SiRNA for inhibiting expression of apolipoprotein C3

CN119421952APending Publication Date: 2025-02-11SHANGHAI RONA THERAPEUTICS CO LTD
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Patent Information

Application Number
CN202380050164.2
Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
Priority Date
2023-05-05
Filing Date
2023-06-27
Publication Date
2025-02-11

AI Technical Summary

Technical Problem

Existing siRNAs have poor stability in vivo and are difficult to be absorbed by tissues and cells, making it difficult to effectively inhibit APOC3 gene expression and limiting their clinical application.

Method used

siRNAs containing specific nucleotide sequences and modifications, with sense and antisense strands, were designed to target desialyl glycoprotein receptors via partial conjugation of N-acetylgalactosamine ligands with phosphate or thiophosphate groups, thereby enhancing their distribution and targeting within cells.

Benefits of technology

It improves the stability and cellular uptake efficiency of siRNA in vivo, effectively inhibits APOC3 gene expression, and reduces the risk of hypertriglyceridemia and related diseases.

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Abstract

A siRNA that inhibits the expression of apolipoprotein C3 is provided. Provided are a small interfering RNA (siRNA) for inhibiting expression of APOC3 in a cell, a vector and a cell comprising a nucleotide encoding the same, and a method for treating a disease or symptom associated with expression of APOC3 in a subject using the siRNA, the vector or the cell.
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Description

siRNA that inhibits apolipoprotein C3 expression Technical Field

[0001] The present invention relates to the field of RNA interference and also to the field of treatment of hypercholesterolemia and related diseases. Background Art

[0002] Lipoproteins are spherical, micelle-like particles composed of a nonpolar core of acylglycerols and cholesterol esters surrounded by a coating of amphipathic proteins, phospholipids, and cholesterol. Lipoproteins have been classified into five types based on their function and physical properties: chylomicrons, very low-density lipoproteins (VLDL), intermediate-density lipoproteins (IDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL). Chylomicrons transport dietary lipids from the intestine to tissues. VLDL, IDL, and LDL all transport triacylglycerols and cholesterol from the liver to tissues. HDL transports endogenous cholesterol from tissues to the liver.

[0003] Apolipoprotein C-III (ApoCIII) is an important regulator of lipoprotein metabolism. In humans, APOC3 is expressed in the liver and, to a lesser extent, in the intestine. APOC3 is initially expressed as a 99-amino acid protein, which is then converted to the mature 79-amino acid ApoC3 protein in the endoplasmic reticulum after the removal of a 20-amino acid signal peptide.

[0004] The primary role of APOC3 is to regulate lipolysis by noncompetitively inhibiting endothelial-bound lipoprotein lipase (LPL). LPL hydrolyzes triglycerides from triglyceride-rich lipoproteins (TRLs), releasing fatty acids into the plasma and converting large triglyceride-rich particles into smaller triglyceride-poor remnant lipoproteins. Individuals deficient in APOC3 have lower TRL levels and efficient triglyceride lipolysis.

[0005] APOC3 also inhibits hepatic lipase (HL), a lipolytic enzyme synthesized in the liver with triglyceride lipase and phospholipase A1 activities. APOC3's inhibition of HL further reduces lipolysis and uptake of TRL residues in the liver.

[0006] ApoC3 has been shown to inhibit lipolysis by both inhibiting lipoprotein lipase and by interfering with the binding of lipoproteins to the cell surface glycosaminoglycan matrix. An increase in APOC3 levels causes the development of hypertriglyceridemia or a high blood concentration of triglycerides. Elevated triglyceride levels are associated with a variety of diseases, including cardiovascular disease, atherosclerosis, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, polycystic ovary syndrome, kidney disease, obesity, type 2 diabetes (insulin resistance), hypertension, and skin lesions (xanthomas). Very high triglyceride levels can also increase the risk of acute pancreatitis.

[0007] There is a need in the art for modulators of APOC3 expression for the treatment of apolipoprotein C3-related disorders, such as hypertriglyceridemia.

[0008] In recent years, inhibitors targeting APOC3 have emerged as novel therapeutic agents for these diseases. As a novel therapeutic approach, siRNA (siRNA) holds enormous potential. siRNA acts on intracellular mRNA, directly silencing target genes compared to traditional small molecule drugs, thereby more effectively preventing the onset and progression of disease. However, siRNA's poor stability, susceptibility to nuclease degradation in the body, poor tissue and cellular uptake, and the potential for off-target effects limit its clinical application. Therefore, there is an urgent need for siRNA that can effectively inhibit APOC3 gene expression in cells.

[0009] Summary of the Invention

[0010] The present invention relates to small interfering RNA (siRNA) for inhibiting the expression of apolipoprotein C3 (APOC3) in cells and a method for treating diseases using the siRNA.

[0011] In a first aspect, the present invention provides a small interfering ribonucleic acid (siRNA) for inhibiting the expression of apolipoprotein C3 (APOC3) in cells, the siRNA 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 at least 15 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 414-826. In some embodiments, the sense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 1-413.

[0012] In some embodiments, the sense strand and the antisense strand are each independently 17-27 nucleotides in length, preferably 19-25 nucleotides, and more preferably 21-23 nucleotides in length.

[0013] In some embodiments, the length of the double-stranded region is 15-25 nucleotide pairs, preferably 17-21 nucleotide pairs, and more preferably 19 nucleotide pairs.

[0014] 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 specific embodiments, the overhang is selected from U, UU, UUU and AA.

[0015] In some embodiments, the antisense strand comprises 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 NOs: 414-826. Preferably, the antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NOs: 414-826.

[0016] In some embodiments, the sense strand comprises 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 NOs: 1-413, and preferably the antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NOs: 1-413.

[0017] In some embodiments, the siRNA comprises a paired sense strand sequence and antisense strand sequence as shown in Table 3.

[0018] In some preferred embodiments, the antisense strand comprises at least 15 consecutive nucleotides, 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 NOs: 473, 612, 690, 757, 761, 816, 817, 818, 819, 820, 814 and 815, preferably the antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NOs: 473, 612, 690, 757, 761, 816, 817, 818, 819, 820, 814, and 815.

[0019] In some preferred embodiments, the sense strand comprises at least 15 consecutive nucleotides, 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 NOs: 60, 199, 277, 344, 348, 403, 404, 405, 406, 407, 401, and 402, and preferably the antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NOs: 60, 199, 277, 344, 348, 403, 404, 405, 406, 407, 401, and 402.

[0020] In some more preferred embodiments, in the siRNA of the present invention:

[0021] (a) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 60, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 473;

[0022] (b) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 199, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 612;

[0023] (c) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 277, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 690;

[0024] (d) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 344, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 757;

[0025] (e) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 348, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 761;

[0026] (f) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 403, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 816;

[0027] (g) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 404, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 817;

[0028] (h) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 405, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 818;

[0029] (i) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 406, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 819;

[0030] (j) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 407, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 820;

[0031] (k) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 401, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 814; or

[0032] (l) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:402, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:815.

[0033] 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 preferred embodiments, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides.

[0034] 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'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, inosine ribonucleotides, abasic nucleotides, inverted abasic deoxyribonucleotides, phosphorothioate internucleotide linkage modifications, vinylphosphonate modified nucleotides, locked nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing non-natural bases, and a terminal nucleotide linked to a cholesterol derivative or a dodecanoic acid didecylamide group, and a deoxyribonucleotide.

[0035] 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'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, inosine ribonucleotides, abasic nucleotides, inverted abasic deoxyribonucleotides, and phosphorothioate internucleotide linkage modifications.

[0036] In some specific embodiments, the sense strand and / or the antisense strand comprises at least two 2'-fluoro-modified nucleotides.

[0037] In some specific embodiments, the sense strand and / or the antisense strand comprises at least 8 2'-O-methyl modified nucleotides.

[0038] In some specific embodiments, the 5' end of the sense strand and / or the antisense strand comprises 1-5 phosphorothioate groups.

[0039] In some embodiments, the antisense strand comprises a modified nucleotide sequence shown in any one of Table 5, and / or the sense strand comprises a modified nucleotide sequence shown in any one of Table 4.

[0040] In some embodiments, the siRNA comprises a paired modified sense strand sequence and a modified antisense strand sequence shown in any one of Table 6.

[0041] In some preferred embodiments, wherein:

[0042] (1) the sense strand comprises AmsAmAmAmGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmAms-STM1 (SEQ ID NO: 1655), and the antisense strand comprises (CP1a-U)sCfsUmGfAmGfAmAfUmAfCmUfGmUfCmCfCmUfUmsUfsUm (SEQ ID NO: 1651);

[0043] (2) the sense strand comprises STM1s-AmsAmUmUmAmAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmAms-STM1 (SEQ ID NO: 1656), and the antisense strand comprises (CP1a-U)sGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAmsUfsUm;

[0044] (3) The sense chain comprises

[0045] STM1s-AmsAmUmUmAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmAms-STM1 (SEQ ID NO: 1657),

[0046] And the antisense strand comprises

[0047] (VPUm)sGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAmsUfsUm (SEQ ID NO: 1649);

[0048] (4) The sense chain comprises

[0049] IBs-AmsAmUmUmAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmAms-IB (SEQ ID NO: 1658),

[0050] And the antisense strand comprises

[0051] (CP1a-U)sGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAmsUfsUm (SEQ ID NO: 1650);

[0052] (5) The sense chain comprises

[0053] UmsUmsAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmsAm (SEQ ID NO: 1659),

[0054] And the antisense strand comprises

[0055] (CP1a-U)sGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAmsUfsUm (SEQ ID NO: 1650);

[0056] (6) The sense chain comprises

[0057] IBs-AmsAmAmAmGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmAms-IB (SEQ ID NO: 1660),

[0058] And the antisense strand comprises

[0059] (CP1a-U)sCfsUmGfAmGfAmAfUmAfCmUfGmUfCmCfCmUfUmsUfsUm (SEQ ID NO: 1651);

[0060] (7) The sense chain comprises

[0061] AmsAmsGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmsAm (SEQ ID NO: 1661),

[0062] And the antisense strand comprises

[0063] (CP1a-U)sCfsUmGfAmGfAmAfUmAfCmUfGmUfCmCfCmUfUmsUfsUm (SEQ ID NO: 1651);

[0064] (8) The sense chain comprises

[0065] IBs-AmsAmAmAmGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmAms-IBs-GL6 (SEQ ID NO: 1662),

[0066] And the antisense strand comprises

[0067] (CP1a-U)sCfsUmGfAmGf(PCN-

[0068] A)AfUmAfCmUfGmUfCmCfCmUfUmsUfsUm (SEQ ID NO: 1652); or

[0069] (9) The sense chain comprises

[0070] AmsAmsGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmsAm (SEQ ID NO: 1663),

[0071] And the antisense strand comprises

[0072] (CP1a-U)sCfsUmGfAmGf(PCN-A)AfUmAfCmUfGmUfCmCfCmUfUmsUfsUm (SEQ ID NO: 1652);

[0073] (a) the sense strand comprises CmsGmsAmGmGmAmUfGfCfCmUmCmCmCmUmUmCmUmUm, and the antisense strand comprises AmsAfsGmAfAmGfGmGfAmGfGmCfAmUfCmCfUmCfGmsUfsUm;

[0074] (b) the sense strand comprises CmsCmsGmUmUmAmAfGfGfAmCmAmAmGmUmUmCmUmUm, and the antisense strand comprises AmsAfsGmAfAmCfUmUfGmUfCmCfUmUfAmAfCmGfGmsUfsUm;

[0075] (c) the sense strand comprises CmsCmsGmUmUmAmAfGfGfAmCmAmAmGmUmUmCmUmUm, and the antisense strand comprises AmsAfsGmAfAmCfUmUfGmUfCmCfUmUfAmAfCmGfGmsUfsUm;

[0076] (d) the sense strand comprises CmsCmsAmAmGmUmCfCfAfCmCmUmGmCmCmUmAmUmUm, and the antisense strand comprises AmsAfsUmAfGmGfCmAfGmGfUmGfGmAfCmUfUmGfGmsUfsUm;

[0077] (e) The positive strand comprises AmsAmsGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmUm

[0078] , and the antisense strand comprises AmsCfsUmGfAmGfAmAfUmAfCmUfGmUfCmCfCmUfUmsUfsUm;

[0079] (f) the sense strand comprises UmsUmsAmAmAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmUm, and the antisense strand comprises AmsGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAmsUfsUm;

[0080] (g) the sense strand comprises AmsAmsGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmUm, and the antisense strand comprises AmsCfsUmGfAmGfAmAfUmAfCmUfGmUfCmCfCmUfUmsUfsUm;

[0081] (h) the sense strand comprises CmsCmsAmAmGmUmCfCfAfCmCmUmGmCmCmUmAmUmUm, and the antisense strand comprises AmsAfsUmAfGmGfCmAfGmGfUmGfGmAfCmUfUmGfGmsUfsUm;

[0082] (i) the sense strand comprises CmsCmsGmUmUmAmAfGfGfAmCmAmAmGmUmUmCmUmUm-L96, and the antisense strand comprises AmsAfsGmAfAmCfUmUfGmUfCmCfUmUfAmAfCmGfGmsUfsUm;

[0083] (j) the sense strand comprises CmsGmsAmGmGmAmUfGfCfCmUmCmCmCmUmUmCmUmUm, and the antisense strand comprises AmsAfsGmAfAmGfGmGfAmGfGmCfAmUfCmCfUmCfGmsUfsUm;

[0084] (k) the sense strand comprises IBs-AmCmGmGmAmCmAmGfUfAfUmUmCmUmCmAmGmUmimAms-IB, and the antisense strand comprises UmsCfsAmsCfUmGfAmGmAmAmUmAfCmUfGmUfCmCfCmGfsUm; or

[0085] (1) The sense strand comprises AmsAmsGmGmAmCfAmGfUfAfUmUmCmUmCmAmGmUmsGmsCm, and the antisense strand comprises GmsCfsAmCmUmGfAmGmAmAmUmAmCmUfGmUfCmCmCmUmUmsUmsUm.

[0086] In some embodiments, the siRNA is further conjugated to a ligand moiety comprising N-acetylgalactosamine via a phosphate group or a phosphorothioate group, preferably the sense strand of the siRNA is conjugated to the ligand moiety via a phosphate group or a phosphorothioate group.

[0087] In some specific embodiments, the 3' end of the sense strand is conjugated to the ligand moiety via a phosphate group or a phosphorothioate group. In other specific embodiments, the 5' end of the sense strand is conjugated to the ligand moiety via a phosphate group or a phosphorothioate group.

[0088] In some embodiments, the ligand moiety comprises a conjugate group represented by formula (X'):

[0089] in,

[0090] Indicates the position where the siRNA is linked;

[0091] Q is independently H,

[0092] Wherein L1 is a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O- or -NHC(O)-(CH2NHC(O)) a -;

[0093] L2 is a chemical bond or -CH2CH2C(O)-;

[0094] L3 is a chemical bond, -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-;

[0095] L4 is -(OCH2CH2) c -、-(OCH2CH2CH2) c -、-(OCH2CH2CH2CH2) c -、-(OCH2CH2CH2CH2CH2) c -or-NHC(O)-(CH2) d -;

[0096] Where a = 0, 1, 2 or 3;

[0097] b = 1, 2, 3, 4, or 5;

[0098] c = 1, 2, 3, 4, or 5;

[0099] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0100] L is a chemical bond, -CH2O- or -NHC(O)-;

[0101] L' is a chemical bond, -C(O)NH-, -NHC(O)- or -O(CH2CH2O) e -;

[0102] Where e is 1, 2, 3, 4 or 5;

[0103] T is a chemical bond, -CH2-, -C(O)-, -M-, -CH2-M- or -C(O)-M-;

[0104] Where M is

[0105] R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0106] Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H;

[0107] wherein R is -OR', -CH2OR' or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group or a solid phase support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0108] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0109] n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0110] In some embodiments, the conjugated ligand targets the asialoglycoprotein receptor (ASGPR).

[0111] In some preferred embodiments, the conjugated group is selected from Table 1.

[0112] Table 1. Structure of conjugated groups

[0113] In some preferred embodiments, the conjugated group is selected from Table 2.

[0114] Table 2. Structures of conjugated groups

[0115] In some preferred embodiments, the ligand has the following structure:

[0116] in Indicates the position of linkage to the sense strand of the siRNA via a phosphate group or a phosphorothioate group.

[0117] In some preferred embodiments, the ligand has the following structure:

[0118] in Indicates the position of linkage to the sense strand of the siRNA via a phosphate group or a phosphorothioate group.

[0119] In some preferred embodiments, the ligand has the following structure:

[0120] in Indicates the position of linkage to the sense strand of the siRNA via a phosphate group or a phosphorothioate group.

[0121] In some specific embodiments, in the siRNA of the present disclosure, wherein:

[0122] (1) The sense chain comprises

[0123] STM1s-AmsAmAmAmGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmAms-STM1s-GL6 (SEQ ID NO: 1238),

[0124] And the antisense strand comprises

[0125] (CP1a-U)sCfsUmGfAmGfAmAfUmAfCmUfGmUfCmCfCmUfUmsUfsUm (SEQ ID NO: 1651);

[0126] (2) The sense chain comprises

[0127] STM1s-AmsAmUmUmAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmAms-STM1s-GL6 (SEQ ID NO: 1235),

[0128] And the antisense strand comprises

[0129] (CP1a-U)sGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAmsUfsUm (SEQ ID NO: 1650);

[0130] (3) The sense chain comprises

[0131] STM1s-AmsAmUmUmAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmAms-STM1s-GL6 (SEQ ID NO: 1235),

[0132] And the antisense strand comprises

[0133] (VPUm)sGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAmsUfsUm (SEQ ID NO: 1649);

[0134] (4) The sense chain comprises

[0135] IBs-AmsAmUmUmAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmAms-IBs-GL6 (SEQ ID NO: 1236),

[0136] And the antisense strand comprises

[0137] (CP1a-U)sGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAmsUfsUm (SEQ ID NO: 1650);

[0138] (5) The sense chain comprises

[0139] UmsUmsAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmsAms-GL6 (SEQ ID NO: 1237),

[0140] And the antisense strand comprises

[0141] (CP1a-U)sGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAmsUfsUm (SEQ ID NO: 1650);

[0142] (6) The sense chain comprises

[0143] IBs-AmsAmAmAmGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmAms-IBs-GL6 (SEQ ID NO: 1239),

[0144] And the antisense strand comprises

[0145] (CP1a-U)sCfsUmGfAmGfAmAfUmAfCmUfGmUfCmCfCmUfUmsUfsUm (SEQ ID NO: 1651);

[0146] (7) The sense chain comprises

[0147] AmsAmsGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmsAms-GL6 (SEQ ID NO: 1240),

[0148] And the antisense strand comprises

[0149] (CP1a-U)sCfsUmGfAmGfAmAfUmAfCmUfGmUfCmCfCmUfUmsUfsUm (SEQ ID NO: 1651); or

[0150] (8) The sense chain comprises

[0151] IBs-AmsAmAmAmGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmAms-IBs-GL6 (SEQ ID NO: 1239),

[0152] And the antisense strand comprises

[0153] (CP1a-U)sCfsUmGfAmGf(PCN-

[0154] A)AfUmAfCmUfGmUfCmCfCmUfUmsUfsUm (SEQ ID NO: 1652); or

[0155] (9) The sense chain comprises

[0156] AmsAmsGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmsAms-GL6 (SEQ ID NO: 1240),

[0157] And the antisense strand comprises

[0158] (CP1a-U)sCfsUmGfAmGf(PCN-A)AfUmAfCmUfGmUfCmCfCmUfUmsUfsUm (SEQ ID NO: 1652).

[0159] In a second aspect, the present invention provides a vector comprising a nucleotide sequence encoding the siRNA disclosed in the present invention.

[0160] In a third aspect, the present invention provides a cell comprising the siRNA or vector disclosed in the present invention.

[0161] In a fourth aspect, the present invention provides a pharmaceutical composition comprising the siRNA disclosed in the present invention, a vector or a cell, and optionally a pharmaceutically acceptable carrier or excipient.

[0162] In a fifth aspect, the present invention provides a kit comprising the siRNA, vector, cell or pharmaceutical composition disclosed in the present invention.

[0163] In a sixth aspect, the present invention provides a method for treating a disease associated with APOC3 in a subject, comprising the step of administering to the subject the siRNA, vector, cell, or pharmaceutical composition disclosed in the present invention.

[0164] In a seventh aspect, the present invention provides a method for reducing the risk of developing a disease associated with APOC3 in a subject, the method comprising the step of administering to the subject the siRNA, vector, cell, or pharmaceutical composition disclosed herein.

[0165] In some embodiments of the sixth and seventh aspects, the APOC3-related disease is selected from hyperlipidemia and hypertriglyceridemia. In some specific embodiments, the APOC3-related disease is a disease that may be caused by, associated with, or a consequence of hypertriglyceridemia, such as non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, polycystic ovary syndrome, kidney disease, obesity, type 2 diabetes, hypertension, atherosclerosis, cardiovascular disease, or pancreatitis.

[0166] In some embodiments, the method comprises subcutaneously, intravenously, or topically administering to the subject an siRNA, vector, cell, or pharmaceutical composition disclosed herein.

[0167] In some embodiments, the subject is a human patient. DETAILED DESCRIPTION

[0168] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different perspectives and applications without departing from the spirit of the present invention.

[0169] It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific embodiments rather than for limiting the scope of protection of the present invention.

[0170] Throughout the specification and claims of the present invention, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0171] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to implement the present invention, and all fall within the scope of protection of the present invention. The following describes embodiments of the present invention in more detail.

[0172] definition

[0173] The term "siRNA" herein refers to a class of double-stranded RNA molecules that can mediate the silencing of a target RNA (e.g., mRNA, e.g., a transcript of a gene encoding a protein) that is complementary thereto. siRNA is typically double-stranded, comprising an antisense strand complementary to the target RNA and a sense strand complementary to the antisense strand. For convenience, such mRNA is also referred to herein as mRNA to be silenced. Such a gene is also referred to as a target gene.

[0174] As used herein, the term "antisense strand" refers to the strand of an siRNA that contains a region that is fully or substantially complementary to a target sequence.

[0175] As used herein, the term "complementary region" refers to a region on the antisense strand that is completely or substantially complementary to the target mRNA 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, for example, within 5, 4, 3, 2 or 1 nucleotides at 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.

[0176] As used herein, the term "complementary" refers to the ability of a first polynucleotide to hybridize to a second polynucleotide under certain conditions, such as stringent conditions. For example, stringent conditions may include 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA at 50° C. or 70° C. for 12-16 hours.

[0177] As used herein, "complementary" sequences, insofar as they meet the above requirements for their ability to hybridize, may also include or be formed entirely from non-Watson-Crick base pairs and / or base pairs formed from non-natural and modified nucleotides. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble base pairing or Hoogstein base pairing.

[0178] 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 APOC3). For example, a polynucleotide is complementary to at least a portion of an APOC3 mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding APOC3.

