Poster abstracts

Poster number 65 submitted by Marcela Cataldi

recombinant adeno-associated virus (rAAV) hairpin structure does not contribute to chromosomal integration

Marcela P. Cataldi (Molecular, Cellular and Developmental Biology Graduate Program, Biological Sciences, The Ohio State University; Center for Gene Therapy, The Research Institute at Nationwide Childrens Hospital), Douglas M. McCarty (Center for Gene Therapy, The Research Institute at Nationwide Childrens Hospital; Department of Pediatrics, The Ohio State University)

Abstract:
The palindromic terminal repeats (TR) of adeno-associated virus (AAV) form DNA hairpins (HP) that are essential for replication and for priming the conversion of single-stranded virion DNA to double-strand. In recombinant AAV (rAAV) gene-delivery vectors, they are targets for DNA repair pathways leading to circularization, concatemerization and, infrequently, chromosomal integration. Recombination with chromosomal DNA poses a risk for genotoxicity, raising the question as to whether the hairpin structures formed by the TRs are more likely to integrate than other forms of DNA ends. Because we cannot generate rAAV viral vectors without HP ends, we investigated the contribution of AAV TR to chromosomal integration by comparing DNA molecules with the TR sequences constrained in the hairpin conformation to molecules with linear duplex ends, with or without TR sequences. In order to measure integration efficiency, 293 cells were transfected with a high dose of each of these AAV genome-like molecules carrying a GFP-expression cassette, and continuously passaged until a stable percentage of GFP-expressing cells was reached, which represented the percentage of cells with DNA molecules integrated into the host genome after loss of episomal DNA by dilution. The results show no significant difference in terms of integration efficiency between the three DNA molecules, suggesting that AAV TR in a hairpin conformation is not more or less likely to integrate than open blunt ends with or without AAV TR sequences. In addition, we found that a previously reported decrease in gene expression mediated by silencing of HP-containing molecules is not observed when molecules are transfected at high doses, suggesting that the pathway that specifically recognizes the hairpin structure and leads to DNA silencing is saturated under these experimental conditions.

Keywords: AAV vectors, DNA recombination, gene therapy