Prime Editing Rescues Aberrant Splicing Defect Caused by Pathogenic Variants in PRPH2

Abstract

Purpose : Mutations in the human peripherin-2 gene (PRPH2) gene cause a wide variety of disabling inherited retinal diseases (IRDs). There is no treatment available for patients with PRPH2-mediated IRDs. Here, we evaluate if therapeutic prime editing (PE) could restore aberrant splicing defects caused by the prevalent c.828+3A>T PRPH2 splice site mutation and several rarer c.828 splice site mutations.

Methods : Human iPSC lines were generated from PRPH2 patients with c.828 splice site mutations. iPSC lines from patient and control lines were differentiated to retinal organoids and characterized for aberrant splicing. PRPH2 c.828 splice site mutations were installed in HEK293T cells using PE. These cells were used to facilitate high throughput screening of PE correction strategies. A mutation-specific PE strategy for the prevalent c.828+3A>T PRPH2 mutation was compared to a PAN mutation PE strategy capable of correcting all PRPH2 c.828 splice site mutations. Editing efficiency and restoration of canonical splicing were evaluated as outcome measures. An adeno-associated virus (AAV) split-intein PE delivery strategy was developed to evaluate the most promising combinations of pegRNA and nicking sgRNA.

Results : Previously, aberrant splicing of PRPH2 was found in illegitimate transcripts from peripheral white blood cells of c.828+3A>T PRPH2 patients (Shankar et al. 2015, JAMA Ophthalmology). We confirmed this by evaluating splicing in PRPH2 c.828 splice site patient iPSC-derived retinal organoids and knock-in HEK293T cell lines. We evaluated pegRNA and sgRNA combinations designed to correct the c.828+3A>T PRPH2 mutation with PE. This mutation-specific approach led to editing efficiencies as high as ~50%. Further, we tested a PAN mutation PE strategy capable of correcting all PRPH2 c.828 splice site mutations, which also yielded editing efficiencies as high as ~50%. Lastly, the aberrant splicing defect caused by the PRPH2 c.828 splice site mutations was rescued using a split-intein PE delivery strategy.

Conclusions : We show the first proof of principle for a PAN mutation prime editing approach that can correct the prevalent c.828+3A>T PRPH2 mutation in addition to several rarer c.828 splice site mutations. Additionally, we confirm the previous finding that c.828 splice site mutations cause aberrant splicing and that with PE we can restore canonical splicing with a split-intein PE delivery strategy.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.