WO2023208109A9 - Arnsi pour inhiber l'expression de hsd17b13, conjugué et composition pharmaceutique associés, et utilisation associée - Google Patents

Arnsi pour inhiber l'expression de hsd17b13, conjugué et composition pharmaceutique associés, et utilisation associée Download PDF

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WO2023208109A9
WO2023208109A9 PCT/CN2023/091141 CN2023091141W WO2023208109A9 WO 2023208109 A9 WO2023208109 A9 WO 2023208109A9 CN 2023091141 W CN2023091141 W CN 2023091141W WO 2023208109 A9 WO2023208109 A9 WO 2023208109A9
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nucleotide sequence
seq
nucleotide
sequence shown
sirna
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WO2023208109A1 (fr
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王书成
黄河
王岩
林国良
耿玉先
荣梅
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北京福元医药股份有限公司
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    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01051—3 (or 17)-Beta-hydroxysteroid dehydrogenase (1.1.1.51)

Definitions

  • the present application relates to siRNA, siRNA conjugates, pharmaceutical compositions containing the same, preparation methods and uses thereof capable of inhibiting HSD17B13 gene expression.
  • the 17 ⁇ -hydroxysteroid dehydrogenase (17 ⁇ -HSD) family consists of 15 enzymes, most of which are related to the activation or inactivation of sex hormones (such as HSD17B1, HSD17B2, HSD17B3, HSD17B5, HSD17B6), and other members are involved in fatty acid metabolism, cholesterol Biosynthesis and bile acid production, etc.
  • sex hormones such as HSD17B1, HSD17B2, HSD17B3, HSD17B5, HSD17B6
  • HSD17B6 sex hormones
  • HSD17B family differ in tissue distribution, subcellular localization, catalytic priority, and have different substrate specificities (Marchais Oberwinkler, et al. (2011) J Steroid Biochem Mol Biol 125(1-2): 66 -82)).
  • HSD17B13 a member of the 17 ⁇ -hydroxysteroid dehydrogenase family, is primarily localized in hepatocytes, with the highest expression levels known to be found in hepatocytes of the liver, and in the ovary, bone marrow, kidney, brain, lung, skeletal muscle, bladder and testis. Detectable only at lower levels, it is a hepatocyte-specific lipid droplet (LD)-associated protein, and increasing evidence suggests that it plays a key role in hepatic lipid metabolism.
  • LD hepatocyte-specific lipid droplet
  • the function of HSD17B13 is not fully understood, however, several 17 ⁇ -HSD family members, including 17 ⁇ -HSD-4, -7, -10, and -12, have been shown to be involved in carbohydrate and fatty acid metabolism.
  • HSD17B13 may also play a role in lipid metabolism pathways. Hepatic upregulation of HSD17B13 has been reported to have been observed in patients with fatty liver disease, supporting a role for this enzyme in the pathogenesis of non-alcoholic fatty liver disease (NAFLD).
  • NAFLD non-alcoholic fatty liver disease
  • Non-alcoholic fatty liver disease also known as metabolic (dysfunction)-associated fatty liver disease (MAFLD)
  • NAFLD is the excessive accumulation of fat in the liver in the absence of another clear cause, such as alcohol consumption.
  • NAFLD is the most common liver disease in the world, affecting approximately 25% of the world's population. The prevalence of NAFLD is still showing an upward trend, which will undoubtedly increase the economic burden and lead to a sharp increase in the number of patients with end-stage liver disease requiring liver transplantation and the number of people suffering from hepatocellular carcinoma.
  • There is currently no specific treatment for NAFLD which mainly involves weight loss through dietary changes and exercise.
  • Preliminary research shows that pioglitazone and vitamin E have therapeutic potential.
  • HSD17B13 as a lipid droplet (LD)-associated protein in NAFLD patients and reported HSD17B13 to be one of the most abundantly expressed LD proteins specifically localized on the surface of LDs (Wen Su, et al. ., Comparative proteomic study reveals 17 ⁇ -HSD13 as a pathogenic protein in nonalcoholic fatty live disease, 111 PNAS 11437-11442 (2014)). Further, HSD17B13 levels were found to be upregulated in the livers of patients and mice with NAFLD. Overexpression leads to LD increase in number and size, whereas gene silencing of HSD17B13 attenuated oleic acid-induced LD formation in cultured hepatocytes.
  • LD lipid droplet
  • HSD17B13 protein in C57BL/6 mice has also been shown to significantly increase lipogenesis and triglyceride (TG) content in the liver, leading to a fatty liver phenotype.
  • NSAbul-Husn et al. provide additional evidence implicating HSD17B13 gene expression in the pathogenesis of NAFLD and nonalcoholic steatohepatitis (NASH) (NSAbul-Husn et al., A Protein-Truncating HSD17B13 Variant and Protection from Chronic Liver Disease , 378 N. Eng. J. Med. 1096-1106 (2018)).
  • HSD17B13 splice variant rs72613567:TA
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • the present invention aims to provide siRNA, siRNA conjugates and pharmaceutical compositions thereof, which can affect the RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the HSD17B13 gene, thereby inhibiting the expression of the HSD17B13 gene in the liver. , to achieve the purpose of disease treatment.
  • RISC RNA-induced silencing complex
  • the present invention provides a siRNA capable of inhibiting HSD17B13 gene expression.
  • the siRNA includes a sense strand and an antisense strand, wherein each nucleotide in the siRNA is independently a modified or unmodified nucleotide, wherein The sense strand contains nucleotide sequence I, the antisense strand contains nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region, wherein the Nucleotide sequence I and nucleotide sequence II are selected from the following sequences:
  • nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 296, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 297:
  • nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 298, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 299:
  • nucleotide sequence I is not SEQ ID NO: 17 and said nucleotide sequence II is not SEQ ID NO: 18;
  • the nucleotide sequence I is not SEQ ID NO: 21 and the nucleotide sequence II is not SEQ ID NO: 22;
  • nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 300
  • nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 301:
  • nucleotide sequence I is not SEQ ID NO: 33 and the nucleotide sequence II is not SEQ ID NO: 34;
  • the nucleotide sequence I is not SEQ ID NO: 35 and the nucleotide sequence II is not SEQ ID NO: 36;
  • the nucleotide sequence I is not SEQ ID NO: 39 and the nucleotide sequence II is not SEQ ID NO: 40;
  • nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 23, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 24;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 302, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 303:
  • N1 is A or U
  • N2 is A or U
  • nucleotide sequence I is not SEQ ID NO: 25 and the nucleotide sequence II is not SEQ ID NO: 26;
  • nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 304
  • nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 305:
  • nucleotide sequence I is not SEQ ID NO: 51 and the nucleotide sequence II is not SEQ ID NO: 52;
  • nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 306, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 307:
  • nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 308, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 309:
  • nucleotide sequence I is not SEQ ID NO: 71 and the nucleotide sequence II is not SEQ ID NO: 72;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 310, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 311:
  • nucleotide sequence I is not SEQ ID NO: 77 and the nucleotide sequence II is not SEQ ID NO: 78;
  • nucleotide sequence I is not SEQ ID NO: 79 and the nucleotide sequence II is not SEQ ID NO: 80;
  • nucleotide sequence I is not SEQ ID NO: 81 and the nucleotide sequence II is not SEQ ID NO: 82;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 312, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 313:
  • nucleotide sequence I is not SEQ ID NO: 89 and the nucleotide sequence II is not SEQ ID NO: 90;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 314, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 315:
  • nucleotide sequence I is not SEQ ID NO: 95 and the nucleotide sequence II is not SEQ ID NO: 96;
  • the nucleotide sequence I is not SEQ ID NO: 99 and the nucleotide sequence II is not SEQ ID NO: 100;
  • nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 316
  • nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 317:
  • nucleotide sequence I is not SEQ ID NO: 121 and the nucleotide sequence II is not SEQ ID NO: 122;
  • nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 318
  • nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 319:
  • nucleotide sequence I is not SEQ ID NO: 139 and the nucleotide sequence II is not SEQ ID NO: 140;
  • nucleotide sequence I is not SEQ ID NO: 141 and the nucleotide sequence II is not SEQ ID NO: 142;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 320, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 321:
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 322, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 323:
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 324, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 325:
  • nucleotide sequence I is not SEQ ID NO: 169 and the nucleotide sequence II is not SEQ ID NO: 170;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 65, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 66;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 75, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 76;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 93, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 94;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 103, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 104;
  • nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 109
  • nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 110;
  • nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 111
  • nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 112;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 115, and the nucleotide sequence Column II contains the nucleotide sequence shown in SEQ ID NO: 116;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 41, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 42;
  • nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 41, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 188;
  • nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 189, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 188;
  • nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 41, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 190;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 41, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 191;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 41, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 192;
  • nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 27
  • nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 28;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 27, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 183;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 184, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 183;
  • nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 27, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 185;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 27, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 186;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 27, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 187;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 73, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 74;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 83, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 84;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 83, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 193;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 194, and the nucleotide sequence Column II contains the nucleotide sequence shown in SEQ ID NO: 193;
  • nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 83
  • nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 195;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 83, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 196;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 83, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 197;
  • nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 97
  • nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 98;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 97, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 198;
  • nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 199, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 198;
  • nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 97
  • nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 200;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 97, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 201;
  • nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 97, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 202;
  • nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 143
  • nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 144;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 19, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 20;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 19, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 178;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 179, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 178;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 19, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 180;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 19, and the nucleotide sequence II includes the nucleotide sequence shown in SEQ ID NO: 181;
  • the nucleotide sequence I includes the nucleotide sequence shown in SEQ ID NO: 19, and the nucleotide sequence II contains the nucleotide sequence shown in SEQ ID NO:182.
