Development of a Prime Editing Strategy to Treat Mutations in the Crumbs Homologue 1 (CRB1) Gene

Abstract

Purpose : Mutations in Crumbs homologue 1 (CRB1) cause chronic and disabling autosomal recessive inherited retinal diseases. Currently, there are ~80,000 CRB1 patients worldwide with no available treatment to date. The retina contains three major CRB1 isoforms: CRB1-A, CRB1-B and CRB1-C. CRB1-A and CRB1-B have predominately cell-type specific expressions, making a gene augmentation approach more challenging. Prime editing (PE) is capable of installing all types of edits, including transitions, transversions, small deletions and insertions. In this study, we tested the efficacy of PE for the correction of CRB1 mutations in patient induced pluripotent stem cell (iPSC) lines.

Methods : To begin optimizing PE for correction of CRB1 mutations, 30 combinations of pegRNA and nicking sgRNA design were tested per mutation on the corresponding CRB1 patient iPSC line using nucleofection or Stem Cell lipofectamine. Combinations showing efficient editing were taken forward for further optimization. Editing efficiency of different PE designs were evaluated by NGS or Sanger sequencing followed by ICE analysis. Currently, we are adapting this PE strategy to a viral delivery system in which we have optimized promoters to highly express PE machinery in postmitotic cells of iPSC-derived retinal organoids. Retinal organoids were differentiated using a previously published protocol (Tso et al., 2022).

Results : Based on our analysis of the CRB1 LOVD database and our cohort of CRB1 patient iPSC lines we chose to initially develop prime editing for p.(Cys948Tyr) (Most prevalent CRB1 mutation) and p.(Gly1103Arg) (within the top 10 most prevalent CRB1 mutations) mutations. We found that CRB1 patient iPSC lines were amenable to prime editing. Editing efficiencies were as high as 72%, dependent on the combination of pegRNA and nicking sgRNA used. This editing methodology is now being modified to a viral system for the transduction of photoreceptor and Müller glial cells in patient iPSC-derived retinal organoids.

Conclusions : Prime editing is amenable for the correction of CRB1 patient mutations and achieves high editing efficiencies. Further, we have developed a viral system, which we are adapting for our PE strategy. Together this work provides a platform for the testing of CRB1 therapeutic editing in CRB1 patient iPSC-derived retinal organoid disease models.

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