Isolation of the gene responsible for Cystic Fibrosis (CF) in 1989 suggested new therapies based on somatic gene transfer into epithelial cells of affected organs. The subsequent demonstration of correction of the CF defect in cultured cells by viral mediated transduction of the CF gene (called CFTR) was the first step toward this goal. However, many important questions remain. We describe in this proposal a human xenograft model of CF that may be extremely important in answering these questions. Epithelial cells isolated from airways of normal and CF patients (nasal, trachea, and bronchus) are seeded into denuded rat trachea which are implanted into the flanks of nu/nu mice. Over the ensuing 6 weeks a fully differentiated human epithelium develops in the seeded grafts which is indistinguishable morphologically from normal airway epithelium. We have revised this system in the following way to form a genetically modified epithelium. The isolated epithelial cells are plated in primary culture, exposed to recombinant retroviruses, harvested, and seeded into denuded grafts. We have achieved transgene expression in up to 50% of the fully differentiated epithelium in vivo using lacZ retroviruses. Primary CF epithelial cells exposed to CFTR transducing retroviruses will be used to reconstitute denuded trachea. The resulting epithelium will be analyzed at the light and electron microscopic level to identify any morphological consequences of constitutive and high-level CFTR expression. Electrophysiological analysis of the grafts will address important issues of complementation in this relevant setting. Fully reconstituted trachea will also be used to study and compare strategies for in vivo gene delivery. Recombinant lacZ expressing retroviruses, adenoassociated viruses, and adenoviruses will be directly introduced into the lumen of the reconstituted grafts as they reside subcutaneously in the nu/nu mice. The grafts will subsequently be analyzed for efficiency of gene transfer and stability of gene expression. Virus systems which show the most promise will be used to directly introduce CFTR into the reconstituted grafts in vivo. Grafts transduced with CFTR in vivo will be analyzed using the morphological and functional criteria described above.
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