Reduced histone deacetylase 7 activity restores function to misfolded CFTR in cystic fibrosis.
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ABSTRACT:
SUBMITTER: Hutt DM
PROVIDER: S-EPMC2901172 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Class Ia/b cystic fibrosis transmembrane regulator (CFTR) variants cause severe lung disease in 10% of cystic fibrosis (CF) patients and are untreatable with small-molecule pharmaceuticals. Genetic replacement of CFTR offers a cure, but its effectiveness is limited in vivo. We hypothesized that enhancing protein levels (using codon optimization) and/or activity (using gain-of-function variants) of CFTR would more effectively restore function to CF bronchial epithelial cells. Three different variants of the CFTR protein were tested: codon optimized (high codon adaptation index [hCAI]), a gain-of-function (GOF) variant (K978C), and a combination of both (hˆK978C). In human embryonic kidney (HEK293T) cells, initial results showed that hCAI and hˆK978C produced greater than 10-fold more CFTR protein and displayed ∼4-fold greater activity than wild-type (WT) CFTR. However, functionality was profoundly different in CF bronchial epithelial cells. Here, K978C CFTR more potently restored essential epithelial functions (anion transport, airway surface liquid height, and pH) than WT CFTR. hCAI and hˆK978C CFTRs had limited impact because of mislocalization in the cell. These data provide a proof of principle showing that GOF variants may be more effective than codon-optimized forms of CFTR for CF gene therapy.Video abstract
| S-EPMC10494266 | biostudies-literature
Project description:Background and purposeCystic fibrosis transmembrane conductance regulator (CFTR) potentiators are small molecules developed to treat the genetic disease cystic fibrosis (CF). They interact directly with CFTR Cl- channels at the plasma membrane to enhance channel gating. Here, we investigate the action of a new CFTR potentiator, CP-628006 with a distinct chemical structure.Experimental approachUsing electrophysiological assays with CFTR-expressing heterologous cells and CF patient-derived human bronchial epithelial (hBE) cells, we compared the effects of CP-628006 with the marketed CFTR potentiator ivacaftor.Key resultsCP-628006 efficaciously potentiated CFTR function in epithelia from cultured hBE cells. Its effects on the predominant CFTR variant F508del-CFTR were larger than those with the gating variant G551D-CFTR. In excised inside-out membrane patches, CP-628006 potentiated wild-type, F508del-CFTR, and G551D-CFTR by increasing the frequency and duration of channel openings. CP-628006 increased the affinity and efficacy of F508del-CFTR gating by ATP. In these respects, CP-628006 behaved like ivacaftor. CP-628006 also demonstrated notable differences with ivacaftor. Its potency and efficacy were lower than those of ivacaftor. CP-628006 conferred ATP-dependent gating on G551D-CFTR, whereas the action of ivacaftor was ATP-independent. For G551D-CFTR, but not F508del-CFTR, the action of CP-628006 plus ivacaftor was greater than ivacaftor alone. CP-628006 delayed, but did not prevent, the deactivation of F508del-CFTR at the plasma membrane, whereas ivacaftor accentuated F508del-CFTR deactivation.Conclusions and implicationsCP-628006 has distinct effects compared to ivacaftor, suggesting a different mechanism of CFTR potentiation. The emergence of CFTR potentiators with diverse modes of action makes therapy with combinations of potentiators a possibility.
| S-EPMC9304199 | biostudies-literature
Project description:Cystic fibrosis is the most common genetically determined, life-limiting disorder in populations of European ancestry. The genetic basis of cystic fibrosis is well established to be mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that codes for an apical membrane chloride channel principally expressed by epithelial cells. Conventional approaches to cystic fibrosis care involve a heavy daily burden of supportive treatments to combat lung infection, help clear airway secretions and maintain nutritional status. In 2012, a new era of precision medicine in cystic fibrosis therapeutics began with the licensing of a small molecule, ivacaftor, which successfully targets the underlying defect and improves CFTR function in a subgroup of patients in a genotype-specific manner. Here, we review the three main targeted approaches that have been adopted to improve CFTR function: potentiators, which recover the function of CFTR at the apical surface of epithelial cells that is disrupted in class III and IV genetic mutations; correctors, which improve intracellular processing of CFTR, increasing surface expression, in class II mutations; and production correctors or read-through agents, which promote transcription of CFTR in class I mutations. The further development of such approaches offers great promise for future therapeutic strategies in cystic fibrosis.
