siRNA targeting the RecQL1 helicase gene

Abstract

The present invention addresses the problem of providing siRNA which targets a RecQL1 helicase gene and is based on a novel target sequence that brings about better effects than conventional siRNA.　Provided is siRNA targeting a RecQL1 helicase gene, the siRNA comprising: a sense strand containing the base sequence represented by SEQ ID NO: 1; and an antisense strand containing the base sequence represented by SEQ ID NO: 2.

Description

[Technical Field]

[0001] The present invention relates to an siRNA targeting the RecQL1 helicase gene, a cancer treatment agent, and a pharmaceutical composition for cancer treatment. [Background technology]

[0002] DNA helicases are enzymes that have the activity of dissociating double-stranded DNA into single-stranded DNA. Various types of DNA helicases are known, and DNA helicases similar to the RecQ helicase derived from E. coli are collectively called RecQ-type helicases. In humans, five types of RecQ-type helicases (RecQL1, WRN, RTS, BLM, and RecQ5) are known.

[0003] The specific functions of RecQL1 helicase (also known as RecQ1 or RecQL) are known to include resolving higher-order DNA structures called holiday structures during genome replication and participating in mismatch repair. Furthermore, RecQL1 helicase is known to be highly expressed in actively proliferating cells such as cancer cells, while its expression level is low in cells in the resting phase.

[0004] Based on the above facts, the inventors have developed an siRNA targeting RecQL1 helicase as a cancer treatment agent. This siRNA has the activity to specifically induce mitotic catastrophe and mitotic cell death in cancer cells (Patent Documents 1-3 and Non-Patent Documents 1-3). This is thought to be because the function of RecQL1 helicase is suppressed in cancer cells, and as a result of proceeding to cell division with the DNA higher-order structure and DNA damage generated during DNA replication remaining, cell death is induced due to abnormalities in chromosome segregation, etc. The inventors have also found that the above siRNA exhibits antitumor activity in cancer-bearing animal models, demonstrating that RecQL1 helicase can be an excellent therapeutic target.

[0005] Patent Document 3 discloses that RNAi activity based on siRNA targeting the RecQL1 helicase gene can be enhanced by introducing chemical modifications. The siRNA described in Patent Document 3 targets the nucleotide sequence corresponding to positions 784-802 in the mRNA of the human RecQL1 helicase gene (NCBI accession number NM_002907.4). This target sequence is identical to the siRNA target sequence disclosed in Patent Document 1 and was selected based on the highest overall evaluation, including efficacy, in previous studies.

[0006] No target sequences capable of achieving a greater effect than the above-mentioned target sequences have been found in the RecQL1 helicase gene. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] International Publication No. 2004 / 100990 [Patent Document 2] International Publication No. 2006 / 054625 [Patent Document 3] International Publication No. 2017 / 022650 [Non-patent literature]

[0008] [Non-Patent Document 1] Futami, K., et al. (2008) Cancer Sci., 99(1): 71-80 [Non-Patent Document 2] Futami, K., et al. (2008) Cancer Sci., 99(6): 1227-1236 [Non-Patent Document 3] Futami, K., et al. (2010) Int. J. Mol. Med., 25: 537-545 [Overview of the project] [Problems that the invention aims to solve]

[0009] An object is to provide an siRNA based on a novel target sequence that targets the RecQL1 helicase gene and has an effect superior to that of a conventional siRNA.

Means for Solving the Problem

[0010] In order to solve the above problems, the present inventors searched for a novel siRNA target sequence that can exhibit properties superior to those of a conventional siRNA. As a result, an siRNA based on a novel target sequence was found, which has properties significantly superior to those of a conventional siRNA in terms of RNAi activity against cancer cells, low toxicity against normal cells, and stability in human serum. Furthermore, as a result of administering the siRNA of the present invention to a mouse model of peritoneal dissemination of a human-derived ovarian cancer cell line ES-2, it was revealed that the siRNA exhibits a high therapeutic effect on tumor cells in vivo. The present invention is based on this finding and provides the following.

[0011] (1) An siRNA targeting the RecQL1 helicase gene, comprising a sense strand containing the nucleotide sequence shown in SEQ ID NO: 1, and an antisense strand containing the nucleotide sequence shown in SEQ ID NO: 2 and comprising the siRNA. (2) The siRNA according to (1), comprising a natural ribonucleoside, a natural deoxyribonucleoside, and / or a modified nucleoside. (3) The siRNA according to (2), wherein the modified nucleoside is a 2'-modified nucleoside and / or a bridged nucleoside. (4) The siRNA according to (3), wherein the 2'-modifying group of the 2'-modified nucleoside is a 2'-O-methyl group or a 2'-fluoro group. (5) The siRNA according to any one of (1) to (4), wherein all or part of the internucleoside linkage of the sense strand and / or the antisense strand is a modified internucleoside linkage. (6) The siRNA according to any one of (1) to (5), wherein the antisense strand contains a 2'-modified nucleoside at the 2nd position in the nucleotide sequence represented by SEQ ID NO: 2. (7) The siRNA according to any one of (1) to (6), wherein the nucleoside at the 14th position in the nucleotide sequence represented by SEQ ID NO: 2 in the antisense strand is not modified. (8) The siRNA according to any one of (1) to (7), wherein the sense strand and / or the antisense strand contains a 2'-modified nucleoside or a bridged nucleoside at the 3'-end. (9) The siRNA according to any one of (1) to (8), wherein the nucleoside at the 7th, 9th, and / or 11th position in the nucleotide sequence represented by SEQ ID NO: 1 in the sense strand is not modified or is a 2'-fluoro modified nucleoside, and / or the nucleoside at the 6th and / or 12th position in the nucleotide sequence represented by SEQ ID NO: 2 in the antisense strand is not modified or is a 2'-fluoro modified nucleoside, and / or the nucleoside at the 7th position in the nucleotide sequence represented by SEQ ID NO: 2 is a 2'-O-methyl modified nucleoside. (10) The siRNA according to any one of (1) to (9), wherein the sense strand contains a 2'-modified nucleoside at one or more positions from the 9th to the 11th position in the nucleotide sequence represented by SEQ ID NO: 1, or the antisense strand contains a 2'-modified nucleoside at one or more positions from the 9th to the 11th position in the nucleotide sequence represented by SEQ ID NO: 2. (11) The sense strand and the antisense strand are each (a) the nucleotide sequence represented by SEQ ID NO: 3 and the nucleotide sequence represented by SEQ ID NO: 4, (b) the nucleotide sequence represented by SEQ ID NO: 5 and the nucleotide sequence represented by SEQ ID NO: 6, (c) the nucleotide sequence represented by SEQ ID NO: 7 and the nucleotide sequence represented by SEQ ID NO: 8, (d) the nucleotide sequence represented by SEQ ID NO: 9 and the nucleotide sequence represented by SEQ ID NO: 10, or (e) the nucleotide sequence represented by SEQ ID NO: 11 and the nucleotide sequence represented by SEQ ID NO: 12 and the siRNA according to (1). (12) The siRNA according to (1), wherein each of the sense strand and the antisense strand consists of a natural ribonucleoside linked by an internucleoside bond. (13) A cell death inducer containing any of the siRNAs described in (1) to (12) as an active ingredient. (14) A cancer treatment agent containing any of the siRNAs described in (1) to (12) as an active ingredient. (15) A pharmaceutical composition for cancer treatment comprising any of the siRNAs described in (1) to (12). (16) The cancer treatment pharmaceutical composition according to (15), 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 includes the disclosures of Japanese Patent Application No. 2022-105409, which forms the basis of the priority claim of this application. [Effects of the Invention]

[0012] According to the present invention, a novel siRNA based on a target sequence that targets the RecQL1 helicase gene and exhibits superior efficacy compared to conventional siRNAs is provided. [Brief explanation of the drawing]

