Compositions, methods and uses for exosome-associated biomarkers for early diagnosis and treatment of health conditions

Abstract

Embodiments of the instant disclosure relate to diagnosis and / or early diagnosis, intervention and / or treatment of Alzheimer's disease (AD). Certain embodiments relate to methods for identifying and isolating / analyzing an enriched population of neuronal, microglial and / or astrocytic exosomes from a sample of a subject and detecting biomarkers of interest on one or more exosomes in the enriched population to diagnose a neurodegenerative condition, Alzheimer's disease (AD), an AD-related dementia / condition, Down Syndrome (DS) or the like at an earlier stage than afforded by current therapies using minimally invasive technologies at reduced costs in time and expenses. Other embodiments relate to assessing interventions and evaluating treatment regimens for neurodegenerative disorders using approaches disclosed herein.

Description

COMPOSITIONS, METHODS AND USES FOR EXOSOME-ASSOCIATED BIOMARKERS FOR EARLY DIAGNOSIS AND TREATMENT OF HEALTH CONDITIONS PRIORITY

[0001] This International Application claims priority to U.S. Provisional Application No. 63 / 529,259 filed July 27, 2023. This provisional application, appendices and figures are incorporated herein by reference in their entireties for all purposes. FIELD

[0002] Embodiments of the instant disclosure relate to minimally invasive methods and uses for diagnosis (e.g., early) and intervention and / or treatment of health conditions. Certain embodiments relate to analyzing makers on plasma-derived exosomes from a subject that correlate with health-related condition risk, onset, progression, and assessing amelioration due to therapeutic intervention in the subject. In certain embodiments, the health condition can include, but is not limited to, Alzheimer’s Disease (AD) or other neurodegenerative disorder. BACKGROUND

[0003] An estimated 6.5 million Americans are living with Alzheimer’s Disease (AD), a number that is expected to more than double by 2050. AD is the fifth leading cause of death among adults aged 65 and older, and the seventh leading cause of death among all adults. In 2021, the U.S. spent over $355 billion on AD / dementia, while 11 million Americans provided billions of hours of unpaid care for those affected. And yet, there remains no cure, and much information is still needed for us to more fully understand how AD pathology is initiated and propagated in a manner that results in neurodegeneration, memory loss and death. To reduce onset or progression of AD in humans and provide more sensitive and selective methods for early diagnosis are needed. SUMMARY

[0004] Embodiments of the instant disclosure relate to minimally invasive cost-effective methods for diagnosing risk, onset, therapeutic responsiveness, or progression of a health condition. In certain embodiments, methods can include (a) obtaining an enriched population of exosomes from the subject; (b) contacting the enriched population of exosomes with an antibody for a targeted marker to detect marker specific exosomes (e.g., an amyloid protein or peptide to detect amyloid positive neuronal exosomes); (c) diagnosing risk, onset, progression 1 96555783.1or therapeutic responsiveness of the health condition (e.g., Alzheimer’s disease (AD) or an Alzheimer’s disease-related condition) in the subject if marker-positive neuronal, astrocytic or microglial exosomes (e.g., amyloid positive neuronal exosomes) are detected. In certain embodiments, the subject having been diagnosed with early indications of the health condition (e.g., AD) can be monitored and / or treated to reduce risk, onset, or progression of the health condition (e.g., AD) in the subject.

[0005] In certain embodiments and further to paragraph

[0004] above, health conditions contemplated herein can include, but are not limited to, AD, AD-related condition, mild cognitive impairment (MCI), Down Syndrome (DS), other neurodegenerative disorders, combinations thereof, and the like. In certain embodiments, neurodegenerative health conditions can be diagnosed and / or assessed for treatment efficacy using compositions and methods disclosed herein. In some embodiments, the health conditions include, but are not limited to, Alzheimer’s disease (AD), an Alzheimer’s disease-related condition, Alzheimer’s disease related dementia (ADRD) condition, or Down Syndrome (DS) in a subject.

[0006] In some embodiments and further to paragraphs

[0004] -

[0005] above, the enriched population of exosomes can include, but is not limited to, neuronal, astrocytic, or microglial exosomes derived from a population of total exosomes purified from cells of a plasma sample originating from various cell types obtained from the subject to be analyzed. In certain embodiments, the cells that generated exosomes found in plasma are neuronal cells and neuronal exosomes can be isolated from total exosomes for further marker analysis and correlation with risk of onset, onset, presence, progression and or amelioration of a health condition (e.g., AD or AD-related condition).

[0007] In some embodiments and further to paragraphs

[0004] -

[0006] above, markers to be detected on enriched exosome populations disclosed herein can include, but are not limited to, one or more cluster of differentiation (CD) markers. In certain embodiments, at least one exosome in an enriched population of neuronal exosomes can be positive for CD63, CD81, and / or CD9 and / or IgG signatures. For example, in some embodiments, at least one exosome in the enriched population of neuronal exosomes can be positive for CD81 when used for assessing risk of onset, onset, presence, progression and / or amelioration due to treatment intervention of AD or AD-related condition (e.g., mild cognitive impairment (MCI)). 2 96555783.1

[0008] In some embodiments and further to paragraphs

[0004] -

[0007] above, embodiments of the instant disclosure relate to methods for diagnosing risk of onset, onset, presence, amelioration, or progression of Alzheimer’s disease (AD) or an Alzheimer’s disease-related condition or other condition in a subject, the method including, but not limited to: (a) isolating a population of exosomes derived from a plasma sample obtained from the subject suspected to be at risk for having or developing Alzheimer’s disease or an Alzheimer’s Disease related condition or other health condition, (b) contacting the population of plasma exosomes with an immobilized antibody to a neuronal, microglial, or astrocyte specific marker to obtain an enriched population of neuronal, microglial, and / or astrocytic exosomes, (c) analyzing the population of neuronal, microglial, or astrocytic enriched exosomes for the presence of exosomes expressing or containing one or more additional biomarkers associated with developing Alzheimer’s Disease or an Alzheimer’s Disease related condition or other condition; and (d) determining whether the subject is at risk for onset, onset, presence, progression or amelioration of Alzheimer’s Disease or an Alzheimer’s Disease related dementia or other condition if an alteration in exosomes expressing the one or more additional biomarkers is observed relative to a neuronal, microglial, and / or astrocyte enriched exosome population obtained from control (e.g., a healthy individual or other control population).

[0009] In other embodiments and further to paragraphs

[0004] -

[0008] above, the enriched neuronal, microglial and / or astrocyte exosome populations of CD81+, CD9+ and / or CD63+ and / or IgG exosomes can further include, but is not limited to, another biomarker specific for diagnosing health condition status such as that of AD or AD-related condition. In accordance with these embodiments, additional biomarkers can include, but are not limited to, NCAM, ATP1A3, pTau181, NfL, GFAP, UCHL1, SSC5D, Emilin, VDAC, MCT4 or the like or a combination thereof. In further embodiments, neuronal enriched exosome population specific markers can include NCAM. As disclosed herein, NCAM can be used as a specific neuronal exosome marker to enrich for neuronal exosomes.

[0010] In certain embodiments and further to the paragraphs above, one or more biomarkers can include or can further include one or more of, Aβ, amyloid, other AD peptide or molecule and any combination thereof. For example, in some embodiments, the one or more biomarkers can include CD81 and an amyloid protein or peptide. In another embodiment, the one or more biomarkers can include Aβ or amyloid. 3 96555783.1

[0011] In certain embodiments and further to paragraphs

[0004] -

[0010] above, the methods described herein can further include administering a treatment to a subject diagnosed with increased risk of onset, onset, presence, or progression of Alzheimer’s disease (AD) or an Alzheimer’s disease-related condition. In some embodiments, a treatment can include treating the subject with granulocyte-macrophage colony-stimulating factor (GM-CSF) or the like, or other AD treatment known in the art.

[0012] In other embodiments and further to paragraphs

[0004] -

[0011] above, methods for identifying a neuronal, microglial, and / or astrocyte specific marker for use in preparing an enriched population of neuronal, microglial, and / or astrocytic exosomes is disclosed. In accordance with these embodiments, these methods can include, but are not limited to, identifying and / or detecting proteins and / or peptides and / or other molecules in exosomes isolated from neuronal, microglial, and / or astrocytic cell cultures in vitro, where at least one of the proteins and / or peptides and / or molecules identified or detected is a neuronal, microglial and / or astrocytic specific marker for an enriched population of these exosomes.

[0013] In certain embodiments and further to paragraphs

[0004] -

[0012] above, methods disclosed herein can include identifying and / or detecting one or more proteins or peptides or other molecules in or on the surface of exosomes using mass spectrometry or other method known in the art to derive a proteome of enriched neuronal, microglial, and / or astrocytic exosomes. In some embodiments, methods disclosed herein can further include use of R analysis of the proteome and / or bioinformatic analysis of the proteome identified by mass spec analysis or similar analysis. In other embodiments, methods disclosed herein further include identifying molecules (e.g., proteins or peptides) expressed on a surface of an exosome, where the surface expressed molecule is a signature of a neuronal, microglial, and / or astrocyte exosome making the expressed molecule specific for identifying a sought-after exosome population for correlative analysis of health conditions disclosed herein.

[0014] Some embodiments of the instant disclosure and further to paragraphs

[0004] -

[0013] above relate to methods of preparing an enriched population of neuronal, microglial, and / or astrocytic exosomes, the method including, but not limited to, (a) contacting a population of exosomes derived from a sample obtained from a subject with an antibody immobilized on a structure, the antibody having affinity for a neuronal, microglial, and / or astrocyte specific marker identified according to a method described herein, (b) allowing exosomes to bind to the 4 96555783.1immobilized antibody, (c) eluting bound exosomes from the immobilized antibody to form an enriched population of neuronal, microglial, and / or astrocytic exosomes. In certain embodiments, the neuronal and / or astrocytic specific marker can include NCAM, ATP1A3, SSC5D, Emilin, pTau181, NfL, GFAP, UCHL1, VDAC, MCT4, Iba1, CD63, CD81, and / or CD9 and / or IgG and the like.

