Successful gene transfer to treat cystic fibrosis (CF) requires a gene transfer vector that mediates long term expression of the CF transmembrane conductance regulator (CFTR) coding sequences in a large fraction of the cells of the airway epithelium. A promising strategy to solve this challenge is to utilize serotypes of adeno-associated virus (AAV) vectors that have a high tropism for lung epithelium. However, all AAV vectors are limited for use for CF gene therapy because the 4.4 kb CFTR cDNA barely fits into the AAV capsid, permitting only minimal controlling sequences in the expression cassette. This proposal focuses on a novel strategy termed """"""""segmental trans-splicing"""""""" (STS) which solves the space problem in AAV vectors by using two vectors to carry in the 5' and 3' fragments of the expression cassette, and designer hybridization and splicing sequences to efficiently join the 5' and 3' fragments at the mRNA level with high fidelity. STS doubles the carrying capacity of AAV vectors, allowing for the use of highly efficient promoters or, the ultimate goal, the true endogenous CFTR controlling regions. We will test the hypothesis that novel recombinant AAV vectors with high tropism for the airway epithelium can deliver segmental trans-splicers to airway epithelial cells and correct the CFTR defect. The focus will be on three AAV serotypes, AAV5 (a human serotype known to be efficient in transferring genes to the lung via the epithelial surface), and AAV9 and AAVrh. 10, two newly discovered AAV serotypes that show remarkable efficiency in gene transfer to the lung via the epithelial route. AAV2, the human serotype extensively studied for CFTR gene transfer to the lung, wilt be used for comparison. Using in vitro human airway epithelial cell culture and in vivo murine CFTR-/- and non-human primate models, the ultimate goal of the 2 specific aims is to capitalize on the space afforded by segmental trans-splicing and the efficiency of these alternative AAV serotypes to include in the expression cassette the natural human CFTR promoter, thus providing to the airway epithelium persistent expression of the normal CFTR coding sequences that can respond in a normal fashion to signals in the airway milieu.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
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Heart, Lung, and Blood Initial Review Group (HLBP)
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Weill Medical College of Cornell University
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Ryan, Dorothy M; Vincent, Thomas L; Salit, Jacqueline et al. (2014) Smoking dysregulates the human airway basal cell transcriptome at COPD risk locus 19q13.2. PLoS One 9:e88051
Dvorak, Anna; Tilley, Ann E; Shaykhiev, Renat et al. (2011) Do airway epithelium air-liquid cultures represent the in vivo airway epithelium transcriptome? Am J Respir Cell Mol Biol 44:465-73
Krause, Anja; Whu, Wen Zhu; Xu, Yaqin et al. (2011) Protective anti-Pseudomonas aeruginosa humoral and cellular mucosal immunity by AdC7-mediated expression of the P. aeruginosa protein OprF. Vaccine 29:2131-9
Limberis, Maria P; Bell, Christie L; Heath, Jack et al. (2010) Activation of transgene-specific T cells following lentivirus-mediated gene delivery to mouse lung. Mol Ther 18:143-50
Limberis, Maria P; Vandenberghe, Luk H; Zhang, Liqun et al. (2009) Transduction efficiencies of novel AAV vectors in mouse airway epithelium in vivo and human ciliated airway epithelium in vitro. Mol Ther 17:294-301
Tertilt, Christine; Joh, Ju; Krause, Anja et al. (2009) Expression of B-cell activating factor enhances protective immunity of a vaccine against Pseudomonas aeruginosa. Infect Immun 77:3044-55
Limberis, M P; Bell, C L; Wilson, J M (2009) Identification of the murine firefly luciferase-specific CD8 T-cell epitopes. Gene Ther 16:441-7
Calcedo, Roberto; Vandenberghe, Luk H; Gao, Guangping et al. (2009) Worldwide epidemiology of neutralizing antibodies to adeno-associated viruses. J Infect Dis 199:381-90
Song, Yuhu; Lou, Howard H; Boyer, Julie L et al. (2009) Functional cystic fibrosis transmembrane conductance regulator expression in cystic fibrosis airway epithelial cells by AAV6.2-mediated segmental trans-splicing. Hum Gene Ther 20:267-81
Vandenberghe, L H; Breous, E; Nam, H-J et al. (2009) Naturally occurring singleton residues in AAV capsid impact vector performance and illustrate structural constraints. Gene Ther 16:1416-28

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