[0013] [Figure 1] Figure 1 shows the structure of the siRNA prepared in this embodiment. Figure 1A shows the conventional unmodified siRNA QL46 and the conventional modified siRNA QL46-19. Figure 1B shows the unmodified siRNA QL201 of the present invention and the modified siRNAs QL201-16, QL201-29, QL201-30, QL201-13, and QL201-15 of the present invention. [Figure 2] Figure 2 shows the results of measuring RNAi activity against the RecQL1 gene in ES-2 cells. It shows the results of introducing the conventional unmodified siRNA, QL46, and the unmodified siRNA of the present invention, QL201, into ES-2 cells at concentrations of 0.0005 nM to 5 nM. Error bars indicate the standard deviation. [Figure 3] Figure 3 shows the results of measuring RNAi activity against the RecQL1 gene in HeLa cells. Each siRNA was introduced into HeLa cells at a concentration of 0.005 nM. Error bars indicate the standard deviation. [Figure 4] Figure 4 shows the results of measuring RNAi activity against the RecQL1 gene in ES-2 cells. Each siRNA was introduced into ES-2 cells at concentrations of 0.05 nM to 5 nM. Error bars indicate the standard deviation. [Figure 5] Figure 5 shows the results of measuring RNAi activity by the siRNA of the present invention in various cancer cell lines. Figure 5A shows the results for A549 cells. Figure 5B shows the results for Lovo cells. Figure 5C shows the results for MKN45 cells. Figure 5D shows the results for Panc-1 cells. Error bars indicate the standard deviation. [Figure 6] Figure 6 shows the results of measuring the immune response of siRNA in the TIG-3 cell line. Figure 6A shows the results of measuring the expression level of the IFN-β gene. Figure 6B shows the results of measuring the expression level of the OAS-1 gene. Figure 6C shows the results of measuring the expression level of the OAS-2 gene. Error bars indicate the standard deviation. [Figure 7] Figure 7 shows the results of electrophoresis of siRNA incubated in human serum. Figure 7A shows the results for QL46 and QL46-19. Figure 7B shows the results for QL201, QL201-16, QL201-29, and QL201-30. The areas shown in black within each band in Figures 7A and 7B indicate the presence of undegraded siRNA beyond the upper limit of detection sensitivity. [Figure 8] Figure 8 shows the cell death induction activity of QL201 in cancer cells. It shows the relative survival rate of ES-2 cells after QL201 introduction. Error bars indicate the standard deviation. [Figure 9]Figure 9 shows the survival rate of human-derived ovarian cancer cell line ES-2 peritoneal dissemination model mice after administration of physiological saline, carboplatin, or QL201-16-encapsulated LNPs. The horizontal axis in the figure represents the cell transplantation day as Day 0, and the p-values ​​indicate statistical significance obtained using the log-rank test. [Modes for carrying out the invention]

[0014] <sirna> In one embodiment of the present invention, an siRNA targeting the RecQL1 helicase gene is provided. In this specification, "siRNA" (short-interfering RNA) means a double-stranded nucleic acid having approximately 19 to 25 base pairs that can induce the repression of target gene expression by RNAi. siRNA consists of two nucleic acid strands, a sense strand and an antisense strand, as described below. The two nucleic acid strands constituting the siRNA of the present invention may include not only ribonucleosides but also deoxynucleosides and / or any modified nucleosides. This is because siRNA capable of inducing RNAi activity is not limited to those consisting of ribonucleosides; siRNA containing deoxynucleosides or modified nucleosides can also be incorporated into RISC (as described below) and recognize target mRNA. siRNA may further include a single-stranded portion (overhang).

[0015] In this specification, "RNAi" (RNA interference) refers to the phenomenon in which the expression of a target gene is specifically suppressed in a cell into which a double-stranded nucleic acid strand, such as siRNA, containing a sequence complementary to the target gene sequence has been introduced. RNAi by siRNA 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 mRNA of the target gene having a highly complementary sequence. The mRNA of the target gene is cleaved by RISC at the central region of the highly complementary sequence. Subsequently, the cleaved mRNA can be degraded.

[0016] The siRNA of the present invention targets a sequence containing the 21-nucleotide sequence 5'-AGCAAUGAAUAUGAUUCUUCA-3' (Sequence ID 18) at positions 683-703 in the mRNA sequence of the human RecQL1 gene (Sequence ID 17).

[0017] The siRNA of the present invention comprises a sense strand containing the nucleotide sequence shown in SEQ ID NO: 1, and an antisense strand containing the nucleotide sequence shown in SEQ ID NO: 2. The nucleotide sequence shown in SEQ ID NO: 2 is a sequence complementary to the above nucleotide sequence (SEQ ID NO: 18) at positions 683-703 in the mRNA sequence of the human RecQL1 gene. The nucleotide sequence shown in SEQ ID NO: 1 corresponds to positions 685-705 in the mRNA sequence of the human RecQL1 gene. Here, "nucleotide sequence shown in SEQ ID NO: 1" means including the nucleotide sequence shown in SEQ ID NO: 1, and does not refer to the state of nucleoside modification at each position (the presence or absence of modification at each position in the nucleotide sequence and the type of modification). The same applies to SEQ ID NO: 2. Therefore, it is understood that "a sense strand containing the nucleotide sequence shown in SEQ ID NO: 1" includes either (i) a sense strand containing the nucleotide sequence shown in SEQ ID NO: 1 and consisting of natural ribonucleosides linked by phosphodiester bonds and / or modified nucleoside bonds, or (ii) a sense strand containing the nucleotide sequence shown in SEQ ID NO: 1 and consisting of natural deoxyribonucleosides or modified nucleosides linked by phosphodiester bonds and / or modified nucleoside bonds. Furthermore, it is understood that "an antisense strand containing the nucleotide sequence shown in SEQ ID NO: 2" includes either (i) an antisense strand containing the nucleotide sequence shown in SEQ ID NO: 2 and consisting of natural ribonucleosides linked by phosphodiester bonds and / or modified nucleoside bonds, or (ii) an antisense strand containing the nucleotide sequence shown in SEQ ID NO: 2 and consisting of natural deoxyribonucleosides or modified nucleosides linked by phosphodiester bonds and / or modified nucleoside bonds.

[0018] In this specification, "antisense strand" means a nucleic acid strand containing a sequence complementary to the mRNA of the target gene. In this specification, "sense strand" means a nucleic acid strand containing a sequence complementary to the antisense strand (i.e., containing a sequence homologous to the mRNA of the target gene). The antisense strand anneals with the sense strand to produce siRNA. The antisense strand can bind to the mRNA of the target gene to induce RNAi. In this invention, the antisense strand constituting the siRNA can bind to positions 683-703 of the RecQL1 mRNA to induce RNAi. Thus, the siRNA of this invention can induce repression of the RecQL1 gene expression.

[0019] The siRNA of the present invention may include natural ribonucleosides, natural deoxyribonucleosides, and / or modified nucleosides.

[0020] In this specification, "natural nucleoside" refers to a nucleoside that exists in nature. Examples include ribonucleosides ("natural ribonucleosides") composed of ribose and a base such as adenine, cytosine, guanine, or uracil, and deoxyribonucleosides ("natural deoxyribonucleosides") composed of deoxyribose and a base such as adenine, cytosine, guanine, or thymine.

[0021] In one embodiment, the siRNA of the present invention is an unmodified siRNA. In this specification, "unmodified siRNA" means an siRNA composed of a deoxyribonucleoside and / or ribonucleoside that has no modifications to sugar, base, or phosphate. When the siRNA of the present invention is an unmodified siRNA, its sense strand and antisense strand may consist of natural ribonucleoside and / or natural deoxyribonucleoside linked by nucleoside bonds.

[0022] In one embodiment, the unmodified siRNA of the present invention comprises a sense strand consisting of a natural ribonucleoside linked by a phosphodiester bond and containing the nucleotide sequence shown in SEQ ID NO: 1, and an antisense strand consisting of a natural ribonucleoside linked by a phosphodiester bond and containing the nucleotide sequence shown in SEQ ID NO: 2.

[0023] In one embodiment, the siRNA of the present invention is a modified siRNA. In this specification, "modified siRNA" means siRNA containing one or more modified nucleosides and / or modified nucleoside bonds.