[0179] As used herein, the terms "complementary," "fully complementary," and "substantially complementary" can be used with respect to base pairing between the sense and antisense strands of an siRNA, or can be used with respect to base pairing between the antisense strand of an siRNA and a target sequence.

[0180] As used herein, the term "sense strand" refers to the strand of an siRNA that includes a region that is substantially complementary to a region as that term is defined herein as the "antisense strand."

[0181] A "nucleoside" is a compound composed of a purine or pyrimidine base and ribose or deoxyribose. A "nucleotide" is a compound composed of a purine or pyrimidine base, ribose or deoxyribose, and phosphate. An "oligonucleotide" is a nucleic acid molecule (RNA or DNA) with a length of, for example, fewer than 100, 200, 300, or 400 nucleotides.

[0182] "Bases" are the fundamental building blocks of nucleosides, nucleotides, and nucleic acids. They contain nitrogen and are also called "nitrogenous bases." Unless otherwise specified, the capital letters A, U, T, G, and C represent the bases of nucleotides, representing adenine, uracil, thymine, guanine, and cytosine, respectively.

[0183] As used herein, the term "nucleotide overhang" or "overhang" refers to at least one unpaired nucleotide that protrudes from the duplex structure of an siRNA. For example, a nucleotide overhang exists when the 3'-end of one strand of an siRNA extends beyond the 5'-end of the other strand, or vice versa. The siRNA may comprise an overhang having at least one nucleotide; alternatively, the overhang may comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more. The nucleotide overhang may comprise or consist of nucleotide / nucleoside analogs (including deoxynucleotides / nucleosides). The one or more overhangs may be on the sense strand, the antisense strand, or any combination thereof. Additionally, the one or more nucleotides of the overhang may be present on the 5'-end, the 3'-end, or both ends of the antisense strand or the sense strand of the siRNA.

[0184] "Blunt end" or "blunt end" means that there are no unpaired nucleotides at that end of the double-stranded siRNA, i.e., there are no nucleotide overhangs at that end. A "blunt-ended siRNA" is a siRNA that is double-stranded throughout its entire length, i.e., there are no nucleotide overhangs at either end of the molecule. The siRNAs of the present invention include siRNAs that have a nucleotide overhang at one end (i.e., an agent having one overhang and one blunt end) or siRNAs that have nucleotide overhangs at both ends.

[0185] Substantially all of the nucleotides of the iRNA of the present invention are modified. For example, substantially all of the nucleotides of the sense strand are modified nucleotides, and / or substantially all of the nucleotides of the antisense strand are modified nucleotides, and / or substantially all of the nucleotides of both the sense strand and the antisense strand are modified nucleotides. In other embodiments of the present invention, all of the nucleotides of the iRNA of the present invention are modified nucleotides. For example, all of the nucleotides of the sense strand are modified nucleotides, and / or all of the nucleotides of the antisense strand are modified nucleotides, and / or all of the nucleotides of both the sense strand and the antisense strand are modified nucleotides. Wherein "substantially all of the nucleotides are modified" means that the siRNA of the present invention is mostly but not entirely modified, and may include no more than 5, 4, 3, 2 or 1 unmodified nucleotides.

[0186] " modified nucleotide " herein includes but is not limited to 2 '-O-methyl modified nucleotides, 2 '-fluoro modified nucleotides, 2 '-deoxy-modified nucleotides, inosine ribonucleotides, abasic nucleotides, reverse abasic deoxyribonucleotides, phosphorothioate internucleotide linkage modifications, vinylphosphonate modified nucleotides, locked nucleotides, 2 '-amino-modified nucleotides, 2 '-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides comprising non-natural bases and terminal nucleotides, deoxyribonucleotides or conventional protecting group protections, etc., that are connected to a cholesterol derivative or a dodecanoic acid didecylamide group. For example, the 2 '-fluoro modified nucleotides refer to nucleotides in which the hydroxyl group at the 2 ' position of the ribose group of the nucleotide is replaced by fluorine. The 2 '-deoxy-modified nucleotides refer to nucleotides in which the 2 '-hydroxyl group of the ribose group is replaced by methoxy and formed.

[0187] As used herein, "ligand moiety" refers to a chemical moiety conjugated to an siRNA that is capable of altering the distribution, targeting, or lifetime of the siRNA. In preferred embodiments, such a ligand provides enhanced affinity for a selected target (e.g., a molecule, a cell or cell type, a compartment (e.g., a cell or organ compartment, a tissue, an organ, or a region of the body) compared to, for example, an siRNA in the absence of such a ligand.

[0188] As used herein, the term "inhibit" is used interchangeably with "reduce," "silence," "downregulate," and other similar terms and includes any level of inhibition.

[0189] The phrase "inhibiting the expression of APOC3" is intended to refer to inhibiting the expression of any APOC3 gene, as well as variants or mutants of the APOC3 gene. Thus, the APOC3 gene can be a wild-type APOC3 gene, a mutant APOC3 gene, or a transgenic APOC3 gene in the context of a genetically manipulated cell, cell group, or organism.

[0190] "Inhibiting APOC3 gene expression" includes any level of inhibition of the APOC3 gene, such as at least partial suppression of APOC3 gene expression. APOC3 gene expression can be assessed based on the level or change in level of any variable associated with APOC3 gene expression, such as APOC3 mRNA level, APOC3 protein level, or lipid level. This level can be assessed in an individual cell or in a group of cells (including, for example, a sample derived from a subject).

[0191] Inhibition can be assessed by a decrease in the absolute or relative level of one or more variables associated with APOC3 expression compared to a control level. The control level can be any type of control level utilized in the art, such as a pre-dose baseline level or a level determined from a similar untreated or control (e.g., a buffer-only control or an inert agent control) treated subject, cell, or sample.

[0192] "Hydroxy protecting group" refers to a group that can prevent the hydroxyl group from chemical reaction and can be removed under specific conditions to restore the hydroxyl group. It mainly includes silane type protecting groups, acyl type protecting groups or ether type protecting groups, preferably the following: trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), acetyl (Ac), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, p-methoxybenzoyl, 9-fluorenylmethoxycarbonyl ( Fmoc), allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), bis-p-methoxytrityl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzyloxymethyl (PMBM).

[0193] "Halo" or "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).

[0194] “C 1-6 "Haloalkyl" refers to the above-mentioned "C 1-6 Alkyl", which is substituted by one or more halogen groups. In some embodiments, C 1-4 Halogenated alkyl is particularly preferred, more preferably C 1-2Haloalkyl. Exemplary haloalkyl groups include, but are not limited to, -CF3, -CH2F, -CHF2, -CHFCH2F, -CH2CHF2, -CF2CF3, -CCl3, -CH2Cl, -CHCl2, 2,2,2-trifluoro-1,1-dimethyl-ethyl, and the like. The haloalkyl group can be substituted at any available point of attachment, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0195] “C 1-6 "Alkylene" refers to the removal of C 1-6 In some embodiments, C 1-4 Alkylene, C 2-4 Alkylene and C 1-2 Alkylene is preferred. Unsubstituted alkylene includes, but is not limited to, methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), butylene (-CH2CH2CH2CH2-), pentylene (-CH2CH2CH2CH2CH2-), hexylene (-CH2CH2CH2CH2CH2CH2-), and the like. Exemplary substituted alkylenes, for example, alkylenes substituted with one or more alkyl (methyl) groups, include, but are not limited to, substituted methylene (-CH(CH3)-, -C(CH3)2-), substituted ethylene (-CH(CH3)CH2-, -CH2CH(CH3)-, -C(CH3)2CH2-, -CH2C(CH3) 2- ), substituted propylene (-CH(CH3)CH2CH2-, -CH2CH(CH3)CH2-, -CH2CH2CH(CH3)-, -C(CH3)2CH2CH2-, -CH2C(CH3)2CH2-, -CH2CH2C(CH3)2-), and the like.

[0196] As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying or expressing another nucleic acid to which it is linked.

[0197] I.siRNA

[0198] The present invention provides a small interfering RNA (siRNA) for inhibiting the expression of apolipoprotein C3 (APOC3) in cells, wherein the siRNA comprises 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 at least 15 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 414-826.

[0199] 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 and may comprise 1, 2, 3, 4 or 5 non-complementary sites.

[0200] In some specific embodiments, the sense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 1-413.

[0201] In some embodiments, the sense strand and the antisense strand are each independently 17-27 nucleotides in length, preferably 19-25 nucleotides, and more preferably 21-23 nucleotides in length.

[0202] In some embodiments, the length of the double-stranded region is 15-25 nucleotide pairs, preferably 17-21 nucleotide pairs, and more preferably 19 nucleotide pairs.

[0203] One or both of the sense strand and the antisense strand comprise 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 comprise a 3' overhang and / or a 5' overhang of at least 2 nucleotides. In some preferred embodiments, the antisense strand has a 3' overhang and / or a 5' overhang of at least 1 nucleotide. For example, the antisense strand comprises a 3' overhang and / or a 5' overhang of 1, 2, or 3 nucleotides. In some preferred embodiments, the sense strand has a 3' overhang and / or a 5' overhang of at least 1 nucleotide. For example, the sense strand comprises a 3' overhang and / or a 5' overhang of 1, 2, or 3 nucleotides.

[0204] In some embodiments, the sense strand and the antisense strand are of the same length. In some embodiments, the full length of the sense strand is complementary to the full length of the antisense strand to form a double strand, i.e., having blunt ends. In other embodiments, the sense strand and the antisense strand are of the same length, and 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 5' overhangs.

[0205] In some embodiments, the sense strand and the antisense strand are different lengths. In preferred embodiments, the 5' end of the antisense strand has an overhang of at least 1 nucleotide, more preferably an overhang of 2 or 3 nucleotides.

[0206] In some embodiments, the antisense strand comprises 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 NOs: 414-826. Preferably, the antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NOs: 414-826.

[0207] In some embodiments, the sense strand comprises 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 NOs: 1-413, and preferably the antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NOs: 1-413.

[0208] In some embodiments, the siRNA comprises a paired sense strand sequence and antisense strand sequence as shown in Table 3.

[0209] In some embodiments, the antisense strand comprises at least 15 consecutive nucleotides, 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 NOs: 473, 612, 690, 757, 761, 816, 817, 818, 819, 820, 814, and 815. Preferably, the antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NOs: 473, 612, 690, 757, 761, 816, 817, 818, 819, 820, 814, and 815.

[0210] In some embodiments, the sense strand comprises at least 15 consecutive nucleotides, 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 NOs: 60, 199, 277, 344, 348, 403, 404, 405, 406, 407, 401 and 402, and preferably the antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NOs: 60, 199, 277, 344, 348, 403, 404, 405, 406, 407, 401 and 402.

[0211] In some embodiments,

[0212] (a) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 60, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 473;

[0213] (b) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 199, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 612;

[0214] (c) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 277, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 690;

[0215] (d) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 344, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 757;

[0216] (e) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 348, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 761;

[0217] (f) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 403, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 816;

[0218] (g) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 404, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 817;

[0219] (h) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 405, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 818;

[0220] (i) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 406, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 819;

[0221] (j) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 407, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 820;

[0222] (k) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 401, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 814; or

[0223] (l) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:402, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:815.

[0224] II. Nucleotide Modification

[0225] 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% of the nucleotides of the antisense strand are modified nucleotides.

[0226] In some embodiments, all of the nucleotides of the sense strand and / or all of the nucleotides of the antisense strand are modified nucleotides.

[0227] The modification of the nucleotides described in the present invention may be modification of the phosphate group, ribose group and / or base group of the nucleotides.

[0228] 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'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, inosine ribonucleotides, abasic nucleotides, inverted abasic deoxyribonucleotides, phosphorothioate internucleotide linkage modifications, vinylphosphonate modified nucleotides, locked nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing non-natural bases, and terminal nucleotides linked to a cholesterol derivative or a dodecanoic acid didecylamide group, and deoxyribonucleotides.

[0229] 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'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, inosine ribonucleotides, abasic nucleotides, inverted abasic deoxyribonucleotides, and phosphorothioate internucleotide linkage modifications.

[0230] In some preferred embodiments, the sense strand and / or the antisense strand comprises at least two 2'-fluoro-modified nucleotides.

[0231] In some preferred embodiments, the sense strand and / or the antisense strand comprises at least 8 2'-O-methyl modified nucleotides.

[0232] In some preferred embodiments, the 3' end and / or 5' end of the sense strand and / or the antisense strand comprises 1-5 phosphorothioate groups, preferably 2-3 phosphorothioate groups. In some more preferred embodiments, the 5' end of the sense strand and / or the antisense strand comprises 1-5 phosphorothioate groups.

[0233] In some preferred embodiments, the antisense strand comprises a modified nucleotide sequence shown in any one of Table 5, and / or the sense strand comprises a modified nucleotide sequence shown in any one of Table 4. In some preferred embodiments, the siRNA comprises a paired modified sense strand sequence and a modified antisense strand sequence shown in any one of Table 6.

[0234] In some embodiments, in the siRNA for inhibiting APOC3 expression in cells of the present invention:

[0235] (1) the sense strand comprises AmsAmAmAmGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmAms-STM1 (SEQ ID NO: 1655), and the antisense strand comprises (CP1a-U)sCfsUmGfAmGfAmAfUmAfCmUfGmUfCmCfCmUfUmsUfsUm (SEQ ID NO: 1651);

[0236] (2) the sense strand comprises STM1s-AmsAmUmUmAmAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmAms-STM1 (SEQ ID NO: 1656), and the antisense strand comprises (CP1a-U)sGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAmsUfsUm;

[0237] (3) The sense chain comprises

[0238] STM1s-AmsAmUmUmAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmAms-STM1 (SEQ ID NO: 1657),

[0239] And the antisense strand comprises

[0240] (VPUm)sGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAmsUfsUm (SEQ ID NO: 1649);

[0241] (4) The sense chain comprises

[0242] IBs-AmsAmUmUmAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmAms-IB (SEQ ID NO: 1658),

[0243] And the antisense strand comprises

[0244] (CP1a-U)sGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAmsUfsUm (SEQ ID NO: 1650);

[0245] (5) The sense chain comprises

[0246] UmsUmsAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmsAm (SEQ ID NO: 1659),

[0247] And the antisense strand comprises

[0248] (CP1a-U)sGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAmsUfsUm (SEQ ID NO: 1650);

[0249] (6) The sense chain comprises

[0250] IBs-AmsAmAmAmGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmAms-IB (SEQ ID NO: 1660),

[0251] And the antisense strand comprises

[0252] (CP1a-U)sCfsUmGfAmGfAmAfUmAfCmUfGmUfCmCfCmUfUmsUfsUm (SEQ ID NO: 1651);

[0253] (7) The sense chain comprises

[0254] AmsAmsGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmsAm (SEQ ID NO: 1661),

[0255] And the antisense strand comprises

[0256] (CP1a-U)sCfsUmGfAmGfAmAfUmAfCmUfGmUfCmCfCmUfUmsUfsUm (SEQ ID NO: 1651);

[0257] (8) The sense chain comprises

[0258] IBs-AmsAmAmAmGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmAms-IBs-GL6 (SEQ ID NO: 1662),

[0259] And the antisense strand comprises

[0260] (CP1a-U)sCfsUmGfAmGf(PCN-A)AfUmAfCmUfGmUfCmCfCmUfUmsUfsUm (SEQ ID NO: 1652); or

[0261] (9) The sense chain comprises

[0262] AmsAmsGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmsAm (SEQ ID NO: 1663),

[0263] And the antisense strand comprises

[0264] (CP1a-U)sCfsUmGfAmGf(PCN-A)AfUmAfCmUfGmUfCmCfCmUfUmsUfsUm (SEQ ID NO: 1652);

[0265] (a) the sense strand comprises CmsGmsAmGmGmAmUfGfCfCmUmCmCmCmUmUmCmUmUm, and the antisense strand comprises AmsAfsGmAfAmGfGmGfAmGfGmCfAmUfCmCfUmCfGmsUfsUm;

[0266] (b) the sense strand comprises CmsCmsGmUmUmAmAfGfGfAmCmAmAmGmUmUmCmUmUm, and the antisense strand comprises AmsAfsGmAfAmCfUmUfGmUfCmCfUmUfAmAfCmGfGmsUfsUm;

[0267] (c) the sense strand comprises CmsCmsGmUmUmAmAfGfGfAmCmAmAmGmUmUmCmUmUm, and the antisense strand comprises AmsAfsGmAfAmCfUmUfGmUfCmCfUmUfAmAfCmGfGmsUfsUm;

[0268] (d) the sense strand comprises CmsCmsAmAmGmUmCfCfAfCmCmUmGmCmCmUmAmUmUm, and the antisense strand comprises AmsAfsUmAfGmGfCmAfGmGfUmGfGmAfCmUfUmGfGmsUfsUm;

[0269] (e) the sense strand comprises AmsAmsGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmUm, and the antisense strand comprises AmsCfsUmGfAmGfAmAfUmAfCmUfGmUfCmCfCmUfUmsUfsUm;

[0270] (f) the sense strand comprises UmsUmsAmAmAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmUm, and the antisense strand comprises AmsGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAmsUfsUm;

[0271] (g) the sense strand comprises AmsAmsGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmUm, and the antisense strand comprises AmsCfsUmGfAmGfAmAfUmAfCmUfGmUfCmCfCmUfUmsUfsUm;

[0272] (h) the sense strand comprises CmsCmsAmAmGmUmCfCfAfCmCmUmGmCmCmUmAmUmUm, and the antisense strand comprises AmsAfsUmAfGmGfCmAfGmGfUmGfGmAfCmUfUmGfGmsUfsUm;

[0273] (i) the sense strand comprises CmsCmsGmUmUmAmAfGfGfAmCmAmAmGmUmUmCmUmUm, and the antisense strand comprises AmsAfsGmAfAmCfUmUfGmUfCmCfUmUfAmAfCmGfGmsUfsUm;

[0274] (j) the sense strand comprises CmsGmsAmGmGmAmUfGfCfCmUmCmCmCmUmUmCmUmUm, and the antisense strand comprises AmsAfsGmAfAmGfGmGfAmGfGmCfAmUfCmCfUmCfGmsUfsUm;

[0275] (k) the sense strand comprises IBs-AmCmGmGmAmCmAmGfUfAfUmUmCmUmCmAmGmUmimAms-IB, and the antisense strand comprises UmsCfsAmsCfUmGfAmGmAmAmUmAfCmUfGmUfCmCfCmGfsUm; or

[0276] (1) The sense strand comprises AmsAmsGmGmAmCfAmGfUfAfUmUmCmUmCmAmGmUmsGmsCm, and the antisense strand comprises GmsCfsAmCmUmGfAmGmAmAmUmAmCmUfGmUfCmCmCmUmUmsUmsUm.

[0277] III. Ligand

[0278] The siRNA of the present invention is further conjugated to a ligand moiety comprising N-acetylgalactosamine via a phosphate group or a thiophosphate group. In a preferred embodiment, the sense strand of the siRNA is conjugated to the ligand moiety via a phosphate group or a thiophosphate group. In some preferred embodiments, the 3' end of the sense strand is conjugated to the ligand moiety via a phosphate group or a thiophosphate group. In other preferred embodiments, the 5' end of the sense strand is conjugated to the ligand moiety via a phosphate group or a thiophosphate group.

[0279] In some embodiments, the ligand moiety comprises a conjugate group represented by formula (X'):

[0280] in,

[0281] Indicates the position where the siRNA is linked;

[0282] Q is independently H,

[0283] Wherein L1 is a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O- or -NHC(O)-(CH2NHC(O)) a -;

[0284] L2 is a chemical bond or -CH2CH2C(O)-;

[0285] L3 is a chemical bond, -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-;

[0286] L4 is -(OCH2CH2) c -、-(OCH2CH2CH2) c -、-(OCH2CH2CH2CH2) c -、-(OCH2CH2CH2CH2CH2) c -or-NHC(O)-(CH2)d -;

[0287] Where a = 0, 1, 2 or 3;

[0288] b = 1, 2, 3, 4, or 5;

[0289] c = 1, 2, 3, 4, or 5;

[0290] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0291] L is a chemical bond, -CH2O- or -NHC(O)-;

[0292] L' is a chemical bond, -C(O)NH-, -NHC(O)- or -O(CH2CH2O) e -;

[0293] Where e is 1, 2, 3, 4 or 5;

[0294] T is a chemical bond, -CH2-, -C(O)-, -M-, -CH2-M- or -C(O)-M-;

[0295] Where M is

[0296] R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0297] Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H;

[0298] wherein R is -OR', -CH2OR' or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group or a solid phase support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0299] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0300] n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0301] In some embodiments, the conjugated group is represented by formula (I'):

[0302] in,

[0303] Indicates the position where the siRNA is linked;

[0304] Q is independently H,

[0305] Wherein L1 is a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O- or -NHC(O)-(CH2NHC(O)) a -;

[0306] L2 is a chemical bond or -CH2CH2C(O)-;

[0307] L3 is a chemical bond, -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-;

[0308] L4 is -(OCH2CH2) c -、-(OCH2CH2CH2) c -、-(OCH2CH2CH2CH2) c -、-(OCH2CH2CH2CH2CH2) c -or-NHC(O)-(CH2) d -;

[0309] Where a = 0, 1, 2 or 3;

[0310] b = 1, 2, 3, 4, or 5;

[0311] c = 1, 2, 3, 4, or 5;

[0312] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0313] L is -CH2O- or -NHC(O)-;

[0314] L' is a chemical bond, -C(O)NH- or -NHC(O)-;

[0315] R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0316] Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H;

[0317] wherein R is -OR', -CH2OR' or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group or a solid phase support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0318] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0319] n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0320] In some specific embodiments, wherein,

[0321] Q is independently H or

[0322] Where L1 is -CH2O- or -NHC(O)-(CH2NHC(O)) a -;

[0323] L2 is -CH2CH2C(O)-;

[0324] L3 is -(NHCH2CH2) b -or-(NHCH2CH2CH2) b -;

[0325] L4 is -(OCH2CH2) c -or-NHC(O)-(CH2) d -;

[0326] Where a = 0, 1, 2 or 3;

[0327] b = 1, 2, 3, 4, or 5;

[0328] c = 1, 2, 3, 4, or 5;

[0329] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0330] L is -CH2O-;

[0331] L' is a chemical bond;

[0332] R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0333] Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H;

[0334] wherein R is -OR', -CH2OR' or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group or a solid phase support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0335] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0336] n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0337] In some embodiments, the conjugated group is represented by Formula (I'-1), Formula (I'-2), or Formula (I'-3):

[0338] in,

[0339] Indicates the position where the siRNA is linked;

[0340] Q is

[0341] Wherein L1 is -CH2O- or -NHC(O)-;

[0342] L2 is -CH2CH2C(O)-;

[0343] L3 is -(NHCH2CH2) b -or-(NHCH2CH2CH2) b -;

[0344] L4 is -(OCH2CH2) c -or-NHC(O)-(CH2) d -;

[0345] Where b = 1, 2, 3, 4 or 5;

