Cystic fibrosis (CF) is a fatal inherited disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) which disrupts chloride ion transport in epithelial cells. Altered ion transport results in thickened mucous in secretory organs causing pathology in the respiratory, digestive, and reproductive tracts. Gene therapy efforts have focused on transferring the normal gene to respiratory epithelial cells since the leading cause of premature death is opportunistic lung infections secondary to the respiratory tract pathology. However, the metabolic dysfunction in the digestive tract causes malabsorption syndromes and, in about 10% of newborn CF patients, meconium ileus which can be fatal. Mice carrying mutations in the CFTR gene (cf/cf) have little respiratory pathology but have extensive pathology in the intestine, which is often fatal. To develop approaches for correcting the intestinal disease in CF this project will explore the feasibility of stably transferring a foreign gene to the gut epithelium. In contrast to lung epithelium, intestinal epithelium turns over at a rapid rate, thus stable gene transfer will require integration into the genome of the progenitor cells in the crypts of Lieberkuhn. We will use retroviral vectors to infect crypt cells and evaluate whether long-term gene transfer can be achieved. As a test system to develop methods and determine feasibility, we will use the beta-glucuronidase-negative mouse, a model of the lysosomal storage disease mucopolysaccharidosis (MPS) VII (Sly disease). beta-glucuronidase is expressed at high levels in the normal gut and the MPS VII mouse is a null mutant. With no interfering background expression of beta-glucuronidase, the fate of the transduced cells will be followed in vivo using in situ histochemical assays to detect the transferred enzymatic activity. Methods that work in the MPS VII mouse will then be tested for efficacy in the CF mouse models. The development of methods for stable gene transfer to the progenitor cells of the crypts in the gut will also be applicable to other genetic deficiencies that affect the gut epithelium, such as sucrose-isomaltase deficiency or receptor-mediated malabsorption syndromes.
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