2-11 nucleotides from the SNP position nucleotide that is mismatched to a nucleotide in the gene. In some cases, the MM position is located 2-10 nucleotides from the SNP position. In some cases, the SNP position nucleotide is any one of positions 2-6 from the 5' end. In exemplary embodiments, the SNP position nucleotide is at positions 2, 4, or 6 from the 5' end, and the mismatch (MM) position nucleotide is located 2-6 nucleotides from the SNP position nucleotide. In some cases, the SNP position nucleotide is any one of positions 2-6 from the 5' end, and the mismatch (MM) position nucleotide is located 2-6 nucleotides from the SNP position nucleotide.

[0105] As used herein, "single nucleotide polymorphism position nucleotide" or "SNP position nucleotide" refers to a position in an RNA (e.g., a first strand of a dsRNA) described herein that corresponds to a polymorphic position in a target nucleic acid sequence (i.e., either a mutant nucleotide corresponding to a SNP allele or a wild-type nucleotide corresponding to a wild-type allele). For example, a strand can be labeled "SNP2," "SNP3," or "SNP3" ​​to indicate that the position of the SNP is 2, 3, or 4 nucleotides from the 5' end of the strand.

[0106] In certain exemplary embodiments, the SNP position nucleotide is located within the seed region. In certain exemplary embodiments, the SNP position nucleotide is located at position 2-7 from the 5' end, position 2-6 from the 5' end, or position 2-5 from the 5' end. In certain exemplary embodiments, the SNP position nucleotide is located at position 2 from the 5' end, position 3 from the 5' end, position 4 from the 5' end, position 5 from the 5' end, position 6 from the 5' end, or position 7 from the 5' end of an RNA described herein (e.g., the first strand of a dsRNA). In certain exemplary embodiments, the SNP position nucleotide is located at a position described in Tables 5-7.

[0107] As used herein, the term "seed region" refers to a 6 nucleotide stretch corresponding to positions 2 to 7 from the 5' end of an RNA strand. It is believed that siRNA discrimination of a target mRNA is conferred by the seed region of its antisense strand.

[0108] As used herein, a "mismatch position nucleotide" or an "MM position nucleotide" refers to a position of an RNA described herein (e.g., the first strand of a dsRNA) that is at a position that does not correspond to a SNP position nucleotide. The MM position nucleotide can be defined by its position from the 5' or 3' end of an RNA described herein (e.g., the 5' or 3' end of the first strand of a dsRNA), or it can be defined by its position relative to the SNP position nucleotide of an RNA described herein (e.g., the first strand of a dsRNA).

[0109] In certain exemplary embodiments, the MM position nucleotide is located 2-11 nucleotides, 2-10 nucleotides, 2-9 nucleotides, 2-8 nucleotides, 2-7 nucleotides, or 2-6 nucleotides from the SNP position nucleotide. In certain exemplary embodiments, the MM position nucleotide is located 11 nucleotides, 10 nucleotides, 9 nucleotides, 8 nucleotides, 7 nucleotides, 6 nucleotides, 5 nucleotides, 4 nucleotides, 3 nucleotides, or 2 nucleotides from the SNP position nucleotide. In certain exemplary embodiments, the MM position nucleotide is located at a position set forth in Tables 5-7.

[0110] In one embodiment, the RNA (e.g., the first strand of dsRNA) described herein is homologous to an allele polymorphism, except for one mismatch oligonucleotide at a specific position relative to the nucleotide corresponding to the allele polymorphism.In certain embodiments, the mismatch is within about 6 nucleotides of the SNP position nucleotide, within about 5 nucleotides of the SNP position nucleotide, within about 4 nucleotides of the SNP position nucleotide, within about 3 nucleotides of the SNP position nucleotide, within about 2 nucleotides of the SNP position nucleotide, or within about 1 nucleotide of the SNP position nucleotide.In certain embodiments, the mismatch is not adjacent to the SNP position nucleotide.

[0111] In another embodiment, the SNP position nucleotide is at position 2, 3, 4, 5 or 6 from the 5' end. In some embodiments, the SNP position nucleotide is at position 2 from the 5' end. In some embodiments, the SNP position nucleotide is at position 3 from the 5' end. In some embodiments, the SNP position nucleotide is at position 4 from the 5' end. In some embodiments, the SNP position nucleotide is at position 5 from the 5' end. In some embodiments, the SNP position nucleotide is at position 6 from the 5' end.

[0112] In certain exemplary embodiments, an RNA described herein (e.g., the first strand of a dsRNA) comprises an MM position nucleotide that is 5, 7, 8, 11, 14, 15, or 16 nucleotides from the 5' end. In certain exemplary embodiments, an RNA described herein (e.g., the first strand of a dsRNA) comprises an MM position nucleotide that is 1, 2, 3, 4, 5, 8, 9, 10, or 11 nucleotides from the SNP position nucleotide.

[0113] In certain exemplary embodiments, the RNA described herein (e.g., the first strand of a dsRNA) comprises a combination of SNP position nucleotide (referenced from the 5' end)-MM position nucleotide (referenced from the 5' end) selected from the group consisting of 2-7, 4-7, 4-8, 4-15, 6-5, 6-8, 6-11, 6-14, 6-16, 3-5, 3-7, and 3-8.

[0114] In certain exemplary embodiments, an RNA described herein (e.g., the first strand of a dsRNA) comprises a SNP position nucleotide at position 6 from the 5' end and a MM position nucleotide at position 11 from the 5' end. In another specific exemplary embodiment, an RNA described herein (e.g., the first strand of a dsRNA) comprises a SNP position nucleotide at position 4 from the 5' end and a mismatch at position 7 from the 5' end.

[0115] In one aspect, the double-stranded RNA provided herein selectively silences the mutant allele with allele polymorphism.In an embodiment, the double-stranded RNA provided herein silences the mutant allele with allele polymorphism and does not affect the wild-type allele of the same gene.In another embodiment, the double-stranded RNA provided herein silences the mutant allele with allele polymorphism and silences the wild-type allele of the same gene to a lower degree than the mutant allele.

[0116] Thus, in one aspect, the invention provides a method of treating a subject having or at risk for a disease characterized or caused by a mutant protein associated with an allelic polymorphism by administering to the subject an effective amount of an RNAi agent that targets an allelic polymorphism in a gene encoding a mutant protein (e.g., a huntingtin protein), such that sequence-specific interference with the gene provides an effective treatment for the disease.

[0117] In one aspect, the RNA silencing agent disclosed herein preferentially silences mutant alleles that contain polymorphisms more efficiently than corresponding wild-type alleles.In some exemplary embodiments, the dsRNA disclosed herein silences the alleles that contain polymorphisms that are about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80% or about 90% more than corresponding wild-type alleles.In some embodiments, the RNA silencing agent disclosed herein silences the alleles that contain polymorphisms that are at least about 50% more than corresponding wild-type alleles. In certain exemplary embodiments, the dsRNA disclosed herein silences an allele containing a polymorphism at least about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 55-fold, about 60-fold, about 65-fold, about 70-fold, about 75-fold, about 80-fold, about 85-fold, about 90-fold, about 95-fold, about 100-fold, about 110-fold, about 120-fold, about 130-fold, about 140-fold, about 150-fold, about 160-fold, about 170-fold, about 180-fold, about 190-fold, about 200-fold, about 250-fold, about 300-fold, about 350-fold, about 400-fold, about 450-fold, or up to about 500-fold above the level of silencing of the corresponding wild-type allele.

[0118] As used herein, the term "antisense strand" of an RNA silencing agent, e.g., an siRNA or RNA silencing agent, refers to a strand that is substantially complementary to a portion of about 10-50 nucleotides, e.g., about 15-30, 16-25, 18-23, or 19-22 nucleotides, of the mRNA of a gene targeted for silencing. The antisense strand, or first strand, has a sequence that is sufficiently complementary to a desired target mRNA sequence to induce target-specific silencing, e.g., sufficiently complementary to induce destruction of the desired target mRNA by the RNAi machinery or process (RNAi interference) or sufficiently complementary to induce translational repression of the desired target mRNA.

[0119] The term "sense strand" or "second strand" of an RNA silencing agent, such as an siRNA or RNA silencing agent, refers to the strand complementary to the antisense strand or the first strand. Antisense and sense strands may also be referred to as first or second strands, which are the first or second strands having complementarity with the target sequence and the second or first strand, respectively, having complementarity with the first or second strand. The miRNA duplex intermediate or siRNA-like duplex includes an miRNA strand having sufficient complementarity with an approximately 10-50 nucleotide portion of the mRNA of the gene targeted for silencing and an miRNA* strand having sufficient complementarity to form a duplex with the miRNA strand.

[0120] As used herein, the term "antisense oligonucleotide" or "ASO" refers to a nucleic acid (e.g., RNA) having sufficient sequence complementarity to target an RNA (e.g., an SNP-containing mRNA or an SNP-containing pre-mRNA) to block a region of the target RNA in an effective manner, such as to inhibit translation of the target mRNA and / or splicing of the target pre-mRNA. An antisense oligonucleotide having a "sequence sufficiently complementary to the target RNA" means that the antisense agent has a sufficient sequence to mask a binding site for a protein that would otherwise regulate splicing, and / or that the antisense agent has a sufficient sequence to mask a binding site for the ribosome, and / or that the antisense agent has a sufficient sequence to alter the three-dimensional structure of the targeted RNA to prevent splicing and / or translation.

[0121] As used herein, the "5' end," as in the case of the 5' end of the antisense strand, refers to the 5' terminal nucleotide, e.g., 1 to about 5 nucleotides at the 5' end of the antisense strand. As used herein, the "3' end," as in the case of the 3' end of the sense strand, refers to a region, e.g., 1 to about 5 nucleotides, that is complementary to the 5' terminal nucleotide of the complementary antisense strand.

[0122] As used herein, the term "base pair" refers to an interaction between a pair of nucleotides (or nucleotide analogs) on opposite strands of an oligonucleotide duplex (e.g., a duplex formed by a strand of an RNA silencing agent and a target mRNA sequence), primarily due to H-bonds, van der Waals interactions, etc. between the nucleotides (or nucleotide analogs). As used herein, the term "binding strength" or "base pair strength" refers to the strength of the base pair.

[0123] As used herein, the term "mismatched base pair" refers to a base pair consisting of a non-complementary or non-Watson-Crick base pair that is not, for example, the normal complementary G:C, A:T or A:U base pair. As used herein, the term "ambiguous base pair" (also known as a non-discriminating base pair) refers to a base pair formed by a universal nucleotide.

[0124] Linkers useful in the conjugated compounds of the invention include glycol chains (e.g., polyethylene glycol), alkyl chains, peptides, RNA, DNA, and combinations thereof. As used herein, the abbreviation "TEG" refers to triethylene glycol.

[0125] Oligonucleotide Design In certain embodiments, the oligonucleotides of the invention, e.g., siRNAs, are double-stranded, consisting of a sense strand and a complementary antisense strand, where the antisense strand has sufficient complementarity with a target mRNA containing an allelic polymorphism that mediates RNAi. In exemplary embodiments, the siRNA molecules have a length of about 10-50 or more nucleotides, i.e., each strand comprises 10-50 nucleotides (or nucleotide analogs). In certain exemplary embodiments, the siRNA molecules have a length of about 15-35, e.g., about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, or about 35 nucleotides in each strand, where one of the strands is sufficiently complementary to the target region. In exemplary embodiments, the strands are aligned such that there are at least 1, 2, or 3 bases at the end of the unaligned strand (i.e., there are no bases complementary to the opposite strand), such that an overhang of 1, 2, or 3 residues results at one or both ends of the duplex when the strands are annealed. In exemplary embodiments, the siRNA molecule has a length of about 10-50 or more nucleotides, i.e., each strand contains 10-50 nucleotides (or nucleotide analogs). In certain exemplary embodiments, the siRNA molecule has a length of about 15 to 35, for example about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34 or about 35 nucleotides in each strand, one of the strands being substantially complementary to the target sequence containing the allelic polymorphism, and the other strand being complementary or substantially complementary to the first strand. In certain embodiments, the siRNA molecule is completely complementary to the target sequence containing the allelic polymorphism, except for one additional mismatch, also known as the second mismatch.

[0126] In general, siRNAs can be designed by using any method known in the art, for example, by using the following protocol.

[0127] 1. The siRNA may be specific to a target sequence containing an allelic polymorphism. In an exemplary embodiment, the first strand is substantially complementary to a target sequence containing an allelic polymorphism with one mismatch to the target sequence containing an allelic polymorphism, and the other strand is substantially complementary to the first strand. In an embodiment, the target sequence is outside the coding region of the target gene. Exemplary target sequences are selected from the 5' untranslated region (5'-UTR) or intron regions of the target gene. Cleavage of the mRNA at these sites should eliminate translation of the corresponding mutant protein. Target sequences from other regions of the htt gene are also suitable for targeting. The sense strand is designed based on the target sequence. Furthermore, siRNAs with lower G / C content (35-55%) may be more active than those with a G / C content higher than 55%. Thus, in one embodiment, the present invention includes a nucleic acid molecule with a G / C content of 35-55%.

[0128] 2. The sense strand of siRNA is designed based on the sequence of the selected target site and the position of allele polymorphism.In an exemplary embodiment, the RNA silencing agent of the present invention does not induce PKR response (i.e., is short enough in length).However, longer RNA agents may be useful, for example, in cell types that cannot generate PKR response or in situations where PKR response is downregulated or weakened by alternative means.

[0129] The siRNA molecule of the present invention has sufficient complementarity with the target sequence so that the siRNA can mediate RNAi. In general, siRNA is used that contains a nucleotide sequence that is sufficiently identical to the target sequence portion of the target gene to cause RISC-mediated cleavage of the target gene. Thus, in an exemplary embodiment, the sense strand of the siRNA is designed to have a sequence that is sufficiently identical to the portion of the target that contains an allelic polymorphism. The present invention has the advantage that it can tolerate certain sequence variants that increase the efficiency and specificity of RNAi. In one aspect, the sense strand has one mismatched nucleotide with the target region that contains an allelic polymorphism, such as a target region that contains at least one base pair difference between wild type and mutant alleles, and the other strand is identical or substantially identical to the first strand. In some embodiments, the mismatch is 4 nucleotides upstream, 3 nucleotides upstream, 2 nucleotides upstream, 1 nucleotide upstream, 1 nucleotide downstream, 2 nucleotides downstream, 3 nucleotides downstream, 4 nucleotides downstream, or 5 nucleotides downstream of the nucleotide corresponding to the allele polymorphism. In certain embodiments, the mismatch is not adjacent to the nucleotide corresponding to the allele polymorphism. In addition, siRNA sequences with small insertions or deletions of 1 or 2 nucleotides can also be effective for mediating RNAi. Alternatively, siRNA sequences with nucleotide analogue substitutions or insertions can be effective for inhibition.

[0130] Sequence identity can be determined by sequence comparison and alignment algorithms known in the art. To determine the percent identity of two nucleic acid sequences (or two amino acid sequences), the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced into the first sequence or the second sequence for optimal alignment). The nucleotides (or amino acid residues) at corresponding nucleotide (or amino acid) positions are then compared. If a position in the first sequence is occupied by the same residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., percent (%) homology = number of identical positions / total number of positions x 100), optionally penalizing the score for the number of gaps introduced and / or the length of the gap introduced.

[0131] Comparison of sequences and determination of percent identity between two sequences can be accomplished using mathematical algorithms. In one embodiment, alignment occurs to a specific portion of the sequence that is aligned with sufficient identity, rather than to a portion with a lower degree of identity (i.e., local alignment). An illustrative, non-limiting example of a local alignment algorithm utilized for sequence comparison is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithm is incorporated into the BLAST program (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.

[0132] In another embodiment, the alignment is optimized by introducing appropriate gaps and the percent identity is determined over the length of the aligned sequences (i.e., gapped alignment). To obtain a gapped alignment for comparison purposes, Gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. In another embodiment, the alignment is optimized by introducing appropriate gaps and the percent identity is determined over the entire length of the aligned sequences (i.e., global alignment). An illustrative, non-limiting example of a mathematical algorithm used for global comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When using the ALIGN program to compare amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0133] 3. The antisense or guide strand of the siRNA is usually the same length as the sense strand and contains complementary nucleotides. In one embodiment, the guide and sense strands are fully complementary, i.e., the strands are blunt-ended when aligned or annealed. In another embodiment, the strands of the siRNA may be paired to have a 3' overhang of 1-4, e.g., 2 nucleotides. The overhang may comprise (or consist of) nucleotides corresponding to the target gene (or its complement). Alternatively, the overhang may comprise (or consist of) deoxyribonucleotides, e.g., dT or nucleotide analogs or other suitable non-nucleotide material. Thus, in another embodiment, the nucleic acid molecule may have a 3' overhang of 2 nucleotides, such as TT. The overhang nucleotides may be either RNA or DNA. As mentioned above, it is desirable to select a target region where the mutant:wild type mismatch is a purine:purine mismatch.

[0134] 4. Compare potential targets against the appropriate genome database (human, mouse, rat, etc.) using any method known in the art, and eliminate from consideration any target sequences that have significant homology to other coding sequences. One such method for such sequence homology searches is known as BLAST, available at the National Center for Biotechnology Information website.

[0135] 5. Select one or more sequences that meet the criteria for evaluation.

[0136] Further general information about the design and use of siRNAs can be found in "The siRNA User Guide" available at the Max-Plank-Institut fur Biophysikalishe Chemie website.

[0137] Alternatively, siRNA may be functionally defined as a nucleotide sequence (or oligonucleotide sequence) capable of hybridizing to a target sequence (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, hybridization at 50° C. or 70° C. for 12-16 hours, followed by washing). Additional exemplary hybridization conditions include hybridization at 70° C. in 1×SSC or at 50° C. in 1×SSC, 50% formamide followed by washing at 70° C. in 0.3×SSC or at 70° C. in 4×SSC or at 50° C. in 4×SSC, 50% formamide followed by washing at 67° C. in 1×SSC. Hybridization temperatures for hybrids predicted to be less than 50 base pairs in length should be 5-10° C. lower than the melting temperature (Tm) of the hybrid, where Tm is determined according to the following formula: For hybrids less than 18 base pairs long, Tm(°C)=2(number of A+T bases)+4(number of G+C bases). For hybrids 18-49 base pairs long, Tm(°C)=81.5+16.6(log10[Na+])+0.41(%G+C)-(600 / N), where N is the number of bases in the hybrid and [Na+] is the concentration of sodium ions in the hybridization buffer ([Na+] for 1xSSC=0.165M). Additional examples of stringency conditions for polynucleotide hybridization are provided in Sambrook, J., E. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, Chapters 9 and 11, and Current Protocols in Molecular Biology, 1995, FMAusubel et al., eds., John Wiley & Sons, Inc., Sections 2.10 and 6.3-6.4, which are incorporated herein by reference in their entirety for all purposes.

[0138] Negative control siRNA has the same nucleotide composition as selected siRNA, but should not have significant sequence complementarity to appropriate genome.Such negative control can be designed by randomly mixing the nucleotide sequence of selected siRNA.Homology search can be performed to ensure that negative control lacks homology to any other gene in appropriate genome.In addition, negative control siRNA can be designed by introducing one or more base mismatches into sequence.

[0139] 6. To verify the effectiveness of siRNA in destroying target mRNA (e.g., wild-type or mutant huntingtin mRNA), the siRNA can be incubated with target cDNA (e.g., huntingtin cDNA) in an in vitro mRNA expression system using Drosophila. 32 Newly synthesized target mRNA (e.g., huntingtin mRNA) radiolabeled with P is detected by autoradiography on an agarose gel. The presence of cleaved target mRNA indicates mRNA nuclease activity. Suitable controls include omitting siRNA and using non-targeting cDNA. Alternatively, a control siRNA is selected that has the same nucleotide composition as the selected siRNA but does not have significant sequence complementarity to the appropriate target gene. Such a negative control can be designed by randomly shuffling the nucleotide sequences of the selected siRNA. A homology search can be performed to ensure that the negative control lacks homology to any other genes in the appropriate genome. In addition, a negative control siRNA can be designed by introducing one or more base mismatches into the sequence.

[0140] siRNA can be designed to target any of the above target sequences. Said siRNA comprises an antisense strand that is sufficiently complementary to the target sequence to mediate the silencing of the target sequence. In some embodiments, the RNA silencing agent is siRNA.

[0141] The siRNA-mRNA complementary site that results in optimal mRNA specificity and maximal mRNA cleavage is selected.

[0142] siRNA-like molecules The siRNA-like molecules of the present invention have a sequence (i.e., have a strand with a sequence) that is "sufficiently complementary" to the target sequence of an mRNA (e.g., htt mRNA) that induces gene silencing by either RNAi or translational repression. siRNA-like molecules are designed in the same manner as siRNA molecules, but the degree of sequence identity between the sense strand and the target RNA is closer to that observed between an miRNA and its target. In general, the degree of sequence identity between an miRNA sequence and the corresponding target gene sequence decreases, thus increasing the tendency to mediate post-transcriptional gene silencing by translational repression rather than RNAi. Thus, in an alternative embodiment, when post-transcriptional gene silencing by translational repression of the target gene is desired, the miRNA sequence has partial complementarity with the target gene sequence. In certain embodiments, the miRNA sequence has partial complementarity with one or more short sequences (complementary sites) dispersed within the target mRNA (e.g., within the 3'-UTR of the target mRNA) (Hutvagner and Zamore, Science, 2002; Zeng et al., Mol. Cell, 2002; Zeng et al., RNA, 2003; Doench et al., Genes & Dev., 2003). Because the mechanism of translational repression is cooperative, multiple complementary sites (e.g., 2, 3, 4, 5, or 6) can be targeted in certain embodiments.

[0143] The ability of an siRNA-like duplex to mediate RNAi or translational repression can be predicted by the distribution of non-identical nucleotides between the target gene sequence at the complementary site and the silencing agent nucleotide sequence. In one embodiment, when gene silencing by translational repression is desired, at least one non-identical nucleotide is present in the central portion of the complementary site, so that the duplex formed by the miRNA guide strand and the target mRNA contains a central "bulge" (Doench JG et al., Genes&Dev., 2003). In another embodiment, 2, 3, 4, 5 or 6 consecutive or non-consecutive non-identical nucleotides are introduced. The non-identical nucleotides can be selected to form wobble base pairs (e.g., G:U) or mismatch base pairs (G:A, C:A, C:U, G:G, A:A, C:C, U:U). In a further exemplary embodiment, the "bulge" is centered at nucleotides 12 and 13 from the 5' end of the miRNA molecule.

[0144] Modified RNA silencing agents In certain aspects of the present invention, the RNA silencing agent of the present invention (or any part thereof) as described above can be modified to further improve the activity of the agent.For example, the RNA silencing agent described above can be modified with any of the following modifications.Modifications can be useful in part to further improve target discrimination, improve the stability of the agent (e.g., prevent degradation), promote intracellular uptake, increase targeting efficiency, improve the efficacy of binding (e.g., to target), improve the patient's tolerance to the agent and / or reduce toxicity.

[0145] 1) Modifications that enhance target discrimination In certain embodiments, the RNA silencing agents of the present invention may be substituted with destabilizing nucleotides that enhance single nucleotide target discrimination (see U.S. Patent Application Publication No. 11 / 698,689, filed January 25, 2007, and U.S. Provisional Patent Application No. 60 / 762,225, filed January 25, 2006, both of which are incorporated herein by reference). Such modifications may be sufficient to eliminate the specificity of the RNA silencing agent for non-target mRNAs (e.g., wild-type mRNAs), but do not appreciably affect the specificity of the RNA silencing agent for target mRNAs (e.g., gain-of-function mutant mRNAs).

