Targeted correction of singlebase-pair mutations with adeno-associated virus vectors under nonselective conditions

Recombinant adeno-associated virus (rAAV) vectors possess the unique ability to introduce genetic alter-ations at sites of homology in genomic DNA through a mechanism thought to predominantly involve homol-ogous recombination. We have investigated the efficiency of this approach using a mutant enhanced green fluorescent protein (eGFP) fluorescence recovery assay that facilitates detection of gene correction events in living cells under nonselective conditions. Our data demonstrate that rAAV infection can correct a mutant eGFP transgene at an efficiency of 0.1 % in 293 cells, as determined by fluorescence-activated cell-sorting analysis. Gene repair was also confirmed using clonal expansion of GFP-positive cells and sequencing of the eGFP transgene. These results support previous findings demonstrating the efficacy of rAAV for gene targeting. In an effort to improve gene-targeting efficiencies, we evaluated several agents known to increase rAAV transduction (i.e., expression of an expressed gene), including genotoxic stress and proteasome inhibitors, but observed no correlation between the level of gene repair and rAAV transduction. Interestingly, however, our results demonstrated that enrichment of G1/S-phase cells in the target population through the addition of thymidine moderately (2-fold) increased gene correction compared to cells in other cell cycle phases, including G0/G1, G1, and G2/M. These results suggest that the S phase of the cell cycle may more efficiently facilitate gene repair by rAAV. Transgenic mice expressing the mutant GFP were used to evaluate rAA