Oligonucleotides for modulation of the IFN-γ signaling pathway

Abstract

The present disclosure relates to novel IFN-γ signaling pathway target gene targeting sequences. Also provided are novel IFNGR1, JAK1, JAK2, and STAT1 targeting oligonucleotides for the treatment of vitiligo.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 213,506, filed June 22, 2021, and U.S. Provisional Patent Application No. 63 / 331,563, filed April 15, 2022, the disclosures of which are incorporated by reference in their entireties herein.

[0002] The present disclosure relates to novel IFN-γ signaling pathway target gene targeting sequences, novel branched oligonucleotides, and novel methods for treating and preventing IFN-γ associated vitiligo. [Background technology]

[0003] Vitiligo is caused by the CD8 + Vitiligo is an autoimmune skin disease mediated by cytotoxic T cells that causes white spots on affected skin areas. IFN-γ signaling is involved in the pathogenesis of vitiligo. Specifically, autoimmunity activates IFN-γ signaling in epidermal keratinocytes via the JAK-STAT pathway, inducing the expression of chemotactic factors CXCL9 and CXCL10, which in turn upregulate CD8, resulting in skin depigmentation. + Promotes further infiltration of cytotoxic T cells.

[0004] Currently, there are no drugs approved by the U.S. Food and Drug Administration for the treatment of vitiligo. Off-label treatments, including phototherapy, topical steroids, and small molecule drugs, often require time-consuming repeated administration and may be associated with long-term safety issues due to high drug exposure. Recent advances in the understanding of the pathogenic role of IFN-γ signaling in vitiligo have led to small molecule JAK inhibitor treatments with acceptable efficacy and substantial improvement in patients' quality of life. However, these JAK inhibitors are "pan-JAK inhibitors" that block multiple cytokine receptor signaling dependent on subtypes JAK1, JAK2, JAK3, and Tyk2. Thus, targeted treatments against IFN-γ signaling with long-lasting efficacy and improved selectivity remain elusive.

[0005] Therefore, to treat vitiligo and related disorders, there is a need to reduce the expression of proteins involved in IFN-γ signaling. Summary of the Invention

[0006] In one aspect, the disclosure provides an oligonucleotide targeting an IFN-γ signaling pathway target gene selected from the group consisting of IFNGR1, JAK1, JAK2, or STAT1, comprising a sequence substantially complementary to any one of SEQ ID NOs:1-96.

[0007] In one aspect, the disclosure provides an oligonucleotide targeting an IFN-γ signaling pathway target gene selected from the group consisting of IFNGR1, JAK1, JAK2, or STAT1, comprising a sequence substantially complementary to any one of SEQ ID NOs:1-6.

[0008] In one aspect, the present disclosure provides an RNA molecule comprising a sequence substantially complementary to the nucleic acid sequence of any one of SEQ ID NOs: 1-96.

[0009] In certain embodiments, the RNA molecule is from about 8 nucleotides to about 80 nucleotides in length (e.g., 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39 nucleotides, 40 nucleotides, 41 nucleotides, 42 nucleotides, nucleotide, 43 nucleotide, 44 nucleotide, 45 nucleotide, 46 nucleotide, 47 nucleotide, 48 nucleotide, 49 nucleotide, 50 nucleotide, 51 nucleotide, 52 nucleotide, 53 nucleotide, 54 nucleotide, 55 nucleotide, 56 nucleotide, 57 nucleotide, 58 nucleotide, 59 nucleotide, 60 nucleotide, 61 nucleotide, 62 nucleotide, 63 nucleotide, 64 nucleotide, 65 nucleotide, 66 nucleotide, 67 nucleotide, 68 nucleotide, 69 nucleotide, 70 nucleotide, 71 nucleotide, 72 nucleotide, 73 nucleotide, 74 nucleotide, 75 nucleotide, 76 nucleotide, 77 nucleotide, 78 nucleotide, 79 nucleotide, or 80 nucleotide).

[0010] In certain embodiments, the RNA molecule is 10 to 50 nucleotides in length (e.g., 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39 nucleotides, 40 nucleotides, 41 nucleotides, 42 nucleotides, 43 nucleotides, 44 nucleotides, 45 nucleotides, 46 nucleotides, 47 nucleotides, 48 ​​nucleotides, 49 nucleotides, or 50 nucleotides in length).

[0011] In certain embodiments, the RNA molecule comprises from about 15 nucleotides to about 25 nucleotides in length. In certain embodiments, the RNA molecule is 15 to 25 nucleotides in length (e.g., 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, or 25 nucleotides in length).

[0012] In certain embodiments, the RNA molecule has a nucleic acid sequence substantially complementary to the nucleic acid sequence of any one of SEQ ID NOs: 143-244.

[0013] In certain embodiments, the RNA molecule has a nucleic acid sequence that is at least 85% identical to any one of the sequences listed in Tables 10-15 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences listed in Tables 10-15). In certain embodiments, the RNA molecule has a nucleic acid sequence that is at least 90% identical to any one of the sequences listed in Tables 10-15 (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences listed in Tables 10-15). In certain embodiments, the RNA molecule has a nucleic acid sequence that is at least 95% identical to any one of the sequences set forth in Tables 10-15 (e.g., 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences set forth in Tables 10-15). In certain embodiments, the RNA molecule has a nucleic acid sequence that is any one of the sequences set forth in Tables 10-15.

[0014] In certain embodiments, the RNA molecule comprises single-stranded (ss) RNA or double-stranded (ds) RNA.

[0015] In certain embodiments, the RNA molecule is a dsRNA comprising a sense strand and an antisense strand. The antisense strand may comprise a nucleic acid sequence substantially complementary to any one of the nucleic acid sequences of SEQ ID NOs: 1 to 6. For example, in certain embodiments, the antisense sequence is substantially complementary to the nucleic acid sequence of SEQ ID NO: 1. In certain embodiments, the antisense sequence is substantially complementary to the nucleic acid sequence of SEQ ID NO: 2. In certain embodiments, the antisense sequence is substantially complementary to the nucleic acid sequence of SEQ ID NO: 3. In certain embodiments, the antisense sequence is substantially complementary to the nucleic acid sequence of SEQ ID NO: 4. In certain embodiments, the antisense sequence is substantially complementary to the nucleic acid sequence of SEQ ID NO: 5. In certain embodiments, the antisense sequence is substantially complementary to the nucleic acid sequence of SEQ ID NO: 6.

[0016] In certain embodiments, the dsRNA comprises an antisense strand having complementarity to at least 10, 11, 12, or 13 contiguous nucleotides of any one of the nucleic acid sequences of SEQ ID NOs: 1-6. For example, in certain embodiments, the dsRNA comprises an antisense strand having complementarity to a segment of 10-25 contiguous nucleotides of any one of the nucleic acid sequences of SEQ ID NOs: 1-6 (e.g., a segment of 10-25 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 1, a segment of 10-25 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 2, a segment of 10-25 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 3, a segment of 10-25 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 4, a segment of 10-25 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 5, or a segment of 10-25 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 6).

[0017] In certain embodiments, the dsRNA comprises an antisense strand having complementarity to a segment of 15-25 contiguous nucleotides of the nucleic acid sequence of any one of SEQ ID NOs: 1-6. For example, the antisense strand may have complementarity to a segment of 15 contiguous nucleotides, 16 contiguous nucleotides, 17 contiguous nucleotides, 18 contiguous nucleotides, 19 contiguous nucleotides, 20 contiguous nucleotides, 21 contiguous nucleotides, 22 contiguous nucleotides, 23 contiguous nucleotides, 24 contiguous nucleotides, or 25 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 1. In certain embodiments, the antisense strand has complementarity to a segment of 15 contiguous nucleotides, 16 contiguous nucleotides, 17 contiguous nucleotides, 18 contiguous nucleotides, 19 contiguous nucleotides, 20 contiguous nucleotides, 21 contiguous nucleotides, 22 contiguous nucleotides, 23 contiguous nucleotides, 24 contiguous nucleotides, or 25 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 2. In certain embodiments, the antisense strand has complementarity to a segment of 15 contiguous nucleotides, 16 contiguous nucleotides, 17 contiguous nucleotides, 18 contiguous nucleotides, 19 contiguous nucleotides, 20 contiguous nucleotides, 21 contiguous nucleotides, 22 contiguous nucleotides, 23 contiguous nucleotides, 24 contiguous nucleotides, or 25 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 3. In certain embodiments, the antisense strand has complementarity to a segment of 15 contiguous nucleotides, 16 contiguous nucleotides, 17 contiguous nucleotides, 18 contiguous nucleotides, 19 contiguous nucleotides, 20 contiguous nucleotides, 21 contiguous nucleotides, 22 contiguous nucleotides, 23 contiguous nucleotides, 24 contiguous nucleotides, or 25 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 4. In certain embodiments, the antisense strand has complementarity to a segment of 15 contiguous nucleotides, 16 contiguous nucleotides, 17 contiguous nucleotides, 18 contiguous nucleotides, 19 contiguous nucleotides, 20 contiguous nucleotides, 21 contiguous nucleotides, 22 contiguous nucleotides, 23 contiguous nucleotides, 24 contiguous nucleotides, or 25 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:5.In certain embodiments, the antisense strand has complementarity to a segment of 15 contiguous nucleotides, 16 contiguous nucleotides, 17 contiguous nucleotides, 18 contiguous nucleotides, 19 contiguous nucleotides, 20 contiguous nucleotides, 21 contiguous nucleotides, 22 contiguous nucleotides, 23 contiguous nucleotides, 24 contiguous nucleotides, or 25 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:6.

[0018] In certain embodiments, the dsRNA comprises an antisense strand having three or less mismatches with any one of the nucleic acid sequences of SEQ ID NO: 1 to 6. For example, the antisense strand has 0 to 3 mismatches (e.g., 0 mismatches, 1 mismatch, 2 mismatches, or 3 mismatches) with the nucleic acid sequence of SEQ ID NO: 1. In certain embodiments, the antisense strand has 0 to 3 mismatches (e.g., 0 mismatches, 1 mismatch, 2 mismatches, or 3 mismatches) with the nucleic acid sequence of SEQ ID NO: 2. In certain embodiments, the antisense strand has 0 to 3 mismatches (e.g., 0 mismatches, 1 mismatch, 2 mismatches, or 3 mismatches) with the nucleic acid sequence of SEQ ID NO: 3. In certain embodiments, the antisense strand has 0 to 3 mismatches (e.g., 0 mismatches, 1 mismatch, 2 mismatches, or 3 mismatches) with the nucleic acid sequence of SEQ ID NO: 4. In certain embodiments, the antisense strand has 0 to 3 mismatches (e.g., 0 mismatches, 1 mismatch, 2 mismatches, or 3 mismatches) with the nucleic acid sequence of SEQ ID NO: 5. In certain embodiments, the antisense strand has 0 to 3 mismatches (e.g., 0 mismatches, 1 mismatch, 2 mismatches, or 3 mismatches) with the nucleic acid sequence of SEQ ID NO: 6.

[0019] In certain embodiments, the dsRNA comprises an antisense strand that is completely complementary to the nucleic acid sequence of any one of SEQ ID NOs:1-6.

[0020] In certain embodiments, the dsRNA molecule has an antisense strand that is at least 85% identical to the nucleic acid sequence of any one of SEQ ID NOs: 1-6 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence of any one of SEQ ID NOs: 1-6). In certain embodiments, the dsRNA molecule has an antisense strand that is at least 90% identical to the nucleic acid sequence of any one of SEQ ID NOs: 1-6 (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence of any one of SEQ ID NOs: 1-6). In certain embodiments, the dsRNA molecule has an antisense strand that is at least 95% identical to the nucleic acid sequence of any one of SEQ ID NOs: 1-6 (e.g., 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence of any one of SEQ ID NOs: 1-6). In certain embodiments, the dsRNA comprises an antisense strand that has the nucleic acid sequence of any one of SEQ ID NOs: 1-6.

[0021] In certain embodiments, the antisense strand and / or the sense strand comprises between about 15 and 25 nucleotides in length, for example, in certain embodiments, the antisense strand and / or the sense strand is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length.

[0022] In certain embodiments, the antisense strand is 20 nucleotides long. In certain embodiments, the antisense strand is 21 nucleotides long. In certain embodiments, the antisense strand is 22 nucleotides long. In certain embodiments, the sense strand is 15 nucleotides long. In certain embodiments, the sense strand is 16 nucleotides long. In certain embodiments, the sense strand is 18 nucleotides long. In certain embodiments, the sense strand is 20 nucleotides long.

[0023] In certain embodiments, the antisense strand is 20 nucleotides in length and the sense strand is 15 or 16 nucleotides in length.

[0024] In certain embodiments, the antisense strand is 21 nucleotides in length and the sense strand is 15 or 16 nucleotides in length.

[0025] In certain embodiments, the antisense strand is 20 or 21 nucleotides in length and the sense strand is 15 nucleotides in length.

[0026] In certain embodiments, the antisense strand is 20 or 21 nucleotides in length and the sense strand is 16 nucleotides in length.

[0027] In certain embodiments, the antisense strand is 20 nucleotides in length and the sense strand is 15 nucleotides in length.

[0028] In certain embodiments, the antisense strand is 21 nucleotides in length and the sense strand is 16 nucleotides in length.

[0029] In certain embodiments, the dsRNA comprises a double-stranded region of 15 to 20 base pairs (e.g., 15, 16, 17, 18, 19, or 20 base pairs). In certain embodiments, the dsRNA comprises a double-stranded region of 15 base pairs. In certain embodiments, the dsRNA comprises a double-stranded region of 16 base pairs. In certain embodiments, the dsRNA comprises a double-stranded region of 18 base pairs. In some embodiments, the dsRNA comprises a double-stranded region of 20 base pairs.

[0030] In certain embodiments, the dsRNA comprises a blunt end. In certain embodiments, the dsRNA comprises at least one single-stranded nucleotide overhang. In certain embodiments, the dsRNA comprises a single-stranded nucleotide overhang of about 2 to 5 nucleotides.

[0031] In certain embodiments, the dsRNA comprises naturally occurring nucleotides.

[0032] In certain embodiments, the dsRNA comprises at least one modified nucleotide.

[0033] In certain embodiments, the modified nucleotides include 2'-O-methyl modified nucleotides, 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, non-natural base containing nucleotides, or mixtures thereof.

[0034] In certain embodiments, the dsRNA comprises at least one modified internucleotide linkage.

[0035] In certain embodiments, the modified internucleotide linkage comprises a phosphorothioate internucleotide linkage. In certain embodiments, the dsRNA comprises 4-16 phosphorothioate internucleotide linkages (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphorothioate linkages). In certain embodiments, the dsRNA comprises 8-13 phosphorothioate internucleotide linkages (e.g., 9, 10, 11, 12, or 13 phosphorothioate linkages).

[0036] In certain embodiments, the dsRNA comprises at least one modified internucleotide linkage of Formula I: [ka] (In the formula, B is a base pair moiety; W is selected from the group consisting of O, OCH2, OCH, CH2, and CH; X is halo, hydroxy, or C 1-6 alkoxy; Y is O - , OH, OR, NH - , NH2, S - and SH, Z is selected from the group consisting of O and CH2; R is a protecting group; [ka] is any double bond).

[0037] In certain embodiments, when W is CH, [ka] is a double bond.

[0038] In certain embodiments, when W is selected from the group consisting of O, OCH2, OCH, CH2, [ka] is a single bond.

[0039] In certain embodiments, dsRNA comprises at least 80% chemically modified nucleotides (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% chemically modified nucleotides).In certain embodiments, dsRNA is completely chemically modified. In certain embodiments, the dsRNA contains at least 70% 2'-O-methyl nucleotide modifications (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% 2'-O-methyl modifications).

[0040] In certain embodiments, the dsRNA comprises about 80% to about 90% 2'-O-methyl nucleotide modifications (e.g., about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% 2'-O-methyl nucleotide modifications). In certain embodiments, the dsRNA comprises about 83% to about 86% 2'-O-methyl modifications (e.g., about 83%, 84%, 85%, or 86% 2'-O-methyl modifications).

[0041] In certain embodiments, the dsRNA comprises about 70% to about 80% 2'-O-methyl nucleotide modifications (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80% 2'-O-methyl nucleotide modifications). In certain embodiments, the dsRNA comprises about 75% to about 78% 2'-O-methyl modifications (e.g., about 75%, 76%, 77%, or 78% 2'-O-methyl modifications).

[0042] In certain embodiments, the antisense strand comprises at least 80% chemically modified nucleotides (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% chemically modified nucleotides). In certain embodiments, the antisense strand is completely chemically modified. In certain embodiments, the antisense strand contains at least 70% 2'-O-methyl nucleotide modifications (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% 2'-O-methyl nucleotide modifications). In certain embodiments, the antisense strand comprises about 70% to about 90% 2'-O-methyl nucleotide modifications (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% 2'-O-methyl modifications). In certain embodiments, the antisense strand comprises about 85% to about 90% 2'-O-methyl modifications (e.g., about 85%, 86%, 87%, 88%, 89%, or 90% 2'-O-methyl modifications).

[0043] In certain embodiments, the antisense strand comprises about 75% to about 85% 2'-O-methyl nucleotide modifications (e.g., about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, or 85% 2'-O-methyl modifications). In certain embodiments, the antisense strand comprises about 76% to about 80% 2'-O-methyl modifications (e.g., about 76%, 77%, 78%, 79%, or 80% 2'-O-methyl modifications).

[0044] In certain embodiments, the sense strand comprises at least 80% chemically modified nucleotides (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% chemically modified nucleotides). In certain embodiments, the sense strand is completely chemically modified. In certain embodiments, the sense strand comprises at least 65% 2'-O-methyl nucleotide modifications (e.g., 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% 2'-O-methyl modifications). In certain embodiments, the sense strand comprises 100% 2'-O-methyl nucleotide modifications.

[0045] In certain embodiments, the sense strand comprises about 70% to about 85% 2'-O-methyl nucleotide modifications (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, or 85% 2'-O-methyl nucleotide modifications). In certain embodiments, the sense strand comprises about 75% to about 80% 2'-O-methyl nucleotide modifications (e.g., about 75%, 76%, 77%, 78%, 79%, or 80% 2'-O-methyl nucleotide modifications).

[0046] In certain embodiments, the sense strand comprises about 65% to about 75% 2'-O-methyl nucleotide modifications (e.g., about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, or 75% 2'-O-methyl nucleotide modifications). In certain embodiments, the sense strand comprises about 67% to about 73% 2'-O-methyl nucleotide modifications (e.g., about 67%, 68%, 69%, 70%, 71%, 72%, or 73% 2'-O-methyl nucleotide modifications).

[0047] In certain embodiments, the sense strand comprises one or more nucleotide mismatches between the antisense strand and the sense strand. In certain embodiments, the one or more nucleotide mismatches are present at positions 2, 6, and 12 from the 5' end of the sense strand. In certain embodiments, the nucleotide mismatches are present at positions 2, 6, and 12 from the 5' end of the sense strand.

[0048] In certain embodiments, the antisense strand comprises a 5' phosphate, a 5'-alkyl phosphonate, a 5' alkylene phosphonate, or a 5' alkenyl phosphonate.

[0049] In certain embodiments, the antisense strand comprises a 5' vinyl phosphonate.

[0050] In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, wherein (1) the antisense strand has a nucleic acid sequence substantially complementary to the nucleic acid sequence of any one of SEQ ID NOs: 1-6; (2) the antisense strand comprises alternating 2'-methoxy-ribonucleotides and 2'-fluoro-ribonucleotides; (3) the nucleotides at positions 2 and 14 from the 5' end of the antisense strand are not 2'-methoxy-ribonucleotides; (4) the nucleotides at positions 1-2 through 1-7 from the 3' end of the antisense strand are linked to each other via phosphorothioate internucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand comprises alternating 2'-methoxy-ribonucleotides and 2'-fluoro-ribonucleotides; (7) the nucleotides at positions 1-2 from the 5' end of the sense strand are linked to each other via phosphorothioate internucleotide linkages.

[0051] In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, wherein (1) the antisense strand has a nucleic acid sequence substantially complementary to a nucleic acid sequence of any one of SEQ ID NOs: 1-6; (2) the antisense strand contains at least 70% 2'-O-methyl modifications (e.g., about 75% to about 80% or about 85% to about 90% 2'-O-methyl modifications); and (3) the nucleotide at position 14 from the 5' end of the antisense strand is not a 2'-methoxy-ribonucleotide. (4) nucleotides 1-2 through 1-7 from the 3' end of the antisense strand are linked to each other via phosphorothioate internucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand contains at least 65% 2'-O-methyl modifications (e.g., about 65% to about 75% or about 75% to about 80% 2'-O-methyl modifications); (7) nucleotides 1-2 from the 5' end of the sense strand are linked to each other via phosphorothioate internucleotide linkages.

[0052] In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, wherein: (1) the antisense strand has a nucleic acid sequence substantially complementary to a nucleic acid sequence of any one of SEQ ID NOs: 1-6; (2) the antisense strand comprises at least 85% 2'-O-methyl modifications; (3) the nucleotides at positions 2 and 14 from the 5' end of the antisense strand are not 2'-methoxy-ribonucleotides; (4) the nucleotides at positions 1-2 through 1-7 from the 3' end of the antisense strand are linked to each other via phosphorothioate internucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand comprises 100% 2'-O-methyl modifications; and (7) the nucleotides at positions 1-2 from the 5' end of the sense strand are linked to each other via phosphorothioate internucleotide linkages.

[0053] In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, wherein: (1) the antisense strand has a nucleic acid sequence substantially complementary to a nucleic acid sequence of any one of SEQ ID NOs: 1-6; (2) the antisense strand comprises at least 75% 2'-O-methyl modifications; (3) the nucleotides at positions 4, 5, 6, and 14 from the 5' end of the antisense strand are not 2'-methoxy-ribonucleotides; (4) the nucleotides at positions 1-2 through 1-7 from the 3' end of the antisense strand are linked to each other via phosphorothioate internucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand comprises 100% 2'-O-methyl modifications; and (7) the nucleotides at positions 1-2 from the 5' end of the sense strand are linked to each other via phosphorothioate internucleotide linkages.

[0054] In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, wherein: (1) the antisense strand has a nucleic acid sequence substantially complementary to any one of the nucleic acid sequences of SEQ ID NOs: 1-6; (2) the antisense strand contains at least 85% 2'-O-methyl modifications (e.g., about 85% to about 90% 2'-O-methyl modifications); (3) the nucleotides at positions 2 and 14 from the 5' end of the antisense strand are not 2'-methoxy-ribonucleotides (e.g., the nucleotides at positions 2 and 14 from the 5' end of the antisense strand can be 2'-fluoronucleotides); (4) the nucleotides at positions 1-2 through 1-7 from the 3' end of the antisense strand are not 2'-methoxy-ribonucleotides (e.g., the nucleotides at positions 2 and 14 from the 5' end of the antisense strand can be 2'-fluoronucleotides); (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand contains at least 75% 2'-O-methyl modifications (e.g., about 75% to about 80% 2'-O-methyl modifications); (7) the nucleotides at positions 7, 10, and 11 from the 3' end of the sense strand are not 2'-methoxyribonucleotides (e.g., the nucleotides at positions 7, 10, and 11 from the 3' end of the sense strand are 2'-fluoronucleotides); (8) the nucleotides at positions 1 and 2 from the 5' end of the sense strand are linked to each other via phosphorothioate internucleotide linkages.

[0055] In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, wherein: (1) the antisense strand has a nucleic acid sequence substantially complementary to a nucleic acid sequence of any one of SEQ ID NOs: 1-6; (2) the antisense strand contains at least 75% 2'-O-methyl modifications (e.g., about 75% to about 80% 2'-O-methyl modifications); (3) the nucleotides at positions 2, 4, 5, 6, and 14 from the 5' end of the antisense strand are not 2'-methoxy-ribonucleotides (e.g., the antisense strand is not 2'-methoxy-ribonucleotides). (4) nucleotides at positions 1-2 through 1-7 from the 3' end of the antisense strand are linked to each other via phosphorothioate internucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand contains 100% 2'-O-methyl modifications; and (7) nucleotides at positions 1-2 from the 5' end of the sense strand are linked to each other via phosphorothioate internucleotide linkages.

[0056] In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, wherein: (1) the antisense strand has a nucleic acid sequence substantially complementary to a nucleic acid sequence of any one of SEQ ID NOs: 1-6; (2) the antisense strand contains at least 75% 2'-O-methyl modifications (e.g., about 75% to about 80% 2'-O-methyl modifications); (3) the nucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand are not 2'-methoxy-ribonucleotides (e.g., the nucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand can be 2'-fluoronucleotides); (4) the nucleotides at positions 1 to 2 from the 3' end of the antisense strand are not 2'-methoxy-ribonucleotides (e.g., the nucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand can be 2'-fluoronucleotides); (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand contains at least 65% 2'-O-methyl modifications (e.g., about 65% to about 75% 2'-O-methyl modifications); (7) the nucleotides at positions 7, 9, 10, and 11 from the 3' end of the sense strand are not 2'-methoxyribonucleotides (e.g., the nucleotides at positions 7, 9, 10, and 11 from the 3' end of the sense strand are 2'-fluoronucleotides); (8) the nucleotides at positions 1 and 2 from the 5' end of the sense strand are linked to each other via phosphorothioate internucleotide linkages.

[0057] In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, wherein: (1) the antisense strand comprises a sequence substantially complementary to a nucleic acid sequence of SEQ ID NOs: 1-6; (2) the antisense strand comprises at least 75% 2'-O-methyl modifications; (3) the nucleotides at positions 2, 6, and 14 from the 5' end of the antisense strand are not 2'-methoxy-ribonucleotides; (4) the nucleotides at positions 1-2 through 1-7 from the 3' end of the antisense strand are not 2'-methoxy-ribonucleotides; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand contains at least 80% 2'-O-methyl modifications; (7) the nucleotides at positions 7, 10, and 11 from the 3' end of the sense strand are not 2'-methoxyribonucleotides; and (8) the nucleotides at positions 1 and 2 from the 5' end of the sense strand are connected to each other by phosphorothioate internucleotide linkages.

[0058] In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, wherein: (1) the antisense strand has a nucleic acid sequence substantially complementary to a nucleic acid sequence of any one of SEQ ID NOs: 1-6; (2) the antisense strand contains at least 75% 2'-O-methyl modifications (e.g., about 75% to about 80% 2'-O-methyl modifications); (3) the nucleotides at positions 2, 6, 14, 16, and 20 from the 5' end of the antisense strand are not 2'-methoxy-ribonucleotides (e.g., the nucleotides at positions 2, 6, 14, 16, and 20 from the 5' end of the antisense strand can be 2'-fluoronucleotides); (4) the nucleotides at positions 1-7 and (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand contains at least 65% 2'-O-methyl modifications (e.g., about 65% to about 75% 2'-O-methyl modifications); (7) the nucleotides at positions 7, 9, 10, and 11 from the 3' end of the sense strand are not 2'-methoxy-ribonucleotides (e.g., the nucleotides at positions 7, 9, 10, and 11 from the 3' end of the sense strand are 2'-fluoronucleotides); (8) the nucleotides at positions 1-2 and 14-15 from the 5' end of the sense strand are linked to each other via phosphorothioate internucleotide linkages.

[0059] In certain embodiments, the functional moiety is linked to the 5'-end and / or 3'-end of the antisense strand. In certain embodiments, the functional moiety is linked to the 5'-end and / or 3'-end of the sense strand. In certain embodiments, the functional moiety is linked to the 3'-end of the sense strand.

[0060] In certain embodiments, the functional moiety comprises a hydrophobic moiety.

[0061] In certain embodiments, the hydrophobic moiety is selected from the group consisting of fatty acids, steroids, secosteroids, lipids, gangliosides, nucleoside analogs, endocannabinoids, vitamins, and mixtures thereof.

[0062] In certain embodiments, the steroid is selected from the group consisting of cholesterol and lithocholic acid (LCA).

[0063] In certain embodiments, the fatty acid is selected from the group consisting of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosanoic acid (DCA).

[0064] In certain embodiments, the vitamin is selected from the group consisting of choline, vitamin A, vitamin E, and derivatives and metabolites thereof.

[0065] In certain embodiments, the vitamin is selected from the group consisting of retinoic acid and alpha-tocopherol succinate.

[0066] In certain embodiments, the functional moiety is myristic acid (Myr). In certain embodiments, the functional moiety is tri-myristic acid (Myr-t).

[0067] In certain embodiments, the functional moiety is linked to the antisense strand and / or the sense strand by a linker.

[0068] In certain embodiments, the linker comprises a bivalent or trivalent linker.

[0069] In certain embodiments, the bivalent or trivalent linker is selected from the group consisting of: [ka] (wherein n is 1, 2, 3, 4, or 5).

[0070] In certain embodiments, the linker comprises an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, a carbamate, or a combination thereof.

[0071] In certain embodiments, when the linker is a trivalent linker, the linker further links a phosphodiester or phosphodiester derivative.

[0072] In certain embodiments, the phosphodiester or phosphodiester derivative is selected from the group consisting of: [ka] (wherein X is O, S or BH3).

[0073] In certain embodiments, the nucleotides 1 and 2 from the 3' end of the sense strand and the nucleotides 1 and 2 from the 5' end of the antisense strand are linked to adjacent ribonucleotides via phosphorothioate linkages.

[0074] In one aspect, the present disclosure provides a pharmaceutical composition for inhibiting expression of an IFN-γ signaling pathway target gene selected from the group consisting of IFNGR1, JAK1, JAK2, or STAT1 in an organism, comprising the above-mentioned dsRNA and a pharma- ceutically acceptable carrier.

[0075] In certain embodiments, the dsRNA inhibits expression of the gene by at least 50%. In certain embodiments, the dsRNA inhibits expression of the gene by at least 80%.

[0076] In certain embodiments, the dsRNA reduces expression of the chemokine CSCL9 by at least 20% to at least 80%.

[0077] In one aspect, the disclosure provides a method for inhibiting expression of an IFN-γ signaling pathway target gene selected from the group consisting of IFNGR1, JAK1, JAK2, or STAT1 in a cell, the method comprising: (a) introducing into the cell a double-stranded ribonucleic acid (dsRNA) as described above; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the gene, thereby inhibiting expression of the gene in the cell.