[0146] In certain exemplary embodiments, the RNA silencing agent of the present invention is modified by the introduction of at least one universal nucleotide in its antisense strand. A universal nucleotide comprises a base moiety that can indiscriminately base pair with any of the four common nucleotide bases (e.g., A, G, C, U). A universal nucleotide is typically utilized because it has a relatively low effect on the stability of the RNA duplex or duplex formed by the RNA silencing agent and the guide strand of the target mRNA. Exemplary universal nucleotides include those having an inosine base moiety or an inosine analog base moiety selected from the group consisting of deoxyinosine (e.g., 2'-deoxyinosine), 7-deaza-2'-deoxyinosine, 2'-aza-2'-deoxyinosine, PNA-inosine, morpholino-inosine, LNA-inosine, phosphoramidate-inosine, 2'-O-methoxyethyl-inosine, and 2'-OMe-inosine. In certain exemplary embodiments, the universal nucleotide is an inosine residue or a naturally occurring analog thereof.

[0147] In certain embodiments, the RNA silencing agent of the present invention is modified by the introduction of at least one destabilizing nucleotide within 11 nucleotides of the specificity-determining nucleotide (e.g., within 11 nucleotides of the nucleotide that recognizes the disease-associated polymorphism (e.g., SNP position nucleotide)). For example, the destabilizing nucleotide can be introduced at a position that is within 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotides of the specificity-determining nucleotide. In an exemplary embodiment, the destabilizing nucleotide is introduced at a position that is 3 nucleotides from the specificity-determining nucleotide (i.e., such that there are two stabilizing nucleotides between the destabilizing nucleotide and the specificity-determining nucleotide). In an RNA silencing agent having two strands or strand portions (e.g., siRNA and shRNA), the destabilizing nucleotide can be introduced in the strand or strand portion that does not contain the specificity-determining nucleotide. In certain exemplary embodiments, the destabilizing nucleotide is introduced in the same strand or strand portion that contains the specificity-determining nucleotide.

[0148] In certain embodiments, an RNA silencing agent of the invention has Formula 1: [ka] (In the formula, D is selected from the group consisting of O, OCH2, OCH, CH2, and CH; C is O - , O.H., O.R. 1 , N.H. - , NH2, S - and SH; A is selected from the group consisting of O and CH2; R 1 is a protecting group; [ka] is an optional double bond; and The subunits bridge two optionally modified nucleosides. is modified by the introduction of modified intersubunit linkages.

[0149] In one embodiment, C is O - and either A or D is not O.

[0150] In one embodiment, D is CH2. In another embodiment, the modified intersubunit linkage of formula VIII is represented by formula 2: [ka] is a modified intersubunit bond.

[0151] In certain embodiments, D is O. In another embodiment, the modified intersubunit linkage of formula VIII is represented by formula 3: [ka] is a modified intersubunit bond.

[0152] In one embodiment, D is CH2. In another embodiment, the modified intersubunit linkage of formula VIII is represented by formula 4: [ka] is a modified intersubunit bond.

[0153] In another embodiment, the modified intersubunit linkage has formula 5: [ka] is a modified intersubunit bond.

[0154] In one embodiment, D is OCH. In another embodiment, the modified intersubunit linkage is represented by formula 6: [ka] is a modified intersubunit bond.

[0155] In another embodiment, the modified intersubunit linkage of formula VII is represented by formula 7: [ka] is a modified intersubunit bond.

[0156] In certain embodiments, an RNA silencing agent of the invention is modified by the introduction of one or more of the intersubunit linkers of Figure 43. In an exemplary embodiment, the intersubunit linkers of Figure 43 are inserted between the SNP position nucleotide and the nucleotides immediately adjacent to and on either side of the SNP position nucleotide of the antisense strand.

[0157] In certain embodiments, the RNA silencing agent of the present invention has the following formula: [ka] The amino acid sequence is modified by the introduction of one or more vinylphosphonate (VP) motifs in the intersubunit linker having the formula:

[0158] In certain embodiments, the VP motif is inserted at any position of the oligonucleotide, e.g., RNA. For example, for an oligonucleotide having a length of 20 nucleotides, the VP motif can be inserted at positions 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14, 14-15, 15-16, 16-17, 17-18, 18-19, or 19-20, and any combination thereof.

[0159] In certain exemplary embodiments, VP motifs are inserted at one or more of positions 1-2, 5-6, 6-7, 10-11, 18-19, and / or 19-20 of the antisense strand.

[0160] In other exemplary embodiments, VP motifs are inserted at one or more of positions 1-2, 6-7, 10-11, and / or 19-20 of the antisense strand.

[0161] In an exemplary embodiment, the VP motif is inserted next to the SNP position nucleotide in the antisense strand (i.e., between the SNP position nucleotide and the nucleotides immediately adjacent to and on either side of it). In another exemplary embodiment, the VP motif is inserted next to the MM position nucleotide in the antisense strand (i.e., between the MM position nucleotide and the nucleotides immediately adjacent to and on either side of it).

[0162] 2) Modifications to increase potency and specificity In certain embodiments, the RNA silencing agent of the present invention can be modified according to asymmetric design rules to promote the enhancement of the efficacy and specificity of mediating RNAi (see U.S. Patent Nos. 8,309,704, 7,750,144, 8,304,530, 8,329,892 and 8,309,705). Such modification promotes the entry of the antisense strand of siRNA (e.g., the siRNA designed using the method of the present invention or the siRNA generated from shRNA) into RISC in favor of the sense strand, so that the antisense strand preferentially induces the cleavage or translational suppression of target mRNA, thus increasing or improving the efficiency of target cleavage and silencing. In an exemplary embodiment, the asymmetry of an RNA silencing agent is increased by decreasing the base pair strength between the 5' end of the antisense strand (AS5') and the 3' end of the sense strand (S3') of the RNA silencing agent relative to the binding strength or base pair strength between the 3' end of the antisense strand (AS3') and the 5' end of the sense strand (S'5) of the silencing agent.

[0163] In one embodiment, the asymmetry of the RNA silencing agent of the present invention can be increased such that there are fewer G:C base pairs between the 5' end of the first or antisense strand and the 3' end of the sense strand portion than between the 3' end of the first or antisense strand and the 5' end of the sense strand portion. In another embodiment, the asymmetry of the RNA silencing agent of the present invention can be increased such that there is at least one mismatched base pair between the 5' end of the first or antisense strand and the 3' end of the sense strand portion. In an exemplary embodiment, the mismatched base pair is selected from the group consisting of G:A, C:A, C:U, G:G, A:A, C:C, and U:U. In another embodiment, the asymmetry of the RNA silencing agent of the present invention can be increased such that there is at least one wobble base pair, e.g., G:U, between the 5' end of the first or antisense strand and the 3' end of the sense strand portion. In another embodiment, the asymmetry of the RNA silencing agent of the present invention can be increased such that there is at least one base pair that includes a rare nucleotide, e.g., inosine (I). In exemplary embodiments, the base pair is selected from the group consisting of I:A, I:U and I:C. In yet another embodiment, the asymmetry of the RNA silencing agent of the present invention can be increased such that there is at least one base pair that includes a modified nucleotide. In certain embodiments, the modified nucleotide is selected from the group consisting of 2-amino-G, 2-amino-A, 2,6-diamino-G and 2,6-diamino-A.

[0164] In certain embodiments, the RNA silencing agent of the present invention has the following formula: [ka] The oligonucleotide is modified at one or more intersubunit linkages by the introduction of a vinylphosphonate (VP) motif having the formula:

[0165] 3) RNA silencing agents with enhanced stability The RNA silencing agent of the present invention can be modified to improve stability in serum or growth medium for cell culture.To enhance stability, 3' residues can be stabilized against degradation, for example, they can be selected to be composed of purine nucleotides, particularly adenosine or guanosine nucleotides.Alternatively, the substitution of pyrimidine nucleotides with modified analogs, for example, the substitution of uridine with 2'-deoxythymidine, is tolerated and does not affect the efficiency of RNA interference.

[0166] In certain aspects, the invention features an RNA silencing agent comprising a first and a second strand, wherein the second strand and / or the first strand are modified by substitution of an internal nucleotide with a modified nucleotide, such that the stability in vivo is enhanced compared to the corresponding unmodified RNA silencing agent. As defined herein, an "internal" nucleotide is one that is present at any position other than the 5' or 3' end of a nucleic acid molecule, polynucleotide, or oligonucleotide. An internal nucleotide may be present within a single-stranded molecule or within a strand of a double-stranded or duplex molecule. In one embodiment, the sense strand and / or the antisense strand are modified by substitution of at least one internal nucleotide. In another embodiment, the sense strand and / or the antisense strand are modified by substitution of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more internal nucleotides. In another embodiment, the sense and / or antisense strand is modified by substitution of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the internal nucleotides. In yet another embodiment, the sense and / or antisense strand is modified by substitution of all of the internal nucleotides.

[0167] In certain embodiments of the present invention, RNA silencing agent can contain at least one modified nucleotide analogue.Nucleotide analogue can be located at the position where target-specific silencing activity, such as RNAi-mediated activity or translation suppression activity, does not occur substantially, for example, in the region of 5'-end and / or 3'-end of siRNA molecule.In particular, end can be stabilized by incorporating modified nucleotide analogue.

[0168] In certain embodiments, the RNA silencing agent of the present invention has the following formula: [ka] The oligonucleotide is modified at one or more intersubunit linkages by the introduction of a vinylphosphonate (VP) motif having the formula:

[0169] Various oligonucleotide types (e.g., gapmers, mixmers, miRNA inhibitors, splice switching oligonucleotides ("SSOs"), phosphorodiamidate morpholino oligonucleotides ("PMOs"), peptide nucleic acids ("PNAs"), etc.) may be used in the oligonucleotides described herein, optionally utilizing various combinations of modifications (e.g., chemical modifications) and / or conjugates as described herein and in, for example, U.S. Patent Application Publication Nos. 15 / 089,423; 15 / 236,051; 15 / 419,593; 15 / 697,120 and U.S. Patent No. 9,809,817; and 15 / 814,350 and U.S. Patent No. 9,862,350, each of which is incorporated by reference in its entirety for all purposes.

[0170] Exemplary nucleotide analogs include sugar- and / or backbone-modified ribonucleotides (i.e., containing modifications to the phosphate-sugar backbone). For example, the phosphodiester linkage of natural RNA can be modified to include at least one of a nitrogen or sulfur heteroatom. In exemplary backbone-modified ribonucleotides, the phosphoester group linking an adjacent ribonucleotide is replaced by a modified group, such as a phosphothioate group. In exemplary sugar-modified ribonucleotides, the 2'-OH group is replaced by a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2, or ON, where R is C1-C6 alkyl, alkenyl, or alkynyl, and halo is F, Cl, Br, or I.

[0171] In certain embodiments, the modifications are 2'-fluoro, 2'-amino and / or 2'-thio modifications. In particular, exemplary modifications include 2'-fluoro-cytidine, 2'-fluoro-uridine, 2'-fluoro-adenosine, 2'-fluoro-guanosine, 2'-amino-cytidine, 2'-amino-uridine, 2'-amino-adenosine, 2'-amino-guanosine, 2,6-diaminopurine, 4-thio-uridine and / or 5-amino-allyl-uridine. In certain embodiments, the 2'-fluoro ribonucleotides are any uridine and cytidine. Additional exemplary modifications include 5-bromo-uridine, 5-iodo-uridine, 5-methyl-cytidine, ribo-thymidine, 2-aminopurine, 2'-amino-butyryl-pyrene-uridine, 5-fluoro-cytidine and 5-fluoro-uridine. 2'-deoxy-nucleotides and 2'-Ome nucleotides can also be used within the modified RNA silencing agent moiety of the present invention.Additional modified residues include deoxy-abasic, inosine, N3-methyl-uridine, N6,N6-dimethyl-adenosine, pseudouridine, purine ribonucleoside and ribavirin.In certain exemplary embodiments, the 2' moiety is a methyl group, so that the linking moiety is a 2'-O-methyl oligonucleotide.

[0172] In an exemplary embodiment, the RNA silencing agent of the present invention comprises locked nucleic acid (LNA). LNA comprises sugar-modified nucleotides that resist nuclease activity (highly stable) and have single nucleotide discrimination for mRNA (Elmen et al., Nucleic Acids Res., (2005), 33(1): 439-447; Braasch et al. (2003) Biochemistry 42: 7967-7975, Petersen et al. (2003) Trends Biotechnol 21: 74-81). These molecules have 2'-O, 4'-C-ethylene bridged nucleic acid with possible modifications such as 2'-deoxy-2''-fluorouridine. Furthermore, LNA increases the specificity of oligonucleotides by restricting the sugar moiety to a 3'-endo conformation, thereby pre-structuring the nucleotide for base pairing and increasing the melting temperature of oligonucleotides by 10°C per base.

[0173] In another exemplary embodiment, the RNA silencing agent of the present invention comprises peptide nucleic acid (PNA). PNA comprises modified nucleotides in which the sugar-phosphate moiety of the nucleotide is replaced with a neutral 2-aminoethylglycine moiety, which can form a polyamide backbone that is highly resistant to nuclease digestion and improves binding specificity to molecules (Nielsen, et al., Science, (2001), 254: 1497-1500).

[0174] In certain exemplary embodiments, nucleobase-modified ribonucleotides are used, i.e., ribonucleotides containing at least one non-naturally occurring nucleobase instead of naturally occurring nucleobases. The base can be modified to block the activity of adenosine deaminase. Exemplary modified nucleobases include, but are not limited to, uridine and / or cytidine modified at 5-position, such as 5-(2-amino)propyluridine, 5-bromouridine; adenosine and / or guanosine modified at 8-position, such as 8-bromoguanosine; deazanucleotides, such as 7-deaza-adenosine; O- and N-alkylated nucleotides, such as N6-methyladenosine, which are preferred. It should be noted that the above modifications can be combined.

[0175] In other embodiments, crosslinking can be used to modify the pharmacokinetics of RNA silencing agents, for example to increase their half-life in the body.Thus, the present invention includes RNA silencing agents with two complementary strands of nucleic acid, and the two strands are crosslinked.The present invention also includes RNA silencing agents that are conjugated (e.g., at their 3' ends) or not conjugated to another moiety (e.g., a non-nucleic acid moiety such as a peptide), an organic compound (e.g., a dye), etc.Modifying siRNA derivatives in this way may improve the cellular uptake or enhance the targeting activity of the resulting siRNA derivatives in cells compared to the corresponding siRNAs, be useful for tracking the siRNA derivatives in cells, or improve the stability of the siRNA derivatives compared to the corresponding siRNAs.

[0176] Other exemplary modifications include: (a) 2' modifications, such as providing a 2'OMe moiety on the sense or antisense strand, particularly on a U on the sense strand, or providing a 2'OMe moiety on a 3' overhang, such as at the 3' end (3' end means the 3' atom or the 3'-most part of the molecule, such as the 3'-most P or 2' position as indicated by the context); (b) backbone modifications, such as providing phosphorothioate modifications at U or A or both, such as having a substitution of O with S in the phosphate backbone, particularly on the antisense strand; such as having a substitution of P with S; (c) substitution of U with a C5 amino linker; (d) substitution of A with G (in most cases the sequence change is located on the sense strand and not the antisense strand); and (d) modifications at the 2', 6', 7' or 8' positions. Exemplary embodiments are those in which one or more of these modifications are present on the sense strand and not the antisense strand, or in which the antisense strand has fewer such modifications. Further exemplary modifications include the use of a 3' overhang, e.g., a methylated P at the 3' end; a combination of 2' modifications, e.g., providing a 2'OMe moiety and a backbone modification, e.g., having a substitution of P with S, e.g., providing a phosphorothioate modification or using a 3' overhang, e.g., a methylated P at the 3' end; modifications with 3' alkyls; modifications with 3' overhangs, e.g., abasic pyrrolidone at the 3' end; modifications with naproxen, ibuprofen or other moieties that inhibit degradation at the 3' end.

[0177] 4) Modifications to enhance cellular uptake In other embodiments, the RNA silencing agent may be modified with a chemical moiety, for example, to enhance cellular uptake by a target cell (e.g., a neuronal cell). Thus, the invention includes RNA silencing agents that are conjugated (e.g., at their 3' termini) or unconjugated to another moiety (e.g., a non-nucleic acid moiety such as a peptide), an organic compound (e.g., a dye), and the like. Conjugation can be carried out by methods known in the art, for example, using the methods of Lambert et al., Drug Deliv. Rev.: 47(1), 99-112 (2001) (describing nucleic acids loaded onto polyalkylcyanoacrylate (PACA) nanoparticles); Fattal et al., J. Control Release 53(1-3): 137-43 (1998) (describing nucleic acids bound to nanoparticles); Schwab et al., Ann. Oncol. 5 Suppl. 4: 55-8 (1994) (describing nucleic acids linked to intercalating agents, hydrophobic groups, polycations or PACA nanoparticles); and Godard et al., Eur. J. Biochem. 232(2): 404-10 (1995) (describing nucleic acids linked to nanoparticles).

[0178] In certain embodiments, a modification to an RNA silencing agent of the invention comprises a vinylphosphonate (VP) motif in one or more intersubunit linkers of the oligonucleotide, the VP motif having the following formula: [ka] has.

[0179] In certain embodiments, the RNA silencing agent of the present invention is conjugated to a lipophilic moiety. In one embodiment, the lipophilic moiety is a ligand comprising a cationic group. In another embodiment, the lipophilic moiety is attached to one or both strands of the siRNA. In an exemplary embodiment, the lipophilic moiety is attached to one end of the sense strand of the siRNA. In another exemplary embodiment, the lipophilic moiety is attached to the 3' end of the sense strand. In certain embodiments, the lipophilic moiety is selected from the group consisting of cholesterol, vitamin E, vitamin K, vitamin A, folic acid, or a cationic dye (e.g., Cy3). In an exemplary embodiment, the lipophilic moiety is cholesterol. Other lipophilic moieties include cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl or phenoxazine.

[0180] 5) Tethered Ligand Other elements can be tethered to the RNA silencing agent of the present invention. For example, a ligand tethered to the RNA silencing agent to improve stability, hybridization thermodynamics with the target nucleic acid, targeting to a specific tissue or cell type, or cell permeability, for example, by endocytosis-dependent or -independent mechanisms. The ligand and associated modifications can also increase sequence specificity, resulting in reduced off-site targeting. The tethered ligand can include one or more modified bases or sugars that can function as interferences. In certain exemplary embodiments, these are located in internal regions, such as bulges, of the RNA silencing agent / target duplex. The interferences can be aromatic, such as polycyclic aromatic or heterocyclic aromatic compounds. Polycyclic interferences can have stacking capabilities and can include systems with two, three, or four fused rings. A universal base as described herein can be included on the ligand. In one embodiment, the ligand can include a cleavage group that contributes to target gene inhibition by cleavage of the target nucleic acid. The cleavage group can be, for example, bleomycin (e.g., bleomycin-A5, bleomycin-A2, or bleomycin-B2), pyrene, phenanthroline (e.g., O-phenanthroline), polyamine, tripeptide (e.g., lys-tyr-lys tripeptide), or a metal ion chelating group. The metal ion chelating group can include, for example, Lu(III) or EU(III) macrocyclic complexes, Zn(II) 2,9-dimethylphenanthroline derivatives, Cu(II) terpyridine, or acridine, which can promote selective cleavage of the target RNA at the site of the bulge by a free metal ion such as Lu(III). In some embodiments, the peptide ligand can be tethered to an RNA silencing agent that promotes cleavage of the target RNA, for example, at the bulge region. For example, 1,8-dimethyl-1,3,6,8,10,13-hexaazacyclotetradecane (cyclam) can be conjugated (e.g., by an amino acid derivative) to a peptide that promotes target RNA cleavage. The tethered ligand can be an aminoglycoside ligand, which can result in an RNA silencing agent with improved hybridization properties or improved sequence specificity.Exemplary aminoglycosides include glycosylated polylysine, galactosylated polylysine, neomycin B, tobramycin, kanamycin A, and acridine conjugates of aminoglycosides, such as neo-N-acridine, neo-S-acridine, neo-C-acridine, tobra-N-acridine, and kanaAN-acridine. The use of acridine analogs can increase sequence specificity. For example, neomycin B has a high affinity for RNA compared to DNA, but low sequence specificity. The acridine analog neo-5-acridine has an increased affinity for the HIV Rev response element (RRE). In some embodiments, the guanidine analog (guanidinoglycoside) of the aminoglycoside ligand is tethered to the RNA silencing agent. In guanidinoglycosides, the amine group on the amino acid is replaced with a guanidine group. The attachment of the guanidine analog can enhance the cell permeability of the RNA silencing agent. The tethered ligand can be a poly-arginine peptide, peptoid, or peptidomimetic that can enhance cellular uptake of an oligonucleotide agent.

[0181] Exemplary ligands are directly or indirectly bound to the ligand-conjugated carrier, typically covalently, via an intervening tether. In exemplary embodiments, the ligands are bound to the carrier via an intervening tether. In exemplary embodiments, the ligands modify the distribution, targeting or life span of the RNA silencing agent incorporated therein. In exemplary embodiments, the ligands provide enhanced affinity to selected targets, such as molecules, cells or cell types, compartments, such as cell or organ compartments, tissues, organs or body regions, for example, compared to species lacking such ligands.

[0182] Exemplary ligands can improve the transport, hybridization and specificity properties, and can also improve the nuclease resistance of the resulting natural or modified RNA silencing agent or polymer molecule comprising any combination of monomers and / or natural or modified ribonucleotides described herein. Ligands can generally include therapeutic modifiers, for example, for enhancing uptake; diagnostic compounds or reporter groups, for example, for monitoring distribution; cross-linking agents; nuclease resistance-conferring moieties; and natural or unconventional nucleobases. Common examples include lipophilic substances, lipids, steroids (e.g., uvaol, hesigenin, diosgenin), terpenes (e.g., triterpenes, such as sarsasapogenin, friedelin, epifriederanol-derivatized lithocholic acid), vitamins (e.g., folic acid, vitamin A, biotin, pyridoxal), carbohydrates, proteins, protein binding agents, integrin targeting molecules, polycationic substances, peptides, polyamines, and peptidomimetics. Ligands may include naturally occurring substances (e.g., human serum albumin (HSA), low density lipoprotein (LDL) or globulins); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); amino acids or lipids. Ligands may also be recombinant or synthetic molecules such as synthetic polymers, e.g., synthetic polyamino acids. Examples of polyamino acids include polyamino acids that are polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic anhydride copolymers, poly(L-lactide-co-glycolide) copolymers, divinyl ether-maleic anhydride copolymers, N-(2-hydroxypropyl) methacrylamide copolymers (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethanes, poly(2-ethylacrylic acid), N-isopropylacrylamide polymers or polyphosphazines.Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimeric polyamines, arginine, amidines, protamines, cationic lipids, cationic porphyrins, quaternary salts of polyamines or alpha-helical peptides.

