Methods for isolating selected populations of exosomes

Abstract

The present invention relates to the isolation of selected populations of exosomes with high specificity, thereby enabling accurate determination of exosomal protein content useful for predicting and identifying subjects with Parkinson's disease, and distinguishing Parkinson's disease from atypical parkinsonism, including MSA.

Description

[Technical field]

[0001] The present invention relates to methods for isolating selected populations of exosomes and determining the exosomal protein content. [Background technology]

[0002] Parkinson's disease (PD) is the most common movement disorder with a long prodromal phase (1, 2) and risk of progression to dementia (3). These disease phases are widely correlated with the development of Lewy bodies and neuritic lesions (5) accompanied by the accumulation and aggregation of α-synuclein (4).

[0003] The earliest phase of PD is also called preclinical PD, during which neurodegeneration begins but there are no obvious symptoms or signs of disease.The disease then progresses to the prodromal phase, during which symptoms and signs of disease are present but are not yet sufficient to define the disease.The prodromal phase is extremely long (more than 10 years in many patients) and surprisingly diverse, with multiple non-motor and motor symptoms, including hyposmia, anxiety, constipation, fatigue and slight bradykinesia.The clinical diagnosis of PD is usually made when classical motor signs are present, and the three basic motor signs of PD are resting tremor, rigidity and bradykinesia.

[0004] However, there is a significant rate of PD misdiagnosis, while many patients with PD in society remain undiagnosed. Definitive diagnosis of the disease can only be made at autopsy. In the early stages of the disease, PD and other forms of degenerative parkinsonism share common features, and clinical distinction can be difficult (6).

[0005] Currently, no test exists in clinical practice that can predict risk or reliably distinguish PD from unrelated neurodegenerative conditions. Such a test would provide significant clinical benefit by allowing a more accurate diagnosis of PD at an early stage, so that appropriate treatment therapy could be initiated earlier, thereby offering individuals a greater chance of maintaining long-term independence and a high quality of life.

[0006] Given that abnormal α-synuclein accumulation is a major component of PD pathology, α-synuclein has been investigated as a potential biomarker for the diagnosis of PD and / or an indicator of disease progression. α-synuclein can be found in cerebrospinal fluid (CSF). Although total α-synuclein in cerebrospinal fluid (CSF) has been found to be decreased in PD patients compared to controls (7), meta-analyses showed insufficient diagnostic accuracy, with pooled sensitivity between 78–88% and specificity between 40–57% (8). Furthermore, the invasiveness of CSF sample collection by lumbar puncture means that this approach is not ideal for routine monitoring.

[0007] α-synuclein can also be found in peripheral body fluids (9). The concentration of α-synuclein in blood is strongly influenced by red blood cells, which are the source of >99% of the protein (10). Therefore, the blood content of total free α-synuclein in PD patients is of limited usefulness, in part due to contamination by red blood cell hemolysis (11).

[0008] α-synuclein can be found associated with exosomes. The composition and function of circulating exosomes are altered in PD (12). Although reports on whether the total α-synuclein content of exosomes is increased in PD patients are inconsistent (12, 13, 14), analysis of a population of exosomes released from neural tissues in plasma (i.e., plasma neuron-derived exosomes) showed that α-synuclein content was increased in PD patients, with a weak correlation with disease severity (15). However, this study only shows the utility of α-synuclein as a biomarker in patients already diagnosed with PD.

[0009] There is a need for new, minimally invasive tests to provide an accurate diagnosis of PD at an early stage, and in particular improved discrimination between PD and other forms of degenerative parkinsonism. It is an object of the present invention to meet these needs. Summary of the Invention [Means for solving the problem]

[0010] The present inventors have surprisingly confirmed that certain proteins in exosomes released from neural tissues in blood (i.e., neuron-derived exosomes) may be useful as biomarkers for Parkinson's disease, especially in the early phase of the disease. In particular, the present inventors have found that increased α-synuclein release in serum neural exosomes precedes the diagnosis of PD and persists with disease progression. In combination with clusterin, α-synuclein is an evolving predictive marker of α-synucleinopathy that may be clinically considered in stratifying at-risk patient groups or monitoring α-synuclein-targeted therapy.

[0011] We evaluated the protein content of neuron-derived exosomes in blood samples from subjects with neurodegenerative conditions spanning the spectrum of Lewy body pathology, i.e. conditions characterized by α-synucleinopathy including early to late stage PD, and non-α-synucleinoproteinopathies (e.g., frontotemporal dementia (FTD), progressive supranuclear palsy (PSP) and corticobasal syndrome (CBS)). We found that the mean α-synuclein content in neuron-derived exosomes in blood was increased 2-fold in prodromal and clinical PD when compared to controls or other neurodegenerative conditions (p<0.0001). In 314 subjects in the training group and 105 subjects in the validation group, α-synuclein content in neuron-derived exosomes in blood showed consistent performance (AUC=0.86) in separating clinical PD from controls across populations. Longitudinal sample analysis showed that, in contrast to previous observations ( 15 ), α-synuclein in neuron-derived exosomes in the blood remained stably elevated with PD progression.

[0012] Without wishing to be bound by theory, data suggest that release of α-synuclein from neural tissue is a specific pathophysiological response in PD that precedes clinical diagnosis and persists as the disease progresses. Thus, α-synuclein may be a useful biomarker for predicting PD and distinguishing conditions characterized by α-synuclein (such as PD and related conditions (e.g., PD with dementia and MSA)) from conditions characterized by non-α-synucleinoproteinopathies.

[0013] Furthermore, the inventors found that clusterin content in neuron-derived exosomes in blood was elevated in subjects with neurodegenerative conditions characterized by non-α-synucleinoproteinopathies (e.g., frontotemporal dementia (FTD), progressive supranuclear palsy (PSP) and corticobasal syndrome (CBS)) (p<0.0001), but not in subjects with conditions characterized by Lewy body pathology, i.e., α-synucleinopathies (e.g., prodromal, motor and cognitive stages of PD). Thus, clusterin may be a useful biomarker for predicting and diagnosing neurodegenerative conditions characterized by non-α-synucleinoproteinopathies, particularly tauopathies.

[0014] Combined measurement of α-synuclein and clusterin in neuron-derived exosomes in blood differentiated subjects with basal α-synucleinopathy from those with non-α-synucleinoproteinopathies with an AUC=0.98. Thus, clusterin can be used in combination with α-synuclein to improve diagnostic power for predicting PD and to distinguish conditions characterized by α-synuclein (such as PD and related conditions (e.g., PD with dementia and MSA)) from conditions characterized by non-α-synucleinoproteinopathies.

[0015] We also found that the average exosomal α-synuclein was increased by 2-fold in prodromal and clinical Parkinson's disease when compared with MSA. Furthermore, the combined measurement of neuron-derived exosomal α-synuclein and clusterin predicted Parkinson's disease from MSA with AUC=0.94. Therefore, exosomal α-synuclein alone or in combination with clusterin can also be used to distinguish PD from its related conditions (e.g., conditions with similar signs and symptoms, such as atypical Parkinson's syndromes, including MSA).

[0016] Thus, the inventors have found that levels of alpha-synuclein and clusterin provide a diagnostic indication of a subject susceptible to or having PD, and that this can be determined in a method for analyzing a blood sample from a subject comprising determining the levels of alpha-synuclein and clusterin in neuron-derived exosomes in the blood sample.

[0017] These methods require the extraction of a selected population of exosomes from blood samples and the analysis of certain protein content in exosomes. Immunoassays involving binding to exosomes have been described in the art. For example, reference 16 describes a method involving immunoaffinity beads designed to capture exosomes through the recognition of epithelial cell adhesion molecule (EpCAM), a biomarker protein of exosomes. The beads are coated with polyacrylic acid to provide functional binding sites, and then conjugated with sulfobetaine (an antifouling zwitterion). Anti-EpCAM antibodies are then conjugated to the sulfobetaine molecules.

[0018] However, the present inventors have found that using prior art methods, such as those described in 15, determining the level of a particular protein in neural exosomes is not accurate enough to provide a useful diagnostic indicator for predicting PD. In particular, the present method requires the isolation of only a specific selected population of exosomes. This requires an assay with a high level of specificity for the desired exosomes. Furthermore, the determination of a certain protein level in this selected population of exosomes requires that the exosome sample is extracted with very low levels of interfering biomolecules. Therefore, the present inventors have recognized that existing methodologies would not be sufficient to study the protein content of neuron-derived exosomes, and have improved a method for selectively isolating this population of exosomes from blood samples.

[0019] The inventors have found that by growing a zwitterionic polymer on the surface of the particle and conjugating a ligand having affinity for a selected population of exosomes to the zwitterionic polymer, greater selectivity for the desired exosomes can be achieved. Thus, the present invention also provides coated particles having a coating comprising a zwitterionic polymer coupled to a ligand having affinity for a selected population of exosomes.

[0020] The present invention also provides a method of isolating exosomes from a sample comprising the steps of contacting the sample with coated particles of the present invention; removing unbound sample; and separating the captured exosomes.

[0021] Zwitterionic materials are effective in preventing non-selective binding of biological materials due to their ability to bind water molecules and provide a high degree of hydration. The coated particles described herein have a high surface coverage of zwitterionic polymers, minimizing the available surface to which biological molecules can bind. Furthermore, the polymer typically grows outward from the surface of the polymer in a brush-like manner. This results in a higher degree of hydration around the particle than can be achieved using a coating of non-polymeric zwitterionic molecules. It also results in a high degree of conformational entropy due to the motion of the polymer chains. All of these factors result in coated particles that are highly effective in minimizing interactions with non-specific biological molecules.

[0022] It is not trivial to attach zwitterionic polymers to small particles such as nanoparticles. Thus, previous methods for isolating exosomes have used simpler processes involving, for example, the attachment of a monolayer of zwitterionic molecules. However, the inventors have identified the need for greater selectivity, without which the predictive value of the identified markers is significantly reduced. The coated particles and methods for capturing exosomes described herein provide effective isolation of the desired exosomes, thereby allowing accurate determination of their protein content. Using these methods, the protein content of exosomes can be measured down to pg / mL levels.

[0023] The present invention also provides kits comprising coated particles of the invention for isolating a selected population of exosomes from a blood sample, and / or reagents for determining levels of alpha-synuclein and clusterin in neuron-derived exosomes in a blood sample.

[0024] The present invention also provides a method for analyzing a blood sample from a subject, comprising determining levels of alpha-synuclein and clusterin in neuron-derived exosomes in the blood sample, wherein the levels of alpha-synuclein and clusterin provide a diagnostic indication for a subject susceptible to or having PD.

[0025] The present invention also provides a method for analyzing a blood sample from a subject, comprising determining levels of α-synuclein and clusterin in neuron-derived exosomes in the blood sample.

[0026] The present invention also provides a method for analyzing a blood sample from a subject having one or more signs or symptoms of Parkinsonism and not diagnosed with PD, comprising determining the level of alpha-synuclein in neuron-derived exosomes in the blood sample, wherein the level of alpha-synuclein provides a diagnostic indication of the subject being susceptible to PD.

[0027] The invention also provides methods of distinguishing conditions characterized by α-synuclein (such as PD and related conditions (e.g., PD with dementia and MSA)) from conditions characterized by non-α-synucleinoproteinopathies comprising analyzing a blood sample from a subject according to any of the methods of the invention.

[0028] The invention also provides a method for identifying a subject susceptible to PD comprising analysing a blood sample from the subject according to any of the methods of the invention.

[0029] The present invention also provides a method of preventing and / or treating PD in a subject comprising identifying a subject susceptible to PD according to any of the methods of the present invention and treating the subject with a therapy for PD.

[0030] The invention also provides methods for monitoring the effectiveness of an alpha-synuclein targeted therapy, such as a therapy for PD, being administered to a subject comprising analysing a blood sample from the subject according to the methods of the invention, wherein each biomarker is measured at two or more different time points, and wherein changes in the level of each biomarker over time indicate whether the disease is improving or worsening.

[0031] The present invention also provides the use of alpha-synuclein, and optionally clusterin, as biomarker(s) to provide a diagnostic indication of subjects susceptible to PD and / or to distinguish conditions characterized by alpha-synuclein (such as PD and related conditions (e.g., PD with dementia and MSA)) from conditions characterized by non-alpha-synucleinoproteinopathies.

[0032] The present invention also provides the use of alpha-synuclein and clusterin as biomarkers to provide a diagnostic indication of subjects with Parkinson's disease.

[0033] The present invention also provides the use of clusterin as a biomarker to provide a diagnostic indication of subjects susceptible to or having a tauopathy.

[0034] The present invention also provides a method for analyzing a blood sample from a subject comprising determining the level of clusterin in neuron-derived exosomes, where an increased level of clusterin provides a diagnostic indication of a subject susceptible to or having a tauopathy.

[0035] The present invention also provides a method for analyzing a blood sample from a subject, comprising the step of determining the level of clusterin in neuron-derived exosomes. [Brief description of the drawings]

[0036] [Figure 1]α-synuclein content in neuron-derived exosomes in blood samples from patients across the spectrum of Lewy body pathology (i.e., with conditions characterized by α-synucleinopathy). (A) Boxplot of mean total α-synuclein across the spectrum of conditions with Lewy body pathology (REM sleep behavior disorder (RBD), motor PD, PD dementia (PDD), dementia with Lewy bodies (DLB)) and unrelated neurodegenerative conditions (frontotemporal dementia (FTD), progressive supranuclear palsy (PSP), corticobasal syndrome (CBS)), as well as age- and sex-matched controls. A two-fold increase in α-synuclein content was detected in L1CAM-positive exosomes isolated from conditions characterized by α-synuclein pathology. (B) At the limit of detection (0.5 pg / ml), PSer129 α-synuclein was only detected in a small subgroup (28.6%) of PD patients tested. No significant correlation was found between exosomal α-synuclein and either the Unified Parkinson's Disease Rating Scale (UPDRS) (Panel C, r=0.0267) or the Montreal Cognitive Assessment (MoCA) (Panel D, r=0.0621). **p<0.01, ****p<0.0001. Means with interquartile ranges and whisker ranges using standard deviations with coefficient 1 for exosomal markers were used in box plots. [Diagram 2]Samples blood neuron-derived exosome clusterin content increased in tauopathies and improved differential diagnosis when combined with α-synuclein. (A) Serum neuroexosome clusterin (clu) release is increased in FTD, PSP and CBS, but not in RBD, PD, PDD, DLB or age- and sex-matched controls. (B) The ratio of α-synuclein to clusterin improved the separation between α-synucleinopathies and other proteinopathies. Receiver operating characteristic (ROC) analysis of individual markers and their ratio or linear regression analysis of the combined measure revealed improved predictive power of the two biomarkers in differentiating prodromal or clinical PD from other proteinopathies, as shown in panels C and D. Clinical PD refers to the combined group of PD and PDD (***p<0.001, ****p<0.0001). [Diagram 3] Cut-off value estimation of α-synuclein in neuron-derived exosomes in blood across cohorts. Box plots of mean exosomal α-synuclein levels and corresponding ROC curves in the training (A) and validation (B) groups. When the cut-off value of exosomal α-synuclein (≥ 14.21 pg / mL) estimated from the training (Keil and Brescia) group was applied to the validation group (Oxford), assay performance analysis revealed consistent results across populations with similar area under the curve (AUC), sensitivity (Sens), specificity (Spec), positive (PPV) and negative (NPV) predictive values ​​in distinguishing clinical PD from controls, as shown in panel C. [Figure 4]Longitudinal analysis of α-synuclein and clusterin content in neuron-derived exosomes in blood samples. Linear mixed models of exosomal α-synuclein (A) and clusterin (B) were fitted to longitudinal values ​​over time from first sampling as covariants, and patients were stratified by levels at first visit relative to the median. When clinical PD was compared with control samples, persistent separation between disease subgroups and controls was confirmed, but no overall significant difference in slope from 0 was identified. Clinical PD refers to the combined PD and PDD group. Patient characteristics and p-values ​​are summarized in panel C. [Diagram 5] 1 shows the molecular structure of carboxybetaine methacrylate (CBMA) monomer and the nuclear magnetic resonance (NMR) spectrum of CBMA in DO. [Figure 6] (A) Fourier transform infrared attenuated total reflectance (FTIR-ATR) spectra of pCBMA-coated beads along with bare iron oxide beads and CBMA monomer used as a control, showing reduced adsorption of BSA (B) or serum proteins (C) on pCBMA-coated beads compared to commercial epoxy beads, both of which were conjugated to anti-HA antibodies. [Figure 7A] Preparation of pCBMA-based zwitterionic magnetic beads and immunocapture of exosomes. (A) Synthesis and application of pCBMA-coated magnetic microbeads for immunocapture of L1CAM-positive neural exosomes in serum. [Figure 7B] (B) SEM of anti-L1CAM-conjugated or control pCBMA-coated beads demonstrating immunocapture of exosomes from serum (scale bar, 200 nm). [Figure 7C] Preparation of pCBMA-based zwitterionic magnetic beads and immunocapture of exosomes. (C) Lysates of immunocaptured vesicles contain transmembrane (CD81 and L1CAM) and internal exosomal proteins (Tsg101 and syntenin-1) as shown by immunoblotting. [Figure 7D] Preparation of pCBMA-based zwitterionic magnetic beads and immunocapture of exosomes. (D) GO analysis of proteins identified by mass spectrometry revealed enriched terms for exosome and related extracellular vesicle functions. [Figure 7E] Preparation of pCBMA-based zwitterionic magnetic beads and immunocapture of exosomes. (E) List of bona fide exosomal proteins and top hits identified by mass spectrometry. [Figure 8A] Specific detection by triplex electrochemiluminescence of α-synuclein in serum exosomes immunocaptured with anti-CD9 (total exosome population), anti-L1CAM (neural exosome subpopulation) or anti-HA (control antibody against an epitope not present on exosomes). [Figure 8B] Specific detection by triplex electrochemiluminescence of syntenin-1 in serum exosomes immunocaptured with anti-CD9 (total exosome population), anti-L1CAM (neural exosome subpopulation) or anti-HA (control antibody against an epitope not present on exosomes). [Figure 8C] Specific detection by triplex electrochemiluminescence of clusterin in serum exosomes immunocaptured with anti-CD9 (total exosome population), anti-L1CAM (neural exosome subpopulation) or anti-HA (control antibody against an epitope not present on exosomes). [Figure 9] Figure 1 shows syntenin-1 content in neuron-derived exosomes in blood samples from subjects across disease groups. No disease-specific distribution patterns were detected across groups that could contribute significantly to biomarker development. [Figure 10]The development of an electrochemiluminescence assay for the detection of pSer129 α-synuclein is shown. (A) Information of the antibody pair used, (B) specificity test and (C) reproducibility. The LLOD of pSer129 α-synuclein is 2.11 pg / mL. It should be pointed out that the protein in the exosome lysate was concentrated 10-fold: 500 μL of serum input was used for exosome capture and dissolved in 50 μL lysis buffer (concentration factor is 10). A calibration curve was used to detect the biomarker in the lysate (e.g., if the concentration of the marker in the lysate is 5 pg / mL, the concentration of the marker in the serum is 5 / 10 pg / mL = 0.5 pg exosome marker / mL serum). For pSer129 α-synuclein, the LLOD is 2.11 pg / mL in the lysate and 0.211 pg / mL of exosomal pSer129 α-synuclein in serum. Therefore, to compare the results between groups, 0.5 pg / mL was considered as the cutoff for detection of exosomal pSer129 a-synuclein in serum. [Figure 11] Figure 1 shows exosomal syntenin-1 levels across disease groups. No disease-specific distribution patterns were detected across groups that could contribute significantly to biomarker development. [Figure 12] An exemplary method for performing surface-initiated RAFT polymerization on the surface of a particle is provided. [Figure 13]Neuron-derived exosomal α-synuclein is increased across the spectrum of Lewy body pathology. (A) Boxplots of mean total α-synuclein across the spectrum of conditions with Lewy body pathology (RBD, motor PD, PDD, DLB), MSA and unrelated neurodegenerative diseases (FTD, PSP, CBS), and age- and sex-matched controls. A two-fold increase in α-synuclein content was detected in L1CAM-positive exosomes isolated from conditions characterized by Lewy body pathology. (B) At the lowest detectable concentration (0.32 pg / ml), pSer129 α-synuclein was detected in a subgroup (55.8%) of PD patients tested. No significant correlation was found between total exosomal α-synuclein and either UPDRS (panel C, r=0.0267) or MoCA (panel D, r=0.0621) in PD patient samples. **p<0.01, ***p<0.001, ****p<0.0001. Means with interquartile ranges and whisker ranges with SD of coefficient 1 for exosomal markers were used in box plots. [Figure 14]Neuron-derived exosomal clusterin is increased in tauopathies and improved differential diagnosis when combined with α-synuclein. (A) Serum neuroexosomal clusterin (clu) release is increased in FTD, PSP and CBS, but not in RBD, PD, PDD, DLB, MSA or age- and sex-matched controls. (B) The ratio of α-synuclein to clusterin improved the separation between Lewy body pathology and another proteinopathies. (C) Heatmap illustration of exosomal profiles using α-Syn, Clu or α-Syn / Clu to differentiate between diseases. The change in concentration of each exosomal marker was normalized to the value of HC. ROC analysis of individual markers and their ratios or linear regression analysis of the combined measurements revealed an additive effect of the two biomarkers in differentiating prodromal or clinical PD from another proteinopathies as shown in panels D and F, and from MSA as shown in panels E and G. Clinical PD refers to the combined group of PD and PDD (**p<0.01, ***p<0.001, ****p<0.0001). [Figure 15]

