WO2023208109A1

WO2023208109A1 - Sirna for inhibiting hsd17b13 expression, conjugate and pharmaceutical composition thereof, and use thereof

Info

Publication number: WO2023208109A1
Application number: PCT/CN2023/091141
Authority: WO
Inventors: 王书成; 黄河; 王岩; 林国良; 耿玉先; 荣梅
Original assignee: 北京福元医药股份有限公司
Priority date: 2022-04-29
Filing date: 2023-04-27
Publication date: 2023-11-02
Also published as: WO2023208109A9; CN116515835A

Abstract

The present invention relates to an siRNA capable of inhibiting HSD17B13 gene expression, an siRNA conjugate, a pharmaceutical composition comprising same, and a use thereof. Each nucleotide in the siRNA is independently a modified or unmodified nucleotide, and the siRNA contains a sense strand and an antisense strand. The siRNA and the conjugate and pharmaceutical composition thereof can effectively treat and/or prevent diseases related to overexpression of the HSD17B13 gene.

Description

siRNA, conjugates and pharmaceutical compositions thereof for inhibiting HSD17B13 expression and uses thereof

Technical field

The present application relates to siRNA, siRNA conjugates, pharmaceutical compositions containing the same, preparation methods and uses thereof capable of inhibiting HSD17B13 gene expression.

Background technique

The 17β-hydroxysteroid dehydrogenase (17β-HSD) family consists of 15 enzymes, most of which are related to the activation or inactivation of sex hormones (such as HSD17B1, HSD17B2, HSD17B3, HSD17B5, HSD17B6), and other members are involved in fatty acid metabolism, cholesterol Biosynthesis and bile acid production, etc. Members of the HSD17B family differ in tissue distribution, subcellular localization, catalytic priority, and have different substrate specificities (Marchais Oberwinkler, et al. (2011) J Steroid Biochem Mol Biol 125(1-2): 66 -82)).

HSD17B13, a member of the 17β-hydroxysteroid dehydrogenase family, is primarily localized in hepatocytes, with the highest expression levels known to be found in hepatocytes of the liver, and in the ovary, bone marrow, kidney, brain, lung, skeletal muscle, bladder and testis. Detectable only at lower levels, it is a hepatocyte-specific lipid droplet (LD)-associated protein, and increasing evidence suggests that it plays a key role in hepatic lipid metabolism. The function of HSD17B13 is not fully understood, however, several 17β-HSD family members, including 17β-HSD-4, -7, -10, and -12, have been shown to be involved in carbohydrate and fatty acid metabolism. This suggests that HSD17B13 may also play a role in lipid metabolism pathways. Hepatic upregulation of HSD17B13 has been reported to have been observed in patients with fatty liver disease, supporting a role for this enzyme in the pathogenesis of non-alcoholic fatty liver disease (NAFLD).

Non-alcoholic fatty liver disease (NAFLD), also known as metabolic (dysfunction)-associated fatty liver disease (MAFLD), is the excessive accumulation of fat in the liver in the absence of another clear cause, such as alcohol consumption. NAFLD is the most common liver disease in the world, affecting approximately 25% of the world's population. The prevalence of NAFLD is still showing an upward trend, which will undoubtedly increase the economic burden and lead to a sharp increase in the number of patients with end-stage liver disease requiring liver transplantation and the number of people suffering from hepatocellular carcinoma. There is currently no specific treatment for NAFLD, which mainly involves weight loss through dietary changes and exercise. Preliminary research shows that pioglitazone and vitamin E have therapeutic potential.

Wen Su et al. have previously identified HSD17B13 as a lipid droplet (LD)-associated protein in NAFLD patients and reported HSD17B13 to be one of the most abundantly expressed LD proteins specifically localized on the surface of LDs (Wen Su, et al. ., Comparative proteomic study reveals 17β-HSD13 as a pathogenic protein in nonalcoholic fatty live disease, 111 PNAS 11437-11442 (2014)). Further, HSD17B13 levels were found to be upregulated in the livers of patients and mice with NAFLD. Overexpression leads to LD increase in number and size, whereas gene silencing of HSD17B13 attenuated oleic acid-induced LD formation in cultured hepatocytes. Hepatic overexpression of HSD17B13 protein in C57BL/6 mice has also been shown to significantly increase lipogenesis and triglyceride (TG) content in the liver, leading to a fatty liver phenotype. NSAbul-Husn et al. provide additional evidence implicating HSD17B13 gene expression in the pathogenesis of NAFLD and nonalcoholic steatohepatitis (NASH) (NSAbul-Husn et al., A Protein-Truncating HSD17B13 Variant and Protection from Chronic Liver Disease , 378 N. Eng. J. Med. 1096-1106 (2018)). The team performed a genome-wide association study, which revealed an HSD17B13 splice variant (rs72613567:TA) associated with reduced levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) indicative of fat Less liver damage and inflammation in liver patients. The splice variants produce truncated deletions of functional protein, suggesting that HSD17B13 normally produces products that promote hepatocellular damage. ARO-HSD, an RNAi therapy targeting HSD17B13 jointly developed by GlaxoSmithKline and Arrowhead, is used to treat non-alcoholic steatohepatitis and has entered Phase I clinical research. Currently, there are no marketed nucleic acid drugs targeting HSD17B13.

The present invention aims to provide siRNA, siRNA conjugates and pharmaceutical compositions thereof, which can affect the RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the HSD17B13 gene, thereby inhibiting the expression of the HSD17B13 gene in the liver. , to achieve the purpose of disease treatment.

Contents of the invention

The present invention provides a siRNA capable of inhibiting HSD17B13 gene expression. The siRNA includes a sense strand and an antisense strand, wherein each nucleotide in the siRNA is independently a modified or unmodified nucleotide, wherein The sense strand contains nucleotide sequence I, the antisense strand contains nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region, wherein the Nucleotide sequence I and nucleotide sequence II are selected from the following sequences:

(1) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 296, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 297:

5’-AGUCGUUGGUGAAGUU-3’(SEQ ID NO: 296)

5’-AACUUCACCAACGACU-3’(SEQ ID NO: 297);

(2) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 298, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 299:

5’-GUUGGUGAAGUUUUUCA-3’(SEQ ID NO: 298)

5’-UGAAAAACUUCACCAAC-3’(SEQ ID NO: 299);

wherein said nucleotide sequence I is not SEQ ID NO: 17 and said nucleotide sequence II is not SEQ ID NO: 18;

The nucleotide sequence I is not SEQ ID NO: 21 and the nucleotide sequence II is not SEQ ID NO: 22;

(3) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 300, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 301:

5’-GACUACUUAUGAAUU-3’(SEQ ID NO: 300)

5’-AAUUCAUAAGUAGUC-3’(SEQ ID NO: 301);

wherein the nucleotide sequence I is not SEQ ID NO: 33 and the nucleotide sequence II is not SEQ ID NO: 34;

The nucleotide sequence I is not SEQ ID NO: 35 and the nucleotide sequence II is not SEQ ID NO: 36;

The nucleotide sequence I is not SEQ ID NO: 39 and the nucleotide sequence II is not SEQ ID NO: 40;

(4) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 23, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 24;

(5) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 302, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 303:

5’-CAGGCAGACUACUUAUGAN1-3’(SEQ ID NO: 302)

5’-N2UCAUAAGUAGUCUGCCUG-3’(SEQ ID NO: 303);

Where N1 is A or U, N2 is A or U;

wherein the nucleotide sequence I is not SEQ ID NO: 25 and the nucleotide sequence II is not SEQ ID NO: 26;

(6) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 304, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 305:

5’-GGUUCUGUGGGAUAUUA-3’(SEQ ID NO: 304)

5’-UAAUAUCCCACAGAACC-3’(SEQ ID NO: 305);

wherein the nucleotide sequence I is not SEQ ID NO: 51 and the nucleotide sequence II is not SEQ ID NO: 52;

(7) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 306, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 307:

5’-CUGCGCAUGCGUAU-3’(SEQ ID NO: 306)

5’-AUACGCAUGCGCAG-3’(SEQ ID NO: 307);

(8) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 308, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 309:

5’-GAUCUAUCGCUCUCUAA-3’(SEQ ID NO: 308)

5'-UUAGAGAGCGAUAGAUC-3' (SEQ ID NO: 309);

wherein the nucleotide sequence I is not SEQ ID NO: 71 and the nucleotide sequence II is not SEQ ID NO: 72;

(9) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 310, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 311:

5’-GAAAGAAGUGGGUGAU-3’(SEQ ID NO: 310)

5’-AUCACCCACUUCUUUC-3’(SEQ ID NO: 311);

wherein the nucleotide sequence I is not SEQ ID NO: 77 and the nucleotide sequence II is not SEQ ID NO: 78;

The nucleotide sequence I is not SEQ ID NO: 79 and the nucleotide sequence II is not SEQ ID NO: 80;

The nucleotide sequence I is not SEQ ID NO: 81 and the nucleotide sequence II is not SEQ ID NO: 82;

(10) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 312, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 313:

5’-AAGUGGGUGAUGUACAA-3’(SEQ ID NO: 312)

5’-UUGUUACAUCACCCACUU-3’(SEQ ID NO: 313);

wherein the nucleotide sequence I is not SEQ ID NO: 89 and the nucleotide sequence II is not SEQ ID NO: 90;

(11) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 314, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 315:

5’-AGAGAUUACCAAGACA-3’(SEQ ID NO: 314)

5’-UGUCUUGGUAAUCUCU-3’(SEQ ID NO: 315);

wherein the nucleotide sequence I is not SEQ ID NO: 95 and the nucleotide sequence II is not SEQ ID NO: 96;

The nucleotide sequence I is not SEQ ID NO: 99 and the nucleotide sequence II is not SEQ ID NO: 100;

(12) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 316, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 317:

5’-AUCGUAUAUCAAUAU-3’(SEQ ID NO: 316)

5’-AUAUUGAUAUACGAU-3’(SEQ ID NO: 317);

wherein the nucleotide sequence I is not SEQ ID NO: 121 and the nucleotide sequence II is not SEQ ID NO: 122;

(13) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 318, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 319:

5'-GCGCCUCAGCGAUUU-3'(SEQ ID NO: 318)

5’-AAAAUCGCUGAGGCGC-3’(SEQ ID NO: 319);

wherein the nucleotide sequence I is not SEQ ID NO: 139 and the nucleotide sequence II is not SEQ ID NO: 140;

The nucleotide sequence I is not SEQ ID NO: 141 and the nucleotide sequence II is not SEQ ID NO: 142;

(14) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 320, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 321:

5’-CUCAGCGAUUUAAAUCGU-3’(SEQ ID NO: 320)

5’-ACGAUUUAAAAUCGCUGAG-3’(SEQ ID NO: 321);

(15) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 322, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 323:

5’-UAUGCAGAAUAUUCA-3’(SEQ ID NO: 322)

5’-UGAAUAUUCUGCAUA-3’(SEQ ID NO: 323);

(16) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 324, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 325:

5’-UUGGCCACAAAAUCAAA-3’(SEQ ID NO: 324)

5’-UUUGAUUUUGUGGCCAA-3’(SEQ ID NO: 325);

wherein the nucleotide sequence I is not SEQ ID NO: 169 and the nucleotide sequence II is not SEQ ID NO: 170;

(17) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 65, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 66;

(18) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 75, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 76;

(19) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 93, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 94;

(20) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 103, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 104;

(21) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 109, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 110;

(22) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 111, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 112;

(23) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 115, and the nucleotide sequence Column II contains the nucleotide sequence shown in SEQ ID NO: 116;

(24) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 41, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 42;

(25) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 41, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 188;

(26) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 189, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 188;

(27) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 41, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 190;

(28) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 41, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 191;

(29) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 41, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 192;

(30) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 27, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 28;

(31) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 27, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 183;

(32) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 184, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 183;

(33) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 27, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 185;

(34) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 27, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 186;

(35) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 27, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 187;

(36) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 73, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 74;

(37) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 83, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 84;

(38) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 83, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 193;

(39) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 194, and the nucleotide sequence Column II contains the nucleotide sequence shown in SEQ ID NO: 193;

(40) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 83, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 195;

(41) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 83, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 196;

(42) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 83, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 197;

(43) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 97, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 98;

(44) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 97, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 198;

(45) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 199, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 198;

(46) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 97, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 200;

(47) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 97, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 201;

(48) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 97, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 202;

(49) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 143, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 144;

(50) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 19, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 20;

(51) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 19, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 178;

(52) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 179, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 178;

(53) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 19, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 180;

(54) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 19, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 181;

(55) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 19, and the nucleotide sequence II contains the nucleotide sequence shown in SEQ ID NO:182.

In one embodiment, the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, substantially reverse complementary or completely reverse complementary; the substantially reverse complementary refers to two nuclei There are no more than 3 base mismatches between the nucleotide sequences; the substantial reverse complementarity refers to the presence of no more than 1 base mismatch between the two nucleotide sequences; complete reverse complementarity Refers to the absence of mismatches between the two nucleotide sequences.

In one embodiment, the sense strand further contains nucleotide sequence III, the antisense strand further contains nucleotide sequence IV, and the lengths of nucleotide sequence III and nucleotide sequence IV are each independently 0- 6 nucleotides, wherein the nucleotide sequence III is connected to the 5′ end of the nucleotide sequence I, the nucleotide sequence IV is connected to the 3′ end of the nucleotide sequence II, and the nucleotide sequence III It is equal to the length of the IV of the nucleotide sequence and is substantially reverse complementary or completely reverse complementary; the substantially reverse complementarity means that there is no more than 1 base mismatch between the two nucleotide sequences. ;Perfect reverse complementarity means there are no mismatches between the two nucleotide sequences; and/or,

The nucleotide sequence III is connected to the 3′ end of the nucleotide sequence I, the nucleotide sequence IV is connected to the 5′ end of the nucleotide sequence II, and the nucleotide sequence III and the nucleotide sequence The IV lengths are equal and are substantially reverse complementary or completely reverse complementary; the substantially reverse complementarity means that there is no more than 1 base mismatch between the two nucleotide sequences; complete reverse complementarity means that there is no more than 1 base mismatch between the two nucleotide sequences; There are no mismatches between the two nucleotide sequences.

In one embodiment, the sense strand further contains the nucleotide sequence V and/or the antisense strand further contains the nucleotide sequence VI, the nucleotide sequences V and VI being 0 to 3 nucleotides in length , the nucleotide sequence V is connected to the 3' end of the sense strand to form the 3' overhang of the sense strand, and/or the nucleotide sequence VI is connected to the 3' end of the antisense strand to form the 3' end of the antisense strand. 'Protruding end. In a preferred embodiment, the length of the nucleotide sequence V or VI is 2 nucleotides. In a preferred embodiment, the nucleotide sequence V or VI is two consecutive thymine deoxyribonucleotides or two consecutive uracil ribonucleotides. In a preferred embodiment, the nucleotide sequence V or VI mismatches or is complementary to the nucleotide at the corresponding position of the target mRNA.

