WO2024222898A1

WO2024222898A1 - Double-stranded ribonucleic acid for inhibiting marc1 gene expression, and modifier, conjugate, and use thereof

Info

Publication number: WO2024222898A1
Application number: PCT/CN2024/090135
Authority: WO
Inventors: 翟蓓蓓; 黄河; 王岩; 王书成; 荣梅; 周玲玲; 林国良; 产运霞; 耿玉先
Original assignee: 北京福元医药股份有限公司
Priority date: 2023-04-28
Filing date: 2024-04-26
Publication date: 2024-10-31
Also published as: CN118853663A

Abstract

A double-stranded ribonucleic acid for inhibiting MARC1 gene expression, a double-stranded ribonucleic acid modifier, a double-stranded ribonucleic acid conjugate, a prodrug, a pharmaceutical composition, a use, and a method for inhibiting intracellular MARC1 gene expression. The double-stranded ribonucleic acid can bind in cells to form an RNA-induced silencing complex (RISC), is used for cleaving mRNA transcribed by an MARC1 gene, can efficiently and specifically inhibit the expression of the MARC1 gene, is used for treating MARC1 gene-mediated diseases, and has important application prospects in clinical disease treatment.

Description

Double-stranded ribonucleic acid for inhibiting MARC1 gene expression and its modified product, conjugate and use

Technical Field

The present disclosure belongs to the field of biomedicine. Specifically, the present disclosure relates to a double-stranded RNA, a double-stranded RNA modification, a double-stranded RNA conjugate, a prodrug, a pharmaceutical composition and uses for inhibiting MARC1 gene expression, and a method for inhibiting MARC1 gene expression in a cell.

Background Art

Liver disease is a leading cause of death and disability worldwide. Chronic disease progressively impairs liver cell regeneration and even destroys it, leading to liver fibrosis and cirrhosis. Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in the world. Its prevalence has doubled in the past 20 years and is now estimated to affect approximately 20-30% of the world's population. The pathology of NAFLD is a combination of factors. In some individuals, the accumulation of ectopic fat in the liver, called steatosis, triggers inflammation and liver cell damage, leading to a further disease called nonalcoholic steatohepatitis (NASH). NASH is defined as lipid accumulation with cell damage, inflammation, and varying degrees of scarring or fibrosis.

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 in the MARC1 gene has recently been shown to protect the liver from fatty liver disease and cirrhosis of all causes. It was reported (Luukkonen P.K., Juuti A., Sammalkorpi H., et al. MARC1 variant rs2642438 increases hepatic phosphatidylcholines and decreases severity of non-alcoholic fatty liver disease in humans. Journal of Hepatology, 2020, 73: 696–739.) that MARC1 variant carriers (n=53) had higher hepatic polyunsaturated phosphatidylcholine concentrations compared with non-carriers (n=65), which may be related to the pathogenesis associated with NASH. In addition, the study showed that by knocking out the expression of the MARC1 gene in patients, hyperglycemia, diabetes, metabolic syndrome, insulin resistance (insulin insensitivity), impaired glucose tolerance, high blood sugar levels, pulmonary hypertension and/or conditions caused by any of the above in patients can be treated.

In summary, by inhibiting the expression of MARC1 gene in patients, obesity, non-alcoholic fatty liver disease, alcohol-related fatty liver disease, non-alcoholic fatty hepatitis, cirrhosis, liver fibrosis, elevated liver enzyme levels (ALT, AST, ALP), hepatocellular carcinoma, hypercholesterolemia and related cardiovascular diseases, insulin resistance, impaired glucose tolerance, hyperglycemia, type II diabetes and metabolic syndrome can be prevented and treated. Currently, there are no related drugs targeting only the expression of such genes on the market, so it is of great value to develop drugs targeting MARC1 target. The present invention aims to provide siRNA compositions, which are effectively applied to RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of MARC1 genes, so as to selectively and effectively inhibit the expression of MARC1 genes and achieve the purpose of disease treatment.

Summary of the invention

Problem that the invention aims to solve

In view of the problems existing in the prior art, for example, more MARC1 inhibitors need to be developed for the treatment of MARC1-related diseases including obesity, non-alcoholic fatty liver disease, alcohol-related fatty liver disease, non-alcoholic steatohepatitis, cirrhosis, liver fibrosis, elevated liver enzyme levels (ALT, AST, ALP), hepatocellular carcinoma, hypercholesterolemia and related cardiovascular diseases, insulin resistance, impaired glucose tolerance, hyperglycemia, type II diabetes and metabolic syndrome, and other unidentified related conditions, pathologies or syndromes. The present disclosure aims to provide a series of double-stranded RNAs, double-stranded RNA modifications, double-stranded RNA conjugates, prodrugs and pharmaceutical compositions for inhibiting MARC1 gene expression, which can inhibit MARC1 gene expression and have important application prospects in clinical disease treatment.

Solutions for solving problems

[1] A double-stranded RNA, comprising a sense strand and an antisense strand, wherein the sense strand is reverse complementary to the antisense strand and/or substantially reverse complementary to form a double-stranded region of the double-stranded RNA;

The sense strand comprises a sequence A that differs by no more than 3 nucleotides from at least 15 consecutive nucleotides in the target sequence, and the antisense strand comprises a sequence B that differs by no more than 3 nucleotides from the reverse complementary sequence of at least 15 consecutive nucleotides in the target sequence;

The target sequence is selected from a nucleotide sequence shown in any one of SEQ ID NOs: 1 to 9 and 812 to 817 and a sequence consisting of at least 15 consecutive nucleotides contained in any one of SEQ ID NOs: 1 to 9 and 812 to 817.

[2]. The double-stranded RNA according to [1], wherein the target sequence is selected from the nucleotide sequence as shown in any one of SEQ ID NOs: 10-37, 799-811 and 818-830, the sense strand comprises a sequence A consisting of at least 15 consecutive nucleotides in the nucleotide sequence as shown in any one of SEQ ID NOs: 10-37, 799-811 and 818-830, and the antisense strand comprises a sequence B that is reverse complementary and/or substantially reverse complementary to a sequence consisting of at least 15 consecutive nucleotides in the nucleotide sequence as shown in any one of SEQ ID NOs: 10-37, 799-811 and 818-830.

[3] The double-stranded RNA according to [1] or [2], wherein the sense strand consists of 15-28 nucleotides, preferably 19-25 nucleotides, more preferably 19-23 nucleotides, and more preferably 19, 21 or 23 nucleotides.

[4]. The double-stranded RNA according to [3], wherein the nucleotide sequence of the positive strand is a sequence A which differs by no more than 1 nucleotide from a sequence consisting of 15 to 28 consecutive nucleotides in the nucleotide sequence shown in any one of SEQ ID NOs: 10 to 37, 799 to 811 and 818 to 830, preferably 19 to 25 consecutive nucleotides, more preferably 19 to 23 consecutive nucleotides, and more preferably 19, 21 or 23 nucleotides.

[5] The double-stranded ribonucleic acid according to any one of [1] to [4], wherein the antisense strand consists of 15-28 nucleotides, preferably 19-25 nucleotides, more preferably 19-23 nucleotides, and more preferably 19, 21 or 23 nucleotides.

[6]. The double-stranded RNA according to [5], wherein the nucleotide sequence of the antisense strand is a sequence B having a difference of no more than 1 nucleotide compared to the reverse complementary sequence of a sequence consisting of 15 to 28 consecutive nucleotides in the nucleotide sequence shown in any one of SEQ ID NOs: 10 to 37, 799 to 811 and 818 to 830, preferably 19 to 25 consecutive nucleotides, more preferably 19 to 23 consecutive nucleotides, and more preferably 19, 21 or 23 nucleotides.

[7]. The double-stranded ribonucleic acid according to any one of [1] to [6], wherein the length of the double-stranded region is 15-25 nucleotides, preferably 19-23 nucleotides, more preferably 19-21 nucleotides, and more preferably 19, 21 or 23 nucleotides.

[8] The double-stranded RNA according to any one of [1] to [7], wherein

The sense strand and the antisense strand complement each other to form the double-stranded region, and the 3' end of the sense strand has 1-2 protruding nucleotides extending out of the double-stranded region, and the 3' end of the antisense strand forms a blunt end; or,

The sense strand and the antisense strand complement each other to form the double-stranded region, and the 3' end of the antisense strand has 1-2 protruding nucleotides extending out of the double-stranded region, and the 3' end of the sense strand forms a blunt end; or,

The sense strand and the antisense strand complement each other to form the double-stranded region, and the 3' ends of the sense strand and the antisense strand each have 1-2 protruding nucleotides extending out of the double-stranded region; or,

The sense strand and the antisense strand complement each other to form the double-stranded region, and the 3' ends of the sense strand and the antisense strand both form blunt ends.

[9] The double-stranded RNA according to any one of [1] to [8], wherein the sense strand and the antisense strand are selected from the following combinations:

The sense strand comprises the sense strand of any one of the siRNAs shown in Table 1 and Table 1-1 herein, and the antisense strand comprises the antisense strand of the corresponding siRNA;

Preferably, the sense strand and the antisense strand are selected from the following combinations:

The sense chain comprises the sense chain of any one of the siRNAs 262, 264 and 330 shown in Table 1 of the present invention, and the antisense chain comprises the antisense chain of the corresponding siRNA.

[10] The double-stranded RNA according to any one of [1] to [9], wherein each nucleotide in the sense strand is independently a modified nucleotide or an unmodified nucleotide, and/or each nucleotide in the antisense strand is independently is a modified nucleotide or an unmodified nucleotide.

[11]. The double-stranded RNA according to any one of [1] to [10], wherein any two nucleotides connected in the sense strand are connected by a phosphodiester bond or a phosphorothioate diester bond, and/or any two nucleotides connected in the antisense strand are connected by a phosphodiester bond or a phosphorothioate diester bond.

[12] The double-stranded RNA according to any one of [1] to [11], wherein the 5' terminal nucleotide of the antisense strand is linked to a 5' phosphate group or a 5' phosphate derivative group, or the 5' terminal nucleotide of the antisense strand is not linked to a 5' phosphate group or a 5' phosphate derivative group.

[13]. The double-stranded RNA according to any one of [1] to [12], wherein the double-stranded RNA is siRNA.

[14] The double-stranded RNA according to any one of [1] to [13], wherein the double-stranded RNA is siRNA for inhibiting the expression of MARC1 gene.

[15] A modified double-stranded RNA, which is a modified double-stranded RNA as described in any one of [1] to [14], wherein the modified double-stranded RNA comprises at least one of the following chemical modifications:

(1) modification of at least one nucleotide in the sense strand,

(2) modification of the phosphodiester bond at at least one position in the sense strand,

(3) modification of at least one nucleotide in the antisense strand,

(4) modification of the phosphodiester bond at at least one position in the antisense strand;

Optionally, the 3' end of sequence A in the sense strand of the double-stranded RNA is connected to a sequence D consisting of 1-2 nucleotides, preferably a sequence D consisting of 1-2 thymine deoxyribonucleotides; and/or, the 3' end of sequence B in the antisense strand of the double-stranded RNA is connected to a sequence E consisting of 1-2 nucleotides, preferably a sequence E consisting of 1-2 thymine deoxyribonucleotides; and/or, the 3' end of sequence A in the sense strand of the double-stranded RNA excludes 1-2 nucleotides to form sequence A';

Optionally, the sense strand and antisense strand of the double-stranded RNA modification are selected from the following sequence combinations:

The nucleotide sequence of the sense strand is the sequence shown in sequence A, and the nucleotide sequence of the antisense strand is the sequence shown in sequence B;

Alternatively, the nucleotide sequence of the sense strand is the sequence shown in sequence A, and the nucleotide sequence of the antisense strand is the sequence shown in sequence B connected to sequence E;

Alternatively, the nucleotide sequence of the sense strand is the sequence shown in sequence A connected to sequence D, and the nucleotide sequence of the antisense strand is the sequence shown in sequence B;

Alternatively, the nucleotide sequence of the sense strand is the sequence shown by sequence A connected to sequence D, and the nucleotide sequence of the antisense strand is the sequence shown by sequence B connected to sequence E;

Alternatively, the nucleotide sequence of the sense strand is the sequence shown in sequence A', and the nucleotide sequence of the antisense strand is the sequence shown in sequence B;

Alternatively, the nucleotide sequence of the sense strand is the sequence shown in sequence A’, and the nucleotide sequence of the antisense strand is the sequence shown in sequence B connected to sequence E.

[16]. The double-stranded RNA modified substance according to [15], wherein the modification of the nucleotide is selected from 2’-fluoro modification, 2’-alkoxy modification, 2’-substituted alkoxy modification, 2’-alkyl modification, 2’-substituted alkyl modification, 2’-deoxy modification, nucleotide derivative modification or a combination of any two or more thereof.

[17]. The modified double-stranded RNA according to [15] or [16], wherein the modification of the nucleotide is selected from 2'-F modification, 2'-O-CH ₃ modification, 2'-O-CH ₂ -CH ₂ -O-CH ₃ modification, 2'-O-CH ₂ -CH＝CH ₂ modification, 2'-CH ₂ -CH ₂ -CH＝CH ₂ modification, 2'-deoxy modification, nucleotide derivative modification or a combination of any two or more thereof.

[18]. The double-stranded RNA modification according to [16] or [17], wherein the nucleotide derivative in the nucleotide derivative modification is selected from isonucleotides, LNA, ENA, cET, UNA or GNA.

[19]. A double-stranded ribonucleic acid modified substance according to any one of [15] to [18], wherein, from the 5' end to the 3' end, the ribonucleotides at positions 7, 9, 10 and 11 in the sense strand are 2'-F modified ribonucleotides, and the ribonucleotides at the remaining positions in the sense strand are 2'-O-CH ₃ modified ribonucleotides.

[20] The double-stranded RNA modification according to any one of [15] to [19], wherein the sense strand comprises a phosphorothioate diester bond located at the following position along the 5' end to the 3' end:

Between the first nucleotide and the second nucleotide starting from the 5' end of the sense strand;

Between the second nucleotide and the third nucleotide starting from the 5' end of the sense strand;

Between the first nucleotide and the second nucleotide starting from the 3' end of the sense strand;

Between the second nucleotide and the third nucleotide starting from the 3' end of the sense strand;

or,

The sense strand contains phosphorothioate diester bonds located at the positions shown below:

Between the second and third nucleotides starting from the 5' end of the sense strand.

[21] The double-stranded ribonucleic acid modified substance according to any one of [15] to [20], wherein, from the 5' end to the 3' end, the ribonucleotides at any odd-numbered positions in the antisense strand are 2'-O-CH _3- modified ribonucleotides, and the ribonucleotides at any even-numbered positions in the antisense strand are 2'-F-modified ribonucleotides;

Alternatively, along the 5' end to the 3' end, the ribonucleotides at positions 2, 6, 14 and 16 in the antisense strand are 2'-F modified ribonucleotides, and the ribonucleotides at the remaining positions in the antisense strand are 2'-O-CH ₃ modified ribonucleotides;

Alternatively, along the 5' end to the 3' end, the ribonucleotides at positions 2, 6, 8, 9, 14 and 16 in the antisense strand are 2'-F modified ribonucleotides, and the ribonucleotides at the remaining positions in the antisense strand are 2'-O-CH ₃ modified ribonucleotides;

Alternatively, along the 5' end to the 3' end, the ribonucleotides at positions 2, 14 and 16 in the antisense strand are 2'-F modified ribonucleotides, the ribonucleotide at position 6 in the antisense strand is a ribonucleotide modified with the nucleotide derivative GNA, and the ribonucleotides at the remaining positions in the antisense strand are 2'-O-CH ₃ modified ribonucleotides;

Alternatively, along the direction from the 5' end to the 3' end, the ribonucleotides at positions 2, 6, 14 and 16 in the antisense chain are 2'-F modified ribonucleotides, the ribonucleotide at position 7 in the antisense chain is a ribonucleotide modified with the nucleotide derivative GNA, and the ribonucleotides at the remaining positions in the antisense chain are 2'-O- _CH3 modified ribonucleotides.

[22] A double-stranded RNA modification according to any one of [15] to [21], wherein, in the direction from the 5’ end to the 3’ end, the nucleotide at the 5’ end of the antisense strand is not connected to a 5’ phosphate group or a 5’ phosphate derivative group, or the nucleotide at the 5’ end of the antisense strand is connected to a 5’ phosphate group or a 5’ phosphate derivative group.

[23] The double-stranded RNA modification product according to any one of [15] to [22], wherein the antisense strand comprises a phosphorothioate diester bond located at the following position:

Between the first nucleotide and the second nucleotide starting from the 5' end of the antisense strand;

Between the second nucleotide and the third nucleotide starting from the 5' end of the antisense strand;

Between the first nucleotide and the second nucleotide starting from the 3' end of the antisense strand;

Between the second and third nucleotides starting from the 3' end of the antisense strand.

[24] The double-stranded RNA modified substance according to any one of [15] to [23], wherein the sense strand of the double-stranded RNA modified substance has a structure as shown in any one of (a ₁ ) to (a ₅ ):

(a ₁ )5'-mN ₁ -(s)-mN ₂ -(s)-mN ₃ -mN ₄ -mN ₅ -mN ₆ -N ₇ f-mN ₈ -N ₉ fN ₁₀ fN ₁₁ f-mN ₁₂ -mN ₁₃ -mN ₁₄ -mN ₁₅ -mN ₁₆ -mN ₁₇ -mN ₁₈ -mN ₁₉ -(s)-T-(s)-T-3'，

(a ₂ )5'-mN ₁ -(s)-mN ₂ -(s)-mN ₃ -mN ₄ -mN ₅ -mN ₆ -N ₇ f-mN ₈ -N ₉ fN ₁₀ fN ₁₁ f-mN ₁₂ -mN ₁₃ -mN ₁₄ -mN ₁₅ -mN ₁₆ -mN ₁₇ -mN ₁₈ -mN ₁₉ -(s)-mN ₂₀ -(s)-mN ₂₁ -3',

(a ₃ )5'-mN ₁ -(s)-mN ₂ -(s)-mN ₃ -mN ₄ -mN ₅ -mN ₆ -N ₇ f-mN ₈ -N ₉ fN ₁₀ fN ₁₁ f-mN ₁₂ -mN ₁₃ -mN ₁₄ -mN ₁₅ -mN ₁₆ -mN ₁₇ -mN ₁₈ -mN ₁₉ -mN ₂₀ -mN ₂₁ -(s)-mN ₂₂ -(s)-mN ₂₃ -3',

(a ₄ )5'-mN ₁ -(s)-mN ₂ -(s)-mN ₃ -mN ₄ -mN ₅ -mN ₆ -N ₇ f-mN ₈ -N ₉ fN ₁₀ fN ₁₁ f-mN ₁₂ -mN ₁₃ -mN ₁₄ -mN ₁₅ -mN ₁₆ -mN ₁₇ -mN ₁₈ -mN ₁₉ -3',

(a ₅ )5'-mN ₁ -(s)-mN ₂ -(s)-mN ₃ -mN ₄ -mN ₅ -mN ₆ -N ₇ f-mN ₈ -N ₉ fN ₁₀ fN ₁₁ f-mN ₁₂ -mN ₁₃ -mN ₁₄ - mN ₁₅ -mN ₁₆ -mN ₁₇ -mN ₁₈ -mN ₁₉ -mN ₂₀ -mN ₂₁ -3';

wherein N ₁ -N ₂₃ are independently selected from ribonucleotides whose base is A, U, C or G,

The capital letter T represents a deoxyribonucleotide with thymine as its base.

The lowercase letter m indicates that the ribonucleotide adjacent to the right of the letter m is a 2'-O-CH ₃ modified ribonucleotide.

The lowercase letter f indicates that the ribonucleotide adjacent to the left side of the letter f is a 2'-F modified ribonucleotide.

-(s)- indicates that the two adjacent nucleotides are connected by a phosphorothioate diester bond.

