WO2005035744A1 - Tumeur ciblant un virus a deux genes, procedes d'hybridation et utilisation - Google Patents

Tumeur ciblant un virus a deux genes, procedes d'hybridation et utilisation Download PDF

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WO2005035744A1
WO2005035744A1 PCT/CN2004/001173 CN2004001173W WO2005035744A1 WO 2005035744 A1 WO2005035744 A1 WO 2005035744A1 CN 2004001173 W CN2004001173 W CN 2004001173W WO 2005035744 A1 WO2005035744 A1 WO 2005035744A1
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tumor
gene
virus
genes
trail
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French (fr)
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Xinyuan Liu
Zifei Pei
Weiguo Zou
Liang Chu
Songpo Qiu
Zilai Zhang
Binghua Li
Lin Feng
Lanying Sun
Jinfa Gu
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Shanghai Institutes For Biological Sciences, Chinese Academy Of Sciences
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the invention belongs to the field of gene therapy, and in particular, relates to a tumor-targeting double gene-virus, a construction method and application thereof. Background technique
  • Gene therapy is a biological high-tech program that has emerged in the past ten years. In the entire gene therapy program, tumor gene therapy programs accounted for more than 60%. Gene therapy was once considered the hope of humans to eventually conquer tumors.
  • Currently used as gene therapy vectors are divided into viral and non-viral types.
  • Viral vectors include: adenovirus, adeno-associated virus, retrovirus, lentivirus, and herpes virus. The viral vector has a high transfection rate and a long expression time, but is highly immunogenic and has certain risks.
  • Non-viral vectors include: naked DNA, liposomes, and other material-encapsulated DNA. Non-viral vectors are less immunogenic and safe, but have a low transfection rate, poor gene stability, and short expression time.
  • Virus vectors are currently the most used. There are two types of virus vectors: one can be integrated into chromosomes: such as retroviruses, adeno-associated viruses, and lentiviruses. The other type cannot be integrated into chromosomes, such as adenovirus, herpes simplex virus, and EB virus.
  • Adenovirus (Adv) vectors are most commonly used in viral vectors. There are six major categories, including eight, B, C, D, E, F, and 49 serotypes, of which type C serotypes 2 in 5 are used the most.
  • the full length of the adenovirus genome is 36Kb, which is divided into early genes (E) and late genes (L).
  • the early gene E1 region (about 4Kb) is deleted (sometimes a partial deletion of 3.6Kb in the E3 region is also added), it is called The first generation of gene therapy vectors, which are commonly used vectors.
  • the second-generation Adv also lacks the E4 and E2A regions to reduce its immunogenicity, but it is rarely used now.
  • the third-generation enterovirus-free Gutless Adv (GL-Adv) vector removes all the genes of Adv, and only retains the reverse terminal repeat (ITR) and assembly genes at both ends. It has no antigenicity and will not Removed by antibodies, so it has long-term efficacy.
  • Adeno-associated virus is a single-stranded DNA virus with minimal immunogenicity. It can be inserted on the chromosome in a targeted manner. No toxic effects and carcinogenicity have been found in current use. Retroviruses (RTV), lentivirus, herpes simplex virus (HSV), and Epstein-Barr virus are also more commonly used viral vectors.
  • a gene therapy vector is a gene that sends a gene to a destination for a therapeutic (eg, anti-cancer) effect.
  • Gene therapy vectors are the key, and another element is genes.
  • the tumor therapy genes can be tumor suppressor genes and cytokine genes.
  • Gene-virus therapy The treatment with it is called gene-virus therapy (Gene-ViroTherapy gene-virus therapy strategy has been obtained Good results, related content has been patented. When applying this gene-virus strategy, only one gene is used, but it has been proven that one gene is not enough to completely destroy the tumor. Purpose of the invention
  • the purpose of the present invention is to combine the advantages of tumor gene therapy and virus therapy, so as to provide a class of recombinant double genes-viruses that have tumor targeting and can effectively express two anti-cancer genes.
  • Another object of the present invention is to provide a method for constructing a tumor-targeting double gene-virus.
  • Another object of the present invention is to provide a tumor-targeting dual gene-virus for treating tumors.
  • the present invention provides an anti-cancer targeting dual gene-virus, which carries two tumor treatment genes, and the two tumor treatment genes are functionally complementary or have a synergistic effect.
  • the invention also provides a method for constructing an anti-cancer targeted double gene-virus, the steps of which include: (1) cloning two tumor treatment genes into a multicloning site of a plasmid, and the two genes are connected by a linker (ribosome entry) Site (IRES) or cell endogenous restriction site sequence (IETD)); then use restriction enzymes to cut out a two-gene expression box containing the CMV promoter, anti-cancer gene, SV40 poly A The tail is inserted into the modified tumor-specific proliferation virus vector; (2) the transformed virus vector carrying the tumor-targeting double gene is transfected into the cell to generate the double gene-virus.
  • a linker ribosome entry) Site
  • IETD cell endogenous restriction site sequence
  • the anti-cancer targeted dual gene-virus of the present invention can also be constructed by the following methods: (1) cloning two tumor treatment genes into a multicloning site of a plasmid, respectively, and cutting out a gene expression frame with a restriction enzyme, Inserted into different sites of the modified tumor-specific proliferation virus vector; (2) Cotransfect the two plasmids into the cells to generate a double gene-virus containing two foreign genes by homologous recombination.
  • the tumor treatment genes include, but are not limited to, the TRAIL gene, tumor suppressor gene, cytokine gene, apoptosis-promoting gene, vascular inhibitory gene, suicide gene and other genes in the tumor necrosis factor superfamily.
  • tumor necrosis factor gene a member of the tumor necrosis factor superfamily TRAIL, after binding to cell surface receptors, initiates the apoptotic pathway and selectively promotes tumor cell apoptosis;
  • Tumor suppressor genes include p53, PTEN, Rb, NF1, VHL, APC. Tumor suppressor genes can inhibit the growth of tumor cells;
  • cytokine genes interleukin-2, -12, -24, granulocyte-single colony stimulating factor, interferon- ⁇ , - ⁇ >- ⁇ ; cytokines have the ability to kill tumor cells, activate immune cells, and increase hematopoiesis Function, etc.
  • Apoptosis-promoting genes TRAIL, Bax, Caspase, and Smac, etc .; Apoptosis is an important pathway for multicellular biological life activities, and abnormal apoptotic pathways are an important mechanism for tumorigenesis in the body; inhibiting cell death Death, tumors must occur; Pro-apoptotic genes can accelerate tumor cell apoptosis and are effective genes for gene therapy of tumors;
  • Angiostatin genes angiostatin, angiostatin, k5, sflt-1, and endostatin; angiostatin genes can interfere with the formation of new blood vessels, can block the nutritional supply of tumor cells, tumors Atrophy and death due to insufficient nutrition;
  • Suicide genes including E. coli cytosine deaminase gene (cd), herpes simplex virus deoxythymidine kinase gene (HSV-tk) ;
  • the vascular endothelial growth factor soluble receptor sflt-1 gene can competitively inhibit the function of vascular endothelial growth factor.
  • Tumor tissue-specific promoters include, but are not limited to: telomerase reverse transcriptase catalytic subunit (hTERT) promoter, alpha-fetoprotein (ATP) promoter, cancer embryonic antigen (CEA) promoter, prostate specific antigen Promoters and breast cancer tissue-specific promoters.
  • hTERT telomerase reverse transcriptase catalytic subunit
  • ATP alpha-fetoprotein
  • CEA cancer embryonic antigen
  • prostate specific antigen Promoters and breast cancer tissue-specific promoters.
  • the use of tumor tissue-specific promoters allows the expression of anti-oncogenes or virus replication to be performed specifically in tumor cells rather than in normal cells.
  • Tumor-specific proliferation virus vectors include, but are not limited to, tumor-specific proliferation adenoviruses (including the adenovirus hTERT-Adv and ZD55 regulated by tumor-specific promoters), AAV, or GL-Adv.
  • the method for constructing a double gene-virus of the present invention can be used to develop new anti-cancer drugs that can effectively treat tumors, and can also be used to form a pharmaceutical composition with other compounds.
  • the compounds can be: chemotherapeutic drugs; biotoxins; immunosuppressive compounds , Monoclonal antibodies, etc.
  • the present invention provides a recombinant virus carrying two anti-cancer genes. Cell experiments have shown that the anti-cancer genes can be specifically expressed in tumor cells, but not in normal cells. It has been proved by animal tests that it can be used to treat a variety of tumors; 2.
  • the present invention provides a method for constructing a virus carrying two anti-cancer genes, which is easy to master;
  • the viral vector constructed by the present invention can be very conveniently loaded with two exogenous anti-cancer genes; using this vector can construct a variety of dual-gene-viruses carrying anti-cancer genes, providing a good basis for tumor gene-virus therapy. basis;
  • the gene-virus constructed by the present invention is a tumor-specific virus that can selectively replicate, proliferate and express the double genes carried in tumor cells, so the double-gene virus has a high targeting resistance. Cancer effect
  • the multi-gene virus constructed by the present invention has been proved by animal tests to selectively kill tumor cells without affecting normal cells; the gene-virus-expressing anti-oncogene can enhance the anti-tumor effect of the virus; Gene-virus targeted therapy can basically eliminate tumors, laying a good foundation for future human tumor treatment.
  • FIG. 1 is a schematic diagram of the construction of the dual gene-virus ZD55-TRAIL-IETD-Smac of the present invention.
  • Figure 2 shows the construction of plasmid pZhTERT.
  • Fig. 3 is a schematic diagram showing the replication ability of the double-gene virus ZD55-TRAIL-IETD-Smac in normal cells or tumor cells. .
