Methods of diagnosing and treating neurovascular diseases

Abstract

The present disclosure relates to methods for diagnosing and treating patients with neurovascular disease.

Description

[0001] METHODS OF DIAGNOSING AND TREATING NEUROVASCULAR DISEASES

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to, and the benefit of, U.S. Provisional Patent Application No. 63 / 624,718, filed January 24, 2024, which is incorporated by reference herein in its entirety.

[0004] FIELD

[0005] The present disclosure relates to methods for diagnosing and treating patients with neurovascular disease.

[0006] BACKGROUND

[0007] Neurovascular disease, including but not limited to stroke, Moyamoya disease, and brain aneurysm, are a major leading cause of serious disability and death in the United States. Although multiple strategies to prevent these neurovascular diseases have been examined, efficient and precise diagnostic methods and effective targeted treatments and therapies are still lacking. Many of these efforts focus on exploiting agonists and / or antagonists that target cell receptors to protect against neural damage. However, a complete understanding of these cell receptors is still lacking. Exploring and characterizing the expression levels of these cell receptors can be beneficial to preventing, diagnosing, and / or treating neurovascular diseases. Thus, there remains a need to characterize said expression levels of such cell receptors to better direct, and further the development of, treatments and therapies to combat neurovascular diseases. Characterization of altered expression levels can also drive alternative new gene therapies as alternatives to conventional therapies. These methods disclosed herein address these needs and more.

[0008] SUMMARY

[0009] Disclosed herein are methods of diagnosing and treating a subject with a neurovascular disease.

[0010] In some aspects, disclosed herein is a method of treating a subject with a neurovascular disease, comprising: obtaining a biological sample from a subject suspected of having the neurovascular disease immediately after onset of neurovascular disease symptoms; quantifying an expression level of one or more biomarkers, wherein the biomarker comprises one or more of AD0RA3, AD0RA2B, AD0RA2A, or ADRB2; determining the subject as suffering from the neurovascular disease if the expression level of the one or more biomarkers is changed in the subject immediately after onset of neurovascular disease symptoms in comparison to a reference control (or a standard); and administering to the subject a therapeutically effective amount of an agonist or antagonist of AD0RA3, AD0RA2B, AD0RA2A, or ADRB2, a gene therapy, or a thrombolytic; or performing a surgical procedure.

[0011] In some embodiments, the biomarker comprises AD0RA3. In some embodiments, the biomarker comprises AD0RA2B. In some embodiments, the biomarker comprises AD0RA2A. In some embodiments, the biomarker comprises ADRB2.

[0012] In some embodiments, the biological sample comprises a blood sample.

[0013] In some embodiments, the quantifying is carried out by one or a combination of Polymerase Chain Reaction, Real Time-Polymerase Chain Reaction, Real Time Reverse Transcriptase-Polymerase Chain Reaction, Real-time quantitative RT-PCR, Northern blot analysis, in situ hybridization, probe array, or gene sequencing.

[0014] In some embodiments, the reference control is a sample from the subject taken about 24 hours after onset of neurovascular disease symptoms. In some embodiments, the reference control is a sample from the subject taken greater than 24 hours after onset of neurovascular disease symptoms. In some embodiments, the reference control is a sample from a subject that is not suffering from the neurovascular disease. In some embodiments, the reference control is a sample from the subject that is not experiencing neurovascular disease symptoms. In some embodiments, the sample is from a different subject, a second subject, or from a population of subjects. In some embodiments, the sample is from a different subject (or population of subjects) that is healthy, that is not suffering from the neurovascular disease, and / or that is not experiencing neurovascular disease symptoms.

[0015] In some embodiments, the agonist is an AD0RA3 agonist. In some embodiments, the agonist is an AD0RA2B agonist. In some embodiments, the agonist is an AD0RA2A agonist. In some embodiments, the agonist is an ADRB2 agonist.

[0016] In some embodiments, the agonist or antagonist is selected from an agonist or antagonist listed in Table 1 herein. In some embodiments, the ADORA3 agonist is AST-004.

[0017] In some embodiments, the subject is a human.

[0018] In some embodiments, the neurovascular disease is stroke or traumatic brain injury. In some embodiments, the neurovascular disease is Moyamoya Disease.

[0019] In some embodiments, the method further comprises administering to the subject an alternative gene therapy, a thrombolytic therapy, or performing a surgical procedure.

[0020] In some aspects, disclosed herein is a method for diagnosing a subject with a neurovascular disease, comprising: obtaining a biological sample from a subject suspected of having the neurovascular disease immediately after onset of neurovascular disease symptoms; quantifying an expression level of one or more biomarkers immediately after onset of neurovascular disease symptoms, wherein the biomarker comprises one or more of AD0RA3, AD0RA2B, AD0RA2A, or ADRB2; and determining the subject as suffering from the neurovascular disease if the expression level of the one or more biomarkers is changed in the subject immediately after onset of neurovascular disease symptoms in comparison to a reference control.

[0021] In some embodiments, the method further comprises administering an agonist or antagonist of AD0RA3, AD0RA2B, AD0RA2A, or ADRB2.

[0022] BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects described below.

[0024] FIG. 1 shows the level of expression for AD0RA3 in stroke patients is different from the level of expression in patients with other condition in blood draws obtained within a short time after the onset of symptoms (DI). Expression levels appear to return to baseline when expression levels are compared for the same patients from samples obtained immediately after the onset of symptoms versus samples obtained 24 hours after the onset of symptoms (D3). Figure 1 further shows the lowest level of expression for AD0RA3 measured in a stroke mimic patient with Moyamoya early after the onset of stroke-like symptoms.

[0025] FIG. 2 shows that variations in relative expression rank vary by sex, age and racial subgroups (marked in orange in the Stroke plots) in ways that can explain clinically known disparities in neuronal injury between different groups. Measuring gene expression for each patient can improve treatment decisions by improving precision of diagnosis. DETAILED DESCRIPTION

[0026] Disclosed herein are methods of diagnosing and treating a subject with a neurovascular disease. Further disclosed are methods for determining, predicting, or improving the outcome for a subject suffering from neurovascular disease symptoms. The unexpected findings herein are used for determining and treating subjects with a neurovascular disease, allowing treatment with the appropriate therapeutic regimens or surgical interventions.