[0078] In one aspect, the present disclosure provides a method of treating vitiligo in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an oligonucleotide that comprises sufficient complementarity to an IFN-γ signaling pathway target gene, thereby treating the subject.

[0079] In certain embodiments, the IFN-γ signaling pathway target gene is selected from the group consisting of IFNGR1, JAK1, JAK2, or STAT1.

[0080] In certain embodiments, the method of treatment comprises administering a therapeutically effective amount of said dsRNA as described above.

[0081] In certain embodiments, the dsRNA is administered by intravenous (IV) injection, subcutaneous (SQ) injection, or a combination thereof.

[0082] In certain embodiments, the dsRNA inhibits expression of the gene by at least 50%. In certain embodiments, the dsRNA inhibits expression of the gene by at least 80%.

[0083] In certain embodiments, the dsRNA reduces expression of the cytokine CXCL9 by at least 20% to at least 80%.

[0084] In one aspect, the disclosure provides a vector comprising a regulatory sequence operably linked to a nucleotide sequence encoding an RNA molecule substantially complementary to a nucleic acid sequence of SEQ ID NOs: 1-6.

[0085] In certain embodiments, the RNA molecule inhibits expression of the gene of interest by at least 50%. In certain embodiments, the RNA molecule inhibits expression of the gene of interest by at least 80%.

[0086] In certain embodiments, the RNA molecule reduces expression of the cytokine CXCL9 by at least 20% to at least 80%.

[0087] In certain embodiments, the RNA molecule comprises ssRNA or dsRNA.

[0088] In certain embodiments, the dsRNA comprises a sense strand and an antisense strand, and the antisense strand comprises a sequence substantially complementary to a nucleic acid sequence of SEQ ID NOs:1-6.

[0089] In one aspect, the disclosure provides a cell comprising the above-described vector.

[0090] In one aspect, the disclosure provides a recombinant adeno-associated virus (rAAV) comprising the above vector and an AAV capsid.

[0091] In one aspect, the disclosure provides branched RNA compounds comprising two or more RNA molecules, e.g., 15-40 nucleotides in length (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length), each of which comprises a portion having a nucleic acid sequence substantially complementary to a segment of an IFN-γ signaling pathway gene mRNA selected from the group consisting of IFNGR1, JAK1, JAK2, or STAT1. The two RNA molecules may be linked to each other by one or more moieties independently selected from a linker, a spacer, and a branch point.

[0092] In certain embodiments, the branched RNA molecules comprise one or both of ssRNA and dsRNA.

[0093] In certain embodiments, the branched RNA molecule comprises an antisense oligonucleotide.

[0094] In certain embodiments, each RNA molecule comprises a dsRNA comprising a sense strand and an antisense strand, and each antisense strand independently comprises a sequence substantially complementary to the nucleic acid sequence of any one of SEQ ID NOs: 1-6.

[0095] In certain embodiments, the branched RNA compound comprises two or more copies of an RNA molecule of any of the above aspects or embodiments of the present disclosure covalently linked to each other (e.g., by a linker, spacer, or branch point).

[0096] In certain embodiments, a branched RNA compound comprises a portion of a nucleic acid sequence that is substantially complementary to a nucleic acid sequence of any one of SEQ ID NOs: 1-6. For example, a branched RNA compound can comprise two or more dsRNA molecules covalently linked to each other (e.g., by a linker, spacer, or branch point) and comprising antisense strands each having complementarity to at least 10, 11, 12, or 13 consecutive nucleotides of a nucleic acid sequence of any one of SEQ ID NOs: 1-6. For example, in certain embodiments, the dsRNA comprises an antisense strand having complementarity to a segment of 10 to 25 contiguous nucleotides of any one of the nucleic acid sequences of SEQ ID NOs: 1-6 (e.g., a segment of 10 to 25 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 1, a segment of 10 to 25 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 2, a segment of 10 to 25 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 3, a segment of 10 to 25 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 4, a segment of 10 to 25 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 5, or a segment of 10 to 25 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 6).

[0097] In certain embodiments, each dsRNA in the branched RNA compound comprises an antisense strand having complementarity to a segment of 15-25 contiguous nucleotides of the nucleic acid sequence of any one of SEQ ID NOs: 1-6. For example, the antisense strand may have complementarity to a segment of 15 contiguous nucleotides, 16 contiguous nucleotides, 17 contiguous nucleotides, 18 contiguous nucleotides, 19 contiguous nucleotides, 20 contiguous nucleotides, 21 contiguous nucleotides, 22 contiguous nucleotides, 23 contiguous nucleotides, 24 contiguous nucleotides, 25 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 1. In certain embodiments, the antisense strand has complementarity to a segment of 15 contiguous nucleotides, 16 contiguous nucleotides, 17 contiguous nucleotides, 18 contiguous nucleotides, 19 contiguous nucleotides, 20 contiguous nucleotides, 21 contiguous nucleotides, 22 contiguous nucleotides, 23 contiguous nucleotides, 24 contiguous nucleotides, 25 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 2. In certain embodiments, the antisense strand has complementarity to a segment of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 3. In certain embodiments, the antisense strand has complementarity to a segment of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO: 4. In certain embodiments, the antisense strand has complementarity to a segment of 15 contiguous nucleotides, 16 contiguous nucleotides, 17 contiguous nucleotides, 18 contiguous nucleotides, 19 contiguous nucleotides, 20 contiguous nucleotides, 21 contiguous nucleotides, 22 contiguous nucleotides, 23 contiguous nucleotides, 24 contiguous nucleotides, or 25 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:5.In certain embodiments, the antisense strand has complementarity to a segment of 15 contiguous nucleotides, 16 contiguous nucleotides, 17 contiguous nucleotides, 18 contiguous nucleotides, 19 contiguous nucleotides, 20 contiguous nucleotides, 21 contiguous nucleotides, 22 contiguous nucleotides, 23 contiguous nucleotides, 24 contiguous nucleotides, or 25 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:6.

[0098] In certain embodiments, each dsRNA in the branched RNA compound comprises an antisense strand having 3 or less mismatches with any one of the nucleic acid sequences of SEQ ID NO: 1-6. For example, the antisense strand may have 0-3 mismatches (e.g., 0 mismatches, 1 mismatch, 2 mismatches, or 3 mismatches) with the nucleic acid sequence of SEQ ID NO: 1. In certain embodiments, the antisense strand has 0-3 mismatches (e.g., 0 mismatches, 1 mismatch, 2 mismatches, or 3 mismatches) with the nucleic acid sequence of SEQ ID NO: 2. In certain embodiments, the antisense strand has 0-3 mismatches (e.g., 0 mismatches, 1 mismatch, 2 mismatches, or 3 mismatches) with the nucleic acid sequence of SEQ ID NO: 3. In certain embodiments, the antisense strand has 0-3 mismatches (e.g., 0 mismatches, 1 mismatch, 2 mismatches, or 3 mismatches) with the nucleic acid sequence of SEQ ID NO: 4. In certain embodiments, the antisense strand has 0 to 3 mismatches (e.g., 0 mismatches, 1 mismatch, 2 mismatches, or 3 mismatches) with the nucleic acid sequence of SEQ ID NO: 5. In certain embodiments, the antisense strand has 0 to 3 mismatches (e.g., 0 mismatches, 1 mismatch, 2 mismatches, or 3 mismatches) with the nucleic acid sequence of SEQ ID NO: 6.

[0099] In certain embodiments, each dsRNA in the branched RNA compound comprises an antisense strand that is perfectly complementary to the nucleic acid sequence of any one of SEQ ID NOs: 1-6.

[0100] In certain embodiments, the branched RNA compound comprises a portion having a nucleic acid sequence substantially complementary to one or more of the nucleic acid sequences of any one of SEQ ID NOs: 143-154.

[0101] In certain embodiments, the RNA molecule comprises an antisense oligonucleotide.

[0102] In certain embodiments, each RNA molecule comprises between 15 and 25 nucleotides in length.

[0103] In certain embodiments, the antisense strand and / or the sense strand comprises about 15-25 nucleotides in length. For example, in certain embodiments, the antisense strand and / or the sense strand is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In certain embodiments, the antisense strand is 20 nucleotides in length. In certain embodiments, the antisense strand is 21 nucleotides in length. In certain embodiments, the antisense strand is 22 nucleotides in length. In certain embodiments, the sense strand is 15 nucleotides in length. In certain embodiments, the sense strand is 16 nucleotides in length. In certain embodiments, the sense strand is 18 nucleotides in length. In certain embodiments, the sense strand is 20 nucleotides in length.

[0104] In certain embodiments, the antisense strand is 20 nucleotides in length and the sense strand is 15 or 16 nucleotides in length.

[0105] In certain embodiments, the antisense strand is 21 nucleotides in length and the sense strand is 15 or 16 nucleotides in length.

[0106] In certain embodiments, the antisense strand is 20 or 21 nucleotides in length and the sense strand is 15 nucleotides in length.

[0107] In certain embodiments, the antisense strand is 20 or 21 nucleotides in length and the sense strand is 16 nucleotides in length.

[0108] In certain embodiments, the antisense strand is 20 nucleotides in length and the sense strand is 15 nucleotides in length.

[0109] In certain embodiments, the antisense strand is 21 nucleotides in length and the sense strand is 16 nucleotides in length.

[0110] In certain embodiments, the dsRNA comprises a double-stranded region of 15 to 20 base pairs. In certain embodiments, the dsRNA comprises a double-stranded region of 15 base pairs. In certain embodiments, the dsRNA comprises a double-stranded region of 16 base pairs. In certain embodiments, the dsRNA comprises a double-stranded region of 18 base pairs. In certain embodiments, the dsRNA comprises a double-stranded region of 20 base pairs.

[0111] In certain embodiments, the dsRNA comprises blunt ends.

[0112] In certain embodiments, the dsRNA comprises at least one single-stranded nucleotide overhang. In certain embodiments, the dsRNA comprises a single-stranded nucleotide overhang of 2 to 5 nucleotides.

[0113] In certain embodiments, the dsRNA comprises naturally occurring nucleotides.

[0114] In certain embodiments, the dsRNA comprises at least one modified nucleotide.

[0115] In certain embodiments, the modified nucleotide comprises a 2'-O-methyl modified nucleotide, a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, or a nucleotide containing a non-natural base.

[0116] In certain embodiments, the dsRNA comprises at least one modified internucleotide linkage.

[0117] In certain embodiments, the modified internucleotide linkages comprise phosphorothioate internucleotide linkages. In certain embodiments, the branched RNA compound comprises between 4 and 16 phosphorothioate internucleotide linkages. In certain embodiments, the branched RNA compound comprises between 8 and 13 phosphorothioate internucleotide linkages.

[0118] In certain embodiments, the dsRNA comprises at least one modified internucleotide linkage of Formula I: [ka] (In the formula, B is a base pair moiety; W is selected from the group consisting of O, OCH2, OCH, CH2, and CH; X is halo, hydroxy, or C 1-6 alkoxy; Y is O - , OH, OR, NH - , NH2, S - and SH, Z is selected from the group consisting of O and CH2; R is a protecting group; [ka] is any double bond).

[0119] In certain embodiments, when W is CH, [ka] is a double bond.

[0120] In certain embodiments, when W is selected from the group consisting of O, OCH2, OCH, CH2, [ka] is a single bond.

[0121] In certain embodiments, dsRNA comprises at least 80% chemically modified nucleotides (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% chemically modified nucleotides).In certain embodiments, dsRNA is completely chemically modified. In certain embodiments, the dsRNA contains at least 70% 2'-O-methyl nucleotide modifications (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% 2'-O-methyl modifications).

[0122] In certain embodiments, the antisense strand comprises at least 80% chemically modified nucleotides.

[0123] In certain embodiments, the antisense strand is fully chemically modified.

[0124] In certain embodiments, the antisense strand comprises at least 70% 2'-O-methyl nucleotide modifications. In certain embodiments, the antisense strand comprises 70%-90% 2'-O-methyl nucleotide modifications. In certain embodiments, the antisense strand comprises about 85%-90% 2'-O-methyl modifications (e.g., about 85%, 86%, 87%, 88%, 89%, or 90% 2'-O-methyl modifications).

[0125] In certain embodiments, the antisense strand comprises about 75% to about 85% 2'-O-methyl nucleotide modifications (e.g., about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, or 85% 2'-O-methyl modifications). In certain embodiments, the antisense strand comprises about 76% to about 80% 2'-O-methyl modifications (e.g., about 76%, 77%, 78%, 79%, or 80% 2'-O-methyl modifications).

[0126] In certain embodiments, the sense strand comprises at least 80% chemically modified nucleotides. In certain embodiments, the sense strand is fully chemically modified. In certain embodiments, the sense strand comprises at least 65% 2'-O-methyl nucleotide modifications. In certain embodiments, the sense strand comprises 100% 2'-O-methyl nucleotide modifications.

[0127] In certain embodiments, the sense strand comprises one or more nucleotide mismatches between the antisense strand and the sense strand. In certain embodiments, the one or more nucleotide mismatches are present at positions 2, 6, and 12 from the 5' end of the sense strand. In certain embodiments, the nucleotide mismatches are present at positions 2, 6, and 12 from the 5' end of the sense strand.

[0128] In certain embodiments, the antisense strand comprises a 5' phosphate, a 5'-alkyl phosphonate, a 5' alkylene phosphonate, a 5' alkenyl phosphonate, or a mixture thereof.

[0129] In certain embodiments, the antisense strand comprises a 5' vinyl phosphonate.

[0130] In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, wherein (1) the antisense strand has a nucleic acid sequence substantially complementary to the nucleic acid sequence of any one of SEQ ID NOs: 1-6; (2) the antisense strand comprises alternating 2'-methoxy-ribonucleotides and 2'-fluoro-ribonucleotides; (3) the nucleotides at positions 2 and 14 from the 5' end of the antisense strand are not 2'-methoxy-ribonucleotides; (4) the nucleotides at positions 1-2 through 1-7 from the 3' end of the antisense strand are linked to each other via phosphorothioate internucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand comprises alternating 2'-methoxy-ribonucleotides and 2'-fluoro-ribonucleotides; (7) the nucleotides at positions 1-2 from the 5' end of the sense strand are linked to each other via phosphorothioate internucleotide linkages.

[0131] In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, wherein (1) the antisense strand has a nucleic acid sequence substantially complementary to a nucleic acid sequence of any one of SEQ ID NOs: 1 to 6; (2) the antisense strand contains at least 70% 2'-O-methyl modifications (e.g., about 75% to about 80%, or about 85% to about 90% 2'-O-methyl modifications); (3) the nucleotide at position 14 from the 5' end of the antisense strand is not a 2'-methoxy-ribonucleotide; (4) nucleotides 1-2 through 1-7 from the 3' end of the antisense strand are linked to each other via phosphorothioate internucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand contains at least 65% 2'-O-methyl modifications (e.g., about 65% to about 75%, or about 75% to about 80% 2'-O-methyl modifications); (7) nucleotides 1-2 from the 3' end of the antisense strand are linked to each other via phosphorothioate internucleotide linkages.

[0132] In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, wherein: (1) the antisense strand has a nucleic acid sequence substantially complementary to a nucleic acid sequence of any one of SEQ ID NOs: 1-6; (2) the antisense strand comprises at least 85% 2'-O-methyl modifications; (3) the nucleotides at positions 2 and 14 from the 5' end of the antisense strand are not 2'-methoxy-ribonucleotides; (4) the nucleotides at positions 1-2 through 1-7 from the 3' end of the antisense strand are linked to each other via phosphorothioate internucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand comprises 100% 2'-O-methyl modifications; and (7) the nucleotides at positions 1-2 from the 5' end of the sense strand are linked to each other via phosphorothioate internucleotide linkages.

[0133] In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, wherein: (1) the antisense strand has a nucleic acid sequence substantially complementary to a nucleic acid sequence of any one of SEQ ID NOs: 1-6; (2) the antisense strand comprises at least 75% 2'-O-methyl modifications; (3) the nucleotides at positions 4, 5, 6, and 14 from the 5' end of the antisense strand are not 2'-methoxy-ribonucleotides; (4) the nucleotides at positions 1-2 through 1-7 from the 3' end of the antisense strand are linked to each other via phosphorothioate internucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand comprises 100% 2'-O-methyl modifications; and (7) the nucleotides at positions 1-2 from the 5' end of the sense strand are linked to each other via phosphorothioate internucleotide linkages.

[0134] In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, wherein: (1) the antisense strand has a nucleic acid sequence substantially complementary to any one of the nucleic acid sequences of SEQ ID NOs: 1-6; (2) the antisense strand contains at least 85% 2'-O-methyl modifications (e.g., about 85% to about 90% 2'-O-methyl modifications); (3) the nucleotides at positions 2 and 14 from the 5' end of the antisense strand are not 2'-methoxy-ribonucleotides (e.g., the nucleotides at positions 2 and 14 from the 5' end of the antisense strand can be 2'-fluoronucleotides); (4) the nucleotides at positions 1-2 to 1-7 from the 3' end of the antisense strand are not 2'-methoxy-ribonucleotides (e.g., the nucleotides at positions 2 and 14 from the 5' end of the antisense strand can be 2'-fluoronucleotides); (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand contains at least 75% 2'-O-methyl modifications (e.g., about 75% to about 80% 2'-O-methyl modifications); (7) the nucleotides at positions 7, 10, and 11 from the 3' end of the sense strand are not 2'-methoxyribonucleotides (e.g., the nucleotides at positions 7, 10, and 11 from the 3' end of the sense strand are 2'-fluoronucleotides); (8) the nucleotides at positions 1 and 2 from the 5' end of the sense strand are linked to each other via phosphorothioate internucleotide linkages.

[0135] In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, wherein (1) the antisense strand has a nucleic acid sequence substantially complementary to a nucleic acid sequence of any one of SEQ ID NOs: 1-6; (2) the antisense strand contains at least 75% 2'-O-methyl modifications (e.g., about 75% to about 80% 2'-O-methyl modifications); and (3) the nucleotides at positions 2, 4, 5, 6, and 14 from the 5' end of the antisense strand are not 2'-methoxy-ribonucleotides (e.g., ...). (4) nucleotides 1-2 through 1-7 at the 3' end of the antisense strand are linked to each other via phosphorothioate internucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand contains 100% 2'-O-methyl modifications; (7) nucleotides 1-2 from the 5' end of the sense strand are linked to each other via phosphorothioate internucleotide linkages.

[0136] In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, wherein: (1) the antisense strand has a nucleic acid sequence substantially complementary to a nucleic acid sequence of any one of SEQ ID NOs: 1-6; (2) the antisense strand contains at least 75% 2'-O-methyl modifications (e.g., about 75% to about 80% 2'-O-methyl modifications); (3) the nucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand are not 2'-methoxy-ribonucleotides (e.g., the nucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand are not 2'-fluoronucleotides). (4) the nucleotides at positions 1-2 through 1-7 from the 3' end of the antisense strand are linked to each other via phosphorothioate internucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand contains at least 65% 2'-O-methyl modifications (e.g., about 65% to about 75% 2'-O-methyl modifications); (7) the nucleotides at positions 7, 9, 10, and 11 from the 3' end of the sense strand are not 2'-methoxyribonucleotides; (8) the nucleotides at positions 1-2 from the 5' end of the sense strand are linked to each other via phosphorothioate internucleotide linkages.

[0137] In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, wherein (1) the antisense strand has a nucleic acid sequence substantially complementary to a nucleic acid sequence of any one of SEQ ID NOs: 1-6; (2) the antisense strand contains at least 75% 2'-O-methyl modifications; (3) the nucleotides at positions 2, 6, and 14 from the 5' end of the antisense strand are not 2'-methoxy-ribonucleotides; (4) the nucleotides at positions 1-2 to 1-3 from the 3' end of the antisense strand are not 2'-methoxy-ribonucleotides; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand contains at least 80% 2'-O-methyl modifications; (7) the nucleotides at positions 7, 10, and 11 from the 3' end of the sense strand are not 2'-methoxyribonucleotides; (8) the nucleotides at positions 1 and 2 from the 5' end of the sense strand are joined to each other by a phosphorothioate internucleotide linkage.

[0138] In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, wherein: (1) the antisense strand has a nucleic acid sequence substantially complementary to a nucleic acid sequence of any one of SEQ ID NOs: 1-6; (2) the antisense strand contains at least 75% 2'-O-methyl modifications (e.g., about 75% to about 80% 2'-O-methyl modifications); (3) the nucleotides at positions 2, 6, 14, 16, and 20 from the 5' end of the antisense strand are not 2'-methoxy-ribonucleotides (e.g., the nucleotides at positions 2, 6, 14, 16, and 20 from the 5' end of the antisense strand can be 2'-fluoronucleotides); (4) the nucleotides at positions 1-7 and (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand contains at least 65% 2'-O-methyl modifications (e.g., about 65% to about 75% 2'-O-methyl modifications); (7) the nucleotides at positions 7, 9, 10, and 11 from the 3' end of the sense strand are not 2'-methoxy-ribonucleotides (e.g., the nucleotides at positions 7, 9, 10, and 11 from the 3' end of the sense strand are 2'-fluoronucleotides); (8) the nucleotides at positions 1-2 and 14-15 from the 5' end of the sense strand are linked to each other via phosphorothioate internucleotide linkages.

[0139] In certain embodiments, the functional moiety is linked to the 5'-end and / or 3'-end of the antisense strand. In certain embodiments, the functional moiety is linked to the 5'-end and / or 3'-end of the sense strand. In certain embodiments, the functional moiety is linked to the 3'-end of the sense strand.

[0140] In certain embodiments, the functional moiety comprises a hydrophobic moiety.

[0141] In certain embodiments, the hydrophobic moiety is selected from the group consisting of fatty acids, steroids, secosteroids, lipids, gangliosides, nucleoside analogs, endocannabinoids, vitamins, and mixtures thereof.

[0142] In certain embodiments, the steroid is selected from the group consisting of cholesterol and lithocholic acid (LCA).

[0143] In certain embodiments, the fatty acid is selected from the group consisting of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosanoic acid (DCA).

[0144] In certain embodiments, the vitamin is selected from the group consisting of choline, vitamin A, vitamin E, derivatives thereof, and metabolites thereof.

[0145] In certain embodiments, the vitamin is selected from the group consisting of retinoic acid and alpha-tocopherol succinate.

[0146] In certain embodiments, the functional moiety is linked to the antisense strand and / or the sense strand by a linker.

[0147] In certain embodiments, the linker comprises a bivalent or trivalent linker.

[0148] In certain embodiments, the bivalent or trivalent linker is selected from the group consisting of: [ka] (wherein n is 1, 2, 3, 4, or 5).

[0149] In certain embodiments, the linker comprises an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, a carbamate, or a combination thereof.

[0150] In certain embodiments, when the linker is a trivalent linker, the linker further links a phosphodiester or phosphodiester derivative.

[0151] In certain embodiments, the phosphodiester or phosphodiester derivative is selected from the group consisting of: [ka] (wherein X is O, S or BH3).

[0152] In certain embodiments, the nucleotides 1 and 2 from the 3' end of the sense strand and the nucleotides 1 and 2 from the 5' end of the antisense strand are linked to adjacent ribonucleotides via phosphorothioate linkages.

[0153] In one embodiment, the disclosure provides a compound of formula (I): [ka] (In the formula, L comprises an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, or a combination thereof, and formula (I) optionally further comprises one or more branch points B, and one or more spacers S, wherein B is, independently at each occurrence, a polyvalent organic species or derivative thereof; S, independently at each occurrence, comprises an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, or a combination thereof; n is 2, 3, 4, 5, 6, 7 or 8; N is a double-stranded nucleic acid, such as a dsRNA molecule of any of the above aspects or embodiments of the present disclosure. In certain embodiments, each N is 15-40 bases in length. In certain embodiments, each N comprises a sense strand and an antisense strand: the antisense strand comprises a sequence substantially complementary to any one of the nucleic acid sequences of SEQ ID NOs: 1 to 6; The sense and antisense strands each independently contain one or more chemical modifications.

[0154] In certain embodiments, the compound comprises a structure selected from formulas (I-1)-(I-9): [Table 1]

[0155] In certain embodiments, the antisense strand comprises a 5' end group R selected from the group consisting of: [Table 2]

[0156] In certain embodiments, the compound comprises the structure of formula (II): [ka] (In the formula, X is independently selected at each occurrence from adenosine, guanosine, uridine, cytidine, and chemically modified derivatives thereof; Y is independently selected at each occurrence from adenosine, guanosine, uridine, cytidine, and chemically modified derivatives thereof; - represents a phosphodiester internucleoside linkage; = represents a phosphorothioate internucleoside linkage; --- represents, independently at each occurrence, a base pair interaction or a mismatch.

[0157] In certain embodiments, the compound comprises the structure of formula (IV): [ka] (In the formula, X is independently selected at each occurrence from adenosine, guanosine, uridine, cytidine, and chemically modified derivatives thereof; Y is independently selected at each occurrence from adenosine, guanosine, uridine, cytidine, and chemically modified derivatives thereof; - represents a phosphodiester internucleoside linkage; = represents a phosphorothioate internucleoside linkage; --- represents, independently at each occurrence, a base pair interaction or a mismatch.

[0158] In certain embodiments, L is the structure L1: [ka]

[0159] In certain embodiments, R is R 3 where n is 2.

[0160] In certain embodiments, L is structure L2: [ka]

[0161] In certain embodiments, R is R 3 where n is 2.

[0162] In one aspect, the disclosure provides a delivery system for a therapeutic nucleic acid having the structure of formula (VI): [ka] (In the formula, L comprises an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, or a combination thereof, and formula (VI) optionally further comprises one or more branch points B, and one or more spacers S, wherein B, independently at each occurrence, comprises a polyvalent organic species or derivative thereof; S, independently at each occurrence, comprises an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, or a combination thereof; each cNA is independently a carrier nucleic acid that contains one or more chemical modifications; Each cNA independently comprises at least 15 contiguous nucleotides of the nucleic acid sequence of any one of SEQ ID NOs: 1-6; where n is 2, 3, 4, 5, 6, 7, or 8.

[0163] In certain embodiments, the delivery system comprises a structure selected from formulas (VI-1)-(VI-9): [Table 3]

[0164] In certain embodiments, each cNA independently comprises a chemically modified nucleotide.

[0165] In certain embodiments, the delivery system further comprises n therapeutic nucleic acids (NAs), each NA hybridizing to at least one cNA.

[0166] In certain embodiments, each NA independently comprises at least 16 contiguous nucleotides.

[0167] In certain embodiments, each NA independently comprises 16 to 20 contiguous nucleotides.

[0168] In certain embodiments, each NA comprises an unpaired overhang of at least 2 nucleotides.

[0169] In certain embodiments, the nucleotides of the overhang are linked via phosphorothioate linkages.

[0170] In certain embodiments, each NA is independently selected from the group consisting of DNA, siRNA, antagomiR, miRNA, gapmer, mixmer, and guide RNA.

[0171] In certain embodiments, each NA is substantially complementary to the nucleic acid sequence of any one of SEQ ID NOs: 1-6.

[0172] In one aspect, the present disclosure provides a pharmaceutical composition for inhibiting expression of an IFN-γ signaling pathway target gene in an organism, the composition comprising the compound as defined above or the system as defined above and a pharma- ceutically acceptable carrier.

[0173] In certain embodiments, the compound or system inhibits expression of the SYNGR3 gene by at least 50%. In certain embodiments, the compound or system inhibits expression of the SYNGR3 gene by at least 80%.

[0174] In certain embodiments, the compound or system reduces expression of the cytokine CXCL9 by at least 20% to at least 80%.

[0175] In one aspect, the disclosure provides a method for inhibiting expression of an IFN-γ signaling pathway target gene in a cell, the method comprising: (a) introducing into a cell a compound as described above or a system as described above; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of an mRNA transcript of the gene, thereby inhibiting expression of the gene in the cell.

[0176] In one aspect, the present disclosure provides a method of treating vitiligo in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the compound or system described above.

[0177] In certain embodiments, the dsRNA is administered by intravenous (IV) injection, subcutaneous (SQ) injection, or a combination thereof.

[0178] In certain embodiments, the dsRNA inhibits expression of the gene by at least 50%. In certain embodiments, the dsRNA inhibits expression of the gene by at least 80%.

[0179] In certain embodiments, the dsRNA reduces expression of the cytokine CXCL9 by at least 20% to at least 80%.

