Nonviral base editing of KCNJ13 mutation preserves vision in a model of inherited retinal channelopathy
Meha Kabra, Pawan K. Shahi, Yuyuan Wang, Divya Sinha, Allison Spillane, Gregory A. Newby, Shivani Saxena, Yao Tong, Yu Chang, Amr A. Abdeen, Kimberly L. Edwards, Cole O. Theisen, David R. Liu, David M. Gamm, Shaoqin Gong, Krishanu Saha, Bikash R. Pattnaik
Meha Kabra, Pawan K. Shahi, Yuyuan Wang, Divya Sinha, Allison Spillane, Gregory A. Newby, Shivani Saxena, Yao Tong, Yu Chang, Amr A. Abdeen, Kimberly L. Edwards, Cole O. Theisen, David R. Liu, David M. Gamm, Shaoqin Gong, Krishanu Saha, Bikash R. Pattnaik
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Research Article Ophthalmology

Nonviral base editing of KCNJ13 mutation preserves vision in a model of inherited retinal channelopathy

Abstract

Clinical genome editing is emerging for rare disease treatment, but one of the major limitations is the targeting of CRISPR editors’ delivery. We delivered base editors to the retinal pigmented epithelium (RPE) in the mouse eye using silica nanocapsules (SNCs) as a treatment for retinal degeneration. Leber congenital amaurosis type 16 (LCA16) is a rare pediatric blindness caused by point mutations in the KCNJ13 gene, a loss of function inwardly rectifying potassium channel (Kir7.1) in the RPE. SNCs carrying adenine base editor 8e (ABE8e) mRNA and sgRNA precisely and efficiently corrected the KCNJ13W53X/W53X mutation. Editing in both patient fibroblasts (47%) and human induced pluripotent stem cell–derived RPE (LCA16-iPSC-RPE) (17%) showed minimal off-target editing. We detected functional Kir7.1 channels in the edited LCA16-iPSC-RPE. In the LCA16 mouse model (Kcnj13W53X/+ΔR), RPE cells targeted SNC delivery of ABE8e mRNA preserved normal vision, measured by full-field electroretinogram (ERG). Moreover, multifocal ERG confirmed the topographic measure of electrical activity primarily originating from the edited retinal area at the injection site. Preserved retina structure after treatment was established by optical coherence tomography (OCT). This preclinical validation of targeted ion channel functional rescue, a challenge for pharmacological and genomic interventions, reinforced the effectiveness of nonviral genome-editing therapy for rare inherited disorders.

Authors

Meha Kabra, Pawan K. Shahi, Yuyuan Wang, Divya Sinha, Allison Spillane, Gregory A. Newby, Shivani Saxena, Yao Tong, Yu Chang, Amr A. Abdeen, Kimberly L. Edwards, Cole O. Theisen, David R. Liu, David M. Gamm, Shaoqin Gong, Krishanu Saha, Bikash R. Pattnaik

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Figure 4

Evaluation of ABE8e to correct W53X alleles in iPSC-RPEW53X/W53X.

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Evaluation of ABE8e to correct W53X alleles in iPSC-RPEW53X/W53X. (A) Re...
(A) Representative bright-field images of base-editor treated and untreated iPSC-RPEW53X/W53X. Scale bars: 100 μm. (B) Base-editing efficiencies following treatment (BE) with ABE8e mRNA and sgRNA encapsulated in SNC-PEG in iPSC-RPEW53X/W53X as compared with untreated cells. Reads from the untreated and treated cells (n = 3) were categorized into 4 subtypes based on their sequences, unedited, W53*>WT, indels, and substitutions. (C) Reads generated by ABE8e mRNA treatment showing the nucleotide distribution around the cleavage site for sgRNA. Substitutions are highlighted in bold. The scatterplot shows the frequency of alleles observed in treated cells (n = 3). Data are represented as means ± SEM. (D) Manual single-cell patch-clamp assays on iPSC-RPEW53X/W53X cells after treatment with ABE8e. Of the 13 cells assessed for Kir7.1 activity, each could be binned into 1 of 3 classes: low-responding single cells, which appeared to be unedited mutant cells; medium-responding single cells, which showed a low level of Rb+ response; and high-responding single cells, which showed Rb+ response like WT iPSC-RPE cells. The number (n) of cells binned into each class is shown at the top of each graph. (E) Current-sweep plot from a representative cell of each bin across a time course of being exposed to physiological HR solution (gray), Rb+ stimulation (red), and subsequent wash with HR solution (green).