SiRNA molecule for regulating and controlling ANGPTL3 gene activity

Abstract

A small interfering RNA (siRNA) for inhibiting the expression of Angiopoietin-like 3 (ANGPTL3) protein in a cell is provided, as well as a cell comprising the siRNA and a method of treating a disease or condition associated with ANGPTL3 expression in a subject using the siRNA or cell.

Description

siRNA molecules that regulate ANGPTL3 gene activity Technical Field

[0001] The present disclosure relates to the field of RNA interference. Background Art

[0002] Angiopoietins are a family of secreted growth factors. Together with their corresponding endothelial-specific receptors, angiopoietins play an important role in angiogenesis. Angiopoietin-like 3 (also known as angiopoietin-like 3, ANGPTL3, or angiopoietin 5, ANGPT5) is a member of the angiopoietin family that is primarily expressed in the liver and is thought to play a role in regulating lipid metabolism.

[0003] Angiopoietin-like 3 (ANGPTL3) is a lipid metabolism regulator that regulates VLDL triglycerides (TG) by inhibiting the catalytic activity of lipoprotein lipase (LPL). The APOEKO mouse model with the hyp1 mutation (apoEKO-hypl) has reduced Angptl3 expression. This mouse model exhibits significant reductions in VLDL TG, VLDL cholesterol, and plasma apoB levels. Furthermore, plasma LPL and hepatic lipase activity are significantly increased in apoEKO-hypl mice after heparin administration, indicating enhanced lipid metabolism (Ando et al., (2003) J. Lipid Res, 44:1216-1223). In humans, ANGPTL3 plasma concentrations are positively correlated with plasma HDL cholesterol and HDL phospholipid levels (Shimamura et al., (2007) Arterioscler. Thromb. Vasc. Biol., 27:366-372).

[0004] In recent years, inhibitors targeting ANGPTL3 have emerged as novel therapeutic agents for these diseases. As a new therapeutic approach, siRNA (siRNA) holds enormous potential. siRNA targets 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 absorption 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 intracellular ANGPTL3 gene expression.

[0005] Summary of the Invention

[0006] The present disclosure provides novel small interfering RNA (siRNA), kits, and pharmaceutical compositions for inhibiting the expression of angiopoietin-like 3 (ANGPTL3) in cells, as well as methods for inhibiting or reducing ANGPTL3 gene expression or treating diseases or symptoms associated with ANGPTL3 expression using the siRNA, kits, or pharmaceutical compositions.

[0007] In a first aspect, the present disclosure provides a small interfering RNA (siRNA) for inhibiting the expression of angiopoietin-like 3 (ANGPTL3) 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 a nucleotide sequence of at least 15 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 16 to 29. In some specific embodiments, the sense strand comprises a nucleotide sequence of at least 15 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 1-14.

[0008] 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 19-23 nucleotides in length.

[0009] In some embodiments, the length of the double-stranded region is 15-25 nucleotide pairs, preferably 17-23 nucleotide pairs, and more preferably 19-21 nucleotide pairs.

[0010] 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 1 nucleotide. In some preferred embodiments, the antisense strand comprises a 3' overhang and / or a 5' overhang of at least 2 nucleotides, preferably, the antisense strand comprises a 3' overhang and / or a 5' overhang of 2 nucleotides.

[0011] In some embodiments, the antisense strand comprises a nucleotide sequence of at least 16 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 16-29, a nucleotide sequence of at least 17 consecutive nucleotides, a nucleotide sequence of at least 18 consecutive nucleotides, a nucleotide sequence of at least 19 consecutive nucleotides, or a nucleotide sequence of at least 20 consecutive nucleotides. Preferably, the antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NOs: 16-29.

[0012] In some embodiments, the sense strand comprises a nucleotide sequence of at least 16 consecutive nucleotides, a nucleotide sequence of at least 17 consecutive nucleotides, or a nucleotide sequence of at least 18 consecutive nucleotides of any one of the nucleotide sequences shown in SEQ ID NOs: 1-14, and preferably the antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NOs: 1-14.

[0013] In some embodiments, the siRNA comprises a paired sense strand sequence and antisense strand sequence as shown in Table 3.

[0014] 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, or all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides.

[0015] 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, non-natural base containing nucleotides, terminal nucleotides attached to a cholesterol derivative or a dodecanoic acid didecylamide group, deoxyribonucleotides, and STM1.

[0016] 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, reverse abasic deoxyribonucleotides, phosphorothioate internucleotide linkage modifications, and STM1. In some preferred embodiments, the sense strand and / or the antisense strand comprise at least 2 2'-fluoro modified nucleotides. In some preferred embodiments, the sense strand and / or the antisense strand comprise at least 8 2'-O-methyl modified nucleotides. In some preferred embodiments, the 3' end and / or the 5' end of the sense strand and / or the antisense strand comprise 1-5 phosphorothioate groups, preferably 2-3 phosphorothioate groups.

[0017] In some embodiments, in the siRNA of the present disclosure,

[0018] (a) The sense strand comprises

[0019] CmsAmsCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAm (SEQ ID NO: 31),

[0020] The antisense strand comprises

[0021] UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm (SEQ ID NO: 61);

[0022] (b) the sense strand comprises

[0023] CmsAmsAmUmUmAmAfGfCfUmCmCmUmUmCmUmUmUmUm (SEQ ID NO: 32),

[0024] The antisense strand comprises

[0025] AmsAfsAmAfGmAfAmGfGmAfGmCfUmUfAmAfUmUfGmsUfsGm (SEQ ID NO: 62);

[0026] (c) the sense strand comprises

[0027] UmsAmsUmUmGmUmUfCfCfUmCmUmAmGmUmUmAmUmUm (SEQ ID NO: 33),

[0028] The antisense strand comprises

[0029] AmsAfsUmAfAmCfUmAfGmAfGmGfAmAfCmAfAmUfAmsAfsAm (SEQ ID NO: 63);

[0030] (d) the sense strand comprises

[0031] GmsUmsUmAmUmUmUfCfCfUmCmCmAmGmAmAmUmUmAm (SEQ ID NO: 34),

[0032] The antisense strand comprises

[0033] UmsAfsAmUfUmCfUmGfGmAfGmGfAmAfAmUfAmAfCmsUfsAm (SEQ ID NO: 64);

[0034] (e) the sense strand comprises

[0035] AmsGmsAmAmUmUmGfAfUfCmAmAmGmAmCmAmAmUmUm (SEQ ID NO: 35),

[0036] The antisense strand comprises

[0037] AmsAfsUmUfGmUfCmUfUmGfAmUfCmAfAmUfUmCfUmsGfsGm (SEQ ID NO: 65);

[0038] (f) the sense strand comprises

[0039] AmsUmsCmAmAmGmAfCfAfAmUmUmCmAmUmCmAmUmUm (SEQ ID NO: 36),

[0040] The antisense strand comprises

[0041] AmsAfsUmGfAmUfGmAfAmUfUmGfUmCfUmUfGmAfUmsCfsAm (SEQ ID NO: 66);

[0042] (g) the sense strand comprises

[0043] GmsAmsGmCmCmAmAfAfAfUmCmAmAmGmAmUmUmUmAm (SEQ ID NO: 37),

[0044] The antisense strand comprises

[0045] UmsAfsAmAfUmCfUmUfGmAfUmUfUmUfGmGfCmUfCmsUfsGm (SEQ ID NO: 67);

[0046] (h) the sense strand comprises

[0047] UmsGmsAmAmCmUmCfAfAfCmUmCmAmAmAmAmCmUmUm (SEQ ID NO: 38),

[0048] The antisense strand comprises

[0049] AmsAfsGmUfUmUfUmGfAmGfUmUfGmAfGmUfUmCfAmsAfsGm (SEQ ID NO: 68);

[0050] (i) the sense strand comprises

[0051] AmsGmsAmGmCmAmAfCfUfAmAmCmUmAmAmCmUmUmAm (SEQ ID NO: 39),

[0052] The antisense strand comprises

[0053] UmsAfsAmGfUmUfAmGfUmUfAmGfUmUfGmCfUmCfUmsUfsCm (SEQ ID NO: 69);

[0054] (j) the sense strand comprises

[0055] GmsCmsAmAmCmUmAfAfCfUmAmAmCmUmUmAmAmUmUm (SEQ ID NO: 40),

[0056] The antisense strand comprises

[0057] AmsAfsUmUfAmAfGmUfUmAfGmUfUmAfGmUfUmGfCmsUfsCm (SEQ ID NO: 70);

[0058] (k) the sense strand comprises

[0059] CmsUmsAmAmCmUmAfAfCfUmUmAmAmUmUmCmAmAmAm (SEQ ID NO: 41),

[0060] The antisense strand comprises

[0061] UmsUfsUmGfAmAfUmUfAmAfGmUfUmAfGmUfUmAfGmsUfsUm (SEQ ID NO: 71);

[0062] (l) The sense chain comprises

[0063] CmsUmsAmAmCmUmUfAfAfUmUmCmAmAmAmUmCmAm (SEQ ID NO: 42),

[0064] The antisense strand comprises

[0065] UmsGfsAmUfUmUfUmGfAmAfUmUfAmAfGmUfUmAfGmsUfsUm (SEQ ID NO: 72);

[0066] (m) the sense strand comprises

[0067] UmsAmsAmCmUmUmAfAfUfUmCmAmAmAmUmCmAmAm (SEQ ID NO: 43),

[0068] The antisense strand comprises

[0069] AmsAfsUmAfAmCfUmAfGmAfGmGfAmAfCmAfAmUfAmsAfsAm (SEQ ID NO: 73); or

[0070] (n) the sense chain comprises

[0071] GmsGmsAmUmCmAmCfAfAfAmAmCmUmUmCmAmAmUmAm (SEQ ID NO: 44),

[0072] The antisense strand comprises

[0073] UmsAfsUmUfGmAfAmGfUmUfUmUfGmUfGmAfUmCfCmsAfsUm (SEQ ID NO:74).

[0074] In some embodiments, in the siRNA of the present disclosure,

[0075] (a) The sense strand comprises

[0076] UmsGmsAmAmCmUmCfAfAfCmUmCmAmAmAmAmCmUmsUm (SEQ ID NO: 131),

[0077] and the antisense strand comprises

[0078] AmsAfsGmUfUmUfUmGfAmGfUmUfGmAfGmUfUmCfAmsAfsGm (SEQ ID NO: 132);

[0079] (b) the sense strand comprises

[0080] GmsGmsAmUmCmAmCfAfAfAmAmCmUmUmCmAmAmUmsAm (SEQ ID NO: 133),

[0081] and the antisense strand comprises

[0082] UmsAfsUmUfGmAfAmGfUmUfUmUfGmUfGmAfUmCfCmsAfsUm (SEQ ID NO: 134);

[0083] (c) the sense strand comprises

[0084] CmsAmsCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmsAm (SEQ ID NO: 135),

[0085] and the antisense strand comprises

[0086] UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm (SEQ ID NO: 136);

[0087] (d) the sense strand comprises

[0088] STM1s-CmsUmUmGmAmAmCmUmCfAfAfCmUmCmAmAmAmAmCmUmUms-STM1 (SEQ ID NO: 137),

[0089] and the antisense strand comprises

[0090] AmsAfsGmUfUmUfUmGfAmGfUmUfGmAfGmUfUmCfAmsAfsGm (SEQ ID NO: 138);

[0091] (e) the sense strand comprises

[0092] STM1s-AmsUmGmGmAmUmCmAmCfAfAfAmAmCmUmUmCmAmAmUmAms-STM1 (SEQ ID NO: 139),

[0093] and the antisense strand comprises

[0094] UmsAfsUmUfGmAfAmGfUmUfUmUfGmUfGmAfUmCfCmsAfsUm (SEQ ID NO: 140);

[0095] (f) the sense strand comprises

[0096] STM1s-UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAms-STM1 (SEQ ID NO: 141),

[0097] and the antisense strand comprises

[0098] UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm (SEQ ID NO: 142);

[0099] (g) the sense strand comprises

[0100] STM1-STM1-

[0101] UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAm-STM1-STM1 (SEQ ID NO: 143),

[0102] and the antisense strand comprises

[0103] UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm (SEQ ID NO: 144); or

[0104] (h) the sense strand comprises

[0105] IBs-UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAms-IB (SEQ ID NO: 145),

[0106] and the antisense strand comprises

[0107] UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm (SEQ ID NO: 146).

