Cystic Fibrosis (CF) has been at the forefront of gene transfer research for the last decade. This hereditary monogenetic disease, although affecting epithelial cells of multiple organs, results most often in mortality due to complications associated with the lung. Cystic fibrosis lung disease has been considered as a prototypic disease state for """"""""proof of concept"""""""" gene transfer strategies. The lack of an alternative long-term treatment for the pulmonary manifestations of this disease, the accessibility of the lung via the airway lumen, and the fact that viruses known to infect the lung were being developed into non-replicating gene transfer vectors led investigators to believe that administration of gene transfer vectors to the lung could potentially result in an effective treatment of this disease. CF is a disease of defective ion and fluid transport. The identification and cloning of the gene altered in CF demonstrated that this protein codes for a cAMP-mediated CI-channel, CFTR. It has also been demonstrated that defects in CFTR lead to decreased hydration of the airway surface liquid (ASL) and reduced mucus clearance. The assessment of the efficacy of gene transfer for CF requires a reliable assay to verify expression of the functional CFTR protein. In the Correction Core we will provide multiple techniques to detect the efficacy of CFTR gene transfer for correcting the ion and fluid transport defect of CF human and murine airway epithelial cells in vitro and CF murine nasal epithelia in vivo. We will use electrophysiological measurements across confluent monolayers mounted in Ussing chambers to determine the magnitude of both CI- (CFTR) and Na+ (ENaC) currents. Fluid transport rates will be assayed in the same preparations by confocal microscopy and absorption of blue dextran. To assess correction in vivo, we will measure nasal potential difference, the pH of ASL and the cytokine content collected by microdialysis. Gene transfer in the perinatal period may be most effective for treating a number of genetic diseases, including CF. We propose to study freshly excised epithelial sheets from prenatal and neonatal murine airways and gut. This will permit us to determine the effectiveness of in vivo vector dosing in neonatal CF (or other disease model) mice. The techniques available in this core will provide gene transfer investigators routine access to high-quality, high-sensitivity, well-controlled, robust measures of CFTR correction.
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