Isolation and diagnostic methods using cell type-specific and / or organ-specific extracellular vesicle (EV) markers

Abstract

The present invention relates to novel biomarkers for cell type-specific and / or organ-specific extracellular vesicles, in particular brain-specific and / or neuron-specific extracellular vesicles, and combinations thereof. The invention also provides methods for isolating and / or enriching cell type-specific and / or organ-specific extracellular vesicles, methods for identifying cell-derived extracellular vesicles, and methods for diagnosing or prognosing a disorder (e.g., a neurodegenerative disorder) using cell type-specific and / or organ-specific extracellular vesicles. Also provided are compositions in the form of kits for detecting cell type-specific and / or organ-specific extracellular vesicles.

Description

[0001] Related Applications

[0002] This application claims the benefit of priority of U.S. Provisional Application No. 63 / 342,353, filed on May 16, 2022, the entire content of which is incorporated herein by reference.

[0003] Government Support

[0004] This invention was made with government support under Grant No. HG008525 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention. Background of the Invention

[0005] Efforts to understand how the human brain functions have been hindered, in part, because it is not possible to perform a brain biopsy on a living individual. Current understanding of brain diseases relies primarily on the analysis of autopsy tissue after neurodegeneration and cell death have occurred. As a result, fundamental questions remain about the underlying biochemical processes of neurological diseases. The ability to obtain proteomic and transcriptomic profiles of neurons and other brain cells in living human individuals would aid current understanding of neuroscience.

[0006] One way to understand the living brain is to analyze extracellular vesicles (EVs). EVs are released by many cell types and are present in all biological fluids. Since EVs contain RNA and proteins from their donor cells, EVs represent a rich potential source of biomarkers. A major challenge in maximizing the potential of EVs in molecular diagnostics is the isolation of cell type-specific EVs (Shah R, Patel T, et al., The New England Journal of Medicine. 2018;379(10):958-66). First, EVs are heterogeneous and difficult to quantify. In clinically relevant biological samples of limited volume, EVs and their contents are present in low amounts. In addition, and in part due to the lack of suitable quantification methods, there is no consensus on the best method for purifying EVs from plasma and other biological fluids. Moreover, although the total population of EVs can be isolated from plasma or other biological fluids, the analysis of the RNA and protein cargo of these EVs does not distinguish which cargo molecules come from which cell type. Isolating EVs from a specific cell type would enable the analysis of the RNA and proteins within those EVs as a non-invasive "snapshot" of that cell type.

[0007] The ability to isolate EVs from neurons or other cell types in the brain would be particularly useful. Since it is not possible to biopsy the brain, isolating neuron-derived EVs would allow for reading the brain state, as well as the development of biomarkers for the early detection of neurodegenerative diseases (Mustapic M et al., Front Neurosci. 2017; 11:278; Hornung S et al., Front Mol Neurosci. 2020; 13:38). In the past several years, many studies have reported using the transmembrane protein L1CAM, a cell adhesion molecule involved in neural development, as a handle for EV capture. However, given the widespread expression of L1CAM on non-neuronal cells outside the brain, it may not be a suitable marker for neuron-derived EVs (Norman M. et al., Nature Methods. 2021, 18:631-634).

[0008] Accordingly, there is a need in the art to identify novel biomarkers for cell type-specific EVs, particularly brain-specific and / or neuron-specific EVs, which can be used to allow for better diagnosis or prognosis of diseases such as neurodegenerative diseases, as well as for improving the prediction of treatment outcomes. SUMMARY OF THE INVENTION

[0009] The present invention is at least in part based on the discovery of novel biomarkers for isolating cell type-specific and / or organ type-specific EV markers (e.g., brain-specific and / or neuron-specific EVs) from human biological samples such as cerebrospinal fluid (CSF) or plasma. In particular, the present invention is based on the surprising discovery that the markers in any one of Tables 1-5 are specifically expressed in brain-specific and / or neuron-specific EVs, thus providing a way to isolate brain-specific and / or neuron-specific EVs from human samples. In addition, these novel biomarkers can be used to identify EVs derived from a specific organ and / or cell type (e.g., brain cells, e.g., neurons, astrocytes, oligodendrocytes, or microglia) from a sample (e.g., a biological sample) by determining the presence or absence of the markers in any one of Tables 1-5 on the surface of the EVs.

[0010] Accordingly, in one aspect, the present invention provides a method for isolating cell type-specific and / or organ-specific extracellular vesicles from a subject, comprising (a) obtaining a biological sample from the subject; and (b) isolating the cell type-specific and / or organ-specific extracellular vesicles based on the presence of a biomarker on the surface of the extracellular vesicles, wherein the biomarker comprises one or more biomarkers selected from Tables 1-5.

[0011] In some embodiments, the biological sample comprises a liquid biological sample.

[0012] In some embodiments, the liquid biological sample is selected from the group consisting of: whole blood, serum, plasma, cerebrospinal fluid, spinal fluid, amniotic fluid, aqueous humor, vitreous humor, bile, milk, cerumen (earwax), chyle, chyme, endolymph, perilymph, exudate, feces, female ejaculate, gastric acid, gastric juice, lymph, mucus (including nasal drainage and phlegm), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skin oil), semen, sputum, synovial fluid, sweat, tears, urine, vaginal secretion, vomit, and mixtures of one or more thereof.

[0013] In some embodiments, the extracellular vesicles are brain-specific. In some embodiments, the extracellular vesicles are neuron-specific, astrocyte-specific, oligodendrocyte-specific, and / or microglia-specific.

[0014] In some embodiments, the extracellular vesicles are neuron-specific, and wherein the one or more biomarkers are selected from Tables 1 and 5.

[0015] In some embodiments, the one or more biomarkers are selected from the group consisting of: GABRG2, SVOP, SLC32A1, GRM7, GABRB3, CHRNB2, SLC12A5, GRM8, PTPRT, SLC6A17, PCDHAC2, PLPPR4, C11orf87, SORCS3, CALY, PTPRR, KIAA1549L, HCN1, CDH18, TMEM132D, GPR158, FRRS1L, ATP2B3, GRIA4, ST8SIA3, HS6ST3, SEZ6, and NRXN3.

[0016] In some embodiments, the extracellular vesicles are astrocyte-specific, and wherein the one or more biomarkers are selected from Table 2.

[0017] In some embodiments, the extracellular vesicles are oligodendrocyte-specific, and wherein the one or more biomarkers are selected from Table 3.

[0018] In some embodiments, the extracellular vesicles are microglia-specific, and wherein the one or more biomarkers are selected from Table 4.

[0019] In some embodiments, cell type-specific and / or organ-specific EVs are isolated by: immunoaffinity separation, mixed-mode chromatography, size exclusion chromatography, cation exchange chromatography, anion exchange chromatography, gel permeation chromatography, differential centrifugation, sucrose density gradient, organelle electrophoresis, magnetic-activated cell sorting (MACS), or nanofilter ultrafiltration concentrator.

[0020] In some embodiments, immunoaffinity separation includes microfluidic affinity-based separation, magnetic-based separation, pull-down separation, or fluorescence-activated sorting-based separation.

[0021] In one aspect, the present invention provides a method for isolating brain-specific extracellular vesicles from a subject, comprising (a) obtaining a biological sample from the subject; (b) isolating extracellular vesicles from the sample based on the presence of biomarkers on the surface of the extracellular vesicles, wherein the biomarkers include one or more biomarkers selected from Tables 1-5.

[0022] In some embodiments, the biological sample comprises a liquid biological sample.

[0023] In some embodiments, the liquid biological sample is selected from the group consisting of: whole blood, serum, plasma, cerebrospinal fluid, spinal fluid, amniotic fluid, aqueous humor, vitreous humor, bile, milk, cerumen (earwax), chyle, chyme, endolymph, perilymph, exudate, feces, female ejaculate, gastric acid, gastric juice, lymph, mucus (including nasal drainage and sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus, inflammatory secretions, saliva, sebum (skin oil), semen, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vomit, and mixtures of one or more thereof.

[0024] In some embodiments, the extracellular vesicles are neuron-specific, astrocyte-specific, oligodendrocyte-specific, and / or microglia-specific.

[0025] In some embodiments, the extracellular vesicles are neuron-specific, and wherein the one or more biomarkers are selected from Tables 1 and 5.

[0026] In some embodiments, the one or more biomarkers are selected from the group consisting of: GABRG2, SVOP, SLC32A1, GRM7, GABRB3, CHRNB2, SLC12A5, GRM8, PTPRT, SLC6A17, PCDHAC2, PLPPR4, C11orf87, SORCS3, CALY, PTPRR, KIAA1549L, HCN1, CDH18, TMEM132D, GPR158, FRRS1L, ATP2B3, GRIA4, ST8SIA3, HS6ST3, SEZ6, and NRXN3.

[0027] In some embodiments, the extracellular vesicles are astrocyte-specific, and wherein the one or more biomarkers are selected from Table 2.

[0028] In some embodiments, the extracellular vesicles are oligodendrocyte-specific, and wherein the one or more biomarkers are selected from Table 3.

[0029] In some embodiments, the extracellular vesicles are microglia-specific, and wherein the one or more biomarkers are selected from Table 4.

[0030] In some embodiments, the brain-specific EVs are isolated by: immunoisolation, mixed-mode chromatography, size-exclusion chromatography, cation-exchange chromatography, anion-exchange chromatography, gel permeation chromatography, differential centrifugation, sucrose density gradient, organelle electrophoresis, magnetic-activated cell sorting (MACS), or nanofilter ultrafiltration concentrator.

[0031] In some embodiments, immunoisolation includes microfluidics affinity-based separation, magnetic-based separation, pull-down separation, or fluorescence-activated sorting-based separation.

[0032] In one aspect, the present invention provides a method for identifying extracellular vesicles derived from brain cells, comprising (a) obtaining a biological sample comprising the extracellular vesicles; (b) determining the presence or absence of a biomarker on the surface of the extracellular vesicles, wherein the biomarker comprises one or more biomarkers selected from Tables 1-5; and wherein the presence of the biomarker indicates that the extracellular vesicles are derived from brain cells.

[0033] In some embodiments, the brain cells are selected from the group consisting of: neurons, astrocytes, oligodendrocytes, and microglia.

[0034] In one aspect, the present invention provides a method for identifying extracellular vesicles derived from neurons, comprising (a) obtaining a biological sample containing the extracellular vesicles; (b) determining the presence or absence of biomarkers on the surface of the extracellular vesicles, wherein the biomarkers include one or more biomarkers selected from Table 1 and Table 5; and wherein the presence of the biomarkers indicates that the extracellular vesicles are derived from neurons.

[0035] In some embodiments, the one or more biomarkers are selected from the group consisting of: GABRG2, SVOP, SLC32A1, GRM7, GABRB3, CHRNB2, SLC12A5, GRM8, PTPRT, SLC6A17, PCDHAC2, PLPPR4, C11orf87, SORCS3, CALY, PTPRR, KIAA1549L, HCN1, CDH18, TMEM132D, GPR158, FRRS1L, ATP2B3, GRIA4, ST8SIA3, HS6ST3, SEZ6, and NRXN3.

[0036] In another aspect, the present invention provides a method for identifying extracellular vesicles derived from astrocytes, comprising (a) obtaining a biological sample containing the extracellular vesicles; (b) determining the presence or absence of biomarkers on the surface of the extracellular vesicles, wherein the biomarkers include one or more biomarkers selected from Table 2; and wherein the presence of the biomarkers indicates that the extracellular vesicles are derived from astrocytes.

[0037] In one aspect, the present invention provides a method for identifying extracellular vesicles derived from oligodendrocytes, comprising (a) obtaining a biological sample containing the extracellular vesicles; (b) determining the presence or absence of biomarkers on the surface of the extracellular vesicles, wherein the biomarkers include one or more biomarkers selected from Table 3; and wherein the presence of the biomarkers indicates that the extracellular vesicles are derived from oligodendrocytes.

[0038] In another aspect, the present invention provides a method for identifying extracellular vesicles derived from microglia, comprising (a) obtaining a biological sample containing the extracellular vesicles; (b) determining the presence or absence of biomarkers on the surface of the extracellular vesicles, wherein the biomarkers include one or more biomarkers selected from Table 4; and wherein the presence of the biomarkers indicates that the extracellular vesicles are derived from microglia.

[0039] In some embodiments, the biological sample comprises a liquid biological sample.

[0040] In some embodiments, the liquid biological sample is selected from the group consisting of: whole blood, serum, plasma, cerebrospinal fluid, spinal fluid, amniotic fluid, aqueous humor, vitreous humor, bile, milk, cerumen (earwax), chyle, chyme, endolymph, perilymph, exudate, feces, female ejaculate, gastric acid, gastric juice, lymph, mucus (including nasal drainage and sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus, inflammatory secretions, saliva, sebum (skin oil), semen, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vomit, and mixtures of one or more thereof.

[0041] In some embodiments, the biological sample is obtained from a subject.

[0042] In some embodiments, the presence or absence of a biomarker is determined by: RNA sequencing (RNAseq), DNA sequencing, array analysis, reverse transcription polymerase chain reaction (RT-PCR), quantitative reverse transcription polymerase chain reaction (qRT-PCR), proteomic analysis, mass spectrometry, immunoassay, ELISA, fluorescence-activated cell sorting (FACS), SDS-polyacrylamide gel electrophoresis (SDS-PAGE), or Western blot analysis.

[0043] In one aspect, the present invention provides a method for diagnosing, prognosticating a neurodegenerative disorder or identifying a subject at risk of developing a neurodegenerative disorder in a subject, comprising: (a) obtaining a biological sample from the subject; (b) isolating brain-specific extracellular vesicles from the biological sample based on the presence of a biomarker in the isolated extracellular vesicles, wherein the biomarker comprises one or more biomarkers selected from Tables 1-5; (c) extracting proteins and / or nucleic acids from the isolated brain-specific extracellular vesicles; and (d) analyzing the extracted proteins and / or nucleic acids from the isolated brain-specific extracellular vesicles to thereby diagnose, prognosticate a neurodegenerative disorder or identify a subject at risk of developing a neurodegenerative disorder.

[0044] In some embodiments, the biological sample comprises a liquid biological sample.

[0045] In some embodiments, the liquid biological sample is selected from the group consisting of: whole blood, serum, plasma, cerebrospinal fluid, spinal fluid, amniotic fluid, aqueous humor, vitreous humor, bile, milk, cerumen (earwax), chyle, chyme, endolymph, perilymph, exudate, feces, female ejaculate, gastric acid, gastric juice, lymph, mucus (including nasal drainage and sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus, inflammatory secretions, saliva, sebum (skin oil), semen, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vomit, and mixtures of one or more thereof.

[0046] In some embodiments, the extracellular vesicles are neuron-specific, astrocyte-specific, oligodendrocyte-specific, and / or microglia-specific.

[0047] In some embodiments, the extracellular vesicles are neuron-specific, and wherein the one or more biomarkers are selected from Tables 1 and 5.

[0048] In some embodiments, the one or more biomarkers are selected from the group consisting of: GABRG2, SVOP, SLC32A1, GRM7, GABRB3, CHRNB2, SLC12A5, GRM8, PTPRT, SLC6A17, PCDHAC2, PLPPR4, C11orf87, SORCS3, CALY, PTPRR, KIAA1549L, HCN1, CDH18, TMEM132D, GPR158, FRRS1L, ATP2B3, GRIA4, ST8SIA3, HS6ST3, SEZ6, and NRXN3.

[0049] In some embodiments, the extracellular vesicles are astrocyte-specific, and wherein the one or more biomarkers are selected from Table 2.

[0050] In some embodiments, the extracellular vesicles are oligodendrocyte-specific, and wherein the one or more biomarkers are selected from Table 3.

[0051] In some embodiments, the extracellular vesicles are microglia-specific, and wherein the one or more biomarkers are selected from Table 4.

[0052] In some embodiments, the extracted nucleic acids include messenger RNA (mRNA), microRNA (miRNA), long non-coding RNA (lncRNA), small non-coding RNA, DNA, and any other full-length RNA or DNA or fragments thereof.

[0053] In some embodiments, analyzing the extracted nucleic acids from isolated brain-specific extracellular vesicles includes RNA sequencing (RNA seq), DNA sequencing, array analysis, reverse transcription polymerase chain reaction (RT-PCR), or quantitative reverse transcription polymerase chain reaction (qRT-PCR).

[0054] In some embodiments, analyzing the extracted nucleic acids from isolated brain-specific extracellular vesicles includes whole genome analysis or transcriptome analysis.

