Laboratory Evaluation Of Adenovirus-mediated Anti-tumor Gene Therapy Strategy

Survivin,a newly identified antiapoptotic protein of IAP(inhibitor of apoptosis) family,is expressed abundantly in a variety of human tumors but negligibly in their normal adult counterparts.Reactivation of survivin expression is involved in the pathogeneses of solid tumors including liver cancer.To explore the therapeutic application of survivin-targeted cancer therapy,we conducted the adenoviral ribozyme-based inhibition of survivin on hepatocellular cancer.Four ribozymes targeting the exposed regions of the SURVIVIN mRNA were choose and cloned into a non-replicative adenoviral vector to generate catalytically active ribozymes.Cell proliferation,apoptosis,and tumor growth assays were used to score ribozyme efficiency in vitro and in vivo.Infection of hepatocellular cancer cells with adenoviral-ribozymes dramatically suppressed the accumulation of survivin with the greatest suppression seen in R3.Combinatorial expression of R1,R3 and R4 produced a synergistic suppression of survivin than any single ribozyme as the combination targets all three survivin transcripts.Furthermore,the ribozyme-mediated suppression of survivin induced mitotic catastrophe and hepatocellular cancer cell death via caspase 3-dependent pathway.Importantly,administration of the ribozyme prevents tumor formation and growth in a hepatocellular cancer xenograft model in vivo.These studies show that a combined suppression of survivin protein expression by ribozyme induced hepatocellular cancer cell death in vitro and inhibited tumor growth in animal.The adenoviral ribozyme system described in this report offers a potential robust gene therapeutic strategy in liver cancer treatment. Oncolytic adenoviruses which replicate selectively in cancer cells and result in cancer-specific cytotoxicity,are a class of promising anticancer agents.Fruthermroe,this type of adenovirus has no cross-resistance to current treatments.The success is hingered upon how specific the oncolytic adenovirus to tumor cells.Using the natural promoter of the tumor selectively high expressing genes to drive the gene(s) essential to the replication has been widely tested with few promising results,especially in vivo experiments.In this thesis,a novel strategy has been designed and tested systematically.The cis-elements have been bioinformatically identified,that are shared by the over-expressing genes in the tumor vascular endothelial cells versus their normal counterparts.One thousand individual clones from a luciferase reporter library that were made with the trimers of each of six cis elements in combination varying with species,copy number and the arrangement,at the upstream of the human heat-shock gene Hsp70 promoter has been tested,were tested in transient transfection/reporter assay in a panel cell lines of different cancer origins.The cis-elements from the clones consistently give the higher promoter activity in all the cell lines have been transferred to the upstream of the Hsp70 promoter.Then we put the E1A gene encoding a n essential protein for the adenovirus replication,under the control of each of a panel of the Hsp70 promoter with the set of cis-elements.By a comprehensive analysis in cell culture and in animal model,one potentent oncolytic adenovirus has been identified for its potency is over two magnitudes higher than the ONYX-015 equivalent that has been approved by Chinese State FDA.