Engineered extracellular vesicles enable high-efficient delivery of intracellular therapeutic proteins

Abstract

Developing an intracellular delivery system is of key importance in the expansion of protein-based therapeutics acting on cytosolic or nuclear targets. Recently, extracellular vesicles (EVs) have been exploited as next-generation delivery modalities due to their natural role in intercellular communication and biocompatibility. However, fusion of protein of interest to a scaffold represents a widely used strategy for cargo enrichment in EVs, which could compromise the stability and functionality of cargo. Herein, we report intracellular delivery via EV-based approach (IDEA) that efficiently packages and delivers native proteins both in vitro and in vivo without the use of a scaffold. As a proof-of-concept, we applied the IDEA to deliver cyclic GMP-AMP synthase (cGAS), an innate immune sensor. The results showed that cGAS-carrying EVs activated interferon signaling and elicited enhanced antitumor immunity in multiple syngeneic tumor models. Combining cGAS EVs with immune checkpoint inhibition further synergistically boosted antitumor efficacy in vivo. Mechanistically, scRNA-seq demonstrated that cGAS EVs mediated significant remodeling of intratumoral microenvironment, revealing a pivotal role of infiltrating neutrophils in the antitumor immune milieu. Collectively, IDEA, as a universal and facile strategy, can be applied to expand and advance the development of protein-based therapeutics.

Key words： extracellular vesicles; intracellular protein delivery; cGAS; cancer immunotherapy

References

1 Banskota S, Raguram A, Suh S et al. Engineered virus-like particles for efficient in vivo delivery of therapeutic proteins. Cell 2022;185:250–265.e16.

2 Beilstein F, Abou Hamdan A, Raux H et al. Identification of a pH-sensitive switch in VSV-G and a crystal structure of the G pre-fusion state highlight the VSV-G structural transition pathway. Cell Rep 2020;32:108042.

3 Bulcha JT, Wang Y, Ma H et al. Viral vector platforms within the gene therapy landscape. Signal Transduct Target Ther 2021;6:53.

4 Cecchin R, Troyer Z, Witwer K et al. Extracellular vesicles: the next generation in gene therapy delivery. Mol Ther 2023;31:1225–1230.

5 Chin EN, Sulpizio A, Lairson LL. Targeting STING to promote antitumor immunity. Trends Cell Biol 2023;33:189–203.

6 Choi H, Kim Y, Mirzaaghasi A et al. Exosome-based delivery of super-repressor IkappaBalpha relieves sepsisassociated organ damage and mortality. Sci Adv 2020;6:eaaz6980.

7 Cui C, Chakraborty K, Tang XA et al. Neutrophil elastase selectively kills cancer cells and attenuates tumorigenesis. Cell 2021;184:3163–3177.e21.

8 Dooley K, McConnell RE, Xu K et al. A versatile platform for generating engineered extracellular vesicles with defined therapeutic properties. Mol Ther 2021;29:1729–1743.

9 Earley J, Piletska E, Ronzitti G et al. Evading and overcoming AAV neutralization in gene therapy. Trends Biotechnol 2023;41:836–845.

10 Escude Martinez de Castilla P, Tong L, Huang, C et al.. Extracellular vesicles as a drug delivery system: a systematic review of preclinical studies. Adv Drug Deliv Rev 2021;175:113801.

11 Ferreira JV, da Rosa Soares A, Ramalho J et al. LAMP2A regulates the loading of proteins into exosomes. Sci Adv 2022;8:eabm1140.

12 Finkelshtein D, Werman A, Novick D et al. LDL receptor and its family members serve as the cellular receptors for vesicular stomatitis virus. Proc Natl Acad Sci U S A 2013;110:7306–7311.

13 Gao D, Wu J, Wu YT et al. Cyclic GMP-AMP synthase is an innate immune sensor of HIV and other retroviruses. Science 2013;341:903–906.

14 Gao L, Sun Y, Zhang X et al. Wnt3a-loaded extracellular vesicles promote alveolar epithelial regeneration after lung injury. Adv Sci (Weinh) 2023;10:e2206606.

15 Goswami R, Jeon T, Nagaraj H et al. Accessing intracellular targets through nanocarrier-mediated cytosolic protein delivery. Trends Pharmacol Sci 2020;41:743–754.

16 Gouveia MG, Wesseler JP, Ramaekers J et al. Polymersome-based protein drug delivery - quo vadis? Chem Soc Rev 2023;52:728–778.

