Prime editing for Crumbs homologue 1 (CRB1) Inherited Retinal Dystrophies

PROJECT SUMMARY Mutations in Crumbs homologue-1 (CRB1) gene cause severe inherited retinal dystrophies (IRDs). Worldwide ~80,000 CRB1 patients are affected, with a prevalence in the United States of 1 in 86,500. There is no treatment available. Gene augmentation in Crb mouse models has shown mixed results, with successful proof-of-concept (POC) using family member CRB2 but only limited morphological and no functional benefits in addition to adverse effects using CRB1-A. CRB1 proteins localize adjacent to adherens junctions and are essential in maintaining their stability in photoreceptors (PRCs) and Müller glial cells (MGCs). The role of CRB1 in retinal development and disease has been focused on CRB1-A. However, three human retinal CRB1 isoforms exist: CRB1-A, the human specific CRB1-C, and the newly identified CRB1-B. In mice, CRB1-A and CRB1-B operate in different cell types (MGCs and PRCs, respectively). Our long-term goal is to halt the progressive retinal degeneration found in CRB1 IRD patients. Our preliminary data confirm the predominate cell-type-distinct localizations of CRB1-A and CRB1-B in addition to the localization of CRB1-C in human cadaveric retina and induced pluripotent stem cell (iPSC)-derived retinal organoids. Further, the majority of CRB1 mutations affect more than one CRB1 isoform. Consequently, the objective of this grant is to determine an isoform-independent approach to treat CRB1 IRDs. Prime editing is a double-strand break-independent gene editing system that can correct all mutation types. Our central hypothesis is that prime editing is amenable to the correction of CRB1 mutations, allowing us to develop the tools necessary to ascertain its therapeutic efficacy in post-mitotic retinal cells. This hypothesis will be tested by pursuing the following three specific aims. Aim 1 (c.2843>A) and Aim 2 (c.3307G>A) will assess if prime editing is amenable for the installation and correction of CRB1 mutations and define its safety profile by evaluating off-targeting of the most efficient strategies. Further Aim 1 and 2 will characterize phenotypic, histopathological, and molecular changes in the derived retinal organoids. Lastly, in Aim3 we will define if a lentiviral all-in-one or AAV split-intein prime editing strategy is most amenable to perform post-mitotic editing in retinal organoids. Impact: Results of this novel project would create new CRB1 retinal organoid disease models, identify therapeutic outcome measures for CRB1 IRDs, and define the efficiency and safety profile for prime editing tools for the amelioration of CRB1 IRDs. This proposal is innovative, as our approach would correct all CRB1 isoforms affected by a given CRB1 mutation. Excitingly, the successful completion of this project will establish a preclinical pathway for showing POC for CRB1 prime editing therapeutics.