Structure of oncolytic viruses comprising bispecific nucleic acid molecules

Abstract

The present invention relates to an anti-tumor adenovirus and an anti-cancer composition comprising the same, the double-stranded small interfering ribonucleic acid (siRNA) of the present invention promotes the killing of cancer cells by simultaneously inhibiting the expression of a first nucleic acid and a second nucleic acid, and the anti-cancer activity is significantly superior to the anti-cancer activity of treating each small interfering ribonucleic acid (siRNA) together. The effect of synergistically improving the cancer cell killing effect in the combined treatment process with the anti-cancer agent can be achieved; an adenovirus comprising an expression cassette encoding a short hairpin ribonucleic acid (shRNA) for expressing an anti-tumor adenovirus and a human telomerase reverse transcriptase (hTERT) promoter has the effects of avoiding an in vivo immune response to be specifically delivered to cancer cells and achieving systemic therapy, can achieve local delivery, has excellent selectivity, and can be used as an anti-tumor adenovirus. The compound can exhibit a significant anti-cancer effect even through minimal invasive treatment, and can be effectively used as an anti-cancer composition or an anti-cancer adjuvant for various cancers.

Description

[0001] This application is a divisional application of the patent application with the application date of March 22, 2021, application number 202180024163.1, and invention name "Structure of oncolytic virus comprising bispecific nucleic acid molecules". Technical Field

[0002] The present invention relates to an anti-tumor adenovirus and an anti-cancer composition containing the same. Background Art

[0003] Cancer is one of the leading causes of death worldwide. The development of innovative cancer therapeutics can reduce medical costs and create high added value. Furthermore, according to 2008 statistics, molecular therapeutics that can overcome resistance to existing anticancer agents represented a market size of US$17.5 billion in seven major countries (the US, Japan, France, Germany, Italy, Spain, and the UK). By 2018, the market size had reached approximately US$45 billion, with a projected growth rate of 9.5% compared to 2008. Cancer treatment is divided into surgery, radiotherapy, chemotherapy, and biological therapy. Among them, chemotherapy is a treatment method that inhibits the proliferation of cancer cells or kills cancer cells by using chemical substances. Since most of the toxicity exhibited by anticancer drugs is also expressed in normal cells, it has a certain degree of toxicity. Since anticancer drugs will lose their original effects after a certain period of use, drug resistance will occur. Therefore, it is urgent to develop anticancer drugs that selectively act on cancer cells and do not produce drug resistance (Current Status of Cancer Control Biowave 2004.6(19)). Recently, by securing molecular genetic information about cancer, new anticancer drugs that target the molecular characteristics of cancer have been developed. There are reports that anticancer drugs that target specific molecular targets that only cancer cells have will not produce drug resistance.

[0004] Technology that inhibits gene expression is an important tool in the development of therapeutic agents and target testing for treating diseases. RNA interference (hereinafter referred to as RNAi) was discovered and confirmed to act on sequence-specific messenger RNA (mRNA) in a variety of mammalian cells (Silence of the transcripts: RNA interference in medicine. J Mol Med (2005) 83:764-773). RNAi is a phenomenon in which small interfering RNA (hereinafter referred to as siRNA), which is a 21-25 nucleotide double-helical structure, specifically binds to transcripts (mRNA transcripts) with complementary sequences, degrading the transcripts and thereby inhibiting the expression of specific proteins. Inside the cell, double-stranded ribonucleic acid (RNA) is converted into 21-23 basepair (bp) double-stranded small interfering RNA (siRNA) by endonuclease called nuclease (Dicer). siRNA will bind to the RNA-induced silencing complex (RISC), allowing the guide (antisense) chain to recognize the target messenger RNA and decompose it, thereby sequence-specifically inhibiting the expression of the target gene (NUCLEIC-ACID THERAPEUTICS: BASIC PRINCIPLES AND RECENT APPLICATIONS. Nature Reviews Drug Discovery. 2002.1, 503-514). According to a report by Bertrand's research team, compared with antisense oligonucleotides (ASOs), small interfering RNA (siRNA) has a superior inhibitory effect on messenger RNA (mRNA) expression in vitro and in vivo for the same target gene, and this effect can be sustained (Comparison of antisense oligonucleotides and siRNAs in cell culture and in vivo. Biochem. Biophys. Res. Commun. 2002. 296: 1000-1004).According to analysis, the market for therapeutic agents based on RNA interference (RNAi) technology, including small interfering RNA (siRNA), will reach a total market size of more than 12 trillion won worldwide by 2020. The scope of application of this technology will be unprecedentedly expanded, and it is considered a next-generation gene therapy technology that can treat diseases that are difficult to treat with existing antibody-based and compound-based drugs. In addition, the mechanism of action of small interfering RNA (siRNA) is to regulate the expression of target genes in a sequence-specific manner by complementary binding to target messenger RNA (mRNA). Therefore, the development time and cost of existing antibody-based drugs or chemical substances (small molecule drugs) to optimize specific protein targets are very long. However, compared with this, the scope of application will be unprecedentedly expanded, the development time will be shortened, and it has the advantage of being able to develop guide compounds that can optimize all protein targets, including target substances that cannot be drugged (Progress Towards in Vivo Use of siRNAs. MOLECULAR THERAPY. 2006 13(4):664-670). To this end, recent research is underway to address the challenges posed by RNA-mediated interference in the development of existing chemically synthesized pharmaceuticals, while also aiming to develop therapeutics for various diseases, particularly tumors, by selectively inhibiting the expression of specific proteins at the transcriptome level. Furthermore, unlike existing anticancer agents, small interfering RNA (siRNA) therapeutics offer the advantage of predictable side effects due to their well-defined targets. However, this target specificity can also lead to limited therapeutic efficacy in tumors, which are diseases caused by multiple genetic factors. Summary of the Invention

[0005] Technical issues

[0006] The purpose of the present invention is to provide an anti-tumor adenovirus.

[0007] Furthermore, the present invention aims to provide a cancer treatment composition.

[0008] Technical Solution

[0009] To achieve the above-mentioned object, the present invention provides an anti-tumor adenovirus comprising a base sequence targeting a first nucleic acid and a base sequence targeting a second nucleic acid.

[0010] Furthermore, the present invention provides a cancer treatment composition comprising the above-mentioned anti-tumor adenovirus.

[0011] Effects of the Invention

[0012] According to the present invention, the double-stranded small interfering RNA (siRNA) of the present invention promotes the killing of cancer cells by simultaneously inhibiting the expression of the first nucleic acid and the second nucleic acid. The anti-cancer activity is significantly better than that of treating individual small interfering RNAs (siRNA) together, and has the effect of synergistically improving the cancer cell killing efficacy during combined treatment with anticancer agents. The adenovirus containing an expression cassette encoding a short hairpin RNA (shRNA) for expressing an anti-tumor adenovirus and a human telomerase reverse transcriptase (hTERT) promoter has the effect of avoiding the body's immune response to specifically deliver to cancer cells and achieve systemic therapeutic efficacy. It can achieve local delivery, has excellent selectivity, and can exhibit significant anti-cancer effects through minimally invasive treatment. It can be effectively used as an anti-cancer composition or anti-cancer adjuvant for various cancers. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The figure shows a vector map for expressing the short hairpin RNA containing the dual-target small interfering RNA group of the present invention in cells.

[0014] Figure 2 The figures show the mTOR or STAT3 gene expression inhibitory effects of the double-target double-stranded small interfering RNAs of Groups 1 to 9 of the present invention.

[0015] Figure 3 These are diagrams confirming the expression inhibitory effects of the BCL2 gene (left) and BI-1 gene (right) using the dual-target small interfering RNA panel 10 (si-BB1) of the present invention.

[0016] Figure 4 A diagram showing the expression inhibition effect of the BCL2 and BI-1 genes of the dual-target small interfering RNA groups 11 to 15 of the present invention, wherein NC is the control group small interfering RNA, and si-BB2 to si-BB6 are the small interfering RNA groups 11 to 15 of the present invention.

[0017] Figure 5 The figure is a diagram to confirm the expression inhibitory effect of AR gene and mTOR gene based on the dual-target small interfering RNA group 16 of the present invention in cancer cell lines, wherein A is the h460 cell line, B is the pc3 cell line, NC is the control group small interfering RNA, siAR is the small interfering RNA for AR, simTOR is the small interfering RNA for mTOR, and si-AT1 is the AR and mTOR dual-target small interfering RNA group 16 of the present invention.

[0018] Figure 6This figure shows the expression inhibition effect of AR gene and mTOR gene in A549 cell line confirmed by the dual-target small interfering RNA groups 17 to 28 of the present invention, wherein NC is the control small interfering RNA, and si-AT2 to si-AT13 are the small interfering RNA groups 17 to 28 of the present invention.

[0019] Figure 7 The graph shows the expression inhibition effect of c-MET and PD-L1 genes confirmed in various cancer cell lines using the double-stranded small interfering RNA (siRNA) of the present invention that can simultaneously inhibit c-MET and PD-L1.

[0020] Figure 8 This is a graph showing confirmation of the expression levels of mTOR and STAT3 based on the amount of deoxyribonucleic acid (DNA) in the short hairpin RNA expression cassette using a vector containing a sequence encoding the TTGGATCCAA circular short hairpin RNA sequence of SEQ ID NO: 66 or the TTCAAGAGAG circular short hairpin RNA sequence of SEQ ID NO: 67.

[0021] Figure 9 This figure compares the gene expression inhibitory effect of two single-target small interfering RNAs linked in tandem with the gene expression inhibitory effect of the dual-target short hairpin RNA of the present invention.

[0022] Figure 10 This figure shows the confirmation of the cell viability of human lung cancer cell line A549 cells when mTOR and STAT3 are simultaneously inhibited by the dual-target siRNA of the present invention (dual-target siRNA in Groups 1 to 9).

[0023] Figure 11 This figure shows the cell viability of human lung cancer cell line A549 cells when mTOR and STAT3 are simultaneously inhibited by the dual-target small interfering RNA of the present invention after cisplatin treatment.

[0024] Figure 12 This figure shows the cell viability of human lung cancer cell line A549 cells after paclitaxel treatment when mTOR and STAT3 are simultaneously inhibited by the dual-target small interfering RNA of the present invention.

[0025] Figure 13 This figure is a graph confirming the cell viability of human lung cancer cell line A549 cells when mTOR and STAT3 are simultaneously inhibited by the dual-target small interfering RNA of the present invention after 5-FU (5-fluorouracil) treatment.

[0026] Figure 14A diagram is provided to confirm the killing of cancer cells achieved by the combined treatment of the dual-target small interfering RNA group of the present invention and an anticancer agent, wherein A represents the combined treatment of an anticancer agent + Bcl2 small interfering RNA + BI-1 small interfering RNA, and B represents the combined treatment of the dual-target small interfering RNA group 10 (si-BB1) of the present invention + an anticancer agent.