  • the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, substantially reverse complementary or completely reverse complementary; the substantially reverse complementary refers to two nuclei There are no more than 3 base mismatches between the nucleotide sequences; the substantial reverse complementarity refers to the presence of no more than 1 base mismatch between the two nucleotide sequences; complete reverse complementarity refers to the absence of mismatches between the two nucleotide sequences.
  • the sense strand further contains nucleotide sequence III
  • the antisense strand further contains nucleotide sequence IV
  • the lengths of nucleotide sequence III and nucleotide sequence IV are each independently 0- 6 nucleotides, wherein the nucleotide sequence III is connected to the 5′ end of the nucleotide sequence I, the nucleotide sequence IV is connected to the 3′ end of the nucleotide sequence II, and the nucleotide sequence III It is equal in length to the IV of the nucleotide sequence and is substantially reverse complementary or completely reverse complementary; the substantially reverse complementarity means that there is no more than 1 base mismatch between the two nucleotide sequences. ;Perfect reverse complementarity means there are no mismatches between the two nucleotide sequences; and/or,
  • the nucleotide sequence III is connected to the 3′ end of the nucleotide sequence I
  • the nucleotide sequence IV is connected to the 5′ end of the nucleotide sequence II
  • the IV lengths are equal and are substantially reverse complementary or completely reverse complementary; the substantially reverse complementarity means that there is no more than 1 base mismatch between the two nucleotide sequences; complete reverse complementarity means that there is no more than 1 base mismatch between the two nucleotide sequences; There are no mismatches between the two nucleotide sequences.
  • the sense strand further contains the nucleotide sequence V and/or the antisense strand further contains the nucleotide sequence VI, the nucleotide sequences V and VI being 0 to 3 nucleotides in length , the nucleotide sequence V is connected to the 3' end of the sense strand to form the 3' overhang of the sense strand, and/or the nucleotide sequence VI is connected to the 3' end of the antisense strand to form the 3' end of the antisense strand. 'Protruding end.
  • the length of the nucleotide sequence V or VI is 2 nucleotides.
  • the nucleotide sequence V or VI is two consecutive thymine deoxyribonucleotides or two consecutive uracil ribonucleotides. In a preferred embodiment, the nucleotide sequence V or VI mismatches or is complementary to the nucleotide at the corresponding position of the target mRNA.
  • the length of the double-stranded region is 15-30 nucleotide pairs; preferably, the length of the double-stranded region is 17-23 nucleotide pairs; more preferably, the length of the double-stranded region is 19-21 nucleotide pairs.
  • the sense strand or antisense strand has 15-30 nucleotides; preferably, the sense strand or antisense strand has 19-25 nucleotides; more preferably, the sense strand or antisense strand has 15-30 nucleotides; The sense strand has 19-23 nucleotides.
  • At least one nucleotide in the sense strand or the antisense strand is a modified nucleotide
  • at least one phosphate group is a phosphate group with a modifying group; preferably , the phosphate group containing a modified group is a phosphorothioate group formed by replacing at least one oxygen atom in the phosphodiester bond of the phosphate group with a sulfur atom.
  • the siRNA comprises a sense strand that does not include 3' overhanging nucleotides.
  • the 5' terminal nucleotide of the sense strand is connected to a 5' phosphate group or a 5' phosphate derivative group, and/or the 5' terminal nucleotide of the antisense strand is connected to a 5' phosphate group or a 5' phosphate derivative group.
  • the modified nucleotide is selected from the group consisting of 2'-fluoro modified nucleotides, 2'-alkoxy modified nucleotides, 2'-substituted alkoxy modified nucleosides Acid, 2'-alkyl modified nucleotide, 2'-substituted alkyl modified nucleotide, 2'-deoxynucleotide, 2'-amino modified nucleotide, 2'-substituted amino Modified nucleotides, nucleotide analogs or a combination of any two or more thereof.
  • each nucleotide in the sense strand and the antisense strand is independently a 2'-fluoro modified nucleotide or a non-fluoro modified nucleotide.
  • the 2'-fluorinated modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are non-fluorinated modified cores According to the 5' to 3' direction, the 2'-fluorinated modified nucleotides are located at the even-numbered positions of the antisense strand, and the remaining positions are non-fluorinated modified nucleotides.
  • the 2'-fluorinated modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are non-fluorinated modified cores According to the 5' to 3' direction, the 2'-fluorinated modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and the remaining positions are non-fluorinated modified nucleotides.
  • the 2'-fluorinated modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are non-fluorinated modified cores In the 5' to 3' direction, the 2'-fluorinated modified nucleotides are located at positions 2, 6, 8, 9, 14 and 16 of the antisense strand, and the remaining positions are non-fluorinated modified nuclei. glycosides.
  • the 2'-fluorinated modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are non-fluorinated modified cores According to the 5' to 3' direction, the 2'-fluorinated modified nucleotides are located at positions 2, 14 and 16 of the antisense strand, and the remaining positions are non-fluorinated modified nucleotides.
  • each non-fluoro modified nucleotide is a 2'-methoxy modified nucleotide
  • the 2'-methoxy modified nucleotide refers to the 2'- of the ribosyl group Nucleotides formed by replacing the hydroxyl group with a methoxy group.
  • each non-fluorinated modified nucleotide is independently selected from nucleotides or nucleotide analogs in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by a non-fluorinated group.
  • the nucleotide analog is selected from one of isonucleotides, LNA, ENA, cET BNA, UNA and GNA.
  • each nucleotide in the sense strand and the antisense strand is independently a 2'-fluoro modified nucleotide, a 2'-methoxy modified nucleotide, GNA Modified nucleotides or a combination of any two or more thereof.
  • the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups Modified nucleotides; in the 5' to 3' direction, 2'- The fluoro-modified nucleotides are located in even-numbered positions of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides.
  • the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups Modified nucleotides; in the 5' to 3' direction, 2'-fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and the remaining positions are 2'-methoxy modified of nucleotides.
  • the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups Modified nucleotides; in the 5' to 3' direction, 2'-fluoro modified nucleotides are located at positions 2, 6, 8, 9, 14 and 16 of the antisense strand, and the remaining positions are 2'- Methoxy modified nucleotides.
  • the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups Modified nucleotides; in the 5' to 3' direction, 2'-fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and GNA modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand. Position 7, and the remaining positions are 2'-methoxy modified nucleotides.
  • the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups Modified nucleotides; in the 5' to 3' direction, 2'-fluoro modified nucleotides are located at positions 2, 14 and 16 of the antisense strand, and GNA modified nucleotides are located at position 1 of the antisense strand. Position 6, and the remaining positions are 2'-methoxy modified nucleotides.
  • At least one of the linkages between the following nucleotides in the siRNA is a phosphorothioate linkage:
  • the siRNA is directed from the 5' end to the 3' end, and the sense strand contains a phosphorothioate group at a position as follows:
  • the sense strand contains phosphorothioate groups at the positions shown below:
  • the siRNA is directed from the 5' end to the 3' end and the antisense strand contains a phosphorothioate group at a position as follows:
  • each nucleotide in the sense strand and the antisense strand is independently a 2'-fluoro modified nucleotide, a 2'-methoxy modified nucleotide, GNA Modified nucleotides or a combination of any two or more thereof.
