SiRNA for inhibiting expression of LECT2 gene, conjugate and pharmaceutical composition thereof, and applications of siRNA and conjugate and pharmaceutical composition

Abstract

The present invention relates to siRNA for inhibiting expression of LECT2 gene, a siRNA conjugate, a pharmaceutical composition containing the siRNA conjugate, and uses of the siRNA conjugate and the pharmaceutical composition. Each nucleotide in the siRNA is independently modified or unmodified nucleotide, and the siRNA contains a positive-sense strand and an antisense strand. The siRNA as well as the conjugate and the pharmaceutical composition thereof can be used for effectively treating and / or preventing diseases related to LECT2 gene overexpression.

Description

Technical Field

[0001] The present application relates to siRNAs for inhibiting LECT2 gene expression, siRNA conjugates, pharmaceutical compositions containing the same, methods for preparing the same, and uses thereof. Background Art

[0002] Leukocyte cell-derived chemotaxin 2 (LECT2) is a 16 kDa protein mainly produced by hepatocytes. It was initially isolated from phytohaemagglutinin (PHA)-activated human T cell leukemia SKW-3 cells and identified as a new neutrophil chemotactic factor. In recent years, many studies have shown that LECT2 is a pleiotropic protein that not only exerts chemotactic properties as a cytokine but also functions as a hepatic factor regulating glucose metabolism, obesity, and non-alcoholic fatty liver disease, playing multifunctional roles in some physiological conditions and pathological abnormalities. LECT2 binds to several cell surface receptors, CD209a, Tie1, and Met, to regulate inflammatory responses, fibrogenesis, vascular invasion, and tumor metastasis in various cell types.

[0003] As an intracellular molecule, it is associated with LECT2-mediated amyloidosis, in which misfolding of LECT2 results in insoluble fibers in various tissues such as the kidney, liver, and lung. In addition, LECT2 has also been found to be associated with the development of rheumatoid arthritis and osteoarthritis, involving dysregulation of osteoclasts, mesenchymal stem cells, osteoblasts, chondrocytes, and endothelial cells in the bone microenvironment.

[0004] Treatment for LECT2-related diseases is limited, and new treatment methods are needed. The present invention aims to provide siRNAs, siRNA conjugates, and pharmaceutical compositions thereof, which can affect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of the LECT2 gene, thereby being able to selectively and effectively inhibit the expression of the LECT2 gene and achieve the purpose of disease treatment. Summary of the Invention

[0005] The present invention provides an siRNA for inhibiting LECT2 gene expression, the siRNA comprising a sense strand and an antisense strand, wherein each nucleotide in the siRNA is independently a modified or unmodified nucleotide, wherein the sense strand 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:

[0006] (1) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 228, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 229:

[0007] 5’-GAAUAUUCUUCA-3’(SEQ ID NO:228)

[0008] 5’-UGAAGAAUAUUC-3’(SEQ ID NO:229);

[0009] (2) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 230, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 231:

[0010] 5’-CAACUCUAAUCAGAGGA-3’(SEQ ID NO:230)

[0011] 5’-UCCUCUGAUUAGAGUUG-3’(SEQ ID NO:231);

[0012] (3) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 232, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 233:

[0013] 5’-CAAUGAGAUCCGGACGU-3’(SEQ ID NO:232)

[0014] 5’-ACGUCCGGAUCUCAUUG-3’(SEQ ID NO:233);

[0015] (4) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 234, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 235:

[0016] 5’-ACCGCCAUGGCUGUGGA-3’(SEQ ID NO:234)

[0017] 5’-UCCACAGCCAUGGCGGU-3’(SEQ ID NO:235);

[0018] (5) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 236, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 237:

[0019] 5’-CAAUAAUGGU-3’(SEQ ID NO:236)

[0020] 5’-ACCAUUAUUG-3’(SEQ ID NO:237);

[0021] (6) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:238, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:239:

[0022] 5’-GUGUUCGAAUAUCU-3’(SEQ ID NO:238)

[0023] 5’-AGAUAUUCGAACAC-3’(SEQ ID NO:239);

[0024] (7) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:240, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:241:

[0025] 5’-AAGAGGUUUUUGUG-3’(SEQ ID NO:240)

[0026] 5’-CACAAAAACCUCUU-3’(SEQ ID NO:241);

[0027] (8) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:242, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:243:

[0028] 5’-UCCUAUUAAGAAGGGA-3’(SEQ ID NO:242)

[0029] 5’-UCCCUUCUUAAUAGGA-3’(SEQ ID NO:243);

[0030] (9) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:244, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:245:

[0031] 5’-UAUUGCCCUU-3’(SEQ ID NO:244)

[0032] 5’-AAGGGCAAUA-3’(SEQ ID NO:245);

[0033] (10) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 246, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 247:

[0034] 5’-GUUUAUCCUG-3’ (SEQ ID NO: 246)

[0035] 5’-CAGGAUAAAC-3’ (SEQ ID NO: 247);

[0036] (11) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 248, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 249:

[0037] 5’-CAUACAAUCGCAUGUG-3’ (SEQ ID NO: 248)

[0038] 5’-CACAUGCGAUUGUAUG-3’ (SEQ ID NO: 249);

[0039] (12) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 250, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 251:

[0040] 5’-CUCGAGUGACCCUACUG-3’ (SEQ ID NO: 250)

[0041] 5’-CAGUAGGGUCACUCGAG-3’ (SEQ ID NO: 251);

[0042] (13) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 1, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 2;

[0043] (14) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 20, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 21;

[0044] (15) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 22, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 23;

[0045] (16) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:34, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:35;

[0046] (17) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:36, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:37;

[0047] (18) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:224, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:225.

[0048] In one embodiment, the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, essentially reverse complementary or completely reverse complementary; the substantially reverse complementary means that there are no more than 3 base mismatches between the two nucleotide sequences; the essentially reverse complementary means that there is no more than 1 base mismatch between the two nucleotide sequences; the completely reverse complementary means that there are no mismatches between the two nucleotide sequences.

[0049] In one embodiment, the sense strand further contains a nucleotide sequence III, and the antisense strand further contains a nucleotide sequence IV. The lengths of the nucleotide sequence III and the nucleotide sequence IV are each independently 0 - 11 nucleotides. Wherein the nucleotide sequence III is linked to the 5'-end of the nucleotide sequence I, and the nucleotide sequence IV is linked to the 3'-end of the nucleotide sequence II. The nucleotide sequence III and the nucleotide sequence IV are of equal length and are essentially reverse complementary or completely reverse complementary; the essentially reverse complementary means that there is no more than 1 base mismatch between the two nucleotide sequences; the completely reverse complementary means that there are no mismatches between the two nucleotide sequences; and / or, the nucleotide sequence III is linked to the 3'-end of the nucleotide sequence I, and the nucleotide sequence IV is linked to the 5'-end of the nucleotide sequence II. The nucleotide sequence III and the nucleotide sequence IV are of equal length and are essentially reverse complementary or completely reverse complementary; the essentially reverse complementary means that there is no more than 1 base mismatch between the two nucleotide sequences; the completely reverse complementary means that there are no mismatches between the two nucleotide sequences.

[0050] In one embodiment, the siRNA comprises 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 sequences I and III, and the antisense strand contains nucleotide sequences II and IV, and the nucleotide sequences I and III are at least partially reverse complementary to the nucleotide sequences II and IV to form a double-stranded region, wherein the nucleotide sequences I and III, and the nucleotide sequences II and IV are selected from the following sequences:

[0051] (1) The nucleotide sequences I and III comprise or consist of the nucleotide sequence shown in SEQ ID NO: 124, and the nucleotide sequences II and IV comprise or consist of the nucleotide sequence shown in SEQ ID NO: 125;

[0052] (2) The nucleotide sequences I and III comprise or consist of the nucleotide sequence shown in SEQ ID NO: 343, and the nucleotide sequences II and IV comprise or consist of the nucleotide sequence shown in SEQ ID NO: 371.

[0053] In one embodiment, the sense strand further contains nucleotide sequence V and / or the antisense strand further contains nucleotide sequence VI, the lengths of nucleotide sequences V and VI are 0 to 3 nucleotides, nucleotide sequence V is connected to the 3'-end of the sense strand to form a 3'-overhang of the sense strand, and / or nucleotide sequence VI is connected to the 3'-end of the antisense strand to form a 3'-overhang of the antisense strand. In a preferred embodiment, the length of nucleotide sequence V or VI is 2 nucleotides. In a preferred embodiment, the nucleotide sequence V or VI is two consecutive thymidine deoxynucleotides or two consecutive uridine ribonucleotides. In a preferred embodiment, the nucleotide sequence V or VI is mismatched or complementary to the nucleotides at the corresponding position of the target mRNA.

[0054] In one embodiment, the length of the double-stranded region is 15 - 30 nucleotide pairs. In a preferred embodiment, the length of the double-stranded region is 17 - 23 nucleotide pairs. In a more preferred embodiment, the length of the double-stranded region is 19 - 21 nucleotide pairs.

[0055] In one embodiment, the sense strand or the antisense strand has 15 - 30 nucleotides. In a preferred embodiment, the sense strand or the antisense strand has 19 - 25 nucleotides. In a more preferred embodiment, the sense strand or the antisense strand has 19 - 23 nucleotides.

[0056] In one embodiment, at least one nucleotide in the sense strand or the antisense strand is a modified nucleotide, and / or at least one phosphate group is a phosphate group having a modifying group; preferably, the phosphate group having a modifying group is a phosphorothioate group formed by replacing one oxygen atom in the phosphodiester bond of the phosphate group with a sulfur atom.

[0057] In one embodiment, the siRNA includes a sense strand that does not contain 3'-overhanging nucleotides.

[0058] In one embodiment, the 5'-terminal nucleotide of the antisense strand is linked to a 5'-phosphate group or a 5'-phosphate-derived group, or the 5'-terminal nucleotide of the antisense strand is not linked to a 5'-phosphate group or a 5'-phosphate-derived group.

[0059] In one embodiment, the 3'-terminal nucleotide of the sense strand is linked to a reverse abasic deoxyribose residue, and / or the 5'-terminal nucleotide of the sense strand is linked to a reverse abasic deoxyribose residue.

[0060] In one embodiment, the modified nucleotide is selected from 2'-fluoro-modified nucleotides, 2'-alkoxy-modified nucleotides, 2'-substituted alkoxy-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-amino-modified nucleotides, 2'-substituted amino-modified nucleotides, 2'-deoxyribonucleotides, nucleotide analogs, or any combination of two or more thereof.

[0061] In one embodiment, the modified nucleotide is selected from 2'-fluoro-modified nucleotides, 2'-methoxy-modified nucleotides, 2'-O-CH2-CH2-O-CH3-modified nucleotides, 2'-O-CH2-CH=CH2-modified nucleotides, 2'-CH2-CH2-CH=CH2-modified nucleotides, 2'-deoxyribonucleotides, nucleotide analogs, or any combination of two or more thereof.

[0062] In one embodiment, each nucleotide in the sense strand and the antisense strand is independently a 2'-fluoro-modified nucleotide or a non-fluoro-modified nucleotide. In a preferred embodiment, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, and the remaining positions are non-fluoro-modified nucleotides; 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 non-fluoro-modified nucleotides. In a preferred embodiment, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, and the remaining positions are non-fluoro-modified nucleotides; in 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, and the remaining positions are non-fluoro-modified nucleotides. In a preferred embodiment, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, and the remaining positions are non-fluoro-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 14, and 16 of the antisense strand, and the remaining positions are non-fluoro-modified nucleotides. In a preferred embodiment, in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 5, 7, 8, and 9 of the sense strand, and the remaining positions are non-fluoro-modified nucleotides; 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 non-fluoro-modified nucleotides. In a preferred embodiment, in the 3' to 5' direction, the 2'-fluoro-modified nucleotides are located at positions 9, 10, 11, and 13 of the sense strand, and the remaining positions are non-fluoro-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand, and the remaining positions are non-fluoro-modified nucleotides. In a preferred embodiment, in the 3' to 5' direction, the 2'-fluoro-modified nucleotides are located at positions 11, 12, 13, and 17 of the sense strand, and the remaining positions are non-fluoro-modified nucleotides; 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 non-fluoro-modified nucleotides. In a preferred embodiment, in the 3' to 5' direction, the 2'-fluoro-modified nucleotides are located at positions 9, 11, 13, and 17 of the sense strand, and the remaining positions are non-fluoro-modified nucleotides; 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 non-fluoro-modified nucleotides.In a preferred embodiment, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 9, 11, 12, and 13 of the sense strand, and the remaining positions are non-fluoro-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 non-fluoro-modified nucleotides. In a preferred embodiment, in the 3' to 5' direction, 2'-fluoro-modified nucleotides are located at positions 9, 10, 11, and 13 of the sense strand, and the remaining positions are non-fluoro-modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand, and the remaining positions are non-fluoro-modified nucleotides. In a preferred embodiment, in the 3' to 5' direction, 2'-fluoro-modified nucleotides are located at positions 9, 10, 11, and 13 of the sense strand, and the remaining positions are non-fluoro-modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 2, 5, 7, and 14 of the antisense strand, and the remaining positions are non-fluoro-modified nucleotides. In a preferred embodiment, in the 3' to 5' direction, 2'-fluoro-modified nucleotides are located at positions 11, 12, 13, and 17 of the sense strand, and the remaining positions are non-fluoro-modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand, and the remaining positions are non-fluoro-modified nucleotides. In a preferred embodiment, in the 3' to 5' direction, 2'-fluoro-modified nucleotides are located at positions 9, 11, 13, and 17 of the sense strand, and the remaining positions are non-fluoro-modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand, and the remaining positions are non-fluoro-modified nucleotides. In a preferred embodiment, in the 3' to 5' direction, 2'-fluoro-modified nucleotides are located at positions 6, 11, and 13 of the sense strand, and the remaining positions are non-fluoro-modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand, and the remaining positions are non-fluoro-modified nucleotides. In a preferred embodiment, in the 3' to 5' direction, 2'-fluoro-modified nucleotides are located at positions 9, 11, 13, and 17 of the sense strand, and the remaining positions are non-fluoro-modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 2, 3, 5, 7, 10, 12, and 14 of the antisense strand, and the remaining positions are non-fluoro-modified nucleotides. In a preferred embodiment, in the 3' to 5' direction, 2'-fluoro-modified nucleotides are located at positions 9, 11, 13, and 17 of the sense strand, and the remaining positions are non-fluoro-modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 2, 7, and 14 of the antisense strand, and the remaining positions are non-fluoro-modified nucleotides.In one embodiment, each non-fluorinated modified nucleotide is a 2'-methoxy modified nucleotide, and the 2'-methoxy modified nucleotide refers to a nucleotide formed by substituting the 2'-hydroxyl group of the ribose group with a methoxy group.

[0063] In one embodiment, each non-fluorinated modified nucleotide is independently selected from a nucleotide formed by substituting the hydroxyl group at the 2'-position of the ribose group of a nucleotide with a non-fluorine group or a nucleotide analogue, and the nucleotide analogue is selected from one of pseudouracil, an isonucleotide, LNA, ENA, cET BNA, UNA, and GNA.

[0064] In one embodiment, each nucleotide in the sense strand and the antisense strand is independently a 2'-fluoro-modified nucleotide, a 2'-methoxy-modified nucleotide, a GNA-modified nucleotide, a 2'-deoxyribonucleotide, or a combination of any two or more thereof. In a preferred embodiment, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; 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 nucleotides. In a preferred embodiment, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 2, 6, 8, 9, 14, and 16 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides. In a preferred embodiment, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 2, 14, and 16 of the antisense strand, a GNA-modified nucleotide is located at position 6 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides. In a preferred embodiment, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, a GNA-modified nucleotide is located at position 7 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides. In a preferred embodiment, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 5, 7, 8, and 9 of the sense strand, and the remaining positions are 2'-methoxy-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 nucleotides. In a preferred embodiment, in the 3' to 5' direction, 2'-fluoro-modified nucleotides are located at positions 9, 10, 11, and 13 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides.In a preferred embodiment, in the 3' to 5' direction, 2'-fluoro-modified nucleotides are located at positions 11, 12, 13, and 17 of the sense strand, and the remaining positions are 2'-methoxy-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 nucleotides. In a preferred embodiment, in the 3' to 5' direction, 2'-fluoro-modified nucleotides are located at positions 9, 11, 13, and 17 of the sense strand, and the remaining positions are 2'-methoxy-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 nucleotides. In a preferred embodiment, in the 3' to 5' direction, 2'-fluoro-modified nucleotides are located at positions 9, 10, 11, and 13 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand, and a GNA-modified nucleotide is located at position 6 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides. In a preferred embodiment, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 9, 11, 12, and 13 of the sense strand, and the remaining positions are 2'-methoxy-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 nucleotides. In a preferred embodiment, in the 3' to 5' direction, 2'-fluoro-modified nucleotides are located at positions 9, 10, 11, and 13 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides. In a preferred embodiment, in the 3' to 5' direction, 2'-fluoro-modified nucleotides are located at positions 9, 10, 11, and 13 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 2, 5, 7, and 14 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides. In a preferred embodiment, in the 3' to 5' direction, 2'-fluoro-modified nucleotides are located at positions 11, 12, 13, and 17 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides.In a preferred embodiment, in the 3' to 5' direction, 2'-fluoro-modified nucleotides are located at positions 9, 11, 13, and 17 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides. In a preferred embodiment, in the 3' to 5' direction, 2'-fluoro-modified nucleotides are located at positions 6, 11, and 13 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides. In a preferred embodiment, in the 3' to 5' direction, 2'-fluoro-modified nucleotides are located at positions 9, 11, 13, and 17 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 2, 3, 5, 7, 10, 12, and 14 of the antisense strand, GNA-modified nucleotides are located at position 6 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides. In a preferred embodiment, in the 3' to 5' direction, 2'-fluoro-modified nucleotides are located at positions 9, 11, 13, and 17 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 2, 7, and 14 of the antisense strand, 2'-deoxyribonucleotides are located at positions 5 and 12 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides.

