In silico, in vitro and ex vivo characterization of cystic fibrosis transmembrane conductance regulator pathogenic variants localized in the fourth intracellular loop and their rescue by modulators.

Background and purpose: Cystic fibrosis (CF) is due to loss-of-function variants of the CF transmembrane conductance regulator (CFTR) channel. The most effective treatment for people with CF carrying the F508del mutation is the triple combination of elexacaftor-tezacaftor-ivacaftor (ETI). ETI can correct the underlying defect(s) in other CFTR mutants. The use of disease-relevant predictive models such as patient-derived human nasal epithelial cells allow to investigate the response to CFTR modulators of specific genotypes, possibly supporting patients' access to treatment.

Experimental approach: Using computational, biochemical and functional methodologies, a detailed analysis of selected variants in the intracellular loop 4 (ICL4) to understand their impact on CFTR structure and function.

Key results: Mutations affecting L1065, R1066 and L1077 compromise structural stability of CFTR. Analyses of single variants expressed heterologously in immortalized bronchial cells showed that, upon ETI, rescued activity for both L1065P and R1066C was close to 50% of the wild-type CFTR activity. Biochemical studies of ICL4 variants expression pattern in CFBE41o-cells, following treatment for 24 h, demonstrate the appearance of the mature, fully glycosylated band, with no changes in the immature band. Finally, our study provides evidence in primary nasal cells from a cohort of people with CF that L1065P and R1066C can be effectively rescued by ETI up to 25%-45% of the activity measured in non-CF epithelia.

Conclusion and implications: Although the observed rescue for L1065P and R1066C was smaller than that of the F508del, it should fall in a range predicted, by various studies, to provide a clinical benefit.