Methods for biomarker identification for diagnosis of neuropsychiatric disorders

Abstract

Schizophrenia (SCZ) is the most debilitating of the Serious Mental Illnesses, a group of disorders affecting 4.8% of Arizona adults. The lack of a biologically based test makes diagnosing SCZ difficult, particularly for the 40% of Arizonans in areas with inadequate mental health care. Even under ideal circumstances, it can take months to years to accurately diagnose SCZ and identify an effective medication regimen. There is a critical need to identify biological markers to rapidly diagnose SCZ, which will lead to faster symptom resolution and improved patient outcomes. Methods described herein capitalize on the long-recognized association between SCZ risk and immune system dysfunction by stimulating peripheral blood immune cells with immunogenic agents to gain insight into disrupted pathways of gene expression in the brain. The immune stimulation of peripheral blood cells produces a unique pattern of gene expression, which will differ between SCZ and healthy control subjects.

Description

UA24-176, ARIZ 24.33 PCT Gallitano-Mendel and Jakobi September 25, 2025METHODS FOR BIOMARKER IDENTIFICATION FOR DIAGNOSIS OF NEUROPSYCHIATRIC DISORDERSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 63 / 699,066 filed September 25, 2024 and U.S. Provisional Application No. 63 / 712,030 filed October 25, 2024, the specifications of which are incorporated herein in their entirety by reference.FIELD OF THE INVENTION

[0002] The present invention provides methods for diagnosing a psychiatric disorder (e.g., a neuropsychiatric disorder), including the identification of biomarkers for use in diagnostic procedures targeting a psychiatric disorder (e.g., a neuropsychiatric disorder).BACKGROUND OF THE INVENTION

[0003] Schizophrenia is one of the most severe psychiatric disorders, affecting nearly 1% of the global population — approximately 59 million people. Despite its prevalence, the exact cause of schizophrenia remains unknown, with both genetic and environmental factors contributing to its complexity. While at least 685 genes are located in regions associated with an increased risk for schizophrenia, none of these genes has been definitively proven to cause the disorder. Environmental influences, such as in-utero exposure to infections, also heighten the risk. Delays in diagnosis and treatment often lead to worse outcomes and a poor long-term prognosis. Untreated or inadequately managed schizophrenia takes an immense toll, not only on the individual but also on their family and society as a whole. Individuals with schizophrenia face a drastically shortened lifespan — on average by 28.5 years — and nearly one in ten die by suicide. Additionally, the economic burden is significant, with lifetime costs of $3.8 million per person diagnosed at age 25, or roughly $92,000 per year. In response to the immense impact of schizophrenia, identifying biomarkers for the diagnosis of mental illnesses is a key priority.BRIEF SUMMARY OF THE INVENTION

[0004] It is an objective of the present invention to provide systems, compositions, and methods that allow for accurately diagnosing schizophrenia, as specified in the independent claims. Embodiments of the invention are given in the dependent claims. Embodiments of the present invention can be freely combined with each other if they are not mutually exclusive.

[0005] Currently, there are no biomarkers for schizophrenia or any mental illness, largelyUA24-176, ARIZ 24.33 PCTGallitano-Mendel and JakobiSeptember 25, 2025 because the affected organ — the brain — is inaccessible for direct evaluation. Diagnosis is based solely on a clinical interview conducted by a psychiatrist, which often requires months of waiting for an initial appointment. This process is imprecise, as psychiatric disorders are defined entirely by their symptoms, and many symptoms overlap across different conditions. Moreover, schizophrenia itself likely consists of several subtypes that are not yet biologically distinguishable, and these subtypes may respond to different treatments. Consequently, it can take months, or even years, to accurately diagnose an individual and establish an effective medication regimen.

