SiRNA for inhibiting expression of NR1H3 gene and application thereof

Abstract

The present application relates to siRNA for inhibiting NR1H3 gene expression and its modification and conjugate and application, belong to the field of biotechnology. The present application provides a kind of siRNA for inhibiting NR1H3 gene expression, its sense strand includes the first nucleotide sequence as shown in any one of SEQ ID NO.4, SEQ ID NO.15, SEQ ID NO.33 or SEQ ID NO.74-SEQ ID NO.80, its antisense strand includes the second nucleotide sequence as shown in any one of SEQ ID NO.40, SEQ ID NO.51, SEQ ID NO.70 or SEQ ID NO.81-87. Experiments have confirmed that the siRNA of the present application and its modification and conjugate are all higher inhibitory activity to NR1H3, therefore, in the preparation of drug for treating disease related to lipid metabolism has very application prospect.

Description

Technical Field

[0001] This invention relates to an siRNA for inhibiting NR1H3 gene expression and its application, belonging to the field of biotechnology. Background Technology

[0002] Nuclear receptors (NRs) are ligand-activated transcription factors that regulate reproduction, development, metabolism, cell growth, inflammation, and immunity by establishing connections between signaling molecules and transcriptional responses. The liver X receptor (LXR) is a well-known nucleosteroid receptor and a member of the NR family, existing in two homologous isoforms, LXRα (NR1H3) and LXRβ (NR1H2). These are key regulators of inflammation, autoimmunity, cholesterol, and lipid metabolism.

[0003] Specifically, NR1H3 (member 3 of the nuclear receptor subfamily 1H) is mainly expressed in the liver, intestine, adipose tissue, and macrophages, while NR1H2 is widely expressed in the liver, intestine, adrenal glands, brain, and testes, indicating different physiological functions. NR1H3 plays a crucial role in hepatic lipid metabolism, and abnormalities in hepatic lipid metabolism are associated with the development of various liver diseases or conditions, such as non-alcoholic fatty liver disease (NAFLD). Furthermore, NR1H3 plays a role in regulating the reverse cholesterol transport pathway, which can prevent lipid metabolism-related diseases such as dyslipidemia and atherosclerosis.

[0004] In summary, inhibiting the expression of the NR1H3 gene in patients can prevent and treat lipid metabolism-related diseases, such as dyslipidemia, atherosclerosis, and non-alcoholic fatty liver disease. Currently, there are no drugs on the market specifically targeting the regulation of this gene's expression; therefore, developing drugs targeting NR1H3 is of significant value. Summary of the Invention

[0005] To address the above problems, the present invention provides an siRNA for inhibiting NR1H3 gene expression, wherein the siRNA contains a sense strand and an antisense strand; the sense strand and the antisense strand are at least partially inversely complementary to form a double-stranded region;

[0006] The sense strand of the siRNA contains a first nucleotide sequence as shown in any one of SEQ ID NO.4, SEQ ID NO.15, SEQ ID NO.33, or SEQ ID NO.74 to SEQ ID NO.80;

[0007] The antisense strand of the siRNA contains a second nucleotide sequence as shown in any one of SEQ ID NO.40, SEQ ID NO.51, SEQ ID NO.70, or SEQ ID NO.81–87.

[0008] In one embodiment of the present invention, the sense strand of the siRNA comprises a first nucleotide sequence as shown in SEQ ID NO.4, and the antisense strand of the siRNA comprises a second nucleotide sequence as shown in SEQ ID NO.40.

[0009] In one embodiment of the present invention, the sense strand of the siRNA comprises a first nucleotide sequence as shown in SEQ ID NO.15, and the antisense strand of the siRNA comprises a second nucleotide sequence as shown in SEQ ID NO.51.

[0010] In one embodiment of the present invention, the sense strand of the siRNA comprises a first nucleotide sequence as shown in SEQ ID NO.33, and the antisense strand of the siRNA comprises a second nucleotide sequence as shown in SEQ ID NO.70.

[0011] In one embodiment of the present invention, the sense strand of the siRNA comprises a first nucleotide sequence as shown in SEQ ID NO.74, and the antisense strand of the siRNA comprises a second nucleotide sequence as shown in SEQ ID NO.81.

[0012] In one embodiment of the present invention, the sense strand of the siRNA comprises a first nucleotide sequence as shown in SEQ ID NO.75, and the antisense strand of the siRNA comprises a second nucleotide sequence as shown in SEQ ID NO.82.

[0013] In one embodiment of the present invention, the sense strand of the siRNA comprises a first nucleotide sequence as shown in SEQ ID NO.76, and the antisense strand of the siRNA comprises a second nucleotide sequence as shown in SEQ ID NO.83.

[0014] In one embodiment of the present invention, the sense strand of the siRNA comprises a first nucleotide sequence as shown in SEQ ID NO.77, and the antisense strand of the siRNA comprises a second nucleotide sequence as shown in SEQ ID NO.84.

[0015] In one embodiment of the present invention, the sense strand of the siRNA comprises a first nucleotide sequence as shown in SEQ ID NO.78, and the antisense strand of the siRNA comprises a second nucleotide sequence as shown in SEQ ID NO.85.

[0016] In one embodiment of the present invention, the sense strand of the siRNA comprises a first nucleotide sequence as shown in SEQ ID NO.79, and the antisense strand of the siRNA comprises a second nucleotide sequence as shown in SEQ ID NO.86.

[0017] In one embodiment of the present invention, the sense strand of the siRNA comprises a first nucleotide sequence as shown in SEQ ID NO. 80, and the antisense strand of the siRNA comprises a second nucleotide sequence as shown in SEQ ID NO. 87.

[0018] In one embodiment of the present invention, a third nucleotide sequence is further attached to the 5' end and / or the 3' end of the first nucleotide sequence; a fourth nucleotide sequence is further attached to the 5' end and / or the 3' end of the second nucleotide sequence; the length of the third nucleotide sequence and / or the fourth nucleotide sequence is 1 to 15 bp.

[0019] In one embodiment of the present invention, the length of the third nucleotide sequence and / or the fourth nucleotide sequence is 1-12 bp, 1-11 bp, 1-10 bp, 1-9 bp, 1-8 bp, 1-7 bp, 1-6 bp, 1-5 bp, 1-4 bp, 1-3 bp, 1-2 bp, 2-9 bp, 2-8 bp, 2-7 bp, 2-6 bp, 2-5 bp, 2-4 bp, or 2-3 bp.

[0020] In one embodiment of the invention, the sense strand further comprises one or more third nucleotide sequences; the antisense strand further comprises one or more fourth nucleotide sequences.

[0021] In one embodiment of the present invention, a third nucleotide sequence is attached to the 5' end or the 3' end of the first nucleotide sequence; and a fourth nucleotide sequence is attached to the 5' end or the 3' end of the second nucleotide sequence.

[0022] In one embodiment of the present invention, the two or more third nucleotide sequences contained in the sense strand may be the same or different; the two or more fourth nucleotide sequences contained in the antisense strand may be the same or different.

[0023] In one embodiment of the present invention, when a third nucleotide sequence is attached to both the 5' end and the 3' end of the first nucleotide sequence, the third nucleotide sequences may be the same or different; preferably, when a third nucleotide sequence is attached to both the 5' end and the 3' end of the first nucleotide sequence, the third nucleotide sequences are different; the length of each third nucleotide sequence is independently 1 bp, 2 bp, 3 bp, 4 bp, 5 bp, 6 bp, 7 bp, 8 bp or 9 bp.

[0024] In one embodiment of the present invention, when a fourth nucleotide sequence is attached to both the 5' end and the 3' end of the second nucleotide sequence, each fourth nucleotide sequence may be the same or different; preferably, when a fourth nucleotide sequence is attached to both the 5' end and the 3' end of the second nucleotide sequence, each fourth nucleotide sequence is different; the length of each fourth nucleotide sequence is independently 1bp, 2bp, 3bp, 4bp, 5bp, 6bp, 7bp, 8bp, 9bp, 10bp, or 11bp.

[0025] In one embodiment of the present invention, the antisense strand of the siRNA and the mRNA of the NR1H3 gene are partially completely complementary by at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0026] In one embodiment of the present invention, the lengths of the justice chain and the antisense chain are the same, or the lengths of the justice chain and the antisense chain are different;

[0027] When the lengths of the sense and antisense strands are the same, the siRNA has blunt ends; when the lengths of the sense and antisense strands are different, the siRNA has protruding ends.

[0028] In one embodiment of the present invention, the blunt end refers to the absence of unpaired nucleotides in the double-stranded structure of the siRNA; the protruding end refers to the protrusion of one or more unpaired nucleotides in the double-stranded structure of the siRNA.

[0029] In one embodiment of the present invention, when the siRNA has blunt ends, the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence as shown in any one of SEQ ID NO.1 to SEQ ID NO.36, and the antisense strand of the siRNA comprises a nucleic acid molecule that is partially or completely anticomplementary to the sense strand.

[0030] When the siRNA has a protruding end, the sense strand of the siRNA contains a nucleic acid molecule with a nucleotide sequence as shown in any one of SEQ ID NO.1 to SEQ ID NO.36, and, except for the protruding end, the antisense strand of the siRNA contains a nucleic acid molecule that is partially or completely anticomplementary to the sense strand.

[0031] In one embodiment of the present invention, when the siRNA has blunt ends, the nucleotide sequence of the sense strand of the siRNA is as shown in any one of SEQ ID NO.1 to SEQ ID NO.36, and the antisense strand of the siRNA is partially or completely anticomplementary to the sense strand.

[0032] When the siRNA has a protruding end, the nucleotide sequence of the sense strand of the siRNA is as shown in any one of SEQ ID NO.1 to SEQ ID NO.36, and the nucleotide sequence of the antisense strand of the siRNA is as shown in any one of SEQ ID NO.37 to SEQ ID NO.73; or, the nucleotide sequence of the sense strand of the siRNA is as shown in any one of SEQ ID NO.1 to SEQ ID NO.36, and, except for the protruding end, the antisense strand of the siRNA is partially or completely anticomplementary to the sense strand.

[0033] In one embodiment of the present invention, "partially reverse complementary" means that there are no more than 5, 4, 3, 2 or 1 base mismatches between the sense strand and the antisense strand; "completely reverse complementary" means that there are no base mismatches between the sense strand and the antisense strand.

[0034] In one embodiment of the present invention, when the siRNA has blunt ends, the nucleotide sequence of the sense strand of the siRNA is as shown in any one of SEQ ID NO.1 to SEQ ID NO.36, and the antisense strand of the siRNA is completely anticomplementary to the sense strand.

[0035] In one embodiment of the present invention, when the siRNA has blunt ends, the antisense strand of the siRNA can be completely anticomplementary to the sense strand. For example, the nucleotide sequence of the sense strand of the siRNA is 5'-AGGAGAUAGUUGACUUUGA-3' (SEQ ID NO. 9), and the nucleotide sequence of the antisense strand is 5'-UCAAAGUCAACUAUCUCCU-3' (SEQ ID NO. 217).

[0036] In one embodiment of the present invention, when the siRNA has a protruding end, the length of the sense strand of the siRNA is greater than the length of the antisense strand of the siRNA, or the length of the antisense strand of the siRNA is greater than the length of the sense strand of the siRNA.

[0037] When the length of the sense strand of siRNA is greater than the length of the antisense strand of siRNA, the protruding end is located at the 5' end and / or the 3' end of the sense strand of siRNA; when the length of the antisense strand of siRNA is greater than the length of the sense strand of siRNA, the protruding end is located at the 5' end and / or the 3' end of the antisense strand of siRNA.

[0038] In one embodiment of the present invention, when the overhang is located at the 5' end and / or 3' end of the sense strand of the siRNA, the homology between each base on the overhang and the mRNA of the NR1H3 gene is 0% or 100%; when the overhang is located at the 5' end and / or 3' end of the antisense strand of the siRNA, each base on the overhang is complementary or non-complementary to the mRNA of the NR1H3 gene.

[0039] In one embodiment of the present invention, the protruding end is located at the 3' end of the antisense strand of the siRNA.

[0040] In one embodiment of the present invention, the length of the protruding end is 1 to 5 bp.

[0041] In one embodiment of the present invention, the length of the protruding end is 1 to 2 bp.

[0042] In one embodiment of the present invention, 0% homology means that the bases at the same position in the overhang of the NR1H3 gene mRNA are not the same; 100% homology means that the bases at the same position in the overhang of the NR1H3 gene mRNA are the same.

