Self-amplifying RNA (saRNA) represents a promising advancement in RNA-based vaccines and therapeutics development due to its ability to autonomously multiply within cells, reducing the required initial dosage. Traditional saRNA systems, derived from alphaviruses, employ a bicistronic mRNA with a replicase as the first open reading frame (ORF), a gene of interest (GOI) as the second ORF and a subgenomic promoter in between. Replication initiation relies on cap-dependent translation of the replicase, necessitating in vitro capping, which is both complex and costly. To remove the constraints imposed by cap-dependent translation of the replicase, we engineered a novel RNA replicon with replicase translation independent of the 5′-terminal cap. In short, we placed the replicase under the control of an internal ribosome entry site (IRES) thereby the initiation of translation depends on the molecular properties of the IRES to direct the ribosome to the translational start site. This IRES-launched replicon demonstrated expression levels comparable to 5’-capped saRNA in human primary fibroblasts. Additionally, our design facilitated the insertion of a second GOI upstream of the IRES, translated under the control of a 5′ cap added in cells by the replicase. We explored this site by inserting viral immune evasion genes to boost replicon expression by inhibiting cell-autonomous innate immunity. This strategy resulted in a seven-fold increase in expression, highlighting the benefit of application of viral immune evasion genes.

In conclusion, IRES-launched RNA replicons offer significant advantages, including the use of uncapped synthetic saRNA without activity loss, streamlined synthesis processes, and reduced costs.