Improved Semliki Forest virus vectors for receptor research and gene therapy

We have modified Semliki Forest virus (SFV) vectors to broaden their applic ation range. Here we describe a series of site-directed mutagenesis experim ents on the SFV subgenomic 26S promoter to down-regulate the heterologous g ene expression. Several mutants showed a dramatic effect on transgene expre ssion levels in BHK cells. The luciferase activity was reduced to approxima tely 30%, 3%, and 1% compared to the wild type promoter. Similarly, a decre ase in beta -galactosidase activity was observed in BHK cells and after inj ection into the striatum of male Wistar rats. Novel non-cytopathogenic and temperature-sensitive SFV vectors have recently been developed by introduct ion of point mutations in the viral nonstructural genes nsP2 and nsP4. Thes e vectors do not show the typical shut down of host cell protein synthesis after SFV infections and therefore allow for a substantially prolonged surv ival of host cells. Both the mutant vectors demonstrating lower and more ph ysiological expression levels and the non-cytopathogenic vectors should be valuable tools for various applications within receptor research. Furthermo re, recent studies suggest that SFV vectors can be efficient gene delivery vehicles for gene therapy applications.