Sirna for inhibiting ar gene expression and use thereof

Abstract

Provided is an siRNA for inhibiting AR gene expression. Experimental results demonstrate that the siRNA exhibits high inhibitory activity against AR gene expression. Further provided is a modified siRNA for inhibiting AR gene expression. The modification comprises methoxy modification, fluoro modification, phosphorothioate linkage, ethylene glycol nucleic acid modification, reverse abasic nucleotide modification, vinyl phosphate ester modification, deoxyribonucleotide substitution, 2'-O-alkyl modification, and tail sequence modification. Experimental results show that the modified siRNA has good AR inhibitory effects at different concentrations.

Description

siRNA for inhibiting AR gene expression and its applications

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese Patent Application No. 202510301956.3, filed on March 14, 2025, entitled "siRNA for inhibiting AR and its modifications and applications," the entire contents of which are incorporated herein by reference. This application claims priority to Chinese Patent Application No. 202510760204.3, filed on June 9, 2025, entitled "siRNA for inhibiting AR and its modifications and applications," the entire contents of which are incorporated herein by reference. This application claims priority to Chinese Patent Application No. 202610223527.3, filed on February 14, 2026, entitled "siRNA for inhibiting AR gene expression and its applications," the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application relates to siRNA for inhibiting AR gene expression and its applications, and belongs to the field of biotechnology. Background Technology

[0004] Androgenetic alopecia (AGA), also known as seborrheic alopecia or male pattern baldness, is the most common type of hair loss in clinical practice. Epidemiological surveys indicate that the overall prevalence of AGA in my country is approximately 21.3% in men and 6% in women, but significant regional variations exist. The pathogenesis of AGA is complex, with polygenic inheritance being the primary factor contributing to individual differences in incidence.

[0005] The clinical manifestations of male AGA include receding hairlines at the forehead, temples and / or sideburns, or progressive hair loss at the crown, eventually exposing the scalp, accompanied by increased scalp oil secretion. Female AGA is mainly characterized by diffuse thinning and fine hair between the crown and hairline, with no change in the position of the hairline at the forehead, and is usually accompanied by increased scalp oil.

[0006] The androgen receptor (AR) is a member of the nuclear receptor superfamily and is considered to play an important role in androgen response elements (ARs). This is mainly manifested in two ways: First, AGA depends on a functional AR; patients with androgen insensitivity syndrome usually do not develop AGA due to the absence or severe impairment of AR function. Second, AR is specifically expressed on the scalp in AGA patients, typically with increased expression in the frontal and parietal lobes but normal expression in the occipital lobe. Testosterone is the main circulating androgen in the human body. It readily crosses the cell membrane and is converted to 5-α-dihydrotestosterone by intracellular 5-α reductase. Although both testosterone and 5-α-dihydrotestosterone can bind to ARs to form receptor-ligand complexes that enter the cell nucleus to regulate androgen response elements (ARs), 5-α-dihydrotestosterone has a five-fold greater affinity for ARs than testosterone and is ten times more potent in AR signaling. Studies have shown that the expression of AR and 5-α-dihydrotestosterone in bald dermal papilla cells (DPCs) is higher than that in non-bald DPCs, and the AR-5-α-dihydrotestosterone complex and its transactivation activity are associated with the miniaturization of AGA hair follicles.

[0007] Currently, the most commonly used drugs for treating AGA are minoxidil and finasteride. Minoxidil typically takes more than three months to take effect, with little noticeable effect observed in the first three months. Most studies indicate that topical minoxidil usually requires six months to show significant effects (average onset time is 6–9 months). Finasteride generally reduces hair loss after 3 months of use, and the treatment effect is observed after 6 months. If the treatment effect is good, it should be continued to maintain efficacy. Finasteride requires long-term use, and it is best to maintain a therapeutic dose of 1 mg daily for long-term treatment. Finasteride spray (Phase III in China) was developed by Polichem SA in Switzerland. Compared with oral finasteride, the lower systemic bioavailability of topical application is expected to reduce the frequency and severity of systemic side effects associated with oral finasteride. It is evident that existing AGA treatment drugs generally have problems such as poor adherence, sexual dysfunction, and fatigue. Furthermore, due to the potential for fetal malformations, the use of these AGA treatment drugs in women of childbearing age is limited. Therefore, there is a need to develop a safer AGA treatment drug. Summary of the Invention

[0008] To address the aforementioned problems, this application provides an siRNA for inhibiting AR gene expression, wherein the siRNA contains a sense strand and an antisense strand; the sense strand and the antisense strand are at least partially anticomplementary to form a double-stranded region; the sense strand of the siRNA comprises at least 15 consecutive nucleotides that differ from any one of the nucleic acid sequences shown in any one of SEQ ID NO. 1-560 or SEQ ID NO. 1121-1160 by no more than 3 nucleotides; the antisense strand of the siRNA comprises at least 15 consecutive nucleotides that differ from any one of the nucleic acid sequences shown in any one of SEQ ID NO. 561-1120 or SEQ ID NO. 1161-1200 by no more than 3 nucleotides.

[0009] In one embodiment of this application, the nucleotide sequence of the sense strand of the siRNA is shown in any one of SEQ ID NO. 1-560 or SEQ ID NO. 1121-1160; the nucleotide sequence of the antisense strand of the siRNA is shown in any one of SEQ ID NO. 561-1120 or SEQ ID NO. 1161-1200.

[0010] In one embodiment of this application, at least one nucleotide in the sense strand and / or antisense strand of the siRNA is a modified nucleotide.

[0011] In one embodiment of this application, all nucleotides in the sense and / or antisense strands of the siRNA are modified nucleotides, and these modifications on the nucleotide groups do not cause a significant weakening or loss of the function of the siRNA disclosed herein in inhibiting AR gene expression.

[0012] In one embodiment of this application, the modified nucleotide includes compounds formed by replacing the 2' hydroxyl group of the ribosyl group of a nucleotide with other groups, compounds formed by modifying the nucleotide with a thiophosphate group, and / or compounds formed by modifying the bases on the nucleotide.

[0013] In one embodiment of this application, the modified nucleotides include fluorinated nucleotides, methoxylated nucleotides, phosphate-thioester linked nucleotides, glycolic acid (GNA) modified nucleotides, inverse baseless nucleotides, vinyl phosphate modified nucleotides, deoxyribonucleotide-substituted nucleotides, 2'-O-methoxyethyl (MOE) modified nucleotides, 2'-deoxy-nucleotides, 3'-terminal deoxy-thymidine (dT) nucleotides, 3'-deoxy-nucleotides, locked nucleotides, non-locked nucleotides, configuration-restricted nucleotides, restricted ethyl nucleotides, baseless nucleotides, 2'-amino-modified nucleotides, and 2'-O-allyl-modified nucleotides. 2'-C-alkyl-modified nucleotides, 2'-hydroxy-modified nucleotides, 2'-O-alkyl-modified nucleotides, tail sequence-modified nucleotides, morpholinonucleotide-modified nucleotides, aminophosphate-modified nucleotides, nucleotides containing non-natural bases, tetrahydropyran-modified nucleotides, 1,5-dehydrohexyl-modified nucleotides, cyclohexenyl-modified nucleotides, open-ring nucleotides (UNA)-modified nucleotides, nucleotides containing methyl phosphate groups, nucleotides containing 5'-phosphate groups, and / or nucleotides containing 5'-phosphate mimics.

[0014] In one embodiment of this application, the fluorinated nucleotide refers to a nucleotide formed by replacing the hydroxyl group at the 2' position of the ribosome with fluorine, and has the following structure (where Base represents a base):

[0015] In one embodiment of this application, the methoxy-modified nucleotide refers to a nucleotide formed by replacing the hydroxyl group at the 2' position of the ribosome with a methoxy group, having the following structure (where Base represents a base):

[0016] In one embodiment of this application, the nucleotides linked by the thiophosphate group refer to two adjacent nucleotides linked by a thiophosphate group.

[0017] The thiophosphate group has the following structure:

[0018] The nucleotide linked to the thiophosphate group has the following structure (where Base represents a base):

[0019] In one embodiment of this application, the glycol nucleic acid (GNA) modified nucleotide is a polymer similar to DNA or RNA, but its "backbone" is different, consisting of repeating glycerol units linked by phosphodiester bonds; the glycol nucleic acid (GNA) modified nucleotide includes A (GNA), G (GNA), C (GNA), U (GNA) and / or T (GNA);

[0020] The A(GNA) has the following structure:

[0021] The G(GNA) has the following structure:

[0022] The C(GNA) has the following structure:

[0023] The U(GNA) has the following structure:

[0024] The T(GNA) has the following structure:

[0025] In one embodiment of this application, the nucleotide modified with the inverse abase-free nucleotide refers to a nucleotide coupled with an inverse abase-free nucleotide (invAb), wherein the inverse abase-free nucleotide has the following structure:

[0026] In one embodiment of this application, the 5' position of the sugar ring of the vinyl phosphate-modified nucleotide is modified with vinyl phosphate; the vinyl phosphate-modified nucleotide includes VPAms, VPUms, VPGms and / or VPCms (VP ​​represents (E)-vinyl phosphate);

[0027] The VPAms have the following structure:

[0028] The VPUms have the following structure:

[0029] The VPGms has the following structure:

[0030] The VPCms has the following structure:

[0031] In one embodiment of this application, the deoxyribonucleotide-substituted nucleotide refers to a nucleotide formed by replacing the hydroxyl group at the 2' position of the ribosyl group with a hydrogen atom, which has the following structure (where Base represents a base):

[0032] In one embodiment of this application, the deoxyribonucleotide-substituted nucleotides include A(d), C(d), G(d), U(d), and T(d); wherein A(d) represents 2'-deoxyadenosine-3'-phosphate; wherein C(d) represents 2'-deoxycytidine-3'-phosphate; wherein U(d) is 2'-deoxyuridine-3'-phosphate; wherein G(d) represents 2'-deoxyguanosine-3'-phosphate; and wherein T(d) represents 2'-deoxythymidine-3'-phosphate.

[0033] In one embodiment of this application, the 2'-O-alkyl-modified nucleotide is a 2'-O-hexadecyl-modified nucleotide having the following structure (where B represents a base):

[0034] In one embodiment of this application, the fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, following the direction from the 5' end to the 3' end, at least the nucleotides at positions 5, 7, 8, and 9 of the sense strand are fluorinated nucleotides, and at least the nucleotides at positions 2, 6, 14, and 16 of the antisense strand are fluorinated nucleotides; or,

[0035] The fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 7, 8, and 9 of the sense strand are fluorinated nucleotides, and at least the nucleotides at positions 2, 6, 14, and 16 of the antisense strand are fluorinated nucleotides; or,

[0036] The fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 9, 10, and 11 of the sense strand are fluorinated nucleotides, and at least the nucleotides at positions 2, 6, 14, and 16 of the antisense strand are fluorinated nucleotides; or,

[0037] The fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 7, 9, 10, and 11 of the sense strand are fluorinated nucleotides, and at least the nucleotides at positions 2, 6, 14, and 16 of the antisense strand are fluorinated nucleotides; or,

[0038] The fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 9, 10, and 11 of the sense strand are fluorinated nucleotides, and at least the nucleotides at positions 2, 10, 12, 14, and 16 of the antisense strand are fluorinated nucleotides; or,

[0039] The fluorinated nucleotides are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 7, 9, and 11 of the sense strand are fluorinated nucleotides, and at least the nucleotides at positions 2, 12, 14, and 16 of the antisense strand are fluorinated nucleotides.

