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Purification of Circular RNA by Ultrafiltration

超滤纯化环状 RNA

基本信息

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
项目类别：
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

项目摘要

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.

项目总结