[0108] In some embodiments, in the siRNA of the present disclosure,

[0109] (a) The sense strand comprises

[0110] STM1-STM1-

[0111] UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmsAm-STM1-STM1 (SEQ ID NO: 147),

[0112] and the antisense strand comprises

[0113] UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm (SEQ ID NO: 148);

[0114] (b) the sense strand comprises

[0115] STM1s-UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAm-STM1-STM1 (SEQ ID NO: 149),

[0116] and the antisense strand comprises

[0117] UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm (SEQ ID NO: 150);

[0118] (c) the sense strand comprises

[0119] STM1s-CmsAmAmGmAmCmAmAmUfUfCfAmUmCmAmUmUmUmGmAmUms-STM1 (SEQ ID NO: 151),

[0120] and the antisense strand comprises

[0121] AmsUfsCmAfAmAfUmGfAmUfGmAfAmUfUmGfUmCfUmsUfsGm (SEQ ID NO: 152);

[0122] (d) the sense strand comprises

[0123] STM1-STM1-

[0124] CmsAmAmGmAmCmAmAmUfUfCfAmUmCmAmUmUmUmGmAmsUm-STM1-STM1 (SEQ ID NO: 153),

[0125] and the antisense strand comprises

[0126] AmsUfsCmAfAmAfUmGfAmUfGmAfAmUfUmGfUmCfUmsUfsGm (SEQ ID NO: 154);

[0127] (e) the sense strand comprises

[0128] STM1s-CmsAmAmGmAmCmAmAmUfUfCfAmUmCmAmUmUmUmGmAmUm-STM1-STM1 (SEQ ID NO: 155),

[0129] and the antisense strand comprises

[0130] AmsUfsCmAfAmAfUmGfAmUfGmAfAmUfUmGfUmCfUmsUfsGm (SEQ ID NO: 156);

[0131] (f) the sense strand comprises

[0132] STM1s-UmsGmGmUmAmUmUmAmAfAfUfCmCmUmUmAmAmGmAmGmAms-STM1 (SEQ ID NO: 157),

[0133] and the antisense strand comprises

[0134] UmsCfsUmCfUmUfAmAfGmGfAmUfUmUfAmAfUmAfCmsCfsAm (SEQ ID NO: 158);

[0135] (g) the sense strand comprises

[0136] STM1s-CmsCmAmGmAmAmUmUmGfAfUfCmAmAmGmAmCmAmAmUmUms-STM1 (SEQ ID NO: 159),

[0137] and the antisense strand comprises

[0138] AmsAfsUmUfGmUfCmUfUmGfAmUfCmAfAmUfUmCfUmsGfsGm (SEQ ID NO: 160); or

[0139] (h) the sense strand comprises

[0140] STM1s-UmsGmAmUmCmAmAmGmAfCfAfAmUmUmCmAmUmCmAmUmUms-STM1 (SEQ ID NO: 161),

[0141] and the antisense strand comprises

[0142] AmsAfsUmGfAmUfGmAfAmUfUmGfUmCfUmUfGmAfUmsCfsAm (SEQ ID NO: 162).

[0143] In some embodiments, the siRNA is further conjugated to a ligand moiety comprising N-acetylgalactosamine, preferably the sense strand of the siRNA is conjugated to the ligand moiety. In some preferred embodiments, the 3' end of the sense strand is conjugated to the ligand moiety. In other preferred embodiments, the 5' end of the sense strand is conjugated to the ligand moiety.

[0144] In some embodiments, the ligand moiety comprises a conjugate group represented by formula (X'):

[0145] in,

[0146] Indicates the location of attachment to a biomolecule;

[0147] Q is independently H,

[0148] Wherein L1 is a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O- or -NHC(O)-(CH2NHC(O)) a -;

[0149] L2 is a chemical bond or -CH2CH2C(O)-;

[0150] L3 is a chemical bond, -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-;

[0151] L4 is -(OCH2CH2) c -、-(OCH2CH2CH2) c -、-(OCH2CH2CH2CH2) c -、-(OCH2CH2CH2CH2CH2) c -or-NHC(O)-(CH2) d -;

[0152] Where a = 0, 1, 2 or 3;

[0153] b = 1, 2, 3, 4, or 5;

[0154] c = 1, 2, 3, 4, or 5;

[0155] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0156] L is a chemical bond, -CH2O- or -NHC(O)-;

[0157] L' is a chemical bond, -C(O)NH-, -NHC(O)- or -O(CH2CH2O) e -;

[0158] Where e is 1, 2, 3, 4 or 5;

[0159] T is a chemical bond, -CH2-, -C(O)-, -M-, -CH2-M- or -C(O)-M-;

[0160] Where M is

[0161] R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0162] Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H;

[0163] 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;

[0164] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0165] n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0166] In some embodiments, the conjugated ligand targets the asialoglycoprotein receptor (ASGPR).

[0167] In some preferred embodiments, the conjugated group is selected from Table 1:

[0168] Table 1 Structure of conjugated groups

[0169] In some preferred embodiments, the conjugated group is selected from Table 2:

[0170] Table 2 Structure of conjugated groups

[0171] In some embodiments, the ligand contained in the siRNA has the following structure:

[0172] in Indicates the position of linkage to the sense strand of the siRNA via a phosphate group or a phosphorothioate group.

[0173] In some embodiments, the ligand contained in the siRNA has the following structure:

[0174] in Indicates the position of linkage to the sense strand of the siRNA via a phosphate group or a phosphorothioate group.

[0175] In some embodiments, the ligand included in the siRNA of the present disclosure has the following structure:

[0176] in Indicates the position of attachment to the siRNA via a phosphate group or a phosphorothioate group.

[0177] In some embodiments, the ligand included in the siRNA of the present disclosure has the following structure:

[0178] in Indicates the position of linkage to the sense strand of siRNA via a phosphate group or a phosphorothioate group.

[0179] In a second aspect, the present disclosure provides a cell containing the siRNA described in the present disclosure.

[0180] In a third aspect, the present disclosure provides a pharmaceutical composition comprising the siRNA or cells described in the present disclosure, and optionally a pharmaceutically acceptable carrier or excipient.

[0181] In a fourth aspect, the present disclosure provides a kit comprising the siRNA, cells or pharmaceutical composition described in the present disclosure.

[0182] In a fifth aspect, the present disclosure provides a method for reducing ANGPTL3 levels, LDL levels, apoC-III levels, triglyceride levels, cholesterol levels, glucose levels, and fat pad weight in a subject, comprising administering to the subject the siRNA, cell, or pharmaceutical composition of the present disclosure.

[0183] The present disclosure also provides a method for treating a disease or symptom associated with ANGPTL3 expression in a subject, comprising administering to the subject the siRNA, cell, or pharmaceutical composition of the present disclosure.

[0184] In some embodiments, the disease associated with ANGPTL3 expression is a metabolic disease or a cardiovascular disease. In some preferred embodiments, the metabolic disease or the cardiovascular disease is selected from obesity, diabetes, atherosclerosis, dyslipidemia, coronary heart disease, non-alcoholic fatty liver disease (NAFLD), hyperfatty acidemia, metabolic syndrome, or a combination thereof.

[0185] In some embodiments, the disease associated with ANGPTL3 expression is dyslipidemia, and the dyslipidemia is hyperlipidemia. In some embodiments, the hyperlipidemia is hypercholesterolemia, hypertriglyceridemia, or a combination thereof.

[0186] In some embodiments, the disease associated with ANGPTL3 expression is NAFLD, and the NAFLD is hepatic steatosis or steatohepatitis.

[0187] In some embodiments, the disease associated with ANGPTL3 expression is diabetes, and the diabetes is type 2 diabetes or type 2 diabetes with dyslipidemia.

[0188] In some embodiments, the disclosed methods of treating a disease or symptom associated with ANGPTL3 expression in a subject comprise administering the siRNA, cell, or pharmaceutical composition to the subject subcutaneously, topically, or intravenously. In some embodiments, the subject is a human subject.

[0189] Detailed Description of the Invention

[0190] The following describes the embodiments of the present disclosure through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present disclosure from the disclosure herein. The present disclosure 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 disclosure.

[0191] It should be understood that the protection scope of the present disclosure is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present disclosure are for describing specific embodiments rather than for limiting the protection scope of the present disclosure.

[0192] Throughout this disclosure and the claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0193] When the embodiment gives a numerical range, it should be understood that, unless otherwise specified in the present disclosure, the two 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 disclosure 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 examples, according to the grasp of the prior art by those skilled in the art and the record of the present disclosure, 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 disclosure can also be used to implement the present disclosure, all of which fall within the scope of protection of the present disclosure. The following describes the embodiments of the present disclosure in more detail.

[0194] definition

[0195] 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. Typically, the RNA to be silenced is an endogenous gene or a pathogen gene. In addition, RNA (e.g., tRNA) and viral RNA other than mRNA can also be targeted.

[0196] 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.

[0197] 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.

[0198] 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.

[0199] As used herein, "complementary" sequences, insofar as they meet the above requirements with respect to 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.

[0200] 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 ANGPTL3). For example, a polynucleotide is complementary to at least a portion of an ANGPTL3 mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding ANGPTL3.

[0201] The terms "complementary," "fully complementary," and "substantially complementary" herein can be used with respect to base pairing between the sense and antisense strands of an siRNA, or between the antisense strand of an siRNA agent and a target sequence.

[0202] 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.

[0203] 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) having, for example, fewer than 100, 200, 300, or 400 nucleotides in length.

[0204] "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.

[0205] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the duplex structure of an siRNA (e.g., siRNA). For example, a nucleotide overhang is present when the 3'-end of one strand of the 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). 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 or sense strand of the siRNA.

[0206] "Flush end" or "blunt end" or "blunt end" means that there are no unpaired nucleotides at that end of the double-stranded siRNA, i.e., no nucleotide overhangs. 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 disclosure include siRNAs having a nucleotide overhang at one end (i.e., an agent having one overhang and one blunt end) or having nucleotide overhangs at both ends.

[0207] Substantially all of the nucleotides of the iRNA of the present disclosure 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 disclosure, all of the nucleotides of the iRNA of the present disclosure 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. "Substantially all of the nucleotides are modified" means that the siRNA of the present disclosure is mostly but not entirely modified, and can include no more than 5, 4, 3, 2 or 1 unmodified nucleotides.

[0208] As used herein, "modified nucleotides" include, but are not limited to, 2'-O-alkyl modified nucleotides (e.g., 2'-O-methyl modified nucleotides, 2'-methoxyethyl modified nucleotides), 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, inosine ribonucleotides, abasic nucleotides, inverted abasic deoxyribonucleotides, nucleotides containing phosphorothioate groups, phosphorothioate internucleotide linkage modifications, vinylphosphonate modified nucleotides, locked nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, Phosphoramidates, non-natural bases containing nucleotides, terminal nucleotides attached to cholesterol derivatives or dodecanoic acid didecylamide groups, deoxyribonucleotides, 3'-terminal deoxythymine (dT) nucleotides, conformationally restricted nucleotides, restricted ethyl nucleotides, 2'-hydroxyl modified nucleotides, nucleotides containing methylphosphonic acid groups, nucleotides containing 5'-phosphates, nucleotides containing 5'-phosphate mimetics, diol-modified nucleotides (GNAs), and 2-O-(N-methylacetamide) modified nucleotides, and STM1, or conventional protecting group protection, etc.

[0209] For example, the 2'-fluoro-modified nucleotide refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose group is replaced by fluorine, and the 2'-deoxy-modified nucleotide refers to a nucleotide in which the 2'-hydroxyl group at the ribose group is replaced by a methoxy group.

[0210] A "phosphorothioate-containing nucleotide" refers to a nucleotide in which one or more oxygen atoms on the phosphate group of the nucleotide are replaced by a sulfur atom. A "phosphorothioate internucleotide linkage modification" refers to a modification in which two adjacent nucleotides are linked by a phosphorothioate.

[0211] 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.

[0212] In some embodiments, the ligand portion targets the asialoglycoprotein receptor (ASGPR) on hepatocytes. Binding of the ligand portion to the ASGPR mediates internalization via clathrin-coated vesicles. Endosome maturation results in a decrease in lysosomal pH, which promotes the dissociation of the ligand-receptor complex, thereby releasing the siRNA. Conjugation of the ligand portion targeting the asialoglycoprotein receptor (ASGPR) on hepatocytes results in efficacy and stability of the siRNA in vivo or within the cell. This facilitates subcutaneous administration of the siRNA.

[0213] As used herein, the term "inhibit" is used interchangeably with "reduce," "silence," "downregulate," and other similar terms and includes any level of inhibition.

[0214] The phrase "inhibiting the expression of ANGPTL3" is intended to refer to inhibiting the expression of any ANGPTL3 gene, as well as variants or mutants of ANGPTL3 genes. Thus, the ANGPTL3 gene can be a wild-type ANGPTL3 gene, a mutant ANGPTL3 gene, or, in the context of a genetically manipulated cell, cell group, or organism, a transgenic ANGPTL3 gene.

[0215] "Inhibiting ANGPTL3 gene expression" includes any level of inhibition of the ANGPTL3 gene, such as at least partial suppression of ANGPTL3 gene expression. ANGPTL3 gene expression can be assessed based on the level or change in level of any variable associated with ANGPTL3 gene expression, such as ANGPTL3 mRNA level, ANGPTL3 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.

[0216] Inhibition can be assessed by a decrease in the absolute or relative level of one or more variables associated with ANGPTL3 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.

[0217] "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).

[0218] "Halo" or "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).

[0219] “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 1-2 Haloalkyl. 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.

[0220] “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 1-2Alkylene 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.

[0221] As used in this disclosure, the terms "treat", "treat", and the like refer to the administration of an agent or the performance of a procedure in order to obtain an effect. These effects may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in terms of affecting a partial or complete cure of a disease and / or disease symptoms. As used in this disclosure, "treatment" may include treating a disease or condition (e.g., cancer) in a mammal, particularly a human, and includes: (a) preventing the occurrence of the disease or disease symptoms in a subject susceptible to the disease but not yet diagnosed as having the disease (e.g., including diseases that may be associated with or caused by the primary disease); (b) inhibiting the disease, i.e., preventing its development; (c) alleviating the disease, i.e., causing regression of the disease. Treatment may refer to any reference to success in treating or improving or preventing cancer, including any objective or subjective parameter, such as elimination; alleviation; reducing symptoms or making the disease condition more tolerable for the patient; slowing the rate of deterioration or decline; or reducing the end point of deterioration. Treatment or improvement of symptoms is based on one or more objective or subjective parameters; including the results of a doctor's examination. Thus, the term "treatment" includes administering the siRNA or pharmaceutical compositions disclosed herein to prevent or delay, alleviate, arrest, or inhibit the development of symptoms or conditions associated with a disease (e.g., cancer). The term "therapeutic effect" refers to the reduction, elimination, or prevention of a disease, disease symptom, or disease side effect in a subject.

