Double-stranded siRNA analogue for inhibiting DGAT2 expression as well as preparation method and application of double-stranded siRNA analogue

Abstract

The present invention relates to a double-stranded siRNA analogue for inhibiting the expression of diacylglycerol acyltransferase 2 (DGAT2), thereby preventing, treating or alleviating diseases and disorders (e.g., NASH) associated with DGAT2, as well as a preparation method and use thereof.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and more specifically, to a double-stranded siRNA analog acting on diacylglycerol acyltransferase 2, and a preparation method and use thereof. Background Art

[0002] Diacylglycerol acyltransferase 2 (DGAT2) is one of the key enzymes regulating lipid metabolism. DGAT2 is closely related to the synthesis of lipid droplets in adipocytes, the assembly of very low-density lipoproteins, and the initial synthesis of triglycerides. The synthesis of triglycerides in NASH is also mainly driven by the catalytic activity of DGAT2. In short, DGAT2 plays an important role in long-term lipid storage and hepatocyte fat metabolism. It is reported that inhibiting DGAT2 can improve plasma lipoprotein distribution, such as reducing total cholesterol and triacylglycerols, as well as reducing liver lipid load, improving insulin sensitivity and systemic glucose control (Liu, Y. et al., 2008. Biochim Biophys Acta 1781:97-104; Choi, CS et al., 2007. J Biol Chem 282:22678-22688; Yu, XX et al., 2005. Hepatology 42:362-371).

[0003] The beneficial effects of inhibiting this target on controlling blood sugar and plasma cholesterol, among other things, demonstrate its value in treating cardiovascular and metabolic diseases. Currently, there is still a lack of effective DGAT2 inhibitors. Therefore, providing novel siRNA structures to inhibit DGAT2 expression and / or activity for the treatment of related conditions remains an urgent problem in the field. Summary of the Invention

[0004] The present invention provides a double-stranded siRNA analogue, a pharmaceutical composition and use thereof. The double-stranded siRNA analogue is used to specifically control, inhibit or eliminate DGAT2 expression and has good inhibitory activity both in vivo and in vitro.

[0005] In a first aspect, the present invention provides a double-stranded siRNA analog for inhibiting DGAT2 expression, the double-stranded siRNA analog comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand are each independently 15-30 nucleotides in length, the antisense strand comprises a complementary region that complementarily pairs with the sense strand, and the antisense strand comprises at least 15 consecutive nucleotides of a sequence that differs from the sequence shown in any one of SEQ ID NOs. 1058-2114 by no more than 3, 2, 1, or 0 nucleotides.

[0006] As a preferred technical solution of the present invention, the length of the sense strand and the antisense strand is each independently 17-27 nucleotides, preferably 19-25 nucleotides, more preferably 19-23 nucleotides, and most preferably 19-21 nucleotides. In a specific embodiment, the length of the sense strand and the antisense strand is each independently 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides. Wherein, the length of the nucleotides of the sense strand and the antisense strand may be the same or different, for example, the sense strand comprises 19 nucleotides and the antisense strand comprises 21 nucleotides, or the sense strand comprises 19 nucleotides and the antisense strand also comprises 19 nucleotides, or alternatively, the sense strand comprises 21 nucleotides and the antisense strand comprises 19 nucleotides.

[0007] As a preferred embodiment of the present invention, the sense strand and the antisense strand may be partially, substantially, or completely complementary to each other. For example, the sense strand and the antisense strand may be 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary. The region of complementarity is at least 15 nucleotide pairs in length. In a specific embodiment, the region of complementarity is 15-25 nucleotide pairs in length. In a specific embodiment, the region of complementarity is 17-23 nucleotide pairs in length. In a specific embodiment, the region of complementarity is 19 nucleotide pairs in length. Generally, if any nucleotide overhangs as defined herein are present, the sequences of these overhangs are not considered when determining the degree of complementarity between two sequences. For example, a sense strand of 21 nucleotides in length and an antisense strand of 21 nucleotides in length that hybridize to form a 19-nucleotide pair region of complementarity with a 2 nucleotide overhang at the 3' end of each strand would be considered 100% complementary.

[0008] As a preferred technical solution of the present invention, the sense strand and the antisense strand each independently comprise a 3' overhang and / or a 5' overhang having at least one nucleotide, for example, one or both of the sense strand and the antisense strand comprise a 3' overhang and / or a 5' overhang having at least two nucleotides. In a specific embodiment, the nucleotides of the overhang are selected from thymidine deoxyribonucleotides or uracil ribonucleotides, for example, the nucleotides of the overhang are selected from 2 consecutive thymidine deoxyribonucleotides or 2 consecutive uracil ribonucleotides. In certain embodiments, when the overhang is present in the antisense strand, the nucleotides in the overhang can be complementary to the target gene sequence, forming a mismatch with the target gene sequence or comprising some other sequences (for example, polypyrimidine or polypurine sequences, UU, TT, AA, GG, etc.).

[0009] As a preferred technical solution of the present invention, the antisense strand comprises a sequence that differs from any one of SEQ ID NOs: 1058 to 2114 by no more than 3, 2, or 1 nucleotides. In a specific embodiment, the antisense strand comprises or is a sequence shown in any one of SEQ ID NOs: 1058 to 2114, 2121 to 2164.

[0010] As a preferred technical solution of the present invention, the antisense strand includes or is a sequence shown in any one of SEQ ID NOs: 1058-1543, 1818-1819, and 2121-2164. In some specific embodiments, the antisense strand comprises or is a sequence shown in any one of SEQ ID NOs: 1058-1112, 1114-1140, 1142-1162, 1164-1191, 1193-1195, 1197-1226, 1228-1229, 1232-1260, 1262-1295, 1297, 1299-1301, 1303, 1306-1315, 1318-1319, 1322-1331, 1333, 1336-1388, 1390-1447, 1449-1543, 2121-2164.

[0011] As a preferred technical solution of the present invention, the antisense strand is selected from SEQ ID NO: 1110, 1120, 1121, 1133, 1134, 1165, 1166, 1257, 1259, 1163, 1305, 1271, 1307, 1328, 1329, 1332, 1333, 1376, 1391, 1392, 1393, 1394, 1396, 1397, 1162, 1252, 1256, 1094, 1095, 1114, 1122, 1125, 1126, 1127, 1128, 1130, 1132, 1135, 1136, 1137, 1141, 116 8, 1309, 1289, 1300, 1303, 1314, 1315, 1317, 1322, 1330, 1331, 1334, 1337, 1349, 1361, 1367, 1258, 1261, 1318, 1323, 1327, 1390, 1530, 1083, 1084, 1079, 1113, 1118, 1164, 1196, 1200, 1270, 1273, 1279, 1302, 1306, 1324, 1336, 1377, 1255, 1395.

[0012] As a preferred technical solution of the present invention, the antisense strand includes a sequence that differs from any of the sequences shown in SEQ ID NO: 1121, 1133, 1134, 1163, 1165, 1166, 1252, 1257, 1259, 1271, 1305, 1307, 1391-1396, 1328-1332 by no more than 2 or 1 nucleotides.

[0013] As a preferred technical solution of the present invention, the antisense strand includes or is SEQ ID The sequence shown in any one of NO: 1079, 1084, 1110, 1118, 1120, 1121, 1125, 1127, 1128, 1132-1134, 1162, 1164-1166, 1252, 1256-1258, 1270, 1271, 1279, 1289, 1303, 1305-1307, 1318, 1323, 1327-1331, 1333, 1337, 1376, 1390-1397, 1530, 2121-2131, 2133-2138, 2140, 2141, 2147, 2148, 2153-2155, 2157, 2164.

[0014] As a preferred technical solution of the present invention, the sense strand comprises a sequence that differs from any one of SEQ ID NOs: 1-1057, 2115-2120 by no more than 3, 2, or 1 nucleotides. As a preferred technical solution of the present invention, the sense strand comprises or is a sequence shown in any one of SEQ ID NOs: 1-1057, 2115-2120.

