Title: Nonviral nanocapsule-mediated CRISPR base editing rescues ocular Kir7.1 channelopathy
Legend: Silica nanoparticle encapsulated delivery of CRISPR base editor ABE8e mRNA to correct disease mutation of KCNJ13 gene – Schematic representation of the W53X mutation loci in the KCNJ13 exon-2. We report high editing efficiency and negligible on-target indels and substitutions in base-edited patient-derived fibroblasts, iPSC RPE, and in vivo mouse RPE. Patch-clamp electrophysiology results showed restored Kir7.1 channel function in base-edited iPSC RPE. In base-edited mice, RPE function was detected by the presence of light-induced electroretinogram (ERG) c-wave.
Citation: Meha Kabra, Pawan K. Shahi, Yuyuan Wang, Divya Sinha, Allison Spillane, Gregory A. Newby, Shivani Saxena, Amr A. Abdeen, Kimberly L. Edwards, Cole O. Theisen, David M. Gamm, David R. Liu, Shaoqin Gong, Krishanu Saha, Bikash R. Pattnaik. (2022). Nanoparticle mediated CRISPR base editing rescues Kir7.1 function relevant to ocular channelopathy. bioRxiv 2022.07.12.499808; doi: https://doi.org/10.1101/2022.07.12.499808
Abstract: Leber 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.
About the Lab: The Saha Lab is affiliated with several multi-disciplinary centers including the Waisman Center, Wisconsin Institute for Discovery and the Stem Cell and Regenerative Medicine Center at UW-Madison. Our research dedicated to using human stem cells together with emerging engineering methods in material science and synthetic biology to make smarter therapeutics, model human disease, and advance personalized medicine. We are collaborative efforts on campus in biomanufacturing cell and gene therapies, and nationally with the NIH Somatic Cell Genome Editing program and the NSF Center for Cell Manufacturing Technologies.
Investigator: Krishanu Saha, PhD