Use of adenine base editing and homology-independent targeted integration strategies to correct the cystic fibrosis causing variant, W1282X

Small molecule drugs known as modulators can treat similar to 90% of people with cystic fibrosis (CF), but do not work for premature termination codon variants such as W1282X (c.3846G>A). Here we evaluated two gene editing strategies, Adenine Base Editing (ABE) to correct W1282X, and Homology -Independent Targeted Integration (HITI) of a CFTR superexon comprising exons 23-27 (SE23-27) to enable expression of a CFTR mRNA without W1282X. In Flp-In-293 cells stably expressing a CFTR expression minigene bearing W1282X, ABE corrected 24% of W1282X alleles, rescued CFTR mRNA from nonsense mediated decay and restored protein expression. However, bystander editing at the adjacent adenine (c.3847A>G), caused an amino acid change (R1283G) that affects CFTR maturation and ablates ion channel activity. In primary human nasal epithelial cells homozygous for W1282X, ABE corrected 27% of alleles, but with a notably lower level of bystander editing, and CFTR channel function was restored to 16% of wild -type levels. Using the HITI approach, correct integration of a SE23-27 in intron 22 of the CFTR locus in 16HBEge W1282X cells was detected in 5.8% of alleles, resulting in 7.8% of CFTR transcripts containing the 5E(23-27) sequence. Analysis of a clonal line homozygous for the HITI-SE23-27 produced full-length mature protein and restored CFTR anion channel activity to 10% of wild -type levels, which could be increased three -fold upon treatment with the triple combination of CF modulators. Overall, these data demonstrate two different editing strategies can successfully correct W1282X, the second most common class I variant, with a concomitant restoration of CFTR function.

Automated summary

In this study, two gene editing strategies were evaluated for correcting W1282X variant in cystic fibrosis. Adenine Base Editing (ABE) successfully corrected W1282X but caused bystander editing at adjacent adenine. Homology-Independent Targeted Integration (HITI) enabled integration of CFTR superexon in CFTR locus, restoring CFTR function.