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

Abstract

The present invention relates to novel biomarkers and combinations thereof for cell type-specific and / or organ-specific extracellular vesicles, particularly brain-specific and / or neuron-specific extracellular vesicles. The present invention also relates to methods for the isolation and / or enrichment of cell type-specific and / or organ-specific extracellular vesicles, methods for the identification of extracellular vesicles derived from cells, and methods for diagnosing or prognosticating a disorder, such as a neurodegenerative disorder, using cell type-specific and / or organ-specific extracellular vesicles. Compositions in the form of kits of reagents for detecting cell type-specific and / or organ-specific extracellular vesicles are also provided.

Description

Technical Field

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 342,353, filed May 16, 2022, the entire contents of which are hereby incorporated by reference.

[0002] Government Support This invention was made with government support under Grant No. HG008525, awarded by the National Institutes of Health (NIH) of the United States. The government has certain rights in this invention.

Background Art

[0003] Background of the Invention Efforts to understand how the human brain functions are hampered, in part, by the inability to perform brain biopsies on living individuals. Current understanding of brain diseases relies primarily on postmortem tissue analysis after neurodegeneration and cell death have already occurred. Thus, fundamental questions regarding the biochemical processes underlying neurological diseases remain. Access to the proteomic and transcriptomic profiles of neurons and other brain cells in living human individuals would aid current understanding of neuroscience.

[0004] One approach to learning about the living brain is to analyze extracellular vesicles (EVs). EVs are released by many cell types and are found in all biological fluids. Because EVs contain RNA and proteins derived 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. EVs and their contents are present in small amounts in clinically relevant biological samples of limited volume. Furthermore, and partly due to the lack of suitable quantification methods, there is a lack of consensus on the best method for purifying EVs from plasma and other body fluids. Additionally, while the entire population of EVs can be isolated from plasma or other biological fluids, profiling of the RNA and protein cargo of these EVs does not distinguish which cargo molecules are derived from which cell types. Isolating EVs from specific cell types enables the analysis of the RNA and proteins within these EVs as a non-invasive “snapshot” of that cell type.

[0005] The ability to isolate EVs from neurons or other cell types in the brain would be particularly useful. Since biopsies cannot be performed on the brain, the isolation of neuron-derived EVs could enable the reading of brain states and 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). Over the past few years, many studies have reported the use of the transmembrane protein L1CAM, a cell adhesion molecule involved in neurogenesis, as a means for EV capture. However, L1CAM may not be a suitable marker for neuron-derived EVs considering its broad expression on non-neuronal cells outside the brain (Norman M. et al., Nature Methods. 2021, 18: 631-634). Therefore, there is a need in the art for the identification of new biomarkers for cell type-specific EVs, particularly brain-specific and / or neuron-specific EVs, that can be used to enable better diagnosis or prognosis of diseases such as neurodegenerative diseases and to improve the prediction of treatment outcomes.

Prior Art Documents

Non-Patent Documents

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Summary of the Invention

Means for Solving the Problems

[0007] Summary of the Invention The present invention is based, at least in part, on the discovery of novel biomarkers for the isolation of cell type-specific and / or organ-specific EV markers, such as cerebrospinal fluid (CSF) or plasma, from human biological samples, such as brain-specific and / or neuron-specific EVs. 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, and thus provide a way forward for the isolation of brain-specific and / or neuron-specific EVs from human samples. Furthermore, these novel biomarkers are useful for the identification of EVs derived from specific organs and / or cell types, such as brain cells, such as neurons, astrocytes, oligodendrocytes, or microglial cells, from a sample, such as a biological sample, by determining the presence or absence of the marker in any one of Tables 1-5 on the surface of the EVs.

[0008] 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 comprising one or more biomarkers selected from Tables 1-5 on the surface of the extracellular vesicles.

[0009] In some embodiments, the biological sample comprises a liquid biological sample.

[0010] In some embodiments, the liquid biological sample is selected from the group consisting of whole blood, serum, plasma, cerebrospinal fluid, marrow fluid, amniotic fluid, aqueous humor, vitreous humor, bile, breast milk, earwax (cerumen), chyle, chyme, endolymph, perilymph, exudate, feces, vaginal fluid, gastric acid, gastric juice, lymph, mucus (including nasal discharge and sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus, catarrhal secretion, saliva, sebum (skin oil), semen, saliva, synovial fluid, sweat, tears, urine, vaginal secretion, vomit, and one or more mixtures thereof.

[0011] 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.

[0012] In some embodiments, the extracellular vesicles are neuron-specific and one or more biomarkers are selected from Table 1 and Table 5.

[0013] In some embodiments, 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.

[0014] In some embodiments, the extracellular vesicles are astrocyte-specific and one or more biomarkers are selected from Table 2.

[0015] In some embodiments, the extracellular vesicles are oligodendrocyte-specific and one or more biomarkers are selected from Table 3.

[0016] In some embodiments, the extracellular vesicles are microglia-specific and one or more biomarkers are selected from Table 4.

[0017] In some embodiments, cell type-specific and / or organ-specific EVs are isolated by immuno-isolation, 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 a nanomembrane ultrafiltration concentrator.

[0018] In some embodiments, immuno-isolation includes isolation based on microfluidic affinity, magnetic-based isolation, pull-down isolation, or fluorescence-activated sorting.

[0019] In one aspect, the present invention provides a method for isolating brain-specific extracellular vesicles from a subject, the method comprising: (a) obtaining a biological sample from the subject; (b) isolating extracellular vesicles from the sample based on the presence of one or more biomarkers selected from Tables 1-5, which are included on the surface of the extracellular vesicles.

[0020] In some embodiments, the biological sample includes a liquid biological sample.

[0021] In some embodiments, the liquid biological sample is selected from the group consisting of whole blood, serum, plasma, cerebrospinal fluid, marrow fluid, amniotic fluid, aqueous humor, vitreous humor, bile, breast milk, earwax (cerumen), chyle, chyme, endolymph, perilymph, exudate, feces, vaginal fluid, gastric acid, gastric juice, lymph fluid, mucus (including nasal mucus and sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus, catarrhal secretions, saliva, sebum (skin oil), semen, saliva, synovial fluid, sweat, tears, urine, vaginal secretions, vomit, and one or more mixtures thereof.

[0022] In some embodiments, the extracellular vesicles are neuron-specific, astrocyte-specific, oligodendrocyte-specific, and / or microglia-specific.

[0023] In some embodiments, the extracellular vesicles are neuron-specific and the one or more biomarkers are selected from Table 1 and Table 5.

[0024] 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.

[0025] In some embodiments, the extracellular vesicles are astrocyte-specific and the one or more biomarkers are selected from Table 2.

[0026] In some embodiments, the extracellular vesicles are oligodendrocyte-specific and the one or more biomarkers are selected from Table 3.

[0027] In some embodiments, the extracellular vesicles are microglia-specific and the one or more biomarkers are selected from Table 4.

[0028] In some embodiments, brain-specific EVs are isolated by immuno-isolation, 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 a nanomembrane ultrafiltration concentrator.

[0029] In some embodiments, immuno-isolation includes isolation based on microfluidic affinity, isolation based on magnetism, pull-down isolation, or isolation based on fluorescence-activated sorting.

[0030] In one aspect, the present invention provides a method for identifying extracellular vesicles derived from brain cells, comprising: (a) obtaining a biological sample containing 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 Tables 1-5, and the presence of the biomarkers indicates that the extracellular vesicles are derived from brain cells.

[0031] In some embodiments, the brain cells are selected from the group consisting of neurons, astrocytes, oligodendrocytes, and microglial cells.

[0032] In one aspect, the present invention provides a method for identifying extracellular vesicles derived from neurons, comprising: (a) obtaining a biological sample containing 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 Tables 1 and 5, and the presence of the biomarkers indicates that the extracellular vesicles are derived from neurons.

[0033] 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.

[0034] In one aspect, the present invention provides a method for identifying extracellular vesicles derived from astrocytes, comprising: (a) obtaining a biological sample containing 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 Table 2, and the presence of the biomarkers indicates that the extracellular vesicles are derived from astrocytes.

[0035] In one aspect, the present invention provides a method for identifying extracellular vesicles derived from oligodendrocytes, comprising: (a) obtaining a biological sample containing 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 Table 3, and the presence of the biomarkers indicates that the extracellular vesicles are derived from oligodendrocytes.

[0036] In one aspect, the present invention provides a method for identifying extracellular vesicles derived from microglial cells, comprising: (a) obtaining a biological sample containing 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 Table 4, and the presence of the biomarkers indicates that the extracellular vesicles are derived from microglial cells.

[0037] In some embodiments, the biological sample comprises a liquid biological sample.

[0038] In some embodiments, the liquid biological sample is selected from the group consisting of whole blood, serum, plasma, cerebrospinal fluid, myeloid fluid, amniotic fluid, aqueous humor, vitreous humor, bile, breast milk, earwax (cerumen), chyle, chyme, endolymph, perilymph, exudate, feces, vaginal fluid, gastric acid, gastric juice, lymph fluid, mucus (including nasal discharge and sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus, catarrhal secretion, saliva, sebum (skin oil), semen, saliva, synovial fluid, sweat, tears, urine, vaginal secretion, vomit, and one or more mixtures thereof.

[0039] In some embodiments, the biological sample is obtained from a subject.

[0040] In some embodiments, the presence or absence of a 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 profiling, mass spectrometry, immunoassay, ELISA, fluorescence-activated cell sorting (FACS), SDS-polyacrylamide gel electrophoresis (SDS-PAGE), or Western blot analysis.

[0041] In one aspect, the present invention provides a method for diagnosing, prognosticating, or identifying a subject at risk of developing a neurodegenerative disorder in a subject, the method comprising: (a) obtaining a biological sample from the subject; (b) isolating brain-specific extracellular vesicles from the biological sample based on the presence of one or more biomarkers selected from Tables 1-5, the biomarkers being contained in isolated extracellular vesicles; (c) extracting proteins and / or nucleic acids from the isolated brain-specific extracellular vesicles; and (d) analyzing the proteins and / or nucleic acids extracted from the isolated brain-specific extracellular vesicles, thereby diagnosing, prognosticating, or identifying a subject at risk of developing a neurodegenerative disorder.

[0042] In some embodiments, the biological sample includes a liquid biological sample.

[0043] In some embodiments, the liquid biological sample is selected from the group consisting of whole blood, serum, plasma, cerebrospinal fluid, myeloid fluid, amniotic fluid, aqueous humor, vitreous humor, bile, breast milk, earwax (cerumen), chyle, chyme, endolymph, perilymph, exudate, feces, vaginal fluid, gastric acid, gastric juice, lymph fluid, mucus (including nasal discharge and sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus, catarrhal secretion, saliva, sebum (skin oil), semen, saliva, synovial fluid, sweat, tears, urine, vaginal secretion, vomit, and one or more mixtures thereof.

[0044] In some embodiments, the extracellular vesicles are neuron-specific, astrocyte-specific, oligodendrocyte-specific, and / or microglia-specific.

[0045] In some embodiments, the extracellular vesicles are neuron-specific and one or more biomarkers are selected from Tables 1 and 5.

[0046] In some embodiments, 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.

[0047] In some embodiments, the extracellular vesicles are astrocyte-specific and one or more biomarkers are selected from Table 2.

[0048] In some embodiments, the extracellular vesicles are oligodendrocyte-specific and one or more biomarkers are selected from Table 3.

[0049] In some embodiments, the extracellular vesicles are microglia-specific and one or more biomarkers are selected from Table 4.

[0050] 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 or fragment of RNA or DNA.

[0051] In some embodiments, analyzing nucleic acids extracted 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).

[0052] In some embodiments, analyzing nucleic acids extracted from isolated brain-specific extracellular vesicles includes genome-wide analysis or transcriptome profiling.

