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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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 3

Evaluation of ABE8e mRNA+sgRNA combinations to correct the W53X allele in LCA16 patient fibroblasts.

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Evaluation of ABE8e mRNA+sgRNA combinations to correct the W53X allele i...
(A) Design of SNCs used to encapsulate ABE8e mRNA and sgRNA. (B) Base-editing efficiencies are shown as the percentages of total DNA sequencing reads, classified as unedited, correctly edited, or incorrectly edited due to bystander A edits, and with indels in treated and untreated (UT) cells. (C) Percentage editing of the target (A6) and bystander (A–9, A–8, A–4, A–2, A14, A17) A to G by ABE8e mRNA as observed in 3 independent experiments. (D) Amino acid conversion at the respective location was generated due to target and bystander edits. The protospacer sequence is underlined, the pathogenic early stop codon is in a purple box, the target A>G edit is marked in orange, and bystander A edits are in green. (E) The sgRNA location is marked by a black line, PAM is marked by a red line, and mutation is in the blue box. All the A bases within the protospacer are numbered 1–20 based on location. The A bases downstream of the protospacer are numbered from –1 to –9, considering +1 as the first base of the protospacer. The top 10 most frequent alleles generated by ABE8e mRNA treatment show the nucleotide distribution around the cleavage site for sgRNA. Substitutions are highlighted in bold, insertions are shown in the red box, and dashes show deletions. The scatterplot shows the frequency of reads observed in treated cells (n = 3 biological replicates). Data from replicates are represented as means ± SEM.