[0043] In one embodiment of the present invention, the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.1, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.37;

[0044] Alternatively, the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.2, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.38 (wherein, nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO.1 to SEQ ID NO.2 contain a first nucleotide sequence as shown in SEQ ID NO.74, and nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO.37 to SEQ ID NO.38 contain a first nucleotide sequence as shown in SEQ ID NO.81);

[0045] Alternatively, the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.3, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.39 (wherein, the nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.3 contains a first nucleotide sequence as shown in SEQ ID NO.4, and the nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.39 contains a first nucleotide sequence as shown in SEQ ID NO.40);

[0046] Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.4, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.40;

[0047] Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.5, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.41;

[0048] Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO. 6, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO. 42;

[0049] Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.7, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.43;

[0050] Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO. 8, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO. 44;

[0051] Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO. 9, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO. 45;

[0052] Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.10, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.46;

[0053] Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.11, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.47;

[0054] Alternatively, the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO. 12, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO. 48 (wherein, nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO. 5 to SEQ ID NO. 12 contain a first nucleotide sequence as shown in SEQ ID NO. 75, and nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO. 41 to SEQ ID NO. 48 contain a first nucleotide sequence as shown in SEQ ID NO. 82);

[0055] Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.13, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.49;

[0056] Alternatively, the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.14, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.50 (wherein, nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO.13 to SEQ ID NO.14 contain a first nucleotide sequence as shown in SEQ ID NO.76, and nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO.46 to SEQ ID NO.50 contain a first nucleotide sequence as shown in SEQ ID NO.83);

[0057] Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.15, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.51;

[0058] Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.16, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.52;

[0059] Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.17, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.53;

[0060] Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.18, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.54;

[0061] Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.19, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.55;

[0062] Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.20, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.56;

[0063] Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.21, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.57;

[0064] Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.22, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.58;

[0065] Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.23, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.59;

[0066] Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.24, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.60;

[0067] Alternatively, the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO. 25, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO. 61 (wherein, nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO. 16 to SEQ ID NO. 25 contain a first nucleotide sequence as shown in SEQ ID NO. 77, and nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO. 52 to SEQ ID NO. 61 contain a first nucleotide sequence as shown in SEQ ID NO. 84);

[0068] Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.26, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.62;

[0069] Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.27, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.63;

[0070] Alternatively, the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO. 28, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO. 64 (wherein, nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO. 26 to SEQ ID NO. 28 contain a first nucleotide sequence as shown in SEQ ID NO. 78, and nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO. 62 to SEQ ID NO. 64 contain a first nucleotide sequence as shown in SEQ ID NO. 85);

[0071] Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.29, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.65;

[0072] Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.29, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.66;

[0073] Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.30, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.67;

[0074] Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.31, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.68;

[0075] Alternatively, the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO. 32, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO. 69 (wherein, nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO. 29 to SEQ ID NO. 32 contain a first nucleotide sequence as shown in SEQ ID NO. 79, and nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO. 65 to SEQ ID NO. 69 contain a first nucleotide sequence as shown in SEQ ID NO. 86);

[0076] Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.33, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.70;

[0077] Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.34, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.71;

[0078] Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.35, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.72;

[0079] Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO. 36, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO. 73 (wherein, nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO. 34 to SEQ ID NO. 36 contain a first nucleotide sequence as shown in SEQ ID NO. 80, and nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO. 71 to SEQ ID NO. 73 contain a first nucleotide sequence as shown in SEQ ID NO. 87).

[0080] In one embodiment of the present invention, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.1, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.37;

[0081] Alternatively, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.2, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.38;

[0082] Alternatively, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.3, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.39;

[0083] Alternatively, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.4, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.40;

[0084] Alternatively, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.5, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.41;

[0085] Alternatively, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.6, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.42;

[0086] Alternatively, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.7, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.43;

[0087] Alternatively, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.8, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.44;

[0088] Alternatively, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.9, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.45;

[0089] Alternatively, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.10, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.46;

[0090] Alternatively, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.11, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.47;

[0091] Alternatively, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.12, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.48;

[0092] Alternatively, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.13, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.49;

[0093] Alternatively, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.14, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.50;

[0094] Alternatively, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.15, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.51;

[0095] Alternatively, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.16, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.52;

[0096] Alternatively, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.17, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.53;

[0097] Alternatively, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.18, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.54;

[0098] Alternatively, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.19, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.55;

[0099] Alternatively, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.20, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.56;

[0100] Alternatively, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.21, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.57;

[0101] Alternatively, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.22, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.58;

[0102] Alternatively, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.23, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.59;

[0103] Alternatively, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.24, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.60;

[0104] Alternatively, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.25, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.61;

[0105] Alternatively, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.26, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.62;

[0106] Alternatively, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.27, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.63;

[0107] Alternatively, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.28, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.64;

[0108] Alternatively, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.29, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.65;

[0109] Alternatively, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.29, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.66;

[0110] Alternatively, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.30, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.67;

[0111] Alternatively, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.31, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.68;

[0112] Alternatively, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.32, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.69;

[0113] Alternatively, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.33, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.70;

[0114] Alternatively, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.34, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.71;

[0115] Alternatively, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.35, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.72;

[0116] Alternatively, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.36, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.73.

[0117] In one embodiment of the present invention, at least one nucleotide in the sense strand or antisense strand of the siRNA is a modified nucleotide; the modification includes fluorination modification, methoxylation modification, thiophosphate modification, trans-vinylphosphonate modification, reverse debasing deoxyribose residue modification, ethylene glycol nucleic acid (GNA) modification and / or deoxyribonucleotide substitution.

[0118] In one embodiment of the present invention, the fluorinated nucleotide has a structure as shown in Formula I:

[0119]

[0120] In one embodiment of the present invention, the methoxylated nucleotide has a structure as shown in Formula II:

[0121]

[0122] In one embodiment of the present invention, the thiophosphate group modification refers to the thiophosphate group linking two adjacent nucleotides; the thiophosphate group has the structure shown in Formula III:

[0123]

[0124] In this invention, the thiophosphate group, thiophosphate bond, and thiophosphate diester bond are interchangeable and all refer to the structure shown in Formula III.

[0125] In one embodiment of the present invention, the nucleotide modified with a thiophosphate group has a structure as shown in Formula IV:

[0126]

[0127] In one embodiment of the present invention, the nucleotide modified with a trans-vinylphosphonate group has a structure as shown in Formula VI:

[0128]

[0129] In one embodiment of the present invention, the reverse debased deoxyribose residue is modified at the end or middle of the sense strand or the antisense strand;

[0130] When the reverse debased deoxyribose residue is modified at the 5' end of the sense or antisense strand, the reverse debased deoxyribose residue has the structure shown in Formula VII:

[0131]

[0132] When the reverse debased deoxyribose residue is modified at the 3' end of the sense or antisense strand, the reverse debased deoxyribose residue has the structure shown in Formula VIII:

[0133]

[0134] When a reverse debased deoxyribose residue is modified in the middle of the sense or antisense strand, the reverse debased deoxyribose residue has the structure shown in Formula IX:

[0135]

[0136] In one embodiment of the present invention, when a reverse debasedoxyribose residue is modified at the 3' end of the sense strand or the antisense strand, the nucleotide modified with the reverse debasedoxyribose residue has a structure as shown in Formula X-1, where X is S - Or O - :

[0137]

[0138] When a reverse debase deoxyribose residue is applied to the 5' end of either the sense or antisense strand, the nucleotide modified with the reverse debase deoxyribose residue has the structure shown in Formula X-2, where X represents S. - Or O - :

[0139]

[0140] When a reverse debase deoxyribose residue is used to modify the middle of the sense or antisense strand, the nucleotide modified with the reverse debase deoxyribose residue has the structure shown in Formula XI, where X is S - Or O - :

[0141]

[0142] In one embodiment of the present invention, the nucleotide modified with ethylene glycol nucleic acid (GNA) has a structure as shown in Formula XII:

[0143]

[0144] In one embodiment of the present invention, the fluorinated nucleotides are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 7, 9, 10, and 11 of the sense strand are fluorinated nucleotides, and at least the nucleotides at positions 2, 6, 14, and 16 of the antisense strand are fluorinated nucleotides.

[0145] Alternatively, the fluorinated nucleotides are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the 5th, 7th, 8th, and 9th nucleotides of the sense strand are fluorinated nucleotides, and at least the 2nd, 6th, 14th, and 16th nucleotides of the antisense strand are fluorinated nucleotides.

[0146] Alternatively, the fluorinated nucleotides are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 3, 7, 8, and 9 of the sense strand are fluorinated nucleotides, and at least the nucleotides at positions 2, 7, 10, and 14 of the antisense strand are fluorinated nucleotides.

[0147] Alternatively, the fluorinated nucleotides are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 3, 7, 9, and 11 of the sense strand are fluorinated nucleotides, and at least the nucleotides at positions 2, 7, 10, and 14 of the antisense strand are fluorinated nucleotides.

[0148] Alternatively, the fluorinated nucleotides are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the 7th, 9th, and 14th nucleotides of the sense strand are fluorinated nucleotides, and at least the 2nd, 7th, 10th, and 14th nucleotides of the antisense strand are fluorinated nucleotides.

[0149] Alternatively, the fluorinated nucleotides are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 3, 7, 9, and 11 of the sense strand are fluorinated nucleotides, and at least the nucleotides at positions 2, 3, 5, 7, 10, 12, and 14 of the antisense strand are fluorinated nucleotides.

[0150] Alternatively, the fluorinated nucleotides are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 3, 7, 9, and 11 of the sense strand are fluorinated nucleotides, and at least the nucleotides at positions 2, 7, and 14 of the antisense strand are fluorinated nucleotides.

[0151] In one embodiment of the present invention, the methoxylated nucleotides are located in the antisense and sense strands of the nucleotide sequence, and in the sense strand, the nucleotides without fluorination, without ethylene glycol nucleic acid (GNA) modification, and without deoxyribonucleotide substitution are all methoxylated nucleotides; in the antisense strand, the nucleotides without fluorination, without ethylene glycol nucleic acid modification, and without deoxyribonucleotide substitution are all methoxylated nucleotides.

[0152] In one embodiment of the invention, the trans-vinylphosphonate modified nucleotide is located in the antisense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, the 5' end of at least the first nucleotide of the antisense strand is attached to a trans-vinylphosphonate group.

[0153] In one embodiment of the present invention, the nucleotide modified by the reverse debased deoxyribose residue is located in the positive strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, the 3' end of at least the penultimate nucleotide of the positive strand is connected to a reverse debased deoxyribose residue (invAb group).

[0154] Alternatively, the nucleotide modified with the reverse debased deoxyribose residue is located in the positive strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, the 5' end of at least the first nucleotide of the positive strand is attached to a reverse debased deoxyribose residue (invAb group).

[0155] Alternatively, the nucleotide modified with the reverse debased deoxyribose residue is located in the positive strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, the 5' end of at least the first nucleotide of the positive strand and the 3' end of the last nucleotide are connected to a reverse debased deoxyribose residue (invAb group).

[0156] In one embodiment of the present invention, the nucleotide modified with a thiophosphate group is located in the antisense strand of the nucleotide sequence, and the nucleotides at least the first and second, the second and third, the penultimate and penultimate, and the penultimate and penultimate positions of the antisense strand are linked by a thiophosphate group in the direction from the 5' end to the 3' end.

[0157] In one embodiment of the present invention, the nucleotide modified with a thiophosphate group is located in the positive strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at the 1st and 2nd, 2nd and 3rd, penultimate and penultimate, and penultimate and penultimate positions of the positive strand are connected by a thiophosphate group.

[0158] Alternatively, the phosphate-thioester modified nucleotide is located in the positive strand of the nucleotide sequence, and in the positive strand, at least the nucleotides at positions 1 and 2, and positions 2 and 3 are linked by phosphate-thioester groups, in the direction from the 5' end to the 3' end; and if the 3' end of the penultimate nucleotide is connected to a reverse debased deoxyribose residue, then the penultimate nucleotide is linked to the reverse debased deoxyribose residue by phosphate-thioester groups.

[0159] Alternatively, the phosphate-thioester modified nucleotide is located in the positive strand of the nucleotide sequence, and in the positive strand, in the direction from the 5' end to the 3' end, the nucleotide at position 1 is linked to the reverse debased deoxyribose residue by a phosphate-thioester group, at least the nucleotides at positions 1 and 2 are linked by a phosphate-thioester group, and the nucleotide at position 1 to 2 is linked to the reverse debased deoxyribose residue by a phosphate-thioester group.

[0160] In one embodiment of the present invention, the ethylene glycol-modified nucleotide is located in the antisense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the 6th nucleotide of the antisense strand is an ethylene glycol-modified nucleotide.

[0161] In one embodiment of the invention, the deoxyribonucleotide-substituted nucleotide is located in the antisense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 5 and 12 of the antisense strand are deoxyribonucleotide-substituted nucleotides.

[0162] In one embodiment of the present invention, "at least" means that, in the sense strand and the antisense strand, except for the nucleotide at the defined position which is a modified nucleotide, at least one nucleotide at the remaining positions is also a modified nucleotide.

