Structure of anti-cancer viruses containing bispecific nucleic acid molecules

Abstract

The present invention relates to an antitumor adenovirus and an anticancer composition containing the same. The double-stranded siRNA of the present invention promotes the death of cancer cells by simultaneously inhibiting the expression of a first nucleic acid and a second nucleic acid, and has more pronounced anticancer activity than treatment with each siRNA together. When treated in combination with an anticancer drug, it has the effect of synergistically improving the death of cancer cells. An expression cassette encoding an shRNA that expresses this and an hTERT promoter-containing adenovirus evade the body's immune response and are delivered specifically to cancer cells, have a systemic therapeutic effect, can be delivered locally, have excellent selectivity, and show remarkable anticancer effects even as a minimally invasive treatment, and can be usefully used as an anticancer composition or anticancer adjuvant for various cancers.

Description

[Technical Field]

[0001] The present invention relates to an antitumor adenovirus and an anticancer composition containing the same. [Background technology]

[0002] Cancer is one of the world's leading causes of death. The development of innovative cancer drugs could reduce medical costs and generate significant added value. According to 2008 statistics, molecular therapeutics capable of overcoming resistance to conventional anticancer drugs accounted for $17.5 billion in the seven major countries (the United States, Japan, France, Germany, Italy, Spain, and the United Kingdom). The market size is expected to reach approximately $45 billion in 2018, a 9.5% growth rate compared to 2008. Cancer treatments are divided into surgery, radiation therapy, chemotherapy, and biological therapy. Chemotherapy, among others, uses chemicals to suppress or kill cancer cells. The toxicity of anticancer drugs is also felt in a significant proportion of normal cells, leading to the development of resistance, which causes anticancer drugs to lose their effectiveness after a certain period of use. Therefore, there is a pressing need for anticancer drugs that selectively target cancer cells and do not induce resistance (Conquering Cancer: Biowave, June 2004, 19). Recently, development of new anticancer drugs that target the molecular characteristics of cancer has been progressing through the acquisition of molecular genetic information about cancer, and it has been reported that anticancer drugs that target specific molecular targets that only cancer cells have do not develop drug resistance.

[0003] Gene expression suppression is an important tool in the development of therapeutic drugs and target validation for disease treatment. Since its role was discovered, interfering RNA (RNA interference, hereafter referred to as "RNAi") has been found to act on sequence-specific mRNAs in various mammalian cells (Silence of the transcripts: RNA interference in medicine. J Mol Med (2005) 83:764-773). RNAi is a phenomenon in which small interfering ribonucleic acids (small interfering RNAs, hereafter referred to as "siRNAs"), which have a double-stranded structure and are 21-25 nucleotides long, specifically bind to and degrade the mRNA transcripts that have complementary sequences, thereby suppressing the expression of specific proteins. Within the cell, the RNA duplex is processed by an endonuclease called Dicer and converted into 21-23 base pair (bp) double-stranded siRNA. The siRNA then binds to the RNA-induced silencing complex (RISC), where the guide (antisense) strand recognizes and degrades the target mRNA, thereby inhibiting the expression of the target gene in a sequence-specific manner (NUCLEIC-ACID THERAPEUTICS: BASIC PRINCIPLES AND RECENT APPLICATIONS. Nature Reviews Drug Discovery. 2002.1, 503-514). Bertrand's research team has shown that siRNAs targeting the same target gene have superior inhibitory effects on mRNA expression in vitro and in vivo compared to antisense oligonucleotides (ASOs), and that these effects last for a long period of time (Comparison of antisense oligonucleotides and siRNAs in cell culture and in vivo. Biochem. Biophys. Res. Commun. 2002. 296:1000-1004).The market for therapeutic drugs based on RNAi technology, including siRNA, is estimated to reach a total global market size of more than 12 trillion won by 2020. The scope of applicable targets for this technology is expanding dramatically, and it is being recognized as a next-generation gene therapy technology capable of treating diseases that are difficult to treat with conventional antibody- and chemical-based drugs. Furthermore, the mechanism of action of siRNA is to complementarily bind to target mRNA and regulate the expression of target genes in a sequence-specific manner. While conventional antibody-based drugs and small molecule drugs require long development times and costs to be optimized for specific protein targets, siRNA offers the advantages of dramatically expanding the scope of applicable targets, shortening development time, and enabling the development of lead compounds optimized for any protein target, including those that cannot be druggably commercialized (Progress Towards in Vivo Use of siRNAs. MOLECULAR THERAPY. 2006 13(4):664-670). Therefore, recently, this ribonucleic acid-mediated interference phenomenon has presented a solution to the problems that have occurred in the development of conventional chemically synthesized drugs, and research is being conducted to selectively suppress the expression of specific proteins at the transcription level and utilize it to develop therapeutic drugs for various diseases, especially tumor therapeutic drugs.In addition, unlike conventional anticancer drugs, siRNA therapeutic drugs have the advantage of having a clear target and predictable side effects, but in the case of tumors, which are diseases caused by various genetic problems, this target specificity may actually be the cause of poor therapeutic efficacy. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Silence of the transcripts:RNA interference in medicine.J Mol Med(2005)83:764773 [Non-patent document 2] NUCLEIC-ACID THERAPEUTICS:BASIC PRINCIPLES AND RECENT APPLICATIONS.Nature Reviews Drug Discovery.2002.1, 503-514 [Non-patent document 3] Comparison of antisense oligonucleotides and siRNAs in cell culture and in vivo.Biochem.Biophys.Res.Commun.2002.296:1000-1004 [Non-patent document 4] Progress Towards in Vivo Use of siRNAs.MOLECULAR THERAPY.2006 13(4):664-670 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide an antitumor adenovirus.

[0006] Another object of the present invention is to provide a composition for treating cancer. [Means for solving the problem]

[0007] According to the present invention, the double-stranded siRNA of the present invention promotes the death of cancer cells by simultaneously suppressing the expression of the first nucleic acid and the second nucleic acid, and exhibits more pronounced anticancer activity than treatment with each siRNA together. When used in combination with an anticancer drug, it has the effect of synergistically improving the death of cancer cells. An adenovirus containing an expression cassette encoding an shRNA containing this and an hTERT promoter can evade the body's immune response, be delivered specifically to cancer cells, be effective in systemic treatment, can be delivered locally, has excellent selectivity, and exhibits significant anticancer effects even with minimally invasive treatment, and can be useful as an anticancer composition or anticancer adjuvant for various types of cancer. [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 1 shows a map of a vector for intracellular expression of shRNAs comprising the dual-targeting siRNA set of the present invention. [Figure 2] FIG. 1 shows the mTOR or STAT3 gene expression suppression effect of dual-targeting double-stranded siRNAs of Sets 1-9 of the present invention. [Figure 3] FIG. 1 shows the inhibitory effect of the dual-targeting siRNA set 10 (si-BB1) of the present invention on the expression of the BCL2 gene (left) and the BI-1 gene (right). [Figure 4] FIG. 1 shows the inhibitory effect of dual-targeting siRNA sets 11 to 15 of the present invention on the expression of BCL2 gene and BI-1 gene. NC: control siRNA; and si-BB2 to si-BB6: siRNA sets 11 to 15 of the present invention. [Figure 5]

[0033] Figure 1 shows the inhibitory effect of the dual-targeting siRNA set 16 of the present invention on the expression of the AR gene and mTOR gene in cancer cell lines. A: h460 cell line; B: pc3 cell line; NC: control siRNA; siAR: siRNA against AR; simTOR: siRNA against mTOR; and si-AT1: AR and mTOR dual-targeting siRNA set 16 of the present invention. [Figure 6] FIG. 1 shows the inhibitory effect of the dual-targeting siRNA set 17-28 of the present invention on the expression of the AR gene and mTOR gene in the A549 cell line. NC: control siRNA; and si-AT2 to si-AT13: siRNA sets 17 to 28 of the present invention. [Figure 7] FIG. 1 shows the results of confirming the inhibitory effect of the dual-targeting siRNA (double strand) set of the present invention, which can simultaneously suppress c-MET and PD-L1, on the expression of the c-MET and PD-L1 genes in various cancer cell lines. [Figure 8]This figure shows the expression levels of mTOR and STAT3 by vectors containing sequences encoding the TTGGATCCAA loop shRNA sequence of SEQ ID NO: 66 or the TTCAAGAGAG loop shRNA sequence of SEQ ID NO: 67, confirmed by the amount of DNA in the shRNA expression cassette. [Figure 9] FIG. 10 is a graph comparing the gene expression suppression effects of two single-targeting siRNAs linked in tandem with those of the dual-targeting shRNA of the present invention. [Figure 10] FIG. 10 is a graph confirming the cell viability of human lung cancer cell line A549 cells when mTOR and STAT3 were simultaneously suppressed with dual-targeting siRNAs of the present invention (dual-targeting siRNAs of Set 1-9). [Figure 11] FIG. 1 shows the cell viability of human lung cancer cell line A549 cells after cisplatin treatment when mTOR and STAT3 were simultaneously suppressed with the dual-targeting siRNA of the present invention. [Figure 12] FIG. 1 shows the cell viability of human lung cancer cell line A549 cells after paclitaxel treatment when mTOR and STAT3 were simultaneously suppressed with the dual-targeting siRNA of the present invention. [Figure 13] FIG. 1 shows the cell viability of human lung cancer cell line A549 cells when mTOR and STAT3 were simultaneously suppressed with the dual-targeting siRNA of the present invention after treatment with 5-FU (5-fluorouracil). [Figure 14] Figures showing the killing of cancer cells by combined treatment with the dual-targeting siRNA set of the present invention and an anticancer drug. A: Combined treatment of an anticancer drug + Bcl2 siRNA + BI-1 siRNA; and B: Combined treatment of the dual-targeting siRNA set 10 (si-BB1) of the present invention + an anticancer drug. [Figure 15] This figure confirms the cancer cell-killing effects of ABT-737, a Bcl2 inhibitor used as an anticancer agent, and the dual-targeting siRNA set 10 (si-BB1) of the present invention, as well as the synergistic effect of their combined treatment. [Figure 16]FIG. 10 shows the cancer cell killing effect of combined treatment of dual-targeting siRNA set 10 (si-BB1) with an anticancer drug, compared with a group treated with both siRNA against the BCL2 gene and siRNA against the BI-1 gene. [Figure 17] This figure shows the cancer cell-killing effect of the dual-targeting siRNA set 1 in cancer cell lines when combined with an anticancer drug. NC: control siRNA; no treat: control group not treated with an anticancer drug; si-AT1: AR and mTOR dual-targeting siRNA set 16 of the present invention; A: DU145 cell line; and B: H460 cell line. [Figure 18] FIG. 1 is a schematic diagram of the structure of the adenovirus of the present invention. [Figure 19] 1 shows a vector map of the adenoviral vector of the present invention. Bs-shRNA: insertion site of the sequence encoding the dual-targeting shRNA of the present invention. [Figure 20] FIG. 1 shows the inhibitory effect of the recombinant adenovirus CA102 of the present invention, which encodes and expresses the hTERT promoter and dual-targeting shRNA, on the expression of the mTOR and STAT3 genes in the bladder cancer cell lines T24 and 253JBV. [Figure 21] This figure confirms the effect of the recombinant adenovirus CA102 of the present invention, which encodes and expresses the hTERT promoter and dual-targeting shRNA, on suppressing the expression of the mTOR and STAT3 genes in the head and neck cancer cell lines FaDu and HSC-2. [Figure 22] This figure confirms the effect of the recombinant adenovirus CA102 of the present invention, which encodes and expresses the hTERT promoter and dual-targeting shRNA, on suppressing the expression of the mTOR and STAT3 genes in the skin squamous cell carcinoma cell lines A431 and HSC-5. [Figure 23] FIG. 1 shows the protein level confirmation of the inhibitory effect of the recombinant adenovirus CA102 of the present invention on the expression of the mTOR and STAT3 genes in the bladder cancer cell lines T24 and 253J-BV. [Figure 24]FIG. 10 is a graph confirming the inhibitory effect of the recombinant adenovirus CA101 of the present invention, which contains an hTERT promoter and a dual-targeting shRNA expression cassette, on the expression of the BCL2 and BI-1 genes. [Figure 25] FIG. 1 shows the effect of the recombinant adenovirus CA103 of the present invention, which contains an hTERT promoter and a dual-targeting shRNA expression cassette, on suppressing the expression of the AR and mTOR genes in the prostate cancer cell line LNcap. [Figure 26] This figure shows the in vitro confirmation of the effect of the recombinant adenovirus CA103 of the present invention, which contains an hTERT promoter and a dual-targeting shRNA expression cassette, on the expression of AR and mTOR genes in the prostate cancer cell lines C42B and 22Rv1. [Figure 27] FIG. 1 shows the in vivo suppressive effect of the recombinant adenovirus CA103 of the present invention, which contains an hTERT promoter and a dual-targeting shRNA expression cassette, on the expression of the AR and mTOR genes. [Figure 28] FIG. 10 is a graph confirming the inhibitory effect of the recombinant adenovirus CA104 of the present invention, which comprises an hTERT promoter and a dual-targeting shRNA expression cassette, on the expression of the c-MET and PD-L1 genes. [Figure 29] FIG. 10 is a graph confirming the killing effect of cancer cell lines by the recombinant adenovirus CA101 of the present invention, which contains an hTERT promoter and a dual-targeting shRNA expression cassette. [Figure 30] FIG. 1 is a graph confirming the killing effect of the recombinant adenovirus CA102 of the present invention on bladder cancer cell lines RT4, T24, and 253J-BV. [Figure 31] FIG. 1 shows the killing effect of the recombinant adenovirus CA102 of the present invention on the head and neck cancer cell lines FaDu and HSC-2. [Figure 32] FIG. 1 shows the killing effect of the recombinant adenovirus CA102 of the present invention on the skin squamous cell carcinoma cell lines A431 and HSC-5. [Figure 33]This figure confirms the killing effect of the cancer cell line LNcap by the recombinant adenovirus CA103 of the present invention, which contains an hTERT promoter and a dual-targeting shRNA expression cassette. [Figure 34] FIG. 10 is a graph confirming the killing effect of the recombinant adenovirus CA103 of the present invention, which contains an hTERT promoter and a dual-targeting shRNA expression cassette, on the cancer cell lines C42B and 22Rv1. [Figure 35] FIG. 1 is a graph confirming the anticancer effect of the recombinant adenovirus CA102 of the present invention on bladder cancer cells (253J-BV) in vivo. [Figure 36] FIG. 1 is a graph confirming the anticancer effect of the recombinant adenovirus CA102 of the present invention on head and neck cancer cells (FaDu) in vivo. [Figure 37] FIG. 1 is a graph confirming the anticancer effect of the recombinant adenovirus CA102 of the present invention on tumors (bladder cancer) formed in vivo. [Figure 38] FIG. 1 is a graph confirming the anticancer effect of CA102 on tumors (bladder cancer) formed in vivo depending on the number of administrations. [Figure 39] FIG. 1 shows the in vivo therapeutic effect of glioblastoma depending on the dose of the recombinant adenovirus CA102 of the present invention. [Figure 40] FIG. 1 shows the in vivo therapeutic effect of the recombinant adenovirus CA103 of the present invention, which contains an hTERT promoter and a dual-targeting shRNA expression cassette, on prostate cancer. [Figure 41] FIG. 1 shows the in vivo therapeutic effects of the recombinant adenovirus CA102 of the present invention and its combined use with cisplatin on bladder cancer. DETAILED DESCRIPTION OF THE INVENTION

