Extracellular vesicles: The next generation in gene therapy delivery

Extracellular vesicles (EVs) are esteemed as a promising delivery vehicle for various genetic therapeutics. They are relatively inert, non-immunogenic, biodegradable, and biocompatible. At least in rodents, they can even transit challenging bodily hurdles such as the blood-brain barrier. Constitutively shed by all cells and with the potential to interact specifically with neighboring and distant targets, EVs can be engineered to carry and deliver therapeutic molecules such as proteins and RNAs. EVs are thus emerging as an elegant in vivo gene therapy vector. Deeper understanding of basic EV biology—including cellular production, EV loading, systemic distribution, and cell delivery—is still needed for effective harnessing of these endogenous cellular nanoparticles as next-generation nanodelivery tools. However, even a perfect EV product will be challenging to produce at clinical scale. In this regard, we propose that vector transduction technologies can be used to convert cells either ex vivo or directly in vivo into EV factories for stable, safe modulation of gene expression and function. Here, we extrapolate from the current EV state of the art to a bright potential future using EVs to treat genetic diseases that are refractory to current therapeutics. Extracellular vesicles (EVs) are esteemed as a promising delivery vehicle for various genetic therapeutics. They are relatively inert, non-immunogenic, biodegradable, and biocompatible. At least in rodents, they can even transit challenging bodily hurdles such as the blood-brain barrier. Constitutively shed by all cells and with the potential to interact specifically with neighboring and distant targets, EVs can be engineered to carry and deliver therapeutic molecules such as proteins and RNAs. EVs are thus emerging as an elegant in vivo gene therapy vector. Deeper understanding of basic EV biology—including cellular production, EV loading, systemic distribution, and cell delivery—is still needed for effective harnessing of these endogenous cellular nanoparticles as next-generation nanodelivery tools. However, even a perfect EV product will be challenging to produce at clinical scale. In this regard, we propose that vector transduction technologies can be used to convert cells either ex vivo or directly in vivo into EV factories for stable, safe modulation of gene expression and function. Here, we extrapolate from the current EV state of the art to a bright potential future using EVs to treat genetic diseases that are refractory to current therapeutics. All eukaryotic cells release an abundance of extracellular vesicles (EVs): membrane-bound nanoparticles that are roughly spherical and range in diameter from around 50 to 500 nm.1Wang W. Li M. Chen Z. Xu L. Chang M. Wang K. Deng C. Gu Y. Zhou S. Shen Y. et al.Biogenesis and function of extracellular vesicles in pathophysiological processes of skeletal muscle atrophy.Biochem. Pharmacol. 2022; 198: 114954https://doi.org/10.1016/j.bcp.2022.114954Crossref Scopus (15) Google Scholar EVs are diverse, categorized not only by size but also by cell of origin, mode of release, molecular composition, and function. Classical EV subtypes like “ectosomes” (plasma membrane origin) and “exosomes” (endosomal origin) may be important at the cell biology level but belie incredible diversity and are difficult to distinguish after they leave the cell.2Buzas E.I. The roles of extracellular vesicles in the immune system.Nat. Rev. Immunol. 