Rescue of multiple class II CFTR mutations by elexacaftor+tezacaftor+ivacaftor mediated in part by the dual activities of elexacaftor as both corrector and potentiator

Abstract

Positive results in pre-clinical studies of the triple combination of elexacaftor, tezacaftor and ivacaftor, performed in airway epithelial cell cultures obtained from patients harbouring the class II cystic fibrosis transmembrane conductance regulator (CFTR) mutation F508del-CFTR, translated to impressive clinical outcomes for subjects carrying this mutation in clinical trials and approval of Trikafta.Encouraged by this correlation, we were prompted to evaluate the effect of the elexacaftor, tezacaftor and ivacaftor triple combination on primary nasal epithelial cultures obtained from individuals with rare class II CF-causing mutations (G85E, M1101K and N1303K) for which Trikafta is not approved.Cultures from individuals homozygous for M1101K responded better than cultures harbouring G85E and N1303K after treatment with the triple combination with respect to improvement in regulated channel function and protein processing. A similar genotype-specific effect of the triple combination was observed when the different mutations were expressed in HEK293 cells, supporting the hypothesis that these modulators may act directly on the mutant proteins. Detailed studies in nasal cultures and HEK293 cells showed that the corrector, elexacaftor, exhibited dual activity as both corrector and potentiator, and suggested that the potentiator activity contributes to its pharmacological activity.These pre-clinical studies using nasal epithelial cultures identified mutation genotypes for which elexacaftor, tezacaftor and ivacaftor may produce clinical responses that are comparable to, or inferior to, those observed for F508del-CFTR.

FIGURE 1

F508del cystic fibrosis transmembrane conductance regulator (CFTR) mutation is rescued by the triple combination VX-445+VX-661+VX-770 in nasal epithelial cells from three patients homozygous for F508del. DMSO: dimethyl sulfoxide; amil: amiloride; FSK: forskolin; I_eq: transepithelial current; WT: wild-type; CNX: calnexin. a) Representative tracings show Ussing chamber measurements of CFTR function in nasal epithelial cell cultures from a CF patient bearing F508del/F508del in the absence or presence of the small-molecule corrector. The upper line reflects the transepithelial potential difference measurements and the downward deflection reflects the transepithelial resistance. b) Maximal response I_eq after stimulation with FSK (10 µM)±VX-770 (1 µM) of one or two technical replicate experiments of nasal cultures generated from three patients. Different pre-treatments were performed (48 h at 37°C): DMSO (0.1%), VX-809 (3 µM)+VX-770 (1 µM), R-VX-445 (3 µM)+VX-661 (3 µM)+VX-770 (1 µM), S-VX-445 (3 µM)+VX-661 (3 µM)+VX-770 (1 µM), VX-809 (3 µM) or R-VX-445 (3 µM)+VX-661 (3 µM). The data points show single Ussing chamber experiments. Comparative analysis showed statistically significant differences between nasal epithelial cells treated with VX-809+VX-770 and VX-445+VX-661 as well as those treated with acute and chronic VX-770. c) I_eq by the CFTR inhibitor CFTRinh-172 (10 µM) from the same experiments done in b). d) Immunoblots of steady-state expression of WT or F508del following treatments with CFTR modulators. Band C: mature, complex-glycosylated CFTR; band B: immature, core-glycosylated CFTR. Dash-lined boxes provide an example how the area for quantification of the protein abundance was chosen. Markers in kDa. e) Ratio band C/(band C+band B) of measured nasal epithelial cells from three patients. Comparative analysis showed statistically significant differences between nasal epithelial cells treated with DMSO and VX-445+VX-661±VX-770. Data are presented as mean±sd. One-way ANOVA followed by Turkey's post hoc test was used for statistical analysis (*: p<0.05; **: p<0.01; ****: p<0.0001). The paired t-test showed statistically differences between nasal epithelial cells treated with DMSO and VX-809+VX-770 (^#: p<0.05; ^##: p<0.01). ns: nonsignificant.

FIGURE 2

Nasal epithelial cultures derived from patients homozygous for other class II cystic fibrosis transmembrane conductance regulator (CFTR) mutations exhibit low CFTR function and variable levels of rescue by VX-445+VX-661+VX-770. DMSO: dimethyl sulfoxide; amil: amiloride; FSK: forskolin; CNX: calnexin; I_eq: transepithelial current; WT: wild-type. a–f) Representative tracings show Ussing chamber measurements of CFTR function in nasal epithelial cell cultures from CF patients bearing a) M1101K/M1101K, c) G85E/G85E and e) N1303K/N1303K in the absence or presence of the small-molecule corrector. Immunoblots of steady-state expression of WT and b) M1101K, d) G85E and f) N1303K following treatments with CFTR modulators. Markers in kDa. Band C: mature, complex-glycosylated CFTR; band B: immature, core-glycosylated CFTR. g) Maximal response I_eq after stimulation by FSK (10 µM)±VX-770 (1 µM) for nasal epithelial cell cultures from one to three technical replicates of three patients bearing M1101K or G85E and two donors bearing N1303K after pre-treatment (48 h at 37°C) with DMSO (0.1%), VX-809 (3 µM)+VX-770 (1 µM), R-VX-445 (3 µM)+VX-661 (3 µM)+VX-770 (1 µM) or S-VX-445 (3 µM)+VX-661 (3 µM)+VX-770 (1 µM). h) I_eq by the CFTR inhibitor CFTRinh-172 (10 µM) for nasal epithelial cell cultures from one to three technical replicates of three patients bearing M1101K or G85E and two donors bearing N1303K. i) Comparison of I_eqCFTRinh-172 across the class II mutations M1101K, G85E and N1303K for nasal epithelial cell cultures from one to three technical replicates of three patients bearing M1101K or G85E and two donors bearing N1303K. Dashed line in g–i) represents the mean results of rescued (r) F508del/F508del nasal cells treated with VX-445+VX-661+VX-770. j) Ratio band C/(band C+band B) from one technical replicate of three patients bearing M1101K or G85E and two patients bearing N1303K. Data are presented as mean±sd. Analysis was performed using one-way ANOVA followed by Turkey's post hoc test (*: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.000). The paired t-test showed statistically differences between nasal epithelial cells treated with DMSO and VX-809+VX-770 (^#: p<0.05; ^##: p<0.01). ns: nonsignificant.