  • the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups Modified nucleotides, between the 1st and 2nd nucleotides at the 5' end, between the 2nd and 3rd nucleotides at the 5' end, and at the 3' end Between the first nucleotide and the second nucleotide, and between the second nucleotide and the third nucleotide at the 3' end, there is a phosphorothioate group connection; according to the 5' to 3' Orientation, the 2'-fluoro modified nucleotide is located at the even position of the antisense strand, the remaining positions are the 2'-methoxy modified nucleotide, the 1st nucleotide and the 2nd core at the 5' end between nucleotides, between the 2nd and 3rd nucle
  • the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups
  • Modified nucleotides, between the first and second nucleotides at the 5' end, and between the second and third nucleotides at the 5' end are phosphorothioates base connection, and the overhang is removed from the 3'end; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and the remaining positions are 2'- Methoxy-modified nucleotides, between the 1st and 2nd nucleotides at the 5' end, between the 2nd and 3rd nucleotides at the 5' end, 3 There is a
  • the 2'-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups Modified nucleotides, between the 1st and 2nd nucleotides at the 5' end, between the 2nd and 3rd nucleotides at the 5' end, and at the 3' end Between the first nucleotide and the second nucleotide, and between the second nucleotide and the third nucleotide at the 3' end, there is a phosphorothioate group connection; according to the 5' to 3' Orientation, 2'-fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, the first core at the 5' end Between the nucleotide and the second nucleotide, between the second and third nucleotides at the
  • the 2'-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups Modified nucleotides, between the 1st and 2nd nucleotides at the 5' end, between the 2nd and 3rd nucleotides at the 5' end, and at the 3' end Between the first nucleotide and the second nucleotide, and between the second nucleotide and the third nucleotide at the 3' end, there is a phosphorothioate group connection; according to the 5' to 3' direction, the 2'-fluoro modified nucleotides are located at positions 2, 6, 8, 9, 14 and 16 of the antisense strand, the remaining positions are 2'-methoxy modified nucleotides, and the 5' end Between the 1st and 2nd nucleotides, between the 2nd and 3rd nucleotides at the 5'
  • the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups Modified nucleotides, between the 1st and 2nd nucleotides at the 5' end, between the 2nd and 3rd nucleotides at the 5' end, and at the 3' end Between the first nucleotide and the second nucleotide, and between the second nucleotide and the third nucleotide at the 3' end, there is a phosphorothioate group connection; according to the 5' to 3' direction, 2'-fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, GNA modified nucleotides are located at position 7 of the antisense strand, and the remaining positions are 2'-methoxy base-modified nucleotide, between the first and second nucleotides at the
  • the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups Modified nucleotides, between the 1st and 2nd nucleotides at the 5' end, between the 2nd and 3rd nucleotides at the 5' end, and at the 3' end Between the first nucleotide and the second nucleotide, and between the second nucleotide and the third nucleotide at the 3' end, there is a phosphorothioate group connection; according to the 5' to 3' Orientation, 2'-fluoro modified nucleotides are located at positions 2, 14 and 16 of the antisense strand, GNA modified nucleotides are located at position 6 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, between the first and second nucleotides at the 5'
  • the invention provides siRNA selected from Table 1, wherein the siRNA is not N-ER-FY007001, N-ER-FY007002, N-ER-FY007004, N-ER-FY007006, N- ER-FY007031, N-ER-FY007007, N-ER-FY007011, N-ER-FY007056, N-ER-FY007013, N-ER-FY007014, N-ER-FY007016, N-ER-FY007038, N-ER- FY007039, N-ER-FY007022, N-ER-FY007023, N-ER-FY007027, N-ER-FY007005, N-ER-FY007030, N-ER-FY007051, N-ER-FY007032, N-ER-FY007033, N-ER-FY007034, N-ER-FY007035, N-ER-FY0070
  • the present invention also provides an siRNA conjugate, which contains the siRNA of the present invention and a conjugation group conjugated to the siRNA (as shown in the figure below, the double helix structure represents the siRNA, and the The conjugating group is attached to the 3′ end of the sense strand of the siRNA):
  • X can be selected as O or S. In one embodiment, X is O.
  • the conjugation group includes a pharmaceutically acceptable targeting group and a linker, and the siRNA, the linker, and the targeting group are sequentially linked covalently or non-covalently.
  • the sense strand and the antisense strand of the siRNA are complementary to form a double-stranded region of the siRNA conjugate, and the 3' end of the sense strand forms a blunt end, and the antisense strand forms a blunt end.
  • the 3' end of the chain has 1-3 protruding nucleotides extending out of the double-stranded region;
  • the sense strand and the antisense strand of siRNA are complementary to form the double-stranded region of the siRNA conjugate, and the 3' end of the sense strand forms a blunt end, and the 3' end of the antisense strand forms a blunt end. 'The ends form blunt ends.
  • the conjugating group is L96 of the formula:
  • the siRNA conjugate is a siRNA conjugate selected from Table 2.
  • the present invention also provides a pharmaceutical composition, which contains the siRNA of the present invention, or the siRNA conjugate of the present invention, and a pharmaceutically acceptable carrier.
  • the present invention also provides a kit comprising the siRNA of the present invention, or the siRNA conjugate of the present invention, or the pharmaceutical composition of the present invention.
  • the present invention also provides the siRNA of the present invention, or the siRNA conjugate of the present invention, or the pharmaceutical composition of the present invention.
  • the compound is used for preparing a medicament for inhibiting HSD17B13 gene expression.
  • the present invention also provides the use of the siRNA of the present invention, or the siRNA conjugate of the present invention, or the pharmaceutical composition of the present invention for preparing a medicament for preventing and/or treating diseases related to HSD17B13 gene overexpression.
  • the disease is selected from non-alcoholic fatty liver disease, cirrhosis, alcoholic hepatitis, liver fibrosis, liver cancer.
  • the present invention also provides a method for inhibiting HSD17B13 gene expression, which includes contacting a therapeutically effective amount of the siRNA of the present invention, or the siRNA conjugate of the present invention, or the pharmaceutical composition of the present invention with cells expressing HSD17B13 or administering it to a patient in need subjects.
  • the present invention also provides a method for treating and/or preventing diseases related to HSD17B13 gene overexpression, comprising administering a therapeutically effective amount of the siRNA of the present invention, or the siRNA conjugate of the present invention, or the pharmaceutical composition of the present invention to a subject in need thereof.
  • siRNA, pharmaceutical composition and siRNA conjugate provided by this application show excellent HSD17B13 gene expression inhibitory activity in in vitro cell experiments, and have good potential to treat diseases related to HSD17B13 gene overexpression.
  • the siRNA and its conjugate disclosed in this application can reduce the expression of HSD17B13 mRNA in the liver, have low toxic and side effects, good plasma stability, and have good clinical application prospects.
  • the siRNA provided in this application shows good inhibitory effect on HSD17B13 gene in human liver cancer cell Huh7 cells.
  • the siRNA provided in this application has an inhibition rate of up to 89.77% at a concentration of 0.1 nM, and an inhibition rate of up to 76.97% at a concentration of 0.01 nM.
  • the siRNA provided by the present application has higher HSD17B13 gene inhibitory activity in Huh7 cells, for example, the IC 50 is as low as 12pM.
  • the siRNA conjugates provided herein have high HSD17B13 gene inhibition activity in PHH cells.
  • the IC 50 when entering PHH cells by free uptake, the IC 50 can be as low as 82 pM; when entering PHH cells by transfection, the IC 50 can be as low as 1.2 pM.
  • G", “C”, “A”, “T” and “U” usually represent guanine, cytosine, adenine and thymine respectively.
  • the base of uracil but it is also commonly known in the art that "G”, “C”, “A”, “T” and “U” each usually represent guanine, cytosine, adenine, respectively.
  • Thymine and uracil are nucleotides as bases, which is a common way of expressing DNA sequences and/or ribonucleic acid sequences, so in the context of this disclosure, “G”, “C”,
  • the meanings represented by “A”, “T” and “U” include the various possible situations mentioned above.
  • Lowercase letters a, u, c, g represent 2'-methoxy modified nucleotides; Af, Gf, Cf, Uf: represent 2'-fluoro modified nucleotides; lowercase letter s represents the same letter as this letter
  • the two adjacent nucleotides to the left and right of s are connected by phosphorothioate groups; P1: indicates that the adjacent nucleotide to the right of P1 is a 5'-phosphate nucleotide;
  • a , U , C , G (Underline + bold + italics): Indicates GNA modified nucleotides.
  • the "2'-fluoro-modified nucleotide” refers to a nucleotide in which the hydroxyl group at the 2' position of the ribosyl group of the nucleotide is replaced by fluorine.
  • “Non-fluorinated modified nucleotides” refers to nucleotides or nucleotide analogs in which the hydroxyl group at the 2’ position of the ribosyl group of the nucleotide is replaced by a non-fluorinated group.
  • each non-fluorinated modified nucleotide is independently selected from nucleotides or nucleotide analogs formed by replacing the hydroxyl group at the 2' position of the ribose group of the nucleotide with a non-fluorinated group.
  • nucleotides formed by replacing the hydroxyl group at the 2' position of the ribosyl group with a non-fluorine group are well known to those skilled in the art.
  • nucleotides can be selected from 2'-alkoxy modified nucleotides, 2'-Substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-amino modified nucleotides, 2'-substituted One of the amino-modified nucleotides and 2'-deoxynucleotides.
  • Alkyl includes straight chain, branched or cyclic saturated alkyl groups.
  • alkyl groups include, but are not limited to, methyl, ethyl, propyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, cyclobutyl, n-pentyl, cyclohexyl, and the like. group.
  • the "C 1-6 " in "C 1-6 alkyl” refers to a linear, branched or cyclic arrangement containing 1, 2, 3, 4, 5 or 6 carbon atoms. group.
  • Alkoxy as used herein means an alkyl group attached to the remainder of the molecule through an oxygen atom (-O-alkyl), wherein said alkyl group is as defined herein.
  • alkoxy include methoxy, ethoxy, trifluoromethoxy, difluoromethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, n- Pentyloxy etc.
  • Nucleotide analogue refers to a nucleotide that can replace a nucleotide in a nucleic acid, but whose structure is different from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide or thymus Pyrimidine deoxyribonucleotide group.
  • BNA refers to constrained or inaccessible nucleotides.
  • BNA may contain a five-membered ring, a six-membered ring, or a seven-membered ring bridged structure with a "fixed"C3'-endoglycocondensation.
  • the bridge is usually incorporated into the 2'-, 4'-position of the ribose to provide a 2', 4'-BNA nucleotide, such as LNA, ENA, cET BNA, etc., where LNA is as shown in formula (1) shown, ENA is shown in formula (2), cET BNA is shown in formula (3).
  • Acyclic nucleotides are a type of nucleotide formed by opening the sugar ring of a nucleotide, such as unlocked nucleic acid (UNA) or glycerol nucleic acid (GNA).
  • UNA is represented by formula (4)
  • GNA is represented by formula (4). 5 shown.
  • R is selected from H, OH or alkoxy (O-alkyl).