[0183] Ligands may also include targeting groups, such as cell or tissue targeting agents, such as lectins, glycoproteins, lipids or proteins, such as antibodies that bind to specific cell types, such as kidney cells. The targeting group may be thyrotropin, 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, polyglutamates, polyaspartates, lipids, cholesterol, steroids, bile acids, folate, vitamin B12, biotin, or an RGD peptide or RGD peptide mimetic. Other examples of ligands include dyes, intercalating agents (e.g., acridine and substituted acridines), crosslinkers (e.g., psoralens, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine, phenanthroline, pyrene), lys-tyr-lys tripeptides, aminoglycosides, guanidinium aminoglycosides, artificial endonucleases (e.g., EDTA), lipophilic molecules such as cholesterol (and its thio analogues), cholic acid, cholanic acid, lithocholic acid, adamantane acetic acid, 1-pyrenebutyric acid, dihydrotestosterone, glycerol (e.g., esters (e.g., mono, bis or tris fatty acid esters, e.g., C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 Or C 20fatty acids) and their ethers, e.g. C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 Or C 20 alkyl; e.g., 1,3-bis-O(hexadecyl)glycerol, 1,3-bis-O(octadecyl)glycerol), geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, stearic acid (e.g., glyceryl distearate), oleic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl or phenoxazine) and peptide conjugates (e.g., antennape di-peptides, Tat peptides), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, naproxen, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bis-imidazole, histamine, imidazole clusters, acridine-imidazole conjugates, tetraaza macrocycles of Eu 3+ complex), dinitrophenyl, HRP or AP.

[0184] Ligands can be proteins, e.g., glycoproteins or peptides, e.g., molecules with specific affinity for co-ligands or antibodies, e.g., antibodies that bind to specific cell types, e.g., cancer cells, endothelial cells, or bone cells. Ligands can also include hormones and hormone receptors. They can also include lipids, lectins, carbohydrates, vitamins, cofactors, non-peptidic species, such as multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, or multivalent fucose. Ligands can be, for example, lipopolysaccharides, activators of p38 MAP kinase, or activators of NF-κB.

[0185] The ligand can be a substance, such as a drug, that can increase the uptake of the RNA silencing agent into cells, for example by disrupting the cytoskeleton of cells, for example by disrupting the microtubules, microfilaments and / or intermediate filaments of cells.The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine or myoservin.The ligand can increase the uptake of the RNA silencing agent into cells, for example, by activating inflammatory response.Exemplary ligands that will have such an effect include tumor necrosis factor (TNFα), interleukin-1 beta or gamma interferon.

[0186] In one aspect, the ligand is a lipid or lipid-based molecule. Such lipid or lipid-based molecules usually bind to serum proteins, such as human serum albumin (HSA). HSA binding to the ligand allows the distribution of the conjugate to target tissues, such as non-renal target tissues of the body. For example, the target tissue can be the liver, including liver parenchymal cells. Other molecules that can bind to HSA can also be used as ligands. For example, naproxen or aspirin can be used. The lipid or lipid-based ligand can (a) increase the resistance of the conjugate to degradation, (b) increase targeting or transport to target cells or cell membranes, and / or (c) be used to regulate binding to serum proteins, such as HSA. The lipid-based ligand can be used to regulate, e.g., control, the binding of the conjugate to the target tissue. In certain embodiments, the lipid-based ligand binds to HSA. However, it is desired that the affinity is not so strong that the HSA ligand binding cannot be reversed. In another exemplary embodiment, the lipid-based ligand binds weakly or never to HSA.

[0187] In another embodiment, the ligand is a moiety, such as a vitamin, that is taken up by target cells, such as proliferating cells. These are particularly useful for treating disorders characterized by unwanted cell proliferation, such as malignant or non-malignant types, such as cancer cells. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include B vitamins, such as folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients taken up by cancer cells. Also included are HSA and low density lipoprotein (LDL).

[0188] In another aspect, the ligand is a cell-penetrating agent, typically a helical cell-penetrating agent. In certain exemplary embodiments, the agent is amphipathic. An exemplary agent is a peptide, such as tat or antennapedia. If the agent is a peptide, it can be modified, including peptidomimetics, invertomers, non-peptide or pseudopeptide bonds, and the use of D-amino acids. Helical agents are usually alpha-helical agents, typically having a lipophilic face and a lipophobic face.

[0189] The ligand can be a peptide or peptidomimetic. Peptidomimetics (also referred to herein as oligopeptidomimetics) are molecules that can fold into a defined three-dimensional structure similar to natural peptides. Attachment of peptides and peptidomimetics to oligonucleotide agents can affect the pharmacokinetic distribution of RNA silencing agents, such as by enhancing cellular recognition and uptake. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long. The peptide or peptidomimetic can be, for example, a cell penetrating peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide (e.g., consisting primarily of Tyr, Trp, or Phe). The peptide moiety can be a dendrimeric peptide, a regulated peptide, or a cross-linked peptide. The peptide moiety can be an L-peptide or a D-peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocating sequence (MTS). The peptide or peptidomimetic may be encoded by a random sequence of DNA, such as a peptide identified from a phage display library or a one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature 354:82-84, 1991). In an exemplary embodiment, the peptide or peptidomimetic tethered to the RNA silencing agent via an incorporated monomeric unit is a cell targeting peptide, such as an arginine-glycine-aspartic acid (RGD)-peptide or RGD mimic. The peptide portion may range in length from about 5 amino acids to about 40 amino acids. The peptide portion may have structural modifications, for example, to increase stability or induce conformational properties. Any of the structural modifications described below may be utilized.

[0190] 6) Hydrophobic part In certain embodiments of the double-stranded RNA provided herein, the RNA molecule is conjugated to one or more hydrophobic moieties (see WO 2018 / 031933, incorporated herein by reference). In some embodiments, the hydrophobic moiety has an affinity for low density lipoproteins and / or intermediate density lipoproteins. In related embodiments, the hydrophobic moiety is a saturated or unsaturated moiety with less than three double bonds.

[0191] In another embodiment, the hydrophobic moiety has an affinity for high density lipoprotein. In a related embodiment, the hydrophobic moiety is a polyunsaturated moiety having three or more double bonds (e.g., having 3, 4, 5, 6, 7, 8, 9, or 10 double bonds). In a particular embodiment, the hydrophobic moiety is a polyunsaturated moiety having three double bonds. In a particular embodiment, the hydrophobic moiety is a polyunsaturated moiety having four double bonds. In a particular embodiment, the hydrophobic moiety is a polyunsaturated moiety having five double bonds. In a particular embodiment, the hydrophobic moiety is a polyunsaturated moiety having six double bonds.

[0192] In another embodiment, the hydrophobic moiety is selected from the group consisting of fatty acids, steroids, secosteroids, lipids, gangliosides and nucleoside analogs, and endocannabinoids.

[0193] In another embodiment, the hydrophobic moiety is a neuromodulatory lipid, such as an endocannabinoid. Non-limiting examples of endocannabinoids include anandamide, arachidonoylethanolamine, 2-arachidonoylglyceryl ether (nolazine ether), 2-arachidonoylglycerol, and N-arachidonoyldopamine.

[0194] In another embodiment, the hydrophobic moiety is an omega-3 fatty acid.Non-limiting examples of omega-3 fatty acids include, but are not limited to, hexadecatrienoic acid (HTA), alpha-linolenic acid (ALA), stearidonic acid (SDA), eicosatrienoic acid (ETE), eicosatetraenoic acid (ETA), eicosapentaenoic acid (EPA, timnodonic acid), heneicosapentaenoic acid (HPA), docosapentaenoic acid (DPA, sardine acid), docosahexaenoic acid (DHA, cervonic acid), tetracosapentaenoic acid and tetracosahexaenoic acid (heric acid).

[0195] In another embodiment, the hydrophobic moiety is an omega-6 fatty acid.Non-limiting examples of omega-6 fatty acids include, but are not limited to, linoleic acid, gamma-linoleic acid (GLA), eicosadienoic acid, dihomo-gamma-linoleic acid (DGLA), arachidonic acid (AA), docosadienoic acid, adrenic acid, docosapentaenoic acid (osbondoic acid), tetracosatetraenoic acid, and tetracosapentaenoic acid.

[0196] In another embodiment, the hydrophobic moiety is an omega-9 fatty acid. Non-limiting examples of omega-9 fatty acids include, but are not limited to, oleic acid, eicosenoic acid, mead acid, erucic acid, and nervonic acid.

[0197] In another embodiment, the hydrophobic moiety is a conjugated linoleic acid. Non-limiting examples of conjugated linoleic acid include, but are not limited to, α-calendic acid, β-calendic acid, jacaric acid, α-eleostearic acid, β-eleostearic acid, catarpic acid, and punicic acid.

[0198] In another embodiment, the hydrophobic moiety is a saturated fatty acid. Non-limiting examples of saturated fatty acids include, but are not limited to, caprylic acid, capric acid, docosanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, and cerotic acid.

[0199] In another embodiment, the hydrophobic moiety is an acid selected from the group consisting of lumellenic acid, α-parinaric acid, β-parinaric acid, boseopentaenoic acid, pinolenic acid, and podocarpic acid.

[0200] In another embodiment, the hydrophobic moiety is selected from the group consisting of docosanoic acid (DCA), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). In a particular embodiment, the hydrophobic moiety is docosanoic acid (DCA). In another particular embodiment, the hydrophobic moiety is DHA. In another particular embodiment, the hydrophobic moiety is EPA.

[0201] In another embodiment, the hydrophobic moiety is a secosteroid. In a particular embodiment, the hydrophobic moiety is calciferol. In another embodiment, the hydrophobic moiety is a steroid other than cholesterol.

[0202] In certain embodiments, the hydrophobic moiety is not cholesterol.

[0203] In another embodiment, the hydrophobic moiety is an alkyl chain, a vitamin, a peptide, or a bioactive conjugate, including but not limited to, a glycosphingolipid, a polyunsaturated fatty acid, a secosteroid, a steroid hormone, or a sterol lipid.

[0204] In some embodiments, the double-stranded RNA provided herein comprises one or more chemically modified nucleotides. In certain embodiments, the double-stranded RNA comprises alternating 2'-methoxy-nucleotides and 2'-fluoro-nucleotides. In another specific embodiment, one or more nucleotides of the double-stranded RNA are linked to adjacent nucleotides via phosphorothioate bonds. In certain embodiments of the dsRNA disclosed herein, the mismatched nucleotide and the nucleotide adjacent to the mismatched nucleotide are 2'-methoxy-ribonucleotides.

[0205] In another specific embodiment, the nucleotides at positions 1 and 2 from the 3' end of the duplex RNA provided herein are linked to adjacent nucleotides via phosphorothioate bonds. In yet another specific embodiment, the nucleotides at positions 1 and 2 from the 3' end of the duplex RNA and the nucleotides at positions 1 and 2 from the 5' end of the duplex RNA are linked to adjacent nucleotides via phosphorothioate bonds.

[0206] In one embodiment of the double-stranded RNA, the first oligonucleotide comprises at least 16 contiguous nucleotides, a 5' end, a 3' end, and has complementarity to the target; (1) the first oligonucleotide comprises alternating 2'-methoxy-nucleotides and 2'-fluoro-nucleotides; (2) the nucleotides at positions 2 and 14 from the 5' end are not 2'-methoxy-nucleotides; (3) the nucleotides are linked via phosphodiester or phosphorothioate bonds; and (4) The nucleotides at positions 1 to 6 from the 3' end or positions 1 to 7 from the 3' end are linked to adjacent nucleotides via phosphorothioate bonds.

[0207] 7) Advanced stabilization pattern In one embodiment of the double-stranded RNA provided herein, (1) the first oligonucleotide comprises alternating 2'-methoxy-ribonucleotides and 2'-fluoro-ribonucleotides, each nucleotide being a 2'-methoxy-ribonucleotide or a 2'-fluoro-ribonucleotide; and the nucleotides at positions 2 and 14 from the 5' end of the first oligonucleotide are not 2'-methoxy-ribonucleotides; (2) the second oligonucleotide comprises alternating 2'-methoxy-ribonucleotides and 2'-fluoro-ribonucleotides, each nucleotide being a 2'-methoxy-ribonucleotide or a 2'-fluoro-ribonucleotide; and the nucleotides at positions 2 and 14 from the 5' end of the second oligonucleotide are 2'-methoxy-ribonucleotides; (3) the nucleotides of the first oligonucleotide are linked to adjacent nucleotides via phosphodiester or phosphorothioate bonds, and the nucleotides at positions 1 to 6 from the 3' end or positions 1 to 7 from the 3' end are linked to adjacent nucleotides via phosphorothioate bonds; and (4) The nucleotides of the second oligonucleotide are linked to adjacent nucleotides via phosphodiester or phosphorothioate bonds, and the nucleotides at positions 1 and 2 from the 3' end are linked to adjacent nucleotides via phosphorothioate bonds.

[0208] In one embodiment of the double-stranded RNA, the first oligonucleotide has 3 to 7 more ribonucleotides than the second oligonucleotide.

[0209] In one embodiment, the double-stranded RNA comprises an 11-16 base pair duplex, where the nucleotides of each base pair duplex have a different chemical modification (e.g., one nucleotide has a 2'-fluoro modification and the other nucleotide has a 2'-methoxy).

[0210] In one embodiment of the double-stranded RNA, the first oligonucleotide has 3 to 7 more ribonucleotides than the second oligonucleotide.

[0211] In one embodiment, the first oligonucleotide is an antisense strand and the second oligonucleotide is a sense strand. See WO 2016 / 161388, which is incorporated herein by reference.

[0212] In one embodiment, the first or second oligonucleotide has the following formula: [ka] The VP subunits contain one or more intersubunit modifications having the following structure:

[0213] 8) Branched oligonucleotides Two or more RNA silencing agents, such as oligonucleotide constructs, such as siRNAs, as disclosed above can be linked together by one or more moieties independently selected from linkers, spacers, and branching points to form a branched oligonucleotide containing two or more RNA silencing agents. Figure 31 shows an exemplary di-siRNA bibranched backbone for delivering two siRNAs. In a representative embodiment, the nucleic acids of the branched oligonucleotide each comprise an antisense strand (or a portion thereof), and the antisense strand has sufficient complementarity to a heterozygous single nucleotide polymorphism to mediate an RNA-mediated silencing mechanism (e.g., RNAi). In another embodiment, a second type of branched oligonucleotide is provided, characterized by a nucleic acid comprising a sense strand (or a portion thereof) for silencing an antisense transcript, and the sense strand has sufficient complementarity to an antisense transcript to mediate an RNA-mediated silencing mechanism. In a further embodiment, a third type of branched oligonucleotide is provided, comprising a nucleic acid comprising both types of nucleic acids, i.e., an oligonucleotide comprising an antisense strand (or a portion thereof) and a sense strand (or a portion thereof).

[0214] In exemplary embodiments, the branched oligonucleotides may have 2-8 RNA silencing agents attached through a linker. The linker may be hydrophobic. In certain embodiments, the branched oligonucleotides of the present application have 2-3 oligonucleotides. In one embodiment, the oligonucleotides have substantial chemical stabilization, independently (e.g., at least 40% of the component bases are chemically modified). In certain embodiments, the oligonucleotides have complete chemical stabilization (i.e., all of the component bases are chemically modified). In some embodiments, the branched oligonucleotides include one or more single-stranded phosphorothioate tails, each independently having 2-20 nucleotides. In certain embodiments, each single-stranded tail has 8-10 nucleotides.

[0215] In certain embodiments, branched oligonucleotides are characterized by three properties: (1) branched structure, (2) complete metabolic stabilization, and (3) the presence of a single-stranded tail that includes a phosphorothioate linker. In certain embodiments, branched oligonucleotides have two or three branches. It is believed that the increase in overall size of the branched structure promotes increased uptake. Also, without being bound to a particular theory of activity, it is believed that multiple adjacent branches (e.g., two or three) can allow each branch to act cooperatively, thus dramatically increasing the rate of internalization, transport, and release.

[0216] Branched oligonucleotides are provided in a variety of structurally diverse embodiments. For example, in some embodiments, the nucleic acid attached at the branch point is single-stranded and consists of miRNA inhibitors, gapmers, mixmers, SSOs, PMOs, or PNAs, as shown in Figure 36. These single strands can be attached at their 3' or 5' ends. A combination of siRNA and single-stranded oligonucleotides can also be used for dual functions. In another embodiment, short nucleic acids complementary to gapmers, mixmers, miRNA inhibitors, SSOs, PMOs, and PNAs are used to carry these active single-stranded nucleic acids and enhance distribution and cellular internalization. The short double-stranded region has a low melting temperature (Tm approx. 37°C) for rapid dissociation upon internalization of the branched structure into cells.

[0217] As shown in Figure 37, di-siRNA branched oligonucleotides can contain chemically diverse conjugates. Conjugated bioactive ligands can be used to enhance cell specificity and promote membrane association, internalization and serum protein binding. Examples of bioactive moieties that will be used for conjugation include DHAg2, DHA, GalNAc and cholesterol. These moieties can be attached to di-siRNA either through a linker or spacer, or can be added via an additional linker or spacer attached to another free siRNA end.

[0218] The presence of branched structure improves the level of tissue retention in the brain by more than 100 times compared with the unbranched compound of the same chemical composition, suggesting a new mechanism of cell retention and distribution.Unexpectedly, branched oligonucleotides have uniform distribution throughout the spinal cord and brain.In addition, unexpectedly, branched oligonucleotides show efficient systemic delivery to various tissues and very high levels of tissue accumulation.

[0219] Branched oligonucleotides include a variety of therapeutic nucleic acids, including ASO, miRNA, miRNA inhibitors, splice switching, PMO, PNA. In some embodiments, the branched oligonucleotides further comprise a conjugated hydrophobic moiety and exhibit unprecedented silencing and efficacy in vitro and in vivo.

[0220] Linker In certain embodiments of the branched oligonucleotide, each linker is independently selected from an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, and combinations thereof; any carbon or oxygen atom of the linker is optionally replaced with a nitrogen atom, has a hydroxyl substituent, or has an oxo substituent. In one embodiment, each linker is an ethylene glycol chain. In another embodiment, each linker is an alkyl chain. In another embodiment, each linker is a peptide. In another embodiment, each linker is RNA. In another embodiment, each linker is DNA. In another embodiment, each linker is a phosphate. In another embodiment, each linker is a phosphonate. In another embodiment, each linker is a phosphoramidate. In another embodiment, each linker is an ester. In another embodiment, each linker is an amide. In another embodiment, each linker is a triazole. In another embodiment, each linker is a structure selected from the formula of FIG. 37.

[0221] 9) Compound of formula (I) In another aspect, the present invention provides a compound of formula (I): L-(N) n (I) wherein L is selected from an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, and combinations thereof, and formula (I) optionally further comprises one or more branch points B and one or more spacers S; B, independently for each occurrence, is a polyvalent organic species or a derivative thereof; S, independently for each occurrence, is selected from an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, and combinations thereof; N is an RNA duplex comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region of complementarity substantially complementary to a region of a gene comprising an allelic polymorphism, the antisense strand comprising a single nucleotide polymorphism (SNP) position nucleotide located 2-7 from the 5' end that is complementary to the allelic polymorphism; and a mismatch (MM) position nucleotide located 2-11 nucleotides from the SNP position nucleotide that is mismatched to a nucleotide in the gene. In an exemplary embodiment, the SNP position nucleotide is at position 2, 4 or 6 from the 5' end, and the mismatch (MM) position nucleotide is located 2-6 nucleotides from the SNP position nucleotide.

[0222] The sense strand and the antisense strand each independently comprise one or more chemical modifications; and n is 2, 3, 4, 5, 6, 7, or 8.

[0223] In certain embodiments, the compound of formula (I) has a structure selected from formulas (I-1) to (I-9) in Table 1.

[0224] [Table 1]

[0225] In one embodiment, the compound of formula (I) is formula (I-1). In another embodiment, the compound of formula (I) is formula (I-2). In another embodiment, the compound of formula (I) is formula (I-3). In another embodiment, the compound of formula (I) is formula (I-4). In another embodiment, the compound of formula (I) is formula (I-5). In another embodiment, the compound of formula (I) is formula (I-6). In another embodiment, the compound of formula (I) is formula (I-7). In another embodiment, the compound of formula (I) is formula (I-8). In another embodiment, the compound of formula (I) is formula (I-9).

[0226] In some embodiments of the compound of formula (I), each linker is independently selected from an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, and combinations thereof; any carbon or oxygen atom of the linker is optionally replaced with a nitrogen atom, has a hydroxyl substituent, or has an oxo substituent. In one embodiment of the compound of formula (I), each linker is an ethylene glycol chain. In another embodiment, each linker is an alkyl chain. In another embodiment of the compound of formula (I), each linker is a peptide. In another embodiment of the compound of formula (I), each linker is an RNA. In another embodiment of the compound of formula (I), each linker is a DNA. In another embodiment of the compound of formula (I), each linker is a phosphate. In another embodiment, each linker is a phosphonate. In another embodiment of the compound of formula (I), each linker is a phosphoramidate. In another embodiment of the compound of formula (I), each linker is an ester. In another embodiment of the compound of formula (I), each linker is an amide. In another embodiment of the compound of formula (I), each linker is a triazole. In another embodiment of the compound of formula (I), each linker is a structure selected from the formulas in Figure 36.

[0227] In one embodiment of the compound of formula (I), B is a polyvalent organic species. In another embodiment of the compound of formula (I), B is a derivative of a polyvalent organic species. In one embodiment of the compound of formula (I), B is a triol or tetraol derivative. In another embodiment, B is a tri- or tetra-carboxylic acid derivative. In another embodiment, B is an amine derivative. In another embodiment, B is a tri- or tetra-amine derivative. In another embodiment, B is an amino acid derivative. In another embodiment of the compound of formula (I), B is selected from the formulae of FIG. 38.

[0228] Polyvalent organic species are moieties that contain carbon and three or more valencies (i.e., points of attachment to moieties such as S, L, or N as defined above). Non-limiting examples of polyvalent organic species include triols (e.g., glycerol, phloroglucinol, etc.), tetraols (e.g., ribose, pentaerythritol, 1,2,3,5-tetrahydroxybenzene, etc.), tri-carboxylic acids (e.g., citric acid, 1,3,5-cyclohexanetricarboxylic acid, trimesic acid, etc.), tetra-carboxylic acids (e.g., ethylenediaminetetraacetic acid, pyromellitic acid, etc.), tertiary amines (e.g., tripropargylamine, triethanolamine, etc.), triamines (e.g., diethylenetriamine, etc.), tetramines, and species that contain combinations of hydroxyl, thiol, amino, and / or carboxyl moieties (e.g., amino acids such as lysine, serine, cysteine, etc.).

[0229] In some embodiments of the compound of formula (I), each nucleic acid comprises one or more chemically modified nucleotides. In some embodiments of the compound of formula (I), each nucleic acid consists of chemically modified nucleotides. In certain embodiments of the compound of formula (I), >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55%, or >50% of each nucleic acid comprises chemically modified nucleotides.

[0230] In certain embodiments, each antisense strand independently comprises a 5'-terminal group R selected from the group in Table 2.

[0231] [Table 2]

[0232] In one embodiment, R is R1. In another embodiment, R is R2. In another embodiment, R is R3. In another embodiment, R is R4. In another embodiment, R is R5. In another embodiment, R is R6. In another embodiment, R is R7. In another embodiment, R is R8.

