Peter van Mourik

56 Chapter 3 combinations triggered modest reduction in the protein levels of these gating mutants (Fig. 2j,k and 3i,j). Whether a similar reduction in CFTR protein expression occurs upon chronic treatment of F508del organoids is difficult to evaluate, considering the very low intensity of the F508del-CFTR band C in VX-809-pretreated organoids even in the absence of any potentiator (Supplementary Fig. S3c). Moreover, functional studies of CFTR after 48 h of chronic potentiator treatment by the FIS assay are difficult to perform as the chronic presence of a potentiator induces organoid swelling in the absence of forskolin, which by itself suggest increased functional activity (Dekkers et al . manuscript submitted). Because it is impossible to exactly mimic chronic potentiator treatment in vitro in terms of temporal concentrations reaching CFTR in vivo , the clinical implications of small potentiator-dependent reductions in CFTR-S1251N and -G551D C-band levels observed in vitro are hard to predict. However, clinical studies with F508del homozygotes indicated that combination treatment with VX-809 and VX-770 15,16 is more effective than VX-809 treatment alone 52 , suggesting that positive effects on CFTR gating by potentiator combinations likely outweigh possible CFTR destabilizing effects. Previous studies 29 (Dekkers et al. manuscript submitted) already indicated that maximal FIS rates (~3000 AUC units) can be reached at higher forskolin levels, most likely because the basolateral chloride import, and not the apical CFTR function, becomes rate limiting. Therefore, optimal detection of synergy between the potentiators in organoids carrying the S1251N or G551D mutations required a titration of forskolin (Fig. 2 and 3). In general, the residual and potentiator-induced FIS levels of F508del homozygous organoids (Fig. 4) were greatly reduced compared to organoids generated from compound heterozygotes carrying both F508del and a gating mutation (Fig. 2 and 3), indicating that FIS responses in these organoids mainly reflect the activity of CFTR-S1251N (Fig. 2) or -G551D rather than -F508del (Fig. 3). Performing the FIS assay on rectal organoids generated from F508del homozygous patients allowed several important conclusions (Fig. 4): (i) even at saturating forskolin concentrations (5 μM) the response to the potentiators genistein and VX-770 could be increased further by the corrector VX-809, suggesting that maximal potentiation is rate limited by low CFTR-F508del protein levels; (ii) the ranking order of the compound potency (VX-770 > genistein > curcumin) was similar as observed for organoids with a gating mutation (Fig. 2c-e and 3a-c); and (iii) genistein greatly enhanced FIS of F508del organoids that were treated with VX-770 (Fig. 4g),. As these responses are observed upon direct stimulation of the compounds, they rather