[0015] As a preferred technical solution of the present invention, the sense strand comprises a sequence that differs from any one of SEQ ID NOs: 1-486, 761, 762, 2115-2120 by no more than 3, 2, or 1 nucleotides. As a preferred technical solution of the present invention, the sense strand comprises or is a sequence shown in any one of SEQ ID NOs: 1-486, 761, 762, 2115-2120.

[0016] As a preferred technical solution of the present invention, the positive chain is selected from the sequence shown in any one of SEQ ID NO: 22, 27, 53, 61, 63, 64, 68, 70, 71, 75-77, 105, 107-109, 195, 199-201, 213, 214, 222, 232, 246, 248-250, 261, 266, 270-276, 280, 319, 333-340, 473, 2115-2118, 2120.

[0017] As a preferred technical solution of the present invention, the double-stranded siRNA analog comprises any pair of paired sense strand sequence and antisense strand sequence as shown in Table 1, or consists of them.

[0018] As a preferred technical solution of the present invention, the double-stranded siRNA analog is selected from DA0023, DA0027, DA0028, DA0038, DA0039, DA0054, DA0057, DA0080, DA0081, DA0085, DA0106, DA0107, DA0108, DA0109, DA0110, DA0112, DA0140, DA0144, DA0196, DA0199, DA020 0. DA0201, DA0202, DA0203, DA0205, DA0214, DA0215, DA0217, DA0223, DA0233, DA0244, DA0246, DA0247, D A0249, DA0250, DA0251, DA0253, DA0258, DA0259, DA0261, DA0262, DA0266, DA0267, DA0268, DA0271, DA027 2. DA0273, DA0274, DA0275, DA0276, DA0277, DA0278, DA0280, DA0281, DA0293, DA0305, DA0311, DA0320, D A0321, DA0333, DA0334, DA0335, DA0336, DA0337, DA0338, DA0339, DA0340, DA0341, DA0474, DA1059, DA106 0, any one of DA1061, DA1062, DA1063, DA1064, DA1065, DA1066, DA1067, DA1068, DA1069, DA1071, DA1072, DA1073, DA1074, DA1075, DA1076, DA1078, DA1079, DA1085, DA1086, DA1091, DA1092, DA1093, DA1095, and DA1102.

[0019] As a preferred technical solution of the present invention, the double-stranded siRNA analog is selected from any one of Table 4.

[0020] As a preferred technical solution of the present invention, the double-stranded siRNA analog includes at least one modified nucleotide. In a specific embodiment, the sense strand and / or the antisense strand each independently include at least one modified nucleotide. In a specific embodiment, the sense strand includes at least one modified nucleotide and the nucleotides of the antisense strand are not modified, or the nucleotides of the sense strand are not modified and the antisense strand includes at least one modified nucleotide. In some preferred embodiments, the nucleotides of the sense strand and the antisense strand are both modified nucleotides.

[0021] In some embodiments, the modified nucleotides are selected from the group consisting of alkyl-modified nucleotides, methoxy-modified nucleotides (e.g., 2'-O-methyl-modified nucleotides), ethoxy-modified nucleotides (e.g., 2'-O-ethyl-modified nucleotides), 2'-O-allyl-modified nucleotides, 2'-hydroxy-modified nucleotides, methoxyethyl-modified nucleotides, amino-modified nucleotides, fluoro-modified nucleotides (e.g., 2'-fluoro-modified nucleotides), deoxynucleotides, 5'-methylphosphate nucleotides, 5'-C-methylphosphate-based nucleotides, 2'-deoxy-2'-fluoro nucleotides, vinylphosphonate deoxyribonucleotides, nucleotides containing thiophosphate groups, nucleotides containing dithiophosphate groups, locked nucleic acids (LNA), glycol nucleic acids (GNA), threose nucleic acids (TNA), morpholino oligonucleotides (PMO), (E)-vinyl phosphate-modified nucleotides (VP), 3'-O-Me-2', 5'-PS-RNA-modified nucleotides. In some embodiments, the alkyl-modified nucleotides are selected from the group consisting of methyl-modified nucleotides and ethyl-modified nucleotides.

[0022] As a preferred technical solution of the present invention, the sense strand and / or the antisense strand independently include at least one 2'-O-methyl modified nucleotide, at least one 2'-fluoro modified nucleotide and at least one nucleotide containing a phosphorothioate group.

[0023] As a preferred technical solution of the present invention, the sense strand and / or the antisense strand each independently comprises at least one 2'-fluoro-modified nucleotide, preferably, the number of 2'-fluoro-modified nucleotides does not exceed 4, for example, 4, 3 or 2. In a specific embodiment, from the 5' end to the 3' end, the 5th, 7th, 8th and 9th nucleotides of the sense strand are each independently a 2'-fluoro-modified nucleotide, and optionally, the remaining nucleotides of the sense strand are each independently selected from 2'-O-methyl-modified nucleotides and nucleotides containing a phosphorothioate group. In a specific embodiment, in the direction from the 5' end to the 3' end, the 2nd, 6th, 14th and / or 16th nucleotides of the antisense strand are each independently a 2'-fluoro-modified nucleotide, preferably, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 14 and 16, or positions 2, 14 and 16 of the antisense strand; optionally, the remaining nucleotides of the antisense strand are each independently selected from 2'-O-methyl-modified nucleotides, nucleotides containing thiophosphate groups, glycol nucleic acids (GNA), threose nucleic acids (TNA), 3'-O-Me-2',5'-PS-RNA modified nucleotides, (E)-vinyl phosphate modified nucleotides (VP).

[0024] As a preferred technical solution of the present invention, the number of the 2'-O-methyl modified nucleotides is no more than 17, preferably 17, 16 or 15. The number of the nucleotides containing phosphorothioate groups is no more than 4, preferably 4 or 2.

[0025] In a specific embodiment, from the 5' end to the 3' end, the 1st to 4th, 6th, and 10th to 19th nucleotides of the sense strand are each independently a 2'-O-methyl modified nucleotide. In a specific embodiment, from the 5' end to the 3' end, the 1st, 3rd to 5th, 7th to 13th, 15th, and 17th to 21st nucleotides of the antisense strand are each independently a 2'-O-methyl modified nucleotide. In a specific embodiment, the phosphorothioate group is present in at least one position selected from the following positions: from the 5' end to the 3' end, between the 1st and 2nd nucleotides of the sense strand, between the 2nd and 3rd nucleotides of the sense strand, between the 1st and 2nd nucleotides of the antisense strand, between the 2nd and 3rd nucleotides of the antisense strand, between the first to last and the second to last nucleotides of the antisense strand, and between the second to last and the third to last nucleotides of the antisense strand.

[0026] As a preferred technical solution of the present invention, the sense strand and / or the antisense strand each independently comprises at least one GNA. In a specific embodiment, the antisense strand has one GNA, and preferably, from the 5' end to the 3' end, the 6th or 7th nucleotide of the antisense strand is a GNA.

[0027] As a preferred technical solution of the present invention, the sense strand and / or the antisense strand each independently include at least one 3'-O-Me-2', 5'-PS-RNA modified nucleotide. In a specific embodiment, the antisense strand contains a 3'-O-Me-2', 5'-PS-RNA modified nucleotide. Preferably, from the 5' end to the 3' end, the 7th nucleotide of the antisense strand is a 3'-O-Me-2', 5'-PS-RNA modified nucleotide.

[0028] As a preferred technical solution of the present invention, the sense strand and / or the antisense strand each independently comprises at least one threose nucleic acid (TNA). In a specific embodiment, the antisense strand has one TNA, and preferably, from the 5' end to the 3' end, the 7th nucleotide of the antisense strand is a TNA.

[0029] As a preferred embodiment of the present invention, the sense strand and / or the antisense strand each independently comprises at least one (E)-vinyl phosphate-modified nucleotide (VP). In a preferred embodiment, the nucleotide at the 5' end of the antisense strand is VP.

[0030] In some specific embodiments, the sense strand is arranged from the 5' end to the 3' end, and the 5th, 7th, 8th and 9th nucleotides are each independently a 2'-fluoro-modified nucleotide, and the remaining nucleotides are each independently a 2'-O-methyl-modified nucleotide, and a phosphorothioate group is present between the 1st and 2nd nucleotides and between the 2nd and 3rd nucleotides.