[0006] The present invention features a blood-based biomarker assay that identifies functional abnormalities in genes contributing to the pathology of schizophrenia rather than merely detecting markers associated with illness risk. By utilizing peripheral blood, an accessible tissue, the assay employs an innovative approach: immunogenic stimulation of white blood cells in vitro to mimic environmental risk factors for schizophrenia and uncover gene expression abnormalities critical for brain function. A cutting-edge analytic approach enhances statistical power, significantly reducing the number of subjects required to detect differences between schizophrenia and control groups. Additionally, within-subject analysis minimizes the number of genes queried, while pathway analysis captures multiple genes within relevant biological processes, further boosting statistical strength. The integration of machine learning and artificial intelligence (Al) allows for the precise identification of distinct biological differences between groups. This novel solution offers significant benefits, providing biologically based diagnoses that will improve access to treatment, reduce delays, and enable personalized therapies for individuals with schizophrenia. It may also help identify at-risk individuals, allowing for early, potentially preventative interventions, and facilitating the discovery of novel therapeutics targeting dysfunctional biological pathways.

[0007] In some embodiments, the present invention features a method comprising obtaining or having obtained a biological sample from a subject, incubating a first portion of the biological sample obtained with a solution comprising a stimulus, and measuring biomarker levels in the first portion of the biological sample and a second portion of the biological sample. In some embodiments, the method further comprises comparing biomarker levels in the first portion of the biological sample to a second portion of the biological sample. In some embodiments, the subject is diagnosed with a psychiatric disorder. In some embodiments, the second portion of the biological sample is incubated with a control solution.UA24-176, ARIZ 24.33 PCTGallitano-Mendel and JakobiSeptember 25, 2025

[0008] The aforementioned method may further comprise, in some embodiments, obtaining or having obtained a biological sample from a healthy subject; incubating a first portion of the biological sample obtained from the healthy subject with a solution comprising a stimulus; and measuring biomarker levels in the first portion of the biological sample from the healthy subject and a second portion of the biological sample from the healthy subject. In some embodiments, the second portion of the biological sample is incubated with a control solution. In certain embodiments, the method further comprises comparing biomarker levels in the first portion of the subject diagnosed with the psychiatric disorder to biomarker levels in the first portion of a healthy control subject. Alternatively, the method may further comprise identifying biomarkers that differ between the first and second portions of the subject diagnosed with the psychiatric disorder, and comparing those biomarkers to corresponding biomarkers identified between the first and second portions of the healthy control subject.

[0009] In other embodiments, the present invention features a method of identifying one or more biomarkers for diagnosing a psychiatric disorder (e.g., a neuropsychiatric disorder). The method may comprise obtaining or having obtained a biological sample from a subject diagnosed with psychiatric disorders, incubating or having incubated a first portion of the biological sample obtained to a solution comprising a stimulus, and analyzing gene expression from the first stimulated portion of the biological sample and a second non-stimulated portion of the biological sample. In some embodiments, genes that are differentially expressed between the first stimulated portion of the biological sample and the second non-stimulated portion are identified as biomarkers. Additionally, in some embodiments, the method may further comprise obtaining or having obtained a biological sample from a healthy control subject, incubating or having incubated a first portion of the biological sample obtained from the healthy control subject to a solution comprising a stimulus, and analyzing gene expression from the first stimulated portion of the biological sample from the healthy control and a second non-stimulated portion of the biological sample from the healthy control. In certain embodiments, biomarkers are identified by comparing genes differentially expressed between the first stimulated portion and a second non-stimulated portion of a biological sample from a subject diagnosed with the psychiatric disorder and comparing the resulting genes to those differentially expressed between the first stimulated portion and a second non-stimulated portion of a biological sample from a healthy control subject.

[0010] In certain embodiments, the present invention features a method of identifying one orUA24-176, ARIZ 24.33 PCTGallitano-Mendel and JakobiSeptember 25, 2025 more biomarkers for diagnosing schizophrenia. Referring to FIG. 1, in some embodiments, the method comprises a) obtaining or having obtained a blood sample from a subject diagnosed with schizophrenia, b) obtaining or having obtained a blood sample from a healthy control subject, c) individually, subjecting or having subjected a first portion of the blood samples obtained from a) and b) to a solution comprising an immunogenic stimulus, d) individually, subjecting or having subjected a second portion of the blood samples obtained from a) and b) to a control solution; and e) analyzing gene expression from each sample from c) and d). In some embodiments, the method further comprises isolating or having isolated white blood cells from each blood sample obtained. In certain embodiments, the method further comprises freezing each of the samples obtained and thawing each sample before use. In some embodiments, the method further comprises extracting RNA from the samples subjected to either the solution that comprises the immunogenic stimulus or the control solution.

