Putting bicarbonate on the spot: pharmacological insights for CFTR correction in the airway epithelium.

Introduction: Cystic fibrosis (CF) is caused by defective Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) proteins. CFTR controls chloride (Cl-) and bicarbonate (HCO3 -) transport into the Airway Surface Liquid (ASL). We investigated the impact of F508del-CFTR correction on HCO3 - secretion by studying transepithelial HCO3 - fluxes. Methods: HCO3 - secretion was measured by pH-stat technique in primary human respiratory epithelial cells from healthy subjects (WT) and people with CF (pwCF) carrying at least one F508del variant. Its changes after CFTR modulation by the triple combination VX445/661/770 and in the context of TNF-α+IL-17 induced inflammation were correlated to ASL pH and transcriptional levels of CFTR and other HCO3 - transporters of airway epithelia such as SLC26A4 (Pendrin), SLC26A9 and NBCe1. Results: CFTR-mediated HCO3 - secretion was not detected in F508del primary human respiratory epithelial cells. It was rescued up to ∼ 80% of the WT level by VX-445/661/770. In contrast, TNF-α+IL-17 normalized transepithelial HCO3 - transport and increased ASL pH. This was related to an increase in SLC26A4 and CFTR transcript levels. VX-445/661/770 induced an increase in pH only in the context of inflammation. Effects on HCO3 - transport were not different between F508del homozygous and F508del compound heterozygous CF airway epithelia. Conclusion: Our studies show that correction of F508del-CFTR HCO3 - is not sufficient to buffer acidic ASL and inflammation is a key regulator of HCO3 - secretion in CF airways. Prediction of the response to CFTR modulators by theratyping should take into account airway inflammation.

Theratyping cystic fibrosis patients to guide elexacaftor/tezacaftor/ivacaftor out-of-label prescription.

BACKGROUND: Around 20% of people with cystic fibrosis (pwCF) do not have access to the triple combination elexacaftor/tezacaftor/ivacaftor (ETI) in Europe because they do not carry the F508del allele on the CF transmembrane conductance regulator (CFTR) gene. Considering that pwCF carrying rare variants may benefit from ETI, including variants already validated by the US Food and Drug Administration (FDA), a compassionate use programme was launched in France. PwCF were invited to undergo a nasal brushing to investigate whether the pharmacological rescue of CFTR activity by ETI in human nasal epithelial cell (HNEC) cultures was predictive of the clinical response. METHODS: CFTR activity correction was studied by short-circuit current in HNEC cultures at basal state (dimethyl sulfoxide (DMSO)) and after ETI incubation and expressed as percentage of normal (wild-type (WT)) CFTR activity after sequential addition of forskolin and Inh-172 (ΔI ETI/DMSO%WT). RESULTS: 11 pwCF carried variants eligible for ETI according to the FDA label and 28 carried variants not listed by the FDA. ETI significantly increased CFTR activity of FDA-approved CFTR variants (I601F, G85E, S492F, M1101K, R347P, R74W;V201M;D1270N and H1085R). We point out ETI correction of non-FDA-approved variants, including N1303K, R334W, R1066C, Q552P and terminal splicing variants (4374+1G>A and 4096-3C>G). ΔI ETI/DMSO%WT was significantly correlated to change in percentage predicted forced expiratory volume in 1 s and sweat chloride concentration (p<0.0001 for both). G85E, R74W;V201M;D1270N, Q552P and M1101K were rescued more efficiently by other CFTR modulator combinations than ETI. CONCLUSIONS: Primary nasal epithelial cells hold promise for expanding the prescription of CFTR modulators in pwCF carrying rare mutants. Additional variants should be discussed for ETI indication.

Nonsense mutations accelerate lung disease and decrease survival of cystic fibrosis children.

