A neuroblastoma-selective suicide gene therapy approach using the tyrosine hydroxylase promoter

Abstract

In this study, selective expression of therapeutic transgenes was evaluated in neuroblastoma cells. Promoter fragments of the genes for neuron-specific enolase (NSEp), tyrosine hydroxylase (THp), and dopamine-β-hydroxylase (DBHp) were studied in neuroblastoma and nonneuronal cell lines by transient transfection experiments using fluorescence-activated cell sorting (FACS) analysis of enhanced green fluorescent protein (egfp) and luciferase (luc+) assay. Both reporter gene assays revealed a neuroblastoma-selective expression mediated by NSEp and THp, whereas DBHp was active only in a murine neuroblastoma cell line. Reporter gene expression by NSEp in neuroblastoma cells was markedly higher than expression by THp, but NSEp also showed considerable background activity in nonneuronal cells. THp-driven expression of egfp was 35-fold higher in human neuroblastoma MHH-NB11 compared with nonneuronal HeLa cells. Thus, THp was chosen for a neuroblastoma-selective suicide gene therapy approach using the herpes simplex virus type 1 thymidine kinase (HSV-tk)/ganciclovir (GCV) system. A retrovirus vector that contained an expression cassette of a HSV-tk/egfp fusion gene and THp in antisense orientation was generated. Stably transduced human neuroblastoma cells and nonneuronal cell lines were generated, and HSV-tk/egfp expression was measured by FACS and GCV cytotoxicity assay. There was a 2.2-fold difference in green fluorescence and a 1.4-fold difference in cell killing between the human neuroblastoma MHH-NB11 and HeLa cells after HSV-tk/egfp gene transfer. The overall difference in THp-HSV-tk/egfp-mediated cell killing between neuroblastoma and nonneuronal tumor cell lines was statistically significant (P = 0.001). In conclusion, the present study demonstrated the feasibility of a neuroblastoma-selective gene therapy approach using the THp/HSV-tk/egfp expression cassette.