Large scale in vitro production of capped polyadenylated mRNA-based vaccines in solid phase using immobilized enzymes

使用固定化酶在固相中大规模体外生产基于封端聚腺苷酸化 mRNA 的疫苗

基本信息

批准号：
10200307
负责人：
Maria Kireeva
金额：
$ 29.72万
依托单位：
AFFINITY MOLECULES, LLC
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-23 至 2023-09-22
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10200307
关键词：
2019-nCoV Affect Bacteriophages Biomedical Research Catalytic Domain Cultured Cells DNA DNA-Directed RNA Polymerase Deoxyribonuclease I Development Disease Outbreaks Enzymatic Biochemistry Enzymes Escherichia coli Excision Exclusion Foundations General Population Genetic Transcription Human Resources Immobilization Immobilized Enzymes In Vitro Influenza Lysine Mammalian Cell Medical Medicine Messenger RNA Methylation Methyltransferase Modeling Modification Mole the mammal Mutation Natural regeneration Organism Phase Plant Resins Polyadenylation Polynucleotide Adenylyltransferase Preparation Process Production Proteins Protocols documentation Public Health Applications Research Publishing RNA RNA purification RNA vaccine Reaction Reagent Recycling Research Risk SARS-CoV-2 spike protein Sepharose Solid Superhelical DNA T7 RNA polymerase Technology Testing Therapeutic Therapeutic Agents Therapeutic Uses Time Transcription Initiation Translations Untranslated RNA Vaccine Production Vaccines Vaccinia Vaccinia virus Virus absorption analog base clinically relevant coronavirus vaccine cost cost effectiveness dimer gene therapy genetic information his6 tag induced pluripotent stem cell influenza virus vaccine influenzavirus large scale production mRNA capping mRNA delivery novel vaccines personalized medicine polyadenylated messenger RNA protein expression protein purification seasonal influenza therapeutically effective tool vaccine development vector

2019-nCoV Affect Bacteriophages Biomedical Research Catalytic Domain Cultured Cells DNA DNA-Directed RNA Polymerase Deoxyribonuclease I Development Disease Outbreaks Enzymatic Biochemistry Enzymes Escherichia coli Excision Exclusion Foundations General Population Genetic Transcription Human Resources Immobilization Immobilized Enzymes In Vitro Influenza Lysine Mammalian Cell Medical Medicine Messenger RNA Methylation Methyltransferase Modeling Modification Mole the mammal Mutation Natural regeneration Organism Phase Plant Resins Polyadenylation Polynucleotide Adenylyltransferase Preparation Process Production Proteins Protocols documentation Public Health Applications Research Publishing RNA RNA purification RNA vaccine Reaction Reagent Recycling Research Risk SARS-CoV-2 spike protein Sepharose Solid Superhelical DNA T7 RNA polymerase Technology Testing Therapeutic Therapeutic Agents Therapeutic Uses Time Transcription Initiation Translations Untranslated RNA Vaccine Production Vaccines Vaccinia Vaccinia virus Virus absorption analog base clinically relevant coronavirus vaccine cost cost effectiveness dimer gene therapy genetic information his6 tag induced pluripotent stem cell influenza virus vaccine influenzavirus large scale production mRNA capping mRNA delivery novel vaccines personalized medicine polyadenylated messenger RNA protein expression protein purification seasonal influenza therapeutically effective tool vaccine development vector

