WO2024032608A1

WO2024032608A1 - Sirna molecule for regulating angptl3 gene activity

Info

Publication number: WO2024032608A1
Application number: PCT/CN2023/111739
Authority: WO
Inventors: 黄金宇; 岳明
Original assignee: 大睿生物医药科技(上海)有限公司
Priority date: 2022-08-08
Filing date: 2023-08-08
Publication date: 2024-02-15

Abstract

Provided are a small interfering RNA (siRNA) for inhibiting the expression of angiopoietin-like 3 (ANGPTL3) protein in a cell, a cell comprising the siRNA, and a method for treating a disease or symptom associated with ANGPTL3 expression in a subject using the siRNA or the cell.

Description

siRNA molecules that regulate ANGPTL3 gene activity

Technical field

This disclosure relates to the field of RNA interference.

Background technique

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

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

In recent years, inhibitors targeting ANGPTL3 have become new therapeutic drugs for these diseases. siRNA has great potential for development as a new treatment method. siRNA acts on intracellular mRNA. Compared with traditional small molecule drugs, it can directly silence target genes, so it can fundamentally prevent the occurrence and development of diseases more efficiently. develop. However, due to the poor stability of siRNA, it is easily degraded by nucleases in the body, is not easily absorbed by tissues, is difficult to be taken up by cells, and is prone to off-target effects, which limits its clinical application. There is an urgent need for an siRNA that can effectively inhibit the expression of ANGPTL3 gene in cells.

Contents of the invention

The present disclosure provides new small interfering RNA (siRNA), kits and pharmaceutical compositions for inhibiting the expression of angiopoietin-like 3 (ANGPTL3) in cells, and the siRNA, kits or pharmaceutical compositions are effective in inhibiting the expression of angiopoietin-like 3 (ANGPTL3). Or methods for reducing ANGPTL3 gene expression or treating diseases or symptoms related to ANGPTL3 expression.

In a first aspect, the present disclosure provides a small interfering RNA (siRNA) for inhibiting the expression of angiopoietin-like 3 (ANGPTL3) in cells, the siRNA comprising a sense strand and an antisense strand forming a double-stranded region, Wherein the length of the sense strand and the antisense strand is each independently 15-30 nucleotides, and the antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NO: 16-29 A nucleotide sequence of at least 15 consecutive nucleotides. In some specific embodiments, the sense strand comprises a nucleotide sequence of at least 15 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 1-14.

In some embodiments, the length of the sense strand and the antisense strand is each independently 17-27 nucleotides, preferably 19-25 nucleotides, more preferably 19-23 nucleotides.

In some embodiments, the double-stranded region is 15-25 nucleotide pairs in length, preferably 17-23 nucleotide pairs, more preferably 19-21 nucleotide pairs.

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, e.g., the sense strand and the One or both antisense strands contain a 3' overhang and/or a 5' overhang of at least 1 nucleotide. In some preferred embodiments, The antisense strand has a 3' overhang and/or a 5' overhang of at least 2 nucleotides, preferably the antisense strand comprises a 3' overhang and/or a 5' overhang of 2 nucleotides. end.

In some embodiments, the antisense strand comprises a nucleotide sequence of at least 16 contiguous nucleotides of the nucleotide sequence set forth in any one of SEQ ID NOs: 16-29, and at least 17 contiguous nucleotides. A nucleotide sequence of an acid, a nucleotide sequence of at least 18 consecutive nucleotides, a nucleotide sequence of at least 19 consecutive nucleotides, a nucleotide sequence of at least 20 consecutive nucleotides, preferably the reverse The sense strand includes the nucleotide sequence shown in any one of SEQ ID NO:16-29.

In some embodiments, the sense strand comprises a nucleotide sequence of at least 16 contiguous nucleotides to any one of the nucleotide sequences set forth in SEQ ID NOs: 1-14, and at least 17 contiguous nucleotides The nucleotide sequence of the acid, the nucleotide sequence of at least 18 consecutive nucleotides, preferably the antisense strand includes the nucleotide sequence shown in any one of SEQ ID NO: 1-14.

In some embodiments, the siRNA comprises paired sense and antisense strand sequences as shown in Table 3.

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

In some specific embodiments, the sense strand and the antisense strand each independently comprise one or more nucleotide modifications selected from the group consisting of: 2'-O-methyl modified nucleotides, 2'-Fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, inosine ribonucleotides, abasic nucleotides, reverse abasic deoxyribonucleotides, phosphorothioates Ester internucleotide linkage modification, vinylphosphonate modified nucleotides, locked nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholine nucleotides, phosphoramidates, non-natural bases containing nucleotides, terminal nucleotides attached to cholesterol-based derivatives or dodecyl dodecylamide groups, deoxyribonucleotides and STM1.

In some preferred embodiments, the sense strand and the antisense strand each independently comprise one or more nucleotide modifications selected from the group consisting of: 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, reverse abasic deoxyribonucleotides, phosphorothioate internucleotide linkage modifications, and STM1. In some preferred embodiments, the sense strand and/or the antisense strand comprises at least 2 2'-fluoro modified nucleotides. In some preferred embodiments, the sense strand and/or the antisense strand comprises at least 8 2'-O-methyl modified nucleotides. In some preferred embodiments, the 3' end and/or the 5' end of the sense strand and/or the antisense strand comprise 1-5 phosphorothioate groups, preferably 2-3 phosphorothioate groups ester group.

In some embodiments, in the siRNA of the present disclosure,

(a) The chain of justice includes

CmsAmsCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAm(SEQ ID NO:31),

The antisense strand contains

UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:61);

(b) The chain of justice includes

CmsAmsAmUmUmAmAfGfCfUmCmCmUmUmCmUmUmUmUm(SEQ ID NO:32),

The antisense strand contains

AmsAfsAmAfGmAfAmGfGmAfGmCfUmUfAmAfUmUfGmsUfsGm(SEQ ID NO:62);

(c) The chain of justice includes

UmsAmsUmUmGmUmUfCfCfUmCmUmAmGmUmUmAmUmUm(SEQ ID NO:33),

The antisense strand contains

AmsAfsUmAfAmCfUmAfGmAfGmGfAmAfCmAfAmUfAmsAfsAm(SEQ ID NO:63);

(d) The chain of justice includes

GmsUmsUmAmUmUmUfCfCfUmCmCmAmGmAmAmUmUmAm(SEQ ID NO:34),

The antisense strand contains

UmsAfsAmUfUmCfUmGfGmAfGmGfAmAfAmUfAmAfCmsUfsAm(SEQ ID NO:64);

(e) The chain of justice includes

AmsGmsAmAmUmUmGfAfUfCmAmAmGmAmCmAmAmUmUm(SEQ ID NO:35),

The antisense strand contains

AmsAfsUmUfGmUfCmUfUmGfAmUfCmAfAmUfUmCfUmsGfsGm(SEQ ID NO:65);

(f) The chain of justice includes

AmsUmsCmAmAmGmAfCfAfAmUmUmCmAmUmCmAmUmUm(SEQ ID NO:36),

The antisense strand contains

AmsAfsUmGfAmUfGmAfAmUfUmGfUmCfUmUfGmAfUmsCfsAm(SEQ ID NO:66);

(g) The justice chain includes

GmsAmsGmCmCmAmAfAfAfUmCmAmAmGmAmUmUmUmAm(SEQ ID NO:37),

The antisense strand contains

UmsAfsAmAfUmCfUmUfGmAfUmUfUmUfGmGfCmUfCmsUfsGm(SEQ ID NO:67);

(h) The justice chain includes

UmsGmsAmAmCmUmCfAfAfCmUmCmAmAmAmAmCmUmUm(SEQ ID NO:38),

The antisense strand contains

AmsAfsGmUfUmUfUmGfAmGfUmUfGmAfGmUfUmCfAmsAfsGm(SEQ ID NO:68);

(i) The chain of justice includes

AmsGmsAmGmCmAmAfCfUfAmAmCmUmAmAmCmUmUmAm(SEQ ID NO:39),

The antisense strand contains

UmsAfsAmGfUmUfAmGfUmUfAmGfUmUfGmCfUmCfUmsUfsCm(SEQ ID NO:69);

(j) The chain of justice includes

GmsCmsAmAmCmUmAfAfCfUmAmAmCmUmUmAmAmUmUm(SEQ ID NO:40),

The antisense strand contains

AmsAfsUmUfAmAfGmUfUmAfGmUfUmAfGmUfUmGfCmsUfsCm(SEQ ID NO:70);

(k) The justice chain includes

CmsUmsAmAmCmUmAfAfCfUmUmAmAmUmUmCmAmAmAm(SEQ ID NO:41),

The antisense strand contains

UmsUfsUmGfAmAfUmUfAmAfGmUfUmAfGmUfUmAfGmsUfsUm(SEQ ID NO:71);

(l) The justice chain includes

CmsUmsAmAmCmUmUfAfAfUmUmCmAmAmAmUmCmAm(SEQ ID NO:42),

The antisense strand contains

UmsGfsAmUfUmUfUmGfAmAfUmUfAmAfGmUfUmAfGmsUfsUm(SEQ ID NO:72);

(m) The justice chain includes

UmsAmsAmCmUmUmAfAfUfUmCmAmAmAmAmUmCmAmAm(SEQ ID NO:43),

The antisense strand contains

AmsAfsUmAfAmCfUmAfGmAfGmGfAmAfCmAfAmUfAmsAfsAm(SEQ ID NO:73); or

(n) The justice chain includes

GmsGmsAmUmCmAmCfAfAfAmAmCmUmUmCmAmAmUmAm(SEQ ID NO:44),

The antisense strand contains

UmsAfsUmUfGmAfAmGfUmUfUmUfGmUfGmAfUmCfCmsAfsUm (SEQ ID NO:74).

In some embodiments, in the siRNA of the present disclosure,

(a) The chain of justice includes

UmsGmsAmAmCmUmCfAfAfCmUmCmAmAmAmAmCmUmsUm(SEQ ID NO:131),

and the antisense strand contains

AmsAfsGmUfUmUfUmGfAmGfUmUfGmAfGmUfUmCfAmsAfsGm(SEQ ID NO:132);

(b) The chain of justice includes

GmsGmsAmUmCmAmCfAfAfAmAmCmUmUmCmAmAmUmsAm(SEQ ID NO:133),

and the antisense strand contains

UmsAfsUmUfGmAfAmGfUmUfUmUfGmUfGmAfUmCfCmsAfsUm(SEQ ID NO:134);

(c) The chain of justice includes

CmsAmsCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmsAm(SEQ ID NO:135),

and the antisense strand contains

UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:136);

(d) The chain of justice includes

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

and the antisense strand contains

AmsAfsGmUfUmUfUmGfAmGfUmUfGmAfGmUfUmCfAmsAfsGm(SEQ ID NO:138);

(e) The chain of justice includes

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

and the antisense strand contains

UmsAfsUmUfGmAfAmGfUmUfUmUfGmUfGmAfUmCfCmsAfsUm(SEQ ID NO:140);

(f) The chain of justice includes

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

and the antisense strand contains

UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:142);

(g) The justice chain includes

STM1-STM1-

UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAm-STM1-STM1 (SEQ ID NO: 143),

and the antisense strand contains

UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:144); or

(h) The justice chain includes

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

and the antisense strand contains

UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm (SEQ ID NO: 146).

In some embodiments, in the siRNA of the present disclosure,

(a) The chain of justice includes

STM1-STM1-

UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmsAm-STM1-STM1 (SEQ ID NO: 147),

and the antisense strand contains

UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:148);

(b) The chain of justice includes

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

and the antisense strand contains

UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:150);

(c) The chain of justice includes

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

and the antisense strand contains

AmsUfsCmAfAmAfUmGfAmUfGmAfAmUfUmGfUmCfUmsUfsGm(SEQ ID NO:152);

(d) The chain of justice includes

STM1-STM1-

CmsAmAmGmAmCmAmAmUfUfCfAmUmCmAmUmUmUmGmAmsUm-STM1-STM1 (SEQ ID NO:153),

and the antisense strand contains

AmsUfsCmAfAmAfUmGfAmUfGmAfAmUfUmGfUmCfUmsUfsGm(SEQ ID NO:154);

(e) The chain of justice includes

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

and the antisense strand contains

AmsUfsCmAfAmAfUmGfAmUfGmAfAmUfUmGfUmCfUmsUfsGm(SEQ ID NO:156);

(f) The chain of justice includes

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

and the antisense strand contains

UmsCfsUmCfUmUfAmAfGmGfAmUfUmUfAmAfUmAfCmsCfsAm(SEQ ID NO:158);

(g) The justice chain includes

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

and the antisense strand contains

AmsAfsUmUfGmUfCmUfUmGfAmUfCmAfAmUfUmCfUmsGfsGm(SEQ ID NO:160); or

(h) The justice chain includes

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

and the antisense strand contains

AmsAfsUmGfAmUfGmAfAmUfUmGfUmCfUmUfGmAfUmsCfsAm (SEQ ID NO: 162).

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

In some embodiments, the ligand moiety includes a conjugation group represented by formula (X'):

in,

Indicates the location of attachment to biomolecules;

Q is independently H,

Where 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 NHC(O)) _a -;

L ₂ is a chemical bond or -CH ₂ CH ₂ C(O)-;

L ₃ is a chemical bond, -(NHCH ₂ CH ₂ ) _b -, -(NHCH ₂ CH ₂ CH ₂ ) _b - or -C(O)CH ₂ -;

L ₄ is -(OCH ₂ CH ₂ ) _c -, -(OCH ₂ CH ₂ CH ₂ ) _c -, -(OCH ₂ CH ₂ CH ₂ CH ₂ ) _c -, -(OCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ ) _c -or-NHC(O)-(CH ₂ ) _d -;

where a=0, 1, 2 or 3;

b=1, 2, 3, 4 or 5;

c=1, 2, 3, 4 or 5;

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

L is a chemical bond, -CH ₂ O- or -NHC(O)-;

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

where M is

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;

Wherein R is -OR', -CH ₂ OR' or -CH ₂ CH ₂ OR', wherein R' is H, hydroxyl protecting group or 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.

In some embodiments, the conjugation ligand targets asialoglycoprotein receptor (ASGPR).

In some preferred embodiments, the conjugation group is selected from Table 1:

Table 1 Structure of conjugated groups

In some preferred embodiments, the conjugation group is selected from Table 2:

Table 2 Structure of conjugated groups

In some embodiments, the ligand comprised in the siRNA has the following structure:

in Indicates the position connected to the sense strand of the siRNA via a phosphate group or a phosphorothioate group.

In some embodiments, the ligand comprised in the siRNA of the present disclosure has the following structure:

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

in Indicates the position of attachment to the sense strand of siRNA via a phosphate group or phosphorothioate group.

The present disclosure In a second aspect, the present disclosure provides a cell containing the siRNA described in the present disclosure.

In a third aspect, the disclosure provides a pharmaceutical composition comprising the siRNA or cells of the disclosure, and optionally a pharmaceutically acceptable carrier or excipient.

In a fourth aspect, the present disclosure provides a kit comprising the siRNA, cells or pharmaceutical composition of the present disclosure.

In a fifth aspect, the present disclosure provides a method of reducing ANGPTL3 levels, LDL levels, apoC-III levels, triglyceride levels, cholesterol levels, glucose levels, and fat pad weight in a subject, the method comprising: The subject is administered a siRNA, cell, or pharmaceutical composition of the present disclosure.

The present disclosure also provides a method of treating a disease or condition associated with ANGPTL3 expression in a subject, the method comprising the step of administering to the subject a siRNA, cell, or pharmaceutical composition described in the present disclosure.

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

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

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

In some embodiments, the disease associated with ANGPTL3 expression is diabetes, which is type 2 diabetes or type 2 diabetes with dyslipidemia.

In some embodiments, methods of the present disclosure for treating a disease or condition associated with ANGPTL3 expression in a subject include administering the siRNA, cells, or pharmaceutical composition to the subject by subcutaneous administration, topical administration, or intravenous administration. In some embodiments, the subject is a human subject.

