Molecular Genetics of the Cutaneous BMZ in EB

描述（由申请人提供）：拟议研究的长期目标是确定突变前胶原VII分子的存在对皮肤基底膜结构和细胞行为的影响，并确定细胞和基因治疗的目标参数，以改变在前胶原VII突变体存在下形成的皮肤基底膜的异常结构。我们的假设是COL7A1突变对皮肤基底膜的结构有广泛的影响，并且对于不同的前VII型胶原突变，成功治疗方法的关键靶点是不同的。我们制定了以下具体目标：(1)建立一个可控的、生物学相关的实验系统，研究在前胶原VII突变体存在下皮肤基底膜的组装；(2)确定抑制前胶原VII突变体表达对异常皮肤基底膜重塑的影响；(3)确定在基于细胞的方法中必须达到的目标，以纠正由前胶原VII突变引起的皮肤基底膜病理改变。由于诸如治疗药物对皮肤的无效递送等限制，平衡前胶原VII突变的病理效应的方法仅以有限的方式进行了测试。因此，我们建议需要一个简单、控制良好的实验系统来建设性地验证前景，并在模拟“治疗剂”完全有效递送的实验条件下揭示潜在治疗方法的局限性。我们还假设，需要进行全面的研究来确定皮肤特异性条件，以驱动含有前胶原蛋白VII突变的皮肤在细胞或基因治疗下重塑为正常结构。我们将通过创建一个生物学上相关的模型来解决这个问题，这个模型将类似于皮肤结构的复杂性。该模型将由来自col7a1缺失小鼠的工程角质形成细胞和真皮成纤维细胞组成，这些细胞将有条件地表达编码重组前胶原VII突变体的cDNA构建物，类似于营养不良大性表皮松解症患者的突变体，此外还有重组野生型前胶原VII和其他皮肤特异性标记物。这些细胞将用于在细胞培养条件和胸腺裸鼠中创建皮肤样结构。随后，在关闭突变型胶原的表达或模拟表达野生型前胶原VII的细胞的“递送”实验后，将研究在突变型前胶原VII变体存在下构建的皮肤样构建物特征的变化。我们在此提出的研究将扩展我们对营养不良大疱性表皮松解症的分子病理机制的理解，并将为开发治疗这种疾病的方法提供基础。因此，拟议的研究与公共卫生的相关性很高。相关性：大疱性营养不良性表皮松解症是一种由前胶原VII突变引起的皮肤致残遗传性疾病，目前尚无有效的治疗方法可以缓解与该疾病相关的临床症状。我们建议开发和利用一个相对简单的实验模型来测试平衡这种疾病的病理症状的方法。我们的研究结果将建立必须达到的最小目标，以纠正由前胶原VII突变引起的皮肤病理变化。因此，我们提出的研究与公共卫生的相关性很高。

DESCRIPTION (provided by applicant): The long-term objective of the proposed study is to determine effects of the presence of mutant procollagen VII molecules on the structure of the cutaneous basement membrane and behavior of cells and to define target parameters for cell and gene therapies that should be reached to change the abnormal architecture of the cutaneous basement membranes developed in the presence of procollagen VII mutants. Our hypothesis is that mutations in COL7A1 have broad effects on the structure of cutaneous basement membrane and that critical targets for successful therapy approaches differ for various procollagen VII mutants. We formulated the following Specific Aims: (1) To create a controllable and biologically relevant experimental system to study assembly of the cutaneous basement membrane in the presence of procollagen VII mutants, (2) To determine the consequences of suppressed expression of procollagen VII mutants on remodeling of an abnormal cutaneous basement membrane, (3) To define targets which have to be reached in cell- based approaches to correct pathological changes in the cutaneous basement membrane caused by mutations in procollagen VII. Due to limits such as ineffective delivery of therapeutic agents to skin approaches to counterbalance pathological effects of mutations in procollagen VII have been tested only in a limited way. Thus, we propose that a simple, well controlled experimental system is needed to constructively validate prospects and uncover limitations of potential therapy approaches in experimental conditions that simulate fully effective delivery of "therapeutic agents". We also postulate that a comprehensive study is required to define skin-specific conditions needed to drive skin harboring a procollagen VII mutant toward remodeling into normal structure in response to cell or gene therapies. We will address this problem by creating a biologically relevant model, which will resemble the complexity of skin structure. This model will consist of engineered keratinocytes and dermal fibroblasts from Col7a1-null mice, which will conditionally express cDNA constructs encoding recombinant procollagen VII mutants analogous to those found in dystrophic epidermolysis bullosa patients in addition to recombinant wild type procollagen VII and other skin- specific markers. These cells will be employed to create skin-like constructs in cell culture conditions and in athymic nude mice. Subsequently, changes in characteristics of the skin-like constructs preformed in the presence of mutant procollagen VII variants will be studied after switching off expression of mutant collagens or after experiments simulating "delivery" of cells expressing wild type procollagen VII. The study we propose here will extend our understanding of the molecular pathomechanisms underlining dystrophic epidermolysis bullosa and will provide a basis for developing approaches to treating this disease. Thus, the relevance of the proposed study to public health is high. Relevance: Dystrophic epidermolysis bullosa is an incapacitating heritable disease of skin caused by mutations in procollagen VII and there is no effective treatment that could alleviate clinical symptoms associated with this disease. We propose to develop and exploit a relatively uncomplicated experimental model to test approaches to counterbalance pathological symptoms of this disease. Results of our study will establish minimal targets that have to be reached to correct pathological changes in skin caused by mutations in procollagen VII. Thus, the relevance of our proposed research for public health is high.