Sirna for inhibiting GRB14 gene expression, conjugate thereof, pharmaceutical composition and use thereof

Abstract

The present invention relates to a siRNA for inhibiting GRB14 gene expression, a siRNA conjugate, a pharmaceutical composition comprising same, and a use thereof. Each nucleotide in the siRNA is independently a modified or unmodified nucleotide, and the siRNA contains a sense strand and an antisense strand. The siRNA, the conjugate thereof, and the pharmaceutical composition thereof can effectively treat and / or prevent diseases related to GRB14 gene overexpression.

Description

siRNAs that inhibit GRB14 gene expression, their conjugates and pharmaceutical compositions and their uses Technical Field

[0001] This application relates to siRNA that inhibits GRB14 gene expression, siRNA conjugates, prodrugs, pharmaceutical compositions comprising the same, methods of preparation thereof, and uses thereof. Background Technology

[0002] Growth factor receptor connector 14 (GRB14) is a connector protein that interacts with receptor tyrosine kinases, including the insulin receptor (IR) and insulin-like growth factor receptor (IGFR). GRB14 is highly expressed in the liver and is an inhibitor of IR catalytic activity, negatively regulating the signaling of IR and IGFR.

[0003] Insulin is a unique hormone that controls metabolic homeostasis, playing a role in maintaining glucose and lipid metabolism homeostasis. When insulin signaling is inhibited, glucose uptake cannot respond to insulin stimulation, leading to insulin resistance. GRB14, as a negative regulator of insulin activity, can significantly improve insulin signaling in the liver and skeletal muscle by knocking down its gene, thereby enhancing insulin sensitivity and improving glucose homeostasis.

[0004] In summary, inhibiting the expression of the GRB14 gene in patients can prevent and treat conditions related to insulin resistance, such as type 2 diabetes (T2D), diabetic nephropathy, diabetic retinopathy, diabetic vascular disease, diabetic neuropathy, obesity, hepatocellular carcinoma (HCC), hyperinsulinemia, cardiometabolic disorders (CMD), bladder cancer (BC), glioblastoma, non-alcoholic steatohepatitis, hypertension, and hyperlipidemia. Currently, there are no drugs on the market that specifically target the expression of this gene; therefore, developing drugs that target GRB14 is of significant value.

[0005] The present invention aims to provide siRNA, siRNA conjugates and pharmaceutical compositions thereof, which can affect the RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the GRB14 gene, thereby selectively and effectively inhibiting the expression of the GRB14 gene and achieving the purpose of disease treatment. Summary of the Invention

[0006] This invention provides an siRNA for inhibiting GRB14 gene expression. The siRNA comprises a sense strand and an antisense strand, wherein each nucleotide in the siRNA is independently modified or unmodified. The sense strand contains nucleotide sequence I, and the antisense strand contains nucleotide sequence II. Nucleotide sequence I and nucleotide sequence II are at least partially anticomplementary to form a double-stranded region. Nucleotide sequence I and nucleotide sequence II are selected from the following sequences:

[0007] (1) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:399, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:400:

[0008] 5'-GUACCCAGUGA-3'(SEQ ID NO:399)

[0009] 5'-UCACUGGGGUAC-3' (SEQ ID NO:400);

[0010] (2) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:401, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:402:

[0011] 5'-GGCUCGAGAU-3'(SEQ ID NO:401)

[0012] 5'-AUCUCGAGCC-3' (SEQ ID NO:402);

[0013] (3) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:403, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:404:

[0014] 5'-CAGCUGU-3'(SEQ ID NO:403)

[0015] 5'-ACAGCUG-3' (SEQ ID NO:404);

[0016] (4) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:33, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:34:

[0017] 5'-GAAUCAUUACAUUGAUGAA-3'(SEQ ID NO:33)

[0018] 5'-UUCAUCAAUGUAAUGAUUC-3' (SEQ ID NO: 34);

[0019] (5) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:405, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:406:

[0020] 5'-CUGGACCCUUUUUGAG-3'(SEQ ID NO:405)

[0021] 5'-CUCAAAAAGGGUCCAG-3' (SEQ ID NO: 406);

[0022] (6) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:407, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:408:

[0023] 5'-AACCAAUGGU-3'(SEQ ID NO:407)

[0024] 5'-ACCAUUGGUU-3' (SEQ ID NO:408);

[0025] (7) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:409, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:410:

[0026] 5'-CUUACAUGCGA-3'(SEQ ID NO:409)

[0027] 5'-UCGCAUGUAAG-3' (SEQ ID NO: 410);

[0028] (8) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:411, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:412:

[0029] 5'-ACAGGGAAAGAA-3'(SEQ ID NO:411)

[0030] 5'-UUCUUUCCCUGU-3' (SEQ ID NO: 412);

[0031] (9) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:413, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:414:

[0032] 5'-GACCUGAAAAU-3'(SEQ ID NO:413)

[0033] 5'-AUUUUCAGGUC-3' (SEQ ID NO: 414);

[0034] (10) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:415, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:416:

[0035] 5'-GCUGGGUGA-3'(SEQ ID NO:415)

[0036] 5'-UCACCCAGC-3' (SEQ ID NO: 416);

[0037] (11) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:417, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:418:

[0038] 5'-CGCGAUUAGAUU-3'(SEQ ID NO:417)

[0039] 5'-AAUCUAAUCGCG-3' (SEQ ID NO: 418);

[0040] (12) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:419, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:420:

[0041] 5'-AAGUAUGGCAUGCA-3'(SEQ ID NO:419)

[0042] 5'-UGCAUGCCUACUU-3' (SEQ ID NO: 420);

[0043] (13) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:421, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:422:

[0044] 5'-AAAGCAGAGUU-3'(SEQ ID NO:421)

[0045] 5'-AACUCUGCUUU-3' (SEQ ID NO:422);

[0046] (14) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:423, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:424:

[0047] 5'-CUCAGCGAU-3'(SEQ ID NO:423)

[0048] 5'-AAUCGCUGAG-3' (SEQ ID NO: 424);

[0049] (15) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:425, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:426:

[0050] 5'-CGAUUGAUUAUUCAGCA-3'(SEQ ID NO:425)

[0051] 5'-UGCUGAAUAAUCAAUCG-3' (SEQ ID NO: 426);

[0052] (16) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:427, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:428:

[0053] 5'-AAGGACUUGUGGAU-3'(SEQ ID NO:427)

[0054] 5'-AUCCACAAGUCCUU-3' (SEQ ID NO: 428);

[0055] (17) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:429, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:430:

[0056] 5'-AGUCAGAGUAAC-3'(SEQ ID NO:429)

[0057] 5'-GUUACUCUGACU-3' (SEQ ID NO: 430);

[0058] (18) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:431, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:432:

[0059] 5'-UGACGGU-3'(SEQ ID NO:431)

[0060] 5'-ACCGUCA-3' (SEQ ID NO:432);

[0061] (19) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:433, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:434:

[0062] 5'-AUGGCCA-3'(SEQ ID NO:433)

[0063] 5'-UGGCCAU-3' (SEQ ID NO: 434);

[0064] (20) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:435, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:436:

[0065] 5'-GAUUUACA-3'(SEQ ID NO:435)

[0066] 5'-UGUAAAUC-3' (SEQ ID NO:436);

[0067] (21) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:437, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:438:

[0068] 5'-GAAACAUUAUUGU-3'(SEQ ID NO:437)

[0069] 5'-ACAAUAAUGUUUC-3' (SEQ ID NO: 438);

[0070] (22) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:439, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:440:

[0071] 5'-GACUUAUUAAACUAUU-3'(SEQ ID NO:439)

[0072] 5'-AAUAGUUUAAUAAGUC-3' (SEQ ID NO: 440);

[0073] (23) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:441, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:442:

[0074] 5'-CUUUACA-3'(SEQ ID NO:441)

[0075] 5'-UGUAAAG-3' (SEQ ID NO:442);

[0076] (24) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:3, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:4;

[0077] (25) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:57, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:58;

[0078] (26) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:84, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:85;

[0079] (27) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:94, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:95;

[0080] (28) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:114, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:115;

[0081] (29) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:153, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:154;

[0082] (30) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:155, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:156;

[0083] (31) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:157, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:158;

[0084] (32) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:159, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:160;

[0085] (33) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:318, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:319;

[0086] (34) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:342, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:343;

[0087] (35) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:617, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:618:

[0088] 5'-CUGUUGAUCCUGAAGAA-3'(SEQ ID NO:617)

[0089] 5'-UUCUUCAGGAUCAACAG-3' (SEQ ID NO: 618);

[0090] (36) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:619, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:620:

[0091] 5'-GAGUUUUCUUGGUAC-3'(SEQ ID NO:619)

[0092] 5'-GUACCAAGAAACUC-3' (SEQ ID NO: 620);

[0093] (37) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:621, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:622:

[0094] 5'-ACGGGAUAGUCA-3'(SEQ ID NO:621)

[0095] 5'-UGACUAUCCCGU-3' (SEQ ID NO: 622);

[0096] (38) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:623, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:624:

[0097] 5'-GUCAAUGAGUCAU-3'(SEQ ID NO:623)

[0098] 5'-AUGACUCAUUGAC-3' (SEQ ID NO: 624);

[0099] (39) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:625, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:626:

[0100] 5'-CCCUGGUAGCAA-3'(SEQ ID NO:625)

[0101] 5'-UUGCUACCAGGG-3' (SEQ ID NO: 626);

[0102] (40) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:513, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:514;

[0103] (41) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:517, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:518;

[0104] (42) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:573, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:574;

[0105] (43) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:577, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:578;

[0106] (44) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:581, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:582;

[0107] (45) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:593, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:594;

[0108] (46) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:597, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:598;

[0109] (47) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:612, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:613.

[0110] In one embodiment, the nucleotide sequence I and the nucleotide sequence II are substantially anticomplementary, substantially anticomplementary, or completely anticomplementary; substantially anticomplementary means that there are no more than 3 base mismatches between the two nucleotide sequences; substantially anticomplementary means that there are no more than 1 base mismatch between the two nucleotide sequences; completely anticomplementary means that there are no mismatches between the two nucleotide sequences.

[0111] In one embodiment, the sense strand further contains nucleotide sequence III, and the antisense strand further contains nucleotide sequence IV, each of which is independently 0-12 nucleotides in length. Nucleotide sequence III is attached to the 5' end of nucleotide sequence I, and nucleotide sequence IV is attached to the 3' end of nucleotide sequence II. Nucleotide sequences III and IV are of equal length and are substantially anticomplementary or completely anticomplementary. "Substantially anticomplementary" means that there is no more than one base mismatch between the two nucleotide sequences; "completely anticomplementary" means that there is no mismatch between the two nucleotide sequences. And / or, nucleotide sequence III is attached to the 3' end of nucleotide sequence I, and nucleotide sequence IV is attached to the 5' end of nucleotide sequence II. Nucleotide sequences III and IV are of equal length and are substantially anticomplementary or completely anticomplementary. "Substantially anticomplementary" means that there is no more than one base mismatch between the two nucleotide sequences; "completely anticomplementary" means that there is no mismatch between the two nucleotide sequences.

[0112] In one embodiment, nucleotide sequences I and III form a double-stranded region that is at least partially anticomplementary to nucleotide sequences II and IV, wherein nucleotide sequences I and III, and nucleotide sequences II and IV are selected from the following sequences:

[0113] (1) The nucleotide sequences I and III comprise or consist of the nucleotide sequences shown in SEQ ID NO:141, and the nucleotide sequences II and IV comprise or consist of the nucleotide sequences shown in SEQ ID NO:142;

[0114] (2) The nucleotide sequences I and III comprise or consist of the nucleotide sequences shown in SEQ ID NO:266, and the nucleotide sequences II and IV comprise or consist of the nucleotide sequences shown in SEQ ID NO:267;

[0115] (3) The nucleotide sequences I and III comprise or consist of the nucleotide sequences shown in SEQ ID NO:457, and the nucleotide sequences II and IV comprise or consist of the nucleotide sequences shown in SEQ ID NO:627;

[0116] (4) The nucleotide sequences I and III comprise or consist of the nucleotide sequences shown in SEQ ID NO:206, and the nucleotide sequences II and IV comprise or consist of the nucleotide sequences shown in SEQ ID NO:207;

[0117] (5) The nucleotide sequences I and III comprise or consist of the nucleotide sequences shown in SEQ ID NO:521, and the nucleotide sequences II and IV comprise or consist of the nucleotide sequences shown in SEQ ID NO:628;

[0118] (6) The nucleotide sequences I and III comprise or consist of the nucleotide sequences shown in SEQ ID NO:537, and the nucleotide sequences II and IV comprise or consist of the nucleotide sequences shown in SEQ ID NO:629;

[0119] (7) The nucleotide sequences I and III comprise or consist of the nucleotide sequences shown in SEQ ID NO:557, and the nucleotide sequences II and IV comprise or consist of the nucleotide sequences shown in SEQ ID NO:630;

[0120] (8) The nucleotide sequences I and III comprise or consist of the nucleotide sequences shown in SEQ ID NO:577, and the nucleotide sequences II and IV comprise or consist of the nucleotide sequences shown in SEQ ID NO:631;

[0121] (9) The nucleotide sequences I and III comprise or consist of the nucleotide sequences shown in SEQ ID NO:585, and the nucleotide sequences II and IV comprise or consist of the nucleotide sequences shown in SEQ ID NO:632;

[0122] (10) The nucleotide sequences I and III comprise or consist of the nucleotide sequences shown in SEQ ID NO:612, and the nucleotide sequences II and IV comprise or consist of the nucleotide sequences shown in SEQ ID NO:633.

[0123] In one embodiment, the sense strand further contains nucleotide sequence V and / or the antisense strand further contains nucleotide sequence VI, wherein nucleotide sequences V and VI are 0 to 3 nucleotides in length, nucleotide sequence V is attached to the 3' end of the sense strand to form a 3' overhang of the sense strand, and / or nucleotide sequence VI is attached to the 3' end of the antisense strand to form a 3' overhang of the antisense strand. In a preferred embodiment, the length of nucleotide sequence V or VI is 2 nucleotides. In a preferred embodiment, nucleotide sequence V is identical or different from the nucleotide at the corresponding position of the target mRNA, or nucleotide VI is mismatched or complementary to the nucleotide at the corresponding position of the target mRNA. In a preferred embodiment, nucleotide sequence V or VI is two consecutive thymine deoxyribonucleotides or two consecutive uracil ribonucleotides.

[0124] In one embodiment, the length of the double-stranded region is 15-30 nucleotide pairs. In a preferred embodiment, the length of the double-stranded region is 17-23 nucleotide pairs. In a more preferred embodiment, the length of the double-stranded region is 19-21 nucleotide pairs.

[0125] In one embodiment, the sense or antisense strand has 15-30 nucleotides. In a preferred embodiment, the sense or antisense strand has 19-25 nucleotides. In a more preferred embodiment, the sense or antisense strand has 19-23 nucleotides.

[0126] In one embodiment, at least one nucleotide in the sense strand or the antisense strand is a modified nucleotide, and / or at least one phosphate ester group is a phosphate ester group with a modifying group; preferably, the phosphate ester group with a modifying group is a thiophosphate ester group formed by replacing an oxygen atom in the phosphodiester bond of the phosphate ester group with a sulfur atom.

[0127] In one embodiment, the siRNA comprises a positive strand that does not contain a 3' overhang nucleotide.

[0128] In one embodiment, the 5' terminal nucleotide of the antisense strand is linked to a 5' phosphate group or a 5' phosphate derivative group, or the 5' terminal nucleotide of the antisense strand is not linked to a 5' phosphate group or a 5' phosphate derivative group.

[0129] In one embodiment, the 5' end and 3' end of the positive strand are not connected to a reverse debased deoxyribose residue, or the 5' end of the positive strand is connected to a reverse debased deoxyribose residue, or the 3' end of the positive strand is connected to a reverse debased deoxyribose residue, or the 5' end and 3' end of the positive strand are each connected to a reverse debased deoxyribose residue.

[0130] In some preferred embodiments, the reverse debased deoxyribose residue is linked to the 3' terminal nucleotide and / or 5' terminal nucleotide of the positive strand via a phosphodiester bond, a thiophosphate group, or other nucleoside internucleotide bond.

[0131] In one embodiment, the modified nucleotide is selected from 2'-fluoro-modified nucleotides, 2'-alkoxy-modified nucleotides, 2'-substituted alkoxy-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-deoxynucleotides, 2'-amino-modified nucleotides, 2'-substituted amino-modified nucleotides, nucleotide analogs, or any combination of two or more thereof.

