ABSTRACT
BACKGROUND: Non-small cell lung cancer (NSCLC) is the leading cause of cancer related mortality, any improvements in therapeutic strategies are urgently required. In this study we generated a novel 'suicide gene' armed oncolytic adenoviral vector and investigated its antitumor effect both in vitro and in vivo. METHODS: Since the up-regulated expression of human telomerase reverse transcriptase (hTERT) is a hallmark of alltypes of NSCLC, we chose hTERT promoter to transcriptionally control E1A gene expression to obtain adenoviral replication in NSCLC. In order to further enhance anti-tumor effect of this oncolytic adenoviral vector, we inserted a 'suicide gene' i.e. Herpes Simplex Virus Thymidine Kinase (HSV-TK) into oncolytic adenoviral vector to engineer a novel armed oncolytic adenoviral vector 'Ad.hTERT-E1A-TK'. RESULTS: Ad.hTERT-E1A-TK efficiently killed different types of tumor cells including two types of NSCLC cells in vitro, causing no damage to normal primary fibroblasts. Furthermore, Ad.hTERT-E1A-TK infection combined with administration of prodrug gancyclovir (GCV) resulted in more potent cytotoxicity on NSCLC cells, and synergistically suppressed human NSCLC tumor growth in nude mice. CONCLUSION: The results from this study showed that Ad.hTERT-E1A-TK/GCV could be a potent but safe anti-tumor strategy for NSCLC biotherapy.
Subject(s)
Adenoviridae/genetics , Carcinoma, Non-Small-Cell Lung/therapy , Lung Neoplasms/therapy , Oncolytic Virotherapy/methods , Simplexvirus/genetics , Telomerase/genetics , Thymidine Kinase/genetics , Adenoviridae Infections/genetics , Adenoviridae Infections/pathology , Adenoviridae Infections/therapy , Animals , Blotting, Western , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line , Combined Modality Therapy , Ganciclovir/administration & dosage , Genes, Transgenic, Suicide , Genetic Therapy , Humans , In Vitro Techniques , Kidney/cytology , Kidney/metabolism , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Male , Mice , Mice, Inbred BALB C , Mice, NudeABSTRACT
To improve the targeting of adenovirus vector for gene therapy, a fusion gene sCAR-EGF, in which epidermal growth factor gene was fused to the 3' end of extracellular Coxsackie virus-adenovirus receptor gene, was constructed and cloned into shuttle plasmid pDC315 to obtain a recombinant plasmid pDC315-sCAR-EGF. With the AdMax system, AD-293 cells were co-transfected with pDC315-sCAR-EGF and adenovirus genomic plasmid pBHGloxdeltaE13cre. Through high efficiency site specific recombination, a replication-defective adenovirus Ad5-CMV-sCAR-EGF was constructed. The recombinant adenovirus was analyzed by PCR and Western blotting, the results indicated that Ad5-CMV-sCAR-EGF contained the fusion gene sCAR-EGF, and the adenovirus infected cells was induced to produce and secrete the fusion protein into the supernatant. We have demonstrated that the fusion protein sCAR-EGF is helpful for elevating the infection efficiency of Ad5-CMV-luc with the reporter gene in vitro, which providing a new approach to the gene therapy for tumors overexpressing EGFR.