[0015] Certain embodiments of the instant disclosure and further to paragraphs

[0004] -

[0014] above relate to compositions including, but not limited to, isolated neuronal, microglial, and / or astrocytic exosomes. In certain embodiments, the isolated neuronal, microglial, and / or astrocytic exosomes are obtained by methods including: (a) isolating or enriching for specified exosomes from a particular cell type or a plasma sample obtained from a subject, (b) harvesting the exosomes isolated in (a) with an antibody to a neuronal, microglial, and / or astrocyte specific marker, where the antibody is immobilized to a support; and (c) eluting a bound complex of antibody-exosomes to collect the neuronal, microglial, and / or astrocyte specific exosomes. In some embodiments, a kit can be used for creating such a population. In other embodiments, harvested / isolated / enriched populations of exosomes from a subject suspected of having or at risk of developing a neurodegenerative disorder can be compared to a baseline control sample or a control harvested / isolated / enriched populations of exosomes from a control subject not having or suspected of developing a neurodegenerative condition. In certain embodiments, compositions disclosed herein can further include a medium, or a pharmaceutically appropriate carrier or excipient. In certain embodiments, the compositions herein further include at least one additional agent. In some embodiments, the compositions can further include a media. In certain embodiments, the enriched populations of exosomes disclosed herein can further be subjected to a lysing buffer which can further include another antibody to a different biomarker specific to the desired exosome subtype (e.g., neuronal exosomes). In some embodiments, enriched populations of exosomes disclosed herein can be further analyzed for specific cargo within the enriched population to further assess treatment regimens, differences in approaches for treatment based on subject population such as sex of the subject as well as create more personalized treatment and evaluation approaches (e.g., annual or more frequent analysis of a subject using these methods to assess progression, etc.) BRIEF DESCRIPTION OF THE DRAWINGS 5 96555783.1

[0016] The following drawings form part of the present specification and are included to further demonstrate certain embodiments of the present disclosure. Certain embodiments can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0017] FIG.1 represents an exemplary plot indicating particles per milliliter of neuronal specific exosomes obtained from control (PET-ve) and mild cognitively impaired (MCI) (PET+ve) individuals positive for exemplary markers according to certain embodiments of the present disclosure.

[0018] FIG.2 represents an exemplary schematic for isolating subtype specific exosomes from a sample after initial enrichment of exosomes from the sample, according to certain embodiments of the present disclosure.

[0019] FIG.3 is a schematic diagram depicting number of novel molecules identified for astrocytes, microglia, and neurons according to certain embodiments of the present disclosure.

[0020] FIG.4 represents an exemplary bar graph comparing biomarker positive exosomes positive for various CD biomarkers of verified samples containing health condition specific marker (e.g., amyloid) according to certain embodiments of the present disclosure.

[0021] FIGS.5A-5C represent in (5A) an exemplary bar graph comparing levels of a neuronal exosome specific biomarker positive exosomes positive for various CD biomarkers; in (5B) an exemplary bar graph comparing levels of a neuronal exosome specific biomarker positive exosomes positive for various CD biomarkers and further level of an AD-specific marker in the various CD-containing biomarker exosome populations; and in (5C) a plot representing neuronal exosome biomarker expressing populations as they correlate to validated health states of subjects (e.g., control, MCI and AD subject samples) where CD-specific populations are compared for correlation according to certain embodiments of the present disclosure.

[0022] FIG.6 represents a plot representing neuronal exosome biomarker expressing populations as they correlate to validated health states of male versus female subjects (e.g., control, MCI, and AD subject samples) where male versus female subjects are compared to identify differences according to sex of the subject according to certain embodiments of the present disclosure.

[0023] FIG.7 represents a plot representing neuronal exosome biomarker expressing populations as they correlate to validated health states and response to treatment (e.g., End of 6 96555783.1Treatment (EOT)) compared to baseline levels and controls in the presence of an amyloid specific antibody according to certain embodiments of the present disclosure.

[0024] FIG.8 represents a plot representing neuronal exosome biomarker expressing populations as they correlate to validated health states of male versus female subjects (e.g., control, MCI, and AD subject samples) in baseline versus treated subjects (e.g., GM-CSF) where male versus female subjects are compared to identify differences according to sex if present in the subject according to certain embodiments of the present disclosure.

[0025] FIG.9 illustrates an exemplary ROC curve for modeling sensitivity versus specificity of the observed correlations disclosed herein according to certain embodiments of the present disclosure.

[0026] FIG.10 represents an exemplary plot comparing biomarker positive neuronal exosomes positive for various CD biomarkers and a neuronal specific biomarker and further another marker able to distinguish disease state of a subject earlier than standard methods of samples from subjects; baseline and treatment analysis were performed to assess outcome according to certain embodiments of the present disclosure.

[0027] FIG.11 represents an exemplary plot comparing biomarker positive neuronal exosomes positive for various CD biomarkers and a neuronal specific biomarker and further another marker able to distinguish disease state of a subject earlier than standard methods of samples from subjects according to certain embodiments of the present disclosure.

[0028] FIG.12 represents an exemplary plot comparing biomarker positive neuronal exosomes positive for various CD biomarkers and a neuronal specific biomarker and further another marker able different than FIG.11 above, to distinguish disease state of a subject earlier than standard methods of samples from subjects according to certain embodiments of the present disclosure.

[0029] FIG.13 represents an exemplary plot comparing biomarker positive neuronal exosomes positive for various CD biomarkers and a neuronal specific biomarker and further another marker able different than FIG.11 or FIG.12 above, to distinguish disease state of a subject earlier than standard methods of samples from subjects according to certain embodiments of the present disclosure.

[0030] FIG.14 represents an exemplary plot comparing biomarker positive neuronal exosomes positive for various CD biomarkers and a neuronal specific biomarker and further another 7 96555783.1marker able different than FIG.11 or FIG.12 or FIG.13 above, to distinguish disease state of a subject earlier than standard methods of samples from subjects according to certain embodiments of the present disclosure.

[0031] FIG.15 represents an exemplary plot comparing biomarker positive neuronal exosomes positive for various CD biomarkers and a neuronal specific biomarker and further another marker able different than FIG.11 or FIG.12 above but the same marker as FIG.13 above, to distinguish disease state of a subject earlier than standard methods of samples from subjects as well as analyzing baseline and treated samples for modifications in exosome particle trends according to certain embodiments of the present disclosure.

[0032] FIG.16 illustrates comparison of disease-diagnosed samples from control samples in standard method measured (PET scan) samples versus exosome analysis disclosed according to certain embodiments of the present disclosure.

[0033] FIG.17 represents an exemplary bar graph comparing levels of a neuronal exosome specific biomarker positive exosomes positive for various CD biomarkers with astrocyte exosomes demonstrating specificity of the neuronal exosome biomarker for neuronal exosomes and not astrocytic exosomes according to certain embodiments of the present disclosure.

[0034] FIG.18 represents an exemplary bar graph comparing levels of an astrocytic exosome specific biomarker positive exosomes positive for various CD biomarkers with astrocytic exosomes demonstrating specificity of the astrocytic exosome biomarker for astrocyte-derived exosomes and not neuronal exosomes according to certain embodiments of the present disclosure.

[0035] FIG.19 represents an exemplary bar graph comparing levels of an astrocytic exosome specific biomarker different than the biomarker in FIG.18 above where positive astrocytic exosomes positive for various CD biomarkers demonstrate specificity of the astrocytic exosome biomarker for astrocyte-derived exosomes and not neuronal exosomes according to certain embodiments of the present disclosure.

[0036] FIG.20 represents an exemplary plot comparing biomarker positive microglial exosomes positive for various CD biomarkers and a second microglial exosome specific biomarker increased over time of differentiation of macrophage to microglia according to certain embodiments of the present disclosure. 8 96555783.1

[0037] FIGS.21A-21B represents in 21A an exemplary plot comparing biomarker positive microglial exosomes positive for various CD biomarkers and a second microglial exosome specific biomarker different than the analyzed biomarker in FIG.20 above increased over time of differentiation of macrophage to microglia; and 21B represents an exemplary plot of a microglial marker used (MCT4 (microglial-affiliated biomarker)) to immunoprecipitate exosomes from total exosomes isolated from plasma samples of subjects and a representative Exoview analyses of these exosomes having enrichment of CD9+ exosomes stained for a second microglial marker according to certain embodiments of the present disclosure. DEFINITIONS

[0038] Terms, unless defined herein, have meanings as commonly understood by a person of ordinary skill in the art relevant to certain embodiments disclosed herein or as applicable.

[0039] Unless otherwise indicated, all numbers expressing quantities of agents and / or compounds, properties such as molecular weights, reaction conditions, and as disclosed herein are contemplated as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters in the specification and claims are approximations that can vary from about 10% to about 15% plus and / or minus depending upon the desired properties sought as disclosed herein. Numerical values as represented herein inherently contain standard deviations that necessarily result from the errors found in the numerical value's testing measurements.

[0040] As used herein, “individual”, “subject”, “host”, and “patient” can be used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, prophylaxis or therapy is desired, for example, humans, pets, livestock, horses or other animals.

[0041] As used herein, “treat,” “treating” or “treatment” can refer to treating, reversing, ameliorating, or inhibiting onset or inhibiting progression of a health condition or disease or a symptom of the health condition or disease. DETAILED DESCRIPTION OF THE INVENTION

[0042] In the following sections, certain exemplary compositions and methods are described in order to detail certain embodiments of the invention. It will be obvious to one skilled in the art that practicing the certain embodiments does not require the employment of all or even some of the specific details outlined herein, but rather that concentrations, times and other specific 9 96555783.1details can be modified through routine experimentation. In some cases, well known methods, or components have not been included in the description.

[0043] To better understand AD in terms of its causes, progression and response to interventions, more sensitive and selective analysis methods are needed such as biomarkers. While cerebral spinal fluid (CSF; A ^42 / A ^40 ratios) and Amyloid / Tau PET imaging, in research settings, are considered gold standards for determining who with dementia is likely to progress to AD, these approaches are invasive, time-consuming, and costly. An additional challenge is that pathological changes related to AD begin up to 20 years before symptoms are evident, often leaving the medical community with an impossible task of reversing years of pathology at first diagnosis. While Amyloid and Tau PET imaging and CSF Biomarkers could provide earlier identifiers of high-risk individuals, their routine use is neither practical nor affordable in an already taxed healthcare system. Their use in clinical trials that seek to test preventative interventions to stave off AD neuropathological development and associated cognitive impairment also selects for organizations with large funding to use these expensive assessments, preventing research that reflects a more diverse community when smaller research centers do not have access to these types of tools. Therefore, plasma based, specific, sensitive, and selective biomarker measures that allow detection of AD, AD-related dementia / conditions and other neurodegenerative disorders in their earliest stages are essential to improve both understanding of the conditions themselves and the ability to determine the efficacy of interventions as they are developed.

[0044] As disclosed herein, exosomes are a targeted population for use in earlier and more affordable, less invasive diagnosis and analysis of neurodegenerative disorders contemplated herein. Exosomes are 50-150 nm diameter vesicles, secreted from every cell in the body and present in the plasma in large quantities (approximately 1011particles per ml). Exosomes carry nucleic acids, proteins, lipids, and metabolites, the composition of which changes dynamically reflecting the state of living CNS cells and serving as more accurate read-outs of cellular pathological changes in the CNS, a useful property for a tool being utilized to assess disease onset, amelioration, and trajectory. As contemplated herein, while exosomes play important roles in the communication and health of the Central Nervous system (CNS), they are also thought to be co- opted in diseases, such as AD and other neurodegenerative disorders, to spread pathology, reflecting the health and / or disease state of the cell(s) of origin in a more dynamic and real time 10 96555783.1capacity. As demonstrated herein, control and Mild Cognitively Impaired (MCI) subjects from one study demonstrated detection capabilities that are on par with PET imaging to detect neurodegenerative disorder risk of onset, onset, presence, progression and amelioration in subjects, making testing for neurodegenerative disorders more affordable and accessible to a larger community to establish going forward health condition “cut-offs” for when the condition begins and progresses using larger populations for analysis, determine responses to interventions in a more dynamic and feasible manner, facilitating the design of more efficacious and safe therapeutic interventions. Compositions and methods disclosed herein relate to sample collection less invasive than CSF collection (lumbar puncture) or PET imaging, that facilitate earlier and less invasive detection of health conditions such as analysis of plasma samples obtained from a subject as described below.

