In vitro evaluation of mutant HSV-1 thymidine kinases for suicide gene therapy

Herpes Simplex Virus type I (HSV-I) thymidine kinase (TK) is cur currently the most widely used suicide agent for gene therapy of cancel: Tumor cells that express HSV-I thymidine kinase ale rendered sensitive to prodrugs due to preferential phosphorylation by this enzyme. While ganciclovir (GCV) is the prodrug of choice for use with TK, this approach is limited in part by the toxicity of this prodrug. From a random mutngenesis library of over a m illion mutant thymidine kinases, ten thymidine kinase variants were identif ied on the basis of activity towards ganciclovir and acyclovir (Black ME, N ewcomb TG, Wilson H-MP and Loeb LA: Proc. Natl. Acad. Sci. U.S.A. 93: 3525- 3529, 1996). Six mutants described here contain three to sh amino acid chan ges and render mammalian cells more sensitive to acyclovir (ACV) including one that demonstrates an 8.5-fold reduction in IC50 compared to wild-type T K. These novel enzymes could provide benefit to ablative gene therapy by no w making it feasible to use the relatively non-toxic acyclovir at nanomolar concentrations.