[0011] One of the unique and inventive technical features of the present invention is the use of an immunogenic stimulus. Without wishing to limit the invention to any theory or mechanism, it is believed that the technical feature of the present invention advantageously enables the expression of genes typically not expressed in unstimulated blood cells. By stimulating these cells, it is possible to induce the expression of other genes, such as those normally expressed in the brain, which may be linked to mental illness. None of the presently known prior references or works have the unique, inventive technical feature of the present invention.

[0012] Moreover, the prior references teach away from the present invention by emphasizing current approaches that have proven inadequate. For instance, one common method is the Polygenic Risk Score, which measures the number of genetic markers associated with an increased risk for schizophrenia. However, this approach fails to identify variations that alter gene function and has not demonstrated effectiveness for diagnosis or risk prediction. Another approach, pharmacogenomic testing, assesses an individual's genotype for enzymes involved in drug metabolism to guide medication selection, such as determining whether a person is a slow or fast metabolizer of specific drugs. However, this testing does not address the underlying illness or contribute to an accurate diagnosis. These limitations highlight the need for improved methods that go beyond current practices.

[0013] Moreover, the prior references teach away from the present invention. For example, previous studies in the field of blood-based gene expression comparing schizophrenia (SCZ) patients to healthy control (HC) subjects have struggled to demonstrate the reproducibility andUA24-176, ARIZ 24.33 PCT Gallitano-Mendel and Jakobi September 25, 2025 specificity required for reliable biomarkers. In contrast, the present invention features a unique and innovative approach. In certain embodiments, the present invention features immune stimulation that a) mimics environmental risk factors associated with SCZ, b) induces transcription to facilitate the detection of functional gene abnormalities, and c) enhances statistical power by enabling within-subject analyses. This comprehensive strategy allows for a more nuanced understanding of the biological underpinnings of schizophrenia and holds promise for developing effective biomarkers.

[0014] Furthermore, the inventive technical features of the present invention contributed to a surprising result. Specifically, changes were identified in the expression of genes that play critical roles in brain processes, such as synaptic plasticity, and are associated with schizophrenia risk, using cells derived from peripheral blood. This finding is highly innovative and surprising, not only to the general public but also to clinicians and scientists in the field.

[0015] Any feature or combination of features described herein are included within the scope of the present invention provided that the features included in any such combination are not mutually inconsistent as will be apparent from the context, this specification, and the knowledge of one of ordinary skills in the art. Additional advantages and aspects of the present invention are apparent in the following detailed description and claims.BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0016] The features and advantages of the present invention will become apparent from a consideration of the following detailed description presented in connection with the accompanying drawings in which:

[0017] FIG. 1 shows a non-limiting example of a method described herein.

[0018] FIG. 2A shows the stimulation of blood cells, reveals gene expression biomarkers between SCZ and HC subjects. FIG. 2B shows that the approach described herein identifies differences in brain development and plasticity gene pathways from a blood sample. As described herein, the present invention identifies brain & neuronal biomarkers from a sample of blood, demonstrating the feasibility and effectiveness of the approach to identify biomarkers for validation.DETAILED DESCRIPTION OF THE INVENTION

[0019] Disclosed are various peptides, solvents, solutions, carriers, and / or components to be usedUA24-176, ARIZ 24.33 PCTGallitano-Mendel and JakobiSeptember 25, 2025 to prepare compositions to be used within the methods disclosed herein. Also disclosed are the various steps, elements, amounts, routes of administration, symptoms, and / or treatments that are used or observed when performing the disclosed methods, as well as the methods themselves. These and other materials, steps, and / or elements are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed, while specific reference of each various individual and collective combination and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0020] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which a disclosed invention belongs. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The term "comprising" means that other elements can also be present in addition to the defined elements presented. The use of "comprising" indicates inclusion rather than limitation. Stated another way, the term "comprising" means "including principally, but not necessary solely". Furthermore, variation of the word "comprising", such as "comprise" and "comprises", have correspondingly the same meanings. In one respect, the technology described herein related to the herein described compositions, methods, and respective component(s) thereof, as essential to the invention, yet open to the inclusion of unspecified elements, essential or not ("comprising").

