Sirna for inhibiting inhbe gene expression, conjugate, and use thereof

Abstract

Provided are a siRNA for inhibiting INHBE gene expression, a conjugate, and a use thereof. Provided are a set of siRNAs for inhibiting INHBE gene expression, derivative sequences comprising nucleotide modifications, and siRNA-targeting ligand conjugates. Also provided are uses of the siRNAs, the derivative sequences, and the conjugates.

Description

siRNA, conjugate and application for inhibiting expression of INHBE gene TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine. Specifically, the present application relates to siRNA, conjugate and application for inhibiting expression of INHBE gene. BACKGROUND

[0002] Inhibin subunit beta E (INHBE), also known as Activin E, is a member of the transforming growth factor-beta (TGF-beta) superfamily, which is highly specifically expressed in hepatocytes. Through gene analysis of whole-exome sequencing data of 362679 individuals, it was found that 12 genes were significantly associated with waist-to-hip ratio adjusted based on BMI, indicating that Activin is likely to be involved in fat regulation pathway. After Activin E is secreted and matured, it can activate the Smad pathway by binding to the surface receptor of adipocytes. Further studies have shown that INHBE is likely to be a liver factor that changes the whole body metabolic state under the conditions of obesity and insulin resistance. Its liver expression level is positively correlated with human insulin resistance and body mass index.

[0003] The demand for effective treatment of metabolic diseases, such as metabolic syndrome, diabetes, hypertension and cardiovascular-related diseases, has not been met. As a liver-specific expression fat storage negative regulatory factor, the development of RNAi drugs to inhibit the expression of INHBE becomes a potential advantage strategy for treating metabolic diseases related to improper fat distribution and storage. SUMMARY

[0004] The purpose of the present application is to provide a siRNA capable of inhibiting the expression and / or activity of INHBE gene, for treating INHBE-related diseases, such as metabolic syndrome, diabetes, hypertension and cardiovascular diseases related to the expression of INHBE gene.

[0005] In a first aspect of the present application, a siRNA for inhibiting the expression of inhibin subunit beta E (INHBE) gene is provided, the siRNA comprising a sense strand and an antisense strand,

[0006] wherein the antisense strand comprises at least 19 contiguous nucleotides differing by 0, 1, 2 or 3 nucleotides from any of the sequences shown in Table 1;

[0007] the sense strand is complementary to the antisense strand by at least 15, 16, 17, 18, 19, 20, 21 or 22 nucleotides;

[0008] wherein each nucleotide of the sense strand and the antisense strand is independently a modified or unmodified nucleotide.

[0009] In another preferred embodiment, the antisense strand comprises at least 21 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from any of the sequences set forth in Table 1;

[0010] The sense strand is complementary to the antisense strand by at least 15, 16, 17, 18, 19, 20, 21, or 22 nucleotides.

[0011] In another preferred embodiment, the antisense strand comprises at least 19, 20, 21, 22, 23 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from any of the sequences set forth in Table 1.

[0012] In another preferred embodiment, the antisense strand comprises at least 21 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from any of the sequences set forth in Table 1.

[0013] In another preferred embodiment, the antisense strand comprises at least 19, 20, 21, or 22 (preferably 21) contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the nucleotide sequence set forth in any of SEQ ID NOs: 42-82, 221-235, 326-370, 627-747, 1172-1175, and 1187.

[0014] In another preferred embodiment, the antisense strand comprises at least 19, 20, 21, or 22 (preferably 21) contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the nucleotide sequence set forth in any of SEQ ID NOs: 42-53, 55-58, 60-67, 70-75, 80-82, 221-235, 326, 328-332, 336-339, 341-370, 627-659, 661-738, 740-747, 1172-1175, and 1187.

[0015] In another preferred embodiment, the antisense strand is missing 2 nucleotides at the 3' end relative to the nucleotide sequence set forth in any of SEQ ID NOs: 42-82, 221-235, and 326-370, 627-747, 1172-1175, and 1187.

[0016] In another preferred embodiment, the sense strand comprises at least 19, 20, 21, or 22 (preferably 21) contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the nucleotide sequence set forth in any of SEQ ID NOs: 1-41, 206-220, 281-325, 506-626, 1169-1171, and 1186.

[0017] In another preferred embodiment, the sense strand comprises at least 19, 20, 21, or 22 (preferably 21) contiguous nucleotides that differ by 0, 1, 2, or 3 nucleotides from the nucleotide sequence set forth in any one of SEQ ID NOs: 1-12, 14-17, 19-26, 29-34, 39-41, 206-220, 281, 283-287, 291-294, 296-325, 506-586, 588-626, 1169-1171, and 1186.

[0018] In another preferred embodiment, the antisense strand is 21-23 base matches complementary to the human INHBE transcript (NM_031479.5).

[0019] In another preferred embodiment, the sense strand and the antisense strand are each independently 21, 22, or 23 nucleotides in length.

[0020] In another preferred embodiment, the antisense strand is missing 2 nucleotides at the 3’ end relative to the sense strand.

[0021] In another preferred embodiment, the nucleotide sequence of the antisense strand is set forth in any one of SEQ ID NOs: 42-82, 221-235, 326-370, 627-747, 1172-1175, and 1187.

[0022] In another preferred embodiment, the antisense strand is set forth in any one of SEQ ID NOs: 42-53, 55-58, 60-67, 70-75, 80-82, 221-235, 326, 328-332, 336-339, 341-370, 627-659, 661-738, 740-747, 1172-1175, and 1187.

[0023] In another preferred embodiment, the nucleotide sequence of the sense strand is set forth in any one of SEQ ID NOs: 1-41, 206-220, and 281-325, 506-626, 1169-1171, and 1186.

[0024] In another preferred embodiment, the sense strand is set forth in any one of SEQ ID NOs: 1-12, 14-17, 19-26, 29-34, 39-41, 206-220, 281, 283-287, 291-294, 296-325, 506-586, 588-626, 1169-1171, and 1186.

[0025] In another preferred embodiment, at least one nucleotide in the sense strand and the antisense strand is a modified nucleotide.

[0026] In another preferred embodiment, the modified nucleotides are selected from: 2'-0-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxynucleotides, 2'-deoxy-2'-fluoro modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, 2'-alkoxy modified nucleotides, phosphorothioate modified nucleotides, abasic nucleotides, morpholino nucleotides, locked nucleotides, or combinations thereof.

[0027] In another preferred embodiment, the modified nucleotides are selected from: 2'-0-methyl modified nucleotides, 2'-fluoro modified nucleotides, phosphorothioate modified nucleotides, or combinations thereof.

[0028] In another preferred embodiment, the modified nucleotides comprise modifications selected from the group consisting of:

[0029] (1) from the 5' end to the 3' end direction, the 9th, 11th, 13th nucleotides of the sense strand are 2'-fluoro modified nucleotides, and the nucleotides at the remaining positions are 2'-0-methyl modified nucleotides;

[0030] (2) from the 5' end to the 3' end direction, the 2nd, 7th, 12th, 14th, 16th nucleotides of the antisense strand are 2'-fluoro modified nucleotides, and the nucleotides at the remaining positions are 2'-0-methyl modified nucleotides;

[0031] (3) the 5' end and the 3' end of the sense strand independently comprise 0, 1, 2, or 3 phosphorothioate modified nucleotides, respectively;

[0032] (4) the 5' end and the 3' end of the antisense strand independently comprise 0, 1, 2, or 3 phosphorothioate modified nucleotides, respectively;

[0033] (5) any combination of (1)-(4) above.

[0034] In another preferred embodiment, the 1st nucleotide at the 5' end of the antisense strand is a 5'-phosphonucleotide or a 5'-phosphonucleotide analogue modified nucleotide.

[0035] In another preferred embodiment, the antisense strand is directly or indirectly linked to the sense strand via a phosphonucleotide, a phosphorothioate, or a phosphorodithioate.

[0036] In another preferred embodiment, the 1st nucleotide at the 5' end of the antisense strand is a nucleotide selected from the group consisting of:

[0037] wherein B is adenosine-3'-phosphate (A), and n is 2'-0-methyl adenosine-3'-phosphate (a); or

[0038] B is uridine 3 '-phosphate (U), and n is 2'-O-methyluridine-3 '-phosphate (u).

[0039] In another preferred embodiment, the 1st nucleotide at the 5' terminus of the antisense strand is A.

[0040] In another preferred embodiment, the 1st and 2nd nucleotides at the 5' terminus of the antisense strand are connected by a phosphodiester linkage or a phosphorothioate linkage.

[0041] In another preferred embodiment, the antisense strand comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 124-164, 251-265, 416-460, 869-989, 1179-1182, and 1189.

[0042] In another preferred embodiment, the sense strand comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 83-123, 236-250, 371-415, 748-868, 1176-1178, and 1188.

[0043] In another preferred embodiment, the antisense strand comprises or consists of the nucleotide sequence set forth in SEQ ID NO: (m1+41); and the sense strand comprises or consists of the nucleotide sequence set forth in SEQ ID NO: ml; wherein ml is any integer number between 1-41 or 83-123; or

[0044] the antisense strand comprises or consists of the nucleotide sequence set forth in SEQ ID NO: (m2+15); and the sense strand comprises or consists of the nucleotide sequence set forth in SEQ ID NO: m2; wherein m2 is any integer number between 206-220 or 236-250; or

[0045] the antisense strand comprises or consists of the nucleotide sequence set forth in SEQ ID NO: (m3+45); and the sense strand comprises or consists of the nucleotide sequence set forth in SEQ ID NO: m3; wherein m3 is any integer number between 281-325 or 371-415; or

[0046] the antisense strand comprises or consists of the nucleotide sequence set forth in SEQ ID NO: (m4+121); and the sense strand comprises or consists of the nucleotide sequence set forth in SEQ ID NO: m4; wherein m4 is any integer number between 506-626 or 748-868; or

[0047] the antisense strand comprises or consists of a nucleotide sequence as set forth in SEQ ID NO: (m5+3); and the sense strand comprises or consists of a nucleotide sequence as set forth in SEQ ID NO: m5; wherein m5 is any integer number from 1169-1171 or 1176-1178; or

[0048] the antisense strand comprises or consists of a nucleotide sequence as set forth in SEQ ID NO: 1182; and the sense strand comprises or consists of a nucleotide sequence as set forth in SEQ ID NO: 1176; or

[0049] the antisense strand comprises or consists of a nucleotide sequence as set forth in SEQ ID NO: 1182; and the sense strand comprises or consists of a nucleotide sequence as set forth in SEQ ID NO: 1176; or

[0050] the antisense strand comprises or consists of a nucleotide sequence as set forth in SEQ ID NO: 1187; and the sense strand comprises or consists of a nucleotide sequence as set forth in SEQ ID NO: 1186; or

[0051] the antisense strand comprises or consists of a nucleotide sequence as set forth in SEQ ID NO: 1189; and the sense strand comprises or consists of a nucleotide sequence as set forth in SEQ ID NO: 1188.

[0052] In another preferred embodiment, m1 is any integer number from 1-12, 14-17, 19-26, 29-34, 39-41 or 83-123.

[0053] In another preferred embodiment, m3 is any integer number from 281, 283-287, 291-294, 296-325 or 371-415.

[0054] In another preferred embodiment, m4 is any integer number from 506-586 or 588-626.

[0055] In another preferred embodiment, the sense strand further comprises an inverted abasic residue (invAb) at the 3' end and / or 5' end of the nucleotide sequence.

[0056] In a second aspect of the present application, there is provided an siRNA conjugate comprising an siRNA as described in the first aspect of the present application and a targeting ligand.

