Characterization of a bipartite recombinant adeno-associated viral vector for site-specific integration.

Adeno-associated virus type 2 (AAV2) is the only virus known to integrate into a specific locus in the human genome. The locus, AAVS1, is on the q arm of chromosome 19 at position 13.4. AAV is currently a popular vector for human gene therapy. However, current vectors do not contain two important elements needed for site-specific integration, that is, the rep gene or the P5 promoter, although they do integrate with low frequency at random locations in the human genome. We have designed a bipartite vector that does insert the transgene into AAVS1. One component, rAAVSVAV2, contains the rep gene, driven by the simian virus 40 early promoter rather than the P5 promoter. Thus, the integration enhancer element (IEE) within P5, which greatly enhances site-specific integration, has been deleted. The other component, rAAVP5UF11, contains the P5 IEE plus the transgene with associated regulatory elements. We have created clones of transduced HeLa cells, most of which appear to have the transgene inserted in AAVS1. We have not detected any clones that have rep inserted anywhere. With the optimal multiplicity of infection and ratio of rAAVSVAV2 and rAAVP5UF11, the transgene integrated specifically at AAVS1 with high efficiency (>60%). Most importantly, the cloned cell lines with the AAVS1 site-specific integrated green fluorescent protein (GFP) were healthy and stably expressed GFP for 35 passages. An AAV vector that would integrate at a specific site with high frequency could offer significant advantage in the transduction of progenitor cells and stem cells ex vivo and engineered cells could be used for human gene therapy. AAV site-specific integration gene therapy could provide a novel approach for diseases that need long-term gene expression.