[0021] Suitable methods and materials for the practice and / or testing of embodiments of the disclosure are described below. Such methods and materials are illustrative only and are not intended to be limiting. Other methods and materials similar or equivalent to those described herein can be used. For example, conventional methods well known in the art to which the disclosure pertains are described in various general and more specific references, including, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989; Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Press, 2001; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates, 1992 (and Supplements to 2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 4th ed., Wiley & Sons, 1999; Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringUA24-176, ARIZ 24.33 PCTGallitano-Mendel and JakobiSeptember 25, 2025Harbor Laboratory Press, 1990; and Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999, Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, Calif.), "Guide to Protein Purification” in Methods in Enzymology (M. P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, Calif.), Culture of Animal Cells: A Manual of Basic Technique, 2nd Ed. (R. I. Freshney. 1987. Liss, Inc. New York, N.Y.), Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J. Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 1998 Catalog (Ambion, Austin, Tex.), the disclosures of which are incorporated in their entirety herein by reference.

[0022] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety for all purposes. In case of conflict, the present specification, including explanations of terms, will control.

[0023] Although methods and materials similar or equivalent to those described herein can be used to practice or test the disclosed technology, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting.

[0024] As used herein, the terms “subject” and “patient” are used interchangeably. As used herein, a subject can be a mammal such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkey and human). In specific embodiments, the subject is a human. In one embodiment, the subject is a mammal (e.g., a human) having a disease, disorder, or condition described herein. In another embodiment, the subject is a mammal (e.g., a human) at risk of developing a disease, disorder, or condition described herein. In certain instances, the term patient refers to a human.

[0025] As used herein, the terms "treat," “treating,” or "treatment" refer to both therapeutic treatment and prophylactic or preventative measures, with the objective of preventing, reducing, slowing down (lessening), inhibiting, or eliminating an undesired physiological change, symptom, disease, or disorder. For example, the disease may be schizophrenia. For purposes of this invention, beneficial or desired clinical results include but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can alsoUA24-176, ARIZ 24.33 PCT Gallitano-Mendel and Jakobi September 25, 2025 mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented or onset delayed. Optionally, the subject or patient may be identified (e.g., diagnosed) as one suffering from the disease or condition prior to administration of the compositions of the invention. Subjects at risk for the disease can be identified by, for example, any or a combination of appropriate diagnostic or prognostic assays known in the art.

[0026] As used herein, “clinical improvement” may refer to a noticeable reduction in the symptoms of a disorder, or cessation thereof.

[0027] The terms “manage,” “managing,” and “management” refer to preventing or slowing the progression, spread or worsening of a disease or disorder, or of one or more symptoms thereof. In certain cases, the beneficial effects that a subject derives from a prophylactic or therapeutic agent do not result in a cure of the disease or disorder.

[0028] As used herein, a “biological sample” or “clinical sample” may be used interchangeably and may refer to any biological material taken from a subject. Non-limiting examples of a biological material to be taken for a biological sample may include, but are not limited to, saliva, blood, or urine, and nasopharyngeal specimens.

[0029] As used herein, the term “biomarker” refers to a bio-marker, or biological marker, which is a measurable indicator of some biological state or condition. Biomarkers are often measured and evaluated to examine normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention. Biomarkers are used in many scientific fields and can be derived from any tissue or fluid, including blood.

[0030] As used herein, “control” or “vehicle control” may be used interchangeably and may refer to a solution identical in composition to the solution containing the stimulus (e.g., an immunogenic stimulus), except that the stimulus is absent. For example, when the stimulus is diluted in the cell culture medium, the control is the same medium without the stimulus.

[0031] As used herein, the terms “psychiatric disorder” and “neuropsychiatric disorder” may be used interchangeably. The term “neuropsychiatric disorder” refers to brain-related disorders that may include psychiatric manifestations. In addition to traditional psychiatric disorders, this term encompasses neurodevelopmental disorders, such as autism spectrum disorder, and neurologicalUA24-176, ARIZ 24.33 PCT Gallitano-Mendel and Jakobi September 25, 2025 disorders, such as Parkinson’s disease.