[0043] Figure 1 shows exosomal syntenin-1 levels across disease groups. No disease-specific distribution patterns were detected across groups that could contribute significantly to biomarker development. [Figure 16] Histograms showing quantitative assessment of BSA adsorption onto different magnetic bead (MB) surfaces (1 mg bead load). Error bars represent standard deviation of three separately collected experimental data sets. [Figure 17A] Figure 17 includes Figure 17A, Figure 17B, Figure 17C, and Figure 17D. (A) Histogram showing quantified adsorption of recombinant α-Syn on different Ab-modified pCBMA@Fe3O4 MB surfaces. Commercially available carboxylate-terminated MBs were used as controls. [Figure 17B] (B) SEM images of serum-captured exosomes on anti-L1CAM-modified MBs versus anti-HA (control)-modified MBs (inset). Scale bar 1 μm. [Figure 17C](C) Immunoblotting of lysates of immunocaptured vesicles confirming detection of both transmembrane proteins (L1CAM, CD81) and the internal protein Synt-1 from exosomes. [Figure 17D] Specific electrochemiluminescence detection of α-Syn(D) in neural exosomes immunocaptured from serum using anti-L1CAM vs. anti-HA (control) modified pCBMA@Fe3O4 MBs [Figure 18] Relative responses of anti-syntenin-1 modified sensors to 10-3 g / mL CRP, 10-3 g / mL α-Syn, 10-3 g / mL BSA and 10-9 g / mL Synt-1 are shown. Error bars were calculated from nine measurements: three replicates across three experiments using three independent working electrodes. [Figure 19A] FIG. 1 shows Nyquist curves of an anti-α-Syn modified working electrode against α-Syn spiked into 10% human serum at various concentrations as indicated. [Figure 19B] 1 shows Nyquist curves of an anti-synthein-1 modified working electrode against Synt-1 spiked into 10% human serum at various concentrations as indicated. [Figure 20A] Impedance calibration curve for α-Syn spiked into 10% human serum with a dynamic range of 10-104 pg / mL. Error bars were calculated from nine measurements: three replicates across three experiments using three independent working electrodes. [Figure 20B] Impedance calibration curve for Synt-1 spiked into 10% human serum in the concentration range 10-104 ng / mL. Error bars were calculated from 9 measurements: 3 replicates across 3 experiments using 3 independent working electrodes. Error bars were calculated from 9 measurements: 3 replicates across 3 experiments using 3 independent working electrodes. [Figure 21] Box plots of α-synuclein levels across different disease and healthy control groups are shown. **P<0.01, ***P<0.001, ****P<0.0001. Means with IQRs and whisker ranges with SD of coefficient 1 for exosomal markers were used in the box plots. [Figure 22A] 4 shows the ROC curve representing the diagnostic mode using α-synuclein as a separating feature for RBD vs. PSP+CBS. [Figure 22B] 4 shows the ROC curve representing the diagnostic mode using α-synuclein as a feature for separation of RBD vs. MSA. [Figure 22C] 4 shows the ROC curve representing the diagnostic mode using α-synuclein as a separating feature for PD vs. PSP+CBS. [Figure 22D] 4 shows the ROC curve representing the diagnostic mode using α-synuclein as a separating feature for PD vs. MSA. [Figure 23] Box plots of clusterin levels across different disease groups and healthy control groups are shown. **P<0.01, ***P<0.001, ****P<0.0001. Means with IQRs and whisker ranges with SD of coefficient 1 for exosomal markers were used in the box plots. [Figure 24] Box plots of α-synuclein / clusterin levels across different disease groups and healthy control groups are shown. **P<0.01, ***P<0.001, ****P<0.0001. Means with IQRs and whisker ranges with SD of coefficient 1 for exosomal markers were used in the box plots. [Figure 25A] 4 shows the ROC curve representing the diagnostic mode using α-Syn / Clu as the separation feature for RBD vs. MSA. [Figure 25B] 4 shows the ROC curve representing the diagnostic mode using α-Syn / Clu as the separating feature for RBD vs. PSP+CBS. [Figure 25C] 4 shows the ROC curve representing the diagnostic mode using α-Syn / Clu as the separating feature for PD vs. MSA. [Figure 25D] 4 shows the ROC curve representing the diagnostic mode using α-Syn / Clu as features for separation of PD vs. PSP+CBS. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] Biomarkers of the Invention Alpha-synuclein The method of the present invention may include detecting and determining the protein level of α-synuclein in neuron-derived exosomes from blood samples. α-synuclein has been well described in the art (see, for example, 5) and is also known as SNCA, NACP, PARK1, PARK4, PD1, or synuclein alpha. The specific protein sequence of α-synuclein is not limiting to the present invention. The present invention includes detecting and measuring the level of polymorphic variants of these proteins, or modified versions of these proteins, such as post-translationally modified versions, such as phosphorylated α-synuclein at serine 129.

[0038] Alpha-synuclein in neuron-derived exosomes in blood can be used as a predictive and / or diagnostic biomarker for PD. Alpha-synuclein content in neuron-derived exosomes in blood provides a strong distinction between PD (from early to late phases of disease progression) and non-PD (e.g. healthy subjects and subjects with conditions characterized by non-alpha-synuclein proteinopathy) subjects. In particular, the inventors found that the alpha-synuclein content in neuron-derived exosomes in blood of PD subjects (from early to late phases) was significantly increased compared to non-PD subjects. The average alpha-synuclein content in neuron-derived exosomes in blood of non-PD subjects is between about 12-13 pg / ml. For example, in the example below, the average alpha-synuclein content in neuron-derived exosomes in blood samples of non-PD subjects was measured to be 12.91 ± 5.93 pg / mL (+ / - SD).

[0039] Thus, the method for analyzing a subject sample can serve as a method for identifying whether a subject is susceptible to PD, i.e., predicting whether a subject has PD. The method for analyzing a subject sample can serve as a method for diagnosing whether a subject has PD. The method for analyzing a subject sample can also serve as a method for distinguishing conditions characterized by α-synuclein (such as PD and related conditions (e.g., PD with dementia and MSA)) from conditions characterized by PD resulting from neurodegenerative diseases with non-α-synuclein proteinopathy. The method for analyzing a subject sample can also serve as a method for distinguishing PD from conditions with similar signs and symptoms, such as its related conditions, such as atypical parkinsonism including MSA.

[0040] Clusterin The method of the present invention may include detecting and determining the protein level of clusterin in neuron-derived exosomes from blood samples. Clusterin is well known in the art (see, for example, 17) and is also known as CLU, AAG4, APO-J, APOJ, CLI, CLU1, CLU2, KUB1, NA1 / NA2, SGP-2, SGP2, SP-40, or TRPM2. The specific protein sequence of clusterin is not limiting to the present invention. The present invention includes detecting and measuring the level of polymorphic variants of these proteins, or modified versions of these proteins, such as post-translationally modified versions.

[0041] Clusterin in neuron-derived exosomes in blood can also be used as a predictive and / or diagnostic biomarker for PD. It was found that the clusterin content in neuron-derived exosomes in blood remains at a similar level to healthy subjects throughout the disease progression of PD. The average clusterin content in neuron-derived exosomes in blood of healthy subjects is between about 8-9 ng / ml. For example, in the example below, the average clusterin content in neuron-derived exosomes in blood samples of healthy subjects was measured to be 8.67 ± 4.92 ng / mL (+ / - SD).

[0042] Thus, the method for analyzing a subject sample can serve as a method for identifying whether a subject is susceptible to PD, i.e., predicting whether a subject has PD. The method for analyzing a subject sample can serve as a method for diagnosing whether a subject has PD. The method for analyzing a subject sample can also serve as a method for distinguishing conditions characterized by α-synuclein (such as PD and related conditions (e.g., PD with dementia and MSA)) from conditions characterized by PD resulting from neurodegenerative diseases with non-α-synuclein proteinopathy. The method for analyzing a subject sample can also serve as a method for distinguishing PD from conditions with similar signs and symptoms, such as its related conditions, such as atypical parkinsonism including MSA.

[0043] Clusterin in neuron-derived exosomes in blood can be used as a predictive and / or diagnostic biomarker for tauopathy. Clusterin provides a strong distinction between tauopathy and non-tauopathy subjects (e.g., healthy subjects and subjects with symptoms characterized by α-synucleinopathy). In particular, the inventors found that the clusterin content in neuron-derived exosomes in the blood of tauopathy subjects was significantly increased compared to non-tauopathy subjects. The average clusterin content in neuron-derived exosomes in the blood of non-tauopathy subjects, such as α-synucleinopathy subjects, is between about 9-10 ng / ml. For example, in the example below, the average clusterin content in neuron-derived exosomes in blood samples from PD subjects was measured to be 9.72 ± 6.02 ng / mL.

[0044] Thus, the method for analyzing a subject sample may serve as a method for identifying whether a subject is susceptible to a tauopathy, i.e., predicting whether a subject will have a tauopathy, and / or diagnosing whether a subject has a tauopathy.

[0045] Combination of α-synuclein and clusterin To increase the overall confidence that the assay is giving sensitive and specific results across populations, it is advantageous to analyze both α-synuclein and clusterin levels.Thus, the method of the present invention can include detecting and determining the protein levels of α-synuclein and clusterin in neuron-derived exosomes from blood samples.The level of biomarkers can provide a diagnostic indication of whether a subject is susceptible to PD and / or whether a subject has PD.

[0046] We found that the α-synuclein content in neuron-derived exosomes in the blood of PD subjects (from early to late phase) was significantly increased compared to non-PD subjects. The average α-synuclein content in neuron-derived exosomes in the blood of non-PD subjects is between about 10-20 pg / ml. On the other hand, the clusterin content in neuron-derived exosomes in the blood remained at a similar level to healthy subjects throughout the disease progression of PD, and was found to be significantly increased in subjects with neurodegenerative diseases with non-α-synuclein proteinopathy compared to healthy subjects or subjects with α-synucleinopathy (e.g., from early to late phase of PD). The average clusterin content in neuron-derived exosomes in the blood of healthy subjects or subjects with α-synucleinopathy is between about 7-17 ng / ml. The different behavior of the two biomarkers can strengthen the diagnosis of PD if they are evaluated in the same sample. This combination of biomarkers is most useful to strengthen the distinction seen between PD and non-α-synuclein proteinopathy samples.

[0047] Thus, the method for analyzing a subject sample can serve as a method for identifying whether a subject is susceptible to PD, i.e., predicting whether a subject has PD. The method for analyzing a subject sample can serve as a method for diagnosing whether a subject has PD. Furthermore, the method for analyzing a subject sample can serve as a method for distinguishing conditions characterized by α-synuclein (such as PD and related conditions (e.g., PD with dementia and MSA)) from conditions characterized by non-α-synuclein proteinopathy. The method for analyzing a subject sample can also serve as a method for distinguishing PD from its related conditions, such as conditions with similar signs and symptoms, such as MSA.

[0048] sample The present invention analyzes a blood sample from a subject. In some embodiments, the method of the present invention includes the initial step of obtaining a blood sample from a subject. However, in other embodiments, the blood sample is obtained separately from and prior to performing the method of the present invention. After the blood sample is obtained, the method of the present invention can be performed in vitro.

[0049] Biomarker detection may be performed directly on the sample taken from the subject, or the sample may be processed between the time it is taken from the subject and the time it is analyzed. For example, a blood sample may be processed by adding an anticoagulant (e.g., EDTA), followed by removal of cells and cell debris, leaving the plasma containing the exosomes for analysis. Alternatively, the blood sample may be coagulated, followed by removal of cells and various clotting factors, leaving the serum containing the exosomes for analysis. For example, in the example below, the level of the biomarker was determined in a serum sample. Once the plasma or serum is prepared, the sample may be aliquoted and frozen prior to biomarker detection.

[0050] In certain embodiments of the present invention, the subject has one or more signs or symptoms of parkinsonism and has not been diagnosed with PD. The present invention may further comprise identifying a subject having one or more signs or symptoms of parkinsonism and has not been diagnosed with PD. Clinical criteria for diagnosing PD, such as the UK Parkinson's Disease Society Brain Bank (UKPDSBB) criteria (18), Gelb criteria (19) or Movement Disorder Society (MDS) PD criteria (20), are well described in the art. A subject is not diagnosed with PD unless it meets the requirements described in any of these clinical PD criteria.

[0051] Parkinsonism encompasses several conditions, including PD and other conditions with similar symptoms such as tremor, bradykinesia, rigidity and postural instability (e.g., primary progressive aphasia (FTD), progressive supranuclear palsy (PSP), corticobasal syndrome (CBS), drug-induced parkinsonism, multiple system atrophy (MSA) and / or vascular parkinsonism). Signs and symptoms of parkinsonism are well described in the art, see, for example, reference (21). For example, signs and symptoms may include one or more of non-motor signs such as altered handwriting, rolling over, gait disturbance, salivary disturbance, speech disturbance, reduced facial expression, rigidity, balance disturbance, resting tremor, bradykinesia (slow movement), and / or postural instability. The signs and symptoms may include one or more of non-motor signs, such as rapid eye movement sleep behavior disorder (RBD), olfactory dysfunction, constipation, excessive daytime sleepiness, symptomatic hypotension, erectile dysfunction, a diagnosis of urinary dysfunction, and / or a diagnosis of depression. The signs and symptoms may include abnormal tracer uptake in the presynaptic dopaminergic system.

[0052] The subject may be in the early stage of PD, but for example, asymptomatic in the preclinical stage of PD.The subject may be in the prodromal stage of PD, for example, the subject may be presymptomatic of PD or may already show clinical symptoms.The signs and symptoms of the early phase of PD are known in the art, for example, as described in Reference 1.

[0053] For subjects who have already shown some clinical PD symptoms, the present invention can be used to confirm or settle on another diagnosis.For example, subjects can be suspected of having other forms of degenerative parkinism or other symptoms that affect movement.For example, subjects can be suspected of having primary progressive aphasia (FTD), progressive supranuclear palsy (PSP), corticobasal syndrome (CBS), drug-induced parkinsonism, multiple system atrophy (MSA), vascular parkinsonism and / or benign essential tremor.Symptoms of these disorders are known in the art (see, for example, 21).

