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) thymidine kinase (TK) has garnered much interest as a suicide gene for cancer ablation. As a means to improve the overall efficacy of the prodrug-gene activation approach, as well as to reduce ganciclovir-mediated toxicity, a large library of mutant thymidine kinases was generated and screened for the ability to enhance in vitro cell sensitivity to the prodrugs, ganciclovir (GCV) and acyclovir (ACV). Enzyme kinetics of one thymidine kinase mutant from this library that contains six amino acid substitutions at or near the active site reveals a distinct mechanism for providing enhanced prodrug-mediated killing in mammalian cells. In in vitro rat C6 cell prodrug sensitivity assays the TK mutant (mutant 30) achieves nanomolar IC50 values with GCV and ACV, in contrast to IC50values of 30 microM and >100 microM, respectively, for wild-type TK. In a mouse xenograft tumor model, growth of mutant 30 expressing 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 tumor growth is seen in tumors bearing the wild-type TK under the same conditions. The enhanced sensitization to prodrugs conferred by mutant 30 is apparently due to a 35-fold increase in thymidine Km which results in reduced competition between prodrug and thymidine at the active site. This provides mutant 30 a substantial kinetic advantage despite very high Kms for both ganciclovir and acyclovir. Molecular modeling of the mutations within the active site suggests that a tyrosine substitution at alanine 168 (A168) alters thymidine and prodrug interactions by causing catalytically important residues to move. The use of mutant 30 in place of the wild-type TK should provide a more effective gene therapy of cancer.
Kokoris
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MS
Chiroscience R & D, Inc., Bothell, WA 99164-6510, USA.
Sabo
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Adman
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Black
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D000212:Acyclovir / Q000627:therapeutic use; D000595:Amino Acid Sequence; D000818:Animals; D015774:Ganciclovir / Q000627:therapeutic use; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D005910:Glioma / Q000188:drug therapy / Q000628:therapy*; D018259:Herpesvirus 1, Human / Q000235:genetics*; D051379:Mice; D008819:Mice, Nude; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009368:Neoplasm Transplantation; D011355:Prodrugs / Q000627:therapeutic use; D051381:Rats; D013937:Thymidine Kinase / Q000235:genetics*; D014162:Transfection / Q000235:genetics; D014407:Tumor Cells, Cultured
D011355:Prodrugs; D013937:Thymidine Kinase; D015774:Ganciclovir; D000212:Acyclovir