[0032] Referring now to FIGs. 1-2A, the present invention features methods of identifying one or more biomarkers for diagnosing a psychiatric disorder (e.g., schizophrenia) as well as methods for diagnosis and treatment of psychiatric disorders (e.g., schizophrenia), e.g., using the identified biomarker.

[0033] In some embodiments, the purpose of this invention is to elucidate biomarkers for a psychiatric disorder (e.g., schizophrenia). Without wishing to limit the present invention to any theory or mechanism, it is believed that the methods described herein may lead to the development of blood-based diagnostic tests that both distinguish schizophrenia from other disorders and define subtypes of schizophrenia. Such discoveries are critical for rapid and accurate diagnosis and the development of effective and personalized treatments for the range of illnesses that comprise the schizophrenia diagnosis. Since the longer the duration of untreated psychosis, the worse the long-term prognosis is for persons suffering from schizophrenia, the methods herein may improve patient outcomes. Moreover, the identification of biomarkers for schizophrenia creates the possibility of identifying high-risk individuals prior to full symptom onset, thereby providing the possibility of preventative interventions.

[0034] Without wishing to limit the present invention to any theory of mechanism, it is believed that while there are no currently recognized biologically distinguishable subtypes, research suggests the existence of subtypes based on individual genetic variations or other biological characteristics. Recent studies of post-mortem brain gene expression, for example, have begun to reveal such differences.

[0035] Thus, in some embodiments, the methodologies described herein may also be applied to identify specific subtypes of a psychiatric or neuropsychiatric disorder, such as schizophrenia. In certain embodiments, biomarkers identified using the methods described herein may be further utilized in personalized medicine applications, including stratifying patients by subtype, guiding treatment selection, or predicting therapeutic response.

[0036] The present invention may feature a method comprising obtaining or having obtained a biological sample from a subject, incubating a first portion of the biological sample obtained with a solution comprising a stimulus, and measuring biomarker levels in the first portion of the biological sample and a second portion of the biological sample. In some embodiments, theUA24-176, ARIZ 24.33 PCTGallitano-Mendel and JakobiSeptember 25, 2025 method further comprises comparing biomarker levels in the first portion of the biological sample to a second portion of the biological sample. In some embodiments, the subject is diagnosed with a psychiatric disorder. In some embodiments, the second portion of the biological sample is incubated with a control solution.

[0037] The aforementioned method may further comprise, in some embodiments, obtaining or having obtained a biological sample from a healthy subject; incubating a first portion of the biological sample obtained from the healthy subject with a solution comprising a stimulus; and measuring biomarker levels in the first portion of the biological sample from the healthy subject and a second portion of the biological sample from the healthy subject. In some embodiments, the second portion of the biological sample is incubated with a control solution. In certain embodiments, the method further comprises comparing biomarker levels in the first portion of the subject diagnosed with the psychiatric disorder to biomarker levels in the first portion of a healthy control subject. Alternatively, the method may further comprise identifying biomarkers that differ between the first and second portions of the subject diagnosed with the psychiatric disorder, and comparing those biomarkers to corresponding biomarkers identified between the first and second portions of the healthy control subject.

[0038] The present invention may also feature methods of identifying one or more biomarkers for diagnosing a psychiatric disorder (e.g., a neuropsychiatric disorder). In some embodiments, the method comprises a) obtaining or having obtained a biological sample from a subject diagnosed with a psychiatric disorder, subjecting or having subjected a first portion of the biological samples obtained to a solution comprising a stimulus (e.g., an immunogenic stimulus), and analyzing gene expression from the first stimulated portion of the biological sample and a second non-stimulated portion of the biological sample. In some embodiments, genes that are differentially expressed between the first stimulated portion of the biological sample and the second non-stimulated portion are identified as biomarkers. The method may further comprise obtaining or having obtained a biological sample from a healthy control subject, incubating or having incubated a first portion of the biological sample obtained from the healthy control subject to a solution comprising a stimulus, and analyzing gene expression from the first stimulated portion of the biological sample from the healthy control and a second non-stimulated portion of the biological sample from the healthy control. In some embodiments, the second portion of either biological sample is incubated with a control solution.