  • Figure 4 is a schematic diagram of the replication ability of the double-gene virus Ad-hTERT-TRAIL-K5 in normal cells or tumor cells.
  • Figures 5A and 5B are schematic diagrams showing the survival rates of tumor cells (5A) and normal cells (5B) measured by the MTT method after 3 days of virus treatment with different titers (MOI). ⁇
  • Figures 6A and 6B are schematic diagrams showing the survival rates of tumor cells (6A) and normal cells (6B) measured by the MTT method after 3 days of virus treatment with different titers (MOI).
  • Fig. 7 is a schematic diagram showing the results of the treatment of the tumor cell transplantation tumor in nude mice by the double gene virus ZD55-TRAIL-IETD-Smac. detailed description
  • TRAIL protein can mediate tumor cell apoptosis and specifically kill tumor cells (Griffith et al. 2001, Mol. Then 4: 257-266). However, some tumor cells have relatively high resistance to TRAIL protein-mediated apoptosis.
  • the Smac protein can bind to the IAP protein, thereby releasing the inhibition of the IAP protein (Du C. et al. 2000, Cell 102: 33-42.).
  • the use of oncolytic virus-mediated Smac and TRAIL genes can exert their synergistic effects and greatly improve the cure rate of tumors.
  • Xba l A primers 5, GCC GAC ATC ACC TGT G TCT AGA GAA TG 3;
  • Xba l B primers 5, TCA GAT GGG TTT CTT CAC TCC ATT TAT CCT 3 ';
  • Bglll ⁇ primer 5, ATA AAG GAT AAA TGG AGT GAA GAA ACC CAT CTG AG 3 '; (The third codon of the 55KDa gene is mutated to a stop codon, C2024T)
  • BglllB primers 5 GA AGA TCT ATA CAG TTA AGC CAC CTA TAC AAC A 3; (ElB 55Kda gene reading frame mutation, C2252T, G2261T added two stop codons)
  • pXCl plasmid was purchased from Microbix Biosystem Inc, Toronto, Canada.
  • the pXCl plasmid contains the human adenovirus type 5 (Ad5) gene sequence from 22bp to 5790bp (0-16.1mu).
  • the primers Xba IA and Xba IB were used for the first PCR reaction (see Molecular Cloning: A Laboratory Manual, 3 rd ed., Joseph Sambrook and David W. Russell for details), and the electrophoretic recovery was 719 bp Fragment to obtain the first PCR product Zl.
  • Primer Bglll A and primer BglllB were subjected to a second PCR reaction (see Molecular Cloning: A Laboratory Manual, 3 rd ed., Joseph Sambrook and David W. Russell for details), and electrophoresis was performed to recover a 270 bp fragment to obtain the second PCR product Z2 .
  • the product of the two PCR reactions had a 34 bp paired sequence.
  • the two PCR products were mixed as a template, and the third PCR reaction was performed with primer Xba IA and primer Bgl II B (see Molecular Cloning: A Laboratory Manual, 3 rd ed., Joseph Sambrook and David W. Russell), and run the electrophoresis The 955 bp fragment was recovered to obtain the third PCR product Z3.
  • the third PCR product Z3 was digested with Xba I + Bgl II and cloned into the pXCl plasmid digested with Xba I + Bgl II.
  • the new plasmid was named pXCl-D55.
  • the pCA13 plasmid (purchased from Microbix Biosystem Inc, Toronto, Canada) contains a SV40 poly A tailing signal.
  • the PCA13 vector was digested with BamH I + Bgl ll, and a 160 bp fragment was recovered by electrophoresis. That is, the SV40 polyA tail was recovered.
  • the digested pXCl-D55 was identified by enzyme digestion, and the forward cloned plasmid was named pZD55.
  • pZD55 contains deletion mutations from 2268bp to 3328bp, deleting the E1B 55KDa gene.
  • Smac2 5 'AAACTCGAGTCACTTGTCATCGTCGTCCTTGTAATCCTC 3;
  • TRAIL1 5, ACGCGTCGACATGGCTATGATGGAGGTC 3;
  • TRAIL2 5 'CCCAAGCTTGCCAACTAAAAAGGCCCC 3';
  • the pCA13 vector contains multiple cloning sites between the CMV promoter and the tailing signal. Sai l, Hind lll, EcoR I, EcoR V, Xba l, Xho l, and BamH L forward the tumor treatment genes by genetic manipulation. Inserted into the multiple cloning site of pCA13 to construct the plasmid pCA13-gene (for specific procedures, see Molecular Cloning: A Laboratory Manual, 3 rd ed., Joseph Sambrook and David W. Russell). The plasmid pCA13-gene was digested with Bgl II to cut out the gene expression box. This expression cassette contains the CMV promoter, therapeutic gene, and SV40 polyA. Tail. This expression frame was then cloned into the pZD55 plasmid, which was digested with Bgl II and digested with phosphorylation, to construct the plasmid pZD55-gene.
  • the pCDNA3-Smac plasmid (purchased from Wuhan Sanying Biotechnology Co., Wuhan) was used as a template for the PCR reaction.
  • Primers Smacl and Smac2 were used for the first PCR reaction (see Molecular Cloning: A Laboratory Manual, 3 rd ed., Joseph Sambrook for details). and David W. Russell), electrophoresis was performed to recover a 736bp fragment.
  • the PCR product was digested with Hind III + Xho I, cloned into the pCA13 plasmid digested with Hind III + Xho I, and named as pCA13-Smac plasmid.
  • the pCDNA3-TRAIL plasmid (purchased from Wuhan Sanying Biotechnology Co., Wuhan) was used as a template for the PCR reaction. Primers TRAIL1 and TRAIL2 were used for the second PCR reaction (see Molecular Cloning: A Laboratory Manual, 3 rd ed., Joseph Sambrook for details). and David W. Russell), running electrophoresis to recover 866bp fragments.
  • the PCR product was digested with Sal I + Hind III, cloned into the pCA13-Smac plasmid digested with Sal I + Hind III, and named pCA13-TRAIL-IETD-Smac plasmid.
  • the plasmid pCA13-TRAIL-IETD-Smac was digested with Bgl II to cut out the gene expression box.
  • This expression box contains the CMV promoter, the therapeutic genes TRAIL and Smac, and the tail of SV40 polyA.
  • This expression frame was then cloned into the pZD55 plasmid that was digested with Bgl II and digested with phosphorylation to construct the plasmid pZD55-TRAIL-IETD-SmaCo.
  • the plasmids constructed by similar methods are pZD55-TRAIL-IETD-k5, pZD55-TRAIL-IETD-IL-24, pZD55-TRAIL-IETD-IL-12, pZD55-TRAIL-IETD-Omi, pZD55-TRAIL-IETD- Eorf4 and so on.
  • Plasmids pBHG-E3 and 293 cells were purchased from Microbix Biosystem Inc. (Toronto), Canada.
  • the plasmid pBHG-E3 contains the Ad5 gene series but lacks the El region 188bp-1339bp series.
  • 293 cells (Microbix Biosystem Inc. (Toronto), Canada) are transformed from human embryonic kidney cells by shearing type 5 adenovirus DNA. It contains the E1 region that expresses adenovirus type 5, which is highly transfected with adenovirus DNA.
  • Co-transfect pZD55-TRAIL-IETD-Smac (which contains the left arm sequence homologously recombined with adenovirus) and plasmid pBHG-E3 containing adenovirus backbone DNA into a 293 cell line to generate infectious
  • Two foreign genes of the recombinant adenovirus ZD55-TRAIL-IETD-Smac (see Figure 1). For details, refer to the operating instructions of Qiagen. Viral plaques appeared on 7-14 days. After two plaques were purified and amplified, the recombinant adenovirus DNA was extracted, analyzed by DNA digestion, and analyzed by PCR to determine the correct recombinant adenovirus strain.
  • Viral plaque purification, expansion, identification 293 cells were plated in 6-well plates, and cells were nearly full after 24 hours. After adding virus containing different dilutions, 2 ml of low-melting gel (10% FBS, 1.25% Agarose) Empty spots are visible in about 9 days. Pick a single plaque and add a small amount of amplified virus to a 24-well plate with approximately 293 cells. Viral DNA was obtained using the Qiagen Blood Kit, and the gene virus Ad5-ZD55-gene was identified using PCR technology. The primers used for the identification were synthesized by Shanghai Shengong. (Note: The sequence of the primer paired with the pXCl plasmid is indicated on the right side of the sequence).
  • the viral DNA extracted from the Qiagen Blood Kit was used as a template, and wild-type viral DNA was used as a control.
  • the Zd55 sense primer and Zd55 antisense primer were used for PCR reaction. PCR conditions: 9 ° C X lmin, 55 ° C X lmin, 72 ° C X2minl5so If the PCR product contains only gene and does not contain 1113bp wild-type adenovirus DNA, the plaques are successfully purified. Repeat this process once to get the correct recombinant adenovirus.
  • Adenovirus ZD55-TRAIL-IETD-Smac multiplied in 293 cells The virus was purified by cesium chloride gradient centrifugation. For details, see the operating instructions of Microbix Biosystem Inc.
  • Recombinant adenoviruses constructed by similar methods include ZD55-TRAIL-IETD-k5, ZD55-TRAIL-IETD-IL-24, ZD55-TRAIL-IETD-IL- 12, ZD55-TRAIL-IETD-Omi, ZD55-TRAIL- IETD-Eorf4 and so on.
