Dsrna molecule for regulating expression of ttr

Abstract

Provided are a double-stranded RNA for inhibiting the expression of the TTR gene, a cell containing same, and a method for using the dsRNA or the cell to treat a TTR-mediated or related disease or symptoms in a subject.

Description

dsRNA molecules that modulate TTR expression

[0001] This application claims priority to Chinese Patent Application No. 202410867257.0, filed on June 28, 2024, entitled “dsRNA molecules that modulate TTR expression”; and Chinese Patent Application No. 202411853735.9, filed on December 13, 2024, entitled “dsRNA molecules that modulate TTR expression”, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0002] The present application relates to the field of RNA interference. BACKGROUND

[0003] TTR is a protein encoded by the TTR gene. Its abnormal expression is associated with a series of diseases, including senile systemic amyloidosis, systemic familial amyloidosis, familial amyloidosis polyneuropathy, familial amyloid cardiomyopathy, leptomeningeal / central nervous system amyloidosis, hyperthyroxinemia, Sturge's disease, diabetic retinopathy, age-related macular degeneration, insulin resistance and cardiovascular disease associated with type II diabetes.

[0004] There is a need in the art for compositions and methods for treating diseases associated with TTR.

[0005] In this regard, one treatment method is to reduce TTR expression through small interfering RNA (siRNA) based on the RNA interference mechanism to treat diseases associated with TTR. SUMMARY

[0006] The present application provides novel double strand RNA (or simply dsRNA) for inhibiting TTR gene expression, cells, and pharmaceutical compositions and kits comprising the dsRNA or cells, and methods of using the dsRNA, cells, and pharmaceutical compositions and kits to inhibit or reduce TTR gene expression or to treat diseases or conditions that benefit from a reduction in TTR gene expression.

[0007] In a first aspect, the present application provides a double strand RNA (dsRNA) for inhibiting TTR gene expression, the dsRNA comprising a sense strand and an antisense strand forming a double stranded region, wherein the sense strand and the antisense strand are each independently 15-30 nucleotides in length, and the antisense strand comprises a nucleotide sequence of at least 15 contiguous nucleotides of the nucleotide sequence set forth in any one of SEQ ID NOs: 132-262. In some embodiments, the sense strand comprises a nucleotide sequence of at least 15 contiguous nucleotides of the nucleotide sequence set forth in any one of SEQ ID NOs: 1-131.

[0008] In some embodiments, the sense strand and the antisense strand of the dsRNA form a hairpin loop. In other embodiments, the dsRNA is an siRNA.

[0009] In some embodiments, the sense strand and the antisense strand are each independently 15-27 nucleotides in length, preferably 18-25 nucleotides in length, more preferably 19-21 nucleotides in length. In some embodiments, the sense strand is 15-27 nucleotides in length, preferably 17-25 nucleotides in length, more preferably 18-23 nucleotides in length, more preferably 19-21 nucleotides in length, most preferably 19 nucleotides in length. In some embodiments, the antisense strand is 15-27 nucleotides in length, preferably 17-25 nucleotides in length, more preferably 18-23 nucleotides in length, more preferably 19-22 nucleotides in length, most preferably 21 nucleotides in length.

[0010] In some embodiments, the double-stranded region is 15-25 nucleotide pairs in length, preferably 16-23 nucleotide pairs in length, more preferably 18-20 nucleotide pairs in length, most preferably 19 nucleotide pairs in length.

[0011] In some embodiments, one or both of the sense strand and the antisense strand comprises a 3' overhang and / or a 5' overhang of at least 1 nucleotide, for example one or both of the sense strand and the antisense strand comprises a 3' overhang and / or a 5' overhang of at least 2 nucleotides. In some embodiments, the antisense strand has a 3' overhang and / or a 5' overhang of at least 1 nucleotide, preferably the antisense strand comprises a 3' overhang and / or a 5' overhang of 2 nucleotides. In some specific embodiments, the dsRNA has a two nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand.

[0012] In some embodiments, the antisense strand comprises a nucleotide sequence of at least 16 contiguous nucleotides, at least 17 contiguous nucleotides, at least 18 contiguous nucleotides, at least 19 contiguous nucleotides, or at least 20 contiguous nucleotides of the nucleotide sequence set forth in any one of SEQ ID NOs: 132-262. In preferred embodiments, the antisense strand comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 132-262.

[0013] In some embodiments, the sense strand comprises a nucleotide sequence of at least 16 contiguous nucleotides, a nucleotide sequence of at least 17 contiguous nucleotides, or a nucleotide sequence of at least 18 contiguous nucleotides of the nucleotide sequence set forth in any one of SEQ ID NOs: 1-131. In preferred embodiments, the sense strand comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 1-131.

[0014] In some embodiments, the antisense strand comprises a nucleotide sequence of at least 15 contiguous nucleotides, a nucleotide sequence of at least 16 contiguous nucleotides, a nucleotide sequence of at least 17 contiguous nucleotides, a nucleotide sequence of at least 18 contiguous nucleotides, a nucleotide sequence of at least 19 contiguous nucleotides, or a nucleotide sequence of at least 20 contiguous nucleotides of the nucleotide sequence set forth in any one of SEQ ID NOs: 134, 144, 174, 176, 177, 179, 180, 185, 188, 190, 191, 192, 193, 194, 195, 201, 202, 204, 210, 214, 217, 218, 220, 221, 222, 223, 224, 225, 226, 231, 232, 236, 243, 247, 248, and 251. In some preferred embodiments, the antisense strand comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 134, 144, 174, 176, 177, 179, 180, 185, 188, 190, 191, 192, 193, 194, 195, 201, 202, 204, 210, 214, 217, 218, 220, 221, 222, 223, 224, 225, 226, 231, 232, 236, 243, 247, 248, and 251.

[0015] In some embodiments, the sense strand comprises a nucleotide sequence of at least 15 contiguous nucleotides, at least 16 contiguous nucleotides, at least 17 contiguous nucleotides, or at least 18 contiguous nucleotides of the nucleotide sequence set forth in any one of SEQ ID NOs: 3, 13, 43, 45, 46, 48, 49, 54, 57, 59, 60, 61, 62, 63, 64, 70, 71, 73, 79, 83, 86, 87, 89, 90, 91, 92, 93, 94, 95, 100, 101, 105, 112, 116, 117, and 120. In some preferred embodiments, the sense strand comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 3, 13, 43, 45, 46, 48, 49, 54, 57, 59, 60, 61, 62, 63, 64, 70, 71, 73, 79, 83, 86, 87, 89, 90, 91, 92, 93, 94, 95, 100, 101, 105, 112, 116, 117, and 120.

[0016] In some embodiments, the dsRNA comprises any one of the paired sense and antisense strand sequences as shown in Table 3 of the specification.

[0017] In some embodiments, in the dsRNAs of the present application for inhibiting the expression of TTR in a cell:

[0018] (1) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 3, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 134;

[0019] (2) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 13, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 144;

[0020] (3) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 43, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 174;

[0021] (4) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 45, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 176;

[0022] (5) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 46, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 177;

[0023] (6) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 48, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 179;

[0024] (7) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 49, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 180;

[0025] (8) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 54, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 185;

[0026] (9) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 57, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 188;

[0027] (10) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 59, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 190;

[0028] (11) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 60, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 191;

[0029] (12) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 61, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 192;

[0030] (13) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 62, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 193;

[0031] (14) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 63, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 194;

[0032] (15) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 64, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 195;

[0033] (16) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 70, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 201;

[0034] (17) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 71, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 202;

[0035] (18) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 73, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 204;

[0036] (19) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 79, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 210;

[0037] (20) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 83, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 214;

[0038] (21) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 86, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 217;

[0039] (22) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 87, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 218;

[0040] (23) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 89, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 220;

[0041] (24) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 90, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 221;

[0042] (25) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 91, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 222;

[0043] (26) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 92, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 223;

[0044] (27) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 93, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 224;

[0045] (28) the sense strand comprises a nucleotide sequence as set forth in SEQ ID NO: 94, and the antisense strand comprises a nucleotide sequence as set forth in SEQ ID NO: 225;

[0046] (29) the sense strand comprises a nucleotide sequence as set forth in SEQ ID NO: 95, and the antisense strand comprises a nucleotide sequence as set forth in SEQ ID NO: 226;

[0047] (30) the sense strand comprises a nucleotide sequence as set forth in SEQ ID NO: 100, and the antisense strand comprises a nucleotide sequence as set forth in SEQ ID NO: 231 ;

[0048] (31) the sense strand comprises a nucleotide sequence as set forth in SEQ ID NO: 101, and the antisense strand comprises a nucleotide sequence as set forth in SEQ ID NO: 232;

[0049] (32) the sense strand comprises a nucleotide sequence as set forth in SEQ ID NO: 105, and the antisense strand comprises a nucleotide sequence as set forth in SEQ ID NO: 236;

[0050] (33) the sense strand comprises a nucleotide sequence as set forth in SEQ ID NO: 112, and the antisense strand comprises a nucleotide sequence as set forth in SEQ ID NO: 243;

[0051] (34) the sense strand comprises a nucleotide sequence as set forth in SEQ ID NO: 116, and the antisense strand comprises a nucleotide sequence as set forth in SEQ ID NO: 247;

[0052] (35) the sense strand comprises a nucleotide sequence as set forth in SEQ ID NO: 117, and the antisense strand comprises a nucleotide sequence as set forth in SEQ ID NO: 248; or

[0053] (36) the sense strand comprises a nucleotide sequence as set forth in SEQ ID NO: 120, and the antisense strand comprises a nucleotide sequence as set forth in SEQ ID NO: 251.

[0054] In some embodiments, substantially all of the nucleotides of the sense strand and / or substantially all of the nucleotides of the antisense strand are modified nucleotides, or all of the nucleotides of the sense strand and / or all of the nucleotides of the antisense strand are modified nucleotides.

[0055] In some embodiments, the sense strand and the antisense strand each independently comprise one or more modified nucleotides selected from the group consisting of 2'-0-alkyl modified nucleotides (e.g., 2'-0-methyl modified nucleotides), 2'-methoxyethyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, inosine ribonucleotides, abasic nucleotides, inverted abasic deoxyribonucleotides, nucleotides comprising phosphorothioate groups, vinyl phosphonate modified nucleotides, locked nucleotides, unlocked nucleotides, 2'-amino-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-0-allyl modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides comprising non-natural bases, terminal nucleotides linked to a cholesteryl derivative or dodecanoic acid didecanoyl amide group, deoxyribonucleotides, 3'-terminal deoxythymine (dT) nucleotides, conformationally restricted nucleotides, restricted ethyl nucleotides, 2'-hydroxyl modified nucleotides, nucleotides comprising methylphosphonate groups, nucleotides comprising 5'-phosphates, nucleotides comprising 5'-phosphate mimics, glycol modified nucleotides (GNAs), and 2-0-(N-methylacetamide) modified nucleotides.

[0056] In some embodiments, the sense strand and the antisense strand each independently comprise one or more nucleotide modifications selected from the group consisting of 2'-0-methyl modified nucleotides, 2'-fluoro modified nucleotides, and nucleotides comprising phosphorothioate groups.

[0057] In some embodiments, the sense strand and / or the antisense strand comprises at least 2 2'-fluoro modified nucleotides. In some embodiments, the sense strand and / or the antisense strand comprises at least 8 2'-0-methyl modified nucleotides. In some embodiments, the 3' end and / or the 5' end of the sense strand and / or the antisense strand comprises 1-5 phosphorothioate internucleotide linkages, preferably 2-4 phosphorothioate internucleotide linkages.

[0058] In some embodiments, the antisense strand of the dsRNA has a length of 21 nucleotides and has

[0059] (i) (counting from the 5' end) 2'-0-methyl modified nucleotides at positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21, and 2'-fluoro modified nucleotides at positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20; and / or

[0060] (ii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21, counting from the 5' end.

[0061] In some embodiments, the antisense strand of the dsRNA has a length of 21 nucleotides and has

[0062] (i) an SCP modification at position 1 (counting from the 5' end);

[0063] (ii) 2'-fluoro modifications at positions 2, 4, 6, 8, 10, 12, 14, 16, and 18 (counting from the 5' end);

[0064] (iii) 2'-O-methyl modifications at positions 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, and 21 (counting from the 5' end); and / or

[0065] (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21 (counting from the 5' end).

[0066] In some embodiments, the antisense strand of the dsRNA has a length of 21 nucleotides and has

[0067] (i) 2'-deoxy modifications at positions 2, 5, 7, and 12 (counting from the 5' end);

[0068] (ii) an SCP modification at position 1 (counting from the 5' end);

[0069] (iii) a 2'-fluoro modification at position 14 (counting from the 5' end);

[0070] (iv) 2'-O-methyl modifications at positions 3, 4, 6, 8, 9, 10, 11, 13, 15, 16, 17, 18, 19, 20, and 21 (counting from the 5' end); and / or

[0071] (v) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21 (counting from the 5' end).

[0072] In some embodiments, the antisense strand of the dsRNA has a length of 21 nucleotides and has

[0073] (i) 2'-O-methyl modifications at positions 3, 4, 6, 8, 9, 10, 11, 13, 15, 17, 18, 19, 20, and 21 (counting from the 5' end);

[0074] (ii) 2'-fluoro-modified nucleotides at positions 5, 7, 12, 14, and 16, counting from the 5' end;

[0075] (iii) a 2'-deoxy-modified nucleotide at position 2, counting from the 5' end;

[0076] (iv) an SCP-modified nucleotide at position 1, counting from the 5' end; and / or

[0077] (v) phosphorothioate internucleotide linkages between nucleotides 1 and 2, between nucleotides 2 and 3, between nucleotides 19 and 20, and between nucleotides 20 and 21, counting from the 5' end.

[0078] In some embodiments, the antisense strand of the dsRNA has 21 nucleotides in length and has

[0079] (i) 2'-O-methyl-modified nucleotides at positions 3, 4, 6, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, and 21, counting from the 5' end;

[0080] (ii) 2'-fluoro-modified nucleotides at positions 2, 5, 7, 14, and 16, counting from the 5' end;

[0081] (iii) an SCP-modified nucleotide at position 1, counting from the 5' end; and / or

[0082] (iv) phosphorothioate internucleotide linkages between nucleotides 1 and 2, between nucleotides 2 and 3, between nucleotides 19 and 20, and between nucleotides 20 and 21, counting from the 5' end.

[0083] In some embodiments, the antisense strand of the dsRNA has 21 nucleotides in length and has

[0084] (i) 2'-O-methyl-modified nucleotides at positions 3, 4, 5, 6, 8, 9, 10, 11, 13, 15, 17, 18, 19, 20, and 21, counting from the 5' end;

[0085] (ii) 2'-fluoro-modified nucleotides at positions 2, 7, 12, 14, and 16, counting from the 5' end;

[0086] (iii) an SCP-modified nucleotide at position 1, counting from the 5' end; and / or

[0087] (iii) a 2'-O-methyl modified nucleotide at position 1, counting from the 5' end; and / or

[0088] In some embodiments, the antisense strand of the dsRNA has a length of 21 nucleotides and has

[0089] (i) a 2'-O-methyl modified nucleotide at positions 3, 4, 5, 7, 8, 9, 10, 11, 13, 15, 17, 18, 19, 20, and 21, counting from the 5' end;

[0090] (ii) a 2'-fluoro modified nucleotide at positions 2, 6, 12, 14, and 16, counting from the 5' end;

[0091] (iii) a SCP modified nucleotide at position 1, counting from the 5' end; and / or

[0092] (iv) a phosphorothioate intemucleotide linkage between nucleotide positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21, counting from the 5' end.