[0027] Figure 15 The diagram shows the cancer cell-killing effects of ABT-737, a Bcl2 inhibitor used as an anticancer agent, and the dual-target small interfering RNA panel 10 (si-BB1) of the present invention, and the synergistic effect of their combined use.

[0028] Figure 16 The graph compares the cancer cell killing effect of the combined treatment of dual-target small interfering RNA group 10 (si-BB1) and an anticancer agent with the group co-treated with small interfering RNA targeting the BCL2 gene and small interfering RNA targeting the BI-1 gene.

[0029] Figure 17 This figure confirms the killing effect of cancer cells treated with anticancer agents based on dual-target small interfering RNA group 1 in cancer cell lines, wherein NC is the control small interfering RNA, no treat is the control group not treated with anticancer agents, si-AT1 is the AR and mTOR dual-target small interfering RNA group 16 of the present invention, A is the DU145 cell line, and B is the H460 cell line.

[0030] Figure 18 This is a diagram schematically showing the structure of the adenovirus of the present invention.

[0031] Figure 19 This is a diagram showing the vector map of the adenoviral vector of the present invention, wherein Bs-short hairpin RNA is the insertion site of the sequence encoding the dual-target short hairpin RNA of the present invention.

[0032] Figure 20 The graph shows the mTOR and STAT3 gene expression inhibitory effects confirmed in bladder cancer cell lines T24 and 253JBV by the recombinant adenovirus CA102 of the present invention encoding and expressing the human telomerase reverse transcriptase (hTERT) promoter and dual-target short hairpin RNA.

[0033] Figure 21 The graph shows the expression-inhibitory effect of the recombinant adenovirus CA102 of the present invention, which encodes and expresses the human telomerase reverse transcriptase (hTERT) promoter and dual-target short hairpin RNA, on the mTOR and STAT3 genes in the head and neck cancer cell lines FaDu and HSC-2.

[0034] Figure 22 The graph shows the mTOR and STAT3 gene expression inhibitory effects confirmed in the skin squamous cell carcinoma cell lines A431 and HSC-5 by the recombinant adenovirus CA102 of the present invention encoding and expressing the human telomerase reverse transcriptase (hTERT) promoter and dual-target short hairpin RNA.

[0035] Figure 23 The graph shows the expression-inhibitory effect of the recombinant adenovirus CA102 of the present invention on mTOR and STAT3 genes in bladder cancer cell lines T24 and 253J-BV at the protein level.

[0036] Figure 24 This figure confirms the expression inhibitory effect of BCL2 and BI-1 genes by the recombinant adenovirus CA101 of the present invention containing the human telomerase reverse transcriptase (hTERT) promoter and the dual-target short hairpin RNA expression cassette.

[0037] Figure 25 The graph shows the expression inhibitory effect of the recombinant adenovirus CA103 of the present invention containing the human telomerase reverse transcriptase (hTERT) promoter and the dual-target short hairpin RNA expression cassette on AR and mTOR genes in the prostate cancer cell line LNcap.

[0038] Figure 26 The figure shows the expression inhibitory effect of AR and mTOR genes confirmed in vitro in prostate cancer cell lines C42B and 22Rv1 by the recombinant adenovirus CA103 of the present invention containing the human telomerase reverse transcriptase (hTERT) promoter and the dual-target short hairpin RNA expression cassette.

[0039] Figure 27 The diagram shows the in vivo confirmation of the expression inhibitory effect of the AR and mTOR genes by the recombinant adenovirus CA103 of the present invention comprising a human telomerase reverse transcriptase (hTERT) promoter and a dual-target short hairpin RNA expression cassette.

[0040] Figure 28 This figure confirms the expression inhibitory effect of c-MET and PD-L1 genes by the recombinant adenovirus CA104 of the present invention containing the human telomerase reverse transcriptase (hTERT) promoter and the dual-target short hairpin RNA expression cassette.

[0041] Figure 29 This figure shows the killing effect of the recombinant adenovirus CA101 of the present invention containing the human telomerase reverse transcriptase (hTERT) promoter and the dual-target short hairpin RNA expression cassette on cancer cell lines.

[0042] Figure 30This figure shows the killing effect of the recombinant adenovirus CA102 of the present invention on bladder cancer cell lines RT4, T24, and 253J-BV.

[0043] Figure 31 This figure 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.

[0044] Figure 32 This figure 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.

[0045] Figure 33 This figure shows the killing effect of the recombinant adenovirus CA103 of the present invention containing the human telomerase reverse transcriptase (hTERT) promoter and the dual-target short hairpin RNA expression cassette on the cancer cell line LNcap.

[0046] Figure 34 This figure shows the killing effect of the recombinant adenovirus CA103 of the present invention containing the human telomerase reverse transcriptase (hTERT) promoter and the dual-target short hairpin RNA expression cassette on the cancer cell lines C42B and 22Rv1.

[0047] Figure 35 This is a diagram confirming the anticancer effect of the recombinant adenovirus CA102 of the present invention on bladder cancer cells (253J-BV) in vivo.

[0048] Figure 36 This figure shows the confirmation of the anticancer effect of the recombinant adenovirus CA102 of the present invention on head and neck cancer cells (FaDu) in vivo.

[0049] Figure 37 This is a diagram confirming the anticancer effect of the recombinant adenovirus CA102 of the present invention on a tumor (bladder cancer) formed in vivo.

[0050] Figure 38 This is a graph confirming the anticancer effect of CA102 on tumors (bladder cancer) formed in vivo based on the number of administrations.

[0051] Figure 39 This figure shows the in vivo confirmation of the therapeutic effect on glioblastoma based on the dosage of the recombinant adenovirus CA102 of the present invention.

[0052] Figure 40 The figure shows the in vivo confirmation of the prostate cancer therapeutic effect of the recombinant adenovirus CA103 of the present invention comprising a human telomerase reverse transcriptase (hTERT) promoter and a dual-target short hairpin RNA expression cassette.

[0053] Figure 41 This figure shows the in vivo confirmation of the therapeutic effect of the recombinant adenovirus CA102 of the present invention and its combined use with cisplatin on bladder cancer. DETAILED DESCRIPTION

[0054] The present invention will be described in detail below using examples of the present invention. However, the following examples are merely illustrative of the present invention and are not intended to be limiting. Various modifications and applications of the present invention are possible within the scope of the appended claims and their equivalents.

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

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

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

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

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

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

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

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

[0063] In one example, the endogenous gene of the adenovirus may have a 3'ITR structure of 5'ITR-C1-C2-C3-C4-C5, wherein C1 may include E1A (SEQ ID NO: 75), E1B (SEQ ID NO: 77), or E1A-E1B, C2 may include E2B-L1-L2-L3-E2A-L4, C3 may include or exclude E3, C4 may include L5, and C5 may include or exclude E4, and may include the base sequence of SEQ ID NO: 78.

[0064] In one example, the E3 region of the adenovirus may be partially deleted, and the deleted base sequence may include the base sequence of SEQ ID NO: 82.

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

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

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

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

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

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

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

[0072] The term "expression inhibition" as used in the present invention refers to causing a decrease in the expression or translation of a target gene, preferably, to the point where the expression of the target gene is undetectable or insignificant.

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

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

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

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

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

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

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

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

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

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

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

[0084] In one example, the first nucleic acid may be STAT3 (signal transducer and activator of transcription 3), and the second nucleic acid may be mTOR (mammalian target of rapamycin). In this case, the expression cassette may include a nucleic acid in which U is converted to T in the base sequence of SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, or SEQ ID NO: 17 and SEQ ID NO: 18. In the above content, the small interfering RNA (siRNA) of sequence number 1 and sequence number 2 can complementarily bind to 17mer in 21mer, the small interfering RNA (siRNA) of sequence number 3 and sequence number 4 can complementarily bind to 16mer in 20mer, the small interfering RNA (siRNA) of sequence number 5 and sequence number 6 can complementarily bind to 15mer in 19mer, the small interfering RNA (siRNA) of sequence number 7 and sequence number 8 can complementarily bind to 14mer in 18mer, and the small interfering RNA (siRNA) of sequence number 9 and sequence number 10 can complementarily bind to 16mer in 20mer. The siRNA of SEQ ID NOs. 11 and 12 can complementarily bind to the 16mer of a 17mer, the siRNA of SEQ ID NOs. 13 and 14 can complementarily bind to the 16mer of a 19mer, the siRNA of SEQ ID NOs. 15 and 16 can complementarily bind to the 15mer of an 18mer, and the siRNA of SEQ ID NOs. 17 and 18 can complementarily bind to the 14mer of a 17mer. Furthermore, when the first nucleic acid is STAT3 and the second nucleic acid is mTOR, the short hairpin RNA (shRNA)-expressing DNA sequence contained in the expression cassette (a DNA sequence encoding a short hairpin RNA (shRNA) that dually targets STAT3 and mTOR) can include the base sequence of SEQ ID NOs. 66 or 67.

[0085] In one example, the first nucleic acid may be BCL2 (B-cell lymphoma 2) and the second nucleic acid may be BI-1 (BAX inhibitor 1). In this case, the expression cassette may include the base sequence of SEQ ID NO: 19 and SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, or SEQ ID NO: 29 and SEQ ID NO: 30. In the above content, the 21-mer siRNA group 10 composed of sequence numbers 19 and 20 has 15mers complementary to each other, the 20-mer siRNA group 11 composed of sequence numbers 21 and 22 has 14mers complementary to each other, the 20-mer siRNA group 12 composed of sequence numbers 23 and 24 has 14mers complementary to each other, the 19-mer siRNA group 13 composed of sequence numbers 25 and 26 has 13mers complementary to each other, the 19-mer siRNA group 14 composed of sequence numbers 27 and 28 has 13mers complementary to each other, and the 18-mer siRNA group 15 composed of sequence numbers 29 and 30 has 12mers complementary to each other. The small interfering RNA (siRNA) (Antisense Bcl-2) of SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, or SEQ ID NO: 29 in Table 2 below can complementarily bind to Bcl-2 messenger RNA (mRNA), and the small interfering RNA (siRNA) (Antisense BI-1) of SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, or SEQ ID NO: 30 can complementarily bind to BI-1 messenger RNA (mRNA). Furthermore, when the first nucleic acid is BCL2 and the second nucleic acid is BI-1, the short hairpin RNA (shRNA) expression DNA contained in the expression cassette can include the base sequence of SEQ ID NO: 68 or SEQ ID NO: 69.

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

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

[0088] In one example, the first nucleic acid may be BCL2, and the second nucleic acid may be MCL1 (MCL1 apoptosis regulator). In this case, the expression cassette may comprise the base sequences of SEQ ID NO: 59 and SEQ ID NO: 60.