[0045] In accordance with embodiments disclosed herein and further to paragraphs

[0043] -

[0044] above, enriched exosome populations having specific biomarkers can be used as personalized medicine tools and / or to define cut-offs for neurodegenerative disorder stage / severity (e.g., AD and other conditions) as well as onset of the health condition, important to effectively determine therapeutics and diagnose disease more efficiently and accurately. Currently imaging tools such as PET and Cerebrospinal fluid (CSF) measures are expensive and invasive and increase the health disparity given these tools are not amenable to all. The efficacy of interventions will be greatly enhanced if early intervention is possible but first, it can be helpful to visualize disease in its earliest stages, so the timing of intervention can be determined. Biomarkers on subsets of neuronal, astrocytic and / or microglial exosomes (e.g., CD81, CD9, CD63 tetraspanin containing and IgG signatures) as well as containing at least a second marker (e.g., NCAM, ATP1A3, pTau181, NfL, GFAP, UCHL1, SSC5D, Emilin, VDAC, MCT4) are distinguishing biomarkers for diagnosis and analysis presented herein.

[0046] Embodiments of the instant disclosure and further to paragraphs

[0043] -

[0045] above relate to novel biomarkers, compositions, and methods for assessing risk and / or early onset of Alzheimer’s Disease (AD), Alzheimer’s disease related dementia (ADRD), other AD- related condition, Down Syndrome, or other neurodegenerative health condition in a subject. In some embodiments, methods for assessing risk of onset, onset, presence, progression, and / or amelioration due to intervention for Alzheimer’s Disease (AD), Alzheimer’s disease related dementia (ADRD), other AD-related condition, MCI, Down Syndrome, or other 11 96555783.1neurodegenerative health condition is provided. In certain embodiments, methods can include, but are not limited to, obtaining an enriched population of exosomes from a subject to be analyzed; contacting the enriched population of exosomes with an exosome specific antibody (e.g., bound to a substrate); enriching for the population of exosomes by eluting bound exosomes; contacting the enriched population of exosomes with a neurodegenerative specific biomarker antibody (e.g., amyloid protein or peptide to detect amyloid positive neuronal exosomes); and (c) diagnosing onset, amelioration, presence, or progression of Alzheimer’s Disease (AD), Alzheimer’s disease related dementia (ADRD), other AD-related condition, Downs Syndrome, or other neurodegenerative health condition in the subject by comparing the isolated antibody reactive enriched exosome population with a baseline and / or control population of exosomes. In some embodiments, enriched neuronal, astrocytic and / or microglial- specific exosome populations can be further analyzed for cargo carried by the exosomes in order to assess a health condition, novel biomarkers of disease and / or response to interventions in a sex dependent manner in a subject. Biomarkers identified herein can be used to specifically isolate key populations of exosomes for a given neurodegenerative condition assessment in a subject for earlier intervention for example, as well as assessing efficacy of treatment by analyzing changes in these distinct exosome populations due to various interventions providing a relatively non-invasive method to rapidly assess treatment regimens and revise treatment regimens as needed in a reduced amount of time and at a much lower expense than current methods.

[0047] In certain embodiments and further to paragraphs

[0043] -

[0046] above, at least one exosome in the enriched population of exosomes can be neuronal, astrocytic and / or microglial exosomes. In some embodiments, at least one exosome type in the enriched population of exosomes can be neuronal exosomes positive for NCAM and / or ATP1A3 (e.g., exemplary neuronal specific markers). In certain embodiments, at least one exosome in the enriched population of neuronal exosomes can be positive for NCAM. In certain embodiments, at least one exosome in the enriched population of neuronal exosomes can be positive for CD63, CD81, and / or CD9. In certain embodiments, at least one exosome in the enriched population of neuronal exosomes can be positive for CD81. In other embodiments, exosomes in the enriched population of exosomes can be double-positive for CD81 or CD9; and NCAM or ATP1A3. In accordance with these embodiments, these subtypes of neuronal exosomes can be isolated from 12 96555783.1a subject to be analyzed and compared to a baseline or control subject in order to assess a neurodegenerative condition of the subject to be analyzed. In some embodiments, these enriched neuronal cell populations can be further assessed for presence of a neurodegenerative specific biomarker (e.g., amyloid or the like).

[0048] In certain embodiments and further to paragraphs

[0043] -

[0047] above, a neurodegenerative specific biomarker can include any biomarker specific to the neurodegenerative disorder carried by a subtype of exosomes. In some embodiments, an antibody can be used to detect these biomarkers. In accordance with these embodiments, an antibody for an AD-specific biomarker can be used to analyze enriched populations of exosomes for assessment of presence of the AD-specific biomarker or biomarkers. In some embodiments, these are commercially available. For example, for detecting an amyloid protein or peptide a polyclonal or monoclonal antibody can be used. In certain embodiments, monoclonal antibody 6E10 can be used as this is commercially available. It is contemplated that any specific antibody to a biomarker of a neurodegenerative disorder can be used to further classify exosomes of use in methods disclosed herein.

[0049] In some embodiments and further to paragraphs

[0043] -

[0048] above, a method for assessing risk for Alzheimer’s Disease or an Alzheimer’s disease related condition is provided, the method including isolating a population of exosomes derived from a plasma sample obtained from a subject suspected to be at risk for developing Alzheimer’s Disease (AD), Alzheimer’s disease related dementia (ADRD), other AD-related condition, MCI, Down Syndrome, or other neurodegenerative health condition, (b) contacting the population of plasma exosomes with an immobilized antibody to a neuronal, microglial, or astrocyte specific marker to obtain a population of neuronal, microglial, and / or astrocytic exosomes, (c) analyzing the population of neuronal, microglial and / or astrocytic exosomes for the presence of exosomes expressing or containing one or more biomarkers associated with developing Alzheimer’s Disease or an Alzheimer’s Disease related condition, and (d) determining that the subject is at risk for developing Alzheimer’s Disease or an Alzheimer’s Disease related condition if an elevated number of exosomes expressing the one or more biomarkers is observed relative to a neuronal or astrocyte exosome population obtained from a healthy individual.

[0050] Further embodiments of the disclosure herein relate to methods of identifying novel biomarkers associated with risk for developing a neuronal disease or condition. In certain 13 96555783.1embodiments, these methods include identifying a disease-specific neuronal, microglial, and / or astrocyte specific marker that can be used, in certain embodiments, to isolate a neuronal or astrocytic specific population of exosomes for further analysis of a health condition in a subject when compared to a control and / or baseline sample of isolated exosomes, for example, with the same biomarker make-up as the tested subject sample. In certain embodiments, method of identifying a disease-specific neuronal, microglial and / or astrocyte specific marker can include culturing neurons, macrophages and / or astrocytes in vitro and then analyzing over a pre- determined period, exosomes from the diseased versus healthy disease-specific or cultured neurons, macrophages and / or astrocytes using mass spectrometry. For example, in some embodiments, exosomes in the neuronal, microglial and / or astrocyte cultures can be collected and analyzed directly, or indirectly, with mass spectrometry to identify molecules, such polypeptides or proteins enriched in the disease-specific neuronal, microglial, and / or astrocytic exosomes. This general group of neuronal / astrocytic / microglial specific markers can then be further culled to identify markers localized to the surface (e.g., to a membrane surface). In certain embodiments, antibodies or other targeting molecules having affinity for these identified surface markers can be derived. These targeting molecules specific for these surface molecules can be immobilized (e.g., to a bead or other structure) and then be used to “pull-down” neuronal, microglial, and / or astrocyte specific exosomes from a collection of exosomes (e.g., an exosome population obtained from plasma from a subject suspected to be at risk of developing a neurodegenerative condition or related condition). Following elution, the collected neuronal and / or astrocyte exosomes can then be analyzed by Exoview or sorted, such as using FACS or other sorting system known in the art, and then further analyzed using mass spectrometry or single molecular array (SIMOA) or MesoScale Discovery (MSD) assays or other known assays to analyze levels of analytes of interest. In certain embodiments, certain analytes of interest can include one or more of NCAM, ATP1A3, GFAP, UCHL1, NFL, Tau, Aβ40, Aβ42, SSC5D, Emilin, MCT4, VDAC, Iba1, pTau181 and pTau217 or other marker listed in Tables 2-4 for the various exosome subpopulations, as well as chemokines and cytokines or other CD biomarkers including but not limited to CD63, CD81 and CD9. When a potential biomarker is identified, further analysis can be conducted on naïve samples obtained from a population of healthy individuals as well as those diagnosed with a neurodegenerative condition. These assays can verify the specificity and sensitivity of an identified exosome biomarker. 14 96555783.1

[0051] In certain embodiments and further to paragraphs

[0043] -

[0050] above, specific subtypes of neuronal, astrocytic and / or microglial exosomes disclosed herein can be used to identify a subject at risk of, or having MCI and / or evaluation of treatment efficacy in a subject with improved accuracy and at earlier onset than standard methods. In accordance with these embodiments, subsets of neuronal exosomes double positive for at least CD81 and at least one of NCAM and ATP1A3 can be compared to controls or baseline exosome samples in order to assess early onset of MCI and / or intervention or progression thereof in a subject. In accordance with these embodiments, a subject can then be treated, or further assessment be undertaken to ameliorate the condition. In certain embodiments, treatment methods can be assessed or compared to one another to identify a treatment specific to a subject or one with a more focused outcome for a subject. With additional information gained from assessing exosome cargo specific markers in diseased versus non-diseased subjects, mechanisms of disease onset and targets for therapeutic intervention can be performed for improved personalized medicines.

