Engineering of extracellular vesicles for efficient intracellular delivery of multimodal therapeutics including genome editors

Xiuming Liang; Dhanu Gupta; Junhua Xie; Elien Van Wonterghem; Lien Van Hoecke; Justin Hean; Zheyu Niu; Marziyeh Ghaeidamini; Oscar P B Wiklander; Wenyi Zheng; Rim Jawad Wiklander; Rui He; Doste R Mamand; Jeremy Bost; Guannan Zhou; Houze Zhou; Samantha Roudi; H Yesid Estupiñán; Julia Rädler; Antje M Zickler; André Görgens; Vicky W Q Hou; Radka Slovak; Daniel W Hagey; Olivier G de Jong; Aileen Geobee Uy; Yuanyuan Zong; Imre Mäger; Carla Martin Perez; Thomas C Roberts; Dave Carter; Pieter Vader; Elin K Esbjörner; Antonin de Fougerolles; Matthew J A Wood; Roosmarijn E Vandenbroucke; Joel Z Nordin; Samir El Andaloussi

doi:10.1038/s41467-025-59377-y

Engineering of extracellular vesicles for efficient intracellular delivery of multimodal therapeutics including genome editors

Xiuming Liang^*, Dhanu Gupta, Junhua Xie, Elien Van Wonterghem, Lien Van Hoecke, Justin Hean, Zheyu Niu, Marziyeh Ghaeidamini, Oscar P B Wiklander, Wenyi Zheng, Rim Jawad Wiklander, Rui He, Doste R Mamand, Jeremy Bost, Guannan Zhou, Houze Zhou, Samantha Roudi, H Yesid Estupiñán, Julia Rädler, Antje M ZicklerAndré Görgens, Vicky W Q Hou, Radka Slovak, Daniel W Hagey, Olivier G de Jong, Aileen Geobee Uy, Yuanyuan Zong, Imre Mäger, Carla Martin Perez, Thomas C Roberts, Dave Carter, Pieter Vader, Elin K Esbjörner, Antonin de Fougerolles, Matthew J A Wood, Roosmarijn E Vandenbroucke, Joel Z Nordin, Samir El Andaloussi

^*Corresponding author for this work

Research output: Contribution to journal › Article › Academic › peer-review

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Abstract

Intracellular delivery of protein and RNA therapeutics represents a major challenge. Here, we develop highly potent engineered extracellular vesicles (EVs) by incorporating bio-inspired attributes required for effective delivery. These comprise an engineered mini-intein protein with self-cleavage activity for active cargo loading and release, and fusogenic VSV-G protein for endosomal escape. Combining these components allows high efficiency recombination and genome editing in vitro following EV-mediated delivery of Cre recombinase and Cas9/sgRNA RNP cargoes, respectively. In vivo, infusion of a single dose Cre loaded EVs into the lateral ventricle in brain of Cre-LoxP R26-LSL-tdTomato reporter mice results in greater than 40% and 30% recombined cells in hippocampus and cortex respectively. In addition, we demonstrate therapeutic potential of this platform by showing inhibition of LPS-induced systemic inflammation via delivery of a super-repressor of NF-ĸB activity. Our data establish these engineered EVs as a platform for effective delivery of multimodal therapeutic cargoes, including for efficient genome editing.

Original language	English
Article number	4028
Journal	Nature Communications
Volume	16
Issue number	1
DOIs	https://doi.org/10.1038/s41467-025-59377-y
Publication status	Published - 29 Apr 2025

Keywords

Animals
CRISPR-Associated Protein 9/metabolism
CRISPR-Cas Systems
Drug Delivery Systems/methods
Extracellular Vesicles/metabolism
Gene Editing/methods
HEK293 Cells
Humans
Inflammation/chemically induced
Integrases/genetics
Mice
NF-kappa B/metabolism

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s41467-025-59377-yFinal published version, 16.2 MBLicence: CC BY