In one embodiment, the length of the double-stranded region is 15-30 nucleotide pairs; preferably, the length of the double-stranded region is 17-23 nucleotide pairs; more preferably, the length of the double-stranded region It is 19-21 nucleotide pairs.

In another embodiment, the sense strand or antisense strand has 15-30 nucleotides; preferably, the sense strand or antisense strand has 19-25 nucleotides; more preferably, the sense strand or antisense strand has 15-30 nucleotides; The sense strand has 19-23 nucleotides.

In one embodiment, at least one nucleotide in the sense strand or the antisense strand is a modified nucleotide, and/or at least one phosphate group is a phosphate group with a modifying group; preferably , the phosphate group containing a modified group is a phosphorothioate group formed by replacing at least one oxygen atom in the phosphodiester bond of the phosphate group with a sulfur atom.

In one embodiment, the siRNA includes a sense strand that does not include a 3' overhanging nucleotide.

In one embodiment, the 5' terminal nucleotide of the sense strand is connected to a 5' phosphate group or a 5' phosphate derivative group, and/or the 5' terminal nucleotide of the antisense strand is connected to a 5' phosphate group or a 5' phosphate derivative group.

In one embodiment, the modified nucleotide is selected from the group consisting of 2'-fluoro modified nucleotides, 2'-alkoxy modified nucleotides, 2'-substituted alkoxy modified nucleosides Acid, 2'-alkyl modified nucleotide, 2'-substituted alkyl modified nucleotide, 2'-deoxynucleotide, 2'-amino modified nucleotide, 2'-substituted amino Modified nucleotides, nucleotide analogs or a combination of any two or more thereof.

In one embodiment, the modified nucleotides are selected from the group consisting of 2'-fluoro modified nucleotides, 2'-methoxy modified nucleotides, 2'-O-CH ₂ -CH ₂ -O -CH ₃ modified nucleotide, 2'-O-CH ₂ -CH=CH ₂ modified nucleotide, 2'-CH ₂ -CH ₂ -CH=CH ₂ modified nucleotide, 2'-deoxy Nucleotides, nucleotide analogs or a combination of any two or more thereof.

In one embodiment, each nucleotide in the sense strand and the antisense strand is independently a 2'-fluoro modified nucleotide or a non-fluoro modified nucleotide. In a preferred embodiment, the 2'-fluorinated modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are non-fluorinated modified cores According to the 5' to 3' direction, the 2'-fluorinated modified nucleotides are located at the even-numbered positions of the antisense strand, and the remaining positions are non-fluorinated modified nucleotides. In a preferred embodiment, the 2'-fluorinated modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are non-fluorinated modified cores According to the 5' to 3' direction, the 2'-fluorinated modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and the remaining positions are non-fluorinated modified nucleotides. In a preferred embodiment, the 2'-fluorinated modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are non-fluorinated modified cores In the 5' to 3' direction, the 2'-fluorinated modified nucleotides are located at positions 2, 6, 8, 9, 14 and 16 of the antisense strand, and the remaining positions are non-fluorinated modified nuclei. glycosides. In a preferred embodiment, the 2'-fluorinated modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are non-fluorinated modified cores According to the 5' to 3' direction, the 2'-fluorinated modified nucleotides are located at positions 2, 14 and 16 of the antisense strand, and the remaining positions are non-fluorinated modified nucleotides. In one embodiment, each non-fluoro modified nucleotide is a 2'-methoxy modified nucleotide, the 2'-methoxy modified nucleotide refers to the 2'- of the ribosyl group Nucleotides formed by replacing the hydroxyl group with a methoxy group.

In one embodiment, each non-fluorinated modified nucleotide is independently selected from nucleotides or nucleotide analogs in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by a non-fluorinated group. One, the nucleotide analog is selected from one of isonucleotides, LNA, ENA, cET BNA, UNA and GNA.

In one embodiment, each nucleotide in the sense strand and the antisense strand is independently a 2'-fluoro modified nucleotide, a 2'-methoxy modified nucleotide, GNA Modified nucleotides or a combination of any two or more thereof. In a preferred embodiment, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups Modified nucleotides; in the 5' to 3' direction, 2'- The fluoro-modified nucleotides are located in even-numbered positions of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides. In a preferred embodiment, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups Modified nucleotides; in the 5' to 3' direction, 2'-fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and the remaining positions are 2'-methoxy modified of nucleotides. In a preferred embodiment, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups Modified nucleotides; in the 5' to 3' direction, 2'-fluoro modified nucleotides are located at positions 2, 6, 8, 9, 14 and 16 of the antisense strand, and the remaining positions are 2'- Methoxy modified nucleotides. In a preferred embodiment, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups Modified nucleotides; in the 5' to 3' direction, 2'-fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and GNA modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand. Position 7, and the remaining positions are 2'-methoxy modified nucleotides. In a preferred embodiment, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups Modified nucleotides; in the 5' to 3' direction, 2'-fluoro modified nucleotides are located at positions 2, 14 and 16 of the antisense strand, and GNA modified nucleotides are located at position 1 of the antisense strand. Position 6, and the remaining positions are 2'-methoxy modified nucleotides.

In a more preferred embodiment, at least one of the linkages between the following nucleotides in the siRNA is a phosphorothioate linkage:

The connection between the first nucleotide and the second nucleotide at the 5' end of the sense strand;

The connection between the 2nd nucleotide and the 3rd nucleotide at the 5' end of the sense strand;

The connection between the first nucleotide and the second nucleotide at the 3' end of the sense strand;

The connection between the second nucleotide and the third nucleotide at the 3’ end of the sense strand;

The connection between the first nucleotide and the second nucleotide at the 5' end of the antisense strand;

The connection between the 2nd nucleotide and the 3rd nucleotide at the 5' end of the antisense strand;

The connection between the first nucleotide and the second nucleotide at the 3' end of the antisense strand;

The connection between the 2nd nucleotide and the 3rd nucleotide at the 3' end of the antisense strand.

In some embodiments, the siRNA is directed from the 5' end to the 3' end, and the sense strand contains a phosphorothioate group at a position as follows:

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

Between the 2nd and 3rd 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 and third nucleotides starting from the 3’ end of the sense strand;

or,

The sense strand contains phosphorothioate groups at the positions shown below:

Between the 2nd and 3rd nucleotide starting from the 5' end of the sense strand.

In some embodiments, the siRNA is directed from the 5' end to the 3' end and the antisense strand contains a phosphorothioate group at a position as follows:

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

Between the 2nd and 3rd 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 2nd and 3rd nucleotide starting from the 3' end of the antisense strand.

In one embodiment, each nucleotide in the sense strand and the antisense strand is independently a 2'-fluoro modified nucleotide, a 2'-methoxy modified nucleotide, GNA Modified nucleotides or a combination of any two or more thereof. In a preferred embodiment, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups Modified nucleotides, between the 1st and 2nd nucleotides at the 5' end, between the 2nd and 3rd nucleotides at the 5' end, and at the 3' end Between the first nucleotide and the second nucleotide, and between the second nucleotide and the third nucleotide at the 3' end, there is a phosphorothioate group connection; according to the 5' to 3' Orientation, the 2'-fluoro modified nucleotide is located at the even position of the antisense strand, the remaining positions are the 2'-methoxy modified nucleotide, the 1st nucleotide and the 2nd core at the 5' end between nucleotides, between the 2nd and 3rd nucleotides at the 5' end, between the 1st and 2nd nucleotides at the 3' end, and between the 3' end and the 1st nucleotide at the 3' end. There is a phosphorothioate linkage between the 2 nucleotides and the 3rd nucleotide. In a preferred embodiment, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups Modified nucleotides, between the first and second nucleotides at the 5' end, and between the second and third nucleotides at the 5' end are phosphorothioates base connection, and the overhang is removed from the 3'end; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and the remaining positions are 2'- Methoxy-modified nucleotides, between the 1st and 2nd nucleotides at the 5' end, between the 2nd and 3rd nucleotides at the 5' end, 3 There is a phosphorothioate group connection between the first and second nucleotides at the 'end and between the second and third nucleotides at the 3' end. In a preferred embodiment, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups Modified nucleotides, between the 1st and 2nd nucleotides at the 5' end, between the 2nd and 3rd nucleotides at the 5' end, and at the 3' end Between the first nucleotide and the second nucleotide, and between the second nucleotide and the third nucleotide at the 3' end, there is a phosphorothioate group connection; according to the 5' to 3' Orientation, 2'-fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, the first core at the 5' end Between the nucleotide and the second nucleotide, between the second and third nucleotides at the 5' end, between the first and second nucleotides at the 3' end between, the second nucleotide at the 3' end It is connected to the third nucleotide by a phosphorothioate group. In a preferred embodiment, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups Modified nucleotides, between the 1st and 2nd nucleotides at the 5' end, between the 2nd and 3rd nucleotides at the 5' end, and at the 3' end Between the first nucleotide and the second nucleotide, and between the second nucleotide and the third nucleotide at the 3' end, there is a phosphorothioate group connection; according to the 5' to 3' direction, the 2'-fluoro modified nucleotides are located at positions 2, 6, 8, 9, 14 and 16 of the antisense strand, the remaining positions are 2'-methoxy modified nucleotides, and the 5' end Between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides at the 5' end, between the 1st and 2nd nucleotides at the 3' end Between nucleotides, there is a phosphorothioate group connection between the second nucleotide and the third nucleotide at the 3' end. In a preferred embodiment, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups Modified nucleotides, between the 1st and 2nd nucleotides at the 5' end, between the 2nd and 3rd nucleotides at the 5' end, and at the 3' end Between the first nucleotide and the second nucleotide, and between the second nucleotide and the third nucleotide at the 3' end, there is a phosphorothioate group connection; according to the 5' to 3' direction, 2'-fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, GNA modified nucleotides are located at position 7 of the antisense strand, and the remaining positions are 2'-methoxy base-modified nucleotide, between the first and second nucleotides at the 5' end, between the second and third nucleotides at the 5' end, and at the 3' end Between the first and second nucleotides, and between the second and third nucleotides at the 3' end, there is a phosphorothioate group connection. In a preferred embodiment, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups Modified nucleotides, between the 1st and 2nd nucleotides at the 5' end, between the 2nd and 3rd nucleotides at the 5' end, and at the 3' end Between the first nucleotide and the second nucleotide, and between the second nucleotide and the third nucleotide at the 3' end, there is a phosphorothioate group connection; according to the 5' to 3' Orientation, 2'-fluoro modified nucleotides are located at positions 2, 14 and 16 of the antisense strand, GNA modified nucleotides are located at position 6 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, between the first and second nucleotides at the 5' end, between the second and third nucleotides at the 5' end, and between the first and third nucleotides at the 3' end There is a phosphorothioate group connection between the 1st nucleotide and the 2nd nucleotide, and between the 2nd nucleotide and the 3rd nucleotide at the 3' end.

In a specific embodiment, the invention provides siRNA selected from Table 1, wherein the siRNA is not N-ER-FY007001, N-ER-FY007002, N-ER-FY007004, N-ER-FY007006, N- ER-FY007031, N-ER-FY007007, N-ER-FY007011, N-ER-FY007056, N-ER-FY007013, N-ER-FY007014, N-ER-FY007016, N-ER-FY007038, N-ER- FY007039, N-ER-FY007022, N-ER-FY007023, N-ER-FY007027, N-ER-FY007005, N-ER-FY007030, N-ER-FY007051, N-ER-FY007032, N-ER-FY007033, N-ER-FY007034, N-ER-FY007035, N-ER-FY007036, N-ER-FY007015: Preferably, the siRNA is selected from N-ER-FY007001M1, N-ER-FY007004M1, N-ER-FY007006M1, N-ER-FY007011M1, N-ER-FY007033M1, N-ER-FY007034M1, N-ER-FY007020, N-ER-FY007020M1, N-ER-FY007024, N-ER-FY007024M1, N-ER-FY007020M2, N- ER-FY007020M2D2, N-ER-FY007020M3, N-ER-FY007020M4, N-ER-FY007020M5, N-ER-FY007001M2, N-ER-FY007001M2D2, N-ER-FY007001M3, N-ER-FY007001M4 ,N-ER- FY007001M5, N-ER-FY007004M2, N-ER-FY007004M2D2, N-ER-FY007004M3, N-ER-FY007004M4, N-ER-FY007004M5, N-ER-FY007006M2, N-ER-FY007006M2D2, N- ER-FY007006M3、 N-ER-FY007006M4, N-ER-FY007006M5, N-ER-FY007033M2, N-ER-FY007033M2D2, N-ER-FY007033M3, N-ER-FY007033M4, N-ER-FY007033M5, N-ER-FY007034M2, N- N -ER- FY007074M4, N-ER-FY007074M5, N-ER-FY007075, N-ER-FY007075M2, N-ER-FY007075M2D2, N-ER-FY007075M3, N-ER-FY007075M4, N-ER-FY007075M5, N-ER-FY 007078, N-ER-FY007078M2, N-ER-FY007078M2D2, N-ER-FY007078M3, N-ER-FY007078M4, N-ER-FY007078M5, N-ER-FY007079, N-ER-FY007079M2, N-ER-FY007079M2D2, N- ER-FY007079M3, N-ER-FY007079M4, N-ER-FY007079M5, N-ER-FY007080, N-ER-FY007080M2, N-ER-FY007080M2D2, N-ER-FY007080M3, N-ER-FY007080M4, N-ER - FY007080M5, N-ER-FY007082, N-ER-FY007082M2, N-ER-FY007082M2D2, N-ER-FY007082M3, N-ER-FY007082M4, N-ER-FY007082M5, N-ER-FY007083, N-ER-FY00 7083M2, N-ER-FY007083M2D2, N-ER-FY007083M3, N-ER-FY007083M4, N-ER-FY007083M5, N-ER-FY007085, N-ER-FY007085M2, N-ER-FY007085M2D2, N-ER-FY007085M3, N- ER-FY007085M4, N-ER-FY007085M5.

The present invention also provides an siRNA conjugate, which contains the siRNA of the present invention and a conjugation group conjugated to the siRNA (as shown in the figure below, the double helix structure represents the siRNA, and the The conjugating group is attached to the 3′ end of the sense strand of the siRNA):

In the above conjugate structure, X can be selected as O or S. In one embodiment, X is O.

