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 5

Visual function of Kcnj13W53X/+ mice is similar to that of Kcnj13+/+ mice.

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Visual function of Kcnj13W53X/+ mice is similar to that of Kcnj13+/+ mic...
(A and B) Two different sgRNAs targeting the Kcnj13 gene at exon 2 and a ssODN sequence with the desired nucleotide change to generate the Kcnj13W53X/W53X mouse model by CRISPR/Cas9 and HDR genome-editing technique by microinjecting them into the pronuclei of the zygote. Double asterisks indicate postnatal day 1 lethal. (C) RFLP analysis of the Kcnj13 gene from the generated mice digested with Nhe1 enzyme on 2% agarose gel. (D) Chromatograph confirming the mouse genotype. (E) OCT images showing comparison between Kcnj13+/+, Kcnj13W53X/+, and WT allele–disrupted Kcnj13W53X/+ΔR mice. (F) Averaged c wave response confirming WT allele disruption in the RPE of Kcnj13W53X/+ΔR using the targeted guide (T). One-way ANOVA with post hoc Tukey’s HSD test was used for comparisons between the groups. NT, nontargeting sgRNA.