[0055] In some embodiments, analyzing the extracted nucleic acids from isolated brain-specific extracellular vesicles includes analyzing genes of interest, wherein the genes of interest are associated with the neurodegenerative disorder.

[0056] In some embodiments, the method includes testing for the presence or absence of the gene of interest, analyzing one or more allelic variants or mutations of the gene of interest, and testing for the presence or absence of the allelic variant or mutation.

[0057] In some embodiments, analyzing the extracted proteins from isolated brain-specific extracellular vesicles includes proteomic analysis, mass spectrometry, immunoassay, ELISA, fluorescence-activated cell sorting (FACS), SDS-polyacrylamide gel electrophoresis (SDS-PAGE), or Western blot analysis.

[0058] In some embodiments, analyzing the extracted proteins from isolated brain-specific extracellular vesicles includes analyzing a protein of interest, wherein the protein of interest is associated with the neurodegenerative disorder.

[0059] In some embodiments, the method includes detecting the presence or absence of the protein of interest, analyzing one or more mutations in the protein of interest, and detecting the presence or absence of the mutation.

[0060] In some embodiments, the neurodegenerative disorder is selected from the group consisting of Alzheimer's disease (AD), Huntington's disease, multiple sclerosis, vascular dementia, frontotemporal dementia (FTD), corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), dementia with Lewy bodies, tangle-predominant senile dementia, Pick's disease (PiD), argyrophilic grain disease, amyotrophic lateral sclerosis (ALS), other motor neuron diseases, Guam Parkinson's disease-dementia complex, FTDP-17, Lytico-Bodig disease, multiple sclerosis, traumatic brain injury (TBI), and Parkinson's disease.

[0061] In one aspect, the present invention provides a kit for isolating brain-specific extracellular vesicles from a subject, comprising (a) one or more reagents for detecting the presence of a biomarker on the surface of the extracellular vesicles, wherein the biomarker comprises one or more biomarkers selected from Tables 1-5; (b) a device for isolating brain-specific extracellular vesicles based on the presence of the biomarker; and (c) a set of instructions for detecting the presence of the biomarker and / or isolating the brain-specific extracellular vesicles.

[0062] In some embodiments, the one or more reagents for detecting the presence of a biomarker on the extracellular vesicles are antibodies or aptamers that bind to the biomarker.

[0063] In some embodiments, the kit further comprises a device for isolating a biological sample from the subject.

[0064] In another aspect, the present invention provides a kit for detecting a neurodegenerative disorder in a subject, comprising (a) one or more reagents for detecting the presence of a biomarker on the surface of extracellular vesicles, wherein the biomarker comprises one or more biomarkers selected from Tables 1-5; (b) a device for isolating brain-specific extracellular vesicles based on the presence of the biomarker; (c) one or more reagents for detecting the level of a gene associated with a neurodegenerative disorder in the isolated brain-specific extracellular vesicles; and (d) a set of instructions for detecting the presence of the biomarker, isolating the brain-specific extracellular vesicles, and / or detecting the level of the gene associated with the neurodegenerative disorder.

[0065] In some embodiments, the kit further comprises a device for isolating a biological sample from the subject.

[0066] Other features and advantages of the present invention will become apparent from the following detailed description and the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figures 1A - 1E Depicts the identification of neuron-specific transmembrane proteins and the characterization of extracellular vesicles from human iPS-derived neurons. Figure 1A Is a schematic diagram depicting the immunoseparation of neuron-derived EVs from human CSF or plasma. Figure 1B Is a Tau scatter plot of genes, where these genes are expressed maximally in neurons relative to other cell types in the brain and maximally in the brain relative to other organs. Figure 1C Depicts the overlap of transmembrane proteins specifically expressed in neurons (using a Tau specificity score of 0.7 or higher) relative to other cell types in the brain and transmembrane proteins specifically expressed in the brain (also using a Tau specificity score of 0.7 or higher) relative to other organs in the body. Figure 1D Depicts the isolation of EVs from the conditioned medium of undifferentiated human iPS cells or iPS-derived neurons and the analysis of their proteins by mass spectrometry to determine the presence of transmembrane proteins (2 peptides or more) on neuron EVs but not on iPS EVs. Figure 1E Depicts the overlap of brain-specific transmembrane proteins, neuron-specific transmembrane proteins, and transmembrane proteins detected in neuron EVs by mass spectrometry. Figure 1F Depicts the validation of the candidate biomarker SYT1 by Western blot in iNGN neurons and iNGN neuron EVs.

[0068] Figures 2A - 2H Depicts the immunoseparation of EVs from cell cultures and human biological fluids. Figure 2A Is a schematic diagram depicting the immunoseparation of EVs from the cell culture medium of K562 cells. Figure 2BWestern blot images of CD81 after immunoisolation of EVs. Equal amounts of EVs obtained by differential ultracentrifugation of K562 cell culture medium were loaded as input, or immunoisolated using anti-CD81 antibody or control non-specific antibody. PD = pulldown, FT = flow-through. Figure 2C Western blot images of L1CAM after immunoisolation of EVs. Equal amounts of EVs obtained by differential ultracentrifugation of iNGN neuron cell culture medium were loaded as input, or immunoisolated using anti-L1CAM antibody or control non-specific antibody. PD = pulldown, FT = flow-through. Figure 2D Schematic diagram depicting an EV mixing experiment using EVs isolated from conditioned media of iPS cells and iPS-derived iNGN neurons. Figure 2E Western blot images of L1CAM and GJA1 after immunoisolation using anti-L1CAM antibody from pooled iNGN neuron EVs and iPS EVs mixed at different ratios (normalized to cell number). PD = pulldown, FT = flow-through. Figure 2F Schematic diagram depicting immunoisolation of EVs from human plasma or CSF. EVs were immunoisolated from the biological fluid without initial EV purification. Immunoisolation was performed using the target antibody or control antibody (PD1 = pulldown 1), and a second immunoisolation of the flow-through was performed using the target antibody (PD2 = pulldown 2). Figure 2G Western blots of CD9 (bottom), CD63 (middle), or CD81 (top) after immunoisolation from human CSF are shown. In each case, a first immunoisolation (PD1) was performed using an antibody against the target protein or a control antibody, and a second immunoisolation (PD2) was performed using the PD1 flow-through with an antibody against the target protein. Figure 2H Western blots of CD9 (bottom), CD63 (middle), or CD81 (top) after immunoisolation from human plasma are shown. In each case, a first immunoisolation (PD1) was performed using an antibody against the target protein or a control antibody, and a second immunoisolation (PD2) was performed using the PD1 flow-through with an antibody against the target protein.

[0069] Figures 3A - 3G Depicts the purification of EVs from human biological fluids using mixed-mode resin (MMR) slurry. Figure 3A Schematic diagram illustrating the MMR slurry technique. Capto Core 700 beads have pores that allow biomolecules smaller than 700 kDa to enter, and once the free protein enters, it remains in the beads, and these beads can be removed to leave pure EVs. Figure 3B Schematic diagram depicting the EV isolation workflow using MMR slurry. Figure 3CWestern blot of CD9, CD63, CD81, and albumin in human cerebrospinal fluid after MMR slurry purification with increasing amounts of Capto Core beads. Figure 3D Western blot of L1CAM in CSF after MMR slurry purification with increasing amounts of Capto Core beads. Figure 3E Total protein staining of CSF after MMR slurry purification with increasing amounts of Capto Core beads is depicted. Figure 3F Western blot of CD9, CD63, CD81, and albumin in plasma after size exclusion chromatography followed by MMR slurry purification with increasing amounts of Capto Core beads is depicted. Figure 3 depicts total protein staining in plasma after size exclusion chromatography followed by MMR slurry purification with increasing amounts of Capto Core beads. The ratio of Capto Core (CC) beads to protein is expressed as μL CC slurry / μg protein in the sample.

[0070] Figures 4A - 4E Depicts the generation of novel neuron-specific EV markers in human biofluids for EV proteomics. Figure 4A Schematic diagram of EV proteomics depicting the use of MMR slurry for CSF and SEC, followed by MMR slurry in plasma. Figure 4B Depicts the total number of different proteins and the number of transmembrane proteins detected in human (pooled) CSF and plasma. Figure 4C Depicts the general workflow for identifying neuron-specific EV markers: assessing neuron-specific gene expression of transmembrane proteins and their presence on EVs (in EV proteomics data). Figure 4D Depicts the use of a Tau cutoff of 0.7 for neuron- and brain-specific expression in MMR slurry proteomics data, along with proteins found on EVs, to generate 27 candidate neuron-specific EV markers. Figure 4E Depicts a list of 27 candidate neuron-specific EV markers, giving their Tau values for specific expression in neurons relative to other cell types in the brain and for specific expression in the brain relative to other organs in the body, as well as their presence in CSF and / or plasma mass spectrometry data.

[0071] Figure 5 Depicts a heatmap of Tau cutoffs. The number of transmembrane proteins when different Tau cutoffs are used for neurons within the brain and for the brain relative to other cells in the body.

[0072] Figure 6 Depicts a Tau scatter plot for all genes. Tau-specific scores for all genes in neurons (in the brain RNA-Seq dataset) and in the brain (in GTEx).

[0073] Figure 7 Depicts Tau scatter plots for genes with the highest expression in the brain and neurons. Tau-specific scores for genes with the highest expression in neurons (in the brain RNA-Seq dataset) relative to other cells in the brain and in the brain relative to other organs (in GTEx).

[0074] Figures 8A - 8E Depicts microscopic images of the differentiation of iPS iNGN cells into neurons. Figure 8A Depicts iPS NGN cells before the addition of doxycycline (magnification 40x). Figure 8B Depicts iPS NGN cells 1 day after the addition of doxycycline (magnification 40x). Figure 8C Depicts iPS NGN cells 2 days after the addition of doxycycline (magnification 40x). Figure 8D Depicts iPS NGN cells 4 days after the addition of doxycycline (magnification 40x). Figure 8E Depicts iPS NGN cells 5 days after the addition of doxycycline (magnification 40x).

[0075] Figure 9 Depicts that for CD81, the effect of direct immunoisolation of EVs is superior to indirect isolation. For CD81 immunoisolation, a comparison of direct (conjugating the primary antibody to beads and then adding to the sample) versus indirect (first adding the primary antibody to the sample and then adding to the beads) pull-down methods was performed using two different antibodies (clone M38 and clone 1.3.3.22). Note: The band at 150 kDa is the primary antibody detached from the beads.

[0076] Figure 10 Depicts the effect of immunoisolation incubation time and temperature on CD81 capture efficiency. At different temperatures (4°C, room temperature, or 37°C), EVs were incubated with beads conjugated to CD81 or a control (ctrl) non-specific antibody (against mCherry) for 1 hour or overnight (ON), and then western blotting for CD81 and CD63 was performed.

[0077] Figure 11 Depicts that the optimized immunoisolation protocol is effective and specific for K562 EVs with CD81 or CD63. Using beads conjugated to antibodies against CD81, CD63, or mCherry, after incubating EVs with the beads at 37°C for 1 hour, western blotting for CD81 and CD63 was performed. After lysis, the beads were re-boiled to observe if more material was left as an additional control to ensure that all EVs detached from the beads during lysis. In addition, EV pellets were treated with proteinase K (PK) or Triton X and proteinase K.

[0078] Figure 12 Depicts the optimal immunoisolation conditions for CD81, which are not optimal for L1CAM. Immunoisolation was performed for 1 hour at 37°C using CD81 (or control GFP antibody) on K562 EVs and L1CAM (or control GFP antibody) on neuronal EVs, and corresponding western blots were performed for CD81 (top) or L1CAM (bottom). The intensity of the bands on the western blot was quantified as the percentage of the band relative to the sum of pull-down (PD) + flow-through (FT) under that condition.

[0079] Figure 13 Depicts the effects of immunoisolation incubation time, volume, and temperature on L1CAM capture efficiency. After incubating neuronal EVs with antibody-conjugated beads (against L1CAM or GFP) for different times (1, 2, 4, or 24 hours) at different temperatures (4°C or 37°C) in different volumes (0.5 mL or 2 mL), western blots were performed for L1CAM. The intensity of the bands on the western blot was quantified as the percentage of the band relative to the sum of pull-down (PD) + flow-through (FT) under that condition.

[0080] Figures 14A - 14B Depicts a comparison of different immunoisolation beads and conditions for human biological fluids. Figure 14A Depicts a comparison of goat anti-mouse (GAM) dynabeads with sheep anti-mouse (SAM) dynabeads in terms of CD81 pull-down in plasma. The pull-down incubation temperature and the amount of GAM dynabeads were also compared. Figure 14B Depicts a comparison of the goat anti-mouse (GAM) dynabead pull-down incubation temperature and incubation with or without BSA in terms of CD81 pull-down in CSF.

[0081] Figures 15A - 15E Depicts the optimization of the Capto Core slurry purification method conditions to maximize the recovery of albumin-depleted exosomes and superior to other separation methods. Figure 15A Depicts adding 100 μL (low CC) or 300 μL (high CC) of 50% Capto Core slurry to K562 exosomes and incubating once (1x) or twice end-over-end at room temperature for 45 minutes. Figure 15B Depicts the incubation of CSF with 50% Capto Core slurry in a time series to optimize the incubation time. Figure 15CDepicts testing different amounts of 50% capto core slurry and CSF in combination to determine whether binding capacity or volume / volume ratio is more important. CC1: 0.5 mL CSF - 160 μL CC; CC2: 1 mL CSF - 160 μL CC; CC3: 0.1 mL CSF / 0.9 mL PBS - 160 μL CC; CC4: 0.5 mL CSF / 0.5 mL PBS - 160 μL CC; CC5: 0.1 mL CSF - 16 μL CC; CC6: 0.5 mL CSF - 80 μL CC; CC7: 0.1 mL CSF / 0.9 mL PBS - 16 μL CC; CC8: 0.5 mL CSF / 0.5 mL PBS - 80 μL CC; CC9: 0.1 mL CSF / 0.9 mL PBS - 40 μL CC; CC10: 0.5 mL CSF / 0.5 mL PBS - 40 μL CC; CC11: 1 mL CSF - 40 μL CC; CC12: 0.1 mL CSF / 0.9 mL PBS - 320 μL CC; CC13: 0.5 mL CSF / 0.5 mL PBS - 320 μL CC; CC14: 1 mL CSF - 320 μL CC. Figure 15D Depicts comparing the yield of CaptoCore four - transmembrane proteins with the yield of four - transmembrane proteins by size - exclusion chromatography (Izon and Sepharose 4B and 6B columns). Figure 15E Depicts comparing Capto Core albumin depletion with size - exclusion chromatography albumin depletion by Acqua Coomassie Brilliant Blue staining, and evaluating Capto Core albumin depletion by Ponceau S.

[0082] Figure 16A Depicts detecting the NRXN3 recombinant protein standard by Simoa assay. Invitrogen PA5 - 71367 was used as the capture antibody, while CST 480045 was used as the detection antibody. The average enzyme per bead (AEB) was plotted against the concentration of the NRXN3 recombinant protein standard. Figure 16B Depicts detecting NRXN3 in an early size - exclusion chromatography (SEC) fraction from EVs ultra - centrifuged from the conditioned medium of human iPS - derived neurons by Simoa NRXN3 assay. Figure 16C Depicts detecting NRXN3 in an early size - exclusion chromatography (SEC) fraction in human CSF samples by Simoa NRXN3 assay. Detailed implementation

[0083] The present invention is at least partially based on the discovery of novel biomarkers for the isolation of cell type-specific and / or organ-specific EV markers from human biological samples such as cerebrospinal fluid (CSF) or plasma, e.g., brain-specific and / or neuron-specific EVs. In particular, the present invention is based on the surprising discovery that the markers in any of Tables 1-5 are specifically expressed in brain-specific and / or neuron-specific EVs, thus providing a way to isolate brain-specific and / or neuron-specific EVs from human samples. Additionally, these novel biomarkers can be used to identify EVs derived from a specific cell type (e.g., brain cells, e.g., neurons, astrocytes, oligodendrocytes, or microglia) from a sample (e.g., a biological sample) by determining the presence or absence of the markers in any of Tables 1-5 on the surface of the EVs.

[0084] The various aspects of the present invention are described in further detail in the following subsections:

[0085] A. Definitions

[0086] To make the present disclosure more readily understandable, certain terms are first defined. It should also be noted that whenever a value or range of values of a parameter is recited, it is intended that the intermediate values and ranges of the recited values are also part of the present disclosure.

