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

摘要 RNA作为一种很有前途的治疗剂出现，并成为一种越来越受欢迎的递送工具 将遗传信息传递给培养细胞和活的有机体。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。高潮 目前，信使核糖核酸的生产成本限制了基于信使核糖核酸的疗法的广泛使用，例如引入 针对普通人群的基于RNA的流感疫苗或冠状病毒疫苗。用于所有研究和医学 应用，通过体外转录带有单亚基RNA的线性DNA模板来产生mRNA 噬菌体聚合酶(RNAP)。对mrna封顶的要求使其简单明了。 制作。共转录封顶，在转录起始过程中RNAP加入CAP类似物， 降低了转录和封顶的效率，导致mRNA产量显著下降。 或者，可以从转录反应中提纯mrna，然后在转录后用 封端酶，这也是昂贵的生产和纯化。RNA聚合酶与mRNA修饰 在信使核糖核酸的纯化过程中，酶发生不可逆变性和破坏。一种技术的发展 允许酶的重复使用将显著降低mRNA的制造成本，从而支持更多 信使核糖核酸的广泛治疗用途。我们建议为mRNA的生产建立一条连续的管道，在 这些酶被固定并在体外转录的多个连续循环中使用 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)和 利用固定化T7RNAP表达SARS-CoV-2刺突(S)蛋白。我们将建立RNAP的条件 固定化、再生和重复转录循环，与溶液中的间歇反应相比， 将显著提高单位RNAP的信使核糖核酸产量。接下来，HA和SARS-CoV-2 S蛋白的mRNAs 将使用通过其催化亚基固定化的痘苗病毒封端酶来封顶。重复的循环 使用相同制剂的固定化酶封盖将用于确定其稳健性、严密性、 稳定性和酶回收的限度。成功完成拟议的第一阶段研究将 为功能mRNA生产的完整流水线奠定了基础。这将增加信使核糖核酸的产量 并促进最终产品的纯化，消除了每一种酶后蛋白质破坏的需要 周而复始。以体外合成为基础，可应用于生物医学研究和医学的各个领域。 尤其是，它将提高基于mRNA的疫苗生产的成本效益。

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.