[0040] In one embodiment of this application, the fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, following the direction from the 5' end to the 3' end, the nucleotides at positions 5, 7, 8, and 9 of the sense strand are fluorinated nucleotides, and the nucleotides at positions 2, 6, 14, and 16 of the antisense strand are fluorinated nucleotides; or,

[0041] Fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, following the direction from the 5' end to the 3' end, the nucleotides at positions 7, 8, and 9 of the sense strand are fluorinated nucleotides, and the nucleotides at positions 2, 6, 14, and 16 of the antisense strand are fluorinated nucleotides; or,

[0042] Fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, following the direction from the 5' end to the 3' end, the nucleotides at positions 9, 10, and 11 of the sense strand are fluorinated nucleotides, and the nucleotides at positions 2, 6, 14, and 16 of the antisense strand are fluorinated nucleotides; or,

[0043] Fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, following the direction from the 5' end to the 3' end, the nucleotides at positions 7, 9, 10, and 11 of the sense strand are fluorinated nucleotides, and the nucleotides at positions 2, 6, 14, and 16 of the antisense strand are fluorinated nucleotides; or,

[0044] Fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, following the direction from the 5' end to the 3' end, the nucleotides at positions 9, 10, and 11 of the sense strand are fluorinated nucleotides, and the nucleotides at positions 2, 10, 12, 14, and 16 of the antisense strand are fluorinated nucleotides; or,

[0045] The fluorinated nucleotides are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, the nucleotides at positions 7, 9, and 11 of the sense strand are fluorinated nucleotides, and the nucleotides at positions 2, 12, 14, and 16 of the antisense strand are fluorinated nucleotides.

[0046] In one embodiment of this application, the methoxylated nucleotides are located in the antisense and sense strands of the nucleotide sequence. In the sense strand, the nucleotides that are not fluorinated, not glycolic acid (GNA) modified, and not deoxyribonucleotide substituted are all methoxylated nucleotides. In the antisense strand, the nucleotides that are not fluorinated, not glycolic acid modified, and not deoxyribonucleotide substituted are all methoxylated nucleotides.

[0047] In one embodiment of this application, the nucleotide modified with an inverse abase-free nucleotide is located in the positive strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the 3' end of the first nucleotide of the positive strand is attached to an inverse abase-free nucleotide (invAb group); and / or,

[0048] The inverse abase-free nucleotide is located in the positive strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, the 5' end of at least the penultimate nucleotide of the positive strand is attached to an inverse abase-free nucleotide (invAb group).

[0049] In one embodiment of this application, the nucleotide modified with inverse abase nucleotide is located in the positive strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, the 3' end of the first nucleotide of the positive strand is attached to an inverse abase nucleotide (invAb group), and the 5' end of at least the penultimate nucleotide of the positive strand is attached to an inverse abase nucleotide (invAb group).

[0050] In one embodiment of this application, the vinyl phosphate-modified nucleotide is located in the antisense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the 5' end of the first nucleotide of the antisense strand is attached to a vinyl phosphate group (VP group).

[0051] In one embodiment of this application, the vinyl phosphate-modified nucleotide is located in the antisense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, the 5' end of the first nucleotide of the antisense strand is attached to a vinyl phosphate group (VP group).

[0052] In one embodiment of this application, the deoxyribonucleotide-substituted nucleotide is located in the antisense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotide at the 5th position of the antisense strand is a deoxyribonucleotide-substituted nucleotide; or,

[0053] The deoxyribonucleotide-substituted nucleotide is located in the antisense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotide at position 7 of the antisense strand is a deoxyribonucleotide-substituted nucleotide; or,

[0054] The deoxyribonucleotide-substituted nucleotide is located in the antisense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 5 and 7 of the antisense strand are deoxyribonucleotide-substituted nucleotides.

[0055] In one embodiment of this application, the deoxyribonucleotide-substituted nucleotide is located in the antisense strand of the nucleotide sequence, and, following the direction from the 5' end to the 3' end, the nucleotide at position 5 of the antisense strand is the deoxyribonucleotide-substituted nucleotide; or,

[0056] The deoxyribonucleotide-substituted nucleotide is located in the antisense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, the nucleotide at position 7 of the antisense strand is the deoxyribonucleotide-substituted nucleotide; or,

[0057] The deoxyribonucleotide-substituted nucleotide is located in the antisense strand of the nucleotide sequence, and the nucleotides at positions 5 and 7 of the antisense strand are deoxyribonucleotide-substituted nucleotides in the direction from the 5' end to the 3' end.

[0058] In one embodiment of this application, the nucleotide modified with ethylene glycol nucleic acid (GNA) is located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, the nucleotide at least the 7th position of the antisense strand is a nucleotide modified with ethylene glycol nucleic acid.

[0059] In one embodiment of this application, the nucleotide modified with ethylene glycol nucleic acid (GNA) is located in the antisense strand of the nucleotide sequence, and the nucleotide at position 7 of the antisense strand is the nucleotide modified with ethylene glycol nucleic acid, in the direction from the 5' end to the 3' end.

[0060] In one embodiment of this application, the nucleotides linked by thiophosphate groups are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 1 and 2, and positions 2 and 3 of the sense strand are linked by thiophosphate groups, and at least the nucleotides at positions 1 and 2, positions 2 and 3, positions 1 to 1 and 2 to 1, and positions 2 to 1 ...

[0061] The phosphate-thioester-linked nucleotides are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the first and second, and the penultimate and penultimate nucleotides of the sense strand are linked by phosphate-thioester bonds, and at least the first and second, second and third, penultimate and penultimate, and penultimate and penultimate nucleotides of the antisense strand are linked by phosphate-thioester bonds; or,

[0062] The phosphate-thioester linked nucleotides are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the first and second, second and third, penultimate and penultimate, and penultimate and penultimate nucleotides of the sense strand are linked by phosphate-thioester bonds; and at least the first and second, second and third, penultimate and penultimate, and penultimate and penultimate nucleotides of the antisense strand are linked by phosphate-thioester bonds; or,

[0063] The phosphate-thioester-linked nucleotides are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, the first nucleotide of the sense strand is linked to the reverse abasic nucleotide by a phosphate-thioester group, and the penultimate nucleotide is linked to the reverse abasic nucleotide by a phosphate-thioester group. At least the first and second, second and third, penultimate and penultimate, and penultimate and penultimate nucleotides of the antisense strand are linked by a phosphate-thioester bond; or,

[0064] The phosphate-thioester-linked nucleotides are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the first and second, penultimate and penultimate, and penultimate and penultimate nucleotides of the sense strand are linked by phosphate-thioester bonds; similarly, at least the first and second, second and third, penultimate and penultimate, and penultimate and penultimate nucleotides of the antisense strand are linked by phosphate-thioester bonds; or,

[0065] The nucleotides linked by thiophosphate groups are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the first and second, and the penultimate and penultimate nucleotides of the sense strand are linked by thiophosphate bonds, and at least the first and second, second and third, third and fourth, and penultimate and penultimate nucleotides of the antisense strand are linked by thiophosphate bonds.

[0066] In one embodiment of this application, the nucleotides linked by thiophosphate groups are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, the nucleotides at positions 1 and 2, and positions 2 and 3 of the sense strand are linked by thiophosphate groups, and the nucleotides at positions 1 and 2, positions 2 and 3, positions 1 to 1 and 2 to 1, and positions 2 to 1 ...

[0067] The nucleotides linked by thiophosphate groups are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, the nucleotides at positions 1 and 2, and from the 1st to the 2nd, and from the 1st to the 2nd, and from the 1st to the 2nd, and from the 1st to the 2nd, and from the 1st to the 2nd, and from the 1st to the 2nd, and from the 1st to the 2nd, and from the 1st to the 2nd, and from the 1st to the 2nd, and from the 1st to the 3rd, nucleotides in the antisense strand are linked by thiophosphate bonds; or,

[0068] The nucleotides linked by thiophosphate groups are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, the nucleotides at positions 1 and 2, 2 and 3, penultimate and penultimate, and penultimate and penultimate are linked by thiophosphate bonds in the sense strand; similarly, the nucleotides at positions 1 and 2, 2 and 3, penultimate and penultimate, and penultimate and penultimate are linked by thiophosphate bonds in the antisense strand; or,

[0069] The phosphate-thioester linked nucleotides are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, the first nucleotide of the sense strand is linked to the reverse non-base nucleotide by a phosphate-thioester group, and the penultimate nucleotide is linked to the reverse non-base nucleotide by a phosphate-thioester group; the first and second, second and third, penultimate and penultimate, and penultimate and penultimate nucleotides of the antisense strand are linked by a phosphate-thioester bond; or,

[0070] The nucleotides linked by thiophosphate groups are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, the nucleotides at positions 1 and 2, 1-1-2, and 2-1-3 of the sense strand are linked by thiophosphate bonds, and the nucleotides at positions 1 and 2, 2 and 3, 1-1-2, and 2-1-3 of the antisense strand are linked by thiophosphate bonds; or,

[0071] The nucleotides linked by thiophosphate groups are located in the antisense and sense strands of the nucleotide sequence, and the nucleotides at positions 1 and 2, and at positions 1 to ...

[0072] In one embodiment of this application, the nucleotide modified with 2'-O-hexadecyl is located in the antisense or sense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the 6th nucleotide of the sense strand is a nucleotide modified with 2'-O-hexadecyl; or,

[0073] The first nucleotide of the positive strand is a nucleotide modified with 2'-O-hexadecyl; or,

[0074] The penultimate nucleotide of the positive strand is a nucleotide modified with 2'-O-hexadecyl; or,

[0075] The antisense strand has at least one penultimate nucleotide that is a nucleotide modified with 2'-O-hexadecyl.

[0076] In one embodiment of this application, the tail-modified nucleotide is located in the positive strand of the nucleotide sequence; the tail-modified nucleotide is a nucleotide with a lipophilic tail sequence attached to its 3' end; the lipophilic tail sequence is an RNA fragment containing 1 to 3 bases.

[0077] In one embodiment of this application, the lipophilic tail sequence has one base; the base is A or U.

[0078] This application also provides an siRNA conjugate for inhibiting AR gene expression, the siRNA conjugate containing the above-mentioned siRNA and a conjugating group conjugated to the siRNA.

[0079] In one embodiment of this application, the conjugation group comprises a ligand formed from a targeting molecule or a derivative thereof; the targeting molecule comprises galactose and / or N-acetylgalactosamine (GalNAc). N-acetylgalactosamine is a ligand that binds to the asialoglycoprotein receptor (ASGPR) on the liver surface. The ASGPR is an endocytic receptor specifically expressed by hepatocytes, and N-acetylgalactosamine serves as a targeting molecule to deliver small RNA to the liver.

[0080] In one embodiment of this application, the conjugating group is N-glucose amino acid (N-Acetylgalactosamine, GalNAc).

[0081] In one embodiment of this application, the conjugation site of the siRNA and the conjugation group is located at the 3' end of the siRNA sense strand, the 5' end of the siRNA sense strand, the internal sequence of the siRNA sense strand, the 5' end of the siRNA antisense strand, or the internal sequence of the siRNA antisense strand.

[0082] In one embodiment of this application, the conjugation site of the siRNA and the conjugating group is located at the 3' end of the siRNA's positive strand.

[0083] In one embodiment of this application, the conjugating group is conjugated to the 3' end of the siRNA positive strand via a phosphodiester bond.

[0084] In one embodiment of this application, the siRNA conjugate has the following structure:

[0085] This application also provides a recombinant plasmid that expresses the above-mentioned siRNA.

[0086] In one embodiment of this application, the vector of the recombinant plasmid includes at least one of a viral vector or a non-viral vector; the viral vector includes at least one of a flavivirus vector, a retrovirus vector, a bacteriophage vector, adenovirus vector, adeno-associated virus vector, vaccinia virus vector, hybrid virus vector, baculovirus vector, herpes simplex virus vector, or lentivirus vector; the non-viral vector includes a plasmid vector.

[0087] In one embodiment of this application, the recombinant plasmid is prepared by: designing shRNA based on siRNA; and ligating the shRNA with a linearized vector to obtain the recombinant plasmid.

[0088] This application also provides a host cell whose genome integrates the above-mentioned siRNA; or, whose genome integrates the above-mentioned antisense oligonucleotide; or, whose host cell carries the above-mentioned recombinant plasmid.

[0089] In one embodiment of this application, the host cell includes fungi, bacteria, plant cells, and / or animal cells.

[0090] This application also provides the use of the above-mentioned siRNA, the above-mentioned siRNA conjugate, the above-mentioned recombinant plasmid or the above-mentioned host cell in the preparation of medicaments for the prevention and / or treatment of pathological conditions or diseases caused by AR.

[0091] In one embodiment of this application, the pathological condition or disease caused by AR includes androgenetic alopecia (AGA).

