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

> **NIH NIH R43** · AFFINITY MOLECULES, LLC · 2021 · $297,193

## Abstract

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.

## Key facts

- **NIH application ID:** 10200307
- **Project number:** 1R43TR003596-01
- **Recipient organization:** AFFINITY MOLECULES, LLC
- **Principal Investigator:** Maria Kireeva
- **Activity code:** R43 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $297,193
- **Award type:** 1
- **Project period:** 2021-09-23 → 2023-09-22

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10200307

## Citation

> US National Institutes of Health, RePORTER application 10200307, Large scale in vitro production of capped polyadenylated mRNA-based vaccines in solid phase using immobilized enzymes (1R43TR003596-01). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10200307. Licensed CC0.

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