[0054] The invention is particularly useful for distinguishing conditions characterized by alpha-synuclein, such as PD and related conditions (e.g., PD with dementia and MSA), from conditions characterized by non-alpha-synucleinoproteinopathies. Thus, a subject may be suspected of having PD, FTD, PSP or CBS.

[0055] The present invention is particularly useful in distinguishing PD from its related conditions (eg, conditions that have similar signs and symptoms, such as atypical parkinsonian conditions, including MSA).

[0056] The subject may have already begun treatment, for example, an α-synuclein targeted therapy such as immunotherapy (e.g., anti-α-synuclein antibody therapy), phenylbutyrate-triglyceride (PBT), NPT200-11, nilotinib, ambroxol, or ENT-01, which are ongoing clinical trials targeting α-synuclein to protect brain cells and slow PD.

[0057] In certain embodiments of the present invention, it is intended that the present invention can be relatively easy and / or inexpensive to carry out, in that the present invention is not limited to use with subjects who are already suspected of having PD.Rather, it can be used to screen general population or high-risk population, for example subjects who are at least 50 years old (for example, 50 years old or older, 55 years old or older, 60 years old or older, 65 years old or older, 70 years old or older).Subjects who are at least 50 years old tend to develop PD.

[0058] The subject may already be known to be predisposed to developing PD, for example, due to family or genetic relationship. For example, the subject may contain mutations in the following genes: alpha-synuclein (Park1), parkin (Park2), DJ-1 (Park7), UCHL1 (Park5), A.53T, A30P and / or E46K. In other embodiments, the subject may not have such a predisposition and may develop the disease as a result of environmental factors, for example, as a result of exposure to certain chemicals (such as toxins or medicines), as a result of diet, as a result of infection, etc.

[0059] Subjects may be identified by questionnaire to screen for relevant prodromal PD signs and symptoms (eg, sleep disorders, anosmia, anxiety, anorexia) followed by blood testing for genetic mutations associated with PD.

[0060] The subject is typically a human. However, in some embodiments, the present invention is useful in non-human organisms, such as mice, rats, rabbits, guinea pigs, cats, dogs, horses, pigs, cows, or non-human primates (monkeys or apes, such as macaques or chimpanzees). In non-human embodiments, any method for use in detecting proteins according to the present invention is typically based on non-human orthologs related to the human proteins disclosed herein. In some embodiments, animals can be used experimentally to monitor the effect of therapeutic agents on certain biomarkers.

[0061] Exosomes The present invention analyzes biomarker content in exosomes from blood samples of subjects. Exosomes are double-membrane vesicles (40-120 nm) released by most cell types, including neurons (22). The composition and function of circulating exosomes are altered in subjects with PD, especially exosomes released from CNS tissues (e.g., neuron-derived exosomes) (12). The inventors surprisingly found that the composition of circulating exosomes is also altered in subjects susceptible to PD. Thus, protein content in exosomes in blood samples can be used as a biomarker for PD, from early to late phases of PD.

[0062] In some embodiments, the methods of the present invention further comprise the step of isolating exosomes from a blood sample of the subject, however, in other embodiments, the exosomes are isolated separately from and prior to performing the methods of the present invention.

[0063] Exosomes can be isolated from blood samples using multiple methods, including ultracentrifugation, immunomagnetic beads, and / or chromatography.In addition, exosomes have a lipid bilayer, and therefore RNAse treatment before use ensures that the cargo used downstream is encapsulated within the vesicle.Exosomes can be identified using Western blot or mass spectrometry using proteins involved in the biogenesis of intraluminal vesicles, including tetraspanins (e.g., CD9, CD63, and / or CD81) and / or proteins involved in the endosomal sorting and transport complex (ESCRT) machinery required for biogenesis (e.g., PDCD6IP, TSG101, VPS28, VPS37, VPS25, VPS36, SNF8, and / or CHMP).

[0064] The present invention refers to determining the level of a biomarker(s) in a population of selected exosomes in a blood sample. The selected population of exosomes may be exosomes released from CNS tissues, such as neurons. The selected population of exosomes released from neurons is referred to herein as neuron-derived exosomes. Thus, the selected population of exosomes may include neuronal proteins. For example, exosomes released from developing and mature hippocampal neurons contain L1 cell adhesion molecule (L1CAM) and GluR2 / 3 subunit of glutamate receptor, both of which are known neuronal markers (23, 24). Thus, the selected population of exosomes may include L1CAM. The selected population of exosomes may include GluR2 / 3 subunit of glutamate receptor.

[0065] Ligands with affinity for neuronal markers can be used to capture neuron-derived exosomes. Affinity ligands can be any molecule that binds to a target without also binding to other molecules in the sample. Any type of ligand can be used with the present invention. Ligands can be antibodies that can be designed to target neuronal markers via their antigen binding sites, organic compounds that can dock into binding sites on neuronal markers, inorganic metals that form coordination complexes with specific amino acids of the target neuronal marker, hydrophobic molecules that can bind to non-polar pockets of neuronal markers, and / or proteins with specific binding regions that can interact with neuronal markers. For example, the ligand can be an anti-L1CAM antibody (e.g., Abcam clone UJ127, Cambridge, MA, USA).

[0066] The population of selected exosomes isolated from a blood sample using the methods according to the present invention may have a purity of 70% or more (i.e., 70% or more), 80% or more, 90% or more, 95% or more, 97% or more, 99% or more or 100%.

[0067] Coated particles The affinity ligands described above can be immobilized on the coated particles to capture selected populations of exosomes.

[0068] The present invention also provides coated particles having a coating comprising a zwitterionic polymer coupled to a ligand having affinity for a selected population of exosomes. The coated particles can be prepared by growing the zwitterionic polymer from the surface of the particle using surface-initiated reversible addition-fragmentation chain transfer (RAFT) polymerization. The coated particles are particularly useful for isolating neuron-derived exosomes for use in the methods of the present invention.

[0069] Thus, the present invention also provides a method of isolating exosomes from a sample comprising the steps of contacting the sample with coated particles of the present invention; removing unbound sample; and separating captured exosomes.

[0070] Also described herein is a method of making coated particles, comprising the steps of: (a) growing a zwitterionic polymer on a surface of a particle using reversible addition-fragmentation chain transfer (RAFT) to provide a particle having a coating comprising the zwitterionic polymer; (b) optionally activating the zwitterionic polymer to provide active functional groups on the zwitterionic polymer; and (c) conjugating a ligand having affinity for the selected population of exosomes to the zwitterionic polymer.

[0071] The step of growing the zwitterionic polymer on the surface of the particle comprises generating the polymer in situ, which the inventors have found results in improved coverage and improved anti-fouling properties compared to methods comprising generating the polymer and then attaching it to the surface of the particle.

[0072] Moreover, RAFT has advantages over other radical polymerization processes such as ATRP. In particular, RAFT process does not require metal cations, whereas ATRP process generally requires metal-based catalysts, including copper ions. Such metal ions, especially copper ions, can be toxic when administered to subjects, and are therefore preferably avoided. Furthermore, metal ions can interfere with measurement methods, especially electrochemical measurement methods, carried out on samples that include coated particles. Still further, RAFT can be usefully applied to a wider range of monomers than ATRP process.

[0073] Thus, step (a) typically involves (i) providing a monomer and a particle, and (ii) initiating polymerization to grow a zwitterionic polymer on the surface of the particle using reversible addition-fragmentation chain transfer (RAFT).

[0074] The monomer may be any monomer capable of forming a zwitterionic polymer. For example, the monomer typically comprises a carboxybetaine and / or a sulfobetaine, most preferably a carboxybetaine. Preferably, the monomer is a carboxybetaine methacrylate.

[0075] Step (i) may involve providing one or more such monomers.

[0076] Step (a)(i) also generally involves providing a chain transfer RAFT agent. Any suitable RAFT agent may be used. The RAFT agent may be, for example, bis(carboxymethyl)trithiocarbonate (also called Bittc or BisCTTC).

[0077] Step (i)(a) may further include providing an initiator. Any initiator suitable for the RAFT process may be used. For example, the initiator may be 4,4'-azobis(4-cyanovaleric acid) (ACVA).

[0078] Thus, in a preferred embodiment, step (a)(i) comprises providing a RAFT agent, a monomer, an initiator and particles. In a particularly preferred embodiment, step (a)(i) comprises providing BisCTTC, carboxybetaine methacrylate, ACVA and particles.

[0079] Preferably, the ligand used in step (c) is an antibody. Particularly preferably, the ligand used in step (c) is an anti-L1CAM antibody.

[0080] Prior to step (a), the method may include functionalizing the surface of the particle with a RAFT agent. For example, the method may include functionalizing the surface with BisCTTC prior to step (a).

[0081] The coated particles may include particles of metal, magnetic material, paramagnetic material, glass or epoxy. In general, any immunoassay bead can be used as the particle. Magnetic or paramagnetic particles are preferred. Magnetic beads can be, for example, iron oxide particles, e.g., Fe 3 O 4 The iron oxide particles may, for example, be encapsulated within a polymer matrix. Preferred particles for use in the present invention are those described by references 26 and 27.

[0082] The particles are typically about 30 nm to 5 μm in size, more preferably 50 to 3000 nm, for example 1000 nm to 3000 nm. The particles can be nanoparticles with sizes of 30 nm to 1000 nm, preferably 50 nm to 800 nm or 100 nm to 500 nm. In some embodiments, the particles are about 100 nm to 5 μm in size, for example 500 nm to 3 μm in size.

[0083] The zwitterionic polymer may comprise carboxybetaine, sulfobetaine and / or phosphorylcholine moieties, preferably carboxybetaine and / or sulfobetaine moieties, most preferably carboxybetaine moieties. Typically, the zwitterionic polymer comprises repeat units of zwitterionic monomers. Preferably, the zwitterionic monomer comprises carboxybetaine and / or sulfobetaine, most preferably carboxybetaine. The monomer units may be, for example, acrylates, methacrylates, acrylamides or methacrylamides. Acrylates and methacrylates are preferred due to the reactivity of the functional groups of the carboxylic acid groups.

[0084] The polymer may be poly(carboxybetaine methacrylate) (pCMBA). pCBMA is a highly effective antifouling polymer and also has the advantage of convenient functionalization to allow attachment of desired antibodies.

[0085] The polymer may be a brush polymer, with multiple polymer chains radiating out from a central particle. Particles with attached brush polymers exhibit particularly effective antifouling properties due to their high conformational entropy and ability to repel non-specific biological materials.

[0086] Preferred particles have a high level of polymer coating on their surface.Preferably, at least 20% of the particle surface is coated with polymer, more preferably at least 50% of the surface, most preferably at least 80%, 90% or 95% of the surface is coated with polymer.In a preferred embodiment, at least 98% or at least 99% of the particle surface is coated with polymer.The degree of coating can be determined using visual techniques such as SEM.

[0087] The polymer coating typically has a thickness of at least 10 nm, preferably at least 100 nm, for example a thickness of 10 nm to 500 nm, preferably 100 to 300 nm, for example 100 nm to 200 nm. The thickness of the coating can be determined, for example, using visual techniques such as SEM, by comparing the size of the uncoated particles to the size of the particles to which the zwitterionic polymer has been attached.

[0088] The polymer can be obtained by a RAFT polymerization process, which is the process described herein. Thus, the polymer can be obtained by a RAFT process using bis(carboxymethyl)trithiocarbonate (BCMTTC) as a chain transfer agent.

[0089] The coated particles may be obtained or may be obtainable by growing a zwitterionic polymer on the particle. For example, the coated particles may be obtained or may be obtainable by the processes described herein. Thus, (a) growing a zwitterionic polymer on a surface of a particle using reversible addition-fragmentation chain transfer (RAFT) to provide a particle having a coating comprising the zwitterionic polymer; (b) optionally activating the zwitterionic polymer to provide active functional groups on the zwitterionic polymer; and (c) conjugating a ligand having affinity for the selected population of exosomes to the zwitterionic polymer. The coated particles may be obtained or may be obtainable by a process comprising:

[0090] Preferably, the coated particles are obtained by the process described above.

[0091] The antibody may be covalently attached to a functional group on the zwitterionic polymer, for example, if the polymer is pCBMA, the antibody may be attached to the carboxyl group of the pCBMA.

[0092] The ligand has affinity for neuron-derived exosomes, for example, the ligand is an anti-L1CAM antibody.

[0093] Coated particles are typically produced by growing the polymer from the surface of the particle. An intermediate layer may be present between the particle and the zwitterionic coating, or the zwitterionic coating may be directly attached to the particle. By growing the polymer from the particle surface (as opposed to grafting the formed polymer onto the particle), highly dense polymer coverage of the surface can be achieved, with consistent coverage and avoiding large areas lacking the polymer coating.

[0094] The preferred technique for providing polymer coating is reversible addition fragmentation chain transfer (RAFT).Although polymerization techniques for growing polymers on flat surfaces are known in the art, it may be difficult to grow such polymers from the surface of particles smaller than 5 μm.The inventors have found that the RAFT process is more advantageous than other processes previously used (e.g., atom transfer radical polymerization, or ATRP), and that the RAFT process results in the production of polymer-coated particles with (i) good colloidal stability; (ii) good density and structure of polymer film; and (iii) good non-fouling properties.

[0095] The RAFT technique is typically a surface-initiated RAFT polymerization and can be carried out as described in reference 28. 4,4'-azobis(4-cyanovaleric acid (ACVA) can be used as the initiator. A typical example of polymerization using the RAFT technique to prepare coated particles is shown in Figure 12. The first step in RAFT polymerization is the attachment of a chain transfer agent (CTA) to the surface where polymerization will occur. Suitable materials include 4-cyano-4-(((decylthio)carbonothioyl)thio)pentanoic acid, bis(carboxymethyl)trithiocarbonate (BCMTTC) and 4-cyano-4-(phenylcarbonothioyl)thio)pentanoic acid (CPCTTP). When applying RAFT polymerization to the surface of small particles, the selection of the correct chain transfer agent (CTA) is important and we have found that the use of bis(carboxymethyl)trithiocarbonate (BCMTTC) provides the most beneficial polymer films as assessed by the spectroscopic characteristics of the polymer film, the colloidal stability of the coated particles, and the reduction of nonspecific adsorption.

[0096] Polymerization is typically carried out for a period sufficient to grow a coating thickness of at least 10 nm or at least 100 nm, preferably from 10 nm to 500 nm, more preferably from 100 nm to 300 nm thick.

[0097] The antibody may be conjugated to the zwitterionic polymer coating by providing an activated functional group on the polymer surface and reacting with the antibody. Suitable activated functional groups include, for example, N-hydroxysuccinimide (NHS), which is reactive with free amine groups on the antibody. For example, the carboxylic acid groups of the pCBMA coating may be activated by reacting with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide (EDC / NHS). Typically, a single antibody specific for the desired exosome is attached to the particle. Anti-L1CAM is a preferred antibody. In some embodiments, one or more additional molecules may also be attached to the coating.

[0098] The coated particles are highly selective for the desired biological molecules and have a very low level of non-specific adsorption. The degree of non-specific adsorption can be measured by comparing (a) particles without zwitterionic polymer and (b) particles with zwitterionic polymer. Typically, the degree of non-specific adsorption to the particles of the present invention is less than 50% of an equivalent particle lacking zwitterionic polymer. Preferably, the degree of non-specific adsorption is less than 20%, more preferably less than 15%, less than 10%, less than 5%, less than 2% or less than 1% of an equivalent particle lacking zwitterionic polymer.

[0099] Non-specific adsorption can be determined, for example, by measuring the adsorption of selected non-specific particles, such as bovine serum albumin (BSA), to particles conjugated to anti-HA antibodies. The degree of non-specific adsorption can be measured spectroscopically by the level of solution depletion or microscopically (e.g., SEM or optically imaged particle non-specific accumulation on the protein surface).

[0100] Isolation of exosomes can be achieved by contacting a sample, such as a blood sample, with the coated particles described herein. After incubation of the coated particles with the sample, the particle-exosome complexes are isolated by standard techniques. In a preferred embodiment, magnetic or paramagnetic particles are used and the particle-exosome complexes are separated by magnetic separation.

[0101] Use of coated particles The coated particles described herein can be used in isolating exosomes from a sample, in particular in isolating neuron-derived exosomes from a sample. Typically, the sample is a blood sample.

[0102] The coated particles described herein can be used to detect alpha-synuclein and / or clusterin.For example, the coated particles can be used to determine the level of alpha-synuclein and / or clusterin in a sample.In particular, the coated particles can be used to determine the relative level of alpha-synuclein and / or clusterin in a sample.

[0103] In general, the coated particles described herein can be used for the diagnosis and prognosis of Parkinson's disease. For example, the coated particles can be used to determine whether a subject is susceptible to Parkinson's disease (PD) or whether the subject has PD.

[0104] The coated particles described above can be used in the research methods described herein, in particular the diagnostic methods described herein. Thus, the coated particles generally comprise: - contacting the sample with the coated particles described herein: - removing unbound sample; and - isolating the captured exosomes The present invention is suitable for use in a method for isolating exosomes from a sample (particularly a blood sample), comprising:

[0105] In particular, the coated particles can be used in methods including isolating neuron-derived exosomes from a sample and determining levels of alpha-synuclein and / or clusterin in neuron-derived exosomes in a blood sample.

[0106] Biomarker detection Techniques for detecting proteins, such as affinity ligand-dependent methods (e.g., enzyme-linked immunosorbent assay (ELISA), protein immunoprecipitation, immunoelectrophoresis, Western blot or protein immunostaining) and / or spectroscopic methods (e.g., high performance liquid chromatography (HPLC), or liquid chromatography mass spectrometry (LC / MS)), are well known in the art.

