RETROVIRAL TRANSFER OF HUMAN CYTOCHROME-P450 GENES FOR OXAZAPHOSPHORINE-BASED CANCER GENE-THERAPY

Cyclophosphamide (CPA) and ifosfamide (IFA) are widely used anticancer prodrugs that are bioactivated in the liver by specific cytochrome P4 50 enzymes (CYPs), The therapeutic activity of these antitumor agents can be compromised by a low therapeutic index that is, in part, due to the systemic distribution of activated drug metabolites. Here, recomb inant retroviruses mere used to deliver six different CPA- or IFA-meta bolizing human CYP genes to 9L gliosarcoma cells: 2B6, 2C8, 2C9, 2C18 ((MeP385 and Thr(385) alleles), 2C19, and 3A4. Intratumoral cytochrome P450 expression conferred substantial sensitivity to CPA cytotoxicity , with the most dramatic effects seen with CYP2B6, Strong CPA chemosen sitivity was also seen following transduction of CYP2C18-Met, despite a very low level of CYP protein expression (>60-fold lower than that o f 2B6), In contrast to CPA, the cytotoxicity of IFA was greatest towar d tumor cells transduced with CYP3A4, followed by CYPs 2B6 and 2C18-Me t. A substantial further increase in chemosensitivity was achieved upo n transduction of 2B6 or 2C18-Met-expressing tumor cells with P450 red uctase, which provided for more efficient intratumoral prodrug activat ion and cytotoxicity at lower drug concentrations. With 2B6- plus P450 reductase-transduced tumor cells, CPA but not IFA conferred a strong cell contact-independent bystander cytotoxic effect on non-P450-expres sing 9L cells. CPA treatment of tumors that were transduced with 2B6 o r 2C18-Met together with P450 reductase and were grown s.c. in immunod eficient mice resulted in a large enhancement of the liver P450-depend ent antitumor effect seen with control 9L tumors, with no apparent inc rease in host toxicity (growth delay of >25-50 days in P450-expressing tumors versus similar to 5-6 days without P450), CYP2B6 plus P450 red uctase and CYP2C18-Met plus P450 reductase thus appear to be excellent gene combinations for use with CPA in P450/prodrug activation-based c ancer gene therapy.