Improved extracellular vesicle-based mRNA delivery for familial hypercholesterolemia treatment.

Abstract

Extracellular vesicle (EV)-based low-density lipoprotein receptor (Ldlr) mRNA delivery showed excellent therapeutic effects in treating familial hypercholesterolemia (FH). Nevertheless, the loading inefficiency of EV-based mRNA delivery presents a significant challenge. Recently, RNA-binding proteins (RBPs) have been fused to EV membrane proteins for selectively encapsulating targeted RNAs to promote loading efficiency. However, the strong interaction between therapeutic RNAs and RBPs prevents RNA release from endosomes to the cytosol in the recipient cells. In this study, an improved strategy was developed for efficient encapsulation of Ldlr mRNA into EVs in donor cells and controllable release in recipient cells. Methods: The MS2 bacteriophage coat protein (CD9-MCP) fusion protein, Ldlr mRNA, and a customized MS2 containing RNA aptamer base-pair matched with Ldlr mRNA were expressed in donor cells. Cells receiving the above therapeutic EVs were simultaneously treated with EVs containing "Ldlr releaser" with a sequence similar to the recognition sites in Ldlr mRNA. Therapeutic effects were analyzed in Ldlr-/- mice receiving EV treatments via the tail vein. Results: In vitro experiments demonstrated improved loading efficiency of Ldlr mRNA in EVs via MS2-MCP interaction. Treatment of "Ldlr releaser" competitively interacted with MS2 aptamer with higher affinity and released Ldlr mRNA from CD9-MCP for efficient translation. When the combinatory EVs were delivered into recipient hepatocytes, the robust LDLR expression afforded therapeutic benefits in Ldlr-/- mice. Conclusion: We proposed an EV-based mRNA delivery strategy for enhanced encapsulation of therapeutic mRNAs in EVs and RNA release into the cytosol for translation in recipient cells with great potential for gene therapy.