Functional correction of the untreatable CFTR 1717-1G>A mutation through mRNA- and sgRNA-optimized base editing

Area of Science:

• Genetics
• Molecular Biology
• Gene Therapy

Background:

• Cystic Fibrosis (CF) is caused by mutations in the CFTR gene.
• The prevalent 1717-1G>A mutation disrupts CFTR splicing, leading to severe disease.
• Current CF treatments are primarily palliative, lacking a genetic cure.

Purpose of the Study:

• To develop and optimize an adenine base editing (ABE) strategy for correcting the CFTR 1717-1G>A mutation.
• To assess the efficacy and safety of the ABE system in relevant cellular models and patient-derived tissues.
• To evaluate the functional restoration of CFTR channel activity post-correction.

Main Methods:

• Utilized the ABE9 base editor with a relaxed SpCas9 variant (SpRY).
• Delivered base editor mRNA and sgRNA via electroporation into HEK293 cells, CF airway epithelial cells, and intestinal organoids.
• Assessed editing efficiency, bystander effects, and CFTR function using short-circuit current measurements and forskolin-induced swelling assays.

Main Results:

• Achieved up to 30% editing efficiency with limited bystander effects in a cellular model.
• Demonstrated successful genetic repair of the 1717-1G>A mutation in patient-derived airway epithelial cells and intestinal organoids.
• Observed restoration of CFTR channel activity, evidenced by normalized short-circuit current and forskolin-induced swelling.

Conclusions:

• SpRY-ABE9 is a promising genome editing tool for permanently correcting the CFTR 1717-1G>A mutation.
• This strategy holds potential for developing a curative therapy for CF patients with this specific mutation.
• The study validates ABE as a viable approach for addressing genetic defects in CFTR.