Peter van Mourik

53 Potentiator synergy in rectal organoids showed for the first time that (i) the effectiveness of the only clinically available potentiator VX-770 can be greatly enhanced by genistein and curcumin, (ii) combining 3 potentiators enhances CFTR-dependent epithelial fluid secretion to a greater extent compared to the combination of 2 potentiators, and (iii) potentiator combinations exert synergistic effects in a CFTR mutation-specific fashion. These data support the development of potentiator combination therapy in clinical practice, especially for people expressing gating mutations. In line with previous studies 42 , we observed that potentiator activity is detected by ICM in rectal biopsies expressing a CFTR gating mutation (Fig. 1). However, while we did observe a chloride secretory response to forskolin in F508del homozygous biopsies of some CF patients, a further stimulation by CFTR potentiators appeared marginal or absent (Fig. 1a,b). In contrast, the organoid assay (FIS) allowed clear detection of both residual function and potentiator activity, in comparison to ICM. Since ICM was able to detect limited residual function in F508del homozygous biopsies, it appears that especially potentiator delivery is difficult in the fresh rectal biopsies. Organoid measurements furthermore allow generation of large subject- specific datasets with forskolin and potentiator dose-ranges and inter-experimental variance, while ICM is in general restricted to maximal 4 biopsies. Compared to stable drug responses of organoids that can be generated for the individual (Dekkers et al . Manuscript submitted), we have experienced greater variability between different ICM measurements (Fig. 1c) as well as between different biopsies and the limited amount of biopsies severely hinders the inclusion of e.g. control stimulation (data not shown). On the other hand, ICM is one of the few techniques capable of measuring CFTR activity in native epithelium ex vivo , is completely free of potential cell culture artifacts, and can be used as an ex vivo biomarker to study drug effectiveness in vivo 43,44 . Clearly, both methods are complementary and need to be validated further as diagnostic, prognostic, and therapeutic biomarkers. Opening of the CFTR gate is regulated by phosphorylation of the regulatory(R)- domain and ATP-dependent dimerization of the nucleotide binding domain(NBD)1 and NBD2. Intriguingly, both VX-770 and curcumin activate CFTR channels in the absence of ATP, suggesting that they bind directly to the channel pore and bypass the conventional ATP-dependent gating mechanism 21,22,24,25 . In contrast, genistein is known to promote ATP-dependent gating of CFTR, probably by binding to the NBD2 and/or the NBD1-NBD2 interface and inhibiting ATP hydrolysis 20,26,27 . As expected from their different mode of potentiation and previous findings 27,28 , combinations of genistein with VX-770 or with curcumin synergistically repaired FIS of organoids carrying the “pure” gating mutations S1251N and G551D (Fig. 2 and 3). Remarkably, 3