[0092] This application also provides a medicament for preventing and / or treating pathological conditions or diseases caused by AR, wherein the medicament comprises the above-mentioned siRNA, the above-mentioned siRNA conjugate, the above-mentioned recombinant plasmid or the above-mentioned host cell.

[0093] In one embodiment of this application, the pathological condition or disease caused by AR includes androgenetic alopecia (AGA).

[0094] In one embodiment of this application, the drug composition further includes pharmaceutically acceptable excipients; the pharmaceutically acceptable excipients include drug carriers, pH buffers, lyophilization protectants, osmotic pressure regulators, excipients, stabilizers and / or preservatives.

[0095] In one embodiment of this application, the drug carrier includes magnetic nanoparticles (such as 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 phosphate), PPEEA and poly(2-dimethylaminoethylmethacrylate) (PDMAEMA) and its derivatives.

[0096] In one embodiment of this application, the pH buffer includes a tris(hydroxymethyl)aminomethane hydrochloride buffer with a pH of 7.5 to 8.5 and / or a phosphate buffer with a pH of 5.5 to 8.5.

[0097] In one embodiment of this application, the protective agent includes one or more of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose, and glucose.

[0098] In one embodiment of this application, the osmotic pressure regulator includes sodium chloride and / or potassium chloride.

[0099] This application also provides a method for preventing or treating pathological conditions or diseases caused by AR, comprising administering the siRNA, the siRNA conjugate, the recombinant plasmid, the host cell, or the drug to a subject in need.

[0100] In one embodiment of this application, the pathological condition or disease caused by AR includes androgenetic alopecia.

[0101] The technical solution of this application has the following advantages:

[0102] This application provides an siRNA for inhibiting AR gene expression, wherein the siRNA contains a sense strand and an antisense strand; the sense strand and the antisense strand are at least partially anticomplementary to form a double-stranded region; the sense strand of the siRNA comprises at least 15 consecutive nucleotides that differ from any one of the nucleic acid sequences shown in any one of SEQ ID NO. 1-560 or SEQ ID NO. 1121-1160 by no more than 3 nucleotides; the antisense strand of the siRNA comprises at least 15 consecutive nucleotides that differ from any one of the nucleic acid sequences shown in any one of SEQ ID NO. 561-1120 or SEQ ID NO. 1161-1200 by no more than 3 nucleotides. Experiments have shown that the siRNA exhibits high inhibitory activity against AR; therefore, the siRNA has great potential for application in the preparation of drugs for the prevention and / or treatment of pathological conditions or diseases caused by AR (e.g., androgenetic alopecia).

[0103] Furthermore, at least one nucleotide in the sense and / or antisense strands of the siRNA is a modified nucleotide; the modifications include methoxy modification, fluorination modification, thiophosphate linkage, glycol nucleic acid modification, reverse abasic nucleotide modification, vinyl phosphate modification, deoxyribonucleotide substitution, 2'-O-alkyl-modification, and tail sequence modification. Experiments have shown that the modified siRNA exhibits good AR inhibitory effects at different concentrations. Therefore, the modified siRNA shows great promise in the preparation of drugs for the prevention and / or treatment of pathological conditions or diseases caused by AR (e.g., androgenetic alopecia). Attached Figure Description

[0104] Figure 1: qPCR results of single-dose assay (0.25 mg / site) of AR-039M1C16, AR-044M1C16 and AR-013M1C16 in mice.

[0105] Figure 2: qPCR results of single-dose assay (0.5 mg / site) of AR-039M1C16, AR-044M1C16, and AR-013M1C16 in mice.

[0106] Figure 3: WB results of single-dose assays (0.25 mg / site, 0.5 mg / site) of AR-039M1C16 in mice.

[0107] Figure 4: WB results of single-dose assays (0.25 mg / site, 0.5 mg / site) of AR-044M1C16 in mice.

[0108] Figure 5: WB results of single-dose assays (0.25 mg / site, 0.5 mg / site) of AR-013M1C16 in mice.

[0109] Figure 6: Hair regrowth effect of AR-013M1C16 in a mouse model of androgenetic alopecia (AGA).

[0110] Figure 7: Hair regrowth effect of AR-044M1C16 in a mouse model of androgenetic alopecia (AGA).

[0111] Figure 8: Hair regrowth effect of AR-039M1C16 in a mouse model of androgenetic alopecia (AGA).

[0112] Figure 9: Hair regrowth effect of minoxidil positive control group in mouse androgenic alopecia (AGA) model.

[0113] Figure 10: Hair weight results of silenced C57 mice in AR-039M1C16, AR-044M1C16, and AR-013M1C16.

[0114] Figure 11: Hair length results in silenced C57 mice of AR-039M1C16, AR-044M1C16, and AR-013M1C16.

[0115] Figure 12: Results of pigmentation days in silenced C57 mice of AR-039M1C16, AR-044M1C16, and AR-013M1C16.

[0116] Figure 13: HE staining results of skin samples taken on day 9 after administration of AR-039M1C16, AR-044M1C16, and AR-013M1C16.

[0117] Figure 14: HE staining results of skin samples taken from the untreated group on day 21.

[0118] Figure 15: HE staining results of skin samples taken on day 21 in the testosterone propionate + PBS group.

[0119] Figure 16: HE staining results of skin samples taken on day 21 in the testosterone propionate + AR13 group.

[0120] Figure 17: HE staining results of skin samples taken on day 21 in the testosterone propionate + AR44 group.

[0121] Figure 18: HE staining results of skin samples taken on day 21 in the testosterone propionate + AR39 group.

[0122] Figure 19: HE staining results of skin samples taken on day 21 in the testosterone propionate + minoxidil group.

[0123] Figure 20: AR mRNA expression levels in skin tissue at the siRNA administration site 14 days after administration of AR-039L23M2C16VP, AR-039L23M2-5C16VP, AR-039L23M2-3C16VP, AR-039L23M2-reverse3C16VP, AR-039L23M2-3+UC16VP, AR-039J1C16VP, AR-039J1-5C16VP, AR-039J1-3C16VP, AR-039J1-reverse3C16VP, AR-039J1-3+UC16VP, AR-039Invab+reverseC16VP, AR-039Invab+positiveUreverseC16VP, AR-039L23M2-3+AC16VP, and AR-039Invab+positiveAreverseC16VP.

[0124] Figure 21: AR mRNA expression levels in skin tissue at the siRNA administration site 21 days after administration of AR-039L23M2C16VP, AR-039L23M2-5C16VP, AR-039L23M2-3C16VP, AR-039L23M2-reverse3C16VP, AR-039L23M2-3+UC16VP, AR-039J1C16VP, AR-039J1-5C16VP, AR-039J1-3C16VP, AR-039J1-reverse3C16VP, AR-039J1-3+UC16VP, AR-039Invab+reverseC16VP, AR-039Invab+positiveUreverseC16VP, AR-039L23M2-3+AC16VP, and AR-039Invab+positiveAreverseC16VP.

[0125] Figure 22: AR protein expression levels in skin tissue at the siRNA administration site 14 days after administration of AR-039L23M2C16VP, AR-039L23M2-5C16VP, AR-039L23M2-3C16VP, AR-039L23M2-reverse3C16VP, AR-039L23M2-3+UC16VP, AR-039J1C16VP, AR-039J1-5C16VP, AR-039J1-3C16VP, AR-039J1-reverse3C16VP, AR-039J1-3+UC16VP, AR-039Invab+reverseC16VP, AR-039Invab+positiveUreverseC16VP, AR-039L23M2-3+AC16VP, and AR-039Invab+positiveAreverseC16VP.

[0126] Figure 23: AR protein expression levels in skin tissue at the siRNA administration site 21 days after administration of AR-039L23M2C16VP, AR-039L23M2-5C16VP, AR-039L23M2-3C16VP, AR-039L23M2-reverse3C16VP, AR-039L23M2-3+UC16VP, AR-039J1C16VP, AR-039J1-5C16VP, AR-039J1-3C16VP, AR-039J1-reverse3C16VP, AR-039J1-3+UC16VP, AR-039Invab+reverseC16VP, AR-039Invab+positiveUreverseC16VP, AR-039L23M2-3+AC16VP, and AR-039Invab+positiveAreverseC16VP.

[0127] Figure 24: AR mRNA expression levels in skin tissue at the siRNA administration site after different number of days of AR-039J1-5C16VP administration.

[0128] Figure 25: AR mRNA expression levels in skin tissue at the siRNA administration site after different number of days of administration of AR-039Invab+positive U and negative C16VP.

[0129] Figure 26: AR mRNA expression levels in skin tissue at the siRNA administration site after different number of days of AR-039J1-5C16VP administration.

[0130] Figure 27: AR protein expression levels in skin tissue at the siRNA administration site after different number of days of administration of AR-039Invab+positive U and negative C16VP.

[0131] Figure 28: In vivo validation of the therapeutic effect of siRNA in AGA mouse model.

[0132] Figure 29: HE staining results of skin 21 days after administration of AR-039J1-5C16VP injection site.

[0133] Figure 30: Ratio of hair follicles in the anagen phase to those in the telogen phase at the model site 21 days after administration of AR-039J1-5C16VP.

[0134] Figure 31: Weight of newly grown hair at the model site 21 days after administration of AR-039J1-5C16VP.

[0135] Figure 32: Length of new hair growth at the model site 21 days after administration of AR-039J1-5C16VP.

[0136] Figure 33: Diameter of newly formed hair in the modeling site 21 days after administration of AR-039J1-5C16VP.

[0137] Figure 34: Percentage of newly formed coverage area at the model site 21 days after administration of AR-039J1-5C16VP.

[0138] Figure 35: AR mRNA expression level in skin tissue at the treatment site 21 days after administration of AR-039J1-5C16VP.

[0139] Figure 36: AR protein expression level in skin tissue at the treatment site 21 days after administration of AR-039J1-5C16VP. Detailed Implementation

[0140] The following embodiments are provided to better understand this application and are not limited to the preferred embodiments described herein. They do not constitute a limitation on the content and scope of protection of this application. Any product that is the same as or similar to this application, derived by anyone under the guidance of this application or by combining features of this application with other prior art, falls within the scope of protection of this application.

[0141] For any experimental steps or conditions not specified in the following examples, the procedures or conditions described in the literature in this field can be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0142] In the following embodiments, AR mRNA refers to mRNA having the sequence shown in GenBank accession number NM_000044.6. Further, unless otherwise specified, the term "target gene" as used in this disclosure refers to the gene that transcribes the aforementioned AR mRNA, and the term "target mRNA" refers to the aforementioned AR mRNA.

[0143] Unless otherwise specified, the reagents and culture media used in the following examples are all commercially available products, and the nucleic acid electrophoresis, real-time PCR and other operations used are all performed in accordance with the methods described in Molecular Biology (4th Edition) (Alexander McLennan et al., 2019).

[0144] The experimental cells used in the following examples are LNCAP cells, purchased from the Chinese Academy of Sciences Cell Bank.

[0145] The experimental animals were male C57BL / 6J black mice (6 weeks old), purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All animals were housed in the SPF-grade animal facility of Suzhou Gemma Gene Co., Ltd. Animals were kept under a 12-hour light-dark cycle, with free access to food and water. Experiments began after one week of acclimatization. The use and handling of experimental animals complied with the requirements of the Animal Management Committee of Suzhou Gemma Gene Co., Ltd. regarding laboratory animals and animal welfare.

[0146] The siRNAs involved in the following examples are siRNA sequences synthesized via phosphoramide solid-phase synthesis.

[0147] In the examples described below, when transfecting cells with siRNA targeting the AR gene or siRNA used as a negative control, Lipo3000 or RNAiMAX (purchased from Invitrogen) were used as transfection reagents, and the specific procedures were performed according to the manufacturer's instructions. For qPCR detection, HiScript III RT SuperMix for qPCR (purchased from Vazyme) was used as the reverse transcription reagent, and the specific procedures were performed according to the manufacturer's instructions.

[0148] Example 1: A siRNA for inhibiting AR

[0149] This embodiment provides an siRNA for inhibiting AR, the nucleotide sequence of which is designed based on the target mRNA, as shown in Tables 1 and 2.

