Cystic Fibrosis Transmembrane Conductance Regulator Protein Expression Research Articles

Promoting readthrough of nonsense mutations in CF mouse model: Biodistribution and efficacy of NV848 in rescuing CFTR protein expression

Nonsense mutations, often resulting from single-nucleotide substitutions, produce mRNA harboring a premature termination codon (PTC), which causes the premature termination of protein synthesis. This produces truncated and non-functional proteins, which cause different genetic diseases, including cystic fibrosis (CF). This work aims to investigate the ability of NV848 (N-(5-methyl-1,2,4-oxadiazol-3-yl)acetamide), a translational readthrough-inducing drug (TRID), to rescue CF transmembrane conductance regulator (CFTR) protein expression in a murine model characterized by the G542X nonsense mutation in the CFTR gene. Invitro experiments assessed the drug's stability in human hepatic metabolism, and invivo investigations on wild-type mice allowed us to clarify the distribution of the drug to the target organs. Moreover, its efficacy in recovering the CFTR protein after chronic treatment was assessed in G542X homozygous mice. Our results provide valuable insights into the biodistribution and therapeutic attributes of NV848, representing a promising therapeutic tool for enhanced clinical outcomes in individuals affected by CF with nonsense mutations.

Pharmacological and pre-clinical safety profile of rSIV.F/HN, a hybrid lentiviral vector for cystic fibrosis gene therapy.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. CFTR modulators offer significant improvements, but ∼10% of patients remain nonresponsive or are intolerant. This study provides an analysis of rSIV.F/HN, a lentiviral vector optimised for lung delivery, including CFTR protein expression, functional correction of CFTR defects and genomic integration site analysis in preparation for a first-in-human clinical trial. Air-liquid interface cultures of primary human bronchial epithelial cells (HBECs) from CF patients (F508del/F508del), as well as a CFTR-deficient immortalised human lung epithelial cell line mimicking class I (CFTR-null) homozygous mutations, were used to assess transduction efficiency. Quantification methods included a novel proximity ligation assay for CFTR protein expression. For assessment of CFTR channel activity, Ussing chamber studies were conducted. The safety profile was assessed using integration site analysis and in vitro insertional mutagenesis studies. rSIV.F/HN expressed CFTR and restored CFTR-mediated chloride currents to physiological levels in primary F508del/F508del HBECs as well as in a class I cells. In contrast, the latter could not be achieved by small-molecule CFTR modulators, underscoring the potential of gene therapy for this mutation class. Combination of rSIV.F/HN-CFTR with the potentiator ivacaftor showed a greater than additive effect. The genomic integration pattern showed no site predominance (frequency of occurrence ≤10%), and a low risk of insertional mutagenesis was observed in an in vitro immortalisation assay. The results underscore rSIV.F/HN as a promising gene therapy vector for CF, providing a mutation-agnostic treatment option.

A promoter-dependent upstream activator augments CFTR expression in diverse epithelial cell types

The cystic fibrosis transmembrane conductance regulator (CFTR) gene encodes an anion-selective channel found in epithelial cell membranes. Mutations in CFTR cause cystic fibrosis (CF), an inherited disorder that impairs epithelial function in multiple organs. Most men with CF are infertile due to loss of intact genital ducts. Here we investigated a novel epididymis-selective cis-regulatory element (CRE), located within a peak of open chromatin at -9.5kb 5' to the CFTR gene promoter. Activation of the -9.5kb CRE alone by CRISPRa had no impact on CFTR gene expression. However, CRISPRa co-activation of the -9.5kb CRE and the CFTR gene promoter in epididymis cells significantly augmented CFTR mRNA and protein expression when compared to promoter activation alone. This increase was accompanied by enhanced chromatin accessibility at both sites. Furthermore, the combined CRISPRa strategy activated CFTR expression in other epithelial cells that lack open chromatin at the -9.5kb site and in which the locus is normally inactive. However, the -9.5kb CRE does not function as a classical enhancer of the CFTR promoter in transient reporter gene assays. These data provide a novel mechanism for activating/augmenting CFTR expression, which may have therapeutic utility for mutations that perturb CFTR transcription.

