Biomarkers for neuronopathic mucopolysaccharidoses

Abstract

Described are materials and methods for detecting a neuronopathic MPS, such as MPS I or MPS III in a subject, as well as methods of monitoring disease progression, cognitive decline, and for treating a neuronopathic MPS, such as MPS I or MPS III.

Description

BIOMARKERS FOR NEURONOPATHIC MUCOPOLYSACCHARIDOSES

[0001] This application claims benefit of United States provisional patent application number 63 / 549,105, filed February 2, 2024, the entire contents of which are incorporated by reference into this application.BACKGROUND

[0002] Lysosomal storage disorders (LSDs) are rare, systemic, progressive diseases caused by genetic defects in lysosomal enzymes responsible for macromolecule degradation, lysosomal activator proteins, or transporters of lysosomal and non-lysosomal proteins required for proper lysosomal function. Mucopolysaccharidosis disorders (MPSs) comprise -30% of all LSDs and are characterized by mutations leading to defective enzymes required for the breakdown of glycosaminoglycans (GAGs). Seven MPSs, including types I, II, III, IV, VI, VII and IX are caused by deficits in 11 different enzymes. They are characterized by primary storage of heparan, keratan, dermatan, or chondroitin sulfates leading to severe musculoskeletal, systemic (e.g., hepatomegaly, cardiac, and respiratory dysfunction), and neurodegenerative abnormalities. MPS types I, II, III, and VII are classified as neuronopathic because in these types heparan sulfate, the main GAG in the central nervous system, accumulates in brain cells causing severe neuroinflammation and neurodegeneration.

[0003] MPS I, the most common and severe MPS type, is caused by a deficiency in iduronidase (IDUA, EC 3.2.1.76), leading to lysosomal storage of dermatan sulfate in the periphery and heparan sulfate (HS) in the CNS. Prominent peripheral features of the diseases include corneal clouding, upper airway obstruction, joint contractures, cardiomyopathy, and inflammation. CNS pathology causing cognitive decline and developmental delays begin at -9 months of age in the severe form of MPS I, called Hurler syndrome. The etiopathology of the CNS pathology in MPS I is similar other tissues and involves neuroinflammation by GAG-mediated activation of Toll-like receptor 4, mitochondrial dysfunction, impaired autophag34, and aberrant lysosomal function leading to accumulation of toxic aggregates, abnormal vesicle fusion and dysregulated intracellular trafficking. Although enzyme replacement therapy (ERT) is approved by FDA for MPS I, the neurological symptoms are minimally affected by the treatment due to the restricted capacity of ERT IDUA to cross blood-brain barrier. A major impediment to developing efficient therapies for MPS I and other neuronopathic MPS types is the absence of reliable biomarkers for the CNS pathology. HS GAG non-reducing ends (NREs) in the cerebrospinal fluid recently have been recommended by FDA as a biomarker, yet the need for a lumbar puncture and the high expertise required for the measurement limit the use of this biomarker.

[0004] MPS III, also known as Sanfilippo syndrome, was described first in 1963 as a neurocognitive and neurobehavioral disorder caused by defects in enzymes required for catabolism of heparan sulfate (HS). There are four subtypes of MPS III in humans, A, B, C, and D, which share a similar clinical phenotype but differ in the deficient enzyme: N- sulfoglucosamine sulfohydrolase (EC 3.10.1.1) in MPS IIIA, N-acetyl-a-D-Glucosoaminidase (EC 3.2.1.50) in MPS HUB, acetyl-CoA:a-glucosaminide-N-acetyltransferase (EC 2.3.1.78) in MPS IIIC and N-acetylglucosamine-6-sulfate sulfatase (EC 3.1.6.14) in MPS HID. MPS IIIA is the most common type, whereas MPS IIIC and MPS HID are the rarest.

[0005] MPS HI leads to severe CNS neurodegeneration, which starts as early as 20 months of age in a wide range of gray and white matter structures. Defects in the autophagy- lysosomal pathway (ALP) underlie the neurodegeneration in MPS HI and other LSDs. Interestingly, although most LSDs become clinically evident in young children, a similar ALP dysfunction is observed in many aging-related neurodegenerative proteinopathies. Reflecting this mechanistic similarity among many LSDs, secondary storage of amyloidogenic proteins accompanies the primary storage of the defective enzyme’s unprocessed substrate. For example, abundant inclusions of a-synuclein (a-syn), most commonly associated with Parkinson’s disease and other synucleinopathies, have been found in the brain of patients with MPS HI. Another study reported accumulation of amyloid P-protein (A ) 1-40, a protein involved in the pathogenesis of Alzheimer’s disease, in MPS HI postmortem brain tissue. Studies in mouse models of MPS I and MPS HI types, A, B, and C showed lysosomal inclusions of a-syn, tau, and Ap protein in cortical regions, often colocalized with each other.

[0006] ALP dysfunction is countered by increased release of extracellular vesicles (EVs) carrying toxic protein aggregates as an attempted neuronal survival response, a common mechanism in many neurodegenerative proteinopathies. Exosomes, an abundant type of EV (30-200 nm diameter) carry proteins, carbohydrates, lipids, and nucleic acids reflecting the status of the original cell (e.g., healthy, stressed, stimulated) for communication of “messages” to neighboring and distant cells. CNS-originating EVs are thought to egress across the blood-brain barrier into peripheral circulation and thus can be isolated from plasma or serum following a blood-draw and serve as a unique source of biomarkers reflecting biochemical changes in the CNS. Such biomarkers have not been assessed to date in any form of MPS.

[0007] There is a need for blood-based biomarkers for MPSs, particularly neuronopathic MPS types, which could facilitate diagnosis in the future if and when variants of uncertain significance are identified in a newborn screen, help identify patients who have evidence of biochemical CNS disease for enrichment of clinical trials, advance therapy development, andassist in personalizing treatment recommendations. These biomarkers are useful also for natural history studies of these rare diseases and are particularly important for assessment of clinical trial outcomes over multiple time points compared to the invasive lumbar puncture required for CSF analysis.SUMMARY

[0008] The materials and methods described herein meet these needs and more by providing a method of detecting neuronopathic MPS, including MPS-I and / or MPS III (Sanfilippo syndrome) in a subject, as well as a method of monitoring disease progression and cognitive decline, and of treating Sanfilippo syndrome and other forms of neuronopathic MPS. In some embodiments, the method comprises: (a) contacting one or more immunobinding agents with a peripheral blood sample obtained from the subject; (b) measuring the binding of the one or more immunobinding agents to one or more target molecules; and (c) detecting a neuronopathic MPS, such as MPS I or MPS III (Sanfilippo syndrome), when the measured binding is greater than a reference amount of binding. Each of the immunobinding agents specifically binds to one of the target molecules, which are selected from: heparan sulfate, a-synuclein, and tau, wherein the foregoing molecules are obtained from plasma or serum or from CNS-originating extracellular vesicles that can originate in any CNS cell, including neurons, astrocytes, microglia, oligodendrocyte, or epithelial cell, and are isolated from the plasma or the serum. In some embodiments, the method further comprises isolating CNS-originating extracellular vesicles from the plasma or the serum, and the heparan sulfate, tau, and / or a-synuclein is measured in the isolated CNS-originating extracellular vesicles.

[0009] In some embodiments, the peripheral blood sample comprises serum or plasma. In some embodiments, the target molecule is plasma HS, a-synuclein, and / or tau. In some embodiments, the method further comprises measuring the binding of a further immunobinding agent that specifically binds neurofilament light (NfL) and / or glial fibrillary acidic protein (GFAP). In some embodiments, the immunobinding agents are labeled with a detectable marker. In some embodiments, the reference amount is the amount measured in a sample of healthy control subjects.

