siRNA TARGETING WRN HELICASE GENE

Abstract

The present invention addresses the problem of providing siRNA that targets a WRN helicase gene, is based on a novel target sequence, and brings about better effects than conventional types of siRNA.　Provided is siRNA which targets a WRN helicase gene and comprises: (a) a sense strand comprising the nucleotide sequence represented by SEQ ID NO: 1 and an antisense strand comprising the nucleotide sequence represented by SEQ ID NO: 2; (b) a sense strand comprising the nucleotide sequence represented by SEQ ID NO: 3 and an antisense strand comprising the nucleotide sequence represented by SEQ ID NO: 4; (c) a sense strand comprising the nucleotide sequence represented by SEQ ID NO: 5 and an antisense strand comprising the nucleotide sequence represented by SEQ ID NO: 6; (d) a sense strand comprising the nucleotide sequence represented by SEQ ID NO: 7 and an antisense strand comprising the nucleotide sequence represented by SEQ ID NO: 8; (e) a sense strand comprising the nucleotide sequence represented by SEQ ID NO: 9 and an antisense strand comprising the nucleotide sequence represented by SEQ ID NO: 10; (f) a sense strand comprising the nucleotide sequence represented by SEQ ID NO: 11 and an antisense strand comprising the nucleotide sequence represented by SEQ ID NO: 12; or (g) a sense strand comprising the nucleotide sequence represented by SEQ ID NO: 13 and an antisense strand comprising the nucleotide sequence represented by SEQ ID NO: 14.

Description

siRNA targeting the WRN helicase gene

[0001] The present invention relates to siRNAs that target the WRN helicase gene, cancer therapeutic agents, and pharmaceutical compositions for cancer treatment.

[0002] DNA helicases are enzymes that dissociate double-stranded DNA into single strands. Various types of DNA helicases are known, and DNA helicases similar to the RecQ helicase from Escherichia coli are collectively called RecQ helicases. Specific functions of RecQ helicases are known to be the resolution of DNA higher-order structures called Holliday structures during genome replication and their involvement in mismatch repair.

[0003] Five types of RecQ helicases are known in humans, designated RecQL1, WRN, RTS, BLM, and RecQ5. Of these, the BLM, WRN, and RTS genes are causative genes for Bloom syndrome, Werner syndrome, and Rothmond-Thomson syndrome, respectively, and all play an important role in maintaining cellular genome stability.

[0004] Based on the above findings, the present inventors developed siRNAs targeting RecQ helicases, including WRN helicase, as cancer therapeutic agents. These siRNAs specifically induce mitotic catastrophe and mitotic cell death in cancer cells (Patent Documents 1 to 3 and Non-Patent Documents 1 to 3). This is thought to be due to the suppression of RecQ helicase function in cancer cells, which leads to cell division while retaining DNA higher-order structures and DNA damage generated during DNA replication, resulting in the induction of cell death due to abnormal chromosome segregation. Furthermore, the present inventors found that the above siRNAs exhibited antitumor activity in cancer-bearing animal models, demonstrating that RecQ helicases could be an effective therapeutic target for cancer treatment.

[0005] WRN1, described in Patent Document 1, is an siRNA that targets the nucleotide sequence corresponding to positions 2824 to 2843 in the mRNA of the human WRN helicase gene (NCBI accession number NM_000553.6). This target sequence was selected based on the highest overall evaluation, including efficacy, in previous research, and the same target sequence has been used as an siRNA with high therapeutic efficacy in other subsequently published research papers (Non-Patent Document 4).

[0006] No target sequence in the WRN helicase gene has been found to date that can achieve an effect superior to that of WRN1.

[0007] International Publication No. 2004 / 100990 International Publication No. 2006 / 054625 International Publication No. 2017 / 022650

[0008] Futami, K., et al. (2008) Cancer Sci., 99(1): 71-80Futami, K., et al. (2008) Cancer Sci., 99(6): 1227-1236Futami, K., et al. (2010) Int. J. Mol. Med., 25: 537-545Arai, A., et al. (2011) Cancer Res., 71(13): 4598-4607

[0009] The objective of the present invention is to provide an siRNA based on a new target sequence that targets the WRN helicase gene and has a superior effect to conventional siRNAs.

[0010] To solve the above problems, the present inventors searched for new siRNA target sequences that exhibit high gene silencing effects, and as a result, they discovered novel siRNAs based on seven target sequences that exhibit significantly high RNAi activity in cancer cells.

[0011] The present inventors compared the gene silencing effects of these seven types of siRNA with WRN1, which has the highest efficacy among conventional siRNAs targeting the mRNA of the human WRN helicase gene. As a result, they found that all of the seven novel siRNAs exhibited even higher silencing effects than WRN1. The present invention is based on these findings and provides the following:

[0012] (1) An siRNA targeting the WRN helicase gene, comprising: (a) a sense strand consisting of the nucleotide sequence shown in SEQ ID NO: 1 and an antisense strand consisting of the nucleotide sequence shown in SEQ ID NO: 2, for example: (a-1) a sense strand consisting of the nucleotide sequence shown in SEQ ID NO: 1 and consisting of natural ribonucleosides, and an antisense strand consisting of the nucleotide sequence shown in SEQ ID NO: 2 and consisting of natural ribonucleosides; (a-2) a sense strand consisting of the nucleotide sequence shown in SEQ ID NO: 33 and an antisense strand consisting of the nucleotide sequence shown in SEQ ID NO: 34; (a-3) a sense strand consisting of the nucleotide sequence shown in SEQ ID NO: 35 and an antisense strand consisting of the nucleotide sequence shown in SEQ ID NO: 36; (a-4) a sense strand consisting of the nucleotide sequence shown in SEQ ID NO: 37 and an antisense strand consisting of the nucleotide sequence shown in SEQ ID NO: 38; (a-5) a sense strand consisting of the nucleotide sequence shown in SEQ ID NO: 39 and an antisense strand consisting of the nucleotide sequence shown in SEQ ID NO: 40; or (a-6) a sense strand consisting of the nucleotide sequence shown in SEQ ID NO: 41 and an antisense strand consisting of the nucleotide sequence shown in SEQ ID NO: 42, preferably (a-3) a sense strand consisting of the nucleotide sequence shown in SEQ ID NO: 35 and an antisense strand consisting of the nucleotide sequence shown in SEQ ID NO: 36, (b) a sense strand consisting of the nucleotide sequence shown in SEQ ID NO: 9 and an antisense strand consisting of the nucleotide sequence shown in SEQ ID NO: 10, (c) a sense strand consisting of the nucleotide sequence shown in SEQ ID NO: 11 and an antisense strand consisting of the nucleotide sequence shown in SEQ ID NO: 12, (d) a sense strand consisting of the nucleotide sequence shown in SEQ ID NO: 5 and an antisense strand consisting of the nucleotide sequence shown in SEQ ID NO: 6, (e) a sense strand consisting of the nucleotide sequence shown in SEQ ID NO: 7 and an antisense strand consisting of the nucleotide sequence shown in SEQ ID NO: 8, (f) a sense strand consisting of the nucleotide sequence shown in SEQ ID NO: 3 and an antisense strand consisting of the nucleotide sequence shown in SEQ ID NO: 4, or (g) a sense strand consisting of the nucleotide sequence shown in SEQ ID NO: 13 and an antisense strand consisting of the nucleotide sequence shown in SEQ ID NO: 14. (2) The siRNA according to (1), which comprises natural ribonucleosides, natural deoxyribonucleosides, and / or modified nucleosides. (3) The siRNA according to (2), wherein the modified nucleosides are 2'-modified nucleosides and / or bridged nucleosides.(4) The siRNA according to (3), wherein the 2'-modified group of the 2'-modified nucleoside is a 2'-O-methyl group or a 2'-fluoro group. (5) The siRNA according to (1), wherein all or some of the internucleoside linkages of the sense strand and / or the antisense strand are modified internucleoside linkages. (6) The siRNA according to (1), wherein the nucleosides at positions 2 to 5 from the 5'-end in the antisense strand are 2'-modified nucleosides. (7) The siRNA according to (6), wherein at least one nucleoside containing a pyrimidine base in the sense strand is a 2'-modified nucleoside. (8) The siRNA according to (6) or (7), wherein the 2'-modified group of the 2'-modified nucleoside is a 2'-O-methyl group. (9) A cell death inducer comprising the siRNA according to any one of (1) to (7) as an active ingredient. (10) A cancer therapeutic agent comprising, as an active ingredient, the siRNA according to any one of (1) to (7). (11) A pharmaceutical composition for cancer treatment comprising the siRNA according to any one of (1) to (7). (12) The pharmaceutical composition for cancer treatment according to (11), wherein the cancer is ovarian cancer, breast cancer, melanoma, gastric cancer, pancreatic cancer, liver cancer, colorectal cancer, lung cancer, head and neck cancer, peritoneal cancer, or cervical cancer. This specification incorporates the disclosure of Japanese Patent Application No. 2024-080324, from which the present application claims priority.

