Assays of CFTR Function In Vitro, Ex Vivo and In Vivo

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.

Prime editing functionally corrects cystic fibrosis-causing CFTR mutations in human organoids and airway epithelial cells

“…These organoids are a well-established model in the CF field, shown to robustly predict patient responses to CFTR-targeting therapies. 29 , 36 Prime editing in intestinal organoids has been previously reported by Geurts et al. and Schene et al.…”

“…Rectal organoids have been established as a highly translational model for CFTR function and allow to reliably predict the efficacy of therapeutic strategies. 29 CFTR activation through the addition of forskolin leads to rapid volumetric expansion of these organoids 30 (forskolin-induced swelling [FIS], Figure 3 A), providing a functional platform to study CFTR gene correction. 31 , 32 , 33 , 34 For CFTR modulators, FIS responses correlate with non-gastrointestinal clinical endpoints in pwCF, including lung function.…”

Airway and Lung Organoids from Human-Induced Pluripotent Stem Cells Can Be Used to Assess CFTR Conductance

“…For several years, the FIS assay on intestinal organoids has been actively used to assess the effectiveness of CFTR modulators for rare CFTR mutations [ 15 , 29 , 30 ]. However, according to the Human Protein Atlas ( , (accessed on 4 April 2022)), the levels of CFTR expression (both protein and mRNA) in the lungs and intestines are different.…”

Stargardt disease-associated missense and synonymous ABCA4 variants result in aberrant splicing

“…The specialized function of ABCA4 imposes challenges in determining its activity as it is not behaving like many other transmembrane proteins. While its structure resembles the one of the transmembrane conductance regulator (CFTR) from the ABCC7 group ( 36 ), ABCA4’s function is more complex than CFTR’s straightforward ion flux, which allowed setting up protocols for in vitro functional assays ( 37 ). So far, functional analyses of ABCA4 have been performed only in extracellular environments or cultured cells and in conditions that involve the use of mild detergents, which can, regardless of their mild nature, affect the activity of the protein and give a false image of one variant’s effect.…”