EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University Salzburg, Austria; Institute of Cellular Medicine, Framlington Place, Newcastle University, Newcastle, United Kingdom.
EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University Salzburg, Austria.
J Invest Dermatol. 2019 Aug;139(8):1699-1710.e6. doi: 10.1016/j.jid.2019.03.1146. Epub 2019 Apr 15.
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 activator-like 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 editing-mediated 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.
表皮松解性鱼鳞病是一种皮肤脆弱性疾病，由 KRT1 或 KRT10 的显性负突变引起。目前尚无针对这些遗传缺陷的明确修复疗法。基因编辑可用于有效地引入移码突变以失活突变基因。这可用于抵消表皮松解性鱼鳞病等显性遗传疾病的影响。在这项研究中，我们使用转录激活因子样效应物核酸酶技术，在体外细胞方法中破坏导致疾病的突变 KRT10 等位基因，旨在为表皮松解性鱼鳞病患者开发一种治疗方法。设计了一种转录激活因子样效应物核酸酶，专门针对已知诱导基因敲除的提前终止密码子上游的 KRT10 区域。这在患者来源的角质形成细胞系中非常有效地实现了基因破坏。此外，针对脱靶效应的分析表明，没有混杂的基因编辑介导的破坏。通过对正确基因编辑的单细胞克隆进行免疫荧光分析，观察到与表皮松解性鱼鳞病相关的角蛋白中间丝脆弱表型的逆转。这与小鼠异种移植模型的免疫荧光和超微结构分析一致。随后在原代患者角质形成细胞中证实了该方法的效率。我们的数据表明，针对超过 95.6%的显性 KRT10 突变，体外基因编辑治疗是可行的。