[0065] In some embodiments, at least one of the linkages between the following nucleotides in the siRNA is a phosphorothioate linkage:

[0066] The linkage between the 1st nucleotide and the 2nd nucleotide starting from the 5'-end of the sense strand;

[0067] The linkage between the 2nd nucleotide and the 3rd nucleotide starting from the 5'-end of the sense strand;

[0068] The linkage between the 1st nucleotide and the 2nd nucleotide starting from the 3'-end of the sense strand;

[0069] The linkage between the 2nd nucleotide and the 3rd nucleotide starting from the 3'-end of the sense strand;

[0070] The linkage between the 1st nucleotide and the 2nd nucleotide starting from the 5'-end of the antisense strand;

[0071] The linkage between the 2nd nucleotide and the 3rd nucleotide starting from the 5'-end of the antisense strand;

[0072] The linkage between the 1st nucleotide and the 2nd nucleotide starting from the 3'-end of the antisense strand;

[0073] The linkage between the 2nd nucleotide and the 3rd nucleotide starting from the 3'-end of the antisense strand.

[0074] In some embodiments, the siRNA is oriented from the 5'-end to the 3'-end.

[0075] (1) The sense strand comprises phosphorothioate groups located at the following positions:

[0076] Between the 1st nucleotide and the 2nd nucleotide starting from the 5'-end of the sense strand; and

[0077] Between the 2nd nucleotide and the 3rd nucleotide starting from the 5'-end of the sense strand; and

[0078] Between the 1st nucleotide and the 2nd nucleotide starting from the 3'-end of the sense strand; and

[0079] Between the 2nd nucleotide and the 3rd nucleotide starting from the 3'-end of the sense strand;

[0080] Or,

[0081] (2) The sense strand comprises phosphorothioate groups located at the following positions:

[0082] Between the 1st nucleotide and the 2nd nucleotide starting from the 5'-end of the sense strand; and

[0083] Between the 2nd nucleotide and the 3rd nucleotide starting from the 5'-end of the sense strand;

[0084] Or,

[0085] (3) The sense strand comprises phosphorothioate groups located at the following positions:

[0086] Between the 1st nucleotide and the 2nd nucleotide starting from the 5'-end of the sense strand; and

[0087] Between the 2nd nucleotide and the 3rd nucleotide starting from the 5'-end of the sense strand; and

[0088] Between the reverse abasic deoxyribose residue and the 1st nucleotide starting from the 3'-end of the sense strand;

[0089] Or,

[0090] (4) The sense strand comprises phosphorothioate groups located at the following positions:

[0091] Between the reverse abasic deoxyribose residue and the 1st nucleotide starting from the 5'-end of the sense strand; and

[0092] between the first nucleotide and the second nucleotide starting from the 5'-end of the sense strand; and

[0093] between the reverse abasic deoxyribose residue starting from the 3'-end of the sense strand and the first nucleotide.

[0094] In some embodiments, along the 5'-end to 3'-end direction of the siRNA, the antisense strand contains phosphorothioate groups at the following positions:

[0095] between the first nucleotide and the second nucleotide starting from the 5'-end of the antisense strand; and

[0096] between the second nucleotide and the third nucleotide starting from the 5'-end of the antisense strand; and

[0097] between the first nucleotide and the second nucleotide starting from the 3'-end of the antisense strand; and

[0098] between the second nucleotide and the third nucleotide starting from the 3'-end of the antisense strand.

[0099] In one embodiment, each nucleotide in the sense strand and the antisense strand is independently a 2'-fluoro-modified nucleotide, a 2'-methoxy-modified nucleotide, a GNA-modified nucleotide, a 2'-deoxyribonucleotide, or a combination of any two or more thereof.

[0100] In a preferred embodiment, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides, with the 3'-end excluding the overhang; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-phosphate group.

[0101] In a preferred embodiment, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; 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 nucleotides, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-phosphate group.

[0102] In a preferred embodiment, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; 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, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-phosphate group.

[0103] In a preferred embodiment, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 2, 14, and 16 of the antisense strand, GNA-modified nucleotides are located at position 6 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-phosphate group.

[0104] In a preferred embodiment, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, GNA-modified nucleotides are located at position 7 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-phosphate group.

[0105] In a preferred embodiment, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; 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 nucleotides, and the 5'-terminal nucleotide of the antisense strand is not linked to a 5'-phosphate group or a 5'-phosphate-derived group.

[0106] In a preferred embodiment, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; 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, and the 5'-terminal nucleotide of the antisense strand is not linked to a 5'-phosphate group or a 5'-phosphate-derived group.

[0107] In a preferred embodiment, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides. The 3'-end has the overhang removed; 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 nucleotides. The 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0108] In a preferred embodiment, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 5, 7, 8, and 9 of the sense strand, and the remaining positions are 2'-methoxy-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 nucleotides. The 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0109] In a preferred embodiment, in the 3' to 5' direction, 2'-fluoro-modified nucleotides are located at positions 9, 10, 11, and 13 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides. The 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0110] In a preferred embodiment, in the 3' to 5' direction, 2'-fluoro-modified nucleotides are located at positions 11, 12, 13, and 17 of the sense strand, and the remaining positions are 2'-methoxy-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 nucleotides. Between the 1st and 2nd nucleotides at the 5'-end, the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0111] In a preferred embodiment, in the 3' to 5' direction, 2'-fluoro-modified nucleotides are located at positions 9, 11, 13, and 17 of the sense strand, and the remaining positions are 2'-methoxy-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 nucleotides. The 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0112] In a preferred embodiment, in the 3' to 5' direction, 2'-fluoro-modified nucleotides are located at positions 9, 10, 11, and 13 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand, a GNA-modified nucleotide is located at position 6 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0113] In a preferred embodiment, in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 9, 11, 12, and 13 of the sense strand, and the remaining positions are 2'-methoxy-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 nucleotides, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0114] In a preferred embodiment, in the 3' to 5' direction, 2'-fluoro-modified nucleotides are located at positions 9, 10, 11, and 13 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0115] In a preferred embodiment, in the 3' to 5' direction, 2'-fluoro-modified nucleotides are located at positions 9, 10, 11, and 13 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 2, 5, 7, and 14 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0116] In a preferred embodiment, in the 3' to 5' direction, 2'-fluoro-modified nucleotides are located at positions 11, 12, 13, and 17 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0117] In a preferred embodiment, in the 3' to 5' direction, 2'-fluoro-modified nucleotides are located at positions 9, 11, 13, and 17 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0118] In a preferred embodiment, in the 3' to 5' direction, 2'-fluoro-modified nucleotides are located at positions 6, 11, and 13 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0119] In a preferred embodiment, in the 3' to 5' direction, 2'-fluoro-modified nucleotides are located at positions 9, 11, 13, and 17 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 2, 3, 5, 7, 10, 12, and 14 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, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0120] In a preferred embodiment, in the 3' to 5' direction, 2'-fluoro-modified nucleotides are located at positions 9, 11, 13, and 17 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, 2'-fluoro-modified nucleotides are located at positions 2, 7, and 14 of the antisense strand, 2'-deoxyribonucleotides are located at positions 5 and 12 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5'-terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0121] In a specific embodiment, the present invention provides siRNAs selected from Table 1; preferably, the siRNAs are selected from N-ER-FY037090, N-ER-FY037023, N-ER-FY037090M2, N-ER-FY037023M2, N-ER-FY037090M3, N-ER-FY037090M4, N-ER-FY037090M5, N-ER-FY037090M6, N-ER-FY037023M6, N-ER-FY037090M7, N-ER-FY037023M8, N-ER-FY037023M11, N-ER-FY037023M15, N-ER-FY037023M27, N-ER-FY037023M29, N-ER-FY037023M35, N-ER-FY037023M37, N-ER-FY037023M40, N-ER-FY037023M44, N-ER-FY037023M45, N-ER-FY037023M46, N-ER-FY037023M47, N-ER-FY037023M48, N-ER-FY037023M49, N-ER-FY037023M50, N-ER-FY037023M51, N-ER-FY037088, N-ER-FY037088M6, N-ER-FY037088M8, N-ER-FY037088M15, N-ER-FY037088M27, N-ER-FY037088M29, N-ER-FY037088M35, N-ER-FY037088M36, N-ER-FY037088M37, N-ER-FY037088M40, N-ER-FY037088M44, N-ER-FY037088M45, N-ER-FY037088M46, N-ER-FY037088M47, N-ER-FY037088M48, N-ER-FY037088M49, N-ER-FY037088M50, N-ER-FY037088M51.

[0122] The present invention also provides an siRNA conjugate, which contains the siRNA of the present invention and a conjugating group conjugated to the siRNA (as shown in the following formula, the double helix structure represents the siRNA, and the conjugating group is connected to the 3'-end of the sense strand of the siRNA):

[0123]

[0124] In the above conjugate structure, X can be optionally O or S. In one embodiment, X is O. In one embodiment, the conjugating group comprises a pharmaceutically acceptable targeting group and a linker, and the siRNA, the linker, and the targeting group are covalently or non-covalently linked in sequence.

[0125] Preferably, in the siRNA conjugate, the sense strand of the siRNA is complementary to the antisense strand to form a double-stranded region of the siRNA conjugate, and the 3'-end of the sense strand forms a blunt end, and the 3'-end of the antisense strand has 1-3 protruding nucleotides extending out of the double-stranded region;

[0126] Or,

[0127] in the siRNA conjugate, the sense strand of the siRNA is complementary to the antisense strand to form a double-stranded region of the siRNA conjugate, and the 3'-end of the sense strand forms a blunt end, and the 3'-end of the antisense strand forms a blunt end.

[0128] In one embodiment, the conjugating group is selected from:

[0129]

[0130]

[0131] In a specific embodiment, the siRNA conjugate is an siRNA conjugate selected from Table 2; preferably, the siRNA conjugate is N-ER-FY037023M2L96, N-ER-FY037090M3L96, N-ER-FY037090M4L96, N-ER-FY037090M5L96, N-ER-FY037023M6L96, N-ER-FY037023M8L96, N-ER-FY037090M7L96, N-ER-FY037023M37L96, N-ER-FY037023M40L96, N-ER-FY037023M44L96, N-ER-FY037023M45L96, N-ER-FY037023M46L96, N-ER-FY037023M47L96, N-ER-FY037023M48L96, N-ER-FY037023M49L96, N-ER-FY037023M50L96, N-ER-FY037023M51L96, N-ER-FY037088M6L96, N-ER-FY037088M37L96, N-ER-FY037088M40L96, N-ER-FY037088M44L96, N-ER-FY037088M45L96, N-ER-FY037088M46L96, N-ER-FY037088M47L96, N-ER-FY037088M48L96, N-ER-FY037088M49L96, N-ER-FY037088M50L96, N-ER-FY037088M51L96.

[0132] The present invention also provides a pharmaceutical composition comprising the siRNA of the present invention, or the siRNA conjugate of the present invention, and a pharmaceutically acceptable carrier.

[0133] 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.

[0134] 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 the preparation of an agent for inhibiting the expression of the LECT2 gene.

[0135] 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 the preparation of an agent for preventing and / or treating diseases associated with overexpression of the LECT2 gene.

[0136] In a specific embodiment, the disease is liver injury, liver fibrosis, NASH, metabolic disorder, obesity, insulin resistance, and systemic amyloidosis.

[0137] The present invention also provides a method for inhibiting LECT2 gene expression, including 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 LECT2 or administering to a subject in need thereof.

[0138] The present invention also provides a method for treating and / or preventing a disease associated with overexpression of the LECT2 gene, including administering a therapeutically effective amount of the siRNA of the present invention, or the siRNA conjugate of the present invention, or the pharmaceutical composition of the present invention to a subject in need thereof.

[0139] In a specific embodiment, the disease is liver injury, liver fibrosis, NASH, metabolic disorder, obesity, insulin resistance, and systemic amyloidosis.

[0140] Beneficial effects

[0141] The siRNA, pharmaceutical composition, and siRNA conjugate provided in the present application show excellent LECT2 gene expression inhibitory activity in in vitro cell experiments and have good potential for treating diseases associated with overexpression of the LECT2 gene. For example, the siRNA and its conjugate disclosed in the present application can reduce the expression of LECT2 mRNA in the liver, have low toxicity and side effects, good plasma stability, and have good clinical application prospects.

[0142] The siRNA provided in the present application shows a good inhibitory effect on the LECT2 gene in PHH cells.

[0143] In some specific embodiments, the siRNA of the present invention can significantly inhibit the expression of the LECT2 gene at 5 nM and 0.5 nM. Preferably, the inhibition rate of the siRNA is basically above 90% at 48 h at 5 nM and basically above 88% at 48 h at 0.5 nM.

[0144] In some specific embodiments, the siRNA provided in the present application has high LECT2 gene inhibitory activity in PHH cells, and the IC 50 can be as low as 0.0015 nM.

[0145] In some specific embodiments, the siRNA conjugate provided by the present application has high LECT2 gene inhibitory activity in PHH cells. When the siRNA conjugate enters PHH by free uptake, the inhibition rate is basically above 95% at 200 nM for 48 h, and basically above 88% at 10 nM for 48 h; when the siRNA conjugate enters PHH by transfection, the inhibition rate is basically above 90% at 5 nM for 48 h, and basically above 86% at 0.5 nM for 48 h. Specific embodiments

[0146] Definition

[0147] Throughout the specification, unless otherwise specified, in the technical field of the present invention, "G", "C", "A", "T" and "U" generally represent the bases of guanine, cytosine, adenine, thymine and uracil respectively. However, it is also generally known in the art that each of "G", "C", "A", "T" and "U" also generally represents a nucleotide containing guanine, cytosine, adenine, thymine and uracil as bases respectively, which is a common way in representing deoxyribonucleic acid sequences and / or ribonucleic acid sequences. Therefore, in the context of the present disclosure, the meanings represented by "G", "C", "A", "T", "U" include the above various possible situations, and "nucleotide" and "ribonucleotide" can be used interchangeably herein. Lowercase letters a, u, c, g: represent 2'-methoxy-modified nucleotides; Af, Gf, Cf, Uf: represent 2'-fluoro-modified nucleotides; "dG", "dC", "dA", "dT", "dU": represent 2'-deoxyribonucleotides; (invAb) is a reverse abasic deoxyribose residue; lowercase letter s: represents a phosphorothioate linkage between the two nucleotides adjacent to the left and right of the letter s; P1: represents that the nucleotide adjacent to the right of this P1 is a 5'-phosphonucleotide; EVP: represents that the nucleotide adjacent to the right of this EVP is a 5'-trans-vinylphosphonate nucleotide; (Underline + bold + italic): represents a GNA-modified nucleotide; Base represents a base, such as A, U, G, C or T.

[0148] As used hereinabove and hereinafter, the term "2'-fluorinated modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2'-position of the ribose moiety of the nucleotide is replaced by fluorine. The "non-fluorinated modified nucleotide" refers to a nucleotide or nucleotide analogue in which the hydroxyl group at the 2'-position of the ribose moiety of the nucleotide is replaced by a non-fluorine group. In some embodiments, each non-fluorinated modified nucleotide is independently selected from a nucleotide or nucleotide analogue in which the hydroxyl group at the 2'-position of the ribose moiety of the nucleotide is replaced by a non-fluorine group. These nucleotides in which the hydroxyl group at the 2'-position of the ribose moiety is replaced by a non-fluorine group are well known to those skilled in the art, and these nucleotides may be selected from one of 2'-alkoxy modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-amino modified nucleotides, 2'-substituted amino modified nucleotides, and 2'-deoxyribonucleotides.

[0149] "Alkyl" includes straight-chain, branched-chain or cyclic saturated alkyl groups. For example, alkyl includes, but is not limited to, methyl, ethyl, propyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, cyclobutyl, n-pentyl, cyclohexyl and similar groups. Exemplarily, the "C" in " 1-6 alkyl" 1-6 " refers to a group arranged in a straight-chain, branched-chain or cyclic form containing 1, 2, 3, 4, 5 or 6 carbon atoms.

[0150] "Alkoxy" as used herein refers to an alkyl group linked to the remainder of the molecule through an oxygen atom (-O-alkyl), wherein the alkyl is as defined herein. Non-limiting examples of alkoxy include methoxy, ethoxy, trifluoromethoxy, difluoromethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, n-pentyloxy and the like.

