CN117642508A

CN117642508A - Oligonucleotides for IFN-gamma signaling pathway modulation

Info

Publication number: CN117642508A
Application number: CN202280048953.8A
Authority: CN
Inventors: 阿纳斯塔西娅·赫沃罗娃; 约翰·E·哈里斯; 汤奇
Original assignee: University of Massachusetts UMass
Current assignee: University of Massachusetts UMass
Priority date: 2021-06-22
Filing date: 2022-06-21
Publication date: 2024-03-01

Abstract

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

Description

Oligonucleotides for IFN-gamma signaling pathway modulation

RELATED APPLICATIONS

The present application claims the benefit of U.S. provisional patent application Ser. No. 63/213,506 filed on 22 th 6 and 2022, U.S. provisional patent application Ser. No. 63/331,563 filed on 15 th 4 and 2021, the entire disclosures of which are incorporated herein by reference.

Technical Field

The present disclosure relates to novel IFN-gamma signaling pathway target gene targeting sequences, novel branching oligonucleotides, and novel methods for treating and preventing IFN-gamma related vitiligo.

Background

Vitiligo is a type of CD8 ⁺ Cytotoxic T cell mediated autoimmune skin disease, the CD8 ⁺ Cytotoxic T cells attack melanocytes and cause leukoplakia in the affected skin area. IFN-gamma signaling is involved in the pathogenesis of vitiligo. Specifically, autoimmunity activates IFN-gamma signaling in epidermal keratinocytes through the JAK-STAT pathway and induces the expression of chemotactic agents CXCL9 and CXCL10, thereby promoting CD8 ⁺ Further infiltration of cytotoxic T cells to effect skin depigmentation.

There is currently no drug approved by the U.S. food and drug administration (U.S. food and Drug Administration) for the treatment of vitiligo. Off-label treatment (including phototherapy, topical-like solid alcohols, and small molecule drugs) typically requires repeated administration, not only is time consuming, but can be associated with long-term safety issues due to high dose exposure. Recent advances in understanding the pathogenic role of IFN-gamma signaling in vitiligo have led to acceptable efficacy of small molecule JAK inhibitor therapy and significantly improved patient quality of life. However, those JAK inhibitors are "pan JAK inhibitors" that block multiple cytokine receptor signaling according to subtypes JAK1, JAK2, JAK3, and Tyk 2. Thus, targeted therapies for IFN- γ signaling with long-term efficacy and improved selectivity remain to be achieved.

Thus, there is a need to reduce the expression of proteins involved in IFN- γ signaling to treat vitiligo and related conditions.

Disclosure of Invention

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

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

In another aspect, the present disclosure provides an RNA molecule comprising a sequence that is substantially complementary to the nucleic acid sequence of any one of SEQ ID NOS: 1-96.

In certain embodiments, the RNA molecule is 8 nucleotides to 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, 43 nucleotides, 44 nucleotides 45 nucleotides, 46 nucleotides, 47 nucleotides, 48 nucleotides, 49 nucleotides, 50 nucleotides, 51 nucleotides, 52 nucleotides, 53 nucleotides, 54 nucleotides, 55 nucleotides, 56 nucleotides, 57 nucleotides, 58 nucleotides, 59 nucleotides, 60 nucleotides, 61 nucleotides, 62 nucleotides, 63 nucleotides, 64 nucleotides, 65 nucleotides, 66 nucleotides, 67 nucleotides, 68 nucleotides, 69 nucleotides, 70 nucleotides, 71 nucleotides, 72 nucleotides, 73 nucleotides, 74 nucleotides, 75 nucleotides, 76 nucleotides, 77 nucleotides, 78 nucleotides, 79 nucleotides or 80 nucleotides in length.

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 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).

In certain embodiments, the RNA molecule has a nucleic acid sequence that is substantially complementary to the nucleic acid sequence of any one of SEQ ID NOS 143-244.

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

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

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 a nucleic acid sequence of any one of SEQ ID NOS.1-6. 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.

In certain embodiments, the dsRNA comprises an antisense strand having complementarity to at least 10, 11, 12 or 13 consecutive nucleotides of the 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 consecutive nucleotides of the nucleic acid sequence of any one of SEQ ID nos. 1-6 (e.g., a segment of 10 to 25 consecutive nucleotides of the nucleic acid sequence of SEQ ID No. 1, a segment of 10 to 25 consecutive nucleotides of the nucleic acid sequence of SEQ ID No. 2, a segment of 10 to 25 consecutive nucleotides of the nucleic acid sequence of SEQ ID No. 3, a segment of 10 to 25 consecutive nucleotides of the nucleic acid sequence of SEQ ID No. 4, a segment of 10 to 25 consecutive nucleotides of the nucleic acid sequence of SEQ ID No. 5, or a segment of 10 to 25 consecutive nucleotides of the nucleic acid sequence of SEQ ID No. 6).

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

In certain embodiments, the dsRNA comprises an antisense strand having up to 3 mismatches with the nucleic acid sequence of any one of SEQ ID NOS: 1-6. For example, the antisense strand can have 0-3 mismatches (e.g., 0 mismatches, 1 mismatch, 2 mismatches, or 3 mismatches) relative to the nucleic acid sequence of SEQ ID NO: 1. In certain embodiments, the antisense strand can have 0-3 mismatches (e.g., 0 mismatches, 1 mismatch, 2 mismatches, or 3 mismatches) relative to the nucleic acid sequence of SEQ ID NO: 2. In certain embodiments, the antisense strand can have 0-3 mismatches (e.g., 0 mismatches, 1 mismatch, 2 mismatches, or 3 mismatches) relative to the nucleic acid sequence of SEQ ID NO: 3. In certain embodiments, the antisense strand can have 0-3 mismatches (e.g., 0 mismatches, 1 mismatch, 2 mismatches, or 3 mismatches) relative to the nucleic acid sequence of SEQ ID NO: 4. In certain embodiments, the antisense strand can have 0-3 mismatches (e.g., 0 mismatches, 1 mismatch, 2 mismatches, or 3 mismatches) relative to the nucleic acid sequence of SEQ ID NO: 5. In certain embodiments, the antisense strand can have 0-3 mismatches (e.g., 0 mismatches, 1 mismatch, 2 mismatches, or 3 mismatches) relative to the nucleic acid sequence of SEQ ID NO: 6.

In certain embodiments, the dsRNA comprises an antisense strand that is fully complementary to the nucleic acid sequence of any one of SEQ ID NOS.1-6.

In certain embodiments, the dsRNA comprises 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 comprises 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 comprises 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 having the nucleic acid sequence of any one of SEQ ID NOs 1-6.

In certain embodiments, the antisense strand and/or sense strand comprises about 15 nucleotides to 25 nucleotides in length. For example, in certain embodiments, the antisense strand and/or 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.

In certain embodiments, the antisense strand is 20 nucleotides in length, while the sense strand is 15 nucleotides or 16 nucleotides in length.

In certain embodiments, the antisense strand is 21 nucleotides in length, while the sense strand is 15 nucleotides or 16 nucleotides in length.

In some embodiments, the antisense strand is 20 nucleotides or 21 nucleotides in length, while the sense strand is 15 nucleotides in length.

In some embodiments, the antisense strand is 20 nucleotides or 21 nucleotides in length, while the sense strand is 16 nucleotides in length.

In certain embodiments, the antisense strand is 20 nucleotides in length, while the sense strand is 15 nucleotides in length.

In certain embodiments, the antisense strand is 21 nucleotides in length, while the sense strand is 16 nucleotides in length.

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

In certain embodiments, the dsRNA comprises blunt ends. 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 nucleotides to 5 nucleotides.

In certain embodiments, the dsRNA comprises naturally occurring nucleotides.

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

In certain embodiments, the modified nucleotides comprise 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, nucleotides comprising a non-natural base, or mixtures thereof.

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

In certain embodiments, the modified internucleotide linkages comprise phosphorothioate internucleotide linkages. 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).

In certain embodiments, the dsRNA comprises at least one modified internucleotide linkage of formula I:

wherein:

b is a base pairing moiety;

w is selected from O, OCH ₂ 、OCH、CH ₂ And CH;

x is selected from halo, hydroxy and C _1-6 Alkoxy groups;

y is selected from O ^- 、OH、OR、NH-、NH ₂ S-and SH;

z is selected from O and CH ₂ A group of;

r is a protecting group; and is also provided with

Is an optional double bond.

In certain embodiments, when W is CH,is a double bond.

In certain embodiments, when W is selected from O, OCH ₂ 、OCH、CH ₂ In the case of the group of the components,is a single bond.

In certain embodiments, the 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, the dsRNA is entirely chemically modified. In certain embodiments, the dsRNA comprises 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).

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 modification (e.g., about 83%, 84%, 85%, or 86% 2' -O-methyl modification).

In certain embodiments, the dsRNA comprises about 70% to about 80% 2 '-O-methyl nucleotide modifications (e.g., about 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 modification (e.g., about 75%, 76%, 77%, or 78% 2' -O-methyl modification).

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 entirely chemically modified. In certain embodiments, the antisense strand comprises at least 70% 2 '-O-methyl nucleotide modification (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 modification). In certain embodiments, the antisense strand comprises from about 70% to 90% 2 '-O-methyl nucleotide modification (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 modification). In certain embodiments, the antisense strand comprises from about 85% to about 90% 2 '-O-methyl modification (e.g., about 85%, 86%, 87%, 88%, 89%, or 90% 2' -O-methyl modification).

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

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 entirely 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.

In certain embodiments, the sense strand comprises about 70% to 85% 2 '-O-methyl nucleotide modifications (e.g., about 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).

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).

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

In certain embodiments, the antisense strand comprises a 5 'phosphate, a 5' -alkylphosphonate, a 5 'alkylene phosphonate, or a 5' alkenylphosphonate.

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

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 of the 5 'end of the antisense strand are not 2' -methoxy-ribonucleotides; (4) Nucleotides at positions 1-2 to 1-7 of the 3' -end of the antisense strand are linked to each other by 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; and (7) the nucleotides at positions 1-2 of the 5' -end of the sense strand are linked to each other by phosphorothioate internucleotide linkages.

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 at least 70% 2 '-O-methyl modification (e.g., about 75% to about 80% or about 85% to about 90% 2' -O-methyl modification); (3) The nucleotide at position 14 of the 5 'end of the antisense strand is not a 2' -methoxy-ribonucleotide; (4) Nucleotides at positions 1-2 to 1-7 of the 3' -end of the antisense strand are linked to each other by phosphorothioate internucleotide linkages; (5) A portion of the antisense strand is complementary to a portion of the sense strand; (6) The sense strand comprises at least 65% 2 '-O-methyl modification (e.g., about 65% to about 75% or about 75% to about 80% 2' -O-methyl modification); and (7) the nucleotides at positions 1-2 of the 5' -end of the sense strand are linked to each other by phosphorothioate internucleotide linkages.

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 at least 85% 2' -O-methyl modification; (3) The nucleotides at positions 2 and 14 of the 5 'end of the antisense strand are not 2' -methoxy-ribonucleotides; (4) Nucleotides at positions 1-2 to 1-7 of the 3' -end of the antisense strand are linked to each other by 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 modification; and (7) the nucleotides at positions 1-2 of the 5' -end of the sense strand are linked to each other by phosphorothioate internucleotide linkages.

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 at least 75% 2' -O-methyl modification; (3) Nucleotides at positions 4, 5, 6 and 14 of the 5 'end of the antisense strand are not 2' -methoxy-ribonucleotides; (4) Nucleotides at positions 1-2 to 1-7 of the 3' -end of the antisense strand are linked to each other by 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 modification; and (7) the nucleotides at positions 1-2 of the 5' -end of the sense strand are linked to each other by phosphorothioate internucleotide linkages.

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 at least 85% 2 '-O-methyl modification (e.g., about 85% to about 90% 2' -O-methyl modification); (3) The nucleotides at positions 2 and 14 of the 5 'end of the antisense strand are not 2' -methoxy-ribonucleotides (e.g., the nucleotides at positions 2 and 14 of the 5 'end of the antisense strand may be 2' -fluoro nucleotides); (4) Nucleotides at positions 1-2 to 1-7 of the 3' -end of the antisense strand are linked to each other by phosphorothioate internucleotide linkages; (5) A portion of the antisense strand is complementary to a portion of the sense strand; (6) The sense strand comprises at least 75% 2 '-O-methyl modification (e.g., about 75% to about 80% 2' -O-methyl modification); (7) The nucleotides at positions 7, 10 and 11 of the 3 'end of the sense strand are not 2' -methoxy-ribonucleotides (e.g., the nucleotides at positions 7, 10 and 11 of the 3 'end of the sense strand are 2' -fluoro nucleotides); and (8) the nucleotides at positions 1-2 of the 5' -end of the sense strand are linked to each other by phosphorothioate internucleotide linkages.

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 at least 75% 2 '-O-methyl modification (e.g., about 75% to about 80% 2' -O-methyl modification); (3) The nucleotides at positions 2, 4, 5, 6 and 14 of the 5 'end of the antisense strand are not 2' -methoxy-ribonucleotides (e.g., the nucleotides at positions 2, 6, 14 and 16 of the 5 'end of the antisense strand may be 2' -fluoro nucleotides); (4) Nucleotides at positions 1-2 to 1-7 of the 3' -end of the antisense strand are linked to each other by 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 modification; and (7) the nucleotides at positions 1-2 of the 5' -end of the sense strand are linked to each other by phosphorothioate internucleotide linkages.

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 at least 75% 2 '-O-methyl modification (e.g., about 75% to about 80% 2' -O-methyl modification); (3) The nucleotides at positions 2, 6, 14 and 16 of the 5 'end of the antisense strand are not 2' -methoxy-ribonucleotides (e.g., the nucleotides at positions 2, 6, 14 and 16 of the 5 'end of the antisense strand may be 2' -fluoro nucleotides); (4) Nucleotides at positions 1-2 to 1-7 of the 3' -end of the antisense strand are linked to each other by phosphorothioate internucleotide linkages; (5) A portion of the antisense strand is complementary to a portion of the sense strand; (6) The sense strand comprises at least 65% 2 '-O-methyl modification (e.g., about 65% to about 75% 2' -O-methyl modification); (7) The nucleotides at positions 7, 9, 10 and 11 of the 3 'end of the sense strand are not 2' -methoxy-ribonucleotides (e.g., the nucleotides at positions 7, 9, 10 and 11 of the 3 'end of the sense strand are 2' -fluoro nucleotides); and (8) the nucleotides at positions 1-2 of the 5' -end of the sense strand are linked to each other by phosphorothioate internucleotide linkages.

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 the nucleic acid sequence of SEQ ID NOS 1-6; (2) the antisense strand comprises at least 75% 2' -O-methyl modification; (3) The nucleotides at positions 2, 6, and 14 of the 5 'end of the antisense strand are not 2' -methoxy-ribonucleotides; (4) Nucleotides at positions 1-2 to 1-7 of the 3' -end of the antisense strand are linked to each other by phosphorothioate internucleotide linkages; (5) A portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand comprises at least 80% 2' -O-methyl modification; (7) Nucleotides at positions 7, 10 and 11 of the 3 'end of the sense strand are not 2' -methoxy-ribonucleotides; and (8) the nucleotides at positions 1-2 of the 5' -end of the sense strand are linked to each other by phosphorothioate internucleotide linkages.

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 at least 75% 2 '-O-methyl modification (e.g., about 75% to about 80% 2' -O-methyl modification); (3) The nucleotides at positions 2, 6, 14, 16 and 20 of the 5 'end of the antisense strand are not 2' -methoxy-ribonucleotides (e.g., the nucleotides at positions 2, 6, 14, 16 and 20 of the 5 'end of the antisense strand may be 2' -fluoro nucleotides); (4) Nucleotides at positions 1-7 and 19-20 of the 3' end of the antisense strand are linked to each other by phosphorothioate internucleotide linkages; (5) A portion of the antisense strand is complementary to a portion of the sense strand; (6) The sense strand comprises at least 65% 2 '-O-methyl modification (e.g., about 65% to about 75% 2' -O-methyl modification); (7) The nucleotides at positions 7, 9, 10 and 11 of the 3 'end of the sense strand are not 2' -methoxy-ribonucleotides (e.g., the nucleotides at positions 7, 9, 10 and 11 of the 3 'end of the sense strand are 2' -fluoro nucleotides); and (8) the nucleotides at positions 1-2 and 14-15 of the 5' -end of the sense strand are linked to each other by phosphorothioate internucleotide linkages.

In certain embodiments, the functional moiety is attached to the 5 'and/or 3' end of the antisense strand. In certain embodiments, the functional moiety is attached to the 5 'and/or 3' end of the sense strand. In certain embodiments, the functional moiety is attached to the 3' end of the sense strand.

In certain embodiments, the functional moiety comprises a hydrophobic moiety.

In certain embodiments, the hydrophobic moiety is selected from the group consisting of: fatty acids, steroids, ring-opened steroids, lipids, gangliosides, nucleoside analogs, endogenous cannabinoids, vitamins, and mixtures thereof.

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-tocopheryl succinate.

In certain embodiments, the functional moiety is myristic acid (Myr). In certain embodiments, the functional moiety is trimyristate (Myr-t).

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

In certain embodiments, the linker comprises a divalent or trivalent linker.

In certain embodiments, the divalent or trivalent linker is selected from the group consisting of:

wherein n is 1, 2, 3, 4 or 5.

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

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

In certain embodiments, the phosphodiester or phosphodiester derivative is selected from the group consisting of:

wherein X is O, S or BH ₃ 。

In certain embodiments, the nucleotides at positions 1 and 2 of the 3 'end of the sense strand and the nucleotides at positions 1 and 2 of the 5' end of the antisense strand are linked to adjacent ribonucleotides by phosphorothioate linkages.

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, JAK2, or STAT1 in an organism comprising the dsRNA described above and a pharmaceutically acceptable carrier.

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%.

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

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

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 comprising sufficient complementarity to an IFN- γ signaling pathway target gene, thereby treating the subject.

In certain embodiments, the IFN- γ signaling pathway target gene is selected from the group consisting of IFNGR1, JAK2, or STAT 1.

In certain embodiments, the method of treatment comprises administering a therapeutically effective amount of the dsRNA set forth above.

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

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

In one aspect, the present disclosure provides vectors comprising regulatory sequences operably linked to a nucleotide sequence encoding an RNA molecule that is substantially complementary to the nucleic acid sequences of SEQ ID NOs 1-6.

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

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

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

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

In one aspect, the present disclosure provides a cell comprising the vector described above.

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

In one aspect, the disclosure provides branched RNA compounds comprising two or more RNA molecules, such as two or more RNA molecules each comprising 15 to 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), wherein each RNA molecule 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, JAK2, or STAT 1. The two RNA molecules may be interconnected by one or more moieties independently selected from the group consisting of a linker, a spacer and a branching point.

In certain embodiments, the branched RNA molecule comprises one or both of ssRNA and dsRNA.

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

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

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 disclosure covalently bound to each other (e.g., through a linker, spacer, or branching point).

In certain embodiments, the branched RNA compound comprises a portion of a nucleic acid sequence that is substantially complementary to the 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 that are covalently bound to each other (e.g., via a linker, spacer, or branch point) and that comprise an antisense strand that has complementarity to at least 10, 11, 12, or 13 consecutive nucleotides of the nucleic acid sequence of any 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 consecutive nucleotides of the nucleic acid sequence of any one of SEQ ID nos. 1-6 (e.g., a segment of 10 to 25 consecutive nucleotides of the nucleic acid sequence of SEQ ID No. 1, a segment of 10 to 25 consecutive nucleotides of the nucleic acid sequence of SEQ ID No. 2, a segment of 10 to 25 consecutive nucleotides of the nucleic acid sequence of SEQ ID No. 3, a segment of 10 to 25 consecutive nucleotides of the nucleic acid sequence of SEQ ID No. 4, a segment of 10 to 25 consecutive nucleotides of the nucleic acid sequence of SEQ ID No. 5, or a segment of 10 to 25 consecutive nucleotides of the nucleic acid sequence of SEQ ID No. 6).

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

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

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

In certain embodiments, the branched RNA compound comprises a portion having a nucleic acid sequence that is substantially complementary to one or more of the nucleic acid sequences of any one of SEQ ID NOS 143-154.

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

In certain embodiments, each RNA molecule comprises 15 to 25 nucleotides in length.

In certain embodiments, the antisense strand and/or sense strand comprises about 15 nucleotides to 25 nucleotides in length. For example, in certain embodiments, the antisense strand and/or 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.

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

In certain embodiments, the dsRNA comprises blunt ends.

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 nucleotides to 5 nucleotides.

In certain embodiments, the dsRNA comprises naturally occurring nucleotides.

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

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, an phosphoramidate, or a nucleotide comprising a non-natural base.

In certain embodiments, the modified internucleotide linkages comprise phosphorothioate internucleotide linkages. In certain embodiments, the branched RNA compound comprises 4-16 phosphorothioate internucleotide linkages. In certain embodiments, the branched RNA compound comprises 8-13 phosphorothioate internucleotide linkages.

wherein:

b is a base pairing moiety;

w is selected from O, OCH ₂ 、OCH、CH ₂ And CH;

x is selected from halo, hydroxy and C _1-6 Alkoxy groups;

y is selected from O-, OH, OR, NH-, NH ₂ S-and SH;

z is selected from O and CH ₂ A group of;

r is a protecting group; and is also provided with

Is an optional double bond.

In certain embodiments, when W is CH,is a double bond.

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

In certain embodiments, the antisense strand is entirely chemically modified.

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

In certain embodiments, the antisense strand comprises at least 80% chemically modified nucleotides. In certain embodiments, the sense strand is entirely 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.

In certain embodiments, the antisense strand comprises a 5 'phosphate, a 5' -alkylphosphonate, a 5 'alkylene phosphonate, a 5' alkenylphosphonate, or a mixture thereof.

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

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 at least 75% 2 '-O-methyl modification (e.g., about 75% to about 80% 2' -O-methyl modification); (3) The nucleotides at positions 2, 4, 5, 6 and 14 of the 5 'end of the antisense strand are not 2' -methoxy-ribonucleotides (e.g., the nucleotides at positions 2, 4, 5, 6, 14 and 16 of the 5 'end of the antisense strand may be 2' -fluoro nucleotides); (4) Nucleotides at positions 1-2 to 1-7 of the 3' -end of the antisense strand are linked to each other by 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 modification; and (7) the nucleotides at positions 1-2 of the 5' -end of the sense strand are linked to each other by phosphorothioate internucleotide linkages.

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 at least 75% 2 '-O-methyl modification (e.g., about 75% to about 80% 2' -O-methyl modification); (3) The nucleotides at positions 2, 6, 14 and 16 of the 5 'end of the antisense strand are not 2' -methoxy-ribonucleotides (e.g., the nucleotides at positions 2, 6, 14 and 16 of the 5 'end of the antisense strand may be 2' -fluoro nucleotides); (4) Nucleotides at positions 1-2 to 1-7 of the 3' -end of the antisense strand are linked to each other by phosphorothioate internucleotide linkages; (5) A portion of the antisense strand is complementary to a portion of the sense strand; (6) The sense strand comprises at least 65% 2 '-O-methyl modification (e.g., about 65% to about 75% 2' -O-methyl modification); (7) Nucleotides at positions 7, 9, 10 and 11 of the 3 'end of the sense strand are not 2' -methoxy-ribonucleotides; and (8) the nucleotides at positions 1-2 of the 5' -end of the sense strand are linked to each other by phosphorothioate internucleotide linkages.

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 at least 75% 2' -O-methyl modification; (3) The nucleotides at positions 2, 6, and 14 of the 5 'end of the antisense strand are not 2' -methoxy-ribonucleotides; (4) Nucleotides at positions 1-2 to 1-7 of the 3' -end of the antisense strand are linked to each other by phosphorothioate internucleotide linkages; (5) A portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand comprises at least 80% 2' -O-methyl modification; (7) Nucleotides at positions 7, 10 and 11 of the 3 'end of the sense strand are not 2' -methoxy-ribonucleotides; and (8) the nucleotides at positions 1-2 of the 5' -end of the sense strand are linked to each other by phosphorothioate internucleotide linkages.

In certain embodiments, the functional moiety comprises a hydrophobic moiety.

In certain embodiments, the vitamin is selected from the group consisting of choline, vitamin a, vitamin E, derivatives thereof, and metabolites thereof.

In certain embodiments, the linker comprises a divalent or trivalent linker.

wherein n is 1, 2, 3, 4 or 5.

wherein X is O, S or BH ₃ 。

In one aspect, the present disclosure provides compounds of formula (I):

L—(N) _n

(I)

wherein the method comprises the steps of

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, wherein 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 multivalent organic species or derivative thereof;

s independently at each occurrence comprises a 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; and is also provided with

N is a double stranded nucleic acid, e.g., a dsRNA molecule of any of the above aspects or embodiments of the disclosure. In certain embodiments, each N is 15 to 40 bases in length.

In certain embodiments, each N comprises a sense strand and an antisense strand; wherein the method comprises the steps of

The antisense strand comprises a sequence substantially complementary to the nucleic acid sequence of any one of SEQ ID NOs 1 to 6; and is also provided with

Wherein the sense strand and the antisense strand each independently comprise one or more chemical modifications.

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

in certain embodiments, the antisense strand comprises a 5' end group R selected from the group consisting of:

/>

in certain embodiments, the compound comprises a structure of formula (II):

wherein the method comprises the steps of

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 phosphorothioate internucleoside linkages; and is also provided with

-each occurrence independently represents a base pairing interaction or mismatch.

In certain embodiments, the compound comprises a structure of formula (IV):

wherein the method comprises the steps of

-represents a phosphodiester internucleoside linkage;

In certain embodiments, L is structure L1:

in some embodiments of the present invention, in some embodiments,r is R ³ And n is 2.

In certain embodiments, L is structure L2:

in certain embodiments, R is R ³ And n is 2.

In one aspect, the present disclosure provides a delivery system for a therapeutic nucleic acid having the structure of formula (VI):

L—(cNA) _n

(VI)

wherein:

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, wherein 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 multivalent organic species or derivative thereof;

each cNA is independently a vector nucleic acid comprising 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; and is also provided with

n is 2, 3, 4, 5, 6, 7 or 8.

In certain embodiments, the delivery system comprises a structure selected from the group consisting of formulas (VI-1) - (VI-9):

in certain embodiments, each cNA independently comprises a chemically modified nucleotide.

In certain embodiments, the delivery system further comprises n therapeutic Nucleic Acids (NA), wherein each NA hybridizes to at least one cNA.

In certain embodiments, each NA independently comprises at least 16 consecutive nucleotides.

In certain embodiments, each NA independently comprises 16-20 contiguous nucleotides.

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

In certain embodiments, the overhanging nucleotides are linked by phosphorothioate linkages.

In certain embodiments, each NA is independently selected from the group consisting of: DNA, siRNA, miRNA antagonists (antagomiR), mirnas, interstitials (gapmers), cocktails (mixmers), and guide RNAs.

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

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

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

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

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

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.

Drawings

The foregoing and other features and advantages of the disclosure will become more fully apparent from the following detailed description of illustrative embodiments thereof, which is to be read in connection with the accompanying drawings. The patent or application document contains at least one color drawing. The patent office will provide a copy of the color drawings of the patent or patent application publication upon request and payment of the necessary fee.

FIGS. 1A-1B depict the selection of siRNA sequences targeting human and mouse IFNGR1 mRNA in human HeLa cells (FIG. 1A) and mouse N2A cells (FIG. 1B). The percentage of IFNGR1 mRNA expression was determined relative to untreated controls. The siRNA sequences were tested at a concentration of 1.5 μm and expression was measured with a QunatiGene assay after 72 hours incubation. NTC: a non-targeted control; no known gene target disorder (scrambled) siRNA sequences. UNT: untreated controls.

