Integration of active human β-galactosidase gene (100 kb) into genome using HSV/AAV amplicon vector

Vectors based on herpes simplex virus type-1 (HSV-1) permit delivery of transgenes of up to 150 kb, while the inverted terminal repeats and Rep of the adeno-associated virus (AAV) can confer site-specific integration into the AAVS1 site, which allows sustained expression of a transgene. In this study, combination of the viral elements in HSV/AAV hybrid vectors has been applied for the infectious transfer of the human lysosomal beta-galactosidase (BGAL) gene of 100 kb. Temporary expression and functional activity of beta-galactosidase (beta-gal) could be detected in human beta-gal-deficient patient and glioblastoma (Gli36) cells upon infection with the basic BGAL amplicon vector. Sustained expression of b-gal was achieved in Gli36 cells infected with rep-plus, but not rep-minus, HSV/AAV hybrid vectors. None of five clones isolated after rep-minus hybrid vector infection showed elevated beta-gal activity or site-specific integration. In contrast, 80% of the rep-plus clones possessed beta-gal activity at least twofold greater than normal levels for up to 4 months of continuous growth, and 33% of the clones exhibited AAVS1-specific integration of the ITR-flanked transgene. One of the rep-plus clones displayed integration of the ITR cassette only at the AAVS1 site, with no sequences outside the cassette detectable and beta-gal activity fourfold above normal levels. These data demonstrate AAVS1-specific integration of an entire genomic locus and expression of the transgene from the endogenous promoter mediated by an HSV/AAV hybrid vector.