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

Abstract

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 b-galactosidase (BGAL) gene of 100 kb. Temporary expression and functional activity of b-galactosidase (b-gal) could be detected in human b-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 b-gal activity or site-specific integration. In contrast, 80% of the rep-plus clones possessed b-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 b-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.

HSV-1 amplicon viral vector-mediated gene transfer to human bone marrow-derived mesenchymal stem cells

“…The HSV/AAV hybrid vectors has been shown to specifically integrate into the AAVS1 site of the human chromosome 19 and extend transgene expression for up to 12 months. [48][49][50] Long-term retention could also be achieved through the incorporation of the EBV nuclear antigen (EBNA-1) and the origin of DNA replication (oriP). 37 Using HSV/EBV hybrid, transgene expression in 4-week-old mice was observed for over 3 weeks.…”

Progress and prospects: Biological properties and technological advances of herpes simplex virus type 1-based amplicon vectors

“…Transient expression and functional activity of b-galactosidase was detected in human b-galactosidase-deficient patient cells on infection with the BGAL vector in the presence of Rep expression. 16 Most of the Rep-positive clones possessed greater than normal b-galactosidase activity for up to 4 months of continuous growth and, furthermore, 33% of these clones exhibited AAV1 sitespecific integration of the ITR-flanked transgene.…”

Non-Viral Gene Therapy Vectors Carrying Genomic Constructs

“…It has been shown that human transgenes flanked by ITRs can integrate into the AAVS1, with the minimal requirement for expression of viral Rep in cultured human cells (Cortés et al, 2008), and in vivo in transgenic mice carrying the human AAVS1 Recchia et al, 2004) . The AAV system has also been shown to be able to integrate large genes of 100kb in size into the AAVS1 (Oehmig et al, 2007). These studies have demonstrated the effectiveness of AAV-based vectors but again safety issues have emerged.…”