[0087] In the following description, for purposes of explanation, specific numbers, materials, and configurations are set forth in order to provide a thorough understanding of the present disclosure. However, it will be apparent to those of ordinary skill in the art that the present disclosure may be practiced without these specific details. In some instances, well-known features may be omitted or simplified so as not to obscure the present disclosure. Additionally, the phrase such as "one embodiment" or "an embodiment" in the specification means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure, and the phrases such as "in one embodiment" that appear throughout the specification do not necessarily all refer to the same embodiment.

[0088] The articles "a" and "an" are used herein to refer to one or more than one (i.e., at least one) of the grammatical object of the article. By way of example, "an element" refers to one element or more than one element.

[0089] The term "comprising" or "containing" is used herein to refer to the compositions, methods, and their respective components that are essential to the present disclosure, but may include elements not expressly stated, whether essential or not.

[0090] As used herein, the term "one or more" or "at least one" is understood to mean each value of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20, and any value greater than 20.

[0091] As used herein, in one embodiment, the term "biomarker" is a biomolecule or a group of biomolecules or any combination thereof, the level of which in a test sample (e.g., cell type-specific or organ-specific EVs, such as brain and / or neuron-specific EVs) is changed compared to its level in a control sample (e.g., EVs from different cell types or different organs). Examples of biomarkers include, for example, polypeptides, peptides, polypeptide fragments, proteins, antibodies, hormones, polynucleotides, DNA, RNA, or RNA fragments, microRNAs (miRNAs), lipids, metabolites, or polysaccharides. In one embodiment, a biomarker is detected in cell type-specific EVs isolated from a biological sample. In one embodiment, the biomarker is an organ-specific EV biomarker, such as a biomarker from the following: anus, artery, appendix, adrenal gland, brain, bone, bronchus, bladder, bone marrow, bulbourethral gland, colon, cervix, clitoris, capillary, cerebellum, diaphragm, ear, eye, fallopian tube, genitalia, gallbladder, heart, hair follicle, hypothalamus, stroma, kidney, joint, liver, lung, larynx, ligament, lymph node, large intestine, lymphatic vessel, oral cavity, mesentery, mammary gland, nose, nail, nerve, nasal cavity, ovary, esophagus, penis, pancreas, pharynx, placenta, prostate, pineal gland, pituitary gland, parathyroid gland, rectum, skin, spleen, scrotum, stomach, spinal cord, small intestine, salivary gland, skeletal muscle, seminal vesicle, subcutaneous tissue, tooth, tonsil, testis, tendon, tongue, thyroid gland, trachea, thymus, ureter, urethra, uterus, vulva, vein, vagina, vas deferens, or vestigial organ. In one embodiment, a biomarker is detected in brain-specific EVs. In one embodiment, a biomarker is detected in neuron-specific EVs. In one embodiment, a biomarker is detected in astrocyte-specific EVs. In one embodiment, a biomarker is detected in oligodendrocyte-specific EVs. In one embodiment, a biomarker is detected in microglia-specific EVs. In some embodiments, the biomarker comprises one or more biomarkers selected from Tables 1-5.

[0092] Biomarkers can be differentially present at any level, but are generally present at increased or decreased levels in a test sample, e.g., cell type-specific or organ-specific EVs isolated from a biological sample obtained from a subject, e.g., brain and / or neuron-specific EVs, that are increased or decreased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150% or more compared to their levels in a control sample (e.g., EVs from a different cell type or different organ). The biomarker is preferably differentially present at a statistically significant level (e.g., a p-value determined using any statistical test of less than 0.05). Thus, the difference between the level of the biomarker of the invention and the corresponding control or reference value can be a statistically significant value.

[0093] As used herein, a "biological sample" refers to any biological sample obtained from or derived from a subject. In some embodiments, the biological sample comprises EVs. In another embodiment, the biological sample is a liquid biological sample. As used herein, the term "liquid biological sample" refers to a sample that is substantially in liquid form. In some embodiments, the liquid sample is a body fluid. Body fluids include, for example, whole blood (including fresh or frozen), peripheral blood, plasma (including fresh or frozen), serum (including fresh or frozen), cerebrospinal fluid (CSF), ascites, sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, milk, bronchoalveolar lavage fluid, semen (including prostatic fluid), Cowper's fluid or pre-ejaculatory fluid, female ejaculate, sweat, feces, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretions, fecal water, pancreatic juice, sinus lavage fluid, broncho-pulmonary aspirate or other lavage fluid. The biological sample can also include a blastocoel, cord blood or maternal circulation that can be derived from a fetus or a mother. The biological sample can also be a tissue sample or biopsy from which EVs can be obtained. In one embodiment, the biological sample is a brain tissue. In one embodiment, the biological sample is a plasma sample. In another embodiment, the biological sample is a CSF sample.

[0094] As used herein, "subject" refers to any animal. In some embodiments, the subject is a human. Other animals that can be subjects include, but are not limited to, non-human primates (e.g., monkeys, gorillas, and chimpanzees), domesticated animals (e.g., horses, pigs, donkeys, goats, rabbits, sheep, cows, yaks, alpacas, and llamas), and companion animals (e.g., cats, dogs, hamsters, guinea pigs, rats, mice, and birds).

[0095] B. Extracellular vesicles

[0096] The present invention provides biomarkers for cell type-specific and / or organ-specific extracellular vesicles (EVs) (e.g., brain and / or neuron-specific EVs), methods for purifying cell type-specific and / or organ-specific EVs, and diagnostic and prognostic methods for diseases (such as neurodegenerative disorders) using these cell type-specific or organ-specific EVs.

[0097] Extracellular vesicles (EVs) are a class of membrane-bound organelles secreted by various cell types. As used herein, the term "extracellular vesicle" refers to cell-derived vesicles that have a membrane surrounding and enclosing a central internal space. The membrane of an EV can consist of a lipid bilayer that has an outer surface and an inner surface that defines an enclosed volume. EVs are capable of carrying various molecules, such as proteins, lipids, and RNA, on their surface and within their lumen.

[0098] EVs include all membrane-bound vesicles with a cross-sectional diameter smaller than their secreting cells. In some embodiments, EVs can have a longest dimension, such as a longest cross-sectional dimension, with a cross-sectional diameter ranging from 1 nm to 1000 nm, such as 10 nm to 1000 nm, such as 20 nm to 1000 nm, such as 30 nm to 1000 nm, such as 1 to 100 nm, such as 10 to 100 nm, such as 20 to 100 nm, such as 30 to 100 nm, such as 40 to 100 nm, such as 10 to 200 nm, such as 20 to 200 nm, such as 30 to 200 nm, such as 40 to 200 nm, such as 10 to 120 nm, such as 20 to 120 nm, such as 30 to 120 nm, such as 40 to 120 nm, such as 10 to 300 nm, such as 20 to 300 nm, such as 30 to 300 nm, such as 40 to 300 nm, such as 50 to 1000 nm, such as 500 to 2000 nm, such as 100 to 500 nm, such as 500 to 1000 nm, and such as 40 nm to 500 nm, each range being inclusive.

[0099] Depending on their size and density, extracellular vesicles can be divided into three major groups: exosomes (10 - 150 nm), microvesicles (100 - 1000 nm), and apoptotic bodies (1 - 10 μm). As used herein, the term "exosome" refers to a cell-derived vesicle composed of a membrane enclosing an internal space, wherein the vesicle is produced by a cell through the fusion of a late endosome with the plasma membrane or by direct plasma membrane budding. Exosomes are typically produced intracellularly when a portion of the cell membrane spontaneously invaginates and is ultimately secreted extracellularly. As used herein, exosomes can also include any shed membrane-bound particles derived from the plasma membrane or internal membranes. Exosomes can also include cell-derived structures bounded by a lipid bilayer membrane, which are caused by both the budding and sealing of portions of the plasma membrane or by the export of vesicular structures bounded by any internal cell membrane containing various membrane-associated proteins, including surface-binding molecules derived from the host circulation, which selectively bind exosome proteins together with molecules contained within the exosome lumen, including but not limited to mRNA, microRNA, or intracellular proteins. Budding and vesiculation are further described in Charras et al., Nature Reviews Molecular and Cell Biology, Vol. 9, No. 11, p. 730 - 736 (2008). Exosomes can also include membrane fragments.

[0100] EVs contain RNA, such as, for example, microRNA (miRNA), long non-coding RNA (lncRNA), mRNA, DNA fragments, as well as proteins from their donor cells, and are thus important for intercellular communication in the human body and are involved in many pathophysiological disorders, such as neurodegenerative diseases. EVs can also be loaded with various drugs and exogenous nucleic acids or proteins and deliver this cargo to different cells. Importantly, EVs are natural carriers of miRNA and other non-coding RNAs, and direct membrane fusion with target cells allows the contents to be delivered directly into the cytoplasm. This makes EVs an excellent delivery system for small molecules.

[0101] EVs are abundant in various biological samples. In some embodiments, the sample is a sample obtained from a cell culture. In some embodiments, the sample is a liquid sample, e.g., a liquid biological sample. Exemplary liquid samples include but are not limited to body fluids such as whole blood (including fresh or frozen), peripheral blood, plasma (including fresh or frozen), serum (including fresh or frozen), cerebrospinal fluid (CSF), ascites, sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, earwax, milk, bronchoalveolar lavage fluid, semen (including prostatic fluid), Cowper's fluid or pre-ejaculatory fluid, female ejaculate, sweat, feces, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretions, fecal water, pancreatic juice, sinus lavage fluid, bronchoalveolar aspirate or other lavage fluids. The biological sample may also include blastocoel, cord blood or maternal circulation that may be derived from the fetus or the mother. The biological sample may also be a tissue sample or biopsy from which EVs can be obtained. In one embodiment, the biological sample is a brain tissue. In one embodiment, the biological sample is a plasma sample. In another embodiment, the biological sample is a CSF sample. Methods for obtaining biopsies and body fluids from mammals are well known in the art.

[0102] According to the methods of the present invention, detecting EVs in various patient body fluids allows for the assessment of disease progression, immune response, and toxicity. Thus, isolating and detecting EVs can assist in disease diagnosis, prognosis, and monitoring treatment response. A major challenge in maximizing the potential of EVs in molecular diagnostics is the isolation of cell type-specific EVs (Shah R, Patel T et al., The New England Journal of Medicine. 2018; 379(10):958-66). Although the total population of EVs can be isolated from plasma or other biological fluids, the analysis of the RNA and protein cargo of these EVs cannot distinguish which cargo molecules are from which cell type. Isolating EVs from a specific cell type would enable the analysis of the RNA and proteins within these EVs as a non-invasive "snapshot" of that cell type.

[0103] However, the inventors of the present application were able to discover novel biomarkers for isolating cell type- and organ-specific EVs. In some embodiments, the EVs of the present invention are from a specific cell type, e.g., a brain cell type such as a neuron, astrocyte, oligodendrocyte, or microglia. In some embodiments, the EVs include neuron-derived EVs. In some embodiments, the EVs include astrocyte-derived EVs, oligodendrocyte-derived EVs, or microglia-derived EVs.

[0104] C. Biomarkers of the Present Invention

[0105] The present invention is at least in part based on the discovery of novel biomarkers for isolating cell type-specific and / or organ-specific EV markers from human biological samples such as cerebrospinal fluid (CSF) or plasma, e.g., brain-specific and / or neuron-specific EVs. In particular, the present invention is based on the surprising discovery that the markers in any of Tables 1-5 are expressed in brain-specific and / or neuron-specific EVs. Accordingly, these differentially expressed markers can be used to isolate brain-specific and / or neuron-specific EVs from human samples.

[0106] Isolating EVs from a specific cell type enables the analysis of the RNA and proteins within these EVs as a non-invasive “snapshot” of that cell type. In particular, given the inability to biopsy the brain, the ability to isolate EVs from neurons or other cell types of the brain is particularly useful. For example, isolating neuron-derived EVs allows for reading the state of the brain, as well as the development of biomarkers for the early detection of neurodegenerative diseases. Accordingly, identifying novel biomarkers for cell type-specific and / or organ-specific EVs (in particular, brain-specific and / or neuron-specific EVs) can be used to allow for better diagnosis, prognosis, or monitoring of diseases such as neurodegenerative diseases, as well as for improving the prediction of treatment outcomes.

[0107] The present invention provides biomarkers for cell type-specific and / or organ-specific EVs (e.g., brain-specific and / or neuron-specific EVs). The biomarker levels are determined in a biological sample obtained from a subject. The markers of the present invention include, but are not limited to, one or more biomarkers selected from Tables 1-5 or any combination thereof. As used herein, the term “one or more biomarkers” or “at least one” is intended to mean the determination of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) markers selected from Tables 1-5 or any combination thereof. The methods, kits, and panels provided herein also include any combination of, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more markers selected from Tables 1-5, or any combination thereof.

[0108] In one embodiment, the biomarkers for brain-specific EVs include one or more biomarkers from Tables 1-5, or any combination thereof.

[0109] In one embodiment, the biomarkers of neuron-specific EVs include one or more biomarkers in Table 1 and Table 5, or any combination thereof. In one embodiment, the biomarkers of neuron-specific EVs include one or more biomarkers selected from the group consisting of: GABRG2, SVOP, SLC32A1, GRM7, GABRB3, CHRNB2, SLC12A5, GRM8, PTPRT, SLC6A17, PCDHAC2, PLPPR4, C11orf87, SORCS3, CALY, PTPRR, KIAA1549L, HCN1, CDH18, TMEM132D, GPR158, FRRS1L, ATP2B3, GRIA4, ST8SIA3, HS6ST3, SEZ6, and NRXN3.

[0110] In one embodiment, the biomarkers of astrocyte-specific EVs include one or more biomarkers in Table 2, or any combination thereof.

[0111] In one embodiment, the biomarkers of oligodendrocyte-specific EVs include one or more biomarkers in Table 3, or any combination thereof.

[0112] In one embodiment, the biomarkers of microglia-specific EVs include one or more biomarkers in Table 4, or any combination thereof.

[0113] Table 1. Biomarkers of Neuron-Specific EVs

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122] Table 2. Biomarkers of Astrocyte-Specific EVs

[0123]

[0124]

[0125]

[0126] Table 3: Biomarkers of Oligodendrocyte-Specific EVs

[0127]

[0128]

[0129] Table 4: Biomarkers of Microglia-Specific EVs

[0130] Gene Gene Name Accession Number Gene ID SYNDIG1 Synaptogenesis Inducing 1 NM_024893.3 79953 HS3ST4 Heparan Sulfate Glucosamine 3 - Sulfotransferase 4 NM_006040.3 9951 CLEC9A C - type Lectin Domain Containing 9A NM_207345.4 283420

[0131] Table 5: Neuronal Markers of Neuron-Specific EVs

[0132]

[0133]

[0134] Each GenBank accession number is incorporated herein by reference in its version available as of the filing date of the present application for which priority is claimed in the present application.

[0135] The levels of the biomarkers of the present invention can be determined by any suitable means, methods or techniques known in the art.

[0136] In some embodiments, with respect to polypeptide or protein biomarkers, immunoassay devices and methods are often used. These devices and methods can utilize labeled molecules in various sandwich, competitive or non-competitive assay formats to generate a signal related to the presence or amount of the biomarker of interest. In addition, certain methods and devices (such as biosensors and optical immunoassays) can be used to determine the presence or amount of the biomarker without the need for a labeled molecule.

[0137] In certain embodiments, the detection method is an immunoassay method involving an antibody that specifically binds to one or more of the biomarkers in Tables 1-5. The steps of various useful immunoassay methods have been described in the scientific literature, for example, Nakamura et al. (1987), which is incorporated herein by reference. Generally, immuno-binding methods include obtaining a sample suspected of containing the biomarker protein, peptide or antibody and, according to the present invention, contacting the sample with the antibody or protein or peptide, as the case may be, under conditions effective to permit the formation of an immune complex.

[0138] The detection of immune complex formation is well known in the art and can be achieved by applying a variety of methods. These methods generally rely on the detection of a label or marker, such as any radioactive, fluorescent, biological, or enzymatic tag or marker used standardly in the art. U.S. patents regarding the use of such labels include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241, each of which is incorporated herein by reference. Of course, additional advantages can be found by using a second binding ligand known in the art, such as a secondary antibody or a biotin / avidin ligand binding arrangement. The protein itself used in the detection can be linked to a detectable label, whereupon the label can then simply be detected, allowing determination of the amount of the first immune complex in the composition. Alternatively, the first added component bound within the first immune complex can be detected by a second binding ligand having a binding affinity for the encoded protein, peptide, or corresponding antibody. In these cases, the second binding ligand can be linked to a detectable label. The second binding ligand is typically an antibody itself and can thus be referred to as a "secondary" antibody. Under effective conditions, the first immune complex is contacted with the labeled second binding ligand or antibody for a time sufficient to allow formation of a second immune complex. The second immune complex is then generally washed to remove any non-specifically bound labeled secondary antibody or ligand, and the remaining label in the second immune complex is then detected.

