Efficient gene reframing therapy for recessive dystrophic epidermolysis bullosa using CRISPR/Cas9
Efficient gene reframing therapy for recessive dystrophic epidermolysis bullosa using CRISPR/Cas9
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使用 CRISPR/Cas9 有效治疗隐性营养不良性大疱性表皮松解症
DOI：
10.1016/j.jid.2019.02.015
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发表时间：
2019
影响因子：
6.5
通讯作者：
Shimizu Hiroshi
中科院分区：
文献类型：
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作者：
Takashima Shota;Shinkuma Satoru;Fujita Yasuyuki;Nomura Toshifumi;Ujiie Hideyuki;Natsuga Ken;Iwata Hiroaki;Nakamura Hideki;Vorobyev Artem;Abe Riichiro;Shimizu Hiroshi
簇状规则间隔短回文重复序列(CRISPR)/Cas9系统诱导位点特异性双链断裂，通过同源重组或非同源末端连接途径刺激细胞DNA修复。非同源末端连接途径比同源重组更频繁地被激活，容易在双链断裂部位引入小的插入和/或缺失，导致阅读框架的变化。我们假设，非同源末端连接途径适用于由移码突变引起的遗传病，通过恢复阅读框架。隐性营养不良性大疱性表皮松解症是一种由COL7A1基因突变引起的遗传性皮肤病。在这项研究中，我们对COL7A1中一个反复发生的移码突变C.5819delC进行了基因重组治疗，该突变导致了一个提前终止密码子。将针对该突变位点的CRISPR/Cas9基因导入隐性营养不良性大疱性表皮松解症患者成纤维细胞。在对大量基因编辑的成纤维细胞克隆进行基因分型后，我们发现有相当数量的克隆(17/50)恢复了COL7A1的移码。体外三螺旋形成实验表明，重组的COL7具有功能，并正确分布于小鼠的基底膜。我们的数据表明，突变位点特异性非同源末端连接可能是治疗移码突变引起的遗传性疾病的一种高效的基因治疗方法。
The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system induces site-specific double-strand breaks, which stimulate cellular DNA repair through either the homologous recombination or non-homologous end-joining pathways. The non-homologous end-joining pathway, which is activated more frequently than homologous recombination, is prone to introducing small insertions and/or deletions at the double-strand break site, leading to changes in the reading frame. We hypothesized that the non-homologous end-joining pathway is applicable to genetic diseases caused by a frameshift mutation through restoration of the reading frame. Recessive dystrophic epidermolysis bullosa is a hereditary skin disorder caused by mutations inCOL7A1. In this study, we applied gene reframing therapy to a recurrent frameshift mutation, c.5819delC, inCOL7A1, which results in a premature termination codon. CRISPR/Cas9 targeting this specific mutation site was delivered to recessive dystrophic epidermolysis bullosa patient fibroblasts. After genotyping a large collection of gene-edited fibroblast clones, we identified a significant number (17/50) of clones in which the frameshift inCOL7A1was restored. The reframed COL7 was functional, as shown by triple-helix formation assay in vitro, and was correctly distributed in the basement membrane zone in mice. Our data suggest that mutation site-specific non-homologous end-joining might be a highly efficient gene therapy for inherited disorders caused by frameshift mutations.