[0180] The above and other features and advantages of the present disclosure will be more fully understood from the detailed description of illustrative embodiments taken in conjunction with the accompanying drawings. This patent or application document contains at least one drawing executed in color. Copies of this patent or patent application publication containing color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0181] [Figure 1] A-B show the screening of siRNA sequences targeting human and mouse IFNGR1 mRNA in human HeLa cells (A) and mouse N2A cells (B). Percent expression of IFNGR1 mRNA was determined relative to untreated control. siRNA sequences were tested at a concentration of 1.5 μM and expression levels were measured after 72 hours of incubation using the QunatiGene assay. NTC: non-targeting control; a scrambled siRNA sequence with no known gene target. UNT: untreated control. [Diagram 2]A-B show screening of siRNA sequences targeting human and mouse JAK1 mRNA target sites in human HeLa cells (A) and mouse N2A cells (B). Percent expression of JAK1 mRNA was determined relative to untreated control. siRNA sequences were tested at a concentration of 1.5 μM and expression levels were measured after 72 hours of incubation using the QunatiGene assay. NTC: non-targeting control; scrambled siRNA sequence with no known gene target. UNT: untreated control. [Diagram 3] A-B show the screening of siRNA sequences targeting human and mouse JAK2 mRNA target sites in human HeLa cells (A) and mouse N2A cells (B). Percent expression of JAK2 mRNA was determined relative to untreated control. siRNA sequences were tested at a concentration of 1.5 μM and expression levels were measured after 72 hours of incubation using the QunatiGene assay. NTC: non-targeting control; a scrambled siRNA sequence with no known gene target. UNT: untreated control. [Figure 4] A-B show the screening of siRNA sequences targeting human and mouse STAT1 mRNA target sites in human HeLa cells (A) and mouse N2A cells (B). Percent expression of STAT1 mRNA was determined relative to untreated control. siRNA sequences were tested at a concentration of 1.5 μM and expression levels were measured after 72 hours of incubation using the QunatiGene assay. NTC: non-targeting control; a scrambled siRNA sequence with no known gene target. UNT: untreated control. [Figure 5A] Dose-response inhibition curves of IFNGR1_1726, Ifngr1_1641, JAK1_3033, JAK2_1936, Jak2_2076, and STAT1_885 screened in HeLa (human) and N2A (mouse) cells are shown. NTC: non-targeting control. [Figure 5B]Dose-response inhibition curves of IFNGR1_1726, Ifngr1_1641, JAK1_3033, JAK2_1936, Jak2_2076, and STAT1_885 screened in HeLa (human) and N2A (mouse) cells are shown. NTC: non-targeting control. [Figure 5C] Dose-response inhibition curves of IFNGR1_1726, Ifngr1_1641, JAK1_3033, JAK2_1936, Jak2_2076, and STAT1_885 screened in HeLa (human) and N2A (mouse) cells are shown. NTC: non-targeting control. [Figure 5D] Dose-response inhibition curves of IFNGR1_1726, Ifngr1_1641, JAK1_3033, JAK2_1936, Jak2_2076, and STAT1_885 screened in HeLa (human) and N2A (mouse) cells are shown. NTC: non-targeting control. [Figure 5E] Dose-response inhibition curves of IFNGR1_1726, Ifngr1_1641, JAK1_3033, JAK2_1936, Jak2_2076, and STAT1_885 screened in HeLa (human) and N2A (mouse) cells are shown. NTC: non-targeting control. [Figure 5F] Dose-response inhibition curves of IFNGR1_1726, Ifngr1_1641, JAK1_3033, JAK2_1936, Jak2_2076, and STAT1_885 screened in HeLa (human) and N2A (mouse) cells are shown. NTC: non-targeting control. [Figure 5G] Dose-response inhibition curves of IFNGR1_1726, Ifngr1_1641, JAK1_3033, JAK2_1936, Jak2_2076, and STAT1_885 screened in HeLa (human) and N2A (mouse) cells are shown. NTC: non-targeting control. [Figure 5H]Dose-response inhibition curves of IFNGR1_1726, Ifngr1_1641, JAK1_3033, JAK2_1936, Jak2_2076, and STAT1_885 screened in HeLa (human) and N2A (mouse) cells are shown. NTC: non-targeting control. [Figure 6] A-B show duration of efficacy in mice following injection of a single dose of siRNA Ifngr1_1641. Wild-type C57BL6 mice were treated with siRNA for up to 4 weeks and Ifngr1 protein expression levels in the skin were measured by fluorescent flow cytometry (A). B shows normalized levels of Ifngr1 protein expression compared to Ifngr1 knockout and non-targeted control treated mice. Up to 66% targeted protein knockdown was achieved 2 weeks after injection and significant levels of protein knockdown were maintained for 4 weeks (B). [Figure 7] A-B show that siRNA Ifngr1_1641 reduces the expression of chemokines CXCL9 and CXCL10 through inhibition of IFN-γ signaling. In A, the protocol used is shown. Eight 4 mm diameter skin punch biopsies were taken per mouse 4 weeks after subcutaneous tail injection of 2 × 20 mg / kg siRNA (2-week dosing interval, n = 5 mice per group). Tail skin punch samples were cultured in the presence of recombinant mouse IFN-γ protein (2-fold serial dilutions at 25600-400 pg / mL, and untreated control). In B, CXCL9 and CXCL10 levels measured by enzyme-linked immunosorbent assay (ELISA) assay. Data are presented as mean ± SD and analyzed by two-way ANOVA with Dunnett's multiple comparison test; *P < 0.05. [Figure 8]A-B show how siRNA Ifngr1_1641 exerts both systemic and local efficacy in a vitiligo model. A shows the protocol used. Vitiligo was induced by adoptive transfer of PMEL CD8+ T cells isolated from the spleens of PMEL TCR transgenic mice. Subsequent activation of these T cells in recipient mice results in epidermal depigmentation in a spotted pattern similar to that of vitiligo patients within 3-7 weeks. Mice were treated with a first dose of siRNA 2 weeks before vitiligo induction and a second dose 1 week after induction. B shows a plot of quantification of vitiligo scores on the ears and tails. Vitiligo scores were objectively quantified by observers blinded to treatment groups, using a graded scale based on the extent of depigmented area in the ears and tails. Each site was examined as a percentage of the anatomical site. Both left and right ears were determined collectively and therefore considered as a single site. Individual sites were assigned a vitiligo score of 0 to 5 as follows: no evidence of depigmentation (0%) was assigned a score of 0, >0-10%=1 point, >10-25%=2 points, >25-75%=3 points, >75-<100%=4 points, and 100%=5 points. Data are presented as mean ± SD and analyzed by two-way ANOVA with Sidak's multiple comparison test; *P<0.05, **P<0.01, ****P<0.0001. [Figure 9A] Quantitative analysis of tail depigmentation levels among treatment groups. Skin depigmentation levels were objectively quantified by comparing tail photographs using ImageJ Fiji software (NIH) (A). Individual tail pixel intensity distribution profiles were plotted against total pixel count per intensity, with completely white and black defined as intensities of 0 and 255, respectively (B). C, Plot of summary data. Statistics are presented as the mean ± SD of the mean pixel intensity of individual distribution curves and analyzed by Mann-Whitney t-test; *P<0.05. D, Plot showing that dermal infiltration of cytotoxic T cells (measured by CD45+ cells) was reduced by siRNA Ifngr1 1641 in both epidermis and dermis (paired t-test; **P<0.01, *P<0.05). [Figure 9B] Quantitative analysis of tail depigmentation levels among treatment groups. Skin depigmentation levels were objectively quantified by comparing tail photographs using ImageJ Fiji software (NIH) (A). Individual tail pixel intensity distribution profiles were plotted against total pixel count per intensity, with completely white and black defined as intensities of 0 and 255, respectively (B). C, Plot of summary data. Statistics are presented as the mean ± SD of the mean pixel intensity of individual distribution curves and analyzed by Mann-Whitney t-test; *P<0.05. D, Plot showing that dermal infiltration of cytotoxic T cells (measured by CD45+ cells) was reduced by siRNA Ifngr1 1641 in both epidermis and dermis (paired t-test; **P<0.01, *P<0.05). [Figure 9C] Quantitative analysis of tail depigmentation levels among treatment groups. Skin depigmentation levels were objectively quantified by comparing tail photographs using ImageJ Fiji software (NIH) (A). Individual tail pixel intensity distribution profiles were plotted against total pixel count per intensity, with completely white and black defined as intensities of 0 and 255, respectively (B). C, Plot of summary data. Statistics are presented as the mean ± SD of the mean pixel intensity of individual distribution curves and analyzed by Mann-Whitney t-test; *P<0.05. D, Plot showing that dermal infiltration of cytotoxic T cells (measured by CD45+ cells) was reduced by siRNA Ifngr1 1641 in both epidermis and dermis (paired t-test; **P<0.01, *P<0.05). [Figure 9D]Quantitative analysis of tail depigmentation levels among treatment groups. Skin depigmentation levels were objectively quantified by comparing tail photographs using ImageJ Fiji software (NIH) (A). Individual tail pixel intensity distribution profiles were plotted against total pixel count per intensity, with completely white and black defined as intensities of 0 and 255, respectively (B). C, Plot of summary data. Statistics are presented as the mean ± SD of the mean pixel intensity of individual distribution curves and analyzed by Mann-Whitney t-test; *P<0.05. D, Plot showing that dermal infiltration of cytotoxic T cells (measured by CD45+ cells) was reduced by siRNA Ifngr1 1641 in both epidermis and dermis (paired t-test; **P<0.01, *P<0.05). [Figure 10] IFNGR1 protein expression in human HeLa cells and mouse N2a cells incubated with 1.5 μM IFNGR1_1726 and Ifngr1_1641 targeting siRNA for 72 hours is shown. [Figure 11] Dose-response inhibition curves of IFNGR1_1631, 1989, and 2072 in HeLa cells and Ifngr1_378, 947, and 1162 in N2a cells are shown. NTC: non-targeting control. [Figure 12] Expression levels of CXCL9, CXCL10, and CXCL11 mRNA in HeLa and N2a cells. Cells were treated with 1.5 μM IFNGR1_1726 and Ifngr1_1641-targeted siRNA for 72 h and then stimulated with IFN-γ (n=4, mean ± SD, one-way ANOVA, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; ns, not significant). Samples were analyzed 6 h after IFN-γ signaling stimulation. [Figure 13A-1]Figure 1 shows silencing of IFNGR1 in mouse skin with Ifngr1_1641-targeting siRNAs of different chemical makeup. A shows schematic diagrams of the chemical structures of hydrophobically conjugated (docosanoic acid, DCA; tri-myristic acid, Myr-t) siRNAs and bivalent (Dio) siRNAs. The DCA and Myr-t conjugates are covalently linked to the 3' end of the sense strand, and the two sense strands of the Dio scaffold are covalently linked by tetraethylene glycol. The study also included non-conjugated siRNA Ifngr1_1641 and DCA-conjugated non-targeting control (NTC) siRNA. B shows silencing of Ifngr1 mRNA in skin at the injection site. Mice (n=5 per group) were injected subcutaneously (between both shoulders) with a single dose of siRNA (20 mg / kg) or two doses (2x, 24 hours apart; n=5). One week after injection, local skin samples were harvested and mRNA levels were measured using the QuantiGene 2.0 assay. Ifngr1 expression was normalized to the housekeeping gene Ppib. Data are presented as percent of PBS control (mean ± SD) and analyzed by Kruskal-Wallis test (*p<0.05, **p<0.01; ns, not significant). [Figure 13A-2]Figure 1 shows silencing of IFNGR1 in mouse skin with Ifngr1_1641-targeting siRNAs of different chemical makeup. A shows schematic diagrams of the chemical structures of hydrophobically conjugated (docosanoic acid, DCA; tri-myristic acid, Myr-t) siRNAs and bivalent (Dio) siRNAs. The DCA and Myr-t conjugates are covalently linked to the 3' end of the sense strand, and the two sense strands of the Dio scaffold are covalently linked by tetraethylene glycol. The study also included non-conjugated siRNA Ifngr1_1641 and DCA-conjugated non-targeting control (NTC) siRNA. B shows silencing of Ifngr1 mRNA in skin at the injection site. Mice (n=5 per group) were injected subcutaneously (between both shoulders) with a single dose of siRNA (20 mg / kg) or two doses (2x, 24 hours apart; n=5). One week after injection, local skin samples were harvested and mRNA levels were measured using the QuantiGene 2.0 assay. Ifngr1 expression was normalized to the housekeeping gene Ppib. Data are presented as percent of PBS control (mean ± SD) and analyzed by Kruskal-Wallis test (*p<0.05, **p<0.01; ns, not significant). [Figure 13B]Figure 1 shows silencing of IFNGR1 in mouse skin with Ifngr1_1641-targeting siRNAs of different chemical makeup. A shows schematic diagrams of the chemical structures of hydrophobically conjugated (docosanoic acid, DCA; tri-myristic acid, Myr-t) siRNAs and bivalent (Dio) siRNAs. The DCA and Myr-t conjugates are covalently linked to the 3' end of the sense strand, and the two sense strands of the Dio scaffold are covalently linked by tetraethylene glycol. The study also included non-conjugated siRNA Ifngr1_1641 and DCA-conjugated non-targeting control (NTC) siRNA. B shows silencing of Ifngr1 mRNA in skin at the injection site. Mice (n=5 per group) were injected subcutaneously (between both shoulders) with a single dose of siRNA (20 mg / kg) or two doses (2x, 24 hours apart; n=5). One week after injection, local skin samples were harvested and mRNA levels were measured using the QuantiGene 2.0 assay. Ifngr1 expression was normalized to the housekeeping gene Ppib. Data are presented as percent of PBS control (mean ± SD) and analyzed by Kruskal-Wallis test (*p<0.05, **p<0.01; ns, not significant). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0182] Novel IFN-γ signaling pathway gene target sequences are provided. Novel oligonucleotides, RNA molecules (such as siRNAs), and branched RNA compounds comprising same that target IFN-γ signaling pathway gene mRNAs (such as one or more target sequences of the present disclosure) are also provided.

[0183] Unless otherwise specified, the nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein, and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. Unless otherwise specified, the methods and techniques provided herein are performed according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout the specification, unless otherwise specified. Enzymatic reactions and purification techniques are performed as described herein according to manufacturer's specifications or as commonly accomplished in the art. The terminology used in connection with analytical chemistry, synthetic organic chemistry and pharmaceutical and medicinal chemistry described herein, as well as the laboratory procedures and techniques, are those well known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, delivery, and treatment of patients.

[0184] Unless otherwise defined herein, scientific and technical terms used herein have the meanings commonly understood by those skilled 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 the plural and plural terms shall include the singular. The use of "or" means "and / or" unless otherwise stated. The use of the term "including" as well as other forms such as "include" and "included" are not limiting.

[0185] In order that the present invention may be more readily understood, certain terms are first defined.

[0186] The term "nucleoside" refers to a molecule having a purine or pyrimidine base covalently linked to a ribose or deoxyribose sugar. Exemplary nucleosides include adenosine, guanosine, cytidine, uridine, and thymidine. Further exemplary nucleosides include inosine, 1-methylinosine, pseudouridine, 5,6-dihydrouridine, ribothymidine, 2N-methylguanosine, and N2,N2-dimethylguanosine (also called "rare" nucleosides). The term "nucleotide" refers to a nucleoside having one or more phosphate groups attached to the sugar moiety in an ester linkage. Exemplary nucleotides include nucleoside monophosphate, diphosphate, and triphosphate. The terms "polynucleotide" and "nucleic acid molecule" are used interchangeably herein and refer to a polymer of nucleotides linked together by phosphodiester or phosphorothioate linkages between the 5' and 3' carbon atoms.

[0187] The term "RNA" or "RNA molecule" or "ribonucleic acid molecule" refers to a polymer of ribonucleotides (e.g., 2, 3, 4, 5, 10, 15, 20, 25, 30, or more ribonucleotides). The term "DNA" or "DNA molecule" or "deoxyribonucleic acid molecule" refers to a polymer of deoxyribonucleotides. DNA and RNA can be naturally synthesized (e.g., by DNA replication or DNA transcription, respectively). RNA can be modified post-transcriptionally. DNA and RNA can also be chemically synthesized. DNA and RNA can be single-stranded (i.e., ssRNA and ssDNA, respectively) or multi-stranded (e.g., double-stranded, i.e., dsRNA and dsDNA, respectively). "mRNA" or "messenger RNA" is a single-stranded RNA that specifies the amino acid sequence of one or more polypeptide chains. This information is converted during protein synthesis when ribosomes bind to the mRNA.

[0188] As used herein, the term "small interfering RNA" ("siRNA") (also referred to in the art as "short interfering RNA") refers to an RNA (or RNA analog) that comprises about 10-50 nucleotides (or nucleotide analogs) and is capable of directing or mediating RNA interference. In certain embodiments, an siRNA comprises about 15-30 nucleotides or nucleotide analogs, or about 16-25 nucleotides (or nucleotide analogs), or about 18-23 nucleotides (or nucleotide analogs), or about 19-22 nucleotides (or nucleotide analogs) (e.g., 19, 20, 21 or 22 nucleotides or nucleotide analogs). The term "short" siRNA refers to an siRNA that comprises about 21 nucleotides (or nucleotide analogs), e.g., 19, 20, 21 or 22 nucleotides. The term "long" siRNA refers to an siRNA that comprises about 24-25 nucleotides, e.g., 23, 24, 25 or 26 nucleotides. Short siRNA can comprise less than 19 nucleotides, for example, 16, 17 or 18 nucleotides, provided that short siRNA retains the ability to mediate RNAi in some cases.Similarly, long siRNA can comprise more than 26 nucleotides, provided that longer siRNA retains the ability to mediate RNAi without further processing, for example enzymatic processing, into short siRNA in some cases.

[0189] The term "nucleotide analog" or "altered nucleotide" or "modified nucleotide" refers to a non-standard nucleotide, such as a non-naturally occurring ribonucleotide or deoxyribonucleotide. Exemplary nucleotide analogs are modified at any position to alter certain chemical properties of the nucleotide but retain the ability of the nucleotide analog to perform its intended function. Examples of nucleotide positions that can be derivatized include the 5-position, e.g., 5-(2-amino)propyluridine, 5-bromouridine, 5-propyneuridine, 5-propenyluridine; the 6-position, e.g., 6-(2-amino)propyluridine; and the 8-position of adenosine and / or guanosine, e.g., 8-bromoguanosine, 8-chloroguanosine, 8-fluoroguanosine. Nucleotide analogs also include deazanucleotides, such as 7-deaza-adenosine; O- and N-modified (e.g., alkylated, such as N6-methyladenosine, or those 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.

[0190] Nucleotide analogs may also include modifications to the sugar portion of the nucleotide. For example, the 2'OH group may be replaced by a group selected from H, OR, R, F, Cl, Br, I, SH, SR, NH2, NHR, NR2, or COOR, where R is a substituted or unsubstituted C1-C6 alkyl, alkenyl, alkynyl, aryl, etc. Other possible modifications include those described in U.S. Patent Nos. 5,858,988 and 6,291,438.

[0191] The phosphate group of a nucleotide can also be modified, for example, by replacing one or more of the oxygens of the phosphate group with sulfur (e.g., phosphorothioate) or by other substitutions so that the nucleotide can perform its intended function. For example, see Eckstein, Antisense Nucleic Acid Drug Dev. 2000 Apr. 10(2): 117-21, Rusckowski et al. Antisense Nucleic Acid Drug Dev. 2000 Oct. 10(5): 333-45, Stein, Antisense Nucleic Acid Drug Dev. 2001 Oct. 11(5): 317-25, Vorobjev et al. Antisense Nucleic Acid Drug Dev. 2001 Apr. 11(2): 77-85, and U.S. Patent No. 5,684,143. Certain modifications (e.g., phosphate group modifications) referred to above reduce the hydrolysis rate of polynucleotides containing the analogs, for example, in vivo or in vitro.

[0192] The term "oligonucleotide" refers to a short polymer of nucleotides and / or nucleotide analogs.

[0193] 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 by linkages that result in a reduced 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. Some RNA analogs include sugar and / or backbone modified ribonucleotides and / or deoxyribonucleotides. Such alterations or modifications can further include the addition of non-nucleotide material, such as to the end(s) or interior (one or more nucleotides of the RNA) of the RNA. The RNA analog need only be sufficiently similar to natural RNA to have the ability to mediate RNA interference.

[0194] The term "RNA interference" ("RNAi") as used herein refers to the selective intracellular degradation of RNA. RNAi occurs naturally in cells to remove foreign RNA (such as viral RNA). Natural RNAi proceeds through fragments cleaved from free dsRNA, directing the degradation mechanism to other similar RNA sequences. Alternatively, RNAi can be initiated by human hands, for example to silence the expression of target genes.

[0195] An RNAi agent, e.g., an RNA silencing agent, having a strand that is "sufficiently complementary to a target mRNA sequence to direct 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.

[0196] As used herein, "isolated RNA" (e.g., "isolated siRNA" or "isolated siRNA precursor") refers to an RNA molecule that, when produced by recombinant techniques, is substantially free of other cellular material or culture medium, and, when chemically synthesized, is substantially free of chemical precursors or other chemicals.

[0197] 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, and translational repression) that result in the inhibition or "silencing" of expression of corresponding protein-coding genes. RNA silencing has been observed in many types of organisms, including plants, animals, and fungi.

[0198] 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, for example, when both polynucleotide sequences are present in the same cell. In certain embodiments, the target polynucleotide sequence corresponds to a target gene and the non-target polynucleotide sequence corresponds to a non-target gene. In other embodiments, the target polynucleotide sequence corresponds to a target allele and 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.

[0199] The term "in vitro" has its art-recognized meaning, including, for example, purified reagents or extracts, e.g., cellular extracts. The term "in vivo" also has its art-recognized meaning, including, for example, living cells, e.g., immortalized cells, primary cells, cell lines, and / or cells within an organism.

[0200] As used herein, the term "transgene" refers to any nucleic acid molecule that is artificially inserted into a cell and becomes part of the genome of the organism that develops from the cell. Such transgenes may include genes that are partially or completely heterologous (i.e., foreign) to the transgenic organism, or may represent genes that are homologous to endogenous genes of the organism. "Transgene" also refers to a nucleic acid molecule that includes one or more selected nucleic acid sequences, e.g., DNA, that encode one or more engineered RNA precursors that are expressed in a transgenic organism, e.g., an animal, that are partially or completely heterologous, i.e., foreign, to the transgenic animal, or that are homologous to the endogenous genes of the transgenic animal, but that are designed to be inserted into the genome of the animal at a different location than the native gene. A transgene includes one or more promoters and any other DNA, e.g., introns, necessary for expression of the selected nucleic acid sequence, all operably linked to the selected sequence, and may include enhancer sequences.

[0201] A gene "involved in" a disease or disorder includes a gene, the normal or abnormal expression or function of which affects or causes the disease or disorder, or at least one symptom of the disease or disorder.

[0202] The term "gain-of-function mutation" as used herein refers to any mutation in a gene in which the protein encoded by the gene (i.e., mutant protein) gains a function not normally associated with that protein (i.e., wild-type protein) and causes or contributes to a disease or disorder. A gain-of-function mutation can be a deletion, addition, or substitution of a nucleotide(s) 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 causes interaction with other proteins. In another embodiment, a gain-of-function mutation causes a reduction or elimination of the normal wild-type protein, for example, by interaction of the modified mutant protein with the normal wild-type protein.

[0203] The term "target gene" as used herein is a gene whose expression is substantially inhibited or "silenced". This silencing can be achieved, for example, by silencing the RNA by cleavage of the mRNA of the target gene or translational repression of the target gene. The term "non-target gene" is a gene whose expression is not substantially silenced. In one embodiment, the polynucleotide sequences of the target gene and the non-target gene (e.g., the mRNAs encoded by the target gene and the non-target gene) 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.

[0204] A "target allele" is an allele (e.g., an SNP allele) whose expression is 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 by siRNA. The term "non-target allele" is an allele whose expression is not substantially silenced. In certain embodiments, the target allele and the non-target allele can correspond to the same target gene. In other embodiments, the target allele corresponds to or is related to the target gene, and the non-target allele corresponds to or is related to the non-target gene. In one embodiment, the polynucleotide sequences of the target allele and the non-target allele can differ by one or more nucleotides. In another embodiment, the target allele and the non-target allele can differ by one or more allelic polymorphisms (e.g., one or more SNPs). In another embodiment, the target allele and the non-target allele can share less than 100% sequence identity.

[0205] The term "polymorphism" as used herein refers to a variation (e.g., one or more deletions, insertions, or substitutions) in a gene sequence that is identified or detected when comparing the same gene sequence from different sources or subjects (but the same organism). For example, when comparing the same gene sequence from different subjects, a polymorphism can be identified. Identification of such polymorphisms is routine in the art, and the methodology is similar to that used to detect, for example, point mutations in breast cancer. Identification can be performed, for example, from DNA extracted from lymphocytes of a subject, and then the polymorphic region can be amplified using specific primers for the polymorphic region. Alternatively, a polymorphism can be identified when comparing two alleles of the same gene. In certain embodiments, the polymorphism is a single nucleotide polymorphism (SNP).

[0206] Variations in sequence between two alleles of the same gene in an organism are referred to herein as "allelic polymorphisms". In certain embodiments, allelic polymorphisms correspond to SNP alleles. For example, allelic polymorphisms can include single nucleotide variations between two alleles of a SNP. Polymorphisms can be at nucleotides in coding regions, but due to the degeneracy of the genetic code, no changes in amino acid sequence are encoded. Alternatively, polymorphic sequences can encode different amino acids at specific positions, but the amino acid changes do not affect the function of the protein. Polymorphic regions are also found in non-coding regions of genes. In exemplary embodiments, polymorphisms are found in coding regions of genes or in untranslated regions of genes (e.g., 5'UTR or 3'UTR).

[0207] The term "IFNGR1" as used herein refers to a gene encoding the protein interferon gamma receptor 1. The IFNGR1 gene is located on chromosome 6q23.3. The IFNGR1 locus spans 23 kb and consists of 9 exons (NCBI gene ID: 3459). The gene is expressed as two splice variants and is expressed in most tissues. The interferon gamma receptor 1 protein is approximately 489 amino acids long and has a molecular weight of approximately 90 kD (UniprotKB P15260It associates with interferon gamma receptor 2 to form a heterodimeric receptor for interferon gamma).

[0208] The term "JAK1" as used herein refers to the gene encoding Janus kinase 1. The JAK1 gene is located on chromosome 1p31.3. The JAK1 locus spans 235 kb and consists of 29 exons (NCBI gene ID: 3716). The gene is expressed in most tissues. The Janus kinase 1 protein is approximately 1154 amino acids long and has a molecular weight of approximately 133 kD (UniProtKB P23458). It is part of the IFN-γ signaling pathway and plays a role in the phosphorylation of STAT proteins.

[0209] The term "JAK2" as used herein refers to the gene encoding the protein Janus Kinase 2. The JAK2 gene is located on chromosome 9p24.1. The JAK2 locus spans 146 kb and consists of 27 exons (NCBI gene ID: 3717). The gene is expressed in most tissues. The Janus Kinase 2 protein is approximately 1132 amino acids long and has a molecular weight of 131 kD (UniProtKB O60674). It is part of the IFN-γ signaling pathway and plays a role in the phosphorylation of STAT proteins.

[0210] The term "STAT1" as used herein refers to the gene encoding signal transducer and activator of transcription 1. The STAT1 gene is located on chromosome 2q32.2. The STAT1 locus spans 113 kb and consists of 26 exons (NCBI gene ID: 6772). The gene is expressed as two splice variants and is expressed in most tissues. The signal transducer and activator of transcription 1 protein is approximately 750 amino acids long and has a molecular weight of approximately 87 kD (UniProtKB P42224). It is part of the IFN-γ signaling pathway and acts as a transcriptional activator when phosphorylated.

[0211] The phrase "examining the function of a gene in a cell or organism" refers to examining or studying the expression, activity, function or phenotype resulting therefrom.

[0212] The term "RNA silencing agent" as used herein refers to an RNA that can inhibit or "silence" the expression of a target gene. In certain embodiments, the RNA silencing agent can prevent complete processing (e.g., complete translation and / or expression) of an mRNA molecule by a post-transcriptional silencing mechanism. RNA silencing agents include small (<50 b.p.), non-coding RNA molecules, such as RNA duplexes that include paired strands, as well as precursor RNAs that can generate such small non-coding RNAs. Exemplary RNA silencing agents include siRNAs, miRNAs, siRNA-like duplexes, antisense oligonucleotides, GAPMER molecules, and dual-function oligonucleotides, as well as their precursors. In one embodiment, the RNA silencing agent can induce RNA interference. In another embodiment, the RNA silencing agent can mediate translational repression.

[0213] As used herein, the term "rare nucleotide" refers to a naturally occurring nucleotide that occurs infrequently, e.g., a naturally occurring deoxyribonucleotide or ribonucleotide that occurs infrequently, e.g., a naturally occurring ribonucleotide that is not guanosine, adenosine, cytosine, or uridine. Examples of rare nucleotides include, but are not limited to, inosine, 1-methylinosine, pseudouridine, 5,6-dihydrouridine, ribothymidine, 2N-methylguanosine, and 2,2N,N-dimethylguanosine.

[0214] The term "engineered" refers to a precursor or molecule that is not found in nature, in that all or part of the nucleic acid sequence of the precursor or molecule is created or selected by humans, such as an engineered RNA precursor or engineered nucleic acid molecule. Once created or selected, the sequence is replicated, translated, transcribed, or otherwise processed by machinery within the cell. Thus, an RNA precursor produced within a cell from a transgene that contains an engineered nucleic acid molecule is an engineered RNA precursor.

[0215] As used herein, the term "microRNA" ("miRNA"), also known in the art as "small temporal RNA" ("stRNA"), refers to small (e.g., 10-50 nucleotide) RNAs that are genetically encoded (e.g., by viral, mammalian, or plant genomes) and can direct or mediate RNA silencing. "miRNA disorder" shall refer to a disease or disorder characterized by aberrant expression or activity of a miRNA.

[0216] As used herein, the term "bifunctional oligonucleotide" refers to an RNA silencing agent having the formula TL-μ, where T is an mRNA targeting moiety, L is a linking moiety, and μ is an miRNA recruiting moiety. As used herein, the term "mRNA targeting moiety", "targeting moiety", "mRNA targeting moiety" or "targeting moiety" refers to a domain, portion or region of a bifunctional oligonucleotide that has sufficient size and sufficient complementarity to a portion or region of the mRNA selected or targeted for silencing (i.e., the portion has sufficient sequence to capture the target mRNA).

[0217] The term "linking moiety" or "linking portion" as used herein refers to a domain, portion or region of an RNA silencing agent that covalently attaches or links to an mRNA.

[0218] 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 section of about 10-50 nucleotides, e.g., about 15-30 nucleotides, 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 direct target-specific silencing, e.g., sufficiently complementary to cause destruction of the desired target mRNA by the mechanism or process of RNAi (RNAi interference), or sufficiently complementary to cause translational inhibition of the desired target mRNA.

[0219] 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. The antisense strand and the sense strand can also be referred to as the first strand or the second strand, where the first strand or the second strand has complementarity to the target sequence, and the second strand or the first strand, respectively, has complementarity to the first strand or the second strand. The miRNA duplex intermediate or siRNA-like duplex includes an miRNA strand that has sufficient complementarity to a section of about 10-50 nucleotides of the mRNA of the gene targeted for silencing, and an miRNA* strand that has sufficient complementarity to form a duplex with the miRNA strand.

[0220] The term "guide strand" as used herein refers to the strand of an RNA silencing agent, e.g., the antisense strand of an siRNA duplex or siRNA sequence, that enters the RISC complex and directs cleavage of a target mRNA.

