Personalized medicine for cystic fibrosis: the next generation.

Alveolar macrophages (AMs) play a major role in host defense against microbial infections in the lung. To perform this function, these cells must ingest and destroy pathogens, generally in phagosomes, as well as secrete a number of products that signal other immune cells to respond. Recently, we demonstrated that murine alveolar macrophages employ the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel as a determinant in lysosomal acidification (Di, A., Brown, M. E., Deriy, L. V., Li, C., Szeto, F. L., Chen, Y., Huang, P., Tong, J., Naren, A. P., Bindokas, V., Palfrey, H. C., and Nelson, D. J. (2006) Nat. Cell Biol. 8, 933-944). Lysosomes and phagosomes in murine cftr(-/-) AMs failed to acidify, and the cells were deficient in bacterial killing compared with wild type controls. Cystic fibrosis is caused by mutations in CFTR and is characterized by chronic lung infections. The information about relationships between the CFTR genotype and the disease phenotype is scarce both on the organismal and cellular level. The most common disease-causing mutation, DeltaF508, is found in 70% of patients with cystic fibrosis. The mutant protein fails to fold properly and is targeted for proteosomal degradation. G551D, the second most common mutation, causes loss of function of the protein at the plasma membrane. In this study, we have investigated the impact of CFTR DeltaF508 and G551D on a set of core intracellular functions, including organellar acidification, granule secretion, and microbicidal activity in the AM. Utilizing primary AMs from wild type, cftr(-/-), as well as mutant mice, we show a tight correlation between CFTR genotype and levels of lysosomal acidification, bacterial killing, and agonist-induced secretory responses, all of which would be expected to contribute to a significant impact on microbial clearance in the lung.

Cystic fibrosis (CF) is a genetic disease caused by mutations of the gene encoding a cAMP-activated Cl- channel, the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR modulator therapies consist of small-molecule drugs that rescue mutant CFTR. Regimens of single or combinations of CFTR modulators still rely on endogenous levels of cAMP to regulate CFTR activity. We investigated CFTR activation by the natural mediator prostaglandin E2 (PGE2) and lubiprostone (a Food and Drug Administration-approved drug known to target prostaglandin receptors) and tested the hypothesis that receptor-mediated CFTR activators can be used in combination with currently available CFTR modulators to increase function of mutant CFTR. Primary-cultured airway epithelia were assayed in Ussing chambers. Experimental CFTR activators and established CFTR modulators were applied for 24 h and/or acutely and analyzed for their effect on CFTR activity as measured by changes in short-circuit current (ISC). In non-CF airway epithelia, acute application of lubiprostone and PGE2 activated CFTR to the levels comparable to forskolin (Fsk). Pretreatment (24 h) with antagonists to prostaglandin receptors EP2 and EP4 abolished the ability of lubiprostone to acutely activate CFTR. In F508del homozygous airway epithelia pretreated with the triple combination of elexacaftor, tezacaftor, and ivacaftor (ELEXA/TEZ/IVA; i.e., Trikafta), acute application of lubiprostone was able to maximally activate CFTR. Prolonged (24 h) cotreatment of F508del homozygous epithelia with ELEXA/TEZ/IVA and lubiprostone increased acute CFTR activation by ∼60% compared with the treatment with ELEXA/TEZ/IVA alone. This work establishes the feasibility of targeting prostaglandin receptors to activate CFTR on the airway epithelia and demonstrates that cotreatment with lubiprostone can further restore modulator-rescued CFTR.

Relative Contribution of Genetic and Nongenetic Modifiers to Intestinal Obstruction in Cystic Fibrosis

Gene Therapy for Cystic Fibrosis: Lessons Learned and Paths Forward

The G551D CFTR chloride channel spurs the development of personalized medicine.

Rationale: The advent of precision treatment for cystic fibrosis using small-molecule therapeutics has created a need to estimate potential clinical improvements attributable to increases in cystic fibrosis transmembrane conductance regulator (CFTR) function. Objectives: To derive CFTR function of a variety of CFTR genotypes and correlate with key clinical features (sweat chloride concentration, pancreatic exocrine status, and lung function) to develop benchmarks for assessing response to CFTR modulators. Methods: CFTR function assigned to 226 unique CFTR genotypes was correlated with the clinical data of 54,671 individuals enrolled in the Clinical and Functional Translation of CFTR (CFTR2) project. Cross-sectional FEV1% predicted measurements were plotted by age at which measurement was obtained. Shifts in sweat chloride concentration and lung function reported in CFTR modulator trials were compared with function-phenotype correlations to assess potential efficacy of therapies. Measurements and Main Results: CFTR genotype function exhibited a logarithmic relationship with each clinical feature. Modest increases in CFTR function related to differing genotypes were associated with clinically relevant improvements in cross-sectional FEV1% predicted over a range of ages (6-82 yr). Therapeutic responses to modulators corresponded closely to predictions from the CFTR2-derived relationship between CFTR genotype function and phenotype. Conclusions: Increasing CFTR function in individuals with severe disease will have a proportionally greater effect on outcomes than similar increases in CFTR function in individuals with mild disease and should reverse a substantial fraction of the disease process. This study provides reference standards for clinical outcomes that may be achieved by increasing CFTR function.

