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Creation of drug-specific herpes simplex virus type 1 thymidine kinase mutants for gene therapy
M E Black1, T G Newcomb, H M Wilson
1Joseph Gottstein Memorial Cancer Research Laboratory, Department of Pathology, School of Medicine, University of Washington, Seattle, 98195-7705, USA.
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.
Insights
Researchers engineered new versions of Herpes simplex virus type 1 (HSV-1) thymidine kinase to improve cancer gene therapy. These modified enzymes increase cell sensitivity to cancer drugs like ganciclovir and acyclovir, potentially reducing toxicity.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Gene Therapy
Background:
- Herpes simplex virus type 1 (HSV-1) thymidine kinase is a suicide gene agent in cancer therapy.
- This therapy relies on tumor cells phosphorylating nucleoside analogs, but drug toxicity is a limitation.
Purpose of the Study:
- To engineer novel HSV-1 thymidine kinase variants with enhanced sensitivity to specific nucleoside analogs.
- To overcome the toxicity limitations associated with current HSV-1 thymidine kinase gene therapy.
Main Methods:
- Random sequence mutagenesis was applied to a key segment of the HSV-1 thymidine kinase gene.
- Mutant enzymes were selected for preferential phosphorylation of ganciclovir or acyclovir in Escherichia coli.
- Over one million transformants were screened to identify active mutants.
Main Results:
- 426 active HSV-1 thymidine kinase mutants were isolated.
- 26 mutants showed enhanced sensitivity to ganciclovir, and 54 to acyclovir.
- One mutant generated mammalian cell transfectants 43-fold more sensitive to ganciclovir and 20-fold more sensitive to acyclovir.
Conclusions:
- Engineered HSV-1 thymidine kinase variants demonstrate significantly improved drug sensitivity.
- These modified enzymes hold promise for enhancing the efficacy and safety of cancer gene therapy.
- Further development could lead to more effective treatments with reduced side effects.