Molecular therapies for junctional epidermolysis bullosa

XVII型胶原（C17）基因（COL17A1）突变引起的大疱性结缔组织表皮松解症（JEB）表现为皮肤起泡和慢性伤口。治疗包括伤口管理，有一个高度未满足的治疗需求。动物模型是早期致死或不再现人类疾病的情况。本提案基于广泛的前期工作和申请人专业知识的互补，旨在开发分子疗法来恢复缺失的C17，从而为临床试验提供基础。为了以个性化的方式靶向不同类型的致病变异，我们将开发针对COL17A1无义突变（占COL17A1突变的20%）的通读疗法，以及针对所有其他COL17A1突变的蛋白质疗法。具有不同作用机制的翻译读通诱导药物（TRIDs），以及增强剂和无义介导的衰变抑制剂，将在来自患者的细胞上进行测试，以找到每种COL17A1无义突变的最佳浓度和组合。此外，我们将验证细胞外脱落C17外结构域（有或没有高抗原NC16A结构域）能够在伤口愈合过程中整合到基底膜中并促进伤口愈合的假设。TRIDs和重组C17外畴也将一起应用于二维和三维细胞模型。在重建的皮肤模型中，主要的读数将是恢复的C17水平，细胞的活力，真皮-表皮连接处的稳定性以及对伤口愈合的影响。作为次要读出，我们将研究C17的转换及其对表皮结构和信号通路的影响。为了评估，我们将采用生化，细胞生物学和组织形态分析。最后，该项目将确定可能对其他遗传疾病有效的药物组合，并将建立一个可重复的工作流程，以研究分子疗法对大疱性表皮松解症个体突变的精确影响。

Junctional epidermolysis bullosa (JEB) caused by type XVII collagen (C17) gene (COL17A1) mutations manifests with skin blistering and chronic wounds. The treatment consists of wound management and there is a high-unmet therapeutic need. Animal models are early lethal or do not reproduce the human disease situation. This proposal, based on extensive preliminary work and on the complementary expertise of the applicants, aims at developing molecular therapies to restore the missing C17, thus providing the basis for clinical trials. To target different types of pathogenic variants in a personalized manner, we will develop read-through therapy for COL17A1 nonsense mutations (20% of the COL17A1 mutations) and protein therapy for all other COL17A1 mutations. Translational read-through inducing drugs (TRIDs) with different mechanisms of action, as well as enhancers and a nonsense mediated decay inhibitor, will be tested on cells derived from patients to find the optimal concentrations and combinations for each COL17A1 nonsense mutation. Furthermore, we will test the hypothesis that the extracellular shed C17 ectodomain (with and without the highly antigenic NC16A domain) is able to be integrated in the basement membrane during wound healing and to promote wound closure. TRIDs and recombinant C17 ectodomain will be also applied together in two-and three dimensional cell models. The primary read-outs will be the level of restored C17, the viability of the cells, the stabilization of the dermal-epidermal junction and the effect on wound closure in reconstructed skin models. As secondary read-outs, we will investigate C17 turnover, and its effect on epidermal architecture and signaling pathways. For assessment, we will employ biochemical, cell biological and tissue morphological assays. Finally, this project will identify combinations of drugs that might be effective in other genetic disorders, and will establish a reproducible workflow to investigate the precise effect of molecular therapies on individual mutations in epidermolysis bullosa.