[0031] In some specific embodiments, the antisense strand is arranged in the 5'-end to 3'-end direction, and the 2nd, 6th, 14th and 16th nucleotides are each independently a 2'-fluoro-modified nucleotide, and the remaining nucleotides are each independently a 2'-O-methyl-modified nucleotide, and a phosphorothioate group is present between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 1st to last and the 2nd to last nucleotides, and between the 2nd to last and the 3rd to last nucleotides, optionally, the 1st nucleotide contains VP.

[0032] In some specific embodiments, the antisense strand is arranged from the 5' end to the 3' end, and the 2nd, 14th and 16th nucleotides are each independently a 2'-fluoro modified nucleotide, the 6th nucleotide is selected from any one of a 2'-O-methyl modified nucleotide or GNA, the 7th nucleotide is selected from any one of a 2'-O-methyl modified nucleotide, GNA, TNA or a 3'-O-Me-2', 5'-PS-RNA modified nucleotide, and the remaining nucleotides are each independently a 2'-O-methyl modified nucleotide, and a phosphorothioate group is present at the 1st and 2nd positions. between the nucleotides at positions 2 and 3, between the 1st to 2nd nucleotides, and between the 2nd to 3rd nucleotides, optionally, the 1st nucleotide comprises VP; preferably, the modifications of the 6th and 7th nucleotides are different; more preferably, the 6th nucleotide is a 2'-O-methyl modified nucleotide, and the 7th nucleotide is selected from any one of GNA, TNA or 3'-O-Me-2', 5'-PS-RNA modified nucleotides; or, the 6th nucleotide is GNA and the 7th nucleotide is a 2'-O-methyl modified nucleotide.

[0033] As a preferred technical solution of the present invention, the antisense strand comprises a modified antisense strand nucleotide sequence shown in any one of Table 2 or Table 3.1, and / or the sense strand comprises a modified sense strand nucleotide sequence shown in any one of Table 2 or Table 3.1. As a preferred technical solution of the present invention, the double-stranded siRNA analog comprises a paired modified sense strand nucleotide sequence and a modified antisense strand nucleotide sequence shown in any one of Table 2 or Table 3.1. In some specific embodiments, the double-stranded siRNA analog is selected from any one of Table 5 and Table 6.

[0034] As a preferred technical solution of the present invention, the double-stranded siRNA analog is connected to a targeting ligand.

[0035] Wherein, the targeting ligand is selected from:

[0036]

[0037] In some specific embodiments, the targeting group can be connected to the 3' end of the sense strand or antisense strand of a double-stranded siRNA analog. In some specific embodiments, the targeting group can be connected to the 5' end of the sense strand or antisense strand of a double-stranded siRNA analog. In some specific embodiments, the targeting group is connected to the 5' end of the sense strand. In some specific embodiments, the targeting group is connected to the 3' end of the sense strand. In some specific embodiments, the targeting group can also be internally connected to nucleotides on the sense strand and / or antisense strand of a double-stranded siRNA analog. In some specific embodiments, the targeting group can also be connected to the double-stranded siRNA analog through a joint, for example, the targeting group can also be connected to the 3' or 5' end of the sense strand through a joint, or, the targeting group can also be connected to the 3' or 5' end of the antisense strand through a joint, or, the targeting group can also be internally connected to nucleotides on the sense strand and / or antisense strand of a double-stranded siRNA analog through a joint.

[0038] As a preferred technical solution of the present invention, the double-stranded siRNA analog comprises a paired modified sense strand nucleotide sequence and a modified antisense strand nucleotide sequence as shown in any one of Table 3.2. In some specific embodiments, the double-stranded siRNA analog is selected from any one of Table 7.1, Table 7.2, Table 8.1, and Table 8.2.

[0039] In a second aspect, the present invention provides a vector comprising a nucleotide sequence encoding a double-stranded siRNA analog as described above. The vector is capable of amplifying or expressing the nucleotide sequence encoding the double-stranded siRNA analog of the present invention linked thereto. The vector can be a viral vector or plasmid capable of transporting nucleic acid molecules, such as: (a) adenoviral vector; (b) retroviral vector; (c) adeno-associated viral vector; (d) herpes simplex virus vector; (e) SV40 vector; (f) polyoma virus vector; (g) papilloma virus vector; (h) picornavirus vector; (i) poxvirus vector; and (j) helper virus-dependent adenovirus or enterovirus.

[0040] In a third aspect, the present invention provides a cell comprising the double-stranded siRNA analog or the vector described above, wherein the double-stranded siRNA analog or the vector of the present invention can be transcribed in the cell.

[0041] In a fourth aspect, the present invention provides a pharmaceutical composition comprising the double-stranded siRNA analog as described above, or the vector as described above, or the cell as described above.

[0042] As a preferred technical solution of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0043] In a fifth aspect, the present invention provides a kit comprising the double-stranded siRNA analog as described above, or the vector as described above, or the cell as described above.

[0044] In a sixth aspect, the present invention further provides use of the double-stranded siRNA analog, vector, cell, kit, or pharmaceutical composition described above in the preparation of a medicament for reducing DGAT2 expression in a cell or subject. Reducing DGAT2 expression includes reducing the amount of DGAT2 mRNA, protein, or both. The subject benefits from the reduced DGAT2 expression.

[0045] The present invention also provides a use of the double-stranded siRNA analog, vector, cell, kit or pharmaceutical composition described above in preparing a medicament for preventing and / or treating diseases, disorders or symptoms mediated by abnormal or overexpression of DGAT2.

[0046] In some embodiments, the disease, disorder or symptom is selected from the group consisting of liver disease, dyslipidemia, dysglycemia, cardiovascular disease, kidney disease, metabolic syndrome, and obesity. In some embodiments, the disease, disorder or symptom is selected from the group consisting of metabolic liver disease, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), drug-induced liver disease, alcohol-induced liver disease, infectious agent-induced liver disease, inflammatory liver disease, liver disease mediated by immune system dysfunction, dyslipidemia, cardiovascular disease, restenosis, syndrome X, metabolic syndrome, diabetes, obesity, hypertension, chronic bile duct disease, such as primary sclerosing cholangitis (PSC), primary biliary cholangitis (PBC), biliary atresia, progressive familial intrahepatic cholestasis type 3 (PFIC3), inflammatory bowel disease, Crohn's disease, ulcerative colitis, liver cancer, hepatocellular carcinoma, gastrointestinal cancer, gastric cancer, colorectal cancer, metabolic disease-induced liver fibrosis or cirrhosis, NAFLD-induced fibrosis or cirrhosis, NASH-induced fibrosis or cirrhosis, alcohol-induced liver fibrosis or cirrhosis, drug-induced liver Fibrosis or cirrhosis, infectious agent-induced liver fibrosis or cirrhosis, parasitic infection-induced liver fibrosis or cirrhosis, bacterial infection-induced liver fibrosis or cirrhosis, viral infection-induced liver fibrosis or cirrhosis, HBV infection-induced liver fibrosis or cirrhosis, HCV infection-induced liver fibrosis or cirrhosis, HIV infection-induced liver fibrosis or cirrhosis, dual HCV and HIV infection-induced liver fibrosis or cirrhosis, radiation-induced or chemotherapy-induced fibrosis or cirrhosis, biliary fibrosis, liver fibrosis or cirrhosis induced due to any chronic cholestatic disease, intestinal fibrosis of any etiology, Crohn's disease-induced fibrosis, ulcerative colitis-induced fibrosis, intestinal (e.g., small intestinal) fibrosis, colonic fibrosis, gastric fibrosis, pulmonary fibrosis, pulmonary fibrosis subsequent to chronic inflammatory airway disease, such as COPD, asthma, emphysema, smoker's lung, tuberculosis, pulmonary fibrosis, idiopathic pulmonary fibrosis (IPF).

[0047] The double-stranded siRNA analogs for inhibiting DGAT2 expression provided by the present invention have excellent drug safety, reduced "off-target" effects, and improved bioavailability. In the treatment of metabolic diseases with complex pathogenic mechanisms such as NASH, they can effectively lower cholesterol and triglyceride levels, improve liver function and liver histology, and regress liver fibrosis. DETAILED DESCRIPTION

[0048] The present invention will be further described in detail below with reference to examples, but the embodiments of the invention are not limited thereto.

