siRNA that inhibits apolipoprotein C3 expression

Abstract

The present invention relates to siRNAs that inhibit the expression of apolipoprotein C-III. There are provided small interfering RNAs (siRNAs) for inhibiting APOC3 expression in cells, vectors and cells containing nucleotides encoding the same, and methods of treating diseases or conditions associated with APOC3 expression in a subject using the siRNAs, vectors or cells.

Description

Technical Field

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

Background Art

[0002] Lipoproteins are spherical, micelle-like particles consisting of a nonpolar core of acylglycerol and cholesteryl esters coated with amphipathic proteins, phosphatides, and cholesterol. Lipoproteins are classified into five types based on function and physical properties: chylomicrons, very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), low density lipoproteins (LDL), and high density lipoproteins (HDL). Chylomicrons transport dietary lipids from the intestine to tissues. VLDL, IDL, and LDL all transport triacylglycerol and cholesterol from the liver to tissues. HDL transports endogenous cholesterol from tissues to the liver.

[0003] Apolipoprotein C-III (ApoCIII) is an important regulator of lipoprotein metabolism. In humans, APOC3 is expressed in the liver and slightly in the intestine. APOC3 is first expressed as a protein consisting of 99 amino acids, and then after removing a 20-amino acid signal peptide in the endoplasmic reticulum, a mature ApoC3 protein of 79 amino acids is formed.

[0004] The main action of APOC3 is to regulate lipolysis by non-competitively inhibiting endothelial-bound lipoprotein lipase (LPL). LPL hydrolyzes the triglycerides in triglyceride-rich lipoproteins (TRL), releases fatty acids into the plasma, and converts large triglyceride-rich particles into relatively small remnant lipoproteins poor in triglycerides. Individuals lacking APOC3 have relatively low TRL levels and efficient triglyceride lipolysis.

[0005] APOC3 also inhibits hepatic lipase (HL). Hepatic lipase is a lipolytic enzyme having triglyceride lipase and phospholipase A1 activities synthesized in the liver. The inhibitory effect of APOC3 on HL further reduces the lipolysis and uptake of TRL residues in the liver.

[0006] ApoC3 has been shown to inhibit lipolysis by inhibiting both lipoprotein lipase and the binding of lipoproteins to cell surface glycosaminoglycan substrates. An increase in APOC3 levels causes the progression of hypertriglyceridemia or high blood levels of triglycerides. An increase in triglyceride levels is associated with various diseases including cardiovascular disease, atherosclerosis, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, polycystic ovary syndrome, kidney disease, obesity, type 2 diabetes (insulin resistance), hypertension and skin disorders (xanthomas). Very high triglyceride levels also increase the risk of acute pancreatitis.

[0007] In the art, there is a need for Apolipoprotein C3-related disorder modulators, such as APOC3 expression regulators, for treating hypertriglyceridemia.

[0008] In recent years, inhibitors targeting APOC3 have become novel therapeutic agents for these diseases. siRNA has great potential for huge development as a novel therapy. Compared with conventional small molecule pharmaceuticals, siRNA can act on intracellular mRNA and directly silence target genes, thereby more effectively blocking the onset and progression of diseases fundamentally. However, due to defects such as poor stability of siRNA, easy degradation by nucleases in the body, difficulty in being absorbed by tissues, difficulty in being taken up by cells, and easy occurrence of off-target effects, its clinical application is limited. An siRNA that can effectively inhibit intracellular APOC3 gene expression has been expected. Summary of the Invention Means for Solving the Problems

[0009] The present invention relates to small interfering RNA (siRNA) for inhibiting the expression of apolipoprotein C3 (APOC3) in cells and a method for treating diseases using the siRNA.

[0010] In a first aspect, the present invention provides small interfering ribonucleic acid (siRNA) for inhibiting the expression of apolipoprotein C3 (APOC3) in cells, the siRNA comprising a sense strand and an antisense strand forming a double-stranded region, the lengths of the sense strand and the antisense strand being each independently 15 to 30 nucleotides, and the antisense strand comprising at least 15 consecutive nucleotides of a nucleotide sequence represented by any one of SEQ ID NOs: 414 to 826. In some embodiments, the sense strand comprises at least 15 consecutive nucleotides of a nucleotide sequence represented by any one of SEQ ID NOs: 1 to 413.

[0011] In some embodiments, the lengths of the sense strand and the antisense strand are each independently 17 to 27 nucleotides, preferably 19 to 25 nucleotides, more preferably 21 to 23 nucleotides.

[0012] In some embodiments, the length of the double-stranded region is 15 to 25 nucleotide pairs, preferably 17 to 21 nucleotide pairs, more preferably 19 nucleotide pairs.

[0013] In some embodiments, one or both of the sense strand and the antisense strand comprise a 3'-overhang and / or a 5'-overhang having at least 1 nucleotide, for example one or both of the sense strand and the antisense strand comprise a 3'-overhang and / or a 5'-overhang having at least 2 nucleotides. In some specific embodiments, the overhang is selected from U, UU, UUU, and AA.

[0014] In some embodiments, the antisense strand comprises at least 16 consecutive nucleotides, at least 17 consecutive nucleotides, at least 18 consecutive nucleotides, at least 19 consecutive nucleotides, or at least 20 consecutive nucleotides of the nucleotide sequence represented by any one of SEQ ID NOs: 414 to 826, and preferably the antisense strand comprises the nucleotide sequence represented by any one of SEQ ID NOs: 414 to 826.

[0015] In some embodiments, the sense strand comprises at least 16 consecutive nucleotides, at least 17 consecutive nucleotides, at least 18 consecutive nucleotides, at least 19 consecutive nucleotides, or at least 20 consecutive nucleotides of the nucleotide sequence represented by any one of SEQ ID NOs: 1 to 413, and preferably the antisense strand comprises the nucleotide sequence represented by any one of SEQ ID NOs: 1 to 413.

[0016] In some embodiments, the siRNA comprises a sense strand sequence and an antisense strand sequence of the pairing shown in Table 3.

[0017] In some preferred embodiments, the antisense strand comprises at least 15 consecutive nucleotides, at least 16 consecutive nucleotides, at least 17 consecutive nucleotides, at least 18 consecutive nucleotides, at least 19 consecutive nucleotides, or at least 20 consecutive nucleotides of the nucleotide sequence represented by any one of SEQ ID NOs: 473, 612, 690, 757, 761, 816, 817, 818, 819, 820, 814, and 815, and preferably the antisense strand comprises the nucleotide sequence represented by any one of SEQ ID NOs: 473, 612, 690, 757, 761, 816, 817, 818, 819, 820, 814, and 815.

[0018] In some preferred embodiments, the sense strand comprises at least 15 consecutive nucleotides, at least 16 consecutive nucleotides, at least 17 consecutive nucleotides, at least 18 consecutive nucleotides, at least 19 consecutive nucleotides, or at least 20 consecutive nucleotides of the nucleotide sequence represented by any of SEQ ID NO: 60, 199, 277, 344, 348, 403, 404, 405, 406, 407, 401, and 402, and preferably the antisense strand comprises the nucleotide sequence represented by any of SEQ ID NO: 60, 199, 277, 344, 348, 403, 404, 405, 406, 407, 401, and 402.

[0019] In some more preferred embodiments, in the siRNA of the present invention, (a) the sense strand comprises the nucleotide sequence represented by SEQ ID NO: 60, and the antisense strand comprises the nucleotide sequence represented by SEQ ID NO: 473, or (b) the sense strand comprises the nucleotide sequence represented by SEQ ID NO: 199, and the antisense strand comprises the nucleotide sequence represented by SEQ ID NO: 612, or (c) the sense strand comprises the nucleotide sequence represented by SEQ ID NO: 277, and the antisense strand comprises the nucleotide sequence represented by SEQ ID NO: 690, or (d) the sense strand comprises the nucleotide sequence represented by SEQ ID NO: 344, and the antisense strand comprises the nucleotide sequence represented by SEQ ID NO: 757, or (e) the sense strand comprises the nucleotide sequence represented by SEQ ID NO: 348, and the antisense strand comprises the nucleotide sequence represented by SEQ ID NO: 761, or (f) the sense strand comprises the nucleotide sequence represented by SEQ ID NO: 403, and the antisense strand comprises the nucleotide sequence represented by SEQ ID NO: 816, or (g) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 404, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 817, or (h) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 405, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 818, or (i) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 406, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 819, or (j) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 407, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 820, or (k) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 401, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 814, or (l) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 402, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 815.

[0020] In some embodiments, substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides. In some preferred embodiments, all nucleotides of the sense strand and all nucleotides of the antisense strand are modified nucleotides.

[0021] 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'-fluorinated modified nucleotides, 2'-deoxy modified nucleotides, inosine ribonucleotides, deprotonated nucleotides, inverted abasic deoxyribonucleotides, phosphorothioate internucleotide linkage modifications, vinylphosphonate modified nucleotides, locked nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing non-natural bases, and terminal nucleotides conjugated to cholesterol-based derivatives or dodecanedioic acid dodecanamide, and deoxyribonucleotides.

[0022] 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'-fluorinated modified nucleotides, 2'-deoxy modified nucleotides, inosine ribonucleotides, deprotonated nucleotides, inverted abasic deoxyribonucleotides, and phosphorothioate internucleotide linkage modifications.

[0023] In some specific embodiments, the sense strand and / or the antisense strand comprise at least two 2'-fluorinated modified nucleotides.

[0024] In some specific embodiments, the sense strand and / or the antisense strand comprise at least eight 2'-O-methyl modified nucleotides.

[0025] In some specific embodiments, the 5'-end of the sense strand and / or the antisense strand comprises 1 to 5 phosphorothioate groups.

[0026] In some embodiments, the antisense strand comprises a modified nucleotide sequence shown in any of Table 5, and / or the sense strand comprises a modified nucleotide sequence shown in any of Table 4.

[0027] In some embodiments, the siRNA comprises a paired modified sense strand sequence and a modified antisense strand sequence shown in any of Table 6.

[0028] In some preferred embodiments, (1) the sense strand comprises AmsAmAmAmGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmAms-STM1 (SEQ ID NO:1655), and the antisense strand comprises (CP1a-U)sCfsUmGfAmGfAmAfUmAfCmUfGmUfCmCfCmUfUmsUfsUm (SEQ ID NO:1651), or (2) the sense strand comprises STM1s-AmsAmUmUmAmAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmAms-STM1 (SEQ ID NO:1656), and the antisense strand comprises (CP1a-U)sGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAmsUfsUm, or (3) the sense strand comprises STM1s-AmsAmUmUmAmAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmAms-STM1 (SEQ ID NO:1657), and the antisense strand comprises (VPUm)sGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAmsUfsUm (SEQ ID NO:1649), or (4) the sense strand comprises IBs-AmsAmUmUmAmAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmAms-IB (SEQ ID NO:1658), and the antisense strand comprises (CP1a-U)sGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAmsUfsUm (SEQ ID NO:1650). (5) The sense strand contains UmsUmsAmAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmsAm (SEQ ID NO:1659), and the antisense strand contains (CP1a-U)sGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAmsUfsUm (SEQ ID NO:1650), or (6) The sense strand contains IBs-AmsAmAmAmGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmAms-IB (SEQ ID NO:1660), and the antisense strand contains (CP1a-U)sCfsUmGfAmGfAmAfUmAfCmUfGmUfCmCfCmUfUmsUfsUm (SEQ ID NO:1651), or (7) The sense strand contains AmsAmsGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmsAm (SEQ ID NO:1661), and the antisense strand (CP1a-U)sCfsUmGfAmGfAmAfUmAfCmUfGmUfCmCfCmUfUmsUfsUm (SEQ ID NO:1651), or (8) The sense strand contains IBs-AmsAmAmAmGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmAms-IBs-GL6 (SEQ ID NO:1662), and the antisense strand contains (CP1a-U)sCfsUmGfAmGf(PCN-A)AfUmAfCmUfGmUfCmCfCmUfUmsUfsUm (SEQ ID NO:1652), or (9) The sense strand contains AmsAmsGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmsAm (SEQ ID NO:1663), and the antisense strand contains (CP1a-U)sCfsUmGfAmGf(PCN-A)AfUmAfCmUfGmUfCmCfCmUfUmsUfsUm (SEQ ID NO:1652), or (a) The sense strand contains CmsGmsAmGmGmAmUfGfCfCmUmCmCmUmUmCmUmUm, and the antisense strand contains AmsAfsGmAfAmGfGmGfAmGfGmCfAmUfCmCfUmCfGmsUfsUm, or (b) The sense strand contains CmsCmsGmUmUmAmAfGfGfAmCmAmAmGmUmUmCmUmUm, and the antisense strand contains AmsAfsGmAfAmCfUmUfGmUfCmCfUmUfAmAfCmGfGmsUfsUm, or (c) The sense strand contains CmsCmsGmUmUmAmAfGfGfAmCmAmAmGmUmUmCmUmUm, and the antisense strand contains AmsAfsGmAfAmCfUmUfGmUfCmCfUmUfAmAfCmGfGmsUfsUm, or (d) The sense strand contains CmsCmsAmAmGmUmCfCfAfCmCmUmGmCmCmUmAmUmUm, and the antisense strand contains AmsAfsUmAfGmGfCmAfGmGfUmGfGmAfCmUfUmGfGmsUfsUm, or (e) The sense strand contains AmsAmsGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmUm, and the antisense strand contains AmsCfsUmGfAmGfAmAfUmAfCmUfGmUfCmCfCmUfUmsUfsUm, or (f) The sense strand contains UmsUmsAmAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmUm, and the antisense strand contains AmsGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAmsUfsUm, or (g) The sense strand contains AmsAmsGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmUm, and the antisense strand contains AmsCfsUmGfAmGfAmAfUmAfCmUfGmUfCmCfCmUfUmsUfsUm, or (h) The sense strand contains CmsCmsAmAmGmUmCfCfAfCmCmUmGmCmCmUmAmUmUm, and the antisense strand contains AmsAfsUmAfGmGfCmAfGmGfUmGfGmAfCmUfUmGfGmsUfsUm, or (i) The sense strand contains CmsCmsGmUmUmAmAfGfGfAmCmAmAmGmUmUmCmUmUm-L96, and the antisense strand contains AmsAfsGmAfAmCfUmUfGmUfCmCfUmUfAmAfCmGfGmsUfsUm, or (j) The sense strand contains CmsGmsAmGmGmAmUfGfCfCmUmCmCmCmUmUmCmUmUm, and the antisense strand contains AmsAfsGmAfAmGfGmGfAmGfGmCfAmUfCmCfUmCfGmsUfsUm, or (k) The sense strand contains IBs-AmCmGmGmGmAmCmAmGfUfAfUmUmCmUmCmAmGmUmimAms-IB, and the antisense strand contains UmsCfsAmsCfUmGfAmGmAmAmUmAfCmUfGmUfCmCfCmGfsUm, or (l) The sense strand contains AmsAmsGmGmGmAmCfAmGfUfAfUmUmCmUmCmAmGmUmsGmsCm, and the antisense strand contains GmsCfsAmCmUmGfAmGmAmAmUmAmCmUfGmUfCmCmCmUmUmsUmsUm.

[0029] In some embodiments, the siRNA is further conjugated to a ligand moiety containing N-acetylgalactosamine by a phosphate ester group or a phosphorothioate ester group, and preferably, the sense strand of the siRNA is conjugated to the ligand moiety by a phosphate ester group or a phosphorothioate ester group.

[0030] In some specific embodiments, the 3'-end of the sense strand is conjugated to the ligand moiety by a phosphate ester group or a phosphorothioate ester group. In another specific embodiment, the 5'-end of the sense strand is conjugated to the ligand moiety by a phosphate ester group or a phosphorothioate ester group.

[0031] In some embodiments, the ligand moiety contains a conjugate group represented by (X'),

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0032] In some embodiments, the conjugate ligand targets the asialoglycoprotein receptor (ASGPR).

[0033] In some preferred embodiments, the conjugate group is selected from Table 1.

[0034]

Table 1-1

Table 1-2

Table 1-3

Table 1-4

Table 1-5

[0035] In some preferred embodiments, the conjugate group is selected from Table 2.

[0036]

Table 2-1

Table 2-2

Table 2-3

Table 2-4

Table 2-5

[0037] In some preferred embodiments, the ligand has the following structure:

Chemical formula

Chemical formula

[0038] In some preferred embodiments, the ligand has the following structure:

Chemical formula

Chemical formula

[0039] In some preferred embodiments, the ligand has the following structure,

Chemical formula

Chemical formula

[0040] In some specific embodiments, in the siRNA of the present disclosure, (1) the sense strand comprises STM1s-AmsAmAmAmGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmAms-STM1s-GL6 (SEQ ID NO: 1238), the antisense strand comprises (CP1a-U)sCfsUmGfAmGfAmAfUmAfCmUfGmUfCmCfCmUfUmsUfsUm (SEQ ID NO: 1651), or (2) the sense strand comprises STM1s-AmsAmUmUmAmAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmAms-STM1s-GL6 (SEQ ID NO: 1235), the antisense strand comprises (CP1a-U)sGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAmsUfsUm (SEQ ID NO: 1650), or (3) the sense strand comprises STM1s-AmsAmUmUmAmAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmAms-STM1s-GL6 (SEQ ID NO: 1235), The antisense strand is, either contains (VPUm)sGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAmsUfsUm (SEQ ID NO:1649), (4) The sense strand is, contains IBs-AmsAmUmUmAmAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmAms-IBs-GL6 (SEQ ID NO:1236), The antisense strand is, either contains (CP1a-U)sGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAmsUfsUm (SEQ ID NO:1650), (5) The sense strand is, contains UmsUmsAmAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmsAms-GL6 (SEQ ID NO:1237), The antisense strand is, either contains (CP1a-U)sGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAmsUfsUm (SEQ ID NO:1650), (6) The sense strand is, contains IBs-AmsAmAmAmGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmAms-IBs-GL6 (SEQ ID NO:1239), The antisense strand is, either contains (CP1a-U)sCfsUmGfAmGfAmAfUmAfCmUfGmUfCmCfCmUfUmsUfsUm (SEQ ID NO:1651), (7) The sense strand is, contains AmsAmsGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmsAms-GL6 (SEQ ID NO:1240), The antisense strand is, either contains (CP1a-U)sCfsUmGfAmGfAmAfUmAfCmUfGmUfCmCfCmUfUmsUfsUm (SEQ ID NO:1651), or (8) The sense strand is, comprising IBs-AmsAmAmAmGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmAms-IBs-GL6 (SEQ ID NO: 1239), wherein the antisense strand comprises (CP1a-U)sCfsUmGfAmGf(PCN-A)AfUmAfCmUfGmUfCmCfCmUfUmsUfsUm (SEQ ID NO: 1652), or (9) wherein the sense strand comprises AmsAmsGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmsAms-GL6 (SEQ ID NO: 1240), wherein the antisense strand comprises (CP1a-U)sCfsUmGfAmGf(PCN-A)AfUmAfCmUfGmUfCmCfCmUfUmsUfsUm (SEQ ID NO: 1652).