  • Isonucleotides refer to compounds formed by changing the position of the base on the ribose ring in the nucleotide. For example, the base moves from the 1'-position to the 2'-position or 3'-position of the ribose ring.
  • the compound is shown in formula (6) or (7).
  • Base represents a base, such as A, U, G, C or T; R is selected from H, OH, F or the non-fluorine group as mentioned above.
  • the nucleotide analog is selected from one of isonucleotides, LNA, ENA, cET BNA, UNA, and GNA.
  • each non-fluorinated modified nucleotide is a 2'-methoxy modified nucleotide, a GNA modified nucleotide, or a combination of any two or more thereof.
  • each non-fluoro modified nucleotide is a 2'-methoxy modified nucleotide, above and below, the 2'-methoxy modified nucleoside Acid refers to a nucleotide in which the 2'-hydroxyl group of the ribosyl group is replaced by a methoxy group.
  • the "2'-methoxy modified nucleotide” refers to a nucleotide in which the 2'-hydroxyl group of the ribose group is replaced by a methoxy group.
  • the "phosphorothioate group” refers to a phosphorothioate group in which one of the oxygen atoms in the phosphodiester bond in the phosphate group is replaced by a sulfur atom.
  • the "5'-phosphate nucleotide” refers to the following structure:
  • the expressions "complementary” and “reverse complementary” are used interchangeably and have the meaning well known to those skilled in the art, that is, in a double-stranded nucleic acid molecule, the bases of one strand are each associated with the other. The bases in the strand are paired in a complementary manner.
  • the purine base adenine (A) always pairs with the pyrimidine base thymine (T) (or uracil (U) in RNA);
  • the purine base guanine (C) always pairs with the pyrimidine base Pairs with cytosine (G).
  • Each base pair consists of a purine and a pyrimidine.
  • mismatch in this field means that in double-stranded nucleic acids, the bases at corresponding positions do not pair in a complementary manner.
  • substantially reverse complementary means that there are no more than 3 base mismatches between the two nucleotide sequences involved; “substantially reverse complementary” means that there are no more than 3 base mismatches between the two nucleotide sequences involved; “ means that there is no more than one base mismatch between the two nucleotide sequences; “complete reverse complementarity” means that there is no base mismatch between the two nucleotide sequences.
  • nucleotide difference between one nucleotide sequence and another nucleotide sequence means that the base type of the nucleotide at the same position has changed between the former and the latter. For example, when one nucleotide base in the latter is A, and when the corresponding nucleotide base at the same position in the former is U, C, G or T, it is regarded as one of the two nucleotide sequences. There are nucleotide differences at this position. In some embodiments, when the nucleotide at the original position is replaced by an abasic nucleotide or its equivalent, it can also be considered that a nucleotide difference is generated at that position.
  • an "overhang” refers to one or more unpaired nucleotides that protrude from the duplex structure of an siRNA when one 3' end of one strand of the siRNA extends beyond the 5' end of the other strand. , or vice versa.
  • "Blunt end” or “blunt end” means that there are no unpaired nucleotides at that end of the siRNA, ie, no nucleotide overhangs.
  • a “blunt-ended" siRNA is one that is double-stranded throughout its length, ie, it has no nucleotide overhangs at either end of the molecule.
  • the nucleoside monomer refers to the siRNA or siRNA to be prepared according to Type and sequence of nucleotides in siRNA conjugates, modified or unmodified nucleoside phosphoramidite monomers used in solid-phase phosphoramidite synthesis.
  • Solid-phase phosphoramidite synthesis is a method used in RNA synthesis well known to those skilled in the art.
  • the nucleoside monomers used in this application are all commercially available.
  • conjugate means that two or more chemical moieties each having a specific function are connected to each other in a covalent manner; accordingly, “conjugate” is Refers to a compound formed by covalent connections between various chemical parts.
  • siRNA conjugate refers to a compound formed by covalently linking one or more chemical moieties with specific functions to siRNA.
  • siRNA conjugate should be understood as a collective name for multiple siRNA conjugates or a siRNA conjugate represented by a certain chemical formula, depending on the context.
  • conjugation molecule should be understood as a specific compound that can be conjugated to siRNA through a reaction, ultimately forming the siRNA conjugate of the present application.
  • hydroxyl protecting groups can be used in this application. Generally speaking, protecting groups sensitize chemical functional groups to specific are insensitive to certain reaction conditions and can be attached to and removed from the functional group in the molecule without substantially damaging the rest of the molecule. In some embodiments, the protecting group is stable under basic conditions but can be removed under acidic conditions. In some embodiments, non-exclusive examples of hydroxyl protecting groups that may be used herein include dimethoxytrityl (DMT), monomethoxytrityl, 9-phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl (Mox).
  • DMT dimethoxytrityl
  • Azure 9-phenylxanthine-9-yl
  • Mox 9-(p-methoxyphenyl)xanthine-9-yl
  • non-exclusive examples of hydroxyl protecting groups that can be used herein include Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4'-dimethoxy trityl) and TMTr (4,4',4′′-trimethoxytrityl).
  • subject refers to any animal, such as a mammal or a marsupial.
  • the subject of the present application includes, but is 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.
  • 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.
  • treatment refers to a method of obtaining beneficial or desired results, including but not limited to therapeutic benefit.
  • “Therapeutic benefit” means eradication or amelioration of the underlying disorder being treated.
  • therapeutic benefit is obtained by eradicating or ameliorating one or more physiological symptoms associated with the underlying disorder, such that improvement is observed in the subject, although the subject may still be suffering from the underlying disorder.
  • prevention refers to a method of obtaining beneficial or desired results, including but not limited to preventive benefits.
  • a siRNA, siRNA conjugate or pharmaceutical composition may be administered to a subject at risk of developing a particular disease, or to a subject who reports one or more physiological symptoms of the disease, even if possible A diagnosis of the disease has not yet been made.
  • the present application relates to a siRNA capable of inhibiting HSD17B13 gene expression.
  • the siRNA of the present application contains a nucleotide group as a basic structural unit. It is well known to those skilled in the art that the nucleotide group contains a phosphate group, a ribose group and a base. Typically active, ie, functional, siRNAs are about 12-40 nucleotides in length, and in some embodiments are about 15-30 nucleotides in length.
  • the siRNA of the present application contains a sense strand and an antisense strand.
  • Each nucleotide in the siRNA is independently a modified or unmodified nucleotide, wherein the sense strand contains a nucleotide sequence I, and the The antisense strand contains a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region.
  • the double-stranded region is 15-30 nucleotide pairs in length.
  • the double-stranded region is 17-23 nucleotide pairs in length.
  • the double-stranded region is 19-21 nucleotide pairs in length.
  • the double-stranded region is 19 or 21 nucleotide pairs in length.
  • the sense strand further contains nucleotide sequence III
  • the antisense strand further contains nucleotide sequence III
  • the lengths of sequence IV, nucleotide sequence III and nucleotide sequence IV are each independently 0-6 nucleotides, and the nucleotide sequence III is connected to the 5′ end of nucleotide sequence I, nucleotide sequence Sequence IV is connected to the 3′ end of nucleotide sequence II, and said nucleotide sequence III and said nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary; said substantially reverse complement It means that there is no more than one base mismatch between the two nucleotide sequences; perfect reverse complementarity means that there is no mismatch between the two nucleotide sequences.
  • the sense strand further contains nucleotide sequence III
  • the antisense strand further contains nucleotide sequence IV
  • the lengths of nucleotide sequence III and nucleotide sequence IV are each independently 0- 6 nucleotides
  • the nucleotide sequence III is connected to the 3′ end of the nucleotide sequence I
  • the nucleotide sequence IV is connected to the 5′ end of the nucleotide sequence II
  • the nucleotide sequences IV are equal in length and are substantially reverse complementary or completely reverse complementary
  • the substantially reverse complementarity means that there is no more than 1 base mismatch between the two nucleotide sequences
  • Perfect reverse complementarity means there are no mismatches between the two nucleotide sequences.
  • the sense strand further contains nucleotide sequence III
  • the antisense strand further contains nucleotide sequence IV
  • the lengths of nucleotide sequence III and nucleotide sequence IV are each independently 0- 6 nucleotides
  • the nucleotide sequence III is connected to the 5′ end of the nucleotide sequence I
  • the nucleotide sequence IV is connected to the 3′ end of the nucleotide sequence II
  • the nucleotide sequence III and The nucleotide sequence IV is equal in length and substantially reverse complementary or completely reverse complementary
  • the nucleotide sequence III is connected to the 3′ end of the nucleotide sequence I
  • the nucleotide sequence IV is connected to the nucleoside
  • the 5′ end of the acid sequence II, the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary; the substantially reverse complementarity refers to two nucleosides There is no more than 1 base
  • the sense strand further contains the nucleotide sequence V and/or the antisense strand further contains the nucleotide sequence VI, the nucleotide sequences V and VI being 0 to 3 nucleotides in length , the nucleotide sequence V is connected to the 3' end of the sense strand to form the 3' overhang of the sense strand, and/or the nucleotide sequence VI is connected to the 3' end of the antisense strand to form the 3' end of the antisense strand. protruding end.
  • the nucleotide sequence V or VI is 2 nucleotides in length.
  • the nucleotide sequence V or VI is two consecutive thymine deoxyribonucleotides or two consecutive uracil ribonucleotides. In other embodiments, the nucleotide sequence V or VI mismatches or is complementary to the nucleotide at the corresponding position of the target mRNA.
