Improved Exosome Profiling for Therapeutics and Diagnostics

Abstract

The present invention provides assays and methods for the characterization of specific extracellular vesicle (EV) populations, and the preparation of improved EV compositions for therapeutic and diagnostic purposes. In particular, in some embodiments, the present invention provides methods and kits for analyzing blood-derived samples, and the generation and use of tissue-derived circulating EV populations that are characterized by unexpected small size and advantageous properties.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to assays and methods for the simultaneous characterization of extracellular vesicle (EV) populations and the preparation of improved EV compositions for therapeutic and diagnostic purposes. [Background technology]

[0002] Extracellular vesicles (EVs) are released from all cells throughout the body via a wide variety of mechanisms, including outward budding of the plasma membrane, exocytosis (microvesicles) and inward budding of the endosomal membrane (exosomes) (Rak 2010.Semin Thromb Hemost 36:888-906; Colombo et al. 2014.Annu Rev Cell Dev Biol 30:255-289). Regardless of their biogenesis pathway, it is widely accepted that most biological fluids (blood, urine, cerebrospinal fluid, etc.) contain EVs originating from multiple cell types. Moreover, a large body of experimental evidence indicates that the molecular composition of EVs closely reflects that of their cells and tissues of origin (He et al. 2018 Theranostics 8:237-255). Moreover, EV surface proteins also reflect their cellular origin and may elucidate EV biogenesis pathways, destinations, and functions (Colombo et al. 2014. Annu Rev Cell Dev Biol 30:255-289).

[0003] EVs comprise a heterogeneous population of particles that differ in their intracellular origin, size, and composition. The function of EVs has been recognized to be transport between the releasing and receiving cells, where in the latter, endocytosis and (intraluminal) cargo delivery induces phenotypic responses. Although this communication paradigm is promising and supported by the literature, EVs can also act in an autocrine manner and have other "delivery-independent" extracellular functions, such as regulating interactions with extracellular matrix (ECM), cell membrane receptors that transport EV-resident proteins to the receiving cell.

[0004] EVs typically exhibit a wide size distribution that can range from 30 to 5000 nm. EV size has been suggested to be a differentiator of biogenesis. For example, exosomes have been defined as vesicles with diameters between 30 nm and 150 nm that originate from endosomes / multivesicular bodies by reverse-inward budding, microvesicles have been defined as particles generated by outward budding of the plasma membrane ranging from 150 nm to 1000 nm, and apoptotic bodies (derived by whole-cell vesiculation) have been identified as ranging from 500 nm to 5000 nm in diameter. However, other evidence suggests that these definitions are not precise and that there is considerable overlap between different EV types. Thus, molecular techniques for EV profiling are needed to further define and characterize EV populations.

[0005] The development of FACS analysis to characterize surface proteins of immune cells has revolutionized the field of immunology, and the development of the capacity to extend this capability towards the analysis of EVs may prove to be extremely advantageous. In response to this opportunity gap, several assays have been developed for the characterization of intact EVs (Kanwar et al. 2014 Lab Chip 14:1891-1900; Ohmichi et al. 2019 Parkinsonism Relat Disord 61:82-87; Ter-Ovanesyan et al. 2021 bioRxiv:2020.2010.2013.337881). All these assays are based on the sandwich principle, where a sandwich is formed by an EV and two antibodies targeting different proteins on the EV surface. To generate a signal, both proteins need to be linked to each other, as is well envisaged, through the EV membrane. This principle can be incorporated into multiple immunoassay systems, including traditional ELISA, mesoscale discovery electrochemiluminescence, SIMOA, FACS, SPR and many others. However, most of these methods are unsuitable for multiplexing and therefore do not allow the simultaneous evaluation of multiple EV surface proteins (Nolan and Duggan 2018 Methods Mol Biol 1678:79-92; Chiang and Chen 2019 J Biomed Sci 26:9; Kurian et al. 2021 Mol Biotechnol 63:249-266). An exception is the MACSPlex FACS Exosome Kit (Miltenyi Biotech, Cat. No. 130-108-813), which allows the detection of multiple EV surface epitopes (37 in total). However, its simultaneous evaluation capacity is limited by the number of compatible fluorescent dyes and dye interference. In addition, this is a challenging method that requires EV enrichment and skilled operators prior to analysis.

[0006] Microsphere-based suspension array technologies, such as the Luminex® xMAP® system, provide high-throughput detection of protein and nucleic acid targets in multiplex assay chemistry. Attempts to utilize such technologies in a variety of applications ranging from transplant medicine, biomarker discovery and validation, pathogen detection, drug discovery, vaccine development, personalized medicine, neurodegeneration, and cancer research have been reported (Graham et al., 2019. Methods 158:2-11). For example, the use of nucleic acid probes immobilized on fluorescent microbeads that are analyzed using a Luminex detector is suggested in U.S. Patent Nos. 8,232,058 and 7,642,348.

[0007] WO2021231720 discloses the use of EVs to detect complement activation and their use for evaluating and / or monitoring the treatment of complement-mediated diseases. In particular, provided is an immunoprecipitation bead-based immunocapture protocol with immunofluorescence detection that involves a combination of EV enrichment (using a pan-EV antibody) and immunocapture of tissue-specific biomarkers present on the surface of shed EVs, prior to detection of complement activation.

[0008] The high bioavailability and unique properties of EVs have attracted increasing attention as potential diagnostic and therapeutic applications. Several groups have reported EV-based diagnostic methods for diseases including cancer, neurodegenerative diseases, viral infections, etc. For example, Szajnik et al. (Gynecol. Obstet. 2013, Suppl 4:003) suggested the possibility of discrimination between ovarian cancer patients and benign tumor patients or healthy controls based on the characterization of cargo and epitopes of tumor-released exosomes. EV-based biomarkers and their possible applications are disclosed in U.S. Patent No. 9,958,460 and WO 2017193115. Various other publications involving the isolation and / or analysis of vesicles from biological samples include, for example, U.S. Patent Application Publication Nos. 20190219578, 20180340945, 20190361037, 20180080945, 20190137517, and International Publication No. 2016172598.

[0009] EVs are increasingly being used as therapeutic agents encompassing a range of pathological conditions, including, but not limited to, treatment against infectious pathogens, mediating the therapeutic effects of mesenchymal stem / stromal cells (MSCs), modulating immune responses, etc. Characterization of EV-based therapeutics involves a multi-step process complicated by several technical issues, including efficient, convenient, cost-conscious, isolation and capture of relevant EVs.

[0010] In particular, the distinctive attributes of EVs and their involvement in many physiological processes make them attractive candidates as carriers for targeted drug delivery and gene therapy. Various studies suggest that EVs have several advantages over traditional synthetic carriers, but despite extensive research, the clinical translation of EV-based therapeutics remains challenging.

[0011] US Patent No. 11,111,475 relates to a method of treating a human subject suffering from stroke, comprising intravenous or intranasal administration of isolated EVs derived from non-transformed human neural progenitor cells. These progenitor cell-derived EVs may be of different sizes, e.g., ranging from 20 nm to 150 nm, and may optionally further comprise an agent selected from the group consisting of small molecules, antisense oligonucleotides, siRNA, exogenous peptides, exogenous proteins, and antibodies. This publication discloses the isolation of EVs from the medium of cultured neural progenitor cells produced from pluripotent stem cells (e.g., human embryonic stem cells) or induced pluripotent stem cells (iPSCs). Some other examples of therapeutic uses of EV-based compositions include WO2020257710, US Patent No. 10,912,736, and US Patent No. 10,590,417.

[0012] Volpert et al. (inventors and collaborators), 2022, published a report of a modification of the Luminex assay for characterization of extracellular vesicle populations in biological fluids after the priority date of this application (Volpert et al., bioRxiv preprint (posted January 12, 2022)).

[0013] It is quite evident from the diversity of EV-based assays and tests for diagnostic, prognostic, and therapeutic efficacy of therapies that much is still left to be desired in terms of the sensitivity, specificity, and technical utility of the assays. In particular, the development of improved assays and methods for the simultaneous characterization of EV populations that allow a reduction in the number of specimens required for analysis and minimize the time and / or technical complexity associated with currently available assays would be highly advantageous. In addition, the generation of EV compositions characterized by improved pharmacokinetic properties and / or tissue penetration would be beneficial for the development of improved drug delivery platforms and other EV-based therapeutics. Summary of the Invention

[0014] The present invention relates to assays and methods for the simultaneous characterization of extracellular vesicle (EV) populations and the preparation of improved EV compositions for therapeutic and diagnostic purposes. In particular, in some embodiments, the present invention provides methods and kits for analyzing blood-derived samples, as well as the generation and use of tissue-derived circulating EV populations that exhibit advantageous properties and are characterized by unexpectedly small dimensions of less than 30 nm in diameter.

[0015] The present invention is based in part on the development of improved assays for multiplexed detection and analysis of EV-associated biomarkers directly from small amounts of biological fluids such as blood-derived samples. Surprisingly, EV populations of different cellular origins were detected using the developed Luminex-compatible assays that show unique expression profiles of tetraspanin markers characteristic of each population. In addition, the developed Luminex-compatible assays allowed for multiplexed detection, quantification and analysis of multiple synaptic proteins presented on the surface of EVs using extremely small amounts of blood-derived samples. Advantageously, the developed assays are much simpler and more accurate than previously described methods for measuring synaptic proteins in blood-derived samples, which mainly rely on immunoprecipitation followed by mass spectrometry. The assays disclosed herein are therefore particularly suitable and useful for clinical diagnostic applications.

[0016] The present invention is further based in part on the surprising discovery that certain populations of circulating EVs, such as those of neuronal and macrophage origin, are extremely smaller in size than the average size of circulating exosomes or previously identified EV populations (such as erythrocyte-derived EVs). In particular, disclosed herein for the first time are isolated EVs of neural origin with diameters less than 30 nm; these EVs were characterized by a size distribution ranging from about 15 to 25 nm, as assessed by size exclusion chromatography (SEC) followed by transmission electron microscopy (TEM). In contrast, EV populations secreted by cultured neural cells (differentiated ex vivo from induced pluripotent stem cells) did not share the unique size characteristics of their circulating counterparts, despite the observation of similar expression profiles of neural markers and tetraspanins in the two populations. Rather, EVs derived from cultured neural cells exhibited characteristics consistent with an average diameter of 100 to 200 nm, similar to the assessed dimensions of erythrocyte-derived EVs. In addition, isolated EVs from the late SEC fraction (corresponding to reduced EV diameter) were also characterized by a unique lipid profile, contained tissue-specific RNA markers, and were found to exhibit advantageous properties including enhanced stability as assessed by wash resistance.

[0017] Thus, disclosed herein are methods and assays for profiling and characterizing tissue-specific EV populations from blood-derived samples based on newly identified markers and size distributions, and utilizing improved methods that allow multiplexed measurements in small (e.g., <75 μl) sample volumes. Additionally, disclosed herein are newly identified tissue-specific, blood-circulating EV (herein named "nano-EV") populations that are distinguishable by their physicochemical properties and tissue specificity from other blood-circulating EV populations. Further disclosed herein are methods for preparing non-naturally occurring EV compositions, including purified (e.g., pharmaceutical grade purity) nano-EVs, and their improved diagnostic and therapeutic uses, including in drug delivery and gene therapy.

[0018] In one aspect, the invention provides an EV preparation comprising a substantially purified extracellular vesicle (EV) population isolated from serum or plasma derived from cells other than blood cells, the population characterized by a particle size distribution of 10-25 nanometers (nm) in diameter and a marker profile corresponding to differentiated solid tissue cells.

[0019] As used herein, a "substantially purified" EV population isolated from serum or plasma indicates that the EVs are at least 80% separated from other plasma / serum components, e.g., at least 85%, at least 90%, at least 95% separated from other plasma / serum components. EVs isolated from serum or plasma are also referred to herein as circulating EVs and are derived from cells other than blood cells, i.e., cells other than red blood cells, platelets, and white blood cells.

[0020] In some embodiments, the EVs are 15-25 nm in diameter. In further embodiments, the EVs are 15-20 nm in diameter. In still further embodiments, the EVs are 10-15 nm in diameter.

[0021] In some embodiments, the EVs comprise at least one type of EVs selected from neural-derived EVs and tissue macrophage-derived EVs. In some embodiments, the EVs comprise neural-derived EVs and tissue macrophage-derived EVs. In some embodiments, the EV population is characterized by surface presentation of GAP43, CD171 and / or CD68; and lack of CD235a. In some embodiments, the EV population is characterized by membrane lipid composition according to Table A.

[0022] In some embodiments, EVs are obtained from plasma or serum samples by a process comprising a size selection of EVs less than 30 nanometers (nm) in diameter and a separation step of the selected EVs. In some embodiments, the size selection step comprises at least one of size exclusion chromatography, nanoporous membrane filtration, deterministic lateral displacement sorting, dielectrophoretic isolation, acoustic fractionation, and differential centrifugation. In some embodiments, the process further comprises immunoaffinity purification and / or analysis of the EV population.

[0023] In some embodiments, the EV is loaded with an exogenous cargo, hi some embodiments, the exogenous cargo is a therapeutic agent.

[0024] In some embodiments, EVs are modified to present targeted drugs.

[0025] In some embodiments, the purity of the EV population is pharmaceutical grade purity. In some embodiments, the formulation is formulated in the form of a pharmaceutical composition and further comprises a pharma- ceutically acceptable carrier, excipient, or diluent. In some embodiments, the formulation is used in therapy. In some embodiments, the therapy comprises the delivery of an exogenously loaded therapeutic agent to a target cell or tissue of a subject in need thereof. In further embodiments, the formulation is used in diagnosis.

[0026] In a further aspect, the present invention provides a method for analyzing a plasma or serum sample, the method comprising the steps of: a. providing a capture system comprising at least three distinct populations of fluorescently labeled magnetic microspheres, each microsphere population displaying an antibody against a distinct target on the surface of an EV population of distinct cellular origin; b. Providing 1-75 μl of unprocessed plasma or serum sample, or a corresponding amount of intact EVs; c. Incubating the sample with the capture system under conditions such as those that allow specific antigen-antibody binding while substantially maintaining the integrity of the EV membrane, thereby providing a distinct population of EV-microsphere complexes corresponding to each target; d. washing the EV-microsphere complexes using a magnetic device under conditions that allow for selective capture of said complexes; e. Incubating the EV-microsphere complex with multiple detection antibodies under conditions that allow specific antigen-antibody binding while substantially maintaining the integrity of the EV membrane, each antibody being directed to a distinct marker on the surface of the EV, and the multiple detection antibodies being labeled with the same fluorescent marker; f. washing the resulting labeled complex with a magnetic device to remove excess reagent; and g. subjecting the resulting complexes to a microfluidic device capable of simultaneous detection and quantification of fluorescence emission at multiple wavelengths, thereby quantifying the levels of fluorescence emission and providing a separate assessment of the levels of surface targets corresponding to each of the EV populations; The method is carried out using reagents and conditions that preserve the EVs in a substantially intact form.