[0041] In a second aspect, the present invention provides a vector comprising a nucleotide sequence encoding the siRNA disclosed in the present invention.

[0042] In a third aspect, the present invention provides a cell comprising the siRNA or vector disclosed in the present invention.

[0043] In a fourth aspect, the present invention provides a pharmaceutical composition comprising the siRNA, vector or cell disclosed in the present invention, and any pharmaceutically acceptable carrier or excipient.

[0044] In a fifth aspect, the present invention provides a kit comprising the siRNA, vector, cell or pharmaceutical composition disclosed in the present invention.

[0045] In a sixth aspect, the present invention provides a method for treating a disease associated with APOC3 in a subject, the method comprising administering to the subject the siRNA, vector, cell or pharmaceutical composition disclosed in the present invention.

[0046] In a seventh aspect, the present invention provides a method for reducing the progression of the risk of APOC3-related diseases in a subject, the method comprising administering to the subject an siRNA, vector, cell or pharmaceutical composition disclosed in the present invention.

[0047] In some embodiments of the sixth and seventh aspects, the APOC3-related disease is selected from hyperlipidemia and hypertriglyceridemia. In some specific embodiments, the APOC3-related disease can be caused by, associated with, or result from hypertriglyceridemia, such as non-alcoholic fatty liver, non-alcoholic steatohepatitis, polycystic ovary syndrome, kidney disease, obesity, type 2 diabetes, hypertension, atherosclerosis, cardiovascular disease or pancreatitis.

[0048] In some embodiments, the method comprises administering to the subject the siRNA, vector, cell, or pharmaceutical composition disclosed in the present invention subcutaneously, intravenously, or topically.

[0049] In some embodiments, the subject is a human patient.

Mode for Carrying Out the Invention

[0050] Hereinafter, embodiments of the present invention will be described by specific examples, but those skilled in the art can easily know other advantages and effects of the present invention from the content disclosed in this specification. The present invention may be further applied or implemented by different specific embodiments, and various details in this specification can be variously modified and changed without departing from the technical idea of the present invention based on different viewpoints and applications.

[0051] It should be understood that the protection scope of the present invention is not limited to the following specific embodiments, and the terms used in the examples of the present invention are for explaining specific embodiments and are not intended to limit the protection scope of the present invention.

[0052] In this specification and the claims, the singular forms "one", "1", and "this" include plural forms unless the context clearly dictates otherwise.

[0053] When embodiments represent numerical ranges, it should be understood that, unless otherwise specified, any of the two endpoints of each numerical range and any numerical value between the two endpoints are selectable. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art. In addition to the specific methods, apparatuses, and materials used in the examples, based on the understanding of the prior art by those skilled in the art and the description of the present invention, the present invention can also be realized using any methods, apparatuses, and materials of the prior art that are similar or equivalent to the methods, apparatuses, and materials described in the embodiments of the present invention, and they also belong to the technical scope of the present invention. The embodiments of the present invention will be described in more detail below.

[0054] Definitions As used herein, the term "siRNA" refers to a double-stranded RNA molecule capable of mediating the silencing of a complementary target RNA (e.g., mRNA, e.g., the transcript of a gene encoding a protein). siRNA is usually a double-stranded molecule containing an antisense strand complementary to the target RNA and a sense strand complementary to the antisense strand. For convenience, such an mRNA is also referred to herein as the mRNA to be silenced. Such a gene is also referred to as the target gene.

[0055] As used herein, the term "antisense strand" refers to the strand of siRNA that contains a region that is completely or substantially complementary to the target sequence.

[0056] As used herein, the term "complementary region" means a region on the antisense strand that is completely or substantially complementary to the target mRNA sequence. If the complementary region is not completely complementary to the target sequence, the mismatch can be located in the internal or terminal region of the molecule. Generally, the most resistant mismatches are located within 5, 4, 3, 2, or 1 nucleotides of the terminal region, e.g., the 5' and / or 3' termini. The portion of the antisense strand that is most sensitive to mismatches is called the "seed region". For example, in an siRNA containing a 19nt strand, the 19th position (from 5' to 3') can tolerate some mismatches.

[0057] As used herein, the term "complementary" refers to the ability of a first polynucleotide to hybridize to a second polynucleotide under certain conditions, e.g., stringent conditions. For example, stringent conditions can include 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50 °C or 70 °C, for a duration of 12 - 16 hours.

[0058] As used herein, a "complementary" sequence, in terms of meeting the above requirements regarding the ability to hybridize, may include, or may consist solely of, base pairs formed from non-Watson-Crick type base pairs and / or non-natural and modified nucleotides. Such non-Watson-Crick type base pairs include, but are not limited to, G:U wobble base pairs or Hoogstein base pairs.

[0059] As used herein, a polynucleotide that is "at least partially complementary" or "substantially complementary" to messenger RNA (mRNA) means a polynucleotide that is substantially complementary to the binding portion of the target mRNA (e.g., the mRNA encoding APOC3). For example, if a sequence is substantially complementary to a continuous portion of the mRNA encoding APOC3, the polynucleotide is at least partially complementary to APOC3 mRNA.

[0060] As used herein, the terms "complementary," "fully complementary," and "substantially complementary" are used for base pairs between the sense strand and the antisense strand of siRNA, or between the antisense strand of siRNA and the target sequence.

[0061] As used herein, the term "sense strand" means a strand of siRNA that includes a region that is substantially complementary to the region that is the "antisense strand" of the term defined herein.

[0062] A "nucleoside" is a compound composed of two substances, a purine base or a pyrimidine base, and ribose or deoxyribose. A "nucleotide" is a compound composed of three substances, a purine base or a pyrimidine base, ribose or deoxyribose, and phosphoric acid. An "oligonucleotide" means a nucleic acid molecule (RNA or DNA) having a length of less than, for example, 100, 200, 300, or 400 nucleotides.

[0063] A "nucleic acid base" is a basic unit for synthesizing nucleosides, nucleotides, and nucleic acids. Its constituent elements contain nitrogen and are also called "nitrogen bases." In this article, unless otherwise specified, the capital letters A, U, T, G, and C represent the nucleic acid base compositions of nucleotides of adenine, uracil, thymine, guanine, and cytosine, respectively.

[0064] As used herein, the term "nucleotide overhang" or "overhang" means at least one unpaired nucleotide that protrudes from the double-stranded structure of the siRNA. For example, when the 3'-end of one strand of the siRNA exceeds the 5'-end of the other strand, a nucleotide overhang is present, and vice versa. The siRNA may include an overhang having at least one nucleotide, or the overhang may include at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more. The nucleotide overhang can include or consist of nucleotides / nucleotide analogs (including deoxynucleotides / nucleosides). One or more overhangs can be located on the sense strand, the antisense strand, or any combination thereof. Further, one or more nucleotides of the overhang can be present at the 5'-end, 3'-end, or both ends of the antisense or sense strand of the siRNA.

[0065] By "blunt end" or "blunt terminus" is meant that there is no nucleotide at this end of the double-stranded siRNA that pairs with the nucleotide at this end, i.e., there is no nucleotide overhang at this end. "Blunt-ended siRNA" means an siRNA that is double-stranded over its entire length, i.e., there is no nucleotide overhang at either end of the molecule. The siRNAs of the present invention include siRNAs having a nucleotide overhang at one end (i.e., a reagent having one overhang and one blunt end) or nucleotide overhangs at both ends.

[0066] Nearly all nucleotides of the iRNA of the present invention are modified. For example, nearly all nucleotides of the sense strand are modified nucleotides, and / or nearly all nucleotides of the antisense strand are modified nucleotides, and / or nearly all nucleotides of both the sense strand and the antisense strand are modified nucleotides. In other embodiments of the present invention, all nucleotides of the iRNA of the present invention are modified nucleotides. For example, all nucleotides of the sense strand are modified nucleotides, and / or all nucleotides of the antisense strand are modified nucleotides, and / or all nucleotides of both the sense strand and the antisense strand are modified nucleotides. Here, "nearly all nucleotides are modified~" indicates that most but not all of the siRNAs of the present invention are modified and can contain 5, 4, 3, 2, or 1 or fewer unmodified nucleotides.

[0067] As used herein, "modified nucleotide" includes, but is not limited to, 2'-O-methyl modified nucleotide, 2'-fluorinated modified nucleotide, 2'-deoxy-acid modified nucleotide, inosine ribonucleotide, deprotonated nucleotide, inverted abasic deoxyribonucleotide, phosphorothioate internucleotide linkage modification, vinylphosphonate modified nucleotide, locked nucleotide, 2'-amino-modified nucleotide, 2'-alkyl-modified nucleotide, morpholino nucleotide, phosphoramidate, nucleotide containing non-natural base, and cholesterol-based derivative or terminal nucleotide linked to dodecanedioic acid dodecaneamide, deoxyribonucleotide or conventional protecting group protection, etc. For example, the 2'-fluorinated modified nucleotide refers to a nucleotide formed by substitution of the hydroxyl at the 2'-position of the ribose of the nucleotide with fluorine. The 2'-deoxy-acid modified nucleotide refers to a nucleotide formed by substitution of the 2'-hydroxyl of the ribose with methoxy.

[0068] As used herein, "ligand moiety" means a chemical moiety attached to an siRNA that can alter the distribution, target, or lifespan of the siRNA. In preferred embodiments, such a ligand provides enhanced affinity for a selected target (e.g., a molecule, cell, or cell type, compartment (e.g., a cellular or organ compartment, tissue, organ, or body region)), for example, compared to an siRNA in the absence of such a ligand.

[0069] As used herein, the terms "inhibit" and "reduce," "silencing," "downregulation," and other similar terms are used interchangeably and include any level of inhibition.

[0070] The term "inhibiting the expression of APOC3" is intended to inhibit the expression of any APOC3 gene and APOC3 gene variants or mutants. Thus, this APOC3 gene may be a wild-type APOC3 gene, a mutant APOC3 gene, or a transgenic APOC3 gene in the case of a genetically engineered cell, cell population, or organism.

[0071] "Inhibiting APOC3 gene expression" includes any level of inhibition of the APOC3 gene, e.g., at least partial inhibition of APOC3 gene expression. APOC3 gene expression can be evaluated based on any variable level or change in level related to APOC3 gene expression, e.g., APOC3 mRNA level, APOC3 protein level, or lipid number. This level can be evaluated in individual cells or a group of cells (including samples from a subject).

[0072] Inhibition can be evaluated by comparing the absolute or relative level of a variable related to APOC3 expression to a control level. The control level can be any type of control level used in the art, e.g., a pre-dose baseline level, or a level determined from a subject, cell, or sample treated with a similar untreated or control (e.g., buffer-only control or vehicle control).

[0073] The term "hydroxyl protecting group" means a group that can protect hydroxyl from chemical reactions and can be removed under specific conditions to restore hydroxyl. It mainly includes silyl protecting groups, acyl protecting groups or ether protecting groups, preferably trimethylsilyl (TMS), triethylsilanol (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), acetyl (Ac), acetyl chloride, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, methoxybenzoyl, 9-fluorenylmethyloxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), benzyl (Bn), methoxybenzyl (PMB), allyl, trityl (Tr), 4,4-dimethoxytrityl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), methoxybenzyloxymethyl (PMBM).

[0074] The term "halogenation" or "halogen" means fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).

[0075] "C 1-6 alkyl halide" means the above-mentioned "C 1-6 alkyl" substituted with one or more halogens. In some embodiments, C 1-4 alkyl halide is particularly preferred, more preferably C 1-2It is an alkyl halide. Exemplary alkyl halides include, but are not limited to, -CF3, -CH2F, -CHF2, -CHFCH2F, -CH2CHF2, -CF2CF3, -CCl3, -CH2Cl, -CHCl2, 2,2,2-trifluoro-1,1-dimethyl-ethyl, etc. The alkyl halide may be substituted with available bonding sites, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0076] “C 1-6 “alkylene” refers to a divalent group formed by removing another hydrogen of C 1-6 alkyl, and may be substituted or unsubstituted. In some embodiments, C 1-4 alkylene, C 2-4 alkylene, and C 1-2 alkylene are preferred. Exemplary unsubstituted alkylene groups include, but are not limited to, methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), butylene (-CH2CH2CH2CH2-), pentylene (-CH2CH2CH2CH2CH2-), hexanediyl (-CH2CH2CH2CH2CH2CH2-), etc. Examples of substituted alkylene include, for example, the alkylene substituted with one or more alkyl (methyl) groups such as 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-), etc., but are not limited thereto.

[0077] As used herein, the term "vector" means a nucleic acid molecule capable of amplifying or expressing another nucleic acid connected thereto.

[0078] I. siRNA The present invention provides small interfering RNAs (siRNAs) for inhibiting the expression of apolipoprotein C3 (APOC3) in cells. The siRNAs include a sense strand and an antisense strand that form a double-stranded region. The lengths of the sense strand and the antisense strand are each independently 15 to 30 nucleotides, and the antisense strand contains at least 15 consecutive nucleotides of a nucleotide sequence represented by any one of SEQ ID NOs: 414 to 826.

[0079] In some embodiments, the double-stranded region formed by the sense strand and the antisense strand is completely complementary. In some other embodiments, the double-stranded region formed by the sense strand and the antisense strand is substantially complementary and may contain one, two, three, four, or five non-complementary loci.

[0080] In some specific embodiments, the sense strand contains at least 15 consecutive nucleotides of a nucleotide sequence represented by any one of SEQ ID NOs: 1 to 413.

[0081] In some embodiments, the lengths of the sense strand and the antisense strand are each independently 17 to 27 nucleotides, preferably 19 to 25 nucleotides, more preferably 21 to 23 nucleotides.

[0082] In some embodiments, the length of the double-stranded region is 15 to 25 nucleotide pairs, preferably 17 to 21 nucleotide pairs, more preferably 19 nucleotide pairs.

[0083] One or both of the sense strand and the antisense strand include 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 include a 3' overhang and / or a 5' overhang having at least two nucleotides. In some preferred embodiments, the antisense strand has a 3' overhang and / or a 5' overhang of at least one nucleotide. For example, the antisense strand includes a 3' overhang and / or a 5' overhang having one, two, or three nucleotides. In some preferred embodiments, the sense strand has a 3' overhang and / or a 5' overhang of at least one nucleotide. For example, the sense strand includes a 3' overhang and / or a 5' overhang having one, two, or three nucleotides.

[0084] In some embodiments, the lengths of the sense strand and the antisense strand are the same. In some embodiments, the sense strand forms a double strand complementarily to the entire length of the antisense strand, that is, it has blunt ends. In another embodiment, the lengths of the sense strand and the antisense strand are the same, and a part of the sense strand is complementary to a part of the antisense strand, that is, both the sense strand and the antisense strand have 5' overhangs.

[0085] In some embodiments, the sense strand and the antisense strand have different lengths. 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.

[0086] In some embodiments, the antisense strand includes at least 16 consecutive nucleotides, at least 17 consecutive nucleotides, at least 18 consecutive nucleotides, at least 19 consecutive nucleotides, at least 20 consecutive nucleotides of the nucleotide sequence represented by any one of SEQ ID NOs: 414 to 826. Preferably, the antisense strand includes the nucleotide sequence represented by any one of SEQ ID NOs: 414 to 826.

[0087] In some embodiments, the sense strand comprises at least 16 consecutive nucleotides, at least 17 consecutive nucleotides, at least 18 consecutive nucleotides, at least 19 consecutive nucleotides, at least 20 consecutive nucleotides of the nucleotide sequence represented by any one of SEQ ID NOs: 1 to 413, and preferably the antisense strand comprises the nucleotide sequence represented by any one of SEQ ID NOs: 1 to 413.

[0088] In some embodiments, the siRNA comprises a sense strand sequence and an antisense strand sequence of the pairing shown in Table 3.

[0089] In some embodiments, the antisense strand comprises at least 15 consecutive nucleotides, at least 16 consecutive nucleotides, at least 17 consecutive nucleotides, at least 18 consecutive nucleotides, at least 19 consecutive nucleotides, at least 20 consecutive nucleotides of the nucleotide sequence represented by any one of SEQ ID NOs: 473, 612, 690, 757, 761, 816, 817, 818, 819, 820, 814, and 815, and preferably the antisense strand comprises the nucleotide sequence represented by any one of SEQ ID NOs: 473, 612, 690, 757, 761, 816, 817, 818, 819, 820, 814, and 815.

[0090] In some embodiments, the sense strand comprises at least 15 consecutive nucleotides, at least 16 consecutive nucleotides, at least 17 consecutive nucleotides, at least 18 consecutive nucleotides, at least 19 consecutive nucleotides, or at least 20 consecutive nucleotides of the nucleotide sequence represented by any of SEQ ID NOs: 60, 199, 277, 344, 348, 403, 404, 405, 406, 407, 401, and 402, and preferably the antisense strand comprises the nucleotide sequence represented by any of SEQ ID NOs: 60, 199, 277, 344, 348, 403, 404, 405, 406, 407, 401, and 402.

[0091] In some embodiments, (a) the sense strand comprises the nucleotide sequence represented by SEQ ID NO: 60 and the antisense strand comprises the nucleotide sequence represented by SEQ ID NO: 473, or (b) the sense strand comprises the nucleotide sequence represented by SEQ ID NO: 199 and the antisense strand comprises the nucleotide sequence represented by SEQ ID NO: 612, or (c) the sense strand comprises the nucleotide sequence represented by SEQ ID NO: 277 and the antisense strand comprises the nucleotide sequence represented by SEQ ID NO: 690, or (d) the sense strand comprises the nucleotide sequence represented by SEQ ID NO: 344 and the antisense strand comprises the nucleotide sequence represented by SEQ ID NO: 757, or (e) the sense strand comprises the nucleotide sequence represented by SEQ ID NO: 348 and the antisense strand comprises the nucleotide sequence represented by SEQ ID NO: 761, or (f) the sense strand comprises the nucleotide sequence represented by SEQ ID NO: 403 and the antisense strand comprises the nucleotide sequence represented by SEQ ID NO: 816, or (g) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 404, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 817, or (h) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 405, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 818, or (i) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 406, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 819, or (j) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 407, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 820, or (k) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 401, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 814, or (l) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 402, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 815.

[0092] II. Nucleotide Modification In some embodiments, substantially all nucleotides of the sense strand and substantially all 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.

[0093] In some embodiments, all nucleotides of the sense strand and / or all nucleotides of the antisense strand are modified nucleotides.

[0094] The modification of the nucleotides of the present invention is a modification on the phosphonate, ribose group and / or basic group of the nucleotide.

