Extracellular vesicle engineering using a small scaffold protein

1. Herrmann, I. K., Wood, M. J. A. & Fuhrmann, G. Extracellular vesicles as a next-generation drug delivery platform. Nat. Nanotechnol. 16, 748–759 (2021).

   Google Scholar 

2. Liu, Q., Li, D., Pan, X. & Liang, Y. Targeted therapy using engineered extracellular vesicles: principles and strategies for membrane modification. J. Nanobiotechnology 21, 334 (2023).

   Google Scholar 

3. Zhang, Y. et al. Recent advances in exosome-mediated nucleic acid delivery for cancer therapy. J. Nanobiotechnology 20, 279 (2022).

   Google Scholar 

4. Radler, J., Gupta, D., Zickler, A. & Andaloussi, S. E. Exploiting the biogenesis of extracellular vesicles for bioengineering and therapeutic cargo loading. Mol. Ther. 31, 1231–1250 (2023).

   Google Scholar 

5. Zheng, W. et al. Identification of scaffold proteins for improved endogenous engineering of extracellular vesicles. Nat. Commun. 14, 4734 (2023).

   Google Scholar 

6. Dooley, K. et al. A versatile platform for generating engineered extracellular vesicles with defined therapeutic properties. Mol. Ther. 29, 1729–1743 (2021).

   Google Scholar 

7. van Niel, G., D’Angelo, G. & Raposo, G. Shedding light on the cell biology of extracellular vesicles. Nat. Rev. Mol. Cell Biol. 19, 213–228 (2018).

   Google Scholar 

8. Hemler, M. E. Tetraspanin proteins promote multiple cancer stages. Nat. Rev. Cancer 14, 49–60 (2014).

   Google Scholar 

9. Silva, A. M. et al. Quantification of protein cargo loading into engineered extracellular vesicles at single-vesicle and single-molecule resolution. J. Extracell. Vesicles 10, e12130 (2021).

   Google Scholar 

10. Gupta, D. et al. Modulation of pro-inflammatory IL-6 trans-signaling axis by splice switching oligonucleotides as a therapeutic modality in inflammation. Cells 12, https://doi.org/10.3390/cells12182285 (2023).

11. Zhang, Y. Y. & Ning, B. T. Signaling pathways and intervention therapies in sepsis. Signal Transduct. Target. Ther. 6, 407 (2021).

    Google Scholar 

12. Liu, D. et al. Sepsis-induced immunosuppression: mechanisms, diagnosis and current treatment options. Mil. Med. Res. 9, 56 (2022).

    Google Scholar 

13. Li, J. et al. Targeted and responsive biomaterials in osteoarthritis. Theranostics 13, 931–954 (2023).

    Google Scholar 

14. Kapoor, M., Martel-Pelletier, J., Lajeunesse, D., Pelletier, J. P. & Fahmi, H. Role of proinflammatory cytokines in the pathophysiology of osteoarthritis. Nat. Rev. Rheumatol. 7, 33–42 (2011).

    Google Scholar 

15. Liu, C. et al. Cytokines: from clinical significance to quantification. Adv. Sci. 8, e2004433 (2021).

    Google Scholar 

16. Chen, Z., Bozec, A., Ramming, A. & Schett, G. Anti-inflammatory and immune-regulatory cytokines in rheumatoid arthritis. Nat. Rev. Rheumatol. 15, 9–17 (2019).

    Google Scholar 

17. Katz, J. N., Arant, K. R. & Loeser, R. F. Diagnosis and treatment of hip and knee osteoarthritis: a review. JAMA 325, 568–578 (2021).

    Google Scholar 

18. McElvaney, O. J., Curley, G. F., Rose-John, S. & McElvaney, N. G. Interleukin-6: obstacles to targeting a complex cytokine in critical illness. Lancet Respir. Med. 9, 643–654 (2021).

    Google Scholar 

19. Scheller, J., Chalaris, A., Schmidt-Arras, D. & Rose-John, S. The pro- and anti-inflammatory properties of the cytokine interleukin-6. Biochim. Biophys. Acta 1813, 878–888 (2011).

    Google Scholar 

20. Rose-John, S. IL-6 trans-signaling via the soluble IL-6 receptor: importance for the pro-inflammatory activities of IL-6. Int. J. Biol. Sci. 8, 1237–1247 (2012).

