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Background: The forskolin-induced swelling (FIS) assay measures CFTR function on patient-derived intestinal organoids (PDIOs) and may guide treatment selection for individuals with Cystic Fibrosis (CF). The aim of this study is to demonstrate the repeatability and reproducibility of the FIS assay following a detailed Standard Operating Procedure (SOP), thus advancing the validation of the assay for precision medicine (theranostic) applications., Methods: Over a 2-year period, FIS responses to CFTR modulators were measured in four European labs. PDIOs from six subjects with CF carrying different CFTR genotypes were used to assess the repeatability and reproducibility across the dynamic range of the assay., Results: Technical, intra-assay repeatability was high (Lin's concordance correlation coefficient (CCC) 0.95-0.98). Experimental, within-subject repeatability was also high within each lab (CCCs all >0.9). Longer-term repeatability (>1 year) showed more variability (CCCs from 0.67 to 0.95). The reproducibility between labs was also high (CCC ranging from 0.92 to 0.97). Exploratory analysis also found that between-lab percentage of agreement of dichotomized CFTR modulator outcomes for predefined FIS thresholds ranged between 78 and 100 %., Conclusions: The observed repeatability and reproducibility of the FIS assay within and across different labs is high and support the use of FIS as biomarker of CFTR function in the presence or absence of CFTR modulators., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

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.

Individuals with cystic fibrosis (CF) suffer from severe respiratory disease due to a genetic defect in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which impairs airway epithelial ion and fluid secretion. New CFTR modulators that restore mutant CFTR function have been recently approved for a large group of people with CF (pwCF), but ~19% of pwCF cannot benefit from CFTR modulators Restoration of epithelial fluid secretion through non-CFTR pathways might be an effective treatment for all pwCF. Here, we developed a medium-throughput 384-well screening assay using nasal CF airway epithelial organoids, with the aim to repurpose FDA-approved drugs as modulators of non-CFTR-dependent epithelial fluid secretion. From a ~1400 FDA-approved drug library, we identified and validated 12 FDA-approved drugs that induced CFTR-independent fluid secretion. Among the hits were several cAMP-mediating drugs, including β2-adrenergic agonists. The hits displayed no effects on chloride conductance measured in the Ussing chamber, and fluid secretion was not affected by TMEM16A, as demonstrated by knockout (KO) experiments in primary nasal epithelial cells. Altogether, our results demonstrate the use of primary nasal airway cells for medium-scale drug screening, target validation with a highly efficient protocol for generating CRISPR-Cas9 KO cells and identification of compounds which induce fluid secretion in a CFTR- and TMEM16A-indepent manner.

Background: The natural food supplements curcumin and genistein, and the drug ivacaftor were found effective as CFTR potentiators in the organoids of individuals carrying a S1251N gating mutation, possibly in a synergistic fashion. Based on these in vitro findings, we evaluated the clinical efficacy of a treatment with curcumin, genistein and ivacaftor, in different combinations., Methods: In three multi-center trials people with CF carrying the S1251N mutation were treated for 8 weeks with curcumin+genistein, ivacaftor and ivacaftor+genistein. We evaluated change in lung function, sweat chloride concentration, CFQ-r, BMI and fecal elastase to determine the clinical effect. We evaluated the pharmacokinetic properties of the compounds by evaluating the concentration in plasma collected after treatment and the effect of the same plasma on the intestinal organoids., Results: A clear clinical effect of treatment with ivacaftor was observed, evidenced by a significant improvement in clinical parameters. In contrast we observed no clear clinical effect of curcumin and/or genistein, except for a small but significant reduction in sweat chloride and airway resistance. Plasma concentrations of the food supplements were low, as was the response of the organoids to this plasma., Conclusions: We observed a clear clinical effect of treatment with ivacaftor, which is in line with the high responsiveness of the intestinal organoids to this drug. No clear clinical effect was observed of the treatment with curcumin and/or genistein, the low plasma concentration of these compounds emphasizes that pharmacokinetic properties of a compound have to be considered when in vitro experiments are performed., Competing Interests: Declaration of Competing Interest J.M.B. and C.K.v.d.E are inventors on a patent application related to these findings. GHK reports research funding from the Lung Foundation of the Netherlands, GSK, Ubbo Emmius Foundation, Vertex, TEVA the Netherlands, TETRI Foundation, outside the submitted work and participation in advisory boards from GSK and PureIMS, outside the submitted work., (Copyright © 2020. Published by Elsevier B.V.)

