Alyftrek® (vanzacaftor/tezacaftor/deutivacaftor, VTD) is a triple CFTR modulator therapy for patients with cystic fibrosis (CF). In this study, we characterized the pharmacological properties of vanzacaftor and deutivacaftor using functional and biochemical assays in patients derived primary bronchial epithelial (HBE) cells (F508del/F508del, F508del/R1162X, F508del/W1282X) and in CFBE41o⁻ cells expressing F508del-CFTR. Cells were treated with Alyftrek® components across a range of concentrations. The S- and R-enantiomers of vanzacaftor were evaluated separately, and the activity of deutivacaftor was compared with that of ivacaftor. CFTR function was assessed by short-circuit current measurements and whole-cell patch-clamp recordings, while CFTR protein maturation was analyzed by Western blotting. Treatment with S-vanzacaftor alone restored F508del-CFTR-dependent chloride currents with an EC₅₀ of 130 nM, which decreased markedly to 8 nM when combined with tezacaftor. Substitution of ivacaftor with deutivacaftor in triple combinations (VTI vs. VTD) yielded comparable functional responses. Both VTD and VTI promoted the concentration-dependent appearance of the mature, fully glycosylated CFTR C-band with an EC₅₀ of 11 nM. The R-enantiomer of vanzacaftor was markedly less potent in rescuing F508del-CFTR and antagonized the effect of the S-enantiomer. Concentration-reduction experiments further showed that 10% to 50% of the initial VTI/VTD concentrations were sufficient to achieve substantial F508del-CFTR rescue. Collectively, these findings demonstrate the high potency of S-vanzacaftor at nanomolar concentrations, both alone and in combination with tezacaftor. The potentiator deutivacaftor exhibited efficacy comparable to that of ivacaftor.

Since the development of the first cystic fibrosis transmembrane conductance regulator (CFTR) modulator in 2012, these therapies have revolutionized patients’ health. They are now the most effective treatment for people with cystic fibrosis (pwCF). In fact, elexacaftor/tezacaftor/ivacaftor and vanzacaftor/tezacaftor/deutivacaftor, the latest combination therapies consisting of a CFTR potentiator and two CFTR correctors, improved lung function by 14% in pwCF. Other modulator therapies targeting CFTR mRNA and/or protein are currently under preclinical/clinical investigation. However, due to the variant-specific nature of these therapies, about 10% of pwCF in Europe remains without effective treatment, and many treated pwCF experience various adverse events such as headaches, infections, hepatotoxicity, hypertension, and depression. Therefore, mutation-agnostic strategies such as gene therapy are needed. They could expand treatment eligibility for all pwCF and improve outcomes. In fact, nucleic acid delivery (e.g., pDNA, mRNA, oligonucleotides, genome editing) or targeting non-CFTR channels to restore ion transport represent promising future additional directions for CF therapy. This review aims to discuss a potential combination between gene therapy approaches and existing modulators to improve treatment eligibility, safety, and efficacy.

Trikafta (elexacaftor/tezacaftor/ivacaftor; ETI) is approved for cystic fibrosis (CF) patients with at least one F508del mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene or another responsive mutation based on in vitro data. However, the pharmacological effects of ETI on F508del-CFTR remain incompletely defined in vitro. To explore the mechanisms underlying Trikafta's clinical efficacy, we used primary bronchial epithelial cells from F508del homozygous patients and CFBE41o-cells expressing F508del-CFTR. We assessed CFTR maturation, turnover, chloride transport, and thermal stability under various ETI concentrations and treatment durations at physiological temperature using electrophysiology (Ussing chamber, patch-clamp) and biochemical assays. We found that ETI efficacy on F508del-CFTR is strongly influenced by both treatment duration and concentration. Reducing ETI from standard doses, i.e., E (3 µM), T (18 µM), and I (1 µM), to 33%, 11%, 3.3%, and 1.1% decreased function and maturation, but 33% retained most of the corrective effect. After 2 h of treatment, around 50% of the CFTR-dependent current was preserved, unlike in untreated cells. Notably, replacing elexacaftor with bamocaftor further improved F508del-CFTR maturation and function compared with ETI, though it did not affect the rate of current decline over time. These findings highlight the importance of optimizing ETI dose and exposure duration, as both significantly affect F508del-CFTR stability and function. The retained efficacy at reduced concentrations suggests possible individualized dosing strategies, particularly for patients experiencing adverse effects with full-dose ETI.NEW & NOTEWORTHY Our in vitro study underscores that ETI/BTI's efficacy in improving F508del-CFTR function depends on treatment concentration and duration, impacting the protein's metabolic and thermal stability. Although ETI/BTI only partially addresses F508del-CFTR's inherent thermal instability, reduced doses retained significant effectiveness. This finding supports dose optimization as a promising strategy to sustain therapeutic benefits while minimizing side effects, offering a personalized approach to treatment for individuals with cystic fibrosis experiencing adverse effects from standard dosing.

