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使用体外转录有效合成高完整性的 mRNA。

Effective Synthesis of High-Integrity mRNA Using In Vitro Transcription.

机构信息

College of Biology and the Environment, Nanjing Forestry University, Nanjing 210037, China.

Vazyme Biotech Co., Ltd., Nanjing 210037, China.

出版信息

Molecules. 2024 May 23;29(11):2461. doi: 10.3390/molecules29112461.

DOI:10.3390/molecules29112461
PMID:38893337
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11173937/
Abstract

mRNA vaccines are entering a period of rapid development. However, their synthesis is still plagued by challenges related to mRNA impurities and fragments (incomplete mRNA). Most impurities of mRNA products transcribed in vitro are mRNA fragments. Only full-length mRNA transcripts containing both a 5'-cap and a 3'-poly(A) structure are viable for in vivo expression. Therefore, RNA fragments are the primary product-related impurities that significantly hinder mRNA efficacy and must be effectively controlled; these species are believed to originate from either mRNA hydrolysis or premature transcriptional termination. In the manufacturing of commercial mRNA vaccines, T7 RNA polymerase-catalyzed in vitro transcription (IVT) synthesis is a well-established method for synthesizing long RNA transcripts. This study identified a pivotal domain on the T7 RNA polymerase that is associated with erroneous mRNA release. By leveraging the advantageous properties of a T7 RNA polymerase mutant and precisely optimized IVT process parameters, we successfully achieved an mRNA integrity exceeding 91%, thereby further unlocking the immense potential of mRNA therapeutics.

摘要

mRNA 疫苗正进入快速发展阶段。然而，它们的合成仍然受到与 mRNA 杂质和片段（不完全的 mRNA）相关的挑战的困扰。体外转录的 mRNA 产品的大多数杂质都是 mRNA 片段。只有含有 5' 帽和 3' 多聚（A）结构的全长 mRNA 转录本才有在体内表达的活力。因此，RNA 片段是主要的与产品相关的杂质，会显著阻碍 mRNA 的疗效，必须进行有效的控制；这些物质被认为来源于 mRNA 水解或过早的转录终止。在商业 mRNA 疫苗的制造中，T7 RNA 聚合酶催化的体外转录（IVT）合成是合成长 RNA 转录本的一种成熟方法。本研究确定了 T7 RNA 聚合酶上与错误的 mRNA 释放相关的关键结构域。通过利用 T7 RNA 聚合酶突变体的优势特性和精确优化的 IVT 工艺参数，我们成功实现了超过 91%的 mRNA 完整性，从而进一步挖掘了 mRNA 疗法的巨大潜力。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7416/11173937/5b36d2449e75/molecules-29-02461-g009a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7416/11173937/006d3289d3cd/molecules-29-02461-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7416/11173937/463226e3929e/molecules-29-02461-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7416/11173937/c4ee070fe797/molecules-29-02461-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7416/11173937/e4b17a1912b6/molecules-29-02461-g004a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7416/11173937/c5f2db849153/molecules-29-02461-g005a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7416/11173937/e8a80947aaae/molecules-29-02461-g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7416/11173937/fb99dfecef8e/molecules-29-02461-g007.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7416/11173937/0e7763be5e2f/molecules-29-02461-g008.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7416/11173937/5b36d2449e75/molecules-29-02461-g009a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7416/11173937/006d3289d3cd/molecules-29-02461-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7416/11173937/463226e3929e/molecules-29-02461-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7416/11173937/c4ee070fe797/molecules-29-02461-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7416/11173937/e4b17a1912b6/molecules-29-02461-g004a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7416/11173937/c5f2db849153/molecules-29-02461-g005a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7416/11173937/e8a80947aaae/molecules-29-02461-g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7416/11173937/fb99dfecef8e/molecules-29-02461-g007.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7416/11173937/0e7763be5e2f/molecules-29-02461-g008.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7416/11173937/5b36d2449e75/molecules-29-02461-g009a.jpg

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Effective synthesis of circRNA via a thermostable T7 RNA polymerase variant as the catalyst.通过热稳定的T7 RNA聚合酶变体作为催化剂有效合成环状RNA。
Front Bioeng Biotechnol. 2024 Apr 9;12:1356354. doi: 10.3389/fbioe.2024.1356354. eCollection 2024.
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Single-molecule RNA sizing enables quantitative analysis of alternative transcription termination.
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Anal Chem. 2024 Mar 5;96(9):3886-3897. doi: 10.1021/acs.analchem.3c05539. Epub 2024 Feb 20.
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From rejection to the Nobel Prize: Karikó and Weissman's pioneering work on mRNA vaccines, and the need for diversity and inclusion in translational immunology.从被拒之门外到诺贝尔奖：卡里科和魏斯曼在 mRNA 疫苗方面的开创性工作，以及转化免疫学领域多样性和包容性的必要性。
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