Rnai agent targeting MARC1 gene

Abstract

Provided is an RNAi agent targeting an MARC1 gene. Provided are a group of siRNAs for inhibiting MARC1 gene expression, derivative sequences containing nucleotide modifications and siRNA conjugates. Further provided are the use of the siRNAs, derivative sequences, and conjugates.

Description

RNAi agents targeting the MARC1 gene

[0001] This application claims priority to Chinese patent applications 202510387020.7 (filed March 28, 2025), 202510992054.9 (filed July 17, 2025), 202610021490.6 (filed January 18, 2026), and 202610179986.6 (filed February 6, 2026). The full text of the aforementioned Chinese patent applications is incorporated herein by reference. Technical Field

[0002] This invention relates to the field of biomedicine. Specifically, this invention relates to RNAi agents targeting the MARC1 gene. Background Technology

[0003] Mitochondrial amidoxime reducing component 1 (MARC1) is a molybdenum-containing enzyme that reduces N-hydroxylated compounds and is associated with the outer mitochondrial membrane. A common missense variant of the MARC1 gene has recently been shown to protect the liver from fatty liver and cirrhosis from various causes. It has been reported that carriers of the MARC1 variant have higher concentrations of polyunsaturated phosphatidylcholine in the liver, which may be related to NASH-related pathogenesis. Liver disease is a leading cause of death and disability worldwide; chronic disease gradually impairs hepatocyte regeneration and even destroys them, leading to liver fibrosis and cirrhosis. Recent genome-wide association studies (GWAS) on liver disease and autoimmune hepatitis have further validated the association between MARC1 and liver disease, confirming that missense mutations in MARC1 and the resulting loss of function can prevent liver damage and cirrhosis. These data suggest that reducing MARC1 protein may lower blood cholesterol levels and prevent cirrhosis; therefore, inhibiting MARC1 is a potential therapeutic target for liver disease. Currently, there are no drugs on the market that specifically target this type of gene expression, so developing drugs that target MARC1 is of great value. Summary of the Invention

[0004] The purpose of this invention is to provide an siRNA that can inhibit the expression and / or activity of the MARC1 gene for the treatment of MARC1-related conditions, such as metabolic syndrome, hypertension, and cardiovascular diseases related to the expression of the MARC1 gene.

[0005] In a first aspect of the invention, a siRNA is provided that inhibits the expression of the mitochondrial micaceous oxime reducing component 1 (MARC1) gene, said siRNA comprising a sense strand and an antisense strand.

[0006] The antisense strand comprises at least 15 consecutive nucleotides that differ from any of the sequences shown in Table 1 by 0, 1, 2 or 3 nucleotides;

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

[0008] Each nucleotide in the sense strand and antisense strand is independently a modified or unmodified nucleotide.

[0009] In another preferred embodiment, the antisense strand comprises at least 21 consecutive nucleotides that differ from any of the sequences shown in Table 1 by 0, 1, 2 or 3 nucleotides;

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

[0011] In another preferred embodiment, the antisense strand comprises at least 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive nucleotides that differ from any of the sequences shown in Table 1 by 0, 1, 2 or 3 nucleotides.

[0012] In another preferred embodiment, the antisense strand comprises at least 21 consecutive nucleotides that differ from any of the sequences shown in Table 1 by 0, 1, 2 or 3 nucleotides.

[0013] In another preferred embodiment, the antisense strand comprises at least 19, 20, 21, or 22 (preferably 21) consecutive nucleotides that differ from the nucleotide sequences shown in any one of SEQ ID NO:101-200, SEQ ID NO:570-598, and SEQ ID NO:668-678 by 0, 1, 2, or 3 nucleotides.

[0014] In another preferred embodiment, the positive strand comprises at least 19, 20, 21, or 22 (preferably 19 or 21) consecutive nucleotides that differ from the nucleotide sequences shown in any one of SEQ ID NO:1-100, SEQ ID NO:541-569, and SEQ ID NO:657-667 by 0, 1, 2, or 3 nucleotides.

[0015] In another preferred embodiment, the antisense strand is 17-23 base pairs complementary to the human MARC1 transcript (NM 022746.4).

[0016] In another preferred embodiment, the lengths of the sense strand and the antisense strand are each independently 19, 20, 21, 22, or 23 nucleotides.

[0017] In another preferred embodiment, the antisense strand has two nucleotides deleted at the 3' end relative to the sense strand.

[0018] In another preferred embodiment, the nucleotide sequence of the antisense strand is shown in any one of SEQ ID NO:101-200, SEQ ID NO:570-598 and SEQ ID NO:668-678.

[0019] In another preferred embodiment, the nucleotide sequence of the positive strand is shown in any one of SEQ ID NO:1-100, SEQ ID NO:541-569 and SEQ ID NO:657-667.

[0020] In another preferred embodiment, at least one nucleotide in the sense strand and the antisense strand is a modified nucleotide.

[0021] In another preferred embodiment, one or more nucleotides of the sense strand and / or the antisense strand are linked by non-standard linkages or a backbone (i.e., modified internucleotide linkages or modified backbones).

[0022] In another preferred embodiment, the modified internucleotide linkage or skeleton includes, but is not limited to, thiophosphate groups, chiral thiophosphates, thiophosphates, dithiophosphates, phosphate triesters, aminoalkyl-phosphate triesters, chiral phosphonates, hypophosphonates, aminophosphates, thioalkylphosphonates, thioalkyl phosphate triesters, and morpholino linkages.

[0023] In another preferred embodiment, the sense chain and / or the antisense chain comprises one or more thiophosphate-modified nucleoside inter-linking bonds.

[0024] In another preferred embodiment, the sense chain and / or the antisense chain independently comprise 2, 3, 4, or 5 thiophosphate bonds.

[0025] In another preferred embodiment, the sense chain and / or the antisense chain independently comprise 4 or 5 thiophosphate bonds.