[0151] "Nucleotide analogue" refers to a group that can replace a nucleotide in a nucleic acid but has a structure different from adenosine ribonucleotide, guanosine ribonucleotide, cytidine ribonucleotide, uridine ribonucleotide or thymidine deoxyribonucleotide. Such as pseudouridine (Ψ), an isonucleotide, a bridged nucleic acid (BNA) or an acyclic nucleotide.

[0152] Pseudouridine (Ψ) refers to: a natural structural analogue of uridine nucleoside, in which the ribose is not linked to the N1 of uracil but to the C5 of the pyrimidine ring

[0153]

[0154] BNA refers to constrained or inaccessible nucleotides. BNA can contain a bridged structure with a "fixed" C3'-endo sugar puckering having a five-membered, six-membered, or seven-membered ring. This bridge is typically incorporated at the 2'- and 4'-positions of the ribose to provide a 2',4'-BNA nucleotide, such as LNA, ENA, cET BNA, etc., where LNA is shown in Formula (1), ENA is shown in Formula (2), and cET BNA is shown in Formula (3):

[0155]

[0156] Acyclic nucleotides are a class of nucleotides formed by opening the sugar ring of a nucleotide, such as unlocked nucleic acid (UNA) or glycerol nucleic acid (GNA), where UNA is shown in Formula (4) and GNA is shown in Formula (5):

[0157]

[0158] In Formulas (4) and (5) above, Base represents a base, such as A, U, G, C, or T; R is selected from H, OH, or an alkoxy group (O-alkyl).

[0159] An isonucleotide refers to a compound formed by changing the position of the base on the ribose ring of a nucleotide. For example, a compound formed by moving the base from the 1'-position to the 2'-position or 3'-position of the ribose ring, as shown in Formulas (6) or (7):

[0160]

[0161] In the compounds of Formulas (6)-(7) above, Base represents a base, such as A, U, G, C, or T; R is selected from H, OH, F, or a non-fluorinated group as described above.

[0162] In some embodiments, the nucleotide analog is selected from one of pseudouracil, isonucleotide, LNA, ENA, cET BNA, UNA, and GNA. In some embodiments, each non-fluorinated modified nucleotide is a 2'-methoxy modified nucleotide, a GNA modified nucleotide, or a combination of any two or more thereof. In some preferred embodiments, each non-fluorinated modified nucleotide is a 2'-methoxy modified nucleotide. As used herein and hereinafter, the "2'-methoxy modified nucleotide" refers to a nucleotide formed by substituting the 2'-hydroxyl group of the ribose group with a methoxy group.

[0163] The "2'-methoxy modified nucleotide" refers to a nucleotide formed by substituting the 2'-hydroxyl group of the ribose group with a methoxy group. The "thiophosphate group" refers to a thiophosphate group formed by substituting one oxygen atom in the phosphodiester bond of the phosphate group with a sulfur atom.

[0164] The "thiophosphate group" refers to the following formula:

[0165]

[0166] The "5'-phosphonucleotide" refers to the structure of the following formula:

[0167]

[0168] In the context of this specification, the expressions "complementary" and "reverse complementary" can be used interchangeably and have the meanings well-known to those skilled in the art, that is, in a double-stranded nucleic acid molecule, the bases of one strand pair with the bases of the other strand in a complementary manner. In DNA, the purine base adenine (A) always pairs with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (G) always pairs with the pyrimidine base cytosine (C). Each base pair includes a purine and a pyrimidine. When adenine on one strand always pairs with thymine (or uracil) on the other strand, and guanine always pairs with cytosine, the two strands are considered to be complementary to each other, and the sequence of one strand can be inferred from the sequence of its complementary strand. Correspondingly, "mismatch" in the art means that in a double-stranded nucleic acid, the bases at corresponding positions do not pair in a complementary form.

[0169] In the above and below, unless otherwise specified, "substantially reverse complementary" means that there are no more than 3 base mismatches between the two nucleotide sequences involved; "essentially reverse complementary" means that there is no more than 1 base mismatch between the two nucleotide sequences; "fully reverse complementary" means that there are no base mismatches between the two nucleotide sequences.

[0170] In the above and below, when a nucleotide sequence has a "nucleotide difference" from another nucleotide sequence, it means that compared with the latter, the base type of the nucleotide at the same position has changed. For example, when a nucleobase in the latter is A, and the corresponding nucleobase at the same position in the former is U, C, G, or T, it is determined that there is a nucleotide difference at this position between the two nucleotide sequences. In some embodiments, when a nucleotide at the original position is replaced by a non-base nucleotide or its equivalent, it can also be considered that a nucleotide difference has occurred at this position.

[0171] In context, a "protruding end" refers to one or more unpaired nucleotides that protrude from the duplex structure of an siRNA when a 3' end of one strand of the siRNA extends beyond the 5' end of the other strand, or vice versa. A "blunt end" or "blunt terminus" means that there are no unpaired nucleotides at that end of the siRNA, i.e., no nucleotide protruding end. A "blunt-ended" siRNA is an siRNA that is double-stranded throughout its length, i.e., has no nucleotide protruding ends at either end of the molecule.

[0172] In the context of the present application, above and below in the specification, particularly when describing the preparation methods of the siRNAs, pharmaceutical compositions or siRNA conjugates of the present application, unless otherwise specified, the nucleoside monomers refer to modified or unmodified nucleoside phosphoramidite monomers used in solid-phase phosphoramidite synthesis according to the types and sequences of nucleotides in the siRNA or siRNA conjugate to be prepared. Solid-phase phosphoramidite synthesis is a method well-known to those skilled in the art for use in RNA synthesis. The nucleoside monomers used in the present application are all commercially available.

[0173] In the context of the present application, unless otherwise specified, "conjugation" refers to the connection of two or more chemical moieties each having a specific function to each other in a covalent linkage manner; correspondingly, a "conjugate" refers to a compound formed by the covalent linkage between the respective chemical moieties. Further, an "siRNA conjugate" refers to a compound formed by covalently linking one or more chemical moieties having specific functions to an siRNA. An siRNA conjugate should be understood, depending on the context, as the general term for multiple siRNA conjugates or an siRNA conjugate represented by a certain chemical formula. In the context of the specification of the present application, a "conjugating molecule" should be understood as a specific compound that can be conjugated to an siRNA through a reaction to ultimately form the siRNA conjugate of the present application.

[0174] Various hydroxyl protecting groups can be used in the present application. Generally, a protecting group renders a chemical functional group insensitive to specific 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. Non-exclusive examples of hydroxyl protecting groups that can be used in the present application include monomethoxytrityl, 9-phenylxanthine-9-yl (Pixyl), and 9-(p-methoxyphenyl)xanthine-9-yl (Mox) in some embodiments. Non-exclusive examples of hydroxyl protecting groups that can be used in the present application include Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4'-dimethoxytrityl), and TMTr (4,4',4''-trimethoxytrityl) in some embodiments.

[0175] As used in this specification, "optional" or "optionally" means that the event or circumstance described thereafter may or may not occur, and the description includes both the case where the event or circumstance occurs and the case where it does not occur.

[0176] As used in this specification, the term "subject" refers to any animal, such as a mammal or a marsupial. The subjects of this application include, but are not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cows, sheep, rats, rabbits, or any kind of poultry.

[0177] As used in this specification, "treatment" refers to a method of obtaining a beneficial or desired result, including but not limited to a therapeutic benefit. "Therapeutic benefit" means eradicating or ameliorating the underlying disorder being treated. In addition, a therapeutic benefit is obtained by observing an improvement in the subject through eradicating or ameliorating one or more physiological symptoms associated with the underlying disorder, even though the subject may still be afflicted with the underlying disorder.

[0178] As used in this specification, "prevention" refers to a method of obtaining a beneficial or desired result, including but not limited to a preventive benefit. To obtain a "preventive benefit", siRNA, siRNA conjugate, or pharmaceutical composition may be administered to a subject at risk of developing a particular disease, or to a subject presenting one or more physiological symptoms of a reported disease, even if the diagnosis of the disease may not have been made.

[0179] As used in this specification, a "prodrug" refers to a compound that can be converted into an active compound through certain chemical or physiological processes (e.g., enzymatic processes and metabolic hydrolysis). Thus, the term "prodrug" also refers to a precursor of a pharmaceutically acceptable bioactive compound.

[0180] In this context, considering the case where the 5'-terminal nucleotide of the antisense strand is linked to a 5'-hydroxyl group (i.e., without a phosphate group), such an antisense strand will be phosphorylated in vivo to convert the 5'-terminal nucleotide into a nucleotide carrying a 5'-phosphate group and then play a role in vivo. Therefore, in the present invention, such siRNA, siRNA modifiers, and siRNA conjugates are also referred to as prodrugs. For example, the siRNA modifier or conjugate with M6 mode modification in this application is a prodrug of the siRNA modifier or conjugate with M2 mode modification, because the difference between M2 mode modification and M6 mode modification is whether there is P1 at the 5'-end of the antisense strand. Similarly, the relationship between M7 mode modification and M3 mode modification is also like this. Therefore, siRNA in this article includes its corresponding prodrugs.

[0181] siRNA

[0182] This application relates to an siRNA capable of inhibiting the expression of the LECT2 gene. The siRNA of this application contains nucleotide groups as basic structural units. As is well known to those skilled in the art, the nucleotide groups contain phosphate groups, ribose groups, and bases. Usually, the length of an active, i.e., functional, siRNA is about 12 - 40 nucleotides, and in some embodiments, it is about 15 - 30 nucleotides.

[0183] The siRNA of this application contains a sense strand and an antisense strand. Each nucleotide in the siRNA is independently a modified or unmodified nucleotide. The sense strand contains a nucleotide sequence I, and the antisense strand contains a nucleotide sequence II. The nucleotide sequence I and the nucleotide sequence II are at least partially reverse complementary to form a double-stranded region. In some embodiments, the length of this double-stranded region is 15 - 30 nucleotide pairs. In some other embodiments, the length of the double-stranded region is 17 - 23 nucleotide pairs. In some other embodiments, the length of the double-stranded region is 19 - 21 nucleotide pairs. In yet some other embodiments, the length of the double-stranded region is 19 or 21 nucleotide pairs.

[0184] In some embodiments, the sense strand further contains nucleotide sequence III, and the antisense strand further contains nucleotide sequence IV. The lengths of nucleotide sequence III and nucleotide sequence IV are each independently 0 - 11 nucleotides. Nucleotide sequence III is linked to the 5'-end of nucleotide sequence I, and nucleotide sequence IV is linked to the 3'-end of nucleotide sequence II. Nucleotide sequence III and nucleotide sequence IV are of equal length and are substantially reverse complementary or completely reverse complementary; the substantial reverse complementarity means that there is no more than 1 base mismatch between the two nucleotide sequences; the complete reverse complementarity means that there is no mismatch between the two nucleotide sequences. In some embodiments, the sense strand further contains nucleotide sequence III, and the antisense strand further contains nucleotide sequence IV. The lengths of nucleotide sequence III and nucleotide sequence IV are each independently 0 - 11 nucleotides. Nucleotide sequence III is linked to the 3'-end of nucleotide sequence I, and nucleotide sequence IV is linked to the 5'-end of nucleotide sequence II. Nucleotide sequence III and nucleotide sequence IV are of equal length and are substantially reverse complementary or completely reverse complementary; the substantial reverse complementarity means that there is no more than 1 base mismatch between the two nucleotide sequences; the complete reverse complementarity means that there is no mismatch between the two nucleotide sequences. In some embodiments, the sense strand further contains nucleotide sequence III, and the antisense strand further contains nucleotide sequence IV. The lengths of nucleotide sequence III and nucleotide sequence IV are each independently 0 - 11 nucleotides. Nucleotide sequence III is linked to the 5'-end of nucleotide sequence I, and nucleotide sequence IV is linked to the 3'-end of nucleotide sequence II. Nucleotide sequence III and nucleotide sequence IV are of equal length and are substantially reverse complementary or completely reverse complementary; and nucleotide sequence III is linked to the 3'-end of nucleotide sequence I, and nucleotide sequence IV is linked to the 5'-end of nucleotide sequence II. Nucleotide sequence III and nucleotide sequence IV are of equal length and are substantially reverse complementary or completely reverse complementary; the substantial reverse complementarity means that there is no more than 1 base mismatch between the two nucleotide sequences; the complete reverse complementarity means that there is no mismatch between the two nucleotide sequences.

[0185] In some embodiments, the sense strand further contains nucleotide sequence V and / or the antisense strand further contains nucleotide sequence VI. The lengths of nucleotide sequences V and VI are 0 to 3 nucleotides. Nucleotide sequence V is connected to the 3'-end of the sense strand to form a 3'-overhang of the sense strand, and / or nucleotide sequence VI is connected to the 3'-end of the antisense strand to form a 3'-overhang of the antisense strand. In some embodiments, the length of nucleotide sequence V or VI is 2 nucleotides. In other embodiments, nucleotide sequence V or VI is two consecutive thymidine deoxynucleotides or two consecutive uridine ribonucleotides. In other embodiments, nucleotide sequence V or VI is mismatched or complementary to the nucleotides at the corresponding position of the target mRNA.

[0186] The sense strand and the antisense strand provided by the present application may have the same or different lengths. In some embodiments, the sense strand or the antisense strand has 15 - 30 nucleotides. In other embodiments, the sense strand or the antisense strand has 19 - 25 nucleotides. In other embodiments, the sense strand or the antisense strand has 19 - 23 nucleotides. The length ratio of the sense strand to the antisense strand of the siRNA provided by the present application can be 15 / 15, 16 / 16, 17 / 17, 18 / 18, 19 / 19, 19 / 20, 19 / 21, 19 / 22, 19 / 23, 20 / 19, 20 / 20, 20 / 21, 20 / 22, 20 / 23, 21 / 19, 21 / 20, 21 / 21, 21 / 22, 21 / 23, 22 / 19, 22 / 20, 22 / 21, 22 / 22, 22 / 23, 23 / 19, 23 / 20, 23 / 21, 23 / 22, 23 / 23, 24 / 24, 25 / 25, 26 / 26, 27 / 27, 28 / 28, 29 / 29, 30 / 30, 22 / 24, 22 / 25, 22 / 26, 23 / 24, 23 / 25 or 23 / 26, etc. In some embodiments, the length ratio of the sense strand to the antisense strand of the siRNA is 19 / 19, 21 / 21, 19 / 21, 21 / 23 or 23 / 23. At this time, the siRNA of the present disclosure has better cellular mRNA silencing activity.

[0187] It has been found that different modification strategies can have very different effects on indicators such as the stability, biological activity and cytotoxicity of siRNA. For example, various chemical modification strategies of siRNA were studied in CN102140458B, and 7 effective modification methods were confirmed. Compared with the unmodified siRNA, the siRNA obtained by one of the modification methods not only improved the blood stability but also maintained substantially the same inhibitory activity as the unmodified siRNA.

[0188] The nucleotides in the siRNA of the present invention are each independently a modified or unmodified nucleotide. In some embodiments, each nucleotide in the siRNA of the present invention is an unmodified nucleotide; in some embodiments, some or all of the nucleotides in the siRNA of the present invention are modified nucleotides, and these modifications on the nucleotide groups do not significantly weaken or lose the function of the siRNA of the present invention to inhibit LECT2 gene expression.

[0189] In some embodiments, the siRNA of the present application contains at least one modified nucleotide. In the context of the present application, the term "modified nucleotide" refers to a nucleotide or nucleotide analogue formed by replacing the hydroxyl group at the 2'-position of the ribose group of a nucleotide with another group, or a nucleotide having a modified base. The modified nucleotide does not significantly weaken or lose the function of the siRNA to inhibit gene expression. For example, the 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.

[0190] In some embodiments, at least one nucleotide in the sense strand or the antisense strand of the siRNA provided by the present invention is a modified nucleotide, and / or at least one phosphate group is a phosphate group with a modifying group; in other words, at least a part of the phosphate group and / or the ribose group in the phospho-sugar backbone of at least one single strand among the sense strand and the antisense strand is a phosphate group with a modifying group and / or a ribose group with a modifying group. In some embodiments, the phosphate group with a modifying group is a phosphorothioate group formed by replacing one oxygen atom in the phosphodiester bond of the phosphate group with a sulfur atom.

[0191] In some embodiments, the siRNA includes a sense strand that does not contain 3'-overhanging nucleotides; that is, the sense strand of the siRNA may have 3'-overhanging nucleotides, and after excluding the 3'-overhanging nucleotides of the sense strand, a blunt end is formed. In some embodiments, the siRNA includes a modifier in which the 3'-end of the sense strand in Table 1 is modified to a blunt end, optionally obtained by excluding the overhanging end extending from the 3'-end of the sense strand in Table 1 in the double-stranded region.

[0192] In some embodiments, when the nucleotide sequences of the sense strand and the antisense strand are complementary to form a double-stranded region and there are no protruding nucleotides at the 3'-end of the sense strand, a nucleotide sequence V is added to the 3'-end of the sense strand as the protruding nucleotide. Then, when the nucleotide sequence formed by ligating the nucleotide sequence V to the 3'-end of the sense strand is chemically modified, the nucleotide sequence V is excluded, and correspondingly, the sense strand of the siRNA forms a blunt end.

[0193] In some embodiments, when the nucleotide sequences of the sense strand and the antisense strand are complementary to form a double-stranded region and there are protruding nucleotides extending out of the double-stranded region at the 3'-end of the sense strand, the protruding nucleotides at the 3'-end in the sense strand are excluded and used as the nucleotide sequence of the sense strand. Correspondingly, the sense strand of the siRNA forms a blunt end.

