Abstract
BackgroundMutations in the cystic fibrosis transmembrane regulator (CFTR) gene can reduce function of the CFTR ion channel activity and impair cellular chloride secretion. The gold standard method to assess CFTR function of ion transport using the Ussing chamber requires a high number of airway epithelial cells grown at air-liquid interface, limiting the application of this method for high throughput screening of potential therapeutic compounds in primary airway epithelial cells (pAECs) featuring less common CFTR mutations. This study assessed an alternative approach, using a small scale halide assay that can be adapted for a personalized high throughput setting to analyze CFTR function of pAEC.MethodsPediatric pAECs derived from children with CF (pAECCF) were established and expanded as monolayer cultures, before seeding into 96-well plates for the halide assay. Cells were then transduced with an adenoviral construct containing yellow fluorescent protein (eYFP) reporter gene, alone or in combination with either wild-type CFTR (WT-CFTR) or p.Phe508del CFTR. Four days post transduction, cells were stimulated with forskolin and genistein, and assessed for quenching of the eYFP signal following injection of iodide solution into the assay media.ResultsData showed that pAECCF can express eYFP at high efficiency following transduction with the eYFP construct. The halide assay was able to discriminate functional restoration of CFTR in pAECCF treated with either WT-CFTR construct or the positive controls syntaxin 8 and B-cell receptor-associated protein 31 shRNAs.SignificanceThe current study demonstrates that the halide assay can be adapted for pediatric pAECCF to evaluate restoration of CFTR function. With the ongoing development of small molecules to modulate the folding and/or activity of various mutated CFTR proteins, this halide assay presents a small-scale personalized screening platform that could assess therapeutic potential of molecules across a broad range of CFTR mutations.
Highlights
Cystic fibrosis (CF) manifests as a multi-organ disease, lung disease presenting with recurrent infections, chronic neutrophilic inflammation and structural pathologies remain the primary cause of mortality [1,2]
The current study demonstrates that the halide assay can be adapted for pediatric pAECs derived from children with CF (pAECCF) to evaluate restoration of cystic fibrosis transmembrane regulator (CFTR) function
The current gold standard method of using Ussing chamber to measure ion transport through electrophysiology requires a high number of Airway epithelial cells (AECs) for each permeable insert grown at air-liquid interface (ALI), which precludes the use of primary AEC from pediatric CF populations
Summary
Cystic fibrosis (CF) manifests as a multi-organ disease, lung disease presenting with recurrent infections, chronic neutrophilic inflammation and structural pathologies remain the primary cause of mortality [1,2]. With the discovery that small molecules can have the potential to actively correct CFTR and many more that are currently in the pipeline especially for rare mutations of CFTR, a small scale high-throughput screening (HTS) platform is necessary to help realize personalized medicine approaches in early CF One such approach would be to adapt a halide sensitive fluorescent reporter molecule for expression in pAEC and its utilization in an assay that assesses ion channel activity. The gold standard method to assess CFTR function of ion transport using the Ussing chamber requires a high number of airway epithelial cells grown at air-liquid interface, limiting the application of this method for high throughput screening of potential therapeutic compounds in primary airway epithelial cells (pAECs) featuring less common CFTR mutations. This study assessed an alternative approach, using a small scale halide assay that can be adapted for a personalized high throughput setting to analyze CFTR function of pAEC
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