Molecular Genetics of the Cutaneous BMZ in EB

EB 皮肤 BMZ 的分子遗传学

基本信息

批准号：
8213724
负责人：
ANDRZEJ FERTALA
金额：
$ 32.05万
依托单位：
THOMAS JEFFERSON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-02-15 至 2013-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8213724
关键词：
Address Architecture Basement membrane Biological Assay Bioreactors Caliber Cell Culture Techniques Cell Therapy Cells Characteristics Clinical Collagen Complementary DNA Complex Cutaneous Dermal Dermis Disease Elasticity Engineering Epidermis Epidermolysis Bullosa Dystrophica Evaluation Experimental Models Fibroblasts Goals Knockout Mice Laboratories Measurement Measures Mechanics Methodology Modeling Molecular Molecular Genetics Mutation Nature Nude Mice Patients Procollagen Property Proteins Public Health Published Comment Recombinants Research Research Personnel Simulate Skin Skin Substitutes Structure Symptoms System Technology Testing Therapeutic Agents Time Type VII Procollagen Variant base cell behavior density effective therapy gene therapy keratinocyte mutant public health research research study response shear stress skin disorder

项目摘要

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.

描述（由申请人提供）：拟议研究的长期目标是确定突变型前胶原 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应该达到改变在前胶原 VII 突变体存在下发育的皮肤基底膜的异常结构的目的。我们的假设是，COL7A1 的突变对皮肤基底膜的结构具有广泛的影响，并且成功治疗方法的关键目标因不同的 VII 前胶原突变体而异。我们制定了以下具体目标：（1）创建一个可控且生物学相关的实验系统来研究在存在 VII 前胶原突变体的情况下皮肤基底膜的组装，（2）确定 VII 前胶原突变体的抑制表达对皮肤基底膜的影响异常皮肤基底膜的重塑，(3)确定基于细胞的方法必须达到的目标，以纠正由前胶原VII突变引起的皮肤基底膜的病理变化。由于治疗剂向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揭示在模拟“治疗剂”完全有效递送的实验条件下潜在治疗方法的局限性。我们还假设需要进行一项全面的研究来定义皮肤特异性条件，以驱动含有前胶原 VII 突变体的皮肤重塑为正常结构以响应细胞或基因治疗。我们将通过创建一个生物学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 缺失小鼠的工程角质形成细胞和真皮成纤维细胞组成，除了重组野生型 VII 型前胶原和其他皮肤特异性标记物之外，它们还将有条件地表达编码重组 VII 前胶原突变体的 cDNA 构建体，类似于营养不良性大疱性表皮松解症患者中发现的突变体。。这些细胞将用于在细胞培养条件下和无胸腺裸鼠中创建皮肤样结构。随后，在关闭突变胶原的表达后或在模拟“递送”表达野生型前胶原VII的细胞的实验后，将研究在突变前胶原VII变体存在下预形成的皮肤样构建体的特征的变化。我们在此提出的研究将扩展我们对营养不良性大疱性表皮松解症的分子病理机制的理解，并将为开发治疗该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：营养不良性大疱性表皮松解症是一种由 VII 号原胶原突变引起的致残性遗传性皮肤病，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。我们的研究结果将确定纠正由 VII 号原胶原突变引起的皮肤病理变化必须达到的最低目标。因此，我们提出的研究与公共卫生的相关性很高。

项目成果

期刊论文数量（4）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Remodeling of the dermal-epidermal junction in bilayered skin constructs after silencing the expression of the p.R2622Q and p.G2623C collagen VII mutants.

沉默 p.R2622Q 和 p.G2623C VII 胶原蛋白突变体的表达后，双层皮肤结构中真皮-表皮连接处的重塑。

DOI：
发表时间：
2012
期刊：
影响因子：
2.9
作者：
Steplewski, Andrzej;Kasinskas, Anthony;Fertala, Andrzej
通讯作者：
Fertala, Andrzej

Keratinocyte-/fibroblast-targeted rescue of Col7a1-disrupted mice and generation of an exact dystrophic epidermolysis bullosa model using a human COL7A1 mutation.

使用人类 COL7A1 突变对 Col7a1 破坏的小鼠进行角质形成细胞/成纤维细胞靶向拯救，并生成精确的营养不良性大疱性表皮松解症模型。

DOI：
发表时间：
2009-12
期刊：
影响因子：
0
作者：
Ito, Kei;Sawamura, Daisuke;Goto, Maki;Nakamura, Hideki;Nishie, Wataru;Sakai, Kaori;Natsuga, Ken;Shinkuma, Satoru;Shibaki, Akihiko;Uitto, Jouni;Denton, Christopher P;Nakajima, Osamu;Akiyama, Masashi;Shimizu, Hiroshi
通讯作者：
Shimizu, Hiroshi

Early intra-amniotic gene transfer using lentiviral vector improves skin blistering phenotype in a murine model of Herlitz junctional epidermolysis bullosa.

使用慢病毒载体进行早期羊膜内基因转移可改善赫利茨交界性大疱性表皮松解症小鼠模型中的皮肤起泡表型。

DOI：
发表时间：
2012-05
期刊：
影响因子：
5.1
作者：
Endo, M;Zoltick, P W;Radu, A;Jiang, Q;Qiujie, J;Matsui, C;Marinkovich, P M;McGrath, J;Tamai, K;Uitto, J;Flake, A W
通讯作者：
Flake, A W

Fluorescent protein markers to tag collagenous proteins: the paradigm of procollagen VII.

标记胶原蛋白的荧光蛋白标记物：前胶原 VII 的范例。