[0027] In some embodiments, the method further comprises comparing the quantified level to a control level.

[0028] In some embodiments, the multiple detection antibodies are directed against multiple diagnostic markers. In some embodiments, the multiple detection antibodies are directed against multiple tetraspanin markers. In some embodiments, the tetraspanin markers are selected from the group consisting of CD9, CD63 and CD81. In some specific embodiments, the multiple detection antibodies include an antibody against CD9, an antibody against CD63 and an antibody against CD81.

[0029] In some embodiments, the targets of the capture system include at least one neuronal target and targets corresponding to at least two additional cellular origins selected from the group consisting of bone, lung, tissue macrophages, lung macrophages, muscle, adipocytes, epithelium, endothelium, monocytes, microglia, megakaryocytes, T cells, erythrocytes, liver, and oligodendrocytes. In some embodiments, the at least one neuronal target is selected from GAP43 and CD171, and the at least two additional targets include P2RY12 and CD68. In some embodiments, the at least two additional targets further include CD235a.

[0030] In some embodiments, the capture system comprises at least four distinct fluorescently labeled magnetic microsphere populations. In some embodiments, the capture system comprises at least five distinct fluorescently labeled magnetic microsphere populations. In some embodiments, the capture system comprises at least six distinct fluorescently labeled magnetic microsphere populations. In some embodiments, the capture system comprises at least seven distinct fluorescently labeled magnetic microsphere populations. In some embodiments, the capture system comprises at least eight distinct fluorescently labeled magnetic microsphere populations. Each microsphere population presents an antibody against a distinct target on the surface of the EV population of distinct cellular origin.

[0031] In some embodiments, between 1 and 50 μl of unprocessed plasma or serum is provided to the method.

[0032] In a further aspect, there is provided herein a method for simultaneously measuring multiple synaptic proteins in a plasma or serum sample, the method comprising: a. providing an unprocessed plasma or serum sample containing extracellular vesicles (EVs); b. providing a capture system comprising at least two populations of fluorescently labeled magnetic microspheres, each population of microspheres displaying an antibody against a distinct EV surface target, the antibody being labeled with a distinct fluorescent label, the targets being synaptic proteins; c. incubating the plasma or serum sample with the capture system under conditions that allow specific binding between the antibodies and the target synaptic proteins while substantially maintaining the integrity of the EV membrane, thereby providing a distinct population of EV-microsphere complexes corresponding to each synaptic protein; d. Washing the EV-microsphere complexes using a magnetic device under conditions that allow for selective capture of the complexes; e. providing a plurality of detection antibodies against a plurality of standard EV surface markers, the plurality of detection antibodies being directly or indirectly labeled with the same fluorescent label; f. incubating the captured EV-microsphere complexes with a plurality of detection antibodies under conditions that allow specific binding between the antibodies and the EV surface markers while substantially maintaining the integrity of the EV membrane; g. washing the resulting labeled complex with a magnetic device to remove excess reagent; and h. subjecting the labeled complex to a microfluidic device capable of simultaneous detection and quantification of fluorescence emission at multiple wavelengths, and quantifying the fluorescence emission levels to provide a separate assessment of the levels of synaptic proteins corresponding to each of the EV populations. The methods are carried out using reagents and conditions that preserve the EVs in a substantially intact form.

[0033] In some embodiments, the synaptic protein is selected from the group consisting of glutamate receptor subunit 2 (GluR2), neurogranin (NRGN), growth-associated protein 43 (GAP43), postsynaptic density 95 (PSD95) and syntaxin-1 (STXN-1).

[0034] In some embodiments, the at least two populations of fluorescently labeled magnetic microspheres include a first population of fluorescently labeled magnetic microspheres that display antibodies against NRGN, and a second population of fluorescently labeled magnetic microspheres that display antibodies against GAP43.

[0035] In some embodiments, the at least two populations of fluorescently labeled magnetic microspheres include a first population of fluorescently labeled magnetic microspheres presenting antibodies against GluR2, a second population of fluorescently labeled magnetic microspheres presenting antibodies against PSD95, and a third population of fluorescently labeled magnetic microspheres presenting antibodies against STXN.

[0036] In some embodiments, the at least two fluorescently labeled magnetic microsphere populations include a first fluorescently labeled magnetic microsphere population presenting antibodies against NRGN, a second fluorescently labeled magnetic microsphere population presenting antibodies against GAP43, a third fluorescently labeled magnetic microsphere population presenting antibodies against GluR2, a fourth fluorescently labeled magnetic microsphere population presenting antibodies against PSD95, and a fifth fluorescently labeled magnetic microsphere population presenting antibodies against STXN.

[0037] In some embodiments, the standard EV surface marker is a tetraspanin. In some embodiments, the multiple detection antibodies include at least three antibodies, each for a different tetraspanin. In some embodiments, the multiple detection antibodies include an antibody for CD9, an antibody for CD63, and an antibody for CD81.

[0038] In some embodiments, the capture system further comprises a population of fluorescently labeled magnetic microspheres displaying a non-specific control antibody, and providing an assessment of the level of a synaptic protein comprises normalizing the specific signal determined for each synaptic protein relative to the non-specific control signal.

[0039] In some embodiments, the capture system further comprises a population of fluorescently labeled magnetic microspheres displaying an antibody against a canonical EV surface marker, and providing an assessment of the level of a synaptic protein comprises normalizing the signal determined for each synaptic protein relative to the signal of the canonical EV surface marker. In some embodiments, the canonical EV surface marker is selected from CD63, CD9, CD81, or a combination thereof.

[0040] In some embodiments, the method is performed with 1-75 ml of plasma or serum (or a corresponding amount of intact EVs). In some embodiments, the plasma or serum sample is from a subject suspected of having a neurological disorder. In further embodiments, the plasma or serum sample is from a subject suspected of having a neurodegenerative disease. The subject according to the present invention is typically a human subject.

[0041] In a further aspect, the present invention provides a kit for analyzing EVs from a blood-derived sample, the kit comprising: i) a capture system comprising a first population of magnetic microspheres displaying an antibody against GAP43 or CD171 and labeled with a first fluorophore combination, a second population of magnetic microspheres displaying an antibody against CD68 and labeled with a second fluorophore combination, and a third population of magnetic microspheres displaying an antibody against P2RY12 and labeled with a third fluorophore combination; ii) a plurality of detection antibodies, each antibody directed against a distinct tetraspanin marker, the plurality of detection antibodies being directly or indirectly labeled with the same fluorescent marker; and optionally iii) Reagents for carrying out the analysis under conditions that maintain the EVs substantially intact.

[0042] In some embodiments, the capture system comprises a population of magnetic microspheres displaying antibodies against GAP43 and a population of magnetic microspheres displaying antibodies against CD171.

[0043] In a further aspect, the present invention provides a kit for analyzing EVs from a blood-derived sample, the kit comprising: i) a capture system comprising at least two populations of fluorescently labeled magnetic microspheres, each population of microspheres displaying an antibody against a distinct EV surface target, the antibody being labeled with a distinct fluorescent label, the targets being synaptic proteins; ii) multiple detection antibodies against multiple standard EV surface markers, the multiple detection antibodies being directly or indirectly labeled with the same fluorescent label; and optionally iii) Reagents for carrying out the analysis under conditions that maintain the EVs substantially intact.

[0044] In some embodiments, the synaptic protein is selected from the group consisting of glutamate receptor subunit 2 (GluR2), neurogranin (NRGN), growth-associated protein 43 (GAP43), postsynaptic density 95 (PSD95) and syntaxin-1 (STXN-1). In some embodiments, the kit comprises: i) a capture system comprising a first population of magnetic microspheres displaying an antibody against NRGN and labeled with a first combination of fluorophores, and a second population of magnetic microspheres displaying an antibody against GAP43 and labeled with a second combination of fluorophores; and ii) A plurality of detection antibodies, each antibody directed against a distinct tetraspanin marker, which are directly or indirectly labeled with the same fluorescent marker.

[0045] In some embodiments, the kit comprises: i) a capture system comprising a first population of magnetic microspheres displaying an antibody against GluR2 and labeled with a first fluorophore combination, a second population of magnetic microspheres displaying an antibody against PSD95 and labeled with a second fluorophore combination, and a third population of magnetic microspheres displaying an antibody against STXN and labeled with a third fluorophore combination; and ii) A plurality of detection antibodies, each antibody directed against a distinct tetraspanin marker, which are directly or indirectly labeled with the same fluorescent marker.

[0046] In some embodiments, the kit comprises: i) a first magnetic microsphere population presenting an antibody against NRGN and labeled with a first fluorophore combination, a second magnetic microsphere population presenting an antibody against GAP43 and labeled with a second fluorophore combination, a third magnetic microsphere population presenting an antibody against GluR2 and labeled with a third fluorophore combination, a fourth magnetic microsphere population presenting an antibody against PSD95 and labeled with a fourth fluorophore combination, and a fifth magnetic microsphere population presenting an antibody against STXN and labeled with a fifth fluorophore combination; and ii) A plurality of detection antibodies, each antibody directed against a distinct tetraspanin marker, which are directly or indirectly labeled with the same fluorescent marker.

[0047] In some embodiments, the detection antibodies include an antibody to CD9, an antibody to CD63, and an antibody to CD81.

[0048] In some embodiments, the methods and kits disclosed herein further comprise a population of fluorescently labeled magnetic microspheres displaying a non-specific control antibody to normalize the specific signal determined for each target protein (e.g., a synaptic protein) based on the non-specific control signal.

[0049] In some embodiments, the methods and kits disclosed herein further comprise a population of fluorescently labeled magnetic microspheres presenting antibodies against standard EV surface markers to normalize the signals determined for each target protein (e.g., synaptic proteins) based on the signals of the standard EV surface markers. In some embodiments, the standard EV surface markers are selected from CD63, CD9, CD81, or combinations thereof.

[0050] In some embodiments, the reagent is selected from the group consisting of: (i) at least one binding buffer characterized by the lack of detergents and the presence of protease and / or phosphatase inhibitors for incubating the sample with the capture system, thereby providing a distinct population of EV-microsphere complexes; (ii) at least one wash buffer characterized by a significantly elevated salt concentration compared to at least one binding buffer; and (iii) at least one binding buffer and at least one wash buffer as defined in (i) and (ii) above. Other objects, features and advantages of the present invention will become apparent from the following description and drawings. [Brief description of the drawings]

