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Therapeutic peptides have regained interest as they can address unmet medical needs and can be an excellent complement to pharmaceutic small molecules and other macromolecular therapeutics. Over the past decades, correctors and potentiators of the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride ion channel causing cystic fibrosis (CF) when mutated, were developed to reduce the symptoms of the patients. In this context, we have previously designed a CFTR-stabilizing iCAL36 peptide able to further increase the CFTR amount in epithelial cells, thereby resulting in a higher CFTR activity. In the present study, optimization of the peptidyl inhibitor was performed by coupling five different cell-penetrating peptides (CPP), which are Tat, dTat, TatRI (retro-inverso), MPG, and Penetratin. Screening of the internalization properties of these CPP-iCAL36 peptides under different conditions (with or without serum or endocytosis inhibitors, etc.) was performed to select TatRI as the optimal CPP for iCAL36 delivery. More importantly, using this TatRI-iCAL36 peptide, we were able to reveal for the first time an additive increase in the CFTR amount in the presence of VX-445/VX-809 compared to VX-445/VX-809 treatment alone. This finding is a significant contribution to the development of CFTR-stabilizing peptides in addition to currently used treatments (small-molecule correctors or potentiators) for CF patients. [ABSTRACT FROM AUTHOR]

Cystic fibrosis (CF), a genetic disorder, is characterized by chronic lung disease. Small non‐coding RNAs are key regulators of gene expression and participate in various processes, which are dysregulated in CF; however, they remain poorly studied. Here, we determined the complete microRNAs (miRNAs) expression pattern in three CF ex vivo models. The miRNA profiles of air–liquid interface cultures of airway epithelia (bronchi, nasal cells, and nasal polyps) samples from patients with CF and non‐CF controls were obtained by deep sequencing. Compared with non‐CF controls, several miRNAs were deregulated in CF samples; for instance, miR‐181a‐5p and the miR‐449 family were upregulated. Moreover, mature miRNAs often showed variations (i.e. isomiRs) relative to their reference sequence, such as miR‐101, suggesting that miRNAs consist of heterogeneous repertoires of multiple isoforms with different effects on gene expression. Analysis of miR‐181a‐5p and miR‐101‐3p roles indicated that they regulate the expression of WISP1, a key component of cell proliferation/migration programs. We showed that miR‐101 and miR‐181a‐5p participated in aberrant recapitulation of wound healing programs by controlling WISP1 mRNA and protein level. Our miRNA expression data bring new insights into CF physiopathology and define new potential therapeutic targets in CF. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Background: Lung disease progression is variable among cystic fibrosis (CF) patients and depends on DNA mutations in the CFTR gene, polymorphic variations in disease modifier genes, and environmental exposure. The contribution of genetic factors has been extensively investigated, whereas the mechanism whereby environmental factors modulate the lung disease is unknown. In this project, we hypothesized that (i) reiterative stress alters the epigenome in CF-affected tissues and (ii) DNA methylation variations at disease modifier genes modulate the lung function in CF patients. Results: We profiled DNA methylation at CFTR, the disease-causing gene, and at 13 lung modifier genes in nasal epithelial cells and whole blood samples from 48 CF patients and 24 healthy controls. CF patients homozygous for the p.Phe508del mutation and ≥18-year-old were stratified according to the lung disease severity. DNA methylation was measured by bisulfite and next-generation sequencing. The DNA methylation profile allowed us to correctly classify 75% of the subjects, thus providing a CF-specific molecular signature. Moreover, in CF patients, DNA methylation at specific genes was highly correlated in the same tissue sample. We suggest that gene methylation in CF cells may be co-regulated by disease-specific trans-factors. Three genes were differentially methylated in CF patients compared with controls and/or in groups of pulmonary severity: HMOX1 and GSTM3 in nasal epithelial samples; HMOX1 and EDNRA in blood samples. The association between pulmonary severity and DNA methylation at EDNRA was confirmed in blood samples from an independent set of CF patients. Also, lower DNA methylation levels at GSTM3 were associated with the GSTM3*B allele, a polymorphic 3-bp deletion that has a protective effect in cystic fibrosis. Conclusions: DNA methylation levels are altered in nasal epithelial and blood cell samples from CF patients. Analysis of CFTR and 13 lung disease modifier genes shows DNA methylation changes of small magnitude: some of them are a consequence of the disease; other changes may result in small expression variations that collectively modulate the lung disease severity. [ABSTRACT FROM AUTHOR]

The promoter of the cystic fibrosis transmembrane conductance regulator gene CFTR is tightly controlled by regulators including CCAAT/enhancer binding proteins (C/EBPs). We previously reported that the transcription factors YY1 and USF2 affect CFTR expression. We can now demonstrate that C/EBPβ, a member of the CCAAT family, binds to the CFTR promoter and contributes to its transcriptional activity. Our data reveal that C/EBPβ cooperates with USF2 and acts antagonistically to YY1 in the control of CFTR expression. Interestingly, YY1, a strong repressor, fails to repress the CFTR activation induced by USF2 through DNA binding competition. Collectively, the data strongly suggest a model by which USF2 functionally interacts with YY1 blocking its inhibitory activity, in favour of C/EBPβ transactivation. Further investigation into the interactions between these three proteins revealed that phosphorylation of C/EBPβ influences the DNA occupancy of YY1 and favours the interaction between USF2 and YY1. This phosphorylation process has several implications in the CFTR transcriptional process, thus evoking an additional layer of complexity to the mechanisms influencing CFTR gene regulation. [ABSTRACT FROM AUTHOR]

• Nearly 5% of people with CF worldwide carry a nonsense mutation on both alleles resulting in a premature termination codon (PTC). For those patients, no efficient targeted therapy is available yet. Limited information is available on their clinical status. • Our study shows thatPwCF carrying at least one PTC exhibited a significantly faster rate of yearly respiratory decline until ∼ 30 years. • Mortality of children and adolescents with one or two PTC alleles was significantly higher than that of their F508del homozygous pairs. • Infection with Pseudomonas aeruginosa was more frequent in patients carrying 2 PTC when compared to PTC/F508del or F508del homozygous patients. • These data highlight the urgent need to develop targeted interventions for patients carrying nonsense mutations in the CFTR gene. 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+/+). 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. 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 (FEV 1) 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 FEV 1 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. Nonsense mutations decrease the survival and accelerate the course of respiratory disease in children and adolescents with Cystic Fibrosis. [ABSTRACT FROM AUTHOR]