[0049] The term "comprising" herein is used to mean, and is used interchangeably with, the phrase "including but not limited to," unless the context clearly indicates otherwise.

[0050] The term "or" of the present invention is used herein to mean, and is used interchangeably with, the term "and / or," unless the context clearly indicates otherwise.

[0051] The terms "sequence" and "nucleotide sequence" of the present invention mean the order or sequence of nucleobases or nucleotides, described in alphabetical order using standard nomenclature.

[0052] The term "siRNA analogue" of the present invention refers to a complex of ribonucleic acid molecules having a double-stranded structure that can mediate the silencing of a target RNA (e.g., mRNA) that is complementary thereto. The siRNA analogue comprises two reverse-parallel and substantially complementary nucleic acid chains, the nucleic acid chains comprising an antisense strand that is complementary to the target RNA, and a sense strand that is complementary to the antisense strand. When the two nucleic acid chains are "substantially complementary" or "substantially complementary," they can be fully complementary, or they can form one or more, but generally no more than 5, 4, 3, or 2, mismatched nucleotide pairs for a duplex of up to 30 nucleotide pairs after hybridization, while retaining the ability to hybridize under conditions most relevant to their ultimate application, such as inhibition of gene expression in vitro or in vivo.

[0053] As used herein, "complementary" has the meaning known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one strand pair with bases on the other strand in a complementary manner. Adenine (A) always pairs with uracil (U); guanine (C) always pairs with cytosine (G). Each base pair consists of a purine and a pyrimidine. When adenine on one strand always pairs with uracil on the other strand, and guanine always pairs with cytosine, the two strands are considered complementary to each other, and the sequence of that strand can be inferred from the sequence of its complementary strand.

[0054] In the present invention, the term "region of complementarity" refers to a region on the antisense strand that is completely or substantially complementary to a sequence defined herein (e.g., a target sequence). In the case where the complementary region is not completely complementary to the target sequence, mispairing can be located in the interior or terminal regions of the molecule. Typically, the most tolerated mispairing is located in the terminal regions, e.g., within 5, 4, 3, 2, or 1 nucleotides at the 5' and / or 3' ends. The antisense strand portion that is most sensitive to mispairing is referred to as a "seed region." For example, in a siRNA comprising a 19nt chain, the 19th position (from 5' to 3') can tolerate some mispairing.

[0055] In the present invention, the term "overhang" refers to one or more unpaired nucleotides at the ends of a chain that extend beyond the complementary region. When the 3' end of a chain extends beyond the 5' end of another chain or when the 5' end of a chain extends beyond the 3' end of another chain, a nucleotide overhang is typically formed. Double-stranded siRNA analogs can include an overhang with at least one nucleotide, for example, the overhang can include at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more. The nucleotide overhang can include or consist of nucleotide / nucleoside analogs (including deoxynucleotides / nucleosides). One or more overhangs can be on the sense strand, the antisense strand, or any combination thereof. One or more overhangs can be on the 5' end, the 3' end, or both ends of the antisense strand or the sense strand.

[0056] The terms "complementary," "fully complementary," and "substantially complementary" herein can be used with respect to base matching between the sense and antisense strands of a double-stranded siRNA analog, or between two oligonucleotides or polynucleotides (such as the antisense strand of a double-stranded siRNA analog and a target sequence), as understood from the context of their use.

[0057] In the present invention, modified nucleotides include but are not limited to: alkyl nucleotides, methoxy nucleotides, ethoxy nucleotides, methoxyethyl nucleotides, amino nucleotides, fluoro nucleotides, deoxy nucleotides, 5'-methyl phosphate nucleotides, 5'-C-methyl phosphate nucleotides, 2'-deoxy-2'-fluoro nucleotides, (E)-vinyl phosphate-modified nucleotides (VP), phosphorothioate nucleotides, phosphorodithioate nucleotides, locked nucleic acids (LNA), glycol nucleic acids (GNA), morpholino oligonucleotides (PMO), and inverted abasic deoxyribose residues (invAb).

[0058] Among them, alkyl-modified nucleotides, such as 2'-methyl nucleotides, 2'-ethyl nucleotides, 2'-methoxy modified nucleotides, e.g. 2'-methoxyethyl nucleotides, e.g. 2'-methoxyethoxy nucleotides, e.g. 2'-fluoronucleotides, e.g. 5'-C-methylphosphononucleotides, e.g.

[0059] (E)-vinyl phosphate modified nucleotides (VP), e.g. Phosphorothioate nucleotides (PS), e.g. 2'-deoxyribonucleotides, such as: Inverted abasic deoxyribose residues (invAb), for example:

[0060] Wherein, Base represents a base, R represents an alkyl group or an alkoxy group, Me represents a methyl group, and Et represents an ethyl group.

[0061] The term "locked nucleic acid" is a nucleotide with a modified ribose moiety, wherein the ribose moiety includes an additional bridge connecting the 2' carbon and the 4' carbon. This structure effectively "locks" the ribose in a 3'-endo conformation. Adding locked nucleic acids to siRNA has been shown to increase siRNA stability in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O. R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).

[0062] Representative U.S. patents for preparing locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Patent Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207; 7,084,125 and 7,399,845, each of which is incorporated herein by reference in its entirety.

[0063] The locked nucleic acid structure is as follows:

[0064]

[0065] In certain embodiments, the sugar surrogate comprises a ring having more than 5 atoms and more than 1 heteroatom. For example, nucleosides comprising morpholino sugar moieties and their use in oligomeric compounds have been reported (see, e.g., Braasch et al., Biochemistry, 2002, 41, 4503-4510; and U.S. Patents 5,698,685; 5,166,315; 5,185,444; and 5,034,506).

[0066] The term "morpholino" means a sugar surrogate having the formula:

[0067]

[0068] In certain embodiments, the morpholino group can be modified, for example, by adding or changing various substituents according to the above morpholino structure. Such sugar surrogates are referred to herein as "modified morpholinos."

[0069] The term "glycol nucleic acid (GNA)" refers to a glycol-modified nucleotide in which the ribose sugar is replaced by a diol unit linked to a phosphodiester bond. Adenosine-GNA is described, for example, in Zhang et al. (JACS 127(12):4174-75 (2005)). The structure of GNA is shown below:

[0070] Wherein B represents a base, preferably (S)-GNA.

[0071] The term "threose nucleic acid (TNA)" refers to a sugar-modified nucleotide containing an α-L-threofuranosyl moiety. TNA is linked via a 2'->3' internucleoside linkage. The structure of TNA and its two preceding and succeeding nucleotides is shown below:

[0072]

[0073] The 3'-O-Me-2',5'-PS-RNA nucleotide has the following structure:

[0074] Where Bx represents the base.

[0075] In the present invention, unless otherwise specified, capital letters C, G, U, A, and T represent the base composition of nucleotides, but it is also generally known in the art that G, C, A, T, and U each generally represent nucleotides containing guanine, cytosine, adenine, thymine, and uracil as bases, respectively, which is a common way of representing deoxyribonucleic acid sequences and / or ribonucleic acid sequences. Therefore, in the context of the present disclosure, the meanings represented by G, C, A, T, and U include the above-mentioned various possible situations. The lowercase letter m indicates that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; the lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a fluorinated modified nucleotide; LNA indicates that the nucleotide adjacent to the right is a locked nucleic acid (LNA) modification; gna indicates that the nucleotide adjacent to the left is GNA; the lowercase letter s indicates that the two nucleotides on the left and right of the letter are connected by a thiophosphate group; VP indicates that the nucleotide on the right of the letter VP is a (E)-vinyl phosphate-modified nucleotide. invAb represents an inverted abasic deoxyribonucleotide; dN represents any deoxyribonucleotide; dA represents a deoxyadenine nucleotide; dT represents a deoxythymidine nucleotide; dC represents a deoxycytosine nucleotide; and dG represents a deoxyguanine nucleotide.

