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

Abstract

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 ( 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-like capability of site-specific genomic integration in dividing cells.

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

“…Amplicon vectors containing only non-coding elements from HSV-1 can transfer entire genomic loci up to 150 kb 2,9-11 and studies with HSV/AAV vectors have verified sustained expression of the transgenes integrated into the AAVS1 site, so far only with cDNAs under viral promoters. [16][17][18]29 The present study extends this HSV/AAV strategy by demonstrating stable delivery of a 133 kb genomic sequence containing an active BGAL gene. Here, the entire human lysosomal BGAL gene was transferred to cells in culture using an HSV or HSV/AAV hybrid amplicon vector, respectively.…”

“…This is in comparison to HSV/AAV hybrid vectors containing about 10 kb as described by Heister et al 18 where the reduction in titers was much more dramatic (100-1000-fold), perhaps because the smaller size of the amplicon allowed more efficient transfection and higher levels of Rep during packaging which would inhibit replication of amplicon DNA and helper virus genome. A negative effect of Rep on virus replication has been seen with HSV and other HSV/AAV sequences, 17,18,29,46 and even more so with Ad and Ad/AAV sequences. [47][48][49] On the other hand, AAV Rep had a positive effect on stable integration frequency.…”

“…29 In 293 cells most clones displayed site-specific integration with no sequences besides what was contained within the ITR cassette being retained, although in some clones with random integration vector sequences were also detectable. 18 In tests with mouse embryonic fibroblasts containing the human AAVS1 site, 50% of the clones had sitespecific integration of the transgene cassette but only one clone had no detectable vector sequences.…”

“…In several studies using these HSV/AAV hybrid vectors sustained transgene expression was achieved by site-specific integration of the ITR cassette. [16][17][18]29 In one case expression of green fluorescent protein (GFP) encoded in the transgene could be detected in dividing cells for up to 12 months, with GFP sequences integrated into the AAVS1 site and no additional vector sequences detectable in the majority of the 293 clones analyzed. 18 The largest reported insert successfully integrated in the AAVS1 site using an HSV/AAV hybrid vector was 22 kb, but in that case the transgene was not expressed.…”

HSV-1 amplicon vector-mediated expression of ATM cDNA and correction of the ataxia–telangiectasia cellular phenotype

“…AAV vectors have a small transgene capacity (4.5 kb), and stably integrate into the genome of dividing and nondividing human cells, preferentially into the AAVS1 site of human chromosome 19 in the presence of the AAV Rep proteins. 44 Recent studies from our group and collaborators demonstrate that by flanking the transgene in amplicon vectors with the ITR elements and a Rep expression cassette from AAV, site-specific integration of the transgene at the AAVS1 chromosomal site can be achieved in about 10-30% of transduced human cells, 45,46 as predicted from studies of AAV. 44,47 We have now generated transgenic mice bearing the human AAVS1 locus, 48 which can be crossed to A-T heterozygous mice to create ATM K-O mice with AAVS1 sites, as a model of the human genome.…”