Beyond Trikafta: new models to assess tissue dependent rescue of N1303K-CFTR

Abstract

Rationale: Respiratory status of people with Cystic Fibrosis (pwCF) carrying N1303K is improved by Elexacaftor/Tezacaftor/Ivacaftor (ETI) but, contrary to other mutations, the impact on sweat test results is limited.

Methods: To explore this discrepancy, we implemented new sweat gland and respiratory cell lines stably expressing Wild type (WT)-, F508del- and N1303K-CFTR. CFTR dependent chloride (Cl^-) and bicarbonate (HCO₃-) transport was measured by short circuit current in these new models and in primary Human Nasal Epithelial Cells (HNECs). CFTR expression was evaluated by Western blot.

Results: In the airway and the sweat gland cells expressing F508del-CFTR, ETI induced maturation of CFTR and increased Cl^- transport. In the respiratory cell lines and HNECs, N1303K-CFTR generated both immature and mature forms of CFTR. Correction by ETI increased CFTR amounts without promoting its maturation and improved Cl^- secretion. N1303K-CFTR channel activity was markedly increased by co-potentiation of IVA with Apigenin. In the sweat gland, N1303K-CFTR was expressed as a globally misfolded protein, non-rescuable by ETI. API treatment to 2 patients improved FEV1 without lowering sweat Cl- content.

Conclusion: N1303K-CFTR shows tissue specific correction and suboptimal response to ETI which can be improved by API.

Keywords: CF; CFTR (cystic fibrosis transmembrane conductance regulator); CFTR modulator; N1303K-CFTR; airway epithelium; cystic fibrosis; elexacaftor/tezacaftor/ivacaftor (ETI); sweat gland.

FIGURE 1

Change in percent predicted forced expiratory volume in 1 s and sweat chloride concentration in pwCF treated with Elexacaftor/Tezacaftor/Ivacaftor (ETI) carrying the N1303K or the F508del variant. Scatter dot plots indicate the corresponding variation after 2 months ETI in (A) percentage predicted Forced Expiratory Volume in 1 s (ΔppFEV 1) and (B) sweat chloride in mmol/l (Δsweat chloride). Comparison by unpaired t-test. ***: p < 0.001.

FIGURE 2

Expression of CFTR in respiratory and sweat gland cell lines stably expressing WT, F508del and N1303K-CFTR. Representative Western blot images and corresponding quantifications for WT, F508del and N1303K-CFTR in lenti-16HBEge-cells stably expressing CFTR after lentiviral transduction (A) sweat gland lenti-NCL-SG3 stably expressing CFTR after lentiviral transduction (B). Cells were treated for 48 h with vehicle (DMSO) or ELX/TEZ combination (3 µM/10 µM). Protein detection in (B) was performed on a single membrane but image was fragmented and reorganized for sample order as in (A). Detection of CFTR variants was performed on separate membranes and imaged separately. Bar graphs on the right panels correspond to the quantification of CFTR maturation expressed as a C/(C + (B) band ratio for each different cell type. As a reference, the dotted bar on the graph corresponds to the ratio for WT. (C) Comparison of total expression (left) and C band expression (right) of F508del and N1303K-CFTR at baseline and in TEZ/ELX corrected cells in lenti-16HBEge and lenti-NCL-SG3 cells. Data are presented as mean ± standard error (SEM) from a minimum of three independent experiments. *: p < 0.05; **: p < 0.01; ***: p < 0.001.

FIGURE 3

CFTR activity in respiratory and sweat gland cell lines stably expressing WT, F508del and N1303K-CFTR. CFTR activity quantified with short-circuit current technique in respiratory cell lines (lenti-16HBEge) (A,C,E,G) and sweat gland cell lines (lenti-NCL-SG3) (B,D,F,H) stably expressing WT (A,B), F508del (C,D) and N1303K (E,F) after lentiviral transduction, as compared to non-transduced cells (A, B, in grey) or non-corrected epithelia (C,D,E,F, in grey). Cells were treated for 48 h with vehicle (DMSO) or ELX/TEZ combination. During the recordings, the epithelia were sequentially treated with Forskolin (Fsk) (10 µM), to activate CFTR, IVA (1 µM), to potentiate CFTR, API (25 µM), to co-potentiate IVA, CFTR inhibitor-172 (10 µM) to inhibit CFTR, all added on the apical side. Barium (5 mM) was added in lenti-NCL-SG3-N1303K on the basal side, as a quality control. Representative tracings are shown. Summary of the results in G (lenti-16HBEge) and H (lenti-NCL-SG3) from a minimum of three independent experiments. Comparison by Wilcoxon test, *: p < 0.05, **: p < 0.01; ***: p < 0.001; ****: p < 0.0001.

FIGURE 4

Representative biochemical and functional analysis of N1303K-CFTR in Human Epithelial Nasal Epithelial cells. (A) Representative Western blot images and corresponding quantifications for WT, F508del and N1303K-CFTR in Human Nasal Epithelial primary cells (HNECs). (B,C) Representative tracings on the left panel of the effect of vehicle (DMSO), or the elexacaftor/tezacaftor (ELX, 3 µM/TEZ, 10 µM) combination on HNECs with the short-circuit-current technique. During the recordings, the epithelia were sequentially treated (as indicated by downward arrows) with amiloride (10 μM; added on the apical side), CPT-cAMP (100 μM; added on both apical and basolateral sides), ivacaftor (IVA, 1 μM; apical side), apigenin (API, 25 μM; apical side), and the CFTR inhibitor-172 (inh-172; 20 μM; apical side). The dashed line indicates zero current level. Right panel: scatter dot plot showing the summary of results. Data reported are the amplitude of the current blocked by inh-172 (∆Isc_inh-172). For each experimental condition the number of biological replicates were n = 4-6. Short-circuit current performed on: (B) N1303K/711 + 1G>T nasal epithelial cells (derived from donor ID: GE156) (C) N1303K/3659delC nasal epithelial cells (derived from donor ID: GE132).

FIGURE 5

N1303K-CFTR activity potentiation by Ivacaftor and Apigenin in Tezacafor/Elexacaftor corrected Human Epithelial Nasal Epithelial cells. CFTR activity quantified with short-circuit current technique in nasal epithelial cells treated for 24 h with vehicle (DMSO) or elexacaftor/tezacaftor (ELX, 3 µM/TEZ, 10 µM) combination. During the recordings, the epithelia were sequentially treated with amiloride (10 μM; added on the apical side), CPT-cAMP (100 μM; added on both apical and basolateral sides), IVA (1 μM; apical side), API (25 μM; apical side), and the CFTR inhibitor-172 (inh-172; 20 μM; apical side). Data reported are the average amplitude of the current blocked by 20 μM inh-172 (∆Isc_inh-172). (A) Scatter dot plots showing experiments performed on thirty subjects compound heterozygous for N1303K and a minimal function (MF), non-rescuable variant (N1303 K/MF) and six homozygous for N1303K (N1303K/N1303K) under different conditions. (B) Connected dot plots of patients shown in (A).