Retinitis pigmentosa (RP), a leading cause of progressive vision loss, affects over one million people worldwide. Pathogenic variants in the USH2A gene are common in both autosomal recessive and syndromic RP forms. The deep intronic variant USH2A:c.7595-2144A>G, associated with Usher syndrome (RP combined with hearing loss), disrupts mRNA splicing, resulting in defective USH2A protein production.

As a safe therapeutic approach for splicing modulation in this and other genes, we developed the Enhanced Deletion Splicing Correction Editing (EDSpliCE) platform, utilizing Engineered Enhanced Deletion synthetic RNA-Guided Nucleases (EDsRGNs). These small chimeric proteins combine the synthetic RNA-guided endonuclease sRGN3.1 with the human exonuclease TREX2. EDsRGNs create larger deletions at target sites with a single gRNA. While disrupting the disease-related sequence, this nuclease engineering aims to incorporate crucial safety features.

Using minigene splicing rescue assays in HEK293T cells, up to 90% correct USH2A splicing was achieved. Delivery via adeno-associated viruses (AAVs) confirmed efficient transduction and splicing rescue. Interchromosomal junction PCR showed nearly undetectable chromosomal translocations with EDsRGNs as opposed to sRGN3.1. Next-generation sequencing (NGS) revealed that EDsRGNs induced enhanced deletions, predominantly in one direction of the cut site. γH2AX immunofluorescence demonstrated the transient occurrence of DSBs induced by EDsRGNs. GUIDE-Seq analysis showed low off-target editing activity.

In sum, EDSpliCE successfully rescued USH2A splicing in vitro while showing improved safety attributes. EDsRGNs are compatible with all-in-one AAVs, enabling efficient delivery into various cell types. Ongoing work includes optimizing AAV vectors, validating off-targets, and protein rescue experiments in patient-derived photoreceptor precursor cells and retinal organoids expressing 3xFLAG-tagged endogenous USH2A.