[25] The double-stranded RNA modified substance according to any one of [15] to [24], wherein the antisense strand of the double-stranded RNA modified substance has a structure as shown in any one of (b ₁ ) to (b ₂₇ ):

(b ₁ )5'-P1mN ₁ -(s)-N ₂ f-(s)-mN ₃ -N ₄ f-mN ₅ -N ₆ f-mN ₇ -N ₈ f-mN ₉ -N ₁₀ f- mN ₁₁ -N ₁₂ f-mN ₁₃ -N ₁₄ f-mN ₁₅ -N ₁₆ f-mN ₁₇ -N ₁₈ f-mN ₁₉ -(s)-T-(s)-T-3',

(b ₂ )5'-P1mN ₁ -(s)-N ₂ f-(s)-mN ₃ -mN ₄ -mN ₅ -N ₆ f-mN ₇ -mN ₈ -mN ₉ -mN ₁₀ -mN ₁₁ - mN ₁₂ -mN ₁₃ -N ₁ ₄ f-mN ₁₅ -N ₁₆ f-mN ₁₇ -mN ₁₈ -mN ₁₉ -(s)-T-(s)-T-3'，

(b ₃ )5'-P1mN ₁ -(s)-N ₂ f-(s)-mN ₃ -mN ₄ -mN ₅ -N ₆ f-mN ₇ -N ₈ fN ₉ f-mN ₁₀ -mN ₁₁ - mN ₁₂ -mN ₁₃ -N ₁₄ f-mN ₁₅ -N ₁₆ f-mN ₁₇ -mN ₁₈ -mN ₁₉ -(s)-T-(s)-T-3'，

(b ₄ )5'-P1mN ₁ -(s)-N ₂ f-(s)-mN ₃ -mN ₄ -mN ₅ -[GNA]N ₆ -mN ₇ -mN ₈ -mN ₉ -mN ₁₀ -mN ₁₁ -mN ₁₂ -mN ₁₃ -N ₁₄ f-mN ₁₅ -N ₁₆ f-mN ₁₇ -mN ₁₈ -mN ₁₉ -(s)-T-(s)-T-3',

(b ₅ )5'-P1mN ₁ -(s)-N ₂ f-(s)-mN ₃ -mN ₄ -mN ₅ -N ₆ f-[GNA]N ₇ -mN ₈ -mN ₉ -mN ₁₀ - mN ₁₁ -mN ₁₂ -mN ₁₃ -N ₁₄ f-mN ₁₅ -N ₁₆ f-mN ₁₇ -mN ₁₈ -mN ₁₉ -(s)-T-(s)-T-3',

(b ₆ )5'-P1mN ₁ -(s)-N ₂ f-(s)-mN ₃ -N ₄ f-mN ₅ -N ₆ f-mN ₇ -N ₈ f-mN ₉ -N ₁₀ f- mN ₁₁ -N ₁₂ f-mN ₁₃ -N ₁₄ f-mN ₁₅ -N ₁₆ f-mN ₁₇ -N ₁₈ f-mN ₁₉ -(s)-N ₂₀ f-(s)-mN ₂₁ -3',

(b ₇ )5'-P1mN ₁ -(s)-N ₂ f-(s)-mN ₃ -mN ₄ -mN ₅ -N ₆ f-mN ₇ -mN ₈ -mN ₉ -mN ₁₀ -mN ₁₁ - mN ₁₂ -mN ₁₃ -N ₁ ₄ f-mN ₁₅ -N ₁₆ f-mN ₁₇ -mN ₁₈ -mN ₁₉ -(s)-mN ₂₀ -(s)-mN ₂₁ -3',

(b ₈ )5'-P1mN ₁ -(s)-N ₂ f-(s)-mN ₃ -mN ₄ -mN ₅ -N ₆ f-mN ₇ -N ₈ fN ₉ f-mN ₁₀ -mN ₁₁ - mN ₁₂ -mN ₁₃ -N ₁₄ f-mN ₁₅ -N ₁₆ f-mN ₁₇ -mN ₁₈ -mN ₁₉ -(s)-mN ₂₀ -(s)-mN ₂₁ -3',

(b ₉ )5'-P1mN ₁ -(s)-N ₂ f-(s)-mN ₃ -mN ₄ -mN ₅ -[GNA]N ₆ -mN ₇ -mN ₈ -mN ₉ -mN ₁₀ -mN ₁₁ -mN ₁₂ -mN ₁₃ -N ₁₄ f-mN ₁₅ -N ₁₆ f-mN ₁₇ -mN ₁₈ -mN ₁₉ -(s)-mN ₂₀ -(s)-mN ₂₁ -3',

(b ₁₀ )5'-P1mN ₁ -(s)-N ₂ f-(s)-mN ₃ -mN ₄ -mN ₅ -N ₆ f-[GNA]N ₇ -mN ₈ -mN ₉ -mN ₁₀ - mN ₁₁ -mN ₁₂ -mN ₁₃ -N ₁₄ f-mN ₁₅ -N ₁₆ f-mN ₁₇ -mN ₁₈ -mN ₁₉ -(s)-mN ₂₀ -(s)-mN ₂₁ -3',

(b ₁₁ )5'-P1mN ₁ -(s)-N ₂ f-(s)-mN ₃ -N ₄ f-mN ₅ -N ₆ f-mN ₇ -N ₈ f-mN ₉ -N ₁₀ f- mN ₁₁ -N ₁₂ f-mN ₁₃ -N ₁₄ f-mN ₁₅ -N ₁₆ f-mN ₁₇ -N ₁₈ f-mN ₁₉ -N ₂₀ f-mN ₂₁ -(s)-N ₂₂ f-(s)- mN ₂₃ -3',

(b ₁₂ )5'-P1mN ₁ -(s)-N ₂ f-(s)-mN ₃ -mN ₄ -mN ₅ -N ₆ f-mN ₇ -mN ₈ -mN ₉ -mN ₁₀ -mN ₁₁ - mN ₁₂ -mN ₁₃ -N ₁₄ f-mN ₁₅ -N ₁₆ f-mN ₁₇ -mN ₁₈ -mN ₁₉ -mN ₂₀ -mN ₂₁ -(s)-mN ₂₂ -(s)-mN ₂₃ -3',

(b ₁₃ )5'-P1mN ₁ -(s)-N ₂ f-(s)-mN ₃ -mN ₄ -mN ₅ -N ₆ f-mN ₇ -N ₈ fN ₉ f-mN ₁₀ -mN ₁₁ - mN ₁₂ -mN ₁₃ -N ₁₄ f-mN ₁₅ -N ₁₆ f-mN ₁₇ -mN ₁₈ -mN ₁₉ -mN ₂₀ -mN ₂₁ -(s)-mN ₂₂ -(s)-mN ₂₃ -3',

(b ₁₄ )5'-P1mN ₁ -(s)-N ₂ f-(s)-mN ₃ -mN ₄ -mN ₅ -[GNA]N ₆ -mN ₇ -mN ₈ -mN ₉ -mN ₁₀ -mN ₁₁ -mN ₁₂ -mN ₁₃ -N ₁₄ f-mN ₁₅ -N ₁₆ f-mN ₁₇ -mN ₁₈ -mN ₁₉ -mN ₂₀ -mN ₂₁ -(s)-mN ₂₂ -(s)-mN ₂₃ -3' ,

(b ₁₅ )5'-P1mN ₁ -(s)-N ₂ f-(s)-mN ₃ -mN ₄ -mN ₅ -N ₆ f-[GNA]N ₇ -mN ₈ -mN ₉ -mN ₁₀ - mN ₁₁ -mN ₁₂ -mN ₁₃ -N ₁₄ f-mN ₁₅ -N ₁₆ f-mN ₁₇ -mN ₁₈ -mN ₁₉ -mN ₂₀ -mN ₂₁ -(s)-mN ₂₂ -(s)-mN ₂₃ -3 ',

(b ₁₆ )5'-mN ₁ -(s)-N ₂ f-(s)-mN ₃ -mN ₄ -mN ₅ -N ₆ f-mN ₇ -mN ₈ -mN ₉ -mN ₁₀ -mN ₁₁ - mN ₁₂ -mN ₁₃ -N ₁₄ f-mN ₁₅ -N ₁₆ f-mN ₁₇ -mN ₁₈ -mN ₁₉ -(s)-T-(s)-T-3'，

(b ₁₇ )5'-mN ₁ -(s)-N ₂ f-(s)-mN ₃ -mN ₄ -mN ₅ -N ₆ f-mN ₇ -N ₈ fN ₉ f-mN ₁₀ -mN ₁₁ - mN ₁₂ -mN ₁₃ -N ₁₄ f-mN ₁₅ -N ₁₆ f-mN ₁₇ -mN ₁₈ -mN ₁₉ -(s)-T-(s)-T-3'，

(b ₁₈ )5'-EVPmN ₁ -(s)-N ₂ f-(s)-mN ₃ -mN ₄ -mN ₅ -N ₆ f-mN ₇ -mN ₈ -mN ₉ -mN ₁₀ -mN ₁₁ - mN ₁₂ -mN ₁₃ -N ₁₄ f-mN ₁₅ -N ₁₆ f-mN ₁₇ -mN ₁₈ -mN ₁₉ -(s)-T-(s)-T-3'，

(b ₁₉ )5'-EVPmN ₁ -(s)-N ₂ f-(s)-mN ₃ -mN ₄ -mN ₅ -N ₆ f-mN ₇ -N ₈ fN ₉ f-mN ₁₀ -mN ₁₁ - mN ₁₂ - _{mN 13} -N ₁₄ f-mN ₁₅ -N ₁₆ f-mN ₁₇ -mN ₁₈ -mN ₁₉ -(s)-T-(s)-T-3'，

(b ₂₀ )5'-mN ₁ -(s)-N ₂ f-(s)-mN ₃ -mN ₄ -mN ₅ -N ₆ f-mN ₇ -mN ₈ -mN ₉ -mN ₁₀ -mN ₁₁ - mN ₁₂ -mN ₁₃ -N ₁₄ f-mN ₁₅ -N ₁₆ f-mN ₁₇ -mN ₁₈ -mN ₁₉ -(s)-mN ₂₀ -(s)-mN ₂₁ -3',

(b ₂₁ )5'-mN ₁ -(s)-N ₂ f-(s)-mN ₃ -mN ₄ -mN ₅ -N ₆ f-mN ₇ -N ₈ fN ₉ f-mN ₁₀ -mN ₁₁ - mN ₁₂ -mN ₁₃ -N ₁₄ f-mN ₁₅ -N ₁₆ f-mN ₁₇ -mN ₁₈ -mN ₁₉ -(s)-mN ₂₀ -(s)-mN ₂₁ -3',

(b ₂₂ )5'-EVPmN ₁ -(s)-N ₂ f-(s)-mN ₃ -mN ₄ -mN ₅ -N ₆ f-mN ₇ -mN ₈ -mN ₉ -mN ₁₀ -mN ₁₁ - mN ₁₂ -mN ₁₃ -N ₁₄ f-mN ₁₅ -N ₁₆ f-mN ₁₇ -mN ₁₈ -mN ₁₉ -(s)-mN ₂₀ -(s)-mN ₂₁ -3',

(b ₂₃ )5'-EVPmN ₁ -(s)-N ₂ f-(s)-mN ₃ -mN ₄ -mN ₅ -N ₆ f-mN ₇ -N ₈ fN ₉ f-mN ₁₀ -mN ₁₁ - mN ₁₂ -mN ₁₃ -N ₁₄ f-mN ₁₅ -N ₁₆ f-mN ₁₇ -mN ₁₈ -mN ₁₉ -(s)-mN ₂₀ -(s)-mN ₂₁ -3',

(b ₂₄ )5'-mN ₁ -(s)-N ₂ f-(s)-mN ₃ -mN ₄ -mN ₅ -N ₆ f-mN ₇ -mN ₈ -mN ₉ -mN ₁₀ -mN ₁₁ - mN ₁₂ -mN ₁₃ -N ₁₄ f-mN ₁₅ -N ₁₆ f-mN ₁₇ -mN ₁₈ -mN ₁₉ -mN ₂₀ -mN ₂₁ -(s)-mN ₂₂ -(s)-mN ₂₃ -3',

(b ₂₅ )5'-mN ₁ -(s)-N ₂ f-(s)-mN ₃ -mN ₄ -mN ₅ -N ₆ f-mN ₇ -N ₈ fN ₉ f-mN ₁₀ -mN ₁₁ - mN ₁₂ -mN ₁₃ -N ₁₄ f-mN ₁₅ -N ₁₆ f-mN ₁₇ -mN ₁₈ -mN ₁₉ -mN ₂₀ -mN ₂₁ -(s)-mN ₂₂ -(s)-mN ₂₃ -3',

(b ₂₆ )5'-EVPmN ₁ -(s)-N ₂ f-(s)-mN ₃ -mN ₄ -mN ₅ -N ₆ f-mN ₇ -mN ₈ -mN ₉ -mN ₁₀ -mN ₁₁ - mN ₁₂ -mN ₁₃ -N ₁₄ f-mN ₁₅ -N ₁₆ f-mN ₁₇ -mN ₁₈ -mN ₁₉ -mN ₂₀ -mN ₂₁ -(s)-mN ₂₂ -(s)-mN ₂₃ -3',

(b ₂₇ )5'-EVPmN ₁ -(s)-N ₂ f-(s)-mN ₃ -mN ₄ -mN ₅ -N ₆ f-mN ₇ -N ₈ fN ₉ f-mN ₁₀ -mN ₁₁ - mN ₁₂ -mN ₁₃ -N ₁₄ f-mN ₁₅ -N ₁₆ f-mN ₁₇ -mN ₁₈ -mN ₁₉ -mN ₂₀ -mN ₂₁ -(s)-mN ₂₂ -(s)-mN ₂₃ -3';

The capital letter T represents a deoxyribonucleotide whose base is thymine.

P1 means that the nucleotide adjacent to the right of the letter is a 5'-phosphate nucleotide.

EVP means that the nucleotide adjacent to the right side of the letter combination is a 5'-trans vinylphosphonate nucleotide.

[GNA] indicates that the adjacent ribonucleotide on the right is a ribonucleotide with GNA modification.

[26] The double-stranded RNA modified substance according to any one of [15] to [25], wherein the double-stranded RNA modified substance is a siRNA modified substance.

[27]. The double-stranded RNA modified substance according to any one of [15] to [26], wherein the double-stranded RNA modified substance is a siRNA modified substance for inhibiting the expression of the MARC1 gene.

[28] The double-stranded RNA modified substance according to any one of [15] to [27], wherein the sense strand and the antisense strand are selected from the following combinations:

The sense strand comprises the sense strand of any one of the siRNA modifications shown in Table 2 herein, and the antisense strand comprises the antisense strand of the corresponding siRNA modification;

The sense chain comprises the sense chain of any one of the siRNAs siRNA 356-siRNA 363, siRNA 366-siRNA 373 and siRNA 433-siRNA 440 shown in Table 2 of the present invention, and the antisense chain comprises the antisense chain of the corresponding siRNA.

[29]. A double-stranded RNA conjugate, wherein the double-stranded RNA conjugate comprises the double-stranded RNA as described in any one of [1]-[14], or the double-stranded RNA modification as described in any one of [15]-[28]; and a conjugated group conjugated to the double-stranded RNA or the double-stranded RNA modification.

[30] The double-stranded RNA conjugate according to [29], wherein the conjugated group has any of the following structures:

[31]. The double-stranded RNA conjugate according to [29] or [30], wherein the conjugated group is connected to the 3’ end of the sense strand.

[32] The double-stranded RNA conjugate according to [31], wherein the conjugated group is conjugated to the 3' end of the sense strand via a phosphodiester bond;

Preferably, the sense strand and the antisense strand of the double-stranded RNA conjugate are complementary to each other to form a double-stranded region of the double-stranded RNA conjugate, and the 3' end of the sense strand forms a blunt end, and the 3' end of the antisense strand has 1-2 protruding nucleotides extending out of the double-stranded region;

or,

The sense strand and antisense strand of the double-stranded RNA conjugate are complementary to each other to form a double-stranded region of the double-stranded RNA conjugate, and the 3' end of the sense strand forms a blunt end, while the 3' end of the antisense strand forms a blunt end.

[33] The double-stranded RNA conjugate according to any one of [29] to [32], wherein the double-stranded RNA conjugate has the following structure:

The double helix structure is double-stranded RNA or a modified double-stranded RNA.

[34]. The double-stranded RNA conjugate according to any one of [29] to [33], wherein the double-stranded RNA conjugate is a siRNA conjugate.

[35] The double-stranded RNA conjugate according to any one of [29] to [34], wherein the double-stranded RNA conjugate is a siRNA conjugate for inhibiting the expression of the MARC1 gene.

[36]. The double-stranded RNA conjugate according to any one of [29] to [35], wherein the double-stranded RNA conjugate is formed by connecting any one of the siRNAs shown in Table 1 herein to a conjugated group, or the double-stranded RNA conjugate is formed by connecting any one of the siRNAs shown in Table 1-1 herein to a conjugated group, or the double-stranded RNA conjugate is formed by connecting any one of the siRNAs shown in Table 1-1 herein to a conjugated group, The conjugate is formed by connecting any one of the siRNA modifications shown in Table 2 herein to a conjugation group;

Preferably, in the double-stranded RNA conjugate, the sense strand and the antisense strand are selected from the following combinations:

The sense strand comprises the sense strand of any one of the siRNA conjugates shown in Table 3 herein, and the antisense strand comprises the antisense strand of the corresponding siRNA conjugate;

More preferably, the sense strand and the antisense strand are selected from the following combinations:

The sense chain comprises the sense chain of any one of the siRNAs siRNA 451-siRNA 456, siRNA 462-siRNA 467 and siRNA 516-siRNA 521 shown in Table 3 of the present invention, and the antisense chain comprises the antisense chain of the corresponding siRNA.

[37]. A prodrug of the double-stranded RNA described in any one of [1]-[14], the modified double-stranded RNA described in any one of [15]-[28], or the double-stranded RNA conjugate described in any one of [29]-[36].

[38]. A pharmaceutical composition, wherein the pharmaceutical composition comprises at least one of the following: a double-stranded RNA as described in any one of [1]-[14], a double-stranded RNA modification as described in any one of [15]-[28], a double-stranded RNA conjugate as described in any one of [29]-[36], or a prodrug as described in [37].

[39] The pharmaceutical composition according to [38], wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers and optionally one or more additional therapeutic agents.

[40] Use of the double-stranded RNA according to any one of [1] to [14], the double-stranded RNA modified product according to any one of [15] to [28], the double-stranded RNA conjugate according to any one of [29] to [36], the prodrug according to [37] or the pharmaceutical composition according to [38] or [39] in at least one of the following:

(1) Inhibiting MARC1 gene expression in vivo or in vitro, or preparing a drug for inhibiting MARC1 gene expression;

(2) for preventing or treating diseases associated with abnormal expression of the MARC1 gene, or for preparing drugs for preventing or treating diseases associated with abnormal expression of the MARC1 gene;

(3) Use for treating a subject suffering from a disease that would benefit from reduced expression of the MARC1 gene, or for preparing a medicament for treating a subject suffering from a disease that would benefit from reduced expression of the MARC1 gene.

[41]. The use according to [40], wherein the disease associated with abnormal expression of the MARC1 gene is selected from the group consisting of the following diseases:

Obesity, non-alcoholic fatty liver disease, alcohol-related fatty liver disease, non-alcoholic steatohepatitis, cirrhosis, liver fibrosis, elevated liver enzyme levels (ALT, AST, ALP), hepatocellular carcinoma, hypercholesterolemia and related cardiovascular disease, insulin resistance, impaired glucose tolerance, hyperglycemia, type II diabetes, and metabolic syndrome.

[42]. A method for inhibiting the expression of MARC1 gene in cells in vivo or in vitro, wherein the method comprises contacting the cells with a double-stranded RNA according to any one of [1]-[14], a double-stranded RNA modification according to any one of [15]-[28], a double-stranded RNA conjugate according to any one of [29]-[36], a prodrug according to [37], or a pharmaceutical composition according to [38] or [39].

[43] The method according to [42], wherein the cells are in vivo cells or in vitro cells.

[44] The method according to [42] or [43], wherein the cell is in a subject.

[45] The method according to [44], wherein the subject is a mammal, preferably a human.

[46] The method according to [44] or [45], wherein the subject has at least one of the following characteristics:

Abnormal expression of MARC1 gene in vivo, more specifically abnormally high expression of MARC1 gene;

Suffering from diseases associated with abnormal expression of the MARC1 gene;

Having a disease that would benefit from decreased expression of the MARC1 gene.

[47]. A double-stranded RNA as described in any one of [1]-[14], a double-stranded RNA modification as described in any one of [15]-[28], a double-stranded RNA conjugate as described in any one of [29]-[36], a prodrug as described in [37] or a pharmaceutical composition as described in [38] or [39] for use in treatment.

Effects of the Invention

In some embodiments, the double-stranded RNA provided by the present disclosure can combine to form an RNA-induced silencing complex (RISC) in cells, cut the mRNA transcribed by the MARC1 gene, and efficiently and specifically inhibit the expression of the MARC1 gene. Expression, for the treatment of MARC1-related diseases including obesity, non-alcoholic fatty liver disease, alcohol-related fatty liver disease, non-alcoholic steatohepatitis, cirrhosis, liver fibrosis, elevated liver enzyme levels (ALT, AST, ALP), hepatocellular carcinoma, hypercholesterolemia and related cardiovascular diseases, insulin resistance, impaired glucose tolerance, hyperglycemia, type II diabetes and metabolic syndrome, as well as other related conditions, pathologies or syndromes that have not yet been identified.

Furthermore, the double-stranded RNA in the present disclosure is siRNA, which targets and degrades the transcription product mRNA of the MARC1 gene, exerts the effect of RNA interference, and inhibits the protein expression of the MARC1 gene. It is a MARC1 inhibitor with a high inhibition rate and good specificity.

In some embodiments, the present disclosure modifies double-stranded RNA to obtain modified double-stranded RNA, which has high stability and is suitable for use in in vivo disease treatment.

