JP4961549B2 - SiRNA specific for androgen receptor gene - Google Patents

Description

  The present invention relates to siRNA specific for the androgen receptor (AR) gene.

  Prostate cancer is the most common cancer affecting men in the West. In Japan, the number of patients with prostate cancer is increasing year by year with the westernization of eating habits and the aging of the population. In general, the growth of prostate cancer cells is stimulated by androgens. Therefore, hormone therapy that inhibits androgen production and function is often used in the treatment of unresectable advanced prostate cancer. The response rate is extremely high. However, it relapses as androgen-independent prostate cancer within a few years. Therefore, control of androgen-independent cancer is the most important issue in prostate cancer treatment.

  Although the detailed molecular mechanism of progression from androgen-dependent prostate cancer to independent cancer is not clear, the involvement of androgen receptor (AR) has been suggested. That is, it has been clarified that androgen-independent cancers are sensitive to ultra-low concentrations of androgens, antiandrogens, other steroid hormones, etc. due to AR mutation or amplification (Patent Document 1, Non-Patent Documents). 1-5). In addition, AR activation by several types of growth factors has been reported (see Non-Patent Document 6). Then, the treatment of prostate cancer by AR antagonist etc. is tried (refer patent document 2).

  RNA interference (RNAi) technology is frequently used in life science research, and its usefulness has been widely confirmed. RNAi refers to a phenomenon in which mRNA is degraded in a sequence-specific manner by double-stranded RNA, resulting in suppression of gene expression. Since it was reported in 2001 that a 21-base small double-stranded RNA can mediate RNAi in mammalian cells (see Non-Patent Document 7), siRNA (small interface RNA) is used as a method for suppressing the expression of a target gene. It is frequently used. Moreover, the sequence selection criteria of siRNA for obtaining a higher RNAi effect have also been reported (see Non-Patent Document 8). The RNAi technology is expected to be applied to pharmaceuticals and various refractory diseases including cancer.

  However, when siRNA is applied clinically, there is a problem of interferon response. That is, when a high concentration of synthetic siRNA is introduced into a cell or siRNA is expressed at a high concentration in a cell, an interferon response is induced and nonspecific expression inhibition or nonspecific cell growth inhibition occurs. At present, it is known that an interferon response is induced when 10 nM or more of a synthetic siRNA is introduced into a cell.

  Furthermore, when siRNA is applied clinically, there is a problem of off-target effect. That is, siRNA is an excellent technique capable of suppressing the expression of a target gene, but it has been clarified that expression of a gene having a similar target sequence other than the target gene may also be suppressed (non-patent literature). 9).

So far, no siRNA that is specific to the AR gene and that can avoid the above-mentioned problems such as interferon response and off-target effect has been reported.
JP 2004-57180 A JP 2003-252854 JP Taplin ME et al., `` Mutation of the androgen-receptor gene in metastatic androgen-independent prostate cancer. '' N Engl J Med 332: 1393-1398, 1995. Koivisto P et al., `` Androgen receptor gene amplification: a possible molecular mechanism for androgen deprivation therapy failure in prostate cancer. '' Cancer Res 57: 314-319, 1997. Gregory CW et al., `` Androgen receptor stabilization in recurrent prostate cancer is associated with hypersensitivity to low androgen. '' Cancer Res 61: 2892-2898, 2001. Zhao XY et al., `` Glucocorticoids can promote androgen-independent growth of prostate cancer cells through a mutated androgen receptor. '' Nat Med 6: 703-706, 2000. Tan J et al., `` Dehydroepiandrosterone activates mutant androgen receptors expressed in the androgen-dependent human prostate cancer xenograft CWR22 and LNCaP cells. '' Mol Endocrinol 11: 450-459, 1997. Culig Z et al., `` Androgen receptor activation in prostatic tumor cell lines by insulin-like growth factor-I, keratinocyte growth factor, and epidermal growth factor. '' Cancer Res 54: 5474-5478, 1994. Elbashir SM et al., `` Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. '' Nature 411 (6836): 494-498, 2001. Reynolds A et al., “Rational siRNA design for RNA interference.” Nat Biotechnol 22 (3): 326-330, 2004. Jackson AL et al., “Expression profiling reveals off-target gene regulation by RNAi.” Nat Biotechnol 21 (6): 635-637, 2003.

