College of Otolaryngology, Head and Neck Surgery, Chinese PLA General Hospital, Beijing 100853, China.
Department of Plastic Surgery, Chinese PLA General Hospital, Beijing, China.
Theranostics. 2023 Jun 12;13(10):3467-3479. doi: 10.7150/thno.82873. eCollection 2023.
Extracellular vesicle (EV)-based low-density lipoprotein receptor () 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 mRNA into EVs in donor cells and controllable release in recipient cells. The MS2 bacteriophage coat protein (CD9-MCP) fusion protein, mRNA, and a customized MS2 containing RNA aptamer base-pair matched with mRNA were expressed in donor cells. Cells receiving the above therapeutic EVs were simultaneously treated with EVs containing " releaser" with a sequence similar to the recognition sites in mRNA. Therapeutic effects were analyzed in mice receiving EV treatments via the tail vein. experiments demonstrated improved loading efficiency of mRNA in EVs via MS2-MCP interaction. Treatment of " releaser" competitively interacted with MS2 aptamer with higher affinity and released mRNA from CD9-MCP for efficient translation. When the combinatory EVs were delivered into recipient hepatocytes, the robust LDLR expression afforded therapeutic benefits in mice. 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.
基于细胞外囊泡(EV)的低密度脂蛋白受体()mRNA 递呈在治疗家族性高胆固醇血症(FH)方面显示出了极好的疗效。然而,基于 EV 的 mRNA 递呈的载药效率不高仍然是一个重大挑战。最近,RNA 结合蛋白(RBPs)已被融合到 EV 膜蛋白上,用于选择性包裹靶向 RNA,以提高载药效率。然而,治疗性 RNA 与 RBPs 之间的强相互作用阻止了 RNA 从受体细胞的内体释放到细胞质中。在本研究中,开发了一种改进的策略,用于在供体细胞中高效包封 mRNA 并在受体细胞中可控释放。在供体细胞中表达了 MS2 噬菌体衣壳蛋白(CD9-MCP)融合蛋白、mRNA 和一种定制的 MS2 ,其中包含与 mRNA 碱基配对的 RNA 适体。同时用含有与 mRNA 识别位点相似序列的“释放剂”处理接收上述治疗性 EV 的细胞。通过尾静脉向接受 EV 治疗的 小鼠进行治疗效果分析。结果表明,通过 MS2-MCP 相互作用,提高了 MS2-MCP 相互作用的 mRNA 在 EV 中的装载效率。“释放剂”竞争性地与具有更高亲和力的 MS2 适体相互作用,并从 CD9-MCP 上释放 mRNA 以进行有效的翻译。当组合 EV 递送至受体肝细胞时,LDLR 的强烈表达为 小鼠提供了治疗益处。我们提出了一种基于 EV 的 mRNA 递呈策略,用于增强治疗性 mRNAs 在 EV 中的包封和 RNA 向细胞质中的释放,以促进受体细胞中的翻译,具有很大的基因治疗潜力。
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