These were determined to be in the range of 20?g for nrRNA (encoding the vaccine antigen only without any replicase component) and of 1 1.25?g of conventional Semliki Forest-virus-based saRNA construct administered intramuscularly.8 Other studies reported potent immune responses against influenza HA BSc5371 by leveraging lipid nanoparticle (LNP)-formulated RNA. 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. transcribed or generated from transfected plasmids in cells overexpressing T7 RNA polymerase.9, 10, 11 Alternatively, the replicase is provided by stably transfected expression plasmids12 or encoded by an unrelated saRNA.11 Such trans-amplifying RNA (taRNA) systems, which split the TR and the replicase activity onto two vectors, are typically used for studying mechanisms and structural requirements of alphaviral RNA BSc5371 replication9, 11, 12, 13, 14 or for the production of recombinant propagation-defective alphaviral particles for which TRs encoding alphaviral structural genes act as helper RNA.15, 16 As a platform for infectious disease vaccines, these systems have not been systematically explored. From a vaccine development standpoint, taRNA-based split-vector systems may be advantageous over saRNA with regard to safety, versatility, and manufacturing. In this paper, we present a novel RNA vaccine platform based on taRNA. It consists of a TR BSc5371 encoding the vaccine antigen and a second molecule coding for an in replication mediated by nrRNA-encoded replicase activity is far superior to that provided by saRNA-encoded replicase activity. Our data motivate further exploration of taRNA-based vaccines, which bear the promise of enabling fast and cost-efficient production of large numbers of vaccine doses as required for rapidly evolving or emerging viral pathogens. Results Expression from taRNA in Conjunction with nrRNA-Delivered Replicase Activity Is as Efficient as Expression from saRNA We engineered taRNA as a split-vector system with an in to amplify the complete saRNA, and a second open reading frame with a gene of interest (GOI). Trans-amplifying RNA system (taRNA, right) is a split-vector system consisting of a transreplicon (TR), which encodes the GOI only, and a second RNA delivering BSc5371 alphaviral replicase. TR-GOI is amplified in by a replicase either encoded on saRNA (saRNA-REPL) bearing an irrelevant transgene (iTG) or on non-replicative mRNA (nrRNA-REPL). RNA structural elements for replication of saRNA or TRs are located in conserved sequence elements (CSE) at the 5 and 3 ends and in the subgenomic Rabbit polyclonal to AGR3 promotor (SGP) upstream of the GOI. The UTRs (5-human alpha globin UTR [hAG] and 3transcribed and transfected RNA molecules and neutralized saRNA synthesis as a confounding factor. Second, we included a saRNA variant with a mutant SGP and full deletion of the transgene ORF (saRNA-REPL-iTG) to control for the possibility that the large unused second ORF (iTG) downstream BSc5371 of the SGP in saRNA-REPL may impair expression from this construct (e.g., by inducing nonsense-mediated mRNA decay22). The amount of replicase protein generated in cells transfected with nrRNA-REPL was the same for wild-type (WT)- and mut-REPL (Figure?2A), indicating that the mutation did not affect protein stability. The expression of the mutant replicase was higher from nrRNA compared to saRNA encoding an iTG (Figure?2A; Figure?S2A). Furthermore, expression of mutant replicase was higher with saRNA lacking the iTG as compared to saRNA encoding an iTG, irrespective of absence (Figure?2A) or presence of TRs (Figure?S2A), confirming our assumption that nonsense-mediated mRNA decay (NMD) would affect replicase levels. Expression of active replicase was only higher with nrRNA-REPL as compared with saRNA-REPL early after electroporation (3 h) (Figure?S2A). Notably, replicase accumulated faster.