RATIONALE: Limited information is available on the clinical status of people with Cystic Fibrosis (pwCF) carrying 2 nonsense mutations (PTC/PTC). The main objective of this study was to compare disease severity between pwCF PTC/PTC, compound heterozygous for F508del and PTC (F508del/PTC) and homozygous for F508del (F508del+/+). METHODS: Based on the European CF Society Patient Registry clinical data of pwCF living in high and middle income European and neighboring countries, PTC/PTC (n = 657) were compared with F508del+/+ (n = 21,317) and F508del/PTC(n = 4254).CFTR mRNA and protein activity levels were assessed in primary human nasal epithelial (HNE) cells sampled from 22 PTC/PTC pwCF. MAIN RESULTS: As compared to F508del+/+ pwCF; both PTC/PTC and F508del/PTC pwCF exhibited a significantly faster rate of decline in Forced Expiratory Volume in 1 s (FEV1) from 7 years (-1.33 for F508del +/+, -1.59 for F508del/PTC; -1.65 for PTC/PTC, p < 0.001) until respectively 30 years (-1.05 for F508del +/+, -1.23 for PTC/PTC, p = 0.048) and 27 years (-1.12 for F508del +/+, -1.26 for F508del/PTC, p = 0.034). This resulted in lower FEV1 values in adulthood. Mortality of pediatric pwCF with one or two PTC alleles was significantly higher than their F508del homozygous pairs. Infection with Pseudomonas aeruginosa was more frequent in PTC/PTC versus F508del+/+ and F508del/PTC pwCF. CFTR activity in PTC/PTC pwCF's HNE cells ranged between 0% to 3% of the wild-type level. CONCLUSIONS: Nonsense mutations decrease the survival and accelerate the course of respiratory disease in children and adolescents with Cystic Fibrosis.

Isolation, cultivation, and application of primary respiratory epithelial cells obtained by nasal brushing, polyp samples, or lung explants.

Here, we present a standardized protocol for isolation, maintenance, and polarization of the respiratory epithelial primary cells from patient samples acquired from nasal brushing, polyp specimens, or lung explants. This protocol generates a clearly defined polarized layer of epithelial cells on filters, with a good number of ciliated cells and a thin layer of mucus. We detail the steps for samples prepared from patients with cystic fibrosis as well as from subjects without cystic fibrosis.

Primary Human Nasal Epithelial Cells: Biobanking in the Context of Precision Medicine.

Human nasal epithelial (HNE) cells are easy to collect by simple, non-invasive nasal brushing. Patient-derived primary HNE cells can be amplified and differentiated into a pseudo-stratified epithelium in air-liquid interface conditions to quantify cyclic AMP-mediated Chloride (Cl-) transport as an index of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) function. If critical steps such as quality of nasal brushing and cell density upon cryopreservation are performed efficiently, HNE cells can be successfully biobanked. Moreover, short-circuit current studies demonstrate that freeze-thawing does not significantly modify HNE cells' electrophysiological properties and response to CFTR modulators. In the culture conditions used in this study, when less than 2 x 106 cells are frozen per cryovial, the failure rate is very high. We recommend freezing at least 3 x 106 cells per cryovial. We show that dual therapies combining a CFTR corrector with a CFTR potentiator have a comparable correction efficacy for CFTR activity in F508del-homozygous HNE cells. Triple therapy VX-445 + VX-661 + VX-770 significantly increased correction of CFTR activity compared to dual therapy VX-809 + VX-770. The measure of CFTR activity in HNE cells is a promising pre-clinical biomarker useful to guide CFTR modulator therapy.

Systemic bis-phosphinic acid derivative restores chloride transport in Cystic Fibrosis mice.

Mutations in the Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR) are responsible for Cystic Fibrosis (CF). The most common CF-causing mutation is the deletion of the 508th amino-acid of CFTR (F508del), leading to dysregulation of the epithelial fluid transport in the airway's epithelium and the production of a thickened mucus favoring chronic bacterial colonization, sustained inflammation and ultimately respiratory failure. c407 is a bis-phosphinic acid derivative which corrects CFTR dysfunction in epithelial cells carrying the F508del mutation. This study aimed to investigate c407 in vivo activity in the F508del Cftrtm1Eur murine model of CF. Using nasal potential difference measurement, we showed that in vivo administration of c407 by topical, short-term intraperitoneal and long-term subcutaneous route significantly increased the CFTR dependent chloride (Cl-) conductance in F508del Cftrtm1Eur mice. This functional improvement was correlated with a relocalization of F508del-cftr to the apical membrane in nasal epithelial cells. Importantly, c407 long-term administration was well tolerated and in vitro ADME toxicologic studies did not evidence any obvious issue. Our data provide the first in vivo preclinical evidence of c407 efficacy and absence of toxicity after systemic administration for the treatment of Cystic Fibrosis.

Reclassifying inconclusive diagnosis after newborn screening for cystic fibrosis. Moving forward.

