Molecular Genetics of the Cutaneous BMZ in EB

描述（由申请方提供）：拟定研究的长期目标是确定存在突变型VII型前胶原分子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，这些参数应达到改变存在VII型前胶原突变体时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，并且成功治疗方法的关键靶点对于各种VII型前胶原突变体是不同的。我们制定了以下具体目标：（1）建立一个可控的和生物学相关的实验系统，以研究在存在VII型前胶原突变体的情况下皮肤基底膜的组装，（2）确定VII型前胶原突变体的抑制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VII型前胶原突变引起的皮肤基底膜的病理变化。由于限制，例如治疗剂向皮肤的无效递送，仅以有限的方式测试了抵消原胶原VII中突变的病理作用的方法。因此，我们建议，需要一个简单的，控制良好的实验系统，建设性地验证前景，并发现潜在的治疗方法在实验条件下，模拟充分有效的“治疗剂”的交付的限制。我们还假设，需要一个全面的研究来定义皮肤特异性条件需要驱动皮肤窝藏一个前胶原VII突变体向重塑成正常结构的细胞或基因治疗。我们将通过创建一个生物相关模型来解决这个问题，该模型将类似于皮肤结构的复杂性。该模型将由来自Col7a1缺失小鼠的工程化角质形成细胞和真皮成纤维细胞组成，除了重组野生型原胶原VII和其他皮肤特异性标记物之外，其将条件性表达编码类似于在营养不良性大疱性表皮病患者中发现的那些的重组原胶原VII突变体的cDNA构建体。这些细胞将用于在细胞培养条件下和在无胸腺裸鼠中产生皮肤样构建体。随后，将在关闭突变型胶原的表达后或在模拟表达野生型前胶原VII的细胞的“递送”实验后，研究在突变型前胶原VII变体存在下形成的皮肤样构建体的特征变化。我们在这里提出的研究将扩大我们的分子病理机制的理解，强调营养不良性大疱性表皮病，并将提供一个基础，发展的方法来治疗这种疾病。因此，拟议的研究与公共卫生的相关性很高。相关性：营养不良性大疱性表皮病是一种由VII型前胶原突变引起的失能性遗传性皮肤病，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方法来平衡这种疾病的病理症状。我们的研究结果将建立最低限度的目标，必须达到纠正由VII型前胶原突变引起的皮肤病理变化。因此，我们提出的研究对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.