BACKGROUND AND PURPOSE:Cystic fibrosis is an autosomal recessive disease caused by mutations in the CFTR gene, leading to progressive respiratory decline and reduced life expectancy. The most common mutation, CFTR-F508del, results in mislocalised and non-functional protein. Although triple therapy with elexacaftor/tezacaftor/ivacaftor (ETI) is prescribed for patients carrying this mutation, some biological defects remain unresolved. We previously identified COMMD1 as a potential therapeutic target, as its overexpression enhances CFTR-WT plasma membrane localisation. CIGB-552, a cell-penetrating peptide discovered in 2013, stabilises COMMD1. This study evaluates its therapeutic potential in cystic fibrosis. EXPERIMENTAL APPROACH:CIGB-552 was tested, with and without ETI, in CFBE and HEK cells stably expressing CFTR-WT or CFTR-F508del, and in primary human bronchial cells. CFTR function was assessed using YFP quenching and short-circuit current assays. Peptide uptake was evaluated using FITC-labelled CIGB-552 in submerged and air-liquid interface models. Plasma membrane density of CFTR was measured in CFBE CFTR-HA cells, and western blotting assessed CFTR maturation and COMMD1 expression. KEY RESULTS:CIGB-552 was non-toxic and preferentially entered CFBE CFTR-F508del cells rather than CFBE CFTR-WT cells, without altering COMMD1 expression or localisation. Although not a corrector or potentiator alone, CIGB-552 synergised with ETI, enhancing CFTR-F508del-mediated chloride efflux, confirmed in primary cells. CIGB-552 also increased YFP quenching of CFTR-WT and CFTR-G551D, in combination with ivacaftor. This effect required COMMD1. CONCLUSIONS AND IMPLICATIONS:COMMD1 expression was necessary for CIGB-552 to affect CFTR function positively. Its synergy with the triple therapy offers a promising strategy for improving CF treatment.

Despite the importance of the ocular surface in human physiology and diseases, little is known about ion channel expression, properties, and regulation in ocular epithelial cells. Furthermore, human primary epithelial cells have rarely been studied in favor of rat, mouse, and especially rabbit animal models. Here, we developed primary human meibomian gland (hMGEC) and conjunctival (hConEC) epithelial cells. We show that hConEC and hMGEC produce MUC5AC and lipids, respectively. With cell cultures maintained at the air-liquid interface, we recorded transepithelial short-circuit currents (Isc) by the Ussing chamber method. We identified in the apical membrane Na+, Cl-, and K+ ion channels; amiloride-sensitive epithelial sodium channel (ENaC), cAMP-dependent CFTR, UTP-dependent TMEM16a, and chromanol 293B-sensitive KCNQ1. At the basolateral membrane, we identified bumetanide-sensitive NKCC and barium-sensitive K+ channels. We also found that vasoactive intestinal peptide, concentration-dependent (EC50 of 1-8 nM), stimulates the CFTR-dependent Isc in both cells. Western blot analysis confirms the expression in both cell cultures of βENaC subunit, CFTR, TMEM16a, and KCNQ1 proteins. We recorded water influx by quantitative phase microscopy and identified a cAMP-dependent and mercury-sensitive water flux and identified by Western blot AQP3 and AQP5 proteins in hConEC and hMGEC. Taken together, we propose a model of the ion transports of human conjunctival and meibomian gland epithelial cells that will set the stage for future molecular dissection of the regulation of these transport proteins in the context of tear secretion and related diseases.NEW & NOTEWORTHY We generated human meibomian gland and conjunctival epithelial cells producing lipids and mucins. We identified ion channels including ENaC, CFTR, TMEM16a, and KCNQ1, as well as NKCC. We found that electrolyte and water flux are regulated by signaling pathways mediated by purinergic and VIP receptors. Our findings provide valuable insights into epithelial ion and water transport in the human conjunctiva and meibomian gland, enhancing understanding of these processes in both physiological and disease states.