Detailed description of the invention

The following describes the embodiments of the present disclosure through specific examples. Those skilled in the art can easily understand other advantages and effects of the present disclosure from the content disclosed in this specification. The present disclosure can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present disclosure.

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

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

When embodiments give numerical ranges, it should be understood that both endpoints of each numerical range and any number between the two endpoints may be selected unless otherwise stated in the disclosure. Unless defined otherwise, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, equipment, and materials used in the examples, those skilled in the art can also use methods, equipment, and materials described in the embodiments of the present disclosure based on their understanding of the prior art and the description of the present disclosure. Any methods, equipment and materials similar or equivalent to the prior art to implement the present disclosure shall fall within the protection scope of the present disclosure. Embodiments of the present disclosure are described in greater detail below.

definition

The term "siRNA" herein refers to a class of double-stranded RNA molecules that can mediate silencing of a target RNA that is complementary to it (eg, mRNA, eg, the transcript of a gene encoding a protein). siRNA is usually double-stranded, including an antisense strand that is complementary to the target RNA, and a sense strand that is complementary to the antisense strand. For convenience, such mRNA is also referred to herein as the mRNA to be silenced. Such genes are also called target genes. Typically, the RNA to be silenced is an endogenous gene or a pathogen gene. In addition, RNA other than mRNA (e.g. tRNA) as well as viral RNA can also be targeted.

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

As used herein, the term "complementary region" refers to a region on the antisense strand that is completely or substantially complementary to the target mRNA sequence. In cases where the complementary region is not completely complementary to the target sequence, the mismatch can be located in the internal or terminal regions of the molecule. Typically, the most tolerated mismatches are in the terminal region, e.g., within 5, 4, 3, 2 or 1 nucleotide of the 5' and/or 3' end. The portion of the antisense strand that is most sensitive to mismatches is called the "seed region." For example, in a siRNA containing a 19nt strand, some mismatches can be tolerated at position 19 (from 5' to 3').

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

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

As used herein, a polynucleotide that is "at least partially complementary" or "substantially complementary" to a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., the mRNA encoding ANGPTL3). glycosides. For example, a polynucleotide is complementary to at least a portion of ANGPTL3 mRNA if the sequence is substantially complementary to a non-interrupted portion of the mRNA encoding ANGPTL3.

The terms "complementary," "completely complementary," and "substantially complementary" as used herein may be used with respect to base pairing between the sense strand and the antisense strand of an siRNA, or between the antisense strand of an siRNA agent and a target sequence.

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

"Nucleoside" is a compound composed of two substances: purine base or pyrimidine base, and ribose or deoxyribose. "Nucleoside" is a compound composed of three substances: purine base or pyrimidine base, ribose or deoxyribose, and phosphate. "Oligonucleotide" refers to, for example, a nucleic acid molecule (RNA or DNA) having a length of less than 100, 200, 300 or 400 nucleotides.

"Base" is the basic unit for the synthesis of nucleosides, nucleotides and nucleic acids. Its constituent elements contain nitrogen, also known as "nitrogen-containing bases". In this article, unless otherwise specified, the capital letters A, U, T, G and C represent the base composition of nucleotides, which are adenine, uracil, thymine, guanine and cytosine respectively.

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

"Blunt end" or "blunt end" or "blunt end" means that there are no unpaired nucleotides at the end of the double-stranded siRNA, ie, no nucleotide overhangs. A "blunt-ended" siRNA is a siRNA that is double-stranded throughout its length, ie, has no nucleotide overhangs at either end of the molecule. siRNAs of the present disclosure include siRNAs with nucleotide overhangs at one end (i.e., agents with one overhang and one blunt end) or with nucleotide overhangs at both ends.

Substantially all nucleotides of the iRNAs of the present disclosure are modified. For example, basically the chain of justice All nucleotides are modified nucleotides, and/or substantially all nucleotides in the antisense strand are modified nucleotides, and/or substantially all nucleotides in both the sense and antisense strands are Acids are all modified nucleotides. In other embodiments of the disclosure, all nucleotides of the iRNA of the disclosure 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 The nucleotides are all modified nucleotides. "Substantially all nucleotides are modified" means that the siRNAs of the present disclosure are mostly, but not entirely, modified and may include no more than 5, 4, 3, 2, or 1 unmodified nucleotides.

"Modified nucleotides" herein include, but are not limited to, 2'-O-alkyl modified nucleotides (e.g., 2'-O-methyl modified nucleotides, 2'-methoxyethyl modified nucleic acids). nucleotides), 2'-fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, inosine ribonucleotides, abasic nucleotides, reverse abasic deoxyribonucleotides , nucleotides containing phosphorothioate groups, phosphorothioate internucleotide linkage modifications, vinylphosphonate modified nucleotides, locked nucleotides, 2'-amino-modified nucleosides Acids, 2'-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, non-natural bases containing nucleotides, linked to cholesteryl derivatives or dodecyl dodecyl amido groups Terminal nucleotides on the group, deoxyribonucleotides, 3'-terminal deoxythymine (dT) nucleotides, conformationally restricted nucleotides, restricted ethyl nucleotides, 2'-hydroxy modified nucleosides Acids, nucleotides containing a methylphosphonic acid group, nucleotides containing a 5'-phosphate, nucleotides containing a 5'-phosphate mimetic, diol-modified nucleotides (GNA), and 2-O -(N-methylacetamide) modified nucleotides, and STM1, or conventional protecting group protection, etc.

For example, the 2'-fluoro modified nucleotide refers to a nucleotide in which the hydroxyl group at the 2' position of the ribosyl group of the nucleotide is replaced by fluorine. The 2'-deoxy-modified nucleotide refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribose group with a methoxy group.

"Nucleotide containing a phosphorothioate group" refers to a nucleotide in which one or more oxygen atoms on the phosphate are replaced by sulfur atoms. "Modification of phosphorothioate inter-nucleotide bonding" refers to the modification in which two adjacent nucleotides on the left and right are connected through phosphorothioate.

As used herein, a "ligand moiety" refers to a chemical moiety that conjugates to an siRNA and is capable of altering the distribution, targeting, or lifetime of the siRNA. In preferred embodiments, such ligand is a selected target (e.g. molecule, cell or cell type, compartment (e.g. cell or organ compartment, tissue, organ or area of the body) provides enhanced affinity.

In some embodiments, the ligand moiety targets the asialoglycoprotein receptor (ASGPR) on hepatocytes. Binding of the ligand moiety to ASGPR mediates internalization via clathrin-coated vesicles. Maturation of endosomes results in a decrease in lysosomal pH, which promotes dissociation of ligand-receptor complexes, thereby releasing siRNA. Conjugation of a ligand moiety targeting the asialoglycoprotein receptor (ASGPR) on hepatocytes results in siRNA efficacy and stability in vivo or intracellularly. This facilitates subcutaneous administration of the siRNA.

As used herein, the term "inhibition" is used interchangeably with "reduction," "silencing," "downregulation," and other similar terms and includes any level of inhibition.

The phrase "inhibiting the expression of ANGPTL3" is intended to mean inhibiting the expression of any ANGPTL3 gene as well as variants or mutants of the ANGPTL3 gene. Thus, the ANGPTL3 gene may be a wild-type ANGPTL3 gene, a mutant ANGPTL3 gene, or in the case of a genetically manipulated cell, cell population, or organism, a transgenic ANGPTL3 gene.

"Inhibition of ANGPTL3 gene expression" includes any level of inhibition of the ANGPTL3 gene, such as at least partial inhibition of ANGPTL3 gene expression. ANGPTL3 gene expression can be assessed based on the level or change in level of any variable associated with ANGPTL3 gene expression, such as ANGPTL3 mRNA levels, ANGPTL3 protein levels, or lipid levels. This level can be assessed in individual cells or in a group of cells (including, for example, a sample derived from a subject).

Inhibition can be assessed by a reduction in absolute or relative levels of one or more variables associated with ANGPTL3 expression compared to control levels. The control level may be any type of control level utilized in the art, such as a pre-dose baseline level or from a similar untreated or control (e.g., buffer only control or inert control) subject, cell , or the level determined by the sample.

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

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

"C _1-6 haloalkyl" refers to the above-mentioned "C _1-6 alkyl" which is substituted by one or more halogen groups. In some embodiments, C _1-4 haloalkyl is particularly preferred, with C _1-2 haloalkyl being more preferred. Exemplary haloalkyl groups include, but are not limited to: -CF ₃ , -CH ₂ F, -CHF ₂ , -CHFCH ₂ F, -CH ₂ CHF ₂ , -CF ₂ CF ₃ , -CCl ₃ , -CH ₂ Cl , -CHCl ₂ , 2,2,2-trifluoro-1,1-dimethyl-ethyl, etc. Haloalkyl groups may be substituted at any available point of attachment, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

"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. The unsubstituted alkylene group includes, but is not limited to: methylene (-CH ₂ -), ethylene (-CH ₂ CH ₂ -), propylene (-CH ₂ CH ₂ CH ₂ -), ethylene Base (-CH ₂ CH ₂ CH ₂ CH ₂ -), pentylene (-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -), hexylene (-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -) ,etc. Exemplary substituted alkylene groups, for example, the alkylene groups substituted by one or more alkyl (methyl) include, but are not limited to: substituted methylene (-CH(CH ₃ )- , -C(CH ₃ ) ₂ -), substituted ethylene (-CH(CH ₃ )CH ₂ -, -CH ₂ CH(CH ₃ )-, -C(CH ₃ ) ₂ CH ₂ -, -CH ₂ C(CH ₃ ) _2- ), substituted propylene (-CH(CH ₃ )CH ₂ CH ₂ -, -CH ₂ CH(CH ₃ )CH ₂ -, -CH ₂ CH ₂ CH(CH ₃ ) -, -C(CH ₃ ) ₂ CH ₂ CH ₂ -, -CH ₂ C(CH ₃ ) ₂ CH ₂ -, -CH ₂ CH ₂ C(CH ₃ ) ₂ -), etc.

As used in this disclosure, the terms "treatment,""treatment," etc., refer to administering an agent or performing a procedure in order to obtain an effect. These effects may be prophylactic in the sense of completely or partially preventing the disease or its symptoms, and/or may be therapeutic insofar as affecting the partial or complete cure of the disease and/or the symptoms of the disease. As used in this disclosure, "treatment" may include treatment of a disease or condition (eg, cancer) in a mammal, particularly a human, and includes: (a) prophylaxis in a subject susceptible to the disease but who has not yet been diagnosed with the disease. The occurrence of the disease or disease symptoms (for example, including diseases that may be related to or caused by the primary disease); (b) inhibit the disease, that is, prevent its progression; (c) alleviate the disease, that is, cause the regression of the disease. Treatment may refer to any indication of success in treating or ameliorating or preventing cancer, including any objective or subjective parameter, such as elimination; remission; reduction of symptoms or making disease symptoms more tolerable for the patient; slowing of progression or decline ; or reduce the endpoint of worsening frailty. Treatment or improvement of symptoms is based on one or more objective or subjective parameters; including the results of a physician's examination. Accordingly, the term "treating" includes administering an siRNA or pharmaceutical composition disclosed in the present disclosure to prevent or delay, alleviate, or arrest or inhibit the development of symptoms or conditions associated with a disease (eg, cancer). The term "therapeutic effect" refers to the reduction, elimination, or prevention of disease, disease symptoms, or disease side effects in a subject.

The term "therapeutically effective amount" as used in this disclosure refers to an amount that, when administered to a subject to treat a disease, is sufficient to effect treatment of a disease.

As used herein, the term "subject" refers to any mammalian subject for whom diagnosis, treatment, or therapy is desired. "Mammal" for therapeutic purposes means any animal classified as a mammal, including humans, domestic animals, and laboratory, zoo, sporting or pet animals, such as dogs, horses, cats, cattle, sheep, Goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, etc.

I.siRNA

The present disclosure provides a small interfering RNA (siRNA) for inhibiting the expression of angiopoietin-like 3 (ANGPTL3) in cells, the siRNA comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand and the length of the antisense strand is each independently 15-30 nucleotides, and the antisense strand includes at least 15 consecutive nucleotide sequences of the nucleotide sequence shown in any one of SEQ ID NO: 16-29 The nucleotide sequence of a nucleotide.

In some embodiments, the double-stranded region formed by the sense strand and antisense strand is completely complementary. In other embodiments, the double-stranded region formed by the sense strand and the antisense strand is substantially complementary and may contain 1, 2, 3, 4, or 5 non-complementary sites.

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

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 or Both contain 3' overhangs and/or 5' overhangs with at least 1 nucleotide. In some preferred embodiments, the antisense strand has a 3' overhang and/or a 5' overhang of at least 2 nucleotides, preferably the antisense strand includes a 3' overhang of 2 nucleotides. Overhangs and/or 5' overhangs. In some embodiments, the sense strand and the antisense strand are the same length. In some embodiments, the entire length of the sense strand is complementary to the entire length of the antisense strand to form a double strand, ie, has blunt ends. In other embodiments, the sense strand and the antisense strand are the same length, 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 a 5' overhang. In some embodiments, the sense strand and the antisense strand are different lengths. In a preferred embodiment, the 5' end of the antisense strand has an overhang of at least 1 nucleotide, more preferably 2 or 3 nucleotides.

In some embodiments, the sense strand comprises a nucleotide sequence of at least 16 contiguous nucleotides to any one of the nucleotide sequences set forth in SEQ ID NOs: 1-14, at least 17 contiguous nucleotides acid nucleotide sequence, a nucleotide sequence of at least 18 consecutive nucleotides, a nucleotide sequence of at least 19 consecutive nucleotides, a nucleotide sequence of at least 20 consecutive nucleotides, preferably the reverse The sense strand includes the nucleotide sequence shown in any one of SEQ ID NO: 1-1093.

In some embodiments, the siRNA comprises paired sense strand sequences and reverse strand sequences as shown in Table 3 sense strand sequence.

II. Modification of Nucleotides

In some embodiments, substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides. In some embodiments, at least 80% of the nucleotides of the sense strand are modified nucleotides, and/or at least 80% of the nucleotides of the antisense strand are modified nucleotides.

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

Modifications of nucleotides described in the present disclosure may be modifications on the phosphate group, ribose group and/or base group of the nucleotide.

In some preferred embodiments, the sense strand and the antisense strand each independently comprise one or more nucleotide modifications selected from the group consisting of: 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, reverse abasic deoxyribonucleotides, and phosphorothioate internucleotide linkage modifications. In some preferred embodiments, the sense strand and/or the antisense strand comprises at least 2 2'-fluoro modified nucleotides. In some preferred embodiments, the sense strand and/or the antisense strand comprises at least 8 2'-O-methyl modified nucleotides. In some preferred embodiments, the 3' end and/or the 5' end of the sense strand and/or the antisense strand comprise 1-5 phosphorothioate groups, preferably 2-3 phosphorothioate groups ester group. In some preferred embodiments, the sense strand and/or antisense strand comprise adenine deoxyribonucleotides, thymine deoxyribonucleotides, guanine deoxyribonucleotides and/or cytosine deoxyribonucleotides glycosides. In a more preferred embodiment, the sense strand and/or antisense strand comprise thymidine deoxyribonucleotides. In the most preferred embodiment, the sense strand comprises thymidine deoxyribonucleotides.

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

III. Ligand

The siRNAs of the present disclosure are further conjugated to a ligand moiety comprising N-acetylgalactosamine. In a preferred embodiment, the sense strand of the siRNA is conjugated to the ligand moiety. In some preferred embodiments, the 3' end of the sense strand is conjugated to the ligand moiety. In other preferred embodiments, the 5' end of the sense strand is conjugated to the ligand moiety.

in,

Indicates the location of attachment to biomolecules;

Q is independently H,

L ₂ is a chemical bond or -CH ₂ CH ₂ C(O)-;

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)-;

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

where M is

Or R ₁ and R ₃ together form -C _1-2 alkylene-, and R ₂ is H;

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.