In one embodiment, the conjugation group includes a pharmaceutically acceptable targeting group and a linker, and the siRNA, the linker, and the targeting group are sequentially linked covalently or non-covalently.

Preferably, in the siRNA conjugate, the sense strand and the antisense strand of the siRNA are complementary to form a double-stranded region of the siRNA conjugate, and the 3' end of the sense strand forms a blunt end, and the antisense strand forms a blunt end. The 3' end of the chain has 1-3 protruding nucleotides extending out of the double-stranded region;

or,

In the siRNA conjugate, the sense strand and the antisense strand of siRNA are complementary to form the double-stranded region of the siRNA conjugate, and the 3' end of the sense strand forms a blunt end, and the 3' end of the antisense strand forms a blunt end. 'The ends form blunt ends.

In one embodiment, the conjugating group is L96 of the formula:

In a specific embodiment, the siRNA conjugate is a siRNA conjugate selected from Table 2.

The present invention also provides a pharmaceutical composition, which contains the siRNA of the present invention, or the siRNA conjugate of the present invention, and a pharmaceutically acceptable carrier.

The present invention also provides a kit comprising the siRNA of the present invention, or the siRNA conjugate of the present invention, or the pharmaceutical composition of the present invention.

The present invention also provides the siRNA of the present invention, or the siRNA conjugate of the present invention, or the pharmaceutical composition of the present invention. The compound is used for preparing a medicament for inhibiting HSD17B13 gene expression.

The present invention also provides the use of the siRNA of the present invention, or the siRNA conjugate of the present invention, or the pharmaceutical composition of the present invention for preparing a medicament for preventing and/or treating diseases related to HSD17B13 gene overexpression.

In one embodiment, the disease is selected from non-alcoholic fatty liver disease, cirrhosis, alcoholic hepatitis, liver fibrosis, liver cancer.

The present invention also provides a method for inhibiting HSD17B13 gene expression, which includes contacting a therapeutically effective amount of the siRNA of the present invention, or the siRNA conjugate of the present invention, or the pharmaceutical composition of the present invention with cells expressing HSD17B13 or administering it to a patient in need subjects.

The present invention also provides methods for treating and/or preventing diseases related to HSD17B13 gene overexpression, including administering a therapeutically effective amount of the siRNA of the present invention, or the siRNA conjugate of the present invention, or the pharmaceutical composition of the present invention to those in need. of subjects.

beneficial effects

The siRNA, pharmaceutical composition and siRNA conjugate provided by this application show excellent HSD17B13 gene expression inhibitory activity in in vitro cell experiments, and have good potential to treat diseases related to HSD17B13 gene overexpression. For example, the siRNA and its conjugate disclosed in this application can reduce the expression of HSD17B13 mRNA in the liver, have low toxic and side effects, good plasma stability, and have good clinical application prospects.

The siRNA provided in this application shows good inhibitory effect on HSD17B13 gene in human liver cancer cell Huh7 cells. In some specific embodiments, the siRNA provided in this application has an inhibition rate of up to 89.77% at a concentration of 0.1 nM, and an inhibition rate of up to 76.97% at a concentration of 0.01 nM.

In some specific embodiments, the siRNA provided by the present application has higher HSD17B13 gene inhibitory activity in Huh7 cells, for example, the IC ₅₀ is as low as 12pM.

In some specific embodiments, the siRNA conjugate provided by the present application has higher HSD17B13 gene inhibition activity in PHH cells. For example, when entering PHH cells by free uptake, the IC ₅₀ can be as low as 82 pM; when entering PHH cells by transfection, the IC ₅₀ can be as low as 1.2 pM.

Detailed ways

definition

Throughout the specification, unless otherwise specified, in this technical field, "G", "C", "A", "T" and "U" usually represent guanine, cytosine, adenine and thymine respectively. , the base of uracil, but it is also commonly known in the art that "G", "C", "A", "T" and "U" each usually represent guanine, cytosine, adenine, respectively. Thymine and uracil are nucleotides as bases, which is a common way of expressing DNA sequences and/or ribonucleic acid sequences, so in the context of this disclosure, "G", "C", The meanings represented by "A", "T" and "U" include the various possible situations mentioned above. Lowercase letters a, u, c, g: represent 2'-methoxy modified nucleotides; Af, Gf, Cf, Uf: represent 2'-fluoro modified nucleotides; lowercase letter s represents the same letter as this letter The two adjacent nucleotides to the left and right of s are connected by phosphorothioate groups; P1: indicates that the adjacent nucleotide to the right of P1 is a 5'-phosphate nucleotide; A , U , C , G (Underline + bold + italics): Indicates GNA modified nucleotides.

In the above and below, the "2'-fluoro-modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2' position of the ribosyl group of the nucleotide is replaced by fluorine. “Non-fluorinated modified nucleotides” refers to nucleotides or nucleotide analogs in which the hydroxyl group at the 2’ position of the ribosyl group of the nucleotide is replaced by a non-fluorinated group. In some embodiments, each non-fluorinated modified nucleotide is independently selected from nucleotides or nucleotide analogs formed by replacing the hydroxyl group at the 2' position of the ribose group of the nucleotide with a non-fluorinated group. A sort of. Nucleotides formed by replacing the hydroxyl group at the 2' position of the ribosyl group with a non-fluorine group are well known to those skilled in the art. These nucleotides can be selected from 2'-alkoxy modified nucleotides, 2'- Substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-amino modified nucleotides, 2'-substituted One of the amino-modified nucleotides and 2'-deoxynucleotides.

"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. group. For example, the "C _1-6 " in "C _1-6 alkyl" refers to a linear, branched or cyclic arrangement containing 1, 2, 3, 4, 5 or 6 carbon atoms. group.

"Alkoxy" as used herein means an alkyl group attached to the remainder of the molecule through an oxygen atom (-O-alkyl), wherein said alkyl group is as defined herein. Non-limiting examples of alkoxy include methoxy, ethoxy, trifluoromethoxy, difluoromethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, n- Pentyloxy etc.

"Nucleotide analogue" refers to a nucleotide that can replace a nucleotide in a nucleic acid, but whose structure is different from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide or thymus Pyrimidine deoxyribonucleotide group. Such as isonucleotides, bridged nucleic acid (BNA) or acyclic nucleotides.

BNA refers to constrained or inaccessible nucleotides. BNA may contain a five-membered ring, a six-membered ring, or a seven-membered ring bridged structure with a "fixed"C3'-endoglycocondensation. 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 BNA, etc., where LNA is as shown in formula (1) shown, ENA is shown in formula (2), cET BNA is shown in formula (3).

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

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

Isonucleotides refer to compounds formed by changing the position of the base on the ribose ring in the nucleotide. For example, the base moves from the 1'-position to the 2'-position or 3'-position of the ribose ring. The compound is shown in formula (6) or (7).

In the above-mentioned compounds of formula (6) to formula (7), Base represents a base, such as A, U, G, C or T; R is selected from H, OH, F or the non-fluorine group as mentioned above.

In some embodiments, the nucleotide analog is selected from one of isonucleotides, LNA, ENA, cET BNA, UNA, and GNA. In some embodiments, each non-fluorinated modified nucleotide is a 2'-methoxy modified nucleotide, a GNA modified nucleotide, or a combination of any two or more thereof. In some preferred embodiments, each non-fluoro modified nucleotide is a 2'-methoxy modified nucleotide, above and below, the 2'-methoxy modified nucleoside Acid refers to a nucleotide in which the 2'-hydroxyl group of the ribosyl group is replaced by a methoxy group.

The "2'-methoxy modified nucleotide" refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribose group with a methoxy group. The "phosphorothioate group" refers to a phosphorothioate group in which one oxygen atom in the phosphodiester bond of the phosphate group is replaced by a sulfur atom. The "5'-phosphate nucleotide" refers to the structure of the following formula:

In the context of this specification, the expressions "complementary" and "reverse complementary" are used interchangeably and have the meaning well known to those skilled in the art, that is, in a double-stranded nucleic acid molecule, the bases of one strand are each associated with the other. The bases in the strand are paired in a complementary manner. In DNA, the purine base adenine (A) always pairs with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (C) always pairs with the pyrimidine base Pairs with cytosine (G). Each base pair consists of a purine and a pyrimidine. When on a chain When adenine always pairs with thymine (or uracil) on the other strand, and guanine always pairs with cytosine, the two strands are said to be complementary to each other, and can be inferred from the sequence of their complementary strands out the sequence of the chain. Correspondingly, "mismatch" in this field means that in double-stranded nucleic acids, the bases at corresponding positions do not pair in a complementary manner.

In the above and below, unless otherwise specified, "substantially reverse complementary" means that there are no more than 3 base mismatches between the two nucleotide sequences involved; "substantially reverse complementary" means that there are no more than 3 base mismatches between the two nucleotide sequences involved; " means that there is no more than one base mismatch between the two nucleotide sequences; "complete reverse complementarity" means that there is no base mismatch between the two nucleotide sequences.

In the above and below, the "nucleotide difference" between one nucleotide sequence and another nucleotide sequence means that the base type of the nucleotide at the same position has changed between the former and the latter. For example, when one nucleotide base in the latter is A, and when the corresponding nucleotide base at the same position in the former is U, C, G or T, it is regarded as one of the two nucleotide sequences. There are nucleotide differences at this position. In some embodiments, when the nucleotide at the original position is replaced by an abasic nucleotide or its equivalent, it can also be considered that a nucleotide difference is generated at that position.

In this context, an "overhang" refers to one or more unpaired nucleotides that protrude from the duplex structure of an siRNA when one 3' end of one strand of the siRNA 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 siRNA, ie, no nucleotide overhangs. A "blunt-ended" siRNA is one that is double-stranded throughout its length, ie, it has no nucleotide overhangs at either end of the molecule.

In the description above and below of this application, especially when describing the preparation method of siRNA, pharmaceutical composition or siRNA conjugate of this application, unless otherwise specified, the nucleoside monomer refers to the siRNA or siRNA to be prepared according to Type and sequence of nucleotides in siRNA conjugates, modified or unmodified nucleoside phosphoramidite monomers used in solid-phase phosphoramidite synthesis. Solid-phase phosphoramidite synthesis is a method used in RNA synthesis well known to those skilled in the art. The nucleoside monomers used in this application are all commercially available.

In the context of this application, unless otherwise stated, "conjugate" means that two or more chemical moieties each having a specific function are connected to each other in a covalent manner; accordingly, "conjugate" is Refers to a compound formed by covalent connections between various chemical parts. Further, "siRNA conjugate" refers to a compound formed by covalently linking one or more chemical moieties with specific functions to siRNA. siRNA conjugate should be understood as a collective name for multiple siRNA conjugates or a siRNA conjugate represented by a certain chemical formula, depending on the context. In the context of the present specification, "conjugation molecule" should be understood as a specific compound that can be conjugated to siRNA through a reaction, ultimately forming the siRNA conjugate of the present application.

A variety of hydroxyl protecting groups can be used in this application. Generally speaking, protecting groups sensitize chemical functional groups to specific are insensitive to certain reaction conditions and can be attached to and removed from the functional group in the molecule without substantially damaging the rest of the molecule. In some embodiments, the protecting group is stable under basic conditions but can be removed under acidic conditions. In some embodiments, non-exclusive examples of hydroxyl protecting groups that may be used herein include dimethoxytrityl (DMT), monomethoxytrityl, 9-phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl (Mox). In some embodiments, non-exclusive examples of hydroxyl protecting groups that can be used herein include Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4'-dimethoxy trityl) and TMTr (4,4',4″-trimethoxytrityl).

As used in this specification, "optional" or "optionally" means that the subsequently described event or condition may or may not occur, and that the description includes instances in which the event or condition occurs and instances in which it does not. .

The term "subject", as used in this specification, refers to any animal, such as a mammal or marsupial. Subjects of the present application include, but are not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, rabbits, or any species of poultry.

As used in this specification, "treatment" refers to a method of obtaining beneficial or desired results, including but not limited to therapeutic benefit. "Therapeutic benefit" means eradication or amelioration of the underlying disorder being treated. Furthermore, therapeutic benefit is obtained by eradicating or ameliorating one or more physiological symptoms associated with the underlying disorder, such that improvement is observed in the subject, although the subject may still be suffering from the underlying disorder.

As used in this specification, "prevention" refers to a method of obtaining beneficial or desired results, including but not limited to preventive benefits. To obtain a "prophylactic benefit", a siRNA, siRNA conjugate or pharmaceutical composition may be administered to a subject at risk of developing a particular disease, or to a subject who reports one or more physiological symptoms of the disease, even if possible A diagnosis of the disease has not yet been made.

siRNA

The present application relates to a siRNA capable of inhibiting HSD17B13 gene expression. The siRNA of the present application contains a nucleotide group as a basic structural unit. It is well known to those skilled in the art that the nucleotide group contains a phosphate group, a ribose group and a base. Typically active, ie, functional, siRNAs are about 12-40 nucleotides in length, and in some embodiments are about 15-30 nucleotides in length.

The siRNA of the present application contains a sense strand and an antisense strand. Each nucleotide in the siRNA is independently a modified or unmodified nucleotide, wherein the sense strand contains a nucleotide sequence I, and the The antisense strand contains a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region. In some embodiments, the double-stranded region is 15-30 nucleotide pairs in length. In other embodiments, the double-stranded region is 17-23 nucleotide pairs in length. In other embodiments, the double-stranded region is 19-21 nucleotide pairs in length. In yet other embodiments, the double-stranded region is 19 or 21 nucleotide pairs in length.

In some embodiments, the sense strand further contains nucleotide sequence III, and the antisense strand further contains nucleotide sequence III The lengths of sequence IV, nucleotide sequence III and nucleotide sequence IV are each independently 0-6 nucleotides, and the nucleotide sequence III is connected to the 5′ end of nucleotide sequence I, nucleotide sequence Sequence IV is connected to the 3′ end of nucleotide sequence II, and said nucleotide sequence III and said nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary; said substantially reverse complement It means that there is no more than one base mismatch between the two nucleotide sequences; perfect reverse complementarity means that there is no mismatch between the two nucleotide sequences. In some embodiments, the sense strand further contains nucleotide sequence III, the antisense strand further contains nucleotide sequence IV, and the lengths of nucleotide sequence III and nucleotide sequence IV are each independently 0- 6 nucleotides, the nucleotide sequence III is connected to the 3′ end of the nucleotide sequence I, the nucleotide sequence IV is connected to the 5′ end of the nucleotide sequence II, the nucleotide sequence III and The nucleotide sequences IV are equal in length and are substantially reverse complementary or completely reverse complementary; the substantially reverse complementarity means that there is no more than 1 base mismatch between the two nucleotide sequences; Perfect reverse complementarity means there are no mismatches between the two nucleotide sequences. In some embodiments, the sense strand further contains nucleotide sequence III, the antisense strand further contains nucleotide sequence IV, and the lengths of nucleotide sequence III and nucleotide sequence IV are each independently 0- 6 nucleotides, the nucleotide sequence III is connected to the 5′ end of the nucleotide sequence I, the nucleotide sequence IV is connected to the 3′ end of the nucleotide sequence II, the nucleotide sequence III and The nucleotide sequence IV is equal in length and substantially reverse complementary or completely reverse complementary; and the nucleotide sequence III is connected to the 3′ end of the nucleotide sequence I, and the nucleotide sequence IV is connected to the nucleoside The 5′ end of the acid sequence II, the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary; the substantially reverse complementarity refers to two nucleosides There is no more than 1 base mismatch between the acid sequences; perfect reverse complementarity means there are no mismatches between the two nucleotide sequences.