[0010] In some embodiments, the method further comprises treating the subject for a neuronopathic MPS, such as MPS I or MPS III. In some embodiments, the method further comprises treating the subject for Sanfilippo syndrome. In some embodiments, the treatment comprises intravenous and / or intrathecal enzyme replacement therapy, substrate reduction therapy, autologous stem cell-based lentiviral gene therapy, and / or adeno-associated viral vector gene therapy. In some embodiments, the treatment comprises administration ofbiologies, anti-inflammatory agents, interleukin-1 receptor alpha antagonists, such as anakinra, or small-molecule drugs directed against a neuronopathic MPS, such as MPS I or MPS III.

[0011] Also provided is a kit comprising one or more immunobinding agents that specifically bind a target molecule. In some embodiments, the immunobinding agents are labeled with a detectable marker. In some embodiments, the target molecule is selected from one or more of: heparan sulfate, tau, and a-synuclein obtained from plasma or serum or from CNS- originating extracellular vesicles. In some embodiments, the kit further comprises an immunobinding agent that specifically binds neurofilament light (NfL). In some embodiments, the immunobinding agents are labeled with a detectable marker.

[0012] Additionally provided is a method of measuring disease progression, including, but not limited to, neurocognitive and neurobehavioral decline. In some embodiments, provided is a method of measuring cognitive decline or disease progression in a subject suffering from a neuronopathic MPS, such as MPS I or MPS III (Sanfilippo syndrome). In some embodiments, the method comprises: (a) contacting one or more immunobinding agents with a plasma or serum sample obtained from the subject or CNS-originating EVs isolated from the plasma or serum; (b) measuring the binding of the one or more immunobinding agents to one or more target molecules; and (c) assessing the extent of cognitive decline or disease progression whereby a greater amount of the measured binding of (b) is indicative of a greater cognitive decline or disease progression. Each of the immunobinding agents specifically binds to one of the target molecules, and the target molecules are selected from: heparan sulfate, a-synuclein, and tau obtained from plasma or serum or from CNS- originating extracellular vesicles. In some embodiments, the assessing of step (c) is based on a comparison of the measured binding of step (b) to a corresponding measured binding of immunobinding agents to the target molecules in a sample representative of a known severity of cognitive decline or disease progression, or in a sample obtained from the same subject at a previous time.

[0013] Moreover, the markers described herein can be used as pharmacodynamic biomarkers reporting on treatment effect of agents undergoing testing in clinical trials. Accordingly, also provided is a method of measuring response to treatment in a subject suffering from a neuronopathic MPS, such as MPS I or MPS III (Sanfilippo syndrome). In some embodiments, the method comprises (a) contacting one or more immunobinding agents with a plasma, serum or CNS-originating EVs isolated from the plasma or serum sample obtained from a subject undergoing treatment for a neuronopathic MPS, such as MPS I or MPS III (Sanfilippo syndrome); (b) measuring the binding of the one or more immunobinding agents to one or more target molecules; and (c) assessing the response totreatment whereby a lower amount of the measured binding of (b) is indicative of a greater response to treatment. Each of the immunobinding agents specifically binds to one of the target molecules, and the target molecules are selected from: heparan sulfate, a-synuclein, and tau obtained from plasma or serum or from CNS-originating extracellular vesicles. In some embodiments, the target molecules further comprise plasma IL-10 and plasma NfL. In some embodiments, the assessing of step (c) is based on a comparison of the measured binding of step (b) to a corresponding measured binding of immunobinding agents to the target molecules in a sample obtained from the same subject at prior to treatment.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIGS. 1A-1 F. Biomarkers separate the healthy control (HC) and MPS III groups. The data are presented as median ± Cl. (1A) Plasma IL-10. (1 B) Plasma NfL. (1C) nEV HS. (1 D) nEV Tau, samples in which the values were below the detection limit were included as zero. (1 E) Plasma tau. (1 F) nEV a-Syn. The two high points in 1 B, 1 D, 1 F represent different patients in each panel. P-values were calculated using two-tailed Student’s T-test.

[0015] FIG. 2. Receiver operating characteristic (ROC) analysis for the biomarkers shown in FIGS. 1 A-1 F. AUC - area under the curve in ROC analysis. The sensitivity and specificity are the values that maximized their unweighted sum.

[0016] FIGS. 3A-3B. Selected biomarkers change during a clinical trial of anakinra (Kineret®, NCT04018755). (3A) nEV HS. (3B) nEV a-syn. Thin gray lines indicate individual patients. Thick black lines indicate the average in each panel. P-values were calculated using mixed models in GraphPad Prism 10.1.

[0017] FIG. 4. Comparison of CD81 -positive small EV concentration between healthy controls and patients with Sanfilippo Syndrome. Small EV concentration was evaluated indirectly by measuring the concentration of the exosomal marker CD81 using ELISA. The data are presented as median ± Cl. The p-value was calculated by a two-tailed Student’s T- test.

[0018] FIGS. 5A-5D. Disease diagnosis and sex comparison of hemoglobin concentration. (5A) hemoglobin concentration in HCs and patients with MPS III. (5B-5D) hemoglobin concentration in males and females. The data are shown as median ± Cl. P-values were calculated using a two-tailed Student’s T-test.

[0019] FIGS. 6A-6F. Correlation analysis between plasma (6A-6C) or nEV (6D-6F) a-syn and plasma hemoglobin. The analysis was done using Spearman correlation. The curved lines indicate the 95% confidence interval. The insignificant correlation between plasmahemoglobin and nEV a-syn concentrations indicates that the measured a-syn in the nEVs did not originate from erythrocyte hemolysis and likely represented the CNS.

[0020] FIGS. 7A-7C. Biomarker concentrations in the HC and MPS III groups. The data are presented as median ± Cl. (7A) Plasma IL-2Ra, (7B) Plasma HS, (7C) Plasma a-syn. P- values were calculated using a two-tailed Student’s T-test.

[0021] FIGS. 8A-8H Biomarker response to treatment in patients with MPS I (all of them with the severe phonotype). NfL (A,E), GFAP (B,F), Tau (C,G), and HS (D,H) were measured in plasma samples obtained from three infants, two who started treatment at 4 months of age and one who started treatment at 10 months of age (A-D), and one older child (E-H) diagnosed with MPS I. The infant patients were treated with AAV9-hlDUA intracisternally at 4 or 10 months and weekly intravenous aldurazyme 0.58 mg / kg and blood samples were collected for 2 years. The older patient was treated with allogeneic hematopoietic stem cell transplant (HSCT) at age 2 years and AAV9-hlDUA intraventricular once at age 13 years and blood samples were collected after the gene-therapy treatment at 4 time points up to 3 years. All the biomarkers show reduction with treatment.

[0022] FIGS. 9A-9C Biomarker measurement in an MPS I mouse model. NfL (A), GFAP (B), and tau (C) were measured using the MSD Neuro Panel kit in IDUA- / - mice (N = 4) and wildtype control (N = 3) mice. Mouse NfL, GFAP, and tau were used in these measurements to create the standard curves. The data are shown as median ± 95% Cl.DETAILED DESCRIPTION

[0023] The invention provides the identification of new biomarkers measured in nEVs isolated from the plasma or serum and non-EV biomarkers assessed directly in plasma or serum that distinguish patients with MPS III manifesting neurologic disease from healthy control subjects with high sensitivity and specificity. The cross-sectional correlation of these new biomarkers with clinical measures of disease severity is evaluated, and their utility in assessing changes in a clinical trial of anakinra (Kineret®) a recombinant human IL-1 receptor antagonist is described. The biomarkers also show a decline in response to approved and experimental treatments in patients with MPS I. The utility of the biomarkers for animal studies is shown by large differences in the biomarkers level in a mouse model of MPS I compared to wild-type littermate mice.