[0222] As used in this disclosure, the term "therapeutically effective amount" refers to an amount that, when administered to a subject for treating a disease, is sufficient to effect such treatment for the disease.

[0223] As used herein, the term "subject" refers to any mammalian subject for whom diagnosis, treatment, or therapy is desired. "Mammal" for therapeutic purposes refers to any animal classified as a mammal, including humans, livestock, and laboratory, zoo, sport, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, and the like.

[0224] I.siRNA

[0225] The present disclosure provides a small interfering RNA (siRNA) for inhibiting the expression of angiopoietin-like 3 (ANGPTL3) 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 a nucleotide sequence of at least 15 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 16-29.

[0226] 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.

[0227] In some specific embodiments, the sense strand comprises a nucleotide sequence of at least 15 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 1-14.

[0228] 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 19-23 nucleotides in length.

[0229] In some embodiments, the length of the double-stranded region is 15-25 nucleotide pairs, preferably 17-23 nucleotide pairs, and more preferably 19-21 nucleotide pairs.

[0230] One or both of the sense strand and the antisense strand comprise a 3' overhang and / or a 5' overhang having at least one nucleotide, for example, one or both of the sense strand and the antisense strand comprise a 3' overhang and / or a 5' overhang having at least one nucleotide. In some preferred embodiments, the antisense strand has a 3' overhang and / or a 5' overhang having at least two nucleotides, preferably, the antisense strand comprises a 3' overhang and / or a 5' overhang having two nucleotides. In some embodiments, the sense strand and the antisense strand are 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., it has a blunt end. In other embodiments, the sense strand and the antisense strand are the same length, a portion of the sense strand is complementary to a portion of the antisense strand, i.e., both the sense strand and the antisense strand have a 5' overhang. In some embodiments, the sense strand and the antisense strand are different in length. In a preferred embodiment, the 5' end of the antisense strand has an overhang of at least one nucleotide, more preferably an overhang of two or three nucleotides.

[0231] In some embodiments, the antisense strand comprises a nucleotide sequence of at least 16 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 16-29, a nucleotide sequence of at least 17 consecutive nucleotides, a nucleotide sequence of at least 18 consecutive nucleotides, a nucleotide sequence of at least 19 consecutive nucleotides, or a nucleotide sequence of at least 20 consecutive nucleotides. Preferably, the antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NOs: 16-29.

[0232] In some embodiments, the sense strand comprises a nucleotide sequence of at least 16 consecutive nucleotides, a nucleotide sequence of at least 17 consecutive nucleotides, a nucleotide sequence of at least 18 consecutive nucleotides, a nucleotide sequence of at least 19 consecutive nucleotides, or a nucleotide sequence of at least 20 consecutive nucleotides of any one of the nucleotide sequences shown in SEQ ID NOs: 1-14, and preferably the antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NOs: 1-1093.

[0233] In some embodiments, the siRNA comprises a paired sense strand sequence and antisense strand sequence as shown in Table 3.

[0234] II. Modification of Nucleotides

[0235] 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.

[0236] In some embodiments, all of the nucleotides of the sense strand and / or all of the nucleotides of the antisense strand are modified nucleotides.

[0237] The modification of the nucleotides described in the present disclosure may be a modification on the phosphate group, ribose group and / or base group of the nucleotide.

[0238] 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, non-natural base containing nucleotides, terminal nucleotides attached to a cholesterol derivative or a dodecanoic acid didecylamide group, deoxyribonucleotides, and STM1.

[0239] 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, reverse abasic deoxyribonucleotides, and thiophosphate internucleotide linkage modifications. In some preferred embodiments, the sense strand and / or the antisense strand comprise at least 2 2'-fluoro modified nucleotides. In some preferred embodiments, the sense strand and / or the antisense strand comprise at least 8 2'-O-methyl modified nucleotides. In some preferred embodiments, the 3' end and / or the 5' end of the sense strand and / or the antisense strand comprise 1-5 thiophosphate groups, preferably 2-3 thiophosphate groups. In some preferred embodiments, the sense strand and / or the antisense strand comprise adenine deoxyribonucleotides, thymine deoxyribonucleotides, guanine deoxyribonucleotides, and / or cytosine deoxyribonucleotides. In a more preferred embodiment, the sense strand and / or antisense strand comprises thymidine deoxyribonucleotides. In a most preferred embodiment, the sense strand comprises thymidine deoxyribonucleotides.

[0240] In some preferred embodiments, 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. 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 of the specification.

[0241] III. Ligand

[0242] The siRNA described in the present disclosure is further conjugated to a ligand moiety comprising N-acetylgalactosamine. In preferred embodiments, the sense strand of the siRNA is conjugated to the ligand moiety. In some preferred embodiments, the 3' end of the sense strand is conjugated to the ligand moiety. In other preferred embodiments, the 5' end of the sense strand is conjugated to the ligand moiety.

[0243] In some embodiments, the ligand moiety comprises a conjugate group represented by formula (X'):

[0244] in,

[0245] Indicates the location of attachment to a biomolecule;

[0246] Q is independently H,

[0247] Wherein L1 is a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O- or -NHC(O)-(CH2NHC(O)) a -;

[0248] L2 is a chemical bond or -CH2CH2C(O)-;

[0249] L3 is a chemical bond, -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-;

[0250] L4 is -(OCH2CH2) c -、-(OCH2CH2CH2) c -、-(OCH2CH2CH2CH2) c -、-(OCH2CH2CH2CH2CH2) c -or-NHC(O)-(CH2) d -;

[0251] Where a = 0, 1, 2 or 3;

[0252] b = 1, 2, 3, 4, or 5;

[0253] c = 1, 2, 3, 4, or 5;

[0254] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0255] L is a chemical bond, -CH2O- or -NHC(O)-;

[0256] L' is a chemical bond, -C(O)NH-, -NHC(O)- or -O(CH2CH2O) e -;

[0257] Where e is 1, 2, 3, 4 or 5;

[0258] T is a chemical bond, -CH2-, -C(O)-, -M-, -CH2-M- or -C(O)-M-;

[0259] Where M is

[0260] R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0261] Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H;

[0262] 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;

[0263] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0264] n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0265] In some embodiments, the conjugated group is represented by formula (I'):

[0266] in,

[0267] Indicates the location of attachment to a biomolecule;

[0268] Q is independently H,

[0269] Wherein L1 is a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O- or -NHC(O)-(CH2NHC(O)) a -;

[0270] L2 is a chemical bond or -CH2CH2C(O)-;

[0271] L3 is a chemical bond, -(NHCH2CH2) b-、-(NHCH2CH2CH2) b - or -C(O)CH2-;

[0272] L4 is -(OCH2CH2) c -、-(OCH2CH2CH2) c -、-(OCH2CH2CH2CH2) c -、- (OCH2CH2CH2CH2CH2CH2) c -or-NHC(O)-(CH2) d -;

[0273] Where a = 0, 1, 2 or 3;

[0274] b = 1, 2, 3, 4, or 5;

[0275] c = 1, 2, 3, 4, or 5;

[0276] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0277] L is -CH2O- or -NHC(O)-;

[0278] L' is a chemical bond, -C(O)NH- or -NHC(O)-;

[0279] R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0280] Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H;

[0281] 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;

[0282] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0283] n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0284] In some specific embodiments, wherein,

[0285] Q is independently H or

[0286] Where L1 is -CH2O- or -NHC(O)-(CH2NHC(O)) a -;

[0287] L2 is -CH2CH2C(O)-;

[0288] L3 is -(NHCH2CH2) b -or-(NHCH2CH2CH2) b -;

[0289] L4 is -(OCH2CH2) c -or-NHC(O)-(CH2) d -;

[0290] Where a = 0, 1, 2 or 3;

[0291] b = 1, 2, 3, 4, or 5;

[0292] c = 1, 2, 3, 4, or 5;

[0293] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0294] L is -CH2O-;

[0295] L' is a chemical bond;

[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'-1), Formula (I'-2), or Formula (I'-3):

[0302] in,

[0303] Indicates the location of attachment to a biomolecule;

[0304] Q is

[0305] Wherein L1 is -CH2O- or -NHC(O)-;

[0306] L2 is -CH2CH2C(O)-;

[0307] L3 is -(NHCH2CH2) b -or-(NHCH2CH2CH2) b -;

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

[0309] Where b = 1, 2, 3, 4 or 5;

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

[0311] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0312] L is -CH2O-;

[0313] 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;

[0314] n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0315] In some specific embodiments, wherein,

[0316] Q is independently H,

[0317] Wherein L1 is -CH2O-, -CH2O-CH2CH2O- or -NHC(O)-(CH2NHC(O)) a -;

[0318] L2 is -CH2CH2C(O)-;

[0319] L3 is -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-;

[0320] L4 is -(OCH2CH2) c -or-NHC(O)-(CH2) d -;

[0321] Where a = 0, 1, 2 or 3;

[0322] b = 1, 2, 3, 4, or 5;

[0323] c = 1, 2, 3, 4, or 5;

[0324] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0325] L is -CH2O- or -NHC(O)-;

[0326] L' is a chemical bond or -C(O)NH-;

[0327] R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0328] Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H;

[0329] 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;

[0330] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0331] n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0332] In some embodiments, the conjugated group is as shown in Formula (II'-1) or Formula (II'-2):

[0333] in,

[0334] Indicates the location of attachment to a biomolecule;

[0335] Q is independently

[0336] Wherein L1 is -CH2O- or -CH2O-CH2CH2O-;

[0337] L3 is -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-;

[0338] L4 is -(OCH2CH2) c -or-NHC(O)-(CH2) d -;

[0339] Where b = 1, 2, 3, 4 or 5;

[0340] c = 1, 2, 3, 4, or 5;

[0341] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0342] L is -NHC(O)-;

[0343] L' is a chemical bond or -C(O)NH-;

[0344] 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;

[0345] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0346] n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0347] In some specific embodiments, wherein,

[0348] Q is independently H,

[0349] Wherein L1 is -CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O- or -NHC(O)-(CH2NHC(O)) a -;

[0350] L2 is a chemical bond;

[0351] L3 is -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-;

[0352] L4 is -(OCH2CH2) c -or-NHC(O)-(CH2) d -;

[0353] Where a = 0, 1, 2 or 3;

[0354] b = 1, 2, 3, 4, or 5;

[0355] c = 1, 2, 3, 4, or 5;

[0356] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0357] L is -CH2O- or -NHC(O)-;

[0358] L' is a chemical bond or -C(O)NH-;

[0359] R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0360] Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H;

[0361] 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;

[0362] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0363] n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0364] In some embodiments, the conjugated group is represented by formula (II'-2):

[0365] in,

[0366] Indicates the location of attachment to a biomolecule;

[0367] Q is independently

[0368] Wherein L1 is -CH2- or -C(O)-;

[0369] L3 is -(NHCH2CH2) b -;

[0370] L4 is -(OCH2CH2) c -;

[0371] Where b = 1, 2, 3, 4 or 5;

[0372] c = 1, 2, 3, 4, or 5;

[0373] L is -CH2O- or -NHC(O)-;

[0374] 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;

[0375] n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0376] In some specific embodiments, wherein:

[0377] Q is independently H,

[0378] Wherein L1 is a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O- or -NHC(O)-(CH2NHC(O)) a -;

[0379] L2 is a chemical bond or -CH2CH2C(O)-;

[0380] L3 is a chemical bond, -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-;

[0381] L4 is -(OCH2CH2) c -、-(OCH2CH2CH2) c -、-(OCH2CH2CH2CH2) c -、-(OCH2CH2CH2CH2CH2) c -or-NHC(O)-(CH2) d -;

[0382] Where a = 0, 1, 2 or 3;

[0383] b = 1, 2, 3, 4, or 5;

[0384] c = 1, 2, 3, 4, or 5;

[0385] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0386] L is a chemical bond, -CH2O- or -NHC(O)-;

[0387] L' is a chemical bond, -C(O)NH-, -NHC(O)- or -O(CH2CH2O) e -;

[0388] Where e is 1, 2, 3, 4 or 5;

[0389] T is a chemical bond, -CH2-, -M-, -CH2-M- or -C(O)-M-;

[0390] Where M is

[0391] R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0392] Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H;

[0393] 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;

[0394] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0395] n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0396] In some specific embodiments, wherein,

[0397] T is -M-, -CH2-M- or -C(O)-M-, wherein M is

[0398] In some specific embodiments, wherein,

[0399] Q is independently H or

[0400] Where L1 is -CH2O- or -NHC(O)-(CH2NHC(O)) a -;

[0401] L2 is -CH2CH2C(O)-;

[0402] L3 is -(NHCH2CH2) b -or-(NHCH2CH2CH2) b -;

[0403] L4 is -(OCH2CH2) c -or-NHC(O)-(CH2) d -;

[0404] Where a = 0, 1, 2 or 3;

[0405] b = 1, 2, 3, 4, or 5;

[0406] c = 1, 2, 3, 4, or 5;

[0407] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0408] L is a chemical bond or -CH2O-;

[0409] L' is a chemical bond or -O(CH2CH2O) e -;

[0410] Where e is 1, 2, 3, 4 or 5;

[0411] R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0412] Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H;

[0413] 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;

[0414] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0415] n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0416] wherein T is as defined in the above embodiment.

