A team at University Medical Center Utrecht has developed a rapid and quantitative in vitro intestinal cell-based assay for CFTR function that could help prospectively identify responders to targeted cystic fibrosis therapies. 1 The researchers are now planning to test how well the approach can predict drug responses in the clinic. Cystic fibrosis is caused by inherited mutations that reduce the function of the cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel that

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... ps keep the lung and intestinal epithelium hydrated and prevents the mucus buildup that leads to airway obstruction and infection. The only marketed disease-modifying treatment for CF is Kalydeco ivacaftor, a small molecule CFTR potentiator from Vertex Pharmaceuticals Inc. that increases chloride transport through the channel. The drug was approved last year and is indicated to treat only the 4% of patients with CF carrying the G551D CFTR mutation. Additional compounds from Vertex are in late-stage development to treat patients carrying ΔF508 CFTR, the dominant disease-associated mutation, carried by about two-thirds of patients. These include VX-809 and VX-661, which are related compounds that act directly on mutant CFTR to correct its structure. This year, VX-809 in combination with Kalydeco entered Phase III testing to treat patients carrying the ΔF508 CFTR mutation. VX-661 is in Phase II testing with Kalydeco in the same population. Despite these recent clinical advances, a significant fraction of patients with CF are not eligible for existing treatments because they carry rare mutations in which the efficacy of CFTR-targeted compounds has not been confirmed. Even for those patient populations addressed by existing treatments, multiple academic teams and companies are seeking to develop additional compounds and combination therapies that will further improve CFTR function. 2 The development and testing of CFTRtargeted compounds has been hampered by a lack of robust in vitro assays to quantify their activity. The gold standard for preclinical testing relies on cultured bronchial lung epithelial cells taken from lung explants. 3 However, access to these bronchial cells is limited because it requires sampling of patient tissue obtained after a lung transplant or biopsy, and their replicative potential is limited. Instead of primary cells, many phenotypic screens rely on cell lines engineered to express CFTR mutants. But these cells lines are less physiologically relevant than patients' bronchial cells. Indeed, Jeffrey Beekman, principal investigator of pediatric pulmonology at UMC Utrecht, told SciBX the lack of more accessible primary cell models has hindered the development and testing of CF treatments. "A big issue for drug screening is to have a relevant cell model, and the field has generally agreed that it is important to move to primary cells as quickly as possible, " he said. "False positives are commonly identified in cell line-based screening approaches. Up to 90% of hits in these cells could not be validated in primary cells, and there may be false negatives that are simply missed. " Thus, Beekman teamed up with Hans Clevers, professor of molecular genetics at the Hubrecht Institute and president of the Royal Netherlands Academy of Arts and Sciences, to develop a more accessible primary cell culture model for CFTR function. The starting point for the model was a recent culture technique developed in Clevers's lab that enabled intestinal stem cells to be grown into organoids that recapitulate the phenotype of in vivo intestinal tissue architecture. 4,5 "These primary adult stem cell cultures can be cultured for long times in vitro without genetic modifications and can be biobanked, " said Beekman. To measure CFTR function in these organoids, the team treated them with forskolin, a commonly used pharmacological tool that activates the transporter by raising intracellular cyclic AMP (cAMP) levels. By activating CFTR, forskolin induces fluid transport across the organoid's intestinal epithelial barrier. The results were immediate and striking. "In organoids derived from mice, after 40 minutes some just pop because they have so much fluid and tension in them due to the amount of fluid transport. We were amazed by the quantity and the speed of the response and the complete dependency on CFTR, " Beekman said. Because the effect is dependent on CFTR function, organoids expressing defective CFTR should have less forskolin-induced swelling than those expressing normal CFTR. Compounds or conditions that improve CFTR function should restore the normal swelling response. Indeed, in organoids derived from mouse intestinal cells, treatment with CFTR inhibitors or use of cells lacking Cftr or carrying ΔF508 Cftr reduced the forskolin-induced swelling compared with vehicle or wild-type controls. In ΔF508 Cftr organoids, corrector compounds increased forskolininduced swelling compared with vehicle. Beekman said the results were highly quantitative and provided a large signal-to-noise ratio that allowed the relative effects of different compounds to be measured. "We plate 30-70 organoids per well, in triplicate, at several time points. More than 90% of the organoids in a single well respond quickly by swelling, allowing a uniform and accurate measurement between the three wells. The measurements are quantified by fluorescently labeling the organoids, and their relative increase is measured by confocal microscopy and quantified by image "This quantification method is straightforward and fully compatible with high throughput screens."