Genome-Edited LCA16 iPSC-RPE Shows Rescue of Kir7.1 Channel

Abstract

Purpose : CRISPR-Cas9 gene-editing technology efficiently targets specific loci within the human genome using sequence-specific guide RNAs. The present study utilized the gene-editing strategy to treat Leber congenital amaurosis (LCA16) caused by the mutations in the KCNJ13 gene that encodes for the inwardly rectifying potassium channel (Kir7.1). Our primary focus is on the use of a combination of viral and synthetic nanoparticle-mediated delivery of the editing machinery.

Methods : Retinal pigmented epithelial cells differentiated from induced pluripotent stem cells (iPSC-RPE) derived from LCA16 patient fibroblasts were utilized. A mutant-allele specific gRNA was designed using bioinformatic CRISPR tools to specifically target c.158G>A; W53X in the KCNJ13. The gRNA efficiency was tested by next generation sequencing (NGS) using a stable cell line carrying the mutation. The synthesized gRNA was cloned to LentiCRISPRV2 plasmid with a Cas9-2A-mCherry reporter gene. Lentiviral particles were generated to transduce the LCA16 iPSC-RPE cells. After 24 hrs of transduction, single stranded DNA donor template tagged with a fluorescent marker (ssODN-ATTO488) was delivered to the cells with either Lipofectamine or synthetic nanoparticles. The cells expressing both mCherry and GFP were subjected to patch-clamp electrophysiology to evaluate the Kir7.1 channel function.

Results : The CRISPR-Cas9 system edits the mutant allele with 5.1% indel frequency. Expression of mCherry in transduced iPSC-RPE cells confirmed the delivery of the editing machinery. Similarly, delivery of the donor sequence by lipofectamine or synthetic nanoparticles was confirmed by visualization of GFP fluorescence. We found that the delivery of ssODN-ATTO488 was significantly higher by synthetic nanoparticles compared to Lipofectamine. Patch-clamp analysis on the cells with dual expression of mCherry and GFP showed normal Kir7.1 current. The inward current was increased five-fold in the presence of rubidium ion which is a crucial feature of the Kir7.1 channel.

Conclusions : We show that the CRISPR-Cas9 genome editor successfully edited W53X mutation in mature RPE cells in culture, which resulted in the functional recovery of Kir7.1 channel. A functional Kir7.1 is essential for ionic homeostasis of the subretinal space, which is perturbed in diseased retina. These functional studies indicate that precise genome editing may be a promising therapeutic approach for inherited retinopathies.

This is a 2020 ARVO Annual Meeting abstract.