[0026] In another preferred embodiment, the modified nucleotide is selected from: 2'-O-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, phosphate thioester modified nucleotides, debased nucleotides, morpholinonucleotides, locked nucleotides, reverse base modified nucleotides, or combinations thereof.

[0027] In another preferred embodiment, the modified nucleotide is selected from: nucleotides modified with 2'-O-methyl, nucleotides modified with 2'-fluoro, nucleotides modified with thiophosphate, nucleotides modified with reverse bases, or combinations thereof.

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

[0029] (1) From the 5' end to the 3' end, the nucleotides at positions 9, 11, and 13 of the positive strand are nucleotides modified with 2'-fluorine, and the nucleotides at the remaining positions are nucleotides modified with 2'-O-methyl.

[0030] (2) From the 5' end to the 3' end, the nucleotides at positions 7, 9, and 11 of the positive strand are nucleotides modified with 2'-fluorine, and the nucleotides at the remaining positions are nucleotides modified with 2'-O-methyl.

[0031] (3) From the 5' end to the 3' end, the nucleotides at positions 2, 7, 12, 14 and 16 of the antisense strand are nucleotides modified with 2'-fluorine, and the nucleotides at the remaining positions are nucleotides modified with 2'-O-methyl.

[0032] (4) The 5' end and 3' end of the positive strand each independently contain 0, 1, 2 or 3 phosphate-thioester modified nucleotides;

[0033] (5) The 5' end and 3' end of the antisense strand each independently contain 0, 1, 2 or 3 phosphate thioester modified nucleotides;

[0034] (6) Any combination of (1)-(5) above.

[0035] In another preferred embodiment, the antisense strand comprises at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides differing by 0, 1, 2, or 3 nucleotides from the nucleotide sequences shown in any one of SEQ ID NO:301-400, SEQ ID NO:471-540, SEQ ID NO:628-656, SEQ ID NO:690-700, and SEQ ID NO:712-722.

[0036] In another preferred embodiment, the antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NO:301-400, SEQ ID NO:471-540, SEQ ID NO:628-656, SEQ ID NO:690-700 and SEQ ID NO:712-722.

[0037] In another preferred embodiment, the positive strand comprises at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides differing by 0, 1, 2, or 3 nucleotides from the nucleotide sequences shown in any one of SEQ ID NO:201-300, SEQ ID NO:401-470, SEQ ID NO:599-627, SEQ ID NO:679-689, and SEQ ID NO:701-711.

[0038] In another preferred embodiment, the positive strand comprises the nucleotide sequence shown in any one of SEQ ID NO:201-300, SEQ ID NO:401-470, SEQ ID NO:599-627, SEQ ID NO:679-689 and SEQ ID NO:701-711.

[0039] In another preferred embodiment, the antisense strand comprises or is composed of any of the nucleotide sequences shown in SEQ ID NO:(m1+100), SEQ ID NO:471-540, SEQ ID NO:570-598, SEQ ID NO:628-656, SEQ ID NO:668-678, SEQ ID NO:690-700, and SEQ ID NO:712-722; and the sense strand comprises or is composed of any of the nucleotide sequences shown in SEQ ID NO:m1, SEQ ID NO:401-470, SEQ ID NO:541-569, SEQ ID NO:599-627, SEQ ID NO:657-667, SEQ ID NO:679-689, and SEQ ID NO:701-711; wherein m1 is any integer number from 1 to 100 or from 201 to 300.

[0040] In another preferred embodiment, the antisense strand and the sense strand form a double strand of nucleic acid sequence pairs as shown in either Table B or Table C.

[0041] In a second aspect of the invention, an siRNA conjugate is provided, the siRNA conjugate comprising the siRNA as described in the first aspect of the invention and a targeting ligand and / or a reverse debasement nucleotide.

[0042] In another preferred embodiment, the targeting ligand is coupled to the 5' or 3' end of the siRNA's positive strand.

[0043] In another preferred embodiment, the reverse debase nucleotide is conjugated to the 3' end of the sense strand and / or antisense strand of the siRNA.

[0044] 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.

[0045] In another preferred embodiment, the targeting ligand is a lipophilic delivery portion.

[0046] In another preferred embodiment, the targeting ligand is a liver-targeted delivery fraction.

[0047] In another preferred embodiment, the targeting ligand is selected from the group consisting of:

[0048] in, This represents the connection point that is directly or indirectly connected to the siRNA.

[0049] According to embodiments of the present invention, the nucleic acid conjugate (complex) has the following structure:

[0050] in, This indicates the siRNA described in this article; X represents O or S.

[0051] In another preferred embodiment, the structural formula of the reverse-debased nucleotide is shown below:

[0052] Here, 'a' is the 5' end of the chain pointing toward the justice chain or the antisense chain, and 'b' is the 3' end of the chain pointing toward the justice chain or the antisense chain.

[0053] In another preferred embodiment, the oxygen atom in the targeting ligand is attached to the 5' position of the first nucleotide of the 5' strand of the positive chain to form a phosphate ester or a thiophosphate ester.

[0054] In another preferred embodiment, the targeting ligand is 2'-O-hexadecyl or

[0055] In another preferred embodiment, the targeting ligand is one or more of any cell-targeting moieties, preferably lipids, carbohydrates, aptamers, vitamins and / or peptides that bind to specific targets on the cell membrane or cell surface.

[0056] In a third aspect of the invention, an expression vector is provided, the expression vector containing a polynucleotide encoding siRNA as described in the first aspect of the invention.

[0057] In a fourth aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0058] (a) siRNA as described in the first aspect of the invention, and / or conjugates as described in the second aspect of the invention, or expression vectors as described in the third aspect of the invention; and

[0059] (b) Pharmaceutically acceptable carriers.

[0060] In another preferred embodiment, the pharmaceutical composition is used to inhibit the expression of the MARC1 gene.