[0095] In some specific embodiments, the sense strand and the antisense strand each independently contain one or more nucleotide modifications selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-fluorinated modified nucleotides, 2'-deoxy-acid modified nucleotides, inosine ribonucleotides, deprotonated nucleotides, inverted abasic deoxyribonucleotides, phosphorothioate internucleotide linkage modifications, vinylphosphonate modified nucleotides, locked nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing non-natural bases, and terminal nucleotides and deoxyribonucleotides bound to cholesterol-based derivatives or dodecanedioic acid dodecanamide.

[0096] In some preferred embodiments, the sense strand and the antisense strand each independently contain one or more nucleotide modifications selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-fluorinated modified nucleotides, 2'-deoxy-acid modified nucleotides, inosine ribonucleotides, deprotonated nucleotides, inverted abasic deoxyribonucleotides, and phosphorothioate internucleotide linkage modifications.

[0097] In some preferred embodiments, the sense strand and / or the antisense strand contain at least two 2'-fluorinated modified nucleotides.

[0098] In some preferred embodiments, the sense strand and / or the antisense strand contain at least eight 2'-O-methyl modified nucleotides.

[0099] In some preferred embodiments, the 3'-end and / or 5'-end of the sense strand and / or the antisense strand contain 1 to 5 phosphorothioate groups, preferably 2 to 3 phosphorothioate groups. In some more preferred embodiments, the 5'-end of the sense strand and / or the antisense strand contains 1 to 5 phosphorothioate groups.

[0100] In some preferred embodiments, the antisense strand comprises a modified nucleotide sequence shown in any of Table 5, and / or the sense strand comprises a modified nucleotide sequence shown in any of Table 4. In some preferred embodiments, the siRNA comprises a paired modified sense strand sequence and a modified antisense strand sequence shown in any of Table 6.

[0101] In some embodiments, in the siRNA of the present invention that inhibits APOC3 expression in cells: (1) The sense strand comprises AmsAmAmAmGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmAms-STM1 (SEQ ID NO: 1655), and the antisense strand comprises (CP1a-U)sCfsUmGfAmGfAmAfUmAfCmUfGmUfCmCfCmUfUmsUfsUm (SEQ ID NO: 1651), or (2) The sense strand comprises STM1s-AmsAmUmUmAmAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmAms-STM1 (SEQ ID NO: 1656), and the antisense strand comprises (CP1a-U)sGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAmsUfsUm, or (3) The sense strand comprises STM1s-AmsAmUmUmAmAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmAms-STM1 (SEQ ID NO: 1657), and the antisense strand comprises (VPUm)sGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAmsUfsUm (SEQ ID NO: 1649), or (4) The sense strand comprises IBs-AmsAmUmUmAmAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmAms-IB (SEQ ID NO: 1658) The antisense strand is either (CP1a-U)sGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAmsUfsUm (SEQ ID NO:1650), (5) The sense strand is UmsUmsAmAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmsAm (SEQ ID NO:1659), The antisense strand is either (CP1a-U)sGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAmsUfsUm (SEQ ID NO:1650), (6) The sense strand is IBs-AmsAmAmAmGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmAms-IB (SEQ ID NO:1660), The antisense strand is either (CP1a-U)sCfsUmGfAmGfAmAfUmAfCmUfGmUfCmCfCmUfUmsUfsUm (SEQ ID NO:1651), (7) The sense strand is AmsAmsGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmsAm (SEQ ID NO:1661), The antisense strand is either (CP1a-U)sCfsUmGfAmGfAmAfUmAfCmUfGmUfCmCfCmUfUmsUfsUm (SEQ ID NO:1651), (8) The sense strand is IBs-AmsAmAmAmGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmAms-IBs-GL6 (SEQ ID NO:1662), The antisense strand is either (CP1a-U)sCfsUmGfAmGf(PCN-A)AfUmAfCmUfGmUfCmCfCmUfUmsUfsUm (SEQ ID NO:1652), or (9) The sense strand is containing AmsAmsGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmsAm (SEQ ID NO:1663), wherein the antisense strand contains (CP1a-U)sCfsUmGfAmGf(PCN-A)AfUmAfCmUfGmUfCmCfCmUfUmsUfsUm (SEQ ID NO:1652), or (a) the sense strand contains CmsGmsAmGmGmAmUfGfCfCmUmCmCmCmUmUmCmUmUm, and the antisense strand contains AmsAfsGmAfAmGfGmGfAmGfGmCfAmUfCmCfUmCfGmsUfsUm, or (b) the sense strand contains CmsCmsGmUmUmAmAfGfGfAmCmAmAmGmUmUmCmUmUm, and the antisense strand contains AmsAfsGmAfAmCfUmUfGmUfCmCfUmUfAmAfCmGfGmsUfsUm, or (c) the sense strand contains CmsCmsGmUmUmAmAfGfGfAmCmAmAmGmUmUmCmUmUm, and the antisense strand contains AmsAfsGmAfAmCfUmUfGmUfCmCfUmUfAmAfCmGfGmsUfsUm, or (d) the sense strand contains CmsCmsAmAmGmUmCfCfAfCmCmUmGmCmCmUmAmUmUm, and the antisense strand contains AmsAfsUmAfGmGfCmAfGmGfUmGfGmAfCmUfUmGfGmsUfsUm, or (e) the sense strand contains AmsAmsGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmUm, and the antisense strand contains AmsCfsUmGfAmGfAmAfUmAfCmUfGmUfCmCfCmUfUmsUfsUm, or (f) the sense strand contains UmsUmsAmAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmUm, and the antisense strand contains AmsGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAmsUfsUm, or (g) The sense strand contains AmsAmsGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmUm, and the antisense strand contains AmsCfsUmGfAmGfAmAfUmAfCmUfGmUfCmCfCmUfUmsUfsUm, or (h) The sense strand contains CmsCmsAmAmGmUmCfCfAfCmCmUmGmCmCmUmAmUmUm, and the antisense strand contains AmsAfsUmAfGmGfCmAfGmGfUmGfGmAfCmUfUmGfGmsUfsUm, or (i) The sense strand contains CmsCmsGmUmUmAmAfGfGfAmCmAmAmGmUmUmCmUmUm, and the antisense strand contains AmsAfsGmAfAmCfUmUfGmUfCmCfUmUfAmAfCmGfGmsUfsUm, or (j) The sense strand contains CmsGmsAmGmGmAmUfGfCfCmUmCmCmCmUmUmCmUmUm, and the antisense strand contains AmsAfsGmAfAmGfGmGfAmGfGmCfAmUfCmCfUmCfGmsUfsUm, or (k) The sense strand contains IBs-AmCmGmGmGmAmCmAmGfUfAfUmUmCmUmCmAmGmUmimAms-IB, and the antisense strand contains UmsCfsAmsCfUmGfAmGmAmAmUmAfCmUfGmUfCmCfCmGfsUm, or (l) The sense strand contains AmsAmsGmGmGmAmCfAmGfUfAfUmUmCmUmCmAmGmUmsGmsCm, and the antisense strand contains GmsCfsAmCmUmGfAmGmAmAmUmAmCmUfGmUfCmCmCmUmUmsUmsUm.

[0102] III. Ligand The siRNA of the present invention is further conjugated to a ligand moiety containing N-acetylgalactosamine by a phosphate ester group or a phosphorothioate ester group. In a preferred embodiment, the sense strand of the siRNA is conjugated to the ligand moiety by a phosphate ester group or a phosphorothioate ester group. In some preferred embodiments, the 3'-end of the sense strand is conjugated to the ligand moiety by a phosphate ester group or a phosphorothioate ester group. In another preferred embodiment, the 5'-end of the sense strand is conjugated to the ligand moiety by a phosphate ester group or a phosphorothioate ester group.

[0103] In some embodiments, the ligand moiety contains a conjugate group represented by (X'),

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0104] In some embodiments, the conjugate group is as shown in (I’),

Chemical formula

Chemical formula

Chemical formula

[0105] In some specific embodiments, Q is independently H or

Chemical formula

[0106] In some embodiments, the conjugate group is as shown by formula (I'-1), formula (I'-2) or formula (I'-3),

Chemical formula

Chemical formula

Chemical formula

[0107] In some specific embodiments, Q is independently H,

Chemical formula

[0108] In some embodiments, the conjugate group is as shown by formula (II’-1) or formula (II’-2),

Chemical formula

Chemical formula

[0109] In some specific embodiments, Q is independently H, [Chemical formula] and is, where 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-, R3 is H, or R1 and R3 together form -C 1-2 alkylene-, R2 is H, where R is -OR’, -CH2OR’ or -CH2CH2OR’, R’ is H, a hydroxyl protecting group or a solid 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.

[0110] In some embodiments, the conjugate group is as shown in formula (II’-2),

Chemical formula

Chemical formula

Chemical formula

[0111] In some specific embodiments, Q is independently H,

Chemical formula

Chemical formula

[0112] In some specific embodiments, T is -M-, -CH2-M- or -C(O)-M-, where M is

Chemical formula

[0113] In some specific embodiments, Q is independently H or

Chemical formula

[0114] In some embodiments, the conjugate group is as shown by formula (III’-1), formula (III’-2), or formula (III’-3)

Chemical formula

[0115] In some specific embodiments, Q is independently H, [Chemical formula] and here, 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 - and 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, where R is -OR’, -CH2OR’ or -CH2CH2OR’, 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, where T is as defined above.

[0116] In some embodiments, the conjugate group is as shown by formula (IV-1) or formula (IV-2),

Chemical formula

Chemical formula

[0117] In some specific embodiments, Q is independently H,

Chemical formula

Chemical formula

[0118] In some preferred embodiments, the conjugate group is selected from Table 1 and Table 2.

[0119] In some embodiments, the ligand targets asialoglycoprotein receptor (ASGPR).

[0120] In one preferred embodiment, the ligand has the following structure,

Chemical formula

[0121] In one preferred embodiment, the ligand has the following structure: [Chemical formula] Here, [Chemical formula] represents the position where it binds to the sense strand of the siRNA via a phosphate ester group or a thiophosphate ester group.

[0122] In one preferred embodiment, the ligand has the following structure: [Chemical formula] Here, [Chemical formula] represents the position where it binds to the sense strand of the siRNA via a phosphate ester group or a thiophosphate ester group.

[0123] In some specific embodiments, in the siRNA of the present disclosure, (1) The sense strand contains STM1s-AmsAmAmAmGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmAms-STM1s-GL6 (SEQ ID NO: 1238), and the antisense strand contains (CP1a-U)sCfsUmGfAmGfAmAfUmAfCmUfGmUfCmCfCmUfUmsUfsUm (SEQ ID NO: 1651), or (2) The sense strand including STM1s-AmsAmUmUmAmAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmAms-STM1s-GL6 (SEQ ID NO:1235), the antisense strand includes (CP1a-U)sGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAmsUfsUm (SEQ ID NO:1650), or (3) the sense strand includes STM1s-AmsAmUmUmAmAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmAms-STM1s-GL6 (SEQ ID NO:1235), the antisense strand includes (VPUm)sGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAmsUfsUm (SEQ ID NO:1649), or (4) the sense strand includes IBs-AmsAmUmUmAmAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmAms-IBs-GL6 (SEQ ID NO:1236), the antisense strand includes (CP1a-U)sGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAmsUfsUm (SEQ ID NO:1650), or (5) the sense strand includes UmsUmsAmAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmsAms-GL6 (SEQ ID NO:1237), the antisense strand includes (CP1a-U)sGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAmsUfsUm (SEQ ID NO:1650), or (6) the sense strand includes IBs-AmsAmAmAmGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmAms-IBs-GL6 (SEQ ID NO:1239), the antisense strand (CP1a-U)sCfsUmGfAmGfAmAfUmAfCmUfGmUfCmCfCmUfUmsUfsUm (SEQ ID NO:1651), or (7) The sense strand contains AmsAmsGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmsAms-GL6 (SEQ ID NO:1240), and the antisense strand (CP1a-U)sCfsUmGfAmGfAmAfUmAfCmUfGmUfCmCfCmUfUmsUfsUm (SEQ ID NO:1651), or (8) The sense strand contains IBs-AmsAmAmAmGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmAms-IBs-GL6 (SEQ ID NO:1239), and the antisense strand (CP1a-U)sCfsUmGfAmGf(PCN-A)AfUmAfCmUfGmUfCmCfCmUfUmsUfsUm (SEQ ID NO:1652), or (9) The sense strand contains AmsAmsGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmsAms-GL6 (SEQ ID NO:1240), and the antisense strand (CP1a-U)sCfsUmGfAmGf(PCN-A)AfUmAfCmUfGmUfCmCfCmUfUmsUfsUm (SEQ ID NO:1652).

[0124] IV. Inhibition of APOC3 gene expression The siRNA of the present invention can inhibit 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% of APOC3 gene expression.

[0125] Inhibition of APOC3 gene expression can be achieved by a decrease in the amount of mRNA expressed by a first cell or cell population (such cells can be present, for example, in a sample from a subject) in which the APOC3 gene is transcribed and the cell or these cells have been treated, compared to a second cell or cell population (one or more control cells) that is substantially identical to the first cell or cell population but has not been treated in this way. This can be accomplished, for example, by contacting the cell or these cells with the siRNA of the present invention or by administering the siRNA of the present invention to a subject in which these cells are currently or previously present.

[0126] In a preferred embodiment, this inhibition is evaluated by the following formula as the ratio of the mRNA level in the treated cells to the mRNA level in the control cells. In some specific embodiments, 2 -△△Ct values are calculated and converted to a percentage to obtain the residual inhibition rate, where △△Ct = [(Ct experimental group target gene - Ct experimental group internal standard) - (Ct control group target gene - Ct control group internal standard)].

[0127] Inhibition of APOC3 protein expression can be achieved by reducing the level of APOC3 protein expressed by a cell or cell population (e.g., the protein level expressed in a sample from a subject). As described above for the evaluation of mRNA inhibition, inhibition of the protein expression level in the treated cell or cell population can be similarly expressed as a ratio of the protein level in the control cell or cell population.

[0128] Examples of control cells or cell populations that can be used to evaluate inhibition of APOC3 gene expression include cells or cell populations that have not yet been contacted with the siRNA of the present invention. For example, the control cells or cell populations may be derived from individual subjects (e.g., human or animal subjects) before treatment with siRNA.

[0129] V. Vectors The present invention provides vectors comprising nucleotide sequences encoding the siRNA of the present invention. The vectors of the present invention can be ligated to amplify or express nucleotides encoding the siRNA of the present invention.

[0130] SiRNAs targeting the APOC3 gene can be expressed from transcription units inserted into DNA or RNA vectors. Expression can be transient (from a few hours to a few weeks) or persistent (from a few weeks to several months or more) and depends on the specific construct and target tissue or cell type used. The coding nucleotides of siRNAs targeting the APOC3 gene can be introduced into linear constructs, circular plasmids, or viral vectors. Nucleotides encoding siRNAs targeting the PCSK gene can be integrated into the cell genome for stable expression or stably inherited and expressed extrachromosomally. Generally, siRNA expression vectors are usually DNA plasmids or viral vectors.

[0131] Examples of viral vector systems containing the coding sequence of siRNA targeting the APOC3 gene include, but are not limited to, (a) adenoviral vectors, (b) retroviral vectors, (c) adeno-associated viral vectors, (d) herpes simplex viral vectors, (e) SV40 vectors, (f) polyomavirus vectors, (g) papillomavirus vectors, (h) picornavirus vectors, (i) poxvirus vectors, and (j) helper virus-dependent adenoviruses or gutless adenoviruses.

[0132] VI. Cells The present invention provides a cell containing the siRNA or vector of the present invention, and the siRNA or vector of the present invention can be transcribed intracellularly.

[0133] VII. Pharmaceutical Compositions The present invention provides a pharmaceutical composition comprising the siRNA, vector or cell of the present invention, and any pharmaceutically acceptable carrier or excipient.

[0134] As used herein, "pharmaceutically acceptable" refers to compounds, materials, compositions and / or dosage forms that, within the scope of sound medical judgment, have no excessive toxicity, irritation, allergic response, or other problems or complications, have a reasonable benefit / risk ratio, and are suitable for use in contact with the tissues of human and animal subjects.

[0135] As used herein, a pharmaceutically acceptable carrier refers to a drug carrier that contributes to the administration of a carrier or cell containing siRNA or its coding sequence to a human body and / or contributes to its absorption or action. For example, diluents, excipients such as water, fillers such as starch, sucrose, etc., binders such as cellulose derivatives, alginates, gelatin and polyvinylpyrrolidone, wetting agents such as glycerin, disintegrants such as agar, calcium carbonate and sodium bicarbonate, absorption promoters such as quaternary ammonium compounds, surfactants such as hexadecanol, adsorbent carriers such as kaolin and soap clay, lubricants such as talc, calcium stearate / magnesium stearate, polyethylene glycol, etc. In addition, other adjuvants such as flavoring agents, sweetening agents, etc. can be added to the composition.

[0136] For example, a pharmaceutical composition containing the siRNA, vector or cell of the present invention may contain a pharmaceutically acceptable diluent or sustained-release matrix, and the siRNA or vector of the present invention is embedded in the sustained-release matrix.

[0137] VIII. Kit The present invention provides a kit containing the siRNA, vector or cell of the present invention.

[0138] The present invention further provides a kit for carrying out the siRNA and / or the method of the present invention. Such a kit contains one or more siRNAs, vectors or cells of the present invention, and may further contain an instruction manual. The instruction manual can record instructions for inhibiting APOC3 expression in the cell by contacting the cell with the siRNA or vector of the present invention in an amount effective to inhibit APOC3 expression.

[0139] When the siRNA or carrier of the present invention contacts cells in vitro, optionally, the kit of the present invention may further include a tool (e.g., an injection device) for contacting the cells with the siRNA or carrier of the present invention or a tool for measuring the inhibitory effect of APOC3 (e.g., a device for measuring the inhibition of APOC3 mRNA or protein). Such a device for measuring the inhibition of APOC3 may include a device for obtaining a sample (e.g., a plasma sample, etc.) from a subject.

[0140] When administering in vitro the siRNA, vector, or cells into which the siRNA or vector of the present invention has been introduced in vitro, the kit of the present invention may optionally include a device for administering the siRNA, vector, or cells of the present invention to a subject or a device for determining a therapeutically effective amount or a prophylactically effective amount.

[0141] IX. Therapeutic methods, pharmaceutical uses The present invention provides a method capable of inhibiting the expression of APOC3 in cells, comprising (a) contacting the cells with the siRNA, vector, cells, or pharmaceutical composition of the present invention, and (b) culturing the cells.

[0142] The present invention provides a method for treating a disease or condition related to APOC3 expression in a subject, the method comprising administering to the subject the siRNA, vector, cells, or pharmaceutical composition of the present invention.

