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Updated: Sep 26, 2025
Rectal Organoid Morphology Analysis ROMA: A Diagnostic Assay in Cystic Fibrosis
Published on: June 10, 2022
Theratyping of the Rare CFTR Variants E193K and R334W in Rectal Organoid-Derived Epithelial Monolayers
Fabiana Ciciriello1, Marcel J C Bijvelds2, Federico Alghisi1
1Cystic Fibrosis Unit, Department of Pediatric Subspecialties, Bambino Gesù Children's Hospital, IRCCS, 00165 Rome, Italy.
Background:
The effect of presently available CFTR modulator combinations, such as elexacaftor (ELX), tezacaftor (TEZ), and ivacaftor (IVA), on rare CFTR alleles is often unknown. Several assays have been developed, such as forskolin-induced swelling (FIS), to evaluate the rescue of such uncommon CFTR alleles both by established and novel modulators in patient-derived primary cell cultures (organoids). Presently, we assessed the CFTR-mediated electrical current across rectal organoid-derived epithelial monolayers. This technique, which allows separate measurement of CFTR-dependent chloride or bicarbonate transport, was used to assess the effect of ELX/TEZ/IVA on two rare CFTR variants.
Methods:
Intestinal organoid cultures were established from rectal biopsies of CF patients carrying the rare missense mutations E193K or R334W paired with F508del. The effect of the CFTR modulator combination ELX/TEZ/IVA on CFTR-mediated Cl- and HCO3- secretion was assessed in organoid-derived intestinal epithelial monolayers. Non-CF organoids were used for comparison. Clinical biomarkers (sweat chloride, FEV1) were monitored in patients receiving modulator therapy.
Results:
ELX/TEZ/IVA markedly enhanced CFTR-mediated bicarbonate and chloride transport across intestinal epithelium of both patients. Consistent with the rescue of CFTR function in cultured intestinal cells, ELX/TEZ/IVA therapy improved biomarkers of CFTR function in the R334W/F508del patient.
Conclusions:
Current measurements in organoid-derived intestinal monolayers can readily be used to monitor CFTR-dependent epithelial Cl- and HCO3- transport. This technique can be explored to assess the functional consequences of rare CFTR mutations and the efficacy of CFTR modulators. We propose that this functional CFTR assay may guide personalized medicine in patients with CF-like clinical manifestations as well as in those carrying rare CFTR mutations.
Insights
Elexacaftor, tezacaftor, and ivacaftor (ELX/TEZ/IVA) combination therapy effectively restored cystic fibrosis transmembrane conductance regulator (CFTR) function in rare CFTR variants. This study highlights organoid-derived epithelial monolayers as a promising tool for personalized medicine in cystic fibrosis.
Area of Science:
- Biochemistry
- Genetics
- Medical Research
Background:
- The efficacy of current cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapies, like elexacaftor/tezacaftor/ivacaftor (ELX/TEZ/IVA), on rare CFTR mutations remains largely unknown.
- Forskolin-induced swelling (FIS) and other assays are used to evaluate CFTR modulator effectiveness in patient-derived organoids.
Purpose of the Study:
- To assess the impact of the ELX/TEZ/IVA modulator combination on CFTR-mediated electrical current in rectal organoid-derived epithelial monolayers.
- To evaluate the separate transport of chloride (Cl-) and bicarbonate (HCO3-) ions, enabling a detailed functional analysis of rare CFTR variants.
Main Methods:
- Organoid cultures were established from rectal biopsies of cystic fibrosis (CF) patients with rare mutations (E193K or R334W) paired with F508del.
- CFTR-mediated Cl- and HCO3- secretion was measured in organoid-derived intestinal epithelial monolayers using the ELX/TEZ/IVA combination.
- Clinical biomarkers, including sweat chloride levels and FEV1, were monitored in patients undergoing modulator therapy.
Main Results:
- The ELX/TEZ/IVA combination significantly enhanced both bicarbonate and chloride transport across the intestinal epithelium in patients with rare CFTR mutations.
- Functional rescue of CFTR activity in cultured intestinal cells correlated with improvements in clinical biomarkers for the R334W/F508del patient.
Conclusions:
- Electrical current measurements in organoid-derived intestinal monolayers provide a reliable method for monitoring CFTR-dependent epithelial transport.
- This functional assay can be utilized to determine the consequences of rare CFTR mutations and assess the efficacy of novel CFTR modulators.
- The proposed functional CFTR assay holds potential for guiding personalized medicine approaches for patients with CF-like symptoms or rare CFTR mutations.
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