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Amelioration of systemic inflammation via the display of two different decoy protein receptors on extracellular vesicles

Abstract

Extracellular vesicles (EVs) can be functionalized to display specific protein receptors on their surface. However, surface-display technology typically labels only a small fraction of the EV population. Here, we show that the joint display of two different therapeutically relevant protein receptors on EVs can be optimized by systematically screening EV-loading protein moieties. We used cytokine-binding domains derived from tumour necrosis factor receptor 1 (TNFR1) and interleukin-6 signal transducer (IL-6ST), which can act as decoy receptors for the pro-inflammatory cytokines tumour necrosis factor alpha (TNF-α) and IL-6, respectively. We found that the genetic engineering of EV-producing cells to express oligomerized exosomal sorting domains and the N-terminal fragment of syntenin (a cytosolic adaptor of the single transmembrane domain protein syndecan) increased the display efficiency and inhibitory activity of TNFR1 and IL-6ST and facilitated their joint display on EVs. In mouse models of systemic inflammation, neuroinflammation and intestinal inflammation, EVs displaying the cytokine decoys ameliorated the disease phenotypes with higher efficacy as compared with clinically approved biopharmaceutical agents targeting the TNF-α and IL-6 pathways.

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Fig. 1: Systematic screening of multiple endogenous EV display strategies for cytokine decoys.
Fig. 2: Multimerization of decoy fusion proteins improves loading and activity.
Fig. 3: Multimeric decoy receptor EV-sorting protein chimaera is functionalized on several EV subpopulations.
Fig. 4: Benchmarking engineered decoy EVs against clinically approved biologics both in vitro and in vivo.
Fig. 5: Targeting the TNF-α, IL-6 and IL-23 signalling axes with engineered decoy EVs suppresses neuroinflammation.
Fig. 6: Dual-functionalized engineered EVs protect against intestinal inflammation.

Data availability

The main data supporting the results in this study are available within the paper and its Supplementary Information. The raw and analysed datasets are available at Figshare (https://figshare.com/projects/Raw_and_Analysed_data_Gupta_et_al_NBME_2021/119859).

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Acknowledgements

We acknowledge B. Vanneste and G. Van Imschoot for their assistance. A.G. is an International Society for Advancement of Cytometry Marylou Ingram Scholar 2019–2023. S.E.-A. is supported by H2020 EXPERT, the Swedish foundation of Strategic Research (SSF-IRC; FormulaEx), ERC CoG (DELIVER) and the Swedish Medical Research Council.

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Authors and Affiliations

Authors

Contributions

S.E.-A. conceived the idea. D.G., O.P.B.W., J.Z.N. and S.E.-A. designed the research. D.G., Y.S. and J.Z.N. designed the genetic constructs and performed the cloning. D.G., O.P.B.W., A.G., M.C., G.C., X.L., Y.S., U.F., B.B., R.J., D.R.M., Y.X.F.L., M.G., D.K.M. and H.S. performed the in vitro experiments. D.G., O.P.B.W., S.B. and A.B. performed the in vivo experiments. D.G., O.P.B.W. and J.Z.N. analysed the results. J.H., I.M., T.C.R., P.L., A.d.F., C.I.E.S., M.J.A.W., R.E.V. and S.E.-A. provided input on experimental plan and discussion of results. D.G. wrote the manuscript with input from all of the co-authors. J.Z.N. and S.E.-A. co-led the study.

Corresponding authors

Correspondence to Dhanu Gupta, Oscar P. B. Wiklander, Joel Z. Nordin or Samir El-Andaloussi.

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Competing interests

M.J.A.W. and S.E.-A. are founders of and consultants for Evox Therapeutics. D.G., O.P.B.W., A.G. and J.Z.N. are consultants for Evox Therapeutics. D.G., O.P.B.W., A.G., J.Z.N., M.J.A.W., P.L., A.d.F. and S.E.-A. have stock interest in Evox Therapeutics. J.H., P.L. and A.d.F. are employees of Evox Therapeutics. The other authors declare no competing interests.