[0132] In one embodiment, the modified nucleotide is selected from 2'-fluoro-modified nucleotides, 2'-methoxy-modified nucleotides, 2'-O-CH2-CH2-O-CH3-modified nucleotides, 2'-O-CH2-CH=CH2-modified nucleotides, 2'-CH2-CH2-CH=CH2-modified nucleotides, 2'-deoxynucleotides, nucleotide analogs, or any combination of two or more of these.

[0133] In one embodiment, each nucleotide in the sense strand and the antisense strand is independently a 2'-fluorinated nucleotide or a non-fluorinated nucleotide. In a preferred embodiment, 2'-fluorinated nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, with the remaining positions being non-fluorinated nucleotides, in a 5' to 3' orientation; 2'-fluorinated nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, with the remaining positions being non-fluorinated nucleotides. In another preferred embodiment, 2'-fluorinated nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, with the remaining positions being non-fluorinated nucleotides, in a 5' to 3' orientation; 2'-fluorinated nucleotides are located at positions 2, 6, 8, 9, 14, and 16 of the antisense strand, with the remaining positions being non-fluorinated nucleotides. In a preferred embodiment, 2'-fluorinated nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, with the remaining positions occupied by non-fluorinated nucleotides, oriented from 5' to 3'; 2'-fluorinated nucleotides are located at positions 2, 14, and 16 of the antisense strand, oriented from 5' to 3', with the remaining positions occupied by non-fluorinated nucleotides. In another preferred embodiment, 2'-fluorinated nucleotides are located at positions 5, 7, 8, and 9 of the sense strand, oriented from 5' to 3', with the remaining positions occupied by non-fluorinated nucleotides; 2'-fluorinated nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, oriented from 5' to 3', with the remaining positions occupied by non-fluorinated nucleotides. In one preferred embodiment, 2'-fluorinated nucleotides are located at positions 3, 7, 8, and 9 of the sense strand, with the remaining positions occupied by non-fluorinated nucleotides, oriented from 5' to 3'; 2'-fluorinated nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand, with the remaining positions occupied by non-fluorinated nucleotides. In another preferred embodiment, 2'-fluorinated nucleotides are located at positions 3, 7, 9, and 11 of the sense strand, oriented from 5' to 3', with the remaining positions occupied by non-fluorinated nucleotides; 2'-fluorinated nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand, with the remaining positions occupied by non-fluorinated nucleotides. In a preferred embodiment, the 2'-fluorinated nucleotides are located at positions 7, 9, and 14 of the sense strand in a 5' to 3' orientation, with the remaining positions being non-fluorinated nucleotides; and the 2'-fluorinated nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand in a 5' to 3' orientation, with the remaining positions being non-fluorinated nucleotides.In a preferred embodiment, the 2'-fluorinated nucleotides are located at positions 3, 7, 9, and 11 of the sense strand in the 5' to 3' direction, with the remaining positions being non-fluorinated nucleotides; and the 2'-fluorinated nucleotides are located at positions 2, 3, 5, 7, 10, 12, and 14 of the antisense strand in the 5' to 3' direction, with the remaining positions being non-fluorinated nucleotides.

[0134] In one embodiment, each non-fluorinated nucleotide is a 2'-methoxy-modified nucleotide, wherein the 2'-methoxy-modified nucleotide refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribosome with a methoxy group.

[0135] In one embodiment, each non-fluorinated modified nucleotide is independently selected from a nucleotide or nucleotide analog formed by replacing the hydroxyl group at the 2' position of the ribosyl group of the nucleotide with a non-fluorinated group, wherein the nucleotide analog is selected from a pseudouracil, isonucleotide, LNA, ENA, cET BNA, UNA, and GNA.

[0136] In one embodiment, each nucleotide in the sense strand and the antisense strand is independently a 2'-fluoro-modified nucleotide, a 2'-methoxy-modified nucleotide, a GNA-modified nucleotide, or any combination of two or more thereof. In a preferred embodiment, with the 5' to 3' orientation, the 2'-fluoro-modified nucleotide is located at positions 7, 9, 10, and 11 of the sense strand, with the remaining positions being 2'-methoxy-modified nucleotides; with the 5' to 3' orientation, the 2'-fluoro-modified nucleotide is located at positions 2, 6, 14, and 16 of the antisense strand, with the remaining positions being 2'-methoxy-modified nucleotides. In a preferred embodiment, with the 5' to 3' orientation, the 2'-fluoro-modified nucleotide is located at positions 7, 9, 10, and 11 of the sense strand, with the remaining positions being 2'-methoxy-modified nucleotides; with the 5' to 3' orientation, the 2'-fluoro-modified nucleotide is located at positions 2, 6, 8, 9, 14, and 16 of the antisense strand, with the remaining positions being 2'-methoxy-modified nucleotides. In a preferred embodiment, 2'-fluorinated nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, with the remaining positions occupied by 2'-methoxylated nucleotides, oriented from 5' to 3'; 2'-fluorinated nucleotides are located at positions 2, 14, and 16 of the antisense strand, oriented from 5' to 3', and GNA-modified nucleotides are located at position 6 of the antisense strand, with the remaining positions occupied by 2'-methoxylated nucleotides. In another preferred embodiment, 2'-fluorinated nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, oriented from 5' to 3', with the remaining positions occupied by 2'-methoxylated nucleotides; 2'-fluorinated nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, oriented from 5' to 3', and GNA-modified nucleotides are located at position 7 of the antisense strand, with the remaining positions occupied by 2'-methoxylated nucleotides. In one preferred embodiment, 2'-fluorinated nucleotides are located at positions 5, 7, 8, and 9 of the sense strand, with the remaining positions occupied by 2'-methoxylated nucleotides; and 2'-fluorinated nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, with the remaining positions occupied by 2'-methoxylated nucleotides. In another preferred embodiment, 2'-fluorinated nucleotides are located at positions 3, 7, 8, and 9 of the sense strand, with the remaining positions occupied by 2'-methoxylated nucleotides; and 2'-fluorinated nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand, with the remaining positions occupied by 2'-methoxylated nucleotides.In a preferred embodiment, 2'-fluorinated nucleotides are located at positions 3, 7, 9, and 11 of the sense strand, with the remaining positions being 2'-methoxylated nucleotides; and 2'-fluorinated nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand, with the remaining positions being 2'-methoxylated nucleotides. In another preferred embodiment, 2'-fluorinated nucleotides are located at positions 7, 9, and 14 of the sense strand, with the remaining positions being 2'-methoxylated nucleotides; and 2'-fluorinated nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand, with the remaining positions being 2'-methoxylated nucleotides. In a preferred embodiment, 2'-fluorinated nucleotides are located at positions 3, 7, 9, and 11 of the sense strand, with the remaining positions being 2'-methoxylated nucleotides; 2'-fluorinated nucleotides are located at positions 2, 3, 5, 7, 10, 12, and 14 of the antisense strand, with GNA-modified nucleotides located at position 6 of the antisense strand, and the remaining positions being 2'-methoxylated nucleotides.

[0137] In this document, when describing the modification methods of the sense or antisense strand of siRNA, when the base is "T", those skilled in the art generally understand that the base "T" represents deoxyribonucleic acid, and there are no modifications to the base "T" such as 2'-F, 2'-O-CH3, or nucleotide derivatives. In some embodiments, the nucleotides in the siRNA are linked by phosphate thioester groups, and the siRNA is positioned from the 5' end to the 3' end.

[0138] (1) The positive chain contains thiophosphate groups located at the positions shown below:

[0139] Between the first and second nucleotides starting at the 5' end of the positive strand; and

[0140] Between the second and third nucleotides starting at the 5' end of the positive strand; and

[0141] Between the first nucleotide and the second nucleotide starting at the 3' end of the positive strand; and

[0142] Between the second and third nucleotides starting at the 3' end of the positive strand;

[0143] or,

[0144] (2) The positive chain contains thiophosphate groups located at the positions shown below:

[0145] Between the first and second nucleotides starting at the 5' end of the positive strand; and

[0146] Between the second and third nucleotides starting at the 5' end of the positive strand;

[0147] or,

[0148] (3) The positive chain contains thiophosphate groups located at the positions shown below:

[0149] Between the first and second nucleotides starting at the 5' end of the positive strand; and

[0150] Between the second and third nucleotides starting at the 5' end of the positive strand;

[0151] The reverse debasing deoxyribose residue starting at the 3' end of the positive strand is between the first nucleotide and the first nucleotide.

[0152] In some embodiments, the siRNA, oriented from the 5' end to the 3' end, contains a phosphate thioester group located at the following positions:

[0153] Between the first and second nucleotides starting at the 5' end of the antisense strand;

[0154] Between the second and third nucleotides starting at the 5' end of the antisense strand; and

[0155] Between the first nucleotide and the second nucleotide starting at the 3' end of the antisense strand; and

[0156] Between the second and third nucleotides starting at the 3' end of the antisense strand.

[0157] In one embodiment, each nucleotide in the sense strand and the antisense strand is independently a 2'-fluoro-modified nucleotide, a 2'-methoxy-modified nucleotide, a GNA-modified nucleotide, or any combination of two or more thereof.

[0158] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, and the 3' end is free of overhangs; 2'-fluoromodified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5' phosphate group.

[0159] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides; 2'-fluoromodified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5' phosphate group.

[0160] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides; 2'-fluoromodified nucleotides are located at positions 2, 6, 8, 9, 14, and 16 of the antisense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5' phosphate group.

[0161] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, with the remaining positions being 2'-methoxymodified nucleotides; 2'-fluoromodified nucleotides are located at positions 2, 14, and 16 of the antisense strand, with GNA-modified nucleotides located at position 6 of the antisense strand, and the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5' phosphate group.

[0162] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, with the remaining positions being 2'-methoxymodified nucleotides; 2'-fluoromodified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, with GNA-modified nucleotides located at position 7 of the antisense strand, and the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5' phosphate group.

[0163] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, with the remaining positions being 2'-methoxymodified nucleotides; 2'-fluoromodified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, with the remaining positions being 2'-methoxymodified nucleotides; the 5' terminal nucleotide of the antisense strand is not linked to a 5' phosphate group or a 5' phosphate-derived group.

[0164] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, with the remaining positions being 2'-methoxymodified nucleotides; 2'-fluoromodified nucleotides are located at positions 2, 6, 8, 9, 14, and 16 of the antisense strand, with the remaining positions being 2'-methoxymodified nucleotides; the 5' terminal nucleotide of the antisense strand is not linked to a 5' phosphate group or a 5' phosphate-derived group.

[0165] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides; 2'-fluoromodified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0166] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, with the remaining positions being 2'-methoxymodified nucleotides; 2'-fluoromodified nucleotides are located at positions 2, 6, 8, 9, 14, and 16 of the antisense strand, with the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0167] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 5, 7, 8, and 9 of the sense strand, with the remaining positions being 2'-methoxymodified nucleotides; 2'-fluoromodified nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, with the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0168] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 3, 7, 8, and 9 of the sense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides; 2'-fluoromodified nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0169] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 3, 7, 9, and 11 of the sense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides; 2'-fluoromodified nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0170] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 7, 9, and 14 of the sense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides; 2'-fluoromodified nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand in a 5' to 3' orientation, with the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0171] In a preferred embodiment, 2'-fluoromodified nucleotides are located at positions 3, 7, 9, and 11 of the sense strand, with the remaining positions being 2'-methoxymodified nucleotides; 2'-fluoromodified nucleotides are located at positions 2, 3, 5, 7, 10, 12, and 14 of the antisense strand, with GNA-modified nucleotides located at position 6 of the antisense strand, and the remaining positions being 2'-methoxymodified nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

[0172] In one specific embodiment, the present invention provides siRNAs selected from Table 1; preferably, the siRNAs are selected from N-ER-FY047136, N-ER-FY047149, N-ER-FY047150, N-ER-FY047136M49, N-ER-FY047149M49, N-ER-FY047150M49, N-ER-FY047167, N-ER-FY047167M49, and N-ER-FY047. 216. N-ER-FY047216M49, N-ER-FY047226, N-ER-FY047226M49, N-ER-FY047232, N-ER-FY047232M49, N-ER- FY047182, N-ER-FY047182M49, N-ER-FY047207, N-ER-FY047207M49, N-ER-FY047245, N-ER-FY047245M49.

[0173] The present invention also provides an siRNA conjugate containing the siRNA of the present invention and a conjugating group conjugated to the siRNA (as shown in the following formula, where a double helix structure represents the siRNA and the conjugating group is attached to the 3' end of the positive strand of the siRNA):

[0174] In the above conjugate structure, X can be selected as O or S; in one embodiment, X is O. In one embodiment, in the siRNA conjugate, the sense strand and antisense strand of the siRNA are complementary to form the double-stranded region of the siRNA conjugate, and the 3' end of the sense strand forms a blunt end, and the 3' end of the antisense strand has 1-3 protruding nucleotides extending out of the double-stranded region;

[0175] or,

[0176] In the siRNA conjugate, the sense strand and antisense strand of the siRNA are complementary to form the double-stranded region of the siRNA conjugate, and the 3' end of the sense strand is blunt, and the 3' end of the antisense strand is blunt.

[0177] In one embodiment, the conjugating group is selected from:

[0178] In one specific embodiment, the siRNA conjugate is selected from the siRNA conjugates in Table 2; preferably, the siRNA conjugate is selected from N-ER-FY047136M49L96, N-ER-FY047149M49L96, N-ER-FY047150M49L96, N-ER-FY047167M49L96, N-ER-FY047216M49L96, N-ER-FY047226M49L96, N-ER-FY047232M49L96, N-ER-FY047182M49L96, N-ER-FY047207M49L96, and N-ER-FY047245M49L96.

[0179] The present invention also provides a pharmaceutical composition comprising the siRNA of the present invention, or the siRNA conjugate of the present invention, and a pharmaceutically acceptable carrier.

[0180] The present invention also provides a kit comprising the siRNA of the present invention, or the siRNA conjugate of the present invention, or the pharmaceutical composition of the present invention.

[0181] The present invention also provides the use of the siRNA of the present invention, or the siRNA conjugate of the present invention, or the pharmaceutical composition of the present invention for the preparation of an agent for inhibiting the expression of the GRB14 gene.

[0182] The present invention also provides the use of the siRNA of the present invention, or the siRNA conjugate of the present invention, or the pharmaceutical composition of the present invention for the preparation of medicaments for the prevention and / or treatment of diseases related to GRB14 gene overexpression.

[0183] In the specific implementation plan, the diseases mentioned are type II diabetes (T2D), diabetic nephropathy, diabetic retinopathy, diabetic vascular disease, diabetic neuropathy, obesity, hepatocellular carcinoma (HCC), hyperinsulinemia, cardiometabolic disorder (CMD), bladder cancer (BC), glioblastoma, non-alcoholic steatohepatitis, hypertension, and hyperlipidemia, etc.

[0184] The present invention also provides a method for inhibiting GRB14 gene expression, comprising contacting or administering a therapeutically effective amount of the siRNA of the present invention, or the siRNA conjugate of the present invention, or the pharmaceutical composition of the present invention to cells expressing GRB14 or to a subject in need.

[0185] The present invention also provides methods for treating and / or preventing diseases associated with GRB14 gene overexpression, comprising administering a therapeutically effective amount of the siRNA of the present invention, or the siRNA conjugate of the present invention, or the pharmaceutical composition of the present invention to a subject in need.

[0186] In the specific implementation plan, the diseases mentioned are type II diabetes (T2D), diabetic nephropathy, diabetic retinopathy, diabetic vascular disease, diabetic neuropathy, obesity, hepatocellular carcinoma (HCC), hyperinsulinemia, cardiometabolic disorder (CMD), bladder cancer (BC), glioblastoma, non-alcoholic steatohepatitis, hypertension, and hyperlipidemia, etc. Beneficial effects

[0187] The siRNA, pharmaceutical composition, and siRNA conjugates provided in this application have shown excellent GRB14 gene expression inhibitory activity in in vitro cell experiments, demonstrating promising potential for treating diseases related to GRB14 gene overexpression. For example, the siRNA and its conjugates disclosed in this application can reduce the expression of GRB14 mRNA in the liver, exhibiting low toxicity and good plasma stability, and showing promising clinical application prospects.

[0188] In some specific embodiments, the siRNA provided in this application has high GRB14 gene repressive activity in Huh7 cells.

[0189] In some specific embodiments, the siRNA conjugates provided in this application exhibit high GRB14 gene repressive activity in PHH cells.

[0190] In some specific embodiments, the siRNA conjugate of this application has high inhibitory activity against the hGRB14 gene in vivo and can reduce the expression level of hGRB14 for a long time. Attached Figure Description

[0191] Figure 1 shows the off-target experiment results of N-ER-FY047232M49L96 at a concentration of 5 nM.

