CN116515835A

CN116515835A - siRNA for inhibiting HSD17B13 expression, conjugate and pharmaceutical composition thereof and application thereof

Info

Publication number: CN116515835A
Application number: CN202310474078.6A
Authority: CN
Inventors: 王书成; 黄河; 王岩; 林国良; 耿玉先; 荣梅
Original assignee: Beijing Fuyuan Pharmaceutical Co ltd
Current assignee: Beijing Fuyuan Pharmaceutical Co ltd
Priority date: 2022-04-29
Filing date: 2023-04-27
Publication date: 2023-08-01
Also published as: WO2023208109A9; WO2023208109A1

Abstract

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

Description

siRNA for inhibiting HSD17B13 expression, conjugate and pharmaceutical composition thereof and application thereof

Technical Field

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

Background

The 17β -hydroxysteroid dehydrogenase (17β -HSD) family consists of 15 enzymes, most of which are involved in the activation or inactivation of sex hormones (e.g., HSD17B1, HSD17B2, HSD17B3, HSD17B5, HSD17B 6), other members being involved in fatty acid metabolism, cholesterol biosynthesis, bile acid production, etc. Members of the HSD17B family differ in tissue distribution, subcellular localization, catalytic preference, and have different substrate specificities (Marchais Oberwinkler, et a1. (2011) J Steroid Biochem Mol Biol (1-2): 66-82)).

The 17 beta-hydroxysteroid dehydrogenase family member HSD17B13 is mainly localized to hepatocytes, with highest expression levels found in hepatocytes of the liver known, whereas only lower levels can be detected in ovaries, bone marrow, kidneys, brain, lungs, skeletal muscle, bladder and testes, a Lipid Droplet (LD) -associated protein with hepatocyte specificity, and increasing evidence suggests that it plays a key role in hepatic lipid metabolism. The function of HSD17B13 is not fully understood, however, some 17 beta-HSD family members, including 17 beta-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. Liver upregulation of HSD17B13 has been reported in fatty liver patients, supporting the role of this enzyme in the pathogenesis of nonalcoholic fatty liver disease (NAFLD).

Nonalcoholic fatty liver disease (NAFLD), also known as metabolic (dysfunctional) related fatty liver disease (MAFLD), is the accumulation of excessive fat in the liver without other clear causes such as alcohol consumption. NAFLD is the most common liver disease in the world, accounting for about 25% of the world population. The prevalence of NAFLD is still currently on an upward trend, which will undoubtedly lead to an increase in the economic burden and will lead to a dramatic increase in the number of end-stage liver disease patients requiring liver transplantation and the number of people suffering from hepatocellular carcinoma. No particular treatment is currently available for NAFLD, mainly by weight loss through dietary changes and exercise, and preliminary studies indicate 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 that HSD17B13 is one of the most abundantly expressed LD proteins that is specifically localized on the surface of LD (Wen Su et al, comparative proteomic study reveals. Beta. -HSD 13as a pathogenic protein in nonalcoholic fatty live disease,111PNAS 11437-11442 (2014)). Further, the level of HSD17B13 was found to be up-regulated in the liver of patients and mice with NAFLD. Overexpression results in an increase in the number and size of LD, while gene silencing of HSDl7B13 attenuated oleic acid-induced LD formation in cultured hepatocytes. Liver overexpression of the HSD17B13 protein in C57BL/6 mice has also been shown to significantly increase adipogenesis and Triglyceride (TG) levels in the liver, resulting in a fatty liver phenotype. Additional evidence is provided by N.S. Abul-Husn et al suggesting HSD17B13 gene expression in pathogenesis of NAFLD and non-alcoholic steatohepatitis (NASH) (N.S. Abul-Husn et al, A Protein-therapeutic HSD17B 13Variant and Protectionfrom Chronic Liver Disease,378N.Eng.J.Med.1096-1106 (2018)). The team conducted a genome-wide association study that revealed that HSD17B13 splice variants (rs 72613567: TA) associated with reduced levels of alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) indicated less liver damage and inflammation in fatty liver patients. Splice variants produce truncations of functional proteins suggesting that HSD17B13 generally produces products that may promote hepatocyte damage. The RNAi therapy ARO-HSD targeting HSD17B13, developed in conjunction with Grandin Smith and Arrowhead, was used to treat non-alcoholic steatohepatitis and has entered clinical phase I studies. Currently, there are no marketed nucleic acid drugs targeting HSD17B 13.

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

Disclosure of Invention

The present invention provides 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 comprises nucleotide sequence I, and the antisense strand comprises nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II being at least partially reverse complementary to form a double-stranded region, wherein the nucleotide sequence I and the nucleotide sequence II are selected from the group consisting of:

(1) The nucleotide sequence I comprises SEQ ID NO:296, and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO: 297:

5’-AGUCGUUGGUGAAGUU-3’(SEQ ID NO：296)

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

(2) The nucleotide sequence I comprises SEQ ID NO:298, and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO:299, a nucleotide sequence shown in seq id no:

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:1 8;

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 comprises SEQ ID NO:300, and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO:301, a nucleotide sequence shown in seq id no:

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 said 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, a step of performing a;

(4) The nucleotide sequence I comprises SEQ ID NO:23, and the nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO:24, a nucleotide sequence shown in seq id no;

(5) The nucleotide sequence I comprises SEQ ID NO:302, and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO:303, a nucleotide sequence shown in seq id no:

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

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

wherein N is 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 comprises SEQ ID NO:304, and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO:305, a nucleotide sequence shown in seq id no:

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 comprises SEQ ID NO:306, and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO: 307:

5’-CUGCGCAUGCGUAU-3’(SEQ ID NO：306)

5’-AUACGCAUGCGCAG-3’(SEQ ID NO：307)；

(8) The nucleotide sequence I comprises SEQ ID NO:308, and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO:309, a nucleotide sequence shown in seq id no:

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 said nucleotide sequence II is not SEQ ID NO:72;

(9) The nucleotide sequence I comprises SEQ ID NO:310, and said nucleotide sequence II comprises the nucleotide sequence set forth 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 said nucleotide sequence II is not SEQ ID NO:78;

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

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

(10) The nucleotide sequence I comprises SEQ ID NO:312, and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO:313, a nucleotide sequence shown in seq id no:

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

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

Wherein the 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 comprises SEQ ID NO:314, and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO:315, a nucleotide sequence shown in seq id no:

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 said nucleotide sequence II is not SEQ ID NO:96;

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

(12) The nucleotide sequence I comprises SEQ ID NO:316, and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO:317, nucleotide sequence:

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, a step of;

(13) The nucleotide sequence I comprises SEQ ID NO:318, and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO: 319:

5’-GCGCCUCAGCGAUUUU-3’(SEQ ID NO：318)

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

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

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

(14) The nucleotide sequence I comprises SEQ ID NO:320, and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO:321, a nucleotide sequence shown in seq id no:

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

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

(15) The nucleotide sequence I comprises SEQ ID NO:322, and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO:323, a nucleotide sequence shown in seq id no:

5’-UAUGCAGAAUAUUCA-3’(SEQ ID NO：322)

5’-UGAAUAUUCUGCAUA-3’(SEQ ID NO：323)；

(16) The nucleotide sequence I comprises SEQ ID NO:324, and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO:325, a nucleotide sequence shown in seq id no:

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 said nucleotide sequence II is not SEQ ID NO:170, a step of;

(17) The nucleotide sequence I comprises SEQ ID NO:65, and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO:66, a nucleotide sequence shown in seq id no;

(18) The nucleotide sequence I comprises SEQ ID NO:75, and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO:76, a nucleotide sequence shown in seq id no;

(19) The nucleotide sequence I comprises SEQ ID NO:93, and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO:94, a nucleotide sequence shown in seq id no;

(20) The nucleotide sequence I comprises SEQ ID NO:103, and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO:104, a nucleotide sequence shown in seq id no;

(21) The nucleotide sequence I comprises SEQ ID NO:109, and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO:110, and a nucleotide sequence shown in seq id no;

(22) The nucleotide sequence I comprises SEQ ID NO:111, and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO:112, a nucleotide sequence shown in seq id no;

(23) The nucleotide sequence I comprises SEQ ID NO:115, and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO:116, a nucleotide sequence shown in seq id no;

(24) The nucleotide sequence I comprises SEQ ID NO:41, and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO:42, a nucleotide sequence shown in seq id no;

(25) The nucleotide sequence I comprises SEQ ID NO:41, and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO:188, a nucleotide sequence shown in seq id no;

(26) The nucleotide sequence I comprises SEQ ID NO:189 and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO:188, a nucleotide sequence shown in seq id no;

(27) The nucleotide sequence I comprises SEQ ID NO:41, and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO:190, a nucleotide sequence shown in seq id no;

(28) The nucleotide sequence I comprises SEQ ID NO:41, and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO:191, a nucleotide sequence shown in seq id no;

(29) The nucleotide sequence I comprises SEQ ID NO:41, and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO:192, and a nucleotide sequence shown in seq id no;

(30) The nucleotide sequence I comprises SEQ ID NO:27, and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO:28, a nucleotide sequence shown in seq id no;

(31) The nucleotide sequence I comprises SEQ ID NO:27, and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO: 183.

(32) The nucleotide sequence I comprises SEQ ID NO:184, and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO: 183.

(33) The nucleotide sequence I comprises SEQ ID NO:27, and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO:185, a nucleotide sequence set forth in seq id no;

(34) The nucleotide sequence I comprises SEQ ID NO:27, and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO:186, a nucleotide sequence shown as seq id no;

(35) The nucleotide sequence I comprises SEQ ID NO:27, and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO: 187;

(36) The nucleotide sequence I comprises SEQ ID NO:73, and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO:74, a nucleotide sequence shown in seq id no;

(37) The nucleotide sequence I comprises SEQ ID NO:83, and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO: 84.

(38) The nucleotide sequence I comprises SEQ ID NO:83, and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO: 193;

(39) The nucleotide sequence I comprises SEQ ID NO:194, and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO: 193;

(40) The nucleotide sequence I comprises SEQ ID NO:83, and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO:195, a nucleotide sequence shown in seq id no;

(41) The nucleotide sequence I comprises SEQ ID NO:83, and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO:196, a nucleotide sequence shown as seq id no;

(42) The nucleotide sequence I comprises SEQ ID NO:83, and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO: 197;

(43) The nucleotide sequence I comprises SEQ ID NO:97, and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO: 98.

(44) The nucleotide sequence I comprises SEQ ID NO:97, and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO:198, a nucleotide sequence as set forth in seq id no;

(45) The nucleotide sequence I comprises SEQ ID NO:199, and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO:198, a nucleotide sequence as set forth in seq id no;

(46) The nucleotide sequence I comprises SEQ ID NO:97, and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO: 200;

(47) The nucleotide sequence I comprises SEQ ID NO:97, and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO:201, a nucleotide sequence shown in seq id no;

(48) The nucleotide sequence I comprises SEQ ID NO:97, and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO:202, a nucleotide sequence shown in seq id no;

(49) The nucleotide sequence I comprises SEQ ID NO:143, and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO:144, a nucleotide sequence shown as seq id no;

(50) The nucleotide sequence I comprises SEQ ID NO:19, and the nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO:20, a nucleotide sequence shown in seq id no;

(51) The nucleotide sequence I comprises SEQ ID NO:19, and the nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO:178, a nucleotide sequence shown as 178;

(52) The nucleotide sequence I comprises SEQ ID NO:179, and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO:178, a nucleotide sequence shown as 178;

(53) The nucleotide sequence I comprises SEQ ID NO:19, and the nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO:180, a nucleotide sequence shown in seq id no;

(54) The nucleotide sequence I comprises SEQ ID NO:19, and the nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO: 181;

(55) The nucleotide sequence I comprises SEQ ID NO:19, and the nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO:182, and a nucleotide sequence shown as seq id no.