[0139] Further methods include detecting the first immune complex by a two-step method. As described above, a second immune complex is formed using a second binding ligand (such as an antibody) having a binding affinity for the encoded protein, peptide, or corresponding antibody. After washing, again under effective conditions, the second immune complex is contacted with a third binding ligand or antibody having a binding affinity for the secondary antibody for a time sufficient to allow formation of an immune complex (the third immune complex). The third ligand or antibody is linked to a detectable label, allowing detection of the third immune complex thus formed. If desired, the system can provide signal amplification.

[0140] Any suitable immunoassay can be utilized, e.g., enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), competitive binding assay, planar waveguide technology, and the like. Specific immunobinding of an antibody to a marker can be detected directly or indirectly. Direct labels include fluorescent or luminescent tags, metals, dyes, radionuclides, etc. attached to the antibody. Indirect labels include various enzymes well known in the art, such as alkaline phosphatase, horseradish peroxidase, and the like. In some embodiments, the biomarkers of the present invention can be identified by other techniques, such as Western blot, dot blot, and FACS analysis.

[0141] The biomarker of the present invention can also be measured, quantified, detected, and otherwise analyzed using proteomic methods and instruments. Proteomics refers to the application of mass spectrometry to the study of proteins. Although not intended to be limiting, two methods are generally used to characterize proteins by mass spectrometry. In the first method, intact proteins are ionized and then introduced into a mass analyzer. This method is referred to as the "top-down" strategy for protein analysis. The two main methods for ionizing intact proteins are electrospray ionization (ESI) and matrix-assisted laser desorption / ionization (MALDI). In the second method, a protease (such as trypsin) is used to proteolytically digest the protein into smaller peptides. Subsequently, these peptides are introduced into a mass spectrometer and identified by peptide mass fingerprinting or tandem mass spectrometry. Thus, the latter method (also known as "bottom-up" proteomics) uses peptide-level identification to infer the presence of proteins.

[0142] The protein biomarkers of the present invention can also be measured in a complex mixture of proteins and molecules co-existing in a biological medium or sample. However, it may be necessary to fractionate the sample, which is also covered herein. It will be understood that ionization of a complex protein mixture can result in a situation where proteins with higher abundance tend to "drown out" or suppress signals from proteins with lower abundance in the same sample. In addition, due to the large number of mixture components, the mass spectrum of a complex mixture may be difficult to interpret. Fractionation can be used to first separate any complex protein mixture prior to mass spectrometry analysis. Two methods are widely used to fractionate proteins or their peptide products from proteolytic digestion. The first method fractionates intact proteins and is called two-dimensional gel electrophoresis. The second method is high performance liquid chromatography (LC or HPLC), which is used to fractionate peptides after proteolytic digestion. In some cases, it may be necessary to combine these two techniques. Any other suitable methods known in the art for fractionating protein mixtures are also covered herein.

[0143] Characterization of protein mixtures using HPLC / MS is also known in the art as "shotgun proteomics" and MuDPIT (multidimensional protein identification technology). The peptide mixture generated from digestion of a protein mixture is fractionated by one or two steps of liquid chromatography (LC). The eluate from the chromatographic stage can be directly introduced into a mass spectrometer by electrospray ionization or placed on a series of small spots for later mass spectrometry analysis using MALDI.

[0144] In certain embodiments, the present invention relates to detecting nucleic acid biomarkers, e.g., the corresponding gene or mRNA of the protein biomarker of the present invention.

[0145] In various embodiments, the methods of the present invention generally relate to determining the expression levels of a set of genes in extracellular vesicles isolated from a biological sample. In the practice of the methods of the present invention, determining gene expression levels can be performed by any suitable method. For example, determining gene expression levels can be performed by detecting the expression of mRNA expressed by a gene of interest and / or by detecting the expression of a polypeptide encoded by the gene by PCR, such as reverse transcription polymerase chain reaction (RT-PCR), quantitative reverse transcription polymerase chain reaction (qRT-PCR), RNA sequencing (RNA seq), or array analysis. In some embodiments, the expression level of a biomarker is determined by measuring the mRNA or miRNA level of the biomarker.

[0146] Analysis of multiple markers can be performed individually or simultaneously on a single test sample. Several markers can be combined into one test for efficient processing of multiple samples. In addition, those skilled in the art will recognize the value of testing multiple samples from the same individual (e.g., at consecutive time points). Such testing of serial samples will allow identification of changes in marker levels over time. Increases or decreases in marker levels, as well as no change in marker levels, will provide useful information regarding disease state, including but not limited to identifying the approximate time of an event, the suitability of a drug therapy, the effectiveness of various therapies, identifying the severity of an event, identifying the severity of a disease, and identifying the outcome of a patient (including the risk of future events).

[0147] Assays can be constructed consisting of combinations of markers mentioned in the present invention to provide information relevant to differential diagnosis. Such groups can be constructed using 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more individual markers. Individual markers or subsets of markers containing larger marker groups can be analyzed using the methods described within the present invention to optimize clinical sensitivity or specificity in various clinical settings.

[0148] Analysis of markers can also be performed in a variety of physical formats. For example, the use of microtiter plates or automation can be used to facilitate the processing of large numbers of test samples. Alternatively, single sample formats can be developed to facilitate timely point-of-care treatment and diagnosis (e.g., in an ambulance transport or emergency room setting). Particularly useful physical formats include surfaces having multiple discrete addressable locations for detecting multiple different markers. Such formats include protein microarrays, or "protein chips," and capillary devices.

[0149] D. Methods for Purifying, Isolating, and / or Enriching Extracellular Vesicles

[0150] The present invention provides methods for purifying extracellular vesicles (EVs), as well as methods for isolating and / or enriching cell type-specific and / or organ-specific EVs (e.g., brain and / or neuron-specific EVs) from a sample (e.g., a biological sample such as plasma or cerebrospinal fluid).

[0151] Suitable methods for purifying or isolating EVs are known in the art and include, but are not limited to, differential centrifugation, anion exchange and / or gel permeation chromatography, sucrose density gradient or organelle electrophoresis, magnetic-activated cell sorting (MACS), nanofilter ultrafiltration concentrators (see, e.g., U.S. Patent Nos. 6,899,863 and 6,812,023; U.S. Patent No. 7,198,923; Taylor and Gercel-Taylor, 2008; Cheruvanky et al., 2007). In some embodiments, EVs can also be identified and isolated from a subject's body fluid by microchip technology that uses a unique microfluidic platform to effectively and selectively isolate tumor-derived microvesicles. Each of the foregoing references is incorporated herein by reference for teaching these methods.

[0152] In some embodiments, as described herein, mixed-mode resins (MMRs) or size-exclusion chromatography (SEC) can be used to purify EVs from a sample.

[0153] Purification of EVs with mixed-mode resin

[0154] Mixed-mode resin (MMR) can be used to purify EVs from a sample (e.g., a biological sample) from a subject. An exemplary mixed-mode resin is Capto TM Core 700. This resin comprises beads with an inert outer shell and pores that exclude molecules larger than 700 kDa. The core of the MMR beads contains an octylamine ligand that is both hydrophobic and positively charged and effectively captures proteins that enter the beads.

[0155] MMR beads can "capture" contaminants within the beads, thereby enabling the purification of extracellular vesicles from a sample (e.g., a biological sample such as a plasma or CSF sample). The disclosed method does not rely on columns and other chromatographic equipment and is compatible with high-throughput purification of extracellular vesicles.

[0156] In some embodiments, the MMR beads exclude molecules larger than the size of the target EVs, e.g., larger than about 400, 500, 600, or 700 kDa. In some embodiments, the beads include an outer or shell that is inactive. In some embodiments, the outer shell includes pores. In some embodiments, the beads contain a ligand-activated core, such as an octylamine ligand. In some embodiments, the size-exclusion beads comprise a binding and elution resin. In some embodiments, the size-exclusion beads are Capto TMCore resin beads, e.g., Capto TM Core 700 resin beads or Capto TM Core resin 400 beads.

[0157] In some embodiments, the method does not require the use of a column to purify EVs from a biological sample.

[0158] In some aspects, the present disclosure provides a method for purifying extracellular vesicles from a biological sample, comprising binding a liquid biological sample containing extracellular vesicles to mixed-mode resin (MMR) beads capable of capturing molecules smaller than the target EV size (e.g., greater than about 700 kDa) to produce a mixture, and separating and removing the MMR beads from the mixture such that the extracellular vesicles are retained, or by removing the supernatant from the mixture, thereby purifying the extracellular vesicles.

[0159] In some embodiments, the biological sample is obtained from a subject. In some embodiments, the biological sample is a liquid biological sample. Those skilled in the art will recognize that the biological sample can be, but is not limited to, the following body fluids: peripheral blood, plasma, serum, cerebrospinal fluid (CSF), ascites, sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, earwax, milk, bronchoalveolar lavage fluid, semen (including prostatic fluid), Cowper's fluid or pre-ejaculatory fluid, female ejaculate, sweat, feces, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal discharge, mucosal secretion, fecal water, pancreatic juice, sinus lavage fluid, broncho-pulmonary aspirate or other lavage fluid. The biological sample can also include blastocoel, cord blood or maternal circulation that can be derived from the fetus or the mother. The biological sample can also be a tissue sample or biopsy from which EVs can be obtained. In one embodiment, the biological sample is a brain tissue. In one embodiment, the biological sample is a plasma sample. In another embodiment, the biological sample is a CSF sample.

[0160] In some embodiments, the method of the present disclosure is carried out as in a slurry method. The MMR beads can be suspended in a buffer to produce a slurry. In some embodiments, the MMR beads can be suspended in an equal volume of buffer to produce a 50% slurry, or suspended in any volume of buffer effective to produce a slurry, the any volume of buffer having an effective amount of MMR beads to purify EVs from the sample. For example, the slurry can be a 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% slurry. Any effective buffer can be used to produce the slurry. In some embodiments, PBS buffer is used.

[0161] MMR beads can be mixed with a biological sample in an amount of, for example, 20 μL, 25 μL, 50 μL, 75 μL, 100 μL, 150 μL, 200 μL, 250 μL, 300 μL, 350 μL, or 400 μL per 1 mL of liquid biological sample.

[0162] The combination or mixture of MMR beads and the sample containing EVs can be mixed, stirred, or rotated, and then the MMR beads can be separated and removed. For example, the mixture can be mixed, stirred, or rotated for about 10, 20, 30, 40, 45, 50, 55, 60, 65, 70, 75, or more minutes, and then the MMR beads can be separated and removed. In some embodiments, the mixture is mixed, stirred, or rotated between about 30 minutes and about 1 hour. In some embodiments, the mixture is mixed, stirred, or rotated for about 45 minutes. The mixture can be mixed, stirred, or rotated at room temperature or at any temperature conducive to capturing impurities in the sample by the MR beads.

[0163] MMR beads can be separated from the mixture using any effective means known in the art. In some embodiments, centrifugation can be used to separate the MMR beads from the mixture. In some embodiments, the mixture is centrifuged at a speed between about 600 g, 700 g, 800 g, 900 g, or higher. In some embodiments, the mixture is centrifuged at about 800 g. In some embodiments, the mixture is centrifuged for a sufficient length of time to allow the MMR beads to be sufficiently separated from the remainder of the mixture to obtain EVs in the supernatant. In some embodiments, the mixture is centrifuged for about 10 minutes, or about 10, 15, 20, 25, 30, 40, or more minutes.

[0164] In some embodiments, the MMR beads used in the methods of the present disclosure can include an outer or shell of inactive beads. The bead outer shell can include pores that allow molecules smaller than a certain size to pass through the shell and be captured in the core of the bead. In some embodiments, the bead can include a core that includes a bead interior containing a ligand (such as a multimodal ligand, such as an octylamine ligand). In a preferred embodiment, the total shell bead (i.e., shell plus core) thickness is preferably 40 - 100 microns in diameter, and the shell thickness is preferably 2 - 10 microns.

[0165] In some preferred embodiments, the MMR beads used in the methods of the present disclosure include an internal porous core and an external porous shell, wherein the internal core is provided with an octylamine ligand and the shell is inactive, and wherein the porosity of the shell and the core does not allow molecules larger than about 700 kD to enter.

[0166] In some embodiments, the MMR beads contain a binding - elution resin. In some embodiments, the MMR beads are Capto TMCore Binding - Elution Beads. In some embodiments, the MMR beads are Capto TM Core 700 Binding - Elution Beads. Capto TM Core 700 chromatography resin (GE Healthcare Biosciences AB) contains octylamine ligands within the Capto TM Core 700 "beads" and is designed to have hydrophobic and positively charged properties, which can capture molecules below 700 daltons. Since extracellular vesicles are over 700 kDa and since there is no activity outside the beads, Capto Core 700 allows for the purification of extracellular vesicles by size exclusion. Using standard gel filtration (size exclusion chromatography), smaller - sized molecules take more time to penetrate the pores of the stationary phase and thus exhibit higher retention (slower elution) relative to larger molecules. In contrast, the ligand - activated pores of Capto TM Core 700 have electrostatic and hydrophobic interactions that "capture" molecules below 700 kDa.

[0167] In some embodiments, a biological sample (e.g., plasma or CSF) is placed in a size exclusion chromatography (SEC) column before being combined with MMR beads capable of capturing molecules less than about 700 kDa. Any size exclusion chromatography resin known in the art is suitable for the methods of the present invention. In some embodiments, the size exclusion chromatography column includes a stationary phase that comprises a 6% cross - linked agarose size exclusion chromatography matrix. In some embodiments, the 6% cross - linked agarose size exclusion chromatography matrix is Sepharose TM CL - 6B resin.

[0168] In some embodiments, after the size exclusion chromatography column and before being combined with MMR beads capable of capturing molecules less than about 700 kDa, a biological sample (e.g., plasma or CSF) is further placed in a cation exchange chromatography resin. Any cation exchange chromatography resin known in the art is suitable for the methods of the present invention. Exemplary cation exchange chromatography resins include, but are not limited to, thiol, sulfonate, sulfate, carboxymethyl, sulfethyl, sulfopropyl, phosphate, and sulfonate. In some embodiments, the cation exchange chromatography resin is EMD - SO3 - resin.

[0169] The methods for EV purification disclosed herein can also be optimized for high - throughput applications.

[0170] Purifying EVs by Size Exclusion Chromatography

[0171] Size exclusion chromatography (SEC) can also be used to purify EVs from a sample from a subject (e.g., a biological sample). In some embodiments, size exclusion chromatography (SEC) columns can be used to purify EVs from a biological sample, the columns having a stationary phase material comprising an agarose size exclusion chromatography matrix (e.g., a matrix having a 6% agarose content). In some embodiments, the stationary phase material is a 6% cross-linked agarose size exclusion chromatography matrix. In some embodiments, the stationary phase material is Sepharose TM resin, e.g., Sepharose TM cross-linked resin, such as Sepharose TM CL-6B resin. In SEC, a porous stationary phase is utilized to classify macromolecules and particulate matter according to their size. Components in a sample with a smaller hydrodynamic radius are able to pass through the pores, resulting in a later elution. Components with a larger hydrodynamic radius (including EVs) are excluded from entering the pores. Compared to other columns, the SEC columns used in the methods of the present invention result in a significant improvement in EV yield, while the cost is only a fraction of that of other columns.

[0172] In some aspects, the present disclosure provides a method for purifying extracellular vesicles from a biological sample, the method comprising providing an SEC column comprising a stationary phase material comprising a 6% cross-linked agarose size exclusion chromatography matrix (e.g., Sepharose TM CL-6B resin), introducing a sample comprising extracellular vesicles into the column, flowing the sample through the stationary phase material, and collecting a fraction containing extracellular vesicles from the SEC column to thereby purify the extracellular vesicles.

[0173] In some embodiments, the sample is a biological sample obtained from a subject. In some embodiments, the biological sample is a liquid biological sample. Those skilled in the art will recognize that biological samples can be, but are not limited to, the following body fluids: peripheral blood, plasma, serum, cerebrospinal fluid (CSF), ascites, sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, earwax, milk, bronchoalveolar lavage fluid, semen (including prostatic fluid), Cowper's fluid or pre-ejaculatory fluid, female ejaculate, sweat, feces, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretions, fecal water, pancreatic juice, sinus lavage fluid, broncho-pulmonary aspirate or other lavage fluids. Biological samples can also include blastocoels, cord blood or maternal circulation that can be derived from a fetus or a mother. Biological samples can also be tissue samples or biopsies from which EVs can be obtained. In one embodiment, the biological sample is a brain tissue sample. In one embodiment, the biological sample is a plasma sample. In another embodiment, the liquid biological sample is a CSF sample.

