Skin-targeted Cell Therapy for Recessive Dystrophic Epidermolysis Bullosa

描述（由申请人提供）：隐性营养不良性大疱性表皮病（RDEB）是由VII型胶原蛋白（C7）基因（COL 7A 1）的功能缺失突变引起的，并导致潜在的致命性和严重的衰弱性疾病。患有严重全身性RDEB的个体发展为涉及大部分体表面积的水疱和糜烂、毁损性瘢痕、关节挛缩和食管狭窄，机会性感染和侵袭性鳞状细胞癌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中的C7蛋白，并改善许多疾病的表现。不幸的是，并非所有的粘膜皮肤病变愈合，与HSCT相关的毒性是显着的。我们的首要目标是通过建立一种可靠的方法来解决这些问题，该方法用于递送C7表达细胞，这些细胞归巢到真皮-表皮连接处并分泌C7蛋白。为了实现这一目标并克服同种异体HSCT的局限性，我们将：1）使用人间充质基质/干细胞和诱导多能干细胞（iPSC）鉴定用于靶向RDEB中受损皮肤的最佳细胞载体和归巢信号，2）通过C7基因扩增来定义系统性RDEB基因疗法的功效，以及3）使用核酸酶对个体突变进行基因编辑，以及C7基因的整个区域。我们建议确定有利于这种严重起泡性遗传性皮肤病伤口愈合的条件，但这些研究的影响可能不仅限于RDEB。C7的功能取决于细胞外聚合，这使得RDEB成为理解细胞外基质中结构蛋白缺陷的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中导致皮肤干细胞小生境的耗尽，进而提供了一种使用供体再生细胞进行皮肤组织修复的独特模型。通过使用强大的工具来研究和操纵生物系统的信息基础-工程化限制性内切酶（核酸酶），皮肤靶向和诱导细胞谱系转换（iPSC）-我们将专注于为RDEB患者开发个性化的细胞治疗，我们的研究结果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。我们的提议同样是出于希望更好地了解受伤皮肤的生物学机制，以及需要通过最大化潜在新疗法的益处和降低风险来改善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.