In some embodiments, the sense strand further contains the nucleotide sequence V and/or the antisense strand further contains the nucleotide sequence VI, the nucleotide sequences V and VI being 0 to 3 nucleotides in length , the nucleotide sequence V is connected to the 3' end of the sense strand to form the 3' overhang of the sense strand, and/or the nucleotide sequence VI is connected to the 3' end of the antisense strand to form the 3' end of the antisense strand. protruding end. In some embodiments, the nucleotide sequence V or VI is 2 nucleotides in length. In other embodiments, the nucleotide sequence V or VI is two consecutive thymine deoxyribonucleotides or two consecutive uracil ribonucleotides. In other embodiments, the nucleotide sequence V or VI mismatches or is complementary to the nucleotide at the corresponding position of the target mRNA.

The lengths of the sense strand and the antisense strand provided by the application are the same or different. In some embodiments, the sense strand or the antisense strand has 15-30 nucleotides. In other embodiments, the sense or antisense strand has 19-25 nucleotides. In other embodiments, the sense or antisense strand has 19-23 nucleotides. The length ratio of the siRNA sense strand and the antisense strand provided in this application can be 15/15, 16/16, 17/17, 18/18, 19/19, 19/20, 19/21, 19/22, 19/ 23, 20/19, 20/20, 20/21, 20/22, 20/23, 21/19, 21/20, 21/21, 21/22, 21/23, 22/19, 22/20, 22/21, 22/22, 22/23, 23/19, 23/20, 23/21, 23/22, 23/23, 24/24, 25/25, 26/26, 27/27, 28/ 28, 29/29, 30/30, 22/24, 22/25, 22/26, 23/24, 23/25 or 23/26 etc. In some embodiments, the length ratio of the siRNA sense strand and antisense strand is 19/19, 21/21, 19/21, 21/23 or 23/23. In this case, the siRNA of the present disclosure has better Cellular mRNA silencing activity.

Studies have found that different modification strategies will have completely different effects on the stability, biological activity, cytotoxicity and other indicators of siRNA. For example, CN102140458B studied various chemical modification strategies of siRNA and confirmed 7 effective modification methods. Compared with unmodified siRNA, the siRNA obtained by one of the modification methods while improving blood stability, It also maintained inhibitory activity that was essentially equivalent to that of unmodified siRNA.

Each nucleotide in the siRNA of the present invention is independently a modified or unmodified nucleotide. In some embodiments, each nucleotide in the siRNA of the present invention is an unmodified nucleotide; in some embodiments, some or all of the nucleotides in the siRNA of the present invention are modified nucleosides. These modifications on the acid and nucleotide groups will not cause the siRNA of the present invention to significantly weaken or lose the function of inhibiting HSD17B13 gene expression.

In some embodiments, the siRNA of the present application contains at least 1 modified nucleotide. In the context of this application, the term "modified nucleotide" is used to refer to a nucleotide or nucleotide analogue in which the 2' hydroxyl group of the ribosyl group of a nucleotide is replaced by another group, or has a modified Modified bases of nucleotides. The modified nucleotides will not cause significant weakening or loss of the function of siRNA to inhibit gene expression. For example, modified nucleotides disclosed in J.K. Watts, G.F. Deleavey, and M.J. Damha, Chemically modified siRNA: tools and applications. Drug Discov Today, 2008, 13(19-20): 842-55, can be selected.

In some embodiments, at least one nucleotide in the sense strand or the antisense strand of the siRNA provided by the present invention is a modified nucleotide, and/or at least one phosphate group has a modified group. Phosphate group; in other words, at least part of the phosphate group and/or ribose group in the phosphate-sugar backbone of at least one single chain in the sense strand and the antisense strand is a phosphate group with a modifying group and/or ribosyl groups with modifying groups. In some embodiments, the phosphate group containing a modifying group is a phosphorothioate group formed by replacing at least one oxygen atom in the phosphodiester bond of the phosphate group with a sulfur atom.

In some embodiments, the siRNA includes a sense strand that does not include a 3' overhanging nucleotide; that is, the sense strand of the siRNA may have a 3' overhanging nucleotide, excluding the 3' overhanging nucleotide of the sense strand. Then a flat end is formed.

In some embodiments, when the nucleotide sequences of the sense strand and the antisense strand are complementary to form a double-stranded region, and there is no overhanging nucleotide at the 3' end of the sense strand, nucleosides are added to the 3' end of the sense strand. Acid sequence V, as overhanging nucleotide. Then, when the nucleotide sequence V is connected to the 3' end of the sense strand, the nucleotide sequence formed is chemically modified to exclude the nucleotide sequence V. Correspondingly, the sense strand of siRNA forms a blunt end.

In some embodiments, when the nucleotide sequences of the sense strand and the antisense strand are complementary to form a double-stranded region, and the 3' end of the sense strand has a protruding nucleotide extending out of the double-stranded region, the sense strand is located at The protruding nucleotide at the 3' end is excluded as the nucleotide sequence of the sense strand, and accordingly, the sense strand of siRNA forms a blunt end.

In some embodiments, the 5' terminal nucleotide of the sense strand is linked to a 5' phosphate group or a 5' phosphate derivative group. In some embodiments, the 5' terminal nucleotide of the antisense strand is linked to a 5' phosphate group or a 5' phosphate derivative group. An exemplary 5' phosphate group structure is: The structures of the 5' phosphate derivative group include but are not limited to: wait.

After the 5' terminal nucleotide located in the sense strand or antisense strand is connected to the 5' phosphate group or 5' phosphate derivative group, the following structure is formed:

Among them, Base represents a base, such as A, U, G, C or T. R' is hydroxyl or 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'-amino modified nucleotides, 2 '-Substituted amino-modified nucleotide, 2'-deoxynucleotide.

Exemplary modified nucleotides have the structure shown below:

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

In some embodiments, the 2'-alkoxy modified nucleotide is a 2'-methoxy (2'OMe, 2'-O- _CH3 ) 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 nucleoside Acid, 2'-O-CH ₂ -CH=CH ₂ modified nucleotide, etc.

In some embodiments, the 2'-substituted alkyl modified nucleotide is a 2'- _CH2- CH2 _- CH= _CH2 modified nucleotide, and the like.

In some embodiments, all nucleotides in the sense strand and/or the antisense strand are modified nucleosides acid. In some embodiments, each nucleotide in the sense strand and the antisense strand of the siRNA provided by the invention is independently a 2'-fluorinated modified nucleotide or a non-fluorinated modified nucleotide. In some embodiments, each non-fluoro modified nucleotide is a 2'-methoxy modified nucleotide or a GNA modified nucleotide, the 2'-methoxy modified nucleotide being A nucleotide formed by replacing the 2'-hydroxyl group of the ribose group with a methoxy group.

In some embodiments, the 2'-fluoromodified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand in a 5' to 3' direction, and the remaining positions are non-fluoromodified nucleotides ; According to the 5' to 3' direction, the 2'-fluorinated modified nucleotides are located at the even-numbered positions of the antisense strand, and the remaining positions are non-fluorinated modified nucleotides. In some embodiments, the 2'-fluoromodified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand in a 5' to 3' direction, and the remaining positions are non-fluoromodified nucleotides ; According to the 5' to 3' direction, the 2'-fluorinated modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and the remaining positions are non-fluorinated modified nucleotides. In some embodiments, the 2'-fluoromodified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand in a 5' to 3' direction, and the remaining positions are non-fluoromodified nucleotides ; According to the 5' to 3' direction, the 2'-fluorinated modified nucleotides are located at positions 2, 6, 8, 9, 14 and 16 of the antisense strand, and the remaining positions are non-fluorinated modified nucleotides . In a preferred embodiment, the 2'-fluorinated modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are non-fluorinated modified cores According to the 5' to 3' direction, the 2'-fluorinated modified nucleotides are located at positions 2, 14 and 16 of the antisense strand, and the remaining positions are non-fluorinated modified nucleotides. In some preferred embodiments, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups Modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at the even-numbered positions of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides. In some preferred embodiments, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups Modified nucleotides; in the 5' to 3' direction, 2'-fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and the remaining positions are 2'-methoxy modified of nucleotides. In some preferred embodiments, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups Modified nucleotides; in the 5' to 3' direction, 2'-fluoro modified nucleotides are located at positions 2, 6, 8, 9, 14 and 16 of the antisense strand, and the remaining positions are 2'- Methoxy modified nucleotides. In some preferred embodiments, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups Modified nucleotides; in the 5' to 3' direction, 2'-fluoro modified nucleotides are located at positions 2, 14 and 16 of the antisense strand, and GNA modified nucleotides are located at position 1 of the antisense strand. Position 6, and the remaining positions are 2'-methoxy modified nucleotides. In some preferred embodiments, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups Modified nucleotides; in the 5' to 3' direction, 2'-fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and GNA modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand. Position 7, and the remaining positions are 2'-methoxy modified nucleotides. In some more preferred embodiments, each non-fluorinated modification The nucleotides are all 2'-methoxy modified nucleotides. The 2'-methoxy modified nucleotides refer to nucleotides formed by replacing the 2'-hydroxyl group of the ribose group with a methoxy group. .

In some embodiments, at least one of the linkages between the following nucleotides in the siRNA is a phosphorothioate linkage:

In some embodiments, the siRNA is directed from the 5' end to the 3' end, and the sense strand contains a phosphorothioate group at a position as shown below:

or,

The sense strand contains phosphorothioate groups at the positions shown below:

In some embodiments, the siRNA is directed from the 5' end to the 3' end, and the antisense strand contains a phosphorothioate group at a position as shown below:

siRNA conjugates

The present application relates to a siRNA conjugate, which contains the above-mentioned siRNA and a conjugation group conjugated to the siRNA.

In the present application, the sense strand and the antisense strand of the siRNA conjugate form the double-stranded region of the siRNA conjugate, and a blunt end is formed at the 3' end of the sense strand of the siRNA conjugate. In some embodiments, the 3' end of the sense strand of the siRNA conjugate forms a blunt end and the 3' end of the antisense strand of the siRNA conjugate has 1-3 protruding nucleosides extending out of the double-stranded region acid. In other embodiments, the 3' end of the sense strand of the siRNA conjugate is blunt-ended, and the 3' end of the antisense strand of the siRNA conjugate is blunt-ended.

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

In some preferred embodiments, the 3' end of the sense strand of siRNA has a protruding nucleotide extending out of the double-stranded region, and the protruding nucleotide located at the 3' end of the sense strand is excluded to form a structure with 3' The blunt-ended sequence serves as the nucleotide sequence for connecting the conjugation group, and the conjugation group is connected to the 3' blunt end of the sense strand to form an siRNA conjugate.

In some more preferred embodiments, when the nucleotide sequences of the sense strand and the antisense strand are complementary to form a double-stranded region, and there is no overhanging nucleotide at the 3' end of the sense strand, add Nucleotide sequence V, as overhanging nucleotide. The sequence with a 3' blunt end formed by excluding the protruding nucleotide at the 3' end of the sense strand is used as the nucleotide sequence for connecting the conjugation group, and the conjugation is connected to the 3' blunt end of the sense strand. groups to form siRNA conjugates.

In some more preferred embodiments, when the nucleotide sequences of the sense strand and the antisense strand are complementary to form a double-stranded region, and the 3' end of the sense strand has a protruding nucleotide that extends out of the double-stranded region, it will be located in the sense strand. The sequence with a 3' blunt end formed after excluding the protruding nucleotides at the 3' end of the chain is used as the nucleotide sequence for connecting the conjugation group, and the conjugation group is connected to the 3' blunt end of the sense strand. Formation of siRNA conjugates.

Exemplarily, for a siRNA with a sequence such as N-ER-FY007006M1, the 3' end of the sense strand of the siRNA has a protruding nucleotide extending out of the double-stranded region, and the protruding - located at the 3' end of the sense strand will be The gsascuacUfuAfUfGfaauuugca blunt-end sequence formed after sTsT nucleotide exclusion serves as the nucleotide sequence used to connect the L96 conjugation group. Therefore, the sequence to form the siRNA conjugate is: the sense strand is gsascuacUfuAfUfGfaauuugcaL96, and the antisense strand is PlusGfscAfaAfuUfcAfuAfaGfuAfgUfcsTsT.

Generally speaking, the conjugation group includes at least one pharmaceutically acceptable targeting group, or further includes a linker, and the siRNA, the linker and the targeting group are connected in sequence . In some embodiments, the number of targeting groups is 1-6. In some embodiments, the number of targeting groups is 2-4. The siRNA molecule may be non-covalently or covalently conjugated to the conjugation group, eg, may be covalently conjugated to the conjugation group. The conjugation site of siRNA and the conjugation group can be at the 3′ end or 5′ end of the siRNA sense strand, or it can At the 5' end of the antisense strand, it can also be within the internal sequence of the siRNA. In some embodiments, the conjugation site of the siRNA and the conjugation group is at the 3′ end of the sense strand of the siRNA.

In some embodiments, the conjugation group can be attached to the phosphate group, the 2'-hydroxyl group, or the base of the nucleotide. In some embodiments, the conjugation group can also be connected to the 3'-position hydroxyl group, in which case the nucleotides are connected via a 2'-5' phosphodiester bond. When the conjugation group is attached to the end of the siRNA chain, the conjugation group is usually attached to the phosphate group of the nucleotide; when the conjugation group is attached to the internal sequence of the siRNA, the conjugation group Usually attached to the ribose sugar ring or base. For various connection methods, please refer to 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 some embodiments, the siRNA and the conjugation group can be connected through acid-labile or reducible chemical bonds. In the acidic environment of cellular endosomes, these chemical bonds can be degraded, thereby leaving the siRNA in a free state. For non-degradable conjugation methods, the conjugation group can be connected to the sense strand of siRNA to minimize the impact of conjugation on siRNA activity.

