Correction of CFTR function in nasal epithelial cells from cystic fibrosis patients predicts improvement of respiratory function by CFTR modulators

Clinical studies with modulators of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) protein have demonstrated that functional restoration of the mutated CFTR can lead to substantial clinical benefit. However, studies have shown highly variable patient responses. The objective of this study was to determine a biomarker predictive of the clinical response. CFTR function was assessed in vivo via nasal potential difference (NPD) and in human nasal epithelial (HNE) cultures by the response to Forskolin/IBMX and the CFTR potentiator VX-770 in short-circuit-current (∆IscF/I+V) experiments. CFTR expression was evaluated by apical membrane fluorescence semi-quantification. Isc measurements discriminated CFTR function between controls, healthy heterozygotes, patients homozygous for the severe F508del mutation and patients with genotypes leading to absent or residual function. ∆IscF/I+V correlated with CFTR cellular apical expression and NPD measurements. The CFTR correctors lumacaftor and tezacaftor significantly increased the ∆IscF/I+V response to about 25% (SEM = 4.4) of the WT-CFTR level and the CFTR apical expression to about 22% (SEM = 4.6) of the WT-CFTR level in F508del/F508del HNE cells. The level of CFTR correction in HNE cultures significantly correlated with the FEV1 change at 6 months in 8 patients treated with CFTR modulators. We provide the first evidence that correction of CFTR function in HNE cell cultures can predict respiratory improvement by CFTR modulators.


Primary bronchial and nasal epithelial cells sampling and culture
HNE cells were sampled by nasal brushing of the medial wall and the inferior turbinate of both nostrils and placed in DMEM/F-12 (Dulbecco's Modified Eagle's /Ham's F-12 media 50/50).
For air-liquid interface (ALI) culture of non-amplified cells, about 330,000 HBE or HNE cells suspended in amplification medium were seeded on type IV collagen-coated porous filter with a 0.33-cm 2 surface (Transwell, Corning). UG (Ultroser G) 2% medium (DMEM/F-12, supplemented with 2% Ultroser G) containing Piperacillin/Tazobactam 90 µg/10 µg/ml and Amphotericin B 5 µg/ml was added to the basal side of filters. After two days, apical medium was aspirated and cells were cultured in ALI with UG 2% basal medium changed daily for 3-4 weeks to establish a differentiated epithelium 2,3 .

3
The protocol for cell expansion was adapted from Suprynowicz et al. 4 . Conditional reprogramming was performed by co-culturing the cells with irradiated fibroblasts and Rho-Kinase inhibition and to minimize phenotypic changes, cells were differentiated after passage 1. Briefly, freshly isolated HNE cells were first grown on a feeder layer, co-cultured with irradiated NIH-3T3 fibroblasts in DMEM/F-12 cell culture medium supplemented with 10% newborn calf serum, Rho kinase inhibitor Y-27632 (10 μM), Piperacillin/Tazobactam 90 µg/10 µg/ml and Amphotericin B 5 µg/m. Cells were then supplemented with fresh culture medium every other day and after achieving ~2-3 x10 6 cell expansion, cultures were trypsinized to separate feeders and epithelial expanded cells. epithelial Na + channel (ENaC); cAMP agonists Forskolin (10 µM) and 3-isobutyl-1methylxanthine (IBMX 100 µM) to activate the transepithelial cAMP-dependent current (including Cltransport through CFTR channels); VX-770 (10 µM) to potentiate CFTR activity; CFTR inhibitor CFTRinh172 (5 µM) to specifically inhibit CFTR; and ATP (100 µM) to challenge the purinergic calcium-dependent Clsecretion. The following parameters were then calculated: ΔIsc Amiloride as the difference between Isc after Amiloride and baseline Isc; ΔIsc F/I as the difference between Isc after Forskolin/IBMX and Isc after Amiloride; ΔIsc VX-770 as the difference between Isc after VX-770 and Isc after Forskolin/IBMX; ΔIsc CFTRinh172 as the difference between Isc after CFTRinh172 and Isc after VX-770.
The sum of the change after Forskolin/IBMX and VX-770 (∆Isc F/I+V) served as an index of CFTR function.

Nasal potential difference measurements
Nasal potential difference (NPD) measurements were performed as described previously 6 , according to sequential perfusion of 100 µM Amiloride in saline solution (∆Amiloride); Amiloride in low-chloride solution to drive chloride secretion (∆low chloride); Amiloride plus 10 µM Isoproterenol in low-chloride solution to stimulate the cAMP-dependent Clconductance related to CFTR (∆Isoproterenol). The sum of ∆low chloride and ∆Isoproterenol (∆LowCl --Isoproterenol) served as an index of CFTR function. 6

Correlation between fluorescence intensity and percentage of apical CFTR staining
Supplementary Figure S2 shows the correlation between the average corrected apical fluorescence intensity and the percentage of cells displaying apical staining according to genotypes in the whole population (R 2 =0.7, p<0.0001; simple regression analysis).

Detailed response to Forskolin/IBMX + VX-770 in HNE cells in non F508del/F508del CF genotypes
ΔIscF/I+V was barely detectable, below 5% of the WT-CFTR response, for the cultures with genotypes homozygous for a premature termination codon (PTC) (Y122X and G542X) or compound heterozygote F508del in trans with a nonsense mutation (G542X), a deletion generating a PTC (394delTT), a mutation associated with abnormal protein folding (N1303K), or splicing mutations resulting in little protein production (711+1G>T, 1717-1G>A). In contrast, the F508del/E1418X genotype, with a PTC in the penultimate exon 7 , displayed a residual CFTR function at 10.3% of the WT-CFTR level. Similarly, when F508del was associated with 2789+5G>A, a mild class V mutation associated with residual normal exon 16 splicing 8 , this provided a residual Cltransport at 14.6% of the WT-CFTR value. Finally, the cells compound heterozygotes for F508del and D1152H, a class IV regulation defect associated with atypical CF 9 , displayed a strong residual Cltransport after Forskolin/IBMX+VX-770 stimulation. This was also the case for the F508del-R117H-T7 genotype, which has been demonstrated to retain a Clconductance 10 , where a normal ∆IscF/I+V change was evidenced.
VX-809 did not modify the ΔIscF/I+V in genotypes with absent basal CFTR function associating F508del CFTR with PTC mutations, 711+1G>T or N1303K. In contrast, the F508del/1717-7 1G>A genotype was strongly corrected, reaching 15.4% of the WT-CFTR level. Similarly, the CFTR activity of the F508del/394delTT HBE cells was increased to 30% of WT-CFTR activity.
Interestingly, the genotypes with strong initial residual Clsecretion, such as F508del/D1152H and L997F/R258G, displayed considerable correction, allowing the WT-CFTR value to be reached. The only exception was the F508del/2789+5G>A, which displayed only a 7.7% increase of ∆IscF/I+V upon VX-809, however, this reached 22% of the average WT-CFTR level because of the basal residual function.