FIGS. 2A-2B depict the selection of siRNA sequences targeting the target sites of human and mouse JAK1 mRNA in human HeLa cells (FIG. 2A) and mouse N2A cells (FIG. 2B). Percentage JAK1 mRNA expression was determined relative to untreated controls. The siRNA sequences were tested at a concentration of 1.5 μm and expression was measured with a QunatiGene assay after 72 hours incubation. NTC: a non-targeted control; no known gene target disorder (scrambled) siRNA sequences. UNT: untreated controls.

FIGS. 3A-3B depict the selection of siRNA sequences targeting the target sites of human and mouse JAK2 mRNA in human HeLa cells (FIG. 3A) and mouse N2A cells (FIG. 3B). Percentage JAK2 mRNA expression was determined relative to untreated controls. The siRNA sequences were tested at a concentration of 1.5 μm and expression was measured with a QunatiGene assay after 72 hours incubation. NTC: a non-targeted control; no known gene target disorder (scrambled) siRNA sequences. UNT: untreated controls.

FIGS. 4A-4B depict the selection of siRNA sequences targeting the target sites of human and mouse STAT1 mRNA in human HeLa cells (FIG. 4A) and mouse N2A cells (FIG. 4B). The percent STAT1 mRNA expression was determined relative to untreated controls. The siRNA sequences were tested at a concentration of 1.5 μm and expression was measured with a QunatiGene assay after 72 hours incubation. NTC: a non-targeted control; no known gene target disorder (scrambled) siRNA sequences. UNT: untreated controls.

Fig. 5A-5H depict dose response inhibition curves for ifngr1_1726, ifngr1_1641, jak1_3033, jak2_1936, jak2_2076 and stat1_885 screened in HeLa (human) and N2A (mouse) cells. NTC: non-targeted controls.

Fig. 6A-6B depict duration of efficacy in mice after single dose siRNA ifngr1_1641 injection. Wild-type C57BL6 mice were treated with siRNA for up to 4 weeks and Ifngr1 protein expression levels in the skin were measured by fluorescence flow cytometry (fig. 6A). FIG. 6B shows normalized levels of Ifngr1 protein expression compared to Ifngr1 knockout mice and non-target control treated mice. Maximum 66% target protein knockdown was achieved 2 weeks after injection, and significant levels of protein knockdown were maintained for 4 weeks (fig. 6B).

Figures 7A to 7B show that siRNAIfngr1_1641 reduces chemokine CXCL9 and CXCL10 expression by inhibiting IFN- γ signaling. The scheme used is depicted in fig. 7A. At week 4 after 2 subcutaneous injections of 20mg/kg siRNA (dosing interval: 2 weeks, n=5 mice per group), 8 skin biopsies of 4-mm diameter were collected per mouse. Tail skin perforations (2-fold serial dilutions of 25600-400pg/mL, as well as untreated controls) were incubated in the presence of recombinant mouse IFN-gamma protein. Fig. 7B depicts CXCL9 and CXCL10 levels measured by an enzyme-linked immunosorbent assay (ELISA) assay. Data are presented as mean ± SD and analyzed by two-way ANOVA with Dunnett multiple comparison test; * P <0.05.

Figures 8A to 8B show how sirnas ifngr1_1641 shows systemic and local efficacy in vitiligo models. Fig. 8A depicts a scheme used. The induction of vitiligo by adoptive transfer of PMEL cd8+ T cells isolated from spleens of PMEL TCR transgenic mice, followed by activation of these T cells in recipient mice resulted in patchy discoloration of the epidermis within 3-7 weeks, similar to vitiligo patients. Mice received a first dose of siRNA 2 weeks prior to vitiligo induction and a second dose of siRNA 1 week after induction. Figure 8B plots the quantified vitiligo scores for ears and tails. Vitiligo scores were objectively quantified by an observer blinded to the treatment group, using a spot scale based on the extent of the decolorized areas at the ear and tail. Each site was examined as a percentage of the anatomical site; the left ear and the right ear are both commonly defined and are therefore considered to be a single site. The score of vitiligo at each part is 0-5 as follows: no evidence of discoloration (0%) was scored as 0,>0 to 10% = 1 min,>10 to 25% = 2 minutes,>25% to 75% = 3 minutes,>75% to<100% = 4 min, and 100% = 5 min. Data are presented as mean ± SD, and by havingAnalyzing by using a two-way ANOVA of multiple comparison tests; * P (P) <0.05，**P<0.01，****P<0.0001。

Fig. 9A-9D depict quantitative analysis of tail discoloration levels between treatment groups. Skin discoloration levels were objectively quantified by comparing tail photographs using ImageJ Fiji software (NIH) (fig. 9A). Drawing a pixel intensity distribution curve of each tail part according to the total pixel number under each intensity; absolute white and black define intensities of 0 and 255, respectively (fig. 9B). Fig. 9C is a graph summarizing data. Statistical data are presented as mean ± SD of mean pixel intensities of individual distribution curves and analyzed by Mann-Whitney t-test; * P <0.05. Fig. 9D is a graph showing reduction of skin infiltration of cytotoxic T cells in epidermis and dermis (as measured by cd45+ cells) with sirna ifngr1 1641 (unpaired T-test; × P <0.01 × P < 0.05).

FIG. 10 depicts IFNGR1 protein expression in human HeLa cells and mouse N2a cells incubated for 72h with 1.5. Mu.M siRNA targeting IFNGR 1-1726 and Ifngr 1-1641.

FIG. 11 depicts dose response inhibition curves for IFNGR1__1631, 1989 and 2072 in HeLa cells and Ifngr1_378, 947 and 1162 in N2a cells. NTC: non-targeted controls.

FIG. 12 depicts CXCL9, CXCL10 and CXCL11 mRNA expression levels in HeLa cells and N2a cells. Cells were treated with 1.5 μm siRNA targeting ifngr1_1726 and ifngr1_1641 for 72h (n=4, mean ± SD, one-way ANOVA, < p <0.05, < p <0.01, < p <0.001, < p <0.0001; ns, not significant) prior to IFN- γ stimulation. Samples were analyzed 6h after stimulation of IFN-gamma signaling.

Fig. 13A-13B depict Ifngr1 silencing in mouse skin with ifngr1_1641-targeted siRNA with a different chemical configuration. FIG. 13A depicts a schematic representation of the chemical structure of hydrophobically conjugated (behenic acid, DCA; trimyritic acid, myr-t) and bivalent (Dio) siRNA; DCA and Myr-t conjugates are covalently linked to the 3' end of the sense strand; the two sense strands of the Dio scaffold are covalently linked by tetraethylene glycol; the study also included unconjugated sirnas ifngr1_1641 and DCA conjugated non-targeted control (NTC) siRNA. FIG. 13B depicts Ifngr1 mRNA silencing in skin at the injection site; mice (n=5 per group) were injected subcutaneously (between shoulders) with either a single dose of siRNA (20 mg/kg) or two doses (2 times, 24 hours apart; n=5); local skin was collected 1 week after injection and mRNA levels were measured using the QuantiGene 2.0 assay; ifngr1 expression is normalized to housekeeping gene Ppib; data are expressed as a percentage of PBS control (mean ± SD) and analyzed by Kruskal-Wallis test (p <0.05, < p <0.01; ns, not significant).

Detailed Description

Novel IFN-gamma signaling pathway gene target sequences are provided. Also provided are novel oligonucleotides, RNA molecules (such as siRNA and branched RNA compounds containing the same) that target IFN- γ signaling pathway gene mRNA (such as one or more target sequences of the present disclosure).

Unless otherwise indicated, nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein is 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 various general and more specific references cited and discussed throughout the present specification, unless otherwise specified. Enzymatic reactions and purification techniques are performed according to manufacturer's instructions, as commonly done in the art, or as described herein. Unless defined otherwise, the nomenclature used in connection with the analytical chemistry, synthetic organic chemistry, and medicinal chemistry described herein and the laboratory procedures and techniques in these chemical arts are those well known and commonly employed in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, delivery and patient treatment.

Unless defined otherwise herein, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. If there are any potential ambiguities, the definitions provided herein take precedence over any dictionary or external definitions. Furthermore, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. The use of "or" means "and/or" unless stated otherwise. The use of the term "include" and other forms such as "include" and "include" are not limiting.

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

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

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 may be synthesized naturally (e.g., by DNA replication or DNA transcription, respectively). RNA may be post-transcriptionally modified. 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 translated when ribosomes bind to mRNA during protein synthesis.

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) comprising about 10-50 nucleotides (or nucleotide analogs) that is capable of directing or mediating RNA interference. In certain embodiments, the siRNA comprises about 15 to 30 nucleotides or nucleotide analogs, or about 16 to 25 nucleotides (or nucleotide analogs), or about 18 to 23 nucleotides (or nucleotide analogs), or about 19 to 22 nucleotides (or nucleotide analogs) (e.g., 19, 20, 21, or 22 nucleotides or nucleotide analogs). The term "short" siRNA refers to an siRNA comprising about 21 nucleotides (or nucleotide analogs), e.g., 19, 20, 21 or 22 nucleotides. The term "long" siRNA refers to an siRNA comprising about 24-25 nucleotides, e.g., 23, 24, 25 or 26 nucleotides. In some cases, a short siRNA can comprise less than 19 nucleotides, such as 16, 17, or 18 nucleotides, so long as the shorter siRNA retains the ability to mediate RNAi. Also, in some cases, long sirnas may include more than 26 nucleotides, so long as longer sirnas retain the ability to mediate RNAi without further processing (e.g., enzymatic processing) to short sirnas.

The term "nucleotide analog" or "altered nucleotide" or "modified nucleotide" refers to a non-standard nucleotide, including non-naturally occurring ribonucleotides or deoxyribonucleotides. Exemplary nucleotide analogs are modified at any position to alter certain chemical properties of the nucleotide, but still retain the ability of the nucleotide analog to perform its intended function. Examples of nucleotide positions that may be derivatized include: position 5, e.g., 5- (2-amino) propyluridine, 5-bromouridine, 5-propynyluridine, 5-propenyl uridine, etc.; position 6, e.g., 6- (2-amino) propyluridine; and adenosine and/or guanosine at the 8-position, e.g., 8-bromoguanosine, 8-chloroguanosine, 8-fluoroguanosine, etc. Nucleotide analogs also include deazanucleotides, such as 7-deazaadenosine; o-and N-modified (e.g., alkylated, e.g., N6-methyladenosine, or as known in the art) nucleotides; and other heterocycle modified nucleotide analogs such as those described in herdiewijn, antisense Nucleic Acid Drug dev, 8 months of 2000 10 (4): 297-310.

Nucleotide analogs may also include modifications to the sugar portion of the nucleotide. For example, the 2' OH-group may be selected from H, OR, R, F, cl, br, I, SH, SR, NH ₂ 、NHR、NR ₂ Or COOR, wherein R is a substituted or unsubstituted C ₁ -C ₆ Alkyl, alkenyl, alkynyl, aryl, and the like. Other possible modifications include those described in U.S. patent nos. 5,858,988 and 6,291,438.

The phosphate group of a nucleotide may also be modified, for example, by replacing one or more of the oxygens of the phosphate group (e.g., phosphorothioates) with sulfur, or by making other substitutions, which allow the nucleotide to perform its intended function, such as described, for example, in Eckstein, antisense Nucleic Acid Drug Dev.2000, month 10 (2): 117-21, ruscoowski et al Antisense Nucleic Acid Drug Dev.2000, month 10 (5): 333-45,Stein,Antisense Nucleic Acid Drug Dev.2001, month 10 (5): 317-25, vorobjev et al Antisense Nucleic Acid Drug Dev.2001, month 11 (2): 77-85, and U.S. Pat. No. 5,684,143. Some of the above-described modifications (e.g., phosphate group modifications) reduce the rate of hydrolysis of, for example, a polynucleotide comprising the analog in vivo or in vitro.

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

The term "RNA analog" refers to a polynucleotide (e.g., a chemically synthesized polynucleotide) that has at least one altered or modified nucleotide as compared to a 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, which results in a lower rate of hydrolysis of the RNA analog than RNA molecules with phosphodiester linkages. For example, the nucleotide of the analog may comprise methylene glycol, ethylene glycol, oxymethyl, oxyethyl-thio, oxycarbonyloxy, phosphorodiamidate, phosphoramide, and/or phosphorothioate linkages. Some RNA analogs include sugar and/or backbone modified ribonucleotides and/or deoxyribonucleotides. Such changes or modifications may further include the addition of non-nucleotide materials, such as to the end or interior of the RNA (at one or more nucleotides of the RNA). The RNA analog need only be sufficiently similar to the native RNA that it has the ability to mediate RNA interference.

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

An RNAi agent, e.g., an RNA silencing agent, having a strand "sufficiently complementary to a target mRNA sequence to direct target-specific RNA interference (RNAi)" means that the strand has a sequence sufficient to trigger destruction of the target mRNA by an RNAi mechanism or process.

The term "isolated RNA" (e.g., "isolated siRNA" or "isolated siRNA precursor") as used herein refers to an RNA molecule that is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other compounds when chemically synthesized.

As used herein, the term "RNA silencing" refers to a set of sequence-specific regulatory mechanisms mediated by RNA molecules (e.g., RNA interference (RNAi), transcriptional Gene Silencing (TGS), post-transcriptional gene silencing (PTGS), suppression, co-suppression, and translational inhibition), resulting in the inhibition or "silencing" of expression of the corresponding protein-encoding gene. RNA silencing has been observed in many types of organisms, including plants, animals, and fungi.

The term "differential RNA silencing" refers to the ability of an RNA molecule to substantially inhibit expression of a "first" or "target" polynucleotide sequence without substantially inhibiting expression of a "second" or "non-target" polynucleotide sequence, e.g., when both polynucleotide sequences are present in the same cell. In certain embodiments, the target polynucleotide sequence corresponds to a target gene, while the non-target polynucleotide sequence corresponds to a non-target gene. In certain embodiments, the target polynucleotide sequence corresponds to a target allele, while the non-target polynucleotide sequence corresponds to a non-target allele. In certain embodiments, the target polynucleotide sequence is a DNA sequence encoding 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.

The term "in vitro" has its art-recognized meaning, for example, as it relates to a purified agent or extract, such as a cell extract. The term "in vivo" also has its art-recognized meaning, e.g., involving living cells, e.g., immortalized cells, primary cells, cell lines, and/or cells in an organism.

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 an organism that develops from the cell. Such transgenes may include genes that are partially or completely heterologous (i.e., exogenous) to the transgenic organism, or may represent genes that are homologous to endogenous genes of the organism. The term "transgenic" also means a nucleic acid molecule comprising one or more selected nucleic acid sequences, such as DNA, encoding one or more engineered RNA precursors to be expressed in a transgenic organism, such as an animal, that are partially or completely heterologous, i.e., exogenous, or homologous to an endogenous gene of the transgenic animal, but designed to be inserted into a location in the animal genome that is different from the native gene. Transgenes include one or more promoters and any other DNA, such as introns, necessary for expression of a selected nucleic acid sequence, all of which are operably linked to the selected sequence and may include enhancer sequences.

Genes "related to" a disease or disorder include genes whose normal or abnormal expression or function affects or results in the disease or disorder or at least one symptom of the disease or disorder.

As used herein, the term "function-obtaining mutation" refers to any mutation in a gene, wherein a protein encoded by the gene (i.e., a mutant protein) obtains a function not normally associated with the protein (i.e., a wild-type protein) and results in or contributes to a disease or disorder. The function-obtaining mutation may be a deletion, addition or substitution of one or more nucleotides in the gene, which results in a change in the function of the encoded protein. In one embodiment, the function-gain mutation alters the function of the mutant protein or causes an interaction with other proteins. In another embodiment, the function-obtaining mutation results in a reduction or removal of the normal wild-type protein, e.g., by an altered interaction of the mutant protein with the normal wild-type protein.

As used herein, the term "target gene" is a gene whose expression is to be substantially inhibited or "silenced". Such silencing may be achieved by RNA silencing, for example by cleavage of mRNA of the target gene or translational inhibition 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 mRNA encoded by the target gene and the non-target gene) may differ by one or more nucleotides. In another embodiment, the target gene and the non-target gene may differ by one or more polymorphisms (e.g., single nucleotide polymorphisms or SNPs). In another embodiment, the target gene and the non-target gene may share less than 100% sequence identity. In another embodiment, the non-target gene may be a homolog (e.g., an ortholog or paralog) of the target gene.

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

As used herein, the term "polymorphism" refers to a change (e.g., one or more deletions, insertions, or substitutions) in an identified or detected gene sequence when comparing identical gene sequences from different sources or subjects (but from the same organism). For example, polymorphisms can be identified when comparing identical gene sequences from different subjects. The identification of such polymorphisms is routine in the art, and the methods are similar to those used to detect, for example, breast cancer point mutations. For example, the polymorphic region may be identified from DNA extracted from lymphocytes from a subject and then amplified using primers specific for the polymorphic region. Alternatively, polymorphisms can be identified when two alleles of the same gene are compared. In certain embodiments, the polymorphism is a Single Nucleotide Polymorphism (SNP).

Sequence variation between two alleles of the same gene in an organism is referred to herein as "allelic polymorphism". In certain embodiments, the allelic polymorphism corresponds to a SNP allele. For example, an allelic polymorphism may comprise a single nucleotide variation between two alleles of a SNP. Polymorphisms can be at a nucleotide within a coding region, but due to the degeneracy of the genetic code, the encoded amino acid sequence does not change. Alternatively, polymorphic sequences may encode different amino acids at specific positions, but amino acid changes do not affect protein function. Polymorphic regions may also be found in non-coding regions of a gene. In exemplary embodiments, the polymorphism is found in the coding region of the gene or in an untranslated region of the gene (e.g., the 5'UTR or the 3' UTR).

As used herein, the term "IFNGR1" refers to a gene encoding the protein interferon gamma receptor 1. The IFNGR1 gene is located on chromosome 6q23.3. The IFNGR1 locus spans 23kb and consists of 9 exons (NCBI gene ID: 3459). The gene is expressed as 2 splice variants and in most tissues. The interferon gamma receptor 1 protein is about 489 amino acids in length and has a molecular weight of about 90kD (UniprotKB P15260). It associates with interferon gamma receptor 2 to form a heterodimeric receptor for interferon gamma.

As used herein, the term "JAK1" refers to a gene encoding janus kinase 1. The JAK1 gene is located on chromosome 1p31.3. The JAK1 locus spans 235kb and consists of 29 exons (NCBI gene ID: 3716). The gene is expressed in most tissues. The janus kinase 1 protein is about 1154 amino acids in length and has a molecular weight of about 133kD (UniProtKB P23458). It is part of the IFN-gamma signaling pathway and plays a role in phosphorylating STAT proteins.

As used herein, the term "JAK2" refers to a gene encoding the protein janus kinase 2. The JAK2 gene is located on chromosome 9p24.1. The JAK2 locus spans 146kb and consists of 27 exons (NCBI gene ID: 3717). The gene is expressed in most tissues. The janus kinase 2 protein is about 1132 amino acids in length and has a molecular weight of about 131kD (UniProtKB O60674). It is part of the IFN-gamma signaling pathway and plays a role in phosphorylating STAT proteins.

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

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.

As used herein, the term "RNA silencing agent" refers to an RNA that is capable of inhibiting or "silencing" the expression of a target gene. In certain embodiments, the RNA silencing agent is capable of preventing complete processing (e.g., complete translation and/or expression) of the mRNA molecule by a post-transcriptional silencing mechanism. RNA silencing agents include small (< 50 b.p.), non-coding RNA molecules, such as RNA duplex comprising paired strands, and precursor RNAs from which such small non-coding RNAs can be produced. Exemplary RNA silencing agents include siRNA, miRNA, siRNA-like duplex, antisense oligonucleotides, GAPMER molecules, and bifunctional oligonucleotides, as well as precursors thereof. In one embodiment, the RNA silencing agent is capable of inducing RNA interference. In another embodiment, the RNA silencing agent is capable of mediating translational inhibition.

As used herein, the term "rare nucleotide" refers to naturally occurring nucleotides that occur infrequently, including naturally occurring deoxyribonucleotides or ribonucleotides that occur infrequently, such as naturally occurring ribonucleotides that are not guanosine, adenosine, cytosine, or uridine. Examples of rare nucleotides include, but are not limited to, inosine, 1-methyl inosine, pseudouridine, 5, 6-dihydrouridine, ribothymidine, 2N-methyl guanosine, and 2,2N, N-dimethyl guanosine.

The term "engineered" as in an engineered RNA precursor or an engineered nucleic acid molecule means that the precursor or molecule is not present in nature because all or part of the nucleic acid sequence of the precursor or molecule is produced or selected by a human. Once the sequences are generated or selected, they may be replicated, translated, transcribed, or otherwise processed by intracellular mechanisms. Thus, an RNA precursor that is produced within a cell from a transgene comprising an engineered nucleic acid molecule is an engineered RNA precursor.

As used herein, the term "microrna" ("miRNA"), also referred to in the art as "chronologically regulated microrna" ("stRNA") refers to a small (10-50 nucleotide) RNA that is genetically encoded (e.g., by a viral, mammalian, or plant genome) and is capable of directing or mediating RNA silencing. "miRNA disorder" shall refer to a disease or disorder characterized by aberrant expression or activity of miRNAs.

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

As used herein, the term "linking moiety" or "linking moiety" refers to a domain, portion or region of an RNA silencing agent that is covalently linked or linked to mRNA.

As used herein, the term "antisense strand" of an RNA silencing agent, e.g., siRNA or RNA silencing agent, refers to a strand that is substantially complementary to a portion of about 10-50 nucleotides, e.g., about 15-30, 16-25, 18-23, or 19-22 nucleotides, of an mRNA targeted for silencing a gene. The antisense strand or first strand has a sequence sufficiently complementary to the desired target mRNA sequence to direct target-specific silencing, e.g., sufficient to trigger an RNAi mechanism or process (RNAi interference) to disrupt the complementarity of the desired target mRNA or sufficient to trigger translational inhibition of the desired target mRNA.

The term "sense strand" or "second strand" of an RNA silencing agent, e.g., siRNA or RNA silencing agent, refers to a strand that is complementary to the antisense strand or first strand. The antisense strand and the sense strand may also be referred to as a first strand or a second strand, which has complementarity to a target sequence, and the corresponding second strand or first strand has complementarity to the first strand or second strand. The miRNA duplex intermediate or siRNA-like duplex comprises a miRNA strand having sufficient complementarity to a portion of about 10-50 nucleotides of mRNA of the gene targeted for silencing and a miRNA strand having sufficient complementarity to form a duplex with the miRNA strand.

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

As used herein, the term "asymmetric", as in the asymmetry of a duplex region of an RNA silencing agent (e.g., a stem of a shRNA), refers to an inequality in bond strength or base pairing strength between ends of the RNA silencing agent (e.g., between a terminal nucleotide on a first strand or stem portion and a terminal nucleotide on an opposing second strand or stem portion) such that the 5 'end of one strand of the duplex is more frequently in a transient unpaired state, e.g., a single stranded state, than the 5' end of the complementary strand. This structural difference determines the preferential incorporation of one strand of the duplex into the RISC complex. The less compact strand pairing of the 5' end with the complementary strand will preferentially incorporate RISC and mediate RNAi.

As used herein, the term "bond strength" or "base pair strength" refers to the strength of interaction between pairs of nucleotides (or nucleotide analogs) on opposite strands of an oligonucleotide duplex (e.g., siRNA duplex), primarily due to hydrogen bonding, van der waals interactions, etc. between the nucleotides (or nucleotide analogs).

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

As used herein, the term "destabilizing nucleotide" refers to a first nucleotide or nucleotide analogue capable of forming a base pair with a second nucleotide or nucleotide analogue such that the bond strength of the base pair is lower than conventional base pairs (i.e., watson-Crick base pairs). In certain embodiments, the destabilizing nucleotide is capable of forming a mismatched base pair with the second nucleotide. In other embodiments, the destabilizing nucleotide is capable of forming a wobble base pair with the second nucleotide. In other embodiments, the destabilizing nucleotide is capable of forming a ambiguous base pair with the second nucleotide.

As used herein, the term "base pair" refers to interactions between pairs of nucleotides (or nucleotide analogs) on opposite strands of an oligonucleotide duplex (e.g., a duplex formed by a strand of an RNA silencing agent and a target mRNA sequence), primarily due to hydrogen bonding, van der waals interactions, etc. between the nucleotides (or nucleotide analogs). As used herein, the term "bond strength" or "base pair strength" refers to the strength of a base pair.

As used herein, the term "mismatched base pair" refers to a base pair consisting of non-complementary or non-Watson-Crick base pairs, e.g., an abnormally complementary G: C, A:T or A:U base pair. As used herein, the term "ambiguous base pair" (also referred to as non-discriminating base pairs) refers to base pairs formed from universal nucleotides.

As used herein, the term "universal nucleotide" (also referred to as "neutral nucleotide") includes nucleotides (e.g., certain destabilizing nucleotides) that have bases that do not significantly distinguish between bases on complementary polynucleotides when formed into base pairs ("universal bases" or "neutral bases"). Universal nucleotides are predominantly hydrophobic molecules that can efficiently assemble into antiparallel double-stranded nucleic acids (e.g., double-stranded DNA or RNA) due to stacking interactions. The base portion of a universal nucleotide typically comprises a nitrogen-containing aromatic heterocyclic moiety.

As used herein, the term "sufficient complementarity" or "sufficient degree of complementarity" refers to an RNA silencing agent having a sequence (e.g., in the antisense strand, the mRNA targeting portion, or the miRNA recruiting portion) sufficient to bind a desired target RNA, respectively, and trigger RNA silencing of the target mRNA.

As used herein, the term "translational inhibition" refers to selective inhibition of mRNA translation. Natural translational inhibition is performed by mirnas cleaved from shRNA precursors. RNAi and translational inhibition are both RISC-mediated. RNAi and translational inhibition are both naturally occurring or may be initiated manually, e.g., silencing expression of the target gene.

Various methods of the invention include steps involving comparing values, levels, characteristics, properties, etc. to "suitable controls," which are interchangeably referred to herein as "suitable controls. An "appropriate control" or "appropriate control" is any control or standard familiar to one of ordinary skill in the art for comparison purposes. In one embodiment, a "suitable control" or "suitable control" is a value, level, characteristic, property, etc., determined prior to performing an RNAi method as described herein. For example, the transcription rate, mRNA level, translation rate, protein level, biological activity, cellular characteristics or properties, genotype, phenotype, etc., can be determined prior to introducing the RNA silencing agent of the invention into a cell or organism. In another embodiment, a "suitable control" or "suitable control" is a value, level, feature, characteristic, property, etc., determined in a cell or organism that exhibits, for example, a normal feature (e.g., a control or normal cell or organism). In yet another embodiment, a "suitable control" or "suitable control" is a predefined value, level, characteristic, property, or the like.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill 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 patents, patent applications, and other references cited 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 not intended to be limiting.

Various aspects of the invention are described in more detail in the following subsections.

I. Novel target sequences

In certain exemplary embodiments, the RNA silencing agents of the present invention are capable of targeting the IFNGR1, JAK2 or STAT1 nucleic acid sequences of any one of SEQ ID NOs 1-6, as described in tables 6 and 8. In certain exemplary embodiments, the RNA silencing agents of the present invention are capable of targeting one or more of the IFNGR1, JAK2 or STAT1 nucleic acid sequences selected from the group consisting of SEQ ID NOS: 143-154, as described in tables 7, 9, 10 and 11.

The genomic sequence of each target sequence can be found in, for example, the public database maintained by NCBI.