In one embodiment, the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, or fully reverse complementary; by substantially reverse complement is meant that there are no more than 3 base mismatches between the two nucleotide sequences; by substantially reverse complement is meant that there is no more than 1 base mismatch between the two nucleotide sequences; complete reverse complement refers to the absence of mismatches between two nucleotide sequences.

In one embodiment, the sense strand further comprises a nucleotide sequence III, the antisense strand further comprises a nucleotide sequence IV, the nucleotide sequence III and the nucleotide sequence IV are each independently 0-6 nucleotides in length, wherein the nucleotide sequence III is attached at the 5 'end of the nucleotide sequence I, the nucleotide sequence IV is attached at the 3' end of the nucleotide sequence II, the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or fully reverse complementary; by substantially reverse complement is meant that there is no more than 1 base mismatch between the two nucleotide sequences; complete reverse complement refers to the absence of mismatches between two nucleotide sequences; and/or the number of the groups of groups,

the nucleotide sequence III is connected at the 3 '-end of the nucleotide sequence I, the nucleotide sequence IV is connected at the 5' -end of the nucleotide sequence II, and the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary; by substantially reverse complement is meant that there is no more than 1 base mismatch between the two nucleotide sequences; complete reverse complement refers to the absence of mismatches between two nucleotide sequences.

In one embodiment, the sense strand further comprises nucleotide sequence V and/or the antisense strand further comprises nucleotide sequence VI, nucleotide sequences V and VI being 0 to 3 nucleotides in length, nucleotide sequence V being linked at the 3 'end of the sense strand constituting the 3' overhang of the sense strand and/or nucleotide sequence VI being linked at the 3 'end of the antisense strand constituting the 3' overhang of the antisense strand. In a preferred embodiment, the nucleotide sequence V or VI is 2 nucleotides in length. In a preferred embodiment, the nucleotide sequence V or VI is two consecutive thymidylate nucleotides or two consecutive uracil ribonucleotides. In a preferred embodiment, the nucleotide sequence V or VI is mismatched or complementary to a nucleotide at the corresponding position of the target mRNA.

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

In another embodiment, the sense strand or antisense strand has 15 to 30 nucleotides; preferably, the sense strand or antisense strand has 19-25 nucleotides; more preferably, the sense strand or the antisense 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 having a modification group; preferably, the phosphate group containing a modifying group is a phosphorothioate group formed by substitution of at least one oxygen atom of a phosphodiester bond in the phosphate group with a sulfur atom.

In one embodiment, the siRNA comprises a sense strand that does not comprise a 3' overhang nucleotide.

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

In one embodiment, the modified nucleotide is selected from the group consisting of a 2 '-fluoro modified nucleotide, a 2' -alkoxy modified nucleotide, a 2 '-substituted alkoxy modified nucleotide, a 2' -alkyl modified nucleotide, a 2 '-substituted alkyl modified nucleotide, a 2' -deoxy nucleotide, a 2 '-amino modified nucleotide, a 2' -substituted amino modified nucleotide, a nucleotide analog, or a combination of any two or more thereof.

In one embodiment, the modified nucleotide is selected from the group consisting of 2' -fluoro modified nucleotide, 2' -methoxy modified nucleotide, 2' -O-CH ₂ -CH ₂ -O-CH ₃ Modified nucleotides, 2' -O-CH ₂ -CH＝CH ₂ Modified nucleotides, 2' -CH ₂ -CH ₂ -CH＝CH ₂ Modified nucleotides, 2' -deoxynucleotides, 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' -fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, the remaining positions being non-fluoro modified nucleotides; the 2' -fluoro modified nucleotide is located at the even position of the antisense strand in the 5' to 3' direction, and the remaining positions are non-fluoro 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, the remaining positions being non-fluoro modified nucleotides; the 2' -fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand in the 5' to 3' direction, the remaining positions being non-fluoro 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, the remaining positions being non-fluoro modified nucleotides; the 2' -fluoro modified nucleotides are located at positions 2, 6, 8, 9, 14 and 16 of the antisense strand in the 5' to 3' direction, the remaining positions being non-fluoro 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, the remaining positions being non-fluoro modified nucleotides; the 2' -fluoro modified nucleotides are located at positions 2, 14 and 16 of the antisense strand in the 5' to 3' direction, with the remaining positions being non-fluoro modified nucleotides. In one embodiment, each non-fluoro modified nucleotide is a 2 '-methoxy modified nucleotide, which refers to a nucleotide formed by substitution of the 2' -hydroxy group of the ribosyl group with a methoxy group.

In one embodiment, each non-fluoro modified nucleotide is independently selected from one of a nucleotide or nucleotide analog formed by substitution of the hydroxyl group at the 2' -position of the ribosyl of the nucleotide with a non-fluoro group, the nucleotide analog being selected from one of an iso-nucleotide, 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, a GNA modified nucleotide, 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, the remaining positions being 2' -methoxy modified nucleotides; the 2 '-fluoro modified nucleotide is located at the even position of the antisense strand in the 5' to 3 'direction, with the remaining positions being 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, the remaining positions being 2' -methoxy modified nucleotides; the 2 '-fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand in the 5' to 3 'direction, with the remaining positions being 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, the remaining positions being 2' -methoxy modified nucleotides; the 2 '-fluoro modified nucleotides are located at positions 2, 6, 8, 9, 14 and 16 of the antisense strand in the 5' to 3 'direction, with the remaining positions being 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, the remaining positions being 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, GNA modified nucleotides are located at position 7 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, the remaining positions being 2' -methoxy modified nucleotides; according to the 5 'to 3' direction, 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.

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

a linkage between nucleotide 1 and nucleotide 2 at the 5' end of the sense strand;

a linkage between nucleotide 2 and nucleotide 3 at the 5' end of the sense strand;

a linkage between nucleotide 1 and nucleotide 2 at the 3' end of the sense strand;

a linkage between nucleotide 2 and nucleotide 3 at the 3' end of the sense strand;

a linkage between nucleotide 1 and nucleotide 2 at the 5' end of the antisense strand;

a linkage between nucleotide 2 and nucleotide 3 at the 5' end of the antisense strand;

a linkage between nucleotide 1 and nucleotide 2 at the 3' end of the antisense strand;

the 3' -end of the antisense strand is linked between nucleotide 2 and nucleotide 3.

In some embodiments, the siRNA is directed along the 5 'end toward the 3' end, and the sense strand comprises phosphorothioate groups at the positions shown below:

between nucleotide 1 and nucleotide 2 from the 5' end of the sense strand;

between nucleotide 2 and nucleotide 3 from the 5' end of the sense strand;

Between nucleotide 1 and nucleotide 2 from the 3' end of the sense strand;

between nucleotide 2 and nucleotide 3 from the 3' end of the sense strand;

or alternatively, the process may be performed,

the sense strand comprises phosphorothioate groups at the positions shown below:

between nucleotide 1 and nucleotide 2 from the 5' end of the sense strand;

between nucleotide 2 and nucleotide 3, starting at the 5' end of the sense strand.

In some embodiments, the siRNA is directed along the 5 'end toward the 3' end, and the antisense strand comprises phosphorothioate groups at the positions shown below:

between nucleotide 1 and nucleotide 2 from the 5' end of the antisense strand;

between nucleotide 2 and nucleotide 3 from the 5' end of the antisense strand;

between nucleotide 1 and nucleotide 2 from the 3' end of the antisense strand;

the antisense strand is between nucleotide 2 and nucleotide 3 from the 3' terminus.

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, a GNA modified nucleotide, 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, the remaining positions being between the 1 st and 2 nd nucleotides of the 5' end, between the 2 nd and 3 rd nucleotides of the 5' end, between the 1 st and 2 nd nucleotides of the 3' end, and between the 2 nd and 3 rd nucleotides of the 3' end being phosphorothioate linkages; according to the 5 'to 3' direction, the 2 '-fluoro modified nucleotide is located at the even number position of the antisense strand, the rest positions are 2' -methoxy modified nucleotides, the 1 st nucleotide and the 2 nd nucleotide of the 5 'end, the 2 nd nucleotide and the 3 rd nucleotide of the 5' end, the 1 st nucleotide and the 2 nd nucleotide of the 3 'end, and phosphorothioate group connection is formed between the 2 nd nucleotide and the 3 rd nucleotide of 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, the remaining positions being 2' -methoxy modified nucleotides, between nucleotide 1 and nucleotide 2 of the 5' end, phosphorothioate linkage between nucleotide 2 and nucleotide 3 of the 5' end, the 3' end being removed; the 2 '-fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand in the 5' to 3 'direction, the remaining positions being 2' -methoxy modified nucleotides, between the 1 st and 2 nd nucleotides at the 5 'end, between the 2 nd and 3 rd nucleotides at the 5' end, between the 1 st and 2 nd nucleotides at the 3 'end, and phosphorothioate linkages between the 2 nd and 3 rd 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, the remaining positions being between the 1 st and 2 nd nucleotides of the 5' end, between the 2 nd and 3 rd nucleotides of the 5' end, between the 1 st and 2 nd nucleotides of the 3' end, and between the 2 nd and 3 rd nucleotides of the 3' end being phosphorothioate linkages; the 2 '-fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand in the 5' to 3 'direction, the remaining positions being 2' -methoxy modified nucleotides, between the 1 st and 2 nd nucleotides at the 5 'end, between the 2 nd and 3 rd nucleotides at the 5' end, between the 1 st and 2 nd nucleotides at the 3 'end, and phosphorothioate linkages between the 2 nd and 3 rd 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, the remaining positions being between the 1 st and 2 nd nucleotides of the 5' end, between the 2 nd and 3 rd nucleotides of the 5' end, between the 1 st and 2 nd nucleotides of the 3' end, and between the 2 nd and 3 rd nucleotides of the 3' end being phosphorothioate linkages; the 2 '-fluoro modified nucleotides are located at positions 2, 6, 8, 9, 14 and 16 of the antisense strand in the 5' to 3 'direction, with the remaining positions being 2' -methoxy modified nucleotides, between the 1 st and 2 nd nucleotides at the 5 'end, between the 2 nd and 3 rd nucleotides at the 5' end, between the 1 st and 2 nd nucleotides at the 3 'end, and phosphorothioate linkages between the 2 nd and 3 rd nucleotides at the 3' end. In a preferred embodiment, the 2' monofluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, the remaining positions being between the 1 st and 2 nd nucleotides of the 5' end, between the 2 nd and 3 rd nucleotides of the 5' end, between the 1 st and 2 nd nucleotides of the 3' end, and between the 2 nd and 3 rd nucleotides of the 3' end being phosphorothioate linkages; 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, the rest positions are 2' -methoxy modified nucleotides, between 1 st and 2 nd nucleotides at the 5 'end, between 2 nd and 3 rd nucleotides at the 5' end, between 1 st and 2 nd nucleotides at the 3 'end, and phosphorothioate linkage between 2 nd and 3 rd 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, the remaining positions being between the 1 st and 2 nd nucleotides of the 5' end, between the 2 nd and 3 rd nucleotides of the 5' end, between the 1 st and 2 nd nucleotides of the 3' end, and between the 2 nd and 3 rd nucleotides of the 3' end being phosphorothioate linkages; according to the 5 'to 3' direction, 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, the rest positions are 2' -methoxy modified nucleotides, between 1 st and 2 nd nucleotides at the 5 'end, between 2 nd and 3 rd nucleotides at the 5' end, between 1 st and 2 nd nucleotides at the 3 'end, and phosphorothioate linkage between 2 nd and 3 rd nucleotides at the 3' end.

