Nucleolin-mediated cell entry of DNA nanoparticles

描述（由申请人提供）：DNA纳米颗粒从质粒DNA和CK30共价连接到聚乙二醇中自组装，并有效转染气道上皮、视网膜和神经元，但它们如何进入细胞尚不清楚。最近，这些DNA纳米颗粒的表面受体被确定为核仁蛋白。DNA纳米颗粒的基因表达与细胞培养中表面核蛋白的含量直接相关，而体内具有表面核蛋白的组织是最佳靶组织。核仁蛋白的表面表达是如何被调节的，或者缺乏膜跨越结构域或糖脂锚定的蛋白质是如何被保持在膜上的尚不清楚。没有建立从细胞表面到细胞核的非降解途径，然后是核仁蛋白/DNA纳米颗粒复合物。该项目将确定DNA纳米颗粒受体核蛋白到达并停留在细胞表面的机制，以及DNA纳米颗粒进入细胞并被运送到细胞核而不被降解的机制。假设是：1)核蛋白在磷酸化作用下到达表面，并通过与其他蛋白质的相互作用保持在表面，其中一些蛋白质对DNA纳米颗粒的结合和摄取很重要；2)核蛋白/DNA纳米颗粒复合物到达细胞核的非降解途径容易受到药理学操纵。为了验证这些假设，将确定细胞表面DNA纳米颗粒和核蛋白的蛋白质组，并通过siRNA敲除测试鉴定的蛋白质的重要性。荧光显微镜、细胞分离、抑制剂研究和siRNA分析将用于确定通过细胞的运输。细胞表面核蛋白的磷酸化状态将用质谱法检测。通过这种方式，DNA纳米颗粒进入细胞的途径调控的全面图景将会出现，并提出可行的药物靶点。如果我们取得成功，我们将提高对进入细胞核的途径的理解，而不审查货物-病原体以及治疗药物（如DNA纳米粒子）所利用的途径。如果我们成功了，我们将拥有一个治疗设备来改善DNA纳米粒子的基因传递。

DESCRIPTION (provided by applicant): DNA nanoparticles self assemble from plasmid DNA and CK30 covalently linked to polyethylene glycol, and effectively transfect airway epithelium, retina, and neurons, but how they access cells has been obscure. Recently the surface receptor for these DNA nanoparticles has been identified as nucleolin. Gene expression from DNA nanoparticles varies directly with the amount of surface nucleolin in cell culture, and the best target tissues in vivo are those with surface nucleolin. How surface expression of nucleolin is regulated, or a protein lacking membrane spanning domain or glycolipid anchor is held at the membrane is unclear. The non-degradative route from cell surface to nucleus followed by the nucleolin/DNA nanoparticle complex is not established. This project will determine the mechanisms by which the DNA nanoparticle receptor, nucleolin, arrives at and is held at the cell surface, as well as the mechanism by which DNA nanoparticles enter the cell and are transported to the nucleus without degradation. The hypotheses are: 1) nucleolin reaches the surface in response to phosphorylation and is held at the surface by interaction with other proteins, some of which are important for DNA nanoparticle binding and uptake 2) the non degradative pathway taken by the nucleolin/DNA nanoparticle complex to the nucleus is susceptible to pharmacologic manipulation. To test these hypotheses, the proteome of DNA nanoparticles and nucleolin at the cell surface will be determined and the importance of the proteins identified tested by siRNA knockout. Fluorescence microscopy, cell fractionation, inhibitor studies, and siRNA analysis will be used to determine the trafficking through the cell. THe phosphorylation state of nucleolin at the cell surface will be tested by mass spectrometry. In this way, a comprehensive picture of the regulation of the route into the cell for DNA nanoparticles will emerge and drug able targets will be suggested. If we are successful, we will improve understanding of pathways into the cell that reach the nucleus without censoring the cargo - pathways which are utilized by pathogens as well as therapeutic agents such as DNA nanoparticles. If we are successful, we will have at our disposal a therapeutic armamentarium to improve gene delivery from DNA nanoparticles. PUBLIC HEALTH RELEVANCE: DNA nanoparticles are promising for gene therapy because they transfect nondividing cells, are non-toxic, non- immunogenic, efficient in some tissues, and can be prepared for high level, long term expression. Manipulating their trafficking to improve transfection improves their versatility, cost-effectiveness, and suitability for human use. In addition, the non-degradative pathway they follow in the cell is probably shared by some pathogens. Further understanding of this route may be important in regulating it. .