[0417] In some embodiments, the conjugated group is represented by Formula (III'-1), Formula (III'-2), or Formula (III'-3):

[0418] in,

[0419] Q is

[0420] Wherein L1 is -CH2O- or -NHC(O)-;

[0421] L2 is -CH2CH2C(O)-;

[0422] L3 is -(NHCH2CH2) b -or-(NHCH2CH2CH2) b -;

[0423] L4 is -(OCH2CH2) c -or-NHC(O)-(CH2) d -;

[0424] Where b = 1, 2, 3, 4 or 5;

[0425] c = 1, 2, 3, 4, or 5;

[0426] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0427] L is a chemical bond or -CH2O-;

[0428] 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;

[0429] n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0430] wherein T is as defined in the above embodiment.

[0431] In some specific embodiments, wherein,

[0432] Q is independently H,

[0433] Wherein L1 is -CH2-, -CH2O- or -C(O)-;

[0434] L2 is a chemical bond;

[0435] L3 is -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-;

[0436] L4 is -(OCH2CH2) c -or-NHC(O)-(CH2) d -;

[0437] Where b = 1, 2, 3, 4 or 5;

[0438] c = 1, 2, 3, 4, or 5;

[0439] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0440] L is a chemical bond or -NHC(O)-;

[0441] L' is a chemical bond;

[0442] R1 and R2 together form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0443] Or R1 and R3 together form -C 1-2 Alkylene-, and R2 is H;

[0444] 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;

[0445] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0446] n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0447] wherein T is as defined in the above embodiment.

[0448] In some embodiments, the conjugated group is represented by formula (IV-1) or formula (IV-2):

[0449] in,

[0450] Q is independently

[0451] Wherein L1 is -CH2-, -CH2O- or -C(O)-;

[0452] L3 is -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-;

[0453] L4 is -(OCH2CH2) c -or-NHC(O)-(CH2) d -;

[0454] Where b = 1, 2, 3, 4 or 5;

[0455] c = 1, 2, 3, 4, or 5;

[0456] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0457] L is a chemical bond or -NHC(O)-;

[0458] L' is a chemical bond;

[0459] 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;

[0460] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0461] n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0462] wherein T is as defined in the above embodiment.

[0463] In some specific embodiments, wherein:

[0464] Q is independently H,

[0465] Wherein L1 is a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O- or -NHC(O)-(CH2NHC(O)) a -;

[0466] L2 is a chemical bond or -CH2CH2C(O)-;

[0467] L3 is a chemical bond, -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-;

[0468] L4 is -(OCH2CH2) c -、-(OCH2CH2CH2) c -、-(OCH2CH2CH2CH2) c -、-(OCH2CH2CH2CH2CH2) c -or-NHC(O)-(CH2) d -;

[0469] Where a = 0, 1, 2 or 3;

[0470] b = 1, 2, 3, 4, or 5;

[0471] c = 1, 2, 3, 4, or 5;

[0472] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0473] L is a chemical bond, -CH2O- or -NHC(O)-;

[0474] L' is -O(CH2CH2O) e -;

[0475] Where e is 1, 2, 3, 4 or 5;

[0476] T is a chemical bond, -CH2-, -C(O)-, -M-, -CH2-M- or -C(O)-M-;

[0477] Where M is

[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] In some preferred embodiments, the conjugated group is selected from the following:

[0484] In some preferred embodiments, the conjugated group is selected from the following:

[0485] In some embodiments, the ligand targets the asialoglycoprotein receptor (ASGPR).

[0486] In a preferred embodiment, the ligand has the following structure:

[0487] in Indicates the position of linkage to the sense strand of siRNA via a phosphate group or a phosphorothioate group.

[0488] In a preferred embodiment, the ligand has the following structure:

[0489] in Indicates the position of linkage to the sense strand of siRNA via a phosphate group or a phosphorothioate group.

[0490] In a preferred embodiment, the ligand has the following structure:

[0491] in Indicates the position of linkage to the sense strand of the siRNA via a phosphate group or a phosphorothioate group.

[0492] In a preferred embodiment, the ligand has the following structure:

[0493] in Indicates the position of linkage to the sense strand of the siRNA via a phosphate group or a phosphorothioate group.

[0494] IV. Inhibition of ANGPTL3 Gene Expression

[0495] The siRNAs of the present disclosure are capable of inhibiting the expression of the ANGPTL3 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%.

[0496] Inhibition of ANGPTL3 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 ANGPTL3 gene is transcribed and which cell or cells have been treated (e.g., by contacting the cell or cells with an siRNA of the disclosure, or by administering an siRNA of the disclosure to a subject in which such cells are or were present) such that ANGPTL3 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.

[0497] 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 difference between the experimental group and the control group was compared by ΔΔCt, where ΔΔCt = [(Ct target gene of experimental group - Ct internal reference of experimental group) - (Ct target gene of control group - Ct internal reference of control group)].

[0498] Alternatively, inhibition of ANGPTL3 gene expression, e.g., ANGPTL3 protein expression, can be assessed in terms of a reduction in parameters functionally related to ANGPTL3 gene expression, such as lipid levels, cholesterol levels, e.g., LDLc levels. ANGPTL3 gene silencing can be determined in any cell expressing ANGPTL3, either constitutively or through genome engineering, and by any assay known in the art. The liver is a major site of ANGPTL3 expression. Other important sites of expression include the pancreas, kidney, and intestine.

[0499] Inhibition of ANGPTL3 protein expression can be demonstrated by a decrease in the level of ANGPTL3 protein expressed by a cell or cell group (e.g., the level of protein expressed in a sample derived 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.

[0500] Control cells or cell groups that can be used to assess inhibition of ANGPTL3 gene expression include cells or cell groups that have not been contacted with the siRNA disclosed herein. For example, the control cells or cell groups can be derived from an individual subject (e.g., a human or animal subject) prior to treatment of the subject with the siRNA.

[0501] V. Cells

[0502] The present disclosure provides a cell containing the siRNA of the present disclosure, wherein the siRNA of the present disclosure is capable of being transcribed in the cell.

[0503] VI. Pharmaceutical Compositions

[0504] The present disclosure provides a pharmaceutical composition comprising the siRNA or cells described herein, and optionally a pharmaceutically acceptable carrier or excipient.

[0505] 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.

[0506] As used herein, a pharmaceutically acceptable carrier refers to a pharmaceutical carrier that facilitates administration of siRNA or cells containing the siRNA into the human body and / or facilitates its absorption or function. Examples include diluents, 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.

[0507] For example, a pharmaceutical composition comprising the siRNA or cells of the present disclosure may comprise a pharmaceutically acceptable diluent or a sustained-release matrix in which the siRNA of the present disclosure is embedded.

[0508] VIII. Kit

[0509] The present disclosure provides a kit comprising the siRNA or cells described in the present disclosure.

[0510] The present disclosure also provides kits for using the siRNAs described herein and / or performing the methods described herein. Such kits include one or more siRNAs or cells described herein and may further include instructions for use. The instructions may include instructions for inhibiting ANGPTL3 expression in cells by contacting the cells with the siRNAs described herein in an amount effective to inhibit ANGPTL3 expression.

[0511] When the siRNA described herein is contacted with cells in vitro, the kits of the present disclosure may optionally further include a means for contacting cells with the siRNA described herein (e.g., an injection device) or a means for measuring the inhibitory effect of ANGPTL3 (e.g., a device for measuring the inhibition of ANGPTL3 mRNA or protein). Such a device for measuring the inhibition of ANGPTL3 may include a device for obtaining a sample (e.g., a plasma sample) from a subject.

[0512] When the siRNA described herein or cells into which siRNA has been introduced in vitro are administered in vivo, the kit of the present disclosure may optionally further comprise a device for administering the siRNA or cells described herein to a subject or a device for determining a therapeutically effective amount or a prophylactically effective amount.

[0513] IX. Methods of treatment and pharmaceutical uses

[0514] The present disclosure provides a method for reducing ANGPTL3 levels, LDL levels, apoC-III levels, triglyceride levels, cholesterol levels, glucose levels, and fat pad weight in a subject, comprising administering to the subject the siRNA, cell, or pharmaceutical composition of the present disclosure.

[0515] The present disclosure provides a method for treating a disease or symptom associated with ANGPTL3 expression in a subject, comprising administering to the subject the siRNA, cell, or pharmaceutical composition of the present disclosure.

[0516] In some embodiments, the disease associated with ANGPTL3 expression is a cardiovascular disease. In some preferred embodiments, the cardiovascular disease is selected from obesity, diabetes, atherosclerosis, dyslipidemia, coronary heart disease, non-alcoholic fatty liver disease (NAFLD), hyperfatty acidemia, or metabolic syndrome, or a combination thereof.

[0517] In some embodiments, the disease associated with ANGPTL3 expression is dyslipidemia, and the dyslipidemia is hyperlipidemia. In some embodiments, hyperlipidemia is hypercholesterolemia, hypertriglyceridemia, or a combination thereof.

[0518] In some embodiments, the disease associated with ANGPTL3 expression is NAFLD, and the NAFLD is hepatic steatosis or steatohepatitis.

[0519] In some embodiments, the disease associated with ANGPTL3 expression is diabetes, and the diabetes is type 2 diabetes or type 2 diabetes with dyslipidemia.

[0520] In some embodiments, the disclosed methods of treating a disease or symptom associated with ANGPTL3 expression in a subject comprise administering the siRNA or pharmaceutical composition to the subject, including subcutaneous administration or intravenous administration. In some embodiments, the subject is a human patient.

[0521] The present disclosure also relates to the siRNA, cells, or pharmaceutical compositions of the present disclosure for use in treating a disease or symptom associated with ANGPTL3 expression in a subject.

[0522] The present disclosure also relates to the use of the siRNA, cells, or pharmaceutical compositions described herein in the preparation of a medicament for treating a disease or symptom associated with ANGPTL3 expression in a subject. The medicament disclosed herein can be prepared into an emulsion, microemulsion, or microparticle.

[0523] sequence

[0524] The RNA sequences provided in the present disclosure target the human ANGPTL3 gene (or target gene, target mRNA sequence, target sequence).

[0525] Table 3 Nucleotide sequences of the sense and antisense strands targeting the ANGPTL3 gene

[0526] Tables 4 to 6 show the modified RNA sequences used in the present disclosure.

[0527] In this article, the meanings of the abbreviations are as follows:

[0528] The A, U, G, and C distribution represents the natural adenine, uracil, guanine, and cytosine ribonucleotides.

[0529] d indicates that the nucleotide adjacent to its right is a deoxyribonucleotide. For example, dA, dT, dG, and dC represent adenine deoxyribonucleotide, thymine deoxyribonucleotide, guanine deoxyribonucleotide, and cytosine deoxyribonucleotide, respectively.

[0530] i represents inosine ribonucleotide.

[0531] 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.

[0532] 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.

[0533] "s" or "s-" indicates that the two adjacent nucleotides and / or delivery vectors are linked by phosphorothioate.

[0534] 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.

[0535] IB represents an inverted abasic deoxyribonucleotide, which can include the following three structures depending on its location / connection method in the siRNA (used for the 5' end, middle and 3' end of the nucleic acid chain respectively):

[0536] 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.

[0537] L96 represents a GalNAc delivery vector of the following structure well known in the art, wherein The position of the siRNA is indicated by a phosphate group or a phosphorothioate group, as described in, for example, PCT Publication Nos. WO2009073809 and WO2009082607.

[0538] NAG37 represents a GalNAc delivery vector of the following structure well known in the art, wherein Indicates the position where the siRNA is linked via a phosphate group or a phosphorothioate group, see, for example, PCT Publication No. WO2018044350

[0539] GL6 represents a GalNAc delivery vector of the following structure, wherein Indicates the position where the phosphate group or phosphorothioate group is attached to the siRNA

[0540] GL12 represents a GalNAc delivery vector of the following structure, wherein Indicates the position where the phosphate group or phosphorothioate group is attached to the siRNA

[0541] STM1 represents a nucleotide substitute of the following structure. Depending on the position of STM1 in the nucleic acid chain, Can be linked to adjacent nucleotides, 3' end structure or 5' end structure

[0542] Table 4 Modified siRNA sense chain sequences targeting the ANGPTL3 gene

[0543] Table 5 Modified siRNA antisense strand sequences targeting the ANGPTL3 gene

[0544] Table 6 Paired siRNA sense and antisense strands targeting the ANGPTL3 gene Example

[0545] Unless otherwise specified, the sources of materials used in the examples are as follows:

[0546] Huh7 cell line was purchased from Nanjing Kebai, catalog number CBP60202;

[0547] Hep3B cell line was purchased from Nanjing Kebai, catalog number CBP60197;

[0548] PHH cells were purchased from Shanghai Xuanyi, catalog number QYLF-HPMC;

[0549] HEK293A cell line was purchased from Nanjing Kebai, catalog number CBP60436;

[0550] Balb / c mice were obtained from Zhejiang Weitonglihua, catalog number Balb / c.

[0551] Example 1 Preparation of Compound E7

[0552] 1. Preparation of Intermediates 3-4

[0553] 1.1 Preparation of Compound 2

[0554] 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.

[0555] 1.2 Preparation of compound 2A

[0556] 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.

[0557] 1.3 Preparation of compound 2B

[0558] Three reactions were performed in parallel.

[0559] 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.

[0560] 1.4 Preparation of compound 3

[0561] 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.

[0562] 1 H NMR: (400 MHz, DMSO).

[0563] 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).

[0564] 1.5 Preparation of Intermediate 3-4 (TFA Salt)

[0565] Three reactions were performed in parallel.

