Cell-Based Therapy for Dystrophic Epidermolysis Bullosa

描述（由申请人提供）：营养不良大疱性表皮松解症（DEB）是一种遗传性机械大疱性疾病，其特征是皮肤和粘膜脆弱。DEB的形态学特征包括真皮-表皮交界处的基底膜区分裂，乳突真皮内有一个卵裂面。锚定原纤维的异常组装是由编码VII型胶原的基因（COL7A1）的基因突变引起的，该基因由上皮基底角质形成细胞和乳头状真皮成纤维细胞合成。目前，还没有针对任何形式的遗传性DEB的特异性治疗方法。该疾病的日常管理侧重于通过明智地使用绷带或宽松的服装和局部抗生素来预防机械创伤和感染。准确了解不同亚型DEB的遗传病变，并阐明这种突变在蛋白质水平上的后果，使我们能够继续尝试开发治疗方法，以对抗这种破坏性皮肤病的临床表现。干细胞研究的最新进展提出了一种可能性，即使用来自骨髓的成体干细胞可能为治疗遗传和获得性疾病提供引人注目的新疗法。骨髓中含有间充质祖细胞，称为间充质干细胞（MSC），具有几个独特的特性，表明它们作为治疗载体的可行性：(1)相对简单的分离；(2)在最低条件下体外培养并扩大到治疗所需数量的能力；(3)体外转导能力；(4)可塑性，外源刺激下的分化潜能；(5)再引种后的移植物能力；(6)高代谢活性和以分泌形式表达治疗性蛋白的高效机制；(7)系统或局部交付的能力。拟议的研究是我们在非病毒基因治疗领域工作的后勤延伸。这些研究的主要目的是验证基因正常的间充质干细胞的移植会导致移植细胞在DEB小鼠皮肤中足够数量的扩增，并且VII型胶原的水平足以减少病理表型的假设。以下参数将被研究：(1)骨髓间充质干细胞在DEB皮肤中的传递路线、移植细胞的分布和数量；(2)间充质干细胞在DEB皮肤中的分布动力学；(3)移植骨髓间充质干细胞的表型和功能特征；(4)成熟VII型胶原在完整小鼠皮肤中的分布和释放动力学；(5)间充质干细胞产生VII型胶原形成锚定原纤维样结构的能力；(6) VII型胶原纤维沉积到BMZ；(7)新形成的VII型胶原纤维的结构；(8)新形成的纤维与BMZ大分子相互作用的能力。这些研究的成功结果可能为开发治疗结缔组织疾病以及其他人类遗传疾病的新治疗方法提供平台。在目前的项目中，我们建议验证从骨髓中分离的基因正常的间充质干细胞的移植会导致移植细胞在DEB小鼠的受损皮肤中足够数量的扩增，并且VII胶原的水平足以降低病理表型的假设。如果成功，建议的策略可能成为治疗结缔组织疾病，特别是营养不良大疱性表皮松解症的有力治疗方法，并有益于公众健康。

DESCRIPTION (provided by applicant): Dystrophic epidermolysis bullosa (DEB) is an inherited mechanobullous disorder characterized by fragility of the skin and mucous membranes. The morphological hallmark of DEB includes splitting of the basement membrane zone of the dermal-epidermal junction, with a cleavage plane lying within the papillary dermis. The abnormal assembly of anchoring fibrils is caused by genetic mutations in the gene (COL7A1) encoding collagen type VII synthesized by the basal keratinocytes of the epithelium and the fibroblast of the papillary dermis. At present, there are no specific therapies for any form of inherited DEB. Day-to-day management of the disease is concentrated on the prevention of mechanical trauma, infections via judicious use of bandages or loose-fitting garments and topical antibiotics. Precise understanding of the genetic lesions underlying different subtypes of DEB, and elucidation of the consequences of such mutations at the protein level, enabled us to continue our attempts to develop therapy approaches towards counteracting the clinical manifestations in this devastating skin disease. Recent advances in stem cell research have raised the possibility that use of adult stem cells from bone marrow may provide dramatic new therapies for treatment of inherited and acquired diseases. Bone marrow contains mesenchymal progenitor cells, called mesenchymal stem cells (MSC), that have several unique properties suggesting the feasibility of their use as a therapeutic vehicle: (1) a relatively simple isolation; (2) the ability to be cultured in vitro in minimal conditions and to expand to quantities required for therapy; (3) the ability for ex vivo transduction; (4) plasticity, the potential to differentiate under exogenous stimuli; (5) the ability to engraft after reintroduction; (6) high metabolic activity and efficient machinery to express therapeutic proteins in secretory form; and, (7) the ability to be delivered systematically or locally. The proposed research is a logistical extension of our work in the field of non-viral gene therapy. The main objective of these studies is to test the hypothesis that engraftment of genetically normal MSC will lead to expansion of the transplanted cells in sufficient numbers in the skin of DEB mice and the level of collagen VII will be sufficient to reduce the pathologic phenotype. The following parameters are will be investigated: (1) the rout of MSC delivery, distribution and quantity of transplanted cells in DEB skin; (2) the kinetics of MSC distribution in DEB skin; (3) the phenotypic and functional features of engrafted MSC; (4) the kinetics of distribution and release of mature collagen VII in the intact mouse skin; (5) the ability of MSC to produce collagen VII to form anchoring fibril-like structures; (6) the deposition of collagen VII fibers into the BMZ; (7) the structure of the newly formed collagen VII fibers; and, (8) the ability of newly formed fibers to interact with BMZ's macromolecules. A successful outcome of these studies may form a platform for development of a novel therapeutic approach for curing connective tissue disorders, as well as other human genetic disorders. In the current project, we suggested to test the hypothesis that engraftment of genetically normal mesenchymal stem cells isolated from bone marrow will lead to expansion of transplanted cells in sufficient numbers in the affected skin of DEB mice and the level of collagen VII will be sufficient to reduce the pathologic phenotype. If successful, the suggested strategy may become powerful therapeutic approach for treatment of connective tissue disorders, in particular dystrophic epidermolysis bullosa, and benefit public health.