Functional Profiling of CFTR-Directed Therapeutics Using Pediatric Patient-Derived Nasal Epithelial Cell Models

Abstract

Functional profiling of CFTR-directed therapeutics offers the potential to provide significant benefits to young people with cystic fibrosis (CF). However, the development of 2D airway epithelial cell models for individual response tests in CF children remains a central task. The objective of this study was to determine the utility of EpiX TM technology for expansion of nasal epithelial cells for use in electrophysiological CFTR function measurements. An initial harvest of as few as 20,000 cells was sufficient to expand up to 50 million cells that were used to generate air-liquid interface (ALI) cultures for ion transport studies with the Ussing assay. CFTR function was assessed by measuring responses to forskolin and the CFTR potentiator VX-770 (ivacaftor) in ALI cultures generated from passage 3 and 4 cells. Short-circuit current (Isc) measurements of blocked CFTR currents (I CFTRinh) discriminated CFTR function between healthy control (wild type, WT) and patients with intermediate (F508del/R117H-7T: 56% WT) and severe (F508del/F508del: 12% WT) CF disease. For the mixed genotypes, CFTR activity for F508del/c.850dupA was 12% WT, R334W/406-1G>A was 24% WT, and CFTRdele2,3(21 kb)/CFTRdele2,3(21 kb) was 9% WT. The CFTR correctors VX-809 (lumacaftor) and VX-661 (tezacaftor) significantly increased CFTR currents for F508del/R117H to 73 and 67% WT, respectively. Cultures with the large deletion mutation CFTRdele2,3(21 kb) unexpectedly responded to VX-661 treatment (20% WT). Amiloride-sensitive sodium currents were robust and ranged between 20-80 µA/cm 2 depending on the subject. In addition to characterizing the electrophysiological profile of mutant CFTR activity in cultures for five genotypes, our study exemplifies the promising paradigm of bed-to-bench side cooperation and personalized medicine.

Nasal Epithelial Cell-Based Models for Individualized Study in Cystic Fibrosis

“…There is additional uncertainty regarding the relevance of the in vitro environment to the actual human nasal or respiratory epithelium. While the studies above have linked nasal cells to individual or unrelated cohort patient studies, the numbers in these comparisons are small, and the precision of this linkage remains unclear [70][71][72]74,75]. Primary human nasal cell cultures are limited by the number of times they can Electrophysiologic assessment of CFTR activity in HNE ALI cultures has the capacity to discriminate between CF, WT, and CF disease with partial CFTR function, and modulator corrected mutant CFTR [72,73].…”

“…While the studies above have linked nasal cells to individual or unrelated cohort patient studies, the numbers in these comparisons are small, and the precision of this linkage remains unclear [70][71][72]74,75]. Primary human nasal cell cultures are limited by the number of times they can Electrophysiologic assessment of CFTR activity in HNE ALI cultures has the capacity to discriminate between CF, WT, and CF disease with partial CFTR function, and modulator corrected mutant CFTR [72,73]. Among subjects with CF, modulated, ex vivo CFTR activity in HNE cells has been shown to correlate with clinical improvements ppFEV 1 and sweat chloride, both at the individual level and against historical trial cohort data [32,[70][71][72]74,75].…”

“…Primary human nasal cell cultures are limited by the number of times they can Electrophysiologic assessment of CFTR activity in HNE ALI cultures has the capacity to discriminate between CF, WT, and CF disease with partial CFTR function, and modulator corrected mutant CFTR [72,73]. Among subjects with CF, modulated, ex vivo CFTR activity in HNE cells has been shown to correlate with clinical improvements ppFEV 1 and sweat chloride, both at the individual level and against historical trial cohort data [32,[70][71][72]74,75]. While Ussing chamber studies represent the gold standard in electrophysiological assessment of modulator-rescued CFTR function they are limited by the need for highly specialized equipment and a time and labor intensive process, resulting in a low-throughput assay.…”

“…There is additional uncertainty regarding the relevance of the in vitro environment to the actual human nasal or respiratory epithelium. While the studies above have linked nasal cells to individual or unrelated cohort patient studies, the numbers in these comparisons are small, and the precision of this linkage remains unclear [ 70 , 71 , 72 , 74 , 75 ]. Primary human nasal cell cultures are limited by the number of times they can be passaged as well as by a risk for contamination as compared to immortalized cell lines, though the relevance of primary culture likely offsets this downside for human studies.…”

“…There is a growing body of evidence that suggests that there is sufficient similarity between the morphology and function of nasal cell models and bronchial epithelial tissue to allow HNE to be used broadly as surrogates for HBE in future research in CF and other lower airways disease [ 31 , 32 , 73 , 85 , 86 ]. Several studies have already reported correlations between the results of studies in nasal cell culture models and meaningful clinical outcomes [ 70 , 71 , 72 , 74 , 75 ]. However, it is important to note that there may be specific situations in which HNE cultures would not be suitable for use as a surrogate for bronchial epithelium and the criteria to determine their suitability is ultimately contingent upon the exact question being asked.…”