2022; : 1-15https://doi.org/10.1038/s41577-022-00763-8Crossref Scopus (23) Google Scholar EVs are thought to function in cell-to-cell communications by delivering nucleic acids, proteins, small molecules, and lipids between cells,3Li S.P. Lin Z.X. Jiang X.Y. Yu X.Y. Exosomal cargo-loading and synthetic exosome-mimics as potential therapeutic tools.Acta Pharmacol. Sin. 2018; 39: 542-551https://doi.org/10.1038/aps.2017.178Crossref PubMed Scopus (182) Google Scholar but other modes of interaction can also be envisioned. Notably, these molecules have been observed to retain their function in recipient cells following being transported in EVs, suggesting that EVs containing active proteins, RNAs, proteins, or DNAs can alter the biology of cells that are distant from the EV producer cells. These characteristics confer unparalleled potential to EVs in terms of safety and biocompatibility; as such, they have been the subject of extensive experimentation and captured the interest of both the public and private sectors.4Yang M. Wu S.Y. The advances and challenges in utilizing exosomes for delivering cancer therapeutics.Front. Pharmacol. 2018; 9: 735https://doi.org/10.3389/fphar.2018.00735Crossref PubMed Scopus (28) Google Scholar To date, several therapeutic biomolecules have been repeatedly loaded in EVs and delivered to target cells and experimentally validated in both in vitro and in vivo models. RNA therapeutics offer distinct advantages over zinc finger or CRISPR therapeutics, as RNAs function by endogenous cellular pathways in a transient manner and are programmable and thus relatively easy to engineer for specific diseases, and are generally not immunogenic, as is unfortunately the case with many of the emerging recombinant protein technologies. Various RNA biotypes with biological functions and therapeutic potential, such as small interfering RNAs (siRNAs), have been discovered and investigated, leading to the development of new classes of therapeutic drugs.5Damase T.R. Sukhovershin R. Boada C. Taraballi F. Pettigrew R.I. Cooke J.P. The limitless future of RNA therapeutics.Front. Bioeng. Biotechnol. 2021; 9: 628137https://doi.org/10.3389/fbioe.2021.628137Crossref PubMed Scopus (136) Google Scholar RNAs can be used to impart short-term transient and longer-term epigenetic silencing, which is based on the target, e.g., targeting gene promoters can induce transcriptional gene silencing.6Weinberg M.S. Morris K.V. Transcriptional gene silencing in humans.Nucleic Acids Res. 2016; 44: 6505-6517https://doi.org/10.1093/nar/gkw139Crossref PubMed Scopus (61) Google Scholar Notably, mRNA-based vaccines are also now being used effectively to combat the COVID-19 pandemic.7Kiaie S.H. Majidi Zolbanin N. Ahmadi A. Bagherifar R. Valizadeh H. Kashanchi F. Jafari R. Recent advances in mRNA-LNP therapeutics: immunological and pharmacological aspects.J. Nanobiotechnology. 2022; 20: 276https://doi.org/10.1186/s12951-022-01478-7Crossref Scopus (3) Google Scholar However, although therapeutic RNAs can be rapidly altered and produced, they must reach their intended target to be effective. For example, lipid nanoparticles (LNPs) are used in the Pfizer-BioNTech COVID-19 vaccine and for treatment of polyneuropathy targeted to the liver, but these approaches may be cytotoxic, unstable in circulation, and unsuited for delivery to other tissues.8Hou X. Zaks T. Langer R. Dong Y. Lipid nanoparticles for mRNA delivery.Nat. Rev. Mater. 2021; 6: 1078-1094https://doi.org/10.1038/s41578-021-00358-0Crossref PubMed Scopus (455) Google Scholar Moreover, cellular and subcellular delivery of RNA-based drugs is also a formidable challenge, with less than 1% of payloads reaching the cytosol of the cell.9Maugeri M. Nawaz M. Papadimitriou A. Angerfors A. Camponeschi A. Na M. Hölttä M. Skantze P. Johansson S. Sundqvist M. et al.Linkage between endosomal escape of LNP-mRNA and loading into EVs for transport to other cells.Nat. Commun. 