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

[0347] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0348] L is -CH2O-;

[0349] R' is H, a hydroxyl protecting group or a solid phase support, wherein the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0350] n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0351] In some specific embodiments, wherein,

[0352] Q is independently H,

[0353] Wherein L1 is -CH2O-, -CH2O-CH2CH2O- or -NHC(O)-(CH2NHC(O)) a -;

[0354] L2 is -CH2CH2C(O)-;

[0355] L3 is -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-;

[0356] L4 is -(OCH2CH2) c -or-NHC(O)-(CH2) d -;

[0357] Where a = 0, 1, 2 or 3;

[0358] b = 1, 2, 3, 4, or 5;

[0359] c = 1, 2, 3, 4, or 5;

[0360] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0361] L is -CH2O- or -NHC(O)-;

[0362] L' is a chemical bond or -C(O)NH-;

[0363] R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0364] Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H;

[0365] wherein R is -OR', -CH2OR' or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group or a solid phase support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0366] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

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

[0368] In some embodiments, the conjugated group is as shown in Formula (II'-1) or Formula (II'-2):

[0369] in,

[0370] Indicates the position where the siRNA is linked;

[0371] Q is independently

[0372] Wherein L1 is -CH2O- or -CH2O-CH2CH2O-;

[0373] L3 is -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-;

[0374] L4 is -(OCH2CH2) c -or-NHC(O)-(CH2) d -;

[0375] Where 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 -NHC(O)-;

[0379] L' is a chemical bond or -C(O)NH-;

[0380] R' is H, a hydroxyl protecting group or a solid phase support, wherein the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0381] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0382] n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0383] In some specific embodiments, wherein,

[0384] Q is independently H,

[0385] Wherein L1 is -CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O- or -NHC(O)-(CH2NHC(O)) a -;

[0386] L2 is a chemical bond;

[0387] L3 is -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-;

[0388] L4 is -(OCH2CH2) c -or-NHC(O)-(CH2) d -;

[0389] Where a = 0, 1, 2 or 3;

[0390] b = 1, 2, 3, 4, or 5;

[0391] c = 1, 2, 3, 4, or 5;

[0392] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0393] L is -CH2O- or -NHC(O)-;

[0394] L' is a chemical bond or -C(O)NH-;

[0395] R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0396] Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H;

[0397] wherein R is -OR', -CH2OR' or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group or a solid phase support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0398] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0399] n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0400] In some embodiments, the conjugated group is represented by formula (II'-2):

[0401] in,

[0402] Indicates the position where the siRNA is linked;

[0403] Q is independently

[0404] Wherein L1 is -CH2- or -C(O)-;

[0405] L3 is -(NHCH2CH2) b -;

[0406] L4 is -(OCH2CH2) c -;

[0407] Where b = 1, 2, 3, 4 or 5;

[0408] c = 1, 2, 3, 4, or 5;

[0409] L is -CH2O- or -NHC(O)-;

[0410] R' is H, a hydroxyl protecting group or a solid phase support, wherein the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0411] n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0412] In some specific embodiments, wherein:

[0413] Q is independently H,

[0414] Wherein L1 is a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O- or -NHC(O)-(CH2NHC(O)) a -;

[0415] L2 is a chemical bond or -CH2CH2C(O)-;

[0416] L3 is a chemical bond, -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-;

[0417] L4 is -(OCH2CH2) c -、-(OCH2CH2CH2) c -、-(OCH2CH2CH2CH2) c -、-(OCH2CH2CH2CH2CH2) c -or-NHC(O)-(CH2) d -;

[0418] Where a = 0, 1, 2 or 3;

[0419] b = 1, 2, 3, 4, or 5;

[0420] c = 1, 2, 3, 4, or 5;

[0421] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0422] L is a chemical bond, -CH2O- or -NHC(O)-;

[0423] L' is a chemical bond, -C(O)NH-, -NHC(O)- or -O(CH2CH2O) e -;

[0424] Where e is 1, 2, 3, 4 or 5;

[0425] T is a chemical bond, -CH2-, -M-, -CH2-M- or -C(O)-M-;

[0426] Where M is

[0427] R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0428] Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H;

[0429] wherein R is -OR', -CH2OR' or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group or a solid phase support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0430] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0431] n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0432] In some specific embodiments, wherein,

[0433] T is -M-, -CH2-M- or -C(O)-M-, wherein M is

[0434] In some specific embodiments, wherein,

[0435] Q is independently H or

[0436] Where L1 is -CH2O- or -NHC(O)-(CH2NHC(O)) a -;

[0437] L2 is -CH2CH2C(O)-;

[0438] L3 is -(NHCH2CH2) b -or-(NHCH2CH2CH2) b -;

[0439] L4 is -(OCH2CH2) c-or-NHC(O)-(CH2) d -;

[0440] Where a = 0, 1, 2 or 3;

[0441] b = 1, 2, 3, 4, or 5;

[0442] c = 1, 2, 3, 4, or 5;

[0443] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0444] L is a chemical bond or -CH2O-;

[0445] L' is a chemical bond or -O(CH2CH2O) e -;

[0446] Where e is 1, 2, 3, 4 or 5;

[0447] R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0448] Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H;

[0449] wherein R is -OR', -CH2OR' or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group or a solid phase support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0450] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0451] n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0452] wherein T is as defined above.

[0453] In some embodiments, the conjugated group is represented by Formula (III'-1), Formula (III'-2), or Formula (III'-3):

[0454] in,

[0455] Q is

[0456] Wherein L1 is -CH2O- or -NHC(O)-;

[0457] L2 is -CH2CH2C(O)-;

[0458] L3 is -(NHCH2CH2)b -or-(NHCH2CH2CH2) b -;

[0459] L4 is -(OCH2CH2) c -or-NHC(O)-(CH2) d -;

[0460] Where b = 1, 2, 3, 4 or 5;

[0461] c = 1, 2, 3, 4, or 5;

[0462] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0463] L is a chemical bond or -CH2O-;

[0464] Wherein R' is H, a hydroxyl protecting group or a solid phase support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0465] n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0466] wherein T is as defined above.

[0467] In some specific embodiments, wherein,

[0468] Q is independently H,

[0469] Wherein L1 is -CH2-, -CH2O- or -C(O)-;

[0470] L2 is a chemical bond;

[0471] L3 is -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-;

[0472] L4 is -(OCH2CH2) c -or-NHC(O)-(CH2) d -;

[0473] Where b = 1, 2, 3, 4 or 5;

[0474] c = 1, 2, 3, 4, or 5;

[0475] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0476] L is a chemical bond or -NHC(O)-;

[0477] L' is a chemical bond;

[0478] R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0479] Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H;

[0480] wherein R is -OR', -CH2OR' or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group or a solid phase support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0481] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0482] n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0483] wherein T is as defined above.

[0484] In some embodiments, the conjugated group is represented by formula (IV-1) or formula (IV-2):

[0485] in,

[0486] Q is independently

[0487] Wherein L1 is -CH2-, -CH2O- or -C(O)-;

[0488] L3 is -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-;

[0489] L4 is -(OCH2CH2) c -or-NHC(O)-(CH2) d -;

[0490] Where b = 1, 2, 3, 4 or 5;

[0491] c = 1, 2, 3, 4, or 5;

[0492] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0493] L is a chemical bond or -NHC(O)-;

[0494] L' is a chemical bond;

[0495] Wherein R' is H, a hydroxyl protecting group or a solid phase support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0496] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0497] n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0498] wherein T is as defined above.

[0499] In some specific embodiments, wherein:

[0500] Q is independently H,

[0501] Wherein L1 is a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O- or -NHC(O)-(CH2NHC(O)) a -;

[0502] L2 is a chemical bond or -CH2CH2C(O)-;

[0503] L3 is a chemical bond, -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-;

[0504] L4 is -(OCH2CH2) c -、-(OCH2CH2CH2) c -、-(OCH2CH2CH2CH2) c -、-(OCH2CH2CH2CH2CH2) c -or-NHC(O)-(CH2) d -;

[0505] Where a = 0, 1, 2 or 3;

[0506] b = 1, 2, 3, 4, or 5;

[0507] c = 1, 2, 3, 4, or 5;

[0508] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0509] L is a chemical bond, -CH2O- or -NHC(O)-;

[0510] L' is -O(CH2CH2O)e -;

[0511] Where e is 1, 2, 3, 4 or 5;

[0512] T is a chemical bond, -CH2-, -C(O)-, -M-, -CH2-M- or -C(O)-M-;

[0513] Where M is

[0514] R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0515] Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H;

[0516] wherein R is -OR', -CH2OR' or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group or a solid phase support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0517] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0518] n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0519] In some preferred embodiments, the conjugated group is selected from Table 1 and Table 2.

[0520] In some embodiments, the ligand targets the asialoglycoprotein receptor (ASGPR).

[0521] In a preferred embodiment, the ligand has the following structure:

[0522] in Indicates the position of linkage to the sense strand of the siRNA via a phosphate group or a phosphorothioate group.

[0523] In a preferred embodiment, the ligand has the following structure:

[0524] in Indicates the position of linkage to the sense strand of the siRNA via a phosphate group or a phosphorothioate group.

[0525] In a preferred embodiment, the ligand has the following structure:

[0526] in Indicates the position of linkage to the sense strand of the siRNA via a phosphate group or a phosphorothioate group.

[0527] In some specific embodiments, in the siRNA of the present disclosure, wherein:

[0528] (1) The sense chain comprises

[0529] STM1s-AmsAmAmAmGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmAms-STM1s-GL6 (SEQ ID NO: 1238),

[0530] And the antisense strand comprises

[0531] (CP1a-U)sCfsUmGfAmGfAmAfUmAfCmUfGmUfCmCfCmUfUmsUfsUm (SEQ ID NO: 1651);

[0532] (2) The sense chain comprises

[0533] STM1s-AmsAmUmUmAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmAms-STM1s-GL6 (SEQ ID NO: 1235),

[0534] And the antisense strand comprises

[0535] (CP1a-U)sGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAmsUfsUm (SEQ ID NO: 1650);

[0536] (3) The sense chain comprises

[0537] STM1s-AmsAmUmUmAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmAms-STM1s-GL6 (SEQ ID NO: 1235),

[0538] And the antisense strand comprises

[0539] (VPUm)sGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAmsUfsUm (SEQ ID NO: 1649);

[0540] (4) The sense chain comprises

[0541] IBs-AmsAmUmUmAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmAms-IBs-GL6 (SEQ ID NO: 1236),

[0542] And the antisense strand comprises

[0543] (CP1a-U)sGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAmsUfsUm (SEQ ID NO: 1650);

[0544] (5) The sense chain comprises

[0545] UmsUmsAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmsAms-GL6 (SEQ ID NO: 1237),

[0546] And the antisense strand comprises

[0547] (CP1a-U)sGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAmsUfsUm (SEQ ID NO: 1650);

[0548] (6) The sense chain comprises

[0549] IBs-AmsAmAmAmGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmAms-IBs-GL6 (SEQ ID NO: 1239),

[0550] And the antisense strand comprises

[0551] (CP1a-U)sCfsUmGfAmGfAmAfUmAfCmUfGmUfCmCfCmUfUmsUfsUm (SEQ ID NO: 1651);

[0552] (7) The sense chain comprises

[0553] AmsAmsGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmsAms-GL6 (SEQ ID NO: 1240),

[0554] And the antisense strand comprises

[0555] (CP1a-U)sCfsUmGfAmGfAmAfUmAfCmUfGmUfCmCfCmUfUmsUfsUm (SEQ ID NO: 1651); or

[0556] (8) The sense chain comprises

[0557] IBs-AmsAmAmAmGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmAms-IBs-GL6 (SEQ ID NO: 1239),

[0558] And the antisense strand comprises

[0559] (CP1a-U)sCfsUmGfAmGf(PCN-

[0560] A)AfUmAfCmUfGmUfCmCfCmUfUmsUfsUm (SEQ ID NO: 1652); or

[0561] (9) The sense chain comprises

[0562] AmsAmsGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmsAms-GL6 (SEQ ID NO: 1240),

[0563] And the antisense strand comprises

[0564] (CP1a-U)sCfsUmGfAmGf(PCN-A)AfUmAfCmUfGmUfCmCfCmUfUmsUfsUm (SEQ ID NO: 1652).

[0565] IV. Inhibition of APOC3 gene expression

[0566] The siRNA of the present invention is capable of inhibiting the expression of the APOC3 gene 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%.

[0567] Inhibition of APOC3 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, for example, in a sample derived from a subject) in which the APOC3 gene is transcribed and which cell or cells have been treated (for example, by contacting the cell or cells with an siRNA of the invention, or by administering an siRNA of the invention to a subject in which the cells are or were present) such that APOC3 gene expression is inhibited compared to a second cell or group of cells (one or more control cells) that is substantially identical to the first cell or group of cells but has not been so treated.

[0568] In preferred embodiments, inhibition is assessed by expressing the level of mRNA in treated cells as a percentage of the level of mRNA in control cells using the formula: -△△Ct The values ​​were converted into percentages to obtain the residual inhibition rate, where ΔΔCt = [(Ct experimental group target gene - Ct experimental group internal reference) - (Ct control group target gene - Ct control group internal reference)].

[0569] Inhibition of APOC3 protein expression can be demonstrated by a decrease in the level of APOC3 protein expressed by a cell or cell group (e.g., the level of protein expressed in a sample from a subject). As explained above with respect to the assessment of mRNA repression, inhibition of protein expression levels in treated cells or cell groups can similarly be expressed as a percentage of the level of protein in control cells or cell groups.

[0570] Control cells or cell groups that can be used to assess inhibition of APOC3 gene expression include cells or cell groups that have not been contacted with the siRNA of the present invention. For example, the control cells or cell groups can be derived from an individual subject (e.g., a human or animal subject) before the subject is treated with the siRNA.

[0571] V. Vector

[0572] The present invention provides a vector comprising a nucleotide sequence encoding the siRNA of the present invention. The vector of the present invention is capable of amplifying or expressing the nucleotide sequence encoding the siRNA of the present invention linked thereto.

[0573] siRNA targeting the APOC3 gene can be expressed from a transcription unit inserted into a DNA or RNA vector. Expression can be short-lived (within hours to weeks) or sustained (weeks to months or longer), depending on the specific construct used and the target tissue or cell type. The coding nucleotides for the siRNA targeting the APOC3 gene can be introduced into a linear construct, a circular plasmid, or a viral vector. The nucleotides encoding the siRNA targeting the PCSK gene can be integrated into the cell genome for stable expression, or stably inherited and expressed outside the chromosome. In general, siRNA expression vectors are typically DNA plasmids or viral vectors.

[0574] Viral vector systems containing the coding sequence of siRNA targeting the APOC3 gene include, but are not limited to: (a) adenoviral vectors; (b) retroviral vectors; (c) adeno-associated viral vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) poxvirus vectors; and (j) helper virus-dependent adenovirus or gut-free adenovirus.

[0575] VI. Cells

[0576] The present invention provides a cell comprising the siRNA or vector of the present invention, wherein the siRNA or vector of the present invention can be transcribed in the cell.

[0577] VII. Pharmaceutical Compositions

[0578] The present invention provides a pharmaceutical composition comprising the siRNA, vector or cell of the present invention, and optionally a pharmaceutically acceptable carrier or excipient.

[0579] As used herein, "pharmaceutically acceptable" refers to those compounds, materials, compositions, and / or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human and animal subjects without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0580] As used herein, a pharmaceutically acceptable carrier refers to a pharmaceutical carrier that facilitates administration of siRNA or vectors or cells containing its coding sequence into the human body and / or facilitates its absorption or function. Examples include diluents and excipients such as water; fillers such as starch and sucrose; binders such as cellulose derivatives, alginates, gelatin, and polyvinyl pyrrolidone; humectants such as glycerol; disintegrants such as agar, calcium carbonate, and sodium bicarbonate; absorption enhancers such as quaternary ammonium compounds; surfactants such as cetyl alcohol; adsorption carriers such as kaolin and bentonite; and lubricants such as talc, calcium / magnesium stearate, and polyethylene glycol. Other adjuvants such as flavoring agents and sweeteners may also be added to the composition.

[0581] For example, a pharmaceutical composition comprising the siRNA, vector or cell of the present invention may comprise a pharmaceutically acceptable diluent or a sustained-release matrix, in which the siRNA or vector of the present invention is embedded.

[0582] VIII. Kit

[0583] The present invention provides a kit comprising the siRNA, vector or cell of the present invention.

[0584] The present invention also provides kits for using the siRNAs described herein and / or performing the methods described herein. Such kits include one or more siRNAs, vectors, or cells described herein, and may further include instructions for use. The instructions may include instructions for inhibiting APOC3 expression in cells by contacting the cells with the siRNAs or vectors described herein in an amount effective to inhibit APOC3 expression.

[0585] When the siRNA or vector described herein is contacted with cells in vitro, the kit of the present invention may optionally further include a means for contacting cells with the siRNA or vector described herein (e.g., an injection device) or a means for measuring the inhibitory effect of APOC3 (e.g., a device for measuring the inhibition of APOC3 mRNA or protein). Such a device for measuring the inhibition of APOC3 may include a device for obtaining a sample (e.g., a plasma sample) from a subject.

[0586] When the siRNA, vector, or cells into which the siRNA or vector has been introduced in vitro of the present invention are administered in vivo, the kit of the present invention may optionally further comprise a device for administering the siRNA, vector, or cells of the present invention to a subject or a device for determining a therapeutically effective amount or a prophylactically effective amount.

[0587] IX. Methods of treatment and pharmaceutical uses

[0588] The present invention provides a method for inhibiting APOC3 expression in a cell, comprising: (a) contacting the cell with the siRNA, vector, cell, or pharmaceutical composition of the present invention; and (b) culturing the cell.

[0589] The present invention provides a method for treating a disease or symptom associated with APOC3 expression in a subject, comprising the step of administering to the subject the siRNA, vector, cell, or pharmaceutical composition of the present invention.

[0590] The siRNA provided by the present invention can inhibit the expression of APOC3, thereby reducing triglyceride levels, particularly serum triglyceride levels, and is used to treat APOC3-related diseases and conditions. The APOC3-related diseases and conditions are hypertriglyceridemia and diseases that may be caused by, associated with, or a consequence of hypertriglyceridemia. Diseases that may be caused by, associated with, or a consequence of hypertriglyceridemia include, but are not limited to, pancreatitis, metabolic syndrome, type II diabetes, familial chylomicronemia syndrome (FCS), chylomicronemia, multifactorial chylomicronemia, lipodystrophy syndrome (e.g., familial partial lipodystrophy), obesity, dyslipidemia, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, hyperlipidemia, hypertriglyceridemia, abnormal lipid and / or cholesterol metabolism, atherosclerosis, cardiovascular disease, coronary artery disease, polycystic ovary syndrome, kidney disease, and other dyslipidemia and metabolic-related conditions and diseases. The hypertriglyceridemia includes, but is not limited to, non-familial hypertriglyceridemia, familial hypertriglyceridemia, heterozygous familial hypertriglyceridemia, and homozygous familial hypertriglyceridemia.

[0591] In some embodiments, the method of treating a disease or symptom associated with APOC3 expression in a subject of the present invention comprises administering the siRNA or pharmaceutical composition to the subject subcutaneously, intravenously, or topically. In some embodiments, the subject is a human patient.

[0592] The present invention also relates to the siRNA, vector, cell or pharmaceutical composition of the present invention for treating a disease or symptom associated with APOC3 expression in a subject.

[0593] The present invention also relates to the use of the siRNA, vector, cell, or pharmaceutical composition of the present invention in the preparation of a medicament for treating a disease or symptom associated with APOC3 expression in a subject. The medicament of the present invention can be prepared into an emulsion, microemulsion, or microparticle.

[0594] The present invention also provides a method for reducing the risk of developing APOC3-related diseases and disorders in a subject, comprising the step of administering to the subject the siRNA, vector, cell, or pharmaceutical composition of the present invention.

[0595] The siRNA of the present invention can significantly reduce triglyceride levels and thereby reduce the risk of developing APOC3-related diseases and conditions. The APOC3-related diseases and conditions are hypertriglyceridemia and diseases that can be caused by, associated with, or are a consequence of hypertriglyceridemia. Diseases that can be caused by, associated with, or are a consequence of hypertriglyceridemia include, but are not limited to, pancreatitis, metabolic syndrome, type II diabetes, familial chylomicronemia syndrome (FCS), chylomicronemia, multifactorial chylomicronemia, lipodystrophy syndrome (e.g., familial partial lipodystrophy), obesity, dyslipidemia, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, hyperlipidemia, hypertriglyceridemia, abnormal lipid and / or cholesterol metabolism, atherosclerosis, cardiovascular disease, coronary artery disease, polycystic ovary syndrome, kidney disease, and other dyslipidemia and metabolic-related conditions and diseases. The hypertriglyceridemia includes, but is not limited to, non-familial hypertriglyceridemia, familial hypertriglyceridemia, heterozygous familial hypertriglyceridemia, and homozygous familial hypertriglyceridemia.

[0596] In some embodiments, the method of the present invention for reducing the risk of developing APOC3-related diseases and disorders in a subject administers the siRNA or pharmaceutical composition to the subject subcutaneously, intravenously, or topically. In some embodiments, the subject is a human patient.

[0597] The present invention also relates to the siRNA, vector, cell or pharmaceutical composition of the present invention for reducing the risk of developing APOC3-related diseases and disorders in a subject.

[0598] The present invention also relates to the use of the siRNA, vector, cell, or pharmaceutical composition of the present invention in the preparation of a medicament for reducing the risk of developing APOC3-related diseases and conditions in a subject. The medicament of the present invention can be prepared into an emulsion, microemulsion, or microparticle.

[0599] sequence

[0600] The RNA sequence provided by the present invention targets the APOC3 gene (or target gene, target mRNA sequence, target sequence).

[0601] Table 3. Nucleotide sequences of the sense and antisense strands targeting the APOC3 gene used in the present invention

[0602] Table 4 below shows the modified RNA sequences used in the present invention.

[0603] In this article, the meanings of the abbreviations are as follows:

[0604] The A, U, G, and C distribution represents the natural adenine, uracil, guanine, and cytosine ribonucleotides.

[0605] i or I represents inosine ribonucleotide.

[0606] m indicates 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.

[0607] f indicates that the nucleotide adjacent to its left is a 2'-F modified nucleotide. For example, Af, Uf, Gf, and Cf represent 2'-F modified A, U, G, and C, respectively.

[0608] "s" or "s-" indicates that the two adjacent nucleotides and / or delivery vectors are linked by phosphorothioate.

[0609] VP indicates that the adjacent nucleotide on its right side is a vinylphosphonate-modified nucleotide, which is well known in the art, and can be found in, for example, PCT Publication Nos. WO2011139702, WO2013033230, and WO2019105419.

[0610] IB represents an inverted abasic deoxyribonucleotide, which can include the following three structures depending on its location / connection mode in the siRNA (used at the 5' end, interchain, and 3' end of the nucleic acid chain respectively):

[0611] IB is well known in the art, see, for example, F. Czauderna, Nucleic Acids Res., 2003, 31(11), 2705-16 and PCT Publication Nos. WO2016011123 and WO2019051402.