[0024] The biomarkers identified herein are: CNS-originating EV heparan sulfate, CNS- originating EV tau, CNS-originating EV a-synuclein, plasma heparan sulfate, plasma tau, plasma a-synuclein, serum heparan sulfate, serum tau, and serum a-synuclein. Thesebiomarkers separate between patients with MPS III and healthy controls with high sensitivity and specificity, correlate cross-sectionally with clinical measures of disease severity and may change as a pharmacodynamic measure of a treatment in patients with MPS III. In some cases, plasma neurofilament light (NfL) also achieved this feat and recently has been used in a clinical trial for MPS III. Plasma IL-10 also shows cross-sectional correlation with clinical measures of disease severity.

[0025] To different extents, the biomarkers differentiated between patients with MPS III and healthy control groups. Specifically, plasma IL-10 separated the groups with receiver operating characteristic (ROC) area under the curve (AUC) = 0.729, plasma NfL separated the groups with AUC = 1.000, plasma tau separated the groups with AUC = 0.951, nEV heparan sulfate and nEV tau separated the groups with AUC = 0.797, and nEV a-synuclein separated the groups with AUC = 0.986.

[0026] To different extents, the biomarkers correlated cross-sectionally with disease symptoms. Specifically, nEV heparan sulfate correlated strongly (p < 0.05, r > |0.4|) with Sanfilippo Behavior Rating Scale - Mood and Vineland III GSV Expressive Communication. It also correlated moderately (0.05 < p < 0.1, |0.3| < r < |0.4| with Vineland III GSV Personal Daily Living Skills and Domestic Daily Living Skills. In addition, nEV tau correlated moderately with Vineland III GSV Interpersonal Socialization, Play & Leisure Socialization, and Gross Motor Skills. Plasma tau correlated strongly with Total Stool Habit Severity. Plasma IL-10 correlated strongly with Sanfilippo Behavior Rating Scale - Mood and moderately with Sanfilippo Behavior Rating Scale - Total, Movement, and Social / Emotional. Plasma heparan sulfate correlated strongly with Child Sleep Health Questionnaire Total, Sleep Probability, Vineland III GSV Expressive Communication, Interpersonal Socialization, and Play & Leisure Socialization, and moderately with Vineland III GSV Receptive Communication. Plasma a-synuclein correlated strongly with Sanfilippo Behavior Rating Scale - Orality and Vineland III GSV Expressive Communication and Personal Daily Living Skills and moderately with Vineland III GSV Receptive Communication.

[0027] In addition, nEV heparan sulfate and nEV a-synuclein showed significant changes in a clinical trial testing the IL-1Ra antagonist anakinra, suggesting that they could be used as pharmacodynamic biomarkers reporting on treatment effect.

[0028] Also reported herein is the discovery that plasma NfL, GFAP, and Tau are increased in a mouse model of MPS I. Moreover, plasma NfL, GFAP, Tau, and HS all decrease in MPS I human patients in response to treatment, suggesting that they could be used as pharmacodynamic biomarkers reporting on treatment effect.

[0029] The biomarkers described herein can be used for diagnosis, as part of newborn screening for neuronopathic MPS, , prognosis, monitoring progression of disease, assessment of outcome measures in clinical trials, and assessment of therapeutic effects in clinical use.Definitions

[0030] All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified. As used in this application, the following words or phrases have the meanings specified.

[0031] As used herein, a “control” or “reference” sample means a sample that is representative of normal measures of the respective marker, such as would be obtained from normal, healthy control subjects or wildtype animals, or a baseline amount of marker to be used for comparison. Typically, a baseline will be a measurement taken from the same subject or patient. The sample can be an actual sample used for testing, or a reference level or range, based on known normal measurements of the corresponding marker.

[0032] As used herein, a “significant difference” means a difference that can be detected in a manner that is considered reliable by one skilled in the art, such as a statistically significant difference, or a difference that is of sufficient magnitude that, under the circumstances, can be detected with a reasonable level of reliability. In one example, an increase or decrease of 10% relative to a reference sample is a significant difference. In other examples, an increase or decrease of 20%, 30%, 40%, or 50% relative to the reference sample is considered a significant difference. In yet another example, an increase of two-fold relative to a reference sample is considered significant.

[0033] As used herein, "pharmaceutically acceptable carrier" or “excipient” includes any material which, when combined with an active ingredient, allows the ingredient to retain biological activity and is non-reactive with the subject's immune system. Examples include, but are not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solution, water, emulsions such as oil / water emulsion, and various types of wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate buffered saline or normal (0.9%) saline.

[0034] Compositions comprising such carriers are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A.Gennaro, ed., Mack Publishing Co., Easton, PA, 1990).

[0035] As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, horses, sheep, dogs, cows, pigs, chickens, and other veterinary subjects. In a typical embodiment, the subject is a human. In some embodiments, the human is an infant.

[0036] As used herein, “a” or “an” means at least one, unless clearly indicated otherwise.Methods

[0037] The invention provides methods for diagnosis, including as part of Newborn Screening, for prognosis, and for monitoring progression of MPS I and MPS III. In addition, provided are methods of treatment, as well as methods for assessment of outcome measures in clinical trials, and for assessment of therapeutic effects in clinical use. In a typical embodiment, the method of detecting Sanfilippo syndrome (MPS III) comprises: (a) contacting one or more immunobinding agents with a peripheral blood sample obtained from the subject; (b) measuring the binding of the one or more immunobinding agents to one or more target molecules; and (c) detecting Sanfilippo syndrome when the measured binding is greater than a reference amount of binding. Likewise, prognosis and disease monitoring can comprise comparison of the measured binding to a reference or baseline amount. Similarly, treatment of Sanfilippo syndrome (MPS III) can comprise administering treatment to a subject for whom the measured binding is greater than a reference amount.

[0038] Each of the immunobinding agents specifically binds to one of the target molecules, and the target molecules are selected from: heparan sulfate, a-synuclein, and tau, wherein the heparan sulfate and a-synuclein are obtained from CNS-originating neuronal extracellular vesicles (nEVs), and wherein the tau is either obtained from nEVs or plasma. In some embodiments, the method further comprises isolating nEVs from the plasma, wherein the heparan sulfate, tau, and / or a-synuclein is measured in the isolated nEVs.

[0039] In some embodiments, the peripheral blood sample comprises serum or plasma. In some embodiments, the target molecule is plasma tau. In some embodiments, the method further comprises measuring the binding of a further immunobinding agent that specifically binds neurofilament light (NfL) or IL-10. In some embodiments, the immunobinding agents are labeled with a detectable marker. In some embodiments, the reference amount is the amount measured in a sample of healthy control subjects.

[0040] In some embodiments, the method further comprises treating the subject for a neuronopathic MPS, such as MPS I or MPS III. In some embodiments, the treatment comprises administration of biologies, anti-inflammatory agents, interleukin-1 receptor alphaantagonists, such as anakinra, or small-molecule drugs directed against a neuronopathic MPS, such as MPS I or MPS III.

[0041] Currently there is no approved treatment for Sanfilippo syndrome. The expectation is that when a treatment is approved, the disease will be added to the Newborn Screening, at which point the biomarkers will be used for making the diagnosis and starting the treatment. At present, an intracerebroventricular AAV gene therapy trial is in advanced stages.