BACKGROUND: Newborn screening for Cystic Fibrosis (CF) is associated with situations where the diagnosis of CF or CFTR related disorders (CFTR-RD) cannot be clearly ruled out. MATERIALS/PATIENTS AND METHODS: We report a case series of 23 children with unconclusive diagnosis after newborn screening for CF and a mean follow-up of 7.7 years (4-13). Comprehensive investigations including whole CFTR gene sequencing, in vivo intestinal current measurement (ICM), nasal potential difference (NPD), and in vitro functional studies of variants of unknown significance, helped to reclassify the patients. RESULTS: Extensive genetic testing identified, in trans with a CF causing mutation, variants with varying clinical consequences and 3 variants of unknown significance (VUS). Eighteen deep intronic variants were identified by deep resequencing of the whole CFTR gene in 13 patients and were finally considered as non-pathogenic. All patients had normal CFTR dependent chloride transport in ICM. NPD differentiated 3 different profiles: CF-like tracings qualifying the patients as CF, such as F508del/D1152H patients; normal responses, suggesting an extremely low likelihood of developing a CFTR-RD such as F508del/TG11T5 patients; partial CFTR dysfunction above 20% of the normal, highlighting a remaining risk of developing CFTR-RD such as F508del/F1052V patients. The 3 VUS were reclassified as variant with defective maturation (D537N), defective expression (T582I) or with no clinical consequence (M952T). CONCLUSION: This study demonstrates the usefulness of combining genetic and functional investigations to assess the possibility of evolving to CF or CFTR-RD in babies with inconclusive diagnosis at neonatal screening.

Correlating genotype with phenotype using CFTR-mediated whole-cell Cl- currents in human nasal epithelial cells.

Dysfunction of the epithelial anion channel cystic fibrosis transmembrane conductance regulator (CFTR) causes a wide spectrum of disease, including cystic fibrosis (CF) and CFTR-related diseases (CFTR-RDs). Here, we investigate genotype-phenotype-CFTR function relationships using human nasal epithelial (hNE) cells from a small cohort of non-CF subjects and individuals with CF and CFTR-RDs and genotypes associated with either residual or minimal CFTR function using electrophysiological techniques. Collected hNE cells were either studied directly with the whole-cell patch-clamp technique or grown as primary cultures at an air-liquid interface after conditional reprogramming. The properties of cAMP-activated whole-cell Cl- currents in freshly isolated hNE cells identified them as CFTR-mediated. Their magnitude varied between hNE cells from individuals within the same genotype and decreased in the rank order: non-CF > CFTR residual function > CFTR minimal function. CFTR-mediated whole-cell Cl- currents in hNE cells isolated from fully differentiated primary cultures were identical to those in freshly isolated hNE cells in both magnitude and behaviour, demonstrating that conditional reprogramming culture is without effect on CFTR expression and function. For the cohort of subjects studied, CFTR-mediated whole-cell Cl- currents in hNE cells correlated well with CFTR-mediated transepithelial Cl- currents measured in vitro with the Ussing chamber technique, but not with those determined in vivo with the nasal potential difference assay. Nevertheless, they did correlate with the sweat Cl- concentration of study subjects. Thus, this study highlights the complexity of genotype-phenotype-CFTR function relationships, but emphasises the value of conditionally reprogrammed hNE cells in CFTR research and therapeutic testing. KEY POINTS: The genetic disease cystic fibrosis is caused by pathogenic variants in the cystic fibrosis transmembrane conductance regulator (CFTR), an ion channel, which controls anion flow across epithelia lining ducts and tubes in the body. This study investigated CFTR function in nasal epithelial cells from people with cystic fibrosis and CFTR variants with a range of disease severity. CFTR function varied widely in nasal epithelial cells depending on the identity of CFTR variants, but was unaffected by conditional reprogramming culture, a cell culture technique used to grow large numbers of patient-derived cells. Assessment of CFTR function in vitro in nasal epithelial cells and epithelia, and in vivo in the nasal epithelium and sweat gland highlights the complexity of genotype-phenotype-CFTR function relationships.

Sweat Chloride Testing and Nasal Potential Difference (NPD) Are Primary Outcome Parameters in Treatment with Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Modulators.

