Background. The most common CFTR mutation, F508del, presents with multiple cellular defects. However, the possible multiple defects caused by many rarer CFTR mutations are not well studied. We investigated four rare CFTR mutations E60K, G85E, E92K and A455E against well-characterized mutations, F508del and G551D, and their responses to corrector VX-809 and/or potentiator VX-770. Methods. Using complementary assays in HEK293T stable cell lines, we determined maturation by Western blotting, trafficking by flow cytometry using extracellular 3HA-tagged CFTR, and function by halide-sensitive YFP quenching. In the forskolin-induced swelling assay in intestinal organoids, we validated the effect of tagged versus endogenous CFTR. Results. Treatment with VX-809 significantly restored maturation, PM localization and function of both E60K and E92K. Mechanistically, VX-809 not only raised the total amount of CFTR, but significantly increased the traffic efficiency, which was not the case for A455E. G85E was refractory to VX-809 and VX-770 treatment. Conclusions. Since no single model or assay allows deciphering all defects at once, we propose a combination of phenotypic assays to collect rapid and early insights into the multiple defects of CFTR variants.

IntroductionCystic fibrosis (CF) is a severe monogenic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Several types of CFTR modulators (correctors/potentiators) have been developed to overcome protein dysfunction associated with these mutations. CFTR modulator therapy is now available for the major CF-causing mutations, however 10% of people with CF remain without causal treatments. By combining investigational and market approved CFTR modulators, we aimed to maximise functional rescue of iva-, luma- and tezacaftor refractory mutants G85E and N1303K.MethodsWe used the well-established forskolin induced swelling (FIS) in primary rectal organoids to assess responses to different CFTR corrector and potentiator types. The FIS analysis was performed with brightfield microscopy, allowing both 1 h and 24 h follow-up. Corrector and potentiator activity of elexacaftor was investigated.ResultsFor G85E, maximal rescue was observed by a combination of elexacaftor and corr4a. For N1303K, the quadruple combination teza-elexa-ivacaftor with apigenin was required to obtain a rescue similar to that of luma-ivacaftor rescued F508del. Elexacaftor rescued G85E and N1303K by different mechanisms, with chronic corrector effects on G85E and acute potentiation of N1303K only in the presence of ivacaftor. Synergy in N1303K rescue for iva-elexacaftor and apigenin suggests at least three potentiator mechanisms for this mutant. 24 h FIS identified ivacaftor as the main CFTR modulator for N1303K and elexacaftor and apigenin as co-potentiators.ConclusionsNovel combinations of CFTR modulators can further improve functional rescue of G85E and N1303K in rectal organoids, although for N1303K more effective CFTR modulators are still needed.

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