[0009] The present invention will be described in detail below with reference to examples thereof. However, the following examples are presented as examples of the present invention and are not intended to limit the present invention, and various modifications and applications of the present invention are possible within the scope of the claims set forth below and the scope of equivalents construed therefrom.

[0010] Unless otherwise specified, nucleic acids are written from left to right in 5' to 3' orientation. Numerical ranges recited herein are inclusive of the numbers defining the range, and include each integer or any non-integer portion within the defined range.

[0011] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein.

[0012] In one aspect, the present invention relates to an antitumor adenovirus comprising an expression cassette comprising a human telomerase promoter (hTERT); a base sequence targeting a first nucleic acid; and a base sequence targeting a second nucleic acid.

[0013] In one embodiment, the base sequence targeting the first nucleic acid and the base sequence targeting the second nucleic acid can be partially or 100% reverse complementary sequences, and when expressed in vivo, the sequence targeting the first nucleic acid and the sequence targeting the second nucleic acid can form a duplex, preferably an shRNA.

[0014] In one embodiment, the base sequence targeting the first nucleic acid and the base sequence targeting the second nucleic acid can bind partially or 100% complementary to each other to form a double strand during gene expression.

[0015] In one embodiment, the human telomerase promoter can be operably linked to an endogenous gene of an adenovirus.

[0016] In the present invention, the term "operably linked" refers to a functional connection between a gene expression control sequence (e.g., a promoter, a signal sequence, or an array of transcriptional regulator binding sites) and another gene sequence, whereby said control sequence regulates the transcription and / or detoxification of the other gene sequence.

[0017] In one embodiment, the hTERT promoter can comprise the base sequence of SEQ ID NO: 74. Furthermore, the hTERT promoter sequence can comprise various known modified sequences.

[0018] In one embodiment, the endogenous gene of the adenovirus has a 5'ITR-C1-C2-C3-C4-C5 3'ITR structure; C1 includes E1A (sequence number 75), E1B (sequence number 77) or E1A to E1B; C2 includes E2B-L1-L2-L3-E2A-L4; C3 does not include or includes E3; C4 includes L5; and C5 may not include or include E4, and may include the base sequence of SEQ ID NO: 78.

[0019] In one embodiment, the adenovirus may have a partial deletion of the E3 region, and the deleted nucleotide sequence may include the nucleotide sequence of SEQ ID NO:82.

[0020] In one embodiment, the expression cassette can be located in the C3 site of an endogenous gene of the adenovirus.

[0021] In one embodiment, the hTERT promoter can be operably linked to the endogenous E1A and E1B genes of an adenovirus.

[0022] In one embodiment, the endogenous adenoviral gene may further comprise an IRES sequence (SEQ ID NO: 76) between E1A and E1B.

[0023] In one embodiment, the expression cassette is capable of encoding and expressing an shRNA.

[0024] In one embodiment, the shRNA is capable of simultaneously inhibiting expression of the first nucleic acid and the second nucleic acid.

[0025] In one embodiment, the antitumor adenovirus of the present invention can suppress expression by degrading the mRNA of a nucleic acid through RNA interference or by inhibiting translation.

[0026] In one embodiment, the expression cassette of the present invention can simultaneously suppress a first nucleic acid and a second nucleic acid by expressing a double-stranded siRNA in which a sense strand specific to the first nucleic acid or the second nucleic acid and an antisense strand specific to the second nucleic acid or the first nucleic acid are partially complementary to each other.

[0027] As used herein, the term "inhibition of expression" means causing a decrease in the expression or translation of a target gene, preferably such that target gene expression is undetectable or present at insignificant levels.

[0028] The term "small interfering RNA (siRNA)" used in the present invention refers to a short double-stranded RNA that can induce RNAi (RNA interference) through the cleavage of a specific mRNA. Generally, siRNA consists of a sense RNA strand with a sequence homologous to the mRNA of a target gene and an antisense RNA strand with a sequence complementary thereto. In the double-stranded siRNA of the present invention, the sense RNA strand is an siRNA specific to a first nucleic acid or a second nucleic acid (antisense strand to the first nucleic acid or the second nucleic acid), and the antisense RNA strand is an siRNA specific to a second nucleic acid or a first nucleic acid (antisense strand to the second nucleic acid or the first nucleic acid), so that the double-stranded siRNA can simultaneously inhibit the first nucleic acid or the second nucleic acid or their expression, respectively.

[0029] The term "shRNA (short hairpin RNA)" as used herein refers to a single-stranded RNA that contains a partially palindromic base sequence, thereby forming a double-stranded structure in the 3' region and forming a hairpin-like structure. After intracellular expression, the RNA can be cleaved by dicer, a type of RNase present in the cell, and converted into siRNA. The length of the double-stranded structure is not particularly limited, but is preferably 10 nucleotides or more, and more preferably 20 nucleotides or more. In the present invention, the shRNA may be contained in an expression cassette. The shRNA can be produced by converting U to T in a set sequence consisting of an siRNA antisense strand and a sense strand for each gene, and then ligating TTGGATCCAA (TTGGATCCAA loop) or TTCAAGAGAG (TTCAAGAGAG loop), the antisense strand, and TT to the 3' end of the sense strand to create an expression cassette encoding the shRNA, and then expressing the expression cassette in cells.

[0030] In one embodiment, the first nucleic acid can comprise a base sequence having 60% or more complementarity to the reverse complementary sequence of the second nucleic acid, and the second nucleic acid can comprise a base sequence having 60% or more complementarity to the reverse complementary sequence of the first nucleic acid.

[0031] In one embodiment, the first nucleic acid can comprise a base sequence having 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more complementarity to the reverse complementary sequence of the second nucleic acid, and the second nucleic acid can comprise a base sequence having 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more complementarity to the reverse complementary sequence to the first nucleic acid.

[0032] The present invention encompasses variants of the base sequence targeting the first or second nucleic acid contained in the expression cassette. The expression cassette of the present invention encompasses functional equivalents of the nucleic acid molecules that comprise it, such as variants in which a portion of the base sequence of the nucleic acid molecule has been modified by deletion, substitution, or insertion, but which can function in the same way as the base sequence molecule. The "percent sequence identity" for a nucleic acid molecule is determined by comparing two optimally aligned sequences with a comparison region, and the portion of the nucleic acid molecule sequence in the comparison region may contain additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) for the optimal alignment of the two sequences.

[0033] In one embodiment, the nucleic acid may be a gene associated with cancer.

[0034] In one embodiment, the cancer-associated gene may be an oncogene whose expression increases in cancer, and the oncogene may be an apoptosis-associated gene, a transcription factor gene, a metastasis-associated gene, an angiogenesis-associated gene, a cancer cell-specific gene, or a tyrosine-kinase gene.

[0035] In one embodiment, the apoptosis-related gene is selected from the group consisting of 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, E2F5, may be ESPL1, FOXO1, HDAC1, HSPA5, IGF1R, IGF2, 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, or YWHAZ;The transcription factor genes include 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, and FOXO. 1, FOXP1, FOXQ1, GATA1, GATA6, GFI1, GFI1B, GLI1, GLI2, GLI3, HES6, HHEX, HLF, HMGA1, HMGA2, HOXA1, HOXA9, HOXD13, HOXD9, ID 1, 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, PA X3, PAX4, PAX8, PBX1, PBX2, PITX2, PLAG1, PLAGL2, PPARG, PPP1R13L, PRDM10, PRDM13, PRDM14, PRDM15, PRDM16, PRDM6, PRDM8, P may be RDM9, 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-associated genes include 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, and TCF3. or WT1; 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 tyrosine-kinase gene may 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; In one embodiment, the cancer genes are 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, BCL9, BCL9L, BCR, BMI1, BMP7, BOC, BRD4, BRF2, CABIN1, CAMK1D, CAPG, CBFB, CBLB, CBLL1, CBX7, CBX8, CCDC28A, CCDC6, CCNB1, CCNB2, CCND1, CCNE1, CCNL1, CD24, CDC25C, CDC6, CDH17, CDK1, CDK14, CDK4, CDK5R2, CDK6, CDK8, CDKN1B, CDKN3, CDON, CEACAM6, CENPW, CHD1L, CHIC1, CHL1, CKS1B, CMC4, CNTN2, COPS3, 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, RUNX 1T1, ​​S100A4, S100A7, S100A8, SAG, SART3, SBSN, SEA, SEC62, SERTAD1, SERTAD2, SERTAD3, SET, SETBP1, SETDB1, SGK1, SIRT1, SI RT6, SKI, SKIL, SKP2, SLC12A5, SLC3A2, SMR3B, SMURF1, SNCG, SNORA59A, SNORA80E, SPAG9, SPATA4, SPRY2, SQSTM1, SRSF1, SRSF 2, 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, TMPR SS2, TNS4, TPD52, TPR, TRE17, TREH, TRIB1, TRIB2, TRIM28, TRIM32, TRIM8, TRIO, TRIP6, TSPAN1, TSPY1, TXN, TYMS, TYRP1, UBE2 C, 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, or ZNHIT6.