[0013] According to the present invention, there are provided siRNAs based on novel target sequences that target the WRN helicase gene and have superior effects compared to conventional siRNAs.

[0014] Figure 1 shows the structures of the siRNAs prepared in this example. Figure 1A shows the conventional unmodified siRNA WRN1. Figure 1B shows the unmodified siRNAs of the present invention, WRN101, WRN102, WRN103, WRN104, WRN105, WRN106, and WRN107. Figure 2 shows the results of measuring RNAi activity against the WRN gene in ES-2 cells. The results are shown for ES-2 cells transfected with conventional unmodified siRNA (WRN1) and unmodified siRNAs of the present invention (WRN101 to WRN107) at concentrations ranging from 0.05 nM to 50 nM. "Lipo" indicates a group treated with Lipofectamine only. "NT" indicates a group treated with neither siRNA nor Lipofectamine. Error bars indicate standard deviation. Figure 3 shows the results of measuring RNAi activity against the WRN gene in HeLa-S3 cells. Figure 4 shows the results of transfecting HeLa-S3 cells with conventional unmodified siRNA (WRN1) and unmodified siRNAs of the present invention (WRN101-WRN107) at concentrations ranging from 0.05 nM to 50 nM. "Lipo" indicates a group treated with Lipofectamine only. "NT" indicates a group treated with neither siRNA nor Lipofectamine. Error bars indicate standard deviation. Figure 4 shows the results of measuring RNAi activity against the WRN gene in HeLa-S3 cells. Figure 4 shows the results of transfecting HeLa-S3 cells with conventional unmodified siRNA (WRN1) and unmodified siRNAs of the present invention (WRN101-WRN107) at concentrations ranging from 0.05 nM to 50 nM. "Lipo" indicates a group treated with Lipofectamine only. "NT" indicates a group treated with neither siRNA nor Lipofectamine. Error bars indicate standard deviation. Figure 5 shows the results of measuring RNAi activity against the WRN gene in HCT-116 cells. "Lipo" indicates the group treated with Lipofectamine only. "NT" indicates the group treated with neither siRNA nor Lipofectamine. Error bars indicate standard deviation. Figure 6 shows the relative positions of the target sequences of WRN101, WRN-X1, WRN-X2, WRN-X3, and WRN8.Figure 7 shows the results of measuring the RNAi activity of WRN101, WRN-X1, WRN-X2, WRN-X3, and WRN8 against the WRN gene in HCT-116 cells. "Lipo" indicates the group treated with Lipofectamine only. "NT" indicates the group treated with neither siRNA nor Lipofectamine. Error bars indicate standard deviation. Figure 8 shows the results of measuring the RNAi activity of the unmodified siRNA WRN101 and the modified siRNAs WRN101-1 to WRN101-5 against the WRN gene in HCT-116 cells. "Lipo" indicates the group treated with Lipofectamine only. "NT" indicates the group treated with neither siRNA nor Lipofectamine. Error bars indicate standard deviation. Figure 9 shows the results of evaluating the IC50 concentrations of WRN101, WRN101-2, WRN8, and WRN1. FIG. 10 shows the results of electrophoresis to assess the remaining amounts of each of WRN8, WRN1, WRN101, and WRN101-2 siRNAs after incubation in human serum for 0 to 35 days.

[0015] <siRNA> In one embodiment of the present invention, siRNA targeting the WRN helicase gene is provided.

[0016] "WRN helicase (Werner syndrome ATP-dependent helicase)" is a DNA helicase belonging to the RecQ helicase family and is known to be the causative gene for Werner syndrome. WRN helicase is also known as RecQ-like type 3 (RECQ3). The mRNA sequence of the human WRN gene targeted by the siRNA of the present invention is shown in SEQ ID NO: 17.

[0017] WRN helicase is known to play an important role in cellular genome stability. Specifically, it has been reported to be involved in homologous recombination repair, non-homologous end joining, and base excision repair. Furthermore, it has been reported to contribute to recovery from replication arrest and maintenance of telomere structure, and is known to be required for immortalization (cancer transformation) of lymphoid cell lines. These findings indicate that WRN helicase plays an important role in DNA repair during DNA replication.

[0018] As used herein, "siRNA" (short / small-interfering RNA) refers to a double-stranded nucleic acid having approximately 19 to 25 base pairs that can induce the suppression of target gene expression by RNAi. siRNA is composed of two nucleic acid strands, a sense strand and an antisense strand, which will be described later. The two nucleic acid strands that make up the siRNA of the present invention can contain not only ribonucleosides but also deoxynucleosides and / or any modified nucleosides. siRNA that can induce RNAi activity is not limited to those composed of ribonucleosides; siRNA containing deoxynucleosides or modified nucleosides can also be incorporated into the RISC described below and recognize the target mRNA. siRNA may further contain a single-stranded portion (overhang).

[0019] As used herein, "RNAi" (RNA interference) refers to the phenomenon in which target gene expression is specifically suppressed in cells into which a double-stranded nucleic acid strand, such as siRNA, containing a sequence complementary to the target gene sequence has been introduced. siRNA-mediated RNAi can be explained as follows: First, one strand of the siRNA introduced into the cell is incorporated into a complex called RISC (RNA-induced silencing complex), which recognizes the target gene mRNA, which has a highly complementary sequence. The target gene mRNA is cleaved by RISC at the center of the highly complementary sequence. The cleaved mRNA can then be degraded.

[0020] As used herein, "antisense strand" refers to a nucleic acid strand containing a sequence complementary to the mRNA of a target gene. As used herein, "sense strand" refers to a nucleic acid strand containing a sequence complementary to the antisense strand (i.e., containing a sequence homologous to the mRNA of a target gene). The antisense strand anneals to the sense strand to produce siRNA. The antisense strand can bind to the mRNA of a target gene to induce RNAi. In the present invention, the antisense strand constituting the siRNA can bind to WRN mRNA to induce RNAi, thereby inducing suppression of WRN gene expression.

[0021] The siRNA of the present invention comprises or consists of natural ribonucleosides, natural deoxyribonucleosides, and / or modified nucleosides.

[0022] As used herein, the term "natural nucleoside" refers to a nucleoside that exists in nature. Examples include ribonucleosides consisting of ribose and a base such as adenine, cytosine, guanine, or uracil (referred to as "natural ribonucleosides"), and deoxyribonucleosides consisting of deoxyribose and a base such as adenine, cytosine, guanine, or thymine (referred to as "natural deoxyribonucleosides").

[0023] In one embodiment, the siRNA of the present invention is an unmodified siRNA. As used herein, "unmodified siRNA" refers to an siRNA composed of deoxyribonucleosides and / or ribonucleosides that have no modifications in the sugar, base, or phosphate. When the siRNA of the present invention is an unmodified siRNA, its sense strand and antisense strand can be composed of natural ribonucleosides and / or natural deoxyribonucleosides linked by internucleoside bonds.

[0024] In one embodiment, the unmodified siRNA of the present invention comprises or consists of a sense strand consisting of natural ribonucleosides linked by phosphodiester bonds and consisting of the base sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, or 13, respectively, and an antisense strand consisting of natural ribonucleosides linked by phosphodiester bonds and consisting of the base sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, or 14.

[0025] In one embodiment, the siRNA of the present invention is a modified siRNA. As used herein, the term "modified siRNA" refers to an siRNA containing one or more modified nucleosides and / or modified internucleoside linkages.

[0026] As used herein, "modified nucleoside" refers to a nucleoside having a modified sugar and / or a modified nucleobase.

[0027] As used herein, the term "modified sugar" refers to a sugar having a substitution and / or any change from a natural sugar moiety (i.e., a sugar moiety found in DNA (2'-H) or RNA (2'-OH)). As used herein, a nucleic acid strand may optionally contain one or more modified nucleosides, including modified sugars. As used herein, examples of nucleosides having modified sugar moieties include, but are not limited to, nucleosides containing 2'-F (2'-fluoro), 2'-OCH (2'-OMe or 2'-O-methyl), 2'-O(CH)OCH (2'-O-MOE or 2'-O-methoxyethyl), 5'-methyl (R or S), or 4'-S.