[0061] In a fifth aspect of the invention, the use of siRNA as described in the first aspect of the invention, conjugates as described in the second aspect of the invention, expression vectors as described in the third aspect of the invention, or pharmaceutical compositions as described in the fourth aspect of the invention in the preparation of medicaments for the prevention and / or treatment of MARC1-mediated diseases is provided.

[0062] In another preferred embodiment, the drug is used to inhibit the expression of the MARC1 gene.

[0063] In another preferred embodiment, the MARC1-mediated disease is a disease of MARC1 overexpression.

[0064] In another preferred embodiment, the disease is a metabolic system disease or a cardiovascular disease.

[0065] In another preferred embodiment, the metabolic system disease includes: lipid metabolism disorder.

[0066] In another preferred embodiment, the lipid metabolism disorder includes: weight imbalance, elevated triglycerides, fatty liver disease, hypercholesterolemia, or non-alcoholic steatohepatitis.

[0067] In another preferred embodiment, the cardiovascular disease includes: hypertension, atherosclerosis, cardiomyopathy, and heart failure.

[0068] In a sixth aspect of the present invention, a method for inhibiting MARC1 expression in cells in vitro is provided, the method comprising the following steps:

[0069] The cells are co-cultured with an effective amount of siRNA as described in the first aspect of the invention, a conjugate as described in the second aspect of the invention, an expression vector as described in the third aspect of the invention, or a pharmaceutical composition as described in the fourth aspect of the invention.

[0070] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0071] In another preferred embodiment, the cell is a cell expressing the MARC1 gene, such as a hepatocyte.

[0072] In a seventh aspect of the invention, a method for treating MARC1-mediated diseases is provided, comprising the steps of administering a therapeutically effective amount of siRNA as described in the first aspect of the invention, a conjugate as described in the second aspect of the invention, an expression vector as described in the third aspect of the invention, or a pharmaceutical composition as described in the fourth aspect of the invention to a subject.

[0073] In another preferred embodiment, the MARC1-mediated disease is a disease of MARC1 overexpression.

[0074] In another preferred embodiment, the disease is a metabolic system disease or a cardiovascular disease.

[0075] In another preferred embodiment, the metabolic system disease includes: lipid metabolism disorder.

[0076] In another preferred embodiment, the lipid metabolism disorder includes: weight imbalance, elevated triglycerides, fatty liver disease, hypercholesterolemia, or non-alcoholic steatohepatitis.

[0077] In another preferred embodiment, the cardiovascular disease includes: hypertension, atherosclerosis, cardiomyopathy, and heart failure.

[0078] In another preferred embodiment, the subject is a human or a non-human mammal.

[0079] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Detailed Implementation

[0080] the term

[0081] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing the invention, it should be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is intended only to describe particular embodiments and is not intended to be restrictive; the scope of the invention will be limited only by the appended claims.

[0082] As used herein, when referring to a specific enumerated value, the term “about” means that the value can vary by no more than 1% from the enumerated values. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0083] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0084] As used herein, the term "pharmaceutically acceptable carrier" refers to a substance that is suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a reasonable benefit / risk ratio.

[0085] As used herein, the term "therapeutic effective amount" refers to an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals. Those skilled in the art will understand that the "therapeutic effective amount" can vary depending on the form of the pharmaceutical composition, the route of administration, the excipients used, the severity of the disease, and whether it is used in combination with other drugs.

[0086] MARC1

[0087] The mitochondrial amylopectin reducing component (MARC) protein was first discovered and described in 2006 as a molybdenum-containing cofactor component in the mitochondrial amylopectin prodrug conversion system. The human genome contains two MARC genes: MARC1 and 2, whose encoded MARC1 and MARC2 proteins share significant sequence and functional homology.

[0088] Researchers have discovered a rare missense variant (called the p.A165T mutation) in MARC1 that leads to loss of function of the MARC1 protein. This mutation is associated with the prevention of cirrhosis, reduction of liver fat, and decrease in various other liver disease biomarkers. Individuals homozygous for this loss-of-function mutation in MARC1 exhibit lower levels of liver fat. Loss-of-function mutations in MARC1 are also associated with low levels of alanine aminotransferase, alkaline phosphatase, total cholesterol, and LDL cholesterol in the blood.

[0089] Recent genome-wide association studies (GWAS) on liver disease and autoimmune hepatitis have further validated the association between MARC1 and liver disease, confirming that missense mutations in MARC1 and the resulting loss of function can prevent liver damage and cirrhosis. These data suggest that reducing MARC1 protein may lower blood cholesterol levels and prevent cirrhosis; therefore, inhibiting MARC1 is a potential therapeutic target for liver disease.

[0090] siRNA

[0091] The terms "interfering RNA" or "RNAi" or "interfering RNA sequence" include single-stranded RNA (e.g., mature miRNA, ssRNAi oligonucleotide, ssDNAi oligonucleotide) or double-stranded RNA (i.e., double-stranded RNA such as siRNA, dsRNA, shRNA, aiRNA, or precursor miRNA) that, when the interfering RNA is in the same cell as the target gene or sequence, can reduce or inhibit the expression of the target gene or sequence (e.g., by mediating degradation and inhibiting the translation of mRNA complementary to the interfering RNA sequence). Interfering RNA therefore refers to a single-stranded RNA complementary to the target mRNA sequence or a double-stranded RNA formed by two complementary strands or a single self-complementary strand.

[0092] Interfering RNA includes “small interfering RNA” or “siRNA”, each strand of which contains 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 and lengths listed above, as well as intermediate values, are also conceivable to be part of this invention. In one specific embodiment, the siRNA is chemically synthesized. The siRNA of this invention is capable of silencing the expression of target sequences in vitro and / or in vivo. In other embodiments, the siRNA contains at least one modified nucleotide, for example, the siRNA contains one, two, three, four, five, six, seven, eight, nine, ten, or more modified nucleotides in the double-stranded region.

