Androgen receptor-targeting sirna, and modified sirna and vector system thereof and use thereof

Abstract

Disclosed are an androgen receptor-targeting siRNA, and a modified siRNA and a vector system thereof and a use thereof. By performing siRNA drug design on an androgen receptor gene sequence, a plurality of siRNAs composed of sense strand and antisense strand RNAs are screened for, and the nucleotide sequences of the siRNAs are as shown in SEQ ID Nos. 1-128. These siRNAs can effectively knock down the mRNA expression level of androgen receptors, some sequences can knock down the mRNA expression level thereof to about 30% on human cells, and these siRNAs exhibit an obvious concentration effect. After the nucleotide chain is subjected to nucleic acid backbone thio-modification and base nucleoside modification, the stability of the siRNA sequence can be effectively improved, and the IC50 for the knock-down of the androgen receptor gene can reach 0.064 nM. The present invention has the potential of preparing a gene therapy drug for treating androgen receptor-related diseases caused by the overexpression of the androgen receptor gene, such as androgenic alopecia or prostate cancer.

Description

siRNA targeting androgen receptor, modified siRNA and carrier system and application thereof TECHNICAL FIELD

[0001] The present application relates to a gene medicine for treating androgen alopecia, in particular to siRNA targeting androgen receptor, modified siRNA and carrier system thereof and application thereof in preparing a gene therapy medicine for preventing or treating androgen alopecia, and belongs to the field of gene therapy medicines for androgen alopecia. BACKGROUND

[0002] Androgen alopecia (AGA) is also known as seborrheic alopecia, early baldness and male baldness, and is a kind of hair loss disease with progressive miniaturization of hair follicles. According to statistics, the prevalence rate of male patients in China is as high as 21.3%, and the prevalence rate of female patients is 6%. Meanwhile, the alopecia population is becoming younger and younger, and the scalp of androgen alopecia patients will show disease characteristics such as yellow dots, pigmentation, perifolliculitis, variation of hair shaft diameter and vellus hair.

[0003] The mechanism of androgen alopecia can be simply summarized as follows: testosterone in the human body can be reduced to dihydrotestosterone (DHT) under the action of 5α-reductase, the expression of type II 5α-reductase gene in the hair follicle cells of the scalp hair loss area of androgen alopecia patients is increased, testosterone is converted into dihydrotestosterone (DHT) and combined with androgen receptor protein (ARP) in the scalp, which leads to normal hair follicles becoming susceptible hair follicles, the expression of androgen receptor (AR) gene in the hair follicle cells of the scalp hair loss area of patients is increased, which makes the effect of androgens on susceptible hair follicles increase, and then a series of symptoms such as miniaturization of hair follicles, shortening of hair follicle growth period and stagnation of resting period occur, and finally different degrees of hair loss are caused. Androgen alopecia is related to heredity and belongs to a polygenic recessive genetic disease.

[0004] The current treatment of AGA mainly includes drug therapy, low-energy laser and hair transplantation. The oral drugs finasteride and dutasteride used in clinic belong to 5α-reductase inhibitors, which can prevent testosterone from converting to dihydrotestosterone by inhibiting 5α-reductase, thereby achieving the purpose of treatment. The main external drug in clinic is minoxidil, which can stimulate hair follicle cell proliferation and differentiation by dilating peripheral blood vessels to promote hair growth. Other drugs under clinical research include androgen receptor antagonists, prostaglandins and prostaglandin analogs, Wnt pathway modulators, and type A botulinum toxin. There is no marketed drug for RNA interference (RNAi) therapy. A siRNA drug OLX72021 developed by OliX Company in South Korea was used in a phase I clinical trial in Australia in March 2023, indicating that nucleic acid drugs have a large market space in the prevention and treatment of AGA. The expression level of AR gene in hair follicle cells of AGA patients is high, which leads to excessive TGF-β, elevated IL-6 levels, and increased DKK levels, ultimately leading to hair loss. Therefore, reducing the expression level of AR gene can effectively solve AGA.

[0005] The working principle of AR protein in hair follicle cells in the hair loss area of AGA patients is that dihydrotestosterone binds to the functional domain LBD of AR protein, and then is transported to the cell nucleus to regulate gene expression. Among the five isoforms of AR protein, AR-3, AR-4 and AR-8 mainly exist in prostate cancer cells, AR-45 has a high expression level in the heart, and its expression in skin cells is not clear. AR-B has the largest molecular weight and is the most common, which can be an effective target for preventing and treating AGA.

[0006] Prostate cancer is the second leading cause of death worldwide and one of the most common cancers in men, usually manifested as a malignant tumor of the prostate epithelium. The development of prostate cancer is highly related to androgens, and studies have shown that mutations in the AR gene can lead to the occurrence of prostate cancer. Clinically, androgen deprivation therapy (ADT) is used to block androgen synthesis and inhibit the AR signaling pathway to achieve the purpose of treatment. However, this treatment can further worsen the patient's condition to castration-resistant prostate cancer (CRPC), which seriously affects the patient's quality of life and survival rate. Androgen antagonistic therapy, as one of the main treatment methods, can be used alone to treat early prostate cancer or in combination with surgery for adjuvant therapy. Clinically, AR inhibitors can be divided into steroidal AR inhibitors and non-steroidal AR inhibitors according to their structure.

[0007] The siRNA is a double-stranded RNA with negative charge of about 21-25 bp, which realizes high efficiency and high specificity binding to the target mRNA in the cell by inducing the formation of RISC complex, and then triggers the specific degradation of the target mRNA, so as to play the RNAi role to regulate the expression of the gene. Screening of the effective siRNA can reduce the expression level of the AR gene, so as to realize the purposes of preventing and treating androgen alopecia and treating prostate cancer. SUMMARY

[0008] One of the purposes of the present application is to provide siRNA targeting the androgen receptor;

[0009] The second purpose of the present application is to modify the siRNA targeting the androgen receptor to obtain modified siRNA;

[0010] The third purpose of the present application is to provide an expression vector containing the siRNA targeting the androgen receptor or the modified siRNA;

[0011] The fourth purpose of the present application is to prepare the siRNA targeting the androgen receptor or the modified siRNA targeting the androgen receptor into a gene therapy drug for treating androgen alopecia or treating prostate cancer.

[0012] The above purposes of the present application are realized by the following technical solutions:

[0013] One aspect of the present application is to provide siRNA targeting the androgen receptor, which specifically binds to the mRNA of the AR gene; preferably, the siRNA is designed to target the 3384 region, the 5032 region, the 6102-6145 region, the 6609-6613 region, the 9209-9506 region or the 10398-10406 region in the mRNA of the full length of the AR gene of 1127-3889 regions.

[0014] In a preferred embodiment of the present application, the siRNA contains a sense strand and an antisense strand, which is selected from any one of the following (1)-(64) siRNAs:

[0015] (1) an siRNA consisting of a positive strand containing the nucleotide sequence shown in SEQ ID No. 1 and an antisense strand containing the nucleotide sequence shown in SEQ ID No. 2; (2) an siRNA consisting of a positive strand containing the nucleotide sequence shown in SEQ ID No. 3 and an antisense strand containing the nucleotide sequence shown in SEQ ID No. 4; (3) an siRNA consisting of a positive strand containing the nucleotide sequence shown in SEQ ID No. 5 and an antisense strand containing the nucleotide sequence shown in SEQ ID No. 6; (4) an siRNA consisting of a positive strand containing the nucleotide sequence shown in SEQ ID No. 7 and an antisense strand containing the nucleotide sequence shown in SEQ ID No. 8; (5) an siRNA consisting of a positive strand containing the nucleotide sequence shown in SEQ ID No. 9 and an antisense strand containing the nucleotide sequence shown in SEQ ID No. 10; (6) an siRNA consisting of a positive strand containing the nucleotide sequence shown in SEQ ID No. 11 and an antisense strand containing the nucleotide sequence shown in SEQ ID No. 12; (7) an siRNA consisting of a positive strand containing the nucleotide sequence shown in SEQ ID No. 13 and an antisense strand containing the nucleotide sequence shown in SEQ ID No. 14. (8) siRNA composed of the antisense strand of the nucleotide sequence shown in SEQ ID No. 14; (9) siRNA composed of the positive strand containing the nucleotide sequence shown in SEQ ID No. 15 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 16; (10) siRNA composed of the positive strand containing the nucleotide sequence shown in SEQ ID No. 17 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 18; (11) siRNA composed of the positive strand containing the nucleotide sequence shown in SEQ ID No. 19 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 20; (12) siRNA composed of the positive strand containing the nucleotide sequence shown in SEQ ID No. 23 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 24; (13) siRNA composed of the positive strand containing the nucleotide sequence shown in SEQ ID No. 25 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 26; (14) siRNA composed of the positive strand containing the nucleotide sequence shown in SEQ ID No. 15 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 26; (15) An siRNA consisting of a sense strand containing the nucleotide sequence shown in SEQ ID No. 27 and an antisense strand containing the nucleotide sequence shown in SEQ ID No. 28; (16) An siRNA consisting of a sense strand containing the nucleotide sequence shown in SEQ ID No. 29 and an antisense strand containing the nucleotide sequence shown in SEQ ID No. 30;(17) siRNA composed of the antisense strand of the nucleotide sequence shown in SEQ ID No. 33 and the antisense strand of the nucleotide sequence shown in SEQ ID No. 34; (18) siRNA composed of the positive strand of the nucleotide sequence shown in SEQ ID No. 35 and the antisense strand of the nucleotide sequence shown in SEQ ID No. 36; (19) siRNA composed of the positive strand of the nucleotide sequence shown in SEQ ID No. 37 and the antisense strand of the nucleotide sequence shown in SEQ ID No. 38; (20) siRNA composed of the positive strand of the nucleotide sequence shown in SEQ ID No. 39 and the antisense strand of the nucleotide sequence shown in SEQ ID No. 40; (21) siRNA composed of the positive strand of the nucleotide sequence shown in SEQ ID No. 41 and the antisense strand of the nucleotide sequence shown in SEQ ID No. 42; (22) siRNA composed of the positive strand of the nucleotide sequence shown in SEQ ID No. 43 and the antisense strand of the nucleotide sequence shown in SEQ ID No. 44; (23) siRNA composed of the positive strand of the nucleotide sequence shown in SEQ ID No. 32 and the antisense strand of the nucleotide sequence shown in SEQ ID No. 43; (24) An siRNA composed of the positive strand of the nucleotide sequence shown in SEQ ID No. 45 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 46; (25) An siRNA composed of the positive strand of the nucleotide sequence shown in SEQ ID No. 47 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 48; (26) An siRNA composed of the positive strand of the nucleotide sequence shown in SEQ ID No. 49 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 50; (27) An siRNA composed of the positive strand of the nucleotide sequence shown in SEQ ID No. 51 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 52; (28) An siRNA composed of the positive strand of the nucleotide sequence shown in SEQ ID No. 53 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 54; (29) An siRNA composed of the positive strand of the nucleotide sequence shown in SEQ ID No. 55 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 56; (30) siRNA consisting of an antisense strand containing the nucleotide sequence shown in SEQ ID No. 58; (31) siRNA consisting of a sense strand containing the nucleotide sequence shown in SEQ ID No. 59 and an antisense strand containing the nucleotide sequence shown in SEQ ID No. 60; (32) siRNA consisting of a sense strand containing the nucleotide sequence shown in SEQ ID No. 61 and an antisense strand containing the nucleotide sequence shown in SEQ ID No. 60.(32) siRNA composed of the antisense strand of the nucleotide sequence shown in SEQ ID No. 63 and the antisense strand of the nucleotide sequence shown in SEQ ID No. 64; (33) siRNA composed of the positive strand of the nucleotide sequence shown in SEQ ID No. 65 and the antisense strand of the nucleotide sequence shown in SEQ ID No. 66; (34) siRNA composed of the positive strand of the nucleotide sequence shown in SEQ ID No. 67 and the antisense strand of the nucleotide sequence shown in SEQ ID No. 68; (35) siRNA composed of the positive strand of the nucleotide sequence shown in SEQ ID No. 69 and the antisense strand of the nucleotide sequence shown in SEQ ID No. 70; (36) siRNA composed of the positive strand of the nucleotide sequence shown in SEQ ID No. 71 and the antisense strand of the nucleotide sequence shown in SEQ ID No. 72; (37) siRNA composed of the positive strand of the nucleotide sequence shown in SEQ ID No. 73 and the antisense strand of the nucleotide sequence shown in SEQ ID No. 74; (38) siRNA composed of the positive strand of the nucleotide sequence shown in SEQ ID No. 62 and the antisense strand of the nucleotide sequence shown in SEQ ID No. 74; (39) An siRNA consisting of the positive strand of the nucleotide sequence shown in SEQ ID No. 75 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 76; (40) An siRNA consisting of the positive strand of the nucleotide sequence shown in SEQ ID No. 77 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 78; (41) An siRNA consisting of the positive strand of the nucleotide sequence shown in SEQ ID No. 79 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 80; (42) An siRNA consisting of the positive strand of the nucleotide sequence shown in SEQ ID No. 83 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 84; (43) An siRNA consisting of the positive strand of the nucleotide sequence shown in SEQ ID No. 85 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 86; (44) An siRNA consisting of the positive strand of the nucleotide sequence shown in SEQ ID No. 87 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 78. (45) An siRNA consisting of an antisense strand containing the nucleotide sequence shown in SEQ ID No. 88; (46) An siRNA consisting of a sense strand containing the nucleotide sequence shown in SEQ ID No. 89 and an antisense strand containing the nucleotide sequence shown in SEQ ID No. 90;(47) siRNA composed of the antisense strand of the nucleotide sequence shown in SEQ ID No. 93 and the antisense strand of the nucleotide sequence shown in SEQ ID No. 94; (48) siRNA composed of the positive strand of the nucleotide sequence shown in SEQ ID No. 95 and the antisense strand of the nucleotide sequence shown in SEQ ID No. 96; (49) siRNA composed of the positive strand of the nucleotide sequence shown in SEQ ID No. 97 and the antisense strand of the nucleotide sequence shown in SEQ ID No. 98; (50) siRNA composed of the positive strand of the nucleotide sequence shown in SEQ ID No. 99 and the antisense strand of the nucleotide sequence shown in SEQ ID No. 100; (51) siRNA composed of the positive strand of the nucleotide sequence shown in SEQ ID No. 101 and the antisense strand of the nucleotide sequence shown in SEQ ID No. 102; (52) siRNA composed of the positive strand of the nucleotide sequence shown in SEQ ID No. 103 and the antisense strand of the nucleotide sequence shown in SEQ ID No. 94. (53) An siRNA composed of the antisense strand of the nucleotide sequence shown in SEQ ID No. 104; (54) An siRNA composed of the positive strand containing the nucleotide sequence shown in SEQ ID No. 105 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 106; (55) An siRNA composed of the positive strand containing the nucleotide sequence shown in SEQ ID No. 107 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 108; (56) An siRNA composed of the positive strand containing the nucleotide sequence shown in SEQ ID No. 109 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 110; (57) An siRNA composed of the positive strand containing the nucleotide sequence shown in SEQ ID No. 111 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 112; (58) An siRNA composed of the positive strand containing the nucleotide sequence shown in SEQ ID No. 113 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 114; (59) An siRNA composed of the positive strand containing the nucleotide sequence shown in SEQ ID No. 115 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 106; (59) An siRNA consisting of an antisense strand containing the nucleotide sequence shown in SEQ ID No. 117 and an antisense strand containing the nucleotide sequence shown in SEQ ID No. 118; (60) An siRNA consisting of a positive strand containing the nucleotide sequence shown in SEQ ID No. 119 and an antisense strand containing the nucleotide sequence shown in SEQ ID No. 120; (61) An siRNA consisting of a positive strand containing the nucleotide sequence shown in SEQ ID No. 121 and an antisense strand containing the nucleotide sequence shown in SEQ ID No. 120.(62) An siRNA composed of an antisense strand containing the nucleotide sequence shown in SEQ ID No. 122; (63) An siRNA composed of a sense strand containing the nucleotide sequence shown in SEQ ID No. 123 and an antisense strand containing the nucleotide sequence shown in SEQ ID No. 124; (64) An siRNA composed of a sense strand containing the nucleotide sequence shown in SEQ ID No. 125 and an antisense strand containing the nucleotide sequence shown in SEQ ID No. 126; (65) An siRNA composed of a sense strand containing the nucleotide sequence shown in SEQ ID No. 127 and an antisense strand containing the nucleotide sequence shown in SEQ ID No. 128.

