Fig. 2: Designing self-replicating viral particles for ASO therapy: a JEV genome re-engineering approach.

a, b Structural organization of the JEV with surface and inner views highlighting the viral components, including capsid (C), membrane (M), envelope (E), and viral RNA. The WT JEV genome encodes specific structural and non-structural proteins, as illustrated in its genomic organization. This genome can be genetically engineered to incorporate a miRNA insertion between the 5′-UTRs and the structural proteins. Additionally, an ASO can be designed and inserted between the structural proteins and the 3′-UTR, flanked on its right by a proteolytic sequence recognized by one of JEV’s own proteases, such as NS3. c In this modified genome, upon cellular entry, viral replication initiates as the positive-strand JEV genome functions directly as mRNA, producing the typical JEV polyprotein via IRES (i). JEV proteases, including NS3, cleave the polyprotein at specific sites to generate individual viral proteins. Importantly, the JEV protease also recognizes and cleaves the proteolytic site linker engineered to flank the ASO, releasing it into the cytoplasm. This released ASO targets mutated SOD1 mRNA, promoting its degradation and preventing translation of the dysfunctional SOD1 protein via RNAi (ii). Concurrently, the viral RdRp (NS5) transcribes the positive-strand genome into a negative-strand template, facilitating synthesis of a new positive-strand genome. This newly replicated genome, along with the transcribed ASO, is subsequently assembled and packaged into a new virion capable of infecting bystander cells (iii).