[0143] The siRNA according to the present invention can inhibit the expression of APOC3, thereby reducing the level of triglycerides, particularly the level of serum triglycerides, and is used for the treatment of diseases and conditions related to APOC3. The diseases and conditions related to APOC3 are diseases that can be caused by, associated with, or result from hypertriglyceridemia and hypertriglyceridemia. Diseases that can be caused by, associated with, or result from hypertriglyceridemia include, but are not limited to, pancreatitis, metabolic syndrome, type II diabetes, familial chylomicronemia syndrome (FCS), chylomicronemia, multiple factor chylomicronemia, lipodystrophy syndrome (e.g., familial partial lipodystrophy), obesity, dyslipidemia, non-alcoholic fatty liver, non-alcoholic steatohepatitis, hyperlipidemia, hypertriglyceridemia, abnormal lipid and / or cholesterol metabolism, atherosclerosis, cardiovascular disease, coronary artery disease, polycystic ovary syndrome, kidney disease, and other dyslipidemias and symptoms and diseases related to metabolism. The hypertriglyceridemia includes, but is not limited to, non-familial hypertriglyceridemia, familial hypertriglyceridemia, heterozygous familial hypertriglyceridemia, and homozygous familial hypertriglyceridemia.

[0144] In some embodiments, a method for treating a disease or condition related to APOC3 expression in a subject according to the present invention is to administer the siRNA or pharmaceutical composition to the subject, including subcutaneous administration, intravenous administration, or topical administration. In some embodiments, the subject is a human patient.

[0145] The present invention also relates to the siRNA, vector, cell, or pharmaceutical composition of the present invention for treating a disease or condition related to APOC3 expression in a subject.

[0146] The present invention also relates to the use of the siRNA, vector, cell, or pharmaceutical composition of the present invention in the preparation of a medicament for treating a disease or condition related to APOC3 expression in a subject. The medicament of the present invention can be prepared as an emulsion, microemulsion, or microparticle.

[0147] The present invention provides a method for reducing the risk of progression of diseases and conditions related to APOC3 expression in a subject, the method comprising administering to the subject the siRNA, vector, cell or pharmaceutical composition of the present invention.

[0148] The siRNA of the present invention can reduce the risk of progression of APOC3-related diseases and conditions by significantly reducing the level of triglycerides. The diseases and conditions related to APOC3 are diseases that can be caused by, associated with, or resulting from hypertriglyceridemia and hypertriglyceridemia. Diseases that can be caused by, associated with, or resulting from hypertriglyceridemia include, but are not limited to, pancreatitis, metabolic syndrome, type II diabetes, familial chylomicronemia syndrome (FCS), chylomicronemia, multiple factor chylomicronemia, lipodystrophy syndrome (e.g., familial partial lipodystrophy), obesity, dyslipidemia, non-alcoholic fatty liver, non-alcoholic steatohepatitis, hyperlipidemia, hypertriglyceridemia, abnormal lipids and / or cholesterol metabolism, atherosclerosis, cardiovascular disease, coronary artery disease, polycystic ovary syndrome, kidney disease, and other dyslipidemias and symptoms and diseases related to metabolism. Examples of the hypertriglyceridemia include, but are not limited to, non-familial hypertriglyceridemia, familial hypertriglyceridemia, heterozygous familial hypertriglyceridemia, and homozygous familial hypertriglyceridemia.

[0149] In some embodiments, a method for reducing the risk of progression of diseases and symptoms related to APOC3 expression in a subject according to the present invention is to administer the siRNA or pharmaceutical composition to the subject, including subcutaneous administration, intravenous administration or topical administration. In some embodiments, the subject is a human patient.

[0150] The present invention also relates to the siRNA, vector, cell or pharmaceutical composition of the present invention for reducing the risk of progression of diseases and symptoms related to APOC3 in a subject.

[0151] The present invention also relates to the use of the siRNA, vector, cell, or pharmaceutical composition of the present invention in the preparation of a medicament for reducing the risk of progression of diseases and symptoms related to APOC3 in a subject. The medicament of the present invention can be prepared into an emulsion, a microemulsion, or microparticles.

[0152] Sequence The RNA sequence according to the present invention targets the APOC3 gene (or target gene, target mRNA sequence, target sequence).

[0153]

Table 3-1

Table 3-2

Table 3-3

Table 3-4

Table 3-5

Table 3-6

Table 3-7

Table 3-8

Table 3-9

Table 3-10

[0154] The modified RNA sequences used in the present invention are shown in Table 4 below.

[0155] In this specification, the meanings of the respective abbreviations are as follows.

[0156] A, U, G, and C represent natural adenine ribonucleotide, uracil ribonucleotide, guanine ribonucleotide, and cytosine ribonucleotide, respectively.

[0157] i or I represents inosine ribonucleotide.

[0158] m represents that the nucleotide adjacent to its left is a nucleotide modified with 2'-OCH3. For example, Am, Um, Gm, and Cm represent A, U, G, and C modified with 2'-OCH3.

[0159] f represents that the nucleotide adjacent to its left is a nucleotide modified with 2'-F. For example, Af, Uf, Gf, and Cf represent A, U, G, and C modified with 2'-F, respectively.

[0160] "s" or "s-" represents that the two nucleotides and / or introduction carriers adjacent to its left and right are connected via a thiophosphate ester.

[0161] VP represents that the nucleotide adjacent to its right is a nucleotide modified with vinylphosphonate. For publicly known information, reference can be made to, for example, PCT Publication Nos. WO2011139702, WO2013033230, and WO2019105419.

[0162] IB represents an inverted abasic deoxyribonucleotide and may include the following three structures depending on the position / binding method within the siRNA (used at the 5'-end, interstrand, and 3'-end of the nucleic acid strand, respectively).

Chemical Structure

[0163] IB is known. See, for example, F. Czauderna, Nucleic Acids Res., 2003, 31(11), 2705-16 and PCT Publication Nos. WO2016011123 and WO2019051402.

[0164] L96 represents a GalNAc-introducing carrier having the following known structure, among which

Chemical Structure

Chemical Structure

[0165] NAG37 represents a GalNAc-introducing carrier having the following known structure, among which

Chemical Structure

Chemical Structure

[0166] GL6 represents a GalNAc-introducing carrier having the following structure, among which

Chemical Structure

Chemical formula

[0167] GL12 represents a GalNAc-introduced carrier having the following structure, among which,

Chemical formula

Chemical formula

[0168] STM1 represents a substitute for a nucleotide having the following structure. The related intermediate synthesis method is described in Example 15 below.

Chemical formula

[0169] PCN represents a substitute for a nucleotide having the following structure, where Base may be any base. For example, PCN-A represents that Base is adenine. The related intermediate synthesis method is described in Example 17 below.

Chemical formula

[0170] CP1a represents a substitute for a nucleotide having the following structure, where Base may be any base. For example, CP1a-U represents that Base is uracil. The related intermediate synthesis method is described in Example 16 below.

Chemical formula

[0171]

Table 4-1

Table 4-2

Table 4-3

Table 4-4

Table 4-5

Table 4-6

Table 4-7

Table 4-8

Table 4-9

Table 4-10

Table 4-11

[0172]

Table 5-1

Table 5-2

Table 5-3

Table 5-4

Table 5-5

Table 5-6

Table 5-7

Table 5-8

Table 5-9

Table 5-10

[0173]

Table 6-1

Table 6-2

Table 6-3

Table 6-4

Table 6-5

Table 6-6

Table 6-7

Table 6-8

Table 6-9

Table 6-10

Example

[0174] The sources of the materials used in the examples are as follows.

[0175] The Huh7 cell line was from Nanjing Kebai with product number CBP60202.

[0176] The Hep3B cell line was from Nanjing Kebai with product number CBP60197.

[0177] The PHH cells were from Shanghai Xuanyi with product number QYLF-HPMC.

[0178] The HEK293A cell line was from Nanjing Kebai with product number CBP60436.

[0179] The Balb / c mice were purchased from Zhejiang vitalriver with product number Balb / c.

[0180] Preparation of Compound E7 in Example 1 1. Preparation of Intermediate 3-4 1.1 Preparation of Compound 2 [Chemical formula] To DCM (1.80 L) of Compound 1 (300 g, 2.01 mol), benzyl (2,5-dioxopyrrolidin-1-yl) carbonate (600 g, 2.40 mol) was slowly added at 15 °C, and TEA (203 g, 2.01 mol, 280 mL) was added dropwise. After addition, the mixture was stirred at 25 °C for 16 hours. TLC (dichloromethane:methanol = 10:1) showed that Reactant 1 (R f = 0.32) was retained, and one important new spot (R f = 0.52) was detected. The reaction mixture (1.00 L x 2) was washed with saturated sodium bicarbonate solution, 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 a yellow oil.

[0181] 1.2 Preparation of Compound 2A [Chemistry] DMAP (19.8 g, 162 mmol) was added all at once to a solution of Compound 4 (350 g, 1.62 mol, HCl), Ac2O (994 g, 9.74 mol, 912 mL), and pyridine (1.75 L) at 0 - 15 °C, and TEA (164 g, 1.62 mol, 226 mL) was added dropwise. The mixture was stirred at 25 °C for 16 hours. LCMS (product: RT = 0.687 min) indicated complete consumption of the starting reactants. EtOAc (1.40 L) was added to the mixture at 25 °C and stirred for 30 minutes, then the mixture was filtered and the cake was washed with EtOAc (300 mL). The cake was triturated with water (1.45 L) at 25 °C for 30 minutes. The mixture was filtered, the cake was washed with water (175 mL x 3), and the cake was collected to obtain Compound 2A as a white solid (approx. 580 g).

[0182] 1.3 Preparation of Compound 2B [Chemistry] The three reactions proceeded in parallel.

[0183] TMSOTf (137 g, 616 mmol, 111 mL) was added dropwise to a solution of Compound 2A (200 g, 514 mmol) in DCM (800 mL) at 10 - 15 °C over 0.5 hour. Then, the mixture was stirred at 25 °C for 3 hours. TLC (dichloromethane:methanol = 20:1) indicated complete consumption of Compound 2A (R f = 0.54) and formation of a new spot (R f = 0.24). The three reactions were combined. The mixture was cooled to 0 - 15 °C, and a solution of NaHCO3 (300 g dissolved in 3.00 L of water) was slowly poured in at 0 - 5 °C. The organic phase was separated, 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, the resulting yellow oil, Compound 2B (approx. 507 g), was used directly in the next step.

[0184] 1.4 Preparation of Compound 3 [Chemical formula] To a mixture of Compound 2B (250 g, 759 mmol) and Compound 2 (151 g, 531 mmol) in DCM (1.00 L), TMSOTf (84.4 g, 380 mmol, 69.0 mL) was added dropwise at 0 - 10 °C, and the mixture was stirred at 20 °C for 12 hours. By TLC (dichloromethane:methanol = 20:1), Compound 2 (R f = 0.33) was completely consumed, indicating the formation of a new spot (R f = 0.03). After cooling the combined reactants to 0 - 5 °C, the reactants were poured into NaHCO3 (aqueous solution, 100 g dissolved in 1 L of water), stirred at 5 - 10 °C for 10 minutes, and the phases were separated. The phases were extracted with DCM (500 mL x 2), the combined organic phases were dried over Na2SO4, filtered, and the filtrate was concentrated in vacuo. Without purification, Compound 3 (approx. 360 g) was obtained as a yellow oil. 1 1H NMR: (400 MHz, DMSO). 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).

[0185] 1.5 Preparation of Intermediate 3-4 (TFA Salt) [Chemical formula] The three reactions proceeded in parallel.

[0186] Under an argon atmosphere, compound 3 (180 g, 293 mmol) and TFA (33.5 g, 293 mmol, 21.8 mL) were added to a mixture of Pd / C (18.0 g, 16.3 mmol, 10% content) in THF (1.80 L). The suspension was degassed and purged with hydrogen gas three times. The mixture was stirred under H2 (50 Psi) at 30 °C for 2 hours. LCMS (product: RT = 0.697 min) indicated the consumption of compound 3 and the detection of the product peak. Three reactions were combined. The mixture was filtered through celite, and the filtrate was concentrated under reduced pressure to remove the solvent. Without purification, yellow solid intermediate 3-4 (TFA salt) (393 g, 660 mmol, 74.8% yield, 99.6% purity, TFA) was obtained. 1 H NMR: (400 MHz, DMSO-d6) δ = 7.92 (d, J = 9.1 Hz, 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).

[0187] 2. Preparation of Intermediate 3-5 2.1 Preparation of Compound 5

Chemical Structure

[0188] 417.0 g of compound 3 - 4 was converted to compound 5 in 9 batches.

[0189] 2.2 Preparation of Intermediate 3-3 [Chemical formula] Under an argon atmosphere, 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 THF (300 mL) of Pd / C (3.00 g, 10% content). The suspension was degassed and purged with hydrogen gas three times. Stirred with H2 (20 Psi) at 20 °C for 16 h. TLC (dichloromethane:methanol = 8:1, R fThe reaction was shown to be complete by [[ID=]], and the mixture was filtered through celite. The filtrate was concentrated under pressure to remove the solvent, and white solid compound 3-3 (about 33.4 g + 129 g + 75.0 g) was obtained. 1 H NMR: (400 MHz, DMSO) δ = 8.53 (t, J = 5.2 Hz, 1H), 8.18 (d, J = 2.4 Hz, 3H), 8.03 (t, J = 5.2 Hz, 1H), 7.84 (dd, J = 3.6 Hz, 2H), 5.22 (d, J = 3.2 Hz, 2H), 4.96 (dd, J = 3.2 Hz, 2H), 4.55 (d, J = 8.4 Hz, 2H), 4.02 (t, J = 8.8 Hz, 6H), 3.77 - 3.59 (m, 5H), 3.58 - 3.45 (m, 21H), 3.40 - 3.20 (m, 4H), 2.18 (t, J = 7.6 Hz, 2H), 2.17 (d, J = 8.0 Hz, 6H), 2.10 (s, 6H), 1.99 (s, 6H), 1.90 - 1.80 (m, 8H), 1.77 (s, 6H).

[0190] 3. Preparation of Compound E7 3.1 Preparation of Compound 3

Chemical Structure

[0191] 3.2 Preparation of Compound 4

Chem.

[0192] 3.3 Preparation of Compound 6

Chem.

[0193] 4. Preparation of Compound E7

Chem.

[0194] Example 2 Preparation of siRNA The siRNA of the present invention was prepared by a known solid-phase phosphoramidite method. For specific methods, reference can be made to, for example, PCT Publication Nos. WO2016081444 and WO2019105419, and the outline is as follows.

[0195] 1. Preparation of siRNA not bound to a ligand 1.1 Synthesis of sense strand (SS strand) Using the solid-phase phosphoramidite synthesis method, with a blank CPG solid support as the starting cycle, nucleoside monomers are connected one by one from the 3'-5' direction according to the sense strand nucleotide sequence order. Each time a nucleoside monomer is connected, it includes four process reactions: deprotection, coupling, capping, and oxidation or thiolation. Regarding the synthesis scale, the synthesis conditions for 5 μmol of oligonucleic acid are as follows.

[0196] The nucleoside monomer provides a 0.05 mol / L acetonitrile solution. The reaction conditions for each step are the same, that is, the temperature is 25 °C. In deprotection, it is deprotected 3 times with a 3% trichloroacetic acid - dichloromethane solution. The activator used in the coupling reaction is a 0.25 mol / L ETT - acetonitrile solution, and it is coupled 2 times. In capping, it is capped 2 times with 10% acetic anhydride - acetonitrile and pyridine / N - methylimidazole / acetonitrile (10:14:76, v / v / v). In oxidation, it is oxidized 2 times with a 0.05 mol / L iodine / tetrahydrofuran / pyridine / water (70 / 20 / 10, v / v / v) solution. In thiolation, it is thiolated 2 times with a 0.2 mol / L PADS acetonitrile / 3 - methylpyridine (1 / 1, v / v) solution.

[0197] 1.2 Synthesis of antisense strand (AS strand) Using the solid - phase phosphoramidite synthesis method, starting from a blank CPG solid support as the starting cycle, it is connected to the nucleoside monomer one by one in the 3‘ - 5’ direction according to the antisense strand nucleotide sequence order. Each time a nucleoside monomer is connected, it includes four process reactions: deprotection, coupling, capping, oxidation or thiolation. The synthesis conditions for 5 μmol of the oligonucleic acid of the antisense strand are the same as those of the sense strand.

[0198] 1.3 Purification and annealing of oligonucleotides 1.3.1 Aminolysis The synthesized solid support (sense strand or antisense strand) is put into a 5 mL centrifuge tube, 3% diethylamine / ammonia water (v / v) is added, and it is reacted under a constant temperature water bath at 35 °C (or 55 °C) for 16 hours (or 8 hours), filtered, the solid support is washed 3 times with ethanol / water, 1 mL each time, and after the filtrate is concentrated by centrifugation, the crude product is purified.

[0199] 1.3.2 Purification The methods of purification and desalination are well-known to those skilled in the art. For example, it may be eluted and washed with a strong anion exchange column and a sodium chloride - sodium hydroxide system, or the product may be collected and desalted with a gel filtration purification column, and the elution system was pure water.

[0200] 1.3.3 Annealing 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 to 70 - 95 °C in a water bath, held for 3 - 5 min, cooled naturally to room temperature, and the system was freeze-dried to obtain the product. Finally, double-stranded DR000001 - DR000400 were obtained.

[0201] 2. Preparation of siRNA with the sense strand bound to the ligand 2.1 Binding of Compound E7 and the CPG vector Compound E7 (53 mg, 0.018 mmol) and HBTU (13.3 mg, 0.035 mmol) were mixed, acetonitrile (5 mL) was added and shaken to dissolve, then DIEA (9.0 mg, 0.07 mmol) and DMAP (2.1 mg, 0.018 mmol) were added and shaken to dissolve until clear. A blank vector Resin (550 mg, CPG pore size 1000 Å) was weighed and added to the reaction solution, the temperature was controlled at 20 °C, and the reaction was carried out overnight on a shaker. Sampling and monitoring were performed, and thin layer chromatography TLC was carried out. The reaction was complete. Here, the developing solvent was DCM / methanol = 4 / 1 and it was colored with phosphomolybdic acid. It was filtered through a sintered glass funnel, the cake was washed with anhydrous acetonitrile (20 mL * 5), the cake was collected, and vacuum extraction filtration was carried out with an oil pump for 6 hours to obtain 530 mg of a quasi-white solid.

[0202] 530 mg of the above-condensed product was put into a 50 mL round-bottom flask, and CapC (DMAP / acetonitrile), CapB (N-methylimidazole / pyridine / acetonitrile), and CapA (acetic anhydride / acetonitrile) were sequentially added, and the reaction was carried out overnight at room temperature with a shaker. It was filtered, the obtained cake was washed with acetonitrile (20 mL * 4), the cake was collected, and after extraction filtration under reduced pressure with an oil pump for 8 hours, 200 mg of a pseudo-white solid (GL6 solid phase support) was obtained and used for solid-phase synthesis.

