Peter van Mourik

21 R117H function and VX-770 response in organoids INTRODUCTION Cystic fibrosis (CF) patients with identical cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations can display heterogeneity in disease severity, which is particularly evident in patients harbouring the R117H-CFTR (p.Arg117His, c.350G>A) mutation. Disease severity in these individuals ranges from being asymptomatic to severe lung function decline comparable to CF patients with homozygous F508del-CFTR (p.Phe508del, c.1521_1523delCTT) mutations 1,2 . A genetic influence contributing to disease heterogeneity is the length of a poly- thymidine (poly-T) tract (5T, 7T or 9T) present in cis with R117H-CFTR 1,3 , which affects splicing efficiency and mRNA transcript availability 4,5 . A 5T tract is considered to be a contributor to disease severity, whereas 7T has been associated with ‘milder’ CF. In this latter (sub)group widely differing symptoms have been reported 3,6 resulting in problems related to patient counselling and treatment strategies. Currently, the R117H-CFTR mutation is one of 33 mutations for which the CFTR potentiator ivacaftor (VX-770) has been approved 7 . Its efficacy, however, differs between individuals possessing similar mutations 8 , which indicates the need for individual drug response prediction. To gain insight into mechanisms contributing to variability in response to CFTR modulator treatment we characterized (i) transcription, (ii) translation and (iii) function of R117H-CFTR using patient-specific intestinal organoid cultures and correlated these datasets with CFTR function restoration upon VX-770 treatment. MATERIAL AND METHODS Colon biopsies were obtained for diagnostic purposes as part of CF care, or study participation with approval of the Ethics Committees of the University Medical Center Utrecht and the Erasmus Medical Centre Rotterdam. Organoid generation and culturing was performed as described previously 9–11 . Paired samples of seven or eight days old organoid cultures from multiple passages (8-12 wells of a 24-wells cluster) were generated of which (i) 30% was used for RNA isolation, (ii) 30% for protein extraction and (iii) 40% for organoid seeding, the forskolin-induced swelling (FIS) assay and continuation of the organoid cultures. RNA/protein isolation, gene expression studies, Western Blot analysis and the FIS assay are described in the Supplementary Methods. Experiments were repeated at three different culturing time points. 2