Structure of oncolytic viruses containing bispecific nucleic acid molecules

Abstract

The present invention relates to an anti-tumor adenovirus and an anti-cancer composition comprising the same. The double-stranded small interfering ribonucleic acid (siRNA) of the present invention promotes the killing of cancer cells by simultaneously inhibiting the expression of a first nucleic acid and a second nucleic acid, and has an anti-cancer activity that is significantly better than treating individual small interfering ribonucleic acids (siRNA) together, and has the effect of synergistically improving the cancer cell killing efficacy during the combined treatment with an anti-cancer agent. The adenovirus comprising an expression cassette encoding a short hairpin ribonucleic acid (shRNA) for expressing an anti-tumor adenovirus and a human telomerase reverse transcriptase (hTERT) promoter has the effect of avoiding the immune response in the body to specifically deliver to cancer cells and achieve systemic treatment, can achieve local delivery, has excellent selectivity, and can present a significant anti-cancer effect through minimally invasive treatment, and can be effectively used as an anti-cancer composition or anti-cancer adjuvant for various cancers.

Description

Technical Field

[0001] The present invention relates to an anti-tumor adenovirus and an anti-cancer composition containing the same. Background Art

[0002] Cancer is one of the diseases with the highest number of deaths worldwide. The development of innovative cancer therapeutics can create high added value while reducing the medical expenses incurred by cancer treatment. In addition, according to statistics in 2008, molecular therapeutics that can overcome the resistance of existing anticancer drugs accounted for a market size of US$17.5 billion in seven major countries (US, Japan, France, Germany, Italy, Spain, and UK). In 2018, the market size was approximately US$45 billion, and it is expected to increase by 9.5% compared to 2008. Cancer treatment is divided into surgery, radiotherapy, chemotherapy, and biological therapy. Among them, chemotherapy is a treatment method that inhibits the proliferation of cancer cells or kills cancer cells by using chemical substances. Since most of the toxicity exhibited by anticancer drugs is also exhibited in normal cells, they have a certain degree of toxicity. Since anticancer drugs will develop drug resistance after a certain period of use, which means that the original effect is lost, there is an urgent need to develop anticancer drugs that selectively act on cancer cells and do not produce drug resistance (Current Status of Cancer Control Biowave 2004.6(19)). Recently, by securing molecular genetic information about cancer, new anticancer drugs that target the molecular characteristics of cancer have been developed. It has been reported that anticancer drugs that target specific molecular targets that only cancer cells have will not produce drug resistance.

[0003] The technology of inhibiting gene expression is an important tool in the development of therapeutic agents for treating diseases and in target testing. After its action was discovered, RNA interference (hereinafter referred to as RNAi) was confirmed to act on sequence-specific messenger RNA (mRNA) in a variety of mammalian cells (Silence of the transcripts: RNA interference in medicine. J Mol Med (2005) 83: 764-773). RNAi is a phenomenon in which a small interfering RNA (hereinafter referred to as siRNA) with a double helical structure of 21 to 25 nucleotides is specifically bound to a transcript (mRNA transcript) with a complementary sequence to decompose the transcript and thereby inhibit the expression of a specific protein. In cells, double-stranded ribonucleic acid (RNA) is converted into 21-23 double-stranded (basepair, bp) small interfering RNA by endonuclease called nuclease (Dicer). Small interfering RNA will combine with RNA-induced silencing complex (RISC) to enable the guide (antisense) strand to recognize the target messenger RNA and decompose it, thereby sequence-specifically inhibiting the expression of the target gene (NUCLEIC-ACID THERAPEUTICS: BASIC PRINCIPLES AND RECENT APPLICATIONS. Nature Reviews Drug Discovery. 2002.1, 503-514). According to the report of Bertrand's research team, compared with antisense oligonucleotides (ASO), small interfering RNA (siRNA) for the same target gene has excellent inhibitory effect on messenger RNA (mRNA) expression in vitro and in vivo, and this effect can be sustained (Comparison of antisense oligonucleotides and siRNAs in cell culture and in vivo. Biochem. Biophys. Res. Commun. 2002. 296: 1000-1004).According to analysis, the market for therapeutic agents based on RNA interference (RNAi) technology including small interfering RNA (siRNA) will reach a total market size of more than 12 trillion won worldwide by 2020. The targets of application of this technology will be unprecedentedly expanded, and it is considered to be a next-generation gene therapy technology that can treat diseases that are difficult to treat with existing antibody-based and compound-based drugs. In addition, the mechanism of action of small interfering RNA (siRNA) is to regulate the expression of target genes in a sequence-specific manner by complementary binding to target messenger RNA (mRNA). Therefore, it takes a long time and a lot of development costs to optimize existing antibody-based drugs or chemical substances (small molecule drugs) for specific protein targets. However, compared with this, the targets of application will be unprecedentedly expanded, the development time will be shortened, and there is an advantage that guide compounds that can be optimized for all protein targets including target substances that cannot be drugged can be developed (Progress Towards in Vivo Use of siRNAs. MOLECULAR THERAPY. 2006 13(4):664-670). For this reason, the following research has been conducted recently, that is, while being able to provide solutions to the problems caused by the RNA-mediated interference phenomenon in the development of existing chemical synthetic drugs, by selectively inhibiting the expression of specific proteins at the transcript level, it can be used for the development of various disease therapeutic agents, especially for the development of tumor therapeutic agents. In addition, unlike existing anticancer drugs, small interfering RNA (siRNA) therapeutic agents have the advantage of predictable side effects due to clear targets. In terms of this target specificity, for tumors, which are diseases caused by various genetic problems, this target specificity may instead become a reason for the lack of obvious therapeutic effects. Summary of the invention

[0004] Technical issues

[0005] The purpose of the present invention is to provide an anti-tumor adenovirus.

[0006] Furthermore, the present invention aims to provide a cancer treatment composition.

[0007] Technical Solution

[0008] In order to achieve the above-mentioned object, the present invention provides an anti-tumor adenovirus comprising a base sequence that uses a first nucleic acid as a target sequence and a base sequence that uses a second nucleic acid as a target sequence.

[0009] Furthermore, the present invention provides a cancer treatment composition comprising the above anti-tumor adenovirus.

[0010] Effects of the Invention

[0011] According to the present invention, the double-stranded small interfering ribonucleic acid (siRNA) of the present invention promotes the killing of cancer cells by simultaneously inhibiting the expression of the first nucleic acid and the second nucleic acid, and its anti-cancer activity is significantly better than that of treating each small interfering ribonucleic acid (siRNA) together, and it has the effect of synergistically improving the cancer cell killing efficacy during the combined treatment with anticancer agents. The adenovirus containing an expression cassette encoding a short hairpin ribonucleic acid (shRNA) for expressing an anti-tumor adenovirus and a human telomerase reverse transcriptase (hTERT) promoter has the effect of avoiding the in vivo immune response to specifically deliver to cancer cells and achieve systemic therapeutic efficacy, can achieve local delivery, has excellent selectivity, and can present significant anti-cancer effects through minimally invasive treatment, and can be effectively used as an anti-cancer composition or anti-cancer adjuvant for various cancers. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The figure shows a vector map for expressing the short hairpin RNA containing a double-target small interfering RNA group of the present invention in cells.

[0013] Figure 2 The diagrams are for confirming the mTOR or STAT3 gene expression inhibitory effects of the double-target double-stranded small interfering RNAs of Groups 1 to 9 of the present invention.

[0014] Figure 3 The diagrams show the expression inhibitory effects of the BCL2 gene (left) and the BI-1 gene (right) by the dual-target small interfering RNA set 10 (si-BB1) of the present invention.

[0015] Figure 4 A diagram to confirm the expression inhibitory effect of BCL2 gene and BI-1 gene based on the dual-target small interfering RNA group 11 to group 15 of the present invention, wherein NC is the control group small interfering RNA, and si-BB2 to si-BB6 are the small interfering RNA groups 11 to group 15 of the present invention.

[0016] Figure 5 The figure is a diagram to confirm the expression inhibition effect of AR gene and mTOR gene based on the dual-target small interfering RNA group 16 of the present invention in cancer cell lines, wherein A is h460 cell line, B is pc3 cell line, NC is control group small interfering RNA, siAR is small interfering RNA for AR, simTOR is small interfering RNA for mTOR, and si-AT1 is AR and mTOR dual-target small interfering RNA group 16 of the present invention.

[0017] Figure 6The figure is a diagram to confirm the expression inhibition effect of AR gene and mTOR gene in A549 cell line based on the dual-target small interfering RNA group 17 to group 28 of the present invention, wherein NC is the control group small interfering RNA, and si-AT2 to si-AT13 are the small interfering RNA groups 17 to group 28 of the present invention.

[0018] Figure 7 The graphs show the expression inhibition effects of c-MET and PD-L1 genes confirmed in various cancer cell lines by the double-target small interfering RNA (double strand) of the present invention that can simultaneously inhibit c-MET and PD-L1.

[0019] Figure 8 This is a graph showing the expression levels of mTOR and STAT3 based on the amount of deoxyribonucleic acid (DNA) in the short hairpin RNA expression cassette, based on a vector containing a sequence encoding the TTGGATCCAA circular short hairpin RNA sequence of sequence number 66 or the TTCAAGAGAG circular short hairpin RNA sequence of sequence number 67.

[0020] Fig. 9 The figure compares the gene expression inhibitory effect after tandem connection of two single-target small interfering RNAs and the gene expression inhibitory effect of the dual-target short hairpin RNA of the present invention.

[0021] Fig.10 This is a diagram to confirm the cell survival rate of human lung cancer cell line A549 cells when mTOR and STAT3 are simultaneously inhibited by the dual-target small interfering RNA of the present invention (dual-target small interfering RNA in Groups 1 to 9).

[0022] Fig.11 This is a diagram for confirming the cell viability of human lung cancer cell line A549 cells when mTOR and STAT3 are simultaneously inhibited by the dual-target small interfering RNA of the present invention after cisplatin treatment.

[0023] Fig.12 This is a diagram for confirming the cell viability of human lung cancer cell line A549 cells when mTOR and STAT3 are simultaneously inhibited by the dual-target small interfering RNA of the present invention after paclitaxel treatment.

[0024] Fig.13 This is a diagram for confirming the cell viability of human lung cancer cell line A549 cells when mTOR and STAT3 are simultaneously inhibited by the dual-target small interfering RNA of the present invention after 5-FU (5-fluorouracil) treatment.

[0025] Fig.14A diagram is provided to confirm the killing of cancer cells achieved by the combined treatment of the dual-target small interfering RNA group of the present invention and an anticancer agent, wherein A represents the combined treatment of anticancer agent + Bcl2 small interfering RNA + BI-1 small interfering RNA, and B represents the combined treatment of the dual-target small interfering RNA group 10 (si-BB1) of the present invention + an anticancer agent.

[0026] Fig.15 The diagrams show the cancer cell killing effects of ABT-737, a Bcl2 inhibitor used as an anticancer agent, and the dual-target small interfering RNA set 10 (si-BB1) of the present invention, and the synergistic effect of their combined use.

[0027] Fig.16 The graph compares the cancer cell killing effect of the combined treatment of dual-target siRNA group 10 (si-BB1) and an anticancer agent and the group co-treated with siRNA for the BCL2 gene and siRNA for the BI-1 gene.

[0028] Fig.17 The figure is a diagram to confirm the killing effect of cancer cells treated with anticancer agents based on dual-target small interfering RNA group 1 in cancer cell lines, wherein NC is the control group small interfering RNA, no treat is the control group not treated with anticancer agents, si-AT1 is the AR and mTOR dual-target small interfering RNA group 16 of the present invention, A is the DU145 cell line, and B is the H460 cell line.

[0029] Fig.18 This is a diagram schematically showing the structure of the adenovirus of the present invention.

[0030] Fig.19 It is a diagram showing the vector map of the adenoviral vector of the present invention, wherein Bs-short hairpin RNA is the insertion site of the sequence encoding the double-target short hairpin RNA of the present invention.

[0031] Fig. 20 The diagram shows the expression inhibitory effect of the recombinant adenovirus CA102 of the present invention, which encodes and expresses the human telomerase reverse transcriptase (hTERT) promoter and the dual-target short hairpin RNA, on the mTOR and STAT3 genes in the bladder cancer cell lines T24 and 253JBV.

[0032] Fig.21 The graph shows the expression inhibitory effect of mTOR and STAT3 genes confirmed in head and neck cancer cell lines FaDu and HSC-2 by the recombinant adenovirus CA102 of the present invention encoding and expressing human telomerase reverse transcriptase (hTERT) promoter and double-target short hairpin RNA.

[0033] Fig. 22 The diagram shows the expression inhibitory effect of the recombinant adenovirus CA102 of the present invention, which encodes and expresses the human telomerase reverse transcriptase (hTERT) promoter and the dual-target short hairpin RNA, on the mTOR and STAT3 genes in the skin squamous cell carcinoma cell lines A431 and HSC-5.

[0034] Fig.23 The diagram shows the expression-suppressing effect of the recombinant adenovirus CA102 of the present invention on mTOR and STAT3 genes in bladder cancer cell lines T24 and 253J-BV at the protein level.

[0035] Fig.24 This is a diagram confirming the expression inhibitory effect of BCL2 and BI-1 genes by the recombinant adenovirus CA101 of the present invention comprising a human telomerase reverse transcriptase (hTERT) promoter and a dual-target short hairpin RNA expression cassette.

[0036] Fig.25 The figure shows the expression inhibition effect of the recombinant adenovirus CA103 of the present invention comprising the human telomerase reverse transcriptase (hTERT) promoter and the dual-target short hairpin RNA expression cassette on the AR and mTOR genes confirmed in the prostate cancer cell line LNcap.

[0037] Fig.26 The figure shows the expression inhibitory effect of AR and mTOR genes confirmed in vitro in prostate cancer cell lines C42B and 22Rv1 based on the recombinant adenovirus CA103 of the present invention comprising a human telomerase reverse transcriptase (hTERT) promoter and a dual-target short hairpin RNA expression cassette.

[0038] Fig. 27 The diagram shows the in vivo confirmation of the expression inhibitory effect of AR and mTOR genes by the recombinant adenovirus CA103 of the present invention comprising a human telomerase reverse transcriptase (hTERT) promoter and a dual-target short hairpin RNA expression cassette.

[0039] Fig.28 This is a diagram confirming the expression inhibitory effect of c-MET and PD-L1 genes by the recombinant adenovirus CA104 of the present invention comprising a human telomerase reverse transcriptase (hTERT) promoter and a dual-target short hairpin RNA expression cassette.

[0040] Fig.29 The diagram is a diagram for confirming the killing effect of the cancer cell line by the recombinant adenovirus CA101 of the present invention comprising a human telomerase reverse transcriptase (hTERT) promoter and a dual-target short hairpin RNA expression cassette.

[0041] Fig.30The diagram is a diagram showing the killing effect of the recombinant adenovirus CA102 of the present invention on bladder cancer cell lines RT4, T24, and 253J-BV.

[0042] Fig.31 This is a diagram showing the killing effect of the recombinant adenovirus CA102 of the present invention on the head and neck cancer cell lines FaDu and HSC-2.

[0043] Fig.32 The diagrams show the killing effect of the recombinant adenovirus CA102 of the present invention on the skin squamous cell carcinoma cell lines A431 and HSC-5.

[0044] Fig.33 This is a diagram showing the killing effect of the recombinant adenovirus CA103 of the present invention comprising a human telomerase reverse transcriptase (hTERT) promoter and a dual-target short hairpin RNA expression cassette on the cancer cell line LNcap.

[0045] Fig.34 The diagram shows the killing effect of the recombinant adenovirus CA103 of the present invention comprising the human telomerase reverse transcriptase (hTERT) promoter and the dual-target short hairpin RNA expression cassette on the cancer cell lines C42B and 22Rv1.

[0046] Fig.35 The graphs show the confirmation of the anticancer effect of the recombinant adenovirus CA102 of the present invention on bladder cancer cells (253J-BV) in vivo.

[0047] Fig.36 The graphs show the confirmation of the anticancer effect of the recombinant adenovirus CA102 of the present invention on head and neck cancer cells (FaDu) in vivo.

[0048] Fig.37 This is a diagram confirming the anticancer effect of the recombinant adenovirus CA102 of the present invention on a tumor (bladder cancer) formed in vivo.

[0049] Fig.38 This is a graph confirming the anticancer effect of CA102 on a tumor (bladder cancer) formed in vivo based on the number of administrations.

[0050] Fig.39 This is a diagram showing the in vivo confirmation of the therapeutic effect on glioblastoma based on the dosage of the recombinant adenovirus CA102 of the present invention.

[0051] Fig.40 The figure shows the in vivo confirmation of the prostate cancer therapeutic effect of the recombinant adenovirus CA103 of the present invention comprising a human telomerase reverse transcriptase (hTERT) promoter and a dual-target short hairpin RNA expression cassette.

[0052] Fig.41 The diagram shows the in vivo confirmation of the therapeutic effect of the recombinant adenovirus CA102 of the present invention and its combined use with cisplatin on bladder cancer.

[0053] Preferred Embodiments

[0054] The present invention is described in detail below by way of examples of the present invention. However, the following examples are illustrative of the present invention, and the present invention is not limited thereto, and various modifications and applications of the present invention can be made within the description in the attached claims and the equivalent scope interpreted thereby.

[0055] Unless otherwise indicated, nucleic acids are recorded from left to right in the direction of 5'→3'. Numerical ranges listed in the specification are inclusive of the numbers defining the range, including each integer or any non-integer division within the defined range.

[0056] Unless otherwise explained, all technical terms and scientific terms used in this application have the same meaning as those commonly understood by those of ordinary skill in the art to which the present invention belongs. Any methods and materials similar or equivalent to those described in this application may be used in the operations used to test the present invention, but preferred materials and methods will be described in this application.

[0057] In one embodiment, the present invention relates to an anti-tumor adenovirus comprising: a human telomerase reverse transcriptase (hTERT) promoter; and an expression cassette comprising a base sequence that uses a first nucleic acid as a target sequence and a base sequence that uses a second nucleic acid as a target sequence.

[0058] In one example, the base sequence of the first nucleic acid as the target sequence and the base sequence of the second nucleic acid as the target sequence can be partially reverse complementary sequences or 100% reverse complementary sequences. When expressed in vivo, the sequence of the first nucleic acid as the target sequence and the sequence of the second nucleic acid as the target sequence form a double strand, preferably, a short hairpin ribonucleic acid (shRNA) can be formed.

[0059] In one example, the base sequence of the first nucleic acid as the target sequence and the base sequence of the second nucleic acid as the target sequence can be combined in a partially complementary or 100% complementary manner to form a double strand during gene expression.

[0060] In one example, the human telomerase reverse transcriptase (hTERT) promoter can be operably linked to an endogenous gene of an adenovirus.

[0061] In the present invention, the term "operably linked" refers to the functional combination between a gene expression regulatory sequence (e.g., a promoter, a signal sequence, or an array of transcriptional regulatory factor binding sites) and other gene sequences, so that the above-mentioned regulatory sequence will regulate the transcription and / or interpretation of the above-mentioned other gene sequences.