[0053] In some embodiments, analyzing nucleic acids extracted from isolated brain-specific extracellular vesicles includes analyzing a gene of interest, wherein the gene of interest is associated with a neurodegenerative disorder.

[0054] In some embodiments, the method includes testing for the presence or absence of a 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.

[0055] In some embodiments, analyzing proteins extracted from isolated brain-specific extracellular vesicles includes proteomic profiling, mass spectrometry, immunoassay, ELISA, fluorescence-activated cell sorting (FACS), SDS-polyacrylamide gel electrophoresis (SDS-PAGE), or Western blot analysis.

[0056] In some embodiments, analyzing proteins extracted from isolated brain-specific extracellular vesicles includes analyzing a protein of interest, wherein the protein of interest is associated with a neurodegenerative disorder.

[0057] In some embodiments, the method includes testing for the presence or absence of a protein of interest, analyzing one or more mutations of the protein of interest, and testing for the presence or absence of the mutation.

[0058] 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), Lewy body dementia, neurofibrillary change predominant senile dementia, Pick's disease (PiD), argentophilic granular disease, amyotrophic lateral sclerosis (ALS), other motor neuron diseases, Guam Parkinson dementia complex, FTDP-17, Lytocercus body disease, multiple sclerosis, traumatic brain injury (TBI), and Parkinson's disease.

[0059] In one aspect, the present invention provides a kit for isolating brain-specific extracellular vesicles from a subject, the kit comprising: (a) one or more reagents for detecting the presence of one or more biomarkers selected from Tables 1-5 on the surface of the extracellular vesicles; (b) means for isolating brain-specific extracellular vesicles based on the presence of the biomarkers; and (c) a set of instructions for detecting the presence of the biomarkers and / or for isolating brain-specific extracellular vesicles.

[0060] In some embodiments, the one or more reagents for detecting the presence of the biomarkers on the extracellular vesicles are antibodies or aptamers that bind to the biomarkers.

[0061] In some embodiments, the kit further comprises means for isolating a biological sample from the subject.

[0062] In one aspect, the present invention provides a kit for detecting neurodegenerative disorders in a subject, the kit comprising: (a) one or more reagents for detecting the presence of one or more biomarkers selected from Tables 1-5 on the surface of extracellular vesicles; (b) means 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 using the kit for detecting the presence of the biomarker, isolating brain-specific extracellular vesicles, and / or detecting the level of a gene associated with a neurodegenerative disorder.

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

[0064] Other features and advantages of the present invention will be apparent from the following detailed description and the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

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Mode for Carrying Out the Invention

[0081] Detailed Description of the Invention The present invention is based, at least in part, on the discovery of novel biomarkers for the isolation of cell type-specific and / or organ-specific EV markers, such as 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, and thus provide a way forward for the isolation of brain-specific and / or neuron-specific EVs from human samples. Furthermore, these novel biomarkers are useful for the identification of EVs derived from specific cell types, such as brain cells, such as neurons, astrocytes, oligodendrocytes, or microglial cells, from samples, such as biological samples, by determining the presence or absence of the markers in any one of Tables 1-5 on the surface of the EVs.

[0082] Various aspects of the present invention are described in more detail in the following subsections.

[0083] A. Definitions To make the present disclosure easier to understand, certain terms are first defined. It should also be noted that whenever a value or range of values of a parameter is listed, intermediate values and ranges within the listed values are also intended to be part of the present disclosure.

[0084] In the following description, for the sake of explanation, specific numbers, materials, and arrangements are set forth to provide a complete understanding of the present disclosure. However, it will be apparent to those skilled in the art that the present disclosure can 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. Further, references in this specification to phrases such as "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the present disclosure. The appearances of the phrase "in one embodiment" in various places in this specification are not necessarily all referring to the same embodiment.

[0085] The articles "a" and "an" are used herein to refer to one or more than one (i.e., at least one) of the grammatical objects of the article. For example, "an element" refers to one element or more than one element.

[0086] The term "comprising" or "comprises" is used herein with reference to the compositions, methods, and their respective components that are necessary for the present disclosure, but is also open to the inclusion of unspecified elements, whether necessary or not.

[0087] As used herein, the terms "one or more" or "at least one" are understood to be each of the values 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.

[0088] As used herein, the term "biomarker" in one embodiment is a biomolecule, or a panel of biomolecules, or any combination thereof, wherein the level in a test sample, e.g., cell type-specific or organ-specific EVs, e.g., brain and / or neuron-specific EVs, is altered as compared to its level in a control sample, e.g., EVs derived from a different cell type or a different organ. Examples of biomarkers include, for example, polypeptides, peptides, polypeptide fragments, proteins, antibodies, hormones, polynucleotides, DNA, RNA or RNA fragments, microRNA (miRNA), lipids, metabolites, or polysaccharides. In one embodiment, the biomarker is detected in cell type-specific EVs isolated from a biological sample. In one embodiment, the biomarker is an organ-specific EV marker, e.g., a marker derived from the anus, artery, appendix, adrenal gland, brain, bone, bronchus, bladder, bone marrow, bulbourethral gland, colon, neck, clitoris, capillary, cerebellum, diaphragm, ear, eye, fallopian tube, genitalia, gallbladder, heart, hair follicle, hypothalamus, interstitial, kidney, joint, liver, lung, larynx, ligament, lymph node, large intestine, lymphatic vessel, mouth, 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, the biomarker is detected in brain-specific EVs. In one embodiment, the biomarker is detected in neuron-specific EVs. In one embodiment, the biomarker is detected in astrocyte-specific EVs. In one embodiment, the biomarker is detected in oligodendrocyte-specific EVs. In one embodiment, the biomarker is detected in microglia-specific EVs. In some embodiments, the biomarker comprises one or more markers selected from Tables 1-5.

[0089] Biomarkers may be differentially present at any level, but generally are present at a level that is 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, when compared to its level in a control sample, e.g., an EV derived from a different cell type or different organ, in a test sample, e.g., a cell type-specific or organ-specific EV, e.g., a brain and / or neuron-specific EV, isolated from a biological sample obtained from a subject. The biomarker is preferably differentially present at a level that is statistically significant (e.g., a p-value of less than 0.05 when determined using any statistical test). Thus, the difference between the level of the biomarker of the present invention and the corresponding control or reference value may be a statistically significant value.

[0090] As used herein, "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. Examples of 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, breast milk, bronchoalveolar lavage fluid, semen (including prostatic fluid), Cowper's gland fluid or bulbourethral gland fluid, vaginal fluid, sweat, fecal matter, hair, tears, cyst fluid, pleural fluid and peritoneal fluid, pericardial fluid, lymph, chyle, chyle, bile, interstitial fluid, menstruation, pus, sebum, vomit, vaginal secretion, mucosal secretion, fecal water, pancreatic juice, washing fluid derived from the nasal sinuses, bronchoalveolar aspirate or other washing fluid. The biological sample may also be of fetal or maternal origin and may include blastocoel, umbilical cord blood, or the maternal circulatory system. 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.

[0091] 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), livestock (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).

[0092] B. Extracellular Vesicles The present invention provides biomarkers for cell type-specific and / or organ-specific extracellular vesicles (EVs), such as 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.

[0093] Extracellular vesicles (EVs) are a class of membrane-bound organelles secreted by various cell types. As used herein, the term "extracellular vesicles" refers to vesicles derived from cells that have a membrane surrounding a central internal space and enclosing it. The membrane of EVs may be composed of a lipid bilayer having an outer surface and an inner surface that binds to the enclosed volume. EVs can carry various molecules such as proteins, lipids, and RNA on their surface and within their lumen.

[0094] EVs include all membrane-bound vesicles having a cross-sectional diameter smaller than the cells from which they are secreted. In some embodiments, the EVs have a longest diameter, such as a longest cross-sectional diameter, in the range of 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 (including each range).

[0095] 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, where the vesicle is generated from the cell by fusion of a late endosome with the plasma membrane or by direct plasma membrane budding. Exosomes are typically produced intracellularly when segments of the cell membrane spontaneously invaginate and are ultimately exocytosed. As used herein, exosomes may also include any budding membrane-bound particle derived from either the plasma membrane or an internal membrane. Exosomes may also be derived from both blebbing and sealing of portions of the plasma membrane or, without limitation, may include any intracellular membrane-bound vesicular structure containing various membrane-associated proteins that selectively bind to exosome proteins together with molecules contained within the exosome lumen, including mRNA, microRNA, or intracellular proteins, and surface-bound molecules derived from the host circulatory system. Exosomes may also include membrane fragments. Blebbing and bleb formation are further described in Charras et al, Nature Reviews Molecular and Cell Biology, Vol. 9, No. 11, p. 730 - 736 (2008). Exosomes may also include membrane fragments.

[0096] EVs contain RNA, such as microRNA (miRNA), long non-coding RNA (lncRNA), mRNA, DNA fragments, and proteins derived from their donor cells, and are thus important for intercellular communication in the human body and are involved in many pathophysiological states such as neurodegenerative diseases. Also, EVs can be loaded with various drugs and exogenous nucleic acids or proteins, and EVs deliver this cargo to different cells. Importantly, EVs are natural carriers for miRNA and other non-coding RNAs, and direct membrane fusion with target cells enables the direct delivery of the contents into the cytosol. Thereby, EVs are an excellent delivery system for small molecules.

[0097] EVs are abundantly present in various biological samples. In some embodiments, the sample is a sample obtained from cell culture. In some embodiments, the sample is a liquid sample, such as 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, breast milk, bronchoalveolar lavage fluid, semen (including prostatic fluid), Cowper's gland fluid or bulbourethral gland fluid, vaginal fluid, sweat, fecal matter, hair, tears, cyst fluid, pleural fluid and peritoneal fluid, pericardial fluid, lymph fluid, chyle, chylous fluid, bile, interstitial fluid, menstruation, pus, sebum, vomit, vaginal secretion, mucosal secretion, fecal water, pancreatic juice, washing fluid derived from the nasal cavity, bronchopulmonary aspirate fluid or other washing fluids, etc. The biological sample may also include a blastocoel cavity, umbilical cord blood, or maternal circulation system of fetal or maternal origin. 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 tissue biopsies and body fluids from mammals are well known in the art.

[0098] According to the method of the present invention, the detection of EVs in biological fluids of various patients enables the evaluation of disease progression, immune response, and toxicity. Therefore, the isolation and detection of EVs can be useful for 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). The entire population of EVs can be isolated from plasma or other biological fluids, but profiling the RNA and protein cargo of these EVs does not distinguish which cargo molecules are derived from which cell types. By isolating EVs from specific cell types, the RNA and proteins within these EVs can be analyzed as a non-invasive "snapshot" of that cell type.

[0099] However, the inventors of the present application have been able to discover novel biomarkers for the isolation of cell type- and organ-specific EVs. In some embodiments, the EVs of the present invention are derived from specific cell types, such as brain cell types, such as neurons, astrocytes, oligodendrocytes, or microglial cells. 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.

[0100] C. Biomarkers of the Present Invention The present invention is based, at least in part, on the discovery of novel biomarkers for the isolation of cell type-specific and / or organ-specific EV markers, such as 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 expressed in brain-specific and / or neuron-specific EVs. Therefore, these differentially expressed markers are useful for isolating brain-specific and / or neuron-specific EVs from human samples.

[0101] By isolating EVs from specific cell types, the RNA and proteins within these EVs can be analyzed as a non-invasive "snapshot" of that cell type. In particular, considering that the brain cannot be biopsied, the ability to isolate EVs from brain neurons or other cell types is particularly useful. For example, the isolation of neuron-derived EVs enables the reading of brain states and the development of biomarkers for the early detection of neurodegenerative diseases. Thus, the identification of new biomarkers for cell type-specific and / or organ-specific EVs, particularly brain-specific and / or neuron-specific EVs, can be useful for better diagnosis, prognosis, or monitoring of diseases such as neurodegenerative diseases, as well as for improving the prediction of treatment outcomes.