[0093] In some embodiments, the antisense strand of the dsRNA has a length of 21 nucleotides and has

[0094] (i) a 2'-O-methyl modified nucleotide at positions 3, 4, 6, 8, 9, 10, 11, 13, 15, 16, 17, 18, 19, 20, and 21, counting from the 5' end;

[0095] (ii) a 2'-fluoro modified nucleotide at positions 2, 5, 7, 12, and 14, counting from the 5' end;

[0096] (iii) a SCP modified nucleotide at position 1, counting from the 5' end; and / or

[0097] (iv) a phosphorothioate intemucleotide linkage between nucleotide positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21, counting from the 5' end.

[0098] In some embodiments, the antisense strand of the dsRNA has a length of 21 nucleotides and has

[0099] (i) 2’-O-methyl modified nucleotides at positions (counting from the 5’ end) 3, 4, 6, 8, 9, 10, 11, 13, 15, 16, 17, 18, 19, 20, and 21;

[0100] (ii) 2’-fluoro modified nucleotides at positions (counting from the 5’ end) 2, 5, 7, 12, and 14;

[0101] (iii) an SCP modified nucleotide at position (counting from the 5’ end) 1; and / or

[0102] (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21 (counting from the 5’ end).

[0103] In some embodiments, the antisense strand of the dsRNA has a length of 21 nucleotides and has

[0104] (i) 2’-O-methyl modified nucleotides at positions (counting from the 5’ end) 3, 4, 5, 6, 8, 9, 10, 11, 13, 15, 17, 18, 19, 20, and 21;

[0105] (ii) 2’-fluoro modified nucleotides at positions (counting from the 5’ end) 2, 7, 12, 14, and 16;

[0106] (iii) an SCP modified nucleotide at position (counting from the 5’ end) 1; and / or

[0107] (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21 (counting from the 5’ end).

[0108] In some embodiments, the antisense strand of the dsRNA has a length of 21 nucleotides and has

[0109] (i) 2’-O-methyl modified nucleotides at positions (counting from the 5’ end) 3, 4, 6, 8, 9, 10, 11, 13, 15, 16, 17, 18, 19, 20, and 21;

[0110] (ii) 2’-fluoro modified nucleotides at positions (counting from the 5’ end) 2, 7, 12, 14, and 16;

[0111] (iii) 2’-deoxy modified nucleotides at positions (counting from the 5’ end) 2, 5, 7, and 12;

[0112] (iv) a SCP-modified nucleotide at position 1 (counting from the 5' end); and / or

[0113] (v) a phosphorothioate internucleotide linkage between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21 (counting from the 5' end).

[0114] In some embodiments, the sense strand of the dsRNA has a length of 19 nucleotides and has:

[0115] (i) 2'-0-methyl modified nucleotides at positions 1 to 6, 10 to 19 (counting from the 5' end), and 2'-fluoro modified nucleotides at positions 7-9; and / or

[0116] (ii) a phosphorothioate internucleotide linkage between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, and between nucleotide positions 18 and 19 (counting from the 5' end).

[0117] In some embodiments, the sense strand of the dsRNA has a length of 19 nucleotides and has:

[0118] (i) 2'-0-methyl modified nucleotides at positions 1 to 6, 10 to 19 (counting from the 5' end), and 2'-fluoro modified nucleotides at positions 7-9; and / or

[0119] (ii) a phosphorothioate internucleotide linkage between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 17 and 18, and between nucleotide positions 18 and 19 (counting from the 5' end).

[0120] In some embodiments, the sense strand of the dsRNA has a length of 19 nucleotides and has

[0121] (i) 2'-0-methyl modified nucleotides at positions 1, 2, 3, 4, 5, 6, 8, 10, 12, 13, 14, 15, 16, 17, 18, and 19 (counting from the 5' end);

[0122] (ii) 2'-fluoro modified nucleotides at positions 7, 9, and 11 (counting from the 5' end); and / or

[0123] (iii) a phosphorothioate internucleotide linkage between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, and between nucleotide positions 18 and 19 (counting from the 5' end).

[0124] In some embodiments, the sense strand of the dsRNA has a length of 19 nucleotides and has

[0125] (i) 2'-0-methyl modified nucleotides at positions (counting from the 5' end) 1, 2, 3, 4, 5, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19;

[0126] (ii) 2'-fluoro modified nucleotides at positions (counting from the 5' end) 7, 8, and 9; and / or

[0127] (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, and between nucleotide positions 18 and 19 (counting from the 5' end).

[0128] In some embodiments, the sense strand of the dsRNA has a length of 23 nucleotides and has

[0129] (i) 2'-0-methyl modified nucleotides at positions (counting from the 5' end) 2, 3, 4, 5, 6, 7, 8, 9, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22;

[0130] (ii) 2'-fluoro modified nucleotides at positions (counting from the 5' end) 10, 11, and 12;

[0131] (iii) IB modified nucleotides at positions (counting from the 5' end) 1 and 23; and / or

[0132] (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, and between nucleotide positions 22 and 23 (counting from the 5' end).

[0133] In some embodiments, the sense strand of the dsRNA has a length of 23 nucleotides and has

[0134] (i) 2'-0-methyl modified nucleotides at positions (counting from the 5' end) 2, 3, 4, 5, 6, 7, 8, 9, 11, 13, 15, 16, 17, 18, 19, 20, 21, and 22;

[0135] (ii) 2'-fluoro modified nucleotides at positions (counting from the 5' end) 10, 12, and 14;

[0136] (iii) IB modified nucleotides at positions (counting from the 5' end) 1 and 23; and / or

[0137] (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, and between nucleotide positions 22 and 23, counting from the 5' end.

[0138] In some embodiments, the sense strand of the dsRNA has a length of 23 nucleotides, and has

[0139] (i) 2'-0-methyl modified nucleotides at positions 2, 3, 4, 5, 6, 7, 8, 9, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22, counting from the 5' end;

[0140] (ii) 2'-fluoro modified nucleotides at positions 10, 11, and 12, counting from the 5' end;

[0141] (iii) IB modified nucleotides at positions 1 and 23, counting from the 5' end; and / or

[0142] (iv) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, and between nucleotide positions 22 and 23, counting from the 5' end.

[0143] In some embodiments, the sense strand of the dsRNA has a length of 19 nucleotides, and has

[0144] (i) 2'-0-methyl modified nucleotides at positions 1, 2, 3, 4, 5, 6, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19, counting from the 5' end;

[0145] (ii) 2'-fluoro modified nucleotides at positions 7 and 9, counting from the 5' end; and / or

[0146] (iii) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, and between nucleotide positions 18 and 19, counting from the 5' end.

[0147] In some embodiments, the antisense strand of the dsRNA comprises any one of the nucleotide sequences set forth in Table 5, and / or the sense strand comprises any one of the nucleotide sequences set forth in Table 4A.

[0148] In some embodiments, the sense strand and antisense strand of the dsRNA respectively comprise the sense strand sequence and the antisense strand sequence of any one of the siRNAs set forth in Table 6B, or the dsRNA comprises a sense strand and an antisense strand selected from the following:

[0149] (1) the sense strand comprises GmsUmsAmUmUmCmCfAfUfUmUmUmUmAmCmUmAmAmsAm (SEQ ID NO: 394); and the antisense strand comprises (SCP-U)sdTsUmAmdGUmdAAmAmAmAmdTGmGfAmAmUmAmCmsUmsCm (SEQ ID NO: 613),

[0150] (2) the sense strand comprises AmsGmsAmGmUmAmUfUfCfCmAmUmUmUmUmUmAmCmsUm (SEQ ID NO: 395), and the antisense strand comprises (SCP-U)sdGsUmAmAfAmAfAmUmGmGmAfAmUfAmCfUmCmUmsUmsGm (SEQ ID NO: 639), and

[0151] (3) the sense strand comprises GmsUmsAmUmUmCmCfAfUfUmUmUmUmAmCmUmAmAmsAm (SEQ ID NO: 396), and the antisense strand comprises (SCP-U)sUfsUmAmGmUmAfAmAmAmAmUfGmGfAmAfUmAmCmsUmsCm (SEQ ID NO: 634)

[0152] In some embodiments, the dsRNA is further conjugated to a ligand moiety that targets asialoglycoprotein receptor (ASGPR). In some preferred embodiments, the sense strand of the dsRNA is conjugated to the ligand moiety.

[0153] In some embodiments, the ligand moiety that targets asialoglycoprotein receptor (ASGPR) comprises N-acetylgalactosamine.

[0154] In one preferred embodiment, the ligand moiety has the following structure:

[0155] wherein represents the position of attachment to the sense strand of the siRNA via a phosphoester group or a thiophosphoester group.

[0156] DR009760, DR009761, DR009764, DR009765, DR009766, DR009767, DR009789, DR009768, DR009833, DR009834, DR009790, DR009769, DR009770, DR009771, DR009772, DR009773, DR009774, DR009776, DR009777, DR009778, DR009779, DR009780, DR009781, DR009782, DR009783, DR009784, DR009785, DR009791, DR009835, DR009786, DR009787, DR009797, DR009836, DR009788, DR009792, DR009793, DR009881, DR009882, DR009886, DR009887, DR009888, DR009891, DR009892, DR009915, DR009916, and DR009917.

[0157] DR011568, DR011569, DR011570, DR011572, DR011573, DR011574, DR011575, DR011576, DR011577, DR011578, DR011579, DR011580, DR011581, DR011582, DR011583, DR011584, DR011585, DR011627, DR011628, DR011682, DR011683, DR011684, DR011685, DR011686, DR011706, DR011707, DR011708, DR011709, DR011710, and DR011711.

[0158] DR009917, DR011707, and DR011683.

[0159] In a second aspect, the present application provides a cell comprising the dsRNA of the first aspect of the present application.

[0160] In a third aspect, the present application provides a pharmaceutical composition comprising the dsRNA of the first aspect of the present application or the cell of the second aspect of the present application, and optionally a pharmaceutically acceptable carrier or excipient.

[0161] In a fourth aspect, the present application provides a kit comprising the dsRNA of the first aspect of the present application, the cell of the second aspect of the present application, or the pharmaceutical composition of the third aspect of the present application.

[0162] In a fifth aspect, the present application provides a method of inhibiting expression of a TTR gene in a cell, the method comprising contacting the cell with the dsRNA of the first aspect of the present application or the pharmaceutical composition of the third aspect of the present application. In some embodiments, the method is performed in vitro.

[0163] In a sixth aspect, the present application provides a method of inhibiting expression of a TTR gene in a cell in a subject, the method comprising administering to the subject the dsRNA of the first aspect of the present application, the cell of the second aspect of the present application, or the pharmaceutical composition of the third aspect of the present application.

[0164] The present application also provides a method of treating a disease or disorder in a subject that benefits from a reduction in TTR gene expression, the method comprising administering to the subject the dsRNA of the first aspect of the present application, the cell of the second aspect of the present application, or the pharmaceutical composition of the third aspect of the present application.

[0165] The present application also provides a method of preventing at least one symptom in a subject having a disease or disorder that benefits from a reduction in TTR gene expression, the method comprising administering to the subject the dsRNA of the first aspect of the present application, the cell of the second aspect of the present application, or the pharmaceutical composition of the third aspect of the present application.

[0166] The present application also provides a method of preventing progression of a disease or disorder in a subject that benefits from a reduction in TTR gene expression, the method comprising administering to the subject the dsRNA of the first aspect of the present application, the cell of the second aspect of the present application, or the pharmaceutical composition of the third aspect of the present application.

[0167] In some embodiments, the disease or disorder that benefits from a reduction in TTR gene expression is a TTR-associated disease. In some preferred embodiments, the TTR-associated disease is selected from the group consisting of senile systemic amyloidosis, systemic familial amyloidosis, familial amyloidotic polyneuropathy, familial amyloid cardiomyopathy, leptomeningeal / central nervous system amyloidosis, hyperthyroxinemia, Sturge-Weber disease, diabetic retinopathy, age-related macular degeneration, insulin resistance and cardiovascular disease associated with type II diabetes.

[0168] In some embodiments, the dsRNA, cell or pharmaceutical composition is administered subcutaneously.

[0169] In some embodiments, the subject is a human.

[0170] DETAILED DESCRIPTION

[0171] The advantages and benefits of the present application will become apparent to those skilled in the art upon consideration of the disclosure. The disclosure will be described with reference to the accompanying drawings, of which:

[0172] It should be understood that the scope of the present application is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present application is intended to describe particular embodiments and is not intended to limit the scope of the present application.

[0173] In the specification and claims of this application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

[0174] When numerical ranges are given, it should be understood that every numerical value between the lower and upper bounds of the range is also explicitly stated. The use of "about" in connection with a numerical value herein can refer to plus or minus a margin of error of 10% of the value. Unless otherwise defined, 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 application belongs. Except as otherwise indicated, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. Although methods, devices, and materials similar or equivalent to those described herein can be used in the practice of the present application, the preferred materials, devices and methods are described herein. The following detailed description is made with reference to the accompanying drawings.

[0175] DEFINITIONS

[0176] A "double-stranded region" refers herein to a region comprising two nucleic acid strands that are antiparallel and complementary or substantially complementary.

[0177] As used herein, the term "double-stranded RNA" or "dsRNA" refers to a ribonucleic acid molecule or complex of ribonucleic acid molecules comprising a double-stranded region as defined above. The two parts forming the double-stranded region can be two different parts of one larger RNA molecule or they are separate RNA molecules.

[0178] When the two parts are separate RNA molecules, the dsRNA herein is referred to as a small interfering RNA or short interfering RNA, siRNA for short.

[0179] When the two parts are two different parts of one larger molecule, i.e. when the 3' end of one part is linked to the 5' end of the other part by one or more uninterrupted nucleotides between them forming the double-stranded region, the uninterrupted nucleotides used for the linkage are referred to as "hairpin loop". When the two parts are covalently linked to form the double-stranded region by other means than a hairpin loop, the linkage structure is referred to as "linker". Such dsRNAs are cleaved into siRNAs by an endoribonuclease in the cell, referred to as Dicer enzyme, after introduction into the cell.

[0180] The term "siRNA" herein is a class of double-stranded RNA molecules comprising a sense strand and an antisense strand that can mediate silencing of a target RNA (e.g. mRNA, e.g. a transcript of a gene encoding a protein) that is complementary or substantially complementary to the antisense strand. The siRNA is typically double-stranded, comprising an antisense strand that is complementary to a target RNA, and a sense strand that is complementary or substantially complementary to the antisense strand. For convenience, such mRNA is also referred to herein as mRNA to be silenced. Such gene is also referred to as target gene. Typically, the RNA to be silenced is an endogenous gene or a pathogen gene. In addition, RNA other than mRNA (e.g. tRNA) and viral RNA can also be targeted.

[0181] The term "antisense strand" as used herein refers to a strand in a dsRNA (in particular siRNA) that comprises a region that is fully complementary or substantially complementary to a target sequence.

[0182] As used herein, the term "complementary region" refers to a region on the antisense strand that is fully or substantially complementary to a target mRNA sequence. Where the complementary region is not fully complementary to the target sequence, mismatches can be located in the internal or terminal regions of the molecule. Generally, the most tolerated mismatches are located in the terminal regions, e.g., within 5, 4, 3, 2, or 1 nucleotides of the 5' and / or 3' end. The portion of the antisense strand that is most sensitive to mismatches is referred to as the "seed region." For example, in an siRNA comprising a 19 nt strand, the 19th position (from 5' to 3') can tolerate some mismatches.

[0183] As used herein, the term "complementary" refers to the ability of a first polynucleotide to hybridize to a second polynucleotide under certain conditions, e.g., stringent conditions. For example, stringent conditions can include 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA at 50°C or 70°C for 12-16 hours.

[0184] As used herein, "complementary" sequences can also include or be formed entirely from non-Watson-Crick base pairs and / or base pairs formed from non-natural and modified nucleotides, insofar as they meet the above requirements with respect to their ability to hybridize. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble base pairing or Hoogstein base pairing.

