Impact of mRNA chemistry and manufacturing process on innate immune activation

Abstract

Messenger RNA (mRNA) represents an attractive therapeutic modality for potentially a wide range of clinical indications but requires uridine chemistry modification and/or tuning of the production process to prevent activation of cellular innate immune sensors and a concomitant reduction in protein expression. To decipher the relative contributions of these factors on immune activation, here, we compared, in multiple cell and in vivo models, mRNA that encodes human erythropoietin incorporating either canonical uridine or N1-methyl-pseudouridine (1mΨ), synthesized by either a standard process shown to have double-stranded RNA (dsRNA) impurities or a modified process that yields a highly purified mRNA preparation. Our data demonstrate that the lowest stimulation of immune endpoints was with 1mΨ made by the modified process, while mRNA containing canonical uridine was immunostimulatory regardless of process. These findings confirm that uridine modification and the reduction of dsRNA impurities are both necessary and sufficient at controlling the immune-activating profile of therapeutic mRNA.

SARS-CoV-2 mRNA vaccine design enabled by prototype pathogen preparedness

“…Pre-clinical mRNA-1273 mRNA and LNP Production Process A sequence-optimized mRNA encoding prefusion-stabilized SARS-CoV-2 S-2P protein was synthesized in vitro using an optimized T7 RNA polymerase-mediated transcription reaction with complete replacement of uridine by N1m-pseudouridine 34 . The reaction included a DNA template containing the immunogen open-reading frame flanked by 5' UTR and 3' UTR sequences and was terminated by an encoded polyA tail.…”

Safe and effective aerosolization of in vitro transcribed mRNA to the respiratory tract epithelium of horses without a transfection agent

“…Delivery systems, predominately based on lipid nanoparticles 4,5,16,19 , have been developed to protect mRNA administered parenterally or topically for delivery to targeted cells both in vitro and in vivo. These transfection delivery agents are primarily designed to shield the mRNA from enzymatic degradation, increase mRNA uptake by targeted cells, and promote subsequent escape from intracellular endosomes into the cytoplasm, thereby increasing translation into the desired protein products.…”

Advancements in mRNA Encoded Antibodies for Passive Immunotherapy

“…First and foremost, mRNA is an unstable molecule that is degradable by ribonucleases in the body resulting in a very short half-life. Additionally, naked mRNA is not efficiently delivered to cells and mRNA can activate innate immune pathways that may decrease protein expression [ 52 ]. In the last decade, significant progress has been made in all these areas.…”

“…Also, higher levels of in vivo expression are needed to compete with recombinant antibodies and enable antibody combinations. Nevertheless, improvements in mRNA delivery and design are rapidly expanding the application of mRNA-based therapies and it is likely that the remaining challenges will be overcome in the near future [ 45 , 51 , 52 ].…”