SiRNA design

In some embodiments, the siRNA is designed as follows. First, a portion of a target gene (e.g., IFNGR1, JAK2, or STAT1 gene), such as one or more of the target sequences listed in tables 6 and 8, is selected. Cleavage of the mRNA at these sites should eliminate translation of the corresponding protein. 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 and sense strand forms an siRNA duplex. The antisense strand comprises about 19 to 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 to 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 other embodiments, the sense strand is 18 nucleotides. In other embodiments, the sense strand is 20 nucleotides. However, those skilled in the art will appreciate that siRNAs less than 19 nucleotides in length or greater than 25 nucleotides in length may also function to mediate RNAi. Thus, sirnas of such length are also within the scope of the invention, so long as they retain the ability to mediate RNAi. Longer RNAi agents have been shown to elicit an interferon or PKR response in certain mammalian cells, which may be undesirable. In certain embodiments, RNAi agents of the invention do not elicit a PKR response (i.e., have a sufficiently short length). However, longer RNAi agents may be useful, for example, in cell types that are unable to produce a PKR response, or where the PKR response has been down-regulated or inhibited by alternative methods.

The sense strand sequence may be designed such that the target sequence is located substantially in the middle of the strand. In some cases, moving the target sequence to an off-center position reduces the cleavage efficiency of the siRNA. If shut down silencing of wild-type mRNA is detected, it may be desirable to use such compositions, i.e., less efficient compositions.

The antisense strand can be the same as the sense strand length and include complementary nucleotides. In one embodiment, the strands are fully complementary, i.e., the strands are blunt-ended when aligned or annealed. In another embodiment, strand alignment or annealing is such that a 1, 2, 3, 4,5, 6, 7, or 8 nucleotide overhang is created, i.e., the 3 'end of the sense strand extends 1, 2, 3, 4,5, 6, 7, or 8 nucleotides over 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 over 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 overhangs may comprise (or consist of) deoxyribonucleotides, such as dT, or nucleotide analogs, or other suitable non-nucleotide materials.

To facilitate entry of the antisense strand into the RISC (thereby increasing or enhancing the efficiency of target cleavage and silencing), the base pair strength between the 5 'end of the sense strand and the 3' end of the antisense strand may be varied, e.g., reduced or decreased, as described in detail in U.S. Pat. nos. 7,459,547, 7,772,203 and 7,732,593 (filed 6/2/2003) entitled "Methods and Compositions for Controlling Efficacy of RNASilencing" and U.S. Pat. nos. 8,309,704, 7,750,144, 8,304,530, 8,329,892 and 8,309,705 (filed 6/2/2003) entitled "Methods and Compositions for Enhancing the Efficacy and Specificity ofRNAi," the contents of which are incorporated herein by reference in their entirety. In one embodiment of these aspects of the invention, the base pair strength is less 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 there are G: C base pairs 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 intensities are lower 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 intensity is less 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 intensities are lower because at least one base pair comprises a rare nucleotide, such as inosine (I). In certain exemplary embodiments, the base pairs are selected from the group consisting of: a, I, U and C. In yet another embodiment, the base pair intensities are lower because at least one base pair comprises 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.

The design of sirnas suitable for targeting the IFNGR1, JAK2 or STAT1 target sequences listed in tables 6 and 8 is described in detail below. siRNA can be designed against any other target sequence found in the IFNGR1, JAK2 or STAT1 genes according to the above exemplary teachings. Furthermore, the techniques are applicable to targeting any other target sequence, such as non-pathogenic target sequences.

To verify the effectiveness of siRNA to destroy mRNA (e.g., IFNGR1, JAK2, or STAT1 mRNA), the siRNA can be incubated with cDNA (e.g., IFNGR1, JAK2, or STAT1 cDNA) in a drosophila-based in vitro mRNA expression system. By using ³² The P radiolabeled newly synthesized mRNA (e.g., IFNGR1, JAK2, or STAT1 mRNA) is detected by autoradiography on agarose gel. The presence of cleaved mRNA indicates mRNA nuclease activity. Suitable controls include omission of siRNA. Alternatively, a control siRNA is selected that has the same nucleotide composition as the selected siRNA, but no significant sequence complementarity to the appropriate target gene. Such negative controls can be designed by randomly scrambling the nucleotide sequence of the selected siRNA; homology searches can be performed to ensure that the negative control lacks any other genes in the appropriate genome Lack of homology. In addition, negative control siRNA can be designed by introducing one or more base mismatches into the sequence. The siRNA-mRNA complementary sites that result in optimal mRNA specificity and maximal mRNA cleavage are selected.

RNAi agents

The invention includes RNAi molecules, such as, for example, siRNA molecules designed as described above. The siRNA molecules of the invention can be chemically synthesized, or can be transcribed in vitro from DNA templates, or in vivo from shRNA, for example, or by cleavage of the in vitro transcribed dsRNA templates into pools of 20, 21 or 23bp duplex RNAs that mediate RNAi using recombinant human DICER enzymes. The siRNA molecules may be designed using any method known in the art.

In one aspect, the RNAi agent is not an interfering ribonucleic acid, such as an siRNA or shRNA as described above, but the RNAi agent can encode an interfering ribonucleic acid, such as an shRNA as described above. In other words, the RNAi agent can be a transcription template that interferes with ribonucleic acid. Thus, RNAi agents of the invention can also include small hairpin RNAs (shrnas), and expression constructs engineered to express shrnas. Transcription of the shRNA starts with the polymerase III (Pol III) promoter and is thought to terminate at position 2 of the 4-5-thymine transcription termination site. Upon expression, shRNA is thought to fold into a stem-loop structure with a 3' uu overhang; subsequently, the ends of these shRNAs are processed to convert the shRNAs into siRNA-like molecules of about 21-23 nucleotides (Brummelkamp et al, 2002; lee et al, 2002, supra; miyagishi et al, 2002; paddison et al, 2002, supra; sui et al, 2002, supra; yu et al, 2002, supra. More information about the design and use of shRNAs can be found at the following addresses on the Internet: katanin. Cshl. Org:9331/RNAi/docs/BseRI-BamHI_Strategy. Pdf and katanin. Cshl. Org: 9331/RNAi/docs/version_of_PCR_strate1. Pdf).

Expression constructs of the present invention include any construct suitable for use in a suitable expression system, and include, but are not limited to, retroviral vectors, linear expression cassettes, plasmids, and viral or viral-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 comprise one or two siRNA strands. Expression constructs expressing both strands may also include a loop structure linking the two strands, or each strand may be transcribed separately from a different promoter in the same construct. Each strand may also be transcribed from a separate expression construct. (Tuschl, t.,2002, supra).

Synthetic siRNA can be delivered into cells by methods known in the art, including cationic liposome transfection and electroporation. To obtain long-term inhibition of a target gene (e.g., IFNGR1, JAK2, or STAT1 genes) and in some cases to facilitate delivery, one or more sirnas may be expressed from the recombinant DNA construct within the cell. Such methods for expressing siRNA duplex from recombinant DNA constructs in cells to allow long term inhibition of a target gene in a cell are known in the art and include mammalian Pol III promoter systems (e.g., H1 or U6/snRNA promoter systems capable of expressing functional double stranded siRNA (Tuschl, T.,2002, supra); (Bagellan 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)). Transcription termination of RNApol III occurs during the run of four consecutive T residues in the DNA template, providing a mechanism for ending the siRNA transcript at a specific sequence. The siRNA is complementary to the target gene sequence in the 5'-3' and 3'-5' directions, and the two strands of the siRNA can be expressed in the same construct or in different constructs. Hairpin siRNAs driven by H1 or U6 snRNA promoters and expressed in cells can inhibit target gene expression (Bagellan 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 comprising siRNA sequences under the control of the T7 promoter also produce functional siRNA when co-transfected into cells with vectors expressing T7 RNA polymerase (Jacque et al, 2002, supra). A single construct may contain multiple sequences encoding sirnas targeting the same gene or genes, such as multiple regions of the gene encoding IFNGR1, JAK2, or STAT1, and may be driven by, for example, separate PolIII promoter sites.

Animal cells express a series of approximately 22 nucleotide non-coding RNAs known as micrornas (mirnas) that regulate gene expression at post-transcriptional or translational levels during animal development. A common feature of mirnas is that they are all excised from a precursor RNA stem loop of about 70 nucleotides, possibly by an rnase type III enzyme Dicer or a homologue thereof. By replacing the stem sequence of the miRNA precursor with a sequence complementary to the target mRNA, vector constructs expressing the engineered precursor can be used to generate siRNA to initiate RNAi against a specific mRNA target in mammalian cells (Zeng et al, 2002, supra). The hairpin(s) designed by the microRNA can silence gene expression when expressed from a DNA vector containing the polymerase III promoter (McManus et al, 2002, supra). Micrornas that target polymorphisms can also be used to block translation of mutant proteins without siRNA mediated gene silencing. Such applications may be useful in certain circumstances, for example, where the designed siRNA results in off-target silencing of wild-type proteins.

Viral-mediated delivery mechanisms can also be used to induce specific silencing of target genes by expression of siRNA, for example, by production of recombinant adenoviruses containing siRNA under transcriptional control of the RNAPol II promoter (Xia et al, 2002, supra). Infection of HeLa cells with these recombinant adenoviruses can reduce expression of endogenous target genes. Injection of the recombinant adenovirus vector into transgenic mice expressing the siRNA target gene resulted in reduced expression of the target gene in vivo. As above. In animal models, whole embryo electroporation can be effective in delivering synthetic siRNA into post-implantation mouse embryos (Calegari et al, 2002). In adult mice, efficient delivery of siRNA can be achieved by "high pressure" delivery techniques, i.e., rapid (within 5 seconds) injection of a large amount of a solution containing siRNA into an animal via the tail vein (Liu et al, 1999, supra; mcCaffrey et al, 2002, supra; lewis et al, 2002. Nanoparticles and liposomes can also be used to deliver siRNA into an animal. In certain exemplary embodiments, recombinant adeno-associated virus (rAAV) and its associated vectors can be used to deliver one or more siRNA into cells, such as skin cells (U.S. patent applications 2014/0296486, 2010/0186103, 2008/0269149, 2006/0078442, and 2005/0220766).

Nucleic acid compositions of the invention include unmodified siRNAs and modified siRNAs such as cross-linked siRNA derivatives or derivatives having a non-nucleotide moiety attached to, for example, their 3 'or 5' ends. Modifying the siRNA derivative in this manner can improve cellular uptake or enhance cellular targeting activity of the resulting siRNA derivative as compared to the corresponding siRNA, and can be used to track the siRNA derivative in a cell or increase stability of the siRNA derivative as compared to the corresponding siRNA.

The introduction of an engineered RNA precursor into a cell or whole organism as described herein will result in the production of a desired siRNA molecule. Such siRNA molecules will then bind to endogenous protein components of the RNAi pathway to bind to and target specific mRNA sequences for cleavage and disruption. In this way, the mRNA targeted by the siRNA produced by the engineered RNA precursor will be depleted from the cell or organism, resulting in a decrease in the concentration of the protein encoded by the mRNA in the cell or organism. RNA precursors are typically nucleic acid molecules that encode one strand of dsRNA alone or the entire nucleotide sequence encoding the hairpin loop structure of RNA.

The nucleic acid compositions of the invention may be unconjugated or may be conjugated to another moiety such as a nanoparticle to enhance properties of the composition, e.g., pharmacokinetic parameters such as absorption, efficacy, bioavailability, and/or half-life. Conjugation may be achieved by methods known in the art, for example, using the following methods: lambert et al, drug Deliv.Rev.:47 (1), 99-112 (2001) (describing nucleic acids loaded into Polyalkylcyanoacrylate (PACA) nanoparticles); fattal et al, J.control Release 53 (1-3): 137-43 (1998) (describing nucleic acids binding to nanoparticles); schwab et al, ann.Oncol.5 support.4:55-8 (1994) (describing nucleic acids linked to intercalators, hydrophobic groups, polycations or PACA nanoparticles); and Godard et al, eur. J. Biochem.232 (2): 404-10 (1995) (describing nucleic acids linked to nanoparticles).

Nucleic acid molecules of the inventionThe labeling may also be performed using any method known in the art. For example, the nucleic acid composition may be labeled with a fluorophore, such as Cy3, fluorescein, or rhodamine. Labelling can be carried out using a kit, e.g. SILENCER ^TM siRNA labelling kit (Ambion). In addition, the siRNA may be radiolabeled, e.g., using ³ H、 ³² P or another suitable isotope.

Furthermore, because RNAi is believed to proceed through at least one single-stranded RNA intermediate, those skilled in the art will appreciate that ss-sirnas (e.g., the antisense strand of ds-sirnas) can also be designed (e.g., for chemical synthesis), produced (e.g., enzymatically produced) or expressed (e.g., from vectors or plasmids) as described herein and used according to the claimed methods. Furthermore, in invertebrates, RNAi can be effectively triggered by long dsRNA (e.g., dsRNA about 100-1000 nucleotides in length, such as about 200-500, e.g., dsRNA about 250, 300, 350, 400, or 450 nucleotides in length) that acts as an effector of RNAi. (Brondani et al Proc Natl Acad Sci USA. 12, 2001; 98 (25): 14428-33.Epub, 2001, 11, 27).

anti-IFNGR 1, anti-JAK 2 and anti-STAT 1 RNA silencing agents

In certain embodiments, the invention provides novel anti-IFNGR 1, anti-JAK 2, and anti-STAT 1 RNA silencing agents (e.g., siRNA, shRNA, and antisense oligonucleotides), methods of making the RNA silencing agents, and methods of using the improved RNA silencing agents (or portions thereof) for RNA silencing of IFNGR1, JAK2, or STAT1 proteins (e.g., research and/or therapeutic methods). The RNA silencing agent comprises an antisense strand (or portion thereof), wherein the antisense strand has sufficient complementarity to a target IFNGR1, JAK2, or STAT1 mRNA to mediate an RNA-mediated silencing mechanism (e.g., RNAi).

In certain embodiments, siRNA compounds are provided that have one or any combination of the following properties: (1) Completely chemically stable (i.e., without unmodified 2' -OH residues); (2) asymmetry; (3) 11-20 base pair duplex; (4) More than 50% 2 '-methoxy modification, such as 70% -100% 2' -methoxy modification, although alternating patterns of chemically modified nucleotides (e.g., 2 '-fluoro and 2' -methoxy modification) are also contemplated; (5) Single-stranded, fully phosphorothioated tails of 5-8 bases. In certain embodiments, the number of phosphorothioate modifications varies from 4 to 16 total. In certain embodiments, the number of phosphorothioate modifications varies from 8 to 13 total.

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), benzathine, cortisol, vitamin a, vitamin D, N-acetylgalactosamine (GalNac), and gangliosides. In a broad range of cell types (e.g., heLa cells, neurons, hepatocytes, trophoblasts), cholesterol modified versions exhibit 5-10 fold improvement in efficacy in vitro compared to the previously used pattern of chemical stability (e.g., where all purines, but not pyrimidines, are modified).

Certain compounds of the invention having the structural properties described above and herein may be referred to as "hsiRNA-ASP" (small interfering RNAs characterized by advanced stable patterns of hydrophobic modification). Furthermore, this hsiRNA-ASP pattern shows a significant improvement in distribution in brain, spinal cord, liver, placenta, kidney, spleen and several other tissues, making it useful for therapeutic intervention.

The compounds of the present invention may be described in the following aspects and embodiments.

In a first aspect, provided herein is a double-stranded RNA (dsRNA) comprising an antisense strand and a sense strand, each strand comprising at least 14 consecutive nucleotides having a 5 'end and a 3' end, wherein:

(1) The antisense strand comprises a sequence substantially complementary to the nucleic acid sequence of any one of SEQ ID NOs 1 to 6;

(2) The antisense strand comprises alternating 2 '-methoxy-ribonucleotides and 2' -fluoro-ribonucleotides;

(3) The nucleotides at positions 2 and 14 of the 5 'end of the antisense strand are not 2' -methoxy-ribonucleotides;

(4) Nucleotides at positions 1-2 to 1-7 of the 3' -end of the antisense strand are linked to each other by 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; and is also provided with

(7) The nucleotides at positions 1-2 of the 5' end of the sense strand are linked to each other by phosphorothioate internucleotide linkages.

In a second aspect, provided herein is a dsRNA comprising an antisense strand and a sense strand, each strand comprising at least 14 consecutive nucleotides having a 5 'end and a 3' end, wherein:

(2) The antisense strand comprises at least 70% 2' -O-methyl modification;

(3) The nucleotide at position 14 of the 5 'end of the antisense strand is not a 2' -methoxy-ribonucleotide;

(6) The sense strand comprises at least 70% 2' -O-methyl modification; and is also provided with

In a third aspect, provided herein is a dsRNA comprising an antisense strand and a sense strand, each strand comprising at least 14 consecutive nucleotides having a 5 'end and a 3' end, wherein:

(2) The antisense strand comprises at least 85% 2' -O-methyl modification;

(6) The sense strand comprises 100% 2' -O-methyl modification; and is also provided with

In a fourth aspect, provided herein is a dsRNA comprising an antisense strand and a sense strand, each strand comprising at least 14 consecutive nucleotides having a 5 'end and a 3' end, wherein:

(2) The antisense strand comprises at least 75% 2' -O-methyl modification;

(3) Nucleotides at positions 4, 5, 6 and 14 of the 5 'end of the antisense strand are not 2' -methoxy-ribonucleotides;

In a fifth aspect, provided herein is a dsRNA comprising an antisense strand and a sense strand, each strand comprising at least 14 consecutive nucleotides having a 5 'end and a 3' end, wherein:

(2) The antisense strand comprises at least 75% 2' -O-methyl modification;

(3) The nucleotides at positions 2, 4, 5, 6 and 14 of the 5 'end of the antisense strand are not 2' -methoxy-ribonucleotides;

In a sixth aspect, provided herein is a dsRNA comprising an antisense strand and a sense strand, each strand comprising at least 14 consecutive nucleotides having a 5 'end and a 3' end, wherein:

(2) The antisense strand comprises at least 75% 2' -O-methyl modification;

(3) The nucleotides at positions 2, 6, 14 and 16 of the 5 'end of the antisense strand are not 2' -methoxy-ribonucleotides;

(6) The sense strand comprises at least 70% 2' -O-methyl modification;

(7) Nucleotides at positions 7, 9, 10 and 11 of the 3 'end of the sense strand are not 2' -methoxy-ribonucleotides; and is also provided with

(8) The nucleotides at positions 1-2 of the 5' end of the sense strand are linked to each other by phosphorothioate internucleotide linkages.

In a seventh aspect, provided herein is a dsRNA comprising an antisense strand and a sense strand, each strand comprising at least 14 consecutive nucleotides having a 5 'end and a 3' end, wherein:

(2) The antisense strand comprises at least 75% 2' -O-methyl modification;

(3) The nucleotides at positions 2, 6 and 14 of the 5 'end of the antisense strand are not 2' -methoxy-ribonucleotides;

(6) The sense strand comprises at least 80% 2' -O-methyl modification;

(7) Nucleotides at positions 7, 10 and 11 of the 3 'end of the sense strand are not 2' -methoxy-ribonucleotides; and is also provided with

In an eighth aspect, provided herein is a dsRNA comprising an antisense strand and a sense strand, each strand comprising at least 14 consecutive nucleotides having a 5 'end and a 3' end, wherein:

(2) The antisense strand comprises at least 50% 2' -O-methyl modification;

(3) Nucleotides at positions 2, 4, 5, 6, 8, 10, 12, 14, 16 and 20 of the 5 'end of the antisense strand are not 2' -methoxy-ribonucleotides;

(4) Nucleotides at positions 1-2 to 1-8 of the 3' -end of the antisense strand are linked to each other by phosphorothioate internucleotide linkages;

(6) The sense strand comprises at least 65% 2' -O-methyl modification;

(7) The nucleotides at positions 3, 7, 9, 11 and 13 of the 3 'end of the sense strand are not 2' -methoxy-ribonucleotides; and is also provided with

(8) The nucleotides at positions 1-3 of the 5' end of the sense strand are linked to each other by phosphorothioate internucleotide linkages.

In a ninth aspect, provided herein is a dsRNA comprising an antisense strand and a sense strand, each strand comprising at least 14 consecutive nucleotides having a 5 'end and a 3' end, wherein:

(2) The antisense strand comprises at least 75% 2' -O-methyl modification;

(3) Nucleotides at positions 2, 6, 14, 16 and 20 of the 5 'end of the antisense strand are not 2' -methoxy-ribonucleotides;

(4) Nucleotides at positions 1-7 and 19-20 of the 3' end of the antisense strand are linked to each other by phosphorothioate internucleotide linkages;

(6) The sense strand comprises at least 65% 2' -O-methyl modification;

(8) The nucleotides at positions 1-2 and 14-15 of the 5' end of the sense strand are linked to each other by phosphorothioate internucleotide linkages.

In a tenth aspect, provided herein is a dsRNA comprising an antisense strand and a sense strand, each strand comprising at least 14 consecutive nucleotides having a 5 'end and a 3' end, wherein:

(1) The antisense strand comprises a sequence substantially complementary to an IFNGR1, JAK2, or STAT1 nucleic acid sequence;

(2) The antisense strand comprises at least 50% 2' -O-methyl modification;

(3) The nucleotide at any one or more of positions 2, 4, 5, 6, 8, 10, 12, 14, 16 and 20 of the 5 'end of the antisense strand is not a 2' -methoxy-ribonucleotide;

(6) The sense strand comprises at least 65% 2' -O-methyl modification;

(7) The nucleotide at any one or more of positions 3, 7, 9, 11 and 13 of the 3 'end of the sense strand is not a 2' -methoxy-ribonucleotide; and is also provided with

a) anti-IFNGR 1, anti-JAK 2 and anti-STAT 1siRNA molecules Meter with a meter body

The siRNA molecules of the present application are duplexes consisting of a sense strand and a complementary antisense strand, the antisense strand having sufficient complementarity to IFNGR1, JAK2 or STAT1 mRNA to mediate RNAi. In certain embodiments, the siRNA molecule has a length of about 10 to 50 or more nucleotides, i.e., each strand comprises 10 to 50 nucleotides (or nucleotide analogs). In other embodiments, the siRNA molecule has a length of about 15 to 30, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in each strand, wherein one strand is substantially complementary to the target region. In certain embodiments, the strands are aligned such that at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases at the ends of the strands are not aligned (i.e., no complementary base for the bases occurs in the opposite strand) such that when the strand anneals, an overhang of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues occurs at one or both ends of the duplex.

In general, siRNA can be designed by using any method known in the art, for example, by using the following scheme:

sirna should be specific for a target sequence, such as the target sequences listed in the examples. The first strand should be complementary to the target sequence, while the other strand is substantially complementary to the first strand. (see examples for exemplary sense and antisense strands.) exemplary target sequences are selected from any region of a target gene that results in effective gene silencing. The region of the target gene includes, but is 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 should eliminate translation of the corresponding IFNGR1, JAK2 or STAT1 proteins. Target sequences of IFNGR1, JAK2 or other regions of STAT1 genes are also suitable for targeting. The sense strand is designed based on the target sequence.

The sense strand of sirna is designed according to the sequence of the selected target site. In certain embodiments, the sense strand comprises about 15 to 25 nucleotides, e.g., 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 in length. In certain embodiments, the sense strand is 18 nucleotides in length. In certain embodiments, the sense strand is 20 nucleotides in length. However, those skilled in the art will appreciate that siRNAs less than 15 nucleotides in length or greater than 25 nucleotides in length may also function to mediate RNAi. Thus, sirnas of such length are also within the scope of the invention, so long as they retain the ability to mediate RNAi. Longer RNA silencing agents have been shown to elicit interferon or Protein Kinase R (PKR) responses in certain mammalian cells, which may be undesirable. In certain embodiments, the RNA silencing agents of the invention do not elicit a PKR response (i.e., have a sufficiently short length). However, longer RNA silencing agents may be useful, for example, in cell types that are not capable of producing a PKR response, or where the PKR response has been down-regulated or inhibited by alternative methods.

The siRNA molecules of the invention have sufficient complementarity to the target sequence that the siRNA can mediate RNAi. In general, siRNAs comprising nucleotide sequences sufficiently complementary to a target sequence portion of a target gene to effect RISC-mediated cleavage of the target gene are contemplated. Thus, in a certain embodiment, 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 may have 100% complementarity to the target site. However, 100% complementarity is not required. Greater than 80% identity between the antisense strand and the target RNA sequence is contemplated, e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% complementarity. The present application has the advantage of being able to tolerate certain sequence variations to improve the efficiency and specificity of RNAi. In one embodiment, the antisense strand has 4, 3, 2, 1 or 0 mismatched nucleotides from the target region, e.g., a target region that differs by at least one base pair between the wild-type and mutant alleles, e.g., a target region comprising a function-obtaining 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 may also be effective in mediating RNAi. Alternatively, siRNA sequences with nucleotide analog substitutions or insertions may be effectively inhibited.

Sequence identity may 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), sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the first sequence or the second sequence to achieve optimal alignment). The nucleotides (or amino acid residues) at the corresponding nucleotide (or amino acid) positions are then compared. When a position in the first sequence is occupied by the same residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e.,% homology = number of identical positions/total number of positions x 100), optionally penalizing the number of gaps introduced and/or the length of gaps introduced.

Comparison of sequences and determination of percent identity between two sequences can be accomplished using mathematical algorithms. In one embodiment, the alignment is made over a specific portion of the aligned sequences that has sufficient identity, but not over a portion that has a low degree of identity (i.e., a local alignment). One non-limiting example of a local alignment algorithm for sequence comparison is the algorithm of Karlin and Altschul (1990) Proc.Natl. Acad.Sci.USA87:2264-68, modified in Karlin and Altschul (1993) Proc.Natl. Acad.Sci.USA90:5873-77. This algorithm is incorporated into the BLAST program of Altschul et al (1990) J.mol.biol.215:403-10 (version 2.0).

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

The antisense or guide strand of an sirna is typically the same as the sense strand length and comprises complementary nucleotides. In one embodiment, the guide strand and sense strand are perfectly complementary, i.e., the strands are blunt-ended when aligned or annealed. In another embodiment, the strands of the siRNA can be paired in a manner having a 3' overhang of 1 to 7 (e.g., 2, 3, 4, 5, 6, or 7) or 1 to 4, e.g., 2, 3, or 4 nucleotides. The overhang may comprise (or consist of) nucleotides corresponding to the target gene sequence (or its complement). Alternatively, the overhangs may comprise (or consist of) deoxyribonucleotides, such as dT, or nucleotide analogs, or other suitable non-nucleotide materials. Thus, in another embodiment, the nucleic acid molecule may have a 2 nucleotide 3' overhang, such as TT. The protruding nucleotides may be RNA or DNA. As described above, it is desirable to select a target region in which the mutation: wild type mismatch is a purine: purine mismatch.

4. Potential targets are compared to the appropriate genomic database (human, mouse, rat, etc.) using any method known in the art, and any target sequences with significant homology to other coding sequences are excluded from consideration. One such method for such sequence homology searches is known as BLAST, and is available at the national center for biotechnology information website.

5. One or more sequences meeting the evaluation criteria are selected.

More general information about the design and use of siRNA can be found in "The siRNA User Guide" on the Max-plane-Institut fur Biophysikalische Chemie website.

