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

• 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
• 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

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递送工具 培养细胞和活生物体的遗传信息。值得注意的是，mRNA 被用作新疫苗的基础 开发和个性化基因治疗，并在各种应用中取代 DNA 载体。高 目前，mRNA 生产成本限制了基于 mRNA 的疗法的广泛使用，例如引入 用于普通人群的基于 RNA 的流感疫苗或冠状病毒疫苗。对于所有研究和医疗 应用中，mRNA是通过线性DNA模板与单亚基RNA的体外转录产生的 来自噬菌体的聚合酶（RNAP）。 mRNA 加帽的要求使其简单的方法变得复杂 生产。共转录加帽，RNAP 在转录起始过程中掺入帽类似物， 损害转录和加帽的效率，导致 mRNA 产量显着降低。 或者，可以从转录反应中纯化 mRNA，然后用 封端酶的生产和纯化成本也很高。 RNA聚合酶和mRNA修饰 在 mRNA 纯化过程中，酶会发生不可逆的变性和破坏。开发一项技术 允许重复使用酶将显着降低 mRNA 制造成本，从而支持更多 mRNA 的广泛治疗用途。我们建议为 mRNA 生产创建一个连续的管道， 其中酶被固定并用于体外转录、mRNA 的多个连续循环 封端和聚腺苷酸化。首先，我们将合成编码流感病毒血凝素（HA）的mRNA和 使用固定化 T7 RNAP 的 SARS-CoV-2 刺突 (S) 蛋白。我们将为 RNAP 创造条件 固定、再生和重复转录循环，与溶液中的批量反应相比， 将显着增加每单位 RNAP 的 mRNA 产量。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痘苗病毒加帽酶进行加帽。重复循环 使用与固定化酶相同的制剂进行封盖将用于确定其稳健性、严谨性、 稳定性和酶回收的限制。拟议的第一阶段研究的成功完成将 作为功​​能性 mRNA 生产完整流程的基础。它将增加 mRNA 产量 并促进最终产品的纯化，无需在每次酶解后破坏蛋白质 循环。依靠体外合成，适用于生物医学研究和医学的各个领域 mRNA，尤其将提高基于 mRNA 的疫苗生产的成本效益。