[0057] In another preferred embodiment, the targeting ligand is conjugated to the 5' end or 3' end of the sense strand of the siRNA.

[0058] In another preferred embodiment, the conjugate further comprises a linker, and the siRNA, the linker, and the targeting ligand are covalently or non-covalently linked in sequence.

[0059] In another preferred embodiment, the linker is a C6NH2(1-amino-n-hexyloxy) linker.

[0060] In another preferred embodiment, the siRNA is linked to the targeting group via a phosphorothioate linkage.

[0061] In another preferred embodiment, the targeting ligand is one or more of any cell targeting moiety, preferably a lipid, a carbohydrate, an aptamer, a vitamin, and / or a peptide that binds to a specific target on the cell membrane or cell surface.

[0062] In another preferred embodiment, the targeting ligand contains a group from a carbohydrate, including a monosaccharide, a disaccharide, a trisaccharide, or a polysaccharide.

[0063] In another preferred embodiment, the targeting ligand contains a monosaccharide selected from the group consisting of D-fucitol, L-fucitol, fucosamine, fucose, galactosamine, D-galactosaminitol, N-acetylgalactosamine (GalNAc), and galactose.

[0064] In another preferred embodiment, the targeting ligand has a structure selected from any one of the following formulae (I)-(XII):

[0065] wherein, represents a point of attachment to the siRNA or the linker.

[0066] In another preferred embodiment, the targeting ligand is attached to the 3' end of the sense strand of the siRNA, and the targeting ligand has a structure selected from any one of the following formulae (I)-(XII).

[0067] In another preferred embodiment, the targeting ligand is attached to the 5' end of the sense strand of the siRNA, and the targeting ligand has a structure selected from any one of the following formulae (I)-(XII).

[0068] In another preferred embodiment, the targeting ligand is attached to the 5' end of the sense strand of the siRNA, and the targeting ligand has a structure selected from any one of the following formulae (I)-(XII).

[0069] In another preferred embodiment, the antisense strand of the conjugate comprises or consists of a nucleotide sequence as set forth in any one of SEQ ID NOs: 124-164, 251-265, 416-460, 869-989, 1140-1168, 1179-1182, and 1189.

[0070] In another preferred embodiment, the antisense strand of the conjugate comprises or consists of a nucleotide sequence as set forth in any one of SEQ ID NOs: 124-164, 251-265, 416-460, 869-989, 1140-1168, 1179-1182, and 1189.

[0071] In another preferred embodiment, the antisense strand of the conjugate comprises or consists of a nucleotide sequence as set forth in any one of SEQ ID NOs: 124-164, 251-265, 416-460, 869-989, 1140-1168, 1179-1182, and 1189.

[0072] the antisense strand comprises or consists of a nucleotide sequence as set forth in SEQ ID NO: p2, and its sense strand comprises or consists of a nucleotide sequence as set forth in SEQ ID NO: (p2+15); wherein p2 is any integer number in 251-265; or

[0073] the antisense strand comprises or consists of a nucleotide sequence as set forth in SEQ ID NO: p3, and its sense strand comprises or consists of a nucleotide sequence as set forth in SEQ ID NO: (p3+45); wherein p3 is any integer number in 416-460; or

[0074] the antisense strand comprises or consists of a nucleotide sequence as set forth in SEQ ID NO: p4, and its sense strand comprises or consists of a nucleotide sequence as set forth in SEQ ID NO: (p4+121); wherein p4 is any integer number in 869-989; or

[0075] the antisense strand comprises or consists of a nucleotide sequence as set forth in SEQ ID NO: p5, and its sense strand comprises or consists of a nucleotide sequence as set forth in SEQ ID NO: (p5-29); wherein p5 is any integer number in 1140-1168; or

[0076] the antisense strand comprises or consists of a nucleotide sequence as set forth in SEQ ID NO: p6, and its sense strand comprises or consists of a nucleotide sequence as set forth in SEQ ID NO: (p6+4); wherein p6 is any integer number in 1179-1181; or

[0077] the antisense strand comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 1182, and the sense strand thereof comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 1183; or

[0078] the antisense strand comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 1189, and the sense strand thereof comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 1190.

[0079] In a third aspect of the present application, there is provided an expression vector comprising a polynucleotide encoding the siRNA as described in the first aspect of the present application.

[0080] In a fourth aspect of the present application, there is provided a pharmaceutical composition comprising:

[0081] (a) the siRNA as described in the first aspect of the present application, and / or the conjugate as described in the second aspect of the present application, or the expression vector as described in the third aspect of the present application; and

[0082] (b) a pharmaceutically acceptable carrier.

[0083] In another preferred embodiment, the pharmaceutical composition is used for inhibiting the expression of the INHBE gene.

[0084] In a fifth aspect of the present application, there is provided use of the siRNA as described in the first aspect of the present application, the conjugate as described in the second aspect of the present application, the expression vector as described in the third aspect of the present application, or the pharmaceutical composition as described in the fourth aspect of the present application in the preparation of a medicament for preventing and / or treating an INHBE-mediated disease.

[0085] In another preferred embodiment, the medicament is used for inhibiting the expression of the INHBE gene.

[0086] In another preferred embodiment, the INHBE-mediated disease is a disease of overexpression of INHBE.

[0087] In another preferred embodiment, the disease is a metabolic system disease or a cardiovascular disease.

[0088] In another preferred embodiment, the metabolic system disease comprises diabetes, lipid metabolism disorder.

[0089] In another preferred embodiment, the diabetes comprises pre-diabetes, type I diabetes, type II diabetes.

[0090] In another preferred embodiment, the lipid metabolism disorder comprises weight disorder, triglyceride elevation, fatty liver disease, hypercholesterolemia, fatty liver disease, or non-alcoholic steatohepatitis.

[0091] In another preferred embodiment, the cardiovascular disease comprises: hypertension, atherosclerosis, cardiomyopathy, heart failure.

[0092] In a sixth aspect of the present application, there is provided a method for inhibiting expression of INHBE in a cell in vitro, the method comprising the steps of:

[0093] co-culturing an effective amount of the siRNA as described in the first aspect of the present application, the conjugate as described in the second aspect of the present application, the expression vector as described in the third aspect of the present application, or the pharmaceutical composition as described in the fourth aspect of the present application with the cell.

[0094] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0095] In another preferred embodiment, the cell is a cell expressing INHBE gene, such as a hepatocyte.

[0096] In a seventh aspect of the present application, there is provided a method for treating a disease mediated by INHBE, comprising the step of: administering a therapeutically effective amount of the siRNA as described in the first aspect of the present application, the conjugate as described in the second aspect of the present application, the expression vector as described in the third aspect of the present application, or the pharmaceutical composition as described in the fourth aspect of the present application to a subject.

[0097] In another preferred embodiment, the disease mediated by INHBE is a disease of overexpression of INHBE.

[0098] In another preferred embodiment, the disease is a disease of metabolic system or a cardiovascular disease.

[0099] In another preferred embodiment, the disease of metabolic system comprises: diabetes, a disorder of lipid metabolism.

[0100] In another preferred embodiment, the diabetes comprises: pre-diabetes, such as type I diabetes, type II diabetes.

[0101] In another preferred embodiment, the disorder of lipid metabolism comprises: body weight disorder, elevated triglyceride, fatty liver disease, hypercholesterolemia, fatty liver disease, or nonalcoholic steatohepatitis.

[0102] In another preferred embodiment, the cardiovascular disease comprises: hypertension, atherosclerosis, cardiomyopathy, heart failure.

[0103] In another preferred embodiment, the subject is a human or a non-human mammal.

[0104] In an eighth aspect of the present application, there is provided the siRNA of the first aspect of the present application, the conjugate of the second aspect of the present application, the expression vector of the third aspect of the present application, or the pharmaceutical composition of the fourth aspect of the present application, for use in the treatment of an INHBE-mediated disease.

[0105] In another preferred embodiment, the INHBE-mediated disease is a disease of overexpression of INHBE.

[0106] In another preferred embodiment, the disease is a metabolic system disease or a cardiovascular disease.

[0107] In another preferred embodiment, the metabolic system disease comprises diabetes, lipid metabolism disorder.

[0108] In another preferred embodiment, the diabetes comprises pre-diabetes, type I diabetes, type II diabetes.

[0109] In another preferred embodiment, the lipid metabolism disorder comprises weight disorder, elevated triglyceride, fatty liver disease, hypercholesterolemia, fatty liver disease, or nonalcoholic steatohepatitis.

[0110] In another preferred embodiment, the cardiovascular disease comprises hypertension, atherosclerosis, cardiomyopathy, heart failure.

[0111] In another preferred embodiment, the subject is a human or a non-human mammal.

[0112] It should be understood that, within the scope of the present application, all combinations of the above-described technical features of the present application and the technical features specifically described hereinafter (e.g., in the examples) can be interchanged, thereby constituting new or preferred technical solutions. Due to the limited space, they are not listed one by one here. DETAILED DESCRIPTION

[0113] Terminology

[0114] For the purposes of the present application, certain technical and scientific terms are specifically defined below. Unless specifically defined herein, all other technical and scientific terms used have the meaning commonly understood by one of ordinary skill in the art to which the present application pertains. Before the present application is described, it is to be understood that this application is not limited to the particular methodology and experimental conditions described, as such methodology and conditions can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present application will be limited only by the appended claims.

[0115] As used herein, the term "about," when used in reference to a numerically recited value, means that the value can vary from the recited value by not more than 1%. For example, as used herein, the expression "about 100" includes all values between 99 and 101, and so on (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0116] As used herein, the terms "comprising," "including," "containing," and variations thereof, are interchangeable and mean that the composition or method described includes, but is not limited to, those elements specifically named.

[0117] As used herein, the term "pharmaceutically acceptable carrier" refers to a material that is suitable for use with humans and / or animals without undue adverse side effects such as toxicity, irritation, and allergic response, commensurate with a reasonable benefit / risk ratio.

[0118] As used herein, the term "therapeutically effective amount" refers to an amount that produces functional or therapeutic effects for which it is administered, and which can be accepted by a human and / or animal. It will be understood by one of ordinary skill in the art that the "therapeutically effective amount" can vary depending on the form of the pharmaceutical composition, the route of administration, the excipient used, the severity of the disease, and the combination with other drugs, and the like.

[0119] INHBE

[0120] The INHBE gene corresponds to a transcript mRNA with accession number NM_031479.5, which is 2460 bp in length, including a 5'-UTR region of 230 bp, a CDS region of 1053 bp, and a 3'-UTR region of 1178 bp. The protein Activin E encoded by this mRNA belongs to the TGF-β superfamily, in which the Activin subgroup includes four human Activin proteins. Activin E is expressed in the form of a dimeric precursor protein, which is further hydrolyzed into a mature protein form, binds to the receptor, and is believed to be associated with fat storage and cardiovascular disease risk.

[0121] siRNA

[0122] The term "interfering RNA" or "RNAi" or "interfering RNA sequence" includes single-stranded RNA (e.g., mature miRNA, ssRNAi oligonucleotide, ssDNAi oligonucleotide) or double-stranded RNA (i.e., duplex RNA such as siRNA, dsRNA, shRNA, aiRNA, or pre-miRNA) that is capable of reducing or inhibiting the expression of a target gene or sequence (e.g., by mediating degradation and inhibiting translation of mRNA complementary to the interfering RNA sequence) when the interfering RNA is in the same cell as the target gene or sequence. Interfering RNA thus refers to single-stranded RNA complementary to a target mRNA sequence or double-stranded RNA formed from two complementary strands or from a single self-complementary strand.

