EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology and Allergology, University Hospital of the Paracelsus Medical University Salzburg, Müllner Hauptstraße 48, 5020 Salzburg, Austria.
Institute for Transfusion Medicine and Gene Therapy, Medical Center - University of Freiburg, Freiburg, Germany; Center for Chronic Immunodeficiency, Medical Center - University of Freiburg, Freiburg, Germany.
Mol Ther. 2022 Aug 3;30(8):2680-2692. doi: 10.1016/j.ymthe.2022.04.020. Epub 2022 Apr 30.
Junctional epidermolysis bullosa (JEB) is a debilitating hereditary skin disorder caused by mutations in genes encoding laminin-332, type XVII collagen (C17), and integrin-α6β4, which maintain stability between the dermis and epidermis. We designed patient-specific Cas9-nuclease- and -nickase-based targeting strategies for reframing a common homozygous deletion in exon 52 of COL17A1 associated with a lack of full-length C17 expression. Subsequent characterization of protein restoration, indel composition, and divergence of DNA and mRNA outcomes after treatment revealed auspicious efficiency, safety, and precision profiles for paired nicking-based COL17A1 editing. Almost 46% of treated primary JEB keratinocytes expressed reframed C17. Reframed COL17A1 transcripts predominantly featured 25- and 37-nt deletions, accounting for >42% of all edits and encoding C17 protein variants that localized accurately to the cell membrane. Furthermore, corrected cells showed accurate shedding of the extracellular 120-kDa C17 domain and improved adhesion capabilities to laminin-332 compared with untreated JEB cells. Three-dimensional (3D) skin equivalents demonstrated accurate and continuous deposition of C17 within the basal membrane zone between epidermis and dermis. Our findings constitute, for the first time, gene-editing-based correction of a COL17A1 mutation and demonstrate the superiority of proximal paired nicking strategies based on Cas9 D10A nickase over wild-type Cas9-based strategies for gene reframing in a clinical context.
交界性大疱性表皮松解症（JEB）是一种衰弱性遗传性皮肤疾病，由编码层粘连蛋白-332、XVII 型胶原（C17）和整合素-α6β4 的基因突变引起，这些基因维持真皮和表皮之间的稳定性。我们设计了基于患者特异性 Cas9 核酸酶和切口酶的靶向策略，用于重新构建与全长 C17 表达缺失相关的 COL17A1 外显子 52 中的常见纯合缺失。随后对蛋白质恢复、插入缺失组成以及治疗后 DNA 和 mRNA 结果的差异进行了特征描述，结果显示基于配对切口的 COL17A1 编辑具有良好的效率、安全性和精准性。经处理的原发性 JEB 角质形成细胞中，近 46%表达了重新构建的 C17。重新构建的 COL17A1 转录本主要表现为 25- 和 37-nt 缺失，占所有编辑的>42%，并编码准确定位于细胞膜的 C17 蛋白变体。此外，校正后的细胞与未经处理的 JEB 细胞相比，能够准确地释放细胞外 120kDa C17 结构域，并改善对层粘连蛋白-332 的黏附能力。三维（3D）皮肤等效物显示 C17 在表皮和真皮之间的基底膜区准确且连续地沉积。我们的研究结果首次证明了基于基因编辑的 COL17A1 突变校正，并证明了基于 Cas9 D10A 切口酶的近端配对切口策略优于野生型 Cas9 策略，用于临床背景下的基因重排。
