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

Maximillian Woodall; Robert Tarran; Rhianna Lee; Hafssa Anfishi; Stella Prins; John Counsell; Paola Vergani; Stephen Hart; Deborah Baines

doi:10.1016/j.omtm.2023.08.006

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

Mol Ther Methods Clin Dev. 2023 Aug 12:30:593-605. doi: 10.1016/j.omtm.2023.08.006. eCollection 2023 Sep 14.

Authors

Maximillian Woodall¹, Robert Tarran², Rhianna Lee², Hafssa Anfishi¹, Stella Prins³, John Counsell⁴, Paola Vergani³, Stephen Hart⁴, Deborah Baines¹

Affiliations

¹ Institute for Infection and Immunity, St George's, University of London, Cranmer Terrace, Tooting, London SW17 0RE, UK.
² Department of Cell Biology & Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7248, USA.
³ Neuroscience, Physiology, & Pharmacology, Division of Biosciences, University College London, London WC1E 6BT, UK.
⁴ Genetics & Genomic Medicine Department, Great Ormond Street Institute of Child Health, London WC1N 1EH, UK.

Abstract

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.

Keywords: CFTR; airway epithelium; airway surface liquid; anion secretion; codon optimisation; cystic fibrosis; gene therapy; sputum.

Associated data

figshare/10.6084/m9.figshare.23915565.v1

Abstract

Associated data

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