[0051] [Figure 1]Figures 1A-1J. Performance of the developed assay as a function of different experimental parameters. Figure 1A - Mean fluorescence intensity (MFI) obtained for loading of CD9 antibodies onto capture beads using staining with increasing amounts of PE-labeled anti-mouse antibody (in the figure, labeled "detection antibody"), for different loading concentrations (μg / 106 beads) ranging from 0.5 to 5 μg / ml. Figures 1B-1C - Analysis of two random untreated plasma samples from healthy donors using beads labeled with capture antibodies against specific targets (CD9, GAP43, P2RY12, CD68 or a non-specific IgG used as a negative control) and a cocktail of biotinylated detection antibodies for tetraspanin detection (CD9, CD63, and CD81 (hereafter referred to as "pan-TSPN"). Figure 1B - Plasma sample obtained from the first healthy donor (labeled "Plasma 1"); Figure 1C - Plasma sample obtained from the second healthy donor (labeled "Plasma 2"). Figure 1D - Illustrates the mean fluorescence intensity measured with a pan-TSPN detection antibody cocktail for EVs captured from plasma samples with CD9, GAP43 or CD171 specific antibodies in intact form ("intact plasma") or after EV depletion using a PEG-based precipitation method ("EV depletion"), or after detergent lysis (Triton X100, 2% final concentration, "detergent"). White dots represent intact plasma, grey dots represent samples treated with precipitation, and hatched represent samples treated with detergent. As shown, P values ​​by two-way ANOVA were less than 0.001 or less than 0.0001. Figure 1E-1F - Illustrates the mean fluorescence intensity obtained for EVs captured by CD171 or GAP43 antibodies in medium conditioned by HEK293 cells overexpressing one of these neural antigens (CD171 or GAP43). Figure 1E shows the detection of GAP43-positive EVs by IEL beads with the addition of anti-GAP43 but not anti-CD171 antibodies.Figure 1F shows the detection of CD171 positive EVs by IEL beads with added anti-CD171 but no anti-GAP43 antibodies; Figures 1G-1J - plasma samples were analyzed using IEL beads specific for GAP43 (1G), CD171 (1H), CD235 (1I) and CD68 (1J) in combination with the indicated detection antibodies (pan-TSPN cocktail, GAP43, CD171, CD68 and CD235). [Diagram 2] Figures 2A-2G show the simultaneous measurement of at least six surface EV proteins. Figure 2A - shows the mean fluorescence intensity measurements at serial plasma dilutions using two canonical EV surface markers (CD9 and CD81), or tissue-specific markers (macrophage marker CD68, erythrocyte marker CD235 (glycophorin A), neuronal markers GAP43 and CD171). Figures 2B-2F show the detection of multiple tissue-specific and canonical EV surface markers measured simultaneously in both multiplexed and single-bead IEL assays. MFI obtained with anti-CD9, anti-CD81, anti-CD235, anti-GAP43 and anti-CD68 capture beads (Figures 2B-2F, respectively). Figure 2G - shows the correlation between single-bead and multiplexed assays measuring the mean fluorescence intensity obtained for CD9 in 25 random plasma samples. [Diagram 3] Figures 3A-3F. Cell type specific differences between tetraspanin profiles of distinct EV populations in plasma. Figure 3A - Whole plasma (10-50 ml / sample) was subjected to IEL with an antibody against the axonal marker, GAP43, followed by detection with biotinylated GAP43 antibody or with a pan-TSPN antibody cocktail. Figure 3B - Plasma IEL with GAP43 antibody followed by detection with individual TSPN antibodies, i.e., CD9, CD63 and CD81 antibodies. Figure 3C - IEL against erythroid EVs with the known marker CD235 glycophorin followed by detection with individual TSPN antibodies, i.e., CD9, CD63 and CD81 antibodies. Figure 3D - IEL against one of the EV markers, CD9, followed by detection with CD235 and GAP43 antibodies. Figure 3E, 3F - IELs for CD63 (E), CD81 (F) followed by GAP43 and CD235 detection show strong positivity for GAP43 and lack of detectable binding to CD235. [Figure 4] Figures 4A-4E. IEL capture and detection analysis of fractions of plasma EVs after size-exclusion chromatography (SEC). 10 ml of plasma-derived EVs were precipitated using a PEG-based technique, resuspended in 2 ml of PBS, loaded onto a QEV2 53 nm column (IZON Sciences), and 2 ml fractions were collected according to the manufacturer's instructions. Protein concentration (A280, grey bars) was measured for each fraction, including three void fractions (labeled -2, -1, and 0), and 50 μl aliquots of each fraction were analyzed by IELs as shown (black bars). Fractions with peak concentrations of specific EVs (detected with pan-TSPN antibody cocktail) are indicated by arrows. (A) IELs against CD9, detected with pan-TSPN antibody cocktail. Protein elution is indicated in grey. (B) IELs against erythrocyte marker CD235, detected with pan-TSPN antibody cocktail. (C) IELs against macrophage marker CD68, detected with pan-TSPN antibody cocktail. (D) IELs against the axonal marker GAP43, detected with a pan-TSPN antibody cocktail. (E) IELs against the neuronal marker CD171, detected with a pan-TSPN antibody cocktail. [Diagram 5] Figures 5A-5G. Intact EV Luminex reveals a continuum of sizes among tetraspanin-positive structures (EVs) in plasma. (Figures 5A-5D) Fractions collected from the QEV2 column were subjected to IELs against the axonal marker GAP43 and probed with a pan-TSPN antibody cocktail (A) or with antibodies against CD9 (B), CD63 (C) and CD81 (D), respectively. (Figures 5E-G) QEV2 fractions were subjected to IELs against CD9 (E), CD63 (F) and CD81 (G) and probed with antibodies against the axonal marker GAP43 or with a pan-TSPN antibody cocktail, as indicated. [Figure 6]Figure 6A-6D. Cell membrane disruption by Triton-X100 pretreatment of plasma samples reduces IEL signal. Plasma samples were concentrated by PEG-based precipitation and reconstituted in 1xPBS. Samples were then pretreated with TX100 (red bars) or mock buffer (blue bars) and subjected to size-exclusion chromatography (IZON qEV 2 column). The fractions were then subjected to IEL with (A) CD9, (B) CD235, (C) CD63 and (D) GAP43 beads combined with a pan-TSPN detection cocktail. [Figure 7] Figures 7A-7F. IEL analysis of neuronal conditioned medium. Serum-free medium conditioned by iPSC-derived neurons was collected, clarified by filtration through Amicon spin filters (100 kDa molecular weight cut-off), reconstituted in 0.5 ml PBS and processed by SEC using QEV Original columns (35 nm, ZON Sciences). Fractions (0.5 ml) were collected and protein concentration (A280) was determined for each fraction (grey bars) according to the manufacturer's instructions. All fractions (1-20, 50 ml per sample) were subjected to IEL with beads against (A) GAP43, (B) CD171 and (C) CD235, followed by detection with a pan-TSPN antibody cocktail. Note the lack of CD235 positive EVs (specificity target) in the medium conditioned by iPSC neurons. (D–F) IELs with beads against EV markers, CD9 (D), CD63 (E) and CD81 (F) in the QEV fraction. [Figure 8] Lipid composition of late and early SEC fractions. Plasma EVs were concentrated and processed by SEC (qEV2, Izon) as described in Methods. Fractions 2–5 and 8–13 were pooled, concentrated using Amicon spin filters (100 kDa molecular weight cut-off) and subjected to lipidomic analysis. Each fraction was analyzed in biological triplicates. Pie charts show the contribution (% of total lipids) of the indicated specific lipid classes in early (left) and late (right) fractions. The "prominent" sectors of the pie charts indicate higher proportions of lipids in both comparisons. For minor lipid components (mean content in early fractions <0.1%), see Table 8. [Figure 9]Figures 9A-9B show transmission electron microscopy (TEM) images of aqueous uranyl acetate stained EVs in fractions 2-5 (Figure 9A) and 8-14 (Figure 9B). [Figure 10] Figures 10A-10F show multiplexed measurements of five synaptic markers, glutamate receptor subunit 2 (GluR2, Figure 10A), neurogranin (NRGN, Figure 10B), growth-associated protein 43 (GAP43, Figure 10C), postsynaptic density (PSD95, Figure 10D) and syntaxin-1 (STXN-1, Figure 10E) as well as the EV marker CD63 (Figure 10F). Assays were performed in three formats: singleplex (1-plex) - using capture beads for individual analytes; 6-plex - using a combination of capture beads for all six analytes; and 3-plex, where two parallel assays with two capture bead combinations (NRGN+GAP43+CD63 and GluR2+PSD95+STXN-1) were performed. [Figure 11] Figures 11A-11D. Quantitative measurements using purified EVs derived from cultured cells are shown. A dilution curve was generated using enriched purified EVs released from cultured glioblastoma cells (U87 cell line, ATCC). For detection, the EV markers CD9 (Figure 11A), CD81 (Figure 11B) and CD63 (Figure 11C) were measured in singleplex (1-plex) and multiplexed (M-plex) formats using appropriate capture beads and a pan-TSPN antibody cocktail. Additionally, non-specific control beads modified with an isotype control antibody (IgG) were added to the assay (Figure 11D). [Figure 12] 12A-12E. Internal specificity controls. Capture beads modified with a non-specific isotype control antibody (IgG) were incorporated into the IEL assay. To ensure the absence of antibody interference, singleplex (1-plex) and multiplexed measurements were compared. For each analyte, including CD63 (FIG. 12A), GAP43 (FIG. 12B), GluR2 (FIG. 12C), PSD95 (FIG. 12D), and NRGN (FIG. 12E), the specific signal exceeded the non-specific signal (IgG) by at least 10-fold. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] The present invention relates to assays and methods for the simultaneous characterization of extracellular vesicles (EVs) and the preparation of improved EV compositions for therapeutic and diagnostic purposes. In particular, in some embodiments, the present invention provides methods and kits for analyzing blood-derived samples, as well as the generation and use of tissue-derived circulating EV populations that exhibit advantageous properties and are characterized by unexpectedly small dimensions of less than 30 nm in diameter.

[0053] The present invention relates in some embodiments to a method for analyzing blood-derived samples (e.g., plasma and serum samples), which method is hereafter referred to as the "analysis method of the present invention". Unlike other biological fluid sources such as urine, the analysis of blood-derived samples is technically complicated by the presence of high concentrations of potential contaminants, including but not limited to circulating proteins (e.g., albumin, fibrinogen and globulins), lipids (e.g., high-density lipoproteins, low-density lipoproteins, and triglycerides) and debris. In addition, blood-derived samples are characterized by high levels of vesicles from various origins, which exacerbates the ability to detect tissue-specific EV markers without cross-contamination by other vesicles, especially in multiplexed assays. In fact, the analysis of blood-derived samples for EV markers usually involves a preliminary EV enrichment step (e.g., centrifugation, precipitation, size exclusion, filtration, and / or immunoprecipitation) to obtain sufficient amounts of purified EVs compatible with existing measurement techniques, thus requiring larger sample volumes and additional labor.

[0054] In contrast, the present invention provides a method for the analysis of EVs from blood-derived samples, which does not require separation or concentration of EVs prior to analysis and can be performed with very small sample volumes, 1-100 ml, even 1-50 ml. The analytical method according to an embodiment of the present invention provides a multiplexed assay using Luminex or similar technology. Typically, and advantageously, the assay is used (or marker evaluation or quantification is performed) under conditions such that EVs remain substantially intact. For example, but not limited to, the analytical assays and methods of the present invention advantageously employ detergent-free buffers that may contain protease and / or phosphatase inhibitors (e.g., when incubating the sample with a capture system according to the present invention, as further detailed below). In additional advantageous embodiments, the assays and methods of the present invention may enhance salt concentrations during the washing steps while simultaneously maintaining a substantially detergent-free environment.

[0055] In some embodiments, the methods and assays employ high salt conditions (e.g., 50 mM to 300 mM NaCl) to reduce specific interactions and improve the specificity of measurements in complex blood-based biofluids. In some embodiments, the methods and assays include a blocking strategy involving competition with negatively charged peptides that reduces interactions of overall negatively charged EVs, thus improving the specificity of measurements in complex blood-based biofluids.

[0056] The blood-derived sample used in connection with the analytical method of the present invention advantageously contains intact EVs. In some embodiments, the sample is a plasma sample. In another embodiment, the sample is a serum sample. As used herein, "unprocessed / untreated plasma or serum sample" indicates that the EVs have not been subjected to any treatment to isolate, separate or concentrate them prior to contacting with the capture system disclosed herein. The sample may be diluted or mixed with various assay reagents. Advantageously, as shown herein, the analytical method is adaptable for use with low input biofluid samples. In some embodiments, 1-100 ml of plasma or serum is sufficient for the method, e.g., 1-75 ml, 1-50 ml of plasma or serum, less than 75 μl per data point (measurement), and typically 1-50 μl of plasma or serum sample is sufficient for the method. In alternative embodiments, the sample comprises 1-25, 15-25, or 10-20 μl (which may be diluted to a final volume compatible with the assay of choice, e.g., 50 μl for a Luminex-based assay), each possibility representing a separate embodiment of the invention, however, it should be understood that in some embodiments larger volumes may also be used in conjunction with the principles of the invention.

[0057] In an exemplary embodiment, an analytical method according to the present invention may include: a. providing a capture system comprising a plurality (e.g., at least three, four, five, six, seven or eight) distinct populations of fluorescently labeled magnetic microspheres, each microsphere population displaying an antibody against a distinct target on the surface of an EV population of distinct cellular origin; b. Providing a blood-derived sample from a subject, the sample comprising less than 75 μl of unprocessed plasma or a corresponding amount of intact EVs; c. incubating the sample with the system under conditions such as those that allow specific antigen-antibody binding while substantially maintaining the integrity of the EV membrane, thereby providing a population of EV complexes with distinct microspheres corresponding to each target (herein designated "EV-microsphere complexes"); d. Washing the EV-microsphere complexes (e.g., to remove non-specific EVs and soluble proteins) using a magnetic device under conditions that allow for selective capture of the complexes; e. Incubating the EV-microsphere complex with multiple detection antibodies under conditions that allow specific antigen-antibody binding while substantially maintaining the integrity of the EV membrane, such as where each antibody is directed to a distinct marker on the surface of the EV, and the multiple detection antibodies are labeled with the same fluorescent marker; f. Optionally and advantageously, washing the resulting labeled complex with a magnetic device to remove excess (e.g., unbound) reagent; and g. subjecting the resulting complexes to a microfluidic device capable of detecting and simultaneously quantitating fluorescent emissions at multiple wavelengths, thereby quantifying the levels of fluorescent emissions and providing a separate assessment of the levels of surface markers corresponding to each of the EV populations; and h. Comparing the quantification levels to control levels (while usually taking into account background levels for each measurement).

[0058] In some embodiments, a method of analyzing a blood derived sample is provided, the method comprising: a. providing an unprocessed plasma or serum sample containing extracellular vesicles (EVs); b. providing a capture system comprising at least two populations of fluorescently labeled magnetic microspheres, each population of microspheres displaying an antibody against a distinct EV surface target, the antibody being labeled with a distinct fluorescent label, the targets being synaptic proteins; c. incubating the plasma or serum sample with the capture system under conditions that allow specific binding between the antibodies and the target synaptic proteins while substantially maintaining the integrity of the EV membrane, thereby providing a distinct population of EV-microsphere complexes corresponding to each synaptic protein; d. Washing the EV-microsphere complexes using a magnetic device under conditions that allow for selective capture of the complexes; e. providing a plurality of detection antibodies against a plurality of standard EV surface markers, the plurality of detection antibodies being directly or indirectly labeled with the same fluorescent label; f. incubating the captured EV-microsphere complexes with a plurality of detection antibodies under conditions that allow specific binding between the antibodies and the EV surface markers while substantially maintaining the integrity of the EV membrane; g. washing the resulting labeled complex with a magnetic device to remove excess reagent; and h. subjecting the labeled complex to a microfluidic device capable of simultaneous detection and quantification of fluorescence emission at multiple wavelengths, and quantifying the fluorescence emission levels to provide a separate assessment of the levels of synaptic proteins corresponding to each of the EV populations.

[0059] As used herein, the term "synaptic protein" refers to a membrane protein known to be highly enriched in the synaptic cleft relative to non-synaptic membrane regions. Synaptic proteins may be involved in regulating neurotransmitter release and reception, maintaining synaptic structure, and / or early neuronal development.

[0060] Synapses are the connections between neurons and are involved in neural interactions. Synaptic function requires many proteins to hold synapses together and transmit messages between neurons. Synapses change in response to a process called synaptic plasticity, which is thought to be involved in learning and memory. Synapse loss and dysfunction occurs early, before cell death, in different neurodegenerative diseases (Lleo et al., 2019, Molecular & Cellular Proteomics, 18:546-560). Synaptic proteins have been suggested as biomarkers for neurodegenerative diseases and have been measured primarily in CSF and plasma (Camporesi et al., 2020, Biomarker Insights, 15:1-17; Vrillon et al., 2022, Alzheimer's Research & Therapy, 14:71). Most attempts to measure synaptic proteins rely on immunoprecipitation followed by mass spectrometry. Developing antibody sets against membrane proteins, such as synaptic proteins, is extremely difficult and reliable assays are not available.

[0061] The Luminex compatible assay disclosed herein advantageously requires only one antibody against the synaptic protein to be analyzed, instead of the antibody pair required by conventional immunoassay methods. Detection is achieved by pairing the synaptic protein antibody with an antibody against a standard EV marker. As exemplified herein below, the assay disclosed herein allows for the simultaneous detection, quantification and analysis of multiple synaptic proteins presented on the surface of EVs using a very small amount of blood-derived sample. The assay disclosed herein is much simpler than previously described methods, and is therefore particularly suitable and useful for clinical diagnostic applications.

