Creation of drug-specific herpes simplex virus type 1 thymidine kinase mutants for gene therapy.
- 16 April 1996
- journal article
- research article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences
- Vol. 93 (8) , 3525-3529
- https://doi.org/10.1073/pnas.93.8.3525
Abstract
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 based on the preferential phosphorylation of nucleoside analogs by tumor cells expressing HSV-1 thymidine kinase. However, the use of HSV-1 thymidine kinase is limited in part by the toxicity of the nucleoside analogs. We have used random sequence mutagenesis to create new HSV-1 thymidine 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 site was substituted with random nucleotide sequences. Mutant enzymes that demonstrate preferential phosphorylation of the nucleoside analogs, ganciclovir or acyclovir, were selected from more than one million Escherichia coli transformants. Among the 426 active mutants we have isolated, 26 demonstrated enhanced sensitivity to ganciclovir, and 54 were more sensitive to acyclovir. Only 6 mutant enzymes displayed sensitivity to both ganciclovir and acyclovir when expressed in E. coli. Analysis of 3 drug-sensitive enzymes demonstrated that 1 produced stable mammalian cell transfectants that are 43-fold more sensitive to ganciclovir and 20-fold more sensitive to acyclovir.Keywords
This publication has 31 references indexed in Scilit:
- The three‐dimensional structure of thymidine kinase from Herpes simplex virus type 1FEBS Letters, 1995
- Gene Therapy for the Treatment of Recurrent Pediatric Malignant Astrocytomas with In Vivo Tumor Transduction with the Herpes Simplex Thymidine Kinase Gene/Ganciclovir System. Childrens Hospital, Los Angeles, CaliforniaHuman Gene Therapy, 1994
- Gene Therapy for the Treatment of Malignant Brain Tumors with In Vivo Tumor Transduction with the Herpes Simplex Thymidine Kinase Gene/Ganciclovir System. Iowa Methodist Medical Center, Des Moines, IowaHuman Gene Therapy, 1994
- Gene Therapy for the Treatment of Brain Tumors Using Intra-Tumoral Transduction with the Thymidine Kinase Gene and Intravenous Ganciclovir. National Institutes of HealthHuman Gene Therapy, 1993
- Identification of important residues within the putative nucleoside binding site of HSV-1 thymidine kinase by random sequence selection: Analysis of selected mutants in vitroBiochemistry, 1993
- Gene therapy of maliganant brain tumors: A rat glioma line bearing the herpes simplex virus type 1‐thymidine kinase gene and wild type retrovirus kills other tumor cellsJournal of Neuroscience Research, 1992
- In Vivo Gene Transfer with Retroviral Vector-Producer Cells for Treatment of Experimental Brain TumorsScience, 1992
- Viral thymidine kinases and their relativesPharmacology & Therapeutics, 1992
- Artificial mutants generated by the insertion of random oligonucleotides into the putative nucleoside binding site of the HSV-1 thymidine kinase geneBiochemistry, 1991
- Herpesviral Deoxythymidine Kinases Contain a Site Analogous to the Phosphoryl-binding Arginine-rich Region of Porcine Adenylate Kinase; Comparison of Secondary Structure Predictions and ConservationJournal of General Virology, 1990