Identification of an ultra-rare Alu insertion in the CFTR gene: Pitfalls and challenges in genetic test interpretation

Cystic fibrosis (CF) is a life-limiting genetic disorder characterized by defective chloride ion transport due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Early detection through newborn screening programs significantly improves outcomes for individuals with CF by enabling timely intervention. Here, we report the identification of an Alu element insertion within the exon 15 of CFTR gene, initially overlooked in standard next-generation sequencing analyses. However, using traditional molecular techniques, based on polymerase chain reaction and Sanger sequencing, allowed the identification of the Alu element and reporting a correct diagnosis. Our analysis, based on bioinformatics tools and molecular techniques, revealed that the Alu element insertion severely affects the gene expression, splicing patterns, and structure of CFTR protein. In conclusion, this study emphasizes the importance of how the integration of human expertise and modern technologies represents a pivotal step forward in genomic medicine, ensuring the delivery of precision healthcare to individuals affected by genetic diseases.

STUB1 exacerbates calcium oxalate-induced kidney injury by modulating reactive oxygen species-mediated cellular autophagy via regulating CFTR ubiquitination.

The formation of calcium oxalate (CaOx) crystals in the kidneys leads to renal epithelial damage and the progression of crystalline nephropathy. This study investigated the role of STIP1 homology and U-box protein 1 (STUB1), an E3 ubiquitin ligase, and cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel, in CaOx-related renal damage and autophagy regulation.HK-2 cells were treated with various doses of CaOx monohydrate (COM) to simulate kidney injury in vitro. Cell viability, reactive oxygen species (ROS) production, and apoptosis were assessed. The regulation of CFTR ubiquitination by STUB1 was confirmed by immunoprecipitation. An in vivo model was established by injecting mice with glyoxylate.COM treatment dose-dependently decreased cell viability, increased TNF-α and ROS production, and induced apoptotic cell death in HK-2 cells. COM-treated cells also showed decreased CFTR protein expression. CFTR overexpression improved cell viability and reduced ROS production in COM-stimulated HK-2 cells. Bioinformatics analysis predicted CFTR's ubiquitination binding site for STUB1. Further analysis confirmed the role of STUB1 as a ubiquitin ligase in CFTR degradation. Knockdown of STUB1 upregulated CFTR expression, while STUB1 overexpression had the opposite effect. Knockdown of CFTR reversed the impact of STUB1 deficiency on autophagy. The in vivo experiments showed that CFTR overexpression attenuated kidney tissue damage and CaOx deposition in mice.STUB1-mediated CFTR ubiquitination plays a crucial role in mitigating calcium oxalate-related renal damage by regulating autophagy. Targeting the STUB1/CFTR axis may hold therapeutic potential for treating kidney injury associated with calcium oxalate deposition.

Lung SORT LNPs enable precise homology-directed repair mediated CRISPR/Cas genome correction in cystic fibrosis models

Approximately 10% of Cystic Fibrosis (CF) patients, particularly those with CF transmembrane conductance regulator (CFTR) gene nonsense mutations, lack effective treatments. The potential of gene correction therapy through delivery of the CRISPR/Cas system to CF-relevant organs/cells is hindered by the lack of efficient genome editor delivery carriers. Herein, we report improved Lung Selective Organ Targeting Lipid Nanoparticles (SORT LNPs) for efficient delivery of Cas9 mRNA, sgRNA, and donor ssDNA templates, enabling precise homology-directed repair-mediated gene correction in CF models. Optimized Lung SORT LNPs deliver mRNA to lung basal cells in Ai9 reporter mice. SORT LNP treatment successfully corrected the CFTR mutations in homozygous G542X mice and in patient-derived human bronchial epithelial cells with homozygous F508del mutations, leading to the restoration of CFTR protein expression and chloride transport function. This proof-of-concept study will contribute to accelerating the clinical development of mRNA LNPs for CF treatment through CRISPR/Cas gene correction.

Differentiation and Characterization of Cystic Fibrosis Transmembrane Conductance Regulator Knockout Human Pluripotent Stem Cells into Salivary Gland Epithelial Progenitors.