[0150] Table 1. siRNAs and their sequences that inhibit AR gene expression

[0151] Table 2. siRNAs and their sequences that inhibit AR gene expression

[0152] Example 2: A modified siRNA for inhibiting AR

[0153] This embodiment provides a modified siRNA for inhibiting AR. The modified siRNA is based on the siRNA of Example 1, with the nucleotides at positions 5, 7, 8, and 9 of the sense strand being fluorinated at the 2' position, and the nucleotides at other positions being methoxylated at the 2' position. A thiophosphate bond connects the nucleotides at positions 1 and 2, and 2 and 3. Similarly, on the antisense strand, the nucleotides at positions 2, 6, 14, and 16 are fluorinated at the 2' position, and the nucleotides at other positions are methoxylated at the 2' position. A thiophosphate bond connects the nucleotides at positions 1 and 2, 2 and 3, the penultimate and penultimate positions, and the penultimate and penultimate positions. Alternatively,

[0154] The modified siRNA is based on the siRNA of Example 1, with the nucleotides at positions 7, 8, and 9 of the sense strand being fluorinated at the 2' position, and the nucleotides at other positions being methoxylated at the 2' position. A thiophosphate bond connects the nucleotides at positions 1 and 2, and 2 and 3. Similarly, on the antisense strand, the nucleotides at positions 2, 6, 14, and 16 are fluorinated at the 2' position, and the nucleotides at other positions are methoxylated at the 2' position. A thiophosphate bond connects the nucleotides at positions 1 and 2, 2 and 3, the penultimate and penultimate positions, and the penultimate and penultimate positions. Alternatively,

[0155] The modified siRNA is based on the siRNA of Example 1, with the nucleotides at positions 9, 10, and 11 of the sense strand being fluorinated at the 2' position, and the nucleotides at other positions being methoxylated at the 2' position. The nucleotide at position 6 is modified with 2'-O-hexadecyl. A thiophosphate bond connects the nucleotides at positions 1 and 2, and the penultimate and penultimate positions. The nucleotides at positions 2, 6, 14, and 16 of the antisense strand are fluorinated at the 2' position, and the nucleotides at other positions are methoxylated at the 2' position. A vinyl phosphate group (VP group) is attached to the 5' end of the nucleotide at position 1. A thiophosphate bond connects the nucleotides at positions 1 and 2, 2 and 3, the penultimate and penultimate positions, and the penultimate and penultimate positions. Alternatively,

[0156] The modified siRNA is based on the siRNA of Example 1, with the nucleotides at positions 7, 9, 10, and 11 of the sense strand being fluorinated at the 2' position, and the nucleotides at other positions being methoxylated at the 2' position. A thiophosphate bond connects the nucleotides at positions 1 and 2, and 2 and 3. Similarly, the nucleotides at positions 2, 6, 14, and 16 of the antisense strand are fluorinated at the 2' position, and the nucleotides at other positions are methoxylated at the 2' position. A thiophosphate bond connects the nucleotides at positions 1 and 2, 2 and 3, the penultimate and penultimate positions, and the penultimate and penultimate positions. Alternatively,

[0157] The modified siRNA is based on the siRNA of Example 1, with the nucleotides at positions 7, 9, 10, and 11 of the sense strand being fluorinated at the 2' position, and the nucleotides at other positions being methoxylated at the 2' position. The nucleotide at position 6 is modified with a 2'-O-hexadecyl group. A thiophosphate bond connects the nucleotides at positions 1 and 2, 2 and 3, penultimate and penultimate, and penultimate and penultimate. The nucleotides at positions 2, 6, 14, and 16 of the antisense strand are fluorinated at the 2' position, and the nucleotides at other positions are methoxylated at the 2' position. A vinyl phosphate group (VP group) is attached to the 5' end of the nucleotide at position 1. A thiophosphate bond connects the nucleotides at positions 1 and 2, 2 and 3, penultimate and penultimate, and penultimate and penultimate. Alternatively,

[0158] The modified siRNA is based on the siRNA of Example 1, with the nucleotides at positions 7, 9, 10, and 11 of the sense strand being fluorinated at the 2' position, and the nucleotides at other positions being methoxylated at the 2' position. The nucleotide at position 1 is modified with 2'-O-hexadecyl. A thiophosphate bond connects the nucleotides at positions 1 and 2, 2 and 3, penultimate and penultimate, and penultimate and penultimate. The nucleotides at positions 2, 6, 14, and 16 of the antisense strand are fluorinated at the 2' position, and the nucleotides at other positions are methoxylated at the 2' position. A vinyl phosphate group (VP group) is attached to the 5' end of the nucleotide at position 1. A thiophosphate bond connects the nucleotides at positions 1 and 2, 2 and 3, penultimate and penultimate, and penultimate and penultimate. Alternatively,

[0159] The modified siRNA is based on the siRNA of Example 1, with the nucleotides at positions 7, 9, 10, and 11 of the sense strand being fluorinated at the 2' position, and the nucleotides at other positions being methoxylated at the 2' position. The nucleotide at the penultimate position is modified with 2'-O-hexadecyl. A thiophosphate bond connects the nucleotides at positions 1 and 2, 2 and 3, penultimate and penultimate, and penultimate and penultimate. The nucleotides at positions 2, 6, 14, and 16 of the antisense strand are fluorinated at the 2' position, and the nucleotides at other positions are methoxylated at the 2' position. A vinyl phosphate group (VP group) is attached to the 5' end of the nucleotide at position 1. A thiophosphate bond connects the nucleotides at positions 1 and 2, 2 and 3, penultimate and penultimate, and penultimate and penultimate. Alternatively,

[0160] The modified siRNA is based on the siRNA of Example 1, with the nucleotides at positions 7, 9, 10, and 11 of the sense strand being fluorinated at the 2' position, and the nucleotides at other positions being methoxylated at the 2' position. A thiophosphate bond connects the nucleotides at positions 1 and 2, 2 and 3, penultimate and penultimate, and penultimate and penultimate. The nucleotides at positions 2, 6, 14, and 16 of the antisense strand are fluorinated at the 2' position, and the nucleotides at other positions are methoxylated at the 2' position. A vinyl phosphate group (VP group) is attached to the 5' end of the nucleotide at position 1. The penultimate nucleotide is modified with 2'-O-hexadecyl, and a thiophosphate bond connects the nucleotides at positions 1 and 2, 2 and 3, penultimate and penultimate, and penultimate and penultimate. Alternatively,

[0161] The modified siRNA is based on the siRNA of Example 1, with the nucleotides at positions 7, 9, 10, and 11 of the sense strand being fluorinated at the 2' position, and the nucleotides at other positions being methoxylated at the 2' position. An additional 2'-O-hexadecyl base U is attached to the end. Phosphothiophosphate bonds connect the nucleotides at positions 1 and 2, 2 and 3, penultimate and penultimate, and penultimate and penultimate. On the antisense strand, the nucleotides at positions 2, 6, 14, and 16 are fluorinated at the 2' position, and the nucleotides at other positions are methoxylated at the 2' position. A vinyl phosphate group (VP group) is attached to the 5' end of the nucleotide at position 1. Phosphothiophosphate bonds connect the nucleotides at positions 1 and 2, 2 and 3, penultimate and penultimate, and penultimate and penultimate. Alternatively,

[0162] The modified siRNA is based on the siRNA of Example 1, with the nucleotides at positions 7, 9, 10, and 11 of the sense strand being fluorinated at the 2' position, and the nucleotides at other positions being methoxylated at the 2' position. An additional 2'-O-hexadecyl base A is attached to the end. Phosphothiophosphate bonds connect the nucleotides at positions 1 and 2, 2 and 3, penultimate and penultimate, and penultimate and penultimate. On the antisense strand, the nucleotides at positions 2, 6, 14, and 16 are fluorinated at the 2' position, and the nucleotides at other positions are methoxylated at the 2' position. A vinyl phosphate group (VP group) is attached to the 5' end of the nucleotide at position 1. Phosphothiophosphate bonds connect the nucleotides at positions 1 and 2, 2 and 3, penultimate and penultimate, and penultimate and penultimate. Alternatively,

[0163] The modified siRNA is based on the siRNA of Example 1, with the following changes: nucleotides at positions 9, 10, and 11 of the sense strand are fluorinated at the 2' position, and nucleotides at other positions are methoxylated at the 2' position. A reverse abasic nucleotide (invAb group) is attached to the 3' end of the first nucleotide, and a reverse abasic nucleotide (invAb group) is attached to the 5' end of the penultimate nucleotide. The first and penultimate nucleotides are linked by a phosphate thioester group, and the penultimate nucleotide is also linked by a phosphate thioester group. Nucleotides at positions 2, 10, 12, 14, and 16 of the antisense strand are fluorinated at the 2' position. The 5th nucleotide is replaced with a deoxyribonucleotide (when the ribonucleotide is uracil ribonucleotide, thymine deoxyribonucleotide is used instead of uracil ribonucleotide, and so on). The 7th nucleotide is a glycol nucleic acid. Nucleotides modified with acid (GNA), where the nucleotides at other positions are methoxy at the 2' position, and a vinyl phosphate group (VP group) is attached to the 5' end of the nucleotide at position 1. The nucleotides at positions 1 and 2, 2 and 3, penultimate and penultimate, and penultimate and penultimate are linked by thiophosphate bonds; or,

[0164] The modified siRNA is based on the siRNA of Example 1, with the nucleotides at positions 9, 10, and 11 of the positive strand being fluorinated at the 2' position, the nucleotides at other positions being methoxylated at the 2' position, the nucleotide at position 6 being modified with 2'-O-hexadecyl, a reverse anucleotide (invAb group) attached to the 3' end of the nucleotide at position 1, a reverse anucleotide (invAb group) attached to the 5' end of the nucleotide at position 1-2, position 1-2-2, and position 2-2-3-2-3-4. The nucleotides are linked by phosphate thioester bonds. The nucleotides at positions 2, 10, 12, 14, and 16 of the antisense strand are fluorinated at the 2' position. The nucleotides at positions 5 and 7 are deoxyribonucleotide substitutions (when the ribonucleotide is uracil ribonucleotide, thymine deoxyribonucleotide replaces uracil ribonucleotide, and so on). The nucleotides at other positions are methoxylated at the 2' position. Phosphothioester bonds link the nucleotides at positions 1 and 2, 2 and 3, penultimate and penultimate, and penultimate and penultimate.

[0165] The modified siRNA is based on the siRNA of Example 1, with the following modifications: nucleotides at positions 9, 10, and 11 of the sense strand are fluorinated at the 2' position, and nucleotides at other positions are methoxylated at the 2' position. The nucleotide at position 6 is modified with 2'-O-hexadecyl. A reverse ablation nucleotide (invAb group) is attached to the 3' end of the nucleotide at position 1, and a reverse ablation nucleotide (invAb group) is attached to the 5' end of the nucleotide at the penultimate position. Phosphothiophosphate bonds connect the nucleotides at positions 1 and 2, penultimate and penultimate, and penultimate and penultimate. Nucleotides at positions 2, 10, 12, 14, and 16 of the antisense strand are fluorinated at the 2' position. The nucleotide at position 5 is replaced with a deoxyribonucleotide (when the ribonucleotide is uracil ribonucleotide, thymine deoxyribonucleotide is used instead of uracil ribonucleotide, and so on). The nucleotide at position 7 is a glycol nucleic acid. Nucleotides modified with acid (GNA), where the nucleotides at other positions are methoxylated at the 2' position, and the nucleotides at positions 1 and 2, 2 and 3, penultimate and penultimate, and penultimate and penultimate are linked by phosphate thioester bonds; or,

[0166] The modified siRNA is based on the siRNA of Example 1, with the nucleotides at positions 7, 9, and 11 of the sense strand being fluorinated at the 2' position, and the nucleotides at other positions being methoxylated at the 2' position. A thiophosphate bond connects the nucleotides at positions 1 and 2, and the penultimate and penultimate positions. Similarly, the nucleotides at positions 2, 12, 14, and 16 of the antisense strand are fluorinated at the 2' position, and the nucleotides at other positions are methoxylated at the 2' position. A thiophosphate bond connects the nucleotides at positions 1 and 2, 2 and 3, 3 and 4, and the penultimate and penultimate positions. Alternatively,

[0167] The modified siRNA is based on the siRNA of Example 1, with the nucleotides at positions 7, 9, and 11 of the sense strand being fluorinated at the 2' position, and the nucleotides at other positions being methoxylated at the 2' position. The nucleotide at position 6 is modified with 2'-O-hexadecyl. A thiophosphate bond connects the nucleotides at positions 1 and 2, and the penultimate and penultimate positions. On the antisense strand, the nucleotides at positions 2, 12, 14, and 16 are fluorinated at the 2' position, and the nucleotides at other positions are methoxylated at the 2' position. A vinyl phosphate group (VP group) is attached to the 5' end of the nucleotide at position 1. A thiophosphate bond connects the nucleotides at positions 1 and 2, 2 and 3, 3 and 4, and the penultimate and penultimate positions. Alternatively,

[0168] The modified siRNA is based on the siRNA of Example 1, with the nucleotides at positions 7, 9, and 11 of the sense strand being fluorinated at the 2' position, and the nucleotides at other positions being methoxylated at the 2' position. The nucleotide at position 1 is modified with 2'-O-hexadecyl. A thiophosphate bond connects the nucleotides at positions 1 and 2, and the nucleotides at positions 1-1-2. In the antisense strand, the nucleotides at positions 2, 12, 14, and 16 are fluorinated at the 2' position, and the nucleotides at other positions are methoxylated at the 2' position. A vinyl phosphate group (VP group) is attached to the 5' end of the nucleotide at position 1. A thiophosphate bond connects the nucleotides at positions 1 and 2, 2 and 3, 3 and 4, and the nucleotides at positions 1-1-2-2.