[0076] It should be emphasized that the "modification" of nucleotides in the present disclosure includes but is not limited to the above examples, and nucleotides can also be replaced with other nucleotides, such as (S)-glycerol nucleic acid.

[0077] The term "targeting ligand" can include naturally occurring substances, such as proteins (e.g., human serum albumin (HAS), low-density lipoprotein (LDL) or globulin); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine or hyaluronic acid); or lipids. The ligand can also be a recombinant or synthetic molecule, such as a synthetic polymer, for example, a synthetic polyamino acid. Examples of polyamino acids include the following polyamino acids: polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene acid-maleic anhydride copolymer, poly-(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl) methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly-(2-ethyl acrylic acid), N-isopropylacrylamide polymer or polyphosphazene. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha helical peptides.

[0078] Targeting ligands can also be cell or tissue targeting agents that bind to a specific cell type, such as renal cells, such as lectins, glycoproteins, lipids or proteins, such as antibodies. The targeting group can be thyroid stimulating hormone, melanocyte stimulating hormone, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, multivalent fucose, glycosylated polyamino acids, multivalent galactose, transferrin, bisphosphonates, polyglutamic acid, polyaspartic acid, lipids, cholesterol, steroids, bile acid, folic acid, vitamin B12, vitamin A, biotin, or RGD peptide or RGD peptide mimetic.

[0079] Targeting ligands can also be proteins, for example, glycoproteins, or peptides, for example, molecules with specific affinity for co-ligands, or antibodies, for example, antibodies that bind to a given cell type, for example, hepatocytes. Ligands can also include hormones and hormone receptors. They can also include non-peptide species, for example, lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose or multivalent fucose. Ligands can be, for example, lipopolysaccharides, activators of p38 MAP kinase, or activators of NF-KKB.

[0080] The targeting ligand can be a substance, e.g., a drug, that can increase the uptake of an iRNA agent into a cell, e.g., by disrupting the cytoskeleton of the cell (e.g., by disrupting cellular microtubules, microfilaments, and / or intermediate filaments). The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, iaplakinolide, erythrosine A, phalloidin, swinholide A, indanocine, or myoservin.

[0081] The term "pharmaceutically acceptable excipient" is a substance that is intentionally included in a drug delivery system other than the active pharmaceutical ingredient (API, therapeutic product, e.g., double-stranded siRNA analog that inhibits INHBE). Excipients do not exert or are not intended to exert a therapeutic effect at the intended dose. Excipients may serve the following functions: a) aid in the handling of the drug delivery system during preparation, b) protect, support, or enhance the stability, bioavailability, or patient acceptability of the API; c) aid in product identification; and / or d) enhance any other attribute of the overall safety, efficacy, or delivery of the API during storage or use.

[0082] Among them, excipients include (but are not limited to): absorption enhancers, anti-adherents, defoaming agents, antioxidants, binders, buffers, carriers, coating agents, colorants, delivery enhancers, delivery polymers, detergents, dextran, dextrose, diluents, disintegrants, emulsifiers, expanders, fillers, flavorings, glidants, wetting agents, oils, polymers, preservatives, saline, salts, solvents, sugars, surfactants, suspending agents, sustained release matrices, sweeteners, thickeners, tonicity agents, vehicles, waterproofing agents, wetting agents, lubricants such as sodium lauryl sulfate and magnesium stearate, flavoring agents and fragrances.

[0083] Pharmaceutical compositions of the present disclosure include those suitable for oral, nasal, topical, buccal, sublingual, rectal and / or parenteral administration. The preparation can be conveniently present in unit dosage form and can be prepared by any method known in the pharmaceutical field. The amount of active ingredient that can be combined with a carrier material to prepare a single dose form is generally the amount of the compound that produces the therapeutic effect. Generally speaking, in percent units, this amount is from about 1% to about 99% active ingredient, preferably from about 5% to about 70%, and most preferably from about 10% to about 30%.

[0084] The term "vector" refers to a nucleic acid molecule capable of amplifying or expressing another nucleic acid to which it is linked.

[0085] As used herein, diacylglycerol acyltransferase 2 (DGAT2) refers to any DGAT2 molecule known to those skilled in the art, including variants of the DGAT2 gene, such as those provided in the SNP database. Many sequence variations within the DGAT2 gene have been identified and can be found, for example, in NCBI dbSNP and UniProt (see, for example, https: / / www.ncbi.nlm.nih.gov / snp / ?term=DGAT2, the entire contents of which are incorporated herein by reference as of the filing date of this application). The mRNA sequence of human DGAT2 can be found, for example, in GenBank Accession Nos. NM_032564.5 and NM_001253891.1; the mRNA sequence of mouse DGAT2 can be found, for example, in GenBank Accession No. NM_026384.3. The mRNA sequence of monkey DGAT2 can be found, for example, in GenBank Accession No. XM_005579118.2. Other examples of DGAT2 mRNA sequences can be obtained using, for example, GenBank, UniProt, OMIM. As of the filing date of this application, the entire contents of each of the above GenBank accession numbers are incorporated herein by reference.

[0086] The term "treatment" is used to refer to obtaining a desired pharmacological and / or physiological effect. The effect may be preventative in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, such as arresting the progression of the disease; or (c) alleviating the disease, such as alleviating the symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug or compound to an individual to treat, cure, alleviate, improve, reduce or inhibit the individual's disease, including but not limited to administering a drug containing a compound described herein to an individual in need.

[0087] The term "inhibit" is used interchangeably with "reduce," "silence," "downregulate," "suppress," and other similar terms and includes any level of inhibition.

[0088] The phrase "inhibiting DGAT2 expression" in the present invention includes inhibiting the expression of any DGAT2 gene as well as gene variants or mutants encoding the protein thereof.

[0089] "Inhibiting DGAT2 expression" includes any level of inhibition of the DGAT2 gene, such as at least partial inhibition of the expression of the DGAT2 gene, such as inhibition of 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%.

[0090] The expression of the DGAT2 gene can be assessed based on the level of any variable associated with the expression of the gene, such as DGAT2 mRNA level, DGAT2 protein level. Inhibition can be assessed by a reduction in the absolute or relative level of one or more of these variables compared to a control level. The control level can be any type of control level used in the art, such as a pre-dose baseline level or a level determined from a similar subject, cell, or sample that has not been treated or that has been treated with a control (e.g., a buffer-only control or an inactive agent control).

[0091] In one embodiment, at least partial inhibition of expression of a DGAT2 gene can be assessed by a decrease in the amount of DGAT2 mRNA isolated or detected from a first cell or a group of first cells in which the DGAT2 gene is transcribed (which has been treated such that expression of the DGAT2 gene is inhibited), as compared to a second cell or a group of second cells (control cells) that are substantially identical to the first cell or cells but have not been so treated. The degree of inhibition can be expressed as follows:

[0092]

[0093] Example 1 Synthesis of Targeting Ligand (L96)

[0094] Step A: Hydroxyproline amine (3.00 g, 7.15 mmol) and monomethyl dodecanedioate (1.748 g, 7.15 mmol) were placed in N,N-dimethylformamide (DMF) (50 mL). The peptide coupling reagent (HBTU) (3.25 g, 8.56 mmol) and N,N-diisopropylethylamine (DIEA) (3.7 mL, 21.24 mmol) were added and the reaction was stirred overnight.

[0095] The reaction mixture was poured into an ice-water mixture and extracted with dichloromethane (DCM). The mixture was washed with bicarbonate solution, water, brine, and dried over sodium sulfate. The solvent was removed and the residue was purified by chromatography (eluting with 50% ethyl acetate / hexane, ethyl acetate, and then 5% methanol / dichloromethane) to afford the desired compound 115 as a white solid (4.30 g, 93%). MS: C 39 H 51 NO7, calculated as 645.37, found as 646.35 (M+H).

[0096]

[0097] Step B: Compound 101 (4.25 g, 6.58 mmol) was dissolved in a mixture of tetrahydrofuran / methanol / water (50 mL, 2:1:1). Lithium hydroxide (LiOH) (1.90 g, 45.2 mmol) was added and the mixture was stirred overnight.