  • the pCDNA3-IL-24 plasmid (purchased from Wuhan Sanying Biotechnology Co., Wuhan) was used as the template for the PCR reaction, and the IL-24 upstream primer and IL-24 downstream primer were used for the first PCR reaction (see Molecular Cloning: A Laboratory for details). Manual, 3 rd ed., Joseph Sambrook and David W. Russell), running electrophoresis to recover 628bp fragment.
  • the PCR product was digested with Sall + Xbal, cloned into the pCA13 plasmid digested by Sall + Xbal, and named as pCA13-IL-24 plasmid.
  • a pIRESPROU plasmid (purchased from Microbix Biosystem Inc, Toronto, Canada) was used as the template for the PCR reaction.
  • the IRES upstream primer and IRES downstream primer were used for the second PCR reaction (see Molecular Cloning: A Laboratory Manual, 3 rd ed., Joseph Sambrook and David W. Russell), electrophoresis was run to recover the 585bp fragment.
  • the Xbal + Xhol-digested PCR product was cloned into the Xbal + Xhol-digested pCAl 3 -IL-24 plasmid and named the pCA13-IL-24-IRES plasmid.
  • the TRAIL upstream primer and the TRAIL downstream primer were subjected to a third PCR reaction (see Molecular Cloning: A Laboratory Manual, 3 rd ed., Joseph Sambrook and David W. Russell), and run electrophoresis The 866bp fragment was recovered.
  • the PCR product was digested with XhoI + BamHI, cloned into the pCA13-IL-24-IRES plasmid digested with XhoI + BamHI, and named as pCA13-IL-24-IRES-TRAIL plasmid.
  • the plasmid pCA13-IL-24-IRES-TRAIL was digested with Bgl II to cut out the gene expression frame.
  • This expression box contains the CMV promoter, the therapeutic genes IL-24 and TRAIL, and the tail of SV40 polyA.
  • This expression box was then cloned into the pZD55 plasmid, which was digested with Bgl II and digested with phosphorylation, to construct the plasmid pZD55-IL-24-IRES-TRAIL.
  • Plasmids constructed in a similar manner also include pZD55-TRAIL-IRES-Smac, pZD55-Smac-IRES-TRAIL, pZD55-k5-IRES-TRAIL, pZD55-TRAIL-IRES-k5, pZD55-sfltl-IRES-k5, pZD55- k5-IRES-sfltl, pZD55-TRAIL-IRES-IL-24, pZD55-IL-24-IRES-TRAIL, pZD55-TRAIL-IRES-IL-12, pZD55-IL-12-IRES-TRAIL, pZD55-TRAIL- IRES-Omi, pZD55-Omi-IRES-TRAIL, pZD55-IL-IRES-12-IL24, pZD55-IL24-IRES-IL-12, pZD55-TRAIL-IRES-Eorf4, pZD55-Eorf
  • Co-transfect pZD55-IL-24-IRES-TRAIL (which contains the left arm sequence homologously recombined with adenovirus) and plasmid pBHG-E3 containing adenovirus backbone DNA into a 293 cell line.
  • Recombinant adenovirus ZD55-IL-24-IRES-TRAIL containing two foreign genes.
  • Viral plaques appeared on 7-14 days. After the virus plaques were purified twice, amplification was performed, the DNA of the recombinant adenovirus was extracted, DNA digestion analysis, and PCR analysis were performed to determine the correct recombinant adenovirus strain. The specific steps are described in Example 1.
  • Recombinant adenoviruses constructed by similar methods include ZD55-TRAIL-IRES-Smac, ZD55-Smac-IRES-TRAIL, ZD55-k5-IRES-TRAIL, ZD55-TRAIL-IRES-k5, ZD55-sfltl-IRES-k5, ZD55-k5-IRES-sfltl, ZD55-TRAIL-IRES-IL-24, ZD55-IL-24-IRES-TRAIL, ZD55-TRAIL-IRES-IL-12, ZD55-IL-12-IRES-TRAIL, ZD55- TRAIL-IRES-Omi, ZD55-Omi-IRES-TRAIL, ZD55-IL-IRES-12-IL24, ZD55-IL24-IRES-IL-12, ZD55-TRAIL-IRES-Eorf4, ZD55-Eorf4-IRES-TRAIL, etc. .
  • TRAIL protein can mediate tumor cell apoptosis and specifically kill tumor cells.
  • tumor growth needs to supply nutrients through abundant blood vessels.
  • K5 is the fifth domain of plasminogen kringle, which can specifically inhibit endothelial cell division by regulating the endogenous angiogenic factor pathway. Proliferation, which has a higher antiangiogenic effect and a significant antitumor effect than angiostatin angiostatin (Chun-xia Luo et al. China J. Cancer Biother, 2003, 10 (1): 9-12,).
  • TRAIL and K5 proteins exert antitumor effects through different mechanisms. Oncolytic virus mediated TRAIL and K5 genes can play complementary roles.
  • pXCl was used as a template, and the site-directed mutagenesis double PCR technique was used to delete the promoter of the E1A gene, and replaced with three single-enzyme cleavage sites.
  • the primers were synthesized by Shanghai Biotech. (Note: The sequence of primer paired with pXCl plasmid is indicated on the right side of the sequence, and the endonuclease site is underlined.)
  • the pXCl plasmid was used as a template for the PCR reaction, and the first PCR reactions of the bowel I objects Bam5 'and Bam3' were performed (see Molecular Cloning: A Laboratory Manual, 3 rd ed., Joseph Sambrook and David W. Russell), and the electrophoresis was recovered. 394 bp fragment. '
  • the pXCl plasmid was used as the template for the PCR reaction.
  • Primer Xba5 'and primer-Xba3' were used for the second PCR reaction (see Molecular Cloning: A Laboratory Manual, 3 rd ed., Joseph Sambrook and David W. Russell for details). 830 bp fragment.
  • the product of the two PCR reactions has a 26bp paired sequence.
  • the PCR product was used as a template to mix the two PCR products.
  • Primer Bam5 and primer Xba3 were used for the third PCR reaction (for details, see Molecular Cloning: A Laboratory Manual, 3 rd ed., Joseph Sambrook and David W. Russell), running electrophoresis to recover the 1198 bp fragment.
  • a 1198 bp PCR product was digested with BamH I + Xba l and cloned into the pXCl plasmid digested with BamH I + Xba l and named pZXC2.
  • the hTERT promoter was pulled out by PCR to generate a suitable restriction site, and then cloned into the plasmid pZXC2 with the deletion of the E1A promoter, as follows:
  • the TRAIL gene was cloned into the pCA13 plasmid, and the gene expression box (including CMV promoter, TRAIL gene, Poly A signal) was cut out with Bgl II enzyme, and cloned into the pZhTERT plasmid cut by Xhol. Partial fill method is used in cloning.
  • the plasmid was named pZhTERT-TRAILo
  • pABS.4 and pBHGlO plasmids were purchased from Microbix.
  • the pBHGlO plasmid was obtained by deleting the 28133bp-30818bp series of the E3 region from the plasmid pBHG-E3, and there was a pad cloning site at the deletion site.
  • K5 gene was cloned into pCA13 plasmid (for specific steps, see Chun-xia Luo et al. Recombinant Kringle 5 of Human Plasminogen for Mammary Cancer Gene Therapy Mediated by Adenovirus. China J.
  • the plasmid pZhTERT-TRAIL (which contains the left arm sequence homologously recombined with the adenovirus) and the plasmid pBHG10-K5 containing the adenovirus backbone DNA were co-transfected into a 293 cell line, and homologous recombination was used to generate an infectious two exogenous Gene Recombinant Adenovirus Ad-hTERT-TRAIL-K5. Viral plaques appeared on 7-14 days. After two plaque purifications and amplification, the recombinant adenovirus DNA was extracted, DNA digestion analysis, and PCR analysis were performed to determine the correct recombinant adenovirus strain. The specific steps are described in Example 1. There are also Ad-hTERT-TRAIL-Smac,
  • Ad-hTERT-TRAIL-sfltl Ad-hTERT-TRAIL-IL-24, Ad-hTERT-TRAIL-IL- 12,
  • Ad-hTERT-TRAIL-Omi Ad-hTERT-TRAIL-Eorf4, Ad-hTERT-IL-24-Omi, Ad-hTERT-IL-24-IL-12, Ad-hTERT-IL-24--Eorf4, Ad -hTERT-IL-24-K5, etc.
  • Adeno-associated virus (AAV) Ad-hTERT-TRAIL-Omi, Ad-hTERT-TRAIL-Eorf4, Ad-hTERT-IL-24-Omi, Ad-hTERT-IL-24-IL-12, Ad-hTERT-IL-24--Eorf4, Ad -hTERT-IL-24-K5, etc.
  • AAV Adeno-associated virus
  • the tumor-specific promoter hTERT is used to replace the HCMV promoter in the TRAIL-IETD-Smac and other expression boxes, so that the expression of the two genes is limited to tumor tissues or cells, and other AAV-double genes-viruses are targeted.
  • the titer can reach 10 12 pfu / ml after packaging.
  • Example 5 Enterovirus-free adenovirus
  • the tumor-specific double-gene hTERT-TRAIL-IETD-Smac and other expression boxes or other hTERT-double-gene expression boxes were inserted into the entero adenovirus vector and packaged as entero adenovirus.
  • Other enteroviruses that target double genes and so on.