[0194] In some embodiments, the 5'-terminal nucleotide of the antisense strand is ligated to a 5'-phosphate group or a 5'-phosphate-derived group.

[0195] The structure of an exemplary 5'-phosphate group is: The structures of 5'-phosphate-derived groups include but are not limited to: etc.

[0196] After the 5'-terminal nucleotide of the antisense strand is ligated to a 5'-phosphate group or a 5'-phosphate-derived group, the following structure is formed:

[0197]

[0198] Wherein, Base represents a base, such as A, U, G, C or T. R' is a hydroxyl group or is substituted by various groups known to those skilled in the art. For example, the modified nucleotide after substitution can be a 2'-fluoro (2'-F) modified nucleotide, a 2'-alkoxy modified nucleotide, a 2'-substituted alkoxy modified nucleotide, a 2'-alkyl modified nucleotide, a 2'-substituted alkyl modified nucleotide, a 2'-amino modified nucleotide, a 2'-substituted amino modified nucleotide, or a 2'-deoxyribonucleotide.

[0199] In some embodiments, the 5'-terminal nucleotide of the sense strand or the antisense strand is not ligated to a 5'-phosphate group or a 5'-phosphate-derived group (i.e., the ribose group of the 5'-terminal nucleotide of the sense strand or the antisense strand is a 5'-hydroxyl group), and its structure is as follows:

[0200]

[0201] Wherein, Base represents a base, such as A, U, G, C or T. R is a hydroxyl group or hydrogen or is substituted by various groups known to those skilled in the art. For example, R can be 2'-fluoro (2'-F), 2'-alkoxy, 2'-substituted alkoxy, 2'-alkyl, 2'-substituted alkyl, 2'-amino, 2'-substituted amino.

[0202] Exemplary modified nucleotides have the structure shown below:

[0203]

[0204] Wherein, Base represents a base, such as A, U, G, C or T. The hydroxyl group at the 2'-position of the ribose group is substituted by R. The hydroxyl groups at the 2'-position of these ribose groups can be substituted by various groups known to those skilled in the art. For example, the modified nucleotides after substitution can be 2'-fluoro (2'-F) modified nucleotides, 2'-alkoxy modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-amino modified nucleotides, 2'-substituted amino modified nucleotides, 2'-deoxyribonucleotides.

[0205] In some embodiments, the sense strand may comprise one or more capping residues or moieties, sometimes referred to in the art as "caps", "terminal caps" or "capping residues". As used herein, a "capping residue" is a non-nucleotide compound or other moiety that can be incorporated at one or both ends of the nucleotide sequence of an siRNA disclosed herein. In some cases, the capping residue can provide certain beneficial properties to the siRNA, such as protection against exonuclease degradation. In some embodiments, an inverted abasic deoxyribose residue (invAb) is added as a capping residue. In some embodiments, the capping residue is present at the 5'-end, 3'-end or both the 5'- and 3'-ends of the sense strand.

[0206] In some embodiments, one or more inverted abasic deoxyribose residues (invAb) are added to the 3'-end of the sense strand. In some embodiments, one or more inverted abasic deoxyribose residues (invAb) are added to the 5'-end of the sense strand. In some embodiments, one or more inverted abasic deoxyribose residues (invAb) are added to the 5'-end and the 3'-end of the sense strand. The inverted abasic deoxyribose residues can be linked via a phosphodiester bond, a phosphorothioate bond or other internucleoside bonds. The chemical structure of the inverted abasic deoxyribose residue is shown below:

[0207] Formula A when (invAb) is located inside the siRNA; Formula B when (invAb) is located at the 3'-end of the siRNA; Formula C when (invAb) is located at the 5'-end of the siRNA:

[0208]

[0209] Among them, the bond of the phosphate bond part of Formula A is a bond facing the 3'-end of the siRNA.

[0210] In some embodiments, the 2'-alkoxy-modified nucleotide is a 2'-methoxy (2'-OMe, 2'-O-CH3)-modified nucleotide, etc.

[0211] In some embodiments, the 2'-substituted alkoxy-modified nucleotide is a 2'-methoxyethoxy (2'-O-CH2-CH2-O-CH3)-modified nucleotide, a 2'-O-CH2-CH=CH2-modified nucleotide, etc.

[0212] In some embodiments, the 2'-substituted alkyl-modified nucleotide is a 2'-CH2-CH2-CH=CH2-modified nucleotide, etc.

[0213] siRNA conjugate

[0214] This application relates to an siRNA conjugate, which contains the above siRNA and a conjugate group conjugated to the siRNA.

[0215] In this application, the sense strand and the antisense strand of the siRNA conjugate form a double-stranded region of the siRNA conjugate, and a blunt end is formed at the 3'-end of the sense strand of the siRNA conjugate. In some embodiments, a blunt end is formed at the 3'-end of the sense strand of the siRNA conjugate, and the 3'-end of the antisense strand of the siRNA conjugate has 1-3 protruding nucleotides extending out of the double-stranded region. In some other embodiments, a blunt end is formed at the 3'-end of the sense strand of the siRNA conjugate, and a blunt end is formed at the 3'-end of the antisense strand of the siRNA conjugate.

[0216] In some preferred embodiments, the siRNA conjugate is obtained by conjugating an siRNA with a conjugate group. Among them, the sense strand and the antisense strand of the siRNA are complementary to form a double-stranded region of the siRNA, and a blunt end is formed at the 3'-end of the sense strand of the siRNA. The conjugate group is conjugated to the 3'-end of the sense strand with a blunt end to form the siRNA conjugate.

[0217] In some preferred embodiments, the 3'-end of the sense strand of the siRNA has protruding nucleotides extending out of the double-stranded region. The sequence with a 3'-flat end formed after excluding the protruding nucleotides at the 3'-end in the sense strand is used as the nucleotide sequence for conjugating a conjugate group. A conjugate group is linked to the 3'-flat end of the sense strand to form an siRNA conjugate.

[0218] In some more preferred embodiments, when the nucleotide sequences of the sense strand and the antisense strand are complementary to form a double-stranded region and there are no protruding nucleotides at the 3'-end of the sense strand, a nucleotide sequence V is added to the 3'-end of the sense strand as the protruding nucleotides. The sequence with a 3'-flat end formed after excluding the protruding nucleotides at the 3'-end in the sense strand is used as the nucleotide sequence for conjugating a conjugate group. A conjugate group is linked to the 3'-flat end of the sense strand to form an siRNA conjugate.

[0219] In some more preferred embodiments, when the nucleotide sequences of the sense strand and the antisense strand are complementary to form a double-stranded region and the 3'-end of the sense strand has protruding nucleotides extending out of the double-stranded region, the sequence with a 3'-flat end formed after excluding the protruding nucleotides at the 3'-end in the sense strand is used as the nucleotide sequence for conjugating a conjugate group. A conjugate group is linked to the 3'-flat end of the sense strand to form an siRNA conjugate.

[0220] Exemplarily, for the siRNA with the sequence as shown in N-ER-FY037002M2, the 3'-end of the sense strand of this siRNA has protruding nucleotides extending out of the double-stranded region. The gsasauauUfcUfUfCfaaagagau flat-end sequence formed after excluding the protruding -sTsT nucleotides at the 3'-end in the sense strand is used as the nucleotide sequence for conjugating the L96 conjugate group. Therefore, the sequence for forming the siRNA conjugate is: the sense strand is

[0221] gsasauauUfcUfUfCfaaagagauL96, and the antisense strand is P1asUfscucUfuugaagaAfuAfuucsTsT.

[0222] Generally speaking, the conjugating group contains at least one pharmaceutically acceptable targeting group, or further contains a linker, and the siRNA, the linker, and the targeting group are connected in sequence. In some embodiments, the number of targeting groups is 1-6. In some embodiments, the number of targeting groups is 2-4. The siRNA molecule can be conjugated to the conjugating group non-covalently or covalently, for example, it can be covalently conjugated to the conjugating group. The conjugation site of the siRNA to the conjugating group can be at the 3'-end or 5'-end of the sense strand of the siRNA, or at the 5'-end of the antisense strand, or also in the internal sequence of the siRNA. In some embodiments, the conjugation site of the siRNA to the conjugating group is at the 3'-end of the sense strand of the siRNA.

[0223] In some embodiments, the conjugating group can be connected to the phosphate group, 2'-hydroxyl group, or base of the nucleotide. In some embodiments, the conjugating group can also be connected to the 3'-hydroxyl group, and in this case, the nucleotides are connected by 2'-5' phosphodiester bonds. When the conjugating group is connected to the end of the siRNA strand, the conjugating group is usually connected to the phosphate group of the nucleotide; when the conjugating group is connected to the internal sequence of the siRNA, the conjugating group is usually connected to the ribose ring or base. Various connection methods can be found in the reference: Muthiah Manoharan et.al. siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatocytes. ACS Chemical biology, 2015, 10(5):1181-7.

[0224] In some embodiments, the siRNA and the conjugating group can be connected by acid-labile or reducible chemical bonds, which can be degraded in the acidic environment of the endosome in the cell, so that the siRNA becomes free. For non-degradable conjugation methods, the conjugating group can be connected to the sense strand of the siRNA to minimize the impact of conjugation on the activity of the siRNA.

[0225] In some embodiments, the pharmaceutically acceptable targeting group can be a ligand commonly used in the field of siRNA administration, such as various ligands described in WO2009082607A2, which is incorporated herein by reference in its entirety.

[0226] In some embodiments, the pharmaceutically acceptable targeting group may be selected from one or more of the ligands formed by the following targeting molecules or their derivatives: 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 cell-penetrating peptides; aptamers; antibodies; quantum dots; saccharides, such as lactose, polylactose, mannose, galactose, N-acetylgalactosamine (GalNAc); folate; receptor ligands expressed on hepatocytes, such as asialoglycoprotein, asialoglycoprotein residues, lipoproteins (such as high-density lipoprotein, low-density lipoprotein, etc.), glucagon, neurotransmitters (such as adrenaline), growth factors, transferrin, etc.

[0227] In some embodiments, each of the ligands is independently selected from a ligand capable of binding to a cell surface receptor. In some embodiments, at least one ligand is a ligand capable of binding to a hepatocyte surface receptor. In some embodiments, at least one ligand is a ligand capable of binding to a mammalian cell surface receptor. In some embodiments, at least one ligand is a ligand capable of binding to a human hepatocyte surface receptor. In some embodiments, at least one ligand is a ligand capable of binding to the asialoglycoprotein receptor (ASGPR) on the liver surface. The types of these ligands are well known to those skilled in the art, and their general function is to bind to specific receptors on the surface of target cells and mediate the delivery of siRNA linked to the ligand to the target cells.

[0228] In some embodiments, the pharmaceutically acceptable targeting group may be any ligand that binds to the asialoglycoprotein receptor (ASGPR) on the surface of mammalian hepatocytes. In some embodiments, each ligand is independently asialoglycoprotein, such as asialoorosomucoid (ASOR) or asialofetuin (ASF). In some embodiments, the ligand is a sugar or a derivative of a sugar.

[0229] In some embodiments, at least one ligand is a sugar. In some embodiments, each ligand is a sugar. In some embodiments, at least one ligand is a monosaccharide, polysaccharide, modified monosaccharide, modified polysaccharide, or sugar derivative. In some embodiments, at least one of the ligands may be a monosaccharide, disaccharide, or trisaccharide. In some embodiments, at least one ligand is a modified sugar. In some embodiments, each ligand is a modified sugar. In some embodiments, each ligand is independently selected from polysaccharides, modified polysaccharides, monosaccharides, modified monosaccharides, polysaccharide derivatives, or monosaccharide derivatives. In some embodiments, each or at least one ligand is selected from the group consisting of the following sugars: glucose and its derivatives, mannan and its derivatives, galactose and its derivatives, xylose and its derivatives, ribose and its derivatives, fucose and its derivatives, lactose and its derivatives, maltose and its derivatives, arabinose and its derivatives, fructose and its derivatives, and sialic acid.

[0230] In some embodiments, each of the ligands may be independently selected from D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, α-D-mannofuranose, β-D-mannofuranose, α-D-mannopyranose, β-D-mannopyranose, α-D-glucopyranose, β-D-glucopyranose, α-D-glucofuranose, β-D-glucofuranose, α-D-fructofuranose, α-D-fructopyranose, α-D-galactopyranose, β-D-galactopyranose, α-D-galactofuranose, β-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine, N-isobutyrylgalactosamine, 2-amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose, N-glycolyl-α-neuraminic acid, 5-thio-β-D-glucopyranose, methyl 2,3,4-tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside, 4-thio-β-D-galactopyranose, ethyl 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucoheptopyranoside, 2,5-anhydro-D-allononitrile, ribose, D-ribose, D-4-thioribose, L-ribose, or L-4-thioribose. Other selections of the ligands can be found, for example, in the description of CN105378082A, which is incorporated herein by reference in its entirety.

[0231] In some embodiments, the pharmaceutically acceptable targeting group in the siRNA conjugate can be galactose or N-acetylgalactosamine, wherein the galactose or N-acetylgalactosamine molecule can be monovalent, divalent, trivalent, or tetravalent. It should be understood that the monovalent, divalent, trivalent, and tetravalent as described herein respectively refer to the molar ratio of the siRNA molecule to the galactose or N-acetylgalactosamine molecule containing the conjugation group as the targeting group in the siRNA conjugate being 1:1, 1:2, 1:3, or 1:4 after the formation of the siRNA conjugate. In some embodiments, the pharmaceutically acceptable targeting group is N-acetylgalactosamine. In some embodiments, when the siRNA described in the present application is conjugated with a conjugation group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent or tetravalent. In some embodiments, when the siRNA described in the present application is conjugated with a conjugation group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent.

[0232] The targeting group can be connected to the siRNA molecule via a suitable linker, and those skilled in the art can select a suitable linker according to the specific type of the targeting group. The types of these linkers, targeting groups, and the connection modes with siRNA can refer to the disclosure of WO2015006740A2, which is incorporated herein by reference in its entirety.

[0233] Synthesis method of siRNA

[0234] By the conventional solid-phase phosphoramidite method in the art, nucleoside monomers are sequentially connected one by one in the 3'-5' direction according to the nucleotide arrangement order. Each connection of a nucleoside monomer includes four reaction steps: deprotection, coupling, oxidation or sulfurization, and capping. Among them, when a phosphoester linkage is used between two nucleotides, when connecting the latter nucleoside monomer, it includes four reaction steps: deprotection, coupling, oxidation, and capping. When a phosphorothioate linkage is used between two nucleotides, when connecting the latter nucleoside monomer, it includes four reaction steps: deprotection, coupling, sulfurization, and capping. The present invention selects nucleotide monomers according to the synthetic target sequence, and the selected nucleotide monomers are the commonly used nucleotide monomers by those skilled in the art. For example, the nucleotide monomer for synthesizing A can be, but is not limited to, adenosine-3-phosphate. It should be understood that these monomers are connected to each other by 5'-3' phosphodiester bonds or 5'-3' phosphorothioate groups when present in oligonucleotides, and when the 3'-position of the last nucleotide in the 5' to 3' direction is a hydroxyl group, it is achieved by conventional means in the art.

[0235] For example, the synthesis conditions of the siRNA of the present application can be as follows:

[0236] The nucleoside monomers are provided in an acetonitrile solution with a concentration of 0.1M. The conditions for the deprotection reaction in each step are the same, i.e., the temperature is 25°C, the reaction time is 70 seconds, the deprotection reagent is a dichloromethane solution of dichloroacetic acid (3% V / V), and the molar ratio of dichloroacetic acid to the 4,4'-dimethoxytrityl protecting group on the solid support is 5:1.

[0237] The coupling reaction conditions include: the reaction temperature is 25°C, the reaction time is 600 seconds, the coupling reagent is selected from a 0.5M acetonitrile solution of 5-ethylthio-1H-tetrazole (ETT), the molar ratio of the nucleic acid sequence linked to the solid support to the nucleoside monomer is 1:10, and the molar ratio of the nucleic acid sequence linked to the solid support and the coupling reagent is 1:65.

[0238] 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 water, and the molar ratio of the oxidation reagent to the nucleic acid sequence linked to the solid support in the coupling step is 30:1. The reaction is carried out in a mixed solvent of tetrahydrofuran: water: pyridine = 3:1:1.

[0239] The sulfurization reaction conditions include: the reaction temperature is 25°C, the reaction time is 300 seconds, the sulfurization reagent is selected from hydrogen xanthate, and the molar ratio of the sulfurization reagent to the nucleic acid sequence linked to the solid support in the coupling step is 120:1. The reaction is carried out in a mixed solvent of acetonitrile: pyridine = 1:1.

[0240] The capping reaction conditions include: the reaction temperature is 25°C, the reaction time is 15 seconds, the capping reagent is selected from a mixed solution of CapA (10% acetic anhydride acetonitrile solution) and CapB (10% N-methylimidazole pyridine / acetonitrile solution) with a molar ratio of 1:1, and the molar ratio of the capping reagent to the nucleic acid sequence linked to the solid support is acetic anhydride: N-methylimidazole: nucleic acid sequence linked to the solid support = 1:1:1.

