Prime editing achieves 58% correction of the East Asian hearing-loss-associated GJB2 c.235delC variant in a cell model
Background
Among the many genetic causes of hereditary hearing loss, variants in the GJB2 gene are a prominent cause. GJB2 c.235delC is reported as a common pathogenic variant, particularly in East Asia. In a cell model carrying this variant, the study optimized prime editing and achieved a correction efficiency of 58.05%.
Hearing aids and cochlear implants can support hearing, but they do not repair the underlying genetic defect. This has prompted research into gene-editing approaches that directly correct the faulty sequence. However, sequence correction in cells must be distinguished from a therapeutic effect that restores hearing.
Prime editing (PE) is a technology for precise genetic modification. In this study, the researchers combined guide RNAs, editors, and nicking strategies to improve correction of the variant.
Key findings
The researchers developed 293T-GJB2mut-EGFP, a cellular disease model containing the c.235delC variant, and optimized gene editing with the aim of restoring the normal gene sequence.
First, they adjusted the structure of the prime editing guide RNA (pegRNA). A combination with a 9-base-pair primer binding site (PBS) and a 16-base-pair reverse transcription template (RTT) achieved 37.15% correction efficiency with the PE2 system. Adding a Csy4 or evopreQ1 motif to the pegRNA’s 3′ end produced a similar efficiency of about 37%.
The pegRNAMMD2 strategy, which introduces two synonymous variants, increased correction efficiency to 48.94%. Among seven prime editor variants, PEmax showed the highest activity at 49.14%. Applying the PE3b nicking strategy with a “+1 nick sgRNA” raised correction efficiency to 58.05%. This figure is an experimental result in the cell model; it does not represent the rate of hearing recovery in patients.
The team also developed a dual-AAV split system to address the packaging limit of adeno-associated virus (AAV). They used a split-intein design that divided the PEmax protein between amino acid residues 1153 and 1154. After nicking optimization, its correction efficiency did not differ statistically significantly from that of a system using the existing split site between residues 1024 and 1025. The absence of a statistically significant difference does not, by itself, establish that the two designs are equivalent.
Meaning and outlook
The study presents editing combinations and a split-delivery design for correcting GJB2 c.235delC, a variant common in East Asia. The 58.05% correction efficiency offers a starting point for further research, but it does not show that sufficient efficacy for patient treatment has been achieved.
In an off-target editing analysis, deep sequencing of the four highest-ranked predicted sites found no statistically significant increase in editing above background levels. This limited result from the sites tested does not establish that off-target editing is absent or that overall genotoxic risk is low.
The study was conducted in an in vitro cell model. Whether the same correction activity occurs in the inner ear, and whether in vivo delivery leads to hearing recovery, remain to be tested separately. The provided abstract reports no efficacy results in animals or patients and no long-term safety results. The researchers describe the work as a foundation for future AAV-based in vivo gene therapy studies.
Source: Gene Therapy article abstract and bibliographic information
Hereditary hearing loss, predominantly attributed to mutations in the GJB2 gene, constitutes a significant global health issue, with the c.235delC mutation being the most prevalent pathogenic allele in East Asian populations. Existing interventions, such as hearing aids and cochlear implants, mitigate symptoms but fail to correct the underlying genetic defects, highlighting the need for precise therapeutic approaches. In this study, we developed a cellular disease model (293T-GJB2mut-EGFP) incorporating the c.235delC mutation and employed prime editing (PE) to restore the GJB2 functionality. We systematically optimized the pegRNA architecture and identified that the optimal pegRNA (PBS 9 bp, RTT 16 bp) achieved 37.15% correction with PE2. Incorporating Csy4 or evopreQ1motifs at the pegRNA 3' end resulted in comparable efficiencies (~37%). The introduction of two synonymous mutations (pegRNAMMD2) increased the efficiency to 48.94%. Among the seven prime editor variants, PEmax showed the highest intrinsic activity (49.14%). The PE3b nicking strategy with a + 1 nick sgRNA further improved the efficiency to 58.05%. To address adeno-associated virus (AAV) packaging limitations, a split-intein dual-AAV system for PEmax (bisected between residues 1153-1154) was developed. After nicking optimization, it showed comparable correction efficiency to a previously reported split site (residues 1024-1025), with no statistically significant difference. Off-target deep sequencing of the top four predicted loci revealed no significant editing above the background. These findings establish a comprehensively optimized prime editing toolkit for GJB2 c.235delC correction and provide a foundation for future AAV-mediated in vivo gene therapy for hereditary hearing loss.
The practical value of this study is that it identifies specific editing design candidates for correcting GJB2 c.235delC. Researchers investigating correction of this variant can consider the pegRNA design, the PEmax and PE3b combination, and the split AAV system as candidates for evaluation. The finding that adding motifs to the guide RNA’s end produced similar efficiency may also inform design choices. However, it remains unknown whether the correction rate in the cell model can be reproduced in the inner ear. Future in vivo studies should assess delivery efficiency, editing in target cells, changes in hearing, and safety as distinct outcomes. The current results cannot predict the effect of a particular inner-ear injection method, whether the approach could replace hearing aids or cochlear implants, or whether it would shorten the time needed to develop a therapy.