Editing the c.3846G>A (p.Trp1282*) mutation in the CFTR gene in iPSCs using adenine editor

Cystic Fibrosis (CF) is an autosomal recessive disease caused by mutations in the CFTR gene, leading to an imbalance of chloride and sodium ions in the epithelial cells of various organs. Nonsense mutations in the CFTR gene are present in 10% of CF patients, with the most common being c.3846G>A (p.Trp1282*, W1282X). Current CFTR-modulator therapy is ineffective against this class of mutations, leaving patients with nonsense mutations in the CFTR gene without effective treatment. Etiotropic therapy for CF can be developed based on the latest genome editing methods, such as base editors, which allow precise changes to nucleotides in the genome. Adenine base editors allow for targeted correction of nonsense mutations. The aim of this study was to evaluate the effectiveness of correcting the c.3846G>A mutation in induced pluripotent stem cells (iPSCs) from a CF patient using an adenine base editor. The xCas9(3.7)-ABE(7.10) editor was used in combination with a guide RNA (in a separate B52-1282 plasmid) to convert c.3846A>G. Plasmids were transfected into iPSCs with the F508del/c.3846G>A genotype in the CFTR gene using electroporation. The effectiveness of correction was evaluated 48 hours later using deep targeted sequencing. The results showed a conversion frequency of 10.9% of c.3846A>G alleles, with no increase in unwanted changes (indels) in the editing locus compared to the untransfected control. Therefore, this study demonstrates that the adenine base editor xCas9(3.7)-ABE(7.10) allows for correction of the c.3846G>A mutation in the CFTR gene in 10.9% of alleles in iPSCs from a CF patient without introducing additional mutations in the editing locus.

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