[0052] In certain embodiments and further to paragraphs

[0043] -

[0051] above, in certain non-limiting examples, a method of identifying whether a subject has or is at risk of developing Alzheimer’s Disease (AD), Alzheimer’s disease related dementia (ADRD), other AD-related condition, MCI, Down Syndrome, or other neurodegenerative health condition is provided. In aspects, this method includes isolating neuronal and / or astrocytic exosomes from a population of plasma exosomes using immobilized antibodies to neuronal specific markers - NCAM and / or ATP1A3. In some embodiments, the neuronal specific marker is NCAM. In further embodiments, the method can further include sorting and quantifying the isolated neuronal and / or astrocytic exosomes based on expression of CD63, CD81 and / or CD9. In some embodiments, the method includes sorting and quantifying the isolated neuronal and / or astrocytic exosomes based on expression of CD81 or CD9. In some embodiments, a subject is identified as at risk for developing Alzheimer’s Disease (AD), Alzheimer’s disease related dementia (ADRD), other AD-related condition, MCI, Down Syndrome, or other neurodegenerative health condition, if the subject has neuronal, microglial and / or astrocytic exosomes enriched for any one of CD63, CD81 and / or CD9 in combination with at least one additional marker compared to a baseline or control sample. In some embodiments, a subject is identified as at risk for developing or having Alzheimer’s Disease (AD), Alzheimer’s disease related dementia (ADRD), other AD-related condition, MCI, Downs Syndrome, or other 15 96555783.1neurodegenerative health condition, if the subject has an alteration or change in number of neuronal CD81+ and NCAM+ or ATP1A3+ exosomes, and optionally, at least one additional neurodegenerative disorder specific marker enriched exosomes compared to a control or baseline subject sample. In accordance with these embodiments, these isolated neuronal exosomes can be used to assess health condition progression and / or amelioration when treated to assess treatment efficacy and to design treatment regimens (e.g., antibody treatment, GM- CSF, or other treatment of these health conditions)

[0053] In certain embodiments and further to paragraphs

[0043] -

[0052] above, compositions disclosed herein can include, but are not limited to, isolated / enriched neuronal, astrocytic, and / or microglial exosome populations in a media, excipient or buffer. In accordance with these embodiments, an isolated neuronal, astrocytic, and / or astrocytic exosome population of CD9, CD81 and / or CD63 positive exosomes can be combined with one or more antibody or binding agent specific to bind to at least one of neuronal, astrocytic, and / or astrocytic exosomes (e.g., an anti- NCAM, ATP1A3, GFAP, UCHL1, NFL, Iba1, Tau, Aβ40, Aβ42, SSC5D, Emilin, MCT4, VDAC, pTau181 and pTau217 antibody or similar). In certain embodiments, the isolated neuronal, microglial, and / or astrocytic exosomes are obtained using any method described herein. For example, they can be obtained by contacting a collection of exosomes obtained from a subject (e.g., a set of plasma derived exosomes) with a neuron or astrocyte-specific antibody immobilized to a support (e.g., a column or a bead). The neuron, microglial (or macrophage) or astrocyte-specific antibody would in some embodiments target a neuronal, microglial, or astrocyte specific marker – such as a marker identified using the mass spectrometry analysis described above. In some embodiments, the neuron, microglia, or astrocyte specific antibody targets one or more of NCAM, ATP1A3, GFAP, UCHL1, NFL, Tau, Aβ40, Aβ42, SSC5D, Emilin, MCT4, VDAC, Iba1, pTau181 and pTau217; or one or more of NCAM, ATP1A3, GFAP, UCHL1, NFL, Tau, Aβ40, Aβ42, SSC5D, Emilin, MCT4, VDAC, and pTau181; or one or more of NCAM, GFAP, UCHL1, NFL, Aβ40, Aβ42, SSC5D, Emilin, MCT4, VDAC, and pTau181; or one or more of NCAM, ATP1A3, GFAP, UCHL1, NFL, SSC5D, Emilin, MCT4, VDAC, and pTau181; alone or in combination with CD81, CD9 or CD63+ exosome subpopulations. In some embodiments, the neuron specific antibody targets NCAM or ATP1A3; optionally using CD81+ enriched neuronal exosomes. In some embodiments, a composition disclosed herein can include isolating exosomes from these plasma-derived cell or health- 16 96555783.1condition populations of interest and identifying unique proteins or peptides to the cell or health-condition population of interest exosome type using identification approaches of at least one of mass spectrometry, lipidomics, metabolomics and transcriptomics of the cell or health- condition population of interest and performing R-analysis and / or bioinformatics to identify unique cell markers or health-condition associated markers on the surface of an isolated exosome population to facilitate enrichment of intact exosomes from a subject’s sample. In other embodiments, compositions can further include, but is not limited to, at least one antibody that binds to an amyloid protein or peptide, or other AD-associated molecule. In some embodiments, compositions disclosed herein include enriched CD81+, NCAM+ and / or ATP1A3+, Amyloid+ neuronal exosomes and a cell media or other buffer. Compositions disclosed herein can in some embodiments include a pharmaceutically appropriate carrier or excipient and therefore be formulated as pharmaceutical compositions, described further below. In certain embodiments disclosed herein, any composition disclosed can be part of a kit and include at least one container and optionally instructions for using the kit. It is contemplated herein the signature assays can be developed and used to test a subject on a regular basis for changes in a targeted enriched exosome population (e.g., using more personalized populations of exosomes to be isolated such as cargo differences in neuronal, astrocyte and / or glial exosome subpopulations) in order to adjust treatment regimens or frequency of evaluation of a particular subject for a more personalized approach.

[0054] In certain embodiments and further to paragraphs

[0043] -

[0053] above, methods for diagnosing risk of onset, onset, presence, progression, or amelioration of Alzheimer’s Disease (AD), Alzheimer’s disease related dementia (ADRD), other AD-related condition, MCI, Down Syndrome, or other neurodegenerative health condition in a subject, the method including, (a) obtaining an enriched population of neuronal exosomes positive for a cluster of differentiation (CD) markers from a sample from the subject; (b) isolating a subpopulation of the population of enriched CD+ neuronal exosomes having at a second marker making the subpopulation of the population of enriched CD+ neuronal exosomes, a double-positive neuronal exosome subpopulation; (c) analyzing the double-positive neuronal exosome subpopulation for amyloid protein or peptide or other peptide to detect amyloid positive double-positive neuronal exosomes in the double-positive neuronal exosome subpopulation; and (d) diagnosing risk of onset, onset, progression, or amelioration of Alzheimer’s Disease (AD), Alzheimer’s disease 17 96555783.1related dementia (ADRD), other AD-related condition, MCI, Downs Syndrome, or other neurodegenerative health condition in the subject based on amyloid positive double-positive neuronal exosomes compared to a control subject. In some embodiments, the enriched population of neuronal exosomes can be derived from a population of exosomes enriched from a plasma sample obtained from the subject. In other embodiments, the enriched population of neuronal exosomes positive for a CD marker or markers in (a) includes at least one of CD63, CD81, and CD9 positive enriched neuronal exosomes. In yet other embodiments, the at least a second marker of the subpopulation of the population of enriched CD+ neuronal exosomes having at least a second marker in (b) comprises at least one of neuronal cell adhesion molecule (NCAM) and ATP1A3. In certain embodiments, the at least a second marker of the subpopulation of the population of enriched CD+ neuronal exosomes having at least the second marker in (b) comprises at least NCAM. In some embodiments, the enriched population of neuronal exosomes positive for a CD marker or markers in (a) can include at least CD81 positive enriched neuronal exosomes. In yet other embodiments, double-positive neuronal exosome subpopulations of (b) can include CD81+ / NCAM+ double-positive neuronal exosome subpopulation. In other embodiments, analyzing the double-positive neuronal exosome subpopulation for amyloid protein or peptide or other peptide to detect amyloid positive double- positive neuronal exosomes in the double-positive neuronal exosome subpopulation in (c) can include, contacting the enriched population of neuronal exosomes with an antibody that binds to an amyloid protein or peptide or other peptide to detect the amyloid positive or peptide thereof, or other peptide double-positive neuronal exosomes; optionally, wherein the other peptides comprises one or more of pTau181, NfL, GFAP, and UCHL1. In some embodiments, antibody that binds the amyloid protein or peptide can be a polyclonal or monoclonal antibody or fragment thereof capable of immobilizing an exosome population, for example. In other embodiments, an antibody that binds the amyloid protein comprises a monoclonal antibody comprising monoclonal antibody 6E10, or other anti-amyloid monoclonal antibody. In certain embodiments, the subject is diagnosed with at least one of mild cognitive impairment (MCI) and AD.

[0055] In yet other embodiments and further to paragraphs

[0043] -

[0054] above, the subject diagnosed can be treated for MCI and / or AD. In yet other embodiments, samples obtained from a treated subject can be analyzed before and after treatment and / or compared to a control 18 96555783.1enriched population of neuronal exosomes for assessing efficacy of treating the subject based on the reduction of enriched population of neuronal exosomes compared to at least one of a control subject or the subject’s own baseline of enriched population of neuronal exosomes. In other embodiments, diagnosing risk of onset, onset, presence, progression, or amelioration of Alzheimer’s Disease (AD), Alzheimer’s disease related dementia (ADRD), other AD-related condition, MCI, Down Syndrome, or other neurodegenerative health condition in the subject reliability of the method is at least essentially equal to that of positron emission tomography (PET) imaging of the subject while avoiding disadvantages of using a PET imaging process such as exposure, cost, efficacy and invasiveness of the process and other standard methods.

[0056] In other embodiments and further to paragraphs

[0043] -

[0055] above, methods diagnosing onset, presence, or progression of Alzheimer’s Disease (AD), Alzheimer’s disease related dementia (ADRD), other AD-related condition, MCI, Down Syndrome, or other neurodegenerative health condition in a subject are disclosed including, but not limited to, a) isolating a population of exosomes derived from a plasma sample obtained from the subject suspected to be at risk for having or developing Alzheimer’s disease or an Alzheimer’s Disease related condition; (b) contacting the population of plasma-derived exosomes with an immobilized antibody to a neuronal, microglial, or astrocyte specific marker to obtain an enriched population of neuronal, microglial, or astrocytic exosomes; (c) analyzing the isolated population of neuronal, microglial, or astrocytic exosomes for the presence of exosomes expressing or containing one or more biomarkers associated with Alzheimer’s Disease (AD), Alzheimer’s disease related dementia (ADRD), other AD-related condition, MCI, Down Syndrome, or other neurodegenerative health condition; (d) determining whether the subject is at risk for developing Alzheimer’s Disease (AD), Alzheimer’s disease related dementia (ADRD), other AD-related condition, MCI, Downs Syndrome, or other neurodegenerative health condition based on whether there is an elevated number of neuronal, microglial, or astrocyte exosomes expressing the one or more biomarkers relative to an isolated neuronal, microglial, or astrocyte exosome population obtained from a control. In some embodiments, the enriched population of neuronal, microglial, or astrocyte exosomes include at least one of CD81+, CD9+ and CD63+ diseased neuronal, microglial, or astrocyte exosomes. In other embodiments, the isolated population of neuronal, microglial, or astrocyte exosome’s specific marker includes at least one of NCAM+ or ATP1A3+ exosomes. In yet other embodiments, the 19 96555783.1isolated population of neuronal, microglial, or astrocyte exosomes includes an isolated population of CD81+ neuronal exosomes and the specific marker comprises NCAM. In some embodiments, the isolated population of neuronal, microglial, or astrocyte exosome specific marker include SSC5D positive-containing astrocytic exosomes. In yet other embodiments, the isolated population of neuronal, microglial, or astrocyte exosome specific marker include Emilin positive-containing astrocytic exosomes. In some embodiments, the isolated population of neuronal, microglial, or astrocyte exosome specific marker includes VDAC positive-containing microglial exosomes. In some embodiments, the isolated population of neuronal, microglial, or astrocyte exosome’s specific marker includes MCT4 positive-containing microglial exosomes. In other embodiments, the one or more biomarkers associated with developing Alzheimer’s Disease or an Alzheimer’s Disease related condition comprise β-amyloid peptide (Aβ), amyloid or similar peptide. In further comprising administering a treatment to a subject diagnosed with onset or progression of Alzheimer’s disease (AD) or an Alzheimer’s disease-related condition.