[0163] In one embodiment of the present invention, in the direction from the 5' end to the 3' end, positions 7, 9, 10, and 11 of the sense strand of the siRNA are fluorinated at 2' positions, and the other positions are methoxylated at 2' positions, with thiophosphate bonds connecting the 1,2 and 2,3 bases; in the direction from the 5' end to the 3' end, positions 2, 6, 14, and 16 of the antisense strand of the siRNA are fluorinated at 2' positions, and the other positions are methoxylated at 2' positions, with thiophosphate bonds connecting the 1,2 and 2,3 bases and the reciprocal 1,2 and 2,3 bases, and the first base is not connected to a 5' phosphate group or a 5' phosphate derivative group;

[0164] Alternatively, in the direction from the 5' end to the 3' end, the siRNA's sense strand has 2' fluorinated positions at positions 7, 9, 10, and 11, and 2' methoxylated positions at other positions, with thiophosphate bonds connecting the 1, 2 and 2, 3 bases; in the direction from the 5' end to the 3' end, the siRNA's antisense strand has 2' fluorinated positions at positions 2, 6, 14, and 16, and 2' methoxylated positions at other positions, with thiophosphate bonds connecting the 1, 2 and 2, 3 bases and the reciprocal 1, 2 and 2, 3 bases, with the first base being a nucleotide linked by a trans-vinylphosphonate group;

[0165] Alternatively, in the direction from the 5' end to the 3' end, the siRNA's sense strand has 2' fluorinated positions at positions 5, 7, 8, and 9, and 2' methoxylated positions at other positions, with thiophosphate bonds connecting the 1, 2 and 2, 3 bases; in the direction from the 5' end to the 3' end, the siRNA's antisense strand has 2' fluorinated positions at positions 2, 6, 14, and 16, and 2' methoxylated positions at other positions, with thiophosphate bonds connecting the 1, 2 and 2, 3 bases and the reciprocal 1, 2 and 2, 3 bases, with the first base being a nucleotide linked by a trans-vinylphosphonate group;

[0166] Alternatively, in the direction from the 5' end to the 3' end, the siRNA's sense strand has 2' fluorinated positions at positions 3, 7, 8, and 9, and 2' methoxylated positions at other positions, with thiophosphate bonds connecting the 1, 2 and 2, 3 bases; in the direction from the 5' end to the 3' end, the siRNA's antisense strand has 2' fluorinated positions at positions 2, 7, 10, and 14, and 2' methoxylated positions at other positions, with thiophosphate bonds connecting the 1, 2 and 2, 3 bases and the reciprocal 1, 2 and 2, 3 bases, with the first base being a nucleotide linked by a trans-vinylphosphonate group;

[0167] Alternatively, in the direction from the 5' end to the 3' end, the siRNA's sense strand has 2' fluorinated positions at positions 3, 7, 9, and 11, and 2' methoxylated positions at other positions, with thiophosphate bonds connecting the 1, 2 and 2, 3 bases; in the direction from the 5' end to the 3' end, the siRNA's antisense strand has 2' fluorinated positions at positions 2, 7, 10, and 14, and 2' methoxylated positions at other positions, with thiophosphate bonds connecting the 1, 2 and 2, 3 bases and the reciprocal 1, 2 and 2, 3 bases, with the first base being a nucleotide linked by a trans-vinylphosphonate group;

[0168] Alternatively, following the direction from the 5' end to the 3' end, the siRNA's sense strand has 2' fluorinated positions at positions 3, 7, 8, and 9, and 2' methoxylated positions at other positions. An invAb group is attached to the 3' end, with thiophosphate bonds connecting the 1, 2 and 2, 3 bases, and between the penultimate base and invAb. Similarly, following the direction from the 5' end to the 3' end, the siRNA's antisense strand has 2' fluorinated positions at positions 2, 7, 10, and 14, and 2' methoxylated positions at other positions. Thiophosphate bonds connect the 1, 2 and 2, 3 bases, and between the penultimate 1, 2, and 2, 3 bases, with the first base being a nucleotide linked by a trans-vinylphosphonate group.

[0169] Alternatively, following the direction from the 5' end to the 3' end, the siRNA's sense strand has 2' fluorinated positions at positions 3, 7, 9, and 11, and 2' methoxylated positions at other positions. An invAb group is attached to the 3' end, with thiophosphate bonds connecting the 1, 2 and 2, 3 bases, and between the penultimate base and invAb. Similarly, following the direction from the 5' end to the 3' end, the siRNA's antisense strand has 2' fluorinated positions at positions 2, 7, 10, and 14, and 2' methoxylated positions at other positions. Thiophosphate bonds connect the 1, 2 and 2, 3 bases, and between the penultimate 1, 2, and 2, 3 bases, with the first base being a nucleotide linked by a trans-vinylphosphonate group.

[0170] Alternatively, in the direction from the 5' end to the 3' end, the siRNA's sense strand has 2' fluorinated positions at positions 7, 9, and 14, and 2' methoxylated positions at other positions, with thiophosphate bonds connecting the 1, 2 and 2, 3 bases; in the direction from the 5' end to the 3' end, the siRNA's antisense strand has 2' fluorinated positions at positions 2, 7, 10, and 14, and 2' methoxylated positions at other positions, with thiophosphate bonds connecting the 1, 2 and 2, 3 bases and the reciprocal 1, 2 and 2, 3 bases, with the first base being a nucleotide linked by a trans-vinylphosphonate group;

[0171] Alternatively, following the direction from the 5' end to the 3' end, the siRNA's sense strand has 2' fluorinated positions at positions 3, 7, 9, and 11, and 2' methoxylated positions at other positions, with thiophosphate bonds connecting the 1, 2 and 2, 3 bases; following the direction from the 5' end to the 3' end, the siRNA's antisense strand has 2' fluorinated positions at positions 2, 3, 5, 7, 10, 12, and 14, GNA modified position 6, and 2' methoxylated positions at other positions, with thiophosphate bonds connecting the 1, 2 and 2, 3 bases and the reciprocal 1, 2 and 2, 3 bases, with the first base being a nucleotide linked by a trans-vinylphosphonate group;

[0172] Alternatively, in the direction from the 5' end to the 3' end, the siRNA's sense strand has 2' fluorinated positions at positions 3, 7, 9, and 11, and 2' methoxylated positions at other positions, with thiophosphate bonds connecting the 1, 2 and 2, 3 bases; in the direction from the 5' end to the 3' end, the siRNA's antisense strand has 2' fluorinated positions at positions 2, 7, and 14, deoxyribonucleotides at positions 5 and 12, and 2' methoxylated positions at other positions, with thiophosphate bonds connecting the 1, 2 and 2, 3 bases and the reciprocal 1, 2 and 2, 3 bases, with the first base being a nucleotide linked by a trans-vinylphosphonate group;

[0173] Alternatively, following the direction from the 5' end to the 3' end, the siRNA's sense strand has 2' fluorinated positions at positions 3, 7, 8, and 9, and 2' methoxylated positions at other positions. An invAb group is attached to both the 5' and 3' ends, with thiophosphate bonds connecting invAb to the first base, to bases 1 and 2, and to the penultimate base. Similarly, following the direction from the 5' end to the 3' end, the siRNA's antisense strand has 2' fluorinated positions at positions 2, 7, 10, and 14, and 2' methoxylated positions at other positions. Thiophosphate bonds connect bases 1 and 2 with bases 2 and 3, and to the penultimate bases 1, 2 with bases 2 and 3. The first base is a nucleotide linked by a trans-vinylphosphonate group.

[0174] In one embodiment of the present invention, any two nucleotides linked together in the positive strand of the siRNA are connected by a phosphodiester bond or a thiophosphate diester bond.

[0175] In one embodiment of the present invention, any two nucleotides linked together in the antisense strand of the siRNA are connected by a phosphodiester bond or a thiophosphate diester bond.

[0176] In one embodiment of the present invention, the modified siRNA contains modification of a phosphodiester bond at at least one position.

[0177] In one embodiment of the present invention, the modification of the phosphodiester bond refers to the substitution of at least one oxygen atom in the phosphodiester bond by a sulfur atom to form a thiophosphate diester bond. The thiophosphate diester bond can stabilize the double-stranded structure of siRNA and maintain the specificity of base pairing.

[0178] In one embodiment of the present invention, the described siRNA shown in N-ER-FY049065、N-ER-FY049004、N-ER-FY049084、N-ER-FY049019、N-ER-FY049086、N-ER-FY049087、N-ER-FY049086、N-ER-FY049087、N-ER-FY049086、N-ER-FY049087 49020、N-ER-FY049088、N-ER-FY049089、N-ER-FY049021、N-ER-FY049090、N-ER-FY049091、N-ER-FY049022、N-ER-FY049023、N-ER-FY049033、N-ER-FY049091 Y049096、N-ER-FY049097、N-ER-FY049098、N-ER-FY049099、N-ER-FY049038、N-ER-FY049039、N-ER-FY049040、N-ER-FY049100、N-ER-FY049101、N-ER-FY049102、N-ER-FY049105、N-ER-FY049106、N-ER-FY049041、N-ER-FY049111、N-ER-FY049128、N-ER-FY049043、N-ER-FY049044、N-ER-FY049113、N -ER-FY049057, N-ER-FY049117, N-ER-FY049062 and N-ER-FY049118, N-ER-FY049065M6, N-ER-FY049065M8, N-ER-FY049004M6, N-ER-FY049084M6, N-ER-FY049084M8, N-ER-FY049019M6, N-ER-FY049086M6, N-ER-FY049086M8, N-ER-FY049020M6, N-ER-FY049089M6, N-ER-FY049089M8, N-ER-FY049089M44 ,N-ER-FY049089M45,N-ER-FY049089M46,N-ER-FY049089M47,N-ER-FY049089M48,N-ER-FY049089M49,N-ER-FY049089M50,N-ER-FY049089M51,N-ER-FY049021M6,N-ER-FY049021M8,N-ER-FY049090M6,N-ER-FY049090M8,N-ER-FY049091M6,N-ER-FY049022M6,N-ER-FY049023M6,N-ER-FY049096M6,N-ER-FY049096M8, N-ER-FY049097M6, N-ER-FY049097M8, N-ER-FY049098M 6. N-ER-FY049098M8, N-ER-FY049099M6, N-ER-FY049099M8, N-ER-FY049038 M6、N-ER-FY049038M8、N-ER-FY049039M6、N-ER-FY049039M8、N-ER-FY04904 0M6、N-ER-FY049100M6、N-ER-FY049100M8、N-ER-FY049105M6、N-ER-FY0491 06M6、N-ER-FY049041M6、N-ER-FY049111M6、N-ER-FY049111M8、N-ER-FY049 128M8、N-ER-FY049043M6、N-ER-FY049044M6、N-ER-FY049044M8、N-ER-FY04 9113M6、N-ER-FY049113M8、N-ER-FY049057M6、N-ER-FY049117M6、N-ER-FY0 One or more of 49117M8, N-ER-FY049062M6, N-ER-FY049118M6 and N-ER-FY049118M8. ,

[0179] In one embodiment of the present invention, the siRNA is selected from one or more of N-ER-FY049089, N-ER-FY049089M44, N-ER-FY049089M46, N-ER-FY049089M48, N-ER-FY049089M50 and N-ER-FY049089M51.

[0180] This invention also provides an siRNA conjugate containing the aforementioned siRNA and a conjugating group attached to the siRNA. The siRNA molecule modified with the conjugating group maintains high inhibitory activity and stability while also exhibiting good tissue and organ targeting and the ability to promote endocytosis. This reduces the impact on other tissues or organs and decreases the amount of siRNA molecule used, thereby achieving the goals of reducing toxicity and lowering costs.

[0181] In one embodiment of the present invention, the conjugation group comprises a ligand formed from a targeting molecule or a derivative thereof; the targeting molecule comprises a lipophilic molecule, a polymer, a polypeptide, an aptamer, an antibody, a quantum dot, a sugar, folic acid, and / or a receptor specifically expressed by hepatocytes.

[0182] In one embodiment of the present invention, the lipophilic molecule includes cholesterol, bile acids, vitamins, and / or lipid molecules of different chain lengths; the polymer includes polyethylene glycol; the polypeptide includes a transmembrane peptide; the carbohydrate includes lactose, polylactose, mannose, galactose, and / or N-acetylgalactosamine; and the receptor expressed by the hepatocytes includes desialyl glycoprotein, desialyl sugar residues, lipoproteins, glucagon, neurotransmitters, growth factors, and / or transferrin.

[0183] In one embodiment of the present invention, the conjugating group is N-glucosamine (N-Acetylgalactosamine, GalNAc).

[0184] In one embodiment of the present invention, the N-glucose amino acid has a structure as shown in Formulas XIII to XX:

[0185]

[0186]

[0187]

[0188]

[0189] In one embodiment of the present invention, the conjugation site of the siRNA and the conjugation group is located at the 3' end of the siRNA sense strand, the 5' end of the siRNA sense strand, the internal sequence of the siRNA sense strand, the 5' end of the siRNA antisense strand, or the internal sequence of the siRNA antisense strand.

[0190] In one embodiment of the present invention, the conjugation site of the siRNA and the conjugating group is located at the 3' end of the positive strand of the siRNA.

[0191] In one embodiment of the present invention, the conjugating group is conjugated to the 3' end of the positive strand of siRNA via a phosphodiester bond. Various ways in which siRNA is conjugated to the conjugating group can be found in the following reference: Muthiah Manoharanet.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.

[0192] In one embodiment of the invention, when the conjugating group is an N-glucose amino acid, the siRNA conjugate has a structure as shown in Formula XXI (the double helix structure represents the siRNA, and the linker is attached to the 3' end of the positive strand of the siRNA):

[0193]

[0194] In one embodiment of the present invention, Base in Formula I, Formula II, Formula IV, Formula VI, Formula X-1, Formula X-2, Formula XI, and Formula XII represents a base; the base includes A, U, G, C, or T.

[0195] In one embodiment of the present invention, R in formulas IV, VI, X-1, X-2, XI, and XII is selected from H, -OH, -F, -OCH3, or a modifying group.

[0196] In one embodiment of the present invention, the siRNA conjugate is selected from N-ER-FY049004M6L96, N-ER-FY049020M6L96, N-ER-FY049020M8L96, N-ER-FY049021M6L96, N-ER-FY049021M8L96, N-ER-FY049022M6L96, N-ER-FY049023M6L96, N-ER-FY049038M6L96, N-ER-FY049038M8L96, N-ER-FY049038M8L96, N-ER-FY0 49039M6L96, N-ER-FY049039M8L96, N-ER-FY049040M6L96, N-ER-FY049040M8L96, N-ER-FY049041M6L96, N-ER-FY049043M6 L96、N-ER-FY049044M6L96、N-ER-FY049044M8L96、N-ER-FY049062M6L96、N-ER-FY049065M8L96、N-ER-FY049084M8L96、N-ER -FY049086M8L96, N-ER-FY049089M8L96, N-ER-FY049090M8L96, N-ER-FY049096M8L96, N-ER-FY049097M8L96, N-ER-FY0490 98M8L96, N-ER-FY049099M8L96, N-ER-FY049100M8L96, N-ER-FY049111M8L96, N-ER-FY049113M8L96, N-ER-FY049117M8L96, One or more of N-ER-FY049118M8L96, N-ER-FY049089M44L96, N-ER-FY049089M45L96, N-ER-FY049089M46L96, N-ER-FY049089M47L96, N-ER-FY049089M48L96, N-ER-FY049089M49L96, N-ER-FY049089M50L96, N-ER-FY049089M51L96 and N-ER-FY049128M8L96.