[0233] Structure of Formula (II) In an embodiment, the compound of formula (I) has the formula (II): [ka] wherein X, independently for each occurrence, is selected from adenosine, guanosine, uridine, cytidine, and chemically modified derivatives thereof; Y, independently for each occurrence, is selected from adenosine, guanosine, uridine, cytidine, and chemically modified derivatives thereof; - represents a phosphodiester internucleoside linkage; = represents a phosphorothioate internucleoside linkage; and ---, independently for each occurrence, represents a base pairing interaction or a mismatch. This is the structure.

[0234] In certain embodiments, the structure of formula (II) contains no mismatches. In one embodiment, the structure of formula (II) contains one mismatch. In another embodiment, the compound of formula (II) contains two mismatches. In another embodiment, the compound of formula (II) contains three mismatches. In another embodiment, the compound of formula (II) contains four mismatches. In an embodiment, each nucleic acid is comprised of chemically modified nucleotides.

[0235] In certain embodiments, >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55%, or >50% of the X in the structure of formula (II) are chemically modified nucleotides. In other embodiments, >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55%, or >50% of the X in the structure of formula (II) are chemically modified nucleotides.

[0236] Structure of formula (III) In an embodiment, the compound of formula (I) has formula (III): [ka] (In the formula, [ka] is, independently for each occurrence, a nucleotide that includes a 2'-deoxy-2'-fluoro modification; X is, independently for each occurrence, a nucleotide that includes a 2'-O-methyl modification; [ka] is, independently for each occurrence, a nucleotide that includes a 2'-deoxy-2'-fluoro modification; and Y is, independently for each occurrence, a nucleotide that includes a 2'-O-methyl modification. It has the structure:

[0237] In certain embodiments, X is selected from the group consisting of 2'-deoxy-2'-fluoro modified adenosine, guanosine, uridine, or cytidine. In certain embodiments, X is selected from the group consisting of 2'-O-methyl modified adenosine, guanosine, uridine, or cytidine. In certain embodiments, Y is selected from the group consisting of 2'-deoxy-2'-fluoro modified adenosine, guanosine, uridine, or cytidine. In certain embodiments, Y is selected from the group consisting of 2'-O-methyl modified adenosine, guanosine, uridine, or cytidine.

[0238] In certain embodiments, the structure of formula (III) contains no mismatches. In one embodiment, the structure of formula (III) contains one mismatch. In another embodiment, the compound of formula (III) contains two mismatches. In another embodiment, the compound of formula (III) contains three mismatches. In another embodiment, the compound of formula (III) contains four mismatches.

[0239] Structure of formula (IV) In an embodiment, the compound of formula (I) has formula (IV): [ka] wherein X, independently for each occurrence, is selected from adenosine, guanosine, uridine, cytidine, and chemically modified derivatives thereof; Y, independently for each occurrence, is selected from adenosine, guanosine, uridine, cytidine, and chemically modified derivatives thereof; - represents a phosphodiester internucleoside linkage; = represents a phosphorothioate internucleoside linkage; and ---, independently for each occurrence, represents a base pairing interaction or a mismatch. It has the structure:

[0240] In certain embodiments, the structure of formula (IV) contains no mismatches. In one embodiment, the structure of formula (IV) contains one mismatch. In another embodiment, the compound of formula (IV) contains two mismatches. In another embodiment, the compound of formula (IV) contains three mismatches. In another embodiment, the compound of formula (IV) contains four mismatches. In an embodiment, each nucleic acid is comprised of chemically modified nucleotides.

[0241] In certain embodiments, >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55%, or >50% of the X in the structure of formula (II) are chemically modified nucleotides. In other embodiments, >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55%, or >50% of the X in the structure of formula (II) are chemically modified nucleotides.

[0242] Structure of formula (V) In an embodiment, the compound of formula (I) has the formula (V): [ka] (In the formula, [ka] is, independently for each occurrence, a nucleotide that includes a 2'-deoxy-2'-fluoro modification; X is, independently for each occurrence, a nucleotide that includes a 2'-O-methyl modification; [ka] is, independently for each occurrence, a nucleotide that includes a 2'-deoxy-2'-fluoro modification; and Y is, independently for each occurrence, a nucleotide that includes a 2'-O-methyl modification. It has the structure:

[0243] In certain embodiments, X is selected from the group consisting of 2'-deoxy-2'-fluoro modified adenosine, guanosine, uridine, or cytidine. In some embodiments, X is selected from the group consisting of 2'-O-methyl modified adenosine, guanosine, uridine, or cytidine. In some embodiments, Y is selected from the group consisting of 2'-deoxy-2'-fluoro modified adenosine, guanosine, uridine, or cytidine. In some embodiments, Y is selected from the group consisting of 2'-O-methyl modified adenosine, guanosine, uridine, or cytidine.

[0244] In certain embodiments, the structure of formula (V) contains no mismatches. In one embodiment, the structure of formula (V) contains one mismatch. In another embodiment, the compound of formula (V) contains two mismatches. In another embodiment, the compound of formula (V) contains three mismatches. In another embodiment, the compound of formula (V) contains four mismatches.

[0245] Flexible Linker In certain embodiments of a compound of Formula (I), L is L1: [ka] It has the structure:

[0246] In certain embodiments of L, R is R 3 and n is 2.

[0247] In some embodiments of the structure of formula (II), L has the structure of L1. In some embodiments of the structure of formula (III), L has the structure of L1. In some embodiments of the structure of formula (IV), L has the structure of L1. In some embodiments of the structure of formula (V), L has the structure of L1. In some embodiments of the structure of formula (VI), L has the structure of L1. In some embodiments of the structure of formula (VI), L has the structure of L1.

[0248] In certain embodiments of a compound of Formula (I), L is L2: [ka] It has the structure:

[0249] In certain embodiments of L2, R is R 3and n is 2. In some embodiments of the structure of Formula (II), L has the structure of L2. In some embodiments of the structure of Formula (III), L has the structure of L2. In some embodiments of the structure of Formula (IV), L has the structure of L2. In some embodiments of the structure of Formula (V), L has the structure of L2. In some embodiments of the structure of Formula (VI), L has the structure of L2. In some embodiments of the structure of Formula (VI), L has the structure of L2.

[0250] 10) Delivery system In a further aspect, the present specification provides a compound of formula (VI): L-(cNA) n (VI) wherein L is selected from an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, and combinations thereof, and formula (VI) optionally further comprises one or more branch points B and one or more spacers S; B is, independently for each occurrence, a polyvalent organic species or a derivative thereof; S is, independently for each occurrence, selected from an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, and combinations thereof; each cNA is independently a carrier nucleic acid comprising one or more chemical modifications; and n is 2, 3, 4, 5, 6, 7, or 8. A delivery system for therapeutic nucleic acids is provided having the structure:

[0251] In one embodiment of the delivery system, L is an ethylene glycol chain. In another embodiment of the delivery system, L is an alkyl chain. In another embodiment of the delivery system, L is a peptide. In another embodiment of the delivery system, L is RNA. In another embodiment of the delivery system, L is DNA. In another embodiment of the delivery system, L is a phosphate. In another embodiment of the delivery system, L is a phosphonate. In another embodiment of the delivery system, L is a phosphoramidate. In another embodiment of the delivery system, L is an ester. In another embodiment of the delivery system, L is an amide. In another embodiment of the delivery system, L is a triazole.

[0252] In one embodiment of the delivery system, S is an ethylene glycol chain. In another embodiment, S is an alkyl chain. In another embodiment of the delivery system, S is a peptide. In another embodiment, S is RNA. In another embodiment of the delivery system, S is DNA. In another embodiment of the delivery system, S is a phosphate. In another embodiment of the delivery system, S is a phosphonate. In another embodiment of the delivery system, S is a phosphoramidate. In another embodiment of the delivery system, S is an ester. In another embodiment, S is an amide. In another embodiment, S is a triazole.

[0253] In one embodiment of the delivery system, n is 2. In another embodiment of the delivery system, n is 3. In another embodiment of the delivery system, n is 4. In another embodiment of the delivery system, n is 5. In another embodiment of the delivery system, n is 6. In another embodiment of the delivery system, n is 7. In another embodiment of the delivery system, n is 8.

[0254] In certain embodiments, each cNA comprises >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55% or >50% chemically modified nucleotides.

[0255] In certain embodiments, the compound of formula (VI) has a structure selected from formulas (VI-1) to (VI-9) in Table 3.

[0256] [Table 3]

[0257] In certain embodiments, the compound of formula (VI) has a structure of formula (VI-1). In certain embodiments, the compound of formula (VI) has a structure of formula (VI-2). In certain embodiments, the compound of formula (VI) has a structure of formula (VI-3). In certain embodiments, the compound of formula (VI) has a structure of formula (VI-4). In certain embodiments, the compound of formula (VI) has a structure of formula (VI-5). In certain embodiments, the compound of formula (VI) has a structure of formula (VI-6). In certain embodiments, the compound of formula (VI) has a structure of formula (VI-7). In certain embodiments, the compound of formula (VI) has a structure of formula (VI-8). In certain embodiments, the compound of formula (VI) has a structure of formula (VI-9).

[0258] In some embodiments, the compound of formula (VI) (e.g., including formulas (VI-1) to (VI-9)), each cNA independently comprises at least 15 consecutive nucleotides. In some embodiments, each cNA independently comprises chemically modified nucleotides.

[0259] In an embodiment, the delivery system further comprises n therapeutic nucleic acids (NAs), each NA comprising a region of complementarity substantially complementary to a region of the gene comprising an allelic polymorphism, and the antisense strand comprises a single nucleotide polymorphism (SNP) position nucleotide at positions 2-7 from the 5' end that is complementary to the allelic polymorphism; and a mismatch (MM) position nucleotide at positions 2-11 from the SNP position nucleotide that is mismatched to a nucleotide in the gene. In an exemplary embodiment, the SNP position nucleotide is at positions 2, 4 or 6 from the 5' end, and the mismatch (MM) position nucleotide is at positions 2-6 from the SNP position nucleotide. Also, each NA is hybridized to at least one cNA. In one embodiment, the delivery system comprises two NAs. In another embodiment, the delivery system comprises three NAs. In another embodiment, the delivery system comprises four NAs. In another embodiment, the delivery system comprises five NAs. In another embodiment, the delivery system comprises six NAs. In another embodiment, the delivery system comprises seven NAs. In another embodiment, the delivery system is composed of 8 NAs.

[0260] In some embodiments, each NA independently comprises at least 16 consecutive nucleotides. In some embodiments, each NA independently comprises 16 to 20 consecutive nucleotides. In some embodiments, each NA independently comprises 16 consecutive nucleotides. In another embodiment, each NA independently comprises 17 consecutive nucleotides. In another embodiment, each NA independently comprises 18 consecutive nucleotides. In another embodiment, each NA independently comprises 19 consecutive nucleotides. In another embodiment, each NA independently comprises 20 consecutive nucleotides.

[0261] In some embodiments, each NA comprises an unpaired overhang of at least 2 nucleotides. In another embodiment, each NA comprises an unpaired overhang of at least 3 nucleotides. In another embodiment, each NA comprises an unpaired overhang of at least 4 nucleotides. In another embodiment, each NA comprises an unpaired overhang of at least 5 nucleotides. In another embodiment, each NA comprises an unpaired overhang of at least 6 nucleotides. In some embodiments, the nucleotides of the overhang are linked via phosphorothioate bonds.

[0262] In some embodiments, each NA is independently selected from the group consisting of DNA, siRNA, antagomir, miRNA, gapmer, mixmer, or guide RNA. In one embodiment, each NA is independently DNA. In another embodiment, each NA is independently antagomir. In another embodiment, each NA is independently miRNA. In another embodiment, each NA is independently gapmer. In another embodiment, each NA is independently mixmer. In another embodiment, each NA is independently guide RNA. In some embodiments, each NA is the same. In some embodiments, each NA is not the same.

[0263] In some embodiments, the delivery system further comprising n therapeutic nucleic acids (NA) has a structure selected from formula (I), (II), (III), (IV), (V), (VI) and embodiments thereof as described herein. In one embodiment, the delivery system further comprises two therapeutic nucleic acids (NA), and has a structure selected from formula (I), (II), (III), (IV), (V), (VI) and embodiments thereof as described herein. In another embodiment, the delivery system further comprises three therapeutic nucleic acids (NA), and has a structure selected from formula (I), (II), (III), (IV), (V), (VI) and embodiments thereof as described herein. In one embodiment, the delivery system further comprises four therapeutic nucleic acids (NA), and has a structure selected from formula (I), (II), (III), (IV), (V), (VI) and embodiments thereof as described herein. In one embodiment, the delivery system has a structure selected from formula (I), (II), (III), (IV), (V), (VI) and embodiments thereof described herein, further comprising five therapeutic nucleic acids (NAs). In one embodiment, the delivery system has a structure selected from formula (I), (II), (III), (IV), (V), (VI) and embodiments thereof described herein, further comprising six therapeutic nucleic acids (NAs). In one embodiment, the delivery system has a structure selected from formula (I), (II), (III), (IV), (V), (VI) and embodiments thereof described herein, further comprising seven therapeutic nucleic acids (NAs). In one embodiment, the delivery system has a structure selected from formula (I), (II), (III), (IV), (V), (VI) and embodiments thereof described herein, further comprising eight therapeutic nucleic acids (NAs).

[0264] In one embodiment, the delivery system has a structure selected from formula (I), (II), (III), (IV), (V), (VI) further comprising a linker of structure L1 or L2, where R is R3, and n is 2. In another embodiment, the delivery system has a structure selected from formula (I), (II), (III), (IV), (V), (VI) further comprising a linker of structure L1, where R is R3, and n is 2. In another embodiment, the delivery system has a structure selected from formula (I), (II), (III), (IV), (V), (VI) further comprising a linker of structure L2, where R is R3, and n is 2.

[0265] Pharmaceutical Compositions and Methods of Administration In one aspect, provided herein is a pharmaceutical composition comprising a therapeutically effective amount of one or more compounds, oligonucleotides or nucleic acids as described herein and a pharma- ceutically acceptable carrier.In one embodiment, the pharmaceutical composition comprises one or more double-stranded chemically modified nucleic acids as described herein and a pharma- ceutically acceptable carrier.In a particular embodiment, the pharmaceutical composition comprises one double-stranded chemically modified nucleic acid as described herein and a pharma- ceutically acceptable carrier.In another particular embodiment, the pharmaceutical composition comprises two double-stranded chemically modified nucleic acids as described herein and a pharma- ceutically acceptable carrier.

[0266] In certain embodiments, the pharmaceutical composition comprises a double-stranded RNA molecule comprising about 15-35 nucleotides complementary to a region of a gene encoding a heterozygous SNP variant protein, said region comprising an allelic polymorphism, and a second strand comprising about 15-35 nucleotides complementary to the first strand, wherein the dsRNA molecule comprises a mismatch that is not at the position of the allelic polymorphism; and a mismatch and nucleotides corresponding to the polymorphism that are not in the center of the dsRNA molecule.

[0267] In certain embodiments, the mismatch is 4 nucleotides upstream, 3 nucleotides upstream, 2 nucleotides upstream of the nucleotide corresponding to the allelic polymorphism, 1 nucleotide upstream, 1 nucleotide downstream of the nucleotide corresponding to the allelic polymorphism, 2 nucleotides downstream of the nucleotide corresponding to the allelic polymorphism, 3 nucleotides downstream of the nucleotide corresponding to the allelic polymorphism, 4 nucleotides downstream of the nucleotide corresponding to the allelic polymorphism, or 5 nucleotides downstream of the nucleotide corresponding to the allelic polymorphism. In certain embodiments, the mismatch is not adjacent to the nucleotide corresponding to the allelic polymorphism.

[0268] In another embodiment of the pharmaceutical composition, the duplexed RNA comprises a nucleotide corresponding to an allelic polymorphism at position 2, 3, 4, 5 or 6 from the 5' end. In one embodiment, the nucleotide corresponding to an allelic polymorphism is at position 2 from the 5' end. In one embodiment, the nucleotide corresponding to an allelic polymorphism is at position 3 from the 5' end. In one embodiment, the nucleotide corresponding to an allelic polymorphism is at position 4 from the 5' end. In one embodiment, the nucleotide corresponding to an allelic polymorphism is at position 5 from the 5' end. In one embodiment, the nucleotide corresponding to an allelic polymorphism is at position 6 from the 5' end.

[0269] In some embodiments of the pharmaceutical composition, the double-stranded RNA selectively silences the mutant allele with allele polymorphism, for example, heterozygous SNP.In some embodiments of the pharmaceutical composition, the double-stranded RNA silences the mutant allele with allele polymorphism, and does not affect the wild-type allele of the same gene.In another embodiment of the pharmaceutical composition, the double-stranded RNA provided herein silences the mutant allele with allele polymorphism, and silences the wild-type allele of the same gene to a lower degree than the mutant allele.

[0270] The pharmaceutical composition of the present invention is formulated to suit its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous (IV), intradermal, subcutaneous (SC or SQ), intraperitoneal, intramuscular, oral (e.g., inhalation), transdermal (topical) and transmucosal administration. Solutions or suspensions used for parenteral, intradermal or subcutaneous application may contain the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, phosphates and agents for adjusting osmolality such as sodium chloride or glucose. The pH may be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral formulations may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0271] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Suitable carriers for intravenous administration include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be desirable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0272] Sterile solution for injection can be prepared by incorporating the active compound in the required amount in the appropriate solvent with one or combination of the components listed above as necessary, and then carrying out sterilization filtration.Generally, dispersion is prepared by incorporating active compound in a sterile solvent that contains a basic dispersion medium and other necessary components from those listed above.For the preparation of sterile powder for preparation of sterile solution for injection, the typical preparation method is vacuum drying and freeze-drying, which produces the powder of active ingredient and any additional desired ingredients from their solution that has been previously sterilized and filtered.

[0273] Data obtained from cell culture assays and animal studies can be used in formulating a range of dosages for use in humans. The dosage of such compounds should usually be within a range of circulating concentrations that include the ED50 with little or no toxicity. Dosages can vary within this range depending on the dosage form used and the route of administration utilized. For any compound used in the methods of the present invention, a therapeutically effective dose can be estimated initially from cell culture assays. Doses can be formulated in animal models to achieve a circulating plasma concentration range that includes the EC50 (i.e., the concentration of the test compound that achieves a half-maximal response) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high performance liquid chromatography.

[0274] Treatment method The present invention provides both preventative and therapeutic methods of treating subjects at risk (or susceptible to) a disease or disorder caused in whole or in part by an allelic polymorphism (e.g., a heterozygous SNP). In one embodiment, the disease or disorder is a trinucleotide repeat disease or disorder. In another embodiment, the disease or disorder is a polyglutamine disorder. In some embodiments, the method comprises administering a therapeutically effective amount of a duplex RNA molecule provided herein. In some embodiments, the disease or disorder is a disorder associated with expression of huntingtin, and alterations in huntingtin, particularly amplification of the copy number of CAG repeats, result in defects in the huntingtin gene (structure or function) or huntingtin protein (structure or function or expression), resulting in clinical symptoms including those seen in Huntington's disease patients.

[0275] In an embodiment of the method, the double-stranded RNA disclosed herein is homologous to an allele polymorphism, except for one mismatch oligonucleotide at a specific position relative to the nucleotide corresponding to the allele polymorphism.In certain embodiments, the mismatch is within about 6 nucleotides of the nucleotide corresponding to the allele polymorphism, within about 5 nucleotides of the nucleotide corresponding to the allele polymorphism, within about 4 nucleotides of the nucleotide corresponding to the allele polymorphism, within about 3 nucleotides of the nucleotide corresponding to the allele polymorphism, within about 2 nucleotides of the nucleotide corresponding to the allele polymorphism, or within about 1 nucleotide of the nucleotide corresponding to the allele polymorphism.In certain exemplary embodiments, the mismatch is not adjacent to the nucleotide corresponding to the allele polymorphism.

[0276] In another embodiment of the method, the duplexed RNA comprises a nucleotide corresponding to an allelic polymorphism at position 2, 3, 4, 5 or 6 from the 5' end. In one embodiment, the nucleotide corresponding to an allelic polymorphism is at position 2 from the 5' end. In one embodiment, the nucleotide corresponding to an allelic polymorphism is at position 3 from the 5' end. In one embodiment, the nucleotide corresponding to an allelic polymorphism is at position 4 from the 5' end. In one embodiment, the nucleotide corresponding to an allelic polymorphism is at position 5 from the 5' end. In one embodiment, the nucleotide corresponding to an allelic polymorphism is at position 6 from the 5' end.

[0277] In some embodiments of the method, the dsRNA contains a nucleotide corresponding to the polymorphism at position 6 from the 5' end and a mismatch at position 11 from the 5' end. In some embodiments of the method, the dsRNA contains a nucleotide corresponding to the polymorphism at position 4 from the 5' end and a mismatch at position 7 from the 5' end.

[0278] In another embodiment of the method, the double-stranded RNA selectively silences the mutant allele with allele polymorphism.In an embodiment, the double-stranded RNA silences the mutant allele with allele polymorphism and does not affect the wild-type allele of the same gene.In another embodiment, the double-stranded RNA silences the mutant allele with allele polymorphism and silences the wild-type allele of the same gene to a lower degree than the mutant allele.

[0279] In certain embodiments of the method, the dsRNA comprises one or more VP intersubunit linkage modifications, the intersubunit linkages having the following formula: [ka] has.

[0280] In additional embodiments, the dsRNA comprises one or more of the intersubunit linkage modifications shown in FIG.

[0281] As used herein, "treatment" or "treating" is defined as the application or administration of a therapeutic agent (e.g., an RNA agent or vector or transgene encoding same) to a patient or to an isolated tissue or cell line derived from a patient having a disease or disorder, a symptom of a disease or disorder, or a predisposition to a disease or disorder, to cure, remedy, alleviate, alter, correct, ameliorate, improve or affect the disease or disorder, a symptom of a disease or disorder, or a predisposition to a disease or disorder.

[0282] In one aspect, the present invention provides a method for preventing a disease or disorder in a subject, as described above, by administering a therapeutic agent (e.g., an RNAi agent or vector or a transgene encoding the same) to the subject. Subjects at risk of the disease can be identified, for example, by any or a combination of diagnostic or prognostic assays as described herein. Administration of the prophylactic agent can occur prior to the manifestation of symptoms characteristic of the disease or disorder, such that the disease or disorder is prevented, or alternatively, its progression is delayed.

[0283] Another aspect of the invention relates to a method of therapeutically treating a subject, i.e., altering the onset of symptoms of a disease or disorder. In an exemplary embodiment, the regulatory method of the invention comprises contacting cells expressing a gain-of-function mutant with a therapeutic agent (e.g., an RNAi agent or vector or transgene encoding the same) specific for one or more target sequences in the gene, thereby achieving sequence-specific interference with the gene. These methods can be performed in vitro (e.g., by culturing the cells with the agent) or alternatively in vivo (e.g., by administering the agent to a subject).

[0284] RNA silencing agents modified for enhanced neuronal uptake may be administered at less than about 1.4 mg / kg body weight, or less than 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001 mg / kg body weight and less than 200 nmoles of RNA agent per kg body weight (e.g., about 4.4×10 16 The unit dose of the RNA silencing agent may be administered at a dose of less than 1500, 750, 300, 150, 75, 15, 7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075, or 0.00015 nmoles per kg of body weight. The unit dose may be administered, for example, by injection (e.g., intravenously or intramuscularly, intrathecally or directly into the brain), inhalation administration, or topical application. In exemplary embodiments, the dosage is less than 2, 1, or 0.1 mg / kg of body weight.