With the advent of CFTR modulators, surrogate outcome parameters that accurately quantify the improvement in CFTR activity are needed. In vivo biomarkers that reflect CFTR ion transport and can serve as outcomes in the treatment of CFTR modulators are the sweat Cl- test (SCT), the nasal potential difference (NPD) measurement or the intestinal current measurement (ICM). This review focus on the SCT and NPD. The SCT displays a low intra-patient variability in contrast to the NPD. It has been used extensively as a biomarker of CFTR function in clinical trials of CFTR modulator therapies and provides evidence for change in the short term. The level of functional rescue in the NPD increases up to 40% of normal CFTR in patients with a Gly551Asp treated with ivacaftor monotherapy, while in F508del homozygous patients treated with ivacaftor-lumacaftor, activity increased on average up to ~20% of normal activity. While both tests provide evidence of the effect on CFTR activity, they cannot be used at an individual level to predict the response to any CFTR modulators. Nevertheless, their rapid modification, reflecting electrophysiological properties, highlight their potential use in proof-of-concept studies for CFTR modulators.

Antisense oligonucleotide-based drug development for Cystic Fibrosis patients carrying the 3849+10 kb C-to-T splicing mutation.

BACKGROUND: Antisense oligonucleotide (ASO)-based drugs for splicing modulation were recently approved for various genetic diseases with unmet need. Here we aimed to develop an ASO-based splicing modulation therapy for Cystic Fibrosis (CF) patients carrying the 3849+10 kb C-to-T splicing mutation in the CFTR gene. METHODS: We have screened, in FRT cells expressing the 3849+10 kb C-to-T splicing mutation, ~30 2'-O-Methyl-modified phosphorothioate ASOs, targeted to prevent the recognition and inclusion of a cryptic exon generated due to the mutation. The effect of highly potent ASO candidates on the splicing pattern, protein maturation and CFTR function was further analyzed in well differentiated primary human nasal and bronchial epithelial cells, derived from patients carrying at least one 3849+10 kb C-to-T allele. RESULTS: A highly potent lead ASO, efficiently delivered by free uptake, was able to significantly increase the level of correctly spliced mRNA and completely restore the CFTR function to wild type levels in cells from a homozygote patient. This ASO led to CFTR function with an average of 43% of wild type levels in cells from various heterozygote patients. Optimized efficiency of the lead ASO was further obtained with 2'-Methoxy Ethyl modification (2'MOE). CONCLUSION: The highly efficient splicing modulation and functional correction, achieved by free uptake of the selected lead ASO in various patients, demonstrate the ASO therapeutic potential benefit for CF patients carrying splicing mutations and is aimed to serve as the basis for our current clinical development.

New insights into structure and function of bis-phosphinic acid derivatives and implications for CFTR modulation.

C407 is a compound that corrects the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein carrying the p.Phe508del (F508del) mutation. We investigated the corrector effect of c407 and its derivatives on F508del-CFTR protein. Molecular docking and dynamics simulations combined with site-directed mutagenesis suggested that c407 stabilizes the F508del-Nucleotide Binding Domain 1 (NBD1) during the co-translational folding process by occupying the position of the p.Phe1068 side chain located at the fourth intracellular loop (ICL4). After CFTR domains assembly, c407 occupies the position of the missing p.Phe508 side chain. C407 alone or in combination with the F508del-CFTR corrector VX-809, increased CFTR activity in cell lines but not in primary respiratory cells carrying the F508del mutation. A structure-based approach resulted in the synthesis of an extended c407 analog G1, designed to improve the interaction with ICL4. G1 significantly increased CFTR activity and response to VX-809 in primary nasal cells of F508del homozygous patients. Our data demonstrate that in-silico optimized c407 derivative G1 acts by a mechanism different from the reference VX-809 corrector and provide insights into its possible molecular mode of action. These results pave the way for novel strategies aiming to optimize the flawed ICL4-NBD1 interface.

Unsolved severe chronic rhinosinusitis elucidated by extensive CFTR genotyping.

Severe chronic rhinosinusitis in children should alert clinicians and extensive CFTR genotyping should be performed. We propose that thorough clinical and functional assessment in severe chronic rhinosinusitis is valuable to discover rare mutations which could be treated by CFTR correctors to postpone pulmonary infection.

Author Correction: Airway surface liquid acidification initiates host defense abnormalities in Cystic Fibrosis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Airway surface liquid acidification initiates host defense abnormalities in Cystic Fibrosis.

