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Self-replicating RNA (repRNA) derived from venezuelan equine encephalitis (VEE) virus is a promising platform for gene therapy and confers prolonged gene expression due to its self-replicating capability, but repRNA suffers from a suboptimal transgene expression level due to its induction of intracellular innate response which may result in inhibition of translation. To improve transgene expression of repRNA, we introduced point mutations in the non-structural protein 1-4 (nsP1-4) coding region of VEE replicon vectors. As a proof of concept, in ammatory cytokines served as genes of interest and were cloned in either their wild type or several mutant replicon vectors, followed by transfection in mammalian cells. Our data show that VEE replicons bearing nsP1GGAC-nsP2T and nsP1GGAC-nsP2AT mutations in the nsP1-4 coding region signi cantly enhance transgene expression.Thus, the newly discovered mutant VEE replicon vectors could serve as promising gene expression platforms to advance VEEderived repRNA-based gene therapies.

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