CRISPR/Cas9-Mediated Treatment Ameliorates the Phenotype of the Epidermolytic Palmoplantar Keratoderma-like Mouse

Abstract

CRISPR/Cas9 has been confirmed as a distinctly efficient, simple-to-configure, highly specific genome-editing tool that has been used to treat monogenetic disorders. Epidermolytic palmoplantar keratoderma (EPPK) is a common autosomal dominant keratin disease resulting from dominant-negative mutation of the KRT9 gene, and it has no effective therapy. We performed CRISPR/Cas9-mediated treatment on a knockin (KI) transgenic mouse model that carried a small indel heterozygous mutation of Krt9, c.434delAinsGGCT (p.Tyr144delinsTrpLeu), which caused a humanized EPPK-like phenotype. The mutation within exon 1 of Krt9 generated a novel protospacer adjacent motif site, TGG, for Cas9 recognition and cutting. By delivering lentivirus vectors (LVs) encoding single-guide RNAs (sgRNAs) and Cas9 that targeted Krt9 sequence into HeLa cells engineered to constitutively express wild-type and mutant keratin 9 (K9), we found the sgRNA was highly effective in reducing expression of the mutant K9 protein in vitro. We injected the LV into the fore-paws of adult KI-Krt9 mice three times every 8 days and found that the expression of K9 decreased ∼14.6%. The phenotypic mitigation was revealed by restoration of the abnormal differentiation and aberrant proliferation of the epidermis. Our data are the first to show that CRISPR/Cas9 is a potentially powerful therapeutic option for EPPK and other PPK subtypes.

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

“…These aspects and the frequently dominant nature of EI engender the development of gene editingebased therapies. Gene editing has been used to permanently correct dominant Luan et al, 2018) and recessive (Hainzl et al, 2017;Webber et al, 2016;Wu et al, 2017) epidermolysis bullosaerelated mutations via knockout and homologous recombination.…”

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.…”

“…Luan et al [33] showed first in vivo data for nuclease-mediated knockout in skin cells in 2018. Correction of a dominant-negative mutation within KRT9, causing epidermolytic palmoplantar keratoderma (EPPK), was achieved using a mutation-specific CRISPR/Cas9-based strategy.…”

“…Following three injections, K9 expression was decreased by approximately 14.6%. The authors also assumed a visible restoration of the abnormal epidermal differentiation and proliferation [33].…”

Ex-vivo-Stammzellgentherapie an der Haut

“…TALENs sowie die CRISPR/Cas9-Nukleasen wurden bereits für therapeutische Ansätze für ausgewählte Genodermatosen als Geneediting-Werkzeuge ausgewählt. 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.…”