[0028] As used herein, "2'-modified sugar" refers to a furanosyl sugar modified at the 2'-position. In a 2'-modified sugar, the 2'-hydroxyl group is replaced by a C1-C 10 Alkyl, aryl, amino, azido, thio, -O-allyl, -O-C1-C 10alkyl, -OCF3, -O(CH2)2SCH3, -O(CH2)2OCH3, -O(CH2)2-ON(Rm)(Rn), O-CH2-C(=O)-N(Rm)(Rn), and -O-(CH2)2-C(=O)-N(Rm)(Rn), and the like, wherein each Rm and Rn is independently H or a substituted or unsubstituted C1-C 10 It is alkyl.

[0029] As used herein, a nucleoside containing a 2'-modified sugar is referred to as a "2'-modified nucleoside" or a "2'-substituted nucleoside." The 2'-position of a 2'-modified nucleoside is -R 1 , -OR 1 , -R 2 OR 1 , -OR 2 OR 1 or -R 3 OR 2 OR 1 where R 1 is C 1-4 is an alkyl group, and R 2 and R 3 is independently C 1-3 It is an alkylene group. In this specification, the term "alkyl group" refers to an optionally substituted, straight-chain or branched, saturated or unsaturated monovalent hydrocarbon group having 1 to 4 carbon atoms. Examples of "alkyl group" include, for example, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, and tert-butyl group. In this specification, the term "alkylene group" refers to an optionally substituted, straight-chain or branched, saturated or unsaturated divalent hydrocarbon group having 1 to 3 carbon atoms. Examples of "alkylene group" include, for example, methylene group, ethylene group, trimethylene group, etc. Substituents that an alkyl group or alkylene group may have include, for example, halogen atoms (e.g., fluorine, chlorine, bromine, iodine), amino group, nitro group, hydroxyl group, etc. R 1 is C 1-3 Alkyl group, C 1-2 It may be an alkyl group or a C1 alkyl group. 2 and R 3 is C 1-2It may be an alkylene group or a C1 alkylene group. Specifically, examples of the substituent at the 2'-position of a 2'-modified nucleoside include, but are not limited to, 2'-F (2'-fluoro group), -OCH3 (methoxy), -OCH2CH3 (ethoxy), -OCH2NH2 (aminomethoxy), -OCH2CH2NH2 (aminoethoxy), -OCH2CH2F (fluoromethylmethoxy), -OCH2CH2CH2F (fluoromethylethoxy), -CH3 (methyl), -CH2CH3 (ethyl), -CH2CH2CH3 (propyl), -CHOCH3 (methoxymethyl; MOM), -CH2CH2OCH3 (methoxyethyl; MOE), -OCHOCH3, -OCH2CH2OCH3, -CH2OCH2OCH3, -CHOCH2CH2OCH3, and -CHOCH2CH2OCH3 (methoxyethoxymethyl; MEM). The 2'-modified nucleoside is preferably a 2'-methoxynucleoside (2'-O-methyl-modified nucleoside) or a 2'-fluoro-modified nucleoside. The sense strand and / or antisense strand constituting the siRNA of the present invention may contain multiple 2'-modified nucleosides as described above, and the substituents at the 2'-position of these nucleosides may be the same or different.

[0030] In one embodiment, the modified nucleosides comprised in the siRNA of the present invention are 2'-modified nucleosides and / or bridged nucleosides.

[0031] As used herein, the term "bridged nucleoside" refers to a nucleoside containing a bicyclic sugar moiety. Nucleic acids containing a bicyclic sugar moiety are commonly referred to as bridged nucleic acids. The bicyclic sugar may be a sugar in which the 2'- and 4'-carbon atoms are bridged by two or more atoms. Examples of bicyclic sugars are known to those skilled in the art. Examples of bicyclic sugar-containing nucleic acids (BNAs) include, but are not limited to, methyleneoxy (4'-CH2-O-2') BNAs (also known as "LNAs"), ethyleneoxy (4'-(CH2)2-O-2') BNAs (also known as "ENAs"), cEt BNAs, cMOE BNAs, AmNAs, and GuNAs.

[0032] As used herein, the term "modified nucleobase" or "modified base" refers to any nucleobase other than adenine, cytosine, guanine, thymine, or uracil. Examples of modified nucleobases include, but are not limited to, 5-methylcytosine, 5-fluorocytosine, 5-bromocytosine, 5-iodocytosine, N4-methylcytosine, N6-methyladenine, 8-bromoadenine, N2-methylguanine, or 8-bromoguanine.

[0033] In one embodiment, all or part of the internucleoside linkages of the sense strand and / or antisense strand of the siRNA of the invention are modified internucleoside linkages.

[0034] As used herein, the term "modified internucleoside linkage" refers to an internucleoside linkage that has a substitution or any change from a naturally occurring internucleoside linkage (i.e., a phosphodiester linkage). Modified internucleoside linkages include phosphorus-containing internucleoside linkages that contain a phosphorus atom and non-phosphorus-containing internucleoside linkages that do not contain a phosphorus atom. Representative phosphorus-containing internucleoside linkages include, but are not limited to, phosphorothioate linkages, phosphorodithioate linkages, phosphotriester linkages, alkylphosphonate linkages, alkylthiophosphonate linkages, and phosphorodiamidates. A phosphorothioate linkage is an internucleoside linkage in which the non-bridging oxygen atom of a phosphodiester bond is replaced with a sulfur atom.

[0035] In another embodiment, all or some of the internucleoside linkages of the sense strand and / or antisense strand of the siRNA of the present invention are phosphodiester linkages.

[0036] The siRNAs of the present invention are selected from the group consisting of: (i) positions 1087 to 1109 in the mRNA sequence of the human WRN gene (SEQ ID NO: 17) (SEQ ID NO: 18; 5'-ATCTTACTAAAGGATATTTCAGA-3'); (ii) positions 1173 to 1195 in the mRNA sequence of the human WRN gene (SEQ ID NO: 17) (SEQ ID NO: 19; 5'-CAGCAATAATTTAAACTTATTAT-3'); (iii) positions 1906 to 1928 in the mRNA sequence of the human WRN gene (SEQ ID NO: 17) (SEQ ID NO: 20; 5'-GTGATTCATTCAGTATTAGAAGA-3'); and (iv) positions 2896 to 2918 in the mRNA sequence of the human WRN gene (SEQ ID NO: 17) (SEQ ID NO: 21; 5'-GAGAAGTTTCGATTATACAAATT-3'). (v) targets positions 3388 to 3410 in the mRNA sequence of the human WRN gene (SEQ ID NO: 17) (SEQ ID NO: 22; 5'-TGGCTTCATAAAGCTAATACAGA-3'), (vi) targets positions 3549 to 3571 in the mRNA sequence of the human WRN gene (SEQ ID NO: 17) (SEQ ID NO: 23; 5'-GTCTAACTTGGAGAAGTTATATT-3'), or (vii) targets positions 3899 to 3921 in the mRNA sequence of the human WRN gene (SEQ ID NO: 17) (SEQ ID NO: 24; 5'-AACATTTCTGCCAAACAAATAGT-3').

[0037] In the following description, the embodiments targeting the above target sequences (i) to (vii) are referred to as embodiment (i) to embodiment (vii), respectively. The target sequences (i) to (vii) correspond to the target sequences WRN101 to WRN107 in the Examples described below.

[0038] In embodiment (i) of the present invention, the siRNA of the present invention targets the target sequence of (i) above. The siRNA of this embodiment consists of a sense strand consisting of the nucleotide sequence shown in SEQ ID NO: 1 and an antisense strand consisting of the nucleotide sequence shown in SEQ ID NO: 2. The nucleotide sequence shown in SEQ ID NO: 1 corresponds to positions 1089 to 1109 in the mRNA sequence of the human WRN gene. The nucleotide sequence shown in SEQ ID NO: 2 is a sequence complementary to the nucleotide sequence of positions 1087 to 1107 in the mRNA sequence of the human WRN gene.

[0039] As used herein, the phrase "consisting of the base sequence shown in SEQ ID NO: 1" means that the base sequence consists of the base sequence shown in SEQ ID NO: 1, regardless of the state of nucleoside modification at each position (presence or absence of modification at each position in the base sequence and the type of modification). Therefore, "sense strand consisting of the base sequence shown in SEQ ID NO: 1" encompasses either (A) a sense strand consisting of the base sequence shown in SEQ ID NO: 1 and natural ribonucleosides linked by phosphodiester bonds and / or modified internucleoside linkages, or (B) a sense strand consisting of the base sequence shown in SEQ ID NO: 1 and containing natural deoxyribonucleosides and / or modified nucleosides linked by phosphodiester bonds and / or modified internucleoside linkages. The same applies to SEQ ID NOs: 2 to 14.