Furthermore, the double-stranded RNA modification is a siRNA modification, which has high stability and good inhibitory activity.

In some embodiments, the present invention discloses a conjugate of double-stranded RNA or double-stranded RNA modified substance obtained by connecting a conjugation group to double-stranded RNA or double-stranded RNA modified substance, which can be used for efficient targeted delivery to tissues and cells, reducing the impact of double-stranded RNA or double-stranded RNA modified substance on non-targeted normal tissues and cells, and improving its safety in clinical disease treatment.

Furthermore, the double-stranded RNA conjugate is a siRNA conjugate, which has organ or tissue targeting while maintaining the inhibitory activity and stability of siRNA, can reduce the impact on other tissues or organs and reduce the amount of siRNA molecules used, thereby achieving the purpose of reducing toxicity and reducing costs.

Furthermore, the conjugated group in the present disclosure is a group (GalNAc) of the structure shown in Formula I, and GalNAc can be used for targeted delivery to liver cells and tissues to effectively inhibit the expression of MARC1 gene in the liver. In addition, the siRNA conjugate disclosed in the present disclosure has low toxicity and an excellent drug safety window.

DETAILED DESCRIPTION

definition

Unless otherwise stated, the terms used in the present invention have the following meanings.

In the claims and/or description of the present invention, the word "a" or "an" or "the" may mean "one", but may also mean "one or more", "at least one" and "one or more than one".

As used in the claims and description, the words "comprising," "having," "including," or "containing" are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

Throughout the application document, the term "about" means that a value includes the standard deviation of the error of the device or method used to determine the value. The numerical ranges and parameters used to define the present invention are all approximate values, and the relevant values in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to the aforementioned test methods or devices. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

The term "MARC1" used in the context of this disclosure refers to the well-known gene and polypeptide. The MARC1 gene and MARC1 mRNA sequence are easily obtained using, for example, the following: Gene Bank (GenBank), database (UniProt), Online Mendelian Inheritance in Man (OMIM), etc.

The term "MARC1 gene" may be a wild-type MARC1 gene, or a MARC1 gene mutant with sequence variation. Many sequence variations in the MARC1 gene have been identified and can be found in, for example, NCBIdbSNP and UniProt (see, for example, ncbi.nlm.nih.gov/snp).

The terms "polypeptide" and "protein" interchangeably refer to a string of at least two amino acid residues linked to each other by covalent bonds (e.g., peptide bonds), and may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide. A polypeptide may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. The term also includes polypeptides that have been modified (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component) amino acid polymers.

The term "target sequence" as used in the context of the present disclosure refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during transcription of a target gene, including mRNA that is a product of RNA processing of a primary transcript.

In some embodiments, the target sequence is a nucleotide sequence consisting of no less than 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 80, 100 or 150 consecutively linked nucleosides. In some optional embodiments, another shorter target sequence may be included in the target sequence. In some embodiments, one or more shorter target sequences may be included in the target sequence. It should be considered that the two or more shorter target sequences included in the same target sequence have the same characteristics.

In some embodiments, the target gene is the MARC1 gene.In some embodiments, the target portion of the sequence will be at least long enough to serve as a substrate for iRNA-directed cleavage at or near the nucleotide sequence portion of the mRNA molecule formed during transcription of the MARC1 gene.

In the art, "G", "C", "A", "T" and "U" generally represent the bases of guanine, cytosine, adenine, thymine and uracil, respectively, but it is also generally known in the art that "G", "C", "A", "T" and "U" each generally represent nucleotides containing guanine, cytosine, adenine, thymine and uracil as bases, respectively, which is a common way to represent deoxyribonucleic acid sequences and/or ribonucleic acid sequences, so in the context of the present disclosure, the meanings represented by "G", "C", "A", "T", "U" include the above-mentioned various possible situations, however, it should be understood that the term "ribonucleotide" or "nucleotide" can also refer to a modified nucleotide (as further described below) or an alternative replacement part. Those skilled in the art can realize that guanine, cytosine, adenine and uracil can be replaced by other parts without substantially changing the base pairing properties of an oligonucleotide (including a nucleotide having such a replacement part). For example, without limitation, a nucleotide comprising inosine as its base can be base paired with a nucleotide comprising adenine, cytosine or uracil. Therefore, a nucleotide containing uracil, guanine or adenine can be replaced by a nucleotide containing, for example, inosine in the nucleotide sequence of the dsRNA characterized by the present invention. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced by guanine and uracil, respectively, to form a G-U wobble base pairing with the target mRNA. Sequences containing such replacement parts are suitable for the compositions and methods characterized by the present invention.

The terms "iRNA", "RNAi agent", "iRNA agent", "RNA interfering agent" used in the context of this disclosure are used interchangeably herein and refer to siRNAs as defined herein and mediate targeted cleavage of RNA transcripts through the RNA induced silencing complex (RISC) pathway. iRNAs direct sequence-specific degradation of mRNAs through a process known as RNA interference (RNAi). iRNAs modulate, e.g., inhibit, expression of a target gene in a cell, such as a cell of a subject, such as a mammalian subject.

The terms "double-stranded ribonucleic acid", "double-stranded RNA (dsRNA) molecule", "dsRNA" used in the context of this disclosure can be used interchangeably. The term "dsRNA" refers to a complex of ribonucleic acid molecules having a double-stranded structure, comprising two antiparallel and substantially complementary nucleic acid strands, referred to as "sense" and "antisense" orientations relative to a target gene, such as the MARC1 gene. In some embodiments, double-stranded ribonucleic acid (dsRNA) triggers degradation of a target RNA, such as mRNA, through a post-transcriptional gene silencing mechanism (referred to herein as RNA interference or RNAi).

Typically, most of the nucleotides of each strand of the dsRNA molecule are ribonucleotides, but as described in detail herein, each or both of the two strands may also include one or more non-ribonucleotides, e.g., deoxyribonucleotides and/or modified nucleotides. In addition, as used in the present disclosure, "double-stranded RNA" may include ribonucleotides, phosphate backbones, etc., with chemical modifications. These modifications may include all types of modifications disclosed herein or known in the art.

The term "isonucleotide" as used in the context of the present disclosure refers to a compound formed by a change in the position of the base on the ribose ring in a nucleotide, for example, a compound formed by a base not being attached to the 1'-position of the ribose ring but being attached to the 2'-position or 3'-position of the ribose ring.

In some embodiments, the double-stranded ribonucleic acid disclosed herein is a siRNA that interacts with an mRNA sequence transcribed from a target gene (e.g., an mRNA sequence transcribed from a MARC1 gene) to guide the cleavage of the target RNA. Constrained, long double-stranded RNA introduced into cells is broken down into siRNA by a type III nuclease called Dicer (Sharp et al., Genes Dev. 2001, 15: 485). Dicer (ribonuclease III-like enzyme) processes dsRNA into 19-23 base pair short interfering RNAs with characteristic two-base 3' overhangs (Bernstein et al., (2001) Nature 409: 363). These siRNAs are then incorporated into RNA-induced silencing complexes (RISC), where one or more helicases unwind the siRNA duplex, making it possible for complementary antisense strands to guide target recognition (Nykanen et al., (2001) Cell 107: 309). Once bound to a suitable target mRNA, one or more nucleases within RISC cleave the target to induce silencing (Elbashir et al., (2001) Genes Dev. 15: 188).

The term "overhanging nucleotides" as used in the context of this disclosure refers to one or more unpaired nucleotides that protrude from the duplex structure of a double-stranded ribonucleic acid when a 3' end of one strand of the dsRNA extends beyond the 5' end of the other strand, or vice versa. "Blunt end" or "blunt end" means that there are no unpaired nucleotides at that end of the double-stranded ribonucleic acid, i.e., no nucleotide overhang. A "blunt-ended" double-stranded ribonucleic acid is a dsRNA that is double-stranded throughout its length, i.e., has no nucleotide overhangs at either end of the molecule.

The term "antisense strand" refers to a strand of a double-stranded RNA that is substantially complementary to a target sequence (e.g., derived from human MARC1 mRNA). Where the region of complementarity is not completely complementary to the target sequence, mismatches are most tolerated in the terminal regions, and if mismatches occur, they are typically within one or more regions of the termini, e.g., 5, 4, 3, 2, or 1 nucleotides of the 5' and/or 3' termini.

The term "sense strand" refers to the nucleic acid strand of a double-stranded RNA that contains a region that is substantially complementary to a region of the antisense strand.

The terms "complementary" or "reverse complement" are used interchangeably and have the meanings known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one strand are paired with bases on the other strand in a complementary manner. In DNA, the purine base adenine (A) is always paired with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (G) is always paired with the pyrimidine base cytosine (C). Each base pair includes a purine and a pyrimidine. When adenine on one strand is always paired with thymine (or uracil) on the other strand, and guanine is always paired with cytosine, the two strands are considered to be complementary to each other, and the sequence of the strand can be inferred from the sequence of its complementary strand. Accordingly, "mismatch" means in the art that in a double-stranded nucleic acid, the bases at corresponding positions are not paired in a complementary form.

The term "substantially reverse complementary" means that there are no more than three base mismatches between the two nucleotide sequences involved, that is, there are 1, 2 or 3 base mismatches between the two nucleotide sequences involved; "completely complementary" means that there are no base mismatches between the two nucleotide sequences.

The terms "complementary," "fully complementary," and "substantially complementary" may be used with respect to base pairing between the sense and antisense strands of a dsRNA, or between the antisense strand of a dsRNA and a target sequence, as will be understood from the context of their use.

The term "inhibit" may be used interchangeably with "reduce," "silence," "downregulate," "suppress," and other similar terms, and includes any level of inhibition.

The term "inhibiting the expression of a MARC1 gene" includes inhibiting the expression of any MARC1 gene (such as, for example, a mouse MARC1 gene, a rat MARC1 gene, a monkey MARC1 gene, or a human MARC1 gene) and variants (such as naturally occurring variants) or mutants of a MARC1 gene. Thus, the MARC1 gene can be a wild-type MARC1 gene, a mutant MARC1 gene, or a transgenic MARC1 gene in the context of a genetically manipulated cell, cell group, or organism.

"Inhibiting MARC1 gene expression" includes any level of inhibition of MARC1 gene, such as at least partial inhibition of MARC1 gene expression, such as inhibition of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

The term "independently" refers to at least two groups (or rings) in the structure that have the same or similar value ranges. The term "substituent" may have the same or different meanings in certain circumstances. For example, if substituent X and substituent Y are each independently hydrogen, hydroxyl, alkyl or aryl, then when substituent X is hydrogen, substituent Y may be either hydrogen, or hydroxyl, alkyl or aryl; similarly, when substituent Y is hydrogen, substituent X may be either hydrogen, or hydroxyl, alkyl or aryl.

The term "alkyl" includes straight chain, branched or cyclic saturated alkyl groups. For example, alkyl groups include, but are not limited to, methyl, ethyl, propyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, cyclobutyl, n-pentyl, cyclohexyl and the like. Exemplarily, "C _1-6 " in "C _1-6 alkyl" refers to a group having 1, 2, 3, 4, 5 or 6 carbon atoms arranged in a straight chain, branched or cyclic form.

The term "alkoxy" herein refers to an alkyl group attached to the remainder of the molecule via an oxygen atom (-O-alkyl), wherein the alkyl group is as defined herein. Non-limiting examples of alkoxy include methoxy, ethoxy, trifluoromethoxy, difluoromethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, n-pentoxy, and the like.

The term "treatment" means that after suffering from a disease, a subject is exposed to (e.g., administered) double-stranded RNA, a double-stranded RNA modified substance, a double-stranded RNA conjugate, a prodrug, or a pharmaceutical composition, thereby alleviating the symptoms of the disease compared to when the subject is not exposed to the disease, and does not necessarily mean that the symptoms of the disease are completely suppressed. Suffering from a disease means that the body has symptoms of the disease.

The term "prevention" means that before a subject develops a disease, by contacting (e.g., administering) the double-stranded RNA, double-stranded RNA modified product, double-stranded RNA conjugate, prodrug, or pharmaceutical composition of the present disclosure, the symptoms after the subject develops the disease are alleviated compared to when the subject does not develop the disease. It does not mean that the disease must be completely suppressed.

The term "effective amount" refers to such an amount or dosage of the double-stranded RNA, double-stranded RNA modification, double-stranded RNA conjugate, prodrug or pharmaceutical composition of the present invention, which produces the desired effect in a patient in need of treatment or prevention after being administered to the patient in a single or multiple doses. The effective amount can be easily determined by the attending physician who is a person skilled in the art by considering a variety of factors such as the species of the mammal; its size, age and general health; the specific disease involved; the extent or severity of the disease; the response of the individual patient; the specific antibody administered; the mode of administration; the bioavailability characteristics of the administered formulation; the selected dosing regimen; and the use of any concomitant therapy.

The term "disease associated with abnormal expression of the MARC1 gene" is a disease or disorder associated with the involvement of complement MARC1. The term "disease associated with abnormal expression of the MARC1 gene" includes diseases, disorders or conditions that will benefit from reducing the expression of MARC1 (i.e., "MARC1-related diseases"). In some embodiments, the disease associated with abnormal expression of the MARC1 gene is selected from the group consisting of: obesity, non-alcoholic fatty liver disease, alcohol-related fatty liver disease, non-alcoholic steatohepatitis, cirrhosis, liver fibrosis, elevated liver enzyme levels (ALT, AST, ALP), hepatocellular carcinoma, hypercholesterolemia and related cardiovascular diseases, insulin resistance, impaired glucose tolerance, hyperglycemia, type II diabetes and metabolic syndrome. Exemplarily, reference is made to the following scientific literature but is not limited thereto: Emdin CA, Haas ME, Khera AV, et al. Amissense variant in Mitochondrial Amidoxime Reducing Component 1 gene and protection against liver disease. PLoS Genet, 2020, 16(4): e1008629.; Ott G., Reichmann D., Boerger C., et al. Functional characterization of protein variants encoded by nonsynonymous single nucleotide polymorphisms in MARC1 and MARC2 in healthy Caucasians. Drug Metab Dispos, 2014, 42: 718–725.; Meroni M., Longo M., Tria G., et al. Genetics is of the essence to face NAFLD. Biomedicines, 2021, 9: 1359.; Parisinos CA, Wilman HR, Thomas EL, et al. al.Genome-wide and Mendelian randomisation studies of liver MRI yield insights into the pathogenesis of steatohepatitis.Journal of Hepatology,2020,73:241–251.; Du MJ Metabolic drivers of non-alcoholic fatty liver disease. Journal of molmet, 2020, 101143.; Luukkonen PK, Juuti A., Sammalkorpi H., et al. MARC1 variant rs2642438 increases hepatic phosphatidylcholines and decreases severity of non-alcoholic fatty liver disease in humans. Journal of Hepatology, 2020, 73:696–739.; Trépo E., Valenti L. Update on NAFLD genetics: from new variants to the clinic. Journal of Hepatology, 2020, 7634.; Sparacino-Watkins CE, Tejero J., Sun B., et al. mistry, 2014, 289(15):10345–10358.; Luukkonen, Panu K et al. "Distinct contributions of metabolic dysfunction and genetic risk factors in the pathogenesis of non-alcoholic fatty liver disease." Journal of hepatology vol.76,3(2022):526-535.doi:10.1016/j.jhep.2021.10.013; Romeo, Stefano. "MARC1 and HNRNPUL1: Two Novel Players in Alcohol-related Liver Disease." Gastroenterology vol.159,4(2020):1231-1232.doi:10.1053/ j.gastro.2020.08.009; Rivera-Paredez, Berenice et al. "Association of MARC1, ADCY5, and BCO1 Variants with the Lipid Profile, Suggests an Additive Effect for Hypertriglyceridemia in Mexican Adult Men." International journal of molecular sciences vol.23,19 11815.5Oct.2022,doi:10.3390/ijms231911815;Gao, Chuan et al. "Genome-wide association analysis of serum alanine and aspartate aminotransferase, and the modifying effects of BMI in 388k European individuals." Genetic epidemiology vol.45,6(2021):664-681.doi:10.1002/ge pi.22392.

The term "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" refers to auxiliary materials widely used in the field of drug production. The main purpose of using excipients is to provide a pharmaceutical composition that is safe to use, stable in nature and/or has specific functionality, and also to provide a method so that after the drug is administered to the subject, the active ingredient can be dissolved at a desired rate, or to promote the effective absorption of the active ingredient in the subject receiving the drug. Pharmaceutically acceptable excipients can be inert fillers or functional ingredients that provide a certain function to the pharmaceutical composition (for example, stabilizing the overall pH value of the composition or preventing the degradation of the active ingredient in the composition). Non-limiting examples of pharmaceutically acceptable excipients include, but are not limited to, binders, suspending agents, emulsifiers, diluents (or fillers), granulating agents, adhesives, disintegrants, lubricants, anti-adhesive agents, glidants, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, sweeteners, etc.

The pharmaceutical compositions of the present disclosure can be prepared using any method known to those skilled in the art, such as conventional mixing, dissolving, granulating, emulsifying, pulverizing, encapsulating, embedding and/or lyophilizing processes.

In the present disclosure, the route of administration can be varied or adjusted in any applicable manner to meet the requirements of the properties of the drug, the convenience of the patient and the medical staff, and other relevant factors.

The terms "individual", "patient" or "subject" used in the context of this disclosure include mammals. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

The term “corresponding siRNA” used in the context of the present disclosure refers to the same siRNA mentioned above. For example, when it is stated that “the sense chain comprises the sense chain of any one siRNA shown in Table 1 and Table 1-1 herein, and the antisense chain comprises the antisense chain of the corresponding siRNA”, it means that the sense chain and antisense chain contained are from the same siRNA shown in Table 1 and Table 1-1 herein. For example, when the sense chain comprises 5’-CCUACACAAAGGACCUACU-3’(SEQ ID NO:38), the antisense chain comprises 5’-AGUAGGUCCUUUGUGUAGG-3’(SEQ ID NO:92). Similarly, the term “corresponding siRNA modifier” refers to the same siRNA modifier mentioned above. For example, when it is stated that “the sense chain comprises the sense chain of any one siRNA modifier shown in Table 2 herein, and the antisense chain comprises the antisense chain of the corresponding siRNA modifier”, it means that the sense chain and antisense chain contained are from the same siRNA modifier shown in Table 2 herein. Similarly, the term "corresponding siRNA conjugate" refers to the same siRNA conjugate mentioned above, for example, when it is mentioned that "the sense strand comprises the sense strand of any one siRNA conjugate shown in Table 3 herein, and the antisense strand comprises the antisense strand of the corresponding siRNA conjugate", it means that the sense strand and antisense strand contained are from the same siRNA conjugate shown in Table 3 herein. In addition, in these contexts, "comprising" includes the case of being composed of these sequences.

Unless otherwise defined or clearly indicated by the context, all technical and scientific terms used in this disclosure have the same meaning as herein. The same meanings as commonly understood by one of ordinary skill in the art are disclosed.

dsRNA

The first aspect of the present disclosure provides a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of the MARC1 gene. One strand of the double-stranded ribonucleic acid is an antisense strand, which is complementary to the mRNA sequence formed during the expression of the target gene (i.e., the MARC1 gene) and is used to guide the cutting of the target mRNA (i.e., the transcription product of the MARC1 gene). The other sense strand in the double-stranded ribonucleic acid includes a double-stranded region that is partially complementary and completely complementary to the antisense strand to form the double-stranded ribonucleic acid.

In some embodiments, the double-stranded RNA is used as a substrate for the endonuclease (Dicer) and is cut into small fragments of dsRNA, i.e., siRNA. In some embodiments, the double-stranded RNA is siRNA. siRNA assembles to form an RNA-induced silencing complex (RISC) RISC complex, cuts the target mRNA, and inhibits the expression of the MARC1 gene.

According to the target sequence derived from human MARC1 mRNA (NM_022746.4), siRNA that binds to the target mRNA is designed. In some embodiments, the target sequence is selected from the nucleotide sequence shown in any one of SEQ ID NOs: 1 to 9 and 812 to 817. In some more specific embodiments, the target sequence is selected from the nucleotide sequence shown in any one of SEQ ID NOs: 10 to 37, 799 to 811, and 818 to 830.

In some specific embodiments, the nucleotide sequence shown in SEQ ID NO:1 includes the nucleotide sequences shown in SEQ ID NO:10-12 and 799-801.

In some specific embodiments, the nucleotide sequence shown in SEQ ID NO:2 includes the nucleotide sequences shown in SEQ ID NO:13-16, 802 and 803.

In some specific embodiments, the nucleotide sequence shown in SEQ ID NO:3 includes the nucleotide sequences shown in SEQ ID NO:17-20 and 804.

In some specific embodiments, the nucleotide sequence shown in SEQ ID NO:4 includes the nucleotide sequences shown in SEQ ID NO:21-24, 805 and 806.

In some specific embodiments, the nucleotide sequence shown in SEQ ID NO:5 contains the nucleotide sequence shown in SEQ ID NO:25 to 28.