In some embodiments, the pharmaceutically acceptable targeting group can be a ligand commonly used in the field of siRNA delivery, such as various ligands described in WO2009082607A2, which is fully incorporated into this specification by reference.

In some embodiments, the pharmaceutically acceptable targeting group can be selected from one or more ligands formed by the following targeting molecules or derivatives thereof: lipophilic molecules, such as cholesterol, bile acids, Vitamins (such as vitamin E), lipid molecules of different chain lengths; polymers, such as polyethylene glycol; polypeptides, such as membrane-penetrating peptides; aptamers; antibodies; quantum dots; sugars, such as lactose, polylactose, and mannose Sugar, galactose, N-acetylgalactosamine (GalNAc); folate; receptor ligands expressed by liver parenchymal cells, such as asialoglycoprotein, asialoglycoside residues, lipoproteins (such as high-density Lipoproteins, low-density lipoproteins, etc.), glucagon, neurotransmitters (such as epinephrine), growth factors, transferrin, etc.

In some embodiments, each ligand is independently selected from a ligand capable of binding to a cell surface receptor. In some embodiments, at least one ligand is a ligand capable of binding to a hepatocyte surface receptor. In some embodiments, at least one ligand is a ligand capable of binding to a mammalian cell surface receptor. In some embodiments, at least one ligand is a ligand capable of binding to a human hepatocyte surface receptor. In some embodiments, at least one ligand is a ligand capable of binding to liver surface asialoglycoprotein receptor (ASGPR). The types of these ligands are well known to those skilled in the art. Their function is generally to bind to specific receptors on the surface of target cells and mediate the delivery of siRNA linked to the ligands to the target cells.

In some embodiments, the pharmaceutically acceptable targeting group can be associated with the mammalian hepatocyte surface Any ligand that binds to the asialoglycoprotein receptor (ASGPR). In some embodiments, each ligand is independently an asialoglycoprotein, such as asialoorosomucoid (ASOR) or asialofetuin (ASF). In some embodiments, the ligand is a sugar or sugar derivative.

In some embodiments, at least one ligand is a sugar. In some embodiments, each ligand is a sugar. In some embodiments, at least one ligand is a monosaccharide, a polysaccharide, a modified monosaccharide, a modified polysaccharide, or a sugar derivative. In some embodiments, at least one of the ligands can be a monosaccharide, a disaccharide, or a trisaccharide. In some embodiments, at least one ligand is a modified sugar. In some embodiments, each ligand is a modified sugar. In some embodiments, each ligand is independently selected from the group consisting of polysaccharides, modified polysaccharides, monosaccharides, modified monosaccharides, polysaccharide derivatives, or monosaccharide derivatives. In some embodiments, each or at least one ligand is selected from the group consisting of glucose and its derivatives, mannan and its derivatives, galactose and its derivatives, xylose and its derivatives substances, ribose and its derivatives, fucose and its derivatives, lactose and its derivatives, maltose and its derivatives, arabinose and its derivatives, fructose and its derivatives and sialic acid.

In some embodiments, each of the ligands may be independently selected from the group consisting of D-mannopyranose, L-mannopyranose, D-arabinose, D-xylfuranose, L-xylfuranose, D- Glucose, L-glucose, D-galactose, L-galactose, α-D-mannofuranose, β-D-mannofuranose, α-D-mannopyranose, β-D-mannopyranose, α-D-glucopyranose, β-D-glucopyranose, α-D-glucofuranose, β-D-glucofuranose, α-D-fructofuranose, α-D-fructopyranose, α-D-pyranose Galactopyranose, β-D-galactopyranose, α-D-galactofuranose, β-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-tris Fluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine, N-isobutyrylgalactosamine, 2-amino-3-O-[(R)-1-carboxyethyl ]-2-Deoxy-β-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-carboxamido-2,3-di-O -Methyl-D-mannopyranose, 2-deoxy-2-sulfonamido-D-glucopyranose, N-glycoloyl-α-neuraminic acid, 5-thio-β-D-glucopyranose, 2,3,4-Tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside methyl ester, 4-thio-β-D-pyran hemi- Lactose, 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucoheptopyranoside ethyl ester, 2,5-anhydro-D-a Rotonitrile, ribose, D-ribose, D-4-thioribose, L-ribose or L-4-thioribose. Other options for the ligand can be found, for example, in the description of CN105378082A, which is fully incorporated into this specification by reference.

In some embodiments, the pharmaceutically acceptable targeting group in the siRNA conjugate can be galactose or N-acetylgalactosamine, wherein the galactose or N-acetylgalactosamine molecule can be a monovalent , divalent, trivalent, quadrivalent. It should be understood that the monovalent, bivalent, trivalent, and tetravalent terms described here respectively refer to the siRNA conjugate formed by a siRNA molecule and a conjugation group containing a galactose or N-acetylgalactosamine molecule as a targeting group. After conjugation, the molar ratio of siRNA molecules to galactose or N-acetylgalactosamine molecules in the siRNA conjugate is 1:1, 1:2, 1:3 or 1:4. In some embodiments, the pharmaceutically acceptable targeting group is N-acetylgalactosamine. In a In some embodiments, when the siRNA described herein is conjugated to a conjugation group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent or tetravalent. In some embodiments, when an siRNA described herein is conjugated to an N-acetylgalactosamine-containing conjugation group, the N-acetylgalactosamine molecule is trivalent.

The targeting group can be connected to the siRNA molecule via a suitable linker, and those skilled in the art can select a suitable linker according to the specific type of the targeting group. The types of these linkers, targeting groups and the connection methods with siRNA can be found in the disclosure of WO2015006740A2, which is fully incorporated into this specification by reference.

siRNA synthesis method

The nucleoside monomers are connected one by one from the 3'-5' direction according to the order of nucleotide arrangement through the conventional solid-phase phosphoramidite method in this field. Each connection of a nucleoside monomer involves four steps of deprotection, coupling, capping, oxidation or sulfation. Among them, when two nucleotides are connected using a phosphate ester, when the next nucleoside monomer is connected, it includes four steps of deprotection, coupling, capping, and oxidation. When two nucleotides are connected using phosphorothioate, when the next nucleoside monomer is connected, it includes four steps of protection, coupling, capping and sulfation.

For example, the synthesis conditions of siRNA of the present application can be as follows:

The conditions for deprotection include: reaction temperature is 25°C, reaction time is 70 seconds, deprotection reagent is selected from dichloroacetic acid in dichloromethane solution (3% V/V), deprotection reagent and protective group on solid phase carrier The molar ratio is 5:1.

The coupling reaction conditions include: the reaction temperature is 25°C, the reaction time is 600 seconds, the coupling reagent is selected from a 0.25M acetonitrile solution of 5-ethylthio-1H-tetrazole (ETT), and the nucleic acid connected to the solid-phase carrier The molar ratio of sequence to nucleoside monomer is 1:10.

The capping reaction conditions include: the reaction temperature is 25°C, the reaction time is 15 seconds, and the capping reagent is selected from CapA (10% acetic anhydride acetonitrile solution) and CapB (10% N-methylimidazole pyridine/ Acetonitrile solution), the molar ratio of the capping reagent to the nucleic acid sequence connected to the solid phase carrier is acetic anhydride:N-methylimidazole:the molar ratio of the nucleic acid sequence connected to the solid phase carrier is 1:1:1.

The oxidation reaction conditions include: the reaction temperature is 25°C, the reaction time is 15 seconds, the oxidation reagent is selected from 0.05M iodine tetrahydrofuran solution, and the molar ratio of the oxidation reagent to the nucleic acid sequence connected to the solid-phase carrier in the coupling step is 30:1.

The sulfidation reaction conditions include: the reaction temperature is 25°C, the reaction time is 300 seconds, the sulfide reagent is selected from hydrogenated xanthogen, and the molar ratio of the sulfide reagent to the nucleic acid sequence connected to the solid-phase carrier in the coupling step is 120:1.

After all the nucleoside monomers are connected, the nucleic acid sequences connected to the solid phase carrier are cut, deprotected, purified, and desalted in sequence to obtain the siRNA sense strand and antisense strand. Finally, the two strands are heated and annealed to obtain the product.

Methods of cleavage, deprotection, purification, desalting and annealing are well known in the art. For example, cleavage and deprotection are carried out by contacting the nucleotide sequence connected to the solid-phase carrier with concentrated ammonia water; purification by chromatography; desalting by reversed-phase chromatography; by mixing the sense strand and the sense strand in equimolar ratios under different stringent conditions. gradually decreases after the antisense strand Cool down.

siRNA conjugate synthesis method

In the first step, compound L96-A is obtained by reacting DMTr-L96 and succinic anhydride:

Preparation process: Add DMTr-L96, succinic anhydride, 4-dimethylaminopyridine and diisopropylethylamine into dichloromethane, stir and react at 25°C for 24 hours, and then wash with 0.5M triethylamine phosphate. The aqueous phase of the reaction solution was washed three times with dichloromethane, and the organic phases were combined and evaporated to dryness under reduced pressure to obtain the crude product; then the pure product L96-A was purified by column chromatography.

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

Preparation process: Mix L96-A, O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU) and diisopropylethylamine (DIPEA) in acetonitrile, stir at room temperature for 5 minutes to obtain a uniform solution , add aminomethyl resin (NH ₂ -SPS, 100-200 mesh) to the reaction liquid, start the shaking reaction at 25°C, filter after 18 hours of reaction, and wash the filter cake with dichloromethane and acetonitrile in sequence to obtain the filter cake . The obtained filter cake is capped with a CapA/CapB mixed solution to obtain L96-B, which is a solid-phase carrier containing the conjugated molecule. Then, the nucleoside monomer is connected to the conjugated molecule under the coupling reaction, and then the nucleoside monomer is connected to the conjugated molecule as described above. The siRNA molecule synthesis method is used to synthesize the siRNA sense strand connected to the conjugate molecule, and the siRNA molecule synthesis method described above is used to synthesize the siRNA antisense strand, and annealed to generate the siRNA conjugate of the present application.

pharmaceutical composition

The present application provides a pharmaceutical composition containing the siRNA as described above as an active ingredient and a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carrier can be a carrier commonly used in the field of siRNA administration, such as but not limited to lipid nanoparticles (Lipid Nanoparticles, LNPs), magnetic nanoparticles (magnetic nanoparticles, such as those based on Fe ₃ O ₄ or Fe ₂ O ₃ nanoparticles), carbon nanotubes, mesoporous silicon silicon), calcium phosphate nanoparticles (calcium phosphate nanoparticles), polyethylenimine (PEI), polyamide dendrimer (polyamidoamine (PAMAM) dendrimer), polylysine (poly(L-lysine), PLL), chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), polyD-type or L-type lactic acid/glycolic acid copolymer Poly(D&L-lactic/glycolic acid)copolymer, PLGA), poly(2-aminoethyl ethylene phosphate), PPEEA, and poly(methacrylic acid-N,N-di Methylaminoethyl ester) (poly(2-dimethylaminoethyl methacrylate), PDMAEMA) and one or more of their derivatives.

In the pharmaceutical composition, there are no special requirements on the contents of siRNA and pharmaceutically acceptable carriers, and they can be the conventional contents of each component.

In some embodiments, the pharmaceutical composition may also contain other pharmaceutically acceptable auxiliary materials, which may be one or more of various preparations or compounds commonly used in the art. For example, the other pharmaceutically acceptable excipients may include at least one of a pH buffer, a protective agent, and an osmotic pressure regulator.

The pH buffer can be a trishydroxymethylaminomethane hydrochloride buffer with a pH of 7.5-8.5 and/or a phosphate buffer with a pH of 5.5-8.5, for example, it can be a phosphate with a pH of 5.5-8.5. Buffer.

The protective agent may be at least one of myo-inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose and glucose. Based on the total weight of the pharmaceutical composition, the content of the protective agent may be 0.01-30% by weight.

The osmotic pressure regulator may be sodium chloride and/or potassium chloride. The content of the osmotic pressure regulator is such that the osmotic pressure of the pharmaceutical composition is 200-700 milliosmole/kg (mOsm/kg). The content of the osmotic pressure regulator can be easily determined by those skilled in the art based on the desired osmotic pressure.

In some embodiments, the pharmaceutical composition can be a liquid preparation, such as an injection; it can also be a freeze-dried powder injection, which is mixed with liquid excipients during administration to prepare a liquid preparation. The liquid preparation may be, but is not limited to, administered by subcutaneous, intramuscular or intravenous injection, may be administered to the lungs by spray, or may be administered to other organs and tissues (such as the liver) through the lungs by spray. In some embodiments, the pharmaceutical composition is for intravenous administration.

In some embodiments, the pharmaceutical composition may be in the form of a liposome formulation. In some embodiments, the pharmaceutically acceptable carrier used in the liposome formulation includes an amine-containing transfection compound (hereinafter also referred to as an organic amine), a helper lipid, and/or a pegylated Lipids.

The following examples are used to further illustrate the present invention, but do not limit the present invention in any way.

Example

Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It is to be understood, however, that the detailed description and specific examples, while indicating specific embodiments of the disclosure, are given for explanatory purposes only because, after reading the detailed description, reasonable explanations will be made within the spirit and scope of the disclosure. Various changes and modifications 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 without specifying specific conditions in the following examples usually follow conventional conditions, such as Sambrook et al., Molecular Cloning: Experiment The conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. The materials, reagents, etc. used in the examples can be obtained through regular commercial channels unless otherwise specified.

Example 1 Preparation of siRNA

The siRNA molecule with the following sequence was synthesized by Tianlin Biotechnology (Shanghai) Co., Ltd.

Table 1 siRNA and its sequence

Among them, the capital letters "G", "C", "A", "T" and "U" each usually represent nucleotides containing guanine, cytosine, adenine, thymine and uracil as bases respectively. ; Lowercase letters a, u, c, g represent: 2'-methoxy modified nucleotides; Af, Gf, Cf, Uf represent: 2'-fluoro modified nucleotides; lowercase letters The letter s indicates that the two nucleotides adjacent to the left and right of the letter s are connected by phosphorothioate groups; P1: indicates that the nucleotide adjacent to the right of P1 is a 5'-phosphate nucleotide; A , U , C , G (underline + bold + italics): indicates GNA modified nucleotides.

The siRNA conjugate with the following sequence was synthesized by Tianlin Biotechnology (Shanghai) Co., Ltd.:

Table 2 siRNA conjugates and their sequences:

Among them, L96 is:

Example 2 siRNA inhibits HSD17B13 gene expression

The inhibitory activity of the siRNA of the present invention on HSD17B13 gene expression was evaluated through dual luciferase reporter gene vectors.