[0057] In some embodiments and further to paragraphs

[0043] -

[0056] above, methods are disclosed for identifying disease-linked neuronal, microglial, or astrocyte specific markers for use in preparing a health-condition-specific or an enriched population of neuronal, microglial, and / or astrocytic exosomes. In accordance with these embodiments, methods can include identifying health condition-specific proteins and / or detecting proteins and / or peptides in exosomes isolated from neuronal, microglial and / or astrocytic cell cultures from a sample in vitro, wherein at least one of the proteins and / or peptides identified or detected is unique or comparatively specific to the health condition and / or the neuronal, microglial, and / or astrocytic exosome-specific marker. In other embodiments, identifying / detecting one or more molecules (e.g., proteins or peptides) in health condition-specific or an enriched population of neuronal, microglial, and / or astrocytic exosomes using mass spectrometry to derive a proteome of the health condition-specific enriched neuronal, microglial, and / or astrocytic exosomes. In other embodiments, methods for analysis can include use of R analysis and bioinformatic analysis or other known method of analysis of the proteome. In other embodiments, methods can further include enriching the health-condition-specific or an enriched population of neuronal, microglial, and / or astrocytic exosomes using at least one antibody to at least one health- condition-specific or neuronal, microglial, and / or astrocytic-specific marker immobilized on a substrate to bind to the health-condition-specific or an enriched population of neuronal, 20 96555783.1microglial, and / or astrocytic exosome-specific marker and eluting the bound health-condition- specific and / or neuronal, microglial, and / or astrocytic exosomes and enriching the population of marker-specific health condition specific and / or neuronal, microglial, and / or astrocytic exosomes. In yet other embodiments, methods can include identifying molecules such as proteins, peptides or other molecules expressed on a surface of a health condition specific and / or a neuronal, microglial, and / or astrocytic exosome associated with a health condition and diagnosing onset, presence, progression, or amelioration of the health condition based on presence of proteins or peptides or other molecules expressed on the surface of a neuronal, microglial, and / or astrocytic exosome associated with a health condition compared to a control population. In some embodiments, the neuronal, microglial, and / or astrocytic exosome specific marker comprises one or more of any one of the markers listing in any one of Tables 2-4 disclosed herein specific for a neuronal, microglial or astrocytic exosome enriched population. In other embodiments, the neuronal, microglial, and / or astrocytic exosome specific marker includes, but is not limited to, one or more of NCAM, ATP1A3, GFAP, UCHL1, Iba1, NFL, Tau, Aβ40, Aβ42, SSC5D, Emilin, MCT4, VDAC, and pTau181. In yet other embodiments, the neuronal, microglial, and / or astrocytic exosome specific marker includes, but is not limited to, one or more of NCAM, ATP1A3, SSC5D, Emilin, VDAC, MCT4, CD63, CD81, and CD9. In other embodiments, the neuronal, microglial, and / or astrocytic exosome specific marker includes, but is not limited to, one or more of NCAM, ATP1A3, SSC5D, Emilin, VDAC, MCT4, CD63, CD81, and CD9 depending on the exosome population of interest and the health condition to be evaluated.

[0058] In certain embodiments, pharmaceutical compositions are contemplated. In accordance with these embodiments, pharmaceutical compositions can include isolated exosomes (e.g., isolated neuronal, microglial, and / or astrocytic exosomes) described herein. In some embodiments, pharmaceutical compositions herein can include isolated exosomes (e.g., isolated neuronal, microglial, and / or astrocytic exosomes) described herein and at least one pharmaceutically acceptable excipient or carrier. As used herein, the term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and / or bodily fluids of a subject without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio. As used herein, 21 96555783.1the term “pharmaceutically acceptable carrier” can refer to solvents, dispersion media, coatings, antibacterial agents, antifungal agents, isotonic and absorption delaying agents, or the like that are physiologically compatible. Pharmaceutically acceptable carriers suitable for use herein, include, but are not limited to, buffers that are well known in the art, and can be phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; amino acids; hydrophobic polymers; monosaccharides; disaccharides; and other carbohydrates; metal complexes; and / or non-ionic surfactants.

[0059] In some embodiments and further to the paragraphs above, pharmaceutical compositions for use in delivering treatments to subject contemplated herein can be formulated for parenteral administration, such as intravenous, intracerebroventricular injection, intra- cisterna magna injection, intra-parenchymal injection, intra-renal, intradermal, subcutaneous, direct introduction to a tumor or a combination thereof. In some embodiments, pharmaceutical compositions for use herein can be formulated for local delivery to specific tissues or other delivery. In some embodiments, pharmaceutical compositions for use herein be formulated for parenteral administration can include pharmaceutically acceptable carriers including sterile liquids, such as water and oil, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and the like. Saline solutions and aqueous dextrose, polyethylene glycol (PEG) and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. In some embodiments, pharmaceutical compositions for use herein can further include additional agents, for example preservatives, buffers, tonicity agents, antioxidants and stabilizers, nonionic wetting or clarifying agents, viscosity-increasing agents, and the like. In some embodiments, pharmaceutical compositions described herein can be packaged in single unit dosages or in multidosage forms.

[0060] In some embodiments, formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which can contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which can include suspending agents and thickening agents. In accordance with some embodiments herein, aqueous solutions can be suitably buffered (preferably to a pH of from 3 to 9). The preparation of suitable 22 96555783.1parenteral formulations for use herein under sterile conditions can be readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.

[0061] In some embodiments, pharmaceutical compositions herein can further include one or more pharmaceutically acceptable salts. Non-limiting examples of pharmaceutically acceptable salts include acid addition salts (formed from a free amino group of a polypeptide with an inorganic acid, or an organic acid. In some embodiments, the salt formed with the free carboxyl groups is derived from an inorganic base, or an organic base. In some embodiments, any of the pharmaceutical compositions herein can be used in therapeutic applications, for example, cancer treatment in human patients, which are also disclosed herein.

[0062] In some embodiments, a subject to any of the methods herein can be any subject for whom analysis of a neurodegenerative disorder and / or treatment or therapy is desired. In some embodiments, a subject can have or can be suspected of having or developing a neurodegenerative disorder. In other embodiments, a subject can be a mammal. In some embodiments, a subject can be a human patient. In yet other embodiments, a human patient such as an adult, child, adolescent, toddler, young adult or infant or fetus who is in need of the methods disclosed herein.

[0063] In some embodiments and further to paragraphs

[0043] -

[0062] above, a subject in any of the methods described herein can be a human patient having, suspected of having, or a risk for developing Alzheimer’s Disease or a disease associated with or related to Alzheimer’s disease. In certain embodiments, a subject in any of the methods described herein can be classified by the 2018 National Institute on Aging and Alzheimer’s Association (NIA-AA) using the AT(N) unbiased system. This system classifies individuals across a range of cognitive stages (ranging from cognitively unimpaired to dementia) as being off or on the Alzheimer spectrum based on measures of A (Aβ biomarker measures of cortical amyloid by PET imaging and / or low CSF A ^42 or A ^42 / A ^40 plaques), T (fibrillar Tau assessed as elevated CSF pTau and cortical Tau PET), and N (Neurodegeneration / Neuronal injury markers: CSF T-tau, hypometabolism (PET), atrophy on MRI). Patients can be categorized by methods and systems known in the art with respect to contemplated neurodegenerative disorders disclosed herein.

[0064] Accordingly in some embodiments and further to paragraphs

[0043] -

[0063] above, subjects can have Alzheimer’s disease, have prodromal Alzheimer’s disease (AD), have Alzheimer’s pathologic change with MCI, have preclinical Alzheimer’s pathologic change, have 23 96555783.1preclinical Alzheimer’s disease, have Alzheimer’s pathologic change with dementia or have Alzheimer’s disease with dementia. Further aspects of the present disclosure relate to methods of treating a patient diagnosed with onset and / or progression of Alzheimer’s disease according to a method disclosed herein. Appropriate treatments for a patient can be determined by one of skill in the art. For example, the treatment can include a small molecule pharmaceutical, a biologic, therapy, physical therapy, occupational therapy, or the like (e.g., to treat MCI, AD or related conditions contemplated herein).

[0065] In some embodiments and further to paragraphs

[0043] -

[0064] above, kits are contemplated of use to distribute components used in any of the methods provided herein such as for further analysis of exosome populations. In other embodiments, kits include therapeutic compositions disclosed herein for storage, transport, and use. constructs or pharmaceutically acceptable formulation disclosed herein. In some embodiments, a kit can further include one or more reagents for storing isolated and / or enriched exosome populations disclosed herein.

[0066] Kits disclosed herein include suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Also contemplated are packages for use in combination with a specific device, such as an inhaler, nasal administration device (e.g., an atomizer) or an infusion device such as a minipump. A kit can have a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The container can also have a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).

[0067] Kits can optionally provide additional components such as buffers and interpretive information. Kits can include a container and a label or package insert(s) on or associated with the container. In some embodiments, the invention provides articles of manufacture including contents of the kits described above. EXAMPLES

[0068] The following examples are included to illustrate certain embodiments. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered to function well in the practice of the claimed methods, compositions and apparatus. However, those of skill in the art should, in light of the present 24 96555783.1disclosure, appreciate that changes can be made in some embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. Example 1