2019; 10: 4333https://doi.org/10.1038/s41467-019-12275-6Crossref PubMed Scopus (123) Google Scholar Potentially, packaging these RNAs into EVs, which naturally carry RNA, could be a safer and more physiologically targeted approach. As such, several attempts have been made to integrate RNA species in EVs and optimize packaging and release efficiency. While EVs are emerging as a promising delivery system, it has proven challenging to effectively load therapeutic cargo into EVs. EVs can be loaded naturally during biogenesis or following EV isolation using physical or chemical methods. Electroporation has been used to load nucleic acids into EVs; however, this deteriorates the intrinsic properties of the EV membrane and causes extensive EV loss.10Johnsen K.B. Gudbergsson J.M. Skov M.N. Christiansen G. Gurevich L. Moos T. Duroux M. Evaluation of electroporation-induced adverse effects on adipose-derived stem cell exosomes.Cytotechnology. 2016; 68: 2125-2138https://doi.org/10.1007/s10616-016-9952-7Crossref PubMed Scopus (94) Google Scholar As such, the most common method for mRNA loading into EVs is to transfect EV-producer cells with plasmids encoding the therapeutic mRNA. The resulting high concentration of cytoplasmic mRNA is sufficient to cause packaging of mRNA into EVs, perhaps because EVs have been found to functionally export cellular components that are in vast surplus.11Shrivastava S. Morris K.V. The multifunctionality of exosomes; from the garbage bin of the cell to a next generation gene and cellular therapy.Genes (Basel). 2021; 12: 173https://doi.org/10.3390/genes12020173Crossref Scopus (4) Google Scholar Villamizar et al. transfected mesenchymal stem cells (MSCs) with a plasmid encoding for a zinc finger transcription factor targeted to the CFTR gene promoter for the treatment of cystic fibrosis (called CFZF). The high expression of CFZF, resulting from the plasmid’s CMV promoter, was sufficient to detect both CFZF mRNA and protein in the isolated EVs.12Villamizar O. Waters S.A. Scott T. Grepo N. Jaffe A. Morris K.V. Mesenchymal Stem Cell exosome delivered Zinc Finger Protein activation of cystic fibrosis transmembrane conductance regulator.J. Extracell. Vesicles. 2021; 10: e12053https://doi.org/10.1002/jev2.12053Crossref PubMed Scopus (13) Google Scholar To increase the RNA loading output, Kojima et al. loaded catalase mRNA into EVs using a loading system called EXOtic,13Kojima R. Bojar D. Rizzi G. Hamri G.C.E. El-Baba M.D. Saxena P. Ausländer S. Tan K.R. Fussenegger M. Designer exosomes produced by implanted cells intracerebrally deliver therapeutic cargo for Parkinson's disease treatment.Nat. Commun. 2018; 9: 1305https://doi.org/10.1038/s41467-018-03733-8Crossref PubMed Scopus (297) Google Scholar consisting of a plasmid construct encoding for CD63, a common transmembrane protein, plus the L7Ae archaeal ribosomal protein that selectively binds to the C/D box RNA structure. Next, they introduced the C/D box into the 3′ UTR of the catalase gene. When producer cells were transfected with these constructs, catalase mRNA was efficiently packed into EVs and transferred to target cell in vitro.13Kojima R. Bojar D. Rizzi G. Hamri G.C.E. El-Baba M.D. Saxena P. Ausländer S. Tan K.R. Fussenegger M. Designer exosomes produced by implanted cells intracerebrally deliver therapeutic cargo for Parkinson's disease treatment.Nat. Commun. 