Correction of CFTR function in intestinal organoids to guide treatment of cystic fibrosis

Anabela S. Ramalho, Eva Fürstová, Annelotte M. Vonk, Marc Ferrante, Catherine Verfaillie, Lieven Dupont, Mieke Boon, Marijke Proesmans, Jeffrey M. Beekman, Ifat Sarouk, Carlos Vazquez Cordero, Francois Vermeulen, Kris De Boeck on behalf of the Belgian Organoid Project
European Respiratory Journal 2021 57: 1902426; DOI: 10.1183/13993003.02426-2019

Abstract

Rationale Given the vast number of cystic fibrosis transmembrane conductance regulator (CFTR) mutations, biomarkers predicting benefit from CFTR modulator therapies are needed for subjects with cystic fibrosis (CF).

Objectives To study CFTR function in organoids of subjects with common and rare CFTR mutations and evaluate correlations between CFTR function and clinical data.

Methods Intestinal organoids were grown from rectal biopsies in a cohort of 97 subjects with CF. Residual CFTR function was measured by quantifying organoid swelling induced by forskolin and response to modulators by quantifying organoid swelling induced by CFTR correctors, potentiator and their combination. Organoid data were correlated with clinical data from the literature.

Results Across 28 genotypes, residual CFTR function correlated (r2=0.87) with sweat chloride values. When studying the same genotypes, CFTR function rescue by CFTR modulators in organoids correlated tightly with mean improvement in lung function (r2=0.90) and sweat chloride (r2=0.95) reported in clinical trials. We identified candidate genotypes for modulator therapy, such as E92K, Q237E, R334W and L159S. Based on organoid results, two subjects started modulator treatment: one homozygous for complex allele Q359K_T360K, and the second with mutation E60K. Both subjects had major clinical benefit.

Conclusions Measurements of residual CFTR function and rescue of function by CFTR modulators in intestinal organoids correlate closely with clinical data. Our results for reference genotypes concur with previous results. CFTR function measured in organoids can be used to guide precision medicine in patients with CF, positioning organoids as a potential in vitro model to bring treatment to patients carrying rare CFTR mutations.

Abstract

Rescue of CFTR function with modulators measured in colon organoids can be used to guide precision medicine in patients with cystic fibrosis. Organoids are an effective model to bring treatment to patients with CF carrying rare CFTR mutations. https://bit.ly/2VHHH6s

Introduction

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene coding for the CFTR protein which functions as an anion channel. More than 2000 CFTR mutations have been reported [1]. Only mutation F508del, occurring on 70% of CF alleles, is frequent. In the European CF patient registry [2], only five mutations have a frequency >1%. Many mutations are ultra-rare, occurring in just a few patients. In the CFTR2 project 412 mutations have been characterised so far [3].

Four CFTR modulators targeting the CFTR protein defect have been approved to treat patients: the correctors lumacaftor, tezacaftor and elexacaftor improve intracellular CFTR trafficking; the potentiator ivacaftor increases CFTR function. Corrector-rescued F508del-CFTR has impaired gating and requires a potentiator to optimise ion transport. The combination of one corrector plus potentiator results in modest clinical benefit for patients homozygous for F508del [4], while the combination of tezacaftor, elexacaftor and ivacaftor improved outcome in patients with one [5] or two F508del mutations [6]. Treatment with the potentiator ivacaftor brings major benefit for subjects with a class III mutation [7, 8], and modest benefit to subjects with a selected list of residual function mutations [9]. For a review of this rapidly expanding area we refer to Cuyx and De Boeck [10].