[0185] As used herein, a polynucleotide that is "at least partially complementary" or "substantially complementary" to a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of an mRNA of interest (e.g., an mRNA encoding apolipoprotein(a)). For example, a polynucleotide is at least partially complementary to an mRNA encoding apolipoprotein(a) if the sequence is substantially complementary to a non-interrupted portion of the mRNA encoding apolipoprotein(a).

[0186] The terms "complementary," "fully complementary," and "substantially complementary," as used herein, can be used in reference to base pairing between the sense strand and the antisense strand of a dsRNA, particularly an siRNA, or between the antisense strand of a dsRNA, particularly an siRNA, and a target sequence.

[0187] The term "sense strand," as used herein, refers to the strand of an siRNA that includes a region that is substantially complementary to a region that is an antisense strand as defined herein.

[0188] A "nucleoside" is a compound consisting of a purine or pyrimidine base, and either ribose or deoxyribose; a "nucleotide" is a compound consisting of a purine or pyrimidine base, ribose or deoxyribose, and a phosphate; and an "oligonucleotide" refers to a nucleic acid molecule (RNA or DNA) having, for example, fewer than 100, 200, 300, or 400 nucleotides in length.

[0189] A "base" is a basic building block of synthetic nucleosides, nucleotides, and nucleic acids, which contains nitrogen in its constituent elements, also known as a "nitrogenous base." As used herein, unless otherwise specified, the capital letters A, U, T, G, and C represent the base composition of a nucleotide, which are adenine, uracil, thymine, guanine, and cytosine, respectively.

[0190] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that overhangs from the double-stranded region of an siRNA. A nucleotide overhang is present, for example, when the 3 '-end of one strand of an siRNA extends beyond the 5 '-end of the other strand, or vice versa. An siRNA can comprise an overhang of at least one nucleotide, an overhang of at least two nucleotides, an overhang of at least three nucleotides, an overhang of at least four nucleotides, an overhang of at least five nucleotides or more. A nucleotide overhang can comprise or consist of nucleotides / modified nucleotides (including deoxynucleotides / nucleosides). One or more overhangs can be on the sense strand or the antisense strand or any combination thereof. An overhang of one or more nucleotides can be present on the 5 '-end, the 3 '-end, or both ends of the antisense or sense strand of an siRNA.

[0191] "Blunt end" or "blunt-ended" or "blunt" means that there are no unpaired nucleotides at that end of the double-stranded siRNA, i.e., no nucleotide overhang. A "blunt-ended siRNA" is an siRNA that is double-stranded over the entire length of the siRNA, i.e., there is no nucleotide overhang at either end of the molecule.

[0192] Substantially all of the nucleotides of a dsRNA (particularly an siRNA) of the present application are modified. For example, substantially all of the nucleotides of the sense strand are modified nucleotides, or substantially all of the nucleotides of the antisense strand are modified nucleotides, or substantially all of the nucleotides of both the sense strand and the antisense strand are modified nucleotides. In other embodiments of the present application, all of the nucleotides of a dsRNA (particularly an siRNA) of the present application are modified nucleotides. For example, all of the nucleotides of the sense strand are modified nucleotides, or all of the nucleotides of the antisense strand are modified nucleotides, or all of the nucleotides of both the sense strand and the antisense strand are modified nucleotides. As used herein, "substantially all of the nucleotides are modified" means that a majority of the nucleotides of a dsRNA (particularly an siRNA) of the present application are modified, but not all of the nucleotides are modified, and can include no more than 5, 4, 3, 2, or 1 unmodified nucleotides.

[0193] "2'-O-methyl modified nucleotide" means a nucleotide in which the 2'-hydroxyl of the ribose group is replaced with a methoxy group. "2'-deoxy-modified nucleotide" means a deoxyribonucleotide.

[0194] For example, "2'-fluoro-modified nucleotide" means a nucleotide in which the hydroxyl group at the 2' position of the ribose group is replaced with a fluorine atom. "2'-O-methyl modified nucleotide" means a nucleotide in which the 2'-hydroxyl of the ribose group is replaced with a methoxy group. "2'-deoxy-modified nucleotide" means a deoxyribonucleotide.

[0195] "Phosphorothioate internucleotide linkage" means a modification in which one or more of the oxygen atoms of the phosphate group of a nucleotide is replaced with a sulfur atom. "Phosphorothioate internucleotide linkage" means a modification in which the two nucleotides to the left and right are linked by a phosphorothioate.

[0196] In some embodiments, the sense strand of a dsRNA (particularly an siRNA) of the present disclosure has 1 or 2 phosphorothioate internucleotide linkage modifications at positions 1 to 5 (counting from the 5' end) and / or has 1 or 2 phosphorothioate internucleotide linkage modifications at positions 1 to 5 (counting from the 3' end), and / or the antisense strand of a dsRNA (particularly an siRNA) of the present disclosure has 1 or 2 phosphorothioate internucleotide linkage modifications at positions 1 to 5 (counting from the 5' end) and / or has 1 or 2 phosphorothioate internucleotide linkage modifications at positions 1 to 5 (counting from the 3' end).

[0197] In some embodiments, the sense strand of a dsRNA (particularly an siRNA) of the present disclosure has 1 or 2 phosphorothioate internucleotide linkage modifications located at positions 1 to 4 (counting from the 5' end) and / or has 1 or 2 phosphorothioate internucleotide linkage modifications located at positions 1 to 4 (counting from the 3' end), and / or the antisense strand of a dsRNA (particularly an siRNA) of the present disclosure has 1 or 2 phosphorothioate internucleotide linkage modifications located at positions 1 to 4 (counting from the 5' end) and / or has 1 or 2 phosphorothioate internucleotide linkage modifications located at positions 1 to 4 (counting from the 3' end).

[0198] In some embodiments, the sense strand of a dsRNA (particularly an siRNA) of the present disclosure has 1 or 2 phosphorothioate internucleotide linkage modifications located at positions 1 to 3 (counting from the 5' end) and / or has 1 or 2 phosphorothioate internucleotide linkage modifications located at positions 1 to 3 (counting from the 3' end), and / or the antisense strand of a dsRNA (particularly an siRNA) of the present disclosure has 1 or 2 phosphorothioate internucleotide linkage modifications located at positions 1 to 3 (counting from the 5' end) and / or has 1 or 2 phosphorothioate internucleotide linkage modifications located at positions 1 to 3 (counting from the 3' end).

[0199] In some embodiments, the sense strand of a dsRNA (particularly an siRNA) of the present disclosure has 1 or 2 phosphorothioate internucleotide linkage modifications located at positions 1 and 2 (counting from the 5' end) and / or has 1 or 2 phosphorothioate internucleotide linkage modifications located at positions 1 and 2 (counting from the 3' end), and / or the antisense strand of a dsRNA (particularly an siRNA) of the present disclosure has 1 or 2 phosphorothioate internucleotide linkage modifications located at positions 1 to 2 (counting from the 5' end) and / or has 1 or 2 phosphorothioate internucleotide linkage modifications located at positions 1 to 2 (counting from the 3' end).

[0200] As used herein, the term "inhibit" is used interchangeably with "reduce," "silence," "down-regulate," and other similar terms, and includes inhibition at any level.

[0201] The phrase "inhibiting expression of a TTR gene" refers to inhibiting expression of any TTR gene as well as variants or mutants of a TTR gene. Thus, the TTR gene can be a wild-type TTR gene, a mutant TTR gene, or a transgenic TTR gene in the context of a genetically manipulated cell, group of cells, or organism.

[0202] "Inhibiting expression of a TTR gene" includes any level of inhibition of a TTR gene, e.g., at least partial inhibition of TTR gene expression. TTR gene expression can be assessed based on the level or change in level of any variable associated with TTR gene expression, e.g., mRNA level of apolipoprotein(a), protein level of apolipoprotein(a). This level can be assessed in a single cell or in a group of cells, including, e.g., a sample derived from a subject.

[0203] Inhibition can be assessed by a decrease in the absolute or relative level of one or more variables associated with TTR gene expression compared to a control level. The control level can be any type of control level utilized in the art, e.g., pre-dose baseline level or a level determined from a similar untreated or control (e.g., buffer control or inert agent control) treated subject, cell, or sample.

[0204] As used herein, the terms "treat," "treatment," and the like, refer to administering an agent or performing a procedure in order to effect an outcome. These outcomes can be prophylactic, in terms of completely or partially preventing a disease or symptom thereof, and / or can be therapeutic, in terms of partially or completely curing a disease and / or symptoms of a disease. As used herein, "treatment" can include treatment of a disease or disorder in a mammal, particularly in a human, and includes: (a) preventing the disease or symptom of the disease from occurring (e.g., preventing a disease associated with or caused by the primary disease) in an individual that can be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; (c) relieving the disease, i.e., causing the disease to regress. Treatment can refer to any indication of success in treating or ameliorating or preventing cancer, including any objective or subjective parameters, e.g., elimination; remission; diminishing of symptoms or making the disease symptoms easier to tolerate; slowing in the rate of worsening or degeneration; or lessening of the disease's end-point. Treatment or amelioration of symptoms is based on one or more objective or subjective parameters; including results of a physician's examination. Thus, the term "treatment" includes the administration of a dsRNA, cell, or pharmaceutical composition disclosed herein to prevent or delay, to alleviate or arrest or inhibit the development of symptoms or conditions associated with a disease. The term "therapeutic effect" refers to the reduction, elimination, or prevention of a disease, disease symptoms, or disease side effects in a subject.

[0205] The term "effective amount" as used herein refers to an amount that is sufficient, when administered to a subject for treatment of a disease, to effect treatment of such disease.

[0206] As used herein, the term "subject" refers to any mammalian subject in whom diagnosis, management or treatment is desired. "Mammalian" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and laboratory and sport animals, such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, and the like.

[0207] I. dsRNA

[0208] The present application provides a double-stranded RNA (dsRNA) for inhibiting TTR gene expression, the dsRNA comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand and the antisense strand are each independently 15-30 nucleotides in length, and the antisense strand comprises a nucleotide sequence of at least 15 contiguous nucleotides of the nucleotide sequence set forth in any one of SEQ ID NOs: 132-262.

[0209] In some embodiments, the double-stranded region formed by the sense strand and the antisense strand is fully complementary. In other embodiments, the double-stranded region formed by the sense strand and the antisense strand is substantially complementary, wherein it can comprise 1, 2, 3, 4, or 5 non-complementary sites.

[0210] In some specific embodiments, the sense strand comprises a nucleotide sequence of at least 15 contiguous nucleotides of the nucleotide sequence set forth in any one of SEQ ID NOs: 1-131.

[0211] In some embodiments, the sense strand and the antisense strand are each independently 15-27 nucleotides in length, for example 16, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 nucleotides, preferably 18-25 nucleotides, more preferably 19-21 nucleotides.

[0212] In some embodiments, the double-stranded region is 15-25 nucleotide pairs in length, for example 15, 16, 17, 18, 19, 20, 21, 22, 24, 3, 24, 25 nucleotide pairs, preferably 16-23 nucleotide pairs, more preferably 18-20 nucleotide pairs.

[0213] In some embodiments, the dsRNA of the present application is an siRNA. In other embodiments, a hairpin loop is formed between the sense strand and the antisense strand of the dsRNA of the present application.

[0214] One or both of the sense strand and the antisense strand comprises a 3' overhang and / or a 5' overhang of at least 1 nucleotide. In some embodiments, one or both of the sense strand and the antisense strand comprises a 3' overhang and / or a 5' overhang of at least 1 nucleotide. In some specific embodiments, one or both of the sense strand and the antisense strand comprises a 3' overhang and / or a 5' overhang of 1 nucleotide. In some specific embodiments, one or both of the sense strand and the antisense strand comprises a 3' overhang and / or a 5' overhang of 2 nucleotides. In some specific embodiments, one or both of the sense strand and the antisense strand comprises a 3' overhang and / or a 5' overhang of 3 nucleotides. In some specific embodiments, one or both of the sense strand and the antisense strand comprises a 3' overhang and / or a 5' overhang of 4 nucleotides.

[0215] In some specific embodiments, the antisense strand comprises a 3' overhang and / or a 5' overhang of 1 nucleotide. In some embodiments, the antisense strand comprises a 3' overhang and / or a 5' overhang of 2 nucleotides. In some embodiments, the antisense strand comprises a 3' overhang and / or a 5' overhang of 3 nucleotides. In some embodiments, the antisense strand comprises a 3' overhang and / or a 5' overhang of 4 nucleotides. In some preferred embodiments, the antisense strand has a 3' overhang and / or a 5' overhang of at least 2 nucleotides.

[0216] Preferably the antisense strand comprises a 3' overhang and / or a 5' overhang of 2 nucleotides.

[0217] In some embodiments, the sense strand and the antisense strand are of the same length.

[0218] In some embodiments, the full length of the sense strand is complementary to the full length of the antisense strand forming a double strand, i.e. having blunt ends.

[0219] In other embodiments, the sense strand and the antisense strand are of the same length, a portion of the sense strand is complementary to a portion of the antisense strand, i.e. both the sense strand and the antisense strand have a 5' overhang. In some embodiments, the sense strand and the antisense strand are of different lengths. In preferred embodiments, the 5' end of the antisense strand has an overhang of at least 1 nucleotide, more preferably 2 or 3 nucleotides.

[0220] The dsRNAs of the application include dsRNAs having a nucleotide overhang at one end (i.e., agents having one overhang and one blunt end) or having a nucleotide overhang at both ends. For example, the 5 '-end of the sense strand of the dsRNA comprises an overhang of one or more nucleotides and the 3 '-end of the sense strand comprises an overhang of one or more nucleotides. For example, the 5 '-end of the antisense strand of the dsRNA comprises an overhang of one or more nucleotides and the 3 '-end of the antisense strand comprises an overhang of one or more nucleotides. For example, the 5 '-end of the sense strand of the dsRNA comprises an overhang of one or more nucleotides and the 5 '-end of the antisense strand comprises an overhang of one or more nucleotides. For example, the 3 '-end of the sense strand of the dsRNA comprises an overhang of one or more nucleotides and the 3 '-end of the antisense strand comprises an overhang of one or more nucleotides. For example, the 5 '-end of the sense strand of the dsRNA comprises an overhang of one or more nucleotides and the 3 '-end of the sense strand comprises a blunt end. For example, the 3 '-end of the sense strand of the dsRNA comprises an overhang of one or more nucleotides and the 5 '-end of the sense strand comprises a blunt end. For example, the 5 '-end of the antisense strand of the dsRNA comprises an overhang of one or more nucleotides and the 3 '-end of the antisense strand comprises a blunt end. For example, the 3 '-end of the antisense strand of the dsRNA comprises an overhang of one or more nucleotides and the 5 '-end of the antisense strand comprises a blunt end.

[0221] In some preferred embodiments, the 3 '-end of the antisense strand of the dsRNA of the application comprises an overhang of one or more nucleotides and the 5 '-end of the antisense strand comprises a blunt end. In some more preferred embodiments, the 3 '-end of the antisense strand of the dsRNA of the application comprises an overhang of 1, 2, 3, or 4 nucleotides and the 5 '-end of the antisense strand comprises a blunt end. In some more preferred embodiments, the 3 '-end of the antisense strand of the dsRNA of the application comprises an overhang of 2 nucleotides and the 5 '-end of the antisense strand comprises a blunt end.

[0222] In some embodiments, the antisense strand comprises a nucleotide sequence of at least 16 contiguous nucleotides, at least 17 contiguous nucleotides, at least 18 contiguous nucleotides, at least 19 contiguous nucleotides, or at least 20 contiguous nucleotides of the nucleotide sequence set forth in any one of SEQ ID NOs: 132-262, preferably the antisense strand comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 132-262.

[0223] In some embodiments, the sense strand comprises a nucleotide sequence of at least 16 contiguous nucleotides, a nucleotide sequence of at least 17 contiguous nucleotides, or a nucleotide sequence of at least 18 contiguous nucleotides of any of the nucleotide sequences set forth in SEQ ID NOs: 1-131, preferably the sense strand comprises the nucleotide sequence set forth in any of SEQ ID NOs: 1-131.

