Use of brain-derived exosomes in neurodegenerative diseases

Abstract

Disclosed herein are methods of assessing changes of expression in patients' nervous systems of molecules responsive to treatments modulating nuclear receptors for retinoid X (RXR alpha, beta, or gamma), Nurr1(NR4A2) nuclear receptors, Nur77 (NR4A1) nuclear receptors, dopamine active transporter (DAT), or dopa decarboxylase (DDC), for nervous system disorders or conditions related to these molecules.

Description

PCT Patent Application Docket 1958481 .00393PCTUSE OF BRAIN-DERIVED EXOSOMES IN NEURODEGENERATIVE DISEASESRELATED APPLICATION(S)

[0001] This application claims priority from U.S. Provisional Patent Application Serial Number 63 / 679,480, filed August 5, 2024, the entirety of which is incorporated herein by reference.FIELD

[0002] The present disclosure relates to methods of assaying for biomarkers associated with neurodegenerative conditions using brain-derived exosomes.SUMMARY

[0003] Described herein are methods of assessing changes of expression in patients’ nervous systems of molecules responsive to treatments modulating nuclear receptors for retinoid X (RXR alpha, beta, or gamma), Nurr1(NR4A2) nuclear receptors, Nur77 (NR4A1) nuclear receptors, dopamine active transporter (DAT), or dopa decarboxylase (DDC), for nervous system disorders or conditions related to these molecules.

[0004] In one example embodiment, a method of assessing changes of expression in patients’ nervous systems of molecules responsive to treatments modulating nuclear receptors for retinoid X (RXR alpha, beta, or gamma), Nurr1 (NR4A2) nuclear receptors, Nur77 (NR4A1) nuclear receptors, dopamine active transporter (DAT), or dopa decarboxylase (DDC), for nervous system disorders or conditions related to these molecules comprises utilizing a quantitative assay for measuring brain derived exosomes released by neurons, glial cells, or nervous system inflammatory cells, wherein the information provided by the assay is useful for development of treatments for at least one nervous system disorder or condition, or for diagnosis, theragnosis, assessment of prognosis, or monitoring of therapeutic response in a patient with a nervous system disorder or condition. In some embodiments, the information provided by the assay is useful for the development of new treatments. In other embodiments, the information provided by the assay is useful for the development of new treatments in that they differ from the initial treatments given to the subject / patient. In any of the example embodiments provided herein, the information provided by the assay can be for the development of one or more treatments.

[0005] The nervous system disorder or condition is Alzheimer’s disease, Parkinson’s disease, a dementia, amyotrophic lateral sclerosis, multiple sclerosis, a stroke, a traumatic CNS injury, a hypoxic CNS injury, a schizophrenia, a depression, an autism, a neuropsychiatric disorder, normal aging related neurodegeneration, or other neurodegenerative condition.PCT Patent ApplicationDocket 1958481 .00393PCT

[0006] In another example embodiment, the nervous system disorder is not amyotrophic lateral sclerosis.

[0007] The assay is used to quantitate the brain derived exosomes in blood, serum, plasma, cerebrospinal fluid, or other biologic fluids. In some embodiments, the assay comprises an enzyme linked immunoadsorption assay (ELISA). In one example embodiment, the assay consists of an enzyme linked immunoadsorption assay (ELISA), utilizing at least one antibody or antibody fragment or selective antigen-binding molecule, which selectively binds to a cell surface molecule selectively expressed on neurons, glial cells, or CNS inflammatory cells; and at least one additional antibody or antibody fragment or selective antigen-binding molecule which separately and selectively binds to a cellular molecule, of which the expression in neurons, glial cells, or CNS inflammatory cells, is assessed by quantitation of brain derived exosomes in a biologic fluid.

[0008] In some embodiments, the assay quantitates brain derived exosomes containing RXR alpha, beta, or gamma nuclear receptors. In other embodiments, the assay quantitates brain derived exosomes containing Nurr1 (NR4A2) nuclear receptors.

[0009] In some embodiments, the assay quantitates brain derived exosomes containing Nur77 (NR4A1) nuclear receptors. In other embodiments, the assay quantitates brain derived exosomes containing dopamine active transporter (DAT). In yet further embodiments, the assay quantitates brain derived exosomes containing dopa decarboxylase (DDC).

[0010] The quantitative brain derived exosomes assay results inform development of one or more therapeutic agents for at least one nervous system disorder or condition. The therapeutic agent(s) for a nervous system disorder or condition is an agonist, antagonist, or inverse agonist of one or more of the RXR nuclear receptors, Nurrl , Nur77, DAT, or DDC.