The differentiation of pluripotent stem cells has been used to study disease mechanisms and development. We previously described a method for differentiating human pluripotent stem cells (hPSCs) into salivary gland epithelial progenitors (SGEPs). Here, cystic fibrosis transmembrane conductance regulator (CFTR) knockout hPSCs were differentiated into SGEPs derived from CFTR knockout hESCs (CF-SGEPs) using the same protocol to investigate whether the hPSC-derived SGEPs can model the characteristics of CF. CF-a disease that affects salivary gland (SG) function-is caused by mutations of the CFTR gene. Firstly, we successfully generated CFTR knockout hPSCs with reduced CFTR protein expression using the CRISPR-Cas9 system. After 16 days of differentiation, the protein expression of CFTR decreased in SGEPs derived from CFTR knockout hESCs (CF-SGEPs). RNA-Seq revealed that multiple genes modulating SG development and function were down-regulated, and positive regulators of inflammation were up-regulated in CF-SGEPs, correlating with the salivary phenotype of CF patients. These results demonstrated that CFTR suppression disrupted the differentiation of hPSC-derived SGEPs, which modeled the SG development of CF patients. In summary, this study not only proved that the hPSC-derived SGEPs could serve as manipulable and readily accessible cell models for the study of SG developmental diseases but also opened up new avenues for the study of the CF mechanism.

Exploring the Potential of Drug Repurposing: Detection of Cystic Fibrosis Transmembrane Conductance Regulator as a Biomarker in Breast Cancer Patients

Despite the advancements in cancer treatment and the benefits of personalized medicine, breast cancer (BC) remains a significant health concern. Moreover, conventional treatment options are costly and often associated with severe side effects, highlighting the need to identify new biomarkers and innovative treatment strategies for BC. Drug repurposing presents an approach that expands the therapeutic window further to mitigate the financial burden and minimize the potential harm caused by existing anticancer drugs. In the present study, we aim to suggest noncancerous FDA-approved drugs for repurposing, through molecular docking studies in BC, after their target cystic fibrosis transmembrane conductance regulator protein (CFTR) detection in BC. We used a drug repurposing approach to identify the target protein CFTR and drugs that target CFTR. MOE (molecular operating environment) was used to analyze the interaction between CFTR and its targeting drugs. For experimental work, we did Immunofluorescence staining on BC tissue. We found that glyburide and tezacaftor were the top antagonist and agonists, respectively. We detected CFTR expression in all ten samples except Triple-Negative Breast Cancer (TNBC). CFTR and estrogen receptor alpha co-expressed at 6.78%, suggesting a potential relationship. High-grade tumors showed CFTR expression below 8%, indicating CFTR down-regulation and its role in tumor aggressiveness. Our study is a preliminary step towards in-vitro and in-vivo experiments on repurposing CFTR-targeting drugs in BC. Glyburide may inhibit CFTR's estrogen production, while tezacaftor can enhance CFTR action to overcome tumor aggressiveness.

Mechanism of Xiaoqinglong Decoction and Qingqi Huatan Pills in improving pathological airway mucus based on nuclear factor-κB/microRNA-494 signaling regulation of mucin 5AC and cystic fibrosis transmembrane conductance regulator