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

[0090] In one example, the first nucleic acid may be c-MET (Homo sapiens MET proto-oncogene) and the second nucleic acid may be PD-L1 (Programmed death-ligand 1). In this case, the expression cassette may include nucleic acids in which U is converted to T in the base sequences of SEQ ID NOs: 63 and 64. In the above context, the small interfering RNAs (siRNAs) of SEQ ID NOs: 63 and 64 may complementarily bind to the 15mer of the 19mer. Furthermore, when the first nucleic acid is c-MET and the second nucleic acid is PD-L1, the short hairpin RNA (shRNA)-expressing deoxyribonucleic acid (DNA) contained in the expression cassette may include the base sequence of SEQ ID NOs: 72 or 73.

[0091] In one example, the expression cassette may 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.

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

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

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

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

[0096] In one embodiment, the composition of the present invention may further comprise an anticancer agent, for example, acivicin, aclarubicin, acodazole, tetracycline, achromycin, adozelesin, alanosin, aldesleukin, allopurinol sodium, altretamine, aminoglutethimide, amonafide, ampligen, amsacrine, androgens, anguidine, aphidicolin glycinate, asaley, asparaginase, 5-azacytidine, azathioprine, Bacillus Calmette-Guérin, or a combination thereof. Calmette-Guerin (BCG), Baker's antifolate, 6-mercapto-2'-deoxythioguanosine, bisantrene HCL, bleomycin sulfate, busulfan, buthioninesulfoximine, BWA773U82, BW 502U83 / HCl, BW 7U85 mesylate, ceracemide, carbetimer, carboplatin, carmustine, chlorambucil, chloroquinoxaline-sulfonamide, chlorozotocin, chromomycin A3 A3), cisplatin, cladribine, corticosteroids, Corynebacterium parvum, CPT-11, crisnatol, cyclocytidine, cyclophosphamide, cytarabine, cytembena, DABIS maleatemaleate), dacarbazine, dactinomycin, danuorubicin HCl, diazauridine, dexrazoxane, dianhydrogalactitol, diaziquone, dibromodulcitol, didemnin B, diethyldithiocarbamate, diglycoaldehyde, dihydro-5-azacytidine, doxorubicin, echinomycin, dedatrexate, edelfosine, eflornithine, Elliott's solution, elsamitrucin, epirubicin, esorubicin, estramustine phosphate, estrogens, etanidazol, ethiofos, etoposide, fadrazole, fazarabine, fenretinide, filgrastim, finasteride, flavoneacetic acid, floxuridine, fludarabine phosphate, 5-fluorouracil, Fluosol TMflutamide, gallium nitrate, gemcitabine, goserelin acetate, hepsulfam, hexamethylene bisacetamide, homoharringtonine, hydrazine sulfate, 4-hydroxyandrostenedione, hydrozyurea, idarubicin HCl, ifosfamide, 4-ipomeanol, iproplatin, isotretinoin, leucovorin calcium, leuprolide acetate, levamisole, liposome daunorubicin, liposome-encapsulated doxorubicin, lomustine, lonidamine, maytansine, mechlorethamine hydrochloride, melphalan, menogaril, merbarone, 6-mercaptopurine, mesna, methanol extraction of Bacillus Calmette-Guerin, methotrexate, N-methylformamide, mifepristone, mitoguazone, mitomycin-C, mitotane, mitoxantrone hydrochloride, monocyte / macrophage colony-stimulating factor factor), nabilone, nafoxidine, neocarzinostatin, octreotide acetateacetate), ormaplatin, oxaliplatin, paclitaxel, Pala, pentostatin, piperazine, pipobroman, pirarubicin, piritrexim, piroxantrone hydrochloride, PIXY-321, plicamycin, porfimer sodium, prednimustine, procarbazine, progestins, pirazofurin, razoxane, sargramostim, semustine, spirogermanium, spiromustine, streptomycin Streptonigrin, streptozocin, sulofenur, suramin sodium, tamoxifen, taxotere, tegafur, teniposide, terephthalamidine, teroxirone, thioguanine, thiotepa, thymidine injection injection), tiazofurine, topotecan, toremifene, tretinoin, trifluoperazine hydrochloride, trifluredine, Trimetrexate, tumor necrosis factor (TNF), uracil mustard, vinblastine sulfate, vincristine sulfate, vindesine, vinorelbine, vinzolidine, Yoshi 864, zorubicin, cytosine arabinosidearabinoside), etoposide, melphalan, paclitaxel, and mixtures thereof. Preferred are cisplatin, paclitaxel, 5-fluorouracil (5-FU), methotrexate, doxorubicin, daunorubicin, cytosine arabinoside, etoposide, melphalan, chlorambucil, cyclophosphamide, vindesine, mitomycin, bleomycin, tamoxifen, and paclitaxel; more preferred are cisplatin, paclitaxel, or 5-fluorouracil (5-FU). The present invention is not limited thereto as long as the combined use with the composition of the present invention can achieve a synergistic anticancer effect.

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

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

[0099] The composition of the present invention may further comprise an adjuvant. Any adjuvant known in the art may be used without limitation, for example, a complete adjuvant or an incomplete adjuvant further comprising Freund's adjuvant may be used to enhance its effect.

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

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

[0102] When formulated, they can be prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules. These solid preparations are prepared by mixing the active ingredients with 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 preparations for oral administration include suspensions, internal solutions, emulsions, and syrups. In addition to the widely used diluents water and liquid paraffin, they can also contain a variety of excipients, such as wetting agents, sweeteners, fragrances, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, oils, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions can use propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, synthetic fatty acid ester (witepsol), polyethylene glycol, Tween 61, cocoa butter, lauryl ester, glycerin gelatin, etc. can be used.

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

[0104] The dosage of the pharmaceutical composition of the present invention can be selected based on the individual's age, weight, sex, and physical condition. The concentration of the single domain antibody in the pharmaceutical composition can be selected in various ways depending on the intended target, but is preferably present in the pharmaceutical composition at a concentration of 0.01 to 5000 μg / ml. Concentrations below 0.01 μg / ml may result in pharmacological inactivity, while concentrations above 5000 μg / ml may cause toxicity in humans.

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

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

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

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

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

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

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

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

[0113] Double-stranded small interfering RNA (siRNA) capable of simultaneously inhibiting STAT3 (signal transducer and activator of transcription 3) and mTOR (mammalian target of rapamycin) were prepared using the sequences in Table 1 (Bioneer, Daejeon, Korea). Specifically, the siRNAs of sequence numbers 1 and 2 in group 1 complementarily bind to the 17mer of the 21mer, the siRNAs of sequence numbers 3 and 4 in group 2 complementarily bind to the 16mer of the 20mer, the siRNAs of sequence numbers 5 and 6 in group 3 complementarily bind to the 15mer of the 19mer, the siRNAs of sequence numbers 7 and 8 in group 4 complementarily bind to the 14mer of the 18mer, and the siRNAs of sequence numbers 9 and 10 in group 5 complementarily bind to the 16mer of the 17mer. Furthermore, the small interfering RNA (siRNA) of sequence number 11 and sequence number 12 of group 6 complementarily binds to 17mer in 20mer, the small interfering RNA (siRNA) of sequence number 13 and sequence number 14 of group 7 complementarily binds to 16mer in 19mer, the small interfering RNA (siRNA) of sequence number 15 and sequence number 16 of group 8 complementarily binds to 15mer in 18mer, and the small interfering RNA (siRNA) of sequence number 17 and sequence number 18 of group 9 complementarily binds to 14mer in 17mer.After the two sequences in each group of Table 1 below enter cells in double-stranded form, the antisense mTOR (antisense_mTOr) small interfering RNA (siRNA) in each group binds complementary to the target site of mTOR messenger RNA (mRNA) (gi|206725550|ref|NM_004958.3|Homo sapiens mechanistic target of rapamycin (serine / threonine kinase) (MTOR), mRNA), and the antisense STAT3 (antisense_STAT3) small interfering RNA (siRNA) in each group binds complementary to the target site of STAT3 messenger RNA (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 the gene expression of mTOR and STAT3.

[0114] Table 1

[0115]

[0116]

[0117] 1-2. Small interfering RNA (siRNA) targeting BCL2 and BI-1

[0118] A 21mer double-strand small interfering RNA (siRNA) capable of simultaneously inhibiting BCL2 (B-cell lymphoma 2) and BI-1 (BA Xinhibitor 1) was prepared according to the sequences in Table 2 (Bioneer, Daejeon, Korea). Specifically, the 21-mer siRNA group 10 consisting of sequence numbers 19 and 20 in Table 2 below makes the 15mers complementary to each other, the 20-mer siRNA group 11 consisting of sequence numbers 21 and 22 makes the 14mers complementary to each other, the 20-mer siRNA group 12 consisting of sequence numbers 23 and 24 makes the 14mers complementary to each other, the 19-mer siRNA group 13 consisting of sequence numbers 25 and 26 makes the 13mers complementary to each other, the 19-mer siRNA group 14 consisting of sequence numbers 27 and 28 makes the 13mers complementary to each other, and the 18-mer siRNA group 15 consisting of sequence numbers 29 and 30 makes the 12mers complementary to each other. The siRNA (Antisense Bcl-2) of SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, or SEQ ID NO: 29 in Table 2 below can complementarily bind to the messenger RNA (mRNA) of Bcl-2, and the siRNA (Antisense BI-1) of SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, or SEQ ID NO: 30 can complementarily bind to the messenger RNA (mRNA) of BI-1. Thus, siRNA Groups 10 to 15 of the present invention simultaneously reduce the expression of the Bcl-2 and BI-1 genes.

[0119] Table 2

[0120]

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

[0122] A double strand siRNA set (Bioneer, Daejeon, Korea) was prepared based on the sequences in Table 3 below, which can simultaneously inhibit AR (androgen receptor) and mTOR (mammalian target of rapamycin). Specifically, the 20-mer siRNA group 16 composed of sequence numbers 31 and 32 makes the 18mers complementary to each other, the 19-mer siRNA group 17 composed of sequence numbers 33 and 34 makes the 17mers complementary to each other, the 18-mer siRNA group 18 composed of sequence numbers 35 and 36 makes the 16mers complementary to each other, the 17-mer siRNA group 19 composed of sequence numbers 37 and 38 makes the 15mers complementary to each other, the 19-mer siRNA group 20 composed of sequence numbers 39 and 40 makes the 15mers complementary to each other, the 18-mer siRNA group 21 composed of sequence numbers 41 and 42 makes the 14mers complementary to each other, and the 17-mer siRNA group 21 composed of sequence numbers 43 and 44 makes the 15mers complementary to each other. siRNA group 22 has 13mers complementary to each other, siRNA group 23, a 23mer consisting of sequence numbers 45 and 46, has 19mers complementary to each other, siRNA group 24, a 22mer consisting of sequence numbers 47 and 48, has 18mers complementary to each other, siRNA group 25, a 22mer consisting of sequence numbers 49 and 50, has 18mers complementary to each other, siRNA group 26, a 21mer consisting of sequence numbers 51 and 52, has 17mers complementary to each other, siRNA group 27, a 20mer consisting of sequence numbers 53 and 54, has 16mers complementary to each other, and siRNA group 28, a 21mer consisting of sequence numbers 55 and 56, has 17mers complementary to each other.The siRNAs (anti-AR) of SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 55 in Table 3 below bind complementary to the messenger RNA (mRNA) of AR, and the siRNAs (anti-mTOR) of SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, or SEQ ID NO: 56 bind complementary to the messenger RNA (mRNA) of mTOR. Thus, siRNAs 16 to 28 of the present invention simultaneously reduce the expression of both AR and mTOR genes.

