Nucleolin-mediated cell entry of DNA nanoparticles

描述(申请人提供)：DNA纳米颗粒由质粒DNA和CK30共价连接到聚乙二醇处自组装，并有效地转染呼吸道上皮、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已被确定为核仁素。DNA纳米粒子的基因表达与细胞培养中表面核仁量的多少直接相关，体内最好的靶向组织是那些具有表面核仁量的组织。核仁的表面表达是如何调节的，或者缺乏跨膜结构域或糖脂锚定的蛋白质是如何被保持在膜上的，目前尚不清楚。核仁/DNA纳米颗粒复合体从细胞表面到细胞核的非降解途径尚未建立。该项目将确定DNA纳米颗粒受体核仁素到达细胞表面并保持在细胞表面的机制，以及DNA纳米粒子进入细胞并被运输到细胞核而不被降解的机制。假设是：1)核仁会通过磷酸化反应到达表面，并通过与其他蛋白质的相互作用而被保持在表面，其中一些蛋白质对DNA纳米颗粒的结合和摄取是重要的。2)核仁素/DNA纳米颗粒复合体到细胞核的非降解途径容易受到药物操纵的影响。为了验证这些假设，将确定DNA纳米颗粒和细胞表面核仁蛋白的蛋白质组，并通过siRNA敲除来测试所识别的蛋白质的重要性。将使用荧光显微镜、细胞分级、抑制剂研究和siRNA分析来确定通过细胞的转运。核仁在细胞表面的磷酸化状态将通过质谱学进行检测。这样，DNA纳米颗粒进入细胞的途径的调控将出现全面的图景，并将提出药物靶点。如果我们成功了，我们将提高对到达细胞核的细胞通路的理解，而不会审查病原体以及DNA纳米颗粒等治疗剂所利用的通路。如果我们成功了，我们将拥有一个治疗性的医疗设备来改善DNA纳米颗粒的基因输送。 与公共健康相关：DNA纳米粒子在基因治疗方面很有希望，因为它们可以转染未分裂的细胞，无毒，无免疫原性，在某些组织中有效，并且可以为高水平的长期表达做好准备。操纵它们的贩运以提高转染率可以提高它们的通用性、成本效益和人类使用的适合性。此外，它们在细胞中遵循的非降解途径可能与一些病原体相同。对这条路线的进一步了解可能对监管它很重要。。

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. .