Alternatively, the siRNA may be functionally defined as a nucleotide sequence (or oligonucleotide sequence) capable of hybridizing to the target sequence (e.g., 400mM NaCl, 40mM PIPES at pH 6.4, 1mM EDTA, hybridization at 50℃or 70℃for 12-16 hours; followed by washing). Additional hybridization conditions include hybridization at 70℃in 1XSSC or 50℃in 1XSSC,50% formamide, followed by washing at 70℃in 0.3XSSC or hybridization at 70℃in 4XSSC or 50℃in 4XSSC,50% formamide, followed by washing at 67℃in 1 XSSC. It is expected thatHybridization temperature of a hybrid of less than 50 base pairs in length should be higher than melting temperature (T _m ) 5-10deg.C lower, wherein T _m Determined according to the following equation. T for hybrids of less than 18 base pairs in length _m (°c) =2 (# -a+t base) +4 (# -g+c base). T for hybrids between 18 and 49 base pairs in length _m (℃)＝81.5+16.6(log 10[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 (1 XSSC [ Na ] ⁺ ]=0.165M). Other examples of stringency conditions for hybridization of polynucleotides are provided in Sambrook, j., e.f. fritsch and T.Maniatis,1989,Molecular Cloning:ALaboratory Manual,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y, chapters 9 and 11 and Current Protocols in Molecular Biology,1995, f.m. ausubel et al, john Wiley&Sons, inc, sections 2.10 and 6.3-6.4, which are incorporated herein by reference.

The negative control siRNA should have the same nucleotide composition as the selected siRNA, but no significant sequence complementarity to the appropriate genome. Such negative controls can be designed by randomly scrambling the nucleotide sequence of the selected siRNA. Homology searches can be performed to ensure that the negative control lacks homology with any other gene in the appropriate genome. In addition, negative control siRNA can be designed by introducing one or more base mismatches into the sequence.

6. To verify the effectiveness of siRNA to destroy target mRNA (e.g., wild-type or mutant IFNGR1, JAK2, or STAT1 mRNA), the siRNA can be incubated with target cDNA (e.g., IFNGR1, JAK2, or STAT1 cDNA) in a drosophila-based in vitro mRNA expression system. By using ³² The P radiolabeled newly synthesized target mRNA (e.g., IFNGR1, JAK2, or STAT1 mRNA) is detected by autoradiography on agarose gel. The presence of the cleaved target mRNA indicates mRNA nuclease activity. Suitable controls include omission of siRNA and use of non-target cDNA. Alternatively, a control siRNA is selected that has the same nucleotide composition as the selected siRNA, but no significant sequence complementarity to the appropriate target gene. Such negative controls can be obtained by randomly scrambling the nucleotides of the selected siRNAThe sequence is designed. Homology searches can be performed to ensure that the negative control lacks homology with any other gene in the appropriate genome. In addition, negative control siRNA can be designed by introducing one or more base mismatches into the sequence.

anti-IFNGR 1, anti-JAK 2, or anti-STAT 1 siRNA can be designed to target any of the target sequences described above. The siRNA comprises an antisense strand sufficiently complementary to a target sequence to mediate silencing of the target sequence. In certain embodiments, the RNA silencing agent is an siRNA.

In certain embodiments, the siRNA comprises a sense strand comprising the sequences listed in table 10 and table 11, respectively, and an antisense strand comprising the sequences listed in table 10 and table 11.

The siRNA-mRNA complementary sites that result in optimal mRNA specificity and maximal mRNA cleavage are selected.

b) siRNA-like molecules

The siRNA-like molecules of the invention have sequences that are "sufficiently complementary" to the target sequence of IFNGR1, JAK2 or STAT1 mRNA (i.e., have strands with sequences) to direct gene silencing by RNAi or translational inhibition. 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 miRNA and its target. In general, as the degree of sequence identity between miRNA sequences and the corresponding target gene sequences decreases, the tendency to mediate post-transcriptional gene silencing by translational inhibition rather than RNAi increases. Thus, in another embodiment, where post-transcriptional gene silencing by translational inhibition of a target gene is desired, the miRNA sequence has partial complementarity to the target gene sequence. In certain embodiments, the miRNA sequence has partial complementarity to one or more short sequences (complementarity 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). Since the translational inhibition mechanisms are cooperative, multiple complementary sites (e.g., 2, 3, 4, 5, or 6) may be targeted in certain embodiments.

The ability of an siRNA-like duplex to mediate RNAi or translational inhibition can be predicted by the distribution of different nucleotides between the target gene sequence and the nucleotide sequence of the silencing agent at complementary sites. In one embodiment, where gene silencing by translational inhibition is desired, at least one different 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 comprises a central "bulge" (Doench J G et al, genes & dev., 2003). In another embodiment, 2, 3, 4, 5 or 6 consecutive or non-consecutive different nucleotides are introduced. The different nucleotides may be selected to form wobble base pairs (e.g., G: U) or mismatched base pairs (G: A, C: A, C: U, G: G, A: A, C: C, U: U). In a further embodiment, the "bulge" is located at nucleotide positions 12 and 13 of the 5' end of the miRNA molecule.

c) Short hairpin RNA (shRNA) molecules

In certain feature embodiments, the invention provides shRNA capable of mediating RNA silencing of IFNGR1, JAK2, or STAT1 target sequences with enhanced selectivity. In contrast to siRNA, shRNA mimics the natural precursors of micrornas (mirnas) and enters the top of the gene silencing pathway. For this reason, shRNA is believed to mediate gene silencing more efficiently through the entire natural gene silencing pathway.

mirnas are non-coding RNAs of approximately 22 nucleotides that regulate gene expression at post-transcriptional or translational levels during plant and animal development. A common feature of mirnas is that they are all excised from a precursor RNA stem loop of about 70 nucleotides, known as a pre-miRNA, possibly by an rnase type III enzyme Dicer or a homologue thereof. Naturally occurring miRNA precursors (pre-mirnas) have a single strand forming a duplex stem comprising two parts, usually complementary, and a loop connecting the two parts of the stem. In a typical pre-miRNA, the stem includes one or more protrusions, e.g., additional nucleotides that create a single nucleotide "loop" in a portion of the stem, and/or one or more unpaired nucleotides that create a void when two portions of the stem hybridize to each other. The short hairpin RNAs or engineered RNA precursors of the present application are artificial constructs based on these naturally occurring pre-mirnas, but which are engineered to deliver the desired RNA silencing agent (e.g., the siRNA of the invention). shRNA is formed by replacing the stem sequence of a pre-miRNA with a sequence complementary to the target mRNA. shRNA is processed through the entire gene silencing pathway of the cell, effectively mediating RNAi.

Essential elements of shRNA molecules 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 parts need not be fully or perfectly complementary. The first and second "stem" portions are joined by a portion having a sequence that is not sufficiently complementary to anneal or hybridize to other portions of the shRNA. The latter part is called the "loop" part of the shRNA molecule. shRNA molecules are processed to generate siRNA. shRNA may also include one or more projections, i.e., additional nucleotides that create small nucleotide "loops" (e.g., mono-, di-, or trinucleotide loops) in a portion of the stem. The stem portions may be the same length, or one portion may include an overhang of, for example, 1-5 nucleotides. The protruding nucleotides may include, for example, uracil (U), e.g., all U. Such U is encoded, inter alia, by thymidine (T) in shRNA-encoding DNA, which signals transcription termination.

In the shRNA (or engineered precursor RNA) of the invention, a portion of the duplex stem is a nucleic acid sequence that is complementary (or antisense) to the IFNGR1, 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 by RNA interference (RNAi). Thus, an engineered RNA precursor includes a duplex 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 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 should be 21 nucleotides or more. When used in mammalian cells, the stem portion should be less than about 30 nucleotides in length to avoid causing a non-specific response such as an interferon pathway. In non-mammalian cells, the stem may be longer than 30 nucleotides. In fact, the stem may include a larger portion (up to and including the entire mRNA) that is complementary to the target mRNA. In fact, the stem portion may include a larger portion (up to and including the entire mRNA) that is complementary to the target mRNA.

The two parts of the duplex stem must be sufficiently complementary to hybridize to form the duplex stem. Thus, the two portions may, but need not, be fully or perfectly complementary. Furthermore, the two stem portions may have the same length, or one portion may comprise an overhang of 1, 2, 3 or 4 nucleotides. The protruding nucleotides may include, for example, uracil (U), e.g., all U. The loops in the shRNA or engineered RNA precursor may be different from the native pre-miRNA sequence by modifying the loop sequence to increase or decrease the number of paired nucleotides, or replacing all or part of the loop sequence with a tetra-or other loop sequence. Thus, the loop length in the shRNA or engineered RNA precursor may be 2, 3, 4, 5, 6, 7, 8, 9 or more, e.g., 15 or 20 or more nucleotides.

The loops in the shRNA or engineered RNA precursor may be different from the native pre-miRNA sequence by modifying the loop sequence to increase or decrease the number of paired nucleotides, or replacing all or part of the loop sequence with a tetra-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, such as 15 or 20 or more nucleotides in length. In certain embodiments, the loop consists of or comprises a "four-loop" sequence. Exemplary tetracyclic sequences include, but are not limited to, the sequences GNRA, GGGG and uuuuu, where N is any nucleotide and R is a purine nucleotide.

In certain embodiments, the shRNA of the present application includes the sequence of the desired siRNA molecule described above. In other embodiments, the sequence of the antisense portion of the shRNA may be designed substantially as described above, or generally by selecting a sequence of 18, 19, 20, 21 nucleotides or more from a region of target RNA (e.g., IFNGR1, JAK2, or STAT1 mRNA), e.g., 100 to 200 or 300 nucleotides upstream or downstream from the start of translation. In general, the sequence can be selected from any portion of a target RNA (e.g., mRNA), including a 5'utr (untranslated region), coding sequence, or 3' utr. This sequence may optionally immediately follow the target gene region comprising two adjacent AA nucleotides. The last two nucleotides of the nucleotide sequence may be selected as UU. These 21 or so nucleotide sequences are used to generate a portion of the duplex stem in the shRNA. This sequence may, for example, be enzymatically substituted for the stem portion of the wild-type pre-miRNA sequence, or be contained in a synthetic complete sequence. For example, DNA oligonucleotides can be synthesized that encode the entire stem-loop engineered RNA precursor, or only the portion to be inserted into the precursor duplex stem, and restriction endonucleases are used to construct an engineered RNA precursor construct, for example from a wild-type pre-miRNA.

In the duplex stem, the engineered RNA precursor includes about 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 comprises 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 and to maximize the efficacy of the resulting siRNA to target the corresponding mRNA in vivo and in vitro for translational inhibition or destruction by RNAi.

In certain embodiments, the shRNA of the invention includes miRNA sequences, optionally end-modified miRNA sequences, to enhance entry into RISC. The miRNA sequence may be similar or identical to the sequence of any naturally occurring miRNA (see, e.g., the miRNA Registry; griffiths-Jones S, nuc. Acids Res., 2004). To date, over one thousand natural mirnas have been identified, which together are considered to include about 1% of all predicted genes in the genome. Many natural mirnas are clustered together in the introns of the pre-mRNA and can be identified on a computer 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 the stem-loop structure of the primary mRNA (Grad et al, mol. Cell.,2003; lim et al Genes dev.,2003; lim et al, science,2003; lai E C et al, genome bio., 2003). On-line registration provides a searchable database of all published miRNA sequences (miRNA Registry on the Sanger institute website; griffiths-Jones S, nuc. Exemplary natural miRNAs include lin-4, let-7, miR-10, mirR-15, miR-16, miR-168, miR-175, miR-196 and homologs thereof, and other natural miRNAs from humans and certain model organisms including Drosophila melanogaster, caenorhabditis elegans, zebra fish, arabidopsis thaliana, mice and brown rats, as described in International PCT publication No. WO 03/029459.

Naturally occurring mirnas are expressed in vivo from endogenous Genes and processed from hairpin or stem-loop precursors (pre-mirnas or primary 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; moureltats 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 may exist transiently in vivo as duplex, but only one strand is taken up by the RISC complex to direct gene silencing. Some mirnas, such as plant mirnas, have perfect or near perfect complementarity with their target mRNA, thus cleaving the target mRNA directly. Other mirnas are not fully complementary to their target mRNA, and thus direct translation inhibits 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 is indicative of a cleavage mechanism (Yekta et al, science, 2004), while non-perfect complementarity is indicative of a translational inhibition mechanism. In certain embodiments, the miRNA sequence is a sequence of a naturally occurring miRNA sequence whose aberrant expression or activity is associated with a miRNA disorder.

d) Bifunctional oligonucleotide tethers

In other embodiments, the RNA silencing agents of the invention include bifunctional oligonucleotide tethers useful for intercellular recruitment of mirnas. Animal cells express a series of mirnas, i.e., non-coding RNAs of about 22 nucleotides, which regulate gene expression at post-transcriptional or translational levels during animal development. By binding to RISC-bound mirnas and recruiting them to the target mRNA, the bifunctional oligonucleotide tether can inhibit expression of genes involved in, for example, the arteriosclerosis process. The use of an oligonucleotide tether provides several advantages over the prior art that inhibit the expression of a particular gene. First, the methods described herein allow for endogenous (often present in large amounts) mirnas to mediate RNA silencing. Thus, the methods described herein eliminate the need to introduce exogenous molecules (e.g., siRNA) to mediate RNA silencing. Second, the RNA silencing agent and linking moiety (e.g., an oligonucleotide such as a 2' -O-methyl oligonucleotide) can be stabilized and resistant to nuclease activity. Thus, the tethers of the present invention can be designed for direct delivery, eliminating the need for indirect delivery (e.g., viral) of a precursor molecule or plasmid designed to produce the desired agent within the cell. Third, the tether and its respective portions may be designed to conform to a particular mRNA site and a particular miRNA. The design may be cell and gene product specific. Fourth, the methods disclosed herein leave the mRNA intact, allowing one to block protein synthesis in short pulses using the mechanisms of the cell itself. Thus, these methods of RNA silencing are highly tunable.

The bifunctional oligonucleotide tethers ("tethers") of the present invention are designed such that they recruit mirnas (e.g., endogenous cellular mirnas) to target mrnas, thereby inducing modulation of genes of interest. In certain embodiments, the tether has the formula T-L- μ, wherein T is an mRNA targeting moiety, L is a linking moiety, and μ is a miRNA recruiting moiety. Any one or more of the moieties may be double stranded. In certain embodiments, each moiety is single stranded.

The moieties within the tether may be aligned or linked (in the 5 'to 3' direction) as shown by formula T-L- μ (i.e., the 3 'end of the targeting 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 miRNA recruiting moiety). Alternatively, these portions may be arranged or attached in a tether as follows: mu-T-L (i.e., the 3 'end of the miRNA recruiting 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).

As described above, the mRNA targeting moiety is capable of capturing a specific target mRNA. According to the present invention, the expression of the target mRNA is not desired, and thus, the inhibition of translation of mRNA is desired. The size of the mRNA targeting portion should be sufficient to effectively bind the target mRNA. The length of the targeting moiety varies greatly, 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 a certain embodiment, the targeting moiety is about 15 to about 25 nucleotides in length.

As described above, the miRNA recruiting moiety is capable of binding to miRNA. According to the present application, the miRNA may be any miRNA capable of inhibiting a target mRNA. Mammals are reported to have 250 more endogenous miRNAs (Lagos-Quantana et al (2002) Current biol.12:735-739; lagos-Quantana et al (2001) Science 294:858-862; and Lim et al (2003) Science 299:1540). In various embodiments, the miRNA may be any art-recognized miRNA.

The linking moiety is any agent capable of linking the targeting moiety so as to maintain the activity of the targeting moiety. The linking moiety may be an oligonucleotide moiety comprising a sufficient number of nucleotides such that the targeting agent can substantially interact with their respective targets. The linking moiety has little or no sequence homology to the cellular mRNA or miRNA sequence. Exemplary linking moieties include one or more 2' -O-methyl nucleotides, such as 2' -beta-methyl adenosine, 2' -O-methyl thymidine, 2' -O-methyl guanosine, or 2' -O-methyl uridine.

e) Gene silencing oligonucleotides

In certain exemplary embodiments, oligonucleotide-based compounds comprising two or more single stranded antisense oligonucleotides linked by their 5 'ends allowing the presence of two or more accessible 3' ends to effectively inhibit or reduce IFNGR1, JAK2, or STAT1 gene expression may be used to modulate gene expression (i.e., IFNGR1, JAK2, or STAT1 gene expression). Such ligated oligonucleotides are also referred to as Gene Silencing Oligonucleotides (GSO). (see, e.g., US 8,431,544, assigned to Idera Pharmaceuticals, inc., which is incorporated herein by reference in its entirety for all purposes.)

The attachment of the 5 'end of the GSO is independent of other oligonucleotide attachments, either directly through the 5', 3 'or 2' hydroxyl groups, or indirectly through non-nucleotide linkers or nucleosides that utilize the 2 'or 3' hydroxyl positions of the nucleosides. Ligation may also utilize functionalized sugars or nucleobases of the 5' terminal nucleotide.

GSOs may comprise two identical or different sequences conjugated at their 5'-5' ends by phosphodiester, phosphorothioate or non-nucleoside linkers. Such compounds may comprise 15 to 27 nucleotides, which are complementary to a specific portion of the mRNA target of interest, for antisense down-regulation of the gene product. GSOs comprising the same sequence can bind to specific mRNAs and inhibit protein expression through Watson-Crick hydrogen bond interactions. GSOs comprising different sequences are capable of binding to two or more different regions of one or more mRNA targets and inhibiting protein expression. Such compounds consist of an oligonucleotide sequence complementary to the target mRNA and form a stable double-stranded structure through Watson-Crick hydrogen bonding. Under certain conditions, GSOs containing two free 3' -ends (5 ' -5' antisense linkages) may inhibit gene expression more effectively than those containing a single free 3' -end or no free 3' -end.

In some embodiments, the non-nucleotide linker is of the formula HO- - (CH) ₂ ) _o --CH(OH)--(CH ₂ ) _p -OH or glycerol homologues, wherein o and p are independently integers from 1 to about 6, from 1 to about 4 or from 1 to about 3. In some other embodiments, the non-nucleotide linker is a derivative of 1, 3-diamino-2-hydroxypropane. Some of these derivatives have the formula HO- - (CH) ₂ )m--C(O)NH--CH ₂ --CH(OH)--CH ₂ --NHC(O)--(CH ₂ ) _m - -OH, wherein m is an integer from 0 to about 10, 0 to about 6, 2 to about 6, or 2 to about 4.

Some non-nucleotide linkers allow for the ligation of more than two GSO components. For example, the non-nucleotide linker glycerol has three hydroxyl groups to which the GSO component may be covalently linked. Thus, some oligonucleotide-based compounds of the invention comprise two or more oligonucleotides linked to a nucleotide or non-nucleotide linker. Such oligonucleotides according to the invention are referred to as "branched".

In certain embodiments, the GSO is at least 14 nucleotides in length. In certain exemplary embodiments, GSO is 15 to 40 nucleotides in length or 20 to 30 nucleotides in length. Thus, the constituent oligonucleotides of GSO may independently be 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.

These oligonucleotides may be prepared by art-recognized methods, such as phosphoramidate or H-phosphonate chemistry, which may be performed manually or by an automated synthesizer. These oligonucleotides can also be modified in a variety of ways without compromising their ability to hybridize to mRNA. Such modifications may include at least one internucleotide linkage of an oligonucleotide that is an alkyl phosphonate, phosphorothioate, phosphorodithioate, methylphosphonate, phosphate, alkyl phosphorothioate, phosphoramidate, carbamate, carbonate, hydroxyl phosphate, 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, wherein the 5' nucleotide phosphodiester linkage has been substituted with any number of chemical groups.

V. modified anti-IFNGR 1, anti-JAK 2 or anti-STAT 1RNA silencing agents

In certain aspects of the invention, as described above, the RNA silencing agent (or any portion thereof) of the present application may be modified such that the activity of the agent is further enhanced. For example, the RNA silencing agent described in section II above can be modified with any of the modifications described below. Modifications may be used, in part, to further enhance target discrimination, enhance stability of an agent (e.g., prevent degradation), promote cellular uptake, increase target efficiency, increase efficacy of binding (e.g., binding to a target), increase tolerance of a patient to a drug, and/or reduce toxicity.

1) Modification to enhance target recognition

In certain embodiments, the RNA silencing agents of the present application may be substituted with destabilizing nucleotides to enhance single nucleotide target discrimination (see U.S. application serial No. 11/698,689 filed on 1 month 25 and U.S. provisional application No. 60/762,225 filed on 1 month 25 in 2007, both of which are incorporated herein by reference). Such modification may be sufficient to eliminate the specificity of the RNA silencing agent for non-target mRNA (e.g., wild-type mRNA) without significantly affecting the specificity of the RNA silencing agent for target mRNA (e.g., functionally-acquired mutant mRNA).

In certain embodiments, the RNA silencing agents of the present application are modified by the incorporation of at least one universal nucleotide in the antisense strand thereof. The universal nucleotide comprises a base moiety capable of base pairing with any of four conventional nucleotide bases (e.g., A, G, C, U). Universal nucleotides are contemplated because they have relatively little effect on the stability of the RNA duplex or duplex formed by the guide strand of the RNA silencing agent and the target mRNA. Exemplary universal nucleotides include those having an inosine base portion or an inosine analog base portion selected from the group consisting of: deoxyinosine (e.g., 2 '-deoxyinosine), 7-aza-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 analog thereof.

In certain embodiments, the RNA silencing agents of the invention are modified by introducing at least one destabilizing nucleotide within 5 nucleotides of a specificity determining nucleotide (i.e., a nucleotide that recognizes a disease-related polymorphism). For example, destabilizing nucleotides may be introduced at a position within 5, 4, 3, 2 or 1 nucleotides of the specificity determining nucleotide. In an exemplary embodiment, the destabilizing nucleotide is introduced 3 nucleotides from the specificity determining nucleotide (i.e., such that there are 2 stabilizing nucleotides between the destabilizing nucleotide and the specificity determining nucleotide). In RNA silencers (e.g., siRNA and shRNA) having two strands or strand portions, destabilizing nucleotides can be incorporated into the strand or strand portion that does not contain a specificity determining nucleotide. In certain embodiments, the destabilizing nucleotide is introduced into the same strand or strand portion comprising the specificity determining nucleotide.

2) Modification to improve efficacy and specificity

In certain embodiments, the RNA silencing agents of the invention may be altered to promote enhanced efficacy and specificity for mediating RNAi according to asymmetric design rules (see U.S. patent nos. 8,309,704, 7,750,144, 8,304,530, 8,329,892, and 8,309,705). Such alterations facilitate entry of the antisense strand of the siRNA (e.g., the siRNA designed using the methods of the present application or the siRNA produced by shRNA) into the RISC, and facilitate the sense strand such that the antisense strand preferentially directs cleavage or translational inhibition of the target mRNA, thereby increasing or enhancing the efficiency of target cleavage and silencing. In certain embodiments, the asymmetry of the RNA silencing agent is enhanced by reducing the base pair strength between the antisense strand 5' end (AS 5 ') and the sense strand 3' end (S3 ') of the RNA silencing agent relative to the bond strength or base pair strength between the antisense strand 3' end (AS 3 ') and the sense strand 5' end (S5).

In one embodiment, the asymmetry of the RNA silencing agents of the present application can be enhanced such that fewer G: C base pairs are present between the 5 'end of the first or antisense strand and the 3' end of the sense strand portion than between the 3 'end of the first or antisense strand and the 5' end of the sense strand portion. In another embodiment, the asymmetry of the RNA silencing agents of the invention may be enhanced such that there is at least one mismatched base pair between the 5 'end of the first strand 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 agents of the invention may be enhanced such that there is at least one wobble base pair, e.g., G: U, between the 5 'end of the first strand or antisense strand and the 3' end of the sense strand portion. In another embodiment, the asymmetry of the RNA silencing agents of the invention may be enhanced such that there is at least one base pair comprising a rare nucleotide, such as inosine (I). In certain embodiments, the base pairs are selected from the group consisting of: a, I, U and C. In yet another embodiment, the asymmetry of the RNA silencing agents of the invention can be enhanced such that there is at least one base pair comprising 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.

3) RNA silencing agents with enhanced stability

The RNA silencing agents of the present application can be modified to improve stability in serum or cell culture growth media. To enhance stability, the 3' -residues may be stabilized against degradation, e.g., they may be selected such that they consist of purine nucleotides, such as adenosine or guanosine nucleotides. Alternatively, substitution of pyrimidine nucleotides with modified analogs, such as substitution of uridine with 2' -deoxythymidine, is acceptable and does not affect the efficiency of RNA interference.

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

In one aspect, the application features an at least 80% chemically modified RNA silencing agent. In certain embodiments, the RNA silencing agent may be chemically modified entirely, i.e., 100% of the nucleotides are chemically modified. In another aspect, the application features an RNA silencing agent comprising 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.

In certain embodiments, the RNA silencing agent may comprise at least one modified nucleotide analog. The nucleotide analogs can be located at a position where target-specific silencing activity (e.g., RNAi-mediated activity or translational inhibitory activity) is substantially unaffected, e.g., in the region of the 5 '-end and/or the 3' -end of the siRNA molecule. Furthermore, the terminal ends can be stabilized by incorporating modified nucleotide analogs.

Exemplary nucleotide analogs include sugar and/or backbone modified ribonucleotides (i.e., including modifications to the phosphate sugar backbone). For example, the phosphodiester linkage of the natural RNA may be modified to include at least one of a nitrogen or sulfur heteroatom. In an exemplary backbone modified ribonucleotide, the phosphate group attached to the adjacent ribonucleotide is replaced with a modified group (e.g., a phosphorothioate group). In exemplary sugar-modified ribonucleotides, the 2' -OH-group can be selected from H, OR, R, halo, SH, SR, NH ₂ 、NHR、NR ₂ Or ON, wherein R is C ₁ -C ₆ Alkyl, alkenyl or alkynyl and halo is F, cl, br or I.

In certain embodiments, the modification is a 2' -fluoro, 2' -amino, and/or 2' -thio modification. 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-thiouridine, and/or 5-aminoallyl uridine. In a certain embodiment, the 2' -fluororibonucleotide is each uridine and cytidine. Other exemplary modifications include 5-bromo-uridine, 5-iodo-uridine, 5-methyl-cytidine, ribose-thymidine, 2-aminopurine, 2' -amino-butyryl-pyrene-uridine, 5-fluoro-cytidine, and 5-fluorouridine. 2 '-deoxynucleotides and 2' -Ome nucleotides may also be used in the modified RNA-silencer moieties of the invention. Other modified residues include deoxyabasic, inosine, N3-methyl-uridine, N6-dimethyl-adenosine, pseudouridine, purine ribonucleoside, and ribavirin. In a certain embodiment, the 2 'moiety is methyl, such that the linking moiety is a 2' -O-methyl oligonucleotide.

In a certain embodiment, the RNA silencing agent of the present application comprises a Locked Nucleic Acid (LNA). LNA comprises sugar modified nucleotides that are resistant to 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 and have possible modifications, such as 2' -deoxy-2 "-fluorouridine. In addition, LNA increases the specificity of the oligonucleotide by confining the sugar moiety in the 3' -internal conformation, thereby pre-organizing the nucleotides for base pairing and increasing the melting temperature of the oligonucleotide by up to 10 ℃/base.

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 by a neutral 2-aminoethylglycine moiety, which is capable of forming a polyamide backbone that is highly resistant to nuclease digestion and confers enhanced binding specificity to the molecule (Nielsen et al, science, (2001), 254:1497-1500).

Nucleobase modified ribonucleotides, i.e., ribonucleotides that contain at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase, are also contemplated. Bases may be modified to block the activity of adenosine deaminase. Exemplary modified nucleobases include, but are not limited to, uridine and/or cytidine modified at the 5-position, e.g., 5- (2-amino) propyluridine, 5-bromouridine; adenosine and/or guanosine modified at position 8, e.g. 8-bromoguanosine; denitrifying nucleotides, such as 7-deaza-adenosine; o-alkylated nucleotides and N-alkylated nucleotides, for example, N6-methyladenosine, are suitable. It should be noted that the above modifications may be combined.

