A Helper-Dependent Capsid-Modified Adenovirus Vector Expressing Adeno-Associated Virus Rep78 Mediates Site-Specific Integration of a 27-Kilobase Transgene Cassette

Abstract

Random integration of viral gene therapy vectors and subsequent activation or disruption of cellular genes poses safety risks. Major efforts in the field are aimed toward targeting vector integration to specific sites in the host genome. The adeno-associated virus (AAV) Rep78 protein is able to target AAV integration to a specific site on human chromosome 19, called AAVS1. We studied whether this ability could be harnessed to achieve site-specific integration of a 27-kb transgene cassette into a model cell line for human hematopoietic cells (Mo7e). To deliver rep78 and the transgene to Mo7e cells, we used helper-dependent adenovirus (Ad) vectors containing Ad serotype 35 fiber knob domains (HD-Ad). An HD-Ad vector containing the rep78 gene under the control of the globin locus control region (LCR) (Ad.LCR-rep78) conferred Rep78 expression on Mo7e cells. Upon coinfection of Ad.LCRrep78 with an HD-Ad vector containing a 27-kb globin-LCR-green fluorescent protein (GFP) transgene cassette flanked by AAV inverted terminal repeats (ITRs) (Ad.AAV-LCR-GFP), transduced cells were cloned and expanded (without selection pressure), and vector integration was analyzed in clones with more than 30% GFP-positive cells. Vector integration into the AAVS1 region was seen in 30% of analyzed integration sites, and GFP expression from these integrants was stable over time. Of the remaining integration sites, 25% were within the genomic globin LCR. In almost 90% of sites, transgene integration occurred via the Ad ITR. This indicates that rescue of the AAV ITR-flanked transgene cassette from Ad.AAV-LCR-GFP is not required for Rep78-mediated integration into AAVS1 and that free ends within the vector genome can be created by breaks within the Ad ITRs, whose structure is apparently recognized by cellular "nicking" enzymes. The finding that 55% of all analyzed integration sites were either within the AAVS1 or globin LCR region demonstrates that a high frequency of targeted integration of a large transgene cassette can be achieved in human hematopoietic stem cell lines.For gene therapy applications, the development of vectors that target transgene integration to specific sites in the host genome is a major focus (7, 32). In this context, the ability of adeno-associated virus (AAV) to integrate into the host genome at a preferred site on chromosome 19q13.4q is of interest (13). This site, called AAVS1, is about 4 kb long and has been mapped to the first exon of myosin binding subunit 85 of the protein phosphatase 1. The AAVS1 site appears to be in an open chromatin conformation in the cell lines tested (31), which is thought to help AAV integration and viral-gene expression (15). Integration into this site is mediated by the large AAV Rep protein Rep68 or Rep78. Rep68/78 demonstrate sequence-specific endonuclease activity and ATP-dependent helicase activity and can mediate rescue/excision of DNA flanked by AAV ITRs in the presence of adenovirus (Ad) superinfection (24). Rep-mediated rescue requires the presence of both a Rep binding site (R...

Tightly regulated gene expression in human hematopoietic stem cells after transduction with helper-dependent Ad5/35 vectors

“…Our group is specifically interested in targeting transgene expression cassettes to specific sites in the genome of human hematopoietic stem cells (HSCs) by transient expression of proteins with site-specific endonuclease activity, such as the AAV Rep78 protein [1] and sequence-specific zinc-finger nucleases [2]. Gene transfer into HSCs by transfection or electroporation is hampered by low efficiency and high cytotoxicity.…”

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

“…In that case, GFP expression from the AAVS1 site was significantly higher than expression from other sites. 55 However, with the experiments conducted in our studies, it was not possible to determine the cause of the different b-gal activities observed.…”

“…However, in the rep-plus BGAL clones detection of green fluorescence or rep-sequences could also be explained by site-specific integrations mediated by the p5IEE in rep. Additionally, recent analysis with Ad/AAV vector, containing a 27 kb globin-LCR-GFP transgene also revealed recombination of the transgene into the genomic globin LCR site in 25% of the clones. 55 In gene therapy, homologous recombination of an introduced transgene with the endogenous gene can correct a mutation in the gene through replacement with the wild-type sequence. The possibility of homologous recombination between the transduced and the endogenous BGAL sequences was not assessed in this study but will be a crucial point in future studies.…”

A role for adeno-associated viral vectors in gene therapy

“…This integration occurs preferentially into a specific site of the q arm of chromosome 19 (AAVS1) in a Rep-dependent manner (Kotin et al, 1990;Kotin et al, 1991;Samulski et al, 1991;McCarty et al, 2004;Philpott et al, 2004;Wang and Lieber, 2006). Specific integration sites for the other AAV serotypes have not yet been identified.…”

“…some not yet identified cellular DNA repair mechanism (reviewed in Stender et al, 2007). These dsDNA intermediates integrate into a specific site of human chromosome 19 (19q13.3q-ter) called AAVS1 (Kotin et al, 1990;Kotin et al, 1991;Samulski et al, 1991;McCarty et al, 2004;Philpott et al, 2004;Wang and Lieber, 2006). In the absence of a co-infection by a helper virus (commonly adenovirus or herpesvirus), the integrated AAV genome remains in a latent state.…”