[0036] In one embodiment, the cancer cell-specific gene may be PD-L1 (Programmed death-ligand 1), which is expressed on the surface of tumor cells.

[0037] In one embodiment, the first and second nucleic acids targeted by the transcripts of the expression cassette of the present invention are selected from the group consisting of 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, IGF 1R, IGF2, JUN, JUNB, JUND, MALT1, MAP3K7, MCL1, MDM2, MDM4, MYB, MYC, NFKB2, NPM1, NTRK1, PAK1, PAX3, PML, PRKCA, PRKCE, PTK2B, RAF1, RHOA, TGFB1, TN FRSF1B, TP73, TRAF6, YWHAG, YWHAQ, YWHAZ, AR, ARID3A, ASCL1, ATF1, ATF3, BCL11A, BCL11B, BCL3, BCL6, CDC5L, CDX2, CREB1, CUX1, DDIT3, DLX5, E2F1, E 2F3, E2F5, ELF4, ELK1, ELK3, EN2, ERG, ETS1, ETS2, ETV1, ETV3, ETV4, ETV6, FEV, FEZF1, FLI1, FOS, FOSL1, FOXA1, FOXG1, FOXM1, FOXO1, FOXP1, FOXQ1, GA TA1, GATA6, 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, PRDM15, PRDM16, PRDM6, PRDM8, PRDM9, RARA, REL, RERE, RUNX1, RUNX3, SALL4, SATB1, SFPQ, SIX1, SNAI1, SOX2, SOX4, SPI1, SRBF1, STAT3, TAF 1、TAL1、TAL2、TBX2、TBX3、TCF3、TFCP2、TFE3、THRA、TLX1、TP63、TP73、TWIS T1、WT1、YBX1、YY1、ZBTB16、ZBTB7A、ZIC2、ZNF217、ZNF268、AKT1、AKT2、AR、C BL、CDH1、CRK、CSF1、CTNNB1、CTTN、CXCR4、EGFR、FGFR1、FLT3、FYN、GLI1、IL K、ITGA3、JAK2、MET、PDGFRB、PLXNB1、PRKCI、PTCH1、PTPN11、RAC1、RHOA、RHO C、ROCK1、SMO、SNAI1、SRC、TCF3、WT1、BRAF、CAV1、CTGF、EGFR、ERBB2、ETS1、 FGF4、FGF6、FGFR1、FGFR3、FGFR4、ID1、NRAS、PDGFB、PDGFRA、PDGFRB、SPARC、 ABL1、ABL2、ALK、AXL、BLK、EGFR、EPHA2、ERBB2、ERBB3、ERBB4、FES、FGFR1、F GFR2、FGFR3、FGFR4、FGR、FLT3、FYN、ITK、JAK1、JAK2、KIT、LCK、MERTK、MET、M ST1R, 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, ARHG EF5、ASPSCR1、AURKA、BAALC、BAIAP2L1、BANP、BCAR4、BCKDHB、BCL9、BCL9L、 BCR、BMI1、BMP7、BOC、BRD4、BRF2、CABIN1、CAMK1D、CAPG、CBFB、CBLB、CBLL1、CBX7、CBX8、CCDC28A、CCDC6、CCNB1、CCNB2、CCND1、CCNE1、CCNL1、CD24、CDC 25C、CDC6、CDH17、CDK1、CDK14、CDK4、CDK5R2、CDK6、CDK8、CDKN1B、CDKN3、CD ON、CEACAM6、CENPW、CHD1L、CHIC1、CHL1、CKS1B、CMC4、CNTN2、COPS3、COPS5 、CRKL、CRLF2、CROT、CRTC1、CRYAB、CSF1R、CSF3、CSF3R、CSNK2A1、CSNK2A2、C T45A1, 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、EIF5A 2、ELAVL1、ELL、EML4、EMSY、ENTPD5、EPCAM、EPS8、ERAS、ERGIC1、ERVW-1、EVI 2A, EVI5, EWSR1, EZH2, FAM189B, FAM72A, FAM83D, FASN, FDPS, FGF10, FGF3, FGF5, FGF8, FR1OP, FHL2, FIP1L1, FNDC3B, FRAT1, FUBP1, FUS, FZD2, GAB2, GA EC1、GALNT10、GALR2、GLO1、GMNN、GNA12、GNA13、GNAI2、GNAQ、GNAS、GOLPH3 、GOPC、GPAT4、GPM6A、GPM6B、GPR132、GREM1、GRM1、GSK3A、GSM1、H19、HAS1、H AX1、HDGFRP2、HMGN5、HNRNPA1、HOTAIR、HOTTIP、HOXA-AS2、HRAS、HSPA1A、H SPA4、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, TBC1D3C, TB C1D7, TCL1A, TCL1B, TCL6, TCP1, TFG, TGM3, TINCR, TKTL1, TLE1, TMEM140, TMPOP2, TMPRSS2, TNS4, TPD52, TPR, TRE17, TREH, TRIB1, TRIB2, TRIM28, TRIM32, TRIM8, TRI They may be different nucleic acids selected from the group consisting of O, 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.

[0038] In one embodiment, 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 which case the expression cassette may comprise 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, the 17mer of the 21mer of the siRNA of SEQ ID NO: 1 and 2, the 16mer of the 20mer of the siRNA of SEQ ID NO: 3 and 4, the 15mer of the 19mer of the siRNA of SEQ ID NO: 5 and 6, the 14mer of the 18mer of the siRNA of SEQ ID NO: 7 and 8, the 16mer of the 17mer of the siRNA of SEQ ID NO: 9 and 10, the 17mer of the 20mer of the siRNA of SEQ ID NO: 11 and 12, the 16mer of the 19mer of the siRNA of SEQ ID NO: 13 and 14, the 15mer of the 18mer of the siRNA of SEQ ID NO: 15 and 16, and the 14mer of the 17mer of the siRNA of SEQ ID NO: 17 and 18. In addition, when the first nucleic acid is STAT3 and the second nucleic acid is mTOR, the shRNA expression DNA (DNA sequence encoding the STAT3 and mTOR dual-targeting shRNA) contained in the expression cassette can comprise the base sequence of SEQ ID NO: 66 or 67.

[0039] In one embodiment, the first nucleic acid may be BCL2 (B-cell lymphoma 2) and the second nucleic acid may be BI-1 (BAX inhibitor 1), in which case the expression cassette may comprise the base sequences of SEQ ID NOs: 19 and 20, SEQ ID NOs: 21 and 22, SEQ ID NOs: 23 and 24, SEQ ID NOs: 25 and 26, SEQ ID NOs: 27 and 28, or SEQ ID NOs: 29 and 30. In the above, the 21-mer siRNA set 10 consisting of sequence numbers 19 and 20 has 15-mers that are complementary to each other, the 20-mer siRNA set 11 consisting of sequence numbers 21 and 22 has 14-mers that are complementary to each other, the 20-mer siRNA set 12 consisting of sequence numbers 23 and 24 has 14-mers that are complementary to each other, the 19-mer siRNA set 13 consisting of sequence numbers 25 and 26 has 13-mers that are complementary to each other, the 19-mer siRNA set 14 consisting of sequence numbers 27 and 28 has 13-mers that are complementary to each other, and the 18-mer siRNA set 15 consisting of sequence numbers 29 and 30 has 12-mers that are complementary to each other. siRNAs of SEQ ID NOs: 19, 21, 23, 25, 27, or 29 (antisense Bcl-2) in Table 2 below can complementarily bind to Bcl-2 mRNA, and siRNAs of SEQ ID NOs: 20, 22, 24, 26, 28, or 30 (antisense BI-1) can complementarily bind to BI-1 mRNA. When the first nucleic acid is BCL2 and the second nucleic acid is BI-1, the shRNA expression DNA contained in the expression cassette can include the base sequence of SEQ ID NO: 68 or 69.

[0040] In one embodiment, the first nucleic acid may be AR (androgen receptor) and the second nucleic acid may be mTOR (mammalian target of rapamycin), in which case the expression cassette may comprise the base sequence of SEQ ID NOs: 31 and 32, SEQ ID NOs: 33 and 34, SEQ ID NOs: 35 and 36, SEQ ID NOs: 37 and 38, SEQ ID NOs: 39 and 40, SEQ ID NOs: 41 and 42, SEQ ID NOs: 43 and 44, SEQ ID NOs: 45 and 46, SEQ ID NOs: 47 and 48, SEQ ID NOs: 49 and 50, SEQ ID NOs: 51 and 52, SEQ ID NOs: 53 and 54, or SEQ ID NOs: 55 and 56. In the above, 20mer siRNA set 16 consisting of SEQ ID NOs: 31 and 32 have complementary 18mers, 19mer siRNA set 17 consisting of SEQ ID NOs: 33 and 34 have complementary 17mers, 18mer siRNA set 18 consisting of SEQ ID NOs: 35 and 36 have complementary 16mers, 17mer siRNA set 19 consisting of SEQ ID NOs: 37 and 38 have complementary 15mers, 19mer siRNA set 20 consisting of SEQ ID NOs: 39 and 40 have complementary 15mers, 18mer siRNA set 21 consisting of SEQ ID NOs: 41 and 42 have complementary 14mers, and 17mer siRNA set 22 consisting of SEQ ID NOs: 43 and 44 have complementary 14mers. siRNA set 22 is complementary to each other at 13mers, 23mer siRNA set 23 consisting of sequence numbers 45 and 46 is complementary to each other at 19mers, 22mer siRNA set 24 consisting of sequence numbers 47 and 48 is complementary to each other at 18mers, 22mer siRNA set 25 consisting of sequence numbers 49 and 50 is complementary to each other at 18mers, 21mer siRNA set 26 consisting of sequence numbers 51 and 52 is complementary to each other at 17mers, 20mer siRNA set 27 consisting of sequence numbers 53 and 54 is complementary to each other at 16mers, and 21mer siRNA set 28 consisting of sequence numbers 55 and 56 is complementary to each other at 17mers.siRNA (antisense AR) of SEQ ID NO: 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, or 55 can complementarily bind to AR mRNA, and siRNA (antisense mTOR) of SEQ ID NO: 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, or 56 can complementarily bind to mTOR mRNA. In addition, when the first nucleic acid is AR and the second nucleic acid is mTOR, the shRNA expression DNA contained in the expression cassette can include the base sequence of SEQ ID NO: 70 or 71.

[0041] In one embodiment, the first nucleic acid may be MGMT (O-6-methylguanine-DNA methyltransferase) and the second nucleic acid may be mTOR, in which case the expression cassette may comprise the base sequences of SEQ ID NOs: 57 and 58.

[0042] In one embodiment, the first nucleic acid may be BCL2 and the second nucleic acid may be MCL1 (MCL1 apoptosis regulator), in which case the expression cassette may comprise the base sequences of SEQ ID NOs: 59 and 60.

[0043] In one embodiment, the first nucleic acid may be STAT3 and the second nucleic acid may be TFEB (transcription factor EB), in which case the expression cassette may comprise the base sequences of SEQ ID NOs: 61 and 62.

[0044] In one embodiment, the first nucleic acid may be c-MET (Homo sapiens MET proto-oncogene) and the second nucleic acid may be PD-L1 (Programmed death-ligand 1). In this case, the expression cassette may comprise nucleic acids in which U is converted to T in the base sequences of SEQ ID NOs: 63 and 64. In the above, the 15-mer of the 19-mer of the siRNA of SEQ ID NOs: 63 and 64 can bind complementarily. Furthermore, when the first nucleic acid is c-MET and the second nucleic acid is PD-L1, the shRNA expression DNA contained in the expression cassette may comprise the base sequence of SEQ ID NO: 72 or 73.

[0045] In one embodiment, the expression cassette can include a base sequence that sequentially encodes a base sequence that targets a first nucleic acid, a loop sequence that can form a hairpin structure, and a base sequence that targets a second nucleic acid.

[0046] In one embodiment, the expression cassette can be regulated by the U6 promoter.

[0047] In one embodiment, the adenovirus may be a group C serotype 5 adenovirus.

[0048] In one embodiment, the antitumor virus of the present invention may have a higher tumor-killing ability than a wild-type adenovirus, and may have a higher tumor-killing ability than an adenovirus in which an hTERT promoter has been introduced into a wild-type adenovirus.