Introduction: Variability in disease severity and CFTR modulator responses exists between patients with identical CFTR genotypes. Here, we characterized transcription, translation and function of R117H-CFTR using intestinal organoids and correlated them with in vitro responses to ivacaftor (VX-770)., Methods: Organoids were generated from individuals possessing at least one R117H-CFTR allele. The forskolin-induced swelling (FIS) assay was used to measure CFTR function and response to VX-770 treatment. R117H-CFTR protein and mRNA expression levels were determined in parallel and Pearson's correlation coefficients were assessed., Results: Variability in R117H-CFTR FIS responses was observed and correlated significantly with mRNA and protein expression. Response to VX-770 treatment in organoids correlated with mRNA and protein expression as well., Conclusions: Our results indicate that gene expression, protein expression and CFTR function are strongly correlated in organoids from people with CFTR-R117H-7T/9T, which may suggest that CFTR gene expression may have consequences for CF diagnosis, prognosis and therapeutic benefit., Competing Interests: Declaration of Competing Interest Cornelis K. van der Ent reports grants from GSK, Nutricia, TEVA, Gilead, Vertex, ProQR, Proteostasis, Galapagos NV, and grants from Eloxx outside the submitted work and has a patent 10006904 with royalties paid. Prof. Dr. Beekman has a patent on organoid swelling for personalized diagnosis in CF with royalties paid to Hubrecht Organoid Technology and received financial support to cover travel costs and preparation time of scientific lectures from Vertex Pharmaceuticals. He also received funding from Galápagos NV for CFTR modulator studies., (Copyright © 2020 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

Background: CFTR function measurements in intestinal organoids may help to better characterise individual disease expression in F508del homozygous people. Our objective was to study correlations between CFTR function as measured with forskolin-induced swelling in rectal organoids with clinical parameters in adult patients with homozygous F508del mutations., Methods: Multicentre observational study. Thirty-four adults underwent rectal biopsy, pulmonary function tests (FEV 1 and FVC), chest X-ray and chest CT. Body-mass index (BMI) was assessed at study visit and exacerbation rate was determined during five years prior to study visit. Organoids were cultured and measured after stimulation with 5 µm forskolin for three hours to quantitate CFTR residual function., Findings: FIS was positively correlated with FEV 1 (r = 0.36, 95% CI 0.02-0.62, p = 0.04) and BMI (r = 0.42, 95% CI 0.09-0.66, p = 0.015). FIS was negatively correlated with PRAGMA-CF CT score for% of disease (r = -0.37, 95% CI -0.62- -0.03, p = 0.049). We found no significant correlation between FIS and chest radiography score for CF (r = -0.16, 95% CI -0.48-0.20, p = 0.44). We observed a trend between higher FIS and a lower mean number of exacerbations over the last 5 years of observation, but this was not statistically significant (Poisson regression, p = 0.089)., Interpretation: FIS of intestinal organoids varied between subjects with homozygous F508del and correlated with pulmonary and nutritional parameters. These findings suggest that differences at low CFTR residual function may contribute to clinical heterogeneity in F508del homozygous patients and small changes in CFTR residual function might impact long-term disease expression., (Copyright © 2019. Published by Elsevier B.V.)

Competing Interests: Conflict of interest: P. van Mourik has nothing to disclose. Conflict of interest: J.M. Beekman has a patent on organoid swelling for personalised diagnosis in CF with royalties paid by Hubrecht Organoid Technology. He received financial support to cover travel costs and preparation time of scientific lectures from Vertex. He also received funding from Galapagos for CFTR modulator studies. Conflict of interest: C.K. van der Ent reports grants from Vertex Pharmaceuticals Inc, Gilead Sciences and Galapagos, grants and other funding from ProQR, and other funding from TEVA pharmaceutical industries, outside the submitted work.

