Cystic Fibrosis CFTR Research Articles

Cystic Fibrosis Transmembrane Conductance Regulator: Roles in Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD) manifests with a variety of clinical presentations, reflecting its complex pathology. Currently, care focuses on symptom amelioration and prevention of complications and thus is generally tailored to disease severity rather than targeting specific pathophysiologic mechanisms. Chronic inflammation and mucus hypersecretion are key features of COPD. Epithelial ion channel dysfunction may be important, as it results in airway dehydration and defective host defense, contributing to chronic airway inflammation. Recent evidence suggests considerable similarities between COPD and cystic fibrosis (CF), a disease in which chloride ion channel dysfunction has been extensively studied (in particular CFTR [CF transmembrane conductance regulator]). Understanding commonalities between CF and COPD, and the role of CFTR in CF, may help in designing strategies targeting ion channel dysfunction and lead to new treatments with potential to alter the natural history of disease progression. Here, we review the roles of airway mucus and CFTR in normal lung function, the previously underestimated contribution of mucus stasis to the development of COPD, and the evidence for targeting CFTR to counteract mucus accumulation.

Furin Inhibition as a Mechanism to Reduce Aberrant ENaC‐Mediated Sodium Transport and Rehydrate the Airways in Cystic Fibrosis Lung Disease

The epithelial sodium channel (ENaC) is found in a number of tissues including lung epithelia and is activated through proteolytic cleavage of its α and γ subunits. Dysregulation of ENaC, secondary to the loss of functional CFTR in cystic fibrosis (CF), leads to increased Na+ absorption which contributes to airways dehydration, thickened mucus and impaired mucociliary clearance (MCC). Pro‐protein convertases such as furin are implicated in the activation of a distinct pool of ENaC as it passes through the biosynthetic pathway (1). Furin cleaves α‐ENaC at two sites releasing a small inhibitory peptide from the extracellular loop, partially activating the channel, and subsequently cleaves γ‐ENaC. These actions ‘prime’ the channel for cleavage at a secondary site on the γ subunit by cell‐surface channel activating proteases such as prostasin, trypsin and neutrophil elastase (2–4). Furin, therefore plays a key role in the proteolytic regulation of ENaC‐mediated sodium absorption and presents a promising therapeutic target for CF lung disease.The aim of this first‐in‐kind study was to determine the effect of two chemically different, highly selective furin inhibitors (BOS‐981 and BOS‐318) on ENaC signaling and airway surface liquid (ASL) height, using primary human CF bronchial epithelial cells (HBEs).Fully differentiated HBEs homozygous for F508‐del, grown at air‐liquid interface, were used to determine ENaC activity via equivalent current (Ieq) readings obtained using a 24‐well Transepithelial Current Clamp (TECC‐24) system. ASL height was measured using confocal microscopy.We report BOS‐981 and BOS‐318 to be potent inhibitors of furin. Both compounds, applied basolaterally, significantly reduced ENaC‐mediated Na+ absorption as measured by an acute reduction in amiloride‐sensitive Ieq after immediate treatment, which was sustained after longer (48 hr) exposure. The compounds also protected ENaC against cell surface activation by neutrophil elastase and CF sputum supernatant. Furthermore, the reduction in ENaC activity after a 48 hr treatment correlated with an increase in ASL height.These ground‐breaking results demonstrate that highly selective inhibition of furin by the novel inhibitors BOS‐981 and BOS‐318 presents a promising mechanism for the reduction of aberrant ENaC‐mediated Na+ absorption. This strategy has the potential to rehydrate the airways, restore effective MCC and reduce the devastating pulmonary decline observed in CF.Support or Funding InformationThis work was supported by funding from the MRC Proximity to Discovery: Industry Engagement FundThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Challenges in Cystic Fibrosis Newborn Screening and Recommendations for Primary Care Physicians.

