Understanding the Role of the Extracellular Matrix on Non-viral Gene Transfer

了解细胞外基质对非病毒基因转移的作用

• 批准号: 7740292
• 负责人: Tatiana Segura
• 金额: $ 17.67万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7740292
• 关键词: Accounting; Actins; Affect; Apoptosis; Bacteria; Basement membrane; Behavior; Binding; Biological Assay; Blood Vessels; Cell physiology; Cells; Clinical; Collagen; Collagen Type IV; Cytoskeleton; DNA; DNA Sequence Rearrangement; Development; Dimensions; Disease; Endocytosis; Endothelial Cells; Engineering; Environment; Extracellular Matrix; Extracellular Matrix Proteins; Fibroblasts; Fibronectins; Gene Delivery; Gene Expression; Gene Transfer; Genes; Goals; Gold; Guided Tissue Regeneration; Homeostasis; Hydrogels; In Vitro; Integrins; Laminin; Mesenchymal Stem Cells; Molecular; Molecular Conformation; Morphology; Natural regeneration; Pathway interactions; Pattern; Plasmids; Play; Polystyrenes; Process; Processed Genes; Proteins; Reporter; Research; Role; Signal Pathway; Signal Transduction; Smooth Muscle Myocytes; Structure; Surface; Technology; Testing; Therapeutic; Tissue Engineering; Tissues; Transfection; Translations; Viral; Viral Genes; Virus; angiogenesis; base; cell type; cellular engineering; design; gene function; in vivo; innovation; interest; matrigel; migration; monolayer; nanoparticle; non-viral gene delivery; particle; pathogen; plasmid DNA; protein complex; public health relevance; receptor; response; scaffold; surface coating; syndecan; vector; viral DNA

DESCRIPTION (provided by applicant): Non-viral gene delivery has been widely investigated over the past decade as a means to guide tissue regeneration, treat disease and study gene function. However, low efficiencies of gene transfer have limited the use of this approach. In this proposal, we are interested in studying the role that the extracellular matrix environment plays on the ability of mesenchymal stem cells, endothelial cells and smooth muscle cells to become transfected. The interaction of cellular receptors with the various components present in the ECM results in a myriad of cellular responses such as proliferation, migration, differentiation, and apoptosis. We hypothesize that the ECM environment is a key modulator of non-viral gene transfer through inducing cell states that make the cells more or less sensitive to gene transfer and that the role of the ECM will be different for cells seeded in two- versus three dimensions. Our approach is divided into two aims. In aim 1 we will engineer cellular microenvironments (eECMs) that contain multiple components of the ECM to study the effect of the eECMs on non-viral gene transfer. The eECMs will be made using self-assembled monolayers on gold in order to be able to control the amount and conformation of the immobilized proteins and DNA particles. Aside from using reporter plasmids and internalization assays to quantify the efficiency of gene transfer and particle internalization, we will investigate if cellular contractility is involved in the observed changes in gene transfer for different eECMs. In aim 2 we will investigate gene transfer when cells are plated inside a matrix (3D) as opposed to in a flat surface (2D). We believe that understanding how the process of gene transfer differs when the cells are plated inside a matrix from when they are plated in a flat surface is critical for the understanding how to enhance gene transfer in vivo and inside tissue engineered constructs. We will use polystyrene nanoparticles and DNA nanoparticles to study the endocytosis rate and overall gene transfer efficiency in 3D and 2D. We will also investigate if the cellular contractile machinery also plays a role when cells are plated in three dimensions. PUBLIC HEALTH RELEVANCE: Non-viral gene delivery offers is an ideal approach to alter cellular behavior at the molecular level through the delivery of plasmid DNA that encodes for a gene of interest. However, the efficiency of gene transfer is too low to achieve the levels required for therapeutic application. In this proposal we are exploring new avenues to enhance the process of gene transfer through exploiting the natural response of cells to the extracellular matrix.

描述（由申请人提供）：在过去十年中，非病毒基因递送作为引导组织再生、治疗疾病和研究基因功能的手段已被广泛研究。然而，基因转移的低效率限制了这种方法的使用。在这个提议中，我们感兴趣的是研究细胞外基质环境对间充质干细胞、内皮细胞和平滑肌细胞被转染的能力所起的作用。细胞受体与ECM中存在的各种组分的相互作用导致无数的细胞反应，例如增殖、迁移、分化和凋亡。我们假设ECM环境是非病毒基因转移的关键调节剂，通过诱导细胞状态使细胞对基因转移或多或少敏感，并且ECM的作用对于二维与三维接种的细胞将是不同的。我们的方法分为两个目标。在目标1中，我们将设计包含ECM多种成分的细胞微环境（eECM），以研究eECM对非病毒基因转移的影响。eECM将使用金上的自组装单层来制造，以便能够控制固定化蛋白质和DNA颗粒的量和构象。除了使用报告质粒和内化测定来量化基因转移和颗粒内化的效率之外，我们还将研究细胞收缩性是否参与不同eECM的基因转移中观察到的变化。在目标2中，我们将研究当细胞接种在基质内（3D）而不是平坦表面（2D）时的基因转移。我们认为，了解细胞接种在基质中与接种在平坦表面时基因转移过程的不同对于了解如何增强体内和组织工程构建体内的基因转移至关重要。我们将使用聚苯乙烯纳米颗粒和DNA纳米颗粒来研究3D和2D中的内吞速率和整体基因转移效率。我们还将研究细胞收缩机制是否也在细胞三维铺板时发挥作用。公共卫生相关性：非病毒基因递送是通过递送编码感兴趣基因的质粒DNA在分子水平上改变细胞行为的理想方法。然而，基因转移的效率太低，无法达到治疗应用所需的水平。在这项提案中，我们正在探索新的途径，通过利用细胞对细胞外基质的自然反应来增强基因转移的过程。