A helper-dependent adenoviral vector rescues CFTR to wild-type functional levels in cystic fibrosis epithelial cells harbouring class I mutations

Abstract

Cystic Fibrosis (CF) is a genetic disorder affecting multiple organs, including the pancreas, hepatobiliary system and reproductive organs however lung disease is responsible for the majority of morbidity and mortality. Management of CF involves CFTR modulator agents including corrector drugs to augment cellular trafficking of mutant CFTR as well as potentiators that open defective CFTR channels. These therapies are poised to help most individuals with CF, with the notable exception of individuals with class I mutations where full length CFTR protein is not produced. For these mutations, gene replacement has been suggested as a potential solution.In this work, we used a helper dependent adenoviral vector (HD-CFTR) to express CFTR in nasal epithelial cell cultures derived from CF subjects with class I CFTR mutations. CFTR function was significantly restored in CF cells by HD-CFTR and reached healthy control functional levels as detected by Ussing chamber and membrane potential (FLIPR) assay. A dose response relationship was observed between the amount of vector used and subsequent functional outcomes; small amounts of HD-CFTR were sufficient to correct CFTR function. At higher doses, HD-CFTR did not increase CFTR function in healthy control cells above baseline values. This latter observation allowed us to use this vector to benchmark in vitro efficacy testing of CFTR-modulator drugs. In summary, we demonstrate the potential for HD-CFTR to inform in vitro testing and to restore CFTR function to healthy control levels in airway cells with class I or CFTR nonsense mutations.

Rescue of multiple class II CFTR mutations by elexacaftor+tezacaftor+ivacaftor mediated in part by the dual activities of elexacaftor as both corrector and potentiator

“…16HBE14o- cells, CRISPR/Cas9 edited to express N1303K-CFTR, were obtained from the Cystic Fibrosis Foundation (CFF 16HBEge N1303K-CFTR) [ 19 ]. Nasal epithelial cell cultures were generated following nasal brushing of the individual patients as previously described [ 20 , 21 ].…”

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

“…HNE models, however, hold promise for adaptation to the development and analysis of next generation therapeutic agents. For example, nasal cells could be used both as a development model for a gene editing or gene therapy approach, as well as an early-phase readout for clinical trials of that therapy or other novel therapies [ 106 , 113 , 117 , 118 , 119 ]. Similar approaches focused on nasal potential difference have already been used with antisense oligonucleotide therapy, and could easily be adapted to culture-based models [ 114 ].…”

Identification of binding sites for ivacaftor on the cystic fibrosis transmembrane conductance regulator

“…The blots were developed with ECL (Amersham) using the Li-Cor Odyssey Fc (LI-COR Biosciences, Lincoln, NE) in a linear range of exposure (2-5min). Relative levels of CFTR protein were quantified by densitometry of immunoblots using ImageStudioLite (LI-COR Biosciences, Lincoln, NE) ( Cao et al, 2020 ; Erwood et al., 2020 ; Wu et al., 2019 ).…”