Requirements for adeno-associated virus-derived non-viral vectors to achieve stable and site-specific integration of plasmid DNA in liver carcinoma cells.

Background And Aims: Adeno-associated virus (AAV) is the only known virus capable of site-specific genomic integration in human cells. Thus, AAV-based vectors may be an attractive option to achieve prolonged transgene expression in human cells. We therefore studied the minimal elements of gene therapy vectors necessary for stable integration and tested the effectiveness of this approach in hepatoma cells.

Methods: Plasmids were constructed that contained a GFPneo fusion transgene with or without the AAV-inverted terminal repeats (ITRs). In addition, Rep protein was either encoded in CIS or supplied in TRANS by co-transfections. Stable clones were analyzed by Southern blotting for site-specific integration.

Results: The ITRs alone conferred neither stable nor site-specific transgene integration. Expression of Rep protein in CIS or TRANS resulted in an increased frequency of integration regardless of the presence of ITRs. It was shown that in the absence of the ITRs, other Rep-binding site (RBS) like sequences such as the ColE1 sequence present in plasmid backbones can function as RBS. Site-specific integration was achieved in up to 26% of clones derived from hepatoma cells.

Conclusion: Both expression of Rep proteins and inclusion of a RBS are necessary for enhanced and stable integration of AAV-based non-viral vectors. A novel two-plasmid system capable of achieving stable and site-specific gene transfer in hepatoma cells is introduced.