ATP-binding cassette (ABC) transporters constitute a 49-member superfamily in humans. These proteins, most of them being transmembrane, allow the active transport of an important variety of substrates across biological membranes, using ATP hydrolysis as an energy source. For an important proportion of these ABC transporters, genetic variations of the loci encoding them have been correlated with rare genetic diseases, including cystic fibrosis and interstitial lung disease (variations in CFTR/ABCC7 and ABCA3) as well as cholestatic liver diseases (variations in ABCB4 and ABCB11). In this review, we first describe these ABC transporters and how their molecular dysfunction may lead to human diseases. Then, we propose a classification of the genetic variants according to their molecular defect (expression, traffic, function and/or stability), which may be considered as a general guideline for all ABC transporters' variants. Finally, we discuss recent progress in the field of targeted pharmacotherapy, which aim to correct specific molecular defects using small molecules. In conclusion, we are opening the path to treatment repurposing for diseases involving similar deficiencies in other ABC transporters.

The accumulation of mucus resulting from the obstruction of bronchi of cystic fibrosis (CF) patients, induces a reduction of the oxygen (O2) pressure and produces a hypoxic environment for the epithelial cells of the lungs. Our study aims to better characterize the impact of hypoxia on CFTR function in the pathophysiological context of cystic fibrosis. We used Human airway epithelial cells from two CF donors and human bronchial epithelial cell lines non-CF and CF, grown and expended in normoxia (21% O2) and then switched to hypoxia (1% O2) for 2 to 24 hours. Cells were treated by dimethyl sulfoxide or Elexacaftor/ Tezacaftor/Ivacaftor for 24 hours. We show that the peak of Hypoxia Inducible Factor 1α is reached in a range of 4 to 6 hours post-hypoxia induction. We also demonstrate that the global amount of ETI corrected F508del-CFTR is significantly decreased after 24 hours of hypoxia. A decreased ETI corrected F508del-CFTR activity was recorded by both patch-clamp and Ussing chamber recordings. Our results show that hypoxia, despite the effectiveness of Elexacaftor/ Tezacaftor/Ivacaftor correction, impacts the downstream effects of the F508del mutation, which suggests that oxygen availability in the lungs is a factor to take into account for the administration of Trikafta to patients.

IntroductionCystic Fibrosis (CF) is an autosomal and recessive disease caused by the mutation of a gene located on the chromosome 7: CFTR (Cystic Fibrosis Transmembrane conductance Regulator). Its codes for CFTR, a protein which plays a role in mucus homeostasis by transporting both chloride ions and water. The most common mutation F508del-CFTR, leads to the absence and malfunction of CFTR at the surface of epithelial cells in various organs especially in lungs. It results in the loss of the mucus clearance properties in the airways, which will cause the obstruction of bronchi and alveola over time. The oxygen (O2) delivery, crucial for aerobic metabolism, is less effective especially for the lung's epithelial cells whose environment is gradually becoming hypoxic. The inability of lungs to realise haematosis, at tissue level is commonly named the respiratory failure.Our project aims to characterise the impact of hypoxia on ion channels, especially CFTR and TRPA1 (an oxygen sensible calcium channel).MethodsCystic Fibrosis Bronchial Epithelial (CFBE) cells-wt (WT) and CFBE F508del (DF) are cultivated in a controlled hypoxic atmosphere (1% O2). Protein expression and quantification have been realised by western blot. CFTR activity have been measured by automated patch-clamp (whole cell recording, WCR) and Ussing chamber while the activity of TRPA1 have been recorded using the Fluo4-AM probe. TRPA1 localisation has been studied by immunostaining.ResultsHere, we show that the change from normoxia (21% O2) to hypoxia (1% O2) is able to induce a fast cellular response with the accumulation of HIF-1α (Hypoxia Inducible Factor) in only 6hours in CFBE WT, CFBE-DF and CFBE-DF corrected by the tri-therapy Kaftrio® (Elexacaftor/Tezacaftor/Ivacaftor, ETI). We also observed, that HIF-1α accumulated is reduced in non-corrected CFBE-F508del. Regarding CFTR, our results shows that only F508del-CFTR is impacted by hypoxia at protein level and activity, despite the correction by ETI. Automated WCR patch-clamp and Ussing chamber recordings both show that Kaftrio® corrected F508del-CFTR activity decreases 24hours after hypoxia induction. F508del-CFTR currents are decreased by 43% in whole cell configuration while short-circuit current (Isc) CFTR dependent are diminished by around 48%. Concerning TRPA1, hypoxia does not impact the protein accumulation but instead decrease the channel activity by 49% in CFBE-wt, 40% in CFBE-F508del non corrected and 30% when corrected by ETI. Finally, it seems that hypoxia plays a role in TRPA1 location inducing its relocation close to the plasma membrane.ConclusionOur data show a reduced amount of CFTR protein accumulated in CFBE F508del corrected or not, which was not observed on the WT form. Electrophysiologic assays show a clear impact of hypoxia on F508del-CFTR activity at a cellular level and at a pseudo-epithelium level. Despite a lack of impact of hypoxia on TRPA1 protein accumulation, both activity (decreased by hypoxia) and his location (closer to the plasma membrane) are affected by a lower O2 concentration. It is also important to note that in hypoxic state, ETI is not as efficient as it could be in normoxia, which raises questions about the intake conditions of treatment for patients.