[0285] Direct delivery of the RNA silencing agent to an organ (e.g., directly to the brain) can be at a dosage of about 0.00001 mg to about 3 mg per organ, or about 0.0001 to 0.001 mg per organ, about 0.03 to 3.0 mg per organ, about 0.1 to 3.0 mg per organ, or about 0.3 to 3.0 mg per organ. The dosage can be an amount effective to treat or prevent a neurological disease or disorder (e.g., Huntington's disease). In one embodiment, the unit dose is administered less frequently than once a day, for example, less than every 2, 4, 8, or 30 days. In another embodiment, the unit dose is not administered at a certain frequency (e.g., not at a regular frequency). For example, the unit dose can be administered once. In one embodiment, the effective dose is administered in conjunction with other conventional therapeutic modalities.

[0286] In one embodiment, the subject is administered an initial dose and one or more maintenance doses of the RNA silencing agent. The maintenance dose is generally less than the initial dose, for example less than half the initial dose. The maintenance regimen may include treating the subject with a dose ranging from 0.01 μg to 1.4 mg / kg of body weight per day, for example 10, 1, 0.1, 0.01, 0.001 or 0.00001 mg / kg of body weight per day. The maintenance dose is usually administered no more than once every 5, 10 or 30 days. Furthermore, the treatment regimen may continue for a period of time that will vary depending on the nature of the particular disease, its severity and the overall condition of the patient. In certain embodiments, the dosage may be delivered no more than once per day, for example no more than once per 24, 36 or 48 hours or more, for example no more than once every 5 or 8 days. Following treatment, the patient may be monitored for changes in the patient's condition and relief of symptoms of the condition. The dosage of the compound may be increased if the patient does not respond significantly to the current dosage level, or the dosage may be decreased if relief of symptoms of the disease state is observed, the disease state is ablated, or undesirable side effects are observed.

[0287] Huntington's disease In certain embodiments of the present invention, RNA silencing agents are designed to target polymorphisms (e.g., heterozygous single nucleotide polymorphisms) in mutant human huntingtin protein (htt) for the treatment of Huntington's disease.Accordingly, in another embodiment, provided herein is a method for treating or treating Huntington's disease, comprising administering to a patient in need of such treatment or treatment a therapeutically effective amount of a compound, oligonucleotide or nucleic acid as described herein, or a pharmaceutical composition comprising said compound, oligonucleotide or nucleic acid.

[0288] Inherited as an autosomal dominant disorder, Huntington's disease causes cognitive impairment and movement disorders. Patients can survive for more than 10 years with severe debilitation before early death from starvation or infection. The disease develops in the 30s or 40s in most cases, but some patients develop the disease in their teens. The genetic mutation in Huntington's disease is an expanded CAG repeat in the huntingtin gene. The CAG repeat varies in number from 8 to 35 copies in normal individuals (Kremer et al., 1994). The genetic mutation (e.g., an increase in the length of the CAG repeat from less than 36 in the normal huntingtin gene to more than 36 in disease) is associated with the synthesis of a mutant huntingtin protein with more than 36 consecutive polyglutamine residues (Aronin et al., 1995). In general, individuals with 36 or more CAG repeats will suffer from Huntington's disease. Huntington's disease, which is the prototype of 20 other diseases with an expanded CAG as the underlying mutation, still lacks an effective therapy. Various interventions (such as blocking apoptotic pathways, adding agents that boost mitochondrial efficiency, and blocking NMDA receptors) have shown promise in cell culture and mouse models of Huntington's disease. However, at best, these approaches show only short extensions of cell or animal survival time.

[0289] The disease gene leading to Huntington's disease is called huntingtin or (htt). The huntingtin locus spans a large area of ​​180 kb and consists of 67 exons. The huntingtin gene is widely expressed and is required for normal development. It is expressed as two alternatively polyadenylated forms that show different relative abundance in various fetal and adult tissues. The larger transcript is approximately 13.7 kb and is expressed mainly in the adult and fetal brain, while the smaller transcript of approximately 10.3 kb is more widely expressed. The two transcripts differ with respect to their 3' untranslated regions (Lin et al., 1993). Both messages are predicted to code for a 348 kilodalton protein containing 3144 amino acids. The genetic abnormality that causes Huntington's disease is thought to confer new properties to the mRNA or to alter the function of the protein.

[0290] Huntington's disease follows the central dogma of genetics: mutant genes serve as templates for the production of mutant mRNA; mutant mRNA then induces the synthesis of mutant proteins (Aronin et al., 1995; DiFiglia et al., 1997). Mutant huntingtin (protein) accumulates in selective neurons in the striatum and cortex, disrupting pending cellular activity, and can cause neuronal dysfunction and death (Aronin et al., 1999; Laforet et al., 2001). Because a single copy of the mutant gene is sufficient to cause Huntington's disease, the most straightforward treatment would be to disable the mutant gene. Rational approaches could include turning off gene transcription of mutant huntingtin, destroying mutant mRNA, and blocking translation. Each has the same consequence: the loss of mutant huntingtin.

[0291] Huntington SNP Exemplary SNPs in the Huntingtin gene sequence suitable for targeting according to certain exemplary embodiments are disclosed in Table 4 below. Genomic sequences for each SNP site can be found, for example, in the publicly available "SNP Entrez" database maintained by NCBI. Heterozygosity frequencies for each SNP site for HD patients and control DNA are further shown in Table 4. Targeting combinations of frequent heterozygous SNPs allows for the treatment of a high percentage of individuals in the HD population using a relatively small number of allele-specific RNA silencing agents.

[0292] [Table 4]

[0293] In one embodiment, the RNA silencing agent of the present invention can target one or more of the SNP sites listed in Table 4. In one embodiment, the RNA silencing agent of the present invention can target the rs363125 SNP site of huntingtin mRNA. In another embodiment, the RNA silencing agent of the present invention can target the rs362273 SNP site of huntingtin mRNA. In another embodiment, the RNA silencing agent of the present invention can target the rs362307 SNP site of huntingtin mRNA. In another embodiment, the RNA silencing agent of the present invention can target the rs362336 SNP site of huntingtin mRNA. In another embodiment, the RNA silencing agent of the present invention can target the rs362331 SNP site of huntingtin mRNA. In another embodiment, the RNA silencing agent of the present invention can target the rs362272 SNP site of huntingtin mRNA. In another embodiment, the RNA silencing agent of the present invention can target the rs362306 SNP site of huntingtin mRNA. In another embodiment, the RNA silencing agent of the present invention can target the rs362268 SNP site of huntingtin mRNA. In another embodiment, the RNA silencing agent of the present invention can target the rs362267 SNP site of huntingtin mRNA. In another embodiment, the RNA silencing agent of the present invention can target the rs363099 SNP site of huntingtin mRNA. In some embodiments, the SNP site targeted by the RNA silencing agent is associated with Huntington's disease. In certain exemplary embodiments, the SNP site targeted by the RNA silencing agent is significantly associated with Huntington's disease.

[0294] In additional exemplary embodiments, the RNA silencing agent comprises one or more of the sequences in Tables 5-7.

[0295] [Table 5]

[0296] [Table 6]

[0297] [Table 7]

[0298] Methods for delivering nucleic acids The RNA silencing agent of the present invention can be directly introduced into cells (e.g., nerve cells) (i.e., intracellularly); or can be introduced extracellularly into the cavity, tissue space, circulation of an organism, can be introduced orally, or can be introduced by immersing cells or organisms in a solution containing nucleic acid. Blood or lymphatic system, which is vascular or extravascular circulation, and cerebrospinal fluid are the sites where nucleic acid can be introduced.

[0299] The RNA silencing agent of the present invention can be introduced using nucleic acid delivery methods known in the art, including injection of a solution containing nucleic acid, irradiation with particles covered with nucleic acid, immersion of cells or organisms in a solution of nucleic acid, or electroporation of cell membrane in the presence of nucleic acid.Other methods known in the art can be used to introduce nucleic acid into cells, such as lipid-mediated carrier transport, chemical-mediated transport, and cationic liposome transfection, such as calcium phosphate.Nucleic acid can be introduced with other components that exert one or more of the following activities: enhancing nucleic acid uptake by cells or increasing the inhibition of target genes by other methods.

[0300] Physical methods of introducing nucleic acids include injection of a solution containing RNA, irradiation with particles covered by RNA, soaking of cells or organisms in a solution of RNA, or electroporation of cell membranes in the presence of RNA. A viral construct packaged in a viral particle will achieve both efficient introduction of the expression construct into cells and transcription of the RNA encoded by the expression construct. Other methods known in the art for introducing nucleic acids into cells can be used, such as lipid-mediated carrier transport, transport mediated by chemical IS such as calcium phosphate. Thus, RNA can be introduced with a component that exerts one or more of the following activities: enhancing RNA uptake by cells, inhibiting single-strand annealing, stabilizing single strands, or otherwise increasing inhibition of the target gene.

[0301] RNA can be introduced directly into a cell (i.e., intracellularly), or extracellularly into a cavity, tissue space, circulation of an organism, orally, or by bathing a cell or organism in a solution containing the RNA. The blood or lymphatic system, vascular or extravascular circulation, and cerebrospinal fluid are sites where RNA can be introduced.

[0302] The cells carrying the target gene may be derived from germline or somatic cells, totipotent or pluripotent, dividing or non-dividing, parenchymal or epithelial, immortalized or transformed, etc. The cells may be stem cells or differentiated cells. Differentiated cell types include adipocytes, fibroblasts, myocytes, cardiomyocytes, endothelium, neurons, glia, blood cells, megakaryocytes, lymphocytes, macrophages, neutrophils, eosinophils, basophils, mast cells, leukocytes, granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts, hepatocytes, and cells of endocrine or exocrine glands.

[0303] Depending on the specific target gene and the dose of double-stranded RNA material delivered, this process may result in partial or complete loss of function of the target gene. A reduction or loss of gene expression in at least 50%, 60%, 70%, 80%, 90%, 95% or 99% or more of the targeted cells is typical. Inhibition of gene expression refers to the absence (or observable reduction) of the levels of protein and / or mRNA products derived from the target gene. Specificity refers to the ability to inhibit the target gene without having an apparent effect on other genes of the cell. The results of inhibition can be confirmed by testing the external properties of the cell or organism (as shown below in the examples) or by biochemical techniques such as RNA solution hybridization, nuclease protection, Northern hybridization, reverse transcription, monitoring gene expression by microarrays, antibody binding, enzyme-linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA), other immunoassays, fluorescence-activated cell analysis (FACS), etc.

[0304] For RNA-mediated inhibition in cell lines or whole organisms, gene expression is conveniently assayed by the use of reporter or drug resistance genes whose protein products are easily assayed. Such reporter genes include acetohydroxyacid synthase (AHAS), alkaline phosphatase (AP), beta-galactosidase (LacZ), beta-glucoronidase (GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase (NOS), octopine synthase (OCS) and derivatives thereof. Multiple selection markers are available that confer resistance to ampicillin, bleomycin, chloramphenicol, gentamicin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin and tetracycline. Depending on the assay, quantification of the amount of gene expression allows to determine a degree of inhibition greater than 10%, 33%, 50%, 90%, 95% or 99% compared to cells not treated according to the present invention. A smaller dose of injected material and a longer time after administration of the RNAi agent may result in inhibition of a smaller percentage of cells (e.g., at least 10%, 20%, 50%, 75%, 90% or 95% of targeted cells). Quantization of gene expression in cells may indicate a smaller amount of inhibition at the level of target mRNA accumulation or target protein translation. As an example, the efficiency of inhibition may be determined by evaluating the amount of gene product in the cells. The mRNA may be detected with a hybridization probe having a nucleotide sequence outside the region used for the inhibitory double-stranded RNA, or the translated polypeptide may be detected with an antibody raised against the polypeptide sequence of the region.

[0305] RNA may be introduced in an amount that allows delivery of at least one copy per cell. Higher doses of material (e.g., at least 5, 10, 100, 500 or 1000 copies per cell) may result in more efficient inhibition; lower doses may also be useful for certain applications.

[0306] In certain embodiments, the efficacy of the RNAi agent of the present invention (e.g., an siRNA targeting a polymorphism in a mutant gene) is tested for its ability to specifically degrade mutant mRNA (e.g., mutant htt mRNA and / or mutant huntingtin protein production) in cells, particularly neurons (e.g., striatal or cortical neuronal clonal lines and / or primary neurons). Other easily transfectable cells, such as HeLa cells or COS cells, are also suitable for cell-based validation assays. Cells are transfected with human wild-type or mutant cDNA (e.g., human wild-type or mutant huntingtin cDNA). Standard siRNA, modified siRNA, or vectors capable of generating siRNA from U-loop mRNA are co-transfected. The selective reduction of mutant mRNA (e.g., mutant huntingtin mRNA) and / or mutant protein (e.g., mutant huntingtin) is measured. The reduction of mutant mRNA or protein can be compared to normal mRNA or protein levels. Exogenously introduced normal mRNA or protein (or endogenous normal mRNA or protein) can be assayed for comparative purposes. When utilizing neural cells, which are known to be somewhat resistant to standard transfection techniques, it may be desirable to introduce an RNAi agent (eg, siRNA) by passive uptake.

[0307] In certain exemplary embodiments, compositions comprising the RNA agent, e.g., dsRNA agent, of the present invention can be delivered to the nervous system of a subject by various routes. Exemplary routes include intrathecal, parenchymal (e.g., intracerebral), nasal and ocular delivery. The compositions can also be delivered systemically, e.g., by intravenous, subcutaneous or intramuscular injection, which is particularly useful for delivery of RNA agents, e.g., dsRNA agents, to peripheral neurons. Exemplary routes of delivery are directly to the brain, e.g., the ventricles or hypothalamus of the brain, or to the lateral or dorsal regions of the brain. RNA agents, e.g., dsRNA agents, for neuronal delivery can be incorporated into pharmaceutical compositions suitable for administration.

[0308] For example, the composition may include one or more species of RNA agent, such as dsRNA agent, and a pharma- ceutical acceptable carrier. The pharmaceutical composition of the present invention may be administered in several ways, depending on whether local or systemic treatment is desired and on the area to be treated. Administration may be topical (including ophthalmic, intranasal, transdermal), oral, or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal, or intramuscular injection, intrathecal, or intraventricular (e.g., intracerebroventricular) administration. In certain exemplary embodiments, the RNA silencing agent of the present invention is delivered through the blood-brain barrier (BBB) ​​using various suitable compositions and methods described herein.

[0309] The route of delivery may depend on the patient's disorder. For example, a subject diagnosed with Huntington's disease may be administered an anti-htt RNA agent, such as a dsRNA agent, of the present invention directly to the brain (e.g., in the globus pallidus of the basal ganglia or in the vicinity of the striatum and medium spiny neurons of the striatum). In addition to the RNA silencing agent of the present invention, the patient may be administered a second therapy, such as a symptomatic therapy and / or a disease-specific therapy. The second therapy may be, for example, symptomatic (e.g., to reduce symptoms), neuroprotective (e.g., to slow or stop disease progression), or restorative (e.g., to reverse the disease process). For the treatment of Huntington's disease, for example, symptomatic therapy may include the drugs haloperidol, carbamazepine, or valproate. Other therapies may include psychotherapy, physical therapy, speech therapy, communication and memory aids, social support services, and dietary advice.

[0310] The RNA agent, e.g., dsRNA agent, can be delivered to neural cells of the brain. Delivery methods that do not require the passage of the composition across the blood-brain barrier can be utilized. For example, a pharmaceutical composition containing an RNA agent, e.g., a dsRNA agent, can be delivered to a patient by direct injection into an area containing disease-affected cells. For example, the pharmaceutical composition can be delivered by direct injection into the brain. The injection can be by stereotactic injection into a specific area of ​​the brain (e.g., substantia nigra, cortex, hippocampus, striatum, or globus pallidus). The RNA agent, e.g., dsRNA agent, can be delivered to multiple areas of the central nervous system (e.g., multiple areas of the brain and / or spinal cord). The RNA agent, e.g., dsRNA agent, can be delivered to diffuse areas of the brain (e.g., diffuse delivery to the cortex of the brain).

[0311] In one embodiment, the RNA agent, e.g., dsRNA agent, can be delivered by means of a cannula or other delivery device with one end implanted in a tissue, e.g., the brain, e.g., the substantia nigra, cortex, hippocampus, striatum, or globus pallidus of the brain. The cannula can be connected to a reservoir of the RNA agent, e.g., dsRNA agent. The flow or delivery can be mediated by a pump, e.g., an osmotic pump such as an Alzet pump (Durect, Cupertino, CA) or a minipump. In one embodiment, the pump and reservoir are implanted in an area remote from the tissue, e.g., the abdomen, and delivery is by a conduit leading from the pump or reservoir to the release site. Devices for delivery to the brain are described, for example, in U.S. Pat. Nos. 6,093,180 and 5,814,014.

[0312] The RNA agent of the present invention, for example, dsRNA agent, can be further modified so that it can cross the blood-brain barrier.For example, the RNA agent, for example, dsRNA agent, can be conjugated to a molecule that allows the agent to cross the barrier.Such modified RNA agent, for example, dsRNA agent, can be administered by any desired method, for example, intracerebroventricular or intramuscular injection or pulmonary delivery.

[0313] In certain embodiments, exosomes are used to deliver RNA agents, e.g., dsRNA agents, of the invention. Exosomes can cross the BBB and can specifically deliver siRNA, antisense oligonucleotides, chemotherapeutic agents, and proteins to neurons after systemic injection (Alvarez-Erviti L, Seow Y, Yin H, Betts C, Lakhal S, Wood MJ. (2011). Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nat Biotechnol. 2011 Apr; 29(4): 341-5. doi: 10.1038 / nbt.1807; El-Andaloussi S, Lee Y, Lakhal-Littleton S, Li J, Seow Y, Gardiner C, Alvarez-Erviti L, Sargent IL, Wood MJ. (2011). Exosome-mediated delivery of siRNA in vitro and in vivo. Nat Protoc. 2012 Dec;7(12):2112-26.doi:10.1038 / nprot.2012.131;EL Andaloussi S,Maeger I,Breakefield XO,Wood MJ.(2013).Extracellular vesicles: biology and emerging therapeutic opportunities.Nat Rev Drug Discov.2013 May;12(5):347-57.doi:10.1038 / nrd3978;El Andaloussi S,Lakhal S,Maeger I,Wood MJ.(2013).Exosomes for targeted siRNA delivery across biological barriers.Adv.Drug Deliv Rev.2013 Mar;65(3):391-7.doi:10.1016 / j.addr.2012.08.008).

[0314] In certain embodiments, one or more lipophilic molecules are used to enable delivery of the RNA agent of the invention, e.g., a dsRNA agent, across the BBB (Alvarez-Ervit (2011)). The RNA silencing agent will then be activated, e.g., by enzymatic degradation of the lipophilic mimic, releasing the drug in its active form.

[0315] In certain embodiments, one or more receptor-mediated permeability compounds can be used to increase the permeability of the BBB to allow the delivery of the RNA silencing agent of the present invention.These drugs temporarily increase the permeability of the BBB by increasing the osmotic pressure in blood, which loosens the tight junctions between endothelial cells (El-Andaloussi (2012)).By loosening the tight junctions, normal intravenous injection of the RNA silencing agent can be performed.

[0316] In certain embodiments, a nanoparticle-based delivery system is used to deliver the RNA agent, e.g., dsRNA agent, of the present invention across the BBB. As used herein, "nanoparticle" refers to polymeric nanoparticles that are typically solid, biodegradable, colloidal, and have been widely studied as drug or gene carriers (SPEgusquiaguirre, M. Igartua, RM Hernandez, and JL Pedraz, "Nanoparticle delivery systems for cancer therapy: advances in clinical and preclinical research," Clinical and Translational Oncology, vol. 14, no. 2, pp. 83-93, 2012). Polymeric nanoparticles are classified into two main categories: natural polymers and synthetic polymers. Natural polymers for siRNA delivery include, but are not limited to, cyclodextrin, chitosan and atelocollagen (Y. Wang, Z. Li, Y. Han, LH Liang, and A. Ji, “Nanoparticle-based delivery system for application of siRNA in vivo,” Current Drug Metabolism, vol. 11, no. 2, pp. 182-196, 2010). Synthetic polymers include, but are not limited to, polyethyleneimine (PEI), poly(dl-lactide-co-glycolide) (PLGA) and dendrimers, which have been intensively studied (X. Yuan, S. Naguib, and Z. Wu, “Recent advances of siRNA delivery by nanoparticles,” Expert Opinion on Drug Delivery, vol. 8, no. 4, pp. 521-536, 2011).For a review of nanoparticles and other suitable delivery systems, see Jong-Min Lee, Tae-Jong Yoon, and Young-Seok Cho, “Recent Developments in Nanoparticle-Based siRNA Delivery for Cancer Therapy,” BioMed Research International, vol. 2013, Article ID 782041, 10 pages, 2013. doi:10.1155 / 2013 / 782041 (incorporated by reference in its entirety).

[0317] The RNA agent, e.g., dsRNA agent, of the present invention can be administered to the eye to treat, for example, retinal disorders, e.g., retinopathy. For example, the pharmaceutical composition can be applied to the surface of the eye or nearby tissues, e.g., inside the eyelid. They can be applied topically, for example, by spraying, in drops, as an eyewash or ointment. Ointments or droppable liquids can be delivered by ocular delivery systems known in the art, such as applicators or eye droppers. Such compositions can include mucus mimetics, such as hyaluronic acid, chondroitin sulfate, hydroxypropyl methylcellulose or poly(vinyl alcohol), preservatives, such as sorbic acid, EDTA or benzyl chromium chloride, and conventional amounts of diluents and / or carriers. The pharmaceutical composition can also be administered inside the eye and introduced by needles or other delivery devices that can introduce it to selected areas or structures. The composition containing the RNA silencing agent can also be applied via an eye patch.

[0318] In general, the RNA agent, e.g., dsRNA agent, of the present invention can be administered by any suitable method. As used herein, local delivery can refer to the direct application of the RNA agent, e.g., dsRNA agent, to any surface or any internal surface of the body, including the surface of the eye, mucous membrane, or body cavity. Preparations for local administration can include transdermal patches, ointments, lotions, creams, gels, drops, sprays, and liquids. Conventional pharmaceutical carriers, aqueous, powder or oil bases, thickeners, and the like may be necessary or desirable. Local administration can also be used as a means to selectively deliver the RNA agent, e.g., dsRNA agent, to the epidermis or dermis of a subject or specific layers thereof or underlying tissues.

[0319] Compositions for intrathecal or intraventricular (e.g., intracerebroventricular) administration can include sterile aqueous solutions, which can contain buffers, diluents, and other suitable additives. Compositions for intrathecal or intraventricular administration typically do not include transfection reagents or additional lipophilic moieties, e.g., lipophilic moieties attached to an RNA agent, e.g., a dsRNA agent.

[0320] Formulations for parenteral administration may include sterile aqueous solutions which may contain buffers, diluents and other suitable additives. Intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir. For intravenous use, the total concentration of solutes should be controlled to render the preparation isotonic.

[0321] The RNA agent of the present invention, for example, dsRNA agent, can be administered to a subject by pulmonary delivery.Pulmonary delivery composition can be delivered by inhalation of dispersion, so that the composition in dispersion can reach the lungs, where it can be easily absorbed directly into blood circulation through alveolar region.Pulmonary delivery can be effective for both systemic delivery and local delivery to treat lung diseases.In one embodiment, the RNA agent, for example, dsRNA agent, administered by pulmonary delivery is modified so that it can cross the blood-brain barrier.

