SUMMARY Almost two decades after identification of the CFTR gene, we lack answers to many questions about the pathogenesis of cystic fibrosis (CF), and it remains a lethal disease. Mice with a disrupted CFTR gene have greatly facilitated CF studies, but they fail to develop the characteristic pancreatic, lung, intestinal, liver, and other CF manifestations. Therefore, we produced pigs with a targeted disruption of both CFTR alleles. These animals exhibited defective chloride transport. They also developed meconium ileus, exocrine pancreatic destruction, and focal biliary cirrhosis, replicating abnormalities seen in newborn patients with CF. This swine model may provide opportunities to address persistent questions about CF pathogenesis and accelerate discovery of treatments and preventions.

NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptLung disease causes most of the morbidity and mortality in cystic fibrosis (CF). However, understanding its pathogenesis has been hindered by lack of an animal model with characteristic features of CF. To overcome this problem, we recently generated pigs with targeted CFTR genes. We now report that, within months of birth, CF pigs spontaneously develop hallmark features of CF lung disease including airway inflammation, remodeling, mucus accumulation, and infection. Their lungs contained multiple bacterial species, suggesting an equal opportunity host defense defect. In humans, the temporal and causal relationships between inflammation and infection have remained uncertain. To investigate these processes, we studied newborn pigs. Their lungs showed no inflammation, but were less often sterile than controls. Moreover, after intrapulmonary bacterial challenge, CF pigs failed to eradicate bacteria as effectively as wild-type pigs. These results suggest that impaired bacterial elimination is the pathogenic event that initiates a cascade of inflammation and pathology in CF lungs. Finding that CF pigs have a bacterial host defense defect within hours of birth provides an opportunity to further investigate pathogenesis and to test therapeutic and preventive strategies before secondary consequences develop.

Interactions between ligands and receptors are central to communication between cells and tissues. Human airway epithelia constitutively produce both a ligand, the growth factor heregulin, and its receptors--erbB2, erbB3 and erbB4 (refs 1-3). Although heregulin binding initiates cellular proliferation and differentiation, airway epithelia have a low rate of cell division. This raises the question of how ligand-receptor interactions are controlled in epithelia. Here we show that in differentiated human airway epithelia, heregulin-alpha is present exclusively in the apical membrane and the overlying airway surface liquid, physically separated from erbB2-4, which segregate to the basolateral membrane. This physical arrangement creates a ligand-receptor pair poised for activation whenever epithelial integrity is disrupted. Indeed, immediately following a mechanical injury, heregulin-alpha activates erbB2 in cells at the edge of the wound, and this process hastens restoration of epithelial integrity. Likewise, when epithelial cells are not separated into apical and basolateral membranes ('polarized'), or when tight junctions between adjacent cells are opened, heregulin-alpha activates its receptor. This mechanism of ligand-receptor segregation on either side of epithelial tight junctions may be vital for rapid restoration of integrity following injury, and hence critical for survival. This model also suggests a mechanism for abnormal receptor activation in diseases with increased epithelial permeability.

Progress toward understanding the pathogenesis of cystic fibrosis (CF) and developing effective therapies has been hampered by lack of a relevant animal model. CF mice fail to develop the lung and pancreatic disease that cause most of the morbidity and mortality in patients with CF. Pigs may be better animals than mice in which to model human genetic diseases because their anatomy, biochemistry, physiology, size, and genetics are more similar to those of humans. However, to date, gene-targeted mammalian models of human genetic disease have not been reported for any species other than mice. Here we describe the first steps toward the generation of a pig model of CF. We used recombinant adeno-associated virus (rAAV) vectors to deliver genetic constructs targeting the CF transmembrane conductance receptor (CFTR) gene to pig fetal fibroblasts. We generated cells with the CFTR gene either disrupted or containing the most common CF-associated mutation (ΔF508). These cells were used as nuclear donors for somatic cell nuclear transfer to porcine oocytes. We thereby generated heterozygote male piglets with each mutation. These pigs should be of value in producing new models of CF. In addition, because gene-modified mice often fail to replicate human diseases, this approach could be used to generate models of other human genetic diseases in species other than mice.

Mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel cause the autosomal recessive disease, cystic fibrosis (CF). The most common mutation is ΔF508, which deletes phenylalanine508. In vitro studies indicate that CFTR-ΔF508 is misprocessed, though in vivo consequences of the mutation are uncertain. To better understand effects of the ΔF508 mutation, we produced CFTRΔF508/ΔF508 pigs. Our biochemical, immunocytochemical and electrophysiological data on CFTR-ΔF508 in newborn pigs paralleled in vitro results. They also indicated that CFTRΔF508/ΔF508 airway epithelia retain a small residual CFTR conductance; maximal stimulation produced ~6% of wild-type function. Interestingly, cAMP agonists were less potent at stimulating current in CFTRΔF508/ΔF508 epithelia, suggesting that quantitative tests of maximal anion current may overestimate transport under physiological conditions. Despite residual CFTR function, four older CFTRΔF508/ΔF508 pigs developed lung disease strikingly similar to human CF. These results suggest that this limited CFTR activity is insufficient to prevent lung or gastrointestinal disease in CF pigs. These data also suggest that studies of recombinant CFTR-ΔF508 misprocessing predict in vivo behavior, which validates its use in biochemical and drug discovery experiments. These findings help elucidate the molecular pathogenesis of the common CF mutation and will guide strategies for developing new therapeutics.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis. The most common mutation, a deletion of the phenylalanine at position 508 (⌬F508), disrupts processing of the protein. Nearly all human CFTR-⌬F508 is retained in the endoplasmic reticulum and degraded, preventing maturation to the plasma membrane. In addition, the F508 deletion reduces the activity of single CFTR channels. Human CFTR-⌬F508 has been extensively studied to better understand its defects. Here, we adopted a cross-species comparative approach, examining human, pig, and mouse CFTR-⌬F508. As with human CFTR-⌬F508, the ⌬F508 mutation reduced the single-channel activity of the pig and mouse channels. However, the mutant pig and mouse proteins were at least partially processed like their wild-type counterparts. Moreover, pig and mouse CFTR-⌬F508 partially restored transepithelial Cl ؊ transport to CF airway epithelia. Our data, combined with earlier work, suggest that there is a gradient in the severity of the CFTR-⌬F508 processing defect, with human more severe than pig or mouse. These findings may explain some previously puzzling observations in CF mice, they have important implications for evaluation of potential therapeutics, and they suggest new strategies for discovering the mechanisms that disrupt processing of human CFTR-⌬F508.airway epithelia ͉ chloride transport ͉ cystic fibrosis ͉ mouse models

Bardet-Biedl syndrome is an autosomal recessive disorder characterized by mental retardation, obesity, retinitis pigmentosa, polydactyly and hypogonadism. Individuals with this disorder also have an increased incidence of hypertension, diabetes mellitus, and renal and cardiac anomalies. We previously identified a locus on chromosome 16 causing this disorder, and provided evidence that Bardet-Biedl syndrome is heterogeneous. In this study, we identify another Bardet-Biedl syndrome locus on chromosome 3 and confirm the non-allelic heterogeneity of this disorder in Bedouin populations. In addition, we demonstrate the feasibility of using pooled DNA samples from members of large kindreds as an efficient approach to homozygosity mapping.

Cystic fibrosis (CF) pigs develop disease with features remarkably similar to those in people with CF, including exocrine pancreatic destruction, focal biliary cirrhosis, micro-gallbladder, vas deferens loss, airway disease, and meconium ileus. Whereas meconium ileus occurs in 15% of babies with CF, the penetrance is 100% in newborn CF pigs. We hypothesized that transgenic expression of porcine CF transmembrane conductance regulator (pCFTR) cDNA under control of the intestinal fatty acid-binding protein (iFABP) promoter would alleviate the meconium ileus. We produced 5 CFTR -/-;TgFABP>pCFTR lines. In 3 lines, intestinal expression of CFTR at least partially restored CFTR-mediated anion transport and improved the intestinal phenotype. In contrast, these pigs still had pancreatic destruction, liver disease, and reduced weight gain, and within weeks of birth, they developed sinus and lung disease, the severity of which varied over time. These data indicate that expressing CFTR in intestine without pancreatic or hepatic correction is sufficient to rescue meconium ileus. Comparing CFTR expression in different lines revealed that approximately 20% of wild-type CFTR mRNA largely prevented meconium ileus. This model may be of value for understanding CF pathophysiology and testing new preventions and therapies.