Main experimental reagents:

Experimental steps:

Day 0: Transfer psiCHECK2-HSD17B13 plasmid into Huh7 cells

Dilute the psiCHECK2-HSD17B13 plasmid to 10ng/μl with Opti-MEM. Take Huh7 cells, wash them with DPBS first, add trypsin for digestion, and adjust the cell density to 1×10 ⁵ cells/ml. Mix according to the ratio of Fugene-HD transfection reagent: 10ng/μl psiCHECK2-HSD17B13 dilution = 3:100 (volume ratio). After mixing, incubate at room temperature for 10 minutes. After the incubation, add it to Huh7 cells, and then A density of 10,000 cells per well was seeded into a 96-well plate, with 100 μl of culture medium per well. Huh7 cells were cultured overnight in a 5% CO ₂ and 37°C incubator.

Day 1: Treatment of siRNA Compounds

Will Mix RNAiMAX transfection reagent and Opti-MEM at a ratio of 1.5:48.5 (volume ratio) to obtain Mixture A. Incubate at room temperature for 15 minutes. Add the siRNA compound to be tested (final concentrations are 0.1nM and 0.01nM respectively) and the above Mixture A. Mix 1:1 and incubate at room temperature for 15 minutes. After incubation, add 20 μl in a ratio of 1:5. The resulting mixture was added to 100 μl of fresh Opti-MEM medium and mixed evenly to obtain Mixed Liquid B. Discard the Huh7 cell supernatant, add 120 μl/well of the above mixture B to the 96-well plate, and place the 96-well plate in a CO ₂ cell culture incubator for 48 hours.

Day 2: Testing the reporter gene

Observe the cell status under a microscope, discard the cell supernatant, and add 75 μl to each well. Add luciferase reagent and 75 μl of fresh 10% FBS-DMEM culture medium, and shake in a plate shaker for 10 minutes in the dark. Take 100 μl of the above sample and transfer it to a 96-well all-white detection plate, and detect the luminescence value of firefly luciferase (Firefly lum) under a multifunctional microplate reader. Then add 50 μl to each well Reagent, shake plate machine in the dark for 10 minutes, and detect the luminescence value of Renilla luciferase (Renilla lum).

A control group was set up in the experiment, and RNA-free H ₂ O was used instead of the above-mentioned siRNA compounds. The other conditions were the same as the experimental group; the blank group was Huh7 cells that were not transfected with psiCHECK2-HSD17B13 plasmid, and no siRNA compounds were added to them.

data processing:

The ratio of the fluorescence value of Renilla luciferase to the fluorescence value of firefly luciferase is recorded as: α, and the formula is:

α=(Renilla lum average in the test hole-Renilla lum average in the blank group)/(Firefly lum average in the test hole-Firefly lum average in the blank group);

Calculated according to the above ratio formula, the ratio of the experimental group is recorded as: α (experimental group), and the ratio of the control group is recorded as: α (control group).

The inhibition rate of siRNA inhibiting the expression of the target gene HSD17B13 is calculated according to the following formula:

Inhibition rate (%) = [1-α (average value of experimental group)/α (average value of control group)] × 100%

Table 3 Inhibition rate of siRNA of the present invention

Table 4 Inhibition rate of control siRNA

It can be seen from Table 3 and Table 4 that the siRNA of the present invention can significantly inhibit the expression of the HSD17B13 gene at both 0.1 nM and 0.01 nM.

Example 3 IC ₅₀ determination of siRNA inhibiting HSD17B13 gene expression

The following final concentrations of siRNA to be tested were 10 nM, 2.5 nM, 0.63 nM, 0.16 nM, 0.04 nM, 0.01 nM, 0.0024 nM and 0.0006 nM, and then the IC ₅₀ was measured in a similar manner to Example 2.

Result analysis:

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

b) Use the following formula to calculate the relative expression of genes:

ΔCt=Ct(HSD17B13 gene)-Ct(GAPDH)

ΔΔCt=ΔCt (test sample group)-ΔCt (Mock group), where the Mock group represents the group in which siRNA is not added compared with the experimental group;

Relative to the mRNA expression of the Mock group = 2 ^-ΔΔCt

Inhibition rate (%) = (Relative expression of mRNA in Mock group - Relative expression of mRNA in sample group)/Relative expression of mRNA in Mock group × 100%

Taking the log value of siRNA concentration as the X-axis and the percentage inhibition rate as the Y-axis, use the "log (inhibitor) vs. response-variable slope" function module of the analysis software GraphPad Prism 8 to fit the dose-effect curve, thus obtaining _IC50 values for individual siRNAs.

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 percent inhibition curve.

The results are shown in Table 5.

Table 5 IC ₅₀ (nM) of siRNA

As can be seen from Table 5, the siRNA provided in this application has high HSD17B13 gene inhibitory activity in Huh7 cells, and the IC ₅₀ can be as low as 12pM.

Example 4 IC ₅₀ determination of siRNA conjugate inhibiting HSD17B13 gene expression

Material:

Human primary hepatocyte PHH cells were provided by WuXi AppTec;

PHH medium: invitroGRO CP Meduim serum free BIOVIT, Cat. No.: S03316

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

RNA extraction kit 96 Kit, item number: QIAGEN-74182;

Reverse transcription kit FastKing RT Kit (With gDNase), product number: Tiangen-KR116-02;

FastStart Universal Probe Mast(Roche-04914058001);

TaqMan Gene Expression Assay (GAPDH, Thermo, Assay ID-Hs02786624_g1;

TaqMan Gene Expression Assay (HSD17B13, Thermo, Assay ID-Hs01068199_m1).

(1) siRNA conjugates (the final concentrations of siRNA conjugates are 10nM, 2.5nM, 0.63nM, 0.16nM, 0.04nM, 0.01nM, 0.0024nM and 0.0006nM, respectively) into PHH cells through transfection, process As follows: take frozen PHH cells, resuscitate, count, adjust the cells to 6 × 10 ⁵ cells/ml, and apply Lipofectamine RNAiMax to transfer the siRNA conjugate into the cells, and inoculate 96 cells at a density of 54,000 cells per well. In the well plate, each well contains 100 μL of culture medium. Cells were cultured in 5% CO ₂ and 37°C incubator. After 48 hours, the medium was removed and cells were collected for total RNA extraction. Use according to kit product instructions 96 Kit to extract total RNA.

(2) siRNA conjugates (the final concentrations of siRNA conjugates are 500nM, 125nM, 31.25nM, 7.81nM, 1.95nM, 0.49nM, 0.12nM and 0.03nM, respectively, in duplicate) enter PHH cells through free uptake. The process is as follows Describe: Take frozen PHH cells, resuscitate, count, adjust the cells to 6×10 ⁵ cells/ml, add siRNA conjugate at the same time, inoculate into a 96-well plate at a density of 54,000 cells per well, and culture in each well The solution is 100μl. Cells were cultured in 5% CO ₂ and 37°C incubator. After 48 hours, the medium was removed and cells were collected for total RNA extraction. Use according to kit product instructions 96 Kit to extract total RNA.

(3) Use the Fastking RT Kit (With gDNase) kit to reverse-transcribe the extracted total RNA into cDNA, follow the following steps:

a) Use gDNAase to remove gDNA according to the table below;

Table 6

Run the program at 42 °C for 3 min, then place the plate at 4 °C.

b) Add the following reagents to the system obtained in step a) and perform reverse transcription:

Table 7

42℃, 15min; 95℃, 3min.

c) Store the reverse transcription product obtained in step b) at -20°C for real-time PCR analysis.

(4) Perform real-time qPCR analysis

a) Prepare the qPCR reaction mixture as shown in the table below, and keep all reagents on ice during the entire operation;

Table 8

Table 9

b) Perform the qPCR procedure as follows

95℃, 10 minutes;

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

(5) Result analysis:

b) Use the following formula to calculate the relative expression of genes:

ΔCt=Ct(HSD17B13 gene)-Ct(GAPDH)

ΔΔCt=ΔCt (test sample group)-ΔCt (Mock group), where the Mock group represents the group in which siRNA was not added compared with the experimental group;

Relative to the mRNA expression of the Mock group = 2 ^-ΔΔCt

Taking the log value of siRNA conjugate concentration as the X-axis and the percentage inhibition rate as the Y-axis, use the "log (inhibitor) vs. response-variable slope" function module of the analysis software GraphPad Prism 8 to fit the dose-effect curve. This resulted in the _IC50 value for each siRNA conjugate.

The fitting formula is: Y=Bottom+(Top-Bottom)/(1+10^((LogIC50-X)*HillSlope))

The experimental results are shown in Table 10.

Table 10 IC ₅₀ value of siRNA conjugates for inhibiting HSD17B13 gene expression

As can be seen from Table 10, the siRNA conjugate of the present application has high HSD17B13 gene inhibition activity.

Example 5 Determination of the inhibition rate of siRNA and its conjugates in inhibiting HSD17B13 gene expression

Material:

Human primary hepatocyte PHH cells were provided by WuXi AppTec;

PHH medium: invitroGRO CP Meduim serum free BIOVIT, Cat. No.: S03316;

RNA extraction kit 96Kit, item number: QIAGEN-74182;

Reverse transcription kit FastQuant RT Kit (With gDNase), product number: Tiangen-KR116-02;

FastStart Universal Probe Mast(Roche-04914058001);

TaqMan Gene Expression Assay (GAPDH, Thermo, Assay ID-Hs02786624_g1;

TaqMan Gene Expression Assay (HSD17b13, Thermo, Assay ID-Hs01068199_m1).

siRNA conjugates (the final concentrations of siRNA conjugates are 1 nM and 0.1 nM, respectively, in duplicate wells) are transfected into PHH cells. The process is as follows: take the frozen PHH cells, resuscitate, count, and adjust the cells to 6×10 ⁵ cells/ml, and Lipofectamine RNAiMax was used to transfer the siRNA conjugate into the cells. The cells were seeded into a 96-well plate at a density of 54,000 cells per well, with 100 μL of culture medium per well. Cells were cultured in 5% CO ₂ and 37°C incubator. After 48 hours, the medium was removed and cells were collected for total RNA extraction. Use according to kit product instructions 96 Kit to extract total RNA.

siRNA conjugates (the final concentrations of siRNA conjugates are 100nM and 10nM, respectively, in duplicate wells) enter PHH cells through free uptake. The process is as follows: take the frozen PHH cells, resuscitate, count, and adjust the cells to 6×10 ⁵ cells/ml, and siRNA conjugate was added at the same time, and seeded into a 96-well plate at a density of 54,000 cells per well, with 100 μl of culture medium per well. Cells were cultured in 5% CO ₂ and 37°C incubator. After 48 hours, the medium was removed and cells were collected for total RNA extraction. Use according to kit product instructions 96 Kit to extract total RNA.

Using a method similar to that in Example 4, the extracted total RNA was reverse transcribed into cDNA through a reverse transcription reaction. HSD17B13 cDNA will be detected by qPCR. GAPDH cDNA will be used as an internal control for parallel testing. The PCR reaction program is: 95°C for 10 minutes, then enter the cycle mode, 95°C for 15 seconds, then 60°C for 60 seconds, a total of 40 cycles.

Result analysis:

b) Use the following formula to calculate the relative expression of genes:

ΔCt=Ct(HSD17B13 gene)-Ct(GAPDH)

Relative to the mRNA expression of the Mock group = 2 ^-ΔΔCt

Table 11 Inhibition rates of siRNA and its conjugates in inhibiting HSD17B13 gene expression

As can be seen from Table 11, the siRNA and its conjugates of the present application have high HSD17B13 gene inhibitory activity.

Example 6 Silencing effect of siRNA conjugates in mice expressing human HSD17B13 (hHSD17B13) gene

(1) AAV constructs mouse model overexpressing hHSD17B13 gene

C57BL/6 mice aged 6-8 weeks (provided by Jiangsu Jicui Yaokang Biotechnology Co., Ltd., SPF grade) entered the facility and were adaptively fed for 3-5 days before a single injection of adeno-associated virus AAV with hHSD17B13 gene into the tail vein. (pAAV-CBh-hHSD17B13-3xFLAG-P2A-Luc2-tWPA, virus provided by Shanghai Heyuan Biotechnology Co., Ltd.) for target gene overexpression modeling, administration volume: 100 μL (3*10 ¹¹ vg)/animal, followed by Feed with ordinary feed.

(2) Investigation of the efficacy of silencing siRNA in vivo in hHSD17B13 mouse model

Fourteen days after AAV virus injection, the mice were examined by in vivo imaging, divided into groups (6 mice in each group), and the mice were administered subcutaneously. A single 3 mg/kg dose of N-ER-FY007001M2L96, N-ER-FY007004M2L96, N-ER-FY007020M2L96, N-ER-FY007033M2L96 and N-ER-FY007034M2L96. In vivo imaging was performed on days 7, 14, 21, 28 and 35 after administration to detect the protein expression of Luciferase (the protein expression indirectly reflects the hHSD17B13 protein expression).

Table 12 The remaining amount of hHSD17B13 protein inhibited by siRNA conjugates

As can be seen from Table 12, the siRNA of the present application has high inhibitory activity on the hHSD17B13 gene in vivo, and can reduce hHSD17B13 protein levels for a long time, with obvious dose effect. Specifically, after a single subcutaneous administration, N-ER-FY007001M2L96 showed 60% inhibition of the hHSD17B13 gene (40% protein remaining) on day 35; N-ER-FY007004M2L96 showed 62% inhibition of the hHSD17B13 gene N-ER-FY007020M2L96 showed 73% inhibition of hHSD17B13 gene (remaining protein of 27%); N-ER-FY007033M2L96 showed 66% inhibition of hHSD17B13 gene (remaining protein of 27%) 34%); N-ER-FY007034M2L96 showed 51% inhibition of hHSD17B13 gene (protein remaining 49%).

Example 7 Kinetic study of siRNA conjugates in CD-1 mouse plasma

Animal: CD-1 mouse, SPF grade, male, about 30g, purchased from Spefford (Beijing) Biotechnology Co., Ltd.

Dosage and mode of administration: The siRNA conjugate of this application is administered at a dose of 3 mg/kg (10 mL/kg), and administered by a single subcutaneous injection after random grouping, with 6 mice in each group.

Sample collection: Collect whole blood samples at 0.0833, 0.25, 0.5, 1, 2, 4, 8, 24, 36, and 48 hours after administration, a total of 10 points. The first three animals in each group were collected at 0.0833, 0.5, 2, 8, and 36 hours, and the last three animals were collected at 0.25, 1, 4, 24, and 48 hours. Whole blood was collected and plasma was centrifuged for detection and analysis.