In a specific embodiment, the present invention provides an siRNA selected from Table 1, wherein said siRNA is not N-ER-FY007031, N-ER-FY007038, N-ER-FY 007026, 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-FY007033, N-ER-FY007027, N-ER-FY007005, N-ER-FY007030, N-ER-FY 051, N-ER-FY007032, N-ER-FY007033, N-ER-F034, N-ER-FY 007036; the method comprises the following steps of N-ER-FY007075M5, N-ER-FY007078M2D2, N-ER-FY007078M3, N-ER-FY007078M4, N-ER-FY007078M5, N-ER-FY007079M2D2, N-ER-FY007079M3, N-ER-FY007079M4, N-ER-FY007079M5, N-ER-FY007078M 080, N-ER-FY007080M2D2, N-ER-FY007080M3, N-ER-FY007079M4, N-ER-FY007080M 5N-ER-FY 007082, N-ER-FY007082M2D2, N-ER-FY007082M3, N-ER-FY007082M4, N-ER-FY007082M5, N-ER-FY007083M2D2, N-ER-FY007083M3, N-ER-FY007083M4, N-ER-FY007083M5, N-ER-FY007085M2D2, N-ER-FY007085M3, N-ER-FY007085M4, N-ER-FY007085M5.

The present invention also provides an siRNA conjugate comprising the siRNA of the present invention and a conjugate group conjugated to the siRNA (as shown below, a duplex structure represents the siRNA, and the conjugate group is attached to the 3' -end of the sense strand of the siRNA):

x in the above conjugate structure may be selected as O or S, and in one embodiment X is O.

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

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, the 3' end of the antisense strand having 1-3 protruding nucleotides extending out of the double-stranded region;

or alternatively, the process may be performed,

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 3' end of the antisense strand forms a blunt end.

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

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

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

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

The invention also provides the use of the siRNA of the invention, or the siRNA conjugate of the invention, or the pharmaceutical composition of the invention, for the preparation of a medicament for inhibiting HSD17B13 gene expression.

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

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

The invention also provides a method of inhibiting HSD17B13 gene expression comprising contacting or administering to a subject in need thereof a therapeutically effective amount of an siRNA of the invention, or an siRNA conjugate of the invention, or a pharmaceutical composition of the invention, with a cell expressing HSD17B 13.

The invention also provides a method for treating and/or preventing a disease associated with overexpression of the HSD17B13 gene, comprising administering to a subject in need thereof a therapeutically effective amount of an siRNA of the invention, or an siRNA conjugate of the invention, or a pharmaceutical composition of the invention.

Advantageous effects

The siRNA, the pharmaceutical composition and the siRNA conjugate provided by the application show excellent HSD17B13 gene expression inhibition activity in vitro cell experiments, and have good potential for treating diseases related to HSD17B13 gene overexpression. For example, the siRNA and the conjugate thereof disclosed by the application can reduce the expression of HSD17B13mRNA in liver, have low toxic and side effects and good plasma stability, and have good clinical application prospect.

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

In some embodiments, the sirnas provided herein have a high HSD17B13 gene inhibitory activity in Huh7 cells, e.g., IC ₅₀ As low as 12pM.

In some embodiments, the siRNA conjugates provided herein have a high HSD17B13 gene inhibitory activity in PHH cells. For example, when entering PHH cells by free uptake, IC ₅₀ As low as 82pM; when (when)IC when entering PHH cells by transfection ₅₀ Can be as low as 1.2pM.

Detailed Description

Definition of the definition

Throughout the specification, unless otherwise indicated, "G", "C", "a", "T" and "U" generally represent bases of guanine, cytosine, adenine, thymine, uracil, respectively, but it is also generally known in the art that "G", "C", "a", "T" and "U" each also generally represent nucleotides containing guanine, cytosine, adenine, thymine and uracil, respectively, as bases, which is a common manner in the expression of deoxyribonucleic acid sequences and/or ribonucleic acid sequences, and thus in the context of the present disclosure, the meaning of "G", "C", "a", "T", "U" includes the various possible scenarios described above. Lowercase letters a, u, c, g: a nucleotide representing 2' -methoxy modification; af. Gf, cf, uf: a 2' -fluoro modified nucleotide; the lower case letter s indicates that phosphorothioate linkages are between two nucleotides adjacent to the letter s; p1: indicating that the adjacent nucleotide to the right of P1 is a nucleotide 5' -phosphate;A、U、C、G(underlined + bold + italic): indicating GNA modified nucleotides.

In the above and in the following, 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 substituted with fluorine. "non-fluoro modified nucleotide" refers to a nucleotide or nucleotide analogue in which the hydroxyl group at the 2' -position of the ribosyl of the nucleotide is replaced with a non-fluoro group. In some embodiments, each non-fluoro modified nucleotide is independently selected from one of the nucleotides or nucleotide analogs formed by substitution of the hydroxyl group at the 2' position of the ribosyl of the nucleotide with a non-fluoro group. Nucleotides in which the hydroxyl group at the 2 '-position of the ribosyl group is substituted with a non-fluorine group are well known to those skilled in the art, and may be selected from one of 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 nucleotides, and 2' -deoxynucleotides.

"alkyl" includes straight, 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. Exemplary, "C _1-6 "C in" alkyl _1-6 "refers to a group comprising an array of straight, branched, or cyclic forms of 1, 2, 3, 4, 5, or 6 carbon atoms.

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

"nucleotide analog" refers to a group that is capable of replacing a nucleotide in a nucleic acid, but that differs in structure from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide. Such as an iso-nucleotide, a bridged nucleotide (bridged nucleic acid, abbreviated as BNA) or an acyclic nucleotide.

BNA refers to a constrained or inaccessible nucleotide. BNA may contain a five-, six-, or seven-membered ring bridging structure with "fixed" C3' -endo-saccharides tucked. The bridge is typically incorporated at the 2'-, 4' -position of the ribose to provide a 2',4' -BNA nucleotide, such as LNA, ENA, cret BNA, etc., where LNA is shown in formula (1), ENA is shown in formula (2), cret BNA is shown in formula (3).

Acyclic nucleotides are a class of nucleotides in which the sugar ring of the nucleotide is opened, such as an Unlocking Nucleic Acid (UNA) or a Glycero Nucleic Acid (GNA), wherein UNA is represented by formula (4) and GNA is represented by formula (5).

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

The term "oligonucleotide" refers to a compound in which the position of a base on a ribose ring is changed, for example, a compound in which a base is shifted from the 1' -position to the 2' -position or the 3' -position of a ribose ring, as shown in formula (6) or (7).

In the compounds of the above formulae (6) to (7), base represents a Base, for example A, U, G, C or T; r is selected from H, OH, F or a non-fluorine group as described above.

In some embodiments, the nucleotide analog is selected from one of an iso-nucleotide, LNA, ENA, cET BNA, UNA, and GNA. In some embodiments, each non-fluoro 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, which in the foregoing and hereinafter refers to a nucleotide formed by substitution of the 2' -hydroxy group of the ribosyl group with a methoxy group.

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

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

In the above and in the following, 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 is no more than 1 base mismatch between two nucleotide sequences; "complete reverse complement" 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 is changed as compared with the former, for example, when one nucleotide base is A in the latter, when the corresponding nucleotide base at the same position in the former is U, C, G or T, it is determined that there is a nucleotide difference between the two nucleotide sequences at the position. In some embodiments, a nucleotide difference is also considered to occur at an original position when the nucleotide is replaced with an abasic nucleotide or its equivalent.

In this context, "overhang" refers to one or more unpaired nucleotides that protrude from the duplex structure of an siRNA when one 3 'end of one strand extends beyond the 5' end of the other strand, or vice versa. By "blunt end" or "blunt end" is meant that there are no unpaired nucleotides at that end of the siRNA, i.e., no nucleotide overhangs. A "blunt-ended" siRNA is one that is double-stranded throughout its length, i.e., has no nucleotide overhangs at either end of the molecule.

In the context of the present application, and in particular in describing the methods of preparation of the siRNA, pharmaceutical compositions or siRNA conjugates of the present application, the nucleoside monomers refer to modified or unmodified nucleoside phosphoramidite monomers used in solid phase phosphoramidite synthesis, depending on the type and order of nucleotides in the siRNA or siRNA conjugate to be prepared, unless otherwise specified. Solid phase phosphoramidite synthesis is a method used in RNA synthesis well known to those skilled in the art. Nucleoside monomers useful in the present application are all commercially available.

In the context of the present application, unless otherwise indicated, "conjugated" means that two or more chemical moieties each having a specific function are linked to each other by covalent linkage; accordingly, "conjugate" refers to a compound formed by covalent linkage between the chemical moieties. Further, "siRNA conjugate" means a compound formed by covalently attaching one or more chemical moieties having specific functions to an siRNA. siRNA conjugates are understood to be, depending on the context, the collective term of multiple siRNA conjugates or siRNA conjugates of a certain chemical formula. In the context of the present specification, a "conjugate molecule" is understood to be a specific compound that can be conjugated to an siRNA by reaction, ultimately forming the siRNA conjugate of the present application.

Various hydroxyl protecting groups may be used in the present application. In general, the protecting group renders the chemical functional group insensitive to specific reaction conditions and may be appended to and removed from the functional group in the molecule without substantially damaging the remainder of the molecule. In some embodiments, the protecting group is stable under alkaline 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-phenylxanthin-9-yl (Pixyl), and 9- (p-methoxyphenyl) xanthin-9-yl (Mox). In some embodiments, non-exclusive examples of hydroxyl protecting groups that may be used herein include Tr (trityl), MMTr (4-methoxytrityl), DMTr (4, 4 '-dimethoxytrityl), and TMTr (4, 4',4 "-trimethoxytrityl).

As used herein, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

The term "subject" as used herein refers to any animal, such as a mammal or a pouched animal. 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 kind of poultry.

As used herein, "treatment" refers to a method of achieving a beneficial or desired result, including but not limited to therapeutic benefit. By "therapeutic benefit" is meant eradication or amelioration of the underlying disorder being treated. In addition, therapeutic benefit is obtained by eradicating or ameliorating one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, although the subject may still be afflicted with the underlying disorder.

As used herein, "preventing" refers to a method of achieving a beneficial or desired result, including but not limited to a prophylactic benefit. To obtain a "prophylactic benefit," the siRNA, siRNA conjugate, or pharmaceutical composition can be administered to a subject at risk of suffering from a particular disease, or to a subject reporting one or more physiological symptoms of the disease, even though a diagnosis of the disease may not have been made.

siRNA

The present application relates to an siRNA capable of inhibiting HSD17B13 gene expression. The siRNA of the present application contains a nucleotide group as a basic structural unit, which is well known to those skilled in the art, and contains a phosphate group, a ribose group, and a base. Typically, active, i.e., functional, siRNAs are about 12 to 40 nucleotides in length, and in some embodiments about 1 5 to 30 nucleotides in length.

The siRNA of the present application comprises a sense strand and an antisense strand, each nucleotide in the siRNA being independently a modified or unmodified nucleotide, wherein the sense strand comprises a stretch of nucleotide sequence I and the antisense strand comprises a stretch of nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II being at least partially reverse complementary to form a double-stranded region. In some embodiments, the double stranded region is 1 5-30 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 comprises nucleotide sequence III, the antisense strand further comprises nucleotide sequence IV, nucleotide sequence III and nucleotide sequence IV are each independently 0-6 nucleotides in length, the nucleotide sequence III is attached at the 5 'end of nucleotide sequence I, the nucleotide sequence IV is attached at the 3' end of nucleotide sequence II, the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or fully reverse complementary; by substantially reverse complement is meant that there is no more than 1 base mismatch between the two nucleotide sequences; complete reverse complement refers to the absence of mismatches between two nucleotide sequences. In some embodiments, the sense strand further comprises nucleotide sequence III, the antisense strand further comprises nucleotide sequence IV, nucleotide sequence III and nucleotide sequence IV are each independently 0-6 nucleotides in length, the nucleotide sequence III is attached at the 3 'end of nucleotide sequence I, the nucleotide sequence IV is attached at the 5' end of nucleotide sequence II, the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or fully reverse complementary; by substantially reverse complement is meant that there is no more than 1 base mismatch between the two nucleotide sequences; complete reverse complement refers to the absence of mismatches between two nucleotide sequences. In some embodiments, the sense strand further comprises nucleotide sequence III, the antisense strand further comprises nucleotide sequence IV, nucleotide sequence III and nucleotide sequence IV are each independently 0-6 nucleotides in length, the nucleotide sequence III is attached at the 5 'end of nucleotide sequence I, the nucleotide sequence IV is attached at the 3' end of nucleotide sequence II, the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or fully reverse complementary; and the nucleotide sequence III is linked at the 3 'end of the nucleotide sequence I, the nucleotide sequence IV is linked at the 5' end of the nucleotide sequence II, the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary; by substantially reverse complement is meant that there is no more than 1 base mismatch between the two nucleotide sequences; complete reverse complement refers to the absence of mismatches between two nucleotide sequences.