  • the lengths of the sense strand and the antisense strand provided by the application are the same or different.
  • the sense strand or the antisense strand has 15-30 nucleotides.
  • the sense or antisense strand has 19-25 nucleotides.
  • the sense or antisense strand has 19-23 nucleotides.
  • the length ratio of the siRNA sense strand and the antisense strand provided in this application can be 15/15, 16/16, 17/17, 18/18, 19/19, 19/20, 19/21, 19/22, 19/ 23, 20/19, 20/20, 20/21, 20/22, 20/23, 21/19, 21/20, 21/21, 21/22, 21/23, 22/19, 22/20, 22/21, 22/22, 22/23, 23/19, 23/20, 23/21, 23/22, 23/23, 24/24, 25/25, 26/26, 27/27, 28/ 28, 29/29, 30/30, 22/24, 22/25, 22/26, 23/24, 23/25 or 23/26 etc.
  • the length ratio of the siRNA sense strand and antisense strand is 19/19, 21/21, 19/21, 21/23 or 23/23.
  • the siRNA of the present disclosure has better Cellular mRNA silencing activity.
  • siRNA obtained by one of the modification methods while improving blood stability, It also maintained inhibitory activity that was essentially equivalent to that of unmodified siRNA.
  • each nucleotide in the siRNA of the present invention is independently a modified or unmodified nucleotide.
  • each nucleotide in the siRNA of the present invention is an unmodified nucleotide; in some embodiments, some or all of the nucleotides in the siRNA of the present invention are modified nucleosides. These modifications on the acid and nucleotide groups will not cause the siRNA of the present invention to significantly weaken or lose the function of inhibiting HSD17B13 gene expression.
  • the siRNA of the present application contains at least 1 modified nucleotide.
  • modified nucleotide is used to refer to a nucleotide or nucleotide analogue in which the 2' hydroxyl group of the ribosyl group of a nucleotide is replaced by another group, or has a modified Modified bases of nucleotides.
  • the modified nucleotides will not cause significant weakening or loss of the function of siRNA to inhibit gene expression.
  • modified nucleotides disclosed in J.K. Watts, G.F. Deleavey, and M.J. Damha, Chemically modified siRNA: tools and applications. Drug Discov Today, 2008, 13(19-20): 842-55 can be selected.
  • At least one nucleotide in the sense strand or the antisense strand of the siRNA provided by the present invention is a modified nucleotide, and/or at least one phosphate group has a modified group.
  • Phosphate group in other words, at least part of the phosphate group and/or ribose group in the phosphate-sugar backbone of at least one single chain in the sense strand and the antisense strand is a phosphate group with a modifying group and/or ribosyl groups with modifying groups.
  • the phosphate group containing a modifying group is a phosphorothioate group formed by replacing at least one oxygen atom in the phosphodiester bond of the phosphate group with a sulfur atom.
  • the siRNA includes a sense strand that does not include a 3' overhanging nucleotide; that is, the sense strand of the siRNA may have a 3' overhanging nucleotide, excluding the 3' overhanging nucleotide of the sense strand. Then a flat end is formed.
  • nucleosides are added to the 3' end of the sense strand.
  • Acid sequence V as overhanging nucleotide.
  • the nucleotide sequence formed is chemically modified to exclude the nucleotide sequence V.
  • the sense strand of siRNA forms a blunt end.
  • the sense strand when the nucleotide sequences of the sense strand and the antisense strand are complementary to form a double-stranded region, and the 3' end of the sense strand has a protruding nucleotide extending out of the double-stranded region, the sense strand is located at The protruding nucleotide at the 3' end is excluded as the nucleotide sequence of the sense strand, and accordingly, the sense strand of siRNA forms a blunt end.
  • the 5' terminal nucleotide of the sense strand is linked to a 5' phosphate group or a 5' phosphate-derived group. In some embodiments, the 5' terminal nucleotide of the antisense strand is connected to a 5' phosphate group or a 5' phosphate derivative group.
  • An exemplary structure of the 5' phosphate group is: The structures of the 5' phosphate derivative group include but are not limited to: wait.
  • Base represents a base, such as A, U, G, C or T.
  • R' is a hydroxyl group or is substituted by various groups known to those skilled in the art, such as 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, and 2'-deoxy nucleotides.
  • 2'-fluoro (2'-F) modified nucleotides such as 2'-fluoro (2'-F) modified nucleotides, 2'-alkoxy modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-sub
  • Exemplary modified nucleotides have the structure shown below:
  • Base represents a base, such as A, U, G, C or T.
  • the hydroxyl group at the 2’ position of the ribose group is replaced by R.
  • the hydroxyl group at the 2' position of these ribose groups can be replaced by various groups known to those skilled in the art, for example, 2'-fluoro (2'-F) modified nucleotides, 2'-alkoxy groups Modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-amino modified Nucleotide, 2'-substituted amino-modified nucleotide, 2'-deoxynucleotide.
  • the 2'-alkoxy modified nucleotide is a 2'-methoxy (2'OMe, 2'-O- CH3 ) modified nucleotide, and the like.
  • all nucleotides in the sense strand and/or the antisense strand are modified nucleosides.
  • each nucleotide in the sense strand and the antisense strand of the siRNA provided by the present invention is independently a 2'-fluoro-modified nucleotide or a non-fluoro-modified nucleotide.
  • each non-fluoro-modified nucleotide is a 2'-methoxy-modified nucleotide or a GNA-modified nucleotide, wherein the 2'-methoxy-modified nucleotide refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribose group with a methoxy group.
  • the 2'-fluoromodified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand in a 5' to 3' direction, and the remaining positions are non-fluoromodified nucleotides ; According to the 5' to 3' direction, the 2'-fluorinated modified nucleotides are located at the even-numbered positions of the antisense strand, and the remaining positions are non-fluorinated modified nucleotides.
  • the 2'-fluoromodified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand in a 5' to 3' direction, and the remaining positions are non-fluoromodified nucleotides ; According to the 5' to 3' direction, the 2'-fluorinated modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and the remaining positions are non-fluorinated modified nucleotides.
  • the 2'-fluoromodified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand in a 5' to 3' direction, and the remaining positions are non-fluoromodified nucleotides ; According to the 5' to 3' direction, the 2'-fluorinated modified nucleotides are located at positions 2, 6, 8, 9, 14 and 16 of the antisense strand, and the remaining positions are non-fluorinated modified nucleotides .
  • the 2'-fluorinated modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are non-fluorinated modified cores According to the 5' to 3' direction, the 2'-fluorinated modified nucleotides are located at positions 2, 14 and 16 of the antisense strand, and the remaining positions are non-fluorinated modified nucleotides.
  • the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups Modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at the even-numbered positions of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides.
  • the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups Modified nucleotides; in the 5' to 3' direction, 2'-fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and the remaining positions are 2'-methoxy modified of nucleotides.
  • the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups Modified nucleotides; in the 5' to 3' direction, 2'-fluoro modified nucleotides are located at positions 2, 6, 8, 9, 14 and 16 of the antisense strand, and the remaining positions are 2'- Methoxy modified nucleotides.
  • the 2'-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups Modified nucleotides; in the 5' to 3' direction, 2'-fluoro modified nucleotides are located at positions 2, 14 and 16 of the antisense strand, and GNA modified nucleotides are located at position 1 of the antisense strand. Position 6, and the remaining positions are 2'-methoxy modified nucleotides.
  • the 2'-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand in the 5' to 3' direction, and the remaining positions are 2'-methoxy groups Modified nucleotides; in the 5' to 3' direction, 2'-fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and GNA modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand. Position 7, and the remaining positions are 2'-methoxy modified nucleotides.
  • each non-fluorinated modification The nucleotides are all 2'-methoxy modified nucleotides.
  • the 2'-methoxy modified nucleotides refer to nucleotides formed by replacing the 2'-hydroxyl group of the ribose group with a methoxy group. .
  • At least one of the linkages between the following nucleotides in the siRNA is a phosphorothioate linkage:
  • the siRNA is directed from the 5' end to the 3' end, and the sense strand contains a phosphorothioate group at a position as shown below:
  • the sense strand contains phosphorothioate groups at the positions shown below:
  • the siRNA is directed from the 5' end to the 3' end, and the antisense strand contains a phosphorothioate group at a position as shown below:
  • the present application relates to a siRNA conjugate, which contains the above-mentioned siRNA and a conjugation group conjugated to the siRNA.
  • 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.
  • the 3' end of the sense strand of the siRNA conjugate forms a blunt end
  • the 3' end of the antisense strand of the siRNA conjugate has 1-3 protruding nucleotides extending out of the double-stranded region.
  • the 3' end of the sense strand of the siRNA conjugate forms a blunt end
  • the 3' end of the antisense strand of the siRNA conjugate forms a blunt end.
  • the siRNA conjugate is obtained by conjugating siRNA with a conjugating group.
  • the sense strand of siRNA and the antisense strand are complementary to form a double-stranded region of siRNA, and the 3' end of the sense strand of siRNA forms a blunt end, and the conjugation group is conjugated to the 3' end of the sense strand with a blunt end. , forming siRNA conjugates.