[0042] Additionally provided is a method of measuring cognitive decline in a subject suffering from a neuronopathic MPS, such as MPS I or MPS III. In some embodiments, the method comprises: (a) contacting one or more immunobinding agents with a plasma sample obtained from the subject; (b) measuring the binding of the one or more immunobinding agents to one or more target molecules; and (c) assessing the extent of cognitive decline whereby a greater amount of the measured binding of (b) is indicative of a greater cognitive decline. Each of the immunobinding agents specifically binds to one of the target molecules, and the target molecules are selected from: IL-10, heparan sulfate, a-synuclein, and tau, wherein the molecules are obtained from plasma or serum or from CNS-originating extracellular vesicles. In some embodiments, the assessing of step (c) is based on a comparison of the measured binding of step (b) to a corresponding measured binding of immunobinding agents to the target molecules in a sample representative of a known severity of cognitive decline or in a sample obtained from the same subject at a previous time.

[0043] In a typical embodiment, the method of detecting MPS I comprises: (a) contacting one or more immunobinding agents with a peripheral blood sample obtained from the subject; (b) measuring the binding of the one or more immunobinding agents to one or more target molecules; and (c) detecting MPS I when the measured binding is greater than a reference amount of binding. Likewise, prognosis and disease monitoring can comprise comparison of the measured binding to a reference or baseline amount. Similarly, treatment of MPS I can comprise administering treatment to a subject for whom the measured binding is greater than a reference amount.

[0044] Each of the immunobinding agents specifically binds to one of the target molecules, and the target molecules are selected from: heparan sulfate, NfL, glial fibrillary acid protein (GFAP), and tau. In some embodiments, the method further comprises treating the subject for MPS I. In some embodiments, the treatment comprises administration of biologies, antiinflammatory agents, interleukin-1 receptor alpha antagonists, such as anakinra, or smallmolecule drugs directed against MPS I. In some embodiments, the treatment comprises ERT, HSCT, and / or gene therapy.

[0045] For use in the methods described herein, representative examples of the sample include, but are not limited to, blood, plasma or serum. In some embodiments, the biomarkers are measured in neuronal extracellular vesicles (nEVs) isolated from patients’ plasma or serum. In some embodiments, the EVs are isolated from neurons. In some embodiments, the EVs are isolated from other brain cells, e.g. astrocytes, microglia, and / or oligodendrocytes. The isolation of neuronal EVs is described in Dutta et al., Acta Neuropathologica 2021 , 142(3): 495-511 ; and Taha et al., Translational Neurodegeneration 2023, 12: 14.

[0046] In some embodiments, the methods described herein employ immunobinding agents that specifically bind to one, two, three, four, or all five of the target molecules described herein: nEV heparan sulfate, nEV a-synuclein, nEV tau, plasma tau, and neurofilament light (NfL). In some embodiments, the method targets detection of nEV heparan sulfate, nEV a- synuclein, nEV tau, and plasma tau. In some embodiments, the method targets any one of the foregoing and NfL. In some embodiments, the method targets detection of at least two of nEV heparan sulfate, nEV a-synuclein, nEV tau, and plasma tau, and optionally, additionally targets detection of NfL. In some embodiments, the method targets detection of at least three of nEV heparan sulfate, nEV a-synuclein, nEV tau, and plasma tau, and optionally, additionally targets detection of NfL.

[0047] In some embodiments, the immunobinding agents additionally or alternatively include any combination of plasma HS, NfL, GFAP, and / or tau. In some embodiments, the method targets detection of at least two of these four markers. In some embodiments, the method targets three or all four of these four markers.

[0048] The detection can comprise a determination that the level detected is above a predetermined threshold for detection, based, for example, on an identified reference level for that marker. In some embodiments, the detection comprises a quantitative measurement that can be used to determine relative severity of the syndrome, its progress, or a level of cognitive decline. The relative severity can be evaluated with respect to a known reference level associated with healthy control subjects, or with respect to a prior measurement obtained for the same subject.Kits

[0049] Also provided is a kit comprising one or more immunobinding agents that specifically bind a target molecule. In some embodiments, the immunobinding agents are labeled with a detectable marker. In some embodiments, the target molecule is selected from one or more of: nEV heparan sulfate, nEV tau, nEV a-synuclein, and plasma tau. In some embodiments, the kit further comprises an immunobinding agent that specifically binds neurofilament light(NfL). In some embodiments, the kit further comprises immunobinding agents directed at plasma GFAP, tau, and / or HS. The kit can comprise immunobinding agents directed at one, two, three, four, or all five of these markers, alone or in combination with one, two, or all three of the additional markers, and / or other markers of interest. In some embodiments, the immunobinding agents are labeled with a detectable marker.

[0050] The invention provides kits comprising a set of reagents as described herein, such as antibodies or fragments thereof that specifically bind one or more markers of the invention (including genes and their expression products), and optionally, one or more suitable containers containing reagents of the invention. Reagents include molecules that specifically bind and / or detect one or more markers of the invention. Such molecules can be provided in the form of a microarray or other article of manufacture for use in an assay described herein. One example of a reagent is an antibody or equivalent immunobinding agent that is specific for the marker(s). Reagents can optionally include a detectable label. Labels can be fluorescent, luminescent, electrochemical, electrochemiluminescent, enzymatic, chromogenic, or radioactive.

[0051] Kits of the invention optionally comprise an assay standard or a set of assay standards, either separately or together with other reagents. An assay standard can serve as a normal control by providing a reference level of normal expression for a given marker that is representative of a healthy individual.

[0052] The kit can optionally include a buffer. Reagents and standards can be provided in combinations reflecting the combinations of markers described herein as useful for detection.EXAMPLES

[0053] The following examples are presented to illustrate the present invention and to assist one of ordinary skill in making and using the same. The examples are not intended in any way to otherwise limit the scope of the invention.Example 1: Novel blood biomarkers for mucopolysaccharidosis type III

[0054] This Example demonstrates that novel biomarkers accessible through a blood test can be used for detection of MPS III, measure MPS III progression, and serve as measures of treatment effect in clinical trials and therapeutic protocols.

[0055] Materials and Methods

[0056] The 24 patients with MPS III donating blood samples for the study were screened for enrolment in a clinical trial of anakinra (NCT04018755). An exclusion criterion for the trialwas age < 4 to ethically exclude children who potentially would qualify for gene-therapy or enzyme-replacement therapy clinical trials. Sixteen healthy control subjects were enrolled in the study. The last subject enrolled toward the end of the study and therefore some biomarker measurements were only performed in samples from the first 15 control subjects.

[0057] Plasma collection

[0058] Plasma was collected in the morning after a minimum 8-hour fast using EDTA tubes, then frozen and stored at -80 °C. Due to attrition or difficulty collecting plasma from some of the patients, complete sets of samples from every visit were available only from 8 patients. The time points and sample numbers available were: baseline samples collected at screening, 8 weeks before the beginning of the trial, the same samples used for comparison with the HC group - 24. Day 1, just before the first dose of anakinra - 22. Week 8 - 18. Week 16, at which point the anakinra dose was adjusted if needed - 15. Week 36, the end of the treatment - 13. week 44, 8 weeks after the treatment ended - 11.

[0059] Demographic and clinical assessment.