In some embodiments, the sense strand further comprises nucleotide sequence V and/or the antisense strand further comprises nucleotide sequence VI, nucleotide sequences V and VI being 0 to 3 nucleotides in length, nucleotide sequence V being attached at the 3 'end of the sense strand to form a 3' overhang of the sense strand and/or nucleotide sequence VI being attached at the 3 'end of the antisense strand to form a 3' overhang of the antisense strand. 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 thymidines or two consecutive uracils. In other embodiments, the nucleotide sequence V or VI is mismatched or complementary to a nucleotide at a corresponding position in the target mRNA.

The sense and antisense strands provided herein are the same or different in length, and in some embodiments, the sense or antisense strand has 15-30 nucleotides. In other embodiments, the sense strand or the antisense strand has 19 to 25 nucleotides. In other embodiments, the sense strand or the antisense strand has 19 to 23 nucleotides. The length ratio of the sense strand and the antisense strand of the siRNA provided herein 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 siRNA has a length ratio of the sense strand to the antisense strand of 19/19, 21/21, 19/21, 21/23 or 23/23, at which time the siRNA of the present disclosure has better cellular mRNA silencing activity.

It was found that different modification strategies can have distinct effects on the stability, bioactivity, cytotoxicity, etc. of siRNA. For example, various strategies for chemical modification of siRNA were studied in CN102140458B, demonstrating 7 effective modifications, one of which resulted in siRNA that improved blood stability while maintaining substantially equivalent inhibition activity as compared to unmodified siRNA.

The nucleotides in the siRNA of the invention are each independently modified or unmodified nucleotides. In some embodiments, each nucleotide in the siRNA of the invention is an unmodified nucleotide; in some embodiments, some or all of the nucleotides in the siRNA of the invention are modified nucleotides, and such modifications on the nucleotide groups do not result in a significant impairment or loss of the function of the siRNA of the invention to inhibit HSD17B13 gene expression.

In some embodiments, the siRNA of the present application contains at least 1 modified nucleotide. In the context of the present application, the term "modified nucleotide" refers to a nucleotide or nucleotide analogue formed by substitution of the hydroxyl group at the 2' -position of the ribosyl of the nucleotide with other groups, or a nucleotide having a modified base. The modified nucleotide does not result in a significant impairment or loss of function of the siRNA to inhibit gene expression. For example, J.K.Watts, G.F.Deleavey and m.j.damha, chemically modified siRNA may be selected: tools and applications. Drug discovery Today,2008, 13 (19-20): 842-55.

In some embodiments, at least one nucleotide in the sense strand or the antisense strand of the siRNA provided herein is a modified nucleotide, and/or at least one phosphate group is a phosphate group having a modifying group; in other words, at least a portion of the phosphate groups and/or ribose groups in at least one single-stranded phosphate-sugar backbone in the sense strand and the antisense strand are phosphate groups and/or ribose groups having a modifying group. In some embodiments, the phosphate group containing a modifying group is a phosphorothioate group formed by substitution of at least one oxygen atom of the phosphodiester bond in the phosphate group with a sulfur atom.

In some embodiments, the siRNA comprises a sense strand that does not comprise a 3' overhang nucleotide; that is, the sense strand of the siRNA may have 3' overhang nucleotides that are removed from the sense strand to form blunt ends.

In some embodiments, when there are no protruding nucleotides at the 3 'end of the sense strand after the nucleotide sequence of the sense strand and the nucleotide sequence of the antisense strand are complementary to form a double-stranded region, a nucleotide sequence V is added at the 3' end of the sense strand as the protruding nucleotide. Then, when the nucleotide sequence V is linked to the 3' -end of the sense strand, the nucleotide sequence V is excluded after the chemical modification is completed, and accordingly, the sense strand of the siRNA forms a blunt end.

In some embodiments, when the 3 'end of the sense strand has a protruding nucleotide extending out of the double-stranded region after the nucleotide sequence of the antisense strand is complementary to the nucleotide sequence of the sense strand, the protruding nucleotide at the 3' end of the sense strand is excluded as the nucleotide sequence of the sense strand, and accordingly, the sense strand of the 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. Exemplary 5' phosphate groups have the structure:the structure of the 5' phosphate derivative group includes, but is not limited toThe method is limited to:etc. />

The nucleotide at the 5' end of the sense or antisense strand is linked to a 5' phosphate group or 5' phosphate derivative group to form the structure shown below:

wherein Base represents a Base, such as A, U, G, C or T. R 'is hydroxyl or substituted with 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 nucleotides, 2' -deoxynucleotides.

Exemplary modified nucleotides have the structure shown below:

wherein Base represents a Base, such as A, U, G, C or T. The hydroxyl group at the 2' -position of the ribose group is substituted by R. The hydroxyl group at the 2 '-position of these ribose groups may be substituted with various groups known to those skilled in the art, such as, 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 nucleotides, 2' -deoxynucleotides.

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

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

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

In some embodiments, all of the nucleotides in the sense strand and/or the antisense strand are modified nucleotides. In some embodiments, each nucleotide in the sense strand and the antisense strand of the siRNA provided herein is independently a 2' -fluoro modified nucleotide or a non-fluoro modified nucleotide. In some embodiments, each non-fluoro modified nucleotide is a 2 '-methoxy modified nucleotide or GNA modified nucleotide, which refers to a nucleotide formed by substitution of the 2' -hydroxy of the ribosyl group with methoxy.

In some 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, with the remaining positions being non-fluoro modified nucleotides; the 2' -fluoro modified nucleotide is located at the even position of the antisense strand in the 5' to 3' direction, and the remaining positions are non-fluoro modified nucleotides. In some 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, with the remaining positions being non-fluoro modified nucleotides; the 2' -fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand in the 5' to 3' direction, the remaining positions being non-fluoro modified nucleotides. In some 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, with the remaining positions being non-fluoro modified nucleotides; the 2' -fluoro modified nucleotides are located at positions 2, 6, 8, 9, 14 and 16 of the antisense strand in the 5' to 3' direction, the remaining positions being non-fluoro 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, the remaining positions being non-fluoro modified nucleotides; the 2' -fluoro modified nucleotides are located at positions 2, 14 and 16 of the antisense strand in the 5' to 3' direction, with the remaining positions being non-fluoro 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, the remaining positions being 2' -methoxy modified nucleotides; the 2 '-fluoro modified nucleotide is located at the even position of the antisense strand in the 5' to 3 'direction, with the remaining positions being 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, the remaining positions being 2' -methoxy modified nucleotides; the 2 '-fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand in the 5' to 3 'direction, with the remaining positions being 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, the remaining positions being 2' -methoxy modified nucleotides; the 2 '-fluoro modified nucleotides are located at positions 2, 6, 8, 9, 14 and 16 of the antisense strand in the 5' to 3 'direction, with the remaining positions being 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, the remaining positions being 2' -methoxy modified nucleotides; according to the 5 'to 3' direction, 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. 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, the remaining positions being 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, GNA modified nucleotides are located at position 7 of the antisense strand, and the remaining positions are 2' -methoxy modified nucleotides. In some more preferred embodiments, each non-fluoro modified nucleotide is a 2 '-methoxy modified nucleotide, which refers to a nucleotide formed by substitution of the 2' -hydroxy group of the ribosyl group with a methoxy group.

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

between nucleotide 1 and nucleotide 2 from the 5' end of the sense strand;

between nucleotide 2 and nucleotide 3 from the 5' end of the sense strand;

Between nucleotide 1 and nucleotide 2 from the 3' end of the sense strand;

between nucleotide 2 and nucleotide 3 from the 3' end of the sense strand;

or alternatively, the process may be performed,

between nucleotide 1 and nucleotide 2 from the 5' end of the sense strand;

between nucleotide 1 and nucleotide 2 from the 5' end of the antisense strand;

between nucleotide 2 and nucleotide 3 from the 5' end of the antisense strand;

between nucleotide 1 and nucleotide 2 from the 3' end of the antisense strand;

siRNA conjugates

The present application relates to an siRNA conjugate comprising the above-described siRNA and a conjugate group conjugated to the siRNA.

In this application, the sense strand and the antisense strand of the siRNA conjugate form a 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 nucleotides extending out of the double-stranded region. In other 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 forms a blunt end.

In some preferred embodiments, the siRNA conjugate is obtained by conjugation of an siRNA to a conjugate group. Wherein the sense strand and the antisense strand of the siRNA are complementary to form a double-stranded region of the siRNA, and the 3 'end of the sense strand of the siRNA forms a blunt end, and the conjugate group is conjugated to the 3' end of the sense strand having the blunt end to form an siRNA conjugate.

In some preferred embodiments, the 3 'end of the sense strand of the siRNA has a protruding nucleotide extending out of the double-stranded region, and the sequence with a 3' blunt end formed after excluding the protruding nucleotide located at the 3 'end in the sense strand is used as the nucleotide sequence for linking the conjugate group, and the conjugate group is linked at the 3' blunt end of the sense strand to form the siRNA conjugate.

In some more preferred embodiments, when there are no protruding nucleotides at the 3 'end of the sense strand after the sense strand is complementary to the nucleotide sequence of the antisense strand to form a double-stranded region, nucleotide sequence V is added at the 3' end of the sense strand as the protruding nucleotide. The 3' -blunt-ended sequence formed after excluding the protruding nucleotide located at the 3' -end in the sense strand is used as a nucleotide sequence for linking the conjugate group, and the conjugate group is linked at the 3' -blunt end of the sense strand to form the siRNA conjugate.

In some more preferred embodiments, when the 3 'end of the sense strand has a protruding nucleotide extending out of the double-stranded region after the nucleotide sequence of the antisense strand is complementary to the nucleotide sequence of the sense strand, the sequence with a 3' blunt end formed after the removal of the protruding nucleotide located at the 3 'end in the sense strand is used as the nucleotide sequence for linking the conjugate group, and the conjugate group is linked at the 3' blunt end of the sense strand to form the siRNA conjugate.

Illustratively, an siRNA of the sequence shown as N-ER-FY 007015M 1, having a protruding nucleotide at the 3 'end of the sense strand extending beyond the double-stranded region, has the gsascuacuufufufugfgffuaugca blunt end sequence formed after exclusion of the protruding-tst nucleotide located at the 3' end in the sense strand as the nucleotide sequence for attachment of the L96 conjugate group, thus forming an siRNA conjugate of the sequence: the sense strand is gsascuafuafufufguaauugcal 96 and the antisense strand is Plusgcaffaafafafafafafafafafafafaffugcsfsst.

In general, the conjugate group comprises at least one pharmaceutically acceptable targeting group, or further comprises a linker (linker), and the siRNA, the linker and the targeting group are sequentially linked. In some embodiments, the targeting group is 1-6. In some embodiments, the targeting group is 2-4. The siRNA molecule may be non-covalently or covalently conjugated to the conjugate group, e.g., may be covalently conjugated to the conjugate group. The conjugation site of the siRNA to the conjugation group may be at the 3' end or the 5' end of the sense strand of the siRNA, or at the 5' end of the antisense strand, or in the internal sequence of the siRNA. In some embodiments, the conjugation site of the siRNA to the conjugation group is at the 3' end of the sense strand of the siRNA.