BACKGROUND:Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) channel. For people with CF (pwCF) affected by the most common pathogenic variant F508del, a tritherapy, named Trikafta/Kaftrio (ETI: elexacaftor (VX-445) /tezacaftor (VX-661) / ivacaftor (VX-770)) was successfully developed. However, in CF airway epithelial cells the calcium homeostasis is also disturbed; it is observed an increased calcium mobilization in CF cells compared to non-CF cells. Here, we studied the effects of ETI on intracellular calcium levels in F508del-CFTR airway epithelial cells to determine whether these compounds, individually or collectively, could normalize intracellular calcium levels. METHODS:We measured intracellular calcium variations using human airway epithelial cells (hAEC) from pwCF, human bronchial epithelial CFBE41o- F508del-CFTR cells and Chinese Hamster Ovary (CHO) cells using the fluorescent probe Fluo4-AM, in the presence or absence of extracellular calcium. The rescue to the plasma membrane of F508del-CFTR protein by ETI was determined by western blot. The SarcoEndoplasmic Reticulum Calcium ATPase (SERCA), was also analysed by western blotting and by interference assay. RESULTS:We show that ETI normalizes calcium homeostasis in our cellular models. However, we also found that (1) each ETI-corrector compound is capable of mobilizing calcium acutely in the absence of CFTR, and (2) tezacaftor mobilizes calcium from the endoplasmic reticulum (ER) probably via inhibition of the SERCA pump. CONCLUSIONS:We show that ETI not only corrects the abnormal trafficking and function of F508del-CFTR but also normalizes calcium homeostasis in our cellular models. Finally, we identified SERCA as a potential intracellular target for tezacaftor.

La mucoviscidose est une pathologie provoquée par une mutation sur le gène CFTR codant pour un canal chlorure. La mutation prépondérante F508del, entraîne l’absence de la protéine CFTR à la surface des cellules épithéliales et par la suite une accumulation du mucus visqueux au niveau des voies aériennes. Dans les stades avancés de cette maladie, une insuffisance respiratoire s’accompagne d’une hypoxie au niveau cellulaire. Notre projet vise à caractériser l’impact de l’hypoxie sur les canaux ioniques ainsi que les voies de signalisation sous-jacentes impliquées, mais aussi de tester l’efficacité des traitements actuels de la mucoviscidose dans de telles conditions.

Cystic fibrosis is a disease caused by a mutation on the CFTR gene coding for a chloride channel. The dominant mutation F508del eliminates the CFTR protein at the surface of epithelial cells, causing an accumulation of viscous mucus in the airways. In advanced stages of the disease, respiratory failure is associated with cellular hypoxia. Our project aims not only to describe the impact of hypoxia on ion channels and to highlight the underlying signaling pathways involved, but also to test the effectiveness of current CF treatments under the above -mentioned conditions.(c) 2023 SPLF. Published by Elsevier Masson SAS. All rights reserved.

