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Functional analysis of gene-edited CF variant G542X

I Rose(1) L Nicosia(3) M Greenwood(2) P Harrison(3) S Hart(2) D Baines(1)

1:St. George's University of London; 2:UCL Institute of Child Health; 3:University College Cork

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 and is amenable to modulator therapy. Plasmid DNA encoding this ABE, guide RNA, and GFP were encapsulated in lipid-based nanocomplexes, which have demonstrated reduced immunogenicity and high uptake in epithelial cells, were delivered to human airway epithelial cells (HAEC) harboring the G542X CFTR variant. Cells with positive GFP fluorescence were then concentrated using fluorescence-activated cell sorting to achieve a cell population enriched with the G542R variant. This rendered the population amenable to treatment with modulators, and provides proof-of principle for the use of ABE to correct G542X with potential for re-engraftment. Cells were assessed for functional improvements demonstrated by improved RNA and protein abundance, increased ion transport (measured as short-circuit current), wild-type-like airway surface liquid height and pH, and decreased time required for wound repair.

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