Artificial primary-miRNAs as a platform for simultaneous delivery of siRNA and antisense oligonucleotide for multimodal gene regulation.

Self-assembled nucleic acid nanostructures have been widely explored for gene therapy applications due to their unique advantages. Their roles are not limited to offer intracellular delivery platforms but additionally provide a biological function to induce targeted gene regulation. Here, we report a self-assembled artificial primary-miRNA (pri-miRNA) for achieving simultaneous multimodal gene regulation. Artificial pri-miRNAs are designed to play a role as substrate RNAs to recruit and interact with Drosha/DGCR8 (Microprocessor). Incorporation of functional RNA motifs and site-specific chemical modification of the primary miRNA are utilized for the biogenesis of two individual gene-regulating oligonucleotides. Once they are cleaved by the endogenous Drosha/DGCR8 complex, basal strands and pre-miRNA can be generated inside of cells. In this study, we integrated basal strands with either SMN2 ASO or anti-miR21 to induce multimodal gene regulation. Microprocessing and subsequent gene regulation were first evaluated by measuring the activity of reporter pre-miRNA. Chemical modification on the primary miRNA was optimized through a series of in vitro Drosha cleavage tests and targeted gene silencing in cells. Primary miRNA with the basal ASO or anti-miR strands showed a successful in vitro activity and resulted in simultaneous multimodal gene regulation in cells. Artificial primary miRNA may offer synergistic therapeutic effects for treating various diseases, including spinal muscular atrophy and cancer.