To observe the effects of Xiaoqinglong Decoction and Qingqi Huatan Pills on interleukin-1β (IL-1β)-induced mucushypersecretion model of human airway epithelial H292 cellsand related molecules of nuclear factor-κB/microRNA-494(NF-κB/miR-494) signaling pathway, and to explore the mechanism of the two medicines in improving pathological airway mucus. Methyl thiazolyl tetrazolium (MTT) colorimetric method was used to detect the effects of different concentrations of Xiaoqinglong Decoction and Qingqi Huatan Pills on the activity of H292 cellsinduced by IL-1β, and the appropriate concentration was selected for subsequent experiments. Cells were randomly divided into blank group, IL-1β model group (5 μg/L IL-1β), NF-κB inhibitor pyrrolidinedithiocarbamate (PDTC) group (5 μg/L IL-1β+100 μmol/L PDTC), Xiaoqinglong Decoction (5 μg/L IL-1β+1 000 mg/L Xiaoqinglong Decoction) and Qingqi Huatan Pill group (5 μg/L IL-1β+1 000 mg/L Qingqi Huatan Pills). 5 μg/L IL-1β was used to induce H292 cells for 24 hours to establish a model of airway epithelial mucus hypersecretion. Enzyme linked immunosorbent assay (ELISA) method was used to detect the levels of mucin 5AC (MUC5AC), tumor necrosis factor-α (TNF-α) and IL-8 and the synthesis of intracellular MUC5AC and cystic fibrosis transmembrane conductance regulator (CFTR). Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression levels of MUC5AC mRNA, CFTR mRNA, miR-494. Western blotting was used to detect protein expression of key proteins (p65) and NF-κB inhibitors (IκB) in NF-κB signaling pathway. Xiaoqinglong Decoction and Qingqi Huatan Pills with the concentration of 1 000 mg/L were selected for the follow-up experiment. Compared with the blank group, the levels of MUC5AC, TNF-α and IL-8 were significantly increased in the model group, intracellular MUC5AC protein content and mRNA expression were also significantly increased, intracellular CFTR protein content and mRNA expression were significantly decreased, and intracellular p65 protein expression was significantly up-regulated, the expression of IκB protein was significantly down-regulated, and the expression of miR-494 was significantly increased. Compared with the model group, the levels of MUC5AC, TNF-α and IL-8 were significantly reduced in PDTC group, Xiaoqinglong Decoction group and Qingqi Huatan Pill group, intracellular MUC5AC protein content and mRNA expression were also significantly decreased, and intracellular p65 protein expression was significantly down-regulated, and IκB protein expression was significantly up-regulated, miR-494 expression was significantly reduced. Intracellular CFTR protein content and mRNA expression were significantly increased in both PDTC group and Qingqi Huatan Pill group. Compared with the PDTC group, the level of TNF-α in the Xiaoqinglong Decoction group was significantly increased (ng/L: 22.77±3.14 vs. 11.09±3.37, P < 0.05),the content and mRNA expression of CFTR and IκB protein expression was significantly decreased [CFTR protein (ng/L): 97.38±6.62 vs. 227.04±19.48, CFTR mRNA (2-ΔΔCt): 0.99±0.08 vs. 1.21±0.08, IκB/β-actin: 1.69±0.11 vs. 2.00±0.18, all P < 0.05], the level of TNF-α in Qingqi Huatan Pill group was significantly higher (ng/L: 19.08±3.71 vs. 11.09±3.37, P < 0.05). Compared with Xiaoqinglong Decoction group, the protein content and mRNA expression of CFTR and IκB protein expression in Qingqi Huatan Pill group were significantly increased [CFTR protein (ng/L): 235.01±22.71 vs. 97.38±6.62, CFTR mRNA (2-ΔΔCt): 1.32±0.15 vs. 0.99±0.08, IκB/β-actin: 1.94±0.16 vs. 1.69±0.11, all P < 0.05]. The effect of Xiaoqinglong Decoctionin improving the hypersecretion of mucus in the airway epithelium may be related to the inhibition of NF-κB/miR-494 inflammatory signal-mediated MUC5AC hypersecretion, while the effect of Qingqi Huatan Pills may be related to the inhibition of NF-κB/miR-494 inflammatory signal-mediated MUC5AC hypersecretion and CFTR dysfunction. Therefore, the difference in the mechanism of the two treatments of airway pathological mucus is mainly in the regulation of CFTR mRNA and protein.