In some specific embodiments, the nucleotide sequence shown in SEQ ID NO:6 includes the nucleotide sequences shown in SEQ ID NO:29-30 and 807.

In some specific embodiments, the nucleotide sequence shown in SEQ ID NO:7 includes the nucleotide sequences shown in SEQ ID NO:31-32 and 808.

In some specific embodiments, the nucleotide sequence shown in SEQ ID NO:8 includes the nucleotide sequences shown in SEQ ID NO:33-35 and 809.

In some specific embodiments, the nucleotide sequence shown in SEQ ID NO:9 includes the nucleotide sequences shown in SEQ ID NO:36~37810, 811.

In some specific embodiments, the nucleotide sequence shown in SEQ ID NO:812 contains the nucleotide sequences shown in SEQ ID NO:818 and 819.

In some specific embodiments, the nucleotide sequence shown in SEQ ID NO:813 contains the nucleotide sequences shown in SEQ ID NO:820 and 821.

In some specific embodiments, the nucleotide sequence shown in SEQ ID NO:814 contains the nucleotide sequences shown in SEQ ID NO:822 and 823.

In some specific embodiments, the nucleotide sequence shown in SEQ ID NO:815 contains the nucleotide sequences shown in SEQ ID NO:824 and 825.

In some specific embodiments, the nucleotide sequence shown in SEQ ID NO:816 contains the nucleotide sequence shown in SEQ ID NO:826-828.

In some specific embodiments, the nucleotide sequence shown in SEQ ID NO:817 includes the nucleotide sequences shown in SEQ ID NO:829 and 830.

In some embodiments, the antisense strand comprises a sequence B that differs by no more than 3 nucleotides from the reverse complementary sequence of at least 15 consecutive nucleotides in the target sequence. Specifically, along the direction from the 5' end to the 3' end, the starting nucleotide is selected in the target sequence, and at least 15 nucleotides extending in the 3' direction including the starting nucleotide are used as the binding region of the siRNA. The antisense strand comprises the reverse complementary sequence of the nucleotide sequence corresponding to the binding region. It should be noted that the starting nucleotide can be a nucleotide at any position of the target sequence, as long as at least 15 consecutive nucleotides (including the nucleotide at the starting position) can be obtained by extending in the 3' direction of the target sequence based on the starting nucleotide.

In the present disclosure, the nucleotide sequence of the antisense strand and the target sequence can be completely complementary or substantially complementary. When the nucleotide sequence of the antisense strand and the target sequence are substantially complementary, there are no more than 3 mismatched bases in the nucleotide sequence of the antisense strand and the target sequence. For example, the mismatched bases are 1, 2 or 3. When the nucleotide sequence of the antisense strand and the target sequence are completely complementary, there are no mismatched bases in the nucleotide sequence of the antisense strand and the target sequence.

Further, the antisense strand consists of at least 15 nucleotides. In some embodiments, the antisense strand consists of 15-28 nucleotides. For example, the length of the antisense strand is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 nucleotides.

Preferably, the antisense strand consists of 19-25 nucleotides, more preferably 19-23 nucleotides, most preferably 19, 21 or 23 nucleotides.

In some optional embodiments, the sequence B included in the antisense strand is identical to the reverse complementary sequence of a sequence consisting of at least 15 consecutive nucleotides on the target sequence.

In some specific embodiments, the sequence B included in the antisense strand is identical to the reverse complementary sequence of a sequence consisting of 15-28 consecutive nucleotides on the target sequence, preferably 19-25 consecutive nucleotides on the target sequence, more preferably 19-23 consecutive nucleotides on the target sequence, and most preferably 19, 21 or 23 consecutive nucleotides.

In some optional embodiments, the sequence B comprised in the antisense strand differs by 1 nucleotide from the reverse complementary sequence of a sequence consisting of at least 15 consecutive nucleotides on the target sequence.

In some specific embodiments, the sequence B included in the antisense strand differs by 1 nucleotide from the reverse complementary sequence of a sequence consisting of 15-28 nucleotides on the target sequence, preferably 19-25 consecutive nucleotides on the target sequence, more preferably 19-23 consecutive nucleotides on the target sequence, and most preferably 19, 21 or 23 consecutive nucleotides.

In some specific embodiments, the different nucleotides are located at the 3' end of sequence B. In other specific embodiments, the different nucleotides are located at the 5' end of sequence B.

In some embodiments, the sense strand comprises a sequence A that differs by no more than 3 nucleotides from at least 15 consecutive nucleotides in the target sequence. The sense strand includes a region complementary to the antisense strand, and the nucleotide sequence of the sense strand is identical or substantially identical to the sequence of the region where the antisense strand binds to the target sequence. Therefore, the nucleotide sequence of the sense strand is at least 15 consecutive nucleotides in the target sequence that bind to the antisense strand; or, the nucleotide sequence of the sense strand is compared with at least 15 consecutive nucleotides in the target sequence that bind to the antisense strand, and there are 1, 2, or 3 different nucleotides in the nucleotide sequence of the sense strand.

Further, the sense strand is composed of at least 15 nucleotides. In some embodiments, the sense strand is composed of 15-28 nucleotides. For example, the length of the sense strand is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 nucleotides.

Preferably, the sense strand consists of 19-25 nucleotides, more preferably 19-23 nucleotides, most preferably 19, 21 or 23 nucleotides.

In some optional embodiments, the sequence A included in the sense strand is identical to a sequence consisting of at least 15 consecutive nucleotides in the target sequence.

In some specific embodiments, the sequence A included in the sense strand is identical to a sequence consisting of 15-28 consecutive nucleotides on the target sequence, preferably 19-25 consecutive nucleotides on the target sequence, more preferably 19-23 consecutive nucleotides on the target sequence, and most preferably 19, 21 or 23 consecutive nucleotides on the target sequence.

In some optional embodiments, the sequence A included in the sense strand differs by 1 nucleotide from a sequence consisting of at least 15 consecutive nucleotides in the target sequence.

In some specific embodiments, the sequence A included in the sense strand differs by 1 nucleotide from a sequence consisting of 15-28 consecutive nucleotides on the target sequence, preferably 19-25 consecutive nucleotides on the target sequence, more preferably 19-23 consecutive nucleotides on the target sequence, and most preferably 19, 21 or 23 consecutive nucleotides on the target sequence.

In some specific embodiments, the different nucleotides are located at the 3' end of sequence A. In other specific embodiments, the different nucleotides are located at the 5' end of sequence A.

In the present disclosure, the length of the sense strand and the length of the antisense strand may be the same or different.

In some embodiments, the sense strand and the antisense strand are the same length, specifically, the sense strand/antisense strand length ratio is 15/15, 16/16, 17/17, 18/18, 19/19, 20/20, 21/21, 22/22, 23/23, 24/24, 25/25, 26/26, 27/27 or 28/28. Preferably, the sense strand/antisense strand length ratio is 19/19, 20/20, 21/21, 22/22, 23/23, 24/24 or 25/25, more preferably 19/19, 20/20, 21/21, 22/22 or 23/23, and most preferably 19/19, 21/21 or 23/23.

In some embodiments, the sense strand is different from the antisense strand in length. For example, the sense strand/antisense strand length ratio is 19/20, 19/21, 19/22, 19/23, 19/24, 19/25, 19/26, 20/19, 20/21, 20/22, 20/23, 20/24, 20/25, 20/26, 21/19, 21/20, 21/22, 21/23, 21/24, 21/25 , 21/26, 22/19, 22/20, 22/21, 22/23, 22/24, 22/25, 22/26, 23/19, 23/20, 23/21, 23/22, 23/24, 23/25 or 23/26, etc.; in some preferred embodiments, the length ratio of the sense strand/antisense strand is 19/21, 20/22 or 21/23.

In the present disclosure, the sense strand and the antisense strand may be completely complementary or substantially complementary. When the two are substantially complementary, there are no more than 3 mismatched bases in the double-stranded region formed by the sense strand and the antisense strand.

In some embodiments, after the sense strand and the antisense strand complement each other to form a double-stranded region, the sense strand, the antisense strand, or a combination thereof has protruding nucleotides extending out of the double-stranded region. The number of protruding nucleotides may be 1 or more, for example, 1 or 2. In addition, the protruding 1-2 nucleotides may be located at the 5' end, 3' end, or both ends of any antisense strand or sense strand, and each protruding nucleotide may be any type of nucleotide.

In some embodiments, the sense strand and the antisense strand are complementary to each other to form the double-stranded region, and the 3' end of the sense strand has 1-2 protruding nucleotides extending out of the double-stranded region, and the 3' end of the antisense strand forms a blunt end.

In some embodiments, the sense strand and the antisense strand are complementary to each other to form the double-stranded region, and the 3' end of the antisense strand has 1-2 protruding nucleotides extending out of the double-stranded region, and the 3' end of the sense strand forms a blunt end.

In some embodiments, the sense strand and the antisense strand are complementary to each other to form the double-stranded region, and the 3' ends of the sense strand and the antisense strand each have 1-2 protruding nucleotides extending out of the double-stranded region.

In some embodiments, the sense strand and the antisense strand are complementary to each other to form the double-stranded region, and the 3' ends of the sense strand and the antisense strand both form blunt ends.

In some specific embodiments, the sense strand comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 38 to 91, 411 to 497, 499 to 502, and the antisense strand comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 92 to 145, 503 to 589, 591 to 594.

In some specific embodiments, the sense strand comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 146 to 183, and the antisense strand comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 184 to 221.

In some specific embodiments, the double-stranded RNA is selected from any siRNA as shown in Table 1 or Table 1-1. In some specific embodiments, the inhibition rate of the siRNA disclosed herein on the MARC1 gene is at least about 20%, and can be at least about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or a value or range between any two of these values.

The siRNA provided by the present disclosure has high specificity in binding to the target mRNA (MARC1 mRNA), has good silencing activity of the target mRNA, can significantly inhibit the expression of the MARC1 gene, and is used to treat MARC1-related diseases including obesity, non-alcoholic fatty liver disease, alcohol-related fatty liver disease, non-alcoholic steatohepatitis, cirrhosis, liver fibrosis, elevated liver enzyme levels (ALT, AST, ALP), hepatocellular carcinoma, hypercholesterolemia and related cardiovascular diseases, insulin resistance, impaired glucose tolerance, hyperglycemia, type II diabetes and metabolic syndrome, as well as other related diseases, pathologies or syndromes that have not yet been identified.

In some embodiments, the present disclosure provides a siRNA composition comprising any one or a combination of two or more of the siRNAs shown in Table 1 or Table 1-1.

In some embodiments, each nucleotide of the sense strand is independently a modified nucleotide or an unmodified nucleotide. In some embodiments, each nucleotide of the antisense strand is independently a modified nucleotide or an unmodified nucleotide.

In some embodiments, any two nucleotides connected in the sense strand are connected by a phosphodiester bond or a phosphorothioate diester bond. In some embodiments, any two nucleotides connected in the antisense strand are connected by a phosphodiester bond or a phosphorothioate diester bond.

In some embodiments, the 5' terminal nucleotide of the antisense strand is not linked to a 5' phosphate group or a 5' phosphate-derived group, or is linked to a 5' phosphate group or a 5' phosphate-derived group.

Herein, when the 5' terminal nucleotide of the antisense strand is not connected to a 5' phosphate group or a 5' phosphate derivative group, the structure of the 5' terminal nucleotide is as shown in Formula X:

Wherein, Base represents a base, such as A, U, G, C or T; R is a hydroxyl group or is substituted by various groups known to those skilled in the art, for example, R can be 2'-fluoro (2'-F), 2'-alkoxy, 2'-substituted alkoxy, 2'-alkyl, 2'-substituted alkyl, 2'-amino, 2'-substituted amino, or 2'-deoxynucleotide.

Exemplarily, the structure of the 5' phosphate group is: The structures of the 5' phosphate derivative group include but are not limited to: wait.

The 5' terminal nucleotide of the antisense strand is connected to a 5' phosphate group or a 5' phosphate derivative group to form the following structure:

Wherein, Base represents a base, such as A, U, G, C or T; R' is a hydroxyl group or is substituted by various groups known to those skilled in the art, for example, 2'-fluoro (2'-F) modified nucleotides, 2'-alkoxy modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-deoxyribonucleotides.

Double-stranded RNA modification

The second aspect of the present disclosure provides a double-stranded RNA modification. Further, the double-stranded RNA modification is a siRNA modification. The siRNA modification can improve the stability of siRNA while maintaining a high MARC1 mRNA inhibitory activity.

In some embodiments, the double-stranded RNA modification comprises at least one modification of a nucleotide. The modification of the nucleotide is selected from at least one of the modification of the ribose group and the modification of the base. In some embodiments, "modification of the nucleotide" refers to a nucleotide or a nucleotide derivative formed by replacing the 2' hydroxyl group of the ribose group of the nucleotide with other groups, or a nucleotide in which the base on the nucleotide is a modified base. The modification of the nucleotide does not cause the siRNA to inhibit gene expression. The function of the siRNA is significantly weakened or lost. For example, the modified nucleotides disclosed in JK Watts, GF Deleavey, and MJ Damha, Chemically modified siRNA: tools and applications. Drug Discov Today, 2008, 13 (19-20): 842-55 can be selected. The stability of the siRNA can be improved by modifying the nucleotides, and its high inhibition efficiency on the MARC1 gene can be maintained.

Exemplarily, the modified nucleotide has the structure shown below:

Wherein, Base represents a base, such as A, U, G, C or T. The hydroxyl group at the 2' position of the ribose group is substituted by R. The hydroxyl group at the 2' position of these ribose groups can be substituted by various groups known to those skilled in the art, for example, 2'-fluoro (2'-F) modified nucleotides, 2'-alkoxy modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-deoxyribonucleotides.

In some embodiments, the 2'-alkoxy modified nucleotide is a 2'-methoxy (2'-OMe, 2'-O-CH ₃ ) modified nucleotide, and the like.

In some embodiments, the 2'-substituted alkoxy modified nucleotide is a 2'-methoxyethoxy (2'-O-CH ₂ -CH ₂ -O-CH ₃ ) modified nucleotide, a 2'-O-CH ₂ -CH=CH ₂ modified nucleotide, and the like.

In some embodiments, the 2'-substituted alkyl modified nucleotide is a 2'- _CH2 - _CH2 -CH= _CH2 modified nucleotide and the like.

In some embodiments, the modification of the nucleotide is the modification of the base. The modification of the base can be various types of modifications known to those skilled in the art. Exemplary, the modification of the base includes but is not limited to m ⁶ A, Ψ, m ¹ A, m ⁵ A, ms ² i ⁶ A, i ⁶ A, m ³ C, m ⁵ C, ac ⁴ C, m ⁷ G, m ^2,2 G, m ² G, m ¹ G, Q, m ⁵ U, mcm ⁵ U, ncm ⁵ U, ncm ⁵ Um, D, mcm ⁵ s ² U, Inosine (I), hm ⁵ C, s ⁴ U, s ² U, azobenzene, Cm, Um, Gm, t ⁶ A, yW, ms ² t ⁶ A or its derivatives.

In some embodiments, a nucleotide derivative refers to a compound that can replace a nucleotide in a nucleic acid but has a structure different from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide or thymine deoxyribonucleotide. In some embodiments, a nucleotide derivative can be an isonucleotide, a bridged nucleotide (BNA for short) or an acyclic nucleotide. BNA refers to a constrained or inaccessible nucleotide. BNA can contain a five-membered ring, a six-membered ring, or a seven-membered ring with a "fixed" C3'-endosugar condensed bridge structure. The bridge is usually incorporated into the 2'-, 4'-position of the ribose to provide a 2',4'-BNA nucleotide, such as LNA, ENA, cET, etc.

LNA is shown in formula (1), ENA is shown in formula (2), and cET is shown in formula (3):

Acyclic nucleotides are a type of nucleotides formed by opening the sugar ring of a nucleotide, such as unlocked nucleic acid (UNA) or glycerol nucleic acid (GNA), wherein UNA is shown in formula (4) and GNA is shown in formula (5):

In the above formula (4) and formula (5), R is selected from H, OH or alkoxy (O-alkyl).

In some embodiments, the nucleotide derivative modification refers to that the nucleotide in the nucleic acid is replaced by a nucleotide derivative. Exemplarily, the nucleotide derivative is selected from isonucleotides, LNA, ENA, cET, UNA or GNA.

In some embodiments, the nucleotides in the nucleic acid are replaced with isonucleotides, which are also referred to as isonucleoside modifications in the context of the present disclosure. In some embodiments, isonucleoside modifications include incorporating isonucleosides at one or more sites of the sense strand and/or antisense strand of the siRNA to be modified to replace natural nucleosides for coupling at the corresponding positions.

In some embodiments, the isonucleoside modification adopts D-isonucleoside modification. In other embodiments, the isonucleoside modification adopts L-isonucleoside modification. In still other embodiments, the isonucleoside modification adopts D-isonucleoside modification and L-isonucleoside modification.

In some embodiments, the double-stranded RNA modification comprises a modification of the phosphodiester bond at at least one position. In some embodiments, the modification of the phosphodiester bond refers to the replacement of at least one oxygen atom in the phosphodiester bond by a sulfur atom to form a thiophosphate diester bond. The thiophosphate diester bond can stabilize the double-stranded structure of the siRNA and maintain the specificity of base pairing. Exemplary, the thiophosphate diester bond structure is shown below:

In some embodiments, the double-stranded RNA modification comprises at least one of the following chemical modifications:

(1) modification of at least one nucleotide in the sense strand,

(3) modification of at least one nucleotide in the antisense strand,

(4) Modification of the phosphodiester bond at at least one position in the antisense strand.

Furthermore, the double-stranded RNA modification product is a siRNA modification product comprising at least one chemical modification among (1) to (4).

In the present disclosure, after sequence A in the sense strand and sequence B in the antisense strand complement each other to form a double-stranded region, the 3' ends of sequence A and sequence B can be any of the following:

(1) The 3' ends of sequence A and sequence B are both blunt-ended;

(2) The 3' end of sequence A has 1-2 protruding nucleotides extending out of the double-stranded region, and the 3' end of sequence B forms a blunt end;

(3) The 3' end of sequence B has 1-2 protruding nucleotides extending out of the double-stranded region, and the 3' end of sequence A forms a blunt end;

(4) The 3’ end of sequence A has 1-2 protruding nucleotides extending beyond the double-stranded region, and the 3’ end of sequence B has 1-2 protruding nucleotides extending beyond the double-stranded region.

In some embodiments, the nucleotide sequence of the sense strand is the sequence shown in sequence A, and the nucleotide sequence of the antisense strand is the sequence shown in sequence B.

In some embodiments, when the nucleotide sequences of the sense strand and the antisense strand complement each other to form a double-stranded region, if there are no protruding nucleotides at the 3' end of the sense strand and the antisense strand, 1-2 nucleotides are added to the 3' end of at least one of the sense strand and the antisense strand as protruding nucleotides. Column D, 1-2 nucleotides connected to the 3' end of the antisense strand constitute sequence E. Accordingly, the nucleotide sequence of the sense strand is the sequence shown in sequence A connected to sequence D, and the nucleotide sequence of the antisense strand is the sequence shown in sequence B connected to sequence E. Alternatively, the nucleotide sequence of the sense strand is the sequence shown in sequence A, and the nucleotide sequence of the antisense strand is the sequence shown in sequence B connected to sequence E. Alternatively, the nucleotide sequence of the sense strand is the sequence shown in sequence A connected to sequence D, and the nucleotide sequence of the antisense strand is the sequence shown in sequence B.

Exemplarily, two deoxyribonucleotides (TT) are added to the 3' end of the sense strand as sequence D, and two deoxyribonucleotides (TT) are added to the 3' end of the antisense strand as sequence E. Alternatively, two deoxyribonucleotides (TT) are added only to the 3' end of the antisense strand as sequence E. Alternatively, two deoxyribonucleotides (TT) are added only to the 3' end of the sense strand as sequence D.

In some embodiments, when the nucleotide sequences of the sense strand and the antisense strand complement each other to form a double-stranded region, if there is no protruding nucleotide at the 3' end of the sense strand, a sequence D consisting of 1-2 nucleotides is added to the 3' end of the sense strand as the protruding nucleotide. Then, after the chemical modification is completed, the nucleotide sequence formed by connecting sequence A and sequence D is excluded. Accordingly, in the double-stranded RNA modified substance, the nucleotide sequence of the sense strand is the sequence shown in sequence A, and the nucleotide sequence of the antisense strand is the sequence shown in sequence B. Alternatively, in the double-stranded RNA modified substance, the nucleotide sequence of the sense strand is the sequence shown in sequence A, and the nucleotide sequence of the antisense strand is the sequence shown in sequence B connected to sequence E. In some embodiments, when sequence A has 1-2 protruding nucleotides extending out of the double-stranded region at the 3' end of sequence A after complementing sequence B to form a double-stranded region, the protruding nucleotides at the 3' end of sequence A are excluded as the nucleotide sequence of the sense strand. The sequence excluding the protruding nucleotides at the 3' end is called sequence A'. Accordingly, the nucleotide sequence of the sense strand of the double-stranded RNA modification is the sequence shown in sequence A', and the nucleotide sequence of the antisense strand of the double-stranded RNA modification is the sequence shown in sequence B. Alternatively, the nucleotide sequence of the sense strand of the double-stranded RNA modification is the sequence shown in sequence A', and the nucleotide sequence of the antisense strand of the double-stranded RNA modification is the sequence shown in sequence B connected with sequence E.