[0062] Synaptic proteins useful as targets for capture of EVs according to the present invention include, for example, glutamate receptor subunit 2 (GluR2), neurogranin (NRGN), growth-associated protein 43 (GAP43), postsynaptic density 95 (PSD95) and syntaxin-1 (STXN-1).

[0063] "Simultaneous" detection / analysis / quantification as disclosed herein means that multiple populations of microspheres utilized by the methods of the invention are subjected to fluorescence analysis and measurement simultaneously. Typically, multiple populations of microspheres are present in the same mixture and are subjected to fluorescence analysis and measurement simultaneously.

[0064] "Tetraspanins", also called transmembrane 4 superfamily (TM4SF) proteins, are primarily transmembrane proteins with a conserved structure that function as membrane protein organizers. Tetraspanins have four transmembrane alpha helices and two extracellular domains, one short (called small extracellular domain or loop, SED / SEL or EC1) and one long, usually 100 amino acid residues domain (large extracellular domain / loop, LED / LEL or EC2). Although some protein families have four transmembrane alpha helices, tetraspanins are defined by a conserved amino acid sequence that includes four or more cysteine ​​residues in the EC2 domain, two of which are in a highly conserved "CCG" motif. Extracellular vesicles are highly enriched in tetraspanins, some of which are characterized by a wide tissue distribution and thus serve as standard markers for EVs. Examples include CD9, CD63 and CD81.

[0065] As used herein, "extracellular vesicles" (abbreviated as EVs / EVs) refer to double-membrane vesicles of various biosynthetic pathways released into bodily fluids and include vesicles released from both live and dead cells. EVs captured and analyzed by the present invention typically range in size from 10 nm to 200 nm in diameter.

[0066] The analysis according to the invention is preferably carried out using the Luminex platform, which uses a series of color-coded magnetic microspheres coated with target-specific capture antibodies. The "capture" of EVs refers to a physical binding / association in which EVs are separated from other components of the plasma or serum sample in which they are present after washing of non-binding substances. The captured EV population is then incubated with a series of detection antibodies, for example, antibodies against standard EV surface markers, to quantify the level of target captured by the capture antibody. The detection antibodies are specific for targets on the surface of EVs that are different from the targets used for capture. For example, antibodies against multiple tetraspanins disclosed herein can be used as detection antibodies. The detection antibodies are conjugated to a label, for example, phycoerythrin (PE) or biotin + streptavidin-phycoerythrin (SAPE).

[0067] In some embodiments, the captured EVs are further purified, e.g., whereby the EVs are at least 80%, at least 85%, at least 90%, at least 95% separated from other components of the plasma or serum sample. Each possibility represents a separate embodiment of the invention. Subsequent steps are performed to obtain purified EVs, including, for example, elution of the EVs from the capture antibodies / beads under conditions including high detergent content and / or reduced pH.

[0068] In some embodiments, multiple populations of magnetic microspheres loaded with antibodies against tissue-specific surface markers indicative of the cellular origin of EVs are used to simultaneously capture multiple populations of EVs from plasma or serum samples.

[0069] In some embodiments, multiple populations of magnetic microspheres loaded with antibodies against multiple synaptic proteins are used to simultaneously capture EVs that display at least one of the multiple synaptic proteins on their surface.

[0070] Multiple capture antibodies (i.e., at least two capture antibodies) against distinct targets are used, a first capture antibody specific for a first target on the surface of the EVs, a second capture antibody specific for a second target on the surface of the EVs, etc. The antibodies are conjugated or otherwise bound to magnetic microspheres to form distinct populations of magnetic microspheres, each presenting an antibody against a distinct target on the surface of the EVs. For example, in some embodiments, multiple populations of magnetic microspheres are provided, each presenting an antibody against a distinct tissue-specific target on the surface of EVs of distinct cellular origin.

[0071] Providing a separate assessment of the levels of surface markers corresponding to each EV population can be performed by generating gates encompassing the range of fluorescence of each microsphere type corresponding to each separate EV population and quantifying the fluorescence emission levels corresponding to the detection antibodies for data points within each gate.

[0072] In another embodiment, the multiple detection antibodies are directed against multiple tetraspanin markers. In another embodiment, the tetraspanin markers are selected from the group consisting of CD9, CD63 and CD81. In another embodiment, the multiple detection antibodies include an antibody against CD9, an antibody against CD63 and an antibody against CD81. In another embodiment, the fluorescent marker for labeling the multiple detection antibodies includes a quantum dot or a combination of multiple fluorophores. Without wishing to be bound by a particular theory or mechanism of action, such a combination of antibodies labeled with the same fluorescent marker results in an enhanced detection signal and sensitivity.

[0073] In another embodiment, the plurality of detection antibodies are directed against a plurality of diagnostic markers. In another embodiment, the plurality of detection antibodies are directed against surface EV markers that identify cell functions or subpopulations, such as, but not limited to, LAMP2, LC3, GAP43, annexins and integrins. In another embodiment, at least one labeled detection antibody is directed against a therapeutic target (also referred to herein as a disease-specific target) that can be found on the EV surface and is present in different EV subpopulations as the target of quantitative or semi-quantitative assessment. In certain embodiments, the use of diagnostic markers that include complement system components (e.g., C3, C5b-9, C4, Clq, C9, C3b, iC3b, TF, CRP, pCRP, MAC, CD59, CD55, CR1, C5aRl, or C5a) is expressly excluded. In yet another embodiment, the marker is not an alpha-synuclein marker.

[0074] In another embodiment, the system includes at least three, typically at least four, populations of fluorescently labeled magnetic microspheres. In another embodiment, each population of distinct fluorescently labeled magnetic microspheres includes a distinct combination of fluorophores, allowing its differentiation from other microsphere populations. In another embodiment, the fluorescently labeled magnetic microspheres are fluorescent magnetic microspheres that are compatible with Luminex detection devices (e.g., MagPlex microspheres).

[0075] In another embodiment, the targets of the capture system include a neuronal target and targets corresponding to at least three additional cell origins selected from the group consisting of bone, lung, tissue macrophages, lung macrophages, muscle, adipocytes, epithelium, endothelium, monocytes, microglia, megakaryocytes, T cells, red blood cells, liver, and oligodendrocytes. In another embodiment, the targets of the capture system include a neuronal target and targets corresponding to at least three additional cell origins selected from the group consisting of bone, lung, tissue macrophages, lung macrophages, muscle, adipocytes, epithelium, endothelium, microglia, liver, and oligodendrocytes. In another embodiment, the targets of the capture system include a neuronal target and targets corresponding to at least three additional cell origins selected from the group consisting of bone, lung, tissue macrophages, lung macrophages, muscle, adipocytes, epithelium, endothelium, microglia.

[0076] In another embodiment, the target of the capture system comprises at least one additional target selected from the group consisting of GAP43 or CD171 and P2RY12, CD68. In another embodiment, the capture system comprises a first population of magnetic microspheres presenting an antibody against GAP43 and labeled with a first combination of fluorophores, a second population of magnetic microspheres presenting an antibody against CD68 and labeled with a second combination of fluorophores, and a third population of magnetic microspheres presenting an antibody against P2RY12 and labeled with a third combination of fluorophores. In another embodiment, the capture system further comprises a fourth population of magnetic microspheres presenting an antibody against CD235a and labeled with a fourth combination of fluorophores. In another embodiment, the target further comprises at least one additional target as shown in Table 4.

[0077] Advantageously, the analytical method according to the invention allows the bypass of concentration and purification steps that require larger sample volumes. Thus, in another embodiment, the method does not include additional steps of EV isolation and / or sample processing intended to concentrate the EV-containing biofluid sample before incubation with the system. In some embodiments, the blood-derived sample analyzed according to the invention is not subjected to a step of isolating or concentrating EVs before incubation with the capture system disclosed herein. For example, the method of the invention is demonstrated herein to obtain high accuracy in multiplex measurements of EV markers using unprocessed plasma samples of, for example, 50 μl of plasma, without employing EV immunoprecipitation, size exclusion chromatography or similar steps required in previously reported assays. In yet other embodiments, for example, when isolation or specific capture of nano-EVs is required, the method may include steps such as size exclusion chromatography, as discussed in more detail below.

[0078] Profiling of EV surface proteins may also serve as an important quality control measure in the generation of therapeutic EVs by providing quality assurance and reproducibility of EV isolation. For diagnostic purposes, such profiling of the EV surface proteome would provide a normalization standard, which is currently sorely lacking in the EV diagnostic space.

[0079] In certain embodiments, the analytical methods of the present invention may also be employed in therapy and diagnosis, e.g., for evaluation of circulating EV profiles in subjects in need thereof, or for quality control purposes, e.g., to elucidate EV origin or validate tissue selectivity of EV compositions intended for drug delivery or other therapeutic uses.

[0080] Thus, in another embodiment, the analysis of the blood-derived sample includes monitoring the tetraspanin profile of the sample (e.g., as a criterion for monitoring the health of the subject). In another embodiment, the analysis of the blood-derived sample includes verifying the assumed cellular origin of the EV preparation. In another embodiment, the EV preparation is used in a treatment. In another embodiment, the assumed origin is neural, and a tetraspanin profile characterized by substantial similarity to the tetraspanin profile of a control neural EV population is of neural origin. In another embodiment, the assumed origin is neural, and a tetraspanin profile characterized by elevated surface levels of CD63 and CD81 compared to surface levels of CD9 indicates that the preparation is of neural origin.

[0081] In some embodiments, the method according to the present invention further comprises: Providing an unprocessed plasma or serum sample containing extracellular vesicles (EVs); providing a capture system comprising a plurality of populations of labeled microspheres, each population of microspheres displaying an antibody against a distinct EV surface target, the antibody being labeled with a distinct fluorescent label; incubating the plasma or serum sample with the capture system under conditions that allow specific binding between the antibodies and the EV surface targets while substantially maintaining the integrity of the EV membrane, thereby providing a distinct population of EV-microsphere complexes corresponding to each target; washing the EV-microsphere complexes under conditions that allow for selective capture of said complexes; providing multiple detection antibodies against multiple EV surface markers, where the multiple detection antibodies are directly or indirectly labeled with the same label; incubating the captured EV-microsphere complexes with a plurality of detection antibodies under conditions that allow specific binding between the antibodies and the EV surface markers while substantially maintaining the integrity of the EV membrane; washing the resulting labeled complex to remove excess reagent; and and subjecting the labeled complex to an instrument capable of simultaneous detection and quantification of fluorescent emissions at multiple wavelengths, and quantifying the multiple signals to provide a separate assessment of the levels of the target protein corresponding to each of the EV populations.

[0082] In yet another aspect, a kit for analyzing a blood derived sample is provided, the kit comprising: i) a capture system comprising a first population of magnetic microspheres displaying an antibody against GAP43 or CD171 and labeled with a first fluorophore combination, a second population of magnetic microspheres displaying an antibody against CD68 and labeled with a second fluorophore combination, and a third population of magnetic microspheres displaying an antibody against P2RY12 and labeled with a third fluorophore combination; ii) a plurality of detection antibodies, each antibody directed against a distinct tetraspanin marker, the plurality of detection antibodies being directly or indirectly labeled with the same fluorescent marker; and optionally iii) reagents for carrying out said assessment under conditions that maintain said EVs substantially intact.

[0083] In another embodiment, the fluorescent marker for labeling the multiple detection antibodies comprises a quantum dot or a combination of multiple fluorophores. (i) at least one binding buffer characterized by the lack of detergents and the presence of protease and / or phosphatase inhibitors for incubating the sample with the capture system, thereby providing a distinct population of EV-microsphere complexes; (ii) at least one wash buffer characterized by a significantly elevated salt concentration compared to at least one binding buffer; and (iii) at least one binding buffer and at least one wash buffer as defined in (i) and (ii) above.

[0084] In additional embodiments, the present invention relates to substantially purified nanoEV populations, processes for their preparation, and methods of their use. The nanoEVs disclosed herein are distinguishable from EVs obtained from respective in vitro cultured cells. As further disclosed herein, the nanoEV populations were identified as presenting mature tissue-specific cells (e.g., neural markers), and thus distinguishable from EVs produced by stem cells or other pluripotent cells. Thus, the nanoEVs can be identified as tissue-specific EVs. In addition, blood-derived EVs - such EVs presenting red blood cell markers - were not included in the identified nanoEV population and were of extremely large size, corresponding to the size of standard EVs. Thus, the nanoEVs can also be identified as non-blood derived or solid tissue derived.

[0085] In another aspect, a process is provided for isolating non-blood derived tissue-specific EVs from a biological sample, typically a blood derived sample. In one embodiment, the process comprises a step of size selection (e.g., by filtration, centrifugation or chromatography-based methods), thereby specifically selecting EVs smaller than 30 nm in diameter. In another embodiment, the selected EVs are 10-25 nm in diameter (e.g., 15-25 nm in diameter, 15-20 nm in diameter or 10-15 nm in diameter). In a particular embodiment, the size selection step comprises size exclusion chromatography. In another embodiment, the size selection step comprises size exclusion chromatography, nanoporous membrane filtration, deterministic lateral displacement methods, electrophoretic separation, ultrasonic fractionation or a combination thereof. In another embodiment, the process further comprises separation, purification or analysis of the EVs using affinity capture methods, e.g., as described herein above for the analytical methods of the invention.

[0086] In one embodiment, the EV population is neuron-specific. In another embodiment, the EV population is macrophage-specific. In another embodiment, the isolated EVs comprise at least one EV population selected from the group consisting of neural-derived EVs and tissue macrophage-derived EVs. In yet another embodiment, the nanoEV population can be isolated from a blood-derived sample and identified, for example, using immunoaffinity capture methods based on tissue-specific and / or tetraspanin profiles as disclosed herein. Each possibility represents a separate embodiment of the present invention.

[0087] In another embodiment, the isolated EVs are characterized by a marker profile corresponding to differentiated solid tissue cells, hi another embodiment, the marker profile comprises protein markers, RNA markers, lipid markers, or a combination thereof (e.g., markers exemplified herein).

[0088] In some embodiments, an extracellular vesicle (EV) preparation is provided that comprises a substantially purified population of circulating neurally-derived EVs characterized by a particle size distribution of 10-25 nanometers (nm) in diameter and a marker profile corresponding to neural cells.

[0089] In some embodiments, provided are extracellular vesicle (EV) preparations comprising a substantially purified population of circulating tissue macrophage-derived EVs characterized by a particle size distribution of 10-25 nanometers (nm) in diameter and a marker profile corresponding to tissue macrophages.

