The Role of Cell Membrane Disruption in Non-Viral Vector Oligonucleotide Delivery

描述（由申请人提供）：用于质粒DNA（pDNA）、反义DNA（asDNA）和小干扰RNA（siRNA）的寡核苷酸递送的非病毒载体的开发仍然是一项重大的科学挑战。目前的方法存在低转基因表达水平（pDNA）和细胞内转运和细胞质中降解的问题（pDNA、asDNA、siRNA）。这项研究计划将测试细胞质膜破坏在触发细胞反应，抑制转染和/或表达的作用。将探讨在膜中诱导的纳米级孔的结构和动力学，并确定它们与触发细胞防御机制的关系。将检查细胞膜中纳米级孔的产生，以观察它们是否引起细胞质核酸酶活性的增加。理解由非病毒载体诱导的细胞应答对于这些寡核苷酸递送剂的合理开发是至关重要的。本研究的具体目的是：1）评估聚合物和复合物细胞膜破坏2）评估和定量质膜渗透性在触发抑制转染和表达的细胞防御机制中的作用。3)复合物结构的设计和定量。 公共卫生相关性：这项工作对公共卫生很重要，因为它将揭示抑制基因疗法有效使用的细胞机制的细节。了解非病毒载体如何激活细胞防御外来寡核苷酸的引入将允许合理优化载体设计。

DESCRIPTION (provided by applicant): The development of non-viral vectors for oligonucleotide delivery of plasmid DNA (pDNA), antisense DNA (asDNA), and small interfering RNA (siRNA) remains a substantial scientific challenge. Current methods suffer from low transgene expression levels (pDNA) and problems with transport inside the cell and degradation in the cytoplasm (pDNA, asDNA, siRNA). This research program will test the role of cell plasma membrane disruption in triggering cellular responses that inhibit transfection and/or expression. The structure and dynamics of the nanoscale pores induced in the membrane will be explored and their relationship to the triggering of cellular defense mechanisms determined. The creation of nanoscale pores in the cell membrane will be examined to see if they cause an increase in cytoplasmic nuclease activity. Understanding the cellular responses induced by the non-viral vectors is critical to rational development of these oligonucleotide delivery agents. The specific aims of this research are: 1) Assessment of polymer and polyplex cell membrane disruption 2) Assessment and quantification of the role of plasma membrane permeability in triggering cellular defense mechanisms that inhibit transfection and expression. 3) Design and quantification of polyplex structure. PUBLIC HEALTH RELEVANCE: This work is important to public health because it will uncover the details of cellular mechanism that inhibit efficient use of gene therapies. Gaining understanding of how non-viral vectors activate cell-based defenses against the introduction of foreign oligonucleotides will allow rational optimization of vector design.