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.

Trans-Amplifying RNA: A Journey from Alphavirus Research to Future Vaccines.

Replicating RNA, including self-amplifying RNA (saRNA) and trans-amplifying RNA (taRNA), holds great potential for advancing the next generation of RNA-based vaccines. Unlike in vitro transcribed mRNA found in most current RNA vaccines, saRNA or taRNA can be massively replicated within cells in the presence of RNA-amplifying enzymes known as replicases. We recently demonstrated that this property could enhance immune responses with minimal injected RNA amounts. In saRNA-based vaccines, replicase and antigens are encoded on the same mRNA molecule, resulting in very long RNA sequences, which poses significant challenges in production, delivery, and stability. In taRNA-based vaccines, these challenges can be overcome by splitting the replication system into two parts: one that encodes replicase and the other that encodes a short antigen-encoding RNA called transreplicon. Here, we review the identification and use of transreplicon RNA in alphavirus research, with a focus on the development of novel taRNA technology as a state-of-the art vaccine platform. Additionally, we discuss remaining challenges essential to the clinical application and highlight the potential benefits related to the unique properties of this future vaccine platform.