[0027] Reference will now be made in detail to the embodiments of the invention, examples of which are illustrated in the drawings and the examples. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

[0028] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of’ and “consisting of” can be used in place of “comprising” and “including” to provide for more specific embodiments and are also disclosed. As used in this disclosure and in the appended claims, the singular forms “a”, “an”, “the”, include plural referents unless the context clearly dictates otherwise.

[0029] The following definitions are provided for the full understanding of terms used in this specification.

[0030] Terminology

[0031] As used herein, the terms “may,” “optionally,” and “may optionally” are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur. Thus, for example, the statement that a formulation “may include an excipient” is meant to include cases in which the formulation includes an excipient as well as cases in which the formulation does not include an excipient.

[0032] As used here, the terms “beneficial agent” and “active agent” are used interchangeably herein to refer to a chemical compound or composition that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, i.e., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, i.e., prevention of a disorder or other undesirable physiological condition. The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, prodrugs, active metabolites, isomers, fragments, analogs, and the like. When the terms “beneficial agent” or “active agent” are used, then, or when a particular agent is specifically identified, it is to be understood that the term includes the agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, conjugates, active metabolites, isomers, fragments, analogs, etc.

[0033] As used herein, the terms “treating” or “treatment” of a subject includes the administration of a drug to a subject with the purpose of curing, healing, alleviating, relieving, altering, remedying, ameliorating, improving, stabilizing or affecting a disease or disorder, or a symptom of a disease or disorder. The terms “treating” and “treatment” can also refer to reduction in severity and / or frequency of symptoms, elimination of symptoms and / or underlying cause, prevention of the occurrence of symptoms and / or their underlying cause, and improvement or remediation of damage.

[0034] As used herein, the term “preventing” a disorder or unwanted physiological event in a subject refers specifically to the prevention of the occurrence of symptoms and / or their underlying cause, wherein the subject may or may not exhibit heightened susceptibility to the disorder or event.

[0035] By the term “effective amount” of a therapeutic agent is meant a nontoxic but sufficient amount of a beneficial agent to provide the desired effect. The amount of beneficial agent that is “effective” will vary from subject to subject, depending on the age and general condition of the subject, the particular beneficial agent or agents, and the like. Thus, it is not always possible to specify an exact “effective amount.” However, an appropriate “effective” amount in any subject case may be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, and unless specifically stated otherwise, an “effective amount” of a beneficial can also refer to an amount covering both therapeutically effective amounts and prophylactically effective amounts.

[0036] An “effective amount” of a drug necessary to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. As used herein, a “therapeutically effective amount” of a therapeutic agent refers to an amount that is effective to achieve a desired therapeutic result, and a “prophylactically effective amount” of a therapeutic agent refers to an amount that is effective to prevent an unwanted physiological condition. Therapeutically effective and prophylactically effective amounts of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, gender, and weight of the subject.

[0037] The term “therapeutically effective amount” can also refer to an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect. The precise desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the drug and / or drug formulation to be administered (e.g., the potency of the therapeutic agent (drug), the concentration of drug in the formulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art.

[0038] As used herein, the term “pharmaceutically acceptable” component can refer to a component that is not biologically or otherwise undesirable, i.e., the component may be incorporated into a pharmaceutical formulation of the invention and administered to a subject as described herein without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the formulation in which it is contained. When the term “pharmaceutically acceptable” is used to refer to an excipient, it is generally implied that the component has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.

[0039] Also, as used herein, the term “pharmacologically active” (or simply “active”), as in a “pharmacologically active” derivative or analog, can refer to a derivative or analog (e.g., a salt, ester, amide, conjugate, metabolite, isomer, fragment, etc.) having the same type of pharmacological activity as the parent compound and approximately equivalent in degree.

[0040] The term “subject” or “host” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician. The subject can be either male or female.

[0041] The terms “peptide,” “protein,” and “polypeptide” are used interchangeably to refer to a natural or synthetic molecule comprising two or more amino acids linked by the carboxyl group of one amino acid to the alpha amino group of another. The term “nucleic acid” refers to a natural or synthetic molecule comprising a single nucleotide or two or more nucleotides linked by a phosphate group at the 3’ position of one nucleotide to the 5’ end of another nucleotide. The nucleic acid is not limited by length, and thus the nucleic acid can include deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

[0042] “Complementary” or “substantially complementary” refers to the hybridization or base pairing or the formation of a duplex between nucleotides or nucleic acids, such as, for instance, between the two strands of a double stranded DNA molecule or between an oligonucleotide primer and a primer binding site on a single stranded nucleic acid. Complementary nucleotides are, generally, A and T / U, or C and G. Two single- stranded RNA or DNA molecules are said to be substantially complementary when the nucleotides of one strand, optimally aligned and compared and with appropriate nucleotide insertions or deletions, pair with at least about 80% of the nucleotides of the other strand, usually at least about 90% to 95%, and more preferably from about 98 to 100%. Alternatively, substantial complementarity exists when an RNA or DNA strand will hybridize under selective hybridization conditions to its complement. Typically, selective hybridization will occur when there is at least about 65% complementary over a stretch of at least 14 to 25 nucleotides, at least about 75%, or at least about 90% complementary. See Kanehisa (1984) Nucl. Acids Res. 12:203.