  Therefore, an object of the present invention is to provide a siRNA that can be applied clinically and targets the androgen receptor (AR) gene.

  In order to achieve the above object, the siRNA of the present invention is a double-stranded siRNA targeting the AR gene, which is a 21-base double-stranded siRNA comprising 19 base pairs and a 2-base 3′-end overhang. Or a blunt-end double-stranded siRNA consisting of 27 base pairs, wherein the 19 base pair sequence is any one of SEQ ID NOs: 1 to 3 in the sequence listing, and the 27 base pair sequence is represented by the sequence listing The double-stranded siRNA which is SEQ ID NO: 4 or 5. The double-stranded siRNAs of the present invention all share a common activity of mediating RNAi specific to the AR gene, and an important structural element essential for this common activity, that is, RNAi. Shares common important structural elements corresponding to the AR gene to mediate.

  The present inventors focused on the role of AR in the progression of prostate cancer from androgen-dependent to independent, and conducted extensive research on siRNA specific to the AR gene. As a result, the present inventors have found a double-stranded siRNA that exhibits a sufficient RNAi effect even at a use concentration of 1 nM and does not show homology to other genes, and has reached the present invention.

  According to the double-stranded siRNA of the present invention, for example, the RNAi effect can be sufficiently exerted even at a low concentration of 1 nM, so that it can be used while avoiding the interferon response. Moreover, since the double-stranded siRNA of the present invention is a sequence selected using a sequence that does not show homology to other genes as a target sequence, it is specific for the AR gene and can be used avoiding the off-target effect. Therefore, the double-stranded siRNA of the present invention can be applied clinically, and can be used, for example, for cancer treatment, pharmaceutical compositions for cancer treatment, AR-specific inhibitors, and the like. Among them, since the AR gene in which the double-stranded siRNA of the present invention exerts an RNAi effect can be a therapeutic target molecule in prostate cancer, the double-stranded siRNA of the present invention is preferably used for prostate cancer treatment or prostate cancer. It can utilize for the pharmaceutical composition of this.

  In the 21-base overhanging double-stranded siRNA of the present invention, the 2 base sequence of the 3 'terminal overhang is preferably TT (thymine / thymine).

  In another embodiment, the double-stranded siRNA of the present invention has one or several bases modified or substituted, added or deleted in the sequence of SEQ ID NOs: 1 to 5 in the sequence listing, and an AR gene. It is a double-stranded siRNA that induces specific RNA interference.

  The AR-specific inhibitor of the present invention is an AR-specific inhibitor that specifically inhibits the expression of the AR gene by RNA interference (RNAi), and includes the double-stranded siRNA of the present invention.

  The pharmaceutical composition of the present invention is a pharmaceutical composition for cancer treatment, which comprises the double-stranded siRNA of the present invention. The pharmaceutical composition of the present invention preferably further contains atelocollagen. In the pharmaceutical composition of the present invention, the cancer to be used for therapeutic use is not particularly limited, but is preferably prostate cancer, for example.

  Next, the double-stranded siRNA of the present invention will be described in detail.

  First, in the present invention, the “androgen receptor (AR) gene” is a gene known to be involved in prostate cancer as described above, and is a gene encoding an androgen receptor (receptor). . Androgen is a general term for steroid hormones having an androgenic action, and includes testosterone synthesized in the testis, dehydroepiandrosterone synthesized in the adrenal cortex, androstenedione, and the like.