[0011] In some embodiments, the quantitative brain derived exosomes assay results inform treatment decisions for patients with nervous system disorders or conditions.

[0012] In other embodiments, the quantitative brain derived exosome assay results provide diagnostic, theragnostic, or prognostic information informing treatment of patients with suspected or documented nervous disorders or conditions. The nervous system disorders or conditions can be for example, but not limited to, Alzheimer’s disease, Parkinson’s disease, a dementia, amyotrophic lateral sclerosis, multiple sclerosis, a stroke, a traumatic CNS injury, a hypoxic CNS injury, a schizophrenia, a depression, an autism, a neuropsychiatric disorder, normal aging relatedPCT Patent ApplicationDocket 1958481 .00393PCT neurodegeneration, or other nervous system disorder or condition. In one example embodiment, the nervous system disorder or condition is not amyotrophic lateral sclerosis.DETAILED DESCRIPTION

[0013] Disclosed herein are assays for neurodegenerative diseases, enabling early detection and characterization of the disease or pathology, for the identification and development of novel therapeutics for neurodegenerative diseases, and methods of monitoring disease progression by analyzing peripheral blood samples.

[0014] Brain derived exosomes (BDEs) are extracellular nanovesicles that are collectively released by all cell lineages of the central nervous system and contain cargo from their original cells. Depending on the cell type from which they are secreted, exosome vesicles themselves also hold particular and varied types of biomarkers that contribute to identifying their origins. Exosomes carrying multiple cargo with valuable biological information can also be released into most bodily fluids such as plasma, cerebrospinal fluid, urine, saliva, amniotic fluid, colostrum, breast milk, synovial fluid, semen, and pleural ascites. Exosomes play key roles in the management of normal physiological environments, such as in the pathological processes associated with several diseases including neurodegenerative diseases. Non-limiting examples of the biomarkers including nuclear receptors such as Retinoid X Receptors (RXR) including RXRa, RXRP, and RXRy; Nurrl nuclear receptor (NR4A2); Nur77 nuclear receptor (NR4A1); dopamine active transporter (DAT); and dopa decarboxylase.

[0015] Neurodegenerative diseases usually develop slowly, and the effects and symptoms tend to appear later in life. Neurodegenerative diseases include dementias such as, but not limited to, Alzheimer's Disease, frontotemporal dementia, chronic traumatic encephalopathy (CTE), Lewy body dementia, vascular dementia, and limbic predominant age-related TDP-43 encephalopathy (LATE); demyelinating diseases such as, but not limited to multiple sclerosis (MS) and neuromyelitis optica spectrum disorder (NMOSD), Parkinson’s disease and Parkinson’s type diseases (parkinsonism); motor neuron diseases such as amyotrophic lateral sclerosis (ALS) and progressive supranuclear palsy (PSP); prion diseases such as Creutzfeldt-Jakob disease; trauma or physical brain damage such as stroke, traumatic CNS injury, or hypoxic CNS injury; aging-related neurodegeneration; depression; schizophrenia; autism; or neuropsychiatric disorders.

[0016] Methods of isolating BDEs can be found in US2023 / 0417773 which is incorporated by reference herein for all it discloses regarding BDEs. Briefly, BDEs are isolated from blood,PCT Patent ApplicationDocket 1958481 .00393PCT plasma, serum, or cerebrospinal fluid samples by contacting the sample with one or more binding molecules capable of specifically binding to target molecules on the surface of BDEs and capturing the BDEs bound to the binding molecules. The target molecule is a synaptic protein present on BDEs such as, but not limited to, one or more of neuroligin-3 (NLGN3), growth- associated protein 43 (GAP43), synaptotagmin-1 (SYT1), glutamate receptor 1 (GluR1 , GRIA1), L1 Cell Adhesion Molecule (L1CAM), and Rab3a.

[0017] Once the BDEs are isolated, they can be assayed for the presence of one or more of the neurodegenerative disease biomarkers selected from retinoid X (RXR alpha, beta, or gamma), Nurr1 (NR4A2) nuclear receptor, Nur77 (NR4A1) nuclear receptor, dopamine active transporter (DAT), or dopa decarboxylase (DDC). The presence, or absence, of one or more of the biomarkers is indicative of a neurodegenerative disease.

[0018] Exemplary assays for the biomarkers include immunoassays, using antibodies (or other affinity molecules) specific to one or more neurodegenerative disease biomarkers. In various embodiments, the immunoassay is selected from the group consisting of dipstick, enzyme-linked immunoassay (ELISA), an antibody array, an antibody chip, a lateral flow test, and multiplex bead immunoassay.

