The Research Of Adenovirus-mediated Cancer Gene Therapy

The technology of transferring genetic materials to the targeted tissues successfully is the main objective in cancer gene therapy.During the past two decades, enormous research in the area of gene delivery has been conducted worldwide.Due to the ability of infecting both dividing and non-dividing cells,the efficient nuclear entry mechanism and its low pathogenicity for humans,adenovirus-based vectors have become widely used vehicles for cancer gene therapy.By now,quite a range of different transductional targeting strategies have been successfully developed in order to improve the efficiency of adenovirus-mediated gene delivery to tumor cells, including genetically encoded or chemically engineered structural modification of the capsid and the use of bispecific adaptor molecules directing vector attachment to alternative cell surface receptors;using some tissue-specific promoters to regulate the gene expression in target cells.According to the mechanism of action,the adenovirus can be divided into two types,replication-defective adenovirus and conditionally replication-competent adenovirus.The replication-defective adenovirus can not replicate in cells.It always acts as a vehicle carrying some exogenous genes,e.g.,tumor suppressor genes, apoptosis inducer genes,immune regulate factors,angiogenesis inhibitors,suicide genes, to target cells and inducing cell death eventually.The conditionally replication-competent adenovirus has the ability of replicating within target cells only.It can infect,usurp host replication machinery,and release newly made progeny to infect other target cells after lysing and killing the host cell.In our study,two types of adenovirus vectors are utilized for cancer gene therapy. Those including:Part one:The construction of a dually-regulated oncolytic adenovirus and its antitumor efficacy on most human solid cancers.Human telomerase reverse transcriptase(hTERT) and cyclooxygenase-2(Cox-2) are always over expressed in tumor cells but not expressed in normal tissue.In our study,we developed a novel therapy strategy using oncolytic adenovirus containing the E1A controlled by hTERT promoter and E1B controlled by Cox-2 promoter.The adenovirus vector pBHGE3 was co-transfected with adenovirus shuttle plasmid pD306/H181-C409 into Hek293 cells using the transfection reagent Lipofectamine^TM2000.After homologously recombining in Hek293 cells,the oncolytic adenovirus Ad5-HC was obtained.A series of in vitro and in vivo assay were performed to evaluate the specificity and cytotoxicity of Ad5-HC,including western blot analysis,adenovirus yield assay,cell viability assay and animal study.The results demonstrated that the oncolytic adenovirus Ad5-HC regulated by both hTERT promoter and Cox-2 promoter has a tumor-specific replication ability and cytolytic ability to a wide range of cancer cells which were positive for telomerase activity and Cox-2 expression.The novel dually-regulated oncolytic adenovirus Ad5-HC has a great potential of destroying tumors and improving the survival of cancer patients.Part two:The construction of replication-defective adenovirus containing artificial multi-microRNAs regulated by AFP enhancer/promoter and its antitumor efficacy on HCC cells.In our study,three types of artificial multi-microRNAs were constructed.Some essential genes for cells metabolism are silenced by these miRNAs, including glyceraldehyde phosphate dehydrogenase(GAPDH),which is an enzyme that catalyzes the process of glycolysis and thus serves to break down glucose for energy and carbon molecules;eukaryotic translation initiation factor 4E(eIF4E), which plays a crucial role in gene expression by controlling protein synthesis at the level of translation initiation;DNA polymerase a,which is a key replicative enzyme, plays an essential role in initiation of both leading- and lagging-strand synthesis.We systematically analyzed the tissue specificity,effect of gene silencing and cytotoxicity of these new-type replication defective adenovirus in order to explore a novel effective strategy for HCC gene therapy.The results demonstrated that the replication-defective adenovirus containing artificial multi-microRNAs regulated by AFP enhancer/promoter can knockdown the target genes which are essential in cells metabolism effectively,and they had a potent ability of inhibiting the HCC cells growth in a tumor-specific manner.The combination of tumor-specific promoter and artificial multi-miRNAs will be a novel strategy in targeted cancer gene therapy and may become a promising approach in future.