Preparation of Exosomes Containing Highly Efficient Encapsulated Tyrosinase Hydroxylase mRNA

doi:10.13523/j.cb.2306035

China Biotechnology

2024, Vol. 44

Issue (2/3): 69-75 DOI: 10.13523/j.cb.2306035

Preparation of Exosomes Containing Highly Efficient Encapsulated Tyrosinase Hydroxylase mRNA

FAN Yuqin¹,LI Zhikang¹,LIANG Zhixuan¹,ZHAO Zihan¹,XIE Qiuling^1,^2,^**(

)

1 School of Life Science and Technology, Jinan University, Guangzhou 510632, China
2 National Engineering Research Center of Genetic Medicine, Guangzhou 510632, China

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Abstract

Objective: Tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis, plays a major role in the pathogenesis of Parkinson disease. Exosomes, vesicles with a diameter ranging from 30 to 200 nm secreted by cells, are considered potential carriers for drug delivery across the blood-brain barrier. To achieve efficient encapsulation of tyrosine hydroxylase mRNA in exosomes (TH-Kt-Exo), the binding properties of the archaeal ribosomal protein L7Ae and the Kt loop are exploited to enable delivery of mRNA-based therapeutics across the blood-brain barrier. Methods: The method involved constructing a recombinant plasmid containing TH mRNA with the Kt loop and a recombinant plasmid, pCMV-CD63-L7Ae-His, expressing the fusion of the exosomal membrane proteins CD63 and L7Ae. HEK293F cells were co-transfected with these plasmids, and exosomes secreted by the cells were harvested by ultracentrifugation. The content of TH mRNA in the exosomes was detected by qPCR, and the exosomes were then transfected into recipient cells. Results: Compared to TH-Exo obtained by transfection with the single tyrosine hydroxylase plasmid, the proposed method resulted in significantly higher levels of TH RNA encapsulation in exosomes (TH-Kt-Exo). In addition, the exosomes were able to deliver the loaded mRNA to recipient cells. Conclusion: The specific binding between L7Ae and the Kt loop effectively enhances the encapsulation of the target mRNA in exosomes.

Key words： Tyrosine hydroxylase Exosome Archaeal ribosomal protein Kt-turn loop

Received: 27 June 2023 Published: 03 April 2024

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	Yuqin FAN
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FAN Yuqin, LI Zhikang, LIANG Zhixuan, ZHAO Zihan, XIE Qiuling. Preparation of Exosomes Containing Highly Efficient Encapsulated Tyrosinase Hydroxylase mRNA. China Biotechnology, 2024, 44(2/3): 69-75.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2306035 OR https://manu60.magtech.com.cn/biotech/Y2024/V44/I2/3/69

Fig.2 Transient expression of recombinant plasmids in HEK293F cells Conduct n = 3 independent replicates. Ensuring uniform experimental conditions for the analysis of all samples

Fig.3 Characterization of exosomes A: Particle size identification B: TEM C: Western blotting. Control: Untransfected exosomes; TH-Exo: pcDNA3.4-TH-Flag-Kt transfection; TH-Kt-Exo:pcDNA3.4hTH-Flag-Kt,pCMV-CD63-L7Ae-His co-transfection

Fig.4 Detection of exosome-coated TH mRNA Conduct n = 3 independent replicates. Ensuring uniform experimental conditions for the analysis of all samples

Fig.5 Expression of TH mRNA and protein in HEK293T cells after exosome incubation A: The level of TH mRNA B: The level of TH protein. Conduct n = 3 independent replicates. Ensuring uniform experimental conditions for the analysis of all samples C: Control 0.1: 0.1×10⁷ Exo/10⁶ cell; 0.25: 0.25×10⁷ Exo/cell; 0.5: 0.5×10⁷ Exo/cell,;1: 1×10⁷Exo/cell


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