[0062] In one example, the human telomerase reverse transcriptase (hTERT) promoter may include the base sequence of SEQ ID NO: 74. In addition, the human telomerase reverse transcriptase (hTERT) promoter sequence may include various known variant sequences.

[0063] In one example, the endogenous gene of the adenovirus may have a structure of 5'ITR-C1-C2-C3-C4-C5 3'ITR, the C1 may include E1A (sequence number 75), E1B (sequence number 77) or E1A-E1B, the C2 may include E2B-L1-L2-L3-E2A-L4, the C3 may not include E3 or include E3, the C4 may include L5, the C5 may not include E4 or include E4, and may include the base sequence of sequence number 78.

[0064] In one example, the E3 region of the adenovirus may be partially deleted, and the deleted base sequence may include the base sequence of sequence number 82.

[0065] In one example, the expression cassette can be located at the C3 site of an endogenous gene of an adenovirus.

[0066] In one example, the human telomerase reverse transcriptase (hTERT) promoter can be operably linked to E1A and E1B of the endogenous genes of the adenovirus.

[0067] In one example, an internal ribosome entry site (IRES) sequence (SEQ ID NO: 76) may be further included between E1A and E1B of the endogenous gene of the adenovirus.

[0068] In one example, the expression cassette can encode and express short hairpin RNA (shRNA).

[0069] In one example, the short hairpin RNA (shRNA) can inhibit the expression of the first nucleic acid and the second nucleic acid at the same time.

[0070] In one example, the anti-tumor adenovirus of the present invention can degrade the messenger RNA (mRNA) of nucleic acid or inhibit translation through RNA interference, thereby inhibiting expression.

[0071] In one example, the expression cassette of the present invention can simultaneously inhibit the first nucleic acid and the second nucleic acid by expressing a double-stranded small interfering ribonucleic acid (siRNA) whose specific sense strand is complementary to the first nucleic acid or the second nucleic acid portion and whose specific antisense strand is complementary to the second nucleic acid or the first nucleic acid portion.

[0072] The term "expression inhibition" used in the present invention refers to causing a decrease in the expression or translation of a target gene, preferably, to the point where the expression of the target gene cannot be detected or reaches a meaningless level.

[0073] The term "small interfering RNA (siRNA)" used in the present invention refers to a short double-stranded RNA (RNA) that causes RNA interference (RNAi, RNA interference) phenomenon by cleavage of a specific messenger RNA (mRNA). Generally, a small interfering RNA (siRNA) is composed of a sense RNA (RNA) chain having the same sequence as the messenger RNA (mRNA) of the target gene and an antisense RNA (RNA) chain having a sequence complementary thereto, but the double-stranded small interfering RNA (siRNA) of the present invention is a small interfering RNA (siRNA) specific to the first nucleic acid or the second nucleic acid (antisense chain to the first nucleic acid or the second nucleic acid) in which the sense RNA (RNA) chain is specific to the second nucleic acid or the first nucleic acid (antisense chain to the second nucleic acid or the first nucleic acid), and thus the double-stranded small interfering RNA (siRNA) can simultaneously inhibit the first nucleic acid or the second nucleic acid or inhibit their expression, respectively.

[0074] The term "short hairpin RNA (shRNA)" in the present invention refers to the following ribonucleic acid, that is, by partially including a striated base sequence in a single-stranded ribonucleic acid (RNA) to form a double-stranded structure in the 3' region and a hairpin-like structure, which can be converted into a small interfering ribonucleic acid (siRNA) by cleavage by dicer, a type of ribonuclease (RNase) present in the cell after expression in the cell. The length of the above-mentioned double-stranded structure is not particularly limited, and is preferably more than 10 nucleotides, and more preferably more than 20 nucleotides. In the present invention, the above-mentioned short hairpin RNA (shRNA) can be included in an expression cassette, and the above-mentioned short hairpin RNA (shRNA) can be generated as follows, that is, in a group sequence consisting of an antisense chain and a sense chain of a small interfering RNA (siRNA) for each gene, after converting U to T, TTGGATCCAA (TTGGATCCAA loop) or TTCAAGAGAG (TTCAAGAGAG loop), the antisense chain and TT are connected to the 3' of the sense chain to prepare an expression cassette encoding the short hairpin RNA (shRNA) so that it is expressed in cells.

[0075] In one example, the first nucleic acid may include a base sequence having 60% or more complementarity with the reverse complementary sequence of the second nucleic acid, and the second nucleic acid may include a base sequence having 60% or more complementarity with the reverse complementary sequence of the first nucleic acid.

[0076] In one example, the first nucleic acid may include a base sequence that is 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% complementary to the reverse complementary sequence of the second nucleic acid, and the second nucleic acid may include a base sequence that is 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% complementary to the reverse complementary sequence of the first nucleic acid.

[0077] Variants of the base sequence of the first nucleic acid contained in the expression cassette as the target sequence or the base sequence of the second nucleic acid as the target sequence are included in the scope of the present invention. The concept of the expression cassette of the present invention is as follows, that is, it includes functional equivalents of the nucleic acid molecules constituting it, for example, including variants that can functionally play the same role as the base sequence molecules although part of the base sequence of the nucleic acid molecule is deformed by deletion, substitution or insertion. The "sequence homology %" for nucleic acid molecules can be confirmed by comparing two optimally arranged sequences and a comparison region, and a part of the nucleic acid molecule sequence in the comparison region may include additions or deletions compared to the reference sequence (excluding additions or deletions) for the optimal arrangement of the two sequences.

[0078] In one example, the nucleic acid can be a gene associated with cancer.

[0079] In one example, the gene associated with cancer can be an oncogene whose expression increases in cancer, and the oncogene can be an apoptosis-related gene, a transcription factor gene, a metastasis-related gene, an angiogenesis-related gene, a cancer cell-specific gene, or a tyrosine-kinase gene.

[0080] In one embodiment, the apoptosis-related gene can be ABL1, AKT1, AKT2, BARD1, BAX, BCL11B, BCL2, BCL2A1, BCL2L1, BCL2L12, BCL3, BCL6, BIRC2, BIRC3, BIRC5, BRAF, CARD11, CAV1, CBL, CDC25A, CDKN1A, CFLAR, CNR2, CTNNB1, CUL4A, DAXX, DDIT3, E2F1, E2F3, E2F 5. ESPL1, FOXO1, HDAC1, HSPA5, IGF1R, IGF2, JUN, JUNB, JUND, MALT1, MAP3K7, MCL1, MDM2, MDM4, MYB, MYC, NFKB2, NP M1, NTRK1, PAK1, PAX3, PML, PRKCA, PRKCE, PTK2B, RAF1, RHOA, TGFB1, TNFRSF1B, TP73, TRAF6, YWHAG, YWHAQ or YWHAZ,The above-mentioned transcription factor gene can be AR, ARID3A, ASCL1, ATF1, ATF3, BCL11A, BCL11B, BCL3, BCL6, CDC5L, CDX2, CREB1, CUX1, DDIT3, DLX5, E2F1, E2F3, E2F5, ELF4, ELK1, ELK3, EN2, ERG, ETS1, ETS2, ETV1, ETV3, ETV4, ETV6, FEV, FEZF1, FLI1, FOS, FOSL1, FOXA1, FOXG1, FOXM1, FO XO1, FOXP1, FOXQ1, GATA1, GATA6, GFI1, GFI1B, GLI1, GLI2, GLI3, HES6, HHEX, HLF, HMGA1, HMGA2, HOXA1, HOXA9, HOXD13, HOXD 9. ID1, ID2, IKZF1, IRF2, IRF4, JUN, JUNB, JUND, KAT6A, KDM2A, KDM5B, KLF2, KLF4, KLF5, KLF6, KLF8, KMT2A, LEF1, LHX1, LMX1B , MAF, MAFA, MAFB, MBD1, MECOM, MEF2C, MEIS1, MITF, MYB, MYC, MYCL, MYCN, NANOG, NCOA3, NFIB, NFKB2, NKX2-1, OTX2, PATZ1, P AX2, PAX3, PAX4, PAX8, PBX1, PBX2, PITX2, PLAG1, PLAGL2, PPARG, PPP1R13L, PRDM10, PRDM13, PRDM14, PRDM15, PRDM16, PRDM6, PRDM8, PRDM9, RARA, REL, RERE, RUNX1, RUNX3, SALL4, SATB1, SFPQ, SIX1, SNAI1, SOX2, SOX4, SPI1, SREBF1, STAT3, TAF1, TAL1, TAL2, TBX2, TBX3, TCF3, TFCP2, TFE3, THRA, TLX1, TP63, TP73, TWIST1, WT1, YBX1, YY1, ZBTB16, ZBTB7A, ZIC2, ZNF217 or ZNF268,The metastasis-related gene may be AKT1, AKT2, AR, CBL, CDH1, CRK, CSF1, CTNNB1, CTTN, CXCR4, EGFR, FGFR1, FLT3, FYN, GLI1, ILK, ITGA3, JAK2, MET, PDGFRB, PLXNB1, PRKCI, PTCH1, PTPN11, RAC1, RHOA, RHOC, ROCK1, SMO, SNAI1, SRC, TCF3 or WT1, and the angiogenesis-related gene may be BRAF, CAV1, CTGF, EGFR, ERBB2, ETS1, FGF4, FGF6, FGFR1, FGFR3 , FGFR4, ID1, NRAS, PDGFB, PDGFRA, PDGFRB or SPARC, the above tyrosine kinase gene can be ABL1, ABL2, ALK, AXL, BLK, EGFR, EPHA2, ERBB2, ERBB3, ERBB4, FES, FGFR1, FGFR2, FGFR3, FGFR4, FGR, FLT3, FYN, ITK, JAK1, JAK2, KIT, LCK, MERTK, MET, MST1R, NTRK1, NTRK3, PDGFRA, PDGFRB, PTK2B, PTK7, RET, ROS1, SRC, SYK, TEC or YES1. ,

[0081] In one example, the oncogenes can be SEPTIN9, ACOD1, ACTN4, ADAM28, ADAM9, ADGRF1, ADRBK2, AFF1, AFF3, AGAP2, AGFG1, AGRN, AHCYL1, AHI1, AIMP2, AKAP13, AKAP9, AKIRIN2, AKTIP, ALDH1A1, ALL1, ANIB1, ANP32C, ANP32D, AQP1, ARAF, ARHGEF1, ARHGEF2, ARHGEF5, ASPSCR1, AURKA, BAALC, BAIAP2L1, BANP, BCAR4, BCKDHB, BC L9, BCL9L, BCR, BMI1, BMP7, BOC, BRD4, BRF2, CABIN1, CAMK1D, CAPG, CBFB, CBLB, CBLL1, CBX7, CBX8, CCDC28A, CCDC6, CCNB1, CCNB2, CCND1, CCNE1, CCNL 1. CD24, CDC25C, CDC6, CDH17, CDK1, CDK14, CDK4, CDK5R2, CDK6, CDK8, CDKN1B, CDKN3, CDON, CEACAM6, CENPW, CHD1L, CHIC1, CHL1, CKS1B, CMC4, CNTN2, C OPS3, COPS5, CRKL, CRLF2, CROT, CRTC1, CRYAB, CSF1R, CSF3, CSF3R, CSNK2A1, CSNK2A2, CT45A1, CTBP2, CTNND2, CTSZ, CUL7, CXCL1, CXCL2, CXCL3, CYGB , CYP24A1, DCD, DCUN1D1DDB2, DDHD2, DDX6, DEK, DIS3, DNPH1, DPPA2, DPPA4, DSG3, DUSP12, DUSP26, ECHS1, ECT2, EEF1A1, EEF1A2, EEF1D, EIF3E, EIF3I, EIF4E, EIF5A2, ELAVL1, ELL, EML4, EMSY, ENTPD5, EPCAM, EPS8, ERAS, ERGIC1, ERVW-1, EVI2A, EVI5, EWSR1, EZH2, FAM189B, FAM72A, FAM83D, FASN, FDPS, FGF10, FGF3, FGF5, FGF8, FR1OP, FHL2, FIP1L1, FNDC3B, FRAT1, FUBP1, FUS, FZD2, GAB2, GAEC1, GALNT10, GALR2, GLO1, GMNN, GNA12, GNA13, GNAI2, GNAQ,GNAS、GOLPH3、GOPC、GPAT4、GPM6A、GPM6B、GPR132、GREM1、GRM1、GSK3A、GSM 1、H19、HAS1、HAX1、HDGFRP2、HMGN5、HNRNPA1、HOTAIR、HOTTIP、HOXA-AS2、HR AS、HSPA1A、HSPA4、HSPB1、HULC、IDH1、IFNG、IGF2BP1、IKBKE、IL7R、INNPPL1 IRS2、IST1、JUP、INTS2、INTS3、INTS4、INTS5、INTS7 KIA0101、KIAA1524、KIF14、KRAS、KSR2、LAMTOR5、LAPTM4B、LCN2、LDHB、LE TMD1、LIN28A、LIN28B、LMO1、LMO2、LMO3、LMO4、LSM1、LUADT1、MACC1、MACROD 1、MAGEA11、MALAT1、MAML2、MAP3K8、MAPRE1、MAS1、MCC、MCF2、MCF2L、MCTS1 、MEFV、MFHAS1、MFNG、MIEN1、MINA、MKL2、MLANA、MLLT1、MLLT11、MLLT3、MLLT 4、MMP12、MMS22L、MN1、MNAT1、MOS、MPL、MPST、MRAS、MRE11A、MSI1、MTCP1、M TDH、Engine、MUC1、MUC4、MUM1、MYD88、NAAA、NANOGP8、NBPF12、NCOA4、NEAT1、N ECTIN4、NEDD4、NEDD9、NET1、NINL、NME1、NOTCH1、NOTCH4、NOV、NSD1、NUAK2 NUP214、NUP98、NUTM1、OLR1、PA2G4、PADI2、PAK7、PARK7、PARM1、PBK、PCAT1 、PCAT5、PDGFA、PDZK1IP1、PELP1、PFN1P3、PIGU、PIK3CA、PIK3R1、PIM1、PIM 2、PIM3、PIR、PIW1、PLAC8、PLK1、PPM1D、PPP1R10、PPP1R14A、PPP2R1A、PRA ME、PRDM12、PRMT5、PSIP1、PSMD10、PTCH2、PTMA、PTP4A1、PTP4A2、PTP4A3、P TTG1、PTTG1IP、PTTG2、PVT1、RAB11A、RAB18、RAB22A、RAB23、RAB8A、RALGDS、RAP1A, RASSF1, RBM14, RBM15, RBM3, RBMY1A1, RFC3, RGL4, RGR, RHO, RING1, RINT1, RIT1, RNF43, RPL23, RRAS, RRAS2, RSF1, RUN X1T1, S100A4, S100A7, S100A8, SAG, SART3, SBSN, SEA, SEC62, SERTAD1, SERTAD2, SERTAD3, SET, SETBP1, SETDB1, SGK1, SIRT1, SIRT6, SKI, SKIL, SKP2, SLC12A5, SLC3A2, SMR3B, SMURF1, SNCG, SNORA59A, SNORA80E, SPAG9, SPATA4, SPRY2, SQSTM1, SRSF1, S RSF2, SRSF3, SRSF6, SS18, SSX1, SSX2, SSX2B, STIL, STMN1, STRA6, STYK1, SUZ12, SWAP70, SYT1, TAC1, TACSTD2, TAF15, TALDO1 , TAZ, TBC1D1, TBC1D15, TBC1D3, TBC1D3C, TBC1D7, TCL1A, TCL1B, TCL6, TCP1, TFG, TGM3, TINCR, TKTL1, TLE1, TMEM140, TMPOP2 , TMPRSS2, TNS4, TPD52, TPR, TRE17, TREH, TRIB1, TRIB2, TRIM28, TRIM32, TRIM8, TRIO, TRIP6, TSPAN1, TSPY1, TXN, TYMS, TYRP 1. UBE2C, UBE3C, UCA1, UCHL1, UHRF1, URI1, USP22, USP4, USP6, VAV1, VAV2, VAV3, VIM, WAPL, WHSC1, WHSC1L1, WISP1, WNT1, WNT 10A, WNT10B, WNT2, WNT3, WNT5A, WWTR1, XCL1, XIAP, YAP1, YEATS4, YY1AP1, ZEB1-AS1, ZFAND4, ZFAS1, ZMYM2, ZNF703, or ZNHIT6. ,

[0082] In one example, the cancer cell specific gene may be PD-L1 (Programmed death-ligand 1) expressed on the surface of tumor cells.