  The AR gene that is the target of the double-stranded siRNA of the present invention is preferably a human AR gene. The mRNA sequence can be obtained from, for example, the registration number NM_000044 in the Nucleotide database of the National Center for Biotechnology Information (NCBI). The AR gene that is the target of the double-stranded siRNA of the present invention includes single nucleotide polymorphism (SNP), other polymorphisms, and mutants. The target sequence of the double-stranded siRNA of the present invention includes known SNP sites, AR polymorphisms registered in the McGill AR gene mutation database (https://www.androgendb.mcgill.ca/), and Since the mutation site is not included, the RNAi effect can be exhibited even for these AR gene polymorphisms.

  In the present invention, “siRNA” is a short RNA molecule capable of mediating RNAi, and generally refers to a double-stranded low-molecular RNA of 21 to 27 bases.

  The double-stranded siRNA of the present invention has two embodiments, and the first embodiment is three types of 21-base double-stranded siRNA comprising 19 base pairs and a 2-base 3 ′ end overhang. Two embodiments are two types of blunt-ended double-stranded siRNAs consisting of 27 base pairs. The 19 base pair and 27 base pair sequences are shown in Table 1 below. In the table, the left column is the code name of each siRNA, and the right column is SeqID, that is, the sequence number of the sequence listing.

本発明の２１塩基オーバーハング二本鎖ｓｉＲＮＡにおいて、前記３’末端のオーバーハングとは、５’末端から１９ｍｅｒが相補的な配列である２本の２１ｍｅｒのＲＮＡ鎖が対合して二本鎖を形成したとき１９塩基対の両端から突出する３’末端の２ｍｅｒの部分をいう。前記３’末端オーバーハングの２塩基の配列は、例えば、両端とも、ＴＴ（チミン・チミン）が好ましい。なお、前記３’オーバーハングの２塩基の配列は、これに制限されず、ＲＮＡｉ効果や細胞増殖抑制効果に実質的に影響を及ぼさない範囲であれば、任意の天然核酸塩基（アデニン、グアニン、チミン、シトシン、ウラシル）、並びに、天然及び人工の公知の修飾塩基であってもよい。また、前記３’末端オーバーハング部分のヌクレオチドは、通常、リボヌクレオチドを使用できるがこれに制限されず、ＲＮＡｉ効果や細胞増殖抑制効果に実質的に影響を及ぼさない範囲であれば、デオキシリボヌクレオチド、修飾リボヌクレオチド、その他の公知のヌクレオチド類似体を使用してもよい。さらに、本発明の２１塩基オーバーハング二本鎖ｓｉＲＮＡは、必要に応じて、前記３’末端オーバーハングに代えて、５’末端オーバーハングとしてもよい。

In the 21-base overhang double-stranded siRNA of the present invention, the 3 ′ end overhang is a double strand obtained by pairing two 21-mer RNA strands having a 19-mer complementary sequence from the 5 ′ end. The 2 mer portion at the 3 ′ end protruding from both ends of 19 base pairs. For example, TT (thymine / thymine) is preferable for both ends of the 3′-terminal overhang sequence. The 2 base sequence of the 3 ′ overhang is not limited to this, and any natural nucleobase (adenine, guanine, guanine, etc.) is not limited as long as it does not substantially affect the RNAi effect and the cell growth inhibitory effect. Thymine, cytosine, uracil ), and natural and artificial known modified bases. In addition, as the nucleotide of the 3 ′ terminal overhang portion, ribonucleotide can be used normally, but is not limited thereto, and deoxyribonucleotide, as long as it does not substantially affect RNAi effect and cell growth inhibitory effect, Modified ribonucleotides and other known nucleotide analogs may be used. Furthermore, the 21-base overhanging double-stranded siRNA of the present invention may have a 5 ′ end overhang instead of the 3 ′ end overhang, if necessary.

  As shown in Table 1 above, three types of AR1, AR24, and AR61 (SEQ ID NOS: 1 to 3, respectively) can be used as the 19 base pair sequences of the 21-base overhanging double-stranded siRNA of the present invention. Is preferably AR1.