Cystic fibrosis (CF) is caused by defective Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein. Morbidity is mainly due to early airway infection. We hypothesized that S. aureus clearance during the first hours of infection was impaired in CF human Airway Surface Liquid (ASL) because of a lowered pH. The ASL pH of human bronchial epithelial cell lines and primary respiratory cells from healthy controls (WT) and patients with CF was measured with a pH microelectrode. The antimicrobial capacity of airway cells was studied after S. aureus apical infection by counting surviving bacteria. ASL was significantly more acidic in CF than in WT respiratory cells. This was consistent with a defect in bicarbonate secretion involving CFTR and SLC26A4 (pendrin) and a persistent proton secretion by ATP12A. ASL demonstrated a defect in S. aureus clearance which was improved by pH normalization. Pendrin inhibition in WT airways recapitulated the CF airway defect and increased S. aureus proliferation. ATP12A inhibition by ouabain decreased bacterial proliferation. Antimicrobial peptides LL-37 and hBD1 demonstrated a pH-dependent activity. Normalizing ASL pH might improve innate airway defense in newborns with CF during onset of S. aureus infection. Pendrin activation and ATP12A inhibition could represent novel therapeutic strategies to normalize pH in CF airways.

Predictive factors for lumacaftor/ivacaftor clinical response.

BACKGROUND: Ivacaftor-lumacaftor combination therapy corrects the F508 del-CFTR mutated protein which causes Cystic Fibrosis. The clinical response of the patients treated with the combination therapy is highly variable. This study aimed to determine factors involved in the individual's response to lumacaftor-ivacaftor therapy. METHODS: Sweat test was assessed at baseline and after 6 months of ivacaftor-lumacaftor treatment in 41 homozygous F508del children and young adults. ß-adrenergic peak sweat secretion, nasal potential difference (NPD) and intestinal current measurements (ICM) were performed in patients accepting these tests. Seric level of lumacaftor and ivacaftor were determined and additional CFTR variant were searched. RESULTS: Sweat chloride concentration significantly decreased after treatment, whereas the ß-adrenergic peak sweat response did not vary in 9 patients who underwent these tests. The average level of F508del-CFTR activity rescue reached up to 15% of the normal level in intestinal epithelium, as studied by ICM in 12 patients (p = .03) and 20% of normal in the nasal epithelium in NPD tests performed in 21 patients (NS). There was no significant correlation between these changes and improvements in FEV1 at 6 months. Serum drug levels did not correlate with changes in FEV1, BMI-Zscore or other CFTR activity biomarkers. Additional exonic variants were identified in 4 patients. The F87L-I1027T-F508del-CFTR complex allele abolished the lumacaftor corrector effect. CONCLUSION: This observational study investigates a number of potential factors linked to the clinical response of F508del homozygous patients treated with lumacaftor-ivacaftor combination therapy. Lumacaftor and ivacaftor blood levels are not associated with the clinical response. Additional exonic variants may influence protein correction.

Characterization of two rat models of cystic fibrosis-KO and F508del CFTR-Generated by Crispr-Cas9.

BACKGROUND: Genetically engineered animals are essential for gaining a proper understanding of the disease mechanisms of cystic fibrosis (CF). The rat is a relevant laboratory model for CF because of its zootechnical capacity, size, and airway characteristics, including the presence of submucosal glands. METHODS: We describe the generation of a CF rat model (F508del) homozygous for the p.Phe508del mutation in the transmembrane conductance regulator (Cftr) gene. This model was compared to new Cftr -/- rats (CFTR KO). Target organs in CF were examined by histological staining of tissue sections and tooth enamel was quantified by micro-computed tomography. The activity of CFTR was evaluated by nasal potential difference (NPD) and short-circuit current measurements. The effect of VX-809 and VX-770 was analyzed on nasal epithelial primary cell cultures from F508del rats. RESULTS: Both newborn F508del and Knock out (KO) animals developed intestinal obstruction that could be partly compensated by special diet combined with an osmotic laxative. The two rat models exhibited CF phenotypic anomalies such as vas deferens agenesis and tooth enamel defects. Histology of the intestine, pancreas, liver, and lungs was normal. Absence of CFTR function in KO rats was confirmed ex vivo by short-circuit current measurements on colon mucosae and in vivo by NPD, whereas residual CFTR activity was observed in F508del rats. Exposure of F508del CFTR nasal primary cultures to a combination of VX-809 and VX-770 improved CFTR-mediated Cl- transport. CONCLUSIONS: The F508del rats reproduce the phenotypes observed in CFTR KO animals and represent a novel resource to advance the development of CF therapeutics.