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 EpiXTM 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 (ΔICFTRinh) 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/cm2 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.
Highlights
Cystic Fibrosis (CF; OMIM 219700) is an autosomal recessive disorder caused by mutations in the gene coding the cystic fibrosis transmembrane conductance regulator (CFTR) which functions as a chloride and bicarbonate-permeable ion channel protein in the apical cell membranes of various epithelia, including the lungs, pancreas, and sweat glands [1, 2]
We report the results of a personalized procedure in which patient-derived cells are used to characterize mutant CFTR function and pharmacodynamic response
We report that the genotypes studied here range from intermediate to low CFTR functionality, with F508del/R117H7T retaining as much as 56% wildtype CFTR function, R334W/406-1G>A retaining 24%, F508del/c.850dupA retaining 12%, F508del/F508del retaining 12%, and CFTRdele2,3(21 kb)/CFTRdele2,3(21 kb) retaining 9%
Summary
Cystic Fibrosis (CF; OMIM 219700) is an autosomal recessive disorder caused by mutations in the gene coding the cystic fibrosis transmembrane conductance regulator (CFTR) which functions as a chloride and bicarbonate-permeable ion channel protein in the apical cell membranes of various epithelia, including the lungs, pancreas, and sweat glands [1, 2]. The majority of CFTR mutations and resulting variety of genotypes have not been experimentally evaluated in terms of their functional consequences on chloride and bicarbonate transport function. We report the results of a personalized procedure in which patient-derived cells are used to characterize mutant CFTR function and pharmacodynamic response. We examined the electrophysiologic properties of rare genotypes of five pediatric CF patients, whose cells were harvested by a swift nasal swab procedure and expanded for in vitro analysis
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