Background

Hereditary hearing loss is a major global health challenge. Among the various genetic factors, mutations in the GJB2 gene are a leading cause. In East Asia, the GJB2 c.235delC mutation is frequently found in patients. This mutation, a single base deletion, disrupts the function of a protein essential for signal transduction between inner ear cells. Currently, hearing aids or cochlear implants are commonly used. These devices provide some hearing improvement but do not correct the underlying genetic defect. Patients must wear and maintain these devices for life, and maintenance issues are also a concern. Therefore, gene therapy, which directly corrects the defective sequence and restores cell function, has emerged as an alternative. Prime editing (PE), a technology that enables precise gene editing, is a powerful tool to address this problem. This technology can correct base sequences without cutting DNA, making it safer than conventional gene editing tools. However, for clinical application, high correction efficiency optimized for the specific mutation and an in vivo delivery system are required.

Key Findings

The researchers developed a cellular disease model (293T-GJB2mut-EGFP) containing the c.235delC mutation and conducted experiments to optimize gene editing for restoring the normal gene sequence. First, the design of the prime editor guide RNA (pegRNA) was adjusted. The analysis revealed that a combination of 9 base pairs in the Primer Binding Site (PBS) and 16 base pairs in the Reverse Transcription Template (RTT) achieved a correction efficiency of 37.15% with the PE2 system. Subsequently, to enhance the stability of the guide RNA, a Csy4 recognition sequence or an evopreQ1 RNA motif was added to the 3' end of the pegRNA. However, even with these improvements, the correction efficiency remained at a similar level of 37%. Therefore, the researchers employed a pegRNAMMD2 strategy, adding two synonymous mutations to the target site, which increased the correction efficiency to 48.94%. In addition, seven prime editor variants were evaluated, and the PEmax variant, which showed the highest activity (49.14% efficiency), was selected. Finally, by combining this with the PE3b nicking strategy, which cleaves the non-target DNA strand, the final correction efficiency was increased to 58.05%. To enable in vivo delivery, a dual-AAV system was also constructed. The large prime editor enzyme has physical limitations that make it difficult to load into a single adeno-associated virus (AAV). The researchers designed an alternative approach by dividing the PEmax protein between amino acid residues 1153 and 1154 and loading it into two AAV vectors. After nicking optimization, this split system showed excellent activity with no statistically significant difference compared to the previously reported split site (residues 1024-1025).

Significance and Prospects

This prime editing system is significant because it provides a concrete therapeutic framework for correcting the GJB2 c.235delC mutation, which is common in East Asian hearing loss patients. The high correction efficiency of over 58% suggests sufficient efficacy for clinical trials in actual patients. Positive indicators were also obtained in terms of safety. Computer analysis predicted the top four gene loci with the highest probability of off-target mutations, and deep sequencing was performed. As a result, no off-target editing above the background level was observed. This indicates a low risk of inducing genotoxicity by modifying bases at unintended sites. However, this study was conducted in vitro, so it remains to be verified whether the same correction activity will be exhibited in the complex environment of inner ear cells. In the future, in vivo efficacy should be verified in animal models of genetic hearing loss, and the immune response after long-term administration should be evaluated. In addition, optimization of the delivery vector injection route should be performed in parallel to enable application to actual patients.