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.
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