Unmet challenges in cystic fibrosis treatment with modulators.

INTRODUCTION: 'Highly effective' modulator therapies (HEMTs) have radically changed the Cystic Fibrosis (CF) therapeutic landscape. AREAS COVERED: A comprehensive search strategy was undertaken to assess impact of HEMT in life of pwCF, treatment challenges in specific populations such as very young children, and current knowledge gaps. EXPERT OPINION: HEMTs are prescribed for pwCF with definite genotypes. The heterogeneity of variants complicates treatment possibilities and around 10% of pwCF worldwide remains ineligible. Genotype-specific treatments are prompting theratyping and personalized medicine strategies. Improvement in lung function and quality of life increase survival rates, shifting CF from a pediatric to an adult disease. This implies new studies addressing long-term efficacy, side effects, emergence of adult co-morbidities and possible drug-drug interactions. More sensitive and predictive biomarkers for both efficacy and toxicity are warranted. As HEMTs cross the placenta and are found in breast milk, studies addressing the potential consequences of treatment during pregnancy and breastfeeding are urgently needed. Finally, although the treatment and expected outcomes of CF have improved dramatically in high- and middle-income countries, lack of access in low-income countries to these life-changing medicines highlights inequity of care worldwide.

Prime editing functionally corrects cystic fibrosis-causing CFTR mutations in human organoids and airway epithelial cells.

Prime editing is a recent, CRISPR-derived genome editing technology capable of introducing precise nucleotide substitutions, insertions, and deletions. Here, we present prime editing approaches to correct L227R- and N1303K-CFTR, two mutations that cause cystic fibrosis and are not eligible for current market-approved modulator therapies. We show that, upon DNA correction of the CFTR gene, the complex glycosylation, localization, and, most importantly, function of the CFTR protein are restored in HEK293T and 16HBE cell lines. These findings were subsequently validated in patient-derived rectal organoids and human nasal epithelial cells. Through analysis of predicted and experimentally identified candidate off-target sites in primary stem cells, we confirm previous reports on the high prime editor (PE) specificity and its potential for a curative CF gene editing therapy. To facilitate future screening of genetic strategies in a translational CF model, a machine learning algorithm was developed for dynamic quantification of CFTR function in organoids (DETECTOR: "detection of targeted editing of CFTR in organoids").

Acute respiratory failure due to pulmonary exacerbation in children with cystic fibrosis admitted in a pediatric intensive care unit: outcomes and factors associated with mortality.

BACKGROUND: Children with advanced pulmonary disease due to cystic fibrosis (CF) are at risk of acute respiratory failure due to pulmonary exacerbations leading to their admission to pediatric intensive care units (PICU). The objectives of this study were to determine short and medium-term outcomes of children with CF admitted to PICU for acute respiratory failure due to pulmonary exacerbation and to identify prognosis factors. METHODS: This retrospective monocentric study included patients less than 18 years old admitted to the PICU of a French university hospital between 2000 and 2020. Cox proportional hazard regression methods were used to determine prognosis factors of mortality or lung transplant. RESULTS: Prior to PICU admission, the 29 patients included (median age 13.5 years) had a severe lung disease (median Forced Expiratory Volume in 1 s percentage predicted at 29%). Mortality rates were respectively 17%, 31%, 34%, 41% at discharge and at 3, 12 and 36 months post-discharge. Survival rates free of lung transplant were 34%, 32%, 24% and 17% respectively. Risk factors associated with mortality or lung transplant using the univariate analysis were female sex and higher pCO2 and chloride levels at PICU admission, and following pre admission characteristics: home respiratory and nutritional support, registration on lung transplant list and Stenotrophomonas Maltophilia bronchial colonization. CONCLUSION: Children with CF admitted to PICU for acute respiratory failure secondary to pulmonary exacerbations are at high risk of death, both in the short and medium terms. Lung transplant is their main chance of survival and should be considered early.

Abnormal functional lymphoid tolerance and enhanced myeloid exocytosis are characteristics of resting and stimulated PBMCs in cystic fibrosis patients.

