Improved long-term expression from helper virus-free HSV-1 vectors packaged using combinations of mutated HSV-1 proteins that include the UL13 protein kinase and specific components of the VP16 transcriptional complex

摘要

Background Herpes Simplex Virus (HSV-1) gene expression is thought to shut off recombinant gene expression from HSV-1 vectors; however, in a helper virus-free HSV-1 vector system, a number of promoters support only short-term expression. These results raise the paradox that recombinant gene expression remains short-term even in the absence of almost all (~99%) of the HSV-1 genome, HSV-1 genes, and HSV-1 gene expression. To resolve this paradox, we hypothesized that specific proteins in the HSV-1 virus particle shut off recombinant gene expression. In two earlier studies, we examined the effects on recombinant gene expression of packaging vectors using specific mutated HSV-1 proteins. We found that vectors packaged using mutated U_L13 (a protein kinase), or VP16, or U_L46 and/or U_L47 (components of the VP16 transcriptional complex) supported improved long-term expression, and vectors packaged using mutated U_L46 and/or U_L47 also supported improved gene transfer (numbers of cells at 4 days). These results suggested the hypothesis that specific proteins in the HSV-1 particle act by multiple pathways to reduce recombinant gene expression. To test this hypothesis, we examined combinations of mutated proteins that included both U_L13 and specific components of the VP16 transcriptional complex. Results A HSV-1 vector containing a neuronal-specific promoter was packaged using specific combinations of mutated proteins, and the resulting vector stocks were tested in the rat striatum. For supporting long-term expression, the preferred combination of mutated HSV-1 proteins was mutated U_L13, U_L46, and U_L47. Vectors packaged using this combination of mutated proteins supported a higher efficiency of gene transfer and high levels expression for 3 months, the longest time examined. Conclusion Vector particles containing this combination of mutated HSV-1 proteins improve recombinant gene expression. Implications of these results for strategies to further improve long-term expression are discussed. Moreover, long-term expression will benefit specific gene therapy applications.