[0221] The term "asymmetry" as used herein refers to the unequal binding strength or base pair strength between the ends of an RNA silencing agent (e.g., between the terminal nucleotide on a first strand or stem portion and the terminal nucleotide on the opposite second strand or stem portion), as in the asymmetry of the double-stranded region of an RNA silencing agent (e.g., the stem of an shRNA). This causes the 5' end of one strand of the duplex to be in a transiently unpaired state, e.g., single-stranded state, more frequently than the 5' end of the complementary strand. This structural difference determines that one strand of the duplex is preferentially incorporated into the RISC complex. The strand that is less tightly paired at the 5' end with the complementary strand will preferentially be incorporated into RISC and mediate RNAi.

[0222] The term "binding strength" or "base pair strength" as used herein refers primarily to the strength of interactions between pairs of nucleotides (or nucleotide analogs) on opposing strands of an oligonucleotide duplex (e.g., a siRNA duplex) and between these nucleotides (or nucleotide analogs), via H-bonds, van der Waals interactions, and the like.

[0223] As used herein, the "5' end" refers to the 5' terminal nucleotide, such as the 5' end of the antisense strand, e.g., between 1 and about 5 nucleotides at the 5' end of the antisense strand. As used herein, the "3' end" refers to the region complementary to the 5' terminal nucleotide of the complementary antisense strand, such as the 3' end of the sense strand, e.g., a region of 1 to about 5 nucleotides.

[0224] The term "destabilizing nucleotide" as used herein refers to a first nucleotide or nucleotide analog that can base pair with a second nucleotide or nucleotide analog such that the base pair has a lower binding strength than a conventional base pair (i.e., Watson-Crick base pair). In certain embodiments, the destabilizing nucleotide can form a mismatch base pair with the second nucleotide. In other embodiments, the destabilizing nucleotide can form a wobble base pair with the second nucleotide. In still other embodiments, the destabilizing nucleotide can form an ambiguous base pair with the second nucleotide.

[0225] The term "base pair" as used herein refers to interactions between pairs of nucleotides (or nucleotide analogs) on opposing strands of an oligonucleotide duplex (e.g., a duplex formed by a strand of an RNA silencing agent and a target mRNA sequence), primarily through H-bonds, van der Waals interactions, etc. between the nucleotides (or nucleotide analogs). The terms "binding strength" or "base pair strength" as used herein refer to the strength of the base pair.

[0226] As used herein, the term "mismatched base pair" refers to a base pair consisting of a non-complementary or non-Watson-Crick base pair, e.g., not a normal complementary G:C, A:T, 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 universal nucleotides.

[0227] As used herein, the term "universal nucleotide" (also known as "neutral nucleotide") includes nucleotides (e.g., certain destabilizing nucleotides) that have bases ("universal bases" or "neutral bases") that do not significantly discriminate between bases on a complementary polynucleotide when base-pairing. Universal nucleotides are primarily hydrophobic molecules that can efficiently pack into antiparallel double-stranded nucleic acids (e.g., double-stranded DNA or RNA) through stacking interactions. The base portion of a universal nucleotide typically includes a nitrogen-containing aromatic heterocyclic moiety.

[0228] As used herein, the term "sufficient complementarity" or "sufficient degree of complementarity" means that an RNA silencing agent has sufficient sequence (e.g., in the antisense strand, mRNA targeting portion, or miRNA recruitment portion) to bind to a desired target and induce RNA silencing of a target mRNA, respectively.

[0229] The term "translational repression" as used herein refers to selective inhibition of mRNA translation.Natural translational repression proceeds through miRNA cleaved from shRNA precursor.Both RNAi and translational repression are mediated by RISC.Both RNAi and translational repression can occur naturally or can be initiated by human hands, for example, to silence the expression of target genes.

[0230] Various methodologies of the present invention include a step that includes comparing a value, level, feature, characteristic, property, etc., to a "suitable control," interchangeably referred to herein as a "suitable control." A "suitable control" or "suitable control" is any control or standard that is useful for comparison purposes and familiar to one of skill in the art. In one embodiment, a "suitable control" or "suitable control" is a value, level, feature, characteristic, property, etc., that is determined prior to performing an RNAi methodology, as described herein. For example, transcription rates, mRNA levels, translation rates, protein levels, biological activities, cellular characteristics or properties, genotypes, phenotypes, etc., can be determined prior to introducing an RNA silencing agent of the present invention into a cell or organism. In another embodiment, a "suitable control" or "suitable control" is a value, level, feature, characteristic, property, etc., that is determined in a cell or organism, e.g., a control or, e.g., a normal cell or organism that exhibits a normal trait. In yet another embodiment, a "suitable control" or "suitable control" is a predefined value, level, feature, characteristic, property, etc.

[0231] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0232] Various aspects of the invention are described in further detail in the following subsections.

[0233] I. Novel Target Sequences In certain exemplary embodiments, the RNA silencing agent of the present invention can target IFNGR1, JAK1, JAK2, or STAT1 nucleic acid sequences of any one of SEQ ID NOs: 1-6 in Tables 6 and 8. In certain exemplary embodiments, the RNA silencing agent of the present invention can target one or more of IFNGR1, JAK1, JAK2, or STAT1 nucleic acid sequences selected from the group consisting of SEQ ID NOs: 143-154 in Tables 7, 9, 10, and 11.

[0234] The genomic sequence of each target sequence can be found, for example, in publicly available databases maintained by NCBI.

[0235] II. siRNA Design In some embodiments, the siRNA is designed as follows. First, a portion of the target gene (e.g., IFNGR1, JAK1, JAK2, or STAT1 gene), such as one or more of the target sequences shown in Tables 6 and 8, is selected. Cleavage of the mRNA at these sites renders translation of the corresponding protein unnecessary. The antisense strand is designed based on the target sequence, and the sense strand is designed to be complementary to the antisense strand. Hybridization of the antisense strand with the sense strand forms an siRNA duplex. The antisense strand comprises about 19-25 nucleotides, e.g., 19, 20, 21, 22, 23, 24, or 25 nucleotides. In other embodiments, the antisense strand comprises 20, 21, 22, or 23 nucleotides. The sense strand comprises about 14-25 nucleotides, e.g., 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. In other embodiments, the sense strand is 15 nucleotides. In certain embodiments, the sense strand is 18 nucleotides. In other embodiments, the sense strand is 20 nucleotides. However, those skilled in the art will understand that siRNAs with lengths less than 19 nucleotides or more than 25 nucleotides can also function to mediate RNAi. Thus, siRNAs of such lengths are also within the scope of the present invention as long as they retain the ability to mediate RNAi. Although longer RNAi agents have been demonstrated to induce interferon or PKR responses in certain mammalian cells, this may not be desirable. In certain embodiments, the RNAi agents of the present invention do not induce PKR responses (i.e., are of sufficiently short length). However, longer RNAi agents may be useful, for example, in cell types that cannot generate PKR responses, or in situations where PKR responses are downregulated or suppressed by alternative means.

[0236] Sense strand sequence can be designed so that target sequence is essentially in the center of strand.By moving target sequence to a position that is off-center, in some cases, the efficiency of cleavage by siRNA can be reduced.This composition, i.e., the composition with low efficiency, may be desirable to use when the off-silencing of wild-type mRNA is detected.

[0237] The antisense strand may be the same length as the sense strand and contain complementary nucleotides. In one embodiment, the strands are fully complementary. That is, the strands are blunt-ended when aligned or annealed. In another embodiment, the strands are aligned or annealed such that a 1, 2, 3, 4, 5, 6, 7, or 8 nucleotide overhang is generated. That is, the 3' end of the sense strand extends 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides beyond the 5' end of the antisense strand and / or the 3' end of the antisense strand extends 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides beyond the 5' end of the sense strand. The overhang may comprise (or consist of) nucleotides corresponding to the target gene sequence (or its complement). Alternatively, the overhang may comprise (or consist of) deoxyribonucleotides, such as dT, or nucleotide analogs, or other suitable non-nucleotide material.

[0238] In order to facilitate the entry of the antisense strand into RISC (and thus increase or improve the efficiency of target cleavage and silencing), the base pairing strength between the 5' end of the sense strand and the 3' end of the antisense strand can be modified (reduced or decreased). These are described in detail below and in U.S. Patent Nos. 7,459,547, 7,772,203 and 7,732,593, entitled "Methods and Compositions for Controlling Efficacy of RNA Silencing", filed June 2, 2003, and U.S. Patent Nos. 8,309,704, 7,750,144, 8,304,530, 8,329,892 and 8,309,705, entitled "Methods and Compositions for Enhancing the Efficacy and Specificity of RNAi", filed June 2, 2003, the entire contents of which are incorporated by reference herein. In one embodiment of these aspects of the invention, the base pair strength is low because there are fewer G:C base pairs between the 5' end of the first or antisense strand and the 3' end of the second or sense strand than between the 3' end of the first or antisense strand and the 5' end of the second or sense strand. In another embodiment, the base pair strength is low due to at least one mismatched base pair between the 5' end of the first or antisense strand and the 3' end of the second or sense strand. In certain exemplary embodiments, 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 base pair strength is low due to 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 second or sense strand. In another embodiment, the base pair strength is low due to at least one base pair containing a rare nucleotide, e.g., inosine (I). In certain exemplary embodiments, the base pairs are selected from the group consisting of I:A, I:U and I:C. In yet other embodiments, the base pair strength is lower due to at least one base pair comprising a modified nucleotide.In certain exemplary 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.

[0239] The design of siRNA suitable for targeting IFNGR1, JAK1, JAK2 or STAT1 target sequence shown in Tables 6 and 8 is described in detail below. siRNA can be designed according to the above exemplary teachings for any other target sequence found in IFNGR1, JAK1, JAK2 or STAT1 gene. Moreover, this technology can be applied to target any other target sequence, such as target sequence that does not cause disease.

[0240] To verify the efficacy of siRNA in disrupting mRNA (e.g., IFNGR1, JAK1, JAK2, or STAT1 mRNA), the siRNA can be incubated with cDNA (e.g., IFNGR1, JAK1, JAK2, or STAT1 cDNA) in a Drosophila melanogaster-based in vitro mRNA expression system. 32 Newly synthesized mRNA (e.g., IFNGR1, JAK1, JAK2, or STAT1 mRNA) radioactively labeled with P is detected by autoradiography on an agarose gel. The presence of cleaved mRNA indicates mRNA nuclease activity. Suitable controls include omitting the siRNA. 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 scrambling the nucleotide sequence of the selected siRNA. A homology search can be performed to ensure that the negative control lacks homology with any other genes in the appropriate genome. Additionally, a negative control siRNA can be designed by introducing one or more base mismatches into the sequence. The siRNA-mRNA complementary site that results in optimal mRNA specificity and maximum mRNA cleavage is selected.

[0241] III. RNAi Agents The present invention includes RNAi molecules, such as the siRNA molecules designed as above.The siRNA molecules of the present invention can be chemically synthesized, or can be transcribed from DNA template in vitro, or from shRNA in vivo, or can be used recombinant human DICER enzyme to cleave in vitro transcribed dsRNA template into a pool of 20, 21 or 23bp double-stranded RNA mediated RNAi.The siRNA molecules can be designed using any method known in the art.

[0242] In one embodiment, instead of the RNAi agent being an interfering ribonucleic acid, for example, the above-mentioned siRNA or shRNA, the RNAi agent can code an interfering ribonucleic acid, for example, the above-mentioned shRNA.In other words, the RNAi agent can be a transcription template for an interfering ribonucleic acid.Thus, the RNAi agent of the present invention can also include small hairpin RNA (shRNA) and the expression construct that is engineered to express shRNA.It is believed that the transcription of shRNA starts at the polymerase III (pol III) promoter and terminates at the 2nd position of the 4-5-thymine transcription termination site. Upon expression, shRNAs are thought to fold into stem-loop structures with 3'UU overhangs and then the ends of these shRNAs are processed converting the shRNAs into siRNA-like molecules of approximately 21-23 nucleotides (Brummelkamp et al., 2002; Lee et al., 2002, supra; Miyagishi et al., 2002; Paddison et al., 2002, supra; Paul et al., 2002, supra; Sui et al., 2002, supra; Yu et al., 2002, supra. Further information regarding the design and use of shRNAs can be found on the Internet at the following addresses: katandin.cshl.org:9331 / RNAi / docs / BseRI-BamHI_Strategy.pdf and katandin.cshl.org:9331 / RNAi / docs / Web_version_of_PCR_strategy1.pdf).

[0243] The expression constructs of the present invention include any construct suitable for use in a suitable expression system, including, but not limited to, retroviral vectors, linear expression cassettes, plasmids, and virus or virus-derived vectors, as known in the art. Such expression constructs can include one or more inducible promoters, RNA Pol III promoter systems, such as U6 snRNA promoters or H1 RNA polymerase III promoters, or other promoters known in the art. The constructs can include one or both strands of the siRNA. Expression constructs that express both strands can also include a loop structure that links both strands, or each strand can be transcribed separately from a separate promoter within the same construct. Each strand can be transcribed from a separate expression construct (Tuschl, T., 2002, supra).

[0244] Synthetic siRNAs can be delivered to cells by methods known in the art, such as cationic liposome transfection and electroporation. To obtain long-term inhibition of a target gene (e.g., IFNGR1, JAK1, JAK2, or STAT1 genes) and to facilitate delivery under certain circumstances, one or more siRNAs can be expressed in cells from recombinant DNA constructs. Such methods for expressing siRNA duplexes in cells from recombinant DNA constructs to allow longer-term target gene inhibition in cells are known in the art, such as mammalian Pol III promoter systems (e.g., H1 or U6 / snRNA promoter systems (Tuschl, T., 2002, supra) that can express functional double-stranded siRNAs (Bagella et al., 1998; Lee et al., 2002, supra; Miyagishi et al., 2002, supra; Paul et al., 2002, supra; Yu et al., 2002, supra; Sui et al., 2002, supra). RNA Transcription termination by PolIII occurs at a series of four consecutive T residues in the DNA template, providing a mechanism for terminating siRNA transcripts at specific sequences. siRNAs are complementary to the sequence of the target gene in the 5'-3' and 3'-5' orientations, and the two strands of the siRNA can be expressed in the same or separate constructs. Hairpin siRNAs expressed in cells driven by H1 or U6 snRNA promoters can inhibit expression of target genes (Bagella et al., 1998; Lee et al., 2002, supra; Miyagishi et al., 2002, supra; Paul et al., 2002, supra; Yu et al., 2002, supra; Sui et al., 2002, supra). Constructs containing siRNA sequences under the control of the T7 promoter also generate functional siRNAs when cotransfected into cells with a vector expressing T7 RNA polymerase (Jacque et al., 2002, supra).A single construct may contain multiple siRNA-encoding sequences, such as multiple regions of the genes encoding IFNGR1, JAK1, JAK2, or STAT1, targeting the same gene or multiple genes, and may be driven, for example, by separate PolIII promoter sites.

[0245] Animal cells express a series of approximately 22 nucleotide non-coding RNAs called microRNAs (miRNAs), which can regulate gene expression at the post-transcriptional or translational level during animal development. One common feature of miRNAs is that they are all excised from a precursor RNA stem loop of approximately 70 nucleotides, presumably by the RNase III type enzyme Dicer, or its homologues. By replacing the stem sequence of the miRNA precursor with a sequence complementary to the target mRNA, vector constructs expressing the engineered precursors can be used to produce siRNAs and initiate RNAi against specific mRNA targets in mammalian cells (Zeng et al., supra, 2002). When expressed by a DNA vector containing a polymerase III promoter, microRNA engineered hairpins can silence gene expression (McManus et al., 2002, supra). MicroRNAs targeting polymorphisms can also be useful to block translation of mutant proteins in the absence of siRNA-mediated gene silencing. Such applications may be useful, for example, in situations where the designed siRNA caused off-target silencing of a wild-type protein.

[0246] Viral-mediated delivery mechanisms can also be used to induce specific silencing of targeted genes through expression of siRNA, for example, by generating recombinant adenoviruses carrying siRNA under the transcriptional control of an RNA Pol II promoter (Xia et al., 2002, supra). Infection of HeLa cells with these recombinant adenoviruses can reduce the expression of endogenous target genes. Injection of recombinant adenoviral vectors into transgenic mice expressing the siRNA target gene results in in vivo reduction of target gene expression. Ibid. In animal models, synthetic siRNA can be efficiently delivered to mouse embryos after implantation by whole embryo electroporation (Calegari et al., 2002). In adult mice, efficient delivery of siRNA can be achieved by "high pressure" delivery techniques, rapidly injecting (within 5 seconds) a large volume of siRNA-containing solution into the tail vein of the animal (Liu et al., 1999, supra; McCaffrey et al., 2002, supra; Lewis et al., 2002. Nanoparticles and liposomes can also be used to deliver siRNA to animals. In certain exemplary embodiments, recombinant adeno-associated viruses (rAAV) and their related vectors can be used to deliver one or more siRNAs to cells, such as skin cells (U.S. Patent Application Nos. 2014 / 0296486, 2010 / 0186103, 2008 / 0269149, 2006 / 0078542, and 2005 / 0220766).

[0247] The nucleic acid composition of the present invention includes both unmodified siRNA and modified siRNA, such as crosslinked siRNA derivatives or derivatives having non-nucleotide moieties linked to their 3'-end or 5'-end. Such modification of siRNA derivatives can improve the uptake into cells or improve the cell targeting activity of the resulting siRNA derivatives compared to the corresponding siRNAs, and is also useful for tracking the siRNA derivatives in cells or improving the stability of the siRNA derivatives compared to the corresponding siRNAs.

[0248] As described herein, engineered RNA precursors introduced into cells or whole organisms lead to the production of desired siRNA molecules.Then, such siRNA molecules associate with endogenous protein components of the RNAi pathway to bind to specific mRNA sequences and target them for cleavage and destruction.In this way, the mRNAs targeted by the siRNAs produced from engineered RNA precursors are depleted from cells or organisms, thereby reducing the concentration of the protein encoded by that mRNA in cells or organisms.RNA precursors are typically nucleic acid molecules that either individually code one strand of dsRNA or code the entire nucleotide sequence of an RNA hairpin loop structure.

[0249] The nucleic acid compositions of the invention may be unconjugated or may be conjugated to another moiety, such as a nanoparticle, which improves the properties of the composition, e.g., pharmacokinetic parameters such as absorption, efficacy, bioavailability and / or half-life. Conjugation can be accomplished using methods known in the art, for example, Lambert et al., Drug Deliv. Rev.: 47(1), 99-112 (2001) (describing nucleic acids attached to 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).

[0250] The nucleic acid molecules of the present invention can also be labeled using any method known in the art. For example, the nucleic acid composition can be labeled with a fluorophore, such as Cy3, fluorescein, or rhodamine. Labeling can be performed using a kit, such as the SILENCER™ siRNA labeling kit (Ambion). In addition, siRNA can be labeled using, for example, 3 H, 32 It may be radiolabeled using P or another suitable isotope.

[0251] Moreover, because RNAi is believed to proceed through at least one single-stranded RNA intermediate, one of skill in the art will appreciate that ss-siRNAs (e.g., the antisense strand of a ds-siRNA) may also be designed (e.g., for chemical synthesis), generated (e.g., enzymatically generated) or expressed (e.g., from a vector or plasmid) as described herein and utilized in accordance with the claimed methodology. Moreover, in invertebrates, RNAi can be effectively induced by long dsRNAs (e.g., dsRNAs of about 100-1000 nucleotides in length, e.g., about 200-500, e.g., about 250, 300, 350, 400 or 450 nucleotides in length, etc.) that act as effectors of RNAi (Brondani et al., Proc Natl Acad Sci USA. 2001 Dec. 4; 98(25):14428-33. Epub 2001 Nov. 27.).

[0252] IV. Anti-IFNGR1, Anti-JAK1, Anti-JAK2, and Anti-STAT1 RNA Silencing Agents In certain embodiments, the present invention provides novel anti-IFNGR1, anti-JAK1, anti-JAK2, and anti-STAT1 RNA silencing agents (e.g., siRNA, shRNA, and antisense oligonucleotides), methods for making the RNA silencing agents, and methods (e.g., research and / or therapeutic methods) for using the improved RNA silencing agents (or portions thereof) for RNA silencing of IFNGR1, JAK1, JAK2, or STAT1 proteins. The RNA silencing agents include an antisense strand (or portions thereof), which has sufficient complementarity to the target IFNGR1, JAK1, JAK2, or STAT1 mRNA to mediate an RNA-mediated silencing mechanism (e.g., RNAi).

[0253] In certain embodiments, siRNA compounds are provided that have one or any combination of the following characteristics: (1) fully chemically stabilized (i.e., no unmodified 2'-OH residues); (2) asymmetric; (3) 11-20 base pair duplex; (4) greater than 50% 2'-methoxy modified, e.g., 70%-100% 2'-methoxy modified, alternating patterns of chemically modified nucleotides (e.g., 2'-fluoro and 2'-methoxy modified) are also contemplated; and (5) single stranded, fully phosphorothioated 5-8 base tail. In certain embodiments, the number of phosphorothioate modifications varies from a total of 4 to 16. In certain embodiments, the number of phosphorothioate modifications varies from a total of 8 to 13.

[0254] In certain embodiments, the siRNA compounds described herein can be conjugated to a variety of targeting agents, including, but not limited to, cholesterol, docosahexaenoic acid (DHA), phenyltropane, cortisol, vitamin A, vitamin D, N-acetylgalactosamine (GalNac), and gangliosides. Cholesterol-modified forms have demonstrated 5-10-fold improved efficacy in vitro compared to previously used chemical stabilization patterns (e.g., all purines but not pyrimidines are modified) in a wide range of cell types (e.g., HeLa, neurons, hepatocytes, trophoblasts).

[0255] Certain compounds of the present invention having the structural characteristics described above and herein may be referred to as "hsiRNA-ASP" (hydrophobically modified small interfering RNA characterized by a highly stabilizing pattern). Moreover, this hsiRNA-ASP pattern has shown dramatically improved distribution via delivery from the brain and spinal cord to the liver, placenta, kidney, spleen, and several other tissues, making it available for therapeutic intervention.

[0256] The compounds of the invention can be illustrated in the following aspects and embodiments.

[0257] In a first aspect, there is provided herein a double-stranded RNA (dsRNA) comprising an antisense strand and a sense strand, each strand comprising at least 14 contiguous nucleotides having a 5' end and a 3' end, (1) the antisense strand comprises a sequence substantially complementary to any one of the nucleic acid sequences of SEQ ID NOs: 1 to 6; (2) the antisense strand contains alternating 2'-methoxy-ribonucleotides and 2'-fluoro-ribonucleotides; (3) the nucleotides at positions 2 and 14 from the 5' end of the antisense strand are not 2'-methoxy-ribonucleotides; (4) nucleotides at positions 1-2 through 1-7 from the 3' end of the antisense strand are joined to each other via phosphorothioate internucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand contains alternating 2'-methoxy-ribonucleotides and 2'-fluoro-ribonucleotides; (7) The nucleotides 1 and 2 from the 5' end of the sense strand are linked to each other via a phosphorothioate internucleotide linkage.

[0258] In a second aspect, there is provided herein a dsRNA comprising an antisense strand and a sense strand, each strand comprising at least 14 contiguous nucleotides having a 5' end and a 3' end, (1) the antisense strand comprises a sequence substantially complementary to any one of the nucleic acid sequences of SEQ ID NOs: 1 to 6; (2) the antisense strand contains at least 70% 2'-O-methyl modifications; (3) the nucleotide at position 14 from the 5' end of the antisense strand is not a 2'-methoxy-ribonucleotide; (4) nucleotides at positions 1-2 through 1-7 from the 3' end of the antisense strand are joined to each other via phosphorothioate internucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand contains at least 70% 2'-O-methyl modifications; (7) The nucleotides 1 and 2 from the 5' end of the sense strand are linked to each other via a phosphorothioate internucleotide linkage.

[0259] In a third aspect, there is provided herein a dsRNA comprising an antisense strand and a sense strand, each strand comprising at least 14 contiguous nucleotides having a 5' end and a 3' end, (1) the antisense strand comprises a sequence substantially complementary to any one of the nucleic acid sequences of SEQ ID NOs: 1 to 6; (2) the antisense strand contains at least 85% 2'-O-methyl modifications; (3) the nucleotides at positions 2 and 14 from the 5' end of the antisense strand are not 2'-methoxy-ribonucleotides; (4) nucleotides at positions 1-2 through 1-7 from the 3' end of the antisense strand are joined to each other via phosphorothioate internucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand contains 100% 2'-O-methyl modifications; (7) The nucleotides 1 and 2 from the 5' end of the sense strand are linked to each other via a phosphorothioate internucleotide linkage.

[0260] In a fourth aspect, there is provided herein a dsRNA comprising an antisense strand and a sense strand, each strand comprising at least 14 contiguous nucleotides having a 5' end and a 3' end, (1) the antisense strand comprises a sequence substantially complementary to any one of the nucleic acid sequences of SEQ ID NOs: 1 to 6; (2) the antisense strand contains at least 75% 2'-O-methyl modifications; (3) the nucleotides at positions 4, 5, 6, and 14 from the 5' end of the antisense strand are not 2'-methoxy-ribonucleotides; (4) nucleotides at positions 1-2 through 1-7 from the 3' end of the antisense strand are joined to each other via phosphorothioate internucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand contains 100% 2'-O-methyl modifications; (7) The nucleotides 1 and 2 from the 5' end of the sense strand are linked to each other via a phosphorothioate internucleotide linkage.

[0261] In a fifth aspect, there is provided herein a dsRNA comprising an antisense strand and a sense strand, each strand comprising at least 14 contiguous nucleotides having a 5' end and a 3' end, (1) the antisense strand comprises a sequence substantially complementary to any one of the nucleic acid sequences of SEQ ID NOs: 1 to 6; (2) the antisense strand contains at least 75% 2'-O-methyl modifications; (3) the nucleotides at positions 2, 4, 5, 6, and 14 from the 5' end of the antisense strand are not 2'-methoxy-ribonucleotides; (4) nucleotides at positions 1-2 through 1-7 from the 3' end of the antisense strand are joined to each other via phosphorothioate internucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand contains 100% 2'-O-methyl modifications; (7) The nucleotides 1 and 2 from the 5' end of the sense strand are linked to each other via a phosphorothioate internucleotide linkage.

[0262] In a sixth aspect, there is provided herein a dsRNA comprising an antisense strand and a sense strand, each strand comprising at least 14 contiguous nucleotides having a 5' end and a 3' end, (1) the antisense strand comprises a sequence substantially complementary to any one of the nucleic acid sequences of SEQ ID NOs: 1 to 6; (2) the antisense strand contains at least 75% 2'-O-methyl modifications; (3) the nucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand are not 2'-methoxy-ribonucleotides; (4) nucleotides at positions 1-2 through 1-7 from the 3' end of the antisense strand are joined to each other via phosphorothioate internucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand contains at least 70% 2'-O-methyl modifications; (7) the nucleotides at positions 7, 9, 10, and 11 from the 3' end of the sense strand are not 2'-methoxy-ribonucleotides; (8) The nucleotides 1 and 2 from the 5' end of the sense strand are linked to each other via a phosphorothioate internucleotide linkage.

[0263] In a seventh aspect, there is provided herein a dsRNA comprising an antisense strand and a sense strand, each strand comprising at least 14 contiguous nucleotides having a 5' end and a 3' end, (1) the antisense strand comprises a sequence substantially complementary to any one of the nucleic acid sequences of SEQ ID NOs: 1 to 6; (2) the antisense strand contains at least 75% 2'-O-methyl modifications; (3) the nucleotides at positions 2, 6, and 14 from the 5' end of the antisense strand are not 2'-methoxy-ribonucleotides; (4) nucleotides at positions 1-2 through 1-7 from the 3' end of the antisense strand are joined to each other via phosphorothioate internucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand contains at least 80% 2'-O-methyl modifications; (7) the nucleotides at positions 7, 10, and 11 from the 3' end of the sense strand are not 2'-methoxy-ribonucleotides; (8) The nucleotides 1 and 2 from the 5' end of the sense strand are linked to each other via a phosphorothioate internucleotide linkage.

[0264] In an eighth aspect, there is provided herein a dsRNA comprising an antisense strand and a sense strand, each strand comprising at least 14 contiguous nucleotides having a 5' end and a 3' end, (1) the antisense strand comprises a sequence substantially complementary to any one of the nucleic acid sequences of SEQ ID NOs: 1 to 6; (2) the antisense strand contains at least 50% 2'-O-methyl modifications; (3) the nucleotides at positions 2, 4, 5, 6, 8, 10, 12, 14, 16, and 20 from the 5' end of the antisense strand are not 2'-methoxy-ribonucleotides; (4) nucleotides at positions 1-2 through 1-8 from the 3' end of the antisense strand are linked to each other via phosphorothioate internucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand contains at least 65% 2'-O-methyl modifications; (7) the nucleotides at positions 3, 7, 9, 11, and 13 from the 3' end of the sense strand are not 2'-methoxy-ribonucleotides; (8) The nucleotides 1 to 3 from the 5' end of the sense strand are linked to each other via phosphorothioate internucleotide linkages.

[0265] In a ninth aspect, there is provided herein a dsRNA comprising an antisense strand and a sense strand, each strand comprising at least 14 contiguous nucleotides having a 5' end and a 3' end, (1) the antisense strand comprises a sequence substantially complementary to any one of the nucleic acid sequences of SEQ ID NOs: 1 to 6; (2) the antisense strand contains at least 75% 2'-O-methyl modifications; (3) the nucleotides at positions 2, 6, 14, 16, and 20 from the 5' end of the antisense strand are not 2'-methoxy-ribonucleotides; (4) nucleotides at positions 1 to 7 and 19 to 20 from the 3' end of the antisense strand are linked to each other via phosphorothioate internucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand contains at least 65% 2'-O-methyl modifications; (7) the nucleotides at positions 7, 9, 10, and 11 from the 3' end of the sense strand are not 2'-methoxy-ribonucleotides; (8) The nucleotides at positions 1-2 and 14-15 from the 5' end of the sense strand are linked to each other via phosphorothioate internucleotide linkages.