Targeted quantitation of CFTR protein expression in vivo using immunoprecipitation &amp; parallel reaction monitoring tandem mass spectrometry

Abstract Background The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a low-abundance membrane protein. The dysfunction of CFTR protein is the fundamental cause of cystic fibrosis (CF), a fatal genetic disease. In recent years, the novel messenger RNA (mRNA)-based therapy shows high potential to treat CF disease, by delivering CFTR mRNA into lung epithelial cells to generate fully functional CFTR replacement protein. To evaluate mRNA drug efficacy, a targeted quantitative proteomics method is needed to estimate the expression level of mRNA encoded CFTR protein. Methods In this paper, a method combining membrane protein extraction, immunoprecipitation (IP), and nanoLC-MS/MS for quantifying CFTR in lung tissue samples was reported for the first time. Absolute quantification was performed by constructing a standard curve by spiking recombinant human CFTR protein in mouse lung tissue matrix. Results This method was qualified, with good linearity of standard curve and lower limit of quantification of human CFTR at 1.4 pg per mg tissue. The coefficient of variation of back calculated concentration of all standards and their back-calculation errors were &lt; 20%. The CFTR expression level in mouse lungs dosed with mRNA encapsulated lipid nanoparticle (LNP), and the endogenous level in wild type human lungs were measured successfully. Conclusions The result demonstrated high sensitivity, precision and accuracy of this proteomics method for quantifying low-abundance CFTR protein in lung tissue sample.

Pinostrobin inhibits renal CFTR-mediated Cl− secretion and retards cyst growth in cell-derived cyst and polycystic kidney disease rats

Cystic fibrosis transmembrane conductance regulator (CFTR) plays crucial role in renal cyst expansion via increase in fluid accumulation. Inhibition of CFTR has been proposed to retard cyst development and enlargement in polycystic kidney disease (PKD). Pinostrobin, a bioactive natural flavonoid, possesses several pharmacological effects. The present study investigated pharmacological effects of pinostrobin on CFTR-mediated Cl- secretion and renal cyst expansion in invitro and invivo models. Pinostrobin (10 and 50μM) reduced number of MDCK cell-derived cyst colonies and inhibited cyst expansion via inhibition of cell proliferation and CFTR-mediated Cl- secretion. The inhibitory effect of pinostrobin was not due to the decrease in cell viability and activity of Na+-K+-ATPase. We also investigated the natural analogue pinocembrin as well as the synthetic analogue pinostrobin oxime. Both pinocembrin and pinostrobin oxime did not reduce CFTR-mediated Cl- secretion. In PKD rats, oral administration of pinostrobin (40mg/kg/day) exhibited a decreasing in cystic area compared to vehicle-treated rats. Pinostrobin treatment inhibited renal expression of CFTR protein in PKD rats. Our findings highlighted the potential therapeutic application of pinostrobin in PKD.

Trikafta Rescues CFTR and Lowers Monocyte P2X7R-induced Inflammasome Activation in Cystic Fibrosis.

Rationale: Cystic fibrosis (CF) is caused by mutations in the CFTR (CF transmembrane conductance regulator) gene and is characterized by sustained inflammation. ATP triggers IL-1β secretion via P2X7R (P2X7 receptor) and activation of the NLRP3 (NOD-, LRR-, and pyrin domain-containing protein 3) inflammasome. Objectives: To explore the effect of the CFTR modulator elexacaftor/tezacaftor/ivacaftor (Trikafta) on CFTR expression and the ATP/P2X7R signaling axis in monocytes and on circulating proinflammatory markers. Methods: Inflammatory mediators were detected in blood from 42 patients with CF before and after 3 months of Trikafta therapy. Markers of inflammasome activation and IL-1β secretion were measured in monocytes before and after stimulation with ATP and LPS, in the presence or absence of the P2X7R inhibitor A438079. Measurements and Main Results: P2X7R is overexpressed in CF monocytes, and receptor inhibition decreased NLRP3 expression, caspase-1 activation, and IL-1β secretion. In vitro and invivo, P2X7R expression is regulated by CFTR function and intracellular chloride (Cl-) levels. Trikafta therapy restored CFTR expression yet decreased P2X7R in CF monocytes, resulting in normalized Cl- and potassium efflux, and reduced intracellular calcium levels. CFTR modulator therapy decreased circulating levels of ATP and LPS and reduced inflammasome activation and IL-1β secretion. Conclusions: P2X7R expression is regulated by intracellular Cl- levels and in CF monocytes promotes inflammasome activation. Trikafta therapy significantly increased CFTR protein expression and reduced ATP/P2X7R-induced inflammasome activation. P2X7R may therefore be a promising target for reducing inflammation in patients with CF who are noneligible for Trikafta or other CFTR modulator therapy.