[0123] Table 3

[0124]

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

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

[0127] Table 4

[0128]

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

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

[0131] Table 5

[0132]

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

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

[0135] Table 6

[0136]

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

[0138] A double-stranded siRNA set (Bioneer, Daejeon, Korea) was prepared using the sequences in Table 7 below to simultaneously inhibit c-MET (Homo sapiens MET proto-oncogene) and PD-L1 (Programmed death-ligand 1). Specifically, the 19-mer siRNA set 32, consisting of SEQ ID NOs: 63 and 64, complements the 15-mers. The siRNA (Antisense c-MET) in SEQ ID NO: 63 in Table 7 below binds complementary to the c-MET messenger RNA (mRNA), while the siRNA (Antisense PD-L1) in SEQ ID NO: 64 binds complementary to the PD-L1 messenger RNA (mRNA). Thus, the siRNA set of the present invention simultaneously reduces the expression of both the c-MET and PD-L1 genes.

[0139] Table 7

[0140]

[0141] Example 2. Preparation of dual-target short hairpin RNA (shRNA)

[0142] 2-1. mTOR and STAT3 target short hairpin RNA (shRNA)

[0143] In order to express the small interfering RNA (siRNA) prepared in the above examples in cells, an expression cassette for expressing short hairpin RNA (shRNA) was prepared. Specifically, a plurality of short hairpin RNA (shRNA) containing the double-stranded sequence of the double-target small interfering RNA (siRNA) (sequence number 1 and sequence number 2) of group 1 among the plurality of small interfering RNA (siRNA) and a plurality of short hairpin RNA (shRNA) containing the circular sequence (TTGGATCCAA circular short hairpin RNA (shRNA) and TTCAAGAGAG circular short hairpin RNA (shRNA)) (Table 8) were representatively prepared. The prepared short hairpin RNA (shRNA) expression cassettes were respectively configured to be inserted into the pE3.1 vector ( Figure 1 ) was located after the U6 promoter (sequence number 65) in the restriction enzyme PstⅠ and EcoRⅤ cutting site, thereby preparing a recombinant expression vector that expresses two short hairpin RNAs (shRNAs) containing dual-target small interfering RNAs (siRNAs) targeting mTOR and STAT3 in cells.

[0144] Table 8

[0145]

[0146] 2-2. Short hairpin RNA (shRNA) targeting BCL2 and BI-1

[0147] In order to express the small interfering RNA (siRNA) prepared in the above examples in cells, an expression cassette for expressing short hairpin RNA (shRNA) was prepared. Specifically, a plurality of short hairpin RNAs (shRNAs) containing the double-stranded sequence of the double-target small interfering RNA (siRNA) (SEQ ID NO: 19 and SEQ ID NO: 20) of group 10 among the plurality of small interfering RNAs (siRNAs) and a circular sequence (TTGGATCCAA circular short hairpin RNA (shRNA) and TTCAAGAGAG circular short hairpin RNA (shRNA)) were representatively prepared (Table 9). The plurality of expression cassettes for expressing each short hairpin RNA (shRNA) were respectively arranged in the pE3.1 vector ( Figure 1 ) was located after the U6 promoter (sequence number 65) in the restriction enzyme PstⅠ and EcoRⅤ cutting site, thereby preparing a recombinant expression vector that expresses two short hairpin RNAs (shRNAs) containing dual-target small interfering RNAs (siRNAs) targeting BCL2 and BI-1 in cells.

[0148] Table 9

[0149]

[0150] 2-3. AR and mTOR target short hairpin RNA (shRNA)

[0151] In order to express the small interfering RNA (siRNA) prepared in the above examples in cells, an expression cassette for expressing short hairpin RNA (shRNA) was prepared. Specifically, a plurality of small interfering RNA (siRNA) double-stranded sequences of the double-target small interfering RNA (siRNA) (SEQ ID NO: 31 and SEQ ID NO: 32) of group 16 and a plurality of short hairpin RNA (shRNA) of circular sequences (TTGGATCCAA circular short hairpin RNA (shRNA) and TTCAAGAGAG circular short hairpin RNA (shRNA)) were representatively prepared (Table 10). The plurality of expression cassettes for expressing each short hairpin RNA (shRNA) were respectively arranged in the pE3.1 vector ( Figure 1 ) was located after the U6 promoter (sequence number 65) in the restriction enzyme PstⅠ and EcoRⅤ cutting site, thereby preparing a recombinant expression vector that expresses two short hairpin RNAs (shRNAs) containing dual-target small interfering RNAs (siRNAs) targeting AR and mTOR in cells.

[0152] Table 10

[0153]

[0154] 2-4. Short hairpin RNA (shRNA) targeting c-MET and PD-L1

[0155] To express the siRNAs prepared in the above examples in cells, multiple short hairpin RNA (shRNA) expression cassettes (TTGGATCCAA circular shRNA and TTCAAGAGAG circular shRNA) containing double-stranded sequences of dual-target siRNAs and loop sequences were prepared. Specifically, TTGGATCCAA (TTGGATCCAA loop) or TTCAAGAGAG (TTCAAGAGAG loop), the antisense strand, and TT were linked to the 3' end of the sense strand of the siRNA set (SEQ ID NOs. 63 and 64) listed in Table 7, from 5' to 3'. DNA sequences encoding the shRNAs were prepared and are shown in Table 11 (small interfering RNAs (siRNAs) are represented by capital letters, and additional sequences are represented by lowercase letters). The prepared multiple short hairpin RNA (shRNA) expression cassettes were respectively configured into pE3.1 vector ( Figure 1) was located after the U6 promoter (sequence number 65) in the cleavage site of the restriction enzymes PstⅠ and EcoRⅤ, thereby preparing a recombinant expression vector that expresses two short hairpin RNAs (shRNAs) containing dual-target small interfering RNAs (siRNAs) targeting c-MET and PD-L1 in cells.

[0156] Table 11

[0157]

[0158] Example 3. Confirmation of the gene expression inhibition effect of dual-target small interfering RNA (siRNA)

[0159] 3-1. Inhibition of mTOR and STAT3 expression

[0160] After injecting Hela cells into 12-well plates, they were cultured in serum-free cell freezing medium (RPMI, Hyclone) supplemented with 10% fetal bovine serum (FBS, Hyclone) at 37°C and 5% CO2 until the cell density reached 50%. Afterwards, the cells were transfected with the double-target small interfering RNA (siRNA) of groups 1 to 9 prepared in Example 1 above using a transfection reagent (lipofectamine3000, Invitrogen, Carlsbad, CA, USA) to knock down mTOR and STAT3 simultaneously. After 48 hours of transfection, total ribonucleic acid (RNA) was extracted using GeneJET RNAPurification Kit (Invitrogen) by disrupting the cells. The extracted total ribonucleic acid (RNA) was used as a template to perform a reverse transcription reaction using RevoScriptTM RT PreMix (iNtRON BIOTECHNOLOGY). Reactions for mTOR (Hs00234522_m1), STAT3 (Hs01047580_m1), and GAPDH (Hs02758991_g1) were performed using 20 μl of sample containing 25 to 200 ng of reverse-transcribed complementary deoxyribonucleic acid (cDNA), AmpONE Taq DNA polymerase (GeneAll), and TaqMan Gene Expression assays (Applied Biosystems) using the ABIPRISM 7700 Sequence Detection System and QS3 Real-time PCR (Biosystems). Real-time polymerase chain reaction (PCR) conditions consisted of a 50°C (2 minutes) followed by a 95°C (10 minutes) cycle, and a 95°C (15 seconds) followed by a 60°C (60 seconds) cycle, for a total of 40 cycles. All reactions were repeated three times, and the average value was calculated. The multiple results thus obtained were used for normalization of the messenger RNA (mRNA) values ​​of GAPDH, which is a housekeeping gene.

[0161] The results showed that the dual-target siRNA in Groups 1 to 9 confirmed that the remaining expression of mTOR and STAT3 was about 20% to 40% compared with the control group, indicating that the dual-target siRNA can simultaneously inhibit the expression of two genes ( Figure 2 ).

[0162] 3-2. Inhibition of BCL2 and BI-1 expression

[0163] After HeLa cells were injected into 12-well plates, they were cultured in serum-free cell freezing medium (RPMI, Hyclone) supplemented with 10% fetal bovine serum (FBS, Hyclone) at 37°C and 5% CO2 until the cell density reached 50%. Subsequently, 80 pmoles of the double-target small interfering RNA (siRNA) group 10 and (si-BB1) and double-target small interfering RNA (siRNA) groups 11 to 15 prepared in Example 1 (Table 2) were transfected into the wells cultured with HeLa cells using 3 μl of transfection reagent (lipofectamine 3000, Invitrogen, Carlsbad, CA, USA) to simultaneously knock down BCL2 and BI-1. 48 hours after transfection, total RNA was extracted using the GeneJET RNA Purification Kit (Invitrogen) by disrupting the cells. After the extracted total RNA was used as a template for reverse transcription into complementary deoxyribonucleic acid (cDNA) by real-time polymerase chain reaction (RT-PCR), the messenger RNA (mRNA) expression levels of BCL2 and BI-1 based on dual-target small interfering RNA (siRNA) were confirmed by quantitative polymerase chain reaction (q-PCR). As a reference, the probes used were Bcl2 (Thermo, Hs00608023_m1), BI-1 (Thermo, Dm01835892_g1), and GAPDH (Thermo, Hs02786624_g1), and the QS3 equipment was used to perform them. All reactions were repeated 3 times and the average value was taken. The multiple results thus obtained were used to normalize the messenger RNA (mRNA) values ​​of GAPDH, a housekeeping gene.

[0164] The results showed that the dual-target siRNA group reduced the expression of BCL2 and BI-1, indicating that the dual-target siRNA of the present invention can simultaneously inhibit the expression of two genes ( Figure 3 and Figure 4 ).

[0165] Therefore, the dual-target small interfering RNA (siRNA) of the present invention can simultaneously inhibit the expression of two genes, and thus has been confirmed to exhibit significant anticancer activity by promoting the killing of cancer cells, showing that it can be effectively used as an anticancer composition or anticancer adjuvant for various tumors.