[0123] Interfering RNA includes "small interfering RNA" or "siRNA," each strand of which comprises about 15 to about 60 nucleotides (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length). The ranges of the above recited ranges and lengths are also contemplated as part of the present application. In a particular embodiment, the siRNA is chemically synthesized. The siRNA of the present application is capable of silencing the expression of a target sequence in vitro and / or in vivo. In other embodiments, the siRNA comprises at least one modified nucleotide, e.g., the siRNA comprises one, two, three, four, five, six, seven, eight, nine, ten or more modified nucleotides in the double-stranded region.

[0124] siRNA molecules mediate the targeted cleavage of RNA transcripts through an RNA-induced silencing complex (RISC) pathway. iRNA directs the sequence-specific degradation of mRNA via a process known as RNA interference (RNAi). The iRNA modulates (e.g., inhibits) the expression of INHBE in a cell (e.g., a cell within a subject such as a mammalian subject). Typically, a majority of the nucleotides of each strand of the siRNA are ribonucleotides, but as detailed herein, each or both of the two strands can also include one or more non-ribonucleotides, e.g., deoxyribonucleotides and / or modified nucleotides. Also, as used in the description, "siRNA" can include ribonucleotides with chemical modifications; the siRNA can include substantial modifications at multiple nucleotides. As used herein, the term "modified nucleotide" refers to a nucleotide independently having a modified sugar moiety, a modified internucleotide linkage, and / or a modified nucleobase. Thus, the term modified nucleotide encompasses substitution, addition, or removal of, e.g., a functional group or atom of an internucleosidic linkage, sugar moiety, or nucleobase. Modifications suitable for use in the present application include all types of modifications disclosed herein or known in the art.

[0125] The term "antisense strand" refers to a strand of an iRNA (e.g., an siRNA) that comprises a region that is substantially complementary to a target sequence (e.g., an INHBE mRNA). As used herein, the term "region of complementarity" refers to a region on the antisense strand that is substantially complementary to a sequence. In cases where the region of complementarity is not perfectly complementary to the target sequence, mismatches can be present in the interior or terminal regions of the molecule. Generally, the most tolerated mismatches are present in the terminal regions, e.g., 5, 4, 3, or 2 nucleotides in the 5'- and / or 3'-terminus of the siRNA.

[0126] The term "sense strand" means a strand of an iRNA that comprises a region that is substantially complementary to a region of an antisense strand as defined herein.

[0127] The antisense strand and the sense strand of an siRNA can be of the same or different length, as described herein and as known in the art.

[0128] As used herein, and unless otherwise indicated, the term "complementary" when used to describe a first nucleotide sequence in relation to a second nucleotide sequence refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize to and form a duplex structure with an oligonucleotide or polynucleotide comprising the second nucleotide sequence under certain conditions. Such conditions can be, for example, stringent conditions, which can include: 400 mM NaCl, 40 mM PIPES, pH 6.4, 1 mM EDTA, 50°C or 70°C for 12-16 hours.

[0129] In siRNAs, the sense strand and the antisense strand are complementary or substantially complementary. As used herein, "substantially complementary" means that the sequence of nucleotides is sufficiently complementary that interaction in a predictable manner can occur, such as forming a secondary structure (e.g., a stem loop structure). Generally, two "substantially complementary" nucleotide sequences will be at least 70% complementary to each other; preferably, at least 80% complementary; more preferably, at least 90% complementary; even more preferably, at least 95% complementary; such as 98%, 99%, or 100%. In the present application, the sense strand and the antisense strand of an siRNA can have up to 4, 3, 2, or 1 non-matching nucleotides.

[0130] Modified nucleotides

[0131] The present application provides siRNAs for inhibiting expression of an INHBE gene. In some embodiments, the siRNA contains one or more modified nucleotides. In some embodiments, the modified nucleotides include, but are not limited to, 2'-0-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-fluoro, 2'-deoxy modified nucleotides, 2'-deoxynucleotides, 2'-methoxyethyl modified nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, 2'-alkoxy modified nucleotides, 2'-F-ara-nucleotides, abasic nucleotides, morpholino nucleotides, and locked nucleotides. It is not necessary that all positions in a given compound be uniformly modified. Rather, one or more modifications can be incorporated into a single siRNA, or even into a single nucleotide thereof. The modification of one nucleotide is independent of the modification of another nucleotide.

[0132] In some embodiments, one or more nucleotides of the siRNA are linked by nonstandard linkages or backbones (i.e., modified internucleoside linkages or modified backbones). Modified internucleoside linkages or backbones include, but are not limited to, phosphorothioate groups, chiral phosphorothioates, phosphorothioate, phosphorodithioate, phosphotriester, aminoalkyl-phosphotriester, chiral phosphine, phosphinate, phosphoramidate, thiophosphoramidate, sulfide, alkyl phosphonate, phosphoranilidate, phosphinate, phosphoramidite, and morpholino linkages.

[0133] In some embodiments, the sense strand of the siRNA can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages (phosphorothioate modified nucleotides), and the antisense strand of the siRNA can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages (phosphorothioate modified nucleotides). In some embodiments, the sense strand of the siRNA can contain 1 or 2 phosphorothioate linkages, and the antisense strand of the siRNA can contain 1, 2, 3, or 4 phosphorothioate linkages.

[0134] In some embodiments, the siRNA sense strand contains 2 phosphorothioate internucleoside linkages. In some embodiments, the phosphorothioate internucleoside linkages are between nucleotides 1-3 from the 5' end of the sense strand. In some embodiments, the phosphorothioate internucleoside linkages are between nucleotides 1-3 from the 3' end of the sense strand. In some embodiments, one phosphorothioate internucleoside linkage is at the 5' end of the sense strand, and the other phosphorothioate linkage is at the 3' end of the sense strand. In some embodiments, the siRNA sense strand contains 1 phosphorothioate internucleoside linkage. In some embodiments, the phosphorothioate internucleoside linkage is between nucleotides 1-2 from the 5' end of the sense strand. In some embodiments, the phosphorothioate internucleoside linkage is between nucleotides 2-3 from the 5' end of the sense strand. In some embodiments, the targeting ligand is linked to the sense strand by a phosphorothioate linkage.

[0135] In some embodiments, the siRNA antisense strand contains 4 phosphorothioate internucleoside linkages. In some embodiments, the 4 phosphorothioate internucleoside linkages are between the nucleotides at positions 1-3 from the 5' end of the antisense strand and between the nucleotides at positions 1-3 from the 5' end. In some embodiments, the siRNA antisense strand contains 3 phosphorothioate internucleoside linkages. In some embodiments, the 3 phosphorothioate internucleoside linkages are between the nucleotides at positions 1-2 from the 5' end of the antisense strand and between the nucleotides at positions 1-3 from the 3' end, respectively. In some embodiments, the 3 phosphorothioate internucleoside linkages are between the nucleotides at positions 1-3 from the 5' end of the antisense strand and between the nucleotides at positions 1-2 from the 3' end, respectively. In some embodiments, the siRNA antisense strand contains 2 phosphorothioate internucleoside linkages. In some embodiments, the 2 phosphorothioate internucleoside linkages are between the nucleotides at positions 1-2 from the 5' end of the antisense strand and between the nucleotides at positions 1-2 from the 5' end.

[0136] In some embodiments, the sense strand can include one or more capping residues or moieties, sometimes referred to in the art as "caps," "terminal caps," or "capping residues." As used herein, a "capping residue" is a non-nucleotide compound or other moiety that can be incorporated at one or more termini of the nucleotide sequence of an RNAi agent disclosed herein. In some cases, a capping residue can provide certain beneficial properties to the RNAi agent, such as protection from exonuclease degradation. In some embodiments, an inverted abasic residue (also referred to in the art as an "inverted abasic site") is added as a capping residue (see, e.g., F. Czauderna, Nucleic Acids Res., 2003, 31(11), 2705-16). Capping residues are generally known in the art and include, for example, inverted abasic residues as well as carbon chains, such as terminal C3H7 (propyl), C6H 13 (alkyl) or C 12 H 25 (dodecyl) groups. In some embodiments, a capping residue is present at the 5' terminus, the 3' terminus, or both the 5' and 3' termini of the sense strand. In some embodiments, the 5' end and / or 3' end of the sense strand can include more than one inverted abasic deoxyribose moiety as a capping residue. The inverted abasic residues can be linked via a phosphate, phosphorothioate (e.g., shown herein as (invAb)s), or other internucleoside linkage.

[0137] Exemplary sense and antisense sequences are shown in Tables 1, 2 below.

[0138] Table 1 (naked sequences)

[0139] Table 2 (modified sequences)

[0140] In one embodiment, the siRNA sense strands shown in Table 2 above further comprise inverted abasic residues (invAb) at the 3' end and / or the 5' end of the nucleotide sequence. Preferably, the inverted abasic residues are linked to the 3' end and the 5' end of the nucleotide sequence by phosphorothioate linkages.

[0141] In one embodiment, the siRNA sense strands shown in Table 2 above are linked to a targeting ligand to form a conjugate. Preferably, the targeting ligand is linked to the 5' end of the sense strand. For example, the sense and antisense strands of the siRNA conjugates of the application can be as shown in the same row of Table 3 below.

[0142] Table 3 (modified sequences + targeting ligand)

[0143] wherein each abbreviation has the following meaning: A = adenosine-3'-phosphate C = cytidine-3'-phosphate G = guanosine-3'-phosphate U = uridine 3'-phosphate a = 2'-O-methyladenosine-3'-phosphate c = 2'-O-methylcytidine-3'-phosphate g = 2'-O-methylguanosine-3'-phosphate u = 2'-O-methyluridine-3'-phosphate as = 2'-O-methyladenosine-3'-phosphorothioate cs = 2'-O-methylcytidine-3'-phosphorothioate gs = 2'-O-methylguanosine-3'-phosphorothioate us = 2'-O-methyluridine-3'-phosphorothioate Af = 2'-fluoroadenosine-3'-phosphate Cf = 2'-fluorocytidine-3'-phosphate Gf = 2'-fluoroguanosine-3'-phosphate Uf = 2'-fluorouridine-3'-phosphate invAb = inverted abasic residue s = phosphorothioate linkage EVp = 5'-ethynylphosphate modification C6NH2= 1-amino-n-hexyloxy modification L1is a targeting ligand of formula (I)

[0144] Conjugate

[0145] The term "conjugate" (or conjugate) as used herein refers to a new compound formed by covalent attachment (conjugation) of two or more compounds through a bivalent or multivalent compound molecule with linking functionality.

[0146] In some embodiments, the siRNAs of the application contain or are conjugated to one or more non-nucleotide groups, including but not limited to targeting groups, linking groups, pharmacokinetic / pharmacodynamic (PK / PD) modulators, delivery polymers or delivery vehicles. The non-nucleotide groups can enhance targeting, delivery or attachment of the siRNA. The non-nucleotide groups can be covalently attached to the 3' end and / or 5' end of the sense strand and / or the antisense strand.

[0147] In some embodiments, the siRNAs of the application contain non-nucleotide groups attached to the 3' end and / or 5' end of the sense strand. In some embodiments, the non-nucleotide groups are attached to the 5' end of the sense strand of the siRNA. The non-nucleotide groups can be attached to the siRNA directly or indirectly via a linker / linking group. In some embodiments, the non-nucleotide groups are attached to the siRNA via a labile, cleavable or reversible bond or linker.

