A three-plasmid system for construction of armed oncolytic ade...

There is growing interest in the use of oncolytic virus as a tool in cancer gene therapy. However, construction of oncolytic adenovirus (Ad) is not an easy task due to lack of convenient, robust methods. A three-plasmid system was introduced for construction of armed oncolytic Ad. Besides the pShuttle-CMV and pAdEasy-1, a third plasmid (pTE-ME1), harboring the E1 region of Ad5, was generated and included in this system. In pTE-ME1, the promoter of E1A was deleted and replaced with a multiple-cloning site (MCS). A therapeutic gene and tissue-specific promoter (TSP) could be inserted routinely into the MCS of pShuttle-CMV and pTE-ME1, respectively. The modified E1 region could then be excised from pTE-ME1 and integrated into the therapeutic gene-containing pShuttle-CMV to form the final shuttle plasmid. This shuttle plasmid was recombined with pAdEasy-1 in Escherichia coli strain BJ5183 to generate Ad plasmid. Finally, the oncolytic Ad could be rescued in Ad plasmid-transfected packaging cells. The GFP gene and the promoter of telomerase reverse transcriptase (TERTp) were chosen as the transgene and TSP, respectively, to test this system. Two oncolytic Ads, Ad-GFP-TPE and Ad-GFP-D19K, were generated successfully. Their oncolytic and replicating abilities were investigated in TERT-positive tumor cells. The results suggest that the three-plasmid system was practicable and could be used to construct other transcriptionally regulated oncolytic Ads carrying a therapeutic gene.