[0016] In order to improve the stability of siRNA in vivo, reduce the degradation of exonucleases, or promote the delivery of siRNA into cells in vivo to better exert the effect of knocking down AR genes, another aspect of the present invention provides modified siRNA obtained by modifying the siRNA. The siRNA provided by the present invention can be modified in various ways to obtain modified siRNA with improved stability or improved AR gene knockdown effect. Those skilled in the art can modify the above-mentioned siRNA according to needs by modifying the backbone, sugar ring, nucleoside or base, etc., including but not limited to one or more of the following types of modifications: (1) replacing the -OH group at the 2' carbon position of the sugar ring in the nucleotide with methoxy, methylethoxy, etc. (1) Modification by substituting methyl, amino, fluorine, -O-2-methylthioethyl, -O-3-aminopropyl or -O-3-dimethylaminopropyl; (2) Modification of the phosphodiester bond between nucleotides, including but not limited to changing the bond to thiophosphate, boron phosphate or methylphosphonate; (3) Nucleoside modification of the sequence, including but not limited to changing siRNA to peptide nucleic acid, locked nucleic acid or unlocked nucleic acid; (4) Base modification, the base modification including but not limited to: base modification or substitution of 5-methylcytosine, 6-methyladenine, ribavirin, pseudouracil and / or inosine of the nucleoside base; (5) No modification of the siRNA sequence end or including but not limited to phosphorylation, thiophosphorylation, vinylphosphonate or other phosphorylation analogs and phosphate prodrug modification;

[0017] To improve the strand selectivity of siRNA to target genes and the silencing effect of target genes, the 5' end of the antisense strand of siRNA can be phosphorylated or modified with a phosphorylation analogue using the following modification methods:

[0018] (6) In order to enable siRNA to have a targeting effect in cells, improve uptake efficiency and exert a silencing effect, the terminal group of siRNA can be modified with cholesterol as needed to improve the efficiency and long-term effectiveness of siRNA entering cells and enhance the therapeutic effect; preferably, the cholesterol-modified structural formula is as follows:

[0019] In one specific embodiment of the present invention, the modified siRNA is a modified siRNA obtained by modification using at least one of the following modification methods (1)-(6):

[0020] (1) Convert a non-ester oxygen bond between the phosphodiester bonds of RNA into sulfur;

[0021] (2) The -OH group at the 2′ carbon position of the nucleic acid base is substituted with, but not limited to, CH3 (methyl), -OCH3 (methoxy), -OCH2CH2OCH3 (methoxyethyl), -F (fluorine), NH2, -O-2-methylthioethyl, -O-3-aminopropyl or -O-3-dimethylaminopropyl and / or the 2′-OH is deoxygenated; preferably, the -OH group at the 2′ carbon position of the nucleic acid base is alternately substituted with methoxy and fluorine;

[0022] (3) The ends of the sense or antisense strands of the siRNA have two or more phosphate-thiolated modifications.

[0023] (4) Replace the TT base at the 3′ end of the sense or antisense strand of the siRNA with the two bases that originally complement the AR mRNA.

[0024] (5) The 5' end of the antisense strand of the RNA sequence is not modified or is modified with phosphate, thiophosphate or its phosphate analogue and phosphate prodrug, and the 5' end of the sense strand is not modified or is modified with an inverted non-basic sugar cap.

[0025] (6) Modify the ends of siRNA with cholesterol.

[0026] The antisense strand described in this invention can be a polynucleotide complementary to the target gene, and its length can be 19 nt or more; in a preferred embodiment, the length of the antisense strand can include the sequence and be between 19 nt and 26 nt; in addition, the antisense strand described in this invention can also have a nucleotide sequence complementary to the sense strand.

[0027] In a more preferred embodiment of the present invention, the modified siRNA is selected from any one of the following (1)-(22) siRNAs composed of nucleotides containing a sense strand and nucleotides containing an antisense strand:

[0028] (1) Justice chain: mG*mU*mUmUmCmUfGmAfGmUfGmAfCmAmUmGmAmUmA*dT*dT; Antisense chain: (Phos)mU*fA*fUfCmAfUmGfUmCfAmCfUmCfAmGfAmAmAmC*dT*dT;

[0029] (2) Justice Chain: mG*mG*mUmGmGmAfGmUfUmUfCmAfUmAmGmUmAmAmA*dT*dT; Antisense Chain: (Phos)mU*fU*fUfAmCfUmAfUmGfAmAfAmCfUmCfCmAmCmC*dT*dT;

[0030] (3) Justice Chain: mC*mC*mUmGmAmUfUmUfCmUfGmCfAmUmUmGmAmUmA*dT*dT; Antisense Chain: (Phos)mU*fA*fUfCmAfAmUfGmCfAmGfAmAfAmUfCmAmGmG*dT*dT;

[0031] (4) Justice Chain: mG*mA*mUmCmCmUfUmCfAmCfCmAfAmUmGmUmCmAmA*dT*dT; Antisense Chain: (Phos)mU*fU*fGfAmCfAmUfUmGfGmUfGmAfAmGfGmAmUmC*dT*dT;

[0032] (5) Justice chain: mG*mG*mAmUmAmUfGmUfUfCfUmGmUmAmAmAmGmAmU*dT*dT; Antisense chain: mA*fU*mCmUmUfUmAfCfAmGmAmAmCfAmUfAmUmCmC*dT*dT;

[0033] (6) Justice chain: mG*mA*mUmAmUmGfUmUfCfUfGmUmAmAmAmGmAmUmU*dT*dT; Antisense chain: mA*fA*mUmCmUfUmUfAfCmAmGmAmAfCmAfUmAmUmC*dT*dT;

[0034] (7) Justice chain: mU*mC*mUmUmUmUfGmUfUfGfCmUmCmUmUmAmAmAmUmA*dT*dT; Antisense chain: mU*fA*mUmUmUfAmGfAfGmCmAmAmCfAmAfAmAmGmA*dT*dT;

[0035] (8) Justice chain: mU*mG*mCmUmCmUfAmAfAfUfAmCmAmAmUmUmAmAmA*dT*dT; Antisense chain: mU*fU*mUmAmAfUmUfGfUmAmUmUmUfAmGfAmGfAmGmCmA*dT*dT;

[0036] (9) Sense strand: mA*mU*mAmUmGmUfUmCfUfGfUmAmAmAmGmAmUmUmU*dT*dT; Antisense strand: mA*fA*mAmUmCfUmUfUfAmCmAmGmAfAmCfAmUmAmU*dT*dT;

[0037] (10) Justice Chain: mC*mA*mCmUmGmAfCmUfGfAfAmUmAmGmUmUmAmAmA*dT*dT Antisense Chain: mU*fU*mUmAmAfCmUfAfUmUmCmAmGfUmCfAmGmUmG*dT*dT;

[0038] (11) Justice chain: mC*mA*mGmUmGmAfAmAfCfAfGmCmAmGmUmGmUmAmA*dT*dT; Antisense chain: mU*fU*mAmCmAfCmUfGfCmUmGmUmUmUfUmCfAmCmUmG*dT*dT;

[0039] (12) Justice chain: mG*mA*mUmUmUmCfUmGfCfAfUmUmGmAmUmAmUmUmA*dT*dT; Antisense chain: mU*fA*mAmUmAfUmCfAfAmUmGmCmAfGmAfAmAmUmC*dT*dT;

[0040] (13) Justice chain: mG*mA*mAmAmCmAfGmCfAfGfUmGmUmAmAmUmUmAmA*dT*dT; Antisense chain: mU*fU*mAmAmUfUmAfCfAmCmUmGmCfUmGfUmUmUmC*dT*dT.