Cystic fibrosis is caused by mutations in the CFTR gene, which are subdivided into six classes. Mutants of classes III and IV reach the cell surface but have limited function. Most class-III and class-IV mutants respond well to the recently approved potentiator VX-770, which opens the channel. We here revisited function and folding of some class-IV mutants and discovered that R347P is the only one that leads to major defects in folding. By this criterion and by its functional response to corrector drug VX-809, R347P qualifies also as a class-II mutation. Other class-IV mutants folded like wild-type CFTR and responded similarly to VX-809, demonstrating how function and folding are connected. Studies on both types of defects complement each other in understanding how compounds improve mutant CFTR function. This provides an attractive unbiased approach for characterizing mode of action of novel therapeutic compounds and helps address which drugs are efficacious for each cystic fibrosis disease variant., (© 2019 van Willigen et al.)

Forskolin-induced swelling (FIS) of intestinal organoids from individuals with cystic fibrosis (CF) measures function of the cystic fibrosis transmembrane conductance regulator (CFTR), the protein mutated in CF.We investigated whether FIS corresponds with clinical outcome parameters and biomarkers of CFTR function in 34 infants diagnosed with CF. Relationships with FIS were studied for indicators of pulmonary and gastrointestinal disease.Children with low FIS had higher levels of immunoreactive trypsinogen (p=0.030) and pancreatitis-associated protein (p=0.039), more often had pancreatic insufficiency (p<0.001), had more abnormalities on chest computed tomography (p=0.049), and had lower z-scores for maximal expiratory flow at functional residual capacity (p=0.033) when compared to children with high FIS values. FIS significantly correlated with sweat chloride concentration (SCC) and intestinal current measurement (ICM) (r= -0.82 and r=0.70, respectively; both p<0.001). Individual assessment of SCC, ICM and FIS suggested that FIS can help to classify individual disease severity.Thus, stratification by FIS identified subgroups that differed in pulmonary and gastrointestinal outcome parameters. FIS of intestinal organoids correlated well with established CFTR-dependent biomarkers such as SCC and ICM, and performed adequately at group and individual level in this proof-of-concept study., Competing Interests: Conflict of interest: J.M. Beekman reports a licensed patent, number CA2859614 A1, for a rapid quantitative assay to measure CFTR function in a primary intestinal culture model. Conflict of interest: J.F. Dekkers reports a licensed patent, number CA2859614 A1, for a rapid quantitative assay to measure CFTR function in a primary intestinal culture model. Conflict of interest: R. Vries reports support from Vertex for drug screening and from CZ for development of diagnostics, outside the submitted work. Conflict of interest: H.A.W.M. Tiddens reports fees/grants from Roche for an industry symposium on treatment in CF, from Novartis for lectures and advisory boards, from CFF for Lung Analysis, from Vertex for Lung Analysis and advisory boards, from Gilead for lectures and advisory boards, and from Chiesi for an investigator-initiated trial, all outside the submitted work. He also reports a licensed combined patent from Vectura on specific targeting with DNase, and an issued patent from the PRAGMA-CF scoring system. He heads the Erasmus Medical Center/Sophia Children's Hospital core laboratory Lung Analysis. Conflict of interest: K.M. de Winter-de Groot has nothing to disclose. Conflict of interest: H.M. Janssens has nothing to disclose. Conflict of interest: R.T. van Uum has nothing to disclose. Conflict of interest: G. Berkers has nothing to disclose. Conflict of interest: A. Vonk has nothing to disclose. Conflict of interest: E. Kruisselbrink has nothing to disclose. Conflict of interest: H. Oppelaar has nothing to disclose. Conflict of interest: H. Clevers has nothing to disclose. Conflict of interest: R.H.J. Houwen has nothing to disclose. Conflict of interest: J.C. Escher has nothing to disclose. Conflict of interest: S.G. Elias has nothing to disclose. Conflict of interest: H.R. de Jonge has nothing to disclose. Conflict of interest: Y.B. de Rijke has nothing to disclose. Conflict of interest: C.K. van der Ent has nothing to disclose., (Copyright ©ERS 2018.)

Background: New functional assays using primary human intestinal adult stem cell cultures can be valuable tools to study epithelial defects in human diseases such as cystic fibrosis., Methods: CFTR-mediated ion transport was measured in rectal organoid-derived monolayers grown from subjects with various CFTR mutations and compared to donor-matched intestinal current measurements (ICM) in rectal biopsies and forskolin-induced swelling of rectal organoids., Results: Rectal organoid-derived monolayers were generated within four days. Ion transport measurements of CFTR function using these monolayers correlated with ICM and organoid swelling (r = 0.73 and 0.79 respectively). Culturing the monolayers under differentiation conditions enhanced the detection of mucus-secreting cells and was accompanied by reduced CFTR function., Conclusions: CFTR-dependent intestinal epithelial ion transport properties can be measured in rectal organoid-derived monolayers of subjects and correlate with donor-matched ICM and rectal organoid swelling., (Copyright © 2018. Published by Elsevier B.V.)