At present, most patients with rare CFTR mutations have no CFTR-directed treatment. Their small number is a hurdle for conclusive clinical trials. For these patients, the development of organoids as a biomarker of CFTR function and its rescue by CFTR modulators is a major breakthrough [11, 12]. Although the culture conditions and techniques to grow organoids are complex, access to subject's rectal tissue via suction biopsies is easy. The procedure is painless and does not require local anaesthesia. Organoids can be expanded over long time periods and biobanked [13]. In the context of CF, CFTR residual function and its rescue by CFTR modulators can be quantified by the forskolin-induced swelling (FIS) assay [11, 14]. Mean improvements seen in the different clinical trials correlate with CFTR rescue assessed via the FIS assay in organoids of subjects with the trialled mutations [14]. In addition, organoid responses in subjects with ultra-rare mutations have predicted clinical benefit [14]. Results so far support that organoids can be used to guide personalised medicine [15].

We established a biobank of intestinal organoids of subjects with CF at the CF research lab of the University of Leuven (Belgium). Our aims were to assess the portability and robustness of the CF organoid model by confirming FIS assay results obtained in subjects with common mutations by others and to expand knowledge on organoid use in patients with rare CFTR mutations.

Materials and methods

Subjects, rectal biopsies and mutations

This study was approved by the ethics review board of the University Hospitals Leuven. All patients/parents gave written informed consent and/or assent. Rectal mucosa tissue was obtained by suction biopsy during a routine clinic visit. The biopsies were stored in ice-cold phosphate buffer and kept on ice until crypt isolation. In this cohort, no major adverse events were reported.

We recruited 97 subjects (supplementary figure S1) with CF [16], with well characterised mutations (F508del, S1251N, R117H and G542X) and with rare CFTR mutations, including mutations not yet characterised in the CFTR2 project [3]. Clinical data retrieved from medical files are summarised in table 1 and supplementary tables S1 and S2.

View this table:
TABLE 1

Baseline characteristics of subjects from whom the rectal organoids are included in the study with lumacaftor and ivacaftor

Isolation of crypts, culturing of organoids and FIS assay

For detailed methods refer to Vonk et al. [17] and the supplementary materials. Crypts were isolated from the rectal biopsies and subsequently mixed with 50% matrigel and plated on 24-well plates; medium was added after the solidification of matrigel, as described previously [11]. The growth medium was changed every other day. The organoids were split by mechanical disruption after every 7 days in culture. Organoids were produced successfully from 87% of the biopsies.

CFTR activity in the organoids was quantified using the FIS assay as described previously [11, 17]. In short, organoids (between 4th and 20th passage) were seeded in 96-well plates (Greiner, Kremsmünster, Austria) in 4-µL matrigel drops containing 15–60 organoids covered with 50 µL of growth medium. The following day, calcein green (Invitrogen, Waltham, MA, USA) was added for staining. Subsequently, to stimulate CFTR, eight dilutions of forskolin from 0.008 to 5 µM were added and the organoids were immediately analysed by confocal live-cell microscopy (LSM800, 5× objective; Zeiss, Oberkochen, Germany). Every 10 min (from 0 to 60 min (t0–t60)), the total organoid area (xy plane) was automatically quantified using Zen blue analysis software (Zeiss), and normalised to the area at t0. To test rescue of CFTR function by correctors, organoids were pre-incubated overnight with 3 µM lumacaftor (VX-809; Selleckchem, Munich, Germany). Ivacaftor at a concentration of 3 µM (VX-770; Selleckchem) was added as a potentiator in combination with forskolin. Within each organoid experiment, every test condition was assessed in duplicate. Per organoid donor, three independent experiments were performed on different days. Values reported correspond to the average area under the curve (AUC) calculated from plots representing the mean percentage of organoids swelling from t0 to t60 (60 min) and standard error of the mean of the three independent experiments (supplementary data and figure S2).

Residual CFTR function was determined from the organoid swelling after addition of forskolin alone. Rescue of CFTR function by CFTR modulator(s) was determined by the increase in AUC after stimulation with forskolin plus modulator(s) subtracting the increase after addition of forskolin alone.