CFTR deficiency aggravates Ang II induced vasoconstriction and hypertension by regulating Ca2+ influx and RhoA/Rock pathway in VSMCs.

Cystic fibrosis transmembrane conductance regulator (CFTR) has been associated with vascular tone and blood pressure (BP), however, its role in the genesis of hypertension remains elusive. In the present study, we investigated the regulating effect of CFTR on angiotensin II (Ang II) -induced hypertension and defined the molecular role of CFTR in vasoconstriction. We found that CFTR mRNA and protein expression were markedly down-regulated in the arteries from Ang II induced hypertensive animals. During the development of hypertension, BP of Cftr-⁣/- mice was significantly higher than that of Cftr+⁣/+ mice. Arteries from Cftr-⁣/- mice or pre-incubated with CFTR specific inhibitor CFTR(inh)-172 exhibited a greater contractile response to Ang II. In vascular smooth muscle cells (VSMCs), the phosphorylation of myosin light chain (MLC), which is the core of VSMCs contraction, was negatively modulated by CFTR. Furthermore, intracellular Ca2+ concentration ([Ca2+]i) rise in response to Ang II was negatively modulated by CFTR, while no alteration was observed in resting VSMCs. Ras homolog family member A/Rho-associated protein kinase (RhoA/Rock) mediated phosphorylation of myosin phosphatase target subunit 1 (MYPT1), a regulator of MLC phosphorylation, was negatively modulated by CFTR in both resting and Ang II-stimulated VSMCs. This study demonstrates that CFTR is a negative regulator of vasoconstriction and hypertension, and the underlying mechanism contains two possible pathways: (1) in resting VSMCs, CFTR altered MLC phosphorylation through RhoA/Rock pathway; (2) in Ang II stimulated VSMCs, the regulating effect was mediated by both Ca2+ influx and RhoA/Rock mediated pathway.

Phosphodiesterase 8A Regulates CFTR Activity in Airway Epithelial Cells.

Cystic fibrosis transmembrane conductance regulator (CFTR), the anion channel that is defective in cystic fibrosis (CF), is phosphorylated and activated by cAMP-dependent protein kinase (PKA). cAMP levels are downregulated by a large family of phosphodiesterases that have variable expression in different cell types. We have previously observed high levels of PDE8A expression in well-differentiated primary human bronchial epithelial (pHBE) cells and thus aimed to assess whether it played a role in cAMP-dependent regulation of CFTR activity. We assessed the effect of the selective PDE8 inhibitor PF-04957325 (PF) on intracellular cAMP levels ([cAMP]i) in well differentiated pHBE cells from non-CF or CF donors and also in CFBE41o- cells that stably express wild-type CFTR (CFBE41o- WT) using ELISA and FRET-FLIM microscopy. CFTR channel function was also measured using electrophysiological recordings from pHBE and CFBE41o- WT cells mounted in Ussing Chambers. PDE8 inhibition elevated [cAMP]i in well-differentiated pHBE cells and stimulated wild-type CFTR-dependent ion transport under basal conditions or after cells had been pre-stimulated with physiological cAMP-elevating agents. The response to PDE8 inhibition was larger than to PDE3 or PDE5 inhibition but smaller and synergistic with that elicited by PDE4 inhibition. CRISPR Cas9-mediated knockdown of PDE8A enhanced CFTR gene and protein expression yet reduced the effect of PDE8 inhibition. Acute pharmacological inhibition PDE8 increased CFTR activity in CF pHBE cells (F508del/F508del and F508del/R117H-5T) treated with clinically-approved CFTR modulators. These results provide the first evidence that PDE8A regulates CFTR and identifies PDE8A as a potential target for adjunct therapies to treat CF.

Evaluation of a novel CFTR potentiator in COPD ferrets with acquired CFTR dysfunction.