In other embodiments, crosslinking may be used to alter the pharmacokinetics of the RNA silencing agent, e.g., increase half-life in vivo. Thus, the present application includes an RNA silencing agent having two complementary nucleic acid strands, wherein the two strands are cross-linked. The present application also includes RNA silencing agents conjugated or unconjugated (e.g., at the 3' end thereof) to another moiety (e.g., a non-nucleic acid moiety such as a peptide), an organic compound (e.g., a dye), or the like. Modifying the siRNA derivative in this manner can improve cellular uptake or enhance cellular targeting activity of the resulting siRNA derivative compared to the corresponding siRNA, can be used to track the siRNA derivative in a cell, or can increase stability of the siRNA derivative compared to the corresponding siRNA.

Other exemplary modifications include: (a) 2 'modification, e.g. on the sense strand or the antisense strand, but especially on the sense strand, or in the 3' overhang, e.g. at the 3 'end (3' end means at the 3 'atom of the molecule or at the most 3' part, e.g. the most 3'P or 2' position as indicated above and below); (b) Modifying the backbone in the phosphate backbone, e.g. replacing 0 with S, e.g. providing phosphorothioate modifications on U or a or both, in particular on the antisense strand; for example, replacing O with S; (C) replacing U with a C5 amino linker; (d) Substitution of G for a (in certain embodiments, sequence changes may be located on the sense strand instead of the antisense strand); and (d) modification at the 2', 6', 7 'or 8' positions. Exemplary embodiments are those in which one or more of these modifications are present on the sense strand but not on the antisense strand, or embodiments in which the antisense strand has fewer such modifications. Other exemplary modifications include the use of methylated P at the 3 'overhang, e.g., at the 3' end; 2 'modification, e.g., a modification providing a 2' ome moiety and a backbone, e.g., replacing O with S, e.g., providing a phosphorothioate modification, or a combination of methylation P at the 3 'overhang, e.g., at the 3' end; modification with 3' alkyl; modification at the 3 'overhang, for example at the 3' end with abasic pyrrolidone; modification with naproxen, ibuprofen or other moieties that inhibit degradation of the 3' end.

Heavily 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 chemically modified entirely, i.e., 100% of the nucleotides are chemically modified.

In certain embodiments, the RNA silencing agent is enriched with 2 '-O-methyl groups, i.e., comprises greater 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 between about 70% and 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 between about 70% and 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 between about 70% and about 90% 2' -O-methyl nucleotide modifications. In certain embodiments, the sense strand comprises 100% 2' -O-methyl nucleotide modifications.

RNA silencing agents rich in 2' -O-methyl and specific chemical modification patterns are further described in U.S. S. N.16/550,076 (filed 8.23 in 2019) and U.S. S. N.16/999,759 (filed 21 in 2020), each of which is incorporated herein by reference.

Internucleotide linkage modification

In certain embodiments, at least one internucleotide linkage, intersubunit linkage, or nucleotide backbone is modified in an RNA silencing agent. In certain embodiments, all internucleotide linkages in the RNA silencing agent are modified. In certain embodiments, the modified internucleotide linkages comprise phosphorothioate internucleotide linkages. 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 oligonucleotide comprises 4-16 phosphorothioate internucleotide linkages. In certain embodiments, the oligonucleotide 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, the nucleotides at positions 1 and 2 of the 5' end of the sense strand are linked to adjacent ribonucleotides by phosphorothioate internucleotide linkages. In certain embodiments, the nucleotides from positions 1 and 2 at the 3' end of the sense strand are linked to adjacent ribonucleotides by phosphorothioate internucleotide linkages. In certain embodiments, the nucleotides at positions 1 and 2 from the 5' end of the antisense strand are linked to adjacent ribonucleotides by phosphorothioate internucleotide linkages. In certain embodiments, the nucleotides from positions 1-2 to 1-8 at the 3' end of the antisense strand are linked to adjacent ribonucleotides by phosphorothioate internucleotide linkages. In certain embodiments, the 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 by phosphorothioate internucleotide linkages. In certain embodiments, the nucleotides from positions 1-2 to 1-7 at the 3' end of the antisense strand are linked to adjacent ribonucleotides by phosphorothioate internucleotide linkages.

In one aspect, the present disclosure provides a modified oligonucleotide complementary to a target, the oligonucleotide having a 5 'end, a 3' end, wherein the oligonucleotide comprises a sense strand and an antisense strand, and at least one modified intersubunit bond of formula (I):

wherein:

b is a base pairing moiety;

w is selected from O, OCH ₂ 、OCH、CH ₂ And CH;

x is selected from halo, hydroxy and C _1-6 Alkoxy groups;

y is selected from O ^- 、OH、OR、NH ^- 、NH ₂ 、S ^- And SH;

z is selected from O and CH ₂ A group of;

r is a protecting group; and is also provided with

Is an optional double bond.

In one embodiment of formula (I), when W is CH,is a double bond.

In one embodiment of formula (I), when W is selected from O, OCH ₂ 、OCH、CH ₂ In the case of the group of the components,is a single bond.

In one embodiment of formula (I), when Y is O ^- When Z or W is not O.

In one embodiment of formula (I), Z is CH ₂ And W is CH ₂ . In another embodiment, the modified intersubunit bond of formula (I) is a modified intersubunit bond of formula (II):

in one embodiment of formula (I), Z is CH ₂ And W is O. In another embodiment, wherein the modified intersubunit bond of formula (I) is a modified intersubunit bond of formula (III):

in one embodiment of formula (I), Z is O and W is CH ₂ . In another embodiment, the modified intersubunit bond of formula (I) is a modified intersubunit bond of formula (IV):

in one embodiment of formula (I), Z is O and W is CH. In another embodiment, the modified intersubunit bond of formula (I) is a modified intersubunit bond of formula V:

in one embodiment of formula (I), Z is O and W is OCH ₂ . In another embodiment, the modified intersubunit bond of formula (I) is a modified intersubunit bond of formula VI:

in one embodiment of formula (I), Z is CH ₂ And W is CH. In another embodiment, the modified intersubunit bond of formula (I) is a modified intersubunit bond of formula VII:

in one embodiment of formula (I), base pairing moiety B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

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

In one embodiment, a modified oligonucleotide is incorporated into an siRNA, the modified siRNA having a 5 'end, a 3' end, complementary to a target and comprising sense and antisense strands, wherein the siRNA comprises at least one modified intersubunit bond of formula VIII:

Wherein:

d is selected from O, OCH ₂ A group consisting of OCH, CH2 and CH;

c is selected from O ^- 、OH、OR ¹ 、NH ^– 、NH ₂ 、S ^- And SH;

a is selected from O and CH ₂ A group of;

R ¹ is a protecting group;

is an optional double bond; and is also provided with

Bridging two optionally modified nucleosides between subunits.

In one embodiment, when C is O ^- When A or D is not O.

In one embodiment, D is CH ₂ . In another embodiment, the modified intersubunit bond of formula VIII is a modified intersubunit bond of formula (IX):

in one embodiment, D is O. In another embodiment, the modified intersubunit bond of formula VIII is a modified intersubunit bond of formula (X):

in one embodiment, D is CH ₂ . In another embodiment, the modified intersubunit bond of formula (VIII) is a modified intersubunit bond of formula (XI):

in one embodiment, D is CH. In another embodiment, the modified intersubunit bond of formula VIII is a modified intersubunit bond of formula (XII):

in another embodiment, the modified intersubunit bond of formula (VII) is a modified intersubunit bond of formula (XIV):

in one embodiment, D is OCH ₂ . In another embodiment, the modified intersubunit bond of formula (VII) is a modified intersubunit bond of formula (XIII):

In another embodiment, the modified intersubunit bond of formula (VII) is a modified intersubunit bond of formula (XXa):

/>

in one embodiment of the modified siRNA bond, each optionally modified nucleoside is independently selected at each occurrence from the group consisting of adenosine, guanosine, cytidine, and uridine.

In certain exemplary embodiments of formula (I), W is O. In another embodiment, W is CH ₂ . In yet another embodiment, W is CH.

In certain exemplary embodiments of formula (I), X is OH. In another embodiment, X is OCH ₃ . In yet another embodiment, X is halo.

In a certain embodiment of formula (I), the modified siRNA does not comprise a 2' -fluoro substituent.

In one 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 ₂ . In another embodiment, Y is S ^– . In yet another embodiment, Y is SH.

In one embodiment of formula (I), Z is O. In another embodiment, Z is CH ₂ 。

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

In one exemplary embodiment of the modified siRNA linkage of formula (VIII), C is O ^- . In another embodiment, C is OH. In another embodiment, C is OR ¹ . In another embodiment, C is NH ^– . In one embodiment, C is NH ₂ . In another embodiment, C is S ^– . In yet another embodiment, C is SH.

In one exemplary embodiment of the modified siRNA bond of formula (VIII), a is O. In another embodiment, A is CH ₂ . In another embodiment, C is OR ¹ . In another embodiment, C is NH ^– . In one embodiment, C is NH ₂ . In another embodiment, C is S ^– . In yet another embodiment, C is SH.

In a certain embodiment of the modified siRNA bond of formula (VIII), the optionally modified nucleoside is adenosine. In another embodiment of the modified siRNA bond of formula (VIII), the optionally modified nucleoside is guanosine. In another embodiment of the modified siRNA bond of formula (VIII), the optionally modified nucleoside is cytidine. In another embodiment of the modified siRNA bond of formula (VIII), the optionally modified nucleoside is uridine.

In one embodiment of the modified siRNA bond, wherein the bond is inserted at positions 1-2 of the antisense strand. In another embodiment, the bond is inserted at positions 6-7 of the antisense strand. In yet another embodiment, the bond is inserted at positions 10-11 of the antisense strand. In another embodiment, the bond is inserted at positions 19-20 of the antisense strand. In one embodiment, the bond is inserted at positions 5-6 and 18-19 of the antisense strand.

In certain embodiments of formula (I), base pairing moiety B is adenine. In certain embodiments of formula (I), base pairing moiety B is guanine. In certain embodiments of formula (I), base pairing moiety B is cytosine. In certain embodiments of formula (I), base pairing moiety B is uracil.

In one embodiment of formula (I), W is O. In one embodiment of formula (I), W is CH ₂ . In one embodiment of formula (I), W is CH.

In one embodiment of formula (I), X is OH. In one embodiment of formula (I), X is OCH ₃ . In one embodiment of formula (I), X is halo.

In an exemplary embodiment of formula (I), the modified oligonucleotide does not comprise a 2' -fluoro substituent.

In one embodiment of formula (I), Y is O ^– . In one embodiment of formula (I), Y is OH. In one embodiment of formula (I), Y is OR. In one embodiment of formula (I), Y is NH ^– . In one embodiment of formula (I), Y is NH ₂ . In one embodiment of formula (I), Y is S ^– . In one embodiment of formula (I), Y is SH.

In one embodiment of formula (I), Z is O. In one embodiment of formula (I), Z is CH ₂ 。

In one embodiment of formula (I), the bond is inserted at positions 1-2 of the antisense strand. In another embodiment of formula (I), the bond is inserted at positions 6-7 of the antisense strand. In another embodiment of formula (I), the bond is inserted at positions 10-11 of the antisense strand. In another embodiment of formula (I), the bond is inserted at positions 19-20 of the antisense strand. In one embodiment of formula (I), the bond is inserted at positions 5-6 and 18-19 of the antisense strand.

Modified intersubunit linkages are further described in U.S. S. n 62/824,136 (filing date 26 of 3.2019), U.S. s.n.62/826,454 (filing date 29 of 3.2019), and U.S. s.n.62/864,792 (filing date 21 of 6.2019), each of which is incorporated herein by reference.

4) Conjugation of functional moieties

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 an RNA silencing agent. In certain embodiments, the functional moiety enhances cellular uptake by target cells (e.g., T cells and epidermal keratinocytes). Thus, the invention includes RNA silencing agents conjugated or unconjugated (e.g., at their 5 'and/or 3' ends) to another moiety (e.g., a non-nucleic acid moiety such as a peptide), an organic compound (e.g., a dye), or the like. Conjugation may be achieved by methods known in the art, for example, using the following methods: lambert et al, drug Deliv.Rev.:47 (1), 99-112 (2001) (describing nucleic acids loaded into Polyalkylcyanoacrylate (PACA) nanoparticles); fattal et al, J.control Release 53 (1-3): 137-43 (1998) (describing nucleic acids binding to nanoparticles); schwab et al, ann.Oncol.5 support.4:55-8 (1994) (describing nucleic acids linked to intercalators, hydrophobic groups, polycations or PACA nanoparticles); and Godard et al, eur. J. Biochem.232 (2): 404-10 (1995) (describing nucleic acids linked to nanoparticles).

In a certain embodiment, the functional moiety is a hydrophobic moiety. In a certain embodiment, the hydrophobic moiety is selected from the group consisting of: fatty acids, steroids, ring-opened steroids, lipids, gangliosides and nucleoside analogues, endogenous cannabinoids and vitamins. In a certain embodiment, the steroid is selected from the group consisting of cholesterol and lithocholic acid (LCA). In a certain embodiment, the fatty acid is selected from the group consisting of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and docosanoic acid (DCA). In a certain embodiment, the vitamin is selected from the group consisting of choline, vitamin a, vitamin E, and derivatives or metabolites thereof. In a certain embodiment, the vitamin is selected from the group consisting of retinoic acid and alpha-tocopheryl succinate.

In a certain embodiment, the RNA silencing agent of the invention is conjugated to a lipophilic moiety. In one embodiment, the lipophilic moiety is a ligand comprising a cationic group. In another embodiment, the lipophilic moiety is linked to one or both strands of the siRNA. In one 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, cationic dyes (e.g., cy 3). In one 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, geranoxyhexyl, hexadecyl glycerol, borneol, menthol, 1, 3-propanediol, heptadecyl, palmitic acid, myristic acid, O3- (oleoyl) lithocholic acid, O3- (oleoyl) cholic acid, dimethoxytrityl or phenoxazine.

In certain embodiments, the functional moiety may comprise one or more ligands linked to the RNA silencing agent to improve stability, hybridization thermodynamics with the target nucleic acid, targeting to a specific tissue or cell type or cell permeability, e.g., by endocytosis-dependent or independent mechanisms. Ligands and related modifications can also increase sequence specificity, thereby reducing ectopic targeting. The tethered ligands can include one or more modified bases or sugars that can be used as intercalators. These may be located in an internal region, for example in the bulge of the RNA silencing agent/target duplex. The intercalator may be an aromatic compound, such as a polycyclic aromatic compound or a heterocyclic aromatic compound. Polycyclic intercalators may have stacking capability and may include systems having 2, 3, or 4 fused rings. The universal bases described herein may be included on a ligand. In one embodiment, the ligand may include a cleavage group that aids in the inhibition of the target gene by cleaving the target nucleic acid. The cleavage group may be, for example, bleomycin (e.g., bleomycin-A5, bleomycin-A2 or bleomycin-B2), pyrene, phenanthroline (e.g., O-phenanthroline), polyamine, tripeptide (e.g., lys-tyr-lys tripeptide), or a metal ion chelating group. The metal ion chelating group can include, for example, lu (III) or EU (III) macrocyclic complexes, zn (II) 2, 9-dimethylphenanthroline derivatives, cu (II) terpyridines or acridines, which can promote selective cleavage of the target RNA at the bulge site by free metal ions such as Lu (III). In some embodiments, the peptide ligand may be linked to an RNA silencing agent to facilitate cleavage of the target RNA, e.g., in raised areas. For example, 1, 8-dimethyl-1,3,6,8,10,13-hexaazacyclotetradecane (cyclam) may be conjugated to peptides (e.g., via amino acid derivatives) to facilitate target RNA cleavage. The tether ligand may be an aminoglycoside ligand that may 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 but low sequence specificity compared to DNA. The acridine analog, new-5-acridine, has increased affinity for the HIV Rev Responsive Element (RRE). In some embodiments, guanidine analogs of aminoglycoside ligands (guanidyl glycosides) are tethered to the RNA silencing agent. In guanidyl glycosides, the amine groups on the amino acids are exchanged by guanidine groups. Attachment of guanidine analogs can enhance cell permeability of RNA silencing agents. The tethering ligand may be a polyarginine peptide, peptoid or peptoid, which may enhance cellular uptake of the oligonucleotide reagent.

Exemplary ligands are coupled to ligand conjugated carriers directly or indirectly via intervening tethers. In certain embodiments, the coupling is by covalent bonds. In certain embodiments, the ligand is attached to the carrier via an intervening tether. In certain embodiments, the ligand alters the distribution, targeting, or lifetime of the RNA silencing agent into which it is incorporated. In certain embodiments, the ligand provides enhanced affinity for a selected target, e.g., a molecule, cell or cell type, compartment, e.g., cell or organ compartment, tissue, organ, or body region, e.g., as compared to a species without such ligand.

Exemplary ligands may improve transport, hybridization, and specificity properties, and may also improve nuclease resistance of the resulting natural or modified RNA silencing agent or polymer molecule comprising any combination of monomers and/or natural or modified ribonucleotides described herein. The ligand may generally include a therapeutic modifier, for example for enhancing absorption; diagnostic compounds or reporter groups, for example, for monitoring distribution; a cross-linking agent; a nuclease resistance conferring moiety; and natural or unusual nucleobases. Typical examples include lipophilic substances, lipids, steroids (e.g., glabrous greenbrier rhizome, hecogenin, diosgenin), terpenes (e.g., triterpenes such as sarsasapogenin, friedelane, epifriedelane alcohol-derived lithocholic acid), vitamins (e.g., folic acid, vitamin a, biotin, pyridoxal), carbohydrates, proteins, protein binding agents, integrin targeting molecules, polycations, peptides, polyamines, and peptidomimetics. The ligand may include naturally occurring substances (e.g., human Serum Albumin (HSA), low Density Lipoprotein (LDL), or globulin); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid); amino acids or lipids. The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g. a synthetic polyamino acid. Examples of the polyamino acid include a polyamino acid which is Polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic anhydride copolymer, poly (L-lactide-co-glycolide) 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, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salts of polyamines, or alpha helical peptides.

The ligand may also include a targeting group, such as a cell or tissue targeting agent, such as a lectin, glycoprotein, lipid or protein, such as an antibody, that binds to a particular cell type, such as a kidney cell. The targeting group may be thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein a, mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine (GalNAc) or a derivative thereof, N-acetyl-glucosamine, multivalent mannose, multivalent fucose, glycosylated polyamino acid, multivalent galactose, transferrin, bisphosphonate, polyglutamic acid, polyaspartic acid, lipid, cholesterol, steroid, bile acid, folic acid, vitamin B12, biotin or RGD peptide mimetic. Other examples of ligands include dyes, intercalators (e.g., acridine and substituted acridine), crosslinkers (e.g., psoralen, mitomycin C), porphyrins (TPPC 4, texas porphyrin (texaphyrin), saphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine, phenanthroline, pyrene), lys-tyr-lys tripeptides, aminoglycosides, guanosine, artificial endonucleases (e.g., EDTA), lipophilic molecules such as cholesterol (and thio-analogues thereof), cholic acid, cholanic acid, lithocholic acid, adamantaneacetic acid, 1-pyrene butyric acid, dihydrotestosterone, glycerol (e.g., esters (e.g., mono-, di-or tri-fatty acid esters, e.g., C) ₁₀ 、C ₁₁ 、C ₁₂ 、C ₁₃ 、C ₁₄ 、C ₁₅ 、C ₁₆ 、C ₁₇ 、C ₁₈ 、C ₁₉ Or C ₂₀ Fatty acids) and ethers thereof, 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 An alkyl group; for example, 1, 3-bis-O (hexadecyl) glycerol, 1, 3-bis-O (octadecyl) glycerol), geranoxyhexyl, hexadecyl glycerol, borneol, menthol, 1, 3-propanediol, heptadecyl, palmitic acid, stearic acid (e.g., glycerol distearate), oleic acid, myristic acid, O3- (oleoyl) lithocholic acid, O3- (oleoyl) cholanic acid, dimethoxytrityl or phenoxazine) and peptide conjugates (e.g., talopoptide, tat peptide), alkylating agents, phosphate esters, amino groups, sulfhydryl groups, PEG (e.g., PEG-40K), MPEG, [ MPEG ]]2. Polyamino groups, alkyl groups, substituted alkyl groups, radiolabelled labels, enzymes, haptens (e.g., biotin), transport/absorption enhancers (e.g., aspirin, naproxen, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, eu of the tetraazamacrocycle) ³⁺ Complex), dinitrophenyl, HRP, or AP. In certain embodiments, the ligand is GalNAc or a derivative thereof.

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

The ligand may be a substance, such as a drug, that may increase the uptake of the RNA silencing agent into the cell, e.g., by disrupting the cytoskeleton of the cell, e.g., by disrupting microtubules, microfilaments, and/or intermediate filaments of the cell. The drug may be, for example, a taxonomic group, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, langchun A (latrunculin A), phalloidin, swinholide a, indannew base (indacene) or myopervin. For example, the ligand may increase uptake of the RNA silencing agent into the cell by activating an inflammatory response. Exemplary ligands having this effect include tumor necrosis factor alpha (tnfα), interleukin 1 beta, or gamma interferon. In one aspect, the ligand is a lipid or lipid-based molecule. Such lipids or lipid-based molecules may bind serum proteins, such as Human Serum Albumin (HSA). HSA binding ligands allow the conjugate to be distributed to target tissue, e.g., non-kidney target tissue of the body. For example, the target tissue may be the liver, including parenchymal cells of the liver. Other molecules that bind HSA may also be used as ligands. For example, neprilysin or aspirin may be used. The lipid or lipid-based ligand may (a) increase resistance to conjugate degradation, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) be used to modulate binding to a serum protein such as HSA. Lipid-based ligands can be used to modulate, for example, control the binding of conjugates to target tissue. For example, a lipid or lipid-based ligand that binds more strongly to HSA will be less likely to target the kidney and therefore less likely to be cleared from the body. Lipids or lipid-based ligands that bind poorly to HSA can be used to target the conjugate to the kidney. In a certain embodiment, the lipid based ligand binds HSA. The lipid-based ligand can bind HSA with sufficient affinity such that the conjugate will be distributed to non-kidney tissue. However, affinity is not expected to be so strong that HSA-ligand binding cannot be reversed. In another embodiment, the lipid-based ligand binds weakly or not at all to HSA, such that the conjugate will distribute to the kidneys. Other moieties that target kidney cells can also be used to replace or supplement lipid-based ligands.

In another aspect, the ligand is a moiety, such as a vitamin, that is taken up by a target cell, such as a proliferating cell. These are useful in the treatment of diseases characterized by unwanted cell proliferation, such as malignant or non-malignant cell types, such as cancer cells. Exemplary vitamins include vitamins A, E and K. Other exemplary vitamins include B vitamins such as folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients absorbed by cancer cells. HSA and Low Density Lipoprotein (LDL) are also included.

In another aspect, the ligand is a cell penetrating agent, such as a helical cell penetrating agent. In certain embodiments, the agent is amphiphilic. Exemplary agents are peptides, such as tat or footlet. If the agent is a peptide, it may be modified, including peptidomimetics, transformants, non-peptide or pseudopeptide bonds, and the use of D-amino acids. The helicant may be an alpha-helicant, which may have a lipophilic phase and a lipophobic phase.

The ligand may be a peptide or a peptidomimetic. Peptide mimetics (also referred to herein as oligopeptide mimetics) are molecules that are capable of folding into a defined three-dimensional structure similar to a natural peptide. The attachment of peptides and peptidomimetics to oligonucleotide reagents can affect the pharmacokinetic profile of the RNA silencing agent, for example, by enhancing cell recognition and uptake. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long. The peptide or peptidomimetic can be, for example, a cell penetrating peptide, a cationic peptide, an amphiphilic peptide, or a hydrophobic peptide (e.g., consisting essentially of Tyr, trp, or Phe). The peptide moiety may be a dendrimer peptide, a constraint peptide or a cross-linked peptide. The peptide moiety may be an L-peptide or a D-peptide. In another alternative, the peptide moiety may include a hydrophobic Membrane Translocation Sequence (MTS). The peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage display library or a single bead-compound (OBOC) combinatorial library (Lam et al, nature 354:82-84,1991). In exemplary embodiments, the peptide or peptidomimetic linked to the RNA silencing agent through the incorporated monomer unit is a cell targeting peptide, such as an arginine-glycine-aspartic acid (RGD) -peptide or RGD mimetic. The peptide portion may range in length from about 5 amino acids to about 40 amino acids. The peptide moiety may have structural modifications, for example, to increase stability or direct conformational properties. Any of the structural modifications described below may be used.

In certain embodiments, the functional moiety is attached to the 5 'and/or 3' end of the RNA silencing agent of the invention. In certain embodiments, the functional moiety is attached to the 5 'and/or 3' end of the antisense strand of the RNA silencing agent of the invention. In certain embodiments, the functional moiety is attached to the 5 'and/or 3' end of the sense strand of the RNA silencing agent of the invention. In certain embodiments, the functional moiety is attached to the 3' end of the sense strand of the RNA silencing agent of the invention.

In certain embodiments, the functional moiety is linked to the RNA silencing agent through a linker. In certain embodiments, the functional moiety is linked to the antisense strand and/or sense strand by a linker. In certain embodiments, the functional moiety is attached to the 3' end of the sense strand by a linker. In certain embodiments, the linker comprises a divalent or trivalent linker. In certain embodiments, the linker comprises a glycol chain, alkyl chain, peptide, RNA, DNA, phosphodiester, phosphorothioate, phosphoramidate, amide, carbamate, or a combination thereof. In certain embodiments, the divalent or trivalent linker is selected from:

/>wherein n is 1, 2, 3, 4 or 5.

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:

Wherein X is O, S or BH ₃ 。

The various functional moieties of the present disclosure and methods of conjugating them to RNA silencing agents are described in further detail in WO2017/030973A1 and WO2018/031933A2, which are incorporated herein by reference.

VI branched oligonucleotides

Two or more RNA silencing agents as disclosed above, e.g., oligonucleotide constructs such as anti-IFNGR 1, anti-JAK 2, or anti-STAT 1 siRNA, may be interconnected by one or more moieties independently selected from the group consisting of a linker, a spacer, and a branching point to form a branched oligonucleotide RNA silencing agent. In certain embodiments, the branched oligonucleotide RNA silencing agent consists of two sirnas to form a dual branched siRNA ("dual siRNA") scaffold for delivering the two sirnas. In representative embodiments, the nucleic acids of the branched oligonucleotides each comprise an antisense strand (or portion thereof), wherein the antisense strand has sufficient complementarity to a target mRNA (e.g., IFNGR1, JAK2, or STAT1 mRNA) to mediate an RNA-mediated silencing mechanism (e.g., RNAi).

In exemplary embodiments, the branched oligonucleotides may have two to eight RNA silencing agents linked by a linker. The linker may be hydrophobic. In one embodiment, the branched oligonucleotides of the present application have two to three oligonucleotides. In one embodiment, the oligonucleotides independently have significant chemical stability (e.g., at least 40% of the constituent bases are chemically modified). In one exemplary embodiment, the oligonucleotide has complete chemical stability (i.e., all constituent bases are chemically modified). In some embodiments, the branched oligonucleotide comprises one or more single stranded phosphorothioate tails, each independently having between two and twenty nucleotides. In one non-limiting embodiment, each single-stranded tail has two to ten nucleotides.

In certain embodiments, the branched oligonucleotides are characterized by three properties: (1) branching structure, (2) complete metabolic stability, and (3) the presence of a single-stranded tail comprising a phosphorothioate linker. In certain embodiments, the branched oligonucleotide has 2 or 3 branches. It is believed that the increased overall size of the branched structure promotes increased absorption. Furthermore, without being bound by a particular theory of activity, multiple adjacent branches (e.g., 2 or 3) are believed to allow each branch to act in concert, thereby significantly increasing the rate of internalization, translocation, and release.

