Recessive dystrophic epidermolysis bullosa is a severe skin fragility disease caused by loss of functional type VII collagen at the dermal-epidermal junction. A frameshift mutation in exon 80 of COL7A1 gene, c.6527insC, is highly prevalent in the Spanish patient population. We have implemented gene editing strategies for COL7A1 frame restoration by NHEJ-induced indels in epidermal stem cells from patients carrying this mutation. TALEN nucleases designed to cut within the COL7A1 exon 80 sequence were delivered to primary patient keratinocyte cultures by non-integrating viral vectors. After genotyping a large collection of vector-transduced patient keratinocyte clones with high proliferative potential, we identified a significant percentage of clones with COL7A1 reading frame recovery and Collagen VII protein expression. Skin equivalents generated with cells from a clone lacking exon 80 entirely were able to regenerate phenotypically normal human skin upon their grafting onto immunodeficient mice. These patient-derived human skin grafts showed Collagen VII deposition at the basement membrane zone, formation of anchoring fibrils and structural integrity when analyzed twelve weeks after grafting. Our data provide a proof-of-principle for recessive dystrophic epidermolysis bullosa treatment through ex vivo gene editing based on removal of pathogenic mutation-containing, functionally expendable COL7A1 exons in patient epidermal stem cells.

隐性营养不良性大疱性表皮松解症是一种严重的皮肤脆弱性疾病，由皮肤-表皮交界处的功能性VII型胶原蛋白丧失引起。在80号外显子的移码突变COL7A1基因，c.6527insC，是在西班牙的患者人群非常普遍。我们已经实施了基因编辑策略COL7A1在表皮干细胞NHEJ诱导的插入缺失携带这种突变的患者恢复框架。TALEN核酸设计成将内切COL7A1外显子80序列是由非整合型病毒载体递送至患者初级角质形成细胞培养。对大量具有高增殖潜能的载体转导的患者角质形成细胞克隆进行基因分型后，我们发现COL7A1阅读框的恢复和VII型胶原蛋白的表达。用完全缺乏外显子80的克隆细胞产生的皮肤等效物在移植到免疫缺陷小鼠上后能够再生表型正常的人皮肤。这些患者来源的人类皮肤移植物在移植后十二周进行分析时显示了胶原蛋白VII在基底膜区域的沉积，锚定原纤维的形成和结构完整性。我们的数据通过基于离体基因编辑，基于去除患者表皮干细胞中含有致病突变的，功能性消耗性COL7A1外显子的离体基因编辑，为隐性营养不良性表皮松解大疱治疗提供了原理证明。