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.