2018; 9: 1305https://doi.org/10.1038/s41467-018-03733-8Crossref PubMed Scopus (297) Google Scholar A tricistronic plasmid encoding for three genes involved in exosome biogenesis (STEAP3, SDC4, L-aspartate oxidase) was also used with the EXOtic system to increase EV release. Introduced into mouse models of Parkinson’s disease, these EVs transgressed the blood-brain barrier and reduced reactive oxygen species in targeted cells in the brain. A constitutively active mutant of gap junction protein Connexin 43 (Cx43) was also included. This protein is responsible for forming gap-junction structures after fusion of two connexon hemichannels, allowing for cellular intercommunication and transfer of materials.14Soares A.R. Martins-Marques T. Ribeiro-Rodrigues T. Ferreira J.V. Catarino S. Pinho M.J. Zuzarte M. Isabel Anjo S. Manadas B. P G Sluijter J. et al.Gap junctional protein Cx43 is involved in the communication between extracellular vesicles and mammalian cells.Sci. Rep. 2015; 5: 13243https://doi.org/10.1038/srep13243Crossref PubMed Scopus (119) Google Scholar It is also the most expressed Cx protein and is naturally present in EVs as hexamers organized in hemichannel structures.14Soares A.R. Martins-Marques T. Ribeiro-Rodrigues T. Ferreira J.V. Catarino S. Pinho M.J. Zuzarte M. Isabel Anjo S. Manadas B. P G Sluijter J. et al.Gap junctional protein Cx43 is involved in the communication between extracellular vesicles and mammalian cells.Sci. Rep. 2015; 5: 13243https://doi.org/10.1038/srep13243Crossref PubMed Scopus (119) Google Scholar,15Gemel J. Kilkus J. Dawson G. Beyer E.C. Connecting exosomes and connexins.Cancers (Basel). 2019; 11: 476https://doi.org/10.3390/cancers11040476Crossref Scopus (15) Google Scholar This protein was engineered into the EV construct and found to increase the release efficiency EV cargo into recipient cells upon contact.12Villamizar O. Waters S.A. Scott T. Grepo N. Jaffe A. Morris K.V. Mesenchymal Stem Cell exosome delivered Zinc Finger Protein activation of cystic fibrosis transmembrane conductance regulator.J. Extracell. Vesicles. 2021; 10: e12053https://doi.org/10.1002/jev2.12053Crossref PubMed Scopus (13) Google Scholar,16Shrivastava S. Ray R.M. Holguin L. Echavarria L. Grepo N. Scott T.A. Burnett J. Morris K.V. Exosome-mediated stable epigenetic repression of HIV-1.Nat. Commun. 2021; 12: 5541https://doi.org/10.1038/s41467-021-25839-2Crossref PubMed Scopus (16) Google Scholar Indeed, CD63-fused L7Ae appears to require co-transfection of the booster tricistronic plasmid, Cx43, and a LAMP2b-fused brain targeting module to transfer nluc-C/D box mRNA. Another method for loading RNA into EVs is to generate lipid-coated RNA particles and integrate these into purified EVs through mixing-induced partitioning.17Sato Y.T. Umezaki K. Sawada S. Mukai S.A. Sasaki Y. Harada N. Shiku H. Akiyoshi K. Engineering hybrid exosomes by membrane fusion with liposomes.Sci. Rep. 2016; 6: 21933https://doi.org/10.1038/srep21933Crossref PubMed Scopus (333) Google Scholar,18Li Y.J. Wu J.Y. Liu J. Xu W. Qiu X. Huang S. Hu X.B. Xiang D.X. Artificial exosomes for translational nanomedicine.J. Nanobiotechnology. 2021; 19: 242https://doi.org/10.1186/s12951-021-00986-2Crossref Scopus (62) Google Scholar As expected, this process leads to a slight increase in EV size and a decrease in EV numbers, but it is efficient and accurate (>90%).19Tsai S.J. Atai N.A. Cacciottolo M. Nice J. Salehi A. Guo C. Sedgwick A. Kanagavelu S. Gould S.J. Exosome-mediated mRNA delivery in vivo is safe and can be used to induce SARS-CoV-2 immunity.J. Biol. Chem. 2021; 297: 101266https://doi.org/10.1016/j.jbc.2021.101266Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar However, the purification and need to pre-coat RNA with lipids introduces expense and time constraints. Therefore, while this process can be used for research purposes, it may prove challenging to scale for clinical or commercial applications. MicroRNAs (miRNAs) are well known to be loaded in EVs and to functionally modulate gene expression in other recipient cell types.20Simeoli R. Montague K. Jones H.R. Castaldi L. Chambers D. Kelleher J.H. Vacca V. Pitcher T. Grist J. Al-Ahdal H. et al.Exosomal cargo including microRNA regulates sensory neuron to macrophage communication after nerve trauma.Nat. Commun. 