[0102] The present invention provides cell type-specific and / or organ-specific EV markers, such as biomarkers for brain-specific and / or neuron-specific EVs. Biomarker levels are determined in a biological sample obtained from a subject. 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 that 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, are assayed. The methods, kits, and panels provided herein also include any combination of any combination of, for example, 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.

[0103] In one embodiment, the biomarker for brain-specific EVs comprises one or more biomarkers in Tables 1-5, or any combination thereof.

[0104] In one embodiment, the biomarker for neuron-specific EVs includes one or more biomarkers in Tables 1 and 5, or any combination thereof. In one embodiment, the biomarker for neuron-specific EVs includes 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.

[0105] In one embodiment, the biomarker for astrocyte-specific EVs includes one or more biomarkers in Table 2, or any combination thereof.

[0106] In one embodiment, the biomarker for oligodendrocyte-specific EVs includes one or more biomarkers in Table 3, or any combination thereof.

[0107] In one embodiment, the biomarker for microglia-specific EVs includes one or more biomarkers in Table 4, or any combination thereof. Table 1. Biomarkers for Neuron-Specific EVs

Table 1-1

Table 1-2

Table 1-3

Table 1-4

Table 1-5

Table 1-6

Table 1-7

Table 1-8

Table 2-1

Table 2-2

Table 2-3

Table 3-1

Table 3-2

Table 4

Table 5-1

Table 5-2

[0108] Each GenBank number is incorporated herein by reference in the version available on the filing date of the application claiming priority to this application.

[0109] The level of the biomarker of the present invention can be determined by any suitable means, method or technique known in the art.

[0110] In some embodiments, immunoassay devices and methods are often used for polypeptide or protein biomarkers. These devices and methods can use labeled molecules in various sandwich, competitive, or non-competitive assay formats to generate a signal related to the presence or amount of the marker of interest. Additionally, certain methods and devices, such as biosensors and optical immunoassays, can be used to determine the presence or amount of the marker without the need for labeled molecules.

[0111] In certain embodiments, the detection method is an immunoassay method comprising an antibody that specifically binds to one or more markers in Tables 1-5. Steps of various useful immunoassay methods are described in scientific literature such as Nakamura et al. (1987), which is incorporated herein by reference, for example. Generally, an immunobinding method includes obtaining a sample suspected of containing a biomarker protein, peptide or antibody, and, optionally, contacting the sample with an antibody or protein or peptide according to the present invention under conditions effective to allow the formation of an immune complex.

[0112] The detection of immune complex formation is well known in the art and can be achieved by the application of several techniques. These methods generally rely on the detection of labels or markers, such as any radioactive, fluorescent, biological, or enzymatic tags or labels that are standardly used in the art. U.S. Patents related to 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, as is known in the art, additional advantages can be found through the use of secondary binding ligands, such as secondary antibodies or biotin / avidin ligand arrangements. The protein itself used in the detection may be conjugated to a detectable label, and one skilled in the art can then simply detect this label, thereby determining the amount of primary immune complexes in the composition. Alternatively, the initially added component that binds within the primary immune complex can be detected using a second binding ligand that has binding affinity for the encoded protein, peptide, or corresponding antibody. In these cases, the second binding ligand can be conjugated to a detectable label. The second binding ligand is often an antibody itself and may thus be referred to as a "secondary" antibody. The primary immune complexes are contacted with a labeled secondary binding ligand, or antibody, under conditions effective to allow the formation of secondary immune complexes and for a sufficient period of time. The secondary immune complexes are then generally washed to remove any non-specifically bound labeled secondary antibody or ligand, and the remaining label in the secondary immune complexes is then detected.

[0113] A further method involves the detection of primary immune complexes by a two-step approach. A second binding ligand, such as an antibody, having a binding affinity for the encoded protein, peptide or corresponding antibody is used to form a secondary immune complex as described above. After washing, the secondary immune complex is contacted with a third binding ligand or antibody having a binding affinity for the second antibody under conditions effective to allow formation of an immune complex (tertiary immune complex) and for a sufficient period of time. The third ligand or antibody is conjugated to a detectable label to enable detection of the thus formed tertiary immune complex. This system can provide signal amplification if desired.

[0114] Any suitable immunoassay can be used, such as an enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), competitive binding assay, planar waveguide technology, etc. Specific immunological binding of the antibody to the marker can be detected directly or indirectly. Examples of direct labels include fluorescent or luminescent tags, metals, dyes, radionuclides, etc. conjugated to the antibody. Examples of indirect labels include various enzymes well known in the art, such as alkaline phosphatase, horseradish peroxidase. In some embodiments, the biomarkers of the invention can be identified by other techniques such as Western blotting, dot blotting, and FACS analysis.

[0115] The biomarkers of the present invention can also be measured, quantified, detected, and analyzed in other forms using protein mass spectrometry and equipment. Protein mass spectrometry refers to the application of mass spectrometry to the study of proteins. Although not intended to be limiting, typically two approaches are used to characterize proteins using mass spectrometry. First, intact proteins are ionized and then introduced into a mass spectrometer. This approach is referred to as the "top-down" strategy for protein analysis. The two main methods for ionization of intact proteins are electrospray ionization (ESI) and matrix-assisted laser desorption / ionization (MALDI). In the second approach, the protein is enzymatically digested into smaller peptides using a protease such as trypsin. Subsequently, these peptides are introduced into a mass analyzer and identified by peptide mass fingerprinting or tandem mass spectrometry. Thus, this latter approach (also called "bottom-up" proteomics) uses identification at the peptide level to infer the presence of proteins.

[0116] The protein biomarkers of the present invention can also be measured in a complex mixture of proteins and molecules that are present simultaneously in a biological medium or sample, although fractionation of the sample may be required and is contemplated herein. Ionization of a complex mixture of proteins can result in a situation where the more abundant proteins tend to "mask" or suppress the signals from the less abundant proteins in the same sample. Furthermore, mass spectra derived from complex mixtures can be difficult to interpret due to the overwhelming number of mixture components. Fractionation can be used to first separate any complex mixture of proteins and then perform mass spectrometry. Two methods are widely used to fractionate proteins or their peptide products derived from enzymatic digestion. The first method fractionates whole proteins and is called two-dimensional gel electrophoresis. The second method, high performance liquid chromatography (LC or HPLC), is used to fractionate peptides after enzymatic digestion. In some situations, it may be desirable to combine both of these techniques. Any other suitable method known in the art for fractionating protein mixtures is also contemplated herein.

[0117] The characterization of protein mixtures using HPLC / MS can also be referred to in the art as "shotgun proteomics" and MuDPIT (multidimensional protein identification technology). The peptide mixture resulting from digestion of the protein mixture is fractionated by one or two steps of liquid chromatography (LC). The eluate from the chromatography step can be introduced directly into a mass spectrometer by electrospray ionization or placed onto a series of small spots for subsequent mass spectrometry using MALDI.

[0118] In certain embodiments, the invention includes the detection of nucleic acid biomarkers, such as the corresponding gene or mRNA of the protein markers of the present invention.

[0119] In various embodiments, the methods of the invention generally include determining the expression levels of a set of genes in extracellular vesicles isolated from a biological sample. Determination of gene expression levels in the practice of the methods of the invention can be carried out by any suitable method. For example, determination of gene expression levels can be by detecting the expression of mRNA expressed from the 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.

[0120] Analysis of multiple markers may be performed separately or simultaneously on one test sample. Some markers may be combined in one test for efficient processing of multiple samples. Furthermore, one of ordinary skill in the art will recognize the value of testing multiple samples (e.g., at successive time points) from the same individual. Such testing of serial samples will allow for the identification of changes in marker levels over time. Increases or decreases in marker levels, as well as the absence of changes in marker levels, will provide useful information regarding the disease state, including, but not limited to, the approximate time from the onset of an event, the appropriateness of drug therapy, the identification of the effectiveness of various therapies, the identification of the severity of an event, the identification of disease severity, and the identification of the patient's outcome, including the risk of future events.

[0121] An assay consisting of a combination of markers referred to in the present invention can be constructed to provide relevant information regarding differential diagnosis. Such panels can be constructed using 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more markers, or individual markers. Analysis of a subset of markers, including a single marker or a larger panel of markers, can be performed in the methods described in the present invention to optimize clinical sensitivity or specificity in various clinical settings.

[0122] Analysis of markers can similarly be performed in a variety of physical formats. For example, microtiter plates or automation can be used to facilitate processing of a large number of test samples. Alternatively, a single-sample format can be developed in a timely manner, for example, in an outpatient transport or emergency room setting, to facilitate immediate treatment and diagnosis. Particularly useful physical formats include surfaces having a plurality of individually addressable positions for detection of multiple different markers. Such formats include protein microarrays, or "protein chips" and capillary devices.

[0123] D. Methods for Purification, Isolation and / or Enrichment of Extracellular Vesicles The present invention provides methods for the purification of extracellular vesicles (EVs) from a sample, such as a biological sample such as plasma or cerebrospinal fluid, as well as methods for the isolation and / or enrichment of cell type-specific and / or organ-specific EVs, such as brain and / or neuron-specific EVs.

[0124] 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), and nanofilter ultrafiltration concentrators (see, e.g., U.S. Pat. Nos. 6,899,863 and 6,812,023, U.S. Pat. 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 using a unique microfluidic platform for efficiently and selectively separating tumor-derived microvesicles. Each of the foregoing references is hereby incorporated by reference herein with respect to its teachings of these methods.

[0125] In some embodiments, as described herein, mixed-mode resin (MMR) or size exclusion chromatography (SEC) can be used to purify EVs from a sample.

[0126] Purification of EVs Using Mixed-Mode Resin Mixed-mode resin (MMR) can be used for the purification of EVs from a sample, such as a biological sample derived from a subject. An exemplary mixed-mode resin is Capto™ Core 700. This resin contains beads with an inert outer shell and pores that exclude molecules larger than 700 kDa. The MMR beads are hydrophobic and have a core containing a positively charged octylamine ligand that efficiently captures proteins entering the beads.

[0127] The MMR beads can "capture" contaminants within the beads, thus enabling the purification of extracellular vesicles from a sample, such as a biological sample, such as a plasma or CSF sample. The disclosed method is independent of columns and other chromatography equipment and is compatible with the high-efficiency purification of extracellular vesicles.

[0128] In some embodiments, the MMR beads exclude molecules that are larger than the size of the target EV, e.g., larger than about 400, 500, 600, or 700 kDa. In some embodiments, the beads include an inert bead exterior or shell. In some embodiments, the 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 include a binding-elution resin. In some embodiments, the size-exclusion beads are Capto™ Core resin beads, e.g., Capto™ Core 700 resin beads or Capto™ Core resin 400 beads.

[0129] In some embodiments, the method does not require the use of a column for the purification of EVs from a biological sample.

[0130] In some aspects, the present disclosure provides a method for purifying extracellular vesicles from a biological sample, comprising mixing a liquid biological sample containing extracellular vesicles with mix-mode resin (MMR) beads capable of capturing molecules that are smaller than the size of the target EV, e.g., larger than about 700 kDa, to create a mixture, and separating and removing the MMR beads from the mixture or removing the supernatant from the mixture such that the extracellular vesicles remain, thereby purifying the extracellular vesicles.

[0131] In some embodiments, the biological sample is obtained from a subject. In some embodiments, the biological sample is a liquid biological sample. One of ordinary skill in the art will recognize that the biological sample may 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, cerumen, breast milk, bronchoalveolar lavage fluid, semen (including prostatic fluid), Cowper's gland fluid or bulbourethral gland fluid, vaginal fluid, sweat, fecal matter, hair, tears, cyst fluid, pleural fluid and peritoneal fluid, pericardial fluid, lymph fluid, chyle, chylous fluid, bile, interstitial fluid, menses, pus, sebum, vomitus, vaginal secretion, mucosal secretion, fecal water, pancreatic fluid, nasal lavage fluid, bronchopulmonary aspirate or other lavage fluid. The biological sample may also be of fetal or maternal origin and may include blastocoel, cord blood, or maternal circulatory system. The biological sample may also be a tissue sample or biopsy from which EVs can be obtained. In one embodiment, the biological sample is brain tissue. In one embodiment, the biological sample is a plasma sample. In another embodiment, the biological sample is a CSF sample.