[0090] In another embodiment, the process further comprises loading the isolated EVs with an exogenous cargo. In another embodiment, the exogenous cargo is a therapeutic agent. In another embodiment, the exogenous cargo is selected from the group consisting of a small molecule drug, a peptide, a polypeptide, an antibody, an oligonucleotide, a nucleic acid construct, and a gene editing agent. In another embodiment, the process further comprises modifying the isolated EVs to present a targeting agent. In another embodiment, the targeting agent is directed to a target selected from the group consisting of a tissue-specific target, a cell-specific target, and a disease-specific target. In another embodiment, the targeting agent is selected from the group consisting of an antibody, a receptor, a ligand, an aptamer, and combinations thereof.

[0091] In another embodiment, an EV preparation is provided that comprises a substantially purified non-blood derived, tissue-specific EV population, a population characterized by a particle size distribution of 10-25 nm in diameter and a marker profile corresponding to differentiated solid tissue cells.

[0092] In another embodiment, an EV preparation is provided comprising a substantially pure EV population produced by the process. In another embodiment, the preparation comprises said EV population of pharmaceutical grade purity. In another embodiment, the preparation is formulated in the form of a pharmaceutical composition and further comprises a pharma- ceutically acceptable carrier, excipient or diluent. In another embodiment, the isolated EVs are further loaded with an exogenous cargo intended for delivery to a target cell or tissue. In one embodiment, the cargo comprises a small molecule drug, antisense RNA, DNA, gene editing system (CRISPR), peptide, protein and / or antibody. In another embodiment, the cargo comprises a gene therapy agent. In another embodiment, the isolated EVs are further modified or engineered to present a targeted drug.

[0093] In another embodiment, the EV formulation is used in therapy. In another embodiment, the EV formulation is used in the treatment of neurological disorders, such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, etc. In another embodiment, the EV formulation is used in the treatment of liver disorders, such as fibrosis and cirrhosis. Without wishing to be bound by a particular theory or mechanism of action, EV formulations including nanoEVs according to the present invention exhibit improved therapeutic properties including, but not limited to, enhanced tissue penetration, improved stability and / or enhanced efficacy.

[0094] In another embodiment, the EV population produced by the process disclosed herein may be used in diagnosis. For example, but not limited to, the EV population may be further analyzed for their biomarker content, such as disease-specific biomarkers, tissue-specific biomarkers, and / or tetraspanin biomarkers, as disclosed herein. For example, but not limited to, biomarkers may include neurogranin (NRGN), orexin (HTCR), SOX1, and Olig2. Each possibility represents a separate embodiment of the present invention.

[0095] In one aspect, a process is provided for the separation of at least one population of non-blood derived, tissue specific EVs from a blood derived sample, the process comprising a size selection step of EVs less than 30 nanometers (nm) in diameter and separation of the selected EVs.

[0096] In one embodiment, the isolated EVs are 10-25 nm in diameter. In another embodiment, the isolated EVs are 15-25 nm in diameter, 15-20 nm in diameter, or 10-15 nm in diameter. Each possibility represents a separate embodiment of the present invention.

[0097] In another embodiment, the size selection step comprises size exclusion chromatography, nanoporous membrane filtration, deterministic lateral displacement, electrophoretic separation, ultrasonic fractionation, or a combination thereof. In another embodiment, the process further comprises immunoaffinity purification and / or analysis of at least one EV population.

[0098] In another embodiment, the isolated EVs are characterized by a marker profile corresponding to differentiated solid tissue cells, hi another embodiment, the marker profile comprises protein markers, RNA markers, lipid markers, or a combination thereof.

[0099] In another embodiment, the isolated EVs comprise at least one EV population selected from the group consisting of neural-derived EVs, and tissue macrophage-derived EVs, and kidney-derived EVs. In another embodiment, the isolated EVs comprise at least one EV population selected from the group consisting of neural-derived EVs, and tissue macrophage-derived EVs. Each possibility represents a separate embodiment of the present invention.

[0100] In another embodiment, the isolated EVs display GAP43. In another embodiment, the isolated EVs do not display CD235a. In another embodiment, the isolated EVs display podocin. In yet another embodiment, the isolated EVs do not display podocin. In another embodiment, the EVs are characterized by higher CD63 levels and lower CD9 levels than standard EVs (e.g., erythrocyte-derived EVs).

[0101] In some embodiments, the EV population is characterized by membrane lipid composition according to Table A. [Table 1]

[0102] In another embodiment, the process further comprises loading the isolated EVs with an exogenous cargo. In another embodiment, the exogenous cargo is a therapeutic agent. In another embodiment, the exogenous cargo is selected from the group consisting of a small molecule drug, a peptide, a polypeptide, an antibody, an oligonucleotide, a nucleic acid construct, and a gene editing agent. Each possibility represents a separate embodiment of the present invention.

[0103] In another embodiment, the process further comprises modifying the isolated EVs to present a targeting agent. In another embodiment, the targeting agent is directed to a target selected from the group consisting of a tissue-specific target, a cell-specific target, and a disease-specific target. In another embodiment, the targeting agent is selected from the group consisting of an antibody, a receptor, a ligand, an aptamer, and combinations thereof. Each possibility represents a separate embodiment of the present invention.

[0104] In another aspect, an EV preparation is provided that comprises a substantially purified population of EVs separated by the processes disclosed herein.

[0105] In another embodiment, the EV preparation comprises said at least one EV population of pharmaceutical grade purity, hi another embodiment, said preparation is formulated in the form of a pharmaceutical composition and further comprises a pharma- ceutically acceptable carrier, excipient or diluent.

[0106] In another embodiment, the EV formulation is used in therapy. In another embodiment, the therapy involves the delivery of an exogenously loaded therapeutic agent to a target cell or tissue of a subject in need thereof. In yet another embodiment, the EV formulation is used in diagnosis.

[0107] In another embodiment, the EV preparation comprises a substantially purified non-blood derived, tissue-specific EV population, which is characterized by a particle size distribution ranging from 10-25 nm in diameter and a marker profile corresponding to differentiated solid tissue cells.

[0108] In another aspect, there is provided a method of analyzing a blood derived sample, the method comprising: a. providing a capture system comprising four distinct populations of fluorescently labeled magnetic microspheres, each microsphere population displaying an antibody against a distinct target on the surface of an EV population of distinct cellular origin; b. Providing a blood-derived sample from a subject, the sample comprising less than 75 μl of unprocessed plasma or a corresponding amount of intact EVs; c. Incubating the sample with the capture system under conditions such as those that allow specific antigen-antibody binding while substantially maintaining the integrity of the EV membrane, thereby providing a distinct population of EV-microsphere complexes corresponding to each target; d. washing the EV-microsphere complexes using a magnetic device under conditions that allow for selective capture of said complexes; e. Incubating the EV-microsphere complex with multiple detection antibodies under conditions that allow specific antigen-antibody binding while substantially maintaining the integrity of the EV membrane, such as where each antibody is directed to a distinct marker on the surface of the EV, and the multiple detection antibodies are labeled with the same fluorescent marker; f. washing the resulting labeled complex using a magnetic device to remove excess reagent; g. subjecting the resulting complexes to a microfluidic device capable of detecting and simultaneously quantitating fluorescent emissions at multiple wavelengths, thereby quantifying the levels of fluorescent emissions and providing a separate assessment of the levels of surface markers corresponding to each of the EV populations; and h. comparing the quantification level to a control level; The method is carried out using reagents and conditions that preserve the EVs in a substantially intact form.

[0109] In another embodiment, the fluorescent markers for labeling the multiple detection antibodies comprise quantum dots or a combination of multiple fluorophores. In another embodiment, the multiple detection antibodies are directed against multiple diagnostic markers. In another embodiment, the multiple detection antibodies are directed against multiple tetraspanin markers. In a particular embodiment, the tetraspanin markers are selected from the group consisting of CD9, CD63 and CD81. In another particular embodiment, the multiple detection antibodies comprise an antibody directed against CD9, an antibody directed against CD63 and an antibody directed against CD81.

[0110] In another embodiment, the targets of the capture system include a neuronal target and targets corresponding to at least three additional cell origins selected from the group consisting of bone, lung, tissue macrophages, lung macrophages, muscle, adipocytes, epithelium, endothelium, monocytes, microglia, megakaryocytes, T cells, erythrocytes, liver, and oligodendrocytes. In another embodiment, the targets of the capture system include GAP43 and at least one additional target selected from the group consisting of P2RY12, CD68, and CD171. In another embodiment, the targets of the capture system include GAP43 or CD171 and at least one additional target selected from the group consisting of P2RY12, CD68. In another embodiment, the capture system comprises a population of magnetic microspheres presenting an antibody against GAP43 and labeled with a first combination of fluorophores, a second population of magnetic microspheres presenting an antibody against CD68 or CD171 and labeled with a second combination of fluorophores, and a third population of magnetic microspheres presenting an antibody against P2RY12 and labeled with a third combination of fluorophores. In another embodiment, the capture system comprises a population of magnetic microspheres presenting an antibody against GAP43 and labeled with a first combination of fluorophores, a second population of magnetic microspheres presenting an antibody against CD68 and labeled with a second combination of fluorophores, and a third population of magnetic microspheres presenting an antibody against P2RY12 and labeled with a third combination of fluorophores.

[0111] In another embodiment, the capture system further comprises a fourth population of magnetic microspheres displaying an antibody against CD235a and labeled with a fourth fluorophore combination. In another embodiment, the target further comprises at least one additional target as set forth in Table 4 below. In another embodiment, the capture system comprises at least five, six, seven or eight distinct populations of fluorescently labeled magnetic microspheres, each microsphere population displaying an antibody against a distinct target on the surface of an EV population of distinct cellular origin (e.g., at least five, at least six, at least seven or at least eight targets as set forth in Table 4).

[0112] In another embodiment, the sample is a plasma or serum sample. In another embodiment, the sample comprises 1-50 μl of plasma or serum (eg, a 1-50 μl unprocessed plasma or serum sample).

[0113] In another embodiment, the analysis of the blood-derived sample includes verifying the assumed cellular origin of the EV preparation. In another embodiment, the EV preparation is used in a treatment. In an exemplary embodiment, the assumed origin of the EV preparation is neural, and a tetraspanin profile characterized by substantial similarity to the tetraspanin profile of a control neural EV population is neural in origin. In another exemplary embodiment, the assumed origin is neural, and a tetraspanin profile characterized by elevated surface levels of CD63 and CD81 compared to surface levels of CD9 indicates that the preparation is neural in origin.

[0114] In another aspect, a kit for analyzing a blood derived sample is provided, the kit comprising: i) a capture system comprising a first population of magnetic microspheres displaying an antibody against GAP43 and labeled with a first fluorophore combination, a second population of magnetic microspheres displaying an antibody against CD68 or CD171 and labeled with a second fluorophore combination, and a third population of magnetic microspheres displaying an antibody against P2RY12 and labeled with a third fluorophore combination; ii) a plurality of detection antibodies, each antibody directed against a distinct tetraspanin marker, the plurality of detection antibodies being directly or indirectly labeled with the same fluorescent marker; and optionally iii) reagents for carrying out said assessment under conditions that maintain said EVs substantially intact.

[0115] In another embodiment, the capture system comprises a first population of magnetic microspheres displaying an antibody against GAP43 or CD171 and labeled with a first fluorophore combination, a second population of magnetic microspheres displaying an antibody against CD68 and labeled with a second fluorophore combination, and a third population of magnetic microspheres displaying an antibody against P2RY12 and labeled with a third fluorophore combination.

[0116] In another embodiment, the fluorescent markers for labeling said multiple detection antibodies comprise quantum dots or a combination of multiple fluorophores.

[0117] In another embodiment, the reagent is selected from the group consisting of: (i) at least one binding buffer characterized by the absence of detergents and the presence of protease and / or phosphatase inhibitors for incubating the sample with the capture system, thereby providing a distinct EV-microsphere complex population; (ii) at least one wash buffer characterized by a significantly elevated salt concentration compared to at least one binding buffer; and (iii) at least one binding buffer and at least one wash buffer as defined in (i) and (ii) above.

[0118] These and other embodiments of the invention are described in further detail below. antibody In some embodiments, the present invention relates in particular to the use of binding reagents, including antibodies. As used herein for embodiments of the present invention, the term antibody relates to at least the antigen-binding portion of an antibody.

[0119] As used herein, an antibody against an antigen (or specific for an antigen) is an antibody that can specifically bind to an antigen. As used herein, the terms "specifically bind" or "specifically recognize" mean that the binding of the antibody to the antigen is not competitively inhibited by the presence of unrelated molecules.

[0120] Intact antibodies include, for example, polyclonal and monoclonal antibodies (mAbs). Exemplary functional antibody fragments (which form the antigen-binding portion) containing the entire or substantially the entire variable region of both the light and heavy chains are defined as: (i) Fv, a genetically engineered fragment consisting of the variable region of the light chain and the variable region of the heavy chain expressed as two chains; (ii) single-chain Fv ("scFv"), a genetically engineered single-chain molecule comprising the variable region of the light chain and the variable region of the heavy chain linked by a suitable polypeptide linker; (iii) Fab, obtained by treating a full-length antibody with the enzyme papain to produce an intact light chain and an Fd fragment of the heavy chain consisting of the variable and its CH1 domains; (iv) Fab', a fragment of an antibody molecule comprising a monovalent antigen-binding portion of the antibody molecule, obtained by treating a full-length antibody with the enzyme pepsin, followed by reduction (yielding two Fab' fragments per antigen molecule); and (v) (Fab')2, a fragment of an antibody molecule comprising a monovalent antigen-binding portion of the antibody molecule, obtained by treating a full-length antibody with the enzyme pepsin (i.e., a dimer of Fab' fragments held together by two disulfide bonds). Further included within the scope of the present invention are chimeric antibodies; recombinant and modified antibodies, single chain antibodies (eg, single chain Fv) and fragments thereof, including antigen-binding portions.

[0121] The pair of variable domains of each of the light and heavy chains forms the antigen-binding site. The domains on the light and heavy chains have the same overall structure, and each domain contains four domains whose sequences are relatively conserved and are divided into three hypervariable domains known as complementarity determining regions (CDRs). 1-3 ) These hypervariable domains contribute to the specificity and affinity of the antigen-binding site.

[0122] As used herein, the term "antigen" refers to a molecule or a part of a molecule that can be bound by an antibody. An antigen can usually induce an animal to produce an antibody that can bind to the epitope of that antigen. An antigen can have one or more epitopes. The specific reaction mentioned above is intended to indicate that an antigen will react highly selectively with its corresponding antibody and not with many other antibodies that may be induced by other antigens.

