CRISPR base editing as a potential therapeutic approach for Kir7.1 channelopathy

Abstract

Purpose : Leber Congenital Amaurosis 16 (LCA16) is a severe form of inherited ocular channelopathy caused by point mutations in KCNJ13, which affect the retinal pigment epithelial (RPE). AAV-gene therapy related immune responses, CRISPR/Cas9 gene editing associated off-targets, and unintended indels pose some challenges in clinical use of such treatments. We used Adenosine and Cytosine CRISPR base editors (ABE and CBE) for proof-of-concept correction of KCNJ13 point mutations (c.158G>A [p.W53X] and c.431T> C [p.L144P]) using nanoparticle-mediated delivery to induced pluripotent stem cell-derived RPE (iPSC-RPE).

Methods : Base editing was carried out using either ABE or CBE mRNA with a guide RNAs specific to the W53X or L144P mutant allele. A HEK293-FRT stable cell line expressing Kir7.1-L144P and Kir7.1- W53X were base edited via electroporation using CBE and ABE mRNA respectively. LCA16-W53X patient-specific fibroblasts and iPS-RPE cells were also targeted with ABE mRNA delivered using nanoparticles. Base editing efficiency, protein expression, localization, and channel function were assessed in edited cells and were compared with non-edited mutant and WT cells. Potential off-targets were screened to evaluate the accuracy and efficacy of CRISPR BEs.

Results : CBE mRNA for L144P correction in HEK cells resulted in 66% editing. The degenerate nature of codons for provides additional flexibility to correct L144P point mutation. Higher rate (~24%) were observed in treated L144P-cells due to multiple bystander cytosines at the targeted locus.

ABE mRNA for W53X (TaG>TgG) correction in HEK stable cells showed 50% editing efficiency than RNP approaches (25% efficiency). Nanoparticle-mediated delivery of ABE-mRNA and sgRNA in fibroblasts (47% editing) and iPSC-RPE (20% editing) established its use for in vivo BE delivery. Control fibroblast cells lacking the mutant allele showed about 1% indels. On target indel mutagenesis (<3%) and deep sequencing of potential off-target sites (<1%) indicated high accuracy of the ABEs. Electrophysiology assays demonstrated robust rescue of channel function in the edited iPSC-RPE cells.

Conclusions : Our results show application of CRISPR base editing for precise correction of point mutations with reduced off-targets compared to CRISPR/Cas9-mediated gene editing. Restoration of channel function in edited iPSC-RPE cells suggests potential of CRISPR BE as a treatment for childhood blindness.

This is a 2021 ARVO Annual Meeting abstract.