[0083] In one example, the first nucleic acid and the second nucleic acid targeted by the transcript of the expression cassette of the present invention can be selected from ABL1, AKT1, AKT2, BARD1, BAX, BCL11B, BCL2, BCL2A1, BCL2L1, BCL2L12, BCL3, BCL6, BIRC2, BIRC3, BIRC5, BRAF, CARD11, CAV1, CBL, CDC25A, CDKN1A, CFLAR, c-MET, CNR2, CTNNB1, CUL4A, DAXX, DDIT3, E2F1, E2F3, E2F5, ESPL1, FOXO1, HDAC1, HSPA5, IGF1R, IGF 2. JUN, JUNB, JUND, MALT1, MAP3K7, MCL1, MDM2, MDM4, MYB, MYC, NFKB2, NPM1, NTRK1, PAK1, PAX3, PML, PRKCA, PRKCE, PTK2B, RAF1, RHOA, TGFB1, TNFRSF1B , TP73, TRAF6, YWHAG, YWHAQ, YWHAZ, AR, ARID3A, ASCL1, ATF1, ATF3, BCL11A, BCL11B, BCL3, BCL6, CDC5L, CDX2, CREB1, CUX1, DDIT3, DLX5, E2F1, E2F3, E2 F5, ELF4, ELK1, ELK3, EN2, ERG, ETS1, ETS2, ETV1, ETV3, ETV4, ETV6, FEV, FEZF1, FLI1, FOS, FOSL1, FOXA1, FOXG1, FOXM1, FOXO1, FOXP1, FOXQ1, GATA1, GA TA6, GFI1, GFI1B, GLI1, GLI2, GLI3, HES6, HHEX, HLF, HMGA1, HMGA2, HOXA1, HOXA9, HOXD13, HOXD9, ID1, ID2, IKZF1, IRF2, IRF4, JUN, JUNB, JUND, KAT6A, KDM2A, KDM5B, KLF2, KLF4, KLF5, KLF6, KLF8, KMT2A, LEF1, LHX1, LMX1B, MAF, MAFA, MAFB, MBD1, MECOM, MEF2C, MEIS1, MITF, MYB, MYC, MYCL, MYCN, NANOG, NCOA3, NFIB, NFKB2, NKX2-1, OTX2, PATZ1, PAX2, PAX3, PAX4, PAX8, PBX1, PBX2, PD-L1, PITX2, PLAG1, PLAGL2, PPARG, PPP1R13L, PRDM10, PRDM13, PRDM14,<h2 style=";text-align:left;direction:ltr">PRDM15、PRDM16、PRDM6、PRDM8、PRDM9、RARA、REL、RERE、RUNX1、RUNX3、SALL 4、SATB1、SFPQ、SIX1、SNAI1、SOX2、SOX4、SPI1、SREBF1、STAT3、TAF1、TAL1、 TAL2、TBX2、TBX3、TCF3、TFCP2、TFE3、THRA、TLX1、TP63、TP73、TWIST1、WT1、 YBX1、YY1、ZBTB16、ZBTB7A、ZIC2、ZNF217、ZNF268、AKT1、AKT2、AR、CBL、CDH1 、CRK、CSF1、CTNNB1、CTTN、CXCR4、EGFR、FGFR1、FLT3、FYN、GLI1、ILK、ITGA3 、JAK2、MET、PDGFRB、PLXNB1、PRKCI、PTCH1、PTPN11、RAC1、RHOA、RHOC、ROCK 1、SMO、SNAI1、SRC、TCF3、WT1、BRAF、CAV1、CTGF、EGFR、ERBB2、ETS1、FGF4、F GF6、FGFR1、FGFR3、FGFR4、ID1、NRAS、PDGFB、PDGFRA、PDGFRB、SPARC、ABL1、A BL2、ALK、AXL、BLK、EGFR、EPHA2、ERBB2、ERBB3、ERBB4、FES、FGFR1、FGFR2、F GFR3、FGFR4、FGR、FLT3、FYN、ITK、JAK1、JAK2、KIT、LCK、MERTK、MET、MST1R、 NTRK1, NTRK3, PDGFRA, PDGFRB, PTK2B, PTK7, RET, ROS1, SRC, SYK, TEC, YES1, SEPTIN9, ACOD1, ACTN4, ADAM28, ADAM9, ADGRF1, ADRBK2, AFF1, AFF3, AGAP2 AGFG1, AGRN, AHCYL1, AHI1, AIMP2, AKAP13, AKAP9, AKIRIN2, AKTIP, ALDH1A1, ALL1, ANIB1, ANP32C, ANP32D, AQP1, ARAF, ARHGEF1, ARHGEF2, ARHGEF5, A SPSCR1、AURKA、BAALC、BAIAP2L1、BANP、BCAR4、BCKDHB、BCL9、BCL9L、BCR、B MI1、BMP7、BOC、BRD4、BRF2、CABIN1、CAMK1D、CAPG、CBFB、CBLB、CBLL1、CBX7、<h2 style=";text-align:left;direction:ltr">CBX8、CCDC28A、CCDC6、CCNB1、CCNB2、CCND1、CCNE1、CCNL1、CD24、CDC25C、C DC6、CDH17、CDK1、CDK14、CDK4、CDK5R2、CDK6、CDK8、CDKN1B、CDKN3、CDON、C EACAM6、CENPW、CHD1L、CHIC1、CHL1、CKS1B、CMC4、CNTN2、COPS3、COPS5、CRK L、CRLF2、CROT、CRTC1、CRYAB、CSF1R、CSF3、CSF3R、CSNK2A1、CSNK2A2、CT45 A1, CTBP2, CTNND2, CTSZ, CUL7, CXCL1, CXCL2, CXCL3, CYGB, CYP24, A1, DCD, DCUN1, D1, DDB2, DDHD2, DDX6, DEK, DIS3, DNPH1, DPPA2, DPPA4, DSG3, DUSP12, D USP26、ECHS1、ECT2、EEF1A1、EEF1A2、EEF1D、EIF3E、EIF3I、EIF4E、EIF5A2、 ELAVL1、ELL、EML4、EMSY、ENTPD5、EPCAM、EPS8、ERAS、ERGIC1、ERVW-1、EVI2A 、EVI5、EWSR1、EZH2、FAM189B、FAM72A、FAM83D、FASN、FDPS、FGF10、FGF3、FG F5、FGF8、FR1OP、FHL2、FIP1L1、FNDC3B、FRAT1、FUBP1、FUS、FZD2、GAB2、GAE C1、GALNT10、GALR2、GLO1、GMNN、GNA12、GNA13、GNAI2、GNAQ、GNAS、GOLPH3、 GOPC、GPAT4、GPM6A、GPM6B、GPR132、GREM1、GRM1、GSK3A、GSM1、H19、HAS1、HA HS PA4、HSPB1、HULC、IDH1、IFNG、IGF2BP1、IKBKE、IL7R、INPPL1、INTS1、INTS2 INTS3, INTS4, INTS5, INTS7, INTS8, IRS2, IST1, JUP, KDM4C, KIAA0101, KIAA1524, KIF14, KRAS, KSR2, LAMTOR5, LAPTM4B, LCN2, LDHB, LETMD1, LIN28ALIN28B、LMO1、LMO2、LMO3、LMO4、LSM1、LUADT1、MACC1、MACROD1、MAGEA11、M ALAT1、MAML2、MAP3K8、MAPRE1、MAS1、MCC、MCF2、MCF2L、MCTS1、MEFV、MFHAS 1、MFNG、MIEN1、MINA、MKL2、MLANA、MLLT1、MLLT11、MLLT3、MLLT4、MMP12、MM S22L、MN1、MNAT1、MOS、MPL、MPST、MRAS、MRE11A、MSI1、MTCP1、MTDH、ENGINE、MU C1、MUC4、MUM1、MYD88、NAAA、NANOGP8、NBPF12、NCOA4、NEAT1、NECTIN4、NED D4、NEDD9、NET1、NINL、NME1、NOTCH1、NOTCH4、NOV、NSD1、NUAK2、NUP214、NUP 98、NUTM1、OLR1、PA2G4、PADI2、PAK7、PARK7、PARM1、PBK、PCAT1、PCAT5、PD- L1、PDGFA、PDZK1IP1、PELP1、PFN1P3、PIGU、PIK3CA、PIK3R1、PIM1、PIM2、PIM 3、PIR、PART1、PLAC8、PLK1、PPM1D、PPP1R10、PPP1R14A、PPP2R1A、PRAME、P RDM12、PRMT5、PSIP1、PSMD10、PTCH2、PTMA、PTP4A1、PTP4A2、PTP4A3、PTTG1、 PTTG1IP、PTTG2、PVT1、RAB11A、RAB18、RAB22A、RAB23、RAB8A、RALGDS、RAP1 A、RASSF1、RBM14、RBM15、RBM3、RBMY1A1、RFC3、RGL4、RGR、RHO、RING1、RINT1 RIT1、RNF43、RPL23、RRAS、RRAS2、RSF1、RUNX1T1、S100A4、S100A7、S100A8 、SAG、SART3、SBSN、SEA、SEC62、certainty1、certainty2、certainty3、SET、SETBP1、SE TDB1、SGK1、SIRT1、SIRT6、SKI、SKIL、SKP2、SLC12A5、SLC3A2、SMR3B、SMURF 1、SNCG、SNORA59A、SNORA80E、SPAG9、SWORD4、SPRY2、SQSTM1、SRSF1、SRSF2、SRSF3, SRSF6, SS18, SSX1, SSX2, SSX2B, STIL, STMN1, STRA6, STYK1, SUZ12, SWAP70, SYT1, TAC1, TACSTD2, TAF15, TALDO1, TAZ, TBC1D1, TBC1D15, TBC1D3, TBC1D 3C, TBC1D7, TCL1A, TCL1B, TCL6, TCP1, TFG, TGM3, TINCR, TKTL1, TLE1, TMEM140, TMPOP2, TMPRSS2, TNS4, TPD52, TPR, TRE17, TREH, TRIB1, TRIB2, TRIM28, TRIM32 、 TRIM8, TRIO, TRIP6, TSPAN1, TSPY1, TXN, TYMS, TYRP1, UBE2C, UBE3C, UCA1, UCHL1, UHRF1, URI1, USP22, USP4, USP6, VAV1, VAV2, VAV3, VIM, WAPL, WHSC1, WHSC1L1, WISP1, WNT1, WNT10A, WNT10B, WNT2, WNT3, WNT5A, WWTR1, XCL1, XIAP, YAP1, YEATS4, YY1AP1, ZEB1-AS1, ZFAND4, ZFAS1, ZMYM2, ZNF703 and ZNHIT6. 、

[0084] In one example, the first nucleic acid may be STAT3 (signal transducer and activator of transcription 3), and the second nucleic acid may be mTOR (mammalian target of rapamycin). In this case, the expression cassette may include a nucleic acid in which U is converted to T in the base sequence of SEQ ID NOs: 1 and 2, SEQ ID NOs: 3 and 4, SEQ ID NOs: 5 and 6, SEQ ID NOs: 7 and 8, SEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 12, SEQ ID NOs: 13 and 14, SEQ ID NOs: 15 and 16, or SEQ ID NOs: 17 and 18. In the above content, the small interfering RNA (siRNA) of sequence number 1 and sequence number 2 can complementarily bind to 17mer in 21mer, the small interfering RNA (siRNA) of sequence number 3 and sequence number 4 can complementarily bind to 16mer in 20mer, the small interfering RNA (siRNA) of sequence number 5 and sequence number 6 can complementarily bind to 15mer in 19mer, the small interfering RNA (siRNA) of sequence number 7 and sequence number 8 can complementarily bind to 14mer in 18mer, and the small interfering RNA (siRNA) of sequence number 9 and sequence number 10 can complementarily bind to 16mer in 20mer. The siRNA of sequence number 11 and sequence number 12 can complementarily bind to 16mer in 17mer, the siRNA of sequence number 13 and sequence number 14 can complementarily bind to 16mer in 19mer, the siRNA of sequence number 15 and sequence number 16 can complementarily bind to 15mer in 18mer, and the siRNA of sequence number 17 and sequence number 18 can complementarily bind to 14mer in 17mer. Furthermore, when the first nucleic acid is STAT3 and the second nucleic acid is mTOR, the short hairpin RNA (shRNA) expression DNA (DNA sequence encoding the dual target short hairpin RNA (shRNA) of STAT3 and mTOR) contained in the expression cassette can include the base sequence of sequence number 66 or sequence number 67.

[0085] In one example, the first nucleic acid may be BCL2 (B-cell lymphoma 2), and the second nucleic acid may be BI-1 (BAX inhibitor 1). In this case, the expression cassette may include the base sequences of sequence numbers 19 and 20, sequence numbers 21 and 22, sequence numbers 23 and 24, sequence numbers 25 and 26, sequence numbers 27 and 28, or sequence numbers 29 and 30. In the above content, the 21mer siRNA group 10 composed of sequence numbers 19 and 20 makes 15mers complementary to each other, the 20mer siRNA group 11 composed of sequence numbers 21 and 22 makes 14mers complementary to each other, the 20mer siRNA group 12 composed of sequence numbers 23 and 24 makes 14mers complementary to each other, the 19mer siRNA group 13 composed of sequence numbers 25 and 26 makes 13mers complementary to each other, the 19mer siRNA group 14 composed of sequence numbers 27 and 28 makes 13mers complementary to each other, and the 18mer siRNA group 15 composed of sequence numbers 29 and 30 makes 12mers complementary to each other. The small interfering RNA (siRNA) (Antisense Bcl-2) of sequence number 19, sequence number 21, sequence number 23, sequence number 25, sequence number 27 or sequence number 29 in Table 2 below can complementarily bind to the messenger RNA (mRNA) of Bcl-2, and the small interfering RNA (siRNA) (Antisense BI-1) of sequence number 20, sequence number 22, sequence number 24, sequence number 26, sequence number 28 or sequence number 30 can complementarily bind to the messenger RNA (mRNA) of BI-1. In addition, when the first nucleic acid is BCL2 and the second nucleic acid is BI-1, the short hairpin RNA (shRNA) expression deoxyribonucleic acid (DNA) contained in the expression cassette can include the base sequence of sequence number 68 or sequence number 69.

[0086] In one example, the first nucleic acid may be AR (androgen receptor) and the second nucleic acid may be mTOR (mammalian target of rapamycin). In this case, the expression cassette may include the base sequence of sequence numbers 31 and 32, sequence numbers 33 and 34, sequence numbers 35 and 36, sequence numbers 37 and 38, sequence numbers 39 and 40, sequence numbers 41 and 42, sequence numbers 43 and 44, sequence numbers 45 and 46, sequence numbers 47 and 48, sequence numbers 49 and sequence numbers 50, sequence numbers 51 and 52, sequence numbers 53 and 54, or sequence numbers 55 and 56. In the above content, the 20mer siRNA group 16 composed of sequence numbers 31 and 32 makes 18mers complementary to each other, the 19mer siRNA group 17 composed of sequence numbers 33 and 34 makes 17mers complementary to each other, the 18mer siRNA group 18 composed of sequence numbers 35 and 36 makes 16mers complementary to each other, the 17mer siRNA group 19 composed of sequence numbers 37 and 38 makes 15mers complementary to each other, the 19mer siRNA group 20 composed of sequence numbers 39 and 40 makes 15mers complementary to each other, the 18mer siRNA group 21 composed of sequence numbers 41 and 42 makes 14mers complementary to each other, and the 17mer siRNA group 22 composed of sequence numbers 43 and 44 makes 15mers complementary to each other. The siRNA group 22 makes 13mers complementary to each other, the siRNA group 23 of 23mer composed of sequence numbers 45 and 46 makes 19mers complementary to each other, the siRNA group 24 of 22mer composed of sequence numbers 47 and 48 makes 18mers complementary to each other, the siRNA group 25 of 22mer composed of sequence numbers 49 and 50 makes 18mers complementary to each other, the siRNA group 26 of 21mer composed of sequence numbers 51 and 52 makes 17mers complementary to each other, the siRNA group 27 of 20mer composed of sequence numbers 53 and 54 makes 16mers complementary to each other, and the siRNA group 28 of 21mer composed of sequence numbers 55 and 56 makes 17mers complementary to each other.The small interfering RNA (siRNA) (Antisense AR) of sequence number 31, sequence number 33, sequence number 35, sequence number 37, sequence number 39, sequence number 41, sequence number 43, sequence number 45, sequence number 47, sequence number 49, sequence number 51, sequence number 53 or sequence number 55 can complementarily bind to the messenger RNA (mRNA) of Ar, and the small interfering RNA (siRNA) (Antisense mTOR) of sequence number 32, sequence number 34, sequence number 36, sequence number 38, sequence number 40, sequence number 42, sequence number 44, sequence number 46, sequence number 48, sequence number 50, sequence number 52, sequence number 54 or sequence number 56 can complementarily bind to the messenger RNA (mRNA) of mTOR. Furthermore, when the first nucleic acid is AR and the second nucleic acid is mTOR, the short hairpin RNA (shRNA) expression deoxyribonucleic acid (DNA) contained in the expression cassette may include the base sequence of SEQ ID NO: 70 or SEQ ID NO: 71.

[0087] In one example, the first nucleic acid may be MGMT (O-6-methylguanine-DNAmethyltransferase), and the second nucleic acid may be mTOR. In this case, the expression cassette may include the base sequences of SEQ ID NO: 57 and SEQ ID NO: 58.

[0088] In one example, the first nucleic acid may be BCL2, and the second nucleic acid may be MCL1 (MCL1 apoptosis regulator). In this case, the expression cassette may include the base sequences of SEQ ID NOs: 59 and 60.

[0089] In one example, the first nucleic acid may be STAT3, and the second nucleic acid may be TFEB (transcription factor EB). In this case, the expression cassette may include the base sequences of SEQ ID NO: 61 and SEQ ID NO: 62.

[0090] In one example, the first nucleic acid may be c-MET (Homo sapiens MET prot o-oncogene) and the second nucleic acid may be PD-L1 (Programmed death-ligand 1). In this case, the expression cassette may include a nucleic acid in which U is converted to T in the base sequence of sequence number 63 and sequence number 64. In the above content, the small interfering RNA (siRNA) of sequence number 63 and sequence number 64 can complementarily bind to 15me in 19mer. In addition, when the first nucleic acid is c-MET and the second nucleic acid is PD-L1, the short hairpin RNA (shRNA) expression deoxyribonucleic acid (DNA) contained in the expression cassette may include the base sequence of sequence number 72 or sequence number 73.

[0091] In one example, the expression cassette may include a base sequence that can sequentially encode a base sequence that uses a first nucleic acid as a target sequence, a loop sequence that can form a hairpin structure, and a base sequence that uses a second nucleic acid as a target sequence.

[0092] In one example, the expression cassette can be expressed by a U6 promoter.

[0093] In one example, the adenovirus can be a group C serotype 5 adenovirus.

[0094] In one example, the anti-tumor adenovirus of the present invention has a higher oncolytic ability than a wild-type adenovirus, and the oncolytic ability may be higher than an adenovirus into which a human telomerase reverse transcriptase (hTERT) promoter is introduced.

[0095] In one embodiment, the present invention relates to a cancer treatment composition comprising the anti-tumor adenovirus of the present invention.

[0096] In one embodiment, the composition of the present invention may further include an anticancer agent, such as acivicin, aclarubicin, acodazole, achromycin, adozelesin, alanosin, aldesleukin, allopurinol sodium, altretamine, aminoglutethimide, amonafide, ampligen, amsacrine, androgens, anguidine, aphidicolin glycinate, asaley, asparaginase, 5-azacytidine, azathioprine, Bacillus Calmette-Guérin, and cytochrome P60 inhibitor. Calmette-Guerin (BCG), Baker's antifolate, 6-mercapto-2'-deoxythioguanosine, bisantrene HCL, bleomycin sulfate, busulfan, buthioninesulfoximine, BWA 773U82, BW 502U83 / HCl, BW 7U85 mesylate, ceracemide, carbetimer, carboplatin, carmustine, chlorambucil, chloroquinoxaline-sulfonamide, chlorozotocin, chromomycin A3 A3), cisplatin, cladribine, corticosteroid, Corynebacterium parvum, CPT-11, crisnatol, cyclocytidine, cyclophosphamide, cytarabine, cytembena, DABIS maleatemaleate, dacarbazine, dactinomycin, danuorubicin HCl, diazauridine, dexrazoxane, dianhydrogalactitol, diaziquone, dibromodulcitol, didemnin B, diethyldithiocarbamate, diglycoaldehyde, dihydro-5-azacytidine, doxorubicin, echinomycin, dedatrexate, edelfosine, eflornithine, Elliott's solution, elsamitrucin, epirubicin, esorubicin, estramustine phosphate, estrogens, etanidazol, ethiofos, etoposide, fadrazole, fazarabine, fenretinide, filgrastim, finasteride, flavoneacetic acid, floxuridine, fludarabine phosphate, 5'-fluorouracil, Fluosol TM, flutamide, gallium nitrate, gemcitabine, goserelin acetate, hepsulfam, hexamethylene bisacetamide, homoharringtonine, hydrazine sulfate, 4-hydroxyandrostenedione, hydrozyurea, idarubicin HCl, ifosfamide, 4-ipomeanol, iproplatin, isotretinoin, leucovorin calcium, leuprolide acetate, levamisole, liposomedaunorubicin, liposome-encapsulated doxorubicin, lomustine, lonidamine, maytansine, mechlorethamine hydrochloride, melphalan, menogaril, merbarone, 6-mercaptopurine, mesna, methanol extraction of Bacillus Calmette-Guerin, methotrexate, N-methylformamide, mifepristone, mitoguazone, mitomycin-C, mitotane, mitoxantrone hydrochloride, monocyte / macrophage colony-stimulating factor factor), nabilone, nafoxidine, neocarzinostatin, octreotide acetateacetate), ormaplatin, oxaliplatin, paclitaxel, Pala, pentostatin, piperazinedione, pipobroman, pirarubicin, piritrexim, piroxantrone hydrochloride, PIXY-321, plicamycin, porfimersodium, prednimustine, procarbazine, progestins, pirazofurin, razoxane, sargramostim, semustine, spirogermanium, spiromustine, streptomycin streptonigrin, streptozocin, sulofenur, suraminsodium, tamoxifen, taxotere, tegafur, teniposide, terephthalamidine, teroxirone, thioguanine, thiotepa, thymidine injection injection), tiazofurine, topotecan, toremifene, tretinoin, trifluoperazine hydrochloride, trifluredine, Trimetrexate, tumor necrosis factor (TNF), uracilmustard, vinblastine sulfate, vincristine sulfate, vindesine, vinorelbine, vinzolidine, Yoshi 864, zorubicin, cytosine arabinosidearabinoside), etoposide, melphalan, taxol and mixtures thereof. Preferred are cisplatin, paclitaxel, 5-fluorouracil (5-FU), methotrexate, doxorubicin, daunorubicin, cytosine arabinoside, etoposide, melphalan, chlorambucil, cyclophosphamide, vindesine, mitomycin, bleomycin, tamoxifen and taxol, more preferred are cisplatin, paclitaxel or 5-fluorouracil (5-FU), but the present invention is not limited thereto as long as the purpose of achieving a synergistic anticancer effect through combined use with the composition of the present invention can be achieved.