In some embodiments, the conjugating group is represented by Formula (I'):

in,

Indicates the location of attachment to biomolecules;

Q is independently H,

L ₂ is a chemical bond or -CH ₂ CH ₂ C(O)-;

L ₄ is -(OCH ₂ CH ₂ ) _c -, -(OCH ₂ CH ₂ CH ₂ ) _c -, -(OCH ₂ CH ₂ CH ₂ CH ₂ ) _c -, - (OCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ ) _c -or-NHC(O)-(CH ₂ ) _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 -CH ₂ O- or -NHC(O)-;

L’ is a chemical bond, -C(O)NH- or -NHC(O)-;

Or R ₁ and R ₃ together form -C _1-2 alkylene-, and R ₂ is H;

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.

In some specific embodiments, wherein,

Q is independently H or

Where L ₁ is -CH ₂ O- or -NHC(O)-(CH ₂ NHC(O)) _a -;

L ₂ is -CH ₂ CH ₂ C(O)-;

L ₃ is -(NHCH ₂ CH ₂ ) _b - or -(NHCH ₂ CH ₂ CH ₂ ) _b -;

L ₄ is -(OCH ₂ CH ₂ ) _c - or -NHC(O)-(CH ₂ ) _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 -CH ₂ O-;

L’ is a chemical bond;

Or R ₁ and R ₃ together form -C _1-2 alkylene-, and R ₂ is H;

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.

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

in,

Indicates the location of attachment to biomolecules;

Q is

Where L ₁ is -CH ₂ O- or -NHC(O)-;

L ₂ is -CH ₂ CH ₂ C(O)-;

L ₃ is -(NHCH ₂ CH ₂ ) _b - or -(NHCH ₂ CH ₂ CH ₂ ) _b -;

L ₄ is -(OCH ₂ CH ₂ ) _c - or -NHC(O)-(CH ₂ ) _d -;

Where b=1, 2, 3, 4 or 5;

c=1, 2, 3, 4 or 5;

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

L is -CH ₂ O-;

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;

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

In some specific embodiments, wherein,

Q is independently H,

Where L ₁ is -CH ₂ O-, -CH ₂ O-CH ₂ CH ₂ O- or -NHC(O)-(CH ₂ NHC(O)) _a -;

L ₂ is -CH ₂ CH ₂ C(O)-;

L ₃ is -(NHCH ₂ CH ₂ ) _b -, -(NHCH ₂ CH ₂ CH ₂ ) _b - or -C(O)CH ₂ -;

L ₄ is -(OCH ₂ CH ₂ ) _c - or -NHC(O)-(CH ₂ ) _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 -CH ₂ O- or -NHC(O)-;

L’ is a chemical bond or -C(O)NH-;

Or R ₁ and R ₃ together form -C _1-2 alkylene-, and R ₂ is H;

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.

In some embodiments, the conjugation group is represented by Formula (II'-1) or Formula (II'-2):

in,

Indicates the location of attachment to biomolecules;

Q is independently

Where L ₁ is -CH ₂ O- or -CH ₂ O-CH ₂ CH ₂ O-;

L ₄ is -(OCH ₂ CH ₂ ) _c - or -NHC(O)-(CH ₂ ) _d -;

Where b=1, 2, 3, 4 or 5;

c=1, 2, 3, 4 or 5;

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

L is -NHC(O)-;

L’ is a chemical bond or -C(O)NH-;

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.

In some specific embodiments, wherein,

Q is independently H,

where L ₁ is -CH ₂ -, -C(O)-, -CH ₂ O-, -CH ₂ O-CH ₂ CH ₂ O- or -NHC(O)-(CH ₂ NHC(O)) _a - ;

L ₂ is a chemical bond;

L ₄ is -(OCH ₂ CH ₂ ) _c - or -NHC(O)-(CH ₂ ) _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 -CH ₂ O- or -NHC(O)-;

L’ is a chemical bond or -C(O)NH-;

Or R ₁ and R ₃ together form -C _1-2 alkylene-, and R ₂ is H;

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.

In some embodiments, wherein the conjugating group is represented by Formula (II'-2):

in,

Indicates the location of attachment to biomolecules;

Q is independently

Where L ₁ is -CH ₂ - or -C(O)-;

L ₃ is -(NHCH ₂ CH ₂ ) _b -;

L ₄ is -(OCH ₂ CH ₂ ) _c -;

Where b=1, 2, 3, 4 or 5;

c=1, 2, 3, 4 or 5;

L is -CH ₂ O- or -NHC(O)-;

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

In some specific embodiments, wherein:

Q is independently H,

L ₂ is a chemical bond or -CH ₂ CH ₂ C(O)-;

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)-;

L' is a chemical bond, -C(O)NH-, -NHC(O)- or -O(CH ₂ CH ₂ O) _e -;

where e is 1, 2, 3, 4 or 5;

T is a chemical bond, -CH ₂ -, -M-, -CH ₂ -M- or -C(O)-M-;

where M is

Or R ₁ and R ₃ together form -C _1-2 alkylene-, and R ₂ is H;

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.

In some specific embodiments, wherein,

T is -M-, -CH ₂ -M- or -C(O)-M-, where M is

In some specific embodiments, wherein,

Q is independently H or

Where L ₁ is -CH ₂ O- or -NHC(O)-(CH ₂ NHC(O)) _a -;

L ₂ is -CH ₂ CH ₂ C(O)-;

L ₃ is -(NHCH ₂ CH ₂ ) _b - or -(NHCH ₂ CH ₂ CH ₂ ) _b -;

L ₄ is -(OCH ₂ CH ₂ ) _c - or -NHC(O)-(CH ₂ ) _d -;

where a=0, 1, 2 or 3;

b=1, 2, 3, 4 or 5;

c=1, 2, 3, 4 or 5;

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

L is a chemical bond or -CH ₂ O-;

L' is a chemical bond or -O(CH ₂ CH ₂ O) _e -;

where e is 1, 2, 3, 4 or 5;

Or R ₁ and R ₃ together form -C _1-2 alkylene-, and R ₂ is H;

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 in the embodiment above.

In some embodiments, wherein the conjugation group is represented by Formula (III'-1), Formula (III'-2) or Formula (III'-3):

in,

Q is

Where L ₁ is -CH ₂ O- or -NHC(O)-;

L ₂ is -CH ₂ CH ₂ C(O)-;

L ₃ is -(NHCH ₂ CH ₂ ) _b - or -(NHCH ₂ CH ₂ CH ₂ ) _b -;

L ₄ is -(OCH ₂ CH ₂ ) _c - or -NHC(O)-(CH ₂ ) _d -;

Where b=1, 2, 3, 4 or 5;

c=1, 2, 3, 4 or 5;

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

L is a chemical bond or -CH ₂ O-;

Wherein 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;

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

where T is as defined in the embodiment above.

In some specific embodiments, wherein,

Q is independently H,

Where L ₁ is -CH ₂ -, -CH ₂ O- or -C(O)-;

L ₂ is a chemical bond;

L ₄ is -(OCH ₂ CH ₂ ) _c - or -NHC(O)-(CH ₂ ) _d -;

Where b=1, 2, 3, 4 or 5;

c=1, 2, 3, 4 or 5;

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

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

L’ is a chemical bond;

Or R ₁ and R ₃ together form -C _1-2 alkylene-, and R ₂ is H;

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 in the embodiment above.

In some embodiments, wherein the conjugating group is represented by Formula (IV-1) or Formula (IV-2):

in,

Q is independently

Where L ₁ is -CH ₂ -, -CH ₂ O- or -C(O)-;

L ₄ is -(OCH ₂ CH ₂ ) _c - or -NHC(O)-(CH ₂ ) _d -;

Where b=1, 2, 3, 4 or 5;

c=1, 2, 3, 4 or 5;

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

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

L’ is a chemical bond;

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 in the embodiment above.

In some specific embodiments, wherein:

Q is independently H,

L ₂ is a chemical bond or -CH ₂ CH ₂ C(O)-;

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)-;

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-;

where M is

Or R ₁ and R ₃ together form -C _1-2 alkylene-, and R ₂ is H;

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.

In some preferred embodiments, wherein said conjugating group is selected from the following:

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

In a preferred embodiment, wherein said ligand has the following structure:

IV.ANGPTL3 gene expression inhibition

The siRNA of the present disclosure can inhibit ANGPTL3 gene expression by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, At least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, At least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

Inhibition of ANGPTL3 gene expression may be manifested by a decrease in the amount of mRNA expressed by a first cell or population of cells (such cells may be present, for example, in a sample derived from the subject) in which the ANGPTL3 gene is transcribed and the cells or these cells have been treated (e.g., by contacting the cell or cells with an siRNA of the present disclosure, or by administering an siRNA of the present disclosure to a subject in which these cells are present or previously present, such that contact with the first cell or cells ANGPTL3 gene expression is inhibited compared to a second group or group of cells (one or more control cells) that are substantially the same but have not been so treated.

In a preferred embodiment, the inhibition is assessed by expressing the level of mRNA in treated cells as a percentage of the level of mRNA in control cells using the following formula. In some specific embodiments, the 2 ^-ΔΔCt value is calculated to compare the difference between the experimental group and the control group, where ΔΔCt=[(Ct experimental group target gene-Ct experimental group internal reference)-(Ct control group Target gene-Ct control group (internal reference)].

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

Inhibition of expression of ANGPTL3 protein may be manifested by a decrease in the level of ANGPTL3 protein expressed by a cell or population of cells (eg, the level of protein expressed in a sample derived from a subject). As explained above with respect to the assessment of mRNA repression, inhibition of protein expression levels in treated cells or populations of cells can similarly be expressed as a percentage of the levels of the protein in control cells or populations of cells.

Control cells or cell populations that may be used to assess inhibition of ANGPTL3 gene expression include cells or cell populations that have not been contacted with the siRNA of the present disclosure. For example, the control cells or population of cells may be derived from an individual subject (eg, a human or animal subject) prior to treatment of the subject with siRNA.

V. cells

The present disclosure provides cells containing the siRNA of the present disclosure, wherein the siRNA of the present disclosure is capable of being transcribed in the cell.

VI. Pharmaceutical compositions

The present disclosure provides pharmaceutical compositions comprising the siRNA or cells of the present disclosure, and optionally a pharmaceutically acceptable carrier or excipient.

As used herein, "pharmaceutically acceptable" refers to those compounds, materials, compositions and/or dosage forms that, within the scope of sound medical judgment, are suitable for contact with tissues of human subjects and animal subjects without undue toxicity, irritation, allergic reactions, or other problems or complications, commensurate with a reasonable benefit/risk ratio.

As used herein, a pharmaceutically acceptable carrier refers to a pharmaceutical carrier that facilitates the administration of siRNA or cells containing the siRNA to the human body and/or facilitates its absorption or effect. 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, carbonic acid Calcium and sodium bicarbonate; absorption accelerators such as quaternary ammonium compounds; surfactants such as cetyl alcohol; adsorption carriers such as kaolin and bentonite; lubricants such as talc, calcium/magnesium stearate, polyethylene glycol, etc. In addition, other auxiliary agents such as flavoring agents, sweeteners, etc. can also be added to the composition.

For example, a pharmaceutical composition containing the siRNA or cells of the present disclosure may include a pharmaceutically acceptable diluent or sustained-release matrix in which the siRNA of the present disclosure is embedded.

VIII. Kit

The present disclosure provides kits comprising the siRNA or cells described in the present disclosure.

The present disclosure also provides kits for using the siRNA described in the present disclosure and/or performing the methods of the present disclosure. Such kits include one or more siRNAs or cells described in the present disclosure and may further include instructions for use. Instructions for inhibiting the expression of ANGPTL3 in the cell by contacting the cell with the siRNA described in the present disclosure in an amount effective to inhibit the expression of ANGPTL3 may be recorded in the instructions for use.

Where the siRNA of the present disclosure is contacted with cells in vitro, optionally, the kit of the present disclosure may also include means (e.g., an injection device) for contacting the cells with the siRNA of the present disclosure or with Tools for measuring the inhibitory effect of ANGPTL3 (e.g., a device for measuring inhibition of ANGPTL3 mRNA or protein). Such a device for measuring inhibition of ANGPTL3 may comprise a device for obtaining a sample (eg, like a plasma sample) from a subject.

In the case where the siRNA of the present disclosure or the cells into which the siRNA has been introduced in vitro are administered into the body, the kit of the present disclosure may also optionally include a method for administering the siRNA or cells of the present disclosure to the subject. Device or device for determining a therapeutically effective amount or a prophylactically effective amount.

IX. Treatment methods and pharmaceutical uses

The present disclosure provides a method of reducing ANGPTL3 levels, LDL levels, apoC-III levels, triglyceride levels, cholesterol levels, glucose levels, fat pad weight in a subject, the method comprising administering to the subject siRNA, cells, or pharmaceutical compositions of the present disclosure.

The present disclosure provides a method of treating a disease or condition associated with ANGPTL3 expression in a subject, the method comprising the step of administering to the subject a siRNA, cell, or pharmaceutical composition described in the present disclosure.

In some embodiments, the disease associated with ANGPTL3 expression is cardiovascular disease. In some preferred embodiments, the cardiovascular disease is selected from obesity, diabetes, atherosclerosis, dyslipidemia, coronary heart disease, non-alcoholic fatty liver disease (NAFLD), hyperlipidemia or metabolic syndrome or its combination.

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

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

In some embodiments, methods of the present disclosure for treating a disease or condition associated with ANGPTL3 expression in a subject include administering the siRNA or pharmaceutical composition to the subject including subcutaneous administration or intravenous administration. In some embodiments, the subject is a human patient.

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

The present disclosure also relates to the use of the siRNA, cells, or pharmaceutical compositions of the present disclosure in the preparation of a medicament for treating a disease or symptom associated with ANGPTL3 expression in a subject. The medicine of the present disclosure can be prepared into emulsions, microemulsions, and microparticles.

sequence

The RNA sequence provided by the present disclosure targets the human ANGPTL3 gene (or target gene, target mRNA sequence, target sequence).

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

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

In this article, the meanings of each abbreviation are as follows:

The A, U, G, and C distributions represent natural adenine ribonucleotides, uracil ribonucleotides, guanine ribonucleotides, and cytosine ribonucleotides.

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

i represents inosine ribonucleotide.

m indicates that the adjacent nucleotide to its left is a 2'-OCH ₃ modified nucleotide. For example, Am, Um, Gm and Cm represent 2'-OCH ₃ modified A, U, G and C.

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

"s" or "s-" means that two adjacent nucleotides and/or delivery vectors are connected through phosphorothioate.

VP indicates that the nucleotide adjacent to the right side is a vinylphosphonate-modified nucleotide, which is well known in the art, see, for example, PCT Publication Nos. WO2011139702, WO2013033230 and WO2019105419.

IB stands for reverse abasic deoxyribonucleotide, which can include the following three structures depending on its position/connection method in siRNA (respectively for the 5' end, middle and 3' end of the nucleic acid chain):

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

L96 represents a GalNAc delivery vector of the following structure, which is well known in the art, wherein Indicates the position of attachment to siRNA via a phosphate group or a phosphorothioate group, see for example PCT Publication Nos. WO2009073809 and WO2009082607.

NAG37 represents a GalNAc delivery vector of the structure well known in the art, wherein Indicates the position of attachment to siRNA via a phosphate group or phosphorothioate group, see for example PCT Publication No. WO2018044350

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

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

STM1 represents the nucleotide substitution of the following structure. Depending on the position of STM1 in the nucleic acid chain, Can be linked to adjacent nucleotides, 3' end structures or 5' end structures

Table 4 Modified siRNA sense strand sequence targeting ANGPTL3 gene

Table 5 Modified siRNA antisense strand sequences targeting ANGPTL3 gene

Table 6 Paired siRNA sense and antisense strands targeting the ANGPTL3 gene

Example

Unless otherwise stated, the sources of materials used in the examples are as follows:

Huh7 cell line was purchased from Nanjing Kebai, product number CBP60202;

Hep3B cell line was purchased from Nanjing Kebai, product number CBP60197;

PHH cells were purchased from Shanghai Xuanyi, product number QYLF-HPMC;

HEK293A cell line was purchased from Nanjing Kebai, product number CBP60436;

Balb/c mice were from Zhejiang Vitong Lever, product number Balb/c.

