Extracellular vesicle compositions and uses thereof

Abstract

The present disclosure relates to compositions comprising a solid surface and two or more capture agents that bind to extracellular vesicles (EVs) for capturing EVs derived from cells of the nervous system. The present disclosure further relates to methods or uses of such compositions for treating, diagnosing and / or assessing the likelihood of a subject suffering from a neurodegenerative disease.

Description

[0001] EXTRACELLULAR VESICLE COMPOSITIONS AND USES THEREOF

[0002] FIELD

[0003] The present disclosure relates to compositions for capturing extracellular vesicles (EVs) and uses thereof. The present disclosure further relates to methods of treating, diagnosing and / or assessing the likelihood of a subject suffering from a neurodegenerative disease.

[0004] BACKGROUND

[0005] Extracellular vesicles (EVs) are membrane vesicles released from a cell, and include exosomes, microvesicles and apoptotic bodies. EVs are categorised into their sub-groups based on their cellular and molecular origins. Exosomes and microvesicles are released from cells and carry a multitude of cargo such as proteins, nucleic acids such as DNA and RNA, and lipids. Upon binding to their recipient cells, EVs release their cargo which can then lead to downstream signalling effects akin to a transfer of information. Thus, EVs are thought to mediate inter-cellular communication and believed to play a critical role in many normal biological processes.

[0006] Isolation of EVs from cells or tissues and characterization of their associated molecular cargo has the potential to inform and expand our knowledge on how this form of inter-cellular communication works. As EVs are also thought to contain a molecular signature from their tissue of origin, some studies have suggested that analysing the cargo of EVs released from pathological tissue could be used to diagnose the underlying disease. The lack of standardized methods for efficient and high-throughput isolation and analysis of EVs, however, has limited their use and clinical application.

[0007] The gold-standard of EV isolation and purification involves differential ultracentrifugation and density gradient ultracentrifugation. However even these methods suffer from reproducibility issues from run-to-run, with inconsistent yields and purity observed even when using the same isolation protocols. Further, generation of cell debris has been reported presumably from the high speeds utilised in ultracentrifugation. Others have utilised size exclusion chromatography and ultrafiltration, however these methods are not compatible with pathology laboratory workflows, requiring long processing times and outputting poor yield and purity.

[0008] Improved compositions and methods for capturing and / or isolating EVs are needed.

[0009] SUMMARY OF INVENTION

[0010] The inventors of the present disclosure have identified a composition for capturing EVs derived from the nervous system such as brain derived EVs (bdEVs). This is a significant advance in the art because they have shown for the first time that capture compositions comprising two or more capture agents can bind, and therefore be used, to specifically capture and / or extract EV subpopulations, such as bdEVs. The inventors’ findings further evidence that EVs can be captured from specific tissue / cells such as neurons. The inventors’ findings are an important advance in the art because, for example, they underpin new applications such as using bdEVs to distinguish diseased from healthy tissue, in particular in the context of neurological disorders.

[0011] The findings by the inventors underpin a multivalent affinity system that can preferentially bind nervous system EVs over other EVs that are present in a sample. Accordingly, in an embodiment, the present disclosure is directed to a solid surface-based (e.g., a bead) immunoaffinity system that targets surface-associated EV epitopes to capture and / or enrich particular subpopulations of EVs.

[0012] In another embodiment, the present disclosure provides a composition for capturing extracellular vesicles (EVs) in a sample, comprising: (i) a solid surface; and (ii) two or more capture agents that bind to extracellular vesicles (EVs) derived from cells of the nervous system, wherein the two or more capture agents are attached to the solid surface.

[0013] In an example, the composition comprises at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 10 or more capture agents attached to the solid surface.

[0014] In one example, the composition comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38 capture agents attached to the solid surface.

[0015] In one example, the composition comprises 2 to 20 capture agents attached to the solid surface. In one example, the composition comprises 3 to 20 capture agents attached to the solid surface. In one example, the composition comprises 4 to 20 capture agents attached to the solid surface. In one example, the composition comprises 5 to 20 capture agents attached to the solid surface. In one example, the composition comprises 2 to 15 capture agents attached to the solid surface. In one example, the composition comprises 3 to 15 capture agents attached to the solid surface. In one example, the composition comprises 4 to 15 capture agents attached to the solid surface. In one example, the composition comprises 5 to 15 capture agents attached to the solid surface. In one example, the composition comprises 2 to 10 capture agents attached to the solid surface. In one example, the composition comprises 3 to 10 capture agents attached to the solid surface. In one example, the composition comprises 4 to 10 capture agents attached to the solid surface. In one example, the composition comprises 5 to 10 capture agents attached to the solid surface.

[0016] In one example, the composition comprises 2 to 10 capture agents that bind to EVs derived from neurons. In one example, the composition comprises 2 to 5 capture agents that bind to EVs derived from neurons. In one example, the composition comprises 2 to 10 capture agents that bind to EVs derived from microglia. In one example, the composition comprises 2 to 5 capture agents that bind to EVs derived from microglia. In one example, the composition comprises 2 to 10 capture agents that bind to EVs derived from oligodendrocytes. In one example, the composition comprises 2 to 5 capture agents that bind to EVs derived from oligodendrocytes. In one example, the composition comprises 2 to 10 capture agents that bind to EVs derived from astrocytes. In one example, the composition comprises 2 to 5 capture agents that bind to EVs derived from astrocytes. In one example, the composition comprises 2 to 10 capture agents that bind to EVs derived from Schwann cells. In one example, the composition comprises 2 to 5 capture agents that bind to EVs derived from Schwann cells. In one example, the composition comprises 2 to 10 capture agents that bind to EVs derived from ependymal cells. In one example, the composition comprises 2 to 5 capture agents that bind to EVs derived from ependymal cells.

[0017] In one example, the cells of the nervous system are from the central nervous system or the peripheral nervous system. In one example, the cells are brain cells.

[0018] In one example, the cells of the nervous system are selected from the group consisting of: neuron, microglia, oligodendrocyte, astrocyte, Schwann cell, and ependymal cell.

[0019] In another example, the two or more capture agents bind to EVs derived from a cell selected from the group consisting of: neuron, microglia, oligodendrocyte, astrocyte, Schwann cell, and ependymal cell.

[0020] In another example, the two or more capture agents bind to EVs derived from a cell selected from the group consisting of: neuron, microglia, oligodendrocyte, astrocyte.

[0021] In one example, the two or more capture agents bind to EVs derived from one or more cells selected from the group consisting of: neuron, microglia, oligodendrocyte, astrocyte, Schwann cell, ependymal cell, and combinations thereof.

[0022] In another example, the two or more capture agents bind to EVs derived from one or more cells selected from the group consisting of: neuron, microglia, oligodendrocyte, astrocyte, and combinations thereof.

[0023] With the present disclosure in hand, those of skill in the art will envisage the capability to prepare compositions which target certain population(s) of bdEVs. For example, compositions of the disclosure encompass compositions which capture bdEVs derived from neurons and other types of brain cells. Accordingly, in one example, the two or more capture agents bind to EVs derived from neurons. In one example, the two or more capture agents bind to EVs derived from microglia. In one example, the two or more capture agents bind to EVs derived from oligodendrocytes. In one example, the two or more capture agents bind to EVs derived from astrocytes. In one example, the two or more capture agents bind to EVs derived from Schwann cells. In one example, the two or more capture agents bind to EVs derived from ependymal cells.

[0024] In another example, the two or more capture agents bind to EVs derived from neurons and microglia. In one example, the two or more capture agents bind to EVs derived from neurons and oligodendrocytes. In one example, the two or more capture agents bind to EVs derived from neurons and astrocytes. In one example, the two or more capture agents bind to EVs derived from neurons and Schwann cells. In one example, the two or more capture agents bind to EVs derived from neurons and ependymal cells. In one example, the two or more capture agents bind to EVs derived from microglia and oligodendrocytes. In one example, the two or more capture agents bind to EVs derived from microglia and Schwann cells. In one example, the two or more capture agents bind to EVs derived from microglia and ependymal cells. In one example, the two or more capture agents bind to EVs derived from oligodendrocytes and astrocytes. In one example, the two or more capture agents bind to EVs derived from oligodendrocytes and astrocytes. In one example, the two or more capture agents bind to EVs derived from oligodendrocytes and Schwann cells. In one example, the two or more capture agents bind to EVs derived from oligodendrocytes and ependymal cells. In one example, the two or more capture agents bind to EVs derived from astrocytes and Schwann cells. In one example, the two or more capture agents bind to EVs derived from astrocytes and ependymal cells. In one example, the two or more capture agents bind to EVs derived from Schwann cells and ependymal cells.

[0025] In one example, the two or more capture agents bind to EVs derived from neurons, microglia and oligodendrocytes. In one example, the two or more capture agents bind to EVs derived from neurons, microglia and astrocytes. In one example, the two or more capture agents bind to EVs derived from neurons, microglia and Schwan cells. In one example, the two or more capture agents bind to EVs derived from neurons, microglia and ependymal cells. In one example, the two or more capture agents bind to EVs derived from neurons, oligodendrocytes and astrocytes. In one example, the two or more capture agents bind to EVs derived from neurons, oligodendrocytes and Schwann cells. In one example, the two or more capture agents bind to EVs derived from neurons, oligodendrocytes and ependymal cells. In one example, the two or more capture agents bind to EVs derived from neurons, astrocytes and Schwann cells. In one example, the two or more capture agents bind to EVs derived from neurons, astrocytes and ependymal cells. In one example, the two or more capture agents bind to EVs derived from neurons, Schwann cells and ependymal cells. In one example, the two or more capture agents bind to EVs derived from microglia, oligodendrocytes, and astrocytes. In one example, the two or more capture agents bind to EVs derived from microglia, oligodendrocytes, and Schwann cells. In one example, the two or more capture agents bind to EVs derived from microglia, oligodendrocytes, and ependymal cells. In one example, the two or more capture agents bind to EVs derived from oligodendrocytes, astrocytes and Schwann cells. In one example, the two or more capture agents bind to EVs derived from oligodendrocytes, astrocytes and ependymal cells. In one example, the two or more capture agents bind to EVs derived from astrocytes, Schwann cells and ependymal cells. In one example, the two or more capture agents bind to EVs derived from neurons, microglia, oligodendrocytes and astrocytes. In one example, the two or more capture agents bind to EVs derived from neurons, microglia, oligodendrocytes and Schwann cells. In one example, the two or more capture agents bind to EVs derived from neurons, microglia, oligodendrocytes and ependymal cells. In one example, the two or more capture agents bind to EVs derived from microglia, oligodendrocytes, astrocytes and Schwann cells. In one example, the two or more capture agents bind to EVs derived from microglia, oligodendrocytes, astrocytes and ependymal cells. In one example, the two or more capture agents bind to EVs derived from oligodendrocytes, astrocytes, Schwann cells and ependymal cells.

[0026] In one example, the capture agents are selected from the group consisting of: antibodies, binding proteins, peptide fragments, engineered proteins and fusion peptides / proteins. In one example, the capture agents are antibodies or aptamers. In one example, the capture agents are binding proteins. In one example, the capture agents are aptamers. In one example, the capture agents are peptides or fragments thereof. In one example, the capture agents are engineered proteins. In one example, the engineered proteins are synthetic proteins such as peptidomimetics. In one example, the capture agents are fusion peptides or proteins.

[0027] In an embodiment, the capture agent comprises:

[0028] (i) a single chain Fv fragment (scFv);

[0029] (ii) a dimeric scFv (di-scFv);

[0030] (iii) a diabody;

[0031] (iv) a triabody;

[0032] (v) a tetrabody; (vi) a nanobody

[0033] (vii) a Fab;

[0034] (viii) a F(ab’)2;

[0035] (ix) a Fv;

[0036] (x) an aptamer;

[0037] (xi) one of (i) to (ix) linked to a constant region of an antibody, Fc or a heavy chain constant domain (CH)2 and / or CH3;

[0038] (xii) one of (i) to (ix) linked to albumin or a functional fragment or variants thereof or a protein that binds to albumin;

[0039] (xiii) an antibody; or

[0040] (xiv) a lectin.

[0041] Examples of antibodies for use in the present disclosure are discussed below. In one example, the two or more antibodies are selected from the group consisting of neurofilament- light chain (Nf-L), CD56 (NCAM), CD90 (Thyl), GD2, SNAP25, OMG, doublecortin, beta III tubulin, neuroDl, P-amyloid, phospho tau 181, phospho tau 217, TDP43, Tau, P-T181-tau, P-S396-tau and CD271. In one example, the two or more antibodies are selected from the group consisting of neurofilament-light chain (Nf-L), CD56 (NCAM), CD90 (Thyl), GD2, SNAP25, OMG, doublecortin, beta III tubulin, neuroDl, P-amyloid, phospho tau 181, phospho tau 217, TDP43, Tau, P-T181-tau, P-S396-tau and CD271, and wherein the cells are neurons. In one example, the two or more antibodies are selected from the group consisting of neurofilament- light chain (Nf-L), CD56 (NCAM), CD90 (Thyl), GD2, SNAP25 and OMG. In one example, the two or more antibodies are selected from the group consisting of neurofilament-light chain (Nf-L), CD56 (NCAM), CD90 (Thyl), GD2, SNAP25 and OMG, and wherein the cells are neurons. In one example, the two or more antibodies comprise neurofilament-light chain (Nf- L), CD56 (NCAM), CD90 (Thyl), GD2, SNAP25 and OMG. In one example, the two or more antibodies comprise neurofilament-light chain (Nf-L), CD56 (NCAM), CD90 (Thyl), GD2, SNAP25 and OMG, and wherein the cells are neurons.

[0042] In one example, the two or more antibodies are selected from the group consisting of CX3CR1, TMEM119, P2ryl2, Hexb, Sall 1, phospho tau 181, phospho tau 217, a-Synuclein, CD63, CD64, CD36, CD 15 and GDNF. In one example, the two or more antibodies are selected from the group consisting of CX3CR1, TMEM119, P2ryl2, Hexb, Sall 1 , phospho tau 181, phospho tau 217, a-Synuclein, CD63, CD64, CD36, CD 15 and GDNF, and wherein the cells are microglia. In one example, the two or more antibodies comprise CX3CR1 and / or TMEM1 19. In one example, the two or more antibodies comprise CX3CR1 and / or TMEM119, and wherein the cells are microglia.

[0043] In one example, the two or more antibodies are selected from the group consisting of VGLUT-2, 04, OMG, TMEM30A, Oligl, Olig2, Olig3, OSP, MBP, phospho tau 181, phospho tau 217 and tau. In one example, the two or more antibodies are selected from the group consisting of VGLUT-2, 04, OMG, TMEM30A, Oligl, Olig2, Olig3, OSP, MBP, phospho tau 181, phospho tau 217 and tau, and wherein the cells are oligodendrocytes. In one example, the two or more antibodies are selected from the group consisting of VGLUT-2, 04, OMG, phospho tau 181, phospho tau 217 and tau. In one example, the two or more antibodies are selected from the group consisting of VGLUT-2, 04, OMG, phospho tau 181, phospho tau 217 and tau, and wherein the cells are oligodendrocytes. In one example, the two or more antibodies comprise VGLUT-2, 04, OMG, phospho tau 181, phospho tau 217 and tau. In one example, the two or more antibodies comprise VGLUT-2, 04, OMG, phospho tau 181, phospho tau 217 and tau, and wherein the cells are oligodendrocytes.

[0044] In one example, the two or more antibodies are selected from the group consisting of CD90 (Thyl), GD2, EAAT1, EAAT2, GFAP, ALDH1L1, S100 beta, Aquaporin-4, P-amyloid, P-T181-tau, P-S396-tau, CD271, CD44 and CD15. In one example, the two or more antibodies are selected from the group consisting of CD90 (Thyl), GD2, EAAT1, EAAT2, GFAP, ALDH1L1, S100 beta, Aquaporin-4, P-amyloid, P-T181-tau, P-S396-tau, CD271, CD44 and CD 15, and wherein the cells are astrocytes. In one example, the two or more antibodies are selected from the group consisting of CD90 (Thyl), GD-2, EAAT1 and EAAT2. In one example, the two or more antibodies are selected from the group consisting of CD90 (Thyl), GD2, EAAT1 and EAAT2, and wherein the cells are astrocytes. In one example, the two or more antibodies comprise CD90 (Thyl), GD-2, EAAT1 and EAAT2. In one example, the two or more antibodies comprise CD90 (Thyl), GD-2, EAAT1 and EAAT2, and wherein the cells are astrocytes.

[0045] In one example, the two or more antibodies are selected from the group consisting of neurofilament-light chain (Nf-L), CD56 (NCAM), CD90 (Thyl), GD-2, SNAP25, OMG, CX3CR1 and TMEM119. In one example, the two or more antibodies are selected from the group consisting of neurofilament-light chain (Nf-L), CD56 (NCAM), CD90 (Thyl), GD-2, SNAP25, OMG, CX3CR1 and TMEM119, and wherein the two or more antibodies bind to EVs derived from a neuron and / or a microglia. In one example, the two or more antibodies comprise neurofilament-light chain (Nf-L), CD56 (NCAM), CD90 (Thyl), GD2, SNAP25, OMG, CX3CR1 and TMEM119, and wherein the two or more antibodies bind to EVs derived from a neuron and a microglia. In one example, the two or more antibodies are selected from the group consisting of neurofilament-light chain (Nf-L), CD56 (NCAM), CD90 (Thyl), GD- 2, SNAP25, OMG, CX3CR1, TMEM119, VGLUT-2 and 04, and wherein the two or more antibodies bind to EVs derived from a neuron, microglia and / or an oligodendrocyte. In one example, the two or more antibodies comprise neurofilament-light chain (Nf-L), CD56 (NCAM), CD90 (Thyl), GD2, SNAP25, OMG, CX3CR1, TMEM119, VGLUT-2 and 04, and wherein the two or more antibodies bind to EVs derived from a neuron, microglia and an oligodendrocyte. In one example, the two or more antibodies are selected from the group consisting of neurofilament-light chain (Nf-L), CD56 (NCAM), CD90 (Thyl), GD2, SNAP25, OMG, CX3CR1, TMEM119, VGLUT-2, 04, EAAT1 and EAAT2, and wherein the two or more antibodies bind to EVs derived from a neuron, microglia, an oligodendrocyte and / or an astrocyte. In one example, the two or more antibodies comprise neurofilament-light chain (Nf- L), CD56 (NCAM), CD90 (Thyl), GD2, SNAP25, OMG, CX3CR1, TMEM119, VGLUT-2, 04, EAAT1 and EAAT2, and wherein the two or more antibodies bind to EVs derived from a neuron, microglia, an oligodendrocyte and an astrocyte.

[0046] In another embodiment, the present disclosure also provides a composition for capturing extracellular vesicles (EVs) in a sample, comprising: (i) a solid surface; and (ii) two or more capture agents that bind to extracellular vesicles (EVs) derived from a neuron, wherein the two or more capture agents are attached to the solid surface. In this example, the capture agents may be antibodies.