The majority of chronic obstructive pulmonary disease (COPD) patients have chronic bronchitis, for which specific therapies are unavailable. Acquired cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction is observed in chronic bronchitis, but has not been proven in a controlled animal model with airway disease. Furthermore, the potential of CFTR as a therapeutic target has not been tested in vivo, given limitations to rodent models of COPD. Ferrets exhibit cystic fibrosis-related lung pathology when CFTR is absent and COPD with bronchitis following cigarette smoke exposure. To evaluate CFTR dysfunction induced by smoking and test its pharmacological reversal by a novel CFTR potentiator, GLPG2196, in a ferret model of COPD with chronic bronchitis. Ferrets were exposed for 6 months to cigarette smoke to induce COPD and chronic bronchitis and then treated with enteral GLPG2196 once daily for 1 month. Electrophysiological measurements of ion transport and CFTR function, assessment of mucociliary function by one-micron optical coherence tomography imaging and particle-tracking microrheology, microcomputed tomography imaging, histopathological analysis and quantification of CFTR protein and mRNA expression were used to evaluate mechanistic and pathophysiological changes. Following cigarette smoke exposure, ferrets exhibited CFTR dysfunction, increased mucus viscosity, delayed mucociliary clearance, airway wall thickening and airway epithelial hypertrophy. In COPD ferrets, GLPG2196 treatment reversed CFTR dysfunction, increased mucus transport by decreasing mucus viscosity, and reduced bronchial wall thickening and airway epithelial hypertrophy. The pharmacologic reversal of acquired CFTR dysfunction is beneficial against pathological features of chronic bronchitis in a COPD ferret model.

Effects of indacaterol on the LPS-evoked changes in fluid secretion rate and pH in swine tracheal membrane

An acquired dysregulation of airway secretion is likely involved in the pathophysiology of chronic bronchitis and chronic obstructive pulmonary disease (COPD). Nowadays, it is widely known that several kinds of long-acting bronchodilators reduce the frequency of COPD exacerbations. However, limited data are available concerning the complementary additive effects on airflow obstruction. Using an optical method and a selective pH indicator, we succeeded in evaluating the gland secretion rate and the pH in swine tracheal membrane. A physiologically relevant concentration of acetylcholine (ACh) 100 nM induced a gradual increase in the amount of gland secretion. Lipopolysaccharides (LPS) accelerated the ACh-induced secretory responses up to around threefold and lowered the pH level significantly. Long-acting β2-agonists (LABAs) including indacaterol (IND), formoterol, and salmeterol restored the LPS-induced changes in both the hypersecretion and acidification. The subsequent addition of the long-acting muscarine antagonist, glycopyrronium, further increased the pH values. Two different inhibitors for cystic fibrosis transmembrane conductance regulator (CFTR), NPPB and CFTRinh172, abolished the IND-mediated pH normalization in the presence of both ACh and ACh + LPS. Both immunofluorescence staining and western blotting analysis revealed that LPS downregulated the abundant expression of CFTR protein. However, IND did not restore the LPS-induced decrease in CFTR expression on Calu-3 cells. These findings suggest that the activation of cAMP-dependent HCO3− secretion through CFTR would be partly involved in the IND-mediated pH normalization in gland secretion and may be suitable for the maintenance of airway defense against exacerbating factors including LPS.

The Effect of Synonymous Single-Nucleotide Polymorphisms on an Atypical Cystic Fibrosis Clinical Presentation

Synonymous single nucleotide polymorphisms (sSNPs), which change a nucleotide, but not the encoded amino acid, are perceived as neutral to protein function and thus, classified as benign. We report a patient who was diagnosed with cystic fibrosis (CF) at an advanced age and presented very mild CF symptoms. The sequencing of the whole cystic fibrosis transmembrane conductance regulator (CFTR) gene locus revealed that the patient lacks known CF-causing mutations. We found a homozygous sSNP (c.1584G>A) at the end of exon 11 in the CFTR gene. Using sensitive molecular methods, we report that the c.1584G>A sSNP causes cognate exon skipping and retention of a sequence from the downstream intron, both of which, however, occur at a relatively low frequency. In addition, we found two other sSNPs (c.2562T>G (p.Thr854=) and c.4389G>A (p.Gln1463=)), for which the patient is also homozygous. These two sSNPs stabilize the CFTR protein expression, compensating, at least in part, for the c.1584G>A-triggered inefficient splicing. Our data highlight the importance of considering sSNPs when assessing the effect(s) of complex CFTR alleles. sSNPs may epistatically modulate mRNA and protein expression levels and consequently influence disease phenotype and progression.

