Bioengineered miRNA Agents are More Efficacious and Selective than Chemo‐Engineered Mimics in the Regulation of Target Gene Expression

Recent investigations have demonstrated the utility of small interfering RNAs (siRNA) and microRNAs (miRNA) as therapeutics for the treatment of many diseases including cancers, viral infections, cardiovascular diseases, and many other disorders. However, current RNAi research and development is limited to the use of chemically‐synthesized RNA agents that are mounted with various artificial modifications. In this study, we aimed to optimize the tRNA/pre‐miRNA‐based technology to achieve in vivo fermentation production of fully‐humanized bioengineered RNAi agents (hBERA). We first achieved high‐level heterogenous overexpression (accounting for >50% of total bacterial RNA) of 20 target hBERAs at 100% success rate, yielding 18‐53 mg high‐purity (≥98.5%) and low endotoxin (<1.5 EU/µg RNA) hBERAs per liter bacterial culture. Further studies demonstrated that target miRNAs (e.g., miR‐124‐3p) are specifically released from hBERAs in human NSCLC cells to modulate protein levels of targeted genes (e.g., VAMP3 and IQGAP1) and control cell viability. Interestingly, hBERA/miRNAs were more effective and selective than the same concentrations of synthetic miRNA mimics in the control of target gene expression and human NSCLC cell viability. In addition, systemic administration of lipid nanoparticle formulated hBERA had no significant effects on cytokine release in both human PBMCs and immunocompetent mice, as well as hepatic and renal functions. This robust RNA bioengineering platform offers a novel class of fully‐humanized, true biologic RNAi agents that are more efficacious than synthetic RNA mimics for research and may be developed as new therapeutics.