    Google Scholar 

21. Garbers, C., Heink, S., Korn, T. & Rose-John, S. Interleukin-6: designing specific therapeutics for a complex cytokine. Nat. Rev. Drug Discov. 17, 395–412 (2018).

    Google Scholar 

22. Jostock, T. et al. Soluble gp130 is the natural inhibitor of soluble interleukin-6 receptor transsignaling responses. Eur. J. Biochem. 268, 160–167 (2001).

    Google Scholar 

23. Sadri, M. et al. Tumor necrosis factor receptor-1 is selectively sequestered into Schwann cell extracellular vesicles where it functions as a TNFalpha decoy. Glia 70, 256–272 (2022).

    Google Scholar 

24. Keller, M. D. et al. Decoy exosomes provide protection against bacterial toxins. Nature 579, 260–264 (2020).

    Google Scholar 

25. Gupta, D. 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).

    Google Scholar 

26. Conceicao, M. et al. Engineered extracellular vesicle decoy receptor-mediated modulation of the IL6 trans-signalling pathway in muscle. Biomaterials 266, 120435 (2021).

    Google Scholar 

27. Saleh, A. F. et al. Extracellular vesicles induce minimal hepatotoxicity and immunogenicity. Nanoscale 11, 6990–7001 (2019).

    Google Scholar 

28. Zhang, H. et al. Surface functionalization of exosomes for chondrocyte-targeted siRNA delivery and cartilage regeneration. J. Control. Release 369, 493–505 (2024).

    Google Scholar 

29. Wu, X., Li, L., Iliuk, A. & Tao, W. A. Highly efficient phosphoproteome capture and analysis from urinary extracellular vesicles. J. Proteome Res. 17, 3308–3316 (2018).

    Google Scholar 

30. Haraszti, R. A. et al. Exosomes produced from 3D cultures of MSCs by tangential flow filtration show higher yield and improved activity. Mol. Ther. 26, 2838–2847 (2018).

    Google Scholar 

31. Corso, G. et al. Systematic characterization of extracellular vesicle sorting domains and quantification at the single molecule—single vesicle level by fluorescence correlation spectroscopy and single particle imaging. J. Extracell. Vesicles 8, 1663043 (2019).

    Google Scholar 

32. Yan, W. et al. Chondrocyte-targeted delivery system of sortase A-engineered extracellular vesicles silencing MMP13 for osteoarthritis therapy. Adv. Healthc. Mater. e2303510. https://doi.org/10.1002/adhm.202303510 (2024).

33. Ye, Y. et al. An engineered exosome for delivering sgRNA:Cas9 ribonucleoprotein complex and genome editing in recipient cells. Biomater. Sci. 8, 2966–2976 (2020).

    Google Scholar 

34. Osteikoetxea, X. et al. Engineered Cas9 extracellular vesicles as a novel gene editing tool. J. Extracell. Vesicles 11, e12225 (2022).

    Google Scholar 

35. Gotzke, H. et al. The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications. Nat. Commun. 10, 4403 (2019).

    Google Scholar 

36. Karanicolas, J. et al. A de novo protein binding pair by computational design and directed evolution. Mol. Cell 42, 250–260 (2011).

    Google Scholar 

37. Yang, Y., Hong, Y., Cho, E., Kim, G. B. & Kim, I. S. Extracellular vesicles as a platform for membrane-associated therapeutic protein delivery. J. Extracell. Vesicles 7, 1440131 (2018).

    Google Scholar 

38. Durocher, Y., Perret, S. & Kamen, A. High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells. Nucleic Acids Res. 30, E9 (2002).

    Google Scholar 

39. Lewis, N. D. et al. Exosome surface display of IL12 results in tumor-retained pharmacology with superior potency and limited systemic exposure compared with recombinant IL12. Mol. Cancer Ther. 20, 523–534 (2021).

    Google Scholar 

40. Pei, W. et al. Multitargeted immunomodulatory therapy for viral myocarditis by engineered extracellular vesicles. ACS Nano 18, 2782–2799 (2024).

    Google Scholar 

41. Nikonorova, I. A. et al. Isolation, profiling, and tracking of extracellular vesicle cargo in Caenorhabditis elegans. Curr. Biol. 32, 1924–1936 e1926 (2022).