[0093] siRNA molecules mediate targeted cleavage of RNA transcripts via an RNA-induced silencing complex (RISC) pathway. Through a process known as RNA interference (RNAi), iRNA guides the sequence-specific degradation of mRNA. iRNA regulates (e.g., inhibits) the expression of MARC1 in cells (e.g., cells within a subject, such as a mammalian subject). Typically, the majority of the nucleotides in each strand of siRNA are ribonucleotides, but as detailed herein, each or both of the two strands may also include one or more non-ribonucleotides, such as deoxyribonucleotides and / or modified nucleotides. Additionally, as used herein, “siRNA” can include chemically modified ribonucleotides; siRNA can include substantial modifications at multiple nucleotide sites. As used herein, the term “modified nucleotide” refers to a nucleotide that independently has a modified sugar moiety, a modified internucleotide link, and / or a modified nucleotide. Thus, the term modified nucleotide encompasses substitution, addition, or removal of, for example, a functional group or atom, in the internucleotide link, sugar moiety, or nucleotide. Modifications suitable for use in this invention include all types of modifications disclosed herein or known in the art.

[0094] The term "antisense strand" refers to a strand of an iRNA (such as a siRNA) that includes a region substantially complementary to a target sequence (such as a MARC1 mRNA). As used herein, the term "complementary region" refers to a region on the antisense strand that is substantially complementary to a sequence. In cases where the complementary region is not perfectly complementary to the target sequence, mismatches can occur within the molecule or in terminal regions. Typically, the most tolerant mismatches are found in terminal regions, such as within the 5' and / or 3' ends of the siRNA, within 5, 4, 3, or 2 nucleotides.

[0095] The term "sense chain" refers to the chain of iRNAs that contains regions substantially complementary to the regions of the antisense chain as defined herein.

[0096] The antisense and sense strands of siRNA can have the same or different lengths, as described herein and as known in the art.

[0097] As used herein, and unless otherwise indicated, when used to describe a first nucleotide sequence associated with a second nucleotide sequence, the term "complementary" means the ability of an oligonucleotide or polynucleotide containing the first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide containing the second nucleotide sequence under certain conditions and form a double-stranded structure.

[0098] In siRNA, the sense and antisense strands are complementary or substantially complementary. As used herein, “substantially complementary” means that the nucleotide sequences are sufficiently complementary to interact in a predictable manner, such as forming secondary structures (e.g., stem-loop structures). Typically, two “substantially complementary” nucleotide sequences have at least 70% complementary nucleotides to each other; preferably, at least 80%; more preferably, at least 90%; and even more preferably, at least 95%; such as 98%, 99%, or 100%. In this invention, the sense and antisense strands of the siRNA may have up to 4, 3, 2, or 1 mismatched nucleotides.

[0099] Modified nucleotides

[0100] This invention provides siRNA for inhibiting MARC1 gene expression. In some embodiments, the siRNA contains one or more modified nucleotides. In some embodiments, the modified nucleotides include, but are not limited to: 2'-O-methyl modified nucleotides, 2'-fluorine modified nucleotides, 2'-fluorine, 2'-deoxy modified nucleotides, 2'-deoxy nucleotides, 2'-methoxyethyl modified nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, 2'-alkoxy modified nucleotides, 2'-F-arabinonucleotides, debased nucleotides, morpholinonucleotides, and locked nucleotides. Not all positions in a given compound need to be modified uniformly. Instead, more than one modification may be added to a single siRNA or even to a single nucleotide. The modification of one nucleotide is independent of the modification of another nucleotide.

[0101] In some implementations, one or more nucleotides of the siRNA are linked via non-standard linkages or a backbone (i.e., modified internucleotide links or modified backbones). Modified internucleotide links or backbones include, but are not limited to, thiophosphate groups, chiral thiophosphates, thiophosphates, dithiophosphates, phosphate triesters, aminoalkyl-phosphate triesters, chiral phosphonates, hypophosphonates, aminophosphates, thioalkylphosphonates, thioalkyl phosphate triesters, and morpholino linkages.

[0102] In some embodiments, the sense strand of the siRNA may contain 1, 2, 3, 4, 5, or 6 phosphate-thioester bonds (phosphate-thioester modified nucleotides), and the antisense strand of the siRNA may contain 1, 2, 3, 4, 5, or 6 phosphate-thioester bonds (phosphate-thioester modified nucleotides). In some embodiments, the sense strand of the siRNA may contain 1 or 2 phosphate-thioester bonds, and the antisense strand of the siRNA may contain 1, 2, 3, or 4 phosphate-thioester bonds.

[0103] In some embodiments, the siRNA positive strand contains two phosphate-thioester nucleoside links. In some embodiments, the phosphate-thioester nucleoside links are located between nucleotides at positions 1-3 starting from the 5' end of the positive strand. In some embodiments, the phosphate-thioester nucleoside links are located between nucleotides at positions 1-3 starting from the 3' end of the positive strand. In some embodiments, one phosphate-thioester nucleoside link is located at the 5' end of the positive strand, and the other phosphate-thioester link is located at the 3' end of the positive strand. In some embodiments, the siRNA positive strand contains one phosphate-thioester nucleoside link. In some embodiments, the phosphate-thioester nucleoside link is located between nucleotides at positions 1-2 starting from the 5' end of the positive strand. In some embodiments, the phosphate-thioester nucleoside link is located between nucleotides at positions 2-3 starting from the 5' end of the positive strand.

[0104] In some embodiments, the siRNA antisense strand contains four phosphate-thioester nucleoside links. In some embodiments, the four phosphate-thioester nucleoside links are located between nucleotides at positions 1-3 from the 5' end of the antisense strand and between nucleotides at positions 1-3 from the 5' end. In some embodiments, the siRNA antisense strand contains three phosphate-thioester nucleoside links. In some embodiments, the three phosphate-thioester nucleoside links are located between nucleotides at positions 1-2 from the 5' end of the antisense strand and between nucleotides at positions 1-3 from the 3' end. In some embodiments, the three phosphate-thioester nucleoside links are located between nucleotides at positions 1-3 from the 5' end of the antisense strand and between nucleotides at positions 1-2 from the 3' end. In some embodiments, the siRNA antisense strand contains two phosphate-thioester nucleoside links. In some embodiments, the two thiophosphate nucleotide links are located between nucleotides 1-2 positions from the 5' end of the antisense strand and between nucleotides 1-2 positions from the 5' end of the antisense strand.

