A New Acycloguanosine-Specific Supermutant of Herpes Simplex Virus Type 1 Thymidine Kinase Suitable for PET Imaging and Suicide Gene Therapy for Potential Use in Patients Treated with Pyrimidine-Based Cytotoxic Drugs

The herpes simplex virus type 1 thymidine kinase (HSV1 -tk) gene is widely used as a suicide gene in combination with ganciclovir (GCV) and as a nuclear imaging reporter gene with an appropriate reporter probe. Wild-type HSV1 -tk recognizes a variety of pyrimidine and acycloguanosine nucleoside analogs, including clinically used antiviral drugs. PET of HSV1-tk reporter gene expression will be compromised in patients receiving nucleoside-based antiviral treatment. With the use of an acycloguanosine-specific mutant of the enzyme, PET of HSV1-tk reporter gene expression can be successfully performed with acyclo-guanosine-based radiotracers without interference from pyrimidine-based antiviral drugs. Methods: The levels of expression of wild-type HSV1-tk and HSV1-A167Ytk, HSV1-sr39tk, and HSV1 -A167Ysr39tk mutants fused with green fluorescent protein (GFP) and transduced into U87 cells were normalized to the mean fluorescence of GFP measured by fluorescence-activated cell sorting. The levels of enzymatic activities of wild-type HSV1-tk and its mutants were compared by 2-h in vitro radiotracer uptake assays with 3 H-2'-fluoro-2'-deoxy-1-β-D-arabinofuranosyl-5-ethyluracil ( 3 H-FEAU), 3 H-pencyclovir ( 3 H-PCV), and 3 H-GCV and by drug sensitivity assays. PET with 18 F-FEAU and 18 F-9-[4-fluoro-3-(hydroxymethyl)butyl]guanine ( 18 F-FHBG) was performed in mice with established subcutaneous tumors, expressing wild-type HSV1-tk and its mutants, followed by tissue sampling. Results: FEAU accumulation was not detected in HSV1-A167Ysr39tk-expressing cells and xenografts. Lack of conversion of pyrimidine derivatives by the HSV1 -A167Ysr39tk supermutant was also confirmed by a drug sensitivity assay, in which the 50% inhibitory concentrations for thymine 1-β-D-arabinofuranoside and bromovinyldeoxyuridine were found to be similar to those in nontransduced cells. In contrast, we found that HSV1-A167Ysr39tk could readily phosphorylate 3 H-GCV at levels similar to those of wild-type HSV1-tk and HSV1-A167Ytk but showed enhanced activity with 3 H-PCV in vitro and with 18 F-FHBG in vivo. Conclusion: We developed a new reporter gene, HSV1-A167Ysr39tk, which exhibits specificity and high phosphorylation activity for acycloguanosine derivatives. The resulting supermutant can be used for PET with 18 F-FHBG and suicidal gene therapy protocols with GCV in patients treated with pyrimidine-based cytotoxic drugs.