[0322] Pulmonary delivery can be achieved by a variety of techniques, including the use of nebulization, aerosolization, micelles, and dry powder-based formulations. Delivery can be achieved by liquid nebulizers, aerosol-based inhalers, and dry powder dispersion devices. Metered dose devices are typical. One advantage of using a nebulizer or inhaler is that the device is self-contained, minimizing the possibility of contamination. Dry powder dispersion devices deliver drugs that can be easily formulated as dry powders, for example. RNA silencing agent compositions can be stably stored as lyophilized or spray-dried powders, alone or in combination with a suitable powder carrier. Delivery of the composition for inhalation can be mediated by a dosing timing element, which can include a timer, dose counter, time measurement device, or time indicator, which allows tracking of doses, monitoring compliance, and / or triggering of doses to patients during administration of aerosolized drugs, when incorporated into the device.

[0323] Types of pharmaceutical excipients useful as carriers include stabilizers such as human serum albumin (HSA), bulking agents such as carbohydrates, amino acids and polypeptides, pH adjusters or buffers, salts such as sodium chloride, etc. These carriers may be in crystalline or amorphous form, or may be a mixture of the two.

[0324] Particularly useful bulking agents include compatible carbohydrates, polypeptides, amino acids or combinations thereof.Suitable carbohydrates include monosaccharides such as galactose, D-mannose, sorbose, etc.; disaccharides such as lactose, trehalose, etc.; cyclodextrins such as 2-hydroxypropyl-beta-cyclodextrin; and polysaccharides such as raffinose, maltodextrin, dextran; alditols such as mannitol, xylitol, etc.Exemplary groups of carbohydrates include lactose, trehalose, raffinose, maltodextrin and mannitol.Suitable polypeptides include aspartame.Amino acids include alanine and glycine, with glycine being typical.

[0325] Suitable pH adjusters or buffers include organic acid salts prepared from organic acids and bases (sodium citrate is typical), such as sodium citrate, sodium ascorbate, and the like.

[0326] The RNA agent of the present invention, for example, dsRNA agent, can be administered by oral and nasal delivery.For example, the drug administered through these membranes has a rapid onset of action, produces therapeutic plasma levels, avoids the first-pass effect of hepatic metabolism, and avoids the exposure of the drug to unsuitable gastrointestinal (GI) environment.Additional advantages include easy access to the membrane site, so that the drug can be easily applied, localized, and removed.In one embodiment, the RNA silencing agent administered by oral or nasal delivery is modified so that it can cross the blood-brain barrier.

[0327] In one embodiment, a unit dose or measured dose of a composition comprising an RNA agent, e.g., a dsRNA agent, is dispensed by an implanted device. The device may include a sensor that monitors a parameter within a subject. For example, the device may include a pump, such as an osmotic pump, and optionally associated electronics.

[0328] It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein may be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. Although certain embodiments have been described in detail, they will be more clearly understood by reference to the following examples, which are included for illustrative purposes only and are not intended to be limiting. EXAMPLES

[0329] Example 1: SNP discrimination varies depending on the position of the mismatch Figure 46 is a flow chart showing the methodology for generating and selecting SNP-discriminating siRNAs constructed for HTT, but also applicable to SNPs in other genes. A primary selection is performed to determine which position the SNP is placed in provides the greatest discrimination. The mismatch position that provides the best results is then selected, and the affinity for non-target alleles is further reduced in a secondary selection where chemical and structural optimization is selected to siRNA molecules with improved selectivity and / or potency.

[0330] There are several SNPs in the HTT gene that have a high rate of heterozygosity in HD patients (Figure 45).To optimize the SNP-specific RNAi-mediated silencing of huntingtin, SNP rs362273 in exon 57 of HTT mRNA was used as a model target for optimizing SNP-selective silencing.This SNP heterozygosity is present in 35% of the HD patient population.

[0331] The psiCHECK reporter plasmid described herein contains the SNP rs362273 within the Rluc 3'UTR and a partial flanking region from exon 57 of htt. The wild-type psiCHECK reporter plasmid contains the same region of htt without the SNP (Figure 1).

[0332] Hydrophobically modified RNAs (hsiRNAs) designed to be complementary to huntingtin (htt) mRNA containing a mutant SNP (2273-1(A)) were screened for potency with the psiCheck reporter plasmid system. The number following the SNP represents the position of the SNP in the siRNA (Figure 47). Figure 2 shows that placing the SNP at positions 2, 4, or 6 resulted in maximum SNP discrimination without compromising potency against the mutant allele. HeLa cells transfected with one of the two reporter plasmids were reverse transfected with 1.5 μM hsiRNA by passive uptake and treated for 72 hours. Luciferase activity was measured 72 hours after transfection (Figure 2).

[0333] The hsiRNAs were further tested for allelic discrimination in a dose-response dual luciferase assay with HeLa cells (Figure 3). Multiple hsiRNAs preferentially silenced the reporter plasmid containing the mutant SNP compared to the wild-type reporter plasmid. HeLa cells transfected with one of the two reporter plasmids were reverse transfected with 1.5 μM hsiRNA by passive uptake and treated for 72 hours. Reporter plasmid expression was measured 72 hours after transfection (Figure 3).

[0334] Example 2: SNP discrimination in endogenous Htt mRNA hsiRNAs were tested for potency against endogenous huntingtin mRNA containing the homozygous rs362273 SNP. Because HeLa cells are homozygous at rs362273 with an A on each allele, allele discrimination was not assessed in this assay. Instead, Figure 4 shows that two hsiRNAs, SNP4-0 and SNP6-0, were highly effective at silencing htt mRNA containing the relevant SNP. mRNA levels were measured using the Quantigene 2.0 bDNA assay after treating HeLa cells with hsiRNAs by passive uptake for 72 hours. Human htt mRNA levels were normalized to human HPRT.

[0335] Example 3: Design of hsiRNAs with a second mismatch for higher allele discrimination For each of the three hsiRNAs previously selected for dose response (SNP2-0, SNP4-0, and SNP6-0, also called mm2, mm4, and mm6, respectively), 16 new hsiRNAs were designed and synthesized with minor sequence modifications (Figure 34). These sequences introduced a single mismatch at every possible position along the original sequence to test whether a second mismatch would impair silencing of the off-target SNP more significantly than before, without significantly affecting silencing of the target SNP. The antisense strand sequence is shown 5' to 3' with the SNP site in red and the new mismatch in blue (Figure 12).

[0336] The primary screening of hsiRNA potency in Figure 12 showed that the position of the second mismatch relative to the nucleotide position corresponding to the SNP resulted in various levels of SNP discrimination in HeLa cells. HeLa cells transfected with one of the two psiCHECK reporter plasmids were reverse transfected with 1.5 μM hsiRNA by passive uptake and treated for 72 hours. Luciferase activity was measured 72 hours after transfection. Figure 5 shows that multiple hsiRNAs preferentially silenced the reporter plasmid containing the SNP mutation compared to the wild-type reporter plasmid.

[0337] The most effective hsiRNA containing a second mismatch was further tested in a dose-response curve to verify the improvement of SNP discrimination. HeLa cells transfected with one of the two reporter plasmids were reverse transfected with hsiRNA by passive uptake and treated for 72 hours. Reporter expression was measured by a dual luciferase assay. Figures 6-8 show the IC50 values ​​of hsiRNA with two mismatches for silencing reporter plasmids containing SNP mutations relative to the wild-type reporter plasmid. SNP6-11 hsiRNA (hsiRNA molecule with a polymorphism at position 6 from the 5' end and a nucleotide corresponding to the mismatch at position 11 from the 5' end) and SNP4-7 hsiRNA (hsiRNA with a polymorphism at position 4 from the 5' end and a nucleotide corresponding to the mismatch at position 7 from the 5' end) were shown to be the most effective (see Figures 7-9). Surprisingly, alteration of the modification pattern around the SNP rescues the loss of potency by introducing a second mismatch without compromising discrimination. SNP6-11 hsiRNA was modified such that it has a 2'O-methyl modification adjacent to the mismatched nucleotide (and the mismatched nucleotide itself has a 2'O-methyl modification) (see FIG. 10).

[0338] Example 4: Additional Modifications Various oligonucleotide types (e.g., gapmers, mixmers, miRNA inhibitors, splice switching oligonucleotides ("SSOs"), phosphorodiamidate morpholino oligonucleotides ("PMOs"), peptide nucleic acids ("PNAs"), etc.) may be used in the oligonucleotides described herein, optionally utilizing various combinations of modifications (e.g., chemical modifications) and / or conjugates as described herein and in, for example, U.S. Patent Application Publication Nos. 15 / 089,423; 15 / 236,051; 15 / 419,593; 15 / 697,120 and U.S. Patent No. 9,809,817; and 15 / 814,350 and U.S. Patent No. 9,862,350, each of which is incorporated by reference in its entirety for all purposes.

[0339] For example, the oligonucleotides described herein may be designed as di-siRNAs (see, e.g., FIG. 14). The oligonucleotides described herein may include one or more different backbone linkages (see, e.g., FIG. 15). The oligonucleotides described herein may include various sugar modifications (see, e.g., FIG. 16). The oligonucleotides described herein may include various internucleotide linkages (see, e.g., FIG. 17). The oligonucleotides described herein may include one or more 5' stabilizing modifications (see, e.g., FIG. 18). The oligonucleotides described herein may include one or more conjugate moieties (see, e.g., FIG. 19). Some exemplary oligonucleotide backbone modifications are shown in FIG. 35.

[0340] The oligonucleotides described herein can be effectively used to target G at the SNP site by simply changing the base at the SNP position.As can be seen in Figure 33, compound SNP6-11 was synthesized a second time, this time targeting G at the SNP site instead of A.This allows selective silencing of either allele, a very useful strategy for patients with different heterozygosity at the same SNP site.

[0341] In certain exemplary embodiments, one or more abasic nucleotides are utilized at the SNP position nucleotide, the MM position nucleotide, the 5' end, the 3' end, or any combination thereof.

[0342] In certain exemplary embodiments, hsiRNAs are synthesized with various sugar modifications around the mismatches that improve allele specificity, e.g., 2'FANA instead of 2'F; triple 2'F or triple 2'OMe around the SNP / mismatch position.

[0343] Example 5: HTT Mouse Model BAC97-HD refers to a transgenic mouse that contains a human bacterial artificial chromosome (BAC) transgenic insert containing the entire disease-causing 170 kb human huntingtin (htt) genomic locus modified by replacing human htt exon 1 with a loxP-flanked human mutant htt exon 1 sequence containing 97 mixed CAA-CAG repeats encoding a consecutive polyglutamine (polyQ) stretch.

[0344] Lead compounds (SNP6-11) were synthesized into a biantennary chemical scaffold with the structure shown in Figure 31 and subsequently tested in vivo via 40 nmol bilateral intracerebroventricular (ICV) injections (20 nmol for each side) in 8-week-old BAC97-HD female mice. Mice carried two copies of transgenic inserts of the normal mouse htt gene with G at SNP rs362273 and the disease-causing human htt gene with A at SNP rs362273A. A nonsense sequence that does not match the target in the RNA transcriptome was also synthesized into the same biantennary chemical scaffold and injected in mice as a negative control (NTC).

[0345] Several brain regions were harvested from the mice for RNA and protein analysis one month after injection, and HTT protein levels were measured by Western blot using Ab1 antibody. Figure 32A is a Western blot performed on harvested striatal tissue, and protein levels normalized to vinculin are shown in Figure 32B.

[0346] Example 6: SNP targeting is sequence independent Whether the SNP discrimination of the lead compounds was sequence-dependent was evaluated. Hydrophobically modified RNAs (hsiRNAs) designed to be complementary to huntingtin (htt) mRNA containing a U to G mismatch or an A to C mismatch at rs362273 were used. Both the 6-11 hsiRNA complementary to the U to G mismatch and the 6-11 hsiRNA complementary to the C to A mismatch preferentially cleaved the target SNP (Figure 20).

[0347] Example 7: Synthesis of vinylphosphonate-modified intersubunit linkages Representative syntheses of the vinylphosphonate modified intersubunit linkages discussed herein are shown in Figures 21 and 29. The synthetic procedure of Figure 21 is detailed below.

[0348] Synthesis of compound 3a To an anhydrous solution of compound 2a (16.6 g, 20.8 mmol) in pyridine (100 mL) was added anhydrous DIPEA (6.5 mL, 37.4 mmol) and benzoyl chloride (3.6 mL, 31.2 mmol). After the mixture was stirred at room temperature for 4 h, excess pyridine was evaporated and diluted with CHCl. ​​The organic solution was washed with saturated aqueous NaHCO. The organic layer was collected, dried over MgSO, filtered and evaporated. The resulting crude material was purified by silica gel column chromatography (hexane-ethyl acetate, 4:1 to 1:1) to give compound 3a (14.5 g, 78%) as a pale yellow foam; 1 H NMR(500MHz,CDCl3)δ 7.88-7.87(m,2H),7.84(d,1H,J=8.3Hz),7.67-7.58(m,5H),7.48-7.45(m,4H),7.39-7.32(m, 4H),7.25-7.23(m,3H),7.18-7.17(m,2H),7.12-7.07(m,4H),6.80-6.75(m,4H),6.08(dd,1H,J HH =1.5Hz,J HF =15.2Hz),5.14,(d,1H,J HH =8.3Hz), 4.59(ddd,1H,J HH =3.7,1.5Hz,J HF =51.9Hz), 4.43(ddd,1H,J HH =7.4,4.0Hz,J HF =19.1Hz),4.24-4.23(m,1H),3.79(s,6H),3.62(dd,1H,J HH =11.2,2.0Hz),3.35(dd,1H,J HH =11.1,2.0Hz),1.00(s,9H); 13C NMR(126Hz,CDCl3)δ 168.4,161.8,158.72,158.66,148.9,143.9,139.4,135.71.135.70,135.1,134.8,134.7,132.3,132.2,131.3,130.4 ,130.2,130.1,129.1,128.2,128.0,127.91,127.89,127.2,113.19,113.16,102.2,92.5(d,JCF=194.4Hz),87.7(d,J CF =34.5Hz), 87.2, 82.4, 70.0(d,J CF =15.4Hz),60.7,60.4,55.2,26.6.

[0349] Synthesis of compound 4a Compound 3a (14.5 g, 16.3 mmol) was dissolved in 3% trichloroacetic acid / CHCl solution (200 mL) containing triethylsilane (8.0 mL, 50.1 mmol) and stirred at room temperature for 1 h. The solution was washed three times with saturated aqueous NaHCO, and the collected organic layer was dried over MgSO, filtered, and evaporated. The resulting crude material was purified by silica gel column chromatography (hexane / ethyl acetate, 4:1 to 3:7) to give compound 4a (8.67 g, 91%) as a white foam; 1 H NMR(500MHz,CDCl3)δ 7.89-7.88(m,2H),7.68-7.64(6H,m),7.51-7.45(m,4H),7.42-7.38(4H,m),5.93(dd,1H,J HH =2.9Hz,J HF =15.1Hz), 5.73(d,1H,J HH =8.2Hz), 4.74(ddd,1H,J HH =4.1,3.2Hz,J HF =52.2Hz), 4.31(ddd,1H,J HH =5.8,4.7,J HF =15.4Hz),4.11-4.09(m,1H),3.82-3.79(m,1H),3.39(ddd,1H,J HH =12.1,5.6,1.5Hz),1.64(br,1H),1.11(s,9H);13 C NMR(126Hz,CDCl3)δ 168.3,161.8,149.0,140.5,135.7,135.2,132.8,132.3,131.3,130.5,130.4,130.3,129.2,128.02,127.96,102.4,91.8(d,J CF =91.8Hz),89.5(d,J CF =33.6Hz),69.5(d,J CF =69.5Hz),60.3,26.8.

[0350] Synthesis of compound 6a To an anhydrous solution of compound 4a (6.5 g, 11.0 mmol) was added IBX (7.7 g, 27.6 mmol) and stirred at 85 °C for 2 h. After cooling the mixture in an ice bath, the precipitate in the solution was filtered off through Celite. The collected eluate was evaporated and coevaporated with anhydrous CH3CN three times under argon atmosphere to give compound 5a as a white foam, which was used without further purification. In a separate flask, PPh3 (11.6 g, 44.2 mmol) was added to a solution of CBr4 (7.3 g, 22.1 mmol) in anhydrous CHCl2 (25 mL) at 0 °C and stirred at 0 °C for 0.5 h. To this solution was added dropwise (10 min) a solution of compound 5a in anhydrous CHCl2 (25 mL) at 0 °C and stirred at 0 °C for 2 h. After dilution with CHCl2, the organic solution was washed with saturated aqueous NH4Cl, dried over MgSO4, filtered, and evaporated. The resulting material was dissolved in a minimum amount of diethyl ether and added dropwise to an excess of diethyl ether solution under vigorous stirring at 0°C. The precipitate in the solution was filtered off through Celite, and the eluent was evaporated. The resulting crude material was purified by silica gel column chromatography (hexane / ethyl acetate, 9:1 to 1:1) to give compound 6a (4.3 g, 52%) as a white foam. 1H NMR(500MHz,CDCl3)δ 7.68-7.84(m,2H),7.70-7.65(m,3H),7.60-7.58(m,2H),7.52-7.49(m,2H),7.42-7.36 (m,4H),7.31-7.28(m,2H),7.09(d,1H,J=8.2Hz),6.25(d,1H,J=8.9Hz),5.75(dd,1H,J HF =8.24Hz), 5.49(dd,1H,J HF =21.4Hz), 4.77(t,1H,J HH =8.5Hz,J HF =8.5Hz), 4.38(dd,1H,J HH =4.1Hz,J HF =52.1Hz), 4.25(ddd,1H,J HH =8.1,4.9Hz,J HF =19.4Hz),1.10(s,9H); 13C NMR(126Hz,CDCl3)δ 167.9,161.6,148.3,141.4,135.8,134.7(d,J C-Br =139.0Hz),132.5,132.2,131.1,130.5,130.3,130.2,129.2,127.9,102.7,97.3,93.3(d,J CF =39.1Hz), 91.5(d,J CF =190.7Hz), 82.4, 73.9(d,J CF =16.4Hz), 26.7.

[0351] Synthesis of compounds 7a-E and 7a-Z To an anhydrous solution of compound 6a (4.2 g, 5.66 mmol) in DMF (25 mL) was added dimethyl phosphite (2.09 mL, 22.6 mmol) and triethylamine (1.58 mL, 11.3 mmol) at 0 °C, followed by stirring at room temperature overnight. After diluting the solution with ethyl acetate, the organic solution was washed with saturated aqueous NH4Cl and brine. Then, the organic solution was dried over MgSO4, filtered and evaporated. The obtained crude material was repeatedly purified by silica gel column chromatography (hexane / ethyl acetate, 9:1 to 1:1) until all the pure isomeric compounds were recovered separately to give compound 7a-E (1.95 g, 52%); 1 H NMR(500MHz,CDCl3)δ 7.87-7.85(m,2H),7.89-7.85(m,3H),7.61-7.59(m,2H),7.52-7.48(m,2H),7.45-7.32(m,6H),7.08(d,1H,J HH =8.2), 6.49(d,1H,J HH =13.7),5.99(dd,1H,J HH =13.7Hz,8.1Hz),5.75(d,1H,J HH = 8.2), 5.63(d,1H,J HF =19.8Hz), 4.43(dd,1H,J HF =52.6Hz,J HH =4.3Hz), 4.42(t,1H,J HH =8.0Hz), 4.07(ddd,J HH =7.8,4.7Hz,JHF = 19.5 Hz), 1.08 (s, 9H); 13 C NMR (126 Hz, CDCl3) δ 148.4, 140.4, 135.8, 135.7, 135.3, 133.3, 132.3, 132.4, 132.1, 131.1, 130.5, 130.4, 130.3, 129.2, 127.95, 127.93, 112.4, 102.7, 91.7 (d, J CF = 36.3 Hz), 91.6 (d, J CF = 191.6 Hz), 82.8, 73.9 (d, J CF = 16.4 Hz), 26.7, 19.1; and 7a-Z (0.58 g, 15%); 1 H NMR (500 MHz, CDCl3) δ 7.87 - 7.85 (m, 2H), 7.68 - 7.65 (m, 3H), 7.61 - 7.59 (m, 2H), 7.52 - 7.48 (m, 2H), 7.42 - 7.39 (m, 2H), 7.34 - 7.29 (m, 4H), 7.12 (d, 1H, J HH = 8.2 Hz), 6.51 (d, 1H, J HH = 7.4 Hz), 5.96 (dd, 1H, J HH = 8.4 Hz, 7.4 Hz), 5.75 (d, 1H, J HH = 8.2 Hz), 5.57 (dd, 1H, J HH = 1.2 Hz, J HF = 20.6 Hz), 5.04 (dd, 1H, J HH = 8.2 Hz), 4.48 (J HH = 3.5 Hz, J HF = 53.1 Hz), 4.24 (ddd, 1H, J HH = 7.8, 4.9 Hz, J HF = 18.6 Hz), 1.09 (s, 9H); 13 C NMR (126 Hz, CDCl3) δ 168.0, 161.7, 148.4, 141.4, 135.9, 135.8, 135.2, 132.6, 132.5, 131.2, 130.6, 130.5, 130.2, 130.1, 129.2, 127.8, 127.7, 114.5, 102.6, 93.0 (d, J CF = 37.2 Hz), 91.6 (d, J CF = 191.6 Hz), 80.3, 74.3 (d, JCF =16.4Hz), 26.7, 19.1.

[0352] Synthesis of compound 9a Anhydrous compound 7a-E (1.95 g, 2.94 mmol), Pd(OAc)2 (125 mg, 0.59 mmol), and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (652 mg, 1.18 mmol) were purged with argon and then dissolved in anhydrous THF (50 mL). Propylene oxide (2.06 mL, 29.4 mmol) was added, followed by compound 8a (2.07 g, 3.24 mmol) in one portion and stirred at 70 °C for 4 h. After removing the solvent under reduced pressure, the crude mixture was purified by silica gel column chromatography (hexane / ethyl acetate, 50:50 to 0:100), and the resulting fraction containing compound 9a was further purified by silica gel column chromatography (CHCl-MeOH, 0% to 5%) to give compound 9a as a mixture of diastereoisomers (2.04 g, 57%); 31 P NMR (202 MHz, CDCl3) δ 18.3.

[0353] Synthesis of compound 10a Compound 9a (2.0 g, 1.64 mmol) in anhydrous THF (22.5 mL) was added with 1.0 M TBAF-THF (2.5 mL, 2.5 mmol) and stirred at ambient temperature for 30 min. After dilution with CHCl (120 mL), the organic layer was washed with brine, dried over MgSO, filtered, and evaporated. The resulting crude material was purified by silica gel column chromatography (1% TEA-CHCl / MeOH, 0%-6%) to give compound 10a (1.52 g, 94%); 31 P NMR(202MHz, CDCl3)δ 19.0,18.7.