[0024] In this specification, "modified nucleoside" means a nucleoside having a modified sugar and / or a modified nucleic acid base.

[0025] In this specification, “modified sugar” refers to a sugar that has been substituted and / or altered from the natural sugar moiety (i.e., the sugar moiety found in DNA(2'-H) or RNA(2'-OH)). In this specification, a nucleic acid chain may optionally contain one or more modified nucleosides, including modified sugars. In this specification, examples of nucleosides having a modified sugar moiety include, but are not limited to, nucleosides containing 2'-F (2'-fluoro group), 2'-OCH3 (2'-OMe group or 2'-O-methyl group), 2'-O(CH2)2OCH3 (2'-O-MOE group or 2'-O-methoxyethyl group), 5'-methyl (R or S), or 4'-S.

[0026] In this specification, "2'-modified sugar" means a furanosyl sugar modified at the 2' position. In 2'-modified sugars, the substitution of the 2'-hydroxyl group is C1-C 10 Alkyl, allyl, amino, azide, thio, -O-allyl, -O-C1-C 10 It can be selected from alkyl, -OCF3, -O(CH2)2SCH3, -O(CH2)2OCH3, -O(CH2)2 - O - N(Rm)(Rn), O - CH2 - C(=O) - N(Rm)(Rn), and -O-(CH2)2 - C(=O) - N(Rm)(Rn), etc. Each Rm and Rn is independently H or substituted or unsubstituted C1 - C 10 is alkyl.

[0027] In this specification, a nucleoside containing a 2'-modified sugar is referred to as a "2'-modified nucleoside" or a "2'-substituted nucleoside". The 2'-position of the 2'-modified nucleoside is -R 1 , -OR 1 , -R 2 OR 1 , -OR 2 OR 1 or -R 3 OR 2 OR 1 and here, R 1 is a C 1-4 alkyl group, and R 2 and R 3 are independently C 1-3 alkylene groups. In this specification, the "alkyl group" means a linear or branched, saturated or unsaturated monovalent hydrocarbon group having 1 to 4 carbon atoms which may be substituted. Examples of the "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 "alkylene group" means a linear or branched, saturated or unsaturated divalent hydrocarbon group having 1 to 3 carbon atoms which may be substituted. Examples of the "alkylene group" include, for example, methylene group, ethylene group, trimethylene group, etc. Substituents that the alkyl group or alkylene group may have include halogen atoms (e.g., fluorine, chlorine, bromine, iodine), amino group, nitro group, and hydroxyl group, etc. R 1 is a C 1-3 alkyl group, a C 1-2 alkyl group, or may also be a C1 alkyl group. R 2 and R 3 are C 1-2 An alkylene group or a C1 alkylene group may also be used. Specifically, examples of substituents at the 2' position of the 2'-modified nucleoside include, but are not limited to, 2'-F (2'-fluoro group), -OCH3 (methoxy), -OCH2CH3 (ethoxy), -OCH2NH2 (aminomethoxy), -OCH2CH2NH2 (aminoethoxy), -OCH2CH2F (methyl methoxy fluoride), -OCH2CH2CH2F (methyl ethoxy fluoride), -CH3 (methyl), -CH2CH3 (ethyl), -CH2CH2CH3 (propyl), -CH2OCH3 (methoxymethyl; MOM), -CH2CH2OCH3 (methoxyethyl; MOE), -OCH2OCH3, -OCH2CH2OCH3, -CH2OCH2OCH3, and -CH2OCH2CH2OCH3 (methoxyethoxymethyl; MEM). The 2'-modified nucleoside is preferably a 2'-methoxynucleoside (2'-O-methyl-modified nucleoside) or a 2'-fluoro-modified nucleoside. The sense and / or antisense strands constituting the siRNA of the present invention may contain a plurality of the above-mentioned 2'-modified nucleosides, but their substituents at the 2' position may be the same or different.

[0028] In one embodiment, the modified nucleoside contained in the siRNA of the present invention is a 2'-modified nucleoside and / or a crosslinked nucleoside.

[0029] In this specification, "crosslinked nucleoside" means a nucleoside containing a bicyclic sugar moiety. Nucleic acids containing a bicyclic sugar moiety are generally referred to as crosslinked nucleic acids. A bicyclic sugar may be a sugar in which the carbon atoms at the 2' and 4' positions are crosslinked by two or more atoms. Examples of bicyclic sugars are known to those skilled in the art. Examples of nucleic acids (BNAs) containing bicyclic sugars are, but are not limited to, methyleneoxy(4'-CH2-O-2')BNA (known as "LNA"), ethyleneoxy(4'-(CH2)2-O-2')BNA (also known as "ENA"), cEt BNA, cMOE BNA, AmNA, GuNA, etc.

[0030] In this specification, “modified nucleic acid base” or “modified base” means any nucleic acid base other than adenine, cytosine, guanine, thymine, or uracil. Examples of modified nucleic acid bases 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.

[0031] In one embodiment, all or part of the nucleoside bonds of the sense strand and / or antisense strand of the siRNA of the present invention are modified nucleoside bonds.

[0032] In this specification, "modified nucleoside bond" refers to a nucleoside bond that has been substituted or modified from a naturally occurring nucleoside bond (i.e., a phosphodiester bond). Modified nucleoside bonds include phosphorus-containing nucleoside bonds containing a phosphorus atom and non-phosphorus-containing nucleoside bonds that do not contain a phosphorus atom. Representative phosphorus-containing nucleoside bonds include, but are not limited to, phosphorothioate bonds, phosphorodithioate bonds, phosphotriester bonds, alkylphosphonate bonds, alkylthiophosphonate bonds, and phosphorodiamidates. A phosphorothioate bond is a nucleoside bond in which the non-bridged oxygen atom of a phosphodiester bond is substituted with a sulfur atom.

[0033] In another embodiment, all or part of the nucleoside bonds of the sense and / or antisense strands of the siRNA of the present invention are phosphodiester bonds.

[0034] In one embodiment, the sense strand and / or antisense strand of the siRNA of the present invention includes a 2'-modified nucleoside or a cross-linked nucleoside at the 3' end. This 2'-modified nucleoside may be, for example, a 2'-O-methyl-modified nucleoside or a 2'-fluoro-modified nucleoside. The cross-linked nucleoside may be, for example, an LNA nucleoside or an ENA nucleoside. By placing a 2'-modified nucleoside or a cross-linked nucleoside at the 3' end, the stability of the siRNA of the present invention can be improved.

[0035] In one embodiment, in the sense strand of the siRNA of the present invention, the nucleoside at position 7 in the nucleotide sequence shown in SEQ ID NO: 1 is not a 2'-O-methyl modified nucleoside. For example, the nucleoside at position 7 in the nucleotide sequence shown in SEQ ID NO: 1 may be a natural ribonucleoside, a natural deoxyribonucleoside, or a 2'-modified nucleoside other than a 2'-O-methyl modified nucleoside (e.g., a 2'-fluoro-modified nucleoside).

[0036] In one embodiment, the sense strand of the siRNA of the present invention contains one or more 2'-modified nucleosides at positions 9 to 11 in the nucleotide sequence shown in SEQ ID NO: 1. These 2'-modified nucleosides may be 2'-O-methyl-modified nucleosides or 2'-fluoro-modified nucleosides.

[0037] In one embodiment, in the sense strand of the siRNA of the present invention, the nucleosides at positions 9 and 11 in the nucleotide sequence shown in SEQ ID NO: 1 are either identical modified nucleosides except for the base portion, or identical natural nucleosides. Here, the natural nucleosides may be natural ribonucleosides or natural deoxyribonucleosides. In a further embodiment, in the sense strand of the siRNA of the present invention, the nucleosides at positions 9 to 11 in the nucleotide sequence shown in SEQ ID NO: 1 are either identical modified nucleosides except for the base portion, or identical natural nucleosides. Here, the natural nucleosides may be natural ribonucleosides or natural deoxyribonucleosides.