* Abbreviations: CF — : cystic fibrosis CFTR — : cystic fibrosis transmembrane conductance regulator gene CFTR2 — : Clinical and Functional Translation of CFTR CRMS — : CFTR-related metabolic syndrome NBS — : newborn screening During the past decade, newborn screening (NBS) for cystic fibrosis (CF) has disseminated worldwide after endorsements by the Centers for Disease Control and Prevention and the CF Foundation. Similar to widespread introduction of other NBS programs, CF NBS implementation occurred on a region-by-region basis by using a wide variety of screening strategies and analytical methods. All strategies begin with measurement of the biomarker, immunoreactive trypsinogen, and should end with a sweat chloride test.1 Although these protocol variations have differing screening efficiency or effectiveness, all CF NBS programs have provided a better opportunity for healthier outcomes than traditional strategies that rely on signs/symptoms for triggering the diagnostic processes associated with delays and discrimination.2 In an ambitious effort to optimize CF NBS for their region’s diverse population, California analyzed and planned their strategy over 2 years, leading to specific goals.3 In essence, California organized a strategy that applied cystic fibrosis transmembrane conductance regulator gene ( CFTR ) sequencing for the first time, required detection of 2 mutations for designation of positive screening, and recommendation of a sweat chloride test. This NBS method relies heavily on the interpretation of CFTR variants, as not every CFTR genetic change will result in CF. CF began as, and remains, a clinical diagnosis. The recognition of the Mendelian inheritance of a well-characterized phenotype allowed the detection of the gene responsible for CF, and of common mutations. These mutations (mostly European derived) were the basis for early CF genetic tests used for diagnosis and screening.4,5 However, as recognition of CF widened, and knowledge of the CF phenotype expanded to include milder and atypical phenotypes, testing for just the common mutations proved inadequate. Carrier screening … Address correspondence to Philip M. Farrell, MD, PhD, Departments of Pediatrics and Population Health Sciences, School of Medicine and Public Health, University of Wisconsin-Madison, 600 Highland Ave, Madison, WI 53726. E-mail: pmfarrell{at}wisc.edu

Genomic Editing of Stem Cells for Modeling and Therapy of Genetic Diseases

The application of advanced genome editing technologies, when combined with induced pluripotent stem cells (iPSC), presents a unique opportunity to develop unprecedented patient‐specific in vitro models of disease‐in‐a‐dish that can be used for the development of more effective and universal stem cell therapies. Towards this aim, we have corrected SCID‐X1 patient‐derived iPSC using TALENs, resulting in the rescue of defective IL2RG expression and in the recovered generation of lymphoid cells derived from these iPSC. We have also corrected the common ΔF508 mutation of CFTR in cystic fibrosis patient‐derived iPSC using a novel, efficient, footprint‐free CRISPR‐mediated approach, and shown the subsequent functional recovery of CFTR function in airway epithelium derived from the corrected iPSC.However, the inability to derive truly transplantable cell types from iPSC limits the long‐term clinical and therapeutic potential of this approach. To address this we have carried out direct CRISPR‐mediated gene correction of CD34+ hematopoietic stem cells (HSC) from the FoxP3 mutant scurfy (sf) NOD mouse, which is defective for the development of regulatory T‐cells. These directly gene‐edited FoxP3sf HSC can then be evaluated for their potential for long‐term engraftment and regeneration of regulatory T‐cells by transplantation into lethally irradiated recipient mice. The ultimate goal is to establish novel pre‐clinical models for stem cell‐based therapy for a variety of genetic disorders.

Potentiator ivacaftor abrogates pharmacological correction of ΔF508 CFTR in cystic fibrosis.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR). Newly developed "correctors" such as lumacaftor (VX-809) that improve CFTR maturation and trafficking and "potentiators" such as ivacaftor (VX-770) that enhance channel activity may provide important advances in CF therapy. Although VX-770 has demonstrated substantial clinical efficacy in the small subset of patients with a mutation (G551D) that affects only channel activity, a single compound is not sufficient to treat patients with the more common CFTR mutation, ΔF508. Thus, patients with ΔF508 will likely require treatment with both correctors and potentiators to achieve clinical benefit. However, whereas the effectiveness of acute treatment with this drug combination has been demonstrated in vitro, the impact of chronic therapy has not been established. In studies of human primary airway epithelial cells, we found that both acute and chronic treatment with VX-770 improved CFTR function in cells with the G551D mutation, consistent with clinical studies. In contrast, chronic VX-770 administration caused a dose-dependent reversal of VX-809-mediated CFTR correction in ΔF508 homozygous cultures. This result reflected the destabilization of corrected ΔF508 CFTR by VX-770, markedly increasing its turnover rate. Chronic VX-770 treatment also reduced mature wild-type CFTR levels and function. These findings demonstrate that chronic treatment with CFTR potentiators and correctors may have unexpected effects that cannot be predicted from short-term studies. Combining these drugs to maximize rescue of ΔF508 CFTR may require changes in dosing and/or development of new potentiator compounds that do not interfere with CFTR stability.