[0224] In some embodiments, the dsRNA comprises any of the paired sense strand sequence and antisense strand sequence as set forth in Table 3.

[0225] II. Modifications of Nucleotides

[0226] In some embodiments, substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides. In some embodiments, at least 80% of the nucleotides of the sense strand are modified nucleotides, and / or at least 80%, at least 85%, at least 90%, at least 92%, at least 95% of the nucleotides of the antisense strand are modified nucleotides. In some embodiments, at least 80% of the nucleotides of the antisense strand are modified nucleotides, and / or at least 80%, at least 85%, at least 90%, at least 92%, at least 95% of the nucleotides of the sense strand are modified nucleotides.

[0227] In some embodiments, all of the nucleotides of the sense strand are modified nucleotides and / or all of the nucleotides of the antisense strand are modified nucleotides.

[0228] The modifications of nucleotides described herein can be modifications on the phosphate group, the ribose group, and / or the base group of the nucleotide.

[0229] In some specific embodiments, the sense strand and the antisense strand each independently comprise one or more nucleotide modifications selected from the group consisting of: 2'-0-alkyl-modified nucleotides (e.g., 2'-0-methyl-modified nucleotides), 2'-methoxyethyl-modified nucleotides, 2'-fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, inosine ribonucleotides, abasic nucleotides, inverted abasic deoxyribonucleotides, nucleotides comprising phosphorothioate groups, vinylphosphonate-modified nucleotides, locked nucleotides, unlocked nucleotides, 2'-amino-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-0-allyl-modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides comprising non-natural bases, terminal nucleotides linked to a cholesteryl derivative or a dodecanoic acid didecanoyl amide group, deoxyribonucleotides, 3'-terminal deoxythymine (dT) nucleotides, conformationally restricted nucleotides, restricted ethyl nucleotides, 2'-hydroxyl-modified nucleotides, nucleotides comprising methylphosphonate groups, nucleotides comprising 5'-phosphates, nucleotides comprising 5'-phosphate mimics, glycol-modified nucleotides (GNAs), and 2-0-(N-methylacetamide)-modified nucleotides.

[0230] In some preferred embodiments, the sense strand and the antisense strand each independently comprise one or more nucleotide modifications selected from the group consisting of: 2'-0-methyl-modified nucleotides, 2'-fluoro-modified nucleotides, and nucleotides comprising phosphorothioate groups. In some preferred embodiments, the sense strand and / or the antisense strand comprises at least 2 2'-fluoro-modified nucleotides. In some preferred embodiments, the sense strand and / or the antisense strand comprises at least 8 2'-0-methyl-modified nucleotides. In some preferred embodiments, the 3' end and / or the 5' end of the sense strand and / or the antisense strand comprises 1-5 phosphorothioate internucleotide linkages, preferably 2-3 phosphorothioate internucleotide linkages.

[0231] In some preferred embodiments, the antisense strand comprises any one of the nucleotide sequences set forth in Table 5, and / or the sense strand comprises any one of the nucleotide sequences set forth in Table 4A.

[0232] III. Ligand Moieties

[0233] In some embodiments, the dsRNA is further conjugated to a ligand moiety, e.g., a carrier that can deliver the dsRNA to the liver; e.g., a ligand targeting the asialoglycoprotein receptor (ASGPR).

[0234] In some embodiments, the dsRNA is conjugated to a ligand moiety comprising N-acetylgalactosamine, preferably via a phosphate group or a phosphorothioate group to a ligand moiety comprising N-acetylgalactosamine.

[0235] In some embodiments, the sense strand of the dsRNA is conjugated to the ligand moiety, preferably the 3' end of the sense strand is conjugated to the ligand moiety.

[0236] In some embodiments, the ligand moiety comprises a conjugation group according to Formula (X'):

[0237] wherein,

[0238] represents the position of attachment to the dsRNA;

[0239] Q is independently H,

[0240] wherein L1is a bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O))a-;

[0241] L2is a bond or -CH2CH2C(O)-;

[0242] L3is a bond, -(NHCH2CH2)b-, -(NHCH2CH2CH2)b-, or -C(O)CH2-;

[0243] L4is -(OCH2CH2)c-, -(OCH2CH2CH2)c-, -(OCH2CH2CH2CH2)c-, -(OCH2CH2CH2CH2CH2)c-, or -NHC(O)-(CH2)d-;

[0244] wherein a = 0, 1, 2, or 3;

[0245] b = 1, 2, 3, 4, or 5;

[0246] c = 1, 2, 3, 4, or 5;

[0247] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0248] L is a bond, -CH2O-, or -NHC(O)-;

[0249] L' is a bond, -C(O)NH-, -NHC(O)-, or -O(CH2CH2O)e-;

[0250] wherein e is 1, 2, 3, 4, or 5;

[0251] T is a chemical bond, -CH2-, -C(O)-, -M-, -CH2-M-, or -C(O)-M-;

[0252] wherein M is

[0253] R1and R2together form -CH2CH2O- or -CH2CH(R)-O-, and R3is H;

[0254] or R1and R3together form -C1-2alkylene-, and R2is H;

[0255] wherein R is -OR', -CH2OR', or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group, or a solid support, the hydroxyl protecting group preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0256] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0257] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0258] In some embodiments, the conjugate group is represented by Formula (I'):

[0259] wherein,

[0260] represents the position of attachment to the dsRNA;

[0261] Q is independently H,

[0262] wherein L1is a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O))a-;

[0263] L2is a chemical bond or -CH2CH2C(O)-;

[0264] L3is a chemical bond, -(NHCH2CH2)b-, -(NHCH2CH2CH2)b-, or -C(O)CH2-;

[0265] L4is -(OCH2CH2)c-, -(OCH2CH2CH2)c-, -(OCH2CH2CH2CH2)c-, -(OCH2CH2CH2CH2CH2)c-, or -NHC(O)-(CH2)d-;

[0266] wherein a = 0, 1, 2, or 3;

[0267] b = 1, 2, 3, 4, or 5;

[0268] c = 1, 2, 3, 4, or 5;

[0269] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0270] L is -CH2O- or -NHC(O)-;

[0271] L' is a bond, -C(O)NH-, or -NHC(O)-;

[0272] R1and R2together form -CH2CH2O- or -CH2CH(R)-O-, and R3is H;

[0273] or R1and R3together form -C1-2alkylene-, and R2is H;

[0274] wherein R is -OR', -CH2OR', or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group, or a solid support, the hydroxyl protecting group preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0275] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0276] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0277] In some specific embodiments, wherein,

[0278] Q is independently H or

[0279] wherein L1is -CH2O- or -NHC(O)-(CH2NHC(O))a-;

[0280] L2is -CH2CH2C(O)-;

[0281] L3is -(NHCH2CH2)b- or -(NHCH2CH2CH2)b-;

[0282] L4is -(OCH2CH2)c- or -NHC(O)-(CH2)d-;

[0283] wherein a = 0, 1, 2, or 3;

[0284] b = 1, 2, 3, 4, or 5;

[0285] c = 1, 2, 3, 4, or 5;

[0286] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0287] L is -CH2O-;

[0288] L' is a bond;

[0289] R1and R2together form -CH2CH2O- or -CH2CH(R)-O-, and R3is H;

[0290] or R1and R3together form -C1-2alkylene-, and R2is H;

[0291] wherein R is -OR', -CH2OR', or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group, or a solid support, the hydroxyl protecting group preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0292] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0293] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0294] In some embodiments, the conjugate group is represented by Formula (I'-1), Formula (I'-2), or Formula (I'-3):

[0295] wherein,

[0296] represents the position of attachment to the dsRNA;

[0297] Q is

[0298] wherein L1is -CH2O- or -NHC(O)-;

[0299] L2is -CH2CH2C(O)-;

[0300] L3is -(NHCH2CH2)b- or -(NHCH2CH2CH2)b-;

[0301] L4is -(OCH2CH2)c- or -NHC(O)-(CH2)d-;

[0302] wherein b = 1, 2, 3, 4, or 5;

[0303] c = 1, 2, 3, 4, or 5;

[0304] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0305] L is -CH2O-.

[0306] R' is H, a hydroxyl protecting group, preferably -C(O)CH2CH2C(O)OH or 4,4'- dimethoxytrityl, or a solid support;

[0307] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0308] In some specific embodiments, wherein,

[0309] Q is independently H,

[0310] wherein L1is -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O))a-;

[0311] L3is -(NHCH2CH2)b-, -(NHCH2CH2CH2)b-, or -C(O)CH2-;

[0312] L4is -(OCH2CH2)c- or -NHC(O)-(CH2)d-;

[0313] wherein a = 0, 1, 2, or 3;

[0314] b = 1, 2, 3, 4, or 5;

[0315] c = 1, 2, 3, 4, or 5;

[0316] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0317] L is -CH2O- or -NHC(O)-;

[0318] L' is a chemical bond or -C(O)NH-;

[0319] R1and R2together form -CH2CH2O- or -CH2CH(R)-O-, and R3is H;

[0320] or R1and R3together form -C1-2alkylene-, and R2is H;

[0321] wherein R is -OR', -CH2OR', or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group, preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl, or a solid support;

[0322] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0323] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0324] In some embodiments, the conjugate group is represented by Formula (II'-1) or Formula (II'-2):

[0325] wherein,

[0326] represents the position of attachment to the dsRNA;

[0327] Q is independently H,

[0328] wherein L1is -CH2O- or -CH2O-CH2CH2O-;

[0329] L3is -(NHCH2CH2)b-, -(NHCH2CH2CH2)b-, or -C(O)CH2-;

[0330] L4is -(OCH2CH2)c- or -NHC(O)-(CH2)d-;

[0331] wherein b = 1, 2, 3, 4, or 5;

[0332] c = 1, 2, 3, 4, or 5;

[0333] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0334] L is -NHC(O)-;

[0335] L' is a chemical bond or -C(O)NH-;

[0336] R' is H, a hydroxyl protecting group, preferably -C(O)CH2CH2C(O)OH or 4,4'- dimethoxytrityl, or a solid support;

[0337] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0338] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0339] In some specific embodiments, wherein,

[0340] Q is independently H,

[0341] wherein L1is -CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O))a-;

[0342] L2is a bond;

[0343] L3is -(NHCH2CH2)b-, -(NHCH2CH2CH2)b- or -C(O)CH2-;

[0344] L4is -(OCH2CH2)c- or -NHC(O)-(CH2)d-;

[0345] wherein a = 0, 1, 2 or 3;

[0346] b = 1, 2, 3, 4 or 5;

[0347] c = 1, 2, 3, 4 or 5;

[0348] d = 1, 2, 3, 4, 5, 6, 7 or 8;

[0349] L is -CH2O- or -NHC(O)-;

[0350] L' is a bond or -C(O)NH-;

[0351] R1and R2together form -CH2CH2O- or -CH2CH(R)-O- and R3is H;

[0352] or R1and R3together form -C1-2alkylene- and R2is H;

[0353] wherein R is -OR', -CH2OR' or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group or a solid support, the hydroxyl protecting group preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0354] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0355] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0356] In some embodiments, wherein the conjugate group is represented by formula (II'-2):

[0357] wherein,

[0358] represents the position of attachment to the dsRNA;

[0359] Q is independently

[0360] wherein L1is -CH2- or -C(O)-;

[0361] L3 is -(NHCH2CH2)b-;

[0362] L4 is -(OCH2CH2)c-;

[0363] wherein b = 1, 2, 3, 4, or 5;

[0364] c = 1, 2, 3, 4, or 5;

[0365] L is -CH2O- or -NHC(O)-;

[0366] R' is H, a hydroxyl protecting group, preferably -C(O)CH2CH2C(O)OH or 4,4'- dimethoxytrityl, or a solid support;

[0367] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0368] In some specific embodiments, wherein:

[0369] Q is independently H,

[0370] wherein L1 is a bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or - NHC(O)-(CH2NHC(O))a-;

[0371] L2 is a bond or -CH2CH2C(O)-;

[0372] L3 is a bond, -(NHCH2CH2)b-, -(NHCH2CH2CH2)b-, or -C(O)CH2-;

[0373] L4 is -(OCH2CH2)c-, -(OCH2CH2CH2)c-, -(OCH2CH2CH2CH2)c-, -(OCH2CH2CH2CH2CH2)c-, or -NHC(O)-(CH2)d-;

[0374] wherein a = 0, 1, 2, or 3;

[0375] b = 1, 2, 3, 4, or 5;

[0376] c = 1, 2, 3, 4, or 5;

[0377] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0378] L is a bond, -CH2O-, or -NHC(O)-;

[0379] L' is a bond, -C(O)NH-, -NHC(O)-, or -O(CH2CH2O)e-;

[0380] wherein e is 1, 2, 3, 4, or 5;

[0381] T is a bond, -CH2-, -M-, -CH2-M-, or -C(O)-M-;

[0382] wherein M is

[0383] R1and R2together form -CH2CH2O- or -CH2CH(R)-O-, and R3is H;

[0384] or R1and R3together form -C1-2alkylene-, and R2is H;

[0385] wherein R is -OR', -CH2OR', or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group, or a solid support, the hydroxyl protecting group preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0386] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0387] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0388] In some specific embodiments, wherein,

[0389] T is -M-, -CH2-M-, or -C(O)-M-, wherein M is

[0390] In some specific embodiments, wherein,

[0391] Q is independently H or

[0392] wherein L1is -CH2O- or -NHC(O)-(CH2NHC(O))a-;

[0393] L2is -CH2CH2C(O)-;

[0394] L3is -(NHCH2CH2)b- or -(NHCH2CH2CH2)b-;

[0395] L4is -(OCH2CH2)c- or -NHC(O)-(CH2)d-;

[0396] wherein a = 0, 1, 2 or 3;

[0397] b = 1, 2, 3, 4 or 5;

[0398] c = 1, 2, 3, 4 or 5;

[0399] d = 1, 2, 3, 4, 5, 6, 7 or 8;

[0400] L is a chemical bond or -CH2O-;

[0401] L' is a chemical bond or -0(CH2CH2O)e-;

[0402] wherein e is 1, 2, 3, 4 or 5;

[0403] R1and R2together form -CH2CH2O- or -CH2CH(R)-O-, and R3is H;

[0404] or R1and R3together form -C1-2alkylene-, and R2is H;

[0405] wherein R is -OR', -CH2OR' or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group or a solid support, the hydroxyl protecting group preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0406] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0407] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0408] wherein T is as defined in the above embodiments.

[0409] In some embodiments, wherein the conjugate group is as shown in formula (III'-1), formula (III'-2) or formula (III'-3):

[0410] wherein,

[0411] Q is

[0412] wherein L1is -CH2O- or -NHC(O)-;

[0413] L2is -CH2CH2C(O)-;

[0414] L3is -(NHCH2CH2)b- or -(NHCH2CH2CH2)b-;

[0415] L4is -(OCH2CH2)c- or -NHC(O)-(CH2)d-;

[0416] wherein b = 1, 2, 3, 4, or 5;

[0417] c = 1, 2, 3, 4, or 5;

[0418] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0419] L is a bond or -CH2O-;

[0420] wherein R' is H, a hydroxyl protecting group, preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl, or a solid support;

[0421] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0422] wherein T is as defined in the above embodiments.