[0612] L96 represents a GalNAc delivery vector of the following structure well known in the art, wherein The position where the siRNA is linked via a phosphate group or a phosphorothioate group can be found in, for example, PCT Publication Nos. WO2009073809 and WO2009082607.

[0613] NAG37 represents a GalNAc delivery vector of the following structure well known in the art, wherein Indicates the position where the siRNA is connected, see for example PCT Publication No. WO2018044350

[0614] GL6 represents a GalNAc delivery vector of the following structure, wherein Indicates the position where the siRNA is linked via a phosphate or phosphorothioate group

[0615] GL12 represents a GalNAc delivery vector of the following structure, wherein Indicates the position where the phosphate group or thiophosphate group is connected to the siRNA. The synthesis method of the related intermediates is shown in the examples below.

[0616] STM1 represents a nucleotide substitute of the following structure. The synthesis method of the related intermediate is shown in Example 15 below.

[0617] PCN represents a nucleotide substitute of the following structure, wherein Base can be any base, for example, PCN-A represents Base is adenine. The synthesis method of the related intermediate is shown in Example 17 below.

[0618] CP1a represents a nucleotide substitute of the following structure, wherein Base can be any base, for example, CP1a-U represents Base is uracil. The synthesis method of the related intermediate is shown in Example 16 below.

[0619] Table 4 Modified RNA sequences of the positive strand of the siRNA targeting the APOC3 gene used in the present invention

[0620] Table 5 Modified RNA sequences of the antisense strand of the siRNA targeting the APOC3 gene used in the present invention

[0621] Table 6 Paired modified sense strands and modified antisense strands of siRNA targeting the APOC3 gene used in the present invention

[0622] Example

[0623] The sources of materials used in the examples are as follows:

[0624] Huh7 cell line was purchased from Nanjing Kebai, catalog number CBP60202;

[0625] Hep3B cell line was purchased from Nanjing Kebai, catalog number CBP60197;

[0626] PHH cells were purchased from Shanghai Xuanyi, catalog number QYLF-HPMC;

[0627] HEK293A cell line was purchased from Nanjing Kebai, catalog number CBP60436;

[0628] Balb / c mice were obtained from Zhejiang Weitonglihua, catalog number Balb / c.

[0629] Example 1. Preparation of Compound E7

[0630] 1. Preparation of Intermediates 3-4

[0631] 1.1 Preparation of Compound 2

[0632] To compound 1 (300 g, 2.01 mol) in DCM (1.80 L) was slowly added benzyl (2,5-dioxopyrrolidin-1-yl) carbonate (600 g, 2.40 mol) at 15°C and TEA (203 g, 2.01 mol, 280 mL) was added dropwise. After the addition, the mixture was stirred at 25°C for 16 hours. TLC (dichloromethane:methanol = 10:1) showed that reactant 1 (R f =0.32) is retained and an important new point is detected (R f=0.52). The reaction mixture was washed with saturated sodium bicarbonate solution (1.00 L x 2), and the organic phase was washed with brine (1.00 L), dried over anhydrous Na2SO4 and concentrated in vacuo. Without purification, compound 2 (about 385 g) was obtained as a yellow oil.

[0633] 1.2 Preparation of compound 2A

[0634] At 0-15 ℃, DMAP (19.8g, 162mmol) was added to a pyridine (1.75L) solution of compound 4 (350g, 1.62mol, HCl) and Ac2O (994g, 9.74mol, 912mL) at a disposable rate and TEA (164g, 1.62mol, 226mL) was added dropwise. The mixture was stirred at 25 ℃ for 16 hours. LCMS (product: RT=0.687min) showed that the starting reactants were completely consumed. EtOAc (1.40L) was added to the mixture at 25 ℃ and stirred for 30 minutes, then the mixture was filtered and the filter cake was washed with EtOAc (300mL). The filter cake was ground with water (1.45L) for 30 minutes at 25 ℃. The mixture was filtered and the filter cake was washed with water (175mL x 3), and the filter cake was collected to obtain compound 2A (about 580g) as a white solid.

[0635] 1.3 Preparation of compound 2B

[0636] Three reactions were performed in parallel.

[0637] To a solution of compound 2A (200 g, 514 mmol) in DCM (800 mL) was added TMSOTf (137 g, 616 mmol, 111 mL) dropwise at 10-15° C. over 0.5 h. The mixture was then stirred at 25° C. for 3 h. TLC (dichloromethane:methanol=20:1) showed that compound 2A (R f =0.54) is completely consumed and the new point (R f =0.24) was formed. The three reactions were combined. The mixture was cooled to 0-15°C and slowly poured into a solution of NaHCO3 (300 g dissolved in 3.00 L water) at 0-5°C. The organic phase was separated and the aqueous phase was extracted with DCM (1.00 L x 3). The organic layers were combined, dried over Na2SO4, filtered and concentrated in vacuo. Without purification, compound 2B (approximately 507 g) was obtained as a yellow oil and used in the next step.

[0638] 1.4 Preparation of compound 3

[0639] To a mixture of compound 2B (250 g, 759 mmol) and compound 2 (151 g, 531 mmol) in DCM (1.00 L) was added TMSOTf (84.4 g, 380 mmol, 69.0 mL) dropwise at 0-10° C. and the mixture was stirred at 20° C. for 12 hours. TLC (dichloromethane:methanol=20:1) showed that compound 2 (R f =0.33) is completely consumed and a new point is formed (R f =0.03). The combined reaction was cooled to 0-5°C and then poured into NaHCO (aqueous solution, 100 g dissolved in 1 L of water) and stirred at 5-10°C for 10 minutes. The phases were separated. The aqueous phase was extracted with DCM (500 mL x 2), the combined organic phases were dried over NaSO, filtered, and the filtrate was concentrated in vacuo. Without purification, compound 3 (approximately 360 g) was obtained as a yellow oil.

[0640] 1 H NMR: (400 MHz, DMSO).

[0641] δ=7.79-7.37(m,1H),7.35-7.26(m,5H),5.21-5.20(m,1H),5.00-4.95(m,3H),4.55-4.53(m,1H),4.03-3.86(m,3H),3.61-3.59(m,1H) ,3.59-3.57(m,1H),3.48-3.40(m,6H),3.39-3.31(m,2H),3.14-3.13(m,2H),2.09(s,3H),1.99(s,3H),1.88(s,3H),1.76-1.74(m,3H).

[0642] 1.5 Preparation of Intermediate 3-4 (TFA Salt)

[0643] Three reactions were performed in parallel.

[0644] To a mixture of Pd / C (18.0 g, 16.3 mmol, 10% content) in THF (1.80 L) was added compound 3 (180 g, 293 mmol) and TFA (33.5 g, 293 mmol, 21.8 mL) under an argon atmosphere. The suspension was vented and vented three times with hydrogen. The mixture was stirred at H2 (50 Psi) and 30 ° C for 2 hours. LCMS (product: RT = 0.697 min) showed that compound 3 was consumed and a product peak was detected. The three reactions were combined. The mixture was filtered through celite and the filtrate was concentrated under reduced pressure to remove the solvent. Intermediate 3-4 (TFA salt) (393 g, 660 mmol, 74.8% yield, 99.6% purity, TFA) was obtained as a yellow solid without purification.

[0645] 1 H NMR: (400 MHz, DMSO-d6)

[0646] δ=7.92(d,J=9.1Hz,4H),5.27-5.17(m,1H),5.03-4.91(m,1H),4.60-4.50(m,1H),4.09-3.97(m,4H),3.85(s ,2H),3.65-3.46(m,10H),3.04-2.92(m,2H),2.10(s,3H),2.00(s,3H),1.94-1.86(m,3H),1.82-1.71(m,4H).

[0647] 2. Preparation of Intermediates 3-5

[0648] 2.1 Preparation of compound 5

[0649] To a solution of compound 4B (10.0 g, 35.5 mmol, 1.00 eq) and compound 3-4 (46.3 g, 78.2 mmol, 2.20 eq, TFA) prepared above in DCM (1.00 L) was added DIEA (30.3 g, 234 mmol, 40.8 mL, 6.60 eq) in one portion at 25°C. The mixture was stirred for half an hour at 25°C. HBTU (30.3 g, 234 mmol, 40.8 mL, 6.60 eq) was added to the mixture. The mixture was stirred at 25°C for 16 hours. LCMS (product: RT = 0.681 mins) indicated the reaction was complete, and the mixture was concentrated in vacuo. At 20 ℃, 0.50N HCl (200mL x 2) was added to the mixture, and extracted with DCM (3 x 500mL), the combined organic layers were washed with saturated NaHCO (3 x 800mL) until pH=8, washed with brine (3 x 500mL), dried over Na SO and concentrated in vacuo for purification. The residue was purified by column chromatography (SiO , DCM:MeOH=50:1-15:1). The residue was concentrated in vacuo at 40 ℃ and purified by preparative-MPLC (column: 800g Agela C18; mobile phase: [water-ACN]; 15-45% 25min; 45% 10min). Vacuum drying was performed to obtain compound 5 (approximately 180g+75.0g+87.0g+40.0g+38.0g) as a yellow solid.

[0650] 417.0 g of compound 3-4 was converted into compound 5 through 9 batches.

[0651] 2.2 Preparation of intermediate 3-3

[0652] Compound 5 (73.0 g, 61.7 mmol, 1.00 eq) and TFA (7.04 g, 61.7 mmol, 4.57 mL, 1.00 eq) were added to a solution of Pd / C (3.00 g, 10% content) in THF (300 mL) under an argon atmosphere. The suspension was degassed and purged with hydrogen three times. The mixture was stirred at 20°C under H2 (20 Psi) for 16 hours. TLC (dichloromethane:methanol = 8:1, R f =0.0) indicated that the reaction was complete. The mixture was filtered through celite, and the filtrate was concentrated under pressure to remove the solvent to obtain compound 3-3 (about 33.4 g + 129 g + 75.0 g) as a white solid.

[0653] 1 H NMR: (400 MHz, DMSO)

[0654] δ=8.53(t,J=5.2Hz,1H),8.18(d,J=2.4Hz,3H),8.03(t,J=5.2Hz,1H),7.84(dd,J=3.6H z,2H),5.22(d,J=3.2Hz,2H),4.96(dd,J=3.2Hz,2H),4.55(d,J=8.4Hz,2H),4.02(t,J= 8.8Hz,6H),3.77-3.59(m,5H),3.58-3.45(m,21H),3.40-3.20(m,4H),2.18(t,J=7.6Hz ,2H),2.17(d,J=8.0Hz,6H),2.10(s,6H),1.99(s,6H),1.90-1.80(m,8H),1.77(s,6H).

[0655] 3. Preparation of Compound E7

[0656] 3.1 Preparation of compound 3

[0657] Compound 1 (2.00 g, 1.87 mmol, prepared according to the method for Intermediate 3-3 above) was dissolved in DCM (20.0 mL) at room temperature. DIEA (0.135 mL, 0.814 mmol) and compound 2 (0.550 g, 0.814 mmol) were added sequentially to the solution, and the nitrogen atmosphere was replaced three times. The reaction mixture was stirred at 25°C for 16 hours. LC-MS / MS detected the product's MS response, and thin-layer chromatography (dichloromethane / methanol = 5 / 1) showed the disappearance of the starting material and the formation of new spots. The reaction solution was concentrated under reduced pressure, and the resulting crude product was purified by column chromatography (dichloromethane / methanol = 5 / 1) to afford compound 3 (approximately 780 mg) as a white solid.

[0658] 1 H NMR (400 MHz, CD3OD)

[0659] δ=7.28-7.42(m,5H),5.30-5.34(m,4H),5.04-5.14(m,6H),4.63-4.67( m,4H),4.36-4.44(m,2H),4.00-4.20(m,23H),3.91-3.95(m,4H),3.69-3 .77(m,9H),3.52-3.67(m,32H),3.34-3.43(m,9H),2.29-2.31(m,4H),2. 14(s,12H),2.03(s,12H),1.92-1.96(m,24H).LCMS:m / z=1221.6(M / 2+H) + .

[0660] 3.2 Preparation of compound 4

[0661] Compound 3 (1.10 g, 0.451 mmol) was dissolved in MeOH (10.0 mL) at room temperature. 10% wet Pd / C (0.050 g, 0.451 mmol) was added to the solution. The hydrogen atmosphere was replaced three times, and the reaction mixture was stirred at 25°C for 18 hours under a hydrogen atmosphere (14.696 psi). Liquid chromatography-mass spectrometry (LC-MS / MS) detected the product. Thin-layer chromatography (dichloromethane / methanol = 10 / 1, phosphomolybdic acid) revealed complete consumption of the starting material and the formation of new spots. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to yield compound 4 (approximately 840 mg) as a white solid.

[0662] 1 H NMR (400 MHz, CD3OD)

[0663] δ=5.32-5.34(m 4H),5.06-5.10(m,4H),4.63-4.65(m,4H),4.38-4.40(m,2H),3.99-4.20 (m,20H),3.90-3.97(m,4H),3.69-3.76(m,6H),3.50-3.68(m,36H),3.35- 3.44(m,11H),2.28-2.38(m,4H),2.15(s,12H),2.03(s,12H),1.90-1.94(m,24H).LCMS:m / z=1154.7(M / 2+H) + .

[0664] 3.3 Preparation of compound 6

[0665] Compound 5 (232 mg, 0.364 mmol) was dissolved in DCM (10.0 mL) at room temperature. HBTU (207 mg, 0.546 mmol), DIEA (0.181 mL, 1.09 mmol), and compound 4 (840 mg, 0.364 mmol) were added sequentially to the solution, and the atmosphere was purged with nitrogen three times. The reaction mixture was stirred at 25°C for 1 hour. LC / MS analysis revealed the disappearance of the starting material, while thin-layer chromatography (dichloromethane / methanol = 5 / 1) showed the disappearance of the starting material and the formation of new spots. The reaction mixture was concentrated under reduced pressure, and the resulting crude product was purified by column chromatography (dichloromethane / methanol = 8 / 1 to 5 / 1) to afford compound 6 (approximately 620 mg) as a white solid.

[0666] 1 H NMR (400 MHz, CD3OD)

[0667] δ=7.41-7.43(m,2H),7.23-7.34(m,7H),6.83-6.90(m,4H),5.31-5.35(m,4H),5.01-5.12(m ,4H),4.63-4.65(m,4H),4.41-4.45(m,2H),4.31-4.33(m,1H),3.99-4.22(m,22H),3.87-3.9 7(m,6H),3.58-3.81(m,45H),3.34-3.43(m,10H),2.19-2.40(m,10H),2.14(s,12H),2.02(s ,12H),1.92-1.96(mz,24H),1.48-1.63(m,4H),1.28-1.38(m,8H).LCMS:m / z=1460.0(M / 2+H) + .

[0668] 4. Preparation of Compound E7

[0669] Compound 6 (300 mg, 0.103 mmol) was dissolved in DCM (10.0 mL) at room temperature. DIEA (0.102 mL, 0.618 mmol), compound 7 (10.3 mg, 0.103 mmol), and DMAP (12.6 mg, 0.103 mmol) were added sequentially to the solution, and the atmosphere was replaced with nitrogen three times. The reaction mixture was stirred at 25°C for 2 hours. LC / MS / MS analysis confirmed the disappearance of the starting material. The reaction solution was concentrated under reduced pressure, and the resulting crude product was separated by preparative HPLC (Preparative-HPLC, column: Waters Xbridge BEH C18 100*30 mm*10 μm; mobile phase: water-ACN; B%: 17%-57%, 5 min) to afford compound E7 (53.0 mg, yield 17.08%, purity 78.94%) as a white solid.

[0670] 1 H NMR (400 MHz, CD3OD)

[0671] δ=7.41-7.45(m,2H),7.17-7.34(m,7H),6.85-6.89(m,4H),5.32-5.36(m,4H),5.03-5.13(m,4H),4.63-4.67(m,4H),4.38-4 .47(m,2H),4.32-4.34(m,1H),4.01-4.26(m,22H),3.88-4.00(m,6H),3.77-3.81(m,7H),3.49-3.76(m,45H),3.33-3.47(m, 10H),2.56-2.62(m,2H),2.45-2.55(m,3H),2.21-2.38(m,7H),2.14(s,12H),2.05- 2.11(m,2H),2.02(s,12H),1.92-1.96(m,24H),1.47-1.68(m,4H),1.28-1.34(m,8H)

[0672] MS: m / z = 3022.36 (M+H) + .

[0673] Example 2 Preparation of siRNA

[0674] The siRNA of the present invention is prepared using the solid phase phosphoramidite method well known in the art. Specific methods can be found in, for example, PCT Publication Nos. WO2016081444 and WO2019105419, and are briefly described below.

[0675] 1. Preparation of siRNA without Ligand

[0676] 1.1 Synthesis of the positive sense chain (SS chain)

[0677] Using solid-phase phosphoramidite synthesis, a blank CPG solid support is used as the starting cycle, and nucleoside monomers are linked one by one in the 3'-5' direction according to the order of the positive chain nucleotides. Each linking of a nucleoside monomer involves four steps: deprotection, coupling, capping, and oxidation or thiolation. The conditions for the synthesis of oligonucleotides at a synthesis scale of 5 μmol are as follows:

[0678] The nucleoside monomer was provided in a 0.05 mol / L acetonitrile solution. The reaction conditions for each step were the same, i.e., the temperature was 25°C, and deprotection was performed three times using a 3% trichloroacetic acid-dichloromethane solution. The coupling reaction was performed twice using a 0.25 mol / L ETT-acetonitrile solution as the activator. Capping was performed twice using 10% acetic anhydride-acetonitrile and pyridine / N-methylimidazole / acetonitrile (10:14:76, v / v / v). Oxidation was performed twice using 0.05 mol / L iodine / tetrahydrofuran / pyridine / water (70 / 20 / 10, v / v / v). Thiolysis was performed twice using 0.2 mol / L PADS in acetonitrile / 3-methylpyridine (1 / 1, v / v).

[0679] 1.2 Synthesis of antisense strand (AS strand)

[0680] Using solid-phase phosphoramidite synthesis, a blank CPG solid support was used as the starting cycle. Nucleoside monomers were linked one by one in the 3'-5' direction according to the nucleotide arrangement of the antisense strand. Each ligation step involved deprotection, coupling, capping, and oxidation or thiolation. Synthesis conditions for 5 μmol of oligonucleotides for the antisense strand were identical to those for the sense strand.

[0681] 1.3 Oligonucleotide purification and annealing

[0682] 1.3.1 Ammonolysis

[0683] The synthesized solid phase carrier (sense chain or antisense chain) 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 (or 55°C) for 16 hours (or 8 hours). After filtering, the solid phase carrier was washed three times with ethanol / water, each time with 1 mL. The filtrate was centrifuged and concentrated, and the crude product was purified.

[0684] 1.3.2 Purification

[0685] Purification and desalination methods are well known in the art. For example, a column packed with a strong anion filler can be used, and a sodium chloride-sodium hydroxide system can be used for elution and purification, and the product can be collected and stored in a tube. A gel-filled purification column can be used for desalination, and the elution system can be pure water.

[0686] 1.3.3 Annealing

[0687] The sense strand (SS strand) and the antisense strand (AS strand) were mixed at a molar ratio of SS strand / AS strand = 1 / 1.05, heated in a water bath to 70-95°C for 3-5 minutes, cooled naturally to room temperature, and lyophilized to obtain the product. Double-stranded DNA sequences DR000001 to DR000400 were obtained.

[0688] 2. Preparation of siRNA with Sense Strand Linked to Ligand

[0689] 2.1 Ligation of compound E7 with CPG carrier

[0690] Compound E7 (53 mg, 0.018 mmol) and HBTU (13.3 mg, 0.035 mmol) were mixed and added to acetonitrile (5 mL) and shaken to dissolve. DIEA (9.0 mg, 0.07 mmol) and DMAP (2.1 mg, 0.018 mmol) were then added and shaken to dissolve until the solution became clear. ) was added to the reaction solution, and the reaction was shaken overnight at 20°C. Samples were taken and monitored for TLC, which indicated the reaction was complete. The developing solvent was DCM / methanol = 4:1, and the color was developed with phosphomolybdic acid. Filtered using a fritted funnel, the filter cake was washed with anhydrous acetonitrile (20 mL x 5), and the filter cake was filtered under reduced pressure using an oil pump for 6 h to obtain 530 mg of an off-white solid.

[0691] 530 mg of the condensed product was placed in a 50 mL round-bottom flask. Cap C (DMAP / acetonitrile), Cap B (N-methylimidazole / pyridine / acetonitrile), and Cap A (acetic anhydride / acetonitrile) were added sequentially. The mixture was shaken overnight at room temperature. Filter and wash the resulting filter cake with acetonitrile (20 mL x 4). The filter cake was removed and filtered under reduced pressure at an oil pump for 8 hours to yield 200 mg of an off-white solid (GL6 solid support) for solid-phase synthesis.

[0692] 2.2 Synthesis of the Sense Chain (SS Chain) Linked to the Ligand

[0693] Using the solid-phase phosphoramidite synthesis method, using the GL6 solid support prepared above as the starting cycle, nucleoside monomers were attached one by one in the 3'-5' direction according to the order of the positive strand nucleotides. Each ligation of a nucleoside monomer involved four steps: deprotection, coupling, capping, and oxidation or thiolation. The synthesis conditions for a 5 μmol oligonucleotide were as follows:

[0694] The nucleoside monomer was provided in a 0.05 mol / L acetonitrile solution. The reaction conditions for each step were the same, i.e., the temperature was 25 degrees Celsius. Deprotection was performed three times using a 3% trichloroacetic acid-dichloromethane solution. The coupling reaction was performed twice using a 0.25 mol / L ETT-acetonitrile solution as the activator. Capping was performed twice using 10% acetic anhydride-acetonitrile and pyridine / N-methylimidazole / acetonitrile (10:14:76, v / v / v). Oxidation was performed twice using 0.05 mol / L iodine / tetrahydrofuran / pyridine / water (70 / 20 / 10, v / v / v). Thiolysis was performed twice using 0.2 mol / L PADS in acetonitrile / 3-methylpyridine (1 / 1, v / v).

[0695] 2.3 Synthesis of antisense strand (AS strand)

[0696] Using solid-phase phosphoramidite synthesis, a blank CPG solid support was used as the starting cycle. Nucleoside monomers were linked one by one in the 3'-5' direction according to the nucleotide arrangement of the antisense strand. Each ligation step involved deprotection, coupling, capping, and oxidation or thiolation. Synthesis conditions for 5 μmol of oligonucleotides for the antisense strand were identical to those for the sense strand.

[0697] 2.4 Oligonucleotide Purification and Annealing

[0698] 2.4.1 Ammonolysis

[0699] The synthesized solid phase carrier (sense chain or antisense chain) 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 (or 55°C) for 16 hours (or 8 hours). After filtering, the solid phase carrier was washed three times with ethanol / water, each time with 1 mL. The filtrate was centrifuged and concentrated, and the crude product was purified.

