318. Development of Optimized AAV Serotype Vectors for the Potential Gene Therapy of Cystic Fibrosis

Cystic fibrosis (CF) is a single gene mutation that it is estimated to affect 30,000 children and adults in the US, and 70,000 patients worldwide. The lack of a functional gene leads to a defective ion conductance, and the associated water transport abnormalities produce increased viscosity of secretions in exocrine epithelial cells. In the respiratory airways, mucus plugging and retention produces severe lung diseases making this the main cause of mortality among CF patients. Several groups have tried to correct this defect using recombinant AAV as a vector to carry the cystic fibrosis transmembrane conductance regulator (CFTR) gene to airway epithelial cells, but without success, mostly because of poor transduction efficiency of these cells.Although AAV vectors have been used successfully in animal models, and shown efficacy in several clinical trials, a number of steps in the life cycle of AAV, including intracellular trafficking, continue to appear to limit the effectiveness of these vectors in gene therapy. We have recently documented that the substitution of surface-exposed specific tyrosine (Y), serine (S), and threonine (T) residues on AAV capsids significantly increases the transduction efficiency of these vectors, both in vitro and in vivo, presumably by preventing phosphorylation, subsequent ubiquitination, and proteasome-mediated degradation. The current studies were undertaken to identify the optimal AAV serotype vectors with improved transduction efficiency in airway epithelial cells. First, we evaluated the transduction efficiency of several commonly used AAV serotype vectors in primary normal (CRL-40111) and CF donor-derived (CRL-4013) human airway epithelial cells. Our data indicated that AAV6 serotype vectors transduce both of these cell lines at high levels, followed by AAV2, AAV5, and AAV1. Interestingly, that transduction efficiency of AAV5 vectors, but not AAV1, 2, and 6, was higher in CRL-4013 cells compared with that in CRL-4011 cells. We have previously developed a number of single- and multiple-mutant capsid-optimized vectors for each serotype (18 for AAV1, 100 for AAV2, 15 for AAV5, and 35 for AAV6). Thus, we next evaluated the transduction efficiency of these capsid-optimized AAV serotype vectors in human airway epithelial cells. The result showed that mutations in a single serine (S) residue at position 663 for AAV1, 662 for AAV2, 651 for AAV5, and 663 for AAV6 to valine (V) increased the transduction efficiency 2-3-fold compared with the wild-type (WT) AAV vectors. Among the multiple-mutants, several beneficial combinations of Y and T residues were identified, such as AAV2-Y444-500-730F+T491V, AAV1-Y705-731F+T492V, AAV5-Y263+719F and AAV6-Y705-731F+T492V, which increased the transduction efficiency 3-5-fold compared with the WT-AAV vectors. Overall, the capsid-optimized AAV6 serotype was identified as the most efficient. Studies are currently underway to generate the optimized AAV6-CFTR vector for its use in the potential gene therapy for CF.