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In vitro evaluation of mutant HSV-1 thymidine kinases for suicide gene therapy
M S Kokoris1, P Sabo, M E Black
1Chiroscience R&D, Inc., Bothell, WA 98021, USA.
Abstract:
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.
Insights
Researchers engineered novel Herpes Simplex Virus type 1 thymidine kinase (HSV-1 TK) variants. These mutants enhance cancer gene therapy by increasing sensitivity to the less toxic prodrug acyclovir (ACV).
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Gene Therapy
Background:
- Herpes Simplex Virus type 1 thymidine kinase (HSV-1 TK) is a key suicide gene therapy agent for cancer.
- Current limitations include the toxicity of the prodrug ganciclovir (GCV).
Purpose of the Study:
- To identify novel HSV-1 TK variants with improved activity against prodrugs.
- To develop more effective and less toxic cancer gene therapy strategies.
Main Methods:
- Random mutagenesis of HSV-1 TK was performed.
- Screening of over a million mutants identified variants with altered activity towards ganciclovir and acyclovir (ACV).
Main Results:
- Ten HSV-1 TK variants were identified.
- Six mutants showed increased sensitivity to ACV, with one exhibiting an 8.5-fold lower IC50 compared to wild-type TK.
- These mutants possess three to six amino acid changes.
Conclusions:
- Novel HSV-1 TK variants offer enhanced sensitivity to acyclovir (ACV).
- These engineered enzymes could enable the use of non-toxic acyclovir at nanomolar concentrations in ablative gene therapy.
- This research advances cancer gene therapy by improving prodrug efficacy and reducing toxicity.