[0038] In one embodiment, the sense strand of the siRNA of the present invention does not contain 2'-O-methyl-modified nucleosides at positions 7, 9, and / or 11 of the nucleotide sequence shown in SEQ ID NO: 1. For example, the sense strand contains 2'-modified nucleosides other than natural ribonucleosides, natural deoxyribonucleosides, and / or 2'-O-methyl-modified nucleosides (e.g., 2'-fluoro-modified nucleosides) at positions 7, 9, and / or 11 of the nucleotide sequence shown in SEQ ID NO: 1.

[0039] In one embodiment, in the sense strand of the siRNA of the present invention, the nucleoside at position 10 in the nucleotide sequence shown in SEQ ID NO: 1 is not a 2'-O-methyl modified nucleoside. For example, the nucleoside at position 10 in the nucleotide sequence shown in SEQ ID NO: 1 may be a natural ribonucleoside, a natural deoxyribonucleoside, or a 2'-modified nucleoside other than a 2'-O-methyl modified nucleoside (e.g., a 2'-fluoro-modified nucleoside). By the fact that the nucleoside at position 10 in the nucleotide sequence shown in SEQ ID NO: 1 is not a 2'-O-methyl modified nucleoside in the sense strand, off-target effects of siRNA can be reduced.

[0040] In one embodiment, the antisense strand of the siRNA of the present invention contains a 2'-modified nucleoside at positions 2 to 5 (preferably at position 2) in the nucleotide sequence shown in SEQ ID NO: 2. This 2'-modified nucleoside may also be a 2'-O-methyl-modified nucleoside. By including a 2'-O-methyl-modified nucleoside at positions 2 to 5 (preferably at position 2) in the nucleotide sequence shown in SEQ ID NO: 2, off-target effects of siRNA can be reduced.

[0041] In one embodiment, the antisense strand of the siRNA of the present invention does not contain a 2'-O-methyl modified nucleoside at positions 6 and / or 12 of the nucleotide sequence shown in SEQ ID NO: 2. For example, the antisense strand contains a natural ribonucleoside, a natural deoxyribonucleoside, and / or a 2'-modified nucleoside other than a 2'-O-methyl modified nucleoside (e.g., a 2'-fluoro-modified nucleoside) at positions 6 and / or 12 of the nucleotide sequence shown in SEQ ID NO: 2.

[0042] In one embodiment, in the antisense strand of the siRNA of the present invention, the nucleoside at position 14 in the nucleotide sequence shown in SEQ ID NO: 2 is not a 2'-O-methyl-modified nucleoside. For example, the nucleoside at position 14 in the nucleotide sequence shown in SEQ ID NO: 2 may be a natural ribonucleoside, a natural deoxyribonucleoside, or a 2'-modified nucleoside other than a 2'-O-methyl-modified nucleoside (e.g., a 2'-fluoro-modified nucleoside).

[0043] In one embodiment, the antisense strand of the siRNA of the present invention contains one or more 2'-modified nucleosides at positions 9 to 11 in the nucleotide sequence shown in SEQ ID NO: 2. These 2'-modified nucleosides may be 2'-O-methyl-modified nucleosides or 2'-fluoro-modified nucleosides.

[0044] In one embodiment, in the antisense strand of the siRNA of the present invention, the nucleosides at positions 9 and 11 in the nucleotide sequence shown in SEQ ID NO: 2 are either identical modified nucleosides except for the base portion, or identical natural nucleosides. Here, the natural nucleosides may be natural ribonucleosides or natural deoxyribonucleosides. In a further embodiment, in the antisense strand of the siRNA of the present invention, the nucleosides at positions 9 to 11 in the nucleotide sequence shown in SEQ ID NO: 2 are either identical modified nucleosides except for the base portion, or identical natural nucleosides. Here, the natural nucleosides may be natural ribonucleosides or natural deoxyribonucleosides.

[0045] In one embodiment, in the antisense strand of the siRNA of the present invention, the nucleoside at position 10 in the nucleotide sequence shown in SEQ ID NO: 2 is not a 2'-O-methyl modified nucleoside. For example, the nucleoside at position 10 in the nucleotide sequence shown in SEQ ID NO: 2 may be a natural ribonucleoside, a natural deoxyribonucleoside, or a 2'-modified nucleoside other than a 2'-O-methyl modified nucleoside (e.g., a 2'-fluoro modified nucleoside). By the fact that the nucleoside at position 10 in the nucleotide sequence shown in SEQ ID NO: 2 is not a 2'-O-methyl modified nucleoside in the antisense strand, off-target effects by siRNA can be reduced.

[0046] In further embodiments, the sense strand and antisense strand of the siRNA of the present invention are, (a) The nucleotide sequence shown in Sequence ID No. 3, and the nucleotide sequence shown in Sequence ID No. 4, (b) The nucleotide sequence shown in Sequence ID No. 5 and the nucleotide sequence shown in Sequence ID No. 6, (c) The nucleotide sequence shown in Sequence ID No. 7, and the nucleotide sequence shown in Sequence ID No. 8, (d) The nucleotide sequence shown in SEQ ID NO: 9 and the nucleotide sequence shown in SEQ ID NO: 10, or (e) The nucleotide sequence shown in SEQ ID NO: 11 and the nucleotide sequence shown in SEQ ID NO: 12 This includes or consists of. Here, "the base sequence shown in SEQ ID NO: 3" means a nucleoside sequence that includes both the base sequence shown in SEQ ID NO: 3 and the state of nucleoside modification shown in SEQ ID NO: 3 (the presence or absence of modification at each position in the base sequence and the type of modification). The same applies to SEQ ID NOs: 4 to 12. All or part of the nucleoside bonds of the sense strand and / or antisense strand in embodiments (a) to (e) may be modified nucleoside bonds (e.g., phosphorothioate bonds) or phosphodiester bonds. Embodiments (a) to (e) are shown in Table 1 below.

[0047] [Table 1]

[0048] It is generally known that siRNA exhibits high RNAi activity when it has a single-stranded portion (overhang) of nucleosides linked by several (e.g., 2 to 5) nucleoside bonds at its terminal. Therefore, the siRNA of the present invention may have an overhang at its terminal consisting of several native or modified nucleosides linked by nucleoside bonds. In one embodiment, the siRNA of the present invention may have a 2-nucleotide-length 3' overhang. For example, the siRNA of the present invention may have a 3' overhang consisting of dithymidylic acid (TT) or diuridylic acid (UU).

[0049] The sense strand and antisense strand constituting the siRNA of the present invention may each be 21 to 25 nucleotides long, and may be the same length or of different lengths. That is, the sense strand may consist of the nucleotide sequence (21 nucleotides long) shown in SEQ ID NO: 1, or in addition to this sequence, it may have, for example, 1 to 4, 1 to 3, 1 to 2, or 1 native nucleoside and / or modified nucleoside (for example, a nucleotide sequence homologous to RecQL1 mRNA, or UU or TT) at the 5' and / or 3' ends. The antisense strand may consist of the nucleotide sequence (21 nucleotides long) shown in SEQ ID NO: 2, or in addition to this sequence, it may have, for example, 1 to 4, 1 to 3, 1 to 2, or 1 native nucleoside and / or modified nucleoside (for example, a nucleotide sequence homologous to RecQL1 mRNA, or UU or TT) at the 5' and / or 3' ends. The sense strand and / or antisense strand are preferably 21 to 23 nucleotides long, and more preferably 21 nucleotides long.

[0050] The inventors of this invention have previously reported that mitotic death and mitotic cell death can be induced in cancer cells by suppressing the expression of the RecQL1 gene 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 RecQL1 gene and can induce cell death in cancer cells by suppressing RecQL1 gene expression via the RNAi mechanism. The siRNA of the present invention has significantly stronger expression suppression activity, reduced toxicity, and high stability in the blood compared to conventional siRNAs, such as QL46 and QL46-19 in the examples described below.