[0039] In some embodiments, the method for identifying one or more biomarkers for diagnosingUA24-176, ARIZ 24.33 PCTGallitano-Mendel and JakobiSeptember 25, 2025 psychiatric disorder comprises obtaining or having obtained a biological sample from a subject diagnosed with a psychiatric disorder and a healthy control subject, individually incubating or having incubated a first portion of the biological samples obtained from the subject diagnosed with a psychiatric disorder and the healthy control subject with a solution comprising a stimulus, and analyzing gene expression from each biological sample from. In some embodiments, the second portion of either biological sample is incubated with a control solution.

[0040] The aforementioned methods may further comprise identifying biomarkers that differ between the first and second portions of the subject diagnosed with the psychiatric disorder (e.g., a neuropsychiatric disorder), and comparing those biomarkers to corresponding biomarkers identified between the first and second portions of the healthy control subject. In some embodiments, the identified biomarkers comprise genes whose expression levels differ between subjects with a psychiatric disorder (e.g., a neuropsychiatric disorder), and healthy controls. In certain embodiments, the expression of these genes is increased in subjects with the disorder relative to controls, while in other embodiments, the expression is decreased.

[0041] In some embodiments, biomarkers are identified using the methods described above by comparing genes differentially expressed between the first stimulated portion and a second non-stimulated portion of a biological sample from a subject diagnosed with the psychiatric disorder and comparing the resulting genes to those differentially expressed between the first stimulated portion and a second non-stimulated portion of a biological sample from a healthy control subject. In certain embodiments, the aforementioned methods further comprise comparing biomarker levels in the first portion of the biological sample from the subject diagnosed with the psychiatric disorder to biomarker levels in the first portion of the biological sample from the healthy control subject.

[0042] In alternate embodiments, the method comprises obtaining or having obtained a first biological sample from a subject diagnosed with a psychiatric disorder during a period of illness (e.g., viral or bacterial infection). In such embodiments, a second biological sample may be obtained from the same subject when the subject is healthy to serve as a control. The same approach may also be applied to a healthy subject for control comparisons.

[0043] Non-limiting examples of psychiatric disorders or neuropsychiatric disorders that may be identified using methods described herein include but are not limited to bipolar disorder, major depressive disorder, schizoaffective disorder, obsessive-compulsive disorder, personalityUA24-176, ARIZ 24.33 PCTGallitano-Mendel and JakobiSeptember 25, 2025 disorders, anxiety disorders, and substance dependence disorders. Also neurocognitive disorders, including autism spectrum disorder and neurodegenerative disorders such as Alzheimer's disease, and other neurological disorders, such as Parkinson’s Disease, may also be identified.

[0044] In certain embodiments, the present invention may feature methods of identifying one or more biomarkers for diagnosing schizophrenia. In some embodiments, the method comprises obtaining or having obtained a biological sample from a subject diagnosed with schizophrenia, subjecting or having subjected a first portion of the biological samples obtained to a solution comprising a stimulus (e.g, an immunogenic stimulus), and analyzing gene expression from the first stimulated portion of the biological sample and a second non-stimulated portion of the biological sample. In some embodiments, genes that are differentially expressed between the first stimulated portion of the biological sample and the second non-stimulated portion are identified as biomarkers. The method may further comprise obtaining or having obtained a biological sample from a healthy control subject, incubating or having incubated a first portion of the biological sample obtained from the healthy control subject to a solution comprising a stimulus, and analyzing gene expression from the first stimulated portion of the biological sample from the healthy control and a second non-stimulated portion of the biological sample from the healthy control. In some embodiments, the second portion of either biological sample is incubated with a control solution. In some embodiments, the method further comprises identifying biomarkers that differ between the first and second portions of the subject diagnosed with schizophrenia, and comparing those biomarkers to corresponding biomarkers identified between the first and second portions of the healthy control subject.