[0266] In a tenth aspect, there is provided herein a dsRNA comprising an antisense strand and a sense strand, each strand comprising at least 14 contiguous nucleotides having a 5' end and a 3' end, (1) the antisense strand comprises a sequence substantially complementary to an IFNGR1, JAK1, JAK2, or STAT1 nucleic acid sequence; (2) the antisense strand contains at least 50% 2'-O-methyl modifications; (3) any one or more of the nucleotides at positions 2, 4, 5, 6, 8, 10, 12, 14, 16, and 20 from the 5' end of the antisense strand is not a 2'-methoxy-ribonucleotide; (4) nucleotides at positions 1-2 through 1-8 from the 3' end of the antisense strand are linked to each other via phosphorothioate internucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand contains at least 65% 2'-O-methyl modifications; (7) any one or more of the nucleotides at positions 3, 7, 9, 11, and 13 from the 3' end of the sense strand is not a 2'-methoxy-ribonucleotide; (8) The nucleotides 1 to 3 from the 5' end of the sense strand are linked to each other via phosphorothioate internucleotide linkages.

[0267] a) Design of anti-IFNGR1, anti-JAK1, anti-JAK2, and anti-STAT1 siRNA molecules The siRNA molecules of the present application are duplexes made of a sense strand and a complementary antisense strand, the antisense strand being sufficiently complementary to IFNGR1, JAK1, JAK2, or STAT1 mRNA to mediate RNAi. In certain embodiments, the siRNA molecules have a length of about 10-50 or more nucleotides, i.e., each strand contains 10-50 nucleotides (or nucleotide analogs). In other embodiments, the siRNA molecules have a length of about 15-30, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in each strand, where one of the strands is sufficiently complementary to the target region. In certain embodiments, the strands are arranged such that there are at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases at the end of the strand that are not aligned (i.e., no complementary base occurs in the opposing strand). This results in an overhang of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues at one or both ends of the duplex when the strands are annealed.

[0268] Generally, siRNAs can be designed using any method known in the art, for example, using the following protocol:

[0269] 1. The siRNA shall be specific to a target sequence, for example, a target sequence described in the Examples. The first strand shall be complementary to the target sequence, and the other strand shall be substantially complementary to the first strand. (See Examples for exemplary sense and antisense strands.) Exemplary target sequences are selected from any region of the target gene that results in strong gene silencing. Regions of the target gene include, but are not limited to, the 5' untranslated region (5'-UTR) of the target gene, the 3' untranslated region (3'-UTR) of the target gene, an exon of the target gene, or an intron of the target gene. Cleavage of the mRNA at these sites renders the translation of the corresponding IFNGR1, JAK1, JAK2, or STAT1 protein unnecessary. Target sequences from other regions of the IFNGR1, JAK1, JAK2, or STAT1 genes are also suitable for targeting. The sense strand is designed based on the target sequence.

[0270] 2. The sense strand of the siRNA is designed based on the sequence of the selected target site. In certain embodiments, the sense strand comprises about 15-25 nucleotides, for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides. In certain embodiments, the sense strand comprises 15, 16, 17, 18, 19, or 20 nucleotides. In certain embodiments, the sense strand is 15 nucleotides long. In certain embodiments, the sense strand is 18 nucleotides long. In certain embodiments, the sense strand is 20 nucleotides long. However, one of skill in the art will appreciate that siRNAs having lengths less than 15 nucleotides or greater than 25 nucleotides may also function to mediate RNAi. Thus, siRNAs of such lengths are also within the scope of the present invention so long as they retain the ability to mediate RNAi. Longer RNA silencing agents have been demonstrated to induce interferon or protein kinase R (PKR) responses in certain mammalian cells, which may not be desirable. In certain embodiments, the RNA silencing agent of the present invention does not induce PKR response (i.e., it is of sufficiently short length).However, longer RNA silencing agents can be useful, for example, in cell types that cannot generate PKR response, or in situations where PKR response is downregulated or suppressed by alternative means.

[0271] The siRNA molecule of the present invention has sufficient complementarity with the target sequence so that the siRNA can mediate RNAi. In general, it is contemplated that the siRNA that comprises a nucleotide sequence that is sufficiently complementary to the target sequence portion of the target gene will cause RISC-mediated cleavage of the target gene. Thus, in certain embodiments, the antisense strand of the siRNA is designed to have a sequence that is sufficiently complementary to a portion of the target. For example, the antisense strand can have 100% complementarity to the target site. However, it is not necessary that the complementarity is 100%. More than 80% identity, for example 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% complementarity is contemplated between the antisense strand and the target RNA sequence. The present application has the advantage that it can tolerate certain sequence modifications to enhance the efficiency and specificity of RNAi. In one embodiment, the antisense strand has 4, 3, 2, 1, or 0 mismatched nucleotides with the target region, such as a target region that differs by at least one base pair between wild-type and mutant alleles. For example, the target region includes a gain-of-function mutation, and the other strand is identical or substantially identical to the first strand. In addition, siRNA sequences with small insertions or deletions of 1 or 2 nucleotides can also be effective in mediating RNAi. Alternatively, siRNA sequences with substitutions or insertions of nucleotide analogs can be effective in inhibition.

[0272] 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 (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, the molecules are identical at that position. The percent identity between two sequences corresponds to the number of identical positions shared by the sequences (i.e., % 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.

[0273] Comparison of sequences and determination of percent identity between two sequences can be accomplished using mathematical algorithms. In one embodiment, alignment is generated at certain portions of the aligned sequences that have sufficient identity and not at portions that have a low degree of identity (i.e., local alignment). A non-limiting example of a local alignment algorithm utilized for comparing sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68, revised Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithm is incorporated into the BLAST program (version 2.0), Altschul, et al. (1990) J. Mol. Biol. 215:403-10.

[0274] 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 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). A non-limiting example of a mathematical algorithm utilized for global comparison of sequences is the Myers and Miller algorithm, CABIOS (1989). Such an algorithm is incorporated in the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing 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.

[0275] 3. The antisense or guide strand of the siRNA is routinely the same length as the sense strand and contains complementary nucleotides. In one embodiment, the guide and sense strands are fully complementary. That is, the strands are blunt-ended when aligned or annealed. In another embodiment, the strands of the siRNA may be paired to have 1-7 (e.g., 2, 3, 4, 5, 6 or 7), or 1-4 3' overhangs, e.g., 2, 3, or 4 nucleotides. The overhangs may comprise (or consist of) nucleotides corresponding to the target gene sequence (or its complement). Alternatively, the overhangs 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 2-nucleotide 3' overhang, such as TT. The overhang nucleotides may be either RNA or DNA. As described above, it is desirable to select a target region in which the mutant:wild type mismatch is a purine:purine mismatch.

[0276] 4. Using any method known in the art, compare potential targets to an appropriate genome database (human, mouse, rat, etc.) to eliminate the need to consider any target sequences that have significant homology to other coding sequences. One such method of sequence homology searching is known as BLAST and is available on the National Center for Biotechnology Information website.

[0277] 5. Select one or more sequences that meet the evaluation criteria.

[0278] General information regarding the design and use of siRNAs can be found in "The siRNA User Guide," available on the website of The Max-Plank-Institut fur Biophysikalische Chemie.

[0279] Alternatively, an siRNA may be functionally defined as a nucleotide sequence (or oligonucleotide sequence) that can hybridize to a target sequence (e.g., hybridize with 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA at 50°C or 70°C for 12-16 hours, followed by washing). Additional hybridization conditions include hybridization at 1xSSC at 70°C or 1xSSC at 50°C, 50% formamide, followed by washing at 0.3xSSC at 70°C, or hybridization at 4xSSC at 70°C or 4xSSC at 50°C, 50% formamide, followed by washing at 1xSSC at 67°C. The hybridization temperature for hybrids predicted to be less than 50 base pairs in length is determined by the melting temperature (T m ) is 5 to 10°C lower than T m is determined according to the formula: For hybrids less than 18 base pairs in length, T m (°C) = 2(# of A+T bases) + 4(# of G+C bases). For hybrids 18-49 base pairs in length, T m (°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+] in 1xSSC). + ]=0.165M). Additional examples of stringency conditions for polynucleotide hybridization are described 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, F.M. Usubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, which are incorporated herein by reference.

[0280] Negative control siRNA shall have the same nucleotide composition as selected siRNA, but no significant sequence complementarity with suitable genome.Such negative control can be designed by randomly scrambling the nucleotide sequence of selected siRNA.Homology search can be carried out to confirm that negative control lacks homology with any other gene in suitable genome.Furthermore, negative control siRNA can be designed by introducing one or more base mismatches into sequence.

[0281] 6. To verify the efficacy of siRNA in disrupting mRNA (e.g., wild-type or mutant IFNGR1, JAK1, JAK2, or STAT1 mRNA), the siRNA can be incubated with target cDNA (e.g., IFNGR1, JAK1, JAK2, or STAT1 cDNA) in a Drosophila melanogaster-based in vitro mRNA expression system. 32 Newly synthesized target mRNA (e.g., IFNGR1, JAK1, JAK2, or STAT1 mRNA) radioactively labeled 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 scrambling the nucleotide sequence of the selected siRNA. A homology search can be performed to confirm that the negative control lacks homology with any other genes in the appropriate genome. Additionally, a negative control siRNA can be designed by introducing one or more base mismatches into the sequence.

[0282] Anti-IFNGR1, anti-JAK1, anti-JAK2 or anti-STAT1 siRNA can be designed to target any of the above target sequences.siRNA comprises an antisense strand that is sufficiently complementary to target sequence to mediate the silencing of target sequence.In certain embodiments, the RNA silencing agent is siRNA.

[0283] In certain embodiments, the siRNA comprises a sense strand comprising a sequence shown in Tables 10 and 11, and an antisense strand comprising a sequence shown in Tables 10 and 11, respectively.

[0284] The siRNA-mRNA complementary site that results in optimal mRNA specificity and maximal mRNA cleavage is selected.

[0285] b) 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 IFNGR1, JAK1, JAK2, or STAT1 mRNA to direct gene silencing by 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 close to that observed between the miRNA and its target. In general, when the degree of sequence identity between the miRNA sequence and the corresponding target gene sequence is decreased, the tendency for post-transcriptional gene silencing to be mediated by translational repression rather than RNAi increases. Thus, in another embodiment where 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, miRNA sequences have partial complementarity with one or more short sequences (complementary sites) dispersed within 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, certain embodiments may target multiple complementary sites (e.g., 2, 3, 4, 5, or 6).

[0286] The ability of siRNA-like duplexes to mediate RNAi or translational repression can be predicted by the distribution of non-identical nucleotides between the target gene sequence and the silencing agent nucleotide sequence at the complementary site. In one embodiment where gene silencing by translational repression is desired, at least one non-identical nucleotide is present in the central portion of the complementary site such 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 embodiment, the "bulge" is centered at nucleotides 12 and 13 from the 5' end of the miRNA molecule.

[0287] c) Short hairpin RNA (shRNA) molecules In a particular characteristic embodiment, the present invention provides shRNA that can mediate the RNA silencing of IFNGR1, JAK1, JAK2 or STAT1 target sequence with high selectivity.In contrast to siRNA, shRNA mimics the natural precursor of microRNA (miRNA) and enters the top of gene silencing pathway.Therefore, shRNA is believed to mediate gene silencing more efficiently by being delivered through the entire natural gene silencing pathway.

[0288] miRNAs are about 22 nucleotide non-coding RNAs that can regulate gene expression at the post-transcriptional or translational level during plant and animal development. One common feature of miRNAs is that they are all excised from a precursor RNA stem loop of about 70 nucleotides, called pre-miRNA, presumably by the RNase III type enzyme Dicer, or its homologues. Naturally occurring miRNA precursors (pre-miRNAs) generally have a single strand that forms a double-stranded stem containing two complementary parts, and a loop that connects the two parts of the stem. In a typical pre-miRNA, the stem contains one or more bulges, e.g., extra nucleotides that create a single nucleotide "loop" in one part of the stem, and / or one or more unpaired nucleotides that create a gap in the mutual hybridization of the two parts of the stem. The short hairpin RNA or engineered RNA precursors of the present application are artificial constructs based on these naturally occurring pre-miRNAs, but engineered to deliver a desired RNA silencing agent (e.g., the siRNA of the present invention). By replacing the stem sequence of the pre-miRNA with a sequence complementary to the target mRNA, an shRNA is formed that is processed through the cellular gene silencing pathway, thereby efficiently mediating RNAi.

[0289] The necessary elements of an shRNA molecule include a first portion and a second portion that have sufficient complementarity to anneal or hybridize to form a duplex or double stranded stem portion. The two portions need not be completely or perfectly complementary. The first and second "stem" portions are joined by a portion that has a sequence that has insufficient sequence complementarity to anneal or hybridize to other portions of the shRNA. This latter portion is referred to as the "loop" portion in the shRNA molecule. The shRNA molecule is processed to generate siRNAs. The shRNA can also include one or more bulges, i.e., extra nucleotides that create small nucleotide "loops" in a portion of the stem, e.g., 1, 2, or 3 nucleotide loops. The stem portions can be of the same length, or some can include overhangs, e.g., 1-5 nucleotides. The overhanging nucleotides can include, e.g., uracils (U), e.g., all Us. Such Us are specifically encoded by thymidines (T) in the shRNA-encoding DNA that signal the termination of transcription.

[0290] In the shRNA (or engineered precursor RNA) of the present invention, a portion of the double-stranded stem is a nucleic acid sequence that is complementary (or antisense) to an IFNGR1, JAK1, JAK2, or STAT1 target sequence. In certain embodiments, one strand of the stem portion of the shRNA is sufficiently complementary (e.g., antisense) to a target RNA (e.g., mRNA) sequence to mediate degradation or cleavage of the target RNA via RNA interference (RNAi). Thus, the engineered RNA precursor comprises a double stem having two portions and a loop connecting the two stem portions. The antisense portion can be at the 5' or 3' end of the stem. The stem portion of the shRNA is about 15 to about 50 nucleotides in length. In certain embodiments, the two stem portions are about 18 or 19 to about 21, 22, 23, 24, 25, 30, 35, 37, 38, 39, or 40 or more nucleotides in length. In certain embodiments, the length of the stem portion shall be 21 nucleotides or more. When used in mammalian cells, the length of the stem portion should be less than about 30 nucleotides to avoid eliciting non-specific responses such as the interferon pathway. In non-mammalian cells, the stem may be more than 30 nucleotides. In fact, the stem may include a much larger section (up to and including the entire mRNA) that is complementary to the target mRNA. In fact, the stem portion may include a much larger section (up to and including the entire mRNA) that is complementary to the target mRNA.

[0291] The two parts of the double stem must be sufficiently complementary to hybridize to form a double stem. Thus, the two parts can be, but do not need to be, completely or perfectly complementary. Furthermore, the two stem parts can be the same length, or one part can include an overhang of 1, 2, 3, or 4 nucleotides. The overhang nucleotides can include, for example, uracil (U), such as all U. The loop in the shRNA or engineered RNA precursor can differ from the natural pre-miRNA sequence by modifying the loop sequence to increase or decrease the number of paired nucleotides, or by replacing all or part of the loop sequence with a tetraloop or other loop sequence. Thus, the loop in the shRNA or engineered RNA precursor can be 2, 3, 4, 5, 6, 7, 8, 9, or more nucleotides long, such as 15 or 20 or more nucleotides long.

[0292] The loop in the shRNA or engineered RNA precursor may differ from the native pre-miRNA sequence by modifying the loop sequence to increase or decrease the number of paired nucleotides, or by replacing all or part of the loop sequence with a tetraloop or other loop sequence. Thus, the loop portion in the shRNA may be about 2 to about 20 nucleotides in length, i.e., about 2, 3, 4, 5, 6, 7, 8, 9, or more, e.g., 15 or 20 nucleotides, or more. In certain embodiments, the loop consists of or includes a "tetraloop" sequence. Exemplary tetraloop sequences include, but are not limited to, the sequences GNRA, where N is any nucleotide and R is a purine nucleotide, GGGG, and UUUU.

[0293] In certain embodiments, the shRNA of the present application comprises the sequence of the desired siRNA molecule described above. In other embodiments, the sequence of the antisense portion of the shRNA can be designed essentially as described above, or generally by selecting a sequence of 18, 19, 20, 21 nucleotides or longer from within the target RNA (e.g., IFNGR1, JAK1, JAK2, or STAT1 mRNA), for example, from a region of 100 to 200 or 300 nucleotides upstream or downstream of the translation start point. In general, the sequence can be selected from any portion of the target RNA (e.g., mRNA), such as the 5'UTR (untranslated region), coding sequence, or 3'UTR. This sequence can optionally immediately follow a region of the target gene that contains two adjacent AA nucleotides. The last two nucleotides of the nucleotide sequence can be selected to be UU. This approximately 21 nucleotide sequence is used to generate a portion of the double-stranded stem of the shRNA. This sequence can replace the stem portion of the wild-type pre-miRNA sequence, for example, enzymatically, or be included in the complete sequence that is synthesized. For example, an engineered RNA precursor construct can be constructed, e.g., from a wild-type pre-miRNA, using restriction enzymes and synthesizing a DNA oligonucleotide that encodes the entire stem-loop engineered RNA precursor, or only the portion to be inserted into the double-stranded stem of the precursor.

[0294] The engineered RNA precursor contains in the duplex stem approximately 21-22 nucleotide sequences of the siRNA or siRNA-like duplex desired to be produced in vivo. Thus, the stem portion of the engineered RNA precursor contains at least 18 or 19 nucleotide pairs corresponding to the sequence of the exon portion of the gene whose expression is to be reduced or inhibited. The two 3' nucleotides flanking this region of the stem are selected to maximize the production of siRNA from the engineered RNA precursor, as well as to maximize the effectiveness of the resulting siRNA in targeting the corresponding mRNA for translational repression or destruction by RNAi in vivo and in vitro.

[0295] In certain embodiments, the shRNA of the present invention comprises miRNA sequence, optionally end-modified miRNA sequence, to enhance entry into RISC. The miRNA sequence can be similar or identical to the sequence of any naturally occurring miRNA (e.g., The miRNA Registry; Griffiths-Jones S, Nuc.Acids Res., 2004). To date, more than 1,000 naturally occurring miRNAs have been identified, and together they are believed to comprise about 1% of all predicted genes in genome. Many natural miRNAs are clustered together in the introns of pre-mRNAs and can be identified in silico using homology-based searches (Pasquinelli et al., 2000; Lagos-Quintana et al., 2001; Lau et al., 2001; Lee and Ambros, 2001) or computer algorithms (e.g., MiRScan, MiRSeeker) that predict the ability of candidate miRNA genes to form stem-loop structures in pri-mRNAs (Grad et al., Mol. Cell., 2003; Lim et al. al., Genes Dev., 2003; Lim et al., Science, 2003; Lai EC et al., Genome Bio., 2003). Online registries provide searchable databases of all published miRNA sequences (The miRNA Registry, Sanger Institute website; Griffiths-Jones S, Nuc. Acids Res., 2004).Exemplary naturally occurring miRNAs include lin-4, let-7, miR-10, mirR-15, miR-16, miR-168, miR-175, miR-196 and their homologs, as well as other naturally occurring miRNAs from humans and certain model organisms, such as Drosophila melanogaster, Caenorhabditis elegans, zebrafish, Arabidopsis thalania, Mus musculus, and Rattus norvegicus (described in PCT International Publication No. WO 03 / 029459).

[0296] Naturally occurring miRNAs are expressed by endogenous genes in vivo and processed from hairpin or stem-loop precursors (pre-miRNAs or pri-miRNAs) by Dicer or other RNAses (Lagos-Quintana et al., Science, 2001; Lau et al., Science, 2001; Lee and Ambros, Science, 2001; Lagos-Quintana et al., Curr. Biol., 2002; Mourelatos et al., Genes Dev., 2002; Reinhart et al., Science, 2002; Ambros et al., Curr. Biol., 2003; Brennecke et al., 2003; Lagos-Quintana et al., RNA, 2003; Lim et al., Genes Dev., 2003; Lim et al., Science, 2003). miRNAs can exist transiently in vivo as double-stranded duplexes, but only one strand is incorporated into the RISC complex to direct gene silencing. Certain miRNAs, such as plant miRNAs, have perfect or near-perfect complementarity to their target mRNAs, and thus directly cleave the target mRNA. Other miRNAs have less than perfect complementarity to their target mRNAs, and thus directly suppress the translation of the target mRNA. It is believed that the degree of complementarity between a miRNA and its target mRNA determines its mechanism of action. For example, perfect or near-perfect complementarity between a miRNA and its target mRNA predicts a cleavage mechanism (Yekta et al., Science, 2004), whereas less than perfect complementarity predicts a translation suppression mechanism. In certain embodiments, the miRNA sequence is that of a naturally occurring miRNA sequence whose abnormal expression or activity correlates with miRNA disorder.

[0297] d) Dual-Functional Oligonucleotide Tethering Factors In other embodiments, the RNA silencing agent of the present invention comprises a dual-functional oligonucleotide tethering factor useful for the intercellular recruitment of miRNA. Animal cells express a set of miRNAs, which are non-coding RNAs of about 22 nucleotides that can regulate gene expression at the post-transcriptional or translational level. The dual-functional oligonucleotide tethering factor can bind the miRNA bound to RISC and recruit it to the target mRNA, thereby suppressing the expression of genes involved in, for example, the atherosclerosis process. The use of oligonucleotide tethering factors offers several advantages over existing techniques for suppressing the expression of specific genes. First, the methods described herein allow an endogenous molecule (often abundant), miRNA, to mediate RNA silencing. Thus, the methods described herein eliminate the need to introduce an exogenous molecule (e.g., siRNA) to mediate RNA silencing. Second, the RNA silencing agent and the linking moiety (e.g., oligonucleotide, e.g., 2'-O-methyl oligonucleotide) can be stabilized and made resistant to nuclease activity. As a result, the tethering factor of the present invention can be designed for direct delivery, eliminating the need for indirect delivery (e.g., virus) of precursor molecules or plasmids designed to make the desired agent in cells. Third, the tethering factor and each part can be designed to match a specific mRNA site and a specific miRNA. The design can be cell and gene product specific. Fourth, in the methods disclosed herein, the mRNA remains intact, which allows the skilled artisan to use the cell's own machinery to block protein synthesis in short pulses. As a result, these methods of RNA silencing are highly tunable.

[0298] The dual-functional oligonucleotide tethering agents of the present invention ("tethering agents") are designed to recruit miRNAs (e.g., endogenous cellular miRNAs) to target mRNAs to induce regulation of genes of interest. In certain embodiments, the tethering agents have the formula TL-μ, where T is the mRNA targeting moiety, L is the linking moiety, and μ is the miRNA recruiting moiety. Any one or more of the moieties can be double-stranded. In certain embodiments, each moiety is single-stranded.

[0299] The moieties within the tethering element can be positioned or linked (5' to 3' direction) as shown in the formula TL-μ (i.e., the 3' end of the targeting moiety linked to the 5' end of the linking moiety and the 3' end of the linking moiety linked to the 5' end of the miRNA recruitment moiety). Alternatively, the moieties can be positioned or linked within the tethering element as follows: μ-TL (i.e., the 3' end of the miRNA recruitment moiety is linked to the 5' end of the linking moiety and the 3' end of the linking moiety is linked to the 5' end of the targeting moiety).

[0300] The above mRNA targeting moieties are capable of capturing a specific target mRNA. According to the present invention, expression of the target mRNA is undesirable, and therefore translational inhibition of the mRNA is desired. The mRNA targeting moiety shall be of a size sufficient to effectively bind to the target mRNA. The length of the targeting moiety varies widely, depending in part on the length of the target mRNA and the degree of complementarity between the target mRNA and the targeting moiety. In various embodiments, the targeting moiety is less than about 200, 100, 50, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 nucleotides in length. In certain embodiments, the targeting moiety is about 15 to about 25 nucleotides in length.

[0301] As mentioned above, the miRNA recruiting portion can be associated with miRNA. According to the present application, the miRNA can be any miRNA that can suppress target mRNA. It has been reported that mammals have more than 250 endogenous miRNAs (Lagos-Quintana et al. (2002) Current Biol. 12: 735-739; Lagos-Quintana et al. (2001) Science 294: 858-862; and Lim et al. (2003) Science 299: 1540). In various embodiments, the miRNA can be any miRNA recognized in the art.

[0302] Linking moiety is any agent that can link targeting moiety so that the activity of targeting moiety is maintained.Linking moiety can be an oligonucleotide moiety that contains a sufficient number of nucleotides so that targeting agent can fully interact with each target.Linking moiety has little or no sequence homology with cellular mRNA or miRNA sequence.Exemplary linking moieties include one or more 2'-O-methyl nucleotides, such as 2'-β-methyl adenosine, 2'-O-methyl thymidine, 2'-O-methyl guanosine or 2'-O-methyl uridine.

[0303] e) Gene Silencing Oligonucleotides In certain exemplary embodiments, gene expression (i.e., IFNGR1, JAK1, JAK2, or STAT1 gene expression) can be regulated using oligonucleotide-based compounds that include two or more single-stranded antisense oligonucleotides linked via their 5' ends, allowing for the presence of two or more accessible 3' ends, to effectively inhibit or reduce IFNGR1, JAK1, JAK2, or STAT1 gene expression. Such linked oligonucleotides are also known as gene silencing oligonucleotides (GSOs) (see, e.g., U.S. Patent No. 8,431,544, assigned to Idera Pharmaceuticals, Inc., which is incorporated herein by reference in its entirety for all purposes).

[0304] The linkage at the 5' end of the GSO is independent of other oligonucleotide linkages and can be direct via the 5', 3', or 2' hydroxyl groups, or indirect, via a non-nucleotidic linker or nucleoside, using either the 2' or 3' hydroxyl position of the nucleoside. Linkages can also utilize functionalized sugars or nucleobases of the 5' terminal nucleotide.

[0305] GSOs can contain two identical or different sequences conjugated at their 5'-5' ends via phosphodiester, phosphorothioate, or non-nucleoside linkers. Such compounds can contain 15-27 nucleotides that are complementary to a specific portion of an mRNA target of interest for antisense downregulation of gene products. GSOs containing identical sequences can bind to a specific mRNA via Watson-Crick hydrogen bonding interactions and inhibit protein expression. GSOs containing different sequences can bind to two or more distinct regions of one or more mRNA targets and inhibit protein expression. Such compounds are composed of heteronucleotide sequences complementary to the target mRNA and form a stable duplex structure by Watson-Crick hydrogen bonding. Under certain conditions, GSOs containing two free 3' ends (5'-5' attached antisense) can be more potent inhibitors of gene expression than those with a single free 3' end or no free 3' ends.

[0306] In some embodiments, the non-nucleotidic linker is glycerol or a linker of the formula HO-(CH2) o -CH(OH)-(CH2) p -OH, where o and p are independently integers from 1 to about 6, 1 to about 4, or 1 to about 3. In some other embodiments, the non-nucleotidic linker is a derivative of 1,3-diamino-2-hydroxypropane. Some such derivatives have the formula HO--(CH2)m--C(O)NH--CH2--CH(OH)--CH2--NHC(O)--(CH2) m --OH, where m is an integer from 0 to about 10, 0 to about 6, 2 to about 6, or 2 to about 4.

[0307] Some non-nucleotide linkers allow attachment of three or more GSO moieties.For example, the non-nucleotide linker glycerol has three hydroxyl groups to which GSO moieties can be covalently attached.Thus, some oligonucleotide-based compounds of the present invention comprise two or more oligonucleotides linked to nucleotide or non-nucleotide linkers.Such oligonucleotides according to the present invention are called "branched".

[0308] In certain embodiments, the GSO is at least 14 nucleotides in length. In certain exemplary embodiments, the GSO is 15-40 nucleotides in length or 20-30 nucleotides in length. Thus, the component oligonucleotides of the GSO can be independently 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length.

[0309] These oligonucleotides can be prepared by art-recognized methods such as phosphoramidate or H-phosphonate chemistry, which can be performed manually or by an automatic synthesizer.These oligonucleotides can also be modified in multiple ways without impairing their ability to hybridize to mRNA.Such modifications can include at least one internucleotide linkage of the oligonucleotide that is an alkyl phosphonate, phosphorothioate, phosphorodithioate, methyl phosphonate, phosphate ester, alkyl phosphonothioate, phosphoramidate, carbamate, carbonate, phosphate hydroxyl, acetamidate, carboxymethyl ester, or a combination of these and other internucleotide linkages between the 5' end of one nucleotide and the 3' end of another nucleotide, where the phosphodiester linkage of the 5' nucleotide is replaced with any number of chemical groups.

[0310] V. Modified anti-IFNGR1, anti-JAK1, anti-JAK2, or anti-STAT1 RNA silencing agents In certain aspects of the present invention, the RNA silencing agent of the present application (or any part thereof) can be modified as described above to further improve the activity of the agent.For example, the RNA silencing agent described in Section II above can be modified with any of the modifications described below.Modifications can act, in part, to further improve target discrimination, to improve the stability of the agent (e.g., to prevent degradation), to promote cellular uptake, to improve targeting efficiency, to improve the effectiveness of binding (e.g., to target), to improve patient tolerance to the agent, and / or to reduce toxicity.

[0311] 1) Modifications to improve target discrimination In certain embodiments, the RNA silencing agents of the present application may be substituted with destabilizing nucleotides to improve single nucleotide target discrimination (see U.S. Application No. 11 / 698,689, filed January 25, 2007, and U.S. Provisional Application No. 60 / 762,225, filed January 25, 2006, both of which are incorporated herein by reference). Such modifications may be sufficient to abolish the specificity of the RNA silencing agent for non-target mRNAs (e.g., wild-type mRNAs) without appreciably affecting the specificity of the RNA silencing agent for target mRNAs (e.g., gain-of-function mutant mRNAs).

[0312] In certain embodiments, the RNA silencing agent of the present application is modified by introducing at least one universal nucleotide into its antisense strand. A universal nucleotide comprises a base moiety that can indiscriminately base pair with any of the four conventional nucleotide bases (e.g., A, G, C, U). A universal nucleotide is contemplated to have only a relatively small effect on the stability of an RNA duplex or the stability of a duplex formed by the guide strand of the RNA silencing agent and the target mRNA. Exemplary universal nucleotides include those having an inosine base moiety or an inosine-like 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 embodiments, the universal nucleotide is an inosine residue or a naturally occurring analogue thereof.