  The 27-base blunt-end double-stranded siRNA of the present invention is a double strand formed by pairing two 27-mer complementary RNA strands, so that the ends are smooth. As shown in Table 1 above, AR1169 and AR4257 (SEQ ID NOS: 4 and 5, respectively) can be used as the 27 base pair sequence of the 27-base blunt-end double-stranded siRNA of the present invention. AR4257.

  In the sequence of the base pair portion of the double-stranded siRNA of the present invention (SEQ ID NO: 1 to 5 in the sequence listing), one or several bases are modified or substituted within a range in which RNAi specific RNAi can be induced, It may be added or deleted. For example, the number is 2, 3, or 4.

  The method for producing the double-stranded siRNA of the present invention may be synthesized in vitro chemically or enzymatically, or synthesized in vivo, and the production method is not particularly limited. It is preferable to manufacture by chemical synthesis. Synthetic double-stranded siRNA facilitates concentration control and facilitates avoidance of interferon responses in clinical applications. Further, it is easy to prevent contamination and there is an advantage in safety. For example, when producing AR1 of the 21-base overhang double-stranded siRNA of the present invention, first, a 21-mer RNA strand in which a 2-base overhang is added to the 3 ′ end of the sequence of SEQ ID NO: 1 in the sequence listing, and A 21-mer RNA strand in which a 2-base overhang is added to the 3 ′ end of the complementary sequence of the sequence of SEQ ID NO: 1 is chemically synthesized. Next, the two RNA strands are paired under the pairing conditions to obtain the 21-base overhanging double-stranded siRNA of the present invention. In use, it is preferable to appropriately purify by a conventionally known method, if necessary.

  Therefore, the present invention provides, in another aspect, a method for producing a double-stranded siRNA, wherein the double-stranded siRNA is the double-stranded siRNA of the present invention, and any one of SEQ ID NOs: 1 to 3 in the sequence listing A synthesis step of synthesizing two RNA strands obtained by adding an overhang of 2 bases to each of the sequence and its complementary sequence, or the sequence of SEQ ID NO: 4 or 5 in the sequence listing and the two RNA strands of its complementary sequence; And a pairing step of pairing the synthesized two RNA strands into a double-stranded RNA.

  Next, a method for using the double-stranded siRNA of the present invention will be described. The double-stranded siRNA of the present invention can be used for suppression of AR gene expression by the RNAi effect. The double-stranded siRNA of the present invention exerts an RNAi effect even with one kind. Moreover, in order to obtain a higher RNAi effect, a plurality of types of the double-stranded siRNA of the present invention may be mixed and used. Examples of the mixture include two types and three types selected from the group consisting of the 21-base overhanging siRNA of AR1, AR24 and AR61 and the 27-base blunt-end double-stranded siRNA of AR1169 and AR4257. The mixture containing 4 types and 5 types is mentioned, The mixture containing said AR4257 and said AR1 is mentioned.

  In addition to being able to be used for cells and tissues in vitro, the double-stranded siRNA of the present invention can be clinically applied to humans. The administration method to human is not particularly limited, and a conventionally known delivery system can be appropriately used. For example, a local administration method and a systemic administration method using atelocollagen are preferable. The atelocollagen has already been clinically applied as, for example, a local hemostatic agent, and the local administration method of siRNA using the atelocollagen is significant in animal experiments using vascular endothelial growth factor (VEGF) as a molecular target. (Takei Y et al., Cancer Res 64 (10): 3365-3370, 2004).

  In addition, as another delivery system, chemical modification for preventing degradation in vivo and enhancing intracellular permeability (Rossi JJ., Nature 432 (7014): 155-156, 2004; Southchek J et al., Nature 432 ( 7014): 173-178, 2004) and delivery systems using cationic liposomes (Yano J et al., Clin Cancer Res 10 (22): 7721-7726, 2004). Furthermore, if necessary, an siRNA expression vector that expresses the double-stranded siRNA of the present invention can be constructed, and a delivery system using gene therapy technology can be used.

