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.

Mutations in β-cardiac myosin, the predominant motor protein for human heart contraction, can alter power output and cause cardiomyopathy. However, measurements of the intrinsic force, velocity, and ATPase activity of myosin have not provided a consistent mechanism to link mutations to muscle pathology. An alternative model posits that mutations in myosin affect the stability of a sequestered, super relaxed state (SRX) of the protein with very slow ATP hydrolysis and thereby change the number of myosin heads accessible to actin. Here we show that purified human β-cardiac myosin exists partly in an SRX and may in part correspond to a folded-back conformation of myosin heads observed in muscle fibers around the thick filament backbone. Mutations that cause hypertrophic cardiomyopathy destabilize this state, while the small molecule mavacamten promotes it. These findings provide a biochemical and structural link between the genetics and physiology of cardiomyopathy with implications for therapeutic strategies.

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.

Objective Case series suggest that Nonalcoholic Fatty Liver Disease (NAFLD) is associated with increased all-cause and cardiovascular mortality. The current study compared the survival of subjects with and without suspected NAFLD in a population-based cohort, and placed the finding in the context of previously published case series. Methods Primary analysis assessed mortality for NHANES-III participants with and without suspected NAFLD using the National Death Index. Suspected NAFLD was based upon unexplained ALT elevation. The Olmsted County and Cleveland Clinic case series were also used for comparison. Survivals were compared using Proportional Hazards Model and direct age standardization. Results The NHANES cohort included 980 with and 6594 subjects without suspected NAFLD. Over a mean of 8.7 years, suspected NAFLD had a hazards ratio of 1.37 (95%CI 0.98–1.91) for all-cause mortality. In the 45–54 age group, suspected NAFLD had significantly higher all-cause (4.40 95%CI 1.27–13.23) and cardiovascular mortality (8.15 , 95%CI 2.00–33.20) after adjusting for conventional cardiovascular risk factors. The age-standardized rate per 10,000 per year was 129 (95%CI 118–140) for the NHANES non-NAFLD cohort, 154 (95%CI 116–198) for the NHANES suspected NAFLD cohort, 214 (95%CI 157–279) for the Olmsted County series, and 426 (95%CI 298–573) for the Cleveland Clinic series. Conclusion The magnitude of mortality risk in NAFLD depends on the setting and method of ascertainment. Suspected NAFLD in the 45–54 age group is a strong independent risk factor for cardiovascular death and warrants further cardiovascular risk management guidelines.

Airway disease currently causes most of the morbidity and mortality in patients with cystic fibrosis (CF). However, understanding the pathogenesis of CF lung disease and developing novel therapeutic strategies have been hampered by the limitations of current models. Although the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) has been targeted in mice, CF mice fail to develop lung or pancreatic disease like that in humans. In many respects, the anatomy, biochemistry, physiology, size, and genetics of pigs resemble those of humans. Thus pigs with a targeted CFTR gene might provide a good model for CF. Here, we review aspects of porcine airways and lung that are relevant to CF.

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

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Somatic cell gene targeting combined with nuclear transfer cloning presents tremendous potential for the creation of new, large-animal models of human diseases. Mouse disease models often fail to reproduce human phenotypes, underscoring the need for the generation and study of alternative disease models. Mice deficient for CFTR have been poor models for cystic fibrosis (CF), lacking many aspects of human CF lung disease. In this study, we describe the production of a CFTR gene-deficient model in the domestic ferret using recombinant adeno-associated virus-mediated gene targeting in fibroblasts, followed by nuclear transfer cloning. As part of this approach, we developed a somatic cell rejuvenation protocol using serial nuclear transfer to produce live CFTR-deficient clones from senescent gene-targeted fibroblasts. We transferred 472 reconstructed embryos into 11 recipient jills and obtained 8 healthy male ferret clones heterozygous for a disruption in exon 10 of the CFTR gene. To our knowledge, this study represents the first description of genetically engineered ferrets and describes an approach that may be of substantial utility in modeling not only CF, but also other genetic diseases.