[0105] In some alternative embodiments of the invention, one or more inverted abasic residues (invAbs) are added to the 3' end of the sense strand and / or antisense strand. In some alternative embodiments of the invention, one or more inverted abasic residues (invAbs) are added to the 5' end of the sense strand and / or antisense strand. In some embodiments, one or more inverted abasic residues or inverted abasic nucleotides are inserted between the nucleotide sequence of the targeting ligand and the sense strand. In some alternative embodiments of the invention, one or more inverted abasic nucleotides are inserted between the ligand and the nucleotide sequence of the sense strand and / or antisense strand. In some alternative embodiments of the invention, the inverted abasic nucleotides may be linked via phosphate esters, phosphate thioesters, or other nucleoside bonds.

[0106] In some optional embodiments of the present invention, the structural formulas of "reverse debasement modification" and "reverse debasement nucleotide" are shown below:

[0107] Here, 'a' is the 5' end of the chain pointing toward the justice chain or the antisense chain, and 'b' is the 3' end of the chain pointing toward the justice chain or the antisense chain.

[0108] In some alternative embodiments of the present invention, when invAb is located at the 5' end:

[0109] When invAb is in the middle of the sequence:

[0110] When invAb is at the 3' end:

[0111] The sense and antisense strand sequences of exemplary siRNAs are shown in Tables A and B below.

[0112] Table A (Naked Sequences)

[0113] Table B (Modification Sequence)

[0114] The abbreviations have the following meanings: 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'-thiophosphate; cs = 2'-O-methylcytidine-3'-thiophosphate; gs = 2'-O-methylguanosine-3'-thiophosphate; us = 2'-O-methyluridine-3'-thiophosphate; Af = 2'-fluoroadenosine-3'-phosphate; Cf = 2'-fluorocytidine-3'-phosphate; Gf = 2'-fluoroguanosine-3'-phosphate; Uf = 2'-fluorouridine-3'-phosphate; s = thiophosphate bond.

[0115] Table C (Conjugate Sequences)

[0116] The abbreviations have the following meanings: 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'-thiophosphate; cs = 2'-O-methylcytidine-3'-thiophosphate; gs = 2'-O-methylguanosine-3'-thiophosphate; us = 2'-O-methyluridine-3'-thiophosphate; Af = 2'-fluoroadenosine-3'-phosphate; Cf = 2'-fluorocytidine-3'-phosphate; Gf = 2'-fluoroguanosine-3'-phosphate; Uf = 2'-fluorouridine-3'-phosphate; s = thiophosphate bond.

[0117] L1 has the following structure, which has been disclosed in patent application PCT / CN2024 / 143392 (Formula II-1):

[0118] L96 has the following structure, which has been disclosed in patent applications WO2009073809 and WO2009082607:

[0119] The structures of EVpu and EVpa are shown below:

[0120] Coupled

[0121] As used in this invention, the term "conjugate" refers to a new compound formed by the covalent connection (coupling) of two or more compound molecules through bivalent or multivalent compound molecules with linking functions. As used in this invention, the terms "nucleic acid conjugate," "siRNA conjugate," "conjugate," and "conjugate" are used interchangeably.

[0122] In some embodiments, the siRNA of the present invention contains or is conjugated to one or more non-nucleotide groups, including but not limited to targeting groups (or targeting ligands, delivery moieties), linker groups, pharmacokinetic / pharmacodynamic (PK / PD) modulators, delivery polymers, or delivery mediators. The non-nucleotide groups can enhance the targeting, delivery, or attachment of the siRNA. The non-nucleotide groups can be covalently linked to the 3' and / or 5' ends of the sense and / or antisense strands.

[0123] In some embodiments, the siRNA of the present invention contains a non-nucleotide group attached to the 3' and / or 5' end of the positive strand. In some embodiments, the non-nucleotide group is attached to the 5' end of the positive strand of the siRNA. The non-nucleotide group can be directly or indirectly attached to the siRNA via a linker / linker group. In some embodiments, the non-nucleotide group is attached to the siRNA via an unstable, cleavable, or reversible bond or linker.

[0124] Targeting groups or targeting moieties enhance the pharmacokinetic or biodistribution properties of the conjugates or siRNAs to which they are attached, thereby improving cell-specific (and in some cases, organ-specific) distribution and cell-specific (or organ-specific) uptake of the conjugates or siRNAs. Targeting groups can be monovalent, divalent, trivalent, tetravalent, or have a higher potency for their target. 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 mimics (which have affinity for cell surface molecules). In some embodiments, the targeting group is linked to the siRNA using a linker such as a PEG linker, or one, two, or three debased and / or ribitol (debased ribose) residues (which can act as linkers in some cases).

[0125] Targeting groups, with or without connectors, can be attached to the 5' or 3' end of any sense and / or antisense strand disclosed in Tables A, B, and C.

[0126] Pharmaceutical Compositions and Administration

[0127] As used herein, the term “effective amount” or “effective dose” means an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals.

[0128] As used herein, the term "pharmaceuticalally acceptable" refers to a substance suitable for human and / or mammalian use without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a reasonable benefit / risk ratio. The term "pharmaceuticalally acceptable carrier" refers to a carrier used for the administration of a therapeutic agent, including various excipients and diluents.

[0129] The pharmaceutical compositions of the present invention contain a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier.

[0130] In this invention, the siRNA or expression vector can be directly applied to the target, or the siRNA or expression vector can be combined with a pharmaceutically acceptable vector to form a drug combination for application.