[0566] 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.

[0567] 1 H NMR: (400 MHz, DMSO-d6)

[0568] 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).

[0569] 2. Preparation of Intermediates 3-5

[0570] 2.1 Preparation of compound 5

[0571] 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 (3x 500mL), the combined organic layers were washed with saturated NaHCO (3x 800mL) until pH=8, washed with brine (3x 500mL), and dried and concentrated in vacuo to purify. 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 used to obtain compound 5 (approximately 180g+75.0g+87.0g+40.0g+38.0g) as a yellow solid.

[0572] 417.0 g of compound 3-4 was converted into compound 5 through 9 batches.

[0573] 2.2 Preparation of intermediate 3-3

[0574] 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.

[0575] 1 H NMR: (400 MHz, DMSO)

[0576] δ=8.53(t,J=5.2Hz,1H),8.18(d,J=2.4Hz,3H),8.03(t,J=5.2Hz,1H),7.84(d d,J=3.6Hz,2H),5.22(d,J=3.2Hz,2H),4.96(dd,J=3.2Hz,2H),4.55(d,J=8.4 Hz,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).

[0577] 3. Preparation of Compound E7

[0578] 3.1 Preparation of compound 3

[0579] 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.

[0580] 1 H NMR (400 MHz, CD3OD)

[0581] 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) + .

[0582] 3.2 Preparation of compound 4

[0583] 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.

[0584] 1 H NMR (400 MHz, CD3OD)

[0585] 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) + .

[0586] 3.3 Preparation of compound 6

[0587] 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.

[0588] 1 H NMR (400 MHz, CD3OD)

[0589] 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) + .

[0590] 4. Preparation of Compound E7

[0591] 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.

[0592] 1 H NMR (400 MHz, CD3OD)

[0593] 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)

[0594] MS: m / z = 3022.36 (M+H) + .

[0595] Example 2 Preparation of Compound E13

[0596] 1. Preparation of Intermediate 2-2

[0597] 1.1 Preparation of compound 3

[0598] Four reactions were performed in parallel.

[0599] To a solution of compound 1 (250 g, 741 mmol) in THF (1.75 L) was added 4-methylmorpholine (434 g, 4.30 mol, 472 mL), followed by cooling to 0°C. Isobutyl chloroformate (243 g, 1.78 mol, 233 mL) was added to the reaction mixture over 10 minutes, maintaining the reaction temperature below 4.0°C. After addition, the mixture was stirred for 40 minutes or longer, and compound 2 (526 g, 1.78 mol) was added to the reaction mixture over 10 minutes, maintaining the reaction temperature below 4.0°C. After addition, the ice bath was removed, and the reactants were then allowed to warm to room temperature for 2 hours. TLC (petroleum ether / ethyl acetate = 1 / 1, compound 1R f=0.43) shows that compound 1 is completely consumed and new spots are formed. 4 reactions were combined. The reaction solution was poured into a stirred cold (0 ℃) 0.50M HCl (aq.) (12.0L) solution and stirred for about 10 minutes. EtOAc (4.00L x 3) was subsequently added, stirred for a period of time, separated, and the organic phase was washed with brine (10.0L), dried with Na2SO4, and concentrated in vacuo to produce a thick colorless oil. Hexane (1.20L) was added to the stirred oil. White smoke appeared in the solution and then disappeared after further stirring. Seed crystals (1.20g, 0.10wt%) were added, and white crystals slowly formed. Within 20 minutes, the suspension was thick enough to hinder stirring, and additional hexane (6.00L) was added and stirred for 12 hours. The suspension was filtered, washed with hexane (1.20L) and dried to obtain compound 3 (about 1.20kg) as a white solid.

[0600] 1 H NMR: (400MHz DMSO)

[0601] δ=8.10-8.08(m,1H),7.62-7.60(m,1H),7.38-7.31(m,5H),5.11-4.94(m,2H),4.12-4.09(m,1H) ,3.92-3.87(m,1H),2.25-2.19(m,4H),1.90-1.88(m,2H),1.73-1.67(m,2H),1.40-1.39(m,27H).

[0602] 1.2 Preparation of compound 4

[0603] Five reactions were performed in parallel.

[0604] A solution of compound 3 (240 g, 415 mmol) in HCOOH (2.40 L) was stirred at 45 ° C for 2 hours. LCMS (compound 4 = 0.570 min) showed that compound 3 was completely consumed and the main peak of the desired m / z appeared. Five reactions were combined. Diluted with toluene and ACN (1.50 L each) and concentrated. Formic acid was removed by azeotropy using 1: 1 ACN and toluene (500 mL) and ACN three times (500 mL each). Compound 4 was dried under high vacuum. The residue was then stirred with DCM (500 mL) and the organic layer was poured off, followed by azeotropic drying with ACN (400 mL) twice, vacuum drying, and then azeotroped with toluene (400 mL) 9 times to obtain compound 4 (about 800 g) as a white solid.

[0605] 1 H NMR: (400MHz DMSO)

[0606] δ=12.4-12.2(m,2H),8.12-8.10(m,1H),7.62-7.60(m,1H),7.38-7.34(m,5H),5.07-5.02(m,2H) ,4.22-4.17(m,1H),3.99-3.96(m,1H),2.31-2.21(m,4H),2.00-1.93(m,2H),1.76-1.75(m,2H).

[0607] 1.3 Preparation of compound 6

[0608] To a stirred solution of compound 4 (80.0 g, 194 mmol) and (384 g, 700 mmol, TFA) in DMF (2.00 L) were added HOBT (103 g, 760 mmol), EDCI (146 g, 760 mmol), and DIEA (113 g, 877 mmol, 153 mL) in sequence. The reaction was stirred at 20°C for 2 hours. TLC (dichloromethane / methanol = 5 / 1, compound 6R f =0.43) showed that compound 4 was completely consumed and a new spot was formed. The reaction mixture was slowly poured into a stirred cold 0.5M HCl aqueous solution (230 mL), stirred for 10 minutes, a white solid was formed and filtered, and the aqueous phase was extracted twice with DCM (1.50 L). The combined organic phase was washed with 5% NaHCO3 (aq.) (200 mL), then dried (Na2SO4), and then concentrated by pressure evaporation. The product was purified by column chromatography (SiO2, dichloromethane / methanol=5 / 1, compound 6R f =0.43) to give compound 6 (about 180 g) as a yellow solid.

[0609] 1 H NMR: (400MHz DMSO)

[0610] δ7.95-7.91(m,3H),7.82-7.80(m,4H),7.39-7.31(m,6H),5.21-5.01 (m,3H),5.00-4.96(m,5H),4.56-4.53(m,3H),4.02(s,1H),3.88(s,9 H),3.85(s,4H),3.76(s,3H),3.50-349(m,9H),3.39-3.36(m,6H),3. 19-3.15(m,6H),2.15-2.05(m,12H),1.99(s,9H),1.89-1.77(m,21H).

[0611] 1.4 Preparation of intermediate 2-2

[0612] In a dry hydrogenation bottle under argon atmosphere, Pd(OH)2 / C (4.94 g, 3.52 mmol, 10% content) was added, MeOH (350 mL) was added, and compound 6 (47.4 g, 28.5 mmol) and TFA (3.26 g, 28.5 mmol, 2.11 mL) were added in sequence, and stirred at H2 (50 psi) and 20°C for 3 hours. By TLC (dichloromethane / methanol = 5 / 1, intermediate 2-2R f =0.25) and the reaction was monitored, showing the consumption of compound 6. The reaction mixture was filtered and the filtrate was concentrated in vacuo to afford intermediate 2-2 (TFA salt) as a white solid (46.3 g, 94.6% yield, 95.7% purity, TFA).

[0613] 1 H NMR: (400MHz DMSO)

[0614] δ=8.56-8.50(m,1H),8.14-8.05(m,4H),7.99(s,1H),7.88-7.82(m,4H),5. 22-5.21(m,3H),4.99-4.96(m,3H),4.55-4.53(m,3H),4.09(s,1H),4.03(s, 9H),3.87-3.79(m,6H),3.59-3.52(m,1H),3.51-347(m,9H),3.39-3.37(m,5 H),3.17-3.16(m,6H),2.22-2.10(m,12H),1.99(s,9H),1.93-1.73(m,21H).

[0615] 2. Preparation of Compound E13

[0616] 2.1 Preparation of compound Int1

[0617] 2.1.1 Preparation of Compound 1-2

[0618] At room temperature, compound 1-1 (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. 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 compound 1-2 (40.0 g) as a colorless oil.

[0619] 1 H NMR (400 MHz, CDCl3)

[0620] δ7.72(d,J=8.4Hz,2H),7.28-7.39(m,7H),4.72-4.86(m,2H),4.42-4.5 6(m,2H),3.52-3.61m,2H),3.14-3.39(m,2H),2.43(s,3H),1.47(s,9H)

[0621] 2.1.2 Preparation of Compounds 1-4

[0622] Compound 1-2 (79.0 g, 181 mmol), compound 1-3 (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) showed that 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 separated and purified by column chromatography (PE / EA=10 / 1-3 / 1) to obtain compound 1-4 (about 40.0 g) as a colorless oil.

[0623] 1 H NMR (400 MHz, CDCl3)

[0624] δ=7.64-7.77(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)

[0625] 2.1.3 Preparation of Compounds 1-5

[0626] Compound 1-4 (77.0 g, 128 mmol) and Et3N (28.6 mL, 205 mmol) were added sequentially to dichloromethane (800 mL) under an ice bath, and methanesulfonyl chloride (24.2 g, 212 mmol) was slowly added dropwise. The reaction was allowed to proceed at 25°C for 2 hours, and LCMS indicated the reaction was complete. The reaction solution was washed with water (800 mL), and the organic phase was concentrated under reduced pressure to afford compound 1-5 (approximately 90.0 g) as a colorless oil.

[0627] LCMS (ESI): m / z = 700.1 (M+Na) + ;

[0628] 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, 4H), 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).LCMS:m / z=700.1(M+Na) + .

[0629] 2.1.4 Preparation of Compounds 1-6

[0630] Compound 1-5 (90.0 g, 133 mmol) and potassium carbonate (91.8 g, 664 mmol) were added to MeOH (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) indicated 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 obtain compound 1-6 (approximately 64.0 g) as a white solid.

[0631] 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)

[0632] 1.1.5 Preparation of Compounds 1-7

[0633] Compound 1-6 (69.0 g, 143 mmol) and magnesium turnings (54.7 g, 2.28 mol) were added to MeOH (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 materials 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 afford compound 1-7 (approximately 32.0 g) as a colorless oil.

[0634] 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)

[0635] 1.1.6 Preparation of Compound 1-8

[0636] At room temperature, compound 1-7 (8.00 g, 24.4 mmol) was added to HCl (100 mL, 12 M) and reacted at 50°C for 2 hours. The reaction solution was concentrated under reduced pressure to obtain a colorless oily compound 1-8 (about 3.60 g, HCl salt).

[0637] 1 H NMR (400MHz, CDCl3) δ4.12-4.22(m,2H),3.78(d,J=4.4Hz,4H),3.17-3.30(m,4H)

[0638] 1.1.7 Preparation of Compound Int-1

[0639] Compound 1-8 (8.29 g, 24.5 mmol) was added to pyridine (50 mL, 618 mmol) at room temperature, followed by the addition of DMTrCl (4.77 g, 12.3 mmol). The reaction system was allowed to react at 25°C for 18 hours. TLC (DCM / MeOH = 10 / 1, UV 254 nm) indicated the formation of the product. The reaction solution was concentrated under reduced pressure, diluted with saturated ammonium chloride (200 mL), extracted with DCM (100 mL x 3), and the organic phase was concentrated under reduced pressure. The crude product was isolated by column chromatography (DCM / MeOH = 99 / 1-10 / 1) to afford Int-1 (approximately 2.6 g, 5.78 mmol, 23.6%) as a yellow solid.

[0640] 1 H NMR (400MHz, CDCl3) δ7.38-7.50(m,2H),7.25-7.36(m,6H),7.18-7.25(m,1H),6.87(d,J=8.8Hz,4H),4.26-4.40(m,1H ),3.94-4.05(m,1H),3.85(d,J=4.4Hz,2H)3.73-3.82(m,6H),3.34-3.41(m,1H),3.23-3.30(m,3H),3.05-3.23(m,2H)

[0641] 2.2 Preparation of compound Int6

[0642] 2.2.1 Preparation of compound 3

[0643] Compound 1 (1.00 g, 3.05 mmol) was dissolved in anhydrous N,N-dimethylformamide (10.0 mL) at 25°C. HOBt (0.54 g, 3.97 mmol), EDCI (0.76 g, 3.97 mmol), and DIEA (1.51 mL, 9.16 mmol) were added sequentially, followed by compound 2 (0.79 g, 3.66 mmol). The mixture was stirred at 25°C for 2 hours. LC / MS analysis showed complete reaction of the starting material and formation of the product. Thin-layer chromatography (dichloromethane:methanol = 10:1) also showed complete reaction of the starting material and formation of the product. Dichloromethane (200 mL) was added to the mixture, and the mixture was washed three times with saturated citric acid solution (20 mL x 3), saturated sodium bicarbonate solution (20 mL x 3), and saturated brine (20 mL x 3). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (dichloromethane:methanol=10:1) to give compound 3 (about 1.00 g) as a yellow oil.