[0060] The following questionnaires were administered to assess study participants: Child Sleep Habits Questionnaire (CSHQ), Sanfilippo Stool Habits Questionnaire (SSHQ), Sanfilippo Behavior Rating Scale (SBRS), Non-communicating Children’s Pain Checklist- Revised (NCCPC-R), NIH Patient Reported Outcomes Measurement Information System (PROMIS) - Fatigue, Autism Parenting Stress Index (APSI), and Individual Clinical Response (ICR). Reporting versions and scoring scales are as follows:

[0061] Child Sleep Habits Questionnaire (CSHQ)

[0062] The CSHQ is a parent questionnaire that has been used in many studies to examine both behavioral based and medical based sleep problems in children. The CSHQ yields a total score and eight subscale scores: 1) Bedtime Resistance; 2) Sleep Onset Delay; 3) Sleep Duration; 4) Sleep Anxiety; 5) Night Wakings; 6) Parasomnias; 7) Sleep-Disordered Breathing; and 8) Daytime Sleepiness. It has been validated in multiple groups including community children, children with diagnosed sleep disorders, children with development delay, and children with autism.

[0063] Sanfilippo Stool Habits Questionnaire (SSHQ)

[0064] Stooling was measured using a newly developed stooling survey titled “Sanfilippo Stool Habit Questionnaire”. The survey covers stool pattern (e.g., consistency, frequency, ease), stooling interference with daily activities, influence on behavior, stooling related medications, and dietary interventions.

[0065] Sanfilippo Behavior Rating Scale (SBRS)

[0066] The SBRS is a 68-item questionnaire, developed by Shapiro et al. to assess the behavioral phenotype of children with MPS III and its progression over time. There are 4 clusters (Movement, Lack of Fear, Emotional / Social, and Executive), and 2 domains (orality and mood). A total score was calculated based on the average of these 6 clusters and domains and this average was used in the correlation analysis.

[0067] Non-communicating Children’s Pain Checklist-Revised (NCCPC-R)

[0068] The NCCPC-R is a validated scale for measuring pain in children with severe cognitive impairment. It includes 7 scales that measure vocal, social, facial, activity, body and limbs, physiological, and eating / sleeping indicators of pain. A combined total score for pain is calculated as well and this total score was used in the correlation analysis.

[0069] NIH Patient Reported Outcomes Measurement Information System (PROMIS) - Fatigue

[0070] Per PROMIS guidance, the 10 most relevant questions for parents of children with MPS III were selected from the PROMIS Parent Proxy Fatigue item bank to make a customized PROMIS Fatigue - Parent Proxy Custom Short Form. Customized short forms were scored using this online scoring service: assessmentcenter.net / ac_scoringservice.

[0071] Autism Parenting Stress Index (APSI)

[0072] The APSI was developed following many interviews of parents of children with autism. The Items fall into three categories: the core social disability, difficult-to-manage behaviors, and physical issues. The APSI measures how much stress related to these three categories the parents are experiencing. The overall APSI scale score has been validated for parents of children with autism and other developmental disabilities.

[0073] Individual Clinical Response (ICR)

[0074] The ICR included the 5 most important clinical problems reported by the caregivers based on previous work (EMBO molecular medicine 2017; 9(1): 112-32.), from a choice including sleep disturbances, hyperactivity, frustration / impulse control / aggressive behaviors, feeding, anxiety, unhappiness, communication, social deficits, digestive issues and toileting, pain, illness / vul nerability to illness, fatigue, seizure, mobility, and gait. The caregivers rated their top 5 outcomes on a 5-point Likert scale as follows:

[0075] 0 - Not stressful

[0076] 1 - Sometimes creates stress

[0077] 2 - Often creates stress

[0078] 3 - Very stressful on a daily basis

[0079] 4 - So stressful sometimes we feel we cannot cope

[0080] EV isolation and immunocapture

[0081] Isolation of EVs and immunocapture of nEVs were done according to protocols described previously (Acta Neuropathol. 2021 ;142(3):495-511).

[0082] Biomarker measurement

[0083] Hemoglobin was measured in the plasma using a commercial kit (Sigma-Aldrich) as described previously (Id.). Exosome concentration was estimated in the plasma using an ExoELISA Ultra CD81 assay (System Biosciences) as described previously (Id.). Heparan Sulfate concentration was measured in plasma or nEVs using an ELISA kit from AMS Bio, Inc. (ELK8454) following the manufacturer’s guidelines. Briefly, 50 pL of plasma, 10 pg of total protein isolated from nEVs, or calibrators were loaded into each well along with 50 pL of a biotinylated detection antibody and incubated for 60 min at 37 °C. The solution then was discarded, and the plate was washed thrice using the Wash Buffer provided with the kit. Subsequently, 100 pL of 1X Streptavidin-HRP (diluted from the solution provided in the kit) were added to each well and incubated for 60 min at 37 °C. After discarding the solution, the plate was washed thrice using the Wash Buffer followed by addition of 90 pL of a 3, 3', 5, 5' tetramethylbenzidine substrate solution to each well. The plate then was incubated for 20 min at 37 °C in the dark, 50 pL of stop reagent were added to each well and the optical density was recorded promptly at 450 nm using a Synergy HTX plate reader (Biotek).

[0084] IL-10 and IL-2Ra were quantified in plasma using an electrochemiluminescence immunoassay (ECLIA), specifically, Meso Scale Discovery (MSD)’s V-PLEX Human IL-10 kit or U-PLEX IL-2Ra kit, respectively, according to the manufacturer’s instructions. For IL-10, the plate was prewashed thrice with a wash buffer (WB) comprising 0.05% (v / v) Tween-20 in 1X PBS, pH 74, and 50 pL of samples or calibrators were added to each well in duplicates and incubated at RT for 2 h with shaking at 700 rpm. The plate then was washed thrice and 50 pL of the SULFO-TAG-conjugated anti-IL-10 detection antibody were added to each well and incubated at RT for 2 h with shaking at 700 rpm. The plate was washed thrice and read immediately with 150 pL of 2X Read Buffer T using a QuickPlex SQ 120 Instrument (MSD).

[0085] For IL-2Ra, the plate was coated with 25 pL biotinylated human anti-IL-2Ra capture antibody and allowed to incubate for 1 h at RT with shaking at 700 rpm. The plate was washed thrice with WB and 25 pL of the assay diluent + 25 pL of samples or calibrators were added to each well and allowed to incubate for 1 h at RT with shaking at 700 rpm. The plate was washed thrice with WB and 50 pL of the SULFO-TAG-conjugated anti-IL-2Ra detection antibody was added to each well and incubated for 1 h at RT with shaking at 700 rpm. The plate was washed thrice and read immediately with 150 pL of MSD’s Gold ReadBuffer B. The data in MSD assays were analyzed using the Discovery Workbench 4.0 software and quantified with reference to freshly prepared standard curves.

[0086] NfL was quantified in plasma using MSD’s R-PLEX NfL kit according to the manufacturer’s recommendations. Briefly, 25 pL of biotinylated anti-human NfL antibody was added to each well and incubated for 1 h at RT with shaking at 700 rpm. Each well then was washed thrice with 150 pL WB and 25 pL of the diluent + 25 pL of either sample or calibrator were added and incubated for 1 h at RT with shaking at 700 rpm. The wells were washed thrice with 150 pL of WB and 50 pL of the SULFO-TAG-conjugated anti-human NfL detection antibody were added to each well and incubated as described above. Finally, the wells were washed thrice with 150 pL WB and the plate was read immediately with 150 pL of MSD Gold Read Buffer B using the QuickPlex SQ 120 instrument.

[0087] a-Syn concentration in plasma or nEVs were determined using ECLIA as described previously (Id.).

[0088] Tau concentrations in plasma or nEVs were determined using an S-Plex ECLIA (MSD).