[0107] Detection of a biomarker of the invention typically involves contacting a sample with an affinity ligand, where a specific (as opposed to non-specific) binding reaction between the sample and the affinity ligand indicates the presence of the biomarker of interest.

[0108] An affinity ligand can be any molecule that binds to a target without also binding to other molecules in the sample. Any type of ligand can be used with the present invention. Ligands can be antibodies that can be designed to target α-synuclein or clusterin through their antigen binding sites, organic compounds that can dock into binding sites on α-synuclein or clusterin, inorganic metals that form coordination complexes with specific amino acids of α-synuclein or clusterin, hydrophobic molecules that can bind to the non-polar pockets of α-synuclein or clusterin, and / or proteins with specific binding regions that can interact with α-synuclein or clusterin.

[0109] For example, the affinity ligand for alpha-synuclein may be an anti-alpha-synuclein antibody, eg from MSD (see examples).

[0110] For example, an affinity ligand for clusterin may be an anti-clusterin antibody, eg from MSD (see examples).

[0111] The affinity ligand can be immobilized on a solid support (eg, a bead, plate, filter, film, slide, microarray support, resin, etc.).

[0112] In embodiments where both biomarkers (i.e., α-synuclein and clusterin) are detected, the sample may be contacted simultaneously with both ligands having affinity for the biomarkers ("multiplexed") in a single reaction compartment, such as a microtiter well, microfluidic chamber, or detection well. Alternatively, the biomarkers may be contacted with their affinity ligands in separate individual reaction compartments, and / or the experiments may be separated over time using different platform technologies, either in a single multiplexed reaction compartment or in separate individual reaction compartments. Multiplex platforms for the detection of proteins by immunoassays are well known in the art, for example, the MSD® Multi-Array Assay System.

[0113] The method and device for detecting binding reaction in immunoassay is standard in the art.For example, fluorescence-based detection method and / or electrochemiluminescence detection method can be used with the present invention.For example, sandwich immunoassay can be used to detect biomarker, and the assay typically includes binding biomarker to the affinity ligand immobilized on glass substrate, followed by binding the second affinity ligand that is fluorescently or electrochemiluminescence-labeled to biomarker, and then detecting fluorescence or electrochemiluminescence.

[0114] The data obtained from the detection of biomarkers can be combined in multivariate analysis. The combination of biomarkers can increase the classification power compared to a single biomarker. The combination of biomarkers can be evaluated simultaneously or sequentially. With regard to sequential evaluation, the data obtained for each biomarker can be combined after analyzing the biomarkers, for example after determining the level of the biomarker. Thus, for example, the sample can be divided into subsamples and the subsamples can be assayed sequentially.

[0115] Data interpretation and manipulation The present invention includes determining the level of the biomarker(s) of the present invention. The present invention may require quantitative or semi-quantitative measurement of each biomarker. The present invention may include relative determinations (e.g., ratios to another marker, or measurements to the same marker in a control sample). The present invention may include threshold determinations (e.g., whether the level is above or below a threshold, yes / no determination).

[0116] The level of the biomarker(s) of the present invention changes in disease cohorts compared to control cohorts.The analysis of the levels of these biomarkers in case and control populations can identify differences that provide diagnostic information.Those skilled in the art can easily determine the relative change (e.g., upregulation or downregulation) of any given biomarker in any given blood sample relative to any particular control of interest (e.g., negative control or positive control).

[0117] The control sample may be a positive control sample or a negative control sample. Typically, the control sample is age-matched to the test subject. The positive control sample includes a sample from a confirmed case of PD. The negative control sample includes a sample from a confirmed case of the absence of PD. The non-PD sample may be a subject with other unrelated neurodegenerative symptoms, such as frontotemporal dementia (FTD), progressive supranuclear palsy (PSP), corticobasal syndrome (CBS). The absolute level of a biomarker in a particular control sample (e.g., a sample from a non-PD subject with FTD) may differ from the absolute level of a biomarker in another control sample (e.g., a sample from a non-PD subject with PSP). It will be understood that the relative expression profile (e.g., up- or down-regulation or fold change) observed for the biomarkers of the present invention in PD samples compared to non-PD samples (i.e., negative control samples) may only relate to the specific control indicated.

[0118] Usually, biomarkers are measured to provide quantitative or semi-quantitative results (whether relative concentration, absolute concentration, fold change, etc.), which provides more data than is used in classifier algorithms. Usually, the raw data obtained from an assay to determine presence, absence or level (absolute or relative) requires some manipulation before their use. For example, the nature of most detection techniques means that some signal is sometimes seen even when the biomarker is not actually present, so this noise can be removed before the results are interpreted. Similarly, there may be background levels of the biomarker in the general population that need to be counterbalanced. Data may require scaling or standardization to facilitate comparison between experiments. These and similar issues, as well as techniques to address them, are well known in the art.

[0119] A variety of techniques are available to offset background signals in a particular experiment. For example, replicate measurements are usually performed (e.g., using duplicate or triplicate reactions) to determine intra-assay variation, and the average values ​​from the replicates (e.g., immunoassay median values) can be compared. Additionally, standard markers can be used to determine inter-assay variation and allow calibration and / or normalization. For example, an immunoassay reaction can include one or more "standards" of known concentration to determine the amplification efficiency of the immunoassay reaction and allow estimation of the total protein content of the unknown sample in comparison with other unknown samples.

[0120] It may be important not only to offset the inherent variation between different experiments, but also to offset the background level of biomarker present in the general population.Again, suitable techniques are well known.For example, the level of a particular biomarker in a sample is usually measured quantitatively or semi-quantitatively to allow comparison with the background level of that biomarker.Various controls can be used to provide a suitable baseline for comparison, and selecting suitable controls is routine in the diagnostic field.

[0121] The measured levels of the biomarker(s), after any offsetting or normalization, can each be converted to a diagnostic result in a variety of ways, which may include an algorithm that provides a diagnostic result as a function of the measured level(s).

[0122] The creation of algorithms for converting measured levels or raw data into scores or results is well known in the art. For example, linear or non-linear classifier algorithms can be used. These algorithms can be trained using data from any particular technique for measuring the marker(s). Appropriate training data will be obtained by measuring biomarkers in "case" and "control" samples, i.e., samples from subjects known to have PD and subjects known not to have PD. Most usefully, control samples also include samples from subjects with unrelated neurodegenerative conditions such as FTD, PSP or CBS that should be differentiated from PD (e.g., it is useful to train the algorithm with data from subjects with prodromal symptoms and / or data from subjects with unrelated neurodegenerative conditions). The classifier algorithm is modified until it can distinguish between case and control samples, for example, by changing the optimal cutoff value. For example, as shown in Example 2 and Figure 3, the optimal cutoff value for using alpha-synuclein to distinguish clinical PD samples from healthy control samples was found to be 14.21 pg / ml.

[0123] Thus, the method of the present invention may include a step of analyzing biomarker levels in a sample of a subject by using a classifier algorithm that distinguishes between PD and non-PD subjects based on the measured biomarker levels in a sample taken from such a subject. A variety of suitable classifier algorithms are available, such as linear discriminant analysis, naive Bayes classifiers, regression modeling, perceptrons, support vector machines (SVMs) and genetic programming (GP), as well as a range of statistical methods such as principal component analysis (PCA) and unsupervised hierarchical clustering and linear modeling.

[0124] Moreover, these approaches can potentially distinguish PD subjects from subjects with unrelated neurodegenerative conditions. The biomarkers of the present invention can be used to train such algorithms to ensure such distinction. The obtained data is analyzed for any potential signatures of differences between patient cohorts, multiple test corrections and fold changes in expression data that can indicate biological effects (usually it is desirable to use techniques that can show changes of at least 1.5-fold, e.g., more than 1.75-fold, more than 2-fold, more than 2.5-fold, more than 5-fold, etc.) with reference to a level of statistical significance (typically p<0.05). The classification performance (sensitivity and specificity (S+S), receiver operating characteristic (ROC) analysis) of any putative biomarker is rigorously evaluated using nested cross-validation and permutation analysis before further validation.

[0125] diagnosis The method of the present invention may include a step of comparing the biomarker level in the subject's sample with a reference. The reference may be (i) a threshold value, (ii) the corresponding biomarker level in the sample from a positive control, and / or (iii) the corresponding biomarker level in the sample from a negative control. The comparison provides a diagnostic indication of whether the subject is susceptible to disease or has disease. As is within the understanding of the skilled artisan, whether the level or biomarker increases or decreases depends on the reference used. For example, in subjects with PD, the α-synuclein content in neuron-derived exosomes in blood is at a higher level than the negative control sample (non-PD sample) and at a similar level to the positive control sample (PD sample).

[0126] Typically, the invention involves comparing the level of a biomarker against a threshold value, where the optimal threshold value can be determined by training a classifier algorithm to distinguish between "case" and "control" samples, as explained above.

[0127] For example, the reference to α-synuclein may be a threshold value between 10-20 pg / ml, such as between 12-16 pg / ml or between 14-15 pg / ml. If the blood sample contains a higher α-synuclein level in neuron-derived exosomes compared to the threshold value, this indicates that the subject is susceptible to or has PD. Conversely, if the blood sample contains an α-synuclein level in neuron-derived exosomes equivalent to the threshold value, this indicates that the subject is susceptible to or does not have PD.

[0128] In some embodiments, if the blood sample contains a higher level of α-synuclein in the neuron-derived exosomes compared to a threshold value, it indicates that the subject has a condition characterized by α-synuclein (such as PD and related conditions (e.g., PD with dementia and MSA)) and does not have a condition characterized by a non-α-synuclein proteinopathy.

[0129] In some embodiments, if the blood sample contains a higher level of alpha-synuclein in the neuron-derived exosomes compared to a threshold value, it indicates that the subject has PD and does not have an associated condition thereof (e.g., a condition with similar signs and symptoms, such as an atypical parkinsonian syndrome, including MSA).

[0130] The reference for clusterin can be a threshold value between 7-17 pg / ml, for example between 10-14 ng / ml or between 12-13 ng / ml. If a blood sample (i) contains a higher α-synuclein level in neuron-derived exosomes compared to the α-synuclein threshold, and (ii) has a clusterin level not higher than the threshold, this indicates that the subject is susceptible to or has PD.

[0131] In some embodiments, if a blood sample (i) contains higher α-synuclein levels in neuron-derived exosomes compared to a threshold value for α-synuclein, and (ii) has clusterin levels not higher than a threshold value, this indicates that the subject has a condition characterized by α-synuclein (such as PD and related conditions (e.g., PD with dementia and MSA)) and does not have a condition characterized by a non-α-synucleinoproteinopathy.

[0132] In some embodiments, if a blood sample (i) contains higher alpha-synuclein levels in neuron-derived exosomes compared to a threshold value for alpha-synuclein, and (ii) has a clusterin level not higher than a threshold value, this indicates that the subject has PD and does not have an associated condition thereof (e.g., a condition with similar signs and symptoms, such as an atypical parkinsonian syndrome, including MSA).

[0133] Alternatively, if a subject contains a higher level of clusterin in neuron-derived exosomes in the blood compared to a threshold value, this indicates that the subject is susceptible to or has a tauopathy.

[0134] When referring to the diagnosis of a subject susceptible to PD, this means predicting whether the subject has clinical PD.Therefore, diagnosis can indicate whether the subject is in the early phase of PD, such as preclinical PD or prodromal PD.Preclinical PD is a disease phase in which neurodegeneration has begun, but there are no obvious symptoms or signs of disease.Prodromal PD is a disease phase in which symptoms and signs of disease are present, but are not yet sufficient to define the disease.The MDS criteria for preclinical and prodromal PD are provided in reference 1.

[0135] When referring to a diagnosis of a tauopathy, the tauopathy may be, for example, frontotemporal dementia (FTD), progressive supranuclear palsy (PSP) and corticobasal syndrome (CBS).

[0136] Advanced statistical tools can be used to determine whether the levels determined for each biomarker in various samples (cases or controls) are the same or different. For example, in vitro diagnosis is rarely based on the comparison of a single measurement. Rather, an appropriate number of measurements are made with an appropriate level of precision to obtain the desired statistical certainty with acceptable sensitivity and / or specificity. Biomarker levels are quantitatively measured to allow for appropriate comparison, and sufficient measurements are made to ensure that any differences in levels can be assigned statistical significance at a level of p<0.05 or better.

[0137] The methods of the present invention may have a sensitivity of at least 50% (e.g., but is not limited to, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more).

[0138] The methods of the present invention may have a specificity of at least 50% (e.g., but is not limited to, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more).

[0139] In particular, we evaluated the ratio of α-synuclein to clusterin and applied a logistic regression model for the combination of these biomarkers. Both analyses showed that the combined measurement of α-synuclein and clusterin showed improved AUC, sensitivity and specificity estimates for differential diagnosis in predicting clinical PD versus other proteinopathies (AUC=0.98 (sensitivity=95%; specificity=93%)), even in the prodromal phase of PD, AUC=0.98 (sensitivity=94%, specificity=96%). The combined measurement of α-synuclein and clusterin also showed high performance in discriminating prodromal or clinical PD from MSA (AUC=0.94 and 0.91, respectively).

[0140] Data obtained from the methods of the present invention and / or diagnostic information based on those data may be stored on a computer medium (e.g., RAM, non-volatile computer memory, CD-ROM, DVD) and / or transmitted between computers, for example via the Internet.

[0141] If the method of the present invention shows that the subject has PD, further steps can follow.For example, the subject can undergo a confirmatory diagnostic procedure, such as one that includes a physical examination of the subject, and / or can be treated with a suitable therapeutic agent(s) for treating PD.Confirmatory diagnostic procedures include known biomarkers for PD and / or non-α-synuclein proteinopathy, other information about the subject; and / or other diagnostic tests or clinical indicators of PD, such as DaTSCAN to determine dopamine uptake, and / or brain imaging scans using MRI-based markers.

[0142] The present invention also provides a method of preventing and / or treating Parkinson's disease in a subject, comprising identifying a subject susceptible to Parkinson's disease according to the methods of the present invention and treating the subject with a Parkinson's disease therapy. Treatment of Parkinson's disease may include administering levodopa, dopamine agonists (e.g., pramipexole, ropinirole) and / or monoamine oxidase B inhibitors (e.g., selegiline and rasagiline).

[0143] Thus, the present invention also provides levodopa for use in a method of preventing and / or treating Parkinson's disease in a subject comprising identifying a subject susceptible to Parkinson's disease according to the methods of the invention and administering to the subject a therapeutically effective amount of levodopa.

[0144] The present invention also provides a dopamine agonist for use in a method of preventing and / or treating Parkinson's disease in a subject comprising identifying a subject susceptible to Parkinson's disease according to the methods of the invention and administering to the subject a therapeutically effective amount of a dopamine agonist.

[0145] The present invention also provides a monoamine oxidase B inhibitor for use in a method of preventing and / or treating Parkinson's disease in a subject comprising identifying a subject susceptible to Parkinson's disease according to the method of the present invention and administering to the subject a therapeutically effective amount of a monoamine oxidase B inhibitor.

[0146] The present invention also provides a method for preventing and / or treating a condition characterized by alpha-synucleinopathy in a subject, comprising treating the subject with an alpha-synuclein-targeted therapy and monitoring the effectiveness of the disease according to the method of the present invention. The alpha-synuclein-targeted therapy may comprise administering an alpha-synuclein-targeted therapeutic agent, such as an anti-alpha-synuclein antibody, phenylbutyrate-triglyceride (PBT), NPT200-11, nilotinib, ambroxol, or ENT-01. Thus, the present invention also provides an alpha-synuclein-targeted therapeutic agent for use in a method for preventing and / or treating a condition characterized by alpha-synucleinopathy in a subject, comprising administering a therapeutically effective amount of an alpha-synuclein-targeted therapeutic agent to a subject and monitoring the effectiveness of the disease according to the method of the present invention.

[0147] Monitoring the effectiveness of treatment The method of the invention may include testing samples from the same subject at two or more different time points. Methods of measuring changes in biomarker(s) over time can be used, for example, to monitor the effectiveness of a treatment being administered to a subject. Thus, the invention also provides a method of monitoring the effectiveness of an alpha-synuclein-targeted therapy being administered to a subject. The invention also provides a method for monitoring the development of a condition characterized by an alpha-synucleinopathy, such as PD, in a subject. Each biomarker of the invention can be measured according to the method of the invention at two or more different time points, and changes in the level of each biomarker over time indicate whether the disease is improving or worsening.

[0148] The treatment may be administered before the first sample is taken, at the same time as the first sample is taken, or after the first sample is taken.The present invention may be used to monitor a subject undergoing alpha-synuclein targeted therapy, for example, a subject may be undergoing treatment such as immunotherapy (e.g., anti-alpha-synuclein antibody therapy), phenylbutyrate-triglyceride (PBT), NPT200-11, nilotinib and ambroxol, ENT-01, which are currently in clinical trials targeting alpha-synuclein to protect brain cells and slow Parkinson's.

[0149] Thus, the method of the present invention may include (i) determining the level of α-synuclein and / or clusterin in a first sample taken from a subject at a first time point; and (ii) determining the level of α-synuclein and / or clusterin in a second sample taken from a subject at a second time point, (a) the second time point being later than the first time point; (b) a change in the level of the biomarker(s) in the second sample compared to the first sample indicates that the condition characterized by α-synucleinopathy, such as PD, is in remission or progression. Thus, the method monitors the biomarker(s) over time, with the changing level indicating whether the disease is improving or worsening. As is within the understanding of one skilled in the art, when the level of the biomarker changes toward the level seen in healthy controls (and away from the level seen in diseased patients), the condition characterized by α-synucleinopathy, such as PD, is in remission. On the other hand, if the levels of the biomarkers change toward or remain at levels seen in diseased patients (and / or move away from levels seen in healthy controls), a condition characterized by an α-synucleinopathy, such as PD, is progressing.