Branched oligonucleotides are provided in various structurally distinct embodiments. In some embodiments, the nucleic acids linked at the branch point are single-stranded or double-stranded and consist of miRNA inhibitors, interstitials, mixtures, SSOs, PMOs, or PNAs. These single strands may be linked at their 3 'or 5' ends. The combination of siRNA and single stranded oligonucleotide can also be used for dual functions. In another embodiment, short nucleic acids complementary to the interstitials, the mixture, the miRNA inhibitors, the SSO, the PMO and the PNA 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-37 ℃) for rapid dissociation upon internalization of the branched structure into the cell.

The double siRNA branched oligonucleotides may comprise chemically diverse conjugates, such as the functional moieties described above. Conjugated bioactive ligands can be used to enhance cell specificity and promote membrane binding, internalization, and serum protein binding. Examples of biologically active moieties for conjugation include DHA, galNAc, and cholesterol. These moieties may be linked to the Di-siRNA by a linker or spacer, or added by an additional linker or spacer linked to the end of another free siRNA.

The presence of branched structures increases tissue retention levels in various tissues (e.g., skin) compared to non-branched compounds of the same chemical composition. The branched oligonucleotides are unexpectedly uniformly distributed throughout the tissue.

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

Joint

In one embodiment of the branched oligonucleotide, each linker is independently selected from the group consisting of a 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 substituted with a nitrogen atom, with a hydroxy substituent, or with an oxo substituent. In one embodiment, each linker is a glycol chain. In another embodiment, each linker is an alkyl chain. In another embodiment, each linker is a peptide. In another embodiment, each linker is RNA. In another embodiment, each linker is DNA. In another embodiment, each linker is a phosphate. In another embodiment, each linker is a phosphonate. In another embodiment, each linker is a phosphoramidate. In another embodiment, each linker is an ester. In another embodiment, each linker is an amide. In another embodiment, each linker is a triazole.

VII Compounds of formula (I)

In another aspect, provided herein are branched oligonucleotide compounds of formula (I):

L—(N) _n

(I)

wherein L is selected from the group consisting of ethylene glycol chains, alkyl chains, peptides, RNA, DNA, phosphate esters, phosphonate esters, phosphoramidates, esters, amides, triazoles, and combinations thereof, wherein formula (I) optionally further comprises one or more branching points B, and one or more spacers S; wherein B is independently at each occurrence a multivalent organic species or derivative thereof; s is independently selected at each occurrence from the group consisting of ethylene glycol chains, alkyl chains, peptides, RNA, DNA, phosphate esters, phosphonate esters, phosphoramidates, esters, amides, triazoles, and combinations thereof.

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

In one embodiment, the compound of formula (I) has a structure selected from formulas (I-1) - (I-9) of Table 1.

TABLE 1

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

In one embodiment of the compound of formula (I), each linker is independently selected from the group consisting of ethylene glycol chain, alkyl chain, peptide, RNA, DNA, phosphate, phosphonate, phosphoramidate, ester, amide, triazole, and combinations thereof; wherein any carbon or oxygen atom of the linker is optionally substituted with a nitrogen atom, with a hydroxy substituent, or with an oxo substituent. In one embodiment of the compounds of formula (I), each linker is an ethylene glycol chain. In another embodiment, each linker is an alkyl chain. In another embodiment of the compounds of formula (I), each linker is a peptide. In another embodiment of the compounds of formula (I), each linker is RNA. In another embodiment of the compounds of formula (I), each linker is DNA. In another embodiment of the compounds of formula (I), each linker is a phosphate. In another embodiment, each linker is a phosphonate. In another embodiment of the compounds of formula (I), each linker is a phosphoramidate. In another embodiment of the compounds of formula (I), each linker is an ester. In another embodiment of the compounds of formula (I), each linker is an amide. In another embodiment of the compounds of formula (I), each linker is a triazole.

In one embodiment of the compounds of formula (I), B is a multivalent organic species. In another embodiment of the compounds of formula (I), B is a derivative of a multivalent organic species. In one embodiment of the compounds of formula (I), B is a triol or tetraol derivative. In another embodiment, B is a tricarboxylic acid or tetracarboxylic acid derivative. In another embodiment, B is an amine derivative. In another embodiment, B is a triamine or tetramine derivative. In another embodiment, B is an amino acid derivative. In another embodiment of the compounds of formula (I), B is selected from the following formulae:

the multivalent organic species is a moiety comprising carbon and three or more valences (i.e., a point of attachment to a S, L or N moiety, etc., as defined above). Non-limiting examples of multivalent organic species include triols (e.g., glycerol, phloroglucinol, and the like), tetraols (e.g., ribose, pentaerythritol, 1,2,3, 5-tetrahydroxybenzene, and the like), tricarboxylic acids (e.g., citric acid, 1,3, 5-cyclohexane tricarboxylic acid, pyromellitic acid, and the like), tetracarboxylic acids (e.g., ethylenediamine tetraacetic acid, pyromellitic acid, and the like), tertiary amines (e.g., tripropylamine, triethanolamine, and the like), triamines (e.g., diethylenetriamine, and the like), tetramines, and species comprising a combination of hydroxyl, thiol, amino, and/or carboxyl moieties (e.g., amino acids, e.g., lysine, serine, cysteine, and the like).

In one embodiment of the compounds of formula (I), each nucleic acid comprises one or more chemically modified nucleotides. In one embodiment of the compounds of formula (I), each nucleic acid consists of chemically modified nucleotides. In certain embodiments of the compounds of formula (I) >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55% or >50% of each nucleic acid comprises a chemically modified nucleotide.

In one embodiment, each antisense strand independently comprises a 5' terminal group R selected from the group of table 2.

TABLE 2

/>

In one embodiment, R is R ₁ . In another embodiment, R is R ₂ . In another embodiment, R is R ₃ . In another embodiment, R is R ₄ . In another embodiment, R is R ₅ . In another embodiment, R is R ₆ . In another embodiment, R is R ₇ . In another embodiment, R is R ₈ 。

Structure of formula (II)

In one embodiment, the compound of formula (I) has the structure of formula (II):

wherein X is independently at each occurrence selected 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 phosphorothioate internucleoside linkages; and each occurrence represents a base pairing interaction or mismatch, respectively.

In certain embodiments, the structure of formula (II) does not comprise a mismatch. In one embodiment, the structure of formula (II) comprises 1 mismatch. In another embodiment, the compound of formula (II) comprises 2 mismatches. In another embodiment, the compound of formula (II) comprises 3 mismatches. In another embodiment, the compound of formula (II) comprises 4 mismatches. In one embodiment, each nucleic acid consists of chemically modified nucleotides.

In certain embodiments >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55%, or >50% of the X' of the structure of formula (II) is a chemically modified nucleotide. In other embodiments >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55%, or >50% of the X' of the structure of formula (II) is a chemically modified nucleotide.

Structure of formula (III)

In one embodiment, the compound of formula (I) has the structure of formula (III):

wherein X is independently at each occurrence a nucleotide comprising a 2 '-deoxy-2' -fluoro modification; x is independently at each occurrence a nucleotide comprising a 2' -O-methyl modification; y is independently at each occurrence a nucleotide comprising a 2 '-deoxy-2' -fluoro modification; and Y is independently at each occurrence a nucleotide comprising a 2' -O-methyl modification.

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.

In certain embodiments, the structure of formula (III) does not comprise a mismatch. In one embodiment, the structure of formula (III) comprises 1 mismatch. In another embodiment, the compound of formula (III) comprises 2 mismatches. In another embodiment, the compound of formula (III) comprises 3 mismatches. In another embodiment, the compound of formula (III) comprises 4 mismatches.

Structure of formula (IV)

In one embodiment, the compound of formula (I) has the structure of formula (IV):

In certain embodiments, the structure of formula (IV) does not comprise a mismatch. In one embodiment, the structure of formula (IV) comprises 1 mismatch. In another embodiment, the compound of formula (IV) comprises 2 mismatches. In another embodiment, the compound of formula (IV) comprises 3 mismatches. In another embodiment, the compound of formula (IV) comprises 4 mismatches. In one embodiment, each nucleic acid consists of chemically modified nucleotides.

In certain embodiments >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55%, or >50% of the X' of the structure of formula (IV) is a chemically modified nucleotide. In other embodiments >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55% or >50% of the X' of the structure of formula (IV) is a chemically modified nucleotide.

Structure of (V)

In one embodiment, the compound of formula (I) has the structure of formula (V):

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.

In certain embodiments, the structure of formula (V) does not comprise a mismatch. In one embodiment, the structure of formula (V) comprises 1 mismatch. In another embodiment, the compound of formula (V) comprises 2 mismatches. In another embodiment, the compound of formula (V) comprises 3 mismatches. In another embodiment, the compound of formula (V) comprises 4 mismatches.

Variable joint

In one embodiment of the compounds of formula (I), L has the structure of L1:

in one embodiment of L1, R is R ³ And n is 2.

In one embodiment of the structure of formula (II), L has the structure of L1. In one embodiment of the structure of formula (III), L has the structure of L1. In one embodiment of the structure of formula (IV), L has the structure of L1. In one embodiment of the structure of formula (V), L has the structure of L1. In one embodiment of the structure of formula (VI), L has the structure of L1. In one embodiment of the structure of formula (VI), L has the structure of L1.

In one embodiment of the structure of formula (I), L has the structure of L2:

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

Delivery system

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

L—(cNA) _n

(VI)

wherein L is selected from the group consisting of ethylene glycol chains, alkyl chains, peptides, RNA, DNA, phosphate esters, phosphonate esters, phosphoramidates, esters, amides, triazoles, and combinations thereof, wherein formula (VI) optionally further comprises one or more branching points B, and one or more spacers S; wherein B is independently at each occurrence a multivalent organic species or derivative thereof; s is independently selected at each occurrence from the group consisting of ethylene glycol chains, alkyl chains, peptides, RNA, DNA, phosphate esters, phosphonate esters, phosphoramidates, esters, amides, triazoles, and combinations thereof; each cNA is independently a vector nucleic acid comprising one or more chemical modifications; n is 2, 3, 4, 5, 6, 7 or 8.

In one embodiment of the delivery system, L is a 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 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 triazole.

In one embodiment of the delivery system, S is a 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 triazole.

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.

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

In one embodiment, the compound of formula (VI) has a structure selected from formulas (VI-1) - (VI-9) of Table 3:

TABLE 3 Table 3

/>

In one embodiment, the compound of formula (VI) has the structure of formula (VI-1). In one embodiment, the compound of formula (VI) has the structure of formula (VI-2). In one embodiment, the compound of formula (VI) has the structure of formula (VI-3). In one embodiment, the compound of formula (VI) has the structure of formula (VI-4). In one embodiment, the compound of formula (VI) has the structure of formula (VI-5). In one embodiment, the compound of formula (VI) has the structure of formula (VI-6). In one embodiment, the compound of formula (VI) has the structure of formula (VI-7). In one embodiment, the compound of formula (VI) has the structure of formula (VI-8). In one embodiment, the compound of formula (VI) has the structure of formula (VI-9).

In one embodiment, the compounds of formula (VI) (including, for example, formulas (VI-1) - (VI-9)), each cNA independently comprises at least 15 consecutive nucleotides, in one embodiment, each cNA independently consists of chemically modified nucleotides.

In one embodiment, the delivery system further comprises n therapeutic Nucleic Acids (NA), wherein each NA comprises a sequence that is substantially complementary to the IFNGR1, JAK2, or STAT1 nucleic acid sequence of any of SEQ ID NOs 1-6, as described in tables 6 and 8. In other embodiments, NA comprises a strand capable of targeting one or more of the IFNGR1, JAK2 or STAT1 nucleic acid sequences selected from the group consisting of SEQ ID NOS: 143-154, as set forth in tables 7, 9, 10 and 11, respectively.

In addition, each NA hybridizes to at least one cNA. In one embodiment, the delivery system consists of 2 NA. In another embodiment, the delivery system consists of 3 NA. In another embodiment, the delivery system consists of 4 NA. In another embodiment, the delivery system consists of 5 NA. In another embodiment, the delivery system consists of 6 NA. In another embodiment, the delivery system consists of 7 NA. In another embodiment, the delivery system consists of 8 NA.

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

In one embodiment, 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 one embodiment, the overhanging nucleotides are linked by phosphorothioate linkages.

In one embodiment, each NA is independently selected from the group consisting of: DNA, siRNA, miRNA antagonists, mirnas, interstitials, mixtures or guide RNAs. In one embodiment, each NA is independently DNA. In another embodiment, each NA is independently an siRNA. In another embodiment, each NA is independently a miRNA antagonist. In another embodiment, each NA is independently a miRNA. In another embodiment, each NA is independently a spacer. In another embodiment, each NA is independently a mixture. In another embodiment, each NA is independently a guide RNA. In one embodiment, each NA is the same. In one embodiment, each NA is different.

In one embodiment, the delivery system further comprising n therapeutic Nucleic Acids (NA) has a structure selected from the group consisting of formulas (I), (II), (III), (IV), (V), (VI), and embodiments described herein. In one embodiment, the delivery system has a structure selected from formulas (I), (II), (III), (IV), (V), (VI) and embodiments described herein, further comprising 2 therapeutic Nucleic Acids (NA). In another embodiment, the delivery system has a structure selected from formulas (I), (II), (III), (IV), (V), (VI) and embodiments described herein, further comprising 3 therapeutic Nucleic Acids (NA). In one embodiment, the delivery system has a structure selected from formulas (I), (II), (III), (IV), (V), (VI) and embodiments described herein, further comprising 4 therapeutic Nucleic Acids (NA). In one embodiment, the delivery system has a structure selected from formulas (I), (II), (III), (IV), (V), (VI) and embodiments described herein, further comprising 5 therapeutic Nucleic Acids (NA). In one embodiment, the delivery system has a structure selected from formulas (I), (II), (III), (IV), (V), (VI) and embodiments described herein, further comprising 6 therapeutic Nucleic Acids (NA). In one embodiment, the delivery system has a structure selected from formulas (I), (II), (III), (IV), (V), (VI) and embodiments described herein, further comprising 7 therapeutic Nucleic Acids (NA). In one embodiment, the delivery system has a structure selected from formulas (I), (II), (III), (IV), (V), (VI) and embodiments described herein, further comprising 8 therapeutic Nucleic Acids (NA).

In one embodiment, the delivery system has a structure selected from the group consisting of formulas (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 the group consisting of formulas (I), (II), (III), (IV), (V), (VI) and further comprises a linker of structure L1 wherein R is R ³ And n is 2. In another embodiment, the delivery system has a structure selected from the group consisting of formulas (I), (II), (III), (IV), (V), (VI) and further comprises a linker of structure L2 wherein R is R ³ And n is 2.

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

In certain embodiments, the compounds of the invention are characterized by the following properties: (1) Two or more branched oligonucleotides, e.g., wherein there are unequal numbers of 3 'and 5' ends; (2) Substantially chemically stable, e.g., wherein more than 40%, optimally 100% of the oligonucleotides are chemically modified (e.g., without RNA and optionally without DNA); and (3) phosphorothioate monooligonucleotides containing at least 3 phosphorothioate linkages. In certain embodiments, phosphorothioate monooligonucleotides contain 4 to 20 phosphorothioate linkages.

It is to be understood that the methods described in this disclosure are not limited to the specific methods and experimental conditions disclosed herein; as 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.

Furthermore, unless otherwise indicated, the experiments described herein employ conventional molecular and cellular biology and immunological techniques within the skill of the art. Such techniques are well known to the skilled person and are well explained in the literature. See, e.g., ausubel et al, current Protocols in Molecular Biology, john Wiley & Sons, inc., NY (1987-2008), including all appendices; MR Green and J.Sambrook et al Molecular Cloning: A Laboratory Manual (fourth edition), antibodies: A Laboratory Manual, chapter 14, cold Spring Harbor Laboratory, cold Spring Harbor (2013, 2 nd edition).

Branched oligonucleotides, including methods of synthesis and use, are described in more detail in WO2017/132669, which is incorporated herein by reference.

Methods for introducing nucleic acids, vectors and host cells

The RNA silencing agents of the invention can be introduced directly into cells (e.g., skin cells) (i.e., intracellular); or introduced extracellularly into the cavity, interstitial space, into the circulation of the organism, orally, or may be introduced by immersing the cell or organism in a solution containing the nucleic acid. Vascular or extravascular circulation, the blood or lymphatic system, and cerebrospinal fluid are sites where nucleic acids can be introduced.

The RNA silencing agents of the invention can be introduced using nucleic acid delivery methods known in the art, including injection of a solution containing the nucleic acid, bombardment with particles covered with nucleic acid, immersing the cell or organism in a solution of the nucleic acid, or electroporation of the cell membrane in the presence of the nucleic acid. Other methods known in the art for introducing nucleic acids into cells, such as lipid-mediated carrier transport, chemical-mediated transport, and cationic liposome transfection, such as calcium phosphate, and the like, may be used. The nucleic acid may be introduced with other components that perform one or more of the following activities: enhancing uptake of nucleic acid by the cell or otherwise increasing inhibition of the target gene.

Physical methods of introducing nucleic acids include injection of a solution containing RNA, bombardment with particles covered with RNA, immersing cells or organisms in the RNA solution, or electroporation of cell membranes in the presence of RNA. The viral construct packaged into a viral particle will allow for efficient introduction of the expression construct into a cell and transcription of the RNA encoded by the expression construct. Other methods known in the art for introducing nucleic acids into cells may be used, such as lipid-mediated carrier transport, chemical-mediated transport such as calcium phosphate, and the like. Thus, RNA may be introduced with components that perform one or more of the following activities: enhancing cellular uptake of RNA, inhibiting single strand annealing, stabilizing single strands, or otherwise increasing inhibition of a target gene.

The RNA can be introduced directly into the cell (i.e., intracellular); or extracellular introduction into cavities, interstitial spaces, into the circulation of organisms, orally, or may be introduced by immersing cells or organisms in a solution containing RNA. Vascular or extravascular circulation, the blood or lymphatic system, and cerebrospinal fluid are sites where RNA can be introduced.

Cells having a target gene may be from germ line or somatic cells, pluripotent or multipotent, dividing or non-dividing, parenchymal or epithelial, immortalized or transformed, and the like. The cells may be stem cells or differentiated cells. Differentiated cell types include adipocytes, fibroblasts, myocytes, cardiomyocytes, endothelial cells, neurons, glia cells, blood cells, megakaryocytes, lymphocytes, macrophages, neutrophils, eosinophils, basophils, mast cells, leukocytes, granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts, hepatocytes, and endocrine or exocrine gland cells.

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. Reducing or losing gene expression in at least 50%, 60%, 70%, 80%, 90%, 95% or 99% or more of the target cells is exemplary. Inhibition of gene expression refers to the absence (or observable decrease) of protein and/or mRNA product levels from the target gene. Specificity refers to the ability to inhibit a target gene without significantly affecting other genes of the cell. The consequences of inhibition can be confirmed by examining the extrinsic properties 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 using microarrays, antibody binding, enzyme-linked immunosorbent assay (ELISA), western blot, radioimmunoassay (RIA), other immunoassays, and Fluorescence Activated Cell Sorting (FACS).

For RNA-mediated inhibition in a cell line or whole organism, gene expression can be conveniently determined by using a reporter gene or drug resistance gene whose protein product is readily determined. Such reporter genes include acetohydroxyacid synthase (AHAS), alkaline Phosphatase (AP), beta-galactosidase (LacZ), beta-Glucuronidase (GUS), chloramphenicol Acetyl Transferase (CAT), green Fluorescent Protein (GFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase (NOS), octopine synthase (OCS), and derivatives thereof. There are a variety of selectable markers that confer resistance to ampicillin, bleomycin, chloramphenicol, gentamicin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin and tetracycline. Based on the assay, quantification of gene expression allows one to determine the extent of inhibition of greater than 10%, 33%, 50%, 90%, 95% or 99% compared to cells not treated according to the invention. A lower dose of the injectable material and a longer period of time after RNAi agent administration may result in a smaller fraction of cells being inhibited (e.g., at least 10%, 20%, 50%, 75%, 90% or 95% of target cells). Quantification of gene expression in cells may exhibit similar amounts of inhibition at the accumulation of target mRNA or at the level of translation of target protein. For example, inhibition efficiency can be determined by assessing the amount of gene product in a cell; mRNA can be detected with hybridization probes having nucleotide sequences outside the region for inhibitory double-stranded RNA, or translated polypeptides can be detected with antibodies raised against the polypeptide sequences of the region.

The RNA can be introduced in an amount that allows for at least one copy to be delivered per cell. Higher doses (e.g., at least 5, 10, 100, 500, or 1000 copies per cell) of material can produce more effective inhibition; lower doses may also be useful for specific applications.

In one exemplary aspect, the RNAi agents of the invention (e.g., sirnas targeting IFNGR1, JAK2, or STAT1 target sequences) are tested for efficacy to determine their ability to specifically degrade mutant mRNA (e.g., IFNGR1, JAK2, or STAT1 mRNA and/or production of IFNGR1, JAK2, or STAT1 protein) in a cell (such as a keratinocyte). Other cells that can be easily transfected are also suitable for cell-based validation assays, such as HeLa cells or COS cells. Cells are transfected with human wild-type or mutant cdnas (e.g., human wild-type or mutant IFNGR1, JAK2, or STAT1 cdnas). Co-transfecting standard siRNA, modified siRNA or a vector capable of producing siRNA from U-ring mRNA. Measurement of a decrease in selectivity of target mRNA (e.g., IFNGR1, JAK2, or STAT1 mRNA) and/or target protein (e.g., IFNGR1, JAK2, or STAT1 protein). The decrease in 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, JAK2, or STAT1 mRNA. For comparison purposes, exogenously introduced mRNA or protein (or endogenous mRNA or protein) can be analyzed. When using neuronal cells known to be resistant to standard transfection techniques, it may be desirable to introduce RNAi agents (e.g., siRNA) by passive uptake.

Recombinant adeno-associated virus and vector

In certain exemplary embodiments, recombinant adeno-associated virus (rAAV) and related vectors thereof can be used to deliver one or more siRNA into a cell (e.g., a skin cell). AAV is capable of infecting many different cell types, although the efficiency of infection varies from serotype to serotype, with serotypes being determined by the sequence of the capsid protein. Several natural AAV serotypes have been identified, with serotypes 1-9 being the most commonly used 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 are generated by DNA shuffling of multiple AAV serotypes to produce AAV with mixed capsids that increase transduction efficiency in a variety of cells and tissues, both in vitro (AAV-DJ) and in vivo (AAV-DJ/8).

The rAAV may be delivered to the subject in the form of a composition according to any suitable method known in the art. The rAAV can be suspended in a physiologically compatible carrier (i.e., in a composition) and can be 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), and the like. In certain embodiments, the host animal is a non-human host animal.

One or more rAAV can be delivered to a mammalian subject, for example, by intramuscular injection or by administration into the blood stream of the mammalian subject. May be administered into the blood stream by injection into a vein, artery or any other vascular conduit. In certain embodiments, the one or more rAAV is administered into the blood stream by isolated limb perfusion, a technique well known in the surgical arts, which essentially enables the skilled artisan to isolate the limb from the systemic circulation prior to administration of the rAAV virions. Variations of the isolated limb perfusion technique described in U.S. patent No. 6,177,403 can also be used by the skilled artisan to apply viral particles into the vasculature of the isolated limb to potentially enhance transduction to muscle cells or tissue.

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

An effective amount of rAAV is an amount sufficient to target an infected animal, targeting the desired tissue. In some embodiments, an effective amount of a rAAV is an amount sufficient to produce a stable somatic transgenic animal model. The effective amount will depend primarily on species, age, weight, health of the subject, and the tissue to be targeted, among other factors, and thus may vary from animal to animal and tissue. For example, an effective amount of one or more rAAV is typically in the range of about 1ml to about 100ml of a solution containing about 10 ⁹ To 10 ¹⁶ And each genome copy. In some cases, about 10 ¹¹ To 10 ¹² Dosages between individual copies of the rAAV genome are appropriate. In certain embodiments, 10 ¹² The rAAV genome copies are effective for targeting heart, liver and pancreatic tissue. In certain instances, the stable transgenic animals are produced from multiple doses of rAAV.

In some embodiments, the rAAV compositions are formulated to reduce aggregation of AAV particles in the composition, particularly in the presence of high rAAV concentrations (e.g., about 10 ¹³ Genome copies/mL or more). Methods for reducing rAAV aggregation are well known in the art and include, for example, adding surfactants, adjusting pH, adjusting salt concentration, and the like (see, e.g., wright et al (2005) Molecular Therapy 12:171-178, the contents of which are incorporated herein by reference).

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 that 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 the regulatory component in a manner that allows the transgene to be transcribed, translated, and/or expressed in cells of the target tissue.

AAV sequences of vectors typically comprise cis-acting 5 'and 3' Inverted Terminal Repeat (ITR) sequences (see, e.g., b.j. Carter, in "Handbook of Parvoviruses", p.tijsser edit, CRC Press, page 155 168 (1990)). The ITR sequence is typically about 145 base pairs in length. In certain embodiments, substantially complete sequences encoding ITRs are used in the molecule, although some minor modifications to these sequences are permitted. The ability to modify these ITR sequences is within the skill of the art. (see, e.g., text such as Sambrook et al, "Molecular cloning. A Laboratory Manual", 2 nd edition, cold Spring Harbor Laboratory, new York (1989); and K.Fisher et al, J Virol.,70:520532 (1996)). An example of such a molecule for use in the present invention is a "cis-acting" plasmid containing a transgene, wherein the selected transgene sequence and associated regulatory elements flank the 5 'and 3' aav ITR sequences. AAV ITR sequences can be obtained from any known AAV, including mammalian AAV types described further herein.

VIII method of treatment

In one aspect, the invention provides prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) vitiligo associated with IFN-gamma signaling. In one embodiment, the disease or disorder is a disease or disorder in which IFNGR1, JAK2 or STAT1 mediates IFN- γ signaling involved in the pathogenesis of vitiligo. In a certain embodiment, the disease or disorder is one in which a reduction in IFNGR1, JAK2 or STAT1 reduces the clinical manifestations observed in vitiligo and potentially other diseases.

As used herein, "treatment" or "treating" is defined as the application or administration of a therapeutic agent (e.g., an RNA agent or vector or transgene encoding the same) to a patient, or to an isolated tissue or cell line from a patient suffering from a disease or disorder, a symptom of a disease or disorder, or a susceptibility to a disease or disorder, for the purpose of curing, treating, alleviating, altering, remediating, improving, ameliorating, or affecting a disease or disorder, a symptom of a disease or disorder, or a predisposition to a disease.

In one aspect, the invention provides a method for preventing a disease or disorder as described above in a subject by administering a therapeutic agent (e.g., an RNAi agent or vector or transgene encoding the same) to the subject. Subjects at risk for disease may be identified by, for example, any of the diagnostic or prognostic assays described herein, or a combination thereof. Administration of the prophylactic agent may occur prior to the manifestation of a symptom characteristic of the disease or disorder, thereby preventing the disease or disorder, or delaying its progression.

Another aspect of the invention relates to a method of therapeutically treating a subject, i.e., altering the onset of symptoms of a disease or disorder. In one exemplary embodiment, the modulation methods of the invention comprise contacting an immune cell expressing IFNGR1, JAK2, or STAT1 with a therapeutic agent specific for a target sequence within the gene (e.g., IFNGR1, JAK2, or STAT1 target sequences of tables 6 and 8), thereby effecting sequence-specific interference with the gene. These methods can be performed in vitro (e.g., by culturing cells with an agent) or in vivo (e.g., by administering an agent to a subject).