[0166] 3-3. Inhibition of AR and mTOR expression

[0167] After PC3, h460, and A549 cells were injected into 12-well plates, they were cultured in serum-free cell freezing medium (RPMI, Hyclone) supplemented with 10% fetal bovine serum (FBS, Hyclone) at 37°C and 5% CO2 until the cell density reached 50%. Then, 80 pmole of the dual-target small interfering RNA (siRNA) groups 16 to 28 (Table 3) prepared in Example 1 were transfected into the wells containing the cells using 3 μl of lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) to simultaneously knock down AR and mTOR. Among them, group 16 was transfected into h460 cells, and group 17 was transfected into PC3 cells, thereby performing knockdown. As its positive control group, the small interfering ribonucleic acid (siRNA) for AR and the small interfering ribonucleic acid (siRNA) for mTOR described in Table 12 were transfected respectively. After 48 hours of transfection, total ribonucleic acid (RNA) was extracted using GeneJET RNA Purification Kit (Invitrogen) by fragmenting the cells. After the extracted total ribonucleic acid (RNA) was reverse transcribed into complementary deoxyribonucleic acid (cDNA) by real-time polymerase chain reaction (RT-PCR) as a template, the expression of messenger ribonucleic acid (mRNA) of AR and mTOR based on small interfering ribonucleic acid (siRNA) and the dual-target small interfering ribonucleic acid (siRNA) groups 16 to 28 (si-AT1 to siAT13) of the present invention was confirmed by quantitative polymerase chain reaction (q-PCR). To confirm the expression level of messenger RNA (mRNA), a primer set for AR or mTOR and a reaction mixture [2 μl of 10X reaction buffer, 2 μl of HQ buffer, 1.6 μl of dNTPs, 1 μl each of Primer (F, R, 10 pmoles / ul), 2 μl of Template (500 ng), 0.2 μl of Taq, 10.2 μl of DW, total volume 20 μl] were used. AR and mTOR mRNA in knocked-down cell fragments were converted into complementary deoxyribonucleic acid (cDNA) under polymerase chain reaction (PCR) conditions [95°C for 2 minutes, 30 cycles of 95°C for 20 seconds, 60°C for 10 seconds, 72°C for 30 to 60 seconds, and 72°C for 5 minutes].Reverse-transcribed complementary deoxyribonucleic acid (cDNA) was used as a template to prepare a reaction mixture [Template (RT-PCR product) 6 μl, Taqman probe 3 μl, 10X reaction buffer 6 μl, HQ buffer 6 μl, dNTP 4.8 μl, Taq 0.6 μl, DW 10.2 μl, total volume 60 μl]. Quantitative polymerase chain reaction (q-PCR) was performed [95°C for 10 minutes, 95°C for 15 seconds, and 60°C for 40 cycles per minute]. For reference, the probes used were AR (Thermo, Hs00171172_m1), mTOR (Thermo, Hs00234508_m1), and GAPDH (Thermo, Hs02786624_g1), using a QS3 instrument. All reactions were repeated three times, and the average value was calculated. The multiple results thus obtained were used for normalization of the messenger RNA (mRNA) values ​​of GAPDH, which is a housekeeping gene.

[0168] Table 12

[0169]

[0170] The results showed that in PC3 cells and h460 cell lines, the expression of AR and mTOr was reduced based on the dual-target small interfering RNA (siRNA) group 16 and group 17 of the present invention ( Figure 5 ), the degree of reduction is similar to or better than the effect of each small interfering RNA (siRNA). In addition, the dual-target small interfering RNA (siRNA) group 17 and group 28 of the present invention reduce the expression of AR and mTOr ( Figure 6 ). Thus, it can be seen that the dual-target small interfering RNA (siRNA) of the present invention can effectively inhibit the expression of two genes at the same time.

[0171] 3-4. Inhibition of c-MET and PD-L1 expression

[0172] Glioblastoma cell line U-87, prostate cancer cell line CWR22Rv-1 (22Rv-1), melanoma cell line A431, and non-small cell lung cancer cell line HCC827 were injected into 12-well plates and cultured in serum-free cell freezing medium (RPMI, Hyclone) supplemented with 10% fetal bovine serum (FBS, Hyclone) at 37°C and 5% CO2 until the cell density reached 50%. Subsequently, 80 pmole of the dual-target small interfering RNA (siRNA) panel prepared in the above example (Table 7) was transfected into the wells containing the cells using 3 μl of lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) to simultaneously knock down c-MET and PD-L1. 48 hours after transfection, total RNA was extracted using the GeneJET RNA Purification Kit (Invitrogen) by disrupting the cells. The extracted total RNA was used as a template for reverse transcription into complementary deoxyribonucleic acid (cDNA) by real-time polymerase chain reaction (RT-PCR). The messenger RNA (mRNA) expression levels of c-MET and PD-L1 based on small interfering RNA (siRNA) and the dual-target small interfering RNA (siRNA) panel of the present invention were confirmed by quantitative polymerase chain reaction (q-PCR). To confirm the expression level of messenger RNA (mRNA), a primer set for PD-L1 or c-MET and a reaction mixture [2 μl of 10× reaction buffer, 2 μl of HQ buffer, 1.6 μl of dNTPs, 1 μl each of Primer (F, R, 10 pmoles / ul), 2 μl of template (500 ng), 0.2 μl of Taq, 10.2 μl of DW, total volume 20 μl] were used. The c-MET and PD-L1 mRNA in knocked-down cell fragments was converted to complementary deoxyribonucleic acid (cDNA) under polymerase chain reaction (PCR) conditions [95°C for 2 minutes, 30 cycles of 95°C for 20 seconds, 60°C for 10 seconds, 72°C for 30 to 60 seconds, and 72°C for 5 minutes].Furthermore, using the reverse-transcribed complementary deoxyribonucleic acid (cDNA) as a template, a reaction mixture was prepared [Template (RT-PCR product) 6μl, Taqman probe 3μl, 10× reaction buffer 6μl, HQ buffer 6μl, dNTP 4.8μl, Taq 0.6μl, DW 10.2μl, total volume 60μl]. Quantitative polymerase chain reaction (q-PCR) was performed using a QS3 instrument [95°C for 10 minutes, 95°C for 15 seconds, and 60°C for 40 cycles per minute]. All reactions were repeated three times and the average value was calculated. The multiple results obtained were used to normalize the messenger RNA (mRNA) values ​​of GAPDH, a housekeeping gene.

[0173] The results showed that in U-87 cell line, 22Rv-1 cell line, A431 cell line and HCC827 cell line, the expression of c-MET and PD-L1 was reduced based on the dual-target small interfering RNA (siRNA) group of the present invention ( Figure 7 a to Figure 7 d). Thus, it can be seen that the dual-target small interfering RNA (siRNA) of the present invention can effectively inhibit the expression of two genes at the same time.

[0174] Example 4. Confirmation of the gene expression inhibition effect of dual-target small interfering RNA (siRNA) and short hairpin RNA (shRNA)

[0175] 4-1. Gene expression inhibition effect of dual-target short hairpin RNA (shRNA)

[0176] A549 cells and U-87 glioblastoma cells (U87MG) were transfected with 0, 1, and 2 μg of a vector containing the TTGGATCCAA circular short hairpin RNA (shRNA) sequence (SEQ ID NO: 66) or the TTCAAGAGAG circular short hairpin RNA (shRNA) sequence (SEQ ID NO: 67) (Table 8), encoding the mTOR and STAT3 target short hairpin RNA (shRNA) prepared in Example 2, respectively, using a transfection reagent (lipofectamine 3000). Forty-eight hours after transfection, the extent of reduction in mTOR and STAT3 gene expression was confirmed using the real-time polymerase chain reaction (PCR) analysis method described in the above example.

[0177] The results showed that the expression of mTOR and STAT3 was reduced in both short hairpin RNAs (shRNAs) containing the dual-target small interfering RNA (siRNA) of the present invention, and showed a tendency to decrease by 20% in proportion to the amount of deoxyribonucleic acid (DNA) of the short hairpin RNA (shRNA) ( Figure 8 ).

[0178] 4-2. Gene expression of single-target small interfering RNA (siRNA) and dual-target short hairpin RNA (shRNA) Comparison of inhibitory effects

[0179] The gene expression inhibition effects of two single-target small interfering RNAs (siRNAs) linked in tandem using two promoters were compared with those of the dual-target short hairpin RNA (shRNA) of the present invention. Specifically, after tandemly linking a small interfering RNA (siRNA) targeting mTOR and a small interfering RNA (siRNA) targeting STAT3 in the order mTOR-STAT3 or STAT3-mTOR, the gene expression inhibition effects of the mTOR and STAT3 genes were compared with those of the mTOR / STAT3 dual-target short hairpin RNA (shRNA) of the present invention.

[0180] The results showed that in 293T cell lines, compared with the case of using a promoter to connect two small interfering RNAs (siRNAs) to genes in series (direct shmTOR-STAT3 or direct shSTAT3-mTOR), the case of using the dual-target short hairpin RNA (shRNA) of the present invention showed a more significant gene expression inhibition effect ( Figure 9 ).

[0181] Example 5. Confirmation of the cancer cell killing effect based on dual-target small interfering RNA (siRNA)

[0182] In order to confirm the cancer cell killing effect of the dual-target small interfering RNA (siRNA) of Groups 1 to 9 of the present invention, the cells were randomly divided into two groups according to the concentration of 5×10 3 After injecting human lung cancer cell line A549 cells into 96-well plates, the cells were transfected with dual-target small interfering RNA (siRNA) from Group 1 to Group 9 using lipofectamine3000. 48 hours after transfection, the cells were additionally treated with 5 mg / mL MTT (Promega, Ltd.) 24 hours later and cultured for 4 hours. Afterwards, the culture medium was removed and 150 μl of solubilization solution and stop solution were treated, and the cells were cultured at 37°C for 4 hours. The absorbance of the reaction solution was measured at 570 nm, and the cell viability was calculated using the following mathematical formula.

[0183] Mathematical formula 1

[0184] Cell survival rate = absorbance of experimental group (570 nm) / absorbance of control group (570 nm) × 100 (%)

[0185] The results showed that when the dual-target small interfering RNA (siRNA) in Groups 1 to 9 of the present invention was used to simultaneously inhibit mTOR and STAT3, the cell survival rate was significantly reduced compared to the control group. Therefore, it was confirmed that the dual-target small interfering RNA (siRNA) in Groups 1 to 9 of the present invention effectively killed cancer cells ( Figure 10 ).

[0186] Example 6. Confirmation of the cancer cell killing effect based on the combined use of dual-target small interfering RNA (siRNA) and anticancer drugs

[0187] 6-1. Combination of mTOR and STAT3 dual-targeting small interfering RNA (siRNA) and anticancer drugs

[0188] 6-1-1. Combination treatment with cisplatin

[0189] In press 5×10 3 After injecting human lung cancer cell line A549 cells into a 96-well plate at 4 μg / well, the cells were transfected with dual-target small interfering RNA (siRNA) (for simultaneous knockdown of mTOR and STAT3) from Groups 1 to 9 of the present invention using lipofectamine 3000. Forty-eight hours after transfection, the cells were treated with 5 μM cisplatin and cultured for 10 hours. Subsequently, an MTT reaction was performed as in the above example, and absorbance was measured at 570 nm to calculate cell viability.

