159 CRISPR/Cas9 nickase mediated gene therapy in primary keratinocytes derived from patients with EPPK

Abstract

Epidermolytic palmoplantar keratoderma (EPPK) is a rare, autosomal dominantly inherited skin disorder. The disease is often caused by heterozygous point mutations in the keratin 9 gene (KRT9) coding for the type I intermediate filament (IF) protein keratin 9 (K9), whose expression is confined to the suprabasal layers of palmoplantar epidermis. Integration of mutant K9 into the IF cytoskeleton has a dominant negative effect resulting in massive thickening and making it prone to develop fissures, infections, and severely impairing the flexibility of hands and feet. The aim of this project is to correct the IF fragility in EPPK patient-derived primary keratinocyte (KC) cultures by establishing a CRISPR/Cas9 D10A double-nicking approach to induce non-homologous end joining (NHEJ) mediated silencing of the mutant disease-causing allele while leaving the wild-type (WT) allele intact. To avoid the risk of integrations into the genome, we used ribonucleoproteins to transfect KCs. We show that mutant K9 massively reduces cytoskeleton stability, resulting in IF clumping upon stress. Moreover, we demonstrate successful targeting and NHEJ of KRT9 using a pair of gRNAs, one of which targets the mutation in an allele specific manner. Using sequencing, we identified single cell clones with a premature STOP-codon on the mutated allele. These clones show a stable K9 IF structure after stress application, similar to the WT controls. Taken together, these results demonstrate CRISPR/Cas9 nickase-mediated rescue of IF fragility due to mutant K9 in primary cultured KCs.