[0043] “Hybridization” refers to the process in which two single- stranded oligonucleotides bind non-covalently to form a stable double-stranded oligonucleotide. The term “hybridization” may also refer to triple-stranded hybridization. The resulting (usually) double- stranded oligonucleotide is a “hybrid” or “duplex.” “Hybridization conditions” will typically include salt concentrations of less than about 1 M, more usually less than about 500 mM and even more usually less than about 200 mM. Hybridization temperatures can be as low as 5° C., but are typically greater than 22° C., more typically greater than about 30° C., and often in excess of about 37° C. In certain exemplary embodiments, hybridization takes place at room temperature. The term “stringent hybridization conditions” as used herein is the binding which occurs within a range from about Tm 5° C. (5° C. below the melting temperature Tm of the probe) to about 20° C. to 25° C. below Tm. The term “highly stringent hybridization conditions” as used herein refers to conditions of: at least about 6xSSC and 1% SDS at 65° C., with a first wash for 10 minutes at about 42° C. with about 20% (v / v) formamide in 0. IxSSC, and with a subsequent wash with 0.2xSSC and 0.1% SDS at 65° C.

[0044] The terms "about" and "approximately" are defined as being “close to” as understood by one of ordinary skill in the art. In one non-limiting embodiment the terms are defined to be within 10%. In another non-limiting embodiment, the terms are defined to be within 5%. In still another non-limiting embodiment, the terms are defined to be within 1%.

[0045] Methods of Diagnosis and Treatment

[0046] In some aspects, disclosed herein is a method of treating a subject with a neurovascular disease, comprising: obtaining a biological sample from a subject suspected of having the neurovascular disease immediately after onset of neurovascular disease symptoms; quantifying an expression level of one or more biomarkers, wherein the biomarker comprises one or more of ADORA3, ADORA2B, ADORA2A, or ADRB2; determining the subject as suffering from the neurovascular disease if the expression level of the one or more biomarkers is changed in the subject immediately after onset of neurovascular disease symptoms in comparison to a reference control; and administering to the subject a therapeutically effective amount of an agonist or antagonist of ADORA3, ADORA2B, ADORA2A, or ADRB2.

[0047] In some embodiments, the biomarker comprises ADORA3. In some embodiments, the biomarker comprises ADORA2B. In some embodiments, the biomarker comprises ADORA2A. In some embodiments, the biomarker comprises ADRB2. Gen information and protein information can be found at NCBI (ncbi.nlm.nih.gov; for example, AD0RA2B (NCBI Gene: 136 Ensembl:ENSG00000170425), AD0RA2A (NCBI Gene: 135

[0048] Ensembl:ENSG00000128271), AD0RA3 (NCBI Gene: 140 Ensembl:ENSG00000282608), ADRB2 (NCBI Gene: 154 EnsemblENSG00000169252)).

[0049] In some aspects, disclosed herein is a method of treating a subject with a neurovascular disease, comprising: obtaining a biological sample from a subject suspected of having the neurovascular disease immediately after onset of neurovascular disease symptoms; quantifying an expression level of one or more biomarkers, wherein the biomarker comprises ADORA3; determining the subject as suffering from the neurovascular disease if the expression level of the one or more biomarkers is changed in the subject immediately after onset of neurovascular disease symptoms in comparison to a reference control; and administering to the subject a therapeutically effective amount of an agonist or antagonist of ADORA3, a gene therapy, or a thrombolytic; or performing a surgical procedure. In some embodiments, the biological sample comprises a blood sample.

[0050] In some embodiments, the quantifying is carried out by one or a combination of Polymerase Chain Reaction, Real Time-Polymerase Chain Reaction, Real Time Reverse Transcriptase-Polymerase Chain Reaction, Real-time quantitative RT-PCR, Northern blot analysis, in situ hybridization, probe array, or gene sequencing.

[0051] In some embodiments, the reference control is a sample from the subject taken about 24 hours after onset of neurovascular disease symptoms. In some embodiments, the reference control is a sample from the subject taken greater than 24 hours after onset of neurovascular disease symptoms. In some embodiments, the reference control is a sample from a subject that is not suffering from the neurovascular disease. In some embodiments, the reference control is a sample from the subject that is not experiencing neurovascular disease symptoms. In some embodiments, the reference control is a sample that provides a baseline for quantifying the expression level of the one or more biomarkers of any preceding aspect. In some embodiments, the sample is from a different subject, a second subject, or from a population of subjects. In some embodiments, the sample is from a different subject (or population of subjects) that is healthy, that is not suffering from the neurovascular disease, and / or that is not experiencing neurovascular disease symptoms.

[0052] As used herein, “immediately after” refers to an action, including but not limited to sample collection, being performed in less than or about 24 hours from onset of any neurovascular disease disclosed herein. In some embodiments, the sample is collected immediately after onset of neurovascular disease symptoms, for example, within 10 minutes, within 30 minutes, within 1 hour, within 2 hours, within 3 hours, within 4 hours, within 5 hours, within 6 hours, or within 7 hours after onset of neurovascular disease symptoms. In some embodiments, the sample is collected up to 24 hours after onset of neurovascular disease. In some embodiments, the sample is collected within 7 hours, within 8 hours, within 9 hours, within 10 hours, within 11 hours, within 12 hours, within 13 hours, within 14 hours, within 15 hours, within 16 hours, within 17 hours, within 18 hours, within 19 hours, within 20 hours, within 21 hours, within 22 hours, within 23 hours, or within 24 hours after onset of neurovascular disease.

[0053] In some embodiments, the agonist is an ADORA3 agonist. In some embodiments, the agonist is an ADORA2B agonist. In some embodiments, the agonist is an ADORA2A agonist. In some embodiments, the agonist is an ADRB2 agonist. In some embodiments, the antagonist is an AD0RA3 antagonist. In some embodiments, the antagonist is an AD0RA2B antagonist. In some embodiments, the antagonist is an AD0RA2A antagonist. In some embodiments, the antagonist is an ADRB2 antagonist.

[0054] In some embodiments, the agonist is selected from an agonist or antagonist listed in Table 1 herein. In some embodiments, the agonist is AST-004.

[0055] In some embodiments, the subject is a human.

[0056] In some embodiments, the neurovascular disease is stroke. In some embodiments, the neurovascular disease is traumatic brain injury (TBI). In some embodiments, the neurovascular disease is Moyamoya Disease.

