Synthetic mRNAs with Superior Translation and Stability Properties
The translational efficiency and stability of synthetic mRNA in both cultured cells and whole animals can be improved by incorporation of modified cap structures at the 5′-end. mRNAs are synthesized in vitro by a phage RNA polymerase transcribing a plasmid containing the mRNA sequence in the presence of all four NTPs plus a cap dinucleotide. Modifications in the cap dinucleotide at the 2′- or 3′-positions of m7Guo, or modifications in the polyphosphate chain, can improve both translational efficiency and stability of the mRNA, thereby increasing the amount and duration of protein expression. In the context of RNA-based immunotherapy, the latter is especially important for antigen production and presentation by dendritic cells. Protocols are presented for synthesis of modified mRNAs, their introduction into cells and whole animals, and measurement of their translational efficiency and stability.
Key wordsBoranophosphate Cap analogs Decapping Gene therapy Immunotherapy mRNA stability Nucleoporation Phosphorothioate Protein expression Translational efficiency
This research was supported by grant GM20818 from the National Institutes of Health of the USA (to R.E.R), by the Combined Project Grant SFB 432 from the German Research Foundation (to U.S.), by GO-Bio funding from the German Federal Ministry of Education and Research (to U.S.), by grants NN204 089438 (to J.J.) and NN301 096339 (to E.D.) from the Polish Ministry of Science and Higher Education, and by grant 55005 604 from the Howard Hughes Medical Institute (to E.D.).
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