Nonviral base editing of KCNJ13 mutation preserves vision in a model of inherited retinal channelopathy
Meha Kabra, … , Krishanu Saha, Bikash R. Pattnaik
Meha Kabra, … , Krishanu Saha, Bikash R. Pattnaik
Published August 10, 2023
Citation Information: J Clin Invest. 2023;133(19):e171356. https://doi.org/10.1172/JCI171356.
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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 1

Evaluation of ABE8e RNP and ABE8e mRNA to correct hKCNJ13W53X/W53X allele in HEK293 FRT stable cells.

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Evaluation of ABE8e RNP and ABE8e mRNA to correct hKCNJ13W53X/W53X allel...
(A) Construct design to generate HEK293 FRT stable cells harboring the KCNJ13 W53X allele. (B) Chromatogram generated from HEK293 FRT stable cells showing the W53X codon marked in the red box and the downward black arrow indicating the specific nucleotide change (G>A).(C) Schematic of the hKCNJ13 locus highlighting the mutation c.158G>A (blue box marked with asterisk) and position of the W53X targeting sgRNA (black line) with TGG PAM (red line). (D) Base-editing efficiencies are shown as the percentages of sequencing reads with the corrected WT allele (and no other silent changes, bystander edits, or indels) in HEK293W53X cells following electroporation of ABE8e protein+sgRNA (RNP) or ABE8e mRNA+sgRNA (n = 3). Markers (diamonds) represent the individual biological replicates (n = 3), and error bars represent SEM by 2-tailed Student’s t test. (E) Percentages of sequencing reads with indels in ABE8e RNP– and ABE8e mRNA–treated stable cells (n = 3). Markers (diamonds) represent the individual biological replicates (n = 3), and error bars represent SEM by 2-tailed Student’s t test. (F) Kir7.1 expression in ABE8e mRNA–treated cells assessed by immunocytochemistry. GFP primary antibody was used to enhance the endogenous signal. DAPI was used to stain the nucleus. Scale bars: 50 μm. White arrows mark membrane localization in cells.