[0148] Targeting groups or targeting moieties enhance the pharmacokinetic or biodistribution properties of the conjugate or siRNA to which they are attached to improve the cell-specific (in some cases, including organ-specific) distribution and cell-specific (or organ-specific) uptake of the conjugate or siRNA. The targeting groups can be monovalent, bivalent, trivalent, tetravalent, or of higher valency for the target to which it is directed. Representative targeting groups include but are not limited to compounds with affinity for cell surface molecules, cell receptor ligands, haptens, antibodies, monoclonal antibodies, antibody fragments and antibody mimetics (with affinity for cell surface molecules). In some embodiments, the targeting groups are attached to the siRNA using a linker, such as a PEG linker, or one, two or three abasic and / or ribose alcohol (abasic ribose) residues, which in some cases can act as linkers.

[0149] The targeting groups, with or without linkers, can be attached to the 5' or 3' end of any of the sense strands and / or antisense strands disclosed in Tables 1, 2 and 3.

[0150] Pharmaceutical compositions and methods of administration

[0151] As used herein, the term "effective amount" or "effective dose" refers to an amount that is functional or active and acceptable to a human and / or animal.

[0152] As used herein, the term "pharmaceutically acceptable" in reference to an ingredient with respect to its suitability or applicability for use in humans and / or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit / risk ratio is intended. The term "pharmaceutically acceptable carrier" refers to a carrier with which the therapeutic agent is administered, including various excipients and diluents.

[0153] The pharmaceutical composition of the present application contains a safe and effective amount of the active ingredient of the present application and a pharmaceutically acceptable carrier.

[0154] In the present application, the expression vector can be administered directly to the subject, or the expression vector can be prepared into a pharmaceutical composition with a pharmaceutically acceptable carrier and then administered.

[0155] Preparation of ligand compound

[0156] 1. Preparation of ligand I-1

[0157] The synthetic route is shown as follows:

[0158] First step: synthesis of ethyl 4-((tert-butoxycarbonyl)(3-((tert- butoxycarbonyl)amino)propyl)amino)butanoate (01B)

[0159] Into a 500 mL single-necked flask, N-tert-butoxycarbonyl-1,3- propanediamine (30.00 g, 172.2 mmol), acetonitrile (250 mL) and potassium carbonate (47.59 g, 344.3 mmol) were added in turn, the mixture was cooled to 0 °C, and 4-bromobutyl acetate (26.87 g, 137.8 mmol) was slowly added dropwise. After the addition was completed, the temperature was allowed to rise to room temperature and the reaction was allowed to proceed for 16 h. The reaction solution was filtered, and di-tert-butyl dicarbonate (56.36 g, 258.2 mmol) was added to the filtrate, and the reaction was allowed to proceed at room temperature for 5 h. The reaction solution was concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography (petroleum ether / ethyl acetate (V / V) = 1:0-7:3) to obtain ethyl 4-((tert-butoxycarbonyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)butanoate (01B) (45.00 g, yield 84.08%).

[0160] LC-MS, M / Z (ESI): 289.4 [M-99] +

[0161] Second step: synthesis of 4-((tert-butoxycarbonyl)(3-((tert- butoxycarbonyl)amino)propyl)amino)butanoic acid (01C)

[0162] Into a 500 mL single neck flask, was added ethyl 4-((tert-butoxycarbonyl)(3- ((tert-butoxycarbonyl)amino)propyl)amino)butanoate (45.00 g, 115.8 mmol), methanol (200 mL) and lithium hydroxide (5.55 g, 231.8 mmol) in water (50 mL) successively. The mixture was stirred at room temperature for 16 h. The reaction solution was concentrated under reduced pressure, water (50 mL) was added, 1 N hydrochloric acid was added to adjust the pH value to 6, and ethyl acetate (150 mL x 3) was added for extraction. The combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 4-((tert-butoxycarbonyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)butanoic acid (01C) (24.00 g, yield 57.49%).

[0163] 1 H NMR (400 MHz, DMSO-d6) δ 6.73 (s, 1H), 3.08 (q, J = 8.6, 8.1 Hz, 4H), 2.85 (q, J = 6.6 Hz, 2H), 2.12 (t, J = 7.3 Hz, 2H), 1.67-1.60 (m, 2H), 1.57-1.49 (m, 2H), 1.36 (s, 9H), 1.35 (s, 9H).

[0164] LC-MS, M / Z (ESI): 383.4 [M+Na] +

[0165] Third step: synthesis of tert-butyl (3-(pent-4-en-1-ylamino)propyl)carbamate (01E)

[0166] Into a 500 mL single neck flask, was added N-tert-butoxycarbonyl-1,3- propanediamine (30.00 g, 172.2 mmol), acetonitrile (250 mL) and potassium carbonate (47.59 g, 344.3 mmol) successively. The mixture was cooled to 0 °C, and 5-bromo-1-pentene (20.53 g, 137.8 mmol) was added dropwise slowly. After the addition was completed, the temperature was allowed to rise to room temperature and stirred for 16 h. The reaction solution was filtered and concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography (dichloromethane / methanol (V / V) = 1:0-20:1) to obtain tert-butyl (3-(pent-4-en-1-ylamino)propyl)carbamate (01E) (16.00 g, yield 38.34%).

[0167] 1H NMR (400 MHz, DMSO-d6) δ 6.78 (t, J = 5.0 Hz, 1H), 5.84 - 5.74 (m, 1H), 5.00 - 4.95 (m, 1H), 4.93 - 4.89 (m, 1H), 3.14 (s, 1H), 2.92 (q, J = 6.7 Hz, 2H), 2.43 (t, J = 7.0 Hz, 4H), 2.01 (q, J = 7.3 Hz, 2H), 1.49 - 1.40 (m, 4H), 1.34 (s, 9H).

[0168] LC-MS, M / Z (ESI): 243.3 [M+H] +

[0169] Fourth Step: Synthesis of tert-butyl (3-((tert-butoxycarbonyl)amino)propyl) (4-((3- ((tert-butoxycarbonyl)amino)propyl) (pent-4-en-1-yl)amino)-4-oxobutyl)carbamate (01F)

[0170] Into a 500 mL single necked flask, was added tert-butyl (3-(pent-4-en-1- ylamino)propyl)carbamate (16.00 g, 66.02 mmol), dichloromethane (200 mL), 4- ((tert-butoxycarbonyl) (3-((tert-butoxycarbonyl)amino)propyl)amino)butanoic acid (23.80 g, 66.03 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (18.98 g, 99.01 mmol), 1-hydroxybenzotriazole (8.92 g, 66.01 mmol) and triethylamine (20.04 g, 198.0 mmol) successively. The mixture was stirred at room temperature for 16 h under nitrogen atmosphere. The reaction mixture was washed with 0.5 N hydrochloric acid (100 mL), saturated aqueous sodium bicarbonate solution (100 mL) successively, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a crude product, which was purified by column chromatography (petroleum ether / ethyl acetate (V / V) = 1:0-0:1) to give tert-butyl (3-((tert-butoxycarbonyl)amino)propyl) (4-((3-((tert- butoxycarbonyl)amino)propyl) (pent-4-en-1-yl)amino)-4-oxobutyl)carbamate (01F) (19.00 g, yield 49.21%).

[0171] 1H NMR (400 MHz, DMSO-d6) δ 6.91 - 6.60 (m, 2H), 5.84 - 5.72 (m, 1H), 5.06 - 4.89 (m, 2H), 3.22 - 3.14 (m, 4H), 3.12 - 3.06 (m, 4H), 2.90 - 2.83 (m, 4H), 2.23 - 2.10 (m, 2H), 1.98 - 1.92 (m, 2H), 1.47 - 1.66 (m, 8H), 1.35 (d, J = 3.8 Hz, 27H).

[0172] LC-MS, M / Z (ESI): 607.6 [M+Na] +

[0173] Fifth Step: Synthesis of N-(3-aminopropyl)-4-((3-aminopropyl)amino)-N- (pent-4-en-1-yl)butanamide (trifluoroacetate salt) (01G)

[0174] To a 250 mL single necked flask was added tert-butyl (3-((tert- butyloxycarbonyl)amino)propyl) (4-((3-((tert-butyloxycarbonyl)amino)propyl) (pent-4-en-1-yl)amino)-4-oxobutyl)carbamate (5.00 g, 8.55 mmol), dichloromethane (200 mL) and trifluoroacetic acid (13.09 mL, 171.1 mmol) and the mixture was stirred at room temperature for 16 h. The reaction was concentrated under reduced pressure to give N-(3-aminopropyl)-4-((3-aminopropyl)amino)-N-(pent-4-en-1-yl)butanamide (trifluoroacetate salt) (01G) (5.34 g, 99.7% yield).

[0175] LC-MS, M / Z (ESI): 285.46 [M+H] +

[0176] Sixth Step: Synthesis of (2R,2'R,3R,3'R,4R,4'R,5R,5'R,6R,6'R)-((15-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6- (acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoyl)-5,11,20-trioxo-10- (pent-4-en-1-yl)-6,10,15,19-tetraazadocosa-1,24-diyl)bis(oxy))bis(5-acetamido-2- (acetyloxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate (01I)

[0177] To a 250 mL single necked flask was added N-(3-aminopropyl)-4-((3- aminopropyl)amino)-N-(pent-4-en-1-yl)butanamide (trifluoroacetate) (01G) (3.30 g, 5.27 mmol), dichloromethane (100 mL), 5-(((2R,3R,4R,5R,6R)-3-acetylamino- 4,5-diacetoxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoic acid (8.26 g, 18.5 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (5.05 g, 26.3 mmol), 1-hydroxybenzotriazole (2.14 g, 15.8 mmol), and triethylamine (5.34 g, 52.8 mmol) were added sequentially. The mixture was stirred at room temperature for 16 h under a nitrogen atmosphere. The reaction was washed sequentially with 0.5 N hydrochloric acid (100 mL), saturated aqueous sodium bicarbonate (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product, which was purified by column chromatography (dichloromethane / methanol (V / V) = 1 :0 to 9:1) to give (2R,2'R,3R,3'R,4R,4'R,5R,5'R,6R,6'R)-((15-(5-(((2R,3R,4R,5R,6R)-3-acetylamino- 4,5-diacetoxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoyl)-5,11,20- trioxo-10-(pent-4-en-1-yl)-6,10,15,19-tetraazadocosan-1,24-diyl)bis(oxy))bis(5- acetylamino-2-(acetyloxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate (7.00 g, 84.5% yield)

[0178] 1 H NMR (600 MHz, DMSO-d6) δ 7.85 - 7.77 (m, 4H), 7.71 - 7.69 (m, 1H), 5.83 - 5.77 (m, 1H), 5.19 (d, J = 3.3 Hz, 3H), 5.05 - 4.91 (m, 5H), 4.46 (dd, J = 8.4, 4.1 Hz, 3H), 4.02 - 3.99 (m, 9H), 3.89 - 3.82 (m, 3H), 3.70 - 3.68 (m, 3H), 3.40 - 3.37 (m, 3H), 3.25 - 3.16 (m, 8H), 3.04 - 2.95 (m, 4H), 2.30 - 2.17 (m, 4H), 2.08 (s, 9H), 2.05 - 1.94 (m, 15H), 1.87 (s, 9H), 1.75 (s, 9H), 1.61 - 1.43 (m, 20H).

