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

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.


Inefficient repair of W53X in patient iPSC-RPE W53X using CRISPR-Cas9 nuclease
We first assessed the correction of the homozygous KCNJ13 W53X mutation in iPSC-derived RPE (iPSC-RPE W53X/W53X ) using Cas9 nuclease-mediated HDR.We delivered Cas9 nuclease and sgRNA via the lentiviral vector, LentiCRISPRv2-mCherry (Supplementary Figure 1), and the HDR donor template (ssODN-ATTO488) via SNC.The size and zeta-potential of donor templated-encapsulated SNC were summarized in Supplementary Table 1.We confirmed the delivery of both constructs using their cognate fluorescent reporters (Supplementary Figure 2A).Deep sequencing analysis of the treated samples indicated that most of the indels (5.05 ± 1.24%) were created downstream of the pathogenic mutation (TAG), resulting in no change to the reading frame (Supplementary Figure 2, B and C).Only a small fraction of reads showed in-frame indel formation (1.03 ± 0.52%) and a corrected wildtype (WT) genotype (0.34%) (Supplementary Figure 2D), both of which are predicted to remove (i.e., repair) the W53X stop codon during translation of the edited KCNJ13 mutant allele.Although the green fluorescence in the treated cells showed a successful delivery of ssODN, we did not observe the inclusion of any silent nucleotide bases from the ssODN (Supplementary Figure 2B).Most reads (94.50 ± 1.25%) were unedited in the treated cells.
Next, we measured the Kir7.1 channel function in gene-edited cells expressing the red (mCherry) and green (ATTO488) fluorescence, indicating that they had received both Cas9-sgRNA and ssODN.
Single-cell patch-clamp recordings revealed a normal Kir7.1 current with an inward current of -101.1 ± 35.54 pA at -150 mV in treated cells.Substitution of extracellular Na + with rubidium (Rb + ), a known activator of Kir7.1, increased the Kir7.1 inward current by 7-fold to -713.5 ± 92.97 pA (Supplementary Figure 2, E-G).These results indicate that delivery of both Cas9-sgRNA and ssODN can sometimes generate function-restoring gene edits.However, the exact nature and frequency of editing outcomes in the single cells we recorded by patch clamp could not be assayed due to the technical challenges of amplifying genomic DNA from a single cell.
Altogether, this nuclease-mediated HDR approach did result in a very small frequency of functional RPE cells-indicating that gene correction is a viable strategy to rescue Kir7.1 function in RPE cells.
However, the low efficiency and high heterogeneity of the edits diminished the translational potential of this strategy.The sgRNAs were designed using PnB Designer available at https://fgcz-shiny.uzh.ch/PnBDesigner/.Letters highlighted in red are the targeted nucleotide base using the protospacer and BE.
FP: Forward primer, RP: Reverse primer.Primers for in-fusion cloning were designed using the Gibson assembly primer design tool available at https://tools.sgidna.com/gibson-assembly-primers.html and ordered from IDT (https://www.idtdna.com).The homology sequence is in uppercase, and the annealing sequence is in lowercase.The primers for Sanger sequencing (GFP FP and Kir7.1 RP) were designed using the NCBI Primer-BLAST tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/).

Table 8 : sgRNA design using CRISPR-RGEN and PnB Designer
The sgRNAs were designed using CRISPR-RGEN tool available at http://www.rgenome.net.The letters highlighted in red are the targeted nucleotide base using the protospacer.Amino acids listed in blue would create a missense mutation, while those in green would produce the desired amino acid change to make a WT Kir7.1 protein.