Novel Separation Methods for exRNA Carriers: Extracellular Vesicles, Lipoprotein Particles, and Protein Aggregates

exRNA 载体的新型分离方法：细胞外囊泡、脂蛋白颗粒和蛋白质聚集体

• 批准号: 10470432
• 负责人: Kenneth W Witwer
• 金额: $ 54.91万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10470432
• 关键词: Novel; Separation; Methods; exRNA; Carriers

ABSTRACT Extracellular RNA (exRNA) is a particularly attractive molecular component of liquid biopsy because RNA species can be specifically amplified. Of the three major classes of exRNA vehicle—extracellular vesicles (EVs), lipoprotein particles (LPPs), and free ribonucleoproteins (RNPs)—EVs have so far received the most attention. Within each class, there is also tremendous diversity by physical characteristics of size, density, and surface charge. Indeed, to our knowledge, no study to date has profiled exRNA in multiple members of the three carrier classes that have been isolated rigorously from the same samples. There is a strong need to develop new strategies and controls to ensure that comparisons of exRNA carriers are not confounded by co- isolation (different classes of carriers present in the same fraction) or contamination (detection of uncomplexed and/or foreign RNAs introduced during sample collection and processing). To this end, we assemble a team of experts on EVs, LPPs, and RNPs, along with experts in cutting-edge separation and characterization methods. In an initial UG3 phase, we will first (Aim 1) use a combination of state-of-the-field physical and biochemical separation methods to separate a library of eight subtypes of EVs, LPPs, and RNPs from the same biological samples and with the best achievable purity. “Gold standard” proteomic, lipidomic, glycomic, and RNomic datasets will be generated. Carefully designed “process” controls will for the first time establish an across-the- board baseline of contaminants and other artifacts that may complicate interpretation. In Aim 2, we will test asymmetric field-flow fractionation (AF4) and affinity capture platforms including the ExoView platform and sensitive electrochemical sensors as superior alternatives to the most commonly used legacy method, differential centrifugation. We will seek gains in speed, resolution, and purity compared with legacy techniques. If go/no-go criteria are met by the end of the second year (UG3), we will proceed to a UH3 phase. This phase will include an Aim 3, validating results in multiple locations and with approximately 6 times the original sample numbers to account for influence of sex and age. The AF4 method will be further developed with additional modifications based on our engineering and analytical chemistry expertise, while ExoView technology will be harnessed to screen antibodies and other affinity materials for rapid isolations and to detect abundant RNA species directly in immobilized exRNA carriers. Finally, an Aim 4 will assess the biological factor of diet with valuable samples from intervention studies, along with the possible desirability of collecting samples in RNase inhibitors to preserve more fragile RNA species. Overall, we hypothesize that 1) AF4, on its own or with methodologic modifications, as well as 2) novel affinity separation approaches will improve substantially on ultracentrifuge-based methods in ease and purity and on current state-of-the-art but tedious and lengthy exRNA carrier separation methods.

摘要 细胞外rna(ExRNA)是液体活检中一个特别吸引人的分子成分，因为rna 物种可以被特别放大。外源RNA载体的三大类--胞外小泡 (EVS)、脂蛋白颗粒(LPP)和游离核糖核蛋白(RNPs)-EVS到目前为止收到的最多 请注意。在每个类别中，还存在着巨大的多样性，其物理特征包括大小、密度和 表面电荷。事实上，据我们所知，到目前为止还没有研究在多个成员中分析exRNA。 从相同样本中严格分离的三个携带者类别。有强烈的需要 制定新的战略和控制措施，以确保外源RNA携带者的比较不会因共同感染而混淆 隔离(同一组分中存在不同类别的载体)或污染(检测未复合的 和/或在样本收集和处理过程中引入的外来RNA)。为此，我们组建了一个团队， 电动汽车、LPP和RNP方面的专家，以及尖端分离和表征方法方面的专家。 在最初的UG3阶段，我们将首先(目标1)使用现场物理和生化状态的组合 从同一生物中分离EVS、LPP和RNPs八个亚型文库的分离方法 样品和可达到的最佳纯度。“黄金标准”蛋白质组、脂组、糖组和核糖体 将生成数据集。精心设计的“过程”控制将首次建立一个全面的 可能使解释复杂化的污染物和其他人工制品的板基线。在目标2中，我们将测试 不对称场流分馏(AF4)和亲和捕获平台，包括ExoView平台和 灵敏的电化学传感器作为最常用的传统方法的卓越替代品， 差速离心法。与传统技术相比，我们将寻求在速度、分辨率和纯度方面的改进。 如果在第二年年底(UG3)达到通过/不通过标准，我们将进入UH3阶段。这一阶段 将包括AIM 3，在多个地点验证结果，大约是原始样本的6倍 考虑性别和年龄影响的数字。AF4方法将进一步发展，还将增加 基于我们的工程和分析化学专业知识进行修改，而ExoView技术将 用于筛选抗体和其他亲和材料，用于快速分离和检测丰富的RNA 物种直接在固定化外源RNA载体中。最后，目标4将通过以下方式评估饮食的生物学因素 来自干预研究的有价值的样本，以及收集核糖核酸酶样本的可能性 抑制剂，以保存更脆弱的RNA物种。总体而言，我们假设1)AF4)本身或与 方法的改进，以及2)新的亲和分离方法将大大改善 超速离心法简单易行，纯度高，目前最先进，但繁琐冗长 外源RNA载体分离方法。