[0423] In some specific embodiments, wherein,

[0424] Q is independently H,

[0425] wherein L1is -CH2-, -CH2O-, or -C(O)-;

[0426] L2is a bond;

[0427] L3is -(NHCH2CH2)b-, -(NHCH2CH2CH2)b-, or -C(O)CH2-;

[0428] L4is -(OCH2CH2)c- or -NHC(O)-(CH2)d-;

[0429] wherein b = 1, 2, 3, 4, or 5;

[0430] c = 1, 2, 3, 4, or 5;

[0431] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0432] L is a bond or -NHC(O)-;

[0433] L' is a bond;

[0434] R1and R2together form -CH2CH2O- or -CH2CH(R)-O-, and R3is H;

[0435] or R1and R3together form -C1-2alkylene-, and R2is H;

[0436] wherein R is -OR', -CH2OR', or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group, or a solid support, the hydroxyl protecting group preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0437] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0438] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0439] wherein T is as defined in the above embodiments.

[0440] In some embodiments, wherein the conjugate group is as shown in Formula (IV-1) or Formula (IV-2):

[0441] wherein,

[0442] Q is independently

[0443] wherein L1is -CH2-, -CH2O-, or -C(O)-;

[0444] L3is -(NHCH2CH2)b-, -(NHCH2CH2CH2)b-, or -C(O)CH2-;

[0445] L4is -(OCH2CH2)c- or -NHC(O)-(CH2)d-;

[0446] wherein b = 1, 2, 3, 4, or 5;

[0447] c = 1, 2, 3, 4, or 5;

[0448] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0449] L is a chemical bond or -NHC(O)-;

[0450] L' is a chemical bond;

[0451] wherein R' is H, a hydroxyl protecting group, or a solid support, the hydroxyl protecting group preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0452] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0453] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0454] wherein T is as defined in the above embodiments.

[0455] In some specific embodiments, wherein:

[0456] Q is independently H,

[0457] wherein L1is a bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O))a-;

[0458] L2is a bond or -CH2CH2C(O)-;

[0459] L3is a bond, -(NHCH2CH2)b-, -(NHCH2CH2CH2)b-, or -C(O)CH2-;

[0460] L4is -(OCH2CH2)c-, -(OCH2CH2CH2)c-, -(OCH2CH2CH2CH2)c-, -(OCH2CH2CH2CH2CH2)c-, or -NHC(O)-(CH2)d-;

[0461] wherein a = 0, 1, 2, or 3;

[0462] b = 1, 2, 3, 4, or 5;

[0463] c = 1, 2, 3, 4, or 5;

[0464] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0465] L is a bond, -CH2O-, or -NHC(O)-;

[0466] L' is -O(CH2CH2O)e-;

[0467] wherein e is 1, 2, 3, 4, or 5;

[0468] T is a bond, -CH2-, -C(O)-, -M-, -CH2-M-, or -C(O)-M-;

[0469] wherein M is

[0470] R1and R2together form -CH2CH2O- or -CH2CH(R)-O-, and R3is H;

[0471] or R1and R3together form -C1-2alkylene-, and R2is H;

[0472] wherein R is -OR', -CH2OR', or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group, or a solid support, preferably -C(O)CH2CH2C(O)OH or 4,4'- dimethoxytrityl;

[0473] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0474] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0475] In some preferred embodiments, wherein the conjugate group is selected from the following Table 1:

[0476] Table 1

[0477] In some preferred embodiments, wherein the conjugate group is selected from the following Table 2:

[0478] Table 2

[0479] In some embodiments, the ligand targets an asialoglycoprotein receptor (ASGPR). In some embodiments, the ligand targets an asialoglycoprotein receptor (ASGPR) on a hepatocyte.

[0480] In one preferred embodiment, wherein the ligand has the following structure:

[0481] wherein represents the position of attachment to the dsRNA via a phosphoester or phosphorothioate group.

[0482] In one preferred embodiment, wherein the ligand has the following structure:

[0483] wherein represents the position of attachment to the dsRNA via a phosphoester or phosphorothioate group.

[0484] In one preferred embodiment, wherein the ligand has the following structure:

[0485] wherein denotes the position of attachment to the sense strand of the dsRNA via a phosphonate or phosphorothioate group. In a preferred embodiment, wherein the ligand has the following structure:

[0486] wherein denotes the position of attachment to the sense strand of the dsRNA via a phosphonate or phosphorothioate group.

[0487] In some embodiments, the sense strand comprises any one of the nucleotide sequences set forth in Table 4B of the specification, preferably the antisense strand comprises any one of the nucleotide sequences set forth in Table 5 of the specification.

[0488] In some preferred embodiments, the sense strand and the antisense strand of the dsRNA comprise the paired sense strand sequence and antisense strand sequence set forth in any one of Table 6C, respectively.

[0489] IV. Inhibition of TTR gene expression

[0490] The dsRNAs of the present application are capable of inhibiting TTR gene expression by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0491] Inhibition of TTR gene expression can be manifested by a decrease in the amount of mRNA expressed by a first cell or group of cells (such cells can be present in, for example, a sample derived from a subject) in which the TTR gene is transcribed and which cell or cells has been treated (e.g., by contacting the cell or cells with a dsRNA of the present application, or by administering a dsRNA of the present application to a subject in which the cells now or formerly exist), such that TTR gene expression is inhibited as compared to a second cell or group of cells (control cell(s)) that is / are substantially identical to the first cell or group of cells but has / have not been so treated.

[0492] In preferred embodiments, the inhibition is assessed by expressing the level of mRNA in the treated cell as a percentage of the level of mRNA in the control cell using the following formula. In some specific embodiments, the 2 -△△CtValues are expressed as differences between experimental and control groups, where ΔΔCt = [(Ct experimental gene of interest - Ct experimental internal control) - (Ct control gene of interest - Ct control internal control)].

[0493] Control cells or groups of cells that can be used to assess inhibition of TTR gene expression include cells or groups of cells that have not been contacted with a dsRNA of the present application. For example, the control cells or groups of cells can be derived from the individual subject (e.g., human or animal subject) prior to treatment of the subject with a dsRNA.

[0494] V. Cells

[0495] The present application provides cells containing a dsRNA of the present application.

[0496] VI. Pharmaceutical Compositions

[0497] The present application provides pharmaceutical compositions comprising a dsRNA or cell of the present application, and optionally a pharmaceutically acceptable carrier or excipient.

[0498] As used herein, "pharmaceutically acceptable" means those compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for contact with the tissues of human subjects and animal subjects without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0499] In the present context, a pharmaceutically acceptable carrier refers to a pharmaceutical carrier that aids in the administration of a dsRNA or cell comprising the same to humans and / or facilitates its absorption or action. For example: diluents, excipients such as water, etc., fillers such as starch, sucrose, etc.; binders such as cellulose derivatives, alginates, gelatin and polyvinylpyrrolidone; humectants such as glycerin; disintegrants such as agar, calcium carbonate and sodium bicarbonate; absorption accelerators such as quaternary ammonium compounds; surfactants such as cetyl alcohol; adsorptive carriers such as kaolin and bentonite clay; lubricants such as talc, calcium / magnesium stearate, polyethylene glycol, etc. Other auxiliary agents such as flavoring agents, sweetening agents, etc. can also be added to the composition.

[0500] The pharmaceutical composition of the present application can comprise a pharmaceutically acceptable diluent or sustained release matrix into which a dsRNA or cell of the present application is embedded.

[0501] The pharmaceutical compositions of the present application can comprise a drug delivery system for delivery of the dsRNA. The drug delivery systems of the present application include, but are not limited to, nanoparticles (e.g., lipid nanoparticles, polymer-based nanoparticles), polymers, PEG, or cationic delivery systems, polylactic acid (PLA) microspheres, poly(lactic-co-glycolic acid) (PLGA) microspheres, liposomes, micelles, inverse micelles, lipid cochleates, lipid microtubules, cholesterol, PEG lipid PEG-2000-C-DMG, PEG-2000-DMG (Moderna), ALC-0159, or DSPC.

[0502] In some embodiments, the dsRNA in the pharmaceutical compositions of the present application can be contained in polymers and polymer-based nanoparticles.

[0503] In some specific embodiments, the polymer is a poly(lactic-co-glycolic acid) (PLGA)-based polymer. In some specific embodiments, the PLGA-based polymer is engineered to contain a cationic group alone.

[0504] In some specific embodiments, the polymer contains amine groups that can become cationic, such as polyethylenimine (PEI) and poly(L-lysine) (PLL), which can form complexes with the dsRNA through electrostatic interactions and deliver the dsRNA into cells. In some embodiments, the PEG and PLL are chemically modified to improve in vivo efficacy and tolerability.

[0505] In some embodiments, the siRNA or cells in the pharmaceutical compositions of the present application can be delivered by the cationic polymer poly(beta-amino ester) (PBAE).

[0506] VII. Kits

[0507] The present application provides kits comprising the dsRNA or cells described herein.

[0508] The present application also provides kits for using the dsRNA or cells described herein and / or performing the methods of the present application. Such kits include one or more of the dsRNA or cells described herein, and can further include instructions for use. The instructions for use can include instructions for inhibiting TTR gene expression in a cell by contacting the cell with the dsRNA described herein in an amount effective to inhibit TTR gene expression.

[0509] In the case where the dsRNA of the present application is contacted with a cell in vitro, optionally, the kit of the present application can further comprise a means for contacting the cell with the dsRNA of the present application (e.g., an injection device) or a means for measuring the inhibitory effect on the TTR gene (e.g., a device for measuring the inhibition of TTR mRNA or protein). Such a device for measuring the inhibition of the TTR gene can comprise a device for obtaining a sample from a subject.

[0510] In the case where the dsRNA of the present application or a cell into which the dsRNA has been introduced in vitro is administered in vivo, the kit of the present application can further optionally comprise a device for administering the dsRNA of the present application or the cell to a subject or a device for determining a therapeutically effective amount or a prophylactically effective amount.

[0511] VIII. Therapeutic methods, pharmaceutical uses

[0512] The present application provides a method of inhibiting expression of a TTR gene in a cell, the method comprising contacting the cell with a dsRNA or a pharmaceutical composition of the present application. In some embodiments, the method is performed in vitro. In some embodiments, the method is performed in vitro. The present application provides a dsRNA or a pharmaceutical composition of the present application for use in inhibiting expression of a TTR gene in a cell.

[0513] The present application provides a method of inhibiting expression of a TTR gene in a cell in a subject, the method comprising administering to the subject a dsRNA, a cell or a pharmaceutical composition of the present application. The present application provides a dsRNA, a cell or a pharmaceutical composition of the present application for use in inhibiting expression of a TTR gene in a cell in a subject. The present application provides use of a dsRNA, a cell or a pharmaceutical composition of the present application in the manufacture of a medicament for inhibiting expression of a TTR gene in a cell in a subject.

[0514] The present application also provides a method of treating a disease or disorder that benefits from a reduction in TTR gene expression in a subject, the method comprising administering to the subject a dsRNA, a cell or a pharmaceutical composition of the present application. The present application also provides a dsRNA, a cell or a pharmaceutical composition of the present application for use in treating a disease or disorder that benefits from a reduction in TTR gene expression in a subject. The present application also provides use of a dsRNA, a cell or a pharmaceutical composition of the present application in the manufacture of a medicament for treating a disease or disorder that benefits from a reduction in TTR gene expression in a subject.

[0515] The present application also provides a method of preventing at least one symptom in a subject having a disease or disorder that benefits from a reduction in TTR gene expression, the method comprising administering to the subject a dsRNA, cell or pharmaceutical composition of the present application. The present application also provides a dsRNA, cell or pharmaceutical composition of the present application for use in preventing a disease or disorder that benefits from a reduction in TTR gene expression. The present application also provides the use of a dsRNA, cell or pharmaceutical composition of the present application in the manufacture of a medicament for preventing a disease or disorder that benefits from a reduction in TTR gene expression.

[0516] The present application also provides a method of preventing the progression of a disease or disorder that benefits from a reduction in TTR gene expression in a subject, the method comprising administering to the subject a dsRNA, cell or pharmaceutical composition of the present application. The present application also provides a dsRNA, cell or pharmaceutical composition of the present application for use in preventing the progression of a disease or disorder that benefits from a reduction in TTR gene expression in a subject. The present application also provides the use of a dsRNA, cell or pharmaceutical composition of the present application in the manufacture of a medicament for preventing the progression of a disease or disorder that benefits from a reduction in TTR gene expression in a subject.

[0517] In some embodiments, the disease or disorder that benefits from a reduction in TTR gene expression is a TTR-associated disease. In some preferred embodiments, the TTR-associated disease is selected from the group consisting of senile systemic amyloidosis, systemic familial amyloidosis, familial amyloidotic polyneuropathy, familial amyloid cardiomyopathy, leptomeningeal / central nervous system amyloidosis, hyperthyroxinemia, Sturge-Weber disease, diabetic retinopathy, age-related macular degeneration, insulin resistance and cardiovascular disease associated with type II diabetes.

[0518] In some embodiments, the dsRNA, cell or pharmaceutical composition is administered subcutaneously.

[0519] In some embodiments, the subject is a mammal. In some embodiments, the subject is a primate mammal. In some embodiments, the subject is a human.

[0520] Sequences

[0521] The RNA sequences provided by the present application target the human TTR gene (or target gene, target mRNA sequence, target sequence). The target TTR mRNA sequence is the gene set forth in Genbank Accession No. NM_000371.4, for example.

[0522] Table 3. Nucleotide sequences of sense and antisense strands targeting TTR mRNA (wherein the sequences of the same row pair the sense and antisense strand sequences that are paired)

[0523] Tables 4A-B and Table 5 show modified RNA sequences used in the present application, respectively.

[0524] Herein, the meaning of each abbreviation is as follows:

[0525] A, U, G, and C represent natural adenosine ribonucleotides, uracil ribonucleotides, guanosine ribonucleotides, and cytosine ribonucleotides, respectively.

[0526] d represents that the nucleotide adjacent to its right is a deoxyribonucleotide. For example, dA, dT, dG, and dC represent adenosine deoxyribonucleotide, thymine deoxyribonucleotide, guanosine deoxyribonucleotide, and cytosine deoxyribonucleotide, respectively.

[0527] i represents an inosine ribonucleotide.

[0528] m represents that the nucleotide adjacent to its left is a 2’-OCH3 modified nucleotide. For example, Am, Um, Gm, and Cm represent 2’-OCH3 modified A, U, G, and C, respectively.

[0529] f represents that the nucleotide adjacent to its left is a 2’-fluoro modified nucleotide. For example, Af, Uf, Gf, and Cf represent 2’-fluoro modified A, U, G, and C, respectively.

[0530] “s” or s- represents that the two nucleotides adjacent to its left and right and / or the delivery vehicle are linked by a phosphorothioate linkage.

[0531] VP represents that the nucleotide adjacent to its right is a vinyl phosphonate modified nucleotide, which is well known in the art, see, e.g., PCT Publication Nos. WO2011139702, WO2013033230, and WO2019105419.

[0532] IB represents an inverted abasic deoxyribonucleotide, which can include the following three structures depending on its position / way of linkage in the siRNA (for the 5’ end, middle, and 3’ end of the nucleic acid strand, respectively):

[0533] IB is well known in the art, see, e.g., F. Czauderna, Nucleic Acids Res., 2003, 31(11), 2705-16, and PCT Publication Nos. WO2016011123 and WO2019051402.

[0534] “SCP modified nucleotide” refers to a modified nucleotide having the following structure: wherein Base is independently selected from H, a modified or unmodified base, or a leaving group. Preferably, Base is an unmodified base, including an adenine base, a guanine base, a uracil base, and a cytosine base. In other embodiments, Base is a modified base.

[0535] (SCP-U) denotes the above structure where Base is a uracil base.

[0536] L96 denotes a GalNAc delivery carrier of the following structure well known in the art, wherein denotes the position of linkage to the dsRNA via a phosphonate or thiophosphonate group, see, e.g., PCT Publication Nos. WO2009073809 and WO2009082607.