[0047] For example, the two or more antibodies may be selected from the group consisting of neurofilament-light chain (Nf-L), CD56 (NCAM), CD90 (Thyl), GD2, SNAP25, OMG, doublecortin, beta III tubulin, neuroDl, P-amyloid, phospho tau 181, phospho tau 217, TDP43, Tau, P-T181-tau, P-S396-tau and CD271. In one example, the two or more antibodies are selected from the group consisting of neurofilament-light chain (Nf-L), CD56 (NCAM), CD90 (Thyl), GD-2, SNAP25 and OMG. In one example, the two or more antibodies comprise neurofilament-light chain (Nf-L), CD56 (NCAM), CD90 (Thyl), GD2, SNAP25 and OMG. The present disclosure also provides a composition for capturing extracellular vesicles (EVs) in a sample, comprising: (i) a solid surface; and (ii) two or more antibodies that bind to extracellular vesicles (EVs) derived from a brain cell, wherein the two or more antibodies are attached to the solid surface.

[0048] For example, the present disclosure also provides a composition for capturing extracellular vesicles (EVs) in a sample, comprising: (i) a solid surface; and (ii) two or more antibodies that bind to extracellular vesicles (EVs) derived from a microglia, wherein the two or more antibodies are attached to the solid surface.

[0049] In one example, the two or more antibodies are selected from the group consisting of CX3CR1, TMEM119, P2ryl2, Hexb, Salll, phospho tau 181, phospho tau 217, P-T181-tau, P- S396-tau, CD271, a-Synuclein, CD63, CD64, CD36, CD15 and GDNF. In one example, the two or more antibodies comprise CX3CR1 and / or TMEM119.

[0050] The present disclosure also provides a composition for capturing extracellular vesicles (EVs) in a sample, comprising: (i) a solid surface; and (ii) two or more antibodies that bind to extracellular vesicles (EVs) derived from an oligodendrocyte, wherein the two or more antibodies are attached to the solid surface.

[0051] In one example, the two or more antibodies are selected from the group consisting of VGLUT-2, 04, OMG, TMEM30A, Oligl, Olig2, Olig3, OSP, MBP, phospho tau 181, phospho tau 217, and tau. In one example, the two or more antibodies are selected from the group consisting of VGLUT-2, 04 and OMG. In one example, the two or more antibodies comprise VGLUT-2, 04 and OMG.

[0052] The present disclosure also provides a composition for capturing extracellular vesicles (EVs) in a sample, comprising: (i) a solid surface; and (ii) two or more antibodies that bind to extracellular vesicles (EVs) derived from an astrocyte, wherein the two or more antibodies are attached to the solid surface.

[0053] In one example, the two or more antibodies are selected from the group consisting of CD90 (Thyl), GD-2, EAAT1, EAAT2, GFAP, ALDH1L1, S100 beta, Aquaporin-4 and CD44. In one example, the two or more antibodies are selected from the group consisting of CD90 (Thyl), GD2, EAAT1 and EAAT2. In one example, the two or more antibodies comprise CD90 (Thyl), GD2, EAAT1 and EAAT2.

[0054] The term “attached” can be used in the context of defining structural features of the compositions disclosed herein to describe direct or indirect binding. For example, a capture agent of the disclosure can be directly bound to a solid support. In another example, the capture agent may be indirectly bound to the solid support. In an example, the term attached may be used to describe a mixture of direct and indirect binding.

[0055] In one example, the two or more capture agents are bound on the solid surface via crosslinkage with a linking agent. In one example, the linking agent is selected from the group consisting of: sulfodisuccinimidyl tartarate, DTSSP (3,3'-Dithiobis(sulfosuccinimidyl) propionate), (1- Ethyl-3 -(3 -dimethylaminopropyl) carbodiimide hyrochloride, N-Sulfosuccinimidyl-6-(4'- azido-2'-nitrophenylamino) hexanoate (sulfo-SANPAH), N-Succinimidyl-6-(4'-azido-2'- nitrophenylamino) hexanoate (ZAMPA), Succinimidyl 4-hydrazinonicotinate acetone hydrazone (SANH), C6-succinimidyl 4-hydrazinonicotinate acetone hydrazone (C6SANH), Succinimidyl 4-hydrazidoterephthalate hydrochloride (SHTH), Succinimidyl 4- formylbenzoate (SFB), C6-succinimidyl 4-formylbenzoate (C6-SFB), Bis-[b-(4-

[0056] Azidosalicylamido)ethyl]disulfide (BASED), p-Azidobenzoyl hydrazide (ABH), N-[k- Maleimidoundecanoic acid]hydrazide (KMUH), 106627-54-7, 4-[p-

[0057] Azidosalicylamido]butylamine (ASBA), [N-e-Maleimidocaproic acid]hydrazide (EMCH), [N- e-Maleimidocaproyloxy]succinimide ester (EMCS), Succinimidyl 6-(4,4'- azipentanamido)hexanoate (LC-SDA), Sulfosuccinimidyl 6-(4,4'-azipentanamido)hexanoate (Sulfo-LC-Sulfo-NHS-LC / SDA), N-[p-Maleimidophenyl]isocyanate (PMPI), 4-(4-N- Maleimidophenyl)butyric acid hydrazide hydrochloride (MPBH), ethylene glycol bisfsuccinimidylsuccinate] (EGS), 5bis-dPEG6 NHS Ester; di-(N-hydroxysuccinimidyl)- 4,7,10,13,16-pentaoxanonadeca-l,19,dioate (BS(PEG)9), 5bis-dPEG6 NHS Ester; di-(N- hydroxy succinimidyl)-4,7, 10,13,16-pentaoxanonadeca- 1 , 19, dioate (B S(PEG)5),

[0058] Succinimidyl-[4-(psorlaen-8-yloxy)]-butyrate (SPB), NHS-(PEG)n-maleamide, NHS- (PEG)n-NHS, B-[Tris(hydroxymethyl)phosphino] propionic acid (THPP), (bisfsulfosuccinimidyl] suberate (BS3), l-Ethyl-10-[10-dimethylaminopropyl]carbodiimide hydrocloride Carbodiimide (EDC), Formaldehyde, glutaraldehyde, Maleamide, and Cyanuric Chloride.

[0059] In an example, the linking agent is Di succinimidyl suberate (DSS), Bis[2-(N- succinimidyl-oxycarbonyloxy) ethyl] sulfone (BSCOES), l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC); dithiobis(succinimidyl propionate) (DSP); disuccinimidyl tartrate (DST). In one example, the linking agent is (bis)[sulfosuccinimidyl] suberate (BS3).

[0060] In one example, the two or more capture agents comprise reactive groups for crosslinking with the linking agent.

[0061] In one example, the composition further comprises a first plurality of capture agents, wherein at least one of the capture agents selected from the first plurality of capture agents is directly bound to the solid surface by one or more linking agents.

[0062] In one example, the composition further comprises a second plurality of capture agents, wherein at least one of the capture agents selected from the second plurality of capture agents is indirectly bound to the solid surface by connecting to a capture agent selected from the first plurality of capture agents by one or more of the linking agents.

[0063] In one example, the composition further comprises a third plurality of capture agents, wherein at least one of the capture agents selected from the third plurality of capture agents is indirectly bound to the solid surface by connecting to a capture agent selected from the second plurality of capture agents by one or more linking agents.

[0064] In one example, the capture agents of the first plurality of capture agents are crosslinked by a linkage comprising at least one linking agent.

[0065] In one example, the capture agents of the second plurality of capture agents are crosslinked by a linkage comprising at least one linking agent.

[0066] In one example, the capture agent of the first plurality of capture agents is crosslinked to the capture agent of the second plurality of capture agents by a linkage comprising at least one linking agent.

[0067] In one example, the first plurality of capture agents is heterogenous and comprises at least a first species of capture agent and a second species of capture agent that bind to EVs derived from different types of cells.

[0068] In one example, each capture agent is capable of releasably binding to at least one EV.

[0069] In one example, the one or more linking agents are heterogenous and comprise at least a first species of linking agent and a second species of linking agent.

[0070] In one example, each linking agent has least two reactive groups, and the first species of linking agent has different reactive groups than the second species of linking agent.

[0071] In one example, no greater than 70% of the capture agents in the composition are capture agents of the first plurality of capture agents.

[0072] In one example, the two or more capture agents form at least one layer of interconnected capture agents.

[0073] In one example, the two or more capture agents form a base layer of interconnected capture agents on the solid support.

[0074] In one example, the two or more capture agents form one, two or three layers of interconnected capture agents.

[0075] In one example, the two or more capture agents form one layer of interconnected capture agents.

[0076] In one example, the two or more capture agents form two layers of interconnected capture agents.

[0077] In one example, the two or more capture agents form three layers of interconnected capture agents.

[0078] In one example, the two or more capture agents form a base layer and at least one additional layer connected to the base layer.

[0079] In one example, the two or more capture agents form a base layer and one, two or three layers connected to the base layer.

[0080] In one example, the solid surface is selected from the group consisting of: a column, a membrane, a magnetic bead, or a non-magnetic bead. In one example, the two or more capture agents form a base layer on the magnetic bead, and at least one additional layer connected to the base layer.

[0081] In one example, the two or more capture agents form a base layer on the magnetic bead, and one, two or three layers connected to the base layer.

[0082] In one example, the solid surface is a magnetic bead or non-magnetic bead having a diameter of about 50 - 1,000 nm, about 100 - 500 nm, about 100 - 250 nm, about 250 - 500 nm, about 500 - 750 nm, about 750 - 1,000 nm, about 200 - 1,000 nm, about 500 - 1,000 nm, about 100 - 5,000 nm, about 500 - 5,000 nm, about 1,000 - 5,000 nm, about 1,000 - 3,000 nm, about 3,000 - 5,000 nm, about 2,000 - 4,000 nm, about 1,000 - 1,500 nm, about 1,500 - 2,000 nm, about 2,000 - 2,500 nm, about 2,500 - 3,000 nm, about 3,000 - 3,500 nm, about

[0083] 3.500 - 4,000 nm, about 4,000 - 4,500 nm, about 4,500 - 5,000 nm, about 1,000 - 2,000 nm, about 2,000 - 3,000 nm, about 3,000 - 4,000 nm, or about 4,000 - 5,000 nm.

[0084] In one example, the solid surface is a magnetic bead or non-magnetic bead having a diameter of about 1 - 5 pm, about 2 - 5 pm, about 3 - 5 pm, about 4 - 5 pm, about 1 - 2 pm, about 2 - 3 pm, about 2 - 4 pm, or about 3 - 4 pm.

[0085] In one example, the magnetic bead or non-magnetic bead has a diameter of about 50 nm, about 100 nm, about 150 nm, about 200 nm, about 250 nm, about 300 nm, about 350 nm, about 400 nm, about 450 nm, about 500 nm, about 550 nm, about 600 nm, about 650 nm, about 700 nm, about 750 nm, about 800 nm, about 850 nm, about 900 nm, about 950 nm about 1,000 nm, about 1,250 nm, about 1,500 nm, about 1,750 nm, about 2,000 nm, about 2,250 nm, about

[0086] 2.500 nm, about 2,750 nm, about 3,000 nm, about 3,250 nm, about 3,500 nm, about 3,750 nm, about 4,000 nm, about 4,250 nm, about 4,500 nm, about 4,750 nm, or about 5,000 nm.

[0087] In one example, the solid surface is a magnetic bead or non-magnetic bead having a diameter of about 1 pm, about 1.5 pm, about 2 pm, about 2.5 pm, about 3 pm, about 3.5 pm, about 4 pm, about 4.5 pm or about 5 pm.

[0088] In one example, the solid surface comprises silica, glass, nitrocellulose, polystyrene, and polyurethane.

[0089] The present disclosure also provides a method of capturing extracellular vesicles (EVs) from a sample, comprising: contacting the sample with the composition according to the present disclosure under conditions that enable binding of the capture agent to the EVs to capture the EVs in the sample.

[0090] The present disclosure also provides a method of isolating extracellular vesicles (EVs) from a sample, comprising: contacting the sample with the composition of the present disclosure under conditions that enable binding of the capture agent to the EVs; and isolating the bound EVs from the sample.

[0091] In one example, the sample is selected from the group consisting of: urine, saliva, nasal swab, faeces, tears, cerebrospinal fluid, ocular fluid, crevicular fluid, amniotic fluid, seminal fluid, vaginal fluid, menstrual fluid, plasma, blood, serum, and conditioned medium of a cell culture. In an example, the sample is plasma or serum.

[0092] The present disclosure also provides a composition comprising extracellular vesicles (EVs) enriched for EVs derived from one or more cells of the nervous system, wherein the EVs are isolated by the method of the present disclosure.

[0093] The present disclosure also provides a population of extracellular vesicles (EVs) enriched for EVs derived from one or more cells of the nervous system, wherein the EVs are isolated by the method of the present disclosure.

[0094] In one example, at least 20% of the EVs in the composition or the population of the present disclosure express a marker indicative of the EVs being derived from a type of nervous system cell such as a neuron. In an example, the nervous system cell is a brain cell.

[0095] In one example, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the EVs in the composition or the population of the present disclosure express a marker indicative of the EVs being derived from a type of nervous system cell.

[0096] In one example, at least 20% of the EVs in the composition or the population of the present disclosure express a marker indicative of the EVs being derived from one or more types of nervous system cell.

[0097] In one example, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the EVs in the composition or the population of the present disclosure express a marker indicative of the EVs being derived from one or more types of nervous system cell.

[0098] In one example, the one or more types of nervous system cell is selected from the group consisting of: neuron, microglia, oligodendrocyte, astrocyte, Schwann cell and ependymal cell.

[0099] In one example, the EV is selected from the group consisting of: an exosome, a microvesicle, and an apoptotic body. In an example, the EV is an exosome. In an example, the EV is membranous. In an example, the EV is non-membranous.

[0100] In one example, the EVs are CD9+, Flotilin- 1+, and / or TSG101+.

[0101] The present disclosure also provides a method of diagnosing a neurodegenerative disease in a subject, comprising: contacting a sample from the subject with the composition of the present disclosure under conditions that enable binding of the capture agent to the EVs; isolating the bound EVs from the sample; and measuring and / or detecting expression of one or more biomarkers of the neurodegenerative disease in the isolated EVs to diagnose the neurodegenerative disease.

[0102] The present disclosure also provides a method of diagnosing a neurodegenerative disease in a subject, comprising: contacting a sample from the subject with the composition of the present disclosure under conditions that enable binding of the capture agent to the EVs; isolating the bound EVs from the sample to generate a composition of EVs enriched for EVs derived from one or more nervous system cells; and measuring and / or detecting expression of one or more biomarkers of the neurodegenerative disease in the composition of EVs enriched for EV derived from one or more nervous system cells to diagnose the neurodegenerative disease.

[0103] The present disclosure also provides a method of treating a neurodegenerative disease in a subject, comprising: contacting a sample from the subject with the composition of the present disclosure under conditions that enable binding of the capture agent to the EVs; isolating the bound EVs from the sample; measuring and / or detecting expression of one or more biomarkers of the neurodegenerative disease in the isolated EVs to diagnose the neurodegenerative disease; and administering a therapeutic agent to treat the neurodegenerative disease, the therapeutic agent selected from the group consisting of: a small molecule inhibitor, induced pluripotent stem cells, a cytokine or chemokine such as GM-CSF, a therapeutic antibody such as a monoclonal antibody or neutralizing antibody, a viral vector, an immunotherapy, a vaccine such as an a-synuclein or amyloid-P vaccine, and a dietary supplement such as an omega-3 fatty acid, eicosapentanoic acid (EP A) or docosahexanoic acid (DHA).

[0104] In one example, the small molecule inhibitor is a cytokine, chemokine or inflammasome inhibitor. In one example, the cytokine inhibitor is an IL-6 or TNFa inhibitor. In one example, the TNFa inhibitor is Etanercept. In one example, the inflammasome inhibitor is an NLRP3 inflammasome inhibitor. In one example, the NLRP3 inflammasome inhibitor is a ketone body. In one example, the cytokine is granulocyte-macrophage colony-stimulating factor (GM- CSF) or recombinant GM-CSF.

[0105] In one example, the therapeutic antibody is a monoclonal antibody or neutralizing antibody. In one example, the monoclonal antibody is selected from the group consisting of: bapineuzumab, solaneuzumab, gantenerubmab, crenezumab, aducanumab, ponezumab, C2n 8E12, gosuranemab, zagotenemab, or semorinemab. In one example, the therapeutic antibody binds or selectively binds to a neuropathic protein or a fragment thereof. In one example, the neuropathic protein or a fragment thereof is selected from the group consisting of: a-synuclein, amyloid-P and tau. In one example, the therapeutic antibody binds or selectively binds to an amyloid-P (AP) protein or a fragment thereof. In one example, the therapeutic antibody binds or selectively binds to a soluble amyloid-P (AP) protein. In one example, the therapeutic antibody binds or selectively binds to an amyloid-P (AP) oligomer, fibril or plaque. In one example, the therapeutic antibody binds or selectively binds to an AP-40 or AP-42 protein. In one example, the therapeutic antibody binds or selectively binds to an amyloid-P (AP) fragment comprising the N-terminus of Ap, the C-terminus of Ap, the mid-domain of Ap, or the Ap 31- 35 sequence. In one example, the therapeutic antibody binds or selectively binds to a tau protein or a fragment thereof. In one example, the therapeutic antibody binds or selectively binds to a tau oligomer. In one example, the therapeutic antibody binds or selectively binds to a soluble tau protein. In one example, the tau protein is a phosphorylated tau protein. In one example, the tau protein fragment comprises the N-terminal projection of tau. In one example, the therapeutic antibody binds or selectively binds to a huntingtin protein or mutant huntingtin protein. In one example, the neutralizing antibody is an IL-12 or IL-23 neutralizing antibody.

[0106] In one example, the dietary supplement is selected from the group consisting of: an omega-3 fatty acid, eicosapentanoic acid (EP A) or docosahexanoic acid (DHA). In one example, the dietary supplement is omega-3 fatty acid. In one example, the dietary supplement is eicosapentanoic acid (EP A). In one example, the dietary supplement is docosahexanoic acid (DHA).

[0107] In one example, the vaccine is selected from the group consisting of: an a-synuclein vaccine, an amyloid-P vaccine and a tau vaccine. In one example, the vaccine is a liposomebased vaccine. In one example, the vaccine comprises an antigen derived from a neuropathic protein or fragment thereof. In one example, the antigen is derived from a neuropathic protein or fragment thereof selected from the group consisting of: a-synuclein, amyloid-P and tau. In one example, the antigen is an amyloid-P (AP) protein or a fragment thereof. In one example, the antigen is a soluble amyloid-P (AP) protein. In one example, the antigen is an amyloid-P (AP) oligomer, fibril or plaque. In one example, the antigen is an AP-40 or AP-42 protein. In one example, the antigen is an amyloid-P (AP) fragment comprising the N-terminus of Ap, the C-terminus of Ap, the mid-domain of Ap, the Api-6 protein, the Api-7 protein or the Ap31- 35 protein. In one example, the antigen is a tau protein or a fragment thereof. In one example, the antigen is a tau oligomer. In one example, the antigen is a soluble tau protein. In one example, the antigen is a phosphorylated tau protein. In one example, the tau protein fragment comprises the N-terminal projection of tau. In one example, the antigen is a huntingtin protein or mutant huntingtin protein.

