Purification of Circular RNA by Ultrafiltration

超滤纯化环状 RNA

• 批准号: 10426638
• 负责人: Scott M Husson
• 金额: $ 21.78万
• 依托单位: CLEMSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10426638
• 关键词: Address; Adopted; Area; Biological Process; Biology; Biomedical Research; COVID-19 prevention; COVID-19 vaccine; Cell Count; Cells; Code; DNA; Data; Development; Disease; Emerging Communicable Diseases; Flow Cytometry; Goals; Half-Life; High Pressure Liquid Chromatography; Hour; Immune response; Knowledge; Lead; Measures; Membrane; Messenger RNA; Methodology; Methods; Molecular Weight; Output; Pathology; Performance; Physiology; Play; Polymers; Production; Property; Proteins; Protocols documentation; RNA; RNA Conformation; RNA Precursors; RNA Splicing; Reaction; Recovery; Research; Resolution; Role; Sampling; Shapes; Structure; Technology; Therapeutic; Ultrafiltration; Vaccine Antigen; Vaccine Production; base; circular RNA; conformer; cytotoxicity; experimental study; feasibility testing; immunogenicity; in vivo; innovation; live cell imaging; macromolecule; novel; novel strategies; plasmid DNA; prevent; protein expression; public health relevance; therapeutic protein; therapeutic vaccine; transmission process

Project Summary Circular RNA (circRNA) has important, understudied roles all across physiology and disease. Additionally, circularization of protein-coding RNA is a promising strategy for increasing the duration and quantity of therapeutic protein production and decreasing immunogenicity relative to linear messenger RNA (mRNA), such as those used to develop the first COVID-19 vaccines. Unfortunately, the synthesis of circRNA produces solutions that contain contaminating linear RNA precursors and nicked RNA that cause cellular immune responses; and no effective purification method has been demonstrated. The goal of this project is to test the feasibility of a novel concept to purify circular RNA with high purity and high yield. The proposed study has strong potential to advance biomedical research aimed at answering questions about the roles of circRNA in disease and biological function, where high sample purity is essential. It also has the potential to have a significant impact on the development of and access to circRNA therapies by demonstrating a high yield approach for their purification that does not exist today. Our research hypothesis is that membrane ultrafiltration can be used to separate RNA based on shape and size. We theorize that RNA transmission through ultrafiltration membranes will begin to occur at a critical flux (quotient of flowrate and membrane area) due to flow-induced elongation. Differences in elongational properties among RNA conformers will lead to differences in critical fluxes, which provides a basis for the purification of circRNA from linear RNA impurities. The Specific Aims are to (1) quantify ultrafiltration critical fluxes for circRNA, linear RNA precursor, and nicked RNA; (2) develop a protocol for purification of circRNA generated from self-splicing reactions, and (3) demonstrate protein production in cells for ultrafiltration-purified circRNA. In Aim 1, we will produce purified fractions of the different RNA conformers and accurately measure their transmission through ultrafiltration membranes to quantify their critical fluxes based on membrane properties. New technical knowledge generated using purified RNA conformers will identify flux conditions that can be used for the separation of circRNA from linear contaminants in self-splicing reaction solutions. In Aim 2, we will establish an easy to implement protocol that exploits differences in these critical fluxes to purify circRNA from self-splicing reaction solutions. This aim will advance knowledge on the key factors influencing circRNA purity and yield. In Aim 3, we will measure the quantity and evaluate the stability of protein production in cells for ultrafiltration-purified circRNA relative to circRNA purified using high-performance liquid chromatography, which represents the state-of-the-art approach. This project will provide a clear proof of concept for ultrafiltration-based purification of circRNA with high purity (95%) and high yield (>70%), which would be a significant advancement over the state of the art.

项目概要 环状 RNA (circRNA) 在生理学和疾病中具有重要的、尚未被研究的作用。此外， 蛋白质编码RNA的环化是一种有前途的策略，可以增加环化的持续时间和数量 治疗性蛋白质的产生并降低相对于线性信使 RNA (mRNA) 的免疫原性，例如 用于开发第一批 COVID-19 疫苗的疫苗。不幸的是，circRNA的合成产生 含有污染性线性 RNA 前体和切口 RNA 的溶液会导致细胞免疫 回应；并且尚未证明有效的纯化方法。该项目的目标是测试 以高纯度和高产量纯化环状RNA的新概念的可行性。拟议的研究有 推进生物医学研究的巨大潜力，旨在回答有关 circRNA 在 疾病和生物功能，其中高样品纯度至关重要。它还具有潜力 通过展示高产量对 circRNA 疗法的开发和获取产生重大影响 当今还不存在的纯化方法。我们的研究假设是膜 超滤可用于根据形状和大小分离 RNA。我们推测 RNA 传输 通过超滤膜将在临界通量（流量与膜面积的商）处开始发生 由于流动引起的伸长。 RNA构象异构体之间伸长特性的差异将导致 临界通量的差异，为从线性RNA杂质中纯化circRNA提供了基础。 具体目标是 (1) 量化 circRNA、线性 RNA 前体和切口 RNA 的超滤临界通量 核糖核酸； (2) 制定纯化自剪接反应生成的 circRNA 的方案，以及 (3) 证明细胞中超滤纯化的 circRNA 的蛋白质生产。在目标 1 中，我们将生产纯化的 不同 RNA 构象异构体的分数，并通过超滤准确测量它们的传输 膜根据膜特性量化其临界通量。新技术知识 使用纯化的 RNA 构象异构体生成的产物将确定可用于分离的通量条件 来自自剪接反应溶液中线性污染物的 circRNA。在目标 2 中，我们将建立一个易于 实施利用这些关键通量的差异从自剪接反应中纯化 circRNA 的方案 解决方案。这一目标将增进对影响 circRNA 纯度和产量的关键因素的了解。在目标 3 中， 我们将测量细胞中蛋白质生产的数量并评估其稳定性，以进行超滤纯化 circRNA相对于使用高效液相色谱纯化的circRNA，代表 最先进的方法。该项目将为基于超滤的纯化提供清晰的概念证明 高纯度（95%）和高产率（>70%）的circRNA，这将是一个显着的进步 最先进的。