[0057] In some aspects, disclosed herein is a method of treating a subject with a stroke, comprising: obtaining a biological sample from a subject suspected of having the stroke immediately after onset of stroke symptoms; quantifying an expression level of one or more biomarkers, wherein the biomarker comprises one or more of AD0RA3, AD0RA2B, AD0RA2A, or ADRB2; determining the subject as suffering from the stroke if the expression level of the one or more biomarkers is changed in the subject immediately after onset of stroke symptoms in comparison to a reference control (or a standard); and administering to the subject a therapeutically effective amount of an agonist or antagonist of AD0RA3, AD0RA2B, AD0RA2A, or ADRB2, a gene therapy, or a thrombolytic; or performing a surgical procedure.

[0058] In some aspects, disclosed herein is a method of treating a subject with a traumatic brain injury, comprising: obtaining a biological sample from a subject suspected of having the traumatic brain injury immediately after onset of traumatic brain injury symptoms; quantifying an expression level of one or more biomarkers, wherein the biomarker comprises one or more of AD0RA3, AD0RA2B, AD0RA2A, or ADRB2; determining the subject as suffering from the traumatic brain injury if the expression level of the one or more biomarkers is changed in the subject immediately after onset of traumatic brain injury symptoms in comparison to a reference control (or a standard); and administering to the subject a therapeutically effective amount of an agonist or antagonist of AD0RA3, AD0RA2B, AD0RA2A, or ADRB2, a gene therapy, or a thrombolytic; or performing a surgical procedure. In some aspects, disclosed herein is a method of treating a subject with Moyamoya Disease, comprising: obtaining a biological sample from a subject suspected of having Moyamoya Disease; quantifying an expression level of one or more biomarkers, wherein the biomarker comprises one or more of AD0RA3, AD0RA2B, AD0RA2A, or ADRB2; determining the subject as suffering from Moyamoya Disease if the expression level of the one or more biomarkers is changed in the subject in comparison to a reference control (or a standard); and administering to the subject a therapeutically effective amount of an agonist or antagonist of AD0RA3, AD0RA2B, AD0RA2A, or ADRB2, a gene therapy, or a thrombolytic; or performing a surgical procedure.

[0059] In some aspects, disclosed herein is a method of treating a subject with a stroke, comprising: obtaining a biological sample from a subject suspected of having the stroke immediately after onset of stroke symptoms; quantifying an expression level of AD0RA3; determining the subject as suffering from the stroke if the expression level of AD0RA3 is changed in the subject immediately after onset of stroke symptoms in comparison to a reference control (or a standard); and administering to the subject a therapeutically effective amount of an agonist or antagonist of AD0RA3, a gene therapy, or a thrombolytic; or performing a surgical procedure.

[0060] In some aspects, disclosed herein is a method of treating a subject with a neurovascular disease, comprising: obtaining a biological sample from a subject suspected of having the neurovascular disease immediately after onset of neurovascular disease symptoms; quantifying an expression level of one or more biomarkers, wherein the biomarkers comprise AD0RA3, AD0RA2B, AD0RA2A, and ADRB2; determining the subject as suffering from the neurovascular disease if the expression level of the one or more biomarkers is changed in the subject immediately after onset of neurovascular disease symptoms in comparison to a reference control (or a standard); and administering to the subject a therapeutically effective amount of an agonist or antagonist of AD0RA3, AD0RA2B, AD0RA2A, or ADRB2, a gene therapy, or a thrombolytic; or performing a surgical procedure. In some aspects, disclosed herein is a method of treating a subject with a neurovascular disease, comprising: obtaining a biological sample from a subject suspected of having the neurovascular disease; quantifying an expression level of one or more biomarkers, wherein the biomarker comprises one or more of AD0RA3, AD0RA2B, AD0RA2A, or ADRB2; determining the subject as suffering from the neurovascular disease if the expression level of the one or more biomarkers is changed in the subject in comparison to a reference control (or a standard); and administering to the subject a therapeutically effective amount of an agonist or antagonist of AD0RA3, AD0RA2B, AD0RA2A, or ADRB2, a gene therapy, or a thrombolytic; or performing a surgical procedure.

[0061] In some aspects, disclosed herein is a method of treating a subject with a neurovascular disease, comprising: obtaining a biological sample from a subject suspected of having the neurovascular disease; quantifying an expression level of AD0RA3; determining the subject as suffering from the neurovascular disease if the expression level of AD0RA3 is changed in the subject in comparison to a reference control (or a standard); and administering to the subject a therapeutically effective amount of an agonist or antagonist of AD0RA3, a gene therapy, or a thrombolytic; or performing a surgical procedure.

[0062] In some aspects, disclosed herein is a method of treating a subject with a traumatic brain injury, comprising: obtaining a biological sample from a subject suspected of having the traumatic brain injury immediately after onset of traumatic brain injury symptoms; quantifying an expression level of AD0RA3; determining the subject as suffering from the traumatic brain injury if the expression level of AD0RA3 is changed in the subject immediately after onset of traumatic brain injury symptoms in comparison to a reference control (or a standard); and administering to the subject a therapeutically effective amount of an agonist or antagonist of AD0RA3, a gene therapy, or a thrombolytic; or performing a surgical procedure.

[0063] In some aspects, disclosed herein is a method of treating a subject with Moyamoya Disease, comprising: obtaining a biological sample from a subject suspected of having Moyamoya Disease; quantifying an expression level of AD0RA3; determining the subject as suffering from Moyamoya Disease if the expression level of AD0RA3 is changed in the subject in comparison to a reference control (or a standard); and administering to the subject a therapeutically effective amount of an agonist or antagonist of AD0RA3, a gene therapy, or a thrombolytic; or performing a surgical procedure.

[0064] In some embodiments, the method further comprises administering to the subject an alternative gene therapy, a thrombolytic therapy, or performing a surgical procedure.