[0098] After checking with thin layer chromatography silica gel plates (TLC), acetic acid was added to neutralize the reaction mixture. The solvent was removed and the residue was extracted with dichloromethane (DCM). Triethanolamine (TEA, excess) was added to the dichloromethane (DCM) solution and the solution was filtered through a small silica gel pad to obtain the desired product 102 as its triethanolamine (TEA) salt (4.15 g, 86%). MS: C 38 H 49 NO7, calculated value is 631.35; found value is 630.34 (MH).

[0099]

[0100] Step C: Compound 102 (1.30 g, 2.06 mmol) and a peptide coupling reagent (HBTU) (0.821 g, 1.05 eq.) were added to N,N-dimethylformamide (DMF) (30 mL). To this N,N-diisopropylethylamine (DIEA) (1.07 ml, 3 eq.) was added and the reaction mixture was stirred for 3-4 minutes. A solution of the amine (3.00 g, 1.58 mmol) was added, followed by 1 eq. of DIEA. The reaction mixture was stirred at room temperature overnight.

[0101] The solvent was removed under reduced pressure and the residue was dissolved in dichloromethane (DCM) and washed with bicarbonate and water. The dichloromethane (DCM) was dried over sodium sulfate and the solvent was removed. The residue was purified by chromatography (eluting first with ethyl acetate and then with 5-20% methanol / dichloromethane) to give the product 103 as a white solid (3.35 g, 88%). MS: for C 117 H 175 N 11 O42 , the calculated value is 2406.19; the measured value is 2429.10 (M+Na).

[0102]

[0103] Among them, ligand 104 (L96) can be linked to siRNA via a phosphate group, a phosphorothioate group, or another linking group.

[0104] For the specific synthesis route, please refer to the document with International Patent Publication No. WO2009073809.

[0105] Example 2 Preparation of double-stranded siRNA analogs for inhibiting DGAT2 expression

[0106] siRNA was prepared using an OligoMaker ApS192 RNA synthesizer (made in Denmark). The specific synthesis route can be found in, for example, patent document CN202180062335.4. The sequences of siRNA analogs used to inhibit DGAT2 expression are shown in Table 1. Furthermore, the double-stranded siRNA analogs shown in Table 1 were prepared into a modified form having the following formula I:

[0107] Justice chain: NmsNmsNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNmNm,

[0108] Antisense strand: NmsNfsNmNmNmNfNmNmNmNmNmNmNmNmNfNmNfNmNmNmsNmsNm;

[0109] Where: "N" represents nucleotides, such as A, G, U and C;

[0110] m indicates that the nucleotide adjacent to its left is a 2'-O-methyl modified nucleotide, for example, Am, Um, Gm and Cm represent 2'-O-methyl modified A, U, G and C respectively;

[0111] f indicates 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;

[0112] s indicates that the two nucleotides adjacent to the letter s are connected by a phosphorothioate group.

[0113] The sequences of exemplary modified double-stranded siRNA analogs of Formula I are shown in Table 2; the sequences of other exemplary modified double-stranded siRNA analogs in Table 1 are listed in Tables 3.1 and 3.2.

[0114] Table 1 is a list of unmodified siRNA analogs used to inhibit DGAT2 expression

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129] Table 2 is an exemplary modified siRNA sequence list

[0130]

[0131]

[0132]

[0133] Table 3.1 is an exemplary modified siRNA sequence table

[0134]

[0135]

[0136] Table 3.2 is an exemplary modified siRNA sequence table

[0137]

[0138]

[0139]

[0140]

[0141] In Tables 3.1-3.2: m indicates that the nucleotide adjacent to its left is a 2'-O-methyl-modified nucleotide; f indicates that the nucleotide adjacent to its left is a 2'-fluoro-modified nucleotide; s indicates that the two nucleotides adjacent to the letter s are connected by a thiophosphate group; gna indicates that the nucleotide adjacent to its left is GNA; VP indicates that the nucleotide adjacent to its right is a (E)-vinyl phosphate-modified nucleotide; TNA indicates that the nucleotide adjacent to its left is TNA; 3OMe-2-5 indicates that the nucleotide adjacent to its left is a 3'-O-Me-2',5'-PS-RNA-modified nucleotide; L96 indicates that the nucleotide adjacent to its left is connected to the L96 ligand.

[0142] Example 3 In vitro testing of double-stranded siRNA analogs in HuH7 cells

[0143] 3.1 In vitro reporter gene-based assays

[0144] The exonic region of the human DGAT2 (NM_032564.5) gene (including 5'UTR and 3'UTR sequences) was cloned into the reporter-based screening plasmid pDRIVE5s-SV40hALB, designated DGAT2-SEAP plasmid, which produces SEAP luciferase / DGAT2 fusion mRNA. HuH7 cells were cultured in DMEM (Gibco-10313021) medium supplemented with 10% fetal bovine serum (Gibco-10099141C), 1% glutamine (Gibco-35050061), 1% non-essential amino acids (Gibco-11140050), and 1% penicillin-streptomycin (Gibco-15070063). DGAT2-SEAP plasmid, RNAi agent and Lipo2000 (Invitrogen-11668019) transfection reagent diluted with Opti-MEM (Gibico-11058021) were added to the HuH7 cell suspension and the cells were cultured at a rate of 1×10 5 The cells were plated in 96-well plates at a density of 100 cells / ml, so that the final concentration of RNAi agent was 1 nM and 0.02 nM. After culturing for 24 hours, the supernatant was taken and analyzed using Phospha-Light TMThe SEAP reporter gene assay system (Invitrogen-T1017) measured relative fluorescence levels normalized to those in cells transfected with the DGAT2-SEAP plasmid alone (control group). The RNAi-mediated inhibition rate of the DGAT2 gene was calculated according to the following formula: DGAT2 gene inhibition rate (%) = (1 - relative SEAP luciferase level in the sample / relative SEAP luciferase level in the control group) × 100. Results for some siRNAs are shown in Table 4.

[0145] Table 4 mRNA inhibition rate of double-stranded siRNA at 1 nM

[0146] Number Average Inhibition% Number Average Inhibition% Number Average Inhibition% DA0023 90.89 DA0215 66.42 DA0339 79.20 DA0028 83.38 DA0223 79.76 DA0340 84.24 DA0054 82.11 DA0233 68.17 DA0341 65.09 DA0062 75.47 DA0247 71.93 DA0474 40.96 DA0064 86.77 DA0250 73.00 DA0065 79.24 DA0251 86.63 DA0069 87.66 DA0262 69.87 DA0071 83.19 DA0267 68.58 DA0072 88.14 DA0271 80.17 DA0076 91.57 DA0272 74.02 DA0077 88.37 DA0273 78.86 DA0078 90.07 DA0274 84.79 DA0106 86.57 DA0275 82.24 DA0108 83.34 DA0277 77.49 DA0109 85.69 DA0281 70.08 ​ 81.57 ​ 73.20 ​ 65.48 ​ 81.17 ​ 75.64 ​ 83.91 ​ 84.62 ​ 82.71 ​ 89.26 ​ 81.71 ​ 73.60 ​ 82.14

[0147] 3.2 In vitro testing based on q-PCR

[0148] q-PCR experiments (Taqman probe method) were used to detect the effect of siRNA on DGAT2 gene expression in HuH7 cells. In order to determine the inhibitory efficiency at the mRNA level, DGAT2 siRNA was transfected into HuH7 cells, and then q-PCR experiments were performed to determine the expression level of DGAT2 mRNA. Specifically, HuH7 cells were cultured in DMEM medium (Gibco11965-092) containing 10% fetal bovine serum (ExCell Bio FSP500), 1% glutamine (Gibco 35050061), 1% NEAA (Gibco 11140050), and 1% penicillin-streptomycin (HyCloneSV30010). HuH7 cells in the logarithmic growth phase were cultured at 1×10 4 The cells were plated in 96-well cell plates at a density of 100 cells / well. siRNA was injected into the plates with Lipofectamine TM RNAiMAX (INVITROGEN 13778150) was mixed to make the final concentration of RNAi agent mixture 1nM, 0.1nM, 0.02nM, and then the RNAi agent mixture was transfected into cells. Incubate overnight in a 37°C 5% CO2 incubator and measure 2 replicates in parallel. At the same time, set up a Lipofectamine TM RNAiMAX compound-free control. 48 hours after transfection, culture medium was removed, and total RNA was extracted (QIAGEN-74182) and reverse transcribed (Vazyme-R323-1). Target cDNA was detected using TaqMan assays, with GAPDH cDNA used in parallel as an internal control.