[0040] The siRNA of embodiment (i) may be a modified siRNA comprising a sense strand consisting of the nucleotide sequence shown in SEQ ID NO: 1 and composed of natural ribonucleotides, and an antisense strand consisting of the nucleotide sequence shown in SEQ ID NO: 2 and composed of natural ribonucleotides. Alternatively, the siRNA of embodiment (i) may be a modified siRNA comprising a sense strand consisting of the nucleotide sequence shown in SEQ ID NO: 33 and an antisense strand consisting of the nucleotide sequence shown in SEQ ID NO: 34; a sense strand consisting of the nucleotide sequence shown in SEQ ID NO: 35 and an antisense strand consisting of the nucleotide sequence shown in SEQ ID NO: 36; a sense strand consisting of the nucleotide sequence shown in SEQ ID NO: 37 and an antisense strand consisting of the nucleotide sequence shown in SEQ ID NO: 38; a sense strand consisting of the nucleotide sequence shown in SEQ ID NO: 39 and an antisense strand consisting of the nucleotide sequence shown in SEQ ID NO: 40; or a sense strand consisting of the nucleotide sequence shown in SEQ ID NO: 41 and an antisense strand consisting of the nucleotide sequence shown in SEQ ID NO: 42. A preferred modified siRNA is a modified siRNA corresponding to WRN101-2 in the Examples described below, which comprises a sense strand consisting of the nucleotide sequence shown in SEQ ID NO: 35 and an antisense strand consisting of the nucleotide sequence shown in SEQ ID NO: 36. The sense strand consisting of the base sequence shown in SEQ ID NO: 35 has 2'-O-methyl modified RNA nucleosides at positions 1 to 6, 8, and 12 to 21, and 2'-O-fluoro modified nucleosides at positions 7 and 9 to 11. The sense strand consisting of the base sequence shown in SEQ ID NO: 36 has 2'-O-methyl modified RNA nucleosides at positions 1, 3 to 5, 7 to 11, 13, and 15 to 21, and 2'-O-fluoro modified nucleosides at positions 2, 6, 12, and 14.

[0041] In embodiment (ii) of the present invention, the siRNA of the present invention targets the target sequence described in (ii) above. The siRNA of this embodiment consists of a sense strand consisting of the nucleotide sequence shown in SEQ ID NO: 3 and an antisense strand consisting of the nucleotide sequence shown in SEQ ID NO: 4. The nucleotide sequence shown in SEQ ID NO: 3 corresponds to positions 1175 to 1195 in the mRNA sequence of the human WRN gene. The nucleotide sequence shown in SEQ ID NO: 4 is a sequence complementary to the nucleotide sequence of positions 1173 to 1193 in the mRNA sequence of the human WRN gene.

[0042] In embodiment (iii) of the present invention, the siRNA of the present invention targets the target sequence of (iii) above. The siRNA of this embodiment consists of a sense strand consisting of the nucleotide sequence shown in SEQ ID NO: 5 and an antisense strand consisting of the nucleotide sequence shown in SEQ ID NO: 6. The nucleotide sequence shown in SEQ ID NO: 5 corresponds to positions 1908 to 1928 in the mRNA sequence of the human WRN gene. The nucleotide sequence shown in SEQ ID NO: 6 is a sequence complementary to the nucleotide sequence of positions 1906 to 1926 in the mRNA sequence of the human WRN gene.

[0043] In embodiment (iv) of the present invention, the siRNA of the present invention targets the target sequence of (iv) above. The siRNA of this embodiment consists of a sense strand consisting of the nucleotide sequence shown in SEQ ID NO: 7 and an antisense strand consisting of the nucleotide sequence shown in SEQ ID NO: 8. The nucleotide sequence shown in SEQ ID NO: 7 corresponds to positions 2898 to 2918 in the mRNA sequence of the human WRN gene. The nucleotide sequence shown in SEQ ID NO: 8 is a sequence complementary to the nucleotide sequence of positions 2896 to 2916 in the mRNA sequence of the human WRN gene.

[0044] In embodiment (v) of the present invention, the siRNA of the present invention targets the target sequence of (v) above. The siRNA of this embodiment consists of a sense strand consisting of the nucleotide sequence shown in SEQ ID NO: 9 and an antisense strand consisting of the nucleotide sequence shown in SEQ ID NO: 10. The nucleotide sequence shown in SEQ ID NO: 9 corresponds to positions 3390 to 3410 in the mRNA sequence of the human WRN gene. The nucleotide sequence shown in SEQ ID NO: 10 is a sequence complementary to the nucleotide sequence of positions 3388 to 3408 in the mRNA sequence of the human WRN gene.

[0045] In embodiment (vi) of the present invention, the siRNA of the present invention targets the target sequence of (vi) above. The siRNA of this embodiment consists of a sense strand consisting of the nucleotide sequence shown in SEQ ID NO: 11 and an antisense strand consisting of the nucleotide sequence shown in SEQ ID NO: 12. The nucleotide sequence shown in SEQ ID NO: 11 corresponds to positions 3551 to 3571 in the mRNA sequence of the human WRN gene. The nucleotide sequence shown in SEQ ID NO: 12 is a sequence complementary to the nucleotide sequence of positions 3549 to 3569 in the mRNA sequence of the human WRN gene.

[0046] In embodiment (vii) of the present invention, the siRNA of the present invention targets the target sequence of (vii) above. The siRNA of this embodiment consists of a sense strand consisting of the nucleotide sequence shown in SEQ ID NO: 13 and an antisense strand consisting of the nucleotide sequence shown in SEQ ID NO: 14. The nucleotide sequence shown in SEQ ID NO: 13 corresponds to positions 3901 to 3921 in the mRNA sequence of the human WRN gene. The nucleotide sequence shown in SEQ ID NO: 14 is a sequence complementary to the nucleotide sequence of positions 3899 to 3919 in the mRNA sequence of the human WRN gene.

[0047] The base sequences of the sense strand and antisense strand in the above-mentioned embodiments (i) to (vii) are shown in Table 1 below.

[0048]

[0049] In one embodiment, in the sense strand and / or antisense strand of the siRNA of the present invention, the nucleosides at positions 2 to 5 from the 5'-terminus are 2'-modified nucleosides. These 2'-modified nucleosides may be, for example, 2'-O-methyl-modified nucleosides or 2'-fluoro-modified nucleosides. By placing 2'-modified nucleosides at positions 2 to 5 from the 5'-terminus, off-target effects can be suppressed in the siRNA of the present invention.

[0050] In one embodiment, in the sense strand and / or antisense strand of the siRNA of the present invention, at least one nucleoside containing a pyrimidine base is a 2'-modified nucleoside.This 2'-modified nucleoside may be, for example, a 2'-O-methyl modified nucleoside or a 2'-fluoro modified nucleoside.By using a nucleoside containing a pyrimidine base as a 2'-modified nucleoside, the stability of the siRNA of the present invention can be improved.

[0051] It is generally known that siRNAs have high RNAi activity when they have a single-stranded portion (overhang) at their termini consisting of several (e.g., 2 to 5) nucleosides linked by internucleoside linkages. Therefore, the siRNAs of the present invention may have an overhang at their termini consisting of several natural or modified nucleosides linked by internucleoside linkages. In one embodiment, the siRNAs of the present invention may have a 2-base 3' overhang. For example, the siRNAs of the present invention may have a 3' overhang consisting of dithymidylic acid (TT) or diuridylic acid (UU).

[0052] The present inventors have previously reported that mitotic catastrophe and mitotic cell death are induced in cancer cells by suppressing WRN gene expression via the RNAi mechanism using siRNA (Futami, K., et al. (2008) Cancer Sci., 99(1): 71-80; Futami, K., et al. (2008) Cancer Sci., 99(6): 1227-1236; Futami, K., et al. (2010) Int. J. Mol. Med., 25: 537-545). The siRNA of the present invention targets the WRN gene and can induce cell death in cancer cells by suppressing WRN gene expression via the RNAi mechanism. The siRNA of the present invention has significantly stronger expression-suppressing activity than conventional siRNAs, such as WRN1 in the Examples described below, and has reduced toxicity and high stability in blood.