[0700] 2.4.2 Purification

[0701] Purification and desalination methods are well known in the art. For example, a column packed with a strong anion filler can be used, and a sodium chloride-sodium hydroxide system can be used for elution and purification. The product can be collected and pipetted, and desalination can be performed using a gel-filled purification column, and the elution system can be pure water.

[0702] 2.4.3 Annealing

[0703] The sense chain (SS chain) and the antisense chain (AS chain) were mixed at a molar ratio of (SS chain / AS chain = 1 / 1.05), heated to 70-95°C in a water bath, maintained for 3-5 minutes, cooled naturally to room temperature, and the system was freeze-dried to obtain the product.

[0704] siRNA conjugated with NAG37 or L96 was obtained in a similar manner.

[0705] Example 3 Activity screening of Huh7 cell line

[0706] Cell transfection

[0707] On the first day, Huh7 cell line (Nanjing Kebai, Cat. No. CBP60202) was digested and resuspended, counted, and plated in 96-well plates at 100 μL / well, 1×10 4 cells / well, and transfection was performed 18 h later.

[0708] On the second day, the 20 μM siRNA stock solution (DR000001 to DR000400) prepared in Example 2 was diluted with Opti-MEM. 198 μL Opti-MEM was added to 2 μL siRNA stock solution, pipetting to mix thoroughly, and set aside. Each experiment was performed according to different experimental requirements.

[0709] The next day, dilute 0.9 μL of RNAiMAX (Thermo, 13778150) with 14.1 μL of Opti-MEM. Gently pipette to mix thoroughly, and let stand at room temperature for 5 minutes. Then, take 15 μL of the prepared RNAi-MAX mixture and 15 μL of the diluted siRNA compound and gently pipette to mix thoroughly, avoiding the introduction of bubbles. Let stand at room temperature for 10 minutes. Add 10 μL / well to a 96-well plate. Incubate in a 37°C, 5% CO2 incubator for 24 hours before RNA extraction.

[0710] RNA extraction

[0711] Cell RNA was extracted using a nucleic acid extractor (Hangzhou Aosheng, Auto-pure96) according to the protocol of the high-throughput cell RNA extraction kit (Fanzhi Medical, FG0412).

[0712] RNA reverse transcription

[0713] For preparation of denaturation reaction mixture, refer to PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B). Each well contains 1 μL of Oligo dT Primer, 1 μL of dNTP Mixture, and 12.5 μL of template RNA. Incubate at 65°C for 5 minutes in a conventional PCR instrument to perform the denaturation reaction. Rapidly cool the mixture on ice for 2 minutes.

[0714] Preparation of reverse transcription reaction solution, refer to PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B). Each well contains 4 μL of 5× PrimeScript II Buffer, 0.5 μL of RNase Inhibitor, and 1 μL of PrimeScript II RTase.

[0715] 14.5 μL of the denatured reaction solution was slowly mixed with the reverse transcription reaction solution, incubated at 42° C. for 45 min in a conventional PCR instrument for reverse transcription, incubated at 95° C. for 5 min to inactivate the enzyme, and the reverse transcription product (cDNA) was cooled at 4° C.

[0716] After reverse transcription, add 30 μL of DNase RNase-Free Distilled Water to the cDNA sample in each well.

[0717] Fluorescence quantitative PCR

[0718] ReferenceTaqMan TM Fast Advanced Master Mix (ABI, 4444965) was used to perform fluorescent quantitative PCR reactions (ABI, QuantStudio3) in 20 μL of the system. The reaction program was: (50°C, 2 min) × 1 cycle; (95°C, 20 s) × 1 cycle; (95°C, 1 s; 60°C, 24 s) × 40 cycles.

[0719] Table 7 Primer information

[0720] Statistics

[0721] Calculation 2 -△△Ct The values ​​were converted into percentages to obtain the residual inhibition rate.

[0722] △△Ct=[(target gene of Ct experimental group-internal reference of Ct experimental group)-(target gene of Ct control group-internal reference of Ct control group)].

[0723] The final concentration of siRNA was 10 nM for high-throughput screening of siRNA compound cell line activity. The experimental screening results are shown in Table 8.

[0724] Table 8 Results of high-throughput screening of Huh7 cell line activity using 10 nM siRNA

[0725] Example 4: Screening Hep3B cell line activity using siRNA at five concentrations diluted 10-fold

[0726] Similar to the above Huh7 cell line activity screening, Hep3B (Nanjing Kebai, catalog number CBP60197) cell line was used for activity screening.

[0727] The starting concentration of siRNA (DR000001 to DR000400) was 10 nM, and 10-fold serial dilution was performed to obtain 5 concentration points (10 nM, 1 nM, 0.1 nM, 0.01 nM, 0.001 nM) for siRNA activity screening in Hep3B cell lines. The screening results are shown in Table 9.

[0728] Table 9 Results of Hep3B cell line activity screening using 5 concentrations of siRNA

[0729] Example 5: Screening Hep3B cell line activity using 11 concentrations of siRNA diluted 3-fold

[0730] The steps in this example were similar to those in Example 4. The siRNAs (DR000001 to DR000400) were screened for activity against Hep3B cells using a 3-fold serial dilution sequence at 11 concentrations (10 nM, 3.33 nM, 1.11 nM, 0.37 nM, 0.123 nM, 0.041 nM, 0.0136 nM, 0.0045 nM, 0.00152 nM, 0.000508 nM, and 0.000169 nM). The results are shown in Table 10.

[0731] Table 10 Results of Hep3B cell line screening using 11 concentrations of siRNA

[0732] Example 6: Screening Huh7 cell line activity using 11 concentrations of siRNA diluted 3-fold

[0733] The steps of this example are similar to those of Example 3. Huh7 cell lines were selected, and the starting concentration of siRNA (DR000001 to DR000400) was 10 nM. A 3-fold serial dilution was performed to obtain 11 concentration points (10 nM, 3.33 nM, 1.11 nM, 0.37 nM, 0.123 nM, 0.041 nM, 0.0136 nM, 0.0045 nM, 0.00152 nM, 0.000508 nM, 0.000169 nM) for siRNA activity screening against Huh7 cell lines. The experimental screening results are shown in the table below.

[0734] Table 11 Results of Huh7 cell line screening using 11 concentrations of siRNA

[0735] Example 7: Screening Hep3B cell line activity using 11 siRNA concentrations diluted 3-fold

[0736] The steps of this example are similar to those of Example 5. Hep3B cell lines were selected, and the starting concentration of siRNAs (DR002222 to DR02226, DR001478, DR002252, DR000344, DR000348, DR000277, DR000199, DR000060, and DR001482) was 10 nM. A three-fold serial dilution was performed to obtain 11 concentration points (10 nM, 3.33 nM, 1.11 nM, 0.37 nM, 0.123 nM, 0.041 nM, 0.0136 nM, 0.0045 nM, 0.00152 nM, 0.000508 nM, and 0.000169 nM) for siRNA activity screening against Hep3B cell lines. The screening results are shown in Table 12.

[0737] Table 12-1 Results of Hep3B cell line screening using 11 concentrations of siRNA

[0738] Table 12-2 Results of Hep3B cell line screening using 11 concentrations of siRNA

[0739] Example 8 psiCHECK2GSSM-5Hits Off-Target Activity Screening

[0740] Plasmid preparation

[0741] The corresponding antisense off-target plasmid was designed according to the siRNA sequence, and the psiCHECK2GSSM-5Hits recombinant plasmid was prepared by Sangon Biotech (Shanghai) Co., Ltd. and diluted to 1000 ng / μL for use.

[0742] Cell transfection

[0743] In each well of a 96-well plate, 100 μL of HEK293A cell suspension (Nanjing Kebai, catalog number CBP60436) was used to plate the cells. 8×10 3 cells / well.

[0744] On the second day, the complete culture medium in the wells was first aspirated and replaced with 80 μL / well of Opti-MEM medium, and the cells were starved for about 1.5 h.

[0745] siRNA preparation: siRNA was diluted 3-fold starting from a final concentration of 40 nM, for a total of 11 concentration points (40 nM, 13.3 nM, 4.44 nM, 1.48 nM, 0.493 nM, 0.164 nM, 0.0548 nM, 0.0182 nM, 0.00609 nM, 0.00203 nM, 0.000677 nM).

[0746] Preparation of plasmid mixture: The preparation volume for a single well is 0.01 μL / well of plasmid and 8.99 μL / well of Opti-MEM.

[0747] Preparation of Lipo mixture: Lipo 2000 0.2 μL and Opti-MEM 9.8 μL were added to each well to dilute Lipo 2000 (Lipofectamine TM 2000 transfection reagent, Thermo, 11668019) to obtain the Lipo mixture, and let it stand at room temperature for 5 minutes.

[0748] Aliquot 22 μL of the prepared Lipo mixture, 2.2 μL of siRNA, and 19.8 μL of the plasmid mixture into corresponding wells, designated well A. Mix thoroughly by pipetting and incubate at room temperature for 20 minutes before co-transfection. Add 20 μL of well A mixture to each well of cells, adding 80 μL of Opti-MEM to the original volume, for a final volume of 100 μL per well. After incubation at 37°C in a 5% CO2 incubator for 4 hours, add 100 μL of DMEM medium supplemented with 20% fetal bovine serum to each well. Incubate at 37°C in a 5% CO2 incubator for 24 hours before testing.

[0749] Result detection

[0750] Before the experiment, mix the ( Luciferase Assay System, Promega, E2940) was reconstituted and after equilibration to room temperature, DMEM was added to each tube at a ratio of 1:1 to prepare substrate I for immediate use. Stop& Buffer was reconstituted and then equilibrated to room temperature. Stop& Prepare substrate II at a 100:1 ratio and use immediately. Use a vacuum pump to remove the existing culture medium from the 96-well culture plate. Add 150 μL of substrate I to each well and incubate on a shaker at room temperature for 10 minutes. Transfer 120 μL of substrate I to a 96-well microplate reader and read the Firefly chemiluminescence value on a microplate reader (Tecan, Infinite 200). Add 60 μL of substrate II to each well and incubate on a shaker at room temperature for 10 minutes. Read the Renilla chemiluminescence value on a microplate reader.

[0751] Data analysis and processing

[0752] Fluorescence activity was measured by a microplate reader. The collected Renilla signal was normalized by the Firefly signal standard. The inhibitory effect of siRNA was calculated by comparing it with the untreated result (residual inhibitory activity). The calculation process is as follows:

[0753] Normalized Ren / Fir ratio: Ratio = Renilla (Renilla luciferase) / Firefly (Firefly luciferase).

[0754] Residual inhibition rate = (RatiosiRNA / Ratiocontrol)*100%, taking the average of two replicate wells: Ratiocontrol is the Ratio value of the control well (without siRNA) (taking the average of two replicate wells).

[0755] Graphing: Graphpad Prism

[0756] Half maximal inhibitory concentration (IC50): In this experiment, Top and Bottom plots were used. The IC50 value was calculated using the formula Y = Bottom + (Top - Bottom) / (1 + 10^((LogIC50 - X) * HillSlope)), where Y = 50 and X = log(concentration).

[0757] The results of psiCHECK2GSSM-5Hits off-target activity screening of siRNA are shown in Table 13.

[0758] Table 13 psiCHECK2GSSM-5Hits off-target activity screening results

[0759] Example 9 Activity screening of primary human hepatocytes (PHH cells)

[0760] Cell transfection

[0761] Take 1.4 mL of rat tail collagen solution (Sigma, C3867), add it to 40.6 mL of DNase RNase-Free Distilled Water, mix well, add 40 μL to each well of a 96-well culture plate, coat the plate at 4°C overnight, and remove the coating solution the next day.

[0762] On the second day, before use, the coated cell plate was rinsed with DPBS and then the DPBS was removed. PHH cells (Shanghai Xuanyi, product number QYLF-HPMC) were revived at 37°C, added to the recovery medium, centrifuged, resuspended and counted. PHH cells were plated in a 96-well plate at 90 μL / well, 2×10 4cells / well; complete culture medium was replaced after 4 hours, and transfection was performed after 18 hours.

[0763] On the third day, 20 μM siRNA (DR002222, DR002223, DR002225, DR002226, DR001478, and DR002252) stock solutions were diluted with Opti-MEM. 198 μL Opti-MEM was added to 2 μL siRNA stock solution and pipetted to mix. This was used as the first concentration point. Gradual dilutions were then performed according to experimental needs.

[0764] On the third day, dilute 0.9 μL of RNAiMAX (Thermo, 13778150) with 14.1 μL of Opti-MEM. Gently pipette to mix thoroughly, and let stand at room temperature for 5 minutes. Then, take 15 μL of the prepared RNAi-MAX mixture and 15 μL of the diluted siRNA compound and gently pipette to mix thoroughly, avoiding the introduction of bubbles. Let stand at room temperature for 10 minutes. Add 10 μL / well to a 96-well plate. Incubate in a 37°C, 5% CO2 incubator for 24 hours before RNA extraction.

[0765] RNA extraction

[0766] Cell RNA was extracted using a nucleic acid extractor (Hangzhou Aosheng, Auto-pure96) according to the protocol of the high-throughput cell RNA extraction kit (Fanzhi Medical, FG0412).

[0767] RNA reverse transcription

[0768] For preparation of denaturation reaction mixture, refer to PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B). Each well contains 1 μL of Oligo dT Primer, 1 μL of dNTP Mixture, and 12.5 μL of template RNA. Incubate at 65°C for 5 minutes in a conventional PCR instrument to perform the denaturation reaction. Rapidly cool the mixture on ice for 2 minutes.

[0769] Preparation of reverse transcription reaction solution, refer to PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B). Each well contains 4 μL of 5× PrimeScript II Buffer, 0.5 μL of RNase Inhibitor, and 1 μL of PrimeScript II RTase.

[0770] 14.5 μL of the denatured reaction solution was slowly mixed with the reverse transcription reaction solution, incubated at 42° C. for 45 min in a conventional PCR instrument for reverse transcription, incubated at 95° C. for 5 min to inactivate the enzyme, and the reverse transcription product (cDNA) was cooled at 4° C.

[0771] After reverse transcription, add 30 μL of DNase RNase-Free Distilled Water to the cDNA sample in each well.

[0772] Fluorescence quantitative PCR

[0773] ReferenceTaqMan TM Fast Advanced Master Mix (ABI, 4444965) was used to perform fluorescent quantitative PCR reactions (ABI, QuantStudio3) in 20 μL of the system. The reaction program was: (50°C, 2 min) × 1 cycle; (95°C, 20 s) × 1 cycle; (95°C, 1 s; 60°C, 24 s) × 40 cycles.

[0774] Table 14 Primer information

[0775] Statistics

[0776] Calculation 2 -△△Ct The values ​​were converted into percentages to obtain the residual inhibition rate.

[0777] △△Ct=[(target gene of Ct experimental group-internal reference of Ct experimental group)-(target gene of Ct control group-internal reference of Ct control group)].

[0778] The starting concentration of siRNA was 10 nM, and 10-fold gradient dilution was used to obtain 5 concentration points (10 nM, 1 nM, 0.1 nM, 0.01 nM, 0.001 nM). The human primary liver cell activity screening of siRNA was performed. The screening results are shown in Table 15.

[0779] Table 15 Results of screening human primary hepatocytes using 5 concentrations of siRNA

[0780] Example 10 Activity screening of mouse HDI model

[0781] HDI animal model

[0782] Six to eight-week-old female Balb / c mice were transfected in vivo using a dual-gene stable transfection system using a tail vein high-pressure injection method. Piggy-Bac transposon plasmids (purchased from Suzhou Bangye) containing target gene cDNA sequences at different mass ratios and Piggy-Bac helper plasmids (purchased from Suzhou Bangye) (mass ratio of 1:1, total plasmid amount 50ug) were injected into the mouse through a 27-gauge needle within 5-7 seconds. Delivery Solution (total volume 10% of animal body weight, Mirusbio-MIR 5240) was injected into mice and placed back in their cages for observation for 30 minutes. Sera were obtained at various time points after modeling (Day 7-Day 35) to measure SEAP expression levels.

[0783] The dual-gene stable transfection system includes a Piggy-Bac helper plasmid and a Piggy-Bac transposon plasmid. The Piggy-Bac helper plasmid provides the Piggy-Bac transposase. The Piggy-Bac transposon plasmid is based on the Piggy-Bac transposon and contains a dual-gene expression element, which contains the secreted alkaline phosphatase gene (SEAP) and the target gene (APOC3).

[0784] Detection of SEAP expression

[0785] Kit (Phospha-Light TM SEAP reporter gene assay system, Invitrogen, T1016) standard was diluted 2-fold with an initial concentration of 15 mU / mL to obtain 7 concentration points.

[0786] Mix the CSPD substrate with Reaction Buffer Diluent at a ratio of 1:20 to form a reaction solution. Dilute 5× Dilution Buffer to 1× Dilution Buffer with DNase- and RNase-Free Distilled Water. Mix serum with 1× Dilution Buffer in a centrifuge tube to form a sample diluent. Incubate the sample diluent at 65°C for 30 minutes and then cool to room temperature. Add 50 μL of the sample diluent to a 96-well plate, then add 50 μL of Assay Buffer to each well and incubate at room temperature for 5 minutes. Add 50 μL of the reaction solution to each well and incubate at room temperature for 20 minutes. Read the SEAP chemiluminescence value on a microplate reader (Tecan, Infinite 200).

[0787] The preventive and / or therapeutic effects of the test sample are evaluated by measuring the SEAP expression level in serum. The test sample that can inhibit the SEAP expression level is selected as a nucleic acid drug.

[0788] On day 15 after modeling, each mouse received a single subcutaneous administration of 200 μl of saline containing 3 mg / kg (mpk) of an APOC3 RNAi agent (DR002222 to DR002226, DR001478, DR002252) according to Table 16; or 200 μl of saline without an APOC3 RNAi agent as a control. The results of the HDI model screening are shown in Tables 17-1 and 17-2.

[0789] Table 16 Single subcutaneous administration of RNAi reagent dosage in mice

[0790] Table 17-1 Experimental results of HDI stable plasmid mouse model

[0791] Table 17-2 Experimental results of HDI stable plasmid mouse model

[0792] Example 11 Activity screening of primary monkey hepatocytes (PCH cells)

[0793] Cell transfection

[0794] Add 100 μL of Coating Medium to each well of a 96-well cell culture plate and coat at 37°C for 30 minutes. After coating, aspirate the Coating Medium.

[0795] Monkey primary hepatocytes (PCH, from Miaoshun (Shanghai) Biotechnology Co., Ltd.) were resuspended in Thawing Medium at 37°C, centrifuged and counted, and plated in 96-well plates at 90 μL / well. 2 × 10 4 cells / well.

[0796] siRNA compound dilution: Dilute 20 μM siRNA compound stock solution with Opti-MEM. Take 198 μL Opti-MEM and add 2 μL compound stock solution. Mix by pipetting. This is used as the first concentration point. Perform corresponding dilution operations according to the actual experimental needs.

[0797] Transfection procedure: Dilute 0.9 μL RNAiMAX (Thermo, 13778150) in 14.1 μL Opti-MEM, gently pipette to mix, and let stand at room temperature for 5 minutes. Then, take 15 μL of the prepared RNAi-MAX mixture and 15 μL of the diluted compound and gently pipette to mix, avoiding the introduction of bubbles. Let stand at room temperature for 10 minutes. Add 10 μL / well to a 96-well plate. Incubate in a 37°C, 5% CO2 incubator for 4 hours, then replace with Culture Medium. RNA will be extracted the next day.

[0798] RNA extraction

[0799] Cell RNA was extracted using a nucleic acid extractor (Hangzhou Aosheng, Auto-pure96) according to the protocol of the high-throughput cell RNA extraction kit (Fanzhi Medical, FG0412).

[0800] RNA reverse transcription

[0801] For preparation of denaturation reaction mixture, refer to PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B) single well preparation volume: Oligo dT Primer 1μL, dNTP Mixture 1μL, template RNA 12.5μL, incubate at 65℃ for 5 minutes in a conventional PCR instrument, then quickly cool on ice for 2 minutes. For reverse transcription reaction solution preparation, refer to PrimeScript TM The single-well preparation volume of II 1st Strand cDNA Synthesis Kit (Takara, 6210B) is as follows: 4 μL of 5× Prime Script II Buffer, 0.5 μL of RNase Inhibitor, 1 μL of PrimeScript II RTase, and 14.5 μL of the denaturation reaction solution from the previous step. Slowly mix and incubate in a conventional PCR instrument at 42°C for 45 min for reverse transcription. Incubate at 95°C for 5 min to inactivate the enzyme. Cool the reverse transcription product (cDNA) at 4°C.

[0802] After the inversion is completed, add 30 μL of DNase RNase-Free Distilled Water to each well of the cDNA sample.

[0803] Fluorescence quantitative PCR

[0804] ReferenceTaqMan TMFluorescence quantitative PCR reaction (ABI, QuantStudio3) was performed using 20 μL of Fast Advanced Master Mix (ABI, 4444965). The reaction procedure was: (50°C, 2 min) × 1 cycle; (95°C, 20 s) × 1 cycle; (95°C, 1 s; 60°C, 24 s) × 40 cycles.

[0805] Table 18 Primer information

[0806] Statistics

[0807] Calculation 2 -△△Ct The values ​​were converted into percentages to obtain the residual inhibition rate.

[0808] △△Ct=[(target gene of Ct experimental group-internal reference of Ct experimental group)-(target gene of Ct control group-internal reference of Ct control group)].

[0809] The starting concentration of the siRNA compound was selected as 10 nM, and 11 concentration points of 3-fold gradient dilution (10 nM, 3.33 nM, 1.11 nM, 0.37 nM, 0.123 nM, 0.041 nM, 0.0136 nM, 0.0045 nM, 0.00152 nM, 0.000508 nM, 0.000169 nM) were used to screen the cell activity of the siRNA compound. The experimental screening results are shown in Table 19.

[0810] Table 19 Results of 11-point IC50 screening experiment on PCH cell activity

[0811] Example 12 Activity screening of mouse HDI model

[0812] According to the experimental method of Example 10, this experiment selected six to eight-week-old female Balb / c mice for HDI model construction and drug administration testing. The mass ratio of the Piggy-Bac helper plasmid and the Piggy-Bac transposon recombinant plasmid was selected at 1:1, and the total plasmid amount was 50ug for model construction. On the 14th day after modeling, serum was collected and blindly grouped (N=6). According to Table 18, each mouse was given a single subcutaneous administration of: 200μl of saline containing 3mg / kg (mpk) Apoc3 RNAi reagent; or 200μl of saline without Apoc3 RNAi reagent as a control. The results of the HDI model experimental screening are shown in Table 20.

