Measurements of Functional Responses in Human Primary Lung Cells as a Basis for Personalized Therapy for Cystic Fibrosis

Abstract

Background: The best investigational drug to treat cystic fibrosis (CF) patients with the most common CF-causing mutation (F508del) is VX-809 (lumacaftor) which recently succeeded in Phase III clinical trial in combination with ivacaftor. This corrector rescues F508del-CFTR from its abnormal intracellular localization to the cell surface, a traffic defect shared by all Class II CFTR mutants. Our goal here is to test the efficacy of lumacaftor in other Class II mutants in primary human bronchial epithelial (HBE) cells derived from CF patients.

Methods: The effect of lumacaftor was investigated in primary HBE cells from non-CF and CF patients with F508del/F508del, A561E/A561E, N1303K/G542X, F508del/G542X and F508del/Y1092X genotypes by measurements of Forskolin plus Genistein-inducible equivalent short-circuit current (Ieq-SC-Fsk + Gen) in perfused open-circuit Ussing chambers. Efficacy of corrector C18 was also assessed on A561E/A561E and F508del/F508del cells.

Results: Our data indicate that A561E (when present in both alleles) responds positively to lumacaftor treatment at equivalent efficacy of F508del in primary HBE cells. Similarly, lumacaftor has a positive impact on Y1092X, but not on N1303K. Our data also show that cells with only one copy of F508del-CFTR respond less to VX-809. Moreover, there is great variability in lumacaftor responses among F508del-homozygous cells from different donors. Compound C18 failed to rescue A561E-CFTR but not in F508del-CFTR, thus plausibly it has a different mechanism of action distinct from lumacaftor.

Conclusions: CF patients with A561E (and likely also those with Y1029X) can potentially benefit from lumacaftor. Moreover, the methodology used here exemplifies how ex vivo approaches may apply personalized therapies to CF and possibly other respiratory diseases.

Keywords: (ΔIeq-sc), equivalent short-circuit currents; CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; ENaC, epithelial Na+ channel; Fsk, forskolin; Gen, Genistein; HBE (cells), human bronchial epithelial cells; Innovative treatments; Mutation-specific therapies; Personalized medicine; Rare diseases; Rte, transepithelial resistance.; SEM, standard error of the mean; TEER, transepithelial electrical resistance; Vte, transepithelial voltage.

Fig. 1

Effect of lumacaftor (VX-809) on cAMP-induced I_sc-eq in primary cultures of HBE cells from CF patients with class II mutations. Original Ussing chamber (open-circuit) recordings showing transepithelial voltage measurements (V_te) obtained for CF primary airway HBE monolayers with different CFTR genotypes: wt/wt control (a, b); F508del/F508del-donor 1 (c, d); F508del/F508del-donor 2 (e, f); and A561E/A561E (g, h). Cells were preincubated for 24 h with either 3 μM/24 h lumacaftor/VX-809 (b, d, f, h) or DMSO (0.1%v/v) vehicle control (a, c, e, g). Amiloride (20 μM) was kept during the whole experiment duration to avoid interference of ENaC-mediated Na⁺ currents. Negative transepithelial voltage (V_te) deflections are observed following the application of luminal forskolin alone (Fsk, 2 μM) or with genistein (Gen, 25 μM). The latter are fully reverted by application of 30 μM Inh₁₇₂, a specific CFTR inhibitor (see also values in Table S1).

Fig. 2

Effect of lumacaftor (VX-809) on cAMP-induced I_sc-eq in primary cultures of HBE cells from CF patients with different CFTR mutations. Original Ussing chamber (open-circuit) recordings showing transepithelial voltage measurements (V_te) obtained for CF primary airway HBE monolayers with different genotypes: N1303K/G542X (a, b); F508del/G542X (c, d); and F508del/Y1092X (e, f). Cells were pre-incubated for 24 h with either 3 μM/24 h lumacaftor/VX-809 (b, d, f) or DMSO (0.1%v/v) vehicle control (a, c, e). Other conditions as described in Fig. 1 legend (see also values in Table S1).

Fig. 3

Summary of the effect of lumacaftor (VX-809) on HBE cells from CF patients with different genotypes. Graphs represent values of I_eq-sc (μA/cm²) calculated from voltage deflection obtained for the responses to Fsk (a) or to Gen + Fsk (b), after 24 h treatment with 0.1% DMSO (white bars) or 3 μM VX-809 (black bars) for HBE cells with different genotypes, as indicated below the graphs. (c) Percentage of I_eq-sc rescue in response to Forskolin plus Genistein (I_{eq-sc-Fsk + Gen}) after VX-809 vs DMSO vs non-CF cells (see also Table S2). *indicates statistically significant (p > 0.05) and “ns” not significant.

Fig. 4

Original tracings and summary of the effect of C18 in A561E/A561E and F508del/F508del primary HBE cells. (a, b) represent original Ussing chamber (open-circuit) recordings obtained for the analysis of CF primary airway HBE monolayers with A561E/A561E and F508del/F508del treated with 5 μM C18 for 24 h. (c) Graph represents summary of I_sc-eq (μA/cm²) values obtained for responses to Fsk (white bars) or Fsk + Gen (black bars) after 24 h-treatment with DMSO, VX-809/lumacaftor or C18 as indicated (see also Table S4). * indicates statistically significant (p > 0.05).