[0537] GL6 denotes a GalNAc delivery carrier of the following structure, wherein denotes the position of linkage to the dsRNA via a phosphonate or thiophosphonate group

[0538] Table 4A. Sense strand sequences of modified siRNAs targeting TTR mRNA

[0539] Table 4B. Sense strand sequences of modified siRNAs targeting TTR mRNA (linkage ligand)

[0540] Table 5. Antisense strand sequences of modified siRNAs targeting TTR mRNA

[0541] The sequences of the positive control molecules used in this application are shown in Table 6A:

[0542] Table 6A Positive control molecule sequences

[0543] Table 6B Paired siRNA sense and antisense strands targeting TTR mRNA

[0544] Table 6C Paired siRNA sense (linker ligand) and antisense strands targeting TTR mRNA

[0545] It will be understood by those skilled in the art that the number and location of phosphorothioate internucleotide linkages in the siRNA compounds of the present application are not limited to the number and location shown in the sequences shown in Tables 4A-B and 5, and that one skilled in the art can adjust the number and location of phosphorothioate internucleotide linkages without changing the sequence and methoxy / fluoro modifications in accordance with the teachings of Tables 4 and 5, with or without the addition of a delivery vehicle, such as a GalNAc-containing delivery vehicle, and that such adjusted sense strands, antisense strands, and paired siRNAs are also included within the scope of the present application.

[0546] The present application will be further illustrated by the following examples. It should be understood that the following examples are illustrative only and should not be taken in a limiting sense on the scope of the present application. Examples

[0547] The materials used in the examples were obtained from the following sources unless otherwise specified:

[0548] The Hep3B cell line was purchased from Nanjing Kebai, catalog number CBP60197;

[0549] The HepG2 cell line was purchased from Nanjing Kebai, catalog number CBP60199.

[0550] Example 1 Preparation of siRNA

[0551] The siRNAs of the present application were prepared using the solid phase phosphoramidite method well known in the art. See, for example, PCT Publication Nos. WO2016081444 and WO2019105419, and summarized as follows.

[0552] 1.1 Synthesis of sense strand (SS strand)

[0553] Using the solid phase phosphoramidite synthesis method, blank CPG solid phase carrier was used as the starting cycle, and nucleoside monomers were connected one by one in the order of nucleotide arrangement of the sense strand from 3 '-5' direction. Each connection of a nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or thio, and the synthesis scale is 5 pmol of oligonucleotide synthesis conditions as follows:

[0554] Commercially available 2'-F, 2'-O-methyl and other modified phosphoramidites were used. Nucleoside monomers were provided as 0.05 M solutions in acetonitrile, and the conditions for each step were the same, i.e., temperature 25°C, deprotection with 3% trichloroacetic acid in dichloromethane, deprotection 3 times; coupling with 0.25 M ETT in acetonitrile, coupling 2 times; capping with 10% acetic anhydride in acetonitrile and pyridine / N-methylimidazole / acetonitrile (10:14:76, v / v / v), capping 2 times; oxidation with 0.05 M iodine / tetrahydrofuran / pyridine / water (70 / 20 / 10, v / v / v), oxidation 2 times; thioation with 0.2 M PADS in acetonitrile / 3-picoline (1 / 1, v / v), thioation 2 times.

[0555] 1.2 Synthesis of antisense strand (AS strand)

[0556] By solid phase phosphoramidite synthesis method, blank CPG solid phase carrier was used as the starting cycle, and nucleoside monomers were connected one by one in the order of antisense strand nucleotide arrangement from 3'-5' direction. Each connection of a nucleoside monomer included four steps of deprotection, coupling, capping, oxidation or thioation. The synthesis conditions of 5 umol of oligonucleic acid of antisense strand were the same as those of sense strand.

[0557] 1.3 Purification and annealing of oligonucleic acid

[0558] 1.3.1 aminolysis

[0559] The synthesized solid phase carrier (sense strand or antisense strand) was added to a 5 mL centrifuge tube, 3% diethylamine / ammonia water (v / v) was added, and the reaction was carried out in a constant temperature water bath at 35°C for 16 hours (or in a constant temperature water bath at 55°C for 8 hours). The solid phase carrier was washed with ethanol / water three times, 1 mL each time, and the filtrate was concentrated by centrifugation. The crude product was purified.

[0560] 1.3.2 purification

[0561] The method of purification and desalting is well known to those skilled in the art. For example, a strong anion filler column can be used, and a sodium chloride-sodium hydroxide system can be used for elution and purification. The product can be collected and purified. A gel filler purification column can be used for desalting, and the elution system is pure water.

[0562] 1.3.3 annealing

[0563] According to Table 6B and Table 6C, the sense strand (SS strand) and the antisense strand (AS strand) were mixed in a molar ratio of (SS strand / AS strand = 1 / 1.05), the water bath pot was heated to 70-95°C and kept for 3-5 min, and then naturally cooled to room temperature. The system was freeze-dried to obtain the product. DR009430 and DR009433 were used as positive controls.

[0564] Example 2 Hep3B cell line activity screening

[0565] Cell transfection

[0566] On the first day, after the cell line was digested and resuspended, it was counted. The cell suspension was plated into a 96-well plate, 100 μL / well, 1 x 10 4 cells / well, and after 18 h, the transfection operation was performed.

[0567] On the second day, the 20 μM siRNA stock solution was diluted with Opti-MEM, 198 μL of Opti-MEM was added to 2 μL of siRNA stock solution, the final concentration of siRNA is shown below, and it was mixed by blowing and sucking, and was ready for use.

[0568] On the second day, 14.1 μL of Opti-MEM was used to dilute 0.9 μL of Lipofectamine TM RNAiMAX (Thermo, 13778150), and it was mixed by blowing and sucking gently, and was left to stand at room temperature for 5 min. Then 15 μL of the prepared RNAi-MAX mixture and 15 μL of the diluted siRNA were mixed by blowing and sucking gently without introducing air bubbles, and were left to stand at room temperature for 10 min, and were added to a 96-well plate, 10 μL / well. Incubation was performed at 37°C in a 5% CO2 incubator for 24 h (the control group did not add siRNA).

[0569] RNA extraction

[0570] Cell RNA extraction was performed according to the operating instructions of the high-throughput cell RNA extraction kit (Fanzhi Medical, FG0417-L) using a nucleic acid extractor (Hangzhou Aosheng, Auto-pure96).

[0571] RNA reverse transcription

[0572] Denaturation reaction mixture preparation: refer to PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B) single-well preparation volume: Oligo dT Primer 1 μL, dNTP Mixture 1 μL, template RNA 12.5 μL, 65°C in a conventional PCR instrument for 5 min, and then quickly cooled on ice for 2 min.

[0573] Reverse transcription reaction solution preparation: refer to PrimeScript TMII 1st Strand cDNA Synthesis Kit(Takara, 6210B). Each well contains 5x Prime Script II Buffer 4 μL, RNase Inhibitor 0.5 μL, PrimeScript II RTase 1 μL.

[0574] After denaturation, mix the reaction solution 14.5 μL slowly with the reverse transcription reaction solution, incubate at 42°C for 45 minutes for reverse transcription, incubate at 95°C for 5 minutes to inactivate the enzyme, and cool the reverse transcription product (cDNA) at 4°C.

[0575] After the reverse transcription is completed, add 30 μL of distilled water without DNase and RNase to the cDNA sample in each well.

[0576] Fluorescence quantitative PCR

[0577] Reference TaqMan TM Fast Advanced Master Mix(ABI, 4444965) operation process, 20 μL system for fluorescence quantitative PCR reaction (ABI, QuantStudio3). The reaction program is: (50°C, 2 minutes) x 1 Cycle; (95°C, 20 seconds) x 1 Cycle; (95°C, 1 second; 60°C, 24 seconds) x 40 Cycles.

[0578] Table 7. Primer information

[0579] Data statistics

[0580] Calculate 2 -△△Ct values and convert them into percentages to get the remaining percentage;

[0581] △△Ct = [(Ct experimental group target gene - Ct experimental group internal reference) - (Ct control group target gene - Ct control group internal reference)].

[0582] The target gene is hTTR, and the internal reference is hACTB.

[0583] The Hep3B cell line (Nanjing Kebai, CBP60197) was used, and the final concentration of the compound was selected as 0.1 nM and 0.01 nM for siRNA compound cell line activity high-throughput screening. The experimental screening results are shown in the following table.

[0584] Table 8. Hep3B cell line activity screening results

[0585] Example 3 In vivo activity evaluation in human TTR Tg mice

[0586] After the adaptation period, male C57BL / 6J human TTR transgenic mice were bled through the orbital vein, and serum samples were collected after centrifugation. The human TTR protein level in each mouse serum was detected by ELISA assay (Abeam, ab231920) as the experimental baseline, and the mice were grouped for administration according to the baseline level (3 mice per group). On the administration day (D0), each group of mice was given normal saline or the siRNA to be tested by subcutaneous injection, with a dose of 1 mg / kg and a volume of 5 ml / kg. Serum samples were collected from the mice on the 14th day (D14) and the 28th day (D28) after administration, and the human TTR protein level was measured by ELISA assay. The siRNA knockdown efficiency was calculated by comparing the human TTR protein level in the serum of each mouse before administration (D-4) and after administration. The remaining percentage was calculated based on the ratio of the human TTR protein level in the serum after administration to that before administration (D-4), and the experimental results are shown in the table below.

[0587] Table 9. Results of activity evaluation in human TTR transgenic mice

[0588] Example 4 In vivo activity evaluation in human TTR Tg mice

[0589] According to the method of Example 3, serum samples were collected from the mice on the 7th day (D7), the 14th day (D14), and the 28th day (D28) after administration, and the human TTR protein level was measured by ELISA assay. The siRNA knockdown efficiency was calculated by comparing the human TTR protein level in the serum of each mouse before administration and after administration. The experimental results are shown in the table below.

[0590] Table 10. Results of activity evaluation in human TTR transgenic mice

[0591] Example 5 Activity screening of Hep3B cell line

[0592] According to the method of Example 2, the Hep3B cell line (Nanjing Kebai, Catalog No. CBP60197) was selected for this experiment, and the compound concentration range was 0.00064-10 nM (5-fold gradient dilution, 7 concentration points). The remaining percentage of each concentration point was tested and the IC 50 was calculated, and the results are shown in the table below.

[0593] Table 11. Results of activity screening of Hep3B cell line

[0594] Example 6 IC in human primary hepatocytes 50 Activity test

[0595] Cell recovery: Human primary hepatocytes (LifeNet Health, Lot: 19148520-01) were recovered according to the instructions, and the cells were diluted to an appropriate density and plated at 30,000 cells / well in a 96-well collagen-coated cell plate. The cells were incubated in a 37°C, 5% CO2 incubator for 24 hours before the experiment was performed.

[0596] Cell transfection: The IC50activity test was performed with a compound concentration range of 0.0002-10 nM (3-fold gradient dilution, 11 concentration points). 4.7 μL Opti-MEM + 0.3 μL lipofectamine RNAiMAX (Invitrogen, 13778150) was added per well, and 5 μL siRNA diluent was added for mixing. The siRNA transfection complex was incubated at room temperature for 10 minutes, and then added to the cells. The cells were incubated for another 24 hours before subsequent operations. Two replicate wells were transfected for each concentration point. TM

[0597] RNA extraction: Cell RNA extraction was performed according to the operation protocol of the high-throughput cell RNA extraction kit (Shanghai Fushen Biotechnology, FSF0035-CS-96T) using a nucleic acid extractor (Hangzhou Aosheng, Auto-pure96).

[0598] Reverse transcription: The denaturation reaction mixture was prepared according to the PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B). Single-well preparation volume: Oligo dT Primer 1 μL, dNTP Mixture 1 μL, Template RNA 12.5 μL. Incubation at 65°C in a conventional PCR instrument for 5 minutes, and then quickly cooled on ice for 2 minutes after the end of the incubation.

[0599] Reverse transcription reaction solution was prepared according to the PrimeScript TM ​II 1st Strand cDNA Synthesis Kit(Takara,6210B) single hole preparation volume: 5x Prime Script II Buffer 4μL, RNase Inhibitor 0.5μL, PrimeScript II RTase 1μL, 14.5μL of the reaction solution after denaturation in the previous step, mix slowly, incubate at 42℃ for 45 minutes for reverse transcription, inactivate the enzyme at 95℃ for 5 minutes, and cool the reverse transcription product (cDNA) at 4℃. After the end of the reverse transcription, add DNase RNase-Free Distilled Water 30μL to each well of the cDNA sample.

[0600] Fluorescence quantitative PCR: reference TaqMan TM Fast Advanced Master Mix(ABI, 4444965) 20μL system for fluorescence quantitative PCR reaction (ABI, QuantStudio3), reaction program: (50℃, 2min) x 1 Cycle; (95℃, 20s) x 1 Cycle; (95℃, 1s; 60℃, 24s) x 40 Cycles.

[0601] Table 12. Primer information

[0602] Data analysis: the relative quantification of the target gene expression is realized by comparing the Ct value. Comparing the Ct value refers to calculating the gene expression difference by comparing the difference between the Ct values of the target gene and the reference gene, and the relative expression amount of the target gene is 2 -△△Ct The relative expression amount of the target gene at each concentration point was tested and converted into percentage, and the results are shown in the table below.

[0603] △Ct experimental group = [(Ct experimental group target gene - Ct experimental group reference gene),

[0604] △Ct blank control group = [(Ct blank control group target gene - Ct blank control group reference gene),

[0605] △△Ct = [(Ct experimental group target gene - Ct experimental group reference gene) - (Ct blank control group target gene - Ct blank control group reference gene)].

[0606] The absolute IC50 value is determined by GraphPad Prism using the "log(inhibitor) vs. response(four parameters)" fitting curve. When Y = 50%, the corresponding X value (X value) is the absolute IC 50Log value (Log absolute IC50), i.e. absolute IC 50 = 10^X.

[0607] The target gene is hTTR, and the reference gene is hACTB.

[0608] Table 13. Results of human primary hepatocyte activity screening

[0609] Example 7 IC in cynomolgus monkey primary hepatocytes 50 Activity test

[0610] Transfection complex preparation: select compound concentration range of 0.00064-10 nM (5-fold gradient dilution, 7 concentration points) for IC50activity test. Mix 4.7 μL Opti-MEM + 0.3 μL lipofectamine RNAiMAX (Invitrogen, 13778150) per well, then add 5 μL siRNA diluent, incubate at room temperature for 10 minutes, and add the siRNA transfection complex to the 96-well collagen-coated cell plate, transfect two replicates for each concentration point.

[0611] Cell recovery: recover the monkey primary hepatocytes (Miaosun Biotechnology, item number: CCH-100CYS-P) according to the instructions, dilute the cells to the appropriate density, and plate 30,000 cells / well in a 96-well collagen-coated cell plate, 90 μL cell suspension per well, 10 μL siRNA transfection complex, total volume 100 μL. Incubate the cells in a 37°C, 5% CO2incubator for 24 hours before conducting the experiment.

[0612] RNA extraction: use the nucleic acid extractor (Hangzhou Aosheng, Auto-pure96) to extract the cell RNA according to the operating instructions of the high-throughput cell RNA extraction kit (Shanghai Fushen Biotechnology, FSF0035-CS-96T).

[0613] Reverse transcription: prepare the denaturation reaction mixture, refer to PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B) single-well preparation volume: Oligo dT Primer 1 μL, dNTP Mixture 1 μL, template RNA 12.5 μL, 65°C in a regular PCR instrument, incubate for 5 minutes, and quickly cool on ice for 2 minutes after the end.

[0614] Reverse transcription reaction solution preparation, refer to PrimeScript TMII 1st Strand cDNA Synthesis Kit(Takara, 6210B) single hole preparation volume: 5x Prime Script II Buffer 4μL, RNase Inhibitor 0.5μL, PrimeScript II RTase 1μL, 14.5μL of the reaction solution after denaturation in the previous step, mix slowly, incubate at 42℃ for 45 minutes for reverse transcription, incubate at 95℃ for 5 minutes to inactivate the enzyme, and cool the reverse transcription product (cDNA) at 4℃. After the end of the reverse transcription, add DNase RNase-Free Distilled Water 30μL to each well of the cDNA sample.

