Thymidine kinase-deleted vaccinia virus expressing purine nucleoside phosphorylase as a vector for tumor-directed gene therapy

Tumor-directed gene therapy faces many obstacles. Lack of tissue targeting and low in vivo transduction efficiency represent some of the limitations f or a successful therapeutic outcome. A thymidine kinase-deleted mutant vacc inia virus has been shown in marker studies to replicate selectively in tum or tissue in animal models. Purine nucleoside phosphorylase (PNP), from E. coil, converts the nontoxic prodrug 6-methylpurine deoxyriboside (6-MPDR) t o the toxic purine 6-methylpurine. In this study, we investigated the cytot oxic properties of PNP, expressed by an optimized synthetic early/late prom oter in a vaccinia virus (vMPPNP). In vitro cytotoxicity of psoralen-inacti vated vMPPNP (1 mu g of psoralen, 4 min of LWUV [365 nm]) at the maximum to lerated dose (MTD) of 6-MPDR (80 mu M) reduced cell viability by day 3 to 1 .7%. At an MOI of 0.002, replication-competent vMPPNP and 6-MPDR (80 mu M) caused reduction of cell viability to 19.8% within 4 days. Furthermore, the re was complete abrogation of viral replication after intracellular convers ion of prodrug into the active toxin. The potency of such a system was simi lar among all histologies tested. Finally, the cytotoxic efficacy has been shown to be more rapid and complete than that of cytosine deaminase (CD), a more established enzyme/prodrug system. When virus was delivered intraperi toneally into athymic mice with hepatic metastases, followed by administrat ion of prodrug, there was a significant prolongation of survival and a 30% cure rate. In summary, owing to its tumor-targeting capabilities, high tran sduction efficiency, and high gene expression, a vaccinia virus expressing PNP could prove to be a potent and valuable vector for tumor-targeted gene therapy.