| S-EPMC4582929 | biostudies-literature
Project description:Despite the addition of cystic fibrosis transmembrane conductance regulator (CFTR) modulators to the cystic fibrosis (CF) treatment regimen, patients with CF continue to suffer from chronic bacterial infections that lead to progressive respiratory morbidity. Host immunity, and macrophage dysfunction specifically, has an integral role in the inability of patients with CF to clear bacterial infections. We sought to characterize macrophage responses to CFTR modulator treatment as we hypothesized that there would be differential effects based on patient genotype. Human CF and non-CF peripheral blood monocyte-derived macrophages (MDMs) were analyzed for CFTR expression, apoptosis, polarization, phagocytosis, bacterial killing, and cytokine production via microscopy, flow cytometry, and ELISA-based assays. Compared to non-CF MDMs, CF MDMs display decreased CFTR expression, increased apoptosis, and decreased phagocytosis. CFTR expression increased and apoptosis decreased in response to ivacaftor or lumacaftor/ivacaftor therapy, and phagocytosis improved with ivacaftor alone. Ivacaftor restored CF macrophage polarization responses to non-CF levels and reduced Pseudomonas aeruginosa bacterial burden, but did not reduce other bacterial loads. Macrophage inflammatory cytokine production decreased in response to ivacaftor alone. In summary, ivacaftor and lumacaftor/ivacaftor have differential impacts on macrophage function with minimal changes observed in CF patients treated with lumacaftor/ivacaftor. Overall improvements in macrophage function in ivacaftor-treated CF patients result in modestly improved macrophage-mediated bacterial killing.
| S-EPMC6244248 | biostudies-literature
Project description: Not available
| S-EPMC4485492 | biostudies-literature
Project description:BackgroundCystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. In this study we assessed the effect of antisense oligonucleotide eluforsen on CFTR biological activity measured by Nasal Potential Difference (NPD) in patients with the most common mutation, F508del-CFTR.MethodsThis multi-centre, exploratory, open-label study recruited adults with CF homozygous or compound heterozygous for the F508del-CFTR mutation. Subjects received intranasal eluforsen three times weekly for 4 weeks. The primary endpoint was the within-subject change from baseline in total chloride transport (Cl-free+iso), as assessed by NPD. Secondary endpoints included within-subject change from baseline in sodium transport.ResultsIn the homozygous cohort (n = 7; per-protocol population), mean change (90% confidence interval) in Cl-free+iso was -3.0 mV (-6.6; 0.6) at day 15, -4.1 mV (-7.8; -0.4, p = .04) at day 26 (end of treatment) and - 3.7 mV (-8.0; 0.6) at day 47. This was supported by improved sodium transport as assessed by an increase in average basal potential difference at day 26 of +9.4 mV (1.1; 17.7, p = .04). The compound heterozygous cohort (n = 7) did not show improved chloride or sodium transport NPD values. Eluforsen was well tolerated with a favourable safety profile.ConclusionsIn F508del-CFTR homozygous subjects, repeated intranasal administration of eluforsen improved CFTR activity as measured by NPD, an encouraging indicator of biological activity.
| S-EPMC7227803 | biostudies-literature
Project description:Restoration of BECN1/Beclin 1-dependent autophagy and depletion of SQSTM1/p62 by genetic manipulation or autophagy-stimulatory proteostasis regulators, such as cystamine, have positive effects on mouse models of human cystic fibrosis (CF). These measures rescue the functional expression of the most frequent pathogenic CFTR mutant, F508del, at the respiratory epithelial surface and reduce lung inflammation in Cftr(F508del) homozygous mice. Cysteamine, the reduced form of cystamine, is an FDA-approved drug. Here, we report that oral treatment with cysteamine greatly reduces the mortality rate and improves the phenotype of newborn mice bearing the F508del-CFTR mutation. Cysteamine was also able to increase the plasma membrane expression of the F508del-CFTR protein in nasal epithelial cells from F508del homozygous CF patients, and these effects persisted for 24 h after cysteamine withdrawal. Importantly, this cysteamine effect after washout was further sustained by the sequential administration of epigallocatechin gallate (EGCG), a green tea flavonoid, both in vivo, in mice, and in vitro, in primary epithelial cells from CF patients. In a pilot clinical trial involving 10 F508del-CFTR homozygous CF patients, the combination of cysteamine and EGCG restored BECN1, reduced SQSTM1 levels and improved CFTR function from nasal epithelial cells in vivo, correlating with a decrease of chloride concentrations in sweat, as well as with a reduction of the abundance of TNF/TNF-alpha (tumor necrosis factor) and CXCL8 (chemokine [C-X-C motif] ligand 8) transcripts in nasal brushing and TNF and CXCL8 protein levels in the sputum. Altogether, these results suggest that optimal schedules of cysteamine plus EGCG might be used for the treatment of CF caused by the F508del-CFTR mutation.
