Programmable RNA Loading of Extracellular Vesicles with Toehold-Release Purification

Mette Galsgaard Malle; Ping Song; Philipp M.G. Löffler; Nazmie Kalisi; Yan Yan; Julián Valero; Stefan Vogel; Jørgen Kjems

doi:10.1021/JACS.3C13123

Programmable RNA Loading of Extracellular Vesicles with Toehold-Release Purification

Mette Galsgaard Malle, Ping Song, Philipp M.G. Löffler, Nazmie Kalisi, Yan Yan, Julián Valero, Stefan Vogel^*, Jørgen Kjems^*

^*Corresponding author for this work

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaper › Journal article › Research › peer-review

Abstract

Synthetic nanoparticles as lipid nanoparticles (LNPs) are widely used as drug delivery vesicles. However, they hold several drawbacks, including low biocompatibility and unfavorable immune responses. Naturally occurring extracellular vesicles (EVs) hold the potential as native, safe, and multifunctional nanovesicle carriers. However, loading of EVs with large biomolecules remains a challenge. Here, we present a controlled loading methodology using DNA-mediated and programmed fusion between EVs and messenger RNA (mRNA)-loaded liposomes. The fusion efficiency is characterized at the single-particle level by real-time microscopy through EV surface immobilization via lipidated biotin-DNA handles. Subsequently, fused EV−liposome particles (EVLs) can be collected by employing a DNA strand-replacement reaction. Transferring the fusion reaction to magnetic beads enables us to scale up the production of EVLs one million times. Finally, we demonstrated encapsulation of mCherry mRNA, transfection, and improved translation using the EVLs compared to liposomes or LNPs in HEK293-H cells. We envision this as an important tool for the EV-mediated delivery of RNA therapeutics.

Original language	English
Journal	Journal of the American Chemical Society
Volume	146
Issue	18
Pages (from-to)	12410-12422
Number of pages	13
ISSN	0002-7863
DOIs	https://doi.org/10.1021/JACS.3C13123
Publication status	Published - May 2024

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@article{555cc42a76444453a96d758976465872,

title = "Programmable RNA Loading of Extracellular Vesicles with Toehold-Release Purification",

abstract = "Synthetic nanoparticles as lipid nanoparticles (LNPs) are widely used as drug delivery vesicles. However, they hold several drawbacks, including low biocompatibility and unfavorable immune responses. Naturally occurring extracellular vesicles (EVs) hold the potential as native, safe, and multifunctional nanovesicle carriers. However, loading of EVs with large biomolecules remains a challenge. Here, we present a controlled loading methodology using DNA-mediated and programmed fusion between EVs and messenger RNA (mRNA)-loaded liposomes. The fusion efficiency is characterized at the single-particle level by real-time microscopy through EV surface immobilization via lipidated biotin-DNA handles. Subsequently, fused EV−liposome particles (EVLs) can be collected by employing a DNA strand-replacement reaction. Transferring the fusion reaction to magnetic beads enables us to scale up the production of EVLs one million times. Finally, we demonstrated encapsulation of mCherry mRNA, transfection, and improved translation using the EVLs compared to liposomes or LNPs in HEK293-H cells. We envision this as an important tool for the EV-mediated delivery of RNA therapeutics.",

author = "Malle, {Mette Galsgaard} and Ping Song and L{\"o}ffler, {Philipp M.G.} and Nazmie Kalisi and Yan Yan and Juli{\'a}n Valero and Stefan Vogel and J{\o}rgen Kjems",

year = "2024",

month = may,

doi = "10.1021/JACS.3C13123",

language = "English",

volume = "146",

pages = "12410--12422",

journal = "Journal of the American Chemical Society",

issn = "0002-7863",

publisher = "ACS Publications",

number = "18",

}

Programmable RNA Loading of Extracellular Vesicles with Toehold-Release Purification. / Malle, Mette Galsgaard ; Song, Ping; Löffler, Philipp M.G. et al.
In: Journal of the American Chemical Society, Vol. 146, No. 18, 05.2024, p. 12410-12422.

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaper › Journal article › Research › peer-review

TY - JOUR

T1 - Programmable RNA Loading of Extracellular Vesicles with Toehold-Release Purification

AU - Malle, Mette Galsgaard

AU - Song, Ping

AU - Löffler, Philipp M.G.

AU - Kalisi, Nazmie

AU - Yan, Yan

AU - Valero, Julián

AU - Vogel, Stefan

AU - Kjems, Jørgen

PY - 2024/5

Y1 - 2024/5

N2 - Synthetic nanoparticles as lipid nanoparticles (LNPs) are widely used as drug delivery vesicles. However, they hold several drawbacks, including low biocompatibility and unfavorable immune responses. Naturally occurring extracellular vesicles (EVs) hold the potential as native, safe, and multifunctional nanovesicle carriers. However, loading of EVs with large biomolecules remains a challenge. Here, we present a controlled loading methodology using DNA-mediated and programmed fusion between EVs and messenger RNA (mRNA)-loaded liposomes. The fusion efficiency is characterized at the single-particle level by real-time microscopy through EV surface immobilization via lipidated biotin-DNA handles. Subsequently, fused EV−liposome particles (EVLs) can be collected by employing a DNA strand-replacement reaction. Transferring the fusion reaction to magnetic beads enables us to scale up the production of EVLs one million times. Finally, we demonstrated encapsulation of mCherry mRNA, transfection, and improved translation using the EVLs compared to liposomes or LNPs in HEK293-H cells. We envision this as an important tool for the EV-mediated delivery of RNA therapeutics.

AB - Synthetic nanoparticles as lipid nanoparticles (LNPs) are widely used as drug delivery vesicles. However, they hold several drawbacks, including low biocompatibility and unfavorable immune responses. Naturally occurring extracellular vesicles (EVs) hold the potential as native, safe, and multifunctional nanovesicle carriers. However, loading of EVs with large biomolecules remains a challenge. Here, we present a controlled loading methodology using DNA-mediated and programmed fusion between EVs and messenger RNA (mRNA)-loaded liposomes. The fusion efficiency is characterized at the single-particle level by real-time microscopy through EV surface immobilization via lipidated biotin-DNA handles. Subsequently, fused EV−liposome particles (EVLs) can be collected by employing a DNA strand-replacement reaction. Transferring the fusion reaction to magnetic beads enables us to scale up the production of EVLs one million times. Finally, we demonstrated encapsulation of mCherry mRNA, transfection, and improved translation using the EVLs compared to liposomes or LNPs in HEK293-H cells. We envision this as an important tool for the EV-mediated delivery of RNA therapeutics.

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Programmable RNA Loading of Extracellular Vesicles with Toehold-Release Purification

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