[0041] (14) Sense strand: mU*mU*mGmGmAmUfAmUfGfUfUmCmUmGmUmAmAmAmG*mA*mU; Antisense strand: mA*fU*mCmUmUfUmAfCfAmGmAmAmCfAmUfAmUmCmC*mA*mA;

[0042] (15) Justice chain: mU*mG*mGmAmUmAfUmGfUfUfCmUmGmUmAmAmAmGmA*mU*mU; Antisense chain: mA*fA*mUmCmUfUmUfAfCmAmGmAmAfCmAfUmAmUmC*mC*mA;

[0043] (16) Sense strand: mA*mA*mUmCmUmUfUmUfGfUfUmGmCmUmCmUmAmAmA*mU*mA; Antisense strand: mU*fA*mUmUmUfAmGfAfGmCmAmAmCfAmAfAmAmGmA*mU*mU;

[0044] (17) Sense strand: mG*mU*mUmGmCmUfCmUfAfAfAmUmAmCmAmAmUmUmA*mA*mA; Antisense strand: mU*fU*mUmAmAfUmUfGfUmAmUmUmUfAmGfAmGmCmA*mA*mC;

[0045] (18) Justice chain: mG*mG*mAmUmAmUfGmUfUfCfUmGmUmAmAmAmGmAmU*mU*mU; Antisense chain: mA*fA*mAmUmCfUmUfUfAmCmAmGmAfAmCfAmUmAmU*mC*mC;

[0046] (19) Sense strand: mG*mA*mCmAmCmUfGmAfCfUfGmAmAmUmAmGmUmUmA*mA*mA; Antisense strand: mU*fU*mUmAmAfCmUfAfUmUmCmAmGfUmCfAmGmUmG*mU*mC;

[0047] (20) Sense strand: mA*mU*mCmAmGmUfGmAfAfAfCmAmGmCmAmGmUmGmU*mA*mA; Antisense strand: mU*fU*mAmCmAfCmUfGfCmUmGmUmUfUmCfAmCmUmG*mA*mU;

[0048] (21) Justice chain: mC*mU*mGmAmUmUfUmCfUfGfCmAmUmUmGmAmUmAmU*mU*mA; Antisense chain: mU*fA*mAmUmAfUmCfAfAmUmGmCmAfGmAfAmAmUmC*mA*mG;

[0049] (22) Justice chain: mG*mU*mGmAmAmAfCmAfGfCfAmGmUmGmUmAmAmUmU*mA*mA; Antisense chain: Antisense chain: mU*fU*mAmAmUfUmAfCfAmCmUmGmCfUmGfUmUmUmC*mA*mC.

[0050] (23) Justice chain: mG*mG*mAmUmAmUfGmUfUfCfUmGmUmAmAmAmGmAmU*dT*dT-Chol; Antisense chain: mA*fU*mCmUmUfUmAfCfAmGmAmAmCfAmUfAmUmCmC*dT*dT;

[0051] In this context, * represents thiophosphate modification, m represents 2′-methoxy modification, f represents 2′-fluoro modification, Phos represents 5′ phosphorylation modification, and Chol represents cholesterol modification.

[0052] Another aspect of the present invention provides a vector system containing the siRNA or modified siRNA, through which the siRNA is delivered to cells in vivo; the vector system is selected from nucleic acid-lipid particles, liposomes, microparticles, viral particles, nucleic acid complexes, or mixtures of one or more. In some cases, the modified siRNA molecule is complexed with lipids such as cationic lipids to form a lipid-nucleic acid complex; in one embodiment, the modified siRNA molecule is complexed with polymers such as cationic polymers to form a polymer-nucleic acid complex; in another embodiment, the modified siRNA molecule may also be complexed with cyclodextrin or a polymer thereof; preferably, the modified siRNA molecule is encapsulated in nucleic acid-lipid particles.

[0053] Another aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of hair loss or for the treatment of prostate cancer, wherein the pharmaceutical composition contains the siRNA or modified siRNA and a pharmaceutically acceptable carrier system and formulation excipients; wherein the carrier system contains any one or a mixture of more than one selected from nucleic acid-lipid particles, liposomes, microparticles, viral particles or nucleic acid complexes; wherein the formulation excipients include, but are not limited to, physiological saline, sterile water, Ringer's solution, buffered saline, dextran solution, maltodextrin solution, glycerol or ethanol; furthermore, by further adding various formulation excipients or carriers, the pharmaceutical composition can be prepared into conventional formulations, such as injectable formulations, lyophilized formulations, tablets, capsules, powders, ointments or syrups, etc., which are all conventional formulation methods in the art.

[0054] The administration method of the pharmaceutical composition of the present invention can be determined according to the patient's symptoms or the severity of the disease. The administration method includes, but is not limited to: oral administration, intramuscular injection, intra-arterial injection, intravenous injection, subcutaneous administration, intracardiac administration, intracavitary administration, etc. The dosage of the pharmaceutical composition of the present invention is determined according to factors such as the severity of the patient's disease, administration time, administration method, age, weight, and gender.

[0055] This invention designs a series of siRNAs targeting the androgen receptor (AR). siRNA drug design was performed on the androgen receptor (AR) gene, and multiple siRNAs composed of sense and antisense RNA strands were screened. The designed siRNA sequences can effectively knock down AR mRNA expression levels. Some sequences can knock down AR mRNA expression levels to approximately 30% in human cells with a significant concentration effect. Further modification of the nucleotide chain through nucleic acid backbone thiolation and base nucleoside modification can effectively improve the stability of the siRNA sequences. The IC50 of the siRNA sequences for AR gene knockdown is [not specified in the original text]. 50 With a potential dose of up to 0.064 nM, it holds promise as a gene therapy drug for treating androgenetic alopecia caused by AR gene overexpression or for treating prostate cancer. Attached Figure Description

[0056] Figure 1 shows the silencing effect of siRNA sequence on AR mRNA.

[0057] Figure 2 shows the IC50 of the siRNA modification sequence. 50 curve.

[0058] Figure 3 shows the silencing effect of siRNA sequences on AR mRNA in MSF cells.

[0059] Figure 4 shows the pharmacodynamic results of siRNA (R-27-E-Chol) in a mouse model of male pattern baldness. Detailed Implementation

[0060] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions to the details and form of the present invention can be made without departing from the spirit and scope of the invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0061] Example 1: Design, screening, and AR gene knockdown experiments using siRNAs and modified siRNAs targeting the androgen receptor.

[0062] 1. siRNA sequence

[0063] Based on the full-length region of the androgen receptor (AR) gene transcript (NG_009014.2), siRNA was designed and screened. TT was added as a prominent base to the 3′ end of the siRNA to increase the stability of the siRNA double-stranded complex. The designed siRNA sequences are shown in Table 1, and the primers used in the experiment are also shown in Table 1.

[0064] Table 1 siRNA sequences

[0065] Table 2 Primer sequences

[0066] 2. Experimental Methods

[0067] 2.1 Cell Culture

[0068] After filtering FBS through a sterile filter, it was added to DMEM medium to prepare DMEM (+,-) medium containing 10% FBS, and stored at 4°C for cell culture. Antibiotics (penicillin and streptomycin) were added to DMEM (+,-) medium to prepare DMEM (+,+) medium containing 1% antibiotics, and stored at 4°C for cell culture.

[0069] HaCaT cells were revived in 25 cm of DMEM (+,+) medium. 2 Cells were proliferated in a gas-permeable cell culture flask placed in a cell culture incubator (constant temperature 37℃, constant CO2 concentration 5%). When the cell density reached approximately 90%, cells were passaged. Cells were washed twice with 1×PBS (2 mL / wash), followed by digestion with 1 mL of trypsin in a cell culture incubator for 2–3 min until complete digestion. Then, 3 mL of DMEM (+,-) was added to terminate the digestion. Cells were pipetted from a flat-bottomed container using a 1 mL pipette, and the cell suspension was transferred to a 15 mL centrifuge tube and centrifuged for 5 min (1000 rpm). The supernatant was discarded, and the cells were resuspended in fresh DMEM (+,-) medium. One-third of the cell resuspended cells was added to a 75 cm⁻¹ centrifuge tube containing 15 mL of DMEM (+,-). 2 Mix well in a breathable cell culture flask and continue proliferation in a cell culture incubator.

