Engineering a CFTR Deficient Porcine Model of CF

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Pulmonary disease is a hallmark of cystic fibrosis (CF) and the most frequent reason for morbidity and mortality. Murine models of CF produced by targeted disruption of the CFTR gene or insertional mutagenesis have been successful in yielding the desired genotype, but significant pulmonary pathology has not been a phenotypic outcome associated with CFTR deficiency in these animals. A pig exhibiting a CF phenotype would provide an attractive model for the study and treatment of CF lung disease. In contrast to mice, the porcine lung shares many anatomical characteristics with humans including similarities in bronchial branching, vascular distribution and structure of the airway mucosa. Because of these similarities, porcine lungs have been used to replicate other pulmonary disease states found in humans and have served as models for the development of transplantation techniques and strategies for immunosuppression. The promise of gene therapy as a means to treat CF has not been realized in part because of the lack of a large animal model that exhibits phenotypic manifestations of CF comparable to that of humans. The central hypothesis of this proposal is that a CFTR deficient pig will exhibit a CF phenotype that closely resembles the human disease and provide an excellent model system for the development of therapeutic approaches for the treatment of CF. To address this hypothesis, our first objective will be to investigate characteristics of porcine CFTR regulation in a chloride secreting epithelium derived from the pig and compare the results to known properties of human CFTR. Results from these studies will provide new information on the regulation of CFTR channel function necessary for interpretation of data obtained from in vivo animal studies designed to track the progression of the CF disease phenotype. In the second objective, genetically modified pigs with impaired CFTR function will be produced by the introduction of the AF508 mutation by homologous recombination, taking advantage of porcine multipotent progenitor cells as a cellular resource for the nuclear transfer experiments. In addition, transgenesis with a transposon for regulated pCFTR RNA interference will also be explored as a means to control the level of CFTR expression. The successful development of this approach will yield important information on levels of CFTR expression necessary for normal secretory function, an issue critical for the development of effective gene therapy The third objective will identify specific in vitro and minimally invasive in vivo physiologic measurements to document the CF phenotype in clinically important epithelial tissues and to follow the progression of CF from post-weaned pigs to when they become sexually mature adults (within 6 months). The successful development of a porcine model for CF will be an important step toward the enhancement of pharmacotherapies and development of new approaches to gene therapy that can be used for the treatment of cystic fibrosis.