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 coding for type I intermediate filament (IF) protein keratin 9 (K9). The integration of mutant K9 into the IF cytoskeleton has a dominant negative effect resulting in IF fragility and massive thickening of palmoplantar skin. The aim of this project was to correct the IF fragility in EPPK patient-derived primary keratinocyte (KC) cultures by gene editing. We identified edited single cell clones with a premature STOP-codon on the mutated allele. In contrast to cultured bulk patient KCs, which exhibited IF clumping upon stress, gene edited clones showed a stable K9 IF structure after heat stress application similar to wild type (WT) controls. Recently, we identified a family carrying a premature STOP-codon in one K9 allele with very mild symptoms. This supports our in vitro data, which shows that knock-out of a single mutated allele strongly ameliorates symptoms of EPPK. Gene editing, however, poses the risk of unintended small insertions or deletions, gross chromosomal rearrangements as well as translocations. The risk for off-target editing was minimized by employing a double nickase approach, designing a gRNA specific for the mutated allele and electroporating ribonucleoproteins into KCs. We evaluated the safety of our approach by performing a CAST-Seq analysis of treated patient cells, which revealed a high safety profile: only on-target effects but no off-target mediated translocations.

ESDR278 - Gene therapy of an EPPK-causing K9 variant using a CRISPR/Cas9 nickase mediated approach in primary keratinocytes

The scientific meeting of the European Society for Dermatological Research (ESDR) is held yearly, usually in September. With an international attendance of more than 1000 skin scientists from around the globe, the four-day event includes short oral presentations from peer-reviewed abstracts, keynote lectures from international experts, sponsored symposia, satellite programs, educational events as well as an important focus on posters, networking and building collaborations amongst scientists. For more information about this year’s meeting visit our website.