[0132] In some embodiments, the methods of the present disclosure are performed by the in-solution method. MMR beads can be suspended in a buffer to create a slurry. In some embodiments, the MMR beads are suspended in an equal volume of buffer to create a 50% slurry, or suspended in any volume of buffer effective to produce a slurry containing an effective amount of MMR beads to purify EVs from a sample. For example, the slurry may 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.

[0133] The MMR beads can be mixed with the biological sample in an amount, for example, of 20 μL, 25 μL, 50 μL, 75 μL, 100 μL, 150 μL, 200 μL, 250 μL, 300 μL, 350 μL, or 400 μL per mL of liquid biological sample.

[0134] After combining or mixing MMR beads with a sample containing EVs and then mixing, stirring, or rotating the combination or mixture, the MMR beads can be separated and removed. For example, after mixing, stirring, or rotating the mixture for about 10, 20, 30, 40, 45, 50, 55, 60, 65, 70, 75 minutes or longer, the MMR beads can be separated and removed. In some embodiments, the mixture is mixed, stirred, or rotated for about 30 minutes to 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 suitable for capturing impurities in the sample by the MR beads.

[0135] The MMR beads can be separated from the mixture using any effective means known in the art. In some embodiments, centrifugation is used to separate the MMR beads from the mixture. In some embodiments, the mixture is centrifuged at between about 600g, 700g, 800g, 900g, or higher g. In some embodiments, the mixture is centrifuged at about 800g. In some embodiments, the mixture is centrifuged for a time sufficient to fully separate the MMR beads from the remaining mixture to obtain the EVs in the supernatant. In some embodiments, the mixture is centrifuged for about 10 minutes or for about 10, 15, 20, 25, 30, 40 minutes or longer.

[0136] In some embodiments, the MMR beads used in the methods of the present disclosure may include an inert bead exterior or shell. The exterior of the bead may include pores that allow molecules smaller than a certain size to pass through the outer shell and be captured within the core of the bead. In some embodiments, the bead may include a core that includes the interior of the bead containing a ligand such as a multimode ligand such as octylamine ligand. In preferred embodiments, the thickness of the whole-shell bead (i.e., shell and core) is preferably 40 - 100 μm in diameter, and the thickness of the shell is preferably 2 - 10 μm.

[0137] 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, the internal core is provided with octylamine ligands, the shell is inert, and the porosity of the shell and core does not allow the entry of molecules larger than about 700 kD.

[0138] In some embodiments, the MMR beads include a bind-elute resin. In some embodiments, the MMR beads are Capto™ Core bind-elute beads. In some embodiments, the MMR beads are Capto™ Core 700 bind-elute beads. Capto™ Core 700 chromatography resin (GE Healthcare Biosciences AB) contains octylamine ligands within the Capto™ Core 700 "beads" and is designed to have both hydrophobic and positively charged properties and can capture molecules below 700 kilodaltons. Since extracellular vesicles are larger than 700 kDa and the outside of the beads is inert, Capto Core 700 enables the purification of extracellular vesicles by size exclusion. When 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) compared to larger molecules. In contrast, the ligand-activated pores of Capto™ Core 700 have electrostatic and hydrophobic interactions that "capture" molecules below 700 kDa.

[0139] In some embodiments, a biological sample, such as plasma or CSF, is applied to a size exclusion chromatography (SEC) column and then mixed with MMR beads capable of capturing molecules smaller than about 700 kDa. Any size exclusion chromatography resin known in the art is suitable for the method of the present invention. In some embodiments, the size exclusion chromatography column comprises a stationary phase comprising a size exclusion chromatography base matrix of 6% cross-linked agarose. In some embodiments, the size exclusion chromatography base matrix of 6% cross-linked agarose is Sepharose™ CL-6B resin.

[0140] In some embodiments, a biological sample, such as plasma or CSF, is further applied to a cation exchange chromatography resin after the size exclusion chromatography column and before mixing with MMR beads capable of capturing molecules smaller than about 700 kDa. Any cation exchange chromatography resin known in the art is suitable for the method of the present invention. Exemplary cation exchange chromatography resins include, but are not limited to, sulfhydryl, sulfonate, sulfate, carboxymethyl, sulfoethyl, sulfopropyl, phosphate, and sulfonate. In some embodiments, the cation exchange chromatography resin is Fractogel® EMD-SO3- resin.

[0141] The methods for EV purification disclosed herein can also be optimized for high-efficiency application.

[0142] Purification of EVs Using Size Exclusion Chromatography Size-exclusion chromatography (SEC) can also be used for the purification of EVs from a sample, such as a biological sample derived from a subject. In some embodiments, size-exclusion chromatography (SEC) columns comprising a stationary phase material comprising an agarose size-exclusion chromatography-based matrix, such as a matrix comprising a 6% agarose content, can be used to purify EVs from a biological sample. In some embodiments, the stationary phase material is a size-exclusion chromatography-based matrix of 6% cross-linked agarose. In some embodiments, the stationary phase material is a Sepharose™ resin, such as a Sepharose™ cross-linked resin such as Sepharose™ CL-6B resin. In SEC, a porous stationary phase is used to sort macromolecules and particulate matter according to their size. Components in a sample with a small hydrodynamic radius can pass through the pores and thus result in a slow elution. Components with a large hydrodynamic radius, including EVs, are excluded from entering the pores. The SEC columns used in the methods of the present invention provide a significant improvement in EV yield at a relatively low cost compared to other columns.

[0143] 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 size-exclusion chromatography-based matrix of 6% cross-linked agarose, such as Sepharose™ 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, thereby purifying the extracellular vesicles.

[0144] In some embodiments, the sample is a biological sample obtained from a subject. In some embodiments, the biological sample is a liquid biological sample. One of ordinary skill in the art will recognize that the biological sample may 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, cerumen, breast milk, bronchoalveolar lavage fluid, semen (including prostatic fluid), Cowper's gland fluid or bulbourethral gland fluid, vaginal fluid, sweat, fecal matter, hair, tears, cyst fluid, pleural fluid and peritoneal fluid, pericardial fluid, lymph fluid, chyle, chylous fluid, bile, interstitial fluid, menses, pus, sebum, vomitus, vaginal secretion, mucosal secretion, fecal water, pancreatic fluid, nasal sinus-derived lavage fluid, bronchopulmonary aspirate fluid or other lavage fluid. The biological sample may also be of fetal or maternal origin and may include blastocoel, cord blood, or maternal circulatory system. 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 sample. In one embodiment, the biological sample is a plasma sample. In another embodiment, the liquid biological sample is a CSF sample.

[0145] In other embodiments, the SEC column is a column with a volume of 5 mL, 7 mL, 10 mL, 12 mL, 15 mL, 20 mL, or 25 mL. In other embodiments, the SEC column is a column with a volume of 5 mL to 25 mL, although columns outside these ranges can also be used. In some embodiments, the SEC column is a column with a volume of 10 mL. In other embodiments, the SEC column is a column with a volume of 20 mL.

[0146] An SEC column containing a size exclusion chromatography-based matrix of 6% cross-linked agarose, such as Sepharose™ CL-6B resin, can be prepared by first washing the resin and then adding it to the column. In some embodiments, a size exclusion chromatography-based matrix of 6% cross-linked agarose, such as Sepharose™ CL-6B resin, is washed in a buffer, such as PBS, and then the column is prepared. After washing the resin multiple times, the column can be prepared. In some embodiments, a size exclusion chromatography-based matrix of 6% cross-linked agarose, such as Sepharose™ CL-6B resin, is washed 4 or more times in a buffer and then the column is prepared.

[0147] Once the resin is washed, it can be added to a suitable column, such as a 10 mL or 20 mL column. In one embodiment, the SEC column includes a housing having at least one wall defining a chamber with an inlet and an outlet. In some embodiments, after washing the stationary phase, the column containing a size exclusion chromatography-based matrix of 6% cross-linked agarose, such as Sepharose™ CL-6B resin, is washed, and then a sample containing extracellular vesicles is introduced into the column. In some embodiments, the stationary phase is washed with PBS.

[0148] Fractions containing EVs are collected from the SEC column, thereby purifying EVs from a liquid biological sample. Depending on the size of the column, for example, fractions 6-21 or fractions 12-27 can be collected. In some embodiments, for a 10 mL column, fractions 6-21 can be collected. In other embodiments, for a 20 mL column, fractions 12-27 can be collected. Fewer fractions can also be collected from the SEC column. For example, higher purity EVs can also be achieved by taking fewer fractions (e.g., 7-9 instead of 7-10), although the yield is lower.

[0149] Although not required, after purification of the EVs using any of the methods of the present disclosure, the purified extracellular vesicles can be further purified by any means known in the art. Further, the methods for purification of 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 be used in conjunction with differential centrifugation, anion exchange and / or gel permeation chromatography, sucrose density gradients, organelle electrophoresis, magnetic activated cell sorting (MACS), or a nanomembrane ultrafiltration concentrator.

[0150] In some embodiments, the fractions collected from the SEC column are further applied to a cation exchange chromatography resin. The cation exchange chromatography resin comprises a stationary phase comprising a functional group selected from the group consisting of sulfhydryl, sulfonate, sulfate, carboxymethyl, sulfoethyl, sulfopropyl, phosphate and sulfonate. In some embodiments, the cation exchange chromatography resin is a Fractogel® EMD-SO3-resin.

[0151] In some embodiments, the fractions collected from the SEC column are further applied to mixed mode resin (MMR) beads capable of capturing molecules smaller than about 700 kDa. In some embodiments, the MMR beads are Capto™ Core 700. In another embodiment, the fractions collected from the SEC column are further applied to a cation exchange chromatography resin and MMR beads capable of capturing molecules smaller than about 700 kDa.

[0152] In some embodiments, the method of the present application recovers 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 a sample, such as a plasma and CSF sample, as compared to current methods in the art.

[0153] High-abundance proteins such as albumin and immunoglobulins can interfere with the purification of EVs from biological samples. Thus, the methods of the present disclosure can be used with a system that utilizes multiple antibodies specific for the most abundant proteins found in blood. Such a system can remove several proteins at once, thus revealing less abundant species such as origin cell-specific exosomes. Other known methods for EV purification include the high-abundance protein removal method described in Chromy et al. J. Proteome Res 2004; 3: 1120-1127. In another embodiment, the purification of EVs from biological samples can also be enhanced by removing serum proteins using glycopeptide capture described in Zhang et al, Mol Cell Proteomics 2005; 4: 144-155.

[0154] Isolation and / or enrichment of cell type-specific EVs 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 selection. As used herein, the terms "isolating" or "enriching" refer to increasing the concentration or density of extracellular vesicles or a subpopulation of extracellular vesicles in a sample and / or removing non-EV substances (e.g., proteins, cells) from the sample.

[0155] EVs can be further isolated and / or enriched based on the source or type of cells. 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 differences in antigens, nucleic acids, metabolism, gene expression, or epigenetics. In some embodiments, magnetic or paramagnetic beads conjugated to an antibody or a fluorescently labeled antibody in a magnetic field gradient are used in conjunction with flow cytometry based on differences in antigens. 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 for EVs. In other embodiments, antibodies, ligands, or soluble receptors are used to enrich a sample for EVs.

