Optimising self-amplifying RNA (saRNA) vaccines: leveraging EEEV replication mechanisms for enhanced mRNA therapy

Abstract

RNA therapeutics has emerged as a transformative modality capable of targeting virtually any gene, and self amplifying RNA (saRNA) platforms offer the promise of potent protein expression at dramatically reduced doses. Central to alphaviral saRNA function are the untranslated regions (UTRs), which contain core promoter elements for both minus and plus strand synthesis, cis acting replication signals, and regulatory motifs that modulate translation, innate immune evasion, and RNA stability. In this study, we focused on the 3′UTR of an Eastern equine encephalitis virus (EEEV) replicon to identify sequence segments that influence RNA replication and translational efficiency. We first performed multiple sequence alignment across New World alphaviruses and predicted secondary structures to map conserved and variable regions. Next, we engineered a series of 50 nucleotide deletion constructs, retaining the key conserved element immediately upstream of the poly(A) tail, and evaluated their performance in HEK293T cells using dual luciferase assays. Among all variants, the WTΔ50bp construct exhibited nearly two fold higher reporter activity than the full length 3′UTR, indicating that this region could possibly repress expression. These findings pinpoint a target for rational optimization of the EEEV 3′UTR and lay the groundwork for next generation saRNA vaccines with enhanced potency and lower-dose requirements.