Construction And Development Of A Conditional Replication-competent Adenovirus Driven By Double Tumor Specific Promoter And Its Oncolytic Effect In Laryngo-carcinoma

Survivin, a member of the inhibitor of apoptosis proteins (IAP) family, is a 142 amino acids protein which inhibits tumor cells apoptosis through inhibiting Caspase-3, Caspase-7 activity and regulates tumor cell division through microtubules and spindle pathway. Telomeres maintain their length stable generally through the action of a cellular reverse transcriptase- telomerase. Telomerase enzyme complex have two major subunits contributing to enzymatic activity: a structural RNA component(hTER) and a catalytic subunit with reverse transcriptase activity (hTERT) which is important in maintaining the length of telomeres and in protecting cells from getting into the process of apoptosis .hTERT and Survivin gene is selectively expressed in a variety of malignant tumors but is undetectable in well differentiated normal cells. In our previous study, we have cloned survivin and hTERT promoter gene successfully, and by using pGL3-Basic luciferase reporter gene expression vector assay, we found that the activity of the two promoters were even higher than CMV promoter in tumor cells and that the two promoters pessessed high specificity in regulating downstream gene expression in tumor cells. With high activity and broad spectrum expression in tumors, Survivin and hTERT promoter were considered more suitable for the constructing of T-SROAd. Therefore, we designed, and developed a noval conditionally replicating adenovirus T-SROAd that the E1A and E1B genes were driven by survivin and hTERT promoters respectively. Meanwhile, the protein transduction domain following a wild-type p53 fusion gene was inserted into this T-SROAd downstream of E1B gene linked by an IRES sequence.【Objective】:By constructing and packaging recombinant adenovirus in that the viral E1A and E1B genes expression, necessary for adenovirus replication, was controlled by two tumor specific promoters of survivin and hTERT, and in that a protein transduction domain (PTD) and wild-type p53 fusion gene was also fused into the construct downstream of E1B gene by an IRES sequence, we expected to provide a better new, higher efficacy, lower toxicity and applicable approach of biological treatment for laryngo-carcinoma as well as for all those malignant metastatic tumors with survivin and hTERT overexpression , and further expand and enrich T-SROAd genetherapy theory.【Methods】:(1).The survivin and hTERT gene promoter fragments and PTD-P53 gene were amplified by polymerase chain reaction and cloned into promoter regions of E1A and E1B gene in pXC1 vector respectively, hTERT promoter also control PTD-P53 fusion gene and the IRES gene to form the HP-PTDP53-IRES fusion gene to construct a dual promoter regulated tumor-specific conditionally replicating adenovirus vector of pXC1-SP-HP53. (2).Recombinant adenovirus vector pXC1-SP-HP53 was cotransfected into 293E cells with adenovirus backbone plasmid pBHGE3 by Lipofectamine TM 2000 for homologous recombination to get packaged recombinant adenovirus of Ad-SP-HP53. Then the plaque technique was used to harvest, enrich, purify the recombinant adenovirus, after that the titers of viruses were tested using Reed-Muench method and the virus was named Ad-SP-HP53.(3). Specific oncolytic effect was investigated in hep-2 laryngo-carcinoma cells infected with Ad-SP-HP53 by MTT and viable count methods and the ctopathic effect was observated under microscope. The normal vascular endothelial cells ECV304 was uesd in this study as control. Finally the FCM method was used to analyze apoptosis and cell cycle changes in the cells infected with Ad-SP-HP53. (4).In vivo tumor growth inhibition effects were investigated in a nude mouse, meanwhile systemic organ pathology changes and positive rate of P53 protein were also detected by HE staining or immunohistochemistry with tumor tissues.【Results】:(1) Survivin and hTERT promoter genes and wild-type P53 gene were obtained by PCR method, the sequences and orientation of inserted genes were verified by DNA sequencing, restriction digestion and PCR methods. Then the dual tumor-specific promoter regulated adenovirus vectors was constructed successfully.(2) The dual tumor-specific promoter regulated adenovirus vectors were efficiently packaged with high-titer recombinant adenoviruses, the titer of the packaged virus was 3.9×1010 TCID50/ml. PCR results demonstrated that p53 gene were expressed in the Hep-2 cells after Hep-2 cells were infected with the recombinant virus. (3) MTT results showed that Ad-SP-HP53 can effectively inhibit cell proliferation in Hep-2 cells but not in normal cells (p <0.05); cell morphology observation showed that recombinant adenovirus in Hep-2 cells selectively replicated and dissolved the infected tumor cells. Apoptosis were observed in recombinant adenovirus infected cells with FCM and AO/EB methods.(4) Recombinant adenoviruses Ad-SP-HP53 inhibited Hep-2 xenograft tumor growth in vivo and prolonged survival of this xenograft nude mouse.【Conclusion】:The successfully constructed recombinant adenovirus bearing the wild type P53 with tumor-specific promoters to control its replication, has significant oncolytic effect on laryngo-carcinoma cells but not normal vascular endothelial cells.This oncolytic effect combined with fused P53 led to the laryngo- carcinoma cells growth inhibition both in vitro and in vivo. The experiment results provide better conditionally replicative adenoviruse mediated laryngo- carcinoma therapy and new targeted therapeutic strategies.