[0192] Figure 2 shows the off-target experiment results of N-ER-FY047232M49L96 at a concentration of 50 nM. Detailed Implementation

[0193] definition

[0194] Throughout this specification, unless otherwise specified, in this technical field, "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 (DNA) sequences and / or ribonucleic acid (RNA) sequences. Therefore, in the context of this disclosure, the meanings of "G", "C", "A", "T", and "U" include all the above-mentioned possible cases. In this disclosure, "nucleotide", "ribonucleic acid", and "ribonucleotide" are used interchangeably, and "deoxyribonucleic acid" and "2'-deoxyribonucleic acid" are used interchangeably. Lowercase letters a, u, c, g: indicate nucleotides modified with 2'-methoxy groups; Af, Gf, Cf, Uf: indicate nucleotides modified with 2'-fluoride groups; lowercase letter s: indicates that the two nucleotides adjacent to s on the left and right are linked by thiophosphate groups; (invAb) indicates a reverse debasing deoxyribose residue; P1: indicates that the nucleotide adjacent to the right of P1 is a 5'-phosphate nucleotide; EVP: indicates a 5'-trans-vinylphosphonate group (i.e., the nucleotide adjacent to the right of EVP is a 5'-trans-vinylphosphonate nucleotide); (Underlined + Bold + Italic): Indicates GNA-modified nucleotides.

[0195] In the foregoing and hereinafter, "2'-fluorinated nucleotide" refers to a nucleotide formed by replacing the hydroxyl group at the 2' position of the ribosyl group with fluorine. "Non-fluorinated nucleotide" refers to a nucleotide or nucleotide analog formed by replacing the hydroxyl group at the 2' position of the ribosyl group with a non-fluorinated group. In some embodiments, each non-fluorinated nucleotide is independently selected from one of the nucleotides or nucleotide analogs formed by replacing the hydroxyl group at the 2' position of the ribosyl group with a non-fluorinated group. These nucleotides formed by replacing the hydroxyl group at the 2' position of the ribosyl group with a non-fluorinated group are well known to those skilled in the art, and these nucleotides may be selected from one of 2'-alkoxy-modified nucleotides, 2'-substituted alkoxy-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-amino-modified nucleotides, 2'-substituted amino-modified nucleotides, and 2'-deoxynucleotides.

[0196] "Alkyl" includes straight-chain, branched, or cyclic saturated alkyl groups. For example, alkyl groups include, but are not limited to, methyl, ethyl, propyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, cyclobutyl, n-pentyl, cyclohexyl, and similar groups. For example, "C 1-6 The "C" in "alkyl" 1-6 "" refers to a group consisting of 1, 2, 3, 4, 5 or 6 carbon atoms arranged in a straight chain, branched chain or cyclic form.

[0197] "Alkoxy" herein refers to an alkyl group that is attached to the remainder of a molecule by an oxygen atom (-O-alkyl), wherein the alkyl group is as defined herein. Non-limiting examples of alkoxy groups include methoxy, ethoxy, trifluoromethoxy, difluoromethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, n-pentoxy, etc.

[0198] "Nucleotide analogues" refer to groups that can replace nucleotides in nucleic acids, but whose structure differs from that of adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, or thymine deoxyribonucleotides. Examples include pseudouracil (Ψ), isonucleotides, bridged nucleic acids (BNA), or acyclic nucleotides.

[0199] Pseudouracil (Ψ) refers to a natural structural analog of a uracil nucleoside, in which the ribose is not linked to the N1 of the uracil ring, but rather to the C5 of the pyrimidine ring. Its structural formula is as follows:

[0200] BNA refers to a restricted or inaccessible nucleotide. BNA can contain a five-membered, six-membered, or seven-membered ring with a "fixed" C3'-endoglycan condensation bridging structure. This bridge is typically incorporated into the 2'-, 4'-position of the ribose to provide a 2',4'-BNA nucleotide, such as LNA, ENA, cET BNA, etc., where LNA is shown in formula (1), ENA in formula (2), and cET BNA in formula (3).

[0201] Acyclic nucleotides are a class of nucleotides formed by opening the sugar ring of a nucleotide, such as unopened nucleic acids (UNA) or glycerol nucleic acids (GNA). UNA is shown in formula (4), and GNA is shown in formula (5).

[0202] In formulas (4) and (5) above, R is selected from H, OH or alkoxy (-O-alkyl).

[0203] Heteronucleotides are compounds formed by changing the position of the bases in the ribose ring of a nucleotide. For example, compounds formed by moving the bases from the 1'-position to the 2'-position or 3'-position of the ribose ring, as shown in formula (6) or (7):

[0204] In the compounds of formulas (6)-(7) above, Base represents a base, such as A, U, G, C or T; R is selected from H, OH, F or non-fluorine groups as described above.

[0205] In some embodiments, the nucleotide analogue is selected from one of pseudouracil, isonucleotides, LNA, ENA, cET BNA, UNA, and GNA. In some embodiments, each non-fluorinated nucleotide is a 2'-methoxy-modified nucleotide, a GNA-modified nucleotide, or any combination of two or more thereof. In some preferred embodiments, each non-fluorinated nucleotide is a 2'-methoxy-modified nucleotide, wherein, as stated above and below, the 2'-methoxy-modified nucleotide refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribosome with a methoxy group.

[0206] The term "2'-methoxy-modified nucleotide" refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribosome with a methoxy group. The term "thiophosphate group" refers to a thiophosphate group formed by replacing an oxygen atom in the phosphodiester bond of the phosphate group with a sulfur atom.

[0207] The "thiophosphate group" refers to the following formula:

[0208] The "5'-phosphate nucleotide" refers to the structure of the following formula:

[0209] In the context of this specification, the terms "complementary" and "reverse complementary" are used interchangeably and have the meanings 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. Correspondingly, "mismatch" in the art means, in the case of a double-stranded nucleic acid, that the bases at corresponding positions are not paired complementaryly.

[0210] Unless otherwise specified above and below, "substantially anticomplementary" means that there are no more than 3 base mismatches between the two nucleotide sequences involved; "substantially anticomplementary" means that there are no more than 1 base mismatch between the two nucleotide sequences; and "completely anticomplementary" means that there are no base mismatches between the two nucleotide sequences.

[0211] In the preceding and following text, a "nucleotide difference" between two nucleotide sequences refers to a change in the type of bases at the same position of the nucleotides compared to the latter. For example, if a nucleotide base in the latter is A, and the corresponding nucleotide base at the same position in the former is U, C, G, or T, then a nucleotide difference at that position is considered to exist between the two nucleotide sequences. In some embodiments, replacing the nucleotide at the original position with a baseless nucleotide or its equivalent can also be considered a nucleotide difference at that position.

[0212] In this context, a "protruding end" refers to one or more unpaired nucleotides that protrude from the double-stranded structure of an siRNA when one 3' end of one strand extends beyond the 5' end of the other strand, or vice versa. A "flat-ended" or "knock-off" siRNA means that there are no unpaired nucleotides at that end of the siRNA, i.e., no nucleotide protrusions. A "flat-ended" siRNA is a double-stranded siRNA that is double-stranded throughout its entire length, meaning there are no nucleotide protrusions at either end of the molecule. A "double-stranded region" refers to the complementary double-stranded region of the siRNA formed by the sense and antisense strands.

[0213] In the preceding and following text, "5'-nucleotide" refers to a nucleotide in which the phosphate group is attached to the 5' carbon of a pentose sugar, and it is the main type of nucleotide that exists freely in organisms. "3'-nucleotide" refers to a nucleotide in which the phosphate group is attached to the 3' carbon of a pentose sugar, and may include, for example, adenosine-3'-phosphate, guanosine-3'-phosphate, cytidine-3'-phosphate, uridine-3'-phosphate, 2'-deoxythymidine-3'-phosphate, 2'-O-methyladenosine-3'-phosphate, 2'-O-methyladenosine-3'-thiophosphate, 2'-fluoroadenosine-3'-phosphate, 2'-fluoroadenosine-3'-thiophosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-O-methylguanosine-3'-phosphate. The terms siRNA, 2'-fluoroguanosine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-O-methyluridine-3'-phosphate, 2'-O-methyluridine-3'-phosphate, 2'-fluorouridine-3'-phosphate, 2'-fluorouridine-3'-phosphate, and 2'-deoxythymidine-3'-phosphate are used interchangeably in the context of this disclosure. The terms "iRNA," "RNAi reagent," "iRNA agent," and "RNA interference agent" as used herein are interchangeable and refer to terms defined herein that include siRNA and mediate targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. iRNAs direct the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). iRNAs regulate, for example, the expression of target genes in cells (such as the cells of a subject, like a mammalian subject).

[0214] Throughout this application, particularly in the description of the preparation methods of the siRNA, pharmaceutical compositions, 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.

[0215] 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.

[0216] 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).

[0217] As used in this specification, "optional" or "optionally" means that the event or condition described thereafter may or may not occur, and the description includes both the occurrence and non-occurrence of the event or condition.

[0218] The term “subject” as used in this specification refers to any animal, such as a mammal or marsupial. Subjects in this application include, but are not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, rabbits, or any kind of poultry.

[0219] As used in this specification, “treatment” means a method of obtaining a beneficial or desired outcome, including but not limited to treatment benefits. A “treatment benefit” means the eradication or improvement of the underlying disorder being treated. Furthermore, a treatment benefit is obtained by eradicating or improving one or more physiological symptoms associated with the underlying disorder, thereby observing improvement in the subject, although the subject may still be suffering from the underlying disorder.

[0220] As used in this specification, “prevention” means a method of obtaining a beneficial or desired outcome, including but not limited to preventive benefits. To obtain a “preventive benefit,” siRNA, siRNA conjugates, or pharmaceutical compositions may be given to subjects at risk of developing a specific disease, or to subjects who report one or more physiological symptoms of a disease, even if a diagnosis of the disease may not have been made.

[0221] As used in this specification, "prodrug" refers to a compound that exerts its pharmacological effect only after being transformed in vivo. For example, in this application, M6 is a prodrug of M2. The difference between the modifications of M2 and M6 is whether there is P1 at the 5' end of the antisense strand. M6 without P1 will be phosphorylated in vivo to become the M2 sequence with P1, thereby exerting its effect. Similarly, the relationship between M7 and M3 is the same. Therefore, in this document, siRNA includes its corresponding prodrug.

[0222] siRNA

[0223] This application relates to an siRNA capable of inhibiting GRB14 gene expression. The siRNA of this application contains nucleotide groups as basic structural units, as is known to those skilled in the art, wherein the nucleotide groups contain phosphate groups, ribose groups, and bases. Typically, an active, i.e., functional siRNA is about 12-40 nucleotides in length, and in some embodiments about 15-30 nucleotides.

[0224] The siRNA of this application contains a sense strand and an antisense strand, each nucleotide in the siRNA being independently modified or unmodified. The sense strand contains a nucleotide sequence I, and the antisense strand contains a nucleotide sequence II. Nucleotide sequence I and nucleotide sequence II are at least partially anticomplementary to form a double-stranded region. In some embodiments, the length of the double-stranded region is 15-30 nucleotide pairs. In other embodiments, the length of the double-stranded region is 17-23 nucleotide pairs. In still other embodiments, the length of the double-stranded region is 19-21 nucleotide pairs. In yet another embodiment, the length of the double-stranded region is 19 or 21 nucleotide pairs.

[0225] In some embodiments, the positive strand further contains nucleotide sequence III, and the antisense strand further contains nucleotide sequence IV. Nucleotide sequence III and nucleotide sequence IV are each independently 0-12 nucleotides in length. Nucleotide sequence III is attached to the 5' end of nucleotide sequence I, and nucleotide sequence IV is attached to the 3' end of nucleotide sequence II. Nucleotide sequence III and nucleotide sequence IV are of equal length and are substantially anticomplementary or completely anticomplementary. Substantially anticomplementary means that there is no more than one base mismatch between the two nucleotide sequences; completely anticomplementary means that there is no mismatch between the two nucleotide sequences. In some embodiments, the sense strand further contains nucleotide sequence III, and the antisense strand further contains nucleotide sequence IV, each of which is independently 0-12 nucleotides in length. Nucleotide sequence III is attached to the 5' end of nucleotide sequence I, and nucleotide sequence IV is attached to the 3' end of nucleotide sequence II. Nucleotide sequences III and IV are of equal length and are substantially anticomplementary or completely anticomplementary. Alternatively, nucleotide sequence III is attached to the 3' end of nucleotide sequence I, and nucleotide sequence IV is attached to the 5' end of nucleotide sequence II. Nucleotide sequences III and IV are of equal length and are substantially anticomplementary or completely anticomplementary. "Substantially anticomplementary" means that there is no more than one base mismatch between the two nucleotide sequences; "completely anticomplementary" means that there is no mismatch between the two nucleotide sequences.

[0226] In some embodiments, the sense strand further contains nucleotide sequence V and / or the antisense strand further contains nucleotide sequence VI, wherein nucleotide sequences V and VI are 0 to 3 nucleotides in length, wherein nucleotide sequence V is attached to the 3' end of the sense strand to form a 3' overhang of the sense strand, and / or nucleotide sequence VI is attached to the 3' end of the antisense strand to form a 3' overhang of the antisense strand. In some embodiments, the length of nucleotide sequence V or VI is 2 nucleotides. In other embodiments, nucleotide sequence V is identical or different from the nucleotide at the corresponding position on the target mRNA, or nucleotide VI is mismatched or complementary to the nucleotide at the corresponding position on the target mRNA. In other embodiments, nucleotide sequence V or VI is two consecutive thymine deoxyribonucleotides or two consecutive uracil ribonucleotides.

[0227] The sense and antisense strands provided in this application may have the same or different lengths. In some embodiments, the sense or antisense strand has 15-30 nucleotides. In other embodiments, the sense or antisense strand has 19-25 nucleotides. In still other embodiments, the sense or antisense strand has 19-23 nucleotides. The length ratio of the sense and antisense strands of the siRNA provided in this application can be 15 / 15, 16 / 16, 17 / 17, 18 / 18, 19 / 19, 19 / 20, 19 / 21, 19 / 22, 19 / 23, 20 / 19, 20 / 20, 20 / 21, 20 / 22, 20 / 23, 21 / 19, 21 / 20, 21 / 21, 21 / 22, 21 / 23. Examples of possible siRNA length ratios include 22 / 19, 22 / 20, 22 / 21, 22 / 22, 22 / 23, 23 / 19, 23 / 20, 23 / 21, 23 / 22, 23 / 23, 24 / 24, 25 / 25, 26 / 26, 27 / 27, 28 / 28, 29 / 29, 30 / 30, 22 / 24, 22 / 25, 22 / 26, 23 / 24, 23 / 25, or 23 / 26. In some embodiments, the length ratio of the sense strand to the antisense strand of the siRNA is 19 / 19, 21 / 21, 19 / 21, 21 / 23, or 23 / 23, in which case the siRNA of this disclosure exhibits better cellular mRNA silencing activity.

[0228] Studies have found that different modification strategies can have drastically different effects on the stability, bioactivity, and cytotoxicity of siRNA. For example, CN201010106762.1 investigated various chemical modification strategies for siRNA, confirming seven effective modification methods. Compared with unmodified siRNA, one of the modification methods resulted in siRNA that improved blood stability while maintaining inhibitory activity essentially equivalent to that of unmodified siRNA.

[0229] The nucleotides in the siRNA of the present invention are each independently modified or unmodified nucleotides. In some embodiments, each nucleotide in the siRNA of the present invention is an unmodified nucleotide; in some embodiments, some or all of the nucleotides in the siRNA of the present invention are modified nucleotides, and these modifications on the nucleotide groups do not cause a significant weakening or loss of the function of the siRNA of the present invention in inhibiting GRB14 gene expression.

[0230] In some embodiments, the siRNA of this application contains at least one modified nucleotide. In the context of this application, the term "modified nucleotide" refers to a nucleotide or nucleotide analog formed by replacing the 2' hydroxyl group of the ribosyl group with another group, or a nucleotide having a modified base. The modified nucleotide does not cause a significant weakening or loss of the siRNA's ability to suppress gene expression. For example, the modified nucleotide disclosed in JKWatts, G.F. Deleavey, and MJDamha, Chemically Modified siRNA: Tools and Applications. Drug Discov Today, 2008, 13(19-20):842-55, can be selected.

