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.

Junyu Fan, Jiajie Pan, Xiaozhe Zhang, Yixi Chen, Yue Zeng, Lihan Huang, Dongwei Ma, Ziqi Chen, Guifu Wu, Wendong Fan

Bioengineered 2022 Mar

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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.

Citation

Junyu Fan, Jiajie Pan, Xiaozhe Zhang, Yixi Chen, Yue Zeng, Lihan Huang, Dongwei Ma, Ziqi Chen, Guifu Wu, Wendong Fan. 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. Bioengineered. 2022 Mar;13(3):4702-4716