[0179] LC-MS, M / Z (ESI): 787.26 [M+2H] 2+

[0180] Step 7: Synthesis of 4-(4-(5-(((2R,3R,4R,5R,6R)-3-acetylamino-4,5- diacetoxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-N-(3-(5-(((2R,3R,4R,5R,6R)- 3-acetylamino-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido) propyl)pentanamido)-N-(3-(5-(((2R,3R,4R,5R,6R)-3-acetylamino-4,5-diacetoxy-6- (acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)butanamido)butanoic acid (01J)

[0181] Into a 250 mL single-necked flask, (2R,2'R,3R,3'R,4R,4'R,5R,5'R,6R,6'R)-((15-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoyl)-5,11,20-trioxo-10-(pent-4-en-1-yl)-6,10,15,19-tetraazadocosa-1,24-diyl)bis(oxy))bis(5-acetamido-2-(acetyloxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate (7.00 g, 4.45 mmol), dichloromethane (30 mL), acetonitrile (30 mL), water (48 mL) were added successively, and after stirring uniformly, sodium periodate (4.76 g, 22.3 mmol) and ruthenium trichloride (18.5 mg, 89.0 μmol) were added. The mixture was stirred at room temperature for 16 h under nitrogen protection. The reaction solution was filtered, saturated aqueous sodium bicarbonate solution was added to the filtrate, and the aqueous phase was adjusted to pH 7-8. After separation, the aqueous phase was washed with dichloromethane (50 mL x 2), 1 N hydrochloric acid was added to adjust the pH to about 5-6, and dichloromethane (100 mL x 3) was added for extraction. The obtained organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 4-(4-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-N-(3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)pentanamido)-N-(3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)butanamido)butanoic acid (01J) (3.50 g, yield 49.4%).

[0182] 1H NMR (600 MHz, DMSO-d6) δ 12.12 (s, 1H), 7.84-7.81 (m, 4H), 7.74-7.71 (m, 1H), 5.19 (d, J = 3.2 Hz, 3H), 4.94 (dd, J = 11.2, 3.3 Hz, 3H), 4.47 (d, J = 8.4 Hz, 3H), 4.00-3.99 (m, 9H), 3.88-3.83 (m, 3H), 3.71-3.66 (m, 3H), 3.40-3.37 (m, 3H), 3.22-3.17 (m, 8H), 3.04-2.95 (m, 4H), 2.30-2.13 (m, 6H), 2.08 (s, 9H), 2.04-2.01 (m, 4H), 1.98 (s, 9H), 1.87 (s, 9H), 1.75 (s, 9H), 1.69-1.43 (m, 20H).

[0183] LC-MS, M / Z (ESI): 796.60 [M+2H] 2+

[0184] Eighth Step: Synthesis of (2R,2'R,3R,3'R,4R,4'R,5R,5'R,6R,6'R)-((15-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6- (acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoyl)-10-(4-(4- nitrophenoxy)-4-oxobutyl)-5,11,20-trioxo-6,10,15,19-tetraazadocosan-1,24- diyl)bis(oxy))bis(5-acetamido-2-(acetyloxymethyl)tetrahydro-2H-pyran-6,3,4- triyl)tetraacetate (I-1)

[0185] Into a 100 mL single necked flask was added 4-(4-(5-(((2R,3R,4R,5R,6R)-3- acetylamino-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-N- (3-(5-(((2R,3R,4R,5R,6R)-3-acetylamino-4,5-diacetoxy-6- (acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)pentanamido)- N-(3-(5-(((2R,3R,4R,5R,6R)-3-acetylamino-4,5-diacetoxy-6- (acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)butanamido)butanoic acid (900 mg, 0.566 mmol), dichloromethane (20 mL), 4-nitrophenol (94.5 mg, 0.679 mmol), N,N'-dicyclohexylcarbodiimide (175.11 mg, 0.85 mmol), the mixture was stirred at room temperature for 16 h under nitrogen protection. The reaction solution was filtered, and the obtained filtrate was concentrated to obtain a crude product, which was purified by column chromatography (dichloromethane / methanol (V / V) = 1:0-9:1) to obtain (2R,2'R,3R,3'R,4R,4'R,5R,5'R,6R,6'R)-((15-(5-(((2R,3R,4R,5R,6R)-3- acetylamino-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoyl)- 10-(4-(4-nitrophenoxy)-4-oxobutyl)-5,11,20-trioxo-6,10,15,19-tetraazadocosan-1,24-diyl)bis(oxy))bis(5-acetylamino-2-(acetyloxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate (I-1) (850 mg, yield 87.8%).

[0186] 1H NMR (400 MHz, DMSO-d6) δ 8.29 (dd, J = 9.2, 2.7 Hz, 2H), 7.82 - 7.67 (m, 4H), 7.74 - 7.66 (m, 1H), 7.44 (dd, J = 9.1, 2.1 Hz, 2H), 5.19 (d, J = 3.3 Hz, 3H), 4.94 (dd, J = 11.2, 3.4 Hz, 3H), 4.46 (d, J = 8.4 Hz, 3H), 4.00 (s, 9H), 3.85 (q, J = 9.2 Hz, 3H), 3.72 - 3.65 (m, 3H), 3.41 - 3.37 (m, 3H), 3.24 - 3.19 (m, 8H), 3.06 - 2.92 (m, 4H), 2.77 - 2.49 (m, 2H), 2.36 - 2.15 (m, 4H), 2.07 - 2.08 (m, 9H), 2.03 - 2.02 (m, 4H), 1.97 (s, 9H), 1.87 (s, 9H), 1.75 (s, 9H), 1.67 - 1.37 (m, 20H).

[0187] LC-MS, M / Z (ESI): 856.5 [M+2H] 2+

[0188] 2. Preparation of Ligand I-2

[0189] The synthetic route is as follows:

[0190] Step 1: Synthesis of methyl 3-((tert-butoxycarbonyl)(3-((tert- butoxycarbonyl)amino)propyl)amino)-2-(((tert-butoxycarbonyl)(3- ((tert-butoxycarbonyl)amino)propyl)amino)methyl)propanoate (02B)

[0191] Into a 100 mL single necked flask was added methyl 3-bromo-2- (bromomethyl)propanoate (2.00 g, 7.69 mmol), N-tert-butoxycarbonyl-1,3- propanediamine (5.36 g, 30.8 mmol) and acetonitrile (30 mL) successively, stirred at room temperature for 16 h. The reaction was concentrated under reduced pressure, dichloromethane (30 mL), di-tert-butyl dicarbonate (6.72 g, 30.8 mmol) and N,N-diisopropyl ethylamine (3.98 g, 30.8 mmol) were added, stirred at room temperature for 16 h. Water 50 mL was added to the reaction, extracted with dichloromethane (50 mL x 3). The combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure to give the crude product. The crude product was purified by column chromatography (petroleum ether / ethyl acetate (V / V) = 1:0-3:2) to give methyl 3-((tert-butoxycarbonyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)-2-(((tert-butoxycarbonyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)methyl)propanoate (02B) (3.50 g, yield 70.7%).

[0192] Second Step: Synthesis of 3-((tert-butoxycarbonyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)-2-(((tert-butoxycarbonyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)methyl)propanoic acid (02C)

[0193] Into a 100 mL single necked flask was added methyl 3-bromo-2- (bromomethyl)propanoate (2.00 g, 7.69 mmol), N-tert-butoxycarbonyl-1,3- propanediamine (5.36 g, 30.8 mmol) and acetonitrile (30 mL) successively, stirred at room temperature for 16 h. The reaction was concentrated under reduced pressure, dichloromethane (30 mL), di-tert-butyl dicarbonate (6.72 g, 30.8 mmol) and N,N-diisopropyl ethylamine (3.98 g, 30.8 mmol) were added, stirred at room temperature for 16 h. Water 50 mL was added to the reaction, extracted with dichloromethane (50 mL x 3). The combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure to give the crude product. The crude product was purified by column chromatography (petroleum ether / ethyl acetate (V / V) = 1:0-3:2) to give methyl 3-((tert-butoxycarbonyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)-2-(((tert-butoxycarbonyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)methyl)propanoate (02B) (3.50 g, yield 70.7%).

[0194] LC-MS, M / Z (ESI): 533.5 [M-99] + .

[0195] Step 3: Synthesis of benzyl 3-((tert-butoxycarbonyl)(3- ((tert-butoxycarbonyl)amino)propyl)amino)-2-(((tert- butoxycarbonyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)methyl)propanoate (02D)

[0196] Into a 500 mL single necked flask, was added 3-((tert- butoxycarbonyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)-2- (((tert-butoxycarbonyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)methyl)propanoic acid (3.00 g, 4.74 mmol), N,N-dimethylformamide (30 mL), potassium carbonate (1.97 g, 14.3 mmol) and benzyl bromide (0.93 g, 5.4 mmol) successively, and stirred at room temperature for 16 h. The reaction solution was filtered, and 30 mL of water was added to the filtrate, which was extracted with ethyl acetate (60 mL x 3). The combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. Purification by column chromatography (petroleum ether / ethyl acetate (V / V) = 1:0-1:1) gave benzyl 3-((tert- butoxycarbonyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)-2-(((tert- butoxycarbonyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)methyl)propanoate (02D) (3.00 g, yield 87.5%).

[0197] 1 H NMR (400 MHz, DMSO-d6) δ 7.39-7.31 (m, 5H), 6.79-6.68 (m, 2H), 5.07 (s, 2H), 3.28-3.25 (m, 4H), 3.15-3.05 (m, 3H), 2.98-2.92 (m, 2H), 2.90-2.82 (m, 4H), 1.54-1.50 (m, 4H), 1.37-1.35 (m, 36H).

[0198] Step 4: Synthesis of benzyl 3-((3-aminopropyl)amino)-2-(((3- aminopropyl)amino)methyl)propanoate tetra(trifluoroacetate) salt (02E)

[0199] Into a 250 mL single necked flask was added 3-((tert-butoxycarbonyl)(3-((tert- butoxycarbonyl)amino)propyl)amino)-2-(((tert-butoxycarbonyl)(3-((tert- butoxycarbonyl)amino)propyl)amino)methyl)propanoic acid benzyl ester (4.50 g, 6.22 mmol), dichloromethane (60 mL), trifluoroacetic acid (21.3 g, 187 mmol), stirred at room temperature for 16 h. The reaction was concentrated under reduced pressure to give 3-((3- aminopropyl)amino)-2-(((3-aminopropyl)amino)methyl)propanoic acid benzyl ester tetra(trifluoroacetic acid) salt (02E) (4.85 g).

[0200] LC-MS, M / Z (ESI): 323.04 [M+H] + .

