Deletion of phenylalanine at position 508 (F508del) in the CFTR chloride channel is the most frequent mutation in cystic fibrosis (CF) patients. F508del impairs the stability and folding of the CFTR protein, thus resulting in mistrafficking and premature degradation. F508del-CFTR defects can be overcome with small molecules termed correctors. We investigated the efficacy and properties of VX-445, a newly developed corrector, which is one of the three active principles present in a drug (Trikafta^®/Kaftrio^®) recently approved for the treatment of CF patients with F508del mutation. We found that VX-445, particularly in combination with type I (VX-809, VX-661) and type II (corr-4a) correctors, elicits a large rescue of F508del-CFTR function. In particular, in primary bronchial epithelial cells of CF patients, the maximal rescue obtained with corrector combinations including VX-445 was close to 60–70% of CFTR function in non-CF cells. Despite this high efficacy, analysis of ubiquitylation, resistance to thermoaggregation, protein half-life, and subcellular localization revealed that corrector combinations did not fully normalize F508del-CFTR behavior. Our study indicates that it is still possible to further improve mutant CFTR rescue with the development of corrector combinations having maximal effects on mutant CFTR structural and functional properties.

Loss-of-function mutations of the CFTR gene cause cystic fibrosis (CF) through a variety of molecular mechanisms involving altered expression, trafficking, and/or activity of the CFTR chloride channel. The most frequent mutation among CF patients, F508del, causes multiple defects that can be, however, overcome by a combination of three pharmacological agents that improve CFTR channel trafficking and gating, namely, elexacaftor, tezacaftor, and ivacaftor. This study was prompted by the evidence of two CF patients, compound heterozygous for F508del and a minimal function variant, who failed to obtain any beneficial effects following treatment with the triple drug combination. Functional studies on nasal epithelia generated in vitro from these patients confirmed the lack of response to pharmacological treatment. Molecular characterization highlighted the presence of an additional amino acid substitution, L467F, in cis with the F508del variant, demonstrating that both patients were carriers of a complex allele. Functional and biochemical assays in heterologous expression systems demonstrated that the double mutant L467F-F508del has a severely reduced activity, with negligible rescue by CFTR modulators. While further studies are needed to investigate the actual prevalence of the L467F-F508del allele, our results suggest that this complex allele should be taken into consideration as plausible cause in CF patients not responding to CFTR modulators.

S737F is a Cystic Fibrosis (CF) transmembrane conductance regulator (CFTR) missense variant. The aim of our study was to describe the clinical features of a cohort of individuals carrying this variant. In parallel, by exploiting ex vivo functional and molecular analyses on nasal epithelia derived from a subset of S737F carriers, we evaluated its functional impact on CFTR protein as well as its responsiveness to CFTR modulators. We retrospectively collected clinical data of all individuals bearing at least one S737F CFTR variant and followed at the CF Centre of Tuscany region (Italy). Nasal brushing was performed in cooperating individuals. At study end clinical data were available for 10 subjects (mean age: 14 years; range 1–44 years; 3 adult individuals). Five asymptomatic subjects had CF, 2 were CRMS/CFSPID and 3 had an inconclusive diagnosis. Ex vivo analysis on nasal epithelia demonstrated different levels of CF activity. In particular, epithelia derived from asymptomatic CF subjects and from one of the subjects with inconclusive diagnosis showed reduced CFTR activity that could be rescued by treatment with CFTR modulators. On the contrary, in the epithelia derived from the other two individuals with an inconclusive diagnosis, the CFTR-mediated current was similar to that observed in epithelia derived from healthy donors. In vitro functional and biochemical analysis on S737F-CFTR expressed in immortalized bronchial cells highlighted a modest impairment of the channel activity, that was improved by treatment with ivacaftor alone or in combination with tezacaftor/elexacaftor. Our study provide evidence towards the evaluation of CFTR function on ex vivo nasal epithelial cell models as a new assay to help clinicians to classify individuals, in presence of discordance between clinical picture, sweat test and genetic profile.

We recently demonstrated that 48 h exposure of primary human bronchial epithelial (hBE) cells, obtained from both CF (F508del homozygous) and non-CF subjects, to the triple drug combination Elexacaftor/Tezacaftor/Ivacaftor (ETI) results in a CFTR genotype-independent modulation of the de novo synthethic pathway of sphingolipids, with an accumulation of dihydroceramides (dHCer). Since dHCer are converted into ceramides (Cer) by the action of a delta-4 sphingolipid desaturase (DEGS) enzyme, we aimed to better understand this off-target effect of ETI (i.e., not related to CFTR rescue) METHODS: hBE cells, both F508del and wild-type, were cultured to create fully differentiated bronchial epithelia. We analyzed Cer and dHCer using an LC-MS based method previously developed by our lab. DEGS expression levels in differentiated hBE cells lysates were quantified by western blot analysis. We demonstrated that 1) dHCer accumulate in hBE with time following prolonged ETI exposure, that 2) similar inhibition occurs in wild-type primary human hepatocytes and that 3) this does not result in an alteration of DEGS expression. We then proved that 4) ETI is a direct inhibitor of DEGS, that 5) Tezacaftor is the molecule responsible for this effect, that 6) the inhibition is concentration dependent. Finally, after repeated oral administration of ETI to naïve, non-CF, mice, we observed a slight accumulation of dHCer in the brain. We believe that further investigations on Tezacaftor should be envisaged, particularly for the use of ETI during pregnancy, breastfeeding and in the early stages of development. DEGS dysfunction and dHCer accumulation causes impairment in the development of the nervous system, due to a derangement in myelin formation and maintenance.