[0156] Since EVs often carry surface molecules such as antigens derived from their donor cells, surface molecules can be used to identify, isolate and / or enrich EVs from specific donor cell types. In some embodiments, surface markers are used to actively enrich subpopulations of EVs derived from one or more cell types. In some embodiments, cell surface markers not found on the EV population are used to negatively enrich for vesicles by depleting the cell population. Flow cytometry sorting can also be used to further enrich for EVs using cell surface markers or intracellular or extracellular markers conjugated to fluorescent labels. Intracellular and extracellular markers may include antibodies against nuclear stains or intracellular or extracellular proteins that are preferentially expressed in vesicles.

[0157] In some embodiments, EVs isolated from a biological sample, such as a body fluid, such as CSF or plasma, are enriched for those originating from a specific tissue, such as the brain, lung, pancreas, stomach, intestine, bladder, kidney, ovary, testis, skin, colorectal, chest, prostate, brain, esophagus, liver, placenta, fetal cells.

[0158] In some embodiments, cell type-specific EVs are isolated and / or enriched. In some embodiments, cell type-specific EVs, such as brain and / or neuron-specific EVs, are isolated and / or enriched from a sample, such as a biological sample like plasma or cerebrospinal fluid, based on one or more biomarkers of the present invention, such as one or more of the biomarkers in Tables 1-5.

[0159] 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 of the biomarkers in Tables 1-5, or any combination thereof.

[0160] 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 of the 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.

[0161] 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 of the biomarkers in Table 2, or any combination thereof.

[0162] 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.

[0163] 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.

[0164] In some embodiments, the isolation of cell-type specific EVs can be achieved by one or more purification or isolation steps. The one or more purification steps may include, but are not limited to, immunoisolation, purification based on microfluidic affinity, purification based on magnetism, pull-down purification, or purification based on fluorescence-activated vesicle sorting. Commercially available precipitation kits such as ExoQuick™ and Total Exosome Isolation™ precipitation solutions are also available. Such kits can be easily used in just one or two steps and do not require any expensive equipment or advanced technical know-how.

[0165] In some embodiments, isolation can be performed using immunoisolation with one or more antibodies against one or more biomarkers of the present invention, such as one or more markers in Tables 1-5. Immunoisolation can be performed using a bait / prey strategy. In some embodiments, the bait molecule may be an antibody against a prey EV biomarker of the present invention, such as a bait protein such as a monoclonal antibody. In some embodiments, the bait molecule may be an RNA aptamer. When attempting to combine several prey EVs for purification, a mix of corresponding monoclonal antibodies against each of the prey EV biomarkers to be pulled up can be used.

[0166] In some embodiments, the bait molecule is recognized by an affinity ligand. The affinity ligand may be a complex based on a divalent metal, a protein, a peptide, such as a fusion protein tag, or more preferably, an antibody.

[0167] In some embodiments, the bait molecule or the affinity ligand is directly or indirectly immobilized or "coupled" to a solid substrate material, such as by the formation of a covalent chemical bond between a specific functional group on the ligand (e.g., a primary amine, thiol, carboxylic acid, aldehyde) and a reactive group on the substrate. The substrate, or matrix, in the affinity purification step of the methods of the invention can be any material to which a biospecific ligand (i.e., the bait molecule or the affinity ligand) can be coupled. Useful affinity supports may have a high ratio of surface area to volume, chemical groups that are easily modified for covalent attachment of ligands, minimal non-specific binding characteristics, good flow characteristics and / or mechanical and chemical stability. For example, several substrates, including agarose, cellulose, dextran, polyacrylamide, latex, or controlled pore glass, can be used as the solid substrate. Instead of beadified agarose or other porous resins, magnetic particles can also be used as the substrate. Their small size provides a sufficient ratio of surface area to volume required for effective ligand immobilization and affinity purification. Magnetic beads can be produced as superparamagnetic iron oxide particles that can be covalently coated with a silane derivative. The coating renders the beads inert (i.e., minimizes non-specific binding) and provides specific chemical groups necessary for binding 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 as a loose slurry. During mixing, the beads remain suspended in the sample solution and undergo an affinity interaction with the immobilized ligand. After allowing sufficient time for binding, the beads are collected and separated from the sample using a strong magnet.

[0168] In some embodiments, for example, by performing a pull-down of one or more specific EV biomarkers of the present invention using one or more antibodies against each of one or more biomarkers in Tables 1-5, a pull-down assay can be performed for the purification or isolation of cell-type specific EVs. The EV biomarker may be specific for at least one cell type, and advantageously results in the enrichment of EVs derived from the selected cell type.

[0169] In some embodiments, at least one or more purification steps for the purification of a cell-type specific EV subpopulation include pull-down purification. In such pull-down purification, the prey EV biomarker is generally a membrane (transmembrane) 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 of the prey EV biomarkers to be pulled up. Magnetic beads such as magnetic nucleic acid binding beads coated with an affinity ligand for the bait protein, or silica beads functionalized with silane (e.g., Dynabeads® from Thermo Fisher Scientific, e.g., Dynabeads® MyOne Silane Beads from Thermo Fisher Scientific) can be used to isolate the bait protein bound to the prey EV biomarker. The affinity ligand is preferably a class-specific or species-specific antibody. As an example, magnetic beads coated with an anti-mouse antibody can be used together with a monoclonal mouse antibody against a specific surface protein of a cell type or cell subtype subpopulation of EVs. Generally, a control antibody such as a mouse mCherry monoclonal antibody can be used.

[0170] Thus, pull-down assays can be used to demonstrate and validate the purification or isolation of one or more EV subpopulations, each expressing at least one specific membrane protein marker. Purification or isolation of EV subpopulations by at least one specific pre-EV biomarker can be further confirmed using Western blot or qRT-PCR.

[0171] E. Methods for disease diagnosis and prognosis The present invention also provides methods for diagnosing or prognosticating a disease, such as a neurodegenerative disorder, in a subject, for identifying a subject at risk of a disorder, or for prescribing a treatment regimen or predicting a benefit from a therapy in a subject having a disorder.

[0172] Once cell type-specific and / or organ-specific EVs, such as brain and / or neuron-derived EVs, are isolated and / or enriched based on the presence of one or more biomarkers of the present invention, profiling of the molecular content of these cell type-specific and / or organ-specific EVs provides deep insights for both early detection of pathology and better understanding. For example, since human brain and / or neurons generally cannot be biopsied, readout of the molecular content of brain and / or neuron-derived EVs from accessible body fluids can provide a unique perspective for understanding brain pathology.

[0173] Accordingly, in one aspect, the present invention provides a method for diagnosing, prognosticating, or identifying a subject at risk of developing a neurodegenerative disorder in a subject, the method comprising: (a) obtaining a biological sample from the subject; (b) isolating brain-specific extracellular vesicles from the biological sample based on the presence of one or more biomarkers selected from Tables 1-5 in the isolated extracellular vesicles; (c) extracting proteins and / or RNA from the isolated brain-specific extracellular vesicles; and (d) analyzing the proteins and / or RNA extracted from the isolated brain-specific extracellular vesicles, thereby diagnosing, prognosticating, or identifying a subject at risk of developing a neurodegenerative disorder.

[0174] In some embodiments, the biological sample comprises a liquid biological sample. In some embodiments, the liquid biological sample is selected from the group consisting of whole blood, serum, plasma, cerebrospinal fluid, myeloid fluid, amniotic fluid, aqueous humor, vitreous humor, bile, breast milk, cerumen (ear wax), chyle, chyme, endolymph, perilymph, exudate, feces, vaginal fluid, gastric acid, gastric juice, lymph fluid, mucus (including nasal discharge and sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus, catarrhal secretions, saliva, sebum (skin oil), semen, saliva, synovial fluid, sweat, tears, urine, vaginal secretions, vomit, and one or more mixtures thereof.

[0175] In some embodiments, the extracellular vesicles are neuron-specific, astrocyte-specific, oligodendrocyte-specific, and / or microglia-specific.

[0176] In some embodiments, the extracellular vesicles are neuron-specific and the one or more biomarkers are selected from Table 1 and Table 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.

[0177] In some embodiments, the extracellular vesicles are astrocyte-specific and the one or more biomarkers are selected from Table 2.

[0178] In some embodiments, the extracellular vesicles are oligodendrocyte-specific and the one or more biomarkers are selected from Table 3.

[0179] In some embodiments, the extracellular vesicles are microglia-specific and the one or more biomarkers are selected from Table 4.

[0180] The protein and / or RNA content of the EVs has been found to correlate with the protein and / or RNA content of the corresponding cells. Thus, analysis of the protein and / or RNA content of the EVs provides both qualitative and quantitative information regarding the cellular RNA content of the corresponding cells. Advantageously, this enables the provision of a non-invasive diagnostic method. Indeed, the analysis (by, for example, DNA / RNA sequencing, transcriptome profiling, qRT-PCR, microarray, proteome profiling, or mass spectrometry) is performed on a biological sample derived from a body fluid such as one derived from blood or cerebrospinal fluid. Such fluids are more readily and immediately available than the corresponding organs, for example, the brain. Consistently, the present invention provides a non-invasive and more reliable diagnostic method.

[0181] 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 or fragment of RNA or DNA. The extraction of proteins and / or nucleic acids can be performed using any known method or commercially available kit in the art.

[0182] Analysis of the RNA content in EVs can be performed using any transcriptomics method 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, analyzing nucleic acids extracted from isolated brain-specific extracellular vesicles includes genome-wide analysis or transcriptome profiling.

[0183] In other embodiments, analyzing nucleic acids extracted from isolated brain-specific extracellular vesicles includes analysis of a gene of interest, where the gene of interest is associated with a neurodegenerative disorder. For example, it is tested for the presence or absence of the gene of interest, for one or more allelic variants or mutations of the gene of interest, and for the presence or absence of allelic variants or mutations.

[0184] Similarly, analysis of the protein content in EVs can be performed using any proteomic method known in the art such as proteomic profiling, mass spectrometry, immunoassay, ELISA, fluorescence-activated cell sorting (FACS), SDS-polyacrylamide gel electrophoresis (SDS-PAGE), or Western blot analysis.

[0185] In some embodiments, the analysis of proteins extracted from isolated brain-specific extracellular vesicles includes the analysis of a protein of interest, which is associated with a neurodegenerative disorder. In some embodiments, the analysis of the extracted proteins includes testing for the presence or absence of the protein of interest, one or more mutations in the protein of interest, such as deletions, additions, substitutions, truncations, or modifications, such as proteins having post-translational modifications in an altered state, or epigenetic changes with respect to the protein of interest, and testing for the presence or absence of the mutation or modification.

[0186] Neurodegenerative disorders for diagnosis or prognosis according to 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), Lewy body dementia, neurofibrillary change predominant senile dementia, Pick's disease (PiD), argyrophilic grain disease, amyotrophic lateral sclerosis (ALS), other motor neuron diseases, Guam Parkinson dementia complex, FTDP-17, corticobasal ganglionic degeneration, multiple sclerosis, traumatic brain injury (TBI), and Parkinson's disease.

[0187] 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, a-synuclein, DJ-1, LRRK2, PINK1, Parkin, UCHL1, synphilin-1, and NURR1. Examples of Alzheimer's disease-related genes / proteins 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 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. Analysis of these genes or proteins of interest in brain- and / or neuron-derived EVs provides insights into the health of the brain of the subject.

[0188] In some embodiments, the analysis of cell type-specific and / or organ-specific extracellular vesicles, e.g., brain and / or neuron-specific EVs, can be performed over a specific time course at various intervals to evaluate the progression and pathology of a subject. For example, as a function of time, the levels and characterization of brain and / or neuron-derived EVs can be tracked by performing analyses at regular intervals such as 1 day, 2 days, 3 days, 1 week, 2 weeks, 1 month, 2 months, 3 months, 6 months, or 1 year. In the case of existing patients, this provides a useful indication of disease progression and aids the physician in making appropriate treatment choices based on increases, decreases, or lack of changes in the content of brain and / or neuron-derived EVs. For those at risk of neurodegenerative diseases, the protein and / or RNA content of brain and / or neuron-derived EVs can provide an early warning or diagnosis.