[0241] After all the nucleoside monomers are linked, the nucleic acid sequence linked to the solid support is subjected to ammonolysis, purification, and desalting in sequence to obtain the sense strand and antisense strand of siRNA. Finally, the two strands are heated and annealed to obtain the product.

[0242] The methods of ammonolysis, purification, desalting, and annealing are well-known in the art. For example, the nucleotide sequence linked to the solid support is contacted with concentrated ammonia water for cleavage and deprotection; purification is carried out by chromatography; desalting is carried out by reverse-phase chromatography; and the sense strand and antisense strand are mixed in an equimolar ratio under different stringent conditions and then gradually cooled.

[0243] The synthesized siRNAs are shown in Table 1, Table 1-1, and Table 1-2.

[0244] Synthesis method of siRNA conjugate

[0245] Taking the synthesis of L96 as an example:

[0246] In the first step, by reacting DMTr-L96 with succinic anhydride, compound L96-A is obtained:

[0247]

[0248] Preparation process: Add DMTr-L96, succinic anhydride, 4-dimethylaminopyridine, and diisopropylethylamine to dichloromethane, stir and react at 25 °C for 24 hours, then wash the reaction solution with 0.5 M triethylamine phosphate, wash the aqueous phase three times with dichloromethane, combine the organic phases and evaporate to dryness under reduced pressure to obtain the crude product. Then purify by column chromatography to obtain the pure product L96-A.

[0249] In the second step, react L96-A with NH2-SPS to obtain L96-B:

[0250]

[0251] Preparation process: Mix L96-A, O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU), and diisopropylethylamine (DIPEA) and dissolve them in acetonitrile, stir at room temperature for 5 minutes to obtain a homogeneous solution, add aminomethyl resin (NH2-SPS, 100 - 200 mesh) to the reaction liquid, start shaking reaction at 25 °C, filter after reacting for 18 hours, wash the filter cake successively with dichloromethane and acetonitrile to obtain the filter cake. The obtained filter cake is subjected to a capping reaction with a CapA / CapB mixed solution to obtain L96-B, which is a solid-phase carrier containing the conjugated molecule.

[0252] In the third step, synthesis of the siRNA conjugate:

[0253] Using L96-B as the solid-phase carrier, synthesize the sense strand of the siRNA conjugate according to the siRNA synthesis method described above, synthesize the antisense strand of the siRNA conjugate using the siRNA synthesis method described above, and anneal to generate the siRNA conjugate of the present application.

[0254] The synthesized siRNA conjugate is shown in Table 2.

[0255] Drug composition

[0256] The present application provides a pharmaceutical composition, which contains the above-mentioned siRNA as an active ingredient and a pharmaceutically acceptable carrier.

[0257] The pharmaceutically acceptable carrier may be a carrier commonly used in the field of siRNA administration, such as, but not limited to, lipid nanoparticles (LNP), magnetic nanoparticles (such as nanoparticles based on Fe3O4 or Fe2O3), carbon nanotubes, mesoporous silicon, calcium phosphate nanoparticles, polyethyleneimine (PEI), polyamidoamine (PAMAM) dendrimer, poly(L-lysine) (PLL), chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), poly(D&L-lactic / glycolic acid) copolymer (PLGA), poly(2-aminoethyl ethylene phosphate) (PPEEA), poly(2-dimethylaminoethyl methacrylate) (PDMAEMA), and one or more of their derivatives.

[0258] (poly(L-lysine), PLL), chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), poly(D&L-lactic / glycolic acid) copolymer (PLGA), poly(2-aminoethyl ethylene phosphate) (PPEEA), poly(2-dimethylaminoethyl methacrylate) (PDMAEMA), and one or more of their derivatives.

[0259] In the pharmaceutical composition, there is no particular requirement for the contents of siRNA and the pharmaceutically acceptable carrier, and they can be the conventional contents of each component.

[0260] In some embodiments, the pharmaceutical composition may further contain other pharmaceutically acceptable excipients, and the excipient may be one or more of various preparations or compounds conventionally used in the art. For example, the other pharmaceutically acceptable excipients may include at least one of a pH buffer, a protective agent, and an osmotic pressure regulator.

[0261] The pH buffer may be a tris(hydroxymethyl)aminomethane hydrochloride buffer with a pH value of 7.5 - 8.5 and / or a phosphate buffer with a pH value of 5.5 - 8.5, for example, a phosphate buffer with a pH value of 5.5 - 8.5.

[0262] The protective agent may be at least one of 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.

[0263] The osmotic pressure regulator may be sodium chloride and / or potassium chloride. The content of the osmotic pressure regulator makes the osmotic pressure of the pharmaceutical composition 200-700 milliosmoles per kilogram (mOsm / kg). Those skilled in the art can easily determine the content of the osmotic pressure regulator according to the required osmotic pressure.

[0264] In some embodiments, the pharmaceutical composition may be a liquid preparation, such as an injection; it may also be a freeze-dried powder injection, which is mixed with liquid excipients during administration to prepare a liquid preparation. The liquid preparation can be used for subcutaneous, intramuscular, or intravenous injection, and can also be used for administration to the lungs by spraying, or for administration to other organ tissues (such as the liver) through the lungs by spraying. In some embodiments, the pharmaceutical composition is used for intravenous injection.

[0265] In some embodiments, the pharmaceutical composition may be in the form of a liposomal preparation. In some embodiments, the pharmaceutically acceptable carrier used in the liposomal preparation includes an amine-containing transfection compound (hereinafter also referred to as an organic amine), a co-lipid, and / or a polyethylene glycolylated lipid.

[0266] The following examples are used to further illustrate the present invention, but do not limit the present invention in any way.

[0267] Examples

[0268] Other objects, features, and advantages of the present disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, although representing specific embodiments of the present disclosure, are given for illustrative purposes only, because various changes and modifications made within the spirit and scope of the present disclosure will become apparent to those skilled in the art after reading this detailed description.

[0269] The experimental techniques and methods used in this example are all conventional technical methods unless otherwise specified. For example, the experimental methods without specific conditions in the following examples are usually carried out under conventional conditions such as those described by 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.

[0270] Preparation of siRNA in Example 1

[0271] The siRNA molecules with the following sequences were synthesized by Tianlin Biotechnology (Shanghai) Co., Ltd. Table 1 shows the synthesized siRNA and their sequences, Table 1-1 shows the comparison sequences, and Table 1-2 shows the core complementary regions of the preferred sequences.

[0272] Table 1 siRNA and Their Sequences

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287] Table 1-1

[0288]

[0289] Among them, N-ER-FY037ZY01 is an antisense oligonucleotide (ASO).

[0290] Table 1-2

[0291]

[0292] Among them, the capital letters "G", "C", "A", "T", and "U" each generally represent nucleotides containing guanine, cytosine, adenine, thymine, and uracil as bases, respectively; the lowercase letters a, u, c, g represent 2'-methoxy-modified nucleotides; Af, Gf, Cf, Uf represent 2'-fluoro-modified nucleotides; "dG", "dC", "dA", "dT", "dU" represent 2'-deoxyribonucleotides; (invAb) is a reverse abasic deoxyribose residue; the lowercase letter s indicates that there is a phosphorothioate linkage between the two nucleotides adjacent to the letter s on its left and right; P1 represents that the nucleotide adjacent to the right of this P1 is a 5'-phosphonucleotide; EVP represents that the nucleotide adjacent to the right of this EVP is a 5'-trans-vinylphosphonate nucleotide; (Underline + bold + italic): represents a GNA-modified nucleotide.

[0293] SiRNA conjugates with the following sequences were synthesized by Tianlin Biotechnology (Shanghai) Co., Ltd.:

[0294] Table 2 SiRNA conjugates and their sequences:

[0295]

[0296]

[0297]

[0298]

[0299] Among them, L96 is connected to the 3'-end of the sense strand in Table 1 or the blunt end formed by the 3'-end of the sense strand through a phosphodiester bond, and L96 is:

[0300]

[0301] In Table 1, Table 1-1, Table 1-2, and Table 2, if there is no P1 or EVP marked on the left of the 5'-terminal nucleotide of the sense strand, the modified sense strand, and the modified sense strand connecting the conjugate group, it means that the 5'-terminal nucleotide is not connected to a 5'-phosphate group or a 5'-phosphate-derived group (i.e., the ribose group of the 5'-terminal nucleotide is a 5'-hydroxyl group), and its structure is as shown in Formula X:

[0302]

[0303] Among them, Base represents a base, such as A, U, G, C or T; R is a hydroxyl group or hydrogen or is substituted by various groups known to those skilled in the art. For example, R can be 2'-fluoro (2'-F), 2'-alkoxy, 2'-substituted alkoxy, 2'-alkyl, 2'-substituted alkyl, 2'-amino, 2'-substituted amino.

[0304] In Table 1, Table 1-1, Table 1-2 and Table 2, if there is no label of P1 or EVP on the left side of the 5'-terminal nucleotide of the antisense strand and the modified antisense strand, it means that the 5'-terminal nucleotide is not linked to a 5'-phosphate group or a 5'-phosphate-derived group, and its structure is also as shown in Formula X.

[0305] In Table 1, Table 1-1, Table 1-2 and Table 2, the 3'-position of the 3'-terminal nucleotide of the sense strand and the modified sense strand, and the 3'-terminal nucleotide of the antisense strand and the modified antisense strand is a hydroxyl group.

[0306] Example 2 siRNA inhibits LECT2 gene expression

[0307] Experimental materials:

[0308] PHH cells were provided by Shanghai Wuxi AppTec New Drug Development Co., Ltd.

[0309] RNA extraction kit, product number QIAGEN-74182;

[0310] RNAiMAX transfection reagent was purchased from Invitrogen, product number 13778-150;

[0311] InvitroGRO CP medium was purchased from BIOIVT, product number S03316;

[0312] Reverse transcription kit (HiScript III RT SuperMix for qPCR(+gDNA wiper)): purchased from Vazyme, R323-01;

[0313] FastStart Universal Probe Master was purchased from Roche, product number 04914058001;

[0314] Opti-medium was purchased from Gibco, product number 31985070;

[0315] Target LECT2 primer and probe set was purchased from Thermo, Hs01040204_m1;

[0316] TaqMan Gene Expression Assay (GAPDH), purchased from Thermo, ID-Hs02786624_g1

[0317] Experimental methods:

[0318] 1. Culture PHH cells in Opti-medium in a 96-well plate for 24 hours. Dilute the cell suspension with 10% InvitroGRO CP medium to a density of 6×10 5 / mL, and seed it into a 96-well plate. Add 90 μL of the cell suspension to each well, i.e., 54,000 cells / well.

[0319] 2. Centrifuge the dry powder of the siRNA to be tested at low temperature and high speed, and then dissolve it with UltraPure Distilled Water to prepare a 50 μM siRNA mother solution.

[0320] 3. Prepare a 0.5 nM siRNA transfection dilution

[0321] (1) Preparation of 0.5 μM siRNA stock solution:

[0322] a. Take 2 μL of the 50 μM siRNA mother solution prepared in step 2 above, and add 18 μL of UltraPure Distilled Water to obtain a siRNA dilution with a final concentration of 5 μM;

[0323] b. Take 2 μL of the 5 μM siRNA dilution prepared in step a, and add 18 μL of UltraPure Distilled Water to obtain a siRNA dilution with a final concentration of 0.5 μM;

[0324] (2) Take 2 μL of the 0.5 μM siRNA stock solution prepared in step (1), and add 98 μL of Opti-medium to obtain a 10 nM siRNA dilution;

[0325] (3) Take 3 μL of RNAiMAX transfection reagent, and add 97 μL of Opti-medium to obtain RNAiMAX transfection reagent dilution; Mix the RNAiMAX transfection reagent dilution with the siRNA dilution at a volume ratio of 1:1, let it stand for 5 minutes, and add 10 μL of the transfection mixture to the PHH cells cultured in the 96-well plate (final volume 100 μL, and the concentration of siRNA in this system is 0.5 nM). A 5 nM siRNA transfection dilution can be prepared according to the above similar operations.

[0326] 5 nM siRNA transfection dilution can be prepared according to the above similar operations.

[0327] 4. Incubate for 48 hours after transfection, with 2 replicates set for each concentration.

[0328] 5. Extract total RNA according to the instructions of the RNA extraction kit.

[0329] 6. Reverse transcribe the extracted total RNA into cDNA using a reverse transcription kit, following the steps below:

[0330] a) Remove gDNA with gDNA enzyme according to the following table;

[0331] Volume / μL 4×gDNA wiper Mix 4 Sample(RNA) 8 <![CDATA[RNase-free ddH2O]]> 4

[0332] 42 °C, 2 min, store at 4 °C;

[0333] b) Add 4 μL of 5×HiScript III qRT SuperMix directly to the reaction plate from step a); 37 °C, 15 min, 85 °C, 5 s;

[0334] c) Store the reverse transcription product at 4 °C for real-time PCR analysis.

[0335] 7. Perform real-time PCR analysis

[0336] a) Prepare the qPCR reaction mixture as shown in the following table. During the whole operation, all reagents are placed on ice;

[0337]

[0338]

[0339] b) Perform the qPCR program as described below

[0340] 95 °C, 10 minutes;

[0341] 95 °C, 15 seconds, 60 °C, 1 minute (this operation is repeated 40 cycles);

[0342] 8. Result analysis

[0343] a) Use Quant Studio 6Flex software with default settings to automatically calculate the Ct value;

[0344] b) Calculate the relative expression level of the gene using the following formula:

[0345] ΔCt = Ct(LECT2 gene) - Ct(GAPDH)

[0346] ΔΔCt = ΔCt(test sample group) - ΔCt(Mock group)

[0347] mRNA expression relative to the Mock group = 2 -ΔΔCt 。

[0348] Mock: The group without siRNA added, compared with the test sample group.

[0349] Inhibition rate (%) = (Relative mRNA expression level of the Mock group - Relative mRNA expression level of the test sample group) / Relative mRNA expression level of the Mock group × 100%

[0350] 9. Experimental results

[0351] Select concentrations of 0.5 nM and 5 nM for testing

[0352] Table 3 Inhibition rates of the siRNA of the present invention

[0353]

[0354]

[0355]

[0356] Table 4 Inhibition rates of the control siRNA

[0357] siRNA ID 5nM - 48h(%) 0.5nM - 48h(%) N-ER-FY037047 -18.95 -9.96 N-ER-FY037ZY01 8.46 -8.49 N-ER-FY037ZY02 87.31 79.30

[0358] As can be seen from Table 3 and Table 4, the siRNA of the present invention can significantly inhibit the expression of the LECT2 gene at 5 nM and 0.5 nM. Preferably, the inhibition rate of the siRNA is basically above 90% at 48 h at 5 nM and basically above 88% at 48 h at 0.5 nM.

[0359] Example 3 IC of siRNA inhibiting LECT2 gene expression 50 Determination

[0360] The final concentrations of the following siRNAs to be tested are 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 IC is carried out according to a method similar to that in Example 2 50 Determination.

[0361] Result analysis:

[0362] a) Use the Quant Studio 6 Flex software with default settings to automatically calculate the Ct value;

[0363] b) Use the following formula to calculate the relative expression level of the gene:

[0364] ΔCt = Ct (LECT2 gene) – Ct (GAPDH)

[0365] ΔΔCt = ΔCt (test sample group) – ΔCt (Mock group), where the Mock group refers to the group without siRNA added compared to the test sample group;

[0366] mRNA expression relative to the Mock group = 2 -ΔΔCt

[0367] Inhibition rate (%) = (relative mRNA expression in the Mock group – relative mRNA expression in the test sample group) / relative mRNA expression in the Mock group × 100%

[0368] Calculation process: Using the log value of the siRNA concentration as the X-axis and the percentage inhibition rate as the Y-axis, the "log(inhibitor) vs. response - variable slope" function module of the analysis software GraphPad Prism 8 is used to fit the dose-response curve, thereby obtaining the IC 50 value.

[0369] The fitting formula is: Y = Bottom + (Top - Bottom) / (1 + 10^((logIC 50 -X)*HillSlope))

[0370] Where: Top represents the percentage inhibition rate at the top plateau, and the Top standard of the curve is generally between 80% and 120%; Bottom represents the percentage inhibition rate at the bottom plateau, and the Bottom of the curve is generally between -20% and 20%; HillSlope represents the slope of the percentage inhibition rate curve.

[0371] The results are shown in Table 5.

[0372] Table 5 IC of siRNA 50 (nM)

[0373] siRNAID <![CDATA[IC 50 (nM)]]> N-ER-FY037002M2 0.0103 N-ER-FY037016M2 0.0058 N-ER-FY037022M2 0.0015 N-ER-FY037090M2 0.0029 N-ER-FY037053M2 0.0375 N-ER-FY037028M2 0.0215 N-ER-FY037031M2 0.0023 N-ER-FY037002M2 0.0103 N-ER-FY037016M2 0.0058

[0374] As can be seen from Table 5, the siRNA provided in this application has high LECT2 gene inhibitory activity in PHH cells, and the IC 50 can be as low as 0.0015 nM.