[0051] The sense strand and antisense strand 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 chemically. When chemically synthesizing RNA or DNA molecules, contract manufacturing services from manufacturers (e.g., GeneDesign, Dharmacon, QIAGEN, Sigma-Aldrich, etc.) may be used. In that case, the type and position of the 2'-modified nucleoside can be specified. The synthesized antisense strand and sense strand may be purified from the mixture by, for example, extraction using a solvent or resin, precipitation, electrophoresis or chromatography. The siRNA of the present invention can be produced by mixing and annealing the sense strand and antisense strand obtained as described above.

[0052] The siRNA of the present invention can be introduced into cells, tissues, or organisms in vitro or in vivo to induce the suppression of the expression of the target gene RecQL1 via RNAi. Furthermore, when the siRNA of the present invention is introduced into cancer cells, cell death can be induced by suppressing the expression of the target gene RecQL1. The introduction of siRNA can be appropriately carried out by a person 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 transport (e.g., lipofection using lipofectamine, etc.) or chemical-mediated transport (e.g., gene transfer methods using polyethyleneimine (PEI), DEAE-Dextran method, calcium phosphate method), or further, by known drug delivery system (DDS) technologies such as liposomes or polymer 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.

[0053] The cells, tissues, or individuals into which the siRNA of the present invention is to be introduced may be derived from primates (e.g., rhesus macaques, cynomolgus macaques, chimpanzees, etc.), but are preferably derived from humans.

[0054] The siRNA of the present invention, based on a novel target sequence in the RecQL1 gene, exhibits improved RNAi activity against cancer cells, low toxicity to normal cells, and / or blood stability compared to conventional siRNAs.

[0055] As used herein, "toxicity" refers to an action that causes unfavorable symptoms or functional abnormalities in a subject. The toxicity may be toxicity in any organ, and may be, for example, hepatotoxicity, nephrotoxicity, or neurotoxicity. As used herein, toxicity includes cytotoxicity and off-target toxicity (off-target effect).

[0056] <RecQL1 Gene Expression Inhibitor, Cell Death Inducer, and Cancer Therapeutic Agent> In one aspect of the present invention, there is provided a RecQL1 gene expression inhibitor comprising the siRNA of the present invention as an active ingredient.

[0057] As used herein, "gene expression inhibition" means that the mRNA expression and / or protein expression of a target gene is suppressed. "Gene expression inhibition" means not only 100% suppression but also 75% or more, 50% or more, 20% or more, or 10% or more suppression as compared to the case where no siRNA is introduced when the expression of a gene is determined using the expression level of the mRNA or protein of that gene as an index. The degree of gene expression inhibition can be determined using the expression level of the mRNA or protein of the target gene as an index. The expression level of mRNA can be determined by Northern hybridization, RT-PCR, or the like, and the expression level of protein can be determined by Western blotting, ELISA, measurement of protein activity, or fluorescence intensity from a fluorescent protein, or the like. Also, as used herein, the "RNAi activity" of siRNA may be determined by the RecQL1 gene expression level (mRNA expression level or protein expression level), or may be determined by the cell death induction activity against cancer cells. The above-described RecQL1 gene expression inhibitor may consist only of the siRNA of the present invention, or may contain other components such as a pharmaceutically acceptable carrier or additive. The RecQL1 gene expression inhibitor may be used as a research reagent (RNAi reagent), or may be used as a medicine for the treatment of diseases.

[0058] Furthermore, in one embodiment of the present invention, a cell death inducer or cancer treatment agent is provided, comprising the siRNA of the present invention as an active ingredient. The cell death inducer or cancer treatment agent of the present invention does not substantially induce cell death in normal cells, but can efficiently induce cell death in cancer cells. The above-mentioned cell death inducer or cancer treatment 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 diseases.

[0059] <Pharmaceutical composition for cancer treatment> In one embodiment of the present invention, a pharmaceutical composition for cancer treatment containing the siRNA of the present invention is provided.

[0060] In this specification, the types of cancer are not limited, but examples include adenocarcinoma, squamous cell carcinoma, small cell carcinoma, and large cell carcinoma. Specific types of cancer include, for example, malignant melanoma, skin cancer, oral cancer, laryngeal cancer, pharyngeal cancer, thyroid cancer, lung cancer, breast cancer, esophageal cancer, stomach cancer, colorectal cancer (including colon 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 and bile duct cancer), brain tumors, head and neck cancers, mesothelioma, osteosarcoma, soft tissue sarcoma, glioma, neuroblastoma and other pediatric tumors, hematological cancers, 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 lymphoma), and myeloma (e.g., multiple myeloma). Ovarian cancer is generally classified into endometrioid adenocarcinoma, serous adenocarcinoma, clear cell adenocarcinoma, and mucinous adenocarcinoma. Ovarian cancer is preferred as the target cancer for treatment, and clear cell adenocarcinoma of ovarian cancer is more preferred. In one embodiment, the target cancers of the cancer treatment pharmaceutical composition of the present invention are 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.

[0061] The pharmaceutical composition of the present invention may optionally further contain a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may include a diluent or excipient, such as maltose, mannitol, lactose, xylose, trehalose, sorbitol, gelatin, gum arabic, guar gum, tragacanth, ethanol, physiological saline, Ringer's solution, etc.

[0062] In addition to the carrier described above, the pharmaceutical composition of the present invention may optionally contain additives such as stabilizers, buffers, emulsifiers, isotonic agents, and preservatives. These additives are preferably those used in pharmaceutical manufacturing.

[0063] Examples of stabilizers include albumin, gelatin, mannitol, and sodium EDTA. Examples of buffering agents include sodium citrate, citric acid, and sodium phosphate. Examples of emulsifiers include sorbitan fatty acid esters and glycerin fatty acid esters. Examples of isotonic agents include sodium chloride, potassium chloride, and sugars. Examples of preservatives include benzalkonium chloride, parahydroxybenzoic acid, and chlorobutanol.

[0064] The pharmaceutical composition of the present invention may also contain other drugs, as long as 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 injectable preparation, a predetermined amount of antibiotics may be included.

[0065] Examples of dosage forms for pharmaceutical compositions 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, or oral dosage forms such as liquids, powders, tablets, granules, suspensions, pills, powders, capsules, sublingual preparations, and lozenges. For example, sprays can be suitably used when targeting head and neck cancer or lung cancer.

[0066] The pharmaceutical composition of the present invention can be administered to a subject or target in a pharmaceutically effective amount for the treatment of the target disease (cancer). In this specification, "pharmaceutically effective amount" means a dose 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 to no harmful side effects on the receiving organism. The specific dose will be determined for each individual subject based on the stage or severity of the disease, overall health, age, weight, sex, and tolerance to treatment, for example, by the judgment of a physician.

[0067] In this specification, "subject" refers to the target to which the siRNA, cancer treatment agent, or cancer treatment pharmaceutical composition of the present invention is applied. A subject includes individuals, organs, tissues, and cells. When the subject is an individual, it may include any animal, including humans. Examples of non-human animals include various livestock, poultry, pets, and laboratory animals. While not limited to humans, the subject may also be a cancer patient.

[0068] The pharmaceutical composition of the present invention may be administered systemically or locally (for example, directly to the affected area). The route of administration may be parenteral or oral, and examples include intraperitoneal, intravenous, intraarterial, intrahepatic, intravaginal, intramuscular, intramedullary, transdermal, subcutaneous, intradermal, intranasal, oral, pharyngeal, transpulmonary (for example, by inhalation through the mouth or nose), transrectal, intraintestinal, intrabronchial, intrapulmonary, or sublingual.

[0069] When a pharmaceutical composition is administered or ingested, the dosage or intake should be such that the amount of siRNA contained is between 0.001 mg / kg / day and 100 mg / kg / day. The single dose of the above 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, for example, any amount within the range of 0.001 mg / kg to 500 mg / kg (e.g., 0.001 mg / kg, 0.01 mg / kg, 0.01 mg / kg). You can choose from mg / kg, 0.1 mg / kg, 1 mg / kg, 5 mg / kg, 10 mg / kg, 50 mg / kg, 100 mg / kg, or 200 mg / kg as appropriate.

