Construction And Application Of A Newly Oncolytic Adenovirus Vector M3 Selectively Blocking STAT3 Of Tumor

PART IConstruction and Application of a Newly Oncolytic Adenovirus Vector M3 Selectively Blocking STAT3 of TumorObjectives The first generation of adenovirus vevtor , Ad5/dE1/TK, vector in our laboratory couldn't replicate and stayed in vivo for a short time. The second generation of adenovirus vector, Ad5/dE1A/6.7K/gp19K, which could replicate and was selective at some degree, was toxical to normal cells because it could lysis tumor cells and released tumor antigens that could stimulate specifically immune response. To aim directly at the shortcomings of two adenovirus vectors above, we planed to construct a newly oncolytic adenovirus vector, Ad5/dE1A/ADP, with more tumor specifity and then combined with an ideal target of cancer therapy.That would be the third genetion of aenovirus vector named M3. The vector would had powerful tumor-specifically effect and provide a potent approach of cancer therapy because of its more selectively replicative ability, high expression of target gene, some ability of immune evasion and minimal toxicity to normal cells. Methods To obtain TP-DNA of adenovirus by chromotography techniques; to delete ADP gene at fix-point in E3 domain of adenovirus genome by two rounds PCR; to acquire recombinant adenovirus by restriction endonuclease digesting, ligase ligating, transformation, cloning, homologous recombination in vitro, Ca-P transfection, packing adenovirus in cells, adenovirus monoclone purification and so on; to verify the recombinant adenovirus by PCR and sequencing; to gain purified recombinant adenovirus by CsCl gradient centrifuge. Results we obtained TP-DNA with high package effect successfully and gained right recombinant adenovirus through homologous recombination of the TP-DNA and shuttle vector. Conclusion Obtained recombinant adenovirus vector M3 for gene therapy that had a variety of anticipated technological characters established fundament for application of the vector. PARTâ…¡Biological character verification of a more tumor-specific and newly oncolytic adenovirus vector M3Objectives We verified the biological characters of the more tumor-specific and newly oncolytic adenovirus vector that had constructed successfully to judge whether the vector satisfied the first expectation of our study. Methods We detected the expression model of exogenous gene (antisense STAT3 cDNA fragment) taken by the vector and detected if the expression of exogenous gene imitated the expression of substituted ADP by PCR. After deletion of ADP or insertion of antisense STAT3 cDNA fragment, effects on other gene expression in E3 domain was determined by real-time PCR. CPE assay detected the ability of the vector that lysised different tumor cells and normal cells. We detected whether deletion of ADP or insertion of antisense STAT3 cDNA fragment would affect on progeny virus production by virus burst assay and TCID50 assay. Results Exogenous gene taken by recombinant adenovirus was expressed in the late stage of the virus replication and the expression, which depended on virus DNA replication, was identified with expression models of substituted ADP. There was no effect on surrounding other gene in E3 domain after deletion of ADP or insertion of antisense STAT3 cDNA fragment. The occurrence of CPE effect of tumor cells by the virus was later than it by wild type adenovirus. And we didn't observed the apparent CPE effect in normal cells when the amount of the recombinant adenovirus was 5000 times as many as that of wild type adenovirus. The production of the recombinant adenovirus was marked increased because of deletion of ADP. Conclusion Obtained recombinant adenovirus vector for gene therapy that had a variety of anticipated biological characters established fundament for application of the vector. PARTâ…¢Analysis of Therapeutic Effects of the More Tumor-specific and Newly Oncolytic Adenovirus Vector M3Objectives To verify the effects of M3 on its target protein, affects of M3 on tumor cells phenotype in vitro and oncolytic effect of M3 in vivo. Methods The blocking effects of M3 on its target protein and the regulations of M3 on its downstream effect proteins were determined by western blot assay and real-time PCR. Lethal effect of M3 on a variety of tumor cells in vitro and sensitization effect by chemotherapy of M3 on drug resistant cell lines were determined by flow cytometry. We observed affects of invasion ability of M3 on different tumor cells by transwell invasion assay in vitro. To detect therapeutic effect of M3 in vivo by dystopic and orthotopic tumor transplanted model. Results Western blot results showed that M3 could effectively inhibit expression of STAT3 protein and down-regulate expression of downstream proteins such as c-myc,Bcl-xL,survivin,VEGF,MMP-9 and Tim3,but had no effect on STAT3 expression of mormal cells HUVEC. Analysis of flow cytometry demonstrated that M3 had potent lethal effect on tumor cells derived from different tissues or organs compared to Ad5/dE1A and Ad5/dE1A/ADP and the maximum apoptosis rate of tumor cells induced by M3 reached to more than 80%. And what's more, M3 still enhanced significantly the sensibility of ovarian cancer cell line C13K to chemotherapeutic drugs. Transwell assay displayed M3 inhibited effectively invasive ability of tumor cells. In subcutaneouly transplant mice model of prostatic carcinoma and gastric cancer, M3 that was intratumorally injected could markedly restrain the growth of tumor cells. In orthotopic mice model of gastric cancer, M3 by intravenous injection not only markedly inhibited the growth of gastric cancer cells, but also prevented enterocoelia implantation metastasis of them. Conclusion Recombinant adenovirus M3 could block validly the expression of STAT3 protein and affect the expression of downstream protein in the mean time. With the potent lethal effect on tumor cells, M3 possessed the potential value of clinical application.