Potentiator ivacaftor abrogates pharmacological correction of F508 CFTR in cystic fibrosis

Potentiator ivacaftor abrogates pharmacological correction of F508 CFTR in cystic fibrosis

Repurposing Amlexanox as a Run the Red Light Cure-All with Readthrough a No-Nonsense Approach to Personalised Medicine

The burden of inherited diseases in terms of congenital defects and cancer predisposition in addition to a multitude of neurological, cardiovascular and other organ diseases arising sporadically during life is enormous on the individual, immediate family and society globally. I approach this major burden to human society by invoking the powerful technology behind read-through, whereby disease-causing substitution nonsense mutations, oftentimes at the root of human diseases, are rescued. Further, the importance of nonsense mutations in the realm of developmental factors linked to cancer stem cell maintenance is presented. The methodology behind the technique is given and proof of concept behind its use ex vivo and in clinical trials. The discovery of a novel read-through drug, Amlexanox, which has been in use for over twenty years in dentistry for oral ulcerations, represents a next generation read-through agent. This agent has been demonstrated in cell lines to correct functional loss in cystic fibrosis CFTR, dystrophin and the tumour suppressor, p53. Its novel ability to inhibit nonsense mediated decay is discussed. Amlexanox is therefore presently ready for testing in a wide variety of in vivo models of human diseases and also in clinical trials. Given the safety profile of Amlexanox and ex vivo efficacy in studies thus far it is envisaged that this accepted medication shall successfully debut as an all-purpose agent for the prevention and management of human diseases.

From a single whole exome read to notions of clinical screening: primary ciliary dyskinesia and RSPH9 p.Lys268del in the Arabian Peninsula.

Primary ciliary dyskinesia (PCD) is a genetic disorder, usually autosomal recessive, causing early respiratory disease and later subfertility. Whole exome sequencing may enable efficient analysis for locus heterogeneous disorders such as PCD. We whole-exome-sequenced one consanguineous Saudi Arabian with clinically diagnosed PCD and normal laterality, to attempt ab initio molecular diagnosis. We reviewed 13 known PCD genes and potentially autozygous regions (extended homozygosity) for homozygous exon deletions, non-dbSNP codon, splice-site base variants or small indels. Homozygous non-dbSNP changes were also reviewed exome-wide. One single molecular read representing RSPH9 p.Lys268del was observed, with no wild-type reads, and a notable deficiency of mapped reads at this location. Among all observations, RSPH9 was the strongest candidate for causality. Searching unmapped reads revealed seven more mutant reads. Direct assay for p.Lys268del (MboII digest) confirmed homozygosity in the affected individual, then confirmed homozygosity in three siblings with bronchiectasis. Our finding in southwest Saudi Arabia indicates that p.Lys268del, previously observed in two Bedouin families (Israel, UAE), is geographically widespread in the Arabian Peninsula. Analogous with cystic fibrosis CFTR p.Phe508del, screening for RSPH9 p.Lys268del (which lacks sentinel dextrocardia) in those at risk would help in early diagnosis, tailored clinical management, genetic counselling and primary prevention.

108. Self-Reactive T Cells to CFTR in Cystic Fibrosis (CF) and Non-CF Humans: Implications for Gene Therapy

108. Self-Reactive T Cells to CFTR in Cystic Fibrosis (CF) and Non-CF Humans: Implications for Gene Therapy

Bioinformatics analysis of homologies between pathogen antigens, autoantigens and the CFTR cystic fibrosis protein: A role for immunoadsorption therapy?