[0174] In other embodiments, the SEC column is a 5 mL, 7 mL, 10 mL, 12 mL, 15 mL, 20 mL, or 25 mL volume column. In other embodiments, the SEC column is a 5 mL to 25 mL volume column, although columns outside of these ranges may also be used. In some embodiments, the SEC column is a 10 mL volume column. In other embodiments, the SEC column is a 20 mL volume column.

[0175] An SEC column containing a 6% cross-linked agarose size exclusion chromatography matrix (e.g., Sepharose TM CL-6B resin) can be prepared by first washing the resin and then adding it to the column. In some embodiments, the 6% cross-linked agarose size exclusion chromatography matrix, e.g., Sepharose TM CL-6B resin, is washed in a buffer (e.g., PBS), and then the column is prepared. The resin can be washed multiple times and then the column is prepared. In some embodiments, the 6% cross-linked agarose size exclusion chromatography matrix (e.g., Sepharose TM CL-6B resin) is washed four or more times in a buffer and then the column is prepared.

[0176] Once the resin has been washed, it can be added to a suitable column, e.g., a 10 mL or 20 mL column. In one embodiment, the SEC column includes a housing having at least one wall that defines a chamber having an inlet and an outlet. In some embodiments, the stationary phase is washed, and then the column containing the 6% cross-linked agarose size exclusion chromatography matrix, e.g., washing Sepharose TM CL-6B resin, and then a sample containing extracellular vesicles is introduced into the column. In some embodiments, the stationary phase is washed with PBS.

[0177] Fractions containing EVs can be collected from the SEC column to purify EVs from a liquid biological sample. For example, fractions 6-21 or fractions 12-27 can be collected, depending on the size of the column. In some embodiments, fractions 6-21 can be collected for a 10 mL column. In other embodiments, fractions 12-27 can be collected for a 20 mL column. Fewer fractions can also be collected from the SEC column. For example, higher purity EVs can be achieved by collecting a smaller number of fractions (e.g., 7-9 instead of 7-10), but the yield is lower.

[0178] Although not required, after purifying EVs using any method of the present disclosure, the purified extracellular vesicles can be further purified by any method known in the art. In addition, the methods for purifying EVs described herein can be combined with each other and with other EV purification methods known in the art. For example, in some embodiments, cation exchange chromatography, size exclusion chromatography (such as gel permeation columns), centrifugation or density gradient centrifugation, and filtration methods can be used in combination with the methods of the present disclosure. As another example, the EV purification methods of the present disclosure can use differential centrifugation, anion exchange and / or gel permeation chromatography, sucrose density gradient, organelle electrophoresis, magnetic-activated cell sorting (MACS), or nanofilter ultrafiltration concentrators.

[0179] In some embodiments, the fractions collected from the SEC column are further placed in a cation exchange chromatography resin. The cation exchange chromatography resin includes a stationary phase containing a functional group selected from the group consisting of: mercapto, sulfonate, sulfate, carboxymethyl, sulfoethyl, sulfopropyl, phosphate, and sulfonate. In some embodiments, the cation exchange chromatography resin is EMD-SO3-resin.

[0180] In some embodiments, the fractions collected from the SEC column are further placed in mixed-mode resin (MMR) beads capable of capturing molecules less than about 700 kDa. In some embodiments, the MMR beads are Capto TM Core 700. In another embodiment, the fractions collected from the SEC column are further placed in a cation exchange chromatography resin and MMR beads capable of capturing molecules less than about 700 kDa.

[0181] In some embodiments, when compared to existing methods in the art, the methods of the present application recover at least about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, or more EVs from samples (e.g., plasma and CSF samples).

[0182] High-abundance proteins (such as albumin and immunoglobulins) may impede the purification of EVs from biological samples. Thus, the methods of the present disclosure can be used with systems that utilize multiple antibodies that are specific for the most abundant proteins found in blood. Such systems can remove up to several proteins at once, thereby revealing less abundant species, such as source cell-specific exosomes. Other known methods for EV purification include high-abundance protein removal methods such as those described in Chromy et al., J. Proteome Res 2004; 3:1120-1127. In another embodiment, serum proteins can also be removed by using glycopeptide capture as described in Zhang et al., Mol Cell Proteomics 2005; 4:144-155, thereby enhancing the purification of EVs from biological samples.

[0183] Isolation and / or enrichment of cell type-specific EVs

[0184] Extracellular vesicles isolated from a biological sample obtained from a subject can be further enriched by positive selection, negative selection, or a combination of positive and negative selections. As used herein, the terms "isolate" or "enrich" refer to increasing the concentration or density of extracellular vesicles or a subset of extracellular vesicles in a sample, or removing non-EV substances (e.g., proteins, cells) from the sample.

[0185] EVs can be further isolated and / or enriched based on the source or type of the cell. In some embodiments, cell type-specific EVs can be isolated and / or enriched based on differences in the biochemical properties of the EVs. For example, cell type-specific EVs can be further isolated and / or enriched based on antigenic, nucleic acid, metabolic, gene expression, or epigenetic differences. In some embodiments, based on antigenic differences, antibody-conjugated magnetic or paramagnetic beads in a magnetic field gradient or fluorescently labeled antibodies in flow cytometry are used. Cell type-specific EVs can also be enriched based on other biochemical properties known in the art. For example, EVs can be enriched based on pH or motility. Further, in some embodiments, more than one method is used to enrich EVs. In other embodiments, antibodies, ligands, or soluble receptors are used to enrich EVs in a sample.

[0186] Because EVs typically carry surface molecules (such as antigens from their donor cells), the surface molecules can be used to identify, isolate, and / or enrich EVs from a specific donor cell type. In some embodiments, surface markers are used to positively enrich EV subpopulations from one or more cell types. In some embodiments, cell surface markers that are not present on the EV population are used to negatively enrich vesicles by depleting the cell population. Flow cytometry sorting can also be used to further enrich EVs, using cell surface markers conjugated to fluorescent labels or intracellular or extracellular markers. Intracellular and extracellular markers can include nuclear staining or antibodies against intracellular or extracellular proteins preferably expressed in vesicles.

[0187] In some embodiments, for EVs isolated from a biological sample (e.g., a body fluid, e.g., CSF or plasma), those EVs derived from a specific tissue (e.g., brain, lung, pancreas, stomach, intestine, bladder, kidney, ovary, testis, skin, colorectal, breast, prostate, brain, esophagus, liver, placenta, fetal cells) are enriched.

[0188] In some embodiments, cell type-specific EVs are isolated and / or enriched. In some embodiments, based on the biomarkers of the present invention (e.g., one or more biomarkers in Tables 1-5), cell type-specific EVs (e.g., brain and / or neuron-specific EVs) are isolated and / or enriched from a sample (e.g., a biological sample such as plasma or cerebrospinal fluid).

[0189] In some embodiments, the EVs enriched in the biological sample are brain-specific EVs. In one embodiment, the biomarkers for brain-specific EVs include one or more biomarkers in Tables 1-5, or any combination thereof.

[0190] In some embodiments, the EVs enriched in the biological sample are neuron-specific EVs. In one embodiment, the biomarkers for neuron-specific EVs include one or more biomarkers in Tables 1 and 5, or any combination thereof. In one embodiment, the biomarkers for neuron-specific EVs include one or more biomarkers selected from the group consisting of: GABRG2, SVOP, SLC32A1, GRM7, GABRB3, CHRNB2, SLC12A5, GRM8, PTPRT, SLC6A17, PCDHAC2, PLPPR4, C11orf87, SORCS3, CALY, PTPRR, KIAA1549L, HCN1, CDH18, TMEM132D, GPR158, FRRS1L, ATP2B3, GRIA4, ST8SIA3, HS6ST3, SEZ6, and NRXN3.

[0191] In some embodiments, the EVs enriched in the biological sample are astrocyte-specific EVs. In one embodiment, the biomarkers for astrocyte-specific EVs include one or more biomarkers in Table 2, or any combination thereof.

[0192] In some embodiments, the EVs enriched in the biological sample are oligodendrocyte-specific EVs. In one embodiment, the biomarkers for oligodendrocyte-specific EVs include one or more biomarkers in Table 3, or any combination thereof.

[0193] In some embodiments, the EVs enriched in the biological sample are microglia-specific EVs. In one embodiment, the biomarkers for microglia-specific EVs include one or more biomarkers in Table 4, or any combination thereof.

[0194] In some embodiments, the isolation of cell type-specific EVs can be achieved by one or more purification or separation steps. One or more purification steps can include, but are not limited to, immunoisolation, microfluidics-based affinity purification, magnetic-based purification, pull-down purification, or fluorescence-activated vesicle sorting-based purification. Commercial precipitation kits are also available, such as ExoQuick TM and Total Exosome Isolation TM precipitation solutions. Such kits are easy to use, with only one or two steps, and do not require any expensive equipment or advanced proprietary technology.

[0195] In some embodiments, immunoisolation can be used for isolation, which utilizes one or more antibodies against one or more biomarkers of the present invention (e.g., one or more markers in Tables 1-5). The immunoisolation can be carried out using a bait / prey strategy. In some embodiments, the bait molecule can be a bait protein, such as an antibody, e.g., a monoclonal antibody against the prey EV biomarker of the present invention. In some embodiments, the bait molecule can also be an RNA aptamer. If several prey EVs are to be combined for purification, a mixture of the corresponding monoclonal antibodies against each of the prey EV biomarkers can be used.

[0196] In some embodiments, the bait molecule is recognized by an affinity ligand. The affinity ligand can be a divalent metal-based complex, a protein, a peptide (such as a fusion protein tag), or more preferably an antibody.

[0197] In some embodiments, a bait molecule or an affinity ligand is immobilized or "coupled" directly or indirectly to a solid substrate material, such as by forming a covalent chemical bond between a specific functional group on the ligand (e.g., primary amine, thiol, carboxylic acid, aldehyde) and a reactive group on the substrate. In the affinity purification step of the methods of the present invention, the substrate or matrix can be any material that is coupled to a biospecific ligand (i.e., a bait molecule or an affinity ligand). Useful affinity supports can be those having a high surface area to volume ratio, chemical groups that are easily modified to covalently attach ligands, minimal non-specific binding properties, good flow characteristics, and / or mechanical and chemical stability. Several substrates can be used as the solid substrate, including, for example, agarose, cellulose, dextran, polyacrylamide, latex, or controlled pore glass. Magnetic particles can also be used as the substrate instead of using bead agarose or other porous resins. Their smaller size provides a sufficient surface area to volume ratio required for efficient ligand immobilization and affinity purification. Magnetic beads can be produced as superparamagnetic iron oxide particles, which can be covalently coated with a silane derivative. This coating renders the beads inert (i.e., to minimize non-specific binding) and provides the specific chemical groups required to attach any affinity ligand of interest. Affinity purification using magnetic particles is generally not performed in a column. Instead, a few microliters of beads can be mixed with several hundred microliters of sample to form a loose slurry. During mixing, the beads remain suspended in the sample solution, allowing for affinity interactions with the immobilized ligand. After allowing sufficient binding time, the beads are collected and separated from the sample using a strong magnet.

[0198] In some embodiments, a pull-down assay can be performed to purify or isolate cell type-specific EVs by pulling down one or more specific EV biomarkers of the present invention (e.g., using one or more antibodies against each of one or more of the biomarkers in Tables 1-5). The EV biomarkers can be specific for at least one cell type and advantageously result in enrichment of EVs from the selected cell type.

[0199] In some embodiments, the at least one or more purification steps for purifying a cell type-specific EV subset include pull-down purification. In such pull-down purification, the prey EV biomarker is generally a (trans)membrane protein that has been found to be expressed in a cell type or cell subtype. The bait protein is preferably a monoclonal antibody against any prey EV biomarker that will be pulled up. Magnetic beads coated with an affinity ligand for the bait protein (such as magnetic nucleic acid-binding beads) or silica beads functionalized with silanes (e.g., from Thermo Fisher Scientific) such as from Thermo Fisher Scientific MyOne silane beads can be used to isolate the bait protein that binds to the prey EV biomarker. The affinity ligand is preferably a class - specific or species - specific antibody. As an example, magnetic beads coated with anti - mouse antibodies can be used together with monoclonal mouse antibodies against specific surface proteins of a subset of cell types or cell subtypes of EVs. Generally, control antibodies such as mouse mCherry monoclonal antibodies can be used.

[0200] Thus, pull - down assays can be used to illustrate and validate the purification or isolation of one or more EV subsets, each of which expresses at least one specific membrane protein biomarker. Using Western blotting or qRT - PCR, the purification and isolation of EV subsets by at least one specific prey EV biomarker can be further confirmed.

[0201] E. Methods for disease diagnosis and prognosis

[0202] The present invention also provides methods for diagnosing or prognosticating a disease (e.g., neurodegenerative disorder) in a subject, identifying a subject at risk of developing a disorder, or prescribing a treatment regimen or predicting therapeutic benefit for a subject suffering from a disorder.

[0203] Once cell type - specific and / or organ - specific EVs (e.g., brain - and / or neuron - derived EVs) are isolated and / or enriched based on the presence of one or more biomarkers of the present invention, analyzing the molecular content of these cell type - specific and / or organ - specific EVs provides very good insights for early detection and better understanding of pathology. For example, since it is generally not possible to biopsy the human brain and / or neurons, reading the molecular content of the brain and / or neurons by isolating brain - and / or neuron - derived EVs from accessible biological fluids can provide a unique perspective through which to understand brain pathology.

[0204] Thus, in one aspect, the present invention provides a method for diagnosing, prognosticating a neurodegenerative disorder or identifying a subject at risk of developing a neurodegenerative disorder in a subject, comprising: (a) obtaining a biological sample from the subject; (b) isolating brain - specific extracellular vesicles from the biological sample based on the presence of a biomarker in the isolated extracellular vesicles, wherein the biomarker comprises one or more biomarkers selected from Tables 1 - 5; (c) extracting proteins and / or RNA from the isolated brain - specific extracellular vesicles; and (d) analyzing the extracted proteins and / or RNA from the isolated brain - specific extracellular vesicles, thereby diagnosing, prognosticating a neurodegenerative disorder or identifying a subject at risk of developing a neurodegenerative disorder.

[0205] In some embodiments, the biological sample includes a liquid biological sample. In some embodiments, the liquid biological sample is selected from the group consisting of: whole blood, serum, plasma, cerebrospinal fluid, spinal fluid, amniotic fluid, aqueous humor, vitreous humor, bile, milk, cerumen (ear wax), chyle, chyme, endolymph, perilymph, exudate, feces, female ejaculate, gastric acid, gastric juice, lymph, mucus (including nasal drainage and sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus, inflammatory secretions, saliva, sebum (skin oil), semen, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vomit, and mixtures of one or more thereof.

[0206] In some embodiments, the extracellular vesicles are neuron-specific, astrocyte-specific, oligodendrocyte-specific, and / or microglia-specific.

[0207] In some embodiments, the extracellular vesicles are neuron-specific, and wherein the one or more biomarkers are selected from Tables 1 and 5. In some embodiments, the one or more biomarkers are selected from the group consisting of: GABRG2, SVOP, SLC32A1, GRM7, GABRB3, CHRNB2, SLC12A5, GRM8, PTPRT, SLC6A17, PCDHAC2, PLPPR4, C11orf87, SORCS3, CALY, PTPRR, KIAA1549L, HCN1, CDH18, TMEM132D, GPR158, FRRS1L, ATP2B3, GRIA4, ST8SIA3, HS6ST3, SEZ6, and NRXN3.

[0208] In some embodiments, the extracellular vesicles are astrocyte-specific, and wherein the one or more biomarkers are selected from Table 2.

[0209] In some embodiments, the extracellular vesicles are oligodendrocyte-specific, and wherein the one or more biomarkers are selected from Table 3.

[0210] In some embodiments, the extracellular vesicles are microglia-specific, and wherein the one or more biomarkers are selected from Table 4.