2017; 8: 1778https://doi.org/10.1038/s41467-017-01841-5Crossref PubMed Scopus (155) Google Scholar This could be by direct interactions with Argonaut 2 (AGO2), which has been found to be packaged into EVs.21Beltrami C. Clayton A. Newbury L.J. Corish P. Jenkins R.H. Phillips A.O. Fraser D.J. Bowen T. Stabilization of urinary MicroRNAs by association with exosomes and argonaute 2 protein.Noncoding. RNA. 2015; 1: 151-166https://doi.org/10.3390/ncrna1020151Crossref PubMed Scopus (35) Google Scholar Alternatively, particular proteins, such as YBX1, have been implicated in loading particular miRNAs into EVs,22Liu X.M. Ma L. Schekman R. Selective sorting of microRNAs into exosomes by phase-separated YBX1 condensates.Elife. 2021; 10: e71982https://doi.org/10.7554/eLife.71982Crossref Scopus (16) Google Scholar while others have suggested that there may not be a specific motif or pathway involved in miRNA recruitment into EVs.23Hung M.E. Leonard J.N. A platform for actively loading cargo RNA to elucidate limiting steps in EV-mediated delivery.J. Extracell. Vesicles. 2016; 5: 31027https://doi.org/10.3402/jev.v5.31027Crossref PubMed Scopus (112) Google Scholar Yet others have found that there does not appear to be a specific miRNA packaging system for loading these RNAs into EVs.24Albanese M. Chen Y.F.A. Hüls C. Gärtner K. Tagawa T. Mejias-Perez E. Keppler O.T. Göbel C. Zeidler R. Shein M. et al.MicroRNAs are minor constituents of extracellular vesicles that are rarely delivered to target cells.PLoS Genet. 2021; 17: e1009951https://doi.org/10.1371/journal.pgen.1009951Crossref Scopus (40) Google Scholar Due to their relatively large range of target genes, miRNAs can significantly alter the phenotype or gene expression of a cell, and therefore they can be a high-value cargo with the potential to promote, trigger, or treat diseases. Simeoli et al. were among the first to describe an endogenous pathway of EV-mediated miRNA transfer from neurons to macrophages in presence of capsaicin.20Simeoli R. Montague K. Jones H.R. Castaldi L. Chambers D. Kelleher J.H. Vacca V. Pitcher T. Grist J. Al-Ahdal H. et al.Exosomal cargo including microRNA regulates sensory neuron to macrophage communication after nerve trauma.Nat. Commun. 2017; 8: 1778https://doi.org/10.1038/s41467-017-01841-5Crossref PubMed Scopus (155) Google Scholar Capsaicin incubation or nerve injury causes an increase in expression of miRNA-21 and milk fat globule-EGF factor 8 protein MFG-E8, a protein responsible for macrophage uptake. The authors demonstrated that EVs derived from capsaicin-treated neurons were taken up more readily by macrophages than the untreated control and promoted inflammatory 13Kojima R. Bojar D. Rizzi G. Hamri G.C.E. El-Baba M.D. Saxena P. Ausländer S. Tan K.R. Fussenegger M. Designer exosomes produced by implanted cells intracerebrally deliver therapeutic cargo for Parkinson's disease treatment.Nat. Commun. 2018; 9: 1305https://doi.org/10.1038/s41467-018-03733-8Crossref PubMed Scopus (297) Google Scholarphenotypes and repression of miR-21 target genes in macrophages. Activated macrophages were more likely to move toward sites of injury where the EV-releasing neurons are situated, thus demonstrating the existence and importance of EV-mediated intercellular communication mediated by miRNAs.20Simeoli R. Montague K. Jones H.R. Castaldi L. Chambers D. Kelleher J.H. Vacca V. Pitcher T. Grist J. Al-Ahdal H. et al.Exosomal cargo including microRNA regulates sensory neuron to macrophage communication after nerve trauma.Nat. Commun. 