[0375] Example 4 Determination of the inhibition rate of siRNA conjugate on LECT2 gene expression

[0376] Experimental materials:

[0377] PHH cells were provided by Shanghai WuXi AppTec New Drug Development Co., Ltd.;

[0378] RNA extraction kit, product number QIAGEN - 74182;

[0379] RNAiMAX transfection reagent, purchased from Invitrogen, catalog number 13778 - 150;

[0380] PPH medium: InvitroGRO CP medium, purchased from BIOIVT, catalog number S03316;

[0381] HiScript III RT SuperMix for qPCR(+gDNA wiper): purchased from Vazyme, R323 - 01;

[0382] FastStart Universal Probe Master: purchased from Roche, catalog number 04914058001;

[0383] Opti - medium: purchased from Gibco, catalog number 31985070;

[0384] Target LECT2 primer and probe set, purchased from Thermo, Hs01040204_m1;

[0385] TaqMan Gene Expression Assay(GAPDH), purchased from Thermo, ID - Hs02786624_g1

[0386] Experimental methods:

[0387] The siRNA conjugate (final concentrations of siRNA conjugate are 5 nM and 0.5 nM, duplicate wells) was transfected into PHH cells as described below: Take the cryopreserved PHH cells, resuscitate, count, and adjust the cell density to 6×10 5 cells / mL, and at the same time apply RNAiMAX transfection reagent to transfer the siRNA conjugate into the cells, inoculate into a 96 - well plate at a density of 54,000 cells per well, and add 100 μL of PHH medium to each well. The cells were cultured in an incubator with 5% CO2 at 37°C. After 48 hours, the medium was removed and the cells were collected for total RNA extraction. Total RNA was extracted using an RNA extraction kit according to the kit product manual.

[0388] The siRNA conjugate (final concentrations of siRNA conjugate are 200 nM and 10 nM, duplicate wells) entered PHH cells by free uptake as described below: Take the cryopreserved PHH cells, resuscitate, count, and adjust the cell density to 6×10 5Cells / mL, and at the same time, siRNA conjugate was added. The cells were seeded into a 96-well plate at a density of 54,000 cells per well, and the culture medium in each well was 100 μL. The cells were cultured in an incubator at 5% CO2 and 37 °C. After 48 hours, the culture medium was removed and the cells were collected for total RNA extraction. Total RNA was extracted using an RNA extraction kit according to the product instructions of the kit.

[0389] Using a method similar to that in Example 2, the extracted total RNA was reverse transcribed into cDNA by reverse transcription reaction, and the LECT2 cDNA obtained by reverse transcription was quantitatively amplified by qPCR. GAPDH cDNA was amplified in parallel as an internal control. The PCR reaction program was: 95 °C for 10 minutes, and then enter the cycling mode, 95 °C for 15 seconds, followed by 60 °C for 60 seconds, for a total of 40 cycles.

[0390] Result analysis:

[0391] a) Using Quant Studio 7 software with default settings, the Ct value was automatically calculated;

[0392] b) The relative expression level of the gene was calculated using the following formula:

[0393] ΔCt = Ct(LECT2 gene) - Ct(GAPDH)

[0394] ΔΔCt = ΔCt(test sample group) - ΔCt(Mock group)

[0395] mRNA expression relative to the Mock group = 2 -ΔΔCt .

[0396] Inhibition rate (%) = (mRNA relative expression level in the Mock group - mRNA relative expression level in the test sample group) / mRNA relative expression level in the Mock group × 100%;

[0397] Where the Mock group represents the group without the addition of siRNA conjugate compared with the test sample group.

[0398] The experimental results are shown in Table 6.

[0399] Table 6 Inhibition rate of siRNA conjugate on LECT2 gene expression

[0400]

[0401]

[0402]

[0403]

[0404] As can be seen from Table 6, the siRNA conjugate provided by the present application has high LECT2 gene inhibitory activity in PHH cells. When the siRNA conjugate enters PHH by free uptake, preferably, the inhibition rate is basically above 95% at 200 nM for 48 h and basically above 88% at 10 nM for 48 h; when the siRNA conjugate enters PHH by transfection, preferably, the inhibition rate is basically above 90% at 5 nM for 48 h and basically above 86% at 0.5 nM for 48 h.

[0405] Example 5 Silencing effect of siRNA conjugate in mice expressing human LECT2 (hLECT2) gene

[0406] (1) Construction of a mouse model overexpressing hLECT2 gene by AAV

[0407] C57BL / 6 mice at 6 - 8 weeks old (provided by Jiangsu Jicui Yakang Biotechnology Co., Ltd.) entered the facility. After 3 - 5 days of adaptive feeding, a single dose of adeno - associated virus AAV of hLECT2 gene (pAAV[Exp]-CBh>SEAP:{LECT2 5utr CDS 3utr}:WPRE, the virus was provided by GenoWay Biotech Co., Ltd., Guangzhou) was injected into the tail vein for overexpression of the target gene. The administration volume: 100 μL (10×10 11 vg) / mouse, and then fed with normal feed.

[0408] (2) Investigation of the efficacy of siRNA for silencing in the hLECT2 mouse model

[0409] 14 days after AAV virus injection, the mice were grouped (5 mice in each group), and a single 3mpk dose of the siRNA conjugate of the present application (as shown by siRNA ID in the following table) was administered subcutaneously. The expression level of SEAP protein (i.e., the expression level of hLECT2 protein) was detected on the 7th, 14th, 21st, 28th, and 35th days after administration.

[0410] (3) Drug preparation

[0411] a. The powder of the drug to be tested was centrifuged for 10 s, the lid was opened in a biosafety cabinet without wind, and 1 mL of enzyme - free and sterile PBS solution was added;

[0412] b. After vortexing, it was centrifuged instantaneously and left to stand for 5 minutes for sufficient dissolution, and 10 μL of the liquid was taken for concentration detection;

[0413] c. The concentration of the stock solution was detected using a calibrated Nanodrop instrument in RNA mode;

[0414] d. Calculate the volume of PBS solution to be supplemented according to the concentration of the mother liquor;

[0415] e. Transfer 900 μL of the mother liquor in the original tube into a 5 mL centrifuge tube, and add the PBS solution to be supplemented;

[0416] f. Vortex and then centrifuge instantaneously, aspirate 10 μL of the liquid for concentration detection, and the concentration this time should be within ±10% of the theoretical concentration;

[0417] g. Transfer the prepared drug to the animal house for administration, and avoid ultraviolet irradiation during this period.

[0418] (4) Detection steps

[0419] a. Preparation of 1×Dilution buffer:

[0420] Reagent name Volume ratio <![CDATA[ddH2O]]> 4 5×Dilution buffer 1

[0421] Vortex and mix well for later use.

[0422] b. Sample dilution

[0423] (Dilute 1000 times)

[0424] Serum volume (μL) 1×Dilution buffer(μL) DNase / RNase-free water(μL) Dilution factor 6 / 54 10 6 / 54 10 5 45 / 10

[0425] (Dilute 300 times)

[0426] Serum volume (μL) 1×Dilution buffer(μL) DNase / RNase-free water(μL) Dilution factor 6 / 114 20 4 56 / 15

[0427] Mix well for later use;

[0428] c. Sample denaturation

[0429] Put the diluted sample into the PCR instrument and incubate at 65 °C for 30 minutes (the PCR program has been set to save at 4 °C after incubation is completed), take it out and put it in an ice box to return to room temperature, and keep it for later use;

[0430] d. Pretreatment of luminescence reaction

[0431] The sample detection layout is carried out according to the layout of Table 8 plate. Add 20 μL of the sample cooled to 4 °C after denaturation to each well, and then add 20 μL of Assay buffer to each well, and incubate with shaking at a frequency of 300 times / minute at 25 °C for 5 minutes.

[0432] e. Dilution of CSPD substrate (prepare it immediately before use and carry out in the dark)

[0433] Reagent name Volume ratio CSPD substrate 1 1×Reaction buffer 19

[0434] Vortex and mix well for later use.

[0435] f. Luminescence reaction

[0436] Add 20 μL of CSPD substrate diluent to each well and incubate with shaking at 25 °C and a shaking frequency of 300 times / min for 5 minutes.

[0437] Measure with an Envision

[0438] Chemiluminescence method: (Detection type: Luminescence; Distance between the plate and the detector: 0.1 mm; Detection time: 0.1 s)

[0439] h. Result calculation

[0440] Calculate the gene expression inhibition rate according to the light intensity value detected by the instrument. The calculation formula is: Inhibition rate % = Average (drug administration group) / Average (blank group) * 100%. The results are shown in Table 7.

[0441] Yang ginseng is AD-81725 from CN114616331

[0442] siRNA ID SS AS AD-81725 gsgsucagAfuCfUfUfcaaaauaaaaL96 usUfsuuaUfuUfUfgaagAfuCfugaccsasu

[0443] Table 7 Inhibition rate of siRNA conjugate on hLECT2

[0444]

[0445]

[0446] It can be concluded from this experiment that the siRNA of the present application has high inhibitory activity on the hLECT2 gene in vivo, can reduce the hLECT2 protein level for a long time. At a drug administration dose of 3 mpk, the inhibition rate of N-ER-FY037023M6L96 is above 60% during the 35-day test period.

[0447] Table 8 Plate layout

[0448] 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 A B C D E F G H I J K L M N O P

[0449] Example 6 Silencing effect of siRNA conjugate in mice expressing human LECT2 (hLECT2) gene

[0450] Refer to the steps described in Example 5, and administer a single dose of 3 mpk or 10 mpk of the siRNA conjugate of the present application (as shown by the siRNA ID in the following table) to mice subcutaneously. Detect the SEAP protein expression level (i.e., the hLECT2 protein expression level) and calculate the inhibition rate on the 7th, 14th, 21st, 28th, 35th, 42nd, 49th, 56th, 63rd, and 70th days after administration. The results are shown in Table 9.

[0451] Table 9 Inhibition rate of siRNA conjugate on hLECT2

[0452]

[0453] It can be concluded from this experiment that the siRNA conjugate of the present application has high inhibitory activity against the hLECT2 gene in vivo, can reduce the hLECT2 protein level for a long time, and at a dosing dose of 10 mpk, N-ER-FY037023M6L96 has an inhibition rate of about 75% or more during the 70-day test period.

[0454] Example 7 Inhibitory Effect of siRNA Conjugate on LECT2 Gene Expression in Wild-Type C57BL / 6 Mice

[0455] Wild-type C57BL / 6 male mice aged 6 - 8 weeks (Beijing Vital River Laboratory Animal Technology Co., Ltd.). After 7 days of adaptive feeding, they were randomly grouped by body weight, with 5 mice in each group. A single dose of 3 mpk of the siRNA conjugate (as shown by the siRNA ID in the following table) and PBS (purchased from Gibco, catalog number 10010 - 023) were administered subcutaneously, with an injection volume of 5 μL / g. The mice were euthanized 2 or 5 weeks after dosing. Two 30-mg samples of the left middle lobe tissue of the mouse liver were collected and immediately frozen in liquid nitrogen. After being ground into a tissue homogenate by freezing, tissue RNA was extracted and the expression of the target gene mRNA was detected. The inhibition rate results are shown in Table 10.

[0456] 1. Experimental reagents:

[0457] RNA extraction kit, purchased from QIAGEN, catalog number: 74106;

[0458] Reverse transcription kit, purchased from Vazyme, catalog number R312 - 02;

[0459] Target LECT2 probe, purchased from Thermo, catalog number Mm00521920_m1 (FAM);

[0460] ACTB probe, purchased from Thermo, catalog number Mm02619580_g1 (VIC);

[0461] TaqMan TM Gene expression premix, purchased from Applied Biosystems, catalog number 4369016.

[0462] 2. Experimental procedures:

[0463] 2.1. Tissue homogenate

[0464] 1) Take the right half of the left lobe tissue of the mouse liver and add 1 mL of lysis buffer;

[0465] 2) Homogenize at 60 Hz for 30 s, pause for 15 s, repeat 10 times, and centrifuge briefly.

[0466] 3) Supplement the remaining RLT (lysis buffer) according to the liver weight to a uniform concentration; vortex to mix well.

[0467] 2.2. RNA Extraction

[0468] 1) Take 350 μL of tissue lysis buffer, add 350 μL of 70% ethanol, and vortex to mix well.

[0469] 2) Transfer 700 μL of the sample mixture to a microcolumn, install the collection tube. Centrifuge at ≥8000 xg for 15 s, and discard the effluent.

[0470] 3) Add 700 μL of RW1 buffer to the microcolumn, close the lid, centrifuge at ≥8000 xg for 15 s, and discard the effluent.

[0471] 4) Add 500 μL of RPE buffer to the microcolumn, close the lid, centrifuge at ≥8000 xg for 15 s, and discard the effluent.

[0472] 5) Add 500 μL of RPE buffer to the microcolumn, close the lid, centrifuge at ≥8000 xg for 2 minutes, and discard the effluent. Install a new collection tube and centrifuge at ≥8000 xg for 1 minute without sample.

[0473] 6) Install the microcolumn into a 1.5 mL collection tube, add 30 μL of RNase-free water, close the lid, and centrifuge at ≥8000 xg for 1 minute.

[0474] 2.3. Reverse Transcription (RNA to cDNA)

[0475] 1) Take 400 ng of RNA for reverse transcription;

[0476] 2) Prepare the first-step reverse transcription reaction mixture as described in the following table and mix well. During the whole operation, all reagents are placed on ice;

[0477] 5×gDNA wiper Mix 2μL Total RNA 400ng <![CDATA[RNase-free ddH2O]]> Make up to 10μL

[0478] 3) Incubate at 42 °C for 2 min;

[0479] 4) Prepare the second-step reverse transcription reaction mixture as described in the following table and mix well;

[0480] The mixture from the previous step 10μL 10×RT Mix 2μL HiScriptⅢ Enzyme Mix 2μL <![CDATA[Oligo(dT) 20 VN]]> 1μL Random hexamers 1μL <![CDATA[RNase-free ddH2O]]> 4μL

[0481] 5) Second-step reverse transcription system

[0482] 50℃, 15min 85℃,5s 4℃

[0483] 3. Amplification and Analysis

[0484] 1) Prepare the qPCR reaction mixture as shown in the following table. Throughout the operation, all reagents are placed on ice.

[0485]

[0486]

[0487]

[0488] 2) Perform the qPCR program as follows:

[0489]

[0490] 3) Data Analysis

[0491] Ct is automatically calculated according to the default settings of the Quant Studio 6Flex software. Export the Ct values to an Excel file

[0492] Calculate the relative expression level of the gene using the following formula:

[0493] ΔCt = Ct(LECT2 gene) - Ct(ACTB)

[0494] ΔΔCt = ΔCt(test sample group) - ΔCt(Mock group)

[0495] mRNA expression relative to the Mock group = 2 -ΔΔCt .

[0496] Mock group: The group without siRNA added compared to the test sample group.

[0497] Inhibition rate (%) = (Mock group mRNA relative expression - Test sample group mRNA relative expression) / Mock group mRNA relative expression × 100%

[0498] Table 10 Inhibition rate of siRNA conjugate on LECT2

[0499] siRNA ID D14(％) PBS group 0.00 N-ER-FY037023M6L96(3mpk) 92.95 N-ER-FY037023M8L96(3mpk) 85.05 N-ER-FY037023M11L96(3mpk) 85.85 N-ER-FY037023M44L96(3mpk) 94.75 N-ER-FY037023M46L96(3mpk) 94.81 N-ER-FY037023M50L96(3mpk) 94.17 N-ER-FY037066M6L96(3mpk) 92.65 N-ER-FY037088M6L96(3mpk) 94.99 N-ER-FY037088M44L96(3mpk) 98.29 N-ER-FY037088M46L96(3mpk) 97.72 N-ER-FY037088M50L96(3mpk) 97.70

[0500] It can be concluded from this experiment that, compared with the PBS control group, the siRNA conjugate of the present application has a high inhibitory activity against the LECT2 gene and can reduce the hLECT2 protein level for a long time. Among them, N-ER-FY037023M44L96, N-ER-FY037023M46L96, N-ER-FY037023M50L96, N-ER-FY037088M44L96, N-ER-FY037088M46L96, N-ER-FY037088M50L96 can achieve an inhibition rate of more than about 95% against the LECT2 gene at a dose of 3mpk on D14.

[0501] Example 8 In vitro Stability Experiment of siRNA Conjugate in Rat Liver Homogenate

[0502] 1. Experimental Reagents and Consumables

[0503]

[0504]

[0505] 2. Experimental Procedures

[0506] 2.1 Preparation of Liver Homogenate

[0507] 2.1.1 Preparation of Grinding Solution

[0508] Reagent name Preparation method 5mM EDTA solution Take 1mL of 0.5M EDTA into a 100mL volumetric flask, make up to the mark with purified water, and shake well 100 mM Magnesium Chloride Solution Take 1 mL of 1 M magnesium chloride into a 10 mL volumetric flask, make up the volume to the mark with purified water, and shake well Grinding Solution 1 mL of EDTA solution + 1 mL of magnesium chloride solution + 98 mL of purified water, adjust pH = 6.0

[0509] 2.1.2 Tissue Homogenization

[0510] The rat liver tissue (collected from SD rats, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) and the grinding solution were prepared into a liver homogenate at a ratio of 100 mg: 5 mL (concentration: 20 mg / mL); after preparation, grinding beads were added and placed in a homogenizer, and the grinding parameters were set as follows.