[0070] Count the cells using a cell counting chamber, prepared with 1×10⁻⁶ cells. 5 Add 0.5 mL of cell resuspension per well to a 24-well plate and 0.25 mL of cell resuspension per well to a 48-well plate. Mix well using the cross-hatching method and incubate in a cell incubator for 24 hours.

[0071] MSF cells (Mouse Skin Fibroblasts Cells) were revived in 25 cm medium containing DMEM (+,+) 2 Cells were proliferated in a gas-permeable cell culture flask placed in a cell culture incubator (constant temperature 37℃, constant CO2 concentration 5%). When the cell density in the culture flask reached approximately 80-90%, cells were passaged. The cells were washed twice with 1×PBS (2 mL / wash), then 1 mL of trypsin was added and the cells were digested in the cell culture incubator for 1 min until complete digestion. 3 mL of DMEM (+,-) was added to terminate the digestion. Cells were pipetted from a flat-bottomed container using a 1 mL pipette, and the cell suspension was transferred to a 15 mL centrifuge tube and centrifuged for 5 min (1000 rpm). The supernatant was discarded, and the cells were resuspended in fresh DMEM (+,-) medium. One-third of the cell resuspended cells was added to a 75 cm⁻¹ centrifuge tube containing 15 mL of DMEM (+,-). 2 Mix well in a breathable cell culture flask and continue proliferation in a cell culture incubator.

[0072] Count the cells using a cell counting chamber, prepared with 2×10⁻⁶ cells per cell count. 5 Add 0.5 mL of cell resuspension per well to each well of a 24-well plate, mix well using the cross-hatching method, and incubate in a cell incubator for 24 hours.

[0073] 2.2 Cell transfection

[0074] Cell transfection experiments were performed when the cell density in the well plates was approximately 70%, using the commercially available transfection reagent Lipofectamine 2000. TM The specific experimental steps for transfecting nucleic acids are as follows:

[0075] According to the 24-well plate Lipofectamine 2000 TM For each experiment, use 2 μL / well. Take 50 μL and dilute it with 1200 μL of Opti-MEM medium to make 1250 μL of Lipofectamine 2000. TM +Opti-MEM stock solution, mix well with a pipette and incubate at room temperature for 5 min. Prepare a 10 μM stock solution of siRNA with DEPC water, then dilute to Opti-MEM medium according to the transfection concentration, 50 μL per well. Add Lipofectamine 2000 after incubation. TM Add an equal volume (50 μL per well) of Opti-MEM stock solution to the nucleic acid transfection stock solution, pipette and mix 10-20 times, and incubate at room temperature for 15 min.

[0076] Remove the DMEM (+, -) medium from the 24-well plate, wash once with 1×PBS, then add 400 μL of Opti-MEM medium to each well. Add 100 μL of the above transfection stock solution to each well according to the experimental design and gently shake to mix. After completion, place the plate in a cell culture incubator. After 6 hours, remove the Opti-MEM medium and replace it with fresh DMEM (+, -) medium. Evaluate the plate 48 hours after the transfection initiation time.

[0077] 2.3 Real-time quantitative PCR

[0078] Total RNA was extracted according to the TRIZOL total RNA extraction reagent instructions. RNA concentration and absorption peak shape were detected using a NanoDrop 2000. The RNA was extracted according to the full-glucan synthesis method. Following the instructions of the Uni All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) kit, 500 ng of total RNA was used for reverse transcription. The resulting cDNA was diluted in 100 μL of enzyme-free water and then processed according to the instructions. Following the instructions for the Green qPCR SuperMix (+Universal Passive Reference Dye) Real-Time PCR Kit, the qPCR system was prepared and then detected on an ABI Real-Time PCR instrument. The fluorescence signal was SYBR Green. The components and their concentrations in the qPCR system are shown in Table 3. GAPDH was selected as the reference gene. The qPCR reaction conditions were: 1) Initial template denaturation at 95℃ for 2 min; 2) PCR cycles of template denaturation, annealing, and extension (40 cycles) at 95℃ for 5 sec and 60℃ for 30 sec; 3) Cooling to 0℃.

[0079] Table 3. Component content of qPCR system

[0080] 3. Experimental Results

[0081] 3.1 siRNA selection and modification

[0082] Evaluation of siRNA requires appropriate concentration and time. A total of 48 hours was selected as the screening time, and 50 nM was selected as the screening concentration (the final concentration of the AR-27-E cholesterol conjugate AR-27-E-Chol was set to 1 μM, allowing it to enter cells via free uptake). The OLX72021 sequence (Sense chain: 5′-CUU UUG ACC UGC UAA U-3′; Antisense chain: 5′-AUU AGC AGG UCA AAA GUG AAC-3′) was used as a control. The silencing effect of siRNA sequences on AR is shown in Table 4 and Figure 1.

[0083] Table 4. Knockdown effect of siRNA sequences on ARmRNA

[0084] Thirteen sequences, AR-5, AR-6, AR-9, AR-23, AR-27, AR-30, AR-33, AR-34, AR-41, AR-44, AR-50, AR-52, and AR-58, were selected for further modification and evaluation. The specific modification schemes are shown in Table 5 below.

[0085] Table 5 Base modification schemes for ARsiRNA

[0086] Where * represents thiophosphate modification, m represents 2′-methoxy modification, f represents 2′-fluorination modification, Phos represents 5′ phosphorylation modification, and Chol represents cholesterol. #Knockdown results represent free uptake (non-transfection reagent packaged).

[0087] During nucleic acid synthesis, the oxidizing agent in the DNA solid-phase synthesizer is replaced with a thioating agent, converting a non-ester oxygen bond between phosphodiester bonds into sulfur. Commercially available fluorinated or methoxylated phosphoramide monomers can be used to achieve fluorination and methoxylation at the 2′ position of nucleic acid bases. Since excessive methoxylation can affect the effectiveness of siRNA, methoxy and fluorination modifications are used alternately. In practice, siRNA performs 5′ phosphorylation of the antisense strand nucleic acid within the cell. Modified or unmodified phosphate groups at the antisense strand end of siRNA have the same gene silencing effect; certain phosphate analog modifications can enhance the gene silencing effect.

[0088] Additionally, sequences AR-27, AR-30, AR-33, AR-34, AR-41, AR-44, AR-50, AR-52, and AR-58 were selected, and the TT bases that were originally hanging over the 3′ end of these sequences were replaced with the two bases that were originally complementary to the ARmRNA (Table 5). This sequencing method had little impact on the effectiveness of siRNA, and no significant difference in activity was observed compared to the original sequences with TT bases at the 3′ end; both sequences possessed the same ability to target and silence the androgen receptor gene.

[0089] 3.2 IC 50 experiment

[0090] Select a concentration of 10 -8 10 -7 10 -6 The silencing effects of siRNAs at concentrations of 0.00001, 0.00005, 0.0001, 0.001, 0.01, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 7.5, 10, 50, 100, 150, and 200 nM at 48 h were compared with the first six modified siRNAs (AR-5-J, AR-6-J, AR-9-J, AR-23-J, AR-27-E, and AR-34-E) in Table 5 using IC50 analysis. 50 The experiment yielded IC 50 The curve is shown in Figure 2, and the obtained IC 50 The values ​​are shown in Table 6.

[0091] Table 6 IC of modified sequences 50 value

[0092] 3.3 Silencing effect of human-mouse homologous siRNA in MSF cells

[0093] Among all siRNA sequences, AR-26 to AR-42 are human-mouse homologous sequences. AR-27, AR-30, AR-33, AR-34, AR-41 and modified sequences AR-27-E, AR-30-E, AR-33-E, AR-34-E, AR-41-E, AR-27-oriE, AR-30-oriE, AR-33-oriE, AR-34-oriE, and AR-41-oriE were selected, with the OLX72021 sequence used as a control. The silencing effect was evaluated on MSF cells.