[0211] The protein and / or RNA content of EVs has been found to be associated with the protein and / or RNA content of the corresponding cells. Thus, analyzing the protein and / or RNA content of EVs provides qualitative and quantitative information about the cellular RNA content of the corresponding cells. Advantageously, this enables the provision of non-invasive diagnostic methods. In fact, the analysis (whether by DNA / RNA sequencing, transcriptome analysis, qRT-PCR, microarray, proteome analysis or mass spectrometry, etc.) is performed on biological samples derived from body fluids such as those derived from blood or cerebrospinal fluid. Such fluids are easier and more accessible than the corresponding organs (e.g., the brain). Accordingly, the present invention provides non-invasive but reliable diagnostic methods.

[0212] In some embodiments, the protein and / or nucleic acid content of cell type-specific and / or organ-specific EVs is extracted and analyzed. In some embodiments, the extracted nucleic acids include messenger RNA (mRNA), microRNA (miRNA), long non-coding RNA (lncRNA), small non-coding RNA, DNA, and any other full-length RNA or DNA or fragments thereof. Protein and / or nucleic acid extraction can be performed using any known method in the art or commercial kits.

[0213] Any transcriptomics method can be used to analyze the RNA content in EVs, such as RNA sequencing, DNA sequencing, reverse transcription polymerase chain reaction (RT-PCR) or quantitative reverse transcription polymerase chain reaction (qRT-PCR) or array analysis. In some embodiments, the analysis of the extracted nucleic acids from isolated brain-specific extracellular vesicles includes whole genome analysis or transcriptome analysis.

[0214] In other embodiments, the analysis of the extracted nucleic acids from isolated brain-specific extracellular vesicles includes analyzing genes of interest, wherein the genes of interest are associated with neurodegenerative disorders. For example, testing for the presence or absence of the genes of interest, analyzing one or more allelic variants or mutations of the genes of interest, and testing for the presence or absence of these allelic variants or mutations.

[0215] Similarly, any proteomics method known in the art can be used to analyze the protein content in EVs, such as proteome analysis, mass spectrometry, immunoassay, ELISA, fluorescence-activated cell sorting (FACS), SDS-polyacrylamide gel electrophoresis (SDS-PAGE) or Western blot analysis.

[0216] In some embodiments, analyzing nucleic acids extracted from isolated brain-specific extracellular vesicles includes analyzing a protein of interest, wherein the protein of interest is associated with a neurodegenerative disorder. In some embodiments, analyzing the extracted protein includes testing for the presence or absence of the protein of interest, analyzing one or more mutations in the protein of interest, e.g., deletions, additions, substitutions, truncations, or modifications, e.g., proteins with altered post-translational modification states, or epigenetic alterations of the protein of interest, and testing for the presence or absence of these mutations or modifications.

[0217] Neurodegenerative disorders diagnosed or prognosed by the methods of the present invention include, but are not limited to, Alzheimer's disease (AD), Huntington's disease, multiple sclerosis, vascular dementia, frontotemporal dementia (FTD), corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), dementia with Lewy bodies, tangle-predominant senile dementia, Pick's disease (PiD), argyrophilic grain disease, amyotrophic lateral sclerosis (ALS), other motor neuron diseases, Guam Parkinson's disease-dementia complex, FTDP-17, Lytico-Bodig disease, multiple sclerosis, traumatic brain injury (TBI), and Parkinson's disease.

[0218] Genes / proteins of interest associated with neurodegenerative disorders are known in the art. Examples of genes / proteins associated with Parkinson's disease include, but are not limited to, alpha-synuclein, DJ-1, LRRK2, PINK1, Parkin, UCHL1, Synphilin-1, and NURR1. Examples of Alzheimer's disease-associated genes / proteins can include, but are not limited to, Tau and related post-translational modifications (p-Tau), very low density lipoprotein receptor protein (VLDLR) encoded by the VLDLR gene, ubiquitin-like modifier activating enzyme 1 (UBA1) encoded by the UBA1 gene, or NEDD8-activating enzyme E1 catalytic subunit protein (UBE1C) encoded by the UBA3 gene. Examples of genes / proteins associated with amyotrophic lateral sclerosis can include SOD1 (superoxide dismutase 1), ALS2 (amyotrophic lateral sclerosis 2), FUS (fused in sarcoma), TARDBP (TAR DNA-binding protein), VAGFA (vascular endothelial growth factor A), VAGFB (vascular endothelial growth factor B), and VAGFC (vascular endothelial growth factor C), and any combination thereof. Analyzing these genes or proteins of interest in brain- and / or neuron-derived EVs can provide insights into the health of a subject's brain.

[0219] In some embodiments, cell type-specific and / or organ-specific extracellular vesicles (e.g., brain and / or neuron-specific EVs) can be analyzed at various time intervals over a specific time course to evaluate the progression and pathology of a subject. For example, the analysis can be performed at regular time intervals, such as once a day, once every two days, once every three days, once a week, once every two weeks, once a month, once every two months, once every three months, once every six months, or once a year, to track changes in the levels and characteristics of brain and / or neuron-derived EVs over time. For existing patients, this provides a useful indication of disease progression and helps medical staff make appropriate treatment choices based on increases, decreases, or lack of changes in the content of brain and / or neuron-derived EVs. For those patients at risk of neurodegenerative diseases, the protein and / or RNA content of brain and / or neuron-derived EVs can provide early warning or diagnosis.

[0220] F. Kits

[0221] Another aspect of the invention encompasses kits for isolating cell type-specific and / or organ-specific extracellular vesicles (e.g., brain and / or neuron-specific EVs) from biological samples obtained from a subject; and kits for detecting neurodegenerative disorders in a subject.

[0222] A kit for isolating brain-specific extracellular vesicles can include one or more of the following: (a) one or more reagents for detecting the presence of a biomarker on the surface of the extracellular vesicles, wherein the biomarker includes one or more biomarkers selected from Tables 1-5; (b) a method for isolating brain-specific extracellular vesicles based on the presence of the biomarker; and (c) a set of instructions for detecting the presence of the biomarker and / or isolating the brain-specific extracellular vesicles. In some embodiments, the one or more reagents for detecting the presence of a biomarker on the extracellular vesicles are antibodies or aptamers that bind to the biomarker. In some embodiments, the kit further includes a device for isolating a biological sample from the subject.

[0223] A kit for detecting a neurodegenerative disorder can include one or more of the following: (a) one or more reagents for detecting the presence of a biomarker on the surface of the extracellular vesicles, wherein the biomarker includes one or more biomarkers selected from Tables 1-5; (b) a method for isolating brain-specific extracellular vesicles based on the presence of the biomarker; (c) one or more reagents for measuring the levels of genes associated with the neurodegenerative disorder in the isolated brain-specific extracellular vesicles; and (d) a set of instructions for detecting the presence of the biomarker, isolating the brain-specific extracellular vesicles, and / or measuring the levels of genes associated with the neurodegenerative disorder. In some embodiments, the kit further includes a device for isolating a biological sample from the subject.

[0224] The present invention encompasses various kits having different components. Generally, a kit will include a device for collecting a biological sample or extracellular vesicles, a device for detecting one or more biomarkers in the extracellular vesicles, and instructions for use of the kit contents. In certain embodiments, the kit includes a device for enriching or isolating a subset of extracellular vesicles (e.g., cell type-specific and / or organ-specific extracellular vesicles) in a biological sample. In further embodiments, the device for enriching or isolating extracellular vesicles includes reagents necessary for enriching or isolating extracellular vesicles from a biological sample. In some embodiments, one or more reagents for detecting the presence of a biomarker on the extracellular surface include an antibody against the biomarker. In certain embodiments, the kit includes a device for detecting and / or quantifying the level of a gene of interest associated with a neurodegenerative disorder in cell type-specific and / or organ-specific extracellular vesicles. In further embodiments, the device for quantifying the amount of the gene of interest includes reagents necessary for detecting the amount of the gene of interest.

[0225] All documents cited or referenced herein and the contents of all documents cited or referenced in the documents cited herein, together with any manufacturer's instructions, descriptions, product specifications, and product manuals for any products mentioned in this document or incorporated herein by reference, GenBank accession numbers and gene numbers, as well as published patents and patent applications, are hereby incorporated by reference and may be used in the practice of the present invention. Those skilled in the art will recognize that the present invention may be practiced with variations in the structures, materials, compositions, and methods disclosed, and such variations are considered to be within the scope of the present invention.

[0226] The present invention is further illustrated by the following examples, which should not be construed as limiting.

[0227] Examples

[0228] Example 1: Identification of Markers for Isolating Neuron-Specific Extracellular Vesicles

[0229] Selection of Neuron-Specific EV Biomarker Candidates Based on Gene Expression

[0230] The potential of all human proteins as handles for isolating neuron-specific EVs from human biological fluids such as CSF and plasma was evaluated ( Figure 1A)。The goal is not to deterministically decide which markers will be most effective, but rather to construct a simple framework for determining which markers should be prioritized for experimental validation. The first requirement is that potential markers must have an annotated transmembrane domain, as this is necessary for the immunoisolation of EVs. Out of the 20,375 reviewed proteins in the protein database UniProt, 4,845 have an annotated transmembrane domain (33).

[0231] The second requirement is cell type-specific gene expression of the marker in the desired cells and source tissues. Thus, the candidate marker should be highly enriched in neurons relative to other cell types in the brain, and highly enriched in the brain relative to other organs in the human body. To evaluate cell type-specific gene expression, RNA-Seq data of the major cell types isolated from human brain tissue by immuno-panning were analyzed (34). To evaluate organ-level expression, the GTEx RNA-Seq dataset of human organs was analyzed (35). The gene expression specificity index Tau was used to calculate the specificity of each gene (36), as it has been shown to be particularly robust relative to other methods (37). A Tau cutoff value of 0.7 ( Figure 1B 、 Figures 5 - 7 ) was selected, and it was found that 291 genes encode transmembrane proteins that are specifically expressed in neurons relative to other cell types in the brain, while 305 genes encode transmembrane proteins that are specific to the brain relative to other organs. Overlapping these two lists, 168 transmembrane proteins were found ( Figure 1C ). Since some of these proteins can be expressed in neurons but are not present on EVs in the biological fluid of interest, it was inferred that transmembrane proteins exhibiting cell and tissue specificity can overlap with the proteomics dataset of EVs in the biological fluid of interest.

[0232] Differentiation of human iPS cells into iNGN neurons for isolation of EVs from conditioned medium

[0233] Before studying neuronal EVs in human biological fluids, an effort was first made to develop a positive control (i.e., a source of pure neuronal EVs). A human induced pluripotent stem (iPS) cell line with doxycycline-inducible expression of the transcription factor Neurogenin 1 / 2 was previously established for the rapid and efficient differentiation of iPS cells into neurons (38). The protocol for differentiating induced Neurogenin (iNGN) cells was scaled up, and EVs were collected from their conditioned medium (39). Mass spectrometry-based proteomic analysis was performed on EVs isolated from neurons, and the uninduced parental iPS line was used as a control. It was found that 197 transmembrane proteins were expressed on EVs from neurons but not on iPS cells ( Figure 1D)。Among these 197 proteins, seven passed the cell- and tissue-specific thresholds ( Figure 1E )。Using Western blot, it was confirmed that one of these markers, SYT1, was present in both neuronal cell lysates and neuronal EVs ( Figure 1F )。

[0234] Developing an EV immunoisolation protocol using EVs from cell cultures

[0235] An immunoisolation protocol was developed to capture a subset of EVs displaying specific markers. Since EVs from neurons were expected to be a small subset of total EVs in biological fluids such as plasma, the protocol needed to be efficient and specific. To simplify this goal, the human K562 cell line was selected ( Figure 2A )。First, immunoisolation was optimized using the widely expressed tetraspanin CD81. After purifying EVs from the conditioned medium of K562 cells using differential ultracentrifugation (39), immunoisolation was performed, and the efficiency was evaluated by Western blot of CD81 in the eluate fraction by pull-down. To ensure that the immunoisolation was specific for the target protein, CD81 was also measured when immunoisolating EVs using the same procedure but with a non-specific antibody. Using this system, several parameters such as antibody conjugation strategy, amount and ratio of beads and antibody, etc. were systematically optimized. The final protocol achieved efficient and specific immunoisolation ( Figure 2B 、 Figures 8A - 8E )。

[0236] Next, the optimized general cell culture EV immunoisolation protocol was applied to EVs from iNGN neurons. Proteomics data detected the presence of L1CAM and NCAM1 on neuronal EVs. Although it did not pass the Tau-specific score, these two proteins, especially L1CAM, have been used by others as targets for immunoisolation in plasma. Although it was ultimately shown that L1CAM was not a viable marker in human CSF and plasma (32), L1CAM was analyzed on iNGN EVs before analyzing L1CAM in biological fluids as a test platform for neuronal EV immunoisolation. A direct comparison of previously used antibodies was performed and further optimized ( Figure 12 and Figure 13 ) to achieve efficient and specific L1CAM pull-down in iNGN EVs ( Figure 2C )。To mimic the capture of cell type-specific EVs from biological fluids, L1CAM-positive EVs from neurons were mixed with L1CAM-negative EVs from undifferentiated iPS cells ( Figure 2D)。GJA1 is a protein present on iPS EVs but not on neuronal EVs. After monitoring EVs mixed from both cell types, it was confirmed that GJA1 was not detected on beads with L1CAM antibody( Figure 2E )。These experiments confirmed that immunoisolation was specific even when the proportion of neuronal EVs relative to non-neuronal EVs was low.

[0237] Immunoisolation of EV subsets from human CSF and plasma

[0238] After developing immunoisolation methods for EVs from cell cultures, these methods were tested in human biological fluids( Figure 2F )。With some optimizations( Figures 9 - 11 、 Figures 14A - 14B and Figures 15A - 15E ), EVs containing the tetraspanins CD9, CD63, and CD81 were immunoisolated from human CSF( Figure 2G ) and plasma( Figure 2H ). Since the effluent contained protein levels too high to run on protein gels, a second immunoisolation (using beads with target or control antibodies) against each tetraspanin was performed on the effluent from the first immunoisolation. Comparing the results of the first immunoisolation with those of the second immunoisolation showed that the protocol was efficient and specific in both CSF and plasma. Thus, although dependent on having good enough antibodies, the protocol enables highly specific immunoisolation from human biological fluids without first purifying the EVs.

[0239] Development of high-purity EV isolation methods

[0240] Next, proteomics was used to evaluate which neuronal EV markers are present in human CSF and plasma. However, contaminating free proteins in EV preparations limit the utility of mass spectrometry for detecting EV proteins because free proteins (such as albumin) in biological fluids are many orders of magnitude more abundant than EV proteins (40). Previous extensive comparisons of different EV isolation methods in CSF and plasma have been made (41). Although size-exclusion chromatography (SEC) has been optimized to remove free proteins (such as albumin) by several orders of magnitude, the EV fraction in SEC still carries substantial amounts of albumin. Therefore, new methods were developed that allow for the purification of EVs from human biological fluids with extremely high purity while still maintaining high yields. After extensive exploration, a mixed-mode resin (MMR) called Capto Core 700 was identified. This resin consists of beads with an inert outer shell and pores that exclude molecules larger than 700 kDa. The MMR beads have a core containing octylamine ligands that are both hydrophobic and positively charged, effectively capturing proteins that enter the beads. This resin was developed for use in chromatography columns for virus purification (42), but a report has described the use of this resin for virus purification “in slurry” without the need for columns used for the purification of non-enveloped infectious viruses from cells (43).

[0241] A simple method of mixing the MMR with biological fluids was developed for the purification of EVs from free proteins. Since the MMR beads bind and capture free proteins, it was inferred that after incubation with biological fluids, the beads could be separated, leaving pure EVs( Figure 3A ). By measuring the levels of the four-transmembrane proteins CD9, CD63, and CD81 (as indicators of EV yield) as well as albumin to measure free protein contamination, after various optimizations, it was able to converge to the most important parameters for EV isolation( Figure 3B ). The most important parameter was found to be the ratio of the resin to the total protein in the sample. An optimized method was developed, which we call MMR Slurry, and it was applied to CSF. It was found that by precisely increasing the ratio of the resin volume to the total protein in the sample, the EV purity could be “regulated”. In this way, albumin could be completely depleted (as measured by Western blotting) while retaining most of the EVs( Figure 3C 、 Figure 3D ). Consistent with previous results (32), as the amount of MMR increased, L1CAM was also found to completely disappear by Western blotting( Figure 3E ).

[0242] After optimizing the separation for CSF, the protocol was applied to plasma. Plasma has two orders of magnitude more protein than CSF, so after initial purification of plasma using SEC, the MMR slurry method was applied to plasma. By applying the MMR slurry method to 1 mL of plasma after SEC, albumin could be depleted to levels that were barely detectable ([ Figure 3F , Figure 3G ). Thus, the MMR slurry method can be applied to CSF as a simple one-step purification or to plasma as a two-step protocol after SEC.