[0108] In one example, the therapeutic agent is a pharmacological agent. In one example, the pharmacological agent is selected from the group consisting of: a non-steroidal antiinflammatory drug, an anti -oxi dative compound, and a neuroprotective compound.

[0109] In one example, the neuroprotective compound is a GLP-1 agonist or a PPAR- y agonist.

[0110] The present disclosure also provides a method for preparing a composition of extracellular vesicles (EVs) enriched for EVs derived from one or more nervous system cells useful for measuring and / or detecting genetic expression of one or more biomarkers associated with a neurodegenerative disease, comprising contacting a sample from the subject with the composition of the present disclosure under conditions that enable binding of the capture agent to the EVs; isolating the bound EVs from the sample to generate the composition of EVs enriched for EV derived from one or more nervous system cells; and measuring and / or detecting expression of one or more biomarkers associated with the neurodegenerative disease in the composition of EVs enriched for EV derived from one or more nervous system cells. The present disclosure also provides a method of assessing the likelihood of a subject suffering from a neurodegenerative disease, comprising contacting a sample from the subject with the composition of the present disclosure under conditions that enable binding of the capture agent to the EVs; isolating the bound EVs from the sample to generate the composition of EVs enriched for EV derived from one or more nervous system cells; measuring and / or detecting expression of one or more biomarkers associated with the neurodegenerative disease in the composition of EVs enriched for EV derived from one or more nervous system cells; comparing the expression of the one or more biomarkers in the sample with a suitable control; and calculating a risk score for the subject based on the expression of the one or more biomarkers to assess the likelihood of the subject suffering from a neurodegenerative disease.

[0111] In one example, the risk score is calculated by allocating a weighted score to the expression of the one or more biomarkers in the sample based on a reference value in the control.

[0112] In one example, the one or more biomarkers are selected from the group consisting of: neurofilament-light chain (Nf-L), CD56 (NCAM), CD90 (Thy 1), GD-2, SNAP25, OMG, doublecortin, beta III tubulin, neuroDl, P-amyloid, phospho tau 181, phospho tau 217, TDP43, Tau, P-T181-tau, P-S396-tau, CD271, CX3CR1, TMEM119, P2ryl2, Hexb, Salll, a- Synuclein, CD63, CD64, CD36, CD15, GDNF, VGLUT-2, 04, TMEM30A, Oligl, Olig2, Olig3, OSP, MBP, EAAT1, EAAT2, GFAP, ALDH1L1, S100 beta, Aquaporin-4 and CD44, CNNM2, CNNM3, IFITM2, LRP10, PEBP4, IGHV4-4, LAMT0R4, LGALS9, DCST1, NCF4, TBC1D10C, NHSL2, FCAR, ARHGAP9, CHST9, CHRDL2, IGLV5-52 IGHV3-33, SPAG6, PIK3AP1, MECP2, PADI4, RPL13A, Hl-10, IGKV3-7, HP1BP3, HNRNPUL2, MMP7, HMGN4, SNRNP70, HAMP, DDX5, PRG3, YY1, RPS29, IGHV4-39, PRG2, H2AC21, ILF2, RALY, NCF2, PRAM1, GNG2, G0LGA3, PCDH12, HINT3, RAB11FIP1, BAIAP3, RASSF2, ALYREF, FPR1, TBC1D10C, NHSL2, ARHGAP9, ARHGAP30, IGLV5-52, IGHV3-33, LILRA6, SPAG6, IGKV5-2, SMIM5, IGKV3-7, TNNI2, LOXL3, GGT1, GSTZ1, SLC12A4, TOR1AIP1, HLA-DRB1, GSTM5, IER5, MR0H2B, H2ST1, NAGA, ERO1B, CARS2, CXCL13, APOL3, GKN2 and HS2ST1.

[0113] In one example, the one or more biomarkers are selected from the group consisting of: neurofilament-light chain (Nf-L), CD56 (NCAM), CD90 (Thyl), GD-2, SNAP25, OMG, doublecortin, beta III tubulin, neuroDl, P-amyloid, phospho tau 181, phospho tau 217, TDP43, Tau, P-T181-tau, P-S396-tau, CD271, CX3CR1, TMEM119, P2ryl2, Hexb, Salll, a- Synuclein, CD63, CD64, CD36, CD15, GDNF, VGLUT-2, 04, TMEM30A, Oligl, Olig2, Olig3, OSP, MBP, EAAT1, EAAT2, GFAP, ALDH1L1, S100 beta, Aquaporin-4 and CD44.

[0114] In one example, the one or more biomarkers are selected from the group consisting of: CNNM2, CNNM3, IFITM2, LRP10, PEBP4, IGHV4-4, LAMT0R4, LGALS9, DCST1, NCF4, TBC1D10C, NHSL2, FCAR, ARHGAP9, CHST9, CHRDL2, IGLV5-52 IGHV3-33, SPAG6, PIK3AP1, MECP2, PADI4, RPL13A, Hl-10, IGKV3-7, HP1BP3, HNRNPUL2, MMP7, HMGN4, SNRNP70, HAMP, DDX5, PRG3, YY1, RPS29, IGHV4-39, PRG2, H2AC21, ILF2 and RALY.

[0115] In one example, the one or more biomarkers are associated with Alzheimer’s disease, and selected from the group consisting of: CNNM2, CNNM3, IFITM2, LRP10, PEBP4, IGHV4-4, LAMTOR4, LGALS9, DCST1, NCF4, TBC1D10C, NHSL2, FCAR, ARHGAP9, CHST9, CHRDL2, IGLV5-52 IGHV3-33, SPAG6, PIK3AP1, MECP2, PADI4, RPL13A, Hl- 10, IGKV3-7, HP1BP3, HNRNPUL2, MMP7, HMGN4, SNRNP70, HAMP, DDX5, PRG3, YY1, RPS29, IGHV4-39, PRG2, H2AC21, ILF2 and RALY.

[0116] In one example, the one or more biomarkers are selected from the group consisting of: NCF2, PRAM1, GNG2, GOLGA3, PCDH12, HINT3, RAB11FIP1, BAIAP3, RASSF2, ALYREF, FPR1, TBC1D10C, NHSL2, ARHGAP9, ARHGAP30, IGLV5-52, IGHV3-33, LILRA6, SPAG6, IGKV5-2, SMIM5, IGKV3-7, TNNI2, LOXL3, GGT1, GSTZ1, SLC12A4, TOR1AIP1, HLA-DRB1, GSTM5, IER5, MROH2B, H2ST1, NAGA, ERO1B, CARS2, CXCL13, APOL3, GKN2 and HS2ST1.

[0117] In one example, the one or more biomarkers are associated with Parkinson’s disease, and selected from the group consisting of: NCF2, PRAM1, GNG2, GOLGA3, PCDH12, HINT3, RABI 1FIP1, BAIAP3, RASSF2, ALYREF, FPR1, TBC1D10C, NHSL2, ARHGAP9, ARHGAP30, IGLV5-52, IGHV3-33, LILRA6, SPAG6, IGKV5-2, SMIM5, IGKV3-7, TNNI2, LOXL3, GGT1, GSTZ1, SLC12A4, TOR1AIP1, HLA-DRB1, GSTM5, IER5, MROH2B, H2ST1, NAGA, ERO1B, CARS2, CXCL13, APOL3, GKN2 and HS2ST1.

[0118] In one example, the one or more biomarkers are selected from the group consisting of: ADAMDEC1, FBLN7, LPL, ASPN, BNC2, LTBP2, C1R, DKK2, CFD, MMP19, CR1, LNPEP,SDCBP, CYSTM1, BASP1, CD53, ALDH1L1, S100A8, S100A9, GYG1, CHMP2B, PTPN6, ARGHAP15, ITGB2, ICAM3, C5AR1, LTB4R, HAGH, FLOT1, FLOT2, DEF6, RPS18, SRP54, TRIR, RPS7, DNAJC8, RPS25, RPL35, RPL9, RPL22, SNRPA, RPL19, RPS20, RPL24, RPL5, RPL13, RPL23A, TRIQK, GCA, BTF3L4, PRAM1, RCSD1, IL16, PAK2, SASH3, CAPG, TES, HNRNPA3, SAMSN1, STK10, TFRC, SLC30A1, SLC1A5, MARCHF8, ATP6V0D1, ATP6V0A1, ESD, ENO1, PDCD6IP, TMCC3, ADGRE3 and TNFRSF10C.

[0119] In one example, the one or more biomarkers are associated with Alzheimer’s disease, and selected from the group consisting of: ADAMDEC1, FBLN7, LPL, ASPN, BNC2, LTBP2, C1R, DKK2, CFD, MMP19, CR1, LNPEP,SDCBP, CYSTM1, BASP1, CD53, ALDH1L1, S100A8, S100A9, GYG1, CHMP2B, PTPN6, ARGHAP15, ITGB2, ICAM3, C5AR1, LTB4R, HAGH, FLOT1, FLOT2, DEF6, RPS18, SRP54, TRIR, RPS7, DNAJC8, RPS25, RPL35, RPL9, RPL22, SNRPA, RPL19, RPS20, RPL24, RPL5, RPL13, RPL23A, TRIQK, GCA, BTF3L4, PRAM1, RCSD1, IL16, PAK2, SASH3, CAPG, TES, HNRNPA3, SAMSN1, STK10, TFRC, SLC30A1, SLC1A5, MARCHF8, ATP6V0D1, ATP6V0A1, ESD, ENO1, PDCD6IP, TMCC3, ADGRE3 and TNFRSF10C. In one example, the one or more biomarkers are selected from the group consisting of: MFNG, PTN, ACSS1, PITPNC1, ERLIN2, GLB1, APLP2, JAGN1, AGPAT1, MAGT1, CTSF, NDUFA12, TPST2, CST7, MYL2, GDF7, MBOAT7, ACOT8, NDUFB4, TNNT1, TMEM33, SELENOT, PDLIM3, GCSAML, MAMDC2, 0RMDL3, FBXO25, ATP5MK, DLAT, PDGFRL, PCSK5, SYAP1, NHEJ1, PLGRKT, IRS2, PTH, PDHA1, BMP2, SFTPD, ECU, ITLN1, ST6GAL2, MICAL3, CHST14, LEFTY1, SCUBE2 and ILF2.

[0120] In one example, the one or more biomarkers are associated with Alzheimer’s disease, and selected from the group consisting of: MFNG, PTN, ACSS1, PITPNC1, ERLIN2, GLB1, APLP2, JAGN1, AGPAT1, MAGT1, CTSF, NDUFA12, TPST2, CST7, MYL2, GDF7, MBOAT7, ACOT8, NDUFB4, TNNT1, TMEM33, SELENOT, PDLIM3, GCSAML, MAMDC2, 0RMDL3, FBXO25, ATP5MK, DLAT, PDGFRL, PCSK5, SYAP1, NHEJ1, PLGRKT, IRS2, PTH, PDHA1, BMP2, SFTPD, ECU, ITLN1, ST6GAL2, MICAL3, CHST14, LEFTY1, SCUBE2 and ILF2.

[0121] In one example, the EV is selected from the group consisting of: an exosome, a microvesicle, and an apoptotic body. In an example, the EV is an exosome. In an example, the EV is a membranous EV. In another example, the EV is a non-membranous EV such as an extracellular nanoparticle.

[0122] In one example, the neurodegenerative disease is a central nervous system disease. In one example, the neurodegenerative disease is a peripheral nervous system disease.

[0123] In one example, the neurodegenerative disease is selected from the group consisting of: Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, motor neuron disease (MND), amyotrophic lateral sclerosis (ALS), peripheral neuropathy and Guillain-Barre syndrome.

[0124] In one example, the sample is substantially cell-free.

[0125] BRIEF DESCRIPTION OF THE DRAWINGS

[0126] Figure 1 is a schematic, cross-sectional diagram of a layered structure formed on a solid surface. The layered structure is formed on base 350 having a top surface 352. Antibodies 354, 358, 360, 3510 and 3512 are interconnected to each other and to the surface 352 by linkers 356. Figure 2 illustrates an exemplary workflow for isolating EVs according to the disclosure, NEURO-NET and comparison of the same with a size exclusion column method of isolating EVs, IZON.

[0127] Figure 3A is a bar chart illustrating the protein yield comparisons between the NEURO-NET method, a non-selective EV capture method, and a further size exclusion column (SEC) method.

[0128] Figure 3B is a bar chart illustrating the reproducibility of protein and RNA yield from NEURO-NET captured EVs in plasma. The error bars shown represent standard deviation. Figure 4 shows the results of Western blot experiments comparing the NEURO-NET method versus the size exclusion column (SEC) method. The Figure shows that NEURO-NET EVs were positive for CD9, TSG101 and Flotilin.

[0129] Figure 5A to 5C shows further results of Western blot experiments comparing the NEURO- NET method versus the size exclusion column (SEC) methods. Figure 5A shows that NEURO- NET EVs were positive for neuron markers and a microglia marker. Figure 5B shows that NEURO-NET EVs were positive for astrocyte markers. Figure 5C shows that NEURO-NET EVs were positive for oligodendrocyte markers and a microglia marker.

[0130] Figure 5D shows results of a comparison between NEURO-NET versus EXO-NET specificity in brain cell line derived EVs. The left panel shows that NEURO-NET EVs were positive for GD2, an astrocyte and neuronal marker. The right panel shows that NEURO-NET EVs were positive for GLAST, an astrocyte marker (right panel).

[0131] Figure 6 is a bar chart illustrating the protein yield comparisons using the NEURO-NET between subjects suffering from Alzheimer’s disease, Parkinson’s disease and normal healthy controls.

[0132] Figure 7A shows the results of Western blot experiments comparing genetic expression of EVs isolated using NEURO-NET in subjects suffering from Alzheimer’s disease and normal healthy controls. Pooled plasma samples from subjects suffering from Alzheimer’s disease had significant upregulation of Phospho-tau 181 and Phospho-tau 217 in comparison to healthy controls.

[0133] Figure 7B shows the results of Western blot experiments comparing protein expression from EVs isolated using NEURO-NET in subjects suffering from Alzheimer’s disease, versus neat plasma protein lysate. EVs isolated using NEURO-NET exhibited enriched expression of Phospho-tau 217, TDP 43 and Neurofilament L, in comparison to levels seen in neat plasma samples.

[0134] Figure 8 is a scatter plot graph showing the results of mass spectrometry analysis of EVs isolated from subjects suffering from Alzheimer’s disease versus healthy controls. Each dot point represents a marker that is either downregulated (darker grey dots on the left-hand side of the Figure) or upregulated (lighter grey dots on the right-hand side of the Figure) in Alzheimer’s samples as compared to healthy controls. White dots at the bottom of the scatter plot represent non-significant proteins.

[0135] Figure 9 is a scatter plot graph showing the results of mass spectrometry analysis of EVs isolated from subjects suffering from Parkinson’s disease versus healthy controls. Each dot point represents a marker that is either downregulated (darker grey dots on the left-hand side of the Figure) or upregulated (lighter grey dots on the right-hand side of the Figure) in Parkinson’s samples as compared to healthy controls. White dots at the bottom of the scatter plot represent non-significant proteins. Figure 10 shows mass spectrometry analysis illustrating the top 20 genes downregulated in EVs isolated using NEURO-NET from subjects having Alzheimer’s disease as compared to healthy controls.

[0136] Figure 11 shows mass spectrometry analysis illustrating the top 20 genes upregulated in EVs isolated using NEURO-NET from subjects having Alzheimer’s disease as compared to healthy controls.

[0137] Figure 12 shows mass spectrometry analysis illustrating the top 20 genes downregulated in EVs isolated using NEURO-NET from subjects having Parkinson’s disease as compared to healthy controls.

[0138] Figure 13 shows mass spectrometry analysis illustrating the top 20 genes upregulated in EVs isolated using NEURO-NET from subjects having Parkinson’s disease as compared to healthy controls.

[0139] Figure 14 is a scatter plot (Volcano plot) graph showing the results of mass spectrometry analysis of EVs isolated from subjects suffering from Alzheimer’s disease versus healthy controls. Each dot point represents a marker that is either downregulated (darker grey dots on the left-hand side of the Figure) (599 proteins) or upregulated (lighter grey dots on the righthand side of the Figure) (255 proteins) in Alzheimer’s samples as compared to healthy controls. White dots at the bottom of the scatter plot represent non-significant proteins.

[0140] Figure 15 shows results of a heatmap analysis illustrating the top 50 significantly, differentially expressed genes between Alzheimer’s disease samples (case) and normal healthy samples (control).

[0141] Figure 16 shows a bar chart illustrating upset plots of shared and unique proteins after filtration. All group, where a protein was counted as identified if it was detected in at least 50%. Case = Alzheimer’s disease sample, Control = normal healthy sample.

[0142] DETAILED DESCRIPTION

[0143] General

[0144] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e., one or more) of those steps, compositions of matter, groups of steps or groups of compositions of matter.

[0145] Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features. The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the present disclosure.

[0146] Any example of the present disclosure herein shall be taken to apply mutatis mutandis to any other example of the disclosure unless specifically stated otherwise. Stated another way, any specific example of the present disclosure may be combined with any other specific example of the disclosure (except where mutually exclusive).

[0147] Any example of the present disclosure disclosing a specific feature or group of features or method or method steps will be taken to provide explicit support for disclaiming the specific feature or group of features or method or method steps.

[0148] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (for example, in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0149] Unless otherwise indicated, the recombinant protein, molecular biology, cell culture, and organic chemistry techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

[0150] The term “and / or”, e.g., “X and / or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

[0151] Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0152] Selected Definitions

[0153] As used herein, the term “nucleotide sequence” or “nucleic acid sequence” will be understood to mean a series of contiguous nucleotides (or bases) covalently linked to a phosphodiester backbone. By convention, sequences are presented from the 5' end to the 3' end, unless otherwise specified.

[0154] As used herein, the term “antigen” refers to a molecule or structure containing one or more epitopes that induce, elicit, augment or boost a cellular and / or humoral immune response. Antigens can include, for example, proteins and peptides related to a neurodegenerative disease, for example tau, a-synuclein, or amyloid-p.

[0155] The present disclosure provides compositions for capturing extracellular vesicles derived from cell(s) in the nervous system (i.e., brain, spinal cord and nerves). It will be appreciated that compositions of the disclosure can capture EVs from cells that are in the brain or other parts of the body, so long as they are part of the nervous system. For example, neurons are components of the nervous system and these cell types persist in the brain as well as in other parts of the body. Accordingly, in certain embodiments, it is envisaged that compositions of the disclosure will capture EVs from cells in the nervous system, wherein the cells are present throughout the body. For example, compositions of the disclosure may be used to capture EVs derived from neurons located in the brain or other parts of the body. In certain examples, compositions of the disclosure will capture EVs from brain cells (i.e., cells that are either located in the brain or are derived from the brain such as a primary culture of brain cells). In an example, the brain cells are neurons. Although, in an example, compositions of the disclosure can capture EVs from neurons and brain cells.

