Sequence simplification of antigen coding IVT mRNA allows accelerated synthetic DNA template generation and epitope...

The recent surge in therapeutic mRNA for vaccination has provided a blueprint for the design of the mRNA molecule, as several complex structural elements indicated as 5' and 3' untranslated regions (UTRs) and poly-A tail have been identified as necessary for in vitro transcribed mRNA stability and reduced immunogenicity. In this work, we investigated the contribution of each structural component on protein expression of antigen-encoding mRNA, in vitro and ex vivo, delivered via electroporation in antigen-presenting cells. After initial investigation, showcasing the effect of sequence, or chemical, modifications on mRNA translation and protein expression, we validated the effect of a structural deconstruction on antigen coding mRNA, observing preserved antigen presentation upon removal of stabilizing structures like the 5' and 3' UTRs and poly-A tail. Moreover, we implemented these findings and established a straightforward method for generating synthetic DNA templates for in vitro transcription, resulting in accelerated mRNA production, facilitating high-throughput screening and evaluation of antigen immunogenicity.