[0203] 2.2 Synthesis of the sense strand (SS strand) bound to the ligand By the solid-phase phosphoramidite synthesis method, using the above-prepared GL6 solid-phase support as the starting cycle, nucleoside monomers are sequentially connected one by one from the 3'-5' direction according to the sense strand nucleotide sequence order. Each time a nucleoside monomer is connected, it includes four-step reactions of deprotection, coupling, capping, and oxidation or thiolation. Regarding the synthesis scale, the synthesis conditions for 5 μmol of oligonucleic acid are as follows.

[0204] The nucleoside monomer provides a 0.05 mol / L acetonitrile solution, and the reaction conditions for each step are the same, that is, the temperature is 25 °C. In deprotection, it is deprotected 3 times with a 3% trichloroacetic acid-dichloromethane solution. The activator used in the coupling reaction is a 0.25 mol / L ETT-acetonitrile solution, and coupling is carried out 2 times. In capping, it is capped 2 times with 10% acetic anhydride-acetonitrile and pyridine / N-methylimidazole / acetonitrile (10:14:76, v / v / v). In oxidation, it is oxidized 2 times with a 0.05 mol / L iodine / tetrahydrofuran / pyridine / water (70 / 20 / 10, v / v / v) solution, and in thiolation, it is thiolated 2 times with a 0.2 mol / L PADS acetonitrile / 3-methylpyridine (1 / 1, v / v) solution.

[0205] 2.3 Synthesis of the antisense strand (AS strand) By the solid-phase phosphoramidite synthesis method, starting from a blank CPG solid support as the initial cycle, nucleoside monomers are sequentially connected one by one from the 3'-5' direction according to the antisense strand nucleotide sequence order. Each time a nucleoside monomer is connected, it involves four process reactions: deprotection, coupling, capping, and oxidation or thiolation. The synthesis conditions for 5 μmol of the oligonucleic acid of the antisense strand are the same as those of the sense strand.

[0206] 2.4 Purification and Annealing of Oligonucleotides 2.4.1 Aminolysis The synthesized solid support (sense strand or antisense strand) is put into a 5 mL centrifuge tube, 3% diethylamine / ammonia water (v / v) is added, and the reaction is carried out in a constant temperature water bath at 35 °C (or 55 °C) for 16 hours (or 8 hours). After filtration, the solid support is washed three times with 1 mL of ethanol / water each time, and after centrifugally concentrating the filtrate, the crude product is purified.

[0207] 2.4.2 Purification The methods of purification and desalting are well-known methods to those skilled in the art. For example, it can be eluted and washed with a strong anion filling column, a sodium chloride-sodium hydroxide system, the product is collected, desalted with a gel filling purification column, and the elution system is pure water.

[0208] 2.4.3 Annealing The sense strand (SS strand) and the antisense strand (AS strand) are mixed at a molar ratio (SS strand / AS strand = 1 / 1.05), heated to 70 - 95 °C in a water bath, held for 3 - 5 min, and naturally cooled to room temperature. The system is freeze-dried to obtain the product.

[0209] In the same way, siRNA conjugated with NAG37 or L96 was obtained.

[0210] Example 3 Activity Screening of Huh7 Cell Line Cell Transfection On the first day, the Huh7 cell line (Nanjing Kebai, product number CBP60202) was digested, resuspended, counted, plated in a 96-well plate at 100 μL / well, 1×10 4 cells / well, and transfection was performed 18 hours later.

[0211] On the next day, a 20 μM stock solution of the siRNA (DR000001 - DR000400) prepared in Example 2 was diluted with Opti-MEM. 198 μL of Opti-MEM was taken and added to 2 μL of the siRNA stock solution, and pipetted to mix uniformly for use. Each time an experiment was conducted, appropriate dilution operations were performed according to different experimental needs.

[0212] On the next day, 0.9 μL of RNAiMAX (Thermo, 13778150) was diluted with 14.1 μL of Opti-MEM, gently pipetted to mix uniformly, and left at room temperature for 5 minutes. Then, 15 μL of the prepared RNAi-MAX mixture and 15 μL of the diluted siRNA compound were gently pipetted to mix uniformly without introducing air bubbles, left at room temperature for 10 minutes, and added to a 96-well plate at 10 μL / well. After culturing in a 37°C, 5% CO2 incubator for 24 hours, RNA was extracted.

[0213] RNA Extraction According to the operation protocol of the high-throughput cell RNA extraction kit (Fanzhi Medical, FG0412), cell RNA extraction was performed using a nucleic acid extraction device (Hangzhou Ausheng, Auto-pure96).

[0214] RNA Reverse Transcription Preparation of the denaturation reaction mixture, refer to PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B). Each well contained 1 μL of Oligo dT Primer, 1 μL of dNTP Mixture, and 12.5 μL of template RNA. A denaturation reaction was performed by incubating at 65°C for 5 minutes using a conventional PCR instrument. The mixture was placed on ice and rapidly cooled for 2 minutes.

[0215] Preparation of reverse transcription reaction solution, PrimeScript TM Refer to PrimeScript II 1st Strand cDNA Synthesis Kit (Takara, 6210B). Each well contains 4 μL of 5×Prime Script II Buffer, 0.5 μL of RNase Inhibitor, and 1 μL of PrimeScript II RTase.

[0216] Gradually and uniformly mix 14.5 μL of the denatured reaction solution with the reverse transcription reaction solution, incubate at 42 °C for 45 minutes for reverse transcription using a conventional PCR instrument, incubate at 95 °C for 5 minutes to inactivate the enzyme, and cool the reverse transcription product (cDNA) at 4 °C.

[0217] After completion of reverse transcription, 30 μL of DNase RNase-Free Distilled Water was added to the cDNA sample in each well.

[0218] Real-Time PCR TaqMan TM Refer to the operation flow of Fast Advanced Master Mix (ABI, 4444965), and perform real-time PCR reaction (ABI, QuantStudio3) in a 20 μL system. The reaction process was set as (50 °C, 2 min) × 1 Cycle; (95 °C, 20 s) × 1 Cycle; (95 °C, 1 s; 60 °C, 24 s) × 40 Cycles.

[0219]

Table 7

[0220] Data Statistics 2 -△△Ct Calculate the value and convert it to a percentage to obtain the residual inhibition rate. △△Ct = [(Ct of target gene in experimental group - Ct of internal standard in experimental group) - (Ct of target gene in control group - Ct of internal standard in control group)]

[0221] The siRNA compound cell line activity high-throughput screening was performed with the final concentration of siRNA being 10 nM, and the experimental screening results are shown in Table 8.

[0222]

Table 8

[0223] Example 4 Hep3B Cell Line Activity Screening Using Five Concentrations of siRNA Diluted in a 10-Fold Gradient Similar to the above-mentioned Huh7 cell line activity screening, activity screening was performed using the Hep3B (Nanjing Kebai, product number CBP60197) cell line.

[0224] With the starting concentration of siRNA (DR000001 - DR000400) being 10 nM, it was diluted in a 10-fold gradient to obtain five concentration points (10 nM, 1 nM, 0.1 nM, 0.01 nM, 0.001 nM), and the Hep3B cell line activity screening of siRNA was carried out. The screening results are shown in Table 9.

[0225]

Table 9

[0226] Example 5 Hep3B Cell Line Activity Screening Using Eleven Concentrations of siRNA Diluted in a 3-Fold Gradient The procedure of this example is the same as that of Example 4. With the starting concentration of siRNA (DR000001 - DR000400) being 10 nM, it was diluted in a 3-fold gradient to obtain eleven concentration points (10 nM, 3.33 nM, 1.11 nM, 0.37 nM, 0.123 nM, 0.041 nM, 0.0136 nM, 0.0045 nM, 0.00152 nM, 0.000508 nM, 0.000169 nM), and the Hep3B cell line activity screening of siRNA was carried out. The screening results are shown in Table 10.

[0227]

Table 10

[0228] Example 6: Activity Screening of Huh7 Cell Line Using siRNAs at 11 Concentrations Diluted in a 3-Fold Gradient The procedure of this example is the same as that of Example 3. The Huh7 cell line was selected, and the starting concentration of siRNAs (DR000001 - DR000400) was set at 10 nM and diluted in a 3-fold gradient to obtain 11 concentration points (10 nM, 3.33 nM, 1.11 nM, 0.37 nM, 0.123 nM, 0.041 nM, 0.0136 nM, 0.0045 nM, 0.00152 nM, 0.000508 nM, 0.000169 nM), and the activity screening of siRNAs on the Huh7 cell line was performed.

[0229] [Table 11]

[0230] Example 7: Activity Screening of Hep3B Cell Line Using siRNAs at 11 Concentrations Diluted in a 3-Fold Gradient The procedure of this example is the same as that of Example 5. The Hep3B cell line was selected, and the starting concentration of siRNAs (DR002222 - DR02226, DR001478, DR002252, DR000344, DR000348, DR000277, DR000199, DR000060, DR001482) was set at 10 nM and diluted in a 3-fold gradient to obtain 11 concentration points (10 nM, 3.33 nM, 1.11 nM, 0.37 nM, 0.123 nM, 0.041 nM, 0.0136 nM, 0.0045 nM, 0.00152 nM, 0.000508 nM, 0.000169 nM), and the activity screening of siRNAs on the Hep3B cell line was performed. The screening results are shown in Table 12.

[0231] [Table 12-1]

[0232] [Table 12-2]

[0233] Example 8 psiCHECK2 GSSM-5 Hits Off-Target Activity Screening Plasmid Preparation Based on the siRNA sequence, the corresponding antisense strand off-target plasmid was designed, and the psiCHECK2 GSSM-5 Hits recombinant plasmid was manufactured by Shanghai Genechem Co., Ltd., and the recombinant plasmid was diluted to 1000 ng / μL for reserve.

[0234] Cell Transfection Each well of the 96-well plate was plated with 100 μL of the resuspended solution of HEK293A cells (Nanjing Kebai, product number CBP60436) at 8×10 3 cells / well.

[0235] The next day, first, the complete medium in the well was aspirated and discarded, and replaced with 80 μL / well of Opti-MEM medium, and starved for about 1.5 hours.

[0236] Preparation of siRNA: The siRNA was diluted 3-fold from the final concentration of 40 nM to a total of 11 concentration points (40 nM, 13.3 nM, 4.44 nM, 1.48 nM, 0.493 nM, 0.164 nM, 0.0548 nM, 0.0182 nM, 0.00609 nM, 0.00203 nM, 0.000677 nM).

[0237] Preparation of plasmid mixture: The preparation amount for a single well was 0.01 μL / well of plasmid and 8.99 μL / well of Opti-MEM.

[0238] Preparation of Lipo mixture: 0.2 μL of Lipo 2000 and 9.8 μL of Opti-MEM were added to each well, and Lipo 2000 (Lipofectamine TM 2000 transfection reagent, Thermo, 11668019) was diluted with Opti-MEM to obtain the Lipo mixture, which was allowed to stand at room temperature for 5 minutes.

[0239] 22 μL of the prepared Lipo mixture, 2.2 μL of siRNA, and 19.8 μL of the plasmid mixture were dispensed into the corresponding identical wells and named the well A mixture. After pipetting and mixing uniformly, it was incubated at room temperature for 20 minutes, and then co-transfection was performed. The well A mixture was added to the cells in each well at 20 μL / well, and the original 80 μL of Opti-MEM was added to make the final volume of each well 100 μL. After culturing in a 37 °C, 5% CO2 incubator for 4 hours, 100 μL of DMEM medium containing 20% fetal bovine serum was added to each well. Detection was performed after culturing in a 37 °C, 5% CO2 incubator for 24 hours.

[0240] Result Detection Before the experiment, the mixed Dual-Glo(R) Luciferase (Dual-Glo(R) Luciferase Assay System, Promega, E2940) was re-melted and balanced to room temperature. Then, at the time of use, DMEM was added to each tube at a ratio of 1:1 to prepare the substrate I. The Dual-Glo(R) Stop&Glo(R) Buffer was re-melted and balanced to room temperature. Then, at the time of use, it was prepared with the Dual-Glo(R) Stop&Glo(R) Substrate at a ratio of 100:1 to be the substrate II. The culture medium in the 96-well culture plate was aspirated and removed with a vacuum pump. 150 μL of the substrate I was added to each well and incubated at room temperature for 10 minutes on a shaker. 120 μL of the substrate I was taken and transferred to a 96-well plate, and the Firefly chemiluminescence value was read from a microplate reader (Tecan, Infinite 200). Furthermore, 60 μL of the substrate II was added to each well and incubated at room temperature for 10 minutes on a shaker, and the Renilla chemiluminescence value was read from the microplate reader.

[0241] Data Analysis and Processing Fluorescence activity was measured by a microplate reader. The collected Renilla signal was normalized by the Firefly signal standard, and the inhibitory effect of siRNA was obtained by comparing with the untreated result (residual inhibitory activity). The calculation process is as follows.

[0242] Normalized Ren / Fir ratio: Ratio = Renilla (Renilla luciferase) / Firefly (Firefly luciferase)

[0243] Residual inhibition rate = (RatiosiRNA / Ratiocontrol)*100%. Take the average value of two wells, where Ratiocontrol is the Ratio value of the control well (without siRNA) (take the average value of two wells).

[0244] Mapping: Mapped with Graphpad Prism.

[0245] Half maximal inhibitory concentration (IC50): In this experiment, mapping was performed at Top and Bottom, and the IC50 value was obtained by the formula Y = Bottom + (Top - Bottom) / (1 + 10^((LogIC50 - X)*HillSlope)), where Y = 50 and X = log(concentration).

[0246] The psiCHECK2 GSSM-5Hits off-target activity screening results of siRNA are shown in Table 13.

[0247]

Table 13

[0248] Example 9 Human primary hepatocyte (PHH cell) activity screening Cell Transfection Add 1.4 mL of rat tail collagen solution (Sigma, C3867) to 40.6 mL of DNase RNase-Free Distilled Water, mix uniformly, 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.

[0249] The next day, before use, rinse the coated cell plate with DPBS, aspirate and remove the DPBS, add PHH cells to the resuscitation medium (Shanghai Xuanyi, product number QYLF-HPMC), resuscitate at 37 °C, centrifuge, resuspend, and count. Plate the PHH cells in a 96-well plate at 90 μL / well, 2×10 4 cells / well, replace the complete medium after 4 hours, and perform transfection operation after 18 hours.

[0250] On the third day, dilute the 20 μM siRNA (DR002222, DR002223, DR002225, DR002226, DR001478, and DR002252) stock solution with Opti-MEM, add 2 μL of the siRNA stock solution to 198 μL of Opti-MEM, pipette to mix uniformly, and use it as the first concentration point, and dilute it with an appropriate gradient according to the actual experimental requirements.

[0251] On the third day, dilute 0.9 μL of RNAiMAX (Thermo, 13778150) with 14.1 μL of Opti-MEM, pipette gently to mix uniformly, and let it stand at room temperature for 5 minutes. Then, gently pipette 15 μL of the prepared RNAi-MAX mixture and 15 μL of the diluted siRNA compound to mix uniformly, without introducing air bubbles, let it stand at room temperature for 10 minutes, and add it to the 96-well plate at 10 μL / well. After culturing in a 37 °C, 5% CO2 incubator for 24 hours, extract the RNA.

[0252] RNA Extraction According to the operation protocol of the high-throughput cell RNA extraction kit (Fanze Medical, FG0412), use a nucleic acid extraction device (Hangzhou Ausheng, Auto-pure96) to extract cell RNA.

[0253] RNA Reverse Transcription Preparation of the denaturation reaction mixture, PrimeScript TM Refer to PrimeScript 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. The denaturation reaction was carried out by incubating at 65 °C for 5 minutes using a conventional PCR instrument. The mixture was placed on ice and rapidly cooled for 2 minutes.

[0254] Preparation of the reverse transcription reaction solution, PrimeScript TM Refer to PrimeScript II 1st Strand cDNA Synthesis Kit (Takara, 6210B). Each well contains 4 μL of 5×Prime Script II Buffer, 0.5 μL of RNase Inhibitor, and 1 μL of PrimeScript II RTase.

[0255] 14.5 μL of the denatured reaction solution and the reverse transcription reaction solution were gradually and uniformly mixed, and reverse transcription was carried out by incubating at 42 °C for 45 minutes using a conventional PCR instrument, and the enzyme was inactivated by incubating at 95 °C for 5 minutes, and the reverse transcription product (cDNA) was cooled at 4 °C.

[0256] After the reverse transcription was completed, 30 μL of DNase RNase-Free Distilled Water was added to the cDNA sample in each well.

[0257] Real-Time PCR TaqMan TM Refer to the operation flow of Fast Advanced Master Mix (ABI, 4444965), and a real-time PCR reaction (ABI, QuantStudio3) was carried out in a 20 μL system. The reaction process was set as (50 °C, 2 min) × 1 Cycle; (95 °C, 20 s) × 1 Cycle; (95 °C, 1 s; 60 °C, 24 s) × 40 Cycles.

[0258] [Table 14]

[0259] Data Statistics 2 -△△Ct Calculate the value and convert it into a percentage to obtain the residual inhibition rate. △△Ct = [(Ct of target gene in experimental group - Ct of internal standard in experimental group) - (Ct of target gene in control group - Ct of internal standard in control group)]

[0260] Set the starting concentration of siRNA to 10 nM, dilute it in a 10-fold gradient, obtain five concentration points (10 nM, 1 nM, 0.1 nM, 0.01 nM, 0.001 nM), perform human primary hepatocyte activity screening for siRNA, and show the screening results in Table 15.

[0261] [Table 15]

[0262] Example 10. Mouse HDI model activity screening HDI Animal Modeling Using the method of high-pressure injection into the tail vein, perform in vivo transfection modeling on 6 - 8-week-old female Balb / c mice with a double-gene stable transfection system. Through the tail vein, within 5 - 7 seconds, use a 27-gauge injection needle to inject Piggy-Bac transposon plasmid (purchased from Suzhou Bangye) and Piggy-Bac helper plasmid (purchased from Suzhou Bangye) containing cDNA sequences of target genes with different mass ratios (mass ratio 1:1, total plasmid amount 50 μg) and TransIT(R)-QR Delivery Solution (total volume 10% of body weight of the animal, Mirusbio-MIR 5240) into the mouse body. After injection, place the mice in a cage and observe for 30 minutes. Taking the modeling day as day 0, obtain sera for detecting SEAP expression levels at each time point (Day7 - Day35) after the start of modeling.

[0263] A dual-gene stable transfection system, comprising a Piggy-Bac helper plasmid and a Piggy-Bac transposon plasmid, wherein the Piggy-Bac helper plasmid provides Piggy-Bac transposase, and the Piggy-Bac transposon plasmid contains a dual-gene expression cassette based on the Piggy-Bac transposon, and the dual-gene expression cassette contains secreted alkaline phosphatase (SEAP) and a target gene (APOC3).