In some embodiments, along the 5' end to the 3' end direction, the sense strand of the siRNA modifier includes the following modifications: the ribonucleotides at positions 7, 9, 10 and 11 in the sense strand are 2'-fluoro-modified ribonucleotides; the ribonucleotides at other positions in the sense strand are 2'-methoxy-modified ribonucleotides.

In this context, the 5' terminal nucleotide of the sense strand is not connected to a 5' phosphate group or a 5' phosphate derivative group structure as shown in Formula X:

In some embodiments, the sense strand of the siRNA modification comprises phosphorothioate diester bonds at the following positions along the 5' end to the 3' end: between the first nucleotide and the second nucleotide starting from the 5' end, between the second nucleotide and the third nucleotide starting from the 5' end, between the first nucleotide and the second nucleotide starting from the 3' end, and between the second and third nucleotides starting from the 3' end.

In some embodiments, the sense strand of the siRNA modification includes phosphorothioate diester bonds at the following positions along the 5' end to the 3' end: between the first nucleotide and the second nucleotide starting from the 5' end, and between the second nucleotide and the third nucleotide starting from the 5' end.

In some specific embodiments, the sense strand of the siRNA modification has a structure as shown in any one of (a ₁ )-(a ₃ ):

(a ₁ )5'-mN ₁ -(s)-mN ₂ -(s)-mN ₃ -mN ₄ -mN ₅ -mN ₆ -N ₇ f-mN ₈ -N ₉ fN ₁₀ fN ₁₁ f-mN ₁₂ -mN ₁₃ -mN ₁₄ -mN ₁₅ -mN ₁₆ -mN ₁₇ -mN ₁₈ -mN ₁₉ -(s)-T-(s)-T-3',

(a ₃ )5'-mN ₁ -(s)-mN ₂ -(s)-mN ₃ -mN ₄ -mN ₅ -mN ₆ -N ₇ f-mN ₈ -N ₉ fN ₁₀ fN ₁₁ f-mN ₁₂ -mN ₁₃ -mN ₁₄ -mN ₁₅ -mN ₁₆ -mN ₁₇ -mN ₁₈ -mN ₁₉ -mN ₂₀ -mN ₂₁ -(s)-mN ₂₂ -(s)-mN ₂₃ -3';

Wherein, _N1 - _N23 are independently selected from ribonucleotides whose base is A, U, C or G, the capital letter T represents a deoxyribonucleotide whose base is thymine, the lowercase letter m represents that a ribonucleotide adjacent to the right of the letter m is a 2'-O- _CH3- modified ribonucleotide, the lowercase letter f represents that a ribonucleotide adjacent to the left of the letter f is a 2'-F-modified ribonucleotide, and -(s)- represents that two adjacent nucleotides are connected by a phosphorothioate diester bond.

In some other specific embodiments, the sense strand of the siRNA modification has a structure as shown in any one of ( _a4 )-( _a5 ):

(a ₅ )5'-mN ₁ -(s)-mN ₂ -(s)-mN ₃ -mN ₄ -mN ₅ -mN ₆ -N ₇ f-mN ₈ -N ₉ fN ₁₀ fN ₁₁ f-mN ₁₂ -mN ₁₃ -mN ₁₄ -mN ₁₅ -mN ₁₆ -mN ₁₇ -mN ₁₈ -mN ₁₉ -mN ₂₀ -mN ₂₁ -3';

Wherein, _N1 - _N21 are independently selected from ribonucleotides whose base is A, U, C or G, the lowercase letter m indicates that the adjacent ribonucleotide to the right of the letter m is a 2'-O- _CH3- modified ribonucleotide, the lowercase letter f indicates that the adjacent ribonucleotide to the left of the letter f is a 2'-F-modified ribonucleotide, and -(s)- indicates that the two adjacent nucleotides are connected by a phosphorothioate diester bond.

In some embodiments, along the 5' end to the 3' end direction, the antisense strand of the siRNA modifier includes the following modifications: the ribonucleotide at any odd position in the antisense strand is a 2'-methoxy-modified ribonucleotide, and the ribonucleotide at any even position in the antisense strand is a 2'-fluoro-modified ribonucleotide.

In some embodiments, along the 5' end to the 3' end direction, the antisense strand of the siRNA modification comprises the following modifications: the ribonucleotides at positions 2, 6, 14 and 16 in the antisense strand are 2'-F modified ribonucleotides, and the ribonucleotides at the remaining positions in the antisense strand are 2'-O-CH ₃ modified ribonucleotides.

In some embodiments, along the 5' end to the 3' end direction, the antisense strand of the siRNA modification comprises the following modifications: the ribonucleotides at positions 2, 6, 8, 9, 14 and 16 in the antisense strand are 2'-F modified ribonucleotides, and the ribonucleotides at the remaining positions in the antisense strand are 2'-O-CH ₃ modified ribonucleotides.

In some embodiments, along the direction from the 5' end to the 3' end, the ribonucleotides at positions 2, 14 and 16 in the antisense chain are 2'-F modified ribonucleotides, the ribonucleotide at position 6 in the antisense chain is a ribonucleotide modified with the nucleotide derivative GNA, and the ribonucleotides at the remaining positions in the antisense chain are 2'-O- _CH3 modified ribonucleotides.

In some embodiments, along the direction from the 5' end to the 3' end, the antisense strand of the siRNA modifier includes the following modifications: the ribonucleotides at positions 2, 6, 14 and 16 in the antisense strand are 2'-F modified ribonucleotides, the ribonucleotide at position 7 in the antisense strand is a ribonucleotide modified with the nucleotide derivative GNA, and the ribonucleotides at the remaining positions in the antisense strand are 2'-O- _CH3 modified ribonucleotides.

In some embodiments, the antisense strand of the siRNA modification comprises phosphorothioate diester bonds at the following positions along the 5' end to the 3' end: between the first nucleotide and the second nucleotide starting from the 5' end, between the second nucleotide and the third nucleotide starting from the 5' end, between the first nucleotide and the second nucleotide starting from the 3' end, and between the second and third nucleotides starting from the 3' end.

In some embodiments, along the 5' end to the 3' end direction, the nucleotide at the 5' end of the antisense strand is not linked to a 5' phosphate group or a 5' phosphate-derivative group, or is linked to a 5' phosphate group or a 5' phosphate-derivative group.

In some specific embodiments, the antisense strand of the siRNA modification has a structure as shown in any one of (b ₁ ) to (b ₂₇ ):

(b ₁₃ )5'-P1mN ₁ -(s)-N ₂ f-(s)-mN ₃ -mN ₄ -mN ₅ -N ₆ f-mN ₇ -N ₈ fN ₉ f-mN ₁₀ -mN ₁₁ - mN ₁₂ - _{mN 13} -N ₁₄ f-mN ₁₅ -N ₁₆ f-mN ₁₇ -mN ₁₈ -mN ₁₉ -mN ₂₀ -mN ₂₁ -(s)-mN ₂₂ -(s)-mN ₂₃ -3',

(b ₁₉ )5'-EVPmN ₁ -(s)-N ₂ f-(s)-mN ₃ -mN ₄ -mN ₅ -N ₆ f-mN ₇ -N ₈ fN ₉ f-mN ₁₀ -mN ₁₁ - mN ₁₂ -mN ₁₃ -N ₁₄ f-mN ₁₅ -N ₁₆ f-mN ₁₇ -mN ₁₈ -mN ₁₉ -(s)-T-(s)-T-3'，

wherein _N1 - _N23 are independently selected from ribonucleotides whose base is A, U, C or G; the capital letter T represents a deoxyribonucleotide whose base is thymine; the lowercase letter m represents that a ribonucleotide adjacent to the right of the letter m is a 2'-O- _CH3- modified ribonucleotide; the lowercase letter f represents that a ribonucleotide adjacent to the left of the letter f is a 2'-F-modified ribonucleotide; P1 represents that a nucleotide adjacent to the right of the letter is a 5'-phosphate nucleotide; EVP represents that a nucleotide adjacent to the right of the letter combination is a 5'-trans-vinylphosphonate nucleotide; -(s)- represents that two adjacent nucleotides are connected by a phosphorothioate diester bond; and [GNA] represents that a ribonucleotide adjacent to the right is a GNA-modified ribonucleotide.

In some optional embodiments, the sense strand comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 222 to 283 and 595 to 650, and the antisense strand comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 284 to 393, 651 to 748, 788 to 798, 498 and 590.

In some embodiments, the double-stranded RNA modifications include, but are not limited to, the siRNA modifications shown in Table 2.

In some specific embodiments, the inhibition rate of the siRNA modification disclosed herein on the MARC1 gene is at least about 20%, and may be at least about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or a value or range between any two of these values. In some embodiments, the IC ₅₀ of the siRNA modification disclosed herein on the MARC1 gene is less than about 0.4 nM, and may be less than About 0.3, about 0.2, about 0.1, about 0.09, about 0.08, about 0.07, about 0.06, about 0.05, about 0.04, about 0.03, about 0.02, about 0.01 nM.

Double-stranded RNA conjugate

The third aspect of the present disclosure provides a double-stranded RNA conjugate, which is obtained by conjugating the double-stranded RNA provided by the first aspect of the present disclosure or the double-stranded RNA modification provided by the second aspect with a conjugation group.

In the present disclosure, the sense strand and the antisense strand of the double-stranded RNA conjugate form a double-stranded region of the double-stranded RNA conjugate, and a blunt end is formed at the 3' end of the sense strand of the double-stranded RNA conjugate. In some embodiments, the 3' end of the sense strand of the double-stranded RNA conjugate forms a blunt end, and the 3' end of the antisense strand of the double-stranded RNA conjugate has 1-2 protruding nucleotides extending out of the double-stranded region. In other embodiments, the 3' end of the sense strand of the double-stranded RNA conjugate forms a blunt end, and the 3' end of the antisense strand of the double-stranded RNA conjugate forms a blunt end.

In some preferred embodiments, the double-stranded RNA conjugate is obtained by conjugating a double-stranded RNA modification with a conjugation group, wherein the sense strand and the antisense strand of the double-stranded RNA modification are complementary to form a double-stranded region of the double-stranded RNA modification, and the 3' end of the sense strand of the double-stranded RNA modification forms a blunt end, and the conjugation group is conjugated with the 3' end of the sense strand with the blunt end to form a double-stranded RNA conjugate.

Exemplarily, the sense strand of the double-stranded RNA modification is the sequence shown in sequence A, and the antisense strand is the sequence shown in sequence B connected to sequence E. In addition, the 3' end of the sense strand of the double-stranded RNA modification forms a blunt end, and the 3' end of the sense strand of the double-stranded RNA modification is connected to a conjugated group to form a double-stranded RNA conjugate.

Exemplarily, the sense strand of the double-stranded RNA modification is the sequence shown in sequence A, and the antisense strand is the sequence shown in sequence B. In addition, the 3' end of the sense strand of the double-stranded RNA modification forms a blunt end, and the 3' end of the sense strand of the double-stranded RNA modification is connected to a conjugated group to form a double-stranded RNA conjugate.

Exemplarily, the sense strand of the double-stranded RNA modification is a sequence A connected to a sequence D, and the antisense strand is a sequence B connected to a sequence E. In addition, the 3' end of the sense strand of the double-stranded RNA modification has a sequence D consisting of 1-2 nucleotides protruding, and after excluding the sequence D at the 3' end of the sense strand in the double-stranded RNA modification, a conjugation group is connected to the 3' end of sequence A to form a double-stranded RNA conjugate.

Exemplarily, the sense strand of the double-stranded RNA modification is the sequence shown by sequence A connected to sequence D, and the antisense strand is the sequence shown by sequence B. In addition, the 3' end of the sense strand of the double-stranded RNA modification has a sequence D consisting of protruding 1-2 nucleotides, and after excluding the sequence D at the 3' end of the sense strand in the double-stranded RNA modification, a conjugation group is connected to the 3' end of sequence A to form a double-stranded RNA conjugate.

Exemplarily, the sense strand of the double-stranded RNA modification is the sequence shown in sequence A, and the antisense strand is the sequence shown in sequence B connected to sequence E. Wherein, the 3' end of sequence A has a protruding nucleotide extending out of the double-stranded region, and the sequence after excluding the protruding nucleotide at the 3' end of sequence A (also known as sequence A') is used as the nucleotide sequence for connecting the conjugated group. Therefore, the nucleotide sequence of the sense strand of the double-stranded RNA conjugate is the sequence shown in sequence A', and the nucleotide sequence of the antisense strand is the sequence shown in sequence B connected to sequence E.

Exemplarily, the sense strand of the double-stranded RNA modification is the sequence shown in sequence A, and the antisense strand is the sequence shown in sequence B. Wherein, the 3' end of sequence A has a protruding nucleotide extending out of the double-stranded region, and the sequence after excluding the protruding nucleotide at the 3' end of sequence A (also known as sequence A') is used as the nucleotide sequence for connecting the conjugated group. Therefore, the nucleotide sequence of the sense strand of the double-stranded RNA conjugate is the sequence shown in sequence A', and the nucleotide sequence of the antisense strand is the sequence shown in sequence B.

For example, in the siRNA conjugate shown as N-ER-FY025102M8L96, the 3' end of the sense strand originally has a protruding nucleotide -smUsmA extending out of the double-stranded region. The mGsmUsmGmAmUmUCfmAGfUfGfmAmUmUmUmCmAmGmA blunt-end sequence formed after the protruding -smUsmA nucleotide at the 3' end of the sense strand is excluded is used as the nucleotide sequence for connecting the L96 conjugation group (i.e., L96 is connected through a phosphodiester bond after the sequence is synthesized to the blunt end). Therefore, the sequence of the siRNA conjugate is: the sense strand is mGsmUsmGmAmUmUCfmAGfUfGfmAmUmUmUmCmAmGmAL96 (SEQ ID NO: 756), and the antisense strand is EVPmUsCfsmUmGmAAfmAmUmCmAmCmUmGAfmAUfmCmAmCsmUsmU (SEQ ID NO: 671).

In some alternative embodiments, the sense strand of the double-stranded RNA conjugate has a structure as shown in any one of (d ₁ )-(d ₂ ):

(d ₁ )5'-mN ₁ -(s)-mN ₂ -(s)-mN ₃ -mN ₄ -mN ₅ -mN ₆ -N ₇ f-mN ₈ -N ₉ fN ₁₀ fN ₁₁ f-mN ₁₂ -mN ₁₃ -mN ₁₄ -mN ₁₅ -mN ₁₆ -mN ₁₇ -mN ₁₈ -mN ₁₉ -L96-3',

(d ₂ )5'-mN ₁ -(s)-mN ₂ -(s)-mN ₃ -mN ₄ -mN ₅ -mN ₆ -N ₇ f-mN ₈ -N ₉ fN ₁₀ fN ₁₁ f-mN ₁₂ -mN ₁₃ -mN ₁₄ -mN ₁₅ -mN ₁₆ -mN ₁₇ -mN ₁₈ -mN ₁₉ -mN ₂₀ -mN ₂₁ -L96-3';

Wherein, _N1 - _N21 are independently selected from ribonucleotides whose bases are A, U, C or G, the lowercase letter m indicates that a ribonucleotide adjacent to the right of the letter m is a 2'-O- _CH3 modified ribonucleotide, the lowercase letter f indicates that a ribonucleotide adjacent to the left of the letter f is a 2'-F modified ribonucleotide, and -(s)- indicates that two adjacent nucleotides are connected by a phosphorothioate diester bond. L96 is the conjugate group GalNAc shown in formula I.

In some alternative embodiments, the antisense strand of the double-stranded RNA conjugate has a structure as shown in any one of (b ₁ )-(b ₂₇ ):

(b ₉ )5'-P1mN ₁ -(s)-N ₂ f-(s)-mN ₃ -mN ₄ -mN ₅ -N ₆ f-[GNA]N ₇ -mN ₈ -mN ₉ -mN ₁₀ - mN ₁₁ -mN ₁₂ -mN ₁₃ -N ₁₄ f-mN ₁₅ -N ₁₆ f-mN ₁₇ -mN ₁₈ -mN ₁₉ -(s)-mN ₂₀ -(s)-mN ₂₁ -3',

(b ₁₀ )5'-P1mN ₁ -(s)-N ₂ f-(s)-mN ₃ -mN ₄ -mN ₅ -[GNA]N ₆ -mN ₇ -mN ₈ -mN ₉ -mN ₁₀ -mN ₁₁ -mN ₁₂ -mN ₁₃ -N ₁₄ f-mN ₁₅ -N ₁₆ f-mN ₁₇ -mN ₁₈ -mN ₁₉ -(s)-mN ₂₀ -(s)-mN ₂₁ -3',

(b ₁₄ )5'-P1mN ₁ -(s)-N ₂ f-(s)-mN ₃ -mN ₄ -mN ₅ -[GNA]N ₆ -mN ₇ -mN ₈ -mN ₉ -mN ₁₀ -mN ₁₁ -mN ₁₂ -mN ₁₃ -N ₁₄ f-mN ₁₅ -N ₁₆ f-mN ₁₇ -mN ₁₈ -mN ₁₉ -mN ₂₀ -mN ₂₁ -(s)-mN ₂₂ -(s)-mN ₂₃ -3'

wherein _N1 - _N23 are independently selected from ribonucleotides whose bases are A, U, C or G; capital letter T indicates a deoxyribonucleotide whose base is thymine; lowercase letter m indicates that a ribonucleotide adjacent to the right of the letter m is a 2'-O- _CH3- modified ribonucleotide; lowercase letter f indicates that a ribonucleotide adjacent to the left of the letter f is a 2'-F-modified ribonucleotide; P1 indicates that a nucleotide adjacent to the right of the letter is a 5'-phosphate nucleotide; EVP indicates that a nucleotide adjacent to the right of the letter combination is a 5'-trans-vinylphosphonate nucleotide; -(s)- indicates that two adjacent nucleotides are connected by a phosphorothioate diester bond; and [GNA] indicates that a ribonucleotide adjacent to the right of the letter is a GNA-modified ribonucleotide.

Further, the double-stranded RNA conjugate is a siRNA conjugate, wherein the siRNA molecule connected to the conjugation group in the siRNA conjugate can be an unmodified siRNA, or a siRNA modification. The siRNA molecule modified with the conjugation group has good tissue and organ targeting and the ability to promote cell endocytosis while maintaining high inhibitory activity and stability, which can reduce the impact on other tissues or organs and reduce the amount of siRNA molecules used, thereby achieving the purpose of reducing toxicity and reducing costs. Optionally, any one of the siRNA molecules shown in Table 1 or Table 1-1 or Table 2 is selected to be connected to the conjugation group to obtain a double-stranded RNA conjugate.

The conjugation site of siRNA and conjugated group can be at the 3' end or 5' end of the siRNA sense strand, or at the 5' end of the antisense strand, or in the internal sequence of siRNA. In some embodiments, the conjugation site of siRNA and conjugated group is at the 3' end of the siRNA sense strand.

In some embodiments, the conjugate group can be connected to the phosphate group, 2'-hydroxyl group or base of the nucleotide. In some embodiments, the conjugate group can also be connected to the 3'-hydroxyl group, in which case the nucleotides are connected by a 2',5'-phosphodiester bond. When the conjugate group is connected to the end of the siRNA chain, the conjugate group is usually connected to the phosphate group of the nucleotide; when the conjugate group is connected to the internal sequence of the siRNA, the conjugate group is usually connected to the ribose sugar ring or the base. Various connection methods can be referred to in the literature: Muthiah Manoharan et.al. siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatocytes. ACS Chemical biology, 2015, 10(5):1181-7.

In the present disclosure, the conjugated group can be a ligand conventionally used in the field of siRNA administration. In some embodiments, the conjugated group can be selected from one or more of the ligands formed by the following targeting molecules or their derivatives: lipophilic molecules, such as cholesterol, bile acid, vitamins (such as vitamin E), lipid molecules of different chain lengths; polymers, such as polyethylene glycol; polypeptides, such as membrane-permeable peptides; aptamers; antibodies; quantum dots; carbohydrates, such as lactose, polylactose, mannose, galactose, N-acetylgalactosamine (GalNAc); folic acid (folate); receptor ligands expressed by hepatocytes, such as asialoglycoproteins, asialosugar residues, lipoproteins (such as high-density lipoproteins, low-density lipoproteins, etc.), glucagon, neurotransmitters (such as adrenaline), growth factors, transferrin, etc.

In some specific embodiments, the conjugated group has any of the following structures:

The conjugated group shown in Formula I is GalNAc. GalNAc has liver targeting property and can deliver siRNA molecules to liver tissue with high specificity, thereby specifically inhibiting the high expression of MARC1 gene in the liver.