[0156] The term “polypeptide” or “polypeptide chain” will be understood to mean a series of contiguous amino acids linked by peptide bonds. For example, a protein shall be taken to include a single polypeptide chain i.e., a series of contiguous amino acids linked by peptide bonds or a series of polypeptide chains covalently or non-covalently linked to one another (i.e., a polypeptide complex). The series of polypeptide chains can be covalently linked using a suitable chemical or a disulfide bond. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, Van der Waals forces, and hydrophobic interactions.

[0157] The term “soluble” shall be understood to mean, when used in the context of a protein or polypeptide, that the protein or polypeptide is in a soluble or secreted form. The soluble protein may be a recombinant soluble protein. The soluble protein may have been truncated to remove one or more regions. For example, the soluble protein may be truncated to lack the transmembrane region. In addition to the removal of the transmembrane region, the soluble protein may additionally comprise one or more additional mutations or modifications to stabilize the refolding of the soluble or secreted protein or polypeptide. For example, one or more sites may be removed to account for the removal of the transmembrane region, and / or one or more mutations or modifications may be introduced that stabilize the soluble protein.

[0158] As used herein, the terms “disease”, “disorder” or “condition” refers to a disruption of or interference with normal function, and is not to be limited to any specific condition and will include diseases or disorders. As used herein, the term “the likelihood of a subject suffering from a disease” or “likely to suffer from a disease” refers to a subject that may or may not have detectable disease or symptoms of disease, and may or may not have displayed detectable disease or symptoms of disease prior to being subjected to the methods according to the present disclosure, but is considered to a reasonable degree of certainty to be suffering from the disease after being subjected to the methods according to the present disclosure. “At risk” denotes that a subject has one or more risk factors, which are measurable parameters that correlate with development of the disease or condition, as known in the art and / or described herein.

[0159] As used herein, the terms “treating”, “treat” or “treatment” include administering a therapeutic agent to thereby reduce or eliminate at least one symptom of a specified disease or condition.

[0160] An “effective amount” refers to at least an amount effective, at dosages and for periods of time necessary, to achieve the desired result. For example, the desired result may be a therapeutic or prophylactic result. An effective amount can be provided in one or more administrations. In some examples of the present disclosure, the term “effective amount” is meant an amount necessary to effect treatment of a disease or condition as hereinbefore described. In some examples of the present disclosure, the term “effective amount” is meant an amount necessary to effect a change associated with a disease or condition as hereinbefore described. The effective amount may vary according to the disease or condition to be treated or factor to be altered and also according to the weight, age, racial background, sex, health and / or physical condition and other factors relevant to the mammal being treated. Typically, the effective amount will fall within a relatively broad range (e.g., a “dosage” range) that can be determined through routine trial and experimentation by a medical practitioner. Accordingly, this term is not to be construed to limit the disclosure to a specific quantity, e.g., dosage or amount of a therapeutic agent. The effective amount can be administered in a single dose or in a dose repeated once or several times over a treatment period.

[0161] A “therapeutically effective amount” is at least the minimum concentration required to effect a measurable improvement of a particular disease or condition. A therapeutically effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the patient, and the ability of the therapeutic agent to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the therapeutic agent are outweighed by the therapeutically beneficial effects.

[0162] As used herein, the term “subject” shall be taken to mean any animal including humans, for example a mammal. Exemplary subjects include but are not limited to humans and nonhuman primates. For example, the subject is a human.

[0163] As used herein, the term “sample” shall be taken to mean any fluid or tissue or material including cells derived from a living or dead animal including humans, or prepared or isolated in the laboratory such as from a cell culture. The sample may contain extracellular vesicles (EVs), nucleic acids, carbohydrates, lipids and / or polypeptides. Samples include, but are not limited to, urine, nasal swab, saliva, faeces, tears, cerebrospinal fluid, ocular fluid, crevicular fluid, amniotic fluid, seminal fluid, vaginal fluid, menstrual fluid, plasma, blood, serum, and conditioned medium of a cell culture. The sample may be a fluid sample, biological sample, or biological fluid sample. In an example, the sample is supernatant from a cell culture. For example, the supernatant can be obtained from a cell population under culture conditions. In an example, the supernatant is purified before contact with a composition of the disclosure.

[0164] The term “culture conditions” is used to refer to cells, such as a population of cells comprising cells of the nervous system, growing in culture. In an example, culture conditions refers to an actively dividing population of cells. Such cells may, in an example, be in exponential growth phase. Alternatively, such cells may be in a stationary phase. In an example, the supernatant is obtained after at least two or three days in culture. In another example, the supernatant is obtained after about 30 to 84 hours of culture. In an example, the cells are a primary cell line. In another example, the cells are an immortalized cell line.

[0165] As used herein, terms such as “separating”, “isolating”, “purifying” shall be taken to mean the removal of one or more molecules in a sample from other molecules in a sample. The term “purifying” may mean removing at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of other molecules. Alternatively, a “purifying” step may mean that the sample is substantially free of other molecules other than the molecule of interest. For example, the molecule of interest may be extracellular vesicles (EVs).

[0166] As used herein, the term “binding” may refer to non-specific binding or specific binding. The term “non-specific binding” refers to binding of one molecule to another in a non-specific and transient manner. Thus, “non-specific binding” may refer to the binding of one molecule to a number of different targets. The term “specific binding” refers to the ability of a molecule (such as a capture agent) to discriminate between possible binding partners, and bind only to one specific binding partner to the exclusion of other molecules. For example, “specific binding” may refer to the binding of a capture agent only to EVs derived from one particular nervous cell type and not EVs derived from other nervous cell types. A capture agent (such as an antibody) that interacts with one particular target at the exclusion of other targets that are present is said to “bind specifically” to the target with which it binds. In one example, the two or more capture agents bind specifically to EVs derived from a cell in the nervous system. In one example, the two or more capture agents do not bind to EVs derived from a non-nervous system cell. In one example, the two or more capture agents bind specifically to EVs derived from a cell in the nervous system, and do not bind to EVs derived from a non-nervous system cell. Specificity of binding can be determined by a number of methods known in the art, such as degree of dissociation of a target-binding moiety-partner complex, or in a competitive binding scenario where the binding ability of the target-binding moiety is determined in comparison to an alternative molecule. Typically, such binding assays are performed using a range of different of concentrations and may be used to calculate a measure of the specificity of binding, e.g., a dissociation constant.

[0167] Extracellular vesicles

[0168] The term “extracellular vesicle” as used herein refers to a lipid-bilayer enclosed vesicle that has been released or secreted by a cell. Examples of extracellular vesicles include exosomes, microvesicles, apoptotic bodies and exosome-like vesicles. The EV subtypes are typically differentiated by characteristics such as size, charge, the nature of their biogenesis and, their contents. Without being bound to theory, it is understood that exosomes are approximately 40 - 120 nm in size, and are generated by inward budding of endosomes. Microvesicles (also known as ectosomes, microparticles or shedding vesicles) are approximately 100 - 1,000 nm in size and form directly from outward budding of the plasma membrane. Apoptotic bodies are approximately 500 - 4,000 nm in size and formed as a consequence of programmed cell death.

[0169] In an example, the extracellular vesicle is or comprises an exosome, microvesicle or apoptotic body. In one example, the extracellular vesicle is an exosome. In one example, the extracellular vesicle is a microvesicle. In one example, the extracellular vesicle is an apoptotic body.

[0170] A subpopulation of EVs includes brain derived EVs (bdEVs), or EVs originating or secreted / released by tissues or cells of the brain. It is envisaged that compositions of the disclosure can also be used to capture / isolate EVs from brain cells in culture. For example, cell culture supernatant can be obtained under culture conditions and then contacted with a composition of the disclosure to capture / isolate certain EV population(s).

[0171] All cells in the central nervous system (CNS) have been shown to release bdEVs, and thus studying bdEVs would be useful in understanding the development of CNS diseases such as neurodegenerative diseases. bdEVs derived from neurons have been shown to regulate development of neuronal circuits and synaptic transmission. bdEVs derived from oligodendrocytes have been shown to support neurons during conditions of stress, and also regulate myelin sheath formation in neurons. bdEVs derived from astrocytes have been shown to regulate dendritic branching and complexity. bdEVs derived from microglia have been shown to modulate neuronal activity by stimulating or inhibiting various ion channels. bdEVs from support cells such as Schwann cells and ependymal cells have also been implicated in modulation of cell signalling, axonal regeneration and protein trafficking.

[0172] EVs or bdEVs may contain cargo, or contents, such as proteins (including chemokines and cytokines, and pathogenic proteins such as a-synuclein, P-amyloid and tau proteins), nucleic acids (such as mRNA, miRNA, tRNA-derived small RNAs (tDRs), Y RNAs, circular RNA (circRNA) and long non-coding RNA (IncRNA)), sugars, amino acids and lipids (such as cholesterol, phosphatidylcholine and sphingomyelin derivatives).

[0173] The present disclosure relates to compositions for capturing extracellular vesicles (EVs). In certain examples such compositions may be characterised based on having improved properties over capture / isolation compositions known in the prior art such as size exclusion techniques. The presently disclosed compositions allow for the capture, isolation or purification of compositions comprising EVs, or populations of EVs, that are derived from a particular tissue, such as the brain. The inventors have advantageously found that the presently claimed combination of capture agents allows for improved tissue-specificity of EVs, such as exosomes. The advantages of the invention are fulfilled by the use of a combination of capture agents that target surface-associated epitopes that allow for capturing and enriching certain subpopulations of EVs, such as brain derived EVs (bdEVs).

[0174] In one example, the capture agents are attached to the solid surface by a combination of electrostatic interactions and covalent bonding in a non-uniform manner.

[0175] In one example, the compositions comprise a heterogenous population of capture agents linked together, and / or bound to the solid surface by a combination of electrostatic interactions and covalent bonding in a non-uniform manner.

[0176] In one example, the arrangement and spacing of the capture agents bound to the solid surface generate a non-uniform, multi-dimensional stable architecture, with different densities of capture agents and linking agents per unit volume in different regions.

[0177] Without being bound to theory, the inventors envisage that such a construction forming a type of 3D matrix increases the density and accessibility of ligand binding sites, thereby, increasing the efficiency and specificity of the EV capture. The 3D matrix may also be described as an affinity matrix.

[0178] In one example, the surface topology of the two or more capture agents when forming a layer of interconnected capture agents bound to the solid surface forms a patchwork of non- uniform, heterogenous textures connected by linkages. For example, the layer of interconnected capture agents may have the structure of a layered lattice or a plurality of staggered lattices. The chemical or physical properties of the solid surface, which may be a polymer or glass, determines the adsorption or repulsion of the first layer of interconnected capture agents. Subsequent layers of interconnected capture agents are preferentially linked to the previous layer of interconnected capture agents. Again without being bound to theory, the inventors envisage that for the capture of certain epitopes, addition of multiple layers alters the accessibility of ligand binding sites can be advantageous for the targeting of certain surface- associated epitopes. However, this must be balanced against the added cost and time for manufacturing a 3D matrix with many layers. The one or more layers of interconnected capture agents may be fabricated using linking agents or crosslinkers of various arm lengths ranging, for example and without limitation, from 2 to 17 Angstroms. In fabricating a layer, linking agents or cross-linkers may be added or may be applied in a sequential fashion to attach, immobilize, and stabilize the capture agents in a random or semi-oriented directionality. The arrangement or distribution of capture agents in the layer will be affected by the size and surface chemistry of a capture agent relative to the neighbouring chemistry of another capture agent or linking agent within the layer. Capture agents of different sizes and shapes are linked or cross-linked together through one or more than one linking agent; whereby the crosslinked capture agents may be surface exposed or may be built into the layer. The overall structure of the one or more layers may be designed to include regions that are specifically formulated and located to identify and segregate particular EVs from the sample.

[0179] Additionally, the arrangement and spacing of the two or more capture agents linked together and / or bound to the solid surface may form a “molecular net” that further improves the ability of the compositions of the present disclosure to capture tissue-specific EVs, such as bdEVs.

[0180] Accordingly, the present disclosure also provides for a multipolymeric molecular net structure, the molecular net structure comprising a solid surface; and two or more capture agents that bind to extracellular vesicles (EVs) derived from a cell of the nervous system, wherein the two or more capture agents are attached to the solid surface.

[0181] In one example, the layered structure or molecular net comprises multiple layers, for example, two or three layers. In one example, the layered structure or molecular net comprises two layers. In one example, the layered structure or molecular net comprises three layers. The net may be built in a layered or striated manner. For example, the initial layer may be a homogenous or heterogenous mixture of a first plurality of capture agents adsorbed to a polymeric surface or glass surface, followed by the addition of one or more linking agents. This may then be followed by addition of homogenous or heterogenous mixture of a second plurality of capture agents, followed by one or more linking agents.

[0182] Accordingly, the present disclosure also provides for a layered multipolymeric molecular net structure, the molecular net structure comprising a solid surface; and two or more capture agents that bind to extracellular vesicles (EVs) derived from a cell of the nervous system, wherein the two or more capture agents are attached to the solid surface.

[0183] In some examples, the molecular net is a large polymeric heterogeneous network containing two or more different capture agents immobilized or linked in three-dimensional space to promote binding to EVs derived from a cell of the nervous system.

[0184] In one example, the one or more layers of interconnected capture agents, or molecular net is attached or fitted to a polymeric device. For example, one or more layers of interconnected capture agents or molecular nets can be formed; placed; adsorbed; adhered; glued; crosslinked; and / or fitted onto a polymeric; and / or non-porous; and / or corona etched; and / or molded; surface with one or more surface chemistry.

[0185] The one or more layers of interconnected capture agents, molecular nets and / or an arrangement of layers of molecular nets can include an array of structures and can be formed or attached to planar or bead surfaces to enable enhanced binding capacity and detection of various EVs from one or more brain cells simultaneously. Also disclosed are devices employing multiple different layers of interconnected capture agents, molecular nets or layers of molecular nets configured within one or more testing area to facilitate the capture, immobilization and isolation of multiple different EVs from one or more cells of the nervous system.

[0186] In one example, a molecular net is a branched pseudorandom copolymer comprising two broad classes of subunits: capture agents and linking agents. The subunits, or “monomers,” self-assemble to form a structure that bind to EVs derived from a cell of the nervous system in a sample.

[0187] Capture agents

[0188] The inventors have for the first time identified compositions for capturing EVs that are specific for cells of the nervous system, for example bdEVs. Accordingly, the present disclosure provides for compositions comprising two or more capture agents that bind to a plurality of brain cells are able to capture and enrich for the bdEV subpopulation.

[0189] Capture agents are macromolecules, typically binding proteins such as antibodies, that specifically bind to EVs. For illustration and not limitation, examples of capture agents include antibodies, polypeptides or proteins such as binding proteins, peptide fragments, engineered proteins and fusion peptides / proteins. An engineered protein may be a synthetic peptide such as one containing non-natural amino acids, e.g., peptidomimetic. A peptidomimetic refers to a mimetic of a peptide wherein the normal peptide chemistry has been altered, e.g., to enhance stability or efficacy of binding. The capture agent may be produced using recombinant techniques, e.g., cloned into an expression vector such as a plasmid and transfected into a host cell (e.g., CHO cells or bacteria cells such as E. coif). In general, the interaction between an EV and a capture agent is non-covalent.

[0190] Capture agents contain one or at least two, and usually several or many, reactive groups with which linking agent functional groups (described below) can react to form a covalent linkage. Exemplary reactive groups are the amino-terminus and lysine-amino groups. Additional examples of reactive groups found on antibodies include amines and carboxyl reactive groups. In some examples, the antibodies are not so derivatized but comprise only the reactive groups normally associated with antibodies.

[0191] In some examples, the one or more layers of interconnected capture agents or molecular net is composed of two or more capture agents that are linked together by one or more than one chemical linking agents or crosslinkers in a manner that preserves the three-dimensional structure and binding capacity of said capture agents.

[0192] In referring to molecular nets, the following terms may be used to describe capture agents in an individual net: One may refer to a single capture agent molecule, a single species (e.g., many capture agent molecules with the same target specificity), or multiple species (e.g., species of capture agents that recognize different targets).

[0193] In some examples, the capture agents are antibodies.

[0194] The compositions may comprise 2 to 38 different antibodies.

[0195] Examples of antibodies suitable for use in the present composition and methods are provided in Table 2.

[0196] The capture agents may bind to EVs derived from cells of the nervous system, such as brain cells. In one example, the capture agents binds to EVs from one type of brain cell, or bind to EVs derived from more than one type of brain cell. Various combinations of capture agents are contemplated that comprise capture agents that bind to EVs derived from one type of cell of the nervous system, and / or bind to EVs derived from more than one type of cell of the nervous system.

[0197] Combinations of capture agents that bind to EVs derived from one type of brain cell are able to capture and enrich for subpopulations of bdEVs derived from that brain cell.

[0198] Combinations of capture agents that bind to EVs derived from more than one type of brain cell, for example, two types of brain cells, are able to capture and enrich for subpopulations of bdEVs derived from the two types of brain cells.

[0199] Linking Agents

[0200] Linking agents (or “linkers” or “crosslinkers”) are used to connect capture agents to each other, and in some cases capture agents to a surface as described herein. A large number of suitable linkers are known in the art and many are commercially available. See, for example, Hermanson, G. T., B IOCONJUGATE TECHNIQUES 2ndEdition, 2008, Elsevier Inc., Oxford, which is incorporated herein by reference. Most often, linking agents are bifunctional (either homo-bifunctional or hetero-bifunctional). Bifunctional linking agents have two functional groups, each of which can bind to reactive groups of a capture agent. (Solely for ease of reference, chemically reactive groups on capture agents are referred to as “reactive groups” while chemically reactive groups on linkers are referred to as “functional groups.”) For illustration and not limitation, examples of bifunctional linking agents are provided in Table 1. Functional groups commonly used in commercially available linkers include amide, azide, imide, and ester. In some examples tri-functional or multifunctional linking agents may be used. Examples of multifunctional linking agents include hydroxymethylphosphino linkers and triazene linkers. In addition to the nature of linker functional groups, an important linker property is effective length, which is approximately the distance between two capture agents linked to the same linker molecule. For purposes of description, effective length can be considered approximately the same as the published extended chain length (in angstroms (A)). For example, the linker succinimidyl 4-formylbenzoate (SFB) has a length of about 6 A while the linker 5bis-dPEG6 NHS ester has a length of about 36 A. Linker length can also be characterized by reference to the number of carbons in a chain, by reference to a number of repeating units (e.g., ethylene glycol units) in the linker, etc. Different layers of interconnected capture agents or molecular nets may be constructed using different linkers or different combinations of linkers and, in certain versions of the layers or molecule nets, linker length(s) may vary from layer to layer of a multilayer structure. In these contexts, it will be appreciated that reference to preferred linker lengths is intended only to be approximate.

