Purification of Circular RNA by Ultrafiltration

超滤纯化环状 RNA

• 批准号: 10629427
• 负责人: Scott M Husson
• 金额: $ 17.91万
• 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 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10629427
• 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; Measures; Membrane; Messenger RNA; Methodology; Methods; Molecular Weight; Output; Pathology; Performance; Physiology; Play; Polymers; Predisposition; Production; Property; Proteins; Protocols documentation; RNA; RNA Conformation; RNA Precursors; RNA Splicing; RNA purification; Reaction; Recovery; Research; Resolution; Role; Sampling; Shapes; Structure; Technology; Therapeutic; Transfection; Ultrafiltration; Vaccine Antigen; Vaccine Production; circular RNA; conformer; cytotoxicity; experimental study; feasibility testing; immunogenicity; in vivo; innovation; live cell imaging; macromolecule; novel; novel strategies; plasmid DNA; prevent; protein expression; protein purification; public health relevance; theories; therapeutic RNA; 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.

项目摘要 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。另外， 蛋白质编码RNA的环化是一种很有前途的策略，可以增加持续时间和数量 与线性信使RNA(MRNA)相关的治疗性蛋白质产生和免疫原性降低，如 就像那些用于开发第一批新冠肺炎疫苗的疫苗一样。不幸的是，CircRNA的合成产生了 含有污染的线性RNA前体和可引起细胞免疫的刻痕RNA的溶液 反应；目前还没有有效的纯化方法。该项目的目标是测试 高纯度、高产率纯化环状RNA新概念的可行性。拟议的研究已经 推动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，其中高样本纯度是必不可少的。它还有可能拥有一种 通过展示高产量，对CircRNA疗法的开发和获得产生重大影响 目前尚不存在的提纯方法。我们的研究假设是膜 超滤可用于根据形状和大小分离RNA。我们理论上认为，RNA的传播 通过超滤膜将开始发生在一个临界通量(流量和膜面积的商) 由于流动诱导的伸长。RNA构象之间的伸长特性的差异将导致 临界通量的差异，这为从线状RNA杂质中纯化CircRNA提供了基础。 具体目的是(1)定量测定CircRNA、线性RNA前体和镍的超滤临界通量。 RNA；(2)制定纯化自剪接反应产生的CircRNA的方案；以及(3) 展示用于超滤纯化的CircRNA在细胞中的蛋白质生产。在目标1中，我们将生产纯化的 不同RNA构象的级分，并通过超滤准确测量它们的透过率 根据膜的性质，确定膜的临界通量。新技术知识 使用纯化的RNA构象产生的将识别可用于分离的熔剂条件 自剪接反应溶液中线性污染物的CircRNA。在目标2中，我们将建立一个简单的 实现利用这些关键通量的差异从自剪接反应中提纯CircRNA的方案 解决办法。这一目标将促进对影响CircRNA纯度和产量的关键因素的了解。在《目标3》中， 我们将测量超滤纯化细胞中蛋白质产量并评估其稳定性。 CircRNA相对CircRNA，用高效液相色谱纯化，它代表 最先进的方法。该项目将为超滤净化提供一个清晰的概念证明。 具有高纯度(95%)和高产率(70%)的CircRNA，这将是比 最先进的技术。