[0065] In some aspects, disclosed herein is a method for diagnosing a subject with a neurovascular disease, comprising: obtaining a biological sample from a subject suspected of having the neurovascular disease immediately after onset of neurovascular disease symptoms; quantifying an expression level of one or more biomarkers immediately after onset of neurovascular disease symptoms, wherein the biomarker comprises one or more of AD0RA3, AD0RA2B, AD0RA2A, or ADRB2; and determining the subject as suffering from the neurovascular disease if the expression level of the one or more biomarkers is changed in the subject immediately after onset of neurovascular disease symptoms in comparison to a reference control.

[0066] In some aspects, disclosed herein is a method for diagnosing a subject with a stroke, comprising: obtaining a biological sample from a subject suspected of having the stroke immediately after onset of stroke symptoms; quantifying an expression level of one or more biomarkers immediately after onset of stroke symptoms, wherein the biomarker comprises one or more of AD0RA3, AD0RA2B, AD0RA2A, or ADRB2; and determining the subject as suffering from the stroke if the expression level of the one or more biomarkers is changed in the subject immediately after onset of stroke symptoms in comparison to a reference control.

[0067] In some aspects, disclosed herein is a method for diagnosing a subject with a traumatic brain injury, comprising: obtaining a biological sample from a subject suspected of having the traumatic brain injury immediately after onset of traumatic brain injury symptoms; quantifying an expression level of one or more biomarkers immediately after onset of traumatic brain injury symptoms, wherein the biomarker comprises one or more of AD0RA3, AD0RA2B, AD0RA2A, or ADRB2; and determining the subject as suffering from the traumatic brain injury if the expression level of the one or more biomarkers is changed in the subject immediately after onset of traumatic brain injury symptoms in comparison to a reference control.

[0068] In some aspects, disclosed herein is a method for diagnosing a subject with Moyamoya Disease, comprising: obtaining a biological sample from a subject suspected of having tMoyamoya Disease; quantifying an expression level of one or more biomarkers, wherein the biomarker comprises one or more of AD0RA3, AD0RA2B, AD0RA2A, or ADRB2; and determining the subject as suffering from Moyamoya Disease if the expression level of the one or more biomarkers is changed in the subject in comparison to a reference control.

[0069] In some aspects, disclosed herein is a method for diagnosing a subject with a neurovascular disease, comprising: obtaining a biological sample from a subject suspected of having the neurovascular disease immediately after onset of neurovascular disease symptoms; quantifying an expression level of one or more biomarkers immediately after onset of neurovascular disease symptoms, wherein the biomarker comprises AD0RA3; and determining the subject as suffering from the neurovascular disease if the expression level of AD0RA3 is changed in the subject immediately after onset of neurovascular disease symptoms in comparison to a reference control.

[0070] In some aspects, disclosed herein is a method for diagnosing a subject with a stroke, comprising: obtaining a biological sample from a subject suspected of having the stroke immediately after onset of stroke symptoms; quantifying an expression level of one or more biomarkers immediately after onset of stroke symptoms, wherein the biomarker comprises AD0RA3; and determining the subject as suffering from the stroke if the expression level of AD0RA3 is changed in the subject immediately after onset of stroke symptoms in comparison to a reference control.

[0071] In some aspects, disclosed herein is a method for diagnosing a subject with a traumatic brain injury, comprising: obtaining a biological sample from a subject suspected of having the traumatic brain injury immediately after onset of traumatic brain injury symptoms; quantifying an expression level of one or more biomarkers immediately after onset of traumatic brain injury symptoms, wherein the biomarker comprises AD0RA3; and determining the subject as suffering from the traumatic brain injury if the expression level of AD0RA3 is changed in the subject immediately after onset of traumatic brain injury symptoms in comparison to a reference control.

[0072] In some aspects, disclosed herein is a method for diagnosing a subject with Moyamoya Disease, comprising: obtaining a biological sample from a subject suspected of having Moyamoya Disease; quantifying an expression level of one or more biomarkers, wherein the biomarker comprises AD0RA3; and determining the subject as suffering from Moyamoya Disease if the expression level of AD0RA3 is changed in the subject in comparison to a reference control.

[0073] In some aspects, disclosed herein is a method for diagnosing a subject with a neurovascular disease, comprising: obtaining a biological sample from a subject suspected of having the neurovascular disease immediately after onset of neurovascular disease symptoms; quantifying an expression level of one or more biomarkers immediately after onset of neurovascular disease symptoms, wherein the biomarker comprises AD0RA3, AD0RA2B, AD0RA2A, and ADRB2; and determining the subject as suffering from the neurovascular disease if the expression level of the one or more biomarkers is changed in the subject immediately after onset of neurovascular disease symptoms in comparison to a reference control.

[0074] In some aspects, disclosed herein is a method for diagnosing a subject with a neurovascular disease, comprising: obtaining a biological sample from a subject suspected of having the neurovascular disease; quantifying an expression level of one or more biomarkers, wherein the biomarker comprises AD0RA3, AD0RA2B, AD0RA2A, or ADRB2; and determining the subject as suffering from the neurovascular disease if the expression level of the one or more biomarkers is changed in the subject in comparison to a reference control.

[0075] In some aspects, disclosed herein is a method for diagnosing a subject with a neurovascular disease, comprising: obtaining a biological sample from a subject suspected of having the neurovascular disease; quantifying an expression level of AD0RA3; and determining the subject as suffering from the neurovascular disease if the expression level of AD0RA3 is changed in the subject in comparison to a reference control.

[0076] In some embodiments, the biomarker comprises AD0RA3. In some embodiments, the biomarker comprises AD0RA2B. In some embodiments, the biomarker comprises AD0RA2A. In some embodiments, the biomarker comprises ADRB2.

[0077] In some embodiments, the biological sample comprises a blood sample.

[0078] In some embodiments, the quantifying is carried out by one or a combination of Polymerase Chain Reaction, Real Time-Polymerase Chain Reaction, Real Time Reverse Transcriptase-Polymerase Chain Reaction, Real-time quantitative RT-PCR, Northern blot analysis, in situ hybridization, probe array, or gene sequencing.