17 Greening DW, Xu R, Ale A et al. Extracellular vesicles as next generation immunotherapeutics. Semin Cancer Biol 2023;90:73–100.

18 Gungabeesoon J, Gort-Freitas NA, Kiss M et al. A neutrophil response linked to tumor control in immunotherapy. Cell 2023;186:1448–1464.e20.

19 Guo J, Huang L. Nanodelivery of cGAS-STING activators for tumor immunotherapy. Trends Pharmacol Sci 2022;43:957–972.

20 Hamilton JR, Tsuchida CA, Nguyen DN et al. Targeted delivery of CRISPR-Cas9 and transgenes enables complex immune cell engineering. Cell Rep 2021;35:109207.

21 Han Z, Liu S, Pei Y et al. Highly efficient magnetic labelling allows MRI tracking of the homing of stem cell-derived extracellular vesicles following systemic delivery. J Extracell Vesicles 2021;10:e12054.

22 Hedrick CC, Malanchi I. Neutrophils in cancer: heterogeneous and multifaceted. Nat Rev Immunol 2022;22:173–187.

23 Herrmann IK, Wood MJA, Fuhrmann G. Extracellular vesicles as a next-generation drug delivery platform. Nat Nanotechnol 2021;16:748–759.

24 Hirschhorn D, Budhu S, Kraehenbuehl L et al. T cell immunotherapies engage neutrophils to eliminate tumor antigen escape variants. Cell 2023;186:1432–1447.e17.

25 Hou X, Zaks T, Langer R et al. Lipid nanoparticles for mRNA delivery. Nat Rev Mater 2021;6:1078–1094.

26 Hu S, Fang Y, Chen X et al. cGAS restricts colon cancer development by protecting intestinal barrier integrity. Proc Natl Acad Sci U S A 2021;118:e2105747118.

27 Ilahibaks NF, Ardisasmita AI, Xie S et al. TOP-EVs: technology of protein delivery through extracellular vesicles is a versatile platform for intracellular protein delivery. J Control Release 2023;355:579–592.

28 Islam MA, Xu Y, Tao W et al. Restoration of tumourgrowth suppression in vivo via systemic nanoparticle-mediated delivery of PTEN mRNA. Nat Biomed Eng 2018;2:850–864.

29 Kalluri R, LeBleu VS. The biology, function, and biomedical applications of exosomes. Science 2020;367:eaau6977.

30 Kasala D, Hong J, Yun CO. Overcoming the barriers to optimization of adenovirus delivery using biomaterials: current status and future perspective. J Control Release 2021;332:285–300.

31 Kitajima S, Tani T, Springer BF et al. MPS1 inhibition primes immunogenicity of KRAS-LKB1 mutant lung cancer. Cancer Cell 2022;40:1128–1144.e8.

32 Kreitz J, Friedrich MJ, Guru A et al. Programmable protein delivery with a bacterial contractile injection system. Nature 2023;616:357–364.

33 Lee KM, Lin CC, Servetto A et al. Epigenetic repression of STING by MYC promotes immune evasion and resistance to immune checkpoint inhibitors in triple-negative breast cancer. Cancer Immunol Res 2022;10:829–843.

34 Li S, Luo M, Wang Z et al. Prolonged activation of innate immune pathways by a polyvalent STING agonist. Nat Biomed Eng 2021;5:455–466.

35 Linde IL, Prestwood TR, Qiu J et al. Neutrophil-activating therapy for the treatment of cancer. Cancer Cell 2023;41:356–372.e10.

36 Ling S, Yang S, Hu X et al. Lentiviral delivery of co-packaged Cas9 mRNA and a Vegfa-targeting guide RNA prevents wet age-related macular degeneration in mice. Nat Biomed Eng 2021;5:144–156.

37 Liu B, Lee BW, Nakanishi K et al. Cardiac recovery via extended cell-free delivery of extracellular vesicles secreted by cardiomyocytes derived from induced pluripotent stem cells. Nat Biomed Eng 2018;2:293–303.

38 Liu S, Mahairaki V, Bai H et al. Highly purified human extra-cellular vesicles produced by stem cells alleviate aging cellular phenotypes of senescent human cells. Stem Cells 2019;37:779–790.

39 Liu S, Wu M, Lancelot M et al. BMI1 enables extensive expansion of functional erythroblasts from human peripheral blood mononuclear cells. Mol Ther 2021;29:1918–1932.