[0190] The results showed that when combined with cisplatin and the dual-target small interfering RNA (siRNA) of Groups 1 to 9 of the present invention was used to simultaneously inhibit mTOR and STAT3, the cell survival rate decreased by about 50% to 70%, and a significant difference was confirmed compared with the control group. Therefore, in the combined treatment with anticancer drugs, when two genes were inhibited simultaneously, it was confirmed that the cell killing effect was significantly improved ( Figure 11 ).

[0191] 6-1-2. Combination treatment with paclitaxel

[0192] In press 5×10 3After injecting human lung cancer cell line A549 cells into a 96-well plate at 4 μg / well, the cells were transfected with dual-target small interfering RNA (siRNA) (for simultaneous knockdown of mTOR and STAT3) from Groups 1 to 9 of the present invention using lipofectamine 3000. Forty-eight hours after transfection, the cells were treated with 5 μM paclitaxel and cultured for 10 hours. Subsequently, an MTT reaction was performed as in Example 4 above, and absorbance was measured at 570 nm to calculate cell viability.

[0193] The results showed that when combined with paclitaxel and the dual-target small interfering RNA (siRNA) of Groups 1 to 9 of the present invention was used to simultaneously inhibit mTOR and STAT3, the cell survival rate decreased by about 30-50%, and a significant difference was confirmed compared with the control group. Therefore, in the combined treatment with anticancer drugs, when two genes were inhibited simultaneously, it was confirmed that the cell killing effect was significantly improved ( Figure 12 ).

[0194] 6-1-3. Combined treatment with 5-fluorouracil (5-FU)

[0195] After injecting human lung cancer cell line A549 cells into 96-well plates at a density of 5×10 cells / well, the cells were transfected with dual-target small interfering RNA (siRNA) (for simultaneous knockdown of mTOR and STAT3) from Groups 1 to 9 of the present invention using lipofectamine 3000. Forty-eight hours after transfection, the cells were treated with 5 μM paclitaxel and cultured for 10 hours. Subsequently, an MTT reaction was performed as described in Example 4 above, and absorbance was measured at 570 nm to calculate cell viability.

[0196] The results showed that when combined with 5-fluorouracil and the dual-target small interfering RNA (siRNA) of Groups 1 to 9 of the present invention was used to simultaneously inhibit mTOR and STAT3, the cell survival rate decreased by about 30%, and a significant difference was confirmed compared to the control group. Therefore, in the combined treatment with anticancer drugs, when two genes were inhibited simultaneously, it was confirmed that the cell killing effect was significantly improved ( Figure 13 ).

[0197] 6-2. Combination treatment with BCL2 and BI-1 dual-targeting small interfering RNA (siRNA) and anticancer drugs

[0198] 6-2-1. Synergistic anticancer effects of BCL2 and BI-1 dual-targeting small interfering RNA (siRNA)

[0199] After transfection of the dual-target small interfering RNA (siRNA) group 1 (si-BB1) of the present invention or individual BCL2 small interfering RNA (siRNA) and BI-1 small interfering RNA (siRNA) as a control group into Hela cells as a human cervical cancer cell line, the anticancer agent was treated for 6 hours according to the type, and then the degree of killing of the cancer cell line was confirmed by MTT analysis. Specifically, 7.5 μl of transfection reagent was used to transfect the Hela cells injected into 6-well plates with 200 pmole per well of small interfering RNA (siRNA) and cultured for 48 hours, and the cell lines were re-injected into 96-well plates and the cell density (2.5×10 4 ) reached 50%. For the control group, the cells were treated with 0.5 μM taxol, 20 μM cisplatin and 10 μM etoposide at different concentrations of anticancer agents, and the cells in the dual-target small interfering RNA (siRNA) group of the present invention were treated with half the concentration of 0.25 μM taxol, 10 μM cisplatin and 5 μM etoposide. After 6 hours, the degree of cancer cell killing was confirmed by performing MTT analysis as in the above embodiment.

[0200] The results showed that for the control group transfected with small interfering RNA (siRNA) against BCL2 or BI-1, almost no cancer cell killing occurred, and only when treated with anticancer drugs did the cancer cell killing effect based on the anticancer drugs partially occur ( Figure 14 In contrast, the dual-target siRNA group 1 (si-BB1) of the present invention significantly killed cancer cells, and compared with the control group treated with both BCL2 siRNA and BI-1 siRNA, a synergistic increase in the cancer cell killing effect was confirmed even when used in combination with an anticancer agent at a significantly lower concentration ( Figure 14 Thus, it can be deduced that the dual-target siRNA of the present invention exhibits anticancer activity itself, and also exhibits specificity for the dual-target siRNA based on the synergistic effect of combined treatment with an anticancer agent.

[0201] 6-2-2. Comparison of the efficacy of Bcl2 inhibitors with small interfering RNA (siRNA) targeting both BCL2 and BI-1 fruit

[0202] The cancer cell killing inhibitory effects of the dual-targeting small interfering RNA (siRNA) targeting BCL2 and BI-1 (Group 1 si-BB1) of the present invention were compared with those of ABT-737, a cancer cell therapeutic agent based on BCL2 inhibition. Specifically, as in the above examples, LnCap cells, which are prostate cancer cells, were injected separately and then transfected with the siRNA Group 1 (si-BB1) targeting BCL2 and BI-1 of the present invention. The cells were then treated with 3 μM ABT-737 and cultured for 12 hours. The degree of cancer cell killing was then confirmed using an MTT assay.

[0203] The results showed that the cell death of LnCap cells increased after treatment with ABT-737 or the present invention's small interfering RNA (siRNA) group 1 (si-BB1) targeting BCL2 and BI-1. In particular, when ABT-737 and the present invention's dual-target small interfering RNA (siRNA) were treated together, the synergistic killing effect of cancer cells was significantly increased ( Figure 15 ).

[0204] 6-2-3. Comparison of BCL2 and BI-1 dual-targeting siRNAs and individual siRNAs Anticancer effects of siRNA

[0205] After culturing PC3 cell lines as human prostate cancer cell lines in 6-well plates, the dual-target small interfering RNA (siRNA) group 10 (si-BB1) of the present invention and each small interfering RNA (siRNA) for BCL2 or BI-1 in Table 13 as a control group were transfected, and after 48 hours, cisplatin was treated with 10 to 20 uM. After 12 hours of cisplatin treatment, 5 mg / mL of MTT (Promega, Ltd.) was treated to the cells and cultured for 4 hours. Afterwards, the culture medium was removed and 150 μl of solubilization solution and stop solution were treated, and cultured at a temperature of 37 ° C for 4 hours. The absorbance of the reaction solution was measured at 570 nm, and the cell survival rate was calculated using the above mathematical formula 1.

[0206] Table 13

[0207]

[0208] The results showed that compared with the control group that was not treated with cisplatin and treated with the control group siRNA, the group treated with the dual-target siRNA group 10 of the present invention and cisplatin showed a significant increase in cancer cell killing. Compared with the group treated with siRNA for BCL2 and BI-1 respectively, the degree of killing was significantly increased ( Figure 16).

[0209] 6-3. Combination of AR and mTOR dual-targeting small interfering RNA (siRNA) and anticancer drugs

[0210] DU145 and H460 cells were cultured in 6-well plates and transfected with the dual-target small interfering RNA (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 cultured for 16 hours. Subsequently, the cells were treated with 5 mg / mL MTT (Promega, Ltd.) and cultured for 4 hours. The culture medium was then removed and treated with 150 μl of solubilization solution and stop solution, followed by incubation at 37°C for 4 hours. The absorbance of the reaction solution was measured at 570 nm, and cell viability was calculated.

[0211] The results showed that the dual-target small interfering RNA (siRNA) of the present invention alone caused cell killing in the DU145 cell line, which is a prostate cancer cell line (no anticancer drug treatment group (no treat)), and also caused cell killing in the cisplatin-treated group that did not show a cell killing effect. In addition, it was confirmed that the dual-target small interfering RNA (siRNA) group 16 (si-AT1) of the present invention significantly improved the DU145 cell killing effect by treating etoposide and paclitaxel, which showed slight anticancer activity. Figure 17 Part A). In addition, similar to the DU145 cell line, the dual-target small interfering RNA (siRNA) of the present invention exhibited a cell-killing effect on H460, a lung cancer cell line. When treated with etoposide and paclitaxel in combination, it was found to exhibit significant anticancer activity ( Figure 17 Part B of the

[0212] Example 7. Preparation of adenovirus encoding dual-target short hairpin RNA (shRNA)

[0213] After the human telomerase reverse transcriptase (hTERT) promoter (SEQ ID NO: 74)-E1A (SEQ ID NO: 75)-internal ribosome entry site (SEQ ID NO: 76)-E1B sequence (SEQ ID NO: 77) (total sequence: SEQ ID NO: 78) was inserted between SpeⅠ and ScaⅠ of the adenovirus vector, the U6 promoter and the BCL2 and BI-1 dual-target short hairpin RNA (shRNA) (U6 promoter + BCL2 and BI-1 dual-target short hairpin RNA (shRNA) encoding sequence: SEQ ID NO: 79), mTOR and STAT3 dual-target short hairpin RNA (shRNA) (U6 promoter + mTOR and STAT3 dual-target short hairpin RNA (shRNA) encoding sequence: SEQ ID NO: 80) prepared in the above example were respectively inserted between SpeⅠ of the E3 region. The human telomerase reverse transcriptase (hTERT) promoter and dual-target short hairpin RNA (shRNA) sequence (U6 promoter + AR and mTOR dual-target short hairpin RNA (shRNA) coding sequence: SEQ ID NO: 81) were used to encode and express the human telomerase reverse transcriptase (hTERT) promoter and dual-target short hairpin RNA (shRNA), and infectious recombinant adenoviruses were prepared (BCL2 and BI-1 dual-target short hairpin RNA (shRNA) encoding and expressing adenovirus: CA101; mTOR and STAT3 dual-target short hairpin RNA (shRNA) encoding and expressing adenovirus: CA102; AR and mTOR dual-target short hairpin RNA (shRNA) encoding and expressing adenovirus: CA103; c-MET and PD-L1 dual-target short hairpin RNA (shRNA) encoding (expression) adenovirus: CA104) (refer to Figure 18 and Figure 19 Furthermore, as a control, a recombinant adenovirus (CA10G) containing only human telomerase reverse transcriptase (hTERT) was prepared. The sequences of the prepared adenoviral vectors were then analyzed, and if no abnormalities were detected, the viral genome was linearized using the PacI restriction enzyme. The resulting viruses were then transduced into 293A cells using the CACl2 method to produce the respective viruses.

