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

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 transcribed mRNA stability and reduced immunogenicity. In this work, we investigated the contribution of each structural component on protein expression of antigen-encoding mRNA, and , 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 transcription, resulting in accelerated mRNA production, facilitating high-throughput screening and evaluation of antigen immunogenicity.