  • Viral replication can be regulated by tumor-specific promoters. After the two-gene TRAIL-IETD-Smac expression frame was inserted into the genome of the virus, the function of the two genes was limited to a specific tumor tissue. Example 7 Analysis of replication ability of the double-gene virus Ad-hTERT-TRAIL-K5 in normal cells or tumor cells
  • the normal cells or tumor cells were plated in 3 xlO 5 6-well plates, after 24 h, the added Ad.TERT 10 4 PFU, Ad-hTERT-TRAIL-K5 , wild-type adenovirus type 5 293 cells were infected with Ad5, liver Cell line BEL7404 (purchased from the Cell Bank of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences), colorectal cancer cell line SW620 (purchased from ATCC) and normal human embryo lung cell lines MRC5 and NHLF (purchased from ATCC). After 48 h, the cells were collected and the cells were repeatedly thawed at -20 ° C and 37 ° C 3 times to release the virus. The virus was diluted and the virus titer was measured.
  • MRC5 cells In MRC5 cells, it is reduced by nearly 1600 times compared with wild-type adenovirus 5 and in NHLF cells by 1200 times.
  • Wild-type adenovirus has strong replication ability in tumor cells and normal cells, and has no selectivity; Ad.TERT, Ad-hTERT-TRAIL-K5 can selectively replicate in tumor cells.
  • Example 8 Detection of Killing Ability of Double Gene Virus Ad-hTERT-TRAIL-K5 on Tumor Cells in Vitro The survival rate of cells after virus treatment was measured by the MTT method (Cancer Research, 1989, 49 (17): 4785-90) .
  • the viruses Ad.TERT, Ad-hTERT-TRAIL-K5 have obvious killing effect on tumor cells, but have little toxicity to normal cells and have tumor selectivity. . Wild-type adenoviruses have strong killing effects on tumor cells and normal cells and are not selective.
  • Example 9 bis viral genes ZD55-TRAIL-IETD-Smac ability to replicate in normal cells or tumor cells or normal cells will be analyzed 3 xlO 5 tumor cells were plated in 6-well plates.
  • Ad5 infect 293 cells, liver cancer cell line BEL7404, colorectal cancer cell line SW620, and normal human embryo lung cells, respectively.
  • the cell supernatant and cells were collected and repeatedly frozen and thawed 3 times at -20 ° C and 37 ° C to release the virus.
  • the virus was diluted and the virus titer was measured.
  • the survival rate of cells after virus treatment was detected by MTT method.
  • the steps are as follows: Hepatoma cell line BEL7404 and normal human embryo lung cells NHLF are plated into a 96-well plate at 5000 per well. After 24 hours of culture, viruses with different titers (MOI) are added, and the virus-containing cells are treated for 3 days. The culture solution was removed and replaced with a normal culture solution containing 5mg / ml MTT. After 4 hours of culture, the culture solution containing MTT was removed, lysed with lysate for 4 hours, and then the absorbance at 655nm was used as a reference to measure the absorbance at 595nm.
  • MOI virus with different titers
  • Cell survival rate (%) A595 (sample) / A595 (control) x 100%.
  • the results are shown in Figs. 6A and 6B.
  • the virus ZD55-TRAIL, the virus ZD55-Smac, and the double-gene virus ZD55-TRAIL-IETD-Smac has a clear killing effect on tumor cells, but has little toxicity to normal cells and has tumor selectivity. Wild-type adenoviruses have strong killing effects on tumor cells and normal cells and are not selective.
  • Example 11 Treatment of tumor cell transplantation tumors in nude mice with the double-gene virus ZD55-TRAIL-IETD-Smac
  • mice 4-5 week old nude mice were subcutaneously inoculated with the liver cancer cell line BEL7404, and animals were divided into groups 12 days later.
  • the treatment group was given 1 X 10 9 pfu of the gene virus ZD55-TRAIL, ZD55-Smac and the double gene virus ZD55-TRAIL-IETD-Smac for treatment.
  • the control group was divided into two groups: the first group was the phosphate buffered saline (PBS) treatment group In group 2, the same dose of ONYX-015 virus was used.
  • PBS phosphate buffered saline
  • the test results are shown in Figure 7. It can be seen that ZD55-TRAIL-IETD-Smac has the best effect. After 9 weeks of treatment, the tumor cell transplantation tumor has completely disappeared, and the treatment effect is much better than ONYX-015.

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Description

肿瘤靶向双基因-病毒、 其构建方法及应用 技术领域
本发明属于基因治疗领域， 具体地说， 是关于一种肿瘤靶向双基因-病毒、 其构建方 法及应用。 背景技术