[0069] In one exemplary method, total plasma exosomes were isolated from blood samples obtained from 5 control subjects and 5 Mild Cognitively Impaired (MCI) subjects. Each subject was previously classified into the control or MCI group by a standard assay where levels of amyloid beta (Aβ) plaques were visualized in the brain using the currently accepted PET scan. Then, neuronal specific exosomes were isolated from total exosomes using for example, immunoprecipitation of neuronal exosomes using neuronal specific antibodies immobilized on nanobeads. In this example, two different neuronal specific antibodies were used – NCAM and ATPA3 as two different “detection tools” for neuronal exosome specificity. Neuronal specific exosomes isolated with NCAM antibodies were classified as obtained using “detection tool 1” and Neuronal specific exosomes isolated with ATPA3 antibodies were classified as obtained using “detection tool 2”. In this example, for the original two groups (control vs. MCI subjects), four exosome populations were isolated for further analysis (e.g., (1) PET- and NCAM+, (2) PET+, NCAM+, (3) PET- ATPA3+ and (4) PET+ ATPA3+). These were then analyzed on an ExoviewTMinstrument which allowed for the examination of subsets of exosomes; CD63+, CD81+ and CD9+ exosomes are indicated. All isolated neuronal exosomes were analyzed with a 647 fluor labelled amyloid antibody against the first 16 amino acids of Ab (e.g., 6E10 antibody, an exemplary monoclonal antibody), and as observed in several preliminary testing experiments, it was found that the CD81+ subpopulation of neuronal exosomes demonstrated particles per ml significantly higher than CD9+ and CD63+ neuronal exosomes in the PET+ve vs the PET-ve individuals which correlate to Ctrl vs MCI individuals (FIG.1). CD9+ neuronal exosomes demonstrated more particles than CD63+ but both were dramatically reduced compared to CD81+ neuronal exosomes. Interestingly, the PET tool AmyvidTMfrom Eli Lilly correlates better with this plasma tool and the amyloid positivity of the CD81+ differentiates positive exosomes from the neuronal exosome subset. Upon analysis by log transformation of the data, NCAM IP was significantly better than the ATP1A3 IP but both demonstrated correlations. This variability appears to be responsible for the lack of significance for the PET+ve vs PET-ve. A summary table of the total exosomes isolated, the numbers of exosomes 25 96555783.1in the subsets that are +ve for amyloid and then the other Biomarkers measured using SIMOA in the plasma of these subjects is included below. Table 1 Subj C PET NTA NCAM ATP Ab40 Ab42 Tau GFAP NfL UCH ID D particles / IP’d IP’d (pg / (pg / (pg / (pg / (pg / L1 R ml 6E10+ve- 6E10+ve ml) ml) ml) ml) ml) ( g / )predominantly on CD81+ neuronal exosomes. When comparing lysed versus not lysed exosomes, the concentration of amyloid detected was the same, implying amyloid is found on the outside of the exosomes. Alpha 2 macroglobulin and 6E10 (amyloid aa 1-16) can also pull down amyloid positive exosomes that include a CD81+ exosome subset. Tau and pTau were also observed to pull down exosomes. Example 2

[0071] In another exemplary method, new biomarkers associated with a disease condition can be identified. In various exemplary methods this will be done by enriching for specific exosome populations from different cell types or health condition samples, analyzing the proteome of isolated or enriched exosomes using mass spectrometry, performing R analysis / bioinformatics of the mass spectrometry data to identify unique neuronal, microglial, 26 96555783.1and astrocytic markers, and further identifying markers expressed on the surface. These newly identified health condition-associated neuronal, microglial, and astrocytic markers can be used to isolate exosomes from human plasma obtained from patients with, at risk of developing or without a health condition. These isolated or enriched exosomes can be further analyzed (by for example, Exoview, SIMOA, Metabolomics, Proteomics, Transcriptomics and Lipidomics) to identify biomarkers that correlate with a health condition. As noted above, this method was validated in Example 1 above by identifying NCAM and ATP1A3 as neuronal specific markers that were used to further identify CD81+ as an Alzheimer’s Disease specific biomarkers.

[0072] FIG.2 illustrates exemplary methods for enrichment of targeted exosome populations disclosed herein. Initially, 1) total exosomes can be isolated from models of disease or cell types (control and diseased); then mass spectrometry (proteomics) performed on the samples and analyzed by 3) R-analysis and / or bioinformatics to identify unique signatures of a health condition or a cell type and then 4) the markers on the surface of an exosome can be determined and then as illustrated in FIG.2, antibodies to these exosome surface targets can be used to specifically enrich for the various exosome populations from human plasma. Eluting these enriched exosomes for further analysis represented in FIG.2 can identify markers of health condition from carried cargo and surface markers of these health-condition isolated and cell type specific exosomes that can be used to diagnose health condition status, risk, onset, presence, progression and / or amelioration of the health condition following therapeutic intervention as well as sex differences in the disease status and sex specific responses to therapeutic interventions affiliated with enriched exosomes of interest. Example 3

[0073] To date, to isolate and examine exosomes as biomarkers of health conditions, researchers have assumed that what is in / on the cells that produce the exosomes, must be in / on the exosomes they produce in the same proportions and that this correlates with a disease condition, etc. It has been discovered herein that more rigorous assessments of what is contained in / on an exosome released by a certain cell type and in a diseased versus a Control condition is essential, as is the determination of which of these markers are on the surface so that these exosomes can be isolated or enriched from plasma for even deeper analyses of their cargo so that additional understanding of disease as well as responses to therapeutic intervention of the disease or health condition. Therefore, these refined methods are essential for analyzing 27 96555783.1subpopulations of exosomes as they relate to health conditions serving as powerful tools to assess disease onset and progression as well as to assess efficacy of treatment or other interventions.

[0074] In this example, AD was analyzed. As an initial step, the following was commenced, (1) identify uniquely expressed proteins / polypeptides on an enriched population of neuronal, astrocytic, and / or microglial exosomes by enriching exosomes from cell cultures from each of these cell types, (2) analyzing the exosomes isolated from these cells by Mass Spectrometry and (3) using R-analysis / bioinformatics, identify proteins / polypeptides common to each cell type as well as unique to a neuronal, astrocytic, and / or microglial exosome. As disclosed herein, 65 unique neuronal markers, 355 microglial markers and 90 astrocytic markers (See Tables 2-4 below) were discovered in screening of cell type specific exosomes. Shared markers were also assessed for overlap. (FIG.3).

[0075] Table 2 28 96555783.1Astrocyte Exosomes Markers LAMA2 IGFBP7 SRPX2 129 96555783.1Microglia Exosome Markers PSMD11 RPN1 RPS12 COPB1 HUWE1 SUPT16H TFRC GART DDOST PSMA6 RPL18 PDCD6IP EIF3F EIF2S1 PSMA1 CTSC KTN1 COPG1 F11 PAICS CCT6A CDC42 DHX9 TNPO1 PSMD14 CLEC3B PSMA3 EIF3B SFRP1 MTCH2 VWF GPX3 RPL13A DSTN GPS1 EXT2 7B10 0A1 1 P N11 N7 B11 L2 1C 13 3 4 L2 L1 1 1 0CNeuronal Exosome Markers30 96555783.1ALDH1A1 UGDH CNTN2 BHMT2 plary mass spectrometry analysis overview of exosomesanalyzed for unique or comparatively distinguishing markers of each enriched neuronal, astrocytic or microglial exosome population.

[0077] FIG.4 illustrates a representative graph of anti-amyloid positive CD positive exosomes where superior numbers (particles per ml) of CD81+ amyloid+ exosomes were identified using NCAM (versus ATP1A3) to isolate the neuronal exosomes that can ultimately distinguish control and MCI from AD subjects. Example 4

[0078] In another exemplary method, to deduce which molecules are on surfaces of exosomes, proteins from the unique sets identified in FIG.3 and the corresponding table were (4) manually curated by determining from the literature which of these are present on the surface of a cell and therefore, which are most likely on the surface of an exosome based on their presence / orientation in a cellular plasma membrane. Then, using these surface targets, exosomes were isolated from a plasma sample of Control versus MCI vs AD subjects. MCI and AD status was confirmed by psychological testing and standard PET imaging. It was observed that plasma exosome-based biomarker tests mirrored ability of PET imaging to determine AD subjects from control and MCI subjects; therefore, providing a less invasive (compared to PET or lumbar puncture CSF collection) plasma-based diagnostic tool (FIGS.5A-5C). 31 96555783.1

[0079] From this information, tests were commenced initially using two of these neuronal markers, one of which is demonstrated here (FIGS.5A-5C).

[0080] FIG.5A represents an exemplary bar graph illustrating correlation of CD+ neuronal exosomes with NCAM surface expression. In this example, when a neuronal surface marker (e.g., NCAM) was used to isolate exosomes from a plasma sample (e.g., human), the percentage of exosomes in total plasma were CD63 (%), CD81 (~0.1%) and CD9 (0.09%) positive. When these exosomes were stained for amyloid content, it was discovered (FIG.5B) that 100% of the NCAM+ve CD81+ve exosomes were positive for amyloid while only about 30% of the CD9+ and about 10% of the CD63+ subsets of exosomes demonstrated this positivity. Therefore, it was assessed that a neuronal exosome biomarker for AD correlated with an NCAM+ve CD81+ve Amyloid+ve neuronal exosome.

[0081] Further, as illustrated in FIG.5C, when testing use of these biomarkers in a pilot study of 5 Control, 5 MCI and 5 AD individuals (whose status had been confirmed by a battery of psychological testing and PET imaging) the numbers of the NCAM+ve CD81+ve Amyloid+ve plasma neuronal exosomes increased correlating with a health status moving from Control (no AD, no MCI) to MCI then to AD. Therefore, a much less invasive plasma exosome- based biomarker has been developed for assessing onset, risk and progression of AD that is at least equal in its ability to determine AD status as is amyloid PET imaging and psychological testing (FIGS.5A-5C).

[0082] FIGS.5A-5C illustrate in (5A), more CD81+ and CD9+ exosomes are isolated by immunoprecipitation with the neuronal (NCAM) exosome marker; (5B) demonstrates that 100% of neuronal CD81+ exosomes are positive for amyloid and (5C) illustrates that neuronal (NCAM+ve) CD81+ve Amyloid+ve (6E10) plasma derived exosome numbers (particles per ml) increase as AD pathology increases and the health condition progresses.

[0083] FIG.4 illustrates testing of another marker other than NCAM, ATP1A3, where CD81+ neuronal exosomes correlate more closely than CD63+ and / or CD9+ neuronal exosomes to amyloid+ exosomes and verified subjects having AD.\

[0084] FIG.6 illustrates that number of particles per ml of NCAM+ve CD81+ve 6E10+ve is significantly different when comparing control (CTRL) or MCI and AD (increased particles per ml as the health condition progresses). This marker selection further distinguishes sex differences (e.g., in these studies, males have higher numbers (particles / ml) than females tested). 32 96555783.1

[0085] FIG.7 illustrates a graph where GM-CSF treatment of the subject demonstrated improvements in plasma biomarkers and cognition in participants has been introduced and AD progression can be assessed. In this example, a biomarker associated with the enriched exosomes can identify whether a treatment effect with the biomarker demonstrating a reduction towards control levels post intervention can affect AD disease state in a subject. It is noted here that the NCAM+ / CD81+ neuronal exosomes correlate with differences between control and AD and progression or amelioration of AD. It is noted that there are significant differences between Control and AD, Control and AD treated (EOT, end of treatment), and AD baseline vs AD treated subjects. A reduction in NCAM+ / CD81+ neuronal exosomes correlates with an improved state of the observed conditions and side effects of AD, indicating that the measures of NCAM+ / CD81+ neuronal exosomes can be used to assess disease state and treatment efficacy. Example 5

[0086] In another exemplary method, analysis of male versus female subject were assessed for differences in correlations of isolated / enriched exosome populations as they relate to AD and / or AD progression and / or treatment.

