Somatic mosaicism in skin fragility disorders: mechanisms and therapeutic perspectives

反向镶嵌现象是发生在具有致病种系突变的个体中的一种现象，其中细胞亚群通过自然发生的重组、回位或二位点突变重新获得野生型表型。这种所谓的天然基因疗法已被报道用于几种遗传性疾病，包括单例大疱性表皮松解症（EB）。EB包括一组异质的皮肤脆弱性疾病，其特征是轻微创伤后皮肤起泡，由真皮-表皮粘附复合物成分突变引起。在我们的700多名分子确诊EB患者的大队列中，我们迄今已在15名重度和轻度交界性EB、营养不良性EB和Kindler综合征患者中发现了逆转嵌合体。这一建议的目的是产生新的知识，在分子机制的基础上，在皮肤易碎性疾病的逆转镶嵌和在恢复细胞的治疗潜力。我们将发现更多的具有逆转嵌合和不同EB形式的患者，并揭示潜在的分子机制。通过仔细的临床调查和定量文件，将定义EB中恢复皮肤斑块的形状和不同模式以及恢复镶嵌的自然历史。作为在基于细胞的治疗方法中应用这些细胞的先决条件，这些细胞将通过迁移、增殖和凋亡检测进行功能表征。将优化恢复角质形成细胞的分离和生长条件，目的是利用患者自身的自然校正细胞建立细胞疗法。器官型共培养物与皮肤的三维结构和细胞组成非常相似；它们将被用来评估恢复角质形成细胞的行为以及恢复和突变细胞之间的竞争。这项研究将有助于提高我们对蛋白质对皮肤完整性的重要作用的理解，以及对恢复细胞作为疾病修饰因子和治疗剂的意义的理解。

Revertant mosaicism is a phenomenon occurring in an individual with a disease-causing germline mutation, wherein a subpopulation of cells re-acquires the wild-type phenotype through a naturally occurring recombination, back or second-site mutation. This so called natural gene therapy has been reported in several inherited disorders, including single cases of epidermolysis bullosa (EB). EB comprises a heterogeneous group of skin fragility disorders, characterized by blistering of the skin after minor trauma and caused by mutations in components of the dermal-epidermal adhesion complexes. In our large cohort of more than 700 patients with molecularly confirmed EB, we have so far identified revertant mosaicism in 15 patients with severe and mild forms of junctional EB, dystrophic EB and Kindler syndrome. The objective of this proposal is to generate new knowledge on the molecular mechanisms that underlie revertant mosaicism in skin fragility disorders and on therapeutic potential of the reverted cells. We will identify more patients with revertant mosaicism and different EB forms and disclose the underlying molecular mechanisms. With careful clinical investigation and quantitative documentation, the shape and different patterns of the reverted skin patches and the natural history of revertant mosaicism in EB will be defined. As a prerequisite for application of the reverted cells in cell-based therapy approaches, these cells will be functionally characterized with migration, proliferation and apoptosis assays. Isolation and growth conditions for reverted keratinocytes will be optimized, with the goal of establishing cell therapy with the patients' own naturally corrected cells. Organotypic co-cultures closely resemble the three-dimensional structure and cellular composition of the skin; they will be used to assess the behaviour of the reverted keratinocytes and the competition between reverted and mutant cells. This research will help improve our understanding on the role of the proteins that are important for skin integrity and on the significance of the reverted cells as disease modifying factors and therapeutic agents.