[0243] Mass spectrometry of EVs from human plasma and CSF using MMR slurry

[0244] The MMR slurry was applied to establish EV proteomes for CSF and plasma. The protocol was further optimized to increase the number of transmembrane proteins detected and to combine this high-purity separation with methods previously developed for low-input proteomics. Using the one-step MMR slurry method for CSF or the two-step SEC and MMR slurry method for plasma, high-quality proteomes were obtained from 1 mL of biological fluid ([ Figure 4A ). These proteomes were comparable or deeper (in terms of the number of proteins detected) compared to previous studies using much larger volumes of CSF and plasma. One limitation of using two-step SEC followed by MMR slurry is that lipoproteins, being larger than 700 kDa, are not removed from plasma. However, it was found that ApoB100 levels were reduced by replacing SEC with dual-mode chromatography (DMC), which combines SEC with cation-exchange resin (44). Using two-step DMC + MMR slurry to isolate EVs from plasma, even more proteins were detected than with SEC + MMR slurry (Figure SI). Various mass spectrometry runs were combined on 1 mL of pooled human plasma or CSF to generate reference EV proteomes, yielding 2104 proteins for CSF and 1862 proteins for plasma.

[0245] Pipeline for identification of neuron-specific EV markers

[0246] After establishing high-quality reference EV proteomes for CSF and plasma, this data was inserted into an in silico pipeline. A list of candidate transmembrane proteins meeting the gene expression cut-off was intersected with the MMR slurry proteomics data ([ Figure 4A ). Overlapping these datasets, 27 candidate markers were found to meet all established criteria: transmembrane, specific cell type and tissue expression, and detected in the generated EV CSF or plasma data ([ Figure 4B ). Of these 27 markers, two were detected in both CSF and plasma, and the rest were present in one of the two biological fluids ([ Figure 4C)。To further rank these candidates, existing datasets of proteins found in CSF (45, 46) and plasma (47) or EVs isolated from these biological fluids (47 - 51) were analyzed (Tables 1 - 5). This comprehensive analysis allowed the identification of candidate markers for neuron - specific EVs isolated from human biological fluids.

[0247] Discussion

[0248] In this work, based on gene expression and EV proteomics data, a systematic and unbiased framework was provided for the identification of markers of neuron - specific EVs. The expression levels of all human transmembrane proteins were analyzed, their enrichment in neurons relative to other cell types in the brain, and their enrichment in the brain relative to other organs. Based on proteomics data, the markers were further ranked according to the importance of experimental validation to ensure the discovery of markers of interest on EVs. To this end, a more extensive reference human CSF and plasma EV proteomics dataset was generated than in previous studies. In addition, EVs from human iNGN neuron conditioned medium were established as a "positive control" for endogenous neuron EVs in human biological fluids, and their EV proteome was analyzed.

[0249] In addition to generating new reference proteomics EV datasets and constructing a computational marker prediction pipeline, several technical advances were made in the use of EVs as biomarkers. First, a highly optimized EV immunoisolation protocol was developed that is effective in both plasma and CSF without the need to pre - isolate EVs as is commonly done in other studies. Second, a novel MMR slurry method was developed that allows the isolation of EVs with unprecedented purity, which is particularly important for the proteomics analysis of EVs. This method can purify EVs from a clinically relevant volume of CSF by simply incubating the resin with the biological fluid. The MMR slurry was also applied to purify high - purity EVs from plasma after SEC or DMC (to remove lipoproteins). Since it has recently been shown that Capto Core resin can remove dyes or small amounts of proteins when used in 96 - well filter plates (52), it is envisioned that the MMR slurry is suitable for high - throughput formats. This would make the MMR slurry, together with the ease of use and low cost of the method, particularly suitable for EV diagnostics.

[0250] The present invention provides important guidance for the great potential of separating neuron-specific EVs and cell type-specific EVs from biological fluids as a non-invasive readout of cell states in health and disease. The resources and framework introduced in the present invention should be widely applicable to the identification of cell type-specific EV markers of other cell types in the brain and other organs. Analyzing the molecular cargo of neuron-derived EVs provides exciting opportunities for both early detection and a better understanding of brain pathologies. Since it is generally not possible to biopsy human neurons, reading the molecular content of neurons by separating neuron-derived EVs from accessible biological fluids can provide an unprecedented perspective through which to understand problems in the brain and disease.

[0251] Materials and Methods

[0252] Computational Marker Pipeline

[0253] Cell type-specific Tau was calculated using brain RNA-Seq expression data (log scale) of the five major cell types of the brain (neurons, astrocytes, oligodendrocytes, microglia, and endothelial cells). For determining cell type-specific expression, genes with the highest expression within a cell type (neurons) relative to the other four cell types were selected if the cell type-specific Tau score of the gene was 0.7 or above. Organ-specific Tau was calculated using GTEx organ-specific RNA sequencing data. Since the GTEx data contains several regions or tissues for each organ, all regions or tissues of a particular organ were averaged to obtain an organ-level measurement. The pituitary, tibial nerve, and testis were removed for determining organ-level Tau. For determining organ-specific expression, genes with the highest expression within an organ (brain) relative to other organs were selected if the cell type-specific Tau score of the gene was 0.7 or above. The Uniprot accession IDs of all human proteins were filtered to find those annotated as containing transmembrane domains. A list of candidate EV cell type-specific markers for neurons was determined by identifying genes with a Tau score of 0.7 in both neuron-specific expression in brain RNA-Seq and brain-specific expression in GTEx, and then filtering these genes to select only those that produce transmembrane proteins. The presence of these candidates was then evaluated in proteomics datasets. The Python script for the computational marker pipeline is available on Github.

[0254] Cell Culture and Isolation of EVs from Cell Culture Medium

[0255] K562 cells (from ATCC) were grown in Glutamax (Thermo Fisher Scientific) Gibco IMDM supplemented with Gibco heat-inactivated fetal bovine serum (Thermo Fisher Scientific) and Gibco penicillin-streptomycin (Thermo Fisher Scientific). For EV isolation, cells were switched to EV-depleted medium (obtained by ultracentrifuging the medium at 120,000 x g for 16 h and then filtering through a Corning 0.22 μm filter). Previously described iNGN cells were grown in mTeSR1 medium (STEMCELL Technologies) on Matrigel (Corning)-coated plates. Doxycycline (Sigma Aldrich) was diluted in PBS and added to mTeSR1 at a final concentration of 0.5 μg / mL to initiate differentiation. On day 4 after Dox addition, the medium was switched to Glutamax (Thermo Fisher Scientific) Gibco IMDM supplemented with B27 serum-free supplement (Thermo Fisher Scientific) and Gibco penicillin-streptomycin (Thermo Fisher Scientific). On day 6 or 7 after Dox addition, EVs were collected from neurons. EVs were isolated from cell cultures by differential ultracentrifugation as described in detail (REF). Cell culture medium (240 mL per isolation) was centrifuged at 300 x g for 10 min, and the supernatant was centrifuged again at 2000 x g for 10 min. The supernatant was centrifuged at 16,500 x g for 20 min at 4°C and filtered through a 0.22 μm Steriflip filter (Millipore Sigma). The sample was then ultracentrifuged at 120,000 x g for 70 min at 4°C, washed with PBS, and ultracentrifuged again. The pellet was then resuspended in PBS.

[0256] EVs were isolated from plasma or CSF by MMR slurry

[0257] Human plasma (collected in K2-EDTA tubes) or CSF was ordered from BioIVT. All biological fluids were spun down at 2000 x g for 10 minutes and the pellet was discarded. The mixed-mode chromatography resin slurry was prepared as follows: Take Capto Core 700 resin (Cytiva) and centrifuge the resin at 800 x g for 5 minutes, wash it 3 times with PBS in a 50 mL falcon tube, and resuspend it in an equal volume of PBS as the resin volume to produce a 50% slurry. For CSF EV isolation, the samples were centrifuged at 2000 x g for 10 minutes to remove any potential residual cells. Then the protein concentration of the CSF was determined using a Qubit protein assay kit (ThermoFisher Scientific) and the MMR slurry volume corresponding to the protein content of the added sample. The ratio of the MMR slurry was changed as detailed. For mass spectrometry experiments, the MMR slurry ratio used was 1 uL slurry / 0.3 ug protein.

[0258] The samples were mixed end-to-end at room temperature for 45 minutes and then centrifuged at 800 x g for 10 min. Finally, the supernatant was transferred and centrifuged at 2000 x g for 10 minutes in a Corning CoStar X 0.45 μm filter to separate the CSF from the Capto Core beads.

[0259] Total protein staining

[0260] The protein samples were denatured in LDS (ThermoFisher Scientific) at 70 °C for 10 minutes and then loaded onto a polyacrylamide protein gel for total protein staining or western blotting. A Bolt Bis-Tris Plus 4 to 12% gel was used and the samples were run at 150 V for 60 minutes. Coomassie blue total protein staining was performed on the gel using Acqua dye (Bulldog Bio). The gel was incubated in the dye overnight, washed in deionized water, and then imaged using a Gel DocEZ imager (BioRad).

[0261] Western blotting

[0262] The western blotting of EVs was previously described in detail (46). The iBlot2 Dry Blotting System (ThermoFisher Scientific) was used for transfer at 20 V for three to seven minutes, depending on the size of the protein marker. The following primary antibodies were used for western blotting at the corresponding dilutions: M38 (Thermo Fisher Scientific) against CD81 at 1:666, H5C6 (BD) against CD63 at 1:1000, CD9 (Millipore) at 1:1000, EPR18998 (Abcam) against L1CAM at 1:500, ab47441 against GJA1 at 1:500, F-10 (Santa Cruz) against albumin at 1:1000, 41 (BD) against SYT1 at 1:500. The blots were incubated overnight at 4 °C on a shaker in milk (5% w / v) dissolved in PBS-T solution (PBS containing 0.1% Tween) containing the primary antibody. The next day, the blots were washed three times with PBS-T, incubated for 2 hours with a secondary antibody of TrueBlot HRP (Rockland) or cross-adsorbed HRP (Bethyl) at a concentration of 1:2000 in milk buffer, and then washed three more times. The blots were developed with Protein Bright ECL-Spray (Advansta) and imaged on a Sapphire Biomolecular Imager (Azure Biosystems).

[0263] Immuno-isolation of EVs from cell culture EVs

[0264] The separation buffer was prepared as follows: BSA was added to PBS at pH 7.4 to a final concentration of 1 mg / mL and filtered through a 0.22 μm Steriflip filter (Millipore). 500 μL (2x10^8) Dynabeads goat anti-mouse IgG beads (Thermo Fisher Scientific) were placed in 2 mL and placed on a magnetic rack. The supernatant was removed and replaced with 250 μL of separation buffer, and the magnet was disengaged. 10 μg of the primary antibody was end-over-end rotated at 4 °C and coupled to the beads overnight. The following antibodies were used for immunoprecipitation: 5G3 (BD) against L1CAM, 1C51 (Abcam) against mCherry, 9F9.F9 (Abcam) against GFP, 1.3.3.22 (Thermo Fisher Scientific) against CD81, and H5C6 (BD) against CD63. The next day, the beads were washed twice, each time with 1 mL of separation buffer. Then EVs (usually one pellet in 150 μL) were added and separation buffer was added to make the volume up to 0.5 mL. Immunoprecipitation was carried out on a rotator, for 24 hours at 4 °C for L1CAM, or for 1 hour at 37 °C for CD81 or CD63.

[0265] Immunoprecipitation of EVs from human biological fluids

[0266] Similarly to the immunoisolation of EVs from cell culture EVs, EVs were immunoisolated from CSF or plasma with some minor modifications. PBS pH 7.4 was used as the isolation buffer without the addition of BSA. 250 μL (1x10^8) Dynabeads goat anti-mouse IgG beads (Thermo Fisher Scientific) or 50 μL (1.5 mg) Dynabeads protein A (Thermo Fisher Scientific) were placed in a 2 mL tube and placed on a magnetic rack. The supernatant was removed, and the beads were washed with 1 mL of isolation buffer and then brought to a final incubation volume of 0.5 mL with PBS pH 7.4 and coupled overnight by end-to-end rotation at 4°C with 10 μg of primary antibody. The following antibodies were used for immunoisolation: mouse monoclonal CD81 (clone 1.3.3.22, Thermo Fisher Scientific), CD63 (clone H5C6, BD Biosciences), CD9 (clone CBL162, Millipore), GFP (clone 1GFP63, Biolegend or clone 9F9.F9, Rockland), and mCherry (clone EPR20579, Abcam). The next day, the beads were washed twice, each time with 1 mL of isolation buffer. CSF or plasma was centrifuged at 2000 x g for 10 minutes, and the supernatant was passed through a CoStar Spin-X 0.45 μm filter (Corning) at 2000 x g for 10 minutes. Then, CSF and plasma were spin-filtered in an Amicon Ultra 2 mL 10K centrifugal filter unit (Millipore) for 2 hours to reduce the volume so that 1 mL of CSF or plasma could be incubated with each bead isolate to a final volume of 0.5 mL. For CD9, CD63, and CD81, immunoisolation was performed on a rotator at 4°C for 1 hour.

[0267] Mass spectrometry

[0268] Mass spectrometry analysis of neuron EVs isolated from cell culture was performed on the Broad Institute Proteomics Platform. EVs were lysed in RIPA buffer (Thermo Fisher Scientific). Samples were then run on an SDS gel, and the band corresponding to albumin in size was excised and discarded. The remaining samples were prepared for TMT labeling and run on a mass spectrometer. Mass spectrometry analysis of EVs isolated from CSF or plasma was performed using MMR slurry at the Harvard Proteomics Center.

[0269] Example 2: Detection of NRXN3 in human CSF and neuron-specific extracellular vesicles This example provides experimental validation for the neuron-specific EV markers identified in Example 1 above. Specifically, one of the markers in Table 1, NRXN3, was detected in EVs from the conditioned medium of human iPS-derived neurons and in human CSF samples.

[0270] As described above, EVs were isolated from the conditioned medium of human iPS-derived neurons. Briefly, iNGN cells (Busskamp et al., 2014) were grown in mTeSR1 medium on Matrigel-coated plates. Doxycycline (Dox) was diluted in PBS and added to mTeSR1 at a final concentration of 0.5 μg / mL to initiate differentiation. On day 4 after the addition of Dox, the medium was switched to Glutamax DMEM supplemented with B27 serum-free supplement and penicillin-streptomycin. On day 6 or 7 after the addition of Dox, EVs were collected from the neurons. EVs were isolated from the cell culture by differential ultracentrifugation. The cell culture medium (240 mL per isolation) was centrifuged at 300 x g for 10 minutes, and the supernatant was centrifuged again at 2000 x g for 10 minutes. The supernatant was centrifuged at 16500 x g for 20 minutes at 4°C and filtered through a 0.22 μm Steriflip filter. Then the sample was ultracentrifuged at 120000 x g for 70 minutes at 4°C, washed with PBS, and ultracentrifuged again. Then the pellet was resuspended in PBS.

[0271] Human CSF samples (from Brigham and Women’s Hospital) were centrifuged at 2000 x g for 10 minutes. Next, the supernatant was centrifuged at 2000 x g for 10 minutes through a 0.45 μm Corning Costar SPIN-X centrifuge tube filter (Sigma-Aldrich) to remove any remaining cells or cell debris. 1 mL of CSF was loaded onto a size exclusion chromatography (SEC) column.

[0272] Briefly, Sepharose CL-6B resin was washed with PBS in a glass bottle. Before use, the resin volume was washed three times with an equal volume of PBS. The resin was used to pack an Econo-Pac chromatography column, and a frit was inserted into the column above the resin. Before loading the sample, each column was washed with 10 mL of PBS (twice, 5 mL each time). For the SEC column, resin was added until the bed volume (resin without liquid) reached 10 mL. Once the PBS from the wash had completely passed through the column, the sample (1 mL of CSF or neuron EVs) was loaded. Once the sample had completely entered the column, 0.5 mL fractions were collected.

[0273] NRXN3 Detection by Simoa Assay( Figure 16A )。The candidate capture antibody (Invitrogen PA5-71367) was conjugated to carboxylated paramagnetic beads from the Simoa Homebrew Assay Development Kit (Quanterix) using EDC chemistry (Thermo Fisher Scientific). The candidate detection antibody (CST 480045) was conjugated to biotin using EZ-Link NHS-PEG4 Biotin (Thermo Fisher Scientific). All samples were measured in duplicate using the HD-X Analyzer (Quanterix). NRXN3 was measured using a two-step assay. The average enzyme per bead (AEB) value was calculated by the HD-X software.

