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Zika virus (ZIKV) is a type of RNA virus that belongs to the Flaviviridae family. We have reported the construction of a DNA-launched replicon of the Asian-lineage Natal RGN strain and the production of single-round infectious particles (SRIPs) via the combination of prM/E virus-like particles with the replicon. The main objective of the study was to engineer the ZIKV replicon as mammalian expression vectors and evaluate the potential of ZIKV mini-replicon-based SRIPs as delivery vehicles for heterologous gene expression in vitro and in vivo. The mini-replicons contained various genetic elements, including NS4B, an NS5 methyltransferase (MTase) domain, and an NS5 RNA-dependent RNA polymerase (RdRp) domain. Among these mini-replicons, only ZIKV mini-replicons 2 and 3, which contained the full NS5 and NS4B-NS5 genetic elements, respectively, exhibited the expression of reporters (green fluorescent protein (GFP) and cyan fluorescent protein–yellow fluorescent fusion protein (CYP)) and generated self-replicating RNAs. When the mini-replicons were transfected into the cells expressing ZIKV prM/E, this led to the production of ZIKV mini-replicon-based SRIPs. ZIKV mini-replicon 3 SRIPs showed a significantly higher yield titer and a greater abundance of self-replicating replicon RNAs when compared to ZIKV mini-replicon 2 SRIPs. Additionally, there were disparities in the dynamics of CYP expression and cytotoxic effects observed in various infected cell types between ZIKV mini-replicon 2-CYP and 3-CYP SRIPs. In particular, ZIKV mini-replicon 3-CYP SRIPs led to a substantial decrease in the survival rates of infected cells at a MOI of 2. An in vivo gene expression assay indicated that hACE2 expression was detected in the lung and brain tissues of mice following the intravenous administration of ZIKV mini-replicon 3-hACE2 SRIPs. Overall, this study highlights the potential of ZIKV mini-replicon-based SRIPs as promising vehicles for gene delivery applications in vitro and in vivo. [ABSTRACT FROM AUTHOR]