[0070] Furthermore, the pharmaceutical composition of the present invention can be administered to a patient in one to several or even dozens of doses at regular time intervals, for example, 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.

[0071] The present invention also provides a method for treating cancer, or a method for inducing cell death of cancer cells in a cancer patient, which includes the step of administering siRNA, a cancer treatment agent, or a pharmaceutical composition for cancer treatment to a target. The use of siRNA in the manufacture of pharmaceuticals for treating cancer is also provided. [Examples]

[0072] The present invention will be described in detail below with reference to examples. These examples are for illustrative purposes only and do not limit the scope of the present invention.

[0073] <Example 1: Production of siRNA targeting the RecQL1 helicase gene> (the purpose) We will create siRNA targeting the RecQL1 helicase gene. (Methods and Results) The composition of the siRNA prepared in this example is shown in Table 2 and Figure 1 below.

[0074] [Table 2]

[0075] The siRNAs prepared in this example target the RecQL1 helicase gene and contain a base sequence complementary to a portion of the mRNA of the human RecQL1 helicase gene as an antisense strand. QL46 corresponds to RERQ1-1 (Sequence ID 1 in the said international publication) and the "unmodified siRNA" described in International Publication No. 2004 / 100990, and QL46-19 corresponds to "QL-19" in International Publication No. 2017 / 022650. All of these are used as conventional siRNAs for comparison with the siRNAs of the present invention. QL201, QL201-16, QL201-29, QL201-30, QL201-13, and QL201-15 are siRNAs newly prepared in this invention. The specific composition of each siRNA is as follows.

[0076] QL46 contains 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 QL46 contains the nucleotide sequence corresponding to positions 784-802 of the mRNA of the human RecQL1 helicase gene (Homo sapiens RecQ like helicase (RECQL), transcript variant 1, mRNA; NCBI accession number NM_002907.4; SEQ ID NO: 17), and has a structure in which 19 natural ribonucleosides at the 5' end and 2 natural deoxyribonucleosides at the 3' end are linked by phosphodiester bonds. The antisense strand of QL46 contains a nucleotide sequence complementary to positions 784-802 of the above mRNA, and has a structure in which 19 natural ribonucleosides at the 5' end and 2 natural deoxyribonucleosides at the 3' end are linked by phosphodiester bonds.

[0077] QL46-19 contains 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 QL46-19 contains 2'-O-methyl modified nucleosides at positions 2-4, 12-14, and 17-19 of the sense strand of QL46. The antisense strand of QL46-19 contains 2'-O-methyl modified nucleosides at positions 13, 15, and 19 of the antisense strand of QL46.

[0078] QL201 includes 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 QL201 contains the nucleotide sequence corresponding to positions 685-705 in the mRNA of the human RecQL1 helicase gene described above, and has a structure in which natural ribonucleosides are linked by phosphodiester bonds. The antisense strand of QL201 contains the nucleotide sequence complementary to positions 683-703 of the mRNA described above, and has a structure in which natural ribonucleosides are linked by phosphodiester bonds.

[0079] QL201-16 includes 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. In the sense strand of QL201, positions 1-6, 8, and 12-21 are 2'-O-methyl-modified nucleosides, and positions 7 and 9-11 are 2'-fluoro-modified nucleosides. In the antisense strand of QL201, positions 1, 3-5, 7-11, 13, and 15-21 are 2'-O-methyl-modified nucleosides, and positions 2, 6, 12, and 14 are 2'-fluoro-modified nucleosides.

[0080] QL201-29 includes 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. In the sense strand of QL201, positions 1-6, 8, 9, 10, and 12-21 are 2'-O-methyl-modified nucleosides, and positions 7, 9, and 11 are natural ribonucleosides. In the antisense strand of QL201, positions 1, 3-5, 7-9, 11, 13, and 15-21 are 2'-O-methyl-modified nucleosides, positions 2, 10, 12, and 14 are 2'-fluoro-modified nucleosides, and position 6 is a natural ribonucleoside.

[0081] QL201-30 includes 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. In the sense strand of QL201, positions 1-6, 8, 10, and 12-21 are 2'-O-methyl modified nucleosides, and positions 7, 9, and 11 are natural ribonucleosides. In the antisense strand of QL201, positions 1, 3-5, 7-11, 13, and 15-21 are 2'-O-methyl modified nucleosides, positions 2, 12, and 14 are 2'-fluoro modified nucleosides, and position 6 is a natural ribonucleoside.

[0082] QL201-13 includes 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. In the sense strand of QL201, positions 1, 4, 8, 10, 13-14, and 20-21 are 2'-O-methyl modified nucleosides, and positions 2-3, 5-7, 9-12, and 15-19 are natural ribonucleosides. In the antisense strand of QL201, positions 2-5, 8-11, 13-15, 17-18, and 20-21 are 2'-O-methyl modified nucleosides, and positions 1, 6-7, 12, 16, and 19 are natural ribonucleosides.

[0083] QL201-15 includes 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. In the sense strand of QL201, positions 2, 4, 6, 8, 12, 14, 16, 18, and 20 of the sense strand of QL201 are 2'-O-methyl modified nucleosides, and positions 1, 3, 5, 7, 9-11, 13, 15, 17, 19, and 21 are 2'-fluoro modified nucleosides. In the antisense strand of QL201, positions 1, 3, 5, 7, 9-11, 13, 15, 17, and 19-21 of the antisense strand of QL201 are 2'-O-methyl modified nucleosides, and positions 2, 4, 6, 8, 12, 14, 16, and 18 are 2'-fluoro modified nucleosides.

[0084] The above siRNAs were synthesized, purified, and annealed by Gene Design, and were dissolved in Nuclease-Free Water before use.

[0085] <Example 2: Evaluation of RNAi activity against the RecQL1 helicase gene> (the purpose) To identify novel siRNA target sequences that exhibit superior RNAi activity compared to conventional siRNAs, we will introduce the siRNA prepared in Example 1 into various cancer cell lines and examine its effect on the expression level of RecQL1 helicase gene mRNA.

[0086] In the following examples, QL46 used for comparison corresponds to the "unmodified siRNA" described in International Publication No. 2017 / 022650, and QL46-19 corresponds to the "QL-19" described in the same international publication (collectively referred to as "conventional siRNA" in the following examples).

[0087] (method) (1) Culture of cancer cell lines The following human cell lines were used: ES-2 cells (ovarian cancer), HeLa cells (cervical cancer), A549 cells (lung cancer), Lovo cells (colorectal cancer), MKN45 cells (stomach cancer), and Panc-1 cells (pancreatic cancer). The cell lines were obtained from ATCC (American Type Culture Collection) for ES-2 cells (ATCC, product number CRL-1978), HeLa cells (ATCC, product number CCL-2.2), and Panc-1 cells (ATCC, product number CRL-1469); from RIKEN for A549 cells (RIKEN, product number RCB0098) and LOVO cells (RIKEN, product number RCB1639); and from JCRB for MKN45 cells (JCRB, product number JCRB0254).

[0088] The cells were measured in a 24-well plate at a rate of 2.5 × 10⁶ per well, one day before siRNA transfection. 4 pieces~4×10 4 After seeding to form individual cells, they were cultured. ES-2 cells were cultured in McCoy5A (Gibco BRL) + 10% NBCS (Newborn Calf Serum, Gibco). HeLa cells were cultured in DMEM (high glucose, Nacalai Tesque) + 10% FBS (Fetal Bovine Serum, Gibco). A549 cells were cultured in EMEM (Wako Pure Chemical Industries) + 10% FBS + NEAA (non-Essential Amino Acids, Nacalai Tesque). Lovo cells were cultured in EMEM (Wako Pure Chemical Industries) + 10% FBS. MKN45 cells were cultured in RPMI1640 (Nacalai Tesque) + 10% FBS. Panc-1 cells were cultured in DMEM (high glucose, Nacalai Tesque) + 20% FBS.