[0197] In one embodiment of the present invention, the siRNA conjugate is selected from one or more of N-ER-FY049089M44L96, N-ER-FY049089M46L96, N-ER-FY049089M48L96, N-ER-FY049089M50L96 and N-ER-FY049089M51L96.

[0198] The present invention also provides a pharmaceutical composition comprising the above-described siRNA and / or the above-described siRNA conjugate.

[0199] In one embodiment of the invention, the pharmaceutical composition further comprises pharmaceutically acceptable excipients; said pharmaceutically acceptable excipients include binders, suspending agents, emulsifiers, diluents (or fillers), granulating agents, adhesives, disintegrants, lubricants, anti-adhesion agents, flow aids, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, and / or flavoring agents (e.g., sweeteners).

[0200] The present invention also provides an inhibitor for suppressing NR1H3 gene expression, the inhibitor comprising the above-mentioned siRNA and / or the above-mentioned siRNA conjugate.

[0201] In one embodiment of the invention, the inhibitor further comprises pharmaceutically acceptable excipients; the pharmaceutically acceptable excipients include binders, suspending agents, emulsifiers, diluents (or fillers), granulating agents, adhesives, disintegrants, lubricants, anti-adhesion agents, flow aids, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, and / or flavoring agents (e.g., sweeteners).

[0202] The present invention also provides a medicament for the prevention and / or treatment of a disease related to NR1H3 gene expression; the medicament comprises the above-mentioned siRNA and / or the above-mentioned siRNA conjugate.

[0203] In one embodiment of the present invention, the diseases associated with NR1H3 gene expression include diseases associated with lipid metabolism.

[0204] In one embodiment of the present invention, the diseases associated with NR1H3 gene expression include dyslipidemia, atherosclerosis, and / or non-alcoholic fatty liver disease.

[0205] In one embodiment of the invention, the medicament further comprises pharmaceutically acceptable excipients; the pharmaceutically acceptable excipients include binders, suspending agents, emulsifiers, diluents (or fillers), granulating agents, adhesives, disintegrants, lubricants, anti-adhesion agents, flow aids, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, and / or flavoring agents (e.g., sweeteners).

[0206] The present invention also provides the use of the above-mentioned siRNA, the above-mentioned siRNA conjugate, the above-mentioned pharmaceutical composition, the above-mentioned inhibitor, or the above-mentioned drug in the preparation of a medicament for the prevention and / or treatment of a disease related to NR1H3 gene expression.

[0207] In one embodiment of the present invention, the diseases associated with NR1H3 gene expression include diseases associated with lipid metabolism.

[0208] In one embodiment of the present invention, the diseases associated with NR1H3 gene expression include dyslipidemia, atherosclerosis, and / or non-alcoholic fatty liver disease.

[0209] The present invention also provides a treatment method for a disease, comprising administering to a subject or patient in need the above-described siRNA, the above-described siRNA conjugate, the above-described pharmaceutical composition, the above-described inhibitor, and / or the above-described drug.

[0210] The technical solution of this invention has the following advantages:

[0211] This invention provides an siRNA for inhibiting NR1H3 gene expression, wherein the siRNA contains a sense strand and an antisense strand; the sense strand and the antisense strand are at least partially anticomplementary to form a double-stranded region; the sense strand of the siRNA contains a first nucleotide sequence as shown in any one of SEQ ID NO.4, SEQ ID NO.15, SEQ ID NO.33, or SEQ ID NO.74–SEQ ID NO.80; the antisense strand of the siRNA contains a second nucleotide sequence as shown in any one of SEQ ID NO.40, SEQ ID NO.51, SEQ ID NO.70, or SEQ ID NO.81–87. This invention aims to provide an siRNA composition that is effective against RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the NR1H3 gene, thereby selectively and effectively inhibiting NR1H3 gene expression and achieving the purpose of disease treatment. In vitro and in vivo experiments have confirmed that the siRNA, its modifications, and conjugates provided by this invention all possess high inhibitory activity against NR1H3. Therefore, the siRNA, its modifiers and conjugates provided by this invention have great application potential in the preparation of drugs for treating diseases related to lipid metabolism. Attached Figure Description

[0212] Figure 1 Results of the inhibition rate of the siRNA conjugate against hNR1H3 protein in this application. Detailed Implementation

[0213] definition

[0214] Unless otherwise specified, the singular forms “a” and “the” as used herein include plural references. For example, an antibody includes one or more antibodies. The terms “a” (or “an”) and “one or more” and “at least one” are used interchangeably herein.

[0215] The one or more instances following the terms "for example" are not intended to be exhaustive or limiting.

[0216] In this document, unless otherwise stated, the terms “comprising,” “including,” “containing,” or “having” indicate a non-exclusive inclusion relationship, meaning that when a substance, composition, or structure is described as “comprising” certain specific ingredients or features, the substance, composition, or structure may also contain other unlisted ingredients or features, and these unlisted ingredients or features should not be considered beyond the scope of protection of this invention. It should be understood that wherever the terms “comprising,” “including,” “containing,” or “having” are used to describe aspects herein, other similar aspects described by “consisting of” and / or “is…” and / or “substantially is…” are also provided. The explicit use of the phrase “consisting of” herein indicates the exclusion of any unspecified elements, steps, or ingredients. The explicit use of the phrase “substantially consists of” herein indicates that the scope is limited to the specified materials or steps and those materials or steps that do not substantially affect one or more essential and novel features of the claimed invention.

[0217] As used herein, “and / or” is considered to be each of two specified features or components disclosed with or without the other. Therefore, the term “and / or” as used herein in phrases such as “A and / or B” is intended to include “A and B”, “A or B”, “A” (alone), and “B” (alone). Similarly, the term “and / or” as used in phrases such as “A, B, and / or C” is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0218] Units, prefixes, and symbols are represented in their internationally recognized (SI) form. Numerical ranges include the numerical values ​​that define that range. The headings provided herein are not limiting to the aspects which can be obtained by referring to the entire specification. Therefore, the terms defined immediately below are more fully defined by referring to the full contents of the specification.

[0219] In this paper, "core sequence" refers to a common sequence between at least two justice chains or between at least two antisense chains.

[0220] In the context of this application, unless otherwise specified, "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" generally also represents a nucleotide containing guanine, cytosine, adenine, thymine and uracil as a base, respectively. This is a common practice in representing deoxyribonucleic acid sequences and / or ribonucleic acid sequences. Therefore, in the context of this application, the meanings of "G", "C", "A", "T" and "U" include all the above-mentioned possible situations.

[0221] In the context of this application, the terms "complementary" and "reverse complementary" are used interchangeably and have the meaning known to those skilled in the art: in a double-stranded nucleic acid molecule, the bases of one strand are paired complementaryly with the bases of the other strand. 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 consists of one purine and one pyrimidine. When adenine on one strand always pairs with thymine (or uracil) on the other strand, and guanine always pairs with cytosine, the two strands are considered complementary, and the sequence of the complementary strand can be inferred from its sequence.

[0222] In the context of this application, and particularly in describing the preparation method of siRNA or siRNA conjugates, unless otherwise specified, the nucleoside monomer refers to the modified or unmodified nucleoside phosphorus amide monomer used in solid-phase phosphorus amide synthesis, depending on the type and sequence of nucleotides in the desired siRNA or siRNA conjugate. Solid-phase phosphorus amide synthesis is a method known to those skilled in the art for RNA synthesis. All nucleoside monomers used in this application are commercially available.

[0223] In the context of this application, unless otherwise stated, "conjugation" refers to the covalent connection between two or more chemical parts, each with a specific function; correspondingly, "conjugated compound" refers to a compound formed by the covalent connection of these chemical parts. Further, "siRNA conjugated compound" refers to a compound formed by the covalent attachment of one or more chemical parts with specific functions to siRNA. siRNA conjugated compound should be understood, depending on the context, as a collective term for multiple siRNA conjugated compounds or a siRNA conjugated compound represented by a specific chemical formula. In the context of this application, "conjugated molecule" should be understood as a specific compound that can be reactively conjugated to siRNA to ultimately form the siRNA conjugated compound of this application.

[0224] In the context of this application, the term "disease related to NR1H3 gene expression" refers to any disease whose occurrence, progression, or pathophysiological process is at least partially mediated, promoted, or maintained by the abnormal expression or activity of the NR1H3 gene and / or its encoded products. If a causal or functional association between NR1H3 gene expression and the disease state is observed in a particular disease, then that disease falls under the category of "diseases related to NR1H3 gene expression" as described in this invention.

[0225] Various hydroxyl protecting groups may be used in this application. Generally, protecting groups insensitize chemical functional groups to specific reaction conditions and can be added to and removed from the functional group in the molecule without substantially impairing the rest of the molecule. In some embodiments, protecting groups are stable under basic conditions but can be removed under acidic conditions. In some embodiments, non-exclusive examples of hydroxyl protecting groups that may be used in this application include monomethoxytriphenylmethyl, 9-phenylxanthine-9-yl (Pixyl), and 9-(p-methoxyphenyl)xanthine-9-yl (Mox). In some embodiments, non-exclusive examples of hydroxyl protecting groups that may be used in this application include Tr (triphenylmethyl), MMTr (4-methoxytriphenylmethyl), DMTr (4,4'-dimethoxytriphenylmethyl), and TMTr (4,4',4”-trimethoxytriphenylmethyl).

[0226] In the context of this application, the same siRNA referred to by the same number refers to the same siRNA in Table 1. For example, when referring to number N-ER-FY049065, the sense strand of the siRNA is 5'-GGCUUCCACUACAAUGUUCUA-3' (SEQ ID NO: 1), and the antisense strand is 5'-UAGAACAUUGUAGUGGAAGCCCG-3' (SEQ ID NO: 37). Similarly, throughout this specification, the same modified siRNA referred to by the same number refers to the same modified siRNA in Table 3, and the same siRNA conjugate referred to by the same number refers to the same siRNA conjugate in Table 4.

[0227] siRNA synthesis methods

[0228] Nucleoside monomers are linked sequentially from 3' to 5' along the nucleotide arrangement using the conventional solid-phase phosphorous amide method. Each linkage of a nucleoside monomer involves four steps: deprotection, coupling, oxidation or sulfidation, and capping. When two nucleotides are linked using a phosphate ester, the linkage of the next nucleoside monomer involves these four steps. When two nucleotides are linked using a thiophosphate ester, the linkage of the next nucleoside monomer involves these four steps. The present invention selects nucleotide monomers based on the target synthetic sequence. The selected nucleotide monomers are those commonly used 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, when present in oligonucleotides, are linked together via 5'-3' phosphodiester bonds or 5'-3' thiophosphate groups. When, for example, the last nucleotide in the 5'-3' direction has a hydroxyl group at the 3' position, this is achieved using conventional methods in the art.

[0229] For example, the synthesis conditions for the siRNA in this application can be as follows:

[0230] The nucleoside monomer was provided in a 0.1 M acetonitrile solution. The deprotection reaction conditions were the same for each step: 25 °C, 70 seconds, and the deprotection reagent was a dichloromethane solution of dichloroacetic acid (3% v / v). The molar ratio of dichloroacetic acid to the 4,4'-dimethoxytriphenylmethyl protecting group on the solid support was 5:1.

[0231] The coupling reaction conditions included: a reaction temperature of 25°C, a reaction time of 600 seconds, a coupling reagent selected from a 0.5M acetonitrile solution of 5-ethylthio-1H-tetrazole (ETT), a molar ratio of nucleic acid sequence to nucleoside monomer linked on the solid-phase support of 1:10, and a molar ratio of nucleic acid sequence to coupling reagent linked on the solid-phase support of 1:65.

[0232] The oxidation reaction conditions included: a reaction temperature of 25°C, a reaction time of 15 seconds, and the oxidizing agent being selected from 0.05M iodine water. The molar ratio of the oxidizing agent to the nucleic acid sequence linked on the solid-phase support in the coupling step was 30:1. The reaction was carried out in a mixed solvent of tetrahydrofuran:water:pyridine = 3:1:1 (volume ratio).

[0233] The sulfidation reaction conditions included: a reaction temperature of 25°C, a reaction time of 300 seconds, and the sulfidation reagent being selected from hydroflavin. The molar ratio of the sulfidation reagent to the nucleic acid sequence linked on the solid-phase support in the coupling step was 120:1. The reaction was carried out in a mixed solvent of acetonitrile and pyridine at a volume ratio of 1:1.

[0234] The capping reaction conditions included: a reaction temperature of 25°C, a reaction time of 15 seconds, and a capping reagent selected from a 1:1 molar ratio of CapA (10% acetic anhydride acetonitrile solution) and CapB (10% N-methylimidazolium pyridine / acetonitrile solution). The molar ratio of the capping reagent to the nucleic acid sequence linked on the solid-phase support was acetic anhydride:N-methylimidazolium:the nucleic acid sequence linked on the solid-phase support was 1:1:1. After all nucleoside monomers were linked, the nucleic acid sequence linked on the solid-phase support was sequentially subjected to ammonolysis, purification, and desalting to obtain the siRNA sense and antisense strands. Finally, the two strands were heated and annealed to obtain the product.

[0235] Methods for ammonolysis, purification, desalting, and annealing are well known in the art. For example, ammonolysis is performed by contacting the nucleotide sequence linked to a solid-phase support with concentrated ammonia; purification is performed by chromatography; desalting is performed by reversed-phase chromatography; and cooling is performed by gradually cooling after mixing sense and antisense strands in equimolar ratios under different stringent conditions.