[0511] Running Speed 60 Hz Running Time 30s Pause Time 15s Number of Runs 4 times Running Temperature -20℃

[0512] 2.2 Sample Preparation

[0513] The siRNA conjugate sample was prepared into a 1 mg / mL solution with enzyme-free water for later use. The internal standard sample was prepared into a concentration of 0.125 mg / mL with enzyme-free water.

[0514] 2.3 Sample Incubation

[0515] (1) Add 250 μL of the prepared liver homogenate to a 2 mL enzyme-free tube,

[0516] (2) On the basis of step (1), add 50 μL of the nucleic acid sample;

[0517] (3) The system is 300 μL of biological sample, vortexed and left standing for 5 min;

[0518] (4) Divide into 2 tubes, 100 μL per tube;

[0519] (5) Incubation time of the system: 48 h.

[0520] 2.4 Biological sample processing

[0521] Vortex and mix each 100 μL of biological sample system, add 300 μL of clarity OTX lysis buffer, vortex, leave standing for 30 min, add 100 μL of internal standard solution, vortex, leave standing for 5 min, centrifuge for 1 min, and set aside for use (total sample volume is about 500 μL).

[0522] 2.5 Solid-phase extraction:

[0523] (1) Preparation of solid-phase extraction reagents

[0524] Activator: Take 200 mL of methanol and add it to the mobile phase bottle, label it as activator;

[0525] Equilibration solution: Prepare 1 M phosphate buffer solution [877 mL of sodium dihydrogen phosphate (1.56 g / L) + 123 mL of disodium hydrogen phosphate (3.58 g / L)], dilute 100 times, adjust the pH to 5.5 with phosphoric acid, and label it as equilibration solution;

[0526] Rinse solution: Take 500 mL of equilibration solution and add it to a 1 L mobile phase bottle, add 500 mL of acetonitrile, adjust the pH to 5.5 with phosphoric acid, mix well, and label it as rinse solution;

[0527] Elution solution: Weigh 7.9 g of ammonium bicarbonate and add it to a 1 L mobile phase bottle, add 1 L of water, take 500 mL of ammonium bicarbonate solution and add it to a 1 L mobile phase bottle, add 500 mL of acetonitrile, adjust the pH = 9 with sodium hydroxide, mix well, and label it as elution solution;

[0528] (2) The extraction steps are as follows:

[0529] Step Process Activation 1 mL of methanol, 10 min Equilibration 2 * 1 mL of 10 mM phosphate (pH = 5.5), 10 min for the first time, 10 min for the second time Sample Loading 400 μL (4 / 5 of the total volume) Rinsing 2 * 1 mL of 10 mM phosphate (pH = 5.5): 50% acetonitrile, 20 min for the first time, 20 min for the second time Elution 2 * 0.75 mL of 100 mM ammonium bicarbonate (pH = 9): 50% acetonitrile, 20 min for the first time, 20 min for the second time

[0530] 2.6 Post-treatment

[0531] Take the elution solution (take it in two times, 600 mL each time, a total of 1200 mL) and place it in a 2 mL EP tube, vacuum concentrate for 6 hours at a speed of 1800 rpm; add 100 μL of mobile phase (initial ratio) to the concentrated sample for reconstitution, centrifuge at low speed for 2 min, take 10 μL of the supernatant and inject it into a high-resolution mass spectrometer. The LC-MS / MS method is used for semi-quantitative detection of the antisense strand ratio of the siRNA conjugate of this application. The metabolic results after in vitro incubation for 48 hours in rat liver homogenates at different concentrations are shown in Table 11 below.

[0532] Table 11 Percentage of remaining antisense strand of siRNA conjugate

[0533] siRNA ID AS% N-ER-FY037023M44L96 99.75 N-ER-FY037023M46L96 99.73 N-ER-FY037023M50L96 99.91 N-ER-FY037088M44L96 94.02 N-ER-FY037088M46L96 97.52 N-ER-FY037088M50L96 97.17

[0534] Wherein, AS represents the antisense strand of siRNA. The greater the remaining amount of AS, the better the stability of the drug and the better the long-acting property of the drug. It can be seen from Table 11 that the siRNA conjugates of the present disclosure, N-ER-FY037023M44L96, N-ER-FY037023M46L96, N-ER-FY037023M50L96, N-ER-FY037088M44L96, N-ER-FY037088M46L96, N-ER-FY037088M50L96 have excellent in vitro stability in rat liver homogenate.

[0535] The above-mentioned embodiments of the present disclosure are only examples for clearly illustrating the present disclosure, rather than limiting the implementation manners of the present disclosure. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation manners here. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present disclosure shall be included in the protection scope of the claims of the present disclosure.

[0536] References:

[0537] 1. Takata Noboru, et al. LECT2 as a hepatokine links liver steatosis to inflammation via activating tissue macrophages in NASH[J]. Scientific Reports 11.1(2021). doi:10.1038 / S41598-020-80689-0.

[0539] 2. Merrill D. Benson. LECT2 amyloidosis[J]. Kidney International(2010)77, 757-759. doi:10.1038 / ki.2010.18.

[0540] 3. Tae Woo Jung, Yoon Hee Chung, Hyoung-Chun Kim, et al. LECT2 promotes inflammation and insulin resistance in adipocytes via P38 pathways. [J]. Journal of molecular endocrinology, 2018, 61(1).

[0541] 4. Honghai Xu, Xutong Li, Zihao Wu, et al. LECT2, A Novel and Direct Biomarker of Liver Fibrosis in Patients With CHB [J]. Frontiers in Molecular Biosciences, 2021, 8.

[0542] 5. Yuan Xie, KaiWei Fan, ShiXing Guan, et al. LECT2: A pleiotropic and promising hepatokine, from bench to bedside. [J]. Journal of cellular and molecular medicine, 2022, 26(13).

Claims

1. An siRNA for inhibiting LECT2 gene expression, the siRNA comprising a sense strand and an antisense strand, wherein each nucleotide in the siRNA is independently a modified or unmodified nucleotide, wherein the sense strand comprises a nucleotide sequence I, and the antisense strand comprises a nucleotide sequence II, wherein the nucleotide sequence I and the nucleotide sequence II are at least partially reverse-complemented to form a double-stranded region, wherein the nucleotide sequence I and the nucleotide sequence II are selected from the following sequences: (1) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 228, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 229: 5'-GAAUAUUCUUCA-3'(SEQ ID NO:228) 5'-UGAAGAAUAUUC-3' (SEQ ID NO: 229); (2) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 230, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 231: 5'-CAACUCUAAUCAGAGGA-3'(SEQ ID NO:230) 5'-UCCUCUGAUUAGAGUUG-3' (SEQ ID NO: 231); (3) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 232, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 233: 5'-CAAUGAGAUCCGGACGU-3'(SEQ ID NO:232) 5'-ACGUCCGGAUCUCAUUG-3' (SEQ ID NO: 233); (4) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 234, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 235: 5'-ACCGCCAUGGCUGUGGA-3'(SEQ ID NO:234) 5'-UCCACAGCCAUGGCGGU-3' (SEQ ID NO: 235); (5) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 236, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 237: 5'-CAUAAUGGU-3'(SEQ ID NO:236) 5'-ACCAUUAUUG-3' (SEQ ID NO:237); (6) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 238, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 239: 5'-GUGUUCGAAUAUCU-3'(SEQ ID NO:238) 5'-AGAUAUUCGAACAC-3' (SEQ ID NO: 239); (7) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 240, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 241: 5'-AAGAGGUUUUUGUG-3'(SEQ ID NO:240) 5'-CACAAAAACCUCUU-3' (SEQ ID NO: 241); (8) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 242, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 243: 5'-UCCUAUUAAGAAGGGA-3'(SEQ ID NO:242) 5'-UCCCUUCUUAAUAGGA-3' (SEQ ID NO: 243); (9) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 244, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 245: 5'-UAUUGCCCUU-3'(SEQ ID NO:244) 5'-AAGGGCAUA-3' (SEQ ID NO: 245); (10) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 246, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 247: 5'-GUUUUACCUG-3'(SEQ ID NO:246) 5'-CAGGAUAAAC-3' (SEQ ID NO: 247); (11) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 248, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 249: 5'-CAUACAAUCGCAUGUG-3'(SEQ ID NO:248) 5'-CACAUGCGAUUGUAUG-3' (SEQ ID NO: 249); (12) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 250, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 251: 5'-CUCGAGUGACCCUACUG-3'(SEQ ID NO:250) 5'-CAGUAGGGUCACUCGAG-3' (SEQ ID NO: 251); (13) the nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 1, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 2; (14) the nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 20, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 21; (15) the nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO: 22, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO: 23; (16) the nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:34, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:35; (17) the nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:36, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:37; (18) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:224, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:

225.

2. The siRNA according to claim 1, wherein the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, essentially reverse complementary or completely reverse complementary; the substantially reverse complementary means that there are no more than 3 base mismatches between the two nucleotide sequences; the substantially reverse complementary means that there are no more than 1 base mismatch between the two nucleotide sequences; and the completely reverse complementary means that there is no mismatch between the two nucleotide sequences.

3. The siRNA according to claim 1 or 2, wherein the sense strand further contains a nucleotide sequence III, and the antisense strand further contains a nucleotide sequence IV, and the lengths of the nucleotide sequence III and the nucleotide sequence IV are each independently 0-11 nucleotides, wherein the nucleotide sequence III is connected to the 5' end of the nucleotide sequence I, and the nucleotide sequence IV is connected to the 3' end of the nucleotide sequence II, and 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 complementary means that there is no more than one base mismatch between the two nucleotide sequences; and the completely reverse complementary means that there is no mismatch between the two nucleotide sequences; and / or, the nucleotide sequence III is connected to the 3' end of the nucleotide sequence I, and the nucleotide sequence IV is connected to the 5' end of the nucleotide sequence II, and the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary; the substantially reverse complementary means that there is no more than one base mismatch between the two nucleotide sequences; and the completely reverse complementary means that there is no mismatch between the two nucleotide sequences.

4. The siRNA according to any one of claims 1 to 3, wherein the siRNA comprises 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 sequences I and III, and the antisense strand contains nucleotide sequences II and IV, and the nucleotide sequences I and III are at least partially reverse complementary to the nucleotide sequences II and IV to form a double-stranded region, wherein the nucleotide sequences I and III, the nucleotide sequences II and IV are selected from the following sequences: (1) the nucleotide sequences I and III comprise or consist of the nucleotide sequence shown in SEQ ID NO: 124, and the nucleotide sequences II and IV comprise or consist of the nucleotide sequence shown in SEQ ID NO: 125; (2) The nucleotide sequences I and III comprise or consist of the nucleotide sequence shown in SEQ ID NO: 343, and the nucleotide sequences II and IV comprise or consist of the nucleotide sequence shown in SEQ ID NO:

371.

5. The siRNA according to any one of claims 1 to 4, wherein the sense strand further comprises a nucleotide sequence V and / or the antisense strand further comprises a nucleotide sequence VI, the length of the nucleotide sequences V and VI is 0 to 3 nucleotides, the nucleotide sequence V is connected to the 3' end of the sense strand to form a 3' overhang of the sense strand and / or the nucleotide sequence VI is connected to the 3' end of the antisense strand to form a 3' overhang of the antisense strand; preferably, the length of the nucleotide sequence V or VI is 2 nucleotides; more preferably, the nucleotide sequence V or VI is two consecutive thymine deoxyribonucleotides or two consecutive uracil ribonucleotides; Alternatively, the nucleotide sequence V or VI is mismatched or complementary to the nucleotide at the corresponding position of the target mRNA.

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

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

8. The siRNA according to any one of claims 1 to 7, wherein at least one nucleotide in the sense strand or the antisense strand is a modified nucleotide, and / or at least one phosphate group is a phosphate group having a modified group; preferably, the phosphate group having a modified group is a thiophosphate group formed by replacing one oxygen atom in the phosphodiester bond in the phosphate group with a sulfur atom; and / or, the siRNA comprises a sense strand that does not include a 3' overhanging nucleotide.

9. The siRNA according to any one of claims 1 to 8, wherein The 5' terminal nucleotide of the antisense strand is linked to a 5' phosphate group or a 5' phosphate-derivative group, or the 5' terminal nucleotide of the antisense strand is not linked to a 5' phosphate group or a 5' phosphate-derivative group.

10. The siRNA according to any one of claims 1 to 9, wherein The 3' terminal nucleotide of the sense strand is linked to an inverted abasic deoxyribose residue, and / or the 5' terminal nucleotide of the sense strand is linked to an inverted abasic deoxyribose residue.

11. The siRNA according to any one of claims 1 to 10, wherein the modified nucleotides are selected from 2'-fluoro-modified nucleotides, 2'-alkoxy-modified nucleotides, 2'-substituted alkoxy-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-amino-modified nucleotides, 2'-substituted amino-modified nucleotides, 2'-deoxyribonucleotides, nucleotide analogs or a combination of any two or more thereof; Preferably, the modified nucleotide is selected from 2'-fluoro modified nucleotides, 2'-methoxy modified nucleotides, 2'-O-CH2-CH2-O-CH3 modified nucleotides, 2'-O-CH2-CH=CH2 modified nucleotides, 2'-CH2-CH2-CH=CH2 modified nucleotides, 2'-deoxyribonucleotides, nucleotide analogs or a combination of any two or more thereof.

12. The siRNA according to any one of claims 1 to 11, wherein each nucleotide in the sense strand and the antisense strand is independently a 2'-fluoro-modified nucleotide or a non-fluoro-modified nucleotide; Preferably, from 5' to 3', the 2'-fluorinated modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand, and the remaining positions are non-fluorinated modified nucleotides; from 5' to 3', 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; Alternatively, from 5' to 3', the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand, and the remaining positions are non-fluoro-modified nucleotides; from 5' to 3', the 2'-fluoro-modified nucleotides are located at positions 2, 6, 8, 9, 14 and 16 of the antisense strand, and the remaining positions are non-fluoro-modified nucleotides; Alternatively, from 5' to 3', the 2'-fluoro-modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand, and the remaining positions are non-fluoro-modified nucleotides; from 5' to 3', the 2'-fluoro-modified nucleotides are located at positions 2, 14 and 16 of the antisense strand, and the remaining positions are non-fluoro-modified nucleotides; Alternatively, from 5' to 3', the 2'-fluoro-modified nucleotides are located at positions 5, 7, 8 and 9 of the sense strand, and the remaining positions are non-fluoro-modified nucleotides; from 5' to 3', the 2'-fluoro-modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and the remaining positions are non-fluoro-modified nucleotides; Alternatively, from 3' to 5', the 2'-fluorinated modified nucleotides are located at positions 9, 10, 11 and 13 of the sense strand, and the remaining positions are non-fluorinated modified nucleotides; from 5' to 3', the 2'-fluorinated modified nucleotides are located at positions 2, 3, 4, 5, 7, 10 and 14 of the antisense strand, and the remaining positions are non-fluorinated modified nucleotides; Alternatively, from 3' to 5', the 2'-fluorinated modified nucleotides are located at positions 11, 12, 13 and 17 of the sense strand, and the remaining positions are non-fluorinated modified nucleotides; from 5' to 3', 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; Alternatively, from 3' to 5', the 2'-fluorinated modified nucleotides are located at positions 9, 11, 13 and 17 of the sense strand, and the remaining positions are non-fluorinated modified nucleotides; from 5' to 3', 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; Alternatively, from 5' to 3', the 2'-fluorinated modified nucleotides are located at positions 9, 11, 12 and 13 of the sense strand, and the remaining positions are non-fluorinated modified nucleotides; from 5' to 3', 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; Alternatively, from 3' to 5', the 2'-fluoro-modified nucleotides are located at positions 9, 10, 11 and 13 of the sense strand, and the remaining positions are non-fluoro-modified nucleotides; from 5' to 3', the 2'-fluoro-modified nucleotides are located at positions 2, 7, 10 and 14 of the antisense strand, and the remaining positions are non-fluoro-modified nucleotides; Alternatively, from 3' to 5', the 2'-fluoro-modified nucleotides are located at positions 9, 10, 11 and 13 of the sense strand, and the remaining positions are non-fluoro-modified nucleotides; from 5' to 3', the 2'-fluoro-modified nucleotides are located at positions 2, 5, 7 and 14 of the antisense strand, and the remaining positions are non-fluoro-modified nucleotides; Alternatively, from 3' to 5', the 2'-fluorinated modified nucleotides are located at positions 11, 12, 13 and 17 of the sense strand, and the remaining positions are non-fluorinated modified nucleotides; from 5' to 3', the 2'-fluorinated modified nucleotides are located at positions 2, 7, 10 and 14 of the antisense strand, and the remaining positions are non-fluorinated modified nucleotides; Alternatively, in the 3' to 5' direction, the 2'-fluoro-modified nucleotides are located at positions 9, 11, 13 and 17 of the sense strand, and the remaining positions are non-fluoro-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 7, 10 and 14 of the antisense strand, and the remaining positions are non-fluoro-modified nucleotides; Alternatively, in the 3' to 5' direction, the 2'-fluoro-modified nucleotides are located at positions 6, 11 and 13 of the sense strand, and the remaining positions are non-fluoro-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 7, 10 and 14 of the antisense strand, and the remaining positions are non-fluoro-modified nucleotides; Alternatively, from 3' to 5', the 2'-fluorinated modified nucleotides are located at positions 9, 11, 13 and 17 of the sense strand, and the remaining positions are non-fluorinated modified nucleotides; from 5' to 3', the 2'-fluorinated modified nucleotides are located at positions 2, 3, 5, 7, 10, 12 and 14 of the antisense strand, and the remaining positions are non-fluorinated modified nucleotides; Alternatively, in the 3' to 5' direction, the 2'-fluoro-modified nucleotides are located at positions 9, 11, 13 and 17 of the sense strand, and the remaining positions are non-fluoro-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 7 and 14 of the antisense strand, and the remaining positions are non-fluoro-modified nucleotides; Further preferably, each non-fluorinated modified nucleotide is a 2'-methoxy modified nucleotide, wherein 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.

