Functional Differentiation Between Rep-Mediated Site-Specific Integration and Transcriptional Repression of the Adeno-Associated Viral p5 Promoter

In this article, Yue et al. sought to increase the safety profile of site-specific integration strategies that employ the AAV Rep protein and its cognate DNA rep binding site (RBS) which is contained in the p5 promoter that controls Rep expression. In this mutational study, the authors attempted to dissect out the sequence requirements in RBS for site-specific integration from the Rep-mediated transcriptional suppression in order to minimize the former while preserving the latter. The adeno-associated virus (AAV) p5 promoter controls expression of Rep68 and Rep78, which are responsible for specific integration of the viral genome into the AAVS1 site of the human genome. The p5 promoter contains a Rep-binding element (RBE) sequence that acts as a substrate of the Rep proteins for both site-specific integration of p5 itself and transcriptional suppression of the p5 promoter. To differentiate these two Rep-mediated functions, we dissected the p5 core structure TATA/RBE/YY1+1 through a series of mutations. Mutations in the TATA box or YY1+1 region of p5IEE significantly reduced Rep-mediated site-specific integration (RMSSI) and p5 promoter transcriptional activity, but only the TATA box is involved in Rep-mediated transcriptional suppression (RMTS). Point mutations at nucleotides 266, 267, 268, 270, and 273 of the GAGTGAGC motif in p5 RBE significantly reduced RMSSI efficiency. However, only p5G270T lost the affinity of Rep binding and had significant reduction of RMTS. It appears that RMTS is determined by the affinity of p5RBE for Rep whereas RMSSI requires more stringent conditions. Thus, RMTS and RMSSI can be differentiated by point mutations in the p5 promoter, which is useful in gene therapy in a helper vector to drive Rep expression, as the mutant promoters seldom integrate themselves but remain the RMTS feature for reduced cytotoxicity caused by a high level of Rep protein.