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Herpes simplex virus-1 thymidine kinase mutants created by semi-random sequence mutagenesis improve prodrug-mediated
M E Black1, M S Kokoris, P Sabo
1Department of Pharmaceutical Sciences, Washington State University, Pullman 99164-6534, USA. blackm@mail.wsu.edu
Abstract:
Cancer suicide gene therapy affords the prospect of using the most optimal genes available because the source of the therapeutic gene is often irrelevant. Currently, there are numerous preclinical and clinical trials to develop tumor ablative therapies that use viral, yeast, or bacterial genes. One such gene, the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) is widely used as a suicide gene in combination with ganciclovir. In the study reported here, a restricted set of random sequences (semi-random) was introduced into the active site of HSV-1 TK, and the resulting variants were selected on the basis of their ability to confer increased ganciclovir or acyclovir sensitivity to Escherichia coli. Sequence analysis demonstrated that functional mutants contained three to five amino acid substitutions that are unique and novel combinations. On the basis of enzyme assay results, three mutants were identified for further analysis in vitro. These three mutants conferred substantial increased sensitivity to both ganciclovir and acyclovir when compared with IC50s of wild-type TK expressing rat C6 glioma cells. One mutant, SR39, was further evaluated in a xenograft tumor model in nude mice. Expression of SR39 in tumors was shown to prevent tumor growth at prodrug dosages that did not affect wild-type HSV-1 TK-expressing tumors. The use of any of these mutants as a suicide gene should provide a more effective and safer alternative to wild-type TK, because lower, less immunosuppressive doses of ganciclovir will be necessary for tumor ablation, and the use of acyclovir may now be possible.
Insights
New herpes simplex virus type 1 thymidine kinase (HSV-1 TK) mutants show enhanced cancer suicide gene therapy. These novel TK variants increase sensitivity to ganciclovir and acyclovir, offering a safer, more effective tumor ablation alternative.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Cancer suicide gene therapy aims to develop effective tumor ablation strategies.
- Herpes simplex virus type 1 thymidine kinase (HSV-1 TK) is a commonly used suicide gene with ganciclovir.
- Optimization of HSV-1 TK is crucial for improving therapeutic efficacy and safety.
Purpose of the Study:
- To engineer novel HSV-1 TK variants with enhanced sensitivity to prodrugs.
- To evaluate the in vitro and in vivo efficacy of these engineered TK mutants.
- To assess the potential of these mutants as improved suicide genes for cancer therapy.
Main Methods:
- Semi-random mutagenesis was applied to the active site of HSV-1 TK.
- Variants were selected for increased sensitivity to ganciclovir or acyclovir in Escherichia coli.
- Enzyme assays and in vitro studies were performed on selected mutants.
- In vivo efficacy was tested using a xenograft tumor model in nude mice.
Main Results:
- Novel functional TK mutants with 3-5 amino acid substitutions were identified.
- Three mutants demonstrated significantly increased sensitivity to ganciclovir and acyclovir compared to wild-type TK.
- One mutant, SR39, effectively prevented tumor growth in vivo at non-toxic prodrug doses.
- SR39 showed superior efficacy compared to wild-type HSV-1 TK in a xenograft model.
Conclusions:
- Engineered HSV-1 TK mutants offer a more effective and potentially safer approach to cancer suicide gene therapy.
- These mutants enable tumor ablation with lower, less immunosuppressive doses of ganciclovir.
- The use of acyclovir as a prodrug may become feasible with these novel TK variants.