[0231] In some embodiments, at least one nucleotide in the sense strand or antisense strand of the siRNA provided by the present invention is a modified nucleotide, and / or at least one phosphate ester group is a phosphate ester group with a modifying group; in other words, at least a portion of the phosphate ester group and / or ribosome in the phosphate-sugar backbone of at least one single strand of the sense strand and the antisense strand is a phosphate ester group and / or a ribosome with a modifying group. In some embodiments, the phosphate ester group with a modifying group is a thiophosphate ester group formed by replacing an oxygen atom in the phosphodiester bond of the phosphate ester group with a sulfur atom.

[0232] In some embodiments, the siRNA comprises a positive strand that does not contain a 3' overhanging nucleotide; that is, the positive strand of the siRNA may contain a 3' overhanging nucleotide, and excluding the 3' overhanging nucleotide of the positive strand forms a blunt end.

[0233] In some implementations, when the nucleotide sequences of the sense and antisense strands are complementary to form a double-stranded region, and there is no protruding nucleotide at the 3' end of the sense strand, a nucleotide sequence V is added to the 3' end of the sense strand as the protruding nucleotide. Then, after the nucleotide sequence formed by linking nucleotide sequence V to the 3' end of the sense strand is chemically modified, nucleotide sequence V is excluded, and correspondingly, the sense strand of the siRNA forms a blunt end.

[0234] In some implementations, when the nucleotide sequences of the sense strand and the antisense strand are complementary to form a double-stranded region, and the 3' end of the sense strand has a protruding nucleotide extending out of the double-stranded region, the protruding nucleotide at the 3' end of the sense strand is excluded and the nucleotide sequence of the sense strand is obtained. Accordingly, the sense strand of the siRNA forms a blunt end.

[0235] In some embodiments, the 5' terminal nucleotide of the antisense strand is linked to a 5' phosphate group or a 5' phosphate-derived group.

[0236] When the 5' terminal nucleotide of the antisense strand is attached to a 5' phosphate group or a 5' phosphate derivative, the following structure is formed:

[0237] Formula (8) shows a structure formed by linking a 5' nucleotide to a 5' phosphate group, Formula (9) shows a structure formed by linking a 5' nucleotide to a 5' phosphate-derived group (EVP), and Formula (10) shows a structure formed by linking a 5' nucleotide to a 5' phosphate-derived group (5' methylene phosphate group). Here, Base represents a base, such as A, U, G, C, or T. R' is a hydroxyl group or hydrogen, or is substituted by various groups known to those skilled in the art. For example, the substituted modified nucleotide can be a 2'-fluoro(2'-F) modified nucleotide, a 2'-alkoxy modified nucleotide, a 2'-substituted alkoxy modified nucleotide, a 2'-alkyl modified nucleotide, a 2'-substituted alkyl modified nucleotide, a 2'-amino modified nucleotide, a 2'-substituted amino modified nucleotide, or a 2'-deoxy nucleotide.

[0238] In some embodiments, the 5' terminal nucleotide of the sense or antisense strand is not linked to a 5' phosphate group, a 5' phosphate-derived group, or (invAb) (i.e., the ribonucleotide of the 5' terminal nucleotide of the sense or antisense strand is a 5' hydroxyl group), and its structure is shown below:

[0239] Wherein, Base represents a base, such as A, U, G, C, or T. R is a hydroxyl group or hydrogen, or is substituted by various groups known to those skilled in the art. For example, R can be 2'-fluoro(2'-F), 2'-alkoxy, 2'-substituted alkoxy, 2'-alkyl, 2'-substituted alkyl, 2'-amino, or 2'-substituted amino.

[0240] An exemplary modified nucleotide has the following structure:

[0241] Wherein, Base represents a base, such as A, U, G, C, or T. The hydroxyl group at the 2' position of the ribose group is replaced by R. These hydroxyl groups at the 2' position of the ribose group can be replaced by various groups known to those skilled in the art, for example, the substituted modified nucleotide can be a 2'-fluoro(2'-F) modified nucleotide, a 2'-alkoxy modified nucleotide, a 2'-substituted alkoxy modified nucleotide, a 2'-alkyl modified nucleotide, a 2'-substituted alkyl modified nucleotide, a 2'-amino modified nucleotide, a 2'-substituted amino modified nucleotide, or a 2'-deoxynucleotide.

[0242] In some embodiments, the 2'-alkoxy modified nucleotide is a nucleotide modified with 2'-methoxy (2'-OMe, 2'-O-CH3), etc.

[0243] In some embodiments, the 2'-substituted alkoxy modified nucleotide is a nucleotide modified with 2'-methoxyethoxy (2'-O-CH2-CH2-O-CH3), a nucleotide modified with 2'-O-CH2-CH=CH2, etc.

[0244] In some embodiments, the 2'-substituted alkyl-modified nucleotide is a nucleotide modified with 2'-CH2-CH2-CH=CH2, etc.

[0245] In some embodiments, the positive strand may contain one or more capping residues or portions, sometimes referred to in the art as a “cap,” “terminal cap,” or “capping residue.” As used herein, a “capping residue” is a nonnucleotide compound or other portion that may be incorporated at one or more ends of the nucleotide sequence of the siRNA disclosed herein. In some cases, capping residues may provide certain beneficial properties to the siRNA, such as protection against exonuclease degradation. In some embodiments, an inverse debased deoxyribose residue (invAb) is added as a capping residue. In some embodiments, the capping residue appears at the 5' end, the 3' end, or both the 5' and 3' ends of the positive strand.

[0246] In some embodiments, one or more inverted debased deoxyribose residues (invAb) are added to the 3' end of the positive strand. In some embodiments, one or more inverted debased deoxyribose residues (invAb) are added to the 5' end of the positive strand. In some embodiments, one or more inverted debased deoxyribose residues (invAb) are added to both the 5' end and the 3' end of the positive strand. The inverted debased deoxyribose residues may be linked via phosphate ester bonds, thiophosphate ester groups, or other nucleoside internucleotide bonds. When describing modification sites in modification methods, (invAb) is not counted as the first site of the sequence. In some embodiments, inverted debased deoxyribose residues (invAb) (also referred to in the art as "inverted debasing sites") may be added. The (invAb) may have the following structure:

[0247] Formula B is used when (invAb) is located at the 3' end of the siRNA; Formula C is used when (invAb) is located at the 5' end of the siRNA.

[0248] siRNA conjugates

[0249] This application relates to an siRNA conjugate containing the aforementioned siRNA and a conjugate group conjugated to the siRNA.

[0250] In this application, the sense and antisense strands of the siRNA conjugate form a double-stranded region of the siRNA conjugate, and a blunt end is formed at the 3' end of the sense strand of the siRNA conjugate. In some embodiments, the 3' end of the sense strand of the siRNA conjugate is blunt, and the 3' end of the antisense strand of the siRNA conjugate has 1-3 protruding nucleotides extending out of the double-stranded region. In other embodiments, the 3' end of both the sense and antisense strands of the siRNA conjugate is blunt. In this disclosure, "the 3' end of the sense strand is blunt" includes cases where the 3' end of the motif sense strand is inherently blunt, and cases where the 3' end of the motif sense strand has a protruding end, but the protruding end is excluded to form a blunt end.

[0251] In some preferred embodiments, the siRNA conjugate is obtained by conjugating siRNA with a conjugating group. Specifically, the sense and antisense strands of the siRNA are complementary to form a double-stranded region of the siRNA, and the 3' end of the sense strand of the siRNA forms a blunt end. The conjugating group is conjugated to the 3' end of the sense strand with the blunt end to form the siRNA conjugate.

[0252] For example, the siRNA shown in N-ER-FY047213M49 has a blunt-end sequence at the 3' end of its sense strand. This blunt end serves as the nucleotide sequence for linking the L96 conjugate group (i.e., L96 is linked via a phosphodiester bond after the sequence is synthesized to (invAb)). Therefore, the sequence forming the siRNA conjugate is: sense strand gscsUfcagCfGfAfuugauuauuas(invAb)L96, antisense strand EVPusAfsauaaUfcaAfucgCfugagcscsu. In some preferred embodiments, the 3' end of the siRNA's sense strand has a protruding nucleotide extending into a double-stranded region. The sequence with a blunt 3' end, formed by excluding the protruding nucleotide at the 3' end of the sense strand, serves as the nucleotide sequence for linking the conjugate group. The conjugate group is then linked to the blunt end of the sense strand to form the siRNA conjugate.

[0253] In some preferred embodiments, when the nucleotide sequences of the sense and antisense strands are complementary to form a double-stranded region, and there is no protruding nucleotide at the 3' end of the sense strand, a nucleotide sequence V is added to the 3' end of the sense strand as the protruding nucleotide. The sequence with a blunt 3' end formed after excluding the protruding nucleotide at the 3' end of the sense strand is used as the nucleotide sequence for attaching the conjugate group. The conjugate group is attached to the blunt 3' end of the sense strand to form an siRNA conjugate.

[0254] In some preferred embodiments, when the nucleotide sequences of the sense strand and the antisense strand are complementary to form a double-stranded region, and the 3' end of the sense strand has a protruding nucleotide extending out of the double-stranded region, the sequence with a blunt 3' end formed after excluding the protruding nucleotide at the 3' end of the sense strand is used as the nucleotide sequence for connecting the conjugate group. The conjugate group is connected to the blunt 3' end of the sense strand to form an siRNA conjugate.

[0255] For example, the siRNA sequence shown as N-ER-FY047116M2 has a protruding nucleotide extending into a double-stranded region at the 3' end of the sense strand. The blunt-ended sequence asasagcaGfaGfUfUfauagaaaa formed by excluding the protruding -sTsT nucleotide at the 3' end of the sense strand is used as the nucleotide sequence for connecting the L96 conjugate group. Therefore, the sequence forming the siRNA conjugate is: sense strand asasagcaGfaGfUfUfauagaaaaL96, antisense strand P1usUfsuucUfauaacucUfgCfuuusTsT.

[0256] Generally, the conjugation group comprises at least one pharmaceutically acceptable target group, or further comprises a linker, and the siRNA, the linker, and the target group are sequentially linked. In some embodiments, there are 1-6 target groups. In some embodiments, there are 2-4 target groups. The siRNA molecule can be non-covalently or covalently conjugated to the conjugation group, for example, it can be covalently conjugated to the conjugation group. The conjugation site of the siRNA and the conjugation group can be at the 3' end or 5' end of the siRNA's sense strand, at the 5' end of the antisense strand, or within the siRNA's internal sequence. In some embodiments, the conjugation site of the siRNA and the conjugation group is at the 3' end of the siRNA's sense strand.

[0257] In some embodiments, the conjugate group may be attached to a phosphate group, a 2'-hydroxyl group, or a base of a nucleotide. In some embodiments, the conjugate group may also be attached to a 3'-hydroxyl group, in which case the nucleotides are linked by a 2'-5' phosphodiester bond. When the conjugate group is attached to the end of the siRNA chain, it is usually attached to a phosphate group of the nucleotide; when the conjugate group is attached to the inner sequence of the siRNA, it is usually attached to a ribose ring or a base. Various connection methods can be found in the reference: Muthiah Manoharan et al. siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatocytes. ACS Chemical biology, 2015, 10(5): 1181-7.

[0258] In some embodiments, the siRNA and the conjugate group are linked by acid-labile or reducible chemical bonds. These bonds can degrade in the acidic environment of the endosomes, thus freeing the siRNA. For non-degradable conjugates, the conjugate group can be attached to the positive and negative strands of the siRNA to minimize the impact of the conjugate on the siRNA's activity.

[0259] In some embodiments, the pharmaceutically acceptable targeting group may be a ligand commonly used in the field of siRNA delivery, such as the various ligands described in WO2009082607A2, which are incorporated herein by reference in their entirety.

[0260] In some embodiments, the pharmaceutically acceptable targeting group may be selected from one or more ligands formed from the following targeting molecules or their derivatives: lipophilic molecules, such as cholesterol, bile acids, vitamins (e.g., vitamin E), lipid molecules of different chain lengths; polymers, such as polyethylene glycol; polypeptides, such as transmembrane peptides; aptamers; antibodies; quantum dots; carbohydrates, such as lactose, polylactose, mannose, galactose, N-acetylgalactosamine (GalNAc); folic acid; receptor ligands expressed by hepatocytes, such as desialyl glycoprotein, desialyl sugar residues, lipoproteins (e.g., high-density lipoprotein, low-density lipoprotein, etc.), glucagon, neurotransmitters (e.g., adrenaline), growth factors, transferrin, etc.

[0261] In some embodiments, each ligand is independently selected from a ligand capable of binding to a cell surface receptor. In some embodiments, at least one ligand is capable of binding to a hepatocyte surface receptor. In some embodiments, at least one ligand is capable of binding to a mammalian cell surface receptor. In some embodiments, at least one ligand is capable of binding to a human hepatocyte surface receptor. In some embodiments, at least one ligand is capable of binding to the liver surface desialylate glycoprotein receptor (ASGPR). The types of these ligands are well known to those skilled in the art, and their function is generally to bind to specific receptors on the surface of target cells, mediating the delivery of ligand-linked siRNA to the target cells.

[0262] In some embodiments, the pharmaceutically acceptable targeting group can be any ligand that binds to the desialyl glycoprotein receptor (ASGPR) on the surface of mammalian hepatocytes. In some embodiments, each ligand is independently a desialyl glycoprotein, such as asialolesomucoid (ASOR) or asialofetin (ASF). In some embodiments, the ligand is a sugar or a sugar derivative.

[0263] In some embodiments, at least one ligand is a sugar. In some embodiments, each ligand is a sugar. In some embodiments, at least one ligand is a monosaccharide, polysaccharide, modified monosaccharide, modified polysaccharide, or sugar derivative. In some embodiments, at least one of the ligands may be a monosaccharide, disaccharide, or trisaccharide. In some embodiments, at least one ligand is a modified sugar. In some embodiments, each ligand is a modified sugar. In some embodiments, each ligand is independently selected from polysaccharides, modified polysaccharides, monosaccharides, modified monosaccharides, polysaccharide derivatives, or monosaccharide derivatives. In some embodiments, each or at least one ligand is selected from the group consisting of glucose and its derivatives, mannan and its derivatives, galactose and its derivatives, xylose and its derivatives, ribose and its derivatives, fucose and its derivatives, lactose and its derivatives, maltose and its derivatives, arabinose and its derivatives, fructose and its derivatives, and sialic acid.

[0264] In some embodiments, each of the ligands may be independently selected from D-mannose, L-mannose, D-arabinose, D-xylfuranose, L-xylfuranose, D-glucose, L-glucose, D-galactose, L-galactose, α-D-mannose, β-D-mannose, α-D-mannose, β-D-mannose, α-D-glucose pyranose, β-D-mannose pyranose, α-D-glucose pyranose, β-D-glucose pyranose, β-D-glucose pyranose, β-D-glucose pyranose Sugars, α-D-furanose glucose, β-D-furanose glucose, α-D-furanofructose, α-D-fructose pyranose, α-D-galactopyranose, β-D-galactopyranose, α-D-galactopyranose, β-D-galactopyranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-butyrylgalactosamine, N-isobutyrylgalactosamine 2-Amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-carboxamido-2,3-di-O-methyl-D-mannpyranose, 2-deoxy-2-sulfonamido-D-glucopyranose, N-ethanolyl-α-neuraminic acid, 5-thio-β-D-glucopyranose, 2, 3,4-Tri-O-acetyl-1-thio-6-O-triphenylmethyl-α-D-glucopyranoside methyl ester, 4-thio-β-D-galactopyranose, 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucopyranoside ethyl ester, 2,5-dehydrated-D-aloxonitrile, ribose, D-ribose, D-4-thioribose, L-ribose, or L-4-thioribose. Other options for the ligands may be found, for example, in CN105378082A, which is incorporated herein by reference in its entirety.

[0265] In some embodiments, the pharmaceutically acceptable targeting group in the siRNA conjugate can be galactose or N-acetylgalactosamine, wherein the galactose or N-acetylgalactosamine molecule can be monovalent, divalent, trivalent, or tetravalent. It should be understood that the terms monovalent, divalent, trivalent, and tetravalent refer to the molar ratio of siRNA molecules to galactose or N-acetylgalactosamine molecules in the siRNA conjugate after the siRNA molecule forms a conjugate with a conjugate group containing galactose or N-acetylgalactosamine as a targeting group, respectively, being 1:1, 1:2, 1:3, or 1:4. In some embodiments, the pharmaceutically acceptable targeting group is N-acetylgalactosamine. In some embodiments, when the siRNA described in this application is conjugated with a conjugate group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent or tetravalent. In some embodiments, when the siRNA described in this application is conjugated with a conjugating group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent.

[0266] The targeting group can be linked to the siRNA molecule via a suitable adapter. Those skilled in the art can select a suitable adapter based on the specific type of the targeting group. For details on these adapters, the types of targeting groups, and the connection methods with siRNA, please refer to the disclosure of WO2015006740A2, which is incorporated herein by reference in its entirety.

