Efficient in vivo gene editing using ribonucleoproteins in skin stem cells of recessive dystrophic epidermolysis bullosa mouse model
Efficient in vivo gene editing using ribonucleoproteins in skin stem cells of recessive dystrophic epidermolysis bullosa mouse model
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DOI：
10.1073/pnas.1614775114
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发表时间：
2017-02-14
影响因子：
11.1
通讯作者：
Chen, Ting
中科院分区：
文献类型：
--
作者：
Wu, Wenbo;Lu, Zhiwei;Chen, Ting
原核CRISPR/Cas9系统最近成为哺乳动物细胞基因组编辑的强大工具，有可能为遗传疾病患者带来治愈性治疗。然而，这种基因组编辑机制的有效体内递送以及实际上在体内使用这些技术的可行性对于大多数组织类型仍然具有挑战性。在这里，我们表明，不可复制的Cas9/sgRNA核糖核蛋白可用于纠正出生后隐性营养不良性大疱性表皮细胞（RDEB）小鼠的皮肤干细胞中的遗传缺陷。我们开发了一种将Cas9/sgRNA核糖核蛋白局部递送到出生后小鼠皮肤中的方法。这种方法导致表皮干细胞中的快速基因编辑。使用这种方法，我们表明Cas9/sgRNA核糖核蛋白有效地切除了外显子80，其覆盖了我们的RDEB小鼠模型中的点突变，从而恢复了胶原VII蛋白在体内的正确定位。治疗后皮肤起泡表型也显着改善。这项研究提供了一种体内基因校正策略，使用核糖核蛋白作为皮肤和其他体组织中遗传性疾病的治愈性治疗。
The prokaryotic CRISPR/Cas9 system has recently emerged as a powerful tool for genome editing in mammalian cells with the potential to bring curative therapies to patients with genetic diseases. However, efficient in vivo delivery of this genome editing machinery and indeed the very feasibility of using these techniques in vivo remain challenging for most tissue types. Here, we show that non-replicable Cas9/sgRNA ribonucleoproteins can be used to correct genetic defects in skin stem cells of postnatal recessive dystrophic epidermolysis bullosa (RDEB) mice. We developed a method to locally deliver Cas9/sgRNA ribonucleoproteins into the skin of postnatalmice. This method results in rapid gene editing in epidermal stem cells. Using this method, we show that Cas9/sgRNA ribonucleoproteins efficiently excise exon80, which covers the point mutation in our RDEB mousemodel, and thus restores the correct localization of the collagen VII protein in vivo. The skin blistering phenotype is also significantly ameliorated after treatment. This study provides an in vivo gene correction strategy using ribonucleoproteins as curative treatment for genetic diseases in skin and potentially in other somatic tissues.