基因治疗是近十多年兴起的一种生物高技术方案， 在整个基因治疗方案中， 肿瘤基因 治疗方案占 60%以上，基因治疗曾被认为是人类最终征服肿瘤的希望。 目前作为基因治疗 的载体分为病毒型和非病毒型两大类。病毒载体包括：腺病毒、腺相关病毒、逆转录病毒、 慢病毒、和疱疹病毒等。病毒载体转染率高，表达时间长， 但免疫原性强， 有一定危险性。 非病毒载体包括： 裸露 DNA、 脂质体和其它物质包奉的 DNA。 非病毒载体则免疫原性小 及安全性好， 但转染率低， 基因稳定性差， 表达时间短。 目前使用最多还是病毒载体。 病 毒载体又有两类： 一类可以整合到染色体： 如逆转录病毒、 腺相关病毒、 慢病毒。 另一类 则不能整合到染色体， 如腺病毒、 单纯疱疹病毒及 EB病毒。 在病毒载体中使用最多的还 是腺病毒 （Adv) 载体， 八^有八、 B、 C、 D、 E、 F等 6大类， 49个血清型， 其中 C类 血清型 2合 5用得最多， 腺病毒基因组全长 36Kb， 分早期基因（E)和晚期基因（L), 如 果将早期基因 E1区 （约 4Kb)缺失掉 （有时还加上 E3区 3.6Kb的部分缺失）， 则称为第 一代基因治疗载体， 这是常用的载体。 第二代 Adv除 E1区等缺失外， 还有 E4、 E2A区 缺失以降低其免疫原性， 但现在很少有人用。 第三代无肠腺病毒 Gutless Adv (简称为 GL-Adv)载体则是将 Adv的所有基因全部去除， 仅保留两端的反向末端重复顺序（ITR) 及装配基因， 它没有抗原性， 不会被抗体清除掉， 故有长期疗效。 腺相关病毒（AAV)是 一种免疫原性极小的单链 DNA病毒， 它可以定点插入到染色体上， 在目前使用中未发现 任何毒付作用和致癌性。 逆转录病毒（RTV)、 慢病毒、 单纯疱疹病毒 （HSV)、 EB病毒 也是较为常用的病毒载体。
基因治疗的载体是将基因送到目的处， 以发挥治疗（如抗癌）作用。基因治疗载体是 关键， 另一要素是基因， 有关肿瘤治疗基因可以是抑癌基因、 细胞因子基因等。
临床上肿瘤的基因治疗方案有几百种，但没有重大进展， 而肿瘤的病毒治疗却取得重 大突破。 没有 ONYX医药公司研制出的突变腺病毒（ONYX- 015， 也叫 dll520)， 乃将腺 病毒基因组的 Elb55K蛋白基因缺失， 使得该突变的腺病毒能在 p53缺失的肿瘤细胞内进 行大量复制， 而在正常细胞内不能复制。应用 ONYX-015, 联合常规的化学疗法治疗头颈 癌患者， 总有效率达 63% (Nature Medicine, 6:879, 2000 )0但单独使用 ONYX-015(不与化 疗结合)， 疗效只有 15-20%。 为了克服单独使用 ONYX-015疗效不理想的缺点， 刘新垣院 士于 2000年就提出了肿瘤的基因-病毒治疗策略： 肿瘤的基因治疗与病毒治疗相结合的策 略（中国肿瘤生物治疗杂志， 8(1):1, 2001 )。 即我们首先构建 ElB55Kda基因缺失的腺病 毒载体 ZD55, 它与 ONYX-015相似， 只将基因靶向导入 p53缺失的肿瘤细胞， 而不进入 正常组织细胞， 但 ZD55含有克隆位点， 可以插入外源基因， 故比 ONYX-015好， 我们将 不同抗癌杀伤基因装入 ZD55载体中构成 ZD55-gene，用它进行治疗称之为基因-病毒治疗 (Gene-ViroTherapy 基因-病毒治疗策略已经获得了很好效果， 相关内容已经申请了专 利。 应用此基因-病毒策略时， 只用一个基因， 但实践证明一个基因还不足以全部消灭肿 瘤。 发明目的
本发明的目的就在于将肿瘤的基因治疗与病毒治疗二者的优势结合起来，从而提供一 类具有肿瘤靶向， 同时又能有效表达两个抗癌基因的重组双基因-病毒。
本发明的另一个目的在于提供一种肿瘤靶向双基因 -病毒的构建方法。
本发明的另一个目的在于提供一种肿瘤靶向双基因-病毒用于治疗肿瘤的用途。 发明概要
为达到上述目的， 本发明提供了一种抗癌靶向双基因-病毒， 该双基因病毒携带有两 个肿瘤治疗基因， 且两个肿瘤治疗基因在功能上互补或具有协同效应。
本发明并提供了抗癌靶向双基因 -病毒的构建方法， 其步骤包括： （1 )将两个肿瘤治 疗基因克隆进质粒的多克隆位点， 两基因之间以连接物（核糖体进入位点（IRES)或细胞 内源性酶切位点序列 （IETD))连接； 然后用限制性内切酶切出双基因表达框， 该表达框 包含 CMV启动子、 抗癌基因、 SV40 poly A尾巴， 插入改造好的肿瘤特异性增殖病毒载 体中； （2)将改造好的携带肿瘤靶向双基因的病毒载体转染细胞以产生双基因-病毒。
本发明的抗癌靶向双基因 -病毒也可以通过以下方法构建： （1 ) 将两个肿瘤治疗基因 分别克隆进质粒的多克隆位点， 分别以限制性内切酶切出基因表达框， 插入改造好的肿瘤 特异性增殖病毒载体的不同位点中； （2)将两个质粒共转染细胞， 通过同源重组产生含有 两个外源基因的双基因-病毒。
其中， 所述肿瘤治疗基因包括但不限于： 肿瘤坏死因子超家族中的 TRAIL基因， 抑 癌基因， 细胞因子基因， 促细胞凋亡基因， 血管抑制基因， 自杀基因和其它基因。 其中， （1 )肿瘤坏死因子基因： 肿瘤坏死因子超家族中的一员 TRAIL, 与细胞表面受 体结合后， 启动细胞凋亡途径， 并选择性的促使肿瘤细胞凋亡；
(2)抑癌基因： 抑癌基因包括 p53、 PTEN、 Rb、 NF1、 VHL、 APC, 抑癌基因能够 抑制肿瘤细胞的生长；
(3 ) 细胞因子基因： 白细胞介素 -2、 -12、 -24， 粒 -单集落刺激因子， 干扰素 - α、 - β > - Υ ; 细胞因子具有杀伤肿瘤细胞， 激活免疫细胞， 增加造血功能等；
(4)促细胞凋亡基因： TRAIL、 Bax、 Caspase以及 Smac等； 细胞凋亡是多细胞生物 生命活动的重要途径， 细胞凋亡途径的异常是机体肿瘤发生的一个重要机制； 抑制了细胞 凋亡， 肿瘤势必要发生； 促细胞凋亡基因可加速肿瘤细胞的凋亡， 是基因治疗肿瘤的有效 基因；
(5) 血管抑制基因： 血管生成抑素基因 (angiostatin), k5、 sflt-l、 血管内皮抑素基因 (endostatin); 血管抑制基因能干涉新生血管形成， 可阻断肿瘤细胞的营养供应， 肿瘤因营 养不足而萎缩、 死亡；
(6) 自杀基因： 包括大肠杆菌胞嘧啶脱氨酶基因 (cd)， 单纯疱疹病毒的脱氧胸腺嘧啶 核苷激酶基因 (HSV-tk);
(7)其它基因： 血管内皮生长因子可溶性受体 sflt-1基因， 可竞争性抑制血管内皮生 长因子发挥作用。
肿瘤组织特异性启动子包括但不限于：端粒酶逆转录酶催化亚基基团 (hTERT)启动子， 甲胎蛋白 （ATP) 启动子， 癌胚抗原 (CEA)启动子， 前列腺特异性抗原启动子和乳腺癌组 织特异性启动子。肿瘤组织特异性启动子的使用， 使抗癌基因的表达或病毒的复制特异性 地在肿瘤细胞中进行而不在正常细胞内进行。
肿瘤特异性增殖病毒载体包括但不限于： 肿瘤特异性增殖腺病毒（包括肿瘤特异性启 动子调控的腺病毒 hTERT-Adv以及 ZD55)、 AAV、 或者 GL-Adv等。
本发明的构建双基因-病毒的方法， 可以用于开发有效治疗肿瘤的抗癌新药， 也可以 与其它化合物组成药用组合物， 所述化合物可以是： 化学治疗药物； 生物毒素； 免疫抑制 化合物， 单克隆抗体等。
本发明的有益效果：
1、 本发明提供携带两种抗癌基因的重组病毒， 经细胞实验证明， 抗癌基因可以特异 性地在肿瘤细胞中表达， 而不在正常细胞中表达。经动物试验证明， 可以用于治疗多种肿 瘤； 2、 本发明提供了携带两种抗癌基因的病毒构建方法， 该方法易于掌握；
3、 本发明构建的病毒载体， 能够非常方便的装入两种外源抗癌基因； 使用这个载体 能够构建多种携带抗癌基因的双基因-病毒， 为肿瘤的基因-病毒治疗提供良好的基础；
4、本发明构建的基因-病毒，是一种肿瘤特异性病毒， 能选择性地在肿瘤细胞内复制、 增殖和表达所携带的双基因， 故该双基因 -病毒具有很高的靶向抗癌作用；
5、 本发明构建的多种基因病毒经动物试验证明能选择性的杀死瘤细胞， 而不影响正 常细胞； 基因-病毒表达的抗癌基因能加强病毒的抗肿瘤效果； 这种新型的双基因-病毒靶 向治疗， 能基本上全部消除肿瘤， 为今后用于人类肿瘤治疗打下了良好基础。 附图说明
图 1为本发明的双基因 -病毒 ZD55-TRAIL-IETD-Smac的构建示意图。
图 2为质粒 pZhTERT的构建示意图。
图 3为双基因病毒 ZD55-TRAIL-IETD-Smac在正常细胞或肿瘤细胞中复制能力的示 '意图。 .
图 4为双基因病毒 Ad-hTERT-TRAIL-K5在正常细胞或肿瘤细胞中复制能力的示意 图。
图 5A、 5B为 MTT法检测肿瘤细胞（5A)和正常细胞（5B)经不同滴度（MOI) 的 病毒处理后 3天的存活率示意图。 ·
图 6A、 6B为 MTT法检测肿瘤细胞（6A)和正常细胞（6B)经不同滴度 （MOI) 的 病毒处理后 3天的存活率示意图。
图 7为双基因病毒 ZD55-TRAIL-IETD-Smac在裸鼠体内治疗肿瘤细胞移植瘤的结果 示意图。 具体实施方式
以下结合具体实施例对本发明作进一步说明， 应理解， 以下实施例仅用于说明本发明 而不用于限定本发明的范围。
实施例 1、 双基因 -病毒 ZD55-TRAIL-IETD-Smac的构建
TRAIL蛋白可介导肿瘤细胞凋亡， 特异性地杀伤肿瘤细胞 （Griffith et al. 2001, Mol. Then 4:257-266 但部分肿瘤细胞对 TRAIL蛋白介导的细胞凋亡存在相当高的抗性， 相 关研究表明， IAP蛋白的髙表达是引起诸多肿瘤细胞对 TRAIL蛋白不敏感的原因之一。 而 Smac蛋白可与 IAP蛋白结合， 从而解除 IAP蛋白的抑制性 （Du C. et al. 2000, Cell 102:33-42.)。采用融瘤病毒介导 Smac以及 TRAIL两种基因， 可发挥其协同作用， 大大提 高对肿瘤的治愈率。
A、 肿瘤靶向载体质粒 pZD55的构建：
先设计如下引物：
Xba l A 引物： 5， GCC GAC ATC ACC TGT G TCT AGA GAA TG 3，；
Xba l B 引物： 5，TCA GAT GGG TTT CTT CAC TCC ATT TAT CCT 3';
Bglll Λ引物： 5， ATA AAG GAT AAA TGG AGT GAA GAA ACC CAT CTG AG 3'； (55KDa基因第三个密码子突变成终止密码子， C2024T)
BglllB 引物： 5，GA AGA TCT ATA CAG TTA AGC CAC CTA TAC AAC A 3，； (ElB 55Kda基因读码框突变， C2252T, G2261T加入了两个终止密码子）
