Abstract
For in vivo application of mRNA therapeutics, the development of mRNA nanocarriers that protect mRNA from enzymatic degradation is needed. While current nanocarrier development focuses on fine-tuning the chemical structure of its components, including lipids and polymers, herein, we propose a novel strategy to design stable mRNA nanocarriers 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). 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 a denser PEG layer and a core with more condensed mRNA compared to naïve/m. As a result, NA/m induced more efficient protein expression after introduction to cultured cells and mouse brain, compared to naïve/m. While newly developed materials need long processes before their clinical approval, our strategy is effective in improving stability and the mRNA introduction efficiency of existing mRNA nanocarriers just by structuring mRNA without the use of additional materials.
Highlights
In vitro transcribed messenger RNA garners much attention as next-generation therapeutics, especially in its application to vaccination against cancer and pandemic, protein replacement therapy and genome editing [1,2,3,4]
polyplex micelles (PMs) were prepared from Gaussia luciferase (gLuc) naïve messenger RNA (mRNA) (0.8 kb) and gLuc NAs and designated as naïve/m and NA/m, respectively
All PM formulations exhibited the average sizes below 100 nm with polydispersity index around 0.15 in dynamic light scattering (DLS) analyses
Summary
In vitro transcribed messenger RNA (mRNA) garners much attention as next-generation therapeutics, especially in its application to vaccination against cancer and pandemic, protein replacement therapy and genome editing [1,2,3,4]. Hybridization of mRNA with 23 nt or longer RNA oligonucleotides resulted in reduced translational activity and increased immunogenicity of mRNA, 17 nt RNA oligonucleotide did not influence such properties of mRNA [18] Using this strategy, we introduced cholesterol moieties to mRNA to stabilize polymer-based mRNA nanocarriers [18], and poly(ethylene glycol) (PEG) to mRNA to prevent aggregation of lipid-based mRNA nanocarriers during their preparation and after their in vivo administration [19]. We introduced cholesterol moieties to mRNA to stabilize polymer-based mRNA nanocarriers [18], and poly(ethylene glycol) (PEG) to mRNA to prevent aggregation of lipid-based mRNA nanocarriers during their preparation and after their in vivo administration [19] These reports showed the utility and versatility of our mRNA architectonics approach
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