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.
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.