[0131] The siRNA sequences were designed based on the mRNA encoding MARC1 as the target sequence, and the siRNA sequences and conjugates in Tables A, B and C above were finally obtained.

[0132] Example 1: Synthesis of siRNA

[0133] The specific steps for designing and synthesizing siRNA sequences without conjugation groups (hereinafter referred to as siRNA) are as follows:

[0134] 1) Synthesis of single-stranded oligonucleotides: Oligonucleotides were synthesized using phosphoramide solid-phase synthesis technology (Lingkun 48-channel synthesizer). This was achieved using a general-purpose controllable porous glass CPG system. Synthesized on CPG after ligand coupling. All 2'-modified RNA, DNA, phosphoramidite ligands, and auxiliary reagents were commercially available (Tangzhi Pharmaceutical). All phosphoramidite was dissolved in anhydrous acetonitrile and sieved with molecular sieves. The coupling time using 5-ethylthio-1H-tetrazole (ETT, Suzhou Kelama Biotechnology Co., Ltd.) as the activator was 200 seconds, and the coupling ligand time was 10 minutes. Phosphate esters were generated using a pyridine (Sinopharm) / aqueous solution containing 50 mM iodine (Suzhou Kelama Biotechnology Co., Ltd.) for 5 minutes. Thiophosphate ester bonds were generated using a pyridine (Sinopharm) solution containing 0.2 M hydroflavin (Shanghai Zhaowei Technology Development Co., Ltd.) for 3 minutes. The synthesis was completed after the final removal of the DMT group from all sequences.

[0135] 2) Cleavage and deprotection of oligonucleotides bound to CPG: After solid-phase synthesis, the dried solid support was treated with ammonia solution at 55°C for 16 hours. Some of the oligonucleotides were deprotected using a mixture of DMSO and triethylamine hydrogen fluoride (Beijing Bailingwei Technology Co., Ltd.) (5:1) at 25°C for 4 hours. The solution was evaporated and the solid residue was redissolved in water.

[0136] 3) Purification of single-stranded oligonucleotides: The crude product was purified by reversed-phase HPLC using a Waters XBridge C18 column and an Autotide 100 system (Insys). Buffer A was a 100 mM aqueous solution of triethylamine acetate, pH 7.5, containing 5% acetonitrile, and buffer B was 100% acetonitrile. UV traces were recorded at 260 nm, and appropriate fractions were collected.

[0137] 4) Annealing of single-stranded oligonucleotides to produce siRNA: The single-stranded oligonucleotides to be annealed were prepared to 200 μM using sterile RNase-free water (free of RNase). The annealing reaction system was set up as follows: 10 nmol of the 100 μL mixture was placed in a 95°C water bath for 5 minutes (≥100 nmol requires 20 minutes at high temperature). The mixture was then quickly placed in a 60°C water bath and allowed to cool naturally to 20–30°C. The annealed solution should not be stored at high temperatures. By combining equimolar amounts of the single-stranded oligonucleotide solutions to form complementary strands, the siRNAs in Tables A and B were finally obtained. The molecular weight of the siRNAs was determined using liquid chromatography-mass spectrometry (LC-MS). Comparing the measured and theoretical molecular weights, the results showed that the measured values ​​were approximately equal to the theoretical values, confirming the successful extraction of the siRNAs in Tables A and B.

[0138] Example 2: Design and synthesis of modified sequences and siRNA conjugates

[0139] 1.1 The synthetic steps for siRNA conjugates with conjugating groups attached to the 5' end of the siRNA positive strand ultimately yield the modified or conjugated siRNA sequences shown in Table C above. Taking C001 as an example, the specific steps are as follows:

[0140] 1) Synthesis of single-stranded oligonucleotides: Oligonucleotides were synthesized using phosphoramide solid-phase synthesis technology. This was achieved using a universally controllable porous glass CPG... The synthesis was performed using a Linkon 48-channel synthesizer. All phosphoramidite monomers (Tangzhi Pharmaceutical & Shanghai Zhaowei) and auxiliary reagents were commercially available. The phosphoramidites containing conjugated groups were prepared from the corresponding compounds in Example 1. All phosphoramidites were dissolved in anhydrous acetonitrile (Suzhou Kelama) and molecular sieves were added. The coupling time using 5-ethylthio-1H-tetrazole (ETT) as an activator (Suzhou Kelama) was 8–12 minutes. Phosphate bonds were constructed using a 0.05 M iodine solution (dissolved in pyridine / water = 9:1, Suzhou Kelama); thiophosphate bonds were generated using a 0.2 M hydroflavin (Suzhou Kelama) solution in anhydrous acetonitrile / pyridine (v / v = 1 / 1), with a reaction time of 5 minutes. The synthesis was complete after the final removal of the DMT group from all sequences.

[0141] 2) Cleavage and deprotection of oligonucleotides bound to CPG: After the solid-phase synthesis was terminated, the protecting groups were removed by treating with an acetonitrile solution containing 20% ​​diethylamine (Sinopharm) for 10 minutes. The obtained CPG carrier was then subjected to ammonolysis with concentrated ammonia (Sinopharm) to remove the protecting groups on the carrier and bases. After filtration, a solution containing the product was obtained.

[0142] 3) Purification of single-stranded oligonucleotides: Oligomers were obtained by HPLC purification using NanoQ anion exchange. Buffer A was a 20 mM sodium hydroxide solution; and buffer B contained 20 mM sodium hydroxide solution and 3 M sodium chloride, from which the target product was separated. The obtained target product was then desalted by gel column chromatography (Cytiva).