[0150] The progression of the disease can be either improved or worsened, and the method can be used in a variety of ways, for example, to monitor the natural progression of a condition characterized by alpha-synucleinopathy, such as PD, or to monitor the effectiveness of an alpha-synuclein-targeted therapy administered to a subject.Thus, the subject may receive a therapeutic agent before the first time point, at the first time point, or between the first and second time points.

[0151] When the method includes a first time point and a second time point, these time points can be at least one day, one week, one month or one year apart.Samples can be taken periodically.The method can include measuring biomarkers in more than two samples taken at more than two time points, i.e., there can be a third sample, a fourth sample, a fifth sample, etc.

[0152] kit The invention also provides diagnostic devices and kits for detecting the biomarkers of the invention.

[0153] The present invention also provides a diagnostic device for use in providing a diagnostic indication of a subject susceptible to or having Parkinson's disease, which allows the determination of levels of alpha-synuclein and / or clusterin in a sample.

[0154] The present invention also provides diagnostic devices for use in distinguishing conditions characterized by alpha-synuclein (such as PD and related conditions (e.g., PD with dementia and MSA)) from conditions characterized by non-alpha-synuclein proteinopathies in a subject, which allow for the determination of levels of alpha-synuclein and / or clusterin.

[0155] The present invention also provides a diagnostic device for use in distinguishing PD from its related conditions in a subject (e.g. conditions with similar signs and symptoms, such as MSA) which allows for the determination of levels of alpha-synuclein and / or clusterin.

[0156] The present invention also provides a kit comprising (i) the diagnostic device of the present invention and (ii) instructions for using the device to detect α-synuclein and / or clusterin. The kit is useful for providing a diagnostic indication of subjects susceptible to or having Parkinson's disease. The kit is particularly useful for distinguishing conditions characterized by α-synuclein in subjects (PD and related conditions (e.g., PD with dementia and MSA)) from conditions characterized by non-α-synucleinoproteinopathies. The kit is particularly useful for distinguishing PD from its related conditions (e.g., conditions with similar signs and symptoms, such as atypical parkinsonism including MSA).

[0157] The invention also provides an article of manufacture comprising (i) one or more detection reagents that allow for the measurement of synuclein and / or clusterin, and (ii) a sample from a subject.

[0158] The present invention also provides kits comprising coated particles of the invention for isolating a selected population of exosomes from a blood sample, and / or reagents for determining levels of alpha-synuclein and clusterin in neuron-derived exosomes in a blood sample.

[0159] Certain embodiments of the present invention The present invention provides the following embodiments: 1. A method for analyzing a blood sample from a subject, comprising determining levels of alpha-synuclein and clusterin in neuron-derived exosomes in the blood sample, wherein the levels of alpha-synuclein and clusterin provide a diagnostic indication of a subject susceptible to or having Parkinson's disease (PD).

[0160] 2. The method of embodiment 1, wherein the levels of alpha-synuclein and clusterin provide a diagnostic indication for subjects with prodromal PD.

[0161] 3. The method of any of embodiments 1 or 2, wherein an increase in the level of alpha-synuclein compared to the reference indicates that the subject is susceptible to or has PD, optionally with the reference being a threshold value between 10 and 20 pg / ml.

[0162] 4. The method of any one of embodiments 1 to 3, wherein a lack of increased levels of clusterin compared to the reference indicates that the subject is susceptible to PD, optionally with the reference being a threshold value between 7 and 17 ng / ml.

[0163] 5. A method for analyzing a blood sample from a subject having one or more signs or symptoms of Parkinsonism and not diagnosed with PD, comprising determining a level of alpha-synuclein in neuron-derived exosomes in the blood sample, wherein the level of alpha-synuclein provides a diagnostic indication of the subject being susceptible to PD.

[0164] 6. Signs or symptoms of Parkinsonism: - one or more of the following non-motor symptoms: diagnosis of rapid eye movement sleep behavior disorder (RBD), olfactory dysfunction, constipation, excessive daytime sleepiness, symptomatic hypotension, erectile dysfunction, urinary dysfunction, and / or a diagnosis of depression; - one or more of the following non-motor signs: altered handwriting, rolling over, gait disturbance, salivary disturbance, speech disturbance, reduced facial expression, rigidity, balance disturbance, resting tremor, bradykinesia (slow movements), and / or postural instability; and / or -Abnormal tracer uptake in the presynaptic dopaminergic system 6. The method of embodiment 5, comprising:

[0165] 7. The method of embodiment 5 or embodiment 6, further comprising determining the level of clusterin in the neuron-derived exosomes, wherein the level of clusterin provides a diagnostic indication of the subject being susceptible to PD.

[0166] 8. The method of any one of embodiments 1 to 7, wherein the neuron-derived exosomes contain a neuronal protein, such as L1CAM.

[0167] 9. The method of embodiment 8, further comprising isolating exosomes using a ligand having affinity for L1CAM.

[0168] 10. The method of any one of embodiments 1 to 9, wherein the biomarker level(s) are determined in serum obtained from the subject's blood sample.

[0169] 11. A method for distinguishing conditions characterized by alpha-synuclein (such as PD and related conditions (e.g., PD with dementia and MSA)) from conditions characterized by PD of neurodegenerative diseases associated with non-alpha-synucleinoproteinopathies, comprising analyzing a blood sample from a subject according to any of the methods of embodiments 1 to 10.

[0170] 12. A method for identifying a subject susceptible to PD, comprising analyzing a blood sample from the subject according to any of the methods of embodiments 1 to 10.

[0171] 13.(a) Known biomarkers for Parkinson's disease; (b) known biomarkers of non-α-synucleinopathies; (c) other information about the subject; and (d) Other diagnostic tests or clinical indicators for PD 13. The method of embodiment 11 or embodiment 12, further comprising determining at least one of:

[0172] 14. A method for preventing and / or treating PD in a subject, comprising identifying a subject susceptible to PD according to the method of embodiment 12 or 13, and treating the subject with a therapy for PD.

[0173] 15. A method for monitoring the effectiveness of an alpha-synuclein targeted therapy, such as a therapy for PD, being administered to a subject, comprising analyzing a blood sample from the subject according to the method of any one of embodiments 1 to 10, wherein each biomarker is measured at two or more different time points, and changes in the level of each biomarker over time indicate whether the disease is improving or worsening.

[0174] 16. A coated particle having a coating comprising a zwitterionic polymer coupled to a ligand having affinity for a selected population of exosomes.

[0175] 17. The coated particle of embodiment 16, wherein the zwitterionic polymer comprises a carboxybetaine, sulfobetaine and / or phosphorylcholine moiety.

[0176] 18. The coated particle of embodiment 16 or 17, wherein the ligand has affinity for neuron-derived exosomes, for example, the ligand is an anti-L1CAM antibody.

[0177] 19. A method for isolating exosomes from a sample, comprising: - contacting a sample with coated particles according to any one of embodiments 16 to 18; - removing unbound sample; and - isolating the captured exosomes A method comprising:

[0178] 20. The method according to any one of embodiments 1 to 10, comprising isolating neuron-derived exosomes from a sample according to the method according to any one of embodiments 17 to 19.

[0179] 21. A kit comprising reagents for determining levels of alpha-synuclein and clusterin in neuron-derived exosomes in a blood sample.

[0180] 22. Use of alpha-synuclein, and optionally clusterin, as biomarkers to provide a diagnostic indication of subjects susceptible to PD and / or to distinguish conditions characterized by alpha-synuclein (such as PD and related conditions (e.g., PD with dementia and MSA)) from conditions characterized by PD, a neurodegenerative disease with a non-alpha-synucleino proteinopathy.

[0181] 23. The use of alpha-synuclein and clusterin as biomarkers to provide a diagnostic indication for subjects with PD.

[0182] 24. The use of clusterin as a biomarker to provide a diagnostic indicator of subjects susceptible to or having a tauopathy.

[0183] 25. A method for analyzing a blood sample from a subject, comprising determining the level of clusterin in neuron-derived exosomes, wherein an increased level of clusterin provides a diagnostic indication of a subject susceptible to or having a tauopathy.

[0184] 26. A method for distinguishing PD from its associated conditions, such as MSA, comprising analyzing a blood sample from a subject according to any of the methods of embodiments 1 to 10.

[0185] 27.(a) Known biomarkers for Parkinson's disease; (b) known biomarkers of non-α-synucleinopathies; (c) other information about the subject; and (d) Other diagnostic tests or clinical indicators for PD 27. The method of embodiment 26, further comprising determining at least one of:

[0186] 28. A method for preventing and / or treating PD in a subject, comprising identifying a subject susceptible to PD according to the method of embodiment 26 or 27, and treating the subject with a therapy for PD.

[0187] 29. The use of alpha-synuclein, and optionally clusterin, as biomarkers to provide a diagnostic indication of subjects susceptible to PD, and to distinguish PD from its associated conditions, such as MSA.

[0188] others It is to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

[0189] Additionally, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the content clearly dictates otherwise. Thus, for example, reference to "a bacteria strain" includes two or more "bacteria strains."

[0190] Additionally, references herein to "≧x" mean greater than or equal to x.

[0191] The term "comprising" encompasses "including" and "consisting", e.g., a composition "comprising" X may consist only of X, or it may include something additional, e.g., X+Y.

[0192] Reference to the "level" of a biomarker means the amount of analyte (e.g., alpha-synuclein or clusterin) measured in a sample, including relative and absolute concentrations of the analyte, titers of the analyte, relationship to thresholds, rankings, percentiles, and the like.

[0193] The "sensitivity" of an assay is the proportion of true positives that are correctly identified, i.e., the proportion of subjects with PD that test positive by the method of the present invention.This can be applied to individual biomarkers, both biomarkers (α-synuclein and clusterin), a single assay, or an assay that combines data integrated from multiple sources.It can refer to the ability of the method to identify samples that contain a specific analyte (e.g., α-synuclein or clusterin), or the ability of the method to correctly identify samples from subjects that are susceptible to or have the disease.

[0194] The "specificity" of an assay is the proportion of true negatives that are correctly identified, i.e., the proportion of subjects without PD that are tested negative by the method of the present invention.This can be applied to individual biomarkers, both biomarkers (α-synuclein and clusterin), a single assay, or an assay that combines data integrated from multiple sources.It can relate to the ability of the method to identify samples that contain a specific analyte (e.g., α-synuclein or clusterin), or the ability of the method to correctly identify samples from subjects that are susceptible to or have a disease.

[0195] All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0196] The following examples illustrate the invention.

[0197] example Example 1 This example aims to develop a method to specifically isolate neuron-specific exosomes.

[0198] Synthesis of Carboxybetaine Methacrylate (CBMA) CBMA was synthesized according to an adapted literature procedure (25). 3.16 g of 2-(dimethylamino)ethyl methacrylate (DMAEMA; 20 mmol, 1 equiv; Sigma Aldrich) was dissolved in 50 mL of dry dichloromethane (DCM) and cooled to 0–5 °C. Then, 1.72 g of β-propiolactone (24 mmol, 1.2 equiv; Alfa Aesar) dissolved in 10 mL of dry DCM was slowly added. The solution was stirred at 0–5 °C for 8 h. The resulting white precipitate was isolated by filtration and washed with DCM and EtO to give 1.91 g (42%) of pure CBMA.

number

[0199] Preparation of poly(carboxybetaine methacrylate)-based zwitterionic magnetic beads and antibody conjugation The magnetic beads were used to detect the formation of ferrihydrite / formaldehyde composite microbeads and the subsequent desorption of Fe from ferrihydrite. 3 O 4 (26, 27) Poly(carboxybetaine methacrylate) was then formed and the Fe was added using the reversible addition-fragmentation chain transfer (RAFT) method. 3 O 4pCBMA magnetic beads were generated by coating on 1000 μg / ml of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide (EDC / NHS, Sigma) with 0.1% NaCl (2.0% NaCl) on 0.1% NaCl (2.0% NaCl) to generate pCBMA magnetic beads (28). Bis(carboxymethyl)trithiocarbonate (Bittc, Sigma) and 4,4'-azobis(4-cyanovaleric acid) (ACVA) were used as the RAFT agent and initiator, respectively. For antibody conjugation, the carboxylic acid groups of the pCBMA beads were activated with 2-morpholinoethanesulfonic acid (MES) buffer (50 mM, pH 5.5) containing 50 mg / mL of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide (EDC / NHS, Sigma) for 1 h at room temperature. The beads were then rinsed with MES buffer and PBS, after which 8 μg of anti-L1CAM (ab80832, Abcam, UK) was added per mg of beads. The mixture was incubated on a rotator at room temperature for 1.5 h. The resulting pCBMA-anti-L1CAM beads were washed twice with PBS and used for immunocapture.

[0200] Assay development for the isolation and detection of neuron-derived exosomes in blood To specifically isolate exosomes derived from neural cells, we used an immunoaffinity-based capture approach with an antibody against neural L1 adhesion molecule (L1CAM) covalently coupled to magnetic microbeads. L1CAM belongs to a group of cell adhesion molecules expressed primarily in the nervous system and was previously shown to be a surface marker for neuron-derived exosomes isolated from multiple sources, including blood

[15] . We further developed this assay to minimize contamination from peripheral sources. To this end, we fabricated magnetic beads (~2.4 μm) pre-coated with the zwitterionic polymer poly(carboxybetaine methacrylate) pCBMA using reversible addition-fragmentation chain transfer (Figure 5). Successful polymerization of pCBMA on the beads was demonstrated by attenuated total IR reflectance spectroscopy in comparison to iron oxide beads (Figure 6A). The antifouling properties of the coated beads were confirmed by the reduction of nonspecific adsorption of bovine serum albumin or total serum proteins compared to commercial epoxy beads, both of which were conjugated to anti-HA antibodies (Figures 6B and 6C). The carboxylic acid groups of pCBMA were then activated, cross-linked to anti-L1CAM antibodies, and evaluated for immunocapture of neural exosomes in serum (Figure 7A). First, we showed by SEM that exosomes bound to anti-L1CAM-conjugated pCBMA-coated beads, but not to control beads (Figure 7B). Second, we tested and confirmed the presence of both surface (L1CAM, CD81) and internal (syntenin-1, tsg101) exosome markers in the lysates of vesicles captured by anti-L1CAM-conjugated pCBMA-coated magnetic beads by immunoblotting (Figure 7C). Third, we profiled the whole proteome composition in L1CAM-captured exosomes from pooled human serum by mass spectrometry and identified 512 proteins. We used gene ontology (GO) term analysis to define enriched functions or components within these proteins.The enrichment score (the degree to which a GO term is represented within the protein list, compared to the total list of proteins tested) was plotted against the GO terms that were significant ( 10 -3 p-value threshold of 0.01). The analysis revealed terms enriched for exosome and related extracellular vesicle functions (Figure 7D). Among the identified proteins were multiple bona fide exosome markers such as CD9, syntenin-1, 14-3-3 zeta / delta (YWHAZ), neural cell adhesion protein (N1CAM) and protein clusterin (Figure 7E). For targeted analysis of protein concentrations in immunocaptured exosomes, we developed a triplex analysis of L1CAM-positive exosomes for total α-synuclein, clusterin and syntenin-1 and demonstrated specific detection of these markers in immunocaptured exosomes (Figure 8).

[0201] Fourier Transform Infrared Attenuated Total Reflectance (FTIR-ATR) An appropriate amount of the prepared pCBMA magnetic beads was washed with ethanol and ultrapure water and dried at 50 °C for FTIR-ATR analysis (Bruker Vertex 80, Bruker Corporation, Ettlingen, Germany). CBMA monomer and non-coated magnetic beads were used as controls.

[0202] Immunoblotting Immunocaptured exosomes were dissolved in LDS buffer (Thermo Fisher), resolved using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride membranes (PVDF, Invitrogen), and immunoblotted with antibodies against syntenin-1 (ab133267, Abcam), CD81 (sc-5275, Santa Cruz), Tsg101 (ab125011, Abcam), and L1CAM (ab80832, Abcam). All antibodies were used at a 1:1,000 dilution. After incubation with horseradish peroxidase-conjugated secondary antibodies (GE Healthcare) (1:10,000 dilution), chemiluminescence was used for immunodetection (ChemiDoc, Bio-Rad).

[0203] SEM Immunocaptured exosomes were fixed in 2% glutaraldehyde on clean silicon wafers and washed twice with PBS. After natural evaporation, samples were coated with approximately 5 nm of platinum using a sputter coater (Cressington) and imaged under a scanning electron microscope (Zeiss Crossbeam 540) at 5 kV.

[0204] mass spectrometry Immunocaptured exosomes were lysed in RIPA buffer for 15 min at room temperature. Lysates were reduced using dithiothreitol and alkylated with iodoacetamide. Exosomal proteins were isolated by methanol-chloroform precipitation and digested using 0.1 μg / μL sequencing-grade modified porcine trypsin (Promega) diluted in NH4HCO3. Peptides were purified using C18 spin columns (Pierce). Eluted peptides containing acetonitrile were evaporated to 10 μL in a Speedvac (Thermo scientific) and then adjusted to 10 μL with 2% acetonitrile, 0.1% formic acid in ultrapure water. Samples were subsequently analyzed by nanoUPLC-MS / MS using a Waters, nanoAcquity column, 75 μm × 250 mm, 1.7 μm particle size, and a gradient of 1–40% acetonitrile for 60 min at a flow rate of 250 nL / min. Mass spectrometry was performed on a Thermo LTQ Orbitrap Velos (60,000 resolution, Top20, CID, Waltham, MA, USA). Raw MS data were analyzed using Progenesis QI for Proteomics software (v3.0; Nonlinear Dynamics, Newcastle upon Tyne, UK). MS / MS spectra were searched against the UniProt Homo Sapiens Reference proteome (read on 6 January 2017) using Mascot (v2.5.1; Matrix Science, Inc., Boston, MA), allowing a precursor mass tolerance of 10 ppm and a fragment ion tolerance of 0.05 Da.