[0236] Synthesis methods of siRNA conjugates

[0237] The first step involves reacting DMTr-L96 with succinic anhydride to obtain compound L96-A:

[0238] Preparation process: DMTr-L96, succinic anhydride, 4-dimethylaminopyridine, and diisopropylethylamine were added to dichloromethane and stirred at 25°C for 24 hours. The reaction solution was then washed with 0.5M triethylamine phosphate, and the aqueous phase was washed three times with dichloromethane. The combined organic phases were evaporated to dryness under reduced pressure to obtain the crude product. Then, column chromatography was used to purify the crude product L96-A. The synthetic route is shown below:

[0239]

[0240] The second step involves reacting L96-A with NH2-SPS to obtain L96-B:

[0241] Preparation process: L96-A, O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU), and diisopropylethylamine (DIPEA) were mixed and dissolved in acetonitrile. The mixture was stirred at room temperature for 5 minutes to obtain a homogeneous solution. Aminomethyl resin (NH2-SPS, 100-200 mesh) was added to the reaction solution, and the reaction was initiated at 25°C in a shaker. After 18 hours of reaction, the mixture was filtered. The filter cake was washed successively with dichloromethane and acetonitrile to obtain the filter cake. The obtained filter cake was subjected to a capping reaction with a CapA / CapB mixed solution to obtain L96-B (synthetic route shown below), which is the solid-phase support containing the conjugated molecules.

[0242]

[0243] The third step is the synthesis of siRNA conjugates:

[0244] Preparation process: Using L96-B as a solid-phase support, the siRNA sense strand linked to the conjugate molecule was synthesized according to the siRNA molecule synthesis method described above. The siRNA antisense strand was synthesized using the siRNA molecule synthesis method described above. Annealing was performed to generate the siRNA conjugate of this application.

[0245] Example

[0246] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0247] For any experimental steps or conditions not specified in the following examples, the procedures or conditions described in the literature in this field can be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0248] Example 1: A siRNA for inhibiting NR1H3 gene expression

[0249] This embodiment provides an siRNA for inhibiting NR1H3 gene expression. The nucleotide sequence of the siRNA is designed based on the target mRNA (SEQ ID NO.92). The siRNA and its sequence are shown in Table 1, and the core sequence of the siRNA is shown in Table 2.

[0250] SEQ ID NO.92:

[0251]

[0252] Table 1. siRNAs and their sequences that inhibit NR1H3 gene expression

[0253]

[0254]

[0255]

[0256] Table 2. Core sequence of siRNA that inhibits NR1H3 gene expression

[0257]

[0258] Example 2: A modified siRNA for inhibiting NR1H3 gene expression

[0259] This embodiment provides a modified siRNA for inhibiting NR1H3 gene expression. The modified siRNA is based on Example 1, with the following modifications: on the sense strand, positions 7, 9, 10, and 11 are fluorinated at 2', and the other positions are methoxylated at 2'; the bases 1 and 2 are linked by a thiophosphate bond. On the antisense strand, positions 2, 6, 14, and 16 are fluorinated at 2', and the other positions are methoxylated at 2'; the bases 1 and 2 are linked by a thiophosphate bond, and the bases from the end of the 1-2-3 strand are linked by a thiophosphate bond. The first base is not linked to a 5' phosphate group or a 5' phosphate derivative group, as shown in the section marked M6 in Table 3.

[0260] Alternatively, the modified siRNA is based on Example 1, with the following configuration: from the 5' end to the 3' end, positions 7, 9, 10, and 11 of the sense strand are 2' fluorinated, and the other positions are 2' methoxylated; the bases 1 and 2 are linked by a thiophosphate bond; positions 2, 6, 14, and 16 of the antisense strand are 2' fluorinated, and the other positions are 2' methoxylated; the bases 1 and 2 are linked by a thiophosphate bond, and the bases from the end of the 1, 2, and 2 are linked by a thiophosphate bond; the first base is a nucleotide linked by a trans-vinylphosphonate group, as shown in the section marked M8 in Table 3.

[0261] Alternatively, the modified siRNA is based on Example 1, with the following configuration: from the 5' end to the 3' end, the 5, 7, 8, and 9 positions of the sense strand are 2' fluorinated, and the other positions are 2' methoxylated; the 1, 2 bases are linked to the 2, 3 bases by a thiophosphate bond; the 2, 6, 14, and 16 positions of the antisense strand are 2' fluorinated, and the other positions are 2' methoxylated; the 1, 2 bases are linked to the 2, 3 bases and the reciprocal 1, 2, and 2, 3 bases by a thiophosphate bond; and the first base is a nucleotide linked by a trans-vinylphosphonate group, as shown in the section marked M11 in Table 3.

[0262] Alternatively, the modified siRNA is based on Example 1, with the following configuration: from the 5' end to the 3' end, positions 3, 7, 8, and 9 of the sense strand are 2' fluorinated, and the other positions are 2' methoxylated; the bases 1 and 2 are linked by a thiophosphate bond; the bases 2, 7, 10, and 14 of the antisense strand are 2' fluorinated, and the other positions are 2' methoxylated; the bases 1 and 2 are linked by a thiophosphate bond, and the bases from the end of the 1, 2, and 2 are linked by a thiophosphate bond; the first base is a nucleotide linked by a trans-vinylphosphonate group, as shown in the section marked M44 in Table 3.

[0263] Alternatively, the modified siRNA is based on Example 1, with the following configuration: from the 5' end to the 3' end, positions 3, 7, 9, and 11 of the sense strand are 2' fluorinated, and the other positions are 2' methoxylated; the bases 1 and 2 are linked by a thiophosphate bond; the bases 2, 7, 10, and 14 of the antisense strand are 2' fluorinated, and the other positions are 2' methoxylated; the bases 1 and 2 are linked by a thiophosphate bond, and the bases from the end of the 1, 2, and 2 are linked by a thiophosphate bond; the first base is a nucleotide linked by a trans-vinylphosphonate group, as shown in the section marked M45 in Table 3.

[0264] Alternatively, the modified siRNA is based on Example 1, with the following orientation from the 5' end to the 3' end: positions 3, 7, 8, and 9 of the sense strand are 2' fluorinated, and the other positions are 2' methoxylated; an invAb group is attached to the 3' end; thiophosphate bonds connect the 1, 2 and 2, 3 bases and the penultimate base to invAb; positions 2, 7, 10, and 14 of the antisense strand are 2' fluorinated, and the other positions are 2' methoxylated; thiophosphate bonds connect the 1, 2 and 2, 3 bases and the penultimate 1, 2, and 2, 3 bases; the first base is a nucleotide linked by a trans-vinylphosphonate group, see the part marked M46 in Table 3;

[0265] Alternatively, the modified siRNA is based on Example 1, with the following orientation from the 5' end to the 3' end: positions 3, 7, 9, and 11 of the sense strand are 2' fluorinated, and the other positions are 2' methoxylated; an invAb group is attached to the 3' end; thiophosphate bonds connect the 1, 2 and 2, 3 bases and the penultimate base to invAb; positions 2, 7, 10, and 14 of the antisense strand are 2' fluorinated, and the other positions are 2' methoxylated; thiophosphate bonds connect the 1, 2 and 2, 3 bases and the penultimate 1, 2 and 2, 3 bases; the first base is a nucleotide linked by a trans-vinylphosphonate group, see the part marked M47 in Table 3;

[0266] Alternatively, the modified siRNA is based on Example 1, with the following configuration: from the 5' end to the 3' end, positions 7, 9, and 14 of the sense strand are 2' fluorinated, and the other positions are 2' methoxylated; the bases 1 and 2 are linked by a thiophosphate bond; the bases 2, 7, 10, and 14 of the antisense strand are 2' fluorinated, and the other positions are 2' methoxylated; the bases 1 and 2 are linked by a thiophosphate bond, and the bases from the end 1, 2, and 2 are linked by a thiophosphate bond; the first base is a nucleotide linked by a trans-vinylphosphonate group, as shown in the section marked M48 in Table 3.

[0267] Alternatively, the modified siRNA is based on Example 1, with the following configuration: from the 5' end to the 3' end, positions 3, 7, 9, and 11 of the sense strand are fluorinated at the 2' position, and the other positions are methoxylated at the 2' position; the bases 1 and 2 are linked by a thiophosphate bond; the bases 2, 3, 5, 7, 10, 12, and 14 of the antisense strand are fluorinated at the 2' position, position 6 is modified with GNA, the other positions are methoxylated at the 2' position, and the bases 1, 2 and 2, 3 are linked by a thiophosphate bond, and the first base is a nucleotide linked by a trans-vinylphosphonate group, as shown in the part marked M49 in Table 3;

[0268] Alternatively, the modified siRNA is based on Example 1, with the following configuration: from the 5' end to the 3' end, positions 3, 7, 9, and 11 of the sense strand are 2' fluorinated, and the other positions are 2' methoxylated; the bases 1 and 2 are linked by a thiophosphate bond; positions 2, 7, and 14 of the antisense strand are 2' fluorinated, positions 5 and 12 are deoxyribonucleotides, and the other positions are 2' methoxylated; the bases 1 and 2 are linked by a thiophosphate bond, and the bases from the end 1, 2, and 2 are linked by a thiophosphate bond; the first base is a nucleotide linked by a trans-vinylphosphonate group, as shown in the section marked M50 in Table 3.

[0269] Alternatively, the modified siRNA is based on Example 1, with the following configuration: positions 3, 7, 8, and 9 of the sense strand are fluorinated at 2', and the other positions are methoxylated at 2'. An invAb group is attached to both the 5' and 3' ends. The invAb is linked to the first base, the 1st and 2nd bases, and the penultimate base to the invAb via a thiophosphate bond. The antisense strand has positions 2, 7, 10, and 14 of the antisense strand being fluorinated at 2', and the other positions are methoxylated at 2'. The 1st and 2nd bases are linked to the 2nd and 3rd bases, and the penultimate 1st and 2nd bases are linked via a thiophosphate bond. The first base is a nucleotide linked to a trans-vinylphosphonate group, as shown in the section marked M51 in Table 3.

[0270] Table 3. Modified siRNAs and their sequences that inhibit NR1H3 gene expression

[0271]

[0272]

[0273]

[0274]

[0275] In Table 3, uppercase letters C, G, U, and A represent ribonucleotides; lowercase letters c, g, u, and a all represent methoxylated nucleotides; lowercase letter f indicates that the nucleotide adjacent to the left of f is fluorinated; lowercase letter s indicates that the two nucleotides adjacent to s are linked by a thiophosphate group; the string EVP indicates that the 5' end of the nucleotide adjacent to the right of the string is connected to a trans-vinylphosphonate group; the string (invAb) indicates that the 3' or 5' end of the nucleotide adjacent to the left or right of the string is connected to a reverse debased deoxyribose residue (invAb group); the string s( The string (invAb) indicates that the 3' end of the nucleotide adjacent to the left of the string is connected to a reverse debased deoxyribose residue (invAb group), and the nucleotide adjacent to the left of the lowercase letter s is connected to the reverse debased deoxyribose residue by a thiophosphate group; the string (invAb)s indicates that the 5' end of the nucleotide adjacent to the right of the string is connected to a reverse debased deoxyribose residue (invAb group), and the nucleotide adjacent to the right of the lowercase letter s is connected to the reverse debased deoxyribose residue by a thiophosphate group; the lowercase letter n indicates that the nucleotide adjacent to the left of the letter n is a nucleotide modified with ethylene glycol nucleic acid (GNA); the lowercase letter d indicates that the nucleotide adjacent to the right of the letter d is replaced by a deoxyribonucleotide.

[0276] Example 3: A siRNA conjugate for inhibiting NR1H3 gene expression

[0277] This embodiment provides an siRNA conjugate for inhibiting NR1H3 gene expression. The siRNA conjugate is composed of the siRNA of Example 1 and a GalNAc group conjugated to the siRNA; or, the siRNA conjugate is composed of the modified siRNA of Example 2 and a GalNAc group conjugated to the modified siRNA, as shown in Table 4.

[0278] The siRNA conjugate has a structure as shown in Formula XXI (the double helix structure represents the siRNA, and the linker is attached to the 3' end of the positive strand of the siRNA):

[0279]

[0280] In Formula XXI, the conjugated group N-glucose amino acid is also called L96, and has the structure shown in Formula XIII:

[0281]

[0282] Table 4. siRNA conjugates and their sequences that inhibit NR1H3 gene expression

[0283]

[0284]

[0285]

[0286] Experimental Example 1: Verification of the inhibitory activity of siRNA, its modifications, and conjugates for inhibiting NR1H3 gene expression

[0287] This experimental example provides a validation experiment of the inhibitory activity of siRNA, its modifications, and conjugates for inhibiting NR1H3 gene expression. The experimental procedure is as follows:

[0288] 1. Experimental materials

[0289] HepG2 cells were purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number SCSP-510.

[0290] RNA extraction kit 96 Kit, purchased from Qiagen, item number 74182;

[0291] RNAiMAX transfection reagent, purchased from Invitrogen, catalog number 13778-150;

[0292] MEM medium, purchased from Gibco, catalog number 41090036;

[0293] Reverse transcription kit ( III. 1st Strand cDNA Synthesis Kit (+gDNA wiper), purchased from Vazyme, catalog number R312-02;

[0294] TaqMan TM Fast Advanced Master Mix, purchased from Applied Biosystems, product number 4369016;

[0295] Opti-MEM: serum-reduced culture medium, purchased from Gibco, catalog number 31985070;

[0296] Random hexamers, purchased from Thermo, item number N8080127;

[0297] NR1H3 probe, purchased from Thermo, part number HS00172885_m1;

[0298] GAPDH probe, purchased from Thermo, part number Hs99999905_m1.

[0299] 2. Experimental Methods

[0300] 2.1 Detection of inhibition rate

[0301] HepG2 cells were seeded in fresh MEM medium in 96-well plates and cultured for 48 hours in a 5% (v / v) CO2 incubator at 37°C. After culture, the HepG2 cells were digested with trypsin (purchased from GIBCO). After digestion, the cells were rinsed with PBS buffer and then resuspended in MEM medium without penicillin-streptomycin mixture (PS) to prepare a density of 1.11 × 10⁶ cells / well. 5 Cell suspension per mL.