Example 1 Preparation of Compound E7

1. Preparation of intermediate 3-4

1.1 Preparation of compound 2

To compound 1 (300 g, 2.01 mol) in DCM (1.80 L) was added slowly dropwise benzyl (2,5-dioxopyrrolidin-1-yl) carbonate (600 g, 2.40 mol) at 15°C. Add TEA (203g, 2.01mol, 280mL). 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 an important new spot was detected (R _f =0.52). The reaction mixture was washed with saturated sodium bicarbonate solution (1.00L x 2), the organic phase was washed with brine (1.00L), dried over _anhydrous _Na2SO4 and concentrated in vacuo. Without purification, compound 2 (approximately 385 g) was a yellow oil.

1.2 Preparation of compound 2A

To a solution of compound 4 (350 g, 1.62 mol, HCl) and Ac ₂ O (994 g, 9.74 mol, 912 mL) in pyridine (1.75 L) at 0-15°C, DMAP (19.8 g, 162 mmol) was added dropwise in one portion Add TEA (164g, 1.62mol, 226mL). The mixture was stirred at 25°C for 16 hours. LCMS (product: RT = 0.687 min) showed complete consumption of 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 filter cake was washed with EtOAc (300 mL). The filter cake was ground with water (1.45 L) at 25°C for 30 minutes. The mixture was filtered and the filter cake was washed with water (175 mL x 3), and the filter cake was collected to obtain compound 2A as a white solid (about 580 g).

1.3 Preparation of compound 2B

Three reactions were carried out in parallel.

To a solution of compound 2A (200 g, 514 mmol) in DCM (800 mL) was added dropwise TMSOTf (137 g, 616 mmol, 111 mL) at 10-15 °C over 0.5 h. The mixture was then stirred at 25°C for 3 hours. TLC (dichloromethane:methanol=20:1) showed that compound 2A (R _f =0.54) was completely consumed and a new spot (R _f =0.24) was formed. Three reactions were combined. Cool the mixture to 0-15°C, slowly pour into a solution of NaHCO ₃ (300g dissolved in 3.00L water) at 0-5°C, separate the organic phase and extract the aqueous phase with DCM (1.00L x 3), combine the organic layers , dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. Without purification, compound 2B (approximately 507 g) was obtained as a yellow oil and used in the next step.

1.4 Preparation of compound 3

To a mixture of compound 2B (250 g, 759 mmol) and compound 2 (151 g, 531 mmol) in DCM (1.00 L) was added dropwise TMSOTf (84.4 g, 380 mmol, 69.0 mL) at 0-10 °C and the mixture was added Stir at 20°C for 12 hours. TLC (dichloromethane:methanol=20:1) showed complete consumption of compound 2 (R _f =0.33) and the formation of new spots (R _f =0.03). The combined reactants were cooled to 0-5°C, then the reaction was poured into _NaHCO3 (aq, 100 g in 1 L water) and stirred at 5-10°C for 10 min and the phases separated. The aqueous phase was 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 (approximately 360 g) was a yellow oil.

¹ H NMR: (400MHz, 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).

1.5 Preparation of intermediate 3-4 (TFA salt)

Three reactions were carried out in parallel.

To a mixture of Pd/C (18.0 g, 16.3 mmol, 10% content) in THF (1.80 L) was added compound 3 (180 g, 293 mmol) and TFA (33.5 g, 293 mmol, 21.8 mL) under an argon atmosphere. . Mix up The suspension was vented and vented three times with hydrogen gas. The mixture was stirred under _H2 (50 Psi) and 30°C for 2 hours. LCMS (Product: RT = 0.697 min) showed consumption of compound 3 and a product peak was detected. Three reactions were combined. The mixture was filtered through celite and the filtrate was concentrated under reduced pressure to remove the solvent. Without purification, intermediate 3-4 (TFA salt) was obtained as a yellow solid (393 g, 660 mmol, 74.8% yield, 99.6% purity, TFA).

¹ H NMR: (400MHz, DMSO-d6)

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

2. Preparation of intermediate 3-5

2.1 Preparation of compound 5

DIEA ( 30.3g, 234mmol, 40.8mL, 6.60eq). Stir at 25°C for half an hour. HBTU (30.3g, 234mmol, 40.8mL, 6.60eq) was added to the mixture. Stir at 25°C for 16 hours. LCMS (Product: RT = 0.681 mins) showed the reaction was complete and the mixture was concentrated in vacuo. 0.50N _HCl (200mL Dry over _Na2SO4 and concentrate in vacuo _for purification. The residue was purified by column chromatography (SiO ₂ , DCM:MeOH=50:1-15:1). The residue was concentrated in vacuo at 40°C and purified by preparative-MPLC (column: 800 g Agela C18; mobile phase: [water-ACN]; 15-45% 25 min; 45% 10 min). Dry under vacuum to obtain compound 5 as a yellow solid (approximately 180g+75.0g+87.0g+40.0g+38.0g).

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

2.2 Preparation of intermediate 3-3

To Pd/C (3.00g, 10% content) in THF (300mL), compound 5 (73.0g, 61.7mmol, 1.00eq) and TFA (7.04g, 61.7mmol, 4.57mL, 1.00eq) were added under an argon atmosphere. ). The suspension was degassed and vented three times with hydrogen gas. Stir in H ₂ (20 Psi) and 20°C for 16 hours. TLC (dichloromethane:methanol=8:1, R _f =0.0) showed that the reaction was complete. The mixture was filtered through celite, and the filtrate was concentrated under pressure to remove the solvent to obtain compound 3-3 (approximately 33.4g+129g+75.0g) as a white solid.

¹ H NMR: (400MHz, DMSO)

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

3. Preparation of compound E7

3.1 Preparation of compound 3

Compound 1 (2.00g, 1.87mmol, prepared according to the method of intermediate 3-3 above) was dissolved in DCM (20.0mL) at room temperature, DIEA (0.135mL, 0.814mmol), compound 2 ( 0.550g, 0.814mmol), replace nitrogen 3 times. The reaction mixture was stirred at 25°C for 16 hours. Liquid mass spectrometry detected the MS response of the product, and thin layer chromatography (dichloromethane/methanol=5/1) showed that the raw material disappeared and new spots were generated. The reaction solution was concentrated under reduced pressure, and the obtained crude product was purified by column chromatography (dichloromethane/methanol=5/1) to obtain compound 3 (approximately 780 mg) as a white solid.

¹ H NMR (400MHz, CD ₃ OD)

δ＝7.28-7.42(m,5H),5.30-5.34(m,4H),5.04-5.14(m,6H),4.63-4.67(m,4H),4.36-4.44(m,2H),4.00-4.20 (m,23H),3.91-3.95(m,4H),3.69-3.77(m,9H),3.52-3.67(m,32H),3.34-3.43(m,9H),2.29-2.31(m,4H) ,2.14(s,12H),2.03(s,12H),1.92-1.96(m,24H).LCMS:m/z＝1221.6(M/2+H) ⁺ .

3.2 Preparation of compound 4

Dissolve compound 3 (1.10g, 0.451mmol) in MeOH (10.0mL) at room temperature, add 10% mass fraction of wet Pd/C (0.050g, 0.451mmol) to the solution, and replace hydrogen gas three times. The reaction mixture was stirred at 25°C for 18 hours under a hydrogen atmosphere (14.696 psi). The MS response of the product was detected by liquid mass spectrometry, and thin layer chromatography (dichloromethane/methanol=10/1, phosphomolybdic acid color development) showed that the raw materials had been completely consumed and new spots were generated. The reaction liquid was filtered, and the filtrate was concentrated under reduced pressure to obtain compound 4 (approximately 840 mg) as a white solid.

¹ H NMR (400MHz, CD ₃ OD)

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

3.3 Preparation of compound 6

Compound 5 (232mg, 0.364mmol) was dissolved in DCM (10.0mL) at room temperature, and HBTU (207mg, 0.546mmol), DIEA (0.181mL, 1.09mmol)) and compound 4 (840mg, 0.364) were added to the solution in sequence. mmol), replaced with nitrogen three times. The reaction mixture was stirred at 25°C for 1 hour. Liquid mass spectrometry detected the disappearance of the raw material, and thin layer chromatography (dichloromethane/methanol=5/1) showed that the raw material disappeared and new spots were generated. The reaction solution was concentrated under reduced pressure, and the obtained crude product was purified by column chromatography (dichloromethane/methanol = 8/1 to 5/1) to obtain compound 6 (approximately 620 mg) as a white solid.

¹ H NMR (400MHz, CD ₃ OD)

δ＝7.41-7.43(m,2H),7.23-7.34(m,7H),6.83-6.90(m,4H),5.31-5.35(m,4H),5.01-5.12(m,4H),4.63-4.65 (m,4H),4.41-4.45(m,2H),4.31-4.33(m,1H),3.99-4.22(m,22H),3.87-3.97(m,6H),3.58-3.81(m,45H) ,3.34-3.43(m,10H),2.19-2.40(m,10H),2.14(s,12H),2.02(s,12H),1.92-1.96(mz,24H),1.48-1.63(m,4H) ,1.28-1.38(m,8H).LCMS:m/z＝1460.0(M/2+H) ⁺ .

4. Preparation of compound E7

Compound 6 (300 mg, 0.103 mmol) was dissolved in DCM (10.0 mL) at room temperature, and DIEA (0.102 mL, 0.618 mmol), compound 7 (10.3 mg, 0.103 mmol) and DMAP (12.6 mg) were added to the solution in sequence. 0.103mmol), replacing nitrogen three times. The reaction mixture was stirred at 25°C for 2 hours. The disappearance of raw material was detected by LC/MS. The reaction solution was concentrated under reduced pressure, and the crude product obtained was prepared by preparative high-performance liquid chromatography (preparative-HPLC, column: Waters Xbridge BEH C18 100*30mm*10um; mobile phase: water-ACN; B%: 17% -57%, 5min) to obtain white solid compound E7 (53.0mg, yield 17.08%, purity 78.94%).

¹ H NMR (400MHz, CD ₃ OD)

δ＝7.41-7.45(m,2H),7.17-7.34(m,7H),6.85-6.89(m,4H),5.32-5.36(m,4H),5.03-5.13(m,4H),4.63-4.67 (m,4H),4.38-4.47(m,2H),4.32-4.34(m,1H),4.01-4.26(m,22H),3.88-4.00(m,6H),3.77-3.81(m,7H) ,3.49-3.76(m,45H),3.33-3.47(m,10H),2.56-2.62(m,2H),2.45-2.55(m,3H),2.21-2.38(m,7H),2.14(s, 12H),2.05-2.11(m,2H),2.02(s,12H),1.92-1.96(m,24H),1.47-1.68(m,4H),1.28-1.34(m,8H)

MS: m/z＝3022.36(M+H) ⁺ .

Example 2 Preparation of Compound E13

1. Preparation of intermediate 2-2

1.1 Preparation of compound 3

Four reactions were carried out in parallel.

To a solution of compound 1 (250 g, 741 mmol) in THF (1.75 L) was added 4-methylmorpholine (434 g, 4.30 mol, 472 mL), followed by cooling to 0°C. Isobutyl chloroformate (243g, 1.78mol, 233mL) was added to the reaction mixture within 10 minutes, maintaining the reaction temperature below 4.0°C. After addition, the mixture was stirred for 40 minutes or more, and compound 2 (526g, 1.78mol) was gradually added to the reaction mixture within 10 minutes, maintaining the reaction temperature less than 4.0°C. After addition, the ice bath was removed and the reaction was allowed to warm to room temperature for 2 hours. TLC (petroleum ether/ethyl acetate=1/1, compound 1R _f =0.43) showed that compound 1 was completely consumed and new spots were formed. The 4 reactions were combined. The reaction solution was poured into a stirring cold (0°C) 0.50 M HCl (aq.) (12.0 L) solution and stirred for about 10 minutes. _EtOAc ( _4.00L Hexane (1.20L) was added to the stirring oil. White smoke appeared in the solution and subsequently disappeared after further stirring. Seed crystals (1.20g, 0.10wt%) were added, and white crystals slowly formed. Within 20 minutes, the suspension was thick enough to prevent stirring, at which time additional hexane (6.00 L) was added and stirred for 12 hours. The suspension was filtered, washed with hexane (1.20 L) and dried to obtain compound 3 as a white solid (approximately 1.20 kg).

¹ H NMR: (400MHz DMSO)

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

1.2 Preparation of compound 4

Five reactions were performed in parallel.

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

¹ H NMR: (400MHz DMSO)

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

1.3 Preparation of compound 6

To a stirred DMF (2.00L) solution of compound 4 (80.0g, 194mmol) and (384g, 700mmol, TFA), HOBT (103g, 760mmol), EDCI (146g, 760mmol) and DIEA (113g, 877mmol, 153mL) were added successively ). The reaction was stirred at 20°C for 2 hours. TLC (dichloromethane/methanol=5/1, compound 6R _f =0.43) showed that compound 4 was completely consumed and new spots formed. The reaction mixture was slowly poured into a stirred cold 0.5 M aqueous HCl solution (230 mL), stirred for 10 minutes, a white solid formed and filtered, and the aqueous phase was extracted twice with DCM (1.50 L). The combined organic phases were washed with 5% NaHCO ₃ (aq.) (200 mL), dried (Na ₂ SO ₄ ), and concentrated by evaporation under pressure. The residue was purified by column chromatography (SiO ₂ , dichloromethane/methanol=5/1, compound 6R _f =0.43) to obtain compound 6 as a yellow solid (approximately 180 g).

¹ H NMR: (400MHz DMSO)

δ7.95-7.91(m,3H),7.82-7.80(m,4H),7.39-7.31(m,6H),5.21-5.01(m,3H),5.00-4.96(m,5H),4.56-4.53 (m,3H),4.02(s,1H),3.88(s,9H),3.85(s,4H),3.76(s,3H),3.50-349(m,9H),3.39-3.36(m,6H ),3.19-3.15(m,6H),2.15-2.05(m,12H),1.99(s,9H),1.89-1.77(m,21H).

1.4 Preparation of intermediate 2-2

Add Pd(OH) ₂ /C (4.94g, 3.52mmol, 10% content) to the dry hydrogenation bottle under an argon atmosphere, add MeOH (350mL), and add compound 6 (47.4g, 28.5mmol) and TFA in sequence. (3.26g, 28.5mmol, 2.11mL), stirred in _H2 (50psi) and 20°C for 3 hours. The reaction was monitored by TLC (dichloromethane/methanol=5/1, intermediate 2-2R _f =0.25), showing consumption of compound 6. The reaction mixture was filtered and the filtrate was concentrated in vacuo to obtain Intermediate 2-2 (TFA salt) as a white solid (46.3 g, 94.6% yield, 95.7% purity, TFA).

¹ H NMR: (400MHz DMSO)

δ＝8.56-8.50(m,1H),8.14-8.05(m,4H),7.99(s,1H),7.88-7.82(m,4H),5.22-5.21(m,3H),4.99-4.96(m ,3H),4.55-4.53(m,3H),4.09(s,1H),4.03(s,9H),3.87-3.79(m,6H),3.59-3.52(m,1H),3.51-347(m ,9H),3.39-3.37(m,5H),3.17-3.16(m,6H),2.22-2.10(m,12H),1.99(s,9H),1.93-1.73(m,21H).

