Assessments of ribosome load and mRNA half-life for synthetic mRNAs

Developing an effective mRNA therapeutic often requires maximizing protein output per delivered mRNA molecule. We previously found that coding sequence (CDS) design can substantially affect protein output, with mRNA variants containing more optimal codons and higher secondary structure content yielding the highest protein outputs due to their slow rates of mRNA decay. Here we demonstrate that CDS-dependent differences in translation initiation and elongation rates lead to differences in translation- and deadenylation-dependent mRNA decay rates, thus explaining the effect of CDS on mRNA half-life. Surprisingly, the most stable and highest expressing mRNAs that we tested had modest initiation/elongation rates and ribosome loads, which led to minimal translation-dependent mRNA decay. The direct implication of this discovery is that optimizing protein output from therapeutic mRNAs is best achieved by attenuating rather than maximizing ribosome load.