[0189] F. Kit 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 a biological sample obtained from a subject, and kits for detecting neurodegenerative disorders in a subject.

[0190] A kit for isolating brain-specific extracellular vesicles may include the following: (a) one or more reagents for detecting the presence of one or more biomarkers selected from Tables 1-5 on the surface of the extracellular vesicles; (b) means for isolating brain-specific extracellular vesicles based on the presence of the biomarker; and (c) one or more of a set of instructions for detecting the presence of the biomarker and / or for isolating brain-specific extracellular vesicles. In some embodiments, 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. In some embodiments, the kit further includes means for isolating a biological sample from the subject.

[0191] A kit for detecting neurodegenerative disorders comprises the following: (a) one or more reagents for detecting the presence of one or more biomarkers selected from Tables 1-5 on the surface of extracellular vesicles; (b) means for isolating brain-specific extracellular vesicles based on the presence of the biomarker; (c) one or more reagents for detecting the level of genes associated with neurodegenerative disorders in the isolated brain-specific extracellular vesicles; and (d) may include one or more of a set of instructions for use for measuring the presence of the biomarker, isolating brain-specific extracellular vesicles, and / or measuring the level of genes associated with neurodegenerative disorders. In some embodiments, the kit further comprises means for isolating a biological sample from a subject.

[0192] Various kits with different components are contemplated by the present invention. Generally, the kit will include means for collecting a biological sample or extracellular vesicles, means for detecting one or more biomarkers in the extracellular vesicles, and instructions for use of the contents of the kit. In certain embodiments, the kit includes means for enriching or isolating a subpopulation of extracellular vesicles, such as cell type-specific and / or organ-specific extracellular vesicles, in a biological sample. In further embodiments, the means for enriching or isolating extracellular vesicles includes reagents necessary to enrich or isolate extracellular vesicles from a biological sample. In some embodiments, the one or more reagents for detecting the presence of a biomarker on the extracellular surface includes an antibody for the biomarker. In certain embodiments, the kit includes means for detecting and / or quantifying the level of a gene of interest associated with neurodegenerative disorders in cell type-specific and / or organ-specific extracellular vesicles. In further embodiments, the means for quantifying the amount of the gene of interest includes reagents necessary to detect the amount of the gene of interest.

[0193] The content of all documents cited or referenced in this specification and all documents cited or referenced in the documents cited in this specification is incorporated herein by reference, together with any manufacturer's instructions, descriptions, product specifications, and product sheets regarding any product described in this specification or in any document incorporated herein by reference, GenBank accession numbers and gene numbers, as well as published patents and patent applications, and can be used in the practice of the present invention. Those skilled in the art will recognize that the present invention can be practiced with modifications to the disclosed structures, materials, compositions, and methods, and that such modifications are considered to be within the scope of the present invention.

[0194] The present invention is further illustrated by the following examples, which should not be construed as limiting.

Examples

[0195] (Example 1) Identification of Markers for Isolating Neuron-Specific Extracellular Vesicles Selection of Neuron-Specific EV Marker Candidates Based on Gene Expression All human proteins were evaluated for their potential as means for isolating neuron-specific EVs from human biological fluids such as CSF and plasma (Figure 1A). The goal was not to definitively determine which marker functions best, but rather to construct a simple framework for determining which markers should be prioritized for experimental validation. The first requirement was that potential markers must have an annotated transmembrane domain. This is because it is necessary to use it in the immunoisolation of EVs. Of the 20,375 reviewed proteins in the protein database UniProt, 4,845 have an annotated transmembrane domain (33).

[0196] The second requirement is cell-type specific gene expression of the marker in the desired cells and tissues of origin. Thus, candidate markers should be highly enriched in neurons compared to other cell types in the brain and highly enriched in the brain compared to other organs in the human body. To evaluate cell-type specific gene expression, RNA-Seq data of major cell types isolated from human brain tissue by immunopanning were analyzed (34). To evaluate organ-level expression, the GTEx RNA-Seq dataset of human organs was analyzed (35). Tau, an indicator of gene expression specificity, has been shown to be particularly robust compared to other methods (37), and it was used to calculate the specificity for each gene (36). Selecting a tau cutoff of 0.7 (Figures 1B, 5 - 7), we found that there are 291 genes encoding transmembrane proteins that are specifically expressed in neurons compared to other cell types in the brain and 305 genes encoding transmembrane proteins that are brain-specific compared to other organs. Overlaying these two lists, we found 168 transmembrane proteins (Figure 1C). Since some of these proteins may be expressed in neurons but not present on EVs in the target biofluid, it was reasonable to overlay transmembrane proteins showing cell and tissue specificity with the proteomics dataset of EVs in the target biofluid.

[0197] Differentiation of human iPS cells into iNGN neurons for isolation of EVs from conditioned medium Before scrutinizing neuronal EVs in human body fluids, it was first necessary to develop a positive control, i.e., a source of pure neuronal EVs. A human induced pluripotent stem (iPS) cell line that exhibits doxycycline-inducible expression of the transcription factors Neurogenin 1 / 2 had been previously established for the rapid and highly efficient differentiation of iPS cells into neurons (38). The protocol for differentiating induced Neurogenin (iNGN) cells was expanded, and EVs were collected from their conditioned media (39). Proteomic analysis based on mass spectrometry of EVs isolated from neurons was performed, using the uninduced parental iPS line as a control. 197 transmembrane proteins were found to be expressed on EVs derived from neurons but not from iPS cells (Figure 1D). Of these 197 proteins, 7 passed the cell- and tissue-specificity thresholds (Figure 1E). Western blotting was used to confirm the presence of one of these markers, SYT1, in both neuronal cell lysates and neuronal EVs (Figure 1F).

[0198] Development of an EV immunoisolation protocol using EVs derived from cell cultures An immune isolation protocol was developed to capture a subset of EVs that exhibit specific markers. Since EVs derived from neurons were expected to be a small subset of total EVs in biological fluids such as plasma, this protocol needed to be highly efficient and specific. To simplify this goal, the human K562 cell line was selected (Figure 2A). Immune isolation was first optimized using the widely expressed tetraspanin CD81. After purifying EVs from the conditioned medium of K562 cells using differential ultracentrifugation (39), immune isolation was performed and the efficiency was evaluated by Western blotting of CD81 in the pull-down versus flow-through fractions. To ensure that the immune isolation was specific for the target protein, the same procedure was used and CD81 was also measured when EVs were immunoisolated using a non-specific antibody. Using this system, several parameters such as antibody conjugation strategy, amount and ratio of beads and antibody were systematically optimized. The final protocol resulted in immune isolation showing high efficiency and specificity (Figure 2B, Figures 8A - 8E).

[0199] Next, the optimized general cell culture EV immunoisolation protocol was applied to EVs derived from iNGN neurons. Proteomic data detected the presence of L1CAM and NCAM1 on neuronal EVs. They did not pass the tau specificity score, but these two proteins, and in particular, L1CAM, have been used by others as targets for immunoisolation in plasma. It was ultimately shown that L1CAM is not a viable marker in human CSF and plasma (32), but L1CAM was analyzed on iNGN EVs as a platform for immunoisolation of neuronal EVs before analyzing L1CAM in biofluids. By directly comparing previously used antibodies and performing further optimization (Figures 12 and 13), we achieved a highly efficient and specific L1CAM pulldown in iNGN EVs (Figure 2C). To model cell type-specific EV capture from biofluids, neuron-derived L1CAM-positive EVs were mixed with L1CAM-negative EVs derived from undifferentiated iPS cells (Figure 2D). When GJA1, a protein present on iPS EVs but not neuronal EVs, was monitored after mixing EVs from both cell types, we confirmed that GJA1 was not detected on beads when using the L1CAM antibody (Figure 2E). These experiments confirmed that immunoisolation is specific even when the proportion of neuronal EVs is low compared to non-neuronal EVs.

[0200] Immunoisolation of EV subsets from human CSF and plasma After developing an immuno-isolation method for EVs derived from cell culture, the method was tested in human biological fluids (Figure 2F). After some optimization (Figures 9-11, 14A-B and 15A-E), EVs containing tetraspanins CD9, CD63, and CD81 were immuno-isolated from human CSF (Figure 2G) and plasma (Figure 2H). The flow-through contained protein levels that were too high to migrate on protein gels, so a second immuno-isolation for each tetraspanin was performed on the flow-through from the first immuno-isolation (using beads containing either the target antibody or a control antibody). Comparison of the results of the first immuno-isolation with those of the second immuno-isolation suggested that the protocol was highly efficient and specific in both CSF and plasma. Thus, provided that a sufficiently good antibody was available, this protocol was able to perform highly specific immuno-isolation of EVs from human biological fluids without first purifying the EVs.

[0201] Development of a high-purity EV isolation method Next, proteomics was used to evaluate which neuron EV markers are present in human CSF and plasma. However, free proteins (such as albumin) are present in body fluids in far greater abundance than EV proteins, so contamination of EV preparations with free proteins limits the usefulness of mass spectrometry in detecting EV proteins (40). Previously, a large-scale comparison of different EV isolation methods from CSF and plasma was performed (41). Size exclusion chromatography (SEC) was optimized to remove free proteins such as albumin by several orders of magnitude, but the EV fraction in SEC still carries substantial amounts of albumin. Therefore, a new method was developed that enables the purification of EVs from human body fluids with very high purity while still maintaining a high yield. After extensive searching, a mixed-mode resin (MMR) called Capto Core 700 was identified. This resin contains beads with an inert outer shell and pores that exclude molecules larger than 700 kDa. The MMR beads are hydrophobic and have a core containing a positively charged octylamine ligand that efficiently captures proteins entering the beads. This resin was developed for use in chromatography columns for virus purification (42), but reports have shown the use of the resin for virus purification “in-line” without the use of columns for non-enveloped infectious viruses purified from cells (43).

[0202] A simple method of mixing MMR was developed using biological fluids to purify EVs away from free proteins. Since MMR beads bind to free proteins and capture them, it was reasonable that after incubation with biological fluids, the beads could be separated to leave pure EVs (Figure 3A). To measure free protein contamination, by measuring the levels of tetraspanins CD9, CD63, and CD81 (as a surrogate for EV yield) and albumin, after various optimizations, we could focus on the most important parameters for EV isolation (Figure 3B). The most important parameter was found to be the ratio of resin to total protein in the sample. We developed an optimized method, which we called the MMR slurry, and applied it to CSF. It was found that by precisely increasing the ratio of resin volume to total protein in the sample, the EV purity could be "tuned". Thus, albumin could be completely depleted while retaining a large amount of EVs (as measured by Western blot) (Figure 3C, D). Consistent with previous results (32), L1CAM also completely disappeared by Western blot when the amount of MMR was increased (Figure 3E).

[0203] After optimizing the isolation for CSF, the protocol was applied to plasma. Since plasma has two orders of magnitude more protein than CSF, plasma was first purified using SEC before applying the MMR slurry method to plasma. By applying the MMR slurry method to 1 mL of plasma after SEC, albumin could be depleted to levels that were almost undetectable (Figure 3F, G). Thus, the MMR slurry method could be applied to CSF as a simple one-step purification or to plasma as a two-step protocol after SEC.

[0204] Mass spectrometry of EVs derived from human plasma and CSF using MMR slurry MMR slurry was applied to establish the proteomes of EVs related to CSF and plasma. The protocol was further optimized to increase the number of transmembrane proteins detected and combined this high-purity isolation with a method previously developed for low-input proteomics. High-quality proteomes were obtained from 1 mL of biofluid using a one-step MMR slurry method for CSF or a two-step SEC and MMR slurry method for plasma (Figure 4A). These proteomes were equivalent to or deeper than previous studies that used much larger volumes of CSF and plasma (with respect to the number of proteins detected). One limitation of using two-step SEC followed by MMR slurry is that lipoproteins are not removed from plasma because they are larger than 700 kDa. However, by replacing SEC with dual-mode chromatography (DMC) that combines SEC and cation exchange resin, a decrease in ApoB100 levels was found (44). Isolation of EVs from plasma using two-step DMC + MMR slurry detected more proteins than SEC + MMR slurry (Figure SI). Various mass spectrometry runs were combined and used on 1 mL of pooled human plasma or CSF to generate a reference EV proteome, yielding 2104 proteins for CSF and 1862 proteins for plasma.