The cystic fibrosis CFTR chloride channel is involved in pathogen entry into epithelial cells, and provides the glutathione and hypochlorous acid necessary for bactericidal and viricidal actions. CFTR mutations block these effects, diminishing pathogen defence and allowing pathogen accumulation in the extracellular space, where antibody encounter is likely. The pathogen antigens observed in cystic fibrosis (including P. Aeruginosa, S.Aureus and S.Maltophilia proteins) are homologous to the autoantigens reported in cystic fibrosis and all are homologous to the CFTR protein itself. Antibodies to pathogens and autoantigens may also target the CFTR protein, acting as antagonists, further compromising its function. The tripartite relationship between pathogen antigens, autoantigens and the CFTR protein creates a feed forward cycle, diminishing the function of the CFTR protein and increasing the probability of pathogen accumulation and further antibody encounters at every turn. Kegg pathway analysis of the CFTR/autoantigen interactome indicates that the CFTR protein is also involved in pathogen entry pathways, diabetes and pancreatic and gastric acid secretion pathways, in pathways related to cardiac myopathy, and in the gonadotrophin signalling network, all which are relevant to cystic fibrosis. Interruption of this cycle by antigen and antibody adsorption, and possibly by immunosuppressant therapy may perhaps be of clinical benefit in cystic fibrosis.

Bioinformatics analysis of homologies between pathogen antigens, autoantigens and the CFTR cystic fibrosis protein: A role for immunoadsorption therapy?

AbstractThe cystic fibrosis CFTR chloride channel is involved in pathogen entry into epithelial cells, and provides the glutathione and hypochlorous acid necessary for bactericidal and viricidal actions. CFTR mutations block these effects, diminishing pathogen defence and allowing pathogen accumulation in the extracellular space, where antibody encounter is likely. The pathogen antigens observed in cystic fibrosis (including P. Aeruginosa, S.Aureus and S.Maltophilia proteins) are homologous to the autoantigens reported in cystic fibrosis and all are homologous to the CFTR protein itself. Antibodies to pathogens and autoantigens may also target the CFTR protein, acting as antagonists, further compromising its function. The tripartite relationship between pathogen antigens, autoantigens and the CFTR protein creates a feed forward cycle, diminishing the function of the CFTR protein and increasing the probability of pathogen accumulation and further antibody encounters at every turn. Kegg pathway analysis of the CFTR/autoantigen interactome indicates that the CFTR protein is also involved in pathogen entry pathways, diabetes and pancreatic and gastric acid secretion pathways, in pathways related to cardiac myopathy, and in the gonadotrophin signalling network, all which are relevant to cystic fibrosis. Interruption of this cycle by antigen and antibody adsorption, and possibly by immunosuppressant therapy may perhaps be of clinical benefit in cystic fibrosis.

Reduced histone deacetylase 7 activity restores function to misfolded CFTR in cystic fibrosis

Reduced histone deacetylase 7 activity restores function to misfolded CFTR in cystic fibrosis

CFTR Delivery to 25% of Surface Epithelial Cells Restores Normal Rates of Mucus Transport to Human Cystic Fibrosis Airway Epithelium

Author SummaryThe ciliated epithelium that lines the conducting airways of the lung normally functions to transport hydrated mucus secretions out of the airways to maintain respiratory sterility. Cystic fibrosis (CF) lung disease results from reduced airway surface hydration leading to decreased mucus clearance that precipitates bacterial infection and progressive obstructive lung disease. CF is a genetic disease, and the mutant protein is a chloride ion channel (CFTR) that normally regulates ion and fluid transport on the airway surface. Restoration of corrected CFTR function to the airway epithelium of CF patients by delivering a new CFTR gene to airway epithelial cells has long been envisioned as a therapeutic strategy for CF lung disease. Towards this goal, we use a novel viral vector to deliver CFTR to a culture model that represents the ciliated airway epithelium of CF patients and show that this strategy restores airway surface hydration and mucus transport to levels of that in non-CF individuals. This study demonstrates efficient and efficacious CFTR delivery to CF ciliated airway epithelium and that CFTR delivered to approximately 25% of the surface epithelial cells restores normal levels of airway surface hydration and mucus transport. These studies serve as a benchmark for the efficiency of CFTR gene delivery to CF airways for future CF gene therapy studies in vivo.