[0274] As Figure 16B and Figure 16C shown, the NRXN3 marker was detected in the early SEC fractions of EVs isolated from the conditioned medium of human iPS-derived neurons and in the early SEC fractions of human CSF samples.

[0275] These data validate and confirm that the markers of the present invention are brain-specific and / or neuron-specific EV markers, and that these markers can be used to isolate brain-specific and / or neuron-specific EVs from human samples.

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[0330] Equivalents

[0331] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments and methods described herein. Such equivalents are intended to be encompassed by the scope of the appended claims.

Claims

1. A method for isolating cell type-specific and / or organ-specific extracellular vesicles from a subject, comprising (a) obtaining a biological sample from the subject; and (b) isolating the cell type-specific and / or organ-specific extracellular vesicles based on the presence of biomarkers on the surface of the extracellular vesicles, wherein the biomarkers comprise one or more biomarkers selected from Tables 1-5.

2. The method according to claim 1, wherein the biological sample comprises a liquid biological sample.

3. The method according to claim 1 or claim 2, wherein the liquid biological sample is selected from the group consisting of: whole blood, serum, plasma, cerebrospinal fluid, spinal fluid, amniotic fluid, aqueous humor, vitreous humor, bile, milk, cerumen (earwax), chyle, chyme, endolymph, perilymph, exudate, feces, female ejaculate, gastric acid, gastric juice, lymph, mucus (including nasal drainage and sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus, inflammatory secretions, saliva, sebum (skin oil), semen, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vomit, and mixtures of one or more thereof.

4. The method according to any one of claims 1-3, wherein the extracellular vesicles are brain-specific.

5. The method according to any one of claims 1-4, wherein the extracellular vesicles are neuron-specific, astrocyte-specific, oligodendrocyte-specific, and / or microglia-specific.

6. The method according to any one of claims 1-5, wherein the extracellular vesicles are neuron-specific, and wherein the one or more biomarkers are selected from Tables 1 and 5.

7. The method according to claim 6, wherein the one or more biomarkers are selected from the group consisting of: GABRG2, SVOP, SLC32A1, GRM7, GABRB3, CHRNB2, SLC12A5, GRM8, PTPRT, SLC6A17, PCDHAC2, PLPPR4, C11orf87, SORCS3, CALY, PTPRR, KIAA1549L, HCN1, CDH18, TMEM132D, GPR158, FRRS1L, ATP2B3, GRIA4, ST8SIA3, HS6ST3, SEZ6, and NRXN3.

8. The method according to any one of claims 1-5, wherein the extracellular vesicles are astrocyte-specific, and wherein the one or more biomarkers are selected from Table 2.

9. The method according to any one of claims 1-5, wherein the extracellular vesicles are oligodendrocyte-specific, and wherein the one or more biomarkers are selected from Table 3.

10. The method according to any one of claims 1-5, wherein the extracellular vesicles are microglia-specific, and wherein the one or more biomarkers are selected from Table 4.

11. The method according to any one of claims 1-10, wherein the cell type-specific and / or organ-specific EVs are isolated by: immunoisolation, mixed-mode chromatography, size exclusion chromatography, cation exchange chromatography, anion exchange chromatography, gel permeation chromatography, differential centrifugation, sucrose density gradient, organelle electrophoresis, magnetic-activated cell sorting (MACS), or nanofilter ultrafiltration concentrator.

12. The method according to claim 11, wherein the immunoisolation comprises microfluidics affinity-based separation, magnetic-based separation, pull-down separation, or fluorescence-activated sorting-based separation.

13. A method for isolating brain-specific extracellular vesicles from a subject, comprising (a) obtaining a biological sample from the subject; (b) isolating extracellular vesicles from the sample based on the presence of biomarkers on the surface of the extracellular vesicles, wherein the biomarkers comprise one or more biomarkers selected from Tables 1-5.

14. The method according to claim 13, wherein the biological sample comprises a liquid biological sample.

15. The method according to claim 13 or claim 14, wherein the liquid biological sample is selected from the group consisting of: whole blood, serum, plasma, cerebrospinal fluid, spinal fluid, amniotic fluid, aqueous humor, vitreous humor, bile, milk, cerumen (earwax), chyle, chyme, endolymph, perilymph, exudate, feces, female ejaculate, gastric acid, gastric juice, lymph, mucus (including nasal drainage and sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus, inflammatory secretions, saliva, sebum (skin oil), semen, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vomit, and mixtures of one or more thereof.

16. The method according to any one of claims 13-15, wherein the extracellular vesicles are neuron-specific, astrocyte-specific, oligodendrocyte-specific, and / or microglia-specific.

17. The method according to any one of claims 13-16, wherein the extracellular vesicles are neuron-specific, and wherein the one or more biomarkers are selected from Tables 1 and 5.

18. The method according to claim 17, wherein the one or more biomarkers are selected from the group consisting of: GABRG2, SVOP, SLC32A1, GRM7, GABRB3, CHRNB2, SLC12A5, GRM8, PTPRT, SLC6A17, PCDHAC2, PLPPR4, C11orf87, SORCS3, CALY, PTPRR, KIAA1549L, HCN1, CDH18, TMEM132D, GPR158, FRRS1L, ATP2B3, GRIA4, ST8SIA3, HS6ST3, SEZ6, and NRXN3.

19. The method according to any one of claims 13-16, wherein the extracellular vesicles are astrocyte-specific, and wherein the one or more biomarkers are selected from Table 2.

20. The method according to any one of claims 13 - 16, wherein the extracellular vesicles are oligodendrocyte - specific, and wherein the one or more biomarkers are selected from Table 3.

21. The method according to any one of claims 13 - 16, wherein the extracellular vesicles are microglia - specific, and wherein the one or more biomarkers are selected from Table 4.

22. The method according to any one of claims 13 - 21, wherein the brain - specific EVs are isolated by: immunoisolation, mixed - mode chromatography, size - exclusion chromatography, cation - exchange chromatography, anion - exchange chromatography, gel - permeation chromatography, differential centrifugation, sucrose density gradient, organelle electrophoresis, magnetic - activated cell sorting (MACS), or nanofilter ultrafiltration concentrator.

23. The method according to claim 22, wherein the immunoisolation comprises separation based on microfluidic affinity, magnetic - based separation, pull - down separation, or separation based on fluorescence - activated sorting.

24. A method for identifying extracellular vesicles derived from brain cells, comprising (a) obtaining a biological sample comprising the extracellular vesicles; (b) determining the presence or absence of biomarkers on the surface of the extracellular vesicles, wherein the biomarkers comprise one or more biomarkers selected from Tables 1 - 5; and wherein the presence of the biomarkers indicates that the extracellular vesicles are derived from brain cells.

25. The claim according to claim 24, wherein the brain cells are selected from the group consisting of neurons, astrocytes, oligodendrocytes, and microglia.

26. A method for identifying extracellular vesicles derived from neurons, comprising (a) obtaining a biological sample comprising the extracellular vesicles; (b) determining the presence or absence of biomarkers on the surface of the extracellular vesicles, wherein the biomarkers comprise one or more biomarkers selected from Tables 1 and 5; and wherein the presence of the biomarkers indicates that the extracellular vesicles are derived from neurons.

27. The method according to claim 26, wherein the one or more biomarkers are selected from the group consisting of GABRG2, SVOP, SLC32A1, GRM7, GABRB3, CHRNB2, SLC12A5, GRM8, PTPRT, SLC6A17, PCDHAC2, PLPPR4, C11orf87, SORCS3, CALY, PTPRR, KIAA1549L, HCN1, CDH18, TMEM132D, GPR158, FRRS1L, ATP2B3, GRIA4, ST8SIA3, HS6ST3, SEZ6, and NRXN3.

28. A method for identifying extracellular vesicles derived from astrocytes, comprising (a) obtaining a biological sample comprising the extracellular vesicles; (b) Determine the presence or absence of a biomarker on the surface of the extracellular vesicle, wherein the biomarker comprises one or more biomarkers selected from Table 2; and wherein the presence of the biomarker indicates that the extracellular vesicle is derived from astrocytes.

29. A method for identifying extracellular vesicles derived from oligodendrocytes, comprising (a) Obtaining a biological sample comprising the extracellular vesicles; (b) Determine the presence or absence of a biomarker on the surface of the extracellular vesicle, wherein the biomarker comprises one or more biomarkers selected from Table 3; and wherein the presence of the biomarker indicates that the extracellular vesicle is derived from oligodendrocytes.

30. A method for identifying extracellular vesicles derived from microglia, comprising (a) Obtaining a biological sample comprising the extracellular vesicles; (b) Determine the presence or absence of a biomarker on the surface of the extracellular vesicle, wherein the biomarker comprises one or more biomarkers selected from Table 4; and wherein the presence of the biomarker indicates that the extracellular vesicle is derived from microglia.

31. The method according to any one of claims 24 - 30, wherein the biological sample comprises a liquid biological sample.

32. The method according to any one of claims 24 - 31, wherein the liquid biological sample is selected from the group consisting of: whole blood, serum, plasma, cerebrospinal fluid, spinal fluid, amniotic fluid, aqueous humor, vitreous humor, bile, milk, cerumen (earwax), chyle, chyme, endolymph, perilymph, exudate, feces, female ejaculate, gastric acid, gastric juice, lymph, mucus (including nasal drainage and sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus, inflammatory secretions, saliva, sebum (skin oil), semen, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vomit, and mixtures of one or more thereof.

33. The method according to any one of claims 24 - 32, wherein the biological sample is obtained from a subject.

34. The method according to any one of claims 24 - 33, wherein the presence or absence of the biomarker is determined by: RNA sequencing (RNA seq), DNA sequencing, array analysis, reverse transcription polymerase chain reaction (RT-PCR), quantitative reverse transcription polymerase chain reaction (qRT-PCR), proteomic analysis, mass spectrometry, immunoassay, ELISA, fluorescence-activated cell sorting (FACS), SDS-polyacrylamide gel electrophoresis (SDS-PAGE), or Western blot analysis.

35. A method for diagnosing, prognosticating a neurodegenerative disorder or identifying a subject at risk of developing a neurodegenerative disorder in a subject, which comprises: (a) Obtaining a biological sample from the subject; (b) Isolating brain-specific extracellular vesicles from the biological sample based on the presence of a biomarker in the isolated extracellular vesicles, wherein the biomarker comprises one or more biomarkers selected from Tables 1 - 5; (c) Extract proteins and / or nucleic acids from the isolated brain-specific extracellular vesicles; and (d) Analyze the extracted proteins and / or nucleic acids from the isolated brain-specific extracellular vesicles so as to diagnose, prognose a neurodegenerative disorder or identify a subject at risk of developing a neurodegenerative disorder.

36. The method according to claim 35, wherein the biological sample comprises a liquid biological sample.

37. The method according to claim 35 or claim 36, wherein the liquid biological sample is selected from the group consisting of whole blood, serum, plasma, cerebrospinal fluid, spinal fluid, amniotic fluid, aqueous humor, vitreous humor, bile, milk, cerumen (earwax), chyle, chyme, endolymph, perilymph, exudate, feces, female ejaculate, gastric acid, gastric juice, lymph, mucus (including nasal drainage and sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus, inflammatory secretions, saliva, sebum (skin oil), semen, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vomit, and mixtures of one or more thereof.

38. The method according to any one of claims 35 - 37, wherein the extracellular vesicles are neuron-specific, astrocyte-specific, oligodendrocyte-specific, and / or microglia-specific.

39. The method according to any one of claims 35 - 38, wherein the extracellular vesicles are neuron-specific, and wherein the one or more biomarkers are selected from Table 1 and Table 5.

40. The method according to claim 39, wherein the one or more biomarkers are selected from the group consisting of GABRG2, SVOP, SLC32A1, GRM7, GABRB3, CHRNB2, SLC12A5, GRM8, PTPRT, SLC6A17, PCDHAC2, PLPPR4, C11orf87, SORCS3, CALY, PTPRR, KIAA1549L, HCN1, CDH18, TMEM132D, GPR158, FRRS1L, ATP2B3, GRIA4, ST8SIA3, HS6ST3, SEZ6, and NRXN3.

41. The method according to any one of claims 35 - 38, wherein the extracellular vesicles are astrocyte-specific, and wherein the one or more biomarkers are selected from Table 2.

42. The method according to any one of claims 35 - 38, wherein the extracellular vesicles are oligodendrocyte-specific, and wherein the one or more biomarkers are selected from Table 3.

43. The method according to any one of claims 35 - 38, wherein the extracellular vesicles are microglia-specific, and wherein the one or more biomarkers are selected from Table 4.

44. The method according to any one of claims 35 - 43, wherein the extracted nucleic acids comprise messenger RNA (mRNA), microRNA (miRNA), long non-coding RNA (lncRNA), small non-coding RNA, DNA, and any other full-length RNA or DNA or fragments thereof.

45. The method according to any one of claims 35 - 44, wherein analyzing the extracted nucleic acids from the isolated brain-specific extracellular vesicles comprises RNA sequencing (RNA seq), DNA sequencing, array analysis, reverse transcription polymerase chain reaction (RT-PCR), or quantitative reverse transcription polymerase chain reaction (qRT-PCR).

46. The method according to any one of claims 35 - 45, wherein analyzing the extracted nucleic acids from the isolated brain-specific extracellular vesicles comprises whole genome analysis or transcriptome analysis.

47. The method according to any one of claims 35 - 46, wherein analyzing the extracted nucleic acids from the isolated brain-specific extracellular vesicles comprises analyzing genes of interest, wherein the genes of interest are associated with the neurodegenerative disorder.

48. The method according to claim 47, which comprises testing for the presence or absence of the genes of interest, analyzing one or more allelic variants or mutations of the genes of interest, and testing for the presence or absence of the allelic variants or mutations.

49. The method according to any one of claims 35 - 48, wherein analyzing the extracted proteins from the isolated brain-specific extracellular vesicles comprises proteomic analysis, mass spectrometry, immunoassay, ELISA, fluorescence-activated cell sorting (FACS), SDS-polyacrylamide gel electrophoresis (SDS-PAGE), or western blot analysis.

50. The method according to any one of claims 35 - 49, wherein analyzing the extracted proteins from the isolated brain-specific extracellular vesicles comprises analyzing proteins of interest, wherein the proteins of interest are associated with the neurodegenerative disorder.

51. The method according to claim 50, which comprises testing for the presence or absence of the proteins of interest, analyzing one or more mutations in the proteins of interest, and testing for the presence or absence of the mutations.

52. The method according to any one of claims 35 - 51, wherein the neurodegenerative disorder is selected from the group consisting of: Alzheimer's disease (AD), Huntington's disease, multi-infarct dementia, frontotemporal dementia (FTD), corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), dementia with Lewy bodies, tangle-predominant senile dementia, Pick's disease (PiD), argyrophilic grain disease, amyotrophic lateral sclerosis (ALS), other motor neuron diseases, Guam Parkinson-dementia complex, FTDP-17, Lytico-Bodig disease, multiple sclerosis, traumatic brain injury (TBI), and Parkinson's disease.

53. A kit for isolating brain-specific extracellular vesicles from a subject, comprising (a) one or more reagents for detecting the presence of a biomarker on the surface of the extracellular vesicles, wherein the biomarker comprises one or more biomarkers selected from Tables 1-5; (b) a device for isolating the brain-specific extracellular vesicles based on the presence of the biomarker; and (c) a set of instructions for detecting the presence of the biomarker and / or isolating the brain-specific extracellular vesicles.

54. The kit according to claim 53, wherein the one or more reagents for detecting the presence of the biomarker on the extracellular vesicles are antibodies or aptamers that bind to the biomarker.

55. The kit according to claim 53 or claim 54, further comprising a device for isolating a biological sample from the subject.

56. A kit for detecting a neurodegenerative disorder in a subject, comprising (a) one or more reagents for detecting the presence of a biomarker on the surface of extracellular vesicles, wherein the biomarker comprises one or more biomarkers selected from Tables 1-5; (b) a device for isolating brain-specific extracellular vesicles based on the presence of the biomarker; (c) one or more reagents for detecting the level of a gene associated with the neurodegenerative disorder in the isolated brain-specific extracellular vesicles; and (d) a set of instructions for detecting the presence of the biomarker, isolating the brain-specific extracellular vesicles, and / or detecting the level of the gene associated with the neurodegenerative disorder.

57. The kit according to claim 56, further comprising a device for isolating a biological sample from the subject.

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