Nanoparticle mediated CRISPR base editing rescues Kir7.1 function relevant to ocular channelopathy

Abstract

AbstractLeber Congenital Amaurosis (LCA16) is a progressive vision loss disorder caused by point mutations in the KCNJ13 gene, which encodes an inward-rectifying potassium channel, Kir7.1. A nonsense mutation, W53X (c.158G>A), leads to premature truncation of the protein, which disrupts K+ conductance and makes retinal pigmented epithelium (RPE) non-functional. While there are no current treatments for LCA16, here we explore CRISPR base editing as a strategy to correct the single base pair pathogenic variant. We show that silica nanoparticle (SNC)-mediated delivery of an adenine base editor (ABE8e) mRNA and single-guide RNA can precisely and efficiently correct the KCNJ13W53X mutation to restore Kir7.1 channel function in an induced pluripotent stem cell-derived RPE (iPSC-RPE) model of LCA16, as well as in patient-derived fibroblasts and in a new LCA16 mouse model. We observed a higher editing efficiency in LCA16 patient-derived fibroblasts (47.38% ± 1.02) than in iPSC-RPE (16.90% ± 1.58) with no detectable off-target editing. The restored channel function in the edited cells resulted in a phenotype comparable to wild-type cells, suggesting the possibility that this treatment could restore vision in LCA16 patients. Injection of ABE8e-carrying SNCs decorated with all-trans retinoic acid (ATRA) ligand into the subretinal space in LCA16 mice showed specific delivery only to RPE and resulted in gene correction in approximately 10% of RPE cells that received the editing machinery. We observed marginal recovery of electroretinogram (ERG) following correction of the W53X allele at the injection site with no further degeneration of RPE. These findings provide a foundation and a proof-of-concept to transition from bench to bedside and support its further development for treating pediatric blindness using a safer mode of SNC-mediated delivery of base editors.