2. Preparation of compound E13

2.1 Preparation of compound Int1

2.1.1 Preparation of compound 1-2

At room temperature, compound 1-1 (38.0g, 231mmol), TsNHBoc (75.4g, 278mmol), K ₂ CO ₃ (6.40g, 46.3mmol), and TEBA (5.27g, 23.1mmol) were added to a three-necked flask. React at 95°C for 2 hours, then cool to 25°C. TLC (PE/EA=2/1, UV 254nm) shows that the reaction is complete. The reaction solution is diluted with water (500mL), extracted with dichloromethane (200mL x 3), and organically The phase was concentrated under reduced pressure, and the crude product was separated and purified by column (PE/EA=10/1-3/1) to obtain compound 1-2 (40.0g) as a colorless oil.

¹ H NMR (400MHz, CDCl ₃ )

δ7.72(d,J=8.4Hz,2H),7.28-7.39(m,7H),4.72-4.86(m,2H),4.42-4.56(m,2H),3.52-3.61m,2H),3.14 -3.39(m,2H),2.43(s,3H),1.47(s,9H)

2.1.2 Preparation of compounds 1-4

Compound 1-2 (79.0g, 181mmol), compound 1-3 (4.21g, 30.5mmol), TEBA (3.47g, 15.2mmol) were reacted at 95°C for 3 hours, TLC (PE/EA=2/1, UV 254nm) indicates that the reaction is complete, The reaction solution was diluted with water (500mL), extracted with dichloromethane (200mL (approximately 40.0g).

¹ H NMR (400MHz, CDCl ₃ )

δ＝7.64-7.77(m,2H),7.27-7.38(m,13H),5.05-5.16(m,1H),4.50-4.59(m,4H),4.03-4.11(m,1H),3.62-3.76 (m,2H),3.44-3.58(m,3H),3.25-3.33(m,2H),3.15-3.20(m,1H),2.42(s,3H),1.46(s,9H)

2.1.3 Preparation of compounds 1-5

Under ice bath conditions, compound 1-4 (77.0g, 128mmol) and Et ₃ N (28.6mL, 205mmol) were sequentially added to dichloromethane (800mL), and methanesulfonyl chloride (24.2g, 212mmol) was slowly added dropwise. . After 2 hours of reaction at 25°C, LCMS showed that the reaction was complete. The reaction solution was washed with water (800 mL), and the organic phase was concentrated under reduced pressure to obtain compound 1-5 (approximately 90.0 g) as a colorless oil.

LCMS (ESI): m/z=700.1(M+Na) ⁺ ;

¹ H NMR (400MHz, CDCl ₃ ) δ7.68 (d, J = 8.4Hz, 2H), 7.28-7.39 (m, 12H), 5.10-5.20 (m, 1H), 4.93-5.02 (m, 1H), 4.52-4.61(m,4H),3.72-3.88(m,2H),3.52-3.67(m,4H),3.27-3.36(m,1H),3.15-3.22(m,1H),3.04(s,3H ),2.43(s,3H),1.43(s,9H).LCMS: m/z＝700.1(M+Na) ⁺ .

2.1.4 Preparation of compounds 1-6

Compound 1-5 (90.0 g, 133 mmol) and potassium carbonate (91.8 g, 664 mmol) were added to MeOH (900 mL) at room temperature. React for 2 hours at 66°C. TLC (PE/EA=2/1, UV 254nm) shows the formation of new spots. The organic phase is concentrated under reduced pressure. The reaction solution is diluted with water (200mL) and extracted with dichloromethane (200mL x 3). , the organic phase was concentrated under reduced pressure, and the crude product was separated and purified by column (PE/EA=30/1-2/1) to obtain white solid compound 1-6 (about 64.0g).

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

1.1.5 Preparation of compounds 1-7

At room temperature, compound 1-6 (69.0g, 143mmol) and magnesium shavings (54.7g, 2.28mol) were added to MeOH (400mL), and the mixture was reacted at 66°C for 1 hour. TLC (DCM/MeOH=10/1, UV 254nm) showed that the raw material reaction was complete and new spots were generated. The reaction solution was diluted with water (3000mL) and saturated aqueous ammonium chloride solution (3000mL), extracted with dichloromethane (1000mL x 3), the organic phase was washed with saturated sodium bicarbonate (300mL x 3), and the organic phase was concentrated under reduced pressure to obtain a colorless oily compound. 1-7 (about 32.0g).

¹ H NMR (400MHz, CDCl ₃ ) δ7.27-7.40(m,10H),4.57(s,4H),3.90-4.00(m,2H),3.59-3.69(m,4H),2.77-3.04(m ,4H)

1.1.6 Preparation of compounds 1-8

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

¹ H NMR (400MHz, CDCl ₃ ) δ4.12-4.22 (m, 2H), 3.78 (d, J = 4.4Hz, 4H), 3.17-3.30 (m, 4H)

1.1.7 Preparation of compound Int-1

Compound 1-8 (8.29g, 24.5mmol) was added to pyridine (50mL, 618mmol) at room temperature, and then DMTrCl (4.77g, 12.3mmol) was added. The reaction system reacted at 25°C for 18 hours, and TLC (DCM/MeOH=10/1, UV 254nm) showed that the product was generated. The reaction solution was concentrated under reduced pressure, diluted with saturated ammonium chloride (200 mL), extracted with DCM (100 mLx3), and the organic phase was concentrated under reduced pressure. The crude product was subjected to column chromatography (DCM/MeOH=99/1-10/1) to obtain yellow solid Int-1 (about 2.6g, 5.78mmol, 23.6%).

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

2.2 Preparation of compound Int6

2.2.1 Preparation of compound 3

Dissolve compound 1 (1.00g, 3.05mmol) into anhydrous N,N-dimethylformamide (10.0mL) at 25°C, and add HOBt (0.54g, 3.97mmol) and EDCI (0.76g, 3.97 mmol), DIEA (1.51 mL, 9.16 mmol), and then compound 2 (0.79 g, 3.66 mmol) was added. The mixture was stirred at 25°C for 2 hours. Liquid mass spectrometry showed that the raw material reacted completely and the product was produced. The thin layer chromatography plate (methylene chloride: methanol = 10:1) showed that the raw material reaction was complete and the product was generated. Add methylene chloride (200ml) to the mixture, and wash the mixture three times with saturated citric acid solution (20ml x 3), saturated sodium bicarbonate solution (20ml x 3), and saturated brine (20ml x 3). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (dichloromethane:methanol=10:1) to obtain compound 3 (approximately 1.00g) as a yellow oil.

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

LCMS: m/z＝526.8(M+H) ⁺ .

2.2.2 Preparation of compound 4

Compound 3 (380 mg, 0.20 mmol) was dissolved in anhydrous methanol (10.0 mL) at 25°C, and 10% wet Pd/C (500 mg, 4.70 mmol) with a mass fraction of 10% was added thereto. The reaction solution was stirred at 50°C for 12 hours. LC/MS showed product formation. The reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to obtain compound 4 (approximately 600 mg) as a yellow oil.

¹ H NMR (400MHz, CD ₃ OD) δ3.79-3.89 (m, 2H), 3.73 (dd, J = 3.6, 13.2Hz, 1H), 3.64-3.68 (m, 5H), 3.57-3.62 (m, 2H),3.48-3.56(m,2H),3.38-3.47(m,1H),1.58-1.62(m,5H),1.34(d,J＝2.8Hz,11H),1.30-1.37(m,1H) .LCMS: m/z＝346.5(M+H) ⁺ .

2.2.3 Preparation of compound 5

Compound 4 (600 mg, 1.74 mmol) was dissolved in pyridine (3.00 mL) at 25°C, and DMTrCl (883 mg, 2.61 mmol) was added thereto. The reaction solution was stirred at 25°C for 1 hour. LC/MS showed product formation. Thin layer chromatography (dichloromethane: methanol = 10:1) showed that the raw material reaction was complete and the product was generated. Add dichloromethane (200ml) to the mixture, and wash the mixture three times with saturated sodium bicarbonate solution (20ml x 3) and brine (20ml x 3). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (petroleum ether:ethyl acetate=3:1) to obtain colorless liquid compound 5 (about 475 mg).

¹ H NMR (400MHz, CD ₃ OD) δ7.41-7.43(m,2H),7.16-7.34(m,7H),6.83-6.87(m,4H),3.88-4.04(m,2H),3.77- 4.09(m,7H),3.61-3.68(m,4H),3.43-3.58(m,3H),3.33-3.38(m,1H),3.14-3.20(m,1H),3.05(dd,J＝8.4 ,13.2Hz,1H),2.20-2.42(m,4H),1.51-1.60(m,4H),1.25-1.39(m,8H).LCMS:m/z＝648.3(M+H) ⁺ .

2.2.4 Preparation of compound Int-6

Compound 5 (475 mg, 0.73 mmol) was dissolved in tetrahydrofuran (8.00 mL) and H ₂ O (2.00 mL) at 25°C, and lithium hydroxide monohydrate (36.9 mg, 0.89 mmol) was added thereto. The reaction solution is at 25 Stir for 12 hours at Celsius. Thin layer chromatography (dichloromethane: methanol = 10:1) showed that the raw material reaction was complete and the product was generated. After the reaction solution was concentrated under reduced pressure, it was directly lyophilized on a freeze dryer to obtain compound 6 (approximately 430 mg) as a white solid product.

2.3 Preparation of compound E13

2.3.1 Preparation of compound 7

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

¹ H NMR (400MHz, CD ₃ OD) δ7.43 (d, J = 8.4Hz, 2H), 7.18-7.34 (m, 7H), 6.81-6.91 (m, 4H), 5.34 (d, J = 2.8Hz ,3H),5.09-5.11(m,3H),4.62-4.66(m,3H),4.27-4.35(m,2H),4.07-4.17(m,9H),4.00-4.07(m,4H),3.89 -3.98(m,5H),3.79(d,J＝2.4Hz,6H),3.69-3.75(m,5H),3.60-3.64(m,9H),3.52-3.56(m,10H),3.43-3.50 (m,4H),3.39-3.40(m,1H),2.25-2.40(m,8H),2.13-2.15(m,9H),2.02(s,9H),1.94-1.96(m,18H),1.48 -1.66(m,4H),1.21-1.36(m,11H)

2.3.2 Preparation of E13

Compound 7 (400mg, 0.187mmol) was dissolved in anhydrous dichloromethane (5.00mL), DIEA (0.18mL, 1.12mmol), DMAP (5.71mg, 0.047mmol) were added in sequence, and finally compound 8 (112mg, 1.12 mmol) was added. The mixture was stirred at 25°C for 3 hours. Thin layer chromatography (dichloromethane:methanol=10:1) showed the formation of new spots. Add methylene chloride (200ml) to the mixture, wash with saturated brine three times (15.0ml x 3), dry the organic phase over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain a crude product. The crude product was separated by high performance liquid chromatography (chromatographic column: Welch Xtimate C18 150*25mm*5um; mobile phase: water-ACN; gradient: 27%-57%/11min; flow rate: 25ml/min) to obtain white solid compound E13 ( 74.8 mg, 0.033 mmol, yield 17.9%).

LCMS (ESI): m/z=1149.5 (M/2+H) ⁺ ; ¹ H NMR (400MHz, CD ₃ OD) δ7.42-7.44 (m, 2H), 7.18-7.34 (m, 7H), 6.83 -6.90(m,4H),5.33-5.34(m,3H),5.08-5.11(m,3H),4.61-4.68(m,3H),4.27-4.36(m,2H),4.19-4.27(m, 1H),4.10-4.19(m,7H),4.08(s,2H),4.00-4.07(m,4H),3.90-3.94(m,4H),3.83-3.89(m,1H),3.79(d, J＝2.4Hz,6H), 3.70-3.72(m,4H),3.53-3.54(m,15H),3.44-3.48(m,3H),3.35-3.40(m,4H),3.10-3.23(m,2H),2.53-2.63(m ,4H),2.22-2.40(m,8H),2.11-2.16(m,9H),2.05-2.10(m,2H),2.02(s,9H),1.90-1.97(m,19H),1.48-1.65 (m,4H),1.29-1.32(m,8H)

Example 3 Preparation of Compound E2

1. Preparation of compound 2b

At room temperature, compound 2a (38.0g, 231mmol), TsNHBoc (75.4g, 278mmol), K ₂ CO ₃ (6.40g, 46.3mmol), and TEBA (5.27g, 23.1mmol) were added to the three-necked flask. React at 95°C for 2 hours, then cool to 25°C. TLC (PE/EA=2/1, UV 254nm) shows that the reaction is complete. The reaction solution is diluted with water (500mL), extracted with dichloromethane (200mL x 3), and organically The phase was concentrated under reduced pressure, and the crude product was separated and purified by column (PE/EA=10/1-3/1) to obtain the title compound 2b (40.0g). ¹ H NMR (400MHz, CDCl ₃ ) δ7.72 (d, J = 8.4Hz, 2H), 7.28-7.39 (m, 7H), 4.72-4.86 (m, 2H), 4.42-4.56 (m, 2H), 3.52-3.61m,2H),3.14-3.39(m,2H),2.43(s,3H),1.47(s,9H)

2. Preparation of compound 2d

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

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

3. Preparation of compound 2e

Under ice-water bath conditions, compound 2d (77.0g, 128mmol) and triethylamine (28.6mL, 205mmol) were sequentially added to dichloromethane (800mL), and methylsulfonyl chloride (24.2g, 212mmol) was slowly added dropwise. After 2 hours of reaction at 25°C, LCMS showed that the reaction was complete. The reaction solution was washed with water (800 mL), and the organic phase was concentrated under reduced pressure to obtain crude compound 2e (90.0 g).

m/z：ES+[M+Na]+700.1

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

4. Preparation of compound 2f

Compound 2e (90.0g, 133mmol) and potassium carbonate (91.8g, 664mmol) were added to methanol (900mL) at room temperature. React at 66°C for 2 hours, TLC (PE/EA=2/1, UV 254nm) It shows that new spots are generated, the organic phase is concentrated under reduced pressure, the reaction solution is diluted with water (200mL), extracted with dichloromethane (200mL x 3), the organic phase is concentrated under reduced pressure, and the crude product is separated and purified by column (PE/EA=30/1- 2/1) to obtain compound 2f (64.0 g, yield 99.9%).

5. Preparation of compound 2g

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

6. Preparation of compound 2h

At room temperature, compound 2g (3.00g, 9.16mmol), compound 1b (1.90mL, 18.3mmol), and acetic acid (1.01mL, 18.3mmol) were added to methanol (30mL). React at 25°C for 18 hours, then add sodium cyanoborohydride (2.30g, 36.7mmol), and react at 50°C for 4 hours. TLC (DCM/MeOH=10/1) shows that new spots are generated. The reaction solution was diluted with water (50mL), extracted with dichloromethane (30mL x3), and the organic phase was concentrated under reduced pressure. The crude product was purified by column chromatography (dichloromethane/methanol=99/1-5/1) to obtain compound 2h (3.00g, yield 79.9%).

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

7. Preparation of Compound 2i

At room temperature, compound 2h (3.00g, 7.32mmol) was added to concentrated hydrochloric acid (10mL, 12M), and the reaction was carried out at 50°C for 18 hours. TLC (DCM/MeOH=10/1) showed that new spots were formed, and the reaction solution Concentrate under reduced pressure to obtain crude compound 2i (2.00g).

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

8. Preparation of compound 2j

Dissolve compound 2i (2.00g, 8.72mmol) into pyridine (40mL) at room temperature, then add DMTrCl (2.96g, 8.72mmol), react at 25°C for 18 hours, TLC (PE/EA=1/1, UV 254nm) ) indicates that the reaction is complete. The reaction solution was concentrated under reduced pressure, added with saturated aqueous ammonium chloride solution (200mL), extracted with DCM (100mLx3), the organic phase was concentrated under reduced pressure, and the crude product was purified by column chromatography (dichloromethane/methanol=99/1-10/1) Compound 2j (1.40g, yield 30.2%) was obtained.

9. Preparation of compound E2

Add compound 2j (660mg, 1.24mmol), compound 1h (374mg, 1.24mmol), DCI (73.3mg, 0.621mmol) to DCM (10mL), react at 25°C for 1 hour, TLC (PE/EA=5/1, PMA) shows that the raw material reaction is complete. Add saturated sodium bicarbonate (50mL), extract with DCM (30mL .