In some embodiments, the conjugate group may be attached to the phosphate group, the 2' -hydroxyl group, or the base of the nucleotide. In some embodiments, the conjugate group may also be attached to the 3' -hydroxyl group, in which case the nucleotides are linked using a 2' -5' phosphodiester linkage. When a conjugate group is attached to the end of the siRNA strand, the conjugate group is typically attached to the phosphate group of the nucleotide; when a conjugate group is attached to the internal sequence of the siRNA, the conjugate group is typically attached to a ribose sugar ring or base. Various connection means can be referred to as: muthiah Manoharan et al 1.SiRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatoducts. ACS Chemical biology,2015, 10 (5): 1181-7.

In some embodiments, the siRNA and the conjugate group may be linked by acid labile, or reducible, chemical bonds that degrade in the acidic environment of the intracellular inclusion bodies, thereby allowing the siRNA to be in a free state. For non-degradable conjugation, the conjugation group can be attached to the sense strand of the siRNA, thereby minimizing the effect of conjugation on siRNA activity.

In some embodiments, the pharmaceutically acceptable targeting group can be a ligand conventionally used in the art of siRNA administration, such as the various ligands described in WO2009082607A2, which is incorporated herein by reference in its entirety.

In some embodiments, the pharmaceutically acceptable targeting group may be selected from one or more of the following ligands formed by the targeting molecule or derivative thereof: lipophilic molecules, such as cholesterol, bile acids, vitamins (e.g. vitamin E), lipid molecules of different chain lengths; polymers, such as polyethylene glycol; polypeptides, such as permeabilizing peptides; an aptamer; an antibody; a quantum dot; sugars, such as lactose, mannose, galactose, N-acetylgalactosamine (GalNAc); folic acid (folate); receptor ligands expressed by hepatic parenchymal cells, such as asialoglycoproteins, asialoglycoresidues, lipoproteins (e.g., high density lipoproteins, low density lipoproteins, etc.), glucagon, neurotransmitters (e.g., 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 class of these ligands is well known to those skilled in the art and generally functions to bind to specific receptors on the surface of target cells, mediating delivery of siRNA linked to the ligand to the target cells.

In some embodiments, the pharmaceutically acceptable targeting group may be any ligand that binds to an asialoglycoprotein receptor (ASGPR) on the surface of mammalian hepatocytes. In some embodiments, each ligand is independently an asialoglycoprotein, such as an asialooomolecular mucin (ASOR) or an Asialofetuin (ASF). In some embodiments, the ligand is a sugar or a derivative of a sugar.

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 may be a monosaccharide, disaccharide, or 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 a polysaccharide, a modified polysaccharide, a monosaccharide, a modified monosaccharide, a polysaccharide derivative, or a monosaccharide derivative. In some embodiments, each or at least one ligand is selected from the group consisting of: glucose and its derivatives, mannans and its derivatives, galactose and its derivatives, xylose and its derivatives, 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-xylose furanose, L-xylose furanose, D-glucose, L-glucose, D-galactose, L-galactose, alpha-D-mannopyranose, beta-D-glucopyranose, alpha-D-glucopyranose, beta-D-glucopyranose, alpha-D-fructofuranose, alpha-D-fructopyranose, alpha-D-galactopyranose, beta-D-galactopyranose, alpha-D-galactofuranose, beta-D-galactosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-N-galactosamine, N-isobutyramide, 2-amino-O-3-carboxyethyl-2-deoxy2-D-deoxygalactopyranose, 2-deoxy2-D-deoxygalactopyranose, 4-D-deoxy2-deoxygalactopyranose 2-deoxy-2-sulphonamino-D-glucopyranose, N-glycolyl- 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-galactopyranose, 3,4,6, 7-tetra-O-acetyl-2-deoxy-1, 5-dithio- α -D-glucoheptopyranoside ethyl ester, 2, 5-anhydro-D-allose nitrile, ribose, D-4-thioribose, L-ribose or L-4-thioribose. Further choices of the ligands can be found in, for example, the description of CN105378082a, incorporated by reference in its entirety.

In some embodiments, the pharmaceutically acceptable targeting group in the siRNA conjugate may be galactose or N-acetylgalactosamine, wherein the galactose or N-acetylgalactosamine molecule may be monovalent, divalent, trivalent, tetravalent. It should be understood that monovalent, divalent, trivalent, tetravalent, as used herein, refer to the molar ratio of siRNA molecule to galactose or N-acetylgalactosamine molecule in the siRNA conjugate being 1:1, 1:2, 1:3, or 1:4, respectively, after the siRNA molecule forms an siRNA conjugate with a conjugate group containing galactose or N-acetylgalactosamine molecule as a targeting group. In some embodiments, the pharmaceutically acceptable targeting group is N-acetylgalactosamine. In some embodiments, when the siRNA described herein is conjugated to a conjugate group comprising N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent or tetravalent. In some embodiments, the N-acetylgalactosamine molecule is trivalent when the siRNA described herein is conjugated to a conjugate group comprising N-acetylgalactosamine.

The targeting group can be attached to the siRNA molecule via a suitable linker, which can be selected by one skilled in the art depending on the particular type of targeting group. The types of these linkers, targeting groups and the manner of attachment to the siRNA can be found in the disclosure of WO2015006740A2, which is incorporated by reference in its entirety.

Method for synthesizing siRNA

The nucleoside monomers are linked one by one in the 3'-5' direction according to the nucleotide arrangement sequence by a solid-phase phosphoramidite method conventional in the art. Each nucleoside monomer attached includes four steps of deprotection, coupling, capping, oxidation or vulcanization. Wherein, when two nucleotides are connected by phosphate, the connection of the latter nucleoside monomer comprises deprotection, coupling, capping and oxidation. When phosphorothioate is adopted to connect two nucleotides, the following nucleoside monomer is connected, and the four steps of protection, coupling, capping and vulcanization are included.

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

the deprotection conditions include: the reaction temperature is 25 ℃, the reaction time is 70 seconds, the deprotection reagent is selected from dichloromethane solution (3%V/V) of dichloroacetic acid, and the molar ratio of the deprotection reagent to the protecting groups on the solid phase carrier is 5:1.

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

The capping reaction conditions include: the reaction temperature was 25℃and the reaction time was 1 5 seconds, the capping reagent was selected from a mixed solution of CapA (10% acetic anhydride acetonitrile solution) and CapB (10% N-methylimidazole pyridine/acetonitrile solution) in a molar ratio of 1:1, and the molar ratio of the capping reagent to the nucleic acid sequence attached to the solid support was acetic anhydride to N-methylimidazole to the nucleic acid sequence attached to the solid support was 1:1:1.

The oxidation reaction conditions include: the reaction temperature was 25℃and the reaction time was 15 seconds, the oxidizing agent was selected from a 0.05M solution of iodotetrahydrofuran, and the molar ratio of the oxidizing agent to the nucleic acid sequence attached to the solid support in the coupling step was 30:1.

The vulcanization reaction conditions include: the reaction temperature was 25℃and the reaction time was 300 seconds, the sulfiding reagent was selected from the group consisting of hydrogenation Huang Yuansu, and the molar ratio of sulfiding reagent to nucleic acid sequence attached to the solid support in the coupling step was 120:1.

After all nucleoside monomers are connected, the nucleic acid sequences connected on the solid phase carrier are sequentially subjected to cutting, deprotection, purification and desalination to obtain siRNA sense strand and antisense strand, and finally the two strands are subjected to heating annealing 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 to which the solid phase carrier is attached with concentrated ammonia water; purification by chromatography; desalting by reverse phase chromatography; by mixing the sense strand and the antisense strand in equimolar ratio under different strict conditions, the temperature is gradually reduced and cooled.

siRNA conjugate synthesis method

In the first step, DMTR-L96 is reacted with succinic anhydride to give compound L96-A:

The preparation process comprises the following steps: adding DMTR-L96, succinic anhydride, 4-dimethylaminopyridine and diisopropylethylamine into dichloromethane, stirring and reacting for 24 hours at 25 ℃, then washing the reaction liquid with 0.5M triethylamine phosphate, washing the water phase with dichloromethane for three times, merging the organic phases, and evaporating the organic phases under reduced pressure to obtain a crude product; then purifying by column chromatography to obtain the pure L96-A.

Second, L96-A is reacted with NH ₂ SPS reaction gives L96-B:

the preparation process comprises the following steps: L96-A, O-benzotriazol-tetramethyluronium Hexafluorophosphate (HBTU) and Diisopropylethylamine (DIPEA) were mixed and dissolved in acetonitrile, stirred at room temperature for 5 minutes to give a homogeneous solution, and aminomethyl resin (NH) was added ₂ SPS,100-200 meshes) into a reaction liquid, starting a shaking table reaction at 25 ℃, filtering after 18 hours of reaction, and washing a filter cake by dichloromethane and acetonitrile in sequence to obtain the filter cake. Capping the filter cake with CapA/CapB mixed solution to obtain L96-B, namely a solid phase carrier containing conjugate molecules, connecting nucleoside monomers to the conjugate molecules under the coupling reaction, synthesizing siRNA sense strand connected to the conjugate molecules according to the siRNA molecule synthesis method, synthesizing siRNA antisense strand by adopting the siRNA molecule synthesis method, and annealing to generate the siRNA conjugate.

Pharmaceutical composition

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

The pharmaceutically acceptable carrier may be a carrier conventionally used in the field of siRNA administration, such as, but not limited to, lipid nanoparticles (Lipid Nanoparticle, LNP),Magnetic nanoparticles (magnetic nanoparticles, e.g. based on Fe ₃ O ₄ Or Fe (Fe) ₂ O ₃ Carbon nanotubes (carbon nanotubes), mesoporous silica (mesosilica), calcium phosphate nanoparticles (calcium phosphate nanoparticles), polyethylenimine (PEI), polyamide dendrimers (polyamidoamine (PAMAM) dendrimer), polylysine (poly L-lysine), PLL), chitosan (chitosan), 1,2-dioleoyl-3-trimethylammonium propane (1, 2-dioleoyl-3-trimethylum-propane, DOTAP), poly D-or L-lactic acid/glycolic acid copolymers (poly (D)&L-lactic/glycolic acid) copolymer, PLGA), poly (aminoethylethylene phosphate) (poly (2-aminoethyl ethylene phosphate), PPEEA) and poly (N, N-dimethylaminoethyl methacrylate) (poly (2-dimethylaminoethyl methacrylate), PDMAEMA) and derivatives thereof.

The content of the siRNA and the pharmaceutically acceptable carrier in the pharmaceutical composition is not particularly required, and can be the conventional content of each component.

In some embodiments, the pharmaceutical composition may further comprise other pharmaceutically acceptable excipients, which may be one or more of various formulations or compounds conventionally employed in the art. For example, the pharmaceutically acceptable additional excipients may include at least one of a pH buffer, a protectant, and an osmolality adjusting agent.

The pH buffer solution can be a tris hydrochloride buffer solution with the pH value of 7.5-8.5 and/or a phosphate buffer solution with the pH value of 5.5-8.5, for example, the pH value of 5.5-8.5.

The protective agent may be at least one of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose, and glucose. The protective agent may be present in an amount of 0.01 to 30% by weight, based on the total weight of the pharmaceutical composition.

The osmolality adjusting agent may be sodium chloride and/or potassium chloride. The osmolality adjusting agent is present in an amount such that the osmolality of the pharmaceutical composition is 200-700 milliosmoles per kilogram (mOsm/kg). The amount of osmolality adjusting agent can be readily determined by one skilled in the art based on the desired osmolality.

In some embodiments, the pharmaceutical composition may be a liquid formulation, such as an injection; or freeze-dried powder injection, and is mixed with liquid adjuvant to make into liquid preparation. The liquid formulation may be administered, but is not limited to, for subcutaneous, intramuscular or intravenous injection, and may be administered, but is not limited to, by spraying to the lungs, or by spraying through the lungs to other visceral tissues such as the liver. In some embodiments, the pharmaceutical composition is for intravenous administration.

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

The following examples serve to further illustrate the invention without however limiting it in any way.

Examples

Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

The experimental techniques and methods used in this example are conventional techniques unless otherwise specified, such as those not specified in the following examples, and are generally performed under conventional conditions such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Materials, reagents and the like used in the examples are all available from a regular commercial source unless otherwise specified.

