Development and application of intestinal organoids for theratyping in cystic fibrosis

Even though in recent year CFTR modulators targeting primary molecular defect of CFTR mutations have been developed and adopted by the clinic, the main obstacle to treat CF remains that of predicting the drug response of patients due to its genetic complexity and heterogeneity. Characterization of functional response of individual variants to CFTR modulators in cell lines does not always reflect what is observed in vivo. In the present study, we characterized the effect of the most promising triple-combination treatment Elexacaftor/Tezacaftor/Ivacaftor (ELX/TEZ/IVA, corresponding to the Trikafta® formulation) on patient-derived intestinal organoids (OGs), which are recently used as a valuable tool for personalized medicine. More specifically the OGs used were characterized by unique genotypes with: - a nonsense allele, that severely reduce or totally impede the production of functional CFTR, - a rare allele with uncharacterized mutation (I507del, R1066H, F1074L, L383S, L1065P). Our aim was to predict the contribution of each of the rare (non-class I) mutations to the phenotypic expression. Intestinal OGs were obtained from rectal biopsies of CF patients carrying the following genotypes: I507del/2183AA>G, R1162X/R1066H, 2183AA>G/F1074L, 2183AA>G/L383S and L1065P/CFTRdele2. The rescue of mutated CFTR activity was evaluated by electrophysiology measurements in Ussing chamber, measuring separately CFTR mediated Cl- and HCO3- transport on two-dimensional epithelial monolayers from dissociated 3D OGs. CFTR protein expression was evaluated by Western-blotting and CFTR mRNA was evaluated by qPCR. Non-CF OGs were used for comparison. ELX/TEZ/IVA markedly enhanced CFTR-mediated bicarbonate and chloride transport across intestinal epithelium of patients with R1162X/R1066H and L1065P/CFTRdele2 genotypes, which counted to about 39% and 59%, respectively, of the HCO3- current normally observed in non-CF monolayers and to about 39% and 37%, respectively, of the Cl- current observed in non-CF monolayers. Consistent with the rescue of CFTR function in intestinal OGs, ELX/TEZ/IVA therapy improved CFTR C-band protein. Patient-derived OGs with 2183AA>G/F1074L and 2183AA>G/L383S genotypes showed a modest residual function and expression of CFTR, that is in line with their 3D OGs cystic phenotype morphology. Synthesis of fully glycosylated CFTR protein increased in both patients at the same level of WT CFTR protein expression, but surprisingly a minimal CFTR functional restoration was detected in ELX/TEZ/IVA treated OGs only in the latter patient. The patient with I507del/2183AA>G mutations was the most critical. In untreated OGs there was no expression of CFTR protein at plasma membrane in accordance with null chloride and bicarbonate current. There was no increased CFTR expression with ELX/TEZ/IVA and, consequently, no increased CFTR activity was observed. As expected, there was no significant change in CFTR transcript abundance for each genotype before and after ELX/TEZ/IVA treatment. To avoid treatments that could be ineffective or harmful, it is important to determine whether rare CFTR mutations respond to existing CFTR modulators by pre-evaluating them directly on the patient’s derived materials. Intestinal OGs represent a valuable tool to assess the functional consequences of rare CFTR variants and the in vitro efficacy of CFTR modulators. Data obtained from 3D OGs culture indicated this in vitro model as a promising tool for predicting the response to available therapies of CF patients with a special relevance for those carrying rare, uncharacterized genotypes.