  The double-stranded siRNA of the present invention is particularly preferably used for a pharmaceutical composition used for the treatment of prostate cancer or for the treatment of prostate cancer. That is, cell growth of prostate cancer cells can be suppressed by inhibiting the expression of the AR gene by the RNAi effect using the double-stranded siRNA of the present invention. The double-stranded siRNA of the present invention can treat both androgen-dependent and independent prostate cancer.

  The double-stranded siRNA of the present invention is not limited to prostate cancer, and includes, for example, breast cancer, salivary gland cancer, squamous cell carcinoma of the head and neck, kidney cancer, bladder cancer, gastric cancer, colon cancer, testicular cancer, lung cancer involving AR, It can be used for general cancer treatments such as liver cancer and pancreatic cancer, and pharmaceutical compositions generally used for cancer treatment.

  As described above, there have conventionally been two major problems in clinical application of siRNA. That is, the problem of inducing an interferon response and the problem of off-target effects. In order to avoid the interferon response, it is necessary that the concentration of intracellular introduction is at least less than 10 nM. However, the double-stranded siRNA of the present invention exhibits a sufficient RNAi effect on the AR gene at an ultra-low concentration of 1 nM. can do. In addition, with regard to the off-target effect, the target sequence of the double-stranded siRNA of the present invention has a possibility that a gene having a mismatch of 3 bases or less does not exist by using BLAST search, or a cross reaction is possible. The off-target effect can be avoided by selecting the gene with the smallest number of genes. Thus, the double-stranded siRNA of the present invention has cleared two major barriers in clinical application and can be clinically applied. In clinical applications, the double-stranded siRNA of the present invention is preferably a synthetic double-stranded siRNA.

  Therefore, the present invention includes, as another aspect, an AR-specific inhibitor that specifically inhibits the expression of the AR gene by RNA interference, and includes an AR-specific inhibitor that includes the double-stranded siRNA of the present invention. The double-stranded siRNA of the present invention to be contained may be one kind or plural kinds. If there are multiple types, a higher inhibitory effect can be obtained. Examples of the plurality of types include two types, three types, four types, and five types selected from the group consisting of AR1, AR24, AR61, AR1169, and AR4257. The AR-specific inhibitor of the present invention can be used in vitro and in vivo for the purpose of specifically knocking down expression of an AR gene in a cell. The dosage form is not particularly limited.

  Furthermore, as another aspect, the present invention includes a method for treating cancer, preferably prostate cancer, which comprises using the double-stranded siRNA of the present invention. In yet another aspect, the present invention provides use of double-stranded siRNA in the treatment of cancer, preferably prostate cancer, wherein the double-stranded siRNA is the double-stranded siRNA of the present invention. Including. The double-stranded siRNA of the invention in these uses may be one kind or plural kinds. With multiple types, higher therapeutic effects can be obtained. Examples of the plurality of types include two types, three types, four types, and five types selected from the group consisting of AR1, AR24, AR61, AR1169, and AR4257.

  Next, the pharmaceutical composition of the present invention will be described. The pharmaceutical composition of the present invention is a pharmaceutical composition for treating cancer and comprises the double-stranded siRNA of the present invention. The double-stranded siRNA of the present invention to be contained may be one kind or plural kinds. With multiple types, higher therapeutic effects can be obtained. Examples of the plurality of types include two types, three types, four types, and five types selected from the group consisting of AR1, AR24, AR61, AR1169, and AR4257. The pharmaceutical composition of the present invention preferably further contains atelocollagen. As described above, the cancer targeted for therapeutic use of the pharmaceutical composition of the present invention is not particularly limited, but is preferably prostate cancer.

  The pharmaceutical composition of the present invention may further contain a pharmaceutically acceptable carrier. The pharmaceutical carrier is not particularly limited, and examples thereof include carriers that can increase the efficiency with which the double-stranded siRNA of the present invention enters the target site, tissue, cell, etc., such as liposomes and cationic liposomes. Can be mentioned. The dosage form of the pharmaceutical composition of the present invention is not particularly limited, and examples thereof include injections, creams, ointments, tablets, suspensions and the like. Also, the administration method is not particularly limited, and examples thereof include injection, oral, topical, intranasal, and rectal administration.

