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

描述(申请人提供)：发展非病毒载体用于寡核苷酸递送质粒DNA(PDNA)、反义DNA(AsDNA)和小干扰RNA(SiRNA)仍然是一个重大的科学挑战。目前的方法存在转基因表达水平低(PDNA)、细胞内转运和细胞质降解(PDNA、asDNA、siRNA)等问题。这项研究计划将测试细胞质膜破坏在触发抑制转染和/或表达的细胞反应中的作用。将探索膜中诱导的纳米级气孔的结构和动力学，并确定它们与触发细胞防御机制的关系。将检查细胞膜上纳米尺度的孔的产生，以确定它们是否导致细胞质核酸酶活性的增加。了解非病毒载体诱导的细胞反应对于合理开发这些寡核苷酸递送剂至关重要。这项研究的具体目的是：1)评估聚合物和复合细胞膜的破坏；2)评估和量化质膜通透性在触发细胞防御机制中的作用，从而抑制转染和表达。3)复合结构的设计与量化。 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针对外来寡核苷酸引入的基于细胞的防御，将有助于合理优化载体设计。

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.