EXAMPLE 1 preparation of siRNA

siRNA molecules having the following sequences were synthesized by tenlin biotechnology (shanghai) limited.

TABLE 1siRNA and sequences thereof

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Wherein the capital letters "G", "C", "A", "T" and "U" each generally represent nucleotides containing guanine, cytosine, adenine, thymine and uracil, respectively, as bases; lowercase letters a, u, c, g indicate: 2' -methoxy modified nucleotide; af. Gf, cf, uf denote: 2' -fluoro modified nucleotide; the lower case letter s indicates that phosphorothioate linkages are between two nucleotides adjacent to the letter s; p1: represents the right side phase of P1 One nucleotide in the neighborhood is a 5' -phosphonucleotide;A、U、C、G(underlined + bold + italic): indicating GNA modified nucleotides.

siRNA conjugates were synthesized by tenlin biotechnology (Shanghai) limited with the following sequences:

table 2siRNA conjugates and sequences thereof:

/>

wherein, L96 is:

EXAMPLE 2siRNA inhibiting HSD17B13 Gene expression

The inhibition activity of the siRNA of the present invention on HSD17B13 gene expression was evaluated by a double luciferase reporter vector.

Main experimental reagent:

the experimental steps are as follows:

day 0: transformation of psiCHECK2-HSD17B13 plasmid into Huh7 cells

The psiCHECK2-HSD17B13 plasmid was diluted to 10 ng/. Mu.l with Opti-MEM. Taking Huh7 cells, washing with DPBS, adding trypsin for digestion, and adjusting cell density to 1×10 ⁵ Cells/ml. The Fugene-HD transfection reagent was used as follows: mixing 10 ng/. Mu.l of psiCHECK2-HSD17B13 dilution=3:100 (volume ratio), incubating at room temperature for 10 min after mixing, incubatingAfter the completion, the cells were added to Huh7 cells, and inoculated into 96-well plates at a density of 10,000 cells per well, with 100. Mu.l of culture medium per well. Huh7 cells were placed in 5% CO ₂ Incubate overnight at 37 ℃.

Day 1: treatment with siRNA compounds

Will beMixing RNAiMAX transfection reagent and Opti-MEM uniformly according to the proportion of 1.5:48.5 (volume ratio) to obtain a mixed solution A, incubating for 15 minutes at room temperature, mixing the siRNA compound to be detected (the final concentration is 0.1nM and 0.01nM respectively) with the mixed solution A1:1, incubating for 15 minutes at room temperature, adding 20 mu l of the obtained mixed solution into 100 mu l of fresh Opti-MEM culture medium according to the proportion of 1:5 after incubating, and uniformly mixing to obtain a mixed solution B. Huh7 cell supernatant was discarded, 120. Mu.l/well of the above mixture B was added to a 96-well plate, and the 96-well plate was placed in CO ₂ The cells were cultured in a cell incubator for 48 hours.

Day 2: detection reporter gene

The cell status was observed under a microscope, the cell supernatant was discarded, and 75. Mu.l was added to each well&/>The luciferase reagent and 75. Mu.l fresh 10% FBS-DMEM medium were shaken in the presence of light for 10 minutes. 100 μl of the above sample was transferred to a 96 Kong Quanbai assay plate, and the luminescence value (Firefly lum) of Firefly luciferase was measured under a multifunctional microplate reader. Then 50. Mu.l of +.>Stop&/>The reagent, shake the trigger and shake for 10 minutes in dark, detect the luminescence value (Renilla lum) of Renilla luciferase.

In the experiment, a control group was set up to be RNA-free H ₂ O replaces the siRNA compound described above, the remainderThe conditions were the same as the experimental group; blank, huh7 cells not transfected with psiCHECK2-HSD17B13 plasmid, and no siRNA compound added thereto.

And (3) data processing:

the ratio of renilla luciferase fluorescence values to firefly luciferase fluorescence values was noted as: alpha, the formula is:

α= (test well Renilla lum mean-blank Renilla lum mean)/(test well Firefly lum mean-blank Firefly lum mean);

the experimental group ratios calculated according to the above ratio formula were noted as: alpha (experimental group), and control group ratio were noted as: alpha (control).

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

inhibition ratio (%) = [1- α (experimental group average value)/α (control group average value) ]×100%

TABLE 3 inhibition of siRNA of the invention

/>

TABLE 4 inhibition of control siRNA

Sequence number	0.1nM(48h)	0.01nM(48h)
			N-ER-FY007039	33.3％	7.2％

As can be seen from tables 3 and 4, the siRNA of the present invention significantly inhibited the expression of HSD17B13 gene at both 0.1nM and 0.01 nM.

EXAMPLE 3siRNA inhibiting HSD17B13 Gene expression IC ₅₀ Measurement

The final concentrations of the following siRNAs to be tested were 10nM, 2.5nM, 0.63nM, 0.16nM, 0.04nM, 0.01nM, 0.0024nM and 0.0006nM, and then IC was carried out in a similar manner to example 2 ₅₀ And (5) measuring.

Analysis of results:

a) Using Quant Studio 7 software to automatically calculate Ct value by default;

b) The relative expression amount of the gene was calculated using the following formula:

Delta ct=ct (HSD 17B13 gene) -Ct (GAPDH)

ΔΔct=Δct (detection of sample) group) -deltact (Mock group), wherein Mock groups represent groups to which no siRNA was added compared to the experimental groups;

mRNA expression = 2 relative to Mock group ^-ΔΔCt

Inhibition ratio (%) = (relative expression amount of mRNA in Mock group-relative expression amount of mRNA in sample group)/relative expression amount of mRNA in Mock group×100%

The log value of siRNA concentration is taken as X axis, the percentage inhibition rate is taken as Y axis, and the "log (inhibitor) vs. response-variable slope" functional module of analysis software GraphPad Prism 8 is adopted to fit a quantitative effect curve, thereby obtaining the IC of each siRNA ₅₀ Values.

The fitting formula is: y=bottom+ (Top-Bottom)/(1+10

Wherein: top represents percent inhibition at the Top plateau, the Top criterion of the curve is typically 80% to 120%; bottom represents the percent inhibition at the Bottom plateau, with Bottom of the curve typically between-20% and 20%; hillSlope represents the slope of the percent inhibition curve.

The results are shown in Table 5.

TABLE 5 IC of siRNA ₅₀ (nM)

siRNAID	IC ₅₀ (nM)
		N-ER-FY007001M1	0.024
N-ER-FY007004M1	0.023
		N-ER-FY007006M1	0.014
N-ER-FY007020M1	0.012
		N-ER-FY007024M1	0.013

As can be seen from Table 5, the siRNA provided by the present application has higher HSD17B13 gene inhibition activity in Huh7 cells, IC ₅₀ And may be as low as 12pM.

EXAMPLE 4siRNA conjugates IC that inhibit expression of HSD17B13 Gene ₅₀ Measurement

Materials:

human primary hepatocytes PHH cells, supplied by the medicine Mingkang;

PHH medium: invitroGRO CP Meduim serum free BIOVIT, cargo number: s03316RNAiMAX transfection reagent, available from Invitrogen, cat: 13778-150;

RNA extraction kit96Kit, cat No.: QIAGEN-74182;

reverse transcription Kit FastKing RT Kit (With gDNase), cat: tiangen-KR 116-02;

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 (final siRNA conjugate concentrations of 10nM, 2.5nM, 0.63nM, 0.16nM, 0.04nM, 0.01nM, 0.0024nM and 0.0006nM, compound wells), respectively) were transfected into PHH cells as follows: the cryopreserved PHH cells were recovered, counted, and the cells were adjusted to 6X 105 cells/ml, while the siRNA conjugates were transferred to the cells using Lipofectamine RNAiMax, and inoculated into 96-well plates at a density of 54,000 cells per well, 100. Mu.L per well of culture broth. Cells were exposed to 5% CO ₂ Culturing in incubator at 37 ℃. After 48 hours, the medium was removed and the cells were collected for total RNA extraction. Use according to the kit product instructionsTotal RNA was extracted at 96 Kit.

(2) The siRNA conjugates (final siRNA conjugate concentrations 500nM, 125nM, 31.25nM, 7.81nM, 1.95nM, 0.49nM, 0.12nM and 0.03nM, compound wells) entered PHH cells by free uptake as follows: the cryopreserved PHH cells were recovered, counted, and the cells were adjusted to 6X 105 cells/ml while siRNA conjugates were added and inoculated into 96-well plates at a density of 54,000 cells per well, 100. Mu.l per well of culture broth. Cells were exposed to 5% CO ₂ Culturing in incubator at 37 ℃. After 48 hours, the medium was removed and the cells were harvested for total RNA extractionTaking. Use according to the kit product instructionsTotal RNA was extracted at 96 Kit.

(3) Reverse transcription of the total RNA extracted into cDNA was performed using the Fastking RT Kit (With gDNase) Kit, according to the following steps:

a) gDNA was removed with gDNase according to the following table;

TABLE 6

	Volume/. Mu.l
		5×gDNA Buffer	2
Sample(RNA)	8

The procedure was run at 42 ℃ for 3 minutes, and then the plate was placed at 4 ℃.

b) Adding the reagents described below to the system obtained in step a) and performing reverse transcription:

TABLE 7

	Volume/. Mu.l
		Fastking RT Enzyme Mix	1
FQ-RT Primer Mix	2
		10×King RT Buffer	2
RNase-Free ddH ₂ O	5

42℃，15min；95℃，3min。

c) Storing the reverse transcription product obtained in step b) at-20 ℃ for real-time PCR analysis.

(4) Real-time qPCR analysis

a) qPCR reaction mixtures were prepared as shown in the following table, with all reagents placed on ice throughout the run;

TABLE 8

TABLE 9

b) qPCR procedure was performed as follows

95 ℃ for 10 minutes;

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

(5) Analysis of results:

delta ct=ct (HSD 17B13 gene) -Ct (GAPDH)

mRNA expression = 2 relative to Mock group ^-ΔΔCt

The log value of siRNA conjugate concentration is taken as X axis, the percent inhibition rate is taken as Y axis, and the "log (inhibitor) vs. response-variable slope" functional module of analytical software GraphPad Prism 8 is adopted to fit the dose-response curve, thereby obtaining the IC of each siRNA conjugate ₅₀ Values.

The fitting formula is: y=bottom+ (Top-Bottom)/(1+10

The experimental results are shown in table 10.

TABLE 10 IC for siRNA conjugates to inhibit HSD17B13 Gene expression ₅₀ Value of

As can be seen from Table 10, the siRNA conjugates of the present application have very high HSD17B13 gene inhibitory activity.

Example 5 inhibition rate assay of siRNA and its conjugate to inhibit HSD17B13 Gene expression

Materials:

human primary hepatocytes PHH cells, supplied by the medicine Mingkang;

PHH medium: invitroGRO CP Meduim serumfree BIOVIT, cargo number: s03316;RNAiMAX transfection reagent, available from Invitrogen, cat: 13778-150;

RNA extraction kit96Kit, cat No.: QIAGEN-74182;

reverse transcription Kit FastQuant RT Kit (With gDNase), cat: tiangen-KR 116-02;

FastStart Universal Probe Mast (Roche-0491 4058001)；

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

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

siRNA conjugates (final concentration of siRNA conjugate 1nM and 0.1nM, respectively, in duplicate wells) were transfected into PHH cells by the following procedure: the cryopreserved PHH cells were recovered, counted, and the cells were adjusted to 6X 105 cells/ml, while the siRNA conjugates were transferred to the cells using Lipofectamine RNAiMax, and inoculated into 96-well plates at a density of 54,000 cells per well, 100. Mu.L per well of culture broth. Cells were exposed to 5% CO ₂ Culturing in incubator at 37 ℃. After 48 hours, the medium was removed and the cells were collected for total RNA extraction. Use according to the kit product instructionsTotal RNA was extracted at 96 Kit.

siRNA conjugates (final siRNA conjugate concentrations of 100nM and 10nM, respectively, in duplicate wells) were entered into PHH cells by free uptake, as follows: the cryopreserved PHH cells were recovered, counted, and the cells were adjusted to 6X 105 cells/ml while siRNA conjugates were added and inoculated into 96-well plates at a density of 54,000 cells per well, 100. Mu.l per well of culture broth. Cells were exposed to 5% CO ₂ Culturing in incubator at 37 DEG C. After 48 hours, the medium was removed and the cells were collected for total RNA extraction. Use according to the kit product instructionsTotal RNA was extracted at 96 Kit.