| S-EPMC4502695 | biostudies-literature
Project description:The treatment of cystic fibrosis (CF) patients homozygous for the F508del mutation with Orkambi®, a combination of a corrector (lumacaftor) and a potentiator (ivacaftor) of the mutated CFTR protein, resulted in some amelioration of the respiratory function. However, a great variability in the clinical response was also observed. The aim of this study was to evaluate the response to Orkambi® in a small cohort of F508del/F508del patients (n = 14) in terms of clinical and laboratory parameters, including ex vivo CFTR activity in mononuclear cells (MNCs), during a 12-month treatment. Patients responded with an increase in percent predicted forced expiratory volume in 1 s (FEV1%) and body mass index (BMI) as well as with a decrease in white blood cell (WBC) total counts and serum C-reactive protein (CRP) levels, although not significantly. Sweat chloride and CFTR-dependent chloride efflux were found to decrease and increase, respectively, as compared with pre-therapy values. CFTR and BMI showed a statistically significant correlation during Orkambi® treatment. Clustering analysis showed that CFTR, BMI, sweat chloride, FEV1%, and WBC were strongly associated. These data support the notion that CFTR-dependent chloride efflux in MNCs should be investigated as a sensitive outcome measure of Orkambi® treatment in CF patients.
| S-EPMC7177453 | biostudies-literature
Project description:BACKGROUND:Chronic lung disease resulting from dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR) and NF?B-mediated neutrophilic-inflammation forms the basis of CF-related mortality. Here we aimed to evaluate if HDAC inhibition controls Pseudomonas-aeruginosa-lipopolysaccharide (Pa-LPS) induced airway inflammation and CF-lung disease. METHODS:For in vitro experiments, HEK293-cells were transfected with IL-8 or NF?B-firefly luciferase, and SV40-renilla- luciferase reporter constructs or ?F508-CFTR-pCEP, followed by treatment with suberoylanilide hydroxamic acid (SAHA), Trichostatin-A (TSA) and/or TNF?. For murine studies, Cftr +/+ or Cftr -/- mice (n?=?3) were injected/instilled with Pa-LPS and/or treated with SAHA or vehicle control. The progression of lung disease was monitored by quantifying changes in inflammatory markers (NF?B), cytokines (IL-6/IL-10), neutrophil activity (MPO, myeloperoxidase and/or NIMP-R14) and T-reg numbers. RESULTS:SAHA treatment significantly (p?<?0.05) suppresses TNF?-induced NF?B and IL-8 reporter activities in HEK293-cells. Moreover, SAHA, Tubacin (selective HDAC6-inhibitor) or HDAC6-shRNAs controls CSE-induced ER-stress activities (p?<?0.05). In addition, SAHA restores trafficking of misfolded-?F508-CFTR, by inducing protein levels of both B and C forms of CFTR. Murine studies using Cftr +/+ or Cftr -/- mice verified that SAHA controls Pa-LPS induced IL-6 levels, and neutrophil (MPO levels and/or NIMP-R14), NF?B-(inflammation) and Nrf2 (oxidative-stress marker) activities, while promoting FoxP3+ T-reg activity. CONCLUSION:In summary, SAHA-mediated HDAC inhibition modulates innate and adaptive immune responses involved in pathogenesis and progression of inflammatory CF-lung disease.
| S-EPMC5755330 | biostudies-literature
Project description:Growth failure in cystic fibrosis (CF) patients has been well-documented and shown to correlate with poorer disease outcomes. This observation is also true in CF animal models, including mouse, pig, rat, and ferret. The etiology underlying growth deficits is unknown, and our previous work demonstrated reduced tubulin acetylation in CF cell models and tissue that is correctable by inhibition of histone deacetylase-6 (HDAC6). Here, we hypothesize that loss of HDAC6 will improve growth phenotype in a CF mouse model. Hdac6 knockout mice were crossed with F508del (CF) mice to generate F508del/Hdac6 (CF/HDA) mice. Growth, fat deposits, survival, and bioelectric measurements were analyzed. CF/HDA mice displayed improvements in length and weight with no correction of CFTR function. Mechanistically, Igf1 levels likely account for increased length and improvements in fertility. Weight gain is attributed to increased fat deposits potentially mediated by increased adipocyte differentiation. CF-related growth deficits can be improved via inhibition of HDAC6, further implicating it as a potential therapeutic target for CF.
| S-EPMC5473831 | biostudies-literature