The design, manufacture and LNP formulation of mRNA for research use

Abstract

The delivery of mRNA provides a versatile platform to achieve rapid and robust protein expression within cells. mRNA delivery has therefore been widely adopted in research, and mRNA medicines are now being developed to treat a range of diseases. However, high-quality mRNA must be produced to ensure the safety, performance and effectiveness of these medicines. Here, we provide a validated, end-to-end protocol describing the production of mRNA for research and preclinical use. This protocol includes primary sequence design, DNA template production, mRNA synthesis by in vitro transcription, formulation into lipid nanoparticles and transfection into cultured cells. Each step is supported by a range of quality control tests to analyze mRNA integrity and purity and is illustrated with example results to provide information about expected performance. The protocol prioritizes simple production steps that are suitable for small-scale mRNA manufacture, such as PCR preparation of the DNA template, and that can be performed within a laboratory and avoid specialized equipment. The protocol produces high-quality mRNA that can be used for in vitro and in vivo preclinical studies, including for vaccine, protein and gene- and cell-therapy applications. Together, this provides a fast and reliable protocol to produce high-quality mRNA suitable for laboratory research and preclinical development.