The total RNA extracted was reverse transcribed into cDNA by reverse transcription reaction in a similar manner as in example 4. The HSD17B13cDNA will be detected by qPCR. GAPDH cDNA will be tested in parallel as an internal control. The PCR reaction procedure was: 10 minutes at 95℃and then enter a cyclic mode, 95℃for 15 seconds followed by 60℃for 60 seconds for a total of 40 cycles.

Analysis of results:

delta ct=ct (HSD 17B13 gene) -Ct (GAPDH)

mRNA expression=2- ^ΔΔC t

TABLE 11 inhibition of HSD17B13 Gene expression by siRNA and its conjugate

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

EXAMPLE 6 silencing Effect of siRNA conjugates in mice expressing the human HSD17B13 (hHSD 17B 13) Gene

(1) AAV construction of mice model over-expressing hHSD17B13 Gene

C57BL/6 mice (SPF grade, supplied by Jiangsu Jiuyaokang Biotechnology Co., ltd.) of 6-8 weeks old were entered into the facility, and after 3-5 days of adaptive feeding, adenovirus AAV (pAAV-CBh-hHSD 17B13-3xFLAG-P2A-Luc2-tWPA, virus supplied by Shanghai and Yuan Biotechnology Co., ltd.) of the hHSD17B13 gene was injected into the tail vein in a single injection, and the administration volume was subjected to target gene overexpression molding: 100 mu L (3 x 10) ¹¹ vg)/animals, followed by normal feed feeding.

(2) In vivo silencing siRNA drug efficacy investigation aiming at hHSD17B13 mouse model

14 days after AAV injection, mice were subjected to in vivo imaging detection, and the mice were subcutaneously administered with a single 3mg/kg dose of N-ER-FY 007051M 2L96, N-ER-FY 0070004M 2L96, N-ER-FY007020M2L96, N-ER-FY007033M2L96, and N-ER-FY007034M2L96, in groups (6 mice per group). Living imaging was performed at 7, 14, 21, 28 and 35 days after administration to detect the protein expression level of Luciferase (which indirectly reflects the protein expression level of hHSD17B 13).

TABLE 12 inhibition of hHSD17B13 protein residual amount by siRNA conjugates

From Table 12, it can be seen that the siRNA of the present application has higher inhibitory activity to hHSD17B13 gene in vivo, can reduce the protein level of hHSD17B13 for a long time, and has obvious dose effect. Specifically, N-ER-FY 0070001M 2L96 showed 60% inhibition (40% protein remaining) of hHSD17B13 gene at day 35 after single subcutaneous administration; N-ER-FY 0070004M 2L96 showed 62% inhibition (protein residual 38%) of the hHSD17B13 gene; N-ER-FY007020M2L96 showed 73% inhibition (protein residual 27%) of the hHSD17B13 gene; N-ER-FY007033M2L96 showed 66% inhibition of the hHSD17B13 gene (protein remaining 34%);

N-ER-FY007034M2L96 showed 51% inhibition of the hHSD17B13 gene (protein remaining 49%).

EXAMPLE 7 plasma kinetic study of siRNA conjugates in CD-1 mice

Animals: CD-1 mice, SPF grade, male, about 30g, purchased from Si Bei Fu (Beijing) Biotechnology Co., ltd.

Dosage and mode of administration: the siRNA conjugates of the present application were administered at a dose of 3mg/kg (10 mL/kg), with a single subcutaneous injection following randomization, and 6 mice per group.

Sample collection: samples of whole blood were collected at 10 points at 0.0833, 0.25, 0.5, 1, 2, 4, 8, 24, 36, 48h post-administration. The front 3 of each group is collected for 0.0833, 0.5, 2, 8 and 36 hours, and the rear 3 is collected for 0.25, 1, 4, 24 and 48 hours, and the whole blood is collected and then the blood plasma is centrifugally separated for detection and analysis.

Sample detection and analysis: the concentration of the original drug in plasma samples at each time point was measured by LC-MS/MS method and PK parameters were calculated using WinNonlin software: c (C) _max 、T _max 、AUC、MRT、t _1/2 。

From this experiment, it can be seen that the siRNA conjugates of the present application have a shorter half-life in plasma and a faster clearance.

EXAMPLE 8 tissue distribution assay of siRNA conjugates in CD-1 mice

Dosage and mode of administration: the siRNA conjugates of the present application were administered at a dose of 3mg/kg (10 mL/kg), with a single subcutaneous injection following randomization, 3 animals at each time point, and a total of 24 mice.

Sample collection:

24h after administration: collecting plasma, liver, kidney and spleen; 72h after administration: collecting plasma, liver, kidney and spleen;

168h (1 week) after administration: collecting plasma, liver, kidney, spleen, brain, heart, lung, stomach, small intestine, muscle, testis; 336h (2 weeks) post-dose: collecting plasma, liver, kidney and spleen;

672h (4 weeks) post-dose: collecting plasma, liver, kidney, spleen, brain, heart, lung, stomach, small intestine, muscle, testis; 1008h (6 weeks) after dosing: collecting plasma, liver, kidney and spleen;

1344h (8 weeks) after dosing: collecting plasma, liver, kidney and spleen;

1680h (10 weeks) post-dose: plasma, liver, kidney, spleen, brain, heart, lung, stomach, small intestine, muscle, testis were collected.

Sample detection and analysis: the concentration of the original drug in the plasma and tissue samples at each time point was detected by LC-MS/MS method, and AUC in the plasma and tissue was calculated by trapezoidal area method.

From the experiment, the siRNA conjugate is mainly enriched in the liver, has long retention time in tissues and has good stability.

EXAMPLE 9 Single subcutaneous injection of siRNA conjugate C57 mice administration MTD assay

C57 mice, SPF grade, male, about 25g, purchased from si Bei Fu (beijing) biotechnology limited. Animals adopt a method of random body weight block according to the body weight of the last 1 day of the adaptation period, and the specific dosage design and grouping are as follows:

detecting the index:

(1) Clinical observation: the administration day is continuously observed for 4 hours, and at least one clinical observation is carried out every day in the recovery period

(2) Weight of: all surviving animals were weighed 2 times per week.

(3) Immunotoxicity: MTD dose animals were alternately bled 1 h.+ -. 2min,4 h.+ -. 5min,8 h.+ -. 10min,24 h.+ -. 20min after D1 dosing, 3 animals per sex/group were harvested at each time point and tested for cytokines (IFN-. Gamma., TNF-. Alpha., IL-2/6/8).

(4) Toxicological kinetics: MTD dose animals are alternately sampled before and 30min 2min,1h 2min,4h 5min,8h 10min and 24h 20min after D1 administration, and blood concentration is detected by collecting 3 animals/sex/animal group at each time point.

(5) Chemistry of blood generation: the animals of the main test group are subjected to R28 sectioning, and the animals of the satellite group are subjected to R7, R14, R21 and R28 sectioning in batches, so as to detect the biochemical property of blood.

(6) Tissue distribution: the main test animals are subjected to R28 sectioning, the satellite animals are subjected to R7, R14, R21 and R28 sectioning in batches, blood and liver are collected, and the tissue drug concentration is detected.

(7) Histopathological examination: animals of the main test group were examined by R28 dissection, and the main organs (heart, liver, spleen, lung, kidney, brain, adrenal gland, thymus, stomach, uterus/testis, ovary/epididymis) and the tissues or organs found abnormal were collected, fixed, and subjected to histopathological examination.

From this experiment, it can be seen that the siRNA conjugates of the present application have low toxicity and a large safety window.

Claims

1. 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 comprises nucleotide sequence I, and the antisense strand comprises nucleotide sequence II, which 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 group consisting of:

wherein N is ₁ Is A or U, N ₂ Is A or U;

(11) The nucleotide sequence I comprises SEQ ID NO:314, and said nucleotide sequence II comprises the nucleotide sequence set forth in SEQ ID NO:31 5, a nucleotide sequence shown in seq id no:

2. The siRNA of claim 1, wherein the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, substantially reverse complementary or fully reverse complementary; by substantially reverse complement is meant that there are no more than 3 base mismatches between the two nucleotide sequences; by substantially reverse complement is meant that there is no more than 1 base mismatch between the two nucleotide sequences; complete reverse complement refers to the absence of mismatches between two nucleotide sequences.

3. The siRNA of claim 1 or 2, wherein the sense strand further comprises a nucleotide sequence III, the antisense strand further comprises a nucleotide sequence IV, each of the nucleotide sequence III and the nucleotide sequence IV being independently 0-6 nucleotides in length, wherein the nucleotide sequence III is linked at the 5 'end of the nucleotide sequence I, the nucleotide sequence IV is linked at the 3' end of the nucleotide sequence II, the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse-complementary or fully reverse-complementary; by substantially reverse complement is meant that there is no more than 1 base mismatch between the two nucleotide sequences; complete reverse complement refers to the absence of mismatches between two nucleotide sequences; and/or, the nucleotide sequence III is connected at the 3 'end of the nucleotide sequence I, the nucleotide sequence IV is connected at the 5' end of the nucleotide sequence II, and the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary; by substantially reverse complement is meant that there is no more than 1 base mismatch between the two nucleotide sequences; complete reverse complement refers to the absence of mismatches between two nucleotide sequences.

4. A siRNA according to any one of claims 1-3, said sense strand further comprising nucleotide sequence V and/or said antisense strand further comprising nucleotide sequence VI, nucleotide sequences V and VI being 0 to 3 nucleotides in length, nucleotide sequence V being linked at the 3 'end of said sense strand to form a 3' overhang of the sense strand and/or nucleotide sequence VI being linked at the 3 'end of said antisense strand to form a 3' overhang of the antisense strand; preferably, the nucleotide sequence V or VI is 2 nucleotides in length; more preferably, the nucleotide sequence V or VI is two consecutive thymidines or two consecutive uracils ribonucleotides;

alternatively, the nucleotide sequence V or VI is mismatched or complementary to a nucleotide at the corresponding position of the target mRNA.

5. The siRNA according to any one of claims 1-4, wherein the double stranded region is 15-30 nucleotide pairs in length; preferably, the double-stranded region is 17-23 nucleotide pairs in length; more preferably, the double stranded region is 19-21 nucleotide pairs in length.

6. The siRNA of any one of claims 1-5, wherein the sense strand or the antisense strand has 15-30 nucleotides; preferably, the sense strand or antisense strand has 19-25 nucleotides; more preferably, the sense strand or the antisense strand has 19-23 nucleotides.

7. The siRNA of 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 is a phosphate group having a modification group; preferably, the phosphate group containing a modifying group is a phosphorothioate group formed by substitution of at least one oxygen atom of a phosphodiester bond in the phosphate group with a sulfur atom; and/or, the siRNA comprises a sense strand that does not comprise a 3' overhang nucleotide.

8. The siRNA of any of claims 1-7, wherein the 5 'terminal nucleotide of the sense strand is linked to a 5' phosphate group or a 5 'phosphate derivative group, and/or the 5' terminal nucleotide of the antisense strand is linked to a 5 'phosphate group or a 5' phosphate derivative group.

9. The siRNA of any one of claims 1-8, wherein the modified nucleotide is selected from the group consisting of a 2 '-fluoro modified nucleotide, a 2' -alkoxy modified nucleotide, a 2 '-substituted alkoxy modified nucleotide, a 2' -alkyl modified nucleotide, a 2 '-substituted alkyl modified nucleotide, a 2' -deoxy nucleotide, a 2 '-amino modified nucleotide, a 2' -substituted amino modified nucleotide, a nucleotide analog, or a combination of any two or more thereof.