[0813] Table 20 Experimental results of RNAi reagents in HDI stable plasmid mouse model

[0814] ND: Not Detected

[0815] Example 13: Verification of drug efficacy in hAPOC3 transgenic mouse model

[0816] hAPOC3 transgenic mice (B6-hAPOC3-Tg, T055510, male, 6-8 weeks, GemPharmatech) were tested for triglyceride (TG), low-density lipoprotein cholesterol (LDL-c), high-density lipoprotein cholesterol (HDL-c), and total cholesterol (TC). Mice were randomly divided into five groups based on TG levels, with five animals in each group. The dose for each animal was calculated based on body weight, and the siRNA conjugate was administered as a single subcutaneous injection in a 3 mg / mL solution (0.9% sodium chloride aqueous solution as the solvent). Prior to the experiment, the siRNA conjugate was dissolved in 0.9% sodium chloride aqueous solution and diluted to the desired solution concentration and volume. The administration volume of physiological saline and siRNA conjugate was 5 mL / kg.

[0817] Blood was collected before administration (recorded as Day -3) and the mice were grouped according to the TG level on Day -3. Blood was collected from the orbital venous plexus of the mice on days 7, 14, 28, 42, 56, and 70 after administration (recorded as Day 0) (the mice were starved for 5 hours before each blood collection). Serum was collected by centrifugation to test TG, LDL-c, TC, and the target protein was detected by ELISA (Human APOC3 ELISA Kit, Abcam, ab154131). The experimental results are shown in Tables 21-24.

[0818] Table 21: Pharmacological efficacy test results in hAPOC3 transgenic mouse model - TG inhibition rate

[0819] Table 22: Efficacy test results in hAPOC3 transgenic mouse model - TC inhibition rate

[0820] Table 23: Pharmacological efficacy results in hAPOC3 transgenic mouse model - LDL-c inhibition rate

[0821] Table 24: Efficacy test results in hAPOC3 transgenic mouse model - Apoc3 protein inhibition rate

[0822] Example 14 psiCHECK2GSSM-5Hits Off-Target Activity Screening

[0823] With reference to Example 8, the HEK293A cell line was selected in this experiment, and the starting concentration of the siRNA compound was 40 nM. A 3-fold gradient dilution with 11 concentration points (40 nM, 13.3 nM, 4.44 nM, 1.48 nM, 0.493 nM, 0.164 nM, 0.0548 nM, 0.0182 nM, 0.00609 nM, 0.00203 nM, 0.000677 nM) was used to screen the off-target activity of the siRNA compound psiCHECK2GSSM-5Hits. The experimental screening results are shown in Table 25.

[0824] Table 25 psiCHECK2GSSM-5Hits off-target activity screening experimental results

[0825] Example 15 Preparation of Compound E2

[0826] 1. Preparation of Compound 2b

[0827] Compound 2a (38.0 g, 231 mmol), TsNHBoc (75.4 g, 278 mmol), K2CO3 (6.40 g, 46.3 mmol), and TEBA (5.27 g, 23.1 mmol) were added to a three-necked flask at room temperature. The reaction was allowed to proceed at 95°C for 2 hours, then cooled to 25°C. TLC (PE / EA = 2 / 1, UV 254 nm) indicated the reaction was complete. The reaction solution was diluted with water (500 mL) and extracted with dichloromethane (200 mL x 3). The organic phase was concentrated under reduced pressure, and the crude product was purified by column chromatography (PE / EA = 10 / 1 to 3 / 1) to afford the title compound 2b (40.0 g).

[0828] 1 H NMR (400MHz, CDCl3) δ7.72 (d, J = 8.4Hz, 2H), 7.28-7.39 (m, 7H), 4.72-4.86 (m, 2H) ,4.42-4.56(m,2H),3.52-3.61m,2H),3.14-3.39(m,2H),2.43(s,3H),1.47(s,9H)

[0829] 2. Preparation of Compound 2d

[0830] Compound 2b (79.0 g, 181 mmol), compound 2c (24.7 g, 150.8 mmol), potassium carbonate (4.21 g, 30.5 mmol), and TEBA (3.47 g, 15.2 mmol) were reacted at 95°C for 3 hours. TLC (PE / EA = 2 / 1, UV 254 nm) indicated the reaction was complete. The reaction solution was diluted with water (500 mL) and extracted with dichloromethane (200 mL x 3). The organic phase was concentrated under reduced pressure, and the crude product was purified by column chromatography (PE / EA = 10 / 1 to 3 / 1) to afford compound 2d (40.0 g, 43.8% yield).

[0831] 1H NMR (400MHz, CDCl3) δ7.64-7.77(m,2H),7.27-7.38(m,13H),5.05-5.16(m,1H),4.50-4.59(m,4H),4.03-4.11 (m,1H),3.62-3.76(m,2H),3.44-3.58(m,3H),3.25-3.33(m,2H),3.15-3.20(m,1H),2.42(s,3H),1.46(s,9H)

[0832] 3. Preparation of Compound 2e

[0833] Compound 2d (77.0 g, 128 mmol) and triethylamine (28.6 mL, 205 mmol) were added sequentially to dichloromethane (800 mL) in an ice-water bath, followed by the slow dropwise addition of methylsulfonyl chloride (24.2 g, 212 mmol). The reaction was allowed to proceed at 25°C for 2 hours, with LCMS indicating completion. The reaction mixture was washed with water (800 mL), and the organic phase was concentrated under reduced pressure to afford crude compound 2e (90.0 g).

[0834] m / z: ES+[M+Na]+700.1

[0835] 1 H NMR (400MHz, CDCl3) δ7.68 (d, J = 8.4Hz, 2H), 7.28-7.39 (m, 12H), 5.10-5.20 (m, 1H), 4.93-5.02 (m, 1H), 4.52-4.61 (m, 4 H),3.72-3.88(m,2H),3.52-3.67(m,4H),3.27-3.36(m,1H),3.15-3.22(m,1H),3.04(s,3H),2.43(s,3H),1.43(s,9H).

[0836] 4. Preparation of Compound 2f

[0837] Compound 2e (90.0 g, 133 mmol) and potassium carbonate (91.8 g, 664 mmol) were added to methanol (900 mL) at room temperature. The reaction was allowed to proceed at 66°C for 2 hours. TLC (PE / EA = 2 / 1, UV 254 nm) showed the formation of new spots. The organic phase was concentrated under reduced pressure, the reaction mixture was diluted with water (200 mL), and extracted with dichloromethane (200 mL x 3). The organic phase was concentrated under reduced pressure, and the crude product was purified by column chromatography (PE / EA = 30 / 1-2 / 1) to afford compound 2f (64.0 g, 99.9% yield).

[0838] 1 H NMR (400MHz, CDCl3) δ7.62 (d, J = 8.4Hz, 2H), 7.27-7.39 (m, 12H), 4.48-4.62 ( m,4H),3.93-4.08(m,2H),3.55-3.69(m,4H),2.84-3.10(m,4H),2.44(s,3H)

[0839] 5. Preparation of Compound 2g

[0840] Compound 2f (69.0 g, 143 mmol) and magnesium turnings (54.7 g, 2.28 mol) were added to methanol (400 mL) at room temperature and reacted at 66°C for 1 hour. TLC (DCM / MeOH = 10 / 1, UV 254 nm) showed complete reaction of the starting material with the formation of new spots. The reaction mixture was diluted with water (3000 mL) and saturated aqueous ammonium chloride (3000 mL), extracted with dichloromethane (1000 mL x 3), and the organic phase was washed with saturated sodium bicarbonate (300 mL x 3). The organic phase was concentrated under reduced pressure to yield crude compound 2g (32.0 g).

[0841] 1 H NMR (400MHz, CDCl3) δ7.27-7.40(m,10H),4.57(s,4H),3.90-4.00(m,2H),3.59-3.69(m,4H),2.77-3.04(m,4H)

[0842] 6. Preparation of Compound 2h

[0843] Compound 2g (3.00 g, 9.16 mmol), compound 1b (1.90 mL, 18.3 mmol), and acetic acid (1.01 mL, 18.3 mmol) were added to methanol (30 mL) at room temperature. The reaction was allowed to proceed at 25°C for 18 hours. Sodium cyanoborohydride (2.30 g, 36.7 mmol) was then added and the mixture was allowed to react at 50°C for 4 hours. TLC (DCM / MeOH = 10 / 1) showed the formation of new spots. The reaction solution was diluted with water (50 mL) and extracted with dichloromethane (30 mL x 3). The organic phase was concentrated under reduced pressure. The crude product was purified by column chromatography (dichloromethane / methanol = 99 / 1 to 5 / 1) to afford compound 2h (3.00 g, 79.9% yield).

[0844] 1 H NMR (400MHz, CDCl3) δ7.28-7.41(m,10H),4.51-4.71(m,4H),4.00-4.35(m,2H ),3.49-3.77(m,4H),2.69-2.98(m,2H),1.63-2.12(m,7H),1.16-1.44(m,6H)

[0845] 7. Preparation of Compound 2i

[0846] Compound 2h (3.00 g, 7.32 mmol) was added to concentrated hydrochloric acid (10 mL, 12 M) at room temperature and reacted at 50°C for 18 h. TLC (DCM / MeOH = 10 / 1) showed the formation of new spots. The reaction solution was concentrated under reduced pressure to give crude compound 2i (2.00 g).

[0847] 1 H NMR (400MHz, CD3OD) δ4.27-4.38(m,1H),4.11-4.20(m,1H),3.96-4.04(m,2H),3.60-3.68(m,3H),3.36-3.43(m,1H),3.19-3.29(m, 2H),3.11(s,1H),2.02-2.17(m,2H),1.90-2.00(m,2H),1.69-1.78(m,1H),1.57-1.69(m,1H),1.35-1.52(m,3H),1.20-1.31(m,1H)

[0848] 8. Preparation of Compound 2j

[0849] Compound 2i (2.00 g, 8.72 mmol) was dissolved in pyridine (40 mL) at room temperature, and DMTrCl (2.96 g, 8.72 mmol) was added. The mixture was allowed to react at 25°C for 18 hours. TLC (PE / EA = 1 / 1, UV 254 nm) indicated the reaction was complete. The reaction solution was concentrated under reduced pressure, and saturated aqueous ammonium chloride (200 mL) was added. The mixture was extracted with DCM (100 mL x 3). The organic phase was concentrated under reduced pressure, and the crude product was purified by column chromatography (dichloromethane / methanol = 99 / 1-10 / 1) to afford compound 2j (1.40 g, 30.2% yield).

[0850] 9. Preparation of Compound E2

[0851] Compound 2j (660 mg, 1.24 mmol), compound 1h (374 mg, 1.24 mmol), and DCI (73.3 mg, 0.621 mmol) were added to DCM (10 mL) and reacted at 25°C for 1 hour. TLC (PE / EA = 5 / 1, PMA) indicated complete reaction. Saturated sodium bicarbonate (50 mL) was added, and the mixture was extracted with DCM (30 mL x 3). The organic phase was concentrated under reduced pressure, and the crude product was separated by column chromatography (PE / EA = 99 / 1-30 / 1) to afford compound E2 (350 mg, 19.3% yield).

[0852] m / z:ES+[M+H]+732.2

[0853] 1 H NMR (400MHz, CDCl3) δ7.40-7.50(m,2H),7.28-7.37(m,6H),7.21(d,J=7. 2Hz,1H),6.77-6.89(m,4H),3.67-4.07(m,11H),3.56-3.66(m,2H),3.04- 3.33(m,2H),2.30-2.96(m,6H),2.10-2.28(m,1H),1.63-1.88(m,4H),1. 47-1.52(m,1H),1.24-1.31(m,3H),1.15-1.23(m,12H),1.11-1.14(m,1H)

[0854] Example 16 Preparation of Compound E1-1

[0855] 1. Preparation of Compound 1b

[0856] Compound 1a (200 g, 1.33 mol, 1.00 eq) was suspended in a mixture of anhydrous acetone (1.00 L) and anhydrous methanol (1.00 L). Concentrated sulfuric acid (20.0 mL, 0.27 eq) was added dropwise and allowed to react at 25°C for 24 hours. The reaction solution was neutralized with saturated sodium bicarbonate and concentrated. The resulting residue was dissolved in ethyl acetate and washed three times with saturated brine (500 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated to yield compound 1b (242 g, 88.9%).

[0857] 1 H NMR:400MHz CDCl3δ4.97(s,1H),4.84(d,J=6.0Hz,1H),4.59(d,J=6.0Hz,1H),4.44-4.43(m,1H),3.72-3.59(m,2H),3.44(s,3H),1.49(s,3H),1.32(s,3H).

[0858] 2. Preparation of Compound 1c

[0859] Compound 1b (310 g, 1.52 mol, 1.00 eq), imidazole (206 g, 3.02 mol, 2.00 eq), and triphenylphosphine (476 g, 1.82 mol, 1.20 eq) were dissolved in toluene (2.1 L). Elemental iodine (446 g, 1.76 mmol, 1.16 eq) was added portionwise at room temperature. After addition, the temperature was raised to 70°C and stirred for 1.5 hours. TLC indicated the reaction was complete. Methanol (60 mL) was added to the reaction solution to quench it, and the temperature was then lowered. A saturated aqueous sodium thiosulfate solution (2.1 L) was added. The organic phase was separated, collected, washed twice with saturated brine (1.5 L), dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was filtered through a slurry of methyl tert-butyl ether. The filtrate was concentrated, and the resulting crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate) to yield compound 1c (401 g, 1.27 mol, 91.1%).

[0860] 1 H NMR:400MHz CDCl3δ4.98(s,1H),4.69(d,J=6.0Hz,1H),4.56(d,J=6.0Hz,1H),4.39-4.35(m,1H) ,3.30(s,3H),3.24-3.20(m,1H),3.09(t,J=10.0Hz,1H),1.41(s,3H),1.26(s,3H).

[0861] 3. Preparation of Compound 1d

[0862] Dissolve compound 1c (203 g, 646 mmol, 1.00 eq) in tetrahydrofuran (2.03 L), and add potassium tert-butoxide (145 g, 1.29 mol, 1.29 eq) in portions at 0°C. Stir at 25°C for 16 hours. Cool to 5°C and add ice water.

[0863] The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate) to give compound 1d (160 g, 91.3%).

[0864] 1 H NMR:400MHz CDCl3δ5.11(s,1H),5.02(d,J=5.6Hz,1H),4.06(d,J=1.6Hz,1H),4.50(d,J=5.6Hz,1H),4.39(d, J=1.6Hz,1H),3.41(s,3H),3.24-3.20(m,1H),3.09(t,J=10.0Hz,1H),1.47(s,3H),1.35(s,3H).

[0865] 4. Preparation of Compound 1e

[0866] A zinc copper reagent (90.0 g, 1.37 mol, 5.50 eq) was dispersed in diethyl ether (200 mL). Compound 1d (60 g, 241 mmol, 1.00 eq) was added at 25°C. A solution of trichloroacetyl chloride (61.5 g, 338 mmol, 1.40 eq) in diethyl ether (200 mL) was added dropwise, and stirring was continued for 1 hour. The mixture was filtered, and the filter cake was rinsed with methyl tert-butyl ether (500 mL). The filtrate was poured into saturated aqueous sodium bicarbonate solution (2.50 L), filtered, and washed three times with saturated brine (500 mL). The mixture was dried over anhydrous sodium sulfate, filtered, and concentrated to give crude compound 1e (71.8 g).

[0867] 1 H NMR: 400MHz CDCl3δ5.12(d,J=5.6Hz,1H),5.09(s,1H),4.70(d,J=6.0Hz,1H),3.70-3.55(m,2H),3.54(s,3H),1.45(s,3H),1.36(s,3H).

[0868] 5. Preparation of Compound 1f

[0869] Compound 1e (71.8 g, 242 mmol, 1.00 eq) was dissolved in tetrahydrofuran (1.60 L), and glacial acetic acid (69.1 mL, 1.20 mol, 5.00 eq) was added. Zinc powder (142 g, 2.17 mol, 9.00 eq) was then added portionwise. The mixture was stirred at 25°C for 18 hours. After completion of the reaction, the mixture was filtered and the filtrate was concentrated to obtain a crude product. The crude product was dissolved in methyl tert-butyl ether and poured into a saturated aqueous sodium bicarbonate solution (2.0 L). The product was filtered and the filtrate was washed twice with saturated brine (500 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate / dichloromethane) to obtain compound 1f (31 g, 56.3% yield over two steps).

[0870] 1 H NMR:400MHz CDCl3δ4.97(s,1H),4.70-4.67(m,2H),3.54-3.48(m,2H),3.37-3.31(m,4H),3.16-3.10(m,1H),3.09-3.03(m,1H),1.43(s,3H),1.34(s,3H).

[0871] 6. Preparation of Compound 1g

[0872] Compound 1f (50 g, 219 mmol, 1.00 eq) and diethyl phosphite (33.2 g, 240 mmol, 1.20 eq) were dissolved in dichloromethane (100 mL). 1,8-diazabicycloundec-7-ene (DBU, 6.67 g, 43.8 mmol, 0.20 eq) was added at 0°C and stirred at 25°C for 18 hours. The reaction solution was washed three times with saturated NH4Cl (100 mL) and once with saturated brine (100 mL), dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / dichloromethane) to obtain compound 1g (70 g, 87.2%).

[0873] 1 H NMR:400MHz CDCl3δ5.27(s,1H),5.01(brs,1H),4.78(s,1H),4.65(d,J=5.6Hz,1H),4.49(d,J=6.0Hz,1H),4.20-4.05(m,5H ),3.31(s,1H),3.29-3.05(m,1H),2.75-2.68(m,1H),2.50-2.40(m,1H),2.35-2.25(m,1H),1.36-1.25(m,12H).

[0874] 7. Preparation of Compound 1h

[0875] Compound 1g (80.2 g, 219 mmol, 1.00 eq) and DMAP (40.1 g, 328 mmol, 1.50 eq) were dissolved in acetonitrile (562 mL), and monomethyl oxalyl chloride (40.2 g, 328 mmol, 1.50 eq) was added at 5°C. The mixture was stirred at 25°C for half an hour. The reaction solution was concentrated, and the crude product was dissolved in ethyl acetate and washed five times with saturated ammonium chloride (300 mL), once with water (200 mL), and once with brine (200 mL). The mixture was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain crude compound 1h (99 g).

[0876] 1 H NMR:400MHz CDCl3δ4.88-4.80(m,2H),4.67-4.53(m,2H),4.24-4.12(m,4H),3.89(s,3H),3.51-3.43(m,1H), 3.35-3.33(m,3H),3.15-3.07(m,1H),2.92-2.80(m,1H),2.69-2.61(m,1H),1.39-1.29(m,12H).

[0877] 8. Preparation of Compound 1i

[0878] Compound 1h (99 g, 218 mmol, 1.00 eq) was dissolved in anhydrous toluene (1.00 L). Tri-n-butyltin hydride (76.4 g, 262 mmol, 1.20 eq) and azobisisobutyronitrile (AIBN, 1.08 g, 6.56 mmol, 0.03 eq) were added. Refluxed for 2 hours. The reaction mixture was concentrated, and the crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate) to obtain crude compound 1i (146 g).

[0879] 1 H NMR: 400MHz CDCl3δ4.75-4.49(m,3H),4.05-3.95(m,4H),3.26-3.24(m,3H),2.76-2.07(m,5H),1.39-1.29(m,12H).

[0880] 9. Preparation of Compound 1j

[0881] Compound 1i (141 g, 201 mmol, 1.00 eq) was dissolved in methanol (1.41 L) and aqueous HCl (704 mL, 1.40 mol, 2 M, 7.00 eq) was added. The reaction mixture was stirred at 60°C for 1 hour. The reaction mixture was extracted with a mixture of methyl tert-butyl ether and petroleum ether. The aqueous phase was removed and the pH was adjusted to 8 with saturated sodium bicarbonate. The aqueous phase was concentrated, and tetrahydrofuran was added to the resulting residue, which was filtered. The filtrate was concentrated to afford crude compound 1j (62.4 g, 201 mmol).

[0882] 10. Preparation of Compound 1k

[0883] Compound 1j (62.4 g, 201 mmol, 1.00 eq) was dissolved in pyridine (300 mL), and acetic anhydride (47.4 mL, 502 mmol, 2.50 eq) was added. The mixture was stirred at 25°C for 12 hours. The reaction solution was diluted with saturated sodium bicarbonate (1.00 L) and extracted twice with ethyl acetate (600 mL). The mixture was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate) to afford compound 1k (74 g, 93.3%).

[0884] 1 H NMR: 400MHz CDCl3δ5.38-5.10(m,3H),4.07-4.03(m,4H),3.36-3.32(m,3H),2.41-2.05(m,5H),2.04-1.98(m,9H),1.27-1.23(m,6H).

[0885] 11. Preparation of Compound 11

[0886] Compound 1k (79.3 g, 201 mmol, 1.00 eq) was dissolved in ethyl acetate (476 mL), and acetic anhydride (62.6 mL, 663 mmol, 3.30 eq) and concentrated sulfuric acid (5.38 mL, 100 mmol, 0.50 eq) were added. The mixture was stirred at 25°C for 3 hours. The reaction solution was neutralized with saturated aqueous sodium bicarbonate solution and extracted twice with ethyl acetate (1.00 L). The organic phases were combined, washed twice with saturated brine (500 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography to yield compound 1l (43 g, 50.6%).

[0887] 1H NMR:400MHz CDC13δ6.11(d,J=2.8Hz,1H),5.44-5.39(m,1H),5.33-5.32(m,1H),4.10-4.05(m,5H),2.80-2.35(m,4H),2.12-2.02(m,9H),1.32-1.23(m,6H).

[0888] 12. Preparation of Compound 1n

[0889] Compound 1m (8.55 g, 76.2 mmol, 2.30 eq) and bis(trimethylsilyl)acetamide (BSA, 34.3 g, 169 mmol, 5.10 eq) were suspended in acetonitrile (200 mL) and stirred at 85°C for 1 hour. After cooling, a solution of compound 11 (14.0 g, 33.1 mmol, 1.00 eq) in acetonitrile (50.0 mL) was added, followed by tin tetrachloride (37.1 g, 142 mmol, 4.30 eq). The mixture was stirred at 25°C for 15 minutes and then stirred in an oil bath at 85°C for 45 minutes. After cooling, the reaction solution was poured into a saturated aqueous sodium bicarbonate solution (1.50 L) and extracted three times with dichloromethane (700 mL). The organic phases were combined, washed with saturated brine (500 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol) to give compound 1n (14.4 g, 84.3%).

[0890] 1 H NMR:400MHz CDCl3δ9.70(brs,0.46H),9.60(brs,0.60H),7.17(d,J=8.0Hz,0.60H),7.12(d,J=8.0Hz,0.49H),5.98(d,J=6.4Hz,0.43H),5.93(d,J=5.2Hz,0.55H), 5.78(dt,J1=8.0Hz,J2=2.0Hz,1H),5.50-5.45(m,2H),4.45-4.02(m,5H),2.77-2.42(m,6 H),2.18(s,1.22H),2.16(s,1.74H),2.04(s,1.31H),2.02(s,1.67H),1.32-1.28(m,6H).