[0169] The modified siRNA is based on the siRNA of Example 1, with the nucleotides at positions 7, 9, and 11 of the sense strand being fluorinated at the 2' position, and the nucleotides at other positions being methoxylated at the 2' position. The nucleotide at the penultimate position is modified with 2'-O-hexadecyl. A thiophosphate bond connects the nucleotides at positions 1 and 2, and the penultimate and penultimate positions. The nucleotides at positions 2, 12, 14, and 16 of the antisense strand are fluorinated at the 2' position, and the nucleotides at other positions are methoxylated at the 2' position. A vinyl phosphate group (VP group) is attached to the 5' end of the nucleotide at position 1. A thiophosphate bond connects the nucleotides at positions 1 and 2, 2 and 3, 3 and 4, and the penultimate and penultimate positions. Alternatively,

[0170] The modified siRNA is based on the siRNA of Example 1, with the nucleotides at positions 7, 9, and 11 of the sense strand being fluorinated at the 2' position, and the nucleotides at other positions being methoxylated at the 2' position. A thiophosphate bond connects the nucleotides at positions 1 and 2, and the penultimate and penultimate positions. On the antisense strand, the nucleotides at positions 2, 12, 14, and 16 are fluorinated at the 2' position, and the nucleotides at other positions are methoxylated at the 2' position. The penultimate nucleotide is modified with a 2'-O-hexadecyl group. A vinyl phosphate group (VP group) is attached to the 5' end of the nucleotide at position 1. Thiophosphate bonds connect the nucleotides at positions 1 and 2, 2 and 3, 3 and 4, and the penultimate and penultimate positions. Alternatively,

[0171] The modified siRNA is based on the siRNA of Example 1, with the nucleotides at positions 7, 9, and 11 of the sense strand being fluorinated at the 2' position, and the nucleotides at other positions being methoxylated at the 2' position. An additional 2'-O-hexadecyl base U is attached to the end. A thiophosphate bond connects the nucleotides at positions 1 and 2, and the penultimate and penultimate positions. On the antisense strand, the nucleotides at positions 2, 12, 14, and 16 are fluorinated at the 2' position, and the nucleotides at other positions are methoxylated at the 2' position. A vinyl phosphate group (VP group) is attached to the 5' end of the nucleotide at position 1. A thiophosphate bond connects the nucleotides at positions 1 and 2, 2 and 3, 3 and 4, and the penultimate and penultimate positions. Alternatively,

[0172] The modified siRNA is based on the siRNA of Example 1, with the 7th, 9th, 10th, and 11th nucleotides of the positive strand having a 2' fluorination, and the other nucleotides having a 2' methoxylation. A reverse abasic nucleotide (invAb group) is attached to the 3' end of the 1st nucleotide, and a reverse abasic nucleotide (invAb group) is attached to the 5' end of the penultimate nucleotide. The 1st nucleotide and the reverse abasic nucleotide are linked by a phosphate thioester group. The penultimate nucleotide... The antisense strand is linked to the non-base nucleotide via a thiophosphate group. The nucleotides at positions 2, 6, 14, and 16 of the antisense strand are fluorinated at the 2' position, while the nucleotides at other positions are methoxylated at the 2' position. The nucleotide at the penultimate position is modified with a 2'-O-hexadecyl group. A vinyl phosphate group (VP group) is attached to the 5' end of the nucleotide at position 1. The nucleotides at positions 1 and 2, 2 and 3, penultimate and penultimate, and penultimate and penultimate are linked by thiophosphate bonds; or...

[0173] The modified siRNA is based on the siRNA of Example 1, with the nucleotides at positions 7, 9, 10, and 11 of the positive strand being fluorinated at the 2' position, and the nucleotides at other positions being methoxylated at the 2' position. A reverse abstinent nucleotide (invAb group) is attached to the 3' end of the first nucleotide, and an additional 2'-O-hexadecyl base U is attached to the end. The first nucleotide and the reverse abstinent nucleotide are linked by a phosphate thioester group, and the penultimate nucleotide is linked to the reverse abstinent nucleotide. The nucleotides are linked by thiophosphate groups. The nucleotides at positions 2, 6, 14, and 16 of the antisense strand are fluorinated at the 2' position, while the nucleotides at other positions are methoxylated at the 2' position. The nucleotide at the penultimate position is modified with a 2'-O-hexadecyl group. A vinyl phosphate group (VP group) is attached to the 5' end of the nucleotide at position 1. The nucleotides at positions 1 and 2, 2 and 3, penultimate and penultimate, and penultimate and penultimate are linked by thiophosphate bonds; or...

[0174] The modified siRNA is based on the siRNA of Example 1, with the 7th, 9th, 10th, and 11th nucleotides of the sense strand being 2' fluorinated, and the other nucleotides being 2' methoxylated. The 3' end of the 1st nucleotide is connected to an inverse abase (invAb group), and an additional 2'-O-hexadecyl base A is attached to the end. The 1st nucleotide and the inverse abase are linked by a thiophosphate group, and the penultimate nucleotide and the inverse abase are also linked by a thiophosphate group. The 2nd, 6th, 14th, and 16th nucleotides of the antisense strand are 2' fluorinated, and the other nucleotides are 2' methoxylated. The penultimate nucleotide is modified with 2'-O-hexadecyl. The 5' end of the 1st nucleotide is connected to a vinyl phosphate group (VP group). The 1st and 2nd, 2nd and 3rd, penultimate and penultimate, and penultimate and penultimate nucleotides are linked by thiophosphate bonds.

[0175] The modified siRNAs are shown in Tables 3 to 8.

[0176] Table 3. Modified siRNAs and their sequences that inhibit AR gene expression

[0177] Table 4. Modified siRNAs and their sequences that inhibit AR gene expression

[0178] Table 5. Modified siRNAs and their sequences that inhibit AR gene expression

[0179] Table 6. Modified siRNAs and their sequences that inhibit AR gene expression

[0180] Table 7. Modified siRNAs and their sequences that inhibit AR gene expression

[0181] Table 8. Modified siRNAs and their sequences that inhibit AR gene expression

[0182] In Tables 3-8, uppercase letters C, G, U, and A represent ribonucleotides; lowercase letter m indicates that the nucleotide adjacent to the left of letter m is a methoxy-modified nucleotide; lowercase letter f indicates that the nucleotide adjacent to the left of letter f is a fluorinated nucleotide; lowercase letter s indicates that the two nucleotides adjacent to the left and right of letter s are linked by thiophosphate groups; the string C16 indicates that the nucleotide adjacent to the left of this string is a 2'-O-hexadecyl-modified nucleotide (2'-O-hexadecyl is linked by an ether bond to the hexadecyl group (C16)). 16 H 33 () is attached to the 2'-oxygen atom of the ribose ring of the nucleotide; string (d) indicates that the nucleotide adjacent to the left of this string is replaced by a deoxyribonucleotide. The nucleotides replaced by deoxyribonucleotides include five types: A(d), C(d), G(d), U(d), and T(d). Among them, A(d) represents 2'-deoxyadenosine-3'-phosphate, C(d) represents 2'-deoxycytidine-3'-phosphate, U(d) represents 2'-deoxyuridine-3'-phosphate, G(d) represents 2'-deoxyguanosine-3'-phosphate, and T(d) represents 2'-deoxythymidine-3'-phosphate; string (GNA) indicates that the nucleotide adjacent to the left of this string is a nucleotide modified with glycerol nucleic acid (GNA); string (invAb) indicates that the nucleotide adjacent to the left or right of this string is attached to an inverse abase-free nucleotide (invAb group); string VP indicates that the nucleotide adjacent to the right of this string is a nucleotide modified with vinyl phosphate.

[0183] Experiment 1: Validation of the activity of siRNA used to inhibit AR in LNCAP cells

[0184] This experimental example provides a validation experiment of the activity of siRNA for inhibiting AR in LNCAP cells. The experimental procedure is as follows:

[0185] LNCAP cells were seeded into MEM medium (Gibco, catalog number 11095-080) containing 10% (v / v) fetal bovine serum (FBS, purchased from Hyclone) and 1% (v / v) penicillin-streptomycin mixture (Penicillin-Streptomycin, purchased from Gibco, catalog number 15140122) and cultured for 48 h in a 5% (v / v) CO2 incubator at 37°C. After culture, the LNCAP cells were digested with trypsin (Gibco, catalog number 25200-072). After digestion, the cells were rinsed with PBS buffer and then resuspended in MEM medium to obtain a cell concentration of 4 × 10⁶ cells / year. 5 Cell suspension of cells / mL;

[0186] Different siRNAs were diluted separately using opti-MEM (Gibco, catalog number 31985-070) to obtain siRNA dilutions containing different siRNAs. 25 μL of opti-MEM was mixed with 0.25 μL of Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific, catalog number 11668-019) to obtain transfection reagent dilutions. 25 μL of each siRNA dilution was then mixed with the transfection reagent dilutions and incubated at room temperature (25°C) for 15 min to obtain transfection solutions containing different siRNAs.

[0187] Cell suspension was seeded into 96-well plates at a seeding rate of 50 μL / well. A transfection reagent control group (MOCK) and an siRNA experimental group were set up in each well, with 3 replicates per group. After setup, 50 μL of transfection buffer containing different siRNAs was added to the wells of the siRNA experimental group (hAR_1173M3 experimental group received transfection buffer containing hAR_1173M3, hAR_1174M3 experimental group received transfection buffer containing hAR_1174M3, and so on; the final concentration of siRNA in the wells was 0.1 nM or 1 nM, and the final siRNA concentration was adjusted using Opti-MEM dilution). 50 μL of transfection buffer without any siRNA was added to the wells of the transfection reagent control group (MOCK). Cells were cultured in a 5% (v / v) CO2, 37°C cell culture incubator for 48 h for transfection.

[0188] After transfection, discard the liquid in the wells, collect the cells, and extract total RNA from the cells in each well using lysis working solution. The lysis working solution was prepared according to the required volume of the well plate (see Table 9 for the lysis working solution formula), and the ratio of each component was FlysisAmp Cells Lysis Buffer (purchased from Vazyme, catalog number CL101-01): DNase I (purchased from Vazyme, catalog number GMP4104PC): Enhancer Solution = 23:1:1. After preparation, invert and mix 10-15 times to avoid vigorous vortexing. Add 50 μL of the prepared lysis buffer to each well of the cell culture plate, gently pipette 8-10 times to mix thoroughly, and let stand at room temperature for 5 min to lyse the cells. After lysis, add 5 μL of stop solution to each well of the cell culture plate, gently pipette 8-10 times to mix thoroughly, and let stand at room temperature for 2 min to stop the reaction.