Additional information

Peer review information Nature Biomedical Engineering thanks Ke Cheng, Gregor Fuhrmann and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Functionalization of the EV surface with cytokine decoy receptors.

A) Schematic illustration showing the generation of engineered decoy EVs at the cellular level. Producer cells are genetically modified to express cytokine receptors without the signalling domain fused to an EV sorting domain for efficient display of cytokine receptors on the surface of the secreted EVs (decoy EVs), which can decoy cytokines specifically. B-C) Mode size determined by NTA of indicated engineered HEK293T EVs purified from cells transfected with various B) TNFR1 display constructs and C) IL6ST display constructs. Central Value: Mean, Error bars: S.D (Technical replicate, n=5). D) Fold change in signal over untreated HEK293T NF-κB reporter cells, upon incubation of different doses of TNFα for 4 hours. Central Value: Mean, Error bars: S.D (Biological replicate, n= 3). E) Fold change in signal over untreated HEK293T STAT3 reporter cells, upon incubation of different doses of IL-6/sIL-6R for 12 hours. Central Value: Mean, Error bars: S.D (Biological replicate, n= 3).

Extended Data Fig. 2 Amelioration of neuroinflammation by cytokine-decoy-engineered EVs.

Relative mRNA expression of pro inflammatory cytokines determined by qPCR (A) TNFα, (B) IL-6 in the spinal cord at day 16 in mice induced with EAE using MOG35-55 peptide and treated with S.C administration of either 4×1010 MSC TNFR1∆∆-FDN-NST EVs (n= 5) or MSC Ctrl EVs (n= 5) or Saline (n= 5) (On day 7, 10 & 13). For the same dataset as shown in Fig. 5b,c. Central Value: Mean, Error bars: S.D, Statistical significance calculated by one-way ANOVA compared with response of Saline treated animal. C) Relative change in clinical score of disease progression between day 16 and 13 in mice induced with EAE using MOG35-55 peptide and treated with I.V administration of either 1×1010 HEK293T IL-23B-LZ-NST EVs pre symptomatic (On day 5, 7 & 10) or 6×1010 HEK293T IL-23B-LZ-NST EVs post symptomatic (On day 13) or Saline. For the same dataset as shown in Fig. 5h, i. Central Value: Mean, Error bars: S.D, Statistical significance calculated by two-way ANOVA with Dunnett’s post-test compared with response of Saline treated animal.

Extended Data Fig. 3 The surface proteome is unaltered by the dual functionalization of engineered EVs.

A) Imaging flow cytometry analysis (IFCM) with dot plots and example event images in the double positive (DP) gate of MSC TNFR1∆∆-FDN-NST and MSC double decoy EVs stained with mIL6ST APC conjugated and hTNFR1 PE conjugated antibody. PBS + antibodies were used for background adjustment and for determining the gating strategy. B) Multiplex EV surface characterization of PAN (CD63, CD81, and CD9) positive, hTNFR1 positive and mIL6ST positive population in MSC double decoy EVs and MSC Ctrl EVs. Data represented as background corrected median APC fluorescence intensity determined by flow cytometry of EVs bound different capture beads and upon using APC labelled detection antibody. Central Value: Mean, Error bars: S.D, (Technical replicate, n=5000-15000).

Extended Data Fig. 4 Engineered EVs show therapeutic benefit in intestinal inflammation.

A) Increment in EV accumulation in various tissues in animal induced with IBD over healthy animal. B) Change in body weight 24 hours after treatment with different groups in mice induced with TNBS colitis. For the same data set as shown in Fig. 6g, h. C) Survival curve and D) percent change in relative bodyweight to initial weight over the disease course in mice induced with colitis by intrarectal injection of TNBS and treated with I.V administration (at 24 hours post disease induction) of either 3×1011 MSC double decoy EVs (n= 8) or PBS (n= 9).

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Gupta, D., Wiklander, O.P.B., Görgens, A. et al. Amelioration of systemic inflammation via the display of two different decoy protein receptors on extracellular vesicles. Nat Biomed Eng 5, 1084–1098 (2021). https://doi.org/10.1038/s41551-021-00792-z

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