[0644] 1H NMR (400MHz, CD3OD) δ7.21-7.41(m,10H),4.47-4.60(m,4H),3.88-4.01(m,2H),3.75(dd,J=3.2,13.2 Hz,1H),3.63-3.65(m,3H),3.40-3.63(m,7H),2.27-2.34(m,4H),1.50-1.60(m,4H),1.28-1.34(m,8H)

[0645] LCMS: m / z=526.8 (M+H) + .

[0646] 2.2.2 Preparation of compound 4

[0647] Compound 3 (380 mg, 0.20 mmol) was dissolved in anhydrous methanol (10.0 mL) at 25°C, and 10% wet Pd / C (500 mg, 4.70 mmol) was added. The reaction was stirred at 50°C for 12 hours. LC / MS analysis indicated the formation of the product. The reaction mixture was filtered through celite, and the filtrate was concentrated under reduced pressure to yield compound 4 (approximately 600 mg) as a yellow oil.

[0648] 1 H NMR (400MHz, CD3OD) δ3.79-3.89(m,2H),3.73(dd,J=3.6,13.2Hz,1H),3.64-3.68(m,5H),3.57-3.62(m,2H),3.48-3 .56(m,2H),3.38-3.47(m,1H),1.58-1.62(m,5H),1.34(d,J=2.8Hz,11H),1.30-1.37(m,1H).LCMS:m / z=346.5(M+H) + .

[0649] 2.2.3 Preparation of compound 5

[0650] Compound 4 (600 mg, 1.74 mmol) was dissolved in pyridine (3.00 mL) at 25°C, and DMTrCl (883 mg, 2.61 mmol) was added. The reaction mixture was stirred at 25°C for 1 hour. LC-MS / MS indicated the formation of the product. Thin-layer chromatography (dichloromethane:methanol = 10:1) showed complete reaction of the starting materials and the formation of the product. Dichloromethane (200 mL) was added to the mixture, and the mixture was washed three times with saturated sodium bicarbonate solution (20 mL x 3) and then with brine (20 mL x 3). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 3:1) to obtain compound 5 (approximately 475 mg) as a colorless liquid.

[0651] 1 H NMR(400MHz,CD3OD)δ7.41-7.43(m,2H),7.16-7.34(m,7H),6.83-6.87(m,4 H),3.88-4.04(m,2H),3.77-4.09(m,7H),3.61-3.68(m,4H),3.43-3.58(m,3 H),3.33-3.38(m,1H),3.14-3.20(m,1H),3.05(dd,J=8.4,13.2Hz,1H),2.2 0-2.42(m,4H),1.51-1.60(m,4H),1.25-1.39(m,8H).LCMS:m / z=648.3(M+H) + .

[0652] 2.2.4 Preparation of Compound Int-6

[0653] Compound 5 (475 mg, 0.73 mmol) was dissolved in tetrahydrofuran (8.00 mL) and H₂O (2.00 mL) at 25°C, and lithium hydroxide monohydrate (36.9 mg, 0.89 mmol) was added. The reaction mixture was stirred at 25°C for 12 hours. Thin-layer chromatography (dichloromethane:methanol = 10:1) indicated complete reaction of the starting materials and formation of the product. The reaction mixture was concentrated under reduced pressure and lyophilized directly to yield compound 6 (approximately 430 mg) as a white solid.

[0654] 2.3 Preparation of compound E13

[0655] 2.3.1 Preparation of compound 7

[0656] Compound Int-6 (100 mg, 0.156 mmol) was dissolved in anhydrous N,N-dimethylformamide (3.00 mL) at 25°C. HBTU reagent (89.0 mg, 0.234 mmol), DIEA (0.078 mL, 0.47 mmol), and Intermediate C (254 mg, 0.156 mmol, synthesized according to the method for Intermediate 2-2 in Example 2) were added sequentially. The reaction mixture was stirred at 25°C for 12 hours. Thin-layer chromatography (dichloromethane:methanol = 10:1) showed the formation of a new spot. Dichloromethane (200 mL) was added to the mixture, and the mixture was washed three times with saturated sodium bicarbonate solution (20 mL x 3) and three times with brine (20 mL x 3). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (dichloromethane:methanol = 10:1) to obtain Compound 7 (approximately 140 mg) as a yellow oil.

[0657] 1 H NMR (400MHz, CD3OD) δ7.43(d,J=8.4Hz,2H),7.18-7.34(m,7H),6.81-6.91(m,4H),5.34(d,J=2.8Hz,3H),5.09-5 .11(m,3H),4.62-4.66(m,3H),4.27-4.35(m,2H),4.07-4.17(m,9H),4.00-4.07(m,4H),3.89-3.98(m,5H),3.79 (d,J=2.4Hz,6H),3.69-3.75(m,5H),3.60-3.64(m,9H),3.52-3.56(m,10H),3.43-3.50(m,4H),3.39-3.40(m,1H ),2.25-2.40(m,8H),2.13-2.15(m,9H),2.02(s,9H),1.94-1.96(m,18H),1.48-1.66(m,4H),1.21-1.36(m,11H)

[0658] 2.3.2 Preparation of E13

[0659] Compound 7 (400 mg, 0.187 mmol) was dissolved in anhydrous dichloromethane (5.00 mL). DIEA (0.18 mL, 1.12 mmol) and DMAP (5.71 mg, 0.047 mmol) were added sequentially, followed by compound 8 (112 mg, 1.12 mmol). The mixture was stirred at 25°C for 3 hours. Thin-layer chromatography (dichloromethane:methanol = 10:1) revealed the formation of a new spot. Dichloromethane (200 mL) was added to the mixture, and the mixture was washed three times with saturated brine (15.0 mL x 3). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to yield the crude product. The crude product was separated by HPLC (column: Welch Xtimate C18 150*25mm*5um; mobile phase: water-ACN; gradient: 27%-57% / 11min; flow rate: 25ml / min) to give white solid compound E13 (74.8mg, 0.033mmol, yield 17.9%).

[0660] LCMS (ESI): m / z = 1149.5 (M / 2+H) + ; 1 H NMR (400MHz, CD3OD) δ7.42-7.44(m,2H),7.18-7.34(m,7H),6.83-6.90(m,4H),5.33-5.34(m,3H),5.08-5.11(m,3H),4.61-4.68(m,3H),4.27-4 .36(m,2H),4.19-4.27(m,1H),4.10-4.19(m,7H),4.08(s,2H),4.00-4. 07(m,4H),3.90-3.94(m,4H),3.83-3.89(m,1H),3.79(d,J=2.4Hz,6H), 3.70-3.72(m,4H),3.53-3.54(m,15H),3.44-3.48(m,3H),3.35-3.40(m,4H),3.10-3.23(m,2H),2.53-2.63(m,4H),2.22- 2.40(m,8H),2.11-2.16(m,9H),2.05-2.10(m,2H),2.02(s,9H),1.90-1.97(m,19H),1.48-1.65(m,4H),1.29-1.32(m,8H)

[0661] Example 3 Preparation of Compound E2

[0662] 1. Preparation of Compound 2b

[0663] 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). 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)

[0664] 2. Preparation of Compound 2d

[0665] 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).

[0666] 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)

[0667] 3. Preparation of Compound 2e

[0668] 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).

[0669] m / z: ES+[M+Na]+700.1

[0670] 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).

[0671] 4. Preparation of Compound 2f

[0672] 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) indicated 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 to 2 / 1) to afford compound 2f (64.0 g, 99.9% yield).

[0673] 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)

[0674] 5. Preparation of Compound 2g

[0675] 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).

[0676] 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)

[0677] 6. Preparation of Compound 2h

[0678] 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).

[0679] 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)

[0680] 7. Preparation of Compound 2i

[0681] 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).

[0682] 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)

[0683] 8. Preparation of Compound 2j

[0684] 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).

[0685] 9. Preparation of Compound E2

[0686] 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).

[0687] m / z:ES+[M+H]+732.2

[0688] 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)

[0689] Example 4 Preparation of siRNA

[0690] The siRNA of the present disclosure was 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.

[0691] 1. Preparation of siRNA without Ligand

[0692] 1.1 Synthesis of the positive sense chain (SS chain)

[0693] Using solid-phase phosphoramidite synthesis, a blank CPG solid support is used as the starting cycle. Nucleoside monomers or nucleotide analog monomers are linked one by one in the 3'-5' direction according to the order of the positive strand nucleotides. Each linking step involves deprotection, coupling, capping, and oxidation or thiolation. The conditions for oligonucleotide synthesis at a synthesis scale of 5 μmol are as follows:

[0694] Nucleoside monomers or nucleotide analog monomers were provided in 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 3% trichloroacetic acid-dichloromethane solution. The coupling reaction used an activator of 0.25 mol / L ETT-acetonitrile solution and was performed twice. 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] The nucleotide monomers of IB were purchased from Shanghai Zhaowei Technology Development Co., Ltd., product number OP-040.

[0696] 1.2 Synthesis of antisense strand (AS strand)

[0697] Using solid-phase phosphoramidite synthesis, a blank CPG solid support is used as the starting cycle. Nucleoside monomers or nucleotide analogs are linked one by one in the 3'-5' direction according to the nucleotide arrangement of the antisense strand. Each ligation step involves deprotection, coupling, capping, and oxidation or thiolation. Synthesis conditions for 5 μmol of oligonucleotides for the antisense strand are the same as for the sense strand.

[0698] 1.3 Oligonucleotide purification and annealing

[0699] 1.3.1 Ammonolysis

[0700] 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.

[0701] 1.3.2 Purification

[0702] 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.

[0703] 1.3.3 Annealing

[0704] According to Table 6, the sense strand (SS strand) and the antisense strand (AS strand) were mixed at a molar ratio (SS strand / AS strand = 1 / 1.05), heated in a water bath to 70-95 degrees, maintained for 3-5 minutes, naturally cooled to room temperature, and the system was lyophilized to obtain the product.

[0705] 2. Preparation of siRNA with Sense Strand Linked to Ligand

[0706] 2.1 Ligand-CPG Carrier Connection

[0707] 2.1.1 Ligation of compound E7 to CPG carrier

[0708] 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.

[0709] Place 530 mg of the condensed product in a 50 mL round-bottom flask. Add Cap C (DMAP / acetonitrile), Cap B (N-methylimidazole / pyridine / acetonitrile), and Cap A (acetic anhydride / acetonitrile) in that order. Shake overnight at room temperature. Filter, wash the filter cake with acetonitrile (20 mL x 4). Remove the filter cake and filter under reduced pressure with an oil pump for 8 hours to obtain 200 mg of an off-white solid, which was used for solid-phase synthesis.

[0710] 2.1.2 Ligation of compound E13 to CPG carrier

[0711] Compound E13 was linked to the CPG carrier in a manner similar to that of compound E7.

[0712] 2.2 Synthesis of the positive sense chain (SS chain)

[0713] Using the solid-phase phosphoramidite synthesis method, using the GL6 solid support prepared in Section 2.1.1 of Example 4 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 step involved deprotection, coupling, capping, and oxidation or thiolation. The conditions for oligonucleotide synthesis at a 5 μmol scale were as follows:

[0714] 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).

[0715] 2.3 Synthesis of antisense strand (AS strand)

[0716] 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.

[0717] 2.4 Oligonucleotide Purification and Annealing

[0718] 2.4.1 Ammonolysis

[0719] 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.

[0720] 2.4.2 Purification

[0721] 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.

[0722] 2.4.3 Annealing

[0723] According to Table 6, the sense chain (SS chain) and the antisense chain (AS chain) were mixed at a molar ratio (SS chain / AS chain = 1 / 1.05), heated in a water bath to 70-95° C., maintained for 3-5 min, naturally cooled to room temperature, and the system was lyophilized to obtain the product.

[0724] L96-conjugated siRNA and NAG37-conjugated siRNA were obtained in a similar manner.

[0725] Example 5 Activity screening of Huh7 cell line

[0726] Cell transfection

[0727] On the first day, Huh7 cell line (Nanjing Kebai, Cat. No. CBP60202) was digested and resuspended, counted, and 1×10 4 Cells / well were plated in 96-well plates and transfection was performed 18 h later.

[0728] On the second day, the 20 μM siRNA stock solution prepared in Example 4 was diluted with Opti-MEM. 198 μL of Opti-MEM was added to 2 μL of the siRNA stock solution, and the mixture was mixed by pipetting. The dilution was performed according to the experimental requirements for each experiment.

[0729] 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. Next, take 15 μL of the prepared RNAi-MAX mixture and 15 μL of the diluted compound and gently pipette to mix thoroughly, avoiding the introduction of bubbles. Let stand at room temperature for 10 minutes, then add 10 μL / well to a 96-well plate. Incubate in a 37°C, 5% CO2 incubator for 24 hours before RNA extraction.

[0730] RNA extraction

[0731] Cell RNA was extracted using a nucleic acid extractor (Hangzhou Aosheng, Auto-pure96) according to the operating procedures of the high-throughput cell RNA extraction kit (Fanzhi Medical, FG0412).

[0732] RNA reverse transcription

[0733] 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.

[0734] 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.

[0735] 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.

[0736] After reverse transcription, 30 μL of DNase- and RNase-free distilled water was added to the cDNA sample in each well.

[0737] Fluorescence quantitative PCR

[0738] ReferenceTaqMan TMFast 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.

[0739] Table 7 Primer information

[0740] Statistics

[0741] Calculation 2 -△△Ct The values ​​were converted into percentages to obtain the residual inhibition rate.

[0742] △△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)].

[0743] The final concentration of siRNA was 1 nM for high-throughput screening of siRNA compound cell line activity. The experimental screening results are shown in Table 8.