[0264] SEAP Expression Detection Kit (Phospha-Light TM The standard product of the SEAP reporter gene detection system (Phospha-Light SEAP Reporter Gene Detection System, Invitrogen, T1016) was serially diluted 2-fold starting from an initial concentration of 15 mU / mL to obtain 7 concentration points.

[0265] CSPD substrate was mixed with Reaction Buffer Diluent at a ratio of 1:20 to obtain a reaction solution. 5× Dilution Buffer was diluted with DNase RNase-Free Distilled Water to 1× Dilution Buffer. Serum and 1× Dilution Buffer were mixed in a centrifuge tube to obtain a sample diluent, and the sample diluent was incubated at 65 °C for 30 minutes and then cooled to room temperature. 50 μL of the sample diluent was added to a 96-well plate, then 50 μL of Assay Buffer was added to each well and incubated at room temperature for 5 minutes. 50 μL of the reaction solution was added to each well, incubated at room temperature for 20 minutes, and the SEAP chemiluminescence value was read using a microplate reader (Tecan, Infinite 200).

[0266] The preventive and / or therapeutic effect of the test sample was evaluated by measuring the SEAP expression level in the serum. Test samples capable of inhibiting the SEAP expression level as nucleic acid drugs were selected.

[0267] On the 15th day after modeling, according to Table 16, each mouse was subcutaneously administered once with 200 μl of physiological saline containing 3 mg / kg (mpk) of APOC3 RNAi reagent (DR002222 - DR002226, DR001478, DR002252), or 200 μl of physiological saline without APOC3 RNAi reagent was used as a control. The HDI model screening results are shown in Table 17-1 and Table 17-2.

[0268]

Table 16

[0269]

Table 17-1

[0270]

Table 17-2

[0271] Example 11 Screening of the Activity of Primary Monkey Hepatocytes (PCH Cells) Cell Transfection A 96-well cell culture plate was taken, 100 μL of Coating Medium was added to each well, and coated at 37°C for 30 minutes. After completion, the Coating Medium was aspirated and removed.

[0272] Primary monkey hepatocytes (derived from Miaoshun (Shanghai) Biotechnology Co., Ltd.) were revived at 37°C, the cells were resuspended with Thawing Medium, centrifuged and counted, and plated into a 96-well plate at 90 μL / well, 2×10 4 cells / well.

[0273] siRNA Compound Dilution: 20 μM of siRNADilute the compound stock solution with Opti-MEM. Take 198 μL of Opti-MEM, add 2 μL of the compound stock solution, pipette to mix uniformly, and perform corresponding dilution operations according to the actual needs of the experiment as the first concentration point.

[0274] Transfection procedure: Dilute 0.9 μL of RNAiMAX (Thermo, 13778150) with 14.1 μL of Opti-MEM, pipette gently to mix uniformly, and let it stand at room temperature for 5 minutes. Then, gently pipette and mix 15 μL of the prepared RNAi-MAX mixture and 15 μL of the diluted compound without introducing air bubbles, let it stand at room temperature for 10 minutes, and dispense into a 96-well plate at 10 μL / well. After culturing in a 37 °C, 5% CO₂ incubator for 4 h, change the Culture Medium and extract RNA the next day.

[0275] RNA Extraction Cell RNA extraction was performed using a nucleic acid extraction device (Hangzhou Ausheng, Auto-pure96) according to the operation protocol of the high-throughput cell RNA extraction kit (Fanze Medical, FG0412).

[0276] RNA Reverse Transcription Preparation of the denaturation reaction mixture, refer to PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B). Preparation volume for a single well: 1 μL of Oligo dT Primer, 1 μL of dNTP Mixture, 12.5 μL of template RNA. After heating at 65 °C for 5 minutes by conventional PCR and then rapidly cooling on ice for 2 minutes.

[0277] Preparation of the reverse transcription reaction solution, refer to PrimeScript TMRefer to the II 1st Strand cDNA Synthesis Kit (Takara, 6210B). Preparation volume for a single well: 4 μL of 5× Prime Script II Buffer, 0.5 μL of RNase Inhibitor, 1 μL of PrimeScript II RTase, 14.5 μL of the reaction solution after the previous step of denaturation. Mix gradually and uniformly, incubate at 42 °C for 45 minutes by conventional PCR for reverse transcription, incubate at 95 °C for 5 minutes to inactivate the enzyme, and cool the reverse transcription product (cDNA) at 4 °C.

[0278] After the completion of reverse transcription, 30 μL of DNase RNase-Free Distilled Water was added to each well of the cDNA sample.

[0279] Real-time PCR TaqMan TM Refer to Fast Advanced Master Mix (ABI, 4444965), and perform a real-time PCR reaction (ABI, QuantStudio3) in a 20 μL system. The reaction process was: (50 °C, 2 min) × 1 Cycle; (95 °C, 20 s) × 1 Cycle; (95 °C, 1 s; 60 °C, 24 s) × 40 Cycles.

[0280]

Table 18

[0281] Data statistics 2 -△△Ct Calculate the value and convert it to a percentage to obtain the residual inhibition rate. △△Ct = [(Ct of the target gene in the experimental group - Ct of the internal standard in the experimental group) - (Ct of the target gene in the control group - Ct of the internal standard in the control group)]

[0282] siRNAThe starting concentration of the compound was set at 10 nM, and 11 concentration points (10 nM, 3.33 nM, 1.11 nM, 0.37 nM, 0.123 nM, 0.041 nM, 0.0136 nM, 0.0045 nM, 0.00152 nM, 0.000508 nM, 0.000169 nM) were diluted in a 3-fold gradient, and siRNA compound cell activity screening was performed. The experimental screening results are shown in Table 19.

[0283]

Table 19

[0284] Example 12 Mouse HDI Model Activity Screening According to the experimental method of Example 10, in this experiment, female Balb / c mice aged 6 - 8 weeks were selected for HDI model construction and administration detection. Piggy-Bac helper plasmid and Piggy-Bac transposon recombinant plasmid were selected, and modeling was performed at a mass ratio of 1:1 and a total plasmid amount of 50 μg. On the 14th day after modeling, after collecting serum, the mice were blindly grouped, and (N = 6) according to Table 18, each mouse was subcutaneously administered once with 200 μl of physiological saline containing 3 mg / kg (mpk) Apoc3 RNAi reagent, or 200 μl of physiological saline without Apoc3 RNAi reagent was used as a control. The experimental screening results of the HDI model are shown in Table 20.

[0285]

Table 20

[0286] Example 13 Efficacy Verification in hAPOC3 Transgenic Mouse Model Detect triglyceride (TG), low-density lipoprotein cholesterol (LDL-c), HDL cholesterol (HDL-c), and total cholesterol (TC) in hAPOC3 transgenic mouse model (B6-hAPOC3-Tg, T055510, male, 6 - 8 weeks old, GemPharmatech). Based on the TG levels, randomly divide the mice into 5 groups with 5 mice in each group. Calculate the dosage according to the body weight for each animal, and administer a single dose by subcutaneous injection. Administer the siRNA conjugate as a 3 mg / mL solution (using 0.9% sodium chloride aqueous solution as the solvent). Before the experiment, dissolve the siRNA conjugate in 0.9% sodium chloride aqueous solution and make up to the required solution concentration and volume. Set the administration volume of physiological saline and siRNA conjugate to 5 mL / kg.

[0287] Collect blood samples for detection respectively before administration (recorded as -3 days). Group the mice according to the TG levels on Day -3. After administration (recorded as Day 0), collect blood from the orbital venous plexus of the mice on days 7, 14, 28, 42, 56, and 70. (Before each blood collection, subject the mice to a 5-hour fasting treatment), centrifuge to collect serum, detect TG, LDL-c, and TC, and detect the target protein (Human APOC3 ELISA Kit, Abcam, ab154131) by ELISA method. The experimental results are shown in Tables 21 - 24.

[0288]

Table 21

[0289]

Table 22

[0290]

Table 23

[0291]

Table 24

[0292] Example 14 psiCHECK2 GSSM-5 Hits Off-Target Activity Screening Referring to Example 8, in this experiment, the HEK293A cell line was selected, siRNA the initial concentration of the compound was set at 40 nM, and 11 concentration points (40 nM, 13.3 nM, 4.44 nM, 1.48 nM, 0.493 nM, 0.164 nM, 0.0548 nM, 0.0182 nM, 0.00609 nM, 0.00203 nM, 0.000677 nM) were diluted in a 3-fold gradient, and siRNA compound psiCHECK2 GSSM-5 Hits off-target activity screening was performed. The experimental screening results are shown in Table 25.

[0293]

Table 25

[0294] Example 15 Preparation of Compound E2

Chem.

[0295] 1. Preparation of Compound 2b

Chem.

[0296] 2. Preparation of Compound 2d

Chemical formula

[0297] 3. Preparation of Compound 2e

Chemical formula

[0298] 4. Preparation of compound 2f

Chemical formula

[0299] 5 Preparation of Compound 2g [Chemical formula] Under room temperature conditions, Compound 2f (69.0 g, 143 mmol) and magnesium turnings (54.7 g, 2.28 mol) were added to methanol (400 mL), and the mixture was reacted at 66 °C for 1 hour. TLC (DCM / MeOH = 10 / 1, UV254 nm) indicated that the reaction of the raw materials was complete and new spots were generated. Water (3000 mL) and saturated aqueous ammonium chloride solution (3000 mL) were added to the reaction solution for dilution, and the mixture was extracted with dichloromethane (1000 mL × 3). The organic phase was washed with saturated sodium bicarbonate (300 mL × 3), and the organic phase was concentrated under reduced pressure to obtain crude Compound 2g (32.0 g). 1 H NMR (400 MHz, CDCl3) δ 7.27 - 7.40 (m, 10 H), 4.57 (s, 4 H), 3.90 - 4.00 (m, 2 H), 3.59 - 3.69 (m, 4 H), 2.77 - 3.04 (m, 4 H)

[0300] 6. Preparation of Compound 2h [Chemical formula] Under room temperature conditions, 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). After reacting at 25 °C for 18 hours, sodium cyanoborohydride (2.30 g, 36.7 mmol) was added and the reaction was carried out at 50 °C for 4 hours, showing the formation of a new spot by TLC (DCM / MeOH = 10 / 1). Water (50 mL) was added to the reaction solution for dilution, and it was 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 obtain compound 2h (3.00 g, yield 79.9%). 1 H NMR (400 MHz, CDCl3) δ 7.28 - 7.41 (m, 10 H), 4.51 - 4.71 (m, 4 H), 4.00 - 4.35 (m, 2 H), 3.49 - 3.77 (m, 4 H), 2.69 - 2.98 (m, 2 H), 1.63 - 2.12 (m, 7 H), 1.16 - 1.44 (m, 6 H)

[0301] 7. Preparation of compound 2i

Chemical formula

[0302] 8. Preparation of Compound 2j

Chemical Structure

[0303] 9. Preparation of Compound E2

Chemical Structure

[0304] Example 16 Preparation of Compound E1-1

Chemical Structure

[0305] 1. Preparation of Compound 1b

Chemical Structure

[0306] 2. Preparation of Compound 1c

Chemical Structure

[0307] 3. Preparation of Compound 1d [Chemical formula] Compound 1c (203 g, 646 mmol, 1.00 eq) was dissolved in tetrahydrofuran (2.03 L), and potassium tert-butoxide (145 g, 1.29 mol, 1.29 eq) was added portionwise at 0 °C. The mixture was stirred at 25 °C for 16 h. It was cooled to 5 °C, quenched with ice water (1.1 L), extracted with methyl tert-butyl ether (2.0 L), washed with saturated brine (2.0 L), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate) to obtain Compound 1d (160 g, 91.3%). 1 1H NMR: 400 MHz CDCl3 δ 5.11 (s, 1H), 5.02 (d, J = 5.6 Hz, 1H), 4.06 (d, J = 1.6 Hz, 1H), 4.50 (d, J = 5.6 Hz, 1H), 4.39 (d, J = 1.6 Hz, 1H), 3.41 (s, 3H), 3.24 - 3.20 (m, 1H), 3.09 (t, J = 10.0 Hz, 1H), 1.47 (s, 3H), 1.35 (s, 3H).

[0308] 4. Preparation of Compound 1e [Chemical formula] Zinc copper reagent (90.0 g, 1.37 mol, 5.50 eq) was dispersed in ether (200 mL), compound 1d (60 g, 241 mmol, 1.00 eq) was added at 25 °C, and a solution of trichloroacetyl chloride (61.5 g, 338 mmol, 1.40 eq) in ether (200 mL) was added dropwise, followed by continuous stirring for 1 hour. It was filtered, the cake was washed away with methyl-t-butyl ether (500 mL), the filtrate was poured into a saturated aqueous sodium hydrogen carbonate solution (2.50 L), filtered, the filtrate was washed three times with saturated brine (500 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain crude compound 1e (71.8 g). 1 H NMR: 400 MHz CDCl3 δ 5.12 (d, J = 5.6 Hz, 1H), 5.09 (s, 1H), 4.70 (d, J = 6.0 Hz, 1H), 3.70 - 3.55 (m, 2H), 3.54 (s, 3H), 1.45 (s, 3H), 1.36 (s, 3H).

[0309] 5. Preparation of Compound 1f

Chemical formula

[0310] 6. Preparation of Compound 1g

Chemical Structure

[0311] 7. Preparation of Compound 1h

Chemical Structure

[0312] 8. Preparation of Compound 1i

Chemical Structure

[0313] 9. Preparation of Compound 1j [Chemistry] Compound 1i (141 g, 201 mmol, 1.00 eq) was dissolved in methanol (1.41 L), and an aqueous solution of HCl (704 mL, 1.40 mol, 2 M, 7.00 eq) was added. The reaction mixture was stirred at 60 °C for 1 hour. The reaction mixture was extracted with a methyl-t-butyl ether / petroleum ether mixture. The aqueous phase was collected, adjusted to pH 8 with saturated sodium hydrogen carbonate, concentrated, tetrahydrofuran was added to the resulting residue and filtered, and the filtrate was concentrated to obtain crude compound 1j (62.4 g, 201 mmol).

[0314] 10. Preparation of Compound 1k [Chemistry] Compound 1j (62.4 g, 201 mmol, 1.00 eq) was dissolved in pyridine (300 mL), acetic anhydride (47.4 mL, 502 mmol, 2.50 eq) was added, and the mixture was stirred at 25 °C for 12 hours. The reaction mixture was diluted with saturated sodium hydrogen carbonate (1.00 L), extracted twice with ethyl acetate (600 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate) to obtain compound 1k (74 g, 93.3%). 1 H NMR: 400 MHz CDCl3 δ 5.38 - 5.10 (m, 3H), 4.07 - 4.03 (m, 4H), 3.36 - 3.32 (m, 3H), 2.41 - 2.05 (m, 5H), 2.04 - 1.98 (m, 9H), 1.27 - 1.23 (m, 6H).

[0315] 11. Preparation of Compound 1l [Chemistry] Compound 1k (79.3 g, 201 mmol, 1.00 eq) was dissolved in ethyl acetate (476 mL), and acetic anhydride (62.6 mL, 663 mmol, 3.30 eq) and concentrated sulfuric acid (5.38 mL, 100 mmol, 0.50 eq) were added. The mixture was stirred at 25 °C for 3 h. The reaction mixture was neutralized with saturated aqueous sodium hydrogen carbonate solution and extracted twice with ethyl acetate (1.00 L). The combined organic phases were washed twice with saturated brine (500 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography to give compound 1l (43 g, 50.6%). 1 H NMR: 400 MHz CDCl3 δ 6.11 (d, J = 2.8 Hz, 1H), 5.44 - 5.39 (m, 1H), 5.33 - 5.32 (m, 1H), 4.10 - 4.05 (m, 5H), 2.80 - 2.35 (m, 4H), 2.12 - 2.02 (m, 9H), 1.32 - 1.23 (m, 6H).

[0316] 12. Preparation of Compound 1n

Chemical Structure

[0317] 13. Preparation of Compound 1o

Chemical Structure

[0318] 14. Preparation of Compound 1p

Chem.

[0319] 15. Preparation of Compound 1q

Chem.

[0320] 16. Preparation of Compounds 1q-1 and 1q-2

Chemical Structure

[0321] m / z: ES+ [M+H]+ 525.2 m / z: ES+ [M+H]+ 525.2 HPLC: Retention time 0.820 min (Column: XBridge C18 2.1*50mm, 5um; Mobile phase: A: 10mM NH4HCO3 aqueous solution B: Acetonitrile; Gradient: 0 - 0.01 min 5% B, 0.01 - 0.7 min 5 - 95% B, 0.7 - 1.16 min 95% B, 1.16 - 1.5min, 95% - 5% B; Flow rate: 1.5 mL / min; Column temp.: 40℃) 1 1H NMR: 400 MHz CDCl3 δ 7.37 - 7.08 (m, 5H), 7.09 (d, J = 8.0 Hz, 1H), 5.76 - 5.74 (m, 2H), 5.49 - 5.44 (m, 2H), 4.73 - 4.67 (m, 2H), 4.15 - 4.06 (m, 4H), 4.02 (d, J = 4.8 Hz, 1H), 3.87 - 3.85 (m, 1H), 3.56 (s, 3H), 2.81 - 2.64 (m, 2H), 2.50 - 2.39 (m, 2H), 2.36 - 2.27 (m, 1H), 1.34 - 1.30 (m, 6H).

[0322] Compound 1q - 2: m / z: ES+ [M + H]+ 525.2 HPLC: Retention time 0.836 min (Column: XBridge C18 2.1*50mm, 5um; Mobile phase: A: 10mM NH4HCO3 aqueous solution B: Acetonitrile; Gradient: 0 - 0.01 min 5% B, 0.01 - 0.7 min 5 - 95% B, 0.7 - 1.16 min 95% B, 1.16 - 1.5min, 95% - 5% B; Flow rate: 1.5 mL / min; Column temp.: 40℃) 11H NMR: 400 MHz CDCl3 δ 7.37 - 7.27 (m, 5H), 7.08 (d, J = 8.0 Hz, 1H), 5.75 - 5.73 (m, 2H), 5.50 - 5.45 (m, 2H), 4.73 - 4.67 (m, 2H), 4.18 - 4.08 (m, 4H), 4.06 (d, J = 4.8 Hz, 1H), 3.88 - 3.86 (m, 1H), 3.56 (s, 3H), 3.06 - 2.95 (m, 1H), 2.83 - 2.41 (m, 5H), 1.34 - 1.31 (m, 6H).