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

¹ H NMR (400MHz, CDCl ₃ ) δ7.40-7.50 (m, 2H), 7.28-7.37 (m, 6H), 7.21 (d, J = 7.2Hz, 1H), 6.77-6.89 (m, 4H), 3.67-4.07(m,11H),3.56-3.66(m,2H),3.04-3.33(m,2H),2.30-2.96(m,6H),2.10-2.28(m,1H),1.63-1.88(m ,4H),1.47-1.52(m,1H),1.24-1.31(m,3H),1.15-1.23(m,12H),1.11-1.14(m,1H)

Example 4 Preparation of siRNA

The siRNAs of the present disclosure are prepared using the solid-phase phosphoramidite method, which is well known in the art. Specific methods can be found, for example, in PCT publication numbers WO2016081444 and WO2019105419, and are briefly described below.

1. Preparation of siRNA without ligand attached

1.1 Synthesis of justice chain (SS chain)

Through the solid-phase phosphoramidite synthesis method, the blank CPG solid-phase carrier is used as the starting cycle, and the nucleoside monomers or nucleotide analogues are connected one by one from the 3'-5' direction according to the sequence of the sense strand nucleotides. monomer. Each connection of a nucleoside monomer or nucleotide analog monomer includes a four-step reaction of deprotection, coupling, capping, oxidation or sulfation. The synthesis conditions for an oligonucleotide with a synthesis scale of 5 μmol are as follows:

The nucleoside monomer or nucleotide analog monomer is provided with a 0.05 mol/L acetonitrile solution. The reaction conditions of each step are the same, that is, the temperature is 25°C, and 3% trichloroacetic acid-dichloromethane solution is used for deprotection. , deprotection 3 times; the activator used in the coupling reaction was 0.25 mol/L ETT-acetonitrile solution, coupled 2 times; the capping reaction used 10% acetic anhydride-acetonitrile and pyridine/N-methylimidazole/acetonitrile (10: 14:76, v/v/v), blocked twice; oxidized using 0.05mol/L iodine/tetrahydrofuran/pyridine/water (70/20/10, v/v/v), oxidized twice; used sulfide 0.2mol/L PADS acetonitrile/3-methylpyridine (1/1, v/v), sulfide 2 times.

The nucleotide monomer of IB was purchased from Shanghai Zhaowei Technology Development Co., Ltd., product number OP-040.

1.2 Synthesis of antisense chain (AS chain)

Through the solid-phase phosphoramidite synthesis method, the blank CPG solid-phase carrier is used as the starting cycle, and the nucleoside monomers or nucleotide analogues are connected one by one from the 3'-5' direction according to the sequence of the antisense strand nucleotides. single object. Each linkage of a nucleoside monomer or nucleotide analog monomer involves deprotection, coupling, capping, oxidation or sulfation. For a four-step reaction, the synthesis conditions of 5 μmol of oligonucleotide for the antisense strand are the same as those for the sense strand.

1.3 Purification and annealing of oligonucleotides

1.3.1 Ammonolysis

Add the synthesized solid phase carrier (sense strand or antisense strand) into a 5mL centrifuge tube, add 3% diethylamine/ammonia water (v/v), and react in a constant temperature water bath at 35°C (or 55°C) for 16 hour (or 8 hours), filter, and wash the solid phase carrier three times with ethanol/water, 1 mL each time. The filtrate is centrifuged and concentrated and the crude product is purified.

1.3.2 Purification

Methods of purification and desalting are well known to those in the art. For example, a strong anion packing column can be used, a sodium chloride-sodium hydroxide system can be used for elution and purification, and the products can be collected and tubed. A gel packing purification column can be used for desalting, and the elution system is pure water.

1.3.3 Annealing

According to Table 6, mix the sense strand (SS chain) and the antisense strand (AS chain) at a molar ratio (SS chain/AS chain = 1/1.05), heat the water bath to 70-95 degrees, keep for 3-5 minutes, naturally Cool to room temperature and freeze-dry the system to obtain the product.

2. Preparation of siRNA with sense strand connected to ligand

2.1 Connection of ligand to CPG carrier

2.1.1 Connection of compound E7 to CPG carrier

Compound E7 (53 mg, 0.018 mmol) and HBTU (13.3 mg, 0.035 mmol) were mixed, acetonitrile (5 mL) was added with shaking to dissolve, and then DIEA (9.0 mg, 0.07 mmol) and DMAP (2.1 mg, 0.018 mmol) were added with shaking until dissolved. become clear. Weigh the blank carrier Resin (550 mg, CPG pore size ) was added to the reaction solution, the temperature was controlled at 20°C, and the reaction was carried out overnight on a shaking table. Samples were taken and monitored, and thin-layer chromatography and TLC were performed. The results showed that the reaction was complete. The developing agent was DCM/methanol=4/1, and phosphomolybdic acid was used to develop the color. Use a sand core funnel to filter, wash the filter cake with anhydrous acetonitrile (20 mL*5), take the filter cake, use an oil pump to filter under reduced pressure for 6 hours, and obtain 530 mg of an off-white solid.

Put 530 mg of the above-mentioned condensation product into a 50 mL round-bottomed bottle, add CapC (DMAP/acetonitrile), CapB (N-methylimidazole/pyridine/acetonitrile), CapA (acetic anhydride/acetonitrile) in sequence, and shake at room temperature. overnight. Filter, wash the filter cake with acetonitrile, 20mL*4), take the filter cake, and filter under reduced pressure with an oil pump for 8 hours to obtain 200 mg of off-white solid, which is used for solid phase synthesis.

2.1.2 Connection of compound E13 to CPG carrier

Compound E13 was connected to the CPG carrier in a manner similar to the method for connecting compound E7 to the CPG carrier.

2.2 Synthesis of justice chain (SS chain)

Through the solid-phase phosphoramidite synthesis method, the GL6 solid-phase carrier prepared in Section 2.1.1 of Example 4 is used as the initial cycle, and the nucleosides are connected one by one from the 3'-5' direction in the sequence of the sense strand nucleotides. monomer. Each connection of a nucleoside monomer includes four-step reactions of deprotection, coupling, capping, oxidation or sulfation. The synthesis conditions for an oligonucleotide with a synthesis scale of 5 μmol are as follows:

The nucleoside monomer is provided with a 0.05 mol/L acetonitrile solution. The reaction conditions for each step are the same, that is, the temperature is 25°C. For deprotection, use 3% trichloroacetic acid-dichloromethane solution, and deprotect 3 times; coupling The activator used in the reaction was 0.25 mol/L ETT-acetonitrile solution, coupled twice; the capping agent used 10% acetic anhydride-acetonitrile and pyridine/N-methylimidazole/acetonitrile (10:14:76, v/v/ v), cap twice; use 0.05mol/L iodine for oxidation /tetrahydrofuran/pyridine/water (70/20/10, v/v/v), oxidized twice; for sulfide, use 0.2 mol/L PADS acetonitrile/3-methylpyridine (1/1, v/v). Thiogeneration 2 times.

2.3 Synthesis of antisense chain (AS chain)

Through the solid-phase phosphoramidite synthesis method, a blank CPG solid-phase carrier is used as the starting cycle, and the nucleoside monomers are connected one by one from the 3'-5' direction in the order of the antisense strand nucleotide arrangement. Each connection of a nucleoside monomer involves a four-step reaction of deprotection, coupling, capping, oxidation or sulfation. The synthesis conditions of 5 μmol of oligonucleotide of the antisense strand are the same as those of the sense strand.

2.4 Purification and annealing of oligonucleotides

2.4.1 Ammonolysis

2.4.2 Purification

Methods of purification and desalting are well known to those in the art. For example, a strong anion packing column can be used, a sodium chloride-sodium hydroxide system can be used for elution and purification, the products can be collected and tubed, and a gel packing purification column can be used for desalting. The elution system is pure water.

2.4.3 Annealing

According to Table 6, mix the sense strand (SS chain) and the antisense strand (AS chain) at a molar ratio (SS chain/AS chain = 1/1.05), heat the water bath to 70-95°C, keep for 3-5 minutes, and naturally Cool to room temperature and freeze-dry the system to obtain the product.

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

Example 5 Huh7 cell line activity screening

cell transfection

On the first day, the Huh7 cell line (Nanjing Kebai, Cat. No. CBP60202) was digested, resuspended, counted, and plated in a 96-well plate at 100 μL/well and 1×10 ⁴ cells/well. Transfection was performed 18 hours later.

The next day, 20 μM of the siRNA stock solution prepared in Example 4 was diluted with Opti-MEM, 198 μL of Opti-MEM was added to 2 μL of the siRNA stock solution, piped and mixed, and set aside. In each experiment, corresponding dilution operations are performed according to different experimental needs.

The next day, take 14.1 μL Opti-MEM and dilute 0.9 μL RNAiMAX (Thermo, 13778150), mix gently by pipetting, and let stand at room temperature for 5 minutes. Then take 15 μL of the prepared RNAi-MAX mixture and 15 μL of the diluted compound by gently pipetting and mixing without bringing in air bubbles. Let stand at room temperature for 10 minutes, and then add 10 μL/well to a 96-well plate. After culturing for 24 hours in a 37°C, 5% _CO2 incubator, RNA was extracted.

RNA extraction

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

RNA reverse transcription

To prepare the denaturation reaction mixture, refer to PrimeScript ^TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B). Each well contains 1 μL of Oligo dT Primer, 1 μL of dNTP Mixture, and 12.5 μL of template RNA. Denaturation reaction was performed by incubating at 65°C for 5 min in a conventional PCR machine. Place the mixture Cool quickly on ice for 2 minutes.

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

Slowly mix 14.5 μL of the denatured reaction solution with the reverse transcription reaction solution, incubate in a conventional PCR machine at 42°C for 45 minutes to perform reverse transcription, incubate at 95°C for 5 minutes to inactivate the enzyme, and remove the reverse transcription product at 4°C. (cDNA) cooling.

After reverse transcription, add 30 μL of DNase- and RNase-free distilled water to the cDNA sample in each well.

Fluorescent quantitative PCR

Refer to the operating procedure of TaqMan ^TM Fast Advanced Master Mix (ABI, 4444965), and perform a fluorescence quantitative PCR reaction (ABI, QuantStudio3) with a 20 μL system. The reaction program is: (50°C, 2min)×1Cycle; (95°C, 20s)×1Cycle; (95°C, 1s; 60°C, 24s)×40Cycles.

Table 7 Primer information

Statistics

Calculate the 2 ^-ΔΔCt value and convert to percentage to obtain the remaining inhibition rate.

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

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

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

Example 6 Screening of Huh7 cell line activity using 10-fold gradient dilutions of 5 concentrations of siRNA

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

The starting concentration of siRNA was 10 nM, and 10-fold gradient dilution was performed to obtain 5 concentration points (10 nM, 1 nM, 0.1 nM, 0.01 nM, 0.001 nM), and the Hep3B cell line activity of siRNA was screened. The screening results are shown in Table 9, in which columns 2-6 are the remaining inhibition rates and column 7 is the IC50 value.

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

Example 7 Activity Screening of Primary Human Hepatocytes (PHH Cells)

cell transfection

Take 1.4 mL of rat tail collagen solution (Sigma, C3867), add 40.6 mL of DNase- and RNase-free distilled water, mix well, add 40 μL to each well of a 96-well culture plate, coat overnight at 4°C, and remove the mixture the next day. Coating fluid.

The next day, before use, the coated cell plate was rinsed with DPBS and the DPBS was aspirated off. PHH cells (Shanghai Xuanyi, Cat. No. QYLF-HPMC) were revived at 37°C, added to the recovery medium, centrifuged, resuspended and counted. PHH cells were plated in a 96-well plate at 90 μL/well, 2×10 ⁴ cells/well; complete medium was replaced after 4 hours, and transfection was performed after 18 hours.

On the third day, 20 μM siRNA stock solution was diluted with Opti-MEM. Add 198 μL Opti-MEM to 2 μL siRNA stock solution, mix by pipetting, and use it as the first concentration point. Perform corresponding gradient dilutions according to actual experimental needs.

On the third day, take 14.1 μL Opti-MEM and dilute 0.9 μL RNAiMAX (Thermo, 13778150), mix gently by pipetting, and let stand at room temperature for 5 minutes. Then take 15 μL of the prepared RNAi-MAX mixture and 15 μL of the diluted compound by gently pipetting and mixing without bringing in air bubbles. Let it stand at room temperature for 10 minutes. Add 10 μL/well to a 96-well plate. After culturing for 24 hours in a 37°C, 5% _CO2 incubator, RNA was extracted.

RNA extraction

RNA reverse transcription

To prepare the denaturation reaction mixture, refer to PrimeScript ^TM II 1st Strand cDNA Synthesis Kit (Takara, 6210B). Each well contains 1 μL of Oligo dT Primer, 1 μL of dNTP Mixture, and 12.5 μL of template RNA. Denaturation reaction was performed by incubating at 65°C for 5 min in a conventional PCR machine. Place the mixture on ice to cool quickly for 2 minutes.

Fluorescent quantitative PCR

Table 10 Primer information

Statistics

The starting concentration of siRNA was 10 nM, and it was diluted 10 times to obtain 5 concentration points (10 nM, 1 nM, 0.1 nM, 0.01 nM, 0.001 nM). The activity of siRNA in human liver primary cells was screened. The screening results are shown in Table 11. , where columns 2-6 are the remaining inhibition rates and column 7 is the IC50 value.

Table 11 Results of human primary hepatocyte screening using 5 concentrations of siRNA

Example 8 Screening of off-target activity of psiCHECK2 GSSM-5Hits

Plasmid preparation

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

cell transfection

In each well of a 96-well plate, 100 μL of HEK293A cell (Nanjing Kebai, Cat. No. CBP60436) resuspension was plated, with 8 × 10 ³ cells/well.

The next day, first remove the complete culture medium in the well, replace it with 80 μL/well of Opti-MEM culture medium, and starve for about 1.5 h.

siRNA preparation: Dilute siRNA 3 times starting from a final concentration of 40nM, with a total of 11 concentration points (10nM, 3.3333nM, 1.1111nM, 0.37037nM, 0.12346nM, 0.04115nM, 0.01372nM, 0.00457nM, 0.00152nM, 0.00051nM, 0.0 0017 nM).

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

Preparation of Lipo mixture: Add 0.2 μL of Lipo 2000 and 9.8 μL of Opti-MEM to each well to dilute Lipo 2000 (Lipofectamine ^TM 2000 transfection reagent, Thermo, 11668019) with Opti-MEM to obtain a Lipo mixture, and let it stand at room temperature for 5 minutes.

Dispense 22 μL of prepared Lipo mixture, 2.2 μL of siRNA, and 19.8 μL of plasmid mixture into the corresponding wells and name them well A mixture. After mixing by pipetting, incubate at room temperature for 20 minutes before co-transfection. Add the well A mixture to the cells in each well, 20μL/well, plus the original 80μL Opti-MEM, the final volume of each well is 100μL. After culturing for 4 hours in a 37°C 5% CO2 incubator, 100 μL of DMEM medium containing 20% fetal calf serum was added to each well. Test after 24 hours of incubation in a 37°C 5% CO2 incubator.

Result detection

Before the experiment, mix the ( Luciferase Assay System, Promega, E2940) was remelted, and after it was equilibrated to room temperature, DMEM was added to each tube at a ratio of 1:1 to prepare substrate I, which was ready for use. Will Stop& Remelt the Buffer and wait until it equilibrates to room temperature. Stop& Prepared as Substrate II at 100:1, ready for immediate use. Use a vacuum pump to remove the original culture medium in the 96-well culture plate. Add 150 μL of substrate I to each well and incubate on a shaker at room temperature for 10 min. Take 120 μL of substrate I, transfer it to a 96-well microplate, and read the Firefly chemiluminescence value on a microplate reader (Tecan, Infinite 200). Then add 60 μL of substrate II to each well, incubate at room temperature on a shaker for 10 min, and read the Renilla chemiluminescence value on a microplate reader.