[0267] siRNA synthesis methods

[0268] Nucleoside monomers are linked sequentially from 3' to 5' along the nucleotide arrangement using the conventional solid-phase phosphoramide 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 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 3' position of the last nucleotide in the 5′ to 3′ direction is a hydroxyl group, this is achieved using conventional methods in the art.

[0269] For example, the synthesis conditions for the siRNA in this application can be as follows:

[0270] 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.

[0271] 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.

[0272] The oxidation reaction conditions included: a reaction temperature of 25°C, a reaction time of 15 seconds, and the oxidizing agent being 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.

[0273] The sulfidation reaction conditions included: a reaction temperature of 25°C, a reaction time of 300 seconds, and the sulfidation reagent was 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 in a ratio of 1:1.

[0274] The capping reaction conditions included: a reaction temperature of 25°C, a reaction time of 15 seconds, and the capping reagent being a mixed solution of CapA (10% acetic anhydride acetonitrile solution) and CapB (10% N-methylimidazolium pyridine / acetonitrile solution) in a molar ratio of 1:1. 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.

[0275] After linking all nucleoside monomers, the nucleic acid sequences linked on the solid-phase support were 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.

[0276] 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.

[0277] The synthesized siRNAs are shown in Table 1 and Table 1-1.

[0278] siRNA conjugate synthesis method

[0279] Taking the synthesis of the L96 conjugate group as an example:

[0280] [Revised according to Rule 26, 09.05.2026] Step 1: By reacting DMTr-L96 with succinic anhydride, compound L96-A is obtained:

[0281] 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 product to obtain pure L96-A.

[0282] [Revised according to Rule 26, 09.05.2026] Step 2: React L96-A with NH2-SPS to obtain L96-B:

[0283] 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 on 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, which is the solid-phase support containing the conjugated molecules.

[0284] The third step is to prepare siRNA conjugates.

[0285] Using L96-B as a solid-phase carrier, the siRNA was synthesized and linked to the siRNA sense strand according to the siRNA synthesis method described above. The siRNA antisense strand was then synthesized using the same method, and the resulting siRNA conjugate was annealed to produce the siRNA conjugate of this application.

[0286] The synthesized siRNA conjugates are shown in Table 2.

[0287] Pharmaceutical Composition

[0288] This application provides a pharmaceutical composition containing siRNA as an active ingredient and a pharmaceutically acceptable carrier as described above.

[0289] The pharmaceutically acceptable carrier can be a carrier commonly used in the field of siRNA delivery, such as, but not limited to, lipid nanoparticles (LNP), magnetic nanoparticles (e.g., Fe3O4 or Fe2O3-based nanoparticles), carbon nanotubes, mesoporous silicon, calcium phosphate nanoparticles, polyethylenimine (PEI), polyamidoamine (PAMAM) dendrimer, poly(L-lysine) (PLL), chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), poly(D&L-lactic / glycolic acid) copolymer (PLGA), and poly(2-aminoethyl ethylene phosphate). One or more of the following: phosphate), PPEEA, and poly(2-dimethylaminoethyl methacrylate), PDMAEMA, and their derivatives.

[0290] The pharmaceutical composition does not have specific requirements for the content of siRNA and pharmaceutically acceptable carriers; the content of each component can be the conventional content.

[0291] In some embodiments, the pharmaceutical composition may also contain other pharmaceutically acceptable excipients, which may be one or more of a variety of formulations or compounds conventionally used in the art. For example, the other pharmaceutically acceptable excipients may include at least one of pH buffers, protectants, and osmotic pressure regulators.

[0292] The pH buffer solution can be a tris(hydroxymethyl)aminomethane hydrochloride buffer with a pH of 7.5-8.5 and / or a phosphate buffer with a pH of 5.5-8.5, for example, a phosphate buffer with a pH of 5.5-8.5.

[0293] The protective agent may be at least one selected from inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose, and glucose. Based on the total weight of the pharmaceutical composition, the content of the protective agent may be 0.01-30% by weight.

[0294] The osmotic pressure regulator may be sodium chloride and / or potassium chloride. The content of the osmotic pressure regulator results in an osmotic pressure of 200-700 milliosm / kg (mOsm / kg) for the pharmaceutical composition. The content of the osmotic pressure regulator can be readily determined by those skilled in the art based on the desired osmotic pressure.

[0295] In some embodiments, the pharmaceutical composition may be a liquid formulation, such as an injection; or it may be a lyophilized powder for injection, which is mixed with liquid excipients to form a liquid formulation for administration. The liquid formulation may be used, but is not limited to, for subcutaneous, intramuscular, or intravenous injection, or may be administered via a spray to the lungs, or via a spray to other organs or tissues (such as the liver). In some embodiments, the pharmaceutical composition is used for intravenous injection.

[0296] In some embodiments, the pharmaceutical composition may be in the form of a liposomal formulation. In some embodiments, the pharmaceutically acceptable carrier used in the liposomal formulation comprises an amine-containing transfection compound (hereinafter also referred to as an organic amine), a cofactor lipid, and / or a polyethylene glycol-modified lipid.

[0297] The following examples are used to further illustrate the present invention, but do not limit the present invention in any way.

[0298] Example

[0299] Other objects, features, and advantages of this disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific embodiments (although illustrating specific implementations of this disclosure) are given for illustrative purposes only, as various changes and modifications that can be made within the spirit and scope of this disclosure will become apparent to those skilled in the art upon reading this detailed description.

[0300] Unless otherwise specified, the experimental techniques and methods used in this embodiment are conventional techniques and methods. For example, experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in the embodiments can be obtained through legitimate commercial channels.

[0301] Example 1: Preparation of siRNA

[0302] Tianlin Biotechnology (Shanghai) Co., Ltd. synthesized siRNA molecules with the following sequence.

[0303] Table 1 siRNA and its sequence

[0304] Table 1-1 siRNAs and their sequences

[0305] In this context, the uppercase letters “G”, “C”, “A”, “T”, and “U” typically represent nucleotides containing guanine, cytosine, adenine, thymine, and uracil as bases, respectively; the lowercase letters a, u, c, and g indicate nucleotides modified with a 2'-methoxy group; Af, Gf, Cf, and Uf indicate nucleotides modified with a 2'-fluoride group; the lowercase letter s indicates that the two nucleotides adjacent to it are linked by a thiophosphate group; (invAb) indicates a reverse debasing deoxyribose residue; P1 indicates that the nucleotide adjacent to the right of P1 is a 5'-phosphate nucleotide; EVP indicates a 5'-trans-vinylphosphonate group (i.e., the nucleotide adjacent to the right of EVP is a 5'-trans-vinylphosphonate nucleotide). (Underlined + Bold + Italic): Indicates GNA-modified nucleotides.

[0306] Tianlin Biotechnology (Shanghai) Co., Ltd. synthesized siRNA conjugates with the following sequences:

[0307] Table 2 siRNA conjugates and their sequences:

[0308] L96 is connected to the 3' end of the positive chain in Table 1 or the blunt end formed by the 3' end of the positive chain via a phosphodiester bond. L96 is:

[0309] In Tables 1, 1-1, and 2, if the left side of the 5' terminal nucleotide of the positive strand, the modified positive strand, and the modified positive strand with the connecting conjugate is not marked with P1, EVP, or (invAb), it means that the ribosome of the 5' terminal nucleotide is a 5' hydroxyl group, and its structure is shown in Formula X:

[0310] Wherein, Base represents a base, such as A, U, G, C or T; R is a hydroxyl group or hydrogen or is substituted by various groups known to those skilled in the art, for example, R can be 2'-fluoro(2'-F), 2'-alkoxy, 2'-substituted alkoxy, 2'-alkyl, 2'-substituted alkyl, 2'-amino, 2'-substituted amino.

[0311] In Tables 1, 1-1, and 2, if the left side of the 5' terminal nucleotide of the antisense strand and the modified antisense strand is not marked with P1 or EVP, it means that the ribose group of the 5' terminal nucleotide is 5' hydroxyl, and its structure is as shown in Formula X.

[0312] In Tables 1, 1-1, and 2, when the 3' ends of the sense strand and the modified sense strand are not connected (invAb), the 3' position of the nucleotide at the 3' end of the sense strand and the modified sense strand is a hydroxyl group. Similarly, the 3' position of the nucleotide at the 3' end of the antisense strand and the modified antisense strand is a hydroxyl group.

[0313] Example 2: siRNA inhibits GRB14 gene expression

[0314] Experimental materials:

[0315] Huh7 cells, provided by Shanghai WuXi AppTec Co., Ltd., catalog number JCRB0403;

[0316] RNA extraction kit 96 Kit, purchased from Qiagen, item number 74182;

[0317] Transfection reagent, purchased from Invitrogen, catalog number 13778-150;

[0318] FastStart Universal SYBR Green Master, purchased from Roche, item number 04914058001;

[0319] FastStart Universal Probe Master, purchased from Roche, item number: 04914058001;

[0320] FBS, purchased from Gibco, item number 10099141;

[0321] DMEM, purchased from Gibco, item number 11965-092;

[0322] Fastking RT Kit (with gDNase), purchased from TIANGEN, item number KR116-02;

[0323] Opti-medium: serum-reduced culture medium, purchased from Gibco, catalog number 31985-070;

[0324] AO / PI, purchased from Countstar, item number RE010212;

[0325] GRB14 forward primer, reverse primer and probe: provided by Chengdu WuXi AppTec New Drug Development Co., Ltd.;

[0326] GAPDH forward primers were purchased from Sangon Biotech, catalog number 2800263525.

[0327] GAPDH reverse primers, purchased from Sangon Biotech, catalog number 2800263526;

[0328] GAPDH probe: provided by WuXi AppTec Co., Ltd.

[0329] Experimental methods:

[0330] 1. Digest Huh7 cells to prepare a cell suspension. Take 20 μL of the cell suspension, mix it with AO / PI, and count the cells using Countstar. Dilute the cell suspension with DMEM containing 10% FBS to the desired final cell density of 2 × 10⁻⁶ cells / mL. 5 Cells / mL.

[0331] 2. Centrifuge the dry powder of the siRNA to be tested at low temperature and high speed, and then dissolve it in ultrapure distilled water to prepare a 100 μM siRNA stock solution.

[0332] 3. Prepare 20 nM siRNA dilution solution Z and 0.2 nM siRNA dilution solution W.

[0333] (1) Preparation of 0.1 μM siRNA stock solution Y and 0.001 μM siRNA stock solution F:

[0334] a) Take 2 μL of the 100 μM siRNA stock solution obtained in step 2 above, add 18 μL of ultrapure distilled water to obtain a siRNA dilution solution with a final concentration of 10 μM.

[0335] b) Take 2 μL of the 10 μM siRNA dilution solution obtained in step a), add 18 μL of ultrapure distilled water to obtain siRNA stock solution Q with a final concentration of 1 μM.

[0336] c) Take 2 μL of the 1 μM siRNA stock solution Q obtained in step b), add 18 μL of ultrapure distilled water to obtain siRNA stock solution Y with a final concentration of 0.1 μM.

[0337] d) Take 2 μL of the 0.1 μM siRNA stock solution Y obtained in step c), add 18 μL of ultrapure distilled water to obtain siRNA stock solution E with a final concentration of 0.01 μM.

[0338] e) Take 2 μL of the 0.01 μM siRNA stock solution E obtained in step d), add 18 μL of ultrapure distilled water to obtain siRNA stock solution F with a final concentration of 0.001 μM.

[0339] (2) Take 2 μL of each of the above-prepared siRNA stock solution Y and siRNA stock solution F, and add 8 μL of Opti-medium to obtain 20 nM siRNA dilution Z and 0.2 nM siRNA dilution W respectively.

[0340] 4. Transfection of Huh7 cells

[0341] (1) Take 3 μL of transfection reagent was added to 97 μL of Opti-medium to obtain... Transfection reagent dilution solution; The transfection reagent dilution solution and the 20nM siRNA dilution solution Z prepared in step 3 were mixed at a volume ratio of 1:1 to prepare a transfection mixture. After standing for 5 minutes, 10 μL of the transfection mixture was added to a 96-well plate to transfect the Huh7 cells cultured in step 1 (final volume 100 μL, the concentration of siRNA in this system is 1 nM).

[0342] (2) Take 3 μL of transfection reagent was added to 97 μL of Opti-medium to obtain... Transfection reagent dilution solution; The transfection reagent diluent and the 0.2 nM siRNA diluent W prepared in step 3 were mixed at a volume ratio of 1:1 to prepare a transfection mixture. After standing for 5 minutes, 10 μL of the transfection mixture was added to a 96-well plate to transfect the Huh7 cells cultured in step 1 (final volume 100 μL, the concentration of siRNA in this system is 0.01 nM).

[0343] The cells were cultured for 48 hours after transfection; two replicates were set up for each concentration (1 nM and 0.01 nM).

[0344] 5. Utilize 96Kit instructions: Extract total RNA from Huh7 cells obtained in step 4;

[0345] 6. Reverse transcribe the extracted total RNA into cDNA using the Fastking RT Kit (with gDNase), following these steps:

[0346] a) Remove gDNA using gDNAase according to the table below;

[0347] 42℃, 3 min; 4℃, let stand.

[0348] b) Add the reagents described below to the system obtained in step a) and perform reverse transcription;

[0349] 42℃, 15min; 95℃, 3min.

[0350] c) Store the reverse transcription product obtained in step b) at -20°C for real-time PCR analysis.

[0351] 7. Perform real-time PCR analysis

[0352] a) Prepare the qPCR reaction mixture as shown in the table below. Throughout the entire process, all reagents should be kept on ice.

[0353] b) Perform the qPCR procedure as described below.

[0354] 95℃, 10 minutes;

[0355] 95℃, 15 seconds, 60℃, 1 minute (this operation is repeated 40 times).

[0356] 8. Results Analysis

[0357] a) Use Quant Studio 6Flex software with default settings to automatically calculate the Ct value;

[0358] b) Calculate the relative expression level of the gene using the following formula:

[0359] ΔCt = Ct(GRB14 gene) - Ct(GAPDH)

[0360] ΔCt = ΔCt(sample group) - ΔCt(mock group)

[0361] mRNA expression relative to the Mock group = 2 -ΔΔCt .

[0362] The Mock group stated: compared to the test sample group, the group without added siRNA.

[0363] 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%

[0364] 9. Experimental Results

[0365] siRNA concentrations of 1 nM and 0.01 nM were selected for testing.

[0366] Table 3 Inhibition rate of the siRNA of the present invention

[0367] Table 4 Inhibition rate of control siRNA

[0368] Table 4-1 Control siRNA

[0369] As can be seen from Tables 3 and 4, the siRNA of the present invention can significantly inhibit the expression of the GRB14 gene at both 1 nM and 0.01 nM.

[0370] Example 3: IC50 of siRNA inhibiting GRB14 gene expression 50 Measurement

[0371] The following concentration ranges (nM) for the siRNA assay were set as follows: 10, 2.5, 0.625, 0.156, 0.039, 0.0097, 0.0024, and 0.0006. IC50 assays were then performed using a method similar to that in Example 2. 50 Measurement.

[0372] Results analysis:

[0373] a) Use Quant Studio 6 Flex software with default settings to automatically calculate the Ct value;

[0374] b) Calculate the relative expression level of the gene using the following formula:

[0375] ΔCt = Ct(GRB14 gene) - Ct(GAPDH)

[0376] ΔCt = ΔCt(test sample group) - ΔCt(Mock group), where the Mock group represents the group without siRNA compared to the test sample group;

[0377] mRNA expression relative to the Mock group = 2 -ΔΔCt

[0378] 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%

[0379] Calculation process: Using the log value of siRNA concentration as the X-axis and the percentage inhibition rate as the Y-axis, the dose-response curve was fitted using the "log (inhibitor) vs. response-variable slope" function module of GraphPad Prism 8 software to obtain the IC50 of each siRNA. 50 value.

[0380] The fitting formula is: Y = Bottom + (Top - Bottom) / (1 + 10^(logIC)) 50 -X)*HillSlope))

[0381] Where: Top represents the percentage inhibition rate at the top plateau, and the standard for the Top of the curve is generally between 80% and 120%; Bottom represents the percentage inhibition rate at the bottom plateau, and the Bottom of the curve is generally between -20% and 20%; HillSlope represents the slope of the percentage inhibition rate curve.

[0382] The results are shown in Table 5.

[0383] Table 5 IC50 of siRNA 50 (nM)

[0384] As can be seen from Table 5, the siRNA provided in this application has high GRB14 gene repressive activity in Huh7 cells.

