The Killing Effect Of Trail Targeted Adeno-associated Virus As A Carrier Of Tumor-specific Promoter-driven Research

Gene therapy represents an attractive approach to treating cancers.For a good cancer gene therapy delivery system,an ideal vector is absolutely necessary for effective and safe therapeutic.Among quite a few viral vectors which have been tested in recent years,Adeno-associated virus(AAV) is considered to be the most promising vector due to its nonpathogenic property,wide tropisms,non immunogenecity to host and long-term transgene expression.Experimental and clinical trials using recombinant AAV(rAAV) as the delivery vehicles have been proved to be an effective way in gene therapy.However, in gene therapy,especially in treating tumors,the most concerned problem is how to limit the cytotoxic exogenous gene expression in the tumors without harm to normal cells.Since the wide host range infectivity of rAAV may become a disadvantage for its applications in cancer gene therapy,due to its nonselective tissue transduction,safety issue of rAAV as the vector of carrying exogenous genes,which are often toxic to both cancer and normal cells in treating tumors,must be considered.Using tumor-specific promoters has been proved to be a potential way to control the exogenous genes in limited sites.Human telomerase reverse transcriptase(hTERT) is considered to be the most crucial unit in telomerase activity,which is present in a majority of human cancers.The hTERT promoter has been proved to be able to controlling the expression and effect of exogenous genes in tumors without implication of normal tissues.Survivin is the member of the inhibitors of apoptosis(IAP),which is expressed in various human cancers but not in normal adult human tissues.As its expression is activated transcriptionally,the promoter of survivin was used as a cancer-specific promoter in cancer gene therapy.In recent years,TNF-related apoptosis-inducing ligand(TRAIL),as an inducer of tumor cell apoptosis,has been proven to be a promising agent for cancer treatment. However,the potential utility and safety of TRAIL has been questioned because its potential toxicity on normal human cells(e.g.hepatocytes) and the mechanism of the related toxicity still hangs in doubt.From a point of clinical view,safety is the most critical problem in developing a drug.Thus,how to control the effect of TRAIL specifically on tumor cells appears to be imperative.Several studies have proved the tumor-specific efficiency of TRAIL mediated by hTERT promoter using adenovirus or plasmids.However,the capability of rAAV-mediated TRAIL expression driven by hTERT promoter to lead tumor-specific killing effect is unknown.Therefore,in the present study,by using rAAV2 as the vector,we tested whether hTERT promoter and survivin promoter are promising candidates in rAAV-mediated tumor-specific therapy.First all,we constructed the EGFP report gene and soluble TRAIL gene controlled by hTERT promoter(about 284bp) or survivin promoter(about 980bp) in rAAV2 vector, repectively.The specificity of EGFP or TRAIL expression driven by the two promoters was tested in human hepatocellular carcinoma(HCC) cells and primary human hepatocyte(PHH).It showed that hTERT and survivin promoter were both efficient in driving tumor-specific expression of EGFP gene in HCC cells and the activity of hTERT promoter seemed higher compared with that of survivin,but both the two promoters had no acitivity in PHH.The apoptosis of HCC induced by TRAIL expression under the control of tumor-specific promoters was proved that hTERT promoter was more efficient in driving the exogenous gene expression than that of survivin promoter.The second,we tested the activity of hTERT promoter in immortal cell line HEK293 and various tumor cell lines,including human carcinoma of lung,breast,colon, cervix,and lymphoma etc.and human kidney proximal tubular cells(HKC).The result demonstrated that hTERT promoter was active in all the tumor cells although they were not same.Moreover,the AAV.hTERT.TRAIL leaded to apoptosis of various tumor cells, including A549,Hela,SMMC-7721,but not in PHH.Thirdly,in vivo study was carried out and proved that hTERT promoter mediated the surppression of tumor formation and growth by apoptosis without toxic to normal tissues,suggesting it is a promising construct for cancer gene therapy.Fatherly,we studied the effect of combination of AAV.hTERT.TRAIL and chemotherapeutic drugs 5-Fu in vivo.The result showed that the combination of the AAV.hTERT.TRAIL and 5-Fu suppressed the tumor growth more efficiently than either one agent,indicating a novel strategy of AAV-mediated gene threpay combined with chemotherapeutic drugs for cancer treatment.