[0205] Pipeline for the identification of neuron-specific EV markers Once high-quality reference EV proteomes for CSF and plasma were established, these data were connected to a computer pipeline. A list of candidate transmembrane proteins that met the gene expression cutoff was partially overlapped with the MMR slurry proteomics data (Figure 4A). When these datasets were overlaid, 27 candidate markers were found to meet the established criteria: transmembrane, specific cell type, and tissue expression, and were found in both 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 body fluids (Figure 4C). To further rank these candidates, datasets containing the proteins found in CSF (45, 46) and plasma (47) or EVs isolated from these body fluids (47 - 51) were analyzed (Tables 1 - 5). This comprehensive analysis made it possible to identify candidate markers for the isolation of neuron-specific EVs from human body fluids.

[0206] Discussion In this study, a systematic and unbiased framework for the identification of markers for neuron-specific EVs based on gene expression and EV proteomics data was provided. The expression levels of all human transmembrane proteins were analyzed for enrichment in neurons compared to other cell types in the brain and in the brain compared to other organs. Markers were further prioritized for experimental validation based on proteomic data to ensure that the markers of interest were found on EVs. Towards this end, reference human CSF and plasma EV proteomics datasets that were much larger than those in previous studies were generated. Additionally, EVs derived from the conditioned medium of human iNGN neurons were established as a "positive control" for endogenous neuron EVs in human body fluids, and their EV proteomes were profiled.

[0207] In addition to the generation of a new reference proteomics EV dataset and the construction of a computer marker prediction pipeline, several technical advancements were also made for the use of EVs as biomarkers. First, a highly optimized EV immuno-isolation protocol was developed that functions in both plasma and CSF without using previous EV isolation as commonly done in other studies. Second, a novel MMR slurry method was developed that enables the isolation of EVs with exceptional purity, which is particularly important for the proteomic profiling of EVs. This method involves simply incubating the resin with the biological fluid to purify EVs from clinically relevant volumes of CSF. The MMR slurry was also applied to the purification of high-purity EVs from plasma after SEC or DMC (to remove lipoproteins). The Capto Core resin was recently shown to remove dyes or small amounts of proteins when used in a 96-well filter plate (52), so the MMR slurry was assumed to be adaptable to high-throughput formats. This has made the MMR slurry particularly well-suited for EV diagnostics, along with the ease and low cost of the method.

[0208] The present invention provides important guidelines for the isolation of neuron-specific EVs from biological fluids and great potential for cell-type-specific EVs 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 for other cell types in the brain and in other organs. Profiling the molecular cargo of neuron-derived EVs presents exciting opportunities for both the early detection and better understanding of brain pathologies. Since human neurons generally cannot be biopsied, reading out the molecular content of neurons by isolating neuron-derived EVs from accessible biological fluids can provide an exceptional view by understanding what is going wrong in the brain and in diseases.

[0209] Materials and Methods Computer-Aided Marker Pipeline Using brain RNA-Seq expression data (logarithmic scale) of five major cell types in the brain: neurons, astrocytes, oligodendrocytes, microglia, and endothelial cells, cell type-specific tau was calculated. For cell type-specific expression determination, when the cell type-specific tau score was 0.7 or higher, genes showing the highest expression within that cell type (neurons) compared to the other four cell types were selected. 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 related to a specific organ were averaged to obtain one organ-level measurement. For organ-level tau determination, the pituitary gland, tibial nerve, and testis were removed. For organ-specific expression determination, when the cell type-specific tau score was 0.7 or higher, genes showing the highest expression within that organ (brain) compared to other organs were selected. Uniprot accession IDs for all human proteins were filtered for those annotated as containing transmembrane domains. After determining which genes had a tau score of 0.7 for both neuron-specific expression in brain RNA-Seq and brain-specific expression in GTEx, these genes were filtered to select only those that produce transmembrane proteins, thereby determining a list of candidate EV cell type-specific markers for neurons. Subsequently, the presence of these candidates was evaluated in the proteomics dataset. The Python script for the computer-based marker pipeline is available on Github.

[0210] Cell culture and isolation of EVs from cell culture media K562 cells (from ATCC) were grown in Gibco IMDM supplemented with Glutamax (Thermo Fisher Scientific) 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 ultracentrifugation of the medium at 120,000 x g for 16 h followed by filtration through a Corning 0.22 μm filter). Previously described iNGN cells were grown on plates coated with Matrigel (Corning) in mTeSR1 medium (STEMCELL Technologies). Doxycycline (Sigma Aldrich) was diluted in PBS and added to mTeSR1 at a final concentration of 0.5 μg / mL to initiate differentiation. Four days after Dox addition, the medium was switched to Gibco DMEM containing Glutamax (Thermo Fisher Scientific) supplemented with B27 serum-free supplement (Thermo Fisher Scientific) and Gibco penicillin streptomycin (Thermo Fisher Scientific). EVs derived from neurons were collected on day 6 or 7 after Dox addition. Isolation of EVs from cell cultures was performed by fractionated 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 at 4 °C for 20 min and filtered through a 0.22 μm Steriflip filter (Millipore Sigma). The sample was then ultracentrifuged at 120,000 x g at 4 °C for 70 min, washed with PBS, and ultracentrifuged again. The pellet was then resuspended in PBS.

[0211] Isolation of EVs from plasma or CSF with MMR slurry 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. Capto Core 700 resin (Cytiva) was taken, the resin was centrifuged at 800 x g for 5 minutes, washed three times with PBS in a 50 mL Falcon tube, and resuspended to a volume equal to the resin volume in PBS to produce a 50% slurry, thereby preparing a mixed-mode chromatography resin slurry. For the isolation of CSF EVs, the samples were centrifuged at 2000 x g for 10 minutes to remove any potential residual cells. The protein concentration of the CSF was then determined using the Qubit Protein Assay Kit (ThermoFisher Scientific), and the volume of the MMR slurry corresponding to the protein content of the sample was added. The ratio of the MMR slurry was varied as specified. For the mass spectrometry experiment, the ratio of the MMR slurry used was 1 μL of slurry / 0.3 μg of protein.

[0212] The samples were mixed by inverting at room temperature for 45 minutes and then centrifuged at 800 x g for 10 minutes. 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.

[0213] Total protein staining 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 4–12% 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 overnight in the dye, washed in deionized water, and then imaged using a Gel Doc EZ Imager (BioRad).

[0214] Western blotting Western blotting of EVs has been described in detail previously (46). The iBlot2 Dry Blotting System (ThermoFisher Scientific) was used for transfer at 20 V for 3 - 7 minutes depending on the size of the protein marker. The following primary antibodies were used for Western blotting at the corresponding dilution ratios: 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% weight / volume) 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 and then incubated for 2 hours with TrueBlot HRP (Rockland) or cross-adsorbed HRP (Bethyl) secondary antibody at a concentration of 1:2000 in milk buffer and washed three times again. The blots were developed with WesternBright ECL spray (Advansta) and imaged on a Sapphire Biomolecular Imager (Azure Biosystems).

[0215] Immunoisolation of EVs from cell culture EVs Isolation buffer was prepared by adding BSA 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 8Dynabeads goat anti-mouse IgG beads (Thermo Fisher Scientific) (number of beads not specified) were placed in 2 mL and positioned on a magnetic rack. The supernatant was removed and replaced with 250 μL of isolation buffer, and the magnet was removed. 10 μg of the primary antibody was coupled to the beads at 4°C overnight with inversion rotation. The following antibodies were used for immunoisolation: 5G3 (BD) for L1CAM, 1C51 (Abcam) for mCherry, 9F9.F9 (Abcam) for GFP, 1.3.3.22 (Thermo Fisher Scientific) for CD81, and H5C6 (BD) for CD63. The next day, the beads were washed twice with 1 mL of isolation buffer each time. Then, EVs were added (usually 150 μL for one pellet), and isolation buffer was added to make the volume 0.5 mL. Immunoisolation was performed on a rotary rack either for 24 hours at 4°C for L1CAM or for 1 hour at 37°C for CD81 or CD63.

[0216] Immunoisolation of EVs from human biological fluids Immunoisolation of EVs from CSF or plasma was performed with some minor modifications similar to that from EVs in cell culture. PBS pH 7.4 was used as the isolation buffer without adding BSA. 250 μL (1x10 8Either 50 μL (1.5 mg) of Dynabeads goat anti-mouse IgG beads (Thermo Fisher Scientific) or 50 μL (1.5 mg) of Dynabeads Protein A (Thermo Fisher Scientific) was 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 10 μg of primary antibody bound to the beads in PBS pH 7.4 and inverted and rotated overnight at 4°C. The following antibodies were used for immunoisolation: mouse monoclonal CD81 (clone 1.3.3.22, ThermoFisher 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 with 1 mL of isolation buffer each time. 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 isolation to a final volume of 0.5 mL. Immunoisolation was performed on a rotating rack at 4°C for 1 hour each for CD9, CD63 and CD81 respectively.

[0217] Mass spectrometry Mass spectrometry on neuron EVs isolated from cell cultures was performed at the Broad Institute Proteomics Platform. The EVs were lysed in RIPA buffer (Thermo Fisher Scientific). The samples were then run on an SDS gel, and the band corresponding to the size of albumin was excised and discarded. The remaining samples were prepared for TMT labeling and run on a mass spectrometer. Mass spectrometry on EVs isolated from CSF or plasma using MMR slurry was performed at the Harvard Center for Proteomics.

[0218] (Example 2) Detection of NRXN3 in Human CSF and Neuron-Specific Extracellular Vesicles This example provides experimental validation of the neuron-specific EV markers identified in Example 1 above. Specifically, NRXN3, one of the markers in Table 1, was detected in EVs derived from the conditioned medium of human iPS-derived neurons, as well as in human CSF samples.

[0219] EVs were isolated from the conditioned medium of human iPS-derived neurons as described above. 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. Four days after Dox addition, the medium was switched to DMEM containing Glutamax supplemented with B27 serum-free supplement and penicillin streptomycin. EVs derived from neurons were collected on day 6 or 7 after Dox addition. Isolation of EVs from cell cultures was performed by differential ultracentrifugation. Cell culture medium (240 mL per isolation) was centrifuged at 300 xg for 10 minutes, and the supernatant was centrifuged again at 2000 xg for 10 minutes. The supernatant was centrifuged at 16,500 xg at 4°C for 20 minutes and filtered through a 0.22 μm Steriflip filter. The sample was then ultracentrifuged at 120,000 xg at 4°C for 70 minutes, washed with PBS, and ultracentrifuged again. The pellet was then resuspended in PBS.

[0220] Human CSF samples (from Brigham and Women’s Hospital) were centrifuged at 2000 xg for 10 minutes. Next, the supernatant was centrifuged at 2000 xg 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.

[0221] Briefly, Sepharose CL-6B resin was washed with PBS in a glass bottle. A certain volume of the resin was washed three times with an equal volume of PBS before use. The Econo-Pac chromatography column was filled with the resin, and a frit was inserted into the column on top of the resin. Each column was washed with 10 mL of PBS (5 mL twice) and then the sample was loaded. For the SEC column, resin was added until the bed volume (resin without liquid) reached 10 mL. When the PBS from the wash finished passing through the column, the sample (1 mL of CSF or neuronal EV) was loaded. When the sample had completely entered the column, 0.5 mL fractions were collected.