[0201] Linkers may be divided, for example, into four categories based on length: i) Zero length linkers (1 -Ethyl- 10-[10-dimethylaminopropyl] carbodiimide hydrochloride (EDC)) ii) Short, non-selective linkers (e.g., formaldehyde, glutaraldehyde) iii) Linkers with lengths in the range of about 6 to about 40 A iv) Linkers with lengths>40 A

[0202] EDC promotes the linkage of amino and carboxy moieties, but does not result in a physical linker, per se. Short, non-selective linkers may be used to stabilize molecular net layers. Numerous linkers with lengths in the range of about 6 to about 40 A are described in the scientific literature; examples are provided in Table 1. Linkers with lengths>40 A are described in the scientific literature or can be made as described in W02014 / 001673.

[0203] In some examples a layer of interconnected capture agents, molecular net, or molecular net layer, contains at least one linker with length in the range of about 6 to about 40 A. In some examples a molecular net, or molecular net layer, contains at least one, at least two, at least three, or more than three linkers with length in the range of about 6 to about 40 A and / or one or more linkers with length greater than 40 A. In some examples a layer of interconnected capture agents, a molecular net, or molecular net layer, contains at least one linker that is not formaldehyde or glutaraldehyde and / or at least one linker that is not a zero-length linker.

[0204] It will be appreciated that preferably linkers are used which do not form homopolymers. When multiple species of linkers are used in fabrication of a molecular net, the linkers and / or linking conditions are selected to minimize formation of linker-only heterooligomers or polymers.

[0205] Long linkers may be useful in certain layers of capture agents, nets or net layers, such as molecular nets in which EVs are captured. In some cases, long linkers can be prepared as described in W02014 / 001673. Briefly, in this method a linear biopolymer (polysaccharide, polypeptide, or polynucleotide) is linked by two heterobifunctional linkers so that the resulting molecule comprises two reactive groups separated by the length of the biopolymer plus linkers.

[0206] The crosslinkers may bind more than one capture agent; and may bind more than one type of capture agent in a manner that preserves the two or three-dimensional structure of the capture agents, and may preserve the secondary; tertiary; or quaternary structures of the capture agents. Preferably the binding of at least one recognition motif or binding site of the capture agents is preserved.

[0207] Exemplary chemical crosslinkers include (bisfsulfosuccinimidyl] suberate (BS3), [N-e- Maleimidocapropyl] succinimide ester (EMCS), ethylene glycol bisfsuccinimidylsuccinate] (EGS), NHS-(PEG)n-maleamide, NHS-(PEG)n-NHS, and where n can be 1 to 50; whereby the chemical crosslinkers can be between 2 and 200 Angstroms in length.

[0208] Cross-linkers may be applied at various concentrations ranging from 1 nanomolar to 1 millimolar. Cross-linkers and concentrations will be selected to achieve molecular net stability under various conditions such as, for example temperatures of 0 to 45 degrees Celsius, buffer conditions such as 0 to 800 millimolar salt and 0 to 11% detergent. Cross-linkers are selected so that the fabricated nets withstand lyophilization followed by rehydration.

[0209] In some examples, the linking agent is degradable, for example, either due to chemical or thermal instability. Exemplary degradable linking agents include PEG derivatives, poly(alkylene oxides), hydroxyalkyl acid esters, polyethylenimine triethyleneglycol (PEI- TEG), poly(spermine ketal ester), poly(spermine ester), amide linkers, thiol conjugation chemistry and the like. Exemplary degradable linkers include BS(PEG)9, poly(alkylene oxides), hydroxyalkyl acid esters, polyethylenimine triethyleneglycol (PEI-TEG), poly(spermine ketal ester), poly(spermine ester) or BS3.

[0210] In some examples, the properties of the linking agents are responsive to environmental conditions (pH, temperature, redox, chemistry, etc.) such that affinity of capture agents for EVs is altered. As the length and configuration changes, the properties of the molecular net may change. Exemplary linking agents of this type include poly(N-isopropylacrylamide), polyethylene glycol)diacrylate, N,N'-methylenebisacrylamide, N-vinyl-2-pyrrolidone, and 2- hydroxy-ethyl methacrylate.

[0211] Table 1: List of linking agents or linkers

[0212] Fabrication of the Interconnected Layers or Molecular Nets

[0213] To produce the layers or molecular nets of the invention, capture agents with reactive groups (“capture agent reactive groups”) are combined with linking agents or linkers having complementary functional groups (“linking agent functional groups”). In this context, “complementary” means the two groups interact to form a covalent chemical bond.

[0214] In one approach, the layer or molecular net is prepared by combining two or more capture agents or species of capture agents and at least one (and often more than one) species of linking agent. Assuming bifunctional linking agents are used, the capture agents and linking agents are combined under conditions in which most of the linking agent molecules in the net are conjugated to two capture agents and each capture agent is linked to one or more other capture agents via a linker. It will be expected that, depending on the linking agent(s) and capture agents, some linking agents may form intramolecular bonds with a single capture agent, but conditions of solvent, pH, buffers, temperature, monomer concentration and the like may be selected to promote linking of different capture agents to each other. It will be recognized that unlinked or singly linked capture agents may be enmeshed in the molecular net if not washed out during fabrication, but most or essentially all (e.g., >95% or >99%) of capture agents are linked to at least one other capture agent.

[0215] As a result of this process, the two or more capture agents become bound to the solid surface, solid support, substrate or polymer strand(s) by the linking agent.

[0216] In one example, the composition further comprises a first plurality of capture agents, wherein at least one of the capture agents selected from the first plurality of capture agents is directly bound to the solid surface by one or more linking agents. It will be recognized that the remaining capture agents of the first plurality of capture agents are still bound to the solid surface by virtue of being indirectly bound to the solid surface by being connected to the at least one capture agent and the one or more linking agents. Thus, in essence, all capture agents are in some form bound to the solid surface.

[0217] It will also be recognized that if multifunctional linking agents are used, individual linking agents may be conjugated to more than two capture agents. In the resulting layer or molecular net, capture agents are directly or indirectly connected to each other to form an amorphous three-dimensional structure, in contrast to structures in which, for example, a plurality of capture agents are individually linked or attached to an underlying solid surface. The resulting molecular net may be described, for purposes of description rather than definition, as a branched pseudorandom copolymer in which the monomers are capture agents and linking agents.

[0218] The molecular net may be called a random copolymer because given a mixture of multiple species of bi- or multi-functional linking agents, capture agents with multiple reactive groups, or multiple species of capture agents, the resulting branched polymer has an unpredictable, irregular, networked structure.

[0219] The molecular net may be called a pseudorandom copolymer because in contrast to a truly random polymer (a) linking agents bind capture agents but preferably do not bind linking agents, and capture agents bind linking agents but not other capture agents and (b) not every reactive group on a linker may bind to reactive groups on any capture agent.

[0220] For illustration, a layer or molecular net may be formed by (1) depositing a solution containing the capture agents (typically a pooled capture agent composition) at a site, and (2) adding the linking agent(s) (typically a pooled linking agent composition) to the capture agent containing solution, under conditions in which the linking agents and capture agents form a cross-linked net. The capture agents may, for example, be deposited on a solid planar surface (e.g., on a hydrophobic substrate). Typically, the net is self-assembling following mixture of the linking agents and capture agents. However, it is not excluded that in some examples a chemical catalyst or other agent (e.g., light activation of photoactivatable linking agents) might be used to promote formation of the net. The fabrication process is described in more detail below.

[0221] After the monomers are combined and the layer or net forms, it is sometimes useful to quench the binding reaction (e.g., using small molecules that render the linking agents unreactive) and / or remove any unlinked capture agent and / or linker. For example, unlinked monomers can be removed by washing the net using an aqueous buffer, optionally containing detergent.

[0222] Exemplary solid surfaces (also termed “solid supports” or “substrates”) are discussed below. The solid surface may be an inert material (e.g., glass or silica), a porous material (e.g., nitrocellulose), a material derivatized to bind the layer or net, etc. Suitable solid surfaces for making layers or molecular nets include, but are not limited to silica, glass, nitrocellulose, polystyrene, and polyurethane. If the substrate includes reactive groups the first layer of the net may bind covalently with the substrate. In some cases, the monomers are combined within a removable mold.

[0223] In some examples, the solid support is a column, a membrane, a magnetic bead, or a non-magnetic bead. In one example, the magnetic bead is a ferromagnetic bead, paramagnetic bead, or a super-paramagnetic bead.

[0224] In some examples, as discussed below, the layer or net may be formed over an “elevated molecular base” or “underlayer” such as a base of uncrosslinked polypeptides. When the underlayer is proteinaceous it may comprise one or more or all of the capture agents present in the first layer or first layer of the net, or may comprise other proteins (e.g., BSA) or molecules (e.g., carbohydrates). In another example, the “underlayer” does not include capture agents (e.g., does not include capture agents found in the layers). In another example, the “underlayer” is cross-linked only with glutaraldehyde or formaldehyde.

[0225] In an alternative approach, layers or nets may be formed by combining linking agents and capture agents in a syringe or other container and extruding the mixture into a liquid whereupon cross-linking occurs.

[0226] Optionally, a layer or molecular net (or molecular net layer) can be stabilized by contacting the layer or net with formaldehyde or glutaraldehyde, typically 0.037 mM for 15 min at ambient temperature.

[0227] In some examples, one or more layers, or layers of molecular net can be extruded, cut into with a sharp edge, a laser or other method; and fitted together.

[0228] Multiple layers of interconnected capture agents or molecular nets

[0229] Simple layers or net structures, made in one step, can be referred to as “one-region” or “one-layer” nets, the terms being used interchangeably. More complex layer or net structures can be referred to as “multiple layer,” “two-layer,” “three-layer” etc. structures. Multilayer may refer to the structure having 2, 3, 4, 5, 6, 7, 8, 9, 10 or more layers. Layers of interconnected capture agents or molecular nets used for medical or diagnostic purposes usually have at least two layers and may have more than two layers (e.g., 3-8 layers).

[0230] A two-layer structure is prepared by fabricating a first layer of interconnected capture agents, or a one-layer net, as described above, and fabricating a second layer by combining the first layer structure with at least one (and more often more than one) species of capture agent and at least one (and more often more than one) species of linking agent. It is convenient, although not entirely accurate, to visualize the process of making additional layers as pipetting solutions of capture agents and linking agents on top of the first layer to form a distinct second layer disposed above the first layer, optionally making a third layer, etc., resulting in a generally cylindrical structure or in a generally concentric series of hemispherical layers. Figure 1 illustrates a multilayer molecular net.

[0231] In some examples, the linking agents and capture agents are selected so that the composition of the one layer of a multilayer structure differs from the composition of at least one other layer. In some examples the at least one other layer is an adjacent layer. In some examples the linking agents and capture agents are selected so that the composition of the one layer of a multilayer net differs from all other layer of the net. For example, in some versions the composition of a second layer is distinct from the composition of a first layer. Thus, a second layer may differ from a first layer in terms of the capture agent composition and / or linker composition. Two layers may have different porosity as a result of the selection of capture agents and linking agents.

[0232] As will be apparent to the skilled person, the porous nature of the multi-layer structure or molecular net means that linking agents and capture agents added in the second step will not necessarily remain entirely “on top” of the first layer. Rather, linking agents and capture agents may migrate into the “pores” of the first layer. However, such migration can be limited so that while both populations of linking agents and capture agents may be somewhat intermingled at the interface between the first and second layers, the bottom of the first layer (assuming for the moment a cylindrical structure) of the resulting multilayer net will have the composition of the linking agents and capture agents added in the first step, and the top of top-most or outer-most layer of the multilayer net will have the composition of the linking agents and capture agents added in the second step.

[0233] A result of this intermingling is that the composition at the interface between two layers may be somewhat different from components used to form either of the two layers.

[0234] The interface between two layers with the same capture agent and linker composition (such as a multilayer net in which each layer has the same composition) will also differ from non-interface regions of the net. Typically, the concentration of capture agents and linking agents is higher in the interface because of the fabrication process. That is, forming a first layer using 1 microliter each of capture agent and linker solutions and then forming a second layer (and interface) by adding 1 microliter each of capture agent and linker solutions to the preformed first layer will result in a different density and distribution of capture agents and linking agents than forming a single layer from 2 microliters each of capture agents and linking agents.

[0235] In one example, a multilayer structure or net may have one layer as a core and additional layer(s) formed as concentric hemispheres or shell(s) surrounding the core. However, other shapes are possible. For example, multilayer molecular nets may have a roughly cylindrical or roughly rectangular shape. For example, a multilayer net may be narrower at one end than at the other (e.g., cone shaped). In some examples, the layers are configured so that a liquid (e.g., sample) contacting with the uppermost (or outermost) layer can flow consecutively through each subsequent layer. That is, not all “layers” are simultaneously or equally accessible.

[0236] It will be recognized that the linking agents added in the second step, in addition to cross-linking capture agents added in the second step to each other, will also link capture agents in the first layer with those added in the second layer, thus connecting the layers to each other.

[0237] For example, layers or molecular nets can simultaneously capture different multiple classes of EVs of different sizes and with different chemistries. For example, EVs belonging to multiple subtypes but all being derived from a particular tissue could be isolated. As another example, EVs being derived from multiple cell types but originating from a particular tissue could be isolated. This allows for simultaneous detection of multiple indicia of disease progression (for example) which in turn provides higher confidence that the results being measured are related to pathogenesis within the originating tissue and not confounded by measuring changes in EVs unrelated to disease progression. In addition, we have observed that multiple layer structures or molecular nets achieve high signal-to-noise ratios and low nonspecific binding relative to other detection approaches. For a given footprint, the multiple layer structure or molecular net configuration facilitates binding and detection of more analyte per unit area, resulting in a more easily detectable signal (e.g., a visually detectable color signal). In addition, the multiple layer structures or molecular nets are readily adaptable to a variety of device formats and are stable and storable.

[0238] In one example, the composition of the present invention comprises two layers of interconnected capture agents bound to the solid surface, wherein the first layer comprises a first plurality of capture agents directly bound to the solid surface by one or more linking agents, and wherein the second layer comprises a second plurality of capture agents indirectly bound to the solid surface, wherein at least one of the capture agents selected from the second plurality of capture agents is indirectly bound to the solid surface by connecting to a capture agent selected from the first plurality of capture agents by one or more of the linking agents.

[0239] In one example, the composition of the present invention comprises two layers of interconnected capture agents bound to the solid surface, wherein the first layer comprises a first plurality of capture agents directly bound to the solid surface by one or more linking agents, and wherein the second layer comprises a second plurality of capture agents indirectly bound to the solid surface, wherein at least one of the capture agents selected from the second plurality of capture agents is indirectly bound to the solid surface by (a) no more than one capture agent selected from the first plurality of capture agents and (b) one or more of the linking agents.

[0240] In one example, the composition of the present invention comprises three layers of interconnected capture agents bound to the solid surface, wherein the first layer comprises a first plurality of capture agents directly bound to the solid surface by one or more linking agents, wherein the second layer comprises a second plurality of capture agents indirectly bound to the solid surface, wherein at least one of the capture agents selected from the second plurality of capture agents is indirectly bound to the solid surface by connecting to a capture agent selected from the first plurality of capture agents by one or more of the linking agents, and wherein the third layer comprises a third plurality of capture agents is indirectly bound to the solid surface by connecting to a capture agent selected from the second plurality of capture agents by one or more linking agents.

[0241] In one example, the composition of the present invention comprises three layers of interconnected capture agents bound to the solid surface, wherein the first layer comprises a first plurality of capture agents directly bound to the solid surface by one or more linking agents, wherein the second layer comprises a second plurality of capture agents indirectly bound to the solid surface, wherein at least one of the capture agents selected from the second plurality of capture agents is indirectly bound to the solid surface by (a) no more than one capture agent selected from the first plurality of capture agents and (b) one or more of the linking agents, and wherein the third layer comprises a third plurality of capture agents is indirectly bound to the solid surface by (a) no more than one capture agent selected from the second plurality of capture agents, (b) no more than one antibody selected from the first plurality of capture agents, and (c) at least two linking agents.

[0242] In one example, there is provided a layered molecular net comprising a solid surface, a first plurality of capture agents directly bound to the solid surface by one or more linking agents, and a second plurality of capture agents indirectly bound to the solid surface, wherein at least one of the capture agents selected from the second plurality of capture agents is indirectly bound to the solid surface by connecting to a capture agent selected from the first plurality of capture agents by one or more of the linking agents.

[0243] In one example, there is provided a layered molecular net comprising a solid surface, a first plurality of capture agents directly bound to the solid surface by one or more linking agents, and a second plurality of capture agents indirectly bound to the solid surface, wherein at least one of the capture agents selected from the second plurality of capture agents is indirectly bound to the solid surface by (a) no more than one capture agent selected from the first plurality of capture agents and (b) one or more of the linking agents.

[0244] In one example, there is provided a layered molecular net comprising a solid surface, a first plurality of capture agents directly bound to the solid surface by one or more linking agents, a second plurality of capture agents indirectly bound to the solid surface, wherein at least one of the capture agents selected from the second plurality of capture agents is indirectly bound to the solid surface by connecting to a capture agent selected from the first plurality of capture agents by one or more of the linking agents, and a third plurality of capture agents indirectly bound to the solid surface by connecting to a capture agent selected from the second plurality of capture agents by one or more linking agents.

[0245] In one example, there is provided a layered molecular net comprising a solid surface, a first plurality of capture agents directly bound to the solid surface by one or more linking agents, a second plurality of capture agents indirectly bound to the solid surface, wherein at least one of the capture agents selected from the second plurality of capture agents is indirectly bound to the solid surface by (a) no more than one capture agent selected from the first plurality of capture agents and (b) one or more of the linking agents, and a third plurality of capture agents indirectly bound to the solid surface by (a) no more than one capture agent selected from the second plurality of capture agents, (b) no more than one antibody selected from the first plurality of capture agents, and (c) at least two linking agents.

[0246] In one example, the first plurality of capture agents forms a first layer.

[0247] In one example, the second plurality of capture agents forms a second layer. In one example, the third plurality of capture agents forms a third layer.

[0248] In these examples, the plurality of capture agents may be homogenous (i.e., the plurality may comprise only one particular capture agent) or heterogenous (i.e., the plurality may comprise one or more different types of capture agents). For example, wherein the capture agents are antibodies, and wherein the compositions comprise two antibodies, e.g., MBP and GFAP, a homogenous plurality of antibodies refers to the plurality only comprising MBP or GFAP antibodies; and a heterogenous plurality of antibodies refers to the plurality comprising a mixture of MBP or GFAP antibodies.

[0249] The three-dimensional structure of layers or molecular nets

[0250] The layers or molecular nets described herein may be understood as three-dimensional structures with a pseudo-random composition including at least capture agents and linking agents. The structure and dimensionality of the layers or molecular nets may be better understood with reference to certain exemplary configurations that layers or molecular nets may assume.

[0251] The term “direct binding” or “directly bound” when used in the context of capture agents being bound to the surface means that there are no intervening capture agents between the surface and the capture agent in question. However, a capture agent is still considered to be directly bound to the surface if intervening linkers lay in between the surface and the capture agent in question.

