CREATION OF DRUG-SPECIFIC HERPES-SIMPLEX VIRUS TYPE-1 THYMIDINE KINASE MUTANTS FOR GENE-THERAPY

Herpes simplex virus type 1 (HSV-1) thymidine kinase is currently used as a suicide agent in the gene therapy of cancer, This therapy is bas ed on the preferential phosphorylation of nucleoside analogs by tumor cells expressing HSV 1 thymidine kinase, However, the use of HSV-1 thy midine kinase is limited in part by the toxicity of the nucleoside ana logs, We have used random sequence mutagenesis to create new HSV-1 thy midine kinases that, compared with wild-type thymidine kinase, render cells much more sensitive to specific nucleoside analogs, A segment of the HSV-1 thymidine kinase gene at the putative nucleoside binding si te was substituted with random nucleotide sequences, Mutant enzymes th at demonstrate preferential phosphorylation of the nucleoside analogs, ganciclovir or acyclovir, were selected from more than one million Es cherichia coli transformants, Among the 426 active mutants we have iso lated, 26 demonstrated enhanced sensitivity to ganciclovir, and 54 wer e more sensitive to acyclovir, Only 6 mutant enzymes displayed sensiti vity to both ganciclovir and acyclovir when expressed in E. coli, Anal ysis of 3 drug-sensitive enzymes demonstrated that 1 produced stable m ammalian cell transfectants that are 43-fold more sensitive to gancicl ovir and 20-fold more sensitive to acyclovir.