[0089] Neurology Panel (NfL, tau, and GFAP multiplex)

[0090] NfL, tau, and glial fibrillary acidic protein (GFAP) were measured in the plasma using MSD’s Neurology Panel 1 Kit (K15639S) following the manufacturer’s guidelines. Briefly, the plate underwent three washes with PBS-T and was subsequently coated with S-PLEX Neurology Panel 1 (human) diluted in Diluent 100. The sealed plate was incubated at RT for 1 h with shaking at 700 rpm and then washed thrice with PBS-T. Subsequently, 25 pL of blocking solution was added to each well along with 25 pL of either sample or calibrator. The plate was re-sealed and incubated at RT for 1.5 h with shaking at 700 rpm. The plate then was washed thrice with PBS-T and 50 pL of TURBO-BOOST antibody solution were added to each well. The plate was re-sealed and incubated for 1 h at RT with shaking at 700 rpm. The plate was washed thrice again with PBS-T and incubated with 50 pL / well of enhance solution for 30 min at RT with shaking at 700 rpm. The plate was washed thrice with PBS-T and 50 pL of TURBO-TAG detection solution was added to each well. The plate was resealed and incubated at 27 °C for 1 h with shaking at 700 rpm. The plate was washed thrice with PBS-T, 150 pL of MSD GOLD read buffer B was added into each well, and the plate was read immediately using the QuickPlex SQ 120 instrument.

[0091] Statistical analysis

[0092] Comparison of continuous variables between the MPS III and HC groups was conducted using Student’s T-test whereas categorical variables were compared usingFisher’s exact test. Sensitivity and specificity were estimated at the best threshold, defined as the value that maximized the unweighted sum of the sensitivity and specificity in receiver operator characteristic (ROC) analyses for each biomarker. Correlations across individual biomarkers or between biomarkers and clinical test scores were evaluated using Spearman’s method. Biomarker changes over time in the clinical trial samples were analyzed using mixed models in GraphPad Prism. Analyses were performed using Prism 10.1.

[0093] Results

[0094] Participant Demographics

[0095] All the patients included in the study (n = 24) were screened and when appropriate, included in a recent clinical trial testing anakinra (n = 23).14Patients were 10.6 ± 4.2 years old, 12 females and 12 males. HCs (n = 16) were 14.3 ± 3.7 years old, 9 females and 7 males. Most of the patients identified as non-Hispanic or Latino Whites (87.5%) whereas in the HC group, among non-Hispanic or Latino (69%) Asians or Pacific Islanders comprised 25% and persons of other / unknown / mixed race were 38%. Demographic data are provided in Table 1.

[0096] Table 1. Demographic and genotype DataMPS III HC P-valueAge at baseline 10.6 ± 4.2 (6.3- 14.3 ± 3.7 (5.5-178) 0.011(mean ± SD, range, years) 26.1)SexFemale 12 (50%) 9 (56%) 0.762Male 12 (50%) 7 (44%)RaceBlack 1 1Asian / Pacific Islander 1 4 0.00062Other / mixed / unknown 1 6White 21 5EthnicityHispanic or Latino 3 5 0.232Not Hispanic or Latino 21 11MPS Type NAMPS I HA 20MPS I IIB 3MPS me1Student’s T-test,2Fisher’s exact test.

[0097] Quality-control analyses

[0098] EV concentration in the plasma using the exosomal marker CD81 showed moderately higher median concentration in the patients (4.1 x 1O10± 4.7 x 1O10) compared to the HCs (2.2 x 1O10± 3.4 x 1010, Fig. 4) suggesting that lysosome dysfunction in MPS III increases CNS clearance of accumulating undegraded biomolecules via exosome release.15

[0099] Hemolysis of erythrocytes may lead to release of erythrocyte a-syn making analysis of nEV a-syn impractical.16There were insignificant differences between the hemoglobin concentrations in the HC and MPS III groups (Fig. 5A). Hemoglobin concentrations were similar in males and females in the whole cohort (Fig. 5B). They were lower in males in the MPS III group at baseline (Fig. 5C) and higher in the HC male (Fig. 5D), yet these differences were not observed in subsequent timepoints in the patients and likely represent a statistical anomaly due to the small N rather than a real sex difference.

[0100] IL-10 and IL-2Ra - markers of neuroinflammation

[0101] Neuroinflammation is thought to be a major contributor to the cognitive deterioration in patients with MPS III.4Therefore, whether plasma neuroinflammation markers distinguish between the patient and HC groups was tested. Plasma IL-10 concentrations were significantly higher in MPS III (0.27 ± 0.14 pg / mL) compared to HCs (0.14 ± 0.07 pg / mL, p = 0.002, Fig. 1A). Overlap between the groups yielded a moderate separation (AUC = 0.764, Fig. 2). The difference in IL-2Ra concentration between the groups was small and insignificant (MPS III - 1643 ± 1047, HC - 2247 ± 1556, p = 0.16, Fig. 7A).

[0102] Neurofilament light chain (NfL) - a marker of neurodegeneration

[0103] NfL is a widely used neurodegeneration marker in neurodegenerative diseases and CNS injuries.18It has been reported to be elevated in several LSDs, including MPS II (in serum)19and recently has been used to assess treatment effect in patients with MPS 11 IA receiving intracerebral AAVrh10-based gene therapy (NCT03612869). Thus, plasma NfL was expected to be significantly higher in the MPS III group than in the HCs. Indeed, patients’ median plasma NfL concentration was 152 ± 125 pg / mL, whereas that of HCs was 26 ± 11 pg / mL, p < 0.0001 (Fig. 1 B). There was no overlap between the groups (AUC = 1.000, Fig. 2).

[0104] Heparan sulfate (HS) - a marker of primary lysosomal storage

[0105] HS was measured using a commercial ELISA in the patient and HC groups, both directly in the plasma and in nEVs. Although the median plasma HS concentration was higher in the MPS III patients than in the HC group (Fig. 7B), there was substantial overlap resulting in an insignificant difference between the groups (p = 0.44). In contrast, nEV HSconcentrations, which were substantially lower than plasma concentrations, differed significantly (p < 0.0001 , Fig. 1 C), resulting in high separation between the groups (AUC = 0.979, Fig. 2). This result suggests that nEV HS may provide a minimally invasive alternative to CSF for sensitive measurement of HS changes in the brain.

[0106] Tau, and a-synuclein - markers of secondary lysosomal storage

[0107] As amyloidogenic proteins accumulate as secondary storage in the brain of patients6-8and animal models9-11of MPS III, their concentration in nEVs might be elevated in patients compared to HCs.

[0108] nEV tau (Fig. 1 D) was not detected in any of the HCs despite the high sensitivity of the assay (lower limit of detection 14 fg / mL). In contrast, it was detected in 23 of the 24 patients with MPS III at 23 ± 48 fg / mL. This difference provided an AUC = 0.979 (Fig. 2). Plasma tau also showed significant differences between the groups (p < 0.0001 , Fig 1 E), resulting in an AUC = 0.951 (Fig. 2).

[0109] nEV a-Syn was significantly higher in patients with MPS III compared to HCs (594 ± 430 vs. 99.6 ± 42.4 pg / mL, p < 0.0001 , Fig. 1 F) resulting in AUC = 1.000 (Fig. 2). In contrast, plasma a-syn did not differ significantly between the groups (Fig. 7C).

[0110] In total, out of the 12 biomarkers tested, 5 provided strong separation between the MPS III and HC groups - plasma NfL and tau, and nEV HS, tau, and a-syn.