Statistics

SPSS 23.0 (IBM, Armonk, NY, USA) or GraphPad (San Diego, CA, USA) were used for figures and statistical analysis. For correlations, Pearson correlation coefficients were calculated. A general linear model including subject, day of testing and their interaction was used for the variance component analysis of the response to modulators.

Results

The effect of lumacaftor and ivacaftor in organoids from subjects with mutations with known responses to CFTR modulators

FIS assays were performed in organoids from subjects with the following reference genotypes: F508del/S1251N (n=4; S1251N, the most common class III mutation in Belgium); F508del/R117H (n=2; R117H, a class IV mutation); F508del/F508del (n=35; a class II mutation); G542X/G542X (n=1; a class I mutation). In F508del/S1251N and F508del/R117H organoids, maximal FIS was obtained after activation of CFTR by ivacaftor (figure 1a), without additive effect from lumacaftor. F508del/R117H organoids showed a high residual CFTR function, as evidenced by the marked FIS without exposure to modulators. In F508del/F508del organoids, a modest FIS was seen after exposure to the combination of ivacaftor–lumacaftor. G542X/G542X organoids showed no response to CFTR modulators and no residual function. Figure 1b shows representative images of the swelling after 1 h compared to baseline.

FIGURE 1
FIGURE 1

Forskolin-induced swelling (FIS) assay in the organoids from patients with reference genotypes. a) Organoids were incubated overnight with lumacaftor (VX-809 3 µM). Stimulation with forskolin (increasing concentrations from 0.008 to 5 µM) was done after 24 h, either alone or in combination with ivacaftor (VX-770 3 µM) over 1 h. Each condition was tested in duplicate on three different days. Values plotted are the average and sem of the area under the curve (AUC); b) representative images of the organoids at baseline (t0) and after stimulation for 1 h (t60) with forskolin without dimethyl sulfoxide (DMSO) and with modulators. Scale bar=200 µm.

Correlating clinical trial results to organoid results

To isolate the drug effect from the residual CFTR function we subtracted the AUC with forskolin alone from the AUC with forskolin plus modulators. The mean improvements in forced expiratory volume in 1 s (FEV1) and sweat chloride from published clinical trials [4, 8, 9, 18–22] correlated closely with the mean FIS modulator responses seen in organoids from subjects with the same mutations as those in these trials (figure 2b and c). The best correlations were found at a forskolin concentration of 0.8 µM: r2=0.90 between the responses in organoids and the mean changes in FEV1 (figure 2b), and r2=0.95 between the responses in organoids and the mean changes in sweat chloride (figure 2c). Correlations at forskolin concentrations of 0.32 µM were in the same range (supplementary figure S3).

FIGURE 2
FIGURE 2

Correlation between responses to modulators in vitro in the forskolin (Fsk)-induced swelling assay and in vivo from published clinical trials. a) Responses to lumacaftor (VX-809) and/or ivacaftor (VX-770) in organoids with the F508del/F508del, F508del/S1251N, F508del/R117H and F508del/minimal function (MF) were compared to the clinical responses (forced expiratory volume in 1 s (FEV1) and sweat chloride) obtained in published clinical trials with patients having the same genotypes; b) correlation between the absolute change in FEV1 in clinical trials and the organoid responses in our cohort at 0.8 µM forskolin with the same modulators; c) correlation between the absolute change in sweat chloride in clinical trials and the organoid responses in our cohort at 0.8 µM forskolin with the same modulators. AUC: area under the curve. #: reference for FEV1 value; : reference for sweat chloride value.

Between- and within-subject differences in modulator responses for reference genotypes

FIS responses to the combination lumacaftor–ivacaftor in organoids from 35 F508del/F508del subjects ranged from very low, close to the absent response in the G542X homozygous subject, to as high as those seen with ivacaftor in F508del/S1251N organoids (figure 3a). Subtraction of the forskolin effect had the most profound influence on the results from the R117H organoids (figure 3b).

