Expression of gain-of-function CFTR in cystic fibrosis airway cells restores epithelial function better than wild-type or codon-optimized CFTR

• Published in: Molecular therapy. Methods & clinical development 2023-09, Vol.30, p.593-605
• Main Authors: Woodall, Maximillian, Tarran, Robert, Lee, Rhianna, Anfishi, Hafssa, Prins, Stella, Counsell, John, Vergani, Paola, Hart, Stephen, Baines, Deborah
• Format: Article
• Language: English
• Subjects: airway epithelium; airway surface liquid; anion secretion; CFTR; codon optimisation; cystic fibrosis; gene therapy; Original; sputum
• ISSN: 2329-0501; 2329-0501
• DOI: 10.1016/j.omtm.2023.08.006

Class Ia/b cystic fibrosis transmembrane regulator (CFTR) variants cause severe lung disease in 10% of cystic fibrosis (CF) patients and are untreatable with small-molecule pharmaceuticals. Genetic replacement of CFTR offers a cure, but its effectiveness is limited in vivo. We hypothesized that enhancing protein levels (using codon optimization) and/or activity (using gain-of-function variants) of CFTR would more effectively restore function to CF bronchial epithelial cells. Three different variants of the CFTR protein were tested: codon optimized (high codon adaptation index [hCAI]), a gain-of-function (GOF) variant (K978C), and a combination of both (hˆK978C). In human embryonic kidney (HEK293T) cells, initial results showed that hCAI and hˆK978C produced greater than 10-fold more CFTR protein and displayed ∼4-fold greater activity than wild-type (WT) CFTR. However, functionality was profoundly different in CF bronchial epithelial cells. Here, K978C CFTR more potently restored essential epithelial functions (anion transport, airway surface liquid height, and pH) than WT CFTR. hCAI and hˆK978C CFTRs had limited impact because of mislocalization in the cell. These data provide a proof of principle showing that GOF variants may be more effective than codon-optimized forms of CFTR for CF gene therapy. [Display omitted] [Display omitted] Baines and colleagues show that viral delivery of cystic fibrosis transmembrane regulator (CFTR) gain-of-function variant cDNA (K978C-CFTR) to cystic fibrosis bronchial epithelial (CFBE) cells in the presence of CF sputum was more effective at restoring anion secretion, ASL hydration, and pH than wild-type CFTR.