[0097] The above-mentioned cancer can be one selected from the group consisting of colorectal cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, brain tumor, head and neck tumor, melanoma, myeloma, leukemia, lymphoma, gastric cancer, lung cancer, pancreatic cancer, non-small cell lung cancer, liver cancer, esophageal cancer, small intestine cancer, perianal cancer, fallopian tube tumor, endometrial tumor, vaginal tumor, vulvar tumor, Hodgkin's disease, bladder cancer, kidney cancer, ureteral cancer, renal cell tumor, renal pelvic tumor, bone cancer, skin cancer, head cancer, neck cancer, skin melanoma, intraocular melanoma, endocrine gland cancer, thyroid cancer, parathyroid cancer, pararenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, glioblastoma multimorphic and pituitary adenoma.

[0098] In the present invention, the term "promoter" refers to a non-decoding nucleic acid sequence upstream of an encrypted region that has transcription initiation activity for the binding site of RNA polymerase to the messenger RNA (mRNA) of the gene downstream of the promoter. In the expression cassette of the present invention, the above-mentioned promoter can use any promoter that can induce the expression of short hairpin RNA (shRNA). Specifically, the promoter of the present invention can use a promoter that can always cause the expression of the target gene at all time periods (constitutive promoter) or a promoter that causes the expression of the target gene at a specific position and time (inducible promoter), such as U6 promoter, H1 promoter, CMV (cytomegalovirus) promoter, SV40 promoter, CAG promoter (Hitoshi Niwa et al., Gene, 108: 193-199, 1991), CaMV35S promoter (Odell et al., Nature 313: 810-812, 1985), Rsyn7 promoter (U.S. Patent Application No. 08 / 991,601), rice actin promoter (McElroy et al., Plant Cell 2: 163-171, 1990), ubiquitin promoter (Christensen et al., Plant Cell 2: 163-171, 1990), Mol.Biol.12:619-632,1989), ALS promoter (U.S. Patent Application No. 08 / 409,297), etc. In addition, all known promoters known to those skilled in the art, such as promoters disclosed in U.S. Patents 5,608,149, 5,608,144, 5,604,121, 5,569,597, 5,466,785, 5,399,680, 5,268,463, and 5,608,142, etc., can be used, but are not limited thereto. Preferably, the promoter of the present invention can be a U6 promoter, a HI promoter, or a CMV promoter. According to a preferred embodiment of the present invention, a U6 promoter can be used.

[0099] The composition of the present invention may further comprise an adjuvant. As for the above-mentioned adjuvant, as long as it is an adjuvant well known in the art, it can be used in an unrestricted manner, for example, the effect can be increased by further comprising a complete adjuvant or an incomplete adjuvant such as Freund's adjuvant.

[0100] The composition of the present invention can be prepared into a form in which an effective ingredient is mixed into a pharmaceutically acceptable carrier. Wherein, the pharmaceutically acceptable carrier includes carriers, excipients and diluents conventionally used in the pharmaceutical field. The pharmaceutically acceptable carriers that can be used for the composition of the present invention are not limited thereto, but examples thereof include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0101] The composition of the present invention can be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, sprays and other oral dosage forms, external dosage forms, suppositories or sterile injection solutions by conventional methods.

[0102] In the case of configuration, it can be prepared by using commonly used fillers, extenders, binders, wetting agents, disintegrants, surfactants and other diluents or excipients. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. Such solid preparations are prepared by mixing one or more excipients such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. with the above-mentioned active ingredients. In addition, lubricants such as magnesium stearate and talcum powder can also be used. Liquid preparations for oral administration include suspensions, internal solutions, emulsions, syrups, etc., and in addition to the widely used diluents of water and liquid paraffin, they can also contain a variety of excipients, such as wetting agents, sweeteners, aromatics, preservatives, etc. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, oils, freeze-dried preparations, suppositories, etc. Non-aqueous solvents and suspensions can use propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. As the base of the suppository, synthetic fatty acid ester (witepsol), polyethylene glycol, Tween 61, cocoa butter, lauryl ester, glycerin gelatin and the like can be used.

[0103] The composition of the present invention can be administered to an individual via a variety of routes. All modes of administration are contemplated, for example, administration can be performed by oral, intravenous, intramuscular, subcutaneous, intraperitoneal injection, and the like.

[0104] The dosage of the pharmaceutical composition of the present invention can be selected by considering the age, weight, sex, physical condition, etc. of the individual. It goes without saying that the concentration of the single domain antibody contained in the above-mentioned pharmaceutical composition can be selected in various ways according to the object, and preferably, it is contained in the pharmaceutical composition at a concentration of 0.01 to 5000 μg / ml. When the concentration is less than 0.01 μg / ml, there may be no pharmaceutical activity, and when the concentration is greater than 5000 μg / ml, toxicity may occur in the human body.

[0105] The composition of the present invention can be used for the prevention or treatment of cancer and its syndrome, and can also be used as an anti-cancer adjuvant.

[0106] Furthermore, the present invention provides a method for preventing and treating cancer, comprising the step of administering a pharmaceutically effective amount of the composition of the present invention to an individual.

[0107] The composition of the present invention will be administered in a therapeutically effective amount or a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio for medical treatment, and the effective amount is determined based on factors including the type and condition of the individual, age, sex, drug activity, sensitivity to the drug, administration time, route of administration and excretion rate, treatment time, drugs used simultaneously, and other factors known in the medical field.

[0108] In one embodiment, the present invention relates to the use of the anti-tumor adenovirus of the present invention for tumor prevention or treatment.

[0109] In one embodiment, the present invention relates to a method for treating tumors using the anti-tumor adenovirus of the present invention. DETAILED DESCRIPTION

[0110] Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are only used to specifically describe the content of the present invention, and the present invention is not limited to the following examples.

[0111] Example 1. Preparation of dual-target small interfering RNA (siRNA)

[0112] 1-1. mTOR and STAT3 dual-target small interfering RNA (siRNA)

[0113] Double-target siRNAs (double strand) (Bioneer, Daejeon, Korea) that can simultaneously inhibit STAT3 (signal transducer and activator of transcription 3) and mTOR (mammalian target of rapamycin) were prepared according to the sequences in Table 1. Specifically, the siRNAs of sequence numbers 1 and 2 of group 1 complementarily bind to 17mer in 21mer, the siRNAs of sequence numbers 3 and 4 of group 2 complementarily bind to 16mer in 20mer, the siRNAs of sequence numbers 5 and 6 of group 3 complementarily bind to 15mer in 19mer, the siRNAs of sequence numbers 7 and 8 of group 4 complementarily bind to 14mer in 18mer, and the siRNAs of sequence numbers 9 and 10 of group 5 complementarily bind to 16mer in 17mer. Furthermore, the small interfering RNA (siRNA) of sequence number 11 and sequence number 12 of group 6 complementarily binds to 17mer among 20mer, the small interfering RNA (siRNA) of sequence number 13 and sequence number 14 of group 7 complementarily binds to 16mer among 19mer, the small interfering RNA (siRNA) of sequence number 15 and sequence number 16 of group 8 complementarily binds to 15mer among 18mer, and the small interfering RNA (siRNA) of sequence number 17 and sequence number 18 of group 9 complementarily binds to 14mer among 17mer.After the two sequences of each group in the following Table 1 enter the cell in a double-stranded form, the small interfering RNA (siRNA) of each group's antisense mTOr (antisense_mTOr) complementarily binds to the target site of mTOR messenger RNA (mRNA) (gi|206725550|ref|NM_004958.3|Homo sapiensmechanistic target of rapamycin(serine / threonine kinase)(MTOR), mRNA), and the small interfering RNA (siRNA) of each group's antisense STAT3 (antisense_STAT3) complementarily binds to the target site of STAT3 messenger RNA (mRNA) (gi|47080104|ref|NM_139276.2|Homo sapiens signal transducer and activator of transcription3(acute-phase response factor)(STAT3), transcriptvariant 1, mRNA), thereby reducing the gene expression of mTOR and STAT3.

[0114] Table 1

[0115]

[0116] 1-2. Small interfering RNA targeting BCL2 and BI-1

[0117] A 21mer double-strand siRNA (Bioneer, Daejeon, Korea) that can simultaneously inhibit BCL2 (B-cell lymphoma 2) and BI-1 (BAXinhibitor 1) was prepared according to the sequence in Table 2 below (Bioneer, Daejeon, Korea). Specifically, the 21mer siRNA group 10 composed of sequence numbers 19 and 20 in the following Table 2 makes 15mers complementary to each other, the 20mer siRNA group 11 composed of sequence numbers 21 and 22 makes 14mers complementary to each other, the 20mer siRNA group 12 composed of sequence numbers 23 and 24 makes 14mers complementary to each other, the 19mer siRNA group 13 composed of sequence numbers 25 and 26 makes 13mers complementary to each other, the 19mer siRNA group 14 composed of sequence numbers 27 and 28 makes 13mers complementary to each other, and the 18mer siRNA group 15 composed of sequence numbers 29 and 30 makes 12mers complementary to each other. The siRNA (Antisense Bcl-2) of sequence number 19, sequence number 21, sequence number 23, sequence number 25, sequence number 27 or sequence number 29 in the following Table 2 can complementarily bind to the messenger RNA (mRNA) of Bcl-2, and the siRNA (Antisense BI-1) of sequence number 20, sequence number 22, sequence number 24, sequence number 26, sequence number 28 or sequence number 30 can complementarily bind to the messenger RNA (mRNA) of BI-1, so that the siRNA group 10 to the siRNA group 15 of the present invention simultaneously reduce the expression of Bcl-2 and BI-1 genes.

[0118] Table 2

[0119]

[0120]

[0121] 1-3. AR and mTOR dual-target small interfering RNA (siRNA)

[0122] A double strand siRNA set (Bioneer, Daejeon, Korea) that can simultaneously inhibit AR (androgen receptor) and mTOR (mammalian target of rapamycin) was prepared according to the sequences in Table 3 below (Bioneer, Daejeon, Korea). Specifically, the 20mer siRNA group 16 composed of sequence numbers 31 and 32 makes 18mers complementary to each other, the 19mer siRNA group 17 composed of sequence numbers 33 and 34 makes 17mers complementary to each other, the 18mer siRNA group 18 composed of sequence numbers 35 and 36 makes 16mers complementary to each other, the 17mer siRNA group 19 composed of sequence numbers 37 and 38 makes 15mers complementary to each other, the 19mer siRNA group 20 composed of sequence numbers 39 and 40 makes 15mers complementary to each other, the 18mer siRNA group 21 composed of sequence numbers 41 and 42 makes 14mers complementary to each other, the 17mer siRNA group 22 composed of sequence numbers 43 and 44 makes 15mers complementary to each other, The siRNA group 22 makes 13mers complementary to each other, the siRNA group 23 of 23mer composed of sequence numbers 45 and 46 makes 19mers complementary to each other, the siRNA group 24 of 22mer composed of sequence numbers 47 and 48 makes 18mers complementary to each other, the siRNA group 25 of 22mer composed of sequence numbers 49 and 50 makes 18mers complementary to each other, the siRNA group 26 of 21mer composed of sequence numbers 51 and 52 makes 17mers complementary to each other, the siRNA group 27 of 20mer composed of sequence numbers 53 and 54 makes 16mers complementary to each other, and the siRNA group 28 of 21mer composed of sequence numbers 55 and 56 makes 17mers complementary to each other.The siRNA (Antisense AR) of sequence number 31, sequence number 33, sequence number 35, sequence number 37, sequence number 39, sequence number 41, sequence number 43, sequence number 45, sequence number 47, sequence number 49, sequence number 51, sequence number 53 or sequence number 55 in the following Table 3 is complementary to the messenger RNA (mRNA) of Ar, and the siRNA (Antisense mTOR) of sequence number 32, sequence number 34, sequence number 36, sequence number 38, sequence number 40, sequence number 42, sequence number 44, sequence number 46, sequence number 48, sequence number 50, sequence number 52, sequence number 54 or sequence number 56 is complementary to the messenger RNA (mRNA) of mTOR. Therefore, the siRNA group 16 to the siRNA group 28 of the present invention simultaneously reduce the expression of AR and mTOR genes.

[0123] Table 3

[0124]

[0125] 1-4. MGMT and mTOR dual-target small interfering RNA

[0126] A double-target small interfering RNA (siRNA) (double strand) set (Bioneer, Daejeon, Korea) that can simultaneously reduce (inhibit) the expression of MGMT (O-6-methylguanine-DNAmethyltransferase, NM_002412.5) and mTOR (NM_004958.3) genes was prepared according to the sequences in Table 4 below.

[0127] Table 4

[0128]

[0129] 1-5. Small interfering RNA (siRNA) targeting BCL2 and MCL1

[0130] A double-stranded siRNA set (Bioneer, Daejeon, Korea) that can simultaneously reduce (inhibit) the expression of BCL2 (NM_000633.2) and MCL1 (MCL1 apoptosis regulator, NM_021960.5) genes was prepared according to the sequences in Table 5 below (Bioneer, Daejeon, Korea).

[0131] Table 5

[0132]

[0133] 1-6. STAT3 and TFEB dual-target small interfering RNA (siRNA)

[0134] A double-stranded siRNA set (Bioneer, Daejeon, Korea) that can simultaneously reduce (inhibit) the expression of STAT3 (NM_139276.2) and TFEB (transcription factor EB, NM_007162.2) genes was prepared according to the sequences in Table 6 below.

[0135] Table 6

[0136]

[0137] 1-7.c-MET and PD-L1 dual-target small interfering RNA (siRNA)

[0138] A double-target siRNA (double strand) set (Bioneer, Daejeon, Korea) that can simultaneously inhibit c-MET (Homo sapiens MET proto-oncogene) and PD-L1 (Programmed death-ligand 1) was prepared according to the sequences in Table 7 below (Bioneer, Daejeon, Korea). Specifically, the 19-mer siRNA set 32 ​​consisting of sequence numbers 63 and 64 makes the 15mers complementary to each other, and the siRNA (Antisense c-MET) of sequence number 63 in Table 7 below (antisense c-MET) is complementary to the messenger RNA (mRNA) of c-MET, and the siRNA (Antisense PD-L1) of sequence number 64 is complementary to the messenger RNA (mRNA) of PD-L1. Therefore, the siRNA set of the present invention simultaneously reduces the expression of c-MET and PD-L1 genes.

[0139] Table 7

[0140]

[0141]

[0142] Example 2. Preparation of dual-target short hairpin RNA (shRNA)

[0143] 2-1. mTOR and STAT3 target short hairpin RNA (shRNA)

[0144] In order to express the small interfering RNA (siRNA) prepared in the above example in cells, an expression cassette for expressing short hairpin RNA (shRNA) was prepared. Specifically, multiple short hairpin RNAs (shRNAs) (TTGGATCCAA circular short hairpin RNA (shRNA) and TTCAAGAGAG circular short hairpin RNA (shRNA)) containing double-stranded sequences of double-target small interfering RNA (siRNA) (sequence number 1 and sequence number 2) of group 1 among multiple small interfering RNAs (siRNAs) were representatively prepared (Table 8). Each of the prepared short hairpin RNA (shRNA) expression cassettes was respectively configured in the pE3.1 vector ( Figure 1 ) was located after the U6 promoter (sequence number 65) in the restriction enzyme PstⅠ and EcoRⅤ cutting site, thereby preparing a recombinant expression vector that expresses two short hairpin RNAs (shRNAs) containing dual-target small interfering RNAs (siRNAs) targeting mTOR and STAT3 in cells.

[0145] Table 8

[0146]

[0147] 2-2. Short hairpin RNA (shRNA) targeting BCL2 and BI-1

[0148] In order to express the small interfering RNA (siRNA) prepared in the above examples in cells, an expression cassette for expressing short hairpin RNA (shRNA) was prepared. Specifically, multiple short hairpin RNAs (shRNAs) (TTGGATCCAA circular short hairpin RNA (shRNA) and TTCAAGAGAG circular short hairpin RNA (shRNA)) containing double-stranded sequences of the double-target small interfering RNA (siRNA) (sequence number 19 and sequence number 20) of group 10 in multiple small interfering RNAs (siRNA) were representatively prepared (Table 9). The prepared multiple expression cassettes for expressing each short hairpin RNA (shRNA) were respectively configured in the pE3.1 vector ( Figure 1 ) was located after the U6 promoter (sequence number 65) in the restriction enzyme PstⅠ and EcoRⅤ cutting site, thereby preparing a recombinant expression vector that expresses two short hairpin RNAs (shRNAs) containing dual-target small interfering RNAs (siRNAs) targeting BCL2 and BI-1 in cells.

[0149] Table 9

[0150]

[0151] 2-3. AR and mTOR target short hairpin RNA (shRNA)

[0152] In order to express the small interfering RNA (siRNA) prepared in the above examples in cells, an expression cassette for expressing short hairpin RNA (shRNA) was prepared. Specifically, multiple short hairpin RNAs (shRNA) of double-stranded sequences of double-target small interfering RNA (siRNA) (sequence number 31 and sequence number 32) of group 16 among multiple small interfering RNAs (siRNA) were representatively prepared (TTGGATCCAA circular short hairpin RNA (shRNA) and TTCAAGAGAG circular short hairpin RNA (shRNA)) (Table 10). The prepared multiple expression cassettes for expressing each short hairpin RNA (shRNA) were respectively configured in the pE3.1 vector ( Figure 1 ) was located after the U6 promoter (sequence number 65) in the restriction enzyme PstⅠ and EcoRⅤ cutting site, thereby preparing a recombinant expression vector that expresses two short hairpin RNAs (shRNAs) containing dual-target small interfering RNAs (siRNAs) targeting AR and mTOR in cells.