Cystic fibrosis (CF) is caused by mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. The combination of the CFTR modulators elexacaftor, tezacaftor, and ivacaftor (ETI) enables the effective rescue of CFTR function in people with the most prevalent F508del mutation. However, the functional restoration of rare CFTR variants remains unclear. Here, we use patient-derived intestinal organoids (PDIOs) to identify rare CFTR variants and potentially individuals with CF that might benefit from ETI. First, steady-state lumen area (SLA) measurements were taken to assess CFTR function and compare it to the level observed in healthy controls. Secondly, the forskolin-induced swelling (FIS) assay was performed to measure CFTR rescue within a lower function range, and to further compare it to ETI-mediated CFTR rescue in CFTR genotypes that have received market approval. ETI responses in 30 PDIOs harboring the F508del mutation served as reference for ETI responses of 22 PDIOs with genotypes that are not currently eligible for CFTR modulator treatment, following European Medicine Agency (EMA) and/or U.S. Food and Drug Administration (FDA) regulations. Our data expand previous datasets showing a correlation between in vitro CFTR rescue in organoids and corresponding in vivo ppFEV1 improvement upon a CFTR modulator treatment in published clinical trials, and suggests that the majority of individuals with rare CFTR variants could benefit from ETI. CFTR restoration was further confirmed on protein levels using Western blot. Our data support that CFTR function measurements in PDIOs with rare CFTR genotypes can help to select potential responders to ETI, and suggest that regulatory authorities need to consider providing access to treatment based on the principle of equality for people with CF who do not have access to treatment.

Regulation of Chemokine Expression by NaCl Occurs Independently of Cystic Fibrosis Transmembrane Conductance Regulator in Macrophages

Designer pharmacotherapy for the treatment of cystic fibrosis: commentary on Zegarra-Moran et al.

Intestinal current measurement (ICM) provides a sensitive bioassay for assessment of cystic fibrosis transmembrane conductance regulator (CFTR) function in rectal biopsies ex vivo and is used as a diagnostic tool for cystic fibrosis (CF). Furthermore, ICM was shown to be sensitive to detect pharmacological rescue of CFTR function by CFTR modulators in people with CF carrying responsive CFTR mutations. Results from clinical trials of CFTR modulators across age groups indicate that CFTR function in the sweat duct may be age-dependent with children reaching higher levels than adults. However, little is known about age dependency of CFTR function in the intestinal epithelium. We investigated CFTR-mediated chloride secretion in rectal biopsies from 258 people without CF and 72 people with pancreatic-insufficient CF from 1 month to 68years of age. Change in transepithelial short-circuit current in response to cyclic adenosine monophosphate (cAMP)-mediated (100μM IBMX, 1µM forskolin, basolateral) and cholinergic (100μM carbachol, basolateral) stimulation was assessed as a readout for CFTR function using perfused micro-Ussing chambers. Furthermore, quantitative real-time PCR of CFTR and morphometric analysis of epithelial cells lining the crypts and surface of the rectal mucosa were performed to assess regulation at the levels of gene expression and epithelial cell densities. We found that CFTR-mediated chloride secretion across rectal tissues, as determined from cAMP-mediated as well as cholinergic chloride-secretory responses was highest during infancy and early childhood and declined with age in people without CF (both P < 0.001). Although, there was no difference in cAMP-mediated currents in people with CF, potassium-secretory responses induced by cholinergic stimulation were also reduced with increasing age. Transcript analyses showed that CFTR mRNA expression was slightly increased with increasing age in people without CF (P < 0.05). Morphometric analyses demonstrated that CFTR expressing colonocytes at the crypt base were decreased with age (P < 0.05). A secondary analysis of the ICM data of our previous studies on the effects of lumacaftor/ivacaftor on CFTR function in F508del -homozygous people with CF aged 12years and older and 2-11year old children showed correlations of the change in cAMP-mediated and cholinergic chloride secretory response with the age of people with CF (P < 0.01 and P < 0.05, respectively). These results demonstrate that CFTR function in the rectal epithelium is reduced with increasing age and indicate that this change is likely due to a decline in the number of secretory colonocytes at the crypt base. These findings suggest that differences in CFTR expressing cells may explain increased functional responses to CFTR modulator therapies in children compared to adult people with CF.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the atypical ATP-binding cassette (ABC) family that functions as a phosphorylation-regulated epithelial anion channel. Cystic fibrosis (CF) is characterised by variants in the CFTR gene that lead to impaired epithelial chloride-ion transport and increased mucus viscosity. Although CFTR modulators such as Trikafta® have transformed the care of many CF patients, individuals harbouring rare CFTR variants still have no effective treatment options. In this study, we used primary air-liquid interface (ALI) airway cultures obtained from 21 CF patients (pwCF) and 21 healthy controls (HC) to evaluate the therapeutic efficacy of CFTR restoration based on chitosan-mediated CFTR mRNA and modulators. While modulators restored CFTR channel function in most cultures derived from CF patients, those with class I or other rare variants showed no improvement. Chitosan-mediated CFTR mRNA delivery successfully restored CFTR function in ALI cultures of patients carrying rare CFTR variants with limited or no observed clinical response to modulator therapy, assessed by electrophysiology using our newly developed Multi Transepithelial Current Clamp (MTECC) Ussing chamber. This was then confirmed by morphological visualisation of CFTR protein expression in modulator-responsive patient samples using immunofluorescence (IF) staining. IF revealed an increase in CFTR signal and the restoration of epithelial barrier integrity following chitosan-mRNA and modulator treatment as a secondary outcome alongside CFTR functional measurements. Notably, MUC5AC expression, a major gel-forming mucin expressed by airway goblet cells and mucus viscosity were elevated in CF cultures, but were markedly reduced following successful intervention, approaching the levels seen in HCs. These findings establish the potential of chitosan-mRNA delivery as a therapeutic approach for CF patients, particularly those who do not respond to modulators. They also provide a practical, comparative evaluation of advanced mRNA-based treatments in patient-derived airway models.