FIGURE 3
FIGURE 3

Between- and within-subject variability of forskolin (Fsk)-induced swelling in organoids of 42 patients with the reference genotypes. a) Organoid swelling (area under the curve (AUC)) with lumacaftor and ivacaftor at 0.8 µM forskolin (without correction for the response to forskolin alone). Data are presented as mean±sem for the three experiments per subject; b) same as a) after subtraction of the swelling (AUC) induced by forskolin alone; c) AUC at 0.8 µM forskolin for the combination of lumacaftor plus ivacaftor (corrected for the AUC at 0.8 µM forskolin without modulators) obtained in each of the three individual experiments on different days (y-axis), according to the mean AUC of the three experiments (x-axis). Error bars represent standard deviation of the two replicates within each experiment.

The within-subject repeatability of the FIS response to modulators is shown in figure 3c: a high responder repeatedly is a high responder; a low responder repeatedly is a low responder, even within the group of F508del homozygous subjects (p<0.001). Variability in the response is mainly between subjects (68%), rather than between tests (24%) or within test (8%).

Residual CFTR function measured in organoids in vitro correlates with sweat chloride measured in vivo

In 74 subjects with 28 different genotypes we found an excellent semi-logarithmic correlation (r2=0.87) between the mean sweat chloride per genotype and the mean residual CFTR function for each genotype assessed in organoids (figure 4a). For five subjects, sweat test results were not available.

FIGURE 4
FIGURE 4

Correlation between residual cystic fibrosis transmembrane conductance regulator (CFTR) function and sweat chloride. a) Correlation between residual CFTR function measured in organoids after stimulation with 0.8 µM forskolin alone and sweat chloride from subjects with the same genotype (mean values per genotype). Sweat chloride was missing for five subjects with five different genotypes; b) i) residual CFTR function (mean and standard error of the area under the curve (AUC) at 0.8 µM forskolin without modulators) in organoids from 33 different genotypes (79 subjects) (table 1). Genotypes are ordered from high to low residual CFTR function; ii) the sweat chloride for the same genotypes (individual value or mean±sd). Ranges of sweat chloride per genotype are reported in table 1. The horizontal line represents the diagnostic cut-off for cystic fibrosis (60 mmol·L−1). Error bars represent sem of the three experiments (n=1) or the sem of the different subjects (n>1). n=1 unless stated otherwise; c) correlation between residual CFTR function measured in organoids after stimulation with 0.8 µM forskolin alone and forced expiratory volume in 1 s (FEV1) % predicted from subjects with the same genotype (mean values per genotype). FEV1 % predicted was missing for 25 subjects with 10 different genotypes.

Figure 4b(i) displays residual CFTR function (FIS responses at 0.8 µM forskolin) in organoids from the 79 subjects with the 33 different genotypes studied (table 1). Figure 4b(ii) represents the sweat chloride per genotype (with median and ranges reported in table 1). Organoids with the highest residual function were from subjects with the lowest sweat chloride, the majority having values below the diagnostic threshold of 60 mmol·L−1 (figure 4b). Correlation was also observed with pancreatic status and age at diagnosis (supplementary table S1). No correlation was found between the mean FEV1 % predicted and the mean residual CFTR function (figure 4c and supplementary table S1).

Organoid swelling induced by CFTR modulators in subjects with rare CFTR mutations and F508del/minimal function mutations

FIS responses to CFTR modulators in organoids from 26 subjects with 23 different rare mutations and from 11 subjects heterozygous for F508del and a known minimal function mutation are shown in figure 5. The responses in the four reference genotypes are displayed for comparison. The organoid swelling results induced by ivacaftor, lumacaftor and ivacaftor–lumacaftor combination (after subtracting the contribution of forskolin) are ranked from highest to lowest responders.