[0053] In the research of the present inventors, the following siRNAs [1] to

[0012] targeting the WRN gene have been previously developed.[1] siRNA targeting positions 293-311 in the mRNA sequence of the human WRN gene (SEQ ID NO: 17) (WO 2004 / 100990, WRN5; SEQ ID NO: 55 in the document) [2] siRNA targeting positions 664-683 in the above mRNA sequence (WO 2004 / 100990, WRN2; SEQ ID NO: 35 in the document) [3] siRNA targeting positions 1079-1097 in the above mRNA sequence (WO 2004 / 100990, WRN8; SEQ ID NO: 58 in the document) [4] siRNA targeting positions 2458-2476 in the above mRNA sequence (WO 2004 / 100990, WRN7; SEQ ID NO: 57 in the document) [5] siRNA targeting positions 2825-2843 in the above mRNA sequence (WO 2004 / 100990, WRN1; SEQ ID NO: 2 in the document) (WRN1 in the Examples below) [6] siRNA targeting positions 3328-3346 in the above mRNA sequence (WO 2004 / 100990, WRN12; SEQ ID NO: 62 in the document) [7] siRNA targeting positions 3426-3444 in the above mRNA sequence (WO 2004 / 100990, WRN3; SEQ ID NO: 53 in the document) [8] siRNA targeting positions 3548-3568 in the above mRNA sequence (WO 2004 / 100990, WRN4; SEQ ID NO: 54 in the document) [9] An siRNA targeting positions 3666 to 3684 in the above mRNA sequence (WO 2004 / 100990, WRN10; SEQ ID NO: 60 in the document).

[0010] An siRNA targeting positions 3881 to 3900 in the above mRNA sequence (WO 2004 / 100990, WRN11; SEQ ID NO: 61 in the document).

[0011] An siRNA targeting positions 4507 to 4524 in the above mRNA sequence (WO 2004 / 100990, WRN6; SEQ ID NO: 56 in the document).

[0012] An siRNA targeting positions 4876 to 4894 in the above mRNA sequence (WO 2004 / 100990, WRN9; SEQ ID NO: 59 in the document).

[0054] Among the above [1] to

[0012] , the siRNA shown in [5] (WRN1 in the Examples below) has the highest gene silencing activity. The gene expression silencing effects of the siRNAs of the above embodiments (i) to (vii) are even higher than that of WRN1.

[0055] The sense and antisense strands constituting the siRNA of the present invention can be produced by methods well known in the art, for example, by manual or automated reactions, enzymatically, or by chemical synthesis. When chemically synthesizing RNA or DNA molecules, contract manufacturing services from manufacturers (e.g., Gene Design, Dharmacon, QIAGEN, Sigma-Aldrich, etc.) may be used. In this case, the type and position of 2'-modified nucleosides can be specified. The synthesized antisense and sense strands may be purified from the mixture by, for example, extraction with a solvent or resin, precipitation, electrophoresis, or chromatography. The siRNA of the present invention can be produced by mixing and annealing the sense and antisense strands obtained as described above.

[0056] The siRNA of the present invention can be introduced into cells, tissues, or individuals in vitro or in vivo and used to induce RNAi-mediated suppression of target WRN gene expression. Furthermore, when the siRNA of the present invention is introduced into cancer cells, suppression of target WRN gene expression can induce cell death. Introduction of siRNA can be appropriately performed by those skilled in the art using methods known in the art. The siRNA may be introduced, for example, by physical methods, such as direct injection of a solution containing the siRNA (e.g., microinjection), bombardment using particles coated with the siRNA, or electroporation in the presence of the siRNA. Alternatively, the siRNA may be introduced by other methods known in the art for introducing nucleic acids into cells, such as lipid-mediated carrier delivery (e.g., lipofection using lipofectamine) or chemical-mediated delivery (e.g., gene transfer using polyethyleneimine (PEI), DEAE-Dextran method, calcium phosphate method). Furthermore, the siRNA may be introduced using known drug delivery system (DDS) technologies, such as liposomes or polymeric micelles. Further examples of gene transfer methods (transformation methods) can be found in Green & Sambrook, 2012, Molecular Cloning: A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, etc.

[0057] The cells, tissues, or individuals into which the siRNA of the present invention is to be introduced may be derived from primates (eg, rhesus monkeys, cynomolgus monkeys, chimpanzees, etc.), but are preferably derived from humans.

[0058] The siRNA of the present invention, based on a novel target sequence in the WRN gene, has improved RNAi activity against cancer cells, low toxicity against normal cells, and / or stability in the blood compared to conventional siRNAs.

[0059] As used herein, "toxicity" refers to an effect that causes undesirable symptoms or functional abnormalities in a subject. Toxicity may be toxicity in any organ, such as hepatotoxicity, nephrotoxicity, or neurotoxicity. As used herein, toxicity also includes cytotoxicity and off-target toxicity (off-target effects).

[0060] <WRN Gene Expression Inhibitor, Cell Death Inducer, and Cancer Therapeutic Agent> In one aspect of the present invention, there is provided an agent for inhibiting WRN gene expression, which comprises the siRNA of the present invention as an active ingredient.

[0061] As used herein, "gene expression suppression" refers to suppression of mRNA expression and / or protein expression of a target gene. When gene expression is assessed based on the expression level of the gene's mRNA or protein, "gene expression suppression" refers not only to 100% suppression but also to suppression by 75% or more, 50% or more, 20% or more, or 10% or more relative to the level without siRNA. The degree of gene expression suppression can be determined based on the expression level of the target gene's mRNA or protein. The mRNA expression level can be determined by Northern hybridization or RT-PCR, etc., and the protein expression level can be determined by Western blotting, ELISA, protein activity measurement, or fluorescence intensity from fluorescent proteins, etc. Furthermore, as used herein, the "RNAi activity" of an siRNA can be determined by the expression level of the WRN gene (mRNA expression level or protein expression level) or by its cell death-inducing activity in cancer cells. The WRN gene expression suppressor may consist solely of the siRNA of the present invention or may contain other components such as pharmaceutically acceptable carriers or additives. The WRN gene expression inhibitor may be used as a research reagent (RNAi reagent) or as a pharmaceutical for the treatment of disease.

[0062] In one embodiment of the present invention, a cell death inducer or cancer therapeutic agent is provided, which comprises the siRNA of the present invention as an active ingredient. The cell death inducer or cancer therapeutic agent of the present invention does not substantially induce cell death in normal cells, but can efficiently induce cell death in cancer cells. The cell death inducer or cancer therapeutic agent may consist solely of the siRNA of the present invention, or may contain other components such as pharmaceutically acceptable carriers or additives. The cell death inducer may be used as a research reagent or as a pharmaceutical for the treatment of disease.

[0063] <Pharmaceutical Composition for Cancer Treatment> In one embodiment of the present invention, a pharmaceutical composition for cancer treatment comprising the siRNA of the present invention is provided.

[0064] The type of "cancer" used herein is not limited, and examples include adenocarcinoma, squamous cell carcinoma, small cell carcinoma, and large cell carcinoma. Specific types of cancer include malignant melanoma, skin cancer, oral cancer, laryngeal cancer, pharyngeal cancer, thyroid cancer, lung cancer, breast cancer, esophageal cancer, gastric cancer, colorectal cancer (including colon cancer and rectal cancer), small intestine cancer, pancreatic cancer, bladder cancer, prostate cancer, testicular cancer, uterine cancer, cervical cancer, endometrial cancer, ovarian cancer, kidney cancer, liver cancer, pancreatic cancer, biliary tract cancer (including gallbladder cancer and bile duct cancer), brain tumor, head and neck cancer, mesothelioma, osteosarcoma, soft tissue sarcoma, glioma, pediatric tumors such as neuroblastoma, blood cancer, lymphoma, and myeloma. Examples of blood cancers include leukemia (e.g., B-cell leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia), lymphoma (e.g., non-Hodgkin's lymphoma), and myeloma (e.g., multiple myeloma). Ovarian cancer is generally classified into endometrioid adenocarcinoma, serous adenocarcinoma, clear cell adenocarcinoma, and mucinous adenocarcinoma. The cancer to be treated is preferably ovarian cancer, and in particular, ovarian clear cell adenocarcinoma is more preferred. In one embodiment, the cancer to be treated with the pharmaceutical composition for cancer treatment of the present invention is ovarian cancer, breast cancer, malignant melanoma, gastric cancer, pancreatic cancer, liver cancer, colorectal cancer, lung cancer, head and neck cancer, peritoneal cancer, or cervical cancer. Gastric cancer, pancreatic cancer, colorectal cancer, and ovarian cancer are known to cause peritoneal dissemination.

[0065] The pharmaceutical composition of the present invention may further contain a pharmaceutically acceptable carrier, if necessary. Pharmaceutically acceptable carriers include diluents or excipients, such as maltose, mannitol, lactose, xylose, trehalose, sorbitol, gelatin, gum arabic, guar gum, tragacanth, ethanol, physiological saline, Ringer's solution, etc.

[0066] In addition to the above-mentioned carrier, the pharmaceutical composition of the present invention may contain additives such as stabilizers, buffers, emulsifiers, isotonicity agents, preservatives, etc. These additives are preferably those used in pharmaceutical preparations.

