Adeno-Associated Virus-5 for Gene Transfer to CF Airway

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Gene transfer to the airways of patients with cystic fibrosis (CF) could represent a major therapeutic advance for this lethal disease. Recombinant adeno-associated viruses (AAV) vectors offer several advantages for achieving this goal, and the recent discovery that the AAV5 serotype targets the apical surface of differentiated airway epithelia makes this a particularly attractive vector. Thus, the overall goal of this project is to test the hypothesis that an AAV5 vector can transfer the CFTR cDNA to airway epithelia and complement the CF defect. Aim 1. Will a short expression cassette placed in an AAV5 vector drive expression in differentiated airway epithelia? A major challenge in developing an AAV vector for CF gene transfer is its limited packaging capacity, which places severe constraints on the size of the expression cassette. We will construct regulatory sequences for AAV5 vectors, with a goal of obtaining a promoter that is short, that drives transgene expression in airway epithelia, that generates persistent expression, and that resists attenuation by inflammatory mediators. We will test vectors in the context of AAV5 and in differentiated human airway epithelia. Aim 2. Will an AAV5 vector expressing CFTR complement the CF defect? Using an additional approach to meet packaging size constraints, we developed a CFTR containing a partial deletion of the R domain (CFTR-deltaR). We are excited by our preliminary work showing that an AAV5 containing a short promoter and a CFTR-deltaR transgene partially corrected the CF Cl- transport defect in CF airway epithelia. This validates our general strategy. However, our current AAV5 vectors have limitations, and many questions remain. Therefore, we will test the hypothesis that a CFTR-deltaR expression cassette in AAV5 will correct the CF Cl- transport defect in vitro in differentiated CF airway epithelia. We will also test the hypothesis that CFTR-deltaR can correct clinical intestinal disease in CF mice. Aim 3. Will an AAV5 vector generate long-lasting correction in airway epithelia in vivo and in vitro? An important reason to develop an AAV5 vector is the possibility of generating prolonged transgene expression. We will test persistence in differentiated airway epithelia and in mice using reporters, and we will test the hypothesis that an AAV5 vector will yield long-lasting complementation of the CF Cl- transport defect in vivo in nasal epithelia of CF mice. These studies will take us closer to our long-term goal of developing new therapies for people who suffer from CF.