A peptide derived from the N-terminus of charged multivesicular body protein 6 (CHMP6) promotes the secretion of gene editing proteins via small extracellular vesicle production.
A peptide derived from the N-terminus of charged multivesicular body protein 6 (CHMP6) promotes the secretion of gene editing proteins via small extracellular vesicle production.
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DOI：
10.1080/21655979.2022.2030571
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发表时间：
2022-03
期刊：
影响因子：
4.9
通讯作者：
Fan W
中科院分区：
文献类型：
--
作者：
Fan J;Pan J;Zhang X;Chen Y;Zeng Y;Huang L;Ma D;Chen Z;Wu G;Fan W
细胞外囊泡是一种很有前途的新型治疗平台。然而，低货物装载效率限制了它们的临床转化。在这项研究中，我们开发了一种高产量的EV货物装载设备，并探索了其封装基因编辑蛋白的能力。构建了一系列基于融合蛋白的系统，并通过NanoGlo荧光素酶测定比较了它们的货物装载效率。从带电多泡体蛋白6（CHMP 6）的N-末端区域克隆的豆蔻酰化（Myr）肽标签，称为Myr（CHMP 6），作为活性包装装置胜过CD 9、ARRDC 1和其他短多肽。如通过纳米颗粒跟踪分析和透射电子显微镜所确定的，Myr（CHMP 6）的过表达增加了Lenti-X 293 T细胞中的小EV（sEV）产生，而不改变sEV形态。还阐明了Myr（CHMP 6）的高被动包装效率用于未修饰的货物装载。Western印迹显示，Myr（CHMP 6）促进Cre和Cas9加载到sEV中，而不产生包装装置-货物融合蛋白。此外，Myr（CHMP 6）修饰的载有Cre或Cas9的sEV促进了受体细胞中的基因编辑，如使用荧光报告系统所观察到的。随后的研究证明了Myr（CHMP 6）标签诱导的货物负载的剂量依赖性效应。从机制上讲，单独的N-豆蔻酰化是必要的，但不足以将蛋白质有效包装到电动汽车中。因此，我们的研究结果表明，Myr（CHMP 6）诱导sEV的产生，并可能有效地将基因编辑蛋白加载到sEV中用于治疗目的。
Extracellular vesicles (EVs) are a promising new therapeutic platform. However, the low cargo-loading efficiency limits their clinical translation. In this study, we developed a high-yield EV cargo-loading device and explored its ability to encapsulate gene editing proteins. A series of fusion protein-based systems were constructed and their cargo loading efficiencies were compared by a NanoGlo luciferase assay. A myristoylated (Myr) peptide tag cloned from the N-terminal region of charged multivesicular body protein 6 (CHMP6), termed Myr(CHMP6), outcompeted CD9, ARRDC1, and other short polypeptides as an active packaging device. As determined by nanoparticle tracking analysis and transmission electron microscopy, the overexpression of Myr(CHMP6) increased small EV (sEV) production in Lenti-X 293T cells without altering sEV morphology. The high passive packaging efficiency of Myr(CHMP6) was also elucidated for unmodified cargo loading. Western blotting revealed that Myr(CHMP6) facilitated the loading of Cre and Cas9 into sEVs without the generation of packaging device-cargo fusion proteins. Furthermore, Myr(CHMP6)-modified sEVs loaded with Cre or Cas9 promoted gene-editing in recipient cells, as observed using a fluorescence reporter system. Subsequent investigation demonstrated a dose-dependent effect of Myr(CHMP6) tag-induced cargo-loading. Mechanistically, N-myristoylation alone was necessary but not sufficient for the effective packaging of proteins into EVs. Thus, our results indicated that Myr(CHMP6) induces sEV production and may be effective in loading gene editing proteins into sEVs for therapeutic purposes.
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影响因子：
5.2
作者：
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通讯作者：
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作者：
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通讯作者：
Wood MJA
DOI：
10.1002/stem.3140
发表时间：
2020-04
期刊：
Stem cells (Dayton, Ohio)
影响因子：
--
作者：
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通讯作者：
Antonyak MA
影响因子：
11.8
作者：
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通讯作者：
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