[0615] Fluorescent quantitative PCR: fluorescent quantitative PCR reaction (ABI, QuantStudio3) was carried out in a 20μL system with reference to 2x ColorSYBR mix Green qPCR Master Mix(ROX2)(EZB, A0012-R2). The reaction program was as follows: (95℃, 2.5min) x 1 Cycle; (94℃, 15s; 60℃, 30s) x 40 Cycles; Melting curve: according to the instrument settings. The primer information is as follows.

[0616] Table 14. Primer information

[0617] Data analysis: the relative quantification of the expression of the target gene was realized by comparing the Ct values. Comparing the Ct values means calculating the difference in gene expression by comparing the difference between the Ct values of the target gene and the internal reference gene. The relative expression of the target gene is 2 -△△Ct The relative expression of the target gene at each concentration point was tested and converted into percentage, and the results are shown in the following table.

[0618] △Ct experimental group = [(Ct experimental group target gene - Ct experimental group internal reference gene),

[0619] △Ct blank control group = [(Ct blank control group target gene - Ct blank control group internal reference gene),

[0620] △△Ct = [(Ct experimental group target gene - Ct experimental group internal reference gene) - (Ct blank control group target gene - Ct blank control group internal reference gene)].

[0621] Absolute IC50 values were determined by GraphPad Prism using a "log(inhibitor) vs. response (four parameters)" curve fit. The X value when Y = 50% is the log value of its absolute IC50 (Log absolute IC50), i.e. absolute IC50 = 10^X.

[0622] The target gene is Monkey TTR and the reference gene is Monkey GAPDH.

[0623] Table 15. Activity screening results in cynomolgus monkey primary hepatocytes

[0624] Example 8 In vivo activity evaluation in hTTR transgenic mice

[0625] After the adaptation period, 2-3 month old male homozygous B6 hTTR transgenic mice (Sino-British Biotechnology Co., Ltd., C001512) were subjected to blood collection from the orbital vein on the 4th day before administration (D-4), and serum samples were collected after centrifugation. The TTR protein level in the serum of each mouse was detected by ELISA (Abeam, ab231920) to determine the experimental baseline, and the mice were grouped for administration according to the baseline level (3 mice per group). On the administration day (D0), each group of mice was administered with normal saline or the test siRNA by subcutaneous injection, with a dose of 1 mg / kg and a volume of 5 ml / kg. Serum samples were collected from the mice on the 7th day (D7), 14th day (D14) and 28th day (D28) after administration, and the TTR protein level in the serum was measured by ELISA. The inhibition efficiency of the compound was calculated by comparing the TTR protein level in the serum of each mouse before administration (D-4) and after administration. The percentage was calculated based on the ratio of the human TTR protein level in the serum after administration to that before administration (D-4), and the experimental results are shown in the following table. The values in the table represent the percentage.

[0626] Table 16. Activity evaluation results of the compounds in hTTR transgenic mice

[0627] Table 17. Activity evaluation results of the compounds in hTTR transgenic mice

[0628] Example 9 In vivo efficacy evaluation in cynomolgus monkeys

[0629] Male cynomolgus monkeys were randomly grouped according to serum TTR level, body weight and age, 3-4 animals per group. Single subcutaneous injection was given, the dose was 1 mg / kg, and the volume was 0.5 ml / kg. Serum was collected at 15 days before administration (D-15), 8 days before administration (D-8), the day of administration (D0), 3, 7, 14, 21, 28, 35, 42, 56, 70, 84 days after administration, and the level of TTR protein in serum was detected by ELISA assay (Abeam, ab231920). The inhibition efficiency of the compound on TTR protein was calculated by comparing the level of TTR protein in serum of each monkey at D0 before administration and at each time point after administration, and was expressed in percentage. The inhibition degree of each group of compounds on serum TTR protein is shown in the following table.

[0630] Table 18. Evaluation results of activity of compounds in cynomolgus monkeys

[0631] The results show that the siRNA of the application has a better inhibition effect on TTR protein compared with the positive control DR009433.

Claims

1. A double-stranded RNA (dsRNA) for inhibiting TTR gene expression, said dsRNA comprising a sense strand and an antisense strand forming a double-stranded region, wherein the length of the sense strand and the antisense strand is each independently 15-30 nucleotides, and said antisense strand comprises a nucleotide sequence of at least 15 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NO: 132-262, preferably said antisense strand comprising SEQ ID NO: 132-262. NO: A nucleotide sequence of at least 15 consecutive nucleotides of any one of the nucleotide sequences shown in NO: 134, 144, 174, 176, 177, 179, 180, 185, 188, 190, 191, 192, 193, 194, 195, 201, 202, 204, 210, 214, 217, 218, 220, 221, 222, 223, 224, 225, 226, 231, 232, 236, 243, 247, 248 and 251.

2. The dsRNA of claim 1, wherein the positive strand comprises a nucleotide sequence of at least 15 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NO: 1-131, preferably the positive strand comprises a nucleotide sequence of at least 15 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NO: 3, 13, 43, 45, 46, 48, 49, 54, 57, 59, 60, 61, 62, 63, 64, 70, 71, 73, 79, 83, 86, 87, 89, 90, 91, 92, 93, 94, 95, 100, 101, 105, 112, 116, 117 and 120.

3. The dsRNA of claim 1 or 2, wherein the dsRNA is siRNA.

4. The dsRNA of any one of claims 1-3, wherein the length of the sense strand and the antisense strand is each independently 15-27 nucleotides, preferably 18-25 nucleotides, more preferably 19-21 nucleotides.

5. The dsRNA of any one of claims 1-4, wherein the length of the double-stranded region is 15-25 nucleotide pairs, preferably 16-23 nucleotide pairs, more preferably 18-20 nucleotide pairs, and most preferably 19 nucleotide pairs.

6. The dsRNA of any one of claims 1-5, wherein the antisense strand comprises a nucleotide sequence of at least 16 consecutive nucleotides, at least 17 consecutive nucleotides, at least 18 consecutive nucleotides, at least 19 consecutive nucleotides, or at least 20 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NO: 132-262; or the antisense strand comprises a nucleotide sequence shown in any one of SEQ ID NO: 132-262. Preferably, the antisense strand comprises a nucleotide sequence of at least 16 consecutive nucleotides, at least 17 consecutive nucleotides, at least 18 consecutive nucleotides, at least 19 consecutive nucleotides, or at least 20 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NO: 134, 144, 174, 176, 177, 179, 180, 185, 188, 190, 191, 192, 193, 194, 195, 201, 202, 204, 210, 214, 217, 218, 220, 221, 222, 223, 224, 225, 226, 231, 232, 236, 243, 247, 248, and 251, or comprises SEQ ID NO: 134, 144, 174, 176, 177, 179, 180, 185, 188, 190, 191, 192, 193, 194, 205, 201, 202, 204, 210, 214, 217, 218, 220, 221, 222, 223, 224, 225, 226, 231, 232, 236, 243, 247, 248, and 251, or comprises SEQ ID NO: 134, 144, 174, 176, 177, 179, 180, 185, 188, 190, 191, 1 The nucleotide sequence shown is one of the following: NO: 134, 144, 174, 176, 177, 179, 180, 185, 188, 190, 191, 192, 193, 194, 195, 201, 202, 204, 210, 214, 217, 218, 220, 221, 222, 223, 224, 225, 226, 231, 232, 236, 243, 247, 248, and 251.

7. The dsRNA of any one of claims 1-6, wherein the sense strand comprises a nucleotide sequence of at least 16 consecutive nucleotides, at least 17 consecutive nucleotides, or at least 18 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NO: 1-131, or the sense strand comprises a nucleotide sequence shown in any one of SEQ ID NO: 1-131. Preferably, the positive strand comprises a nucleotide sequence of at least 16 consecutive nucleotides, at least 17 consecutive nucleotides, or at least 18 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NO: 3, 13, 43, 45, 46, 48, 49, 54, 57, 59, 60, 61, 62, 63, 64, 70, 71, 73, 79, 83, 86, 87, 89, 90, 91, 92, 93, 94, 95, 100, 101, 105, 112, 116, 117, and 120, or comprises SEQ ID NO:

120. The nucleotide sequence shown is one of the following: NO: 3, 13, 43, 45, 46, 48, 49, 54, 57, 59, 60, 61, 62, 63, 64, 70, 71, 73, 79, 83, 86, 87, 89, 90, 91, 92, 93, 94, 95, 100, 101, 105, 112, 116, 117, and 120.

8. The dsRNA of any one of claims 1-7, wherein the siRNA comprises any pair of paired sense and antisense sequences as shown in Table 3 of the specification, preferably the siRNA comprises any pair of the following sense and antisense sequences: (1) The sense strand contains the nucleotide sequence shown in SEQ ID NO:3, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:134; (2) The sense strand contains the nucleotide sequence shown in SEQ ID NO:13, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:144; (3) The sense strand contains the nucleotide sequence shown in SEQ ID NO:43, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:174; (4) The sense strand contains the nucleotide sequence shown in SEQ ID NO:45, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:176; (5) The sense strand contains the nucleotide sequence shown in SEQ ID NO:46, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:177; (6) The sense strand contains the nucleotide sequence shown in SEQ ID NO:48, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:179; (7) The sense strand contains the nucleotide sequence shown in SEQ ID NO:49, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:180; (8) The sense strand contains the nucleotide sequence shown in SEQ ID NO:54, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:185; (9) The sense strand contains the nucleotide sequence shown in SEQ ID NO:57, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:188; (10) The sense strand contains the nucleotide sequence shown in SEQ ID NO:59, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:190; (11) The sense strand contains the nucleotide sequence shown in SEQ ID NO:60, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:191; (12) The sense strand contains the nucleotide sequence shown in SEQ ID NO:61, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:192; (13) The sense strand contains the nucleotide sequence shown in SEQ ID NO:62, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:193; (14) The sense strand contains the nucleotide sequence shown in SEQ ID NO:63, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:194; (15) The sense strand contains the nucleotide sequence shown in SEQ ID NO:64, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:195; (16) The sense strand contains the nucleotide sequence shown in SEQ ID NO:70, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:201; (17) The sense strand contains the nucleotide sequence shown in SEQ ID NO:71, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:202; (18) The sense strand contains the nucleotide sequence shown in SEQ ID NO:73, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:204; (19) The sense strand contains the nucleotide sequence shown in SEQ ID NO:79, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:210; (20) The sense strand contains the nucleotide sequence shown in SEQ ID NO:83, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:214; (21) The sense strand contains the nucleotide sequence shown in SEQ ID NO:86, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:217; (22) The sense strand contains the nucleotide sequence shown in SEQ ID NO:87, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:218; (23) The sense strand contains the nucleotide sequence shown in SEQ ID NO:89, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:220; (24) The sense strand contains the nucleotide sequence shown in SEQ ID NO:90, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:221; (25) The sense strand contains the nucleotide sequence shown in SEQ ID NO:91, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:222; (26) The sense strand contains the nucleotide sequence shown in SEQ ID NO:92, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:223; (27) The sense strand contains the nucleotide sequence shown in SEQ ID NO:93, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:224; (28) The sense strand contains the nucleotide sequence shown in SEQ ID NO:94, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:225; (29) The sense strand contains the nucleotide sequence shown in SEQ ID NO:95, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:226; (30) The sense strand contains the nucleotide sequence shown in SEQ ID NO:100, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:231; (31) The sense strand contains the nucleotide sequence shown in SEQ ID NO:101, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:232; (32) The sense strand contains the nucleotide sequence shown in SEQ ID NO:105, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:236; (33) The sense strand contains the nucleotide sequence shown in SEQ ID NO:112, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:243; (34) The sense strand contains the nucleotide sequence shown in SEQ ID NO:116, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:247; (35) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:117, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:248; or (36) The sense strand contains the nucleotide sequence shown in SEQ ID NO:120, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO:

251.

9. The dsRNA of any one of claims 1-8, wherein substantially all nucleotides of the sense strand and / or substantially all nucleotides of the antisense strand are modified nucleotides, or wherein all nucleotides of the sense strand and / or all nucleotides of the antisense strand are modified nucleotides.

10. The dsRNA of claim 9, wherein the sense strand and the antisense strand each independently comprise one or more modified nucleotides selected from the group consisting of: 2'-O-alkyl modified nucleotides (e.g., 2'-O-methyl modified nucleotides), 2'-methoxyethyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, inosine ribonucleotides, abase-free nucleotides, reverse abase-free deoxyribonucleotides, nucleotides containing a thiophosphate group, vinylphosphonate modified nucleotides, locked nucleotides, unlocked nucleotides, 2'-amino-modified nucleotides, 2'-C-alkyl-modified nucleonucleotides. Nucleotides, 2'-O-allyl modified nucleotides, morpholinonucleotides, aminophosphates, nucleotides containing non-natural bases, terminal nucleotides linked to cholesterol derivatives or dodecanoic acid didecylamide groups, deoxyribonucleotides, 3'-terminal deoxythymidine (dT) nucleotides, conformation-restricted nucleotides, restricted ethyl nucleotides, 2'-hydroxyl modified nucleotides, nucleotides containing methylphosphonic acid groups, nucleotides containing 5'-phosphate, nucleotides containing 5'-phosphate mimics, diol modified nucleotides (GNA), SCP modified nucleotides, and 2-O-(N-methylacetamide) modified nucleotides.

11. The dsRNA of claim 10, wherein the sense strand and the antisense strand each independently comprise one or more modified nucleotides selected from the group consisting of: SCP-modified nucleotides, 2'-deoxy-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-fluoro-modified nucleotides, and nucleotides containing a thiophosphate group.

12. The dsRNA of any one of claims 9-11, wherein the antisense strand has a length of 21 nucleotides and has (i) (counting from the 5' end) nucleotides with 2'-O-methyl modification at positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21, and nucleotides with 2'-fluoro modification at positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20; and / or (ii) (Counting from the 5' end) Phosphophosphate nucleotide bonds located between nucleotide positions 1 and 2, nucleotide positions 2 and 3, nucleotide positions 19 and 20, and nucleotide positions 20 and 21.

13. The dsRNA of any one of claims 9-11, wherein the antisense strand has a length of 21 nucleotides and has (i) (counting from the 5' end) SCP-modified nucleotides located at position 1; (ii) (counting from the 5' end) nucleotides with 2'-fluorinated modification at positions 2, 4, 6, 8, 10, 12, 14, 16 and 18; (iii) (counting from the 5' end) nucleotides with a 2'-O-methyl modification at positions 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, and 21; and / or (iv) (Counting from the 5' end) Phosphophosphate nucleotide bonds located between nucleotide positions 1 and 2, nucleotide positions 2 and 3, nucleotide positions 19 and 20, and nucleotide positions 20 and 21.

14. The dsRNA of any one of claims 9-11, wherein the antisense strand has a length of 21 nucleotides and has (i) (counting from the 5' end) nucleotides with 2'-deoxy modification at positions 2, 5, 7 and 12; (ii) (Counted from the 5' end) SCP-modified nucleotides located at position 1; (iii) (Counting from the 5' end) nucleotides with 2'-fluorine modification at position 14; (iv) (counting from the 5' end) nucleotides with a 2'-O-methyl modification at positions 3, 4, 6, 8, 9, 10, 11, 13, 15, 16, 17, 18, 19, 20, and 21; and / or (v) (counting from the 5' end) Phosphophosphate nucleotide bonds located between nucleotide positions 1 and 2, nucleotide positions 2 and 3, nucleotide positions 19 and 20, and nucleotide positions 20 and 21.

