Skin-targeted Cell Therapy for Recessive Dystrophic Epidermolysis Bullosa

摘要 隐性营养不良性大疱性表皮松解症(RDEB)是由双等位基因缺失引起的一种典型的遗传性皮肤病。 COL7A1基因功能外突变。这些突变导致皮肤中缺乏III型胶原(C7)， 粘膜，导致水泡、纤维化、假指、关节等复杂表型 宫缩、食道狭窄、角膜擦伤、营养不良、自身免疫、贫血和鳞状细胞 癌症。尽管在过去的十年里做出了巨大的努力来建立治疗这种严重和 潜在的致命疾病，到目前为止还没有治疗方法可以可靠地向多个部位供应C7蛋白 受全身性重度RDEB影响。为了解决这个问题，我们建议更机械化地理解 如何在不对基因组其余部分造成附带损害的情况下恢复COL7A1的完整性，以及如何 细胞的特殊取向作用是将完整的、有功能的C7输送到全身。为了 为了实现这些目标并克服当前基因和细胞疗法的局限性，我们将研究 以下问题：[I]基础编辑优于CRISPR/CAS9-COL7A1的更正编辑 突变？因为碱基编辑不会像传统的基因编辑那样导致双链断裂 DNA核酸酶起作用，它避免了遗传毒性应激。[ii]是皮肤特化细胞，如ABCB5间充质细胞 基质/干细胞(MSCs)，在C7水平的表达上优于其他来源的MSCs RDEB中C7缺乏症的交叉矫正？我们将评估特定于皮肤的基质细胞，如间充质 表达ATP结合盒B亚家族成员5(ABCB5)表面蛋白的基质细胞，来源 直接来自皮肤或间接来自患者特异性诱导的多能干细胞，这些干细胞具有COL7A1 通过基本编辑恢复了功能。[III]COL7A1编辑的人ABCB5间充质干细胞是否在体内介导伤口愈合 临床前RDEB？小鼠模型？使用我们的接受人类异种移植的RDEB小鼠模型，我们将 量化碱基编辑校正的ABCB5 MSCs和诱导的多能干细胞来源的MSCs的价值。 我们建议在这种严重的水泡性遗传性皮肤病中定义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库(即， 可编程脱氨酶用于碱基编辑介导的基因治疗；诱导细胞谱系转换；以及皮肤 取向)。我们的目标是为患有全身性重度RDEB的个人进行个性化的细胞治疗，想法是 我们的发现可能会为管理其他遗传性皮肤病的方法以及治疗 皮肤粘膜溃疡，以及化学和热烧伤。我们的建议同样受到想要更好的 了解损伤皮肤的生物学机制，并通过需要改善患者的生活 RDEB通过降低潜在的新基因和细胞疗法的风险和最大限度地发挥其效益。

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.