SERCA Pump Inhibitors Do Not Correct Biosynthetic Arrest of ΔF508 CFTR in Cystic Fibrosis

SERCA Pump Inhibitors Do Not Correct Biosynthetic Arrest of ΔF508 CFTR in Cystic Fibrosis

Basolateral localization of native ClC-2 chloride channels in absorptive intestinal epithelial cells and basolateral sorting encoded by a CBS-2 domain di-leucine motif

The Cl- channel ClC-2 is expressed in transporting epithelia and has been proposed as an alternative route for Cl- efflux that might compensate for the malfunction of CFTR in cystic fibrosis. There is controversy concerning the cellular and membrane location of ClC-2, particularly in intestinal tissue. The aim of this paper is to resolve this controversy by immunolocalization studies using tissues from ClC-2 knockout animals as control, ascertaining the sorting of ClC-2 in model epithelial cells and exploring the possible molecular signals involved in ClC-2 targeting. ClC-2 was exclusively localized at the basolateral membranes of surface colonic cells or villus duodenal enterocytes. ClC-2 was sorted to the basolateral membranes in MDCK, Caco-2 and LLC-PK1-mu1B, but not in LLC-PK1-mu1A cells. Mutating a di-leucine motif (L812L813) to a di-alanine changed the basolateral targeting of ClC-2 to an apical location. The basolateral membrane localization of ClC-2 in absorptive cells of the duodenum and the colon is compatible with an absorptive function for this Cl- channel. Basolateral targeting information is contained in a di-leucine motif (L812L813) within CBS-2 domain at the C-terminus of ClC-2. It is speculated that ClC-2 also contains an apical sorting signal masked by L812L813. The proposal that CBS domains in ClC channels might behave as regulatory sites sensing intracellular signals opens an opportunity for pharmacological modulation of ClC-2 targeting.

Development and validation of a screening test for 12 common mutations of the cystic fibrosis CFTR gene.

The results obtained using deoxyribonucleic acid (DNA) amplification-based tests must be accurate and reproducible. One such test that simultaneously detects any of 12 of the most common mutations of the cystic fibrosis transmembrane conductance regulator gene is presented in this report. An investigation was conducted into how changes of primer, DNA template and Taq DNA polymerase concentrations and of polymerase chain reaction annealing temperatures affect the test. A total of 383 DNA samples obtained from different laboratories was then examined. The preliminary studies defined the conditions under which accurate results are obtained even if the test is performed under suboptimal conditions. Subsequently, 377 (98.4%) of the DNA samples analysed were in full agreement with DNA typing results derived by other methods. The remaining 1.6% of samples were not mistyped, rather they were not scored owing to failure to detect control DNA sequences. These were also archival DNA preparations rather than freshly prepared samples from venous blood. Careful primer design and optimization of reaction conditions are important in the development of multiplex deoxyribonucleic acid amplification-based diagnostic tests. Providing the recommended protocols are followed, the test described here is simple to carry out, gives accurate results and works well if performed within defined operational windows for each reaction variable.

A mouse model to test the in vivo efficacy of chemical chaperones

In vitro studies in transfected cells have indicated that chemical chaperones including glycerol (0.5–1.2 M) and trimethylamine oxide (TMAO, 50–100 mM) can correct defective trafficking of some proteins, including ΔF508 CFTR in cystic fibrosis and AQP2 mutants in nephrogenic diabetes insipidus. To develop a mouse model to test the efficacy of chemical chaperones in vivo, glycerol and TMAO were administered by intraperitoneal (i.p.), subcutaneous (s.c.), and oral routes. Glycerol and TMAO assays that utilized 1–5 μL of tail vein blood were developed. Administration by the i.p. and s.c. routes gave maximum serum glycerol concentrations of ∼100 mM, levels that were well below the effective in vitro concentrations. Single i.p. or s.c. doses of TMAO (7 g/kg, 8% solution in water) resulted in serum [TMAO] greater than 50 mM, with a long half-life (t 1/2 ∼18–21 h). Sustained high serum and tissue [TMAO] > 52 mM for 3 days was achieved by s.c. administration of TMAO (7 g/kg) in water every 8 h. Although ∼50% of the mice died with this multiple-dose regimen, the remaining mice had nearly normal liver, renal, and pancreatic function. A lower dose of TMAO (5 g/kg) given by the s.c. route every 8 h resulted in serum [TMAO] concentration of 22 mM, a level that was well tolerated by all mice for 72 h. These mice also had high [TMAO] in urine, 400 mM. These results demonstrate that potentially therapeutic concentrations of TMAO can be sustained in mice in vivo, permitting the testing of chemical chaperones in transgenic mouse models of diseases caused by defective protein trafficking.