[0123] Methods for producing monoclonal and polyclonal antibodies are well known in the art. Antibodies can be produced by any one of several known methods, which may employ inducing in vivo production of antibody molecules, screening immunoglobulin libraries, or producing monoclonal antibody molecules by continuous cell lines in culture. These include, but are not limited to, hybridoma technology, human hybridoma technology, and Epstein-Barr Virus (EBV) hybridoma technology. In addition to traditional methods of producing antibodies in vivo, antibodies can be produced in vitro using phage display technology by methods well known in the art (e.g., Current Protocols in Immunology, Colligan et al (Eds.), John Wiley & Sons, Inc. (1992-2000), Chapter 17, Section 17.1).

[0124] The following examples are provided in order to more fully illustrate some embodiments of the present invention, but these examples should not be construed in any way as limiting the broad scope of the invention.

[0125] Working Example Materials and Methods Preparation and characterization of intact exosome Luminex (IEL) beads: 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) chemistry (He et al. 2007, Bioconjug Chem 18:983-988) was used to conjugate the carboxyl groups (MagPlexR Antibodies were conjugated to functionalized magnetic microspheres of the desired emission range functionalized with 500 mM NaCl (Luminex Corp., Cat. No. MC1XXXX-01). Reusable chamber slides (Thermo Fisher Scientific, Cat. No. A25750) were used to determine bead recovery / enrichment after conjugation on a Countess™ 3 FL Automated Cell counter (Thermo Fisher Scientific, Cat. No. A49866). Beads were diluted at 1x10 in blocking buffer containing 0.01-0.5% sodium azide and 0.5-5% bovine serum albumin (BSA) at pH 7.5-9. 6 / ml for a maximum of 3 months.

[0126] To measure antibody loading, plate 5.0x10 IEL beads in assay diluent containing protease and phosphatase inhibitors, no detergent, and 0.1-2% bovine serum albumin in phosphate-buffered saline, pH 7.5-8.5. 6 The beads were resuspended at 100 μL / ml and loaded in duplicate into 96 black well plates (BrandTech., Cat. No. 781671) with at least 200 beads / well and an equal volume of the appropriate serially diluted phycoerythrin (PE)-labeled secondary antibody. Assay diluent alone was used as background control. Staining was performed on a Genie® Microplate Mixer / Shaker (600 RPM) for at least 20 minutes at room temperature, and the beads were washed three times in PBS-containing wash buffer pH 7.5-8, containing 10-100 mM Tris, 100-500 mM NaCl, and resuspended in xMAP Sheath Fluid Plus (100 uL / well, Thermo Fisher Scientific, Cat. No. 4050021). Plates were analyzed on a Luminex 200 plate reader with at least 100 beads per condition.

[0127] Preparation of biotinylated detection antibodies: Antibodies were biotinylated using EZ-link™ (Thermo Fisher Scientific) at 20-fold molar excess by overnight incubation at -4° C. according to the manufacturer's instructions. Excess reagent was removed using a 7K molecular weight cut-off Zeba™ Spin Desalting Column (Thermo Fisher Scientific, Cat. No. 89882).

[0128] IEL procedure: 50 μl of test sample was diluted to the desired concentration in assay diluent and placed in duplicate into wells of a 96 black well plate. Assay diluent alone was used as a background control. A working IEL bead / microsphere suspension in assay diluent was generated for up to seven IEL analytes and 50 μl of working suspension was added to each well, with 2.5-5.0x10 dilutions for each antibody / bead combination. 3 beads / ml. Pull-down assays were performed overnight (16-18 h) at 4°C on a Genie microplate shaker (400-1200 RPM) or for 3 h at room temperature or 2 h at 37°C. Plates were washed three times in PBS-containing wash buffer pH 7.5-8, containing 10-100 mM Tris, 100-500 mM NaCl in a magnetic plate holder, and beads were resuspended in assay diluent containing biotinylated detection antibody (1-2 μg / ml, 50 μl / well). After 1-4 h of incubation at room temperature (Genie shaker), beads were washed three times, resuspended in 50 μl of streptavidin-PE reagent (SAPE, 6 μg / ml) or streptavidin-quantum dots in PBS, incubated at room temperature for 20-60 min, washed three times, and resuspended in xMAP Sheath Fluid. Plates were analyzed in a Luminex 200 reader as above.

[0129] Size Exclusion Chromatography: Extracellular vesicles (EVs) from cell culture conditioned medium were concentrated using a 100 kDa molecular weight cut-off Amicon concentrator (EMD Millipore, Cat. No. UFC910096) and plasma EVs were concentrated by PEG-based precipitation. Concentrated samples (0.5 and 2 ml, as required) were loaded onto SEC columns (IZON qEV Original, 35 nm and IZON qEV2, 35 nm, for conditioned medium and plasma, respectively). The excluded volume and up to 25 fractions (0.5 and 2 ml, respectively) were collected and the protein concentration (A280 absorbance) of each fraction was measured using a NanoDrop2000 spectrophotometer (Thermo Fisher Scientific).

[0130] EV isolation from conditioned medium: Conditioned medium (120 ml) was collected on the BrainXell from induced pluripotent stem cells (iPSCs) differentiated into cortical neurons according to the BrainXell protocol. HEK293 cells were grown in 10% FBS-containing medium; upon reaching 50-70% confluency, cells were thoroughly washed and transferred into EV-free serum-free basal medium and incubated for a further 48 h. The medium was collected and clarified by two centrifugation rounds (10 min at 3000 g), and EVs were collected by ion exchange chromatography on DEAE Sephadex A-50 (20 mL) as previously described (Kosanovic et al. 2017, Biotechniques 63:65-71). After washing off unbound material with equilibration buffer (0.05 M Tris-HCl, pH 7.6), weakly bound proteins and EVs were eluted with a step gradient of 0.25 and 0.5 M NaCl in 0.05 M Tris-HCl, pH 7.6. Finally, the EV-containing high-salt fraction was concentrated and buffer-exchanged into PBS in a 100 kDa molecular weight cut-off spin filter (Amicon).

[0131] Detergent treatment of crude EV fractions: Crude EV fractions generated by PEG-based precipitation (plasma EVs) or by centrifugation / ultrafiltration through 100 kDa molecular weight cut-off spin filters (Amicon) were supplemented with Triton X100 to a final concentration of 2% and incubated for 1–2 h at room temperature or 30 min at 50°C.

[0132] For RNA isolation, SEC fractions 2-5 and 8-13 were pooled and concentrated using a 100 kDa molecular weight cut-off Amicon filtration device. RNA was isolated using the miRNeasy Serum / Plasma Kit (Qiagen, Cat. No. 217184) according to the manufacturer's instructions with modifications (treated with RNase-free DNase). cDNA was generated using the SuperScript™ VILO™ cDNA Synthesis Kit (Cat. No. 11754050) according to the manufacturer's instructions. qPCR was performed using Taqman kits and primers (Thermo Fisher Scientific, Cat. Nos. 44-445-56 and 4331182; see Table 1 below for specific primers). [Table 2]

[0133] Lipidomic analysis: Lipidomic profiling was performed by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MSMS). Lipid extracts were prepared using a modified Bligh and Dyer method (Bligh and Dyer 1959), followed by addition of appropriate internal standards and analysis using an Agilent 1260 Infinity HPLC integrated with an Agilent 6490A QQQ mass spectrometer controlled by Masshunter V7.0 (Agilent Technologies, Santa Clara, CA). Glycerophospholipids and sphingolipids were separated by normal phase HPLC (Chan et al. 2012) at 25°C using an Agilent Zorbax Rx-Sil column (2.1x100mm, 1.8um) maintained as follows: mobile phase A (chloroform:methanol:ammonium hydroxide, 89.9:10:0.1, v / v) and mobile phase B (chloroform:methanol:water:ammonium hydroxide, 55:39:5.9:0.1, v / v); 95% A for 2 min, linearly reduced to 30% A over 18 min, and further reduced to 25% A over 3 min, then returned to 95% over 2 min and held for 6 min. Separation of sterols and glycerolipids was performed on a reversed-phase Agilent Zorbax Eclipse XDB-C18 column (4.6x100mm, 3.5um) using an isocratic mobile phase of chloroform, methanol, 0.1M ammonium acetate (25:25:1) at a flow rate of 300μl / min.

[0134] Appropriate internal standards: PA 14:0 / 14:0, PC 14:0 / 14:0, PE 14:0 / 14:0, PG 15:0 / 15:0, PI 17:0 / 20:4, PS 14:0 / 14:0, BMP 14:0 / 14:0, APG 14:0 / 14:0, LPC 17:0, LPE 14:0, LPI 13:0, Cer d18:1 / 17:0, SM d18:1 / 12:0, dhSM d18:0 / 12:0, GalCer d18:1 / 12:0, GluCer d18:1 / 12:0, LacCer d18:1 / 12:0, D7-cholesterol, CE 17:0, MG 17:0, 4ME 16:0 diether Quantification of lipid species was performed by multiple reaction monitoring transitions (Hsu et al. 2004; Guan et al. 2007; Chan et al. 2012) using both positive and negative ionization modes with DG, D5-TG 16:0 / 18:0 / 16:0 (Avanti Polar Lipids, Alabaster, AL) as the standard. Lipid levels in each sample were calculated as the sum of the total molar amounts of all lipid species measured by all three LC-MS methods, normalized to mole % and presented as the mean mole % ± SEM.

[0135] Example 1. EV capture and detection method using Luminex technology allows specific detection of surface EV proteins in low volume plasma samples. Antibody-conjugated beads for intact exosome Luminex (IEL) were generated as described above for the following surface antigens: CD9, a canonical EV marker, CD68, a macrophage marker, the purinergic receptor P2RY12, a microglial marker, and the neuronal marker axonal protein GAP43. These and other capture antibodies are listed in Table 2. [Table 3]

[0136] Increasing concentrations of antibody (0.5, 1.0, 2.0 and 5.0 μg / 10 6 The beads were loaded using 100 µL of ...6 Loading was assessed by staining with increasing concentrations of PE-labeled secondary antibodies (as detailed in Table 3 below) and measurement on a Luminex 200 reader (Thermo Fisher Scientific). [Table 4]

[0137] Figure 1A shows anti-CD9 loading as an example. The figure shows the mean fluorescence intensity (MFI) obtained for loading of CD9 antibody onto capture beads using staining with increasing amounts of PE-labeled anti-mouse antibody (labeled as "detection antibody" in the figure) at various loading concentrations (μg / 10 6 For further bead preparations, as antibody concentration increases, the MFI increases proportionally to the antibody concentration, but not to saturation levels (1 μg / 10 for CD9). 6 For beads, 1-5 μg / 10 for other antibodies 6 The upper end of the linear range was chosen to be less than 100 μm (beads).

[0138] Beads generated by this protocol were tested for EV capture in unprocessed serially diluted plasma samples from healthy donors (BioIVT). Detection was performed with a cocktail of biotinylated detection antibodies against standard EV markers: tetraspanin surface antigens CD9, CD63 and CD81 (hereafter referred to as pan-TSPN; see Table 2 for detection antibodies). Analysis of two random unprocessed plasma samples with beads conjugated to antibodies against specific targets showed a strong dose-dependent signal (1 / 5 to 1 / 1 plasma dilution, Figure 1B, 1C) in the linear assay range of 10-50 μl plasma input. In contrast, beads supplemented with non-specific IgG (negative control) generated a much lower signal, which may be due to non-specific binding (Figure 1B, 1C). To further evaluate assay specificity, EV depletion using PEG-based precipitation was performed (essentially as described in Rider et al., Sci Rep 6, 23978 (2016)) or detergent lysis (Triton X100, 2% final concentration) prior to analysis. As shown in Figure 1D, the signal was significantly reduced in both cases, indicating that the signal can be specifically attributed to plasma EVs.

[0139] Finally, medium conditioned by HEK293 cells overexpressing the neural antigens, GAP43 or CD171 (see Methods) was analyzed as described above using beads loaded with anti-CD171 or anti-GAP43 antibodies (Figure 1E and 1F). As can be seen in Figure 1E, EVs from medium conditioned by GAP43-overexpressing cells were detected with GAP43-specific, but not CD171-specific beads. Conversely, CD171-positive EVs were detected by IEL beads loaded with CD171 but not with GAP43 antibodies (Figure 1F), thus demonstrating the specificity of detection. Taken together, these results show that the newly developed method detects both EV-specific and antigen-specific signals in extremely small amounts of untreated human plasma or conditioned medium.

[0140] Example 2. Specific EV pulldown with minimal cross-population contamination. EVs of different cellular origin were then captured using IEL beads conjugated with appropriate capture antibodies and detected using the pan-TSPN cocktail or specific detection antibodies against GAP43, CD171, CD68 and CD235 (see Table 2 for specific antibodies) essentially as described in Example 1. The results are shown in Figures 1G-1J. As can be seen in Figure 1G, EVs captured with IEL beads supplemented with an antibody against the neuronal marker GAP43 generated a strong positive signal when detected with the pan-TSPN cocktail, GAP43 antibody and an antibody against another neuronal marker CD171, but did not generate a positive signal with antibodies against macrophage (CD68) and erythrocyte (CD235) markers. Similarly, EVs captured with CD171 IEL beads were detectable by the pan-TSPN cocktail, GAP43 antibody and CD171 antibody. The slight positivity with CD68 detection is consistent with the low level CD171 expression on monocytes (Figure 1H). Conversely, EV populations generated by CD235 IEL bead pulldown were detectable only with pan-TSPN and CD235 antibodies, with no positivity for GAP43, CD171, or CD68 (Figure 1I). Finally, CD68-positive EVs were detectable with a strong signal using the pan-TSPN cocktail and CD68 antibodies, and to a lesser extent with the CD171 antibody (Figure 1J). Thus, the results demonstrate remarkable specificity in the capture and detection of specific EV populations, including erythrocyte-derived EVs and neuron-derived EVs, characterized by minimal cross-reactivity with antigens specific for non-blood cell types.

