692. A Novel AAV Vector for the Treatment of Cystic Fibrosis Airway Disease

Abstract

Cystic fibrosis (CF) is one of the most common life-threatening autosomal recessive disorders affecting the Caucasian population. The malfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) gene results in both severe chronic lung disease, which currently accounts for more than 95% of the morbidity and mortality of CF patients, and manageable pancreatic disease. Up to date CFTR gene transfer to the lung for the treatment of CF airway disease has been limited by poor vector performance combined with the significant barriers that the airway epithelium poses to effective gene transfer. Recently, our group isolated a novel AAV family (AAV9). In an attempt to evaluate its tropism and transfection efficiency we tested the AAV2 genome packaged in the AAV9 capsid (AAV2/9) and compared it to AAV2/5 in various airway model systems. A 50 ml single dose of 1 × 1011 genome copies (gc) of AAV2/9 expressing either the nuclear targeted b-galactosidase (nLacZ) gene or the green fluorescence protein (GFP) gene under the transcriptional control of the chicken b-actin promoter was instilled intranasally into nude and also C57Bl/6 mice. Twenty-one days later, the lung and nose were processed for gene expression. In control animals transduced with AAV2/9-GFP no LacZ positive cells were seen. AAV2/9-nLacZ successfully transduced mainly airways, whereas AAV2/5-nLacZ transduced mainly alveoli and few airways. Across the nasal airway epithelium both AAV2/5 and AAV2/9 transduced ciliated and non-ciliated epithelial cells. We are currently evaluating epithelial cell specific promoters to improve targeting to the airway cells in vivo. Based on the in vivo findings we next tested the gene transfer efficiency of AAV2/9 to human airway epithelial cells. Airway epithelial cells were isolated from human trachea and bronchi and grown at air-liquid-interface (ALI) on collagen coated membrane supports. Once the cells polarized and differentiated they were transduced with AAV2/9 or AAV2/5 expressing GFP from the apical as well as the basolateral side. Both AAV2/5 and AAV2/9 were successful at transducing epithelial cells from the basolateral surface. However, when applied onto the apical surface AAV2/9 resulted in a 10-fold increase in the number of transduced cells compared to AAV2/5. Currently, we are assessing the gene transfer performance of AAV2/9 in the lungs and nasal airways of nonhuman primates. We have demonstrated that AAV2/9 can efficiently transduce the airways of murine lung and well-differentiated human airway epithelial cells grown at ALI. These encouraging results have prompted the evaluation of AAV2/9 in nonhuman primates as well as the evaluation of AAV2/9-CFTR gene transfer to the nasal airway epithelium of cystic fibrosis knockout mice in an attempt to evaluate physiological correction of the CF defect. This novel family of AAV vectors shows great promise for the treatment of CF airway disease.