[0313] In certain embodiments, the RNA silencing agent of the present invention is modified by introducing at least one destabilizing nucleotide within 5 nucleotides of the specificity-determining nucleotide (i.e., the nucleotide that recognizes the disease-associated polymorphism). For example, the destabilizing nucleotide can be introduced within 5, 4, 3, 2, or 1 nucleotide of the specificity-determining nucleotide. In an exemplary embodiment, the destabilizing nucleotide is introduced at a position 3 nucleotides away 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 embodiments, the destabilizing nucleotide is introduced in the same strand or strand portion that contains the specificity-determining nucleotide.

[0314] 2) Modifications to increase efficacy and specificity In certain embodiments, the RNA silencing agent of the present invention can be modified according to asymmetric design rules to easily improve its effectiveness and specificity in mediating RNAi (see U.S. Patent Nos. 8,309,704, 7,750,144, 8,304,530, 8,329,892 and 8,309,705). Such modifications facilitate the antisense strand of siRNA (e.g., siRNA designed using the method of the present invention, or siRNA produced from shRNA) to enter RISC in favor of the sense strand. This allows the antisense strand to preferentially induce the cleavage or translational suppression of target mRNA, thus increasing or improving the efficiency of target cleavage and silencing. In certain embodiments, the asymmetry of an RNA silencing agent is improved by decreasing the base pairing 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 pairing strength between the 3' end of the antisense strand (AS3') and the 5' end of the sense strand (S'5) of the RNA silencing agent.

[0315] In one embodiment, the asymmetry of the RNA silencing agent of the present application can be improved 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 there are G:C base pairs 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 improved 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 certain embodiments, 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 improved such that there is at least one wobble base pair, for example, 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 improved such that there is at least one base pair that includes a rare nucleotide, such as inosine (I). In certain 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 improved 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.

[0316] 3) RNA silencing agents with improved stability The RNA silencing agent of the present application can be modified to improve stability in serum or in cell culture growth medium.To improve stability, 3'-residue can be stabilized against degradation and can be selected to be composed of purine nucleotides, such as adenosine or guanosine nucleotides.Alternatively, the substitution of pyrimidine nucleotides with modified analogs, such as the substitution of uridine with 2'-deoxythymidine, is tolerated and does not affect the efficiency of RNA interference.

[0317] In one aspect, the present application features an RNA silencing agent comprising a first and a second strand, wherein the second strand and / or the first strand are modified by replacing an internal nucleotide with a modified nucleotide to enhance in vivo stability 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 within a single-stranded molecule or within a strand of a double-stranded or double-stranded molecule. In one embodiment, the sense strand and / or the antisense strand are modified by replacement of at least one internal nucleotide. In another embodiment, the sense strand and / or the antisense strand are modified by 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 strand and / or the antisense strand are modified by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more internal nucleotides, hi yet another embodiment, the sense strand and / or the antisense strand are modified by substitution of all of the internal nucleotides.

[0318] In one aspect, the present application features an RNA silencing agent that is at least 80% chemically modified.In certain embodiments, the RNA silencing agent can be completely chemically modified, i.e., 100% of the nucleotides are chemically modified.In another aspect, the present application features an RNA silencing agent that comprises at least 80% chemically modified 2'-OH ribose groups.In certain embodiments, the RNA silencing agent comprises about 80%, 85%, 90%, 95% or 100% chemically modified 2'-OH ribose groups.

[0319] In certain embodiments, RNA silencing agent can comprise 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, is not substantially affected, for example, at the 5'-end and / or 3'-end of siRNA molecule.Furthermore, end can be stabilized by incorporating modified nucleotide analogue.

[0320] Exemplary nucleotide analogs include sugar- and / or backbone-modified ribonucleotides (i.e., containing modifications to the phosphate sugar backbone). For example, the phosphodiester linkages of natural RNA can be modified to include at least one nitrogen or sulfur heteroatom. In exemplary backbone-modified ribonucleotides, the phosphate ester group that is linked to adjacent ribonucleotides is replaced by a modified group, for example, a phosphothioate group. In exemplary sugar-modified ribonucleotides, the 2'OH-group is replaced with 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.

[0321] In certain embodiments, the modifications are 2'-fluoro, 2'-amino, and / or 2'-thio modifications. 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 all uridines and cytidines. 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-fluorouridine. 2'-deoxy-nucleotide and 2'-Ome nucleotide can also be used in the modified RNA silencing agent portion of the present invention.Additional modified residues include deoxyabasic, inosine, N3-methyl-uridine, N6,N6-dimethyl-adenosine, pseudouridine, purine ribonucleoside, and ribavirin.In certain embodiments, the 2' moiety is a methyl group, so that the linking moiety is a 2'-O-methyl oligonucleotide.

[0322] In certain embodiments, the RNA silencing agent of the present application 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 acids that can be modified, such as 2'-deoxy-2''-fluorouridine. In addition, LNA increases the specificity of oligonucleotides by constraining the sugar moiety in a 3'-endo conformation, thereby pre-organizing the nucleotide for base pairing and increasing the melting temperature of oligonucleotides by as much as 10°C per base.

[0323] In another exemplary embodiment, the RNA silencing agent of the present application comprises a 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 that can form a polyamide backbone, making it highly resistant to nuclease digestion and conferring improved binding specificity to the molecule (Nielsen, et al., Science, (2001), 254: 1497-1500).

[0324] Also contemplated are nucleobase-modified ribonucleotides, i.e. ribonucleotides, which contain at least one non-naturally occurring nucleobase instead of naturally occurring nucleobases. Bases can be modified to block the activity of adenosine deaminase. Examples of 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. Note that the above modifications can also be combined.

[0325] In other embodiments, crosslinking can be used to change the pharmacokinetics of the RNA silencing agent, for example, to extend its half-life in the body. Thus, the present application includes an RNA silencing agent having two complementary strands of nucleic acid, the two strands being crosslinked. Thus, the present application includes an RNA silencing agent that is conjugated or not conjugated (e.g., at its 3' end) to another moiety (e.g., a non-nucleic acid moiety such as a peptide) or an organic compound (e.g., a dye), etc. Modification of the siRNA derivative in this way can improve the uptake into cells or improve the cell targeting activity of the resulting siRNA derivative compared to the corresponding siRNA, and is useful for tracking the siRNA derivative in cells or improving the stability of the siRNA derivative compared to the corresponding siRNA.

[0326] Other exemplary modifications include: (a) 2' modifications, such as providing a 2'OMe moiety on a U in the sense or antisense strand, particularly in the sense strand, or providing a 2'OMe moiety in a 3' overhang, for example at the 3' end (3' end means the 3' atom or the 3' most part of the molecule, for example the 3' most P or 2' position as indicated by the context); (b) backbone modifications, such as by replacing O with S in the phosphate backbone, for example providing a phosphorothioate modification to U or A or both, particularly in the antisense strand; for example by replacing O with S; (c) replacing U with a C5 amino linker; (d) replacing A with G (the sequence change may in certain embodiments be located in the sense strand but not in 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 but not the antisense strand, or in which the antisense strand has few such modifications. Further exemplary modifications include the use of a methylated P in the 3' overhang, e.g., at the 3' end; combinations of 2' modifications, e.g., providing a 2'OMe moiety and modifying the backbone, e.g., replacing O with S, e.g., providing a phosphorothioate modification, or the use of a methylated P in the 3' overhang, e.g., at the 3' end; modifications with 3' alkyls; modifications with abasic pyrrolidone in the 3' overhang, e.g., at the 3' end; modifications with naproxen, ibuprofen, or other moieties that inhibit degradation at the 3' end.

[0327] Highly modified RNA silencing agents In certain embodiments, the RNA silencing agent comprises at least 80% chemically modified nucleotides. In certain embodiments, the RNA silencing agent is fully chemically modified, i.e., 100% of the nucleotides are chemically modified.

[0328] In certain embodiments, the RNA silencing agent is 2'-O-methyl rich, i.e., comprises more than 50% 2'-O-methyl content. In certain embodiments, the RNA silencing agent comprises at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% 2'-O-methyl nucleotide content. In certain embodiments, the RNA silencing agent comprises at least about 70% 2'-O-methyl nucleotide modifications. In certain embodiments, the RNA silencing agent comprises about 70% to about 90% 2'-O-methyl nucleotide modifications. In certain embodiments, the RNA silencing agent is a dsRNA comprising an antisense strand and a sense strand. In certain embodiments, the antisense strand comprises at least about 70% 2'-O-methyl nucleotide modifications. In certain embodiments, the antisense strand comprises about 70% to about 90% 2'-O-methyl nucleotide modifications. In certain embodiments, the sense strand comprises at least about 70% 2'-O-methyl nucleotide modifications. In certain embodiments, the sense strand comprises about 70% to about 90% 2'-O-methyl nucleotide modifications. In certain embodiments, the sense strand comprises 100% 2'-O-methyl nucleotide modifications.

[0329] 2'-O-methyl-rich RNA silencing agents and specific chemical modification patterns are further described in USSN 16 / 550,076 (filed August 23, 2019) and USSN 16 / 999,759 (filed August 21, 2020), each of which is incorporated by reference herein.

[0330] Internucleotide linkage modifications In certain embodiments, at least one internucleotide linkage, intersubunit linkage, or nucleotide backbone is modified in the RNA silencing agent. In certain embodiments, all of the internucleotide linkages in the RNA silencing agent are modified. In certain embodiments, the modified internucleotide linkage comprises a phosphorothioate internucleotide linkage. In certain embodiments, the RNA silencing agent comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 phosphorothioate internucleotide linkages. In certain embodiments, the RNA silencing agent comprises 4-16 phosphorothioate internucleotide linkages. In certain embodiments, the RNA silencing agent comprises 8-13 phosphorothioate internucleotide linkages. In certain embodiments, the RNA silencing agent is a dsRNA comprising an antisense strand and a sense strand, each comprising a 5' end and a 3' end. In certain embodiments, nucleotides at positions 1 and 2 from the 5' end of the sense strand are linked to adjacent ribonucleotides via phosphorothioate internucleotide linkages. In certain embodiments, nucleotides at positions 1 and 2 from the 3' end of the sense strand are linked to adjacent ribonucleotides via phosphorothioate internucleotide linkages. In certain embodiments, nucleotides at positions 1 and 2 from the 5' end of the antisense strand are linked to adjacent ribonucleotides via phosphorothioate internucleotide linkages. In certain embodiments, nucleotides at positions 1-2 and 1-8 from the 3' end of the antisense strand are linked to adjacent ribonucleotides via phosphorothioate internucleotide linkages. In certain embodiments, nucleotides at positions 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, or 1-8 from the 3' end of the antisense strand are linked to adjacent ribonucleotides via phosphorothioate internucleotide linkages. In certain embodiments, nucleotides 1-2 and 1-7 from the 3' end of the antisense strand are linked to adjacent ribonucleotides via phosphorothioate internucleotide linkages.

[0331] In one aspect, the disclosure provides a modified oligonucleotide, the oligonucleotide having a 5' end, a 3' end complementary to a target, the oligonucleotide comprising a sense and an antisense strand, and at least one modified intersubunit linkage of formula (I): [ka] (In the formula, B is a base pair moiety; W is selected from the group consisting of O, OCH2, OCH, CH2, and CH; X is halo, hydroxy, or C 1-6 alkoxy; Y is O - , OH, OR, NH - , NH2, S - and SH, Z is selected from the group consisting of O and CH2; R is a protecting group; [ka] is any double bond).

[0332] In an embodiment of formula (I), when W is CH: [ka] is a double bond.

[0333] In an embodiment of formula (I), when W is selected from the group consisting of O, OCH2, OCH, CH2: [ka] is a single bond.

[0334] In an embodiment of formula (I), Y is O - Then either Z or W is not O.

[0335] In an embodiment of Formula (I), Z is CH2 and W is CH2. In another embodiment, the modified intersubunit linkage of Formula (I) is a modified intersubunit linkage of Formula (II): [ka]

[0336] In one embodiment of Formula (I), Z is CH and W is O. In another embodiment, the modified intersubunit linkage of Formula (I) is a modified intersubunit linkage of Formula (III): [ka]

[0337] In an embodiment of Formula (I), Z is O and W is CH. In another embodiment, the modified intersubunit linkage of Formula (I) is a modified intersubunit linkage of Formula (IV): [ka]

[0338] In one embodiment of Formula (I), Z is O and W is CH. In another embodiment, the modified intersubunit linkage of Formula (I) is a modified intersubunit linkage of Formula V: [ka]

[0339] In an embodiment of Formula (I), Z is O and W is OCH. In another embodiment, the modified intersubunit linkage of Formula (I) is a modified intersubunit linkage of Formula VI: [ka]

[0340] In one embodiment of Formula (I), Z is CH and W is CH. In another embodiment, the modified intersubunit linkage of Formula (I) is a modified intersubunit linkage of Formula VII: [ka]

[0341] In an embodiment of Formula (I), the base pair moiety B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0342] In one embodiment, the modified oligonucleotide is incorporated into an siRNA, the modified siRNA having a 5' end, a 3' end complementary to the target, and the siRNA comprises a sense strand and an antisense strand, and at least one modified intersubunit linkage of any one or more of Formula (I), Formula (II), Formula (III), Formula (IV), Formula (V), Formula (VI), or Formula (VII).

[0343] In one embodiment, the modified oligonucleotide is incorporated into an siRNA, the modified siRNA has a 5' end, a 3' end, is complementary to a target, comprises a sense and an antisense strand, and the siRNA comprises at least one modified intersubunit linkage and is of Formula VIII: [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; Between the subunits there is a bridge connecting two optionally modified nucleosides.

[0344] In one embodiment, C is O - If so, then either A or D is not O.

[0345] In one embodiment, D is CH. In another embodiment, the modified intersubunit linkage of formula VIII is a modified intersubunit linkage of formula (IX): [ka]

[0346] In one embodiment, D is O. In another embodiment, the modified intersubunit linkage of formula VIII is a modified intersubunit linkage of formula (X): [ka]

[0347] In one embodiment, D is CH. In another embodiment, the modified intersubunit linkage of formula (VIII) is a modified intersubunit linkage of formula (XI): [ka]

[0348] In one embodiment, D is CH. In another embodiment, the modified intersubunit linkage of formula VIII is a modified intersubunit linkage of formula (XII): [ka]

[0349] In another embodiment, the modified intersubunit linkage of formula (VII) is a modified intersubunit linkage of formula (XIV): [ka]

[0350] In one embodiment, D is OCH. In another embodiment, the modified intersubunit linkage of formula (VII) is a modified intersubunit linkage of formula (XIII): [ka]

[0351] In another embodiment, the modified intersubunit linkage of formula (VII) is a modified intersubunit linkage of formula (XXa): [ka]

[0352] In one embodiment of the modified siRNA conjugate, each optionally modified nucleoside is independently selected at each occurrence from the group consisting of adenosine, guanosine, cytidine, and uridine.

[0353] In certain exemplary embodiments of Formula (I), W is O. In another embodiment, W is CH. In yet another embodiment, W is CH.

[0354] In certain exemplary embodiments of Formula (I), X is OH. In other embodiments, X is OCH. In yet other embodiments, X is halo.

[0355] In certain embodiments of Formula (I), the modified siRNA does not contain a 2'-fluoro substituent.

[0356] In an embodiment of formula (I), Y is O - In another embodiment, Y is OH. In yet another embodiment, Y is OR. In yet another embodiment, Y is NH -In one embodiment, Y is NH. In another embodiment, Y is S - In yet another embodiment, Y is SH.

[0357] In one embodiment of Formula (I), Z is O. In another embodiment, Z is CH.

[0358] In one embodiment, the modified intersubunit linkage is inserted at positions 1-2 of the antisense strand. In another embodiment, the modified intersubunit linkage is inserted at positions 6-7 of the antisense strand. In yet another embodiment, the modified intersubunit linkage is inserted at positions 10-11 of the antisense strand. In yet another embodiment, the modified intersubunit linkage is inserted at positions 19-20 of the antisense strand. In one embodiment, the modified intersubunit linkage is inserted at positions 5-6 and 18-19 of the antisense strand.

[0359] In an exemplary embodiment of the modified siRNA linkage of formula (VIII), C is O - In another embodiment, C is OH. In yet another embodiment, C is OR 1 In yet another embodiment, C is NH - In one embodiment, C is NH. In another embodiment, C is S - In yet another embodiment, C is SH.

[0360] In an exemplary embodiment of the modified siRNA linkage of formula (VIII), A is O. In another embodiment, A is CH. In yet another embodiment, C is OR. 1 In yet another embodiment, C is NH - In one embodiment, C is NH. In another embodiment, C is S - In yet another embodiment, C is SH.

[0361] In certain embodiments of the modified siRNA linkage of formula (VIII), the optionally modified nucleoside is adenosine.In another embodiment of the modified siRNA linkage of formula (VIII), the optionally modified nucleoside is guanosine.In certain embodiments of the modified siRNA linkage of formula (VIII), the optionally modified nucleoside is cytidine.In certain embodiments of the modified siRNA linkage of formula (VIII), the optionally modified nucleoside is uridine.

[0362] In an embodiment of the modified siRNA linkage, the linkage is inserted at position 1-2 of the antisense strand. In another embodiment, the linkage is inserted at position 6-7 of the antisense strand. In yet another embodiment, the linkage is inserted at position 10-11 of the antisense strand. In yet another embodiment, the linkage is inserted at position 19-20 of the antisense strand. In one embodiment, the linkage is inserted at position 5-6 and 18-19 of the antisense strand.

[0363] In certain embodiments, base pair moiety B is adenine. In certain embodiments of formula (I), base pair moiety B is guanine. In certain embodiments of formula (I), base pair moiety B is cytosine. In certain embodiments of formula (I), base pair moiety B is uracil.

[0364] In one embodiment of Formula (I), W is O. In one embodiment of Formula (I), W is CH. In one embodiment of Formula (I), W is CH.

[0365] In an embodiment of formula (I), X is OH. In an embodiment of formula (I), X is OCH. In an embodiment of formula (I), X is halo.

[0366] In an exemplary embodiment of Formula (I), the modified oligonucleotide does not include a 2'-fluoro substituent.

[0367] In an embodiment of formula (I), Y is O -In an embodiment of formula (I), Y is OH. In an embodiment of formula (I), Y is OR. In an embodiment of formula (I), Y is NH - In an embodiment of formula (I), Y is NH2. In an embodiment of formula (I), Y is S - In an embodiment of formula (I), Y is SH.

[0368] In an embodiment of Formula (I), Z is O. In one embodiment of Formula (I), Z is CH2.

[0369] In an embodiment of Formula (I), the linkage is inserted at positions 1-2 of the antisense strand. In another embodiment of Formula (I), the linkage is inserted at positions 6-7 of the antisense strand. In yet another embodiment of Formula (I), the linkage is inserted at positions 10-11 of the antisense strand. In yet another embodiment of Formula (I), the linkage is inserted at positions 19-20 of the antisense strand. In an embodiment of Formula (I), the linkage is inserted at positions 5-6 and 18-19 of the antisense strand.

[0370] Modified intersubunit linkages are further described in USSN 62 / 824,136 (filed March 26, 2019), USSN 62 / 826,454 (filed March 29, 2019), and USSN 62 / 864,792 (filed June 21, 2019), each of which is incorporated herein by reference.

[0371] 4) Conjugate functional moiety In other embodiments, the RNA silencing agent may be modified with one or more functional moieties. A functional moiety is a molecule that confers one or more additional activities to the RNA silencing agent. In certain embodiments, the functional moiety improves cellular uptake by target cells (e.g., T cells and epidermal keratinocytes). Thus, the present invention includes RNA silencing agents that are conjugated or unconjugated (e.g., at their 5' and / or 3' ends) to other moieties (e.g., non-nucleic acid moieties such as peptides) or organic compounds (e.g., dyes), etc. Conjugation can be accomplished using methods known in the art, for example, Lambert et al., Drug Deliv. Rev.: 47(1), 99-112 (2001) (describing nucleic acids attached to 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).

[0372] In certain embodiments, the functional moiety is a hydrophobic moiety. In certain embodiments, the hydrophobic moiety is selected from the group consisting of fatty acids, steroids, secosteroids, lipids, gangliosides, and nucleoside analogs, endocannabinoids, and vitamins. In certain embodiments, the steroid is selected from the group consisting of cholesterol and lithocholic acid (LCA). In certain embodiments, the fatty acid is selected from the group consisting of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosanoic acid (DCA). In certain embodiments, the vitamin is selected from the group consisting of choline, vitamin A, vitamin E, and derivatives or metabolites thereof. In certain embodiments, the vitamin is selected from the group consisting of retinoic acid and alpha-tocopherol succinate.

[0373] In certain embodiments, the RNA silencing agent of the invention is conjugated to a lipophilic moiety. In one embodiment, the lipophilic moiety is a ligand that includes 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, and cationic dyes (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.

[0374] In certain embodiments, the functional moiety may include one or more ligands tethered to the RNA silencing agent to improve stability, hybridization thermodynamics with the target nucleic acid, targeting to specific tissues or cell types, or cell permeability, for example, by endocytosis-dependent or -independent mechanisms. The ligand and associated modifications can also increase sequence specificity and, as a result, reduce off-site targeting. The tethering factor ligand can include one or more modified bases or sugars that can function as intercalators. These can also be located within an internal region, such as within a bulge, of the RNA silencing agent / target duplex. The intercalator can be aromatic, e.g., a polycyclic aromatic or heterocyclic aromatic compound. Polycyclic intercalators can have stacking capabilities and can include systems with two, three, or four fused rings. The universal bases described herein can be included in the ligand. In one embodiment, the ligand can include a cleavage group that contributes to inhibition of the target gene 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. Metal ion chelating groups 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 target RNA at the bulge site by free metal ions such as Lu(III). In some embodiments, a peptide ligand can be tethered to the RNA silencing agent to promote cleavage of target RNA, for example, at the bulge region. For example, 1,8-dimethyl-1,3,6,8,10,13-hexaazacyclotetradecane (cyclam) can be conjugated (eg, via an amino acid derivative) to a peptide to promote target RNA cleavage.The tethered ligand can be an aminoglycoside ligand, which can provide the 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 KanaA-N-acridine. The use of acridine analogs can increase sequence specificity. For example, neomycin B has a high affinity for RNA compared to DNA, but with lower sequence specificity. The acridine analog neo-5-acridine has a higher affinity for the HIV Rev-response element (RRE). In some embodiments, a guanidine analog (guanidinoglycoside) of the aminoglycoside ligand is tethered to the RNA silencing agent. In guanidinoglycosides, the amine group on amino acid is replaced with a guanidine group.The attachment of guanidine analogues can increase the cell permeability of RNA silencing agents.The anchoring ligand can be a polyarginine peptide, peptoid or peptidomimetic, which can increase the cellular uptake of oligonucleotide agents.

[0375] Exemplary ligands are coupled to the ligand-conjugated carrier directly or indirectly through an intermediate tethering agent. In certain embodiments, coupling is via covalent bond. In certain embodiments, the ligand is attached to the carrier through an intermediate tethering agent. In certain embodiments, the ligand changes the distribution, targeting or life span of the RNA silencing agent in which it is incorporated. In certain embodiments, the ligand provides, for example, higher affinity to a selected target, for example, a molecule, a cell or cell type, a compartment, for example, a cell or organ compartment, a tissue, an organ or an area of ​​the body, compared to a species in which such ligand is not present.

[0376] Exemplary ligands can improve the transport, hybridization, and specificity properties, and may also improve the nuclease resistance of the resulting natural or modified RNA silencing agent, or polymeric molecule comprising any combination of monomers described herein and / or natural or modified ribonucleotides. Ligands can generally include therapeutic modifiers, e.g., to increase uptake, diagnostic compounds or reporter groups, e.g., to monitor distribution; crosslinkers; nuclease resistance-conferring moieties; and natural or unusual nucleobases. Common examples include lipophilic substances, lipids, steroids (e.g., uvaol, hecigenin, diosgenin), terpenes (e.g., triterpenes, e.g., sarsasapogenin, friedelin, epifriedelanol-derivatized lithocholic acid), vitamins (e.g., folic acid, vitamin A, biotin, pyridoxal), carbohydrates, proteins, protein-binding agents, integrin-targeting molecules, polycations, peptides, polyamines, and peptidomimetics. Ligands can 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 can also be recombinant or synthetic molecules, such as synthetic polymers, e.g., synthetic polyamino acids. Examples of polyamino acids include the polyamino acid polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolized) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl) methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphazine.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.

[0377] The ligand can also include a targeting group that binds to a particular cell type, such as a kidney cell, e.g., a cell or tissue targeting agent, e.g., a lectin, a glycoprotein, a lipid, or a protein, e.g., an antibody. The targeting group can also be thyroid stimulating hormone, melanotropin, a lectin, a glycoprotein, surfactant protein A, a mucin carbohydrate, polyvalent lactose, polyvalent galactose, N-acetylgalactosamine (GalNAc) or a derivative thereof, N-acetylglucosamine, polyvalent mannose, polyvalent fucose, a glycosylated polyamino acid, polyvalent galactose, transferrin, a bisphosphonate, a polyglutamate, a polyaspartate, a lipid, cholesterol, a steroid, a bile acid, a folate, vitamin B12, biotin, or an RGD peptide or an 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, guanidium 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 , C18 , C 19 or C 20 fatty 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., anteoxycholic acid, acetylcholine ... napedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K, etc.), 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, tetraazamacrocyclic Eu 3+ complex), dinitrophenyl, HRP or AP. In certain embodiments, the ligand is GalNAc or a derivative thereof.

[0378] The ligand can be a protein, such as a glycoprotein, or a peptide, such as a molecule with specific affinity for a co-ligand, or an antibody, such as an antibody that binds to a particular cell type, such as a cancer cell, an endothelial cell, or a bone cell. Ligands can also include hormones and hormone receptors. They can also include non-peptide species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-kB.

[0379] The ligand can be a substance, e.g., a drug, that can increase the uptake of the RNA silencing agent into the cell, e.g., by disrupting the cytoskeleton of the cell, e.g., by disrupting the microtubules, microfilaments, and / or intermediate filaments of the cell. The drug can be, e.g., 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 the cell, e.g., by activating an inflammatory response. Exemplary ligands that have such an effect include tumor necrosis factor alpha (TNF), interleukin-1 beta, or gamma interferon. In one embodiment, the ligand is a lipid or lipid-based molecule. Such lipid or lipid-based molecule can bind to serum proteins, e.g., human serum albumin (HSA). HSA-binding ligands allow the distribution of the conjugate to target tissues, e.g., non-renal target tissues of the body. For example, the target tissue can be the liver, such as liver parenchymal cells. Other molecules capable of binding to HSA can also be used as ligands. For example, naproxen or aspirin can be used. The lipid or lipid-based ligand can be used to (a) improve the degradation resistance of the conjugate, (b) increase targeting or transport to the target cell or cell membrane, and / or (c) 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. For example, a lipid or lipid-based ligand that binds more tightly to HSA is less likely to target the kidney and therefore less likely to be excreted from the body. A lipid or lipid-based ligand that binds less tightly to HSA can be used to target the conjugate to the kidney. In certain embodiments, the lipid-based ligand binds to HSA. The lipid-based ligand can bind to HSA with sufficient affinity so that the conjugate is distributed to tissues other than the kidney.However, it is contemplated that the affinity is not so strong that it cannot reverse HSA-ligand binding.In another embodiment, the lipid-based ligand binds weakly or not at all to HSA, so that the conjugate distributes to the kidney.In place of or in addition to the lipid-based ligand, other moieties that target kidney cells can also be used.

[0380] In another embodiment, the ligand is a moiety, e.g., a vitamin, that is taken up by target cells, e.g., proliferating cells. These may be particularly useful in treating disorders characterized by unwanted cell proliferation, e.g., malignant or non-malignant, e.g., cancer cells. Representative vitamins include vitamins A, E, and K. Examples of other vitamins include vitamin B, e.g., folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients taken up by cancer cells. Also included are HSA and low-density lipoprotein (LDL).

[0381] In another aspect, the ligand is a cell-penetrating agent, such as a helical cell-penetrating agent. In certain embodiments, the agent is amphiphilic. An exemplary agent is a peptide, such as tat or antennopedia. If the agent is a peptide, it may be modified, including peptidyl mimics, invertomers, non-peptide or pseudopeptide bonds, and the use of D-amino acids. The helical agent may be an alpha-helical agent, which may have a lipophilic phase and a lipophobic phase.

[0382] The ligand can be a peptide or peptidomimetic. Peptidomimetics (also referred to herein as oligopeptidomimetics) are molecules capable of folding into a defined three-dimensional structure similar to a natural peptide. Attachment of peptides and peptidomimetics to an oligonucleotide agent can affect the pharmacokinetic distribution of an RNA silencing agent, such as by improving 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 constrained 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 translocation sequence (MTS). The peptide or peptidomimetic can be encoded by random DNA sequences, such as peptides identified from a phage display library, or 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 the incorporated monomer unit is a cell targeting peptide, such as an arginine-glycine-aspartic acid (RGD)-peptide or RGD mimic. A peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moiety can have structural modifications, such as, for example, to improve stability or direct conformation. Any of the structural modifications described below can be used.

[0383] In certain embodiments, the functional moiety is linked to the 5'-end and / or 3'-end of the RNA silencing agent of the present disclosure. In certain embodiments, the functional moiety is linked to the 5'-end and / or 3'-end of the antisense strand of the RNA silencing agent of the present disclosure. In certain embodiments, the functional moiety is linked to the 5'-end and / or 3'-end of the sense strand of the RNA silencing agent of the present disclosure. In certain embodiments, the functional moiety is linked to the 3'-end of the sense strand of the RNA silencing agent of the present disclosure.

[0384] In certain embodiments, the functional moiety is linked to the RNA silencing agent by a linker. In certain embodiments, the functional moiety is linked to the antisense strand and / or the sense strand by a linker. In certain embodiments, the functional moiety is linked to the 3' end of the sense strand by a linker. In certain embodiments, the linker comprises a bivalent or trivalent linker. In certain embodiments, the linker comprises an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, a carbamate, or a combination thereof. In certain embodiments, the bivalent or trivalent linker is selected from the following: [ka] wherein n is 1, 2, 3, 4 or 5.