[0354] Synthesis of compound 11a Compound 10a (589.7 mg, 0.6 mmol) was dried by repeated coevaporation with anhydrous CH3CN and subsequently dissolved in anhydrous CHCl2 (6.0 mL). To this solution, N,N-diisopropylethylamine (0.31 mL, 1.8 mmol) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.16 mL, 0.72 mmol) were added at 0 °C. After stirring at 0 °C for 30 min, the reaction mixture was diluted with excess CHCl2. The organic layer was repeatedly washed with saturated aqueous NaHCO3, dried over MgSO4, filtered, and evaporated. The resulting crude material was purified by silica gel column chromatography (1% TEA-CH2Cl2 / MeOH, 100%-4%) to give compound 11a (570 mg, 80%) as a white foam; 31 P NMR(202MHz, CDCl3)δ 150.3,151.2,151.1,151.0,18.72,18.65,18.55,18.3.

[0355] Synthesis of compound 4b To an anhydrous solution of compound 3b (1.35 g, 2.0 mmol) in pyridine (10 mL) was added DIPEA (0.63 mL, 3.6 mmol) and benzoyl chloride (0.35 mL, 3.0 mmol) and stirred at room temperature for 3 h. After dilution with excess CHCl, the organic solution was washed with saturated aqueous NaHCO and brine. After drying with MgSO, filtration, evaporation, and the obtained crude material was used for the next reaction without further purification. To the obtained crude material containing compound 3b was added 3% trichloroacetic acid in CHCl (25 mL) and triethylsilane (1 mL, 6.26 mmol) and stirred at room temperature for 1 h. After dilution of the reaction mixture with CHCl, the solution was washed three times with saturated aqueous NaHCO, dried with MgSO, filtration, and subsequent evaporation. The obtained crude material was purified by silica gel column chromatography (hexane / ethyl acetate, 4:1 to 1:4) to give pure compound 4b (596.7 mg, 63% for two steps); 1 H NMR(500MHz,DMSO-d6)δ 8.13(d,1H,J HH =8.2Hz), 7.95(d,2H,J HH= 7.3 Hz), 7.81 (t, 1H, J HH = 7.5 Hz), 7.69 - 7.68 (m, 2H), 7.64 - 7.59 (m, 4H), 7.49 - 7.42 (m, 6H), 5.93 (d, 1H, J HH = 4.6 Hz), 5.26 (t, 1H, J HH = 4.6 Hz), 4.36 (dd, 1H, J HH = 4.6, 4.6 Hz), 4.02 - 4.00 (m, 1H), 3.65 - 3.61 (m, 1H), 3.54 (dd, 1H, J HH = 4.6, 4.6 Hz), 3.09 (s, 3H), 1.03 (s, 9H); 13 C NMR (126 Hz, DMSO-d6) 169.8, 162.1, 149.5, 141.3, 136.1, 135.9, 135.8, 133.4, 133.2, 131.5, 130.7, 130.52, 130.48, 130.0, 128.4, 128.3, 102.1, 86.7, 85.6, 82.8, 79.7, 70.8, 60.2, 57.8, 27.2, 19.4; HRMS (ESI) C 33 H 35 N2O7Si - [M - H] - Calculated m / z for [M - H] m / z 599.2219, found m / z 599.2258.

[0356] Synthesis of Compound 6b To an anhydrous solution of compound 4b (300.4 mg, 0.5 mmol) in CH3CN (5 mL) was added IBX (350 mg, 1.3 mmol) and stirred at 85 °C for 2 h. After cooling the solution at 0 °C, the precipitate was filtered off by celite filtration. The resulting eluate containing compound 5b was evaporated, anhydrous by repeated coevaporation with anhydrous CH3CN, and used for the next reaction without further purification. To the prepared anhydrous solution of CBr4 (331.6 mg, 1.0 mmol) in CHCl2 (5.0 mL) for separation was added triphenylphosphine (524.6 mg, 2.0 mmol) in one portion at 0 °C and stirred at 0 °C for 30 min. To this solution was added compound 5b in anhydrous CHCl2 (1.5 mL) dropwise at 0 °C (10 min) and stirred at 0 °C for 2 h. The solution was then diluted with CHCl2 and washed with saturated aqueous NaHCO3 and brine. The organic solution was dried over MgSO4, filtered, evaporated, and the resulting crude material was purified by silica gel column chromatography (hexane / ethyl acetate, 9:1 to 4:6) to give compound 6b (210.9 mg, 56%); 1 H NMR(500MHz,CDCl3)δ 7.88(d,2H,J HH =7.3Hz),7.70-7.62(5H,m),7.51-7.38(m,9H),7.08(d,1H,J HH =8.2Hz), 6.26(d,1H,J HH =8.6Hz), 5.75(d,1H,J HH =8.2Hz), 5.68(d,1H,J HH =0.8Hz), 4.84(dd,1H,J HH =8.6Hz,8.6Hz),3.86(dd,1H,J HH =7.5Hz,5.0Hz),3.30(s,3H),3.18(br,1H),1.11(s,9H); 13 C NMR(126Hz,CDCl3)168.3,161.7,148.6,138.9,135.9,135.8,134.3,132.6,132.4,131.2,130.5,130 .4,130.3,129.2,128.0,127.9,102.4,97.5,90.0,82.44,82.39,74.4,58.2,26.7,19.1;HRMS(ESI)C34 H 33 Br2N2O6Si - [MH] - Calculated m / z value for m / z 751.0480[MH] - ,Actual value m / z 753.6495.

[0357] Synthesis of 7b-E and 7b-Z To an anhydrous solution of compound 6b (6.11 g, 8.1 mmol) in DMF (35 mL) was added dimethyl phosphite (2.97 mL, 34.0 mmol) and triethylamine (2.26 mL, 17.0 mmol) at 0 °C, followed by stirring at room temperature overnight. After diluting the solution with ethyl acetate, the organic solution was washed with saturated aqueous NH4Cl and brine. The organic solution was then dried over MgSO4, filtered and evaporated to give the crude material, which was repeatedly purified by silica gel column chromatography (hexane / ethyl acetate, 9:1 to 1:1) until all the pure isomeric compounds were recovered separately to give compound 7b-E (3.0 g, 55%); 1 H NMR(500MHz,CDCl3)δ 7.89-7.87(m,2H),7.70-7.62(m,5H),7.51-7.39(m,8H),7.10(d,1H,J HH =8.3Hz), 6.47(dd,1H,J HH =13.6,0.8Hz),6.01(dd,1H,J HH =13.6,7.9Hz),5.76-5.74(m,2H),4.51(dd,1H,J HH =7.8,7.8Hz),7.36(dd,1H,J HH =7.8Hz,4.9Hz),3.34(s,3H),3.17(dd,1H,J HH =4.7,1.2Hz),1.09(s,9H); 13C NMR(126Hz,CDCl3)δ 168.3,161.7,148.7,138.4,135.9,135.8,135.3,133.8,132.6,132.4,131.2,130.5,130.4,130.3,12 9.2,128.0,127.9,112.1,102.3,88.9,82.8,82.6,77.2,74.2,58.1,26.8,19.1; and び7b-Z (1.23g, 22%); 1 H NMR(500MHz,CDCl3)δ 7.89-7.87(m,2H),7.72-7.70(m,2H),7.68-7.63(m,3H),7.51-7.44(m,4H),7.41-7.37(m,4H),7.16(d,1H,J-8.2Hz),6.53(dd,1H,J HH= 7.4,0.6Hz),6.03(dd,1H,J HH =8.5,7.4Hz),5.75-5.73(m,2H),5.12(t,1H,J HH =8.1Hz),3.93(dd,1H,J HH =6.9,5.0Hz),3.32(br,1H),3.26(s,3H),1.10(s,9H); 13 C NMR(126Hz,CDCl3)δ 168.3,161.8,148.7,139.3,135.91,135.85,135.22,132.74,132.71,131.2,130.8,130.5,130. 23,130.16,129.2,127.78,127.75,114.6,102.2,90.1,82.4,80.6,77.2,74.8,58.1,26.8,19.2.

[0358] Synthesis of compound 8b Anhydrous 5'-O-DMTr-2'-deoxy-2'-fluoro-3'-[methyl-N,N-(diisopropyl)amino]phosphoramidite (4.26 g, 6.0 mmol) was dissolved in 0.45 M 1H-tetrazole / CH3CN solution (27 mL, 12 mmol) and stirred at room temperature for 30 min. H2O (3.6 mL) was added to the solution and stirred at room temperature for 30 min. After dilution with ethyl acetate, the organic solution was washed six times with brine, dried over MgSO4, filtered, and subsequently evaporated. Compound 8b obtained with a small amount of impurity was used for the next reaction without further purification; 31 P NMR(CDCl3,202MHz)δ 8.92,8.28.

[0359] Synthesis of compound 9b Anhydrous compound 7b-E (2.84 g, 4.20 mmol), Pd(OAc)2 (188.6 mg, 0.84 mmol) and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (931.4 mg, 1.68 mmol) were purged with argon and then dissolved in anhydrous THF (50 mL). Propylene oxide (2.94 mL, 42.0 mmol) was added, followed by compound 9b (3.16 g, 5.04 mmol) in one portion and stirred at 70 °C for 4 h. After removing the solvent under reduced pressure, the crude mixture was purified by silica gel column chromatography (hexane-ethyl acetate, 50:50-0:100), and the resulting fraction containing compound 9b was further purified by silica gel column chromatography (1% TEA-CH2Cl2 / MeOH, 0%-5%) to obtain compound 9a as a mixture of diastereoisomers (3.3 g, 64%); 31 P NMR(202MHz, CDCl3)δ 19.31,18.72.

[0360] Synthesis of compound 10b Compound 9b (3.3 g, 2.70 mmol) in anhydrous THF (36.5 mL) was added with 1.0 M TBAF-THF (4.05 mL, 4.05 mmol) and stirred at ambient temperature for 30 min. After dilution with CHCl (150 mL), the organic layer was washed with brine, dried over MgSO, filtered, and subsequently evaporated. The resulting crude material was purified by silica gel column chromatography (1% TEA-CHCl / MeOH, 0%-8%) to give compound 10b (1.25 g, 47%); 31 P NMR(202MHz, CDCl3)δ 19.8,19.1.

[0361] Synthesis of compound 11b Compound 10b (393.2 mg, 0.4 mmol) was dried by repeated coevaporation with anhydrous CH3CN and subsequently dissolved in anhydrous CHCl2 (4.0 mL). To this solution, N,N-diisopropylethylamine (0.21 mL, 1.2 mmol) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.11 mL, 0.48 mmol) were added at 0 °C. After stirring at 0 °C for 30 min, the reaction mixture was diluted with excess CHCl2. The organic layer was repeatedly washed with saturated aqueous NaHCO3, dried over MgSO4, filtered, and evaporated. The resulting crude material was purified by silica gel column chromatography (1% TEA-CH2Cl2 / MeOH, 100%-4%) to give compound 11b (319.6 mg, 68%) as a white foam; 31 P NMR (202MHz, CDCl3) δ150.7,150.4,150.3,19.9,19.5,19.4,18.8.

[0362] Example 8: Solid support mediated synthesis of vinylphosphonate modified oligonucleotides A representative synthesis of oligonucleotides with vinylphosphinate modified intersubunit linkages is shown in Figure 22. Examples of synthesized VP modified sequences can be found in Figures 28A and 28B.

[0363] Synthesis of internucleotide (E)-vinylphosphonate modified RNA oligonucleotides. Synthetic RNA oligonucleotides with one vinyl phosphonate linkage were performed on a MerMade 12 automated RNA synthesizer (BioAutomation) using 0.1 M anhydrous CH3CN solutions of 2'-modified (2'-fluoro, 2'-O-methyl) phosphoramidites and vinyl phosphonate-linked dimeric phosphoramidites. For the solid support, UnyLinker support (ChemGenes) was used. The synthesis was carried out by a standard 1.0 μmol scale RNA phosphoramidite synthesis cycle consisting of (i) detritylation, (ii) coupling, (iii) capping, and (iv) iodine oxidation. 5-(benzylthio)-1H-tetrazole in anhydrous CH3CN was used for the phosphoramidite activation reagent, and 3% dichloroacetic acid in CHCl2 was used for detritylation. 16% N-methylimidazole in tetrahydrofuran (Cap A) and 80:10:10 (v / v / v) tetrahydrofuran-Ac2O-2,6-lutidine (Cap B) were used for the capping reaction. 0.02M I2 in THF-pyridine-H2O (7:2:1, v / v / v) was used for oxidation, and 0.1M 3-[(dimethylamino-methylidene)amino]-3H-1,2,4-dithiazole-3-thione in pyridine:CH3CN (9:1, v / v) was used for sulfurization. Bis(2-cyanoethyl)-N,N-diisopropyl phosphoramidite was used for 5'-end phosphorylation. Cholesterol 3'-lcaa CPG 500Å (ChemGenes) was used for 3'-cholesterol modified RNA oligonucleotide synthesis, and RNA synthesis was carried out in the same conditions as those used for VP modified RNA. After chemical chain elongation, deprotection and cleavage from the solid support was carried out with NH4OH-EtOH (3:1, v / v) for 48 h at 26° C. For vinylphosphonate-modified RNA, the RNA on the solid support was first treated with TMSBr-pyridine-CHCl2 (3:1:18, v / v / v) in an RNA synthesis column for 1 h at ambient temperature.The solid support was then washed with running solutions of water (1 mL x 3), CH3CN (1 mL x 3) and CHCl2 (1 mL x 3) through a synthesis column, followed by drying under vacuum. After transferring the solid support to a screw-capped sample tube, a base treatment with NH4OH-EtOH (3:1, v / v) was carried out at 26 °C for 48 h. Crude RNA oligonucleotides without cholesterol conjugates were purified by standard anion exchange HPLC, whereas RNA with cholesterol conjugates was purified by reversed-phase HPLC. All the obtained purified RNAs were desalted by Sephadex G-25 (GE Healthcare) and characterized by electrospray ionization mass spectrometry (ESI-MS) analysis.

[0364] Example 9: Silencing efficacy Figures 23 and 24 provide a visual representation of the VP-modified siRNA tested herein. Figure 25 illustrates the effect that one or more vinyl phosphonate modifications at intersubunit linkages at various positions on guide strand have on silencing. As can be seen from the data in Figure 25, RISC is highly sensitive to VP modifications, and can destroy siRNA potency by having mismatched base pairs at various positions.

[0365] Figures 26, 27A and 27B also show the ability of VP-modified siRNA to silence mutant alleles. As can be seen by Figures 27A and 27B, adding mismatches in the siRNA sequence can improve allele discrimination without affecting the silencing of mutant alleles. Figure 30 demonstrates that the introduction of VP-modified bonds next to the SNP site significantly enhanced the target / non-target discrimination of SNP-selective siRNAs. Compounds containing the primary (position 6) and secondary (position 11) SNPs were synthesized with or without VP modifications between positions 5 and 6. As can be seen in Figure 30, the presence of VP modifications does not affect "on-target" activity, but completely eliminates any detectable silencing for non-target mRNAs. The methods for generating the data in Figures 25, 26, 27A and 27B are described below.

[0366] Passive delivery of hsiRNA. Cells were seeded in Dulbecco's Modified Eagle Medium containing 6% FBS at 8,000 cells per well in 96-well cell culture plates. hsiRNA was diluted to twice the final concentration in OptiMEM (Carlsbad, CA; 31985-088) and 50 μL of diluted hsiRNA was added to 50 μL of cells, resulting in a final of 3% FBS. Cells were incubated at 37° C. and 5% CO2 for 72 hours. The maximum dose in the in vitro dose-response assay was 1.5 μM compound.

[0367] Methods for quantitative analysis of target mRNA. mRNA was quantified from cells using QuantiGene 2.0 assay kit (Affymetrix, QS0011). Cells were lysed for 30 minutes at 55°C in 250 μL diluted lysis mixture consisting of one part lysis mixture (Affymetrix, 13228), two parts H2O and 0.167 μg / μL proteinase K (Affymetrix, QS0103). Cell lysates were mixed thoroughly and 40 μL of each lysate was added per well of the capture plate along with 20 μL diluted lysis mixture without proteinase K. Probe sets for human HTT and HPRT (Affymetrix; #SA-50339, SA-10030) were diluted and used according to the manufacturer's recommended protocol. Data sets were normalized to HPRT.

[0368] A method for creating a bar graph. Data were analyzed using GraphPad Prism 7 software (GraphPad Software, Inc., San Diego, CA). Concentration-dependent IC 50 Curves were fitted using a log(inhibitor)-variable slope (4 parameters) for the response. For each cell treatment plate, the level of knockdown at each dose was normalized to the mean of the control (untreated) group. The lower limit of the curve was set to less than 5 and the upper limit of the curve was set to greater than 95. To generate the bar graphs, the percentage of difference was calculated using the IC of each corresponding control compound. 50 IC value for each compound 50 The IC value of the control compound was subtracted. 50 The percent difference was calculated by dividing by the value and multiplying by 100. If the percent difference was less than -500%, the percent difference was artificially set to -500%. The lower limit of the graph was clipped at -300%.

[0369] The contents of all references (including literature references, patents, patent applications, and websites) that may be cited throughout this application, as well as the references cited therein, are hereby expressly incorporated by reference in their entirety for all purposes. This disclosure will employ, unless otherwise indicated, conventional techniques of immunology, molecular biology, and cell biology that are well known in the art.

[0370] The present disclosure may be embodied in other specific forms without departing from its spirit or essential characteristics. Accordingly, the above-described embodiments are to be considered in all respects as illustrative and not limiting of the present disclosure. Accordingly, the scope of the present disclosure is indicated by the appended claims, rather than the foregoing description, and therefore, all modifications that come within the spirit and scope of the claims are intended to be embraced herein.

[0371] Example 10: Primary screening yields multiple effective siRNA sequences for SNP rs362307 heterozygosity All of the siRNAs designed to be complementary to HTT mRNA containing another mutant SNP (rs362307) (Figure 39) were screened with a reporter plasmid containing the target region for the SNP of interest (Figure 40). HeLa cells transfected with one of the two reporter plasmids were reverse transfected with 1.5uM hsiRNA by passive uptake and treated for 72 hours. The number following the SNP represents the position of the SNP in the siRNA. It was expected that this SNP would be more difficult to target based on the high G / C content of the region around it. Replacing the SNP at position 3 appears to result in the greatest SNP discrimination without losing potency against the mutant allele, indicating that the optimal SNP position is sequence specific (Figure 41). Thus, this primary screening process can be performed to select the optimal SNP position for any SNP.

[0372] Example 11: Following a second mismatch improves allele discrimination when applied to SNP rs362307 As reported in Figure 42, the initial selection of new sequences with mismatches introduced at all possible positions yields multiple effective hsiRNAs with increased SNP discrimination even at position rs362307. The introduction of mismatches at positions 7 and 8 appears to increase selectivity, but maintains target silencing potency. Other second mismatches provide better discrimination, but reduce overall activity.

[0373] Example 12: Measuring SNP discrimination in sequences containing SNPs To measure SNP discrimination by each of the sequences disclosed in Tables 5-7 (i.e., each hsiRNA with a particular SNP position nucleotide and mismatch (MM) position nucleotide combination), psiCHECK reporter plasmids containing either the wild type region of htt or the same region of htt with the SNP of the sequence were prepared and tested using dual luciferase. HeLa cells transfected with one of the two reporter plasmids were reverse transfected with hsiRNA by passive uptake and treated for 72 hours. Luciferase activity was measured in assays with or without additional mismatches and then plotted in a dose-response curve and compared to reveal the sequence that gives the best results in terms of silencing discrimination and potency.

[0374] Example 13: Synthesis of phosphinate-modified intersubunit linkages A method for preparing the phosphinate modified intersubunit linkages of the present invention is summarized in Figures 44A-44C. The method involves Jones oxidation of the free alcohol to the corresponding ketone followed by Wittig olefination to give the exomethylene moiety shown in intermediate compound 3. Hydroboration-oxidation after protection of the amide with BOM gives the free alcohol intermediate 5. A modified Finkelstein reaction followed by mesylation produces the iodide intermediate 7 which undergoes further functionalization to give the methylphosphinate monomer 9.

[0375] Various protection and deprotection steps are utilized to give intermediate 13 to afford monomer 18. IBX oxidation generates the corresponding ketone, followed by Wittig olefination to give the methylene. Once again, hydroboration-oxidation is followed by mesylation and Finkelstein reaction to give monomer 18.