[0252] The term “indirect binding” or “indirectly bound” when used in the context of capture agents being bound to the surface means that there is one or more intervening capture agents between the surface and the capture agent in question.

[0253] The term “shortest linkage” when used in the context of capture agents being bound to the surface refers to the shortest path (i.e., the path of least steps or jumps) of the multiple alternative paths leading from the capture agent in question to the surface.

[0254] Figure 1 is a schematic diagram of interconnected layers of capture agents, or a molecular net formed on base 350, a polystyrene base having a top surface 352. The layers or molecular net includes two species of capture agents, a number of each species are depicted. While the capture agent of the first species depicted as triangles are identical in structure, their relationship, or linkage, to surface 352 varies. Capture agent 354 is directly bound to the surface via linker 356. Capture agent 354 can be considered a first-order capture agent, as it is attached to surface 352 by a shortest linkage path (via linker 356) that does not include any other capture agents. Additionally, capture agent 360 is directly bound to the surface by two linkers. Capture agent 354 is linked to surface 352 by alternative paths that do include one or more other capture agents (e.g., capture agent 358); however, the linkage having the least number of intervening capture agents is defined as the shortest linkage and defines the order of a capture agent, with respect to the surface of the base. Thus, capture agent 3510 is a second-order capture agent (albeit, belonging to the second species of capture agents denoted as squares), as its shortest linkage includes one additional capture agent (capture agent 354 or 358). Capture agent 3510 is thus indirectly bound to the surface via its linkage to first order capture agent 354 or 358, both of which are directly bound to the surface via linkers 356. Similarly, capture agent 3512 also belonging to the second species of capture agents is a third-order capture agent having a shortest linkage including two other capture agents.

[0255] It should be noted that the exemplary configuration of Figure 1 illustrates that the capture agents do not have to be directly bound to the surface. The third order capture agent 3512 is only indirectly bound to the surface via its linkage to linkers that are cross-linked to second order capture agents 3510. While the capture agents in Figure 1 are shown only to be linked to a maximum of two other capture agents, it is possible that the capture agents bind to more than two other capture agents in the multilayer structure.

[0256] Exemplary methods of making a layered structure according to the disclosure include preparing a first layer by incubating a mixture of capture agents with a bead and a linking agent for around 15 minutes at room temperature. A second layer is then produced by incubating a mixture of capture agents with a linking agent for around 15 minutes at room temperature. The second layer is then attached to the first layer by incubating it with the second layer and a linking agent for around 15 minutes at room temperature. This process can then be repeated to add third and subsequent layers as required.

[0257] Methods of capturing or isolating EVs

[0258] The compositions of the present disclosure can also be used in various research and / or medical applications, such as methods of capturing or isolating EVs from a sample. The present methods may be called a selective EV capture method, in that the compositions result in selective capture of bdEVs from a sample. The present methods are distinguished from other prior art methods including those that describe non-selective EV capture methods. In prior art non-selective EV capture methods, such methods may capture EVs indiscriminately, and without distinguishing between tissue and / or cell from which the EV is derived from. Prior art methods, while being able to successfully capture EVs, suffer from a lack of tissue / cell specificity and thus have limited utility in understanding tissue / cell-specific changes that arise due to disease development.

[0259] Advantages of selective capture methods such as those of the present disclosure include the ability to capture particular EV subtypes, such as bdEVs, in order to study the otherwise inaccessible central nervous system (CNS).

[0260] Accordingly, the present disclosure relates to a method for capturing or isolating EVs from a sample, comprising contacting a sample from the subject with the composition of the present disclosure under conditions that enable binding of the capture agents to the EVs; and capturing or isolating the bound EVs from the sample. The term “capturing” when used in the context of EVs refers to the binding of EVs in preferably a releasable manner, for example, in order to remove EVs from a sample of interest and washing away the remaining unbound components. In an example, an EV is “captured” once it is bound by a composition of the disclosure.

[0261] The term “isolating” when used in the context of EVs refers to the steps of “capturing” EVs, but further includes one or more steps to isolate the EVs, such as exosomes, from the sample. For example, once the unbound components are washed away, the bound EVs could be permeabilized and brought into solution, and subsequently pelleted and stored for future use.

[0262] Exemplary conditions that enable binding of the capture agent to the EVs to capture the EVs in the sample are incubation for about 15 minutes at room temperature.

[0263] In one example, the method comprises contacting the sample from the subject with the composition of the present disclosure for about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes.

[0264] In one example, the method comprises contacting the sample from the subject with the composition of the present disclosure for about 1, 2 or 3 hours.

[0265] In one example, the method comprises contacting the sample from the subject with the composition of the present disclosure for about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes at about 20°C.

[0266] In one example, the method comprises contacting the sample from the subject with the composition of the present disclosure for about 1, 2 or 3 hours at about 20°C.

[0267] In one example, the method comprises contacting the sample from the subject with the composition of the present disclosure for about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes at about 22.5°C.

[0268] In one example, the method comprises contacting the sample from the subject with the composition of the present disclosure for about 1, 2 or 3 hours at about 22.5°C.

[0269] In one example, the method comprises contacting the sample from the subject with the composition of the present disclosure for about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes at about 25°C.

[0270] In one example, the method comprises contacting the sample from the subject with the composition of the present disclosure for about 1, 2 or 3 hours at about 25°C.

[0271] In one example, the method comprises contacting the sample from the subject with the composition of the present disclosure for about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes at about 20 to 25°C.

[0272] In one example, the method comprises contacting the sample from the subject with the composition of the present disclosure for 1, 2 or 3 hours at about 20 to 25°C.

[0273] In some examples, the solid surface is a magnetic or paramagnetic bead, and the EVs are isolated by magnetic isolation. In one example, the methods comprise a step of magnetic isolation to capture or isolate the bound EVs from the sample.

[0274] Accordingly, the present disclosure relates to a method for capturing or isolating EVs from a sample, comprising contacting a sample from the subject with the composition of the present disclosure to bind the capture agents to the EVs; and magnetically capturing or isolating the bound EVs from the sample.

[0275] Accordingly, the present disclosure also relates to a method for capturing or isolating EVs from a sample, comprising contacting a sample from the subject with the composition of the present disclosure to bind the capture agents to the EVs; and applying a magnetic force to capture or isolate the bound EVs from the sample.

[0276] In one example, the methods further comprise one or more steps of washing the sample following magnetic isolation or application of the magnetic force.

[0277] The samples may be freshly collected samples or samples that have been stored, e.g., frozen or refrigerated. Samples may be prior processed before being subjected to the methods of the present disclosure, such as clarifying, filtering or debris / contaminants removal. Suitable methods include centrifugation, ultracentrifugation, filtration, or ultrafiltration.

[0278] In some examples, the sample is processed e.g., to remove cell debris or cells prior to the capturing or isolating steps.

[0279] Suitable filters for use include those having a size in the range about 0.1 to about 1.0 pm may be employed, for example, about 0.8 pm or 0.22 pm. One or more filtration steps may be performed including with successive filtrations using filters with decreasing porosity.

[0280] In one example, the sample is substantially cell-free.

[0281] The methods and uses of the present disclosure may have utility in a number of applications, including but not limited to, (i) the isolation of EVs derived from one or more particular nervous system cell types or (ii) the isolation of EVs specifically derived from the brain, i.e., containing EVs derived from one or more brain cell types for further analysis.

[0282] Accordingly, the present disclosure also relates to a method for preparing a composition of extracellular vesicles enriched for EVs derived from one or more nervous system cells comprising contacting a sample from the subject with the composition of the present disclosure under conditions that enable binding of the capture agents to the EVs; isolating the bound EVs from the sample to generate the composition of EVs enriched for EVs derived from one or more brain cells.

[0283] The term “enriched” refers to increasing the concentration of a desired subtype of EV, and / or EV originating from a particular tissue type, for example the brain. For example, enriched may refer to the composition having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% EVs derived from one or more brain cells of interest. Put differently, enriched may mean that the composition is substantially free of EVs that are not derived from the one or more brain cells of interest. Taking the example wherein the brain cells of interest are neurons, a composition enriched for EVs derived from neurons may refer to the composition having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% EVs derived from neurons; or alternatively a composition that is substantially free of EVs that are not derived from neurons.

[0284] Accordingly, the present disclosure also relates to a composition comprising EVs, or a population of EVs enriched for EVs derived from one or more cells of the nervous system.

[0285] In one example, the composition comprising EVs or the population of EVs is enriched for EVs derived from a cell of the nervous system selected from the group consisting of neurons, microglia, astrocytes, oligodendrocytes, Schwann cells or ependymal cells. In one example, the composition comprising EVs or the population of EVs is enriched for EVs derived from neurons. In one example, the composition comprising EVs or the population of EVs is enriched for EVs derived from microglia. In one example, the composition comprising EVs or the population of EVs is enriched for EVs derived from oligodendrocytes. In one example, the composition comprising EVs or the population of EVs is enriched for EVs derived from astrocytes. In one example, the composition comprising EVs or the population of EVs is enriched for EVs derived from Schwann cells. In one example, the composition comprising EVs or the population of EVs is enriched for EVs derived from ependymal cells.

[0286] Various ways of determining if a composition or population is enriched for particular EVs could be envisaged by a person skilled in the art. For example, the EVs could be subjected to further analysis to determine the magnitude or strength of expression of particular markers indicative of a particular EV subtype, that the EVs are derived from one or more types of brain cells or to determine that the EVs are derived from a particular tissue, such as the brain.

[0287] In one example, the markers are selected from the group comprising CD56 (NCAM-1), GD2, CXCR1, CD90 (THY1), MOG, GLAST, 04, TDP-43, VGLUT, TMEM119, SNAP-25. In another example, the markers are selected from the group consisting of CD56 (NCAM-1), GD2, CXCR1, CD90 (THY1), MOG, GLAST, 04, TDP-43, VGLUT, TMEM119, SNAP-25.

[0288] Markers for specific types of EVs may also be assessed to confirm whether the composition or population is enriched for EVs generally.

[0289] In one example the EVs are CD9+, Flotilin-1+, and / or TSG101+.

[0290] Treatment methods with diagnostic step

[0291] The compositions of the present disclosure can also be used in various research and / or medical applications, such as methods of diagnosis, treatment, preparation of compositions useful in research or medicine, or methods of risk assessment. The methods may involve measurement of one or more biomarkers associated with a disease, disorder or condition, the creation of molecular or biomarker signatures that are indicative of a disease, disorder or condition, screening of patients for a specific disease, disorder or condition, monitoring progression of a disease, disorder or condition, and / or evaluation and / or monitoring efficacy of a treatment administered to a subject in need thereof. Examples diseases, disorders or conditions are typically neurodegenerative diseases, including but not limited to Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, motor neuron disease (MND), amyotrophic lateral sclerosis (ALS), peripheral neuropathy and Guillain-Barre syndrome.

[0292] The term “biomarkers” refers to a molecule wherein a detectable change can be measured, and such a change is correlated or associated with an underlying disease, disorder or condition. For example, a biomarker may be a gene or protein, and changes in e.g., genetic expression or protein content are correlated or associated with underlying disease.

[0293] The term “associated with” when used in the context of two different molecules or entities refers to when the level, change and / or magnitude of one is correlated to the other. For example, a specific biomarker is associated with a particular disease if its presence correlates with the incidence / presence and / or risk / likelihood / susceptibility of the disease. In another example, a specific biomarker is associated with a particular disease if a change in its expression or content (relative to a suitable control, such as a healthy subject) correlates with the incidence / presence and / or risk / likelihood / susceptibility of the disease.

[0294] It will be understood that the methods or uses in the above applications are not intended to be limited to any particular disease, disorder or condition, nor any biomarkers associated with any disease, disorder or condition. Rather, the compositions and methods of the present disclosure advantageously allow for the capturing and isolation of particular EVs of interest, such as exosomes, and allow for the downstream analysis of their contents or cargo. While studies have previously reported differences in EV contents or cargo between the disease or healthy states, there have been no reports of a reliable test analysing samples containing EVs and associating these samples with a disease. It is likely that these prior art methods have suffered from an inability to capture EVs specific from a particular tissue type, such as bdEVs from the brain. For example, when examining neurodegenerative diseases, it would be preferable to specifically capture and isolate bdEVs, that is EVs originating from the brain, as it is likely that, for a particular timepoint in the development of the neurodegenerative disease, any pathogenic changes in EV cargo or content would only be found in bdEVs.

[0295] The inventors here for the first time have developed improved methods of capturing or isolating EVs that allow for the capturing or isolating of EVs with higher tissue-specificity than prior art methods. As disclosed above, the inventors have discovered specific combinations of capture agents for capturing or isolating EVs derived from one or more brain cells that provide for this improved effect. Thus, samples containing EVs derived from various types of brain cells can be captured or isolated and used for further study, in particular in relation to studying and understanding their roles in the development of diseases, such as neurodegenerative disease. Accordingly, the present disclosure also relates to a method of diagnosing a neurodegenerative disease in a subject.

[0296] The methods and uses of the present disclosure may have utility in a number of applications, including but not limited to, (i) the isolation of EVs derived from one or more particular nervous system cell types (e.g., where a particular nervous system cell type is known to or has been reported to have a role in disease development) or (ii) the isolation of EVs specifically derived from the brain, i.e., containing EVs derived from one or more brain cell types (e.g., where there is interest to understand tissue-wide changes that may be related to disease development).

[0297] Accordingly, the present disclosure also relates to a method for preparing a composition of extracellular vesicles enriched for EVs derived from one or more brain cells for diagnosing or treating a neurodegenerative disease in a subject.

[0298] The above methods and uses may also be used in the treatment of neurodegenerative disease. Where a subject has been diagnosed with, or has been determined to likely be suffering from a neurodegenerative disease, a therapeutic agent may be administered to the subject in order to treat the neurodegenerative disease.

[0299] Accordingly, the present disclosure also relates to a method of treating a neurodegenerative disease in a subject comprising administering a therapeutic agent to a subject.

[0300] For example, the therapeutic agent may be a small molecule inhibitor, induced pluripotent stem cells, a cytokine such as GM-CSF, a therapeutic antibody such as a monoclonal antibody, a viral vector, an immunotherapy, a vaccine such as an a-synuclein or amyloid-P vaccine, and a dietary supplement such as an omega-3 fatty acid, eicosapentanoic acid (EP A) or docosahexanoic acid (DHA).

[0301] The present disclosure also provides a method of assessing the likelihood of a subject suffering from a neurodegenerative disease, comprising contacting a sample from the subject with the composition of the present disclosure under conditions that enable binding of the capture agent to the EVs; isolating the bound EVs from the sample to generate the composition of EVs enriched for EV derived from one or more cells of the nervous system; measuring and / or detecting expression of one or more biomarkers associated with the neurodegenerative disease in the composition of EVs enriched for EV derived from one or more cells of the nervous system; comparing the expression of the one or more biomarkers in the sample with a suitable control; and calculating a risk score for the subject based on the expression of the one or more biomarkers to assess the likelihood of the subject suffering from a neurodegenerative disease.

[0302] For example, a biomarker could be a change in genetic expression correlated with an increased or decreased expression of a protein found in the contents or cargo of an EV, such as an exosome. In one exemplary method, a sample containing EVs may be subjected to the methods or compositions of the present disclosure and the EV subtypes / subpopulations of interest isolated from the sample. An analyte of interest, for example proteins, may be isolated from the EV subtypes / subpopulations of interest and subjected to downstream analysis, such as mass spectrometry analysis to identify and quantify the proteins within the EV subtypes / subpopulations of interest. Screening of the enriched EV associated proteins could then be performed and the extent of upregulation / downregulation of genes associated with the protein quantification calculated or measured. In this way, protein biomarkers that are for example known to be upregulated in a disease state could be measured, and if a sample were found to have upregulation of this protein biomarker, it may be concluded that the subject from which the sample was extracted from has, or is likely to have the disease. Similarly, if a downregulation of the biomarker was associated with a disease state, then a finding of downregulation of that specific biomarker in a sample may indicate that the subject has, or is likely to have the disease.

[0303] Additionally, or in the alternative, a number of protein biomarkers could be measured and a molecular signature or risk score generated based on the upregulation or downregulation of the protein biomarkers in comparison to reference values in a suitable control, such as a healthy subject. In one example, each protein biomarker could be allocated a weighted score based on the measurement or detection of each biomarker based on a reference value (e.g., a suitable healthy control) and the combined weighted scores of each biomarker being assayed combined to generate a molecular signature. The weighted score is based on the indicative or diagnostic power of the biomarker, for example, a biomarker of higher diagnostic power is allocated a greater weighted score. In some circumstances, a scoring matrix may be utilised to allocate the weighted scores. It is also possible that certain biomarkers are better predictors of a specific neurodegenerative disease, compared to other biomarkers. Another possibility envisaged by the inventors is that different panels of biomarkers, comprising different combinations of specific biomarkers may be measured or detected depending on the specific circumstances. For example, measuring or detecting the expression levels for certain biomarkers having greater indicative or diagnostic power may be more challenging or difficult in certain samples. This could be due to, for example, those biomarkers having a relatively low expression in those samples. Alternatively, it is possible that the sample needs to be subjected to complicated processing steps before those biomarkers can be detected. In those circumstances, it may be more desirable to assemble a panel of biomarkers having relatively lower indicative or diagnostic power. Nonetheless, the inventors envisage that as a result of the enrichment of tissue-specific EVs, the person skilled in the art would be able to apply known ways of measuring expression of the biomarkers, then diagnosing or classifying a subject as suffering from or being likely to suffer from a neurodegenerative disease based on a clear differentiation between healthy subjects and subjects having a neurodegenerative disease. The present disclosure also provides for methods of classifying a subject as having or being likely to suffer from a neurodegenerative disease, comprising contacting a sample from the subject with the composition of the present disclosure under conditions that enable binding of the capture agent to the EVs; isolating the bound EVs from the sample to generate a composition of EVs enriched for EVs derived from one or more cells of the nervous system; measuring and / or detecting expression of one or more biomarkers associated with the neurodegenerative disease in the composition of EVs enriched for EV derived from one or more cells of the nervous system; classifying the subject as having or being likely to suffer from a neurodegenerative disease if the sample shows an elevated or reduced level of the one or more biomarkers relative to the reference threshold level.

[0304] Means for measuring or detecting changes to the expression levels of gene biomarkers in a sample can be achieved by any suitable method, which would be well known to the person of skill in the art, such as but not limited to PCR including real-time PCR (RT-PCR), quantitative PCR (qPCR), RNA sequencing (RNASeq), Next-generation sequencing (NGS), or microarrays. Such methods are known in the art and described in, for example, Sambrook, J., Russet D.W., ed. Molecular Cloning: A Laboratory Manual Volume 1, Chapter 7, pages 7.1 to 7.88. 2001. Cold Spring Harbor Laboratory Press; Rio, D. C. et al. RNA: A Lab Manual 2011, Cold Spring Harbor Laboratory Press.

