Synthetic mRNAs with Superior Translation and Stability Properties

Abstract The translational ef Þ ciency and stability of synthetic mRNA in both cultured cells and whole animals can be improved by incorporation of modi Þ ed 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. Modi Þ cations in the cap dinucleotide at the 2 ¢- or 3 ¢-positions of m 7 Guo, or modi Þ cations in the polyphosphate chain, can improve both translational ef Þ ciency 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 modi Þ ed mRNAs, their introduction into cells and whole animals, and measurement of their translational ef Þ ciency and stability. Key words: Boranophosphate , Cap analogs , Decapping , Gene therapy , Immunotherapy , mRNA stability , Nucleoporation , Phosphorothioate , Protein expression , Translational ef Þ ciency