Peter van Mourik

42 Chapter 3 We recently developed a functional CFTR assay in human intestinal CF organoids 29,30 that was used to study CFTR gene-editing 31 and CFTR modulator mechanisms of action 32-34 . One of these studies reported the robust repair of CFTR-F508del trafficking by the combination of structure-guided correctors 33 , but data on repair of CFTR gating by potentiator combinations in primary CF cells is lacking. Rapid swelling of organoids induced by forskolin is used to measure the residual and drug-corrected CFTR activity in a subject-specific manner. The robust organoid growth 35-38 and CFTR assay conditions 29,30 allow us to generate large and accurate datasets using drug combinations and forskolin-dose-response curves (Dekkers et al . manuscript submitted). FIS was previously shown to be fully CFTR-dependent, as indicated by CFTR null mutations and pharmacological CFTR inhibitors 29,33 . We here tested the impact of potentiator combinations on mutant CFTR function in freshly excised rectal biopsies and in organoids generated from these tissues. Results indicated synergy between VX-770, genistein and curcumin in enhancing CFTR- dependent epithelial fluid secretion in a mutation-specific manner, and support the combination of potentiators as therapeutic strategy for CF. METHODS Compounds DMSO stock solutions of VX-770 (20mM; Selleck Chemicals LLC, Houston, USA), genistein (50 mM; Sigma; G6649-25MG) and curcumin (50mM; Fluka; 08511-10MG) were prepared and stored at -80 for a maximum of 6 months. Stock solutions were disposed directly after use and curcumin was protected from light during all procedures. Human material Study approval was obtained by the Ethics Committee of the University Medical Centre Utrecht and the Erasmus Medical Centre Rotterdam and informed consent was obtained from all participating subjects. Rectal biopsies were obtained (i) during standard cystic fibrosis care (ii) for diagnostic purposes or (iii) during voluntary participation in studies and used for intestinal current measurements (ICM) and generation of rectal organoids. Intestinal current measurement (ICM) Transepithelial, CFTR-dependent anion secretion in human rectal suction or forceps biopsies (in general 4 per subject, for some subjects 1 - 3) was measured using an amendment 56 of the ICM protocol described in detail previously 57 . In short, the biopsies were collected in phosphate-buffered saline on ice and directly mounted