Adenovirus-mediated gene transfer of enhanced Herpes simplex virus thymidine kinase mutants improves prodrug-mediated tumor cell killing

Abstract

The Herpes simplex virus 1 (HSV) thymidine kinase (tk) suicide gene together with ganciclovir (GCV) have been successfully used for the in vivo treatment of various solid tumors and for the ablation of unwanted transfused stem cells in recent clinical trials. With the aim of improving this therapeutic system, we compared the potential efficacy of adenoviral (Ad) vectors expressing enhanced tk mutants in vitro and in vivo. The previously created HSV-tk mutants dm30 and sr39, created by random sequence mutagenesis, were inserted into a standard Ad.RSV E1 À E3À backbone using homologous recombination. GCV killing of Ad.HSV-tk, Ad.dm30-tk and Ad.sr39-tk was assessed in various tumor cell lines with a cell proliferation assay. Cells expressing the two TK mutants were two-to-five-fold more sensitive to GCV when compared with Ad.HSV-tk transduced cells in all cell lines tested (five human mesotheliomas, one human lung cancer, a human cervical carcinoma, a mouse fibrosarcoma, and a rat glioma line) at equal TK expression levels. Flank tumor models, including cell-mixing studies, assessed the in vivo efficacy of the engineered viruses in BALB/C and SCID mice. In all animal studies, Ad.dm30-tk and Ad.sr39-tk showed more tumor growth inhibition than Ad.HSV-tk when GCV was administered. The use of adenovirus-mediated gene transfer of both tk mutants dm30-tk and sr39-tk for cancer suicide gene therapy should provide a more effective and safer alternative to wild-type HSV-tk.

The carcinoma-specific epithelial glycoprotein-2 promoter controls efficient and selective gene expression in an adenoviral context

“…SR39TK is a mutant TK displaying superior sensitivity to GCV compared to wild-type TK. 20,21 In this study, we demonstrate that the EGP-2 promoter mediates efficient and selective killing of tumor cells in vitro and maintains activity and selectivity in vivo in an adenoviral context. We show that the EGP-2 promoter is a promising tool in cancer gene therapy to tackle a broad range of tumor types.…”

“…SR39TK can convert the nontoxic prodrug GCV resulting in toxic metabolites, ultimately causing cell death and displays a superior sensitivity to GCV compared to wild-type TK. 20,21 The 1.2 kb EGP-2 promoter controlled selective cell killing of EGP-2-positive cells in a GCV dosedependent manner. However, no toxicity was seen in an EGP-2-negative cell line in contrast to the CMV promoter, which controlled efficient cell killing in all cell lines tested.…”

Frontiers in Suicide Gene Therapy of Cancer

“…The adenovirus engineered with the H19 enhancer / DMD-H19 promoter complex induces apoptosis only in the cancer cells with loss of imprinting of the insulin-like growth factor 2 gene (IGF2). Artificial "death switches" are introduced into cancer cells by adenoviruses to initiate apoptosis [124][125][126][127][128][129][130]. Replication-competent adenovirusmediated suicide gene therapy (ReCAP) is in the clinical trials for newly-diagnosed prostate cancer.…”

Genetic strategies for brain tumor therapy

“…32 Strategies to improve therapy include optimizing delivery regimens, the timing of gene and drug delivery being a crucial factor in therapeutic success. 33 TK with increased enymatic activity, generated by mutagenesis, enhanced GCV sensitivity in flank tumor models, 34 and new GCV formulations with improved bioavailability are in development. 35 The TK bystander effect is enhanced by gap junctions, and codelivery of the gap junction protein connexin-43 synergistically increased effectiveness.…”