742. Generation and Evaluation of a New Replication-Competent Retrovirus (RCR) Vector Coding for the SuperCD Suicide Gene

Abstract

Limited efficiency of in vivo gene transfer constitutes the main problem in cancer gene therapy applications employing replication-defective retroviral (RDR) vectors based on Moloney murine leukaemia virus (MoMLV). Therefore, replication-competent retroviral (RCR) vectors have been developed being able to transmit inserted transgenes very efficiently not only in cell culture, but also throughout entire tumor tissues. For efficient elimination of transduced tumor cells RCRs encoding well-known suicide genes like herpes simplex virus thymidine kinase (HSV TK) or yeast cytosine deaminase (YCD) have been made. Here, we generated a new RCR carrying the highly effective SuperCD gene which is composed of a bifunctional fusion suicide gene of YCD and yeast uracil phosphoribosyltransferase (YUPRT). We then compared the in vitro killing potential of this viral vector with the parental one (coding for YCD only). Viral vectors were produced by transient transfection of 293T producer cells with plasmids containing the proviral DNA. Viral supernatant was harvested and titered using real-time-PCR. Human hepatoma cells were transduced with each vector with equal multiplicity of infections; subsequently, incubation with the prodrug 5-FC was carried out. We observed enhanced cell cytotoxicity for the new RCR-SuperCD vector compared to parental RCR-YCD vector. Our results demonstrate that RCR-SuperCD vectors are more effective in toxifying the 5-FC prodrug.