FIGURE 5
FIGURE 5

Response to modulator(s) at 0.8 µM forskolin corrected for the residual function (response to forskolin alone), with the residual function stacked on top of the response to modulator(s). The four reference genotypes are ordered by the (mean) response to lumacaftor plus ivacaftor. The horizontal line corresponds to the mean response to lumacaftor plus ivacaftor in the organoids of the 35 F508del/F508del subjects. Error bars represent the sem of the three experiments (n=1) or the sem of the different subjects (n>1). Characteristics of the subjects are described in table 1. n=1 unless stated otherwise.

There was no correlation between residual function and response to CFTR modulators (p=0.96). The organoid response was higher than the average response to the combination lumacaftor–ivacaftor in F508del/F508del organoids for subjects with the E92K mutation and with the very rare mutations Q237E and Q359K_T360K. Responses similar to those in F508del homozygous organoids were seen for L159S (not explored in CFTR2) and for R334W.

For most genotypes, the swelling induced in organoids was higher with the combination ivacaftor–lumacaftor than with either drug alone. Only for mutations Q237E, D1152H, 3849+10 kb(C>T) and R117C the response to ivacaftor equalled the response to the combination. E92K shows the particularity that CFTR function is mainly rescued by lumacaftor, with only a slight further increase on addition of ivacaftor (figure 5).

Very high residual CFTR function, but low additional responses to modulators were observed for mutation T5, classified as “of varying clinical consequence” in the CFTR2 database.

Residual CFTR function was absent in all organoids with genotypes F508del/minimal function, and at best low responses to lumacaftor plus ivacaftor were found.

Almost no response to modulators was observed in I1234V/W1282X organoids. For two mutations (g.3464_3471dupTCATTGCT_V1198M and K464E), not reported in CFTR1 nor in CFTR2, there was no swelling observed with the CFTR modulators tested.

Informing precision medicine: two examples

A 38-year-old pancreatic-insufficient man with CF, homozygous for complex allele Q359K_T360K, received a donation for 1-month OrkambiTM (combination ivacaftor–lumacaftor) treatment based on the organoid response (supplementary figure S4). 2 weeks after starting treatment his FEV1 rose from 49% to 65%, sweat conductivity decreased from 86 to 33 mmol·L−1 and he gained 1.8 kg.

A high response to modulators was found in organoids from a subject with the E60K/I507del genotype (supplementary figure S4 and table S2), convincing the health authorities to approve treatment with SymkeviTM (combination tezacaftor–ivacaftor). 6 weeks after starting SymkeviTM, sweat chloride improved from 73 to 36 mmol·L−1, FEV1 increased from 32% to 47%, and CFQ-Rresp (a CF-specific quality-of-life score to a maximum of 100 and a minimal clinically important difference of 4) rose from 24 to 78.

Discussion

We confirm and expand on previous findings by other labs in organoids from subjects with CF [11, 12, 14, 23], showing that assessment of CFTR function in intestinal organoids is feasible, portable and repeatable, as these new results were obtained in a cohort of unique subjects, in a different lab by different researchers.

The magnitude of organoid responses seen in the reference genotypes is in line with those reported by Dekkers et al. [14]. In addition, we found a correlation between the mean clinical benefit from modulators in clinical trials and the mean rescue of CFTR function measured in organoids from subjects with the same mutations. However, the correlation with clinical trial data reported here is stronger than reported previously. By only taking into account the modulator response rather than the modulator plus forskolin response, we “correct” for the high residual function seen in some organoids, mainly R117H. The closer correlation with clinical trial data in this paper (r2=0.90 for changes in FEV1) compared to Dekkers et al. (r2=0.76) [14] might also be related to larger numbers in the present study.

The within-subject repeatability of the FIS assay was acceptable, and much smaller than the between-subject variability. Organoid responses vary between subjects even within the same genotype. This mimics findings in the lumacaftor plus ivacaftor clinical trial in patients homozygous for F508del: the waterfall plot of improvement in FEV1 shows large benefits in some subjects and hardly any in others [4]. Whether subjects whose response to modulators in organoids is higher than others with the same genotype can also be expected to have a better clinical response is still to be investigated.