[0385] Example 4: Determination of the inhibition rate of siRNA conjugates on GRB14 gene expression

[0386] 4.1 Test Materials:

[0387] Human primary hepatocytes (PHH cells) were provided by Chengdu WuXi AppTec Co., Ltd.

[0388] PHH culture medium: invitroGRO CP Medium, purchased from Bioreclamation, catalog number: S03316;

[0389] Transfection reagent, purchased from Invitrogen, catalog number: 13778-150;

[0390] 96 Kit, purchased from Qiagen, item number: Qiagen-74182;

[0391] FastStart Universal SYBR Green Master, purchased from Roche, item number 04914058001;

[0392] AceQ Universal U Probe Master Mix V2, purchased from Vazyme, item number: 7F680J2;

[0393] HiScript III RT SuperMix for qPCR (+gDNA wiper), purchased from Vazyme, catalog number R323-01;

[0394] The GRB14 primer and probe were synthesized by Sangon Biotech.

[0395] TaqMan Gene Expression Assay (GAPDH), purchased from Thermo, ID-Hs02786624_g1.

[0396] 4.2 Test Methods

[0397] siRNA conjugates (final concentrations of siRNA conjugates were 5 nM and 0.5 nM, in duplicate) were transfected into PHH cells as follows: Frozen PHH cells were harvested, thawed, counted, and adjusted to a cell volume of 6 × 10⁶ cells / well. 5 cells / mL, simultaneously applied The siRNA conjugate was transfected into cells using the transfection reagent, and the cells were seeded at a density of 54,000 cells per well in a 96-well plate. 100 μL of PHH culture medium was added to each well. The cells were incubated in a 5% CO2, 37°C incubator. After 48 hours, the culture medium was removed and the cells were collected for total RNA extraction. Use according to the kit instructions. Total RNA was extracted using 96Kit.

[0398] siRNA conjugates (final concentrations of siRNA conjugates were 200 nM and 10 nM, in duplicate) were introduced into PHH cells via free uptake, as described below: Frozen PHH cells were harvested, thawed, counted, and adjusted to a cell volume of 6 × 10⁶ cells / well. 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 PHH culture medium added to each well. Cells were incubated in a 5% CO2, 37°C incubator. After 48 hours, the culture medium was removed and cells were collected for total RNA extraction. Use according to the kit instructions. Total RNA was extracted using 96Kit.

[0399] Reverse transcription to cDNA was performed using a reverse transcription kit, following these steps:

[0400] (1) Remove gDNA using gDNAase according to the table below;

[0401] 42℃, 2 min; 4℃, let stand.

[0402] (2) Add 4 μL of 5×HiScript III qRT SuperMix directly to the reaction plate from step (1). Run the program: 37°C for 15 minutes, then 85°C for 5 seconds.

[0403] (3) Detect the target gene cDNA by qPCR.

[0404] The qPCR procedure is described below.

[0405] 95℃, 10 minutes;

[0406] 95℃, 15 seconds, 60℃, 1 minute (this operation is repeated 40 times).

[0407] Results analysis:

[0408] a) Use Quant Studio 7 software with default settings to automatically calculate the Ct value;

[0409] b) Calculate the relative expression level of the gene using the following formula:

[0410] ΔCt = Ct(GRB14 gene) - Ct(GAPDH)

[0411] ΔCt = ΔCt(test sample group) - ΔCt(Mock group), where the Mock group represents the group without siRNA conjugate compared to the test sample group;

[0412] mRNA expression relative to the Mock group = 2 -ΔΔCt

[0413] 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%

[0414] Table 6. Inhibition rate of siRNA conjugates on GRB14 gene expression Note: "--" indicates that the result is not shown.

[0415] As can be seen from Table 6, the siRNA conjugate provided in this application has high GRB14 gene repression activity in PHH cells.

[0416] Example 5

[0417] The experimental methods in this embodiment are basically the same as those in Embodiment 2, and the results of the inhibition rate measurement are shown in Table 7.

[0418] Table 7. Inhibition rate of siRNA in inhibiting GRB14 gene expression Note: "--" indicates that the result is not shown.

[0419] As can be seen from Table 7, the siRNA provided in this application has high GRB14 gene repressive activity in Huh7 cells.

[0420] Example 6

[0421] The experimental methods in this embodiment are basically the same as those in Example 4. Under free uptake conditions, the inhibition rate was measured and the results are shown in Table 8.

[0422] Table 8. Inhibition rate of siRNA conjugates on GRB14 gene expression

[0423] As can be seen from Table 8, the siRNA conjugate provided in this application has high GRB14 gene repression activity in PHH cells.

[0424] Example 7: IC50 of siRNA conjugate inhibiting GRB14 gene expression 50 Measurement

[0425] The following concentration ranges (nM) for the siRNA conjugate assay were set as follows: 500, 125, 31.25, 7.8125, 1.9531, 0.4883, 0.1221, and 0.03052 nM (replicas). The siRNA was allowed to enter PHH cells via free uptake, and then IC50 was performed using a method similar to that in Example 4. 50 Measurement.

[0426] Results analysis:

[0427] a) Use Quant Studio 6Flex software with default settings to automatically calculate the Ct value;

[0428] b) Calculate the relative expression level of the gene using the following formula:

[0429] ΔCt = Ct(GRB14 gene) - Ct(GAPDH)

[0430] ΔCt = ΔCt(test sample group) - ΔCt(Mock group), where the Mock group represents the group without siRNA conjugate compared to the test sample group;

[0431] mRNA expression relative to the Mock group = 2 -ΔΔCt

[0432] 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%

[0433] Calculation process: Using the log value of siRNA concentration as the X-axis and the percentage inhibition rate 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 conjugate. 50 value.

[0434] The fitting formula is: Y = Bottom + (Top - Bottom) / (1 + 10^(logIC)) 50 -X)*HillSlope))

[0435] Where: Top represents the percentage inhibition rate at the top plateau, and the standard for the Top of the curve is generally between 80% and 120%; Bottom represents the percentage inhibition rate at the bottom plateau, and the Bottom of the curve is generally between -20% and 20%; HillSlope represents the slope of the percentage inhibition rate curve.

[0436] The results are shown in Table 9.

[0437] Table 9 IC50 of siRNA conjugates50 (nM)

[0438] As can be seen from Table 9, the siRNA conjugate provided in this application has high GRB14 gene repression activity in PHH cells.

[0439] Example 8: In vitro stability test of rat liver homogenate

[0440] 1. Experimental reagents and consumables

[0441] 2. Experimental Procedure

[0442] 2.1 Preparation of liver homogenate

[0443] 2.1.1 Preparation of grinding fluid

[0444] 2.1.2 Tissue Homogenization

[0445] Rat liver tissue (collected from SD rats, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) was mixed with homogenizing solution at a ratio of 100mg:5mL to prepare liver homogenate (concentration of 20mg / mL). After preparation, the homogenate was added to a homogenizer and the homogenization parameters were set as follows.

[0446] 2.2 Sample Preparation

[0447] The siRNA conjugate sample was prepared into a 1 mg / mL solution using enzyme-free water and set aside for later use. The internal standard sample was prepared into a 0.125 mg / mL solution using enzyme-free water.

[0448] 2.3 Incubation of biological samples

[0449] (1) Add 250 μL of the prepared liver homogenate to a 2 mL enzyme-free tube.

[0450] (2) Add 50 μL of siRNA conjugate sample solution based on step (1);

[0451] (3) The system consists of 300 μL of biological sample. Vortex and let stand for 5 min.

[0452] (4) After standing, take 2 tubes of the solution obtained in step (3), 100 μL in each tube;

[0453] (5) The system was incubated at 37°C for 48 hours.

[0454] 2.4 Biological Sample Processing

[0455] Vortex each 100 μL biological sample system, mix well, add 300 μL of Clarity OTX Lysis-loading Buffer (purchased from Agilent-FinnoMed, catalog number AL0-8579), vortex, let stand for 30 min, add 100 μL of internal standard solution, vortex, let stand for 5 min, centrifuge for 1 min, and set aside for use (total sample volume approximately 500 μL).

[0456] 2.5 Solid-phase extraction:

[0457] (1) Preparation of solid phase extraction reagent

[0458] Activator: Add 200 mL of methanol to the mobile phase bottle and label it as activator;

[0459] Equilibrium buffer: Prepare 1M phosphate buffer solution [877mL sodium dihydrogen phosphate (1.56g / L) + 123mL disodium hydrogen phosphate (3.58g / L)], dilute 100 times, adjust pH to 5.5 with phosphate, and label as equilibrium buffer;

[0460] 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;

[0461] 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;

[0462] (2) The extraction steps are as follows:

[0463] 2.6 Post-processing

[0464] The eluent (600 μL each time, totaling 1200 μL) was placed in a 2 mL EP tube and concentrated under vacuum for 6 hours at 1800 rpm. The concentrated sample was then reconstituted with 100 μL of mobile phase (initial ratio), centrifuged at low speed for 2 min, and 10 μL of the supernatant was injected into a high-resolution mass spectrometer. The antisense strand ratio of the siRNA conjugate was semi-quantitatively detected using LC-MS / MS, calculated as: AS strand remaining percentage % = AS strand MS intensity / (MS intensity of AS strand and all related degradation products) × 100%; where MS intensity is the mass spectrometry signal value. The metabolic results after in vitro incubation in rat liver homogenate for 48 hours are shown in the table below.

[0465] Table 10. Remaining antisense strand percentage of siRNA conjugates

[0466] Where AS represents the antisense strand of the siRNA conjugate, a larger remaining AS indicates better drug stability and longer-lasting effect. Table 10 shows that the siRNA conjugate disclosed in this invention exhibits excellent in vitro stability in rat liver homogenate.

[0467] Example 9: In vitro off-target experiment

[0468] 1. Main reagents and consumables

[0469] InvitroGRO CP Medium, serum-free (Bioreclamation IVT-S03316), fetal bovine serum (ExCellBio-FSP500), penicillin / streptomycin (Hyclone-SV30010), AO / PI staining solution (Countstar-RE010212), nuclease-free water (Invitrogen-AM9932), RNA extraction kit (miRNeasy Tissue / Cells Advanced Mini Kit, Qiagen-217604), Agilent RNA analysis kit (Agilent RNA 6000 Nano Kit, Agilent-5067-1511), transcriptome library construction kit ( The following reagents were provided by WuXi AppTec Co., Ltd.: Universal V10RNA-seq Library Prep Kit for Illumina (Vazyme-NR606-02), Double-stranded Deoxyribonucleic Acid High Sensitivity Concentration Assay Kit (Qubit 1X dsDNA HS Assay Kit, Thermo Fisher Scientific-Q33231), High Sensitivity Deoxyribonucleic Acid Pre-prepared Electrophoresis Strips (High Sensitivity D1000 ScreenTape, Agilent-5067-5584), Next-Generation Sequencing High-Throughput Kit (NovaSeq 6000 S4 Reagent Kit v1.5 (300 cycles) (Illumina-20028312)), and Human Primary Hepatocytes (PHH) (Batch: JMJ).

[0470] 2. Main Instruments

[0471] Biosafety cabinet (Lixin (Shanghai) Instrument Co., Ltd., Hfsafe-1800LCA2), micro-volume UV-Vis spectrophotometer (NanoDrop One, Thermo Fisher Scientific), Agilent 2100 Bioanalyzer (Agilent), thermal cycler (T100 thermal cycler, Bio-Rad), fluorescence meter (Qubit 4fluorometer, Thermo Fisher Scientific), Agilent 4200 TapeStation automated electrophoresis system, sequencer (NovaSeq 6000 (Illumina)), fully automated cell fluorescence analyzer (Rigel S2, Countstar).

[0472] 3. Culture medium formulation

[0473] The cell culture medium preparation system containing 10% fetal bovine serum in 20 mL consists of: 17.8 mL of serum-free in vitro culture medium InvitroGRO CP Medium (Bioreclamation IVT-S03316), 2 mL of fetal bovine serum (ExCellBio-FSP500), and 0.2 mL of penicillin / streptomycin (Hyclone-SV30010).

[0474] 4. Experimental Procedure

[0475] 4.1 Cell Plating

[0476] N-ER-FY047232M49L96 enters human primary hepatocytes via free uptake, as described below:

[0477] 1) Dilute the conjugate N-ER-FY047232M49L96 with nuclease-free water to a final concentration of 10 times (final concentrations are 5nM and 50nM).

[0478] 2) Remove one vial of cryopreserved cells from the liquid nitrogen tank, gently agitate in a water bath until the cryopreservation solution thaws, then transfer the cells to a cell culture medium containing 10% fetal bovine serum, adjusting the final cell density to 6.7*102. 5 cells / mL.

[0479] 3) Take 50 μL of the conjugate from step 1) into a collagen-coated 24-well cell plate, and then add 450 μL of the cell suspension from step 2), resulting in a final cell density of 300,000 per well. The final concentrations of N-ER-FY047232M49L96 are 5 nM and 50 nM. Each conjugate is tested in triplicate, and the wells without the conjugate serve as the control group.

[0480] 4) After culturing for 72 hours, wash each well with PBS, then add 260 μL of lysis buffer to lyse the cells for subsequent experiments.

[0481] 4.2 Next-Generation Sequencing

[0482] 1) Purify total RNA from cells according to the manufacturer's (Qiagen-217604_miRNeasy Tissue / Cells Advanced Mini Kit) instructions.

[0483] 2) The total RNA concentration was detected using NanoDrop One, and RNA integrity was analyzed according to the manufacturer's (Agilent 2100 Bioanalyzer\Agilent-5067-1511_Agilent RNA 6000Nano Kit) guidelines.

[0484] 3) According to the manufacturer (Vazyme-NR606_ Library preparation was performed using the Universal V10RNA-seq Library Prep Kit for Illumina.

[0485] 4) Detect the library concentration according to the manufacturer's (Thermo Fisher Scientific-Qubit 4fluorometer\Thermo Fisher Scientific-Q33231_Qubit 1X dsDNA HS Assay Kit) guidelines, and analyze the library fragment size according to the manufacturer's (Agilent 4200TapeStation\Agilent-5067-5584_High Sensitivity D1000 ScreenTape) guidelines.

[0486] 5) Perform library sequencing according to the manufacturer's (Illumina-NovaSeq 6000\Illumina-20028312_NovaSeq 6000S4 Reagent Kit v1.5(300cycles)) guidelines.

[0487] This study, based on next-generation sequencing technology, investigated the effect of free uptake of the test sample (N-ER-FY047232M49L96) by human hepatocytes on transcriptome expression. |log2(FoldChange)|>1 & padj.<0.05 was set as the screening criterion for differentially expressed genes.

[0488] 5. IC50 in PHH cells50 test

[0489] The concentration range (nM) for the N-ER-FY047232M49L96 assay of the siRNA conjugate to be tested was set as follows: 200.0, 50, 12.5, 3.125, 0.78125, 0.1953125, 0.048828125, 0.012207031. The IC50 assay was then performed using the same method as in Example 7. 50 The free uptake in PHH cells was determined, and the IC50 of N-ER-FY047232M49L96 in human hepatocytes was obtained. 50 It is 0.11 nM.

[0490] Sequencing of N-ER-FY047232M49L96 at concentrations of 5 nM and 50 nM revealed that only one differentially expressed gene conforming to the criteria |log2(FoldChange)|>1 & padj<0.05 was observed at both concentrations, with no overlap (as shown in Figures 1 and 2). This indicates that the differentially expressed gene did not exhibit a dose-dependent effect, meaning that no off-target genes were found with N-ER-FY047232M49L96 in in vitro PHH cell experiments. The IC50 of N-ER-FY047232M49L96 in PHH cells was also measured. 50 The concentration was 0.11 nM, and the two concentrations selected for the off-target experiment were IC50 and IC50, respectively. 50 The values ​​were 45.45 times and 454.54 times higher, indicating that the conjugate has a safety window of several hundred times, thus the risk of off-target effects in vivo is low, the safety is good, and it has good drug-like properties.

[0491] Example 10: Silent effect of siRNA conjugates on mice expressing the human GRB14 (hGRB14) gene.

[0492] Experimental materials:

[0493] 1. AAV-based mouse model overexpressing the hGRB14 gene was constructed.

[0494] 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 hGRB14 gene (pAAV[Exp]-CBh>SEAP(ns):T2A:hGRB14[NM_004490.3], virus provided by Yunzhou Biotechnology (Guangzhou) Co., Ltd.) was administered to induce target gene overexpression. The administration volume was 100 μL (10 × 10⁻⁶). 11 vg) / animal, then fed with regular feed.