[0045] In some embodiments, biomarkers are identified using the methods described above by comparing genes differentially expressed between the first stimulated portion and a second non-stimulated portion of a biological sample from a subject diagnosed with schizophrenia and comparing the resulting genes to those differentially expressed between the first stimulated portion and a second non-stimulated portion of a biological sample from a healthy control subject. In certain embodiments, the aforementioned methods further comprise comparing biomarker levels in the first portion of the biological sample from the subject diagnosed with schizophrenia with those in the first portion of the biological sample from the healthy control subject.

[0046] In related embodiments, biological samples (e.g., blood samples, including the PBMC fraction) may be collected from the same individual (e.g., a subject with schizophrenia) atUA24-176, ARIZ 24.33 PCT Gallitano-Mendel and Jakobi September 25, 2025 different clinical states, such as during symptomatic and asymptomatic periods and / or while on and off medication. Gene expression differences between these samples may reveal genes whose expression is similarly altered in the brain and may serve as biomarkers for schizophrenia or for a particular subtype of the disorder in that individual.

[0047] In some embodiments, the obtained samples may be incubated for about 12 hours. In some embodiments, the obtained samples may be incubated for about 24 hours. In some embodiments, the obtained samples may be incubated for about 48 hours. The present invention is not limited to these incubation times, and other durations may be used as appropriate.

[0048] Any of the aforementioned methods may further comprise isolating white blood cells from each obtained sample. In certain embodiments, the method further comprises freezing the obtained samples and subsequently thawing them prior to use. In additional embodiments, the method further comprises extracting RNA from the samples that have been subjected to either the solution containing the stimulus or the control solution.

[0049] In some embodiments, the present invention may feature a Microarray “Chip” to provide a quantitative readout of genes comprising key biological processes validated to distinguish schizophrenia subjects from controls. In certain embodiments, the present invention may also include a method involving the isolation of Peripheral Blood Mononuclear Cells (PBMCs), immunogenic stimulation, RNA extraction, chip-based assay analysis, and subsequent reporting of results.

[0050] In some embodiments, the stimulus is an immunogenic stimulus. In some embodiments, the immunogenic stimulus comprises lipopolysaccharide (LPS). In some embodiments, the immunogenic stimulus comprises polyinosinic-polycytidylic acid (Poly I:C). In some embodiments, the immunogenic stimulus comprises viruses (e.g., influenza virus, Epstein-Barr virus, Herpes simplex viruses, cytomegalovirus, norovirus, coronaviruses including SARS-CoV-2), and other bacterial stimuli. In other embodiments, the immunogenic stimulus comprises hormones, such as hormones released in response to stress (e.g., cortisol) or starvation, both states that are associated with increased risk for schizophrenia, particularly in utero exposure, and hormones and hormone mimics that are increased in the environment, e.g., chemical hormone disruptors. Other circulating factors present in people exposed to stress or starvation, such as ketone bodies and altered free fatty acids, may be used as immunogenic stimuli.UA24-176, ARIZ 24.33 PCT Gallitano-Mendel and Jakobi September 25, 2025

[0051] In some embodiments, the stimulus is an environmental factor, such as a chemical, drug, pesticide, component of air pollution, or industrial contaminant. In some embodiments, the stimulus is a physical condition that mimics a disease-associated state, such as incubation at an elevated temperature to simulate fever.

[0052] The present invention is not limited to the aforementioned stimuli and may include any other stimuli that may now or in the future be associated with an increased risk of a psychiatric disorder (e.g., schizophrenia), neurodevel opmental disorder, or neurodegenerative disorder.

[0053] In some embodiments, the biological sample comprises a blood sample. In some embodiments, the blood sample comprises peripheral blood mononuclear cells (white blood cells), red blood cells, plasma, and serum. In other embodiments, the biological sample comprises extracellular vesicles (EVs) that may be collected from a blood sample, e.g., from plasma, or alternatively, EVs may be released from blood cells in vitro. EVs are particles that bud off of, or are otherwise released from, cells and contain proteins, nucleic acids (e.g., DNA & RNA), and other cellular components. The contents of EVs can be compared from a single subject under different conditions, such as following an immunogenic stimulus, or other conditions described above and / or below. In other embodiments, the biological sample comprises skin fibroblasts. In some embodiments, the biological sample comprises buccal mucosal cells. In some embodiments, the biological sample comprises bone marrow.