[0079] In some embodiments, the reference control is a sample from the subject taken about 24 hours after onset of neurovascular disease symptoms. In some embodiments, the reference control is a sample from the subject taken greater than 24 hours after onset of neurovascular disease symptoms. In some embodiments, the reference control is a sample from a subject that is not suffering from the neurovascular disease. In some embodiments, the reference control is a sample from the subject that is not experiencing neurovascular disease symptoms. In some embodiments, the reference control is a sample that provides a baseline for quantifying the expression level of the one or more biomarkers of any preceding aspect. In some embodiments, the sample is from a different subject, a second subject, or from a population of subjects. In some embodiments, the sample is from a different subject (or population of subjects) that is healthy, that is not suffering from the neurovascular disease, and / or that is not experiencing neurovascular disease symptoms.

[0080] In some embodiments, the method further comprises administering an agonist or antagonist of AD0RA3, AD0RA2B, AD0RA2A, or ADRB2. In some embodiments the agonist or antagonist are replaced by alternative therapies including gene therapies that affect the expression of AD0RA3, AD0RA2B, AD0RA2A, or ADRB2 receptors.

[0081] In some embodiments, the agonist is an AD0RA3 agonist. In some embodiments, the agonist is an AD0RA2B agonist. In some embodiments, the agonist is an AD0RA2A agonist. In some embodiments, the agonist is an ADRB2 agonist.

[0082] In some embodiments, the agonist is selected from an agonist or antagonist listed in Table 1 herein. In some embodiments, the AD0RA3 agonist is AST-004. AST-004

[0083] Chemical formula: C13H17N5O3S MW: 323 g / mol

[0084] AST-004

[0085] (l ?,21?,35,4 ?,5S)-4-(6-ammo-2-(methylthio)-9 / / -purin-9-yr)-l- (hydroxymethyl)bicyclo[3.1.0]hexane-2,3-diol

[0086] See Listen, T. et al. Purinergic Signal. 2020 Dec; 16(4): 543-559.

[0087] In some embodiments, the agonist or antagonist of ADORA3, ADORA2B, ADORA2A, or ADRB2 can be a nucleic acid, for example, an siRNA, shRNA, or an antisense oligonucleotide. In some embodiments, the antagonist is an ADORA3 antagonist. In some embodiments, the antagonist is an ADORA2B antagonist. In some embodiments, the antagonist is an ADORA2A antagonist. In some embodiments, the antagonist is an ADRB2 antagonist.

[0088] In some embodiments, the subject is a human.

[0089] In some embodiments, the neurovascular disease is stroke. In some embodiments, the neurovascular disease is traumatic brain injury (TBI). In some embodiments, the neurovascular disease is Moyamoya Disease.

[0090] In some embodiments, the quantifying is carried out to detect protein expression levels instead of gene expression levels. In some embodiments, the quantifying is carried out by one or a combination of Western blot, ELISA, flow cytometry, immunohistochemistry, and other methods of detection using antibodies. In some embodiments, the quantifying is carried out to detect gene methylation and gene copy number.

[0091] In some embodiments, the reference control is a healthy subject. In some embodiments, the reference control is not suffering from the neurovascular disease. In some embodiments, the reference control is not experiencing neurovascular disease symptoms. In some embodiments, the reference control is from a pooled population of patient samples or biological samples. In some embodiments, the reference control is a standard. In some aspects, disclosed herein are methods for determining, predicting, or improving the outcome for a subject suffering from neurovascular disease symptoms, comprising: obtaining a biological sample from a subject; quantifying an expression level of one or more biomarkers, wherein the biomarker comprises one or more of an adenosine receptor; determining the subject as suffering from the neurovascular disease if the expression level of the one or more biomarkers is changed in the subject immediately after onset of neurovascular disease symptoms in comparison to a reference control; and administering to the subject a therapeutically effective amount of an agonist or antagonist of an adenosine receptor.

[0092] Gene expression measured in blood can act as a surrogate for the level of expression of various receptors on cell surfaces that may respond to treatments designed to trigger protective mechanisms or stop damaging mechanisms within the cell. To this end, measuring the level of expression before, during or after such treatment can be useful to help diagnose the patient with respect to requiring the treatment, determining the level of treatment necessary and monitoring the effect of the treatment. In some embodiments, the level of expression of the biomarker is useful in maximizing dose for maximizing drug effectiveness or for reducing side effects.

[0093] In some embodiments, the method further comprises administering a therapeutic agent for treating a stroke. In some embodiments, the method further comprises administering a therapeutic agent for treating Moyamoya Disease. In some embodiments, the therapeutic agent comprises an antiplalelet agent or an anticoagulant agent. In some embodiments, the therapeutic agent comprises aspirin. In some embodiments, the therapeutic agent comprises a blood thinner. In some embodiments, the therapeutic agent comprises warfarin. In some embodiments, the therapeutic agent comprises a gene therapy compound, including but not limited to plasmid DNA, viral vectors, bacterial vectors, nanoparticles lipid nanoparticles, cationic polymers, and non-coding RNAs (such as, for example microRNA (miRNA), shortinterfering RNA (siRNA)).

[0094] In some embodiments, the method further comprises administering a surgical treatment to the subject. In some embodiments, the surgical treatment comprises a revascularization surgery. EXAMPLES

[0095] The following examples are set forth below to illustrate the methods and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention which are apparent to one skilled in the art.

[0096] Example 1. Diagnosis and Treatment of Neurovascular Conditions

[0097] Conditions such as stroke cause injury to the central nervous system which can trigger various damaging as well as protective mechanisms within the human body [1]. Research has shown that agonists that target certain cell receptors can protect against neuronal damage. Some of these agonists specifically bind to the Adenosine A3 Receptor (associated with the gene identified with GeneCards Symbol: AD0RA3) [2,2,4]. The level of expression of these receptors can vary in the human body based on physiological conditions and may have baseline levels of expression that differ in different individuals or groups of individuals.