[0049] In one aspect, the present invention relates to a composition for treating cancer, comprising the antitumor virus of the present invention.

[0050] In one embodiment, the composition of the present invention can further comprise an anti-cancer agent, such as acivicin, aclarubicin, acodazole, acronicin, adozelesin, alanosin, aldesleukin, allopurinol sodium, altretamine, aminoglutethimide, amonafide, ampligen, amsacrine, androgen, anguidine, aphidicolin glycinate, asalei, asparaginase, 5-azacytidine, azathioprine, bacillus Calmette-Guerin (BCG), Baker's antifol, beta-2-deoxythioguanosine, bisantrene HCl, bleomycin sulfate, busulfan, buthionine sulfoximine, BWA 773U82, BW 502U83 / HCl, BW 7U85 mesylate, cerasemide, carbetimer, carboplatin, carmustine, chlorambucil, chloroquinoxaline-sulfonamide, chlorozotocin, chromomycin A3, cisplatin, cladribine, corticosteroids, Corynebacterium parvum, CPT-11, crisnatol, cyclocytidine, cyclophosphamide, cytarabine, sitembena, davismaleate, dacarbazine, dactinomycin, daunorubicin HCl, deazauridine, dexrazoxane, dianhydrogalactitol, diaziconazole, dibromodalcitrate le, didemnin B, diethyldithiocarbamate, diglycaldehyde, dihydro-5-azacytidine, doxorubicin, echinomycin, edatrexate, edelfosine, eflornithine, Elliott's solution, elsamitrucin, epirubicin, esorubicin, estramustine phosphate, estrogen, etanidazole, etiophos, etoposide, fadrazole, fazarabine, fenretinide, filgrastim, finasteride, flavone acetic acid, floxuridine, fludarabine phosphate, 5'-fluorouracil, Fluosol TM, flutamide, gallium nitrate, gemcitabine, goserelin acetate, hepsulfam, hexamethylene bisacetamide, homoharringtonine, hydrazine sulfate, 4-hydroxyandrostenedione, hydroxyurea, idarubicin HCl, ifosfamide, 4-ipomeanol, iproplatin, isotretinoin, leucovorin calcium, leuprolide acetate, levamisole, liposomal daunorubicin, liposomal doxorubicin, lomustine, lonidamine, maytansine, mechlorethamine hydrochloride, melphalan, menogaril, melbarone, 6-mercaptopurine, mesna, methanol extract residue of Bacillus Calmette-Guerin, methotrexate, N-methylformamide, mifepristone, mitoguazone, mitomycin-C, mitotane, mitoxantrone hydrochloride, monocyte / macrophage colony-stimulating factor, nabilone , nafoxidine, neocarzinostatin, octreotide acetate, ormaplatin, oxaliplatin, paclitaxel, para, pentostatin, piperazinedione, pipobroman, pirarubicin, piritrexim, piroxantrone hydrochloride, PIX-321, plicamycin, porfimer sodium, prednimustine, procarbazine, progestin, pirazofurin, razoxane, sargramostim, sems In some embodiments, the active ingredient may be benzodiazepine, benzodiazepine, benzocaine ...Preferred are cisplatin, paclitaxel, 5-FU (5-fluorouracil), methotrexate, doxorubicin, daunorubicin, cytosine arabinoside, etoposide, melphalan, chlorambucil, cyclophosphamide, vindesine, mitomycin, bleomycin, tamoxifen, and taxol, and more preferred are cisplatin, paclitaxel, or 5-FU (5-fluorouracil). However, the present invention is not limited to these, as long as the objective of achieving a synergistic anti-cancer effect when used in combination with the composition of the present invention is achieved.

[0051] The cancer may be any selected from the group consisting of colon cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, brain cancer, head and neck cancer, 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 cancer, endometrial cancer, vaginal cancer, vulva cancer, Hodgkin's disease, bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvis cancer, bone cancer, skin cancer, head cancer, neck cancer, cutaneous melanoma, intraocular melanoma, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, central nervous system (CNS) tumors, primary CNS lymphoma, spinal cord tumors, glioblastoma multiforme, and pituitary adenoma.

[0052] In the present invention, the term "promoter" refers to a non-translated nucleic acid sequence upstream of a coding region that contains an RNA polymerase binding site and has transcription initiation activity into mRNA of a downstream gene. In the expression cassette of the present invention, the promoter can be any promoter that can initiate the expression of shRNA. Specifically, the promoter of the present invention can be a constitutive promoter that constantly induces the expression of a gene of interest at all times, or an inducible promoter that induces the expression of a gene of interest at a specific location or time. Examples of such promoters include the U6 promoter, H1 promoter, CMV (cytomegalovirus) promoter, SV40 promoter, CAG promoter (Hitoshi Niwa et al., Gene, 108:193-199, 1991), CaMV 35S 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), and 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, any promoter known to those skilled in the art may be used, including, but not limited to, promoters disclosed in U.S. Patent Nos. 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. Preferably, the promoter of the present invention may be a U6 promoter, an HI promoter, or a CMV promoter. According to a preferred embodiment of the present invention, the U6 promoter may be used.

[0053] The composition of the present invention may further contain an adjuvant. Any adjuvant known in the art may be used without limitation, and the composition may further contain, for example, complete Freund's adjuvant or incomplete Freund's adjuvant to enhance the effect.

[0054] The composition of the present invention can be prepared by mixing the active ingredient with a pharmaceutically acceptable carrier. Here, pharmaceutically acceptable carriers include carriers, excipients, and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers that can be used in the composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginic acid, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0055] The composition of the present invention can be formulated into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, topical preparations, suppositories, or sterile injectable solutions according to the usual methods.

[0056] When formulated, they can be prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like. These solid formulations can be prepared by mixing the active ingredient with at least one or more excipients, such as starch, calcium carbonate, sucrose, lactose, and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid formulations for oral administration include suspensions, oral solutions, emulsions, syrups, and the like. In addition to commonly used diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives can be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. Examples of non-aqueous solvents and suspending agents that can be used include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Examples of suppository bases that can be used include witepsol, tween 61, cacao butter, laurin butter, and glycerogelatin.

[0057] The compositions of the present invention can be administered to an individual by any route, including oral, intravenous, intramuscular, subcutaneous, and intraperitoneal injection, although all modes of administration are contemplated.

[0058] The dosage of the pharmaceutical composition of the present invention is selected taking into consideration the age, weight, sex, physical condition, etc. of the individual. It is obvious that the concentration of the single domain antibody contained in the pharmaceutical composition can be selected in various ways depending on the subject, and preferably, the single domain antibody is contained in the pharmaceutical composition at a concentration of 0.01 to 5,000 μg / ml. If the concentration is less than 0.01 μg / ml, no pharmacological activity may be observed, and if it exceeds 5,000 μg / ml, it may be toxic to the human body.

[0059] The composition of the present invention can be used for the prevention or treatment of cancer and its complications, and can also be used as an anti-cancer adjuvant.

[0060] The present invention also provides a method for preventing and treating cancer, which comprises administering to an individual a pharmaceutically effective amount of a composition of the present invention.

[0061] The composition of the present invention is administered in a therapeutically effective amount or a pharmaceutically effective amount.The term "pharmacologically effective amount" means an amount sufficient to treat disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level can be determined according to factors including individual type and severity, age, sex, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment duration, concurrently used drugs, and other factors well known in the medical field.

[0062] In one aspect, the present invention relates to the use of the antitumor adenovirus of the present invention for tumor prevention or treatment.

[0063] In one aspect, the present invention relates to a method for treating tumors using the antitumor adenovirus of the present invention. [Example]

[0064] The present invention will be described in more detail with reference to the following examples, which are merely for illustrative purposes and are not intended to limit the scope of the present invention.

[0065] Example 1. Construction of dual-targeting siRNA 1-1.mTOR and STAT3 dual-targeting siRNA Dual-targeting siRNAs (double strands) capable of simultaneously inhibiting STAT3 (signal transducer and activator of transcription 3) and mTOR (mammalian target of rapamycin) were prepared (Bioneer, Daejeon, Korea) according to the sequences shown in Table 1. Specifically, the 17-mer of the 21-mer siRNAs of SEQ ID NOS: 1 and 2 in Set 1 complementarily binds, the 16-mer of the 20-mer siRNAs of SEQ ID NOS: 3 and 4 in Set 2 complementarily binds, the 15-mer of the 19-mer siRNAs of SEQ ID NOS: 5 and 6 in Set 3 complementarily binds, the 14-mer of the 18-mer siRNAs of SEQ ID NOS: 7 and 8 in Set 4 complementarily binds, and the 16-mer of the 17-mer siRNAs of SEQ ID NOS: 9 and 10 in Set 5 complementarily binds. In addition, the 17mers of the 20mers of the siRNAs of sequence numbers 11 and 12 in set 6, the 16mers of the 19mers of the siRNAs of sequence numbers 13 and 14 in set 7, the 15mers of the 18mers of the siRNAs of sequence numbers 15 and 16 in set 8, and the 14mers of the 17mers of the siRNAs of sequence numbers 17 and 18 in set 9 bind complementarily. After the two sequences in each set (Table 1) enter the cell in double-strand form, the antisense_mTOR siRNA in each set binds to the target site of mTOR mRNA (gi|206725550|ref|NM_004958.3|Homo sapiens mechanistic target of rapamycin (serine / threonine kinase) (MTOR), mRNA), and the antisense_STAT3 siRNA in each set binds to the target site of STAT3 mRNA (gi|47080104|ref|NM_139276.2|Homo sapiens signal transducer and activator of transcription 3 (acute-phase response factor) (STAT3), transcript variant 1, mRNA), thereby reducing mTOR and STAT3 gene expression.

[0066] [Table 1]

[0067] 1-2.BCL2 and BI-1 dual-targeting siRNA 21-mer dual-targeting siRNAs (double strands) capable of simultaneously inhibiting BCL2 (B-cell lymphoma 2) and BI-1 (BAX inhibitor 1) were prepared (Bioneer, Daejeon, Korea) according to the sequences shown in Table 2 below. Specifically, 21-mer siRNA set 10 consisting of SEQ ID NOs: 19 and 20 in Table 2 below has complementary 15-mers, 20-mer siRNA set 11 consisting of SEQ ID NOs: 21 and 22 has complementary 14-mers, 20-mer siRNA set 12 consisting of SEQ ID NOs: 23 and 24 has complementary 14-mers, 19-mer siRNA set 13 consisting of SEQ ID NOs: 25 and 26 has complementary 13-mers, 19-mer siRNA set 14 consisting of SEQ ID NOs: 27 and 28 has complementary 13-mers, and 18-mer siRNA set 15 consisting of SEQ ID NOs: 29 and 30 has complementary 12-mers. Since siRNAs of sequence numbers 19, 21, 23, 25, 27 or 29 (Antisense Bcl-2) in Table 2 below bind complementarily to Bcl-2 mRNA, and siRNAs of sequence numbers 20, 22, 24, 26, 28 or 30 (Antisense BI-1) bind complementarily to BI-1 mRNA, siRNA sets 10-15 of the present invention simultaneously reduce the expression of the Bcl-2 and BI-1 genes.