[0385] In certain embodiments, the linker further comprises a phosphodiester or phosphodiester derivative. In certain embodiments, the phosphodiester or phosphodiester derivative is selected from the group consisting of: [ka] (wherein X is O, S or BH3).

[0386] Various functional moieties of the present disclosure and means for conjugating them to RNA silencing agents are described in further detail in WO2017 / 030973A1 and WO2018 / 031933A2, which are incorporated herein by reference.

[0387] VI. Branched Oligonucleotides Two or more RNA silencing agents disclosed above, such as oligonucleotide constructs such as anti-IFNGR1, anti-JAK1, anti-JAK2 or anti-STAT1 siRNA, can be linked together by one or more moieties independently selected from linker, spacer and branch point to form a branched oligonucleotide RNA silencing agent. In certain embodiments, the branched oligonucleotide RNA silencing agent is composed of two siRNAs to form a bi-branched siRNA ("di-siRNA") scaffold for delivering two siRNAs. In a representative embodiment, the nucleic acid of the branched oligonucleotide each comprises an antisense strand (or a part thereof), and the antisense strand has sufficient complementarity to the target mRNA (e.g., IFNGR1, JAK1, JAK2 or STAT1 mRNA) to mediate an RNA-mediated silencing mechanism (e.g., RNAi).

[0388] In an exemplary embodiment, the branched oligonucleotide may have 2-8 RNA silencing agents attached via a linker. The linker may be hydrophobic. In one embodiment, the branched oligonucleotide of the present application has 2-3 oligonucleotides. In one embodiment, the oligonucleotides have substantial independent chemical stabilization (e.g., at least 40% of the constituent bases are chemically modified). In an exemplary embodiment, the oligonucleotides have complete chemical stabilization (i.e., all of the constituent 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 a non-limiting embodiment, each single-stranded tail has 2-10 nucleotides.

[0389] In certain embodiments, the branched oligonucleotide is characterized by three properties: (1) a branched structure, (2) complete metabolic stabilization, and (3) the presence of a single-stranded tail that includes a phosphorothioate linker. In certain embodiments, the branched oligonucleotide has 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) allow each branch to act in concert, thus dramatically improving the rate of internalization, transport, and release.

[0390] Branched oligonucleotides are provided in a variety of structurally diverse embodiments. In some embodiments, the nucleic acid attached at the branch point is single-stranded or double-stranded and consists of miRNA inhibitors, gapmers, mixmers, SSOs, PMOs, or PNAs. These single strands can be attached at the 3' or 5' end. Combinations 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 about 37°C) to allow rapid dissociation when the branched structure is taken up into the cell.

[0391] Di-siRNA branched oligonucleotides can contain chemically diverse conjugates such as the functional moieties described above. Conjugated bioactive ligands can be used to improve cell specificity and promote membrane binding, internalization, and serum protein binding. Examples of bioactive moieties used for conjugation include DHA, GalNAc, and cholesterol. These moieties can be attached to Di-siRNA via a connecting linker or spacer, or can be added via an additional linker or spacer attached to another free siRNA end.

[0392] The presence of branched structures results in improved levels of tissue retention in various tissues (e.g., skin) compared to unbranched compounds of the same chemical composition. Branched oligonucleotides are unexpectedly uniformly distributed throughout tissues.

[0393] Branched oligonucleotides include a variety of therapeutic nucleic acids, including siRNA, ASO, miRNA, miRNA inhibitors, splice switching, PMO, PNA, etc. In some embodiments, the branched oligonucleotides further comprise conjugated hydrophobic moieties and exhibit unprecedented silencing and efficacy in vitro and in vivo.

[0394] Linker In one embodiment 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; where any carbon or oxygen atom of the linker is optionally replaced with a nitrogen atom, carries a hydroxyl substituent, or carries 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 an RNA. In another embodiment, each linker is a 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.

[0395] VII. Compounds of formula (I): In another aspect, provided herein are branched oligonucleotide compounds of formula (I): [ka] 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; wherein B is, independently at each occurrence, a polyvalent organic species or derivative thereof, and S is, independently at 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.

[0396] The moiety N is an RNA duplex comprising a sense strand and an antisense strand; n is 2, 3, 4, 5, 6, 7 or 8. In one embodiment, the antisense strand of N comprises a sequence substantially complementary to any one of the IFNGR1, JAK1, JAK2 or STAT1 nucleic acid sequences of SEQ ID NOs: 1-6 in Tables 6 and 8. In a further embodiment, N comprises a strand capable of targeting one or more of the IFNGR1, JAK1, JAK2 or STAT1 nucleic acid sequences selected from the group consisting of SEQ ID NOs: 143-154 in Tables 7, 9, 10 and 11. The sense strand and the antisense strand may each independently comprise one or more chemical modifications.

[0397] In one embodiment, the compound of formula (I) has a structure selected from formulas (I-1) to (I-9) in Table 1. [Table 4]

[0398] 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).

[0399] In an embodiment 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; wherein any carbon or oxygen atom of the linker is optionally replaced with a nitrogen atom, carries a hydroxyl substituent, or carries 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.

[0400] 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 tetrol derivative. In another embodiment, B is a tri- or tetracarboxylic acid derivative. In another embodiment, B is an amine derivative. In another embodiment, B is a tri- or tetraamine derivative. In another embodiment, B is an amino acid derivative. In another embodiment of the compound of formula (I), B is selected from the formula consisting of: [ka]

[0401] 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.), tetrols (e.g., ribose, pentaerythritol, 1,2,3,5-tetrahydroxybenzene, etc.), tricarboxylic acids (e.g., citric acid, 1,3,5-cyclohexanetricarboxylic acid, trimesic acid, etc.), tetracarboxylic 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.).

[0402] In an embodiment of the compound of formula (I), each nucleic acid comprises one or more chemically modified nucleotides. In an embodiment of the compound of formula (I), each nucleic acid consists of chemically modified nucleotides. In certain embodiments of the compound of formula (I), more than 95%, more than 90%, more than 85%, more than 80%, more than 75%, more than 70%, more than 65%, more than 60%, more than 55% or more than 50% of each nucleic acid comprises chemically modified nucleotides.

[0403] In one embodiment, each antisense strand independently comprises a 5'-terminal group R selected from the group in Table 2. [Table 5]

[0404] 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.

[0405] Structure of Formula (II) In certain embodiments, the compound of Formula (I) has the structure of Formula (II): [ka] (wherein X, independently at each occurrence, is selected from adenosine, guanosine, uridine, cytidine, and chemically modified derivatives thereof; Y, independently at 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 at each occurrence, represents a base pairing interaction or a mismatch).

[0406] In certain embodiments, the structure of formula (II) does not contain a mismatch. 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 one embodiment, each nucleic acid is composed of chemically modified nucleotides.

[0407] In certain embodiments, more than 95%, more than 90%, more than 85%, more than 80%, more than 75%, more than 70%, more than 65%, more than 60%, more than 55%, or more than 50% of the X's in the structure of formula (II) are chemically modified nucleotides. In other embodiments, more than 95%, more than 90%, more than 85%, more than 80%, more than 75%, more than 70%, more than 65%, more than 60%, more than 55%, or more than 50% of the X's in the structure of formula (II) are chemically modified nucleotides.

[0408] Structure of formula (III) In certain embodiments, the compound of Formula (I) has the structure of Formula (III): [ka]

[0409] where X, independently at each occurrence, is a nucleotide that includes a 2'-deoxy-2'-fluoro modification; X, independently at each occurrence, is a nucleotide that includes a 2'-O-methyl modification; Y, independently at each occurrence, is a nucleotide that includes a 2'-deoxy-2'-fluoro modification; and Y, independently at each occurrence, is a nucleotide that includes a 2'-O-methyl modification.

[0410] In one embodiment, X is selected from the group consisting of 2'-deoxy-2'-fluoro modified adenosine, guanosine, uridine, or cytidine. In one embodiment, X is selected from the group consisting of 2'-O-methyl modified adenosine, guanosine, uridine, or cytidine. In one embodiment, Y is selected from the group consisting of 2'-deoxy-2'-fluoro modified adenosine, guanosine, uridine, or cytidine. In one embodiment, Y is selected from the group consisting of 2'-O-methyl modified adenosine, guanosine, uridine, or cytidine.

[0411] In certain embodiments, the structure of formula (III) does not contain a mismatch. 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.

[0412] Structure of formula (IV) In one embodiment, the compound of formula (I) has the structure of formula (IV): [ka] (wherein X, independently at each occurrence, is selected from adenosine, guanosine, uridine, cytidine, and chemically modified derivatives thereof; Y, independently at 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 at each occurrence, represents a base pairing interaction or a mismatch).

[0413] In certain embodiments, the structure of formula (IV) does not contain a mismatch. 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 one embodiment, each nucleic acid is comprised of chemically modified nucleotides.

[0414] In certain embodiments, more than 95%, more than 90%, more than 85%, more than 80%, more than 75%, more than 70%, more than 65%, more than 60%, more than 55%, or more than 50% of the X's in the structure of formula (IV) are chemically modified nucleotides. In other embodiments, more than 95%, more than 90%, more than 85%, more than 80%, more than 75%, more than 70%, more than 65%, more than 60%, more than 55%, or more than 50% of the X's in the structure of formula (IV) are chemically modified nucleotides.

[0415] Structure of formula (V) In certain embodiments, the compound of Formula (I) has the structure of Formula (V): [ka] where X, independently at each occurrence, is a nucleotide that includes a 2'-deoxy-2'-fluoro modification; X, independently at each occurrence, is a nucleotide that includes a 2'-O-methyl modification; Y, independently at each occurrence, is a nucleotide that includes a 2'-deoxy-2'-fluoro modification; and Y, independently at each occurrence, is a nucleotide that includes a 2'-O-methyl modification.

[0416] In certain embodiments, X is selected from the group consisting of 2'-deoxy-2'-fluoro modified adenosine, guanosine, uridine, or cytidine. In one embodiment, X is selected from the group consisting of 2'-O-methyl modified adenosine, guanosine, uridine, or cytidine. In one embodiment, Y is selected from the group consisting of 2'-deoxy-2'-fluoro modified adenosine, guanosine, uridine, or cytidine. In one embodiment, Y is selected from the group consisting of 2'-O-methyl modified adenosine, guanosine, uridine, or cytidine.

[0417] In certain embodiments, the structure of formula (V) does not contain a mismatch. 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.

[0418] Flexible Linker In an embodiment of the compound of formula (I), L has the structure L1: [ka] In an embodiment of L1, R is R 3 and n is 2.

[0419] In an embodiment of the structure of formula (II), L has structure L1. In an embodiment of the structure of formula (III), L has structure L1. In an embodiment of the structure of formula (IV), L has structure L1. In an embodiment of the structure of formula (V), L has structure L1. In an embodiment of the structure of formula (VI), L has structure L1. In an embodiment of the structure of formula (VI), L has structure L1.

[0420] In an embodiment of the compound of Formula (I), L has the structure L2: [ka]

[0421] In one embodiment of L2, R is R3 and n is 2. In one embodiment of the structure of formula (II), L has structure L2. In an embodiment of the structure of formula (III), L has structure L2. In an embodiment of the structure of formula (IV), L has structure L2. In an embodiment of the structure of formula (V), L has structure L2. In an embodiment of the structure of formula (VI), L has structure L2. In an embodiment of the structure of formula (VI), L has structure L2.

[0422] delivery system In a third aspect, provided herein is a delivery system for a therapeutic nucleic acid having the structure of formula (VI): [ka]

[0423] wherein L is selected from ethylene glycol chain, alkyl chain, peptide, RNA, DNA, phosphate, phosphonate, phosphoramidate, ester, amide, triazole, and combinations thereof, and formula (VI) optionally further comprises one or more branch points B, and one or more spacers S; wherein B is, independently at each occurrence, a polyvalent organic species or its derivative, and S is, independently at each occurrence, selected from ethylene glycol chain, alkyl chain, peptide, RNA, DNA, phosphate, phosphonate, phosphoramidate, ester, amide, 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.

[0424] 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.

[0425] 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.

[0426] 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.

[0427] In certain embodiments, each cNA comprises more than 95%, more than 90%, more than 85%, more than 80%, more than 75%, more than 70%, more than 65%, more than 60%, more than 55%, or more than 50% chemically modified nucleotides.

[0428] In one embodiment, the compound of formula (VI) has a structure selected from formulas (VI-1) to (VI-9) in Table 3. [Table 6]

[0429] 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).

[0430] In one embodiment, the compound of formula (VI) (e.g., formulas (VI-1) to (VI-9) etc.) has each cNA independently comprising at least 15 consecutive nucleotides. In one embodiment, each cNA independently comprises chemically modified nucleotides.

[0431] In one embodiment, the delivery system further comprises n therapeutic nucleic acids (NAs), wherein each NA comprises a sequence substantially complementary to any one of IFNGR1, JAK1, JAK2, or STAT1 nucleic acid sequences of SEQ ID NOs: 1-6 set forth in Tables 6 and 8. In a further embodiment, the NA comprises a strand capable of targeting one or more of IFNGR1, JAK1, JAK2, or STAT1 nucleic acid sequences selected from the group consisting of SEQ ID NOs: 143-154 set forth in Tables 7, 9, 10, and 11, respectively.

[0432] Also, each NA hybridizes to at least one cNA. In one embodiment, the delivery system is comprised of two NAs. In another embodiment, the delivery system is comprised of three NAs. In another embodiment, the delivery system is comprised of four NAs. In another embodiment, the delivery system is comprised of five NAs. In another embodiment, the delivery system is comprised of six NAs. In another embodiment, the delivery system is comprised of seven NAs. In another embodiment, the delivery system is comprised of eight NAs.

[0433] In one embodiment, each NA independently comprises at least 15 contiguous nucleotides. In one embodiment, each NA independently comprises 15 to 25 contiguous nucleotides. In one embodiment, each NA independently comprises 15 contiguous nucleotides. In one embodiment, each NA independently comprises 16 contiguous nucleotides. In another embodiment, each NA independently comprises 17 contiguous nucleotides. In another embodiment, each NA independently comprises 18 contiguous nucleotides. In another embodiment, each NA independently comprises 19 contiguous nucleotides. In another embodiment, each NA independently comprises 20 contiguous nucleotides. In one embodiment, each NA independently comprises 21 contiguous nucleotides. In one embodiment, each NA independently comprises 22 contiguous nucleotides. In one embodiment, each NA independently comprises 23 contiguous nucleotides. In one embodiment, each NA independently comprises 24 contiguous nucleotides. In one embodiment, each NA independently comprises 25 contiguous nucleotides.

[0434] 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 linkages.

[0435] In an embodiment, each NA is independently selected from the group consisting of DNA, siRNA, antagomiR, miRNA, gapmer, mixmer, or guide RNA. In an embodiment, each NA is independently DNA. In another embodiment, each NA is independently siRNA. 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 an embodiment, each NA is the same. In an embodiment, each NA is not the same.

[0436] 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 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 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 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 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).

[0437] In one embodiment, the delivery system has a structure selected from formula (I), (II), (III), (IV), (V), (VI), and further comprises a linker of structure L1 or L2, wherein R is R 3 and n is 2. In another embodiment, the delivery system has a structure selected from formula (I), (II), (III), (IV), (V), (VI), and further comprises a linker of structure L1, wherein R is R 3and n is 2. In another embodiment, the delivery system has a structure selected from formula (I), (II), (III), (IV), (V), (VI), and further comprises a linker of structure L2, wherein R is R 3 where n is 2.

[0438] In one embodiment of the delivery system, the target of delivery is selected from the group consisting of brain, liver, skin, kidney, spleen, pancreas, colon, fat, lung, muscle, and thymus, hi one embodiment, the target of delivery is skin.

[0439] In certain embodiments, the compounds of the present invention are characterized by the following properties: (1) two or more branched oligonucleotides, e.g., unequal numbers of 3' and 5' ends; (2) substantially chemically stabilized, e.g., greater than 40%, optimally 100%, of the oligonucleotides are chemically modified (e.g., free of RNA and optionally free of DNA); (3) phosphorothioate single oligonucleotides containing at least three phosphorothioate linkages. In certain embodiments, the phosphorothioate single oligonucleotides contain 4 to 20 phosphorothioate linkages.

[0440] It should be understood that the methods described in this disclosure are not limited to the specific methods and experimental conditions disclosed herein. Therefore, the methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0441] Furthermore, the experiments described herein use conventional molecular and cell biological and immunological techniques within the scope of those skilled in the art, unless otherwise indicated. Such techniques are well known to those skilled in the art and are fully described in the literature. For example, see Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley&Sons, Inc., NY (1987-2008), including all supplements, Molecular Cloning: A Laboratory Manual (Fourth Edition); MR Green and J. Sambrook and Harlow et al., Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (2013, 2nd edition).

[0442] Branched oligonucleotides, including their synthesis and methods of use, are described in further detail in WO2017 / 132669, which is incorporated herein by reference.

[0443] Nucleic acids, vectors and methods for introducing into host cells The RNA silencing agent of the present invention can be directly introduced into cells (e.g., skin cells) (i.e., intracellularly). Or it can be introduced extracellularly into cavities, interstitial spaces, circulation of organisms, orally, or by immersing cells or organisms in a solution containing nucleic acid. Vascular or extravascular circulation, blood or lymphatic system, and cerebrospinal fluid are sites where nucleic acid can be introduced.

[0444] The RNA silencing agent of the present invention can be introduced using nucleic acid delivery methods known in the art, such as injection of a solution containing nucleic acid, bombardment 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 for introducing nucleic acid into cells can also be used, such as lipid-mediated carrier transport, chemical-mediated transport, and cationic liposome transfection such as calcium phosphate.Nucleic acid can be introduced together with other components that perform one or more of the following activities: enhance the uptake of nucleic acid by cells, or otherwise increase the inhibition of target genes.

[0445] Physical methods of introducing nucleic acid include injection of a solution containing RNA, bombardment with particles covered with RNA, soaking a cell or organism in a solution of RNA, or electroporation of cell membranes in the presence of RNA. The viral construct packaged in the viral particle achieves both efficient introduction of the expression construct into the cell and transcription of the RNA encoded by the expression construct. Other methods known in the art for introducing nucleic acid into cells may also be used, such as lipid-mediated carrier transport, chemical-mediated transport, such as calcium phosphate. Thus, RNA may be introduced with a component that performs 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.

[0446] RNA can be directly introduced into a cell (i.e., intracellularly), or can be introduced extracellularly into a cavity, interstitial space, or into the circulation of an organism, or can be introduced orally, or by bathing a cell or organism in a solution containing the RNA. Vascular or extravascular circulation, the blood or lymphatic system, and cerebrospinal fluid are sites where RNA can be introduced.

[0447] 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, endothelial cells, neurons, glial cells, blood cells, megakaryocytes, lymphocytes, macrophages, neutrophils, eosinophils, basophils, mast cells, leukocytes, granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts, hepatocytes, and cells of endocrine or exocrine glands.

[0448] Depending on the particular 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 target cells is typical. Inhibition of gene expression refers to the absence (or observable reduction) in the levels of protein and / or mRNA product from the target gene. Specificity refers to the ability to inhibit the target gene without clearly affecting other genes of the cell. The results of inhibition can be confirmed by examining the external characteristics of the cell or organism (as shown in the examples below) or by biochemical techniques such as RNA solution hybridization, nuclease protection, Northern hybridization, reverse transcription, gene expression monitoring by microarray, antibody binding, enzyme-linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA), other immunoassays, and fluorescence-activated cell sorting (FACS).

[0449] For RNA-mediated inhibition in cell lines or whole organisms, gene expression is conveniently assayed by using 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, gentarnycin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, and tetracycline. Depending on the assay, quantification of gene expression can determine inhibition of more than 10%, 33%, 50%, 90%, 95% or 99% compared to cells not treated according to the present invention. If the dose of injected material is small and the time after administration of the RNAi agent is long, a smaller percentage of cells (e.g., at least 10%, 20%, 50%, 75%, 90%, or 95% of the target cells) may be inhibited. Quantification of gene expression in cells may show a similar 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 by 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.

[0450] 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 / cell) may result in more effective inhibition, while lower doses may also be useful for certain applications.

[0451] In an exemplary embodiment, the effect of the RNAi agent of the present invention (e.g., siRNA targeting IFNGR1, JAK1, JAK2, or STAT1 target sequence) is tested for its ability to specifically degrade mutant mRNA (e.g., IFNGR1, JAK1, JAK2, or STAT1 mRNA and / or IFNGR1, JAK1, JAK2, or STAT1 protein production) in cells (such as keratinocytes). Also suitable for cell-based validation assays are other easily transfectable cells, such as HeLa cells or COS cells. The cells are transfected with human wild-type or mutant cDNA (e.g., human wild-type or mutant IFNGR1, JAK1, JAK2, or STAT1 cDNA). Standard siRNA, modified siRNA, or vectors capable of generating siRNA from U-loop mRNA are co-transfected. The selective reduction of target mRNA (e.g., IFNGR1, JAK1, JAK2, or STAT1 mRNA) and / or target protein (e.g., IFNGR1, JAK1, JAK2, or STAT1 protein) is measured. The reduction of target mRNA or protein can be compared to the level of target mRNA or protein in the absence of RNAi agent or in the presence of RNAi agent that does not target IFNGR1, JAK1, JAK2, or STAT1 mRNA. Exogenously introduced mRNA or protein (or endogenous mRNA or protein) can be assayed for comparison purposes. When utilizing neural cells, which are known to be somewhat resistant to standard transfection techniques, it may be desirable to introduce RNAi agent (e.g., siRNA) by passive uptake.

[0452] Recombinant adeno-associated viruses and vectors In certain exemplary embodiments, recombinant adeno-associated viruses (rAAV) and their associated vectors can be used to deliver one or more siRNAs to cells, such as skin cells. AAV can infect many different cell types, with different infection efficiencies based on the serotype determined by the sequence of the capsid protein. Several native AAV serotypes have been identified, with serotypes 1-9 being the most commonly used for recombinant AAV. AAV-2 is the most well-studied and published serotype. AAV-DJ systems include serotypes AAV-DJ and AAV-DJ / 8. These serotypes were created by DNA shuffling of multiple AAV serotypes to produce AAVs containing hybrid capsids with improved transduction efficiency in various cells and tissues in vitro (AAV-DJ) and in vivo (AAV-DJ / 8).

[0453] rAAV can be delivered to a subject in a composition according to any suitable method known in the art.rAAV can be suspended in a physiologically compatible carrier (i.e., composition) and administered to a subject, i.e., a host animal such as a human, mouse, rat, cat, dog, sheep, rabbit, horse, cow, goat, pig, guinea pig, hamster, chicken, turkey, non-human primate (e.g., macaque).In certain embodiments, the animal is a non-human host animal.

[0454] Delivery of one or more rAAVs to a mammalian subject can be performed, for example, by intramuscular injection or by administration to the bloodstream of a mammalian subject. Administration to the bloodstream can be by injection into a vein, an artery, or any other vascular conduit. In certain embodiments, one or more rAAVs are administered to the bloodstream by isolated limb perfusion, a technique well known in the surgical arts, which essentially allows a person skilled in the art to isolate a limb from the general circulation prior to administration of rAAV virions. A variation of the isolated limb perfusion technique described in U.S. Patent No. 6,177,403 can also be used by a person skilled in the art to administer virions to the vasculature of an isolated limb to potentially enhance transduction into muscle cells or tissues.

[0455] The compositions of the invention may include rAAV alone or in combination with one or more other viruses (e.g., a second rAAV encoding one or more different transgenes). In certain embodiments, the compositions include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different rAAVs, each with one or more different transgenes.

[0456] An effective amount of rAAV is an amount sufficient to target infection of an animal and target a desired tissue. In some embodiments, an effective amount of rAAV is an amount sufficient to generate a stable somatic transgenic animal model. The effective amount mainly depends on factors such as the subject's species, age, weight, health status, and the tissue to be targeted, and therefore may vary from animal to animal and tissue. For example, an effective amount of one or more rAAVs is generally about 10 9 ~10 16 The volume ranges from about 1 ml to about 100 ml of solution containing about 10 genome copies. 11 ~10 12 A dosage of 10 rAAV genome copies is appropriate. In certain embodiments, 12 rAAV genome copies are effective in targeting heart, liver, and pancreatic tissues. In some cases, stable transgenic animals are produced by multiple administrations of rAAV.

[0457] In some embodiments, the rAAV composition is particularly effective when high rAAV concentrations are present (e.g., about 10 13 The composition is formulated to reduce aggregation of AAV particles (genome copies / mL or more). Methods for reducing aggregation of rAAV are well known in the art and include, for example, the addition of detergents, pH adjustment, salt concentration adjustment, etc. (e.g., Wright et al. (2005) Molecular Therapy 12:171-178, the contents of which are incorporated herein by reference).

[0458] A "recombinant AAV (rAAV) vector" comprises at least a transgene and its regulatory sequences, as well as 5' and 3' AAV inverted terminal repeats (ITRs). It is this recombinant AAV vector that is packaged into a capsid protein and delivered to a selected target cell. In some embodiments, the transgene is a nucleic acid sequence heterologous to the vector sequence, which encodes a polypeptide, protein, functional RNA molecule (e.g., siRNA) or other gene product of interest. The nucleic acid coding sequence is operably linked to regulatory components in a manner that allows transcription, translation, and / or expression of the transgene in cells of the target tissue.

[0459] The AAV sequence of the vector typically includes cis-acting 5' and 3' inverted terminal repeat (ITR) sequences (see, for example, BJ Carter, "Handbook of Parvoviruses" ed., P. Tijsser, CRC Press, pp. 155 168 (1990)). The ITR sequences are usually about 145 base pairs in length. In certain embodiments, substantially the entire sequence encoding the ITRs is used in the molecule, although some minor modifications of these sequences are tolerated. It is within the skill of the art that these ITR sequences can be modified (see, for example, the texts Sambrook et al., "Molecular Cloning. A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J Virol., 70:520 532 (1996)). An example of such a molecule for use in the present invention is a "cis-acting" plasmid containing a transgene, in which the selected transgene sequence and associated regulatory elements are flanked by 5' and 3' AAV ITR sequences, which can be obtained from any known AAV, such as the mammalian AAV types further described herein.

[0460] VIII. Treatment method In one aspect, the invention provides both prophylactic and therapeutic methods of treating a subject at risk (or susceptible) for developing vitiligo associated with IFN-γ signaling. In one embodiment, the disease or disorder is one in which IFNGR1, JAK1, JAK2, or STAT1 mediate IFN-γ signaling that is involved in the pathogenesis of vitiligo. In certain embodiments, the disease or disorder is one in which decreasing IFNGR1, JAK1, JAK2, or STAT1 reduces the clinical symptoms seen in vitiligo and potentially other diseases.

[0461] "Treatment" or "treating" as used herein is defined as the application or administration of a therapeutic agent (e.g., an RNA agent or a vector or transgene encoding same) to a patient, or to a tissue or cell line isolated from a patient having a disease or disorder, a symptom of the disease or disorder, or a predisposition to a disease or disorder, in order to cure, cure, alleviate, palliate, alter, repair, ameliorate, ameliorate or affect the disease or disorder, the symptom of the disease or disorder, or the predisposition to a disease.

[0462] In one aspect, the present invention provides a method for preventing the above-mentioned disease or disorder in a subject 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 disease can be identified, for example, by any or a combination of diagnostic or prognostic assays described herein.Administration of the prophylactic agent can be performed before the onset of symptoms characteristic of the disease or disorder, thereby preventing or slowing the progression of the disease or disorder.

[0463] Another aspect of the present invention relates to a method of therapeutically treating a subject, i.e., a method of altering the onset of a disease or disorder symptom. In an exemplary embodiment, the modulatory method of the present invention comprises contacting an immune cell expressing IFNGR1, JAK1, JAK2, or STAT1 with a therapeutic agent (e.g., an RNAi agent, vector, or transgene encoding the same) specific for a target sequence in the gene (e.g., an IFNGR1, JAK1, JAK2, or STAT1 target sequence in Tables 6 and 8), thereby resulting in sequence-specific interference with the gene. These methods can be performed in vitro (e.g., by culturing the cells with the agent) or in vivo (e.g., by administering the agent to the subject).

[0464] IX. Pharmaceutical Compositions and Methods of Administration The present invention relates to the use of said agents for preventive and / or therapeutic treatment as described below. Thus, the modulators (e.g., RNAi agents) of the present invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically include nucleic acid molecules, proteins, antibodies, or modulatory compounds and pharmaceutically acceptable carriers. As used herein, the term "pharmaceutically acceptable carriers" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agents are incompatible with the active compounds, their use in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0465] The pharmaceutical composition of the present disclosure is formulated to be compatible with its intended route of administration.Examples of routes of administration include parenteral, such as intravenous, intradermal, subcutaneous, intraperitoneal, intramuscular, oral (e.g., inhalation), transdermal (topical), and transmucosal administration.In certain embodiments, the route of administration is transdermal (topical).

[0466] The nucleic acid molecules of the present invention can be inserted into an expression construct, such as, for example, a viral vector, a retroviral vector, an expression cassette, or a plasmid viral vector, using methods known in the art, such as, for example, but not limited to, those described in Xia et al., (2002), supra. The expression construct can be delivered to a subject, for example, by inhalation, oral administration, intravenous injection, topical administration (see, for example, U.S. Pat. No. 5,328,470), or stereotactic injection (see, for example, Chen et al. (1994), Proc. Natl. Acad. Sci. USA, 91, 3054-3057). A pharmaceutical preparation of the delivery vector can include the vector in an acceptable diluent, or can include a slow release matrix in which the delivery vehicle is embedded. Alternatively, if the complete delivery vector can be produced intact from a recombinant cell, such as a retroviral vector, the pharmaceutical preparation can include one or more cells that produce the gene delivery system.

