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

描述（由申请人提供）：在过去的十年里，非病毒基因传递作为一种指导组织再生、治疗疾病和研究基因功能的手段得到了广泛的研究。然而，低效率的基因转移限制了这种方法的使用。在本课题中，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转染能力的影响。细胞受体与ECM中存在的各种成分的相互作用导致无数的细胞反应，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的作用在二维和三维细胞中是不同的。我们的方法分为两个目标。在目标1中，我们将设计含有多种细胞微环境（eecm）成分的细胞微环境（eecm）来研究eecm对非病毒基因转移的影响。为了能够控制固定的蛋白质和DNA颗粒的数量和构象，eecm将在金上使用自组装的单层来制造。除了使用报告质粒和内化实验来量化基因转移和颗粒内化的效率外，我们还将研究细胞收缩性是否与观察到的不同eecm基因转移的变化有关。在目标2中，我们将研究当细胞被镀在基质（3D）而不是平面（2D）中时的基因转移。我们认为，了解细胞在基质内和在平面上的基因转移过程的不同，对于理解如何增强基因在体内和组织工程构建体内的转移至关重要。我们将使用聚苯乙烯纳米粒子和DNA纳米粒子在3D和2D上研究内吞速率和整体基因转移效率。我们还将研究当细胞被镀在三维空间中时，细胞收缩机制是否也起作用。公共卫生相关性：通过传递编码感兴趣基因的质粒DNA，非病毒基因传递提供了一种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。在这个建议中，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。

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.