Gene Editing–Mediated Disruption of Epidermolytic Ichthyosis–Associated KRT10 Alleles Restores Filament Stability in Keratinocytes

Abstract

Epidermolytic ichthyosis is a skin fragility disorder caused by dominant-negative mutations in KRT1 or KRT10. No definitive restorative therapies exist that target these genetic faults. Gene editing can be used to efficiently introduce frameshift mutations to inactivate mutant genes. This can be applied to counter the effect of dominantly inherited diseases such as epidermolytic ichthyosis. In this study, we used transcription activatorlike effector nuclease technology, to disrupt disease-causing mutant KRT10 alleles in an ex vivo cellular approach, with the intent of developing a therapy for patients with epidermolytic ichthyosis. A transcription activator-like effector nuclease was designed to specifically target a region of KRT10, upstream of a premature termination codon known to induce a genetic knockout. This proved highly efficient at gene disruption in a patient-derived keratinocyte cell line. In addition, analysis for off-target effects indicated no promiscuous gene editingemediated disruption. Reversion of the keratin intermediate filament fragility phenotype associated with epidermolytic ichthyosis was observed by the immunofluorescence analysis of correctly gene-edited single-cell clones. This was in concurrence with immunofluorescence and ultrastructure analysis of murine xenograft models. The efficiency of this approach was subsequently confirmed in primary patient keratinocytes. Our data demonstrate the feasibility of an ex vivo gene-editing therapy for more than 95.6% of dominant KRT10 mutations.

Context-Dependent Strategies for Enhanced Genome Editing of Genodermatoses

“…However, the associated phenotypes of many mutations may be treated through the implementation of several approaches. The simplest and most efficient genome editing strategies employ EJ-based DSB repair for gene disruption [31][32][33][34] and gene reframing [35][36][37]. As EJ-based strategies do not require a DNA donor template, potentially detrimental integrations and DNA-associated toxicity are avoided.…”

“…Genome editing-mediated disruption of pathogenic alleles is an effective therapeutic strategy for the treatment of dominant-negative skin diseases [32,34]. Gene disruption can be achieved via the generation of a single DSB, followed by error-prone EJ-based repair, to introduce indels into the target gene.…”

“…In many cases, induction of PTCs leads to nonsense-mediated mRNA decay (NMD) [53]. As approximately two thirds of all nuclease-induced indels lead to PTC induction, this approach can result in very high gene disruption efficiencies, without extensive screening [34]. Within genodermatoses, gene disruption is only suitable for the phenotypic correction of heterozygous autosomal dominant disease forms, whereby knockout of a single mutant allele would provide phenotypic alleviation.…”

Ex-vivo-Stammzellgentherapie an der Haut

“…individuelle Patienten entwickelt. Der Einsatz von Nukleasen als Gentherapiewerkzeuge begann in den 1990er-Jahren mit der Entwicklung von genspezifischen Zinkfingernukleasen, die in weiterer Folge in vielen Einsatzgebieten aufgrund der erhöhten Spezifität und Effizienz von TALENs ("transcription activator-like effector nuclease") [1,15,19] verdrängt wurden. Die kürzlich (2012) entdeckten CRISPR ("clustered regularly interspaced short palindromic repeats")/Cas9("CRISPR associated protein 9")-Nukleasen stellen aktuell vielleicht die am vielseitigsten einsetzbaren Gene-editing-Werkzeuge dar.…”

“…Darunter fallen die monogenetischen Hauterkrankungen EB [1,3,10,12,13], epidermolytische Palmoplantarkeratose [14] oder epidermolytische Ichthyose [15]. Gene-Editing mittels Designer-Nukleasen ermöglicht einerseits die Deletion von Genen, in denen eine dominantnegative Mutation den Phänotyp am Patienten verursacht [1,15], aber auch die potenziell spurenlose Reparatur von Mutationen über die sog. homologe Rekombination (HR) [10,12,13].…”