An estimated 25 million people worldwide have one deleterious variant in the cystic fibrosis transmembrane conductance regulator ( CFTR ) gene. Chronic respiratory disease symptoms are at an increased prevalence in cystic fibrosis (CF) heterozygotes. Determine the level of CFTR function in CF heterozygotes compared to individuals without CF-causing variants. Establish whether CFTR function differs between asymptomatic and symptomatic CF heterozygotes. Individuals without respiratory symptoms or CF family history were recruited as controls. Heterozygotes were recruited from families with a CF individual harboring null alleles or c.1521_1523del (F508del) in CFTR . CFTR function was measured by short circuit current in primary human nasal epithelial cells (HNEs) from participants. Cell composition was assessed by single cell RNA sequencing. CFTR function was variable in cells from control and heterozygous individuals. Mean CFTR function in asymptomatic null (8.8±0.5µA/cm 2 (SEM); n=30) and F508del (8.7±1.0µA/cm 2 ; n=22) heterozygotes was similar and significantly lower at 54.6% and 53.9% than controls (16.1±1.1µA/cm 2 ; n=24; p<0.0001). Mean CFTR function in symptomatic heterozygotes (8.4±1.0µA/cm 2 ; n =15) was 52.1% of controls and did not differ from asymptomatic heterozygotes (p=0.7803). Cell identities and proportions were equivalent between control and heterozygous cultures. HNEs from CF heterozygotes showed variable response to CFTR modulators. CFTR function in primary airway cells exhibits substantial interindividual variability and overlaps between controls and CF heterozygotes. CF heterozygotes exhibit approximately 50% of CFTR function in controls, regardless of symptom status. These findings suggest that respiratory symptoms in CF heterozygotes are influenced by factors beyond CFTR dysfunction. Scientific Knowledge on the Subject: A growing body of evidence indicates that cystic fibrosis (CF) heterozygotes are at increased risk for a range of common and chronic respiratory diseases. Given that an estimated 10 million individuals in the United States are CF heterozygotes, this population may represent a substantial and underappreciated burden of CFTR-associated disease. However, CFTR function has not been well characterized in CF heterozygotes, and it remains uncertain whether observed clinical phenotypes reflect reduced CFTR activity. Resolving these issues will be essential for clarifying the pathobiology of common respiratory diseases and for evaluating the potential role of CFTR modulator therapy in symptomatic CF heterozygotes.What This Study Adds to the Field: Analysis of 24 controls and 67 CF heterozygotes revealed substantial interindividual variability in CFTR function, as measured ex vivo in differentiated nasal airway epithelial cells. Mean CFTR function in CF heterozygotes was approximately 50% of that observed in controls. CFTR function did not differ significantly between symptomatic and asymptomatic heterozygotes. These findings suggest that reduced CFTR activity may contribute to symptom susceptibility in CF heterozygotes, but additional factors beyond CFTR dysfunction are likely required for the development of CF-like features. Ethical approval and participant consent statement: All participants provided written consent to the research study under IRB00116966 and/or IRB00235883 and consented to have their anonymized data published. All research was conducted in a fair and ethical manner.