[0094] Experimental methods: Cell transfection was performed when the cell density in the well plates was approximately 70%, using the commercially available transfection reagent Lipofectamine 3000. TM Transfection, using Lipofectamine 3000 in a 24-well plate. TMFor experiments, use 1 μL / well. Take 25 μL and add it to 1225 μL of Opti-MEM medium to dilute to 1250 μL of Lipofectamine 3000. TM +Opti-MEM stock solution, mix well with a pipette and incubate at room temperature for 5 min. Prepare a 10 μM stock solution of siRNA with DEPC water, then dilute to a final concentration of 50 nM in Opti-MEM medium, 50 μL per well. Add the incubated Lipofectamine 2000... TM Add an equal volume (50 μL per well) of Opti-MEM stock solution to the nucleic acid transfection stock solution, pipette and mix 10-20 times, then incubate at room temperature for 15 min. Remove the DMEM (+, -) medium from the 24-well plate, wash once with 1×PBS, add 400 μL of Opti-MEM medium to each well, and add 100 μL of the above transfection stock solution to each well according to the experimental design. Gently shake to mix, then place in a cell culture incubator. After 6 h, remove the Opti-MEM medium and replace it with fresh DMEM (+, -) medium. 48 h after the transfection initiation time, remove the medium from the well plate and perform real-time quantitative PCR evaluation according to the experimental method in section 2.3. The final concentration of the AR-27-E cholesterol conjugate (AR-27-E-Chol) was set at 1 μM, allowing it to enter MSF cells (non-transfection reagent packaged) via free uptake.

[0095] Table 7. Silent effect of human-mouse homologous sequences on AR gene in mouse MSF cells.

[0096] The experimental results (Table 7, Figure 3) show that sequences AR-27, AR-30, AR-33, AR-34, AR-41 and modified sequences AR-27-E, AR-30-E, AR-33-E, AR-34-E, AR-41-E, AR-27-oriE, AR-30-oriE, AR-33-oriE, AR-34-oriE, AR-41-oriE and AR-27-E-Chol all have a good silencing effect on the AR gene in MSF cells.

[0097] 3.4 Therapeutic effect of human-mouse homologous siRNA (AR-27-E-Chol) on androgenetic alopecia model mice

[0098] A male C57BL / 6J mouse model of androgenetic alopecia was established by intraperitoneal injection of dihydrotestosterone (1 mg / day / mouse). On the fourth day after dihydrotestosterone injection, hair was removed from the backs of the mice to synchronize the hair cycle. The mice were randomly divided into three groups of six: a normal group (NC) receiving no modeling treatment and intradermal injection of PBS on the backs on days 1, 5, 9, 13, and 17 post-hair removal; a model group (Model) receiving continuous modeling treatment and intradermal injection of PBS on the backs on days 1, 5, 9, 13, and 17 post-hair removal; a low-dose AR-27-E-Chol siRNA group (AR-LD) receiving continuous modeling treatment and intradermal injection of 5 mg / kg AR-27-E-Chol on the backs on days 1, 5, 9, 13, and 17 post-hair removal; and an AR-27-E-Chol siRNA group (AR-LD) receiving continuous modeling treatment and intradermal injection of 25 mg / kg AR-27-E-Chol on the backs on days 1, 5, 9, 13, and 17 post-hair removal. High-dose siRNA group (AR-HD); PC siRNA low-dose group (PC-LD) with continuous modeling and intradermal injection of 5 mg / kg OLX72021 (Sense chain: 5′-mCUmU UmUG mACmC UmGC mUA*mA*U(Chol)-3′; Antisense chain: 5′-mA*fU*mU fAmGfC mAfGmG fUmCfA mAfAmA fGmU*fG*mA*fA*mC-3′, where * represents thiophosphate modification, m represents 2′-methoxy modification, f represents 2′-fluorination modification, and Chol represents cholesterol) on the back on days 1, 5, 9, 13, and 17 after hair removal; continuous modeling and intradermal injection of 25 mg / kg OLX72021 siRNA on the back on the back on days 1, 5, 9, 13, and 17 after hair removal. The mice were divided into three groups: a high-dose siRNA group (PC-HD); a minoxidil group (Minoxidil) receiving continuous modeling and daily intradermal application of minoxidil gel (2.2%) on the back of the mouse on days 1, 5, 9, 13, and 17 after hair removal; and a finasteride group (Finasteride) receiving continuous modeling and daily gavage administration of finasteride corn oil solution (finasteride dose 10 mg / kg) on ​​the back of the mouse on days 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19 after hair removal. Hair growth on the back of the mice was recorded on days 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19 after hair removal.

[0099] The experimental results are shown in Figure 4: During the 7-11 days, the model group showed significant hair growth retardation compared to the normal group, indicating that the androgenic alopecia model was successfully established. At the end of the treatment, the newly grown hair in the high- and low-dose AR siRNA administration groups completely covered the back, similar to the high- and low-dose OLX72021 administration groups and the minoxidil administration groups, and there was no significant difference from normal mice, indicating that the AR siRNA provided by this invention can promote hair growth.