40 Lokugamage MP, Vanover D, Beyersdorf J et al. Optimization of lipid nanoparticles for the delivery of nebulized therapeutic mRNA to the lungs. Nat Biomed Eng 2021;5:1059–1068.

41 Low JT, Chandramohan V, Bowie ML et al. Epigenetic STING silencing is developmentally conserved in gliomas and can be rescued by methyltransferase inhibition. Cancer Cell 2022;40:439–440.

42 Ma D, Liu S, Lal B et al. Extracellular matrix protein tenascin c increases phagocytosis mediated by CD47 loss of function in glioblastoma. Cancer Res 2019;79:2697–2708.

43 Machtakova M, Therien-Aubin H, Landfester K. Polymer nano-systems for the encapsulation and delivery of active biomacromolecular therapeutic agents. Chem Soc Rev 2022;51:128–152.

44 Meric-Bernstam F, Sweis RF, Hodi FS et al. Phase I dose-escalation trial of MIW815 (ADU-S100), an intratumoral STING agonist, in patients with advanced/ metastatic solid tumors or lymphomas. Clin Cancer Res 2022;28:677–688.

45 Meric-Bernstam F, Sweis RF, Kasper S et al. Combination of the STING agonist MIW815 (ADU-S100) and PD-1 inhibitor spartalizumab in advanced/metastatic solid tumors or lymphomas: an open-label, multicenter, phase Ib study. Clin Cancer Res 2023;29:110–121.

46 Milone MC, O’Doherty U. Clinical use of lentiviral vectors. Leukemia 2018;32:1529–1541.

47 Morad G, Helmink BA, Sharma P et al. Hallmarks of response, resistance, and toxicity to immune checkpoint blockade. Cell 2021;184:5309–5337.

48 Morshedi Rad D, Alsadat Rad M, Razavi Bazaz S et al. A comprehensive review on intracellular delivery. Adv Mater 2021;33:e2005363.

49 Muttenthaler M, King GF, Adams DJ et al. Trends in peptide drug discovery. Nat Rev Drug Discov 2021;20:309–325.

50 Nikolic J, Belot L, Raux H et al. Structural basis for the recognition of LDL-receptor family members by VSV glycoprotein. Nat Commun 2018;9:1029.

51 Nusse R, Clevers H. Wnt/beta-catenin signaling, disease, and emerging therapeutic modalities. Cell 2017;169:985–999.

52 Paunovska K, Loughrey D, Dahlman JE. Drug delivery systems for RNA therapeutics. Nat Rev Genet 2022;23:265–280.

53 Ponzetta A, Carriero R, Carnevale S et al. Neutrophils driving unconventional T cells mediate resistance against murine sarcomas and selected human tumors. Cell 2019;178:346–360.e244.

54 Qin X, Yu C, Wei J et al. Rational design of nanocarriers for intracellular protein delivery. Adv Mater 2019;31:e1902791.

55 Quail DF, Amulic B, Aziz M et al. Neutrophil phenotypes and functions in cancer: a consensus statement. J Exp Med 2022;219:e20220011.

56 Ren Q, Cheng Y, Lv J. Boronate building blocks for intracellular protein delivery. Adv Healthc Mater 2023;12:e2202049.

57 Routledge D, Scholpp S. Mechanisms of intercellular Wnt transport. Development 2019;146:dev176073.

58 Russell JO, Monga SP. Wnt/beta-catenin signaling in liver development, homeostasis, and pathobiology. Annu Rev Pathol 2018;13:351–378.

59 Samson N, Ablasser A. The cGAS-STING pathway and cancer. Nat Cancer 2022;3:1452–1463.

60 Sanchez-Navarro M. Advances in peptide-mediated cytosolic delivery of proteins. Adv Drug Deliv Rev 2021;171: 187–198.

61 Schunk SJ, Floege J, Fliser D et al. WNT-beta-catenin signalling – a versatile player in kidney injury and repair. Nat Rev Nephrol 2021;17:172–184.

62 Segel M, Lash B, Song J et al. Mammalian retrovirus-like protein PEG10 packages its own mRNA and can be pseudotyped for mRNA delivery. Science 2021;373:882–889.

63 Shae D, Becker KW, Christov P et al. Endosomolytic polymersomes increase the activity of cyclic dinucleotide STING agonists to enhance cancer immunotherapy. Nat Nanotechnol 2019;14:269–278.