[0205] Example 2 The aim of this study was to evaluate the clinical utility of serum neuroexosomes in stratifying or predicting patients across the spectrum of Parkinson's disease for neurodegenerative conditions characterized by non-α-synuclein proteinopathies.

[0206] method Patient population A total of 638 subjects were included in the study (Table 1). Serum samples and clinical data were collected from patients with polysomnographically confirmed RBD (n=53), PD (n=275), dementia with Lewy bodies (n=21, DLB), frontotemporal dementia including behavioral type or primary progressive aphasia (n=65 FTD), progressive supranuclear palsy, PSP (n=35) and corticobasal syndrome, CBS (n=45). Healthy controls (n=144, HC) were of similar age and sex.

[0207] Patients and controls were recruited from three different centers: the Oxford Parkinson's Disease Centre Discovery cohort, the Kiel-PD cohort and the Bresica cohort.

[0208] Serum from neuropathologically confirmed cases of DLB ​​with relatively pure α-synuclein pathology (n=10) and healthy controls (n=10) were used.

[0209] Longitudinal serum samples were evaluated from Parkinson's (n=40) and control (n=14) individuals. [Table 1]

[0210] Exosome immunocapture Blood samples were collected and serum was isolated, aliquoted and frozen at -80°C until further use.

[0211] For exosome isolation, a three-step sequential spin (300g for 10 min, 2000g for 20 min, and 10,000g for 30 min) was used to remove cell debris, protein aggregates, and fatty substances in the serum. The supernatant, i.e., pre-cleared serum, was obtained for immunocapture using coated beads as described in Example 1. The immunobeads were incubated overnight at 4°C, and the bead-exosome complexes were collected and washed. The isolated exosomes were dissolved in 1% triton X-100 in PBS with 4% protease inhibitors at room temperature for quantification of exosomal proteins.

[0212] Exosomal protein detection Electrochemiluminescence (ECL) was performed in 96-well Meso Scale Discovery (MSD) U-Plex plates, which allow multiplexing of markers in the same exosome preparation. All steps were performed at room temperature. Three unique linkers for selected markers (syntenin-1, clusterin and α-synuclein) were used according to the manufacturer's protocol (MSD). Plates were coated with biotinylated capture antibodies and exosome lysates or recombinant protein standards were added, followed by detection antibodies with sulfo-TAG labels. Plates were read and data were analyzed using the MSD-ECL platform (QuickPlex SQ 120).

[0213] Antibody pairs against clusterin and α-synuclein were provided by MSD and pre-conjugated with biotin and ruthenium tags. Additive-free anti-syntenin-1 goat polyclonal antibody (PAB7132, Abnova) and anti-syntenin-1 rabbit monoclonal antibody (ab236071, Abcam) were conjugated with biotin and ruthenium and used as capture and detection antibodies, respectively. For phosphorylated α-synuclein at serine 129 (pSer129) detection, the antibody pair used consisted of a biotinylated antibody against pSer129 α-synuclein (11A5, purified from PTA-8222 hybridoma cell line, ATCC) acting as the capture antibody, and a ruthenium-labeled antibody against total α-synuclein (4B12, Biolegend) acting as the detection antibody.

[0214] For the combination of exosomal α-synuclein, clusterin and syntenin-1, we developed a triplex MSD and demonstrated specific detection of these markers in immunocaptured exosomes (Figure 8), and for all assays we evaluated the dynamic range and lower limit of detection (Figures 9 and 10).

[0215] Study design and statistical analysis Regarding multiple comparisons, we performed non-parametric statistical tests (Kruskal-Wallis one-way ANOVA with Dunn's test for post-hoc comparisons between individual pairings) since the data were not normally distributed. The relationship between exosomal markers and disease duration, sex, MoCA score and UPDRS motor score was analyzed with bivariate correlations using Pearson's correlation coefficient. To evaluate the performance of the proposed biomarkers in separating α-synucleinopathy from controls and to define cut-off values, we used the Kiel and Brescia cohort as the training group (n=314) and the Oxford cohort as the validation group (n=105). Data from these groups were analyzed using receiver operating characteristics (ROC). The "optimal" cut-off point was determined by Youden's index, i.e. the value associated with the maximum value of sensitivity + specificity -1. A value of p<0.05 was considered significant. Logistic regression analysis was used to determine the best combination of different protein markers (clusterin and α-synuclein) to discriminate between sets of diagnostic groups or subgroups. To investigate the correlation between biomarker concentrations and duration, linear mixed models were used to analyze longitudinal samples, treating the sample at the first visit as the baseline. Robust regression and outlier removal methods (ROUT) were applied to test for outliers.

[0216] Logistic regression and linear mixed models were performed using MATLAB® (MATLAB and Statistics Toolbox Release 2014a The MathWorks, Inc, Natick, Massachusetts, United States).

[0217] result Neuron-derived exosomal α-synuclein is increased across the spectrum of Lewy body pathology We blindly analyzed serum samples from 638 subjects across three multinational cohorts to investigate the role of neuron-derived exosomal α-synuclein as a biomarker across the spectrum of Lewy body pathology by comprehensively evaluating blood-based assays and assaying patients at prodromal, motor and cognitive stages. To this end, we separated PD subjects into those with pure motor PD or PD dementia according to their MoCA scores corrected for education history. Dementia in the context of PD was defined as a MoCA screening score below 21 / 30 at the time of sample collection (29). Thus, we subsequently blindly analyzed subgroups of motor PD (n=230) or PD with dementia (n=45). We also included a group of 21 cases with a clinical diagnosis of DLB, 10 of which were confirmed at autopsy. We also used a group of idiopathic RBD without motor signs (n ​​= 53) as a surrogate for prodromal PD, since prospective cohort studies observed a very strong association between RBD and subsequent clinically defined α-synucleinopathy, with up to 80% of cases converting to primary PD or DLB ( 30 , 31 ).

[0218] We found that α-synuclein was elevated approximately 2-fold in RBD, PD and DLB exosomes compared to controls or other proteinopathies (Figure 1A). Specifically, α-synuclein content in L1CAM-positive exosomes was similarly elevated in RBD (26.44±12.64pg / mL), motor PD (27.44±18.82pg / mL) and PD with dementia (PDD 27.76±17.25pg / mL) when compared to healthy subjects (HC, 12.91±5.93pg / mL) (data shown as mean±SD). α-synuclein was also elevated in DLB (17.23±4.58pg / mL). We demonstrated an association between increased release of α-synuclein in neural exosomes and Lewy body pathology by testing serum collected before death in autopsy-confirmed controls and DLB cases (n=10 / group). In these two subgroups, mean neural exosome-associated α-synuclein was 17.60±5.86 pg / mL in DLB and 10.50±4.60 pg / mL in controls (1.7-fold increase, p=0.0097). As expected, exosomal α-synuclein concentrations were much lower compared to reported levels of total free α-synuclein in blood (10-17 ng / mL) (7, 10).

[0219] To assess the abundance of α-synuclein in neuron-derived exosomes in unrelated neurodegenerative conditions, we included FTD patients (n=65), whose pathology is characterized primarily by tau or TDP-43 aggregation, as well as PSP patients (n=35) and CBS patients (n=45), whose pathology is characterized primarily by fibrillar aggregates of 4-repeat tau. We found that α-synuclein content in L1CAM-positive exosomes from these diseases was similar to HC (FTD, 12.60±4.03pg / mL; PSP, 9.20±4.90pg / ml; CBS, 9.93±3.68pg / mL), as shown in FIG. 1A.

[0220] In the first 226 subjects, we also determined whether phosphorylated α-synuclein at serine 129 (pSer129) was detected in L1CAM-positive exosomes and whether it had value as a blood-based biomarker. pSer129α-synuclein is the major disease-associated modification that accounts for more than 90% of α-synuclein found in Lewy bodies (32). This analysis showed that only a minority of individuals had detectable levels of pSer129α-synuclein in neural exosomes. Interestingly, when applying a cutoff value of 0.5 pg / ml (Figure 1B), which is within the detection limit of the assay (Figure 10), pSer129α-synuclein was elevated in a subgroup of PD patients (33) (28.6% of all PD patients tested). In this subpopulation, pSer129 α-synuclein correlated with disease duration longer than 7.3 years (r = 0.26, p = 0.0263) and UPDRS (r = 0.34, p = 0.0495), but not with MoCA (r = 0.006, p = 0.3643). Unlike previous studies (15, 13), we did not detect any significant correlation between exosomal α-synuclein and either UPDRS (r = 0.0267) or MoCA (r = 0.0621), as shown in Figure 1C and Figure 1D.

[0221] Multiplexed measurement of α-synuclein and clusterin improved the predictive value of exosome testing across α-synucleinopathies. To evaluate the value of multiplexed exosome measurements, we selected clusterin as an additional marker since it was the most abundant exosome-associated protein detected by mass spectrometry (Figure 7E). Clusterin was previously identified as a risk gene for dementia (33, 34). Therefore, we hypothesized that quantification of clusterin in neural exosomes might aid in stratifying patients with cognitive impairment or separating patients with another pathology. Surprisingly, we found that clusterin was elevated in FTD (20.22 ± 10.47 ng / mL), PSP (18.42 ± 8.84 ng / mL) and CBS (16.16 ± 6.07 ng / mL) (Figure 2A), but not in RBD (9.55 ± 3.71 ng / mL), clinical PD (9.72 ± 6.02 ng / mL) and HC (8.67 ± 4.92 ng / mL). This differential abundance of clusterin in unrelated proteinopathies suggests that incorporation of clusterin in blood-based PD exosome tests may be valuable in distinguishing patients with primary non-α-synuclein pathology. In contrast, syntenin-1, a common exosomal protein, did not show disease-specific distribution with sufficient separation to serve as a biomarker (Figure 11).

[0222] To further evaluate the clinical potential of the combined measurement of α-synuclein and clusterin in L1CAM-positive exosomes as a biomarker, we evaluated the ratio of α-synuclein to clusterin and applied a logistic regression model to the combination of these markers. Both analyses showed that the combined measurement of α-synuclein and clusterin showed improved AUC, sensitivity, and specificity estimates for the differential diagnosis in predicting clinical PD vs. other proteinopathies (AUC=0.98 (sensitivity 0.95; specificity 0.93)), even in the prodromal phase of PD, RBD vs. other proteinopathies, AUC=0.98, sensitivity 0.94, specificity 0.96, as shown in Figure 2 and Table 2.

[0223] Table 2 Summary of ROC analyses in patient groups across cohorts comparing synucleinopathies and controls or other proteinopathies using α-synuclein, clusterin and combined markers (α-synuclein and clusterin). Combined markers were analyzed with logistic regression. ROC-based separation was applied when there was a significant difference between the two groups. High-performing markers are shown in bold. [Table 2]

[0224] To evaluate the consistency of exosomal α-synuclein in distinguishing clinical PD from healthy subjects across populations, we applied a two-stage design model: a training set of 314 subjects from the Kiel and Brescia cohort was used to identify the optimal cutoff value, which was then applied to an independent validation set of 105 subjects from the Oxford cohort. This revealed that at 14.21 pg / ml, the assay showed consistent performance (training vs. validation), with an AUC of 0.86, sensitivity of 0.82 vs. 0.85, specificity of 0.71 vs. 0.74, positive predictive value of 0.83 vs. 0.89, and negative predictive value of 0.72 vs. 0.68, as shown in Figure 3.

[0225] Longitudinal trajectories of exosome-associated α-synuclein and clusterin with disease progression To investigate the variability of neuron-associated exosomal markers within individuals over the course of the disease, we blindly analyzed prospective, longitudinal samples from the Oxford cohort. A linear mixed model was applied to fit the longitudinal values ​​of exosomal α-synuclein and clusterin over time from first sampling as covariants, and patients were stratified by levels at first visit relative to the median. The longitudinal sample sizes of PD, PDD and controls are summarized in Figure 4. Overall, the slopes from 0 for either α-synuclein or clusterin strata were not significantly different when comparing clinical PD (PD or PDD or combined) or controls. This analysis shows that neuron-derived exosomal α-synuclein levels remain elevated in individuals with PD over a 5-year period, with a sustained separation from controls, as shown in Figure 4.

[0226] Consideration This study presents a blood-based test of clinical utility in α-synucleinopathies such as PD. This analysis is the largest multicenter study of neuroexosomal proteins in serum with defined parameters for potential utility in clinical practice: As single cross-sectional measurements, serum neuroexosome-associated α-synuclein and clusterin perform best as predictive markers of basal α-synucleinopathy versus healthy subjects in another proteinopathy or clinical and prodromal PD, outperforming any previously reported blood-based assay or CSF total α-synuclein or pathogenic α-synuclein (7, 35). This improved performance in serum neuroexosome testing across samples collected at multiple sites is due, at least in part, to improved specific immunocapture using a zwitterionic coating that resists nonspecific binding (36). The assay consistency of exosomal α-synuclein across populations and its stability against disease progression when assessed within individuals suggests that it could be considered a pharmacodynamic biomarker for α-synuclein-targeted therapies in PD and related diseases.

[0227] The finding that neural exosomal α-synuclein levels were increased approximately two-fold in PD and PDD compared to controls across three studies firmly establishes that increased exosomal α-synuclein is a valid disease-related finding in PD. In addition, we demonstrated that neural exosomal α-synuclein levels are elevated in patients with RBD, a group at high risk of developing PD, but not in other neurodegenerative conditions (FTD / PSP / CBS). In this context, pSer129 α-synuclein was not consistently detected in blood-derived neural exosomes, except in the PD subgroup. Thus, at least in the early stages of the disease, exosomal release appears to be primarily related to non-pathogenic forms of α-synuclein, whereas exosome-associated pathogenic α-synuclein may occur at advanced stages, implying a more severe motor phenotype.

[0228] Interestingly, exosomal clusterin, but not α-synuclein, was elevated in FTD, PSP, and CBS, three neurodegenerative conditions pathologically characterized by primarily tau or TDP-43 proteinopathies and minimal α-synuclein pathology (37). Total serum clusterin is elevated in Alzheimer's disease (AD), although this association is controversial (38, 39) and may involve Aβ-independent pathways (40). The data in this study suggest that the neuron-associated exosomal fraction of clusterin may be useful as a diagnostic biomarker for neurodegenerative conditions characterized by tauopathies. In the context of this study, the incorporation of clusterin quantification may aid in the separation of patients with primarily non-α-synuclein pathology. Given that concomitant proteinopathies are frequent in dementia (3, 37), clusterin in combination with α-synuclein may be particularly useful for stratifying patients with cognitive involvement (e.g., PDD, DLB) who are most likely to benefit from α-synuclein-targeted therapies. In support of this view, we found that combined measurement of serum neuroexosomal α-synuclein and clusterin or their ratio improved the sensitivity and specificity of blood-based exosome testing with an AUC of 0.98.

[0229] We have previously shown that serum exosome number or size does not differ between PD patients and controls (12). This finding, and the differential protein patterns between groups reported herein (i.e., α-synuclein is highest in RBD / PD / PDD / DLB, whereas clusterin is highest in FTD / PSP / CBS), suggest that alterations in L1CAM-positive exosome composition are the most likely explanation for these observations. Genome-wide association studies and functional investigations of monogenic causes of PD indicate that protein trafficking to endosomes and lysosomes is associated with pathogenic cascades (41). Exosomes originate from intraluminal vesicles within mature (late) endosomes, also known as multivesicular bodies (MVBs). The contents of MVBs are typically destined for degradation upon fusion with lysosomes. Another purpose of MVBs is the release of plasma membrane and exosomes. Thus, progressive impairment of intraneuronal transport from endosomes to lysosomes may result in increased exosomal α-synuclein release. This model would be consistent with several cell-based studies that showed that α-synuclein is transported to endosomes and undergoes lysosomal degradation (42, 43, 44), whereas inhibition of lysosomal function increases α-synuclein release in exosomes in conditioned medium (45, 46, 47). Based on this model, the reported decrease in CSF total α-synuclein in PD (7, 8) may be due to adaptive efflux into serum exosomes in response to defective neuronal handling of the protein.

[0230] Strengths of this study include the multicenter nature and large sample size across the spectrum of α-synucleinopathies, as well as the inclusion of unrelated proteinopathies beyond any previous exosomal investigations in PD. This allowed us to use training and validation groups from different cohorts, establish cutoff values ​​for exosomal α-synuclein, and demonstrate consistent performance of the assay. The availability of longitudinal samples allowed us to show the stability of the marker over time. Limitations include the need to replicate the association of clusterin in additional patient cohorts.

[0231] The finding that neuron-derived exosomal α-synuclein is consistently elevated across populations and remains elevated in individuals with PD when tested over a 5-year period suggests that measurements of the neural exosomal content of α-synuclein in serum can be used as a surrogate for its intraneuronal processing and therefore as a marker for monitoring disease-modifying therapies targeting α-synuclein in the brain, especially in the early stages of PD. Given the high risk of RBD conversion to PD (48) and the widespread acceptance of RBD patients as potential candidates for neuroprotective therapies for PD, this study also defined parameters for an easily accessible objective readout of the underlying Lewy pathology in this group of prodromal PD. Notably, combined measurements of the neural exosomal content of α-synuclein and clusterin improved the predictive test value of primary α-synucleinopathy versus another proteinopathy (AUC0.98). Thus, assaying neuron-derived exosomal α-synuclein and clusterin in serum is a blood-based predictive test for ongoing α-synuclein pathology such as PD, which could be implemented in clinical trials of α-synuclein-targeted therapies targeted to at-risk populations.