[0019] In certain embodiments, the detection of the biomarkers (gene products) can be performed using other immunoassays such as an ELISA testing kit. In such assays, for example, samples are typically incubated in the presence of an immobilized first specific binding agent (e g. an antibody) capable of specifically binding the biomarker. Binding of the biomarker to the first specific binding agent can be measured using any one of a variety of known methods, such as using a labeled second specific binding agent capable of specifically binding the biomarker (at a different epitope) or the first specific binding agent. Exemplary specific binding agents include, but are not limited to, monoclonal antibodies, polyclonal antibodies, and antibody fragments such as recombinant antibody fragments, and single-chain antibodies (scFv). In some embodiments, various conventional tags or labels can be used, such as, but not limited to, a radioisotope, an enzyme, a chromophore, or a fluorophore. A typical radioisotope is iodine125or sulfur35. Typical enzymes for this purpose include horseradish peroxidase, horseradish galactosidase, and alkaline phosphatase. Conveniently, other detection methods involving chemiluminescence, electrochemiluminescence (ECL), or fluorescence, can readily be used for evaluating the amount of bound detection antibody.

[0020] Also within the scope of the present disclosure are methods of determining the levels of one or more biomarkers.PCT Patent ApplicationDocket 1958481 .00393PCT

[0021] In some embodiments, the level of biomarkers quantitated from the assay(s) can be compared to control levels found in healthy subjects. Levels of biomarkers quantitated from more than one assay taken from an individual can be compared against each other to determine if a treatment plan is effective. For example, if an individual’s initial assay shows low levels of biomarkers associated with a neurodegenerative disease, and an individual’s secondary assay shows the same biomarkers at elevated levels, then this can be indicative of a positive treatment response. Alternatively, if an individual’s initial assay shows low levels of biomarkers associated with a neurodegenerative disease, and an individual’s secondary assay shows slightly lower levels of the same biomarkers from the initial assay, then this can be indicative of a negative / null treatment response.EXAMPLESEXAMPLE 1

[0022] An enzyme linked immunoadsorption assay (ELISA) is performed on a blood sample provided by a 60-year-old female subject. The subject has been previously diagnosed with any nervous system disorder or condition. The ELISA quantitates brain derived exosomes containing low levels of Nurr1 (NR4A2) nuclear receptors when compared to control levels of a healthy subject. As such, the results of the ELISA inform the physician that the female subject may be suffering from a nervous system disorder and / or condition. The physician prepares a treatment plan with a therapeutic agent to target the Nurr1(NR4A2) nuclear receptors. The physician monitors the subject quarterly. After the first quarter, an ELISA is performed on another blood sample provided by the female subject. The ELISA quantitates brain derived exosomes containing Nurr1 (NR4A2) nuclear receptors but in a higher quantity than the initial assay. Thus, the physician continues the subject on the treatment plan and continues to monitor her progress.EXAMPLE 2

[0023] An enzyme linked immunoadsorption assay (ELISA) is performed on a sample of blood provided by a 72-year-old male subject. The subject has been previously diagnosed with Alzheimer’s disease. The ELISA quantitates brain derived exosomes containing low levels of Nurr1 (NR4A2) nuclear receptors and dopamine active transporter (DAT) when compared to control levels of a healthy subject. As such, the results of the ELISA confirm and inform the physician that the male subject is suffering from Alzheimer’s disease. The physician prepares a treatment plan with a therapeutic agent to target the Nurr1 (NR4A2) nuclear receptors and DAT. The physician monitors the subject quarterly.PCT Patent ApplicationDocket 1958481 .00393PCT

[0024] After the first quarter, an ELISA is performed on another blood sample provided by the male subject. The ELISA quantitates brain derived exosomes containing Nurr1(NR4A2) nuclear receptors in a slightly lower quantity than the initial assay. As such, the physician prepares a different treatment plan with a different therapeutic agent to target the target the Nurr1(NR4A2) nuclear receptors and DAT. After the second quarter, an ELISA is performed on another blood sample provided by the male subject. The ELISA quantitates brain derived exosomes containing Nurr1 (NR4A2) nuclear receptors ata higher quantity than the previous assays. The male subject’s caregivers also notice a reduction in visible neurological symptoms associated with Alzheimer’s Disease. Thus, the physician continues the subject on the current treatment plan and continues to monitor his progress.