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项目摘要

ABSTRACT RNA emerges as a promising therapeutic agent and is becoming an increasingly popular tool for delivery of genetic information to cultured cells and living organisms. Notably, mRNA is used as a basis for new vaccine development and personalized gene therapy and is replacing DNA vectors in a variety of applications. The high cost of mRNA production currently limits the widespread use of mRNA-based therapeutics, such as introduction of RNA-based flu vaccine or coronavirus vaccine for general population. For all research and medical applications, mRNA is produced by in vitro transcription of linear DNA templates with single-subunit RNA polymerases (RNAPs) from bacteriophages. The requirement for mRNA capping complicates its straightforward production. Co-transcriptional capping, with RNAP incorporating a cap analogue during transcription initiation, compromises efficiency of both transcription and capping, resulting in significantly decreased mRNA yield. Alternatively, mRNA can be purified from transcription reaction and then modified post-transcriptionally with capping enzymes, which are also expensive to produce and purify. RNA polymerases and mRNA modifying enzymes are irreversibly denatured and destroyed during mRNA purification. Development of a technology that allows reusing of the enzymes will significantly decrease the mRNA manufacturing costs, thus supporting more widespread therapeutic uses of mRNA. We propose to create a sequential pipeline for mRNA production, in which the enzymes are immobilized and used in multiple consecutive cycles of in vitro transcription, mRNA capping, and polyadenylation. First, we will synthesize mRNA encoding influenza virus haemagglutinin (HA) and SARS-CoV-2 spike (S) protein using immobilized T7 RNAP. We will establish the conditions for RNAP immobilization, regeneration, and repeated transcription cycles which, compared to a batch reaction in solution, will significantly increase the mRNA yield per unit of RNAP. Next, the HA and SARS-CoV-2 S protein mRNAs will be capped using the vaccinia virus capping enzyme immobilized via its catalytic subunit. Repeated cycles of capping using the same preparation of the immobilized enzyme will be used to determine its robustness, rigor, stability and the limits of the enzyme recycling. The successful completion of the proposed Phase I research will serve as a foundation for the complete pipeline of functional mRNA production. It will increase the mRNA yield and promote purification of the final product, eliminating the need for protein destruction after each enzymatic cycle. It is applicable in various fields of biomedical research and medicine relying on the in vitro synthesis of mRNA and, particularly, will enhance the cost-effectiveness of mRNA-based vaccine manufacturing.

抽象的 RNA成为一种有前途的治疗剂，并且正在成为越来越受欢迎的工具培养细胞和生物体的遗传信息。值得注意的是，mRNA被用作新疫苗的基础开发和个性化基因疗法，正在替代各种应用中的DNA载体。高 mRNA生产的成本目前限制了基于mRNA的治疗剂的广泛使用，例如介绍用于普通人群的基于RNA的流感疫苗或冠状病毒疫苗。用于所有研究和医学应用，mRNA是通过用单sumunit RNA的线性DNA模板在体外转录产生的来自噬菌体的聚合酶（RNAP）。 mRNA封盖的要求使其简单明了生产。共转录封顶，RNAP在转录启动过程中包含帽类似物，损害了转录和封盖的效率，导致mRNA产量显着降低。或者，可以从转录反应中纯化mRNA，然后在转录后与上限酶，生产和净化也很昂贵。 RNA聚合酶和mRNA修饰在mRNA纯化过程中，酶是不可逆转的变性和破坏的。开发技术允许重复酶会大大降低mRNA制造成本，从而支持更多 mRNA的广泛治疗用途。我们建议创建一个用于mRNA产生的顺序管道，酶被固定并用于多个连续的体外转录循环上限和聚腺苷酸化。首先，我们将合成编码流感病毒血凝素（HA）和使用固定的T7 RNAP使用SARS-COV-2尖峰蛋白。我们将建立RNAP的条件固定，再生和重复的转录周期，与溶液中的批处理反应相比每单位RNAP的mRNA产量将显着增加。接下来，HA和SARS-COV-2 S蛋白mRNA 将使用通过其催化亚基固定的疫苗病毒限制酶上限。重复的周期使用固定酶的相同制剂的封盖将用于确定其稳健性，严格性，稳定性和酶回收的极限。拟议的第一阶段研究的成功完成将作为功能mRNA产生的完整管道的基础。它将增加mRNA产量并促进最终产品的纯化，消除了每种酶促后对蛋白质破坏的需求循环。它适用于依赖于体外合成的生物医学研究和医学领域 mRNA，尤其是将增强基于mRNA的疫苗制造的成本效益。