Abstract
Purpose :
CRISPR/Cas9-mediated genome editing is a promising modality for the treatment of various age-related and inherited retinal disorders. The recently developed prime editor (PE) is the most versatile and safest Cas9-based genome editor. However, there are major challenges to delivering PE in vivo for therapeutic editing of tissues in situ, especially in viral vector form. A major way to solve issues around safety and delivery of PE is through the use of engineered virus-like particles (eVLPs).
Methods :
All animal procedures were approved by the UCI IACUC and conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
Many of the optimizations previously performed on base editing eVLPs did not support efficient PE-eVLP editing. We systematically optimized PE and epegRNA components to achieve a 28- to 57-fold improvement over the base editor architecture with v3 and v3b systems, respectively.
Results :
For the rd12 and rd6 mouse models, whose genetic defects include a point mutation and a small deletion, we screened epegRNAs and ngRNAs to correct the mutations efficiently and precisely. Single subretinal injections of v3b PE3-eVLPs resulted in therapeutically relevant levels of prime editing, with precise editing reaching up to 24.5%, restored protein expression in the RPE, and partially restored visual function. We demonstrated safety of the eVLPs, with minimal indels and no off-target editing detected by CIRCLE-seq analysis.
Conclusions :
We achieved therapeutic in vivo prime editing with our optimized, engineered v3- and v3b-PE-eVLPs. We restored protein expression after a single dose of eVLP administration by subretinal injection and also partially restored visual function. Our optimized PE-eVLP architecture can therefore be quickly adapted for PE in the RPE and potentially other ocular tissues to address genetic diseases previously untargetable by gene therapy, nuclease Cas9, and base editing.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.