Data analysis and processing

The fluorescence activity is measured by a microplate reader, and the collected Renilla signals are normalized by the Firefly signal standard. The inhibitory effect of siRNA is obtained by comparing the unprocessed results (residual inhibitory activity). The calculation process is as follows:

Normalized Ren/Fir ratio: Ratio=Renilla (Renilla luciferase)/Firefly (firefly luciferase).

The remaining inhibition rate = (RatiosiRNA/Ratiocontrol) * 100%, take the average of two duplicate wells: Ratiocontrol is the Ratio value of the control well (excluding siRNA) (take the average of two duplicate wells).

Drawing: Drawing using Graphpad Prism

Half maximal inhibitory concentration (IC50): This experiment is plotted with Top and Bottom. The IC50 value is based on the formula Y=Bottom+(Top-Bottom)/(1+10^((LogIC50-X)*HillSlope)) Obtained, where Y=50, X=log (concentration).

The results of the off-target activity screening of siRNA's psiCHECK2 GSSM-5Hits are shown in Table 12, in which columns 2-12 are the remaining inhibition rates and column 13 is the IC50 value. The results show that DR000405, DR000424, DR000430, DR000442, DR000447, DR000619, and DR000621 of the present disclosure have lower off-target activity against HEK293A cells compared to the positive control DR001483.

Table 12 Screening results of psiCHECK2 GSSM-5Hits off-target activity

Example 9 Mouse HDI model activity screening

HDI animal modeling

Using high-pressure tail vein injection, six- to eight-week-old female Balb/c mice were treated with double-gene stabilized The transfection system performs in vivo transfection modeling. Piggy-Bac transposon plasmids containing target gene cDNA sequences (Genbank registration number NM_014495.2) with different mass ratios are transferred through the tail vein through a 27-gauge needle within 5-7 seconds. (purchased from Suzhou Bangye) and Piggy-Bac helper plasmid (purchased from Suzhou Bangye) (mass ratio 1:1, total plasmid amount 100ug) The delivery solution (total volume is 10% of the animal body weight, Mirusbio-MIR 5240) was injected into the mice, and after injection, they were returned to the cage for observation for 30 min. Taking the day of modeling as day 0, serum was obtained at various time points after modeling (day 7 to day 35) for detecting SEAP expression levels.

Dual-gene stable transfection system, including Piggy-Bac auxiliary plasmid and Piggy-Bac transposon plasmid, in which Piggy-Bac auxiliary plasmid provides Piggy-Bac transposase; Piggy-Bac transposon plasmid uses Piggy-Bac transposon Based on it, it contains a dual-gene expression element, which contains the secreted alkaline phosphatase gene (SEAP) and the target gene (ANGPTL3). SEAP and ANGPTL3 are co-expressed.

Detection of SEAP expression

The standard of the kit (Phospha-Light ^TM SEAP Reporter Gene Detection System, Invitrogen, T1016) was diluted 2-fold with an initial concentration of 15mU/mL to obtain 7 concentration points.

Mix CSPD substrate and reaction buffer diluent at a ratio of 1:20 to form a reaction solution. Dilute the 5× dilution buffer to 1× dilution buffer with DNase- and RNase-free distilled water. Mix the serum and 1× dilution buffer in a centrifuge tube to form a sample dilution, incubate the sample dilution at 65°C for 30 minutes, and then cool to room temperature. Add 50 μL of sample dilution into the 96-well plate, then add 50 μL of assay buffer to each well, and incubate at room temperature for 5 minutes. Add 50 μL reaction solution to each well, incubate at room temperature for 20 min, and read the SEAP chemiluminescence value on a microplate reader (Tecan, Infinite 200).

The effect of siRNA compounds on inhibiting the expression of target genes was evaluated by measuring SEAP expression levels in serum. The lower the SEAP chemiluminescence value, the better the siRNA compound is at inhibiting the expression of the target gene. Select the test sample that can inhibit the expression level of SEAP as a nucleic acid drug.

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

Table 13 Single subcutaneous administration of RNAi reagent dosage to mice

Table 14 Experimental results of HDI stably transfected plasmid mouse model

Example 10 Mouse HDI model activity screening

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

Table 15.siRNA sequences

Table 16. Experimental results of HDI stably transfected plasmid mouse model

Example 11 Mouse HDI model activity screening

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

Table 17. Sequence of siRNA

Table 18. Experimental results of HDI stably transfected plasmid mouse model

Claims

A small interfering RNA (siRNA) for inhibiting the expression of angiopoietin-like 3 (ANGPTL3) protein in cells, the siRNA comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand and the The length of the antisense strands is each independently 15-30 nucleotides, and the antisense strands comprise at least 15 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 16-29 nucleotide sequence.
The siRNA of claim 1, wherein the sense strand comprises a nucleotide sequence of at least 15 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NOs: 1-14.
The siRNA of any one of claims 1-2, wherein the length of the double-stranded region is 15-25 nucleotide pairs, preferably 17-23 nucleotide pairs, more preferably 19-21 nucleotide pairs .
The siRNA of any one of claims 1-3, wherein the antisense strand comprises a nucleotide sequence of at least 16 consecutive nucleotides of the nucleotide sequence shown in any one of SEQ ID NO: 16-29 , a nucleotide sequence of at least 17 contiguous nucleotides, a nucleotide sequence of at least 18 contiguous nucleotides, a nucleotide sequence of at least 19 contiguous nucleotides, a nucleoside of at least 20 contiguous nucleotides Acid sequence, preferably the antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NO: 16-29.
The siRNA of any one of claims 1-4, wherein the sense strand comprises a nucleotide sequence of at least 16 consecutive nucleotides with any one of the nucleotide sequences shown in SEQ ID NO: 1-14 , a nucleotide sequence of at least 17 consecutive nucleotides, a nucleotide sequence of at least 18 consecutive nucleotides, preferably the antisense strand includes the nucleoside shown in any one of SEQ ID NO: 1-14 acid sequence.
The siRNA of any one of claims 1-5, wherein the siRNA comprises paired sense strand sequences and antisense strand sequences as shown in Table 3.
The siRNA of any one of claims 1-6, wherein substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, or the sense strand All nucleotides of the strand and all nucleotides of the antisense strand are modified nucleotides.
The siRNA of claim 7, wherein the sense strand and the antisense strand each independently comprise one or more nucleotide modifications selected from the group consisting of: 2'-O-methyl modified nucleotides, 2 '-Fluoromodified nucleotides, 2'-deoxy-modified nucleotides, inosine ribonucleotides, abasic nucleotides, reverse abasic deoxyribonucleotides, phosphorothioates Internucleotide linkage modification, vinylphosphonate-modified nucleotides, locked nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino Nucleotides, phosphoramidates, non-natural bases containing nucleotides, terminal nucleotides linked to cholesteryl derivatives or dodecyl dodecylamide groups, deoxyribonucleotides and STM1.
The siRNA of claim 7, wherein the sense strand and the antisense strand each independently comprise one or more nucleotide modifications selected from the group consisting of: 2'-O-methyl modified nucleotides, 2 '-fluorinated modified nucleotides, reverse abasic deoxyribonucleotides, phosphorothioate internucleotide linkage modifications, and STM1.
siRNA according to claim 7, wherein,

(a) The chain of justice includes

CmsAmsCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAm(SEQ ID NO:31),

The antisense strand contains

UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:61);

(b) The chain of justice includes

CmsAmsAmUmUmAmAfGfCfUmCmCmUmUmCmUmUmUmUm(SEQ ID NO:32),

The antisense strand contains

AmsAfsAmAfGmAfAmGfGmAfGmCfUmUfAmAfUmUfGmsUfsGm(SEQ ID NO:62);

(c) The chain of justice includes

UmsAmsUmUmGmUmUfCfCfUmCmUmAmGmUmUmAmUmUm(SEQ ID NO:33),

The antisense strand contains

AmsAfsUmAfAmCfUmAfGmAfGmGfAmAfCmAfAmUfAmsAfsAm(SEQ ID NO:63);

(d) The chain of justice includes

GmsUmsUmAmUmUmUfCfCfUmCmCmAmGmAmAmUmUmAm(SEQ ID NO:34),

The antisense strand contains

UmsAfsAmUfUmCfUmGfGmAfGmGfAmAfAmUfAmAfCmsUfsAm(SEQ ID NO:64);

(e) The chain of justice includes

AmsGmsAmAmUmUmGfAfUfCmAmAmGmAmCmAmAmUmUm(SEQ ID NO:35),

The antisense strand contains

AmsAfsUmUfGmUfCmUfUmGfAmUfCmAfAmUfUmCfUmsGfsGm(SEQ ID NO:65);

(f) The chain of justice includes

AmsUmsCmAmAmGmAfCfAfAmUmUmCmAmUmCmAmUmUm(SEQ ID NO:36),

The antisense strand contains

AmsAfsUmGfAmUfGmAfAmUfUmGfUmCfUmUfGmAfUmsCfsAm(SEQ ID NO:66);

(g) The justice chain includes

GmsAmsGmCmCmAmAfAfAfUmCmAmAmGmAmUmUmUmAm(SEQ ID NO:37),

The antisense strand contains

UmsAfsAmAfUmCfUmUfGmAfUmUfUmUfGmGfCmUfCmsUfsGm(SEQ ID NO:67);

(h) The justice chain includes

UmsGmsAmAmCmUmCfAfAfCmUmCmAmAmAmAmCmUmUm(SEQ ID NO:38),

The antisense strand contains

AmsAfsGmUfUmUfUmGfAmGfUmUfGmAfGmUfUmCfAmsAfsGm(SEQ ID NO:68);

(i) The chain of justice includes

AmsGmsAmGmCmAmAfCfUfAmAmCmUmAmAmCmUmUmAm(SEQ ID NO:39),

The antisense strand contains

UmsAfsAmGfUmUfAmGfUmUfAmGfUmUfGmCfUmCfUmsUfsCm(SEQ ID NO:69);

(j) The chain of justice includes

GmsCmsAmAmCmUmAfAfCfUmAmAmCmUmUmAmAmUmUm(SEQ ID NO:40),

The antisense strand contains

AmsAfsUmUfAmAfGmUfUmAfGmUfUmAfGmUfUmGfCmsUfsCm(SEQ ID NO:70);

(k) The justice chain includes

CmsUmsAmAmCmUmAfAfCfUmUmAmAmUmUmCmAmAmAm(SEQ ID NO:41),

The antisense strand contains

UmsUfsUmGfAmAfUmUfAmAfGmUfUmAfGmUfUmAfGmsUfsUm(SEQ ID NO:71);

(l) The justice chain includes

CmsUmsAmAmCmUmUfAfAfUmUmCmAmAmAmUmCmAm(SEQ ID NO:42),

The antisense strand contains

UmsGfsAmUfUmUfUmGfAmAfUmUfAmAfGmUfUmAfGmsUfsUm(SEQ ID NO:72);

(m) The justice chain includes

UmsAmsAmCmUmUmAfAfUfUmCmAmAmAmAmUmCmAmAm(SEQ ID NO:43),

The antisense strand contains

AmsAfsUmAfAmCfUmAfGmAfGmGfAmAfCmAfAmUfAmsAfsAm(SEQ ID NO:73); or

(n) The justice chain includes

GmsGmsAmUmCmAmCfAfAfAmAmCmUmUmCmAmAmUmAm(SEQ ID NO:44),

The antisense strand contains

UmsAfsUmUfGmAfAmGfUmUfUmUfGmUfGmAfUmCfCmsAfsUm (SEQ ID NO:74).
The siRNA of claim 7, wherein:

(a) The chain of justice includes

UmsGmsAmAmCmUmCfAfAfCmUmCmAmAmAmAmCmUmsUm(SEQ ID NO:131),

and the antisense strand contains

AmsAfsGmUfUmUfUmGfAmGfUmUfGmAfGmUfUmCfAmsAfsGm(SEQ ID NO:132);

(b) The chain of justice includes

GmsGmsAmUmCmAmCfAfAfAmAmCmUmUmCmAmAmUmsAm(SEQ ID NO:133),

and the antisense strand contains

UmsAfsUmUfGmAfAmGfUmUfUmUfGmUfGmAfUmCfCmsAfsUm(SEQ ID NO:134);

(c) The chain of justice includes

CmsAmsCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmsAm(SEQ ID NO:135),

and the antisense strand contains

UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:136);

(d) The chain of justice includes

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

and the antisense strand contains

AmsAfsGmUfUmUfUmGfAmGfUmUfGmAfGmUfUmCfAmsAfsGm(SEQ ID NO:138);

(e) The chain of justice includes

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

and the antisense strand contains

UmsAfsUmUfGmAfAmGfUmUfUmUfGmUfGmAfUmCfCmsAfsUm(SEQ ID NO:140);

(f) The chain of justice includes

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

and the antisense strand contains

UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:142);

(g) The justice chain includes

STM1-STM1-

UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAm-STM1-STM1 (SEQ ID NO: 143),

and the antisense strand contains

UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:144); or

(h) The justice chain includes

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

and the antisense strand contains

UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm (SEQ ID NO: 146).
The siRNA of claim 7, wherein:

(a) The chain of justice includes

STM1-STM1-

UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmsAm-STM1-STM1 (SEQ ID NO: 147),

and the antisense strand contains

UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:148);

(b) The chain of justice includes

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

and the antisense strand contains

UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:150);

(c) The chain of justice includes

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

and the antisense strand contains

AmsUfsCmAfAmAfUmGfAmUfGmAfAmUfUmGfUmCfUmsUfsGm(SEQ ID NO:152);

(d) The chain of justice includes

STM1-STM1-

CmsAmAmGmAmCmAmAmUfUfCfAmUmCmAmUmUmUmGmAmsUm-STM1-STM1 (SEQ ID NO:153),

and the antisense strand contains

AmsUfsCmAfAmAfUmGfAmUfGmAfAmUfUmGfUmCfUmsUfsGm(SEQ ID NO:154);

(e) The chain of justice includes

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

and the antisense strand contains

AmsUfsCmAfAmAfUmGfAmUfGmAfAmUfUmGfUmCfUmsUfsGm(SEQ ID NO:156);

(f) The chain of justice includes

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

and the antisense strand contains

UmsCfsUmCfUmUfAmAfGmGfAmUfUmUfAmAfUmAfCmsCfsAm(SEQ ID NO:158);

(g) The justice chain includes

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

and the antisense strand contains

AmsAfsUmUfGmUfCmUfUmGfAmUfCmAfAmUfUmCfUmsGfsGm(SEQ ID NO:160); or

(h) The justice chain includes

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

and the antisense strand contains

AmsAfsUmGfAmUfGmAfAmUfUmGfUmCfUmUfGmAfUmsCfsAm (SEQ ID NO: 162).
The siRNA of any one of claims 1-12, wherein the siRNA is further conjugated with a ligand moiety comprising N-acetylgalactosamine, preferably the 3' end of the sense strand of the siRNA is conjugated with the ligand moiety. combine.
The siRNA of claim 13, wherein the ligand moiety comprises a conjugation group represented by formula (X'):

in,

Indicates the location of attachment to biomolecules;

Q is independently H,

Where 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 NHC(O)) a -;

L 2 is a chemical bond or -CH 2 CH 2 C(O)-;

L 3 is a chemical bond, -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;

L 4 is -(OCH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 CH 2 CH 2 ) c -or-NHC(O)-(CH 2 ) d -;

where a=0, 1, 2 or 3;

b=1, 2, 3, 4 or 5;

c=1, 2, 3, 4 or 5;

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

L is a chemical bond, -CH 2 O- or -NHC(O)-;

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

where M is

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;

Wherein R is -OR', -CH 2 OR' or -CH 2 CH 2 OR', wherein R' is H, hydroxyl protecting group or 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.
The siRNA of claim 14, wherein the conjugation group is represented by formula (I'):

in,

Indicates the location of attachment to biomolecules;

Q is independently H,

Where 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 NHC(O)) a -;