[0232] Example 3 This example further demonstrates the clinical utility of measuring α-synuclein, optionally in combination with clusterin, in serum and neuroexosomes as a biomarker across the spectrum of Parkinson's disease, multiple system atrophy and other proteinopathies.

[0233] material and method A total of 664 subjects were included in the study (Table 3). Serum samples and clinical data were collected from patients with polysomnographically confirmed RBD (n=65), PD (n=275), dementia with Lewy bodies (n=14, DLB), multiple system atrophy (n=14, MSA), frontotemporal dementia including behavioral type or primary progressive aphasia (n=65, FTD), progressive supranuclear palsy (n=35, PSP) and corticobasal syndrome (n=45, CBS). Healthy controls (n=144, HC) were of similar age and sex.

[0234] Levels of α-synuclein, clusterin and syntenin-1 in L1CAM-positive exosomes from serum samples were determined as detailed in Example 2.

[0235] Statistical analysis was performed as detailed in Example 2.

[0236] Table 3 Summary of individual cohort characteristics and exosomal marker concentrations. Data represent the mean at time of sampling. UPDRS and MoCA were available in 48% of healthy controls. RBD=Rapid Eye Movement Sleep Behavior Disorder, PD=Parkinson's Disease, PDD=Parkinson's Disease with Dementia, DLB=Dementia with Lewy Bodies, MSA=Multiple System Atrophy, HC=Healthy Controls, FTD=Frontotemporal Dementia with Behavioral or Primary Progressive Aphasia, PSP=Progressive Supranuclear Gaze Palsy, CBS=Corticobasal Syndrome. * Postmortem cases. [Table 3-1] [Table 3-2]

[0237] result Neuron-derived exosomal α-synuclein is increased across the spectrum of Lewy body diseases Serum samples from 664 subjects spanning the spectrum of Lewy body pathology were analyzed by assaying patients in the prodromal, motor and cognitive stages. For this purpose, PD subjects were separated into those with pure motor PD or PD dementia according to the MoCA score corrected for educational history. Dementia within the PD cohort was defined as a MoCA screening score below 21 / 30 at the time of sample collection (29). Therefore, subgroups of motor PD (n=230) or PD with dementia (n=45) were subsequently analyzed blindly. We also included a group of 21 cases with a clinical diagnosis of DLB, 10 of which were confirmed at autopsy. Since prospective cohort studies observed a very strong association between RBD and subsequent clinically defined α-synucleinopathy, with up to 80% of cases converting to primarily PD or DLB, a group of idiopathic RBD without motor signs (n=65) was also used as a surrogate for prodromal PD. (30,31)

[0238] We found that α-synuclein was elevated approximately two-fold in RBD, PD and DLB exosomes compared to controls, MSA or other proteinopathies (Figure 13A and Table 3). Specifically, α-synuclein content in L1CAM-positive exosomes was similarly elevated in RBD (26.69 ± 12.82 pg / mL), motor PD (27.44 ± 18.82 pg / mL) and PD with dementia (PDD 26.76 ± 17.25 pg / mL) when compared to healthy subjects (HC, 12.71 ± 5.93 pg / mL) (data shown as mean + / - SD). α-synuclein was also elevated in DLB (17.23 ± 4.58 pg / mL). By testing serum collected before death in autopsy-confirmed controls and DLB cases (n=10 / group), we demonstrated an association between increased release of α-synuclein in neural exosomes and Lewy body pathology. In these two subgroups, mean neural exosome-associated α-synuclein was 17.60±5.86 pg / mL in DLB and 10.50±4.60 pg / mL in controls (1.7-fold increase, p=0.0097). As expected, exosomal α-synuclein concentrations were much lower compared to reported levels of total free α-synuclein in blood (10-17 ng / mL) (7,15). Interestingly, neuron-derived exosomal α-synuclein was not elevated in any of the MSA cases (10.72 ± 4.49 pg / mL), a disease primarily characterized by oligodendroglial lesions, despite the fact that the MSA samples were collected and processed using identical procedures as the PD samples.

[0239] To assess α-synuclein abundance in neuron-derived exosomes in unrelated neurodegenerative conditions, we tested the following patient groups: FTD patients (n=65), whose pathology is characterized primarily by tau or TDP-43 aggregation, and PSP patients (n=35) and CBS patients (n=45), who presented with atypical parkinsonism and whose pathology is characterized by fibrillar aggregates of 4-repeat tau. We found that α-synuclein content in L1CAM-positive exosomes from these diseases was similar to HC (FTD, 12.60±4.03pg / mL; PSP, 9.20±4.90pg / ml; CBS, 9.93±3.68pg / mL), as shown in Figure 13A.

[0240] In 226 subjects (18 RBD, 77 PD, 36 PDD, 11 DLB, 69 HC, 15 FTD), we also investigated whether increased α-synuclein in Lewy body disease is phosphorylated at serine 129 (pSer129) in L1CAM-positive exosomes and has value as a blood-based biomarker. pSer129α-synuclein is the major disease-associated modification that accounts for more than 90% of the α-synuclein found in Lewy bodies (32). This analysis showed that only a minority of individuals had detectable levels of pSer129α-synuclein in neural exosomes. Interestingly, when applying a cutoff value of 0.5 pg / ml, which is within the detection limit of the assay (Figure 13B), pSer129α-synuclein was elevated in a subgroup of 22 PD patients (28.6% of all PD tested). In this PD subpopulation, pSer129 α-synuclein correlated with disease duration longer than 7.3 years (r = 0.26, p = 0.0263) and UPDRS (r = 0.34, p = 0.0495), but not with MoCA (r = 0.006, p = 0.3643). Unlike previous studies (13, 15), no significant correlation was detected between exosomal α-synuclein and either UPDRS (r = 0.0267) or MoCA (r = 0.0621), as shown in Figures S4C and S4D.

[0241] α-Synuclein and clusterin measurements improved the predictive value of exosome testing We found that clusterin was elevated in FTD (20.22 ± 10.47 ng / mL), PSP (18.42 ± 8.84 ng / mL) and CBS (16.16 ± 6.07 ng / mL) (Figure 14A), but not in RBD (10.01 ± 5.22 ng / mL), clinical PD (9.72 ± 6.02 ng / mL), MSA (6.84 ± 3.24 ng / mL) and HC (8.67 ± 4.92 ng / mL). The differential abundance of clusterin in unrelated proteinopathies suggests that incorporation of clusterin in blood-based exosome testing may be valuable in discriminating PD patients from tau-related atypical parkinsonism (Figure 14B). This is demonstrated in a heatmap (Figure 14C) summarizing the overall trend of the biomarker (using the mean concentration) across different patient groups when normalized to HC. In contrast, syntenin-1, a common exosomal protein, did not show disease-specific distribution with sufficient separation to serve as a biomarker (Figure 15).

[0242] To further evaluate the clinical potential of the combined measurement of α-synuclein and clusterin in L1CAM-positive exosomes as a biomarker, the ratio of α-synuclein to clusterin was evaluated and a logistic regression model was applied to the combination of these markers. The combined measurement of α-synuclein and clusterin showed improved AUC, sensitivity and specificity estimates for differential diagnosis in predicting clinical PD vs. other proteinopathies (AUC=0.98 (sensitivity 0.94; specificity 0.96)), as shown in Figure 14D and Figure 14F and Table 4, and even in the prodromal phase of PD, RBD vs. other proteinopathies, AUC=0.98, sensitivity 0.95, specificity 0.93. This measurement also showed high performance in discriminating prodromal or clinical PD from MSA (AUC=0.94 and 0.91, respectively), as summarized in Figure 14E and Figure 14G.

[0243] Table 4. Summary of ROC analyses of patient groups across cohorts comparing synucleinopathies with controls or other proteinopathies using α-synuclein, clusterin and combined markers (α-synuclein and clusterin). Composite markers were analyzed by logistic regression. ROC-based separation was applied when there was a significant difference between the two groups (p<0.001). High-performing (AUC≧0.90) markers are shown in bold and underlined. [Table 4-1] [Table 4-2]

[0244] conclusion We found that mean exosomal α-synuclein was increased 2-fold in prodromal and clinical Parkinson's disease when compared to multiple system atrophy (MSA), controls or other neurodegenerative diseases. In 314 subjects in the training group and 105 subjects in the validation group, exosomal α-synuclein showed consistent performance (AUC=0.86) in separating clinical Parkinson's disease from controls across populations. Exosomal clusterin was elevated in subjects with non-α-synuclein proteinopathies. Combined measurements of neuron-derived exosomal α-synuclein and clusterin predicted Parkinson's disease with AUC=0.98 from other proteinopathies and AUC=0.94 from MSA.

[0245] In conclusion, increased α-synuclein release in serum neuroexosomes precedes Parkinson's disease diagnosis, persists with disease progression, and, in combination with clusterin, predicts and distinguishes Parkinson's disease from atypical parkinsonism.

[0246] Example 4 This example demonstrates the superior antifouling properties of the coated particles described herein, and the improved sensitivity of these assays compared to commercially available electrochemiluminescence kits.

[0247] material All chemical reagents were used as received. Potassium ferricyanide, potassium ferrocyanide, 3-mercaptopropionic acid (3-MPA), 2-mercaptoethanol (2-MU), 1-ethyl 3-(3-(dimethylamino)propyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), Triton X-100 (TX), bis(carboxymethyl)trithiocarbonate (BisCTTC) were obtained from Sigma-Aldrich (Gillingham, UK). Commercially available electrochemiluminescence (ECL) detection plates with linkers and ruthenium tags were ordered from Meso Scale Discovery (MSD, United States). Sera-Mag carboxylate modified magnetic beads (24152105050250) were purchased from GE Healthcare and used as a control. (Buckinghamshire, UK). Nanoparticle tracking analysis was performed using a Malvern NanoSight NS500 (Malvern, UK) configured with a 405 nm laser and a highly sensitive CMOS camera (OrcaFlash2.8, Hamamatsu C11440, NanoSight Ltd.). Footage was collected and analyzed using NTA software (version 2.3, build 0025) with camera level and detection threshold set to 14 and 5, respectively. All analyses were performed at a controlled temperature of 23 °C.

[0248] Fetal bovine serum (FBS), C-reactive protein (CRP), bovine serum albumin (BSA) and human serum albumin (HSA) were purchased from Sigma-Aldrich. α-Synuclein (αSyn), syntenin-1 (Synt-1) standards, anti-α-Syn, anti-Syn-1, anti-L1CAM and anti-hemagglutinin (HA) antibodies were obtained from Abcam (Cambridge, UK). All protein samples were diluted in filtered PBS buffer (pH 7.4).

[0249] Parkinson's disease (PD) and healthy controls (HC) were recruited and whole blood samples were collected in accordance with institutional guidelines and ethical approval. Full details of the Kiel-PD cohort are published in reference 49.

[0250] method Preparation of antifouling pCBMA-coated MBs Magnetic microbeads were prepared by a two-step approach involving the formation of ferrihydrite / formaldehyde composite microbeads followed by hydrothermal reduction of ferrihydrite to magnetite. Iron hydroxide was synthesized by hydrolysis of ferric chloride salt solution at room temperature as described in refs. 26 and 27. Briefly, 25 g of FeCl 3 6H 2 A total of 16 g of NaHCO3 was dissolved in 100 mL of ultrapure water. 3 was slowly added. The mixture was stirred for 1 h to obtain a reddish-brown ferrihydrite solution, after which 1.05 g urea was added and then the pH was adjusted to 2.0 with 2 M nitric acid. This was followed by the addition of 1.57 mL of aqueous formaldehyde (37 wt%) under stirring. After the addition was complete, the mixture was left without stirring at ambient temperature. Within 10 min, a yellowish gel was formed. The resulting microspheres were aged overnight before recovery by filtration and washing with Milli-Q water (18.2 MΩ, Millipore UK Ltd). Finally, the particle sample was suspended in 130 mL of 0.1 M sodium borohydride solution (pH 9.0) and the suspension was transferred to an autoclave. The reaction was carried out at 80 °C for 2 h, during which the initially yellowish microspheres turned black and could be easily magnetically extracted before being washed extensively with EtOH and Milli-Q water. They are then oven dried at 40° C. and resuspended in Milli-Q water at a concentration of 50 mg / mL.

[0251] The bead surface was functionalized with the bifunctional RAFT agent BisCTTC as follows: 1 mL of Fe 3 O 4The suspension was added to 10 mL of a water / ethanol (3 / 7, v / v) mixture under sonication for 10 min at room temperature, followed by the addition of 10 mg of BisCTTC (0.044 mmol). The water / ethanol solvent was chosen to ensure the dispersion of the magnetic beads and the solubilization of BisCTTC. The mixture was left under magnetic stirring and nitrogen flow for 24 h. Fe 3 O 4 The final product of @BisCTTC was isolated and purified by magnetic collection and washed three times with ethanol and Milli-Q water.

[0252] In the final step, BisCTTC and 4,4'-azobis(4-cyanovaleric acid) (ACVA) were used as monomer, free chain transfer agent (in solution phase) and initiator, respectively. 3 O 4 The synthesis of was carried out by standard RAFT polymerization procedures. Typically, Fe 3 O 4 A 1 mL suspension containing pCBMA@Fe@BisCTTC beads was mixed with CBMA (360 mg, 1.568 mmol), ACVA (1.1 mg, 0.00392 mmol) and the free CTA BisCTTC (3.55 mg, 0.01568 mmol) dissolved in 10 mL of ethanol / water (1:1). After purging the reaction mixture with nitrogen for 1 h, the glass flask was heated in an oil bath at 70 °C and left under S-5 nitrogen with mechanical stirring for 8 h. The reaction was terminated by inserting the reaction flask into an ice bath followed by exposure to air (quenching). The final pCBMA@Fe@BisCTTC was 3 O 4 The bead products were magnetically separated and washed several times with ethanol and water.

[0253] Preparation of immunobeads. Antifouling immunobeads were prepared by injecting anti-L1CAM Ab (ab20148, Abcam) into pCBMA@Fe 3 O 4 Specifically, pCBMA@Fe 3 O 4The carboxylate groups of the beads (1 mg / mL) were activated with 50 mg / mL EDC / NHS in MES buffer and then reacted with 8 μg / mL (final concentration) of anti-L1CAM or CD9 antibodies for 1.5 h at room temperature. After washing with PBS using a magnet, the beads were mixed in 1 mL of PBS containing 5 mg / mL BSA for 30 min at room temperature (to quench remaining activation sites and backfill remaining spaces). The immunobeads were magnetically collected and stored at 4°C until further use. All such immunobeads were prepared and consumed on the same day.

[0254] Fourier transform infrared attenuated total reflectance (FTIR-ATR) of the prepared pCBMA magnetic beads was washed with ethanol and Milli-Q water and dried at 50 °C before testing. 3 O 4 Magnetic beads were used as a control. All spectra were recorded using a Bruker Vertex 80 spectrometer equipped with a mercury cadmium telluride (MCT) detector and an ATR unit (DuraSamplIR II diamond ATR) at 2 cm -1 The spectra were recorded between 4000 and 400 cm-1 at a resolution of 0.01 μm and evaluated using OPUS 6.5 software.

[0255] Antifouling test of pCBMA beads To test the antifouling performance of pCBMA beads, 1 mg of AbpCBMA@Fe 3 O 4 or 1 mg of pCBMA@Fe 3 O 4 (Uncoated Fe 3 O 4 Beads were used as a control) were added separately to 10 mg / mL BSA solution and incubated at room temperature for 1 h. The supernatant containing unbound proteins was collected and subjected to the bicinchoninic acid (BCA) test to determine the adsorbed protein from: Adsorbed amount = input amount - unbound amount in supernatant

[0256] To assess the level of non-specific adsorption of free α-synuclein onto the immunobeads, 1 mg of pCBMA magnetic beads coated with anti-L1CAM antibody (anti-HA antibody or no antibody as control) was added to 500 μL of PBS containing 20 ng / mL of α-synuclein standard protein (i.e., a concentration reflecting clinically relevant levels of free α-synuclein in blood). The mixture was gently shaken overnight at 4 °C. After incubation, the supernatant fraction was collected using a magnetic rack. Control beads (commercially available carboxylate magnetic beads) with the same experimental setup were run in parallel. The amount of α-synuclein adsorption onto the beads was quantified using the ECL kit using the following formula: Amount adsorbed = input amount - unbound amount in the supernatant.

[0257] Zeta potential Surface zeta potential analysis was performed on a Malvern Zetasizer Nano using a 532 nm laser as the light source, with uncoated Fe in PBS (10 mM, pH = 7.4). 3 O 4 This was performed using beads and pCBMA-coated MBs (approximately 1 mg / mL).

[0258] For exosome isolation, a three-step sequential spin (300g for 10 min, 2000g for 20 min, and 10,000g for 30 min) was used to remove cell debris, protein aggregates, and fatty materials from the serum. An appropriate volume (0.5 mL for commercial ECL plates and 0.1 mL for EIS sensors) of the supernatant, i.e., pre-cleared serum, was transferred to a protein low-binding tube (Eppendorf) for immunocapture using anti-L1CAM antibody pre-conjugated to pCBMA beads generated to reduce non-specific adsorption. The immunobeads were incubated overnight at 4°C on a rotating mixer, and the bead-exosome complexes were collected by magnetic separation and washed successively with 0.05% Tween-20 in PBS (PBST) and PBS. For quantification of exosomal proteins, isolated exosomes were lysed in lysis buffer (50 μL for commercial ECL plates and 10 μL for EIS sensors) containing 1% triton X-100 in PBS with 4% protease inhibitors for 15 min at room temperature for quantification of exosomal proteins.

