Skin-targeted Cell Therapy for Recessive Dystrophic Epidermolysis Bullosa

描述（由申请人提供）：隐性营养不良大疱性表皮松解症（RDEB）是由胶原蛋白VII型（C7）基因（COL7A1）的功能丧失突变引起的，导致潜在的致命和可怕的衰弱性疾病。患有严重全身性RDEB的个体出现大面积体表水疱和糜烂、致残疤痕、关节挛缩和食道狭窄，并伴有机会性感染和侵袭性鳞状细胞癌，这是早期死亡的主要原因。我们已经证明造血干细胞移植（HSCT）可以增加皮肤和粘膜的C7蛋白，并改善许多疾病的表现。不幸的是，并不是所有的粘膜皮肤病变都能愈合，并且与HSCT相关的毒性是显著的。我们的首要目标是通过建立一种可靠的方法，将表达C7的细胞递送到真皮-表皮交界处并分泌C7蛋白，从而解决这些问题。为了实现这一目标并克服同种异体造血干细胞移植的局限性，我们将：1)利用人间充质基质/干细胞和诱导多能干细胞（iPSCs），确定针对RDEB损伤皮肤的最佳细胞载体和归家信号，2)通过C7基因增强确定系统性RDEB基因治疗的疗效，以及3)使用核酸酶对单个突变以及C7基因的整个区域进行基因编辑。我们建议确定有利于伤口愈合的条件在这种严重的起泡性遗传性皮肤病，但这些研究的影响可能不限于RDEB。C7的功能依赖于细胞外聚合，这使得RDEB成为理解细胞外基质中结构蛋白缺乏交叉校正机制的理想模型。此外，伤口愈合将皮肤细胞外基质和皮肤细胞整合到一个动态系统中，这在RDEB中导致皮肤干细胞壁龛的耗竭，反过来，提供了一个独特的皮肤组织修复模型与供体再生细胞。通过使用强大的工具来研究和操纵生物系统的信息基础——工程限制性内切酶（核酸酶）、皮肤靶向和诱导细胞系转化（iPSCs）——我们将专注于开发针对RDEB患者的个性化细胞疗法，并认为我们的发现可能对理解其他结缔组织细胞外基质疾病具有广泛的意义。我们的建议同样是为了更好地了解受伤皮肤的生物学机制，并需要通过最大化益处和降低潜在新疗法的风险来改善RDEB患者的生活。

DESCRIPTION (provided by applicant): Recessive dystrophic epidermolysis bullosa (RDEB) is caused by loss-of-function mutations in the collagen type VII (C7) gene (COL7A1), and results in a potentially fatal and terribly debilitating disorder. Individuals with severe generalied RDEB develop blisters and erosions involving large portions of the body surface area, mutilating scarring, joint contractures, and strictures of the esophagus, with opportunistic infections and aggressive squamous cell carcinoma as principal causes of early death. We have shown that hematopoietic stem cell transplantation (HSCT) can increase C7 protein in skin and mucous membranes, and ameliorate many of the disease manifestations. Unfortunately not all mucocutaneous lesions heal, and the toxicities associated with HSCT are significant. Our overarching goal is to solve these problems by establishing a reliable method for delivering C7-expressing cells that home to the dermal-epidermal junction and secrete C7 protein. In order to accomplish this goal and to overcome the limitations of allogeneic HSCT, we will: 1) identify the optimal cellular vector and homing signal for targeting injured skin in RDEB, using human mesenchymal stromal/stem cells and induced pluripotent stem cells (iPSCs), 2) define the efficacy of systemic RDEB gene therapy by C7 gene augmentation, and 3) use nucleases for gene editing of individual mutations, as well as whole regions of the C7 gene. We propose to define the conditions conducive to wound healing in this severe blistering genodermatosis, but the impact of these studies may not be limited to RDEB. The function of C7 depends on extracellular polymerization, which makes RDEB an ideal model for understanding the mechanisms of cross- correction of structural protein deficiency in the extracellular matrix. In addition, wound healing integrates skin extracellular matrix and skin cells into a dynamic system, which in RDEB results in the depletion of skin stem cell niches and, in turn, provides a unique model of skin tissue repair with donor regenerative cells. By using powerful tools for studying and manipulating the information basis of biological systems-engineered restriction enzymes (nucleases), skin- targeting, and induced cell lineage conversion (iPSCs)-we will focus on developing personalized cell therapy for individuals with RDEB with the idea that our findings may have broad implications for understanding other connective tissue extracellular matrix diseases. Our proposal is equally motivated by wanting a better understanding of the biological mechanisms in injured skin and by needing to improve the lives of people with RDEB through maximizing the benefits and reducing the risks of potential novel therapies.