IX. pharmaceutical compositions and methods of administration

The present invention relates to the use of the above agents for prophylactic and/or therapeutic treatment as described below. Thus, modulators (e.g., RNAi agents) of the invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise a nucleic acid molecule, protein, antibody or regulatory compound and a pharmaceutically acceptable carrier. As used herein, the phrase "pharmaceutically acceptable carrier" refers to 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. Unless any conventional medium or agent is incompatible with the active compound, it is contemplated that it will be used in the composition. Supplementary active compounds may also be incorporated into the compositions.

The pharmaceutical compositions of the present invention are formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral administration, such as intravenous, intradermal, subcutaneous, intraperitoneal, intramuscular, oral (e.g., inhalation), transdermal (topical), and transmucosal administration. In certain embodiments, the intended use is transdermal (topical).

The nucleic acid molecules of the invention may be inserted into an expression construct, such as a viral vector, retroviral vector, expression cassette or plasmid viral vector, for example using methods known in the art, including but not limited to, xia et al (2002), as described above. The expression construct may be delivered to the subject by, for example, inhalation, oral administration, intravenous injection, topical administration (see U.S. Pat. No. 5,328,470), or by stereotactic injection (see, for example, chen et al (1994), proc. Natl. Acad. Sci. USA,91,3054-3057). The pharmaceutical formulation of the delivery vehicle may include the vehicle in an acceptable diluent, or may include a slow release matrix embedded in the delivery vehicle. Alternatively, the pharmaceutical formulation may include one or more cells that produce the gene delivery system, as the complete delivery vector may be produced intact from recombinant cells (e.g., retroviral vectors).

The nucleic acid molecules of the invention may also include small hairpin RNAs (shrnas), as well as expression constructs engineered to express shrnas. Transcription of the shRNA starts with the polymerase III (PolIII) promoter and is thought to terminate at position 2 of the 4-5-thymine transcription termination site. Upon expression, shRNA is thought to fold into a stem-loop structure with a 3' uu overhang; subsequently, the ends of these shRNA are processed to convert the shRNA into an siRNA-like molecule of about 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) is as above; yu et al (2002), supra.

The expression construct may be any construct suitable for use in a suitable expression system and includes, but is not limited to, retroviral vectors, linear expression cassettes, plasmids, and viral 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 comprise one or two siRNA strands. Expression constructs expressing both strands may also include a loop structure linking the two strands, or each strand may be transcribed separately from a different promoter in the same construct. Each strand may also be transcribed from a separate expression construct, tuschl (2002), supra.

For example, a composition may comprise one or more compounds of the invention and a pharmaceutically acceptable carrier. The pharmaceutical compositions of the present invention may be administered in a variety of ways depending on whether local or systemic treatment is desired and on the area to be treated. Administration may be topical (including ocular, intranasal, transdermal) administration, oral administration, or parenteral administration. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, intrathecal or intraventricular (e.g., intraventricular) administration.

The route of delivery may depend on the condition of the patient. For example, a subject diagnosed with vitiligo may be administered an anti-IFNGR 1, anti-JAK 2, or anti-STAT 1 compound of the invention directly to the skin. In addition to the compounds of the invention, a second treatment, such as palliative treatment and/or disease-specific treatment, may be administered to the patient. The second treatment may be, for example, symptomatic (e.g., for symptomatic relief) or restorative (e.g., for reversal of the disease process).

Lipid Nanoparticle (LNP) formulation

The RNA silencing agents of the present disclosure can be formulated in Lipid Nanoparticles (LNPs). LNP refers to lipid vesicles that coat an aqueous interior, which may comprise nucleic acids such as RNAi silencing agents or plasmids from which RNAi silencing agents are transcribed. LNP typically contains at least one cationic lipid, at least one non-cationic lipid, a lipid that prevents aggregation of particles (e.g., PEG-lipid conjugate), and optionally cholesterol or derivatives thereof.

The cationic lipid may be, for example, N, N-dioleyl-N, N-dimethylammonium chloride (DODAC), N, N-distearyl-N, N-dimethylammonium bromide (DDAB), N- (I- (2, 3-dioleyloxy) 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-dioleyloxy-N, N-dimethylaminopropane (DLINDMA), 1, 2-dioleyloxy-N, N-dimethylaminopropane (DLenDMA), 1, 2-dioleoylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1, 2-dioleyloxy-3- (dimethylamino) acetylpropane (DLin), 1, 2-dioleyloxy-3-dioleyloxy-propan-1, 2-dioleyloxy-2, 3-dioleyloxy-propan-DLN, N-dimethylaminopropane (DLIn DMA), 1, 2-dioleyloxy-3-dioleyloxy-propan (DLN, N-D-N-Dimethylaminopropane (DLA) 1, 2-dioleoyloxy-3-trimethylaminopropane chloride (DLin-TMA. Cl), 1, 2-dioleoyloxy-3-trimethylaminopropane chloride (DLin-TAP. Cl), 1, 2-dioleoyloxy-3- (N-methylpiperazine) propane (DLin-MPZ), or 3- (N, N-dioleoylamino) -1, 2-propanediol (DLinaP), 3- (N, N-dioleoylamino) -1, 2-propanediol (DOAP), 1, 2-dioleoyloxy-3- (2-N, N-dimethylamino) ethoxypropane (DLin-EG-DMA), 1, 2-dioleoyloxy-N, N-dimethylaminopropane (DLinDMA), 2-diimine-4-dimethylaminomethyl- [1,3] -dioxolane (DLin-K-DMA) or analogues thereof, (3 aR,5s,6 aS) -N, N-dimethyl-2, 2-bis ((9Z, 12Z) -octadeca-9, 12-dienyl) tetrahydro-3 aH-cyclopenta [ d ] [1,3] dioxin-5-amine (ALN 100), (6Z, 9Z,28Z, 31Z) -hepta-hexa-enoic acid-6,9,28,31-tetraen-19-yl 4- (dimethylamino) butyrate (MC 3), 1,1' - (2- (4- (2- ((2- (bis (2-hydroxydodecyl) amino) ethyl) piperazin-1-yl) ethylazadiyl) dodecane-2-ol (Tech Gl), or a mixture thereof. The cationic lipid may comprise from about 20mol% to about 50mol% of the total lipid present in the particle.

The non-cationic lipid may be an anionic lipid or a neutral lipid including, but not limited to, distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl phosphatidylethanolamine (DOPE), palmitoyl Oleoyl Phosphatidylcholine (POPC), palmitoyl Oleoyl Phosphatidylethanolamine (POPE), dioleoyl phosphatidylethanolamine 4- (-maleimidomethyl) cyclohexane-l-carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), distearoyl phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl phosphatidylethanolamine (SOPE), cholesterol, and mixtures thereof.

The conjugated lipid that inhibits aggregation of particles may be, for example, a polyethylene glycol (PEG) -lipid, including but not limited to PEG-Diacylglycerol (DAG), PEG-Dialkoxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), or mixtures thereof. The PEG-DAA conjugate may be, for example, PEG-dilauroxypropyl (C12), PEG-dimyristoxypropyl (Ci 4), PEG-dipalmitoxypropyl (Ci 6) or PEG-distearoyloxypropyl (C ] s). Conjugated lipids that prevent aggregation of the particles may comprise from 0mol% to about 20mol% or about 2mol% of the total lipids present in the particles. In some embodiments, the nucleic acid-lipid particles further comprise, for example, about 10mol% to about 60mol% or about 48mol% cholesterol, based on total lipids present in the particles.

The LNPs of the invention typically have an average diameter of about 50nm to about 200nm, about 60nm to about 130nm, about 70nm to about 110nm, or about 60nm to about 80 nm. Furthermore, when present in LNP, the nucleic acids are resistant to degradation using nucleases in aqueous solutions.

In one embodiment, the lipid to drug ratio (mass/mass ratio; w/w ratio) (e.g., lipid to dsRNA ratio) will be within the following range: 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.

LNP formulations are further described, for example, in U.S. patent No. 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; in international application number WO 2009082817. These patents and applications are incorporated herein by reference in their entirety.

It will be apparent to those skilled in the art that other suitable modifications and adaptations to the methods described herein can be made using the appropriate equivalents without departing from the scope of the embodiments disclosed herein. Having now described certain embodiments in detail, the embodiments will be more clearly understood by reference to the following examples, which are included herein for purposes of illustration only and are not intended to be limiting of the invention.

Examples

Example 1 in vitro identification of ifngr1, JAK2 and STAT1 targeting sequences

IFNGR1, JAK2 and STAT1 genes were used as targets for mRNA knockdown. A set of sirnas targeting several different sequences of human and mouse IFNGR1, JAK2 or STAT1 mRNA were developed and screened in vitro in human HeLa cells and mouse N2A cells and compared to untreated control cells. The siRNAs were each tested at a concentration of 1.5. Mu.M and mRNA was assessed at the 72 hour time point using the QuantiGene gene expression assay (ThermoFisher, waltham, mass.). FIG. 1A depicts the results of evaluating 22 IFNGR1 siRNAs against human IFNGR1 mRNA in human HeLa cells. FIG. 1B depicts the results of evaluating the selection of 22 IFNGR1 siRNAs against mouse IFNGR1 mRNA in mouse N2A cells. FIG. 2A depicts the results of evaluating the selection of 24 JAK1 siRNAs against human JAK1 mRNA in human HeLa cells. FIG. 2B depicts the results of evaluating the selection of 24 JAK1 siRNAs against mouse JAK1 mRNA in mouse N2A cells. FIG. 3A depicts the results of evaluating the selection of 24 JAK2 siRNAs against human JAK2 mRNA in human HeLa cells. FIG. 3B depicts the results of evaluating the selection of 24 JAK2 siRNAs against mouse JAK2 mRNA in mouse N2A cells. Fig. 4A depicts the results of evaluating the screening of 24 STAT1 sirnas against human STAT1 mRNA in human HeLa cells. Fig. 4B depicts the results of evaluating the selection of 24 STAT1 sirnas against mouse STAT1 mRNA in mouse N2A cells.

Relative to untreated controls％Six sites were identified that produced potent and effective silencing of IFNGR1, JAK2 and STAT1 mRNA. Dose response curves for six identified siRNAs (oligonucleotides ID IFNGR1_1726, JAK1_3033, JAK2_1936, STAT1_885, ifngr1_1641 and Jak2_2076) are shown in FIGS. 5A-5H. Two of the 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. IFNGR1 protein expression was also tested in human HeLa cells and mouse N2a cells. Target(s)siRNA to ifngr1_1726 reduced IFNGR1 expression in HeLa cells and ifngr1-1641-targeting siRNA reduced IFNGR1 expression in N2a cells. Cells were treated with 1.5 μm of fully modified cholesterol conjugated siRNA for 72h (n=4, mean ± SD). Protein expression was determined by ELISA and normalized to total protein level (quantified by Bradford assay). Data are expressed as mean ± SD and analyzed by unpaired t-test (×p)<0.001，****p<0.0001 (fig. 10).

Additional human and mouse targets for IFNGR1 were tested in the dose response curve (1631, 1989 and 2072 in HeLa cells, and 378, 947 and 1162 in N2a cells). A 7-point dose response curve (n=3, mean ± SD) was generated by treating cells with 1.5 μm of fully modified cholesterol conjugated siRNA and stepwise 2-fold serial dilutions for 72 h. M represents the molar concentration of siRNA (n=3, mean ± SD). As shown in fig. 11, siRNA against the target was effective to silence human or mouse IFNGR1.

Tables 6 and 7 set forth the 45 nucleotide gene region and 20 nucleotide target sequence of the human IFNGR1, JAK2 and STAT1 target sequences tested in the screening and dose response curves described above, respectively. Tables 8 and 9 set forth the 45 nucleotide gene region and the 20 nucleotide target sequence of the mouse IFNGR1, JAK2 and STAT1 target sequences tested in the screening and dose response curves described above, respectively. The sense and antisense strands of the human IFNGR1, JAK2 and STAT1 siRNA duplex screened in fig. 1 are shown in table 10. The sense and antisense strands of the mouse IFNGR1, JAK2 and STAT1 siRNA duplex screened in fig. 2 are shown in table 11. Table 12 sets forth antisense and sense strands of 12 sirnas that result in potent and effective silencing of IFNGR1, JAK2, and STAT1 mRNA. The antisense strand contains 5' uracil to enhance loading into RISC and may or may not be complementary to the target IFNGR1, JAK2 and STAT1 mRNA sequences.

Tables 13-15 list modified sense and antisense strands of IFNGR1, JAK2, and STAT1 mRNA target sequences set forth in other embodiments.

Example 2.siRNA Ifngr1_1641 in vivo target protein knockdown

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 skin were measured by fluorescence flow cytometry. Fig. 6A shows the results of fluorescence flow cytometry, and fig. 6B shows summary data. Maximum 66% target protein knockdown was achieved 2 weeks after injection, and significant levels of protein knockdown were maintained for 4 weeks. These data indicate that a single dose of siRNA ifngr1_1641 provides a duration of action in skin of at least 4 weeks. The data also indicate that a dosing interval of 2 weeks can provide maximum target knockdown and rationalize the subsequent experiments as follows.

Example 3 in vitro skin culture model for testing inhibition of IFN-gamma signalling

To test the efficacy of siRNA ifngr1_1641 in inhibiting IFN- γ signaling, expression of chemokines CXCL9 and CXCL10 was measured in an ex vivo skin culture model. CXCL9 and CXCL10 are IFN- γ signaling downstream chemoattractants that are involved in recruiting cd8+ T cells to the skin and amplifying vitiligo autoimmunity. Knock down of the IFN- γ receptor IFNGR1 inhibits signal transduction, resulting in reduced expression of downstream CXCL9 and CXCL 10. FIG. 7A shows a procedure for testing the effect of Ifngr1_1641siRNA on IFN- γ signaling. At week 4 after 2 injections of 20mg/kg siRNA (dosing interval: 2 weeks, n=5 mice per group), 8 skin biopsies with a diameter of 4-mm were collected per mouse. Tail skin punctures (2-fold serial dilutions of 25600-400pg/mL, and untreated controls) were incubated in the presence of recombinant mouse IFN-gamma protein. CXCL9 and CXCL10 levels were measured by enzyme-linked immunosorbent assay (ELISA). Fig. 7B shows the result. Data are presented as mean ± SD and analyzed by two-way ANOVA with Dunnett multiple comparison test; * P <0.05. These data indicate that functional inhibition of IFN-gamma signaling at the protein level is achieved by target gene silencing. The siRNA used was conjugated to DCA and either scaffold 1 or scaffold 2 used, as shown below:

Bracket 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 'to 3'

(mU)#(mA)#(mG)(mC)(fU)(fA)(fA)(mU)(fA)(mC)(mU)(mA)(mA)#(mC)#(mA)(dT)(dT)-DCA

And (2) a bracket:

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 'to 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 = behenic acid

CXCL9, CXCL10 and CXCL11mRNA expression levels in HeLa cells and N2a cells were measured. Cells were treated with 1.5 μm siRNA targeting ifngr1_1726 and ifngr1_1641 for 72h (n=4, mean ± SD, one-way ANOVA, < p <0.05, < p <0.01, < p <0.001, < p <0.0001; ns, not significant) prior to IFN- γ stimulation. Samples were analyzed 6h after stimulation of IFN-gamma signaling. As shown in fig. 12, siRNA effectively reduced expression of CXCL9, 10 and 11 in the presence of IFN- γ signaling stimulation.

Example 4.siRNA Ifngr1_1641 systemic and local efficacy in vitiligo mouse model

In order to further improve the efficacy of siRNA targeting IFN-gamma signaling in treating vitiligo, a vitiligo mouse model is developed. Fig. 8A shows how PMEL cd8+ T cells isolated from spleens of PMEL TCR transgenic mice induced vitiligo by adoptive transfer. Subsequent activation of these T cells in recipient mice resulted in a patchy discoloration of the epidermis within 3-7 weeks, similar to vitiligo patients. Mice received a first dose of siRNA treatment 2 weeks prior to vitiligo induction and a second dose 1 week after induction. For efficacy evaluation, vitiligo scores were objectively quantified by an observer blinded to the treatment group, using a spot scale based on the extent of the decolorized areas at the ear and tail. Each site is advanced as a percentage of the anatomical site Performing row inspection; the left and right ears are both commonly defined and are therefore considered to be a single site. The score of vitiligo at each part is 0-5 as follows: no evidence of discoloration (0%) was scored as 0,>0 to 10% = 1 min,>10 to 25% = 2 minutes,>25% to 75% = 3 minutes,>75% to<100% = 4 min, and 100% = 5 min. Fig. 8B shows the result. Data are presented as mean ± SD, and by havingAnalyzing by using a two-way ANOVA of multiple comparison tests; * P (P)<0.05，**P<0.01，****P<0.0001。

Figure 9 shows a quantitative analysis of the level of tail discoloration between treatment groups. Fig. 9A shows objectively quantified skin depigmentation levels by comparing tail photographs using ImageJ Fiji software (NIH). In fig. 9B, the pixel intensity distribution curves of the respective tails are plotted for the total number of pixels at each intensity. Absolute white and black define intensities of 0 and 255, respectively. Fig. 9C plots the average pixel intensity for each tail. Statistical data are presented as mean ± SD of mean pixel intensities of individual distribution curves and analyzed by Mann-Whitney t-test; * P <0.05. Fig. 9D is a graph showing reduction of skin infiltration of cytotoxic T cells in both epidermis and dermis (as measured by cd45+ cells) with sirna ifngr1 1641 (unpaired T-test; × P <0.01 × P < 0.05).

These data indicate that siRNA ifngr1_1641 significantly prevented decolorization during vitiligo disease progression, consistent with the result of reduced tail vitiligo score.

These data indicate that siRNA ifngr1_1641 is capable of achieving systemic and local efficacy of vitiligo treatment, and that the platform technology is also likely to be applied to other disease gene targets of interest.

Example 5 siRNA targeting Ifngr1 in various chemical configurations

Ifngr1 silencing in mouse skin with sirnas targeting ifngr1_1641 with different chemical configurations was tested. FIG. 13A depicts a schematic representation of the chemical structure of hydrophobically conjugated (behenic acid, DCA; trimyritic acid, myr-t) and bivalent (Dio) siRNA; DCA and Myr-t conjugates are covalently linked to the 3' end of the sense strand; the two sense strands of the Dio scaffold are covalently linked by tetraethylene glycol; the study also included unconjugated siRNA ifngr1_1641 and DCA conjugated non-targeted control (NTC) siRNA. FIG. 13B depicts Ifngr1 mRNA silencing in skin at the injection site; mice (n=5 per group) were injected subcutaneously (between shoulders) with either a single dose of siRNA (20 mg/kg) or two doses (2 times, 24 hours apart; n=5); local skin was collected 1 week after injection and mRNA levels were measured using the QuantiGene 2.0 assay; ifngr1 expression is normalized to housekeeping gene Ppib; data are expressed as a percentage of PBS control (mean ± SD) and analyzed by Kruskal-Wallis test (p <0.05, < p <0.01; ns, not significant).

The data indicate that IFNGR1 silencing in all configurations tested is effective.

Table 5-dose response screening results for six sirnas that resulted in potent and effective silencing of IFNGR1, JAK2 and STAT1 mRNA.

TABLE 6 human IFNGR1, JAK2 and STAT1 Gene 45 nucleotide target sequences

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TABLE 7 human IFNGR1, JAK2 and STAT1 mRNA20 nucleotide target sequences

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TABLE 8 mouse IFNGR1, JAK2 and STAT1 Gene 45 nucleotide target sequences

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TABLE 9 mouse IFNGR1, JAK2 and STAT1 mRNA20 nucleotide target sequences

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Table 10-mouse IFNGR1, JAK2 and STAT1 siRNA sequences, which were used for the screens depicted in fig. 1-4.

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Table 11-mouse IFNGR1, JAK2 and STAT1 siRNA sequences, which were used for the screens depicted in fig. 1-4.

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Table 12-guide human and mouse IFNGR1, JAK2 and STAT1 siRNA sequences for dose response assays depicted in fig. 3 and 4.

Table 13-modified human IFNGR1, JAK2 and STAT1 mRNA targeting sequences, sense and antisense strands, additional embodiments.

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Table 14-modified IFNGR1, JAK2 and STAT1 mouse mRNA targeting sequences, sense and antisense strands, additional embodiments.

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Table 15-modified guide IFNGR1, JAK2 and STAT1 human and mouse mRNA targeting sequences, sense and antisense strands, additional embodiments.

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Incorporated by reference

The contents of all documents cited throughout this application, including references, patents, patent applications, and websites, are hereby expressly incorporated by reference, particularly for the references cited herein. Unless otherwise indicated, the present disclosure will employ conventional techniques of immunology, molecular biology, and cell biology, which are well known in the art.

The present disclosure is also incorporated by reference in its entirety into techniques well known in the fields of molecular biology and drug delivery. These techniques include, but are not limited to, those described in the following publications:

atwell et al J.mol.biol.1997,270:26-35;

ausubel et al (eds.), C _URRENT P _ROTOCOLS _IN M _OLECULAR B _IOLOGY ,John Wiley&Sons,NY(1993)；

Ausubel, F.M. et al, S _HORT P _ROTOCOLS I _N M _OLECULAR B _IOLOGY (4 th edition 1999) John Wiley&Sons,NY.(ISBN 0-471-32938-X)；

C _ONTROLLED D _RUG B _{IOAVAILABILITY} ,D _RUG P _RODUCT D _ESIGN _AND P _ERFORMANCE Smolen and Ball (ed.), wiley, new York (1984);

giege, R. and Ducruix, A.Barrett, C _{RYSTALLIZATION} _OF N _UCLEIC A _CIDS _AND P _ROTEINS A Practical Approach, 2 nd edition, pages 20-16, oxford University Press, new York, new York, (1999);

Goodson,in M _EDICAL A _PPLICATIONS _OF C _ONTROLLED R _ELEASE roll 2, pages 115-138 (1984);

hammerling et al, in: M _ONOCLONAL A _NTIBODIES _AND T-C _ELL HYBRIDOMAS 563-681(Elsevier,N.Y.,1981；

Harlow et al, A _NTIBODIES :A L _ABORATORY M _ANUAL (Cold Spring Harbor Laboratory Press, 2 nd edition 1988);

kabat et al S _EQUENCES _OF P _ROTEINS _OF I _MMUNOLOGICAL I _NTEREST (National Institutes of Health, bethesda, md. (1987) and (1991);

kabat, E.A. et al (1991) S _EQUENCES _OF P _ROTEINS _OF I _MMUNOLOGICAL I _NTEREST 5 th edition, U.S. Pat. No. of Health and Human Services, NIH Publication No.91-3242;

Kontermann and Dubel, A _NTIBODY E _NGINEERING (2001) Springer-Verlag. New York. Page 790 (ISBN 3-540-41354-5).

Kriegler,Gene Transfer and Expression,A Laboratory Manual,Stockton Press,NY(1990)；

Lu and Weiner, C _LONING _AND E _XPRESSION V _ECTORS _FOR G _ENE F _UNCTION A _NALYSIS (2001) BioTechniques Press.Westborough, MA. page 298 (ISBN 1-881299-21-X).

M _EDICAL A _PPLICATIONS _OF C _ONTROLLED R _ELEASE Langer and Wise (ed), CRC pres., boca Raton, fla. (1974);

Old,R.W.&S.B.Primrose,P _RINCIPLES _OF G _ENE M _ANIPULATION :A _N I _NTRODUCTION T _O G _ENETIC E _NGINEERING (3 rd edition 1985) Blackwell Scientific Publications, boston. Studies in Microbiology; volume 2, page 409 (ISBN 0-632-01318-4).

Sambrook, J et al, M _OLECULAR C _LONING :A L _ABORATORY M _ANUAL (2 nd edition 1989) Cold Spring Harbor Laboratory Press, NY., volumes 1-3 (ISBN 0-87969-309-6).

S _USTAINED _AND C _ONTROLLED R _ELEASE D _RUG D _ELIVERY S _YSTEMS Robinson, marcel Dekker, inc., new York,1978

Winnacker,E.L.F _ROM G _ENES T _O C _LONES :I _NTRODUCTION T _O G _ENE T _ECHNOLOGY (1987) VCH Publishers, NY (Horst Ibelgaufts translation). Page 634 (ISBN 0-89573-614-4).

Equivalent scheme

The present disclosure may be embodied in other specific forms without departing from its spirit or essential characteristics. The above embodiments are therefore to be considered in all respects as illustrative and not restrictive. The scope of the disclosure is, therefore, indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims

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

2. The oligonucleotide of claim 1, comprising a sequence substantially complementary to the nucleic acid sequence of any one of SEQ ID NOS 143-244.

3. The oligonucleotide of any one of claims 1 or 2, wherein the oligonucleotide is an RNA molecule comprising a length of about 15 nucleotides to 25 nucleotides.

4. The RNA molecule of claim 3, wherein the RNA molecule comprises single-stranded (ss) RNA or double-stranded (ds) RNA.

5. The dsRNA of claim 4 comprising a sense strand and an antisense strand, wherein said antisense strand comprises a sequence that is substantially complementary to a nucleic acid sequence of any one of SEQ ID NOs 1-96.

6. The dsRNA of claim 4 comprising complementarity to at least 10, 11, 12 or 13 consecutive nucleotides of the nucleic acid sequence of any one of SEQ ID NOs 1-96.

7. The dsRNA of claim 4 comprising NO more than 3 mismatches to the nucleic acid sequence of any one of SEQ ID NOs 1-96.

8. The dsRNA of claim 4 comprising complete complementarity to the nucleic acid sequence of any one of SEQ ID NOs 1-96.

9. The dsRNA of any one of claims 5-8, wherein said antisense strand and/or sense strand comprises a length of about 15 nucleotides to 25 nucleotides.

10. The dsRNA of any one of claims 5-9, wherein said antisense strand is 20 nucleotides in length.

11. The dsRNA of any one of claims 5-9, wherein said antisense strand is 21 nucleotides in length.

12. The dsRNA of any one of claims 5-9, wherein said antisense strand is 22 nucleotides in length.

13. The dsRNA of any one of claims 5-9, wherein said sense strand is 15 nucleotides in length.

14. The dsRNA of any one of claims 5-9, wherein said sense strand is 16 nucleotides in length.

15. The dsRNA of any one of claims 5-9, wherein said sense strand is 18 nucleotides in length.

16. The dsRNA of any one of claims 5-9, wherein said sense strand is 20 nucleotides in length.

17. The dsRNA of any one of claims 4-16 comprising a double stranded region of 15 base pairs to 20 base pairs.

18. The dsRNA of any one of claims 4-17 comprising a double stranded region of 15 base pairs.

19. The dsRNA of any one of claims 4-17 comprising a double stranded region of 16 base pairs.

20. The dsRNA of any one of claims 4-17 comprising a double stranded region of 18 base pairs.

21. The dsRNA of any one of claims 4-17 comprising a double stranded region of 20 base pairs.

22. The dsRNA of any one of claims 4-21, wherein said dsRNA comprises blunt ends.

23. The dsRNA of any one of claims 4-22, wherein said dsRNA comprises at least one single stranded nucleotide overhang.

24. The dsRNA of claim 23, wherein said dsRNA comprises a single-stranded nucleotide overhang of about 2 nucleotides to 5 nucleotides.

25. The dsRNA of any one of claims 4-24, wherein said dsRNA comprises naturally occurring nucleotides.

26. The dsRNA of any one of claims 4-25, wherein said dsRNA comprises at least one modified nucleotide.

27. The dsRNA of claim 26, wherein said 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, an phosphoramidate, a nucleotide comprising a non-natural base, or a mixture thereof.

28. The dsRNA of any one of claims 4-27, wherein said dsRNA comprises at least one modified internucleotide linkage.

29. The dsRNA of claim 28, wherein said modified internucleotide linkages comprise phosphorothioate internucleotide linkages.