FIGURE 3

VX-445 increased channel activation of wild-type (WT) cystic fibrosis transmembrane conductance regulator (CFTR) in nasal and bronchial epithelial cells. Amil: amiloride; DMSO: dimethyl sulfoxide; FSK: forskolin; I_eq: transepithelial current. a) Representative tracings show Ussing chamber measurements of CFTR function in nasal epithelial cell cultures from a non-CF donor (WT-CFTR). b, c) Fold increase in FSK (10 µM)+S-VX-445 (3 µM)-activated ΔI_eq compared with FSK (10 µM) control in b) one to two technical replicates of nasal epithelial cells generated from four healthy controls and c) bronchial epithelial cells from four donors (n=4). d, e) Fold increase in I_eq by the CFTR inhibitor CFTRinh-172 (10 µM) in d) nasal epithelial cultures and e) bronchial epithelial cell cultures. f) Representative tracings show Ussing chamber measurements of CFTR function in nasal epithelial cell cultures from a CF patient homozygous for the F508del mutation following treatment with 48 h of VX-661 (3 µM) (F508del-CFTR). CFTRinh-172 (10 µM) was given repeatedly to ensure complete inhibition of CFTR. g) Fold increase in FSK (10 µM)+S-VX-445 (3 µM)-activated ΔI_eq compared with FSK (10 µM) control in two technical replicates of nasal epithelial cells generated from two CF donors homozygous for F508del. h) Corresponding fold increase in I_eq by CFTRinh-172 (10 µM). Data are presented as mean±sd. Comparative analysis was performed using the paired two-tailed t-test (*: p<0.05; ***: p<0.001). Statistically significant differences are shown between untreated cells and those with acute treatment of S-VX-445.

FIGURE 4

VX-445 potentiates temperature-rescued F508del, M1101K and N1303K cystic fibrosis transmembrane conductance regulator (CFTR) mutations in HEK293 cells. RFU: relative fluorescence units; DMSO: dimethyl sulfoxide; FSK: forskolin; EC₅₀: half-maximal effective concentration (µM). a) Representative traces of F508del-CFTR-dependent chloride efflux (membrane depolarisation assay, 3 µM S-VX-445+10 µM FSK) in HEK293 cells stably transfected with F508del-CFTR after 24 h incubation at 27°C. b–d) Dose–response curves of VX-770 or S-VX-445 (0.001–3 µM)+FSK (10 µM) in b) F508del-, c) M1101K- or d) N1303K-CFTR HEK293 cells (n=5 biological replicates and four technical replicates for each experiment). The peak changes in fluorescence to CFTR agonists were normalised relative to the baseline fluorescence (ΔF/F₀). Data are presented as mean±sd. Analysis was performed using one-way ANOVA followed by Turkey's post hoc test (*: p<0.05; **: p<0.01; ***: p<0.001).

FIGURE 5

F508del cystic fibrosis transmembrane conductance regulator (CFTR) mutation and other rare CFTR mutations expressed in HEK293 cells rescued by VX-445+VX-661+VX-770. RFU: relative fluorescence units; DMSO: dimethyl sulfoxide; FSK: forskolin; CNX: calnexin. a) Representative traces of F508del-CFTR-dependent chloride efflux (membrane depolarisation assay) in HEK293 cells stably transfected with F508del-CFTR pre-treated with DMSO or S-VX-445 (3 µM)+VX-661 (3 µM) for 24 h at 37°C. b) Maximal activation of F508del-CFTR after stimulation by FSK (1 0 µM)±VX-770 (1 µM) and/or S-VX-445 (1 µM). c) HEK293 cells were transiently transfected with M1101K-, G85E- or N1303K-CFTR and treated for 24 h at 37°C with DMSO, VX-809 (3 µM), VX-661 (3 µM) or S-VX-445 (3 µM)+VX-661 (3 µM). Maximal activation of M1101K-, G85E- and N1303K-CFTR after stimulation by FSK (10 µM)±VX-770 (1 µM) and/or S-VX-445 (1 µM) (n=3–10). d) Immunoblots of steady-state expression of F508del-, M1101K-, G85E- or N1303K-CFTR following treatments for 24 h at 37°C with DMSO, VX-809 (3 µM), VX-661 (3 µM) or S-VX-445 (3 µM)+VX-661 (3 µM). Markers in kDa. Band C: mature, complex-glycosylated CFTR; band B: immature, core-glycosylated CFTR. e) Ratio band C/(band C+band B) (n=3). Data are presented as mean±sd. Comparative analysis was performed using one-way ANOVA followed by Turkey's post hoc test (*: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001).