Sample detection and analysis: LC-MS/MS method was used to detect the concentration of prototype drug in plasma samples at each time point, and WinNonlin software was used to calculate PK parameters: C _max , T _max , AUC, MRT, t _1/2 .

It can be concluded from this experiment that the siRNA conjugate of the present application has a shorter half-life in plasma and is cleared faster.

Example 8 siRNA conjugate tissue distribution test in CD-1 mice

Dosage and method of administration: The siRNA conjugate of this application is administered at a dose of 3 mg/kg (10 mL/kg). After random grouping, it is administered by a single subcutaneous injection. There are 3 animals at each time point, for a total of 24 mice. .

Sample Collection:

24 hours after administration: collect plasma, liver, kidney, and spleen; 72 hours after administration: collect plasma, liver, kidney, and spleen;

168h after administration (1 week): collect plasma, liver, kidney, spleen, brain, heart, lung, stomach, small intestine, muscle, and testes; 336h (2 weeks) after administration: collect plasma, liver, kidney, and spleen;

672h after administration (4 weeks): Collect plasma, liver, kidney, spleen, brain, heart, lung, stomach, small intestine, muscle, and testis; 1008h (6 weeks) after administration: Collect plasma, liver, kidney, and spleen;

1344h (8 weeks) after administration: plasma, liver, kidney, and spleen were collected;

1680h (10 weeks) after administration: plasma, liver, kidney, spleen, brain, heart, lung, stomach, small intestine, muscle, and testis were collected.

Sample detection and analysis: The LC-MS/MS method was used to detect the concentration of the prototype drug in plasma and tissue samples at each time point, and the trapezoidal area method was used to calculate the AUC in plasma and tissue.

It can be concluded from this experiment that the siRNA conjugate of the present application is mainly enriched in the liver, has a long retention time in the tissue, and has good stability.

Example 9 Single subcutaneous injection of siRNA conjugate into C57 mice for MTD test

C57 mice, SPF grade, male, about 25 g, purchased from Spefford (Beijing) Biotechnology Co., Ltd. The animals were randomly grouped according to their body weight on the last day of the adaptation period. The specific dose design and grouping are as follows:

Detection Indicator:

(1) Clinical observation: Continuous observation for 4 hours on the dosing day, and at least one clinical observation per day during the recovery period

(2) Body weight: All surviving animals were weighed twice a week.

(3) Immunotoxicity: Blood samples were collected from animals in the MTD dose group alternately at 1h±2min, 4h±5min, 8h±10min, and 24h±20min after D1 administration. 3 animals/sex/group were collected at each time point to detect cytokines ( IFN-γ, TNF-α, IL-2/6/8).

(4) Toxicokinetics: Blood samples were collected from animals in the MTD dose group alternately before D1 administration and at 30min±2min, 1h±2min, 4h±5min, 8h±10min, and 24h±20min after D1 administration. Three animals were collected at each time point. /Gender/ group of animals to detect blood drug concentration.

(5) Blood biochemistry: The animals in the main experimental group were necropsied at R28, and the animals in the satellite group were necropsied in batches at R7, R14, R21, and R28 to detect blood biochemistry.

(6) Tissue distribution: The animals in the main test group were necropsied at R28, and the animals in the satellite group were necropsied in batches at R7, R14, R21, and R28. Blood and liver were collected to detect tissue drug concentrations.

(7) Histopathological examination: The animals in the main test group were necropsied at R28, and the main organs (heart, liver, spleen, lung, kidney, brain, adrenal gland, thymus, stomach, uterus/testis, ovary/epididymis) and findings were collected Abnormal tissues or organs are collected and fixed for histopathological examination.

It can be concluded from this experiment that the siRNA conjugate of the present application is less toxic and has a large safety window.

Claims

An siRNA capable of inhibiting HSD17B13 gene expression, the siRNA comprising a sense strand and an antisense strand, wherein each nucleotide in the siRNA is independently a modified or unmodified nucleotide, wherein the sense strand Containing nucleotide sequence I, the antisense strand contains nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence I and nucleotide sequence II are selected from the following sequences:

(1) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 296, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 297:

5’-AGUCGUUGGUGAAGUU-3’(SEQ ID NO: 296)

5’-AACUUCACCAACGACU-3’(SEQ ID NO: 297);

(2) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 298, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 299:

5’-GUUGGUGAAGUUUUUCA-3’(SEQ ID NO: 298)

5’-UGAAAAACUUCACCAAC-3’(SEQ ID NO: 299);

wherein the nucleotide sequence I is not SEQ ID NO: 17 and the nucleotide sequence II is not SEQ ID NO: 18;

The nucleotide sequence I is not SEQ ID NO: 21 and the nucleotide sequence II is not SEQ ID NO: 22;

(3) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 300, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 301:

5’-GACUACUUAUGAAUU-3’(SEQ ID NO: 300)

5’-AAUUCAUAAGUAGUC-3’(SEQ ID NO: 301);

wherein the nucleotide sequence I is not SEQ ID NO: 33 and the nucleotide sequence II is not SEQ ID NO: 34;

The nucleotide sequence I is not SEQ ID NO: 35 and the nucleotide sequence II is not SEQ ID NO: 36;

The nucleotide sequence I is not SEQ ID NO: 39 and the nucleotide sequence II is not SEQ ID NO: 40;

(4) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 23, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 24;

(5) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 302, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 303:

5'-CAGGCAGACUACUUAUGAN 1 -3' (SEQ ID NO: 302)

5'- N 2UCAUAAGUAGUCUGCCUG-3' (SEQ ID NO: 303);

Where N 1 is A or U, N 2 is A or U;

wherein the nucleotide sequence I is not SEQ ID NO: 25 and the nucleotide sequence II is not SEQ ID NO: 26;

(6) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 304, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 305:

5’-GGUUCUGUGGGAUAUUA-3’(SEQ ID NO: 304)

5’-UAAUAUCCCACAGAACC-3’(SEQ ID NO: 305);

wherein the nucleotide sequence I is not SEQ ID NO: 51 and the nucleotide sequence II is not SEQ ID NO: 52;

(7) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 306, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 307:

5’-CUGCGCAUGCGUAU-3’(SEQ ID NO: 306)

5’-AUACGCAUGCGCAG-3’(SEQ ID NO: 307);

(8) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 308, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 309:

5’-GAUCUAUCGCUCUCUAA-3’(SEQ ID NO: 308)

5’-UUAGAGAGCGAUAGAUC-3’(SEQ ID NO: 309);

wherein the nucleotide sequence I is not SEQ ID NO: 71 and the nucleotide sequence II is not SEQ ID NO: 72;

(9) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 310, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 311:

5’-GAAAGAAGUGGGUGAU-3’(SEQ ID NO: 310)

5’-AUCACCCACUUCUUUC-3’(SEQ ID NO: 311);

wherein the nucleotide sequence I is not SEQ ID NO: 77 and the nucleotide sequence II is not SEQ ID NO: 78;

The nucleotide sequence I is not SEQ ID NO: 79 and the nucleotide sequence II is not SEQ ID NO: 80;

The nucleotide sequence I is not SEQ ID NO: 81 and the nucleotide sequence II is not SEQ ID NO: 82;

(10) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 312, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 313:

5’-AAGUGGGUGAUGUACAA-3’(SEQ ID NO: 312)

5’-UUGUUACAUCACCCACUU-3’(SEQ ID NO: 313);

wherein said nucleotide sequence I is not SEQ ID NO: 89 and said nucleotide sequence II is not SEQ ID NO: 90;

(11) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 314, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 315:

5’-AGAGAUUACCAAGACA-3’(SEQ ID NO: 314)

5’-UGUCUUGGUAAUCUCU-3’(SEQ ID NO: 315);

wherein the nucleotide sequence I is not SEQ ID NO: 95 and the nucleotide sequence II is not SEQ ID NO: 96;

The nucleotide sequence I is not SEQ ID NO: 99 and the nucleotide sequence II is not SEQ ID NO: 100;

(12) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 316, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 317:

5’-AUCGUAUAUCAAUAU-3’(SEQ ID NO: 316)

5’-AUAUUGAUAUACGAU-3’(SEQ ID NO: 317);

wherein the nucleotide sequence I is not SEQ ID NO: 121 and the nucleotide sequence II is not SEQ ID NO: 122;

(13) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 318, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 319:

5’-GCGCCUCAGCGAUUU-3’(SEQ ID NO: 318)

5’-AAAAUCGCUGAGGCGC-3’(SEQ ID NO: 319);

wherein the nucleotide sequence I is not SEQ ID NO: 139 and the nucleotide sequence II is not SEQ ID NO: 140;

The nucleotide sequence I is not SEQ ID NO: 141 and the nucleotide sequence II is not SEQ ID NO: 142;

(14) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 320, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 321:

5’-CUCAGCGAUUUAAAUCGU-3’(SEQ ID NO: 320)

5’-ACGAUUUAAAAUCGCUGAG-3’(SEQ ID NO: 321);

(15) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 322, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 323:

5’-UAUGCAGAAUAUUCA-3’(SEQ ID NO: 322)

5’-UGAAUAUUCUGCAUA-3’(SEQ ID NO: 323);

(16) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 324, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 325:

5’-UUGGCCACAAAAUCAAA-3’(SEQ ID NO: 324)

5’-UUUGAUUUUGUGGCCAA-3’(SEQ ID NO: 325);

wherein the nucleotide sequence I is not SEQ ID NO: 169 and the nucleotide sequence II is not SEQ ID NO: 170;

(17) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 65, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 66;

(18) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 75, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 76;

(19) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 93, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 94;

(20) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 103, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 104;

(21) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 109, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 110;

(22) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 111, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 112;

(23) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 115, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 116;

(24) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 41, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 42;

(25) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 41, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 188;

(26) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 189, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 188;

(27) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 41, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 190;

(28) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 41, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 191;

(29) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 41, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 192;

(30) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 27, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 28;

(31) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 27, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 183;

(32) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 184, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 183;

(33) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 27, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 185;

(34) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 27, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 186;

(35) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 27, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 187;

(36) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 73, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 74;

(37) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 83, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 84;

(38) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 83, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 193;

(39) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 194, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 193;

(40) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 83, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 195;

(41) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 83, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 196;

(42) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 83, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 197;

(43) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 97, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 98;

(44) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 97, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 198;

(45) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 199, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 198;

(46) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 97, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 200;

(47) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 97, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 201;

(48) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 97, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 202;

(49) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 143, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 144;

(50) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 19, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 20;

(51) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 19, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 178;

(52) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 179, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 178;

(53) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 19, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 180;

(54) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 19, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 181;

(55) The nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 19, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 182.
siRNA according to claim 1, wherein said nucleotide sequence I and said nucleotide sequence II are substantially reverse complementary, substantially reverse complementary or completely reverse complementary; said substantially reverse complement is Refers to the presence of no more than 3 base mismatches between the two nucleotide sequences; the substantial reverse complementarity refers to the presence of no more than 1 base mismatch between the two nucleotide sequences; Perfect reverse complementarity means there are no mismatches between the two nucleotide sequences.
siRNA according to claim 1 or 2, wherein the sense strand further contains nucleotide sequence III, the antisense strand further contains nucleotide sequence IV, the length of nucleotide sequence III and nucleotide sequence IV Each independently is 0-6 nucleotides, wherein the nucleotide sequence III is connected to the 5′ end of the nucleotide sequence I, and the nucleotide sequence IV is connected to the 3′ end of the nucleotide sequence II, so The nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary; the substantially reverse complementarity means that there is no more than 1 between the two nucleotide sequences. base mismatch; complete reverse complementarity means that there is no mismatch between the two nucleotide sequences; and/or, the nucleotide sequence III is connected to the 3′ end of the nucleotide sequence I, and the nucleoside The acid sequence IV is connected to the 5′ end of the nucleotide sequence II, and the nucleotide sequence III and the nucleotide sequence IV are equal in length and substantially reverse complementary. Or completely reverse complementary; the substantial reverse complementarity means that there is no more than 1 base mismatch between the two nucleotide sequences; the complete reverse complementarity means that there is no base mismatch between the two nucleotide sequences. mismatch.
According to the siRNA according to any one of claims 1-3, the sense strand further contains the nucleotide sequence V and/or the antisense strand further contains the nucleotide sequence VI, the nucleotide sequences V and VI The length is 0 to 3 nucleotides. The nucleotide sequence V is connected to the 3' end of the sense strand to form the 3' overhang of the sense strand and/or the nucleotide sequence VI is connected to the 3' end of the antisense strand. The 'end constitutes the 3' overhang of the antisense strand; preferably, the length of the nucleotide sequence V or VI is 2 nucleotides; more preferably, the nucleotide sequence V or VI is two consecutive nucleotides. A thymine deoxyribonucleotide or two consecutive uracil ribonucleotides;

Alternatively, the nucleotide sequence V or VI mismatches or is complementary to the nucleotide at the corresponding position of the target mRNA.
The siRNA according to any one of claims 1-4, wherein the length of the double-stranded region is 15-30 nucleotide pairs; preferably, the length of the double-stranded region is 17-23 nucleotide pairs; More preferably, the length of the double-stranded region is 19-21 nucleotide pairs.
The siRNA according to any one of claims 1-5, wherein the sense strand or the antisense strand has 15-30 nucleotides; preferably, the sense strand or the antisense strand has 19-25 nucleotides; more Preferably, the sense or antisense strand has 19-23 nucleotides.
The siRNA according to any one of claims 1-6, wherein at least one nucleotide in the sense strand or the antisense strand is a modified nucleotide, and/or at least one phosphate group has A phosphate group of a modifying group; preferably, the phosphate group containing a modifying group is a phosphorothioate group formed by replacing at least one oxygen atom in the phosphodiester bond of the phosphate group with a sulfur atom; And/or, the siRNA includes a sense strand that does not include a 3' overhanging nucleotide.
The siRNA according to any one of claims 1 to 7, wherein the 5' terminal nucleotide of the sense strand is connected to a 5' phosphate group or a 5' phosphate derivative group, and/or the antisense strand The 5' terminal nucleotide is attached to a 5' phosphate group or a 5' phosphate derivative group.
The siRNA according to any one of claims 1-8, wherein the modified nucleotide is selected from the group consisting of 2'-fluoro modified nucleotides, 2'-alkoxy modified nucleotides, 2' -Substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-deoxy nucleotides, 2'-amino modified Nucleotide, 2'- Substituted amino-modified nucleotides, nucleotide analogs or a combination of any two or more thereof.
The siRNA according to any one of claims 1-9, wherein the modified nucleotide is selected from the group consisting of 2'-fluoro modified nucleotides, 2'-methoxy modified nucleotides, 2 '-O-CH 2 -CH 2 -O-CH 3 modified nucleotide, 2'-O-CH 2 -CH＝CH 2 modified nucleotide, 2'-CH 2 -CH 2 -CH＝CH 2 modified nucleotides, 2'-deoxynucleotides, nucleotide analogs or a combination of any two or more thereof.
The siRNA according to any one of claims 1-10, wherein each nucleotide in the sense strand and the antisense strand is independently a 2'-fluorinated modified nucleotide or a non-fluorinated nucleotide. modified nucleotides;

