Rep-mediated Site-specific Integration System Of Cis-elements And Applications

Nonviral plasmid DNA is a promising vector for achieving ex vivo and in vivo gene transfer. However, transgene expression is usually transient, especially in dividing target cells due to loss of vector genomes. Insertion of gene transfer vectors into the human genome results in long-term gene expression usually, but uncontrolled insertion is raising significant safty concerns for their clinical use. Adeno-associated virus (AAV) is a non-pathogenic virus and the only known eukaryotic virus capable of targeting human chromosome 19 for integration at a well-characterized AAVS1 site. Its site-specific integration is mediated by Rep68 and Rep78, viral proteins that bind to both the viral genome and AAVS1 site on chl9 through a specific Rep-binding element (RBE) located in both the viral genome and AAVS1. There are three RBEs in the AAV genome: two identical ones in both Inverted Terminal Repeats (ITR) and another one in a recently discovered region termed the P5 Integration Efficiency Element (P5IEE) that encompasses the viral P5 promoter.In order to identify the viral cis-acting sequence essential for Rep-mediated integration, we tested a series of constructs containing various lengths of P5IEE and compared the two RBEs from ITR (RBE_itr) and P5IEE (RBE_p5) in terms of their efficiency in Rep-dependent integration. Methods employed included a colony-forming assay, a PCR-based assay and Southern blotting analysis. We found that 16-bp of the RBE cis-element was sufficient for mediating Rep-dependent site-specific integration. Furthermore, RBE_itr was both more effective and specific than the RBE_p5 in Rep-dependent integration at the AAVS1 site.We further describe transfer of plasmid containing Rep binding element (RBE) and human coagulating factor IX (hFIX) together with rep gene expression plasmid into transgenic mouse carrying the full-length human AAVS1 locus in vivo by using hydrodynamic-based procedure. Long-term (over 240 day) gene expression of hFIX (150～200ng/ml) was achieved from mouse livers. In contrast, a hFIX plasmid without integration element produced initially high-level gene expression that rapidly declined to undetectable levels. PCR analyses of the cellular DNA indicated that hFIX persistent in heart, liver and kidney for more than 240 days. RT-PCR analyses indicated that the majority transcripts of hFIX were in the liver. Transaminase levels and liver histological results showed that rapid intravenous plasmid injection intomice induced transient focal acute liver damage, which was rapidly repaired within 10 days and resulted thereafter in histologically normal tissue. Neither cytotoxic effects nor long-term toxicity from expressing of Rep have been observed. Very low level antibodies directed against hFIX did not prevent the circulation of therapeutic levels of the protein.The data established in this study point to important viral cis-acting sequences required for targeting. Our results showed that plasmid vector carrying the RBE was capable of site-specific integration in the 293 cell and long-term expression of hFIX can be achieved in the AAVS1 transgenic mice using this system. Our findings added new information on the mechanism of Rep-dependent AAV genome insertion at the AAVS1 site and The ITRRBE integrating plasmid vector described in this study may be important in complementing efficient plasmid delivery systems currently compromised by transient transduction and should greatly facilitate development of site-specific in vitro integration systems.