Background: The majority of variants of unknown clinical significance (VUCS) in the CFTR gene are missense variants. While change on the CFTR protein structure or function is often suspected, impact on splicing may be neglected. Such undetected splicing default of variants may complicate the interpretation of genetic analyses and the use of an appropriate pharmacotherapy. Methods: We selected 15 variants suspected to impact CFTR splicing after in silico predictions on 319 missense variants (214 VUCS), reported in the CFTR -France database. Six specialized laboratories assessed the impact of nucleotide substitutions on splicing (minigenes), mRNA expression levels (quantitative PCR), synthesis and maturation (western blot), cellular localization (immunofluorescence) and channel function (patch clamp) of the CFTR protein. We also studied maturation and function of the truncated protein, consecutive to in-frame aberrant splicing, on additional plasmid constructs. Results: Six of the 15 variants had a major impact on CFTR splicing by in-frame ( n = 3) or out-of-frame ( n = 3) exon skipping. We reclassified variants into: splicing variants; variants causing a splicing defect and the impairment of CFTR folding and/or function related to the amino acid substitution; deleterious missense variants that impair CFTR folding and/or function; and variants with no consequence on the different processes tested. Conclusion: The 15 variants have been reclassified by our comprehensive approach of in vitro experiments that should be used to properly interpret very rare exonic variants of the CFTR gene. Targeted therapies may thus be adapted to the molecular defects regarding the results of laboratory experiments. (c) 2022 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Trikafta, currently the leading therapeutic in cystic fibrosis (CF), has demonstrated a real clinical benefit. This treatment is the triple combination therapy of two folding correctors elexacaftor/tezacaftor (VX445/VX661) plus the gating potentiator ivacaftor (VX770). In this study, our aim was to compare the properties of F508del-CFTR in cells treated with either lumacaftor (VX809), tezacaftor, elexacaftor, elexacaftor/tezacaftor with or without ivacaftor. We studied F508del-CFTR function, maturation and membrane localisation by Ussing chamber and whole-cell patch-clamp recordings, Western blot and immunolocalisation experiments. With human primary airway epithelial cells and the cell lines CFBE and BHK expressing F508del, we found that, whereas the combination elexacaftor/tezacaftor/ivacaftor was efficient in rescuing F508del-CFTR abnormal maturation, apical membrane location and function, the presence of ivacaftor limits these effects. The basal F508del-CFTR short-circuit current was significantly increased by elexacaftor/tezacaftor/ivacaftor and elexacaftor/tezacaftor compared to other correctors and nontreated cells, an effect dependent on ivacaftor and cAMP. These results suggest that the level of the basal F508del-CFTR current might be a marker for correction efficacy in CF cells. When cells were treated with ivacaftor combined to any correctors, the F508del-CFTR current was unresponsive to the subsequently acute addition of ivacaftor, unlike the CFTR (cystic fibrosis transmembrane conductance regulator) potentiators genistein and Cact-A1 which increased elexacaftor/tezacaftor/ivacaftor and elexacaftor/tezacaftor-corrected F508del-CFTR currents. These findings show that ivacaftor reduces the correction efficacy of Trikafta. Thus, combining elexacaftor/tezacaftor with a different potentiator might improve the therapeutic efficacy for treating CF patients.

Visual deficit is one of the complications of Huntington disease (HD), a fatal neurological disorder caused by CAG trinucleotide expansions in the Huntingtin gene, leading to the production of mutant Huntingtin (mHTT) protein. Transgenic HD R6/1 mice expressing human HTT exon1 with 115 CAG repeats recapitulate major features of the human pathology and exhibit a degeneration of the retina. Our aim was to gain insight into the ultrastructure of the pathological HD R6/1 retina by electron microscopy (EM). We show that the HD R6/1 retina is enriched with unusual organelles myelinosomes, produced by retinal neurons and glia. Myelinosomes are present in all nuclear and plexiform layers, in the synaptic terminals of photoreceptors, in the processes of retinal neurons and glial cells, and in the subretinal space. In vitro study shows that myelinosomes secreted by human retinal glial Müller MIO-M1 cells transfected with EGFP-mHTT-exon1 carry EGFP-mHTT-exon1 protein, as revealed by immuno-EM and Western-blotting. Myelinosomes loaded with mHTT-exon1 are incorporated by naive neuronal/neuroblastoma SH-SY5Y cells. This results in the emergence of mHTT-exon1 in recipient cells. This process is blocked by membrane fusion inhibitor MDL 28170. Conclusion: Incorporation of myelinosomes carrying mHTT-exon1 in recipient cells may contribute to HD spreading in the retina. Exploring ocular fluids for myelinosome presence could bring an additional biomarker for HD diagnostics.

The medical and scientific history of cystic fibrosis will have a lasting impact on human medicine. It will take 50 years of scientific experiments and medical observations, favored by advances in genetics, molecular biology and physiology to go from the ionic theory showing that this disease is the consequence of a generalized defect of the transepithelial transport of NaCl until the cloning of the CFTR gene in 1989. The discovery of the gene and its mutations, the description of the CFTR protein and its role in the disease have revolutionized the physiology and the pathophysiology of ionic transports in epithelial cells of the respiratory and digestive systems.

We present a low-cost, 3D-printed, and biocompatible fluidic device, engineered to produce laminar and homogeneous flow over a large field-of-view. Such a fluidic device allows us to perform multiplexed temporal monitoring of cell cultures compatible with the use of various pharmacological protocols. Therefore, specific properties of each of the observed cell cultures can be discriminated simultaneously during the same experiment. This was illustrated by monitoring the agonists-mediated cellular responses, with digital holographic microscopy, of four different cell culture models of cystic fibrosis. Quantitatively speaking, this multiplexed approach provides a time saving factor of around four to reveal specific cellular features.