Herpes simplex virus-1 thymidine kinase mutants created by semi-random sequence mutagenesis improve produrg-mediated tumor cell killing

Cancer suicide gene therapy affords the prospect of using the most optimal genes available because the source of the therapeutic gene is often irrelev ant, Currently, there are numerous preclinical and clinical trials to devel op tumor ablative therapies that use viral, yeast, or bacterial genes. One such gene, the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) is widely used as a suicide gene in combination with ganciclovir, In the stud y reported here, a restricted set of random sequences (semi-random) was int roduced into the active site of HSV-1 TK, and the resulting variants were s elected on the basis of their ability to confer increased ganciclovir or ac yclovir sensitivity to Escherichia coli, Sequence analysis demonstrated tha t functional mutants contained three to five amino acid substitutions that are unique and novel combinations. On the basis of enzyme assay results, th ree mutants were identified for further analysis in vitro, These three muta nts conferred substantial increased sensitivity to both ganciclovir and acy clovir when compared with IC(50)s of wild-type TR expressing rat C6 glioma cells. One mutant, SR39, was further evaluated in a xenograft tumor model i n nude mice. Expression of SR39 in tumors was shown to prevent tumor growth at prodrug dosages that did not affect wild-type HSV-I TK-expressing tumor s. The use of any of these mutants as a suicide gene should provide a more effective and safer alternative to wild-type TK, because lower, less immuno suppressive doses of ganciclovir will be necessary for tumor ablation, and the use of acyclovir may now be possible.