[0214] Example 8. Confirmation of gene expression inhibition by adenovirus encoding dual-target short hairpin RNA (shRNA)

[0215] 8-1. Confirmation of CA102 inhibition of mTOR and STAT3 expression

[0216] 8-1-1. Confirmation of messenger RNA (mRNA) levels

[0217] 8-1-1-1. Bladder cancer

[0218] 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, T24 cells (0.5×10 5 / well) and 253JBV cells (1×10 5 After incubation at 4 °C for 1 hour, CA10G and CA102 were added to each well at an MOI of 10. After 72 hours, RNA was prepared using an RNA prep kit (Takara, 9767A). RNA was then quantified using a Nanodirp spectrophotometer. 400 ng / 20 μl of the premix was added to each tube using a real-time premix (Intron, 25081) and mixed uniformly with the premix. The mixture was then reacted using a polymerase chain reaction (PCR) instrument at 45°C for 1 hour and 95°C for 5 minutes to synthesize complementary deoxyribonucleic acid (cDNA). Using 2 μl of the synthesized complementary deoxyribonucleic acid (cDNA) as a template, a polymerase chain reaction (PCR) mixture (total volume 20 μl) was prepared for the experimental group (2 μl of template, 0.5 μl (10 pmoles / μl) of forward primer, 0.5 μl (10 pmoles / μl) of reverse primer, 10 μl of 2X master mix (Bioline, BIO-94005), and 7 μl of DW). The PCR mixture was mixed by vortexing and centrifuged. The reaction was then performed using a quantitative PCR instrument (Applied Biosystems, QS3) for 40 cycles of 95°C for 5 minutes, 95°C for 10 seconds, and 60°C for 30 seconds. The results were analyzed using the qPCR instrument's program.

[0219] The results showed that the recombinant adenovirus CA102 of the present invention encoding and expressing the human telomerase reverse transcriptase (hTERT) promoter and mTOR and STAT3 dual-target short hairpin RNA (shRNA) significantly inhibited the expression of mTOR and STAT3 genes in both T24 cells and 253JBV cells compared with the recombinant adenovirus CA10G containing only the human telomerase reverse transcriptase (hTERT) promoter ( Figure 20 ).

[0220] 8-1-1-2. Head and neck cancer

[0221] The method described in the above examples was used to confirm the inhibitory effect of the recombinant adenovirus CA102 prepared in the above example 7 on the expression of the target genes mTOR and STAT3 in HSC-2 and Fadu, which are head and neck cancer cell lines. The results showed that in both cell lines, the recombinant adenovirus CA102 of the present invention encoding and expressing the human telomerase reverse transcriptase (hTERT) promoter and the mTOR and STAT3 dual-target short hairpin RNA (shRNA) significantly inhibited the expression of the mTOR and STAT3 genes, compared with the recombinant adenovirus CA10G containing only the human telomerase reverse transcriptase (hTERT) promoter ( Figure 21 ).

[0222] 8-1-1-3. Skin squamous cell carcinoma

[0223] The method described in the above examples was used to confirm the inhibitory effect of the recombinant adenovirus CA102 prepared in the above example 7 on the expression of the target genes mTOR and STAT3 in A431 and HSC-5, which are skin squamous cell carcinoma cell lines. The results showed that in both cell lines, the recombinant adenovirus CA102 of the present invention encoding and expressing the human telomerase reverse transcriptase (hTERT) promoter and the mTOR and STAT3 dual-target short hairpin RNA (shRNA) significantly inhibited the expression of the mTOR and STAT3 genes, compared with the recombinant adenovirus CA10G containing only the human telomerase reverse transcriptase (hTERT) promoter ( Figure 22 ).

[0224] 8-1-2. Confirmation of protein expression inhibition

[0225] After the recombinant adenovirus CA102 prepared in Example 7 was treated with bladder cancer cell lines T24 and 253JBV, the expression inhibition effect on the target genes mTOR and STAT3 was confirmed at the protein level by Western blot analysis. The results showed that in both cell lines, the recombinant adenovirus CA102 of the present invention inhibited the protein expression of mTOR and STAT3 ( Figure 23 ).

[0226] 8-2. Confirmation of CA101 Inhibition of BCL2 and BI-1 Expression

[0227] The inhibitory effect of the recombinant adenovirus CA101 prepared in Example 7 on the expression of target genes BCL2 and BI-1 was confirmed. Specifically, U-87 cells (1×10 5After incubation at 4 °C / well, CA10G and CA101 were added to each well at an MOI of 10 one hour later. After 72 hours, RNA was prepared using an RNA prep kit (Takara, 9767A). RNA was then quantified using a Nanodirp spectrophotometer. 400 ng / 20 μl of the premix was added to each tube using a real-time premix (Intron, 25081) and mixed uniformly with the premix. The mixture was then reacted using a polymerase chain reaction (PCR) instrument at 45°C for one hour and at 95°C for five minutes to synthesize complementary deoxyribonucleic acid (cDNA). Using 2 μl of the synthesized complementary deoxyribonucleic acid (cDNA) as a template, a polymerase chain reaction (PCR) mixture (total volume 20 μl) was prepared for the experimental group (2 μl of template, 0.5 μl (10 pmoles / μl) of forward primer, 0.5 μl (10 pmoles / μl) of reverse primer, 10 μl of 2× master mix (Bioline, BIO-94005), and 7 μl of DW). The PCR mixture was vortexed and centrifuged. The reaction was then performed using a quantitative PCR instrument (Applied Biosystems, QS3) for 40 cycles of 95°C for 5 minutes, 95°C for 10 seconds, and 60°C for 30 seconds. The results were analyzed using the qPCR instrument's program.

[0228] The results showed that in U-87 cells, the recombinant adenovirus CA101 of the present invention encoding and expressing the human telomerase reverse transcriptase (hTERT) promoter and BCL2 and BI-1 dual-target short hairpin RNA (shRNA) significantly inhibited the expression of BCL2 and BI-1 genes compared with the recombinant adenovirus CA10G containing only the human telomerase reverse transcriptase (hTERT) promoter ( Figure 24 ).

[0229] 8-3. Confirmation of CA103 Inhibition of AR and mTOR Expression

[0230] 8-3-1. In vitro

[0231] The inhibitory effect of the recombinant adenovirus CA103 prepared in Example 7 on the expression of target genes AR and mTOR was confirmed. 5Human prostate cancer cell lines LNcap, C42B, and 22Rv1 were injected into a 12-well plate per well. One hour later, CA10G and CA103 were added to each well at an MOI of 2 or 5. Seventy-two hours later, RNA was prepared using an RNA prep kit (Takara, 9767A). RNA was then quantified using a spectrophotometer (Nanodirp). 400 ng / 20 μl of the premix was added to each tube using a real-time premix (Intron, 25081) and mixed uniformly with the premix. The mixture was then reacted using a polymerase chain reaction (PCR) instrument at 45°C for 1 hour and 95°C for 5 minutes to synthesize complementary deoxyribonucleic acid (cDNA). Using 2 μl of the synthesized complementary deoxyribonucleic acid (cDNA) as a template, a polymerase chain reaction (PCR) mixture (total volume 20 μl) was prepared for the experimental group (2 μl of template, 0.5 μl (10 pmoles / μl) of forward primer, 0.5 μl (10 pmoles / μl) of reverse primer, 10 μl of 2× master mix (Bioline, BIO-94005), and 7 μl of DW). The PCR mixture was vortexed and centrifuged. The reaction was then performed using a quantitative PCR instrument (Applied Biosystems, QS3) for 40 cycles of 95°C for 5 minutes, 95°C for 10 seconds, and 60°C for 30 seconds. The results were analyzed using the qPCR instrument's program.

[0232] The results showed that in the LNcap cell line, the recombinant adenovirus CA103 of the present invention encoding and expressing the human telomerase reverse transcriptase (hTERT) promoter and AR and mTOR dual-target short hairpin RNA (shRNA) significantly inhibited the expression of AR and mTOR genes compared with the recombinant adenovirus CA10G containing only the human telomerase reverse transcriptase (hTERT) promoter ( Figure 25 ). Moreover, in C42B and 22Rv1, the recombinant adenovirus CA103 of the present invention also significantly inhibited the expression of AR and mTOR genes compared with CA10G ( Figure 26 ).

[0233] 8-3-2. In vivo

[0234] Prostate cancer cell line (22Rv-1) was transplanted subcutaneously into balb c nu / nu mice to construct a prostate cancer mouse model. Afterwards, the recombinant adenoviruses CA10G and CA103 (2×10 8 pfu / spot, 3 times). After 21 days, the tumor was removed and the expression levels of AR and mTOR genes in the tumor were confirmed by Western blot analysis and IHC analysis. The results of Western blot analysis showed that in the group administered with CA103, the expression of mTOR and Ar was reduced compared with the control group and the CA10G administration group. The results of IHC analysis also showed that the fluorescence expression of mTOR and Ar in the group administered with CA103 was reduced by 70% to 790% or more than that in the control group and the CA10G administration group, confirming that CA103 effectively inhibited the expression of mTOR and Ar as target genes in the tumor 9( Figure 27 ).

[0235] 8-4. Confirmation of inhibition of c-MET and PD-L1 expression by CA104

[0236] The inhibitory effect of the recombinant adenovirus CA104 prepared in Example 7 on the expression of target genes c-MET and PD-L1 was confirmed. Specifically, A431 cells (1×10 5After 1 hour, CA10G and CA104 were added to each well at an MOI of 2 or 5. After 72 hours, RNA was prepared using an RNAprep kit (Takara, 9767A). RNA was then quantified using a spectrophotometer (Nanodirp). 400 ng / 20 μl of the premix was added to each tube using a real-time premix (Intron, 25081) and mixed evenly with the premix. The mixture was then reacted using a polymerase chain reaction (PCR) instrument at 45°C for 1 hour and 95°C for 5 minutes to synthesize complementary deoxyribonucleic acid (cDNA). Using 2 μl of the synthesized complementary deoxyribonucleic acid (cDNA) as a template, a polymerase chain reaction (PCR) mixture (total volume 20 μl) was prepared for the experimental group (2 μl of template, 0.5 μl (10 pmoles / μl) of forward primer, 0.5 μl (10 pmoles / μl) of reverse primer, 10 μl of 2X master mix (Bioline, BIO-94005), and 7 μl of DW). The PCR mixture was mixed by vortexing and centrifuged. The reaction was then performed using a quantitative PCR instrument (Applied Biosystems, QS3) for 40 cycles of 95°C for 5 minutes, 95°C for 10 seconds, and 60°C for 30 seconds. The results were analyzed using the qPCR instrument's program.

[0237] The results showed that in A431 cells, the recombinant adenovirus CA104 of the present invention encoding and expressing the human telomerase reverse transcriptase (hTERT) promoter and short hairpin RNA (shRNA) targeting both c-MET and PD-L1 significantly inhibited the expression of c-MET and PD-L1 genes compared with the recombinant adenovirus CA10G containing only the human telomerase reverse transcriptase (hTERT) promoter ( Figure 28 ).