[0087] FIG.8 illustrates an exemplary plot of male versus female subject’s amyloid positive NCAM+ / CD81+ neuronal exosomes in treated versus untreated subjects. As disclosed herein, one exemplary intervention for treating AD is granulocyte-macrophage colony-stimulating factor (GM-CSF). It is understood that any MCI and / or AD treatment known in the art can be used to treat a subject diagnosed and / or assessed for AD using compositions and / or methods disclosed herein. It was observed that intervention with GM-CSF significantly reduced particles per ml of amyloid positive NCAM+ / CD81+ neuronal exosomes compared to baseline control where no treatment was administered. In this study, females seemed to respond better and demonstrated lower particles / ml of the NCAM+CD81+Amyloid+ exosomes. Example 6

[0088] In another exemplary method, particles per ml of NCAM+ve CD81+ve Amyloid+ve in Control vs AD individuals were assessed. These data were graphed and analyzed for sensitivity and specificity and demonstrated that the analysis, assays, and methods disclosed herein are both sensitive and specific. In this example, it is understood that ROC curves assess both sensitivity and specificity of a test of interest. For example, sensitivity relates to ability of a 33 96555783.1test in question to identify those with the disease e.g., subjects known to have AD demonstrated a positive result with a test for AD at the earliest stage in the disease process possible; while specificity, demonstrates ability of a test in question to not detect non-diseased subjects or that those subjects not having AD would not be mistakenly identified as having AD (e.g., reduced to no false positives). When interferences are introduced to the test in question, the test remains true to the outcome despite the introduction of interferences, again, e.g., the test in question does not detect non-AD subjects as having AD. As observed herein, the instantly disclosed methods are both sensitive and specific (See FIG.9). FIG.9 illustrates an exemplary ROC curve for modeling sensitivity versus specificity of the observed correlations disclosed herein. Example 7

[0089] In another exemplary method, other health condition related molecules or analytes of use to diagnose state of a health condition can also present on NCAM+ve CD81+ve neuronal exosomes. Other molecules assessed for correlation with AD and / or AD progression or amelioration can include, but is not limited to, NfL (FIG.10 and 12), GFAP (FIG.13), pTau181 (FIG.14) and UCHL1 (FIG.11). It was observed herein that some of these analytes on NCAM+ve CD81+ve can differentiate control subjects from MCI subjects which the assessed amyloid marker is not as accurate at distinguishing due to variability. Further, some of these analytes on NCAM+ve CD81+ve neuronal exosome subtypes can distinguish treatment effects (FIG.15).

[0090] FIG.13 and 14 illustrate CD marker variability when assessing additional markers GFAP (FIG.13) and pTau181 (FIG.14) in enriched neuronal exosome populations demonstrating the significant difference in CD81+ exosomes compared to CD9+ and / or CD63+ enriched exosomes affiliated with GFAP (FIG.13) and pTau181 (FIG.14) for assessing control versus MCI subjects. These data provide additional information not provided using amyloid marker data alone for additional sensitivity in assessing MCI onset, presence, progression and / or amelioration even when only a small cohort was assessed. This supports using staining with panels of markers in combination to provide an even further enhanced sensitivity and specificity to the NCAM+ CD81+ exosome assays described.

[0091] FIG.15 illustrates an exemplary use of marker GFAP to further distinguish control from MCI subjects using NCAM+ CD81+ neuronal exosomes. The NCAM+ exosomes were divided into their subtypes and stained for biomarkers, GFAP, NfL, pTau181 or UCHL1 and the 34 96555783.1particles per ml assessed. All staining observed was predominantly observed on the CD81 subset of exosomes with GFAP and NfL distinguishing between control and MCI subjects double positive exosomes were analyzed. Here, baseline versus treated’ leukine subjects received treatment (GM-CSF) for a period (about 3 weeks). FIG.15 illustrates CD marker variability when assessing additional markers GFAP in enriched neuronal exosome populations demonstrating the significant difference in CD81+ exosomes compared to CD9+ and / or CD63+ enriched exosomes. FIG 15 also illustrates an exemplary use of marker GFAP to further distinguish control from MCI subjects using CD81+ / NCAM+ neuronal exosomes. GFAP can also help distinguish Control from MCI and AD and demonstrates a trend in distinguishing untreated (baseline) AD from GM-CSF(Leukine) treated AD.

[0092] Fig.16 represents an exemplary plot of particles per ml comparing exosome testing technologies disclosed herein to verified PET scanned subjects having or not having AD in support that the technologies disclosed herein provide accurate diagnoses without issues caused using PET scans, for example. This is another version of a neuronal exosome NCAM vs ATP1A3 test. Subset 1 are CD63+, subset 2 are CD81+ and subset 3 are CD9+. Example 8

[0093] In other exemplary methods, astrocytic enriched populations of exosomes were examined for marker correlation in a neurodegenerative health conditions. Astrocyte marker SSC5D was determined by the methods described herein to be a specific marker of astrocyte- derived exosomes. FIG.17 represents a bar graph demonstrating supporting evidence that neuronal exosomes derived by immunoprecipitation of plasma exosomes with an anti-NCAM antibody provides a population of exosomes that does not bind to anti-SSC5D antibodies, indicating that astrocyte-derived and neuron-derived exosomes are distinguishable from one another and can provide additional information related to health conditions and treatment thereof.

[0094] In another exemplary method, when plasma exosomes were immunoprecipitated with a SSC5D antibody to enrich for astrocytic exosomes and then analyzed for the ability to bind to an anti-NCAM antibody, again, as supported in FIG.17 above, FIG.18 demonstrates that the astrocytic exosomes have distinct features and specificity and that astrocyte (SSC5D+ve) enriched exosomes do not have the neuronal specific marker NCAM. In these astrocytic exosomes, tetraspanins, CD63 and CD9 predominate compared to CD81 which is in 35 96555783.1contrast to neuronal exosomes where CD81 is predominant compared to CD9 and CD63 positive populations.

[0095] Further, FIG.19 illustrates that biomarker, Emilin, was determined by the methods described herein to also be a specific marker of astrocyte enriched exosomes isolated from plasma samples containing a mix of exosomes. In this example, plasma exosomes were precipitated with anti-Emilin antibody that were not subsequently detected with anti-NCAM antibody, demonstrating that Emilin +ve exosomes are astrocyte-specific and do not contain the neuronal marker NCAM. Emilin positive exosomes, like SSC5D positive exosomes predominantly also carry CD63 and CD9 compared to CD81 and CD9 predominant compared to CD63 positive populations. On staining the neuronal exosomes for amyloid, the NCAM+ CD81+ double positive populations were found to be the predominant subpopulation. Example 9

[0096] In other exemplary methods, microglial enriched populations of exosomes were examined for marker correlation using exosomes from a model system in which macrophages were differentiated to microglia over time (e.g., Days 4, 7, 10 and 14) with the cells being almost exclusively microglia by Day 14. As illustrated in FIG.20, microglial marker VDAC was determined by the methods described herein to be a specific marker of microglia as more exosome particles per ml were produced by the cells as they became microglia. Therefore, VDAC became a predominant marker during the differentiation, with CD63 and CD9 being the predominant exosome subtype over the course of differentiation compared to CD81 for neuronal exosomes. These data confirm that VDAC is microglia specific marker, especially on CD63 and CD9 exosomes.

[0097] In another exemplary method, when MCT4 expressing exosomes were measured using the same system described above, microglial exosomes also had an increased number of exosome particles per ml that expressed MCT4; therefore, it was determined that this biomarker is specific for microglial exosomes. FIG.21A represents a plot over time where exosome differentiation was assess for microglial exosomes where MCT4 was determined by the methods described herein to be a specific marker of these microglia enriched exosomes populations. In this experiment, activation of a cell line to differentiate from macrophage to a microglial phenotype over a period of 14 days, demonstrated that MCT4 became a predominant marker during the change, with CD 9 being the predominant exosome tetraspanin marker. These 36 96555783.1data confirm that MCT4 is microglia specific marker, especially on CD9 exosomes. As macrophages differentiated into microglia, VDAC and MCT4 were the most prominent markers (see Day 14). It was observed that these exosomes are predominantly CD9+ demonstrating that different subtypes of exosomes can be produced from different starting cell types (e.g., macrophages to microglia differentiation). In addition, these exosomes were then immunoprecipitated from control vs DS-AD human brain exosomes and were stained for VDAC and MCT4. These exosomes were found positive for both markers and in addition the MCT4 stained positively for Iba1 which is a known marker of microglia. Given astrocytic and microglial activation are common features of neurodegenerative type diseases being able to detect these cell types of exosomes in the plasma of individuals dramatically simplifies the measurement of changes in these cell types in the CNS but using plasma samples instead, in turn indicating the disease status in a less invasive manner. The tetraspanins, CD63, CD81, and CD9 are not consistently predominant over the course of differentiation, although by day 14, with CD63 and CD9 are more prevalent than is CD81 as these are predominantly microglial at this stage.

[0098] Because microglia are prevalent and activated in MCI and AD, an exosome-based biomarker of microglia can be useful for assessing the disease state (MCI and / or AD) of a subject and for assessing the efficacy of treatment, which would be expected to reduce the number of microglial derived exosomes as demonstrated for exosomes carrying another activated glial marker (GFAP), see FIG.15.

[0099] In another exemplary method, MCT4 (microglial-affiliated biomarker) was used to immunoprecipitate exosomes from total exosomes isolated from plasma samples of subjects of a control human brain or DS-AD human brain. In this example, exoview analyses of the IP’d populations of the immunoprecipitated microglial exosomes demonstrated a significant enrichment of CD9+ve exosomes that stained positively for the microglia biomarker Iba1 (Iba1 is a distinguishing microglial marker) while CD81+ enriched subpopulations can differentiate DS-AD from a control sample. (FIG.21B). This method was used to confirm that identified microglial biomarker MCT4 isolates microglial exosomes where a gold standard microglial marker (Iba1) was selected to stain the exosomes post isolation with MCT4. Here, MCT4 was used to immunoprecipitate microglial exosomes from a population of total human brain cell- derived exosomes (Control and DS-AD brain), and the MCT4+ exosomes separated into the 37 96555783.1CD63+ CD81 and CD9+ exosomes using Exoview analysis. These MCT4+ exosomes were confirmed to be Iba1+ and therefore are microglial exosomes. In addition, these microglial exosomes were capable of distinguishing control from DS-AD exosomes pointing to the use of these microglial exosomes as diagnostic / treatment evaluation tools. **************************************************** All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods have been described in terms of embodiments, it is apparent to those of skill in the art that variations maybe applied to the compositions and methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope herein. More specifically, certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept as defined by the appended claims. 38 96555783.1

Claims

What is Claimed:

1. A method for diagnosing risk of onset, onset, presence, progression, or amelioration of Alzheimer’s disease (AD) or an Alzheimer’s disease-related condition in a subject, the method comprising (a) obtaining an enriched population of neuronal exosomes positive for a cluster of differentiation (CD) markers from a sample from the subject; (b) isolating a subpopulation of the population of enriched CD+ neuronal exosomes having at a second marker making the subpopulation of the population of enriched CD+ neuronal exosomes, a double-positive neuronal exosome subpopulation; (c) analyzing the double-positive neuronal exosome subpopulation for amyloid protein or peptide or other peptide to detect amyloid positive double-positive neuronal exosomes in the double-positive neuronal exosome subpopulation; and (d) diagnosing risk of onset, onset, progression, or amelioration of AD or an AD- related condition in the subject based on amyloid positive double-positive neuronal exosomes compared to a control subject.