In some specific embodiments, GalNAc is conjugated to the 3' end of the sense strand via a phosphodiester bond to obtain a siRNA conjugate with the following structure:

The double helix structure is unmodified siRNA or siRNA modification.

In some embodiments, the double-stranded ribonucleic acid conjugates include, but are not limited to, siRNA conjugates as shown in Table 3.

In some specific embodiments, the inhibition rate of the siRNA conjugates disclosed herein on the MARC1 gene is at least about 20%, and may be at least about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or a value or range between any two of these values.

Prodrug

The fourth aspect of the present disclosure provides a prodrug. The double-stranded RNA described in the first aspect of the present disclosure, the double-stranded RNA modified product described in the second aspect, and the double-stranded RNA conjugate described in the third aspect can also exist in the form of a prodrug, and the prodrug can be converted into the double-stranded RNA, double-stranded RNA modified product, and double-stranded RNA conjugate of the present disclosure in vivo or in vitro.

As used in this specification, "prodrug" refers to a compound that exerts its pharmacological effect after transformation in the organism. For example, in this application, M6 is a prodrug of M2. The difference between the modification of M2 and M6 is whether there is P1 at the 5' end of the antisense strand. M6 without P1 will be phosphorylated in the body to become the M2 sequence with P1, thereby exerting its effect. Similarly, the relationship between M7 and M3 is the same. Therefore, siRNA in this article includes its corresponding prodrug.

Pharmaceutical composition

The fifth aspect of the present disclosure provides a pharmaceutical composition, comprising one or more of the double-stranded RNA described in the first aspect, the double-stranded RNA modification described in the second aspect, the double-stranded RNA conjugate described in the third aspect, and the prodrug described in the fourth aspect.

In some embodiments, the pharmaceutical composition contains siRNA or prodrug as described above as an active ingredient and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition also contains one or more additional therapeutic agents, such as therapeutic agents that are beneficial to the prevention or treatment of diseases, disorders or symptoms mediated at least in part by MARC1 gene expression. In the present disclosure, the purpose of using the pharmaceutical composition is to promote administration to an organism, which is conducive to the absorption of the active ingredient and thus exerts biological activity. The pharmaceutical composition of the present disclosure can be administered in any form, including injection (intra-arterial, intravenous, intramuscular, intraperitoneal, subcutaneous), mucosal, oral (oral solid preparations, oral liquid preparations), rectal, inhalation, implantation, topical (e.g., ophthalmic) administration, etc. Non-limiting examples of oral solid preparations include, but are not limited to, powders, capsules, lozenges, granules, tablets, etc. Non-limiting examples of liquid preparations for oral or mucosal administration include, but are not limited to, suspensions, tinctures, elixirs, solutions, etc. Non-limiting examples of topical preparations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops or serum preparations. Non-limiting examples of parenteral preparations include, but are not limited to, solutions for injection, dry powders for injection, suspensions for injection, emulsions for injection, etc. The pharmaceutical compositions of the present disclosure can also be prepared into controlled-release or delayed-release dosage forms (eg, liposomes or microspheres).

Medical Uses

The sixth aspect of the present disclosure provides at least one of the following uses of double-stranded RNA, double-stranded RNA modifications, double-stranded RNA conjugates, prodrugs, and pharmaceutical compositions:

(1) Inhibiting the expression of MARC1 gene, or preparing a drug for inhibiting the expression of MARC1 gene;

The present disclosure further provides the use of siRNA molecules (including unmodified siRNA, siRNA modifications, siRNA conjugates) or prodrugs or pharmaceutical compositions thereof in at least one of the above (1)-(3).

In the present disclosure, abnormal expression of MARC1 gene causes one or more of the following diseases related to abnormal expression of MARC1 gene: obesity, non-alcoholic fatty liver disease, alcohol-related fatty liver disease, non-alcoholic fatty hepatitis, cirrhosis, liver fibrosis, elevated liver enzyme levels (ALT, AST, ALP), hepatocellular carcinoma, hypercholesterolemia and related cardiovascular diseases, insulin resistance, impaired glucose tolerance, hyperglycemia, type II diabetes and metabolic syndrome, etc.

The siRNA molecule causes the expression of the MARC1 gene to be inhibited by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%, thereby achieving the treatment of diseases related to abnormal expression of the MARC1 gene.

In some embodiments, the present disclosure provides a method for inhibiting MARC1 gene expression in a cell, comprising contacting a double-stranded RNA, a double-stranded RNA modification, a double-stranded RNA conjugate, a prodrug or a pharmaceutical composition with the cell.

Furthermore, the method for inhibiting the expression of MARC1 gene in cells is to introduce siRNA molecules (including unmodified siRNA, siRNA modifications, siRNA conjugates), prodrugs or pharmaceutical compositions into cells.

In some embodiments, the cell is an in vivo cell or an in vitro cell. In some specific embodiments, the cell is in a subject.

In some embodiments, the present disclosure provides a method for preventing or treating a disease, comprising administering a double-stranded RNA, a double-stranded RNA modification, a double-stranded RNA conjugate, a prodrug, or a pharmaceutical composition to a subject or patient.

Furthermore, the method for preventing or treating a disease is to administer siRNA molecules (including unmodified siRNA, siRNA modifications, siRNA conjugates), prodrugs or pharmaceutical compositions to a subject or patient.

In the present disclosure, "subject" includes either a human or a non-human animal, preferably a vertebrate, and more preferably a mammal. The subject may include a transgenic organism. Most preferably, the subject is a human. Further, the subject has at least one of the following characteristics:

(1) Abnormal expression of the MARC1 gene in vivo, more specifically, abnormally high expression of the MARC1 gene;

(2) suffering from diseases related to abnormal expression of MARC1 gene;

(3) People who suffer from diseases that would benefit from reduced MARC1 gene expression, such as people who suffer from or are prone to diseases associated with abnormal MARC1 gene expression.

The dosage of the siRNA molecules (including unmodified siRNA, siRNA modifications, siRNA conjugates), prodrugs or pharmaceutical compositions disclosed herein can be determined according to the patient's weight, age, gender, severity of the disease, etc. Based on the amount of double-stranded ribonucleic acid contained therein, the dosage of the siRNA molecules (including unmodified siRNA, siRNA modifications, siRNA conjugates), prodrugs or pharmaceutical compositions disclosed herein is about 1-300 mg/kg body weight.

The frequency of administration can be daily, weekly, every two weeks, every three weeks, every 1 month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months or yearly, once or more.

The total number of times the siRNA molecules (including unmodified siRNA, siRNA modifications, siRNA conjugates), prodrugs or pharmaceutical compositions of the present disclosure are administered can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50. For example, the siRNA molecules (including unmodified siRNA, siRNA modifications, siRNA conjugates), prodrugs or pharmaceutical compositions of the present disclosure can be administered about 1, 2, 3, 4 times.

In some embodiments, the siRNA molecules (including unmodified siRNA, siRNA modifications, siRNA conjugates), prodrugs, pharmaceutical compositions, and optionally other therapeutic agents disclosed herein may be packaged in a kit, wherein the siRNA molecules (including unmodified siRNA, siRNA modifications, siRNA conjugates), prodrugs, pharmaceutically acceptable carriers, and optionally other therapeutic agents may be provided in liquid form or dry form. In some embodiments, the kit Instructions are included to describe how to mix the siRNA molecule or prodrug with a pharmaceutically acceptable carrier or other ingredients.

Table 1 siRNA sequence information

Table 1-1 siRNA sequence information

Table 2 siRNA modifications

In the above table, each of the capital letters "G", "C", "A", "T" and "U" generally represents a nucleotide containing guanine, cytosine, adenine, thymine and uracil as a base, respectively; mA, mU, mC, mG: represent 2-methoxy modified nucleotides; Af, Gf, Cf, Uf: represent 2-fluoro modified nucleotides; the lowercase letter s indicates that the two nucleotides adjacent to the letter s are connected by a thiophosphate diester bond; P1: indicates that the nucleotide adjacent to the right of P1 is a 5'-phosphate nucleotide; EVP indicates that the nucleotide adjacent to the right is a 5'-trans vinylphosphonate nucleotide; [GNA] indicates that the ribonucleotide adjacent to the right is a ribonucleotide modified with GNA.

Table 3 siRNA conjugates

In the above table, L96 is the conjugate group GalNAc shown in Formula I.

In Table 1, Table 1-1, Table 2 and Table 3, if the left side of the 5' terminal nucleotide of the sense strand, the modified sense strand and the modified sense strand connected to the conjugated group is not marked with P1 or EVP, it means that the 5' terminal nucleotide is not connected to a 5' phosphate group or a 5' phosphate derivative group, and its structure is shown in Formula X:

In Table 1, Table 1-1, Table 2 and Table 3, if the left side of the 5' terminal nucleotide of the antisense strand and the modified antisense strand is not marked with P1 or EVP, it means that the 5' terminal nucleotide is not connected to a 5' phosphate group or a 5' phosphate derivative group, and its structure is also shown in Formula X.

In Table 1, Table 1-1, Table 2 and Table 3, the 3' position of the 3' terminal nucleotide of the sense strand and the modified sense strand, and the 3' terminal nucleotide of the antisense strand and the modified antisense strand is a hydroxyl group.

Example

Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples (although representing specific embodiments of the present disclosure) are given for illustrative purposes only, because after reading the detailed description, various changes and modifications made within the spirit and scope of the present disclosure will become apparent to those skilled in the art.

The experimental techniques and experimental methods used in this example are all conventional technical methods unless otherwise specified. For example, the experimental methods in the following examples that do not specify specific conditions are usually carried out according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. The materials, reagents, etc. used in the examples can be obtained through regular commercial channels unless otherwise specified.

The siRNA, siRNA modifications, and siRNA conjugates involved in the following examples were synthesized by Tianlin Biotechnology (Shanghai) Co., Ltd. The cells and reagents used in the examples are shown in Table 4:

Table 4

Example 1: Synthesis of siRNA

1.1 siRNA sequence design

Based on the human MARC1 gene mRNA sequence, multiple pairs of MARC1 siRNAs were designed at different sites. All the designed single siRNAs can target all transcripts of the target gene (as shown in Table 5). These multiple pairs of siRNAs have the lowest homology with all other non-target gene sequences after sequence similarity software alignment.

Table 5

The target sequence used to design siRNA is shown below. The target sequence is derived from the mRNA sequence of the MARC1 gene (see NM_022746.4).

Target sequence I:

Target sequence I-1:

Target sequence I-2:

Target sequence I-3:

Target sequence I-4:

Target sequence I-5:

Target sequence I-6:

Target sequence II:

Target sequence II-1:

Target sequence II-2:

Target sequence II-3:

Target sequence II-4:

Target sequence II-5:

Target sequence II-6:

Target sequence III:

Target sequence III-1:

Target sequence III-2:

Target sequence III-3:

Target sequence III-4:

Target sequence III-5:

Target sequence IV:

Target sequence IV-1:

Target sequence IV-2:

Target sequence IV-3:

Target sequence IV-4:

Target sequence IV-5:

Target sequence IV-6:

Target sequence V:

Target sequence V-1:

Target sequence V-2:

Target sequence V-3:

Target sequence V-4:

Target sequence VI:

Target sequence VI-1:

Target sequence VI-2:

Target sequence VI-3:

Target sequence VII:

Target sequence VII-1:

Target sequence VII-2:

Target sequence VII-3:

Target sequence VIII:

Target sequence VIII-1:

Target sequence VIII-2:

Target sequence VIII-3:

Target sequence VIII-4:

Target sequence IX:

Target sequence IX-1:

Target sequence IX-2:

Target sequence IX-3:

Target sequence IX-4:

Target sequence X:

Target sequence X-1:

Target sequence X-2:

Target sequence XI:

Target sequence XI-1:

Target sequence XI-2:

Target sequence XII:

Target sequence XII-1:

Target sequence XII-2:

Target sequence XIII:

Target sequence XIII-1:

Target sequence XIII-2:

Target sequence XIV:

Target sequence XIV-1:

Target sequence XIV-2:

Target sequence XIV-3:

Target sequence XV:

Target sequence XV-1:

Target sequence XV-2:

1.2 Description of synthesis method:

By solid phase phosphoramidite method, nucleoside monomers are connected one by one from 3'-5' direction according to the order of nucleotide arrangement. Each connection of a nucleoside monomer includes four steps of deprotection, coupling, oxidation or sulfidation, and capping. Among them, when phosphate is used to connect two nucleotides, when the latter nucleoside monomer is connected, four steps of deprotection, coupling, oxidation, and capping are included. When thiophosphate is used to connect two nucleotides, when the latter nucleoside monomer is connected, four steps of deprotection, coupling, sulfidation, and capping are included. The present invention selects nucleotide monomers according to the target sequence of synthesis, and the selected nucleotide monomers are nucleotide monomers commonly used by those skilled in the art. For example, the nucleotide monomers for synthesizing A can be, but are not limited to, adenosine-3-phosphate. It should be understood that these monomers, when present in oligonucleotides, are interconnected by 5'-3' phosphodiester bonds or 5'-3' thiophosphate groups. When, for example, the 3' position of the last nucleotide in the 5' to 3' direction is a hydroxyl group, it is achieved according to conventional means of the art.

1.3 The synthesis conditions are given as follows:

The nucleoside monomer was provided in a 0.1 M acetonitrile solution. The conditions for the deprotection reaction in each step were the same, i.e., the temperature was 25°C, the reaction time was 70 seconds, the deprotection reagent was a dichloroacetic acid solution in dichloromethane (3% V/V), and the molar ratio of dichloroacetic acid to the 4,4'-dimethoxytrityl protecting group on the solid support was 5:1.

The reaction conditions for each step of the coupling reaction were the same, including a temperature of 25°C, a molar ratio of the nucleic acid sequence connected to the solid phase support to the nucleoside monomer of 1:10, a molar ratio of the nucleic acid sequence connected to the solid phase support to the coupling reagent of 1:65, and a reaction time of The reaction time was 600 seconds and the coupling reagent was a 0.5 M acetonitrile solution of 5-ethylthio-1H-tetrazole.

The oxidation reaction conditions in each step were the same, including a temperature of 25°C, a reaction time of 15 seconds, and an oxidizing agent of 0.05 M iodine water. The molar ratio of iodine to the nucleic acid sequence connected to the solid phase support in the coupling step was 30:1. The reaction was carried out in a mixed solvent of tetrahydrofuran: water: pyridine = 3:1:1.

The conditions of each step of the sulfurization reaction are the same, including a temperature of 25°C, a reaction time of 300 seconds, and a sulfurization reagent of hydrogenated xanthan. The molar ratio of the sulfurization reagent to the nucleic acid sequence connected to the solid phase support in the coupling step is 120:1. The reaction is carried out in a mixed solvent of acetonitrile:pyridine=1:1.

The capping conditions are the same for each step, including a temperature of 25°C and a reaction time of 15 seconds. The capping reagent solution is a mixed solution of CapA and CapB in a molar ratio of 1:1, and the molar ratio of the capping reagent to the nucleic acid sequence connected to the solid phase carrier is acetic anhydride: N-methylimidazole: nucleic acid sequence connected to the solid phase carrier = 1:1:1. After the last nucleoside monomer is connected, the nucleic acid sequence connected to the solid phase carrier is cut, deprotected, purified, desalted, and then freeze-dried to obtain the sense chain and the antisense chain; finally, the two chains are heated and annealed to obtain the product, freeze-dried, and freeze-dried to obtain freeze-dried powder.

The synthesized siRNAs are shown in Table 1 and Table 1-1, and the synthesized siRNA modifications are shown in Table 2.

Example 2: Synthesis of siRNA conjugate (GalNAc-siRNA)

2.1 The siRNA conjugate has the structure shown in the following formula II:

2.2 Synthesis of siRNA conjugates

In the first step, DMTr-L96 and succinic anhydride are reacted to obtain compound L96-A:

Preparation process: DMTr-L96, succinic anhydride, 4-dimethylaminopyridine and diisopropylethylamine were added to dichloromethane, stirred at 25°C for 24 hours, and then the reaction solution was washed with 0.5M triethylamine phosphate, the aqueous phase was washed three times with dichloromethane, and the organic phases were combined and evaporated to dryness under reduced pressure to obtain a crude product. Then, column chromatography was used for purification to obtain pure L96-A.

In the second step, L96-A is reacted with NH ₂ -SPS to obtain L96-B:

Preparation process: L96-A, O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU) and diisopropyl ethyl Amine (DIPEA) was mixed and dissolved in acetonitrile, stirred at room temperature for 5 minutes to obtain a uniform solution, and aminomethyl resin (NH2 _- SPS, 100-200 mesh) was added to the reaction liquid, and the shaking reaction was started at 25°C. After the reaction for 18 hours, the filter cake was filtered and washed with dichloromethane and acetonitrile in sequence to obtain a filter cake. The obtained filter cake was capped with a CapA/CapB mixed solution to obtain L96-B, which is a solid phase carrier containing a conjugate molecule, and then the nucleoside monomer was connected to the conjugate molecule under a coupling reaction, and then the siRNA sense chain connected to the conjugate molecule was synthesized according to the siRNA molecule synthesis method described above, and the siRNA antisense chain was synthesized using the siRNA molecule synthesis method described above, and annealing was performed to generate the siRNA conjugate of the present application.

The synthesized siRNA conjugates are shown in Table 3.

Example 3: siRNA and siRNA modifications inhibit MARC1 gene expression

3.1 Experimental Materials:

HepG2 cells were purchased from the Cell Bank of Type Culture Collection Committee of the Chinese Academy of Sciences, catalog number SCSP-510;

RNA extraction kit, 96 Kit, Catalog No. QIAGEN-74182;

RNAiMAX transfection reagent, purchased from Invitrogen, catalog number 13778-150;

MEM medium, purchased from Gibco, catalog number 41090036;

Reverse transcription kit ( Ⅲ1st Strand cDNA Synthesis Kit (+gDNA wiper), purchased from Vazyme, catalog number R312-02;

TaqMan ^TM Gene Expression Master Mix, purchased from Applied Biosystems, catalog number 4369016;

Opti-MEM: Reduced serum medium, purchased from Gibco, catalog number 31985070;

Target MARC1 primer and probe set, purchased from Thermo, Hs00224227_m1;

TaqMan Gene Expression Assay (GAPDH), purchased from Thermo, ID-Hs99999905_m1.

3.2 Experimental methods:

3.2.1 HepG2 cells were plated in fresh MEM medium in a 96-well plate and cultured for 48 hours. The cultured cells were resuspended in MEM medium without PS (penicillin-streptomycin mixture) to prepare a cell suspension with a density of 1.11×10 ⁵ /mL. The cell suspension was plated in a 96-well plate and 90 μL of cell suspension was added to each well, i.e., 10,000 cells/well.

3.2.2 Centrifuge the dry powder of the siRNA to be tested and siRNA modifications (for ease of description, collectively referred to as siRNA in the experimental process of this example) at low temperature and high speed, and then dissolve it with ultrapure distilled water (ULtraPure Distilled Water) to prepare a 100 μM siRNA stock solution.

3.2.3 Preparation of 200 nM siRNA transfection diluent Z and 2 nM siRNA diluent W

(1) Preparation of 10 μM siRNA stock solution Q and 0.1 μM siRNA stock solution E:

a. Take 2 μL of the 100 μM siRNA stock solution prepared in step 3.2.2 above, add 18 μL of ultrapure distilled water to obtain a siRNA stock solution Q with a final concentration of 10 μM;

b. Take 2 μL of the 10 μM siRNA stock solution Q prepared in step a), add 18 μL of ultrapure distilled water to obtain a siRNA stock solution Y with a final concentration of 1 μM;

c. Take 2 μL of the 1 μM siRNA stock solution Y prepared in step b), add 18 μL of ultrapure distilled water to obtain a siRNA stock solution E with a final concentration of 0.1 μM;

(2) Take 2 μL of the above-prepared siRNA stock solution E and add 98 μL of Opti-MEM to obtain 2 nM siRNA dilution solution W; take 2 μL of the above-prepared siRNA stock solution Q and add 98 μL of Opti-MEM to obtain 200 nM siRNA dilution solution Z;

3.2.4 Transfection of HepG2 cells

(1) Take 3 μL RNAiMAX transfection reagent was added to 97 μL Opti-MEM to obtain RNAiMAX transfection reagent diluent; RNAiMAX transfection reagent diluent was mixed with the 2 nM siRNA diluent W prepared in step 3.2.3 at a volume ratio of 1:1, allowed to stand for 5 minutes, and 10 μL of the transfection mixture was added to a 96-well plate to transfect the HepG2 cells cultured in step 3.2.1 (final volume 100 μL, siRNA concentration in this system was 0.1 nM).

(2) Take 3 μL RNAiMAX transfection reagent was added to 97 μL Opti-MEM to obtain RNAiMAX Transfection reagent diluent; RNAiMAX transfection reagent diluent and 200nM siRNA diluent Z prepared in step 3.2.3 were mixed in a volume ratio of 1:1 to prepare a transfection mixture, which was allowed to stand for 5 minutes. 10μL of the transfection mixture was added to a 96-well plate to transfect the HepG2 cells cultured in step 3.2.1 (final volume 100μL, the concentration of siRNA in this system was 10nM).