Claims

1. siRNA targeting the androgen receptor, characterized in that, The siRNA contains a sense strand and an antisense strand, and the siRNA is selected from any one of the following (1)-(64): (1) siRNA consisting of a sense strand containing the nucleotide sequence shown in SEQ ID No. 1 and an antisense strand containing the nucleotide sequence shown in SEQ ID No. 2; (2) siRNA consisting of a sense strand containing the nucleotide sequence shown in SEQ ID No. 3 and an antisense strand containing the nucleotide sequence shown in SEQ ID No. 4; (3) siRNA consisting of a sense strand containing the nucleotide sequence shown in SEQ ID No. 5 and an antisense strand containing the nucleotide sequence shown in SEQ ID No. 6; (4) siRNA consisting of a sense strand containing the nucleotide sequence shown in SEQ ID No. 7 and an antisense strand containing the nucleotide sequence shown in SEQ ID No. 8; (5) siRNA consisting of a sense strand containing the nucleotide sequence shown in SEQ ID No. 9 and an antisense strand containing the nucleotide sequence shown in SEQ ID No. 10; (6) siRNA consisting of a sense strand containing the nucleotide sequence shown in SEQ ID No. 11 and an antisense strand containing the nucleotide sequence shown in SEQ ID No. 12; (7) siRNA consisting of a sense strand containing the nucleotide sequence shown in SEQ ID No. 13 and an antisense strand containing the nucleotide sequence shown in SEQ ID No. 14; (8) siRNA consisting of a sense strand containing the nucleotide sequence shown in SEQ ID No. 15 and an antisense strand containing the nucleotide sequence shown in SEQ ID No. 16; (9) siRNA consisting of a sense strand containing the nucleotide sequence shown in SEQ ID No. 17 and an antisense strand containing the nucleotide sequence shown in SEQ ID No. 18; (10) siRNA consisting of a sense strand containing the nucleotide sequence shown in SEQ ID No. 19 and an antisense strand containing the nucleotide sequence shown in SEQ ID No. 20; (11) siRNA consisting of a sense strand containing the nucleotide sequence shown in SEQ ID No. 21 and an antisense strand containing the nucleotide sequence shown in SEQ ID No. 22; (12) siRNA consisting of a sense strand containing the nucleotide sequence shown in SEQ ID No. 23 and an antisense strand containing the nucleotide sequence shown in SEQ ID No. 24; (13) siRNA consisting of a sense strand containing the nucleotide sequence shown in SEQ ID No. 25 and an antisense strand containing the nucleotide sequence shown in SEQ ID No. 26; (14) siRNA consisting of a sense strand containing the nucleotide sequence shown in SEQ ID No. 27 and an antisense strand containing the nucleotide sequence shown in SEQ ID No. 28; (15) siRNA consisting of a sense strand containing the nucleotide sequence shown in SEQ ID No. 29 and an antisense strand containing the nucleotide sequence shown in SEQ ID No. 30; (16) siRNA consisting of a sense strand containing the nucleotide sequence shown in SEQ ID No. 31 and an antisense strand containing the nucleotide sequence shown in SEQ ID No.32 the nucleotide sequence shown in SEQ ID No. 33 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 34; (18) siRNA consisting of the sense strand containing the nucleotide sequence shown in SEQ ID No. 35 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 36; (19) siRNA consisting of the sense strand containing the nucleotide sequence shown in SEQ ID No. 37 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 38; (20) siRNA consisting of the sense strand containing the nucleotide sequence shown in SEQ ID No. 39 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 40; (21) siRNA consisting of the sense strand containing the nucleotide sequence shown in SEQ ID No. 41 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 42; (22) siRNA consisting of the sense strand containing the nucleotide sequence shown in SEQ ID No. 43 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 44; (23) siRNA consisting of the sense strand containing the nucleotide sequence shown in SEQ ID No. 45 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 46; (24) siRNA consisting of the sense strand containing the nucleotide sequence shown in SEQ ID No. 47 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 48; (25) siRNA consisting of the sense strand containing the nucleotide sequence shown in SEQ ID No. 49 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 50; (26) siRNA consisting of the sense strand containing the nucleotide sequence shown in SEQ ID No. 51 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 52; (27) siRNA consisting of the sense strand containing the nucleotide sequence shown in SEQ ID No. 53 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 54; (28) siRNA consisting of the sense strand containing the nucleotide sequence shown in SEQ ID No. 55 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 56; (29) siRNA consisting of the sense strand containing the nucleotide sequence shown in SEQ ID No. 57 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 58; (30) siRNA consisting of the sense strand containing the nucleotide sequence shown in SEQ ID No. 59 and the antisense strand containing the nucleotide sequence shown in SEQ ID No. 60; (31) siRNA consisting of the sense strand containing the nucleotide sequence shown in SEQ ID No. 61 and the antisense strand containing the nucleotide sequence shown in SEQ ID No.62 the antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 64; (33) an siRNA consisting of the sense strand comprising the nucleotide sequence set forth in SEQ ID No. 65 and the antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 66; (34) an siRNA consisting of the sense strand comprising the nucleotide sequence set forth in SEQ ID No. 67 and the antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 68; (35) an siRNA consisting of the sense strand comprising the nucleotide sequence set forth in SEQ ID No. 69 and the antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 70; (36) an siRNA consisting of the sense strand comprising the nucleotide sequence set forth in SEQ ID No. 71 and the antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 72; (37) an siRNA consisting of the sense strand comprising the nucleotide sequence set forth in SEQ ID No. 73 and the antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 74; (38) an siRNA consisting of the sense strand comprising the nucleotide sequence set forth in SEQ ID No. 75 and the antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 76; (39) an siRNA consisting of the sense strand comprising the nucleotide sequence set forth in SEQ ID No. 77 and the antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 78; (40) an siRNA consisting of the sense strand comprising the nucleotide sequence set forth in SEQ ID No. 79 and the antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 80; (41) an siRNA consisting of the sense strand comprising the nucleotide sequence set forth in SEQ ID No. 81 and the antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 82; (42) an siRNA consisting of the sense strand comprising the nucleotide sequence set forth in SEQ ID No. 83 and the antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 84; (43) an siRNA consisting of the sense strand comprising the nucleotide sequence set forth in SEQ ID No. 85 and the antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 86; (44) an siRNA consisting of the sense strand comprising the nucleotide sequence set forth in SEQ ID No. 87 and the antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 88; (45) an siRNA consisting of the sense strand comprising the nucleotide sequence set forth in SEQ ID No. 89 and the antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 90; (46) an siRNA consisting of the sense strand comprising the nucleotide sequence set forth in SEQ ID No. 91 and the antisense strand comprising the nucleotide sequence set forth in SEQ ID No.92 the antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 94; (48) an siRNA consisting of the sense strand comprising the nucleotide sequence set forth in SEQ ID No. 95 and the antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 96; (49) an siRNA consisting of the sense strand comprising the nucleotide sequence set forth in SEQ ID No. 97 and the antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 98; (50) an siRNA consisting of the sense strand comprising the nucleotide sequence set forth in SEQ ID No. 99 and the antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 100; (51) an siRNA consisting of the sense strand comprising the nucleotide sequence set forth in SEQ ID No. 101 and the antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 102; (52) an siRNA consisting of the sense strand comprising the nucleotide sequence set forth in SEQ ID No. 103 and the antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 104; (53) an siRNA consisting of the sense strand comprising the nucleotide sequence set forth in SEQ ID No. 105 and the antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 106; (54) an siRNA consisting of the sense strand comprising the nucleotide sequence set forth in SEQ ID No. 107 and the antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 108; (55) an siRNA consisting of the sense strand comprising the nucleotide sequence set forth in SEQ ID No. 109 and the antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 110; (56) an siRNA consisting of the sense strand comprising the nucleotide sequence set forth in SEQ ID No. 111 and the antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 112; (57) an siRNA consisting of the sense strand comprising the nucleotide sequence set forth in SEQ ID No. 113 and the antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 114; (58) an siRNA consisting of the sense strand comprising the nucleotide sequence set forth in SEQ ID No. 115 and the antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 116; (59) an siRNA consisting of the sense strand comprising the nucleotide sequence set forth in SEQ ID No. 117 and the antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 118; (60) an siRNA consisting of the sense strand comprising the nucleotide sequence set forth in SEQ ID No. 119 and the antisense strand comprising the nucleotide sequence set forth in SEQ ID No. 120; (61) an siRNA consisting of the sense strand comprising the nucleotide sequence set forth in SEQ ID No. 121 and the antisense strand comprising the nucleotide sequence set forth in SEQ ID No.122 the antisense strand comprising the nucleotide sequence shown in SEQ ID No. 124; (63) an siRNA consisting of the sense strand comprising the nucleotide sequence shown in SEQ ID No. 125 and the antisense strand comprising the nucleotide sequence shown in SEQ ID No. 126; (64) an siRNA consisting of the sense strand comprising the nucleotide sequence shown in SEQ ID No. 127 and the antisense strand comprising the nucleotide sequence shown in SEQ ID No.

128.

2. A siRNA, characterized in that, The siRNA has 15 or more consecutive identical nucleotide sequences in succession with the siRNA sequence of claim 1.

3. The siRNA of claim 1, wherein The TT bases at the 3' end of the sense strand or the antisense strand of the siRNA are replaced by two bases that are complementary to the mRNA of the androgen receptor gene.

4. A modified siRNA, characterized in that, The modified siRNA is a modified siRNA obtained by modifying the siRNA of claim 1, 2 or 3 in the backbone, sugar ring, nucleoside or base; preferably, the modification is selected from one or more of the following modification methods: (1) substitution of the 2' carbon position of the sugar ring of the nucleotide, including but not limited to methoxy, methyl ethoxy, methyl, amino, fluorine, -O-2-methylthioethyl, -O-3-aminopropyl or -O-3-dimethylaminopropyl substitution modification; (2) modification of the phosphodiester bond between nucleotides, including but not limited to phosphorothioate, boranophosphonate or methylphosphonate; (3) modification of the nucleosides of the sequence, including but not limited to peptide nucleic acid, locked nucleic acid or unlocked nucleic acid; (4) base modification, including but not limited to base modification or substitution of 5-methylcytosine, 6-methyladenine, ribavirin, pseudouracil and / or inosine of the nucleoside base; (5) phosphorylation, phosphorothioation, vinylphosphonation or other phosphonation analogue modification of the ends of the siRNA sequence; (6) cholesterol modification of the end groups of the siRNA; Preferably, the modified siRNA is a modified siRNA obtained by modification using at least one of the following (1)-(6): (1) converting one non-ester oxygen bond between the phosphodiester of RNA to sulfur; (2) substituting the -OH group at the 2' carbon position of the nucleic acid base with CH3, -OCH3, -OCH2CH2OCH3, -F, NH2, and / or deoxy-modifying the 2'-OH; preferably, the -OH group at the 2' carbon position of the nucleic acid base is alternately modified by substituting with methoxy and fluorine; (3) both the sense strand and the antisense strand of the siRNA have two or more phosphorothioate modifications at the ends; (4) the TT bases at the 3' end of the sense strand or the antisense strand of the siRNA are replaced by two bases that are originally complementary to the AR mRNA; (5) the 5' end of the antisense strand of the RNA sequence is not modified or is modified with a phosphate, phosphorothioate or phosphonic analogue or prodrug of the phosphate; the 5' end of the sense strand is not modified or is modified with an inverted non-base sugar cap; (6) cholesterol modification of the end groups of the siRNA.

