mRNA structuring for stabilizing mRNA nanocarriers and improving their delivery efficiency

For in vivo application of mRNA therapeutics, development of mRNA nanocarriers that protect mRNA from enzymatic degradation is needed. While current nanocarrier development focuses on fine-tuning chemical structure of its components, including lipids and polymers, herein, we propose a novel strategy to design stable mRNA carriers by structuring mRNA inside the nanocarriers. Firstly, several mRNA strands were crosslinked with each other using RNA crosslinkers that hybridize to mRNA strands, to prepare mRNA nanoassemblies (NAs). Interestingly, NAs preserved their translational activity, because of selective NA dissociation inside cells through 5´ cap-dependent translation. Then, we mixed NAs with poly(ethylene glycol) (PEG)-polycation block copolymers to prepare core-shell-structured polyplex micelles (PMs), composed of PEG shell and mRNA containing core. Notably, PM loading NAs (NA/m) exhibited enhanced stability against enzymatic attack and polyion exchange reaction compared to that loading naïve mRNA (naïve/m). According to mechanistic analyses, NA/m possessed a shell with denser PEG layer and a core with more condensed mRNA compared to naïve/m, which may contribute to PM stabilization. As a result, NA/m induced more efficient protein expression after introduction to cultured cells and mouse brain, compared to naïve/m. Further notably, the improved functionality of NA/m was observed in 3 types of mRNA, demonstrating versatility of our strategy. While newly developed materials need long processes before their clinical approval, our strategy is effective in improving stability and mRNA introduction efficiency of existing mRNA carriers just by structuring mRNA without the use of additional materials, holding great promises for future clinical applications.