15. The dsRNA of any one of claims 9-11, wherein the antisense strand has a length of 21 nucleotides and has (i) (counting from the 5' end) nucleotides with 2'-O-methyl modification at positions 3, 4, 6, 8, 9, 10, 11, 13, 15, 17, 18, 19, 20 and 21; (ii) (counting from the 5' end) nucleotides with 2'-fluorinated modification at positions 5, 7, 12, 14 and 16; (iii) (Counting from the 5' end) nucleotides with 2'-deoxy modification at position 2; (iv) (counting from the 5' end) SCP-modified nucleotides located at position 1; and / or (v) (counting from the 5' end) Phosphophosphate nucleotide bonds located between nucleotide positions 1 and 2, nucleotide positions 2 and 3, nucleotide positions 19 and 20, and nucleotide positions 20 and 21.

16. The dsRNA of any one of claims 9-11, wherein the antisense strand has a length of 21 nucleotides and has (i) (counting from the 5' end) nucleotides with 2'-O-methyl modification at positions 3, 4, 6, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20 and 21; (ii) (counting from the 5' end) nucleotides with 2'-fluorinated modification at positions 2, 5, 7, 14 and 16; (iii) (counting from the 5' end) SCP-modified nucleotides located at position 1; and / or (iv) (Counting from the 5' end) Phosphophosphate nucleotide bonds located between nucleotide positions 1 and 2, nucleotide positions 2 and 3, nucleotide positions 19 and 20, and nucleotide positions 20 and 21.

17. The dsRNA of any one of claims 9-11, wherein the antisense strand has a length of 21 nucleotides and has (i) (counting from the 5' end) nucleotides with 2'-O-methyl modification at positions 3, 4, 5, 6, 8, 9, 10, 11, 13, 15, 17, 18, 19, 20 and 21; (ii) (counting from the 5' end) nucleotides with 2'-fluorinated modification at positions 2, 7, 12, 14 and 16; (iii) (counting from the 5' end) SCP-modified nucleotides located at position 1; and / or (iv) (Counting from the 5' end) Phosphophosphate nucleotide bonds located between nucleotide positions 1 and 2, nucleotide positions 2 and 3, nucleotide positions 19 and 20, and nucleotide positions 20 and 21.

18. The dsRNA of any one of claims 9-11, wherein the antisense strand has a length of 21 nucleotides and has (i) (counting from the 5' end) nucleotides with 2'-O-methyl modification at positions 3, 4, 5, 7, 8, 9, 10, 11, 13, 15, 17, 18, 19, 20 and 21; (ii) (counting from the 5' end) nucleotides with 2'-fluorinated modifications at positions 2, 6, 12, 14 and 16; (iii) (counting from the 5' end) SCP-modified nucleotides located at position 1; and / or (iv) (Counting from the 5' end) Phosphophosphate nucleotide bonds located between nucleotide positions 1 and 2, nucleotide positions 2 and 3, nucleotide positions 19 and 20, and nucleotide positions 20 and 21.

19. The dsRNA of any one of claims 9-11, wherein the antisense strand has a length of 21 nucleotides and has (i) (counting from the 5' end) nucleotides with 2'-O-methyl modification at positions 3, 4, 6, 8, 9, 10, 11, 13, 15, 16, 17, 18, 19, 20 and 21; (ii) (counting from the 5' end) nucleotides with 2'-fluorinated modification at positions 2, 5, 7, 12 and 14; (iii) (counting from the 5' end) SCP-modified nucleotides located at position 1; and / or (iv) (Counting from the 5' end) Phosphophosphate nucleotide bonds located between nucleotide positions 1 and 2, nucleotide positions 2 and 3, nucleotide positions 19 and 20, and nucleotide positions 20 and 21.

20. The dsRNA of any one of claims 9-11, wherein the antisense strand has a length of 21 nucleotides and has (i) (counting from the 5' end) nucleotides with 2'-O-methyl modification at positions 3, 4, 6, 8, 9, 10, 11, 13, 15, 16, 17, 18, 19, 20 and 21; (ii) (counting from the 5' end) nucleotides with 2'-fluorinated modification at positions 2, 5, 7, 12 and 14; (iii) (counting from the 5' end) SCP-modified nucleotides located at position 1; and / or (iv) (Counting from the 5' end) Phosphophosphate nucleotide bonds located between nucleotide positions 1 and 2, nucleotide positions 2 and 3, nucleotide positions 19 and 20, and nucleotide positions 20 and 21.

21. The dsRNA according to any one of claims 9-11, wherein the antisense strand has a length of 21 nucleotides and has (i) (counting from the 5' end) nucleotides with 2'-O-methyl modification at positions 3, 4, 5, 6, 8, 9, 10, 11, 13, 15, 17, 18, 19, 20 and 21; (ii) (counting from the 5' end) nucleotides with 2'-fluorinated modification at positions 2, 7, 12, 14 and 16; (iii) (counting from the 5' end) SCP-modified nucleotides located at position 1; and / or (iv) (Counting from the 5' end) Phosphophosphate nucleotide bonds located between nucleotide positions 1 and 2, nucleotide positions 2 and 3, nucleotide positions 19 and 20, and nucleotide positions 20 and 21.

22. The dsRNA of any one of claims 9-11, wherein the antisense strand has a length of 21 nucleotides and has (i) (counting from the 5' end) nucleotides with 2'-O-methyl modification at positions 3, 4, 6, 8, 9, 10, 11, 13, 15, 16, 17, 18, 19, 20 and 21; (ii) (counting from the 5' end) nucleotides with 2'-fluorine modification at position 14; (iii) (Counting from the 5' end) nucleotides with 2'-deoxy modification at positions 2, 5, 7 and 12; (iv) (counting from the 5' end) SCP-modified nucleotides located at position 1; and / or (v) (counting from the 5' end) Phosphophosphate nucleotide bonds located between nucleotide positions 1 and 2, nucleotide positions 2 and 3, nucleotide positions 19 and 20, and nucleotide positions 20 and 21.

23. The dsRNA of any one of claims 9-22, wherein the sense strand has a length of 19 nucleotides and has: (i) (counting from the 5' end) nucleotides with 2'-O-methyl modifications at positions 1 to 6 and 10 to 19, and nucleotides with 2'-fluoro modifications at positions 7 to 9; and / or (ii) (Counting from the 5' end) Phosphophosphate nucleotide bonds located between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, and between nucleotide positions 18 and 19.

24. The dsRNA of any one of claims 9-22, wherein the sense strand has a length of 19 nucleotides and has: (i) (counting from the 5' end) nucleotides with 2'-O-methyl modifications at positions 1 to 6 and 10 to 19, and nucleotides with 2'-fluoro modifications at positions 7 to 9; and / or (ii) (Counting from the 5' end) Phosphophosphate nucleotide bonds located between nucleotide positions 1 and 2, nucleotide positions 2 and 3, nucleotide positions 17 and 18, and nucleotide positions 18 and 19.

25. The dsRNA of any one of claims 9-22, wherein the sense strand has a length of 19 nucleotides and has (i) (counting from the 5' end) nucleotides with 2'-O-methyl modification at positions 1, 2, 3, 4, 5, 6, 8, 10, 12, 13, 14, 15, 16, 17, 18 and 19; (ii) (counting from the 5' end) nucleotides with 2'-fluorinated modifications at positions 7, 9, and 11; and / or (iii) (Counting from the 5' end) Phosphophosphate nucleotide bonds located between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, and between nucleotide positions 18 and 19.

26. The dsRNA of any one of claims 9-22, wherein the sense strand has a length of 19 nucleotides and has (i) (counting from the 5' end) nucleotides with 2'-O-methyl modification at positions 1, 2, 3, 4, 5, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18 and 19; (ii) (counting from the 5' end) nucleotides with 2'-fluorinated modifications at positions 7, 8, and 9; and / or (iii) (Counting from the 5' end) Phosphophosphate nucleotide bonds located between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, and between nucleotide positions 18 and 19.

27. The dsRNA of any one of claims 9-22, wherein the sense strand has a length of 23 nucleotides and has (i) (counting from the 5' end) nucleotides with 2'-O-methyl modification at positions 2, 3, 4, 5, 6, 7, 8, 9, 13, 14, 15, 16, 17, 18, 19, 20, 21 and 22; (ii) (counting from the 5' end) nucleotides with 2'-fluorinated modification at positions 10, 11 and 12; (iii) (Counting from the 5' end) IB-modified nucleotides located at positions 1 and 23; and / or (iv) (Counting from the 5' end) Phosphophosphate nucleotide bonds located between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, and between nucleotide positions 22 and 23.

28. The dsRNA of any one of claims 9-22, wherein the sense strand has a length of 23 nucleotides and has (i) (counting from the 5' end) nucleotides with 2'-O-methyl modification at positions 2, 3, 4, 5, 6, 7, 8, 9, 11, 13, 15, 16, 17, 18, 19, 20, 21 and 22; (ii) (counting from the 5' end) nucleotides with 2'-fluorinated modifications at positions 10, 12 and 14; (iii) (Counting from the 5' end) IB-modified nucleotides located at positions 1 and 23; and / or (iv) (Counting from the 5' end) Phosphophosphate nucleotide bonds located between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, and between nucleotide positions 22 and 23.

29. The dsRNA of any one of claims 9-22, wherein the sense strand has a length of 23 nucleotides and has (i) (counting from the 5' end) nucleotides with 2'-O-methyl modification at positions 2, 3, 4, 5, 6, 7, 8, 9, 13, 14, 15, 16, 17, 18, 19, 20, 21 and 22; (ii) (counting from the 5' end) nucleotides with 2'-fluorinated modification at positions 10, 11 and 12; (iii) (Counting from the 5' end) IB-modified nucleotides located at positions 1 and 23; and / or (iv) (Counting from the 5' end) Phosphophosphate nucleotide bonds located between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, and between nucleotide positions 22 and 23.

30. The dsRNA of any one of claims 9-22, wherein the sense strand has a length of 19 nucleotides and has (i) (counting from the 5' end) nucleotides with 2'-O-methyl modification at positions 1, 2, 3, 4, 5, 6, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18 and 19; (ii) (counting from the 5' end) nucleotides with 2'-fluorinated modifications at positions 7 and 9; and / or (iii) (Counting from the 5' end) Phosphophosphate nucleotide bonds located between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, and between nucleotide positions 18 and 19.

31. The dsRNA of claim 9, wherein the antisense strand comprises any nucleotide sequence shown in Table 5 of the specification, and / or the sense strand comprises any nucleotide sequence shown in Table 4A of the specification.

32. The dsRNA of claim 18, wherein the sense strand and antisense strand of the dsRNA respectively comprise the sense strand sequence and antisense strand sequence of any one of the siRNAs shown in Table 6B of the specification, or the dsRNA comprises a sense strand and antisense strand selected from the following: (1) The justice chain contains GmsUmsAmUmUmCmCfAfUfUmUmUmUmAmCmUmAmAmsAm (SEQ ID NO:394); and the antisense chain contains (SCP-U)sdTsUmAmdGUmdAAmAmAmAmdTGmGfAmAmUmAmCmsUmsCm (SEQ ID NO:613). (2) The justice chain contains AmsGmsAmGmUmAmUfUfCfCmAmUmUmUmUmUmAmCmsUm (SEQ ID NO:395), and the antisense chain contains (SCP-U)sdGsUmAmAfAmAfAmUmGmGmAfAmUfAmCfUmCmUmsUmsGm (SEQ ID NO:639), and (3) The justice chain contains GmsUmsAmUmUmCmCfAfUfUmUmUmUmAmCmUmAmAmsAm (SEQ ID NO:396), and the antisense chain contains (SCP-U)sUfsUmAmGmUmAfAmAmAmAmUfGmGfAmAfUmAmCmsUmsCm (SEQ ID NO:634).

33. The dsRNA of any one of claims 1-18, further conjugated to a ligand portion targeting the desialylate glycoprotein receptor (ASGPR), preferably with the sense strand of the dsRNA conjugated to the ligand portion.

34. The dsRNA of claim 33, wherein the ligand moiety comprises N-acetylgalactosamine, preferably the ligand moiety having the following structure: in This indicates the position where the siRNA is attached to the positive strand of the siRNA via a phosphate ester group or a thiophosphate ester group.

35. The dsRNA of claim 33 or 34, wherein the sense strand and antisense strand of the dsRNA respectively comprise the sense strand and antisense strand sequences of any one of the following dsRNAs: DR009760, DR009761, DR009764, DR009765, DR009766, DR009767, DR009789, DR009768, DR009833, DR009834, DR009790, DR009769, DR009770, DR009771, DR009772, DR009773, DR009774, DR009776, DR009777, DR009778, DR00977.

9. DR009780, DR009781, DR009782, DR009783, DR009784, DR009785, DR009791, DR009835, DR009786, DR009787, DR009797, DR009836, DR009788, DR009792, DR009793, DR009881, DR009882, DR009886, DR009887, DR009888, DR009891, DR009892, DR009915, DR009916 and DR009917, preferably including the positive and negative chain sequences in DR009917.

36. The dsRNA of claim 33 or 34, wherein the sense strand and antisense strand of the dsRNA respectively comprise the sense strand and antisense strand sequences of any one of the following dsRNAs: DR011568, DR011569, DR011570, DR011572, DR011573, DR011574, DR011575, DR011576, DR011577, DR011578, DR011579, DR011580, DR011581, DR011568, DR011569, DR011579, DR011580, DR011581, DR011568, DR011569, DR011569, DR011578, DR011579, DR011580, DR011581 ...69, DR011578, DR011579, DR011580, DR011581, DR011569, DR011569, DR011569, DR011578, DR011569, DR01 011582, DR011583, DR011584, DR011585, DR011627, DR011628, DR011682, DR011683, DR011684, DR011685, DR011686, DR011706, DR011707, DR011708, DR011709, DR011710 and DR011711, preferably containing the positive and negative chain sequences from DR011707 or DR011683.

37. A cell containing dsRNA as described in any one of claims 1-36.

38. A pharmaceutical composition comprising dsRNA as described in any one of claims 1-36, or cells as described in claim 37, and optionally a pharmaceutically acceptable carrier or excipient.

39. A kit comprising dsRNA as described in any one of claims 1-36, cells as described in claim 37, or a pharmaceutical composition as described in claim 38.

40. A method for inhibiting TTR gene expression in cells, the method comprising contacting the cells with dsRNA as described in any one of claims 1-36 or a pharmaceutical composition as described in claim 38.

41. The method of claim 40, wherein the method is performed in vitro.

42. A method for inhibiting TTR gene expression in cells of a subject, the method comprising administering to the subject dsRNA as described in any one of claims 1-36, cells as described in claim 37, or a pharmaceutical composition as described in claim 38.

43. A method for treating a subject who benefits from reduced TTR gene expression, the method comprising administering to the subject the dsRNA of any one of claims 1-36, the cell of claim 37, or the pharmaceutical composition of claim 38.

44. A method for preventing at least one symptom in a subject suffering from a disease or condition that benefits from reduced TTR gene expression, the method comprising administering to the subject the dsRNA of any one of claims 1-36, the cell of claim 37, or the pharmaceutical composition of claim 38.

45. A method for preventing the progression of a disease or condition in a subject that benefits from reduced TTR gene expression, the method comprising administering to the subject the dsRNA of any one of claims 1-36, the cell of claim 37, or the pharmaceutical composition of claim 38.

46. ​​The method of any one of claims 43-45, wherein the disease or condition that benefits from reduced TTR gene expression is a TTR-related disease.

47. The method of claim 46, wherein the TTR-related disease is selected from the group consisting of: senile systemic amyloidosis, systemic familial amyloidosis, familial amyloid polyneuropathy, familial amyloid cardiomyopathy, meningeal / central nervous system amyloidosis, hyperthyroxineemia, Sturgeon's disease, diabetic retinopathy, age-related macular degeneration, insulin resistance associated with type II diabetes, and cardiovascular disease.

48. The method of any one of claims 43-47, wherein the dsRNA, cell, or pharmaceutical composition is administered subcutaneously.

49. The method of any one of claims 43-48, wherein the subject is a human being.

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