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Liang, X., Gupta, D., Xie, J., Van Wonterghem, E., Van Hoecke, L., Hean, J., Niu, Z., Ghaeidamini, M., Wiklander, O. P. B., Zheng, W., Wiklander, R. J., He, R., Mamand, D. R., Bost, J., Zhou, G., Zhou, H., Roudi, S., Estupiñán, H. Y., Rädler, J., ... El Andaloussi, S. (2025). Engineering of extracellular vesicles for efficient intracellular delivery of multimodal therapeutics including genome editors. Nature Communications, 16(1), Article 4028. https://doi.org/10.1038/s41467-025-59377-y

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abstract = "Intracellular delivery of protein and RNA therapeutics represents a major challenge. Here, we develop highly potent engineered extracellular vesicles (EVs) by incorporating bio-inspired attributes required for effective delivery. These comprise an engineered mini-intein protein with self-cleavage activity for active cargo loading and release, and fusogenic VSV-G protein for endosomal escape. Combining these components allows high efficiency recombination and genome editing in vitro following EV-mediated delivery of Cre recombinase and Cas9/sgRNA RNP cargoes, respectively. In vivo, infusion of a single dose Cre loaded EVs into the lateral ventricle in brain of Cre-LoxP R26-LSL-tdTomato reporter mice results in greater than 40\% and 30\% recombined cells in hippocampus and cortex respectively. In addition, we demonstrate therapeutic potential of this platform by showing inhibition of LPS-induced systemic inflammation via delivery of a super-repressor of NF-ĸB activity. Our data establish these engineered EVs as a platform for effective delivery of multimodal therapeutic cargoes, including for efficient genome editing.",

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author = "Xiuming Liang and Dhanu Gupta and Junhua Xie and \{Van Wonterghem\}, Elien and \{Van Hoecke\}, Lien and Justin Hean and Zheyu Niu and Marziyeh Ghaeidamini and Wiklander, \{Oscar P B\} and Wenyi Zheng and Wiklander, \{Rim Jawad\} and Rui He and Mamand, \{Doste R\} and Jeremy Bost and Guannan Zhou and Houze Zhou and Samantha Roudi and Estupi{\~n}{\'a}n, \{H Yesid\} and Julia R{\"a}dler and Zickler, \{Antje M\} and Andr{\'e} G{\"o}rgens and Hou, \{Vicky W Q\} and Radka Slovak and Hagey, \{Daniel W\} and \{de Jong\}, \{Olivier G\} and Uy, \{Aileen Geobee\} and Yuanyuan Zong and Imre M{\"a}ger and Perez, \{Carla Martin\} and Roberts, \{Thomas C\} and Dave Carter and Pieter Vader and Esbj{\"o}rner, \{Elin K\} and \{de Fougerolles\}, Antonin and Wood, \{Matthew J A\} and Vandenbroucke, \{Roosmarijn E\} and Nordin, \{Joel Z\} and \{El Andaloussi\}, Samir",

note = "Publisher Copyright: {\textcopyright} The Author(s) 2025.",

year = "2025",

month = apr,

day = "29",

doi = "10.1038/s41467-025-59377-y",

language = "English",

volume = "16",

journal = "Nature Communications",

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Liang, X, Gupta, D, Xie, J, Van Wonterghem, E, Van Hoecke, L, Hean, J, Niu, Z, Ghaeidamini, M, Wiklander, OPB, Zheng, W, Wiklander, RJ, He, R, Mamand, DR, Bost, J, Zhou, G, Zhou, H, Roudi, S, Estupiñán, HY, Rädler, J, Zickler, AM, Görgens, A, Hou, VWQ, Slovak, R, Hagey, DW, de Jong, OG, Uy, AG, Zong, Y, Mäger, I, Perez, CM, Roberts, TC, Carter, D, Vader, P, Esbjörner, EK, de Fougerolles, A, Wood, MJA, Vandenbroucke, RE, Nordin, JZ & El Andaloussi, S 2025, 'Engineering of extracellular vesicles for efficient intracellular delivery of multimodal therapeutics including genome editors', Nature Communications, vol. 16, no. 1, 4028. https://doi.org/10.1038/s41467-025-59377-y

TY - JOUR

T1 - Engineering of extracellular vesicles for efficient intracellular delivery of multimodal therapeutics including genome editors