[0141] Example 3. Multiplexed measurement of EV surface proteins. One of the greatest advantages of the Luminex platform is its multiplexing capabilities. Multiplexed and single-bead IEL assays were compared against multiple tissue-specific and canonical EV surface markers, including the erythrocyte marker CD235 (glycophorin A), macrophage marker CD68, EV markers CD9 and CD81, and neuronal markers GAP43 and CD171. Results are shown in Tables 2A-2G. Capture was performed with Luminex beads containing the indicated antibodies, and detection was performed with pan-TSPN. As can be seen in Figure 2A, measurements at serial plasma dilutions showed a dose-dependent signal decline for each analyte, with a clear linear range from 10 to 50 μl. To assess the possible loss of sensitivity due to multiplexing, dose / response curves were generated for each analyte in both single-bead and multiplexed assays. As can be seen in Figures 2B-2F, both types of assays generated similar curves within their linear range. As can be seen in Figure 2G, measurements of CD9 in 25 random plasma samples yielded significant differences between individual donors, but strong correlations between single-bead and multiplexed assays. Thus, the results demonstrate the utility of the newly developed method for multiplexed measurements.

[0142] The assay was further tested and validated to be useful for multiplexed capture of EVs of various additional tissue origins. The targets for the various capture antibodies used are summarized in Table 4 below. [Table 5]

[0143] Example 4. Exosomes of different cellular origins exhibit unique characteristic tetraspanin profiles. Next, we investigated possible differences in distinct surface protein expression by EVs of various cellular origins using multiple combinations of IEL capture beads with detection antibodies. The results are shown in Tables 3A-3F. As can be seen from Figure 3A, when GAP43 (neuron-specific) IEL beads were used for detection in combination with a pan-TSPN antibody cocktail or GAP43 antibody, similar signal intensities were recovered, indicating that most GAP43 is bound to EVs. When GAP43 IEL beads were applied with CD9, CD63, or CD81 detection antibodies, the signal generated with the CD9 antibody was extremely weak (Figure 3B), suggesting that CD63 and CD81 are the two main tetraspanins expressed by GAP43-positive neural EVs. Interestingly, a lower CD9 signal, compared to the signals for CD63 and CD81, was also generated by EVs derived from neuronal cultures, but not by HEK293 cells (Figures 7D-7F). When using CD235 IEL beads to capture erythroid EVs, only the CD9 detection antibody produced a strong dose-dependent signal, whereas the CD63 or CD81 detection antibodies did not (Figure 3C), suggesting that the majority of CD235-positive EVs contain low levels of CD63 and CD81, but high levels of CD9. This correlation between surface markers and EV types suggests that the differences in IEL signal may be due to differences in surface presentation of specific antigens by different EV types, rather than differences in antibody affinity. Furthermore, EVs captured by CD9 IEL beads were positive for CD235, but not GAP43 (Figure 3D), and EVs positive for CD63 or CD81 did not contain detectable CD235, but produced a weak signal with the GAP43 detection antibody (Figure 3E,F). These results indicate that the methods disclosed herein are useful for characterizing EV surface protein composition, for evaluating the coexistence of markers on the EV surface, and, potentially, for identifying EV subpopulations. Indeed, the data presented herein unexpectedly reveal a wide variety of unique characteristic profiles of EVs depending on their cellular origin, which is a useful tool in the utilization of EVs as diagnostics.

[0144] Example 5. Intact EV Luminex coupled with size-exclusion chromatography reveals cell-specific variations in EV size. Size exclusion chromatography (SEC) of plasma EVs was then performed and the collected fractions were subjected to IEL capture and detection analysis essentially as described in Example 4. To ensure that the signal was generated by EVs, initially only the pan-TSPN cocktail was used for detection. To this end, plasma EVs were concentrated by PEG precipitation (as described in the Methods section above), resuspended in PBS and loaded onto a qEV2 column (IZON Sciences). Several void fractions (negative numbers) and all eluted fractions were analyzed by IEL using beads supplemented with antibodies against CD9, CD235, CD68, GAP43 and CD171. The results are shown in Tables 4A-4E and reveal the size range of EVs detected by the assay.

[0145] As can be seen in Figure 4A, the combination of CD9 IEL beads and the pan-TSPN detection cocktail produced the expected peak that continued from fraction 3 to fraction 9. CD235-positive EVs detected with the pan-TSPN cocktail also peaked in fraction 3 (Figure 4B). Thus, CD9- and CD235-positive EVs cluster in early fractions, consistent with average diameters above 100 nm, e.g., 150-200 nm. Surprisingly, IEL beads specific for the tissue macrophage marker CD68 and for the neuronal markers GAP43 and CD171, detectable with the same pan-TSPN cocktail, produced strong signals in fractions 11-20, 8-15, and 11-20, respectively (Figure 4C-4E), indicating smaller particle sizes, less than 30-50 nm, depending on the fraction.

[0146] To further characterize the origin of these late fraction signals, the analysis was repeated using GAP43 IEL beads followed by detection with a pan-TSPN antibody cocktail or specific TSPN antibodies (Figures 5A-5D). Interestingly, the pan-TSPN antibody cocktail generated a strong positive signal in fractions 7-15 (Figure 5A), whereas detection of CD9 alone produced a weak (<50 mean fluorescence intensity) positive signal in fractions 11-16 (Figure 5B). Both CD63 and CD81 detection generated much stronger signals in fractions 9-15 and 7-16, respectively (Figures 5C, 5D). Consistent with this, CD9 IEL beads combined with GAP43 detection antibodies generated a weak signal in fractions 10-16, whereas CD63 and CD81 IEL beads combined with GAP43 detection antibodies generated signals in fractions greater than 10 (Figures 5E-5G).

[0147] To confirm whether the late fraction signal detectable with the pan-TPSN antibody could be due to EVs, fractions of the crude EV formulation were subjected to detergent treatment to disrupt the binding between EVs and cell type-specific antigens via the cell membrane, following the MISEV guidelines (Welsh et al. 2020, J Extracell Vesicles 9:1713526). Treatment with Triton X100 at 50°C resulted in a marked reduction in signal for both the early, CD9 and CD235 positive fractions (Figures 6A, 6B), and the late, CD63 and GAP43 positive fractions (Figures 6C, 6D). Interestingly, pretreatment at room temperature with the same TX100 concentration resulted in a reduced signal in the early fractions, but not in the late fractions: this relative resistance to TX100 represents an increased lipid raft content within the smaller EV membrane (see Example 6 below for lipid content analysis). Interestingly, IEL beads against CD63 and CD81 generated positive signals dispersed towards the late fractions in addition to the strong peaks in the early fractions (Figure 5F-G). Collectively, the results presented herein demonstrate that plasma-derived EVs of different cellular origins are, unexpectedly, characterized by strikingly different size distribution profiles, with EVs of neural and macrophage origin exhibiting substantially smaller mean particle sizes. Thus, the capture and analysis method disclosed herein unexpectedly allows the elucidation of cell-specific variations in EV size and their diverse characteristic profiles, unique attributes applicable to their diagnostic and therapeutic applications.

[0148] Example 6. SEC late fraction contains EV-specific mRNAs and lipids. Previously, it has been assumed that late SEC fractions contain soluble proteins and not membranous vesicles. Therefore, experiments were performed to investigate whether the IEL signal detected in the late fractions is EV-specific. To this end, RNA and lipid analyses of pooled fractions 2–5 and 8–13 were performed.

[0149] Cell-free mRNA is found in plasma in association with HDL, protein particles and EVs, among which EVs are considered the major mRNA carriers. To obtain sufficient material for quantitative analysis, early (2–5) and late (8–13) fractions were mixed and concentrated using 100 kDa molecular weight cut-off Amicon spin filters prior to RNA extraction. Levels of several cell-specific mRNAs were measured by qPCR using Taqman primers (Table 1 above). Results are shown in Tables 5–7 below. In the tables, values ​​are expressed as qPCR cycle threshold (Ct) and / or as cycle threshold difference (ΔCt) compared to a control transcript (GAPDH, HBB or PF4, respectively).

[0150] The observed approximately two-fold decrease in GAPDH content from early to late fractions (Ct values ​​of 26.33 and 27.34, respectively) suggests higher total RNA levels in the early fraction, which is consistent with the predicted larger EV size and absence of soluble protein; however, the clearly detectable mRNA levels in the late fraction (see Tables 5-7 below) suggest the presence of EVs. In addition, normalization to housekeeping transcripts (GAPDH) showed a two- to four-fold enrichment of neuron-specific transcripts such as neurogranin (NRGN) and orexin (HTCR) as well as oligodendrocyte markers SOX1 and OLIG2 (Late / Early ΔCt ratio (2 ΔCt初期 - ΔCt後期 )). Data for NRGN, SOX1 and OLIG2 are shown in Table 5 below. [Table 6]

[0151] The observed enrichment of neuron-specific mRNAs together with the high abundance of neuron-specific protein markers, such as GAP43 and CD171, in the late SEC fraction, as detected by IELs (see above), indicates a distinct cellular origin of EVs in the late fraction. In addition, the ratio of neuron-specific mRNAs (NRGN) to the mRNA encoding the erythrocyte protein hemoglobin β (HBB) was significantly higher in the late fraction (Table 6), suggesting that in plasma, the majority of EVs of neural origin are in the smaller size range. [Table 7]

[0152] Similar results were obtained when the ratio of neuron-specific mRNA to platelet mRNA was determined (Platelet Factor 4, PF4, Table 7 below): PF4 mRNA was also found predominantly in the early fraction, and the ratio between NRGN and PF4 mRNA was much higher in the late fraction, suggesting a strong enrichment of neural transcripts. [Table 8]

[0153] To further confirm the presence of EVs in the late SEC fractions, pooled and enriched fractions 2-5 and 8-13 were subjected to lipidomics analysis (see Methods). The lipid composition of the late fractions was consistent with published data generated by lipidomics analysis of EV membranes (Skotland et al. 2020, Adv Drug Deliv Rev 159:308-321), where for the major lipid classes (cholesterol, sphingomyelin, phosphatidylcholine and PC ethers, phosphatidylserine, phosphatidylethanolamine, PE ethers, diglycerides, phosphoglycerate, phosphatidic acid, phosphatidylinositol, ceramide and lactoceramide), the measured percentages of total lipid content in the late and early EV fractions were within the ranges previously observed. Interestingly, the early and late SEC fractions also showed notable differences in some lipid content (Table 8 below and Figure 8). [Table 9]

[0154] First, the free cholesterol content in the late fractions was approximately two-fold lower than in the early fractions, suggesting that some cholesterol in these fractions may be sequestered in protein complexes involved in its transport in the circulation. This coincided with a 2-3 fold increase in the amount of phosphatidylethanolamine (PE), PE esters, phosphatidylcholine, and phosphatidylserine (Table 8). On the other hand, the rather large 2.5 fold increase in sphingomyelin (SM) content and the increased SM / cholesterol ratio indicate a higher proportion of lipid raft domains. In addition to the resistance to Triton X100 (TSPN positive signal in the late fraction decreased by 30 min after TX100 treatment of crude plasma EV preparations at 50°C, but not at room temperature as reported above), these observations suggest a higher lipid raft content in the smaller EVs found in the late SEC fraction, consistent with their distinct cellular origin, compared to standard EVs eluted in the early fraction.

[0155] Taken together, these findings further indicate that the signal detected by IELs in the late SEC fraction represents an atypical EV subpopulation rather than a protein contaminant.

[0156] Example 7. IEL analysis of SEC fractions of neuronal conditioned medium detects an EV peak in early fractions. To compare the size distribution of EVs released into plasma and tissue culture media, EVs were isolated from media conditioned with iPSC-derived cortical neurons (BrainXell, see Methods). Conditioned media concentrated by ultrafiltration (see Methods) was subjected to SEC (qEV Original, IZON Sciences), and fractions were collected and analyzed by IEL with beads against GAP43, CD171 and CD235 in combination with pan-TSPN detection (Figures 7A-7C). Consistent with their neuronal origin, EVs in the early fractions were strongly positive for GAP43 and CD171 (Figures 7A, 7B); however, approximately 10% of the total signal was localized to the late fractions. Consistent with neuronal differentiation, CD235 IEL beads did not generate a detectable signal in any fraction (Figure 7C), further confirming the specificity of the method.

[0157] In addition, the same qEV fractions were analyzed using CD9, CD63 and CD81 IEL beads followed by GAP43 antibody detection (Figures 7D-7F). All tetraspanins eluted in a similar pattern, with a larger peak in the early fraction followed by a lower scattered signal in the late fraction, suggesting a predominance of larger EVs in the conditioned medium. The CD63 and CD81 signals in EVs from iPSC-derived neurons were an order of magnitude larger than the signal for CD9, and although the NDE size distribution differed between cell culture and plasma, their TSPN composition remained similar, with a relative paucity of CD9-positive EVs.

[0158] Example 8. Transmission electron microscopy (TEM) observation of EVs in early and late SEC fractions The results described above in Examples 5-7 illustrate the presence of tissue-specific EV populations in the late SEC fractions, previously considered to be substantially EV-free. To further evaluate the vesicles identified in the early and late SEC fractions, TEM analysis was performed on pooled early (2-5) and late (8-13) fractions generated as described in Example 5 (qEV Original, IZON Sciences).

[0159] To this end, pooled EV suspensions were fixed overnight in 1% paraformaldehyde. Then, 4 ml of the suspension was loaded onto glow-discharged copper mesh formvar-coated carbon grids and allowed to adsorb for approximately 30 s. The grids were washed briefly with double-distilled water and subsequently stained with 2.5% aqueous uranyl acetate and allowed to dry completely before imaging. The grids were imaged using an EFI Morgani transmission electron microscope (FEI, Hillsboro, OR) operated at 80 kV and equipped with a Nanosprint5 CMOS camera (AMT, Woburn, MA). The results are shown in Figure 9A-9B (three representative images from each sample for early and late fractions, respectively).

[0160] TEM analysis revealed spherical nanoscale particles in both samples, as seen in Figures 9A-9B. Figure 9A shows EVs with diameters of 75-125 nm in fractions 2-5. Figure 9B illustrates smaller particles in fractions 8-14, less than 30 nm in diameter, ranging from 15-25 nm.

[0161] Taken together, these findings provide visual proof that the signal detected by IELs in the late SEC fraction represents intact EVs, not surface antigen fragments or other artifacts. Furthermore, these results revealed a previously unknown neuron-specific EV population derived from plasma samples, herein named "nanoEVs", with a distinct size distribution profile of remarkably small diameters. Without wishing to be bound by a particular theory or mechanism of action, the properties of these newly identified EVs found herein, including their small size, membrane lipid composition, and tissue specificity, may provide useful advantages for therapy and diagnosis.