[0089] (2) Introduction of siRNA In each of the following examples, siRNA at the concentrations described was introduced into cells using Lipofectamine® RNAi MAX Reagent (ThermoFisher) according to the manufacturer's protocol. In the following examples, cells treated only with Lipofectamine® RNAi MAX Reagent (ThermoFisher) without the introduction of siRNA were used as the control group.

[0090] (3) Evaluation of gene expression repression activity Twenty-four hours after siRNA introduction, cells were harvested and total RNA was extracted using NucleoZOL (MACHERY-NAGEL). The amount of RecQL1 mRNA (RecQL1 gene expression level) in the extracted RNA was determined by quantitative RT-PCR. Quantitative RT-PCR was performed using the Rotor-Gene Q 2plex System (QIAGEN). RT-PCR primers for RecQL1 and β-actin genes, as well as TaqMan probes, were purchased from Applied Biosystems. The RT-PCR reaction was performed using the QuantiFast Probe RT-PCR Kit (QIAGEN) according to the manufacturer's protocol.

[0091] RecQL1 gene expression levels were standardized relative to β-actin gene expression levels, and then the relative expression level was calculated with the control group's expression level set to 1. Experiments with each siRNA were conducted with n=2 or n=3, while the control group was conducted with n=4 to n=6.

[0092] (result) (1) RNAi activity of unmodified siRNA targeting novel sequences We searched for novel siRNA target sequences that exhibit superior RNAi efficacy compared to conventional siRNAs. Specifically, we introduced each unmodified siRNA into ES-2 cells at concentrations of 0.0005 nM, 0.005 nM, 0.05 nM, 0.5 nM, and 5 nM, and searched for novel siRNA target sequences that could induce stronger RNAi efficacy compared to the conventional unmodified siRNA QL46. As a result, we identified QL201 as a novel siRNA based on a target sequence that could induce overwhelmingly higher RNAi efficacy compared to QL46.

[0093] Figure 2 shows the results of introducing QL46 or QL201 into ES-2 cells at various concentrations and measuring the relative expression level of the RecQL1 gene. The introduction of QL201 significantly reduced the relative expression level of the RecQL1 gene. QL201 showed a more pronounced gene expression repression effect compared to QL46. The difference between the two was particularly large when used at low concentrations (0.0005 nM to 0.05 nM). These results clearly demonstrate that QL201, based on a novel target sequence, exhibits remarkable efficacy compared to conventional sequences.

[0094] (2) RNAi activity of modified siRNA The modified nucleosides were introduced into QL201, as identified in (1) above, to obtain QL201-16, QL201-29, QL201-30, QL201-13, and QL201-15. These were then introduced into HeLa cells, and their effects on the expression level of the RecQL1 gene were examined.

[0095] Figure 3 shows the results of measuring the relative expression level of the RecQL1 gene after introducing 0.005 nM siRNA into HeLa cells. The modified siRNAs QL201-16, QL201-29, QL201-30, QL201-13, and QL201-15 showed gene expression repression effects equivalent to those of the unmodified siRNA QL201. This effect was significantly higher compared to both the conventional unmodified siRNA QL46 and the conventional modified siRNA QL46-19.

[0096] Next, Figure 4 shows the results of introducing 0.05 nM to 5 nM siRNAs into ES-2 cells and measuring the relative expression level of the RecQL1 gene. The modified siRNAs QL201-13 and QL201-15 showed a gene expression repression effect equivalent to that of the unmodified siRNA QL201.

[0097] (3) RNAi activity in various cancer cell lines Figure 5 shows the results of introducing each siRNA (QL201, QL201-16, QL201-29, and QL201-30) with a molecular weight of 0.05 nM to 5 nM into A549, Lovo, MKN45, and Panc-1 cell lines, and measuring the relative expression levels of the RecQL1 gene. Each siRNA effectively suppressed gene expression in all cancer cell lines.

[0098] These results demonstrate that unmodified QL201 and modified siRNAs based on QL201 exhibit significant RNAi activity against the RecQL1 gene in cancer cells derived from various cancer types.

[0099] <Example 3: Examination of toxicity to normal cells> (the purpose) This study investigates the toxicity of QL201 and modified siRNAs based on QL201 to normal cells. Specifically, it compares the immune response induced when siRNA is introduced into normal cells with that of conventional siRNA. In particular, siRNA will be introduced into the TIG-3 cell line (human-derived normal fibroblasts), and changes in the expression levels of the IFN-β gene, OAS-1 gene, and OAS-2 gene will be examined.

[0100] (method) To investigate toxicity to normal cells, this example used the TIG-3 cell line (JCRB, product name TIG-3-20, product number JCRB0506).

[0101] The cells were placed in a 24-well plate with 2 × 10⁶ cells per well one day before siRNA transfection. 4 After seeding to a single cell, the cells were cultured. Culture was performed using EMEM (Wako Pure Chemical Industries) + 10% FBS + NEAA (non-essential amino acids, Nacalai Tesque). After changing to fresh medium, 5 nM or 50 nM siRNA was introduced into the cells using Lipofectamine® RNAi MAX Reagent (ThermoFisher) according to the manufacturer's protocol.

[0102] In this example, cells treated only with Lipofectamine® RNAi MAX Reagent (ThermoFisher) without the introduction of siRNA were used as the control group. Cells treated with 0.5 ng of Poly(I:C) were also used as a control.

[0103] 48 hours after siRNA introduction, cells were harvested and total RNA was extracted using NucleoZOL (MACHERY-NAGEL). The expression levels of the IFN-β gene, OAS-1 gene, and OAS-2 gene in the extracted RNA were determined by quantitative RT-PCR. Quantitative RT-PCR was performed using RNA-direct SYBR Green Realtime PCR Master Mix (TOYOBO, QRT-201).

[0104] The expression levels of the IFN-β, OAS-1, and OAS-2 genes were standardized against the hTubulinB gene expression level, and then the relative expression levels were calculated by setting the control group's expression level to 1. Experiments were conducted with n=3 for each group.

[0105] (result) Figure 6 shows the results of measuring the expression levels of the IFN-β gene, OAS-1 gene, and OAS-2 gene. It was revealed that QL201, QL201-16, QL201-29, and QL201-30 exhibited lower immune responsiveness compared to the conventional unmodified siRNA QL46 and the conventional modified siRNA QL46-19. These results demonstrate that QL201, QL201-16, QL201-29, and QL201-30 have reduced toxicity compared to conventional siRNAs.

[0106] <Example 4: Investigation of siRNA blood stability> (the purpose) The stability of the siRNA prepared in Example 1 will be evaluated in human serum.

[0107] (Methods and Results) 10 μL each of siRNA (20 μM) QL46, QL46-19, QL201, QL201-16, QL201-29, and QL201-30 was mixed with 10 μL of human serum and 80 μL of PBS, and incubated in a 37°C CO2 incubator. 20 μL samples were collected at time points from 0 hours to 3 days, mixed with 10 μL of 0.1 M EDTA, and temporarily stored at -20°C. 2 μL of 6× Loading Buffer was added to 10 μL of each collected sample, applied to a 20% acrylamide gel (Native PAGE), and electrophoresis was performed at 100 V for 3 hours. The gel was stained with ethylene bromide, and the remaining amount of siRNA was examined by electrophoresis.

[0108] Figure 7 shows the electrophoresis results after incubation in human serum. The conventional unmodified siRNA, QL46, showed complete degradation, with the band disappearing after 2 hours of incubation. The conventional modified siRNA, QL46-19, showed almost complete degradation, with the band disappearing within 24 hours (Figure 7A).

[0109] On the other hand, the modified siRNAs of the present invention, QL201-16, QL201-29, and QL201-30, all showed no band disappearance for at least 3 days. This result demonstrates that the modified siRNAs of the present invention are stable in serum (Figure 7B).

[0110] These results demonstrate that the modified siRNA of the present invention exhibits significantly increased stability in human serum compared to conventional modified siRNA.

[0111] <Example 5: Evaluation of cell death induction activity> (the purpose) The cell death-inducing activity of QL201 prepared in Example 1 against ovarian cancer-derived ES-2 cells will be evaluated.

