Peter van Mourik

51 Potentiator synergy in rectal organoids Incubation of VX-770, genistein and curcumin in rectal organoids derived from F508del / F508del CF subjects Direct stimulations by VX-770, genistein and curcumin were also assessed in organoids homozygous for the most common CFTR mutation F508del. The hierarchy of the compound potency (VX-770 > genistein > curcumin) was similar as observed for organoids with a gating mutation (Fig. 2c-e and 3a-c), but potentiator- induced FIS rates were much lower in F508del homozygous organoids, even when a saturating forskolin dose was used (5 μM) (Fig. 4a-c). Synergy between VX-770 and genistein was observed using near-saturating potentiator dosages (0.05 - 0.8 μM forskolin; Fig. 4d) and VX-770 and genistein additively increased FIS using suboptimal dosages (0.32 – 2 μM forskolin; Fig. 4e). Importantly, genistein greatly enhanced FIS of organoids that were treated with VX-770 or VX-770 and VX-809, even at genistein levels < 10 μM (Fig. 4f and Supplementary Fig. S3a,b). In VX- 809-corrected organoids, using saturating (3 and 50 μM; Fig. 4g) and suboptimal (0.1 and 10 μM; Fig. 4h) potentiator concentrations, we detected synergy between VX-770 and genistein at near-saturating dose, but not for any other combination (Fig. 4g,h). Western blot assays were performed to assess the effects of chronic stimulations on protein levels, but the lack of robust detection of a CFTR-F508del C-band prevented proper quantification and interpretation of the results (Fig. S3c). In conclusion, these data indicate that FIS of VX-809-corrected or non-corrected CFTR-F508del homozygous organoids is synergistically repaired by VX-770 and genistein, but not by curcumin. 3