Skin-targeted Cell Therapy for Recessive Dystrophic Epidermolysis Bullosa

摘要 隐性营养不良性大疱性表皮病（RDEB）是由双等位基因缺失引起的典型遗传性皮肤病， COL 7A 1的功能缺失突变。这些突变导致皮肤中缺乏VII型胶原蛋白（C7）， 粘膜，导致水疱、纤维化、假并指、关节 挛缩、食管狭窄、角膜擦伤、营养不良、自身免疫、贫血和鳞状细胞癌 carcinoma.尽管在过去十年中作出了巨大努力，为这种严重和严重的疾病制定治疗措施， 潜在的致命性疾病，目前还没有可靠的治疗方法，提供C7蛋白的多个网站 受全身性严重RDEB影响。为了解决这个问题，我们建议获得一个更机械的理解， 如何恢复COL 7A 1的完整性，而不对其余基因组造成附带损害，以及如何 细胞的特化趋向性用于将完整的、功能性的C7递送到整个身体。为了 为了实现这些目标，并克服目前基因和细胞疗法的局限性，我们将研究 [i]碱基编辑是否上级CRISPR/Cas9编辑以校正COL 7A 1 突变？因为碱基编辑不会像经典的基因编辑那样导致双链断裂， DNA核酸酶，它避免了遗传毒性压力。[ii]是皮肤特化细胞，如ABCB 5+间充质细胞 基质/干细胞（MSC），在C7表达水平方面优于MSC的替代来源，足以 RDEB中C7缺陷的交叉校正？我们将评估皮肤特异性基质细胞，如间充质干细胞， 表达ATP结合盒亚家族B成员5（ABCB 5+）表面蛋白的基质细胞， 直接来自皮肤或间接来自患者特异性诱导多能干细胞，其具有COL 7A 1 恢复基本编辑功能。[iii]COL 7A 1编辑的人ABCB 5 + MSC介导创伤愈合吗？ RDEB的临床前小鼠模型？使用我们接受人类异种移植物的RDEB小鼠模型，我们将 定量碱基编辑校正的ABCB 5 + MSC和诱导的多能干细胞衍生的MSC的价值。 我们建议通过以下方式来定义这种严重起泡性遗传性皮肤病中有利于伤口愈合的条件： 使用强大的工具来研究和操纵生物系统的信息基础（即， 用于碱基编辑介导的基因治疗的可编程脱氨酶;诱导的细胞谱系转化;和皮肤 向性）。我们的目标是为全身性重度RDEB患者提供个性化的细胞治疗， 我们的研究结果可能会为管理其他遗传性皮肤病以及治疗 皮肤粘膜溃疡以及化学和热烧伤。我们的提议同样是出于希望有一个更好的 了解受伤皮肤的生物学机制，并需要改善患有 RDEB通过降低风险和最大化潜在的新型基因和细胞疗法的益处。

Abstract Recessive dystrophic epidermolysis bullosa (RDEB) is a prototypical genodermatosis caused by biallelic loss- of-function mutations of COL7A1. These mutations lead to a lack of type VII collagen (C7) in the skin and mucosal membranes, resulting in a complex phenotype of blistering, fibrosis, pseudosyndactyly, joint contractures, esophageal strictures, corneal abrasions, malnutrition, autoimmunity, anemia, and squamous cell carcinoma. Despite tremendous efforts over the last decade to establish curative measures for this severe and potentially fatal disorder, there are as yet no therapies that reliably supply C7 protein to the multiple sites affected by generalized severe RDEB. To address this, we propose to gain a more mechanistic understanding of how to restore the integrity of COL7A1 without causing collateral damage to the rest of the genome, and of how the specialized tropism of cells works to deliver intact, functional C7 throughout the body. In order to accomplish these goals and to overcome the limitations of current gene and cell therapies, we will investigate the following questions: [i] Is base editing superior to CRISPR/Cas9-editing for correction of COL7A1 mutations? Because base editing does not cause double-strand breaks in the way that classic gene editing with DNA nucleases does, it avoids genotoxic stress. [ii] Are skin-specialized cells, such as ABCB5+ mesenchymal stromal/stem cells (MSCs), superior to alternative sources of MSCs in expression of C7 levels adequate for cross-correction of C7 deficiency in RDEB? We will evaluate skin-specific stromal cells, such as mesenchymal stromal cells expressing ATP-binding cassette sub-family B member 5 (ABCB5+) surface protein, derived directly from skin or indirectly from patient-specific induced pluripotent stem cells, which have had COL7A1 restored to function with base editing. [iii] Do COL7A1-edited human ABCB5+ MSCs mediate wound healing in a preclinical murine model of RDEB? Using our murine model of RDEB that accepts human xenografts, we will quantify the value of base editing-corrected ABCB5+ MSCs and induced pluripotent stem cell-derived MSCs. We propose to define the conditions conducive to wound healing in this severe blistering genodermatosis by using powerful tools for studying and manipulating the information bases of biological systems (i.e., programmable deaminases for base editing-mediated gene therapy; induced cell lineage conversion; and skin tropism). We will aim for personalized cell therapy for individuals with generalized severe RDEB, with the idea that our findings may provide insights into ways to manage other genodermatoses, as well as treatment of mucocutaneous ulcers, and chemical and thermal burns. Our proposal is equally motived by wanting a better understanding of the biological mechanisms in injured skin and by needing to improve the lives of people with RDEB through reducing the risks and maximizing the benefits of potential novel gene and cell therapies.