    Google Scholar 

42. Li, Z., He, Q., Peng, J., Yan, Y. & Fu, C. Identification of downregulated exosome-associated gene ENPP1 as a novel lipid metabolism and immune-associated biomarker for hepatocellular carcinoma. J. Oncol. 2022, 4834791 (2022).

    Google Scholar 

43. An, Y. et al. Tumor exosomal ENPP1 hydrolyzes cGAMP to inhibit cGAS-STING signaling. Adv. Sci. 11, e2308131 (2024).

    Google Scholar 

44. Ferreira, C. R., Carpenter, T. O. & Braddock, D. T. ENPP1 in blood and bone: skeletal and soft tissue diseases induced by ENPP1 deficiency. Annu. Rev. Pathol. 19, 507–540 (2024).

    Google Scholar 

45. Jansen, S. et al. Structure of NPP1, an ectonucleotide pyrophosphatase/phosphodiesterase involved in tissue calcification. Structure 20, 1948–1959 (2012).

    Google Scholar 

46. Borza, R., Salgado-Polo, F., Moolenaar, W. H. & Perrakis, A. Structure and function of the ecto-nucleotide pyrophosphatase/phosphodiesterase (ENPP) family: tidying up diversity. J. Biol. Chem. 298, 101526 (2022).

    Google Scholar 

47. Corbeil, D. et al. Uptake and fate of extracellular membrane vesicles: nucleoplasmic reticulum-associated late endosomes as a new gate to intercellular communication. Cells 9, https://doi.org/10.3390/cells9091931 (2020).

48. Sun, M., Luong, G., Plastikwala, F. & Sun, Y. Control of Rab7a activity and localization through endosomal type Igamma PIP 5-kinase is required for endosome maturation and lysosome function. FASEB J. 34, 2730–2748 (2020).

    Google Scholar 

49. Escude Martinez de Castilla, P. et al. Extracellular vesicles as a drug delivery system: a systematic review of preclinical studies. Adv. Drug Deliv. Rev. 175, 113801 (2021).

    Google Scholar 

50. Grange, C. et al. Biodistribution of mesenchymal stem cell-derived extracellular vesicles in a model of acute kidney injury monitored by optical imaging. Int. J. Mol. Med. 33, 1055–1063 (2014).

    Google Scholar 

51. Driedonks, T. et al. Pharmacokinetics and biodistribution of extracellular vesicles administered intravenously and intranasally to Macaca nemestrina. J. Extracell. Biol. 1, https://doi.org/10.1002/jex2.59 (2022).

52. Kamerkar, S. et al. Exosomes facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer. Nature 546, 498–503 (2017).

    Google Scholar 

53. Murphy D.E. et al. Extracellular vesicle-based therapeutics: natural versus engineered targeting and trafficking. Exp. Mol. Med. 51, 1–12 (2019).

    Google Scholar 

54. Kang, M., Jordan, V., Blenkiron, C. & Chamley, L. W. Biodistribution of extracellular vesicles following administration into animals: a systematic review. J. Extracell. Vesicles 10, e12085 (2021).

    Google Scholar 

55. Zhang, H. et al. Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow field-flow fractionation. Nat. Cell Biol. 20, 332–343 (2018).

    Google Scholar 

56. Liam-Or, R. et al. Cellular uptake and in vivo distribution of mesenchymal-stem-cell-derived extracellular vesicles are protein corona dependent. Nat. Nanotechnol. 19, 846–855 (2024).

    Google Scholar 

57. Zhu, X. et al. Comprehensive toxicity and immunogenicity studies reveal minimal effects in mice following sustained dosing of extracellular vesicles derived from HEK293T cells. J. Extracell. Vesicles 6, 1324730 (2017).

    Google Scholar 

58. Hung, M. E. & Leonard, J. N. A platform for actively loading cargo RNA to elucidate limiting steps in EV-mediated delivery. J. Extracell. Vesicles 5, 31027 (2016).

    Google Scholar 

59. Kojima, R. et al. Designer exosomes produced by implanted cells intracerebrally deliver therapeutic cargo for Parkinson’s disease treatment. Nat. Commun. 9, 1305 (2018).

    Google Scholar 

60. Watanabe, E. et al. Extremely high interleukin-6 blood levels and outcome in the critically ill are associated with tumor necrosis factor- and interleukin-1-related gene polymorphisms. Crit. Care Med. 33, 89–97 (2005).