  • the 3' end of the sense strand of siRNA has a protruding nucleotide extending out of the double-stranded region, and the protruding nucleotide located at the 3' end of the sense strand is excluded to form a structure with 3'
  • the blunt-ended sequence serves as the nucleotide sequence for connecting the conjugation group, and the conjugation group is connected to the 3' blunt end of the sense strand to form an siRNA conjugate.
  • nucleotide sequences of the sense strand and the antisense strand are complementary to form a double-stranded region
  • a nucleotide sequence V is added to the 3' end of the sense strand as a protruding nucleotide.
  • the sequence with a 3' blunt end formed after the protruding nucleotide at the 3' end of the sense strand is excluded is used as the nucleotide sequence for connecting the conjugated group, and the conjugated group is connected to the 3' blunt end of the sense strand to form a siRNA conjugate.
  • the nucleotide sequences of the sense strand and the antisense strand are complementary to form a double-stranded region, and the 3' end of the sense strand has a protruding nucleotide that extends out of the double-stranded region, it will be located in the sense strand.
  • the sequence with a 3' blunt end formed after excluding the protruding nucleotides at the 3' end of the chain is used as the nucleotide sequence for connecting the conjugation group, and the conjugation group is connected to the 3' blunt end of the sense strand. Formation of siRNA conjugates.
  • the 3' end of the sense strand of the siRNA has a protruding nucleotide extending out of the double-stranded region, and the protruding - located at the 3' end of the sense strand will be
  • the gsascuacUfuAfUfGfaauuugca blunt-end sequence formed after sTsT nucleotide exclusion serves as the nucleotide sequence used to connect the L96 conjugation group.
  • the sequence to form the siRNA conjugate is: the sense strand is gsascuacUfuAfUfGfaauuugcaL96, and the antisense strand is PlusGfscAfaAfuUfcAfuAfaGfuAfgUfcsTsT.
  • the conjugated group includes at least one pharmaceutically acceptable targeting group, or further includes a linker, and the siRNA, the linker and the targeting group are sequentially connected.
  • the targeting groups are 1-6.
  • the targeting groups are 2-4.
  • the siRNA molecule can be non-covalently or covalently conjugated to the conjugated group, for example, it can be covalently conjugated to the conjugated group.
  • the conjugation site of the siRNA and the conjugated group can be at the 3' end or the 5' end of the siRNA sense strand, or it can be at the 5' end. 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 and the conjugation group is at the 3' end of the siRNA sense strand.
  • the conjugation group can be attached to the phosphate group, the 2'-hydroxyl group, or the base of the nucleotide. In some embodiments, the conjugation group can also be connected to the 3'-position hydroxyl group, in which case the nucleotides are connected via a 2'-5' phosphodiester bond.
  • the conjugation group is usually attached to the phosphate group of the nucleotide; when the conjugation group is attached to the internal sequence of the siRNA, the conjugation group Usually attached to the ribose sugar ring or base.
  • the siRNA and the conjugation group can be connected through acid-labile or reducible chemical bonds. In the acidic environment of cellular endosomes, these chemical bonds can be degraded, thereby leaving the siRNA in a free state.
  • the conjugation group can be connected to the sense strand of siRNA to minimize the impact of conjugation on siRNA activity.
  • the pharmaceutically acceptable targeting group can be a ligand commonly used in the field of siRNA delivery, such as various ligands described in WO2009082607A2, which is fully incorporated into this specification by reference.
  • the pharmaceutically acceptable targeting group can be selected from one or more ligands formed by the following targeting molecules or derivatives thereof: lipophilic molecules, such as cholesterol, bile acids, Vitamins (such as vitamin E), lipid molecules of different chain lengths; polymers, such as polyethylene glycol; polypeptides, such as membrane-penetrating peptides; aptamers; antibodies; quantum dots; sugars, such as lactose, polylactose, and mannose Sugar, galactose, N-acetylgalactosamine (GalNAc); folate; receptor ligands expressed by liver parenchymal cells, such as asialoglycoprotein, asialoglycoside residues, lipoproteins (such as high-density Lipoproteins, low-density lipoproteins, etc.), glucagon, neurotransmitters (such as epinephrine), growth factors, transferrin, etc.
  • lipophilic molecules such as cholesterol, bile acids, Vitamins (such
  • each ligand is independently selected from a ligand capable of binding to a cell surface receptor.
  • at least one ligand is a ligand capable of binding to a hepatocyte surface receptor.
  • at least one ligand is a ligand capable of binding to a mammalian cell surface receptor.
  • at least one ligand is a ligand capable of binding to a human hepatocyte surface receptor.
  • at least one ligand is a ligand capable of binding to liver surface asialoglycoprotein receptor (ASGPR).
  • ASGPR liver surface asialoglycoprotein receptor
  • the types of these ligands are well known to those skilled in the art. Their function is generally to bind to specific receptors on the surface of target cells and mediate the delivery of siRNA linked to the ligands to the target cells.
  • the pharmaceutically acceptable targeting group can be associated with the mammalian hepatocyte surface Any ligand that binds to the asialoglycoprotein receptor (ASGPR).
  • each ligand is independently an asialoglycoprotein, such as asialoorosomucoid (ASOR) or asialofetuin (ASF).
  • the ligand is a sugar or sugar derivative.
  • At least one ligand is a sugar. In some embodiments, each ligand is a sugar. In some embodiments, at least one ligand is a monosaccharide, a polysaccharide, a modified monosaccharide, a modified polysaccharide, or a sugar derivative. In some embodiments, at least one of the ligands can be a monosaccharide, a disaccharide, or a trisaccharide. In some embodiments, at least one ligand is a modified sugar. In some embodiments, each ligand is a modified sugar.
  • each ligand is independently selected from the group consisting of polysaccharides, modified polysaccharides, monosaccharides, modified monosaccharides, polysaccharide derivatives, or monosaccharide derivatives.
  • each or at least one ligand is selected from the group consisting of glucose and its derivatives, mannan and its derivatives, galactose and its derivatives, xylose and its derivatives substances, ribose and its derivatives, fucose and its derivatives, lactose and its derivatives, maltose and its derivatives, arabinose and its derivatives, fructose and its derivatives and sialic acid.
  • each of the ligands may be independently selected from the group consisting of D-mannopyranose, L-mannopyranose, D-arabinose, D-xylfuranose, L-xylfuranose, D- Glucose, L-glucose, D-galactose, L-galactose, ⁇ -D-mannofuranose, ⁇ -D-mannofuranose, ⁇ -D-mannopyranose, ⁇ -D-glucopyranose, ⁇ -D-glucopyranose, ⁇ -D-glucofuranose, ⁇ -D-glucofuranose, ⁇ -D-fructofuranose, ⁇ -D-fructopyranose, ⁇ -D-pyranose Galactopyranose, ⁇ -D-galactopyranose, ⁇ -D-galactofuranose, ⁇ -D-galactofuranose, gluco
  • the pharmaceutically acceptable targeting group in the siRNA conjugate can be galactose or N-acetylgalactosamine, wherein the galactose or N-acetylgalactosamine molecule can be a monovalent , divalent, trivalent, quadrivalent. It should be understood that the monovalent, bivalent, trivalent, and tetravalent terms described here respectively refer to the siRNA conjugate formed by a siRNA molecule and a conjugation group containing a galactose or N-acetylgalactosamine molecule as a targeting group.
  • the molar ratio of siRNA molecules to galactose or N-acetylgalactosamine molecules in the siRNA conjugate is 1:1, 1:2, 1:3 or 1:4.
  • the pharmaceutically acceptable targeting group is N-acetylgalactosamine.
  • the siRNA described herein when the siRNA described herein is conjugated to a conjugation group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent or tetravalent.
  • the N-acetylgalactosamine molecule is trivalent.
  • the targeting group can be connected to the siRNA molecule via a suitable linker, and those skilled in the art can select a suitable linker according to the specific type of the targeting group.
  • suitable linker those skilled in the art can select a suitable linker according to the specific type of the targeting group.
  • the types of these linkers, targeting groups and the connection methods with siRNA can be found in the disclosure of WO2015006740A2, which is fully incorporated into this specification by reference.
  • the nucleoside monomers are connected one by one from the 3'-5' direction according to the order of nucleotide arrangement through the solid-phase phosphoramidite method conventional in this field.
  • Each connection of a nucleoside monomer involves four steps of deprotection, coupling, capping, oxidation or sulfation.
  • deprotection, coupling, capping, oxidation or sulfation when two nucleotides are connected using a phosphate ester, when the next nucleoside monomer is connected, it includes four steps of deprotection, coupling, capping, and oxidation.
  • two nucleotides are connected using phosphorothioate, when the next nucleoside monomer is connected, it includes four steps of protection, coupling, capping and sulfation.
  • siRNA of the present application can be as follows:
  • reaction temperature is 25°C
  • reaction time is 70 seconds
  • deprotection reagent is selected from dichloroacetic acid in dichloromethane solution (3% V/V)
  • deprotection reagent and protective group on solid phase carrier The molar ratio is 5:1.
  • the coupling reaction conditions include: the reaction temperature is 25°C, the reaction time is 600 seconds, the coupling reagent is selected from a 0.25M acetonitrile solution of 5-ethylthio-1H-tetrazole (ETT), and the nucleic acid connected to the solid-phase carrier
  • ETT 5-ethylthio-1H-tetrazole
  • the molar ratio of sequence to nucleoside monomer is 1:10.