  Hereinafter, the present invention will be described using examples.

  Three types of 21-base overhanging double-stranded siRNAs (AR1, AR24 and AR61; respectively, SEQ ID NOS: 1 to 3) and two types of 27-base blunt-end double-stranded siRNAs (AR1169 and AR4257) in Table 1 above. Each prepared by synthesizing 5 types of double-stranded siRNA of SEQ ID NO: 4 or 5) in the sequence listing, and using each synthetic double-stranded siRNA at a concentration of 1 nM, and the RNAi effect on the AR gene; The cell growth inhibitory effect was confirmed. Specifically, it was performed as follows.

Preparation of synthetic double-stranded siRNA The above-mentioned three types of 21-base overhanging double-stranded siRNA (AR1, AR24 and AR61) are the 19-base sequences of SEQ ID NOs: 1 to 3 in the sequence listing and their 3′-ends. An RNA strand having a two-base overhang portion consisting of a TT sequence added thereto was chemically synthesized by a conventional method and prepared by pairing them together. The two types of 27-base blunt-end double-stranded siRNAs (AR1169 and AR4257) are obtained by chemically synthesizing the sequences of SEQ ID NO: 4 or 5 in the sequence listing and RNA strands of their complementary sequences by a conventional method, and combining them. Prepared. When the following RNAi effect and cell growth inhibitory effect were confirmed, those purified by HPLC or the like were used as appropriate.

Cell and culture method For confirmation of RNAi effect, human prostate cancer cell line LNCaP cells were used. RPMI1640 (Sigma-Aldrich) containing 10% fetal bovine serum (FBS; manufactured by Biosource International), 100 μg / ml streptomycin, 100 U / ml penicillin, 0.25 mg / ml amphotericin B (produced by Invitrogen) is used for the culture of the cells. Was used as a growth medium, and was carried out at 37 ° C. in an incubator containing carbon dioxide gas at a rate of 5% in the air.

Intracellular introduction of synthetic double-stranded siRNA In a 60 mm plastic petri dish (trade name: Falcon, manufactured by BD Biosciences), 8 × 10 ⁵ LNCaP cells were implanted, and cultured for 24 hours, then Opti-MEM (manufactured by Invitrogen). After washing twice, Lipofectamine 2000 (manufactured by Invitrogen) solution containing 1 nM synthetic double-stranded siRNA was added. After 5 hours, the medium was changed to DMEM containing 10% FBS.

Western blotting synthetic double-stranded siRNA was introduced as described above and cultured for 48 hours, and then cells were solubilized using CelLytic M Cell Lys Reagent (manufactured by Sigma-Aldrich). The solubilized sample is developed by SDS-PAGE (sodium dodecyl sulfate polyacrylamide electrophoresis), and PDVF (polyvinylidene difueide) membrane (manufactured by Millipore) is used for 2 hours using Mini-PROTEAN II (manufactured by Bio-Rad). Transcribed to. After the transfer, blocking was performed overnight at 4 ° C. with T-TBS (25 mM Tris-HCl, 125 mM NaCl, 0.1% Tween 20; Sigma-Aldrich) containing 5% skim milk (Wako Pure Chemical Industries). Further, the primary antibody was diluted with a blocking solution and reacted at room temperature for 1 hour. After washing with the T-TBS solution three times, the secondary antibody was similarly reacted for 1 hour. Color was developed with an ECLplus kit (Amersham Biosciences), and then digitalized using LAS3000 (Fuji Film).

Cell Proliferation Evaluation Method 5 × 10 ⁴ cells in which the synthetic double-stranded siRNA has been introduced into the LNCaP cells are implanted in a 6-well microplate (trademark: Falcon, manufactured by BD Biosciences), and cultured for 4 days. Cells were collected with 05% trypsin-0.53 mM EDTA (manufactured by Invitrogen), and the number of cells was counted using a Z1 Coulter Counter (manufactured by Beckman Coulter).