[0067] Examples of stabilizers include albumin, gelatin, mannitol, sodium EDTA, etc. Examples of buffers include sodium citrate, citric acid, sodium phosphate, etc. Examples of emulsifiers include sorbitan fatty acid esters, glycerin fatty acid esters, etc. Examples of isotonic agents include sodium chloride, potassium chloride, sugars, etc. Examples of preservatives include benzalkonium chloride, parahydroxybenzoic acid, chlorobutanol, etc.

[0068] The pharmaceutical composition of the present invention may contain other drugs to the extent that the RNAi activity of the siRNA of the present invention, which is the active ingredient, is not lost. For example, in the case of an injection, it may contain a predetermined amount of an antibiotic.

[0069] Examples of dosage forms of the pharmaceutical composition include, but are not limited to, parenteral dosage forms such as injections, sprays, mists, aerosols, eye drops, creams, nasal drops, nasal sprays, gels, ointments, transmucosal preparations, plasters, and suppositories, and oral dosage forms such as liquids, powders, tablets, granules, suspensions, pills, powders, capsules, sublingual tablets, and lozenges. For example, when head and neck cancer or lung cancer is the target, a spray can be preferably used.

[0070] The pharmaceutical composition of the present invention can be administered to a subject or object in a pharmaceutically effective amount for treating the target disease (cancer). As used herein, the term "pharmaceutically effective amount" refers to the amount of siRNA contained in the pharmaceutical composition of the present invention that is necessary to treat the target cancer or alleviate its symptoms, and that has little or no harmful side effects on the recipient organism. The specific dosage will be determined for each individual subject based on factors such as the progression or severity of the disease, overall health, age, weight, sex, and tolerance to treatment, and will be determined, for example, by a physician's judgment.

[0071] As used herein, the term "subject" refers to a subject to which the siRNA, cancer therapeutic agent, or pharmaceutical composition for cancer treatment of the present invention is applied. Subjects include not only individuals but also organs, tissues, and cells. When the subject is an individual, it may be any animal, including humans. For example, non-human subjects include various livestock, poultry, pets, laboratory animals, etc. The subject may be, but is not limited to, a cancer patient.

[0072] The pharmaceutical composition of the present invention may be administered systemically or locally (e.g., directly to an affected area). The route of administration may be parenteral or oral, and examples thereof include intraperitoneal, intravenous, intraarterial, intrahepatic, intravaginal, intramuscular, intramedullary, intrathecal, transdermal, subcutaneous, intradermal, intranasal, oral, intrapharyngeal, pulmonary (e.g., inhalation via the mouth or nose), rectal, intestinal, intrabronchial, intrapulmonary, or sublingual.

[0073] When the pharmaceutical composition is administered or ingested, the dosage or intake amount may be, for example, such that the amount of siRNA contained therein is 0.001 mg / kg / day to 100 mg / kg / day. The single dose of the siRNA can be, for example, 0.001 mg / kg or more, 0.005 mg / kg or more, 0.01 mg / kg or more, 0.1 mg / kg or more, 0.25 mg / kg or more, 0.5 mg / kg or more, 1.0 mg / kg or more, 2.0 mg / kg or more, 2.5 mg / kg or more, 3.0 mg / kg or more, 4.0 mg / kg or more, 5 mg / kg or more, 10 mg / kg or more, 20 mg / kg or more, 30 mg / kg or more, 40 mg / kg or more, 50 mg / kg or more, 75 mg / kg or more, 100 mg / kg or more, 150 mg / kg or more, 200 mg / kg or more, 300 mg / kg or more, 400 mg / kg or more, or 500 mg / kg or more, and can be, for example, any amount within the range of 0.001 mg / kg to 500 mg / kg (e.g., 0.001 mg / kg, 0.01 The dose may be selected appropriately from the group consisting of 0.1 mg / kg, 1 mg / kg, 5 mg / kg, 10 mg / kg, 50 mg / kg, 100 mg / kg, and 200 mg / kg.

[0074] Furthermore, the pharmaceutical composition of the present invention can be administered to a patient in one to several or several dozen divided doses at regular time intervals, for example, at intervals of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 6 months, or 1 year, based on a treatment plan determined by a physician, for example.

[0075] Also provided is a method for treating cancer or a method for inducing cell death of cancer cells in a cancer patient, which comprises the step of administering the siRNA, cancer therapeutic agent, or pharmaceutical composition for cancer treatment of the present invention to a subject.

[0076] Also provided is the use of an siRNA of the invention in the manufacture of a medicament for treating cancer.

[0077] The present invention will be specifically described below with reference to examples. Note that these examples are for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention.

[0078] Example 1: Preparation of siRNA targeting the WRN helicase gene (Objective) To prepare siRNA targeting the WRN helicase gene.

[0079] (Methods and Results) The structures of the siRNAs prepared in this example are shown in Table 2 below and FIG.

[0080] The siRNAs prepared in this example target the WRN helicase gene and contain a base sequence complementary to a portion of the mRNA of the human WRN helicase gene as the antisense strand. WRN101, WRN102, WRN103, WRN104, WRN105, WRN106, and WRN107 are siRNAs newly prepared in this invention. WRN1 corresponds to WRN1 described in International Publication No. 2004 / 100990 (SEQ ID NO: 2 in that publication). WRN1 is a conventional siRNA with the highest gene silencing activity and will be used as a comparison in the following examples. The specific composition of each siRNA is as follows:

[0081] WRN101 consists of a sense strand consisting of the nucleotide sequence shown in SEQ ID NO: 1 and an antisense strand consisting of the nucleotide sequence shown in SEQ ID NO: 2. The sense strand of WRN101 has a structure in which 21 natural ribonucleosides are linked by phosphodiester bonds and consist of a nucleotide sequence corresponding to positions 1089 to 1109 of the mRNA of the human WRN helicase gene (Homo sapiens WRN RecQ like helicase (WRN), mRNA; NCBI accession number NM_000553.6; SEQ ID NO: 17). The antisense strand of WRN101 has a structure in which 21 natural ribonucleosides are linked by phosphodiester bonds and consist of a nucleotide sequence complementary to positions 1087 to 1107 of the above mRNA.

[0082] WRN102 consists of a sense strand consisting of the nucleotide sequence shown in SEQ ID NO: 3 and an antisense strand consisting of the nucleotide sequence shown in SEQ ID NO: 4. The sense strand of WRN102 has a structure in which 21 natural ribonucleosides consisting of a nucleotide sequence corresponding to positions 1175 to 1195 of the above mRNA are linked by phosphodiester bonds. The antisense strand of WRN102 has a structure in which 21 natural ribonucleosides consisting of a nucleotide sequence complementary to positions 1173 to 1193 of the above mRNA are linked by phosphodiester bonds.

[0083] WRN103 consists of a sense strand consisting of the nucleotide sequence shown in SEQ ID NO: 5 and an antisense strand consisting of the nucleotide sequence shown in SEQ ID NO: 6. The sense strand of WRN103 has a structure in which 21 natural ribonucleosides consisting of a nucleotide sequence corresponding to positions 1908 to 1928 of the above mRNA are linked by phosphodiester bonds. The antisense strand of WRN103 has a structure in which 21 natural ribonucleosides consisting of a nucleotide sequence complementary to positions 1906 to 1926 of the above mRNA are linked by phosphodiester bonds.

[0084] WRN104 consists of a sense strand consisting of the nucleotide sequence shown in SEQ ID NO: 7 and an antisense strand consisting of the nucleotide sequence shown in SEQ ID NO: 8. The sense strand of WRN104 has a structure in which 21 natural ribonucleosides consisting of a nucleotide sequence corresponding to positions 2898 to 2918 of the above mRNA are linked by phosphodiester bonds. The antisense strand of WRN104 has a structure in which 21 natural ribonucleosides consisting of a nucleotide sequence complementary to positions 2896 to 2916 of the above mRNA are linked by phosphodiester bonds.

[0085] WRN105 consists of a sense strand consisting of the nucleotide sequence shown in SEQ ID NO: 9 and an antisense strand consisting of the nucleotide sequence shown in SEQ ID NO: 10. The sense strand of WRN105 has a structure in which 21 natural ribonucleosides consisting of a nucleotide sequence corresponding to positions 3390 to 3410 of the above mRNA are linked by phosphodiester bonds. The antisense strand of WRN105 has a structure in which 21 natural ribonucleosides consisting of a nucleotide sequence complementary to positions 3388 to 3408 of the above mRNA are linked by phosphodiester bonds.