Cystic fibrosis (CF) is a lethal recessive genetic disease caused primarily by the F508del mutation in the CF transmembrane conductance regulator (CFTR). The potentiator VX-770 was the first CFTR modulator approved by the FDA for treatment of CF patients with the gating mutation G551D. Orkambi is a drug containing VX-770 and corrector VX809 and is approved for treatment of CF patients homozygous for F508del, which has folding and gating defects. At least 30% of CF patients are heterozygous for the F508del mutation with the other allele encoding for one of many different rare CFTR mutations. Treatment of heterozygous F508del patients with VX-809 and VX-770 has had limited success, so it is important to identify heterozygous patients that respond to CFTR modulator therapy. R117H is a more prevalent rare mutation found in over 2,000 CF patients. In this study we investigated the effectiveness of VX-809/VX-770 therapy on restoring CFTR function in human bronchial epithelial (HBE) cells from R117H/F508del CF patients. We found that VX-809 stimulated more CFTR activity in R117H/F508del HBEs than in F508del/F508del HBEs. R117H expressed exclusively in immortalized HBEs exhibited a folding defect, was retained in the ER, and degraded prematurely. VX-809 corrected the R117H folding defect and restored channel function. Because R117 is involved in ion conductance, VX-770 acted additively with VX-809 to restore CFTR function in chronically treated R117H/F508del cells. Although treatment of R117H patients with VX-770 has been approved, our studies indicate that Orkambi may be more beneficial for rescue of CFTR function in these patients.

Cystic fibrosis (CF), a common lethal pulmonary disorder in Caucasians, is caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) that disturbs fluid homeostasis and host defense in target organs. The effects of CFTR and delta508-CFTR were assessed in transgenic mice that 1) lack CFTR expression (Cftr-/-); 2) express the human delta508 CFTR (CFTR(delta508)); 3) overexpress the normal human CFTR (CFTR(tg)) in respiratory epithelial cells. Genes were selected from Affymetrix Murine Gene-Chips analysis and subjected to functional classification, k-means clustering, promoter cis-elements/modules searching, literature mining, and pathway exploring. Genomic responses to Cftr-/- were not corrected by expression of CFTR(delta508). Genes regulating host defense, inflammation, fluid and electrolyte transport were similarly altered in Cftr-/- and CFTR(delta508) mice. CFTR(delta508) induced a primary disturbance in expression of genes regulating redox and antioxidant systems. Genomic responses to CFTR(tg) were modest and were not associated with lung pathology. CFTR(tg) and CFTR(delta508) induced genes encoding heat shock proteins and other chaperones but did not activate the endoplasmic reticulum-associated degradation pathway. RNAs encoding proteins that directly interact with CFTR were identified in each of the CFTR mouse models, supporting the hypothesis that CFTR functions within a multiprotein complex whose members interact at the level of protein-protein interactions and gene expression. Promoters of genes influenced by CFTR shared common regulatory elements, suggesting that their co-expression may be mediated by shared regulatory mechanisms. Genes and pathways involved in the response to CFTR may be of interest as modifiers of CF.

Introduction: Cystic fibrosis (CF) transmembrane conductance regulator (CFTR) is expressed in human skeletal muscle cells and CFTR dysfunction may present an important determinant of aerobic exercise capacity in CF. Previous studies on the relationship between CFTR genotype and aerobic exercise capacity are scarce and contradictory. Aims and objectives: This study was designed to explore factors influencing aerobic exercise capacity, expressed as peak oxygen consumption (VO 2peak , primary outcome measure) with a specific focus on CFTR genotype in children and adults with CF. Methods: In an international, multicenter cross-sectional study we collected data on CFTR genotype and cardiopulmonary exercise tests (CPET) in patients with CF age 8 years and older. CFTR mutations were classified into functional classes I-V. Results: 513 patients (45% females) from 10 CF centers in North America and Europe had both valid maximal CPET and complete CFTR genotype data and were included in the analysis. Overall, patients had reduced VO 2peak (mean±SD, 81.5 ± 19.2% predicted), but values were comparable among different CFTR classes. Using multilevel mixed-effects models adjusted for study center and relevant confounders, lung function and body mass index were the main predictors of VO 2peak , independent of CFTR genotype. Conclusions: Lung disease severity and reduced nutritional status rather than CFTR genotype are the major determinants of maximal exercise capacity in CF patients.