30. The dsRNA of any one of claims 4-29 comprising 4-16 phosphorothioate internucleotide linkages.

31. The dsRNA of any one of claims 4-29 comprising 8-13 phosphorothioate internucleotide linkages.

32. The dsRNA of any one of claims 4-28, wherein said dsRNA comprises at least one modified internucleotide linkage of formula I:

wherein:

b is a base pairing moiety;

w is selected from O, OCH ₂ 、OCH、CH ₂ And CH;

x is selected from halo, hydroxy and C _1-6 Alkoxy groups;

y is selected from O-, OH, OR, NH-, NH ₂ 、S ^- And SH;

z is selected from O and CH ₂ A group of;

r is a protecting group; and is also provided with

Is an optional double bond.

33. The dsRNA of any one of claims 4-32, wherein said dsRNA comprises at least 80% chemically modified nucleotides.

34. The dsRNA of any one of claims 4-33, wherein said dsRNA is fully chemically modified.

35. The dsRNA of any one of claims 4-33, wherein said dsRNA comprises at least 70% 2' -O-methyl nucleotide modifications.

36. The dsRNA of any one of claims 5-33, wherein said antisense strand comprises at least 50% 2 '-O-methyl nucleotide modifications or at least 70% 2' -O-methyl nucleotide modifications.

37. The dsRNA of claim 36, wherein said antisense strand comprises about 70% to 90% 2' -O-methyl nucleotide modifications.

38. The dsRNA of any one of claims 5-33, wherein said sense strand comprises at least 65% 2 '-O-methyl nucleotide modifications or at least 70% 2' -O-methyl nucleotide modifications.

39. The dsRNA of claim 38, wherein said sense strand comprises 100% 2' -O-methyl nucleotide modifications.

40. The dsRNA of any one of claims 5-39, wherein said sense strand comprises one or more nucleotide mismatches between said antisense strand and said sense strand.

41. The dsRNA of claim 40, wherein said one or more nucleotide mismatches are present at positions 2, 6 and 12 of the 5' end of said sense strand.

42. The dsRNA of claim 40, wherein said nucleotide mismatch is present at positions 2, 6 and 12 of the 5' end of said sense strand.

43. The dsRNA of any one of claims 5-42 wherein said antisense strand comprises a 5 'phosphate, 5' -alkylphosphonate, 5 'alkylenephosphonate, or 5' alkenylphosphonate.

44. The dsRNA of claim 43, wherein said antisense strand comprises a 5' vinylphosphonate.

45. The dsRNA of claim 4 comprising 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 the nucleic acid sequence of any one of SEQ ID NOs 1-96;

(4) Nucleotides at positions 1-2 to 1-7 of the 3' end of the antisense strand are linked to each other by phosphorothioate internucleotide linkages;

46. The dsRNA of claim 4 comprising an antisense strand and a sense strand, each strand having a 5 'end and a 3' end, wherein:

(2) The antisense strand comprises at least 70% 2' -O-methyl modification;

47. The dsRNA of claim 4 comprising an antisense strand and a sense strand, each strand having a 5 'end and a 3' end, wherein:

(2) The antisense strand comprises at least 85% 2' -O-methyl modification;

48. The dsRNA of claim 4 comprising an antisense strand and a sense strand, each strand having a 5 'end and a 3' end, wherein:

(2) The antisense strand comprises at least 75% 2' -O-methyl modification;

(3) The nucleotides at positions 4, 5, 6 and 14 of the 5 'end of the antisense strand are not 2' -methoxy-ribonucleotides;

49. The dsRNA of claim 4 comprising an antisense strand and a sense strand, each strand having a 5 'end and a 3' end, wherein:

(2) The antisense strand comprises at least 75% 2' -O-methyl modification;

50. The dsRNA of claim 4 comprising an antisense strand and a sense strand, each strand having a 5 'end and a 3' end, wherein:

(2) The antisense strand comprises at least 75% 2' -O-methyl modification;

(6) The sense strand comprises at least 70% 2' -O-methyl modification;

(7) The nucleotides at positions 7, 9, 10 and 11 of the 3 'end of the sense strand are not 2' -methoxy-ribonucleotides; and is also provided with

51. The dsRNA of claim 4 comprising an antisense strand and a sense strand, each strand having a 5 'end and a 3' end, wherein:

(2) The antisense strand comprises at least 75% 2' -O-methyl modification;

(6) The sense strand comprises at least 80% 2' -O-methyl modification;

(7) The nucleotides at positions 7, 10 and 11 of the 3 'end of the sense strand are not 2' -methoxy-ribonucleotides; and is also provided with

52. The dsRNA of claim 4 comprising an antisense strand and a sense strand, each strand having a 5 'end and a 3' end, wherein:

(2) The antisense strand comprises at least 50% 2' -O-methyl modification;

(3) The nucleotides at positions 2, 4, 5, 6, 8, 10, 12, 14, 16 and 20 of the 5 'end of the antisense strand are not 2' -methoxy-ribonucleotides;

(4) Nucleotides at positions 1-2 to 1-8 of the 3' end of the antisense strand are linked to each other by phosphorothioate internucleotide linkages;

(6) The sense strand comprises at least 65% 2' -O-methyl modification;

53. The dsRNA of claim 4 comprising an antisense strand and a sense strand, each strand having a 5 'end and a 3' end, wherein:

(2) The antisense strand comprises at least 75% 2' -O-methyl modification;

(3) The nucleotides at positions 2, 6, 14, 16 and 20 of the 5 'end of the antisense strand are not 2' -methoxy-ribonucleotides;

(6) The sense strand comprises at least 65% 2' -O-methyl modification;

54. A double-stranded RNA (dsRNA) molecule comprising 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 an IFN- γ signaling pathway target gene nucleic acid sequence;

(2) The antisense strand is 21 nucleotides in length;

(3) The antisense strand comprises at least 50% 2' -O-methyl modification;

(4) The nucleotide at any one or more of positions 2, 4, 5, 6, 8, 10, 12, 14, 16 and 20 of the 5 'end of the antisense strand is not a 2' -methoxy-ribonucleotide;

(5) Nucleotides at positions 1-2 to 1-8 of the 3' end of the antisense strand are linked to each other by phosphorothioate internucleotide linkages;

(6) A portion of the antisense strand is complementary to a portion 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 of the 3 'end of the sense strand are not 2' -methoxy-ribonucleotides; and is also provided with

(10) The nucleotides at positions 1-2 of the 5' end of the sense strand are linked to each other by phosphorothioate internucleotide linkages.

55. The dsRNA of any one of claims 45-54, wherein a functional moiety is attached to the 5 'and/or 3' end of said antisense strand.

56. The dsRNA of any one of claims 45-54, wherein a functional moiety is attached to the 5 'and/or 3' end of said sense strand.

57. The dsRNA of any one of claims 45-54, wherein a functional moiety is attached to the 3' end of said sense strand.

58. The dsRNA of any one of claims 55-57, wherein said functional moiety comprises a hydrophobic moiety.

59. The dsRNA of claim 58, wherein said hydrophobic moiety is selected from the group consisting of: fatty acids, steroids, ring-opened steroids, lipids, gangliosides, nucleoside analogs, endogenous cannabinoids, vitamins, and mixtures thereof.

60. The dsRNA of claim 59, wherein said steroid is selected from the group consisting of cholesterol and lithocholic acid (LCA).

61. The dsRNA of claim 59, wherein said fatty acid is selected from the group consisting of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosanoic acid (DCA).

62. The dsRNA of claim 59, wherein said vitamin is selected from the group consisting of choline, vitamin a, vitamin E, and derivatives or metabolites thereof.

63. The dsRNA of claim 62, wherein said vitamin is selected from the group consisting of retinoic acid and alpha-tocopheryl succinate.

64. The dsRNA of any one of claims 5-63, wherein said functional moiety is linked to said antisense strand and/or sense strand by a linker.

65. The dsRNA of claim 64, wherein said linker comprises a bivalent or trivalent linker.

66. The dsRNA of claim 65, wherein said bivalent or trivalent linker is selected from the group consisting of:

wherein n is 1, 2, 3, 4 or 5.

67. The dsRNA of claim 64 or 65, wherein said linker comprises a glycol chain, alkyl chain, peptide, RNA, DNA, phosphodiester, phosphorothioate, phosphoramidate, amide, carbamate, or a combination thereof.

68. The dsRNA of claim 65 or 66, wherein when said linker is a trivalent linker, said linker is further linked to a phosphodiester or phosphodiester derivative.

69. The dsRNA of claim 68, wherein said phosphodiester or phosphodiester derivative is selected from the group consisting of:

wherein X is O, S or BH ₃ 。

70. The dsRNA of any one of claims 5-69, wherein the nucleotides at positions 1 and 2 of the 3 'end of the sense strand and the nucleotides at positions 1 and 2 of the 5' end of the antisense strand are linked to adjacent ribonucleotides by phosphorothioate linkages.

71. A pharmaceutical composition for inhibiting expression of an IFN- γ signaling pathway gene selected from the group consisting of IFNGR1, JAK2, or STAT1 in an organism, the composition comprising the dsRNA of any one of claims 4-70 and a pharmaceutically acceptable carrier.

72. The pharmaceutical composition of claim 71, wherein said dsRNA inhibits expression of said gene by at least 50%.

73. The pharmaceutical composition of claim 71, wherein said dsRNA inhibits expression of said gene by at least 80%.

74. The pharmaceutical composition of claim 71, wherein the dsRNA reduces expression of chemokine CXCL9 by at least 20% to at least 80%.

75. A method for inhibiting IFN- γ signaling pathway gene expression in a cell selected from the group consisting of IFNGR1, JAK2, or STAT1, the method comprising:

(a) Introducing the double-stranded ribonucleic acid (dsRNA) of any one of claims 4-70 into the cell; and

(b) Maintaining the cell produced in step (a) for a time sufficient to obtain degradation of mRNA transcripts of the gene, thereby inhibiting expression of the gene in the cell.

76. 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 comprising sufficient complementarity to an IFN- γ signaling pathway target gene, thereby treating the subject.

77. The method of claim 76 comprising administering a therapeutically effective amount of the dsRNA of any one of claims 4-70.

78. The method of claim 77, wherein the dsRNA is administered by Intravenous (IV) injection, subcutaneous (SQ) injection, or a combination thereof.

79. The method of any one of claims 75-78, wherein said dsRNA inhibits expression of said gene by at least 50%.

80. The method of any one of claims 75-78, wherein said dsRNA inhibits expression of said gene by at least 80%.

81. The method of any one of claims 75-78, wherein the dsRNA reduces expression of cytokine CXCL9 by at least 20% to at least 80%.

82. A vector comprising a regulatory sequence operably linked to a nucleotide sequence encoding an RNA molecule that is substantially complementary to a nucleic acid sequence of any one of SEQ ID NOs 1-96.

83. The vector of claim 82, wherein said RNA molecule inhibits expression of said gene by at least 50%.

84. The vector of claim 82, wherein said RNA molecule inhibits expression of said gene by at least 80%.

85. The vector of claim 82, wherein the RNA molecule reduces expression of cytokine CXCL9 by at least 20% to at least 80%.

86. The vector of any one of claims 82-85, wherein said RNA molecule comprises ssRNA or dsRNA.

87. The vector of claim 86, wherein the dsRNA comprises a sense strand and an antisense strand, wherein the antisense strand comprises a sequence that is substantially complementary to a nucleic acid sequence of any one of SEQ ID NOs 1-96.

88. A cell comprising the vector of any one of claims 82-87.

89. A recombinant adeno-associated virus (rAAV) comprising the vector of any one of claims 82-87 and an AAV capsid.

90. A branched RNA compound, the branched RNA compound comprising:

two or more RNA molecules having a length of 15 to 35 nucleotides, and

a sequence substantially complementary to an IFN-gamma signaling pathway target gene mRNA selected from the group consisting of IFNGR1, JAK2 or STAT1,

wherein the two RNA molecules are linked to each other by one or more moieties independently selected from the group consisting of a linker, a spacer, and a branching point.

91. The branched RNA compound of claim 90 comprising a sequence substantially complementary to the nucleic acid sequence of any one of SEQ ID NOs 1-96.

92. The branched RNA compound of claim 90 comprising a sequence substantially complementary to one or more of the nucleic acid sequences of any one of SEQ ID NOs 143-244.

93. The branched RNA compound of any one of claims 90-92, wherein the RNA molecule comprises one or both of ssRNA and dsRNA.

94. The branched RNA compound of any one of claims 90-92, wherein the RNA molecule comprises an antisense oligonucleotide.

95. The branched RNA compound of any one of claims 90-92, wherein each RNA molecule comprises a length of 15 to 25 nucleotides.

96. The branched RNA compound of any one of claims 90-92, wherein each RNA molecule comprises a dsRNA comprising a sense strand and an antisense strand, wherein each antisense strand independently comprises a sequence substantially complementary to a nucleic acid sequence of any one of SEQ ID NOs 1-96.

97. The branched RNA compound of claim 96 comprising complementarity to at least 10, 11, 12 or 13 consecutive nucleotides of the nucleic acid sequence of any one of SEQ ID NOs 1-96.

98. The branched RNA compound of claim 96, wherein each RNA molecule comprises NO more than 3 mismatches with the nucleic acid sequence of any one of SEQ ID NOs 1-96.

99. The branched RNA compound of claim 96 comprising complete complementarity to the nucleic acid sequence of any one of SEQ ID NOs 1-96.

100. The branched RNA compound of any one of claims 96-99, wherein the antisense strand and/or sense strand comprises a length of about 15 nucleotides to 25 nucleotides.

101. The branched RNA compound of any one of claims 96-100, wherein the antisense strand is 20 nucleotides in length.

102. The branched RNA compound of any one of claims 96-100, wherein the antisense strand is 21 nucleotides in length.

103. The branched RNA compound of any one of claims 96-100, wherein the antisense strand is 22 nucleotides in length.

104. The branched RNA compound of any one of claims 96-100, wherein the sense strand is 15 nucleotides in length.

105. The branched RNA compound of any one of claims 96-100, wherein the sense strand is 16 nucleotides in length.

106. The branched RNA compound of any one of claims 96-100, wherein the sense strand is 18 nucleotides in length.

107. The branched RNA compound of any one of claims 96-100, wherein the sense strand is 20 nucleotides in length.

108. The branched RNA compound of any one of claims 93-107, wherein the dsRNA comprises a double stranded region of 15 base pairs to 20 base pairs.

109. The branched RNA compound of any one of claims 93-107, wherein the dsRNA comprises a double stranded region of 15 base pairs.

110. The branched RNA compound of any one of claims 93-107, wherein the dsRNA comprises a double stranded region of 16 base pairs.

111. The branched RNA compound of any one of claims 93-107, wherein the dsRNA comprises a double stranded region of 18 base pairs.

112. The branched RNA compound of any one of claims 93-107, wherein the dsRNA comprises a double stranded region of 20 base pairs.

113. The branched RNA compound of any one of claims 93-112, wherein the dsRNA comprises a blunt end.

114. The branched RNA compound of any one of claims 93-112, wherein the dsRNA comprises at least one single stranded nucleotide overhang.

115. The branched RNA compound of any one of claims 93-114, wherein the dsRNA comprises a single stranded nucleotide overhang of between 2 nucleotides and 5 nucleotides.

116. The branched RNA compound of any one of claims 93-115, wherein the dsRNA comprises a naturally occurring nucleotide.

117. The branched RNA compound of any one of claims 93-116, wherein the dsRNA comprises at least one modified nucleotide.

118. The branched RNA compound of claim 117, wherein 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 comprising a non-natural base.

119. The branched RNA compound of any one of claims 93-118, wherein the dsRNA comprises at least one modified internucleotide linkage.

120. The branched RNA compound of claim 119, wherein the modified internucleotide linkage comprises a phosphorothioate internucleotide linkage.

121. The branched RNA compound of any one of claims 93-120, comprising 4-16 phosphorothioate internucleotide linkages.

122. The branched RNA compound of any one of claims 93-120, comprising 8-13 phosphorothioate internucleotide linkages.

123. The branched RNA compound of any one of claims 93-118, wherein the dsRNA comprises at least one modified internucleotide linkage of formula I:

wherein:

b is a base pairing moiety;

w is selected from O, OCH ₂ 、OCH、CH ₂ And CH;

x is selected from halo, hydroxy and C _1-6 Alkoxy groups;

y is selected from O ^- 、OH、OR、NH ^- 、NH ₂ 、S ^- And SH;

z is selected from O and CH ₂ A group of;

r is a protecting group; and is also provided with

Is an optional double bond.

124. The branched RNA compound of any one of claims 93-123, wherein the dsRNA comprises at least 80% chemically modified nucleotides.

125. The branched RNA compound of any one of claims 93-123, wherein the dsRNA is fully chemically modified.

126. The branched RNA compound of any one of claims 93-123, wherein the dsRNA comprises at least 70% 2' -O-methyl nucleotide modifications.

127. The branched RNA compound of any one of claims 96-123, wherein the antisense strand comprises at least 50% 2' -O-methyl nucleotide modification.

128. The branched RNA compound of any one of claims 96-123, wherein the antisense strand comprises at least 70% 2' -O-methyl nucleotide modification.

129. The branched RNA compound of claim 128 wherein the antisense strand comprises about 70 to 90% 2' -O-methyl nucleotide modification.

130. The branched RNA compound of any one of claims 96-123, wherein the sense strand comprises at least 65% 2' -O-methyl nucleotide modification.

131. The branched RNA compound of any one of claims 96-123, wherein the sense strand comprises at least 70% 2' -O-methyl nucleotide modification.

132. The branched RNA compound of claim 131 wherein the sense strand comprises 100% 2' -O-methyl nucleotide modifications.

133. The branched RNA compound of any one of claims 96-132, wherein the sense strand comprises one or more nucleotide mismatches between the antisense strand and the sense strand.

134. The branched RNA compound of claim 133, wherein the one or more nucleotide mismatches are present at positions 2, 6 and 12 of the 5' end of the sense strand.

135. The branched RNA compound of claim 133, wherein the nucleotide mismatch is present at positions 2, 6 and 12 of the 5' end of the sense strand.

136. The branched RNA compound of any one of claims 96-135, wherein the antisense strand comprises a 5 'phosphate, a 5' -alkylphosphonate, a 5 'alkylenephosphonate, a 5' alkenylphosphonate, or a mixture thereof.

137. The branched RNA compound of claim 136 wherein the antisense strand comprises a 5' vinyl phosphonate.

138. The branched RNA compound of claim 93, wherein the dsRNA comprises an antisense strand and a sense strand, each strand having a 5 'end and a 3' end, wherein:

139. The branched RNA compound of claim 93, wherein the dsRNA comprises an antisense strand and a sense strand, each strand having a 5 'end and a 3' end, wherein:

(2) The antisense strand comprises at least 70% 2' -O-methyl modification;

140. The branched RNA compound of claim 93, wherein the dsRNA comprises an antisense strand and a sense strand, each strand having a 5 'end and a 3' end, wherein:

(2) The antisense strand comprises at least 85% 2' -O-methyl modification;

141. The branched RNA compound of claim 93, wherein the dsRNA comprises an antisense strand and a sense strand, each strand having a 5 'end and a 3' end, wherein:

(2) The antisense strand comprises at least 75% 2' -O-methyl modification;

142. The branched RNA compound of claim 93, wherein the dsRNA comprises an antisense strand and a sense strand, each strand having a 5 'end and a 3' end, wherein:

(2) The antisense strand comprises at least 75% 2' -O-methyl modification;

143. The branched RNA compound of claim 93, wherein the dsRNA comprises an antisense strand and a sense strand, each strand having a 5 'end and a 3' end, wherein:

(2) The antisense strand comprises at least 75% 2' -O-methyl modification;

(6) The sense strand comprises at least 70% 2' -O-methyl modification;

144. The branched RNA compound of claim 93, wherein the dsRNA comprises an antisense strand and a sense strand, each strand having a 5 'end and a 3' end, wherein:

(2) The antisense strand comprises at least 75% 2' -O-methyl modification;

(6) The sense strand comprises at least 80% 2' -O-methyl modification;

145. The branched RNA compound of claim 93, wherein the dsRNA comprises an antisense strand and a sense strand, each strand having a 5 'end and a 3' end, wherein:

(2) The antisense strand comprises at least 50% 2' -O-methyl modification;

(6) The sense strand comprises at least 65% 2' -O-methyl modification;

146. The branched RNA compound of claim 93, wherein the dsRNA comprises an antisense strand and a sense strand, each strand having a 5 'end and a 3' end, wherein:

(2) The antisense strand comprises at least 75% 2' -O-methyl modification;

(6) The sense strand comprises at least 65% 2' -O-methyl modification;

147. A branched RNA compound, the branched RNA compound comprising:

two or more RNA molecules having a length of 15 to 35 nucleotides, and

sequences substantially complementary to the IFN-gamma signaling pathway target gene mRNA,

wherein the two RNA molecules are linked to each other by one or more moieties independently selected from the group consisting of a linker, a spacer and a branching point,

and wherein the RNA molecule comprises a dsRNA, wherein the dsRNA comprises an antisense strand and a sense strand, each strand having a 5 'end and a 3' end, wherein:

(2) The antisense strand comprises at least 50% 2' -O-methyl modification;

(6) The sense strand comprises at least 65% 2' -O-methyl modification;

148. The branched RNA compound of any one of claims 96-147, wherein a functional moiety is attached to the 5 'and/or 3' end of the antisense strand.

149. The branched RNA compound of any one of claims 96-147, wherein a functional moiety is attached to the 5 'and/or 3' end of the sense strand.

150. The branched RNA compound of any one of claims 96-147, wherein a functional moiety is attached to the 3' end of the sense strand.

151. The branched RNA compound of any one of claims 148-150, wherein the functional moiety comprises a hydrophobic moiety.

152. The branched RNA compound of claim 151, wherein the hydrophobic moiety is selected from the group consisting of: fatty acids, steroids, ring-opened steroids, lipids, gangliosides and nucleoside analogs, endogenous cannabinoids, vitamins, and mixtures thereof.

153. The branched RNA compound of claim 152, wherein the steroid is selected from the group consisting of cholesterol and lithocholic acid (LCA).

154. The branched RNA compound of claim 152, wherein the fatty acid is selected from the group consisting of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosanoic acid (DCA).

155. The branched RNA compound of claim 152, wherein the vitamin is selected from the group consisting of choline, vitamin a, vitamin E, and derivatives or metabolites thereof.

156. The branched RNA compound of claim 152, wherein the vitamin is selected from the group consisting of retinoic acid and alpha-tocopheryl succinate.

157. The branched RNA compound of any one of claims 148-156, wherein the functional moiety is linked to the antisense strand and/or sense strand by a linker.

158. The branched RNA compound of claim 157, wherein the linker comprises a divalent or trivalent linker.

159. The branched RNA compound of claim 158, wherein the divalent or trivalent linker is selected from the group consisting of:

wherein n is 1, 2, 3, 4 or 5.

160. The branched RNA compound of claim 157 or 158, wherein the linker comprises a glycol chain, alkyl chain, peptide, RNA, DNA, phosphodiester, phosphorothioate, phosphoramidate, amide, carbamate, or a combination thereof.

161. The branched RNA compound of claim 158, wherein when the linker is a trivalent linker, the linker is further linked to a phosphodiester or phosphodiester derivative.

162. The branched RNA compound of claim 161, wherein the phosphodiester or phosphodiester derivative is selected from the group consisting of:

/>

wherein X is O, S or BH ₃ 。

163. The branched RNA compound of any one of claims 96-162, wherein the nucleotides at positions 1 and 2 of the 3 'end of the sense strand and the nucleotides at positions 1 and 2 of the 5' end of the antisense strand are linked to adjacent ribonucleotides by phosphorothioate linkages.

164. A compound of formula (I):

L-(N) _n

(I)

wherein the method comprises the steps of

s independently at each occurrence comprises a 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 is also provided with

N is a double-stranded nucleic acid comprising 15 to 35 bases in length, comprising a sense strand and an antisense strand; wherein the method comprises the steps of

The antisense strand comprises a sequence substantially complementary to the nucleic acid sequence of any one of SEQ ID NOs 1-96;

The sense strand and the antisense strand each independently comprise one or more chemical modifications; and is also provided with

n is 2, 3, 4, 5, 6, 7 or 8.

165. The compound of claim 164 having a structure selected from formulas (I-1) - (I-9):

。

166. the compound of claim 165, wherein the antisense strand comprises a 5' terminal group R selected from the group consisting of:

167. the compound of claim 164 having the structure of formula (II):

/>

wherein the method comprises the steps of

-represents a phosphodiester internucleoside linkage;

168. The compound of claim 164 having the structure of formula (IV):

wherein the method comprises the steps of

-represents a phosphodiester internucleoside linkage;

169. The compound of any one of claims 164-168, wherein L belongs to structure L1:

170. the compound of claim 169, wherein R is R ³ And n is 2.

171. The compound of any one of claims 164-168, wherein L belongs to structure L2:

172. the compound of claim 171, wherein R is R ³ And n is 2.

173. A delivery system for a therapeutic nucleic acid having the structure of formula (VI):

L-(cNA) _n

(VI)

wherein the method comprises the steps of

Each cNA independently comprises at least 15 contiguous nucleotides of the nucleic acid sequence of any one of SEQ ID NOs 1-96; and is also provided with

n is 2, 3, 4, 5, 6, 7 or 8.

174. The delivery system of claim 173, having a structure selected from formulas (VI-1) - (VI-9):

175. the delivery system of claim 173, wherein each cNA independently comprises a chemically modified nucleotide.

176. The delivery system of claim 173, further comprising n therapeutic Nucleic Acids (NA), wherein each NA hybridizes to at least one cNA.

177. The delivery system of claim 176, wherein each NA independently comprises at least 16 contiguous nucleotides.

178. The delivery system of claim 177, wherein each NA independently comprises 16-20 contiguous nucleotides.

179. The delivery system of claim 176, wherein each NA comprises an unpaired overhang of at least 2 nucleotides.

180. The delivery system of claim 179, wherein the overhanging nucleotides are linked by phosphorothioate linkages.

181. The delivery system of claim 176, wherein each NA is independently selected from the group consisting of: DNA, siRNA, miRNA antagonists, mirnas, interstitials, mixtures and guide RNAs.

182. The delivery system of claim 176, wherein each NA is substantially complementary to the nucleic acid sequence of any one of SEQ ID NOs 1-96.

183. A pharmaceutical composition for inhibiting IFN- γ signaling pathway target gene expression in an organism comprising a compound of any one of claims 90-172 or a system of any one of claims 173-182, and a pharmaceutically acceptable carrier.

184. The pharmaceutical composition of claim 183, wherein the compound or system inhibits expression of the gene by at least 50%.

185. The pharmaceutical composition of claim 183, wherein the compound or system inhibits expression of the gene by at least 80%.

186. The pharmaceutical composition of claim 183, wherein the compound or system reduces expression of cytokine CXCL9 by at least 20% to at least 80%.

187. A method for inhibiting IFN- γ signaling pathway target gene expression in a cell, the method comprising:

(a) Introducing the compound of any one of claims 90-172 or the system of any one of claims 173-182 into the cell; and

188. 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 of any one of claims 90-172 or the system of any one of claims 173-182.

189. The method of claim 188, wherein the dsRNA is administered by Intravenous (IV) injection, subcutaneous (SQ) injection, or a combination thereof.

190. The method of any one of claims 187-189 wherein the dsRNA inhibits expression of the gene by at least 50%.

191. The method of any one of claims 187-189 wherein the dsRNA inhibits expression of the gene by at least 80%.

192. The method of any one of claims 187-189, wherein the dsRNA reduces expression of cytokine CXCL9 by at least 20% to at least 80%.