[0098] The current example characterizes variations in gene expression for AD0RA3 measured in blood samples collected from patients who enter the emergency room with symptoms that raise suspicion for stroke. These symptoms typically trigger an alert to mobilize neurology personnel to properly assess the patient and confirm if the patient is experiencing a stroke event. Ischemic stroke specifically is caused by disruption of the blood supply to the brain because of some blockage that affects the vessels that carry blood to the brain tissue. Lacking oxygen and nutrients, brain cells begin to experience stress and are susceptible to damage that can lead to cell death.

[0099] Gene expression measured in blood can act as a surrogate for the level of expression of various receptors on cell surfaces that may respond to treatments designed to trigger protective mechanisms or stop damaging mechanisms within the cell. To this end, measuring the level of expression before, during or after such treatment can be useful to help diagnose the patient with respect to requiring the treatment, determining the level of treatment necessary and monitoring the effect of the treatment.

[0100] The gene expression level for AD0RA3 was specifically found to be different for patients who were confirmed to have a stroke versus patients with other conditions that have similar symptoms when measured immediately after a code stroke alert (Figure 1). Further, the level of gene expression changed significantly for patients who experienced a stroke in a subsequent blood draw obtained 24 hours after the onset of their symptoms. This change was not observed in patients later confirmed to have other conditions. This supports the fact that the expression of AD0RA3 changed in the hyperacute stage in patients with stroke (Figure 1 and Table 2).

[0101] The AD0RA3 expression patterns were also found to vary by age for male versus female patients and for different racial groups (Figure 2) showing that changes in expression of the receptor explain some of the known differences in the risk for stroke as well as the outcome for the disease among racial and demographic groups. Understanding these differences and providing means to measure the expression level of AD0RA3 for each patient can lead to more informed and more precise treatment for each patient.

[0102] For the system to improve precision of diagnosis in neurovascular and related conditions that can cause neurological damage to the central nervous system, the method comprises:

[0103] 1- Taking a human fluid sample.

[0104] 2- Targeting mRNA for a receptor of interest from blood representing transcripts (for example, AD0RA3, AD0RA2B, AD0RA2A, or ADRB2) for the purpose of estimating abundance of transcript.

[0105] 3- Targeting mRNA from blood representing a control transcript for the purpose of determining the abundance of control.

[0106] 4- Providing measurement(s) derived from abundance of transcripts to a medical professional for the purpose of improving the precision of predicting, diagnosing or affecting the type, frequency or amount of treatment required for the condition. Example neurovascular conditions include common and rare conditions including stroke, or Moyamoya disease.

[0107] 5- Providing a reference for interpreting the measurements where the measurements may be interpreted alone, or in the context of other information including patient gender, age, race, body weight, fat composition, prior neurological injury or illness.

[0108] 6- Measurements can be interpreted with respect to the time from onset of the neurological symptoms.

[0109] 7- The receptor of interest is a receptor with agonists or antagonists that can have a neuroprotective or damaging effect, respectively, on the central nervous system after neurological injury (e.g. AD0RA3, AD0RA2B, AD0RA2A, Adrenoceptor Beta 2: ADRB2). Example 2. ADORA3 Agonists and Adenosine Receptor Agonists and Antagonists

[0110] Specific AD0RA3 agonists include:

[0111] AST-004, which binding data indicated was an adenosine A3 receptor (A3R) agonist [6]. Adenosine A3 receptor (A3R) agonist AST-004 was synthesized, in part, at the National Institute of Health (Bethesda, MD), as previously reported [7] and in part, was a gift of Astrocyte Pharmaceuticals, Inc. Unbound brain concentrations of AST-004 were determined following dosing using a specific LC / MS / MS assay [7]. Maximum unbound brain concentrations were measured at 5 ng / gm, demonstrating that AST-004 was distributed in the brain and available to interact with A3R.

[0112] Additional compounds with a measure of their affinity to various adenosine receptors are listed in Table 1 (Table adapted from Reference 4 (below)).

[0113] References

[0114] 1- Shehjar, Faheem et al. “Stroke: Molecular mechanisms and therapies: Update on recent developments.” Neurochemistry international vol. 162 (2023)

[0115] 2- Zheng J, Wang R, Zambraski E, Wu D, Jacobson KA, Liang BT. Protective roles of adenosine Al, A2A, and A3 receptors in skeletal muscle ischemia and reperfusion injury. Am J Physiol Heart Circ Physiol. 2007 Dec;293(6)

[0116] 3- Effendi WI, Nagano T, Kobayashi K, Nishimura Y. Focusing on Adenosine Receptors as a Potential Targeted Therapy in Human Diseases. Cells. 2020 Mar 24 ;9(3)

[0117] 4- Jacobson KA, Tosh DK, Jain S and Gao Z-G Historical and Current Adenosine Receptor Agonists in Preclinical and Clinical Development. Front. Cell. Neurosci. 13:124, (2019)

[0118] 5- Monik C. Jimenez, et al., Racial Variation in Stroke Risk Among Women by Stroke Risk Factors, Stroke 2019-04-01 50(4): 797-804.

[0119] 6- Bozdemir E, Vigil FA, Chun SH, Espinoza L, Bugay V, Khoury SM, Holstein DM, Stoja A, Lozano D, Tunca C, Sprague SM, Cavazos JE, Brenner R, Liston TE, Shapiro MS, Lechlei ter JD. Neuroprotective Roles of the Adenosine A3 Receptor Agonist AST-004 in Mouse Model of Traumatic Brain Injury. Neurotherapeutics. 2021 Oct;18(4):2707-2721

[0120] 7- Theodore E. Liston, PhD, Aldric Hama, PhD, Johannes Boitze, MD, PhD, Russell B. Poe, PhD, Takahiro Natsume, PhD, Ikuo Hayashi, PhD, Hiroyuki Takamatsu, PhD, William S.

[0121] Korinek, PhD, James D. Lechleiter PhD, Adenosine A1R / A3R (Adenosine Al and A3 Receptor) Agonist AST-004 Reduces Brain Infarction in a Nonhuman Primate Model of Stroke, Stroke 2022-01-01 53(1): 238-248

[0122] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference. Those skilled in the art will appreciate that numerous changes and modifications can be made to the preferred embodiments of the invention and that such changes and modifications can be made without departing from the spirit of the invention. It is, therefore, intended that the appended claims cover all such equivalent variations as fall within the true spirit and scope of the invention.