Background: Combination treatment with the cystic fibrosis transmembrane conductance regulator (CFTR) modulators tezacaftor (VX-661) and ivacaftor (VX-770) was designed to target the underlying cause of disease in patients with cystic fibrosis., Methods: In this phase 3, randomized, double-blind, multicenter, placebo-controlled, parallel-group trial, we evaluated combination therapy with tezacaftor and ivacaftor in patients 12 years of age or older who had cystic fibrosis and were homozygous for the CFTR Phe508del mutation. Patients were randomly assigned in a 1:1 ratio to receive either 100 mg of tezacaftor once daily and 150 mg of ivacaftor twice daily or matched placebo for 24 weeks. The primary end point was the absolute change in the percentage of the predicted forced expiratory volume in 1 second (FEV 1 ) through week 24 (calculated in percentage points); relative change in the percentage of the predicted FEV 1 through week 24 (calculated as a percentage) was a key secondary end point., Results: Of the 510 patients who underwent randomization, 509 received tezacaftor-ivacaftor or placebo, and 475 completed 24 weeks of the trial regimen. The mean FEV 1 at baseline was 60.0% of the predicted value. The effects on the absolute and relative changes in the percentage of the predicted FEV 1 in favor of tezacaftor-ivacaftor over placebo were 4.0 percentage points and 6.8%, respectively (P<0.001 for both comparisons). The rate of pulmonary exacerbation was 35% lower in the tezacaftor-ivacaftor group than in the placebo group (P=0.005). The incidence of adverse events was similar in the two groups. Most adverse events were of mild severity (in 41.8% of patients overall) or moderate severity (in 40.9% overall), and serious adverse events were less frequent with tezacaftor-ivacaftor (12.4%) than with placebo (18.2%). A total of 2.9% of patients discontinued the assigned regimen owing to adverse events. Fewer patients in the tezacaftor-ivacaftor group than in the placebo group had respiratory adverse events, none of which led to discontinuation., Conclusions: The combination of tezacaftor and ivacaftor was efficacious and safe in patients 12 years of age or older who had cystic fibrosis and were homozygous for the CFTR Phe508del mutation. (Funded by Vertex Pharmaceuticals; EVOLVE ClinicalTrials.gov number, NCT02347657 .).

We hypothesized that people with cystic fibrosis (CF) who express CFTR (cystic fibrosis transmembrane conductance regulator) gene mutations associated with residual function may benefit from G-protein coupled receptor (GPCR)-targeting drugs that can activate and enhance CFTR function.We used intestinal organoids to screen a GPCR-modulating compound library and identified β2-adrenergic receptor agonists as the most potent inducers of CFTR function.β2-Agonist-induced organoid swelling correlated with the CFTR genotype, and could be induced in homozygous CFTR-F508del organoids and highly differentiated primary CF airway epithelial cells after rescue of CFTR trafficking by small molecules. The in vivo response to treatment with an oral or inhaled β2-agonist (salbutamol) in CF patients with residual CFTR function was evaluated in a pilot study. 10 subjects with a R117H or A455E mutation were included and showed changes in the nasal potential difference measurement after treatment with oral salbutamol, including a significant improvement of the baseline potential difference of the nasal mucosa (+6.35 mV, p<0.05), suggesting that this treatment might be effective in vivo Furthermore, plasma that was collected after oral salbutamol treatment induced CFTR activation when administered ex vivo to organoids.This proof-of-concept study suggests that organoids can be used to identify drugs that activate CFTR function in vivo and to select route of administration., (Copyright ©ERS 2016.)

