Low transduction efficiencies of viral and non-viral vectors still remain a major limitation in suicide gene therapy. The HSV-1 tegument protein VP22 can spread from cells where it is produced to surrounding recipient cells, thus making it a promising tool for compensation of inadequate gene transfer efficiencies. In our previous study, we focused on the optimization of the cytosine deaminase (CD) suicide gene system for the treatment of hepatocellular carcinoma. The fusion of yeast cytosine deaminase (YCD) to yeast uracil-phosphoribosyltransferase designated SuperCD was shown to be catalytically superior to the YCD gene in our previous study. The aim of our study was to investigate whether fusion of the bifunctional SuperCD suicide gene to VP22 could further potentiate suicide gene therapy efficiency. C- and N-terminal fusions of SuperCD linked in-frame with VP22 were created and cloned into recombinant adenoviral vectors. Under incubation with the prodrug 5-fluorocytosine (5-FC) a strong enhancement in suicide gene induced target cell cytotoxicity was observed whereby the C-terminal fusion of VP22 to SuperCD (VP22-SuperCD) caused the most tremendous decrease in IC50 compared to both Ad-SuperCD transduced and uninfected hepatoma control cells. Optimization of the bystander effect mediated by the intercellular transport of VP22-fusion proteins was demonstrated by cytotoxicity assays performed with a mixture of adenoviral transduced cells and naïve uninfected cells. Immunofluorescence analysis of adenoviral transduced COS-1 cells coplated with naïve HeLa cells further confirmed the unique property of VP22 for intercellular trafficking.

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