Gene therapy to human diseases

The identification of defective genes associated with a number of human disorders (tyrosine hydroxylase for Parkinson's disease, aspartylglucosaminidase in lysosomal storage disease, CFTR in cystic fibrosis, and LDL receptor in familial hypercholesterolemia) has promoted the development of strategies aimed at transferring to the somatic cells of the patient or of animal models vectors carrying the corrected gene. The obstacles to overcome include targeting the specific cell type or organ (liver for Factors VIII and IX in hemophilia), enhancing entry to cells into non-lysosomal compartments, nuclear import, percentage of cells transduced with the therapeutic gene, sustained expression of the transgene in human tissues, and immunogenicity of the transduced cells expressing the recombinant or viral proteins. Improvements in each single of these steps are likely to enhance enormously the potential of gene transfer for the treatment of human diseases. A number of human diseases including HIV infections and hypertension are approached by somatic gene transfer. VEGF regulating vascular permeability, growth of endothelial cells and angiogenesis, and TGF-B implicated in wound healing and in stimulation in synthesis of extracellular matrix, are potential targets for restenosis, atherosclerosis, and cancer.

Mapping the human acetylcholinesterase gene to chromosome 7q22 by fluorescent in situ hybridization coupled with selective PCR amplification from a somatic hybrid cell panel and chromosome-sorted DNA libraries

To establish the chromosomal location of the human ACHE gene encoding the acetylcholine hydrolyzing enzyme acetylcholinesterase (ACHE, acetylcholine acetylhydrolase, E.C. 3.1.1.7), a human-specific polymerase chain reaction (PCR) procedure that supports the selective amplification of ACHE DNA fragments from human genomic DNA was employed with 19 human-hamster somatic cell hybrids carrying one or more human chromosomes. Informative ACHE-specific PCR fragments were produced from two cell lines, both of which include human chromosome 7, but not with DNA from 17 cell hybrids carrying various combinations of all human chromosomes other than 7. Fluorescent in situ hybridization of biotinylated ACHE DNA with metaphase chromosomes from human peripheral blood lymphocytes revealed prominent labeling on the 7q22 position. Therefore, further tests were performed to confirm the chromosome 7 location. DNA samples from the two cell lines including chromosome 7 and the ACHE gene were positive with PCR primers informative for the human cystic fibrosis CFTR gene, known to reside at the 7q31.1 position, but negative for the ACHE-related butyrylcholinesterase (BCHE, acylcholine acylhydrolase, E.C. 3.1.1.8) gene, mapped at the 3q26-ter position, confirming that these lines contain chromosome 7 but not chromosome 3. In contrast, three other cell lines including chromosome 3, but not 7, were BCHE-positive and ACHE-negative. In addition, genomic DNA from a sorted chromosome 7 library supported the production of ACHE- but not BCHE-specific PCR products, whereas with DNA from a sorted chromosome 3 library, the BCHE but not the ACHE fragment was amplified. These findings assign the human ACHE gene to a single locus on chromosome 7q22 and should assist in establishing linkage between the in vivo amplification of the ACHE gene in ovarian tumors and leukemias and the phenomenon of tumor-related breakage in the long arm of chromosome 7.

Characterization of the hemolysin transporter, HlyB, using an epitope insertion.

The prokaryotic hlyB gene product is a member of a superfamily of ATP-binding transport proteins that include the eukaryotic multidrug-resistance P-glycoprotein, the yeast STE6, and the cystic fibrosis CFTR gene products (Juranka, P. F., Zastawny, R. L., and Ling, V. (1989) FASEB J. 3, 2583-2592). Previous genetic studies have indicated that HlyB is involved in the transport of the 107-kDa HlyA protein from Escherichia coli; however, the HlyB protein has not been purified for biochemical studies due to its low abundance. In this study, we have engineered a monoclonal antibody epitope into the C-terminal end of HlyB that did not destroy its function. This has allowed us to use immunological methods to identify and localize various molecular forms of the HlyB protein present in vivo.