Time-ordered-expression mRNA (TOE mRNA) for melanoma RNA vaccines

Abstract

Modified mRNA technology has transformed vaccine development by enabling rapid and precise antigen production. The incorporation of adjuvants may further enhance innate immune activation, thereby improving the efficacy of mRNA vaccines. However, inappropriate vaccine kinetics leading to excessive activation of the innate immune system can inhibit the mTORC1 pathway and impair antigen mRNA translation, ultimately limiting vaccine potency. Herein, we present the development of a time-ordered-expression mRNA (TOE mRNA) based on ADAR-mediated A-to-I base editing. Upon cellular entry, TOE mRNA initiates immediate antigen translation, while the adjuvant encoded within the same mRNA is translated approximately 12 hours post-antigen expression. This delayed adjuvant expression ensures sustained activation of the mTORC1 pathway and robust antigen expression, effectively overcoming the limitations imposed by suboptimal vaccine kinetics. We demonstrate that vaccines utilizing TOE mRNA encoding a tumor neoantigen (for normal translation) and an IL-12 adjuvant (with delayed translation) elicit significantly enhanced antitumor immune responses. TOE mRNA technology represents a promising platform for advancing next-generation mRNA vaccines with improved efficacy.