The potentiator VX-770 (ivacaftor/KALYDECO™) targets defective gating of CFTR and has been approved for treatment of cystic fibrosis (CF) subjects carrying G551D, S1251N or one of 8 other mutations. Still, the current potentiator treatment does not normalize CFTR-dependent biomarkers, indicating the need for development of more effective potentiator strategies. We have recently pioneered a functional CFTR assay in primary rectal organoids and used this model to characterize interactions between VX-770, genistein and curcumin, the latter 2 being natural food components with established CFTR potentiation capacities. Results indicated that all possible combinations of VX-770, genistein and curcumin synergistically repaired CFTR-dependent forskolin-induced swelling of organoids with CFTR-S1251N or CFTR-G551D, even under suboptimal CFTR activation and compounds concentrations, conditions that may predominate in vivo. Genistein and curcumin also enhanced forskolin-induced swelling of F508del homozygous organoids that were treated with VX-770 and the prototypical CFTR corrector VX-809. These results indicate that VX-770, genistein and curcumin in double or triple combinations can synergize in restoring CFTR-dependent fluid secretion in primary CF cells and support the use of multiple potentiators for treatment of CF., (Copyright © 2016 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

Small-molecule therapies that restore defects in cystic fibrosis transmembrane conductance regulator (CFTR) gating (potentiators) or trafficking (correctors) are being developed for cystic fibrosis (CF) in a mutation-specific fashion. Options for pharmacological correction of CFTR-p.Phe508del (F508del) are being extensively studied but correction of other trafficking mutants that may also benefit from corrector treatment remains largely unknown.We studied correction of the folding mutants CFTR-p.Phe508del, -p.Ala455Glu (A455E) and -p.Asn1303Lys (N1303K) by VX-809 and 18 other correctors (C1-C18) using a functional CFTR assay in human intestinal CF organoids.Function of both CFTR-p.Phe508del and -p.Ala455Glu was enhanced by a variety of correctors but no residual or corrector-induced activity was associated with CFTR-p.Asn1303Lys. Importantly, VX-809-induced correction was most dominant for CFTR-p.Phe508del, while correction of CFTR-p.Ala455Glu was highest by a subgroup of compounds called bithiazoles (C4, C13, C14 and C17) and C5.These data support the development of mutation-specific correctors for optimal treatment of different CFTR trafficking mutants, and identify C5 and bithiazoles as the most promising compounds for correction of CFTR-p.Ala455Glu., (Copyright ©ERS 2016.)

Identifying subjects with cystic fibrosis (CF) who may benefit from cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs is time-consuming, costly, and especially challenging for individuals with rare uncharacterized CFTR mutations. We studied CFTR function and responses to two drugs-the prototypical CFTR potentiator VX-770 (ivacaftor/KALYDECO) and the CFTR corrector VX-809 (lumacaftor)-in organoid cultures derived from the rectal epithelia of subjects with CF, who expressed a broad range of CFTR mutations. We observed that CFTR residual function and responses to drug therapy depended on both the CFTR mutation and the genetic background of the subjects. In vitro drug responses in rectal organoids positively correlated with published outcome data from clinical trials with VX-809 and VX-770, allowing us to predict from preclinical data the potential for CF patients carrying rare CFTR mutations to respond to drug therapy. We demonstrated proof of principle by selecting two subjects expressing an uncharacterized rare CFTR genotype (G1249R/F508del) who showed clinical responses to treatment with ivacaftor and one subject (F508del/R347P) who showed a limited response to drug therapy both in vitro and in vivo. These data suggest that in vitro measurements of CFTR function in patient-derived rectal organoids may be useful for identifying subjects who would benefit from CFTR-correcting treatment, independent of their CFTR mutation., (Copyright © 2016, American Association for the Advancement of Science.)