[0201] Fifth Step: Synthesis of (2R,2'R,3R,3'R,4R,4'R,5R,5'R,6R,6'R)-(((1,23-bis(((2R,3R,4R,5R,6R)- 3-acetamido-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-12- ((benzyloxy)carbonyl)-5,19-dioxo-6,10,14,18-tetraazatridecan-10,14-diyl)bis(5- oxopentane-5,1-diyl))bis(oxy))bis(5-acetamido-2-(acetyloxymethyl)tetrahydro-2H-pyran- 6,3,4-triyl)tetraacetate (02F)

[0202] To a 250 mL single necked flask was added 3-((3-aminopropyl)amino)-2-(((3- aminopropyl)amino)methyl)benzyl propanoate tetra(trifluoroacetate) salt (4.85 g, 6.23 mmol), dichloromethane (100 mL), 5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5- diacetoxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoic acid (12.55 g, 28.05 mmol), l-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (7.17 g, 37.4 mmol), 1-hydroxybenzotriazole (3.37 g, 24.9 mmol), and N,N-diisopropylethylamine (12.1 g, 93.5 mmol) sequentially. The reaction was stirred at room temperature for 16 h under nitrogen. The reaction was washed with 0.5 N aqueous hydrochloric acid (100 mL) and saturated sodium bicarbonate solution (100 mL) sequentially. The combined organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product. The crude product was purified by column chromatography (dichloromethane / methanol (V / V) = 1:0-9:1) to give (2R,2'R,3R,3'R,4R,4'R,5R,5'R,6R,6'R)-(((1,23-bis(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6- (acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-12-((benzyloxy)carbonyl)-5,19- dioxo-6,10,14,18-tetraazatridecan-10,14-diyl)bis(5-oxopentane-5,1-diyl))bis(oxy))bis(5- acetamido-2-(acetyloxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate (02F) (9.00 g, 70.8% yield)

[0203] 1 H NMR (400 MHz, DMSO-d6) δ 7.82 - 7.64 (m, 6H), 7.37 - 7.25 (m, 5H), 5.17 (d, J = 3.3 Hz, 4H), 4.98 (d, J = 10.7 Hz, 2H), 4.93 (dd, J = 11.2, 3.3 Hz, 4H), 4.45 (d, J = 8.4 Hz, 4H), 3.98 (s, 12H), 3.84 (q, J = 9.2 Hz, 4H), 3.69 - 3.64 (m, 4H), 3.57 - 3.36 (m, 8H), 3.10 - 2.90 (m, 9H), 2.20 - 2.11 (m, 4H), 2.06 (s, 12H), 2.03 - 1.98 (m, 4H), 1.95 (s, 12H), 1.85 (s, 12H), 1.73 (s, 12H), 1.58 - 1.37 (m, 20H).

[0204] LC-MS, M / Z (ESI): 1020.9 [M+2H] 2+ .

[0205] Step 6: Synthesis of 3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6- (acetyloxymethyl) tetrahydro-2H-pyran-2-yl)oxy)-N-(3-(5-(((2R,3R,4R,5R,6R)-3- acetamido-4,5-diacetoxy-6-(acetyloxymethyl) tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)pentanamido)-2-((5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5- diacetoxy-6-(acetyloxymethyl) tetrahydro-2H-pyran-2-yl)oxy)-N-(3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetyloxymethyl) tetrahydro-2H-pyran-2- yl)oxy)pentanamido)propyl)pentanamido)methyl)propanoic acid (02G)

[0206] To a 100 mL single necked flask was added (2R,2'R,3R,3'R,4R,4'R,5R,5'R,6R,6'R)-(((1,23-bis(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6- (acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-12-((benzyloxy)carbonyl)-5,19- dioxo-6,10,14,18-tetraazatridecan-10,14-diyl)bis(5-oxopentane-5,1-diyl))bis(oxy))bis(5- acetamido-2-(acetyloxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate (0.18 g, 88 μmol), methanol (5 mL), ethyl acetate (5 mL), and 10% palladium on carbon (18 mg) were added sequentially. The mixture was stirred under a hydrogen atmosphere (1 atm) at room temperature for 16 h. The reaction was filtered through celite, the filter cake was washed with methanol and the filtrates were combined. The filtrates were concentrated under reduced pressure to give 3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6- (acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-N-(3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5- diacetoxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)pentanamido)- 2-((5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2- yl)oxy)-N-(3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydro-2H- pyran-2-yl)oxy)pentanamido)propyl)pentanamido)methyl)propanoic acid (02G) (0.12 g, 70% yield).

[0207] 1H NMR (400 MHz, DMSO-d6) δ 7.83 - 7.81 (m, 5H), 7.72 - 7.68 (m, 1H), 5.21 (d, J = 3.3 Hz, 4H), 4.96 (dd, J = 11.2, 3.3 Hz, 4H), 4.49 (dd, J = 8.4, 3.3 Hz, 4H), 4.04 - 3.99 (m, 12H), 3.87 (q, J = 10.5, 9.8 Hz, 4H), 3.75 - 3.66 (m, 4H), 3.59 - 3.39 (m, 8H), 3.27 - 3.13 (m, 3H), 3.08 - 2.90 (m, 6H), 2.34 - 2.15 (m, 4H), 2.10 (s, 12H), 2.07 - 2.02 (m, 4H), 1.99 (s, 12H), 1.88 (s, 12H), 1.77 (s, 12H), 1.61 - 1.43 (m, 20H).

[0208] LC-MS, M / Z (ESI): 975.9 [M+2H] 2+ .

[0209] Step 7: Synthesis of (2R,2'R,3R,3'R,4R,4'R,5R,5'R,6R,6'R)-(((1,23-bis(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6- (acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-12-((4-nitrophenoxy)carbonyl)- 5,19-dioxo-6,10,14,18-tetraazatridecan-10,14-diyl)bis(5-oxopentane-5,1-diyl))bis(oxy))bis(5-acetamido-2- (acetyloxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate (I-2)

[0210] To a 100 mL single necked flask was added 3-(5-(((2R,3R,4R,5R,6R)-3- acylamino-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-N-(3-(5- (((2R,3R,4R,5R,6R)-3-acylamino-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydro-2H-pyran- 2-yl)oxy)pentanamido)propyl)pentanamido)-2-((5-(((2R,3R,4R,5R,6R)-3-acylamino-4,5- diacetoxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-N-(3-(5-(((2R,3R,4R,5R,6R)- 3-acylamino-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido) propyl)pentanamido)methyl)propanoic acid (200 mg, 0.103 mmol), dichloromethane (10 mL), 4-nitrophenol (21.4 mg, 0.154 mmol) and N,N'-dicyclohexylcarbodiimide (31.7 mg, 0.154 mmol), the mixture was stirred at room temperature for 16 h under nitrogen protection. The reaction solution was filtered, and the obtained filtrate was concentrated to give a crude product. Purification by column chromatography (dichloromethane / methanol (V / V) = 1:0-8:2) gave (2R,2'R,3R,3'R,4R,4'R,5R,5'R,6R,6'R)-(((1,23-bis(((2R,3R,4R,5R,6R)-3- acylamino-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-12-((4- nitrophenoxy)carbonyl)-5,19-dioxo-6,10,14,18-tetraazatridecan-10,14-diyl)bis(5- oxopentane-5,1-diyl))bis(oxy))bis(5-acylamino-2-(acetyloxymethyl)tetrahydro-2H-pyran- 6,3,4-triyl)tetraacetate (I-2) (120 mg, yield 56.5%).

[0211] 1H NMR (400 MHz, DMSO-d6) δ 8.33 - 8.29 (m, 2H), 7.79 - 7.69 (m, 6H), 7.43 - 7.31 (m, 2H), 5.18 (d, J = 3.3 Hz, 4H), 4.94 (dd, J = 11.2, 3.3 Hz, 4H), 4.45 (d, J = 8.4 Hz, 4H), 3.99 (s, 12H), 3.89 - 3.80 (m, 4H), 3.69 - 3.61 (m, 5H), 3.49 - 3.31 (m, 7H), 3.29 - 3.13 (m, 5H), 3.09 - 2.94 (m, 4H), 2.35 - 2.17 (m, 4H), 2.07 (d, J = 2.7 Hz, 12H), 2.04 - 2.01 (m, 4H), 1.96 (d, J = 2.0 Hz, 12H), 1.86 (s, 12H), 1.74 (s, 12H), 1.65 - 1.39 (m, 20H).

[0212] 3. Preparation of Ligand I-3

[0213] The synthesis route is as follows:

[0214] Step 1: Synthesis of (2R,2'R,3R,3'R,4R,4'R,5R,5'R,6R,6'R)-((15-(5-(((2R,3R,4R,5R,6R)-3- acetylamino-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoyl)- 10-(4-((2S,4R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxypyrrolidin-1-yl)- 4-oxobutyl)-5,11,20-trioxo-6,10,15,19-tetraazadocosan-1,24-diyl)bis(oxy))bis(5- acetylamino-2-(acetyloxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate (03B)

[0215] To a 100 mL single necked flask was added 4-(4-(5-(((2R,3R,4R,5R,6R)-3- acetylamino-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-N- (3-(5-(((2R,3R,4R,5R,6R)-3-acetylamino-4,5-diacetoxy-6- (acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)pentanamido)- N-(3-(5-(((2R,3R,4R,5R,6R)-3-acetylamino-4,5-diacetoxy-6- (acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)butanamido) butanoic acid (01J, 1.0 g, 0.63 mmol), dichloromethane (20 mL), (3R,5S)-5-{[bis(4- methoxyphenyl)(phenyl)methoxy]methyl}pyrrolidin-3-ol (03A, 0.29 g, 0.69 mmol), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (376 mg, 0.991 mmol), 1- hydroxybenzotriazole (255 mg, 1.89 mmol), and N,N-diisopropylethylamine (244 mg, 1.89 mmol) were added successively under nitrogen protection. The reaction was stirred at room temperature for 16 h. The reaction was washed with saturated sodium bicarbonate solution (20 mL), dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated to give a crude product. The product was purified by column chromatography (dichloromethane (2% triethylamine) / methanol (V / V) = 1:0-9:1) to give (2R,2'R,3R,3'R,4R,4'R,5R,5'R,6R,6'R)-((15-(5-(((2R,3R,4R,5R,6R)-3- acetylamino-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoyl)- 10-(4-((2S,4R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxypyrrolidin-1- yl)-4-oxobutyl)-5,11,20-trioxo-6,10,15,19-tetraazadocosan-1,24-diyl)bis(oxy))bis(5- acetylamino-2-(acetyloxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate (03B) (1.0 g, 80% yield).

[0216] 1H NMR (400 MHz, DMSO-d6) δ 7.86 - 7.65 (m, 5H), 7.32 - 7.29 (m, 4H), 7.21 - 7.17 (m, 5H), 6.90 - 6.86 (m, 4H), 5.21 (s, 3H), 5.03 - 4.89 (m, 4H), 4.48 (d, J = 8.3 Hz, 3H), 4.36 (m, 1H), 4.16 (s, 1H), 4.02 (s, 9H), 3.87 (q, J = 9.8 Hz, 3H), 3.73 - 3.68 (m, 9H), 3.64 - 3.45 (m, 7H), 3.19 - 2.93 (m, 12H), 2.35 - 2.14 (m, 6H), 2.09 (s, 9H), 2.06 - 2.02 (m, 4H), 1.99 (s, 9H), 1.88 (s, 9H), 1.76 (s, 9H), 1.74 - 1.25 (m, 22H).

[0217] Step 2: Synthesis of 4-(((3R,5S)-1-(4-(4-(5-(((2R,3R,4R,5R,6R))-3- acylamido-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-N-(3- (5-(((2R,3R,4R,5R,6R)-3-acylamido-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydro-2H- pyran-2-yl)oxy)pentanamido)propyl)pentanamido)-N-(3-(5-(((2R,3R,4R,5R,6R)-3- acylamido-4,5-diacetoxy)-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)butanamido)butyryl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-3- yl)oxy)-4-oxobutanoic acid (I-3)

[0218] To a 100 mL single neck flask was added (2R,2'R,3R,3'R,4R,4'R,5R,5'R,6R,6'R)-((15-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6- (acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoyl)-10-(4-((2S,4R)-2- ((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxypyrrolidin-1-yl)-4- oxobutyl)-5,11,20-trioxo-6,10,15,19-tetraazadocosan-1,24-diyl)bis(oxy))bis(5- acetamido-2-(acetyloxymethyl)tetrahydro-2H-pyran-6,3,4-triyl)tetraacetate (800 mg, 0.402 mmol), dichloromethane (30 mL), succinic anhydride (321 mg, 3.21 mmol), and 4-dimethylaminopyridine (12.3 mg, 0.101 mmol) and stirred at room temperature for 16 h under nitrogen. The reaction was concentrated under reduced pressure and the crude product was purified by reverse phase preparative HPLC (column: Waters Xbridge Prep C18 (150 mm x 40 cm x 10 pm), mobile phase: A = water (0.1% ammonium bicarbonate), B = acetonitrile; gradient: 15% - 45%) to give 4-(((3R,5S)-1-(4-(4-(5-(((2R,3R,4R,5R,6R))-3-acetamido-4,5-diacetoxy-6- (acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-N-(3-(5-(((2R,3R,4R,5R,6R)-3- acetamido-4,5-diacetoxy-6-(acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)pentanamido)-N-(3-(5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy)-6- (acetyloxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanamido)propyl)butanamido)butanoyl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)pyrrolidin-3-yl)oxy)-4- oxobutanoic acid (I-3) (630 mg, 75.0% yield).