10. The siRNA of any of claims 1-9, wherein the modified nucleotide is selected from the group consisting of a 2' -fluoro modified nucleotide, a 2' -methoxy modified nucleotide, a 2' -O-CH ₂ -CH ₂ -O-CH ₃ Modified nucleotides, 2' -O-CH ₂ -CH＝CH ₂ Modified nucleotides, 2' -CH ₂ -CH ₂ -CH＝CH ₂ Modified nucleotides, 2' -deoxynucleotides, nucleotide analogs, or a combination of any two or more thereof.

11. The siRNA of any one of claims 1-10, wherein each nucleotide in the sense strand and the antisense strand is independently a 2' -fluoro modified nucleotide or a non-fluoro modified nucleotide;

preferably, the 2' -fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, the remaining positions being non-fluoro modified nucleotides; the 2' -fluoro-modified nucleotide is located at the even number position of the antisense strand in the 5' to 3' direction, and the rest positions are non-fluoro-modified nucleotides;

alternatively, the 2' -fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, the remaining positions being non-fluoro modified nucleotides; the 2' -fluoro modified nucleotide is located at positions 2, 6, 14 and 16 of the antisense strand according to the 5' to 3' direction, the remaining positions being non-fluoro modified nucleotides;

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

alternatively, the 2' -fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, the remaining positions being non-fluoro modified nucleotides; the 2' -fluoro modified nucleotide is located at positions 2, 14 and 16 of the antisense strand according to the 5' to 3' direction, and the rest positions are non-fluoro modified nucleotides;

further preferably, each of the non-fluoro modified nucleotides is a 2 '-methoxy modified nucleotide, which is a nucleotide formed by substituting the 2' -hydroxyl group of the ribosyl group with a methoxy group.

12. The siRNA of claim 11, wherein each non-fluoro modified nucleotide is independently selected from one of a nucleotide or a nucleotide analogue formed by substitution of the hydroxyl group at the 2' -position of the ribosyl of the nucleotide with a non-fluoro group, the nucleotide analogue being selected from one of an iso-nucleotide, LNA, ENA, cret BNA, UNA and GNA.

13. The siRNA of 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 nucleotide, a GNA modified nucleotide, or a combination of any two or more thereof;

preferably, the 2 '-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3 'direction, the remaining positions being 2' -methoxy modified nucleotides; the 2 '-fluoro modified nucleotide is located at the even number position of the antisense strand in the 5' to 3 'direction, and the rest positions are 2' -methoxy modified nucleotides;

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

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

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

alternatively, the 2 '-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3 'direction, the remaining positions being 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, GNA modified nucleotides are located at position 7 of the antisense strand, and the remaining positions are 2' -methoxy modified nucleotides.

14. The siRNA of any one of claims 1-13, wherein at least one of the linkages between the following nucleotides in the siRNA is a phosphorothioate linkage:

15. The siRNA of any of claims 1-14, wherein said sense strand comprises phosphorothioate groups in the 5 'to 3' direction at positions shown below:

between nucleotide 1 and nucleotide 2 from the 5' end of the sense strand;

between nucleotide 2 and nucleotide 3 from the 5' end of the sense strand;

between nucleotide 1 and nucleotide 2 from the 3' end of the sense strand;

between nucleotide 2 and nucleotide 3 from the 3' end of the sense strand;

or alternatively, the process may be performed,

Between nucleotide 1 and nucleotide 2 from the 5' end of the sense strand;

16. The siRNA of any of claims 1-15, wherein said antisense strand comprises phosphorothioate groups at positions shown below, in the 5 'to 3' direction:

between nucleotide 1 and nucleotide 2 from the 5' end of the antisense strand;

between nucleotide 2 and nucleotide 3 from the 5' end of the antisense strand;

between nucleotide 1 and nucleotide 2 from the 3' end of the antisense strand;

17. The siRNA of any one of claims 1-16, wherein each nucleotide in the sense strand and the antisense strand is independently a 2 '-fluoro modified nucleotide, a 2' -methoxy modified nucleotide, a GNA modified nucleotide, or a combination of any two or more thereof;

preferably, the 2 '-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3 'direction, the remaining positions being 2' -methoxy modified nucleotides, between the 1 st and 2 nd nucleotides of the 5 'end, between the 2 nd and 3 rd nucleotides of the 5' end, between the 1 st and 2 nd nucleotides of the 3 'end, between the 2 nd and 3 rd nucleotides of the 3' end being phosphorothioate linkages; according to the 5' to 3' direction, the 2' -fluoro modified nucleotide is located at the even number position of the antisense strand, the rest positions are 2' -methoxy modified nucleotides, the 1 st nucleotide and the 2 nd nucleotide of the 5' end, the 2 nd nucleotide and the 3 rd nucleotide of the 5' end, the 1 st nucleotide and the 2 nd nucleotide of the 3' end are connected by phosphorothioate groups;

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, the remaining positions being 2' -methoxy modified nucleotides, between nucleotide 1 and nucleotide 2 of the 5' end, between nucleotide 2 and nucleotide 3 of the 5' end being phosphorothioate linkages, the 3' end being removed from the overhang; the 2 '-fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand in the 5' to 3 'direction, the remaining positions being 2' -methoxy modified nucleotides, between the 1 st and 2 nd nucleotides at the 5 'end, between the 2 nd and 3 rd nucleotides at the 5' end, between the 1 st and 2 nd nucleotides at the 3 'end, and between the 2 nd and 3 rd nucleotides at the 3' end being phosphorothioate linkages;

alternatively, the 2 '-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3 'direction, the remaining positions being 2' -methoxy modified nucleotides, between the 1 st and 2 nd nucleotides at the 5 'end, between the 2 nd and 3 rd nucleotides at the 5' end, between the 1 st and 2 nd nucleotides at the 3 'end, and between the 2 nd and 3 rd nucleotides at the 3' end being phosphorothioate linkages; the 2 '-fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand in the 5' to 3 'direction, the remaining positions being 2' -methoxy modified nucleotides, between the 1 st and 2 nd nucleotides at the 5 'end, between the 2 nd and 3 rd nucleotides at the 5' end, between the 1 st and 2 nd nucleotides at the 3 'end, and between the 2 nd and 3 rd nucleotides at the 3' end being phosphorothioate linkages;

Alternatively, the 2 '-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3 'direction, the remaining positions being 2' -methoxy modified nucleotides, between the 1 st and 2 nd nucleotides at the 5 'end, between the 2 nd and 3 rd nucleotides at the 5' end, between the 1 st and 2 nd nucleotides at the 3 'end, and between the 2 nd and 3 rd nucleotides at the 3' end being phosphorothioate linkages; according to the 5 'to 3' direction, 2 '-fluoro modified nucleotides are positioned at positions 2, 6, 8, 9, 14 and 16 of the antisense strand, the rest positions are 2' -methoxy modified nucleotides, the 1 st nucleotide and the 2 nd nucleotide of the 5 'end, the 2 nd nucleotide and the 3 rd nucleotide of the 5' end, the 1 st nucleotide and the 2 nd nucleotide of the 3 'end and the 2 nd nucleotide and the 3 rd nucleotide of the 3' end are connected by phosphorothioate groups;

alternatively, the 2 '-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3 'direction, the remaining positions being 2' -methoxy modified nucleotides, between the 1 st and 2 nd nucleotides at the 5 'end, between the 2 nd and 3 rd nucleotides at the 5' end, between the 1 st and 2 nd nucleotides at the 3 'end, and between the 2 nd and 3 rd nucleotides at the 3' end being phosphorothioate linkages; 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, the rest positions are 2' -methoxy modified nucleotides, between 1 st and 2 nd nucleotides at the 5 'end, between 2 nd and 3 rd nucleotides at the 5' end, between 1 st and 2 nd nucleotides at the 3 'end, and phosphorothioate linkage between 2 nd and 3 rd nucleotides at the 3' end;

Alternatively, the 2 '-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3 'direction, the remaining positions being 2' -methoxy modified nucleotides, between the 1 st and 2 nd nucleotides at the 5 'end, between the 2 nd and 3 rd nucleotides at the 5' end, between the 1 st and 2 nd nucleotides at the 3 'end, and between the 2 nd and 3 rd nucleotides at the 3' end being phosphorothioate linkages; according to the 5 'to 3' direction, 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, the rest positions are 2' -methoxy modified nucleotides, between 1 st and 2 nd nucleotides at the 5 'end, between 2 nd and 3 rd nucleotides at the 5' end, between 1 st and 2 nd nucleotides at the 3 'end, and phosphorothioate linkage between 2 nd and 3 rd nucleotides at the 3' end.

18. The siRNA of claim 1, selected from the siRNA of table 1, wherein the siRNA is not N-ER-FY007031, N-ER-FY007036, N-ER-FY 007026, 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-FY007036, N-ER-FY007027, N-ER-FY007005, N-ER-FY007030, N-ER-FY007051, N-ER-FY007032, N-FY 007033, N-ER-FY 00704, N-ER-FY007036, N-ER-FY 0336; the method comprises the following steps of N-ER-FY007075M5, N-ER-FY007078M2D2, N-ER-FY007078M3, N-ER-FY007078M4, N-ER-FY007078M5, N-ER-FY007079M2D2, N-ER-FY007079M3, N-ER-FY007079M4, N-ER-FY007079M5, N-ER-FY007078M 080, N-ER-FY007080M2D2, N-ER-FY007080M3, N-ER-FY007079M4, N-ER-FY007080M 5N-ER-FY 007082, N-ER-FY007082M2D2, N-ER-FY007082M3, N-ER-FY007082M4, N-ER-FY007082M5, N-ER-FY007083M2D2, N-ER-FY007083M3, N-ER-FY007083M4, N-ER-FY007083M5, N-ER-FY007085M2D2, N-ER-FY007085M3, N-ER-FY007085M4, N-ER-FY007085M5.

19. An siRNA conjugate comprising the siRNA of any one of claims 1-18 and a conjugate group conjugated to the siRNA.

20. The siRNA conjugate of claim 19, wherein the conjugate group comprises a pharmaceutically acceptable targeting group and a linker, and the siRNA, the linker and the targeting group are sequentially covalently or non-covalently linked;

or alternatively, the process may be performed,

21. The siRNA conjugate of claim 20, wherein the conjugate group is L96 of the formula:

22. the siRNA conjugate of any one of claims 19-21, wherein the siRNA conjugate is an siRNA conjugate selected from table 2.

23. A pharmaceutical composition comprising the siRNA of any one of claims 1-18, or the siRNA conjugate of any one of claims 19-22, and a pharmaceutically acceptable carrier.

24. A kit comprising the siRNA of any one of claims 1-18, or the siRNA conjugate of any one of claims 19-22, or the pharmaceutical composition of claim 23.

25. Use of the siRNA of any one of claims 1-18, or the siRNA conjugate of any one of claims 19-22, or the pharmaceutical composition of claim 23, for the preparation of a medicament for inhibiting HSD17B13 gene expression.

26. Use of the siRNA of any one of claims 1-18, or the siRNA conjugate of any one of claims 19-22, or the pharmaceutical composition of claim 23, for the manufacture of a medicament for preventing and/or treating a disease associated with overexpression of the HSD17B13 gene.

27. 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.

28. A method of inhibiting HSD17B13 gene expression comprising contacting or administering a therapeutically effective amount of the siRNA of any one of claims 1-18, or the siRNA conjugate of any one of claims 19-22, or the pharmaceutical composition of claim 23, with a cell expressing HSD17B13 or to a subject in need thereof.

29. A method of treating and/or preventing a disease associated with overexpression of the HSD17B13 gene, comprising administering a therapeutically effective amount of the siRNA of any one of claims 1-18, or the siRNA conjugate of any one of claims 19-22, or the pharmaceutical composition of claim 23 to a subject in need thereof.