The Cystic Fibrosis (CF) causing variant G542X (GGA>TGA) results in premature termination of translation of the cystic fibrosis transmembrane regulator (CFTR) protein, and nonsense-mediated decay of the CFTR mRNA resulting in almost complete loss of functional CFTR protein expression. This leads to defective anion transport and the development of CF disease pathology. Currently available CF modulator therapies cannot be used to treat this variant, but an Adenine Base Editor (ABE) and guide RNA combination can convert the G542X stop codon to G542R, a variant which retains about 30% of WT activity^[1] and is amenable to CFTR triple modulator therapy (Elexacaftor-Tezacaftor-Ivacaftor ETI). 

Plasmid DNA encoding this ABE and guide RNA with GFP were encapsulated in lipid-based nanocomplexes, which have reduced immunogenicity and high cellular uptake, and were delivered to human airway epithelial cells (HAECs) harboring the G542X CFTR variant. Transfected cells were identified by analysing GFP fluorescence. Ion transport was measured as short circuit current (Isc) across resistive epithelial monolayers. Cultures were equilibrated using standard physiological salt solution before addition of pharmacological drugs, forskolin, 10 μM (bilateral) to activate CFTR; CFTRInh-172, 10 μM (apical) to inhibit CFTR; and ouabain, 100 μM (basolateral) to inhibit Na⁺K⁺ATPase activity. Airway Surface Liquid (ASL) height was visualised using Texas-red dextran as a soluble fluorescent label of volume, and perfluorocarbon-77 to prevent fluid evaporation. CFTR was activated using vasoactive intestinal peptide (VIP)(100 nM, basolateral), and changes were calculated from XZ scans taken at regular intervals. ASL pH was measured using pHrodo-dextran pH-sensitive fluorescent labelling normalised to a pH-insensitive (pHins) in situ control.

48 hours post transfection, next-generation sequencing showed 17% of alleles had been edited from G542X to G542R. Isc measurements showed that even as little as 17% of editing restored CFTR activity to ~ 60 % that of normal levels when in combination with modulators. CFTRInh-172 inhibitable current was ~ four-fold greater in ETI treated G542R compared to G542X samples (Table 1; p=0.015, n = 6). Transfected cells were sorted using fluorescence-activated cell sorting (FACS) to concentrate the population of edited cells (henceforth called edited and sorted). CFTRInh-172 inhibitable Isc was not significantly different to that of wild-type samples (p = 0.97; n = 6, N = 2). VIP stimulated changes in ASL height indicated that edited and sorted cell monolayers displayed wild-type like airway hydration (Table 1). Additionally, VIP stimulated changes in ASL pH were positively correlated to the proportion of G542R cells, suggesting that bicarbonate transport was also being restored by correction of G542X to G542R CFTR protein.

This data provides proof-of-concept for restoration of anion transport by gene editing of G542X.