Single murine and human intestinal stem cells can be expanded in culture over long time periods as genetically and phenotypically stable epithelial organoids. Increased cAMP levels induce rapid swelling of such organoids by opening the cystic fibrosis transmembrane conductor receptor (CFTR). This response is lost in organoids derived from cystic fibrosis (CF) patients. Here we use the CRISPR/Cas9 genome editing system to correct the CFTR locus by homologous recombination in cultured intestinal stem cells of CF patients. The corrected allele is expressed and fully functional as measured in clonally expanded organoids. This study provides proof of concept for gene correction by homologous recombination in primary adult stem cells derived from patients with a single-gene hereditary defect., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Cystic fibrosis (CF) is caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and is characterized by chronic pulmonary infections. The mechanisms underlying chronic infection and inflammation remain incompletely understood. Mutant CFTR in nonepithelial tissues such as immune cells has been suggested to contribute to infection, inflammation, and the resultant lung disease. However, much controversy still exists regarding the intrinsic role of CFTR in immune cells, especially phagocytes. Therefore, we investigated CFTR expression and function in neutrophils and monocytes isolated from human peripheral blood. CFTR function was assessed by comparing non-CF and CF cells, before and after the chemical inhibition of CFTR. We found CFTR protein expression in monocytes, but this expression was limited or undetectable in neutrophils. Furthermore, the phagocytosis and intracellular killing of Pseudomonas aeruginosa was reduced in CF monocytes, and impaired phagocyte effector mechanisms were phenocopied in non-CF monocytes upon the pharmacological inhibition of CFTR. Reduced phagocytosis in CF monocytes relied on the complement-dependent opsonization of Pseudomonas aeruginosa, and was also observed in the context of latex particles labeled with purified C3b. In mechanistic terms, we observed that CFTR function in monocytes is required for the optimal expression of CD11b. We observed no role for CFTR in neutrophil-mediated phagocytosis. These data support an intrinsic role for CFTR in monocytes, and suggest that CFTR-dependent alterations in complement-mediated interactions between Pseudomonas aeruginosa and monocytes may contribute to enhanced susceptibility to infection in patients with CF.

We recently established conditions allowing for long-term expansion of epithelial organoids from intestine, recapitulating essential features of the in vivo tissue architecture. Here we apply this technology to study primary intestinal organoids of people suffering from cystic fibrosis, a disease caused by mutations in CFTR, encoding cystic fibrosis transmembrane conductance regulator. Forskolin induces rapid swelling of organoids derived from healthy controls or wild-type mice, but this effect is strongly reduced in organoids of subjects with cystic fibrosis or in mice carrying the Cftr F508del mutation and is absent in Cftr-deficient organoids. This pattern is phenocopied by CFTR-specific inhibitors. Forskolin-induced swelling of in vitro-expanded human control and cystic fibrosis organoids corresponds quantitatively with forskolin-induced anion currents in freshly excised ex vivo rectal biopsies. Function of the CFTR F508del mutant protein is restored by incubation at low temperature, as well as by CFTR-restoring compounds. This relatively simple and robust assay will facilitate diagnosis, functional studies, drug development and personalized medicine approaches in cystic fibrosis.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis. CFTR-dependent iodide transport measured by fluorescent quenching of ectopically expressed halide-sensitive yellow fluorescent protein (YFP) is widely being used to study CFTR function by microscopy or plate readers. Since YFP fluorescence in these systems is dependent on YFP expression levels and iodide concentration, differences in sensor expression level between experimental units are normalized at the start of each experiment. To allow accurate measurement of CFTR function by flow cytometry, we reasoned that co-expression of an iodide insensitive fluorescent protein would allow for normalization of sensor expression levels and more accurate quantification of CFTR function. Our data indicated that dsRed and mKate fluorescence are iodide insensitive, and we determined an optimal format for co-expression of these fluorescent proteins with halide-sensitive YFP. We showed using microscopy that ratiometric measurement (YFP/mKate) corrects for differences in sensor expression levels. Ratiometric measurements were essential to accurately measure CFTR function by flow cytometry that we here describe for the first time. Mixing of wild type or mutant CFTR expressing cells indicated that addition of approximately 10% of wild type CFTR expressing cells could be distinguished by ratiometric YFP quenching. Flow cytometric ratiometric YFP quenching also allowed us to study CFTR mutants associated with differential residual function upon ectopic expression. Compared with conventional plate-bound CFTR function assays, the flow cytometric approach described here can be used to study CFTR function in suspension cells. It may be further adapted to study CFTR function in heterologous cell populations using cell surface markers and selection of cells that display high CFTR function by cell sorting., (Copyright © 2013 International Society for Advancement of Cytometry.)