[0238] Example 9. Confirmation of the anticancer effect of adenovirus encoding dual-target short hairpin RNA (shRNA)

[0239] 9-1. Confirmation of the anticancer effect of CA101

[0240] The cancer cell killing effects of the recombinant adenoviruses CA10G and CA101 prepared in Example 7 were compared. Specifically, U87MG cells (5×10 3After 1 hour, CA10G and CA101 were added to each well at an MOI of 1, 2, 5, 10, 30, or 50, respectively. After 72 hours, MTT reagent was added and 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 was then measured at a wavelength of 540 nm using a microplate reader to perform the MTT assay.

[0241] The results showed that CA101 of the present invention significantly killed cancer cells compared to CA10G ( Figure 29 ).

[0242] 9-2. Confirmation of the anticancer effect of CA102

[0243] 9-2-1. Bladder cancer

[0244] The cancer cell killing effects of the recombinant adenoviruses CA10G and CA102 prepared in Example 7 were compared. Specifically, T24 cells (2.5×10 3 / well), 253J-BV cells (5×10 3 / well) and human bladder epithelial cell line RT4 cells (5×10 3 After 1 hour, CA10G and CA102 were added to each well at an MOI of 1, 2, 5, 10, 20, or 50, respectively. After 72 hours, MTT reagent was added and 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 was then measured at a wavelength of 540 nm using a microplate reader, and the MTT assay was performed.

[0245] The results showed that CA102 of the present invention significantly killed cancer cells compared to CA10G ( Figure 30 ).

[0246] 9-2-2. Head and neck cancer

[0247] In order 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 HSC-2 and Fadu, which are head and neck cancer cell lines, and the cell killing effects were compared by MTT analysis. The results showed that when treated with CA10G, about 40% of the cells were killed, while CA102, which encodes and expresses short hairpin RNA (shRNA) that targets both mTOR and STAT3, caused more than 70% cell killing ( Figure 31 ).

[0248] 9-2-3. Squamous cell carcinoma of the skin

[0249] In order to confirm the cell-killing effect of the recombinant adenovirus CA102 of the present invention on skin squamous cell carcinoma cells, the recombinant adenoviruses CA10G and CA102 prepared in Example 4 were treated on A431 and HSC-5, which are skin squamous cell carcinoma cell lines, and the cell-killing effects were compared by MTT analysis. The results showed that the cell-killing effect of CA102 was significantly higher than that of CA10G (based on the treatment of 10 MOI). Figure 32 ).

[0250] 9-3. Confirmation of the anticancer effect of CA103

[0251] The cancer cell killing effects of the recombinant adenoviruses CA10G and CA103 prepared in Example 7 were compared. 3 LNcap, C42B, and 22Rv1 cells were seeded into a 96-well plate per well. One hour later, CA10G and CA103 were treated at 1, 2, 5, 10, 20, 40, or 50 MOIs per well. MTT reagent was added 72 hours later 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. The absorbance was then measured at 540 nm using a microplate reader for MTT analysis.

[0252] The results showed that in LNcap cell line, CA103 of the present invention significantly killed cancer cells compared with CA10G ( Figure 33 ), CA103 of the present invention also significantly killed cancer cells in C42B and 22Rv1 cell lines ( Figure 34 ).

[0253] Example 10. Confirmation of the in vivo anticancer effect of adenovirus encoding a dual-target short hairpin RNA (shRNA)

[0254] 10-1. Confirmation of the anticancer effect of CA102

[0255] 10-1-1. Anticancer effect on cancer cells in vivo

[0256] In order to confirm the anticancer effect of the recombinant adenovirus CA102 of the present invention on cancer cells in vivo, the 3 After culturing 1.0×107 cells of the bladder cancer cell line 253J-BV and the head and neck cancer cell line FaDu on plates, the recombinant adenoviruses CA10G and CA102 of the present invention were treated at 2 MOI and 5 MOI, respectively, for 1 hour (the control group was treated with PBS). After replacing the culture medium with fresh culture medium, the cells were cultured for 2 hours. Afterwards, the cells were recovered and mixed with Matrigel at a ratio of 1:1 (v / v). After transplantation (Xenograft) into 6-week-old male Balb / c nu-nu mice, the cells were observed for 32 days. The results showed that in the 253J-BV group, the size of the cancer cells was significantly reduced in the group treated with CA10G at 2 MOI, while the cancer cells were eliminated in the group treated with CA102 at 2 MOI ( Figure 35 Furthermore, in FaDu, the size of cancer cells in the group treated with CA10G at 5 MOI did not show a significant difference compared with the control group, whereas cancer cells were eliminated in the group treated with CA102 at 5 MOI ( Figure 36 ).

[0257] 10-1-2. Anticancer effect on tumors formed in vivo

[0258] 5.0×10 6 After the bladder cancer cell line 253J-BV was isolated, the patients were observed twice a week. When the tumor size reached an average of 150-200 mm 3 The mice were divided into multiple groups so that the average tumor size was constant, and 2.0×10 8 PFU of CA10G and CA102 viruses were administered. Tumor size was then observed twice a week for 43 days. The results showed that when CA10G was administered, tumor size was smaller than that of the control group, but over time, the growth rate of cancer cells increased again. Conversely, when CA102 was administered, tumor size was significantly smaller than that of the control group and CA10G, and even over time, the growth of cancer cells was significantly suppressed. Figure 37 ).

[0259] 10-1-3. Anticancer effect on tumors formed in vivo based on the number of doses

[0260] 5.0×10 6 The bladder cancer cell line 253J-BV was observed twice a week. When the average tumor size reached 200 mm3, the tumors were divided into multiple groups so that the average tumor size remained constant. 1.0×10 8 PFU of CA10G and CA102 viruses. Afterwards, the tumor size was observed twice a week for 42 days. The results showed that the tumor size decreased based on the number of CA102 administrations, confirming that the growth of cancer cells was suppressed based on the number of administrations ( Figure 38 ).

[0261] 10-1-4. Anticancer effect on tumors formed in vivo based on the dosage

[0262] After glioblastoma cell line (U-87) was transplanted into 6-week-old male Balb / c nu-nu mice, the size of the tumor was observed. When the tumor size reached an average of 200 mm 3 The researchers divided the patients into multiple groups so that the average tumor size remained constant. Different CA102 dosages were administered directly into the tumors for each group, and the tumor volume and weight were observed. The results showed that the tumor volume and weight in the CA102-treated group were significantly reduced compared to the CA10G-treated group, and the anticancer effect increased further with increasing dosage. Figure 39 ).

[0263] 10-2. Confirmation of the anticancer effect of CA103

[0264] After subcutaneous transplantation of prostate cancer cell line (22Rv-1) into balb c nu / nu mice to establish a prostate cancer mouse model, the recombinant adenoviruses CA10G and CA103 prepared in Example 7 were directly administered into the tumor (2×10 8 pfu / spot, 3 times) to observe its growth. The results showed that the volume and weight of the tumor in the CA103-treated group were significantly reduced compared with the untreated control group (buffer-treated group) and the vehicle control group (CA10G-treated group). Figure 40 ).

[0265] Example 11. Confirmation of the effect of combined treatment with an anticancer agent encoding adenovirus encoding dual-target short hairpin RNA (shRNA)

[0266] After constructing bladder cancer mouse models by subcutaneously transplanting bladder cancer cell lines (253J-BV) into balb c nu / nu mice, the recombinant adenoviruses CA10G and CA102 prepared in Example 7 were directly administered into the tumors, respectively, to observe tumor growth. The results showed that the volume and weight of the tumors in the CA102-treated group were significantly reduced compared to the untreated control group (buffer-treated group) and the vector control group (CA10G-treated group). When cisplatin was administered in combination as an anticancer agent, a synergistic increase in the anticancer effect was confirmed ( Figure 41 ).

Claims

1. An expression cassette, characterized in that, the expression cassette contains a nucleic acid that converts U to T in the base sequences of a first nucleic acid sequence SEQ ID NO:1 targeting the STAT3 gene and a second nucleic acid sequence SEQ ID NO:2 targeting the mTOR gene.

2. The expression cassette according to claim 1, characterized in that, the expression cassette may contain a base sequence targeting the first nucleic acid as a target sequence, a loop sequence capable of forming a hairpin structure, and a base sequence encoding in sequence a base sequence targeting the second nucleic acid as a target sequence.

3. The expression cassette according to claim 1 or 2, characterized in that, the expression of the expression cassette can be regulated by the U6 promoter.

4. An anti-tumor adenovirus, comprising: a human telomerase reverse transcriptase (hTERT) promoter; and an expression cassette containing a first nucleic acid sequence targeting the mTOR gene and a second nucleic acid sequence targeting the STAT3 gene, characterized in that, when expressed in a cell or tissue, the first nucleic acid sequence and the second nucleic acid sequence are partially double-stranded to form siRNA or shRNA targeting mTOR mRNA and STAT3 mRNA, the expression cassette expresses the nucleic acid sequences shown in SEQ ID NO:1 and SEQ ID NO:2, wherein the human telomerase reverse transcriptase promoter is operably linked to E1A and E1B of the endogenous gene of the adenovirus.

5. The anti-tumor adenovirus according to claim 4, characterized in that, the expression cassette is located at the C3 site of the endogenous gene of the adenovirus.

6. The anti-tumor adenovirus according to claim 4, characterized in that, an internal ribosome entry site sequence (IRES) is further included between E1A and E1B.

7. The anti-tumor adenovirus according to claim 4, characterized in that, the expression cassette encodes short hairpin ribonucleic acid (shRNA).

8. The anti-tumor adenovirus according to claim 7, characterized in that, the short hairpin ribonucleic acid (shRNA) simultaneously inhibits mTOR and STAT3.

9. The anti-tumor adenovirus according to claim 4, characterized in that, the first nucleic acid contains a base sequence having more than 60% complementarity with the reverse complementary sequence of the second nucleic acid.

10. The anti-tumor adenovirus according to claim 4, characterized in that, the second nucleic acid contains a base sequence having more than 60% complementarity with the reverse complementary sequence of the first nucleic acid.

11. The anti-tumor adenovirus according to claim 4, characterized in that, the expression cassette contains a first nucleic acid sequence, a loop sequence capable of forming a hairpin structure, and a second nucleic acid sequence.

12. The anti-tumor adenovirus according to claim 4, characterized in that, the expression of the expression cassette is regulated by the U6 promoter.

13. The anti-tumor adenovirus according to claim 4, characterized in that, the adenovirus is a group C adenovirus.

14. The anti-tumor adenovirus according to claim 4, characterized in that, the serotype is type 5.

15. The anti-tumor adenovirus according to claim 4, characterized in that, the oncolytic ability of the anti-tumor adenovirus is higher than that of the wild-type adenovirus.

16. The oncolytic adenovirus according to claim 4, wherein, the oncolytic ability of the oncolytic adenovirus is higher than that of the adenovirus obtained by introducing the human telomerase reverse transcriptase (hTERT) promoter into wild-type adenovirus.

17. A cancer therapeutic composition comprising the expression cassette according to any one of claims 1-3 or the oncolytic adenovirus according to claim 4.

18. The cancer therapeutic composition according to claim 17, wherein, it further comprises an anti-cancer agent.

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