  • the capping reaction conditions include: the reaction temperature is 25°C, the reaction time is 15 seconds, and the capping reagent is selected from CapA (10% acetic anhydride acetonitrile solution) and CapB (10% N-methylimidazole pyridine/ Acetonitrile solution), the molar ratio of the capping reagent to the nucleic acid sequence connected to the solid phase carrier is acetic anhydride:N-methylimidazole:the molar ratio of the nucleic acid sequence connected to the solid phase carrier is 1:1:1.
  • the oxidation reaction conditions include: the reaction temperature is 25°C, the reaction time is 15 seconds, the oxidation reagent is selected from 0.05M iodine tetrahydrofuran solution, and the molar ratio of the oxidation reagent to the nucleic acid sequence connected to the solid-phase carrier in the coupling step is 30:1.
  • the sulfidation reaction conditions include: the reaction temperature is 25°C, the reaction time is 300 seconds, the sulfide reagent is selected from hydrogenated xanthogen, and the molar ratio of the sulfide reagent to the nucleic acid sequence connected to the solid-phase carrier in the coupling step is 120:1.
  • the nucleic acid sequences connected to the solid phase carrier are cut, deprotected, purified, and desalted in sequence to obtain the siRNA sense strand and antisense strand. Finally, the two strands are heated and annealed to obtain the product.
  • cleavage, deprotection, purification, desalting and annealing are well known in the art.
  • cleavage and deprotection are carried out by contacting the nucleotide sequence connected to the solid-phase carrier with concentrated ammonia water; purification by chromatography; desalting by reversed-phase chromatography; by mixing the sense strand and the sense strand in equimolar ratios under different stringent conditions. gradually decreases after the antisense strand Cool down.
  • compound L96-A is obtained by reacting DMTr-L96 and succinic anhydride:
  • Preparation process Mix L96-A, O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU) and diisopropylethylamine (DIPEA) in acetonitrile, stir at room temperature for 5 minutes to obtain a uniform solution , add aminomethyl resin (NH 2 -SPS, 100-200 mesh) to the reaction liquid, start the shaking reaction at 25°C, filter after 18 hours of reaction, and wash the filter cake with dichloromethane and acetonitrile in sequence to obtain the filter cake .
  • the obtained filter cake is capped with a CapA/CapB mixed solution to obtain L96-B, which is a solid-phase carrier containing the conjugated molecule.
  • the nucleoside monomer is connected to the conjugated molecule under the coupling reaction, and then the nucleoside monomer is connected to the conjugated molecule as described above.
  • the siRNA molecule synthesis method is used to synthesize the siRNA sense strand connected to the conjugate molecule, and the siRNA molecule synthesis method described above is used to synthesize the siRNA antisense strand, and annealed to generate the siRNA conjugate of the present application.
  • the present application provides a pharmaceutical composition, which contains the siRNA described above as an active ingredient and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier can be a carrier commonly used in the field of siRNA administration, such as but not limited to lipid nanoparticles (Lipid Nanoparticles, LNPs), magnetic nanoparticles (magnetic nanoparticles, such as those based on Fe 3 O 4 or Fe 2 O 3 nanoparticles), carbon nanotubes, mesoporous silicon silicon), calcium phosphate nanoparticles (calcium phosphate nanoparticles), polyethylenimine (PEI), polyamide dendrimer (polyamidoamine (PAMAM) dendrimer), polylysine (poly(L-lysine), PLL), chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), polyD-type or L-type lactic acid/glycolic acid copolymer Poly(D&L-lactic/glycolic acid)copolymer, PLGA), poly(2-aminoethyl ethylene phosphate), PP
  • siRNA and pharmaceutically acceptable carriers there are no special requirements on the contents of siRNA and pharmaceutically acceptable carriers, and they can be the conventional contents of each component.
  • the pharmaceutical composition may also contain other pharmaceutically acceptable excipients, which may be one or more of various preparations or compounds commonly used in the art.
  • the other pharmaceutically acceptable excipients may include at least one of a pH buffer, a protective agent, and an osmotic pressure regulator.
  • the pH buffer can be a trishydroxymethylaminomethane hydrochloride buffer with a pH of 7.5-8.5 and/or a phosphate buffer with a pH of 5.5-8.5, for example, it can be a phosphate with a pH of 5.5-8.5. Buffer.
  • the protective agent may be at least one of myo-inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose and glucose. Based on the total weight of the pharmaceutical composition, the content of the protective agent may be 0.01-30% by weight.
  • the osmotic pressure regulator may be sodium chloride and/or potassium chloride.
  • the content of the osmotic pressure regulator is such that the osmotic pressure of the pharmaceutical composition is 200-700 milliosmole/kg (mOsm/kg).
  • the content of the osmotic pressure regulator can be easily determined by those skilled in the art based on the desired osmotic pressure.
  • the pharmaceutical composition can be a liquid preparation, such as an injection; it can also be a freeze-dried powder injection, which is mixed with liquid excipients during administration to prepare a liquid preparation.
  • the liquid preparation may be, but is not limited to, administered by subcutaneous, intramuscular or intravenous injection, may be administered to the lungs by spray, or may be administered to other organs and tissues (such as the liver) through the lungs by spray.
  • the pharmaceutical composition is for intravenous administration.
  • the pharmaceutical composition may be in the form of a liposome formulation.
  • the pharmaceutically acceptable carrier used in the liposome formulation includes an amine-containing transfection compound (hereinafter also referred to as an organic amine), a helper lipid, and/or a pegylated Lipids.
  • the experimental techniques and experimental methods used in this example are all conventional technical methods unless otherwise specified.
  • the experimental methods in the following examples that do not specify specific conditions are usually carried out according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.
  • the materials, reagents, etc. used in the examples can be obtained through regular commercial channels unless otherwise specified.
  • siRNA molecule with the following sequence was synthesized by Tianlin Biotechnology (Shanghai) Co., Ltd.
  • the capital letters "G”, “C”, “A”, “T” and “U” each usually represent nucleotides containing guanine, cytosine, adenine, thymine and uracil as bases respectively.
  • Lowercase letters a, u, c, g represent: 2'-methoxy modified nucleotides
  • Af, Gf, Cf, Uf represent: 2'-fluoro modified nucleotides
  • the letter s indicates that the two nucleotides adjacent to the left and right of the letter s are connected by phosphorothioate groups
  • P1 indicates that the nucleotide adjacent to the right of P1 is a 5'-phosphate nucleotide
  • a , U , C , G indicates GNA modified nucleotides.
  • siRNA conjugate with the following sequence was synthesized by Tianlin Biotechnology (Shanghai) Co., Ltd.:
  • L96 is:
  • the inhibitory activity of the siRNA of the present invention on HSD17B13 gene expression was evaluated through a dual-luciferase reporter gene vector.
  • a control group was set up in the experiment, and RNA-free H 2 O was used instead of the above-mentioned siRNA compounds.
  • the other conditions were the same as the experimental group; the blank group was Huh7 cells that were not transfected with psiCHECK2-HSD17B13 plasmid, and no siRNA compounds were added to them.
  • ⁇ (Renilla lum average in the test hole-Renilla lum average in the blank group)/(Firefly lum average in the test hole-Firefly lum average in the blank group);
  • the ratio of the experimental group is recorded as: ⁇ (experimental group), and the ratio of the control group is recorded as: ⁇ (control group).
  • the inhibition rate of siRNA inhibiting the expression of the target gene HSD17B13 is calculated according to the following formula:
  • Inhibition rate (%) [1- ⁇ (average value of experimental group)/ ⁇ (average value of control group)] ⁇ 100%
  • the siRNA of the present invention can significantly inhibit the expression of the HSD17B13 gene at both 0.1 nM and 0.01 nM.
  • the final concentrations of the following siRNAs to be tested were 10 nM, 2.5 nM, 0.63 nM, 0.16 nM, 0.04 nM, 0.01 nM, 0.0024 nM and 0.0006 nM, and then IC50 determination was performed in a manner similar to Example 2.
  • ⁇ Ct ⁇ Ct (test sample group)- ⁇ Ct (Mock group), where the Mock group represents the group in which siRNA is not added compared with the experimental group;
  • Inhibition rate (%) (Relative expression of mRNA in Mock group - Relative expression of mRNA in sample group)/Relative expression of mRNA in Mock group ⁇ 100%
  • Top represents the percentage inhibition rate at the top platform, and the Top standard of the curve is generally between 80% and 120%;
  • Bottom represents the percentage inhibition rate at the bottom platform, and the Bottom of the curve is generally between -20% and 20%;
  • HillSlope represents the slope of the percentage inhibition rate curve.
  • the siRNA provided in this application has high HSD17B13 gene inhibitory activity in Huh7 cells, and the IC 50 can be as low as 12pM.
  • PHH medium invitroGRO CP Meduim serum free BIOVIT, Cat. No.: S03316
  • RNAiMAX transfection reagent purchased from Invitrogen, product number: 13778-150;
  • RNA extraction kit 96 Kit item number: QIAGEN-74182;
  • Reverse transcription kit FastKing RT Kit (With gDNase), product number: Tiangen-KR116-02;
  • siRNA conjugates (the final concentrations of siRNA conjugates are 10nM, 2.5nM, 0.63nM, 0.16nM, 0.04nM, 0.01nM, 0.0024nM and 0.0006nM, respectively) into PHH cells through transfection, process As follows: take frozen PHH cells, resuscitate, count, adjust the cells to 6 ⁇ 10 5 cells/ml, and apply Lipofectamine RNAiMax to transfer the siRNA conjugate into the cells, and inoculate 96 cells at a density of 54,000 cells per well. In the well plate, each well contains 100 ⁇ L of culture medium. Cells were cultured in 5% CO 2 and 37°C incubator. After 48 hours, the medium was removed and cells were collected for total RNA extraction. Use according to kit product instructions 96 Kit to extract total RNA.