5. The modified siRNA of claim 4, wherein The modified siRNA is selected from any one of the siRNAs consisting of the nucleotides of the sense strand and the nucleotides of the antisense strand in the following (1)-(22): (1) sense: mG*mU*mUmUmCmUfGmAfGmUfGmAfCmAmUmGmAmUmA*dT*dT; antisense: (Phos)mU*fA*fUfCmAfUmGfUmCfAmCfUmCfAmGfAmAmAmC*dT*dT; (2) sense: mG*mG*mUmGmGmAfGmUfUmUfCmAfUmAmGmUmAmAmA*dT*dT; antisense: (Phos)mU*fU*fUfAmCfUmAfUmGfAmAfAmCfUmCfCmAmCmC*dT*dT; (3) sense: mC*mC*mUmGmAmUfUmUfCmUfGmCfAmUmUmGmAmUmA*dT*dT; antisense: (Phos)mU*fA*fUfCmAfAmUfGmCfAmGfAmAfAmUfCmAmGmG*dT*dT; (4) sense: mG*mA*mUmCmCmUfUmCfAmCfCmAfAmUmGmUmCmAmA*dT*dT; antisense: (Phos)mU*fU*fGfAmCfAmUfUmGfGmUfGmAfAmGfGmAmUmC*dT*dT; (5) sense: mG*mG*mAmUmAmUfGmUfUfCfUmGmUmAmAmAmGmAmU*dT*dT; antisense: mA*fU*mCmUmUfUmAfCfAmGmAmAmCfAmUfAmUmCmC*dT*dT; (6) sense: mG*mA*mUmAmUmGfUmUfCfUfGmUmAmAmAmGmAmUmU*dT*dT; antisense: mA*fA*mUmCmUfUmUfAfCmAmGmAmAfCmAfUmAmUmC*dT*dT; (7) sense: mU*mC*mUmUmUmUfGmUfUfGfCmUmCmUmAmAmAmUmA*dT*dT; antisense: mU*fA*mUmUmUfAmGfAfGmCmAmAmCfAmAfAmAmGmA*dT*dT; (8) sense: mU*mG*mCmUmCmUfAmAfAfUfAmCmAmAmUmUmAmAmA*dT*dT; antisense: mU*fU*mUmAmAfUmUfGfUmAmUmUmUfAmGfAmGmCmA*dT*dT; (9) sense: mA*mU*mAmUmGmUfUmCfUfGfUmAmAmAmGmAmUmUmU*dT*dT; antisense: mA*fA*mAmUmCfUmUfUfAmCmAmGmAfAmCfAmUmAmU*dT*dT; (10) sense: mC*mA*mCmUmGmAfCmUfGfAfAmUmAmGmUmUmAmAmA*dT*dT anti-sense: mU*fU*mUmAmAfCmUfAfUmUmCmAmGfUmCfAmGmUmG*dT*dT; (11) sense: mC*mA*mGmUmGmAfAmAfCfAfGmCmAmGmUmGmUmAmA*dT*dT; anti-sense: mU*fU*mAmCmAfCmUfGfCmUmGmUmUfUmCfAmCmUmG*dT*dT; (12) sense: mG*mA*mUmUmUmCfUmGfCfAfUmUmGmAmUmAmUmUmA*dT*dT; anti-sense: mU*fA*mAmUmAfUmCfAfAmUmGmCmAfGmAfAmAmUmC*dT*dT; (13) sense: mG*mA*mAmAmCmAfGmCfAfGfUmGmUmAmAmUmUmAmA*dT*dT; anti-sense: mU*fU*mAmAmUfUmAfCfAmCmUmGmCfUmGfUmUmUmC*dT*dT; (14) sense mU*mU*mGmGmAmUfAmUfGfUfUmCmUmGmUmAmAmAmG*mA*mU; anti-sense: mA*fU*mCmUmUfUmAfCfAmGmAmAmCfAmUfAmUmCmC*mA*mA; (15) sense mU*mG*mGmAmUmAfUmGfUfUfCmUmGmUmAmAmAmGmA*mU*mU; anti-sense: mA*fA*mUmCmUfUmUfAfCmAmGmAmAfCmAfUmAmUmC*cC*mA; (16) sense: mA*mA*mUmCmUmUfUmUfGfUfUmGmCmUmCmUmAmAmA*mU*mA; anti-sense: mU*fA*mUmUmUfAmGfAfGmCmAmAmCfAmAfAmAmGmA*mU*mU; (17) sense: mG*mU*mUmGmCmUfCmUfAfAfAmUmAmCmAmAmUmUmA*mA*mA; anti-sense: mU*fU*mUmAmAfUmUfGfUmAmUmUmUfAmGfAmGmCmA*mA*mC; (18) sense: mG*mG*mAmUmAmUfGmUfUfCfUmGmUmAmAmAmGmAmU*mU*mU; anti-sense: mA*fA*mAmUmCfUmUfUfAmCmAmGmAfAmCfAmUmAmU*mC*mC; (19) sense: mG*mA*mCmAmCmUfGmAfCfUfGmAmAmUmAmGmUmUmA*mA*mA; antisense: mU*fU*mUmAmAfCmUfAfUmUmCmAmGfUmCfAmGmUmG*mU*mC; (20) sense: mA*mU*mCmAmGmUfGmAfAfAfCmAmGmCmAmGmUmGmU*mA*mA; antisense: mU*fU*mAmCmAfCmUfGfCmUmGmUmUfUmCfAmCmUmG*mA*mU; (21) sense: mC*mU*mGmAmUmUfUmCfUfGfCmAmUmUmGmAmUmAmU*mU*mA; antisense: mU*fA*mAmUmAfUmCfAfAmUmGmCmAfGmAfAmAmUmC*mA*mG; (22) sense: mG*mU*mGmAmAmAfCmAfGfCfAmGmUmGmUmAmAmUmU*mA*mA; antisense: mU*fU*mAmAmUfUmAfCfAmCmUmGmCfUmGfUmUmUmC*mA*mC; (23) sense: mG*mG*mAmUmAmUfGmUfUfCfUmGmUmAmAmAmGmAmU*dT*dT-Chol; antisense: mA*fU*mCmUmUfUmAfCfAmGmAmAmCfAmUfAmUmCmC*dT*dT; wherein * represents phosphorothioate modification, m represents 2'-methoxy modification, f represents 2'-fluoro modification, Phos represents 5' phosphorylation modification, and Chol represents cholesteryl modification.

6. A siRNA conjugate, characterized by, The siRNA of claim 1, 2 or 3 is conjugated with a conjugating group, or the modified siRNA of claim 4 is conjugated to obtain a siRNA conjugate; wherein the conjugating group is a cholesteryl group, a cell penetrating peptide or an alkyl chain group.

7. Use of the siRNA of any one of claims 1-3, the modified siRNA of claim 4 or 5, or the siRNA conjugate of claim 6 in the preparation of a gene drug for preventing or treating alopecia or treating prostate cancer.

8. A carrier system comprising the siRNA of any one of claims 1-3, or a carrier system comprising the modified siRNA of claim 4 or 5, or a carrier system comprising the siRNA conjugate of claim 6; preferably, the carrier system is selected from any one of a nucleic acid-lipid particle, a liposome, a micelle, a viral particle, a nucleic acid complex, or a mixture of one or more thereof.

9. Use of the carrier system of claim 8 in the preparation of a gene drug for preventing or treating alopecia or treating prostate cancer.

10. A pharmaceutical composition for preventing or treating alopecia or treating prostate cancer, characterized by, The pharmaceutical composition contains a therapeutically effective amount of the siRNA according to any one of claims 1 to 3, the modified siRNA according to claim 4 or 5 or the siRNA conjugate according to claim 6 and a pharmaceutically acceptable carrier system and formulation adjuvants.

Images