[0376] Combination of monomers 9 and 18 under basic conditions gives the phosphinate-linked dimer 19. Further phosphane amine functionalization after acid-mediated and Pearlman-catalyzed deprotection gives the dimer 22. The present invention includes the following aspects. [Item 1] An oligonucleotide having a 5' end, a 3' end and a seed region, which is complementary to a region of a gene containing an allelic polymorphism; a single nucleotide polymorphism (SNP) position nucleotide at a position within the seed region that is complementary to the allelic polymorphism; and a mismatch (MM) position nucleotide located 2 to 11 nucleotides from the SNP position nucleotide that is mismatched with a nucleotide in the gene; An oligonucleotide comprising: [Item 2] The oligonucleotide according to Item 1, wherein the oligonucleotide is RNA. [Term 3] Formula: [C1] JPEG0007618229000041.jpg29160 The RNA of item 2, further comprising at least one vinylphosphonate (VP) modification at an intersubunit bond having the formula: [Item 4] The RNA described in Item 3, wherein the VP motif is inserted adjacent to the SNP position nucleotide or adjacent to the MM position nucleotide. [Item 5] The oligonucleotide described in Item 1, which is selected from the group consisting of siRNA, miRNA, shRNA, CRISPR guide, DNA, antisense oligonucleotide (ASO), gapmer, mixmer, miRNA inhibitor, splice switching oligonucleotide (SSO), phosphorodiamidate morpholino oligomer (PMO) and peptide nucleic acid (PNA). [Item 6] The oligonucleotide described in Item 1, which is an ASO or double-stranded RNA (dsRNA). [Item 7] A first strand of about 15 to 35 nucleotides complementary to the region of the gene containing an allelic polymorphism; and a second strand of about 15-35 nucleotides complementary to at least a portion of said first strand; Including, the first strand comprises the SNP position nucleotide at positions 2 to 6 from the 5' end that is complementary to the allelic polymorphism; Item 7. The dsRNA according to Item 6, wherein the first strand comprises the MM position nucleotide located 2 to 6 nucleotides from the SNP position nucleotide that is mismatched with a nucleotide in the gene. [Item 8] The dsRNA according to Item 7, wherein the SNP position nucleotide is at position 2 from the 5' end. [Item 9] The dsRNA according to Item 7, wherein the SNP position nucleotide is at position 4 from the 5' end. [Item 10] The dsRNA of Item 7, wherein the SNP position nucleotide is at position 6 from the 5' end of the first strand. [Item 11] The dsRNA according to any one of Items 7 to 10, wherein the MM position nucleotide is located within 2 nucleotides or 3 nucleotides of the SNP position nucleotide. [Item 12] The dsRNA according to any one of Items 7 to 10, wherein the MM position nucleotide is located within 4 nucleotides of the SNP position nucleotide. [Item 13] The dsRNA according to any one of Items 7 to 10, wherein the MM position nucleotide is located within 6 nucleotides of the SNP position nucleotide. [Item 14] The dsRNA according to any one of Items 7 to 13, which has a blunt end. [Item 15] The dsRNA according to any one of Items 7 to 13, comprising at least one single-stranded nucleotide overhang. [Item 16] The dsRNA according to any one of Items 7 to 15, which contains naturally occurring nucleotides. [Item 17] The dsRNA according to any one of Items 7 to 16, which contains at least one modified nucleotide. [Item 18] The dsRNA according to any one of Items 7 to 15, wherein each nucleotide is modified. [Item 19] The dsRNA described in Item 17, wherein the at least one modified nucleotide is selected from the group consisting of a 2'-O-methyl modified nucleotide, a nucleotide containing a 5'-phosphorothioate group, a terminal nucleotide linked to a cholesteryl derivative, and a terminal nucleotide linked to a dodecanoic acid bisdecylamide group. [Item 20] The dsRNA according to Item 17, wherein the modified nucleotide is selected from the group consisting of 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates and nucleotides containing unnatural bases. [Item 21] The dsRNA according to any one of Items 7 to 20, comprising at least one 2'-O-methyl modified nucleotide and a 5'-phosphorothioate group. [Item 22] The dsRNA according to any one of Items 7 to 21, wherein the first strand contains at least three 2'-O-methyl modified nucleotides. [Item 23] The dsRNA of item 22, wherein the first strand comprises 2'-O-methyl modified nucleotides on either side of the SNP position nucleotide. [Item 24] The dsRNA according to any one of Items 7 to 23, which comprises a hydrophobic portion. [Item 25] The dsRNA according to any one of Items 7 to 24, wherein the region of the gene containing the allelic polymorphism comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 10. [Item 26] The dsRNA according to any one of Items 7 to 25, wherein the first strand comprises UUCUGUAGCAUCAGCUUCUC. [Item 27] ​​A double-stranded RNA (dsRNA), a first strand of about 15-35 nucleotides complementary to a region of the gene containing the allelic variant; and a second strand of about 15-35 nucleotides complementary to at least a portion of said first strand; Including, the first strand comprises a single nucleotide polymorphism (SNP) position nucleotide at position 4 from the 5' end that is complementary to the allelic polymorphism; The first strand is a double-stranded RNA (dsRNA) that includes a mismatch (MM) position nucleotide located at position 7 from the 5' end that is mismatched with a nucleotide in the gene. [Section 28] Formula: [C2] JPEG0007618229000042.jpg28160 28. The dsRNA of claim 27, further comprising at least one vinyl phosphonate modification at an intersubunit linkage having the formula: [Item 29] The bifurcated oligonucleotide compound according to Item 28, wherein a VP motif is inserted adjacent to the SNP position nucleotide or adjacent to the MM position nucleotide. [Item 30] The dsRNA of item 27, wherein the first strand comprises 2'-O-methyl modified nucleotides on either side of the SNP position nucleotide. [Item 31] The dsRNA of Item 30, wherein the first strand contains at least three 2'-O-methyl modified nucleotides. [Item 32] The dsRNA according to Item 31, which contains a 5'-phosphorothioate group. [Item 33] The dsRNA according to Item 27, wherein the gene containing an allelic polymorphism is the huntingtin (htt) gene. [Item 34] The dsRNA according to Item 27, wherein the region of the gene containing the allelic polymorphism comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 10. [Item 35] The dsRNA of Item 27, wherein the first strand comprises UUCUGUAGCAUCAGCUUCUC. [Item 36] A double-stranded RNA (dsRNA), a first strand of about 15-35 nucleotides complementary to a region of the gene containing the allelic variant; and a second strand of about 15-35 nucleotides complementary to at least a portion of said first strand; Including, the first strand comprises a single nucleotide polymorphism (SNP) position nucleotide at position 6 from the 5' end that is complementary to the allelic polymorphism; The first strand is a double-stranded RNA (dsRNA) that includes a mismatch (MM) position nucleotide located at position 11 from the 5' end that is mismatched with a nucleotide in the gene. [Section 37] Formula: [C3] JPEG0007618229000043.jpg28160 37. The dsRNA of claim 36, further comprising at least one vinyl phosphonate modification at an intersubunit linkage having the formula: [Item 38] The bifurcated oligonucleotide compound according to Item 37, wherein a VP motif is inserted adjacent to the SNP position nucleotide or adjacent to the MM position nucleotide. [Item 39] The dsRNA of item 36, wherein the first strand comprises 2'-O-methyl modified nucleotides on either side of the SNP position nucleotide. [Item 40] The dsRNA of Item 39, wherein the first strand comprises at least three 2'-O-methyl modified nucleotides. [Item 41] The dsRNA of Item 36, which contains a 5'-phosphorothioate group. [Item 42] The dsRNA according to Item 36, wherein the gene containing an allelic polymorphism is the huntingtin (htt) gene. [Item 43] The dsRNA according to Item 36, wherein the region of the gene containing the allelic polymorphism comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 10. [Item 44] The dsRNA of Item 36, wherein the first strand comprises UUCUGUAGCAUCAGCUUCUC. [Item 45] A pharmaceutical composition comprising the RNA according to any one of items 1 to 44 and a pharma- ceutically acceptable carrier. [Item 46] A method for inhibiting expression of a gene containing an allelic polymorphism in a cell, the method comprising contacting the cell with the RNA according to any one of Items 1 to 44. [Item 47] A method for treating a disease or disorder characterized or caused by a gene comprising an allelic polymorphism in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of RNA having a 5' end, a 3' end and a seed region that are complementary to regions of the gene comprising an allelic polymorphism, wherein the RNA comprises: a single nucleotide polymorphism (SNP) position nucleotide at a position within the seed region that is complementary to the allelic polymorphism; and a mismatch (MM) position nucleotide located 2 to 11 nucleotides from the SNP position nucleotide that is mismatched with a nucleotide in the gene; A method comprising: [Item 48] The RNA has the formula: [C4] JPEG0007618229000044.jpg28160 48. The method of claim 47, further comprising at least one vinyl phosphonate modification at an intersubunit linkage having the formula: [Item 49] The bifurcated oligonucleotide compound according to Item 48, wherein a VP motif is inserted adjacent to the SNP position nucleotide or adjacent to the MM position nucleotide. [Item 50] The method described in Item 47, wherein the RNA is ASO or dsRNA. [Item 51] The dsRNA is a first strand of about 15-35 nucleotides complementary to said region of said gene containing an allelic variant; and a second strand of about 15-35 nucleotides complementary to at least a portion of said first strand; Including, the first strand comprises the SNP position nucleotide at positions 2 to 6 from the 5' end that is complementary to the allelic polymorphism; 48. The method of claim 47, wherein the first strand comprises the MM position nucleotide located 2 to 6 nucleotides from the SNP position nucleotide that is mismatched to a nucleotide in the gene. [Item 52] The method of item 51, wherein the dsRNA is administered to the brain of the subject. [Item 53] The method of item 51, wherein the dsRNA is administered by intrastriatal injection. [Item 54] The method according to Item 52, wherein a reduction in expression of the gene in the striatum is achieved. [Item 55] The method according to Item 52, wherein a reduction in expression of the gene in the cortex is achieved. [Item 56] The method according to any one of Items 47 to 55, wherein the gene containing an allelic polymorphism is the huntingtin (htt) gene. [Item 57] The method according to any one of Items 47 to 56, wherein the disease is Huntington's disease. [Item 58] The RNA according to Item 1, wherein the SNP position nucleotide is located at position 2, 4 or 6 from the 5' end of the RNA, and the MM position nucleotide is located 2 to 6 nucleotides from the SNP position nucleotide. [Item 59] A biantennary oligonucleotide compound comprising two RNAs, the RNAs being linked to each other by one or more moieties selected from a linker, a spacer, and a branch point, each RNA having a 5' end, a 3' end, and a seed region, each RNA being complementary to a region of a gene comprising an allelic polymorphism, each RNA comprising: a single nucleotide polymorphism (SNP) position nucleotide at a position within the seed region that is complementary to the allelic polymorphism; and a mismatch (MM) position nucleotide located 2 to 11 nucleotides from the SNP position nucleotide that is mismatched with a nucleotide in the gene; 1. A biantennary oligonucleotide compound comprising: [Item 60] The RNA has the formula: [5] JPEG0007618229000045.jpg31160 60. The biantennary oligonucleotide compound according to claim 59, further comprising at least one vinyl phosphonate modification at the intersubunit linkage having the formula: [Item 61] The bifurcated oligonucleotide compound according to Item 60, wherein a VP motif is inserted adjacent to the SNP position nucleotide or adjacent to the MM position nucleotide. [Item 62] The bifurcated oligonucleotide according to Item 59, having an hsi-RNA structure. [Item 63] The bifurcated oligonucleotide compound according to Item 59, wherein the SNP position nucleotide is complementary to an allelic polymorphism of an htt SNP selected from the group consisting of rs363125, rs362273, rs362307, rs362336, rs362331, rs362272, rs362306, rs362268, rs362267, and rs363099. [Item 64] The RNA of item 1, wherein the SNP position nucleotide is complementary to an allelic polymorphism of an htt SNP selected from the group consisting of rs363125, rs362273, rs362307, rs362336, rs362331, rs362272, rs362306, rs362268, rs362267, and rs363099. [Item 65] The RNA of item 1, further comprising a 5' stabilizing moiety selected from the group consisting of phosphate, vinyl phosphonate, C5-methyl (R, or S, or racemic), C5-methyl on vinyl, and reduced vinyl. [Item 66] The RNA of item 1, further comprising a conjugate moiety selected from the group consisting of alkyl chains, vitamins, peptides, sphingoglycolipids, polyunsaturated fatty acids, secosteroids, steroid hormones, and steroid lipids. [Item 67] A nucleic acid having a 5' end, a 3' end and a seed region that is complementary to a region of a gene containing an allelic polymorphism, a single nucleotide polymorphism (SNP) position nucleotide at a position within the seed region, the SNP position nucleotide being complementary to the allelic polymorphism; A mismatch (MM) position that is a mismatch with a nucleotide in the gene; and at least one modified nucleotide (X) on either side of the SNP position nucleotide, each X being located within 4, 3, or 2 nucleotides of the SNP position nucleotide; A nucleic acid comprising: [Section 68] Formula: [6] JPEG0007618229000046.jpg28160 68. The nucleic acid of claim 67, further comprising at least one vinyl phosphonate modification at an intersubunit linkage having the formula: [Item 69] The bifurcated oligonucleotide compound according to Item 68, wherein a VP motif is inserted adjacent to the SNP position nucleotide or adjacent to the MM position nucleotide. [Item 70] A nucleic acid having a 5' end, a 3' end and a seed region that is complementary to a region of a gene containing an allelic polymorphism, a single nucleotide polymorphism (SNP) position nucleotide at a position within the seed region, the SNP position nucleotide being complementary to the allelic polymorphism; A mismatch (MM) position that is a mismatch with a nucleotide in the gene; and at least one modified nucleotide (X) on either side of the SNP position nucleotide, each X being located within 4, 3, or 2 nucleotides of the SNP position nucleotide; A nucleic acid comprising: [Section 71] Formula: [7] JPEG0007618229000047.jpg28160 71. The nucleic acid of claim 70, further comprising at least one vinyl phosphonate modification at an intersubunit linkage having the formula: [Item 72] The bifurcated oligonucleotide compound according to Item 71, wherein a VP motif is inserted adjacent to the SNP position nucleotide or adjacent to the MM position nucleotide. [Item 73] A nucleic acid having a 5' end, a 3' end and a seed region that is complementary to a region of a gene containing an allelic polymorphism, a single nucleotide polymorphism (SNP) position nucleotide at a position within the seed region, the SNP position nucleotide being complementary to the allelic polymorphism; A mismatch (MM) position that is a mismatch with a nucleotide in the gene; and at least one modified nucleotide (Y) on either side of the MM position nucleotide, each Y being located within 4, 3, or 2 nucleotides of the MM position nucleotide; A nucleic acid comprising: [Section 74] Formula: [8] JPEG0007618229000048.jpg29160 74. The nucleic acid of claim 73, further comprising at least one vinyl phosphonate modification at an intersubunit linkage having the formula: [Item 75] The bifurcated oligonucleotide compound according to Item 74, wherein a VP motif is inserted adjacent to the SNP position nucleotide or adjacent to the MM position nucleotide. [Item 76] The nucleic acid of item 70, wherein X comprises a sugar modification selected from the group consisting of 2'-O-methyl (2'-OMe), 2'-fluoro (2'-F), 2'-ribo, 2'-deoxyribo, 2'-F-4'-thioarabino (2'-F-ANA), 2-O-(2-methoxyethyl) (2'-MOE), 4'-S-RNA, locked nucleic acid (LNA), 4'-SF-ANA, 2'-O-allyl, 2'-O-ethylamine, 2-cyanoethyl-RNA (CNet-RNA), tricyclo-DNA, cyclohexyl nucleic acid (CeNA), arabino nucleic acid (ANA) and hexitol nucleic acid (HNA). [Item 77] The nucleic acid of Item 73, wherein Y comprises a sugar modification selected from the group consisting of 2'-OMe, 2'-F, 2'-ribo, 2'-deoxyribo, 2'-F-ANA, 2'-MOE, 4'-S-RNA, LNA, 4'-SF-ANA, 2'-O-allyl, 2'-O-ethylamine, CNet-RNA, tricyclo-DNA, CeNA, ANA and HNA. [Item 78] The nucleic acid of Item 70, wherein X is located immediately 5' to the SNP position nucleotide or immediately 3' to the SNP position nucleotide. [Item 79] The nucleic acid of Item 70, wherein X is located immediately 5' to the SNP position nucleotide and immediately 3' to the SNP position nucleotide. [Item 80] The nucleic acid of Item 73, wherein Y is located immediately 5' to the MM position nucleotide or immediately 3' to the MM position nucleotide. [Item 81] The nucleic acid of Item 73, wherein Y is located immediately 5' to the MM position nucleotide and immediately 3' to the MM position nucleotide. [Item 82] The nucleic acid according to item 70 or 73, wherein the SNP position nucleotide is located at positions 2 to 6 from the 5' end. [Item 83] The nucleic acid according to Item 70 or 73, wherein the MM position nucleotide is located 2 to 11 nucleotides from the SNP position nucleotide. [Item 84] The nucleic acid according to item 70 or 73, wherein the MM position nucleotide is located 2 to 6 nucleotides from the SNP position nucleotide. [Item 85] A nucleic acid having a 5' end, a 3' end and a seed region that is complementary to a region of a gene containing an allelic polymorphism, a single nucleotide polymorphism (SNP) position nucleotide at a position within the seed region, the SNP position nucleotide being complementary to the allelic polymorphism; A mismatch (MM) position that is a mismatch with a nucleotide in the gene; and at least one modified nucleotide (X) on either side of the SNP position nucleotide, where each X is located within 4, 3, or 2 nucleotides of the SNP position nucleotide; and at least one modified nucleotide (Y) on either side of the MM position nucleotide, each Y being located within 4, 3, or 2 nucleotides of the MM position nucleotide; A nucleic acid comprising: [Section 86] Formula: [9] JPEG0007618229000049.jpg29160 86. The nucleic acid of claim 85, further comprising at least one vinyl phosphonate modification at an intersubunit linkage having the formula: [Item 87] The bifurcated oligonucleotide compound according to Item 86, wherein a VP motif is inserted adjacent to the SNP position nucleotide or adjacent to the MM position nucleotide. [Item 88] The nucleic acid of item 85, wherein X comprises a sugar modification selected from the group consisting of 2'-OMe, 2'-F, 2'-ribo, 2'-deoxyribo, 2'-F-ANA, 2'-MOE, 4'-S-RNA, LNA, 4'-SF-ANA, 2'-O-allyl, 2'-O-ethylamine, CNet-RNA, tricyclo-DNA, CeNA, ANA, and HNA. [Item 89] The nucleic acid of Item 85, wherein Y comprises a sugar modification selected from the group consisting of 2'-OMe, 2'-F, 2'-ribo, 2'-deoxyribo, 2'-F-ANA, 2'-MOE, 4'-S-RNA, LNA, 4'-SF-ANA, 2'-O-allyl, 2'-O-ethylamine, CNet-RNA, tricyclo-DNA, CeNA, ANA and HNA. [Item 90] The nucleic acid of Item 85, wherein X is located immediately 5' to the SNP position nucleotide or immediately 3' to the SNP position nucleotide. [Item 91] The nucleic acid of Item 85, wherein X is located immediately 5' to the SNP position nucleotide and immediately 3' to the SNP position nucleotide. [Item 92] The nucleic acid of Item 85, wherein Y is located immediately 5' to the MM position nucleotide or immediately 3' to the MM position nucleotide. [Item 93] The nucleic acid of Item 85, wherein Y is located immediately 5' to the MM position nucleotide and immediately 3' to the MM position nucleotide. [Item 94] The nucleic acid according to Item 85, wherein the SNP position nucleotide is located at positions 2 to 6 from the 5' end. [Item 95] The nucleic acid according to Item 85, wherein the MM position nucleotide is located 2 to 11 nucleotides from the SNP position nucleotide. [Item 96] The nucleic acid according to Item 85, wherein the MM position nucleotide is located 2 to 6 nucleotides from the SNP position nucleotide. [Item 97] The nucleic acid of any one of Items 70, 73, or 85, wherein X and Y are the same. [Item 98] The nucleic acid of any one of Items 70, 73, or 85, wherein X and Y are different. [Item 99] A bifurcated oligonucleotide compound comprising two or more nucleic acid sequences, wherein the nucleic acid sequences (N) are linked to each other by one or more moieties selected from a linker (L), a spacer (S) and optionally a branch point (B); each nucleic acid sequence is double-stranded and comprises a sense strand and an antisense strand, the sense strand and the antisense strand having a 5' end and a 3' end, respectively, the sense strand and the antisense strand each comprising one or more chemically modified nucleotides; Each antisense strand has a seed region, each antisense strand is complementary to a region of the gene that contains an allelic variant, and each antisense strand comprises: a single nucleotide polymorphism (SNP) position nucleotide at a position within the seed region that is complementary to the allelic polymorphism; and a mismatch (MM) position nucleotide located 2 to 11 nucleotides from the SNP position nucleotide that is mismatched with a nucleotide in the gene; 1. A biantennary oligonucleotide compound comprising: [Item 100] The RNA has the formula: [C10] JPEG0007618229000050.jpg28160 100. The biantennary oligonucleotide compound according to claim 99, further comprising at least one vinyl phosphonate modification at the intersubunit linkage having the formula: [Item 101] The bifurcated oligonucleotide compound according to Item 100, wherein a VP motif is inserted adjacent to the SNP position nucleotide or adjacent to the MM position nucleotide. [Item 102] The biantennary oligonucleotide compound according to Item 99, wherein the sense strand and the antisense strand each contain >80% chemically modified nucleotides. [Item 103] The bifurcated oligonucleotide compound of item 99, wherein the nucleotides at positions 1 and 2 from the 5' end of the sense and antisense strands are linked to adjacent nucleotides via phosphorothioate bonds. [Item 104] The bifurcated oligonucleotide compound according to Item 99, wherein each antisense strand comprises at least 15 consecutive nucleotides, and each sense strand comprises at least 15 consecutive nucleotides and has complementarity to the antisense strand. [Item 105] The bifurcated oligonucleotide compound according to Item 99, further comprising a hydrophobic moiety attached to the terminal 5' position of the branched oligonucleotide compound. [Item 106] The biantennary oligonucleotide compound of Item 99, wherein each double-stranded nucleic acid sequence is independently linked to a linker, spacer or branch point at the 3' end or the 5' end of the sense strand or the antisense strand. [Item 107] The bifurcated oligonucleotide compound according to Item 99, wherein the SNP position nucleotide is complementary to an allelic polymorphism of an htt SNP selected from the group consisting of rs363125, rs362273, rs362307, rs362336, rs362331, rs362272, rs362306, rs362268, rs362267, and rs363099.

Claims

1. A biantennary oligonucleotide compound comprising two RNAs, said RNAs linked to each other by one or more moieties selected from a linker, a spacer and a branch point, each RNA having a 5' end, a 3' end and a seed region located 2-7 nucleotides from the 5' end of said RNA, each RNA being complementary to a region of a gene comprising an allelic polymorphism, each RNA comprising: a single nucleotide polymorphism (SNP) position nucleotide at a position within the seed region that is complementary to the allelic polymorphism; and a mismatch (MM) position nucleotide located 2-11 nucleotides from the SNP position nucleotide that is mismatched with a nucleotide in the gene; 1. A biantennary oligonucleotide compound comprising:

2. The RNA is linked with nucleotides from the RNA and has the formula: 【Chemistry 1】 and further comprising at least one vinyl phosphonate modification at the intersubunit linkage having the formula: The bifurcated oligonucleotide compound according to claim 1.

3. The biantennary oligonucleotide of claim 1, having an hsi-RNA structure.

4. 2. The bifurcated oligonucleotide compound of claim 1, wherein the SNP position nucleotide is complementary to an allelic polymorphism of an http SNP selected from the group consisting of rs363125, rs362273, rs362307, rs362336, rs362331, rs362272, rs362306, rs362268, rs362267, and rs363099.

5. A biantennary oligonucleotide compound comprising two or more double stranded RNAs (dsRNAs), said dsRNAs being linked together by one or more moieties selected from a linker (L), a spacer (S) and a branch point (B); Each dsRNA comprises a sense strand and an antisense strand, the sense strand and the antisense strand each having a 5' end and a 3' end, the sense strand and the antisense strand each comprising one or more chemically modified nucleotides; Each antisense strand has a seed region located 2-7 nucleotides from the 5' end of the antisense strand, each antisense strand is complementary to a region of a gene that contains an allelic polymorphism, and each antisense strand comprises: a single nucleotide polymorphism (SNP) position nucleotide at a position within the seed region that is complementary to the allelic polymorphism; and a mismatch (MM) position nucleotide located 2-11 nucleotides from the SNP position nucleotide that is mismatched with a nucleotide in the gene; 1. A biantennary oligonucleotide compound comprising:

6. The dsRNA links nucleotides from the dsRNA and has the formula: 【Chemistry 2】 and further comprising at least one vinyl phosphonate modification at the intersubunit linkage having the formula: The bifurcated oligonucleotide compound according to claim 5 .

7. the sense strand and the antisense strand each contain >80% chemically modified nucleotides; The bifurcated oligonucleotide compound according to claim 5 .

8. 3. The bifurcated oligonucleotide compound of claim 2, wherein a vinylphosphonate motif is inserted next to the SNP position nucleotide or next to the MM position nucleotide.

9. 7. The bifurcated oligonucleotide compound of claim 6, wherein a vinylphosphonate motif is inserted next to the SNP position nucleotide or next to the MM position nucleotide.

10. 6. The biantennary oligonucleotide compound of claim 5, wherein the nucleotides at positions 1 and 2 from the 5' end of the sense and antisense strands are linked to adjacent nucleotides via phosphorothioate bonds.

11. 6. The biantennary oligonucleotide compound of claim 5, wherein each antisense strand comprises at least 15 consecutive nucleotides, and each sense strand comprises at least 15 consecutive nucleotides and has complementarity to the antisense strand.

12. 6. The bifurcated oligonucleotide compound of claim 5, further comprising a hydrophobic moiety attached to the terminal 5' position of the branched oligonucleotide compound.

13. 6. The biantennary oligonucleotide compound of claim 5, wherein each double-stranded nucleic acid sequence is independently linked to a linker, spacer or branch point at the 3'-end or the 5'-end of the sense strand or the antisense strand.

14. 6. The bifurcated oligonucleotide compound of claim 5, wherein the SNP position nucleotide is complementary to an allelic polymorphism of an http SNP selected from the group consisting of rs363125, rs362273, rs362307, rs362336, rs362331, rs362272, rs362306, rs362268, rs362267, and rs363099.

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