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Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. Airway disease is the major source of morbidity and mortality. Successful implementation of gene- and cell-based therapies for CF airway disease requires knowledge of relationships among percentages of targeted cells, levels of CFTR expression, correction of electrolyte transport, and rescue of host defense defects. Previous studies suggested that, when ~10-50% of airway epithelial cells expressed CFTR, they generated nearly wild-type levels of Cl- secretion; overexpressing CFTR offered no advantage compared with endogenous expression levels. However, recent discoveries focused attention on CFTR-mediated HCO3- secretion and airway surface liquid (ASL) pH as critical for host defense and CF pathogenesis. Therefore, we generated porcine airway epithelia with varying ratios of CF and wild-type cells. Epithelia with a 50:50 mix secreted HCO3- at half the rate of wild-type epithelia. Likewise, heterozygous epithelia (CFTR+/- or CFTR+/ΔF508) expressed CFTR and secreted HCO3- at ~50% of wild-type values. ASL pH, antimicrobial activity, and viscosity showed similar relationships to the amount of CFTR. Overexpressing CFTR increased HCO3- secretion to rates greater than wild type, but ASL pH did not exceed wild-type values. Thus, in contrast to Cl- secretion, the amount of CFTR is rate-limiting for HCO3- secretion and for correcting host defense abnormalities. In addition, overexpressing CFTR might produce a greater benefit than expressing CFTR at wild-type levels when targeting small fractions of cells. These findings may also explain the risk of airway disease in CF carriers. [ABSTRACT FROM AUTHOR]

Gene transfer could provide a novel therapeutic approach for cystic fibrosis (CF), and adeno-associated virus (AAV) is a promising vector. However, the packaging capacity of AAV limits inclusion of the full-length cystic fibrosis transmembrane conductance regulator (CFTR) cDNA together with other regulatory and structural elements. To overcome AAV size constraints, we recently developed a shortened CFTR missing the N-terminal portion of the R domain (residues 708-759, CFTRΔR) and found that it retained regulated anion channel activity in vitro. To test the hypothesis that CFTRΔR could correct in vivo defects, we generated CFTR-/- mice bearing a transgene with a fatty acid binding protein promoter driving expression of human CFTRΔR in the intestine (CFTR-/-;TgΔR). We found that intestinal crypts of CFTR-/-;TgΔR mice expressed CFTRΔR and the intestine appeared histologically similar to that of WT mice. Moreover, like full-length CFTR transgene, the CFTRΔR transgene produced CFTR Cl- currents and rescued the CFTR-/- intestinal phenotype. These results indicate that the N-terminal part of the CFTR R domain is dispensable for in vivo intestinal physiology. Thus, CFTRΔR may have utility for AAV-mediated gene transfer in CF. [ABSTRACT FROM AUTHOR]

Gene transfer of CFTR cDNA to airway epithelia is a promising approach to treat cystic fibrosis (CF). Most gene transfer vectors use strong viral promoters even though the endogenous CFTR promoter is very weak. To learn whether expressing CFTR at a low level in a fraction of cells would correct Cl- transport, we mixed freshly isolated wild-type and CF airway epithelial cells in varying proportions and generated differentiated epithelia. Epithelia with ∼20% wild-type cells generated ∼70% the transepithelial Cl- current of epithelia containing 100% wild-type cells. These data were nearly identical to those previously obtained with CFTR expressed under control of a strong promoter in a CF epithelial cell line. We also tested high level CFTR expression using the very strong cytomegalovirus (CMV) promoter as well as the cytokeratin-18 (K18) promoter. In differentiated airway epithelia, the CMV promoter generated 50-fold more transgene expression than the K18 promoter, but the K18 promoter generated more transepithelial Cl- current at high vector doses. Using functional studies, we found that with marked overexpression, some CFTR channels were present in the basolateral membrane where they shunted Cl- flow, thereby reducing net transepithelial Cl- transport. These results suggest that very little CFTR is required in a fraction of CF epithelial cells to complement Cl- transport because transepithelial Cl- flow is limited at the basolateral membrane. Thus they suggest a broad leeway in promoter strength for correcting the CF gene transfer, although at very high expression levels CFTR may be mislocalized to the basolateral membrane. [ABSTRACT FROM AUTHOR]

Cystic fibrosis (CF) pigs spontaneously develop sinus and lung disease resembling human CF. The CF pig presents a unique opportunity to use gene transfer to test hypotheses to further understand the pathogenesis of CF sinus disease. In this study, we investigated the ion transport defect in the CF sinus and found that CF porcine sinus epithelia lack cyclic AMP (cAMP)-stimulated anion transport. We asked whether we could restore CF transmembrane conductance regulator gene (CFTR) current in the porcine CF sinus epithelia by gene transfer. We quantified CFTR transduction using an adenovirus expressing CFTR and green fluorescent protein (GFP). We found that as little as 7% of transduced cells restored 6% of CFTR current with 17-28% of transduced cells increasing CFTR current to 50% of non-CF levels. We also found that we could overcorrect cAMP-mediated current in non-CF epithelia. Our findings indicate that CF porcine sinus epithelia lack anion transport, and a relatively small number of cells expressing CFTR are required to rescue the ion transport phenotype. These studies support the use of the CF pig as a preclinical model for future gene therapy trials in CF sinusitis. [ABSTRACT FROM AUTHOR]