[0143] 4) Coupling and purification of the conjugated group: First, the purified nucleic acid sequence with NH2-C6 at the end (prepared and purified using conventional methods in the art) was dissolved in 100 mM PB buffer. Ligand I-1 (see Preparation Example 1 of Patent Application PCT / CN2024 / 143392) was dissolved in DMF or DMSO and then added to the nucleic acid solution. The reaction was carried out at room temperature for 4–16 h. Mass spectrometry was used to monitor the completeness of the reaction. Two volumes of AMA solution (25% ammonia: 40% methylamine aqueous solution = 1:1) were added, and the mixture was stirred at room temperature for 0.5 h, followed by quenching with water. The mixed solution was desalted to remove organic reagents and then purified to finally obtain a nucleic acid chain with a conjugated group at the end.

[0144] 5) The sense and antisense chains obtained by chemical synthesis were subjected to base complementation in a molar ratio of 1:1. The reaction conditions were 70℃ for 10 min, and then slowly restored to room temperature to finally obtain the product.

[0145] The modified or conjugated nucleotide sequences of the sense and antisense strands of the siRNA obtained in this invention are shown in Table C.

[0146] Example 3: Design and synthesis of modified sequences and siRNA conjugates

[0147] Modified siRNA was prepared using a Linkon 48-channel synthesizer. Then, the siRNA modified in Table B of Example 1 was linked to ligand L96 (for the preparation of L96, see patent applications WO2009073809 and WO2009082607). The specific synthetic route can be found in patent application CN201480067917.1, the contents of which can be incorporated into this application by reference.

[0148] The modified or conjugated nucleotide sequences of the sense and antisense strands of the siRNA obtained in this invention are shown in Table C.

[0149] Test Example 1: In vitro screening of HEK293 cells using dual-luciferase psiCHECK-2 vector

[0150] In a 96-well cell culture plate, double-stranded siRNA was added to each well, with two replicates of each type. Then, a plasmid carrying the MARC1 gene from the psiCHECK-2 vector (hereinafter referred to as plasmid) was added to each well. Opti-MEM medium (Gibco, catalog number 31985-070) was used to obtain a mixture containing siRNA and plasmid. Lipofectamine 2000 (Invitrogen, catalog number 11668-019) was added to the Opti-MEM medium and incubated for 5 minutes. This was then added to the mixture containing the double-stranded siRNA and plasmid in each well. After incubation at room temperature for 20 minutes, 100 μL of a solution containing 5 × 10⁵ siRNAs was added to each well. 4 Dulbecco's Modified Eagle Medium (Gibco, catalog number C11995500BT) was added to each well of HEK293 cells (ATCC) into a mixture containing double-stranded siRNA, plasmid, and Lipofectamine 2000 transfection reagent. After incubating the cells for 24 hours, firefly luciferase and Renilla luciferase were measured using a dual-luciferase reporter assay kit (Novizan, catalog number DD1205-02). Different concentrations of double-stranded siRNA were used for testing. The test results for some exemplary compounds are shown in Table 1 below.

[0151] Table 1

[0152] The test results showed that the siRNA and conjugates obtained by this invention have good silencing effects.

[0153] Test Example 2: Evaluation of siRNA Activity in MARC1 Humanized Mice

[0154] The inhibitory activity of the target siRNA on the target gene MARC1 was evaluated using hMARC1 mice. The hMARC1 mice were provided by Shanghai Southern Model Biotechnology Co., Ltd.

[0155] The specific experimental procedure is as follows:

[0156] (1) Animal grouping, drug administration, and tissue sample collection:

[0157] Six- to eight-week-old hMARC1 mice (all males) were randomly divided into groups of five mice each, based on body weight. Each test group received a predetermined dose of siRNA and an additional PBS group. The dosage was calculated based on body weight and administered via a single subcutaneous injection. siRNA was administered in 0.6 mg / mL PBS solution at a volume of 5 mL / kg mouse body weight, meaning each siRNA dose was 3 mg / kg mouse body weight. The PBS control group received the same volume of PBS solution (without siRNA). The day of administration was designated Day 0. Mice were sacrificed on Day 14, and liver tissue was collected, flash-frozen in liquid nitrogen, and stored at -80°C.

[0158] (2) Take an appropriate amount of liver tissue, thaw it on ice, and add VeZol Reagent (Novozymes, catalog number R411-02). Homogenize the tissue using an automated cryogenic homogenizer (Shanghai Jingxin Industrial Development Co., Ltd., model JXFSTPRP-CLN). Then, use the VAMNE Magnetic Universal Total RNA Kit (Novozymes, catalog number ROA3302-02) to extract total RNA from the tissue homogenate.

[0159] (3) For each mouse, 1000 ng of total RNA was collected, and a 20 μL reverse transcription system was prepared using the HiScript III All-in-one RT SuperMix Perfect for qPCR kit (Novizan, catalog number R333) to complete the reverse transcription reaction and obtain cDNA solution. After diluting the cDNA 10-fold, the expression level of MARC1 mRNA in mouse liver tissue was detected using the Taq Pro HighGC U+Multiple Probe qPCR Mix kit (Novizan, catalog number QN211-02). In this real-time quantitative PCR method, mGAPDH was used as an internal reference gene. The expression levels of MARC1 mRNA and mGAPDH were homogenized, and then the expression level of the PBS control group was used as 100% baseline for calculation and comparison.

[0160] The results showed that the siRNA conjugate of the present invention had a significant silencing effect on MARC1 gene expression in MARC1 humanized mice.

[0161] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0162] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. An siRNA that inhibits the expression of a MARC1 gene, characterized in that, The siRNA contains a sense strand and an antisense strand. The antisense strand comprises at least 15 consecutive nucleotides that differ from any of the sequences shown in Table 1 by 0, 1, 2 or 3 nucleotides; Table 1 The sense strand and the antisense strand are complementary to each other by at least 15, 16, 17, 18, 19, 20, 21 or 22 nucleotides; Each nucleotide in the sense strand and antisense strand is independently a modified or unmodified nucleotide.

