In vitro evaluation of mutant HSV-1 thymidine kinases for suicide gene therapy.
Herpes Simplex Virus type 1 (HSV-1) thymidine kinase (TK) is currently the most widely used suicide agent for gene therapy of cancer. Tumor cells that express HSV-1 thymidine kinase are 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 mutagenesis library of over a million mutant thymidine kinases, ten thymidine kinase variants were identified on the basis of activity towards ganciclovir and acyclovir (Black ME, Newcomb 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 six amino acid changes and render mammalian cells more sensitive to acyclovir (ACV) including one that demonstrates an 8.5-fold reduction in IC50 compared to wild-type TK. These novel enzymes could provide benefit to ablative gene therapy by now making it feasible to use the relatively non-toxic acyclovir at nanomolar concentrations.
Kokoris
M S
MS
Chiroscience R&D, Inc., Bothell, WA 98021, USA.
Sabo
P
P
Black
M E
ME
D000212:Acyclovir / Q000633:toxicity*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects*; D003001:Cloning, Molecular; D006224:Cricetinae; D015774:Ganciclovir / Q000633:toxicity*; D015723:Gene Library; D015316:Genetic Therapy; D049452:Green Fluorescent Proteins; D018259:Herpesvirus 1, Human / Q000201:enzymology*; D006641:Histidinol / Q000633:toxicity; D019382:Human Growth Hormone / Q000235:genetics; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry; D013937:Thymidine Kinase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics*; D014162:Transfection
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D019382:Human Growth Hormone; D049452:Green Fluorescent Proteins; D006641:Histidinol; D013937:Thymidine Kinase; D015774:Ganciclovir; D000212:Acyclovir