Introduction: Cystic Fibrosis (CF) is the commonest genetically inherited disease (1 in 4,500 newborns) and 70% of people with CF (pwCF) harbour the F508Del mutation, resulting in misfolding and incorrect addressing of the channel CFTR to the epithelial membrane and subsequent dysregulation of fluid homeostasis. Although studies have underscored the importance and over-activation of myeloid cells, and in particular neutrophils in the lungs of people with CF (pwCF), relatively less emphasis has been put on the potential immunological bias in CF blood cells, at homeostasis or following stimulation/infection. Methods: Here, we revisited, in an exhaustive fashion, in pwCF with mild disease (median age of 15, median % FEV1 predicted = 87), whether their PBMCs, unprimed or primed with a 'non specific' stimulus (PMA+ionomycin mix) and a 'specific' one (live P.a =PAO1 strain), were differentially activated, compared to healthy controls (HC) PBMCs. Results: 1) we analysed the lymphocytic and myeloid populations present in CF and Control PBMCs (T cells, NKT, Tgd, ILCs) and their production of the signature cytokines IFN-g, IL-13, IL-17, IL-22. 2) By q-PCR, ELISA and Luminex analysis we showed that CF PBMCs have increased background cytokines and mediators production and a partial functional tolerance phenotype, when restimulated. 3) we showed that CF PBMCs low-density neutrophils release higher levels of granule components (S100A8/A9, lactoferrin, MMP-3, MMP-7, MMP-8, MMP-9, NE), demonstrating enhanced exocytosis of potentially harmful mediators. Discussion: In conclusion, we demonstrated that functional lymphoid tolerance and enhanced myeloid protease activity are key features of cystic fibrosis PBMCs.

[Therapeutics in cystic fibrosis: Clinical revolution and new challenges]. / Traitements de la mucoviscidose - Révolution clinique et nouveaux défis.

Over time, cystic fibrosis has become a model of synergy between research in pathophysiology and cell biology, and clinical advances. Therapies targeting the CFTR protein, in particular CFTR modulators, have transformed the prognosis of patients, bringing the hope of a normal life with the possibility of starting a family and growing old, challenging established statistics. However, patients are not yet cured, and side effects remain insufficiently documented. Epidemiological changes create new challenges for the management of cystic fibrosis. Approximately 10 % of patients still lack a therapeutic option. The community of researchers, pharmaceutical industries, patient associations, and health authorities remains committed to monitor the long-term effects of these still poorly characterised treatments, and to explore new pharmacological approaches, such as gene therapies.

Title: Traitements de la mucoviscidose - Révolution clinique et nouveaux défis. Abstract: Avec le temps, la mucoviscidose est devenue un exemple de synergie entre la recherche en biologie cellulaire et les progrès cliniques. Les thérapies protéiques ont enfin apporté l'espoir d'une vie normale aux patients, bouleversant ainsi les statistiques épidémiologiques établies. Néanmoins, les patients ne guérissent pas, et l'évolution épidémiologique de la maladie ouvre de nouveaux défis pour la prise en charge des malades. Par ailleurs, environ 10 % des patients demeurent sans solution thérapeutique. De nouvelles stratégies sont ainsi envisagées et la communauté des chercheurs, industriels, patients et autorités de santé reste mobilisée pour suivre les effets à long terme de ces nouveaux traitements et explorer de nouvelles approches pharmacologiques.

Lumacaftor/Ivacaftor Population Pharmacokinetics in Pediatric Patients with Cystic Fibrosis: A First Step Toward Personalized Therapy.

BACKGROUND: A major breakthrough in cystic fibrosis (CF) therapy was achievedAQ1 with CFTR modulators. The lumacaftor/ivacaftor combination is indicated for the treatment of CF in pediatric patients above 6 years old. Pharmacokinetic (PK) studies of lumacaftor/ivacaftor in these vulnerable pediatric populations are AQ2crucial to optimize treatment protocols. OBJECTIVES AND METHODS: The objectives of this study were to describe the population PK (PPK) of lumacaftor and ivacaftor in children with CF, and to identify factors associated with interindividual variability. The association between drug exposure and clinical response was also investigated. RESULTS: A total of 75 children were included in this PPK study, with 191 concentrations available for each compound and known metabolites (lumacaftor, ivacaftor, ivacaftor-M1, and ivacaftor-M6). PPK analysis was performed using Monolix software. A large interindividual variability was observed. The main sources of interpatient variability identified were patient bodyweight and hepatic function (aspartate aminotransferase). Forced expiratory volume in the first second (FEV1) was statistically associated with the level of exposure to ivacaftor after 48 weeks of treatment. CONCLUSIONS: This study is the first analysis of lumacaftor/ivacaftor PPK in children with CF. These data suggest that dose adjustment is required after identifying variability factors to optimize efficacy. The use of therapeutic drug monitoring as a basis for dose adjustment in children with CF may be useful.

Cystic fibrosis in Europe: improved lung function and longevity - reasons for cautious optimism, but challenges remain.