2. The siRNA as described in claim 1, characterized in that, The antisense strand comprises at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides differing from any of the sequences shown in Table 1 by 0, 1, 2, or 3 nucleotides; and / or, The positive strand comprises at least 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive nucleotides that differ from any of the sequences shown in Table 1 by 0, 1, 2 or 3 nucleotides; Preferably, the antisense strand comprises at least 21 consecutive nucleotides that differ from any of the sequences shown in Table 1 by 0, 1, 2 or 3 nucleotides; Preferably, the positive chain comprises at least 19 or 21 consecutive nucleotides that differ from any of the sequences shown in Table 1 by 0, 1, 2 or 3 nucleotides.

3. The siRNA as described in claim 1 or 2, characterized in that, At least one nucleotide in the sense strand and the antisense strand is a modified nucleotide; Preferably, the modified nucleotide is selected from: 2'-O-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, phosphate thioester modified nucleotides, debased nucleotides, morpholinonucleotides, locked nucleotides, reverse base modified nucleotides, or combinations thereof; More preferably, the modified nucleotide is selected from: nucleotides modified with 2'-O-methyl, nucleotides modified with 2'-fluoro, nucleotides modified with thiophosphate, nucleotides modified with reverse bases, or combinations thereof.

4. The siRNA as described in claim 1, characterized in that, The modified nucleotides include modifications selected from the group consisting of: (1) From the 5' end to the 3' end, the nucleotides at positions 9, 11, and 13 of the positive strand are nucleotides modified with 2'-fluorine, and the nucleotides at the remaining positions are nucleotides modified with 2'-O-methyl. (2) From the 5' end to the 3' end, the nucleotides at positions 7, 9, and 11 of the positive strand are nucleotides modified with 2'-fluorine, and the nucleotides at the remaining positions are nucleotides modified with 2'-O-methyl. (3) From the 5' end to the 3' end, the nucleotides at positions 2, 7, 12, 14 and 16 of the antisense strand are nucleotides modified with 2'-fluorine, and the nucleotides at the remaining positions are nucleotides modified with 2'-O-methyl. (4) The 5' end and 3' end of the positive strand each independently contain 0, 1, 2 or 3 phosphate-thioester modified nucleotides; (5) The 5' end and 3' end of the antisense strand each independently contain 0, 1, 2 or 3 phosphate thioester modified nucleotides; (6) Any combination of (1)-(5) above.

5. The siRNA as described in claim 1, characterized in that, The antisense strand comprises at least 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive nucleotides differing by 0, 1, 2 or 3 nucleotides from the nucleotide sequences shown in any one of SEQ ID NO:301-400, SEQ ID NO:471-540, SEQ ID NO:628-656, SEQ ID NO:690-700 and SEQ ID NO:712-722; Preferably, the antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NO:301-400, SEQ ID NO:471-540, SEQ ID NO:628-656, SEQ ID NO:690-700 and SEQ ID NO:712-722.

6. The siRNA as described in claim 1, characterized in that, The positive strand comprises at least 15, 16, 17, 18, 19, 20, 21, 22 or 23 consecutive nucleotides differing by 0, 1, 2 or 3 nucleotides from the nucleotide sequences shown in any one of SEQ ID NO:201-300, SEQ ID NO:401-470, SEQ ID NO:599-627, SEQ ID NO:679-689 and SEQ ID NO:701-711; Preferably, the positive strand comprises the nucleotide sequence shown in any one of SEQ ID NO:201-300, SEQ ID NO:401-470, SEQ ID NO:599-627, SEQ ID NO:679-689 and SEQ ID NO:701-711.

7. The siRNA as described in claim 1, characterized in that, The antisense strand comprises or consists of any of the nucleotide sequences shown in SEQ ID NO:(m1+100), SEQ ID NO:471-540, SEQ ID NO:570-598, SEQ ID NO:628-656, SEQ ID NO:668-678, SEQ ID NO:690-700, and SEQ ID NO:712-722; and the sense strand comprises or consists of any of the nucleotide sequences shown in SEQ ID NO:m1, SEQ ID NO:401-470, SEQ ID NO:541-569, SEQ ID NO:599-627, SEQ ID NO:657-667, SEQ ID NO:679-689, and SEQ ID NO:701-711; wherein m1 is any integer number from 1 to 100 or from 201 to 300; Preferably, the antisense strand and the sense strand form a double strand of nucleic acid sequence pairs as shown in any of Tables B and C.

8. A siRNA conjugate comprising siRNA as described in any one of claims 1-7 and a targeting ligand and / or a reverse debasement nucleotide. Preferably, the targeting ligand is a lipophilic delivery portion; Preferably, the targeting ligand is a liver-targeting delivery fraction; Preferably, the targeting ligand is selected from the group consisting of: in, This represents the connection point that is directly or indirectly connected to the siRNA.

9. The siRNA conjugate as described in claim 8, characterized in that, The targeting ligand is coupled to the 5' or 3' end of the siRNA's positive strand; and / or, The reverse debase nucleotide is conjugated to the 3' end of the sense strand and / or antisense strand of the siRNA.

10. A pharmaceutical composition comprising: (a) the siRNA as described in any one of claims 1-7, and / or the conjugate as described in any one of claims 8-9; and (b) Pharmaceutically acceptable carriers.

11. Use of the siRNA as described in any one of claims 1-7, and / or the conjugate as described in any one of claims 8-9, or the pharmaceutical composition as described in claim 10, in the preparation of a medicament for the prevention and / or treatment of MARC1-mediated diseases; Preferably, the MARC1-mediated disease is a disease of MARC1 overexpression; Preferably, the disease is a metabolic system disease or a cardiovascular disease; Preferably, the metabolic system disease includes: Lipid metabolism disorder; And / or, The cardiovascular diseases mentioned include: hypertension, atherosclerosis, cardiomyopathy, and heart failure; The lipid metabolism disorders mentioned include: weight imbalance, elevated triglycerides, fatty liver disease, hypercholesterolemia, or non-alcoholic steatohepatitis.

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