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
View: Text | PDF
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

×

Figure 6

Phenotypic reversal of RPEW53X mice following in vivo ABE8e treatment.

Options: View larger image (or click on image) Download as PowerPoint
Phenotypic reversal of RPEW53X mice following in vivo ABE8e treatment. (...
(A) Kcnj13W53X allele–specific sgRNA. Black arrow represents the sgRNA spacer sequence, the desired base editing site is indicated by an asterisk, and the PAM is shown in yellow. (B) Workflow of in vivo base-editing strategy. (C) RPE florets after SNC-PEG-ATRA packaged ABE8e mRNA, W53X sgRNA, and GFP mRNA or empty SNC-PEG-ATRA/PBS as a mock treatment subretinal delivery. (D) W53X>WT corrected cell percentages observed in Kcnj13W53X/– mice treated with 2 μg or 3 μg of ABE8e. (E) Indel percentages observed in Kcnj13W53X/– mice treated with 2 μg or 3 μg of ABE8e. (F) In vivo experiment time line. Baseline ERG prior to the disruption of the WT allele and after 6 weeks follow-up. ERG prior to injection of the base editor. Recovery monitored for 10 weeks. (G) Representation of the c wave amplitude in Kcnj13W53X/+ mice with retina OCT image. (H) Reduced c wave amplitude in the Kcnj13W53X/+ mice at 6 weeks after disrupting the WT allele with Cas9 protein and WT-specific sgRNA. (I) The c wave and mfERG traces following the injection of base editor with a nontargeting guide (red) and base editor with a targeting guide (green). The faded traces represent comparisons before the disruption of the WT allele (gray) and injection of the base editor (orange). (J) Average c wave amplitude 6 weeks after the disruption of the WT allele (blue) or after the injection of base editor with a nontargeting guide (red) and targeting guide (green). (K) Normalized c wave amplitude in the eyes injected with nontargeting and targeting guides at weeks 2, 6, and 10. One-way ANOVA with post hoc Tukey’s HSD test was used for comparisons between the groups.