2017; 8: 1778https://doi.org/10.1038/s41467-017-01841-5Crossref PubMed Scopus (155) Google Scholar EV-transferred miRNAs have also been implicated in cancer by promoting metastasis, drug resistance, proliferation, and inflammation.25Dilsiz N. Role of exosomes and exosomal microRNAs in cancer.Future Sci. OA. 2020; 6: FSO465https://doi.org/10.2144/fsoa-2019-0116Crossref Scopus (60) Google Scholar As demonstrated by the existence of EV-loaded miRNA communication pathways, miRNAs seem to be preferentially loaded in EVs relative to other RNA types, suggesting that an endogenous loading system exists within cells. AGO2 is an RNA-binding protein that binds miRNA and may be responsible for miRNA loading in EVs.26McKenzie A.J. Hoshino D. Hong N.H. Cha D.J. Franklin J.L. Coffey R.J. Patton J.G. Weaver A.M. KRAS-MEK signaling controls Ago2 sorting into exosomes.Cell Rep. 2016; 15: 978-987https://doi.org/10.1016/j.celrep.2016.03.085Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar Due to their profound regulatory potential and natural occurrence in EVs, miRNA and AGO2-binding shRNAs appear to be great candidates for EV therapeutics. Another class of regulatory RNAs that have been observed in EVs are circRNAs.27Li Y. Zheng Q. Bao C. Li S. Guo W. Zhao J. Chen D. Gu J. He X. Huang S. Circular RNA is enriched and stable in exosomes: a promising biomarker for cancer diagnosis.Cell Res. 2015; 25: 981-984https://doi.org/10.1038/cr.2015.82Crossref PubMed Scopus (1462) Google Scholar circRNAs are a class of single-stranded circular non-coding RNA resulting from the back splicing of exons in mRNAs.28Conn S.J. Pillman K.A. Toubia J. Conn V.M. Salmanidis M. Phillips C.A. Roslan S. Schreiber A.W. Gregory P.A. Goodall G.J. The RNA binding protein quaking regulates formation of circRNAs.Cell. 2015; 160: 1125-1134https://doi.org/10.1016/j.cell.2015.02.014Abstract Full Text Full Text PDF PubMed Scopus (1298) Google Scholar,29Ragan C. Goodall G.J. Shirokikh N.E. Preiss T. Insights into the biogenesis and potential functions of exonic circular RNA.Sci. Rep. 2019; 9: 2048https://doi.org/10.1038/s41598-018-37037-0Crossref PubMed Scopus (74) Google Scholar Some genes have been observed to express several times the amount of circRNA compared with the protein-coding mRNA, suggesting an important functional role, which includes transcription regulation by absorbing miRNAs, interaction with proteins, competition with pre-mRNA splicing, and, more rarely, as templates for protein translation.30Meng S. Zhou H. Feng Z. Xu Z. Tang Y. Li P. Wu M. CircRNA: functions and properties of a novel potential biomarker for cancer.Mol. Cancer. 2017; 16: 94https://doi.org/10.1186/s12943-017-0663-2Crossref PubMed Scopus (978) Google Scholar The lack of 5′ and 3′ ends protects the circRNAs from degradation by exonucleases, which ultimately confers a longer lifespan of these transcripts in the cytoplasm compared with other RNAs.31Liu L. Wang J. Khanabdali R. Kalionis B. Tai X. Xia S. Circular RNAs: isolation, characterization and their potential role in diseases.RNA Biol. 2017; 14: 1715-1721https://doi.org/10.1080/15476286.2017.1367886Crossref PubMed Scopus (78) Google Scholar This is also confirmed by the negative relation between cell proliferation and circRNA concentration, allegedly because circRNAs can be diluted to daughter cells after proliferation. Recently, functional circRNAs were found to be loaded into and transferred to recipient cells by EVs. The ratio between circRNA and linear RNA in EVs is higher than in the producer cells, indicating an endogenous sorting mechanism.32Lasda E. Parker R. Circular RNAs Co-precipitate with extracellular vesicles: a possible mechanism for circRNA clearance.PLoS