[0495] 2. In vivo efficacy study of siRNA silencing in hGRB14 mouse model

[0496] Fourteen days after AAV virus injection, mice were divided into groups of five. Mice were subcutaneously administered a single 3 mg / kg (mpk) 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 samples were 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. For analysis, the samples were thawed on ice, centrifuged, and the supernatant was used for protein content determination using Phospha-Light assay. TM SEAP reporter gene assay system (Invitrogen) TM (Purchased from Thermo Fisher Scientific, catalog number T1017) SEAP protein expression assay (reflecting hGRB14 protein expression). The inhibition rate (%) of the siRNA conjugate in mice expressing the human GRB14 (hGRB14) gene was calculated according to the formula: inhibition rate % = (1 - mean protein expression level in the treatment group / mean protein expression level in the control group) * 100%, as shown in Table 11.

[0497] Table 11

[0498] As can be seen from Table 11, the siRNA conjugate of this application has high inhibitory activity against the hGRB14 gene in vivo, and can reduce the expression level of hGRB14 for a long time, with long-lasting inhibition in vivo.

[0499] Example 11: Inhibitory effect of siRNA conjugates on GRB14 gene expression in wild-type mice.

[0500] Wild-type C57BL / 6 male mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) aged 6-8 weeks were randomly divided into groups of 5 mice each after 7 days of acclimatization. They were subcutaneously administered a single dose of 3 mg / kg of the siRNA conjugate described in this application and PBS (purchased from Gibco, catalog number 10010-023), with an injection volume of 5 μL / g. Mice were euthanized after 14, 28, or 56 days of administration, and two 30 mg portions of left liver tissue were collected and flash-frozen in liquid nitrogen. For analysis, after removing the tissue, the prepared lysis buffer was added according to the instructions of the RNeasy Mini Kit (250) (purchased from QIAGEN, catalog number 74106), and magnetic beads were added before homogenization using a tissue homogenizer. After homogenization, subsequent RNA extraction steps were performed according to the instructions. The extracted RNA was reverse transcribed using a reverse transcription kit (Vazyme, catalog number R312-02). The cDNA obtained from the reverse transcription was amplified using a SupRealQ Purple Universal SYBR qPCR Master Mix (U+) kit (Vazyme, catalog number Q412-03) to detect the expression level of the target gene. GAPDH was used as an internal control, and the ΔΔCt assay was performed using ABI QuantStudio. TM Real-time fluorescence PCR was performed using the 6 Pro real-time fluorescence PCR system. Two replicates were used for each mouse liver tissue sample.

[0501] In this real-time quantitative PCR method, the ΔΔCt method was used to calculate the relative quantitative levels and inhibition rates of target gene mRNA in each test group. The calculation method is as follows:

[0502] ΔCt(test group) = Ct(target gene in test group) - Ct(internal reference gene in test group)

[0503] ΔCt(control group) = Ct(target gene in control group) - Ct(internal reference gene in control group)

[0504] ΔCt(test group) = ΔCt(test group) - ΔCt(control group average)

[0505] ΔCt(control group) = ΔCt(control group) - ΔCt(control group average)

[0506] Here, ΔCt (control group mean) is the arithmetic mean of the ΔCt (control group) values ​​of each animal that died in the control group at the same time point. Therefore, each animal in both the test group and the control group corresponds to a ΔCt value.

[0507] The relative expression level of the target gene mRNA in the test group was 2. -ΔΔCt (Test Group)

[0508] The inhibition rate of target gene mRNA expression in the test group = (1 - relative expression level of target gene mRNA in the test group) × 100%

[0509] Table 12 Note: "--" indicates that the result is not shown.

[0510] As can be seen from Table 12, the siRNA conjugate of this application has high inhibitory activity against the GRB14 gene in vivo, and can reduce the expression level of GRB14 for a long time, with long-lasting inhibition in vivo.

[0511] Example 12: Inhibitory effect of siRNA conjugates on GRB14 gene expression in wild-type mice.

[0512] Wild-type C57BL / 6 male mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) aged 6-8 weeks were randomly divided into groups of 5 mice each after 7 days of acclimatization. They were subcutaneously administered a single dose of the siRNA conjugate described in this application and PBS (purchased from Gibco, catalog number 10010-023) at a volume of 5 μL / g. Fourteen days after administration, the mice were euthanized. Two 30 mg portions of left lobe liver tissue were collected and rapidly frozen in liquid nitrogen. After being cryogenically homogenized, tissue RNA was extracted, and the mRNA inhibition rate of the target gene was detected using the method described in Example 11. The results are shown in Table 13.

[0513] Table 13

[0514] As can be seen from Table 13, the siRNA conjugate of this application has high inhibitory activity against the GRB14 gene in vivo and can reduce the expression level of GRB14 for a long time.

[0515] Example 13 Hypoglycemic Efficacy of siRNA Conjugate in ob / ob Mice

[0516] Five-week-old male ob / ob mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were acclimatized for 3 days or more. Random blood glucose and random insulin levels were measured on day 4 (4 days before drug administration). On day 1 (1 day before drug administration), mice were randomly divided into groups of 6 mice each based on their body weight and random blood glucose levels.

[0517] The day of administration was defined as D0. The treatment groups received subcutaneous injections of 1 mg / kg or 10 mg / kg of PBS solution containing N-ER-FY047232M49L96, while the PBS control group received the same volume of PBS solution. The administration volume was 5 μL / g, and the frequency was once every two weeks. Random blood samples (tail tip blood) were collected from mice on D-4, D7, and D21, and morning random blood glucose levels were measured using Roche (Zhihang) assay. The results are shown in Table 14.

[0518] Table 14 Blood glucose levels in mice

[0519] Random blood samples (whole blood) were collected from mice on days 4-4, 7-8, and 21. Serum solutions were obtained and stored at -80°C for long-term preservation. Before testing, the samples were thawed on ice, centrifuged, and the supernatant was collected for detection. Insulin was detected using the Insulin Mouse Ultra Sensitive ELISA kit (purchased from Chrystal Chem, catalog number #90082). The results are shown in Table 15.

[0520] Table 15 Insulin levels in mice

[0521] As can be seen from the experimental results in Tables 14 and 15, N-ER-FY047232M49L96 can significantly reduce random blood glucose and random insulin levels in ob / ob mice.

[0522] Example 14: Repeated-dose toxicity test of subcutaneous injection of siRNA conjugate in C57BL / 6 mice

[0523] This experiment used 72 C57BL / 6J mice (sourced from Guoke Saifu Hebei Pharmaceutical Technology Co., Ltd.), which were randomly divided into three groups: a solvent control group, and low- and high-dose groups of the siRNA conjugate N-ER-FY047232M49L96 (administered at doses of 100 and 300 mg / kg, respectively). Each main experimental group and toxicotropic satellite group consisted of 12 mice, half male and half female. Mice in each group were subcutaneously injected with the corresponding concentration of the siRNA conjugate or the solvent control (PBS) at a dose volume of 5 mL / kg. Administration was repeated every two weeks for four consecutive weeks, for a total of three administrations (D1, D15, and D29). The day of administration was defined as day 1 of the experiment (D1).

[0524] During the experiment, general condition was observed at least twice daily and detailed observation was conducted weekly after drug administration; food intake was measured weekly; and animal weight was measured twice weekly. At the end of the 2-week administration period (D16), hematological and blood biochemical tests were performed on 3 animals / groups / sex in the main experimental group; at the end of the 4-week administration period (D30), hematological, coagulation, and blood biochemical tests (with added HbA1C testing) were performed on 3 animals / groups / sex in the main experimental group, followed by euthanasia and gross anatomical observation and histopathological examination (liver, kidneys, spleen, drug administration site, and surrounding tissues). Cytokine (IFN-γ, TNF-α, IL-6, IL-10) levels were measured in 3 animals / groups / sex in the satellite groups before drug administration and 4 hours and 24 hours after the first and last drug administration.

[0525] In summary, under the conditions of this experiment, C57BL / 6J mice were subcutaneously injected with N-ER-FY047232M49L96 at doses of 100 and 300 mg / kg for 4 consecutive weeks (1 time / 2 weeks, 3 times in total). The no obvious adverse reaction level (NOAEL) was 100 mg / kg.

[0526] We conducted repeated-dose toxicity tests on multiple siRNA conjugates in mice, and the results showed that the siRNA conjugates of this application had no obvious toxic reactions.

[0527] Example 15: siRNA conjugate knockdown experiment in cynomolgus monkeys

[0528] Ten male cynomolgus macaques aged 5–7 years (weighing 5–7 kg) (provided by Zhaoyan (Suzhou) New Drug Research Center Co., Ltd.) were housed in a breeding facility. Liver biopsies were performed three days prior to drug administration (D-3) for GRB14 protein expression analysis. A single dose of 3 mg / kg of the siRNA conjugate described in this application was administered subcutaneously to the macaques (injection site: the loose skin area on the scapula / lateral neck). The day of administration was defined as Day 0 (D0). Liver biopsies were performed on D22, D57, and D85 post-administration for GRB14 protein expression analysis.

[0529] The experimental results show that the siRNA conjugate of this application has a high inhibition rate on GRB14 protein expression in cynomolgus monkeys.

[0530] Example 16: Silent effect of siRNA conjugates on mice expressing the human GRB14 (hGRB14) gene.

[0531] A mouse model overexpressing the hGRB14 gene was constructed using the same method as in Example 10. Mice were subcutaneously administered a single 3 mg / kg dose of the siRNA conjugate of this application at a volume of 5 μL / g in sterile PBS without RNase. The control group was injected with the same volume of sterile PBS without RNase. The inhibition rate (%) of the siRNA conjugate in mice expressing the human GRB14 (hGRB14) gene was detected on days 7, 14, 21, and 28 after administration, using the same method as in Example 10. The results are shown in Table 16.

[0532] Table 16

[0533] As can be seen from Table 16, the siRNA conjugate of this application has high inhibitory activity against the GRB14 gene in vivo, and can reduce the expression level of GRB14 for a long time, with long-lasting inhibition in vivo.

Claims

1. A siRNA for inhibiting GRB14 gene expression, the siRNA comprising a sense strand and an antisense strand, wherein each nucleotide in the siRNA is independently modified or unmodified, wherein the sense strand contains nucleotide sequence I, the antisense strand contains nucleotide sequence II, wherein nucleotide sequence I and nucleotide sequence II are at least partially anticomplementary to form a double-stranded region, wherein nucleotide sequence I and nucleotide sequence II are selected from the following sequences: (1) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:399, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:400: 5'-GUACCCAGUGA-3'(SEQ ID NO:399) 5'-UCACUGGGGUAC-3' (SEQ ID NO:400); (2) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:401, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:402: 5'-GGCUCGAGAU-3'(SEQ ID NO:401) 5'-AUCUCGAGCC-3' (SEQ ID NO:402); (3) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:403, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:404: 5'-CAGCUGU-3'(SEQ ID NO:403) 5'-ACAGCUG-3' (SEQ ID NO:404); (4) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:33, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:34: 5'-GAAUCAUUACAUUGAUGAA-3'(SEQ ID NO:33) 5'-UUCAUCAAUGUAAUGAUUC-3' (SEQ ID NO: 34); (5) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:405, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:406: 5'-CUGGACCCUUUUUGAG-3'(SEQ ID NO:405) 5'-CUCAAAAAGGGUCCAG-3' (SEQ ID NO: 406); (6) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:407, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:408: 5'-AACCAAUGGU-3'(SEQ ID NO:407) 5'-ACCAUUGGUU-3' (SEQ ID NO:408); (7) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:409, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:410: 5'-CUUACAUGCGA-3'(SEQ ID NO:409) 5'-UCGCAUGUAAG-3' (SEQ ID NO: 410); (8) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:411, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:412: 5'-ACAGGGAAAGAA-3'(SEQ ID NO:411) 5'-UUCUUUCCCUGU-3' (SEQ ID NO: 412); (9) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:413, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:414: 5'-GACCUGAAAAU-3'(SEQ ID NO:413) 5'-AUUUUCAGGUC-3' (SEQ ID NO: 414); (10) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:415, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:416: 5'-GCUGGGUGA-3'(SEQ ID NO:415) 5'-UCACCCAGC-3' (SEQ ID NO: 416); (11) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:417, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:418: 5'-CGCGAUUAGAUU-3'(SEQ ID NO:417) 5'-AAUCUAAUCGCG-3' (SEQ ID NO: 418); (12) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:419, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:420: 5'-AAGUAUGGCAUGCA-3'(SEQ ID NO:419) 5'-UGCAUGCCUACUU-3' (SEQ ID NO: 420); (13) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:421, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:422: 5'-AAAGCAGAGUU-3'(SEQ ID NO:421) 5'-AACUCUGCUUU-3' (SEQ ID NO:422); (14) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:423, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:424: 5'-CUCAGCGAU-3'(SEQ ID NO:423) 5'-AAUCGCUGAG-3' (SEQ ID NO: 424); (15) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:425, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:426: 5'-CGAUUGAUUAUUCAGCA-3'(SEQ ID NO:425) 5'-UGCUGAAUAAUCAAUCG-3' (SEQ ID NO: 426); (16) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:427, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:428: 5'-AAGGACUUGUGGAU-3'(SEQ ID NO:427) 5'-AUCCACAAGUCCUU-3' (SEQ ID NO: 428); (17) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:429, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:430: 5'-AGUCAGAGUAAC-3'(SEQ ID NO:429) 5'-GUUACUCUGACU-3' (SEQ ID NO: 430); (18) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:431, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:432: 5'-UGACGGU-3'(SEQ ID NO:431) 5'-ACCGUCA-3' (SEQ ID NO:432); (19) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:433, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:434: 5'-AUGGCCA-3'(SEQ ID NO:433) 5'-UGGCCAU-3' (SEQ ID NO: 434); (20) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:435, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:436: 5'-GAUUUACA-3'(SEQ ID NO:435) 5'-UGUAAAUC-3' (SEQ ID NO:436); (21) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:437, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:438: 5'-GAAACAUUAUUGU-3'(SEQ ID NO:437) 5'-ACAAUAAUGUUUC-3' (SEQ ID NO: 438); (22) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:439, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:440: 5'-GACUUAUUAAACUAUU-3'(SEQ ID NO:439) 5'-AAUAGUUUAAUAAGUC-3' (SEQ ID NO: 440); (23) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:441, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:442: 5'-CUUUACA-3'(SEQ ID NO:441) 5'-UGUAAAG-3' (SEQ ID NO:442); (24) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:3, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:4; (25) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:57, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:58; (26) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:84, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:85; (27) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:94, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:95; (28) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:114, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:115; (29) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:153, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:154; (30) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:155, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:156; (31) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:157, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:158; (32) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:159, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:160; (33) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:318, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:319; (34) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:342, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:343; (35) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:617, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:618: 5'-CUGUUGAUCCUGAAGAA-3'(SEQ ID NO:617) 5'-UUCUUCAGGAUCAACAG-3' (SEQ ID NO: 618); (36) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:619, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:620: 5'-GAGUUUUCUUGGUAC-3'(SEQ ID NO:619) 5'-GUACCAAGAAACUC-3' (SEQ ID NO: 620); (37) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:621, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:622: 5'-ACGGGAUAGUCA-3'(SEQ ID NO:621) 5'-UGACUAUCCCGU-3' (SEQ ID NO: 622); (38) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:623, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:624: 5'-GUCAAUGAGUCAU-3'(SEQ ID NO:623) 5'-AUGACUCAUUGAC-3' (SEQ ID NO: 624); (39) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:625, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:626: 5'-CCCUGGUAGCAA-3'(SEQ ID NO:625) 5'-UUGCUACCAGGG-3' (SEQ ID NO: 626); (40) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:513, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:514; (41) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:517, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:518; (42) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:573, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:574; (43) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:577, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:578; (44) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:581, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:582; (45) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:593, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:594; (46) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:597, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:598; (47) The nucleotide sequence I comprises the nucleotide sequence shown in SEQ ID NO:612, and the nucleotide sequence II comprises the nucleotide sequence shown in SEQ ID NO:

613.

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

3. The siRNA according to claim 1 or 2, wherein the sense strand further comprises nucleotide sequence III, and the antisense strand further comprises nucleotide sequence IV, wherein nucleotide sequence III and nucleotide sequence IV are each independently 0-12 nucleotides in length, wherein nucleotide sequence III is attached to the 5' end of nucleotide sequence I, and nucleotide sequence IV is attached to the 3' end of nucleotide sequence II, wherein nucleotide sequence III and nucleotide sequence IV are of equal length and substantially anticomplementary or completely anticomplementary; substantially anticomplementary means that there is no more than one base mismatch between the two nucleotide sequences; completely anticomplementary means that there is no mismatch between the two nucleotide sequences; and / or, nucleotide sequence III is attached to the 3' end of nucleotide sequence I, and nucleotide sequence IV is attached to the 5' end of nucleotide sequence II, wherein nucleotide sequence III and nucleotide sequence IV are of equal length and substantially anticomplementary or completely anticomplementary; substantially anticomplementary means that there is no more than one base mismatch between the two nucleotide sequences; completely anticomplementary means that there is no mismatch between the two nucleotide sequences.