[0149] The expression level of the target gene mRNA in each sample was calculated using the ΔΔCT relative quantification method. The relative expression of the target gene was expressed as 2-ΔΔCt. The specific method is to subtract the CT value of the internal reference gene (GAPDH) from the CT value of the target gene in each sample to obtain the ΔCT value. Then, the ΔCT value of the transfection reagent-only control group (RNAiMAX Control) was subtracted from the ΔCT value of the drug group (sample) to obtain the ΔΔCT value. Finally, the ΔΔCT value was converted to 2-ΔΔCt to obtain the relative expression level of DGAT2 mRNA (value of sample).

[0150] The inhibition rate of DGAT2 gene mediated by siRNA analogs was calculated according to the following formula:

[0151] DGAT2 inhibition rate (%) = (1-value of sample / Average value of RNAiMAX Control)*100.

[0152] The siRNA of the present invention exhibits DGAT2 inhibitory activity, and in particular, the double-stranded siRNA analogs listed in Table 5 have higher DGAT2 mRNA inhibitory activity.

[0153] Table 5 mRNA inhibition rate of double-stranded siRNA at 1 nM

[0154]

[0155] Example 4 In vitro testing of double-stranded siRNA analogs in primary hepatocytes

[0156] To determine the inhibitory efficiency at the mRNA level, DGAT2 siRNA was transfected into primary human hepatocytes (PHH) and primary cynomolgus monkey hepatocytes (PCH), and q-PCR experiments were performed to measure the expression level of DGAT2 mRNA. Specifically, PHH and PCH cells were cultured in CP medium (BIOIVT Catalog No. BIOIVTS03316) containing 10% fetal bovine serum (ExCell Bio FSP500). PHH or PCH cells (5.4×10 4 cells / well) to a 96-well cell plate coated with collagen in advance, and siRNA was added to the plate with Lipofectamine TM RNAiMAX (INVITROGEN 13778150) was mixed to make the final concentration of RNAi agent mixture 10nM, 1nM, 0.1nM, 0.02nM, and then the RNAi agent mixture was transfected into cells. TMRNAiMAX compound-free control. 48 hours after transfection, culture medium was removed, and total RNA was extracted (QIAGEN-74182) and reverse transcribed (Vazyme-R323-1). Target cDNA was detected using TaqMan assays, with GAPDH cDNA used in parallel as an internal control.

[0157] The expression level of the target gene mRNA in each sample was calculated using the ΔΔCT relative quantification method. The relative expression of the target gene was expressed as 2-ΔΔCt. The specific method is to subtract the CT value of the internal reference gene (GAPDH) from the CT value of the target gene in each sample to obtain the ΔCT value. Then, the ΔCT value of the transfection reagent-only control group (RNAiMAX Control) was subtracted from the ΔCT value of the drug group (sample) to obtain the ΔΔCT value. Finally, the ΔΔCT value was converted to 2-ΔΔCt to obtain the relative expression level of DGAT2 mRNA (value of sample).

[0158] The inhibition rate of DGAT2 gene mediated by siRNA analogs was calculated according to the following formula, and the results are shown in Table 6:

[0159] DGAT2 inhibition rate (%) = (1-value of sample / Average value of RNAiMAX Control)*100.

[0160] Table 6 Inhibition rate of siRNA in PHH and PCH cells (1 nM)

[0161]

[0162]

[0163] Example 5 Evaluation of the efficacy of double-stranded siRNA analogs in the mouse AAV model

[0164] 7-8 week old male C57BL / 6 mice were given AAV8 adeno-associated virus (2×10 11vg / mouse / 0.2mL, iv, single) model. One week after modeling, under the action of AAV8 virus, the DGAT2 gene was ubiquitously expressed in mice. At this time, the mice were subcutaneously treated with a dose of 3 mg / kg of GalNAc-conjugated DGAT2 oligonucleotide. After 2 weeks, liver samples were collected for total RNA extraction (Transgen, EC521-96). The extracted total RNA was reverse transcribed (Takara, RR036B), and qRT-PCR analysis of DGAT2 mRNA (Takara, RR820B) was performed. The comparative Ct (ΔΔCt) method was used to determine the expression of the target gene in each test sample by relative quantification; this method measures the Ct difference (ΔCt) between the target gene and the internal reference gene (GAPDH). The formula is as follows:

[0165] ΔCT = average Ct of target gene - average Ct of GAPDH;

[0166] ΔΔCT = ΔCT (sample) - ΔCT (vehicle control);

[0167] Relative amount of target gene mRNA = 2-ΔΔCt;

[0168] % inhibition rate = (relative amount of vehicle control - relative amount of sample) / relative amount of vehicle control × 100%. The inhibition results of some siRNAs are shown in Table 7, which shows that the siRNA of the present invention can still effectively inhibit DGAT2 in vivo.

[0169] Table 7.1 Inhibition rate of siRNA in mouse AAV model

[0170] ​ ​ ​ ​ ​ 0.00 ​ 72.68 ​ 84.95 ​ 75.11 ​ 72.95 ​ 74.32 ​ 91.88 ​ 61.81 ​ 88.66 ​ 79.05 ​ 83.97 ​ 83.05 ​ 84.81 ​ 67.55 ​ 80.61 ​ 79.29 ​ 78.40 ​ 61.00 ​ 80.29 ​ 69.47 ​ 86.90 ​ 74.22 ​ 76.14 ​ 87.12 ​ 85.52 ​ 87.46 ​ 87.48 ​ 78.24 ​ 72.17 ​ 76.65 ​ 77.76 ​ 83.40 ​ 80.31 ​ 82.94 ​ 81.17 ​ 81.29 ​ 81.65 ​ 86.77 ​ 83.58

[0171] Table 7.2 Inhibition rate of siRNA in mouse AAV model

[0172]

[0173]

[0174] Example 6 Evaluation of the in vivo activity of double-stranded siRNA analogs in mice using the DGAT2-SEAP system

[0175] To evaluate the in vivo activity of siRNA compounds, 6-8 week old Balb / C mice were injected with the DGAT2-SEAP system plasmid by hydrodynamic tail vein injection (HDI) at least 7 days in advance, and transient transfection was achieved in mice using this plasmid. The plasmid contains the SEAP (secreted human placental alkaline phosphatase) reporter gene, and the DGAT2 cDNA sequence (NM_032564.5) is inserted into the 3'UTR of the SEAP gene. A saline solution containing 5 μg / mL of the plasmid at a total volume of 10% of the mouse body weight was injected into the mouse via the tail vein within 3-5 seconds to achieve the construction of DGAT2-SEAP model mice. Subsequent treatment of mice with siRNA compounds will inhibit the expression of DGAT2 and SEAP expression. Before the administration of siRNA (day -1), the mouse was treated with Phospha-Light TM The SEAP reporter gene assay system (Invitrogen) was used to measure baseline SEAP expression levels in mouse serum. The mice were then grouped according to baseline SEAP levels. Serum was collected from the mice on days 7, 14, 21, and 28 after administration, and SEAP expression was measured at each time point.

[0176] By comparing the "normalized to pre-treatment" ratio (Ratio Dosed) of individual mice with the average "normalized to pre-treatment" ratio (Average Ratio Vehicle) of the vehicle control group mice, the normalized expression value of the control group at a specific time point can be calculated. Finally, the inhibition rate is calculated as: Inhibition Rate = (1-Ratio Dosed / Average Ratio Vehicle) x 100%.

