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Assays of CFTR Function In Vitro, Ex Vivo and In Vivo
Anabela Santo Ramalho1, Mieke Boon1,2, Marijke Proesmans1,2
1CF Research Lab, Woman and Child Unit, Department of Development and Regeneration, KU Leuven (Catholic University of Leuven), B-3000 Leuven, Belgium.
Abstract:
Cystic fibrosis, a multi-organ genetic disease, is characterized by abnormal function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, a chloride channel at the apical membrane of several epithelia. In recent years, therapeutic strategies have been developed to correct the CFTR defect. To evaluate CFTR function at baseline for diagnosis, or the efficacy of CFTR-restoring therapy, reliable tests are needed to measure CFTR function, in vitro, ex vivo and in vivo. In vitro techniques either directly or indirectly measure ion fluxes; direct measurement of ion fluxes and quenching of fluorescence in cell-based assays, change in transmembrane voltage or current in patch clamp or Ussing chamber, swelling of CFTR-containing organoids by secondary water influx upon CFTR activation. Several cell or tissue types can be used. Ex vivo and in vivo assays similarly evaluate current (intestinal current measurement) and membrane potential differences (nasal potential difference), on tissues from individual patients. In the sweat test, the most frequently used in vivo evaluation of CFTR function, chloride concentration or stimulated sweat rate can be directly measured. Here, we will describe the currently available bio-assays for quantitative evaluation of CFTR function, their indications, advantages and disadvantages, and correlation with clinical outcome measures.
Insights
Accurate measurement of cystic fibrosis transmembrane conductance regulator (CFTR) protein function is crucial for diagnosis and monitoring CFTR-restoring therapies. This review details various in vitro, ex vivo, and in vivo bioassays used to quantify CFTR function, discussing their pros and cons.
Area of Science:
- Biomedical Engineering
- Molecular Biology
- Genetics
Background:
- Cystic fibrosis is a multi-organ genetic disorder caused by defective cystic fibrosis transmembrane conductance regulator (CFTR) protein function.
- CFTR is a chloride channel essential for epithelial cell function.
- Developing effective CFTR-restoring therapies necessitates reliable methods to assess CFTR function.
Purpose of the Study:
- To review and compare available bioassays for quantitative CFTR function evaluation.
- To discuss the indications, advantages, and disadvantages of each assay.
- To correlate assay results with clinical outcomes.
Main Methods:
- In vitro assays: direct/indirect ion flux measurements, fluorescence quenching, patch clamp, Ussing chamber, organoid swelling.
- Ex vivo/in vivo assays: intestinal current measurement, nasal potential difference, sweat tests.
- Evaluation of CFTR function across different biological contexts.
Main Results:
- Various bioassays exist for CFTR function assessment, utilizing different biological systems and measurement techniques.
- Each method offers unique advantages and disadvantages regarding accuracy, invasiveness, and applicability.
- Correlation between bioassay results and clinical outcomes is essential for therapeutic assessment.
Conclusions:
- A range of bioassays are available for quantitative CFTR function assessment.
- Selection of the appropriate assay depends on the specific clinical or research question.
- Continued development and validation of these assays are vital for advancing cystic fibrosis care.
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