Talk abstracts

Talk on Saturday 11:50-12:05pm submitted by Marcela P. Cataldi

The Adeno-Associated Virus (AAV) Terminal Hairpin Structure is a Specific Target for DNA Recombination through the ATM Pathway

Marcela P. Cataldi (OSU, MCDB Graduate Program, The Research Institute at Nationwide Childrens Hospital, Center for Gene Therapy), Douglas M. McCarty (OSU, Department of Pediatrics, The Research Institute at Nationwide Childrens Hospital, Center for Gene Therapy)

Abstract:
Purpose: The AAV terminal repeat (TR) sequences are replication origins and priming sites for conversion of single-stranded virion DNA to double-stranded DNA templates for gene expression. They are essential components of AAV gene-delivery vectors, but also targets for DNA recombination, typically circularization and concatemerization, mediated by any of several DNA repair pathways. Infrequent recombinations with chromosomal DNA pose a risk for genotoxicity raising the question as to whether the TR hairpin (HP) structures are more likely to recombine than other forms of DNA ends. Methods: We investigated TR HP DNA recombination by comparing DNA substrates with hairpin TR ends to substrates with linear TR ends, or duplex ends lacking TR sequences. These were transfected into either normal cells lines, or cells lacking specific recombination factors. A green fluorescent protein (GFP) reporter gene was arranged either as an intact coding region within the linear substrate, or as two half-gene segments at the ends, such that expression depended on circularization of the DNA by recombination. Results: Circularization of the linear TR and hairpin substrates was ~70% at 24 hours. However, the hairpin substrates yielded a lower frequency of GFP expression, sugesting that the hairpin structure is recognized by a specific DNA recombination/repair pathway that can lead to degradation or silencing. In cells lines from ataxia telangiectasia (AT) patients, circularization of hairpin substrates was reduced to 25% while the others were unchanged, suggesting that ATM mediated recombination is targeting the hairpin structure. Further, the loss of expression associated with the HP structure was absent in ATM- cells, suggesting that this pathway leads to significant degradation or silencing of vector genomes. Conclusions: Despite the reliance on ATM, the HP structure is unlikely to contribute to higher rates of chromosomal DNA integration of AAV vectors.

Keywords: Adeno-Associated Virus, DNA repair, DNA recombination