[0111] Correlation with demographic data and clinical measures

[0112] There were no sex differences for any of the measured biomarkers, either in the whole cohort or in the MPS III and HC groups analyzed separately. Plasma NfL and tau and nEV HS and a-syn, correlated inversely with age when the MPS III and HC groups were analyzed together, yet only plasma tau showed this correlation when the groups were analyzed separately (Table 5). In contrast to plasma tau, there was no correlation between nEV tau and age (Table 5) suggesting that the negative correlation with plasma tau may reflect peripheral, but not CNS processes.

[0113] Cross-sectional correlation of the biomarkers with a battery of clinical measures assessed in the MPS III group upon consideration of enrolling these patients in the anakinra clinical trial was examined. Rather than absolute p-value cutoffs, such as 0.05,24combinations of biomarker and clinical measure yielding p < 0.05 and r > |0.4| were considered strong correlations whereas combinations with 0.05 < p < 0.1 and |0.3| < r < |0.5| were considered moderate correlations (Table 2, underlined and italicized, respectively).

[0114] Table 2 appears before the claims below. Correlation between biomarkers and clinical measurements at baseline. P-values (Spearman) are shown in each case. P < 0.05 ishighlighted in bold font and 0.05 < p < 0.1 in italics. R values are given in parenthesis only for correlations with p < 0.1. Table 2 is followed by Tables 3-5. Table 3 presents correlation among biomarkers in the MPS III (baseline) and HC group combined. Table 4 provides Spearman correlation among biomarkers in the MPS III group only. Table 5 provides Spearman correlation of plasma and nEV biomarkers with subject age.

[0115] In all the tests used except for the Vineland Adaptive Behavior Scales, 3rd Edition (Vineland III)25, higher scores reflect higher levels of difficulty / symptom frequency. Thus, positive correlations with the biomarkers were expected, reflecting an increase in symptom severity when the biomarkers are elevated. Conversely, in the Vineland III, a higher growth scale value is associated with a higher ability, so the correlation with elevated biomarkers was expected to be negative.

[0116] Plasma HS showed the highest number of correlations with clinical measures. It correlated positively with the Child Sleep Health Questionnaire and Sleep Problems measures, suggesting that sleep frequency, duration, and quality are main clinical features affected by elevated plasma HS. Plasma HS correlated negatively in the Vineland III with neurocognitive domains including communication and socialization, suggesting that CNS changes affecting these behavioral domains also are reflected in the plasma HS levels. A moderate negative correlation was found between plasma HS and Sanfilippo Behavior Rating Scale (SBRS) movement. nEV HS correlated negatively with Vineland III communication and personal daily living skills, in agreement with the plasma HS results. Surprisingly, this biomarker showed a strong negative correlation with SBRS mood, i.e., higher nEV HS concentrations indicated better mood. This finding is at odds with the improvement in communication and daily living skills and if validated in the future, further studies will be needed to explore the underlying reasons.

[0117] Plasma a-syn correlated positively with SBRS orality and negatively with Vinland III communication and daily living skills. These correlations likely reflect both CNS and blood a- syn in view of the significant correlation found between plasma a-syn and hemoglobin concentrations (Fig. 6B). Plasma IL-10 showed mainly moderate positive correlations with SBRS measures (Table 2), further supporting the notion that certain neurocognitive changes may be reflected in plasma biomarkers. nEV tau showed a moderate positive correlation with Vineland III socialization and motor skills, i.e., better socialization and motor skills were moderately associated with higher nEV tau. A possible explanation is that the higher nEV tau concentrations may reflect better removal of tau from the CNS to the peripheral circulation. In addition, recent work has suggested that the Vineland III Gross Motor Skill scale may not be a sensitive measure to detect the unique gross motor impacts of MPS III26.

[0118] Plasma tau was the only biomarker showing a strong correlation with Stool Habit Severity, further suggesting that this biomarker may be indicative of peripheral, rather than central function. Moreover, this correlation raises the possibility that inflammatory changes in the digestive system, possibly due to dysbiosis of the gut microbiome, may induce tau aggregation as has been reported recently in Alzheimer’s disease,27leading to the observed elevated tau levels in the plasma.

[0119] Testing of select biomarkers for pharmacodynamic response in the anakinra clinical trial

[0120] Following identification of the new biomarkers, whether those showing the strongest differences between the MPS III and HC groups could be used to report pharmacodynamic changes in the patients enrolled in the anakinra trial was tested. Although anakinra acts on IL-1 Ra and was expected to have minimal effects on primary or secondary lysosomal storage, these markers were included, as well as markers of neurodegeneration and neuroinflammation. A high-sensitivity multiplex kit (Meso Scale Discovery) was employed, which includes NfL, tau, and glial fibrillary acidic protein (GFAP), an astrocytic marker used commonly to assess astrogliosis.

[0121] Due to attrition or difficulty collecting blood from some of the participants, complete sets of samples from every visit were available only from 8 patients.

[0122] Plasma IL-10, plasma IL-2Ra, plasma NfL, plasma tau, and plasma GFAP did not change significantly. When the difference between the beginning (day 1) and end (week 36) time points was analyzed separately in 11 of the 12 patients for whom both time points were available (excluding one outlier), a significant reduction was found in plasma tau.

[0123] nEV HS showed a significant increase from 0.06 ± 0.05 to 0.13 ± 0.05 pg / mL, p < 0.0001 , during the 8 weeks preceding the treatment, possibly reflecting a natural course of accumulation in neuronal lysosomes and release via nEVs in patients with MPS III. The levels stabilized at 0.13 ± 0.02 pg / mL for the duration of the treatment, and then continued to climb in the 8 weeks after the treatment was discontinued to 0.18 ± 0.07 pg / mL, p = 0.02 (Fig. 3A). These results support using nEV HS as a pharmacodynamic biomarker in MPS III clinical trials. Moreover, the data suggest that the anakinra treatment stabilized HS release in nEVs, possibly reflecting a relief to HS accumulation in the CNS. In contrast, nEV tau concentrations did not change significantly before, during, or after the treatment.

[0124] Analysis of nEV a-syn showed that one patient had particularly high concentrations during most of the trial, causing strong skewing of the data. When the data were re-analyzed excluding this outlier, during the treatment, the nEV a-syn decreased gradually from 173 ± 115 pg / mL to 93 ± 52 pg / mL by week 36 and then increased to 245 ± 224 pg / mL, p = 0.03 8weeks after the treatment was discontinued (Fig. 3B). Comparing only the start and end points of the treatment (Day 1 vs Week 36) in the 12 patients for whom both time points were available further demonstrated the reduction in nEV a-syn concentration in most of them. Moreover, the magnitude of the change correlated with the baseline level of nEV a- syn, whereas a similar correlation was not found for the other biomarkers.

[0125] Unexpectedly, regardless of whether the outlier patient was included or excluded (Fig. 3B), during the 8 weeks before the start of the treatment, nEV a-syn inexplicably declined from 487 ± 473 to 173 ± 115 pg / mL, p = 0.04 (Fig. 3B). There were no technical or clinical reasons identified to explain these results. Overall, the data suggest that nEV HS, nEV a- syn, and plasma tau may report on treatment effects in MPS III clinical trials during periods as short as 36 weeks.

[0126] Discussion

[0127] Biomarkers assisting MPS III phenotype characterization may become important when newborn screening programs are initiated. Urine and plasma GAGs correlate cross- sectionally with disease severity but not with the rate of cognitive decline, and there is poor correlation between CSF GAGs and urine / plasma GAGs.28The short-term nature of many studies does not allow capturing clinically meaningful outcomes.26

[0128] CSF analyses of HS-GAG-NREs and other biomarkers, e.g., GM2 and GM3 gangliosides, support current clinical trials of disease-modifying enzyme-replacement therapy, gene therapy, or hematopoietic stem-cell therapy yet require an invasive lumbar puncture. NfL measurement in plasma / serum is an attractive alternative but may require nearly a year to show significant change. In this Example, nEV biomarkers, including primary storage of HS and secondary storage of tau and a-syn, are shown for the first time to be promising for assessment of MPS III. These data suggest that they likely will be useful also for other neuropathic LSDs. In addition, plasma tau and a-syn may be useful for monitoring disease progression (Table 2) and treatment outcomes.