[0467] The nucleic acid molecules of the present invention may also include small hairpin RNAs (shRNAs) and expression constructs engineered to express shRNAs. Transcription of shRNAs is believed to initiate at the polymerase III (pol III) promoter and terminate at position 2 of the 4-5-thymine transcription termination site. Upon expression, shRNAs are believed to fold into a stem-loop structure with a 3'UU overhang. The ends of these shRNAs are then processed, which converts the shRNAs into siRNA-like molecules of approximately 21 nucleotides. Brummelkamp et al. (2002), Science, 296, 550-553; Lee et al, (2002). supra; Miyagishi and Taira (2002), Nature Biotechnol., 20, 497-500; Paddison et al. (2002), supra; Paul (2002), supra; Sui (2002) supra; Yu et al. (2002), supra.

[0468] The expression construct may be any construct suitable for use in an appropriate expression system, including, but not limited to, retroviral vectors, linear expression cassettes, plasmids, and virus or virus-derived vectors known in the art. Such expression constructs may include one or more inducible promoters, RNA Pol III promoter systems, such as the U6 snRNA promoter or the H1 RNA polymerase III promoter, or other promoters known in the art. The construct may include one or both strands of the siRNA. Expression constructs expressing both strands may also include a loop structure linking both strands, or each strand may be transcribed separately from a separate promoter within the same construct. Each strand may also be transcribed from a separate expression construct (Tuschl (2002), supra).

[0469] For example, a composition can include one or more species of compounds of the invention and a pharma- ceutically acceptable carrier. The pharmaceutical compositions of the invention can be administered in a number of ways, depending on whether local or systemic treatment is desired and on the area to be treated. Administration can be topical (ophthalmic, intranasal, transdermal, etc.), oral, or parenteral. Parenteral administration includes intravenous infusion, subcutaneous, intraperitoneal or intramuscular injection, intrathecal, or intraventricular (e.g., intracerebroventricular) administration.

[0470] The delivery route can depend on the disorder of the patient. For example, a subject diagnosed with vitiligo can be directly administered the anti-IFNGR1, anti-JAK1, anti-JAK2 or anti-STAT1 compound of the present invention to the skin. In addition to the compound of the present invention, the patient can be administered a second treatment, such as a palliative treatment and / or a disease-specific treatment. The second treatment can be, for example, a symptomatic treatment (e.g., to relieve symptoms) or a remedy (e.g., to reverse the disease process).

[0471] Lipid Nanoparticle (LNP) Formulations The RNA silencing agent of the present disclosure can be formulated in lipid nanoparticles (LNPs).LNPs correspond to lipid vesicles that cover an aqueous internal space that can contain nucleic acid (such as RNAi silencing agent or the plasmid from which RNAi silencing agent is transcribed).LNPs typically contain at least one cationic lipid, at least one non-cationic lipid, lipid that prevents particle aggregation (e.g., PEG-lipid conjugate), and optionally cholesterol or its derivatives.

[0472] Examples of cationic lipids include N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-dilinoleyl kaline, ... 1,2-Dilinoleyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLin-DAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanediol (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or similar. The cationic lipid may be an analogue, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][l,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), l,l'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (Tech Gl), or a mixture thereof. The cationic lipid may comprise about 20 mol% to about 50 mol% of the total lipid present in the particle.

[0473] The non-cationic lipids may be anionic or neutral lipids, including, but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine ...ethanolamine (DPPG), dioleoyl-phosphatidylethanolamine (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylethanolamine (DPPG), dioleoyl-phosphatidylethanolamine (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylethanolamine (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylethanolamine (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylethanolamine (DPPG), diole oleoylamine (POPE), dioleoyl-phosphatidylethanolamine 4-(-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidiethanolamine (SOPE), cholesterol, or a mixture thereof.

[0474] The conjugated lipid that inhibits particle aggregation can be, for example, a polyethylene glycol (PEG)-lipid, including, but not limited to, PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), or mixtures thereof. The PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl (C12), PEG-dimyristyloxypropyl (Ci4), PEG-dipalmityloxypropyl (Ci6), or PEG-distearyloxypropyl (C]s). The conjugated lipid that inhibits particle aggregation can be 0 mol% to about 20 mol% or about 2 mol% of the total lipid present in the particle. In some embodiments, the nucleic acid-lipid particle further comprises cholesterol, for example, about 10 mol% to about 60 mol% or about 48 mol% of the total lipid present in the particle.

[0475] LNPs of the invention typically have an average diameter of about 50 nm to about 200 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, or about 60 nm to about 80 nm. Furthermore, nucleic acids, when present in LNPs, are resistant in aqueous solution to degradation by nucleases.

[0476] In one embodiment, the lipid to drug ratio (mass / mass ratio; w / w ratio) (e.g., lipid to dsRNA ratio) is within the range of about 1:1 to about 50:1, about 1:1 to about 25:1, about 10:1 to about 14:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1.

[0477] LNP formulations are further described, for example, in U.S. Patent Nos. 7,901,708, 7,811,603, 7,030,097, 6,858,224, 6,106,858, 5,478,860, and 5,908,777, U.S. Patent Application Publication Nos. 20060240093 and 20070135372, and International Application No. WO2009082817. These patents and applications are incorporated herein by reference in their entireties.

[0478] It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein can be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. Although certain embodiments have been described in detail above, the same 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

[0479] Example 1. In vitro identification of IFNGR1, JAK1, JAK2, and STAT1 targeting sequences IFNGR1, JAK1, JAK2, and STAT1 genes were targeted for mRNA knockdown. A panel of siRNAs targeting several different sequences of human and mouse IFNGR1, JAK1, JAK2, or STAT1 mRNA was developed and screened in vitro in human HeLa cells and mouse N2A cells and compared to untreated control cells. Each of these siRNAs was tested at a concentration of 1.5 μM, and mRNA was evaluated at 72 hours using QuantiGene gene expression assays (ThermoFisher, Waltham, MA). Figure 1A shows the results of screening against human IFNGR1 mRNA in human HeLa cells to evaluate 22 IFNGR1 siRNAs. Figure 1B shows the results of screening against mouse IFNGR1 mRNA in mouse N2A cells to evaluate 22 IFNGR1 siRNAs. Figure 2A shows the results of screening against human JAK1 mRNA in human HeLa cells to evaluate 24 JAK1 siRNAs. FIG. 2B shows the results of screening against mouse JAK1 mRNA in mouse N2A cells to evaluate 24 JAK1 siRNAs. FIG. 3A shows the results of screening against human JAK2 mRNA in human HeLa cells to evaluate 24 JAK2 siRNAs. FIG. 3B shows the results of screening against mouse JAK2 mRNA in mouse N2A cells to evaluate 24 JAK2 siRNAs. FIG. 4A shows the results of screening against human STAT1 mRNA in human HeLa cells to evaluate 24 STAT1 siRNAs. FIG. 4B shows the results of screening against mouse STAT1 mRNA in mouse N2A cells to evaluate 24 STAT1 siRNAs.

[0480] Six sites were identified that resulted in potent and efficient silencing of IFNGR1, JAK1, JAK2, and STAT1 mRNA compared to % of untreated controls. Dose-response curves for the six identified siRNAs (oligo IDs IFNGR1_1726, JAK1_3033, JAK2_1936, STAT1_885, Ifngr1_1641, and Jak2_2076) are shown in Figures 5A-5H. Two of these siRNAs (JAK1_3033 and STAT1_885) were tested in both human HeLa cells and mouse N2A cells. The results are summarized in Table 5 below. Protein expression of IFNGR1 was also tested in human HeLa cells and mouse N2a cells. IFNGR1_1726-targeted siRNA reduced IFNGR1 expression in HeLa cells, and Ifngr1_1641-targeted siRNA reduced IFNGR1 expression in N2a cells. Cells were treated with 1.5 μM fully modified cholesterol-conjugated siRNA for 72 h (n=4, mean ± SD). Protein expression was determined by ELISA and normalized to total protein levels (quantified by Bradford assay). Data are presented as mean ± SD and analyzed by unpaired t-test ( *** p<0.001, **** p<0.0001) (Figure 10).

[0481] Additional human and mouse targets for IFNGR1 were tested in dose-response curves (1631, 1989, and 2072 in HeLa cells, and 378, 947, and 1162 in N2a cells). Seven-point dose-response curves were generated by treating cells with 1.5 μM fully modified cholesterol-conjugated siRNA for 72 hours with progressive 2-fold serial dilutions (n=3, mean±SD). M represents the molar concentration of siRNA (n=3, mean±SD). As shown in Figure 11, siRNA against the targets listed was effective in silencing human or mouse IFNGR1.

[0482] Table 6 and Table 7 show the 45 nucleotide gene regions and 20 nucleotide target sequences of human IFNGR1, JAK1, JAK2 and STAT1 target sequences tested in the above screening and dose-response curve, respectively. Table 8 and Table 9 show the 45 nucleotide gene regions and 20 nucleotide target sequences of mouse IFNGR1, JAK1, JAK2 and STAT1 target sequences tested in the above screening and dose-response curve, respectively. Table 10 shows the sense and antisense strands of human IFNGR1, JAK1, JAK2 and STAT1 siR duplexes screened in Figure 1. Table 11 shows the sense and antisense strands of mouse IFNGR1, JAK1, JAK2 and STAT1 siR duplexes screened in Figure 2. Table 12 shows the antisense and sense strands of 12 siRNAs that caused potent and efficient silencing of IFNGR1, JAK1, JAK2 and STAT1 mRNA. The antisense strand contains a 5' uracil to enhance incorporation into RISC and can be complementary or non-complementary to the target IFNGR1, JAK1, JAK2, and STAT1 mRNA sequences.

[0483] Tables 13-15 show modified sense and antisense strands of FNGR1, JAK1, JAK2, and STAT1 mRNA target sequences according to additional embodiments.

[0484] Example 2. In vivo target protein knockdown by siRNA Ifngr1_1641 To test the duration of efficacy after a single dose of siRNA Ifngr1_1641, wild-type C57BL6 mice were treated with siRNA for up to 4 weeks, and Ifngr1 protein expression levels in the skin were measured by fluorescent flow cytometry. Figure 6A shows the results of fluorescent flow cytometry, and Figure 6B shows the summary data. A maximum of 66% target protein knockdown was achieved 2 weeks after injection, and a significant level of protein knockdown was maintained for 4 weeks. These data demonstrated that a single dose of siRNA Ifngr1_1641 provided at least a 4-week duration of efficacy in the skin. The data suggested that a 2-week interval between doses could maximize target knockdown, and also rationalized the subsequent experiments described below.

[0485] Example 3. Ex vivo skin culture model for testing IFN-γ signaling inhibition To test the efficacy of siRNA Ifngr1_1641 on inhibition of IFN-γ signaling, the expression of chemokines CXCL9 and CXCL10 was measured in an ex vivo skin culture model. CXCL9 and CXCL10 are IFN-γ signaling downstream chemotactic factors involved in the recruitment of CD8+ T cells to the skin and the amplification of vitiligo autoimmunity. Knocking down the IFN-γ receptor IFNGR1 inhibits signaling, thereby reducing downstream CXCL9 and CXCL10 expression. Figure 7A shows the procedure used to test the effect of Ifngr1_1641 siRNA on IFN-γ signaling. Eight 4 mm diameter skin punch biopsies were taken per mouse 4 weeks after subcutaneous tail injection of 2 × 20 mg / kg siRNA (dosing interval: 2 weeks, n = 5 mice per group). Tail skin punch samples were cultured in the presence of recombinant mouse IFN-γ protein (2-fold serial dilutions from 25600 to 400 pg / mL, and untreated control). The levels of CXCL9 and CXCL10 were measured by enzyme-linked immunosorbent assay (ELISA). Figure 7B shows the results. Data are presented as mean ± SD and analyzed by two-way ANOVA with Dunnett's multiple comparison test; *P<0.05. These data indicated that functional inhibition of IFN-γ signaling at the protein level was achieved by targeted gene silencing. The siRNAs used were conjugated with DCA and used either scaffold 1 or scaffold 2 as shown below. Scaffold 1: Antisense strand, 5' to 3': V(mU)#(fG)#(mU)(mU)(mA)(fG)(mU)(mA)(mU)(mU)(mA)(mG)(mC)#(fU)#(mA)#(fA)#(mU)#(mG)#(mU)#(fA) Sense strand, 5'→3': (mU)#(mA)#(mG)(mC)(fU)(fA)(fA)(mU)(fA)(mC)(mU)(mA)(mA)#(mC)#(mA)(dT)(dT)-DCA Scaffold 2: Antisense strand, 5' to 3': V(mU)#(fG)#(mU)(fU)(fA)(fG)(mU)(fA)(mU)(fU)(mA)(fG)(mC)(fU)#(mA)#(fA)#(mU)#(mG)#(mU)#(fA)#(mU) Sense strand, 5'→3': (mU)#(mU)#(mA)(fG)(mC)(fU)(mA)(fA)(mU)(fA)(mC)(mU)(mA)(fA)#(mC)#(mA)(dT)(dT)-DCA m=2'-O-methyl; f=2'-fluoro; #=phosphorothioate; V=5'-vinyl phosphate; dT=thymidine; DCA=docosanoic acid

[0486] The expression levels of CXCL9, CXCL10, and CXCL11 mRNA were measured in HeLa and N2a cells. Cells were treated with 1.5 μM IFNGR1_1726 and Ifngr1_1641-targeted siRNA for 72 h and then stimulated with IFN-γ (n=4, mean ± SD, one-way ANOVA, * p<0.05, ** p<0.01, ***p<0.001, **** p<0.0001; ns, not significant). Samples were analyzed 6 hours after IFN-γ signaling stimulation. As shown in Figure 12, siRNA efficiently reduced the expression of CXCL9, 10, and 11 in the presence of IFN-γ signaling stimulation.

[0487] Example 4. Systemic and local efficacy of siRNA Ifngr1_1641 in a vitiligo mouse model To further evaluate the efficacy of IFN-γ signaling-targeting siRNA in treating vitiligo, a vitiligo mouse model was developed. Figure 8A shows how vitiligo was induced by adoptive transfer of PMEL CD8+ T cells isolated from the spleens of PMEL TCR transgenic mice. Subsequent activation of these T cells in recipient mice results in epidermal depigmentation in a spotted pattern similar to that of vitiligo patients within 3–7 weeks. Mice were treated with the first dose of siRNA 2 weeks before vitiligo induction and with the second dose 1 week after induction. For efficacy assessment, vitiligo scores were objectively quantified by observers blinded to the treatment groups, using a graded scale based on the extent of depigmented area in the ears and tails. Each site was examined as a percentage of the anatomical site. Both the left and right ears were determined collectively and therefore considered as a single site. Individual sites were assigned a vitiligo score of 0 to 5 as follows: no evidence of depigmentation (0%) was assigned a score of 0, >0-10%=1 point, >10-25%=2 points, >25-75%=3 points, >75-<100%=4 points, and 100%=5 points. Figure 8B shows the results. Data are presented as mean ± SD and analyzed by two-way ANOVA with Sidak's multiple comparison test; * P < 0.05, ** P < 0.01, **** P<0.0001.

[0488] Figure 9 shows the quantitative analysis of the tail depigmentation levels between treatment groups. Figure 9A shows the skin depigmentation levels quantified objectively by comparing tail photographs using ImageJ Fiji software (NIH). In Figure 9B, pixel intensity distribution profiles of individual tails were plotted against the total number of pixels per intensity. Complete white and black were defined as intensities of 0 and 255, respectively. Figure 9C is a plot of the average pixel intensity per tail. Statistical data are presented as the mean ± SD of the average pixel intensity of individual distribution curves and analyzed by Mann-Whitney t-test; * P<0.05. Figure 9D is a plot showing that dermal infiltration of cytotoxic T cells (as measu...

Claims

1. An oligonucleotide targeting an IFN-γ signaling pathway target gene selected from the group consisting of IFNGR1, JAK1, JAK2, or STAT1, comprising a sequence substantially complementary to any one nucleic acid sequence of SEQ ID NOs: 1 to 96.

2. The oligonucleotide according to claim 1, comprising a sequence substantially complementary to any one nucleic acid sequence of SEQ ID NOs: 143 to 244.

3. The oligonucleotide according to claim 1, which is an RNA molecule comprising single-stranded (ss) RNA or double-stranded RNA (dsRNA).

4. The oligonucleotide according to claim 3, wherein the dsRNA comprises at least one modified nucleotide internucleoside linkage of Formula I: 【Chemical 1】 (wherein, B is a base pair moiety; W is selected from the group consisting of O, OCH ２ , OCH, CH ２ , and CH; X is selected from the group consisting of halo, hydroxy, and C １－６ alkoxy, Y is O － , OH, OR, NH － , NH ２ , S － , and is selected from the group consisting of SH Z is selected from the group consisting of O and CH ２ and; R is a protecting group, [Chemical Formula 2] is an optional double bond).

5. The oligonucleotide according to claim 3, wherein the dsRNA comprises an antisense strand and a sense strand, each strand having a 5' end and a 3' end, A: (1) the antisense strand comprises a sequence substantially complementary to any one nucleic acid sequence of SEQ ID NOs: 1 to 96; (2) the antisense strand alternately comprises 2'-methoxy-ribonucleotides and 2'-fluoro-ribonucleotides; (3) the nucleotides at positions 2 and 14 from the 5' end of the antisense strand are not 2'-methoxy-ribonucleotides; (4) the nucleotides at positions 1 to 2 to 1 to 7 from the 3' end of the antisense strand are linked to each other via phosphorothioate nucleotide internucleoside linkages; (5) a part of the antisense strand is complementary to a part of the sense strand; (6) the sense strand alternately comprises 2'-methoxy-ribonucleotides and 2'-fluoro-ribonucleotides; (7) the nucleotides at positions 1 to 2 from the 5' end of the sense strand are linked to each other via phosphorothioate nucleotide internucleoside linkages; B: (1) the antisense strand comprises a sequence substantially complementary to any one nucleic acid sequence of SEQ ID NOs: 1 to 96; (2) the antisense strand comprises at least 70% 2'-O-methyl modification; (3) the nucleotide at position 14 from the 5' end of the antisense strand is not a 2'-methoxy-ribonucleotide; (4) The nucleotides at positions 1 to 7 from positions 1 to 2 counted from the 3'-end of the antisense strand are linked to each other via phosphorothioate internucleotide linkages; (5) A part of the antisense strand is complementary to a part of the sense strand; (6) The sense strand contains at least 70% 2'-O-methyl modification; (7) The nucleotides at positions 1 to 2 counted from the 5'-end of the sense strand are linked to each other via phosphorothioate internucleotide linkages; C: (1) The antisense strand contains a sequence substantially complementary to any one of the nucleic acid sequences of SEQ ID NOs: 1 to 96; (2) The antisense strand contains at least 85% 2'-O-methyl modification; (3) The nucleotides at positions 2 and 14 counted from the 5'-end of the antisense strand are not 2'-methoxy-ribonucleotides; (4) The nucleotides at positions 1 to 7 from positions 1 to 2 counted from the 3'-end of the antisense strand are linked to each other via phosphorothioate internucleotide linkages; (5) A part of the antisense strand is complementary to a part of the sense strand; (6) The sense strand contains 100% 2'-O-methyl modification; (7) The nucleotides at positions 1 to 2 counted from the 5'-end of the sense strand are linked to each other via phosphorothioate internucleotide linkages, D: (1) The antisense strand contains a sequence substantially complementary to any one of the nucleic acid sequences of SEQ ID NOs: 1 to 96; (2) The antisense strand contains at least 75% 2'-O-methyl modification; (3) The nucleotides at positions 4, 5, 6, and 14 counted from the 5'-end of the antisense strand are not 2'-methoxy-ribonucleotides; (4) The nucleotides at positions 1 to 7 from positions 1 to 2 counted from the 3'-end of the antisense strand are linked to each other via phosphorothioate internucleotide linkages; (5) A part of the antisense strand is complementary to a part of the sense strand; (6) The sense strand contains 100% 2'-O-methyl modification; (7) The nucleotides at positions 1 to 2 counted from the 5'-end of the sense strand are linked to each other via phosphorothioate internucleotide linkages; E: (1) The antisense strand contains a sequence substantially complementary to any one of the nucleic acid sequences of SEQ ID NOs: 1 to 96; (2) The antisense strand contains at least 75% 2'-O-methyl modification; (3) The nucleotides at positions 2, 4, 5, 6, and 14 from the 5'-end of the antisense strand are not 2'-methoxy-ribonucleotides; (4) The nucleotides at positions 1 to 2 to 1 to 7 from the 3'-end of the antisense strand are linked to each other via phosphorothioate nucleotide internucleotide linkages; (5) A part of the antisense strand is complementary to a part of the sense strand; (6) The sense strand contains 100% 2'-O-methyl modification; (7) The nucleotides at positions 1 to 2 from the 5'-end of the sense strand are linked to each other via phosphorothioate nucleotide internucleotide linkages; F: (1) The antisense strand contains a sequence substantially complementary to any one of the nucleic acid sequences of SEQ ID NOs: 1 to 96; (2) The antisense strand contains at least 75% 2'-O-methyl modification; (3) The nucleotides at positions 2, 6, 14, and 16 from the 5'-end of the antisense strand are not 2'-methoxy-ribonucleotides; (4) The nucleotides at positions 1 to 2 to 1 to 7 from the 3'-end of the antisense strand are linked to each other via phosphorothioate nucleotide internucleotide linkages; (5) A part of the antisense strand is complementary to a part of the sense strand; (6) The sense strand contains at least 70% 2'-O-methyl modification; (7) The nucleotides at positions 7, 9, 10, and 11 from the 3'-end of the sense strand are not 2'-methoxy-ribonucleotides; (8) The nucleotides at positions 1 to 2 from the 5'-end of the sense strand are linked to each other via phosphorothioate nucleotide internucleotide linkages; G: (1) The antisense strand contains a sequence substantially complementary to any one of the nucleic acid sequences of SEQ ID NOs: 1 to 96; (2) The antisense strand contains at least 75% 2'-O-methyl modification; (3) The nucleotides at positions 2, 6, and 14 from the 5'-end of the antisense strand are not 2'-methoxy-ribonucleotides; (4) The nucleotides at positions 1 to 2 to 1 to 7 from the 3'-end of the antisense strand are linked to each other via phosphorothioate nucleotide internucleotide linkages; (5) A part of the antisense strand is complementary to a part of the sense strand; (6) The sense strand contains at least 80% 2'-O-methyl modification; (7) The nucleotides at positions 7, 10, and 11 from the 3'-end of the sense strand are not 2'-methoxy-ribonucleotides; (8) The nucleotides at positions 1 to 2 from the 5'-end of the sense strand are linked to each other via phosphorothioate nucleotide internucleotide linkages; H: (1) The antisense strand contains a sequence substantially complementary to any one of the nucleic acid sequences of SEQ ID NOs: 1 to 96; (2) The antisense strand contains at least 50% 2'-O-methyl modification; (3) The nucleotides at positions 2, 4, 5, 6, 8, 10, 12, 14, 16, and 20 from the 5'-end of the antisense strand are not 2'-methoxy-ribonucleotides; (4) The nucleotides at positions 1 to 2 to 1 to 8 from the 3'-end of the antisense strand are linked to each other via phosphorothioate nucleotide internucleotide linkages; (5) A part of the antisense strand is complementary to a part of the sense strand; (6) The sense strand contains at least 65% 2'-O-methyl modification; (7) The nucleotides at positions 3, 7, 9, 11, and 13 from the 3'-end of the sense strand are not 2'-methoxy-ribonucleotides; (8) The nucleotides at positions 1 to 2 from the 5'-end of the sense strand are linked to each other via phosphorothioate nucleotide internucleotide linkages; or I: (1) The antisense strand contains a sequence substantially complementary to any one of the nucleic acid sequences of SEQ ID NOs: 1 to 96; (2) The antisense strand contains at least 75% 2'-O-methyl modification; (3) The nucleotides at positions 2, 6, 14, 16, and 20 from the 5'-end of the antisense strand are not 2'-methoxy-ribonucleotides; (4) The nucleotides at positions 1 to 7 and 19 to 20 from the 3'-end of the antisense strand are linked to each other via phosphorothioate nucleotide internucleotide linkages; (5) A part of the antisense strand is complementary to a part of the sense strand; (6) The sense strand contains at least 65% 2'-O-methyl modification; (7) The nucleotides at positions 7, 9, 10, and 11 from the 3'-end of the sense strand are not 2'-methoxy-ribonucleotides; The nucleotides at positions 1 to 2 from the 5'-end of the sense strand are linked to each other via phosphorothioate nucleotide internucleotide linkages. The oligonucleotide. **Claim 6** A double-stranded RNA (dsRNA) molecule comprising an antisense strand and a sense strand, each strand having a 5'-end and a 3'-end, (1) the antisense strand comprises a sequence substantially complementary to the nucleic acid sequence of a target gene in the IFN-γ signaling pathway; (2) the antisense strand is 21 nucleotides in length; (3) the antisense strand comprises at least 50% 2'-O-methyl modification; (4) the nucleotides at any one or more of positions 2, 4, 5, 6, 8, 10, 12, 14, 16, and 20 from the 5'-end of the antisense strand are not 2'-methoxy-ribonucleotides; (5) the nucleotides at positions 1 to 2 to 1 to 8 from the 3'-end of the antisense strand are linked to each other via phosphorothioate nucleotide internucleotide linkages; (6) a part of the antisense strand is complementary to a part of the sense strand; (7) the sense strand is 16 nucleotides in length; (8) the sense strand comprises at least 65% 2'-O-methyl modification; (9) the nucleotides at positions 3, 7, 9, 11, and 13 from the 3'-end of the sense strand are not 2'-methoxy-ribonucleotides; (10) the nucleotides at positions 1 to 2 from the 5'-end of the sense strand are linked to each other via phosphorothioate nucleotide internucleotide linkages, the dsRNA molecule. **Claim 7** A functional moiety is linked to the 3'-end of the sense strand by a linker, and optionally the linker comprises an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, a carbamate, or a combination thereof, the dsRNA molecule according to claim 6. **Claim 8** The dsRNA molecule according to claim 7, wherein the functional moiety is a fatty acid selected from the group consisting of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosanoic acid (DCA). **Claim 9** A pharmaceutical composition for inhibiting the expression of an IFN-γ signaling pathway gene selected from the group consisting of IFNGR1, JAK1, JAK2, and STAT1 in an organism, comprising the oligonucleotide or dsRNA molecule according to any one of claims 3 to 8 and a pharmaceutically acceptable carrier.

10. A method for inhibiting the expression of an IFN-γ signaling pathway gene selected from the group consisting of IFNGR1, JAK1, JAK2, and STAT1 in a cell, comprising: (a) introducing the oligonucleotide or dsRNA molecule according to any one of claims 3 to 8 into the cell; (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the gene, thereby inhibiting the expression of the gene in the cell.

11. A pharmaceutical composition for treating vitiligo, comprising a therapeutically effective amount of an oligonucleotide comprising sufficient complementarity to an IFN-γ signaling pathway target gene.

12. A vector comprising a regulatory sequence operably linked to a nucleotide sequence encoding an RNA molecule substantially complementary to any one of the nucleic acid sequences of SEQ ID NOs: 1 to 96.

13. A cell comprising the vector according to claim 12.

14. Two or more RNA molecules having a length of 15 to 35 nucleotides, and a sequence substantially complementary to the mRNA of an IFN-γ signaling pathway target gene selected from the group consisting of IFNGR1, JAK1, JAK2, and STAT1, wherein the two or more RNA molecules are linked to each other by one or more moieties independently selected from linkers, spacers, and branch points, a branched RNA compound.

15. Two or more RNA molecules having a length of 15 to 35 nucleotides, and a sequence substantially complementary to the mRNA of an IFN-γ signaling pathway target gene, a branched RNA compound comprising: wherein the two or more RNA molecules are linked to each other by one or more moieties independently selected from linkers, spacers, and branch points; wherein the two or more RNA molecules comprise dsRNA, the dsRNA comprises an antisense strand and a sense strand, and each strand comprises a 5' end and a 3' end; (1) the antisense strand comprises a sequence substantially complementary to the nucleic acid sequence of an IFN-γ signaling pathway target gene; (2) the antisense strand contains at least 50% 2'-O-methyl modification; (3) the nucleotides at any one or more of positions 2, 4, 5, 6, 8, 10, 12, 14, 16, and 20 from the 5'-end of the antisense strand are not 2'-methoxy-ribonucleotides; (4) the nucleotides at positions 1-2 to 1-8 from the 3'-end of the antisense strand are linked to each other via phosphorothioate nucleotide internucleotide linkages; (5) a part of the antisense strand is complementary to a part of the sense strand; (6) the sense strand contains at least 65% 2'-O-methyl modification; (7) the nucleotides at any one or more of positions 3, 7, 9, 11, and 13 from the 3'-end of the sense strand are not 2'-methoxy-ribonucleotides; (8) the nucleotides at positions 1-2 from the 5'-end of the sense strand are linked to each other via phosphorothioate nucleotide internucleotide linkages, the branched RNA compound.

16. A compound of formula (I): 【Chemical Formula 3】 (wherein, L includes an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, phosphate, phosphonate, phosphoramidate, ester, amide, triazole, or a combination thereof, and formula (I) further includes one or more branch points B and one or more spacers S, wherein, one or more branch points B are, independently for each occurrence, a polyvalent organic species or a derivative thereof; one or more spacers S are, independently for each occurrence, an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, phosphate, phosphonate, phosphoramidate, ester, amide, triazole, or a combination thereof; N is a double-stranded nucleic acid having a length of 15 to 35 bases including a sense strand and an antisense strand; here the antisense strand contains a sequence substantially complementary to any one of the nucleic acid sequences of SEQ ID NOs: 1 to 96; the sense strand and the antisense strand each independently contain one or more chemical modifications; n is 2, 3, 4, 5, 6, 7, or 8).

17. A pharmaceutical composition for inhibiting the expression of a target gene of the IFN-γ signaling pathway in a living being, comprising the compound according to any one of claims 14 to 16 and a pharmaceutically acceptable carrier.

18. A method for inhibiting the expression of a target gene in the IFN-γ signaling pathway in cells, comprising: (a) introducing the compound according to any one of claims 14 to 16 into the cells; and (b) maintaining the cells produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the gene, thereby inhibiting the expression of the gene in the cells. The method as described above.

19. A pharmaceutical composition for treating vitiligo in a subject in need of treatment for vitiligo, the pharmaceutical composition comprising a therapeutically effective amount of the compound according to any one of claims 14 to 16.

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