[0068] [Table 2]

[0069] 1-3.AR and mTOR dual-targeting siRNA A set of dual-targeting siRNAs (double strands) capable of simultaneously inhibiting AR (androgen receptor) and mTOR (mammalian target of rapamycin) was prepared (Bioneer, Daejeon, Korea) as shown in Table 3. Specifically, 20-mer siRNA set 16 consisting of SEQ ID NOs: 31 and 32 has complementary 18-mers, 19-mer siRNA set 17 consisting of SEQ ID NOs: 33 and 34 has complementary 17-mers, 18-mer siRNA set 18 consisting of SEQ ID NOs: 35 and 36 has complementary 16-mers, 17-mer siRNA set 19 consisting of SEQ ID NOs: 37 and 38 has complementary 15-mers, 19-mer siRNA set 20 consisting of SEQ ID NOs: 39 and 40 has complementary 15-mers, 18-mer siRNA set 21 consisting of SEQ ID NOs: 41 and 42 has complementary 14-mers, and 17-mer siRNA set 22 consisting of SEQ ID NOs: 43 and 44 has complementary 15-mers. iRNA set 22 has 13mers complementary to each other, 23mer siRNA set 23 consisting of SEQ ID NOs: 45 and 46 has 19mers complementary to each other, 22mer siRNA set 24 consisting of SEQ ID NOs: 47 and 48 has 18mers complementary to each other, 22mer siRNA set 25 consisting of SEQ ID NOs: 49 and 50 has 18mers complementary to each other, 21mer siRNA set 26 consisting of SEQ ID NOs: 51 and 52 has 17mers complementary to each other, 20mer siRNA set 27 consisting of SEQ ID NOs: 53 and 54 has 16mers complementary to each other, and 21mer siRNA set 28 consisting of SEQ ID NOs: 55 and 56 has 17mers complementary to each other. siRNAs (antisense AR) of sequence numbers 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53 or 55 in Table 3 below bind complementarily to AR mRNA, and siRNAs (antisense mTOR) of sequence numbers 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54 or 56 bind complementarily to mTOR mRNA.Therefore, the siRNA set 16-28 of the present invention simultaneously reduces the expression of the AR and mTOR genes.

[0070] [Table 3]

[0071] MGMT and mTOR dual-targeting siRNA A set of dual-targeting siRNA (double strand) that can simultaneously reduce (inhibit) the expression of the MGMT (O-6-methylguanine-DNA methyltransferase, NM_002412.5) and mTOR (NM_004958.3) genes was constructed (Bioneer, Daejeon, Korea) as shown in Table 4 below.

[0072] [Table 4]

[0073] 1-5. BCL2 and MCL1 dual-targeting siRNA A set of dual-targeting siRNAs (double strands) that can simultaneously reduce (inhibit) the expression of the BCL2 (NM_000633.2) and MCL1 (MCL1 apoptosis regulator, NM_021960.5) genes was constructed (Bioneer, Daejeon, Korea) according to the sequences shown in Table 5 below.

[0074] [Table 5]

[0075] STAT3 and TFEB dual-targeting siRNA A set of dual-targeting siRNA (double strand) that can simultaneously reduce (inhibit) the expression of the STAT3 (NM_139276.2) and TFEB (transcription factor EB, NM_007162.2) genes was constructed (Bioneer, Daejeon, Korea) as shown in Table 6 below.

[0076] [Table 6]

[0077] 1-7.c-MET and PD-L1 dual-targeting siRNA A dual-targeting siRNA (double strand) set capable of simultaneously inhibiting c-MET (Homo sapiens MET proto-oncogene) and PD-L1 (Programmed death-ligand 1) was constructed (Bioneer, Daejeon, Korea) according to the sequences shown in Table 7 below. Specifically, 19-mer siRNA set 32 ​​consisting of SEQ ID NOs: 63 and 64 has 15-mers complementary to each other, with siRNA SEQ ID NO: 63 (antisense c-MET) in Table 7 below binding to c-MET mRNA and siRNA SEQ ID NO: 64 (antisense PD-L1) binding to PD-L1 mRNA. Therefore, the siRNA set of the present invention simultaneously reduces the expression of the c-MET and PD-L1 genes.

[0078] [Table 7]

[0079] Example 2. Generation of dual-targeting shRNA 2-1.mTOR and STAT3 targeting shRNA To enable intracellular expression of the siRNAs prepared in the above examples, expression cassettes for expressing shRNAs were prepared. Specifically, representative shRNAs (TTGGATCCAA loop shRNA and TTCAAGAGAG loop shRNA) containing the dual-target siRNA (SEQ ID NOs: 1 and 2) siRNA duplex sequence and loop sequence of Set 1 of the siRNAs were prepared (Table 8). Each of the prepared shRNA expression cassettes was placed after the U6 promoter (SEQ ID NO: 65) at the restriction enzyme PstI and EcoRV cleavage sites of the pE3.1 vector (Figure 1), thereby producing recombinant expression vectors for intracellular expression of two types of shRNAs, including dual-target siRNAs targeting mTOR and STAT3.

[0080] [Table 8]

[0081] 2-2. BCL2 and BI-1 targeting shRNA To enable intracellular expression of the siRNAs prepared in the above examples, expression cassettes expressing shRNAs were prepared. Specifically, shRNAs containing the dual-target siRNAs (SEQ ID NOs: 19 and 20) of Set 10 and a loop sequence (TTGGATCCAA loop shRNA and TTCAAGAGAG loop shRNA) were prepared (Table 9). The expression cassettes expressing each of the prepared shRNAs were placed after the U6 promoter (SEQ ID NO: 65) at the restriction enzyme PstI and EcoRV cleavage sites of the pE3.1 vector (Figure 1), respectively, to prepare recombinant expression vectors capable of intracellular expression of two shRNAs, including dual-target siRNAs targeting BCL2 and BI-1.

[0082] [Table 9]

[0083] 2-3.AR and mTOR-targeting shRNA To enable the siRNAs prepared in the above examples to be expressed intracellularly, expression cassettes expressing shRNAs were prepared. Specifically, shRNAs containing the 16 dual-target siRNAs (SEQ ID NOs: 31 and 32) siRNA double-stranded sequences and loop sequences (TTGGATCCAA loop shRNA and TTCAAGAGAG loop shRNA) were prepared as representative examples (Table 10). The expression cassettes expressing each of the prepared shRNAs were placed behind the U6 promoter (SEQ ID NO: 65) at the restriction enzyme PstI and EcoRV cleavage sites of the pE3.1 vector (Figure 1), respectively, to prepare recombinant expression vectors that express two types of shRNAs, including dual-target siRNAs targeting AR and mTOR, in cells.

[0084] [Table 10]

[0085] 2-4.c-MET and PD-L1 targeting shRNA To enable intracellular expression of the siRNAs prepared in the above Examples, shRNA expression cassettes containing dual-targeting siRNA duplex sequences and loop sequences (TTGGATCCAA loop shRNA and TTCAAGAGAG loop shRNA) were prepared. Specifically, TTGGATCCAA (TTGGATCCAA loop) or TTCAAGAGAG (TTCAAGAGAG loop), antisense strand, and TT were ligated from 5' to 3' to the 3' end of the sense strand of the siRNA set (SEQ ID NOs: 63 and 64) in Table 7 above, to prepare the DNA sequences encoding the shRNAs, which are shown in Table 11 (siRNAs are written in uppercase, and additional sequences are written in lowercase). The prepared shRNA expression cassettes were inserted into the pE3.1 vector (Figure 1) at the PstI and EcoRV restriction enzyme cleavage sites, respectively, behind the U6 promoter (SEQ ID NO: 65), to prepare recombinant expression vectors capable of intracellular expression of two shRNAs containing dual-targeting siRNAs targeting c-MET and PD-L1.

[0086] [Table 11]

[0087] Example 3. Confirmation of the gene expression inhibitory effect of dual-targeting siRNA Inhibition of mTOR and STAT3 expression HeLa cells were seeded into 12-well plates and cultured in RPMI medium (Hyclone) supplemented with 10% FBS (Hyclone) at 37°C and 5% CO2 until cell density reached 50%. The cells were then transfected with the dual-targeting siRNAs (Sets 1-9) prepared in Example 1 using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) to simultaneously knockdown mTOR and STAT3. Forty-eight hours after transfection, the cells were lysed, and total RNA was extracted using a GeneJET RNA Purification Kit (Invitrogen). The extracted total RNA was used as a template for reverse transcription using RevoScript™ RT PreMix (iNtRON BIOTECHNOLOGY). A 20 μl sample containing 25–200 ng of reverse-transcribed cDNA was used with AmpONE taq DNA polymerase (GeneAll) and TaqMan Gene Expression assays (Applied Biosystems) to perform PCR reactions for mTOR (Hs00234522_m1), STAT3 (Hs01047580_m1), and GAPDH (Hs02758991_g1) using an ABI PRISM 7700 Sequence Detection System and QS3 Real-time PCR (Biosystems). The real-time PCR reaction conditions were 50°C for 2 minutes, 95°C for 10 minutes, and a two-step cycle consisting of 95°C for 15 seconds and 60°C for 60 seconds, for a total of 40 cycles. All reactions were performed in triplicate, and the average was calculated. Results were normalized to the mRNA level of the housekeeping gene, GAPDH.

[0088] As a result, the dual-targeting siRNAs in Sets 1-9 confirmed that the residual expression of mTOR and STAT3 was approximately 20-40% compared to the control group, indicating that the dual-targeting siRNAs simultaneously inhibited the expression of both genes (Figure 2).

[0089] Inhibition of BCL2 and BI-1 expression HeLa cells were distributed into 12-well plates and cultured in RPMI medium (Hyclone) supplemented with 10% FBS (Hyclone) at 37°C and 5% CO2 until cell density reached 50%. The HeLa cells were then transfected with 80 pmol of dual-targeting siRNA Set 10 (si-BB1) and dual-targeting siRNA Sets 11-15 prepared in Example 1 (Table 2) using 3 μl of Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) per well to simultaneously knockdown BCL2 and BI-1. Forty-eight hours after transfection, cells were disrupted, and total RNA was extracted using the GeneJET RNA Purification Kit (Invitrogen). The extracted total RNA was used as a template and reverse transcribed to cDNA by RT-PCR. The mRNA expression levels of BCL2 and BI-1 induced by the dual-targeting siRNA were then confirmed by q-PCR. For reference, the probes used were Bcl2 (Thermo, Hs00608023_m1), BI-1 (Thermo, Dm01835892_g1), and GAPDH (Thermo, Hs02786624_g1), and the results were analyzed using a QS3 instrument. All reactions were performed in triplicate and the average values ​​were calculated. The results were normalized to the mRNA level of the housekeeping gene, GAPDH.

[0090] As a result, the dual-targeting siRNA set reduced the expression of both BCL2 and BI-1, demonstrating that the dual-targeting siRNA of the present invention simultaneously inhibits the expression of two genes (FIGS. 3 and 4).

[0091] Therefore, the dual-targeting siRNA of the present invention simultaneously inhibits the expression of two genes, thereby promoting the death of cancer cells and demonstrating significant anticancer activity, suggesting that it may be useful as an anticancer composition or anticancer adjuvant for various cancers.

[0092] Inhibition of AR and mTOR expression PC3, h460, and A549 cell lines were each dispensed into a 12-well plate and then cultured in RPMI medium (Hyclone) supplemented with 10% FBS (Hyclone) at 37°C and 5% CO2 until cell density reached 50%. The wells containing the cultured cells were then transfected with 80 pmol of the dual-targeting siRNA Set 16-28 (Table 3) prepared in Example 1 using 3 μl of Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) per well to simultaneously knockdown AR and mTOR. Set 16 was transfected into h460 cells, and Set 17 was transfected into PC3 cells for knockdown. As positive controls, siRNAs against AR and mTOR listed in Table 12 below were transfected, respectively. Forty-eight hours after transfection, cells were disrupted and total RNA was extracted using the GeneJET RNA Purification Kit (Invitrogen). The extracted total RNA was used as a template for reverse transcription to cDNA, followed by q-PCR to confirm the mRNA expression levels of AR and mTOR induced by each siRNA and the dual-targeting siRNA set 16-28 (si-AT1-siAT13) of the present invention. To confirm mRNA expression levels, a primer set for AR or mTOR and a reaction mixture (2 μl 10X reaction buffer, 2 μl HQ buffer, 1.6 μl dNTPs, 1 μl each of primers (F, R, 10 pmole / µL), 2 μl template (500 ng), 0.2 μl Taq, 10.2 μl DW, total volume 20 μl) were used. AR and mTOR mRNA in knockdown cell lysates were converted to cDNA using PCR conditions of 95°C for 2 minutes, 30 cycles of 95°C for 20 seconds, 60°C for 10 seconds and 72°C for 30-60 seconds, and 72°C for 5 minutes.Additionally, reverse-transcribed cDNA was used as a template. A reaction mixture containing 6 μl of template (RT-PCR product), 3 μl of TaqMan probe, 6 μl of 10X reaction buffer, 6 μl of HQ buffer, 4.8 μl of dNTPs, 0.6 μl of TaqMan probe, 10.2 μl of DW, and a total volume of 60 μl was prepared and used for qPCR. The reaction mixture consisted of 10 min at 95°C, 15 s at 95°C, and 40 cycles at 60°C per min. For reference, the probes used were AR (Thermo, Hs00171172_m1), mTOR (Thermo, Hs00234508_m1), and GAPDH (Thermo, Hs02786624_g1), and the reaction was performed using a QS3 instrument. All reactions were performed in triplicate, and the average was calculated. The results thus obtained were normalized to the mRNA value of the housekeeping gene GAPDH.