One. 2016; 11: e0148407https://doi.org/10.1371/journal.pone.0148407Crossref PubMed Scopus (260) Google Scholar Some circRNAs are highly expressed in cancer cells, and EV-packed circRNAs are demonstrated to be partially responsible for the proliferation of various cancers; as such, exosomal circRNAs (exo-circRNAs) have been considered important primarily as biomarkers for screening of cancer in early onset.32Lasda E. Parker R. Circular RNAs Co-precipitate with extracellular vesicles: a possible mechanism for circRNA clearance.PLoS One. 2016; 11: e0148407https://doi.org/10.1371/journal.pone.0148407Crossref PubMed Scopus (260) Google Scholar,33Du W.W. Li X. Ma J. Fang L. Wu N. Li F. Dhaliwal P. Yang W. Yee A.J. Yang B.B. Promotion of tumor progression by exosome transmission of circular RNA circSKA3.Mol. Ther. Nucleic Acids. 2022; 27: 276-292https://doi.org/10.1016/j.omtn.2021.11.027Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar However, due to their increased stability, circRNA can be packaged into EVs and transferred to target cells, where they may support protein translation for longer than a typical mRNA.34Wesselhoeft R.A. Kowalski P.S. Anderson D.G. Engineering circular RNA for potent and stable translation in eukaryotic cells.Nat. Commun. 2018; 9: 2629https://doi.org/10.1038/s41467-018-05096-6Crossref PubMed Scopus (240) Google Scholar Notably, circRNAs can be engineered with an internal ribosome entry site (IRES) to express proteins of interest.34Wesselhoeft R.A. Kowalski P.S. Anderson D.G. Engineering circular RNA for potent and stable translation in eukaryotic cells.Nat. Commun. 2018; 9: 2629https://doi.org/10.1038/s41467-018-05096-6Crossref PubMed Scopus (240) Google Scholar As circRNAs persist longer than linear RNAs, this may be one approach to generating enhanced long-term protein expression. Such an application would be especially useful in vaccine treatments to extend the exposure time of antigens to the immune system or, generally, to produce the most protein out of a therapeutic dose. While naturally occurring open reading frame (ORF)-possessing circRNAs are a minority in cells and have yet to be proven capable of translation, attempts have been made to engineer circRNAs with coding capacity.35Miao Q. Ni B. Tang J. Coding potential of circRNAs: new discoveries and challenges.PeerJ. 2021; 9: e10718https://doi.org/10.7717/peerj.10718Crossref Scopus (7) Google Scholar Wesselhoeft et al. achieved robust expression of luciferase, EGFP, erythropoietin, and CRISPR-associated endonuclease 9 (Cas9) upon transfection of a self-splicing intron-induced circRNA into HEK293 cells.34Wesselhoeft R.A. Kowalski P.S. Anderson D.G. Engineering circular RNA for potent and stable translation in eukaryotic cells.Nat. Commun. 2018; 9: 2629https://doi.org/10.1038/s41467-018-05096-6Crossref PubMed Scopus (240) Google Scholar Qu et al. created a circRNA encoding the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and performed in vivo experiments in mice to test the immunization capacity of circRNA vaccines encapsulated in LNPs.36Qu L. Yi Z. Shen Y. Lin L. Chen F. Xu Y. Wu Z. Tang H. Zhang X. Tian F. et al.Circular RNA vaccines against SARS-CoV-2 and emerging variants.Cell. 2022; 185: 1728-1744.e16https://doi.org/10.1016/j.cell.2022.03.044Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar Mice treated with these particles produced antibodies and T cell responses similar to those of counterparts treated with a linear mRNA.36Qu L. Yi Z. Shen Y. Lin L. Chen F. Xu Y. Wu Z. Tang H. Zhang X. Tian F. et al.Circular RNA vaccines against SARS-CoV-2 and emerging variants.Cell. 2022; 185: 1728-1744.e16https://doi.org/10.1016/j.cell.2022.03.044Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar Overall, these results suggest that circRNAs make more proteins than linear mRNAs and therefore may improve the general efficacy of mRNA therapies and may prove useful in vaccine approaches and in treating cancer and infectious and genetic diseases. Ideally, such therapeutic circRNAs could be engineered to code for therapeutic proteins and be specifically loaded into EVs for long-lasting expression of proteins in target cells. While the EXOtic system allows for packaging protein-coding mRNAs into EVs, others have developed a means of loading unbound therapeutic proteins into EVs. CRY2 is a plant protein that changes conformation upon exposure to blue light, and CIBN is a truncated version of CIB1, a protein with affinity for CRY2 in its excited form.37Kennedy M.J. Hughes R.M. Peteya L.A. Schwartz J.W. Ehlers M.D. Tucker C.L. Rapid blue-light-mediated induction of protein interactions in living cells.Nat. Methods. 2010; 7: 973-975https://doi.org/10.1038/nmeth.1524Crossref PubMed Scopus (749) Google Scholar CIB1 was attached to the cytosolic tail of EV marker CD9 and CRY2 to reporter proteins such as mCherry and GFP.38Yim N. Ryu S.W. Choi K. Lee K.R. Lee S. Choi H. Kim J. Shaker M.R. Sun W. Park J.H. et al.Exosome engineering for efficient intracellular delivery of soluble proteins using optically reversible protein-protein interaction module.Nat. Commun. 2016; 7: 12277https://doi.org/10.1038/ncomms12277Crossref PubMed Scopus (318) Google Scholar This system, named EXPLOR, was demonstrated to cause loading of the cargo-CRY2 into EVs by reversible binding to CIB1 when producer cells are exposed to blue light. In the absence of blue light, however, the cargo-CRY2 complex was freely available in the isolated EVs. Using this approach, Choi et al. successfully loaded Cre recombinase and the nuclear factor κB (NF-κB) pathway suppressor srIκB into EVs.39Choi H. Kim Y. Mirzaaghasi A. Heo J. Kim Y.N. Shin J.H. Kim S. Kim N.H. Cho E.S. Song E. Kim P. Shin E.C. Chung K. Choi K. Choi C. In Yook J. Yoo T.H. Exosome-based delivery of super-repressor IkappaBalpha relieves sepsis-associated organ damage and mortality.Sci. Adv. 2020; 6: eaaz6980https://doi.org/10.1126/sciadv.aaz6980Crossref PubMed Scopus (72) Google Scholar Based on this model, Osteikoetxea et al. tested whether the Cas9 protein could be loaded into EVs and compared it with three other similar loading systems based on heterodimerization upon exposure to an activating stimulus.40Osteikoetxea X. Silva A. Lázaro-Ibáñez E. Salmond N. Shatnyeva O. Stein J. Schick J. Wren S. Lindgren J. Firth M. et al.Engineered Cas9 extracellular vesicles as a novel gene editing tool.J. Extracell. Vesicles. 2022; 11: e12225https://doi.org/10.1002/jev2.12225Crossref Scopus (5) Google Scholar These were PHIB and PIF6, which interact upon exposure to 630 nm light, and the small molecule phycocyanobilin, engineered VVD proteins with nanomagnets that interact in presence of blue light, and finally FKBP and FRB, which interact in the presence of the small molecule rapamycin. The group demonstrated that loading with CRY2-CIB1 resulted in the highest concentration of Cas9 in EV fractions, reaching more than 20 Cas9 molecules per EV.40Osteikoetxea X. Silva A. Lázaro-Ibáñez E. Salmond N. Shatnyeva O. Stein J. Schick J. Wren S. Lindgren J. Firth M. et al.Engineered Cas9 extracellular vesicles as a novel gene editing tool.J. Extracell. Vesicles. 2022; 11: e12225https://doi.org/10.1002/jev2.12225Crossref Scopus (5) Google Scholar A noteworthy observation of this study was the data suggesting that engineering of MysPalm for protein cargo delivery appears to be more advantageous compared with engineering to tetraspanin markers such as CD9. Two possib