[0189] RNA templates were prepared by preparing RNA templates (the RNA template preparation systems are shown in Table 10); the reaction procedure was as follows: the RNA template preparation systems were incubated at 37°C for 15 min, and after the reaction was completed, 1 μL of 50 mM EDTA aqueous solution was added to each RNA template preparation system for reaction;

[0190] For each reverse transcription reaction system, take 3.25 μL of the above RNA template solution as a template, and use the reagents provided in the AceQ Universal SYBR qPCR Master Mix kit (purchased from Vazyme, catalog number Q511-02) to prepare a 13 μL qPCR reaction system on an ice box according to Table 11. The PCR primer sequences are shown in Table 12, where hAR-F, hAR-R, and hAR-P are the primer and probe sequences for amplifying the target gene AR, and HGAPDH-F, HGAPDH-R, and HGAPDH-PHGAPDH-P are the primer and probe sequences for the internal reference gene GAPDH. Place each qPCR reaction system in an ABIStepOnePlus Real-Time... Amplification was performed using a three-step method on a PCR instrument. The amplification program was as follows: pre-denaturation at 50℃ for 10 min, followed by denaturation at 95℃ for 30 s, annealing at 58℃ for 30 s, and extension at 72℃ for 30 s. This denaturation, annealing, and extension process was repeated 40 times to obtain product W containing the amplified target gene AR and the internal reference gene GAPDH. Product W was then incubated sequentially at 95℃ for 15 s, 60℃ for 1 min, and 95℃ for 15 s. The melting curves of the target gene AR and the internal reference gene GAPDH in product W were collected by a real-time quantitative PCR instrument to obtain the Ct values ​​of the target gene AR and the internal reference gene GAPDH.

[0191] The relative quantitative calculation of AR of the target gene in each test group was performed using the comparison Ct(ΔΔCt) method, as follows:

[0192] ΔCt(test group) = Ct(target gene in test group) – Ct(internal reference gene in test group);

[0193] ΔCt(control group) = Ct(target gene in control group) – Ct(internal reference gene in control group);

[0194] ΔCt(test group) = ΔCt(test group) - ΔCt(control group average);

[0195] ΔCt(control group) = ΔCt(control group) - ΔCt(control group average);

[0196] Wherein, ΔCt (control group mean) is the arithmetic mean of ΔCt (control group) for each sample in the control group; thus, each sample in the test group and the control group corresponds to a ΔCt value;

[0197] Using the control group as a baseline, the expression level of AR mRNA in the test group was normalized, and the AR mRNA expression level in the control group was defined as 100%.

[0198] The relative expression level of AR mRNA in the test group = 2 - ΔΔCt(test group) × 100%;

[0199] AR mRNA inhibition rate in the test group = 1 - relative expression level of AR mRNA in the test group;

[0200] AR mRNA levels were compared with the internal reference gene GAPDH, and the values ​​were normalized to the mean of the saline control group. The data are expressed as a percentage relative to the saline control group and presented as the mean plus the standard deviation. The experimental results are shown in Tables 13 and 15–17.

[0201] siRNA concentration gradients were set: 10 nM, 1 nM, 0.333 nM, 0.111 nM, 0.037 nM, 0.012 nM, 0.004 nM, 0.001 nM, and 0.0005 nM. Following the method described above for detecting the relative expression level of AR mRNA, the effect of gradient concentrations of siRNA on the relative expression level of AR mRNA was examined. Then, using GraphPad Prism software, the IC50 of siRNA was calculated using the log(inhibitor) vs. response--Variable slope(four parameters) fitting method. The experimental results are shown in Table 14.

[0202] As shown in Tables 13 to 17, the modified siRNA in Example 2 exhibited good AR inhibition effects at different concentrations.

[0203] Table 9. Cell lysis reaction system

[0204] Table 10. Genome Removal Reaction System

[0205] Table 11. DNA Amplification Reaction System

[0206] Table 12. Primer Information

[0207] Table 13. Inhibitory levels of different modified siRNAs on AR mRNA in LNCAP cells

[0208] Table 14. Median inhibition concentration (IC50) of different modified siRNAs on AR mRNA in LNCAP cells

[0209] Table 15. Inhibitory levels of different modified siRNAs on AR mRNA in LNCAP cells

[0210] Table 16. Inhibitory levels of different modified siRNAs (1 nM) on AR mRNA in LNCAP cells

[0211] Table 17. Inhibitory levels of different modified siRNAs on AR mRNA in LNCAP cells

[0212] Experiment Example 2: Validation of the activity of siRNA for inhibiting AR in mice (qPCR)

[0213] This experimental example provides an experiment to verify the activity of siRNA for inhibiting AR in mice. The experimental procedure is as follows:

[0214] Based on the results of Experiment 1, C57BL / 6J male black mice (6 weeks old) were intradermally administered AR-039M1C16, AR-044M1C16, AR-013M1C16 or PBS buffer.

[0215] Forty mice were divided into two groups: one group received 0.25 mg / site and the other group received 0.5 mg / site.

[0216] 0.25 mg / site: Twenty mice were divided into four groups: AR-039M1C16, AR-044M1C16, AR-013M1C16, and PBS group. Each group contained five mice, and each mouse had four administration sites. 50 μL of siRNA was injected intradermally at a single site, with the administered dose being 0.25 mg. Skin samples were collected from mice on days 1, 3, 7, and 14 using an 8 mm diameter skin sampler to detect mAR mRNA levels.

[0217] 0.5 mg / site: Twenty mice were divided into four groups: AR-039M1C16, AR-044M1C16, AR-013M1C16, and PBS group. Each group contained five mice, and each mouse had four administration sites. 50 μL of siRNA was injected intradermally at a single site. The administered dose was 0.5 mg siRNA. Skin samples were collected from mice on days 1, 3, 7, and 14 using an 8 mm diameter skin sampler to detect mAR mRNA levels.

[0218] The experimental procedure is as follows:

[0219] 1. Drug injection: The back hair of the mice was removed 24 hours in advance. After anesthesia, each mouse was injected intradermally with 0.25 mg at one point, with 4 injection points and each point 2 cm apart.

[0220] 2. Animal observation: After weighing on Day 1, administer the medication according to the dosing regimen and observe once a day.

[0221] 3. Animal sample collection: Skin samples were taken from the injection sites of mice using an 8mm skin sampler. The skin was fixed in tissue preservation solution for subsequent RNA sample extraction. After overnight storage at 4°C, the samples were transferred to -80°C for storage.

[0222] 4. RNA extraction:

[0223] (1) Preparation of DNase I reaction solution: Prepare the solution according to the required amount of DNase I, DNase I Buffer and DEPC water in a volume ratio of 1:1:8, and prepare it according to 110% of the theoretical amount. Prepare it immediately before use.

[0224] (2) Sample lysis: Take an appropriate amount of tissue sample (it is recommended not to exceed 50mg), add 1000μL Ezol, add 2 steel balls to each tube, grind in a low-temperature cryogenic ball mill for 6 minutes, and stop grinding when there are no obvious tissue blocks.

[0225] (3) Sample addition:

[0226] First plate: Add 100 μL of DNase I reaction solution to each well;

[0227] Second plate: Add 400 μL of the supernatant sample after grinding to each well, then add 400 μL of isopropanol, mix by pipetting, and then add 20 μL of magnetic beads (the magnetic beads should be mixed by pipetting to avoid clumping and reducing the adsorption efficiency of the magnetic beads).

[0228] Third plate: Add 900 μL of Buffer CRW 0 to each well;

[0229] Fourth plate: Add 900 μL of Buffer CRW 0 to each well;

[0230] Fifth plate: Add 900 μL of Buffer CRW 1 to each well;

[0231] Plate 6: Add 100 μL of DEPC water to each well.

[0232] (4) RNA extraction: Run the automatic extraction program; after the program is completed, collect the RNA elution buffer from the sixth plate, store it at 4°C, and use it for downstream experiments within one day.

[0233] 5. RT-qPCR detection:

[0234] RNA templates were prepared by preparing RNA templates (the RNA template preparation systems are shown in Table 18); the reaction procedure was as follows: the RNA template preparation systems were incubated at 37°C for 15 min, and after the reaction was completed, 2 μL of 50 mM EDTA aqueous solution was added to each RNA template preparation system for reaction;

[0235] For each reverse transcription reaction system, take 3.25 μL of the above RNA template solution as a template, and use the reagents provided in the AceQ Universal SYBR qPCR Master Mix kit (purchased from Vazyme, catalog number Q231) to prepare a 13 μL qPCR reaction system on an ice box according to Table 19. The PCR primer sequences are shown in Table 20. Place each qPCR reaction system in an ABIStepOnePlus Real-Time... Amplification was performed using a three-step method on a PCR instrument. The amplification program was: pre-denaturation at 55℃ for 10 min, followed by denaturation at 95℃ for 30 s, annealing at 58℃ for 30 s, and extension at 72℃ for 30 s. This denaturation, annealing, and extension process was repeated 40 times to obtain product W containing amplified target gene AR and internal reference gene GAPDH. Product W was then incubated sequentially at 95℃ for 15 s, 60℃ for 1 min, and 95℃ for 15 s. The melting curves of target gene AR and internal reference gene GAPDH in product W were collected using a real-time quantitative PCR instrument to obtain the Ct values ​​of target gene AR and internal reference gene GAPDH. Referring to Experiment 1, the inhibitory effect of different siRNAs on AR mRNA in mice was detected by comparing Ct (ΔΔCt). The experimental results are shown in Figures 1 and 2.

[0236] As shown in Figures 1 and 2, AR-039M1C16, AR-044M1C16, and AR-013M1C16 all exhibited good inhibitory effects on AR mRNA in mice at different concentrations.

[0237] Table 18. Genome Removal Reaction System

[0238] Table 19. DNA Amplification Reaction System

[0239] Table 20. Primer Information

[0240] Experiment Example 3: Validation of the activity of siRNA used to inhibit AR in mice (WB)

[0241] This experimental example provides an experiment to verify the activity of siRNA for inhibiting AR in mice. The experimental procedure is as follows:

[0242] Based on the results of Experiment 1, C57BL / 6J male black mice (6 weeks old) were intradermally administered AR-039M1C16, AR-044M1C16, AR-013M1C16 or PBS buffer.

[0243] Forty mice were divided into two groups: one group received 0.25 mg / site and the other group received 0.5 mg / site.

[0244] 0.25 mg / site: Twenty mice were divided into four groups: AR-039M1C16, AR-044M1C16, AR-013M1C16, and PBS group. Each group contained five mice, and each mouse had four administration sites. 50 μL of siRNA was injected intradermally at a single site. The administered dose was 0.25 mg siRNA. Skin samples were collected from mice on days 1, 3, 7, and 14 using an 8 mm diameter skin sampler to detect mAR protein levels.

[0245] 0.5 mg / site: Twenty mice were divided into four groups: AR-039M1C16, AR-044M1C16, AR-013M1C16, and PBS group. Each group contained five mice, and each mouse had four administration sites. 50 μL of siRNA was injected intradermally at a single site. The administered dose was 0.5 mg siRNA. Skin samples were collected from mice on days 1, 3, 7, and 14 using an 8 mm diameter skin sampler to detect mAR protein levels.

[0246] The experimental procedure is as follows:

[0247] 1. Drug injection: The back hair of the mice was removed 24 hours in advance. After anesthesia, each mouse was injected intradermally with 0.25 mg at one point, with 4 injection points and each point 2 cm apart.

[0248] 2. Animal observation: After weighing on Day 1, administer the medication according to the dosing regimen and observe once a day.

[0249] 3. Animal sample collection: Skin samples were taken from the injection sites of mice using an 8mm skin sampler. The skin was washed with PBS and then transferred to -80℃ for storage for subsequent protein sample extraction.

[0250] 4. Extract protein, add cell lysis buffer (M-PER Mammalian Protein Extraction Reagent, purchased from Thermo), centrifuge at 4℃ and 12000rpm for 10min, and collect the supernatant.

[0251] 5. The total protein concentration was determined using the Bradford method. The OD value was detected at 595 nm using an ELISA reader. The standard curve and the total protein concentration of the samples were calculated. Based on the calculated total protein concentration, the total protein concentration of each group was adjusted with 1×PBS.