[0302] The siRNA to be tested, the modified siRNA to be tested, or the siRNA conjugate to be tested (collectively referred to as siRNA in this embodiment for ease of description) were prepared into a 100 μM siRNA stock solution using ultrapure distilled water. 2 μL of the 100 μM siRNA stock solution was added to 18 μL of ultrapure distilled water to obtain a 10 μM siRNA stock solution Q. 2 μL of the 10 μM siRNA stock solution Q was added to 18 μL of ultrapure distilled water to obtain a 1 μM siRNA stock solution Y. 2 μL of the 1 μM siRNA stock solution Y was added to 18 μL of ultrapure distilled water to obtain a 0.1 μM siRNA stock solution E. 2 μL of the 0.1 μM siRNA stock solution E was added to 98 μL of ultrapure distilled water... Opti-MEM was used to obtain a siRNA dilution W with a concentration of 2 nM; 2 μL of siRNA stock solution Y with a concentration of 1 μM was added to 98 μL of Opti-MEM to obtain a siRNA dilution Z with a concentration of 20 nM.

[0303] The cell suspension was seeded into 96-well plates at a rate of 90 μL per well (i.e., 10,000 cells / well). After seeding, HepG2 cells in the 96-well plates were transfected with low and high concentrations of the target siRNA, respectively, as follows:

[0304] Pick 3 μL of RNAiMAX transfection reagent was added to 97 μL of Opti-MEM to obtain... RNAiMAX transfection reagent dilution solution; The RNAiMAX transfection reagent diluent and 2 nM siRNA diluent W were mixed at a volume ratio of 1:1 and allowed to stand for 5 minutes to obtain the transfection mixture. 10 μL of the transfection mixture was added to a 96-well plate (final volume 100 μL, the concentration of siRNA in this transfection system is 0.1 nM) and cultured in a 5% (v / v) CO2, 37℃ cell culture incubator for 48 h to transfect HepG2 cells.

[0305] Pick 3 μL of RNAiMAX transfection reagent was added to 97 μL of Opti-MEM to obtain... RNAiMAX transfection reagent dilution solution; The RNAiMAX transfection reagent diluent and 20 nM siRNA diluent Z were mixed at a 1:1 volume ratio and allowed to stand for 5 minutes to obtain the transfection mixture. 10 μL of the transfection mixture was added to a 96-well plate (final volume 100 μL, siRNA concentration in this transfection system is 1 nM), and the plate was incubated for 48 h in a 5% (v / v) CO2, 37°C cell culture incubator to transfect HepG2 cells. Two replicates were set for each concentration (1 nM and 0.1 nM).

[0306] After transfection, total RNA was extracted from the transfected HepG2 cells according to the RNA extraction kit instructions. Then, the extracted total RNA was reverse transcribed into cDNA using a reverse transcription kit. The reverse transcription process is as follows:

[0307] (1) Remove gDNA using gDNAase according to the system in Table 5;

[0308] Table 5. gDNA Removal System

[0309] Components Volume / μL 5×gDNA wiper Mix 2 Sample (RNA) 8

[0310] gDNA removal reaction procedure: 42℃, 2 min; 4℃, stand;

[0311] (2) Perform reverse transcription according to the system in Table 6;

[0312] Table 6. Reverse Transcription System

[0313] Components Volume / μL The mixture obtained in step (1) 10 10×RT Mix 2 HiScriptⅢEnzyme Mix 2 <![CDATA[Oligo(dT) 20 VN]]> 1 Random hexamers 1 <![CDATA[RNase-free ddH2O]]> 4

[0314] Reverse transcription reaction program: 50℃, 15 min; 85℃, 5 s.

[0315] (3) Store the reverse transcription product obtained in step (2) at 4°C for real-time PCR analysis.

[0316] After reverse transcription is completed, the reverse transcription product obtained in step (2) is subjected to real-time PCR analysis; the real-time PCR analysis process is as follows:

[0317] (1) Prepare the qPCR reaction system according to Table 7. During the entire operation, all reagents should be placed on ice.

[0318] Table 7. qPCR reaction system for detecting the NR1H3 gene

[0319]

[0320] Table 8. qPCR reaction system for detecting the internal reference gene (GAPDH)

[0321]

[0322] qPCR reaction program: 50℃, 2 min, 95℃, 10 min; 95℃, 15 sec, 60℃, 1 min (40 cycles of this operation).

[0323] (2) Using Quant Studio 6Flex software with default settings, the Ct value was automatically calculated; the relative expression level of the target gene (NR1H3 mRNA) was calculated based on the Ct value; the inhibition rate of the target gene (NR1H3 mRNA) expression level by the test siRNA was calculated based on the relative expression level of the target gene (NR1H3 mRNA); the method for calculating the relative expression level of the target gene (NR1H3 mRNA) is as follows:

[0324] ΔCt=Ct(NR1H3 gene)–Ct(GAPDH);

[0325] ΔCt = ΔCt(sample group) - ΔCt(mock group);

[0326] mRNA expression relative to the Mock group = 2 -ΔΔCt ;

[0327] In the formula, the sample group is the group with the siRNA to be tested added; the mock group is the group without siRNA compared with the test sample group.

[0328] The method for calculating the inhibition rate of the siRNA on the expression level of the target gene (NR1H3 mRNA) is as follows:

[0329] Inhibition rate (%) = (Relative expression level of mRNA in the Mock group – Relative expression level of mRNA in the test sample group) / Relative expression level of mRNA in the Mock group × 100%.

[0330] 2.2, IC 50 Detection

[0331] The concentration range (nM) of the siRNA to be tested was set as follows: 10, 2.5, 0.625, 0.16, 0.04, 0.01, 0.0025, and 0.0006. Based on section 2.1, the inhibition rates of the siRNA to be tested were obtained by adjusting the concentration of the siRNA in the transfection system within the measured concentration range. The log value of the siRNA concentration was used as the X-axis, and the percentage inhibition rate was used as the Y-axis. The dose-response curve was fitted using the "log (inhibitor) vs. response-variable slope" function module of the analysis software GraphPad Prism 8 to obtain the IC50 of each siRNA to be tested. 50 Value; where the fitting formula is as follows:

[0332] Y=Bottom+(Top-Bottom) / (1+10^((logIC 50 -X)*HillSlope));

[0333] In the formula, Top represents the percentage inhibition rate at the top plateau, with the Top standard of the curve ranging from 80% to 120%; Bottom represents the percentage inhibition rate at the bottom plateau, with the Bottom of the curve ranging from -20% to 20%; and HillSlope represents the slope of the percentage inhibition rate curve.

[0334] 3. Experimental Results

[0335] The inhibition rates and IC50 values ​​of the siRNA from Example 1, the modified siRNA from Example 2, and the siRNA conjugate from Example 3 on the target gene (NR1H3 mRNA) were measured. 50 The values ​​and detection results are shown in Tables 9 and 10. As can be seen from Tables 9 and 10, the modified siRNA of Example 2 and the siRNA conjugate of Example 3 both exhibited certain inhibitory activity against NR1H3 mRNA expression; among them, N-ER-FY049089, N-ER-FY049086, N-ER-FY049087, N-ER-FY049020, N-ER-FY049088, N-ER-FY049021, N-ER-FY049089M44L96, N-ER-FY049089M48L96, and N-ER-FY049089M50L96 showed high inhibitory activity against NR1H3.

[0336] Table 9. Inhibition rate of unmodified / modified siRNA on the target gene (NR1H3 mRNA)

[0337]

[0338]

[0339]

[0340] Table 10. IC50 of modified siRNA against the target gene (NR1H3 mRNA) 50 value

[0341] siRNA name <![CDATA[IC 50 (nM)]]> N-ER-FY049020M6 0.1095 N-ER-FY049021M6 0.0600 N-ER-FY049038M6 0.0671 N-ER-FY049039M6 0.0341 N-ER-FY049040M6 0.0167 N-ER-FY049041M6 0.0927 N-ER-FY049044M6 0.1852 N-ER-FY049057M6 0.1579 N-ER-FY049062M6 0.2139

[0342] Experimental Example 2: Verification of the inhibitory activity of siRNA conjugates used to inhibit NR1H3 gene expression

[0343] This experimental example provides a validation experiment of the inhibitory activity of siRNA conjugates used to inhibit NR1H3 gene expression. The experimental procedure is as follows:

[0344] 1. Experimental materials

[0345] Human primary hepatocytes (PHH cells) were provided by Huizhiheyuan.

[0346] PHH culture medium: purchased from Huizhiheyuan, product number: 0193301.32;

[0347] opti-MEM medium, purchased from Gibco, catalog number 31985070;

[0348] RNAiMAX transfection reagent, purchased from Invitrogen, catalog number 13778-150;

[0349] RNA extraction kit, purchased from QIAGEN, catalog number 74106;

[0350] Reverse transcription kit ( III. 1st Strand cDNA Synthesis Kit (+gDNA wiper), purchased from Vazyme, catalog number R312-02;

[0351] TaqMan TM Fast Advanced Master Mix, purchased from Applied Biosystems, product number 4369016;

[0352] Random hexamers, purchased from Thermo, item number N8080127;

[0353] NR1H3 probe, purchased from Thermo, part number HS00172885_m1;

[0354] GAPDH probe, purchased from Thermo, part number Hs99999905_m1.

[0355] 2. Experimental Methods

[0356] 2.1 Transfection

[0357] siRNA conjugates (with final concentrations of 5 nM and 0.5 nM, in duplicate) were transfected into PHH cells, as described below:

[0358] Following the experimental method in Example 1, cryopreserved PHH cells were taken, thawed, counted, and adjusted to a cell count of 6 × 10⁶. 5 cells / mL, simultaneously applied The RNAiMAX transfection reagent was used to transfect the siRNA conjugate into cells, seeding them at a density of 54,000 cells per well in a 96-well plate with 100 μL of culture medium (containing PHH medium and siRNA conjugate) per well. Cells were incubated in a 5% CO2, 37°C incubator. Forty-eight hours after transfection, the culture medium was removed and cells were collected for total RNA extraction. Total RNA was extracted from the transfected HepG2 cells according to the RNA extraction kit instructions, and the inhibition rate of the siRNA conjugate on the expression of the target gene (NR1H3 mRNA) was detected using the same method as in Example 1. The difference in the inhibition rate detection compared to Example 1 was that the PCR reaction program was: 95°C for 10 minutes, followed by cycling at 95°C for 15 seconds, then at 60°C for 60 seconds, for a total of 40 cycles.

[0359] 2.2 Free intake

[0360] siRNA conjugates (with final concentrations of 200 nM and 10 nM, in duplicate) were introduced into PHH cells via free uptake, as described below:

[0361] Following the experimental method in Example 1, cryopreserved PHH cells were taken, thawed, counted, and adjusted to a cell count of 6 × 10⁶. 5 Cells / mL, along with siRNA conjugate, were seeded into 96-well plates at a density of 54,000 cells per well, with 100 μL of culture medium (containing PHH medium and siRNA conjugate) per well. Cells were incubated in a 5% CO2, 37°C incubator. After 48 hours of free uptake, the culture medium was removed and cells were collected for total RNA extraction. Total RNA was extracted from transfected HepG2 cells according to the RNA extraction kit instructions, and the inhibition rate of the siRNA conjugate on the expression of the target gene (NR1H3 mRNA) was detected according to the experimental method in Example 1. The difference in the inhibition rate detection compared to Example 1 was that the PCR reaction program was: 95°C for 10 minutes, then cycling at 95°C for 15 seconds, followed by 60°C for 60 seconds, for a total of 40 cycles.

[0362] 3. Experimental Results

[0363] The inhibition rate of the siRNA conjugate of Example 3 on the expression of the target gene (NR1H3 mRNA) was detected, and the results are shown in Table 11. As can be seen from Table 11, the siRNA conjugate of Example 3 can generally effectively inhibit the expression of NR1H3 mRNA under the conditions of transfection reagent intervention and free uptake.

[0364] Table 11. Inhibition rate of siRNA conjugate on the expression of target gene (NR1H3 mRNA)

[0365]

[0366]

[0367] Note: "--" indicates that the result was not detected.

[0368] Experiment Example 3: In vitro stability experiment of rat liver homogenate of siRNA conjugate

[0369] This experimental example provides an in vitro stability verification experiment for siRNA conjugates used to inhibit NR1H3 gene expression. The experimental procedure is as follows: 1. Experimental materials

[0370] 1M MgCl2, purchased from Beyotime, item number ST269;

[0371] Ammonium bicarbonate, purchased from Tianjin Guangfu Fine Chemical Co., Ltd., item number GB663-78;

[0372] Sodium dihydrogen phosphate, purchased from Tianjin Guangfu Fine Chemical Co., Ltd., item number GB / T1267-1999;

[0373] Disodium hydrogen phosphate, purchased from Tianjin Guangfu Fine Chemical Co., Ltd., item number 10039-32-4;

[0374] Acetonitrile, purchased from Honeywell, product number AH015-4HC;

[0375] Methanol, purchased from Honeywell, product number AH230-4HC;

[0376] 0.5M EDTA, purchased from Beyotime, item number ST066.

[0377] 2. Experimental Procedure

[0378] 2.1. Prepare liver homogenate

[0379] 2.1.1 Preparation of grinding slurry

[0380] Prepare the grinding fluid according to Table 12.

[0381] Table 12. Grinding slurry and its preparation method

[0382]

[0383] 2.1.2. Tissue homogenization

[0384] Rat liver tissue (using SD rats, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) was mixed with homogenizing solution at a ratio of 100 mg: 5 mL to prepare liver homogenate (concentration of 20 mg / mL); after adding grinding beads to the liver homogenate, it was placed in a homogenizer for grinding to obtain liver homogenate homogenate; the grinding parameters are shown in Table 13.

