(414c) Engineering of a Novel AAV Vector In a Human Airway Model System for Cystic Fibrosis Gene Therapy

Cystic Fibrosis is a common autosomal recessive disease caused by mutations in the CFTR gene. Although current treatments have improved the lives of patients, none are directed at the underlying genetic defect, and CF remains a lethal disease. Gene transfer is attractive because it could correct all of the physiologic abnormalities due to the loss of CFTR. However, recent clinical trials with a variety of delivery vehicles, both viral and nonviral, have proven inefficient. In particular, recent clinical trials for CF have shown that adeno-associated virus (AAV2) is extremely safe; however, failed to achieve therapeutic benefits due to a range of barriers to effective delivery, including poor binding to the apical surface ? due to low apical levels of receptors ? and inefficient intracellular trafficking.

Rational efforts to engineer novel AAV vectors have resulted in moderate success; however, lack of sufficient knowledge regarding the structure/function relationships of AAV2 and other serotypes has hindered further rational engineering methods. We thus hypothesized that a directed evolution approach employing recombination and mutagenesis, akin to natural evolutionary mechanisms, have the potential to engineer novel viral capabilities under artificial, clinically relevant selective pressures not found in nature. Specifically, we employed directed evolution of the AAV capsid to generate novel viral variants with enhanced gene transfer to human pulmonary epithelium. PCR based mutagenesis coupled with high-throughput in vitro recombination generated a diverse library of chimeric cap genes with components from two divergent serotypes that utilize distinct receptors, AAV2 and AAV5. Subsequent selection of this library, consisting of two rounds of evolution and five rounds of selection, in polarized primary human airway epithelia (HAE) identified a single novel AAV chimera with a novel point mutation. Impressively, binding to HAE was increased approximately 100-fold and luciferase gene transfer showed 100-fold improvement suggesting that the changes improved both binding and post-binding steps. Furthermore, the novel AAV virus mediates successful CFTR cDNA-gene transfer to correct the Cl-transport defect in human CF epithelia. In summary, we have engineered a novel AAV variant that is capable of significantly greater airway gene transfer and holds the promise of therapy for CF as well as other pulmonary diseases, and these results highlight the potential of directed evolution approaches to engineer ?designer' gene delivery vectors.