[0252] 6. Add 4×SDS loading buffer (200mM Tris-HCl (pH 6.8), 8% w / v SDS, 4% β-mercaptoethanol, 40% glycine, 0.08% w / v bromophenol blue), heat for 10 min. Centrifuge at 12000g for 1 min, and load the supernatant onto the sample.

[0253] 7. Samples were separated using 10% w / v (g / 100mL) SDS-PAGE. The gel was run in a vertical electrophoresis tank under the following conditions: 80V for 30 min, then 120V for 70 min.

[0254] 8. After electrophoresis, wet the PVDF membrane in methanol, then soak the gel, filter paper, and methanol-wetted PVDF membrane in Transfer Buffer (3g Tris base, 14.4g glycine, 200mL methanol, dilute to 1L of distilled water, pH 8.3) for several minutes to prepare a transfer sandwich. Perform the transfer using a transfer electrophoresis tank at 80V for 90 minutes.

[0255] 9. After the transfer is complete, block the transfer membrane with Blocking Buffer (1×PBS, 0.1% Tween-20, 5% w / v non-fat milk) for 2 hours.

[0256] 10. Add diluted primary antibody (nti-Androgen Receptor (Source: Rabbit; abcam, ab133273), 1:1000) and incubate overnight (16 h) at 4 °C. Wash three times with 1×PBST for 10 min each time.

[0257] 11. Add the diluted secondary antibody (HRP-conjugated Goat anti-Rabbit IgG, JIR 111-035-003, 1:10000) and incubate at room temperature (25℃) for 1 hour. Wash three times with 1×PBST for 10 minutes each time.

[0258] 12. Chemiluminescence detection was performed using ECL substrate (Pierce ECL Western Blotting Substrate, purchased from MDBio). After exposure using a fully automated chemiluminescence image analysis system, grayscale analysis was performed using Gel-Pro Analyzer software for quantification. The inhibitory effects of different siRNAs on AR protein in mice were evaluated based on the quantitative results. The experimental results are shown in Figures 3-5.

[0259] As shown in Figures 3 to 5, AR-039M1C16, AR-044M1C16, and AR-013M1C16 all exhibited good inhibitory effects on AR protein in mice at different concentrations.

[0260] Experiment Example 4: Validation of the hair regrowth effect of siRNA used to inhibit AR in a C57 male mouse model of androgenetic alopecia (AGA).

[0261] This experimental example provides a validation experiment on the hair regrowth effect of siRNA used to inhibit AR in a C57 male mouse model of androgenetic alopecia (AGA). The experimental procedure is as follows:

[0262] Based on the results of Experiment 1, C57BL / 6J mice (male, 6 weeks old) with testosterone propionate model were administered AR-039M1C16, AR-044M1C16, AR-013M1C16, PBS buffer, or minoxidil.

[0263] Thirty-six mice were used, and all mice were administered testosterone propionate (25 mg / mL) daily for 7 days prior to Day 0, at a dose of 50 μL (intraperitoneal injection). The experiment was divided into 6 groups, and the administration regimens are shown in Table 21.

[0264] Testosterone propionate (25 mg / mL) was purchased from Ningbo No. 2 Hormone Factory, and 5% minoxidil was purchased from Dafnix. Testosterone propionate was diluted with corn oil (purchased from COFCO). The labeling was done by clipping toes.

[0265] Mice were photographed and their results recorded on days 1, 3, 7, 9, 15, and 21 (Figures 6-9). On day 21, the mice were shaved and weighed (Figure 10). Five hairs were randomly selected from the shaved hair and their length was measured using calipers; the average length was recorded (Figure 11). Pigmentation in the mice was observed and recorded daily, as well as the number of days of pigmentation in C57 mice silenced by siRNA (Figure 12). Skin samples were taken on day 9 and stained with hematoxylin and eosin (HE) to observe hair follicle recovery (Figure 13). Skin samples were taken again on day 21 and stained with HE to observe hair follicle recovery (Figures 14-19).

[0266] The HE staining process is as follows:

[0267] (1) Baking paraffin sections in an oven at 60℃ for 30 minutes.

[0268] (2) Dewaxing: immerse in xylene (I,II,III) for 5 min each, then in anhydrous ethanol (I,II), 90% (v / v) alcohol (I,II), 80% alcohol (I,II), and 70% alcohol (I,II) for 1 min each, wash off the alcohol in water, and then transfer to distilled water for 2 min.

[0269] (3) Staining: Add hematoxylin staining solution to the slices for 5 minutes and wash off the excess color with tap water.

[0270] (4) 1% (v / v) hydrochloric acid alcohol differentiation for a few seconds.

[0271] (5) Rinse with tap water for 5 minutes to bluish the slices.

[0272] (6) Wash with distilled water.

[0273] (7) Add eosin staining solution to 50% alcohol and 70% alcohol for 30 seconds each, and counterstain for 30 seconds to make the cytoplasm red.

[0274] (8) Dehydration: 30s each in 80% alcohol, 90% alcohol, and anhydrous ethanol, and 1min each in xylene (I,II).

[0275] (9) Add neutral resin to the slice and seal it.

[0276] As shown in Figures 6-9, AR-039M1C16 showed significantly better hair regrowth than AR-044M1C16 and AR-013M1C16. Figures 10-12 show that AR-039M1C16 resulted in the highest weight of newly grown hair, the earliest period of pigment deposition, and the longest average length of newly grown hair, surpassing AR-044M1C16 and AR-013M1C16. Figures 14-19 show that AR-039M1C16 achieved better hair follicle recovery, with effects comparable to the positive control group (minoxidil), and superior to AR-044M1C16 and AR-013M1C16 in hair follicle recovery.

[0277] Table 21. Dosing Regimen

[0278] Experiment Example 5: Validation of the activity of siRNA for inhibiting AR in mice (qPCR+WB)

[0279] This experimental example provides an experiment to verify the activity of siRNA for inhibiting AR in mice. The experimental procedure is as follows:

[0280] Based on the results of Experiment 1, male C57BL / 6J black mice (6 weeks old) were intradermally treated with AR-039L23M2C16VP, AR-039L23M2-5C16VP, AR-039L23M2-3C16VP, AR-039L23M2-reverse-3C16VP, AR-039L23M2-3+UC16VP, AR-039J1C16VP, and AR-039J1C16VP. J1-5C16VP, AR-039J1-3C16VP, AR-039J1-reverse3C16VP, AR-039J1-3+UC16VP, AR-039Invab+reverseC16VP, AR-039Invab+positiveUreverseC16VP, AR-039L23M2-3+AC16VP, AR-039Invab+positiveAreverseC16VP, or PBS buffer. The inhibitory effects of different siRNAs on AR mRNA and AR protein in mice were detected using the methods described in Examples 2 and 3 (dose: 0.25 mg siRNA). The experimental results are shown in Figures 20-23.

[0281] As shown in Figures 20-23, AR-039L23M2C16VP, AR-039L23M2-5C16VP, AR-039L23M2-3C16VP, AR-039L23M2-reverse-3C16VP, AR-039L23M2-3+UC16VP, AR-039J1C16VP, AR-039J1-5C16VP, AR-039J1-3C16VP, AR-039J1-reverse-3C16VP, AR-039J1-3+UC16VP, AR-039Invab+reverse-C16VP, AR-039Invab+positive-U-reverse-C16VP, AR-039L23M2-3+AC16VP, and AR-039Invab+positive-A-reverse-C16VP all have an effect on AR in mice. The mRNA and AR protein showed good inhibitory effects.

[0282] Experiment Example 6: Validation of the activity of siRNA for inhibiting AR in mice (qPCR+WB)

[0283] This experimental example provides an experiment to verify the activity of siRNA for inhibiting AR in mice. The experimental procedure is as follows:

[0284] Based on the results of Experiment 1, male C57BL / 6J black mice (6 weeks old) were intradermally administered AR-039J1-5C16VP, AR-039Invab + positive U reverse C16VP, or PBS buffer. The inhibitory effects of different siRNAs on AR mRNA and AR protein in mice were detected using the methods described in Experiments 2 and 3 (dose: 0.125 mg siRNA, 0.25 mg siRNA, or 0.5 mg siRNA). The experimental results are shown in Figures 24–27.

[0285] As shown in Figures 24 to 27, AR-039J1-5C16VP and AR-039Invab+positiveUreC16VP both showed good inhibitory effects on AR mRNA and AR protein in mice at different doses.

[0286] Experiment 7: Validation of the hair regrowth effect of siRNA used to inhibit AR in a C57 male mouse model of androgenetic alopecia (AGA).

[0287] This experimental example provides a validation experiment on the hair regrowth effect of siRNA used to inhibit AR in a C57 male mouse model of androgenetic alopecia (AGA). The experimental procedure is as follows:

[0288] AGA Model Establishment: Thirty male C57 mice, aged 6 weeks, were selected. All mice received intraperitoneal injections of testosterone propionate (50 mg / mL, purchased from Harbin Sanma Veterinary Pharmaceutical Co., Ltd., injection dose 75 μL) daily from Day 0 to Day 7. Hair was removed using a depilatory cream over a 2cm × 4cm area on the back of the mice. Mice with no pigmentation on their backs were selected for the model group to normalize the hair follicle cycle. Subsequently, testosterone propionate (50 mg / mL, injection dose 75 μL) was administered intraperitoneally daily to maintain the AGA model. siRNA was administered intradermally to the back of the AGA model mice according to the intradermal injection protocol.

[0289] AR-039J1-5C16VP Intradermal Injection Dosing Regimen: Each dosing method constitutes a group, with the following dosing methods: 0.5 mg siRNA injected once at three dosing points, 0.25 mg injected once at six dosing points, and 0.25 mg siRNA injected twice at three dosing points. Each group consists of 4 mice. The solvent PBS control group consists of 5 mice per group, and the marketed drug minoxidil for treating AGA serves as a positive control group of 3 mice per group. 50 μL is administered via single intradermal injection at a single point. The dosing regimen is shown in Table 22.

[0290] During the drug administration period, the hair growth on the backs of mice was photographed and recorded. On day 21 after drug administration, newly grown hair was scraped from the drug administration site, and the hair length, weight, diameter, and percentage of hair coverage area were calculated. Skin samples from the drug administration site were collected using an 8mm diameter skin sampler, and the expression levels of AR gene mRNA and protein were detected according to the methods used in Experiments 2 and 3. Simultaneously, paraffin sections were prepared from skin samples collected from the drug administration site using an 8mm diameter skin sampler, and the hair follicle recovery was observed by HE staining. The experimental results are shown in Figures 28-36.

[0291] As shown in Figures 28-36, in the AGA mouse model, intradermal injection of siRNA (0.25 mg / point) twice a day resulted in a 96% hair regrowth coverage rate on the back after 21 days of administration, which was superior to the marketed drug minoxidil (92%).

[0292] In AGA mouse models, siRNA was administered intradermally twice (0.25 mg / dose). Skin samples were collected from the injection site 21 days after administration for paraffin sectioning and HE staining. The ratio of anagen to catagen phases in hair follicles was analyzed. The ratio in the siRNA group was 22, significantly higher than the 1.66 in the minoxidil group. This suggests that the siRNA group was superior to the minoxidil group in hair follicle recovery.

[0293] In AGA mouse models, siRNA was administered intradermally twice (0.25 mg / dose). Hair length and weight were measured 21 days after administration. The mean hair length in the siRNA group was 5.97 mm, while the mean hair length in the minoxidil group was 4.99 mm. The mean diameter of newly grown hair was 0.05 mm in the siRNA group and 0.03 mm in the minoxidil group. Overall, the siRNA group showed superior quality in newly grown hair compared to the minoxidil group.

[0294] After 21 days of administration, the expression levels of AR mRNA and protein in the skin tissue at the siRNA administration site decreased by 83% and 82%, respectively; it showed good long-term efficacy in skin tissue, which is beneficial for prolonging the administration period.

