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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

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.

摘要