O18 Precision genome editing for targeted correction of pathogenic D50N mutation in keratitis–ichthyosis–deafness syndrome using CRISPR/Cas9 and homology-directed repair

Abstract Introduction and aims Keratitis–ichthyosis–deafness (KID) syndrome is an ectodermal disorder that causes blindness, skin inflammation and deafness. It is caused by autosomal dominant mutations in the gap junction beta 2 (GJB2) gene with 86% being a hotspot mutation on c.148G>A, resulting in the amino acid replacement (D50N) in its coding protein connexin 26 (Cx26). There is currently no curative treatment for KID syndrome. We aim to correct the hotspot mutation D50N using a genome-editing approach. Methods Keratinocytes generated from the patient with KID who had a D50N mutation (KID-KCs) were genetically corrected by homologous-directed repair/CRISPR-Cas9 using an electroporation approach. The correction efficiency was determined and analysed using Sanger sequencing and Synthego Inference of CRISPR Edits analysis. The off-target effects were confirmed by whole-exome sequencing [exome next-generation sequencing (NGS)]. Furthermore, the functional recovery following genome editing was assessed by GJB2 mRNA production, Cx26 protein expression, and trafficking and hemichannel activity. Results The correction efficiencies ranged from 95% to 100% with different doses of CRISPR-Cas9 and 8µg CRISPR-Cas9 was the most optimal concentration with the efficiency of 100% and approximately 0% insertion-deletion (INDEL). Fifty days after gene editing, the INDEL increased to only 2%, indicating a minimum residual CRISPR-Cas9 effect in edited cells. The exome-NGS analysis also showed no off-target effects in the outside target area. Functional studies showed that there was a significant increase in wildtype GJB2 mRNA expression and a reduction of mutant GJB2 expression (n = 5, P < 0.01). Increased Cx26 membranous localization in KID-KCs was also observed (n = 3, P < 0.01). Additionally, Cx26 hemichannel activity assessment using the neurobiotin assay showed a significant decrease in ‘leaky’ hemichannel in gene-edited KID-KCs compared with unedited cells (n = 3, P < 0.05). Conclusions We can genome-correct KID keratinocytes harbouring the mutation D50N with 100% editing efficiency, 0–2% INDEL, and undetectable off-target effects. This result indicates a promising gene therapy for KID syndrome with the hotspot mutation D50N.