[0385] Table 13. Grinding parameters

[0386] Running speed 60Hz Runtime 30s Pause time 15s Number of runs 4 times Operating temperature -20℃

[0387] 3. Sample preparation

[0388] The siRNA conjugate was prepared into a 1 mg / mL solution using enzyme-free water and set aside for use (this is the nucleic acid sample). The internal standard sample was prepared into a 0.125 mg / mL solution using enzyme-free water and set aside for use.

[0389] 4. Sample incubation

[0390] Add 250 μL of liver homogenate and 50 μL of nucleic acid sample to a 2 mL enzyme-free tube to obtain a biological sample of 300 μL. Vortex the biological sample and let it stand for 5 min. After standing, divide the biological sample into two tubes of 100 μL each. After dividing the tubes, incubate the biological sample at 37 °C for 48 h to obtain the incubated biological sample.

[0391] 5. Biological sample processing

[0392] After incubation, vortex each 100 μL of biological sample and then mix. After mixing, add 300 μL of Clarity OTX Lysis-loading Buffer (purchased from Agilent-FinnoMed, catalog number AL0-8579) to each 100 μL of biological sample and vortex again. After vortexing, let the biological sample stand for 30 min. After standing, add 100 μL of internal standard solution to each 100 μL of biological sample and vortex again. After vortexing, let the biological sample stand for 5 min. After standing, centrifuge the biological sample at 7000 rpm for 1 min. After centrifugation, collect the supernatant for later use (total supernatant sample volume is approximately 500 μL).

[0393] 6. Solid-phase extraction:

[0394] 6.1 Preparation of solid phase extraction reagents

[0395] Activator: Add 200 mL of methanol to the mobile phase bottle and label it as activator;

[0396] Equilibrium buffer: Prepare a 1M phosphate buffer solution, dilute it 100 times with water, adjust the pH to 5.5 with phosphate, mix well, and label it as equilibrium buffer solution; the formula of the 1M phosphate buffer solution is: 877mL sodium dihydrogen phosphate (1.56g / L) + 123mL disodium hydrogen phosphate (3.58g / L).

[0397] Rinse solution: Take 500 mL of equilibrium solution into a 1 L mobile phase bottle, add 500 mL of acetonitrile, adjust the pH to 5.5 with phosphoric acid, mix well, and label as rinsing solution;

[0398] Eluent: Weigh 7.9 g of ammonium bicarbonate into a 1 L mobile phase bottle, add 1 L of water, take 500 mL of ammonium bicarbonate solution into a 1 L mobile phase bottle, add 500 mL of acetonitrile, adjust the pH to 9 with sodium hydroxide, mix well, and label as eluent;

[0399] 6.2 Extraction Steps

[0400] Extract the supernatant sample according to Table 14 and collect the eluent.

[0401] Table 14. Extraction process of supernatant samples

[0402] step process activation 1 mL methanol, 10 min balance 2 x 1 mL 10 mM phosphate (pH = 5.5), first time 10 min, second time 10 min Sample 400 μL (4 / 5 of the total volume) rinse 2 x 1 mL 10 mM phosphate (pH = 5.5): 50% (v / v) acetonitrile, first time 20 min, second time 20 min. Washout 2 x 0.75 mL 100 mM ammonium bicarbonate (pH = 9): 50% (v / v) acetonitrile, first time 20 min, second time 20 min

[0403] 7. Post-processing

[0404] The eluent (600 μL each time, totaling 1200 μL) was placed in a 2 mL EP tube and concentrated under vacuum at 1800 rpm for 6 hours to obtain the concentrated sample. 100 μL of mobile phase (initial ratio) was added to the concentrated sample for reconstitution, and the sample was centrifuged at 5000 rpm for 2 min. After centrifugation, 10 μL of the supernatant was injected into a high-resolution mass spectrometer. The remaining antisense strand (AS) of the siRNA conjugate was semi-quantitatively detected using LC-MS / MS, calculated as: AS strand remaining percentage % = AS strand percentage MS intensity The sum of / AS chains and all related degradation products MS intensity ×100%; where MS intensity is the mass spectrometry intensity signal value, and the detection results are shown in Table 15.

[0405] Table 15. Remaining antisense strand AS of siRNA conjugates

[0406] siRNA ID AS remaining amount / % N-ER-FY049089M11L96 91.34 N-ER-FY049089M44L96 98.05 N-ER-FY049089M46L96 91.44 N-ER-FY049089M48L96 99.81 N-ER-FY049089M50L96 96.64 N-ER-FY049089M51L96 98.86

[0407] The results in Table 15 show that the siRNA conjugates of Example 3 all exhibited good in vitro stability in rat liver homogenate, with N-ER-FY049089M11L96, N-ER-FY049M44L96, N-ER-FY049089M46L96, N-ER-FY049M48L96, N-ER-FY049M50L96, and N-ER-FY049M51L96 demonstrating excellent in vitro stability in rat liver homogenate.

[0408] Experiment Example 4: Silent Effect of siRNA Conjugates on Mice Expressing the Human NR1H3 (hNR1H3) Gene

[0409] (1) AAV was used to construct a mouse model overexpressing the hNR1H3 gene.

[0410] Six- to eight-week-old male C57BL / 6 mice (provided by Beijing Vital River Laboratory Animal Technology Co., Ltd.) were introduced into the facility. A single tail vein injection of adeno-associated virus (AAV) containing the hNR1H3 gene (pAAV[Exp]-CBh>SEAP(ns):T2A:{LXRACDS+UTR}, virus provided by Yunzhou Biotechnology (Guangzhou) Co., Ltd.) was administered to induce target gene overexpression. The injection volume was 100 μL (5 × 10⁻⁶ mcg). 11 vg) / animal, then fed with regular feed.

[0411] (2) Efficacy study of silencing siRNA in hNR1H3 mouse model

[0412] Fourteen days after AAV virus injection, mice were divided into groups of five. Mice were subcutaneously administered a single 3 mg / kg dose of the siRNA conjugate described in this application at a volume of 5 μL / g in RNase-free sterile PBS. The control group received the same volume of RNase-free sterile PBS. Serum was collected from mice on days 7, 14, 21, 28, 35, 42, 49, 56, and 63 post-administration. The serum solutions were stored at -80°C for long-term preservation. Before testing, the solutions were thawed on ice, centrifuged, and the supernatant was used for protein content analysis using Phospha-Light. TM SEAP reporter gene assay system (Invitrogen) TM (Purchased from Thermo Fisher Scientific, catalog number T1017) SEAP protein expression assay (reflecting hNR1H3 protein expression). The inhibition rate (%) of the siRNA conjugate in mice expressing the human NR1H3 (hNR1H3) gene was calculated according to the formula: Inhibition rate % = (1 - mean protein expression level in the treated group / mean protein expression level in the control group) × 100%. The results are shown in Table 16 and... Figure 1As shown in Table 17. Among them, N-ER-FY049Y01 is a positive reference, whose sequence is derived from NR1H3-1210-1230-1307 in the PCT patent application publication text WO2022 / 223515A2. Specific information is shown in Table 17 (the meaning of each letter is as noted in the previous notes on Table 3). Since the positive control has a large molecular weight, its dosage is 4 mg / kg after conversion based on the molecular weight.

[0413] Table 16. In vivo protein inhibition rate (%) of siRNA conjugates against hNR1H3

[0414] siRNA ID D7 D14 D21 D28 D35 D42 D49 D56 D63 N-ER-FY049Y01-4mg / kg 79.26 86.16 86.10 83.20 74.53 63.46 68.88 42.60 44.66 N-ER-FY049089M44L96-3mg / kg 86.33 88.70 90.89 85.60 74.83 70.94 82.80 67.35 66.04 N-ER-FY049089M46L96-3mg / kg 83.06 88.68 88.63 86.73 79.59 77.52 83.87 72.04 67.39 N-ER-FY049089M50L96-3mg / kg 79.99 83.42 83.40 88.39 81.37 74.27 79.96 57.10 61.21 N-ER-FY049089M51L96-3mg / kg 85.47 92.24 92.18 90.74 82.21 78.41 84.88 80.61 74.39

[0415] From Table 16 and Figure 1 It can be seen that the siRNA conjugates N-ER-FY049089M44L96, N-ER-FY049089M46L96, N-ER-FY049089M50L96, and N-ER-FY049089M51L96 of this application have high inhibitory activity against the hNR1H3 gene in vivo and can reduce the expression level of hNR1H3 for a long time.

[0416] Table 17 Sequence information of positive reference N-ER-FY049Y01

[0417]

[0418] The [ademAGalNAc] structure is as follows:

[0419]

[0420] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A siRNA for inhibiting NR1H3 gene expression, characterized in that, The siRNA contains a sense strand and an antisense strand; the sense strand and the antisense strand are at least partially anti-complementary to form a double-stranded region; The sense strand of the siRNA contains a first nucleotide sequence as shown in any one of SEQ ID NO.4, SEQ ID NO.15, SEQ ID NO.33, or SEQ ID NO.74 to SEQ ID NO.80; The antisense strand of the siRNA contains a second nucleotide sequence as shown in any one of SEQ ID NO.40, SEQ ID NO.51, SEQ ID NO.70, or SEQ ID NO.81–87.

2. The siRNA as described in claim 1, characterized in that, The first nucleotide sequence is further connected to a third nucleotide sequence at its 5' end and / or 3' end; the second nucleotide sequence is further connected to a fourth nucleotide sequence at its 5' end and / or 3' end; the length of the third nucleotide sequence and / or the fourth nucleotide sequence is 1 to 15 bp.

3. The siRNA as described in claim 1 or 2, characterized in that, The antisense strand of the siRNA and the mRNA of the NR1H3 gene are at least 90% partially complementary.

4. The siRNA according to any one of claims 1 to 3, characterized in that, The lengths of the justice chain and the antisense chain are the same, or the lengths of the justice chain and the antisense chain are different; When the lengths of the sense and antisense strands are the same, the siRNA has blunt ends; when the lengths of the sense and antisense strands are different, the siRNA has protruding ends.

5. The siRNA as described in claim 4, characterized in that, When the siRNA has blunt ends, the sense strand of the siRNA contains a nucleic acid molecule with a nucleotide sequence as shown in any one of SEQ ID NO.1 to SEQ ID NO.36, and the antisense strand of the siRNA contains a nucleic acid molecule that is partially or completely anticomplementary to the sense strand. When the siRNA has a protruding end, the sense strand of the siRNA contains a nucleic acid molecule with a nucleotide sequence as shown in any one of SEQ ID NO.1 to SEQ ID NO.36, and, except for the protruding end, the antisense strand of the siRNA contains a nucleic acid molecule that is partially or completely anticomplementary to the sense strand.

6. The siRNA as described in claim 4 or 5, characterized in that, When the siRNA has blunt ends, the nucleotide sequence of the sense strand of the siRNA is as shown in any one of SEQ ID NO.1 to SEQ ID NO.36, and the antisense strand of the siRNA is partially or completely anticomplementary to the sense strand. When the siRNA has a protruding end, the nucleotide sequence of the sense strand of the siRNA is as shown in any one of SEQ ID NO.1 to SEQ ID NO.36, and the nucleotide sequence of the antisense strand of the siRNA is as shown in any one of SEQ ID NO.37 to SEQ ID NO.73; or, the nucleotide sequence of the sense strand of the siRNA is as shown in any one of SEQ ID NO.1 to SEQ ID NO.36, and, except for the protruding end, the antisense strand of the siRNA is partially or completely anticomplementary to the sense strand.

7. The siRNA according to any one of claims 1 to 4, characterized in that, The sense strand of the siRNA contains a first nucleotide sequence as shown in SEQ ID NO.4, and the antisense strand of the siRNA contains a second nucleotide sequence as shown in SEQ ID NO.40; Alternatively, the sense strand of the siRNA contains a first nucleotide sequence as shown in SEQ ID NO.15, and the antisense strand of the siRNA contains a second nucleotide sequence as shown in SEQ ID NO.51; Alternatively, the sense strand of the siRNA contains a first nucleotide sequence as shown in SEQ ID NO.33, and the antisense strand of the siRNA contains a second nucleotide sequence as shown in SEQ ID NO.70; Alternatively, the sense strand of the siRNA contains a first nucleotide sequence as shown in SEQ ID NO.74, and the antisense strand of the siRNA contains a second nucleotide sequence as shown in SEQ ID NO.81; Alternatively, the sense strand of the siRNA contains a first nucleotide sequence as shown in SEQ ID NO.75, and the antisense strand of the siRNA contains a second nucleotide sequence as shown in SEQ ID NO.82; Alternatively, the sense strand of the siRNA contains a first nucleotide sequence as shown in SEQ ID NO.76, and the antisense strand of the siRNA contains a second nucleotide sequence as shown in SEQ ID NO.83; Alternatively, the sense strand of the siRNA contains a first nucleotide sequence as shown in SEQ ID NO.77, and the antisense strand of the siRNA contains a second nucleotide sequence as shown in SEQ ID NO.84; Alternatively, the sense strand of the siRNA contains a first nucleotide sequence as shown in SEQ ID NO.78, and the antisense strand of the siRNA contains a second nucleotide sequence as shown in SEQ ID NO.85; Alternatively, the sense strand of the siRNA contains a first nucleotide sequence as shown in SEQ ID NO.79, and the antisense strand of the siRNA contains a second nucleotide sequence as shown in SEQ ID NO.86; Alternatively, the sense strand of the siRNA may contain a first nucleotide sequence as shown in SEQ ID NO. 80, and the antisense strand of the siRNA may contain a second nucleotide sequence as shown in SEQ ID NO.

