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Theranostics 2023; 13(10):3467-3479. doi:10.7150/thno.82873 This issue Cite

Research Paper

Improved extracellular vesicle-based mRNA delivery for familial hypercholesterolemia treatment

Zheng Yang^1,2,3,*, Panpan Ji^4,*, Zhelong Li^5,*, Rongxin Zhang^3,6, Mengying Wei³, Yang Yang⁷, Lijun Yuan⁵, Yan Han^2✉, Guodong Yang^3,5✉

1. College of Otolaryngology, Head and Neck Surgery, Chinese PLA General Hospital, Beijing 100853, China.
2. Department of Plastic Surgery, Chinese PLA General Hospital, Beijing, China.
3. The State Laboratory of Cancer Biology, Department of Biochemistry and Molecular Biology, Fourth Military Medical University, Xi'an, Shaanxi, China.
4. The State Laboratory of Cancer Biology, Department of Gastrointestinal Surgery, Xijing Hospital of Digestive Diseases, Fourth Military Medical University, Xi'an, Shaanxi, China.
5. Department of Ultrasound Diagnostics, Tangdu Hospital, Fourth Military Medical University, Xi'an, Shaanxi, China.
6. College of Life Sciences, Northwest University, Xi'an, Shaanxi, China.
7. Department of Plastic Surgery, Xi'an Daxing Hospital, Xi'an, Shaanxi, China.
^* These authors contributed equally.

✉ Corresponding authors: Department of Biochemistry and Molecular Biology, Fourth Military Medical University, No. 169 Changlexi Road, Xi'an, Shaanxi 710032, China. Department of Plastic Surgery, The First Medical Center, Chinese PLA General Hospital, No.28 Fuxing Road, Beijing 100853, China. E-mail addresses: yanggdedu.cn (G. Yang): 13720086335com (Y. Han). More

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.

Keywords: Extracellular vesicles, therapeutic mRNA, RNA aptamer, loading efficiency, endosome escape, low-density lipoprotein receptor

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