L 2 is a chemical bond or -CH 2 CH 2 C(O)-;

L 3 is a chemical bond, -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;

L 4 is -(OCH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 CH 2 CH 2 ) c -or-NHC(O)-(CH 2 ) 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 -CH 2 O- or -NHC(O)-;

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-, and R 3 is H;

Or R 1 and R 3 together form -C 1-2 alkylene-, and R 2 is H;

Wherein R is -OR', -CH 2 OR' or -CH 2 CH 2 OR', wherein R' is H, hydroxyl protecting group or 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.
siRNA according to claim 15, wherein,

Q is independently H or

Where L 1 is -CH 2 O- or -NHC(O)-(CH 2 NHC(O)) a -;

L 2 is -CH 2 CH 2 C(O)-;

L 3 is -(NHCH 2 CH 2 ) b - or -(NHCH 2 CH 2 CH 2 ) b -;

L 4 is -(OCH 2 CH 2 ) c - or -NHC(O)-(CH 2 ) 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 -CH 2 O-;

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;

Wherein R is -OR', -CH 2 OR' or -CH 2 CH 2 OR', wherein R' is H, hydroxyl protecting group or 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.
The siRNA of claim 16, wherein the conjugation group is represented by formula (I'-1), formula (I'-2) or formula (I'-3):

in,

Indicates the location of attachment to biomolecules;

Q is

Where L 1 is -CH 2 O- or -NHC(O)-;

L 2 is -CH 2 CH 2 C(O)-;

L 3 is -(NHCH 2 CH 2 ) b - or -(NHCH 2 CH 2 CH 2 ) b -;

L 4 is -(OCH 2 CH 2 ) c - or -NHC(O)-(CH 2 ) d -;

Where b=1, 2, 3, 4 or 5;

c=1, 2, 3, 4 or 5;

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

L is -CH 2 O-;

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;

n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
The siRNA of claim 15, wherein,

Q is independently H,

Where L 1 is -CH 2 O-, -CH 2 O-CH 2 CH 2 O- or -NHC(O)-(CH 2 NHC(O)) a -;

L 2 is -CH 2 CH 2 C(O)-;

L 3 is -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;

L 4 is -(OCH 2 CH 2 ) c - or -NHC(O)-(CH 2 ) 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 -CH 2 O- or -NHC(O)-;

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;

Wherein R is -OR', -CH 2 OR' or -CH 2 CH 2 OR', wherein R' is H, hydroxyl protecting group or 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.
The siRNA of claim 18, wherein the conjugation group is represented by formula (II'-1) or formula (II'-2):

in,

Indicates the location of attachment to biomolecules;

Q is independently

Where L 1 is -CH 2 O- or -CH 2 O-CH 2 CH 2 O-;

L 3 is -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;

L 4 is -(OCH 2 CH 2 ) c - or -NHC(O)-(CH 2 ) d -;

Where b=1, 2, 3, 4 or 5;

c=1, 2, 3, 4 or 5;

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

L is -NHC(O)-;

L’ is a chemical bond or -C(O)NH-;

R' is H, a hydroxyl protecting group or a solid phase 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.
The siRNA of claim 15, wherein,

Q is independently H,

where L 1 is -CH 2 -, -C(O)-, -CH 2 O-, -CH 2 O-CH 2 CH 2 O- or -NHC(O)-(CH 2 NHC(O)) a - ;

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 -;

L 4 is -(OCH 2 CH 2 ) c - or -NHC(O)-(CH 2 ) 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 -CH 2 O- or -NHC(O)-;

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;

Wherein R is -OR', -CH 2 OR' or -CH 2 CH 2 OR', wherein R' is H, hydroxyl protecting group or 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.
The siRNA of claim 20, wherein the conjugation group is represented by formula (II'-2):

in,

Indicates the location of attachment to biomolecules;

Q is independently

Where L 1 is -CH 2 - or -C(O)-;

L 3 is -(NHCH 2 CH 2 ) b -;

L 4 is -(OCH 2 CH 2 ) c -;

Where b=1, 2, 3, 4 or 5;

c=1, 2, 3, 4 or 5;

L is -CH 2 O- or -NHC(O)-;

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;

n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
The siRNA of claim 14, wherein:

Q is independently H,

Where 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 NHC(O)) a -;

L 2 is a chemical bond or -CH 2 CH 2 C(O)-;

L 3 is a chemical bond, -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;

L 4 is -(OCH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 CH 2 CH 2 ) c -or-NHC(O)-(CH 2 ) d -;

where a=0, 1, 2 or 3;

b=1, 2, 3, 4 or 5;

c=1, 2, 3, 4 or 5;

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

L is a chemical bond, -CH 2 O- or -NHC(O)-;

L' is a chemical bond, -C(O)NH-, -NHC(O)- or -O(CH 2 CH 2 O) e -;

where e is 1, 2, 3, 4 or 5;

T is a chemical bond, -CH 2 -, -M-, -CH 2 -M- or -C(O)-M-;

where M is

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;

Wherein R is -OR', -CH 2 OR' or -CH 2 CH 2 OR', wherein R' is H, hydroxyl protecting group or 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.
The siRNA of claim 22, wherein,

T is -M-, -CH 2 -M- or -C(O)-M-, where M is
The siRNA of claim 22 or 23, wherein,

Q is independently H or

Where L 1 is -CH 2 O- or -NHC(O)-(CH 2 NHC(O)) a -;

L 2 is -CH 2 CH 2 C(O)-;

L 3 is -(NHCH 2 CH 2 ) b - or -(NHCH 2 CH 2 CH 2 ) b -;

L 4 is -(OCH 2 CH 2 ) c - or -NHC(O)-(CH 2 ) d -;

where a=0, 1, 2 or 3;

b=1, 2, 3, 4 or 5;

c=1, 2, 3, 4 or 5;

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

L is a chemical bond or -CH 2 O-;

L' is a chemical bond or -O(CH 2 CH 2 O) e -;

where 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;

Wherein R is -OR', -CH 2 OR' or -CH 2 CH 2 OR', wherein R' is H, hydroxyl protecting group or 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 22 or 23.
The siRNA of claim 24, wherein the conjugation group is represented by formula (III'-1), formula (III'-2) or formula (III'-3):

in,

Q is

Where L 1 is -CH 2 O- or -NHC(O)-;

L 2 is -CH 2 CH 2 C(O)-;

L 3 is -(NHCH 2 CH 2 ) b - or -(NHCH 2 CH 2 CH 2 ) b -;

L 4 is -(OCH 2 CH 2 ) c - or -NHC(O)-(CH 2 ) d -;

Where b=1, 2, 3, 4 or 5;

c=1, 2, 3, 4 or 5;

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

L is a chemical bond or -CH 2 O-;

Wherein 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;

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

wherein T is as defined in claim 33 or 34.
The siRNA of claim 22 or 23, wherein,

Q is independently H,

Where L 1 is -CH 2 -, -CH 2 O- or -C(O)-;

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 -;

L 4 is -(OCH 2 CH 2 ) c - or -NHC(O)-(CH 2 ) d -;

Where b=1, 2, 3, 4 or 5;

c=1, 2, 3, 4 or 5;

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

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

L’ is a chemical bond;

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;

Wherein R is -OR', -CH 2 OR' or -CH 2 CH 2 OR', wherein R' is H, hydroxyl protecting group or 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 22 or 23.
The siRNA of claim 22 or 23, wherein the conjugation group is represented by formula (IV-1) or formula (IV-2):

in,

Q is independently

Where L 1 is -CH 2 -, -CH 2 O- or -C(O)-;

L 3 is -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;

L 4 is -(OCH 2 CH 2 ) c - or -NHC(O)-(CH 2 ) d -;

Where b=1, 2, 3, 4 or 5;

c=1, 2, 3, 4 or 5;

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

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

L’ is a chemical bond;

Wherein R' is H, a hydroxyl protecting group or a solid phase 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 22 or 23.
The siRNA of claim 14, wherein:

Q is independently H,

Where 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 NHC(O)) a -;

L 2 is a chemical bond or -CH 2 CH 2 C(O)-;

L 3 is a chemical bond, -(NHCH 2 CH 2 ) b -, -(NHCH 2 CH 2 CH 2 ) b - or -C(O)CH 2 -;

L 4 is -(OCH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 CH 2 ) c -, -(OCH 2 CH 2 CH 2 CH 2 CH 2 ) c -or-NHC(O)-(CH 2 ) d -;

where a=0, 1, 2 or 3;

b=1, 2, 3, 4 or 5;

c=1, 2, 3, 4 or 5;

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

L is a chemical bond, -CH 2 O- or -NHC(O)-;

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-;

where M is

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;

Wherein R is -OR', -CH 2 OR' or -CH 2 CH 2 OR', wherein R' is H, hydroxyl protecting group or 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.
The siRNA of claim 14, wherein said conjugation group is selected from the following:
The siRNA of claim 14, wherein said conjugation group is selected from the following:
The siRNA of any one of claims 13-30, wherein the ligand targets the asialoglycoprotein receptor (ASGPR).
The siRNA of claim 13, wherein said ligand has the following structure:

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

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

in Indicates the position connected to the sense strand of the siRNA via a phosphate group or a phosphorothioate group.
The siRNA of claim 13, wherein

(a) The chain of justice includes

UmsGmsAmAmCmUmCfAfAfCmUmCmAmAmAmAmCmUmsUms-GL6 (SEQ ID NO:91),

The antisense strand contains

AmsAfsGmUfUmUfUmGfAmGfUmUfGmAfGmUfUmCfAmsAfsGm(SEQ ID NO:92);

(b) The chain of justice includes

GmsGmsAmUmCmAmCfAfAfAmAmCmUmUmCmAmAmUmsAms-GL6 (SEQ ID NO:93),

The antisense strand contains

UmsAfsUmUfGmAfAmGfUmUfUmUfGmUfGmAfUmCfCmsAfsUm(SEQ ID NO:94);

(c) The chain of justice includes

CmsAmsCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmsAms-GL6 (SEQ ID NO:95),

The antisense strand contains

UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:96);

(d) The chain of justice includes

STM1s-CmsUmUmGmAmAmCmUmCfAfAfCmUmCmAmAmAmAmCmUmUms-STM1s-GL6 (SEQ ID NO:97),

The antisense strand contains

AmsAfsGmUfUmUfUmGfAmGfUmUfGmAfGmUfUmCfAmsAfsGm(SEQ ID NO:98);

(e) The chain of justice includes

STM1s-AmsUmGmGmAmUmCmAmCfAfAfAmAmCmUmUmCmAmAmUmAms-STM1s-GL6 (SEQ ID NO:99),

The antisense strand contains

UmsAfsUmUfGmAfAmGfUmUfUmUfGmUfGmAfUmCfCmsAfsUm(SEQ ID NO:100);

(f) The chain of justice includes

STM1s-UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAms-STM1s-GL6 (SEQ ID NO:101),

The antisense strand contains

UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:102);

(g) The justice chain includes

STM1-STM1-

UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAm-STM1-STM1-GL6 (SEQ ID NO: 103),

The antisense strand contains

UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:104); or

(h) The justice chain includes

IBs-UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAms- GL6(SEQ ID NO:105),

The antisense strand contains

UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm (SEQ ID NO: 106).
The siRNA of claim 13, wherein

(a) The chain of justice includes

STM1s-UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAms-STM1s-GL6 (SEQ ID NO: 107),

The antisense strand contains

UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:108);

(b) The chain of justice includes

STM1-STM1-

UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmsAm-STM1-STM1s-GL6(SEQ ID NO:109),

The antisense strand contains

UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:110);

(c) The chain of justice includes

STM1s-UmsCmCmAmCmGmUmUmGfCfUfUmGmAmAmAmUmUmGmAmAm-STM1-STM1s-GL6 (SEQ ID NO: 111),

The antisense strand contains

UmsUfsCmAfAmUfUmUfCmAfAmGfCmAfAmCfGmUfGmsGfsAm(SEQ ID NO:112);

(d) The chain of justice includes

STM1s-CmsAmAmGmAmCmAmAmUfUfCfAmUmCmAmUmUmUmGmAmUms-STM1s-GL6 (SEQ ID NO: 113),

The antisense strand contains

AmsUfsCmAfAmAfUmGfAmUfGmAfAmUfUmGfUmCfUmsUfsGm(SEQ ID NO:114);

(e) The chain of justice includes

STM1-STM1-

CmsAmAmGmAmCmAmAmUfUfCfAmUmCmAmUmUmUmGmAmsUm-STM1-STM1s-GL6 (SEQ ID NO:115),

The antisense strand contains

AmsUfsCmAfAmAfUmGfAmUfGmAfAmUfUmGfUmCfUmsUfsGm(SEQ ID NO:116);

(f) The chain of justice includes

STM1s-CmsAmAmGmAmCmAmAmUfUfCfAmUmCmAmUmUmUmGmAmUm-STM1-STM1s-GL6 (SEQ ID NO: 117),

The antisense strand contains

AmsUfsCmAfAmAfUmGfAmUfGmAfAmUfUmGfUmCfUmsUfsGm(SEQ ID NO:118);

(g) The justice chain includes

STM1s-UmsGmGmUmAmUmUmAmAfAfUfCmCmUmUmAmAmGmAmGmAms- STM1s-GL6 (SEQ ID NO:119),

The antisense strand contains

UmsCfsUmCfUmUfAmAfGmGfAmUfUmUfAmAfUmAfCmsCfsAm(SEQ ID NO:120);

(h) The justice chain includes

STM1s-CmsCmAmGmAmAmUmUmGfAfUfCmAmAmGmAmCmAmAmUmUms-STM1s-GL6 (SEQ ID NO: 121),

The antisense strand contains

AmsAfsUmUfGmUfCmUfUmGfAmUfCmAfAmUfUmCfUmsGfsGm(SEQ ID NO:122); or

(i) The chain of justice includes

STM1s-UmsGmAmUmCmAmAmGmAfCfAfAmUmUmCmAmUmCmAmUmUms-STM1s-GL6(SEQ ID NO:123),

The antisense strand contains

AmsAfsUmGfAmUfGmAfAmUfUmGfUmCfUmUfGmAfUmsCfsAm (SEQ ID NO: 124).
Cells containing the siRNA of any one of claims 1-36.
A pharmaceutical composition comprising the siRNA according to any one of claims 1 to 36, or the cell according to claim 37, and optionally a pharmaceutically acceptable carrier or excipient.
A kit comprising the siRNA according to any one of claims 1-36, the cell according to claim 37, or the pharmaceutical composition according to claim 38.
A method of treating a disease or symptom associated with ANGPTL3 expression in a subject, the method comprising administering to the subject the siRNA of any one of claims 1-36, the cell of claim 37, or as 38. The step of the pharmaceutical composition of claim 38.
The method of claim 40, wherein the disease or condition associated with ANGPTL3 expression is a metabolic disease or a cardiovascular disease.
The method of claim 41, wherein the metabolic disease or cardiovascular disease is obesity, diabetes, atherosclerosis, dyslipidemia, coronary heart disease, non-alcoholic fatty liver disease (NAFLD), hyperlipidemia, or Metabolic syndrome or combination thereof.
The method of claim 42, wherein said dyslipidemia is hyperlipidemia.
The method of claim 43, wherein the hyperlipidemia is hypercholesterolemia, hypertriglyceridemia, or a combination thereof.
The method of claim 42, wherein said NAFLD is hepatic steatosis or steatohepatitis.
The method of claim 42, wherein the diabetes is type 2 diabetes or type 2 diabetes with dyslipidemia.
A method of reducing ANGPTL3 levels, LDL levels, apoC-III levels, triglyceride levels, cholesterol levels, glucose levels, and fat pad weight in a subject, the method comprising administering to the subject claims 1- The siRNA of any one of 36, the cell of claim 37, or the pharmaceutical composition of claim 38.
The method of any one of claims 40-47, wherein administering the siRNA, cell or pharmaceutical composition to the subject comprises subcutaneous administration, topical administration or intravenous administration.