Nanomanipulation of analysis of lipoplex assembly

描述（由申请人提供）：基因治疗有望治疗各种遗传疾病，包括癌症、糖尿病和许多其他遗传和获得性疾病。基因治疗的成功临床应用需要开发有效的工具，将核酸靶向递送到受影响的细胞中。目前使用的基于病毒的DNA传递系统会产生严重的免疫和肿瘤副作用。基于脂质的DNA传递系统被广泛认为是一种潜在的更安全的替代方案。有效的基于脂质DNA纳米颗粒（脂质体）的发展受到对DNA如何装入脂质囊泡的了解不足的限制。该项目旨在利用单DNA操作表征脂丛的组装。初步研究揭示了脂质- dna相互作用的一些机制细节，这些细节是使用更传统的批量方法无法检测到的。我们计划评估脂质与DNA的相互作用可以与脂质体组装效率和细胞转染效率相关的想法。该项目的具体目标是(i)通过DOTAP量化单个DNA的变形，（ii）比较一系列脂质压缩DNA的能力；（iii）将单分子数据与组装的脂丛的质量联系起来。本研究的完成将评估单分子数据可以改善脂质体组装和转染效率的想法。如果这是真的，这将使含有纳米颗粒的DNA的组装成为一个更可预测的过程，并为更合理地设计非病毒DNA传递系统奠定基础。这反过来可能有助于发展适合临床应用的脂丛，从而使治疗疾病的新干预成为可能。

DESCRIPTION (provided by applicant): Gene therapy holds a promise to treat a variety of genetic disorders including cancer, diabetes and many other inherited and acquired diseases. Successful clinical application of gene therapy requires the development of efficient tools for targeted delivery of nucleic acids into affected cells. Presently used virus-based DNA delivery systems produce serious immunological and oncological side effects. Lipid-based DNA delivery systems are widely viewed as a potentially safer alternative. The development of effective lipid-based DNA nanoparticles (lipoplexes) is limited by poor understanding of how DNA is packed into the lipid vesicles. This project aims at characterizing the assembly of lipoplexes using single DNA manipulations. Preliminary studies revealed a number of mechanistic details of lipid-DNA interaction that were not detected using more conventional, bulk methods. We plan to evaluate the idea that the interactions of lipids with DNA can be correlated with the efficiency of lipoplex assembly and cell transfection efficacy. Specific aims of the project are (i) Quantify deformation of single DNA by DOTAP, (ii) compare a series of lipids in their ability to compact DNA; (iii) correlate the single molecule data with the quality of assembled lipoplexes. The completion of this study will evaluate the idea that single molecule data can improve the assembly of lipoplexes and transfection efficiency. If true, this would make the assembly of DNA containing nanoparticles a much more predictable process and would lay grounds for more rational design of non-viral DNA delivery systems. This in turn may help develop lipoplexes suitable for clinical applications and thereby enable novel interventions in treating disease. PUBLIC HEALTH RELEVANCE: Gene therapy could potentially be used to treat a variety of inherited and acquired ailments including cancer, diabetes, aging and heart and neurological disorders. The development of non-viral, lipid-based DNA delivery vectors could help design efficient gene therapies with reduced side effects. We plan to evaluate the idea that the design of such vectors can be greatly improved using single DNA nanomanipulation technique.