[0295] Table 22. Dosing Regimen

[0296] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. An siRNA for inhibiting the expression of an AR gene, characterized in that, The siRNA contains a sense strand and an antisense strand; the sense strand and the antisense strand are at least partially reverse complementary to form a double-stranded region; the sense strand of the siRNA comprises at least 15 consecutive nucleotides which differ by no more than 3 nucleotides from any one of the nucleotide sequences as shown in any one of SEQ ID NO. 1-560 or SEQ ID NO. 1121-1160; and the antisense strand of the siRNA comprises at least 15 consecutive nucleotides which differ by no more than 3 nucleotides from any one of the nucleotide sequences as shown in any one of SEQ ID NO. 561-1120 or SEQ ID NO. 1161-1200.

2. The siRNA of claim 1, wherein At least one of the nucleotides in the sense strand and / or the antisense strand of the siRNA is a modified nucleotide.

3. The siRNA of claim 2, wherein The modified nucleotide includes a compound in which the hydroxyl group at the 2' position of the ribose of the nucleotide is replaced by another group, a compound in which the phosphorothioate group of the nucleotide is modified, and / or a compound in which the base of the nucleotide is modified.

4. The siRNA according to any one of claims 2 to 3, wherein The modified nucleotide includes a fluorine-modified nucleotide, a methoxy-modified nucleotide, a phosphorothioate-linked nucleotide, a glycol nucleic acid-modified nucleotide, an inverted abasic nucleotide-modified nucleotide, a vinyl phosphate-modified nucleotide, a deoxyribonucleotide-substituted nucleotide, a 2'-O-methoxyethyl-modified nucleotide, a 2'-deoxy-nucleotide, a 3'-terminal deoxy-thymine nucleotide, a 3'-deoxy-nucleotide, a locked nucleotide, an unlocked nucleotide, a configuration-restricted nucleotide, a restricted ethyl nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide, a 2'-O-allyl-modified nucleotide, a 2'-C-alkyl-modified nucleotide, a 2'-hydroxyl-modified nucleotide, a 2'-O-alkyl-modified nucleotide, a tail sequence-modified nucleotide, a morpholino nucleotide-modified nucleotide, an aminophosphonate-modified nucleotide, a nucleotide containing a non-natural base, a tetrahydropyrane-modified nucleotide, a 1,5-anhydrohexitol-modified nucleotide, a cyclohexenyl-modified nucleotide, an open ring nucleotide-modified nucleotide, a nucleotide containing a methylphosphonate group, a nucleotide containing a 5'-phosphate, and / or a nucleotide containing a 5'-phosphate mimic.

5. The siRNA of claim 4, wherein The fluorine-modified nucleotide is located in the antisense strand and the sense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the 5th, 7th, 8th, or 9th nucleotide of the sense strand is a fluorine-modified nucleotide, and at least the 2nd, 6th, 14th, or 16th nucleotide of the antisense strand is a fluorine-modified nucleotide; or, The fluorine-modified nucleotide is located in the antisense strand and the sense strand of a nucleotide sequence, and, in the direction from the 5' end to the 3'end, at least the 7th, 8th, or 9th nucleotide of the sense strand is the fluorine-modified nucleotide, and at least the 2nd, 6th, or 14th nucleotide of the antisense strand is the fluorine-modified nucleotide; or, The fluorine-modified nucleotide is located in a nucleotide sequence, and, in the direction from the 5' end to the 5' end, at least the 7th, 8th, or 9th nucleotide is the fluorine-modified nucleotide. The fluorine-modified nucleotide is located in the antisense strand and the sense strand of the nucleotide sequence, and at least the 9th, 10th, 11th nucleotide of the sense strand and at least the 2nd, 6th, 14th, 16th nucleotide of the antisense strand are fluorine-modified nucleotides in the direction from the 5' end to the 3' end; or, The fluorine-modified nucleotide is located in the antisense strand and the sense strand of the nucleotide sequence, and at least the 7th, 9th, 10th, 11th nucleotide of the sense strand and at least the 2nd, 6th, 14th, 16th nucleotide of the antisense strand are fluorine-modified nucleotides in the direction from the 5' end to the 3' end; or, The fluorine-modified nucleotide is located in the antisense strand and the sense strand of the nucleotide sequence, and at least the 9th, 10th, 11th nucleotide of the sense strand and at least the 2nd, 10th, 12th, 14th, 16th nucleotide of the antisense strand are fluorine-modified nucleotides in the direction from the 5' end to the 3' end; or, The fluorine-modified nucleotide is located in the antisense strand and the sense strand of the nucleotide sequence, and at least the 7th, 9th, 11th nucleotide of the sense strand and at least the 2nd, 12th, 14th, 16th nucleotide of the antisense strand are fluorine-modified nucleotides in the direction from the 5' end to the 3' end.

6. The siRNA of claim 4 or 5, wherein The methoxy-modified nucleotide is located in the antisense strand and the sense strand of the nucleotide sequence, and in the sense strand, the nucleotide which is not fluorine-modified, the nucleotide which is not glycol nucleic acid modified, and the nucleotide which is not deoxyribonucleotide substituted are all methoxy-modified nucleotides, and in the antisense strand, the nucleotide which is not fluorine-modified, the nucleotide which is not ethylene glycol nucleic acid modified, and the nucleotide which is not deoxyribonucleic acid substituted are all methoxy-modified nucleotides.

7. The siRNA according to any one of claims 4 to 6, wherein The reverse abasic nucleotide-modified nucleotide is located in the sense strand of the nucleotide sequence, and in the direction from the 5' end to the 3' end, the 3' end of at least the 1st nucleotide of the sense strand is connected with a reverse abasic nucleotide; and / or, The reverse abasic nucleotide-modified nucleotide is located in the sense strand of the nucleic acid sequence, and in the direction from the 5' end to the 3' end, at least the 1st nucleotide of the sense strand is connected with a reverse abasic group.

8. The siRNA according to any one of claims 4 to 7, wherein The vinyl phosphate-modified nucleotide is located in the antisense strand of the nucleotide sequence, and in the direction from the 5' end to the3' end, the 5' end of at least the 1st nucleotide of the antisense strand is connected with a vinyl phosphate group.

9. The siRNA according to any one of claims 4 to 8, wherein The deoxyribonucleotide-substituted nucleotide is located in the antisense strand of the nucleotide sequence, and in the direction of the 5' end to the 3' end, at least the 5th nucleotide of the antisense strand is a deoxyribonucleotide-substituted nucleotide; or, The deoxyribonucleotide-substituted nucleotide is located in the antisense strand, and in the direction from the 5' end to the 3' end, at the least 7th nucleotide of the antisense strand is a deoxyribonucleotide-substituted; or, The deoxyribonucleotide-substituted nucleotide is located in the antisense strand of the nucleotide sequence, and at least the 5th and 7th nucleotides of the antisense strand are deoxyribonucleotide-substituted nucleotides in the direction from the 5' end to the 3' end.

10. The siRNA according to any one of claims 4 to 9, wherein The glycol nucleic acid-modified nucleotide is located in the antisense strand of the nucleotide sequence, and at least the 7th nucleotide of the antisense strand is a glycol nucleic acid-modified nucleotide in the direction from the 5' end to the 3' end.

11. The siRNA according to any one of claims 4 to 10, wherein The phosphorothioate group-linked nucleotide is located in the antisense strand and the sense strand of the nucleotide sequence, and in the direction from the 5' end to the 3' end, the nucleotides at least between the 1st and 2nd, the 2nd and 3rd of the sense strand are connected by phosphorothioate group, and the nucleotides at least between the 1st and 2nd, the 2nd and 3rd, the last 1st and the last 2nd, the last 2nd and the last 3rd of the antisense strand are connected by phosphorothioate group; or, The phosphorothioate group-linked nucleotide is located in the antisense strand and the antisense strand of the nucleotide sequence, and in the direction from the 5' end to the3' end, the nucleotides at least between the 1st and 2nd and the last 1st and the last 2nd of the sense strand are connected by phosphorothioate bond, and the nucleotides at least between the 1st and 2nd, the2nd and 3rd, the last 1st and the last 2nd, and the last 2nd and the last 3rd of the antisense strand are connected by the phosphorothioate bond; or, The phosphorothioate group-linked nucleotide is located in the antisense and sense strands of the nucleotide sequence, and in the direction from the 5' end to the 31 end, the nucleotides at least between the 1st and 2nd, the2ndand 3rd, the last 1st and the last 2nd, and the last2ndand the last 3rd of the sense strand are connected by the phosphorothioate bond, and the nucleotides at least between the 1stand 2nd, the 2nd and 3rd, the last 1st and last 2nd, and the last 2nd and the last 3rd of theantisense strand are connected by the phosphorothioate bond; or, The phosphorothiote group-linked nucleotide is located in the antisense and sense strands of the nucleotide sequence, in the direction from the 5' end to the 3' end, the nucleotide at the 1st of the sense strand is connected to the reverse abasic nucleotide by the phosphorothioate group, the nucleotide at the last 1st is connected to the reverse abasic nucleotide by the phosphorothioate group, and the nucleotides at least between the 1stand 2nd, the2ndand 3rd, the last 1st and thelast 2nd, and the last 2nd and the last 3rd of the antisensestrand are connected by the phosphorothioate bond; or, The phosphorothiote-group linked nucleotide is located in the antisense and sense strands of the nucleotide sequence, in the 5' end to the 3' end direction, the nucleotides at least between the 1st and 2nd, the last 1st and the last 2nd, and the last 2ndand the last 3rd of the sense strand are connected by the phosphorothiote bond, and the nucleotides at least between the 1st and 2nd, 2nd and 3rd, the last 1st and the last 2nd and the last 2nd and the last 3rd of the antisense strand are connected through the phosphorothiote bond; or, The phosphorothioate group-linked nucleotides are located in the antisense strand and the sense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the 1st and 2nd, the last 1st and the last 2nd nucleotides of the sense strand are linked by phosphorothioate bond, and at least the 1st and 2nd, the 2nd and 3rd, the 3rd and 4th, the last 1st and the last 2nd nucleotides of the antisense strand are linked by phosphorothioate bond.

12. The siRNA according to any one of claims 4 to 11, wherein The 2'-O-alkyl-modified nucleotide is a 2'-O-hexadecyl-modified nucleotide; The 2'-O-hexadecyl-modified nucleotide is located in the antisense strand or the sense strand of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the 6th nucleotide of the sense strand is a 2'-O-hexadecyl-modified nucleotide; or, at least the 1st nucleotide of the sense strand is a 2'-O-hexadecyl-modified nucleotide; or, at least the last 1st nucleotide of the sense strand is a 2'-O-hexadecyl-modified nucleotide; or, at least the last 1st nucleotide of the antisense strand is a 2'-O-hexadecyl-modified nucleotide.

13. The siRNA according to any one of claims 4 to 12, wherein The tail sequence-modified nucleotide is located in the sense strand of the nucleotide sequence; the tail sequence-modified nucleotide is a nucleotide with a lipophilic tail sequence connected to the 3' end; the lipophilic tail sequence is an RNA fragment containing 1-3 bases.

14. The siRNA of claim 13, wherein The number of bases of the lipophilic tail sequence is 1; the base is A or U.

15. An siRNA conjugate for inhibiting the expression of an AR gene, characterized in that, The siRNA conjugate contains the siRNA according to any one of claims 1-14 and a conjugation group conjugated to the siRNA.

16. A recombinant plasmid, characterized in that, The recombinant plasmid expresses the siRNA according to any one of claims 1-14.

17. A host cell, characterized in that, The genome of the host cell is integrated with the siRNA according to any one of claims 1-14; or the host cell carries the recombinant plasmid according to claim 16.

18. Use of the siRNA according to any one of claims 1-14, the siRNA conjugate according to claim 15, the recombinant plasmid according to claim 16, or the host cell according to claim 17 in the preparation of a medicament for preventing and / or treating a pathological condition or disease caused by AR.

19. The use according to claim 18, wherein the compound is ###00009### The pathological condition or disease caused by AR includes androgenic alopecia.

20. A medicament for preventing and / or treating a pathological condition or disease caused by AR, characterized by, The components of the medicament include the siRNA according to any one of claims 1-14, the siRNA conjugate according to claim 15, the recombinant plasmid according to claim 16, or the host cell according to claim 17.

21. The medicament of claim 20, wherein, The pathological condition or disease caused by AR includes androgenic alopecia. The pathological condition or disease caused by AR includes androgenic alopecia.

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