[0222] Detection of NRXN3 was performed by the Simoa assay (Figure 16A). The candidate capture antibody (Invitrogen PA5-71367) was coupled 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 twice using an 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.

[0223] As shown in Figures 16B and 16C, the NRXN3 marker was detected in the initial SEC fractions derived from EV isolated from the conditioned medium of human iPS-derived neurons, as well as in the initial SEC fractions in human CSF samples.

[0224] These data verify and confirm that the markers of the present invention are brain-specific and / or neuron-specific EV markers and that these markers are useful for isolating brain-specific and / or neuron-specific EV from human samples.

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[0226] Equivalents One of ordinary skill 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 following claims.

Claims

1. A method for isolating cell type-specific and / or organ-specific extracellular vesicles from a biological sample obtained from a subject, Isolating the cell type-specific and / or organ-specific extracellular vesicles based on the presence of a biomarker on the surface of the extracellular vesicle, the biomarker being one or more biomarkers selected from Tables 1 to 5. Methods that include...

2. The method according to claim 1, wherein the biological sample comprises a liquid biological sample, and optionally the biological sample is selected from the group consisting of whole blood, serum, plasma, cerebrospinal fluid, cerebrospinal fluid, amniotic fluid, aqueous humor, vitreous fluid, bile, breast milk, earwax (ear oil), chyle, porridge, endolymph, perilymph, exudate, feces, vaginal fluid, gastric acid, gastric juice, lymph fluid, mucus (including rhinorrhea and sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus, catarrhal secretions, saliva, sebum (skin oil), semen, saliva, synovial fluid, sweat, tears, urine, vaginal secretions, vomit, and one or more mixtures thereof.

3. (a) The extracellular vesicle is brain-specific, (b) The extracellular vesicles are neuron-specific, astrocyte-specific, oligodendrocyte-specific, and / or microglia-specific. (c) The extracellular vesicles are neuron-specific and, if necessary, The one or more biomarkers mentioned above are selected from Tables 1 and 5, or 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. (d) The extracellular vesicle is astrocyte-specific, and, if necessary, one or more biomarkers are selected from Table 2. (e) The extracellular vesicle is oligodendrocyte-specific, and, if necessary, one or more biomarkers are selected from Table 3, and / or (f) The extracellular vesicle is microglia-specific, and, if necessary, one or more biomarkers are selected from Table 4. The method according to claim 1.

4. (a) 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, fractionation centrifugation, sucrose density gradient, organelle electrophoresis, magnetically activated cell sorting (MACS), or nanomembrane ultrafiltration concentrator, and / or (b) The immunoisolation includes isolation based on microfluidic affinity, isolation based on magnetism, pull-down isolation, or isolation based on fluorescence-activated sorting. The method according to claim 1.

5. A method for isolating brain-specific extracellular vesicles from biological samples obtained from a subject, Isolating the extracellular vesicles from the sample based on the presence of a biomarker on the surface of the extracellular vesicles, the biomarker being one or more biomarkers selected from Tables 1 to 5. Methods that include...

6. The method according to claim 5, wherein the biological sample comprises a liquid biological sample, and optionally the biological sample is selected from the group consisting of whole blood, serum, plasma, cerebrospinal fluid, cerebrospinal fluid, amniotic fluid, aqueous humor, vitreous fluid, bile, breast milk, earwax (ear oil), chyle, porridge, endolymph, perilymph, exudate, feces, vaginal fluid, gastric acid, gastric juice, lymph fluid, mucus (including rhinorrhea and sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus, catarrhal secretions, saliva, sebum (skin oil), semen, saliva, synovial fluid, sweat, tears, urine, vaginal secretions, vomit, and one or more mixtures thereof.

7. (a) The extracellular vesicles are neuron-specific, astrocyte-specific, oligodendrocyte-specific, and / or microglia-specific. (b) The extracellular vesicles are neuron-specific and, if necessary, The one or more biomarkers mentioned above are selected from Tables 1 and 5, or 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. (c) The extracellular vesicle is astrocyte-specific, and, if necessary, one or more biomarkers are selected from Table 2. (d) The extracellular vesicle is oligodendrocyte-specific, and, if necessary, one or more biomarkers are selected from Table 3, and / or (e) The extracellular vesicle is microglia-specific, and, if necessary, one or more biomarkers are selected from Table 4. The method according to claim 5.

8. (a) The brain-specific EV is isolated by immunoisolation, mixed-mode chromatography, size exclusion chromatography, cation exchange chromatography, anion exchange chromatography, gel permeation chromatography, fractionation centrifugation, sucrose density gradient, organelle electrophoresis, magnetically activated cell sorting (MACS), or nanomembrane ultrafiltration concentrator, and / or (b) The immunoisolation includes isolation based on microfluidic affinity, isolation based on magnetism, pull-down isolation, or isolation based on fluorescence-activated sorting. The method according to claim 5.

9. A method for identifying extracellular vesicles originating from brain cells, A method comprising determining the presence or absence of a biomarker on the surface of an extracellular vesicle obtained from a biological sample containing the extracellular vesicle, wherein the biomarker comprises one or more biomarkers selected from Tables 1 to 5, and the presence of the biomarker indicates that the extracellular vesicle originates from brain cells.

10. The method according to claim 9, wherein the brain cells are neurons, and optionally the biomarker comprises one or more biomarkers selected from Tables 1 and 5, or 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, and the presence of the biomarker indicates that the extracellular vesicles originate from neurons.

11. The method according to claim 9, wherein the brain cells are astrocytes, and optionally the biomarker comprises one or more biomarkers selected from Table 2, and the presence of the biomarker indicates that the extracellular vesicles originate from astrocytes.

12. The method according to claim 9, wherein the brain cells are oligodendrocytes, and optionally the biomarker comprises one or more biomarkers selected from Table 3, and the presence of the biomarker indicates that the extracellular vesicles originate from oligodendrocytes.

13. The method according to claim 9, wherein the brain cells are microglia cells, and optionally the biomarker comprises one or more biomarkers selected from Table 4, and the presence of the biomarker indicates that the extracellular vesicles originate from microglia cells.

14. (a) The biological sample comprises a liquid biological sample, and optionally the biological sample is selected from the group consisting of whole blood, serum, plasma, cerebrospinal fluid, cerebrospinal fluid, amniotic fluid, aqueous humor, vitreous fluid, bile, breast milk, earwax (ear oil), chyle, porridge, endolymph, perilymph, exudate, feces, vaginal fluid, gastric acid, gastric juice, lymph fluid, mucus (including rhinorrhea and sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus, catarrhal secretions, saliva, sebum (skin oil), semen, saliva, synovial fluid, sweat, tears, urine, vaginal secretions, vomit, and / or mixtures thereof of one or more of these, and / or (b) The biological sample is obtained from the subject and / or (c) 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 profiling, mass spectrometry, immunoassay, ELISA, fluorescence-activated cell sorting (FACS), SDS-polyacrylamide gel electrophoresis (SDS-PAGE), or Western blotting analysis. The method according to claim 9.

15. A method for extracting proteins and / or nucleic acids as an indicator of the risk of a subject developing neurodegenerative disorders, (a) Isolating brain-specific extracellular vesicles from a biological sample obtained from the subject based on the presence of a biomarker containing one or more biomarkers selected from Tables 1 to 5 in the isolated extracellular vesicles; and (b) Extracting proteins and / or nucleic acids from the isolated brain-specific extracellular vesicles. Includes, The extracted proteins and / or nucleic acids from the isolated brain-specific extracellular vesicles indicate that the subject is at risk of developing the neurodegenerative disorder. method.

16. The method according to claim 15, wherein the biological sample comprises a liquid biological sample, and optionally the biological sample is selected from the group consisting of whole blood, serum, plasma, cerebrospinal fluid, cerebrospinal fluid, amniotic fluid, aqueous humor, vitreous fluid, bile, breast milk, earwax (ear oil), chyle, porridge, endolymph, perilymph, exudate, feces, vaginal fluid, gastric acid, gastric juice, lymph fluid, mucus (including rhinorrhea and sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus, catarrhal secretions, saliva, sebum (skin oil), semen, saliva, synovial fluid, sweat, tears, urine, vaginal secretions, vomit, and one or more mixtures thereof.

17. (a) The extracellular vesicle is neuron-specific, and, if necessary, one or more biomarkers are selected from Tables 1 and 5, 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. (b) The extracellular vesicle is astrocyte-specific, and, if necessary, one or more biomarkers are selected from Table 2. (c) The extracellular vesicle is oligodendrocyte-specific, and, if necessary, one or more biomarkers are selected from Table 3, and / or (d) The extracellular vesicle is microglia-specific, and, if necessary, one or more biomarkers are selected from Table 4. The method according to claim 15.

18. (a) The extracted nucleic acid comprises messenger RNA (mRNA), microRNA (miRNA), long non-coding RNA (lncRNA), small non-coding RNA, DNA, and any other full length or fragment of RNA or DNA, (b) The extracted nucleic acids from the isolated brain-specific extracellular vesicles are analyzed using RNA sequencing (RNA seq), DNA sequencing, array analysis, reverse transcription polymerase chain reaction (RT-PCR), or quantitative reverse transcription polymerase chain reaction (qRT-PCR). (c) The extracted nucleic acids from the isolated brain-specific extracellular vesicles are analyzed using genome-wide analysis or transcriptome profiling. (d) The extracted nucleic acids from the isolated brain-specific extracellular vesicles are analyzed by analyzing the gene of interest associated with the neurodegenerative disorder and / or testing for the presence or absence of the gene of interest, analyzing one or more allele variants or mutations of the gene of interest, and testing for the presence or absence of the allele variants or mutations. (e) The extracted proteins from the isolated brain-specific extracellular vesicles are analyzed using proteomic profiling, mass spectrometry, immunoassay, ELISA, fluorescence-activated cell sorting (FACS), SDS-polyacrylamide gel electrophoresis (SDS-PAGE), or Western blotting. (f) The extracted proteins from the isolated brain-specific extracellular vesicles are analyzed by analyzing the protein of interest associated with the neurodegenerative disorder and / or testing for the presence or absence of the protein of interest, analyzing for one or more mutations in the protein of interest, testing for the presence or absence of the mutations, and / or (g) 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), Lewy body dementia, neurofibrillary tangle-dominant senile dementia, Pick's disease (PiD), argyrophilic granule disease, amyotrophic lateral sclerosis (ALS), other motor neuron diseases, Guam Parkinson's dementia complex, FTDP-17, Ritico-Bodig disease, multiple sclerosis, traumatic brain injury (TBI), and Parkinson's disease. The method according to claim 15.

19. A kit for isolating brain-specific extracellular vesicles from a target, (a) One or more reagents for detecting the presence of a biomarker on the surface of the extracellular vesicle, comprising one or more biomarkers selected from Tables 1 to 5; (b) means for isolating the brain-specific extracellular vesicles based on the presence of the biomarker; and (c) A set of instructions for use to detect the presence of the biomarker and / or to isolate the brain-specific extracellular vesicles. A kit that includes this.

20. A kit for detecting neurodegenerative disorders in subjects, (a) One or more reagents for detecting the presence of a biomarker on the surface of an extracellular vesicle, comprising one or more biomarkers selected from Tables 1 to 5; (b) means for isolating brain-specific extracellular vesicles based on the presence of the biomarker; (c) one or more reagents for detecting the level of genes associated with the neurodegenerative disorder in the isolated brain-specific extracellular vesicles; and (d) A set of instructions for use to detect the presence of the biomarker, to isolate the brain-specific extracellular vesicles, and / or to detect the levels of the gene associated with the neurodegenerative disorder. A kit that includes this.

21. The kit according to claim 19 or 20, further comprising means for isolating a biological sample from the subject.

22. The kit according to claim 19 or 20, wherein one or more reagents for detecting the presence of the biomarker on the surface of the extracellular vesicle is an antibody or aptamer that binds to the biomarker.

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