[0891] 13. Preparation of Compound 1o

[0892] Compound 1n (8.30 g, 17.4 mmol, 1.00 eq) was dissolved in DMF (40.0 mL), and DBU (2.93 g, 19.2 mmol, 1.10 eq) was added. After cooling, benzyloxymethyl chloride (BOMCl, 3.01 g, 19.2 mmol, 1.10 eq) was added. The mixture was stirred at 0-5°C for 2.5 hours. The reaction solution was diluted with saturated ammonium chloride (160 mL) and extracted twice with dichloromethane (80.0 mL). The combined organic phases were washed with saturated brine (80 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol) to obtain compound 1o (12.0 g, 92.3%).

[0893] 1 H NMR:400MHz CDCl3δ7.36-7.24(m,5H),7.15-7.09(m,1H),5.96-5.92(m,1H),5.81-5.79(m,1H),5.52-5.42(m,4H),4. 67(s,2H),4.15-4.05(m,4H),2.80-2.45(m,5H),2.19-2.17(m,3H),2.05-2.03(m,3H),1.34-1.30(m,6H).

[0894] 14. Preparation of Compound 1p

[0895] Compound 1o (10.4 g, 17.4 mmol, 1.00 eq) was dissolved in methanolic ammonia (29.9 mL, 7 M) and stirred at 21°C for 1 hour. The reaction solution was concentrated to obtain a crude product, which was purified by silica gel column chromatography (eluent: dichloromethane / methanol) to obtain compound 1p (8.00 g, 62.7%).

[0896] 1 H NMR: 400 MHz CDCl3δ7.34-7.06(m,5H),5.83(s,0.52H),5.71(d,J=8.4Hz,0.41H),5.661(d, J=8.4Hz,0.53H),5.60(d,J=2.0Hz,0.59H),5.53(d,J=5.2Hz,0.41H),5.42-5.3 6(m,2H),4.62(s,2H),4.51(d,J=3.6Hz,0.45H),3.72(d,J=2.8Hz,0.36H),3.5 4(d,J=2.0Hz,0.48H),2.93-2.40(m,5H),2.24-2.13(m,1H),1.27-1.23(m,6H).

[0897] 15. Preparation of Compound 1q

[0898] Compound 1p (1 g, 1.95 mmol, 1.00 eq) was dissolved in acetone (19.6 mL), and silver oxide (3.63 g, 15.6 mmol, 10.0 eq) and iodomethane (1.22 mL, 19.5 mmol, 10.0 eq) were added. The mixture was stirred at 25°C for 24 hours. The reaction mixture was filtered and concentrated to obtain crude compound 1q (1.36 g).

[0899] 1 H NMR:400MHz CDCl3δ5.11(s,1H),5.02(d,J=5.6Hz,1H),4.06(d,J=1.6Hz,1H),4.50(d,J=5.6Hz,1H),4.39(d, J=1.6Hz,1H),3.41(s,3H),3.24-3.20(m,1H),3.09(t,J=10.0Hz,1H),1.47(s,3H),1.35(s,3H).

[0900] 16. Preparation of Compounds 1q-1 and 1q-2

[0901] The crude compound 1q (10.9 g, 20.7 mmol, 1.00 eq) was reacted with C 18 The residue was purified by reverse phase column chromatography (aqueous ammonium bicarbonate / acetonitrile) to obtain compound 1q-1 (1.50 g, 11.5%) and compound 1q-2 (1.50 g, 11.5%).

[0902] Compound 1q-1:

[0903] m / z:ES+[M+H]+525.2

[0904] HPLC: Retention time 0.820min (Column: 95%B, 1.16-1.5min, 95%-5%B; Flow rate: 1.5mL / min; Column temp.: 40℃)

[0905] 1H NMR:400MHz CDC13δ7.37-7.08(m,5H),7.09(d,J=8.0Hz,1H),5.76-5.74(m,2H),5.49-5.44(m,2H),4.73-4.67(m,2H),4.15-4.06(m,4H),4. 02(d,J=4.8Hz,1H),3.87-3.85(m,1H),3.56(s,3H),2.81-2.64(m,2H),2.50-2.39(m,2H),2.36-2.27(m,1H),1.34-1.30(m,6H).

[0906] Compound 1q-2:

[0907] m / z:ES+[M+H]+525.2

[0908] HPLC: retention time 0.836min (Column: 95%B, 1.16-1.5min, 95%-5%B; Flow rate: 1.5mL / min; Column temp.: 40℃)

[0909] 1 H NMR:400MHz CDC13δ7.37-7.27(m,5H),7.08(d,J=8.0Hz,1H),5.75-5.73(m,2H),5.50-5.45(m,2H),4.73-4.67(m,2H),4.18-4.08( m,4H),4.06(d,J=4.8Hz,1H),3.88-3.86(m,1H),3.56(s,3H),3.06-2.95(m,1H),2.83-2.41(m,5H),1.34-1.31(m,6H).

[0910] 17. Preparation of Compound 1r-1

[0911] Compound 1q-1 (1.00 g, 1.90 mmol, 1.00 eq) was dissolved in anhydrous dichloromethane (20.0

[0912] mL) and cooled to -60°C. Boron trichloride (7.62 mL, 7.62 mmol, 1 M dichloromethane solution, 4.00 eq) was added and stirred at -20°C for 1 hour. The reaction solution was quenched with anhydrous ethanol (10.0 mL), neutralized with concentrated ammonia to a pH of about 7, and concentrated. The crude product was added to a mixed solution of dichloromethane and ethanol and filtered. The filtrate was concentrated and the resulting residue was purified by C 18 The residue was purified by reverse phase column chromatography (aqueous ammonium bicarbonate solution / acetonitrile) to obtain compound 1r-1 (400 mg, 51.9%).

[0913] m / z:ES+[M+H]+405.3

[0914] 1 H NMR:400MHz CDCl3δ8.51-8.43(m,1H),7.15(d,J=8.4Hz,1H),5.78(d,J=3.2Hz,1H),5.76(dd,J1=8.0Hz,J2=2.4Hz,1H),4.16-4.07(m,5H),3.94(dd , J1=4.8Hz, J2=3.2Hz,1H),3.55(s,3H),2.96-2.94(m,1H),2.79-2.66(m,2H),2.52-2.41(m,2H),2.35-2.26(m,1H),1.34-1.31(m,6H).

[0915] 18. Preparation of Compound E1-1

[0916] Compound 1r-1 (500 mg, 1.23 mmol, 1.00 eq) was dissolved in dichloromethane (5.00 mL), and compound 1s (0.59 mL, 1.85 mmol, 1.50 eq) was added. The temperature was cooled to 0°C, 4,5-dicyanoimidazole (160 mg, 1.36 mmol, 1.10 eq) was added, and the mixture was stirred at 15°C for 4 hours. The reaction solution was quenched with saturated sodium bicarbonate (10.0 mL) and extracted twice with dichloromethane (10.0 mL). The crude product was concentrated to give a crude product, which was purified by silica gel column chromatography (eluent: dichloromethane / acetone) to give compound E1-1 (530 mg, 70.9%).

[0917] 1H NMR:400MHz CDCl3δ9.17(brs,1H),7.24(d,J=8.4Hz,1H),5.83-5.79(m,1H),5.64-5.61(m,1H),4.43-4.33(m,1 H),4.09-3.62(m,9H),3.43-3.38(m,3H),2.86-2.66(m,3H),2.57-2.21(m,4H),1.28-1.18(m,18H).

[0918] Example 17 Preparation of Compound E1

[0919] 1. Preparation of Compound 2

[0920] Compound 1 (50.0 g, 263 mmol) was dissolved in DCM (800 mL) at 25°C, followed by the addition of imidazole (26.9 g, 394 mmol) and TBDPSCl (75.2 mL, 289 mmol). The reaction was stirred at 25°C for 18 hours. Thin-layer chromatography (DCM / MeOH = 10 / 1, PE / EA = 3 / 1) showed complete consumption of the reactants and the formation of new spots. The reaction solution was dried to give the crude product, which was then purified by MPLC (PE / EA = 1 / 0-5 / 1) to afford compound 2 (95.0 g, 84.31% yield) as a colorless oil.

[0921] 1 H NMR (400MHz, CDCl3) δ7.70(dd,J=7.6,1.6Hz,4H),7.35-7.46(m,6H),5.86(d,J=3.6Hz,1H),4.61(dd,J=4.8,4.0H z,1H),4.15(td,J=8.8,5.2Hz,1H),3.93-4.01(m,1H),3.81-3.92(m,2H),1.60(s,3H),1.39(s,3H),1.06(s,9H).

[0922] 2. Preparation of Compound 3

[0923] Compound 2 (95.0 g, 222 mmol) was dissolved in toluene (1.50 L) at 25°C. Imidazole (30.2 g, 443 mmol), triphenylphosphine (116 g, 443 mmol), and iodine (84.4 g, 322 mmol) were added sequentially. The reaction mixture was stirred at 100°C for 18 hours. Thin-layer chromatography (PE / EA = 5 / 1) showed complete consumption of the reactants and the formation of new spots. 20.0 mL of saturated NaHSO₃ solution and 500 mL of water were added to the reaction mixture. The layers were separated, and the organic phase was washed with saturated NaCl solution (50.0 mL x 3), dried over anhydrous Na₂SO₄, and spin-dried to obtain the crude product. The crude product was purified by MPLC (PE / EA = 1 / 0-10 / 1) to afford compound 3 (115 g, 96.35% yield) as a colorless oil.

[0924] 1 H NMR (400MHz, CD3OD) δ7.64-7.72(m,4H),7.37-7.49(m,6H),5.95(d,J=3.6Hz,1H),5.06(d,J=3.6Hz,1H),4.42(d,J=3.2Hz,1H),3. 87(dd,J=10.4,5.6Hz,1H),3.66(dd,J=10.4,6.4Hz,1H),3.53(td,J=6.0,3.2Hz,1H),1.44(s,3H),1.29(s,3H),1.02-1.07(m,9H).

[0925] 3. Preparation of Compound 4

[0926] Compound 3 (12.5 g, 23.2 mmol) was dissolved in a mixture of MeOH (225 mL) and EtOAc (25 mL) at 25°C. TEA (6.45 mL, 46.4 mmol) and 10% Pd / C (2.00 g, 18.8 mmol) were added. The reaction mixture was stirred at 25°C under a hydrogen atmosphere (14.696 psi) for 14 hours. Thin-layer chromatography (PE / EA = 10 / 1) showed complete consumption of the reactants and the formation of new spots. The reaction mixture was filtered, and the filtrate was dried to give the crude product, which was then purified by MPLC (PE / EA = 1 / 0 to 10 / 1) to afford compound 4 (8.75 g, 83.06% yield) as a colorless oil.

[0927] 1H NMR (400MHz, CD3OD) δ7.64-7.72(m,4H),7.36-7.47(m,6H),5.80(d,J=3.6Hz,1H),4.77(t,J=4.4Hz,1H),4.25-4.32 (m,1H),3.68-3.82(m,2H),2.00(dd,J=13.6,4.8Hz,1H),1.78-1.87(m,1H),1.45(s,3H),1.30(s,3H),1.04(s,9H).

[0928] 4. Preparation of Compound 5

[0929] Compound 4 (35.0 g, 84.8 mmol) was dissolved in THF (500 mL) at 25°C, and tetraethylammonium fluoride (63.3 g, 424 mmol) was added. The reaction was stirred at 25°C for 18 hours. Thin-layer chromatography (PE / EA = 10 / 1, DCM / MeOH = 10 / 1) showed complete consumption of the reactants and the formation of new spots. The reaction mixture was concentrated in vacuo to afford the crude product, which was then purified by MPLC (DCM / MeOH = 1 / 0 to 10 / 1) to afford Compound 5 (12 g, 81.21% yield) as a white solid.

[0930] 1 H NMR (400MHz, CD3OD) δ5.79(d,J=3.6Hz,1H),4.77(t,J=4.0Hz,1H),4.21-4.28(m,1H),3.70(dd,J=12.0,3.6Hz,1H ),3.54(dd,J=12.0,4.8Hz,1H),1.95-2.02(m,1H),1.74(ddd,J=13.2,10.8,4.8Hz,1H),1.46(s,3H),1.30(s,3H).

[0931] 5. Preparation of Compound 6

[0932] Compound 5 (12.0 g, 68.9 mmol) was dissolved in toluene (500 mL) at 25°C. Imidazole (9.38 g, 138 mmol), triphenylphosphine (36.1 g, 138 mmol), and iodine (26.2 g, 103 mmol) were added sequentially. The reaction mixture was stirred at 100°C for 3 hours. Thin-layer chromatography (PE / EA = 5 / 1) showed complete consumption of the reactants and the formation of new spots. 100 mL of saturated NaHSO₃ solution and 100 mL of water were added to the reaction mixture. The layers were separated, and the organic phase was washed with saturated NaCl solution (100 mL x 3), dried over anhydrous Na₂SO₄, and spin-dried to obtain the crude product. The crude product was purified by MPLC (PE / EA = 1 / 0-10 / 1) to afford Compound 6 (16.6 g, 84.82% yield) as a white solid.

[0933] 1 H NMR(400MHz, CDCl3)δ5.88(d,J=3.6Hz,1H),4.77(t,J=4.2Hz,1H),4.13-4.21(m,1H),3.2 5-3.38(m,2H),2.31(dd,J=13.6,4.4Hz,1H),1.61-1.67(m,1H),1.52(s,3H),1.33(s,3H).

[0934] 6. Preparation of Compound 7

[0935] Compound 6 (40.0 g, 141 mmol) was dissolved in trimethyl phosphite (500 mL) at 25°C, and the reaction mixture was stirred at 120°C for 11 hours. Thin-layer chromatography (ethyl acetate / acetone = 3 / 1, PE / EA = 10 / 1) showed residual starting material and the formation of new spots. The reaction mixture was spin-dried to obtain a crude product, which was purified by MPLC (ethyl acetate / acetone = 1 / 0-20 / 1) to afford compound 7 (8.90 g, 23.74% yield) as a pale yellow oil and recovered as a white solid (30.0 g).

[0936] 1 H NMR(400MHz, CDCl3) δ5.81(d,J=4.0Hz,1H),4.74(t,J=4.4Hz,1H),4.40-4.52(m,1H),3.76(dd,J=10.8,0.8H z,6H),2.23-2.35(m,2H),1.95-2.07(m,1H),1.63(ddd,J=13.6,10.8,4.8Hz,1H),1.52(s,3H),1.32(s,3H).

[0937] 7. Preparation of Compound 8

[0938] Compound 7 (13 g, 48.830 mmol), Ac2O (23.036 mL, 244.150 mmol), and H2SO4 (2.615 mL, 48.830 mmol) were added sequentially to AcOH (260 mL). The reaction was allowed to proceed at 25°C for 6 hours. A new spot was detected by TLC (ethyl acetate:acetone = 3:1). The reaction solution was quenched with ice water, extracted with DCM (500 mL x 3), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (ethyl acetate:acetone = 50:1-3:1) to obtain compound 8 (7.9 g, 25.464 mmol, 52.15%) as a yellow oil.

[0939] 1 H NMR (400MHz, CDCl3) δ6.10(d,J=1.2Hz,1H),5.18(d,J=5.2Hz,1H),4.58-4.72(m,1H),3.75-3 .79(m,3H),3.71-3.74(m,3H)2.16-2.38(m,3H),2.07(s,3H),2.05(s,3H),1.96-2.04(m,1H).

[0940] 8. Preparation of Compound 9

[0941] Compound 8 (7.9 g, 25.464 mmol) and compound 8A (6.09 g, 25.464 mmol) were added to CHCN (316 mL). SnCl (8.781 mL, 76.392 mmol) was then slowly added dropwise at 0°C. The mixture was allowed to react at 25°C for 2 hours. TLC (ethyl acetate:acetone = 10:1, PMA) showed the disappearance of the starting compound 8 and the formation of a new spot. LCMS (RW0006-267-P1A) indicated a 31.3% yield of product. The reaction solution was cooled to 0°C and adjusted to pH 8 with saturated aqueous NaHCO3 solution. The aqueous phase was then extracted with DCM (200 mL x 3). The organic phase was dried over anhydrous Na2SO4, filtered, and spin-dried to give a crude product, which was purified by column chromatography (ethyl acetate:acetone = 20:1-3:1) to give compound 9 (6.1 g, 12.464 mmol, 48.95%) as a yellow oil.

[0942] 1H NMR(400MHz, CDCl3)δ8.75-8.84(m,1H),8.17(s,1H),8.01-8.10(m,2H), 7.59-7.70(m,1H),7.50-7.57(m,2H),6.07(s,1H),5.66-5.73(m,1H),4.9 8-5.12(m,1H),4.70-4.81(m,1H),3.71-3.82(m,12H),2.70-2.81(m,1H), 2.34-2.52(m,2H),2.21-2.27(m,1H),2.16(s,3H),2.06(d,J=4.4Hz,4H).

[0943] 9. Preparation of Compound 10

[0944] Compound 9 (6.1 g, 12.259 mmol) was dissolved in pyridine (30 mL) and water (20 mL) and reacted at 60°C for 12 hours. TLC (DCM:MeOH = 10:1) showed the disappearance of the starting material. The reaction solution was directly spin-dried to obtain the crude product, which was then purified by Prep-HPLC (MeCN / H2O = 30 / 1 to 80 / 1; flow rate: 30 mL / min) to afford Compound 10 (3.85 g, 8.098 mmol, 66.06%) as a yellow oil.

[0945] 10. Preparation of Compound 11

[0946] Compound 10 (3.7 g, 7.783 mmol) and compound SM2 (10.24 g, 15.566 mmol) were dissolved in pyridine (37 mL). 2,4,6-Triisopropylbenzenesulfonyl chloride (14.14 g, 46.698 mmol) was added at 0°C. After reacting at 0°C for 60 minutes, N-methylimidazole (5.11 g, 62.263 mmol) was slowly added dropwise to the reaction solution. The reaction was continued at 25°C for 13 hours. The product was observed by LCMS (RW0006-284-P1C). TLC (DCM:MeOH = 10:1, UV) revealed the formation of a new spot. The reaction mixture was cooled to 0°C and quenched by the addition of 50 mL of saturated sodium carbonate solution. The layers were separated, and the organic phase was diluted with 300 mL of DCM. The mixture was washed with water (50 mL x 3) and saturated aqueous NaCl (50 mL x 1). The organic phase was dried over anhydrous NaSO, filtered, and spin-dried to obtain the crude product. The crude product was purified by column chromatography (DCM:MeOH = 2%-4%, TEA) to afford Compound 11 (2100 mg, 1.883 mmol, 24.20%) as a brown oil.

[0947] 11. Preparation of Compound 12

[0948] Compound 11 (2.4 g, 2.152 mmol) was dissolved in MeOH (36 mL), and NH3 / MeOH (12.299 mL, 7 M) was added. The reaction was continued at 0°C for 3 hours. LCMS (RW0006-290-P1A) showed the formation of the product. The reaction solution was diluted with 150 mL of DCM and washed with 30 mL of water. The organic phase was dried over anhydrous Na2SO4, filtered and dried to give a crude product. The crude product was purified by Prep-HPLC (column: 01-Waters Xbridge BEH C18 19*150 mm; mobile phase: TEAA-ACN; gradient: 33%-58.5% / 15 min; flow rate: 15 ml / min) to give a yellow solid compound 12.

[0949] (380 mg, 0.354 mmol, 16.45%).

[0950] 12. Preparation of E1

[0951] Compound 12 (380 mg, 0.354 mmol) was washed three times with acetonitrile and dissolved in DCM (6.0 mL). 5A molecular sieves, DCI (41.82 mg, 0.354 mmol), and compound 13 (426.95 mg, 1.417 mmol) were then added sequentially. The atmosphere was purged with nitrogen three times and the reaction was allowed to proceed at 25°C for 1 hour. LCMS (RW00006-291-P1A) indicated 5.4% starting material remaining. The reaction mixture was quenched by adding dropwise to 20 mL of ice-cold saturated aqueous NaHCO₃ solution, diluted with 30 mL of DCM, and separated. The organic phase was washed with 20 mL of saturated aqueous NaHCO₃ and 20 mL of saturated aqueous NaCl. The organic phase was dried over anhydrous Na2SO4, filtered, and dried to give a crude product, which was purified by reverse phase purification (acetonitrile / water: 20-80%, 30 min, 20 mL / min) to give a yellow solid E1 (210 mg, 0.165 mmol, 46.57%).

[0952] 1H NMR(400MHz,CDCl3)δ11.96-12.03(m,1H),10.07-10.20(m,1H),8.99-9.12(m,1H),8.62-8.80(m,1H),8.07-8.16(m,1H),8.01-8.05(m,2H),7.78(d,J=1.2Hz,1H),7.60-7.66(m,1H),7.51-7.58(m,2H),7.21-7.26(m,2H),7.09-7.20(m,7H),6.66-6.75(m,4H),6.37-6.56(m,1H),5.75-6.11(m,3H),5.07-5.19(m,1H),4.60-4.74(m,1 H),4.16-4.29(m,1H),3.81-3.89(m,1H),3.76-3.81(m,3H),3.70(d,J=1.6Hz,7H),3.54-3.69(m,3H),3.05-3.15(m,1H),2.54-2.65(m,2H),2.03-2.53(m,5H),1.43-1.50(m,1H),1.14-1.21(m,11H),1.01-1.13(m,6H).

Claims

1. A small interfering RNA (siRNA) for inhibiting the expression of apolipoprotein C3 (APOC3) in a cell, the siRNA 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 at least 15 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 414-826.

2. The siRNA of claim 1, wherein the sense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 1-413.

3. The siRNA according to claim 1 or 2, wherein the length of the double-stranded region is 15-25 nucleotide pairs, preferably 17-21 nucleotide pairs, more preferably 19 nucleotide pairs.

4. The siRNA according to any one of claims 1 to 3, wherein the siRNA comprises a paired sense strand sequence and antisense strand sequence as shown in Table 3.

5. The siRNA of any one of claims 1 to 4, wherein the antisense strand comprises at least 15 consecutive nucleotides, 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 NOs: 473, 612, 690, 757, 761, 816, 817, 818, 819, 820, 814, and 815. Preferably, the antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NOs: 473, 612, 690, 757, 761, 816, 817, 818, 819, 820, 814, and 815.

6. The siRNA of any one of claims 1 to 3 and 5, wherein the sense strand comprises at least 15 consecutive nucleotides, 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 NOs: 60, 199, 277, 344, 348, 403, 404, 405, 406, 407, 401 and 402, preferably the sense strand comprises the nucleotide sequence shown in any one of SEQ ID NOs: 60, 199, 277, 344, 348, 403, 404, 405, 406, 407, 401 and 402.