[0744] Table 8 Results of high-throughput screening of Huh7 cell line activity using 1 nM siRNA

[0745] Example 6: Screening Huh7 cell line activity using 5 concentrations of siRNA diluted 10-fold

[0746] Similar to the activity screening in Example 5 above, further activity screening was performed using the Huh7 cell line.

[0747] The siRNA was screened for activity against Hep3B cells using a 10-fold serial dilution to obtain five concentrations (10 nM, 1 nM, 0.1 nM, 0.01 nM, and 0.001 nM). The results are shown in Table 9, where columns 2-6 represent the residual inhibition rate and column 7 represents the IC50 value.

[0748] Table 9 Results of Huh7 cell line activity screening using 5 concentrations of siRNA

[0749] Example 7 Activity screening of primary human hepatocytes (PHH cells)

[0750] Cell transfection

[0751] Take 1.4 mL of rat tail collagen solution (Sigma, C3867), add it to 40.6 mL of distilled water without DNase and RNase, mix well, add 40 μL to each well of a 96-well culture plate, coat overnight at 4°C, and remove the coating solution the next day.

[0752] 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 4 cells / well; complete culture medium was replaced after 4 hours, and transfection was performed after 18 hours.

[0753] On the third day, 20 μM siRNA stock solution was diluted with Opti-MEM. 198 μL Opti-MEM was added to 2 μL siRNA stock solution and mixed by pipetting. This was used as the first concentration point and gradient dilution was performed according to actual experimental needs.

[0754] On the third day, dilute 0.9 μL RNAiMAX (Thermo, 13778150) with 14.1 μL 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 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.

[0755] RNA extraction

[0756] Cell RNA was extracted using a nucleic acid extractor (Hangzhou Aosheng, Auto-pure96) according to the operating procedures of the high-throughput cell RNA extraction kit (Fanzhi Medical, FG0412).

[0757] RNA reverse transcription

[0758] 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.

[0759] Preparation of reverse transcription reaction solution, refer to PrimeScript TMII 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.

[0760] 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.

[0761] After reverse transcription, 30 μL of DNase- and RNase-free distilled water was added to the cDNA sample in each well.

[0762] Fluorescence quantitative PCR

[0763] 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.

[0764] Table 10 Primer information

[0765] Statistics

[0766] Calculation 2 -△△Ct The values ​​were converted into percentages to obtain the residual inhibition rate.

[0767] △△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)].

[0768] 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 11, where columns 2-6 are the residual inhibition rates and column 7 is the IC50 value.

[0769] Table 11 Results of screening human primary hepatocytes using 5 concentrations of siRNA

[0770] Example 8 psiCHECK2 GSSM-5 Hits off-target activity screening

[0771] Plasmid preparation

[0772] The corresponding antisense off-target plasmid was designed according to the siRNA sequence, and the psiCHECK2 GSSM-5Hits recombinant plasmid was prepared by Sangon Biotech (Shanghai) Co., Ltd. and diluted to 1000 ng / μL for later use.

[0773] Cell transfection

[0774] 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.

[0775] 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.

[0776] siRNA preparation: Dilute siRNA 3-fold starting from a final concentration of 40 nM, for a total of 11 concentration points (10 nM, 3.3333 nM, 1.1111 nM, 0.37037 nM, 0.12346 nM, 0.04115 nM, 0.01372 nM, 0.00457 nM, 0.00152 nM, 0.00051 nM, 0.00017 nM).

[0777] 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.

[0778] 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.

[0779] 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.

[0780] Result detection

[0781] 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.

[0782] Data analysis and processing

[0783] 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:

[0784] Normalized Ren / Fir ratio: Ratio = Renilla (Renilla luciferase) / Firefly (Firefly luciferase).

[0785] 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).

[0786] Graphing: Graphpad Prism

[0787] 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).

[0788] The results of the psiCHECK2 GSSM-5 Hits off-target activity screening of siRNA are shown in Table 12, where columns 2-12 represent the residual inhibition rate and column 13 represents the IC50 value. The results show that DR000405, DR000424, DR000430, DR000442, DR000447, DR000619, and DR000621 of the present disclosure have lower off-target activity against HEK293A cells than the positive control DR001483.

[0789] Table 12 psiCHECK2 GSSM-5Hits off-target activity screening results

[0790] Example 9 Mouse HDI model activity screening

[0791] HDI animal model

[0792] 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 (Genbank accession number NM_014495.2) and Piggy-Bac helper plasmids (purchased from Suzhou Bangye) (mass ratio 1:1, total plasmid 100 μg) were injected into the mouse through a 27-gauge needle within 5-7 seconds. The delivery solution (total volume of 10% animal body weight, Mirusbio-MIR 5240) was injected into mice and placed back in their cages for observation for 30 minutes after injection. The day of modeling was designated as day 0, and serum was obtained at various time points after modeling (days 7-35) to detect SEAP expression levels.

[0793] 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. The dual-gene expression element contains the secreted alkaline phosphatase gene (SEAP) and the target gene (ANGPTL3). SEAP and ANGPTL3 are co-expressed.

[0794] Detection of SEAP expression

[0795] 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.

[0796] The CSPD substrate was mixed with the reaction buffer at a ratio of 1:20 to prepare the reaction solution. 5× dilution buffer was diluted to 1× dilution buffer with DNase- and RNase-free distilled water. The serum was mixed with 1× dilution buffer in a centrifuge tube to prepare the sample dilution solution. The sample dilution solution was incubated at 65°C for 30 minutes and then cooled to room temperature. Fifty microliters of the sample dilution solution was added to a 96-well plate, and 50 μL of assay buffer was added to each well. The solution was incubated at room temperature for 5 minutes. Fifty microliters of the reaction solution was added to each well, and the solution was incubated at room temperature for 20 minutes. The SEAP chemiluminescence value was read on a microplate reader (Tecan, Infinite 200).

[0797] The effectiveness of the siRNA compound in inhibiting target gene expression was evaluated by measuring SEAP expression levels in serum. The lower the SEAP chemiluminescence value, the better the siRNA compound's ability to inhibit target gene expression. Samples that inhibit SEAP expression were selected as nucleic acid drugs.

[0798] On day 15 after modeling, each mouse was given a single subcutaneous administration of 200 μl of saline containing 3 mg / kg (mpk) RNAi agent, or 200 μl of saline without RNAi agent as a vehicle control, according to Table 13. The results of the HDI model screening are shown in Table 14.

[0799] Table 13 Single subcutaneous administration of RNAi reagent dosage in mice

[0800] Table 14 Experimental results of HDI stable transgenic plasmid mouse model

[0801] Example 10 Activity screening of mouse HDI model

[0802] According to the method of Example 9 above, the sequences in Table 15 were further screened for activity, and the results are shown in Table 16.

[0803] Table 15. siRNA sequences

[0804] Table 16. Experimental results of HDI stable plasmid mouse model

[0805] Example 11 Activity screening of mouse HDI model

[0806] According to the method of Example 9, the sequences in Table 17 were further screened for activity. The results are shown in Table 18.

[0807] Table 17. siRNA sequences

[0808] Table 18. Experimental results of HDI stable plasmid mouse model

Claims

1. A small interfering RNA (siRNA) for inhibiting the expression of angiopoietin-like 3 (ANGPTL3) protein 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 a nucleotide sequence of at least 15 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 16-29. 2 . The siRNA according to claim 1 , wherein the sense strand comprises a nucleotide sequence of at least 15 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 1-14.

3. The siRNA according to any one of claims 1 to 2, wherein the length of the double-stranded region is 15-25 nucleotide pairs, preferably 17-23 nucleotide pairs, more preferably 19-21 nucleotide pairs.

4. The siRNA according to any one of claims 1 to 3, wherein the antisense strand comprises a nucleotide sequence of at least 16 consecutive nucleotides, a nucleotide sequence of at least 17 consecutive nucleotides, a nucleotide sequence of at least 18 consecutive nucleotides, a nucleotide sequence of at least 19 consecutive nucleotides, or a nucleotide sequence of at least 20 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 16 to 29. Preferably, the antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NOs: 16 to 29.

5. The siRNA according to any one of claims 1 to 4, wherein the sense strand comprises a nucleotide sequence of at least 16 consecutive nucleotides, a nucleotide sequence of at least 17 consecutive nucleotides, or a nucleotide sequence of at least 18 consecutive nucleotides of any one of the nucleotide sequences shown in SEQ ID NOs: 1 to 14, and preferably the antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NOs: 1 to 14. 6 . The siRNA according to claim 1 , wherein the siRNA comprises a paired sense strand sequence and antisense strand sequence as shown in Table 3.

7. The siRNA of any one of claims 1 to 6, wherein substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, or all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides.

8. The siRNA of claim 7, 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, non-natural base containing nucleotides, terminal nucleotides attached to a cholesteryl derivative or a dodecyldiamide group, deoxyribonucleotides, and STM1.

9. The siRNA of claim 7, 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, inverted abasic deoxyribonucleotides, phosphorothioate internucleotide linkage modifications, and STM1.

10. The siRNA according to claim 7, wherein (a) The sense strand comprises CmsAmsCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAm (SEQ ID NO: 31), The antisense strand comprises UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm (SEQ ID NO: 61); (b) the sense strand comprises CmsAmsAmUmUmAmAfGfCfUmCmCmUmUmCmUmUmUmUm (SEQ ID NO: 32), The antisense strand comprises AmsAfsAmAfGmAfAmGfGmAfGmCfUmUfAmAfUmUfGmsUfsGm (SEQ ID NO: 62); (c) the sense strand comprises UmsAmsUmUmGmUmUfCfCfUmCmUmAmGmUmUmAmUmUm (SEQ ID NO: 33), The antisense strand comprises AmsAfsUmAfAmCfUmAfGmAfGmGfAmAfCmAfAmUfAmsAfsAm (SEQ ID NO: 63); (d) the sense strand comprises GmsUmsUmAmUmUmUfCfCfUmCmCmAmGmAmAmUmUmAm (SEQ ID NO: 34), The antisense strand comprises UmsAfsAmUfUmCfUmGfGmAfGmGfAmAfAmUfAmAfCmsUfsAm (SEQ ID NO: 64); (e) the sense strand comprises AmsGmsAmAmUmUmGfAfUfCmAmAmGmAmCmAmAmUmUm (SEQ ID NO: 35), The antisense strand comprises AmsAfsUmUfGmUfCmUfUmGfAmUfCmAfAmUfUmCfUmsGfsGm (SEQ ID NO: 65); (f) the sense strand comprises AmsUmsCmAmAmGmAfCfAfAmUmUmCmAmUmCmAmUmUm (SEQ ID NO: 36), The antisense strand comprises AmsAfsUmGfAmUfGmAfAmUfUmGfUmCfUmUfGmAfUmsCfsAm (SEQ ID NO: 66); (g) the sense strand comprises GmsAmsGmCmCmAmAfAfAfUmCmAmAmGmAmUmUmUmAm (SEQ ID NO: 37), The antisense strand comprises UmsAfsAmAfUmCfUmUfGmAfUmUfUmUfGmGfCmUfCmsUfsGm (SEQ ID NO: 67); (h) the sense strand comprises UmsGmsAmAmCmUmCfAfAfCmUmCmAmAmAmAmCmUmUm (SEQ ID NO: 38), The antisense strand comprises AmsAfsGmUfUmUfUmGfAmGfUmUfGmAfGmUfUmCfAmsAfsGm (SEQ ID NO: 68); (i) the sense strand comprises AmsGmsAmGmCmAmAfCfUfAmAmCmUmAmAmCmUmUmAm (SEQ ID NO: 39), The antisense strand comprises UmsAfsAmGfUmUfAmGfUmUfAmGfUmUfGmCfUmCfUmsUfsCm (SEQ ID NO: 69); (j) the sense strand comprises GmsCmsAmAmCmUmAfAfCfUmAmAmCmUmUmAmAmUmUm (SEQ ID NO: 40), The antisense strand comprises AmsAfsUmUfAmAfGmUfUmAfGmUfUmAfGmUfUmGfCmsUfsCm (SEQ ID NO: 70); (k) the sense strand comprises CmsUmsAmAmCmUmAfAfCfUmUmAmAmUmUmCmAmAmAm (SEQ ID NO: 41), The antisense strand comprises UmsUfsUmGfAmAfUmUfAmAfGmUfUmAfGmUfUmAfGmsUfsUm (SEQ ID NO: 71); (l) The sense chain comprises CmsUmsAmAmCmUmUfAfAfUmUmCmAmAmAmUmCmAm (SEQ ID NO: 42), The antisense strand comprises UmsGfsAmUfUmUfUmGfAmAfUmUfAmAfGmUfUmAfGmsUfsUm (SEQ ID NO: 72); (m) the sense strand comprises UmsAmsAmCmUmUmAfAfUfUmCmAmAmAmUmCmAmAm (SEQ ID NO: 43), The antisense strand comprises AmsAfsUmAfAmCfUmAfGmAfGmGfAmAfCmAfAmUfAmsAfsAm (SEQ ID NO: 73); or (n) the sense chain comprises GmsGmsAmUmCmAmCfAfAfAmAmCmUmUmCmAmAmUmAm (SEQ ID NO: 44), The antisense strand comprises UmsAfsUmUfGmAfAmGfUmUfUmUfGmUfGmAfUmCfCmsAfsUm (SEQ ID NO:74).