2. The method according to claim 1, wherein the enriched population of neuronal exosomes is derived from a population of exosomes enriched from a plasma sample obtained from the subject.

3. The method according to claim 1 or 2, wherein the enriched population of neuronal exosomes positive for a CD marker or markers in (a) comprises at least one of CD63, CD81, and CD9 positive enriched neuronal exosomes.

4. The method according to any one of claims 1-3, wherein the at least a second marker of the subpopulation of the population of enriched CD+ neuronal exosomes having at least a second marker in (b) comprises at least one of neuronal cell adhesion molecule (NCAM) and ATP1A3. 39 96555783.

15. The method according to any one of claims 1-3, wherein the at least a second marker of the subpopulation of the population of enriched CD+ neuronal exosomes having at least the second marker in (b) comprises at least NCAM.

6. The method according to any one of claims 1-5, wherein the enriched population of neuronal exosomes positive for a CD marker or markers in (a) comprises at least CD81 positive enriched neuronal exosomes.

7. The method according to claim 1, wherein the double-positive neuronal exosome subpopulation of (b) comprises a CD81+ / NCAM+ double-positive neuronal exosome subpopulation.

8. The method according to any one of claims 1 to 7, wherein analyzing the double- positive neuronal exosome subpopulation for amyloid protein or peptide or other peptide to detect amyloid positive double-positive neuronal exosomes in the double-positive neuronal exosome subpopulation in (c) comprises contacting the enriched population of neuronal exosomes with an antibody that binds to an amyloid protein or peptide or other peptide to detect the amyloid positive or peptide thereof, or other peptide double-positive neuronal exosomes; optionally, wherein the other peptides comprises one or more of pTau181, NfL, GFAP, and UCHL1.

9. The method according to claim 8, wherein the antibody that binds the amyloid protein or peptide comprises a polyclonal or monoclonal antibody.

10. The method according to claim 8 or 9, wherein the antibody that binds the amyloid protein comprises a monoclonal antibody comprising monoclonal antibody 6E10 or other anti- amyloid monoclonal antibody.

11. The method according to any one of claims 1-10, wherein the subject is diagnosed with at least one of mild cognitive impairment (MCI) and AD. 40 96555783.

112. The method according to claim 11, further comprising treating the subject for MCI and / or AD.

13. The method according to claim 12, further comprising obtaining an additional enriched population of neuronal exosomes according to any one of claim 1-10 and assessing efficacy of treating the subject based on the additional enriched population of neuronal exosomes compared to at least one of a control subject or the subject’s own baseline of enriched population of neuronal exosomes.

14. The method according to any one of claims 1-11, wherein diagnosing the risk of onset, onset, presence, progression, or amelioration of Alzheimer’s disease (AD) or an Alzheimer’s disease-related condition in the subject reliability of the method is at least essentially equal to that of positron emission tomography (PET) imaging of the subject while avoiding disadvantages of using a PET imaging process.

15. A composition comprising an isolated enriched population of exosomes positive for a cluster of differentiation (CD) markers; and at least one additional agent or excipient.

16. The composition according to claim 15, wherein the isolated enriched exosomes comprise at least one of isolated enriched neuronal, microglial, and astrocytic exosomes.

17. The composition according to claim 15 or 16, wherein the isolated enriched population of exosomes is obtained by: (a) isolating total exosomes from a plasma sample obtained from a subject, (b) separating subtypes of exosomes from total exosomes using unique markers identified in (a) with an antibody to a health condition-specific, neuronal, microglial and / or astrocyte specific marker, wherein the antibody is immobilized to a support; (c) collecting antibody-bound neuronal, microglial, and / or astrocyte specific exosomes. 41 96555783.

118. The composition according to claim 17, further comprising prior to (a), identifying unique markers for a cell or health-condition population of interest using model systems of the cell or health condition population, isolating exosomes from these cell or health-condition populations of interest and identifying unique proteins or peptides to the cell or health-condition population of interest exosome type using identification approaches comprising at least one of mass spectrometry, lipidomics, metabolomics and transcriptomics of the cell or health-condition population of interest and performing R-analysis to identify unique cell markers or health- condition associated markers on the surface of an isolated exosome population to facilitate enrichment of intact exosomes from a subject’s sample.

19. The composition according to any one of claims 15-18, further comprising a media.

20. The composition according to any one of claims 15-19, further comprising at least one antibody.

21. The composition according to claim 20, wherein the at least one antibody comprises an antibody that binds to a health condition associated protein or polypeptide.

22. The composition according to claim 15, wherein the enriched population of exosomes positive for a cluster of differentiation (CD) marker or markers comprises at least one of a CD81+, CD9+, and CD63+ enriched population of exosomes.

23. The composition according to claim 22, wherein the enriched population of the at least one of a CD81+, CD9+, and CD63+ neuronal exosomes comprise an enriched population of at least one of a CD81+, and CD9+ neuronal exosomes.

24. The composition according to claim 23, wherein the enriched population of at least one of a CD81+, and CD9+ neuronal exosomes are further NCAM+ and / or ATP1A3+ at least one of a CD81+, and CD9+ neuronal exosomes. 42 96555783.

125. The composition according to claim 15, further comprising at least one antibody wherein the antibody binds to an amyloid protein or peptide.

26. The composition according to claim 15, wherein the isolated enriched population of exosomes positive for a cluster of differentiation (CD) marker or markers; and at least one additional agent or excipient comprises CD81+, NCAM+ and / or ATP1A3+, Amyloid+ neuronal exosomes and a cell media.

27. A method for diagnosing onset, presence, or progression of Alzheimer’s disease (AD) or an Alzheimer’s disease-related condition in a subject, the method comprising: (a) isolating a population of exosomes derived from a plasma sample obtained from the subject suspected to be at risk for having or developing Alzheimer’s disease or an Alzheimer’s Disease related condition; (b) contacting the population of plasma-derived exosomes with an immobilized antibody to a health-condition neuronal, microglial, or astrocyte specific marker to obtain an enriched population of neuronal, microglial, or astrocytic exosomes; (c) analyzing the isolated population of neuronal, microglial, or astrocytic exosomes for the presence of exosomes expressing or containing one or more biomarkers associated with developing Alzheimer’s Disease or an Alzheimer’s Disease related condition; (d) determining whether the subject is at risk for developing Mild Cognitive Impairment (MCI), Alzheimer’s Disease, or an Alzheimer’s Disease related condition based on whether there is an elevated number of neuronal, microglial, or astrocyte exosomes expressing the one or more biomarkers relative to an isolated neuronal, microglial, or astrocyte exosome population obtained from a control.

28. The method according to claim 27, wherein the enriched population of neuronal, microglial, or astrocyte exosomes comprise at least one of CD81+, CD9+ and CD63+ neuronal, microglial, or astrocyte exosomes.

29. The method according to claim 27 or 28, wherein the isolated population of neuronal, microglial, or astrocyte exosome’s specific marker comprises NCAM+ or ATP1A3+ exosomes. 43 96555783.

130. The method according to claim 29, wherein the isolated population of neuronal, microglial, or astrocyte exosomes comprises an isolated population of CD81+ neuronal exosomes and the specific marker comprises NCAM.

31. The method according to claim 27 or 28, wherein the isolated population of neuronal, microglial, or astrocyte exosome specific marker comprises SSC5D positive-containing exosomes.

32. The method according to claim 27 or 28, wherein the isolated population of neuronal, microglial, or astrocyte exosome specific marker comprises Emilin positive-containing exosomes.

33. The method according to claim 27 or 28, wherein the isolated population of neuronal, microglial, or astrocyte exosome specific marker comprises VDAC positive-containing exosomes.

34. The method according to claim 27 or 28, wherein the isolated population of neuronal, microglial, or astrocyte exosome’s specific marker comprises MCT4 positive-containing exosomes.

35. The method according to any one of claims 27-34, wherein the one or more biomarkers associated with developing Alzheimer’s Disease or an Alzheimer’s Disease related condition comprise β-amyloid peptide (Aβ), amyloid or similar peptide.

36. The method of any one of claims 27 to 35, further comprising administering a treatment to a subject diagnosed with onset or progression of Alzheimer’s disease (AD) or an Alzheimer’s disease-related condition.

37. A method of identifying a neuronal, microglial, or astrocyte specific marker for use in preparing a health-condition-specific or an enriched population of neuronal, microglial, and / or 44 96555783.1astrocytic exosomes, the method comprising identifying health condition-specific proteins and / or detecting proteins and / or peptides in exosomes isolated from neuronal, microglial and / or astrocytic cell cultures from a sample in vitro, wherein at least one of the proteins and / or peptides identified or detected is unique or comparatively specific to the health condition and / or the neuronal, microglial, and / or astrocytic exosome-specific marker.

38. The method according to claim 37, further comprising identifying / detecting one or more proteins or peptides in the health condition-specific or an enriched population of neuronal, microglial, and / or astrocytic exosomes using mass spectrometry to derive a proteome of the health condition-specific enriched neuronal, microglial, and / or astrocytic exosomes.

39. The method according to claim 37 or 38, wherein the method further comprises use of R analysis and bioinformatic analysis of the proteome.

40. The method according to claim 37, further comprising enriching the health-condition- specific or an enriched population of neuronal, microglial, and / or astrocytic exosomes using at least one antibody to at least one health-condition-specific or neuronal, microglial, and / or astrocytic-specific marker immobilized on a substrate to bind to the health-condition-specific or an enriched population of neuronal, microglial, and / or astrocytic exosome-specific marker and eluting the bound health-condition-specific and / or neuronal, microglial, and / or astrocytic exosomes and enriching the population of marker-specific health condition specific and / or neuronal, microglial, and / or astrocytic exosomes.

41. The method according to claim 37, further comprising identifying molecules comprising proteins, peptides or other molecules expressed on a surface of a health condition specific and / or a neuronal, microglial, and / or astrocytic exosome associated with a health condition and diagnosing onset, presence, progression, or amelioration of the health condition based on presence of proteins or peptides or other molecules expressed on the surface of a neuronal, microglial, and / or astrocytic exosome associated with a health condition compared to a control population. 45 96555783.

142. The method according to any one of claims 37-41, wherein the neuronal, microglial, and / or astrocytic exosome specific marker comprises one or more of any one of the markers listing in any one of Tables 2-4.

43. The method according to any one of claims 37-41, wherein the neuronal, microglial, and / or astrocytic exosome specific marker comprises one or more of NCAM, ATP1A3, GFAP, UCHL1, NFL, Tau, Aβ40, Aβ42, SSC5D, Emilin, MCT4, VDAC, Iba1, pTau181, and / or CD63, CD81, and CD9.

44. A kit comprising a composition according to any one of claims 15-26; and at least one container. 46 96555783.1

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