[0086] WRN106 consists of a sense strand consisting of the nucleotide sequence shown in SEQ ID NO: 11 and an antisense strand consisting of the nucleotide sequence shown in SEQ ID NO: 12. The sense strand of WRN106 has a structure in which 21 natural ribonucleosides consisting of a nucleotide sequence corresponding to positions 3551 to 3571 of the above mRNA are linked by phosphodiester bonds. The antisense strand of WRN106 has a structure in which 21 natural ribonucleosides consisting of a nucleotide sequence complementary to positions 3549 to 3569 of the above mRNA are linked by phosphodiester bonds.

[0087] WRN107 consists of a sense strand consisting of the nucleotide sequence shown in SEQ ID NO: 13 and an antisense strand consisting of the nucleotide sequence shown in SEQ ID NO: 14. The sense strand of WRN107 has a structure in which 21 natural ribonucleosides consisting of a nucleotide sequence corresponding to positions 3901 to 3921 of the above mRNA are linked by phosphodiester bonds. The antisense strand of WRN107 has a structure in which 21 natural ribonucleosides consisting of a nucleotide sequence complementary to positions 3899 to 3919 of the above mRNA are linked by phosphodiester bonds.

[0088] WRN1 consists of a sense strand consisting of the nucleotide sequence shown in SEQ ID NO: 15 and an antisense strand consisting of the nucleotide sequence shown in SEQ ID NO: 16. The sense strand of WRN1 has a structure in which two natural deoxyribonucleosides having a thymine base at the 3'-terminus of 19 natural ribonucleosides consisting of a nucleotide sequence corresponding to positions 2825 to 2843 of the above-mentioned mRNA are linked by a phosphodiester bond. The antisense strand of WRN1 has a structure in which two natural deoxyribonucleosides having a thymine base at the 3'-terminus of 19 natural ribonucleosides consisting of a nucleotide sequence complementary to positions 2825 to 2843 of the above-mentioned mRNA are linked by a phosphodiester bond.

[0089] The above siRNAs were custom synthesized, purified, and annealed by Gene Design, and were dissolved in nuclease-free water before use.

[0090] Example 2: Evaluation of RNAi activity against the WRN helicase gene (Objective) To identify the target sequence of a new siRNA that exhibits RNAi activity superior to that of WRN1 (hereinafter simply referred to as "conventional siRNA"), which has the highest gene silencing activity among conventional siRNAs. To this end, the siRNAs prepared in Example 1 were introduced into various cancer cell lines, and their effect on the expression level of mRNA of the WRN helicase gene was examined.

[0091] (Methods) (1) Culturing of cancer cell lines The human cancer cell lines used were the ES-2 cell line (ovarian cancer) and the HeLa-S3 cell line (cervical cancer). ES-2 cells (ATCC, product number CRL-1978) and HeLa-S3 cells (ATCC, product number CCL-2.2) were obtained from the American Type Culture Collection (ATCC).

[0092] Cancer cell lines were cultured at approximately 4 × 10 cells per well in 24-well plates one day before siRNA transfection. 4 ES-2 cells were cultured in McCoy's 5A (Gibco BRL) + 10% NBCS (Newborn Calf Serum, Gibco). HeLa-S3 cells were cultured in DMEM (high glucose, Nacalai Tesque) + 10% FBS (Fetal Bovine Serum, Gibco).

[0093] (2) Introduction of siRNA siRNA at the concentration described in each of the following examples was introduced into the cells using 1 μL / well of Lipofectamine™ RNAi MAX Reagent (ThermoFisher) according to the manufacturer's protocol.

[0094] In the following examples, cells without siRNA transfection and treated only with Lipofectamine™ RNAi MAX Reagent (ThermoFisher) were used as a control group (denoted as "Lipo" in the figures). Furthermore, as a further control group, cells without siRNA transfection and treatment with Lipofectamine™ RNAi MAX Reagent (ThermoFisher) were used (denoted as "NT" in the figures).

[0095] (3) Evaluation of gene expression suppression activity One to two days after siRNA transfection, cells were harvested, and total RNA was extracted using NucleoZOL (MACHERY-NAGEL). WRN mRNA levels (WRN gene expression levels) were determined in the extracted RNA by quantitative RT-PCR. Quantitative RT-PCR was performed using the Rotor-Gene Q 2plex System (QIAGEN). RT-PCR primers and TaqMan probes for the WRN and β-actin genes were purchased from Applied Biosystems. RT-PCR reactions were performed using the QuantiFast Probe RT-PCR Kit (QIAGEN) according to the manufacturer's protocol.

[0096] The WRN gene expression level was normalized to that of the β-actin gene, and the relative expression level was calculated by setting the expression level in the control group treated with Lipofectamine alone to 1. Experiments for each siRNA were performed with n = 3 (ES-2 cell line) or n = 2 (HeLa-S3 cell line), and the control group (Control) was performed with n = 4 to 6.

[0097] (Results) Each siRNA was introduced into ES-2 cells at concentrations ranging from 0.05 nM to 50 nM, and the relative expression level of the WRN gene was measured. The results are shown in Figure 2. Furthermore, the relative expression level of the WRN gene was measured after introduction into HeLa-S3 cells. The results are shown in Figure 3.

[0098] WRN101, WRN102, WRN103, WRN104, WRN105, WRN106, and WRN107 exhibited superior gene expression suppression effects compared to the conventional WRN1. Among these, WRN101, WRN102, WRN103, WRN104, WRN105, WRN106, and WRN107, WRN105 in particular exhibited potent suppression effects at all concentrations and in both ES-2 and HeLa-S3 cancer cell lines. Furthermore, WRN101 and WRN105 exhibited excellent suppression effects in ES-2 cells even at a low concentration of 0.05 nM (Figure 2).

[0099] The above results demonstrated that the siRNA of the present invention, which is based on a new target sequence, is superior to WRN1, which is known to have the highest gene suppression activity among conventional siRNAs.

[0100] Example 3: Further evaluation of RNAi activity against the WRN helicase gene (Objective) The siRNA prepared in Example 1 was compared with WRN1 and WRN8, conventional siRNAs disclosed in WO 2004 / 100990. Human cancer cell lines, HeLa-S3 cell line (cervical cancer) and HCT-116 cell line (colon cancer), were used to compare the effects on the mRNA expression level of the WRN helicase gene.

[0101] (Method) HeLa-S3 cells were cultured in DMEM (high glucose, Nacalai Tesque) + 10% FBS (fetal bovine serum). The human cell line HCT-116 (colon cancer) was obtained from Dainippon Pharmaceutical. Cells were grown at 3x10 per well in a 24-well plate 24 hours before siRNA transfection. 4 HCT-116 cells were cultured in McCoy's 5A (GibcoBRL) + 10% FBS (fetal bovine serum).

[0102] WRN8 corresponds to the WRN8 described in WO2004 / 100990. Specifically, WRN8 consists of a sense strand consisting of the nucleotide sequence shown in SEQ ID NO: 25 and an antisense strand consisting of the nucleotide sequence shown in SEQ ID NO: 26. The sense strand of WRN8 has a structure in which two natural deoxyribonucleosides having a thymine base at the 3'-terminus of 19 natural ribonucleosides consisting of a nucleotide sequence corresponding to positions 1079 to 1098 of the mRNA of the WRN helicase gene are linked by a phosphodiester bond. The antisense strand of WRN8 has a structure in which two natural deoxyribonucleosides having a thymine base at the 3'-terminus of 19 natural ribonucleosides consisting of a nucleotide sequence complementary to positions 1079 to 1098 of the mRNA are linked by a phosphodiester bond.

[0103] siRNAs other than WRN8 and the control group were the same as in Examples 1 and 2. The methods for introducing siRNA and evaluating its gene expression-suppressing activity were also the same as in Examples 1 and 2. Experiments for each siRNA were performed with n=3.

[0104] (Results) Each siRNA was introduced into HeLa-S3 cells at concentrations of 0.05 nM to 50 nM, and the relative expression levels of the WRN gene were measured. The results are shown in Figure 4. WRN101, WRN102, WRN103, WRN104, WRN105, WRN106, and WRN107 showed superior gene expression suppression effects compared to the conventional WRN1.

[0105] The relative expression levels of the WRN gene after transfection into HCT-116 cells were measured, and the results are shown in Figure 5. WRN101, WRN102, WRN103, WRN104, WRN105, WRN106, and WRN107 showed superior gene expression suppression effects compared to the conventional WRN1 and WRN8.

[0106] In particular, WRN101 showed a significant inhibitory effect at both concentrations and in both HeLa-S3 and HCT-116 cancer cell lines. Based on these results, WRN101 was selected for further analysis.

[0107] Example 4: siRNA consisting of sequences surrounding WRN101 (Objective) To create new siRNAs targeting sequences located near the target sequence of WRN101, which showed particularly excellent effects in Examples 2 and 3, and evaluate their RNAi activity against the WRN helicase gene in the HCT-116 cell line.