In subjects with 28 different genotypes, we showed an excellent (r2=0.87) nonlinear correlation between sweat chloride and residual CFTR function assessed in organoids. Others also found correlations between in vivo and in vitro biomarkers of CFTR function, including sweat chloride concentration, CFTR mediated chloride transport by intestinal current measurements (ICM) in rectal biopsies and/or FIS on rectal organoids [12, 24, 25].

The logarithmic correlation is in line with McCague et al. [26], who showed a semilogarithmic correlation between sweat chloride of subjects with 226 different genotypes and the CFTR function in Fisher rat thyroid cells (FRT). Although the number of mutations assessed by McCague et al. is much higher, the correlation (r2=0.67) is not as tight as in our dataset (r2=0.87). Indeed, organoids allow assessment of CFTR function in native tissue with the entire genetic background of the subject, compared to heterologous cell lines expressing only a mutant CFTR common to the subject, as is done in FRT cells. Furthermore, in FRT cells, the mutations are in the cDNA context without introns, jeopardising the analysis of mutations that affect splicing and nonsense mRNA-mediated decay [27]. This favours organoids versus heterologous cell line models for the assessment of mutations and modulators on CFTR function. The results obtained for I1234V, E831X and R334W in organoids contradict observations in the FRT cells [28]. Organoid results are more in line with what is known about these mutations (supplementary data). Discrepancies between findings in organoids and FRT cells have been reported before. No benefit of ivacaftor was seen in patients with a G970R mutation [8], behaving as an ivacaftor-responsive gating mutation in FRT cells [27]. Subsequently, no effect of ivacaftor was seen in organoids with the G970R mutation, and the mutation was shown to induce alternative splicing with very limited protein production [29]. This illustrates the superiority of using the patients’ own tissue rather than heterologous expression of mutations in nonhuman cell lines.

The increase in CFTR function by modulators was also captured by means of ICM in native rectal tissue [30, 31], with changes in CFTR-mediated chloride transport correlated to changes in sweat chloride, but not in lung function. Residual CFTR function, measured by means of the FIS assay and of ICM [25], correlates with sweat chloride concentration. A correlation between FEV1 and residual CFTR function on ICM was previously found [25]. The absence of correlation in our cohort could be explained by the larger age heterogeneity of our cohort or by fundamental differences in the physiology of the assay.

In organoids, both the correctors and the potentiators are added in vitro. ICM is performed on fresh biopsies resulting in faster results, the correctors have to be administered to the patients before executing the assay. The FIS assay does not allow comparison to wild-type CFTR function, as pre-swelling of non-CF organoids results in little additional effect of CFTR activators or modulators. Organoid cultures can be stored in biobanks, allowing later retesting when new drugs become available and easy exchanges between labs for remote testing or for research purposes.

We observed high levels of CFTR functional rescue in organoids of several subjects with rare mutations like E92K, Q237E and L159S (the latter is not yet described in the CFTR2 database [3]. E92K is rescued by lumacaftor, concurring with results in heterologous cell systems [32, 33]. FIS in E92K organoids was slightly higher with ivacaftor–lumacaftor than lumacaftor alone, suggesting impaired channel gating of rescued E92K. This common mutation in the Chuvash Russian population [34, 35] would be a good candidate for combination modulator treatment.

We showed direct proof of clinical benefit in two subjects with high CFTR rescue in organoids, one homozygous for Q359K_T360K and the second with genotype E60K/I507del, the rescue being derived from the E60K allele. For the remaining genotypes, responses to modulators were in line with what was already known about the mutations from heterologous cell systems and in silico predictions (supplementary data).

Our study's strengths come from reporting on CFTR function in cultures derived from tissues of individual subjects in a large cohort with different genotypes. We showed excellent correlations between organoid responses to modulators and improvement in clinical trials. In addition, we identified several rare mutations that may be amenable to treatment with the already approved modulators.