[0219] 1H NMR (400 MHz, DMSO-d6) δ 7.90-7.79 (m, 4H), 7.76-7.69 (m, 1H), 7.31-7.27 (m, 4H), 7.23-7.17 (m, 5H), 6.89-6.87 (m, 4H), 5.37-5.28 (m, 1H), 5.21 (d, J = 2.9 Hz, 3H), 4.97 (dd, J = 11.2, 3.0 Hz, 3H), 4.49 (d, J = 8.4 Hz, 3H), 4.21 (s, 1H), 4.02 (s, 9H), 3.91-3.84 (m, 3H), 3.73-3.70 (m, 9H), 3.56-3.39 (m, 7H), 3.23-3.19 (m, 8H), 3.04-2.97 (m, 4H), 2.49-2.40 (m, 4H), 2.29-2.17 (m, 6H), 2.09 (s, 9H), 2.05-2.04 (m, 4H), 1.99 (s, 9H), 1.89 (s, 9H), 1.77 (s, 9H), 1.72-1.40 (m, 22H).

[0220] Example 1: Design and synthesis of siRNA conjugates

[0221] The synthesis step of conjugating group to the siRNA sequence of the 5' end of the siRNA sense strand, for example C1 in Table 3, is shown below:

[0222] 1) Synthesis of single-stranded oligoribonucleotides: Oligoribonucleotides were synthesized according to the phosphoramidite solid-phase synthesis technique. The synthesis was performed on universal controlled-pore glass CPG . All phosphoramidite monomers (Genscript & ZOWE) and auxiliary reagents were commercially available reagents. All phosphoramidites were dissolved in anhydrous acetonitrile (Kelongma, Suzhou) and added with molecular sieves . The coupling time was 8-12 minutes using 5-ethylthio-1H-tetrazole (ETT) as an activator (Kelongma, Suzhou). A 0.05 M iodine solution (dissolved in pyridine / water = 9:1, Kelongma, Suzhou) was used to build phosphate bonds; a 0.2 M solution of hydrogenated xanthate (Kelongma, Suzhou) in anhydrous acetonitrile / pyridine (v / v = 1 / 1) was used to generate thiophosphate bonds, with a reaction time of 5 minutes. All sequences were completed after the DMT group was removed at the end of the synthesis.

[0223] 2) Cleavage and deprotection of oligoribonucleotides bound to CPG: After the solid-phase synthesis was terminated, the protecting group was removed by treating with a 20% diethylamine (Guo Yao) acetonitrile solution for 10 minutes. The resulting CPG carrier was subjected to heated ammonolysis with concentrated ammonia water (Guo Yao) to remove the protecting groups on the carrier and bases, and the solution containing the product was obtained after filtration.

[0224] 3) Purification of single-stranded oligoribonucleotides: The oligomers were purified by HPLC using NanoQ anion exchange. Buffer A was 20 mM sodium hydroxide solution; and buffer B, containing 20 mM sodium hydroxide solution, 3 M sodium chloride, the target product was separated. The target product obtained was further desalted by gel.

[0225] 4) Coupling and purification of conjugated groups: The purified nucleic acid sequence with NH2-C6 at the end (prepared and purified by conventional methods in the art) was dissolved in 100 mM PB buffer, and the ligand I-1 of Preparation Example 1 was dissolved in DMF or DMSO and then added to the nucleic acid solution, and reacted at room temperature for 4-16 h, and then quenched with water. After removing the organic reagent by desalting the mixed solution, purification was performed, and finally the nucleic acid chain with conjugated groups at the end was obtained.

[0226] 5) The sense strand and the antisense strand obtained by chemical synthesis were subjected to base complementary pairing according to a molar ratio of 1:1, and the reaction conditions were 70°C, 10 min, and slow recovery to room temperature, and finally the product was obtained.

[0227] In the siRNA conjugate obtained by the present application, the nucleotide sequences of the sense strand and the antisense strand are shown in Table 3.

[0228] Test Example 1: In vitro dual-luciferase psiCHECK-2 vector HEK293 cell screening

[0229] In a 96-well cell plate, 3 μL of double-stranded siRNA was added to each well, and two replicates of each double-stranded siRNA were added, and then 2 μL of psiCHECK-2 vector plasmid (referred to as plasmid) carrying the INHBE gene at a concentration of 100 ng / μL, 20 μL of Opti-MEM medium (Gibco, item number 31985-070) were added to each well to obtain a mixture containing siRNA and plasmid. 0.5 μL of Lipofectamine 2000 (Invitrogen, item number 11668-019) was added to 24.5 μL of Opti-MEM medium, incubated for 5 minutes, and then added to the mixture containing double-stranded siRNA and plasmid in each well. After incubation at room temperature for 20 minutes, 100 μL of 5 x 10 4Dulbecco's Modified Eagle Medium medium (Gibco, Cat. No. C11995500BT) was added to each well containing a mixture of double-stranded siRNA, plasmid and Lipofectamine 2000 transfection reagent. After incubating the cells for 24 hours, the measurement of firefly luciferase and Renilla luciferase was performed according to the dual luciferase reporter assay kit (Promega, Cat. No. DD1205-02). Among them, the double-stranded siRNA was set at different concentrations, and the concentration of the double-stranded siRNA was 10 nM, 1 nM, 0.1 nM and 0.01 nM in the total volume of each well. The EC 50 value of part of the double-stranded siRNA was tested, and the concentration range was 5 nM to 0.05 pM, with a three-fold concentration step dilution.

[0230] The test results showed that the siRNA of the application had good silencing effect. Among them, the measurement results of part of the siRNA in the test example at part of the concentration were shown in Table 4.

[0231] Table 4

[0232] Table 5

[0233] In the above table, "-" represents not tested.

[0234] Test Example 2: Inhibition of INHBE mRNA expression by free uptake of siRNA conjugate by human primary hepatocytes

[0235] The primary human hepatocytes (PHH) were provided by Chengdu Pharmalink New Drug Development Co., Ltd.

[0236] The main reagents and consumables used in this experiment include: RNA extraction kit (Qiagen-74182), HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme-R323-01), AceQ Universal U Probe Master Mix V2 (Vazyme-Q513-02) and 96-well plate (Costar 3599). Other reagents and consumables were provided by Pharmalink.

[0237] The diluted siRNA conjugate was added to the collagen-coated 96-well cell plate, and then the PHH cell suspension was added. At the same time, an siRNA conjugate-free group containing Nuclease-Free Water was set as a control group. Incubation was performed in a 5% CO2, 37°C incubator for 48 h.

[0238] The culture medium was removed, the cell plate was washed once with PBS, and cell lysis solution was added. Total RNA was extracted using an RNA extraction kit (Qiagen-74182). After removing gDNA, cDNA was synthesized using random primers by reverse transcription kit HiScript III RT SuperMix for qPCR.

[0239] The target gene cDNA was detected by qPCR, and the corresponding internal reference gene (GAPDH cDNA) was detected in parallel. The expression level of the target gene mRNA of each sample was calculated by ΔΔCT quantitative method. The relative expression amount of the target gene was represented by 2-ΔΔCT.

[0240] The calculation formula is as follows:

[0241] ΔCT = average Ct value of target gene - average Ct value of internal reference gene;

[0242] ΔΔCT = ΔCT (drug group) - ΔCT (Nuclease-Free Water control group);

[0243] Relative expression amount of target gene = 2-ΔΔCT;

[0244] Inhibition rate of target gene % = (1-sample relative expression amount / Nuclease-Free Water group average relative expression amount) x 100%;

[0245] GraphPad Prism software was used for graphing and analysis, and the inhibition rate results were expressed as mean ± SD.

[0246] The results show that the siRNA conjugate obtained in Example 1 of the present application has a relatively significant silencing effect on the INHBE gene expression in human primary hepatocytes under free uptake conditions. In the test example, the measurement results of the exemplary part of the siRNA at part of the concentration are shown in Table 6.

[0247] The positive control is AC004285 in WO2025049773A1:

[0248] SS: (NAG37) s (invAb) scuggcuuaUfaCfUfuucuuaauas (invAb) (SEQ ID NO: 1191);

[0249] AS:usAfsuuAfagaaagUfaUfaAfgccassg (SEQ ID NO: 1192).

[0250] Table 6

[0251] Test Example 3: Inhibition of INHBE mRNA expression by free uptake of siRNA conjugates by cynomolgus monkey primary hepatocytes

[0252] The prepared siRNA conjugates were configured into corresponding concentration stock solutions with Nuclease-Free Water.

[0253] The main reagents and consumables used in this experiment include: AceQ Universal U Probe Master Mix V2 (Vazyme-Q513-P8), EZ-Press 96 RNA Purification Kit (EZB-EZ4001-L), HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme-R323), and 96-well cell culture plates (Cellpro-803096). The primers and probes of the target gene INHBE and the internal reference gene β-actin were synthesized by Shanghai Sangon Biological Engineering Co., Ltd. Cynomolgus monkey primary hepatocytes (PCH, Catalog No.: CCH100CY-V10088) were provided by Chengdu Pharmalink New Drug Development Co., Ltd.

[0254] Inoculate PCH cells (4.5 x 10 4The cells were plated into a pre-collagen coated 96-well cell culture plate at a density of 5,000 cells / well, and the siRNA compounds were added at the same time. A Nuclease-Free Water control group without the compounds was also set up. The cells were incubated in a 37℃ 5% CO2 incubator for 48 hours. After 48 hours, the culture medium was removed and the cells were collected for RNA extraction. Total RNA was extracted using the EZ-Press 96 RNA Purification Kit (EZB-EZ4001-L) according to the kit instructions. cDNA was synthesized using the HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme-R323) according to the instructions. The target gene INHBE was detected by Taqman qPCR, and β-actin cDNA was detected as an internal control. 8 μL of prepared PCR reaction solution and 2 μL of sample cDNA were added to a 384-well plate. The qPCR reaction program was as follows: 37℃ preheating for 2 minutes, 95℃ heating for 5 minutes, then entering the cycle mode, 95℃ heating for 10 seconds, followed by 60℃ for 30 seconds, a total of 45 cycles.

[0255] The expression level of each sample target gene mRNA was calculated by the ΔΔCT relative quantification method. The relative expression of the target gene was calculated using 2 -ΔΔCT . The calculation formula is as follows:

[0256] ΔCT = average CT value of target gene - average CT value of internal reference gene

[0257] ΔΔCT = ΔCT (sample group) - ΔCT (control group)

[0258] Relative expression of target gene = 2 -ΔΔCT

[0259] Inhibition rate % = (1 - relative expression of sample / average relative expression of control group) x 100

[0260] The test results are shown in Table 7.