[0153] Table 10

[0154]

[0155] 2-4. c-MET and PD-L1 target short hairpin RNA (shRNA)

[0156] In order to express the small interfering RNA (siRNA) prepared in the above examples in cells, multiple short hairpin RNA (shRNA) expression cassettes (TTGGATCCAA circular short hairpin RNA (shRNA) and TTCAAGAGAG circular short hairpin RNA (shRNA)) containing double-stranded sequences of dual-target small interfering RNA (siRNA) and circular sequences were prepared. Specifically, TTGGATCCAA (TTGGATCCAA loop) or TTCAAGAGAG (TTCAAGAGAG loop), antisense strand and TT were connected to the 3' of the sense strand of the small interfering RNA (siRNA) group (sequence number 63 and sequence number 64) in Table 7 from 5' to 3', to prepare a deoxyribonucleic acid (DNA) sequence encoding short hairpin RNA (shRNA) and shown in Table 11 (small interfering RNA (siRNA) is represented by capital letters, and additional sequences are represented by lowercase letters). The prepared multiple short hairpin RNA (shRNA) expression cassettes were respectively configured in the pE3.1 vector ( Figure 1) was located after the U6 promoter (sequence number 65) in the restriction enzyme PstⅠ and EcoRⅤ cutting site, thereby preparing a recombinant expression vector that expresses two short hairpin RNAs (shRNAs) containing dual-target small interfering RNAs (siRNAs) targeting c-MET and PD-L1 in cells.

[0157] Table 11

[0158]

[0159] Example 3. Confirmation of the gene expression inhibition effect of dual-target small interfering RNA (siRNA)

[0160] 3-1. Inhibition of mTOR and STAT3 expression

[0161] After HeLa cells were injected into 12-well plates, they were incubated in serum-free cell freezing medium (RPMI, Hyclone) supplemented with 10% fetal bovine serum (FBS, Hyclone) at 37°C and 5% CO. 2The cells were cultured under the conditions of 50% and 10% saturation until the cell density reached 50%. Afterwards, the cells were transfected with the double-target small interfering RNA (siRNA) of groups 1 to 9 prepared in the above Example 1 respectively using a transfection reagent (lipofectamine3000, Invitrogen, Carlsbad, CA, USA) to knock down mTOR and STAT3 simultaneously. After 48 hours of transfection, total RNA (RNA) was extracted using GeneJET RNA Purification Kit (Invitrogen) by disrupting the cells. The extracted total RNA (RNA) was used as a template to perform a reverse transcription reaction using RevoScriptTM RT PreMix (iNtRON BIOTECHNOLOGY). The mTOR (Hs00234522_m1), STAT3 (Hs01047580_m1) and GAPDH (Hs02758991_g1) reactions were performed using 20 μl of a sample containing 25 ng to 200 ng of reverse transcribed complementary deoxyribonucleic acid (cDNA), AmpONE taq DNA polymerase (GeneAll) and TaqMan Gene Expression assays (Applied Biosystems) using ABI PRISM 7700 Sequence Detection System and QS3 Real-time PCR (Biosystems). The real-time polymerase chain reaction (Real-time PCR) conditions were 2 cycles of 50°C for 2 minutes and 95°C for 10 minutes and 95°C for 15 seconds and 60°C for 60 seconds, and a total of 40 cycles were performed. All reactions were repeated 3 times and the average value was taken. The multiple results thus obtained were used for normalization of the messenger RNA (mRNA) values ​​of GAPDH as a housekeeping gene.

[0162] The results showed that the residual expression of mTOR and STAT3 was confirmed to be about 20% to 40% in groups 1 to 9 compared with the control group, indicating that the dual-target siRNA can simultaneously inhibit the expression of two genes ( Figure 2 ).

[0163] 3-2. Inhibition of BCL2 and BI-1 expression

[0164] After injecting Hela cells into 12-well plates, the cells were incubated in serum-free cell freezing medium (RPMI, Hyclone) supplemented with 10% fetal bovine serum (FBS, Hyclone) at 37°C and 5% CO. 2 The cells were cultured under the conditions of 50% until the cell density reached 50%. Afterwards, 3 μl of transfection reagent (lipofectamine3000, Invitrogen, Carlsbad, CA, USA) was used to transfect the double-target small interfering RNA (siRNA) group 10 and (si-BB1) and double-target small interfering RNA (siRNA) groups 11 to 15 prepared in Example 1 (Table 2) at 80 pmole per well into the wells in which the Hela cells were cultured, so that BCL2 and BI-1 were knocked down simultaneously. After 48 hours of transfection, total ribonucleic acid (RNA) was extracted using GeneJET RNA Purification Kit (Invitrogen) by disrupting the cells. After the extracted total RNA was used as a template to be reverse transcribed into complementary deoxyribonucleic acid (cDNA) by real-time polymerase chain reaction (RT-PCR), the expression of messenger RNA (mRNA) of BCL2 and BI-1 based on dual-target small interfering RNA (siRNA) was confirmed by quantitative polymerase chain reaction (q-PCR). As a reference, the probes used were Bcl2 (Thermo, Hs00608023_m1), BI-1 (Thermo, Dm01835892_g1), and GAPDH (Thermo, Hs02786624_g1), and the QS3 equipment was used to perform them. All reactions were repeated 3 times and the average value was taken. The multiple results thus obtained were used to normalize the messenger RNA (mRNA) values ​​of GAPDH as a housekeeping gene.

[0165] The results showed that the dual-target siRNA group reduced the expression of BCL2 and BI-1, indicating that the dual-target siRNA of the present invention can simultaneously inhibit the expression of two genes ( Figure 3 and Figure 4 ).

[0166] Therefore, the dual-target small interfering RNA (siRNA) of the present invention can simultaneously inhibit the expression of two genes, and thus it is confirmed that it exhibits significant anticancer activity by promoting the killing of cancer cells, and can be effectively used as an anticancer composition or anticancer adjuvant for various tumors.

[0167] 3-3. Inhibition of AR and mTOR expression

[0168] After PC3 cell lines, h460 cell lines and A549 cell lines were injected into 12-well plates, they were cultured in serum-free cell freezing medium (RPMI, Hyclone) supplemented with 10% fetal bovine serum (FBS, Hyclone) at 37°C and 5% CO. 2The cells were cultured under the conditions of 50% . Afterwards, 3 μl of transfection reagent (lipofectamine) 3000 (Invitrogen, Carlsbad, CA, USA) was used to transfect the double-target small interfering RNA (siRNA) groups 16 to 28 (Table 3) prepared in Example 1 above into the wells in which the cells were cultured at 80 pmole per well, so that AR and mTOR were knocked down at the same time. Among them, group 16 was transfected into h460 cells, and group 17 was transfected into PC3 cells, thereby performing knockdown, and as its positive control group, the small interfering RNA (siRNA) for AR and the small interfering RNA (siRNA) for mTOR recorded in Table 12 were transfected respectively. After 48 hours of transfection, total ribonucleic acid (RNA) was extracted using GeneJET RNAPurification Kit (Invitrogen) by crushing the cells. After the extracted total RNA was used as a template to be reverse transcribed into complementary deoxyribonucleic acid (cDNA) by real-time polymerase chain reaction (RT-PCR), the expression levels of messenger RNA (mRNA) of AR and mTOR based on small interfering RNA (siRNA) and double-target small interfering RNA (siRNA) groups 16 to 28 (si-AT1 to siAT13) of the present invention were confirmed by quantitative polymerase chain reaction (q-PCR). In order to confirm the expression level of messenger RNA (mRNA), a primer set for AR or mTOR and a reaction mixture [10X reaction Buffer 2μl, HQ Buffer 2μl, dNTP 1.6μl, Primer (F, R, 10pmole / ul) 1μl each, Template (500ng) 2μl, Taq 0.2μl, DW 10.2μl, Total vol. 20μl] were used. The messenger RNA (mRNA) of AR and mTOr in the knocked-down cell fragments was converted into complementary deoxyribonucleic acid (cDNA) under polymerase chain reaction (PCR) conditions [95°C for 2 minutes, 95°C for 20 seconds for 30 cycles, 60°C for 10 seconds, 72°C for 30 to 60 seconds, and 72°C for 5 minutes].In addition, the reverse transcribed complementary deoxyribonucleic acid (cDNA) was used as a template to prepare a reaction mixture [Template (RT-PCR product) 6μl, Taqman probe 3μl, 10X reaction buffer 6μl, HQ buffer 6μl, dNTP 4.8μl, Taq 0.6μl, DW 10.2μl, Total vol. 60μl] and perform quantitative polymerase chain reaction (q-PCR) [10 minutes at 95°C, 15 seconds at 95°C and 40 cycles per minute at 60°C]. For reference, the probes used were AR (Thermo, Hs00171172_m1), mTOR (Thermo, Hs00234508_m1), GAPDH (Thermo, Hs02786624_g1), and the QS3 equipment was used for the reaction. All reactions were repeated 3 times and the average value was taken. The multiple results thus obtained were used for normalization of the messenger RNA (mRNA) values ​​of GAPDH as a housekeeping gene.

[0169] Table 12

[0170]

[0171] The results showed that in PC3 cells and h460 cell lines, the expression of AR and mTOr was reduced based on the dual-target small interfering RNA (siRNA) group 16 and group 17 of the present invention ( Figure 5 ), the degree of reduction is similar to or better than the effect based on each small interfering RNA (siRNA). In addition, the dual-target small interfering RNA (siRNA) group 17 and group 28 of the present invention reduce the expression of AR and mTOr ( Figure 6 ). Thus, it can be seen that the dual-target small interfering RNA (siRNA) of the present invention can effectively inhibit the expression of two genes at the same time.

[0172] 3-4. Inhibition of c-MET and PD-L1 expression

[0173] Glioblastoma cell line U-87, prostate cancer cell line CWR22Rv-1 (22Rv-1), melanoma cell line A431, and non-small cell lung cancer cell line HCC827 were injected into 12-well plates, and then incubated in serum-free cell freezing medium (RPMI, Hyclone) supplemented with 10% fetal bovine serum (FBS, Hyclone) at 37°C and 5% CO. 2The cells were cultured under the conditions of 50% RT-PCR until the cell density reached 50%. Afterwards, 3 μl of lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) was used to transfect the double-target small interfering RNA (siRNA) group (Table 7) prepared in the above embodiment into the wells where the above cells were cultured at 80 pmole per well, so that c-MET and PD-L1 were knocked down simultaneously. After 48 hours of transfection, total RNA (RNA) was extracted by crushing the cells using GeneJET RNA Purification Kit (Invitrogen). After the extracted total RNA (RNA) was used as a template to be reverse transcribed into complementary deoxyribonucleic acid (cDNA) by real-time polymerase chain reaction (RT-PCR), the messenger RNA (mRNA) expression levels of c-MET and PD-L1 based on the small interfering RNA (siRNA) and the double-target small interfering RNA (siRNA) group of the present invention were confirmed by quantitative polymerase chain reaction (q-PCR). In order to confirm the expression level of messenger RNA (mRNA), primer sets for PD-L1 or c-MET and reaction mixture [10×reaction buffer 2μl, HQ buffer 2μl, dNTP 1.6μl, Primer (F, R, 10pmole / ul) 1μl each, Template (500ng) 2μl, Taq 0.2μl, DW 10.2μl, Total vol. 20μl] were used. The messenger RNA (mRNA) of c-MET and PD-L1 in the knocked-down cell fragments was converted into complementary deoxyribonucleic acid (cDNA) under polymerase chain reaction (PCR) conditions [2 minutes at 95°C, 30 cycles of 20 seconds at 95°C, 10 seconds at 60°C, 30 to 60 seconds at 72°C, and 5 minutes at 72°C]. In addition, the reverse transcribed complementary deoxyribonucleic acid (cDNA) was used as a template to prepare a reaction mixture [Template (RT-PCR product) 6μl, Taqman probe 3μl, 10× reaction buffer 6μl, HQ buffer 6μl, dNTP 4.8μl, Taq 0.6μl, DW 10.2μl, Total volume 60μl] and quantitative polymerase chain reaction (q-PCR) was performed using QS3 equipment [at 95°C for 10 minutes, at 95°C for 15 seconds, and at 60°C for 40 cycles per minute]. All reactions were repeated 3 times and the average value was taken.The multiple results thus obtained were used for normalization of the messenger RNA (mRNA) values ​​of GAPDH as a housekeeping gene.

[0174] The results showed that in U-87 cell line, 22Rv-1 cell line, A431 cell line and HCC827 cell line, the expression of c-MET and PD-L1 was reduced based on the dual-target small interfering RNA (siRNA) group of the present invention ( Figure 7 a to 7d). Thus, it can be seen that the dual-target small interfering RNA (siRNA) of the present invention can effectively inhibit the expression of two genes at the same time.

[0175] Example 4. Confirmation of the gene expression inhibition effect of dual-target small interfering RNA (siRNA) and short hairpin RNA (shRNA)

[0176] 4-1. Gene expression inhibition effect of dual-target short hairpin RNA (shRNA)

[0177] A549 cells and glioblastoma U-87 (U87MG) cells were transfected with a vector containing the TTGGATCCAA circular short hairpin RNA (shRNA) sequence of sequence number 66 or the TTCAAGAGAG circular short hairpin RNA (shRNA) sequence of sequence number 67 (Table 8) encoding the mTOR and STAT3 target short hairpin RNA (shRNA) prepared in the above Example 2 at 0, 1, and 2 μg using a transfection reagent (lipofectamine3000). 48 hours after transfection, the degree of reduction in gene expression of mTOR and STAT3 was confirmed using the real-time polymerase chain reaction (Real time PCR) analysis method described in the above example.

[0178] The results showed that the expression of mTOR and STAT3 was reduced in both short hairpin RNAs (shRNAs) containing the dual-target small interfering RNA (siRNA) of the present invention, and showed a tendency to decrease by 20% in a manner proportional to the amount of deoxyribonucleic acid (DNA) of the short hairpin RNA (shRNA) ( Figure 8 ).

[0179] 4-2. Gene expression of single-target small interfering RNA (siRNA) and dual-target short hairpin RNA (shRNA) Comparison of inhibitory effects

[0180] The gene expression inhibition effects of two single-target small interfering RNAs (siRNAs) connected in series using two promoters and the dual-target short hairpin RNA (shRNA) of the present invention were compared. Specifically, after connecting the small interfering RNA (siRNA) for mTOR and the small interfering RNA (siRNA) for STAT3 in series in the order of mTOR-STAT3 or STAT3-mTOR, the gene expression inhibition effects on mTOR and STAT3 were compared with the mTOR / STAT3 dual-target short hairpin RNA (shRNA) of the present invention.

[0181] The results showed that in 293T cell lines, compared with the case where two small interfering RNAs (siRNAs) for genes were linked in series using a promoter (direct shmTOR-STAT3 or direct shSTAT3-mTOR), the case where the dual-target short hairpin RNA (shRNA) of the present invention was used showed a more significant gene expression inhibition effect ( Fig. 9 ).

[0182] Example 5. Confirmation of the cancer cell killing effect based on dual-target small interfering RNA (siRNA)

[0183] In order to confirm the cancer cell killing effect of the dual-target small interfering RNA (siRNA) of Groups 1 to 9 of the present invention, 5×10 3 After injecting human lung cancer cell line A549 cells into 96-well plates, the cells were transfected with double-target small interfering RNA (siRNA) of groups 1 to 9 using lipofectamine3000. 48 hours after transfection, 5 mg / mL MTT (Promega, Ltd.) was additionally treated to the cells 24 hours later and cultured for 4 hours. After that, the culture medium was removed and 150 μl of solubilization solution and stop solution were treated, and cultured at 37°C for 4 hours. The absorbance of the reaction solution was measured at 570 nm, and the cell survival rate was calculated using the following mathematical formula.

[0184] Mathematical formula 1

[0185] Cell survival rate = absorbance of experimental group (570nm) / absorbance of control group (570nm) × 100 (%)

[0186] The results showed that when mTOR and STAT3 were simultaneously inhibited using the dual-target siRNAs in Groups 1 to 9 of the present invention, the cell survival rate was significantly reduced compared to the control group. Therefore, it was confirmed that the dual-target siRNAs in Groups 1 to 9 of the present invention effectively killed cancer cells ( Fig.10 ).

[0187] Example 6. Confirmation of the cancer cell killing effect based on the combined treatment of dual-target small interfering RNA (siRNA) and anticancer agents

[0188] 6-1. Combination treatment with mTOR and STAT3 dual-targeting small interfering RNA (siRNA) and anticancer drugs

[0189] 6-1-1. Combined treatment with cisplatin

[0190] In press 5×10 3 After injecting human lung cancer cell line A549 cells into 96-well plates, the cells were transfected with dual-target small interfering RNA (siRNA) (mTOR and STAT3 knocked down simultaneously) of Groups 1 to 9 of the present invention using lipofectamine3000. After 48 hours of transfection, 5 μM cisplatin was treated and cultured for 10 hours. Afterwards, as in the above example, the MTT reaction was performed, and the absorbance was measured at 570 nm to calculate the cell survival rate.

[0191] The results showed that when the combination treatment with cisplatin and the dual-target small interfering RNA (siRNA) of Groups 1 to 9 of the present invention were used to simultaneously inhibit mTOR and STAT3, the cell survival rate was reduced by about 50% to 70%, and a significant difference was confirmed compared with the control group. Therefore, in the combination treatment with anticancer agents, when two genes were inhibited simultaneously, it was confirmed that the cell killing effect was significantly improved ( Fig.11 ).

[0192] 6-1-2. Combination therapy with paclitaxel

[0193] In press 5×10 3 After injecting human lung cancer cell line A549 cells into 96-well plates, the cells were transfected with dual-target small interfering RNA (siRNA) (mTOR and STAT3 knocked down simultaneously) of Groups 1 to 9 of the present invention using lipofectamine3000. 48 hours after transfection, 5 μM paclitaxel was treated and cultured for 10 hours. Afterwards, as in Example 4 above, the MTT reaction was performed, and the absorbance was measured at 570 nm to calculate the cell survival rate.

[0194] The results showed that when paclitaxel was used in combination with the dual-target small interfering RNA (siRNA) of Groups 1 to 9 of the present invention to simultaneously inhibit mTOR and STAT3, the cell survival rate was reduced by about 30-50%, and a significant difference was confirmed compared with the control group. Therefore, in the combined treatment with anticancer agents, when two genes were inhibited simultaneously, it was confirmed that the cell killing effect was significantly improved ( Fig.12 ).

[0195] 6-1-3. Combined treatment with 5-fluorouracil (5-FU, 5-fluorouracil)

[0196] After injecting human lung cancer cell line A549 cells into 96-well plates at 5×103cell / well, the cells were transfected with dual-target small interfering RNA (siRNA) (mTOR and STAT3 knocked down simultaneously) of Groups 1 to 9 of the present invention using lipofectamine3000. 48 hours after transfection, 5μM paclitaxel was treated and cultured for 10 hours. Thereafter, as in Example 4 above, the MTT reaction was performed, and the absorbance was measured at 570nm to calculate the cell survival rate.