BACKGROUND: Prognosis and disease severity in cystic fibrosis (CF) are linked to declining lung function. To characterise lung function by the number of adults in countries with different levels of Gross National Income (GNI), data from the European Cystic Fibrosis Society Patient Registry were utilised. METHODS: Annual data including age, forced expiratory volume in 1 s (FEV1), anthropometry, genotype, respiratory cultures and CF-related diabetes (CFRD) were retrieved between 2011 and 2021. All countries were stratified into GNI per capita to reflect differences within Europe. RESULTS: A consistent improvement in FEV1 % pred and survival was observed among the 47 621 people with CF (pwCF), including subjects with chronic Pseudomonas aeruginosa infection, CFRD and/or undernutrition. Mean values of FEV1 % pred changed from 85% to 94.2% for children and from 63.6% to 74.7% for adults. FEV1 % pred further increased among those carrying the F508del mutation in 2021, when elexacaftor/tezacaftor/ivacaftor was available. The number of adult pwCF increased from 13 312 in 2011 to 21 168 in 2021, showing a 60% increase. PwCF living in European lower income countries did not demonstrate a significant annual increase in FEV1 % pred or in the number of adults. CONCLUSION: This pan-European analysis demonstrates a consistent improvement in FEV1 % pred, number of adult pwCF and survival over the last decade only in European higher and middle income countries. Urgent action is needed in the lower income countries where such improvement was not observed. The notable improvement observed in pwCF carrying the F508del mutation emphasises the need to develop treatments for all CF mutations.

Representation and quantification of module activity from omics data with rROMA.

The efficiency of analyzing high-throughput data in systems biology has been demonstrated in numerous studies, where molecular data, such as transcriptomics and proteomics, offers great opportunities for understanding the complexity of biological processes. One important aspect of data analysis in systems biology is the shift from a reductionist approach that focuses on individual components to a more integrative perspective that considers the system as a whole, where the emphasis shifted from differential expression of individual genes to determining the activity of gene sets. Here, we present the rROMA software package for fast and accurate computation of the activity of gene sets with coordinated expression. The rROMA package incorporates significant improvements in the calculation algorithm, along with the implementation of several functions for statistical analysis and visualizing results. These additions greatly expand the package's capabilities and offer valuable tools for data analysis and interpretation. It is an open-source package available on github at: www.github.com/sysbio-curie/rROMA . Based on publicly available transcriptomic datasets, we applied rROMA to cystic fibrosis, highlighting biological mechanisms potentially involved in the establishment and progression of the disease and the associated genes. Results indicate that rROMA can detect disease-related active signaling pathways using transcriptomic and proteomic data. The results notably identified a significant mechanism relevant to cystic fibrosis, raised awareness of a possible bias related to cell culture, and uncovered an intriguing gene that warrants further investigation.

Airway environment drives the selection of quorum sensing mutants and promote Staphylococcus aureus chronic lifestyle.

Staphylococcus aureus is a predominant cause of chronic lung infections. While the airway environment is rich in highly sialylated mucins, the interaction of S. aureus with sialic acid is poorly characterized. Using S. aureus USA300 as well as clinical isolates, we demonstrate that quorum-sensing dysfunction, a hallmark of S. aureus adaptation, correlates with a greater ability to consume free sialic acid, providing a growth advantage in an air-liquid interface model and in vivo. Furthermore, RNA-seq experiment reveals that free sialic acid triggers transcriptional reprogramming promoting S. aureus chronic lifestyle. To support the clinical relevance of our results, we show the co-occurrence of S. aureus, sialidase-producing microbiota and free sialic acid in the airway of patients with cystic fibrosis. Our findings suggest a dual role for sialic acid in S. aureus airway infection, triggering virulence reprogramming and driving S. aureus adaptive strategies through the selection of quorum-sensing dysfunctional strains.

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.

Standards for the care of people with cystic fibrosis (CF): A timely and accurate diagnosis.

There is considerable activity with respect to diagnosis in the field of cystic fibrosis (CF). This relates primarily to developments in newborn bloodspot screening (NBS), more extensive gene analysis and improved characterisation of CFTR-related disorder (CFTR-RD). This is particularly pertinent with respect to accessibility to variant-specific therapy (VST), a transformational intervention for people with CF with eligible CFTR gene variants. This advance reinforces the need for a timely and accurate diagnosis. In the future, there is potential for trials to assess effectiveness of variant-specific therapy for CFTR-RD. The guidance in this paper reaffirms previous standards, clarifies a number of issues, and integrates emerging evidence. Timely and accurate diagnosis has never been more important for people with CF.

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.