11. The siRNA of claim 7, wherein: (a) The sense strand comprises UmsGmsAmAmCmUmCfAfAfCmUmCmAmAmAmAmCmUmsUm (SEQ ID NO: 131), and the antisense strand comprises AmsAfsGmUfUmUfUmGfAmGfUmUfGmAfGmUfUmCfAmsAfsGm (SEQ ID NO: 132); (b) the sense strand comprises GmsGmsAmUmCmAmCfAfAfAmAmCmUmUmCmAmAmUmsAm (SEQ ID NO: 133), and the antisense strand comprises UmsAfsUmUfGmAfAmGfUmUfUmUfGmUfGmAfUmCfCmsAfsUm (SEQ ID NO: 134); (c) the sense strand comprises CmsAmsCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmsAm (SEQ ID NO: 135), and the antisense strand comprises UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm (SEQ ID NO: 136); (d) the sense strand comprises STM1s-CmsUmUmGmAmAmCmUmCfAfAfCmUmCmAmAmAmAmCmUmUms-STM1 (SEQ ID NO: 137), and the antisense strand comprises AmsAfsGmUfUmUfUmGfAmGfUmUfGmAfGmUfUmCfAmsAfsGm (SEQ ID NO: 138); (e) the sense strand comprises STM1s-AmsUmGmGmAmUmCmAmCfAfAfAmAmCmUmUmCmAmAmUmAms-STM1 (SEQ ID NO: 139), and the antisense strand comprises UmsAfsUmUfGmAfAmGfUmUfUmUfGmUfGmAfUmCfCmsAfsUm (SEQ ID NO: 140); (f) the sense strand comprises STM1s-UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAms-STM1 (SEQ ID NO: 141), and the antisense strand comprises UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm (SEQ ID NO: 142); (g) the sense strand comprises STM1-STM1- UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAm-STM1-STM1 (SEQ ID NO: 143), and the antisense strand comprises UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm (SEQ ID NO: 144); or (h) the sense strand comprises IBs-UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAms-IB (SEQ ID NO: 145), and the antisense strand comprises UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm (SEQ ID NO: 146).

12. The siRNA of claim 7, wherein: (a) The sense strand comprises STM1-STM1- UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmsAm-STM1-STM1 (SEQ ID NO: 147), and the antisense strand comprises UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm (SEQ ID NO: 148); (b) the sense strand comprises STM1s-UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAm-STM1-STM1 (SEQ ID NO: 149), and the antisense strand comprises UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm (SEQ ID NO: 150); (c) the sense strand comprises STM1s-CmsAmAmGmAmCmAmAmUfUfCfAmUmCmAmUmUmUmGmAmUms-STM1 (SEQ ID NO: 151), and the antisense strand comprises AmsUfsCmAfAmAfUmGfAmUfGmAfAmUfUmGfUmCfUmsUfsGm (SEQ ID NO: 152); (d) the sense strand comprises STM1-STM1- CmsAmAmGmAmCmAmAmUfUfCfAmUmCmAmUmUmUmGmAmsUm-STM1-STM1 (SEQ ID NO: 153), and the antisense strand comprises AmsUfsCmAfAmAfUmGfAmUfGmAfAmUfUmGfUmCfUmsUfsGm (SEQ ID NO: 154); (e) the sense strand comprises STM1s-CmsAmAmGmAmCmAmAmUfUfCfAmUmCmAmUmUmUmGmAmUm-STM1-STM1 (SEQ ID NO: 155), and the antisense strand comprises AmsUfsCmAfAmAfUmGfAmUfGmAfAmUfUmGfUmCfUmsUfsGm (SEQ ID NO: 156); (f) the sense strand comprises STM1s-UmsGmGmUmAmUmUmAmAfAfUfCmCmUmUmAmAmGmAmGmAms-STM1 (SEQ ID NO: 157), and the antisense strand comprises UmsCfsUmCfUmUfAmAfGmGfAmUfUmUfAmAfUmAfCmsCfsAm (SEQ ID NO: 158); (g) the sense strand comprises STM1s-CmsCmAmGmAmAmUmUmGfAfUfCmAmAmGmAmCmAmAmUmUms-STM1 (SEQ ID NO: 159), and the antisense strand comprises AmsAfsUmUfGmUfCmUfUmGfAmUfCmAfAmUfUmCfUmsGfsGm (SEQ ID NO: 160); or (h) the sense strand comprises STM1s-UmsGmAmUmCmAmAmGmAfCfAfAmUmUmCmAmUmCmAmUmUms-STM1 (SEQ ID NO: 161), and the antisense strand comprises AmsAfsUmGfAmUfGmAfAmUfUmGfUmCfUmUfGmAfUmsCfsAm (SEQ ID NO: 162).

13. The siRNA according to any one of claims 1 to 12, wherein the siRNA is further conjugated to a ligand moiety comprising N-acetylgalactosamine, preferably the 3' end of the sense strand of the siRNA is conjugated to the ligand moiety.

14. The siRNA of claim 13, wherein the ligand moiety comprises a conjugate group represented by formula (X'): in, Indicates the location of attachment to a biomolecule; 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 -, -(OCH2CH2CH2CH2CH) 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.

15. The siRNA of claim 14, wherein the conjugated group is represented by formula (I'): in, Indicates the location of attachment to a biomolecule; 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.

16. The siRNA according to claim 15, 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.

17. The siRNA of claim 16, wherein the conjugated group is represented by Formula (I'-1), Formula (I'-2), or Formula (I'-3): in, Indicates the location of attachment to a biomolecule; 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.

18. The siRNA according to claim 15, 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.

19. The siRNA of claim 18, wherein the conjugated group is represented by formula (II'-1) or formula (II'-2): in, Indicates the location of attachment to a biomolecule; 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.

20. The siRNA of claim 15, 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.

21. The siRNA of claim 20, wherein the conjugated group is represented by formula (II'-2): in, Indicates the location of attachment to a biomolecule; 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.

22. The siRNA of claim 14, 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.

23. The siRNA of claim 22, wherein T is -M-, -CH2-M- or -C(O)-M-, wherein M is 24. The siRNA according to claim 22 or 23, 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 22 or 23.

25. The siRNA of claim 24, 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 33 or 34.

26. The siRNA according to claim 22 or 23, 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 22 or 23.

27. The siRNA of claim 22 or 23, 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 22 or 23.

28. The siRNA of claim 14, 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.

29. The siRNA of claim 14, wherein the conjugated group is selected from the group consisting of:

30. The siRNA of claim 14, wherein the conjugated group is selected from the group consisting of:

31. The siRNA of any one of claims 13-30, wherein the ligand targets the asialoglycoprotein receptor (ASGPR).

32. The siRNA of claim 13, wherein the ligand has the structure: in Indicates the position of linkage to the sense strand of siRNA via a phosphate group or a phosphorothioate group.

33. The siRNA of claim 13, wherein the ligand has the structure: in Indicates the position of linkage to the sense strand of siRNA via a phosphate group or a phosphorothioate group.

34. The siRNA of claim 13, 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 claim 13, wherein (a) The sense strand comprises UmsGmsAmAmCmUmCfAfAfCmUmCmAmAmAmAmCmUmsUms-GL6 (SEQ ID NO: 91), The antisense strand comprises AmsAfsGmUfUmUfUmGfAmGfUmUfGmAfGmUfUmCfAmsAfsGm (SEQ ID NO: 92); (b) the sense strand comprises GmsGmsAmUmCmAmCfAfAfAmAmCmUmUmCmAmAmUmsAms-GL6 (SEQ ID NO: 93), The antisense strand comprises UmsAfsUmUfGmAfAmGfUmUfUmUfGmUfGmAfUmCfCmsAfsUm (SEQ ID NO: 94); (c) the sense strand comprises CmsAmsCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmsAms-GL6 (SEQ ID NO: 95), The antisense strand comprises UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm (SEQ ID NO: 96); (d) the sense strand comprises STM1s-CmsUmUmGmAmAmCmUmCfAfAfCmUmCmAmAmAmAmCmUmUms-STM1s-GL6 (SEQ ID NO: 97), The antisense strand comprises AmsAfsGmUfUmUfUmGfAmGfUmUfGmAfGmUfUmCfAmsAfsGm (SEQ ID NO: 98); (e) the sense strand comprises STM1s-AmsUmGmGmAmUmCmAmCfAfAfAmAmCmUmUmCmAmAmUmAms-STM1s-GL6 (SEQ ID NO: 99), The antisense strand comprises UmsAfsUmUfGmAfAmGfUmUfUmUfGmUfGmAfUmCfCmsAfsUm (SEQ ID NO: 100); (f) the sense strand comprises STM1s-UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAms-STM1s-GL6 (SEQ ID NO: 101), The antisense strand comprises UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm (SEQ ID NO: 102); (g) the sense strand comprises STM1-STM1- UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAm-STM1-STM1-GL6 (SEQ ID NO: 103), The antisense strand comprises UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm (SEQ ID NO: 104); or (h) the sense strand comprises IBs-UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAms- GL6 (SEQ ID NO: 105), The antisense strand comprises UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm (SEQ ID NO: 106).

36. The siRNA of claim 13, wherein (a) The sense strand comprises STM1s-UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAms-STM1s-GL6 (SEQ ID NO: 107), The antisense strand comprises UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm (SEQ ID NO: 108); (b) the sense strand comprises STM1-STM1- UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmsAm-STM1-STM1s-GL6 (SEQ ID NO: 109), The antisense strand comprises UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm (SEQ ID NO: 110); (c) the sense strand comprises STM1s-UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAm-STM1-STM1s-GL6 (SEQ ID NO: 111), The antisense strand comprises UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm (SEQ ID NO: 112); (d) the sense strand comprises STM1s-CmsAmAmGmAmCmAmAmUfUfCfAmUmCmAmUmUmUmGmAmUms-STM1s-GL6 (SEQ ID NO: 113), The antisense strand comprises AmsUfsCmAfAmAfUmGfAmUfGmAfAmUfUmGfUmCfUmsUfsGm (SEQ ID NO: 114); (e) the sense strand comprises STM1-STM1- CmsAmAmGmAmCmAmAmUfUfCfAmUmCmAmUmUmUmGmAmsUm-STM1-STM1s-GL6 (SEQ ID NO: 115), The antisense strand comprises AmsUfsCmAfAmAfUmGfAmUfGmAfAmUfUmGfUmCfUmsUfsGm (SEQ ID NO: 116); (f) the sense strand comprises STM1s-CmsAmAmGmAmCmAmAmUfUfCfAmUmCmAmUmUmUmGmAmUm-STM1-STM1s-GL6 (SEQ ID NO: 117), The antisense strand comprises AmsUfsCmAfAmAfUmGfAmUfGmAfAmUfUmGfUmCfUmsUfsGm (SEQ ID NO: 118); (g) the sense strand comprises STM1s-UmsGmGmUmAmUmUmAmAfAfUfCmCmUmUmAmAmGmAmGmAms- STM1s-GL6 (SEQ ID NO: 119), The antisense strand comprises UmsCfsUmCfUmUfAmAfGmGfAmUfUmUfAmAfUmAfCmsCfsAm (SEQ ID NO: 120); (h) the sense strand comprises STM1s-CmsCmAmGmAmAmUmUmGfAfUfCmAmAmGmAmCmAmAmUmUms-STM1s-GL6 (SEQ ID NO: 121), The antisense strand comprises AmsAfsUmUfGmUfCmUfUmGfAmUfCmAfAmUfUmCfUmsGfsGm (SEQ ID NO: 122); or (i) the sense strand comprises STM1s-UmsGmAmUmCmAmAmGmAfCfAfAmUmUmCmAmUmCmAmUmUms-STM1s-GL6 (SEQ ID NO: 123), The antisense strand comprises AmsAfsUmGfAmUfGmAfAmUfUmGfUmCfUmUfGmAfUmsCfsAm (SEQ ID NO: 124).

37. A cell comprising the siRNA of any one of claims 1-36.

38. A pharmaceutical composition comprising the siRNA of any one of claims 1 to 36, or the cell of claim 37, and optionally a pharmaceutically acceptable carrier or excipient.

39. A kit comprising the siRNA of any one of claims 1-36, the cell of claim 37, or the pharmaceutical composition of claim 38.

40. A method of treating a disease or condition associated with ANGPTL3 expression in a subject, the method comprising the step of administering to the subject the siRNA of any one of claims 1-36, the cell of claim 37, or the pharmaceutical composition of claim 38.

41. The method of claim 40, wherein the disease or condition associated with ANGPTL3 expression is a metabolic disease or a cardiovascular disease.

42. The method of claim 41, wherein the metabolic disease or cardiovascular disease is obesity, diabetes, atherosclerosis, dyslipidemia, coronary heart disease, non-alcoholic fatty liver disease (NAFLD), hyperfatty acidemia, or metabolic syndrome, or a combination thereof.

43. The method of claim 42, wherein the dyslipidemia is hyperlipidemia.

44. The method of claim 43, wherein the hyperlipidemia is hypercholesterolemia, hypertriglyceridemia, or a combination thereof.

45. The method of claim 42, wherein the NAFLD is hepatic steatosis or steatohepatitis.

46. ​​The method of claim 42, wherein the diabetes is type 2 diabetes or type 2 diabetes with dyslipidemia.

47. A method of reducing ANGPTL3 levels, LDL levels, apoC-III levels, triglyceride levels, cholesterol levels, glucose levels, fat pad weight in a subject, the method comprising the step of administering to the subject the siRNA of any one of claims 1-36, the cell of claim 37, or the pharmaceutical composition of claim 38.

48. The method of any one of claims 40-47, wherein administering the siRNA, cell, or pharmaceutical composition to the subject comprises subcutaneous administration, topical administration, or intravenous administration.