Branched chemically modified poly(A) tails enhance the translation capacity of mRNA

Publication Date: 22 March 2024

Chen, H., et al. (2024). Branched chemically modified poly(A) tails enhance the translation capacity of mRNA. Nature Biotechnology. DOI: 10.1038/s41587-024-02174-7

Advancements within the last several years have seen an increase in interest around messenger RNA (mRNA) therapeutics, likely driven by the effectiveness of the COVID-19 vaccines. While messenger RNA (mRNA) has shown promise in vaccine development, the inherent instability and low translation efficiency have restricted their use in broader therapeutic domains. These need to be addressed before considering the potential use of mRNA's for larger-scale therapeutic protein production. Within the realm of clinical applications, mRNAs have potential in enzyme replacement therapies, antibody therapies, and gene editing.

To overcome these challenges, this study introduces a novel approach involving the synthesis of branched, chemically modified poly(A) tails attached to mRNA. It was proposed that the additional poly (A) tails would improve the stability and length of protein production of mRNA. The pmirGLO Dual-Luciferase miRNA Target Expression Vector was used to obtain firefly (FLuc) and Renilla Luciferase (RLuc) constructs.  The resulting FLuc and RLuc bioluminescence that was quantified with the Dual-Glo® Luciferase Assay. Relative RLuc luminescence was prolonged with the multitail mRNA when compared to both unligated linear mRNA and a chemical modified mRNA control. To compare the protein-production of these branched mRNA to state-of-the-art circRNA technology, the researchers engineered mRNA constructs encoding a secreted form of NanoLuc® luciferase (Nluc). Through the use of the Nano-Glo® Luciferase Assay, they were able to measure NLuc protein in the cell culture medium over time without having to lyse the cells. Branched mRNA outperformed the circRNA by three orders of magnitude with protein production, and impressively, the multitail mRNA also exhibited dramatically slower decay, with protein signal after 14 days, twice the length of time that typical mRNA can be detected. The effectiveness of the multitail mRNA constructs was further validated in vivo after retro-orbital administration of lipid nanoparticles containing the mRNA, and the Nano-Glo® Fluorofurimazine In Vivo Substrate (FFz). Luminescent signal was greater from a dose of multitail mRNA compared to the controls within the first couple days, and that difference continued to increase even after 11 days, up to 47-fold difference. Evaluation of TNF, aspartate transaminase, and alanine transaminase indicate there were no long-term immunogenicity effects. To further test the therapeutic applications in vivo, lipid nanoparticles containing mRNA Cas9 constructs targeting PCSK9 and ANGPTL3 were introduced by retro-orbital injection. These targets are known regulators of cholesterol metabolism. Using the Cholesterol/Cholesterol Ester-Glo™ Assay, the authors found dramatic decreases in target protein levels, serum free cholesterol, and serum total cholesterol in mice treated with the multitail mRNA. 

This study provided compelling evidence that branched, chemically modified poly(A) tails can markedly improve the stability and translational efficiency of mRNA. Furthermore, this study showcased the application of this technology in enhancing the efficiency of multiplexed genome editing in the mouse liver, targeting clinically relevant genes at reduced mRNA dosages. There are challenges associated with these modifications, such as difficulty in scalability, and specific fine-tuning in branching patterns. However, this work shows promise, as enhancing the stability and translation capabilities of mRNA could lead to sustained high-level protein production, minimizing the required dosage, and reducing potential cytotoxic effects associated with high doses of mRNA therapies.

Keywords: Messenger RNA (mRNA), NanoLuc® reporters, in vivo imaging, lipid metabolism