13. The siRNA according to claim 12, wherein each non-fluorinated modified nucleotide is independently selected from a nucleotide or nucleotide analogue formed by replacing the hydroxyl group at the 2' position of the ribose group of the nucleotide with a non-fluorinated group, and the nucleotide analogue is selected from one of pseudouracil, isonucleotide, LNA, ENA, cET BNA, UNA and GNA.

14. The siRNA according to any one of claims 1 to 13, wherein each nucleotide in the sense strand and the antisense strand is independently a 2'-fluoro-modified nucleotide, a 2'-methoxy-modified nucleotide, a GNA-modified nucleotide, a 2'-deoxyribonucleotide, or a combination of any two or more thereof; Preferably, from 5' to 3' direction, the 2'-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides; from 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; Alternatively, from 5' to 3', the 2'-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides; from 5' to 3', the 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; Alternatively, from 5' to 3', the 2'-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides; from 5' to 3', the 2'-fluoro modified nucleotides are located at positions 2, 14 and 16 of the antisense strand, the GNA modified nucleotide is located at position 6 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides; Alternatively, from 5' to 3', the 2'-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides; from 5' to 3', the 2'-fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, the GNA modified nucleotide is located at position 7 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides; Alternatively, in the 5' to 3' direction, the 2'-fluoro modified nucleotides are located at positions 5, 7, 8 and 9 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides; 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; Alternatively, from 3' to 5', the 2'-fluoro modified nucleotides are located at positions 9, 10, 11 and 13 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides; from 5' to 3', the 2'-fluoro modified nucleotides are located at positions 2, 3, 4, 5, 7, 10 and 14 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides; Alternatively, in the 3' to 5' direction, the 2'-fluoro modified nucleotides are located at positions 11, 12, 13 and 17 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides; 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; Alternatively, in the 3' to 5' direction, the 2'-fluoro modified nucleotides are located at positions 9, 11, 13 and 17 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides; 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; Alternatively, from 3' to 5', the 2'-fluoro modified nucleotides are located at positions 9, 10, 11 and 13 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides; from 5' to 3', the 2'-fluoro modified nucleotides are located at positions 2, 3, 4, 5, 7, 10 and 14 of the antisense strand, the GNA modified nucleotide is located at position 6 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides; Alternatively, in the 5' to 3' direction, the 2'-fluoro modified nucleotides are located at positions 9, 11, 12 and 13 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides; 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; Alternatively, in the 3' to 5' direction, the 2'-fluoro modified nucleotides are located at positions 9, 10, 11 and 13 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides; in the 5' to 3' direction, the 2'-fluoro modified nucleotides are located at positions 2, 7, 10 and 14 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides; Alternatively, in the 3' to 5' direction, the 2'-fluoro modified nucleotides are located at positions 9, 10, 11 and 13 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides; in the 5' to 3' direction, the 2'-fluoro modified nucleotides are located at positions 2, 5, 7 and 14 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides; Alternatively, in the 3' to 5' direction, the 2'-fluoro modified nucleotides are located at positions 11, 12, 13 and 17 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides; in the 5' to 3' direction, the 2'-fluoro modified nucleotides are located at positions 2, 7, 10 and 14 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides; Alternatively, from 3' to 5', the 2'-fluoro modified nucleotides are located at positions 9, 11, 13 and 17 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides; from 5' to 3', the 2'-fluoro modified nucleotides are located at positions 2, 7, 10 and 14 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides; Alternatively, in the 3' to 5' direction, the 2'-fluoro modified nucleotides are located at positions 6, 11 and 13 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides; in the 5' to 3' direction, the 2'-fluoro modified nucleotides are located at positions 2, 7, 10 and 14 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides; Alternatively, from 3' to 5', the 2'-fluoro modified nucleotides are located at positions 9, 11, 13 and 17 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides; from 5' to 3', the 2'-fluoro modified nucleotides are located at positions 2, 3, 5, 7, 10, 12 and 14 of the antisense strand, the GNA modified nucleotide is located at position 6 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides; Alternatively, in the 3' to 5' direction, the 2'-fluoro-modified nucleotides are located at positions 9, 11, 13 and 17 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 7 and 14 of the antisense strand, the 2'-deoxyribonucleotides are located at positions 5 and 12 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides.

15. The siRNA according to any one of claims 1 to 14, in the direction from the 5' end to the 3' end, (1) The sense strand comprises a phosphorothioate group located at the following position: Between the first nucleotide and the second nucleotide starting from the 5' end of the sense strand; and Between the second nucleotide and the third nucleotide starting from the 5' end of the sense strand; and Between the first nucleotide and the second nucleotide starting from the 3' end of the sense strand; and Between the second nucleotide and the third nucleotide starting from the 3' end of the sense strand; or, (2) The sense strand comprises a phosphorothioate group located at the following position: Between the first nucleotide and the second nucleotide starting from the 5' end of the sense strand; and Between the second nucleotide and the third nucleotide starting from the 5' end of the sense strand; or, (3) The sense strand comprises a phosphorothioate group located at the following position: Between the first nucleotide and the second nucleotide starting from the 5' end of the sense strand; and Between the second nucleotide and the third nucleotide starting from the 5' end of the sense strand; and between the reverse abasic deoxyribose residue starting from the 3' end of the sense strand and the first nucleotide; or, (4) The sense strand comprises a phosphorothioate group located at the following position: between the reverse abasic deoxyribose residue starting from the 5' end of the sense strand and the first nucleotide; and Between the first nucleotide and the second nucleotide starting from the 5' end of the sense strand; and The positive strand is located between the inverted abasic deoxyribose residue starting from the 3' end and the first nucleotide.

16. The siRNA according to any one of claims 1 to 15, wherein the antisense strand comprises a phosphorothioate group located at the following positions in the direction from the 5' end to the 3' end: Between the first nucleotide and the second nucleotide starting from the 5' end of the antisense strand; and Between the second nucleotide and the third nucleotide starting from the 5' end of the antisense strand; and Between the first nucleotide and the second nucleotide starting from the 3' end of the antisense strand; and The antisense strand is located between the second nucleotide and the third nucleotide starting from the 3' end.

17. The siRNA according to any one of claims 1 to 16, wherein each nucleotide in the sense strand and the antisense strand is independently a 2'-fluoro-modified nucleotide, a 2'-methoxy-modified nucleotide, a GNA-modified nucleotide, a 2'-deoxyribonucleotide or a combination of any two or more thereof; Preferably, in the 5' to 3' direction, the 2'-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand, the remaining positions are 2'-methoxy modified nucleotides, and the 3' end is free of overhangs; in the 5' to 3' direction, the 2'-fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, the remaining positions are 2'-methoxy modified nucleotides, and the 5' terminal nucleotide of the antisense strand is connected to a 5' phosphate group; Alternatively, from 5' to 3', the 2'-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides; from 5' to 3', 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, and the 5' terminal nucleotide of the antisense strand is connected to a 5' phosphate group; Alternatively, from 5' to 3', the 2'-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides; from 5' to 3', the 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, and the 5' terminal nucleotide of the antisense strand is connected to a 5' phosphate group; Alternatively, from 5' to 3', the 2'-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides; from 5' to 3', the 2'-fluoro modified nucleotides are located at positions 2, 14 and 16 of the antisense strand, the GNA modified nucleotide is located at position 6 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, and the 5' terminal nucleotide of the antisense strand is connected to a 5' phosphate group; Alternatively, from 5' to 3', the 2'-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides; from 5' to 3', the 2'-fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, the GNA modified nucleotide is located at position 7 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, and the 5' terminal nucleotide of the antisense strand is connected to a 5' phosphate group; Alternatively, from 5' to 3', the 2'-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides; from 5' to 3', 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, and the 5' terminal nucleotide of the antisense strand is not connected to a 5' phosphate group or a 5' phosphate derivative group; Alternatively, from 5' to 3', the 2'-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides; from 5' to 3', the 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, and the 5' terminal nucleotide of the antisense strand is not connected to a 5' phosphate group or a 5' phosphate derivative group; Alternatively, in the 5' to 3' direction, the 2'-fluoro modified nucleotides are located at positions 7, 9, 10 and 11 of the sense strand, the remaining positions are 2'-methoxy modified nucleotides, and the 3' end is removed from the overhang; in the 5' to 3' direction, the 2'-fluoro modified nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, the remaining positions are 2'-methoxy modified nucleotides, and the 5' terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group; Alternatively, from 5' to 3', the 2'-fluoro modified nucleotides are located at positions 5, 7, 8 and 9 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides; from 5' to 3', 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, and the 5' terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group; Alternatively, from 3' to 5', the 2'-fluoro modified nucleotides are located at positions 9, 10, 11 and 13 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides; from 5' to 3', the 2'-fluoro modified nucleotides are located at positions 2, 3, 4, 5, 7, 10 and 14 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, and the 5' terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group; Alternatively, in the 3' to 5' direction, the 2'-fluoro modified nucleotides are located at positions 11, 12, 13 and 17 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides; 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 first and second nucleotides at the 5' end, and the 5' terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group; Alternatively, in the 3' to 5' direction, the 2'-fluoro modified nucleotides are located at positions 9, 11, 13 and 17 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides; 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, and the 5' terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group; Alternatively, from 3' to 5', the 2'-fluoro modified nucleotides are located at positions 9, 10, 11 and 13 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides; from 5' to 3', the 2'-fluoro modified nucleotides are located at positions 2, 3, 4, 5, 7, 10 and 14 of the antisense strand, the GNA modified nucleotide is located at position 6 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, and the 5' terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group; Alternatively, from 5' to 3', the 2'-fluoro modified nucleotides are located at positions 9, 11, 12 and 13 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides; from 5' to 3', 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, and the 5' terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group; Alternatively, in the 3' to 5' direction, the 2'-fluoro modified nucleotides are located at positions 9, 10, 11 and 13 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides; in the 5' to 3' direction, the 2'-fluoro modified nucleotides are located at positions 2, 7, 10 and 14 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, and the 5' terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group; Alternatively, in the 3' to 5' direction, the 2'-fluoro modified nucleotides are located at positions 9, 10, 11 and 13 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides; in the 5' to 3' direction, the 2'-fluoro modified nucleotides are located at positions 2, 5, 7 and 14 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, and the 5' terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group; Alternatively, in the 3' to 5' direction, the 2'-fluoro modified nucleotides are located at positions 11, 12, 13 and 17 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides; in the 5' to 3' direction, the 2'-fluoro modified nucleotides are located at positions 2, 7, 10 and 14 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, and the 5' terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group; Alternatively, in the 3' to 5' direction, the 2'-fluoro modified nucleotides are located at positions 9, 11, 13 and 17 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides; in the 5' to 3' direction, the 2'-fluoro modified nucleotides are located at positions 2, 7, 10 and 14 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, and the 5' terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group; Alternatively, in the 3' to 5' direction, the 2'-fluoro modified nucleotides are located at positions 6, 11 and 13 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides; in the 5' to 3' direction, the 2'-fluoro modified nucleotides are located at positions 2, 7, 10 and 14 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, and the 5' terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group; Alternatively, from 3' to 5', the 2'-fluoro modified nucleotides are located at positions 9, 11, 13 and 17 of the sense strand, and the remaining positions are 2'-methoxy modified nucleotides; from 5' to 3', the 2'-fluoro modified nucleotides are located at positions 2, 3, 5, 7, 10, 12 and 14 of the antisense strand, the GNA modified nucleotide is located at position 6 of the antisense strand, and the remaining positions are 2'-methoxy modified nucleotides, and the 5' terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group; Alternatively, in the 3' to 5' direction, the 2'-fluoro-modified nucleotides are located at positions 9, 11, 13 and 17 of the sense strand, and the remaining positions are 2'-methoxy-modified nucleotides; in the 5' to 3' direction, the 2'-fluoro-modified nucleotides are located at positions 2, 7 and 14 of the antisense strand, the 2'-deoxyribonucleotides are located at positions 5 and 12 of the antisense strand, and the remaining positions are 2'-methoxy-modified nucleotides, and the 5' terminal nucleotide of the antisense strand is connected to a 5'-trans vinyl phosphonate group.

18. The siRNA according to any one of claims 1 to 17, which is selected from the siRNA of Table 1; preferably, the siRNA is selected from N-ER-FY037090, N-ER-FY037023, N-ER-FY037090M2, N-ER-FY037023M2, N-ER-FY037090M3, N-ER-FY037090M4, N-ER-FY037090M5, N-ER-FY037090M6, N-ER-FY037023M6, N-ER -FY037090M7, N-ER-FY037023M8, N-ER-FY037023M11, N-ER-FY037023M15, N-ER-FY037023M27, N-ER-FY037023M29, N -ER-FY037023M35, N-ER-FY037023M37, N-ER-FY037023M40, N-ER-FY037023M44, N-ER-FY037023M45, N-ER-FY037023M 46. ​​N-ER-FY037023M47, N-ER-FY037023M48, N-ER-FY037023M49, N-ER-FY037023M50, N-ER-FY037023M51, N-ER-FY03 7088, N-ER-FY037088M6, N-ER-FY037088M8, N-ER-FY037088M15, N-ER-FY037088M27, N-ER-FY037088M29, N-ER-FY03 7088M35, N-ER-FY037088M36, N-ER-FY037088M37, N-ER-FY037088M40, N-ER-FY037088M44, N-ER-FY037088M45, N-ER -FY037088M46, N-ER-FY037088M47, N-ER-FY037088M48, N-ER-FY037088M49, N-ER-FY037088M50, N-ER-FY037088M51.

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

20. The siRNA conjugate according to claim 19, wherein the conjugated group comprises a pharmaceutically acceptable targeting group and a linker, and the siRNA, the linker and the targeting group are sequentially covalently or non-covalently linked; Preferably, in the siRNA conjugate, the sense strand and the antisense strand of the siRNA are complementary to each other to form a double-stranded region of the siRNA conjugate, and the 3' end of the sense strand forms a blunt end, and the 3' end of the antisense strand has 1-3 protruding nucleotides extending out of the double-stranded region; Alternatively, in the siRNA conjugate, the sense strand and the antisense strand of the siRNA are complementary to each other to form a double-stranded region of the siRNA conjugate, and the 3' end of the sense strand forms a blunt end, and the 3' end of the antisense strand forms a blunt end.

21. The siRNA conjugate according to claim 19 or 20, wherein the conjugated group is selected from:

22. The siRNA conjugate according to any one of claims 19 to 21, wherein the siRNA conjugate is a siRNA conjugate selected from Table 2; the siRNA conjugate is N-ER-FY037023M2L96, N-ER-FY037090M3L96, N-ER-FY037090M4L96, N-ER-FY037090M5L96, N-ER-FY0370 23M6L96, N-ER-FY037023M8L96, N-ER-FY037090M7L96, N-ER-FY037023M37L96, N-ER-FY0370 23M40L96, N-ER-FY037023M44L96, N-ER-FY037023M45L96, N-ER-FY037023M46L96, N-ER-FY03 7023M47L96, N-ER-FY037023M48L96, N-ER-FY037023M49L96, N-ER-FY037023M50L96, N-ER-F Y037023M51L96, N-ER-FY037088M6L96, N-ER-FY037088M37L96, N-ER-FY037088M40L96, N-ER- FY037088M44L96, N-ER-FY037088M45L96, N-ER-FY037088M46L96, N-ER-FY037088M47L96, N- ER-FY037088M48L96, N-ER-FY037088M49L96, N-ER-FY037088M50L96, N-ER-FY037088M51L96.

23. A pharmaceutical composition comprising the siRNA according to any one of claims 1 to 18, or the siRNA conjugate according to any one of claims 19 to 22, and a pharmaceutically acceptable carrier. 24 . A kit comprising the siRNA according to claim 1 , or the siRNA conjugate according to claim 19 , or the pharmaceutical composition according to claim 23 .

25. Use of the siRNA according to any one of claims 1 to 18, or the siRNA conjugate according to any one of claims 19 to 22, or the pharmaceutical composition according to claim 23 for preparing a medicament for inhibiting LECT2 gene expression.

26. Use of the siRNA according to any one of claims 1 to 18, or the siRNA conjugate according to any one of claims 19 to 22, or the pharmaceutical composition according to claim 23 for preparing a medicament for preventing and / or treating a disease associated with overexpression of the LECT2 gene.

27. The use according to claim 26, wherein the disease is liver damage, liver fibrosis, NASH, metabolic abnormalities, obesity, insulin resistance and systemic amyloidosis.

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