    Google Scholar 

61. Garbers, C. et al. Plasticity and cross-talk of interleukin 6-type cytokines. Cytokine Growth Factor Rev. 23, 85–97 (2012).

    Google Scholar 

62. Lokau, J. & Garbers, C. Biological functions and therapeutic opportunities of soluble cytokine receptors. Cytokine Growth Factor Rev. 55, 94–108 (2020).

    Google Scholar 

63. Liang, M. et al. Replenishing decoy extracellular vesicles inhibits phenotype remodeling of tissue-resident cells in inflammation-driven arthritis. Cell Rep. Med. 4, 101228 (2023).

    Google Scholar 

64. Xu, X. et al. Exosome-mediated delivery of kartogenin for chondrogenesis of synovial fluid-derived mesenchymal stem cells and cartilage regeneration. Biomaterials 269, 120539 (2021).

    Google Scholar 

65. Zhang, X. et al. Long-term 4-nonylphenol exposure drives cervical cell malignancy through MAPK-mediated ferroptosis inhibition. J. Hazard. Mater. 471, 134371 (2024).

    Google Scholar 

66. Brinkman, E. K., Chen, T., Amendola, M. & van Steensel, B. Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res. 42, e168 (2014).

    Google Scholar 

67. Mitrani, M. I. et al. Case Report: administration of amniotic fluid-derived nanoparticles in three severely Ill COVID-19 patients. Front. Med. 8, 583842 (2021).

    Google Scholar 

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Author notes

1. These authors contributed equally: Wenjing Yan, Shizhi Wang, Haibin Hao.

Authors and Affiliations

1. Key Laboratory of Environmental Medicine Engineering, Ministry of Education, School of Public Health, Southeast University, Nanjing, China

   Wenjing Yan, Shizhi Wang, Shuqian Xie, Xing Zhang, Yiran Lu, Xin Ding, Xue Chen, Haohan Liu & Jing Hu

2. School of Public Health, Shandong Second Medical University, Weifang, China

   Wenjing Yan

3. Department of Critical Care Medicine, Jinling Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing, China

   Haibin Hao, Weiqin Li & Hao Zhang

4. School of Medicine, Southeast University, Nanjing, China

   Hong Lin

5. School of Pharmacy, Jiangsu University, Zhenjiang, China

   Chen Wang

6. State Key Laboratory of Digital Medical Engineering, School of Biological Science and Medical Engineering, Southeast University, Nanjing, China

   Guiyuan Zhang, Dong Wei, Zhongze Gu & Hao Zhang

7. Department of Medical Genetics, Nanjing Medical University, Nanjing, China

   ChangYan Ma

8. Department of Orthopaedics, Nanjing First Hospital, Nanjing Medical University, Nanjing, China

   Cheng Tang

9. School of Public Health Administration, Jiangsu Health Vocational College, Nanjing, China

   Xiuting Li & Bingjia Yu

10. Key Laboratory of Environmental Medicine and Engineering of the Ministry of Education, and Department of Epidemiology & Biostatistics, School of Public Health, Southeast University, Nanjing, China

    Evan Yi-Wen Yu

11. Department of Chemistry, The Chinese University of Hong Kong, Hong Kong SAR, China

    Jiang Xia

12. EVLiXiR Biotech Inc., Nanjing, China

    Hao Zhang

Contributions

H.Z. and J.X. conceived and designed the study. H.Z. and S.W. supervised and acquired funding for this project. W.Y., H.H. and S.W. wrote the first draft. H. Lin, C.W., S.X., X.Z., Y.L. and X.D. performed the experiments. X.C., H. Liu, D.W., C.M., C.T., X.L., J.H., Z.G. and B.Y. analyzed the data. G.Z. provided mass spectrometry expertise. W.J., S.W., E.Y., W.L., J.X. and H.Z. revised the manuscript.

Corresponding authors

Correspondence to Shizhi Wang, Evan Yi-Wen Yu, Weiqin Li, Jiang Xia or Hao Zhang.

Competing interests

This research has been filed for a patent under application number CN116769718A with W.Y. and H.Z. named as inventors. J.X. and H.Z. co-founded EVLiXiR Biotech Inc., where H.Z. serves as CEO and J.X. as chief scientist. All other authors declare no conflicts of interest.

Peer review information

Nature Communications thanks Christoph Garbers and the other anonymous reviewers for their contribution to the peer review of this work. A peer review file is available.

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