7. The siRNA of any one of claims 1 to 6, wherein: (a) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 60, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 473; (b) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 199, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 612; (c) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 277, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 690; (d) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 344, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 757; (e) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 348, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 761; (f) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 403, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 816; (g) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 404, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 817; (h) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 405, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 818; (i) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 406, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 819; (j) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 407, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 820; (k) the sense strand comprises the nucleotide sequence shown in SEQ ID NO: 401, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 814; or (l) The sense strand comprises the nucleotide sequence shown in SEQ ID NO:402, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:

815.

8. The siRNA according to any one of claims 1 to 7, wherein substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, preferably all nucleotides of the sense strand and all nucleotides of the antisense strand are modified nucleotides.

9. The siRNA of claim 8, wherein the sense strand and the antisense strand each independently comprise one or more nucleotide modifications selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, inosine ribonucleotides, abasic nucleotides, inverted abasic deoxyribonucleotides, phosphorothioate internucleotide linkage modifications, vinylphosphonate modified nucleotides, locked nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing non-natural bases, and terminal nucleotides linked to a cholesteryl derivative or a dodecanoic acid didecylamide group, and deoxyribonucleotides.

10. The siRNA of claim 8, wherein the sense strand and the antisense strand each independently comprise one or more nucleotide modifications selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, inosine ribonucleotides, abasic nucleotides, inverted abasic deoxyribonucleotides, and phosphorothioate internucleotide linkage modifications.

11. The siRNA according to claim 8, wherein the antisense strand comprises a modified nucleotide sequence shown in any one of Table 5 of the specification, and / or the sense strand comprises a modified nucleotide sequence shown in any one of Table 4 of the specification.

12. The siRNA of claim 8, wherein the siRNA comprises a paired modified sense strand sequence and a modified antisense strand sequence shown in any one of Table 6 of the specification.

13. The siRNA of claim 8, wherein: (1) The sense chain comprises STM1s-AmsAmAmAmGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmAms- STM1 (SEQ ID NO: 1655), The antisense strand comprises (CP1a-U)sCfsUmGfAmGfAmAfUmAfCmUfGmUfCmCfCmUfUmsUfsUm(SEQ ID NO: 1651)； (2) The sense chain comprises STM1s-AmsAmUmUmAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmAms-STM1 (SEQ ID NO: 1656), The antisense strand comprises (CP1a-U)sGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAmsUfsUm (SEQ ID NO: 1650)； (3) The sense chain comprises STM1s-AmsAmUmUmAmAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmAms- STM1 (SEQ ID NO: 1657), And the antisense strand comprises (VPUm)sGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAmsUfsUm(SEQ ID NO: 1649)； (4) The sense chain comprises IBs-AmsAmUmUmAmAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmAms-IB (SEQ ID NO:1658), And the antisense strand comprises (CP1a-U)sGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAmsUfsUm (SEQ ID NO: 1650)； (5) The sense chain comprises UmsUmsAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmsAm (SEQ ID NO: 1659), And the antisense strand comprises (CP1a-U)sGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAmsUfsUm (SEQ ID NO: 1650)； (6) The sense chain comprises IBs-AmsAmAmAmGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmAms-IB (SEQ ID NO:1660), And the antisense strand comprises (CP1a-U)sCfsUmGfAmGfAmAfUmAfCmUfGmUfCmCfCmUfUmsUfsUm(SEQ ID NO: 1651)； (7) The sense chain comprises AmsAmsGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmsAm (SEQ ID NO: 1661), And the antisense strand comprises (CP1a-U)sCfsUmGfAmGfAmAfUmAfCmUfGmUfCmCfCmUfUmsUfsUm(SEQ ID NO: 1651)； (8) The sense chain comprises IBs-AmsAmAmAmGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmAms-IBs- GL6 (SEQ ID NO: 1662), And the antisense strand comprises (CP1a-U)sCfsUmGfAmGf(PCN-A)AfUmAfCmUfGmUfCmCfCmUfUmsUfsUm(SEQ ID NO:1652); or (9) The sense chain comprises AmsAmsGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmsAm (SEQ ID NO: 1663), And the antisense strand comprises (CP1a-U)sCfsUmGfAmGf(PCN-A)AfUmAfCmUfGmUfCmCfCmUfUmsUfsUm(SEQ ID NO:1652); (a) the sense strand comprises CmsGmsAmGmGmAmUfGfCfCmUmCmCmCmUmUmCmUmUm, and the antisense strand comprises AmsAfsGmAfAmGfGmGfAmGfGmCfAmUfCmCfUmCfGmsUfsUm; (b) the sense strand comprises CmsCmsGmUmUmAmAfGfGfAmCmAmAmGmUmUmCmUmUm, and the antisense strand comprises AmsAfsGmAfAmCfUmUfGmUfCmCfUmUfAmAfCmGfGmsUfsUm; (c) the sense strand comprises CmsCmsGmUmUmAmAfGfGfAmCmAmAmGmUmUmCmUmUm, and the antisense strand comprises AmsAfsGmAfAmCfUmUfGmUfCmCfUmUfAmAfCmGfGmsUfsUm; (d) the sense strand comprises CmsCmsAmAmGmUmCfCfAfCmCmUmGmCmCmUmAmUmUm, and the antisense strand comprises AmsAfsUmAfGmGfCmAfGmGfUmGfGmAfCmUfUmGfGmsUfsUm; (e) the sense strand comprises AmsAmsGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmUm, and the antisense strand comprises AmsCfsUmGfAmGfAmAfUmAfCmUfGmUfCmCfCmUfUmsUfsUm; (f) the sense strand comprises UmsUmsAmAmAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmUm, and the antisense strand comprises AmsGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAmsUfsUm; (g) the sense strand comprises AmsAmsGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmUm, and the antisense strand comprises AmsCfsUmGfAmGfAmAfUmAfCmUfGmUfCmCfCmUfUmsUfsUm; (h) the sense strand comprises CmsCmsAmAmGmUmCfCfAfCmCmUmGmCmCmUmAmUmUm, and the antisense strand comprises AmsAfsUmAfGmGfCmAfGmGfUmGfGmAfCmUfUmGfGmsUfsUm; (i) the sense strand comprises CmsCmsGmUmUmAmAfGfGfAmCmAmAmGmUmUmCmUmUm, and the antisense strand comprises AmsAfsGmAfAmCfUmUfGmUfCmCfUmUfAmAfCmGfGmsUfsUm; (j) the sense strand comprises CmsGmsAmGmGmAmUfGfCfCmUmCmCmCmUmUmCmUmUm, and the antisense strand comprises AmsAfsGmAfAmGfGmGfAmGfGmCfAmUfCmCfUmCfGmsUfsUm; (k) the sense strand comprises IBs-AmCmGmGmAmCmAmGfUfAfUmUmCmUmCmAmGmUmimAms-IB (SEQ ID NO: 1014), and the antisense strand comprises UmsCfsAmsCfUmGfAmGmAmAmUmAfCmUfGmUfCmCfCmGfsUm (SEQ ID NO: 1174); or (1) The sense strand comprises AmsAmsGmGmAmCfAmGfUfAfUmUmCmUmCmAmGmUmsGmsCm, and the antisense strand comprises GmsCfsAmCmUmGfAmGmAmAmUmAmCmUfGmUfCmCmCmUmUmsUmsUm.

14. The siRNA according to any one of claims 1 to 13, wherein the siRNA is further conjugated to a ligand moiety comprising N-acetylgalactosamine via a phosphate group or a phosphorothioate group, preferably the sense strand of the siRNA is conjugated to the ligand moiety via a phosphate group or a phosphorothioate group.

15. The siRNA of claim 14, wherein the 3' end of the sense strand is conjugated to the ligand moiety via a phosphate group or a phosphorothioate group.

16. The siRNA of claim 14 or 15, wherein the ligand moiety comprises a conjugated group represented by formula (X'): in, Indicates the position where the siRNA is linked; Q is independently H, Wherein L1 is a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O- or -NHC(O)-(CH2NHC(O)) a -; L2 is a chemical bond or -CH2CH2C(O)-; L3 is a chemical bond, -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-; L4 is -(OCH2CH2) c -, -(OCH2CH2CH2) c -, -(OCH2CH2CH2CH2) c -, -(OCH2CH2CH2CH2CH2) c - or -NHC(O)-(CH2) d -; Where a = 0, 1, 2 or 3; b = 1, 2, 3, 4, or 5; c = 1, 2, 3, 4, or 5; d = 1, 2, 3, 4, 5, 6, 7, or 8; L is a chemical bond, -CH2O- or -NHC(O)-; L' is a chemical bond, -C(O)NH-, -NHC(O)- or -O(CH2CH2O) e -; Where e is 1, 2, 3, 4 or 5; T is a chemical bond, -CH2-, -C(O)-, -M-, -CH2-M- or -C(O)-M-; Where M is R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H; Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H; wherein R is -OR', -CH2OR' or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group or a solid phase support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl; m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

17. The siRNA of claim 16, wherein the conjugated group is represented by formula (I'): in, Indicates the position where the siRNA is linked; Q is independently H, Wherein L1 is a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O- or -NHC(O)-(CH2NHC(O)) a -; L2 is a chemical bond or -CH2CH2C(O)-; L3 is a chemical bond, -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-; L4 is -(OCH2CH2) c -, -(OCH2CH2CH2) c -, -(OCH2CH2CH2CH2) c -, -(OCH2CH2CH2CH2CH2) c - or -NHC(O)-(CH2) d -; Where a = 0, 1, 2 or 3; b = 1, 2, 3, 4, or 5; c = 1, 2, 3, 4, or 5; d = 1, 2, 3, 4, 5, 6, 7, or 8; L is -CH2O- or -NHC(O)-; L' is a chemical bond, -C(O)NH- or -NHC(O)-; R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H; Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H; wherein R is -OR', -CH2OR' or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group or a solid phase support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl; m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

18. The siRNA according to claim 17, wherein Q is independently H or Where L1 is -CH2O- or -NHC(O)-(CH2NHC(O)) a -; L2 is -CH2CH2C(O)-; L3 is -(NHCH2CH2) b -or-(NHCH2CH2CH2) b -; L4 is -(OCH2CH2) c -or-NHC(O)-(CH2) d -; Where a = 0, 1, 2 or 3; b = 1, 2, 3, 4, or 5; c = 1, 2, 3, 4, or 5; d = 1, 2, 3, 4, 5, 6, 7, or 8; L is -CH2O-; L' is a chemical bond; R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H; Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H; wherein R is -OR', -CH2OR' or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group or a solid phase support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl; m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

19. The siRNA of claim 18, wherein the conjugated group is represented by Formula (I'-1), Formula (I'-2), or Formula (I'-3): in, Indicates the position where the siRNA is linked; Q is Wherein L1 is -CH2O- or -NHC(O)-; L2 is -CH2CH2C(O)-; L3 is -(NHCH2CH2) b -or-(NHCH2CH2CH2) b -; L4 is -(OCH2CH2) c -or-NHC(O)-(CH2) d -; Where b = 1, 2, 3, 4 or 5; c = 1, 2, 3, 4, or 5; d = 1, 2, 3, 4, 5, 6, 7, or 8; L is -CH2O-; R' is H, a hydroxyl protecting group or a solid phase support, wherein the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl; n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

20. The siRNA of claim 17, wherein Q is independently H, Wherein L1 is -CH2O-, -CH2O-CH2CH2O- or -NHC(O)-(CH2NHC(O)) a -; L2 is -CH2CH2C(O)-; L3 is -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-; L4 is -(OCH2CH2) c -or-NHC(O)-(CH2) d -; Where a = 0, 1, 2 or 3; b = 1, 2, 3, 4, or 5; c = 1, 2, 3, 4, or 5; d = 1, 2, 3, 4, 5, 6, 7, or 8; L is -CH2O- or -NHC(O)-; L' is a chemical bond or -C(O)NH-; R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H; Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H; wherein R is -OR', -CH2OR' or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group or a solid phase support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl; m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

21. The siRNA of claim 20, wherein the conjugated group is represented by formula (II'-1) or formula (II'-2): in, Indicates the position where the siRNA is linked; Q is independently Wherein L1 is -CH2O- or -CH2O-CH2CH2O-; L3 is -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-; L4 is -(OCH2CH2) c -or-NHC(O)-(CH2) d -; Where b = 1, 2, 3, 4 or 5; c = 1, 2, 3, 4, or 5; d = 1, 2, 3, 4, 5, 6, 7, or 8; L is -NHC(O)-; L' is a chemical bond or -C(O)NH-; R' is H, a hydroxyl protecting group or a solid phase support, wherein the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl; m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

22. The siRNA of claim 17, wherein Q is independently H, Wherein L1 is -CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O- or -NHC(O)-(CH2NHC(O)) a -; L2 is a chemical bond; L3 is -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-; L4 is -(OCH2CH2) c -or-NHC(O)-(CH2) d -; Where a = 0, 1, 2 or 3; b = 1, 2, 3, 4, or 5; c = 1, 2, 3, 4, or 5; d = 1, 2, 3, 4, 5, 6, 7, or 8; L is -CH2O- or -NHC(O)-; L' is a chemical bond or -C(O)NH-; R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H; Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H; wherein R is -OR', -CH2OR' or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group or a solid phase support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl; m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

23. The siRNA of claim 22, wherein the conjugated group is represented by formula (II'-2): in, Indicates the position where the siRNA is linked; Q is independently Wherein L1 is -CH2- or -C(O)-; L3 is -(NHCH2CH2) b -; L4 is -(OCH2CH2) c -; Where b = 1, 2, 3, 4 or 5; c = 1, 2, 3, 4, or 5; L is -CH2O- or -NHC(O)-; R' is H, a hydroxyl protecting group or a solid phase support, wherein the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl; n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

24. The siRNA of claim 16, wherein: Q is independently H, Wherein L1 is a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O- or -NHC(O)-(CH2NHC(O)) a -; L2 is a chemical bond or -CH2CH2C(O)-; L3 is a chemical bond, -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-; L4 is -(OCH2CH2) c -, -(OCH2CH2CH2) c -, -(OCH2CH2CH2CH2) c -, -(OCH2CH2CH2CH2CH2) c -, or -NHC(O)-(CH2) d -; Where a = 0, 1, 2 or 3; b = 1, 2, 3, 4, or 5; c = 1, 2, 3, 4, or 5; d = 1, 2, 3, 4, 5, 6, 7, or 8; L is a chemical bond, -CH2O- or -NHC(O)-; L' is a chemical bond, -C(O)NH-, -NHC(O)- or -O(CH2CH2O) e -; Where e is 1, 2, 3, 4 or 5; T is a chemical bond, -CH2-, -M-, -CH2-M- or -C(O)-M-; Where M is R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H; Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H; wherein R is -OR', -CH2OR' or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group or a solid phase support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl; m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

25. The siRNA of claim 24, wherein T is -M-, -CH2-M- or -C(O)-M-, wherein M is 26. The siRNA according to claim 24 or 25, wherein Q is independently H or Where L1 is -CH2O- or -NHC(O)-(CH2NHC(O)) a -; L2 is -CH2CH2C(O)-; L3 is -(NHCH2CH2) b -or-(NHCH2CH2CH2) b -; L4 is -(OCH2CH2) c -or-NHC(O)-(CH2) d -; Where a = 0, 1, 2 or 3; b = 1, 2, 3, 4, or 5; c = 1, 2, 3, 4, or 5; d = 1, 2, 3, 4, 5, 6, 7, or 8; L is a chemical bond or -CH2O-; L' is a chemical bond or -O(CH2CH2O) e -; Where e is 1, 2, 3, 4 or 5; R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H; Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H; wherein R is -OR', -CH2OR' or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group or a solid phase support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl; m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; wherein T is as defined in claim 24 or 25.

27. The siRNA of claim 26, wherein the conjugated group is represented by Formula (III'-1), Formula (III'-2), or Formula (III'-3): in, Q is Wherein L1 is -CH2O- or -NHC(O)-; L2 is -CH2CH2C(O)-; L3 is -(NHCH2CH2) b -or-(NHCH2CH2CH2) b -; L4 is -(OCH2CH2) c -or-NHC(O)-(CH2) d -; Where b = 1, 2, 3, 4 or 5; c = 1, 2, 3, 4, or 5; d = 1, 2, 3, 4, 5, 6, 7, or 8; L is a chemical bond or -CH2O-; Wherein R' is H, a hydroxyl protecting group or a solid phase support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl; n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; wherein T is as defined in claim 24 or 25.

28. The siRNA according to claim 24 or 25, wherein Q is independently H, Wherein L1 is -CH2-, -CH2O- or -C(O)-; L2 is a chemical bond; L3 is -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-; L4 is -(OCH2CH2) c -or-NHC(O)-(CH2) d -; Where b = 1, 2, 3, 4 or 5; c = 1, 2, 3, 4, or 5; d = 1, 2, 3, 4, 5, 6, 7, or 8; L is a chemical bond or -NHC(O)-; L' is a chemical bond; R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H; Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H; wherein R is -OR', -CH2OR' or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group or a solid phase support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl; m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; wherein T is as defined in claim 24 or 25.

29. The siRNA of claim 24 or 25, wherein the conjugated group is represented by formula (IV-1) or formula (IV-2): in, Q is independently Wherein L1 is -CH2-, -CH2O- or -C(O)-; L3 is -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-; L4 is -(OCH2CH2) c -or-NHC(O)-(CH2) d -; Where b = 1, 2, 3, 4 or 5; c = 1, 2, 3, 4, or 5; d = 1, 2, 3, 4, 5, 6, 7, or 8; L is a chemical bond or -NHC(O)-; L' is a chemical bond; Wherein R' is H, a hydroxyl protecting group or a solid phase support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl; m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; wherein T is as defined in claim 24 or 25.

30. The siRNA of claim 16, wherein: Q is independently H, Wherein L1 is a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O- or -NHC(O)-(CH2NHC(O)) a -; L2 is a chemical bond or -CH2CH2C(O)-; L3 is a chemical bond, -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-; L4 is -(OCH2CH2) c -, -(OCH2CH2CH2) c -, -(OCH2CH2CH2CH2) c -, -(OCH2CH2CH2CH2CH2) c - or -NHC(O)-(CH2) d -; Where a = 0, 1, 2 or 3; b = 1, 2, 3, 4, or 5; c = 1, 2, 3, 4, or 5; d = 1, 2, 3, 4, 5, 6, 7, or 8; L is a chemical bond, -CH2O- or -NHC(O)-; L' is -O(CH2CH2O) e -; Where e is 1, 2, 3, 4 or 5; T is a chemical bond, -CH2-, -C(O)-, -M-, -CH2-M- or -C(O)-M-; Where M is R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H; Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H; wherein R is -OR', -CH2OR' or -CH2CH2OR', wherein R' is H, a hydroxyl protecting group or a solid phase support, and the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl; m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

31. The siRNA of claim 16, wherein the conjugated group is selected from the group consisting of:

32. The siRNA of claim 16, wherein the conjugated group is selected from the group consisting of:

33. The siRNA of any one of claims 1-32, wherein the ligand targets the asialoglycoprotein receptor (ASGPR).

34. The siRNA of claim 14 or 15, wherein the ligand has the structure: in Indicates the position of linkage to the sense strand of the siRNA via a phosphate group or a phosphorothioate group.

35. The siRNA of any one of claims 14 or 15, wherein the ligand has the structure: in Indicates the position of linkage to the sense strand of the siRNA via a phosphate group or a phosphorothioate group.

36. The siRNA of claim 14 or 15, wherein the ligand has the structure: in Indicates the position of linkage to the sense strand of the siRNA via a phosphate group or a phosphorothioate group.

37. The siRNA of claim 13, wherein: (1) The sense chain comprises STM1s-AmsAmAmAmGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmAms-STM1s- GL6 (SEQ ID NO: 1238), And the antisense strand comprises (CP1a-U)sCfsUmGfAmGfAmAfUmAfCmUfGmUfCmCfCmUfUmsUfsUm(SEQ ID NO: 1651)； (2) The sense chain comprises STM1s-AmsAmUmUmAmAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmAms- GL6 (SEQ ID NO: 1235), And the antisense strand comprises (CP1a-U)sGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAmsUfsUm (SEQ ID NO: 1650)； (3) The sense chain comprises STM1s-AmsAmUmUmAmAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmAms- GL6 (SEQ ID NO: 1235), And the antisense strand comprises (VPUm)sGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAmsUfsUm(SEQ ID NO: 1649)； (4) The sense chain comprises IBs-AmsAmUmUmAmAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmAms-IBs-GL6 (SEQ ID NO: 1236), And the antisense strand comprises (CP1a-U)sGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAmsUfsUm (SEQ ID NO: 1650)； (5) The sense chain comprises UmsUmsAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmsAms-GL6 (SEQ ID NO: 1237)， And the antisense strand comprises (CP1a-U)sGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAmsUfsUm (SEQ ID NO: 1650)； (6) The sense chain comprises IBs-AmsAmAmAmGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmAms-IBs-GL6 (SEQ ID NO: 1239), And the antisense strand comprises (CP1a-U)sCfsUmGfAmGfAmAfUmAfCmUfGmUfCmCfCmUfUmsUfsUm(SEQ ID NO: 1651)； (7) The sense chain comprises AmsAmsGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmsAms-GL6 (SEQ ID NO: 1240)， And the antisense strand comprises (CP1a-U)sCfsUmGfAmGfAmAfUmAfCmUfGmUfCmCfCmUfUmsUfsUm(SEQ ID NO: 1651); or (8) The sense chain comprises IBs-AmsAmAmAmGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmAms-IBs- GL6 (SEQ ID NO: 1239), And the antisense strand comprises (CP1a-U)sCfsUmGfAmGf(PCN-A)AfUmAfCmUfGmUfCmCfCmUfUmsUfsUm(SEQ ID NO:1652); or (9) The sense chain comprises AmsAmsGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmsAms-GL6 (SEQ ID NO: 1240)， And the antisense strand comprises (CP1a-U)sCfsUmGfAmGf(PCN-A)AfUmAfCmUfGmUfCmCfCmUfUmsUfsUm(SEQ ID NO:1652).

38. A cell comprising the siRNA of any one of claims 1-37.

39. A pharmaceutical composition comprising the siRNA of any one of claims 1 to 37, or the cell of claim 38, and optionally a pharmaceutically acceptable carrier or excipient.

40. A kit comprising the siRNA of any one of claims 1-37, the cell of claim 38, or the pharmaceutical composition of claim 39.

41. A method of treating a disease associated with APOC3 in a subject, the method comprising the step of administering to the subject the siRNA of any one of claims 1-37, the cell of claim 38, or the pharmaceutical composition of claim 39.

42. A method for reducing the risk of developing a disease associated with APOC3 in a subject, the method comprising the step of administering to the subject the siRNA of any one of claims 1 to 37, the cell of claim 38, or the pharmaceutical composition of claim 39.

43. The method of claim 41 or 42, wherein the APOC3-related disease is selected from hyperlipidemia and hypertriglyceridemia.

44. The method of claim 41 or 42, wherein the APOC3-related disease is a disease that may be caused by, associated with, or a consequence of hypertriglyceridemia, such as non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, polycystic ovary syndrome, kidney disease, obesity, type 2 diabetes, hypertension, atherosclerosis, cardiovascular disease, or pancreatitis.

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