[0323] 17. Preparation of Compound 1r-1

Chemical Structure

[0324] 18. Preparation of Compound E1-1

Chemical Structure

[0325] Example 17 Preparation of Compound E1 [Chemistry]

[0326] 1. Preparation of Compound 2 [Chemistry] At 25 °C, Compound 1 (50.0 g, 263 mmol) was dissolved in DCM (800 mL), and further imidazole (26.9 g, 394 mmol) and TBDPSCl (75.2 mL, 289 mmol) were sequentially added, and the reaction solution was stirred at 25 °C for 18 hours. Thin layer chromatography (DCM / MeOH = 10 / 1, PE / EA = 3 / 1) indicated that the reactants were completely consumed and new spots were generated. The reaction solution was spin-dried to obtain a crude product, and the crude product was purified by MPLC (PE / EA = 1 / 0 - 5 / 1) to obtain a colorless oily liquid Compound 2 (95.0 g, yield 84.31%). 1 H NMR (400 MHz, CDCl3) δ 7.70 (dd, J = 7.6, 1.6 Hz, 4 H), 7.35 - 7.46 (m, 6 H), 5.86 (d, J = 3.6 Hz, 1 H), 4.61 (dd, J = 4.8, 4.0 Hz, 1 H), 4.15 (td, J = 8.8, 5.2 Hz, 1 H), 3.93 - 4.01 (m, 1 H), 3.81 - 3.92 (m, 2 H), 1.60 (s, 3 H), 1.39 (s, 3 H), 1.06 (s, 9 H).

[0327] 2. Preparation of Compound 3 [Chemistry] At 25 °C, compound 2 (95.0 g, 222 mmol) was dissolved in toluene (1.50 L), and imidazole (30.2 g, 443 mmol), triphenylphosphine ((116 g, 443 mmol) and iodine (84.4 g, 322 mmol) were sequentially added. The reaction mixture was stirred at 100 °C for 18 hours. Thin layer chromatography (PE / EA = 5 / 1) indicated that the reactants were completely consumed and new spots were generated. 20.0 mL of saturated NaHSO3 solution was added to the reaction mixture, followed by 500 mL of water. The reaction mixture was separated, and the organic phase was washed with saturated NaCl solution (50.0 mL × 3), dried over anhydrous Na2SO4, and spin-dried to obtain the crude product. The crude product was purified by MPLC (PE / EA = 1 / 0 - 10 / 1) to obtain a colorless oily liquid compound 3 (115 g, yield 96.35%). 1 1H NMR (400 MHz, CD3OD) δ 7.64 - 7.72 (m, 4 H), 7.37 - 7.49 (m, 6 H), 5.95 (d, J = 3.6 Hz, 1 H), 5.06 (d, J = 3.6 Hz, 1 H), 4.42 (d, J = 3.2 Hz, 1 H), 3.87 (dd, J = 10.4, 5.6 Hz, 1 H), 3.66 (dd, J = 10.4, 6.4 Hz, 1 H), 3.53 (td, J = 6.0, 3.2 Hz, 1 H), 1.44 (s, 3 H), 1.29 (s, 3 H), 1.02 - 1.07 (m, 9 H).

[0328] 3. Preparation of compound 4

Chemical formula

[0329] 4. Preparation of Compound 5

Chemical Structure

[0330] 5. Preparation of Compound 6

Chemical Structure

[0331] 6. Preparation of Compound 7 [Chemical formula] At 25 °C, Compound 6 (40.0 g, 141 mmol) was dissolved in trimethyl phosphite (500 mL), and the reaction solution was stirred at 120 °C for 11 hours. Thin layer chromatography (ethyl acetate / acetone = 3 / 1, PE / EA = 10 / 1) indicated that the raw material remained and a new spot was generated. The reaction solution was spin-dried to obtain a crude product, and the crude product was purified by MPLC (ethyl acetate / acetone = 1 / 0 - 20 / 1) to obtain a pale yellow oily liquid Compound 7 (8.90 g, yield 23.74%), and the raw material of white solid Compound 7 (30.0 g) was recovered. 1 H NMR (400 MHz, CDCl3) δ 5.81 (d, J = 4.0 Hz, 1 H), 4.74 (t, J = 4.4 Hz, 1 H), 4.40 - 4.52 (m, 1 H), 3.76 (dd, J = 10.8, 0.8 Hz, 6 H), 2.23 - 2.35 (m, 2 H), 1.95 - 2.07 (m, 1 H), 1.63 (ddd, J = 13.6, 10.8, 4.8 Hz, 1 H), 1.52 (s, 3 H), 1.32 (s, 3 H).

[0332] 7. Preparation of Compound 8 [Chemical formula] Compound 7 (13 g, 48.830 mmol), Ac2O (23.036 mL, 244.150 mmol), and H2SO4 (2.615 mL, 48.830 mmol) were sequentially added to AcOH (260 mL). The reaction was carried out at 25 °C for 6 hours. A new spot was detected by TLC (ethyl acetate:acetone = 3:1). The reaction solution was quenched with ice water, extracted with DCM (500 mL × 3), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by TLC column chromatography (ethyl acetate:acetone = 50:1 - 3:1) to obtain a yellow oily Compound 8 (7.9 g, 25.464 mmol, 52.15%). 1 1H NMR (400 MHz, CDCl3) δ 6.10 (d, J = 1.2 Hz, 1 H), 5.18 (d, J = 5.2 Hz, 1 H), 4.58 - 4.72 (m, 1 H), 3.75 - 3.79 (m, 3 H), 3.71 - 3.74 (m, 3 H), 2.16 - 2.38 (m, 3 H), 2.07 (s, 3 H), 2.05 (s, 3 H), 1.96 - 2.04 (m, 1 H).

[0333] 8. Preparation of Compound 9 [Chemical Structure] Compound 8 (7.9 g, 25.464 mmol) and Compound 8A (6.09 g, 25.464 mmol) were added to CH3CN (316 mL). SnCl4 (8.781 mL, 76.392 mmol) was slowly added dropwise at 0 °C, and the mixture was reacted at 25 °C for 2 hours. TLC (ethyl acetate:acetone = 10:1, PMA) indicated the disappearance of the starting compound 8 and the formation of a new spot. LCMS (RW0006 - 267 - P1A) indicated the formation of 31.3% of the product. The reaction mixture was cooled to 0 °C, adjusted to pH = 8 with an aqueous solution of saturated NaHCO3, and then the aqueous phase was extracted with DCM (200 mL × 3). The organic phase was dried over anhydrous Na2SO4, filtered, and spin - dried to obtain a crude product. The crude product was purified by column chromatography (ethyl acetate:acetone = 20:1 - 3:1) to obtain yellow oily Compound 9 (6.1 g, 12.464 mmol, 48.95%). 11H NMR (400 MHz, CDCl3) δ 8.75 - 8.84 (m, 1 H), 8.17 (s, 1 H), 8.01 - 8.10 (m, 2 H), 7.59 - 7.70 (m, 1 H), 7.50 - 7.57 (m, 2 H), 6.07 (s, 1 H), 5.66 - 5.73 (m, 1 H), 4.98 - 5.12 (m, 1 H), 4.70 - 4.81 (m, 1 H), 3.71 - 3.82 (m, 12 H), 2.70 - 2.81 (m, 1 H), 2.34 - 2.52 (m, 2 H), 2.21 - 2.27 (m, 1 H), 2.16 (s, 3 H), 2.06 (d, J=4.4 Hz, 4 H).

[0334] 9. Preparation of Compound 10

Chem.

[0335] 10. Preparation of Compound 11

Chem.

[0336] 11. Preparation of Compound 12

Chemical Structure

[0337] 12. Preparation of E1

Chemical Structure

Claims

1. A small interfering RNA (siRNA) for inhibiting the expression of apolipoprotein C3 (APOC3) in cells, wherein the siRNA comprises a sense strand and an antisense strand that form a double-stranded region, and the lengths of the sense strand and the antisense strand are each independently 15 to 30 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence represented by any one of SEQ ID NOs: 414 to 826, siRNA.

2. The siRNA according to claim 1, wherein the sense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence represented by any one of SEQ ID NOs: 1 to 413.

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

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

5. The antisense strand of the siRNA according to any one of claims 1 to 4 comprises at least 15 consecutive nucleotides, at least 16 consecutive nucleotides, at least 17 consecutive nucleotides, at least 18 consecutive nucleotides, at least 19 consecutive nucleotides, or at least 20 consecutive nucleotides of the nucleotide sequence represented by any one of SEQ ID NOs: 473, 612, 690, 757, 761, 816, 817, 818, 819, 820, 814, and 815, and preferably the antisense strand comprises the nucleotide sequence represented by any one of SEQ ID NOs: 473, 612, 690, 757, 761, 816, 817, 818, 819, 820, 814, and 815.

6. The sense strand contains at least 15 consecutive nucleotides, at least 16 consecutive nucleotides, at least 17 consecutive nucleotides, at least 18 consecutive nucleotides, at least 19 consecutive nucleotides, or at least 20 consecutive nucleotides of the nucleotide sequence represented by any one of SEQ ID NOs: 60, 199, 277, 344, 348, 403, 404, 405, 406, 407, 401, and 402. Preferably, the sense strand contains the nucleotide sequence represented by any one of SEQ ID NOs: 60, 199, 277, 344, 348, 403, 404, 405, 406, 407, 401, and 402. The siRNA according to any one of claims 1 to 3 and 5. **Claim 7** (a) The sense strand contains the nucleotide sequence represented by SEQ ID NO: 60, and the antisense strand contains the nucleotide sequence represented by SEQ ID NO: 473, or (b) The sense strand contains the nucleotide sequence represented by SEQ ID NO: 199, and the antisense strand contains the nucleotide sequence represented by SEQ ID NO: 612, or (c) The sense strand contains the nucleotide sequence represented by SEQ ID NO: 277, and the antisense strand contains the nucleotide sequence represented by SEQ ID NO: 690, or (d) The sense strand contains the nucleotide sequence represented by SEQ ID NO: 344, and the antisense strand contains the nucleotide sequence represented by SEQ ID NO: 757, or (e) The sense strand contains the nucleotide sequence represented by SEQ ID NO: 348, and the antisense strand contains the nucleotide sequence represented by SEQ ID NO: 761, or (f) The sense strand contains the nucleotide sequence represented by SEQ ID NO: 403, and the antisense strand contains the nucleotide sequence represented by SEQ ID NO: 816, or (g) The sense strand contains the nucleotide sequence represented by SEQ ID NO: 404, and the antisense strand contains the nucleotide sequence represented by SEQ ID NO: 817, or (h) The sense strand contains the nucleotide sequence represented by SEQ ID NO: 405, and the antisense strand contains the nucleotide sequence represented by SEQ ID NO: 818, or (i) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 406, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 819, or (j) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 407, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 820, or (k) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 401, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 814, or (l) The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 402, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 815, the siRNA according to any one of claims 1 to 6.

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

9. 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'-fluorinated modified nucleotides, 2'-deoxy modified nucleotides, inosine ribonucleotides, deprotonated nucleotides, inverted abasic deoxyribonucleotides, thiophosphate ester nucleotide internucleotide linkage modifications, vinylphosphonate modified nucleotides, locked nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing non-natural bases, and terminal nucleotides and deoxyribonucleotides conjugated to cholesterol-based derivatives or dodecanedioic acid dodecaneamide, the siRNA according to claim 8.

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

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

12. The siRNA contains a paired modified sense strand sequence and a modified antisense strand sequence shown in any of Table 6 of the specification, the siRNA according to claim 8.

13. (1) The sense strand contains STM1s - Am sAm Am Am Gm Gm Gm Am Cf Af Gf Um Am Um Um Cm Um Cm Am Gm Am s - STM1 (SEQ ID NO: 1655), and the antisense strand contains (CP1a - U)s Cf sUm Gf Am Gf Am Af Um Af Cm Uf Gm Uf Cm Cf Cm Uf Um sUf sUm (SEQ ID NO: 1651), or (2) The sense strand contains STM1s - Am sAm Um Um Am Am Am Am Gf Gf Gf Am Cm Am Gm Um Am Um Um Cm Am s - STM1 (SEQ ID NO: 1656), and the antisense strand contains (CP1a - U)s Gf sAm Af Um Af Cm Uf Gm Uf Cm Cf Cm Uf Um Uf Um Af Am sUf sUm (SEQ ID NO: 1650), or (3) The sense strand contains STM1s - Am sAm Um Um Am Am Am Am Gf Gf Gf Am Cm Am Gm Um Am Um Um Cm Am s - STM1 (SEQ ID NO: 1657), and the antisense strand contains (VPUm)s Gf sAm Af Um Af Cm Uf Gm Uf Cm Cf Cm Uf Um Uf Um Af Am sUf sUm (SEQ ID NO: 1649), or (4) The sense strand contains IB s - Am sAm Um Um Am Am Am Am Gf Gf Gf Am Cm Am Gm Um Am Um Um Cm Am s - IB (SEQ ID NO: 1658), and the antisense strand comprises (CP1a-U)sGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAm sUfsUm (SEQ ID NO: 1650), or (5) the sense strand comprises UmsUmsAmAmAmAmGfGfGfAmCfAmGmUmAmUmUmCm sAm (SEQ ID NO: 1659), and the antisense strand comprises (CP1a-U)sGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAm sUfsUm (SEQ ID NO: 1650), or (6) the sense strand comprises IBs-Am sAmAmAmGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmAm s-IB (SEQ ID NO: 1660), and the antisense strand comprises (CP1a-U)sCfsUmGfAmGfAmAfUmAfCmUfGmUfCmCfCmUfUm sUfsUm (SEQ ID NO: 1651), or (7) the sense strand comprises Am sAm sGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGm sAm (SEQ ID NO: 1661), and the antisense strand comprises (CP1a-U)sCfsUmGfAmGfAmAfUmAfCmUfGmUfCmCfCmUfUm sUfsUm (SEQ ID NO: 1651), or (8) the sense strand comprises IBs-Am sAmAmAmGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmAm s-IBs-GL6 (SEQ ID NO: 1662), and the antisense strand comprises (CP1a-U)sCfsUmGfAmGf(PCN-A)AfUmAfCmUfGmUfCmCfCmUfUm sUfsUm (SEQ ID NO: 1652), or (9) the sense strand comprises Am sAm sGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGm sAm (SEQ ID NO: 1663), and the antisense strand comprises (CP1a-U)sCfsUmGfAmGf(PCN-A)AfUmAfCmUfGmUfCmCfCmUfUm sUfsUm (SEQ ID NO: 1652), or (a) The sense strand contains CmsGmsAmGmGmAmUfGfCfCmUmCmCmUmUmCmUmUm, and the antisense strand contains Am sAfsGmAfAmGfGmGfAmGfGmCfAmUfCmCfUmCfGmsUfsUm, or (b) The sense strand contains CmsCmsGmUmUmAmAfGfGfAmCmAmAmGmUmUmCmUmUm, and the antisense strand contains Am sAfsGmAfAmCfUmUfGmUfCmCfUmUfAmAfCmGfGmsUfsUm, or (c) The sense strand contains CmsCmsGmUmUmAmAfGfGfAmCmAmAmGmUmUmCmUmUm, and the antisense strand contains Am sAfsGmAfAmCfUmUfGmUfCmCfUmUfAmAfCmGfGmsUfsUm, or (d) The sense strand contains CmsCmsAmAmGmUmCfCfAfCmCmUmGmCmCmUmAmUmUm, and the antisense strand contains Am sAfsUmAfGmGfCmAfGmGfUmGfGmAfCmUfUmGfGmsUfsUm, or (e) The sense strand contains Am sAm sGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmUm, and the antisense strand contains Am sCfsUmGfAmGfAmAfUmAfCmUfGmUfCmCfCmUfUmsUfsUm, or (f) The sense strand contains UmsUmsAmAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmUm, and the antisense strand contains Am sGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUfUmAfAmsUfsUm, or (g) The sense strand contains Am sAm sGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmUm, and the antisense strand contains Am sCfsUmGfAmGfAmAfUmAfCmUfGmUfCmCfCmUfUmsUfsUm, or (h) The sense strand contains CmsCmsAmAmGmUmCfCfAfCmCmUmGmCmCmUmAmUmUm, and the antisense strand contains Am sAfsUmAfGmGfCmAfGmGfUmGfGmAfCmUfUmGfGmsUfsUm, or (i) The sense strand comprises CmsCmsGmUmUmAmAfGfGfAmCmAmAmGmUmUmCmUmUm, and the antisense strand comprises Am sAfsGmAfAmCfUmUfGmUfCmCfUmUfAmAfCmGfGmsUfsUm, or (j) The sense strand comprises CmsGmsAmGmGmAmUfGfCfCmUmCmCmCmUmUmCmUmUm, and the antisense strand comprises Am sAfsGmAfAmGfGmGfAmGfGmCfAmUfCmCfUmCfGmsUfsUm, or (k) The sense strand comprises IBs - AmCmGmGmGmAmCmAmGfUfAfUmUmCmUmCmAmGmUmisAm s - IB (SEQ ID NO: 1014), and the antisense strand comprises UmsCfsAmsCfUmGfAmGmAmAmUmAfCmUfGmUfCmCfCmGfsUm (SEQ ID NO: 1174), or (l) The sense strand comprises Am sAm sGmGmGmAmCfAmGfUfAfUmUmCmUmCmAmGmUmsGmsCm, and the antisense strand comprises GmsCfsAmCmUmGfAmGmAmAmUmAmCmUfGmUfCmCmCmUmUmsUmsUm, the siRNA according to claim 8.