[0108] (Methods and Results) WRN-X1, WRN-X2, and WRN-X3 were constructed as siRNAs targeting sequences located near the target sequence of WRN101. The target sequences of WRN-X1 and WRN-X2 are located between those of WRN8 and WRN101, while the target sequence of WRN-X3 is located downstream of the WRN gene relative to the target sequence of WRN101 (Figure 6). The RNAi activities of WRN101, WRN-X1, WRN-X2, WRN-X3, and WRN8 were compared in the HCT-116 cell line using the same method as in Example 3.

[0109] The results are shown in Figure 7. Although WRN-X1, WRN-X2, and WRN-X3 target sequences located very close to the target sequence of WRN101, their RNAi activity was found to be lower than that of WRN101. This result indicates that WRN101 is an siRNA that exhibits outstanding effects among siRNAs targeting sequences close to its target sequence.

[0110] Example 5: Effect of modified siRNA (Objective) To prepare siRNAs by introducing various chemical modifications into WRN101 and evaluate their effects.

[0111] (Methods and Results) (1) Preparation of modified siRNAs The structures of the siRNAs prepared in this example are shown in Table 3. In WRN101-1 to WRN101-5, some of the RNA nucleotides in WRN101 were replaced with 2'-O-methyl-modified nucleotides and / or 2'-fluoro-modified nucleotides.

[0112] (2) Evaluation of RNAi Activity The RNAi activity of each siRNA shown in Table 3 against the WRN helicase gene was evaluated in the HCT-116 cell line. The activity evaluation method was the same as in Example 3.

[0113] The evaluation results for WRN101-1 to WRN101-5 are shown in Figure 8. WRN101-1 to WRN101-5 showed lower RNAi activity than WRN101 at a low concentration of 0.05 nM, but showed equal or greater RNAi activity than WRN101 at high concentrations of 5 nM and 50 nM. Furthermore, among the modified siRNAs, WRN101-2 was found to exhibit superior RNAi activity.

[0114] (3) Measurement of IC50 Concentration The RNAi activity of each siRNA for WRN101, WRN101-2, WRN8, and WRN1 in HCT-116 cells was measured at concentrations of 0.00005 nM, 0.0005 nM, 0.005 nM, 0.05 nM, 0.5 nM, 5 nM, and 50 nM. The results were analyzed using a four-parameter logistic model to calculate the IC50 concentration.

[0115] The results of IC50 concentration are shown in Figure 9. The IC50 concentration of WRN101 was 0.002 nM, that of WRN101-2 was 0.011 nM, that of WRN8 was 0.057 nM, and that of WRN1 was 0.061 nM. These results demonstrate that the IC50 concentrations of WRN101 and WRN101-2 of the present invention were lower than those of the conventional siRNAs WRN8 and WRN1, demonstrating RNAi activity even at lower concentrations.

[0116] (4) Evaluation of serum stability. 20 μL of each siRNA (20 μM) for WRN8, WRN1, WRN101, and WRN101-2 was mixed with 20 μL of human serum and 160 μL of buffer (10 mM phosphate-100 mM NaCl). 20 μL aliquots were dispensed into Eppendorf tubes, the sampling time was written on the lid, and the tubes were incubated in a CO2 incubator at 37°C. At each time point from 0 to 35 days, the tubes were mixed with 10 μL of 0.1 M EDTA to stop the incubation and stored at -30°C. After obtaining samples at all time points, each sample was thawed, and 7 μL of 2x loading buffer was added. The mixture was denatured at 95°C for 5 minutes and electrophoresed on a 7M urea-20% polyacrylamide gel (Urea PAGE) at 200 V for 1 hour. After electrophoresis, the gel was stained with SYBR™ Green I (LONZA) to assess the amount of remaining siRNA.

[0117] The electrophoresis results are shown in Figure 10. The unmodified siRNAs WRN1, WRN8, and WRN101 were completely degraded, with the bands disappearing after 24 hours of incubation. On the other hand, the modified siRNA WRN101-2 remained stable for up to 21 days. These results demonstrate that the stability of the modified siRNAs in human serum is significantly increased compared to unmodified siRNAs. All publications, patents, and patent applications cited herein are incorporated by reference in their entirety.

Claims

1. An siRNA targeting the WRN helicase gene, comprising: (a) a sense strand consisting of the nucleotide sequence shown in SEQ ID NO: 1 and an antisense strand consisting of the nucleotide sequence shown in SEQ ID NO: 2; (b) a sense strand consisting of the nucleotide sequence shown in SEQ ID NO: 9 and an antisense strand consisting of the nucleotide sequence shown in SEQ ID NO: 10; (c) a sense strand consisting of the nucleotide sequence shown in SEQ ID NO: 11 and an antisense strand consisting of the nucleotide sequence shown in SEQ ID NO: 12; (d) a sense strand consisting of the nucleotide sequence shown in SEQ ID NO: 5 and an antisense strand consisting of the nucleotide sequence shown in SEQ ID NO: 6; (e) a sense strand consisting of the nucleotide sequence shown in SEQ ID NO: 7 and an antisense strand consisting of the nucleotide sequence shown in SEQ ID NO: 8; (f) a sense strand consisting of the nucleotide sequence shown in SEQ ID NO: 3 and an antisense strand consisting of the nucleotide sequence shown in SEQ ID NO: 4; or (g) a sense strand consisting of the nucleotide sequence shown in SEQ ID NO: 13 and an antisense strand consisting of the nucleotide sequence shown in SEQ ID NO:

14.

2. The siRNA of claim 1, comprising natural ribonucleosides, natural deoxyribonucleosides, and / or modified nucleosides.

3. The siRNA of claim 2, wherein the modified nucleoside is a 2'-modified nucleoside and / or a bridged nucleoside.

4. The siRNA of claim 3, wherein the 2'-modified group of the 2'-modified nucleoside is a 2'-O-methyl group or a 2'-fluoro group.

5. The siRNA of claim 1, wherein all or part of the internucleoside linkages of the sense strand and / or the antisense strand are modified internucleoside linkages.

6. The siRNA of claim 1, wherein the nucleosides at positions 2 to 5 from the 5'-end in the antisense strand are 2'-modified nucleosides.

7. The siRNA of claim 6, wherein at least one of the nucleosides containing a pyrimidine base in the sense strand is a 2'-modified nucleoside.

8. The siRNA of claim 6, wherein the 2'-modified group of the 2'-modified nucleoside is a 2'-O-methyl group.

9. The siRNA according to claim 1, wherein the sense strand consisting of the base sequence shown in SEQ ID NO: 1 and the antisense strand consisting of the base sequence shown in SEQ ID NO: 2 in (a) are composed of natural RNA nucleotides.

10. The siRNA of claim 1, wherein the sense strand consisting of the base sequence shown in SEQ ID NO: 1 and the antisense strand consisting of the base sequence shown in SEQ ID NO: 2 in (a) are respectively: (a-1) a sense strand consisting of the base sequence shown in SEQ ID NO: 33 and an antisense strand consisting of the base sequence shown in SEQ ID NO: 34; (a-2) a sense strand consisting of the base sequence shown in SEQ ID NO: 35 and an antisense strand consisting of the base sequence shown in SEQ ID NO: 36; (a-3) a sense strand consisting of the base sequence shown in SEQ ID NO: 37 and an antisense strand consisting of the base sequence shown in SEQ ID NO: 38; (a-4) a sense strand consisting of the base sequence shown in SEQ ID NO: 39 and an antisense strand consisting of the base sequence shown in SEQ ID NO: 40; or (a-5) a sense strand consisting of the base sequence shown in SEQ ID NO: 41 and an antisense strand consisting of the base sequence shown in SEQ ID NO:

42.

11. The siRNA described in claim 10, wherein the sense strand consisting of the base sequence shown in SEQ ID NO: 1 and the antisense strand consisting of the base sequence shown in SEQ ID NO: 2 in (a) are comprised of: (a-2) a sense strand consisting of the base sequence shown in SEQ ID NO: 35 and an antisense strand consisting of the base sequence shown in SEQ ID NO:

36.

12. A cell death inducer comprising the siRNA according to any one of claims 1 to 11 as an active ingredient.

13. A cancer therapeutic agent comprising the siRNA according to any one of claims 1 to 11 as an active ingredient.

14. A pharmaceutical composition for cancer treatment, comprising the siRNA described in any one of claims 1 to 11.

15. The pharmaceutical composition for cancer treatment according to claim 14, wherein the cancer is ovarian cancer, breast cancer, melanoma, gastric cancer, pancreatic cancer, liver cancer, colon cancer, lung cancer, head and neck cancer, peritoneal cancer, or cervical cancer.

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