In vivo cancer gene therapy by adenovirus-mediated transfer of a bifunctional yeast cytosine deaminase/uracil phosphoribosyltransferase fusion gene

Direct transfer of prodrug activation systems into tumors was demonstrated to be an attractive method Fur the selective in vivo elimination of tumor c ells. However, most current suicide gene therapy strategies are still handi capped by a poor efficiency of in vivo gene transfer and a limited bystande r cell killing effect, In this study, we describe a novel and highly potent suicide gene derived from the Saccharomyces cerevisiae cytosine deaminase (FCY1) and uracil phosphoribosyltransferase genes (FUR1). This suicide gene , designated FCU1, encodes a bifunctional chimeric protein that combines th e enzymatic activities of FCY1 and FUR1 and efficiently catalyzes the direc t conversion of 5-FC, a nontoxic antifungal agent, into the toxic metabolit es 5-fluorouracil and 5-fluorouridine-5'-monophosphate, thus bypassing the natural resistance of certain human tumor cells to 5-fluorouracil, Unexpect edly, although the uracil phosphoribosyltransferase activity of FCU1 was eq uivalent to that encoded by FUR1, its cytosine deaminase activity was 100-f old higher than the one encoded by FCY1. As a consequence, tumor cells tran sduced with an adenovirus expressing FCU1 (Ad-FCU1) were sensitive to conce ntrations of 5-FC 1000-fold lower than the ones used for cells transduced w ith a vector expressing FCY1 (Ad-FCY1). Furthermore, bystander cell killing was also more effective in cells transduced with Ad-FCU1 than in cultures infected with Ad-FCY1 or Ad-FUR1, alone or in combination. Finally, intratu moral injections of Ad-FCU1 into allo- or xenogeneic tumors implanted s.c. into mice, with concomitant systemic administration of 5-FC, led to substan tial delays in tumor growth. These unique properties make of the FCU1/5-FC prodrug activation system a novel and powerful candidate for cancer gene th erapy strategies.