Enhancement of tumor ablation by a selected HSV-1 thymidine kinase mutant

With the advent of gene therapy, herpes simplex virus type I (HSV-1) thymid ine kinase (TK) has garnered much interest as a suicide gene for cancer abl ation. As a means to improve the overall efficacy of the prodrug-gene activ ation approach, as well as to reduce ganciclovir-mediated toxicity, a large library of mutant thymidine kinases was generated and screened for the abi lity to enhance in vitro cell sensitivity to the prodrugs, ganciclovir(GCV) and acyclovir (ACV). Enzyme kinetics of one thymidine kinase mutant from t his library that contains six amino acid substitutions at or near the activ e site reveals a distinct mechanism for providing enhanced prodrug-mediated killing in mammalian cells. In in vitro rat C6 cell prodrug sensitivity as says the TK mutant (mutant 30) achieves nanomolar IC50 values with GCV and ACV, in contrast to IC50 values of 30 mu M and > 100 mu M, respectively, fo r wild-type TK. In a mouse xenograft tumor model, growth of mutant 30 expre ssing tumors is restricted by ganciclovir at a dose at least 10-fold lower than one that impedes growth of wild-type TK-expressing tumors. Furthermore , in the presence of GCV a substantial bystander effect is observable when only 20% of the tumor cells express mutant 30 whereas no restriction in tum or growth is seen in tumors bearing the wild-type TK under the same conditi ons. The enhanced sensitization to prodrugs conferred by mutant 30 is appar ently due to a 35-fold increase in thymidine K-m which results in reduced c ompetition between prodrug and thymidine at the active site. This provides mutant 30 a substantial kinetic advantage despite very high K(m)s for both ganciclovir and acyclovir. Molecular modeling of the mutations within the a ctive site suggests that a tyrosine substitution at alanine 168 (A168) alte rs thymidine and prodrug interactions by causing catalytically important re sidues to move. The use of mutant 30 in place of the wild-type TK should pr ovide a more effective gene therapy of cancer.