  • siRNA conjugates enter PHH cells through free uptake.
  • the process is as follows Describe: Take frozen PHH cells, resuscitate, count, adjust the cells to 6 ⁇ 10 5 cells/ml, add siRNA conjugate at the same time, inoculate into a 96-well plate at a density of 54,000 cells per well, and culture in each well The solution is 100 ⁇ l. Cells were cultured in 5% CO 2 and 37°C incubator. After 48 hours, the medium was removed and cells were collected for total RNA extraction. Use according to kit product instructions 96 Kit to extract total RNA.
  • step b) Add the following reagents to the system obtained in step a) and perform reverse transcription:
  • step b) Store the reverse transcription product obtained in step b) at -20°C for real-time PCR analysis.
  • ⁇ Ct ⁇ Ct(test sample group)- ⁇ Ct(Mock group), where the Mock group represents the group without siRNA added compared with the experimental group;
  • Inhibition rate (%) (Relative expression of mRNA in Mock group - Relative expression of mRNA in sample group)/Relative expression of mRNA in Mock group ⁇ 100%
  • Top represents the percentage inhibition rate at the top platform, and the Top standard of the curve is generally between 80% and 120%;
  • Bottom represents the percentage inhibition rate at the bottom platform, and the Bottom of the curve is generally between -20% and 20%;
  • HillSlope represents The slope of the percent inhibition curve.
  • the siRNA conjugate of the present application has a very high HSD17B13 gene inhibitory activity.
  • PHH medium invitroGRO CP Meduim serum free BIOVIT, catalog number: S03316;
  • RNAiMAX transfection reagent purchased from Invitrogen, product number: 13778-150;
  • RNA extraction kit 96Kit item number: QIAGEN-74182;
  • Reverse transcription kit FastQuant RT Kit (With gDNase), product number: Tiangen-KR116-02;
  • siRNA conjugates (the final concentrations of siRNA conjugates are 1 nM and 0.1 nM, respectively, in duplicate wells) are transfected into PHH cells.
  • the process is as follows: take the frozen PHH cells, resuscitate, count, and adjust the cells to 6 ⁇ 10 5 cells/ml, and Lipofectamine RNAiMax was used to transfer the siRNA conjugate into the cells.
  • the cells were seeded into a 96-well plate at a density of 54,000 cells per well, with 100 ⁇ L of culture medium per well. Cells were cultured in 5% CO 2 and 37°C incubator. After 48 hours, the medium was removed and cells were collected for total RNA extraction. Use according to kit product instructions 96 Kit to extract total RNA.
  • siRNA conjugates enter PHH cells through free uptake.
  • the process is as follows: take the frozen PHH cells, resuscitate, count, and adjust the cells to 6 ⁇ 10 5 cells/ml, and siRNA conjugate was added at the same time, and seeded into a 96-well plate at a density of 54,000 cells per well, with 100 ⁇ l of culture medium per well. Cells were cultured in 5% CO 2 and 37°C incubator. After 48 hours, the medium was removed and cells were collected for total RNA extraction. Use according to kit product instructions 96 Kit to extract total RNA.
  • the extracted total RNA was reverse transcribed into cDNA through a reverse transcription reaction.
  • HSD17B13 cDNA will be detected by qPCR.
  • GAPDH cDNA will be used as an internal control for parallel testing.
  • the PCR reaction program is: 95°C for 10 minutes, then enter the cycle mode, 95°C for 15 seconds, then 60°C for 60 seconds, a total of 40 cycles.
  • ⁇ Ct ⁇ Ct (test sample group)- ⁇ Ct (Mock group), where the Mock group represents the group in which siRNA is not added compared with the experimental group;
  • Inhibition rate (%) (Relative expression of mRNA in Mock group - Relative expression of mRNA in sample group)/Relative expression of mRNA in Mock group ⁇ 100%
  • the siRNA and its conjugates of the present application have high HSD17B13 gene inhibitory activity.
  • mice Fourteen days after AAV virus injection, the mice were examined by in vivo imaging, divided into groups (6 mice in each group), and the mice were administered subcutaneously. A single 3 mg/kg dose of N-ER-FY007001M2L96, N-ER-FY007004M2L96, N-ER-FY007020M2L96, N-ER-FY007033M2L96 and N-ER-FY007034M2L96. In vivo imaging was performed on days 7, 14, 21, 28 and 35 after administration to detect the protein expression of Luciferase (the protein expression indirectly reflects the hHSD17B13 protein expression).
  • the siRNA of the present application has high inhibitory activity on the hHSD17B13 gene in vivo, and can reduce hHSD17B13 protein levels for a long time, with obvious dose effect.
  • N-ER-FY007001M2L96 showed 60% inhibition of the hHSD17B13 gene (40% protein remaining) on day 35;
  • N-ER-FY007004M2L96 showed 62% inhibition of the hHSD17B13 gene
  • N-ER-FY007020M2L96 showed 73% inhibition of hHSD17B13 gene (remaining protein of 27%);
  • N-ER-FY007033M2L96 showed 66% inhibition of hHSD17B13 gene (remaining protein of 27%) 34%);
  • N-ER-FY007034M2L96 showed 51% inhibition of hHSD17B13 gene (protein remaining 49%).
  • the siRNA conjugate of this application is administered at a dose of 3 mg/kg (10 mL/kg), and administered by a single subcutaneous injection after random grouping, with 6 mice in each group.
  • Sample collection Whole blood samples were collected at 0.0833, 0.25, 0.5, 1, 2, 4, 8, 24, 36, and 48 hours after administration, for a total of 10 points. The first 3 samples in each group were collected at 0.0833, 0.5, 2, 8, and 36 hours, and the last 3 samples were collected at 0.25, 1, 4, 24, and 48 hours. After whole blood was collected, plasma was separated by centrifugation for detection and analysis.
  • siRNA conjugate of the present application has a shorter half-life in plasma and is cleared faster.
  • the siRNA conjugate of this application is administered at a dose of 3 mg/kg (10 mL/kg). After random grouping, it is administered by a single subcutaneous injection. There are 3 animals at each time point, for a total of 24 mice. .
  • Sample detection and analysis The LC-MS/MS method was used to detect the concentration of the prototype drug in plasma and tissue samples at each time point, and the trapezoidal area method was used to calculate the AUC in plasma and tissue.
  • the siRNA conjugate of the present application is mainly enriched in the liver, has a long retention time in the tissue, and has good stability.
  • mice SPF grade, male, about 25 g, purchased from Spefford (Beijing) Biotechnology Co., Ltd.
  • the animals were randomly grouped according to their body weight on the last day of the adaptation period.
  • the specific dose design and grouping are as follows:
  • Clinical observation Continuous observation for 4 hours on the day of administration, and clinical observation at least once a day during the recovery period
  • Tissue distribution The animals in the main test group were necropsied at R28, and the animals in the satellite group were necropsied in batches at R7, R14, R21, and R28. Blood and liver were collected to detect tissue drug concentrations.
  • Histopathological examination The animals in the main test group were necropsied at R28, and the main organs (heart, liver, spleen, lung, kidney, brain, adrenal gland, thymus, stomach, uterus/testis, ovary/epididymis) and findings were collected Abnormal tissues or organs are collected and fixed for histopathological examination.
  • siRNA conjugate of the present application is less toxic and has a large safety window.

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Abstract

La présente invention concerne un ARNsi capable d'inhiber l'expression du gène HSD17B13, un conjugué d'ARNsi, une composition pharmaceutique le comprenant, et une utilisation associée. Chaque nucléotide de l'ARNsi est indépendamment un nucléotide modifié ou non modifié, et l'ARNsi contient un brin sens et un brin antisens. L'ARNsi et le conjugué et la composition pharmaceutique associée peuvent traiter et/ou prévenir efficacement des maladies liées à la surexpression du gène HSD17B13.
PCT/CN2023/091141 2022-04-29 2023-04-27 Arnsi pour inhiber l'expression de hsd17b13, conjugué et composition pharmaceutique associés, et utilisation associée WO2023208109A1 (fr)

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WO2005116204A1 (fr) * 2004-05-11 2005-12-08 Rnai Co., Ltd. Polynucléotide provoquant l'interférence rna et procédé de regulation d'expression génétique avec l’usage de ce dernier
CA3091146A1 (fr) * 2018-03-21 2019-09-26 Regeneron Pharmaceuticals, Inc. Compositions d'arni de 17s-hydroxysteroide deshydrogenase de type 13 (hsd17b13) et leurs methodes d'utilisation
SG11202101698WA (en) * 2018-09-19 2021-04-29 Arrowhead Pharmaceuticals Inc Rnai agents for inhibiting expression of 17beta-hsd type 13- (hsd17b13), compositions thereof, and methods of use
AU2021284377A1 (en) * 2020-06-01 2022-12-22 Amgen Inc. RNAi constructs for inhibiting HSD17B13 expression and methods of use thereof

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