Purpose of Review: Aspergillus fumigatus is frequently isolated from cystic fibrosis (CF) patients and is notorious for its role in the debilitating condition of allergic bronchopulmonary aspergillosis (ABPA). Although CF patients suffer from perpetual microorganism-related lung disease, it is unclear whether A. fumigatus colonization has a role in causing accelerated lung function decline and whether intervention is necessary., Recent Findings: A. fumigatus morbidity appears to be related to cystic fibrosis transmembrane conductance regulator-dependant function of the innate immune system. A. fumigatus-colonized patients have a lower lung capacity, more frequent hospitalizations and more prominent radiological abnormalities than noncolonized patients. Treatment with antifungal agents can be of value but has several drawbacks and a direct effect on lung function is yet to be shown., Summary: A. fumigatus appears to have an important role in CF lung disease, not exclusive to the context of ABPA. However, a causal relationship still needs to be confirmed. Study observations and trends indicate a need to further elucidate the mechanisms of A. fumigatus interactions with the host innate immune system and its role in CF lung morbidity.

Rationale: Unbiased approaches that study aberrant protein expression in primary airway epithelial cells at single cell level may profoundly improve diagnosis and understanding of airway diseases. We here present a flow cytometric procedure to study CFTR expression in human primary nasal epithelial cells from patients with Cystic Fibrosis (CF). Our novel approach may be important in monitoring of therapeutic responses, and better understanding of CF disease at the molecular level., Objectives: Validation of a panel of CFTR-directed monoclonal antibodies for flow cytometry and CFTR expression analysis in nasal epithelial cells from healthy controls and CF patients., Methods: We analyzed CFTR expression in primary nasal epithelial cells at single cell level using flow cytometry. Nasal cells were stained for pan-Cytokeratin, E cadherin, and CD45 (to discriminate epithelial cells and leukocytes) in combination with intracellular staining of CFTR. Healthy individuals and CF patients were compared., Measurements and Main Results: We observed various cellular populations present in nasal brushings that expressed CFTR protein at different levels. Our data indicated that CF patients homozygous for F508del express varying levels of CFTR protein in nasal epithelial cells, although at a lower level than healthy controls., Conclusion: CFTR protein is expressed in CF patients harboring F508del mutations but at lower levels than in healthy controls. Multicolor flow cytometry of nasal cells is a relatively simple procedure to analyze the composition of cellular subpopulations and protein expression at single cell level.

Epithelial ion and fluid transport studies in patient-derived organoids (PDOs) are increasingly being used for preclinical studies, drug development and precision medicine applications. Epithelial fluid transport properties in PDOs can be measured through visual changes in organoid (lumen) size. Such organoid phenotypes have been highly instrumental for the studying of diseases, including cystic fibrosis (CF), which is characterized by genetic mutations of the CF transmembrane conductance regulator (CFTR) ion channel. Here we present OrgaSegment, a MASK-RCNN based deep-learning segmentation model allowing for the segmentation of individual intestinal PDO structures from bright-field images. OrgaSegment recognizes spherical structures in addition to the oddly-shaped organoids that are a hallmark of CF organoids and can be used in organoid swelling assays, including the new drug-induced swelling assay that we show here. OrgaSegment enabled easy quantification of organoid swelling and could discriminate between organoids with different CFTR mutations, as well as measure responses to CFTR modulating drugs. The easy-to-apply label-free segmentation tool can help to study CFTR-based fluid secretion and possibly other epithelial ion transport mechanisms in organoids. A deep learning model—OrgaSegment—is presented for segmentation of individual intestinal patient-derived organoid structures from bright-field images. This enables quantification of organoid swelling and discrimination between organoids with different levels CFTR function and response to therapy. [ABSTRACT FROM AUTHOR]

Cystic fibrosis is the most prevalent inherited disease caused by a defect in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The impaired electrolyte homeostasis caused by the mutated or absent protein leads to symptoms in multiple organ systems. However, the pulmonary manifestation with chronic infections and eventually respiratory failure remains the most important threat. Until one decade ago, only symptomatic treatment was available. However, since 2012, different combinations of CFTR modulators are available for people with cystic fibrosis (pwCF) that carry different mutations. The advent of these drugs has impressively changed life expectancy and quality of life in people with cystic fibrosis and raised new challenges regarding long-term complications and tapering of conventional therapies.Conclusion: In this review, we provide an update on the latest developments around diagnostics, treatment, and prognosis of pwCF. What is Known: • Cystic fibrosis is an incurable and life-shortening disease asking for life-long symptomatic treatment. • Three combination CFTR modulating drugs has gained marked approval over the last 10 years. What is New: • The emerge of new (modulating) therapies contribute to the increasing life expectancy. • A high unmet need to develop new therapies for people with CF who cannot access or benefit from these drugs remains. This review gives an update on the current status. [ABSTRACT FROM AUTHOR]

The article presents a research study and comparison of organoid swelling and In Vivo Biomarkers of cystic fibrosis transmembrane conductance regulator (CFTR) function. Topics include its aim to determine effects of drug, Lumacaftor–Ivacaftor in patients with cystic fibrosis homozygous for the F508del mutation.

