TOP-EVs: Technology of Protein delivery through Extracellular Vesicles is a versatile platform for intracellular protein delivery

Nazma F. Ilahibaks; Arif I. Ardisasmita; Songpu Xie; Anders Gunnarsson; Joseph Brealey; Pieter Vader; Olivier G. de Jong; Saskia de Jager; Niek Dekker; Ben Peacock; Raymond M. Schiffelers; Joost P.G. Sluijter; Zhiyong Lei

doi:10.1016/j.jconrel.2023.02.003

TOP-EVs: Technology of Protein delivery through Extracellular Vesicles is a versatile platform for intracellular protein delivery

Nazma F. Ilahibaks, Arif I. Ardisasmita, Songpu Xie, Anders Gunnarsson, Joseph Brealey, Pieter Vader, Olivier G. de Jong, Saskia de Jager, Niek Dekker, Ben Peacock, Raymond M. Schiffelers, Joost P.G. Sluijter^*, Zhiyong Lei

^*Corresponding author for this work

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

Abstract

Extracellular vesicles (EVs) have emerged as biocompatible drug delivery vehicles due to their native ability to deliver bioactive cargo to recipient cells. However, the application of EVs as a therapeutic delivery vehicle is hampered by effective methods for endogenously loading target proteins inside EVs and unloading proteins after delivery to recipient cells. Most EV-based engineered loading methods have a limited delivery efficiency owing to their inefficient endosomal escape or cargo release from the intraluminal attachment from the EV membrane. Here, we describe the 'Technology Of Protein delivery through Extracellular Vesicles’ (TOP-EVs) as a tool for efficient intracellular delivery of target proteins mediated via EVs. The vesicular stomatitis virus glycoprotein and the rapamycin-heterodimerization of the FKBP12/T82L mutant FRB proteins were both important for the effective protein delivery through TOP-EVs. We showed that TOP-EVs could efficiently deliver Cre recombinase and CRISPR/Cas9 ribonucleoprotein complex in vitro. Moreover, our results demonstrated that the capacity of TOP-EVs to deliver intracellular proteins in recipient cells was not an artifact of plasmid contamination or direct plasmid loading into EVs. Finally, we showed that TOP-EVs could successfully mediate intracellular protein delivery in the liver in vivo. Taken together, TOP-EVs are a versatile platform for efficient intracellular protein delivery in vitro and in vivo, which can be applied to advance the development of protein-based therapeutics.

Original language	English
Pages (from-to)	579-592
Number of pages	14
Journal	Journal of Controlled Release
Volume	355
Issue number	March 2023
DOIs	https://doi.org/10.1016/j.jconrel.2023.02.003
Publication status	Published - Mar 2023

Bibliographical note

Publisher Copyright:
© 2023 The Authors

Funding

Funding for the present work was provided by the Project EVICARE [No. 725229] of the European Research Council (ERC) to J.P.G.S, co-funded by the Project SMARTCARE-II of the BioMedicalMaterials institute to J.P.G.S, the ZonMw-TAS program [No. 116002016 ] to J.P.G.S./Z.L, PPS grant [No. 2018B014 ] to J.P.G.S./Z.L/P.V./S.J., the Dutch Ministry of Economic Affairs, Agriculture and Innovation and the Netherlands CardioVascular Research Initiative (CVON) : the Dutch Heart Foundation to J.P.G.S, Dutch Federations of University Medical Centers , the Netherlands Organization for Health Research and Development , and the Royal Netherlands Academy of Sciences .

Funders	Funder number
BioMedicalMaterials institute
CVON
Netherlands Cardiovascular Research Initiative
Royal Netherlands Academy of Sciences
University Medical Centers
ZonMw-TAS program	2018B014, 116002016
European Research Council
ZonMw
Hartstichting
Ministerie van Economische Zaken

Keywords

Active protein loading
Extracellular vesicles
Functional intracellular protein delivery
Genome editing
TOP-EV

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Ilahibaks, N. F., Ardisasmita, A. I., Xie, S., Gunnarsson, A., Brealey, J., Vader, P., de Jong, O. G., Jager, S. D., Dekker, N., Peacock, B., Schiffelers, R. M., Sluijter, J. P. G., & Lei, Z. (2023). TOP-EVs: Technology of Protein delivery through Extracellular Vesicles is a versatile platform for intracellular protein delivery. Journal of Controlled Release, 355(March 2023), 579-592. https://doi.org/10.1016/j.jconrel.2023.02.003

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title = "TOP-EVs: Technology of Protein delivery through Extracellular Vesicles is a versatile platform for intracellular protein delivery",

abstract = "Extracellular vesicles (EVs) have emerged as biocompatible drug delivery vehicles due to their native ability to deliver bioactive cargo to recipient cells. However, the application of EVs as a therapeutic delivery vehicle is hampered by effective methods for endogenously loading target proteins inside EVs and unloading proteins after delivery to recipient cells. Most EV-based engineered loading methods have a limited delivery efficiency owing to their inefficient endosomal escape or cargo release from the intraluminal attachment from the EV membrane. Here, we describe the 'Technology Of Protein delivery through Extracellular Vesicles{\textquoteright} (TOP-EVs) as a tool for efficient intracellular delivery of target proteins mediated via EVs. The vesicular stomatitis virus glycoprotein and the rapamycin-heterodimerization of the FKBP12/T82L mutant FRB proteins were both important for the effective protein delivery through TOP-EVs. We showed that TOP-EVs could efficiently deliver Cre recombinase and CRISPR/Cas9 ribonucleoprotein complex in vitro. Moreover, our results demonstrated that the capacity of TOP-EVs to deliver intracellular proteins in recipient cells was not an artifact of plasmid contamination or direct plasmid loading into EVs. Finally, we showed that TOP-EVs could successfully mediate intracellular protein delivery in the liver in vivo. Taken together, TOP-EVs are a versatile platform for efficient intracellular protein delivery in vitro and in vivo, which can be applied to advance the development of protein-based therapeutics.",