[0093] [Table 12]

[0094] As a result, in both PC3 cells and h460 cell lines, the dual-targeting siRNA sets 16 and 17 of the present invention reduced the expression of both AR and mTOR (Figure 5), and the degree of reduction was similar to or superior to the effect of each siRNA. Furthermore, the dual-targeting siRNA sets 17 and 28 of the present invention reduced the expression of AR and mTOR (Figure 6). This demonstrates that the dual-targeting siRNA of the present invention can effectively inhibit the expression of two genes simultaneously.

[0095] Inhibition of c-MET and PD-L1 expression 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 each dispensed into a 12-well plate and cultured in RPMI medium (Hyclone) supplemented with 10% FBS (Hyclone) at 37°C and 5% CO2 until cell density reached 50%. The cells were then transfected with the dual-targeting siRNA set (Table 7) prepared in the previous example at 80 pmol per well using 3 μl of Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) to simultaneously knockdown c-MET and PD-L1. Forty-eight hours after transfection, the cells were lysed and total RNA was extracted using the GeneJET RNA Purification Kit (Invitrogen). Extracted total RNA was used as a template for reverse transcription to cDNA by RT-PCR, followed by q-PCR to confirm the mRNA expression levels of c-MET and PD-L1 induced by each siRNA and the dual-targeting siRNA set of the present invention. To confirm mRNA expression levels, a primer set for PD-L1 or c-MET was used with a reaction mixture containing 2 μl of 10X reaction buffer, 2 μl of HQ buffer, 1.6 μl of dNTPs, 1 μl each of primers (F, R, 10 pmole / µL), 2 μl of template (500 ng), 0.2 μl of Taq, 10.2 μl of DW, and a total volume of 20 μl. c-MET and PD-L1 mRNAs in knockdown cell lysates were converted to cDNA using PCR conditions of 95°C for 2 minutes, followed by 30 cycles of 95°C for 20 seconds, 60°C for 10 seconds, and 72°C for 30-60 seconds, followed by 72°C for 5 minutes.Additionally, reverse-transcribed cDNA was used as a template. A reaction mixture containing 6 μl of template (RT-PCR product), 3 μl of TaqMan probe, 6 μl of 10X reaction buffer, 6 μl of HQ buffer, 4.8 μl of dNTPs, 0.6 μl of TaqMan probe, 10.2 μl of DW, and a total volume of 60 μl was prepared. qPCR was performed using a QS3 instrument at 95°C for 10 minutes, 95°C for 15 seconds, and 60°C for 40 cycles per minute. All reactions were performed in triplicate, and the average was calculated. Results were normalized to the mRNA level of the housekeeping gene, GAPDH.

[0096] The results showed that the dual-targeting siRNA set of the present invention reduced the expression of both c-MET and PD-L1 in all of the U-87, 22Rv-1, A431, and HCC827 cell lines (Figures 7a-7d), demonstrating that the dual-targeting siRNA set of the present invention can effectively inhibit the expression of two genes simultaneously.

[0097] Example 4. Confirmation of gene expression inhibitory effects of dual-targeting siRNA and shRNA 4-1. Gene expression inhibition effect of dual-targeting shRNA A549 cells and U-87 glioblastoma cells (U87MG) were transfected with 0, 1, and 2 μg of the vector containing the TTCAAGAGAG loop shRNA sequence of SEQ ID NO: 66 or the TTCAAGAGAG loop shRNA sequence of SEQ ID NO: 67 (Table 8), encoding the shRNAs targeting mTOR and STAT3, prepared in Example 2, using lipofectamine 3000. 48 hours after transfection, the extent of reduction in mTOR and STAT3 gene expression was confirmed using the real-time PCR analysis method described in the previous example.

[0098] As a result, the expression of mTOR and STAT3 was reduced by both types of shRNA, including the dual-targeting siRNA of the present invention, and tended to decrease by approximately 20% in proportion to the amount of shRNA DNA (Figure 8).

[0099] 4-2. Comparison of gene expression inhibition effects of single-targeting siRNA and dual-targeting shRNA The gene expression inhibitory effect of the dual-targeting shRNA of the present invention was compared with that of two single-targeting siRNAs tandemly linked using two promoters. Specifically, siRNA for mTOR and siRNA for STAT3 were tandemly linked in the order of mTOR-STAT3 or STAT3-mTOR, and the gene expression inhibitory effect of this on mTOR and STAT3 was compared with that of the mTOR / STAT3 dual-targeting shRNA of the present invention.

[0100] As a result, in the 293T cell line, it was shown that the gene expression inhibition effect was significantly higher when the dual-target shRNA of the present invention was used than when two siRNAs for each gene were linked in series using a promoter (direct shmTOR-STAT3 or direct shSTAT3-mTOR) (Figure 9).

[0101] Example 5. Confirmation of the cancer cell killing effect of dual-targeting siRNA To confirm the killing effect of the dual-targeting siRNAs of Sets 1-9 of the present invention on cancer cells, human lung cancer cell line A549 cells were plated in a 96-well plate at 5 × 10 3 After dispensing 1000 cells / well, each of the dual-targeting siRNAs from Sets 1-9 was transfected into the cells using Lipofectamine 3000. Forty-eight hours after transfection, and then 24 hours later, the cells were treated with 5 mg / mL MTT (Promega, Ltd.) and incubated for 4 hours. The medium was then removed, and the cells were treated with 150 μL of solubilization solution and stop solution and incubated at 37°C for 4 hours. The absorbance of the reaction solution was measured at 570 nm, and cell viability was calculated using the following formula:

[0102]

number

[0103] As a result, it was confirmed that when mTOR and STAT3 were simultaneously inhibited with the dual-targeting siRNAs of Sets 1-9 of the present invention, cell viability was significantly reduced compared to the control group, thus confirming that the dual-targeting siRNAs of Sets 1-9 of the present invention effectively killed cancer cells (Figure 10).

[0104] Example 6. Confirmation of the cancer cell killing effect of combined treatment of dual-targeting siRNA and anticancer drug 6-1. Combined treatment of mTOR and STAT3 dual-targeting siRNA with anticancer drugs 6-1-1. Combination treatment with cisplatin Human lung cancer cell line A549 cells were plated in a 96-well plate at 5 x 10 3 After dispensing the cells into 1000 cells / well, the cells were transfected with each of the dual-targeting siRNAs (mTOR and STAT3 simultaneous knockdown) of Sets 1-9 of the present invention using Lipofectamine 3000. Forty-eight hours after transfection, the cells were treated with 5 μM cisplatin and incubated for 10 hours. The MTT reaction was then performed as in the previous example, and the absorbance was measured at 570 nm to calculate cell viability.

[0105] As a result, when mTOR and STAT3 were simultaneously inhibited with the dual-targeting siRNAs of Sets 1-9 of the present invention in combination with cisplatin, cell viability was reduced to approximately 50-70%, demonstrating a significant difference compared to the control group. Therefore, it was confirmed that the apoptotic effect was significantly improved when two genes were simultaneously inhibited in combination with an anticancer drug (Figure 11).

[0106] 6-1-2. Combination treatment with paclitaxel Human lung cancer cell line A549 cells were plated in a 96-well plate at 5 x 10 3After dispensing the solution into 100 cells / well, the cells were transfected with each of the dual-targeting siRNAs (mTOR and STAT3 simultaneous knockdown) of Sets 1-9 of the present invention using Lipofectamine 3000. Forty-eight hours after transfection, the cells were treated with 5 μM paclitaxel and incubated for 10 hours. The MTT reaction was then performed as in Example 4, and the absorbance was measured at 570 nm to calculate cell viability.

[0107] As a result, when mTOR and STAT3 were simultaneously inhibited with the dual-targeting siRNAs of Sets 1-9 of the present invention in combination with paclitaxel, cell viability was reduced to approximately 30-50%, demonstrating a significant difference compared to the control group. Therefore, it was confirmed that the apoptotic effect was significantly improved when two genes were simultaneously inhibited, even in combination with an anticancer drug (Figure 12).

[0108] 6-1-3. Combination treatment with 5-fluorouracil (5-FU, 5-fluorouracil) Human lung cancer cell line A549 cells were plated in a 96-well plate at 5 x 10 3 After dispensing the solution into 100 cells / well, the cells were transfected with each of the dual-targeting siRNAs (mTOR and STAT3 simultaneous knockdown) of Sets 1-9 of the present invention using Lipofectamine 3000. Forty-eight hours after transfection, the cells were treated with 5 μM paclitaxel and incubated for 10 hours. The MTT reaction was then performed as in Example 4, and the absorbance was measured at 570 nm to calculate cell viability.

[0109] As a result, when mTOR and STAT3 were simultaneously inhibited with the dual-targeting siRNAs of Sets 1-9 of the present invention in combination with 5-fluorouracil, cell viability was reduced to approximately 30%, a significant difference compared to the control group. Therefore, it was confirmed that the apoptotic effect was significantly improved when two genes were simultaneously inhibited, even in combination with an anticancer drug (Figure 13).

[0110] 6-2. Combination treatment of BCL2 and BI-1 dual-targeting siRNA with anticancer drugs 6-2-1. Synergistic anti-cancer effect of BCL2 and BI-1 dual-targeting siRNA Hela cells, a human cervical cancer cell line, were transfected with the dual-targeting siRNA set 1 (si-BB1) of the present invention or with individual BCL2 siRNA and BI-1 siRNA as controls. After treatment with different anticancer drugs for 6 hours, the degree of cell death of the cancer cell line was confirmed by MTT assay. Specifically, Hela cells were plated in a 6-well plate and transfected with 200 pmoles of siRNA per well using 7.5 μL of Lipofectamine. After incubation for 48 hours, the cell line was re-plated in a 96-well plate and grown to a 50% cell density (2.5 × 10 4 ), and then the control group was treated with anticancer drug concentrations of taxol 0.5 μM, cisplatin 20 μM, and etoposide 10 μM, respectively, while the cells treated with the dual-targeting siRNA set of the present invention were treated with half the concentrations of taxol 0.25 μM, cisplatin 10 μM, and etoposide 5 μM, respectively. After 6 hours, MTT analysis was performed as in the above example to confirm the extent of cancer cell death.

[0111] As a result, in the control group transfected with siRNA for either BCL2 or BI-1, almost no cancer cell death occurred, and only when treated in combination with an anticancer drug did the anticancer drug partially kill the cancer cells (Figure 14A). In contrast, in the case of the dual-targeted siRNA set 1 (si-BB1) of the present invention, significant cancer cell death occurred, and the cancer cell killing effect was synergistically increased even when treated in combination with an anticancer drug at a significantly lower concentration than in the control group treated with both BCL2 siRNA and BI-1 siRNA (Figure 14B). This suggests that the dual-targeted siRNA of the present invention itself exhibits anticancer activity, and that the synergistic effect of combined treatment with an anticancer drug is specific to the dual-targeted siRNA.

[0112] 6-2-2. Comparison of the effects of BCL2 and BI-1 dual-targeting siRNA with a Bcl2 inhibitor The inhibitory effect of the dual-targeting siRNA set 1 (si-BB1) against BCL2 and BI-1 of the present invention on cancer cell death was compared with that of ABT-737, a drug used as a cancer cell treatment by inhibiting BCL2. Specifically, as in the previous example, the LnCap prostate cancer cell line was injected, transfected with the siRNA set 1 (si-BB1) against BCL2 and BI-1 of the present invention, treated with 3 μM ABT-737, and incubated for 12 hours, after which the degree of cancer cell death was confirmed by MTT analysis.

[0113] As a result, treatment with ABT-737 or the siRNA set 1 (si-BB1) of the present invention against BCL2 and BI-1 increased the death of the LnCap cell line, and in particular, combined treatment with ABT-737 and the dual-targeting siRNA of the present invention significantly increased the death of cancer cells in a synergistic manner (Figure 15).

