Herpes simplex virus type 1/adeno-associated virus rep+ hybrid amplicon vector improves the stability of transgene expression in human cells by site-specific integration

Herpes simplex virus type 1 (HSV-1) amplicon vectors are promising gene delivery tools, but their utility in gene therapy has been impeded to some extent by their inability to achieve stable transgene expression. In this study, we examined the possibility of improving transduction stability in cultured human cells via site-specific genomic integration mediated by adeno-associated virus (AAV) Rep and inverted terminal repeats (ITRs). A rep(-) HSV/AAV hybrid amplicon vector was made by inserting a transgene cassette flanked with AAV ITRs into an HSV-1 amplicon backbone, and a rep(+) HSV/AAV hybrid amplicon was made by inserting rep68/78 outside the rep- vector 3' AAV ITR sequence. Both vectors also had a pair of loxP sites flanking the ITRs. The resulting hybrid amplicon vectors were successfully packaged and compared to a standard amplicon vector for stable transduction frequency (STF) in human 293 and Gli36 cell lines and primary myoblasts. The rep+, but not the rep-, hybrid vector improved STF in all three types of cells; 84% of Gli36 and 40% of 293 stable clones transduced by the rep(+) hybrid vector integrated the transgene into the AAVS1 site. Due to the difficulty in expanding primary myoblasts, we did not assess site-specific integration in these cells. A strategy to attempt further improvement of STF by "deconcatenating" the hybrid amplicon DNA via Cre-loxP recombination was tested, but it did not increase STF. These data demonstrate that introducing the integrating elements of AAV into HSV-1 amplicon vectors can significantly improve their ability to achieve stable gene transduction by conferring the AAV-Iike capability of site-specific genomic integration in dividing cells.