[0123] TABLES

[0124] Table 1. Compounds with a measure of their affinity to various adenosine receptors

Claims

CLAIMSWe claim:

1. A method of treating a subject with a neurovascular disease, comprising: obtaining a biological sample from a subject suspected of having the neurovascular disease immediately after onset of neurovascular disease symptoms; quantifying an expression level of one or more biomarkers, wherein the biomarker comprises one or more of AD0RA3, AD0RA2B, AD0RA2A, or ADRB2; determining the subject as suffering from the neurovascular disease if the expression level of the one or more biomarkers is changed in the subject immediately after onset of neurovascular disease symptoms in comparison to a reference control; and administering to the subject a therapeutically effective amount of an agonist or antagonist of AD0RA3, AD0RA2B, AD0RA2A, or ADRB2, a gene therapy, or a thrombolytic; or performing a surgical procedure.

2. The method of claim 1, wherein the biomarker comprises AD0RA3.

3. The method of claim 1, wherein the biomarker comprises AD0RA2B.

4. The method of claim 1, wherein the biomarker comprises AD0RA2A.

5. The method of claim 1, wherein the biomarker comprises ADRB2.

6. The method of any one of claims 1 to 5, wherein the biological sample comprises a blood sample.

7. The method of any one of claims 1 to 6, wherein the quantifying is carried out by one or a combination of Polymerase Chain Reaction, Real Time-Polymerase Chain Reaction, Real Time Reverse Transcriptase-Polymerase Chain Reaction, Real-time quantitative RT-PCR, Northern blot analysis, in situ hybridization, probe array, or gene sequencing.

8. The method of any one of claims 1 to 7, wherein the reference control is a sample from the subject taken about 24 hours after onset of neurovascular disease symptoms.

9. The method of any one of claims 1 to 8, wherein the reference control is a sample from the subject taken greater than 24 hours after onset of neurovascular disease symptoms.

10. The method of any one of claims 1 to 8, wherein the reference control is a sample from a subject that is not suffering from the neurovascular disease.

11. The method of any one of claims 1 to 8, wherein the reference control is a sample from the subject that is not experiencing neurovascular disease symptoms.

12. The method of any one of claims 1 to 11, wherein the agonist is an ADORA3 agonist.

13. The method of any one of claims 1 to 11, wherein the agonist is an ADORA2B agonist.

14. The method of any one of claims 1 to 11, wherein the agonist is an ADORA2A agonist.

15. The method of any one of claims 1 to 11, wherein the agonist is an ADRB2 agonist.

16. The method of any one of claims 1 to 11, wherein the agonist is selected from an agonist or antagonist listed in Table 1.

17. The method of any one of claims 1 to 11, wherein the agonist is AST-004.

18. The method of any one of claims 1 to 17, wherein the subject is a human.

19. The method of any one of claims 1 to 18, wherein the neurovascular disease is stroke or a traumatic brain injury.

20. The method of any one of claims 1 to 18, wherein the neurovascular disease is Moyamoya Disease.

21. The method of any one of claims 1 to 20, wherein the method further comprises administering to the subject an alternative gene therapy, a thrombolytic therapy, or performing a surgical procedure.

22. A method for diagnosing a subject with a neurovascular disease, comprising: obtaining a biological sample from a subject suspected of having the neurovascular disease immediately after onset of neurovascular disease symptoms; quantifying an expression level of one or more biomarkers immediately after onset of neurovascular disease symptoms, wherein the biomarker comprises one or more of ADORA3, ADORA2B, ADORA2A, or ADRB2; and determining the subject as suffering from the neurovascular disease if the expression level of the one or more biomarkers is changed in the subject immediately after onset of neurovascular disease symptoms in comparison to a reference control.

23. The method of claim 22, wherein the biomarker comprises ADORA3.

24. The method of claim 22, wherein the biomarker comprises ADORA2B.

25. The method of claim 22, wherein the biomarker comprises ADORA2A.

26. The method of claim 22, wherein the biomarker comprises ADRB2.

27. The method of any one of claims 22 to 26, wherein the biological sample comprises a blood sample.

28. The method of any one of claims 22 to 27, wherein the quantifying is carried out by one or a combination of Polymerase Chain Reaction, Real Time-Polymerase Chain Reaction, Real Time Reverse Transcriptase-Polymerase Chain Reaction, Real-time quantitative RT-PCR, Northern blot analysis, in situ hybridization, probe array, or gene sequencing.

29. The method of any one of claims 22 to 28, wherein the reference control is a sample from the subject taken about 24 hours after onset of neurovascular disease symptoms.

30. The method of any one of claims 22 to 29, wherein the reference control is a sample from the subject taken greater than 24 hours after onset of neurovascular disease symptoms.

31. The method of any one of claims 22 to 30, wherein the reference control is a sample from a subject that is not suffering from the neurovascular disease.

32. The method of any one of claims 22 to 31 , wherein the reference control is a sample from the subject that is not experiencing neurovascular disease symptoms.

33. The method of any one of claims 22 to 32, further comprising administering an agonist or antagonist of ADORA3, ADORA2B, ADORA2A, or ADRB2.

34. The method of claim 33, wherein the agonist is an ADORA3 agonist.

35. The method of claim 33, wherein the agonist is an ADORA2B agonist.

36. The method of claim 33, wherein the agonist is an ADORA2A agonist.

37. The method of claim 33, wherein the agonist is an ADRB2 agonist.

38. The method of claim 33, wherein the agonist is selected from an agonist or antagonist listed in Table 1.

39. The method of claim 33, wherein the agonist is AST-004.

40. The method of any one of claims 22 to 39, wherein the subject is a human.

41. The method of any one of claims 22 to 40, wherein the neurovascular disease is stroke or traumatic brain injury.

42. The method of any one of claims 22 to 41, wherein the neurovascular disease is Moyamoya Disease.

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