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. AAV Rep78 is able to target AAV integration vectors to a specific site on human chromosome 19 called AAVS1. We studied whether this ability can be harnessed to achieve site-specific integration of a 27 kb transgene cassette into a model cell line for hematopoietic cells (Mo7e). To deliver Rep78 and the transgene to MO7e cells we used helper-dependent adenovirus vectors containing Ad serotype 35 fibers (HD-Ad). An HD-Ad containing the rep78 gene conferred efficient Rep 78 expression in target cells. Upon co-infection of HD-Ad-rep78 with an HD-Ad vector containing a 27k |[beta]|-globin-LCR-GFP transgene cassette flanked by AAV ITRs (HD-Ad.AAV-LCR), efficient Rep78-mediated rescue/excision of the transgene cassette was observed in 293 cells but not in MO7e cells. We found that rescue in 293 cells was enhanced due to expression of viral proteins, specifically proteins encoded by Ad5 E2a and E4orf6. Although no rescue of the LCR-GFP cassette was detectable in MO7e cells, co-infection of HD-Ad.rep78 increased the overall stable transduction frequency of HD.Ad.AAV-LCR and mediated integration of the transgene cassette into AAV-S1. (40% of all analyzed vector junction were in AAVS1). Interestingly, analysis of integration junctions revealed that Ad integration occurred in 80% of sites via the Ad ITR and only in 20% of sites via the AAV ITR. No rearrangements within AAV-S1 or AAV-S1 specific transgene integration were observed when HD- Ad.AAV-LCR was infected onto MO7e cells without the Rep78-expressing HD Ad vector. Our data indicate that rescue of the AAV-ITR flanked transgene cassette is not necessary for integration into AAV-S1. We are currently evaluating whether rescue can enhance the frequency of site-specific integration. This study provides a basis for the construction of vectors that allow for preferential site-specific transgene integration in hematopoietic cells.