AU - Liang, Xiuming

AU - Gupta, Dhanu

AU - Xie, Junhua

AU - Van Wonterghem, Elien

AU - Van Hoecke, Lien

AU - Hean, Justin

AU - Niu, Zheyu

AU - Ghaeidamini, Marziyeh

AU - Wiklander, Oscar P B

AU - Zheng, Wenyi

AU - Wiklander, Rim Jawad

AU - He, Rui

AU - Mamand, Doste R

AU - Bost, Jeremy

AU - Zhou, Guannan

AU - Zhou, Houze

AU - Roudi, Samantha

AU - Estupiñán, H Yesid

AU - Rädler, Julia

AU - Zickler, Antje M

AU - Görgens, André

AU - Hou, Vicky W Q

AU - Slovak, Radka

AU - Hagey, Daniel W

AU - de Jong, Olivier G

AU - Uy, Aileen Geobee

AU - Zong, Yuanyuan

AU - Mäger, Imre

AU - Perez, Carla Martin

AU - Roberts, Thomas C

AU - Carter, Dave

AU - Vader, Pieter

AU - Esbjörner, Elin K

AU - de Fougerolles, Antonin

AU - Wood, Matthew J A

AU - Vandenbroucke, Roosmarijn E

AU - Nordin, Joel Z

AU - El Andaloussi, Samir

PY - 2025/4/29

Y1 - 2025/4/29

N2 - Intracellular delivery of protein and RNA therapeutics represents a major challenge. Here, we develop highly potent engineered extracellular vesicles (EVs) by incorporating bio-inspired attributes required for effective delivery. These comprise an engineered mini-intein protein with self-cleavage activity for active cargo loading and release, and fusogenic VSV-G protein for endosomal escape. Combining these components allows high efficiency recombination and genome editing in vitro following EV-mediated delivery of Cre recombinase and Cas9/sgRNA RNP cargoes, respectively. In vivo, infusion of a single dose Cre loaded EVs into the lateral ventricle in brain of Cre-LoxP R26-LSL-tdTomato reporter mice results in greater than 40% and 30% recombined cells in hippocampus and cortex respectively. In addition, we demonstrate therapeutic potential of this platform by showing inhibition of LPS-induced systemic inflammation via delivery of a super-repressor of NF-ĸB activity. Our data establish these engineered EVs as a platform for effective delivery of multimodal therapeutic cargoes, including for efficient genome editing.

AB - Intracellular delivery of protein and RNA therapeutics represents a major challenge. Here, we develop highly potent engineered extracellular vesicles (EVs) by incorporating bio-inspired attributes required for effective delivery. These comprise an engineered mini-intein protein with self-cleavage activity for active cargo loading and release, and fusogenic VSV-G protein for endosomal escape. Combining these components allows high efficiency recombination and genome editing in vitro following EV-mediated delivery of Cre recombinase and Cas9/sgRNA RNP cargoes, respectively. In vivo, infusion of a single dose Cre loaded EVs into the lateral ventricle in brain of Cre-LoxP R26-LSL-tdTomato reporter mice results in greater than 40% and 30% recombined cells in hippocampus and cortex respectively. In addition, we demonstrate therapeutic potential of this platform by showing inhibition of LPS-induced systemic inflammation via delivery of a super-repressor of NF-ĸB activity. Our data establish these engineered EVs as a platform for effective delivery of multimodal therapeutic cargoes, including for efficient genome editing.

KW - Animals

KW - CRISPR-Associated Protein 9/metabolism

KW - CRISPR-Cas Systems

KW - Drug Delivery Systems/methods

KW - Extracellular Vesicles/metabolism

KW - Gene Editing/methods

KW - HEK293 Cells

KW - Humans

KW - Inflammation/chemically induced

KW - Integrases/genetics

KW - Mice

KW - NF-kappa B/metabolism

U2 - 10.1038/s41467-025-59377-y

DO - 10.1038/s41467-025-59377-y

M3 - Article

C2 - 40301355

SN - 2041-1723

VL - 16

JO - Nature Communications

JF - Nature Communications

IS - 1

M1 - 4028

ER -

Engineering of extracellular vesicles for efficient intracellular delivery of multimodal therapeutics including genome editors

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