Recessive dystrophic epidermolysis bullosa (RDEB) is a severe disorder caused by mutations to the COL7A1 gene that deactivate production of a structural protein essential for skin integrity. Haematopoietic cell transplantation can ameliorate some of the symptoms; however, significant side effects from the allogeneic transplant procedure can occur and unresponsive areas of blistering persist. Therefore, we employed genome editing in patient-derived cells to create an autologous platform for multilineage engineering of therapeutic cell types. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system facilitated correction of an RDEB-causing COL7A1 mutation in primary fibroblasts that were then used to derive induced pluripotent stem cells (iPSCs). The resulting iPSCs were subsequently re-differentiated into keratinocytes, mesenchymal stem cells (MSCs) and haematopoietic progenitor cells using defined differentiation strategies. Gene-corrected keratinocytes exhibited characteristic epithelial morphology and expressed keratinocyte-specific genes and transcription factors. iPSC-derived MSCs exhibited a spindle morphology and expression of CD73, CD90 and CD105 with the ability to undergo adipogenic, chondrogenic and osteogenic differentiation in vitro in a manner indistinguishable from bone marrow-derived MSCs. Finally, we used a vascular induction strategy to generate potent definitive haematopoietic progenitors capable of multilineage differentiation in methylcellulose-based assays. In totality, we have shown that CRISPR/Cas9 is an adaptable gene-editing strategy that can be coupled with iPSC technology to produce multiple gene-corrected autologous cell types with therapeutic potential for RDEB.

中文翻译：

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基于CRISPR / Cas9的遗传学纠正大隐性营养不良性表皮松解症。

隐性营养不良性大疱性表皮松解症（RDEB）是一种严重的疾病，由COL7A1基因突变引起，该突变使皮肤完整性所必需的结构蛋白的生产失活。造血细胞移植可以缓解某些症状。然而，同种异体移植程序可能会产生明显的副作用，并且无反应的水疱区域会持续存在。因此，我们在患者来源的细胞中采用了基因组编辑功能，为治疗细胞类型的多谱系工程创建了一个自体平台。成簇的规则间隔的回文重复序列（CRISPR）/ Cas9系统有助于纠正原发性成纤维细胞中引起RDEB的COL7A1突变，然后将其用于诱导多能干细胞（iPSC）。随后将生成的iPSC重新分化为角质形成细胞，间充质干细胞（MSCs）和造血祖细胞使用已定义的分化策略。基因校正的角质形成细胞表现出特征性的上皮形态，并表达角质形成细胞特异性基因和转录因子。iPSC衍生的MSC表现出纺锤体形态，并表达CD73，CD90和CD105，并具有与骨髓MSC难以区分的体外脂肪形成，软骨形成和成骨分化的能力。最后，我们使用了血管诱导策略来生成有效的确定性造血祖细胞，该祖细胞能够在基于甲基纤维素的测定中进行多谱系分化。总的来说，