[0197] The results showed that when 5-fluorouracil was used in combination with the dual-target small interfering RNA (siRNA) of Groups 1 to 9 of the present invention to simultaneously inhibit mTOR and STAT3, the cell survival rate was reduced by about 30%, and a significant difference was confirmed compared with the control group. Therefore, in the combined treatment with anticancer agents, when two genes were inhibited simultaneously, it was confirmed that the cell killing effect was significantly improved ( Fig.13 ).

[0198] 6-2. Combination treatment with BCL2 and BI-1 dual-targeting small interfering RNA (siRNA) and anticancer drugs

[0199] 6-2-1. Synergistic anticancer effects of BCL2 and BI-1 dual-targeting small interfering RNA (siRNA)

[0200] After transfecting the dual-target siRNA group 1 (si-BB1) of the present invention or individual BCL2 siRNA and BI-1 siRNA as a control group into Hela cells as human cervical cancer cell lines, the anticancer agents were treated for 6 hours according to the type, and then the killing degree of the cancer cell lines was confirmed by MTT analysis. Specifically, 7.5 μl of transfection reagent was used to transfect siRNA (200 pmole) per well into Hela cells injected into 6-well plates and cultured for 48 hours, and the cell lines were re-injected into 96-well plates and the cell density (2.5×10 4) reached 50%, and the control group was treated with 0.5 μM taxol, 20 μM cisplatin and 10 μM etoposide, respectively, in terms of the concentration of anticancer agents, and the cells of the dual-target small interfering RNA (siRNA) group of the present invention were treated with half the concentration of 0.25 μM taxol, 10 μM cisplatin and 5 μM etoposide, respectively. After 6 hours, the degree of cancer cell killing was confirmed by performing MTT analysis as in the above embodiment.

[0201] The results showed that for the control group transfected with small interfering RNA (siRNA) against BCL2 or BI-1, almost no cancer cell killing occurred, and only when treated with anticancer drugs in combination, the cancer cell killing effect based on the anticancer drugs was partially produced ( Fig.14 In contrast, for the dual-target siRNA group 1 (si-BB1) of the present invention, significant cancer cell killing occurred, and a synergistic increase in cancer cell killing was confirmed even when used in combination with an anticancer agent at a significantly lower concentration, compared to the control group treated with both BCL2 siRNA and BI-1 siRNA ( Fig.14 Thus, it can be deduced that the dual-target siRNA of the present invention exhibits anticancer activity itself, and also exhibits specificity for the dual-target siRNA based on the synergistic effect of combined treatment with anticancer agents.

[0202] 6-2-2. Comparison of the efficacy of Bcl2 inhibitors with small interfering RNA (siRNA) that dually targets BCL2 and BI-1 fruit

[0203] The cancer cell killing inhibitory effect of the dual-target small interfering RNA (siRNA) for BCL2 and BI-1 (group 1 si-BB1) of the present invention was compared with the ABT-737 drug used as a cancer cell therapeutic agent based on the inhibition of BCL2. Specifically, as in the above-mentioned embodiment, after LnCap cell lines as prostate cancer cells were injected separately, the small interfering RNA (siRNA) group 1 (si-BB1) for BCL2 and BI-1 of the present invention was transfected, 3 μM ABT-737 was treated and cultured for 12 hours, and then the killing degree of the cancer cell line was confirmed by MTT analysis.

[0204] The results showed that the death of LnCap cell line increased by treatment with ABT-737 or the small interfering RNA (siRNA) group 1 (si-BB1) of the present invention for BCL2 and BI-1. In particular, when ABT-737 and the dual-target small interfering RNA (siRNA) of the present invention were treated together, the synergistic killing effect of cancer cells was significantly increased ( Fig.15).

[0205] 6-2-3. Comparison of BCL2 and BI-1 dual-target siRNA and individual siRNAs Anticancer effects of siRNA

[0206] After culturing PC3 cell lines as human prostate cancer cell lines in 6-well plates, the dual-target small interfering RNA (siRNA) group 10 (si-BB1) of the present invention and each small interfering RNA (siRNA) for BCL2 or BI-1 in Table 13 as a control group were transfected, and 48 hours later, cisplatin was treated with 10-20uM. After 12 hours of cisplatin treatment, 5mg / mL of MTT (Promega, Ltd.) was treated to the cells and cultured for 4 hours. Afterwards, the culture medium was removed and 150μl of solubilization solution and stop solution were treated, and cultured at 37°C for 4 hours. The absorbance of the reaction solution was measured at 570nm, and the cell survival rate was calculated using the above mathematical formula 1.

[0207] Table 13

[0208]

[0209] The results showed that compared with the control group that was not treated with cisplatin and treated with the control group siRNA, the group treated with the dual-target siRNA group 10 of the present invention and cisplatin showed a significant increase in cancer cell killing, and compared with the group treated with siRNA for BCL2 and BI-1 respectively, the degree was confirmed to be significantly increased ( Fig.16 ).

[0210] 6-3. Combination treatment with AR and mTOR dual-targeting small interfering RNA (siRNA) and anticancer drugs

[0211] After culturing DU145 cell line and H460 cell line in 6-well plates, the dual-target small interfering RNA (siRNA) group 16 (si-AT1) of the present invention was transfected respectively. After 48 hours, 50uM cisplatin, 20uM etoposide or 1uM taxol were treated and cultured for 16 hours. After that, 5mg / mL MTT (Promega, Ltd.) was treated to the cells and cultured for 4 hours. After that, the culture medium was removed and 150μl of solubilization solution and stop solution were treated, and cultured at 37°C for 4 hours. The absorbance of the reaction solution was measured at 570nm, and the cell survival rate was calculated.

[0212] The results showed that the dual-target siRNA of the present invention alone caused cell killing in the DU145 cell line, which is a prostate cancer cell line (no anticancer agent treatment group (no treat)), and in the cisplatin treatment group that did not show a cell killing effect, the dual-target siRNA of the present invention also caused cell killing. In addition, it was confirmed that for etoposide and paclitaxel, which slightly showed anticancer activity, the dual-target siRNA group 16 (si-AT1) of the present invention significantly improved the DU145 cell killing effect ( Fig.17 Part A). In addition, for H460, a lung cancer cell line, similar to the DU145 cell line, the dual-target small interfering RNA (siRNA) of the present invention itself exhibits a cell-killing effect, and when etoposide and paclitaxel are used in combination, it is known that a significant anticancer activity is exhibited ( Fig.17 Part B of the

[0213] Example 7. Preparation of dual-target short hairpin RNA (shRNA) encoding adenovirus

[0214] After human telomerase reverse transcriptase (hTERT) promoter (SEQ ID NO. 74)-E1A (SEQ ID NO. 75)-internal ribosome entry site (SEQ ID NO. 76)-E1B sequence (SEQ ID NO. 77) (total sequence: SEQ ID NO. 78) was inserted between SpeⅠ and ScaⅠ of the adenovirus vector, U6 promoter and BCL2 and BI-1 dual target short hairpin RNA (shRNA) (U6 promoter + BCL2 and BI-1 dual target short hairpin RNA (shRNA) encoding sequence: SEQ ID NO. 79), mTOR and STAT3 dual target short hairpin RNA (shRNA) (U6 promoter + mTOR and STAT3 dual target short hairpin RNA (shRNA) encoding sequence: SEQ ID NO. 80) prepared in the above example were respectively inserted between SpeⅠ in the E3 region. The TOR dual-target short hairpin RNA (shRNA) sequence (U6 promoter + AR and mTOR dual-target short hairpin RNA (shRNA) coding sequence: sequence number 81) was used to encode and express the human telomerase reverse transcriptase (hTERT) promoter and dual-target short hairpin RNA (shRNA), and infectious recombinant adenoviruses were prepared (BCL2 and BI-1 dual-target short hairpin RNA (shRNA) coding and expressing adenovirus: CA101; mTOR and STAT3 dual-target short hairpin RNA (shRNA) coding and expressing adenovirus: CA102; AR and mTOR dual-target short hairpin RNA (shRNA) coding and expressing adenovirus: CA103; c-MET and PD-L1 dual-target short hairpin RNA (shRNA) coding (expressing) adenovirus: CA104) (refer to Fig.19 ). In addition, as a control group, a recombinant adenovirus (CA10G) with only human telomerase reverse transcriptase (hTERT) inserted was prepared. After that, the sequence of the prepared adenovirus vector was analyzed, and the viral genome was linearized using the PacⅠ restriction enzyme if there was no abnormality. 2 Each virus was produced by transducing 293A cells.

[0215] Example 8. Confirmation of gene expression inhibition by adenovirus encoding dual-target short hairpin RNA (shRNA)

[0216] 8-1. Confirmation of inhibition of mTOR and STAT3 expression by CA102

[0217] 8-1-1. Confirmation of messenger RNA (mRNA) levels

[0218] 8-1-1-1. Bladder cancer

[0219] The expression inhibition effect of the recombinant adenovirus CA102 prepared in Example 7 on the target genes mTOR and STAT3 was confirmed in bladder cancer cell lines. Specifically, T24 cells (0.5×10 5 / well) and 253JBV cells (1×10 5 / well), CA10G and CA102 were treated to each well at a MOI of 10 after 1 hour, and RNA prep was performed using an RNA prep kit (Takara, 9767A) 72 hours later. RNA was then quantified using a spectrophotometer (Nanodirp), 400 ng / 20 ul was added to each tube using a real-time premix (RT premix, intron, 25081) and mixed evenly with the premix contents, and then a polymerase chain reaction (PCR) instrument was used to react at 45°C for 1 hour (hr) and at 95°C for 5 minutes, thereby synthesizing complementary deoxyribonucleic acid (cDNA). 2ul of the synthesized complementary deoxyribonucleic acid (cDNA) was used as a template, and a polymerase chain reaction (PCR) mixture (total volume 20ul) was prepared as an experimental group (template 2ul, forward primer 0.5ul (10pmole / ul), reverse primer 0.5ul (10pmole / ul), 10ul 2X master mix (Bioline, BIO-94005) and 7ul DW). After mixing the prepared polymerase chain reaction (PCR) mixture by vortexing and centrifuging, 40 cycles of reaction were performed in a quantitative polymerase chain reaction (qPCR) instrument (Applied Biosystems, QS3) at 95°C for 5 minutes, 95°C for 10 seconds, and 60°C for 30 seconds. The results were analyzed using the program in the quantitative polymerase chain reaction (qPCR) instrument.

[0220] The results showed that in both T24 cells and 253JBV cells, the recombinant adenovirus CA102 of the present invention encoding and expressing the human telomerase reverse transcriptase (hTERT) promoter and mTOR and STAT3 dual-target short hairpin RNA (shRNA) significantly inhibited the expression of mTOR and STAT3 genes compared with the recombinant adenovirus CA10G containing only the human telomerase reverse transcriptase (hTERT) promoter ( Fig. 20 ).

[0221] 8-1-1-2. Head and neck cancer

[0222] The method in the above example was used to confirm the inhibitory effect of the recombinant adenovirus CA102 prepared in the above example 7 on the expression of the target genes mTOR and STAT3 in HSC-2 and Fadu, which are head and neck cancer cell lines. The results showed that in both cell lines, the recombinant adenovirus CA102 of the present invention encoding and expressing the human telomerase reverse transcriptase (hTERT) promoter and the mTOR and STAT3 dual-target short hairpin RNA (shRNA) significantly inhibited the expression of the mTOR and STAT3 genes, compared with the recombinant adenovirus CA10G containing only the human telomerase reverse transcriptase (hTERT) promoter ( Fig.21 ).

[0223] 8-1-1-3. Skin squamous cell carcinoma

[0224] The method in the above example was used to confirm the inhibitory effect of the recombinant adenovirus CA102 prepared in the above example 7 on the expression of the target genes mTOR and STAT3 in A431 and HSC-5, which are skin squamous cell carcinoma cell lines. The results showed that in both cell lines, the recombinant adenovirus CA102 of the present invention encoding and expressing the human telomerase reverse transcriptase (hTERT) promoter and the mTOR and STAT3 dual-target short hairpin RNA (shRNA) significantly inhibited the expression of the mTOR and STAT3 genes, compared with the recombinant adenovirus CA10G containing only the human telomerase reverse transcriptase (hTERT) promoter ( Fig. 22 ).

[0225] 8-1-2. Confirmation of protein expression inhibition

[0226] After the recombinant adenovirus CA102 prepared in Example 7 was treated with bladder cancer cell lines T24 and 253JBV, the expression inhibition effect on the target genes mTOR and STAT3 was confirmed at the protein level by Western blot analysis. The results showed that in both cell lines, the recombinant adenovirus CA102 of the present invention inhibited the protein expression of mTOR and STAT3 ( Fig.23 ).

[0227] 8-2. Confirmation of inhibition of BCL2 and BI-1 expression by CA101

[0228] The recombinant adenovirus CA101 prepared in Example 7 above was confirmed to have an inhibitory effect on the expression of the target genes BCL2 and BI-1. Specifically, U-87 cells (1×10 5 / well), CA10G and CA101 were treated to each well at a MOI of 10 after 1 hour, and RNA prep was performed using an RNA prep kit (Takara, 9767A) 72 hours later. RNA was then quantified using a spectrophotometer (Nanodirp), 400 ng / 20 ul was added to each tube using a real-time premix (RT premix, intron, 25081) and mixed evenly with the premix contents, and then a polymerase chain reaction (PCR) instrument was used to react at 45°C for 1 hour (hr) and at 95°C for 5 minutes, thereby synthesizing complementary deoxyribonucleic acid (cDNA). 2ul of the synthesized complementary deoxyribonucleic acid (cDNA) was used as a template, and a polymerase chain reaction (PCR) mixture (total volume 20ul) was prepared as an experimental group (template 2ul, forward primer 0.5ul (10pmole / ul), reverse primer 0.5ul (10pmole / ul), 10ul 2×master mix (Bioline, BIO-94005) and 7ul DW). After mixing the prepared polymerase chain reaction (PCR) mixture by vortexing and centrifuging, 40 cycles of reaction were performed in a quantitative polymerase chain reaction (qPCR) instrument (Applied Biosystems, QS3) at 95°C for 5 minutes, 95°C for 10 seconds, and 60°C for 30 seconds. The results were analyzed using the program in the quantitative polymerase chain reaction (qPCR) instrument.

[0229] The results showed that in U-87 cells, the recombinant adenovirus CA101 of the present invention encoding and expressing the human telomerase reverse transcriptase (hTERT) promoter and BCL2 and BI-1 dual-target short hairpin RNA (shRNA) significantly inhibited the expression of BCL2 and BI-1 genes compared with the recombinant adenovirus CA10G containing only the human telomerase reverse transcriptase (hTERT) promoter ( Fig.24 ).

[0230] 8-3. Confirmation of inhibition of AR and mTOR expression by CA103

[0231] 8-3-1. In vitro vitro

[0232] The inhibitory effect of the recombinant adenovirus CA103 prepared in Example 7 on the expression of the target genes AR and mTOR was confirmed. 5After LNcap, C42B and 22Rv1, which are human prostate cancer cell lines, were injected into a 12-well plate, CA10G and CA103 were treated to each well at a MOI of 2 or 5 after 1 hour, and RNA prep was performed using an RNA prep kit (Takara, 9767A) 72 hours later. RNA was then quantified using a spectrophotometer (Nanodirp), and 400 ng / 20 ul was added to each tube using a real-time premix (RT premix, intron, 25081) and mixed evenly with the premix contents, and then a polymerase chain reaction (PCR) instrument was used to react at 45°C for 1 hour (hr) and at 95°C for 5 minutes to synthesize complementary deoxyribonucleic acid (cDNA). 2ul of the synthesized complementary deoxyribonucleic acid (cDNA) was used as a template, and a polymerase chain reaction (PCR) mixture (total volume 20ul) was prepared as an experimental group (template 2ul, forward primer 0.5ul (10pmole / ul), reverse primer 0.5ul (10pmole / ul), 10ul 2×master mix (Bioline, BIO-94005) and 7ul DW). After mixing the prepared polymerase chain reaction (PCR) mixture by vortexing and centrifuging, 40 cycles of reaction were performed in a quantitative polymerase chain reaction (qPCR) instrument (Applied Biosystems, QS3) at 95°C for 5 minutes, 95°C for 10 seconds, and 60°C for 30 seconds. The results were analyzed using the program in the quantitative polymerase chain reaction (qPCR) instrument.

[0233] The results showed that in the LNcap cell line, compared with the recombinant adenovirus CA10G containing only the human telomerase reverse transcriptase (hTERT) promoter, the recombinant adenovirus CA103 of the present invention encoding and expressing the human telomerase reverse transcriptase (hTERT) promoter and AR and mTOR dual-target short hairpin RNA (shRNA) significantly inhibited the expression of AR and mTOR genes ( Fig.25 ). Moreover, in C42B and 22Rv1, the recombinant adenovirus CA103 of the present invention also significantly inhibited the expression of AR and mTOR genes compared with CA10G ( Fig.26 ).

[0234] 8-3-2. In vivo

[0235] Prostate cancer cell line (22Rv-1) was subcutaneously transplanted into balb c nu / nu mice to construct a prostate cancer mouse template. Afterwards, the recombinant adenovirus CA10G and CA103 (2×10 8 pfu / spot, 3 times), and the tumor was removed 21 days later and the expression of AR and mTOR genes in the tumor was confirmed by Western blot analysis and IHC analysis. The results of Western blot analysis showed that in the group administered with CA103, the expression of mTOR and Ar was reduced compared with the control group and the CA10G administration group. The results of IHC analysis also showed that the fluorescence expression of mTOR and Ar in the group administered with CA103 was reduced by 70% to 790% or more compared with the control group and the CA10G administration group, confirming that CA103 effectively inhibited the expression of mTOR and Ar as target genes in the tumor by 9 ( Fig. 27 ).

[0236] 8-4. Confirmation of inhibition of c-MET and PD-L1 expression by CA104

[0237] The recombinant adenovirus CA104 prepared in Example 7 above was confirmed to have an inhibitory effect on the expression of target genes c-MET and PD-L1. Specifically, A431 cell lines (1×10 5 / well), CA10G and CA104 were treated to each well at a MOI of 2 or 5 after 1 hour, and RNA prep was performed using an RNA prep kit (Takara, 9767A) 72 hours later. RNA was then quantified using a spectrophotometer (Nanodirp), 400 ng / 20ul was added to each tube using a real-time premix (RT premix, intron, 25081) and mixed evenly with the premix contents, and then a polymerase chain reaction (PCR) instrument was used to react at 45°C for 1 hour (hr) and at 95°C for 5 minutes, thereby synthesizing complementary deoxyribonucleic acid (cDNA). 2ul of the synthesized complementary deoxyribonucleic acid (cDNA) was used as a template, and a polymerase chain reaction (PCR) mixture (total volume 20ul) was prepared as an experimental group (template 2ul, forward primer 0.5ul (10pmole / ul), reverse primer 0.5ul (10pmole / ul), 10ul 2X master mix (Bioline, BIO-94005) and 7ul DW). After mixing the prepared polymerase chain reaction (PCR) mixture by vortexing and centrifuging, 40 cycles of reaction were performed in a quantitative polymerase chain reaction (qPCR) instrument (Applied Biosystems, QS3) at 95°C for 5 minutes, 95°C for 10 seconds, and 60°C for 30 seconds. The results were analyzed using the program in the quantitative polymerase chain reaction (qPCR) instrument.

