Direct retroviral delivery of human cytochrome P4502B6 for gene-directed enzyme prodrug therapy of cancer

Human cytochrome P450 2B6 (CYP2B6) metabolizes the prodrug cyclophosphamide (CPA) to produce phosphoramide mustard that cross-links DNA leading to cel l death. We have constructed a novel retroviral vector encoding CYP2B6 (des ignated "MetXia-P450") and used it to transduce the human tumor cell lines HT29 and T47D. MetXia-P450 transduction sensitised these cells to the cytot oxic effects of the prodrug CPA. Results from in vitro experiments demonstr ated adverse effects on the clonogenic survival of cyclophosphamide-treated cells transduced with MetXia-P450. Cytotoxic activity accompanied by bysta nder effect was particularly evident in 3-D multicellular spheroid models s uggesting that this in vitro system may be a more appropriate model for ass essing the efficacy of gene directed-enzyme prodrug therapy (GDEPT). We hav e applied this approach in a clinically relevant gene therapy protocol on e stablished subcutaneous tumor xenografts. These studies show for the first time the efficacy of a P450-based GDEPT strategy mediated by a direct retro viral gene transfer in vivo.