[0261] Table 7

[0262] The results show that the siRNA conjugates of the present application have a significant silencing effect on the expression of the INHBE gene in cynomolgus monkey primary hepatocytes under free uptake conditions.

[0263] Test Example 4: Inhibition of INHBE mRNA expression in INHBE humanized mouse liver by siRNA conjugates

[0264] This example uses B-hINHBE mice to evaluate the inhibitory activity of siRNA on the target gene INHBE. B-hINHBE mice are provided by BioS&T Jiangsu Gene Biotechnology Co., Ltd.

[0265] Animal grouping, drug administration and tissue sample collection:

[0266] 6-8 week old B-hINHBE mice were randomly grouped by weight (all male), with 5 mice in each group. Each test group was given a predetermined dose of siRNA and a PBS group was added. All mice were calculated for drug dosage according to body weight, and were given a single dose by subcutaneous injection. Each siRNA was administered in the form of a 0.6 mg / mL PBS solution, with a drug volume of 5 mL / kg of mouse body weight, i.e. the drug dosage of each siRNA was 3 mg / kg of mouse body weight. The PBS control group was given the same volume of PBS solution (without siRNA). The day of administration was recorded as day 0 (Day 0). All mice were sacrificed on day 7 after administration (Day 7), and mouse liver tissue was collected, frozen in liquid nitrogen and stored at -80°C.

[0267] An appropriate amount of liver tissue was thawed on ice and VeZol Reagent (Novozyme, Catalog No. R411-02) was added. The tissue homogenate was prepared using a full-automatic sample cryogenic grinder (Shanghai Jingxin Industrial Development Co., Ltd., Model JXFSTPRP-CLN). The VAMNE Magnetic Universal Total RNA Kit (Novozyme, Catalog No. ROA3302-02) nucleic acid extraction kit was then used to extract total RNA from the tissue homogenate.

[0268] For each mouse, 1000 ng of total RNA was taken and a 20 μL reverse transcription system was prepared using the reverse transcription kit HiScript III All-in-one RT SuperMix Perfect for qPCR (Novozyme, Catalog No. R333) to complete the reverse transcription reaction and obtain cDNA solution. After diluting the cDNA 10-fold, the Taq Pro HighGC U+Multiple Probe qPCR Mix (Novozyme, Catalog No. QN211-02) kit was used to detect the expression of hINHBE mRNA in mouse liver tissue. In this real-time fluorescence quantitative PCR method, mGAPDH was used as an internal reference gene, and the expression of hINHBE mRNA was normalized with the expression of mGAPDH. Then, the expression of the PBS control group was taken as the 100% reference to calculate and compare. The test results are shown in Table 8.

[0269] Table 8

[0270] The results show that the siRNA conjugate has a remarkable silencing effect on INHBE gene expression in the INHBE humanized mice.

[0271] All documents referred to in this disclosure are incorporated by reference herein as if each individual document were incorporated by reference. In addition, it is to be understood that various alterations and modifications can be made to the application herein disclosed in the drawing figures and described above, and it is intended that the application encompass all such alterations and modifications as fall within the scope of the appended claims.

Claims

1. An siRNA that inhibits the expression of the inhibin subunit βE (INHBE) gene, characterized in that, The siRNA comprises a sense strand and an antisense strand; The antisense strand comprises at least 19 consecutive nucleotides differing by 0, 1, 2, or 3 nucleotides from any one of the sequences shown in Table 1; Table 1 The sense strand is complementary to the antisense strand by at least 15, 16, 17, 18, 19, 20, 21, or 22 nucleotides; Each nucleotide of the sense strand and the antisense strand is independently a modified or unmodified nucleotide.

2. The siRNA of claim 1, wherein At least one nucleotide in the sense strand and the antisense strand is a modified nucleotide; Preferably, the modified nucleotide is selected from the group consisting of: a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy nucleotide, a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-amino modified nucleotide, a 2'-alkyl modified nucleotide, a 2'-alkoxy modified nucleotide, a phosphorothioate modified nucleotide, an abasic nucleotide, a morpholino nucleotide, a locked nucleotide, or a combination thereof; More preferably, the modified nucleotide is selected from the group consisting of: a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a phosphorothioate modified nucleotide, or a combination thereof.

3. The siRNA of claim 1, wherein The antisense strand comprises at least 19, 20, 21, 22, 23 consecutive nucleotides differing by 0, 1, 2, or 3 nucleotides from any one of the sequences shown in Table 1; Preferably, the antisense strand comprises at least 21 consecutive nucleotides differing by 0, 1, 2, or 3 nucleotides from any one of the sequences shown in Table 1.

4. The siRNA of claim 1, wherein The modified nucleotide comprises a modification selected from the group consisting of: (1) from 5' end to 3' end, the 9th, 11th, 13th nucleotide of the sense strand is a 2'-fluoro modified nucleotide, and the nucleotide at the rest of the positions is a 2'-O-methyl modified nucleotide; (2) from 5' end to 3' end, the 2nd, 7th, 12th, 14th, 16th nucleotide of the antisense strand is a 2'-fluoro modified nucleotide, and the nucleotide at the rest of the positions is a 2'-O-methyl modified nucleotide; (3) the 5' end and the 3' end of the sense strand independently comprises 0, 1, 2, or 3 phosphorothioate modified nucleotides; (4) the 5' end and the 3' end of the antisense strand independently comprises 0, 1, 2, or 3 phosphorothioate modified nucleotides; (5) any combination of (1)-(4) above.

5. The siRNA of claim 1, wherein The antisense strand and the sense strand are directly or indirectly connected by a phosphate, a phosphorothioate, or a phosphorodithioate.

6. The siRNA of claim 1, wherein the 1st nucleotide of the 5' terminus of the antisense strand is a nucleotide selected from the group consisting of: In the formula, B is adenosine-3'-phosphate (A), n is 2'-O-methyladenosine-3'-phosphate (a); or B is uridine 3'-phosphate (U), n is 2'-O-methyluridine-3'-phosphate (u).

7. The siRNA of claim 1, wherein The antisense strand comprises or consists of a nucleotide sequence as set forth in SEQ ID NO: (m1+41); and the sense strand comprises or consists of a nucleotide sequence as set forth in SEQ ID NO: m1; wherein m1 is any integer number from 1-41 or 83-123; or the antisense strand comprises or consists of a nucleotide sequence as set forth in SEQ ID NO: (m2+15); and the sense strand comprises or consists of a nucleotide sequence as set forth in SEQ ID NO: m2; wherein m2 is any integer number from 206-220 or 236-250; or the antisense strand comprises or consists of a nucleotide sequence as set forth in SEQ ID NO: (m3+45); and the sense strand comprises or consists of a nucleotide sequence as set forth in SEQ ID NO: m3; wherein m3 is any integer number from 281-325 or 371-415; or the antisense strand comprises or consists of a nucleotide sequence as set forth in SEQ ID NO: (m4+121); and the sense strand comprises or consists of a nucleotide sequence as set forth in SEQ ID NO: m4; wherein m4 is any integer number from 506-626 or 748-868; or the antisense strand comprises or consists of a nucleotide sequence as set forth in SEQ ID NO: (m5+3); and the sense strand comprises or consists of a nucleotide sequence as set forth in SEQ ID NO: m5; wherein m5 is any integer number from 1169-1171 or 1176-1178; or the antisense strand comprises or consists of a nucleotide sequence as set forth in SEQ ID NO: 1175; and the sense strand comprises or consists of a nucleotide sequence as set forth in SEQ ID NO: 1169; or the antisense strand comprises or consists of a nucleotide sequence as set forth in SEQ ID NO: 1182; and the sense strand comprises or consists of a nucleotide sequence as set forth in SEQ ID NO: 1176; or the antisense strand comprises or consists of a nucleotide sequence as set forth in SEQ ID NO: 1186; and the sense strand comprises or consists of a nucleotide sequence as set forth in SEQ ID NO: 1187.

8. An siRNA conjugate comprising the siRNA of any one of claims 1-7 and a targeting ligand.

9. The siRNA conjugate of claim 8, wherein, the targeting ligand is conjugated to the 5' end or 3' end of the sense strand of the siRNA.

10. The siRNA conjugate as described in claim 8, characterized in that, The targeting ligand has a structure selected from any one of the following formulae (I)-(XII): wherein represents a linking point to the siRNA or linker.

11. The siRNA conjugate as described in claim 8, characterized in that, the targeting ligand is linked to the 3' end or 5' end of the sense strand of the siRNA, and the targeting ligand has a structure as set forth in any one of Formulas (I)-(XII); preferably, the targeting ligand is linked to the 5' end of the sense strand of the siRNA, and the targeting ligand has a structure as set forth in Formula (I).

12. The siRNA conjugate of claim 8, wherein, the antisense strand of the conjugate comprises or consists of a nucleotide sequence as set forth in any one of SEQ ID NOs: 124-164, 251-265, 416-460, 869-989, 1140-1168, 1179-1182, and 1189; and / or the sense strand thereof comprises or consists of a nucleotide sequence as set forth in any one of SEQ ID NOs: 165-205, 266-280, 461-505, 990-1139, 1183-1185, and 1190.

13. A pharmaceutical composition comprising: (a) the siRNA of any one of claims 1-7, and / or the conjugate of any one of claims 8-12; and (b) a pharmaceutically acceptable carrier.

14. Use of the siRNA of any one of claims 1-7, and / or the conjugate of any one of claims 8-12, or the pharmaceutical composition of claim 13 in the preparation of a medicament for preventing and / or treating an INHBE-mediated disease; Preferably, the INHBE-mediated disease is a disease of INHBE overexpression; Preferably, the disease is a metabolic system disease or a cardiovascular disease; Preferably, the metabolic system disease comprises: diabetes, lipid metabolism disorder; and / or, the cardiovascular disease comprises: hypertension, atherosclerosis, cardiomyopathy, heart failure; Preferably, the diabetes comprises: prediabetes, such as type I diabetes, type II diabetes; and / or, the lipid metabolism disorder comprises: body weight disorder, triglyceride elevation, fatty liver disease, hypercholesterolemia, fatty liver disease, or nonalcoholic steatohepatitis. the antisense strand of the conjugate comprises or consists of a nucleotide sequence as set forth in any one of SEQ ID NOs: 124-164, 251-265, 416-460, 869-989, 1140-1168, 1179-1182, and 1189; and / or the sense strand thereof comprises or consists of a nucleotide sequence as set forth in any one of SEQ ID NOs: 165-205, 266-280, 461-505, 990-1139, 1183-1185, and 1190.

13. A pharmaceutical composition comprising: (a) the siRNA of any one of claims 1-7, and / or the conjugate of any one of claims 8-12; and (b) a pharmaceutically acceptable carrier.

14. Use of the siRNA of any one of claims 1-7, and / or the conjugate of any one of claims 8-12, or the pharmaceutical composition of claim 13 in the preparation of a medicament for preventing and / or treating an INHBE-mediated disease; Preferably, the INHBE-mediated disease is a disease of INHBE overexpression; Preferably, the disease is a metabolic system disease or a cardiovascular disease; diabetes, lipid metabolism disorder; and / or, the cardiovascular disease comprises: hypertension, atherosclerosis, cardiomyopathy, heart failure; Preferably, the diabetes comprises: prediabetes, such as type I diabetes, type II diabetes; and / or, the lipid metabolism disorder comprises: body weight disorder, triglyceride elevation, fatty liver disease, hypercholesterolemia, fatty liver disease, or nonalcoholic steatohepatitis.

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