[0162] Example 9. Multiplexed measurement of synaptic proteins on the surface of plasma-derived EVs An IEL assay was used in this experiment for the multiplexed measurement of synaptic proteins on the surface of EVs derived from plasma samples. The IEL beads were supplemented with antibodies against the following five synaptic markers: glutamate receptor subunit 2 (GluR2), neurogranin (NRGN), growth-associated protein 43 (GAP43), postsynaptic density 95 (PSD95) and syntaxin-1 (STXN-1). In addition, when multiple samples with diverse EV content are compared, for normalization purposes, IEL beads supplemented with an antibody against the standard EV marker CD63 were added to the assay. In this experiment, the levels of CD63 were used for normalization. However, various alternative normalization methods may be used. For example, the levels of the analyzed synaptic proteins may be normalized to the sample volume, the amount of protein, and / or the concentration of EVs. The levels of the analyzed synaptic proteins may also be normalized to other standard EV markers, such as CD9, CD81, or a combination of such markers. The levels of the analyzed synaptic proteins may also be normalized to a general neuronal marker, such as CD171.

[0163] Assays were performed in three formats: singleplex (1-plex) - using capture beads for individual analytes; 6-plex - using a combination of capture beads for all six analytes; and 3-plex, where two parallel assays with two capture bead combinations (NRGN+GAP43+CD63 and GluR2+PSD95+STXN-1) were performed. Bead combinations for the 3-plex assays were selected based on signal intensity determined in the 1-plex assay. Detection in all experiments was performed with a pan-TSPN antibody cocktail. Results are summarized in Tables 10A-10F.

[0164] For all analytes measured, clearly detectable concentration-dependent signals were generated for all assay formats, i.e., 1-plex, 3-plex and 6-plex. For some analytes, the signal strength was reduced in the 6-plex format compared to 1-plex and 3-plex. However, even when the signal was reduced it was still clear and significant, again demonstrating the multiplexing capability of the assay. Of note, the strong positive signals obtained with this assay were achieved using extremely small volumes of plasma sample, up to 15 ml.

[0165] Example 10. Quantitative measurement Concentrated purified EVs released from cultured glioblastoma cells (U87 cell line, ATCC) were used to generate a dilution curve as follows: EVs were purified from cultured cells and protein content (mg / ml) was measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific). Purified EVs were serially diluted and subjected to IEL assays to measure EV markers: CD9, CD81 and CD63 (Figures 11A, 11B and 11C, respectively). For detection, EV markers were measured in singleplex (1-plex) and multiplexed (M-plex) formats (see above) using appropriate capture beads and a pan-TSPN antibody cocktail. In addition, non-specific control beads modified with an isotype control antibody (IgG) were added to the assay (Figure 11D). In agreement with published data, U87-derived EVs presented high levels of CD63, lower levels of CD9 and very little amount of CD81. All three markers showed clear dilution linearity, thus indicating that a linear dependence can be established between the levels of EV surface markers and EV proteins, and therefore between EV surface marker levels and particle number. Such linear correlations can be subsequently used to generate standard curves to normalize synaptic marker signals per particle number in plasma samples.

[0166] Example 11. Internal specific controls Capture beads modified with a non-specific isotype control antibody (IgG) were incorporated into the IEL assay and the assay was performed essentially as described in Example 9. To ensure the absence of antibody interference, singleplex (1-plex) and multiplexed (3-plex) measurements were compared. For each analyte, including CD63 (Figure 12A), GAP43 (Figure 12B), GluR2 (Figure 12C), PSD95 (Figure 12D) and NRGN (Figure 12E), the specific signal exceeded the non-specific signal (IgG) by at least 10-fold. To take into account possible matrix effects, such an internal control was incorporated into all samples and the non-specific background could be subtracted from all analytes.

[0167] conclusion In summary, disclosed herein are highly specific, sensitive and accurate methods and assays that allow for the simultaneous identification and evaluation of EV surface markers, the analysis requires only small volumes (≦50 μl) of plasma sample and is directly applied to unprocessed plasma without the need for EV precipitation or other pre-concentration steps. The methods and assays disclosed herein allow for the characterization of specific EV populations, revealing unique molecular and physicochemical properties and allowing for the differentiation between various EV populations in plasma. In particular, and unexpectedly, it was discovered that certain circulating EV populations, such as those of neuronal and macrophage origin, are extremely small compared to the previously assumed average EV size and compared to other EV populations, such as erythrocyte-derived EVs. These EV populations can be differentiated by their tetraspanin profile as well as by their mRNA and lipid content. Moreover, EV populations of neural origin from blood circulation were similar in terms of their marker profile to EVs obtained from cultured neurons, but were notably and unexpectedly smaller than those obtained in culture. These newly identified properties may confer additional advantages in the isolation and characterization of specific EV populations of different origins that are useful for diagnosis and therapy.

[0168] In addition, the methods and assays disclosed herein allow for the simultaneous capture and analysis of multiple synaptic proteins using extremely small amounts of plasma sample. The methods and assays disclosed herein provide easier and more accurate measurement of synaptic proteins in plasma samples, which is extremely useful for clinical diagnostic purposes.

[0169] The foregoing description of specific embodiments fully reveals the general nature of the present invention, such that others, by applying current knowledge, can easily improve and / or adapt such specific embodiments for various applications without undue experimentation and without departing from the general concept, and therefore such adaptations and improvements should and are intended to be interpreted as falling within the meaning and scope of equivalents of the disclosed embodiments. It should be understood that the expressions or terms used herein are for the purpose of description and not for the purpose of limitation. The means, materials, and steps for implementing the various disclosed chemical structures and functions may take various alternative forms without departing from the present invention.

Claims

1. 1. An extracellular vesicle (EV) preparation comprising a substantially purified population of EVs derived from cells other than blood cells and separated from serum or plasma, the population characterized by a particle size distribution of 10 to 25 nanometers (nm) in diameter and a marker profile corresponding to differentiated solid tissue cells.

2. The EV formulation of claim 1, wherein the EVs are 15 to 25 nm in diameter, 15 to 20 nm in diameter, or 10 to 15 nm in diameter.

3. The EV preparation of claim 1, wherein the EVs comprise at least one type of EVs selected from neural-derived EVs and tissue macrophage-derived EVs.

4. The EV preparation according to claim 2, wherein the EVs include neural-derived EVs and tissue macrophage-derived EVs.

5. the EV population is characterized by surface display of GAP43, CD171 and / or CD68, and a lack of CD235a; or 2. The EV formulation of claim 1, wherein the EV population is characterized by a membrane lipid composition according to Table A.

6. 6. The EV preparation of any one of claims 1 to 5, wherein the EVs are obtained from a plasma or serum sample by a process comprising a size selection of EVs having a diameter of less than 30 nanometers (nm) and a separation step of the selected EVs.

7. the size selection step comprises at least one of size exclusion chromatography, nanoporous membrane filtration, deterministic lateral displacement sorting, electrophoretic isolation, acoustic fractionation and differential centrifugation; and / or 7. The EV formulation of claim 6, wherein the process further comprises immunoaffinity purification and / or analysis of the EV population.

8. The EV formulation of claim 1, wherein the EV is loaded with an exogenous cargo.

9. the exogenous cargo is a therapeutic agent, and / or The EV formulation of claim 8, wherein the EV is modified to present a targeted drug.

10. the purity of the EV population is pharmaceutical grade; and / or 10. The EV formulation of claim 9, wherein the formulation is formulated in the form of a pharmaceutical composition and further comprises a pharmaceutically acceptable carrier, excipient or diluent.

11. EV formulation according to claim 10 for use in therapy.

12. 12. The EV formulation for use according to claim 11, wherein the treatment comprises delivery of an exogenously loaded therapeutic agent to a target cell or tissue of a subject in need thereof.

13. EV formulation according to claim 10 for use in diagnosis.

14. 1. A method for analyzing a plasma or serum sample, comprising: a. providing a capture system comprising at least three distinct populations of fluorescently labeled magnetic microspheres, each microsphere population displaying an antibody against a distinct target on the surface of an EV population of distinct cellular origin; b. Providing 1-75 μl of unprocessed plasma or serum sample, or a corresponding amount of intact EV; c. Incubating the sample with the capture system under conditions that allow specific antigen-antibody binding while substantially maintaining the integrity of the EV membrane, thereby providing distinct populations of EV-microsphere complexes corresponding to each target; d. Washing the EV-microsphere complexes using a magnetic device under conditions that allow for selective capture of the complexes; e. Incubating the EV-microsphere complexes with multiple detection antibodies under conditions that allow specific antigen-antibody binding while substantially maintaining the integrity of the EV membrane, each antibody directed against a distinct marker on the surface of the EV, and the multiple detection antibodies labeled with the same fluorescent marker; f. Washing the resulting labeled complex using a magnetic device to remove excess reagent; g. subjecting the resulting complexes to a microfluidic device capable of simultaneous detection and quantification of fluorescence emission at multiple wavelengths, thereby quantifying the fluorescence emission levels and providing separate assessments of the levels of the surface target corresponding to each of the EV populations; h. comparing the quantified level to a control level; The method is carried out using reagents and conditions that preserve the EVs in a substantially intact form.

15. The method of claim 14, wherein the plurality of detection antibodies are directed against a plurality of diagnostic markers.

16. The method of claim 15, wherein the plurality of detection antibodies are directed against a plurality of tetraspanin markers.

17. the tetraspanin marker is selected from the group consisting of CD9, CD63 and CD81, and 17. The method of claim 16, wherein the plurality of detection antibodies comprises an antibody against CD9, an antibody against CD63, and an antibody against CD81.

18. 15. The method of claim 14, wherein the targets of the capture system include at least one neuronal target and targets corresponding to at least two additional cellular origins selected from the group consisting of bone, lung, tissue macrophages, lung macrophages, muscle, adipocytes, epithelium, endothelium, monocytes, microglia, megakaryocytes, T cells, erythrocytes, liver, and oligodendrocytes.

19. 19. The method of claim 18, wherein at least one neuronal target is selected from GAP43 and CD171, and at least two additional targets include P2RY12 and CD68, and optionally further include CD235a.

20. 15. The method of claim 14, wherein the capture system comprises at least four, at least five, at least six, at least seven, or at least eight distinct fluorescently labeled magnetic microsphere populations, each microsphere population displaying an antibody against a distinct target on the surface of an EV population of distinct cellular origin.

21. The method of claim 14, wherein 1 to 50 μl of untreated plasma or serum is provided.

22. 1. A method for simultaneously measuring multiple synaptic proteins in a plasma or serum sample, comprising: a. Providing an unprocessed plasma or serum sample containing extracellular vesicles (EVs); b. Providing a capture system comprising at least two populations of fluorescently labeled magnetic microspheres, each microsphere population displaying an antibody against a distinct EV surface target, the antibody being labeled with a distinct fluorescent label, the target being a synaptic protein; c) incubating the plasma or serum sample with the capture system under conditions that allow specific binding between the antibody and the target synaptic protein while substantially maintaining the integrity of the EV membrane, thereby providing distinct populations of EV-microsphere complexes corresponding to each synaptic protein; d. Washing the EV-microsphere complexes using a magnetic device under conditions that allow for selective capture of the complexes; e. Providing multiple detection antibodies against multiple standard EV surface markers, wherein the multiple detection antibodies are directly or indirectly labeled with the same fluorescent label; f) incubating the captured EV-microsphere complexes with the plurality of detection antibodies under conditions that allow specific binding between the antibodies and EV surface markers while substantially maintaining the integrity of the EV membrane; g. Washing the resulting labeled complex using a magnetic device to remove excess reagent; and h) subjecting the labeled complex to a microfluidic device capable of simultaneous detection and quantification of fluorescence emission at multiple wavelengths, and quantifying the fluorescence emission levels to provide separate assessments of the levels of synaptic proteins corresponding to each of the EV populations.

23. the synaptic protein is selected from the group consisting of glutamate receptor subunit 2 (GluR2), neurogranin (NRGN), growth-associated protein 43 (GAP43), postsynaptic density 95 (PSD95) and syntaxin-1 (STXN-1); or 23. The method of claim 22, wherein the canonical EV surface marker is a tetraspanin.

24. 24. The method of claim 23, wherein the plurality of detection antibodies comprises at least three antibodies, each directed against a different tetraspanin.

25. 25. The method of claim 24, wherein the plurality of detection antibodies comprises an antibody against CD9, an antibody against CD63, and an antibody against CD81.

26. the capture system further comprises a population of fluorescently labeled magnetic microspheres displaying a non-specific control antibody, and providing an assessment of the level of the synaptic protein comprises normalizing the specific signal determined for each synaptic protein relative to the non-specific control signal; or the method is performed on 1 to 75 ml of plasma or serum; or the plasma or serum sample is from a subject suspected of having a neuropathy; or 23. The method of claim 22, wherein the plasma or serum sample is from a subject suspected of having a neurodegenerative disease.

27. A kit for analyzing EV from a blood-derived sample, comprising: i) a capture system comprising a first population of magnetic microspheres displaying antibodies against GAP43 or CD171 and labeled with a first fluorophore combination, a second population of magnetic microspheres displaying antibodies against CD68 and labeled with a second fluorophore combination, and a third population of magnetic microspheres displaying antibodies against P2RY12 and labeled with a third fluorophore combination; ii) a plurality of detection antibodies, each antibody directed against a distinct tetraspanin marker, said plurality of detection antibodies being directly or indirectly labeled with the same fluorescent marker; and optionally iii) reagents for carrying out said assessment under conditions that keep the EV substantially intact.

28. A kit for analyzing EV from a blood-derived sample, comprising: i) a capture system comprising at least two populations of fluorescently labeled magnetic microspheres, each microsphere population displaying an antibody against a distinct EV surface target, the antibody being labeled with a distinct fluorescent label, said target being a synaptic protein selected from the group consisting of GluR2, NRGN, GAP43, PSD95 and STXN-1; ii) multiple detection antibodies against multiple standard EV surface markers, the multiple detection antibodies being directly or indirectly labeled with the same fluorescent label; and optionally iii) reagents for carrying out the assay under conditions that keep the EVs substantially intact.

29. 29. The kit of claim 27 or 28, wherein the reagent is selected from the group consisting of: (i) at least one binding buffer characterized by the absence of detergents and the presence of protease and / or phosphatase inhibitors for incubating the sample with said capture system, thereby providing a distinct population of EV-microsphere complexes; (ii) at least one wash buffer characterized by a significantly elevated salt concentration compared to at least one binding buffer; and (iii) At least one binding buffer and at least one wash buffer as defined in (i) and (ii) above.

Images