87.

8. The siRNA according to any one of claims 1 to 7, characterized in that, The sense strand of the siRNA contains a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.1, and the antisense strand contains a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.37; Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.2, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.38; Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.3, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.39; Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.4, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.40; Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.5, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.41; Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO. 6, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO. 42; Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.7, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.43; Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO. 8, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO. 44; Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO. 9, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO. 45; Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.10, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.46; Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.11, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.47; Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.12, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.48; Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.13, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.49; Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.14, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.50; Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.15, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.51; Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.16, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.52; Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.17, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.53; Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.18, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.54; Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.19, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.55; Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.20, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.56; Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.21, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.57; Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.22, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.58; Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.23, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.59; Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.24, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.60; Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.25, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.61; Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.26, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.62; Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.27, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.63; Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.28, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.64; Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.29, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.65; Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.29, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.66; Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.30, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.67; Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.31, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.68; Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.32, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.69; Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.33, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.70; Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.34, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.71; Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.35, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.72; Alternatively, the sense strand of the siRNA may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.36, and the antisense strand may contain a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.

73.

9. The siRNA according to any one of claims 1 to 8, characterized in that, At least one nucleotide in the sense or antisense strand of the siRNA is a modified nucleotide; the modification includes fluorination, methoxylation, thiophosphate modification, trans-vinylphosphonate modification, reverse debasing deoxyribose residue modification, ethylene glycol nucleic acid modification and / or deoxyribonucleotide substitution.

10. The siRNA as described in claim 9, characterized in that, The fluorinated nucleotides are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 7, 9, 10, and 11 of the sense strand are fluorinated nucleotides, and at least the nucleotides at positions 2, 6, 14, and 16 of the antisense strand are fluorinated nucleotides. Alternatively, the fluorinated nucleotides are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the 5th, 7th, 8th, and 9th nucleotides of the sense strand are fluorinated nucleotides, and at least the 2nd, 6th, 14th, and 16th nucleotides of the antisense strand are fluorinated nucleotides. Alternatively, the fluorinated nucleotides are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 3, 7, 8, and 9 of the sense strand are fluorinated nucleotides, and at least the nucleotides at positions 2, 7, 10, and 14 of the antisense strand are fluorinated nucleotides. Alternatively, the fluorinated nucleotides are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 3, 7, 9, and 11 of the sense strand are fluorinated nucleotides, and at least the nucleotides at positions 2, 7, 10, and 14 of the antisense strand are fluorinated nucleotides. Alternatively, the fluorinated nucleotides are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the 7th, 9th, and 14th nucleotides of the sense strand are fluorinated nucleotides, and at least the 2nd, 7th, 10th, and 14th nucleotides of the antisense strand are fluorinated nucleotides. Alternatively, the fluorinated nucleotides are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 3, 7, 9, and 11 of the sense strand are fluorinated nucleotides, and at least the nucleotides at positions 2, 3, 5, 7, 10, 12, and 14 of the antisense strand are fluorinated nucleotides. Alternatively, the fluorinated nucleotides are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 3, 7, 9, and 11 of the sense strand are fluorinated nucleotides, and at least the nucleotides at positions 2, 7, and 14 of the antisense strand are fluorinated nucleotides.

11. The siRNA as described in claim 9 or 10, characterized in that, The methoxylated nucleotides are located in the antisense and sense strands of the nucleotide sequence. In the sense strand, the nucleotides that are not fluorinated, not ethylene glycol-modified, and not substituted with deoxyribonucleotides are all methoxylated nucleotides. In the antisense strand, the nucleotides that are not fluorinated, not ethylene glycol-modified, and not substituted with deoxyribonucleotides are all methoxylated nucleotides.

12. The siRNA according to any one of claims 9 to 11, characterized in that, The trans-vinylphosphonate-modified nucleotide is located in the antisense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, the 5' end of at least the first nucleotide of the antisense strand is attached to a trans-vinylphosphonate group.

13. The siRNA according to any one of claims 9 to 12, characterized in that, The nucleotide modified with a reverse debased deoxyribose residue is located in the positive strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the penultimate nucleotide of the positive strand is attached to the 3' end of a reverse debased deoxyribose residue. Alternatively, the nucleotide modified with a reverse debased deoxyribose residue is located in the positive strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, the 5' end of at least the first nucleotide of the positive strand is attached to a reverse debased deoxyribose residue. Alternatively, the nucleotide modified with a reverse debased deoxyribose residue is located in the positive strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, the 5' end of at least the first nucleotide of the positive strand and the 3' end of the last nucleotide are connected to a reverse debased deoxyribose residue.

14. The siRNA according to any one of claims 9 to 13, characterized in that, The phosphate-thioester modified nucleotide is located in the antisense strand of the nucleotide sequence, and at least the nucleotides at positions 1 to 2, 2 to 3, penultimate to penultimate, and penultimate to penultimate of the antisense strand are linked by phosphate-thioester groups in the direction from the 5' end to the 3' end.

15. The siRNA according to any one of claims 9 to 14, characterized in that, The phosphate-thioester modified nucleotide is located in the positive strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at the 1st and 2nd, 2nd and 3rd, penultimate and penultimate, and penultimate and penultimate positions of the positive strand are linked by phosphate-thioester groups. Alternatively, the phosphate-thioester modified nucleotide is located in the positive strand of the nucleotide sequence, and in the positive strand, at least the nucleotides at positions 1 and 2, and positions 2 and 3 are linked by phosphate-thioester groups, in the direction from the 5' end to the 3' end; and if the 3' end of the penultimate nucleotide is connected to a reverse debased deoxyribose residue, then the penultimate nucleotide is linked to the reverse debased deoxyribose residue by phosphate-thioester groups. Alternatively, the phosphate-thioester modified nucleotide is located in the positive strand of the nucleotide sequence, and in the positive strand, in the direction from the 5' end to the 3' end, the nucleotide at position 1 is linked to the reverse debased deoxyribose residue by a phosphate-thioester group, at least the nucleotides at positions 1 and 2 are linked by a phosphate-thioester group, and the nucleotide at position 1 to 2 is linked to the reverse debased deoxyribose residue by a phosphate-thioester group.

16. The siRNA according to any one of claims 9 to 15, characterized in that, The glycolic acid-modified nucleotide is located in the antisense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the 6th nucleotide of the antisense strand is a glycolic acid-modified nucleotide.

17. The siRNA according to any one of claims 9 to 16, characterized in that, The deoxyribonucleotide-substituted nucleotide is located in the antisense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 5 and 12 of the antisense strand are deoxyribonucleotide-substituted nucleotides.

18. The siRNA according to any one of claims 1 to 17, characterized in that, The siRNA is selected from N-ER-FY049065, N-ER-FY049004, N-ER-FY049084, N-ER-FY049019, N-ER-FY049086, N-ER-FY049087, N-ER-FY049020, N-ER-FY049088, N-ER-FY049089, N-ER-FY049021, N-ER-FY049090, N-ER-FY049091, N-ER-FY049022, N-ER-FY049023, N-ER-FY049033, N-ER-FY049096, N-ER-FY049097, N- ER-FY049098、N-ER-FY049099、N-ER-FY049038、N-ER-FY049039、N-ER-FY049040、N-ER-FY049100、N-ER-FY049101、N-ER-FY049102、N-ER-FY049105、N-ER-FY049106、N-ER-FY049041、N-ER-FY049111、N-ER-FY049128、N-ER-FY049043、N-ER-FY049044、N-ER-FY049113、N-ER-FY049057、N-ER-FY049117、 N-ER-FY049062 and N-ER-FY049118, N-ER-FY049065M6, N-ER-FY049065M8, N-ER-FY049004M6, N-ER-FY049084M6, N-ER-FY049084M8, N-ER-FY049019M6, N-ER-FY049086M6, N-ER-FY049086M8, N-ER-FY049020M6, N-ER-FY049089M6, N-ER-FY049089M8, N-ER-FY049089M44, N-ER-FY049089M45, N-ER-FY04908 9M46、N-ER-FY049089M47、N-ER-FY049089M48、N-ER-FY049089M49、N-ER-FY049089M50、N-ER-FY049089M51、N-ER-FY049021M6、N-ER-FY049021M8、N-ER-FY049090M6、N-ER-FY049090M8、N-ER-FY049091M6、N-ER-FY049022M6、N-ER-FY049023M6、N-ER-FY049096M6、N-ER-FY049096M8、N-ER-FY049097M6、One or more of N-ER-FY049097M8, N-ER-FY049098M6, N-ER-FY049098M8, N-ER-FY049099M6, N-ER-FY049099M8, N-ER-FY049038M6, N-ER-FY049038M8, N-ER-FY049039M6, N-ER-FY049039M8, N-ER-FY049040M6, N-ER-FY049100M6, N-ER-FY049100M8, N-ER-FY049105M6, N-ER-FY049106M6, N-ER-FY049041M6, N-ER-FY049111M6, N-ER-FY049111M8, N-ER-FY049128M8, N-ER-FY049043M6, N-ER-FY049044M6, N-ER-FY049044M8, N-ER-FY049113M6, N-ER-FY049113M8, N-ER-FY049057M6, N-ER-FY049117M6, N-ER-FY049117M8, N-ER-FY049062M6, N-ER-FY049118M6, and N-ER-FY049118M8; Preferably, the siRNA is selected from one or more of N-ER-FY049089, N-ER-FY049089M44, N-ER-FY049089M46, N-ER-FY049089M48, N-ER-FY049089M50 and N-ER-FY049089M51.

19. A siRNA conjugate, characterized in that, The siRNA conjugate contains the siRNA as described in any one of claims 1 to 18 and a conjugating group conjugated to the siRNA; Preferably, the conjugation group comprises a ligand formed from a targeting molecule or its derivative; the targeting molecule comprises a lipophilic molecule, polymer, polypeptide, aptamer, antibody, quantum dot, sugar, folic acid and / or a receptor specifically expressed by hepatocytes; Preferably, the lipophilic molecules include cholesterol, bile acids, vitamins, and / or lipid molecules of different chain lengths; the polymer includes polyethylene glycol; the polypeptide includes a transmembrane peptide; the carbohydrates include lactose, polylactose, mannose, galactose, and / or N-acetylgalactosamine; and the receptors expressed by the hepatocytes include desialyl glycoprotein, desialyl sugar residues, lipoproteins, glucagon, neurotransmitters, growth factors, and / or transferrin. Preferably, the conjugating group is an N-glucose amino acid; Preferably, the N-glucose amino acid has a structure as shown in any one of formulas XIII to XX:

20. The siRNA conjugate as described in claim 19, characterized in that, When the conjugating group is an N-glucose amino acid, the siRNA conjugate has a structure as shown in Formula XXI:

21. The siRNA conjugate as described in claim 20, characterized in that, The siRNA compounds selected are N-ER-FY049004M6L96, N-ER-FY049020M6L96, N-ER-FY049020M8L96, N-ER-FY049021M6L96, N-ER-FY049021M8L96, N-ER-FY049022M6L96, N-ER-FY049023M6L96, N-ER-FY049038M6L96, N-ER-FY049038M8L96, and N-ER-FY049039M6L96. , N-ER-FY049039M8L96, N-ER-FY049040M6L96, N-ER-FY049040M8L96, N-ER-FY049041M6L96, N-ER-FY049043M6L96, N-ER- FY049044M6L96, N-ER-FY049044M8L96, N-ER-FY049062M6L96, N-ER-FY049065M8L96, N-ER-FY049084M8L96, N-ER-FY0490 86M8L96, N-ER-FY049089M8L96, N-ER-FY049090M8L96, N-ER-FY049096M8L96, N-ER-FY049097M8L96, N-ER-FY049098M8L 96, N-ER-FY049099M8L96, N-ER-FY049100M8L96, N-ER-FY049111M8L96, N-ER-FY049113M8L96, N-ER-FY049117M8L96, N-E R-FY049118M8L96, N-ER-FY049089M44L96, N-ER-FY049089M45L96, N-ER-FY049089M46L96, N-ER-FY049089M47L96, N-ER- FY049089M48L96, N-ER-FY049089M49L96, N-ER-FY049089M50L96, N-ER-FY049089M51L96 and N-ER-FY049128M8L96 neutral one or more kinds; Best choice, the siRNA combination described above N-ER-FY049089M44L96, N-ER-FY049089M46L96, N-ER- FY049089M48L96, N-ER-FY049089M50L96 and N-ER-FY049089M51L96, one or more types.

22. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: (i) the siRNA of any one of claims 1 to 18 and / or the siRNA conjugate of any one of claims 19 to 21; and (ii) pharmaceutically acceptable excipients.

23. An inhibitor for suppressing NR1H3 gene expression, characterized in that, The inhibitor comprises: (i) the siRNA of any one of claims 1 to 18 and / or the siRNA conjugate of any one of claims 19 to 21; and (ii) a pharmaceutically acceptable excipient.

24. A medicine for preventing and / or treating a disease, characterized in that, The disease is a disease associated with NR1H3 gene expression; the drug comprises: (i) the siRNA of any one of claims 1 to 18 and / or the siRNA conjugate of any one of claims 19 to 21; and (ii) pharmaceutically acceptable excipients.

25. The use of the siRNA according to any one of claims 1 to 18, the siRNA conjugate according to any one of claims 19 to 21, the pharmaceutical composition according to claim 22, the inhibitor according to claim 23, or the medicament according to claim 24 in the preparation of a medicament for the prevention and / or treatment of a disease, characterized in that, The disease in question is one related to NR1H3 gene expression.

26. The application as described in claim 25, characterized in that, The diseases associated with NR1H3 gene expression include those related to lipid metabolism; preferably, the diseases related to lipid metabolism include dyslipidemia, atherosclerosis, and / or non-alcoholic fatty liver disease.

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