As a result of the RNAi effect The five kinds of synthetic double-stranded siRNAs were introduced into the cultured human prostate cancer cell line LNCaP, and the RNAi effect was evaluated by the expression level of the AR protein (Western blot) as described above. As a result, it was confirmed that all the five synthetic double-stranded siRNAs significantly suppressed the expression of the target AR gene at a concentration of 1 nM. An example of the result is shown in FIG. In the figure, the leftmost lane is a control when a synthetic double-stranded siRNA (21 base overhang) for the GFP gene is introduced. As shown in the figure, among the five types, four types of synthetic double-stranded siRNAs AR1, AR61, AR1169, and AR4257 showed a remarkable RNAi effect of 80% or more.

As a result of the cell growth inhibitory effect The five types of synthetic double-stranded siRNAs were introduced into the cultured human prostate cancer cell line LNCaP, and the cell growth inhibitory effect was evaluated as described above. As a result, it was confirmed that all the five synthetic double-stranded siRNAs significantly suppressed the proliferation of the prostate cancer cells at a concentration of 1 nM. An example of the result is shown in FIG. In the figure, GFP19 is a control when a 21-base overhang siRNA targeting the GFP gene is introduced, and GFP27 is a control when a 27-base blunt-end siRNA targeting the GFP gene is introduced. In the same figure, the vertical axis represents the number of cells. As shown in the figure, among the five types, the synthetic double-stranded siRNA of AR4257 showed a remarkable prostate cancer cell growth inhibitory effect of 70% or more.

As described above, the five kinds of synthetic double-stranded siRNA, even ultra-low concentration of 1 nM, significant AR gene shows RNAi effect that target, and shows the inhibitory effect of prostate cancer cells. Among them, AR4257 showed particularly excellent RNAi effect and cell growth inhibitory effect.

  As described above, the double-stranded siRNA of the present invention can mediate AR gene-specific RNAi while avoiding the off-target effect and interferon response, so that, for example, clinical application is possible, and AR is used as a target molecule. The present invention is useful in the fields of medical treatment and pharmaceutical composition, for example, treatment related to cancer treatment including prostate cancer and pharmaceutical composition. In addition, the double-stranded siRNA of the present invention is useful, for example, in academic and basic medical research and development fields.

FIG. 1 is an example of the results of confirming the RNAi effect targeting the AR gene of the double-stranded siRNA of the present invention by Western blotting. FIG. 2 is an example of the results of confirming the cell growth inhibitory effect of the double-stranded siRNA of the present invention.

SEQ ID NO: 1 Overhang siRNA AR1
SEQ ID NO: 2 Overhang siRNA AR24
SEQ ID NO: 3 Overhang siRNA AR61
SEQ ID NO: 4 Blunt End siRNA AR1169
SEQ ID NO: 5 Blunt End siRNA AR4257

Claims (6)

A double-stranded siRNA targeting the androgen receptor (AR) gene,
A 21-base double-stranded siRNA consisting of 19 base pairs and a 2-base 3 ′ end overhang, or a blunt-end double-stranded siRNA consisting of 27 base pairs,
The 19 base pair sequence is any one of SEQ ID NOs: 1 to 3 in the sequence listing;
The double-stranded siRNA wherein the 27 base pair sequence is SEQ ID NO: 4 or 5 in the sequence listing.   The double-stranded siRNA according to claim 1, wherein the two-base sequence of the 3 'terminal overhang is TT. An AR-specific inhibitor comprising the double-stranded siRNA according to claim 1 or 2 and specifically inhibiting the expression of an AR gene by RNA interference. A pharmaceutical composition for cancer treatment, comprising the double-stranded siRNA according to claim 1 or 2 . The pharmaceutical composition according to claim 4 , further comprising atelocollagen. The pharmaceutical composition according to claim 4 or 5 , wherein the cancer is prostate cancer.

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