One weakness of our study is that we could correlate in vitro response to clinical improvement in only two subjects. The high cost of modulators and lack of approval for rare mutations are still hurdles to the performance of therapeutic trials. However, for other rare mutations, we provide convincing results of rescue in organoids backed up by knowledge of how these mutations disturb the normal CFTR structure or function.

Overall, our results confirm that the study of CFTR function and its rescue in rectal organoids opens a path to personalised therapies. This is especially relevant for patients with rare mutations unlikely to enter clinical trials, given the high number of rare mutations and the low number of subjects per mutation. Using organoids as biomarker to select responders to modulators opens a new horizon for these patients. We identify several new mutations that respond well to modulator therapies: some are ultra-rare (L159S, Q237E), whereas others occur in hundreds of European patients (R334W and E92K).

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Acknowledgements

We thank the patients and parents who participated in this study. We thank Els Aertgeerts (Department of Pediatric Pulmonology, University Hospital Leuven, Leuven, Belgium); Karolien Bruneel (Department of Pediatric Pneumology, UZ Brussel, Brussels, Belgium); Claire Collard (Service de Pédiatrie, CHU Liège, Liège, Belgium); Liliane Collignon (Department of Pulmonology, Erasme University Hospital, Brussels); Monique Delfosse (Service de Pédiatrie, CHU Liège); Anja Delporte (Department of Paediatrics, Universitair Ziekenhuis, Ghent, Belgium); Nathalie Feyaerts (Department of Pediatric Pulmonology, University Hospital Leuven); Cécile Lambremont (Service de Pédiatrie, CHU Liège); Lut Nieuwborg (Department of Pulmonology, Erasme University Hospital); Nathalie Peeters (Department of Pulmonology, University Hospital Antwerp, Antwerp, Belgium); Ann Raman (Department of Paediatrics, Universitair Ziekenhuis); Pim Sansen (Department of Pulmonology, University Hospital Antwerp); Hilde Stevens (Department of Pediatrics, Antwerp University Hospital); Marianne Schulte (Department of Pediatric Pulmonology, University Hospital Leuven); Els Van Ransbeeck (Department of Pediatric Pneumology, UZ Brussel); Christel Van de Brande (Department of Pediatric Pneumology, UZ Brussel); Geert Van den Eynde (Department of Pulmonology, University Hospital Antwerp); Marleen Vanderkerken (Department of Paediatrics, Universitair Ziekenhuis); Inge Van Dijck (Department of Pulmonology, University Hospital Antwerp); Audrey Wagener (Service de Pédiatrie, CHU Liège); Monika Waskiewicz (Department of Pediatrics, Antwerp University Hospital); Bernard Wenderickx (Hôpital Universitaire des Enfants Reine Fabiola, Brussels) for logistic support. We also thank Stefan Joris and Jan Vanleeuwe from the Mucovereniging/Association Muco for their financial support.

Footnotes

  • This article has supplementary material available from erj.ersjournals.com

  • Belgian Organoid Project: Hedwige Boboli (CHR Citadelle, Liège, Belgium), Linda Boulanger (University Hospital Leuven, Belgium), Georges Casimir (HUDERF, Brussels, Belgium), Senne Cuyx (KU Leuven and UZ Leuven, Belgium), Benedicte De Meyere (University Hospital Ghent, Belgium), Elke De Wachter (University Hospital Brussels, Belgium), Danny De Looze (University Hospital Ghent, Belgium), Isabelle Etienne (CHU Erasme, Brussels, Belgium), Laurence Hanssens (HUDERF, Brussels), Christiane Knoop (CHU Erasme, Brussels, Belgium), Monique Lequesne (University Hospital Antwerp, Belgium), Vicky Nowé (vzw Gasthuiszusters Antwerp, Belgium), Stephanie Van Biervliet (University Hospital Ghent, Belgium), Eva Van Braeckel (University Hospital Ghent, Belgium), Kim Van Hoorenbeeck (University Hospital Antwerp, Belgium), Eef Vanderhelst (University Hospital Brussels, Belgium), Stijn Verhulst (University Hospital Antwerp, Belgium), Stefanie Vincken (University Hospital Brussels, Belgium).