pXCl质粒购自加拿大 Microbix Biosystem Inc, Toronto。 pXCl质粒含有人腺病毒 5型 (Ad5) 从 22bp— 5790bp (0-16.1mu)的基因序列。
以 pXCl质粒为 PCR反应的模板， 引物 Xba I A与 Xba I B进行第一次 PCR反应（详 见 Molecular Cloning: A Laboratory Manual, 3rd ed., Joseph Sambrook and David W. Russell), 跑电泳回收 719 bp片段， 得到第一次 PCR产物 Zl。 引物 Bglll A与引物 BglllB进行第 二次 PCR反应（详见 Molecular Cloning: A Laboratory Manual, 3rd ed., Joseph Sambrook and David W. Russell), 跑电泳回收 270 bp片段， 得到第二次 PCR产物 Z2。 两次 PCR反应的 产物有 34bp的配对序列。
以两次 PCR产物混合作为模板， 以引物 Xba I A与引物 Bgl II B进行第三次 PCR反 应 (详见 Molecular Cloning: A Laboratory Manual, 3rd ed., Joseph Sambrook and David W. Russell), 跑电泳回收 955 bp片段， 得到第三次 PCR产物 Z3。
以 Xba I + Bgl II酶切第三次 PCR产物 Z3， 克隆进 Xba I + Bgl II酶切过的 pXCl质 粒， 得到的新质粒命名 pXCl-D55。
pCA13质粒 （购自加拿大 Microbix Biosystem Inc, Toronto)含有 SV40 poly A加尾信 号， 以 BamH I+Bgl ll酶切 PCA13载体， 跑电泳回收 160bp片段， 即回收 SV40 polyA尾 巴， 将此片段克隆进 Bgl II酶切过的 pXCl-D55， 酶切鉴定， 将正向克隆的质粒命名为 pZD55。 pZD55含有从 2268bp-3328bp的缺失突变， 缺失 E1B 55KDa基因。
B、 质粒 pZD55-TRAIL-IETD-Smac的构建
先设计如下引物： Smacl : 5'CCCAAGCTTATTGAGACAGACGCGGTTCCTATTGCACAGAAA 3'；
Hindlll
Smac2： 5 ' AAACTCGAGTCACTTGTCATCGTCGTCCTTGTAATCCTC 3，；
Xho l
TRAIL1： 5， ACGCGTCGACATGGCTATGATGGAGGTC 3，；
Sai l
TRAIL2: 5' CCCAAGCTTGCCAACTAAAAAGGCCCC 3'；
Hindlll
pCA13载体在 CMV启动子与加尾信号之间含有多克隆位点， Sai l 、 Hind lll 、 EcoR I、 EcoR V 、 Xba l 、 Xho l 、 BamH L 将肿瘤治疗基因， 通过基因操作的方法顺向插 入到 pCA13的多克隆位点，构建成质粒 pCA13-gene(具体操作步骤详见 Molecular Cloning: A Laboratory Manual, 3rd ed., Joseph Sambrook and David W. Russell )。 用 Bgl II酶切质粒 pCA13-gene，切出该 gene的表达框。这个表达框包含 CMV启动子、治疗基因及 SV40 polyA. 尾巴。而后将此表达框克隆进 Bgl II单酶切并去磷的 pZD55质粒，构建成质粒 pZD55-gene。
质粒 pZD55-TRAIL-IETD-Smac的具体构建步骤如下：
以 pCDNA3-Smac质粒（购自武汉三鹰生物技术公司， 武汉） 为 PCR反应的模板， 引物 Smacl与 Smac2进行第一次 PCR反应（详见 Molecular Cloning: A Laboratory Manual, 3rd ed., Joseph Sambrook and David W. Russell), 跑电泳回收 736bp片段。 以 Hind III + Xho I酶切 PCR产物， 克隆进 Hind III + Xho I酶切过的 pCA13质粒， 命名为 pCA13-Smac质 粒。
以 pCDNA3-TRAIL质粒（购自武汉三鹰生物技术公司， 武汉） 为 PCR反应的模板， 引物 TRAIL1与 TRAIL2进行第二次 PCR反应 （详见 Molecular Cloning: A Laboratory Manual, 3rd ed., Joseph Sambrook and David W. Russell), 跑电泳回收 866bp片段。 以 Sal I + Hind III酶切 PCR产物， 克隆进 Sal I + Hind III酶切过的 pCA13-Smac质粒， 命名为 pCA13-TRAIL-IETD-Smac质粒 。 用 Bgl II酶切质粒 pCA13-TRAIL-IETD-Smac质粒， 切 出 gene的表达框。这个表达框包含 CMV启动子、治疗基因 TRAIL和 Smac及 SV40 polyA 尾巴。 而后将此表达框克隆进 Bgl II 单酶切并去磷的 pZD55 质粒， 构建成质粒 pZD55-TRAIL-IETD-SmaCo
用类似方法构建的质粒还有 pZD55-TRAIL-IETD-k5、 pZD55-TRAIL-IETD-IL-24 , pZD55-TRAIL-IETD-IL-12、 pZD55-TRAIL-IETD-Omi、 pZD55-TRAIL-IETD-Eorf4等。 C.双基因 -病毒 ZD55-TRAIL-IETD-Smac的构建
质粒 pBHG-E3 及 293 细胞购于加拿大 Microbix Biosystem Inc. (Toronto)。 质粒 pBHG-E3含有 Ad5基因系列但缺失 El区 188bp-1339bp系列。 293细胞（加拿大 Microbix Biosystem Inc. (Toronto)) 是由剪切的 5型腺病毒 DNA转化人胚胎肾细胞而成。 它含有 表达 5型腺病毒的 E1区， 腺病毒 DNA对其具有高转染率。
将 pZD55-TRAIL-IETD-Smac (它含有与腺病毒同源重组的左臂序列） 与含有腺病毒 骨架 DNA的质粒 pBHG-E3共转染 293细胞株，通过同源重组产生具有感染性的含二外源 基因的重组腺病毒 ZD55-TRAIL-IETD-Smac (见图 1 )。 具体方法参见 Qiagen公司的操作 说明。 7-14天出现病毒空斑，经过 2次病毒空斑纯化，进行扩增，提取重组腺病毒的 DNA， 进行 DNA酶切分析， PCR分析， 确定重组正确的腺病毒株。
病毒空斑纯化、 扩增、 鉴定： 293细胞铺于 6孔板， 24h后细胞接近长满， 加入含不 同稀释度的病毒， 感染 2小时后， 每孔铺 3ml低熔点胶（10%FBS， 1.25% Agarose) 9天 左右就可见空斑。 挑取单个空斑， 加入接近长满 293细胞的 24孔板中小量扩增病毒。 病 毒 DNA用 Qiagen Blood Kit获得， 利用 PCR技术， 鉴定基因病毒 Ad5-ZD55-gene。 鉴定 所用引物由上海生工合成。 （注： 序列右侧注明引物与 pXCl质粒配对的序列）。
Zd55 sense弓 1物：
5 ' AGA GCC CAT GGA ACC CGA GA 3，； bp 2200-2219
Zd55 antisense引物：
5， CAT CGT ACC TCA GCA CCT TCC A 3，； bp 3353-3332
以 Qiagen Blood Kit抽提所得的病毒 DNA作为模板， 同时以野生型病毒 DNA作为对 照， Zd55 sense引物与 Zd55 antisense引物进行 PCR反应。 PCR条件： 9 °C X lmin, 55 °C X lmin, 72°C X2minl5so 如 PCR产物只含 gene, 不含 1113bp野生型腺病毒 DNA， 空斑 纯化成功。 重复此过程一次， 得到重组正确的腺病毒。
腺病毒 ZD55-TRAIL-IETD-Smac在 293细胞中大量繁殖， 应用氯化铯梯度离心纯化 病毒。 具体操作方法见 Microbix Biosystem Inc.的操作说明。
用类似方法构建的重组腺病毒还有 ZD55-TRAIL-IETD-k5、 ZD55-TRAIL-IETD-IL-24、 ZD55-TRAIL-IETD-IL- 12 , ZD55-TRAIL-IETD-Omi、 ZD55-TRAIL-IETD-Eorf4等。 实施例 2、 双基因 -病毒 ZD55-IL-24-IRES-TRAIL的构建
A、 质粒 pZD55-IL-24-IRES-TRAIL的构建
先设计如下引物： IL-24 引物：
上游 ： 5 ' CGTCGACATGAATTTTCAACAGAGGCTGC 3，；
Sail
下游- 5 ' GATCTAGACTAGAC ATTC AGAGCTTGTAG 3 '；
Xbal
IRES引物：
上游： 5 ^AGTCTAGAGCATCTAGGGCGGCCAATTC 3 ?；
Xbal
下游： 5 'TCTCTCGAGCAGATCAGATCCCATACAAT 3 '；
Xhol
Trail引物：
上游： 5 'CTCAGCTCGAGCATGGCTATGATGGAGGTC 3 '；
Xhol
下游： 5 ' CATGGGATCCCAGGTCAGTTAGCCAACTA 3，
BamHI
以 pCDNA3-IL-24质粒 (购自武汉三鹰生物技术公司，武汉）为 PCR反应的模板， IL-24 上游引物与 IL-24下游引物进行第一次 PCR反应（详见 Molecular Cloning: A Laboratory Manual, 3rd ed., Joseph Sambrook and David W. Russell), 跑电泳回收 628bp片段。 以 Sall+ Xbal酶切 PCR产物， 克隆进 Sall+ Xbal酶切过的 pCA13质粒， 命名为 pCA13-IL-24质 粒。