Preferably, according to the 5' to 3' direction, the 2'-fluorinated modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand, and the remaining positions are non-fluorinated modified nucleotides; according to 5 In the 'to 3' direction, the 2'-fluoro-modified nucleotides are located in the even-numbered positions of the antisense strand, and the remaining positions are non-fluoro-modified nucleotides;

Alternatively, according to the 5' to 3' direction, the 2'-fluorinated modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand, and the remaining positions are non-fluorinated modified nucleotides; according to the 5' To the 3' direction, 2'-fluorinated modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and the remaining positions are non-fluorinated modified nucleotides;

Alternatively, according to the 5' to 3' direction, the 2'-fluorinated modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand, and the remaining positions are non-fluorinated modified nucleotides; according to the 5' To the 3' direction, 2'-fluorinated modified nucleotides are located at positions 2, 6, 8, 9, 14 and 16 of the antisense strand, and the remaining positions are non-fluorinated modified nucleotides;

Alternatively, according to the 5' to 3' direction, the 2'-fluorinated modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand, and the remaining positions are non-fluorinated modified nucleotides; according to the 5' To the 3' direction, 2'-fluoro-modified nucleotides are located at positions 2, 14, and 16 of the antisense strand, and the remaining positions are non-fluoro-modified nucleotides;

Further preferably, each non-fluorinated modified nucleotide is a 2'-methoxy modified nucleotide, and the 2'-methoxy modified nucleotide refers to the 2'-hydroxyl group of the ribose group. Nucleotides formed by methoxy substitution.
The siRNA according to claim 11, wherein each non-fluorinated modified nucleotide is independently selected from the group consisting of nucleotides or nucleotides formed by replacing the hydroxyl group at the 2' position of the ribosyl group of the nucleotide with a non-fluorinated group. One of the analogs, the nucleotide analog is selected from one of isonucleotides, LNA, ENA, cET BNA, UNA and GNA.
The siRNA according to any one of claims 1-12, wherein each nucleotide in the sense strand and the antisense strand is independently a 2'-fluoro modified nucleotide, a 2'- Methoxy-modified nucleotides, GNA-modified nucleotides, or a combination of any two or more thereof;

Preferably, the 2'-fluoro-modified nucleotides are located at the 7th, 9th, 10th and 10th positions of the sense strand in the 5' to 3' direction. Position 11, the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at the even-numbered positions of the antisense strand, and the remaining positions are 2' -methoxy modified nucleotides;

Alternatively, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; According to the 5' to 3' direction, 2'-fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides;

Alternatively, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; According to the 5' to 3' direction, 2'-fluoro modified nucleotides are located at positions 2, 6, 8, 9, 14 and 16 of the antisense strand, and the remaining positions are 2'-methoxy modified cores glycolic acid;

Alternatively, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; According to the 5' to 3' direction, the 2'-fluoro modified nucleotides are located at positions 2, 14 and 16 of the antisense strand, the GNA modified nucleotides are located at position 6 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides;

Alternatively, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; According to the 5' to 3' direction, the 2'-fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, the GNA modified nucleotides are located at position 7 of the antisense strand, and the rest The 2'-methoxy modified nucleotide position.
The siRNA according to any one of claims 1-13, wherein at least one of the connections between the following nucleotides in the siRNA is a phosphorothioate group connection:

The connection between the first nucleotide and the second nucleotide at the 5' end of the sense strand;

The connection between the 2nd nucleotide and the 3rd nucleotide at the 5' end of the sense strand;

The connection between the first nucleotide and the second nucleotide at the 3' end of the sense strand;

The connection between the second nucleotide and the third nucleotide at the 3’ end of the sense strand;

The connection between the first nucleotide and the second nucleotide at the 5' end of the antisense strand;

The connection between the 2nd nucleotide and the 3rd nucleotide at the 5' end of the antisense strand;

The connection between the first nucleotide and the second nucleotide at the 3' end of the antisense strand;

The connection between the 2nd nucleotide and the 3rd nucleotide at the 3' end of the antisense strand.
According to the siRNA of any one of claims 1-14, in the 5' to 3' direction, the sense strand includes a phosphorothioate group located at the position 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;

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

Between the second and third nucleotides starting from the 3’ end of the sense strand;

or,

The sense strand contains phosphorothioate groups at the positions shown below:

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

Between the 2nd and 3rd nucleotide starting from the 5' end of the sense strand.
According to the siRNA of any one of claims 1-15, in the 5' to 3' direction, the antisense strand includes a phosphorothioate group located at the position shown below:

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

Between the 2nd and 3rd 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 2nd and 3rd nucleotide starting from the 3' end of the antisense strand.
The siRNA according to any one of claims 1-16, wherein each nucleotide in the sense strand and the antisense strand is independently a 2'-fluoro modified nucleotide, 2'- Methoxy-modified nucleotides, GNA-modified nucleotides or a combination of any two or more thereof;

Preferably, in the 5' to 3' direction, the 2'-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides , between the first and second nucleotides at the 5' end, between the second and third nucleotides at the 5' end, and between the first nucleoside at the 3' end Between the acid and the second nucleotide, there is a phosphorothioate group connection between the second and third nucleotides at the 3' end; in the 5' to 3' direction, the 2'- The fluoro-modified nucleotides are located at the even-numbered positions of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, between the first and second nucleotides at the 5' end, Between the 2nd and 3rd nucleotides at the 5' end, between the 1st and 2nd nucleotides at the 3' end, and the 2nd nucleotide at the 3' end It is connected to the third nucleotide by a phosphorothioate group;

Alternatively, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, with 2'-methoxy-modified nucleotides at the remaining positions, Between the first and second nucleotides at the 5' end, between the second and third nucleotides at the 5' end, there is a phosphorothioate group connection, and at the 3' end Remove the overhang; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, and the remaining positions are 2'-methoxy-modified cores nucleotide, between the 1st and 2nd nucleotides at the 5' end, between the 2nd and 3rd nucleotides at the 5' end, and the 1st nucleotide at the 3' end There is a phosphorothioate group connection between the nucleotide and the second nucleotide, and between the second nucleotide and the third nucleotide at the 3'end;

Alternatively, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, with 2'-methoxy-modified nucleotides at the remaining positions, Between the 1st and 2nd nucleotides at the 5' end, between the 2nd and 3rd nucleotides at the 5' end, the 1st nucleotide at the 3' end and the second nucleotide, and between the second nucleotide and the third nucleotide at the 3' end is a phosphorothioate group; in the 5' to 3' direction, 2'-fluoro The modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, the first nucleotide and the second nucleotide at the 5' end. Between nucleotides, between the 2nd and 3rd nucleotides at the 5' end, between the 1st and 2nd nucleotide at the 3' end, and at the 3' end There is a phosphorothioate group connection between the 2nd nucleotide and the 3rd nucleotide;

Alternatively, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, with 2'-methoxy-modified nucleotides at the remaining positions, Between the 1st and 2nd nucleotides at the 5' end, between the 2nd and 3rd nucleotides at the 5' end, the 1st nucleotide at the 3' end and the second nucleotide, and between the second nucleotide and the third nucleotide at the 3' end is a phosphorothioate group; in the 5' to 3' direction, 2'-fluoro The modified nucleotides are located at positions 2, 6, 8, 9, 14 and 16 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, the first nucleotide at the 5' end and between the 2nd nucleotide, between the 2nd nucleotide and the 3rd nucleotide at the 5' end, between the 1st nucleotide and the 2nd nucleotide at the 3' end, The 2nd and 3rd nucleotides at the 3' end are connected by a phosphorothioate group;

Alternatively, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, with 2'-methoxy-modified nucleotides at the remaining positions, Between the 1st and 2nd nucleotides at the 5' end, between the 2nd and 3rd nucleotides at the 5' end, the 1st nucleotide at the 3' end and the second nucleotide, and between the second nucleotide and the third nucleotide at the 3' end is a phosphorothioate group; in the 5' to 3' direction, 2'-fluoro The GNA-modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, the GNA-modified nucleotides are located at position 7 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides , between the first and second nucleotides at the 5' end, between the second and third nucleotides at the 5' end, and between the first nucleoside at the 3' end There is a phosphorothioate group connection between the acid and the second nucleotide, and between the second nucleotide and the third nucleotide at the 3' end;

Alternatively, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, with 2'-methoxy-modified nucleotides at the remaining positions, Between the 1st and 2nd nucleotides at the 5' end, between the 2nd and 3rd nucleotides at the 5' end, the 1st nucleotide at the 3' end and the second nucleotide, and between the second nucleotide and the third nucleotide at the 3' end is a phosphorothioate group; in the 5' to 3' direction, 2'-fluoro The GNA-modified nucleotides are located at positions 2, 14 and 16 of the antisense strand, the GNA-modified nucleotides are located at position 6 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, 5 Between the 1st and 2nd nucleotides at the 'end, between the 2nd and 3rd nucleotides at the 5' end, the 1st nucleotide at the 3' end and the second nucleotide, and between the second and third nucleotides at the 3' end are phosphorothioate groups.
siRNA according to claim 1, which is selected from the siRNA of Table 1, wherein the siRNA is not N-ER-FY007001, N-ER-FY007002, N-ER-FY007004, N-ER-FY007006, N-ER -FY007031, N-ER-FY007007, N-ER-FY007011, N-ER-FY007056, N-ER-FY007013, N-ER-FY007014, N-ER-FY007016, N-ER-FY007038, N-ER-FY007039 , N-ER-FY007022, N-ER-FY007023, N-ER-FY007027, N-ER-FY007005, N-ER-FY007030, N-ER-FY007051, N-ER-FY007032, N-ER-FY007033, N -ER-FY007034, N-ER-FY007035, N-ER-FY007036, N-ER-FY007015; preferably, the siRNA is selected from N-ER-FY007001M1, N-ER-FY007004M1, N-ER-FY007006M1, N -ER-FY007011M1, N-ER-FY007033M1, N-ER-FY007034M1, N-ER-FY007020, N-ER-FY007020M1, N-ER-FY007024, N-ER-FY007024M1, N-ER-FY007020M2, N-ER -FY007020M2D2, N-ER-FY007020M3, N-ER-FY007020M4, N-ER-FY007020M5, N-ER-FY007001M2, N-ER-FY007001M2D2, N-ER-FY007001M3, N-ER-FY007001M4, N -ER-FY007001M5 . 6M3,N -ER-FY007006M4, N-ER-FY007006M5, N-ER-FY007033M2, N-ER-FY007033M2D2, N-ER-FY007033M3, N-ER-FY007033M4, N-ER-FY007033M5, N-ER-FY007034M2, N -ER -FY007034M2D2, N-ER-FY007034M3, N-ER-FY007034M4, N-ER-FY007034M5, N-ER-FY007074, N-ER-FY007074M2, N-ER-FY007074M2D2, N-ER-FY007074M3, N-ER -FY007074M4 N N -ER -FY007079M3, N-ER-FY007079M4, N-ER-FY007079M5, N-ER-FY007080, N-ER-FY007080M2, N-ER-FY007080M2D2, N-ER-FY007080M3, N-ER-FY007080M4, N-ER-F Y007080M5 , N-ER-FY007082, N-ER-FY007082M2, N-ER-FY007082M2D2, N-ER-FY007082M3, N-ER-FY007082M4, N-ER-FY007082M5, N-ER-FY007083, N-ER-FY007083M2, N N -ER -FY007085M4, N-ER-FY007085M5.
An siRNA conjugate comprising the siRNA of any one of claims 1-18 and a conjugation group conjugated to the siRNA.
The siRNA conjugate of claim 19, wherein the conjugation group comprises a pharmaceutically acceptable targeting group and a linker, and the siRNA, the linker and the targeting group are sequentially covalent or non-covalent linkage;

Preferably, in the siRNA conjugate, the sense strand and the antisense strand of the siRNA are complementary to form a double-stranded region of the siRNA conjugate, and the 3' end of the sense strand forms a blunt end, and the antisense strand forms a blunt end. The 3' end of the chain has 1-3 protruding nucleotides extending out of the double-stranded region;

or,

In the siRNA conjugate, the sense strand and the antisense strand of siRNA are complementary to form the double-stranded region of the siRNA conjugate, and the 3' end of the sense strand forms a blunt end, and the 3' end of the antisense strand forms a blunt end. 'The ends form blunt ends.
The siRNA conjugate according to claim 20, wherein the conjugation group is L96 of the following formula:
The siRNA conjugate according to any one of claims 19-21, wherein the siRNA conjugate is a siRNA conjugate selected from Table 2.
A pharmaceutical composition comprising the siRNA according to any one of claims 1-18, or the siRNA conjugate according to any one of claims 19-22, and a pharmaceutically acceptable carrier.
A kit comprising the siRNA according to any one of claims 1-18, or the siRNA conjugate according to any one of claims 19-22, or the pharmaceutical composition according to claim 23.
The siRNA described in any one of claims 1-18, or the siRNA conjugate described in any one of claims 19-22, or the pharmaceutical composition described in claim 23 is used to prepare an agent that inhibits HSD17B13 gene expression. The purpose of the medicine.
The siRNA according to any one of claims 1-18, or the siRNA conjugate according to any one of claims 19-22, or the pharmaceutical composition according to claim 23 for the preparation of prevention and/or treatment Use of pharmaceuticals for diseases related to HSD17B13 gene overexpression.
The use according to claim 26, wherein the disease is selected from the group consisting of non-alcoholic fatty liver disease, cirrhosis, alcoholic hepatitis, liver fibrosis and liver cancer.
A method for inhibiting HSD17B13 gene expression, comprising adding a therapeutically effective amount of the siRNA according to any one of claims 1-18, or the siRNA conjugate according to any one of claims 19-22, or the siRNA conjugate according to any one of claims 19-22. The pharmaceutical composition described in 23 is contacted with cells expressing HSD17B13 or administered to a subject in need.
A method for treating and/or preventing diseases related to HSD17B13 gene overexpression, comprising using a therapeutically effective amount of the siRNA described in any one of claims 1-18, or the siRNA described in any one of claims 19-22 The siRNA conjugate, or the pharmaceutical composition of claim 23 is administered to a subject in need.