A Scientific Rationale for Using Cystic Fibrosis Transmembrane Conductance Regulator Therapeutics in COVID-19 Patients.

Several pathological manifestations in coronavirus disease 2019 (COVID-19), including thick mucus, poor mucociliary clearance, and bronchial wall thickening, overlap with cystic fibrosis disease patterns and may be indicative of “acquired” cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction. Indeed, tumor necrosis factor (TNF), a key cytokine driving COVID-19 pathogenesis, downregulates lung CFTR protein expression, providing a strong rationale that acquired CFTR dysfunction arises in the context of COVID-19 infection. In this perspective, we propose that CFTR therapeutics, which are safe and generally well-tolerated, may provide benefit to COVID-19 patients. Although CFTR therapeutics are currently only approved for treating cystic fibrosis, there are efforts to repurpose them for conditions with “acquired” CFTR dysfunction, for example, chronic obstructive pulmonary disease. In addition to targeting the primary lung pathology, CFTR therapeutics may possess value-added effects: their anti-inflammatory properties may dampen exaggerated immune cell responses and promote cerebrovascular dilation; the latter aspect may offer some protection against COVID-19 related stroke.

Mechanism-Based Personalized Medicine for Cystic Fibrosis by Suppressing Pseudo Exon Inclusion

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that compromise its chloride channel activity. Here, we present a therapeutic strategy to ameliorate RNA splicing deficiency of CFTR with a small molecule. The 3,849+ 10 kb C>T is the most common splicing mutation in CF, creating a pseudo exon with premature stop codon. We reveal that the 3,849+ 10 kb C>T-induced CFTR pseudo exon is regulated by phosphorylation of serine/arginine-rich splicing factors, and their functional inhibition by a CDC-like kinase inhibitor restores normal splicing of CFTR. Subsequent screening of our focused chemical library identified CaNDY as a rectifier of the aberrant splicing. CaNDY treatment restored normal splicing of CFTR with the 3,849+ 10 kb C>T in CF patient cells and functional CFTR protein expression in the CF model cells. Our findings open the door for mechanism-based personalized medicine for pseudo-exon-type genetic diseases.

Consequences of CRISPR-Cas9-Mediated CFTR Knockout in Human Macrophages.

Macrophage dysfunction is fundamentally related to altered immunity in cystic fibrosis (CF). How genetic deficits in the cystic fibrosis transmembrane conductance regulator (CFTR) lead to these defects remains unknown. Rapid advances in genomic editing such as the clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR/Cas9) system provide new tools for scientific study. We aimed to create a stable CFTR knockout (KO) in human macrophages in order to study how CFTR regulates macrophage function. Peripheral blood monocytes were isolated from non-CF healthy volunteers and differentiated into monocyte-derived macrophages (MDMs). MDMs were transfected with a CRISPR Cas9 CFTR KO plasmid. CFTR KO efficiency was verified and macrophage halide efflux, phagocytosis, oxidative burst, apoptosis, and cytokine functional assays were performed. CFTR KO in human MDMs was efficient and stable after puromycin selection. CFTR KO was confirmed by CFTR mRNA and protein expression. CFTR function was abolished in CFTR KO MDMs. CFTR KO recapitulated known defects in human CF MDM (CFTR class I/II variants) dysfunction including (1) increased apoptosis, (2) decreased phagocytosis, (3) reduced oxidative burst, and (4) increased bacterial load. Activation of the oxidative burst via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase assembly was diminished in CFTR KO MDMs (decreased phosphorylated p47phox). Cytokine production was unchanged or decreased in response to infection in CFTR KO MDMs. In conclusion, we developed a primary human macrophage CFTR KO system. CFTR KO mimics most pathology observed in macrophages obtained from persons with CF, which suggests that many aspects of CF macrophage dysfunction are CFTR-dependent and not just reflective of the CF inflammatory milieu.