[0238] The results showed that in A431 cells, the recombinant adenovirus CA104 of the present invention encoding and expressing the human telomerase reverse transcriptase (hTERT) promoter and c-MET and PD-L1 dual-target short hairpin RNA (shRNA) significantly inhibited the expression of c-MET and PD-L1 genes compared with the recombinant adenovirus CA10G containing only the human telomerase reverse transcriptase (hTERT) promoter ( Fig.28 ).

[0239] Example 9. Confirmation of the anticancer effect of adenovirus encoding dual-target short hairpin RNA (shRNA)

[0240] 9-1. Confirmation of the anticancer effect of CA101

[0241] The cancer cell killing effects of the recombinant adenoviruses CA10G and CA101 prepared in Example 7 were compared. Specifically, U87MG cells (5×10 3 / well), and CA10G and CA101 were treated to each well at 1MOI, 2MOI, 5MOI, 10MOI, 30MOI or 50MOI after 1 hour, and MTT reagent was added after 72 hours and cultured at 37°C for 3 hours. After 3 hours, the culture medium was removed from each well and 100ul of DMSO was added, and then the absorbance was measured at a wavelength of 540nm using a microplate reader and MTT analysis was performed.

[0242] The results showed that CA101 of the present invention significantly killed cancer cells compared with CA10G ( Fig.29 ).

[0243] 9-2. Confirmation of the anticancer effect of CA102

[0244] 9-2-1. Bladder cancer

[0245] The cancer cell killing effects of the recombinant adenoviruses CA10G and CA102 prepared in Example 7 were compared. Specifically, T24 cells (2.5×10 3 / well), 253J-BV cells (5×10 3 / well) and human bladder epithelial cell line RT4 cells (5×10 3 / well), and CA10G and CA102 were treated to each well at 1MOI, 2MOI, 5MOI, 10MOI, 20MOI or 50MOI after 1 hour, and MTT reagent was added after 72 hours and cultured at 37°C for 3 hours. After 3 hours, the culture medium was removed from each well and 100ul of DMSO was added, and then the absorbance was measured at a wavelength of 540nm using a microplate reader and MTT analysis was performed.

[0246] The results showed that CA102 of the present invention significantly killed cancer cells compared to CA10G ( Fig.30 ).

[0247] 9-2-2. Head and neck cancer

[0248] In order to confirm the killing effect of the recombinant adenovirus CA102 of the present invention on head and neck cancer cells, the recombinant adenovirus CA10G and CA102 prepared in the above Example 4 were treated with HSC-2 and Fadu as head and neck cancer cell lines, and the cell killing effect was compared by MTT analysis. The results showed that when treated with CA10G, about 40% of the cells were killed, while CA102 encoding and expressing short hairpin RNA (shRNA) with dual targets of mTOR and STAT3 caused more than 70% of the cells to be killed ( Fig.31 ).

[0249] 9-2-3. Skin squamous cell carcinoma

[0250] In order to confirm the skin squamous cell carcinoma cell killing effect of the recombinant adenovirus CA102 of the present invention, the recombinant adenovirus CA10G and CA102 prepared in Example 4 were treated with A431 and HSC-5 as skin squamous cell carcinoma cell lines, and the cell killing effect was compared by MTT analysis. The results showed that the cell killing effect of CA102 was significantly higher than that of CA10G (based on the treatment of 10 MOI). Fig.32 ).

[0251] 9-3. Confirmation of the anticancer effect of CA103

[0252] The cancer cell killing effects of the recombinant adenoviruses CA10G and CA103 prepared in Example 7 were compared. 3 After LNcap, C42B and 22Rv1 cell lines were injected into 96-well plates, CA10G and CA103 were treated with 1MOI, 2MOI, 5MOI, 10MOI, 20MOI, 40MOI or 50MOI in each well 1 hour later, and MTT reagent was added 72 hours later and cultured at 37°C for 3 hours. After 3 hours, the culture medium was removed from each well and 100ul of DMSO was added, and then the absorbance was measured at a wavelength of 540nm using a microplate reader and MTT analysis was performed.

[0253] The results showed that in LNcap cell line, CA103 of the present invention significantly killed cancer cells compared with CA10G ( Fig.33 ), in C42B and 22Rv1 cell lines, CA103 of the present invention also significantly killed cancer cells ( Fig.34 ).

[0254] Example 10. Confirmation of the in vivo anticancer effect of adenovirus encoding dual-target short hairpin RNA (shRNA)

[0255] 10-1. Confirmation of the anti-cancer effect of CA102

[0256] 10-1-1. Anticancer effect on cancer cells in vivo

[0257] In order to confirm the anticancer effect of the recombinant adenovirus CA102 of the present invention on cancer cells in vivo, the 3 After culturing 1.0×107 cells of bladder cancer cell line 253J-BV and head and neck cancer cell line FaDu on plates, the recombinant adenoviruses CA10G and CA102 of the present invention were treated with 2MOI and 5MOI for 1 hour respectively (the control group was treated with PBS), and then cultured for 2 hours after replacing with a new culture medium. After that, the cells were recovered and mixed with Matrigel at a ratio of 1:1 (v / v), and after transplantation (Xenograft) into 6-week-old male Balb / c nu-nu mice, they were observed for 32 days. The results showed that in 253J-BV, the size of cancer cells was significantly reduced in the group treated with CA10G at 2MOI, while cancer cells were eliminated in the group treated with CA102 at 2MOI ( Fig.35 ). In addition, in FaDu, the size of cancer cells in the group treated with CA10G at 5 MOI did not show a significant difference compared with the control group, but in the group treated with CA102 at 5 MOI, cancer cells were eliminated ( Fig.36 ).

[0258] 10-1-2. Anticancer effect on tumors formed in vivo

[0259] Six-week-old male Balb / c nu-nu mice were transplanted with 5.0×10 6 After the bladder cancer cell line 253J-BV was transplanted, the patients were observed twice a week. When the tumor size reached an average of 150 to 200 mm 3 The mice were divided into multiple groups so that the average tumor size was constant. 2.0×10 8 PFU of CA10G and CA102 viruses. After that, the tumor size was observed twice a week for 43 days. The results showed that when CA10G was administered, the tumor size was smaller than that of the control group, but as time went on, the growth rate of cancer cells increased again. On the contrary, when CA102 was administered, the tumor size was significantly smaller than that of the control group and CA10G, and the growth of cancer cells was significantly inhibited even after the passage of time ( Fig.37 ).

[0260] 10-1-3. Anticancer effect on tumors formed in vivo based on the number of administrations

[0261] Six-week-old male Balb / c nu-nu mice were transplanted with 5.0×10 6 The bladder cancer cell line 253J-BV was observed twice a week. When the average tumor size reached 200 mm3, the tumors were divided into multiple groups in such a way that the average tumor size was constant. 1.0×10 8 PFU of CA10G and CA102 viruses. After that, the tumor size was observed twice a week for 42 days. The results showed that the tumor size decreased based on the number of CA102 administrations, and it was confirmed that the growth of cancer cells was suppressed based on the number of administrations ( Fig.38 ).

[0262] 10-1-4. Anticancer effect on tumors formed in vivo based on the dosage

[0263] After transplanting glioblastoma cell line (U-87) into 6-week-old male Balb / c nu-nu mice, the size of the tumor was observed. When the tumor size reached an average of 200 mm 3 The researchers divided the patients into multiple groups so that the average tumor size would remain constant. Different doses of CA102 were administered directly into the tumors in each group to observe the tumor volume and weight. The results showed that compared with the CA10G-treated group, the tumor volume and weight in the CA102-treated group were significantly reduced, and the anticancer effect increased further with the increase in the dose ( Fig.39 ).

[0264] 10-2. Confirmation of the anti-cancer effect of CA103

[0265] After subcutaneously transplanting prostate cancer cell lines (22Rv-1) into balb c nu / nu mice to construct a prostate cancer mouse model, the recombinant adenoviruses CA10G and CA103 prepared in Example 7 were directly administered into the tumor (2×10 8 pfu / spot, 3 times) to observe its growth. The results showed that the volume and weight of the tumor in the CA103-treated group were significantly reduced compared with the untreated control group (buffer-treated group) and the vehicle control group (CA10G-treated group). Fig.40 ).

[0266] Example 11. Confirmation of the effect of combined treatment with an anticancer agent encoded by adenovirus encoding dual-target short hairpin RNA (shRNA)