  • Author contributions: Conceptualisation: A.S. Ramalho, F. Vermeulen and K. De Boeck; methodology: A.S. Ramalho, E. Fürstová, J.M. Beekman, A.M. Vonk, C. Vazquez Cordero, M. Ferrante, F. Vermeulen, M. Proesmans, M. Boon and L. Dupont; recruiting of subjects and collection of rectal biopsies: M. Boon, M. Proesmans, F. Vermeulen, C. Vazquez Cordero, L. Dupont and I. Sarouk, Belgian Organoid Project participants; culturing the organoids and performing the FIS assay analysis: A.S. Ramalho and E. Fürstová; analysis of the results and figures preparation: A.S. Ramalho and F. Vermeulen; writing the original draft: A.S. Ramalho, F. Vermeulen and K. De Boeck; review and editing: all; supervision: F. Vermeulen and K. De Boeck.

  • Conflict of interest: A.S. Ramalho has nothing to disclose.

  • Conflict of interest: E. Fürstová has nothing to disclose.

  • Conflict of interest: A.M. Vonk has nothing to disclose.

  • Conflict of interest: M. Ferrante reports grants and personal fees from Amgen, grants, personal fees and non-financial support from Biogen, Janssen, Pfizer and Takeda, personal fees and non-financial support from Boehringer Ingelheim, MSD, Falk and Ferring, personal fees from Sandoz, Lamepro, and Mylan, outside the submitted work.

  • Conflict of interest: C. Verfaillie has nothing to disclose.

  • Conflict of interest: L. Dupont has nothing to disclose.

  • Conflict of interest: M. Boon is a member of the European Reference Network for Rare Respiratory Diseases (ERN-LUNG) – Project ID number 739546.

  • Conflict of interest: M. Proesmans has nothing to disclose.

  • Conflict of interest: J.M. Beekman reports personal fees from various industries (Vertex, Proteostasis, Teva, others) for costs related to conference presentations, outside the submitted work; and has a patent WO2013093812A3 with royalties paid by Hubrecht Organoid Technology.

  • Conflict of interest: I. Sarouk has nothing to disclose.

  • Conflict of interest: C. Vazquez Cordero has nothing to disclose.

  • Conflict of interest: F. Vermeulen has nothing to disclose.

  • Conflict of interest: K. De Boeck has provided consultancy for Boehringer, Protalix, Raptor, Novabiotics, Eloxx and Chiesi, has been member of steering committees/advisory boards and been PI in studies for Vertex, has provided consultancy and been PI in studies for Galapagos, and has received speaker fees from Teva, outside the submitted work.

  • Support statement: This study was funded by the Belgian CF patients’ association “Mucovereniging/Association Muco” and the Flemish Research Foundation (FWO – Fonds voor Wetenschappelijk Onderzoek – Vlaanderen – C3 project). Funding information for this article has been deposited with the Crossref Funder Registry.

  • Received December 17, 2019.
  • Accepted June 29, 2020.

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Correction of CFTR function in intestinal organoids to guide treatment of cystic fibrosis
Anabela S. Ramalho, Eva Fürstová, Annelotte M. Vonk, Marc Ferrante, Catherine Verfaillie, Lieven Dupont, Mieke Boon, Marijke Proesmans, Jeffrey M. Beekman, Ifat Sarouk, Carlos Vazquez Cordero, Francois Vermeulen, Kris De Boeck
European Respiratory Journal Jan 2021, 57 (1) 1902426; DOI: 10.1183/13993003.02426-2019
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