以 pIRESPROU质粒（购自加拿大 Microbix Biosystem Inc, Toronto)为 PCR反应的模 板， IRES上游引物与 IRES下游引物进行第二次 PCR反应 （详见 Molecular Cloning: A Laboratory Manual, 3rd ed., Joseph Sambrook and David W. Russell),跑电泳回收 585bp片段。 以 Xbal + Xhol酶切 PCR产物，克隆进 Xbal + Xhol酶切过的 pCAl 3 -IL-24质粒中，命名 为 pCA13-IL-24-IRES质粒。
以 pCDNA3-TRAIL质粒为 PCR反应的模板， TRAIL上游引物与 TRAIL下游引物进 行第三次 PCR反应（详见 Molecular Cloning: A Laboratory Manual, 3rd ed., Joseph Sambrook and David W. Russell), 跑电泳回收 866bp片段。 以 XhoI+ BamHI酶切 PCR产物， 克隆 进 XhoI+ BamHI酶切过的 pCA13-IL-24-IRES质粒， 命名为 pCA13-IL-24-IRES-TRAIL质 粒。 用 Bgl II酶切质粒 pCA13-IL-24-IRES-TRAIL质粒， 切出 gene的表达框。这个表达框 包含 CMV启动子、 治疗基因 IL-24和 TRAIL及 SV40 polyA尾巴。 而后将此表达框克隆 进 Bgl II单酶切并去磷的 pZD55质粒， 构建成质粒 pZD55-IL-24-IRES-TRAIL。
用类似方法构建的质粒还有 pZD55-TRAIL-IRES-Smac、 pZD55-Smac-IRES-TRAIL、 pZD55-k5-IRES-TRAIL 、 pZD55-TRAIL-IRES-k5 、 pZD55-sfltl-IRES-k5 、 pZD55-k5-IRES-sfltl 、 pZD55-TRAIL-IRES-IL-24 、 pZD55-IL-24-IRES-TRAIL 、 pZD55-TRAIL-IRES-IL-12 、 pZD55-IL-12-IRES-TRAIL 、 pZD55-TRAIL-IRES-Omi 、 pZD55-Omi-IRES-TRAIL 、 pZD55-IL-IRES-12-IL24 、 pZD55-IL24-IRES-IL-12 、 pZD55-TRAIL-IRES-Eorf4、 pZD55-Eorf4-IRES-TRAIL等。
B、 双基因 -病毒 ZD55-IL-24-IRES-TRAIL的构建
将 pZD55-IL-24-IRES-TRAIL (它含有与腺病毒同源重组的左臂序列）与含有腺病毒 骨架 DNA的质粒 pBHG-E3共转染 293细胞株，通过同源重组产生具有感染性的含二外源 基因的重组腺病毒 ZD55-IL-24-IRES-TRAIL。 7-14天出现病毒空斑， 经过 2次病毒空斑纯 化， 进行扩增， 提取重组腺病毒的 DNA, 进行 DNA酶切分析， PCR分析， 确定重组正确 的腺病毒株。 具体步骤见实施例 1。
用 类 似 方 法 构 建 的 重 组 腺 病 毒 还 有 ZD55-TRAIL-IRES-Smac 、 ZD55-Smac-IRES-TRAIL 、 ZD55-k5-IRES-TRAIL 、 ZD55-TRAIL-IRES-k5 、 ZD55-sfltl-IRES-k5 、 ZD55-k5-IRES-sfltl 、 ZD55-TRAIL-IRES-IL-24 、 ZD55-IL-24-IRES-TRAIL 、 ZD55-TRAIL-IRES-IL-12 、 ZD55-IL-12-IRES-TRAIL 、 ZD55-TRAIL-IRES-Omi 、 ZD55-Omi-IRES-TRAIL 、 ZD55-IL-IRES-12-IL24 、 ZD55-IL24-IRES-IL-12、 ZD55-TRAIL-IRES-Eorf4、 ZD55-Eorf4-IRES-TRAIL等。 实施例 3、 双基因 -病毒 Ad-hTERT-TRAIL-K5的构建
肿瘤细胞的特征之一是能无限的增殖， 而 TRAIL蛋白可介导肿瘤细胞凋亡， 特异性 地杀伤肿瘤细胞。 另一方面， 肿瘤的生长需要通过丰富的血管供应养分， K5 是血纤维蛋 白溶酶原 kringle的第 5个结构域， 它可通过调控内源性血管形成因子途径特异性地抑制 内皮细胞的分裂增殖， 具有比 angiostatin血管抑素更高的抗血管形成效应和显著的抗肿瘤 效应（ Chun- xia Luo et al. China J. Cancer Biother , 2003, 10(1): 9-12，）。 TRAIL蛋白和 K5 蛋白通过不同的机制发挥抗肿瘤作用，采用融瘤病毒介导 TRAIL以及 K5两种基因，可发 挥其互补作用。 A.肿瘤靶向载体质粒 pZhTERT的构建
为了改造 E1A基因的启动子，以 pXCl为模板，用定点突变双次 PCR技术，缺失 E1A 基因的启动子， 代之以三个单酶切位点。 引物由上海生工合成。 （注： 序列右侧注明引物 与 pXCl质粒配对的序列， 下划线标出内切酶位点）
Bam5' 引物：
5， TCC TGT GGA TCC GGG CCC CCA TTT C 3，； bp 9876-9901；
Bam3' 引物：
5， TTCAG TAG GTA GTC GAC CTC GAG ATA TTA CGC GCT ATG AGT AAC AC 3'； bp 342-319；
Xba5' 引物：
GAG GTC GAC TAC GTA CTG AAA ATG AGA
bp 552-573；
Xba3' 引物：
5， TACT ACT ATT GCA TTC TCT AGA CAC A 3'； bpl359-1334;
以 pXCl质粒为 PCR反应的模板， 弓 I物 Bam5'与 Bam3'进行第一次 PCR反应（详见 Molecular Cloning: A Laboratory Manual, 3rd ed., Joseph Sambrook and David W. Russell), 跑 电泳回收 394 bp片段。 '
以 pXCl质粒为 PCR反应的模板，引物 Xba5'与引物 -Xba3'进行第二次 PCR反应（详 见 Molecular Cloning: A Laboratory Manual, 3rd ed., Joseph Sambrook and David W. Russell), 跑电泳回收 830 bp片段。
两次 PCR反应的产物有 26bp配对的序列， 以两次 PCR产物混合作为模板， 以引物 Bam5，与引物 Xba3，进行第三次 PCR反应（详见 Molecular Cloning: A Laboratory Manual, 3rd ed., Joseph Sambrook and David W. Russell )， 跑电泳回收 1198 bp片段。 以 BamH I + Xba l 酶切 1198 bp的 PCR产物， 克隆进 BamH I + Xba l酶切过的 pXCl质粒， 命名为 pZXC2。
用 PCR拉出 hTERT启动子，产生合适的酶切位点，然后酶切克隆进 E1A启动子缺失 的质粒 pZXC2中， 具体如下：
hTERT 5' 引物：
5， TCTT CTC GAG TGG CCC CTC CCT CGG GTT AC 3'； Xho l;
hTERT 5' 引物:
5， GTA GGG CGG GGC CGC GGA AAG GA 3，； 以质粒 pAd/TERT为模板， hTERT 5' 引物与 hTERT 3' 引物进行 PCR反应（详见 Molecular Cloning: A Laboratory Manual, 3rd ed., Joseph Sambrook and David W. Russell), 跑 电泳回收 460 bp片段。以 Xho l酶切 PCR产物，克隆进 ho l + SnaB I酶切过的 pZXC2 质粒， 命名为 pZhTERT。此质粒缺失了内源性 Ε1Α·启动子， 而由 hTERT启动子取代， 并 含有克隆位点 Xho l, 可插入外源基因表达框 （见图 2)。
B、 质粒 pZhTERT-TRAIL的构建
TRAIL基因克隆至 pCA13质粒，用 Bgl II酶切出基因表达框（含 CMV启动子， TRAIL 基因， Poly A信号）， 克隆进 Xhol酶切过的 pZhTERT质粒。克隆中使用部分补平的方法。 质粒命名为 pZhTERT-TRAILo
C、 质粒 pBHG10-K5的构建
pABS.4及 pBHGlO质粒均购自 Microbix公司。 pBHGlO质粒是由质粒 pBHG-E3缺失 E3区 28133bp-30818bp系列而得到，在其缺失部位有 pad克隆位点。K5基因克隆至 pCA13 质粒 (具体步骤见 Chun-xia Luo et al. Recombinant Kringle 5 of Human Plasminogen for Mammary Cancer Gene Therapy Mediated by Adenovirus. China J. Cancer Biother , 2003, 10(1): 9-12, ), 用 Bgl ll酶切出基因表达框（含 CMV启动子， K5基因， Poly A信号）， 克 隆进 Bgl II+BamH I酶切过的 pABS.4质粒，得到 pABS.4-K5质粒。再用 pad酶切出基因 表达框， 克隆进 pacl酶切过的 pBHGlO质粒， 得到质粒 pBHG10-K5。
D、双基因 -病毒 Ad-hTERT-TRAIL-K5的构建
将质粒 pZhTERT-TRAIL (它含有与腺病毒同源重组的左臂序列)与含有腺病毒骨架 DNA的质粒 pBHG10-K5共转染 293细胞株，通过同源重组产生具有感染性的含二外源基 因的重组腺病毒 Ad-hTERT-TRAIL-K5。 7-14天出现病毒空斑， 经过 2次病毒空斑纯化， 进行扩增， 提取重组腺病毒的 DNA， 进行 DNA酶切分析， PCR分析， 确定重组正确的腺 病毒株。 具体步骤见实施例 1。 用 类 似 方 法 构 建 的 重 组 腺 病 毒 还 有 Ad-hTERT-TRAIL-Smac 、
Ad-hTERT-TRAIL-sfltl 、 、 Ad-hTERT-TRAIL-IL-24 、 Ad-hTERT-TRAIL-IL- 12 、
Ad-hTERT-TRAIL-Omi 、 Ad-hTERT-TRAIL-Eorf4 、 Ad-hTERT-IL-24-Omi 、 Ad-hTERT-IL-24-IL-12、 Ad-hTERT-IL-24--Eorf4、 Ad-hTERT-IL-24-K5等。 实施例 4、 腺相关病毒（AAV)