[0305] Means for measuring or detecting changes to the expression levels of protein biomarkers, or fragments thereof, in a sample can be achieved by any suitable method, which would be well known to the person of skill in the art, such as mass spectrometry. Examples of suitable mass spectrometry include: ionisation sources such as El, CI, MALDI, ESI, and analysis such as Quad, ion trap, TOF, FT or combinations thereof, spectrometry, isotope ratio mass spectrometry (IRMS), thermal ionisation mass spectrometry (TIMS), spark source mass spectrometry, Multiple Reaction Monitoring (MRM) or SRM. The mass spectrometry may be conducted in combination with 2D gel electrophoresis, high performance liquid chromatography (HPLC) or other prefractionation or enrichment techniques. Methods for quantitation of molecules by two-dimensional gel electrophoresis, HPLC and mass spectrometry such as MALDI and SELDI are known in the art and are described in, for example, Simpson R. J., ed. Proteins and Proteomics: a Lab Manual. 2003 Cold Spring Harbor Laboratory Press; Sanchez, J. C. et al. Biomedical Applications of Proteomics 2004, Wiley- Blackwell. 425. Methods such as prefractionation are known and described in Ly and Wasinger (2008) Proteomics, 8(20): pp 4197-4208. Methods such as MRM are known in the art and described in, for example, Anderson and Hunter (2006) MCP, 5(4): pp.573-589.

[0306] In another example, the compositions or methods provided herein may also be useful in managing patient care e.g., for one or more individual subjects or a population of subjects. Accordingly, the present disclosure provides a method of screening a population of subjects for neurodegenerative disease, comprising contacting a sample from one or more of the population of subjects with the composition of the present disclosure under conditions that enable binding of the capture agent to the EVs, isolating the bound EVs from the sample, measuring and / or detecting expression of one or more biomarkers of the neurodegenerative disease in the isolated EVs to diagnose the neurodegenerative disease. In one example, the method may comprise repeating one or more steps of the method over a period of time at a prespecified frequency.

[0307] The present disclosure also provides a method of monitoring or evaluating progression of a neurodegenerative disease in a subject, comprising contacting a sample from the subject with the composition of the present disclosure under conditions that enable binding of the capture agent to the EVs, isolating the bound EVs from the sample, measuring and / or detecting expression of one or more biomarkers of the neurodegenerative disease in the isolated EVs to monitor progression of the neurodegenerative disease. In one example, progression of the neurodegenerative disease comprises measuring and / or detecting a change in one or more symptoms associated with the neurodegenerative disease.

[0308] The present disclosure also provides a method of monitoring or evaluating efficacy of a treatment of a neurodegenerative disease administered to a subject, comprising contacting a sample from the subject with the composition of the present disclosure under conditions that enable binding of the capture agent to the EVs, isolating the bound EVs from the sample, measuring and / or detecting expression of one or more biomarkers of the neurodegenerative disease in the isolated EVs to monitor efficacy of the treatment of the neurodegenerative disease. In one example, monitoring efficacy of the treatment of the neurodegenerative disease comprises measuring and / or detecting a change in one or more symptoms associated with the neurodegenerative disease.

[0309] A change in one or more symptoms may involve an improvement or worsening of symptoms, such as clinical symptoms. Such a change may be determined by observing or measuring changes in clinical symptoms such as determined by a change in a relevant clinical scoring system such as the Alzheimer’s Disease Composite Score (ADCOMS), the Alzheimer’s disease Scale-Cognitive Subscale (ADAS-Cog), the Clinical Dementia Rating (CDR) Scale, the Hoehn and Yahr scale, or the Unified Parkinson’s Disease Rating Scale (UPDRS).

[0310] The compositions or methods herein could be used to screen or monitor or evaluate a subject or population of subjects at a pre-specified frequency, such as monthly, annually, bi- annually, or another frequency as deemed appropriate by a skilled person. The subjects may be those that are pre-disposed or at a higher risk of developing the neurodegenerative disease, such as subjects being older than a specific age (e.g., over 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or over 80). Other subjects may be at higher risk of suffering or developing the neurodegenerative disease, such as based on family history of the disease, identification of certain risk factors for disease, results from other tests such as imaging tests (e.g., ultrasound, computerized tomography (CT) scans or magnetic resonance imaging (MRI) scans).

[0311] In one example, the EV is selected from the group consisting of: an exosome, a microvesicle, and an apoptotic body. In one example, the EV is an exosome.

[0312] Kits

[0313] The present disclosure also relates to systems and kits for use in the methods or uses of the invention. The kit may comprise one or more containers (such as tubes, vials, bottles and the like) either containing or separately containing the reagents of the kit, including but not limited to the compositions of the present disclosure. For example, various components of the compositions of the present disclosure may be separated into individual vials or containers, or combined into a single vial or container. Further, the systems and kits may further comprise an isolation member, such as a magnet. Where systems or kits are intended to be used in methods or uses related to measurement of biomarkers, the system or kits may further one or more chemical reagents and / or detection agents for performing detection or measurement of the biomarkers. Examples of typical reagents suitable for use in the kits according to the invention would be known to a person skilled in the art, and include but are not limited to forward and reverse primers, probes, buffers, polymerase enzymes, digestion enzymes, reverse transcriptase enzymes, detection antibodies or fragments thereof, fluorescent labelling dyes, aptamers, The kit may also comprise reagents for use in suitably preparing the biological samples, for example for pre-processing blood samples or to prepare liver tissue sections for immunohistochemistry purposes. The kits may also comprise washing solutions, diluents and buffers.

[0314] Suitable buffers include those suitable for biological samples such as phosphate, acetate, citrate and TRIS buffers. The buffers may additionally be pH balanced, and typically the pH of the buffer will range between 6 to 8.

[0315] Example detection antibodies contained in the kits may be antibodies capable of binding and allowing for the detection and measurement of expression of the biomarkers. These detection antibodies may suitably be used in binding assays such as an ELISA, and may be either directly labelled or indirectly labelled through the use of a further agent such as fluorophore or secondary antibody.

[0316] The kit may also comprise written instructions for performing the method. The kit may also provide standards or reference points for measuring or detecting the biomarkers in a method according to the invention. The standards may be for example reference proteins or peptides required to establish a standard curve that will allow more accurate determination of expression levels. EXAMPLES

[0317] Example 1: Study aims

[0318] 1. To develop a specific bead-based immunocapture EV isolation method (NEURONET) that can isolate and selectively capture nervous system-derived (e.g., astrocyte, glial, oligodendrocyte and neuronal) EVs from biofluids including urine, plasma, serum, CSF and saliva obtained from normal healthy individuals and patient diagnosed with neurodegenerative diseases including Alzheimer’s Disease (AD), Parkinson Disease (PD) and Amyotrophic Lateral Sclerosis (ALS).

[0319] 2. To confirm the isolation of bdEVs by NEURO-NET in comparison with other non- selective EV isolation methods (e.g., a non-selective EV capture bead-based kit and size exclusion column (SEC)).

[0320] Example 2: Generation of bead-based immunocapture method NEURO-NET

[0321] The inventors sought to design and generate an affinity matrix magnetic nanoparticle system designed specifically for the capture and / or isolation of nervous system-derived EVs from biofluids, such as plasma, serum and CSF which can be used for downstream analysis of EV associated proteins, oligonucleotides (mRNAs and microRNAs) and lipids for biomarker discoveries and diagnostic applications.

[0322] NEURO-NET is a three-dimensional (3D) affinity matrix paramagnetic bead. To prepare NEURO-NET, sequential covalent coupling of a cocktail of multiple brain antibodies (Table 1) was performed on a paramagnetic bead (diameter of 100-600 nm) using a linking agent (Bissulfosuccinimidyl suberate (BS3)) to form one or more layers of interconnected capture agent reactive groups. The sequential construction of the 3D matrix increases the density and accessibility of ligand binding sites, thereby, increasing the efficiency. Certain ligands may benefit from the addition of more layers, while others may work more effectively with less layers. Further, the addition of many layers also increases the costs and time required to produce the NEURO-NET. However, it is expected that the optimal number of layers for a specific ligand can be deduced through experimental testing based on the general guidance provided below, and the exact configuration of the NEURO-NET tailored for each specific use case.

[0323] In short, NEURO-NET is a three-dimensional (3D), affinity matrix which is built out from a paramagnetic bead. NEURO-NET may be constructed as a multilayer, three- dimensional (3D) affinity matrix if desired. In order to construct a two-layer NEURO-NET, the first layer of the matrix is formed by BS3 directed coupling of capture agents to the paramagnetic bead. A second mixture of capture agents are cross-linked using BS3 to form a second layer. The second layer is then coupled to the first-bead bound layer using BS3. It will be appreciated that other layers may then also be formed and added in a similar way if required. To illustrate this process, the methodology for producing a two-layer and three-layer NEURO-NET shown below. However, a similar amended process may be adopted if forming a NEURO-NET with more than three layers, such as four layers, five layers, six layers and so on. For example, a four-layer NEURO-NET could be developed by following Method 2 to develop a three-layer NEURO-NET, then repeating steps 11 to 14 to add an additional layer, prior to completing the process with step 15 of blocking the nets with BSA.

[0324] Method 1: Two-layer NEURO-NET

[0325] Underlayer

[0326] 1) Pre-mix a batch of underlayer: 1 : 1 ratio of albumin and formaldehyde

[0327] 2) Immediately add [pipette] 1 or 2 pl underlayer mixture on a surface

[0328] 3) Let cure for 15 minutes to 1 hour at room temperature First Laver

[0329] 4) Dilute linker stock at 1 :40 into amine-free PBS pH 7.4, use that day only

[0330] 5) Pre -mix a batch of capture and linker at 2:0.5 ratio of capture to linker stocks to generate 1st layer materials

[0331] 6) Immediately add [pipette] 1.25 or 2.5pl of first capture layer onto the top of the underlayer drop (if cured at higher temperature, evaporation occurs; place first capture layer onto the remnant underlayer)

[0332] 7) Let cure for 15 minutes to 1 hour at room temperature

[0333] Second Layer

[0334] 8) Pre-mix a batch of capture and linker at 2:0.5 ratio of capture to linker stocks to generate 2nd layer

[0335] 9) Immediately add [pipette] 1.25 or 2.5 pl of second capture layer onto the top of the 1st layer drop (if cured at higher temperature, evaporation occurs; place second capture layer onto the remnant 1st layer)

[0336] 10) Let cure for 15 minutes to 1 hour at room temperature

[0337] 11) Block nets with 200 of 0.5% BSA in PBS pH 7.4 for 12 hours at 4°C or for 2 hours at 37°C.

[0338] Method 2: Three-layer NEURO-NET

[0339] Underlayer

[0340] 1) Pre-mix a batch of underlayer: 1 : 1 ratio of albumin and formaldehyde

[0341] 2) Immediately add [pipette] 1 or 2 pl underlayer mixture on a surface

[0342] 3) Let cure for 15 minutes to 1 hour at room temperature

[0343] First Laver

[0344] 4) Dilute linker stock at 1 :40 into amine-free PBS pH 7.4, use that day only 5) Pre -mix a batch of capture and linker at 2:0.5 ratio of capture to linker stocks to generate 1st layer materials

[0345] 6) Immediately add [pipette] 1.25 or 2.5pl of first capture layer onto the top of the underlayer drop (if cured at higher temperature, evaporation occurs; place first capture layer onto the remnant underlayer)

[0346] 7) Let cure for 15 minutes to 1 hour at room temperature

[0347] Second Laver

[0348] 8) Pre-mix a batch of capture and linker at 2:0.5 ratio of capture to linker stocks to generate 2nd layer

[0349] 9) Immediately add [pipette] 1.25 or 2.5 pl of second capture layer onto the top of the 1st layer drop (if cured at higher temperature, evaporation occurs; place second capture layer onto the remnant 1st layer)

[0350] 10) Let cure for 15 minutes to 1 hour at room temperature

[0351] Third Laver

[0352] 11) Pre-mix a batch of capture and linker at 2:0.5 ratio of capture to linker stocks to generate 3rd layer

[0353] 12) Immediately add [pipette] 1.25 or 2.5 pl of second capture layer onto the top of the 2nd layer drop (if cured at higher temperature, evaporation occurs; place second capture layer onto the remnant 2nd layer)

[0354] 13) Let cure for 15 minutes to 2 hours at room temperature to quench linkers

[0355] 14) Let absorb to surface by storing overnight at 4°C

[0356] 15) Block nets with 200 of 0.5% BSA in PBS pH 7.4 for 12 hours at 4°C or for 2 hours at 37°C. Example 3: Isolation of extracellular vesicles

[0357] Sample information

[0358] Analytical validation and comparison were performed using 500 pl of pooled normal human plasma (Central BioHub, Germany). For clinical validation, 500 pl plasma from 10 age- matched healthy (ProteoGenex, USA), Alzheimer's (BioIVT, UK), and Parkinson’s (ProteoGenex, USA) patients were used.

[0359] Extracellular vesicles isolation

[0360] EVs were isolated using the NEURO-NET selective EV capture bead-based kit, a non-selective EV capture bead-based kit (EXO-NET, INOVIQ) and size exclusion columns (IZON qEVl 70 nm columns, Izon Science Limited, New Zealand) according to manufacturers’ instructions. Briefly, as shown in Figure 2, for NEURO-NET and EXO-NET, plasma samples were defrosted at room temperature and centrifuged for 5 min at 10000 x g. Cleared plasma (500 pl) was transferred to a new 1.5 mL microfuge tube and 30 pl of beads were added on top of the plasma and mixed by the gentle flicking of the tube until a homogenous suspension is formed. The mixture was incubated for 15 min at room temperature and then placed on the magnetic rack (ThermoFisher, #CS 15000) for 5 min. The supernatant was carefully removed and set aside for nanoparticle tracking analysis. Then 1 mL of PBS was added to wash the beads. The beads were washed an additional two times. After the final wash, beads were resuspended in the relevant lysis buffer for downstream analysis. For IZON columns, ImL of normal human plasma was passed through the SEC columns and fractions that contained EV (according to manufacturer’s instruction) were collected for further downstream analysis.

[0361] Example 4: Protein yield comparison

[0362] NEURO-NET captured EVs from 500pl of pooled normal human plasma was lysed in 30pl of sample lysis buffer (1% SDS) (n=16) and total amount of protein in each sample was measured using Nanodrop One spectrophotometer (ThermoFisher) at an absorbance of 280 nm. EVs isolated using another commercially available bead-based kit (EXO-NET, n=16) and SEC column (n=3) were used as the control groups.

[0363] Comparing NEURO-NET with the non-selective bead-based EV capture kit, NEURO-NET resulted in a slightly higher amount of protein isolated as compared to the non-selective beadbased EV capture kit. However, the amount of protein isolated using SEC was significantly greater than recovery using bead-based methods (Figure 3 A). This is consistent with the lack of EV specificity associated with SEC.

[0364] Based on the above results, the inventors further sought to evaluate the reproducibility of the protein and RNA yield from NEURO-NET captured EVs in plasma. EVs were isolated from 200JJ,1 of pooled normal human plasma with 15 pl of NEURO-NET using high-throughput KingFisher Apex instrument (10 replicates, 2 independent runs performed on different days). For protein yield, NEURO-NET captured EV were lysed in 1% SDS and protein yield was measured using NanoDrop One. For RNA yield, NEURO-NET captured EV were lysed in RNA solution buffer and RNA yield was measured using NanoDrop One. The results are indicated in Tables 3 and 4 below, and shown in Figure 3B.

[0365] Table 3: Protein yield using NEURO-NET over 10 replicates

[0366] Table 4: RNA yield using NEURO-NET over 10 replicates

[0367] The data suggests that protein and RNA yields using NEURO-NET were highly reproducible and resulted in optimal yield and recovery levels.

[0368] Example 5: Characterisation of isolated EVs

[0369] Western Blot

[0370] EV lysate containing same amount of protein isolated using NEURO-NET, another bead based EV isolation kit, and SEC were combined with 7.5 pl of 4X Bolt LDS Sample Buffer (ThermoFisher, #B007) and 3 pl of 1 OX Bolt Sample Reducing Agent (ThermoFisher, #B0009) and incubated for 5 min at 95°C for protein denaturation. The total protein lysate was then separated on 4-12% Bolt Bis-Tris Plus gels (ThermoFisher, #NW04120BOX) and transferred onto nitrocellulose membrane. The membranes were blocked for 1 h at RT with 2.5% BSA (Sigma, #A9647-50G) followed by overnight incubation at 4°C with primary antibodies at 1 :500 dilution in TBS-T. The membranes were then washed with TBST and then incubated with HRP-conjugated secondary antibody for 1 h at RT. Then membranes were washed again and visualised using ChemiDoc imaging system (BioRad, Germany).

[0371] EV markers analysis

[0372] Western blot analysis confirmed the presence of EVs markers such as CD9, Flotillin-1, and TSG101 in EVs isolated by NEURO-NET (Figure 4). Brain cell markers analysis

[0373] Western blot analysis was used to compare presence of markers from brain cells (neuron, microglia, oligodendrocyte, and astrocyte) in the EVs isolated using NEURO-NET, the non- selective EV capture bead-based kit and SEC column.

[0374] Results showed that NEURO-NET isolated EVs were positive for all the tested markers while EVs isolated using the other bead-based kit or SEC column were positive only for one or two markers (Figure 5A to 5C).

[0375] Further, the inventors sought to validate the above findings by testing the specificity of the NEURO-NET in isolating EVs from brain cell lines, in comparison to the non-selective EV capture bead-based kit (EXO-NET).

[0376] Western blot analysis was again used to compare the presence of astrocyte and neuronal markers in brain cell line derived EVs captured using NEURO-NET versus EXO-NET. Two brain cell lines were used, HMC3, which are microglial cells (HMC), and CCF-STTG1 which are astrocyte cells (CCF). EVs were isolated from 2mL of conditioned medium of HMC3 and CCF cell lines using 40pl of NEURO-NET or EXO-NET. Captured EVs were lysed in 1% SDS. A similar amount of protein was loaded (~ 7pg) to detect GD2 and GLAST expression in both scenarios.

[0377] Results showed that NEURO-NET isolated brain cell line derived EVs were positive for astrocyte and neuronal markers, while brain cell line derived EVs isolated using EXO-NET were negative for both astrocyte and neuronal markers (Figure 5D). This confirms the above findings and shows that NEURO-NET was able to isolate EVs from brain cells, at significantly higher levels as compared to standard, non-selective EV capture bead-based kits.

[0378] Example 6: Validation on clinical samples

[0379] Protein yield

[0380] Total protein isolated from 500 pl of plasma from Alzheimer’s, Parkinson’s and age-matched healthy samples were measured using Nano drop as described above. All three samples contained similar amount of protein with no observed statistically significant difference (Figure 6).

[0381] Western blot analysis

[0382] Western blot analysis was performed to compare expression of known Alzheimer’s markers such as Phospho-taul81 and phospho-tau217 in the EVs isolated from Alzheimer's vs healthy plasma using NEURO-NET as described. The results indicated that NEURO-NET was able to enrich EVs positive for Alzheimer’s markers from the Alzheimer’s plasma while the markers were not expressed or only minimally expressed in the healthy plasma (Figure 7A).

[0383] Based on the above findings, the inventors sought to further validate the use of NEURO-NET in enriching EVs positive for other biomarkers.