[0025] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” As used herein the terms "about" and “approximately” means within 10 to 15%, preferably within 5 to 10%. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0026] The terms “a,” “an,” “the” and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminatePCT Patent ApplicationDocket 1958481 .00393PCT the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0027] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0028] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0029] Specific embodiments disclosed herein may be further limited in the claims using consisting of or consisting essentially of language. When used in the claims, whether as filed or added per amendment, the transition term “consisting of” excludes any element, step, or ingredient not specified in the claims. The transition term “consisting essentially of limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s). Embodiments of the invention so claimed are inherently or expressly described and enabled herein.

[0030] Furthermore, numerous references have been made to patents and printed publications throughout this specification. Each of the above-cited references and printed publications are individually incorporated herein by reference in their entirety.

[0031] In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation,PCT Patent Application Docket 1958481 .00393PCT alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.

Claims

PCT Patent ApplicationDocket 1958481 .00393PCTWe Claim:

1. A method of assessing changes of expression in patients’ nervous systems of molecules responsive to treatments modulating nuclear receptors for retinoid X (RXR alpha, beta, or gamma), Nurr1(NR4A2) nuclear receptors, Nur77 (NR4A1) nuclear receptors, dopamine active transporter (DAT), or dopa decarboxylase (DDC), for nervous system disorders or conditions related to these molecules, comprising utilizing a quantitative assay for measuring brain derived exosomes released by neurons, glial cells, or nervous system inflammatory cells, wherein the information provided by the assay is useful for development of treatments for at least one nervous system disorder or condition, or for diagnosis, theragnosis, assessment of prognosis, or monitoring of therapeutic response in a patient with a nervous system disorder or condition.

2. The method of claim 1 , wherein the nervous system disorder or condition is Alzheimer’s disease, Parkinson’s disease, a dementia, amyotrophic lateral sclerosis, multiple sclerosis, a stroke, a traumatic CNS injury, a hypoxic CNS injury, a schizophrenia, a depression, an autism, a neuropsychiatric disorder, normal aging related neurodegeneration, or other neurodegenerative condition.

3. The method of claim 1 , wherein the nervous system disorder is not amyotrophic lateral sclerosis.

4. The method of claim 1 , wherein the assay is used to quantitate the brain derived exosomes in blood, serum, plasma, cerebrospinal fluid, or other biologic fluids.

5. The method of claim 1 , wherein the assay consists of an enzyme linked immunoadsorption assay (ELISA), utilizing at least one antibody or antibody fragment or selective antigen-binding molecule, which selectively binds to a cell surface molecule selectively expressed on neurons, glial cells, or CNS inflammatory cells; and at least one additional antibody or antibody fragment or selective antigen-binding molecule which separately and selectively binds to a cellular molecule, of which the expression in neurons, glial cells, or CNS inflammatory cells, is assessed by quantitation of brain derived exosomes in a biologic fluid.

6. The method of claim 1 , wherein the assay quantitates brain derived exosomes containing RXR alpha, beta, or gamma nuclear receptors.

7. The method of claim 1 , wherein the assay quantitates brain derived exosomes containing Nurr1(NR4A2) nuclear receptors.PCT Patent Application Docket 1958481 .00393PCT8. The method of claim 1 , wherein the assay quantitates brain derived exosomes containing Nur77 (NR4A1) nuclear receptors.

9. The method of claim 1 , wherein the assay quantitates brain derived exosomes containing dopamine active transporter (DAT).

10. The method of claim 1 , wherein the assay quantitates brain derived exosomes containing dopa decarboxylase (DDC).11 . The method of any one of claims 1-9, wherein the quantitative brain derived exosomes assay results inform development of one or more therapeutic agents for at least one nervous system disorder or condition.

12. The method of any one of claims 1-11 wherein the therapeutic agent for nervous system disorder or condition is an agonist, antagonist, or inverse agonist of one or more of the RXR nuclear receptors, Nurrl , Nur77, DAT, or DDC.

13. The method of any one of claims 1-12, wherein the quantitative brain derived exosomes assay results inform treatment decisions for patients with nervous system disorders or conditions.

14. The method of any one of claims 1-13, wherein the quantitative brain derived exosome assay results provide diagnostic, theragnostic, or prognostic information informing treatment of patients with suspected or documented nervous disorders or conditions.

15. The method of any one of claims 11-14, wherein the nervous system disorders or conditions are Alzheimer’s disease, Parkinson’s disease, a dementia, amyotrophic lateral sclerosis, multiple sclerosis, a stroke, a traumatic CNS injury, a hypoxic CNS injury, a schizophrenia, a depression, an autism, a neuropsychiatric disorder, normal aging related neurodegeneration, or other nervous system disorder or condition.

16. The method of any one of claims 11-14, wherein the nervous system disorder or condition is not amyotrophic lateral sclerosis.