A trans-amplifying RNA simplified to essential elements is highly replicative and robustly immunogenic in mice

Trans-amplifying RNA (taRNA) is a split-vector derivative of self-amplifying RNA (saRNA) and a promising vaccine platform. taRNA combines a non-replicating mRNA encoding an alphaviral replicase and a transreplicon (TR) RNA coding for the antigen. Upon translation, the replicase amplifies the antigen-coding TR, thereby requiring minimal amounts of TR for immunization. TR amplification by the replicase follows a complex mechanism orchestrated by genomic and subgenomic promoters (SGPs) and generates genomic and subgenomic amplicons whereby only the latter are translated into therapeutic proteins. This complexity merits simplification to improve the platform. Here, we eliminated the SGP and redesigned the 5' untranslated region to shorten the TR (STR), thereby enabling translation of the remaining genomic amplicon. We then applied a directed evolution approach to select for faster replicating STRs. The resulting evolved STR (eSTR) had acquired A-rich 5' extensions, which improved taRNA expression thanks to accelerated replication. Consequently, we reduced the minimal required TR amount by more than 10-fold without losing taRNA expression in vitro. Accordingly, eSTR-immunized mice developed greater antibody titers to taRNA-encoded influenza HA than TR-immunized mice. In summary, this work points the way for further optimization of taRNA by combining rational design and directed evolution.