Construction Of Tumor-Targeting Adeno-associated Virus Armed With TRAIL And Its Suppression Effect On Hepatocellular Carcinoma

Currently cancer have gradually replaced the cardio-and cerebrovascular diseases and become the most disease with mortality around the worldwide. Cancer incidence and mortality rapidly increase, and the mortality in developed countries accounted for 21.6% of the total deaths from cancer. Hepatocellular carcinoma (HCC) is one of the most common tumor and the uppermost mortality diseases. More than one million patients died for HCC in the world each year and the death rate for HCC in China ranks the first, thus HCC seriously menaces human health. Although traditional treatment methods for HCC such as surgery, radio-and chemotherapy achieved some effort, there are still defects including uncurable, low antitumor ability and tumor-killing effect, large side effects et al.Tumor gene therapy brings hope for cancer treatment through utilizing viruses as vectors to carry the transgenes. It has made great progress for nearly two decades, and some gene therapy drugs have been used in the clinical. Among these used virus vectors, adeno-associated virus (AAV) has been considered as one of the most promising gene therapy vectors due to non-pathogenic to humans, low immunogenicity, as well as long-term and stable exogenous gene expression. However, the most worried problem for AAV is short of tumor targeting ability, which can easily cause damage to normal cells. Therefore it is required to improve the targeting of AAV for the better safety and antitumor effect. At present, there are mainly two strategies including the infectious modification of AAV capsid and the application of cell or tissue-specific promoters.The activity of human telomerase is strictly regulated in normal somatic cells and it don't express under physiological conditions. However, human telomerase can express a high level in most tumor cells, and the activity level is consistent with the deterioration degree of tumor progression. Therefore targeting tumor gene therapy mediated by AAV vector could be achieved through using human telomerase reverse transcriptase (hTERT) promoter to drive AAV-carried therapeutic gene expression.Therefore, the tumor-targeting AAV-hTERT-gene system was firstly designed and constructed by using the hTERT promoter to control transgene expression mediated by AAV in this paper. Then, the feasibility and effectivity of this system on cancer therapy was further investigated by using IFN-P gene as a therapeutic gene. The research results demonstrated that the constructed tumor-targeting virus AAV-hTERT-IFN-βcould restrict IFN-0 gene to effectively express in tumor cells, and induce cancer-specific toxic effects. In treatmental experiments of SW620 colorectal cancer model in vivo, AAV-hTERT-IFN-βsignificantly inhibited the growth of the tumor xenograft in mice. Moreover, the anticancer mechanism of AAV-hTERT-IFN-βwas mainly due to activation of caspase 8 of signaling pathway and release of cytochrome c, and was achieved by inducement of tumor cell apoptosis.Presently, aiming at these difficulties in HCC for great harm, high mortality rate and poor therapeutic efficacy et al, on the basis of tumor-targeting AAV-hTERT-gene system, AAV-hTERT-TRAIL was constructed for the first time through applying TRAIL gene, which was able to specifically induce apoptosis of hepatoma cells, as the therapeutic gene in this paper. The results indicated that AAV-hTERT-TRAIL could specifically mediate TRAIL gene expression in tumor cells and resulted in toxic effects of tumor cells. Since the combination of the targeting of hTERT promoter and the specificity of TRAIL-induced tumor cell apoptosis, the AAV-hTERT-TRAIL not only had the good tumor cell-killing effect and had little impact on normal cells, but also resulted in the activation of caspase 3 and release of cytochrome c. In in vivo experiments, the results showed that AAV-hTERT-TRAIL could effectively inhibit tumor growth of nude mice with subcutaneous human HCC SMMC7721 model, and its antitumor efficacy was consistent with the increase of apoptotic cells in tumor. Moreover, AAV-hTERT-TRAIL showed synergistic tumor cell-killing effect and inhibitory activity on BEL7404 liver cancer xenograft in nude mice when it combined with chemotherapeutic agent cisplatin, which is also named as targeting gene-virus-chemotheray strategy. Simultaneously, the combinational effect improved greatly the survival rate of animals. Thus, AAV-hTERT-TRAIL could become a novel and effective anti-cancer agent.In conclusion, the novel tumor-targeting AAV-hTERT-gene system with therapeutic genes IFN-βand TRAIL, designed and constructed in this paper, showed the effective antitumor activity whether in tumor cells in vitro or in animal model in vivo, especially in its excellent targeting ability. The data suggested that this system may offer a promising approach for future targeting tumor gene therapy.