MicroRNA-124-targeted recombinant Zika virus: a dual-functional and safe candidate for vaccination and oncolytic virotherapy

Zika virus (ZIKV) remains a significant public health threat due to its pronounced neurotropism linked to Congenital Zika Syndrome (CZS). Paradoxically, the same neurotropism also provides a unique opportunity for exploiting ZIKV as an oncolytic agent against malignant brain tumors such as glioma. However, achieving precise control of ZIKV replication in specific cell types or tissues remains a major challenge. Here, we show that the brain-specific and enriched microRNA, miR-124, is highly expressed in neural progenitor cells (NPCs) and neurons, but minimally expressed in glioma stem cells (GSCs) and differentiated glioma stem cells (DGCs). Based on these observations, we engineered a recombinant ZIKV containing a miR-124 target sequence (miR-124T) inserted into the 3′ untranslated region (UTR) of the viral genome, generating the virus designated ZIKV-miR124T. ZIKV-miR124T exhibited a significantly attenuated phenotype across multiple mouse models, including adult A129 mice, BALB/c neonates, and pregnant mice. Importantly, in an orthotopic glioma model, ZIKV-miR124T retained potent oncolytic activity while showing a markedly improved safety profile. Viral replication was strictly confined in the tumor region, with a ~1,000-fold reduction in viral load in non-tumoral brain regions compared to the well-established live-attenuated ZIKV (ZIKV-LAV). Furthermore, a single immunization with ZIKV-miR124T conferred effective protection against lethal ZIKV challenge and significantly reduced vertical transmission in pregnant mice. Collectively, our findings establish a strong proof of concept for a rational, miRNA-guided strategy to generate a next-generation ZIKV platform with dual potential as a safe live-attenuated vaccine and a precisely regulated oncolytic virus. IMPORTANCE This study presents a crucial advancement in controlling the safety and function of neurotropic viruses. We engineered a dual-purpose ZIKV, ZIKV-miR124T, which is regulated by the brain-specific microRNA, miR-124. This design forces the virus to strongly self-suppress in healthy neural tissue, solving a major safety concern for ZIKV-based therapies. ZIKV-miR124T is shown to be a potent oncolytic agent against malignant glioma while also serving as a highly effective, safe live-attenuated vaccine against ZIKV infection, reducing vertical transmission to the fetus. Our work provides a strong demonstration of utilizing microRNA regulation to achieve precise viral tropism and attenuation, offering a valuable, generalizable strategy for the development of safer and more effective viral therapies and vaccines against neurotropic pathogens.