64 Sheller-Miller S, Radnaa E, Yoo JK et al. Exosomal delivery of NF-kappaB inhibitor delays LPS-induced preterm birth and modulates fetal immune cell profile in mouse models. Sci Adv 2021;7:eabd3865.

65 Shirley JL, de Jong YP, Terhorst C et al. Immune responses to viral gene therapy vectors. Mol Ther 2020;28:709–722.

66 Silva AM, Lazaro-Ibanez E, Gunnarsson A et al. Quantification of protein cargo loading into engineered extracellular vesicles at single-vesicle and single-molecule resolution. J Extracell Vesicles 2021;10:e12130.

67 Stewart MP, Sharei A, Ding X et al. In vitro and ex vivo strategies for intracellular delivery. Nature 2016;538:183–192.

68 Tian Y, Tirrell MV, LaBelle JL. Harnessing the therapeutic potential of biomacromolecules through intracellular delivery of nucleic acids, peptides, and proteins. Adv Healthc Mater 2022;11:e2102600.

69 Vargason AM, Anselmo AC, Mitragotri S. The evolution of commercial drug delivery technologies. Nat Biomed Eng 2021;5:951–967.

70 Votteler J, Ogohara C, Yi S et al. Designed proteins induce the formation of nanocage-containing extracellular vesicles. Nature 2016;540:292–295.

71 Wang Q, Yu J, Kadungure T et al. ARMMs as a versatile platform for intracellular delivery of macromolecules. Nat Commun 2018;9:960.

72 Wang F, Su H, Xu D et al. Tumour sensitization via the extended intratumoural release of a STING agonist and camptothecin from a self-assembled hydrogel. Nat Biomed Eng 2020;4:1090–1101.

73 Wang BZ, Luo LJ, Vunjak-Novakovic G. RNA and protein delivery by cell-secreted and bioengineered extracellular vesicles. Adv Healthc Mater 2022;11:e2101557.

74 Wang L, Liu Y, Dai Y et al. Single-cell RNA-seq analysis reveals BHLHE40-driven pro-tumour neutrophils with hyperactivated glycolysis in pancreatic tumour microenvironment. Gut 2023a;72:958–971.

75 Wang Y, Xu M, Sun J et al. Glycolytic neutrophils accrued in the spleen compromise anti-tumour T cell immunity in breast cancer. Nat Metab 2023b;5:1408–1422.

76 Wei X, Zhang L, Yang Y et al. LL-37 transports immunore-active cGAMP to activate STING signaling and enhance interferon-mediated host antiviral immunity. Cell Rep 2022;39:110880.

77 Wiklander OPB, Brennan MA, Lotvall J et al. Advances in therapeutic applications of extracellular vesicles. Sci Transl Med 2019;11:eaav8521.

78 Wolf L, Boutros M. The role of Evi/Wntless in exporting Wnt proteins. Development 2023;150:dev201352.

79 Yang K, Han W, Jiang X et al. Zinc cyclic di-AMP nanoparticles target and suppress tumours via endothelial STING activation and tumour-associated macrophage reinvigoration. Nat Nanotechnol 2022;17:1322–1331.

80 Yang W, Mixich L, Boonstra E et al. Polymer-based mRNA delivery strategies for advanced therapies. Adv Healthc Mater 2023;12:e2202688.

81 Yom-Tov N, Guy R, Offen D. Extracellular vesicles over adeno- associated viruses: Advantages and limitations as drug delivery platforms in precision medicine. Adv Drug Deliv Rev 2022;190:114535.

82 Yonezawa A, Cavrois M, Greene WC. Studies of ebola virus glycoprotein-mediated entry and fusion by using pseudotyped human immunodeficiency virus type 1 virions: involvement of cytoskeletal proteins and enhancement by tumor necrosis factor alpha. J Virol 2005;79:918–926.

83 You Y, Tian Y, Yang Z et al. Intradermally delivered mRNA-encapsulating extracellular vesicles for collagen-replacement therapy. Nat Biomed Eng 2023;7:887–900.

84 Zhang X, Xu Q, Zi Z et al. Programmable extracellular vesicles for macromolecule delivery and genome modifications. Dev Cell 2020;55:784–801.e9.

85 Zheng W, Radler J, Sork H et al. Identification of scaffold proteins for improved endogenous engineering of extracellular vesicles. Nat Commun 2023;14:4734.

86 Zong Y, Lin Y, Wei T et al. Lipid nanoparticle (LNP) enables mRNA delivery for cancer therapy. Adv Mater 2023;35:e2303261.