Nanoparticle mediated in vitro and in vivo CRISPR base correction of LCA16 causing nonsense mutation rescues Kir7.1 channel function

Abstract: Lebers Congenital Amaurosis (LCA16) is caused by point mutations in KCNJ13 gene, which encodes for an inward-rectifying potassium channel, Kir7.1. A nonsense mutation, W53X (c.158G>A), leads to premature truncation of protein which makes retinal pigmented epithelial (RPE) non-functional. Molecular mechanism studies revealed a compromised membrane potential, altered subretinal ionic homeostasis, and loss in phagocytic activity caused autosomal recessive childhood blindness. Since all reported mutations in KCNJ13 gene are single base change, CRISPR base editing offers a potential means to correct gene function endogenously and restores the channel function permanently. Further, base editing is free of double-stranded breaks and homology-directed repair (HDR) to generate minimal to no off-targets and indels. Here, we report the silica-nanoparticle (SNP) mediated delivery of an Adenosine CRISPR base editor (ABE8e) mRNA and single-guide RNA for the correction of KCNJ13-W53X (c.158G>A) mutation in an induced pluripotent stem cell-derived (iPSC)-RPE model of LCA16. We demonstrated that, unlike CRISPR-Cas9 mediated gene editing, base editing by ABE8e can efficiently and precisely correct the gene mutation in post-mitotic cells like RPE. We observed a higher editing efficiency in LCA16-patient-derived fibroblasts (47.38% +/- 1.02) than iPSC-RPE (16.90% +/- 1.58) with no detectable off-target activity at the predicted sites. The SNP-mediated ABE8e delivery and correction of W53X mutation in iPSC-RPE restored the Kir7.1 channel activity with no noticeable toxicity to cells. Restoration of channel function in the edited cells was comparable to a wild-type phenotype, which is a prime requisite for vision restoration in LCA16 patients. Subretinal injections of SNPs decorated with all-trans retinoic acid (ATRA) ligand with a payload of ABE8e and mouse-specific sgRNA in LCA16 mice (with no ERG phenotype) showed specific delivery only to RPE. We observed marginal recovery of ERG generated from the 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 SNP-mediated delivery of CRISPR base editors.