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[0160] Plasma HS, NfL, GFAP, and tau were analyzed in four patients with MPS I. Three of the patients were diagnosed through newborn screening and treated with gene therapy and IDIIA ERT. One patient was treated at age 2 with HSCT and at age 13 with the same gene therapy(Fig. 8). All of the biomarkers showed substantial reduction following an initial period of fluctuations in the infant patients (Fig. 8A-D). Similarly, all the biomarkers were reduced substantially in the older patient following the gene therapy (Fig. 8E-H).

[0161] The infant patients were treated with AAV9-hlDUA intracisternally once, two at age 4 months and one at age 10 months, and received IDIIA ERT weekly. Blood samples from these patients were collected for 2 years. The older patient was treated with HSCT at age 2 and with AAV9-hlDUA intraventricularly once at age 13. Blood samples from this patientwere collected at 4 time points up to 3 years after the gene therapy. All the biomarkers show reduction with treatment.

[0162] These results demonstrate the utility of the biomarkers for evaluating treatment outcome.Example 3: Biomarkers for detection of mucopolysaccharidosis type I

[0163] Measurements were taken of NfL, GFAP, and tau in plasma from MPS-I mice (IDUA- / -) and compared to wild type mice. These biomarkers were increased in the disease model, as shown in Fig. 9. Measurements were obtained using the MSD Neuro Panel kit in IDUA- / - mice (N = 4) and wild-type control (N = 3) mice. Mouse NfL, GFAP, and tau were used in these measurements to create the standard curves.

[0164] Throughout this application various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to describe more fully the state of the art to which this invention pertains.

[0165] Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.Table 2Table 2 (continued)Table 3Table 4Table 5

Claims

What is claimed is:

1. A method of detecting a neuronopathic MPS in a subject, the method comprising:(a) contacting one or more immunobinding agents with a peripheral blood sample obtained from the subject;(b) measuring the binding of the one or more immunobinding agents to one or more target molecules; and(c) detecting a neuronopathic MPS when the measured binding is greater than a reference amount of binding; wherein each of the immunobinding agents specifically binds to one of the target molecules, and wherein the target molecules are selected from: IL-10, heparan sulfate, a- synuclein, and tau, wherein the heparan sulfate and a-synuclein are obtained from CNS- originating neuronal extracellular vesicles (nEVs), and wherein the IL-10 is obtained from the plasma and the tau is either obtained from nEVs or plasma.

2. The method of claim 1, wherein method further comprises isolating nEVs from the plasma, and wherein the heparan sulfate, tau, and / or a-synuclein is measured in the isolated nEVs.

3. The method of claim 1, wherein the peripheral blood sample comprises serum or plasma.

4. The method of claim 1, wherein the target molecule is plasma tau.

5. The method of claim 1, further comprising measuring the binding of a further immunobinding agent that specifically binds neurofilament light (NfL) and / or glial fibrillary acid protein (GFAP).

6. The method of any one of claims 1 to 5, wherein the immunobinding agents are labeled with a detectable marker.

7. The method of any one of claims 1 to 6, wherein the reference amount is the amount measured in a sample of healthy control subjects.

8. The method of any one of claims 1 to 7, further comprising treating the subject for a neuronopathic MPS.

9. The method of claim 8, wherein the treatment comprises intravenous and / or intrathecal enzyme replacement therapy, substrate reduction therapy, autologous stem cellbased lentiviral gene therapy, and / or adeno-associated viral vector gene therapy.

10. A kit comprising one or more immunobinding agents that specifically bind a target molecule, wherein the immunobinding agents are labeled with a detectable marker, and wherein the target molecule is selected from one or more of: nEV heparan sulfate, nEV tau, nEV a-synuclein, and plasma tau.

11. The kit of claim 10, further comprising an immunobinding agent that specifically binds neurofilament light (NfL).

12. The kit of claim 11 , further comprising an immunobinding agent that specifically binds glial fibrillary acid protein (GFAP).

13. A method of measuring cognitive decline or disease progression in a subject suffering from a neuronopathic MPS, the method comprising:(a) contacting one or more immunobinding agents with a plasma sample obtained from the subject;(b) measuring the binding of the one or more immunobinding agents to one or more target molecules; and(c) assessing the extent of cognitive decline or disease progression whereby a greater amount of the measured binding of (b) is indicative of a greater cognitive decline; wherein each of the immunobinding agents specifically binds to one of the target molecules, and wherein the target molecules are selected from: heparan sulfate (HS), a- synuclein, and tau, wherein the heparan sulfate, a-synuclein, and tau are obtained from CNS-originating neuronal extracellular vesicles (nEVs).

14. The method of claim 13, wherein the target molecules further comprise plasma IL-10, plasma HS, plasma tau, and plasma a-synuclein.

15. The method of claim 13 or 14, wherein the assessing of step (c) is based on a comparison of the measured binding of step (b) to a corresponding measured binding of immunobinding agents to the target molecules in a sample representative of a known severity of cognitive decline or disease progression, or in a sample obtained from the same subject at a previous time.

16. A method of measuring response to treatment in a subject suffering from a neuronopathic MPS, the method comprising:(a) contacting one or more immunobinding agents with a plasma sample obtained from a subject undergoing treatment for a neuronopathic MPS;(b) measuring the binding of the one or more immunobinding agents to one or more target molecules; and(c) assessing the response to treatment whereby a lower amount of the measured binding of (b) is indicative of a greater response to treatment; wherein each of the immunobinding agents specifically binds to one of the target molecules, and wherein the target molecules are selected from: heparan sulfate, a- synuclein, and tau, wherein the heparan sulfate and a-synuclein are obtained from CNS- originating neuronal extracellular vesicles (nEVs), and wherein the tau is either obtained from nEVs or plasma.

17. The method of claim 16, wherein the target molecules further comprise plasma IL-10, plasma HS, plasma tau, and plasma a-synuclein.

18. The method of claim 16 or 17, wherein the assessing of step (c) is based on a comparison of the measured binding of step (b) to a corresponding measured binding of immunobinding agents to the target molecules in a sample obtained from the same subject at prior to treatment.

19. The method of any one of claims 1 , 13, or 16, wherein the MPS is MPS I or MPS III (Sanfilippo syndrome).

20. The method of claim 1 , wherein the MPS is MPS I, and wherein the target molecules are selected from: heparan sulfate, NfL, GFAP, and tau.

21. The method of claim 20, further comprising treating a subject for MPS I.

22. A method of detecting Sanfilippo syndrome in a subject, the method comprising:(a) contacting one or more immunobinding agents with a peripheral blood sample obtained from the subject;(b) measuring the binding of the one or more immunobinding agents to one or more target molecules; and(c) detecting Sanfilippo syndrome when the measured binding is greater than a reference amount of binding; wherein each of the immunobinding agents specifically binds to one of the target molecules, and wherein the target molecules are selected from: IL-10, heparan sulfate, a- synuclein, and tau, wherein the heparan sulfate and a-synuclein are obtained from CNS- originating neuronal extracellular vesicles (nEVs), and wherein the IL-10 is obtained from the plasma and the tau is either obtained from nEVs or plasma.

23. The method of claim 22, further comprising treating the subject for Sanfilippo syndrome.

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