Orphan CFTR Mutations: From disease mechanisms to novel therapeutic opportunities

Cystic Fibrosis (CF) is the most common lethal genetic recessive disorder of Northern Europe, affecting between 1/2,500 and 1/6,000 newborns of European ascent. CF is caused by mutations in the CF Transmembrane Conductance Regulator (CFTR) gene, which encodes a cAMP-regulated chloride (Cl-) and bicarbonate (HCO3 -) channel expressed at the apical membrane of epithelial cells, that also regulates other epithelial channels and transporters. Due to its function as an ion channel and as a regulator of other channels, CFTR influences the ion and water content of the airway surface liquid (ASL) and its dysregulation leads to reduced ASL volume and consequently thick and dehydrated airway mucus. This thick mucus impairs mucociliary clearance (MCC) and causes the accumulation of bacteria and other pathogens, leading to persistent infections, inflammation and eventually to airway fibrosis and lung destruction, which is the primary cause of morbidity and mortality among people with CF (pwCF). CFTR is synthesised at the endoplasmic reticulum (ER) where it is co-translationally core-glycosylated, generating an immature form (called band B). The protein is then processed during its trafficking through the Golgi apparatus to produce its fullyglycosylated mature form (band C) that functions as a channel at the cell surface. Although more than 2,100 variants have been reported to date in the CFTR gene, disease liability is only confirmed for about 400. The most common mutation – F508del – is present in ~80% of pw CF worldwide. This mutant causes CFTR misfolding leading to ER retention by the ER quality control (ERQC), premature degradation and failure to reach the apical plasma membrane (PM) of epithelial cells. Identification of small molecules that rescue F508del-CFTR defect resulted in the approval of three corrector drugs currently available for individuals with CF: VX-809 (lumacaftor), VX-661 (tezacaftor) or VX-445 (elexacaftor – combined with VX-661) used in combination with VX-770 (ivacaftor), a gating potentiator. Ultimately, all variants result in abnormal Cl- secretion by epithelial cells and, according to the impact in CFTR, they may result in “classical” or “atypical” forms of CF disease, associated with total absence or residual Cl- transport through CFTR, respectively. CFTR mutations have been grouped into seven classes according to the defect caused. In class I mutations, there is no protein production; class II mutations cause CFTR retention in the ER; class III and IV mutations lead to defects in channel function; class V mutations result in decreased CFTR levels; in class VI mutations, CFTR is poorly stable in the cell membrane; and finally, class VII mutations lead to no CFTR mRNA production, and are therefore very difficult to correct pharmacologically. Elucidation of the molecular and cellular effects of rare mutations on CFTR protein 3Dstructure and function is crucial to predict disease severity and response to CFTR modulators. The use of cellular models is very useful to clarify these mechanisms, particularly because most of the rare mutations are present in heterozygosity. Here we proposed: i) To characterize rare ("orphan") CFTR mutations and to test the efficacy of approved corrective CFTR drugs; ii) To validate results using patient-derived materials (intestinal organoids) – when available; iii) To identify putative therapeutic targets for the correction of the unrescuable class II CFTR mutations. We started by developing cellular models stably expressing CFTR bearing 22 rare variants. Then these variants were characterised in terms of their impact in CFTR expression and function and their response to modulators, particularly to the two correctors (tezacaftor and elexacaftor) – which are part of the approved triple combination Trikafta® (US)/Kaftrio® (EU) - both in cellular models constitutively expressing CFTR mutants and, when available, in patient-derived materials (intestinal organoids). Our results show that 14 of the 22 variants studied present a processing defect, being characterised as class II mutations (i.e., defective traffic), 7 exhibit both immature and mature CFTR forms, suggesting a functional defect (classes III or IV) and 1 complex allele that causes the total abrogation of CFTR function (class VII). Among the 22 variants characterised, 8 were corrected with VX-661 alone, 9 were corrected with the combination VX-661+VX-445 and 5 did not show correction with any of the CFTR modulators tested. Moreover, to further characterise and to find novel therapeutic targets for 4 of the CFTR mutations under study (W57G, R560S, H1079P, Q1100P), we used an siRNA-based assay targeting genes that, when knocked-down, were described to rescue F508del. For N1303K, a similar approach was used, with siRNAs targeting proteins shown to be differentially expressed in a proteomic study performed in our lab. We were able to identify potential drug targets for the rescue of CFTR bearing R560S, H1079P or N13030K mutations. Altogether, the data presented here illustrate how complementary in vitro and ex vivo studies can contribute to understand the defect of rare CFTR mutations and to assess their response to CFTR modulator drugs for possible translation into clinical use. Furthermore, our data shows that the knowledge about CFTR folding is still insufficient to understand the defect caused by many class II mutations