Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The CFTR gene encodes a chloride channel that regulates ion flow across epithelial cell membranes. Most CF patients have a mutation that deletes phenylalanine 508 (?F508) and prevents CFTR from being properly processed and delivered to cell membranes. Failure of CFTR to reach the cell surface in the lung disrupts the composition and quantity of the airway surface liquid, and through mechanisms not yet fully understood, begins a cascade of events that leads to chronic airway infection and inflammation and lung destruction. Despite improvements in the lives of patients with CF, it remains a lethal disease, and current treatments are directed at secondary manifestations rather than the underlying defect. Several studies have demonstrated that the ?F508-CFTR channel retains chloride channel function when it is experimentally delivered to the cell membrane. Thus, the ?F508-CFTR channel has become an important therapeutic target for pharmacological intervention. However, two barriers limit progress toward new treatments. First, lack of an animal model that develops lung disease is a major impediment to understanding disease pathogenesis and the development of effective therapies. Mice with CFTR knockouts and with the ?F508 mutation have been developed, but they fail to exhibit the airway infection and inflammation that cause most of the morbidity and mortality in patients. Second, the ?F508-CFTR protein from other species shows differences in its processing compared to human ?F508-CFTR. Thus, studies focused on understanding CF and developing new treatments require a new animal model expressing the human ?F508-CFTR. In this application, we propose to produce such a model. We will use pigs for this project, because in contrast to mice, the structure, physiology, and biochemistry of their lungs and airways closely resemble those of humans. The ultimate goal of this project is to develop and commercialize a porcine model of CF carrying a human CFTR transgene bearing the common ?F508 mutation. A """"""""humanized"""""""" ?F508 pig would be the ideal model in which to develop and test CF drugs and a valuable tool for answering persistent questions about CF pathogenesis. This proposal outlines the development of such a pig by first stably introducing the entire human CFTR gene bearing the ?F508 mutation into the genome of pig cells lacking endogenous pig CFTR expression. The transgenic cells will then be used as nuclear donors for somatic cell nuclear transfer. The resulting pigs will be characterized molecularly, biochemically, and electrophysiologically to determine ?F508-CFTR expression and function. To validate the human ?F508-CFTR transgenic pig as a relevant model in which to test new therapies, pig airway cultures will be established, and compounds known to partially rescue the processing of ?F508-CFTR in human tissues will be tested. This project will generate an animal model that will provide the CF industrial and academic community with an opportunity to better understand the disease and its pathogenesis and to develop and test new therapeutics and preventative strategies. Thus, this work will accelerate the discovery of novel therapies for this lethal disease.
This project will generate an animal model that will provide the cystic fibrosis academic and industrial community with an opportunity to better understand the disease and its pathogenesis and to develop and test new therapeutics and preventative strategies. Thus, this work will accelerate the discovery of novel therapies for this lethal disease.