502. Advances in the Application of Combinatorial Technology for the Optimization of Adeno-Associated Virus (AAV) Vectors

In vitro evolution protocols have been successfully applied to screen combinatorial AAV libraries in order to obtain mutants that escape neutralization by human sera (Perabo et al. JGM 2006; Maheshri et al. Nat Biotechnol 2006).We have further optimized panning protocols by monitoring selection rounds with Real Time PCR to follow the evolution of the viral pools after each selection round. Application of this technology to new screening experiments allowed in shorter times and at reduced costs the identification of novel capsid variants with higher transduction efficiency and improved ability to circumvent antibody neutralization at the same time. Packaging ability was unaffected by the introduced mutations. Beside providing proof of principle for the advantages of this monitoring procedure, these new variants enrich the pool of alternative vectors that can be employed for the treatment of patients with preexisting immunity to AAV-2 and further extend our knowledge of capsid biology.Our results contribute to the development of combinatorial technology for the engineering of viral particles. This emerging approach is gathering interest as alternative to rational design, overcoming the limit posed by our incomplete understanding of the infectious process, and at the same time offering a powerful tool to dissect viral biology by reverse genetics (see also abstract by Goldnau et al).