WS14.02In silico, in vitro and ex vivo characterization of CFTR pathogenic variants localized in the Fourth Intracellular Loop and their rescue by modulators

The most effective treatment for people with CF (pwCF) carrying the F508del mutation is the triple combination of Elexacaftor-Tezacaftor-Ivacaftor (ETI). Although initially developed for F508del, CFTR modulators can correct the underlying defect(s) in other CFTR mutants. Previous studies have investigated the impact of several missense variants within the Intracellular Loop 4 (ICL4) on CFTR protein maturation and channel activity. We performed a detailed analysis of selected variants in the ICL4 using computational, functional and biochemical methodologies, to understand their impact on CFTR structure and function.

Mutations affecting L1065P, R1066C, and L1077P result in disruptions of hydrophobic interactions, salt bridges, and helical integrity, respectively, leading to compromised 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 band C, corresponding to mature, fully glycosylated protein, with no changes in the immature band. Cell surface measurements of CFTR performed using the HiBiT complementation assay in transiently transfected HEK293 cells confirmed partial restoration of CFTR surface expression, in the same range as observed with F508del. Finally, our study provides evidence in primary nasal cells from a cohort of pwCF that L1065P and R1066C, can be effectively rescued by ETI. Upon treatment with modulators, the CFTR-mediated current averaged around 25%-45% of the activity measured in non-CF epithelia.

Supported by Fondazione Ricerca Fibrosi Cistica grants FFC #9/2019 and FFC #10/2021, Italian Ministry of Health grants GR-2018–12367126 and PNRR-MR1-2023-12378412 and Cystic Fibrosis Foundation grant PEDEMO20G0.