Genomic analysis of Staphylococcus aureus sequential isolates from lungs of patients with cystic fibrosis.

Staphylococcus aureus is the predominant pathogen in children with cystic fibrosis (CF) in France and, around 80% of them harbored S. aureus in their lungs. This study investigated virulence and antimicrobial resistance-associated genes and within-host evolution polymorphisms in 14 S. aureus persistent clones from 14 chronically infected CF children. For each of the 14 patients, we compared genomes of two isogenic sequential isolates separated by 2-9 years. All isolates were methicillin-sensitive and harbored the immune evasion gene cluster, whereas half of them harbored the enterotoxin gene cluster. Most clones were capsule type 8 (8/14) and accessory gene regulator (agr)-specificity group 1 (9/14). We identified convergent mutations in genes involved in carbohydrate metabolism, cell wall metabolism, genetic information processing and adhesion, which are likely to play important role in intracellular invasion and persistence. Further explorations relying notably on proteomics will contribute to improve our understanding of the mechanisms at play in the striking long-term persistence ability of S. aureus.

The French Compassionate Program of elexacaftor-tezacaftor-ivacaftor in people with cystic fibrosis with advanced lung disease and no F508del CFTR variant.

BACKGROUND: The European Medicines Agency has approved the cystic fibrosis transmembrane conductance regulator (CFTR) modulator combination elexacaftor-tezacaftor-ivacaftor (ETI) for people with cystic fibrosis (pwCF) carrying at least one F508del variant. The United States Food and Drug Administration (FDA) also approved ETI for pwCF carrying one of 177 rare variants. METHODS: An observational study was conducted to evaluate the effectiveness of ETI in pwCF with advanced lung disease that were not eligible to ETI in Europe. All patients with no F508del variant and advanced lung disease (defined as having a percent predicted forced expiratory volume (ppFEV1)<40 and/or being under evaluation for lung transplantation) and enrolled in the French Compassionate Program initiated ETI at recommended doses. Effectiveness was evaluated by a centralized adjudication committee at 4-6 weeks in terms of clinical manifestations, sweat chloride concentration and ppFEV1. RESULTS: Among the first 84 pwCF included in the program, ETI was effective in 45 (54%) and 39 (46%) were considered to be non-responders. Among the responders 22/45 (49%) carried a CFTR variant that is not currently approved by FDA for ETI eligibility. Important clinical benefits, including suspending the indication for lung transplantation, a significant decrease in sweat chloride concentration by a median [IQR] -30 [-14;-43]mmol·l-1 (n=42; p<0.0001) and an improvement in ppFEV1 by+10.0 [6.0; 20.5] (n=44, p<0.0001), were observed in those for whom treatment was effective. CONCLUSION: Clinical benefits were observed in a large subset of pwCF with advanced lung disease and CFTR variants not currently approved for ETI.

Proteomic profiling of sweat in patients with cystic fibrosis provides new insights into epidermal homoeostasis.

Background: A high proportion of patients with Cystic Fibrosis (CF) also present the rare skin disease aquagenic palmoplantar keratoderma. A possible link between this condition and absence of a functional CF Transmembrane conductance Regulator protein in the sweat acinus and collecting duct remains unknown. Methods: In-depth characterization of sweat proteome profiles was performed in 25 CF patients compared to 12 healthy controls. A 20 µL sweat sample was collected after pilocarpine iontophoresis and liquid chromatography tandem mass spectrometry (LC-MS/MS) proteomic analysis was performed. Results: Sweat proteome profile of CF patients was significantly different from that of healthy subjects with 57 differentially expressed proteins. Cystic Fibrosis sweat proteome was characterized by an increase in 25 proteins including proteases (Kallikrein 7 and 13, Phospholipase B domain containing 1, Cathepsin A L2 and B, Lysosomal Pro-X carboxypeptidase); proinflammatory proteins (Annexin A2, Chitinase-3-like protein 1); cytochrome c and transglutaminases. Thirty-two proteins were downregulated in CF sweat including proteases (Elastase 2), antioxidative protein FAM129 B; membrane-bound transporter SLC6A14 and regulator protein Sodium-hydrogen antiporter 3 regulator 1. Conclusion: This study is the first to report in-depth characterization of endogenous peptides in CF sweat and could help understand the complex physiology of the sweat gland. The proteome profile highlights the unbalanced proteolytic and proinflammatory activity of sweat in CF. These results also suggest a defect in pathways involved in skin barrier integrity in CF patients. Sweat proteome profile could prove to be a useful tool in the context of personalized medicine in CF.