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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., Competing Interests: Declaration of interests U.S., T.B., and M.P. are inventors on patents and patent applications, which cover parts of this article. U.S. is employee at BioNTech Corporation (Mainz, Germany), a privately owned company developing therapeutic RNA., (Copyright © 2023 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Here, we present a potent RNA vaccine approach based on a novel bipartite vector system using trans-amplifying RNA (taRNA). The vector cassette encoding the vaccine antigen originates from an alphaviral self-amplifying RNA (saRNA), from which the replicase was deleted to form a transreplicon. Replicase activity is provided in trans by a second molecule, either by a standard saRNA or an optimized non-replicating mRNA (nrRNA). The latter delivered 10- to 100-fold higher transreplicon expression than the former. Moreover, expression driven by the nrRNA-encoded replicase in the taRNA system was as efficient as in a conventional monopartite saRNA system. We show that the superiority of nrRNA- over saRNA-encoded replicase to drive expression of the transreplicon is most likely attributable to its higher translational efficiency and lack of interference with cellular translation. Testing the novel taRNA system in mice, we observed that doses of influenza hemagglutinin antigen-encoding RNA as low as 50 ng were sufficient to induce neutralizing antibodies and mount a protective immune response against live virus challenge. These findings, together with a favorable safety profile, a simpler production process, and the universal applicability associated with this bipartite vector system, warrant further exploration of taRNA., (Copyright © 2019. Published by Elsevier Inc.)