[0384] Western blot analysis was again used to compare the levels of Alzheimer’s markers phospho- tau217, TDP43 and neurofilament-light chain (Nf-L, or Neurofilament L) in NEURO-NET isolated EV proteins, as compared to neat plasma protein lysate. EV were isolated from 200 pL of pooled Alzheimer’s plasma samples (n=10) using 15ul of NEURO-NET. Isolated EVs and neat plasma samples were lysed in 1% SDS and equal amount of protein (8pg of protein / lane ) were loaded to detect P-tau 217 = phosphorylated at threonine 217; TDP43 = TAR DNA Binding Protein 43 and Neurofilament L.

[0385] The results indicated that NEURO-NET was able to enrich EVs positive for Alzheimer’s markers phospho-tau217, TDP43 and Nf-L from the Alzheimer’s plasma while the markers were only minimally expressed in the healthy plasma (Figure 7B).

[0386] Notably, TDP43 and Nf-L are also common biomarkers and pathological hallmarks of neurodegenerative diseases in general. Thus, this supports the proposition that NEURO-NET enriches samples for common neurodegenerative disease biomarkers, and has utility in diagnosing neurodegenerative diseases more broadly.

[0387] Pilot mass spectrometry analysis

[0388] EVs from 500 pl plasma samples from 10 Healthy, 10 Alzheimer's, and 10 Parkinson’s was isolated using NEURO-NET. The isolated protein was then quantified and sent for mass spectrometry analysis to identify potential biomarkers in EVs isolated using NEURO-NET for Alzheimer’s and Parkinson’s diseases.

[0389] Results from the mass spectrometry analysis identified hundreds of proteins being significantly up or down regulated in AD and PD samples compared to healthy individuals (Figures 8 and 9). Around 406 proteins were significantly down regulated, whereas 328 markers were significantly up regulated in Alzheimer’s samples compared to healthy ones. List of top 20 up and down regulated markers is shown in Figures 10 and 11. For Parkinson’s samples, 189 markers were significantly down regulated (Figure 12), but 278 markers were significantly up regulated compared to healthy samples (Figure 13). This pilot data surprisingly shows that NEURO-NET has specifically screened and enriched EVs associated proteins and differentiated between healthy and diseased samples. This is a significant advance in the art as the inventors have shown for the first time that enriching for nervous system-derived EVs may be sufficient to discriminate diseased samples from healthy samples simply on the basis that the cargo of nervous system-derived EVs is altered in the disease state. While it has been postulated for some time that EVs from diseased tissue have altered cargo or contents, the inventors hypothesize that such results have not been shown previously as the present compositions and methods for the first time allow sufficiently high specificity of nervous system-derived EVs capture to enable such an analysis and tissue discrimination.

[0390] Example 7: Expanded clinical study

[0391] Based on the above findings, the inventors sought to conduct a follow-up, expanded clinical study to verify the ability of the NEURO-NET to screen and enrich for EV associated proteins.

[0392] EVs were isolated from 500 pL plasma samples from 44 Alzheimer’s patients (34 purchased from ProteoGenex,USA and 10 were purchased from BioIVT, UK) and 48 age matched, healthy donor (purchased from ProteoGenex, USA) using 3 Opl of NEURO-NET. Protein was isolated from NEURO-NET captured EVs and sent for mass spectrometry analysis.

[0393] Mass spectrometry analysis identified 854 of proteins being significantly up or down regulated in AD compared to healthy individuals (Figure 14). Around 599 proteins were significantly down regulated, whereas 255 markers were significantly up regulated in Alzheimer’s samples compared to healthy controls. The list of top 50 up and down regulated markers is shown in Figure 15. Furthermore, there were 47 proteins exclusively expressed in Alzheimer’s sample compared to control samples (Figure 16 and Table 5).

[0394] The data further suggests the ability of the NEURO-NETs to distinguish between healthy and Alzheimer’s disease samples based on the differentiation of nervous-system derived EV cargo and supports the data in Example 6 above. Notably, the data suggests that differentially expressed proteins can differ between various studies; however, the key observation is that the differential expression of these proteins clearly delineates between healthy and diseased patient populations. Table 5: List of the 47 proteins unique to current Alzheimer’s disease samples.

[0395] Example 8: Discussion

[0396] The inventors have shown that the NEURO-NET affinity matrix magnetic nanoparticle technology was able to enrich nervous system-derived EVs from biofluids. Specifically, NEURO-NET was found to capture EVs from pooled human plasma, and these EVs expressed known EV protein markers including CD9, Flotillin, and TSG101. In addition, NEURO-NET captured EVs were positive for 9 out of 10 brain known markers compared to other bead based and SEC method which were shown to be positive for only 2 out of 10 brain markers. When assessing EVs derived from brain cell lines, NEURO-NET captured brain derived EVs successfully while the other non-selective bead based method was unsuccessful. NEURO-NET also resulted in desirable protein and RNA yields and recovery from EVs in plasma, a finding which was highly reproducible over multiple runs.

[0397] NEURO-NET isolated EVs were also enriched for disease-specific biomarkers in neurodegenerative diseases, as compared to neat plasma. Further, NEURO-NET was then applied to perform downstream analysis of associated proteins, oligonucleotides (mRNAs and microRNAs) and lipids specifically from AD and PD patient samples. There were distinct genetic expression profiles or molecular signatures that separated patients suffering from AD or PD and healthy controls. This data supports the use of the present affinity matrix for the diagnosis of these neurodegenerative diseases.

[0398] These surprising data underpin a general approach for selective capture of nervous system- derived EVs such as exosomes. In certain instances, such a platform can be adapted to selectively capture nervous system-derived EVs from desired cell population(s) such as neurons. The approaches underpinned by the present inventors’ findings are particularly advantageous in terms of distinguishing between disease states and open doors for further applications on this front.

[0399] It will be appreciated by persons skilled in the art that numerous variations and / or modifications may be made to the disclosure as shown in the specific embodiments without departing from the spirit or scope of the disclosure as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. The present application claims priority from Australian Patent Application Number 2023903359 filed 20 October 2023, the disclosures of which are incorporated herein by reference. All publications discussed and / or referenced herein are incorporated herein in their entirety.

[0400] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present disclosure. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each claim of this application.

Claims

CLAIMS1. A composition for capturing extracellular vesicles (EVs) in a sample, comprising:(i) a solid surface; and(ii) two or more capture agents that bind to extracellular vesicles (EVs) derived from cells of the nervous system, wherein the two or more capture agents are attached to the solid surface.

2. The composition of claim 1, comprising at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least 10 capture agents that bind to extracellular vesicles (EVs) derived from cells of the nervous system attached to the solid surface, preferably wherein the cells are neurons and / or brain cells.

3. The composition of claim 1 or 2, wherein the two or more capture agents bind to an EV derived from a brain cell selected from the group consisting of: neuron, microglia, oligodendrocyte, astrocyte, Schwann cell, and ependymal cell.

4. The composition of any one of claims 1 to 3, wherein the capture agents are selected from the group consisting of: antibodies, binding proteins, peptide fragments, engineered proteins and fusion peptides / proteins.

5. The composition of claim 4, wherein the capture agents are antibodies.

6. The composition of claim 5, wherein the two or more antibodies are selected from the group consisting of neurofilament-light chain (Nf-L), CD56 (NCAM), CD90 (Thyl), GD-2, SNAP25, OMG, doublecortin, beta III tubulin, neuroDl, [3-amyloid, phospho tau 181, phospho tau 217, TDP43, Tau, P-T181-tau, P-S396-tau and CD271 [and wherein the EVs are derived from neurons],7. The composition of claim 5 or 6, wherein the two or more antibodies are selected from the group consisting of CX3CR1, TMEM119, P2ryl2, Hexb, Sall 1, a-Synuclein, CD63, CD64, CD36, CD 15 and GDNF [and wherein the EVs are derived from microglia],8. The composition of any one of claims 5 to 7, wherein the two or more antibodies are selected from the group consisting of VGLUT-2, 04, TMEM30A, Oligl, Olig2, Olig3, OSP and MBP [and wherein the EVs are derived from oligodendrocytes].

9. The composition of any one of claims 5 to 8, wherein the two or more antibodies are selected from the group consisting of EAAT1, EAAT2, GFAP, ALDH1L1, S100 beta, Aquaporin-4 and CD44 [and wherein the EVs are derived from astrocytes],10. The composition of any one of claims 5 to 9, wherein the two or more antibodies comprise neurofilament-light chain (Nf-L), CD56 (NCAM), CD90 (Thyl), GD2, SNAP25 and OMG [and wherein the EVs are derived from neurons],11. The composition of any one of claims 5 to 10, wherein the two or more antibodies comprise CX3CR1 and TMEM119 [and wherein the EVs are derived from microglia],12. The composition of any one of claims 5 to 11, wherein the two or more antibodies comprise VGLUT-2, 04 and OMG [and wherein the EVs are derived from oligodendrocytes] .

13. The composition of any one of claims 5 to 12, wherein the two or more antibodies comprise CD90 (Thyl), GD2, EAAT1 and EAAT2 [and wherein the EVs are derived from astrocytes],14. A composition for capturing extracellular vesicles (EVs) in a sample, comprising:(i) a solid surface; and(ii) two or more capture agents that bind to extracellular vesicles (EVs) derived from a neuron, wherein the two or more capture agents are attached to the solid surface.

15. The composition of claim 14, wherein the two or more capture agents are antibodies selected from the group consisting of neurofilament-light chain (Nf-L), CD56 (NCAM), CD90 (Thyl), GD2, SNAP25, OMG, doublecortin, beta III tubulin, neuroDl, [3-amyloid, phospho tau 181, phospho tau 217, TDP43, Tau, P-T181-tau, P- S396-tau and CD271.

16. A composition for capturing extracellular vesicles (EVs) in a sample, comprising:(i) a solid surface; and(ii) two or more capture agents that bind to extracellular vesicles (EVs) derived from a microglia, wherein the two or more capture agents are attached to the solid surface.

17. The composition of claim 16, wherein the two or more capture agents are antibodies selected from the group consisting of CX3CR1, TMEM119, P2ryl2, Hexb, Sall 1, a- Synuclein, CD63, CD64, CD36, CD 15 and GDNF.

18. A composition for capturing extracellular vesicles (EVs) in a sample, comprising:(i) a solid surface; and(ii) two or more capture agents that bind to extracellular vesicles (EVs) derived from an oligodendrocyte, wherein the two or more capture agents are attached to the solid surface.

19. The composition of claim 18, wherein the two or more capture agents are antibodies selected from the group consisting of VGLUT-2, 04, TMEM30A, Oligl, Olig2, Olig3, OSP and MBP.

20. A composition for capturing extracellular vesicles (EVs) in a sample, comprising:(i) a solid surface; and(ii) two or more capture agents that bind to extracellular vesicles (EVs) derived from an astrocyte, wherein the two or more capture agents are attached to the solid surface.

21. The composition of claim 20, wherein the two or more capture agents are antibodies selected from the group consisting of EAAT1, EAAT2, GFAP, ALDH1L1, S100 beta, Aquaporin-4 and CD44.

22. The composition of any one of claims 1 to 21, wherein the two or more capture agents are attached to the solid surface by a linking agent.

23. The composition of claim 22, wherein the linking agent is selected from the group consisting of: Bissulfosuccinimidyl suberate (BS3), Disuccinimidyl suberate (DSS), Bis[2-(N-succinimidyl-oxycarbonyloxy)ethyl] sulfone (BSCOES), l-ethyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride (EDC); dithiobis(succinimidyl propionate) (DSP); disuccinimidyl tartrate (DST), preferably wherein the linking agent is BS3.

24. The composition of any one of claims 1 to 23, comprising a first plurality of capture agents, wherein at least one of the capture agents selected from the first plurality of capture agents is directly bound to the solid surface by one or more linking agents.

25. The composition of claim 24, further comprising a second plurality of capture agents, wherein at least one of the capture agents selected from the second plurality of capture agents is indirectly bound to the solid surface by connecting to a capture agent selected from the first plurality of capture agents by one or more linking agents.

26. The composition of claim 25, further comprising a third plurality of capture agents, wherein at least one of the capture agents selected from the third plurality of capture agents is indirectly bound to the solid surface by connecting to a capture agent selected from the second plurality of capture agents by one or more linking agents.

27. The composition of any one of claims 1 to 26, wherein the two or more capture agents form at least one layer of interconnected capture agents.

28. The composition of any one of claims 1 to 27, wherein the two or more capture agents form two layers of interconnected capture agents.

29. The composition of any one of claims 1 to 27, wherein the two or more capture agents form three layers of interconnected capture agents.

30. The composition of any one of claims 1 to 29, wherein the solid surface is selected from the group consisting of: a column, a membrane, a magnetic bead, or a nonmagnetic bead.

31. The composition of claim 30, wherein the solid surface is a magnetic bead or nonmagnetic bead having a diameter of about 50 - 5,000 nm.

32. A method of capturing extracellular vesicles (EVs) from a sample, comprising: contacting the sample with the composition of any one of claims 1 to 31 under conditions that enable binding of the capture agents to the EVs to capture the EVs in the sample.

33. A method of isolating extracellular vesicles (EVs) from a sample, comprising: contacting the sample with the composition of any one of claims 1 to 32 under conditions that enable binding of the capture agents to the EVs; and isolating the bound EVs from the sample.

34. The method of claim 32 or 33, wherein the sample is selected from the group consisting of: urine, saliva, nasal swab, faeces, tears, cerebrospinal fluid, ocular fluid,crevicular fluid, amniotic fluid, seminal fluid, vaginal fluid, menstrual fluid, plasma, blood, serum, and conditioned medium of a cell culture, preferably wherein the sample is plasma or serum.

35. A composition comprising extracellular vesicles (EVs) enriched for EVs derived from one or more neurons and / or brain cells, wherein the EVs are isolated by the method of claim 33 or 34.

36. A population of extracellular vesicles (EVs) enriched for EVs derived from one or more cells of the nervous system, wherein the EVs are isolated by the method of claim 33 or 34.

37. The composition of claim 35, or the population of claim 36, wherein at least 20% of the EVs in the composition or the population express a marker indicative of the EVs being derived from a cell from the nervous system.

38. The composition of claim 35, or the population of claim 36, wherein at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% of the EVs in the composition or the population express a marker indicative of the EVs being derived from one or more cells from the nervous system.

39. The composition of any one of claims 1 to 31, the method of any one of claims 32 to 34, the composition of any one of claims 35 and 37-38, or the population of any one of claims 35 to 37, wherein the EV is a membranous particle, preferably wherein the particle is selected from the group consisting of: an exosome, a microvesicle, and an apoptotic body.

40. The composition of any one of claims 1 to 31, the method of any one of claims 32 to 34, the composition of any one of claims 35 and 37-38, or the population of any one of claims 35 to 37, wherein the EV is an exosome.

41. The composition of any one of claims 1 to 31, the method of any one of claims 32 to 34, the composition of any one of claims 35 and 37-38, or the population of any one of claims 35 to 37, wherein the EV is a non-membranous particle, preferably wherein the non-membranous particle is an extracellular nanoparticle.

42. The composition of any one of claims 1 to 31, the method of any one of claims 32 to 34, the composition of any one of claims 35 and 37-38, or the population of any one of claims 35 to 37, wherein the EVs are CD9+, Flotilin- 1+, and / or TSG101+.

43. A method of diagnosing a neurodegenerative disease in a subject, comprising: contacting a sample from the subject with the composition of any one of claims 1 to 31 under conditions that enable binding of the capture agent to the EVs; isolating the bound EVs from the sample; and measuring and / or detecting expression of one or more biomarkers of the neurodegenerative disease in the isolated EVs to diagnose the neurodegenerative disease.

44. A method of diagnosing a neurodegenerative disease in a subject, comprising: contacting a sample from the subject with the composition of any one of claims 1 to 31 under conditions that enable binding of the capture agent to the EVs; isolating the bound EVs from the sample to generate a composition of EVs enriched for EVs derived from one or more cell types such as neuron and / or a brain cell; and measuring and / or detecting expression of one or more biomarkers of the neurodegenerative disease in the composition of EVs enriched for EV derived from the one or more cells to diagnose the neurodegenerative disease.

45. A method of treating a neurodegenerative disease in a subject, comprising: contacting a sample from the subject with the composition of any one of claims 1 to 31 under conditions that enable binding of the capture agent to the EVs; isolating the bound EVs from the sample; measuring and / or detecting expression of one or more biomarkers of the neurodegenerative disease in the isolated EVs to diagnose the neurodegenerative disease; and administering a therapeutic agent to treat the neurodegenerative disease, the therapeutic agent selected from the group consisting of: a small molecule inhibitor, induced pluripotent stem cells, a cytokine such as GM-CSF, a therapeutic antibody such as a monoclonal antibody, a viral vector, an immunotherapy, a vaccine such as an a-synuclein or amyloid-P vaccine, and a dietary supplement such as an omega-3 fatty acid, eicosapentanoic acid (EP A) or docosahexanoic acid (DHA).

46. A method for preparing a composition of extracellular vesicles (EVs) enriched for EVs derived from one or more neurons and / or brain cells useful for measuring and / ordetecting genetic expression of one or more biomarkers associated with a neurodegenerative disease, comprising contacting a sample from the subject with the composition of any one of claims 1 to 31 under conditions that enable binding of the capture agent to the EVs; isolating the bound EVs from the sample to generate the composition of EVs enriched for EV derived from the one or more cells; and measuring and / or detecting expression of one or more biomarkers associated with the neurodegenerative disease in the composition of EVs enriched for EV derived from one or more cells.

47. A method of assessing the likelihood of a subject suffering from a neurodegenerative disease, comprising contacting a sample from the subject with the composition of any one of claims 1 to 31 under conditions that enable binding of the capture agent to the EVs; isolating the bound EVs from the sample to generate the composition of EVs enriched for EV derived from one or more cells; measuring and / or detecting expression of one or more biomarkers associated with the neurodegenerative disease in the composition of EVs enriched for EV derived from one or more cells; comparing the expression of the one or more biomarkers in the sample with a suitable control; and calculating a risk score for the subject based on the expression of the one or more biomarkers to assess the likelihood of the subject suffering from a neurodegenerative disease.

48. The method of claim 47, wherein the risk score is calculated by allocating a weighted score to the expression of the one or more biomarkers in the sample based on a reference value in the control.

49. The method of any one of claims 43 to 48, wherein the EV is a membranous particle, preferably wherein the particle is selected from the group consisting of: an exosome, a microvesicle, and an apoptotic body.

50. The method of any one of claims 43 to 48, wherein the EV is an exosome.

51. The method of any one of claims 43 to 48, wherein the EV is a non-membranous particle, preferably wherein the non-membranous particle is an extracellular nanoparticle.

52. The method of any one of claims 43 to 51, wherein the neurodegenerative disease is selected from the group consisting of: Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, motor neuron disease (MND), amyotrophic lateral sclerosis (ALS), peripheral neuropathy and Guillain-Barre syndrome.

53. The method of any one of claims 43 to 52, wherein the sample is substantially cell- free.

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