Gene transfer offers the potential of a new therapy to slow or prevent CF lung disease. Yet, despite outstanding progress in several areas of gene transfer, we still do not have answers to many crucial questions that will guide development of gene transfer. The major impediment to answering these and other critical questions is the lack of an animal model that faithfully replicates the lung disease in humans with CF. Therefore, the goal of this project is to develop a porcine model of CF. We chose the pig because their lungs share many anatomical, histological, biochemical and physiologic features with human lungs. In addition, our preliminary data reveal similarities to humans in gene transfer to airway epithelia. Their reproductive characteristics also make them a good model. Tile success of this endeavor is enhanced by collaboration between two laboratories; Dr. Welsh's lab has a long history of CF investigation and Dr. Prather's lab has been at the forefront in developing technology for creating gene-targeted pigs by nuclear transfer.
Aim 1. Target the porcine CFTR gene in fetal fibroblasts. Because deltaF508 is the most common CF mutation, we will generate a targeting vector that encodes this mutation. We will introduce the vector into fetal fibroblasts by nuclear microinjection and identify targeted cells using strategies that maximize the frequency of homologous recombination and obviate the need to include a selectable gene.
Aim 2. Nuclear transfer and generation of a pig with a targeted CFTR gene. We will transfer targeted fetal fibroblasts to enucleated oocytes, fuse and activate them. Nuclear transfer embryos will be implanted into surrogate sows to generate offspring heterozygous for the deltaF508 mutation. Importantly, the targeted pig will be identical to a wild-type pig with the exception of a 3-bp deletion that generates deltaF508. In a second line, we will delete most of CFTR exon 10 to generate a """"""""knockout"""""""" allele.
Aim 3. Do the gene-targeted pigs manifest CF defects and disease? To answer this question, we will use molecular analyses to verify targeting, and will test the hypothesis that the deltaF508 protein is expressed but misprocessed in pigs. Because defective ion transport is a hallmark of CF, we will ask whether CF pig airway epithelia show defective ion transport in vitro and in vivo. A CF pig will provide the opportunity to better understand the disease and its pathogenesis, to develop gene transfer, and to test pharmaceuticals. Thus, it will accelerate the discovery of novel therapies for this lethal disease.
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