Cystic fibrosis (CF) is caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The major manifestations are on the airway epithelial surface, with purulent mucus, opportunistic infections, chronic inflammation and loss of lung function. Consequent to mutations in both parental genes, airway epithelial cells have insufficient CFTR function. Since this is corrected in vitro by transfer of the normal CFTR gene into airway epithelial cells, it is reasonable to hypothesize that the respiratory manifestations of CF could be prevented by transfer of the normal human CFTR cDNA to the airway epithelium in vivo. Experimental studies have demonstrated this is feasible with a replication deficient E1- E3- recombinant adenovirus (Ad) type 5 vector containing a normal human CFTR cDNA, and a clinical study is ongoing to evaluate this in individuals with CF. The objectives of this proposal are to establish fundamentals necessary to maximize the efficacy and safety of in vivo transfer of the human CFTR protein coding sequence to the airway epithelium for the therapy of the respiratory manifestations of CF. The proposed program is centered on Ad vectors, but the information derived will be generally applicable to other vector systems. The focus is to: (1) identify and/or design a promoter(s) for gene therapy for the respiratory manifestations of CF that will maximize safety, is resistent to inappropriate down regulation, and can be regulated in response to local administration of pharmacologic agents; (2) evaluate the dose-response relationship between CFTR-related function and increasing amounts of CFTR gene expression (i.e.., CFTR protein) both at the single cell level and for the intact airway epithelial sheet; (3) identify the Ad receptor by expression cloning, and to characterize the structure and function of the Ad receptor gene product; and (4) modify Ad vector targeting by modifying the ligand for the Ad receptor such that the Ad vector will enter cells via alternative receptors or so that the Ad vector will interact with the natural Ad receptor in a more efficacious and safe fashion for gene therapy. These four major projects will be supported by five core facilities: (l) molecular biology (vector design); (2) tissue culture and adenovirus production; (3) morphology; (4) animal models; and (5) administrative. Ten CF related pilot/feasibility projects are proposed, all related to innovative strategies for gene therapy for CF and/or basic principles that, if successful, will permit gene therapy for CF to proceed in a more efficacious and safe manner.
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