The cells were cultured for 48 hours after the above transfection; 2 replicates were set for each concentration (10 nM and 0.1 nM).

3.2.5 Extract total RNA from HepG2 cells obtained in step 3.2.4 according to the instructions of the RNA extraction kit.

3.2.6 The total RNA obtained in step 3.2.5 was reverse transcribed into cDNA using a reverse transcription kit, following the steps below:

a) Remove gDNA using gDNA enzyme according to the table below;

Table 6

42℃, 2min; 4℃, stand

b) The reverse transcription procedure was performed as follows:

Table 7

50℃, 15min; 85℃, 5s.

c) The reverse transcription product obtained in step b) was stored at 4° C. for real-time PCR analysis.

3.2.7 Real-time PCR analysis

a) Prepare qPCR reaction mixture as shown in the table below. Keep all reagents on ice during the entire operation.

Table 8

b) Perform qPCR procedure as follows

50°C, 2 minutes, 95°C, 10 minutes;

95°C, 15 seconds, 60°C, 1 minute (40 cycles of this operation).

3.2.8 Result Analysis

a) Use Quant Studio 6Flex software with default settings to automatically calculate Ct values;

b) Calculate the relative expression of genes using the following formula:

ΔCt＝Ct(MARC1 gene)–Ct(GAPDH)

ΔΔCt＝ΔCt(test sample group)-ΔCt(Mock group)

mRNA expression relative to the Mock group = 2 ^−ΔΔCt .

Mock group: Compared with the test sample group, the group without siRNA

Inhibition rate (%) = (relative expression of mRNA in the Mock group – relative expression of mRNA in the test sample group) / relative expression of mRNA in the Mock group × 100%

3.3 Results of Silencing Experiment

Concentrations of 0.1 nM and 10 nM were selected for testing, and the results are shown in Tables 9 and 10 below.

Table 9

As can be seen from Table 9, in the HepG2 cell experiment, the inhibition rate of the siRNA disclosed in the present invention on the MARC1 gene is at least about 21%, ranging from about 21% to about 97%, and the concentration of 10nM generally shows a higher inhibition rate on the MARC1 gene than the concentration of 1nM, showing a dose-dependent effect. Among them, the inhibition rate of 0.1nM N-ER-FY025096 on the MARC1 gene is about 89%, and 10nM is about 93%; the inhibition rate of 0.1nM N-ER-FY025102 on the MARC1 gene is about 89%, and 10nM is about 92%; the inhibition rate of 0.1nM N-ER-FY025175 on the MARC1 gene is about 90%, and 10nM is about 94%, indicating that these siRNAs have strong inhibition on the MARC1 gene.

Table 10

As can be seen from Table 10, in the HepG2 cell experiment, the inhibition rate of the siRNA modification disclosed in the present invention on the MARC1 gene is at least about 30%, ranging from about 30% to about 98%, and the concentration of 10nM generally shows a higher inhibition rate on the MARC1 gene than the concentration of 1nM, showing a dose-dependent effect. Among them, the inhibition rates of 0.1 nM N-ER-FY025096M2, N-ER-FY025096M3, N-ER-FY025096M6, N-ER-FY025096M7, N-ER-FY025096M8, and N-ER-FY025096M9 on the MARC1 gene were about 81% to about 85%, and 10 nM was about 90% to about 95%; 0.1 nM N-ER-FY025102M2, N-ER-FY025102M3, N-ER-FY025102M4, N-ER-FY025102M5, N-ER-FY025102M6, N-ER-FY025102M7, N-ER-FY025102M8, and N-ER-FY025102M9 The inhibition rates of 2M8 and N-ER-FY025102M9 on MARC1 gene are about 69%-about 90%, and 10nM is about 90%-about 95%; the inhibition rates of 0.1nM N-ER-FY025175M4, N-ER-FY025175M5, N-ER-FY025175M2, N-ER-FY025175M3, N-ER-FY025175M6, N-ER-FY025175M7, N-ER-FY025175M8, and N-ER-FY025175M9 on MARC1 gene are about 89%-about 94%, and 10nM is about 91%-about 95%, indicating that these siRNA modifications have a strong inhibitory effect on MARC1 gene.

3.4 _IC50 determination results

The concentration range of the following siRNA to be tested was set (nM) as follows: starting from 10 nM, 3-fold dilution, 8 concentration gradients: 10 nM, 3.33 nM, 1.11 nM, 0.37 nM, 0.12 nM, 0.041 nM, 0.014 nM, 0.0045 nM; and IC ₅₀ was determined in a manner similar to that in 3.2.

Result analysis:

b) Calculate the relative expression of genes using the following formula:

ΔCt＝Ct(MARC1 gene)–Ct(GAPDH)

ΔΔCt=ΔCt(test sample group)-ΔCt(Mock group), where the Mock group represents the group without siRNA added compared with the test sample group;

mRNA expression relative to the Mock group = 2 ^{- ΔΔCt}

Calculation process: With the log value of siRNA concentration as the X-axis and the percentage inhibition rate as the Y-axis, the "log (inhibitor) vs. response-variable slope" of the analysis software GraphPad Prism 8 was used to fit the dose-effect curve to obtain the _IC50 value of each siRNA.

The fitting formula is: Y = Bottom + (Top-Bottom) / (1 + 10^(( _logIC50 -X) × HillSlope))

Among them: Top represents the percentage inhibition rate at the top platform, and the Top standard of the curve is generally between 80% and 120%; Bottom represents the percentage inhibition rate at the bottom platform, and the Bottom of the curve is generally between -20% and 20%; HillSlope represents the slope of the percentage inhibition rate curve.

The results are shown in Table 11 below.

Table 11

Example 4: Determination of the inhibition rate of siRNA conjugates in inhibiting MARC1 gene expression

4.1 Test materials:

Human primary hepatocytes PHH cells were provided by Shanghai WuXi AppTec Pharmaceuticals Co., Ltd.;

PHH culture medium: invitroGRO CP Medium, purchased from Bioreclamation, catalog number: S03316;

RNAiMAX transfection reagent, purchased from Invitrogen, catalog number: 13778-150;

96 Kit, purchased from QIAGEN, catalog number: QIAGEN-74182;

FastQuant RT Kit (containing gDNase), purchased from TianGen, catalog number: KR116-02;

FastStart Universal Probe master, purchased from Roche, catalog number: 04914058001;

HiScript III RT SuperMix for qPCR (+gDNA wiper), purchased from Vazyme, catalog number R323-01;

The MARC1 primer and probe were synthesized by Sangon Biotech;

TaqMan Gene Expression Assay (GAPDH), purchased from Thermo, ID-Hs02786624_g1.

4.2 Test methods

siRNA conjugates (final concentrations of siRNA conjugates were 2 nM and 0.2 nM, respectively, in duplicate) were transfected into PHH cells by the following process: frozen PHH cells were taken, revived, counted, and the cells were adjusted to 6 × 10 ⁵ cells/mL, and then applied RNAiMax transfection reagent was used to transfer siRNA conjugates into cells, and 54,000 cells were seeded into 96-well plates at a density of 1:1. 100 μL of PHH culture medium was added to each well. The cells were cultured in a 5% CO ₂ , 37°C incubator. After 48 hours, the culture medium was removed and the cells were collected for total RNA extraction. Use according to the kit product instructions Total RNA was extracted using 96Kit.

siRNA conjugates (final concentrations of siRNA conjugates were 200 nM and 10 nM, respectively, in duplicate) were freely taken into PHH cells, and the process was as follows: frozen PHH cells were taken, revived, counted, and adjusted to 6×10 ⁵ cells/mL, and siRNA conjugates were added at the same time, and inoculated into 96-well plates at a density of 54,000 cells per well, and 100 μL of PHH culture medium was added to each well. The cells were cultured in a 5% CO ₂ , 37°C incubator. After 48 hours, the culture medium was removed and the cells were collected for total RNA extraction. Use according to the kit product instructions Total RNA was extracted using 96Kit.

Perform reverse transcription to cDNA using a reverse transcription kit and follow these steps:

(1) Remove gDNA using gDNA enzyme according to Table 12 below;

Table 12

42℃, 2min; 4℃, stand

(2) Add 4 μL 5×HiScript III qRT SuperMix directly to the reaction plate in step (1). Run the program: 37°C for 15 minutes, 85°C for 5 seconds.

(3) Detect the target gene cDNA by qPCR.

Table 13

Table 14

The qPCR procedure was performed as follows

95°C, 10 minutes;

95°C, 15 seconds, 60°C, 1 minute (40 cycles of this operation).

Result analysis:

a) Use Quant Studio 7 software with default settings to automatically calculate Ct values;

b) Calculate the relative expression of genes using the following formula:

ΔCt＝Ct(MARC1 gene)–Ct(GAPDH)

ΔΔCt=ΔCt(test sample group)−ΔCt(Mock group), where the Mock group represents a group without the addition of siRNA conjugates compared with the test sample group;

mRNA expression relative to the Mock group = 2 ^{- ΔΔCt}

The experimental results are shown in Table 15.

Table 15 Inhibition rate of siRNA conjugates in inhibiting MARC1 gene expression

As can be seen from Table 15, in the pHH cell experiment, the inhibition rate of the siRNA conjugates disclosed in the present invention on the MARC1 gene was at least about 36%, ranging from about 36% to about 94%, and showed a dose-dependent effect in both transfection and free uptake. In the free uptake method, 10 nM N-ER-FY025096M2L96, N-ER-FY025096M3L96, N-ER-FY025096M6L96, N-ER -FY025096M7L96, N-ER-FY025096M8L96, and N-ER-FY025096M9L96 showed an inhibition rate of about 72% to about 77% on the MARC1 gene, and about 84% to about 89% at 200 nM. Under the transfection mode, 0.2 nM of N-ER-FY025096M2L96, N-ER-FY025096M3L96, N-ER-FY025096M6L96, and N-ER-FY025096M7L96 showed an inhibition rate of about 72% to about 77% on the MARC1 gene, and about 84% to about 89% at 200 nM. The inhibition rates of 5096M7L96, N-ER-FY025096M8L96, and N-ER-FY025096M9L96 on the MARC1 gene were about 70% to about 74%, and 2nM was about 87% to about 89%; under the free uptake method, 10nM N-ER-FY025102M2L96, N-ER-FY025102M3L96, N-ER-FY025102M6L96, N-ER-FY025102M The inhibition rate of MARC1 gene by N-ER-FY025102M7L96, N-ER-FY025102M8L96, and N-ER-FY025102M9L96 was about 81% to about 83%, and 200nM was about 85% to about 89%. Under the transfection method, 0.2nM N-ER-FY025102M2L96, N-ER-FY025102M3L96, N-ER-FY025102M6L96, N-ER-FY025102M7L96, The inhibition rate of N-ER-FY025102M8L96 and N-ER-FY025102M9L96 on the MARC1 gene was about 80% to about 84%, and 2nM was about 83% to about 90%; under free uptake mode, the inhibition rate of 10nM N-ER-FY025175M2L96, N-ER-FY025175M3L96, N-ER-FY025175M6L96, N-ER-FY025175M7L96, N-ER-FY025175M8L96, and N-ER-FY025175M9L96 on the MARC1 gene was about 87% - about 93%, 200nM is about 88% - about 94%, under transfection mode, 0.2nM of N-ER-FY025175M2L96, N-ER-FY025175M3L96, N-ER-FY025175M6L96, N-ER-FY025175M7L96, N-ER-FY025175M8L96, N-ER-FY025175M9L96 has an inhibition rate of about 86% - about 88%, and 2nM is about 90% - about 93%, indicating that these siRNA conjugates have a strong inhibitory effect on the MARC1 gene.

Example 5: Silencing effect of siRNA conjugates in mice expressing the human MARC1 (hMARC1) gene

5.1 Construction of hMARC1 gene overexpression mouse model by AAV

6-8 week old C57BL/6 mice (provided by Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were brought into the facility. After 3-5 days of adaptive feeding, a single injection of adeno-associated virus pAAV[Exp]-CBh>SEAP:{MARC1 part 3'UTR} (the virus was prepared by Yunzhou Biotechnology (Guangzhou) Co., Ltd., virus ID: VB230627-1558gqe) expressing hMARC1 gene was performed through the tail vein for target gene overexpression modeling. The administration volume was 100 μL (3*10 ¹¹ vg)/mouse, and then the mice were fed with normal feed.

5.2 In vivo efficacy study of siRNA silencing in hMARC1 mouse model

Fourteen days after AAV virus injection, the mice were divided into groups (6 mice per group) and subcutaneously administered with a single 1, 3, or 10 mg/kg (mpk) dose of N-ER-FY025096M2L96, N-ER-FY025102M2L96, N-ER-FY025103M2L96, N-ER-FY025096M6L96, N-ER-FY025102M6L96, N-ER-FY025172M6L96, N-ER-FY025173M6L96, and N-ER-FY02 The administration volume was 5 μL/g, and the solvent was RNase-free sterile PBS. The blank group was injected with the same volume of RNase-free sterile PBS. The SEAP protein expression (i.e., hMARC1 protein expression) was detected on days 7, 14, 21, 28, 35, 42, and 49 after administration.

The formula for calculating the inhibition rate of hMARC1 is: Inhibition rate (%) = (1-relative expression of SEAP protein in the test sample group/relative expression of SEAP protein in the blank group) * 100%

Table 16 Inhibition rate of siRNA conjugates on hMARC1

As can be seen from Table 16, the siRNA conjugates of the present application have a high inhibitory activity on the hMARC1 gene in vivo and can reduce the hMARC1 protein level for a long time. From the experimental results of drugs N-ER-FY025096M2L96 and N-ER-FY025102M2L96 at different doses, it can be seen that the higher the dose of the siRNA conjugate, the higher the inhibition rate and the longer the inhibition time. In addition, on the 14th day, at a dose of 3mpk, the drugs N-ER-FY025096M6L96, N-ER-FY025096M8L96, N-ER-FY025102M8L96, and N-ER-FY025175M8L96 can all achieve an inhibition rate of more than 80%. The higher the dose of the siRNA conjugate, the higher the inhibition rate and the longer the inhibition time. Among them, the inhibition rate of hMARC1 of 1 mpk dose of N-ER-FY025096M2L96 was about 29%-about 60% within 7-49 days, 3 mpk was about 55%-about 79% within 7-49 days, and 10 mpk was about 71%-about 87% within 7-49 days; the inhibition rate of hMARC1 of 1 mpk dose of N-ER-FY025102M2L96 was about 24%-about 60% within 7-49 days, 3 mpk was about 47%-about 78% within 7-49 days, and 10 mpk was about 76%-about 84% within 7-49 days; the inhibition rate of hMARC1 of 3 mpk dose of N-ER-FY025096M6L96 was about 70%-about 82% within 7-49 days; The inhibition rate of N-ER-FY025102M6L9 on hMARC1 at 3mpk was about 36% to about 71% within 7-49 days; the inhibition rate of N-ER-FY025175M6L96 on hMARC1 at 3mpk was about 53% to about 82% within 7-49 days; the inhibition rate of N-ER-FY025096M8L96 on hMARC1 at 3mpk was about 68% to about 83% within 7-49 days; the inhibition rate of N-ER-FY025102M8L96 on hMARC1 at 3mpk was about 73% to about 87% within 7-49 days; the inhibition rate of N-ER-FY025175M8L96 on hMARC1 at 3mpk was about 68% to about 86% within 7-49 days.

The above embodiments of the present disclosure are merely examples for clearly illustrating the present disclosure, and are not intended to limit the implementation methods of the present disclosure. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all implementation methods here. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present disclosure shall be included in the protection scope of the claims of the present disclosure.

Claims

A double-stranded RNA, comprising a sense strand and an antisense strand, wherein the sense strand is reverse complementary to the antisense strand and/or substantially reverse complementary to form a double-stranded region of the double-stranded RNA;

The sense strand comprises a sequence A that differs by no more than 3 nucleotides from at least 15 consecutive nucleotides in the target sequence, and the antisense strand comprises a sequence B that differs by no more than 3 nucleotides from the reverse complementary sequence of at least 15 consecutive nucleotides in the target sequence;

The target sequence is selected from a nucleotide sequence shown in any one of SEQ ID NO: 1 to 9 and 812 to 817 and a sequence consisting of at least 15 consecutive nucleotides contained in any one of SEQ ID NO: 1 to 9 and 812 to 817.
The double-stranded ribonucleic acid according to claim 1, wherein the target sequence is selected from the nucleotide sequence as shown in any one of SEQ ID NOs: 10-37, 799-811 and 818-830, the positive strand comprises a sequence A consisting of at least 15 consecutive nucleotides in the nucleotide sequence as shown in any one of SEQ ID NOs: 10-37, 799-811 and 818-830, and the antisense strand comprises a sequence B that is reverse complementary and/or substantially reverse complementary to a sequence consisting of at least 15 consecutive nucleotides in the nucleotide sequence as shown in any one of SEQ ID NOs: 10-37, 799-811 and 818-830.
The double-stranded ribonucleic acid according to claim 1 or 2, wherein the sense strand consists of 15-28 nucleotides, preferably 19-25 nucleotides, more preferably 19-23 nucleotides, more preferably 19, 21 or 23 nucleotides.
The double-stranded ribonucleic acid according to claim 3, wherein the nucleotide sequence of the positive chain is a sequence A that differs by no more than 1 nucleotide compared to a sequence consisting of 15-28 consecutive nucleotides in the nucleotide sequence shown in any one of SEQ ID NOs: 10-37, 799-811 and 818-830, preferably 19-25 consecutive nucleotides, more preferably 19-23 consecutive nucleotides, and more preferably 19, 21 or 23 nucleotides.
The double-stranded ribonucleic acid according to any one of claims 1 to 4, wherein the antisense strand consists of 15-28 nucleotides, preferably 19-25 nucleotides, more preferably 19-23 nucleotides, more preferably 19, 21 or 23 nucleotides.
The double-stranded ribonucleic acid according to claim 5, wherein the nucleotide sequence of the antisense strand is a sequence B having a difference of no more than 1 nucleotide compared to the reverse complementary sequence of a sequence consisting of 15-28 consecutive nucleotides in the nucleotide sequence shown in any one of SEQ ID NOs: 10-37, 799-811 and 818-830, preferably 19-25 consecutive nucleotides, more preferably 19-23 consecutive nucleotides, more preferably 19, 21 or 23 nucleotides.
The double-stranded ribonucleic acid according to any one of claims 1 to 6, wherein the length of the double-stranded region is 15-25 nucleotides, preferably 19-23 nucleotides, more preferably 19-21 nucleotides, more preferably 19, 21 or 23 nucleotides.
The double-stranded RNA according to any one of claims 1 to 7, wherein

The sense strand and the antisense strand complement each other to form the double-stranded region, and the 3' end of the sense strand has 1-2 protruding nucleotides extending out of the double-stranded region, and the 3' end of the antisense strand forms a blunt end; or,

The sense strand and the antisense strand complement each other to form the double-stranded region, and the 3' end of the antisense strand has 1-2 protruding nucleotides extending out of the double-stranded region, and the 3' end of the sense strand forms a blunt end; or,

The sense strand and the antisense strand complement each other to form the double-stranded region, and the 3' ends of the sense strand and the antisense strand each have 1-2 protruding nucleotides extending out of the double-stranded region; or,

The sense strand and the antisense strand complement each other to form the double-stranded region, and the 3' ends of the sense strand and the antisense strand both form blunt ends.
The double-stranded RNA according to any one of claims 1 to 8, wherein the sense strand and the antisense strand are selected from the following combinations:

The sense strand comprises the sense strand of any one of the siRNAs shown in Table 1 and Table 1-1 herein, and the antisense strand comprises the antisense strand of the corresponding siRNA;

Preferably, the sense strand and the antisense strand are selected from the following combinations:

The sense chain comprises the sense chain of any one of the siRNAs 262, 264 and 330 shown in Table 1 of the present invention, and the antisense chain comprises the antisense chain of the corresponding siRNA.
The double-stranded ribonucleic acid according to any one of claims 1 to 9, wherein each nucleotide in the sense strand Each nucleotide in the antisense strand is independently a modified nucleotide or an unmodified nucleotide, and/or each nucleotide in the antisense strand is independently a modified nucleotide or an unmodified nucleotide.
The double-stranded ribonucleic acid according to any one of claims 1 to 10, wherein any two nucleotides connected in the sense strand are connected by a phosphodiester bond or a phosphorothioate diester bond, and/or any two nucleotides connected in the antisense strand are connected by a phosphodiester bond or a phosphorothioate diester bond.
The double-stranded ribonucleic acid according to any one of claims 1 to 11, wherein the 5' terminal nucleotide of the antisense strand is connected to a 5' phosphate group or a 5' phosphate derivative group, or the 5' terminal nucleotide of the antisense strand is not connected to a 5' phosphate group or a 5' phosphate derivative group.
The double-stranded ribonucleic acid according to any one of claims 1 to 12, wherein the double-stranded ribonucleic acid is siRNA.
The double-stranded ribonucleic acid according to any one of claims 1 to 13, wherein the double-stranded ribonucleic acid is siRNA for inhibiting the expression of MARC1 gene.
A double-stranded RNA modified substance, which is a double-stranded RNA modified substance according to any one of claims 1 to 14, wherein the double-stranded RNA modified substance comprises at least one of the following chemical modifications:

(1) modification of at least one nucleotide in the sense strand,

(2) modification of the phosphodiester bond at at least one position in the sense strand,

(3) modification of at least one nucleotide in the antisense strand,

(4) modification of the phosphodiester bond at at least one position in the antisense strand;

Optionally, the 3' end of sequence A in the sense strand of the double-stranded RNA is connected to a sequence D consisting of 1-2 nucleotides, preferably a sequence D consisting of 1-2 thymine deoxyribonucleotides; and/or, the 3' end of sequence B in the antisense strand of the double-stranded RNA is connected to a sequence E consisting of 1-2 nucleotides, preferably a sequence E consisting of 1-2 thymine deoxyribonucleotides; and/or, the 3' end of sequence A in the sense strand of the double-stranded RNA excludes 1-2 nucleotides to form sequence A';

Optionally, the sense strand and antisense strand of the double-stranded RNA modification are selected from the following sequence combinations:

The nucleotide sequence of the sense strand is the sequence shown in sequence A, and the nucleotide sequence of the antisense strand is the sequence shown in sequence B;

Alternatively, the nucleotide sequence of the sense strand is the sequence shown in sequence A, and the nucleotide sequence of the antisense strand is the sequence shown in sequence B connected to sequence E;

Alternatively, the nucleotide sequence of the sense strand is the sequence shown in sequence A connected to sequence D, and the nucleotide sequence of the antisense strand is the sequence shown in sequence B;

Alternatively, the nucleotide sequence of the sense strand is the sequence shown by sequence A connected to sequence D, and the nucleotide sequence of the antisense strand is the sequence shown by sequence B connected to sequence E;

Alternatively, the nucleotide sequence of the sense strand is the sequence shown in sequence A', and the nucleotide sequence of the antisense strand is the sequence shown in sequence B;

Alternatively, the nucleotide sequence of the sense strand is the sequence shown in sequence A’, and the nucleotide sequence of the antisense strand is the sequence shown in sequence B connected to sequence E.
The double-stranded RNA modified substance according to claim 15, wherein the modification of the nucleotide is selected from 2'-fluoro modification, 2'-alkoxy modification, 2'-substituted alkoxy modification, 2'-alkyl modification, 2'-substituted alkyl modification, 2'-deoxy modification, nucleotide derivative modification or a combination of any two or more thereof.
The double-stranded ribonucleic acid modified substance according to claim 15 or 16, wherein the modification of the nucleotide is selected from 2'-F modification, 2'-O-CH 3 modification, 2'-O-CH 2 -CH 2 -O-CH 3 modification, 2'-O-CH 2 -CH＝CH 2 modification, 2'-CH 2 -CH 2 -CH＝CH 2 modification, 2'-deoxy modification, nucleotide derivative modification or a combination of any two or more thereof.
The double-stranded RNA modification according to claim 16 or 17, wherein the nucleotide derivative in the nucleotide derivative modification is selected from isonucleotides, LNA, ENA, cET, UNA or GNA.
The double-stranded RNA modified substance according to any one of claims 15 to 18, wherein the Direction, the ribonucleotides at positions 7, 9, 10 and 11 in the sense strand are 2'-F modified ribonucleotides, and the ribonucleotides at the remaining positions in the sense strand are 2'-O-CH 3 modified ribonucleotides.
The double-stranded RNA modification according to any one of claims 15 to 19, wherein, from the 5' end to the 3' end, the sense strand comprises a phosphorothioate diester bond located at the following position:

Between the first nucleotide and the second nucleotide starting from the 5' end of the sense strand;

Between the second nucleotide and the third nucleotide starting from the 5' end of the sense strand;

Between the first nucleotide and the second nucleotide starting from the 3' end of the sense strand;

Between the second nucleotide and the third nucleotide starting from the 3' end of the sense strand;

or,

The sense strand contains phosphorothioate diester bonds located at the positions shown below:

Between the first nucleotide and the second nucleotide starting from the 5' end of the sense strand;

Between the second and third nucleotides starting from the 5' end of the sense strand.
The double-stranded ribonucleic acid modified substance according to any one of claims 15 to 20, wherein, from the 5' end to the 3' end, the ribonucleotide at any odd position in the antisense strand is a 2'-O-CH 3- modified ribonucleotide, and the ribonucleotide at any even position in the antisense strand is a 2'-F-modified ribonucleotide;

Alternatively, along the 5' end to the 3' end, the ribonucleotides at positions 2, 6, 14 and 16 in the antisense strand are 2'-F modified ribonucleotides, and the ribonucleotides at the remaining positions in the antisense strand are 2'-O-CH 3 modified ribonucleotides;

Alternatively, along the 5' end to the 3' end, the ribonucleotides at positions 2, 6, 8, 9, 14 and 16 in the antisense strand are 2'-F modified ribonucleotides, and the ribonucleotides at the remaining positions in the antisense strand are 2'-O-CH 3 modified ribonucleotides;

Alternatively, along the 5' end to the 3' end, the ribonucleotides at positions 2, 14 and 16 in the antisense strand are 2'-F modified ribonucleotides, the ribonucleotide at position 6 in the antisense strand is a ribonucleotide modified with the nucleotide derivative GNA, and the ribonucleotides at the remaining positions in the antisense strand are 2'-O-CH 3 modified ribonucleotides;

Alternatively, along the direction from the 5' end to the 3' end, the ribonucleotides at positions 2, 6, 14 and 16 in the antisense chain are 2'-F modified ribonucleotides, the ribonucleotide at position 7 in the antisense chain is a ribonucleotide modified with the nucleotide derivative GNA, and the ribonucleotides at the remaining positions in the antisense chain are 2'-O- CH3 modified ribonucleotides.
The double-stranded ribonucleic acid modification according to any one of claims 15 to 21, wherein, in the direction from the 5' end to the 3' end, the nucleotide at the 5' end of the antisense strand is not connected to a 5' phosphate group or a 5' phosphate derivative group, or the nucleotide at the 5' end of the antisense strand is connected to a 5' phosphate group or a 5' phosphate derivative group.
The double-stranded RNA modification according to any one of claims 15 to 22, wherein the antisense strand comprises a phosphorothioate diester bond located at the following position:

Between the first nucleotide and the second nucleotide starting from the 5' end of the antisense strand;

Between the second nucleotide and the third nucleotide starting from the 5' end of the antisense strand;

Between the first nucleotide and the second nucleotide starting from the 3' end of the antisense strand;

Between the second and third nucleotides starting from the 3' end of the antisense strand.
The double-stranded RNA modified substance according to any one of claims 15 to 23, wherein the sense strand of the double-stranded RNA modified substance has a structure as shown in any one of (a 1 ) to (a 5 ):

(a 1 )5'-mN 1 -(s)-mN 2 -(s)-mN 3 -mN 4 -mN 5 -mN 6 -N 7 f-mN 8 -N 9 fN 10 fN 11 f-mN 12 -mN 13 -mN 14 -mN 15 -mN 16 -mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3'，

(a 2 )5'-mN 1 -(s)-mN 2 -(s)-mN 3 -mN 4 -mN 5 -mN 6 -N 7 f-mN 8 -N 9 fN 10 fN 11 f-mN 12 -mN 13 -mN 14 -mN 15 -mN 16 -mN 17 -mN 18 -mN 19 -(s)-mN 20 -(s)-mN 21 -3',

(a 3 )5'-mN 1 -(s)-mN 2 -(s)-mN 3 -mN 4 -mN 5 -mN 6 -N 7 f-mN 8 -N 9 fN 10 fN 11 f-mN 12 -mN 13 -mN 14 -mN 15 -mN 16 -mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -(s)-mN 22 -(s)-mN 23 -3',

(a 4 )5'-mN 1 -(s)-mN 2 -(s)-mN 3 -mN 4 -mN 5 -mN 6 -N 7 f-mN 8 -N 9 fN 10 fN 11 f-mN 12 -mN 13 -mN 14 - mN 15 -mN 16 -mN 17 -mN 18 -mN 19 -3',

(a 5 )5'-mN 1 -(s)-mN 2 -(s)-mN 3 -mN 4 -mN 5 -mN 6 -N 7 f-mN 8 -N 9 fN 10 fN 11 f-mN 12 -mN 13 -mN 14 -mN 15 -mN 16 -mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -3';

wherein N 1 -N 23 are independently selected from ribonucleotides whose bases are A, U, C or G,

The capital letter T represents a deoxyribonucleotide with thymine as its base.

The lowercase letter m indicates that the ribonucleotide adjacent to the right of the letter m is a 2'-O-CH 3 modified ribonucleotide.

The lowercase letter f indicates that the ribonucleotide adjacent to the left side of the letter f is a 2'-F modified ribonucleotide.

-(s)- indicates that the two adjacent nucleotides are connected by a phosphorothioate diester bond.
The double-stranded RNA modified substance according to any one of claims 15 to 24, wherein the antisense strand of the double-stranded RNA modified substance has a structure as shown in any one of (b 1 ) to (b 27 ):

(b 1 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -N 4 f-mN 5 -N 6 f-mN 7 -N 8 f-mN 9 -N 10 f- mN 11 -N 12 f-mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -N 18 f-mN 19 -(s)-T-(s)-T-3',

(b 2 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -mN 8 -mN 9 -mN 10 -mN 11 - mN 12 -mN 13 -N 1 4 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3'，

(b 3 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -N 8 fN 9 f-mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3'，

(b 4 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -[GNA]N 6 -mN 7 -mN 8 -mN 9 -mN 10 -mN 11 -mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3',

(b 5 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-[GNA]N 7 -mN 8 -mN 9 -mN 10 - mN 11 -mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3',

(b 6 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -N 4 f-mN 5 -N 6 f-mN 7 -N 8 f-mN 9 -N 10 f- mN 11 -N 12 f-mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -N 18 f-mN 19 -(s)-N 20 f-(s)-mN 21 -3',

(b 7 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -mN 8 -mN 9 -mN 10 -mN 11 - mN 12 -mN 13 -N 1 4 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-mN 20 -(s)-mN 21 -3',

(b 8 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -N 8 fN 9 f-mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-mN 20 -(s)-mN 21 -3',

(b 9 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -[GNA]N 6 -mN 7 -mN 8 -mN 9 -mN 10 -mN 11 -mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-mN 20 -(s)-mN 21 -3',

(b 10 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-[GNA]N 7 -mN 8 -mN 9 -mN 10 - mN 11 -mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-mN 20 -(s)-mN 21 -3',

(b 11 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -N 4 f-mN 5 -N 6 f-mN 7 -N 8 f-mN 9 -N 10 f- mN 11 -N 12 f-mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -N 18 f-mN 19 -N 20 f-mN 21 -(s)-N 22 f-(s)- mN 23 -3',

(b 12 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -mN 8 -mN 9 -mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -(s)-mN 22 -(s)-mN 23 -3',

(b 13 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -N 8 fN 9 f-mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -(s)-mN 22 -(s)-mN 23 -3',

(b 14 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -[GNA]N 6 -mN 7 -mN 8 -mN 9 -mN 10 -mN 11 -mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -(s)-mN 22 -(s)-mN 23 -3' ,

(b 15 )5'-P1mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-[GNA]N 7 -mN 8 -mN 9 -mN 10 - mN 11 -mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -(s)-mN 22 -(s)-mN 23 -3 ',

(b 16 )5'-mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -mN 8 -mN 9 -mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3'，

(b 17 )5'-mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -N 8 fN 9 f-mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3'，

(b 18 )5'-EVPmN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -mN 8 -mN 9 -mN 10 -mN 11 - mN 12 - mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3'，

(b 19 )5'-EVPmN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -N 8 fN 9 f-mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3'，

(b 20 )5'-mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -mN 8 -mN 9 -mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-mN 20 -(s)-mN 21 -3',

(b 21 )5'-mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -N 8 fN 9 f-mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-mN 20 -(s)-mN 21 -3',

(b 22 )5'-EVPmN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -mN 8 -mN 9 -mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-mN 20 -(s)-mN 21 -3',

(b 23 )5'-EVPmN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -N 8 fN 9 f-mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-mN 20 -(s)-mN 21 -3',

(b 24 )5'-mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -mN 8 -mN 9 -mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -(s)-mN 22 -(s)-mN 23 -3',

(b 25 )5'-mN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -N 8 fN 9 f-mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -(s)-mN 22 -(s)-mN 23 -3',

(b 26 )5'-EVPmN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -mN 8 -mN 9 -mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -(s)-mN 22 -(s)-mN 23 -3',

(b 27 )5'-EVPmN 1 -(s)-N 2 f-(s)-mN 3 -mN 4 -mN 5 -N 6 f-mN 7 -N 8 fN 9 f-mN 10 -mN 11 - mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -mN 20 -mN 21 -(s)-mN 22 -(s)-mN 23 -3';

wherein N 1 -N 23 are independently selected from ribonucleotides whose bases are A, U, C or G,

The capital letter T represents a deoxyribonucleotide with thymine as its base.

The lowercase letter m indicates that the ribonucleotide adjacent to the right of the letter m is a 2'-O-CH 3 modified ribonucleotide.

The lowercase letter f indicates that the ribonucleotide adjacent to the left side of the letter f is a 2'-F modified ribonucleotide.

P1 means that the nucleotide adjacent to the right of the letter is a 5'-phosphate nucleotide.

EVP means that the nucleotide adjacent to the right side of the letter combination is a 5'-trans vinylphosphonate nucleotide.

-(s)- indicates that the two adjacent nucleotides are connected by a phosphorothioate diester bond.

[GNA] indicates that the adjacent ribonucleotide on the right is a ribonucleotide with GNA modification.
The double-stranded RNA modified substance according to any one of claims 15 to 25, wherein the double-stranded RNA modified substance is a siRNA modified substance.
The double-stranded RNA modified substance according to any one of claims 15 to 26, wherein the double-stranded RNA modified substance is a siRNA modified substance for inhibiting the expression of the MARC1 gene.
The double-stranded RNA modification according to any one of claims 15 to 27, wherein the sense strand and the antisense strand are selected from the following combinations:

The sense strand comprises the sense strand of any one of the siRNA modifications shown in Table 2 herein, and the antisense strand comprises the antisense strand of the corresponding siRNA modification;

Preferably, the sense strand and the antisense strand are selected from the following combinations:

The sense chain comprises the sense chain of any one of the siRNAs siRNA 356-siRNA 363, siRNA 366-siRNA 373 and siRNA 433-siRNA 440 shown in Table 2 of the present invention, and the antisense chain comprises the antisense chain of the corresponding siRNA.
A double-stranded RNA conjugate, wherein the double-stranded RNA conjugate comprises the double-stranded RNA as described in any one of claims 1-14, or the double-stranded RNA modification as described in any one of claims 15-28; and a conjugated group conjugated to the double-stranded RNA or the double-stranded RNA modification.
The double-stranded RNA conjugate according to claim 29, wherein the conjugated group has a structure as shown in any of the following:
The double-stranded ribonucleic acid conjugate according to claim 29 or 30, wherein the conjugated group is connected to the 3' end of the sense strand.
The double-stranded RNA conjugate according to claim 31, wherein the conjugated group is conjugated to the 3' end of the sense strand via a phosphodiester bond;

Preferably, the sense strand and the antisense strand of the double-stranded RNA conjugate are complementary to each other to form a double-stranded region of the double-stranded RNA conjugate, and the 3' end of the sense strand forms a blunt end, and the 3' end of the antisense strand has 1-2 protruding nucleotides extending out of the double-stranded region;

or,

The sense strand and antisense strand of the double-stranded RNA conjugate are complementary to each other to form a double-stranded region of the double-stranded RNA conjugate, and the 3' end of the sense strand forms a blunt end, while the 3' end of the antisense strand forms a blunt end.
The double-stranded ribonucleic acid conjugate according to any one of claims 29 to 32, wherein the double-stranded ribonucleic acid conjugate has the following structure:

The double helix structure is double-stranded RNA or a modified double-stranded RNA.
The double-stranded ribonucleic acid conjugate according to any one of claims 29 to 33, wherein the double-stranded ribonucleic acid conjugate is a siRNA conjugate.
The double-stranded RNA conjugate according to any one of claims 29 to 34, wherein the double-stranded RNA conjugate is a siRNA conjugate for inhibiting the expression of the MARC1 gene.
The double-stranded ribonucleic acid conjugate according to any one of claims 29 to 35, wherein the double-stranded ribonucleic acid conjugate is formed by connecting any one of the siRNAs shown in Table 1 herein to a conjugation group, or the double-stranded ribonucleic acid conjugate is The acid conjugate is formed by connecting any one of the siRNAs shown in Table 1-1 herein to a conjugation group, or the double-stranded RNA conjugate is formed by connecting any one of the siRNA modifications shown in Table 2 herein to a conjugation group;

Preferably, in the double-stranded RNA conjugate, the sense strand and the antisense strand are selected from the following combinations:

The sense strand comprises the sense strand of any one of the siRNA conjugates shown in Table 3 herein, and the antisense strand comprises the antisense strand of the corresponding siRNA conjugate;

More preferably, the sense strand and the antisense strand are selected from the following combinations:

The sense chain comprises the sense chain of any one of the siRNAs siRNA 451-siRNA 456, siRNA 462-siRNA 467 and siRNA 516-siRNA 521 shown in Table 3 of the present invention, and the antisense chain comprises the antisense chain of the corresponding siRNA.
A prodrug of the double-stranded ribonucleic acid according to any one of claims 1 to 14, the double-stranded ribonucleic acid modification according to any one of claims 15 to 28, or the double-stranded ribonucleic acid conjugate according to any one of claims 29 to 36.
A pharmaceutical composition, wherein the pharmaceutical composition comprises at least one of the following: a double-stranded ribonucleic acid as described in any one of claims 1 to 14, a double-stranded ribonucleic acid modification as described in any one of claims 15 to 28, a double-stranded ribonucleic acid conjugate as described in any one of claims 29 to 36, and a prodrug as described in claim 37.
The pharmaceutical composition according to claim 38, wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers and optionally one or more additional therapeutic agents.
Use of the double-stranded RNA according to any one of claims 1 to 14, the double-stranded RNA modified substance according to any one of claims 15 to 28, the double-stranded RNA conjugate according to any one of claims 29 to 36, the prodrug according to claim 37 or the pharmaceutical composition according to claim 38 or 39 in at least one of the following:

(1) Inhibiting MARC1 gene expression in vivo or in vitro, or preparing a drug for inhibiting MARC1 gene expression;

(2) for preventing or treating diseases associated with abnormal expression of the MARC1 gene, or for preparing drugs for preventing or treating diseases associated with abnormal expression of the MARC1 gene;

(3) Use for treating a subject suffering from a disease that would benefit from reduced expression of the MARC1 gene, or for preparing a medicament for treating a subject suffering from a disease that would benefit from reduced expression of the MARC1 gene.
The use according to claim 40, wherein the disease associated with abnormal expression of the MARC1 gene is selected from the group consisting of the following diseases:

Obesity, non-alcoholic fatty liver disease, alcohol-related fatty liver disease, non-alcoholic steatohepatitis, cirrhosis, liver fibrosis, elevated liver enzyme levels (ALT, AST, ALP), hepatocellular carcinoma, hypercholesterolemia and related cardiovascular disease, insulin resistance, impaired glucose tolerance, hyperglycemia, type II diabetes, and metabolic syndrome.
A method for inhibiting MARC1 gene expression in cells in vivo or in vitro, wherein the method comprises contacting the cells with a double-stranded RNA according to any one of claims 1 to 14, a double-stranded RNA modification according to any one of claims 15 to 28, a double-stranded RNA conjugate according to any one of claims 29 to 36, a prodrug according to claim 37, or a pharmaceutical composition according to claim 38 or 39.
The method according to claim 42, wherein the cell is an in vivo cell or an in vitro cell.
The method of claim 42 or 43, wherein the cell is in a subject.
The method according to claim 44, wherein the subject is a mammal, preferably a human.
The method according to claim 44 or 45, wherein the subject has at least one of the following characteristics:

Abnormal expression of MARC1 gene in vivo, more specifically abnormally high expression of MARC1 gene;

Suffering from diseases associated with abnormal expression of the MARC1 gene;

Having a disease that would benefit from decreased expression of the MARC1 gene.
The double-stranded RNA according to any one of claims 1 to 14, the double-stranded RNA modification according to any one of claims 15 to 28, the double-stranded RNA conjugate according to any one of claims 29 to 36, the prodrug according to claim 37 or the pharmaceutical composition according to claim 38 or 39, for use in treatment.