Targeted gene correction mediated by recombinant adeno-associated virus and DNA double strand breaks

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Recent news of serious adverse events in a clinical trial involving retroviral gene transfer revealed the inherent danger of using randomly integrating vectors. An alternative approach is the correction of the genetic mutation in the chromosome throug homologous recombination, called gene targeting. The use of recombinant adeno-associated virus (AAV) as a packing vector for a repair matrix has led to a significant improvement in mammalian gene targeting, reaching correction frequencies of up to 1%. We have systematically investigated the variables that regulate AAV gene targeting and showed that a local DNA double strand break (DSB) introduced by a site-specific endonuclease stimulated the frequency of gene correction over 300-fold. Moreover, a DSB in the target gene improved the targeting ratio (gene correction to random integration), thus significantly lowering the risk of insertional mutagenesis. This proposal aims at broadening the system by investigating the vector and cell parameters that govern AAV gene targeting ex vivo and in vivo. I further propose to design and apply site-specific zinc finger nucleases capable of creating a DSB at any desired site in the genom with the long-term goal to evaluate the approach in an animal disease model. The combine delivery of a repair matrix and a site-specific endonuclease by AAV vectors holds promise for achieving high gene targeting frequencies for both scientific and therapeutic applications.