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

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

16. The ligand moiety contains a conjugate group represented by (X'), 【Chemical 1】 where 【Chemical 2】 represents the position where it binds to the siRNA, Q is independently H, [Chemical Formula 3] and Here, L １ is a chemical bond, -CH ２ -, -CH ２ CH ２ -, -C(O)-, -CH ２ O-, -CH ２ O-CH ２ CH ２ O- or -NHCO-(CH ２ NHCO) ａ -, and L ２ is a chemical bond or -CH ２ CH ２ C(O)-, and L ３ is a chemical bond, -(NHCH ２ CH ２ ) ｂ -, -(NHCH ２ CH ２ CH ２ ) ｂ - or -C(O)CH ２ -, and L ４ is -(OCH ２ CH ２ ), -(OCH ｃ CH ２ CH ２ CH ２ ), -(OCH ｃ CH ２ CH ２ CH ２ CH ２ ), -(OCH ｃ CH ２ CH ２ CH ２ CH ２ CH ２ ), -(OCH ｃ CH ２ ), - or -NHC(O)-(CH ｄ ), and 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, -CH ２ O- or -NHC(O)-, and L' is a chemical bond, -C(O)NH-, -NHC(O)- or -O(CH ２ CH ２ O) ｅ -, and where e is 1, 2, 3, 4 or 5, T is a chemical bond, -CH ２ -, -C(O)-, -M-, -CH ２ -M- or -C(O)-M-, and where M is 【Chemical 4】 and R １ and R ２ together form -CH ２ CH ２ O- or -CH ２ CH(R)-O- is formed, and R ３ is H, or R １ and R ３ together form -C １－２ alkylene-, and R ２ is H Here, R is -OR', -CH ２ OR' or -CH ２ CH ２ OR', where R' is H, a hydroxyl protecting group or a solid support, and the hydroxyl protecting group is preferably -C(O)CH ２ CH ２ C(O)OH or 4,4'-dimethoxytrityl, m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, The siRNA according to claim 14 or 15, wherein n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

17. The conjugate group is represented by the formula (I'), 【Chemical Formula 5】 where 【Chemical Formula 6】 represents the position of binding to the siRNA, Q is independently H, 【Chemical Formula 7】 and Here, L １ is a chemical bond, -CH ２ -, -CH ２ CH ２ -, -C(O)-, -CH ２ O-, -CH ２ O-CH ２ CH ２ O- or -NHC(O)-(CH ２ NH C(O)) ａ -, and L ２ is a chemical bond or -CH ２ CH ２ C(O)-, and L ３ is a chemical bond, -(NHCH ２ CH ２ ) ｂ -, -(NHCH ２ CH ２ CH ２ ) ｂ - or -C(O)CH ２ -, and L ４ is -(OCH ２ CH ２ ), -(OCH ｃ CH ２ CH ２ CH ２ ), -(OCH ｃ CH ２ CH ２ CH ２ CH ２ ), -(OCH ｃ CH ２ CH ２ CH ２ CH ２ CH ２ ), -(OCH ｃ CH ２ ), - or -NHC(O)-(CH ｄ ), and 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 -CH ２ O- or -NHC(O)-, and L' is a chemical bond, -C(O)NH- or -NHC(O)-, R １ and R ２ together form -CH ２ CH ２ O- or -CH ２ CH(R)-O-, where R ３ is H, or R １ and R ３ together form -C １－２ alkylene-, and R ２ is H Here, R is -OR', -CH ２ OR' or -CH ２ CH ２ OR', where R' is H, a hydroxyl protecting group or a solid support, and the hydroxyl protecting group is preferably -C(O)CH ２ CH ２ C(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, the siRNA according to claim 16.

18. Q is independently H or [Chemical 8] and Here, L １ is -CH ２ O- or -NHC(O)-(CH ２ NHC(O)) ａ - and L ２ is -CH ２ CH ２ C(O)-, and L ３ is -(NHCH ２ CH ２ ) ｂ - or -(NHCH ２ CH ２ CH ２ ) ｂ - and L ４ is -(OCH ２ CH ２ ) ｃ - or -NHC(O)-(CH ２ ) ｄ - and 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 -CH ２ O-, and L' is a chemical bond, R １ and R ２ together form -CH ２ CH ２ O- or -CH ２ CH(R)-O-, and R ３ is H, or R １ and R ３ together form -C １－２ alkylene-, and R ２ is H Here, R is -OR', -CH ２ OR' or -CH ２ CH ２ OR', where R' is H, a hydroxyl protecting group or a solid support, and the hydroxyl protecting group is preferably -C(O)CH ２ CH ２ C(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, the siRNA according to claim 17.

19. The conjugate group is represented by the formula (I'-1), the formula (I'-2) or the formula (I'-3), 【Chemical Formula 9】 where 【Chemical Formula 10】 represents the position of binding to the siRNA, Q is 【Chemical 11】 and Here, L １ is -CH ２ O- or -NHC(O)-, and L ２ is -CH ２ CH ２ C(O)-, and L ３ is -(NHCH ２ CH ２ ) ｂ - or -(NHCH ２ CH ２ CH ２ ) ｂ - and L ４ is -(OCH ２ CH ２ ) ｃ - or -NHC(O)-(CH ２ ) ｄ - and 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 -CH ２ O- and R' is H, a hydroxyl protecting group or a solid support, and the hydroxyl protecting group is preferably -C(O)CH ２ CH ２ C(O)OH or 4,4'-dimethoxytrityl, n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, the siRNA according to claim 18.

20. Q is independently H, 【Chemical 12】 and Here, L １ is -CH ２ O-, -CH ２ O-CH ２ CH ２ O- or -NHC(O)-(CH ２ NH C(O)) ａ - and L ２ is -CH ２ CH ２ C(O)-, and L ３ is -(NHCH ２ CH ２ ) ｂ -, -(NHCH ２ CH ２ CH ２ ) ｂ - or -C(O)CH ２ -, and L ４ is -(OCH ２ CH ２ ) ｃ - or -NHC(O)-(CH ２ ) ｄ - and 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 -CH ２ O- or -NHC(O)-, and L' is a chemical bond or -C(O)NH-, R １ and R ２ together form -CH ２ CH ２ O- or -CH ２ CH(R)-O-, and R ３ is H, or R １ and R ３ together form -C １－２ alkylene-, and R ２ is H Here, R is -OR', -CH ２ OR' or -CH ２ CH ２ OR', where R' is H, a hydroxyl protecting group or a solid support, and the hydroxyl protecting group is preferably -C(O)CH ２ CH ２ C(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, the siRNA according to claim 17.

21. The conjugate group is represented by the formula (II'-1) or the formula (II'-2), 【Chemical 13】 where 【Chemical Formula 14】 represents the position of binding to the siRNA, Q is independently 【Chemical 15】 and Here, L １ is -CH ２ O- or -CH ２ O-CH ２ CH ２ O-, and L ３ is -(NHCH ２ CH ２ ) ｂ -, -(NHCH ２ CH ２ CH ２ ) ｂ - or -C(O)CH ２ -, and L ４ is -(OCH ２ CH ２ ) ｃ - or -NHC(O)-(CH ２ ) ｄ - and 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 support, and the hydroxyl protecting group is preferably -C(O)CH ２ CH ２ C(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, the siRNA according to claim 20.

22. Q is independently H, 【Chemical 16】 where, Here, L １ is -CH ２ -, -C(O)-, -CH ２ O-, -CH ２ O-CH ２ CH ２ O- or -NHC(O)-(CH ２ NH C(O)) ａ - and L ２ is a chemical bond, L ３ is -(NHCH ２ CH ２ ) ｂ -, -(NHCH ２ CH ２ CH ２ ) ｂ - or -C(O)CH ２ -, and L ４ is -(OCH ２ CH ２ ) ｃ - or -NHC(O)-(CH ２ ) ｄ - and here, 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 -CH ２ O- or -NHC(O)-, and L’ is a chemical bond or -C(O)NH-, R １ and R ２ together form -CH ２ CH ２ O- or -CH ２ CH(R)-O- is formed, and R ３ is H, or R １ and R ３ together form -C １－２ alkylene-, and R ２ is H Here, R is -OR', -CH ２ OR' or -CH ２ CH ２ OR', where R' is H, a hydroxyl protecting group or a solid support, and the hydroxyl protecting group is preferably -C(O)CH ２ CH ２ C(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, the siRNA according to claim 17.

23. The conjugate group is represented by formula (II’-2), 【Chemical 17】 where, 【Chemical Formula 18】 represents the position of binding to the siRNA, Q is independently, 【Chemical Formula 19】 where, Here, L １ is -CH ２ - or -C(O)-, and L ３ is -(NHCH ２ CH ２ ) ｂ - and L ４ is -(OCH ２ CH ２ ) ｃ - and here, b = 1, 2, 3, 4 or 5, c = 1, 2, 3, 4 or 5, L is -CH ２ O- or -NHC(O)-, and R' is H, a hydroxyl protecting group or a solid support, and the hydroxyl protecting group is preferably -C(O)CH ２ CH ２ C(O)OH or 4,4'-dimethoxytrityl, n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, the siRNA according to claim 22.

24. Q is independently H, 【Chemical 20】 where, Here, L １ is a chemical bond, -CH ２ -, -CH ２ CH ２ -, -C(O)-, -CH ２ O-, -CH ２ O-CH ２ CH ２ O- or -NHCO-(CH ２ NHCO) ａ -, and L ２ is a chemical bond or -CH ２ CH ２ C(O)-, and L ３ is a chemical bond, -(NHCH ２ CH ２ ) ｂ -, -(NHCH ２ CH ２ CH ２ ) ｂ - or -C(O)CH ２ -, and L ４ is -(OCH ２ CH ２ ), -(OCH ｃ CH ２ CH ２ CH ２ ), -(OCH ｃ CH ２ CH ２ CH ２ CH ２ ), -(OCH ｃ CH ２ CH ２ CH ２ CH ２ CH ２ ), -(OCH ｃ CH ２ ), or -NHC(O)-(CH ｄ ), and here, 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, -CH ２ O- or -NHC(O)-, and L' is a chemical bond, -C(O)NH-, -NHC(O)- or -O(CH ２ CH ２ O) ｅ -, and here, e is 1, 2, 3, 4 or 5, T is a chemical bond, -CH ２ -, -M-, -CH ２ -M- or -C(O)-M-, and here, M is, 【Chemical 21】 where, R １ and R ２ together form -CH ２ CH ２ O- or -CH ２ CH(R)-O- is formed, and R ３ is H, or R １ and R ３ together form -C １－２ alkylene-, and R ２ is H Here, R is -OR', -CH ２ OR' or -CH ２ CH ２ OR', where R' is H, a hydroxyl protecting group or a solid support, and the hydroxyl protecting group is preferably -C(O)CH ２ CH ２ C(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, the siRNA according to claim 16.

25. T is -M-, -CH ２ -M- or -C(O)-M-, where M is 【Chemical 22】 the siRNA according to claim 24.

26. Q is independently H or 【Chemical formula 23】 where, Here, L １ is -CH ２ O- or -NHC(O)-(CH ２ NHC(O)) ａ -, and L ２ is -CH ２ CH ２ C(O)-, and L ３ is -(NHCH ２ CH ２ ) ｂ - or -(NHCH ２ CH ２ CH ２ ) ｂ - and L ４ is -(OCH ２ CH ２ ) ｃ - or -NHC(O)-(CH ２ ) ｄ - and here, 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 -CH ２ O-, and L' is a chemical bond or -O(CH ２ CH ２ O) ｅ - and here, e is 1, 2, 3, 4 or 5, R １ and R ２ together form -CH ２ CH ２ O- or -CH ２ CH(R)-O-, and R ３ is H, or R １ and R ３ together form -C １－２ alkylene-, and R ２ is H Here, R is -OR', -CH ２ OR' or -CH ２ CH ２ OR', where R' is H, a hydroxyl protecting group or a solid support, and the hydroxyl protecting group is preferably -C(O)CH ２ CH ２ C(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, here, T is as defined in claim 24 or 25, the siRNA according to claim 24 or 25.

27. The conjugate group is represented by formula (III’-1), formula (III’-2) or formula (III’-3), 【Chemical 24】 where, Q is, 【Chemical 25】 where, Here, L １ is -CH ２ O- or -NHC(O)-, and L ２ is -CH ２ CH ２ C(O)-, and L ３ is -(NHCH ２ CH ２ ) ｂ - or -(NHCH ２ CH ２ CH ２ ) ｂ - and L ４ is -(OCH ２ CH ２ ) ｃ - or -NHC(O)-(CH ２ ) ｄ - and here, 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 -CH ２ O-, and Here, R' is H, a hydroxyl protecting group, or a solid support, and the hydroxyl protecting group is preferably -C(O)CH ２ CH ２ C(O)OH or 4,4'-dimethoxytrityl, n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, wherein T is as defined in claim 24 or 25, the siRNA according to claim 26.

28. Q is independently H, 【Chemical 26】 wherein, Here, L １ is -CH ２ -, -CH ２ O- or -C(O)-, and L ２ is a chemical bond, L ３ is -(NHCH ２ CH ２ ) ｂ -, -(NHCH ２ CH ２ CH ２ ) ｂ - or -C(O)CH ２ -, and L ４ is -(OCH ２ CH ２ ) ｃ - or -NHC(O)-(CH ２ ) ｄ - and is 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, R １ and R ２ together form -CH ２ CH ２ O- or -CH ２ CH(R)-O- is formed, and R ３ is H, or R １ and R ３ together form -C １－２ alkylene-, and R ２ is H Here, R is -OR', -CH ２ OR' or -CH ２ CH ２ OR', where R' is H, a hydroxyl protecting group or a solid support, and the hydroxyl protecting group is preferably -C(O)CH ２ CH ２ C(O)OH or 4,4'-dimethoxytrityl, m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, wherein T is as defined in claim 24 or 25, the siRNA according to claim 24 or 25.

29. The conjugate group is represented by formula (IV-1) or formula (IV-2), 【Chemical 27】 wherein, Q is independently, 【Chemical Formula 28】 wherein, Here, L １ is -CH ２ -, -CH ２ O- or -C(O)-, and L ３ is -(NHCH ２ CH ２ ) ｂ -, -(NHCH ２ CH ２ CH ２ ) ｂ - or -C(O)CH ２ -, and L ４ is -(OCH ２ CH ２ ) ｃ - or -NHC(O)-(CH ２ ) ｄ - and 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, Here, R' is H, a hydroxyl protecting group, or a solid phase carrier, and the hydroxyl protecting group is preferably -C(O)CH ２ CH ２ C(O)OH or 4,4'-dimethoxytrityl, m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, wherein T is as defined in claim 24 or 25, the siRNA according to claim 24 or 25.

30. Q is independently H, 【Chemical 29】 wherein, Here, L １ is a chemical bond, -CH ２ -, -CH ２ CH ２ -, -C(O)-, -CH ２ O-, -CH ２ O-CH ２ CH ２ O- or -NHCO-(CH ２ NHCO) ａ -, and L ２ is a chemical bond or -CH ２ CH ２ C(O)-, and L ３ is a chemical bond, -(NHCH ２ CH ２ ) ｂ -, -(NHCH ２ CH ２ CH ２ ) ｂ - or -C(O)CH ２ -, and L ４ is -(OCH ２ CH ２ ), -(OCH ｃ CH ２ CH ２ CH ２ ), -(OCH ｃ CH ２ CH ２ CH ２ CH ２ ), -(OCH ｃ CH ２ CH ２ CH ２ CH ２ CH ２ ), -(OCH ｃ CH ２ ), - or -NHC(O)-(CH ｄ ), and 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, -CH ２ O- or -NHC(O)-, and L' is -O(CH ２ CH ２ O) ｅ -. where e is 1, 2, 3, 4 or 5, T is a chemical bond, -CH ２ -, -C(O)-, -M-, -CH ２ -M- or -C(O)-M-, and where M is, 【Chemical Formula 30】 wherein, R １ and R ２ together form -CH ２ CH ２ O- or -CH ２ CH(R)-O- is formed, and R ３ is H, or R １ and R ３ together form -C １－２ alkylene-, and R ２ is H Here, R is -OR', -CH ２ OR' or -CH ２ CH ２ OR', where R' is H, a hydroxyl protecting group or a solid support, and the hydroxyl protecting group is preferably -C(O)CH ２ CH ２ C(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, the siRNA according to claim 16.

31. The conjugate group is, 【Chemical 31】 【Chemical 32】 【Chemical 33】 【Chemical 34】 【Chemical 35】 selected from, the siRNA according to claim 16.

32. The conjugate group is, 【Chemical 36】 【Chemical 37】 【Chemical Formula 38】 【Chemical 39】 【Chemical Formula 40】 selected from, the siRNA according to claim 16.

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

34. The ligand has the following structure, 【Chemical Formula 41】 wherein, 【Chemical Formula 42】 The siRNA according to claim 14 or 15, which represents a position for binding to the sense strand of the siRNA via a phosphate ester group or a thiophosphate ester group.

35. The ligand has the following structure, 【Chemical Formula 43】 where 【Chemical 44】 is the siRNA according to claim 14 or 15, which represents a position for binding to the sense strand of the siRNA via a phosphate ester group or a thiophosphate ester group.

36. The ligand has the following structure, 【Chemical 45】 where 【Chemical Formula 46】 is the siRNA according to claim 14 or 15, which represents a position for binding to the sense strand of the siRNA via a phosphate ester group or a thiophosphate ester group.

37. (1) The sense strand contains STM1s - Am sAmAmAmGmGmGmA mCfAfGfUmAmUmUmCmUmCmAmGmAm s - STM1s - GL6 (SEQ ID NO: 1238), and the antisense strand contains (CP1a - U)sCf sUmGfAmGfAmAfUmAfCmUfGmUfCmCfCmUfUm sUf sUm (SEQ ID NO: 1651), or (2) The sense strand contains STM1s - Am sAmUmUmAmAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmAm s - STM1s - GL6 (SEQ ID NO: 1235), and the antisense strand contains (CP1a - U)sGf sAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAm sUf sUm (SEQ ID NO: 1650), or (3) The sense strand contains STM1s - Am sAmUmUmAmAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmAm s - STM1s - GL6 (SEQ ID NO: 1235), and the antisense strand contains (VPUm)sGf sAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAm sUf sUm (SEQ ID NO: 1649), or (4) The sense strand contains IBs - Am sAmUmUmAmAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmAm s - IBs - GL6 (SEQ ID NO: 1236), and the antisense strand contains (CP1a - U)sGf sAmAfUmAfCmUfGmUfCmCfCmUfUmUfUmAfAm sUf sUm (SEQ ID NO: 1650), or (5) The sense strand comprising UmsUmsAmAmAmAmGfGfGfAmCmAmGmUmAmUmUmCmsAms-GL6 (SEQ ID NO: 1237), wherein the antisense strand comprises (CP1a-U)sGfsAmAfUmAfCmUfGmUfCmCfCmUfUmUfUfUmAfAmsUfsUm (SEQ ID NO: 1650), or (6) wherein the sense strand comprises IBs-AmsAmAmAmGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmAms-IBs-GL6 (SEQ ID NO: 1239), wherein the antisense strand comprises (CP1a-U)sCfsUmGfAmGfAmAfUmAfCmUfGmUfCmCfCmUfUmsUfsUm (SEQ ID NO: 1651), or (7) wherein the sense strand comprises AmsAmsGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmsAms-GL6 (SEQ ID NO: 1240), wherein the antisense strand comprises (CP1a-U)sCfsUmGfAmGfAmAfUmAfCmUfGmUfCmCfCmUfUmsUfsUm (SEQ ID NO: 1651), or (8) wherein the sense strand comprises IBs-AmsAmAmAmGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmAms-IBs-GL6 (SEQ ID NO: 1239), wherein the antisense strand comprises (CP1a-U)sCfsUmGfAmGf(PCN-A)AfUmAfCmUfGmUfCmCfCmUfUmsUfsUm (SEQ ID NO: 1652), or (9) wherein the sense strand comprises AmsAmsGmGmGmAmCfAfGfUmAmUmUmCmUmCmAmGmsAms-GL6 (SEQ ID NO: 1240), wherein the antisense strand comprises (CP1a-U)sCfsUmGfAmGf(PCN-A)AfUmAfCmUfGmUfCmCfCmUfUmsUfsUm (SEQ ID NO: 1652), the siRNA according to claim 13.

38. A cell containing the siRNA according to any one of claims 1 to 37.

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

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

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

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

43. The method according to claim 41 or 42, wherein the disease associated with APOC3 is selected from hyperlipidemia and hypertriglyceridemia.

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

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