4. The siRNA according to any one of claims 1-3, wherein nucleotide sequences I and III form a double-stranded region that is at least partially anticomplementary to nucleotide sequences II and IV, wherein nucleotide sequences I and III, and nucleotide sequences II and IV are selected from the following sequences: (1) The nucleotide sequences I and III comprise or consist of the nucleotide sequences shown in SEQ ID NO:141, and the nucleotide sequences II and IV comprise or consist of the nucleotide sequences shown in SEQ ID NO:142; (2) The nucleotide sequences I and III comprise or consist of the nucleotide sequences shown in SEQ ID NO:266, and the nucleotide sequences II and IV comprise or consist of the nucleotide sequences shown in SEQ ID NO:267; (3) The nucleotide sequences I and III comprise or consist of the nucleotide sequences shown in SEQ ID NO:457, and the nucleotide sequences II and IV comprise or consist of the nucleotide sequences shown in SEQ ID NO:627; (4) The nucleotide sequences I and III comprise or consist of the nucleotide sequences shown in SEQ ID NO:206, and the nucleotide sequences II and IV comprise or consist of the nucleotide sequences shown in SEQ ID NO:207; (5) The nucleotide sequences I and III comprise or consist of the nucleotide sequences shown in SEQ ID NO:521, and the nucleotide sequences II and IV comprise or consist of the nucleotide sequences shown in SEQ ID NO:628; (6) The nucleotide sequences I and III comprise or consist of the nucleotide sequences shown in SEQ ID NO:537, and the nucleotide sequences II and IV comprise or consist of the nucleotide sequences shown in SEQ ID NO:629; (7) The nucleotide sequences I and III comprise or consist of the nucleotide sequences shown in SEQ ID NO:557, and the nucleotide sequences II and IV comprise or consist of the nucleotide sequences shown in SEQ ID NO:630; (8) The nucleotide sequences I and III comprise or consist of the nucleotide sequences shown in SEQ ID NO:577, and the nucleotide sequences II and IV comprise or consist of the nucleotide sequences shown in SEQ ID NO:631; (9) The nucleotide sequences I and III comprise or consist of the nucleotide sequences shown in SEQ ID NO:585, and the nucleotide sequences II and IV comprise or consist of the nucleotide sequences shown in SEQ ID NO:632; (10) The nucleotide sequences I and III comprise or consist of the nucleotide sequences shown in SEQ ID NO:612, and the nucleotide sequences II and IV comprise or consist of the nucleotide sequences shown in SEQ ID NO:

633.

5. The siRNA according to any one of claims 1-4, wherein the sense strand further comprises a nucleotide sequence V and / or the antisense strand further comprises a nucleotide sequence VI, wherein the length of nucleotide sequences V and VI is 0 to 3 nucleotides, wherein nucleotide sequence V is attached to the 3' end of the sense strand to form a 3' overhang of the sense strand and / or nucleotide sequence VI is attached to the 3' end of the antisense strand to form a 3' overhang of the antisense strand; preferably, the length of nucleotide sequence V or VI is 2 nucleotides; more preferably, the nucleotide sequence V is identical or different from the nucleotide at the corresponding position of the target mRNA, or VI is mismatched or complementary to the nucleotide at the corresponding position of the target mRNA.

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

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

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

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

10. The siRNA of any one of claims 1-9, wherein, The positive chain has no reverse debased deoxyribose residues connected to either the 5' end or the 3' end, or the positive chain has only one reverse debased deoxyribose residue connected to the 5' end, or the positive chain has only one reverse debased deoxyribose residue connected to the 3' end, or the positive chain has one reverse debased deoxyribose residue connected to both the 5' end and the 3' end.

11. The siRNA according to any one of claims 1-10, wherein the modified nucleotide is selected from 2'-fluoro-modified nucleotides, 2'-alkoxy-modified nucleotides, 2'-substituted alkoxy-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-deoxynucleotides, 2'-amino-modified nucleotides, 2'-substituted amino-modified nucleotides, nucleotide analogs, or any combination of two or more thereof. Preferably, the modified nucleotide is selected from 2'-fluoromodified nucleotides, 2'-methoxymodified nucleotides, 2'-O-CH2-CH2-O-CH3modified nucleotides, 2'-O-CH2-CH=CH2modified nucleotides, 2'-CH2-CH2-CH=CH2modified nucleotides, 2'-deoxynucleotides, nucleotide analogs, or any combination of two or more thereof.

12. The siRNA according to any one of claims 1-11, wherein each nucleotide in the sense strand and the antisense strand is independently a 2'-fluoro-modified nucleotide or a non-fluoro-modified nucleotide; Preferably, in the 5' to 3' direction, the 2'-fluorinated nucleotides are located at positions 7, 9, 10 and 11 of the sense strand, and the remaining positions are non-fluorinated nucleotides; in the 5' to 3' direction, the 2'-fluorinated nucleotides are located at positions 2, 6, 14 and 16 of the antisense strand, and the remaining positions are non-fluorinated nucleotides. Alternatively, in the 5' to 3' direction, 2'-fluorinated nucleotides are located at positions 7, 9, 10, and 11 on the sense strand, with the remaining positions being non-fluorinated nucleotides; in the 5' to 3' direction, 2'-fluorinated nucleotides are located at positions 2, 6, 8, 9, 14, and 16 on the antisense strand, with the remaining positions being non-fluorinated nucleotides. Alternatively, in the 5' to 3' direction, 2'-fluorinated nucleotides are located at positions 7, 9, 10, and 11 on the sense strand, with the remaining positions being non-fluorinated nucleotides; in the 5' to 3' direction, 2'-fluorinated nucleotides are located at positions 2, 14, and 16 on the antisense strand, with the remaining positions being non-fluorinated nucleotides. Alternatively, in the 5' to 3' direction, 2'-fluorinated nucleotides are located at positions 5, 7, 8, and 9 of the sense strand, with the remaining positions being non-fluorinated nucleotides; in the 5' to 3' direction, 2'-fluorinated nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, with the remaining positions being non-fluorinated nucleotides. Alternatively, in the 5' to 3' direction, 2'-fluorinated nucleotides are located at positions 3, 7, 8, and 9 of the sense strand, with the remaining positions being non-fluorinated nucleotides; in the 5' to 3' direction, 2'-fluorinated nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand, with the remaining positions being non-fluorinated nucleotides. Alternatively, in the 5' to 3' direction, 2'-fluorinated nucleotides are located at positions 3, 7, 9, and 11 on the sense strand, with the remaining positions being non-fluorinated nucleotides; in the 5' to 3' direction, 2'-fluorinated nucleotides are located at positions 2, 7, 10, and 14 on the antisense strand, with the remaining positions being non-fluorinated nucleotides. Alternatively, in the 5' to 3' direction, 2'-fluorinated nucleotides are located at positions 7, 9, and 14 of the sense strand, with the remaining positions being non-fluorinated nucleotides; in the 5' to 3' direction, 2'-fluorinated nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand, with the remaining positions being non-fluorinated nucleotides. Alternatively, in the 5' to 3' direction, 2'-fluorinated nucleotides are located at positions 3, 7, 9, and 11 on the sense strand, with the remaining positions being non-fluorinated nucleotides; in the 5' to 3' direction, 2'-fluorinated nucleotides are located at positions 2, 3, 5, 7, 10, 12, and 14 on the antisense strand, with the remaining positions being non-fluorinated nucleotides. More preferably, each non-fluorinated nucleotide is a 2'-methoxy-modified nucleotide, wherein the 2'-methoxy-modified nucleotide refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribosome with a methoxy group.

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

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

15. The siRNA according to any one of claims 1-14, wherein the siRNA is arranged in the direction from the 5' end to the 3' end. (1) The positive chain contains thiophosphate groups located at the positions shown below: Between the first and second nucleotides starting at the 5' end of the positive strand; and Between the second and third nucleotides starting at the 5' end of the positive strand; and Between the first nucleotide and the second nucleotide starting at the 3' end of the positive strand; and Between the second and third nucleotides starting at the 3' end of the positive strand; or, (2) The positive chain contains thiophosphate groups located at the positions shown below: Between the first and second nucleotides starting at the 5' end of the positive strand; and Between the second and third nucleotides starting at the 5' end of the positive strand; or, (3) The positive chain contains thiophosphate groups located at the positions shown below: Between the first and second nucleotides starting at the 5' end of the positive strand; and Between the second and third nucleotides starting at the 5' end of the positive strand; The reverse debasing deoxyribose residue starting at the 3' end of the positive strand is between the first nucleotide and the first nucleotide.

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

17. The siRNA according to any one of claims 1-16, wherein each nucleotide in the sense strand and the antisense strand is independently a 2'-fluoro-modified nucleotide, a 2'-methoxy-modified nucleotide, a GNA-modified nucleotide, or any combination of two or more thereof. Preferably, in the 5' to 3' direction, the 2'-fluorinated nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, and the remaining positions are 2'-methoxylated nucleotides; in the 5' to 3' direction, the 2'-fluorinated nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, and the remaining positions are 2'-methoxylated nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5' phosphate group; Alternatively, in the 5' to 3' direction, 2'-fluorinated nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, with the remaining positions being 2'-methoxylated nucleotides, and the 3' end is removed; in the 5' to 3' direction, 2'-fluorinated nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, with the remaining positions being 2'-methoxylated nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5' phosphate group; Alternatively, in the 5' to 3' direction, 2'-fluorinated nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, with the remaining positions being 2'-methoxylated nucleotides; in the 5' to 3' direction, 2'-fluorinated nucleotides are located at positions 2, 6, 8, 9, 14, and 16 of the antisense strand, with the remaining positions being 2'-methoxylated nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5' phosphate group; Alternatively, in the 5' to 3' direction, 2'-fluorinated nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, with the remaining positions being 2'-methoxylated nucleotides; in the 5' to 3' direction, 2'-fluorinated nucleotides are located at positions 2, 14, and 16 of the antisense strand, GNA-modified nucleotides are located at position 6 of the antisense strand, with the remaining positions being 2'-methoxylated nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5' phosphate group; Alternatively, in the 5' to 3' direction, 2'-fluorinated nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, with the remaining positions being 2'-methoxylated nucleotides; in the 5' to 3' direction, 2'-fluorinated nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, GNA-modified nucleotides are located at position 7 of the antisense strand, with the remaining positions being 2'-methoxylated nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5' phosphate group; Alternatively, in the 5' to 3' direction, 2'-fluorinated nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, with the remaining positions being 2'-methoxylated nucleotides; in the 5' to 3' direction, 2'-fluorinated nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, with the remaining positions being 2'-methoxylated nucleotides; the 5' terminal nucleotide of the antisense strand is not linked to a 5' phosphate group or a 5' phosphate-derived group; Alternatively, in the 5' to 3' direction, 2'-fluorinated nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, with the remaining positions being 2'-methoxylated nucleotides; in the 5' to 3' direction, 2'-fluorinated nucleotides are located at positions 2, 6, 8, 9, 14, and 16 of the antisense strand, with the remaining positions being 2'-methoxylated nucleotides; the 5' terminal nucleotide of the antisense strand is not linked to a 5' phosphate group or a 5' phosphate-derived group; Alternatively, in the 5' to 3' direction, 2'-fluorinated nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, with the remaining positions being 2'-methoxylated nucleotides; in the 5' to 3' direction, 2'-fluorinated nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, with the remaining positions being 2'-methoxylated nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group; Alternatively, in the 5' to 3' direction, 2'-fluorinated nucleotides are located at positions 7, 9, 10, and 11 of the sense strand, with the remaining positions being 2'-methoxylated nucleotides; in the 5' to 3' direction, 2'-fluorinated nucleotides are located at positions 2, 6, 8, 9, 14, and 16 of the antisense strand, with the remaining positions being 2'-methoxylated nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group; Alternatively, in the 5' to 3' direction, 2'-fluorinated nucleotides are located at positions 5, 7, 8, and 9 of the sense strand, with the remaining positions being 2'-methoxylated nucleotides; in the 5' to 3' direction, 2'-fluorinated nucleotides are located at positions 2, 6, 14, and 16 of the antisense strand, with the remaining positions being 2'-methoxylated nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group; Alternatively, in the 5' to 3' direction, 2'-fluorinated nucleotides are located at positions 3, 7, 8, and 9 of the sense strand, with the remaining positions being 2'-methoxylated nucleotides; in the 5' to 3' direction, 2'-fluorinated nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand, with the remaining positions being 2'-methoxylated nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group; Alternatively, in the 5' to 3' direction, 2'-fluorinated nucleotides are located at positions 3, 7, 9, and 11 of the sense strand, with the remaining positions being 2'-methoxylated nucleotides; in the 5' to 3' direction, 2'-fluorinated nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand, with the remaining positions being 2'-methoxylated nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group; Alternatively, in the 5' to 3' direction, 2'-fluorinated nucleotides are located at positions 7, 9, and 14 of the sense strand, with the remaining positions being 2'-methoxylated nucleotides; in the 5' to 3' direction, 2'-fluorinated nucleotides are located at positions 2, 7, 10, and 14 of the antisense strand, with the remaining positions being 2'-methoxylated nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group; Alternatively, in the 5' to 3' direction, 2'-fluorinated nucleotides are located at positions 3, 7, 9, and 11 of the sense strand, with the remaining positions being 2'-methoxylated nucleotides; in the 5' to 3' direction, 2'-fluorinated nucleotides are located at positions 2, 3, 5, 7, 10, 12, and 14 of the antisense strand, GNA-modified nucleotides are located at position 6 of the antisense strand, with the remaining positions being 2'-methoxylated nucleotides, wherein the 5' terminal nucleotide of the antisense strand is linked to a 5'-trans-vinylphosphonate group.

18. The siRNA according to claims 1-17, wherein the siRNA is selected from the siRNAs in Table 1; preferably, the siRNA is selected from N-ER-FY047136, N-ER-FY047149, N-ER-FY047150, N-ER-FY047136M49, N-ER-FY047149M49, N-ER-FY047150M49, N-ER-FY047167, N-ER-FY047167M49, N-ER-FY 047216, N-ER-FY047216M49, N-ER-FY047226, N-ER-FY047226M49, N-ER-FY047232, N-ER-FY047232M49, N-E R-FY047182, N-ER-FY047182M49, N-ER-FY047207, N-ER-FY047207M49, N-ER-FY047245, N-ER-FY047245M49.

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

20. The siRNA conjugate according to claim 19, wherein the sense strand and antisense strand of the siRNA are complementary to form a double-stranded region of the siRNA conjugate, and the 3' end of the sense strand forms a blunt end, and the 3' end of the antisense strand has 1-3 protruding nucleotides extending out of the double-stranded region; or, In the siRNA conjugate, the sense strand and antisense strand of the siRNA are complementary to form the double-stranded region of the siRNA conjugate, and the 3' end of the sense strand is blunt, and the 3' end of the antisense strand is blunt.

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

22. The siRNA conjugate according to any one of claims 19-21, wherein the siRNA conjugate is selected from the siRNA conjugates in Table 2; preferably, the siRNA conjugate is selected from N-ER-FY047136M49L96, N-ER-FY047149M49L96, N-ER-FY047150M49L96, N-ER-FY047167M49L96, N-ER-FY047216M49L96, N-ER-FY047226M49L96, N-ER-FY047232M49L96, N-ER-FY047182M49L96, N-ER-FY047207M49L96, and N-ER-FY047245M49L96.

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

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

25. Use of the siRNA of any one of claims 1-18, or the siRNA conjugate of any one of claims 19-22, or the pharmaceutical composition of claim 23 for the preparation of an agent for inhibiting GRB14 gene expression.

26. Use of the siRNA of any one of claims 1-18, or the siRNA conjugate of any one of claims 19-22, or the pharmaceutical composition of claim 23 for the preparation of a medicament for the prevention and / or treatment of diseases associated with GRB14 gene overexpression.

27. The use according to claim 26, wherein the disease is type II diabetes (T2D), diabetic nephropathy, diabetic retinopathy, diabetic vascular disease, diabetic neuropathy, obesity, hepatocellular carcinoma (HCC), hyperinsulinemia, cardiometabolic disorder (CMD), bladder cancer (BC), glioblastoma, non-alcoholic steatohepatitis, hypertension, and hyperlipidemia.

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