keywords = "Active protein loading, Extracellular vesicles, Functional intracellular protein delivery, Genome editing, TOP-EV",

author = "Ilahibaks, {Nazma F.} and Ardisasmita, {Arif I.} and Songpu Xie and Anders Gunnarsson and Joseph Brealey and Pieter Vader and {de Jong}, {Olivier G.} and Jager, {Saskia de} and Niek Dekker and Ben Peacock and Schiffelers, {Raymond M.} and Sluijter, {Joost P.G.} and Zhiyong Lei",

note = "Publisher Copyright: {\textcopyright} 2023 The Authors",

year = "2023",

month = mar,

doi = "10.1016/j.jconrel.2023.02.003",

language = "English",

volume = "355",

pages = "579--592",

journal = "Journal of Controlled Release",

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Ilahibaks, NF, Ardisasmita, AI, Xie, S, Gunnarsson, A, Brealey, J, Vader, P, de Jong, OG, Jager, SD, Dekker, N, Peacock, B, Schiffelers, RM, Sluijter, JPG & Lei, Z 2023, 'TOP-EVs: Technology of Protein delivery through Extracellular Vesicles is a versatile platform for intracellular protein delivery', Journal of Controlled Release, vol. 355, no. March 2023, pp. 579-592. https://doi.org/10.1016/j.jconrel.2023.02.003

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T1 - TOP-EVs: Technology of Protein delivery through Extracellular Vesicles is a versatile platform for intracellular protein delivery

AU - Ilahibaks, Nazma F.

AU - Ardisasmita, Arif I.

AU - Xie, Songpu

AU - Gunnarsson, Anders

AU - Brealey, Joseph

AU - Vader, Pieter

AU - de Jong, Olivier G.

AU - Jager, Saskia de

AU - Dekker, Niek

AU - Peacock, Ben

AU - Schiffelers, Raymond M.

AU - Sluijter, Joost P.G.

AU - Lei, Zhiyong

PY - 2023/3

Y1 - 2023/3

N2 - Extracellular vesicles (EVs) have emerged as biocompatible drug delivery vehicles due to their native ability to deliver bioactive cargo to recipient cells. However, the application of EVs as a therapeutic delivery vehicle is hampered by effective methods for endogenously loading target proteins inside EVs and unloading proteins after delivery to recipient cells. Most EV-based engineered loading methods have a limited delivery efficiency owing to their inefficient endosomal escape or cargo release from the intraluminal attachment from the EV membrane. Here, we describe the 'Technology Of Protein delivery through Extracellular Vesicles’ (TOP-EVs) as a tool for efficient intracellular delivery of target proteins mediated via EVs. The vesicular stomatitis virus glycoprotein and the rapamycin-heterodimerization of the FKBP12/T82L mutant FRB proteins were both important for the effective protein delivery through TOP-EVs. We showed that TOP-EVs could efficiently deliver Cre recombinase and CRISPR/Cas9 ribonucleoprotein complex in vitro. Moreover, our results demonstrated that the capacity of TOP-EVs to deliver intracellular proteins in recipient cells was not an artifact of plasmid contamination or direct plasmid loading into EVs. Finally, we showed that TOP-EVs could successfully mediate intracellular protein delivery in the liver in vivo. Taken together, TOP-EVs are a versatile platform for efficient intracellular protein delivery in vitro and in vivo, which can be applied to advance the development of protein-based therapeutics.

AB - Extracellular vesicles (EVs) have emerged as biocompatible drug delivery vehicles due to their native ability to deliver bioactive cargo to recipient cells. However, the application of EVs as a therapeutic delivery vehicle is hampered by effective methods for endogenously loading target proteins inside EVs and unloading proteins after delivery to recipient cells. Most EV-based engineered loading methods have a limited delivery efficiency owing to their inefficient endosomal escape or cargo release from the intraluminal attachment from the EV membrane. Here, we describe the 'Technology Of Protein delivery through Extracellular Vesicles’ (TOP-EVs) as a tool for efficient intracellular delivery of target proteins mediated via EVs. The vesicular stomatitis virus glycoprotein and the rapamycin-heterodimerization of the FKBP12/T82L mutant FRB proteins were both important for the effective protein delivery through TOP-EVs. We showed that TOP-EVs could efficiently deliver Cre recombinase and CRISPR/Cas9 ribonucleoprotein complex in vitro. Moreover, our results demonstrated that the capacity of TOP-EVs to deliver intracellular proteins in recipient cells was not an artifact of plasmid contamination or direct plasmid loading into EVs. Finally, we showed that TOP-EVs could successfully mediate intracellular protein delivery in the liver in vivo. Taken together, TOP-EVs are a versatile platform for efficient intracellular protein delivery in vitro and in vivo, which can be applied to advance the development of protein-based therapeutics.

KW - Active protein loading

KW - Extracellular vesicles

KW - Functional intracellular protein delivery

KW - Genome editing

KW - TOP-EV

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U2 - 10.1016/j.jconrel.2023.02.003

DO - 10.1016/j.jconrel.2023.02.003

M3 - Article

SN - 0168-3659

VL - 355

SP - 579

EP - 592

JO - Journal of Controlled Release

JF - Journal of Controlled Release

IS - March 2023

ER -

TOP-EVs: Technology of Protein delivery through Extracellular Vesicles is a versatile platform for intracellular protein delivery

Abstract

Bibliographical note

Funding

Keywords

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