Cystic fibrosis (CF) is an autosomal recessive genetic disorder impacting approximately 80,000 people of all races and ethnicities world-wide. CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene which encodes a protein of the same name. Protein dysfunction results in abnormal chloride and bicarbonate transport in mucus membranes, including those in the respiratory, gastrointestinal and reproductive tracts. Abnormal anion transport causes viscous secretions at the site of involvement. The majority of people with CF succumb to respiratory failure following recurrent cycles of infection and inflammation in the airways. Historically, providers treated the signs and symptoms of CF, but since 2012, have been able to impact the basic defect for the subset of people with CF who have mutations that respond to the new class of drugs, CFTR protein modulators. With the improved health and longevity afforded by CFTR modulators, more women are interested in parenthood and are becoming pregnant. Furthermore, this class of drugs likely increases fertility in women with CF. However, the safety of CFTR modulators in pregnancy and lactation is only beginning to be established. We summarize available data on the impact of CFTR modulators on fertility, pregnancy and lactation in women with CF. [ABSTRACT FROM AUTHOR]

Refinement of personalized treatment of cystic fibrosis (CF) with emerging medicines targeting the CF basic defect will likely benefit from biomarkers sensitive to detect improvement of cystic fibrosis transmembrane conductance regulator (CFTR) function in individual patients. Intestinal current measurement (ICM) is a technique that enables quantitative assessment of CFTR chloride channel function in rectal tissues or other intestinal epithelia. ICM was originally developed to study the CF ion transport defect in the intestine and has been established as a sensitive biomarker of CFTR function and diagnostic test for CF. With the emergence of CFTR-directed therapeutics, ICM has become an important tool to estimate the level of rescue of CFTR function achieved by approved CFTR modulators, both at the level of CFTR genotype groups, as well as individual patients with CF. In combination with preclinical patient-derived cell culture models, ICM may aid the development of targeted therapies for patients with rare CFTR mutations. Here, we review the principles of ICM and examine how this CFTR biomarker may be used to support diagnostic testing and enhance personalized medicine for individual patients with common as well as rare CFTR mutations in the new era of medicines targeting the underlying cause of CF. [ABSTRACT FROM AUTHOR]

• Breast development was observed as a side effect of treatment with ivacaftor. • Plasma concentration of ivacaftor and clinical effect are not correlated. • Personalized dosing should be considered in patients treated with ivacaftor. Substantial progress has been made in the treatment of Cystic fibrosis due to introduction of CFTR modulators. However, little is known about the long term side effects of treatment with these drugs. We here present a 7 year old girl with CF who presented with breast development as a rare dose dependent side effect of treatment with ivacaftor and we report data on the correlation between drug plasma concentration and clinical effect, bodyweight, and BSA in 16 patients. Higher plasma concentrations did not correlate with clinical effect, as change in FEV1 and sweat chloride concentration. Patients with low bodyweight or BSA tended to have higher plasma concentrations. This might indicate that the current recommended dose of ivacaftor is at the top of the dose-response curve and that some patients can be treated with lower doses of ivacaftor with similar clinical effect. [ABSTRACT FROM AUTHOR]

Abstract: The variation in cystic fibrosis (CF) lung disease and development of CF related complications correlates poorly with the genotype of the CF transmembrane regulator (CFTR) and with environmental factors. Increasing evidence suggests that phenotypic variation in CF can be attributed to genetic variation in genes other than the CFTR gene, so-called modifier genes. In recent years, multiple candidate modifier genes have been investigated in CF, especially genes that are involved in the control of infection, immunity and inflammation. Some of these genes have been rather conclusively identified as modifiers of the CF phenotype, whereas associations found in other genes have not been confirmed or are conflicting. Identification of genetic variation in modifier genes, obtained by genotype–phenotype studies in well-defined patient populations, may be used as an aid to prognosis and may provide the possibility of new therapeutic interventions. [Copyright &y& Elsevier]