用肿瘤特异性启动子 hTERT替换双基因 TRAIL-IETD-Smac等表达框的 HCMV启动 子， 使双基因的表达仅限于肿瘤组织或细胞， 其它靶向 AAV双基因-病毒以此类推。 经过 包装滴度可达 1012pfu/ml。 实施例 5、 无肠腺病毒载体
将肿瘤特异性的双基因 hTERT-TRAIL-IETD-Smac等表达框或其它 hTERT-双基因表 达框插入无肠腺病毒载体中，包装成无肠腺病毒。其它靶向双基因的无肠腺病毒以此类推。 实施例 6、 肿瘤特异性启动子调控的腺病毒
病毒的复制可受肿瘤特异性启动子的调控。 双基因 TRAIL-IETD-Smac表达框插入到 该病毒的基因组中后， 双基因的功能的发挥仅限于某特异性肿瘤组织中。 实施例 7、 双基因病毒 Ad-hTERT-TRAIL-K5在正常细胞或肿瘤细胞中复制能力分析
将 3 xlO5的正常细胞或肿瘤细胞铺于 6孔板， 24 h后， 加入 104 PFU 的 Ad.TERT、 Ad-hTERT-TRAIL-K5、 野生型 5型腺病毒 Ad5分别感染 293细胞， 肝癌细胞株 BEL7404 细胞（购自中国科学院上海生命科学研究院细胞库）， 大肠癌细胞株 SW620 (购自 ATCC) 及正常人胚肺细胞株 MRC5和 NHLF (购自 ATCC)。 48 h后，收集细胞上 与细胞，在 -20 °〇与 37°C反复冻融 3次以释放病毒。将病毒稀释，检测病毒滴度。 293细胞铺于 60mm dish, 24h后细胞接近长满， 加入含不同稀释度的 毒， 37Ό感染 2小时后， 铺 8ml低熔点胶 (5%FBS， 1.25% Agarose) o 9天左右记数。 计算每 PFU病毒产生的病毒数目。 结果如图 4所示， 由图 4可见， 在肿瘤细胞中， Ad.TERT、 Ad-hTERT-TRAIL-K5的复制能力与野 生型病毒相比略有下降。 而在正常细胞中， Ad.TERT、 Ad-hTERT-TRAIL-K5 的复制能力 显著降低， 在 MRC5细胞中， 与野生型 5型腺病毒相比下降近 1600倍， 而在 NHLF细胞 中下降 1200倍。野生型腺病毒在肿瘤细胞和正常细胞都有很强的复制能力， 没有选择性； 而 Ad.TERT、 Ad-hTERT-TRAIL-K5能选择性地在肿瘤细胞中复制。 实施例 8、 双基因病毒 Ad-hTERT-TRAIL-K5在体外对肿瘤细胞的杀伤能力检测 细胞经病毒处理后的存活率由 MTT法检测 （Cancer Research, 1989, 49(17):4785-90)。 步骤如下： 将大肠癌细胞株 SW620及正常人胚肺细胞 NHLF以 5000每孔的量铺入 96孔 板， 培养 24小时后加入不同滴度 （MOI) 的病毒， 作用 3天， 然后将含病毒的培养液移 去， 换成含 5mg/ml MTT的正常培养液， 培养 4小时后将含 MTT的培养液移去， 以裂解 液裂解 4小时， 然后以 655nm处吸光度为参比测定 595nm处吸光度。 细胞存活率 （％) =A595(样品) /A595(对照) χ100%。结果如图 5Α、5Β所示，由图 5Α、5Β可知，病毒 Ad.TERT、 Ad-hTERT-TRAIL-K5对肿瘤细胞有很明显的杀伤作用， 而对正常细胞毒性很小， 具有肿 瘤选择性。 野生型腺病毒对肿瘤细胞和正常细胞都有很强的杀伤作用， 没有选择性。 实施例 9、 双基因病毒 ZD55-TRAIL-IETD-Smac在正常细胞或肿瘤细胞中复制能力分析 将 3 xlO5 的正常细胞或肿瘤细胞铺于 6 孔板， 24 h 后， 加入 104 PFU 的 ZD55-TRAIL-IETD-Smac、 ONYX-015、 野生型 5型腺病毒 Ad5分别感染 293细胞， 肝癌 细胞株 BEL7404细胞， 大肠癌细胞株 SW620及正常人胚肺细胞。 48 h后， 收集细胞上清 与细胞， 在 -20°C与 37Ό反复冻融 3次以释放病毒。 将病毒稀释， 检测病毒滴度。 293细 胞铺于 60mm dish, 24h后细胞接近长满，加入含不同稀释度的病毒， 37°C感染 2小时后， 铺 8ml低熔点胶（5%FBS， 1.25% Agarose )0 9天左右记数。计算每 PFU病毒产生的病毒 数目。 结果如图 3所示， 由图 3可见， 在肿瘤细胞中， ZD55-TRAIL-IETD-SmaC的复制能 力与野生型病毒相比有所下降。而在正常细胞中， ZD55-TRAIL-IETD-SmaC的复制能力显 著降低， 在 MRC5细胞中， 与野生型 5型腺病毒相比下降近 1500倍， 而在 NHLF细胞中 下降 1300倍 （图 3)。 野生型腺病毒在肿瘤细胞和正常细胞都有很强的复制能力， 没有选 择性； 而 ZD55-TRAIL-IETD-Smac能选择性地在肿瘤细胞中复制。 实施例 10、 双基因病毒 ZD55-TRAIL-IETD-Smac在体外对肿瘤细胞的杀伤能力检测
细胞经病毒处理后的存活率由 MTT法检测。步骤如下： 将肝癌细胞株 BEL7404及正 常人胚肺细胞 NHLF以 5000每孔的量铺入 96孔板， 培养 24小时后加入不同滴度（MOI) 的病毒， 作用 3天， 然后将含病毒的培养液移去， 换成含 5mg/ml MTT的正常培养液， 培 养 4小时后将含 MTT的培养液移去， 以裂解液裂解 4小时， 然后以 655nm处吸光度为参 比测定 595nm处吸光度。 细胞存活率 （％) =A595(样品) /A595(对照) χ100%。 结果如图 6A、 6B所示， 由图 6A、 6B可知， 病毒 ZD55-TRAIL、 病毒 ZD55-Smac及双基因病毒 ZD55-TRAIL-IETD-Smac对肿瘤细胞有很明显的杀伤作用， 而对正常细胞毒性很小， 具有 肿瘤选择性。 野生型腺病毒对肿瘤细胞和正常细胞都有很强的杀伤作用， 没有选择性。 实施例 11、 双基因病毒 ZD55-TRAIL-IETD-Smac在裸鼠体内治疗肿瘤细胞移植瘤
将 4-5周龄的裸鼠皮下接种肝癌细胞株 BEL7404, 12天后进行动物分组。 治疗组分 别给予 1 X 109 pfu 的基因病毒 ZD55-TRAIL、 ZD55-Smac 及双基因病毒 ZD55-TRAIL-IETD-Smac进行治疗，对照分 2组：第 1组是磷酸缓冲液 (PBS)处理组，第 2 组用 同剂量的 ONYX-015病毒。 试验结果如图 7所示， 可见 ZD55-TRAIL-IETD-Smac 疗效最好，治疗 9周后，肿瘤细胞移植瘤已完全消失，治疗效果比 ONYX-015好很多很多。

Claims

权利要求书
1、 一种肿瘤靶向双基因-病毒， 其特征在于， 该双基因-病毒包含肿瘤特异性启动子， 并携带有两个肿瘤治疗基因。
2、 如权利要求 1所述的双基因-病毒， 其特征在于， 所述两个肿瘤治疗基因为功能上 互补或具有协同作用的基因。
3、 如权利要求 1所述的双基因-病毒， 其特征在于， 所述两个肿瘤治疗基因以连接物 相连接。
4、 如权利要求 3所述的双基因-病毒， 其特征在于， 所述连接物为核糖体进入位点或 细胞内源性酶切位点序列。
5、 如权利要求 1所述的双基因-病毒， 其特征在于， 所述肿瘤特异性启动子包括但不 限于： 端粒酶逆转录启动子， 甲胎蛋白启动子， 癌胚抗原启动子， 前列腺特异性抗原启动 子和乳腺癌组织特异性启动子。
6、如权利要求 1所述的双基因-病毒，其特征在于，所述肿瘤治疗基因包括但不限于： 肿瘤坏死因子超家族中的 TRAIL基因， 抑癌基因，，细胞因子基因， 促细胞凋亡基因， 血管抑制基因和自杀基因。
7、 一种肿瘤靶向双基因 -病毒的构建方法， 其特征在于包括以下步骤- a、将两个胂瘤治疗基因克隆进载体质粒的多克隆位点， 两基因之间以连接物相连接 ·， 然后用 Bgl II酶切出包含启动子、 抗癌基因以及 poly A加尾信号的双基因表达框， 插入 含有肿瘤特异性启动子的肿瘤特异性增殖病毒载体中；
b、 将改造好的携带肿瘤靶向双基因的病毒载体转染细胞以产生双基因-病毒。
8、 如权利要求 7所述的构建方法， 其特征在于， 所述两基因之间的连接物为核糖体 进入位点或细胞内源性酶切位点序列。
9、 一种肿瘤靶向双基因 -病毒的构建方法， 其特征在于包括以下步骤：
a、 将两个肿瘤治疗基因分别克隆进质粒的多克隆位点， 分别以限制性内切酶切出基 因表达框， 插入改造好的含有肿瘤特异性启动子的肿瘤特异性增殖病毒载体的不同位点 中；
b、 将两个质粒共转染细胞， 通过同源重组产生含有两个外源基因的双基因-病毒。
10、 如权利要求 7或 9所述的肿瘤靶向双基因 -病毒的构建方法， 其特征在于所使用 的两个基因在功能上互补或具有协同作用。 ·
11、如权利要求 7或 9所述肿瘤靶向双基因 -病毒的构建方法，其特征在于，所述基因 包括但不限于： 肿瘤坏死因子超家族中的 TRAIL基因， 抑癌基因， 细胞因子基因， 促细 胞凋亡基因， 血管抑制基因和自杀基因。
12、 如权利要求 7或 9所述的肿瘤靶向双基因 -病毒的构建方法， 其特征在于所使用 的抗癌基因包括但不限于： p53、 PTEN、 Rb、 NF1、 VHL、 APC、 IL-2、 IL-12、 IL-24、 GM-CSF、 IFN- a、 IFN- β、 IFN- Y、 TRAIL、 Smac、 Omi、 Bax、 Caspase-3、 Caspase-7、 Eorf4、 cd、 tk、 endostatin、 angiostatin(kl-4)、 kl-3、 k5、 sflt-l。
13、 如权利要求 7或 9所述的肿瘤靶向双基因 -病毒的构建方法， 其特征在于所使用 的肿瘤特异性增殖病毒载体是肿瘤特异性增殖腺病毒、 AAV或 GL-Adv。
14、 如权利要求 7或 9所述的肿瘤靶向双基因 -病毒的构建方法， 其特征在于肿瘤特 异性启动子包括但不限于： 端粒酶逆转录启动子， 甲胎蛋申启动子， 癌胚抗原启动子，.前 列腺特异性抗原启动子和乳腺癌组织特异性启动子。
15、权利要求 1-6中任一项所述的肿瘤靶向双基因-病毒用于开发治疗肿瘤的药物的用 途。
16、 如权利要求 15所述的用途， 其特征在于， 与其它化合物组成药用组合物， 所述 化合物选自： 化学治疗药物； 生物毒素； 免疫抑制化合物， 单克隆抗体等。 .
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