Molecular Basis Of AAV-2 P5 Promoter

The AAV genome can integrate into a specific region of human chromosome 19, named AAVS1 site, in a rep-dependent way. Use of the Rep-mediated site-specific integration of AAV in gene therapy will enable the long-term stable expression of the transgene. As the Rep protein is cytotoxic when using the Rep-AAVS1 site-specific integration transgene system, it is necessary to keep Rep expression transient and in a low level. In wild type AAV-2, the promoter driving the Rep protein expression is the P5 promoter, which was identified as a multifunctional element. The P5 promoter can be served as the substrate of Rep-mediated site-specific integration and regulate the Rep transcription in a negative feedback way. So when using the P5-Rep expression frame to donor Rep protein in-trans, it can keep the Rep expression in a low level. But the problem is, the P5-Rep will compete with the cis-element, which was together with the transgene, to integrate into the AAVS1 due to the high integration efficiency of P5, and thus decrease the efficiency of transgene delivery. On the other hand, the integrated P5-Rep will express Rep in a long-term way, raising the danger of cytotoxicity. Our research is to explain the molecular mechanism of site-specific integration and negative feedback character of P5 promoter by mutation analysis, and in an attempt to search for a useful P5 mutant, which can eliminate the site-specific integration but maintain the efficient negative feedback activity.Mutation with the TATA/RBE/YYl core structure of p5 promoter reveals that the P5 RBE is essential for both the Rep-mediated site-specific integration and transcriptional suppression of P5 promoter. Though mutations with the TATA box and YY1+1 site will eventually lead to severely decreased site-specific integration efficiency (only 8.3% and 10.4% of these two mutant, versus more than 34.0% of wild type P5), the former resulted in certain decrease of the negative feedback activity, while the later seems to have no influence on the negative feedback activity. When making detailed mutation analysis within the nucleotide of P5 RBE, we found:it is in the internal 266GAGTGAGC273 motif of P5 RBE that can lead to the differentiation of the two functions of P5 RBE, but not in the flanking sequence of 266GAGTGAGC273. Mutation of any single nucleotide in the 266GAGTGAGC273 motif will dramatically compromise the efficiency of Rep-mediated site-specific integration, but still retain a fully negative feedback activity of P5 promoter. Among these mutations, the C273T can most strongly differentiate the two P5 characters (6.5% of site-specific integration efficiency and 76.0% of negative feedback). In vitro Rep-P5 RBE binding affinity assay indicated that the negative feedback activity of P5 is mainly depended on the Rep-P5 RBE affinity. But it is not the case to site-specific integration which may need other factors rather than the Rep-P5 RBE binding affinity. And we propose that the sequence homology of the 266GAGTGAGC273 motif of P5 RBE should be need.Above all, we demonstrated that the Rep-mediated site-specific integration and transcriptional suppression of P5 promoter can be differentiated based on the structure-function analysis. More over, our research provides new insight to the mechanism of Rep-mediated site-specific integration and transcriptional differentiation. At last, in an gene transfer system based on the Rep-AAVS1 site-specific integration, the C273T mutant of P5 should be useful in expressing Rep in-trans in a transient, low-level way.