Correction of the most common Cystic Fibrosis mutation (F508del) using prime editing

Background

Cystic fibrosis is primarily caused by the F508del mutation in the CFTR gene, leading to defective chloride ion transport in epithelial cells. Traditional treatments require lifelong administration of small-molecule drugs, which, while beneficial, do not offer a permanent solution. Prime editing (PE) emerges as a promising gene-editing tool capable of precise DNA modifications without inducing double-stranded breaks.

Methodology and Innovations

The researchers implemented a series of six strategic enhancements to the prime editing system to improve the correction efficiency of the F508del mutation:

1. Engineered pegRNAs (epegRNAs): Incorporation of structured RNA motifs at the 3′ end to enhance stability and editing efficiency.

2. PEmax Architecture: Utilization of an optimized prime editor protein to increase editing activity.

3. MLH1dn Co-expression: Transient expression of a dominant-negative mismatch repair protein to reduce the cellular repair mechanisms that counteract editing.

4. Silent Edits: Introduction of synonymous mutations to prevent re-editing and improve editing outcomes.

5. PE6 Variants: Deployment of evolved prime editor proteins with enhanced performance.

6. Dead sgRNAs (dsgRNAs): Use of non-cutting guide RNAs to modulate chromatin structure and increase target site accessibility.

By systematically combining these enhancements, the team achieved significant improvements in editing efficiency across various cell types.

Results

• HEK293T Cells: Correction efficiency increased from less than 0.5% to 11%.

• Immortalized Bronchial Epithelial Cells (16HBEge-F508del): Achieved up to 58% correction efficiency.

• Patient-Derived Airway Epithelial Cells: Attained a 25% correction rate.

Notably, the functional restoration of CFTR ion channels in primary airway cells exceeded 50% of wild-type levels, comparable to the effects of existing combination drug therapies. The optimized PE system also demonstrated minimal off-target effects and a favorable edit-to-indel ratio, surpassing traditional homology-directed repair methods.

Implications

This study underscores the potential of prime editing as a durable, one-time therapeutic strategy for cystic fibrosis. The successful correction of the F508del mutation without introducing double-stranded breaks or requiring DNA templates simplifies the therapeutic approach and reduces potential risks associated with other gene-editing techniques. The methodologies and optimizations detailed in this research provide a valuable framework for correcting other challenging genetic mutations.

References

Sousa AA, Hemez C, Lei L, Traore S, Kulhankova K, Newby GA, Doman JL, Oye K, Pandey S, Karp PH, McCray PB Jr, Liu DR. Systematic optimization of prime editing for the efficient functional correction of CFTR F508del in human airway epithelial cells. Nat Biomed Eng. 2025 Jan;9(1):7-21. doi: 10.1038/s41551-024-01233-3. Epub 2024 Jul 10. PMID: 38987629; PMCID: PMC11754097.