[0114] 6-2-3. Comparison of the anti-cancer effects of BCL2 and BI-1 dual-targeting siRNA and individual siRNAs PC3 cells, a human prostate cancer cell line, were cultured in 6-well plates and transfected with the dual-targeting siRNA set 10 (si-BB1) of the present invention or siRNAs against BCL2 or BI-1 listed in Table 13 below as controls. After 48 hours, the cells were treated with 10-20 μM cisplatin. Twelve hours after cisplatin treatment, the cells were treated with 5 mg / mL MTT (Promega, Ltd.) and incubated for 4 hours. The medium was then removed, and the cells were treated with 150 μL of solubilization solution and stop solution and incubated at 37°C for 4 hours. The absorbance of the reaction solution was measured at 570 nm, and cell viability was calculated using Equation 1.

[0115] [Table 13]

[0116] As a result, it was found that the group treated with the dual-targeted siRNA set 10 of the present invention in combination with cisplatin showed significantly increased cancer cell death compared to the control group treated with control siRNA without cisplatin, and the degree of death was significantly increased compared to the groups treated with siRNA against BCL2 and BI-1 respectively (Figure 16).

[0117] 6-3. Combination treatment of AR and mTOR dual-targeting siRNA with anticancer drugs DU145 and H460 cell lines were cultured in 6-well plates and transfected with the dual-targeting siRNA set 16 (si-AT1) of the present invention. After 48 hours, the cells were treated with 50 μM cisplatin, 20 μM etoposide, or 1 μM taxol and incubated for 16 hours. The cells were then treated with 5 mg / mL MTT (Promega, Ltd.) and incubated for 4 hours. The medium was then removed, and the cells were treated with 150 μL of solubilization solution and stop solution and incubated at 37°C for 4 hours. The absorbance of the reaction solution was measured at 570 nm to calculate cell viability.

[0118] As a result, it was found that the dual-targeted siRNA of the present invention alone induced cell death in the DU145 prostate cancer cell line (no-treatment group), and also in the cisplatin-treated group, which showed no cell death effect. Furthermore, it was confirmed that the dual-targeted siRNA set 16 (si-AT1) of the present invention significantly improved DU145 cell death when treated with etoposide and taxol, which showed some anticancer activity (Figure 17A). Furthermore, it was found that the dual-targeted siRNA of the present invention itself exhibited cell death in the lung cancer cell line H460, as in the DU145 cell line, and when treated with etoposide and taxol in combination, it exhibited significant anticancer activity (Figure 17B).

[0119] Example 7. Generation of dual-target shRNA-encoding adenovirus The hTERT promoter (SEQ ID NO: 74)-E1A (SEQ ID NO: 75)-IRES (SEQ ID NO: 76)-E1B sequence (SEQ ID NO: 77) (full sequence: SEQ ID NO: 78) was inserted between SpeI and ScaI of the adenoviral vector, and then the vector was incubated with the U6 promoter and the BCL2 and BI-1 dual-targeted shRNA (U6 promoter + BCL2 and BI-1 dual-targeted shRNA coding sequence: SEQ ID NO: 79), mTOR and STAT3 dual-targeted shRNA (U6 promoter + mTOR and STAT3 dual-targeted shRNA coding sequence: SEQ ID NO: 80), and AR and mTOR dual-targeted shRNA sequence (U6 promoter + AR and mTOR dual-targeted shRNA coding sequence: SEQ ID NO: 81) prepared in the previous example. The hTERT promoter and dual-targeting shRNA were encoded and expressed by inserting the hTERT promoter and dual-targeting shRNA (SEQ ID NO: 81) into the E3 region between the SpeI sites, respectively, to generate infectious recombinant adenoviruses (BCL2 and BI-1 dual-targeting shRNA encoding and expression adenovirus: CA101; mTOR and STAT3 dual-targeting shRNA encoding and expression adenovirus: CA102; AR and mTOR dual-targeting shRNA encoding and expression adenovirus: CA103; and c-MET and PD-L1 dual-targeting shRNA encoding (expressing) adenovirus: CA104) (see Figure 19). A control recombinant adenovirus (CA10G) containing only hTERT was also generated. The resulting adenoviral vectors were sequence-analyzed, and if no abnormalities were detected, the viral genome was linearized using PacI restriction enzyme. 293A cells were transduced using the CaCl2 method to produce each virus.

[0120] Example 8. Determining gene expression inhibition of adenovirus encoding dual-targeting shRNA 8-1. Confirmation of inhibition of mTOR and STAT3 expression in CA102 8-1-1.Confirmation of mRNA levels 8-1-1-1. Bladder cancer The inhibitory effect of the recombinant adenovirus CA102 prepared in Example 7 on the expression of target genes mTOR and STAT3 was confirmed in bladder cancer cell lines. Specifically, the expression of T24 cells (0.5×105 / well) and 253JBV cells (1x10 5 100µL / well) was dispensed into a 12-well plate. After 1 hour, CA10G and CA102 were treated at a multiplication factor of 10 (MOI) in each well. After 72 hours, RNA prep was performed using an RNA prep kit (Takara, 9767A). RNA was then quantified using Nanodirp. 20µL of RT premix (intron, 25081) was added to each tube at 400ng / 20µL, mixed thoroughly with the premix, and then incubated at 45°C for 1 hour and 95°C for 5 minutes in a PCR machine to synthesize cDNA. 2µL of the synthesized cDNA was used as a template to prepare a PCR mixture (total volume, 20µL) corresponding to the experimental group (2µL template, 0.5µL forward primer (10pmole / µL), 0.5µL reverse primer (10pmole / µL), 10µL 2X master mix (Bioline, BIO-94005), and 7µL DW). The resulting PCR mixture was mixed thoroughly by vortexing, centrifuged, and then subjected to 40 cycles of 95°C for 5 minutes, 95°C for 10 seconds, and 60°C for 30 seconds in a qPCR instrument (Applied Biosystems, QS3). The results were analyzed using the instrument's program.

[0121] As a result, in both T24 cells and 253JBV cells, the recombinant adenovirus CA102 of the present invention, which encodes and expresses the hTERT promoter and the mTOR and STAT3 dual-targeting shRNA, was shown to significantly inhibit the expression of mTOR and STAT3 compared to the recombinant adenovirus CA10G, which contains only the hTERT promoter (Figure 20).

[0122] 8-1-1-2.Head and neck cancer The expression inhibitory effect of the recombinant adenovirus CA102 prepared in Example 7 on the target genes mTOR and STAT3 was confirmed in the head and neck cancer cell lines HSC-2 and Fadu using the method of the above example. As a result, in both cell lines, the recombinant adenovirus CA102 of the present invention, which encodes and expresses the hTERT promoter and mTOR-STAT3 dual-targeting shRNA, was shown to significantly inhibit the expression of the mTOR and STAT3 genes compared to the recombinant adenovirus CA10G, which contains only the hTERT promoter (Figure 21).

[0123] 8-1-1-3.Skin squamous carcinoma The expression inhibitory effect of the recombinant adenovirus CA102 prepared in Example 7 on the target genes mTOR and STAT3 was confirmed in the skin squamous cell carcinoma cell lines A431 and HSC-5 using the method of the above example. As a result, in both cell lines, the recombinant adenovirus CA102 of the present invention, which encodes and expresses the hTERT promoter and the mTOR and STAT3 dual-targeting shRNA, was shown to significantly inhibit the expression of the mTOR and STAT3 genes compared to the recombinant adenovirus CA10G, which contains only the hTERT promoter (Figure 22).

[0124] 8-1-2. Confirmation of protein expression inhibition The recombinant adenovirus CA102 prepared in Example 7 was treated in bladder cancer cell lines T24 and 253JBV cells, and the inhibitory effect on the expression of the target genes mTOR and STAT3 was confirmed at the protein level by Western blot analysis. As a result, it was confirmed that the recombinant adenovirus CA102 of the present invention inhibited the protein expression of mTOR and STAT3 in both cell lines (Figure 23).

[0125] 8-2. Confirmation of inhibition of BCL2 and BI-1 expression in CA101 The effect of the recombinant adenovirus CA101 prepared in Example 7 on inhibiting the expression of target genes BCL2 and BI-1 was confirmed. Specifically, U-87 cells (1x10 5100µL / well of cDNA was dispensed into a 12-well plate. After 1 hour, CA10G and CA101 were treated at a multiplication factor of 10 (MOI) in each well. After 72 hours, RNA prep was performed using an RNA prep kit (Takara, 9767A). RNA was then quantified using Nanodirp. 20µL of cDNA was added to each tube at 400ng / 20µL with RT premix (intron, 25081). The premix contents were mixed thoroughly and then incubated at 45°C for 1 hour and 95°C for 5 minutes in a PCR machine to synthesize cDNA. Using 2µL of the synthesized cDNA as a template, PCR mixtures (total volume, 20µL) corresponding to each experimental group were prepared (2µL template, 0.5µL forward primer (10pmole / µL), 0.5µL reverse primer (10pmole / µL), 10µL 2X master mix (Bioline, BIO-94005), and 7µL DW). The resulting PCR mixture was mixed thoroughly by vortexing, centrifuged, and then subjected to 40 cycles of 95°C for 5 minutes, 95°C for 10 seconds, and 60°C for 30 seconds in a qPCR instrument (Applied Biosystems, QS3). The results were analyzed using the program provided with the qPCR instrument.

[0126] As a result, it was shown that in U-87 cells, the recombinant adenovirus CA101 of the present invention, which encodes and expresses the hTERT promoter and the BCL2 and BI-1 dual-targeting shRNA, significantly inhibited the expression of the BCL2 and BI-1 genes compared to the recombinant adenovirus CA10G, which contains only the hTERT promoter (Figure 24).

[0127] 8-3. Confirmation of inhibition of AR and mTOR expression in CA103 8-3-1.in vitro The expression inhibitory effect of the recombinant adenovirus CA103 prepared in Example 7 on the target genes AR and mTOR was confirmed. Specifically, human prostate cancer cell lines LNcap, C42B, and 22Rv1 were each incubated at 1×10 5After dispensing the cDNA into a 12-well plate at 1 / well, CA10G and CA103 were treated at 2 or 5 MOIs in each well. After 72 hours, RNA prep was performed using an RNA prep kit (Takara, 9767A). RNA was quantified using Nanodirp, and 400 ng / 20 μl of RNA was added to each tube using RT premix (intron, 25081). The premix contents were mixed thoroughly and then incubated at 45°C for 1 hour and 95°C for 5 minutes in a PCR machine to synthesize cDNA. Using 2 μl of the synthesized cDNA as a template, PCR mixtures (total volume, 20 μl) corresponding to each experimental group were prepared (2 μl template, 0.5 μl forward primer (10 pmole / μl), 0.5 μl reverse primer (10 pmole / μl), 10 μl 2X master mix (Bioline, BIO-94005), and 7 μl DW). The resulting PCR mixture was mixed thoroughly by vortexing, centrifuged, and then subjected to 40 cycles of 95°C for 5 minutes, 95°C for 10 seconds, and 60°C for 30 seconds in a qPCR instrument (Applied Biosystems, QS3). The results were analyzed using the program provided with the qPCR instrument.

[0128] As a result, in the LNcap cell line, the recombinant adenovirus CA103 of the present invention, which encodes and expresses an hTERT promoter and an AR and mTOR dual-targeting shRNA, was shown to significantly inhibit the expression of the AR and mTOR genes compared to the recombinant adenovirus CA10G containing only the hTERT promoter (Figure 25).Furthermore, in the C42B and 22Rv1 cell lines, the recombinant adenovirus CA103 of the present invention was shown to significantly inhibit the expression of the AR and mTOR genes compared to CA10G (Figure 26).

[0129] 8-3-2.in vivo A prostate cancer cell line (22Rv-1) was subcutaneously transplanted into balb / c nu / nu mice to establish a prostate cancer mouse model, and the recombinant adenoviruses CA10G and CA103 prepared in Example 7 were each directly administered once (2 × 108 The tumors were then excised 21 days later and the expression of AR and mTOR genes in the tumors was confirmed by Western blot and IHC analysis. Western blot analysis showed that the expression of mTOR and AR was reduced in the CA103-treated group compared to the control and CA10G-treated groups. IHC analysis also showed that the fluorescent expression of mTOR and AR was reduced by 70-90% or more in the CA103-treated group compared to the control and CA10G-treated groups, confirming that CA103 effectively inhibits the expression of its target genes, mTOR and AR, in the tumors (Figure 27).