[0267] After constructing bladder cancer mouse models by subcutaneously transplanting bladder cancer cell lines (253J-BV) into balb c nu / nu mice, the recombinant adenoviruses CA10G and CA102 prepared in Example 7 were directly administered into the tumors to observe tumor growth. The results showed that the volume and weight of the tumors in the CA102-treated group were significantly reduced compared with the untreated control group (buffer-treated group) and the vector control group (CA10G-treated group). In the case of concurrent administration of cisplatin as an anticancer agent, a synergistic increase in the anticancer effect was confirmed ( Fig.41 ). SEQUENCE LISTING <110> Kulikin Co., Ltd. <120> Structure of oncolytic viruses containing bispecific nucleic acid molecules <130> P22116854WP <160> 82 <170> PatentIn version 3.5 <210> 1 <211> twenty one <212> RNA <213> Artificial Sequence <220> <223> si-MS1 <400> 1 gacuguggca uccaccugca u 21 <210> 2 <211> twenty one <212> RNA <213> Artificial Sequence <220> <223> si-MS1 antisense strand <400> 2 augcagguag gcgccucagu c 21 <210> 3 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> si-MS2 sense strand <400> 3 gacuguggca uccaccugca 20 <210> 4 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> si-MS2 antisense strand <400> 4 ugcagguagg cgccucaguc 20 <210> 5 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> si-MS3 sense strand <400> 5 gacuguggca uccaccugc 19 <210> 6 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> si-MS3 antisense strand <400> 6 gcagguaggc gccucaguc 19 <210> 7 <211> 18 <212> RNA <213> Artificial Sequence <220> <223> si-MS4 sense strand <400> 7 gacuguggca uccaccug 18 <210> 8 <211> 18 <212> RNA <213> Artificial Sequence <220> <223> si-MS4 antisense strand <400> 8 cagguaggcg ccucaguc 18 <210> 9 <211> 17 <212> RNA <213> Artificial Sequence <220> <223> si-MS5 sense strand <400> 9 ucagccacag cagcuug 17 <210> 10 <211> 17 <212> RNA <213> Artificial Sequence <220> <223> si-MS5 antisense strand <400> 10 caagcugcug uagcuga 17 <210> 11 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> si-MS6 sense strand <400> 11 gcagcgcaug cggcccagca 20 <210> 12 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> si-MS6 antisense strand <400> 12 ugcugggccg caguggcugc 20 <210> 13 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> si-MS7 sense strand <400> 13 gcagcgcaug cggcccagc 19 <210> 14 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> si-MS7 antisense strand <400> 14 gcugggccgc aguggcugc 19 <210> 15 <211> 18 <212> RNA <213> Artificial Sequence <220> <223> si-MS8 sense strand <400> 15 gcagcgcaug cggcccag 18 <210> 16 <211> 18 <212> RNA <213> Artificial Sequence <220> <223> si-MS8 antisense strand <400> 16 cugggccgca guggcugc 18 <210> 17 <211> 17 <212> RNA <213> Artificial Sequence <220> <223> si-MS9 sense strand <400> 17 gcagcgcaug cggccca 17 <210> 18 <211> 17 <212> RNA <213> Artificial Sequence <220> <223> si-MS9 antisense strand <400> 18 ugggccgcag uggcugc 17 <210> 19 <211> twenty one <212> RNA <213> Artificial Sequence <220> <223> si-BB1 sense strand <400> 19 aagaagagga gaaaaaaaug a 21 <210> 20 <211> twenty one <212> RNA <213> Artificial Sequence <220> <223> si-BB1 antisense strand <400> 20 ucauuucuuc ucuuucuucu u 21 <210> twenty one <211> 20 <212> RNA <213> Artificial Sequence <220> <223> si-BB2 sense strand <400> twenty one aagaagagga gaaaaaaaug 20 <210> twenty two <211> 20 <212> RNA <213> Artificial Sequence <220> <223> si-BB2 antisense strand <400> twenty two cauuucuucu cuuucuucuu 20 <210> twenty three <211> 20 <212> RNA <213> Artificial Sequence <220> <223> si-BB3 sense strand <400> twenty three agaagaggag aaaaaaauga 20 <210> twenty four <211> 20 <212> RNA <213> Artificial Sequence <220> <223> si-BB3 antisense strand <400> twenty four cauuucuucu cuuucuucuu 20 <210> 25 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> si-BB4 sense strand <400> 25 gaagaggaga aaaaaauga 19 <210> 26 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> si-BB4 antisense strand <400> 26 ucauuucuuc ucuuucuuc 19 <210> 27 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> si-BB5 sense strand <400> 27 agaagaggag aaaaaaaug 19 <210> 28 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> si-BB5 antisense strand <400> 28 cauuucuucu cuuucuucu 19 <210> 29 <211> 18 <212> RNA <213> Artificial Sequence <220> <223> si-BB6 sense strand <400> 29 gaagaggaga aaaaaaug 18 <210> 30 <211> 18 <212> RNA <213> Artificial Sequence <220> <223> si-BB6 antisense strand <400> 30 cauuucuucu cuuucuuc 18 <210> 31 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> si-AT1 sense strand <400> 31 gcugcugcug cugccugggg 20 <210> 32 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> si-AT1 antisense strand <400> 32 ccccaugcag cugcagcagc 20 <210> 33 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> si-AT2 sense strand <400> 33 cugcugcugc ugccugggg 19 <210> 34 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> si-AT2 antisense strand <400> 34 ccccaugcag cugcagcag 19 <210> 35 <211> 18 <212> RNA <213> Artificial Sequence <220> <223> si-AT3 sense strand <400> 35 ugcugcugcu gccugggg 18 <210> 36 <211> 18 <212> RNA <213> Artificial Sequence <220> <223> si-AT3 antisense strand <400> 36 ccccaugcag cugcagca 18 <210> 37 <211> 17 <212> RNA <213> Artificial Sequence <220> <223> si-AT4 sense strand <400> 37 gcugcugcug ccugggg 17 <210> 38 <211> 17 <212> RNA <213> Artificial Sequence <220> <223> si-AT4 antisense strand <400> 38 ccccaugcag cugcagc 17 <210> 39 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> si-AT5 sense strand <400> 39 ccacccccac caccaccac 19 <210> 40 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> si-AT5 antisense strand <400> 40 gugguggcag cgguggugg 19 <210> 41 <211> 18 <212> RNA <213> Artificial Sequence <220> <223> si-AT6 sense strand <400> 41 ccacccccac caccacca 18 <210> 42 <211> 18 <212> RNA <213> Artificial Sequence <220> <223> si-AT6 antisense strand <400> 42 ugguggcagc gguggugg <210> 43 <211> 17 <212> RNA <213> Artificial Sequence <220> <223> si-AT7 switch <400> 43 17. ccccccc cccccc <210> 44 <211> 17 <212> RNA <213> Artificial Sequence <220> <223> si-AT7 drill <400> 44 gguggcagcg gguggg <210> 45 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> si-AT8 switch <400> 45 uggaggcaga gagugagaga 23 years <210> 46 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> si-AT8 holder <400> 46 23. uucucucaga cgcucucccu <210> 47 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> si-AT9 sense strand <400> 47 ggaggcagag agugagagag aa 22 <210> 48 <211> twenty two <212> RNA <213> Artificial Sequence <220> <223> si-AT9 antisense strand <400> 48 uucucucaga cgcucucccu cc 22 <210> 49 <211> twenty two <212> RNA <213> Artificial Sequence <220> <223> si-AT10 sense strand <400> 49 uggaggcaga gagugagaga ga 22 <210> 50 <211> twenty two <212> RNA <213> Artificial Sequence <220> <223> si-AT10 antisense strand <400> 50 ucucucagac gcucucccuc ca 22 <210> 51 <211> twenty one <212> RNA <213> Artificial Sequence <220> <223> si-AT11 sense strand <400> 51 uggaggcaga gagugagaga g 21 <210> 52 <211> twenty one <212> RNA <213> Artificial Sequence <220> <223> si-AT11 antisense strand <400> 52 cucucagacg cucucccucc a 21 <210> 53 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> si-AT12 sense strand <400> 53 ggaggcagag agugagagag 20 <210> 54 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> si-AT12 antisense strand <400> 54 cucucagacg cucucccucc 20 <210> 55 <211> twenty one <212> RNA <213> Artificial Sequence <220> <223> si-AT13 sense strand <400> 55 ggaggcagag agugagagag a 21 <210> 56 <211> twenty one <212> RNA <213> Artificial Sequence <220> <223> si-AT13 antisense strand <400> 56 ucucucagac gcucucccuc c 21 <210> 57 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> si-MM sense strand <400> 57 gcagcuugga ggcagagcg 19 <210> 58 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> si-MM antisense strand <400> 58 cgcucucccu ccaugcugc 19 <210> 59 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> si-BM sense strand <400> 59 gccaaggcca cacagccaa 19 <210> 60 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> si-BM antisense strand <400> 60 uuggcuuugu guccuuggc 19 <210> 61 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> si-ST sense strand <400> 61 ccagccagac ccagaagga 19 <210> 62 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> si-ST antisense strand <400> 62 uccuucuugg acaggcugg 19 <210> 63 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Dual-targeted siRNA (antisense c-MET) <400> 63 accacacauc ugacuuggu 19 <210> 64 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Dual-targeted siRNA (antisense PD-L1) <400> 64 accaauucag cuguauggu 19 <210> 65 <211> 276 <212> DNA <213> Artificial Sequence <220> <223> U6 promoter <400> 65 cctagagtcg acactagata acaacatagg agctgtgatt ggctgttttc agccaatcag 60 cactgactca tttgcatagc ctttacaagc ggtcacaaac tcaagaaacg agcggtttta 120 atagtctttt agaatattgt ttatcgaacc gaataaggaa ctgtgctttg tgattcacat 180 atcagtggag gggtgtggaa atggcacctt gatctcaccc tcatcgaaag tggagttgat 240 gtccttccct ggctcgctac agacgcactt ccgcaa 276 <210> 66 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> MS-TTGGATCCAA loop shRNA <400> 66 gactgtggca tccacctgca tttggatcca aatgcaggta ggcgcctcag tctt 54 <210> 67 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> MS-TTCAAGAGAG loop shRNA <400> 67 gactgtggca tccacctgca tttcaagaga gatgcaggta ggcgcctcag tctt 54 <210> 68 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> BB-TTGGATCCAA loop shRNA <400> 68 aagaagagga gaaaaaaatg attggatcca atcatttctt ctctttcttc tttt 54 <210> 69 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> BB-TTCAAGAGAG loop shRNA <400> 69 aagaagagga gaaaaaaatg attcaagaga gtcatttctt ctctttcttc tttt 54 <210> 70 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> AT-TTGGATCCAA loop shRNA <400> 70 gctgctgctg ctgcctgggg ttggatccaa ccccatgcag ctgcagcagc tt 52 <210> 71 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> AT-TTCAAGAGAG loop shRNA <400> 71 gctgctgctg ctgcctgggg ttcaagagag ccccatgcag ctgcagcagc tt 52 <210> 72 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> c-MET / PD-L1-TTGGATCCAA loop shRNA coding sequence <400> 72 accacacatc tgacttggtt tggatccaaa ccaattcagc tgtatggttt 50 <210> 73 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> c-MET / PD-L1-TTCAAGAGAG loop shRNA coding sequence <400> 73 accacacatc tgacttggtt tcaagagaga ccaattcagc tgtatggttt 50 <210> 74 <211> 455 <212> DNA <213> Artificial Sequence <220> <223> hTERT promoter <400> 74 tggcccctcc ctcgggttac cccacagcct aggccgattc gacctctctc cgctggggcc 60 ctcgctggcg tccctgcacc ctgggagcgc gagcggcgcg cgggcgggga agcgcggccc 120 agacccccgg gtccgcccgg agcagctgcg ctgtcggggc caggccgggc tcccagtgga 180 ttcgcgggca cagacgccca ggaccgcgct ccccacgtgg cggagggact ggggacccgg 240 gcacccgtcc tgccccttca ccttccagct ccgcctcctc ccgcggacc ccgccccgtc 300 ccgacccctc ccgggtcccc ggcccagcccc cctccggggcc ctccccgccc ctccccttcc 360 tttccgcggc cccgccctct cctcgcggcg cgagtttcag gcagcgctgc gtcctgctgc 420 gcacgtggga agccctggcc ccggccacccc ccggcg 455 <210> 75 <211> 900 <212> DNA <213> Artificial Sequence <220> <223> E1A <400> 75 60. acaccgggac tgaaaatgag acatattatc tgccacggag gtgttattac cgaagaaatg gccgccagtc ttttggacca gctgatcga gaggtactgg ctgataatct tccacctcct agccattttg aaccacctac ccttcacgaa ctgtatgatt tagacgtgac ggcccccgaa gatcccaacg aggaggcggt ttcgcagatt tttcccgact ctgtaatgtt ggcggtgcag 240 gaagggattg acttactcac ttttccgccg gcgcccggtt ctccggagcc gcctcacctt 300 tcccggcagc ccgagcagcc ggagcagaga gccttgggtc cggtttctat gccaaacctt 360 gtaccggagg tgatcgatct tacctgccac gaggctggct ttccacccag tgacgacgag 420 gatgaagagg gtgaggagtt tgtgttagat tatgtggagc accccgggca cggttgcagg 480 tcttgtcatt atcaccggag gaatacgggg gacccagata ttatgtgttc gctttgctat 540 atgaggacct gtggcatgtt tgtctacagt cctgtgtctg aacctgagcc tgagcccgag 600 ccagaaccgg agcctgcaag acctacccgc cgtcctaaaa tggcgcctgc tatcctgaga 660 cgcccgacat cacctgtgtc tagagaatgc aatagtagta cggatagctg tgactccggt 720 ccttctaaca cacctcctga gatacacccg gtggtcccgc tgtgccccat taaaccagtt 780 gccgtgagag ttggtgggcg tcgccaggct gtggaatgta tcgaggactt gcttaacgag 840 cctgggcaac ctttggactt gagctgtaaa cgccccaggc cataaggtgt aaacctgtga 900 <210> 76 <211> 572 <212> DNA <213> Artificial Sequence <220> <223> IRES <400> 76 cccctctccc tcccccccct aacgttactg gccgaagccg cttggaataa ggccggtgtg 60 cgtttgtcta tatgttattt tccaccatat tgccgtcttt tggcaatgtg agggcccgga 120 aacctggccc tgtcttcttg acgagcattc ctaggggtct ttcccctctc gccaaaggaa 180 tgcaaggtct gttgaatgtc gtgaaggaag cagttcctct ggaagcttct tgaagacaaa 240 caacgtctgt agcgaccctt tgcaggcagc ggaacccccc acctggcgac aggtgcctct 300 gcggccaaaa gccacgtgta taagatacac ctgcaaaggc ggcacaaccc cagtgccacg 360 ttgtgagttg gatagttgtg gaaagagtca aatggctctc ctcaagcgta ttcaacaagg 420 ggctgaagga tgcccagaag gtaccccatt gtatgggatc tgatctgggg cctcggtgca 480 catgctttac atgtgtttag tcgaggttaa aaaaacgtct aggccccccg aaccacgggg 540 acgtggtttt cctttgaaaa acacgatgat aa 572 <210> 77 <211> 1797 <212> DNA <213> Artificial Sequence <220> <223> E1B <400> 77 catggaggct tgggagtgtt tggaagattt ttctgctgtg cgtaacttgc tggaacagag 60 ctctaacagt acctcttggt tttggaggtt tctgtggggc tcatcccagg caaagttagt 120 ctgcagaatt aaaggaggatt acaagtggga atttgaagag cttttgaaat cctgtggtga 180 gctgtttgat tctttgaatc tgggtcacca ggcgcttttc caagagaagg tcatcaagac 240 tttggtttt tccacaccgg ggcgcgctgc ggctgctgtt gcttttttga gttttataaa 300 ggataaatgg agcgaagaaa cccatctgag cggggggtac ctgctggatt ttctggccat 360 gcatctgtgg agagcggttg tgagacacaa gaatcgcctg ctactgttgt cttccgtccg 420 cccggcgata ataccgacgg aggagcagca gcagcagcag ggagagcca ggcggcggcg 480 gcaggagcag agcccatgga acccgagagc cggcctggac cctcgggaat gaatgttgta 540 caggtggctg aactgtatcc agaactgaga cgcattttga caattacaga ggatgggcag 600 gggctaaagg gggtaaagag ggagcgggg gcttgtgagg ctacagagga ggctaggaat 660 ctagctttta gcttaatgac cagacaccgt cctgagtgta ttacttttca acagatcaag 720 gataattgcg ctaatgagct tgatctgctg gcgcagaagt attccataga gcagctgacc 780 acttactggc tgcagccagg ggatgatttt gaggaggcta ttagggtata tgcaaaggtg 840 gcacttaggc cagattgcaa gtacaagatc agcaaacttg taaatatcag gaattgttgc 900 tacatttctg ggaacggggc cgaggtggag atagatacgg aggatagggt ggcctttaga 960 tgtagcatga taaatatgtg gccgggggtg cttggcatgg acggggtggt tattatgaat 1020 gtaaggttta ctggccccaa ttttagcggt acggttttcc tggccaatac caaccttatc 1080 ctacacggtg taagcttcta tgggtttaac aatacctgtg tggaagcctg gaccgatgta 1140 agggttcggg gctgtgcctt ttactgctgc tggaaggggg tggtgtgtcg ccccaaaagc 1200 agggcttcaa ttaagaaatg cctctttgaa aggtgtacct tgggtatcct gtctgagggt 1260 aactccaggg tgcgccacaa tgtggcctcc gactgtggtt gcttcatgct agtgaaaagc 1320 gtggctgtga ttaagcataa catggtatgt ggcaactgcg aggacagggc ctctcagatg 1380 ctgacctgct cggacggcaa ctgtcacctg ctgaagacca ttcacgtagc cagccactct 1440 cgcaaggcct ggccagtgtt tgagcataac atactgaccc gctgttcctt gcatttgggt 1500 aacaggaggg gggtgttcct accttaccaa tgcaatttga gtcacactaa gatattgctt 1560 gagcccgaga gcatgtccaa ggtgaacctg aacggggtgt ttgacatgac catgaagatc 1620 tggaaggtgc tgaggtacga tgagacccgc accaggtgca gaccctgcga gtgtggcggt 1680 aaacatatta ggaaccagcc tgtgatgctg gatgtgaccg aggagctgag gcccgatcac 1740 ttggtgctgg cctgcacccg cgctgagttt ggctctagcg atgaagatac agattga 1797 <210> 78 <211> 3762 <212> DNA <213> Artificial Sequence <220> <223> hTERT-E1A-IRES-E1B <400> 78 tggcccctcc ctcgggttac cccacagcct aggccgattc gacctctctc cgctggggcc 60 ctcgctggcg tccctgcacc ctgggagcgc gagcggcgcg cgggcgggga agcgcggccc 120 agacccccgg gtccgcccgg agcagctgcg ctgtcggggc caggccgggc tcccagtgga 180 ttcgcgggca cagacgccca ggaccgcgct ccccacgtgg cggagggact ggggacccgg 240 gcacccgtcc tgccccttca ccttccagct ccgcctcctc cgcgcggacc ccgccccgtc 300 ccgacccctc ccgggtcccc ggcccagccc cctccgggcc ctcccagccc ctccccttcc 360 tttccgcggc cccgccctct cctcgcggcg cgagtttcag gcagcgctgc gtcctgctgc 420 gcacgtggga agccctggcc ccggccaccc ccgcgactag tacaccggga ctgaaaatga 480 gacatattat ctgccacgga ggtgttatta ccgaagaaat ggccgccagt cttttggacc 540 agctgatcga agaggtactg gctgataatc ttccacctcc tagccatttt gaaccaccta 600 cccttcacga actgtatgat ttagacgtga cggcccccga agatcccaac gaggaggcgg 660 tttcgcagat ttttcccgac tctgtaatgt tggcggtgca ggaagggatt gacttactca 720 cttttccgcc ggcgcccggt tctccggagc cgcctcacct ttcccggcag cccgagcagc 780 cggagcagag agccttgggt ccggtttcta tgccaaacct tgtaccggag gtgatcgatc 840 ttacctgcca cgaggctggc tttccaccca gtgacgacga ggatgaagag ggtgaggagt 900 ttgtgttaga ttatgtggag caccccgggc acggttgcag gtcttgtcat tatcaccgga 960 ggaatacggg ggacccagat attatgtgtt cgctttgcta tatgaggacc tgtggcatgt 1020 ttgtctacag tcctgtgtct gaacctgagc ctgagcccga gccagaaccg gagcctgcaa 1080 gacctacccg ccgtcctaaa atggcgcctg ctatcctgag acgcccgaca tcacctgtgt 1140 ctagagaatg caatagtagt acggatagct gtgactccgg tccttctaac acacctcctg 1200 agatacaccc ggtggtcccg ctgtgcccca ttaaaccagt tgccgtgaga gttggtgggc 1260 gtcgccaggc tgtggaatgt atcgaggact tgcttaacga gcctgggcaa cctttggact 1320 tgagctgtaa acgccccagg ccataaggtg taaacctgtg acccctctcc ctcccccccc 1380 taacgttact ggccgaagcc gcttggaata aggccggtgt gcgtttgtct atatgttatt 1440 ttccaccata ttgccgtctt ttggcaatgt gagggcccgg aaacctggcc ctgtcttctt 1500 gacgagcatt cctaggggtc tttcccctct cgccaaagga atgcaaggtc tgttgaatgt 1560 cgtgaaggaa gcagttcctc tggaagcttc ttgaagacaa acaacgtctg tagcgaccct 1620 ttgcaggcag cggaaccccc cacctggcga caggtgcctc tgcggccaaa agccacgtgt 1680 ataagataca cctgcaaagg cggcacaacc ccagtgccac gttgtgagtt ggatagttgt 1740 ggaaagagtc aaatggctct cctcaagcgt attcaacaag gggctgaagg atgcccagaa 1800 ggtaccccat tgtatgggat ctgatctggg gcctcggtgc acatgcttta catgtgttta 1860 gtcgaggtta aaaaaacgtc taggcccccc gaaccacggg gacgtggttt tcctttgaaa 1920 aacacgatga taagcttgcc acaacccggg atcctctaga gtcgacatgg aggcttggga 1980 gtgtttggaa gatttttctg ctgtgcgtaa cttgctggaa cagagctcta acagtacctc 2040 ttggttttgg aggtttctgt ggggctcatc ccaggcaaag ttagtctgca gaattaagga 2100 ggattacaag tgggaatttg aagagctttt gaaatcctgt ggtgagctgt ttgattcttt 2160 gaatctgggt caccaggcgc ttttccaaga gaaggtcatc aagactttgg atttttccac 2220 accggggcgc gctgcggctg ctgttgcttt tttgagtttt ataaaggata aatggagcga 2280 agaaacccat ctgagcgggg ggtacctgct ggattttctg gccatgcatc tgtggagagc 2340 ggttgtgaga cacaagaatc gcctgctact gttgtcttcc gtccgcccgg cgataatacc 2400 gacggaggag cagcagcagc agcaggagga agccaggcgg cggcggcagg agcagagccc 2460 atggaacccg agagccggcc tggaccctcg ggaatgaatg ttgtacaggt ggctgaactg 2520 tatccagaac tgagacgcat tttgacaatt acagaggatg ggcaggggct aaagggggta 2580 aagagggagc ggggggcttg tgaggctaca gaggaggcta ggaatctagc ttttagctta 2640 atgaccagac accgtcctga gtgtattact tttcaacaga tcaaggataa ttgcgctaat 2700 gagcttgatc tgctggcgca gaagtattcc atagagcagc tgaccactta ctggctgcag 2760 ccaggggatg atttgagga ggctattagg gtatatgcaa aggtggcact taggccagat 2820 tgcaagtaca agatcagcaa acttgtaaat atcaggaatt gttgctacat ttctgggaac 2880 ggggccgagg tggagataga tacggaggat agggtggcct ttagatgtag catgataaat 2940 atgtggccgg gggtgcttgg catggacggg gtggttatta tgaatgtaag gttactggc 3000 cccaatttta gcggtacggt tttcctggcc aataccaacc ttatcctaca cggtgtaagc 3060 ttctatgggt ttaacaatac ctgtgtggaa gcctggaccg atgtaagggt tcggggctgt 3120 gccttttact gctgctggaa gggggtggtg tgtcgcccca aaagcagggc ttcaattaag 3180 aaatgcctct ttgaaaggtg taccttgggt atcctgtctg agggtaactc cagggtgcgc 3240 cacaatgtgg cctccgactg tggttgcttc atgctagtga aaagcgtggc tgtgattaag 3300 cataacatgg tatgtggcaa ctgcgaggac agggcctctc agatgctgac ctgctcggac 3360 ggcaactgtc acctgctgaa gaccattcac gtagccagcc actctcgcaa ggcctggcca 3420 gtgtttgagc ataacatact gacccgctgt tccttgcatt tgggtaacag gaggggggtg 3480 ttcctacctt accaatgcaa tttgagtcac actaagatat tgcttgagcc cgagagcatg 3540 tccaaggtga acctgaacgg ggtgtttgac atgaccatga agatctggaa ggtgctgagg 3600 tacgatgaga cccgcaccag gtgcagaccc tgcgagtgtg gcggtaaaca tattaggaac 3660 cagcctgtga tgctggatgt gaccgaggag ctgaggcccg atcacttggt gctggcctgc 3720 acccgcgctg agttggctc tagcgatgaa gatacagatt ga 3762 <210> 79 <211> 319 <212> DNA <213> Artificial Sequence <220> <223> U6promoter-Bs-shRNA (CA101) <400> 79 aaggtcgggc aggaagaggg cctatttccc atgattcctt catatttgca tatacgatac 60 aaggctgtta gagagataat tagaattaat ttgactgtaa acacaaagat attagtacaa 120 aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt 180 aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat 240 atcttgtgga aaggacgaaa caccgaagaa gaggagaaaa aaatgattca agagagtcat 300 ttctctcttt tctcttttt 319 <210> 80 <211> 317 <212> DNA <213> Artificial Sequence <220> <223> U6promoter-Bs-shRNA (CA102) <400> 80 aaggtcgggc aggaagaggg cctatttccc atgattcctt catatttgca tatacgatac 60 aaggctgtta gagagataat tagaattaat ttgactgtaa acacaaagat attagtacaa 120 aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt 180 aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat 240 atcttgtgga aaggacgaaa caccggactg tggcatccac ctgcatttca agagagatgc 300 aggtaggcgc ctcagtc 317 <210> 81 <211> 315 <212> DNA <213> Artificial Sequence <220> <223> U6promoter-Bs-shRNA (CA103) <400> 81 aaggtcgggc aggaagaggg cctatttccc atgattcctt catatttgca tatacgatac 60 aaggctgtta gagagataat tagaattaat ttgactgtaa acacaaagat attagtacaa 120 aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt 180 aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat 240 atcttgtgga aaggacgaaa caccggctgc tgctgctgcc tggggttcaa gagagcccca 300 tgcagctgca gcagc 315 <210> 82 <211> 483 <212> DNA <213> Artificial Sequence <220> <223> E3 DEL <400> 82 atgattagt acataatcct aggtttactc acccttgcgt cagcccacgg taccacccaa 60 aaggtggatt ttaaggagcc agcctgtaat gttacattcg cagctgaagc taatgagtgc accactctta taaaatgcac cacagaacat gaaagctgc ttattcgcca caaaaacaaa attggcaagt atgctgttta tgctatttgg cagccaggtg acactacaga gtataatgtt acagttttcc agggtaaaag tcataaaact tttatgtata cttttccatt ttatgaaatg 360. tgcgacatta ccatgtacat gagcaaacag tataagttgt ggcccccaca aaattgtgtg gaaaacactg gcactttctg ctgcactgct atgctaatta cagtgctcgc tttggtctgt 420 accctactct attack caaaagcaga cgcagcttta ttgaggaaaa gaaaatgcct taa 483

Claims

1. An anti-tumor adenovirus, comprising: a human telomerase reverse transcriptase (hTERT) promoter; and an expression cassette comprising a first nucleic acid sequence targeting the mTOR gene and a second nucleic acid sequence targeting the STAT3 gene, The anti-tumor adenovirus is characterized in that: When expressed in cells or tissues, the first nucleic acid sequence and the second nucleic acid sequence are partially double-stranded to form siRNA or shRNA targeting mTOR mRNA and STAT3 mRNA. The expression cassette comprises a first nucleic acid sequence, a loop sequence capable of forming a hairpin structure, and a second nucleic acid sequence, and the nucleotide sequence of the expression cassette comprises the sequence shown in SEQ ID NO: 80, in, The human telomerase reverse transcriptase promoter is operably linked to the E1A and E1B of the endogenous gene of the adenovirus, The adenovirus is adenovirus of serotype 5 of group C.

2. The anti-tumor adenovirus according to claim 1, characterized in that The expression cassette is located at the C3 site of the endogenous gene of the adenovirus.

3. The anti-tumor adenovirus according to claim 1, characterized in that An internal ribosome entry site sequence (IRES) is also included between E1A and E1B.

4. The anti-tumor adenovirus according to claim 1, characterized in that Short hairpin RNA (shRNA) simultaneously inhibits mTOR and STAT3.

5. The anti-tumor adenovirus according to claim 1, characterized in that The oncolytic ability of anti-tumor adenovirus is higher than that of wild-type adenovirus.

6. The anti-tumor adenovirus according to claim 1, characterized in that The oncolytic ability of the anti-tumor adenovirus is higher than that of the adenovirus in which the human telomerase reverse transcriptase (hTERT) promoter is introduced into the wild-type adenovirus.

7. An anticancer composition comprising the anti-tumor adenovirus according to claim 1.

8. The anticancer composition according to claim 7, characterized in that: Also contains anti-cancer agents.

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