[0259] Transmission electron microscopy Transmission electron microscopy (TEM) was used to examine the shape and morphology of captured exosomes eluted from pCBMA beads. Specifically, EVs captured on MBs were eluted by adding 20 μL of glycine solution (pH 2.9) and the pH was quickly returned to neutral with 20 μL of Tris solution (pH 9.5). 10 μl of the resulting eluate sample was applied to a freshly glow-discharged carbon formvar 300 mesh copper grid for 2 min, blotted with filter paper, stained with 2% uranyl acetate (aqueous solution) for 10 s, then blotted and air-dried. Grids were imaged in a TEM operated at 120 kV using a Gatan OneView CMOS camera.

[0260] Scanning Electron Microscopy Immunocaptured exosomes on pCBMA beads were fixed in 2% glutaraldehyde on clean silicon wafers and washed twice with PBS. After natural evaporation, samples were coated with approximately 5 nm of platinum using a sputter coater (Cressington) and imaged with a scanning electron microscope (JEOL 6010LV) at 5 kV.

[0261] Western blot Western blot was used to characterize transmembrane and internal proteins from immunocaptured exosomes. Exosomes captured by anti-L1CAM immunobeads (anti-CD9 as a positive control targeting general exosomes and anti-HA immunobeads as a negative control) were dissolved in LDS buffer (Thermo Fisher), resolved using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride membranes (PVDF, Invitrogen), and immunoblotted with antibodies against Synt-1 (ab133267, Abcam), CD9 (CBL162, Millipore), and L1CAM (ab80832, Abcam). All antibodies were used at a 1:1,000 dilution. After incubation with horseradish peroxidase-conjugated secondary antibodies (GE Healthcare) (1:10,000 dilution), chemiluminescence was used for immunodetection (ChemiDoc, Bio-Rad).

[0262] Commercial electrochemiluminescence detection Electrochemiluminescence (ECL) detection was performed in 96-well Meso Scale Discovery (MSD) U-Plex plates according to the manufacturer's instructions. Two unique linkers (anti-Synt-1, anti-α-synuclein) for the selected capture antibodies were used according to the manufacturer's protocol. Immunocaptured exosome lysates or S-8 standard solution (50 μL) were loaded and incubated for 1 h at room temperature. After three washes, detection antibodies with sulfo-TAG labels were incubated for 1 h. After washing with wash buffer (from Meso Scale Discovery) and addition of MSD Read buffer (from Meso Scale Discovery), the plates were read using the MSDECL platform (QuickPlex SQ 120). Data were analyzed using the MSD Discovery Workbench 3.0 Data Analysis Toolbox. Antibody pairs against α-synuclein (pre-conjugated with biotin and ruthenium tags, provided by Meso Scale Discovery) were provided by MSD. Additive-free anti-Synt-1 goat polyclonal antibody (PAB7132, Abnova) and anti-Synt-1 rabbit monoclonal antibody (ab236071, Abcam) were conjugated with biotin and ruthenium and used as capture and detection antibodies, respectively.

[0263] Exosome capture efficiency. To evaluate the exosome capture efficiency using immunobeads, anti-CD9 antibody-modified pCBMA@Fe 3 O 4 MBs were prepared according to the same procedure as in "Preparation of Immunobeads" above. Immunobeads (0.2 mg) were mixed with 100 μL of pre-cleared serum and incubated overnight at 4°C. After incubation, the supernatant was collected with the aid of an external magnetic rack. The exosome concentration in the input serum and supernatant was then measured using nanoparticle tracking analysis of the particle fraction ranging from 40 to 140 nm (i.e., the typical size of exosomes). The capture efficiency was measured using the following formula: (Input (CD9+exosomes) - Unbound amount) / Input (CD9+exosomes) x 100% = (Total input amount × 75% * - Unbound amount) / total amount input × 75%) × 100% =(2.66-0.51) / 2.66×100%=80.8% (Note: CD9+ exosomes make up approximately 75% of the total exosome population).

[0264] Preparation of the receptor interface and EIS detection Au disk electrodes (diameter 3.0 mm, purchased from BASi®, USA) were mechanically polished with 1.0 μm, 0.3 μm, and 0.05 μm alumina slurries, respectively. The electrodes were sonicated in ethanol for 10 min and then washed with piranha (v / v 3:1, H 2 SO 4 :H 2 O 2 ) for 10 min. After rinsing with Milli-Q water and drying with nitrogen, the electrodes were immersed in 0.5 M KOH aqueous solution for 100 cycles of cyclic voltammetry scans (-1.7 to -0.7 V). They were then induced at 0.5 M KOH vs. a Ag wire reference electrode at 0.1 V / s until the heights and shapes of the anodic and cathodic peaks were constant. 2 SO 4 The electrodes were electrochemically cycled from -0.15V to 1.35V.

[0265] Mixed SAMs of 3-MPA and 2-MU were generated by immersing a clean gold disk electrode in a 50 mM 3-MPA and 10 mM 2-MU solution overnight at room temperature in the dark. The electrode was rinsed with ethanol to remove physically adsorbed molecules and then dried in a stream of argon. The terminal carboxyl groups of 3-MPA were then activated with 0.4 M EDC / NHS solution for 30 min and carefully washed with PBS. 10 μL of antibody solution at an optimized concentration of 100 μg / mL was then incubated on the electrode for 1 h, and the surface was then blocked with FBS solution for 30 min to inactivate the residual carboxyl groups. The stability of the antibody-modified electrode was verified by repeated incubations in PBS for 20 min, followed by 5 mM K 3[Fe(CN) 6 ] and K 4 [Fe(CN) 6

[0036] . Afterwards, 10 μL of α-Syn, Synt-1 spiked into 10% human serum or exosome lysate (obtained by adding 1% triton X-100 in PBS containing 4% protease inhibitors to the exosome-beads complex at room temperature for 15 min) was then incubated on the electrode for an optimized incubation time of 20 min and washed with PBS solution. The sensor electrode was then washed with PBS solution for 10 min. -3 CRP in g / mL, 10 -3 g / mL α-Syn, or 10 -3 Selectivity analysis was performed by incubating with 100 µg / mL BSA for 20 min. EIS measurements were recorded using a PalmSens electrochemical workstation with a standard three-electrode configuration and 5 mM K in PBS solution. 3 [Fe(CN) 6 ] and K 4 [Fe(CN) 6 All measurements were performed at a fixed amplitude of 0.01 V and a fixed frequency range of 100 kHz to 100 mHz. ct (R ct-antibody ) and R when S-10 antigen was added ct (R ct-antigen ) was calculated from the fitting of the equivalent circuit diagram. The relative response is determined from: relative response=R ct-antigen -R ct-antibody .

[0266] Statistical analysis of patient samples was performed by standard Student's t-test.

[0267] result Performance and antifouling properties study As described above, magnetic beads (approximately 2.4 μm) were coated with the zwitterionic polymer pCBMA via the RAFT process and further modified with anti-L1CAM antibody.

[0268] The zeta potential was evaluated before polymerization (Fe 3 O 4 , -33.8±3.2mV) and after polymerization (pCBMA@Fe 3 O 4 , −2.3 ± 1.2 mV), indicating a near-zero total charge, as desired for optimal performance (see references 50 and 51).

[0269] pCBMA@Fe 3 O 4 The antifouling properties of MB are similar to those of Fe 3 O 4 This was confirmed by the significant reduction (about 90%) in nonspecific adsorption of bovine serum albumin (BSA) when compared to beads (see Figure 16). 3 O 4 It is noteworthy that the antifouling performance is not significantly impaired even after MB. 3 O 4 It was further demonstrated that MBs, unlike commercial carboxylate MBs, exhibit good antifouling properties when incubated with soluble recombinant α-synuclein, regardless of the antibody used (anti-L1CAM or anti-HA shown in Figure 17A), which is of great importance in supporting the selective and clean isolation of exosomes from serum samples.

[0270] The anti-L1CAM antibody-coated pCBMA was then evaluated for immunocapture of neural exosomes in serum. SEM image analysis showed that exosomes were bound to the anti-L1CAM-conjugated pCBMA@Fe 3 O 4 The anti-HAAb-coated pCBMA@Fe beads in the inset of FIG. 17B bound to the MB (FIG. 17B) but not to the control beads (i.e., the anti-HAAb-coated pCBMA@Fe beads in the inset of FIG. 17B). 3 O 4 beads).

[0271] To further confirm their molecular composition, the captured vesicles were lysed and processed for immunoblotting (Figure 17C). Transmembrane markers L1CAM and CD81 as well as the internal protein marker Synt-1 were immunoblotted with anti-L1CAM@pCBMA@Fe 3 O 4 was detected in the lysates from the MB samples but not in the control lysates (anti-HA-coated pCBMA@Fe 3 O 4 It was not detected in the samples incubated with MB.

[0272] Anti-L1CAM modified pCBMA Fe 3 O 4 It was also confirmed that MB was effective in isolating serum containing α-Syn from neural exosomes (Figure 17D).

[0273] Comparison of selectivity with commercially available electrochemiluminescence kits Selectively captured exosomes were electrochemically quantified as described above. In particular, the reliability of biomarker quantification was verified by measuring the concentration of exosomes within 10% of the expected marker levels. 6 The assay was tested by replicate analysis of spiked solutions prepared for both α-Syn and Synt-1, including analysis with a 2-fold excess of control proteins (e.g., C-reactive protein (CRP) and BSA) (Figure 18). Reliable triplicate quantification of both markers (Figure 19) was demonstrable within 30 minutes with limits of detection (LOD) and limits of quantification (LOQ) of 0.3 and 0.8 pg / mL for α-Syn, respectively (Figure 20). This is significantly better than most previous exosome analyses. Thus, the assay herein is significantly more sensitive (by almost an order of magnitude), much less expensive, and much faster than commercially available electrochemiluminescence kits, and requires significantly less sample input (100 μL versus 500 μL).

[0274] Example 5 This example further validates the clinical utility of measuring α-synuclein, optionally in combination with clusterin, in serum neuroexosomes as a biomarker across the spectrum of Parkinson's disease, multiple system atrophy and other proteinopathies, by using patients from additional cohorts.

[0275] material and method A total of 288 subjects were included in the study (Table 5). Serum samples and clinical data were collected from patients with polysomnographically confirmed rapid eye movement (REM) sleep behavior disorder (RBD) (n=26), PD (n=45), multiple system atrophy (MSA) (n=36), progressive supranuclear palsy (PSP) (n=81) and corticobasal syndrome (CBS) (n=43). Healthy controls (HC) (n=57) were of similar age and sex.

[0276] L1CAM-positive neural exosomes were isolated as detailed in Example 2, except that a smaller amount of serum was used (250 μL instead of 500 μL).

[0277] Samples were blindly analyzed for α-synuclein, clusterin and syntenin-1 as detailed in Example 2.

[0278] Statistical analyses were performed as detailed in Examples 2 and 3. [Table 5]

[0279] result The results are shown in Figures 21 to 25.

[0280] It can be seen that exosomal α-synuclein was significantly increased in RBD and PD compared to controls (Figure 21), and there was higher clusterin in PSP and CBS (Figure 23). A significant increase in the α-synuclein / clusterin ratio can also be seen in RBD and PD compared to controls (Figure 24).

[0281] Using ROC analysis, it was further confirmed that α-synuclein or the α-synuclein / clusterin ratio independently provided accurate biomarkers predicting neuronal synucleinopathy in RBD and PD versus MSA (glial synucleinopathy) or tauopathy (PSP, CBS) (see Figure 22 and Figure 25).

[0282] These observations are consistent with those from the examples above. The present invention includes the following preferred embodiments. (1) Coated particles having a coating comprising a zwitterionic polymer coupled to a ligand having affinity for a selected population of exosomes. (2) 1. The coated particle of claim 1, wherein the zwitterionic polymer comprises a carboxybetaine, sulfobetaine, and / or phosphorylcholine moiety. (3) 3. The coated particle of claim 1 or 2, wherein the zwitterionic polymer comprises poly(carboxybetaine methacrylate). (4) The coated particle according to any one of claims 1 to 3, wherein the ligand has affinity for neuron-derived exosomes, for example, the ligand is an anti-L1CAM antibody. (5) The coated particles according to any one of 1 to 4 have a size of 30 nm to 5 μm, preferably a size of 100 nm to 5 μm, and more preferably a size of 500 nm to 3 μm. (6) 6. The coated particle according to any one of 1 to 5, wherein at least 80% of the particle surface is coated with a polymer. (7) 7. The coated particle according to any one of 1 to 6, wherein the polymer coating has a thickness of 10 nm to 500 nm. (8) 8. The coated particles according to any one of 1 to 7, having a degree of non-specific adsorption of less than 10%. (9) The coated particle according to any one of claims 1 to 7, wherein the polymer has a brush structure. (10) The coated particles according to any one of claims 1 to 8, wherein the polymer is obtainable by a RAFT polymerization process, optionally using bis(carboxymethyl)trithiocarbonate (BCMTTC) as a chain transfer agent. (11) The coated particles according to any one of claims 1 to 9, optionally obtained or obtainable by the method according to claim 17 or 18, by growing a zwitterionic polymer on the particle. (12) 1. A method for isolating exosomes from a sample, comprising: - contacting a sample with a coated particle according to any one of claims 1 to 10; - removing unbound sample; and - isolating the captured exosomes A method comprising: (13) 13. A method for analyzing a blood sample from a subject, comprising isolating neuron-derived exosomes from the sample according to the method described in (11) and determining a level of alpha-synuclein and / or clusterin in the neuron-derived exosomes in the blood sample. (14) The method of claim 12, wherein the levels of α-synuclein and clusterin provide a diagnostic indicator for a subject susceptible to or having Parkinson's disease (PD). (15) The levels of α-synuclein and clusterin provide a diagnostic indicator for subjects with prodromal PD. (16) An increase in the level of alpha-synuclein compared to the reference indicates that the subject is susceptible to or has PD, optionally with the reference being a threshold value between 10 and 20 pg / ml (13 or 14). (17) A lack of an increase in the level of clusterin compared to the reference indicates that the subject is susceptible to PD, optionally with the reference being a threshold value between 7 and 17 ng / ml. (18) (A method for producing the coated particle according to any one of 1 to 10, (a) growing a zwitterionic polymer on a surface of a particle using reversible addition-fragmentation chain transfer (RAFT) to provide a particle having a coating comprising the zwitterionic polymer; (b) optionally activating the zwitterionic polymer to provide active functional groups on the zwitterionic polymer; and (c) conjugating a ligand having affinity for the selected population of exosomes to the zwitterionic polymer. A method comprising: (19) The method according to (17), wherein step (a) comprises using bis(carboxymethyl)trithiocarbonate (BCMTTC) as a chain transfer agent.

[0283] References JPEG0007680048000009.jpg99130 JPEG0007680048000010.jpg153130

Claims

1. 1. A coated particle having a coating comprising a zwitterionic polymer coupled to a ligand having affinity for a selected population of exosomes, the zwitterionic polymer being obtainable by a reversible addition fragmentation chain transfer (RAFT) polymerization process.

2. The coated particle of claim 1 , wherein the zwitterionic polymer comprises a carboxybetaine, a sulfobetaine and / or a phosphorylcholine moiety.

3. The coated particle of claim 1 or 2, wherein the zwitterionic polymer comprises poly(carboxybetaine methacrylate).

4. The coated particle of claim 1 , wherein the ligand has affinity for neuron-derived exosomes.

5. The coated particle of claim 4 , wherein the ligand is an anti-L1CAM antibody.

6. The coated particle of claim 1, wherein the particle is 30 nm to 5 μm in size, 100 nm to 5 μm in size, or 500 nm to 3 μm in size.

7. At least 80% of the particle surface is coated with a polymer, and / or The coated particle of any one of claims 1 to 6, wherein the polymer coating has a thickness of from 10 nm to 500 nm.

8. 8. The coated particle of claim 1 having a degree of non-specific adsorption of less than 10%.

9. The coated particle of claim 1 , wherein the polymer has a brush structure.

10. The coated particle according to any one of claims 1 to 9, wherein the RAFT polymerization process is a RAFT polymerization process using bis(carboxymethyl)trithiocarbonate (BCMTTC) as a chain transfer agent.

11. 11. The coated particle according to any one of claims 1 to 10, obtained or obtainable by growing a zwitterionic polymer on the particle.

12. 1. A method for isolating exosomes from a sample, comprising: Contacting a sample with the coated particles of any one of claims 1 to 11; Removing unbound sample; and Isolating the captured exosomes The above method.

13. The method of claim 12, wherein the sample is a blood sample from the subject.

14. 14. A method for analyzing a blood sample from a subject, comprising isolating neuron-derived exosomes from the sample according to the method of claim 12 or 13, and measuring levels of alpha-synuclein and / or clusterin in the neuron-derived exosomes in the blood sample.

15. 15. The method of claim 14, wherein the levels of α-synuclein and clusterin provide a diagnostic indication for a subject susceptible to or having Parkinson's disease (PD).

16. the levels of α-synuclein and clusterin provide a diagnostic indication for subjects with prodromal PD; and / or The method of claim 15, wherein an increase in the level of α-synuclein compared to the reference indicates that the subject is susceptible to or has PD.

17. The method of claim 15 or 16, wherein a lack of increased levels of clusterin compared to the reference indicates that the subject is susceptible to PD.

18. A method for producing the coated particles of any one of claims 1 to 11, comprising the steps of: (a) growing a zwitterionic polymer on the surface of a particle using reversible addition-fragmentation chain transfer (RAFT) to provide a particle having a coating comprising the zwitterionic polymer; and (b) conjugating a ligand having affinity for the selected population of exosomes to the zwitterionic polymer. The above method.

19. and / or further comprising the step of activating the zwitterionic polymer to provide active functional groups on the zwitterionic polymer; 20. The method of claim 18, wherein step (a) comprises using bis(carboxymethyl)trithiocarbonate (BCMTTC) as a chain transfer agent.

20. 20. Coated particles obtained or obtainable by the method according to claim 18 or 19.

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