[0130] 8-4. Confirmation of inhibition of c-MET and PD-L1 expression by CA104 The inhibitory effect of the recombinant adenovirus CA104 prepared in Example 7 on the expression of the target genes c-MET and PD-L1 was confirmed. Specifically, the A431 cell line (1x10 5 10 μL of cDNA was added to each well of a 12-well plate. After 1 hour, CA10G and CA104 were treated at 2 or 5 MOIs. 72 hours later, RNA prep was performed using an RNA prep kit (Takara, 9767A). RNA was then quantified using Nanodirp. RT premix (intron, 25081) was added to each tube at 400 ng / 20 μL, mixed thoroughly with the premix, and then incubated at 45°C for 1 hour and 95°C for 5 minutes in a PCR machine to synthesize cDNA. Using 2 μL of the synthesized cDNA as a template, PCR mixtures (total volume, 20 μL) corresponding to each experimental group were prepared (2 μL template, 0.5 μL forward primer (10 pmole / μL), 0.5 μL reverse primer (10 pmole / μL), 10 μL 2X master mix (Bioline, BIO-94005), and 7 μL DW). The resulting PCR mixture was mixed thoroughly by vortexing, centrifuged, and then subjected to 40 cycles of 95°C for 5 minutes, 95°C for 10 seconds, and 60°C for 30 seconds in a qPCR instrument (Applied Biosystems, QS3). The results were analyzed using the program provided with the qPCR instrument.

[0131] The results showed that in A431 cells, the recombinant adenovirus CA104 of the present invention, which encodes and expresses the hTERT promoter and the c-MET and PD-L1 dual-targeting shRNA, significantly inhibited the expression of the c-MET and PD-L1 genes compared with the recombinant adenovirus CA10G, which contains only the hTERT promoter (Figure 28).

[0132] Example 9. Determination of the anti-cancer effect of adenovirus encoding dual-targeting shRNA 9-1. Confirmation of the anti-cancer effect of CA101 The cancer cell killing effects of the recombinant adenoviruses CA10G and CA101 prepared in Example 7 were compared. 3 One hour later, CA10G and CA101 were added to each well at MOIs of 1, 2, 5, 10, 30, or 50. 72 hours later, MTT reagent was added and the wells were incubated at 37°C for 3 hours. After 3 hours, the medium was removed from each well, and 100 μl of DMSO was added. Immediately, the absorbance at 540 nm was measured using a microplate reader for MTT analysis.

[0133] As a result, it was confirmed that CA101 of the present invention significantly killed cancer cells compared to CA10G (FIG. 29).

[0134] 9-2. Confirmation of the anti-cancer effect of CA102 9-2-1. Bladder cancer 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 3Each well was treated with CA10G and CA102 at MOIs of 1, 2, 5, 10, 20, or 50 (MOI = 1 / well). After 72 hours, MTT reagent was added and the wells were incubated at 37°C for 3 hours. After 3 hours, the medium was removed from each well, and 100 μl of DMSO was added. The absorbance at 540 nm was immediately measured using a microplate reader for MTT analysis.

[0135] As a result, it was confirmed that CA102 of the present invention significantly kills cancer cells compared to CA10G (FIG. 30).

[0136] 9-2-2. Head and neck cancer To confirm the killing effect of the recombinant adenovirus CA102 of the present invention on head and neck cancer cells, the recombinant adenoviruses CA10G and CA102 prepared in Example 4 were treated with the head and neck cancer cell lines HSC-2 and Fadu, and the cell killing effects were compared using MTT assay. The results showed that, compared to a 10 MOI treatment, CA10G treatment resulted in approximately 40% cell death, while CA102, encoding and expressing an mTOR and STAT3 dual-targeting shRNA, induced over 70% cell death (Figure 31).

[0137] 9-2-3. Cutaneous squamous cell carcinoma To confirm the killing effect of the recombinant adenovirus CA102 of the present invention on cutaneous squamous cell carcinoma cells, the recombinant adenoviruses CA10G and CA102 prepared in Example 4 were treated with the cutaneous squamous cell carcinoma cell lines A431 and HSC-5, and the cell killing effects were compared using MTT assay. The results showed that the cell killing effect of CA102 was significantly higher than that of CA10G at 10 MOI treatment (Figure 32).

[0138] 9-3. Confirmation of the anti-cancer effect of CA103 The cancer cell-killing effects of the recombinant adenoviruses CA10G and CA103 prepared in Example 7 were compared. Specifically, LNcap, C42B, and 22Rv1 cell lines were each treated with 5x10 3One hour later, CA10G and CA103 cells were treated at MOIs of 1, 2, 5, 10, 20, 40, or 50 in each well. 72 hours later, MTT reagent was added and the cells were incubated at 37°C for 3 hours. After 3 hours, the medium was removed from each well, and 100 μl of DMSO was added. MTT analysis was then performed by immediately measuring the absorbance at 540 nm using a microplate reader.

[0139] As a result, it was confirmed that in the case of the LNcap cell line, the CA103 of the present invention significantly killed cancer cells compared to CA10G (Figure 33), and even in the case of the C42B and 22Rv1 cell lines, it was confirmed that the CA103 of the present invention significantly killed cancer cells (Figure 34).

[0140] Example 10. Determining the in vivo anti-cancer effect of adenovirus encoding dual-targeting shRNA 10-1. Confirmation of the anti-cancer effect of CA102 10-1-1. Anticancer effect on cancer cells in vivo In order to confirm the anticancer effect of the recombinant adenovirus CA102 of the present invention on cancer cells in vivo, 3 1.0 x 10 on the plate 7 The bladder cancer cell line 253J-BV and the head and neck cancer cell line FaDu were cultured and then treated with the recombinant adenoviruses CA10 and CA102 of the present invention at 2 MOI and 5 MOI, respectively, for 1 hour (PBS treatment was used as a control). The cells were then cultured in fresh medium for 2 hours. The cells were then harvested, mixed with Matrigel at a 1:1 (v / v) ratio, and xenografted into 6-week-old male Balb / c nu-nu mice. The xenografts were then observed for 32 days. The results showed that in the case of 253J-BV, the size of cancer cells was significantly reduced in the group treated with CA10G at 2 MOI, while the cancer cells disappeared in the group treated with CA102 at 2 MOI (Figure 35). In addition, in the case of FaDu, the size of cancer cells in the group treated with CA10G at 5 MOI did not show any significant difference compared to the control group, but the group treated with CA102 at 5 MOI showed that cancer cells had disappeared (Figure 36).

[0141] 10-1-2. Anticancer effects on in vivo tumors 5.0 × 10 6 After transplanting bladder cancer cell line 253J-BV, the size of the tumor was observed twice a week. The average size of the tumor was 150-200 mm. 3 When the tumor size reached 2.0 × 10, the tumors were divided into groups based on the average tumor size. 8 PFU of CA10G and CA102 viruses were injected intratumorally. Tumor size was then monitored twice weekly for 43 days. The results showed that tumor size decreased when CA10G was administered compared to the control group, but the cancer cell growth rate increased again over time. In contrast, tumor size decreased significantly when CA102 was administered compared to the control group and CA10G, demonstrating significant inhibition of cancer cell growth over time (Figure 37).

[0142] 10-1-3. Anticancer effect against tumors formed in vivo depending on the number of doses 5.0 × 10 6 After transplantation of the bladder cancer cell line 253J-BV, the size of the tumor was observed twice a week. 3 When the mean size of each tumor was constant, the tumors were divided into groups and the tumors were measured once a day for three days, a total of five times, at 1.0 × 10 8 PFU of CA10G and CA102 viruses were injected intratumorally. Tumor size was then monitored twice weekly for 42 days. The results showed that tumor size decreased with increasing CA102 injection frequency, and cancer cell growth was inhibited with increasing injection frequency (Figure 38).

[0143] 10-1-4. Anticancer effects against tumors formed in vivo depending on the dose After subcutaneously inoculating glioblastoma cell lines (U-87) into 6-week-old male Balb / c nu-nu mice, tumor size was observed and the average tumor size was 200 mm. 3When the tumor size reached 100 mg / kg, the tumors were divided into groups based on the mean tumor size. CA102 was administered directly into the tumors at different doses, and tumor volume and weight were monitored. The results showed that tumor volume and weight were significantly reduced in the CA102-treated group compared to the CA10G-treated group, and the anti-cancer effect increased with increasing dose (Figure 39).

[0144] 10-2. Confirmation of the anti-cancer effect of CA103 A prostate cancer cell line (22Rv-1) was subcutaneously transplanted into Balb / c nu / nu mice to establish a prostate cancer mouse model, and the recombinant adenoviruses CA10G and CA103 prepared in Example 7 were then directly administered into the tumor (2 × 10 8 pfu / spot, 3 times) and observed their growth. As a result, it was confirmed that the tumor volume and weight were significantly reduced in the CA103-treated group compared to the untreated control group (buffer-treated group) and the vector control group (CA10G-treated group) (Figure 40).

[0145] Example 11: Confirmation of the effect of combined treatment of adenovirus encoding dual-targeting shRNA with anticancer drugs Bladder cancer mouse models were constructed by subcutaneously implanting a bladder cancer cell line (253J-BV) into Balb / c nu / nu mice, and the recombinant adenoviruses CA10G and CA102 prepared in Example 7 were then directly administered into the tumors, and tumor growth was observed. The results showed that the tumor volume and weight were significantly reduced in the CA102-treated group compared to the untreated control group (buffer-treated group) and the vector control group (CA10G-treated group). When the anticancer drug cisplatin was administered in combination, the anticancer effect was confirmed to be synergistically increased (Figure 41).

Claims

1. human telomerase promoter (hTERT); and An antitumor adenovirus comprising an expression cassette comprising a first base sequence targeting the mTOR gene and a second base sequence targeting the STAT3 gene, the expression cassette comprises a base sequence sequentially encoding a base sequence targeting the STAT3 gene, a loop sequence capable of forming a hairpin structure, and a base sequence targeting the mTOR gene; wherein, when a base sequence targeting the STAT3 gene and a base sequence targeting the mTOR gene are expressed, the loop sequence forms an shRNA that simultaneously inhibits the expression of the STAT3 gene and the mTOR gene; when the first base sequence and the second base sequence are expressed in a cell or tissue, the first base sequence and the second base sequence partially form a double strand to form an siRNA or shRNA, thereby targeting mTOR mRNA and STAT3 mRNA; An anti-tumor adenovirus, characterized in that the expression cassette expresses the nucleic acids represented by SEQ ID NOs: 1 and 2 and is located in the endogenous E3 region of the adenovirus.

2. The antitumor adenovirus according to claim 1, wherein the human telomerase promoter is operably linked to an endogenous gene of the adenovirus.

3. Adenovirus endogenous genes have the structure 5'ITR-C1-C2-C3-C4-C5 3'ITR; C1 comprises E1A, E1B, or E1A-E1B; C2 comprises E2B-L1-L2-L3-E2A-L4; said C3 does not include or includes E3; said C4 comprises L5; and The antitumor adenovirus according to claim 2, wherein the C5 does not contain E4 or contains E4.

4. The antitumor adenovirus according to claim 1, wherein the hTERT promoter is operably linked to the endogenous E1A and E1B genes of the adenovirus.

5. The antitumor adenovirus according to claim 4, further comprising an IRES sequence between E1A and E1B.

6. The antitumor adenovirus according to claim 1, wherein the expression of the expression cassette is controlled by a U6 promoter.

7. The antitumor adenovirus according to claim 1, wherein the adenovirus is a group C adenovirus.

8. The antitumor adenovirus according to claim 1, characterized in that its serotype is type 5.

9. The antitumor adenovirus according to claim 1, characterized in that the antitumor virus has a higher tumor-killing ability than wild-type adenovirus.

10. The antitumor adenovirus according to claim 1, characterized in that the antitumor virus has a higher tumor-killing ability than an adenovirus in which the hTERT promoter is introduced into a wild-type adenovirus.

11. A composition for cancer treatment, comprising the antitumor adenovirus of claim 1.

12. The composition for cancer treatment according to claim 11, further comprising an anticancer agent.

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