[0177] The sequence listing is compiled in accordance with WIPO Sequence STANDARD ST.26, wherein Table 8 – Conventional Nucleotide Symbols, and Definition:

[0178] Table 8

[0179]

[0180] The above embodiments are preferred implementations of the present invention, but the implementations of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A double-stranded siRNA analog for inhibiting DGAT2 expression, characterized in that: The double-stranded siRNA analog comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand are each independently 15-30 nucleotides in length, the antisense strand comprises a complementary region that is complementary to the sense strand, and the antisense strand comprises at least 15 consecutive nucleotides of a sequence that differs from any one of SEQ ID NOs. 1058-2114 by no more than 3, 2, 1, or 0 nucleotides; Preferably, the length of the sense strand and the antisense strand is independently 17-27 nucleotides, or 19-25 nucleotides, or 19-23 nucleotides, or 19-21 nucleotides; Preferably, the length of the region of complementarity is at least 15 nucleotide pairs, or 15-25 nucleotide pairs, or 17-23 nucleotide pairs, or 19 nucleotide pairs; Preferably, the sense strand and the antisense strand each independently comprise a 3' overhang and / or a 5' overhang of at least 1 nucleotide, or one or both of the sense strand and the antisense strand comprise a 3' overhang and / or a 5' overhang of at least 2 nucleotides, or the antisense strand comprises a 3' overhang of 2 nucleotides; Preferably, the antisense strand comprises a sequence shown in any one of SEQ ID NOs: 1058-1543, 2121-2164.

2. The double-stranded siRNA analog according to claim 1, characterized in that The sense strand comprises a sequence that differs from the sequence shown in any one of SEQ ID NOs: 1-1057, 2115-2120 by no more than 3, 2, or 1 nucleotides; Preferably, the sense strand comprises a sequence shown in any one of SEQ ID NOs: 1-486, 2115-2120.

3. The double-stranded siRNA analog according to claim 1 or 2, characterized in that The double-stranded siRNA analogs include any pair of paired sense strand sequences and antisense strand sequences as shown in Table 1; Preferably, the double-stranded siRNA analog is selected from DA0023, DA0027, DA0028, DA0038, DA0039, DA0054, DA0057, DA0058, DA0062, DA0064, DA0065, DA0066, DA0069, DA0070, DA0071, DA0072, DA0074, DA0076, DA0077, DA0078, DA0079, DA0080, DA0081, DA0085, DA0106, DA0107, DA0108 0108, DA0109, DA0110, DA0112, DA0140, DA0144, DA0196, DA0199, DA0200, DA0201, DA0202, DA0203, DA0205, DA0214, DA021 5. DA0217, DA0223, DA0233, DA0244, DA0246, DA0247, DA0249, DA0250, DA0251, DA0253, DA0258, DA0259, DA0261, DA0262, DA 0266, DA0267, DA0268, DA0271, DA0272, DA0273, DA0274, DA0275, DA0276, DA0277, DA0278, DA0280, DA0281, DA0293, DA030 5. DA0311, DA0320, DA0321, DA0333, DA0334, DA0335, DA0336, DA0337, DA0338, DA0339, DA0340, DA0341, DA0474, DA1059, DA 1093, DA1095, or DA1102; or, the double-stranded siRNA analog is selected from any one of Table 4.

4. The double-stranded siRNA analog according to any one of claims 1 to 3, characterized in that The double-stranded siRNA analog comprises at least one modified nucleotide selected from: Alkyl-modified nucleotides, methoxy-modified nucleotides, ethoxy-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-hydroxy-modified nucleotides, methoxyethyl-modified nucleotides, amino-modified nucleotides, fluoro-modified nucleotides, deoxyribonucleotides, 5'-methylphosphate nucleotides, 5'-C-methylphosphate nucleotides, 2'-deoxy-2'-fluoro nucleotides, vinylphosphonate deoxyribonucleotides, nucleotides containing phosphorothioate groups, nucleotides containing phosphorodithioate groups, locked nucleic acids (LNA), glycol nucleic acids (GNA), threose nucleic acids (TNA), morpholino oligonucleotides (PMO), (E)-vinyl phosphate-modified nucleotides (VP), 3'-O-Me-2', 5'-PS-RNA modified nucleotides; Preferably, the sense strand and / or the antisense strand each independently comprises at least one 2'-O-methyl modified nucleotide, at least one 2'-fluoro modified nucleotide and at least one nucleotide comprising a phosphorothioate group; More preferably, in the 5' to 3' direction, the 5th, 7th, 8th and 9th nucleotides of the sense strand are each independently a 2'-fluoro-modified nucleotide, and optionally, the remaining nucleotides are each independently selected from a 2'-O-methyl-modified nucleotide and a nucleotide containing a phosphorothioate group; and / or, The 2nd, 6th, 14th and / or 16th nucleotides of the antisense strand are each independently a 2'-fluoro-modified nucleotide, and optionally, the remaining nucleotides are each independently selected from 2'-O-methyl-modified nucleotides, nucleotides containing a phosphorothioate group, GNA, TNA, VP, 3'-O-Me-2', 5'-PS-RNA modified nucleotides; Most preferably, in the 5' to 3' direction, the 5th, 7th, 8th and 9th nucleotides of the sense strand are each independently a 2'-fluoro modified nucleotide, the remaining nucleotides are each independently a 2'-O-methyl modified nucleotide, and a phosphorothioate group is present between the 1st and 2nd nucleotides and between the 2nd and 3rd nucleotides; and / or, The 2nd, 6th, 14th and 16th nucleotides of the antisense strand are each independently a 2'-fluoro-modified nucleotide, and the remaining nucleotides are each independently a 2'-O-methyl-modified nucleotide, and the phosphorothioate group is present between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 1st to last and the 2nd to last nucleotides, and between the 2nd to last and the 3rd to last nucleotides; or, the 2nd, 14th and 16th nucleotides of the antisense strand are each independently a 2'-fluoro-modified nucleotide, and the 6th nucleotide is selected from any one of a 2'-O-methyl modified nucleotide or GNA, the 7th nucleotide is selected from any one of a 2'-O-methyl modified nucleotide, GNA, TNA or a 3'-O-Me-2',5'-PS-RNA modified nucleotide, the remaining nucleotides are each independently a 2'-O-methyl modified nucleotide, and a phosphorothioate group is present between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides, between the 1st to last and the 2nd to last nucleotides, and between the 2nd to last and the 3rd to last nucleotides; Optionally, the first nucleotide of the antisense strand comprises VP in the 5' to 3' direction.

5. The double-stranded siRNA analog according to any one of claims 1 to 4, characterized in that The antisense strand comprises a modified antisense strand nucleotide sequence shown in any one of Table 2 or Table 3.1, and / or the sense strand comprises a modified sense strand nucleotide sequence shown in any one of Table 2 or Table 3.1; Preferably, the double-stranded siRNA analog comprises a paired modified sense strand nucleotide sequence and a modified antisense strand nucleotide sequence as shown in any one of Table 2 or Table 3.1; More preferably, the double-stranded siRNA analog is selected from any one of Table 5 and Table 6.

6. The double-stranded siRNA analog according to any one of claims 1 to 5, characterized in that The double-stranded siRNA analog is linked to a targeting ligand; Preferably, the targeting ligand is selected from the following structures: More preferably, the targeting ligand is linked to the 3' or 5' end of the sense strand; Most preferably, the double-stranded siRNA analog comprises a paired modified sense strand nucleotide sequence and a modified antisense strand nucleotide sequence as shown in any one of Table 3.2, or the double-stranded siRNA analog is selected from any one of Table 7.1, Table 7.2, Table 8.1, and Table 8.

2. 7 . A vector comprising a nucleotide sequence encoding the double-stranded siRNA analog according to claim 1 . A cell comprising the double-stranded siRNA analog according to any one of claims 1 to 6 or the vector according to claim 7.

9. A pharmaceutical composition or kit comprising the double-stranded siRNA analog according to any one of claims 1 to 6, or the vector according to claim 7, or the cell according to claim 8.

10. Use of the double-stranded siRNA analog according to any one of claims 1 to 6, or the vector according to claim 7, or the cell according to claim 8, or the pharmaceutical composition or kit according to claim 9 in the preparation of a medicament for preventing and / or treating a disease, condition or symptom mediated by abnormal or overexpression of DGAT2; Preferably, the disease, disorder or symptom is selected from the group consisting of liver disease, dyslipidemia, dysglycemia, cardiovascular disease, kidney disease, metabolic syndrome, obesity.

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