[0112] (Methods and Results) ES-2 cells (ATCC, product number CRL-1978) were cultured in McCoy5A (GibcoBRL) + 10% NBCS (Newborn Calf Serum, Gibco) using the same method as in Example 2. The unmodified siRNA of the present invention, QL201 (0.5 nM, 5 nM, or 50 nM), prepared in Example 1, was introduced into the cells using Lipofectamine® RNAi MAX Reagent (ThermoFisher) according to the manufacturer's protocol. Cells treated with only Lipofectamine® RNAi MAX Reagent (ThermoFisher) without siRNA introduction were used as the control group. After siRNA introduction, the cells were cultured in a CO2 incubator at 37°C for 120 hours. Using Celltiter Glo® 2.0 Assay (Promega, product number G9242), the luminescence intensity of each group (n=6) was measured, and survival rates were evaluated by calculating relative values ​​with the control group set to 100%.

[0113] The results are shown in Figure 8. It was demonstrated that the introduction of the unmodified siRNA of the present invention can efficiently induce cell death in ES-2 cells.

[0114] <Example 6: Survival rate evaluation test using a mouse model of human-derived ovarian cancer cell line ES-2 peritoneal dissemination> (the purpose) The siRNA QL201-16 prepared in Example 1 was encapsulated in lipid nanoparticles (LNPs) and administered to a human-derived ovarian cancer cell line ES-2 peritoneal dissemination model mouse, and the survival rate after administration was evaluated.

[0115] (Methods and Results) (1) Preparation of QL201-16 encapsulated lipid nanoparticles The following four lipids—CLZ-42 (HFX Pharma), DSPC (Nippon Seika, A90195), Cholesterol (hereinafter abbreviated as "Cho") (Nippon Seika, B71155), and DMG-MPEG2000 (Nippon Seika, RHF-MD051)—were each dissolved in ethanol, and then mixed in a weight ratio of CLZ-42:DSPC:Cho:DMG-MPEG2k = 54:14:26:7 to prepare a lipid solution containing 32 mg / mL of lipids. In addition, an siRNA solution containing 0.68 mg / mL of QL201-16 and 1 mM of citrate buffer (pH 4.0) was prepared by mixing QL201-16, the siRNA prepared in Example 1, 100 mM citrate buffer, and RNase-free water.

[0116] Next, the lipid solution and siRNA solution were mixed in a ratio of 1:3.58 and then dialyzed with PBS(-). The sample was collected from the dialysis membrane, and the lipid nanoparticle (LNP) solution obtained after dialysis was adjusted to an siRNA concentration of 1 mg / mL using PBS(-), and then sterile filtered through a 0.2 μm filter. In the following examples, the sample after sterile filtration was used as QL201-16-encapsulated lipid nanoparticles (or QL201-16-encapsulated LNPs) for administration.

[0117] (2) Survival rate evaluation test Human ovarian cancer cell line ES-2 was cultured in culture medium (McCoy's 5A (Modified) Medium + 10% FBS + 1% Penicillin-Streptomycin Solution), and the resulting cell suspension was transplanted into the peritoneal cavity of 5-week-old female nude mice (CAnN.Cg-Foxn1nu / CrlCrlj; purchased from Jackson Laboratory Japan Co., Ltd.). The number of transplanted cells was 2.0 × 10⁶. 6 The cells were divided into cells / mouse units, and 0.2 mL / mouse of the cell suspension was transplanted into the peritoneal cavity.

[0118] The day of cell transplantation was designated as Day 0. The test substance was administered to human-derived ovarian cancer cell line ES-2 peritoneal dissemination model mice using the following method for each treatment group. Eight mice were used in each group. Group 1 (Vehicle group): As a negative control, physiological saline was administered intraperitoneally (ip) at a dose rate of 10 mL / kg. The administration was performed once a day from Day 3 to Day 12 for a total of 10 doses. Group 2 (Carboplatin group): As a positive control, carboplatin (75 mg / 10 mL / kg) was administered intravenously (iv) via tail vein on Day 3, Day 10, and Day 17 for a total of three doses. Group 3 (QL201-16 encapsulated LNP group): QL201-16 encapsulated lipid nanoparticles (QL201-16 encapsulated LNP, 2 mg / 10 mL / kg) were administered intraperitoneally (ip) once a day from Day 3 to Day 12 for a total of 10 doses.

[0119] Figure 9 shows the results of measuring the number of surviving individuals up to Day 56, with the day of cell transplantation designated as Day 0. The average survival days for each group were 18.0 days for Group 1 (Vehicle group), 20.6 days for Group 2 (Carboplatin group), and 32.6 days for Group 3 (QL201-16-encapsulated LNP group). The survival days of Group 3 (QL201-16-encapsulated LNP group) were significantly longer than those of both Group 1 (Vehicle group) and Group 2 (Carboplatin group) (in the figure, p-values ​​are calculated using the log-rank test), demonstrating that QL201-16, the siRNA of the present invention, has excellent therapeutic effects against tumors.

[0120] From these results, it has become clear that the siRNA of the present invention exhibits effective therapeutic effects against tumor cells in vivo. All publications, patents, and patent applications cited herein shall be incorporated herein by direct reference.< / sirna>

Claims

1. An siRNA that targets the RecQL1 helicase gene, A sense strand containing the base sequence shown in Sequence ID No. 1, and Antisense strand containing the base sequence shown in Sequence ID No. 2 The above-mentioned siRNA, which consists of the above.

2. The siRNA according to claim 1, comprising a natural ribonucleoside, a natural deoxyribonucleoside, and / or a modified nucleoside.

3. The siRNA according to claim 2, wherein the modified nucleoside is a 2'-modified nucleoside and / or a crosslinked nucleoside.

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

5. The siRNA according to claim 1, wherein all or part of the nucleoside bonds of the sense strand and / or the antisense strand are modified nucleoside bonds.

6. The siRNA according to claim 1, wherein the antisense strand contains a 2'-modified nucleoside at position 2 in the base sequence shown in Sequence ID No.

2.

7. The siRNA according to claim 1, wherein the nucleoside at position 14 in the base sequence shown in SEQ ID NO: 2 is not modified in the antisense strand.

8. The siRNA according to claim 1, wherein the sense strand and / or the antisense strand comprises a 2'-modified nucleoside or a cross-linked nucleoside at its 3' end.

9. In the sense strand, the nucleosides at positions 7, 9, and / or 11 in the base sequence shown in SEQ ID NO: 1 are either unmodified or 2'-fluoromodified nucleosides, and / or The siRNA according to claim 1, wherein in the antisense strand, the nucleoside at position 6 and / or position 12 in the base sequence shown in SEQ ID NO: 2 is either unmodified or a 2'-fluoromodified nucleoside, and / or the nucleoside at position 7 in the base sequence shown in SEQ ID NO: 2 is a 2'-O-methylmodified nucleoside.

10. The sense chain and the antisense chain are, respectively, (1) The base sequence shown in Sequence ID No. 3, and the base sequence shown in Sequence ID No. 4, (2) The base sequence shown in Sequence ID No. 5, and the base sequence shown in Sequence ID No. 6, (3) The base sequence shown in Sequence ID No. 7, and the base sequence shown in Sequence ID No. 8, (4) The base sequence shown in Sequence ID No. 9 and the base sequence shown in Sequence ID No. 10, or (5) The base sequence shown in Sequence ID No. 11 and the base sequence shown in Sequence ID No. 12 The siRNA according to claim 1, comprising:

11. The siRNA according to claim 1, wherein each of the sense chain and the antisense chain consists of a natural ribonucleoside linked by an internucleoside bond.

12. A cell death inducer comprising the siRNA described in claim 1 as an active ingredient.

13. A cancer treatment agent comprising the siRNA described in claim 1 as an active ingredient.

14. A pharmaceutical composition for cancer treatment comprising the siRNA described in claim 1.

15. The cancer treatment pharmaceutical composition according to claim 14, 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.

Images