[0054] In certain embodiments, cells from the biological sample may be isolated and subsequently transformed in vitro into pluripotent stem cells, brain cell precursors (including neuronal and other brain cell precursors), or even fully differentiated neurons and other brain cell types.

[0055] The aforementioned methods may further comprise obtaining or having obtained a biological sample from a healthy control subject. Additionally, in some embodiments, the method further comprises subjecting or having subjected a first portion of the biological samples obtained from the healthy control subject to a solution comprising an immunogenic stimulus and / or subjecting or having subjected a second portion of the biological samples obtained from the healthy control subject to a control solution.

[0056] In other embodiments, the method comprises a) obtaining or having obtained a biological sample from a subject diagnosed with schizophrenia, b) obtaining or having obtained a biologicalUA24-176, ARIZ 24.33 PCT Gallitano-Mendel and Jakobi September 25, 2025 sample from a healthy control subject, c) individually subjecting or having subjected a first portion of the biological samples obtained from a) and b) to a solution comprising an immunogenic stimulus; and d) analyzing gene expression from each sample from c). In some embodiments, the biological sample comprises a blood sample, e.g., comprising peripheral blood mononuclear cells (white blood cells). The method may further comprise subjecting or having subjected a second portion of the biological samples obtained from a) and b) to a control solution and analyzing gene expression from each of the biological samples obtained from a) and b) subjected to the control solution.

[0057] In some embodiments, gene expression in biological samples (e.g., blood samples) obtained from a subject diagnosed with schizophrenia is analyzed by comparing a first portion of the sample treated with a solution comprising an immunogenic stimulus to a second portion treated with a control solution. Additionally, in some embodiments, gene expression in biological samples (e.g., blood samples) obtained from a healthy control subject is analyzed by comparing a first portion of the sample treated with a solution comprising an immunogenic stimulus to a second portion treated with a control solution. After comparing gene expression between samples treated with a solution containing an immunogenic stimulus and a control solution, gene expression is further compared between the samples obtained from a subject diagnosed with schizophrenia and those from control subjects.

[0058] In some embodiments, gene expression may comprise RNA expression or protein expression. In some embodiments, RNA expression is not limited to mRNA expression and may further include rRNAs, circRNA, and long non-coding RNAs. In other embodiments, gene expression may comprise measurements of intracellular or extracellular molecules and proteins, such as cytokines, hormones, or other products of the cell, including extracellular vesicles.

[0059] EXAMPLE 1

[0060] The following is a non-limiting example of how the methods of the present invention may be used to identify one or more biomarkers for diagnosing a psychiatric disorder (e.g., Schizophrenia (SCZ)). It is to be understood that said example is not intended to limit the present invention in any way. Equivalents or substitutes are within the scope of the present invention.

[0061] In some embodiments, the methods described herein involve a two-stage process. The first stage demonstrates feasibility and proof of concept, leading to the identification of candidate biomarkers for validation. In the second stage, these candidate biomarker pathways are validatedUA24-176, ARIZ 24.33 PCTGallitano-Mendel and JakobiSeptember 25, 2025 using an RNA sequencing approach established in the first stage to confirm differentially expressed gene pathways (DEGPs) that differentiate neuropsychiatric disorders from control subjects.

[0062] In this example, three putative biomarker genes were identified in a pilot study involving three subjects with schizophrenia (SCZ) and three healthy controls using the methods described herein. Briefly, blood samples were collected and peripheral blood mononuclear cells (PBMCs) were isolated and cryopreserved until use. At the time of the experiment, the PBMC samples were thawed, live cells were counted microscopically, and equivalent numbers of cells were suspended in cell culture media containing either (1) Poly I:C (immunogenic stimulus), (2) LPS (immunogenic stimulus), or (3) culture media alone (control). The cells were then plated and incubated at 37°C for about 24 hours. Following incubation, cells were collected and lysed, and RNA was isolated for subsequent RNA sequencing.

[0063] RNA sequencing data were analyzed by comparing gene expression between Poly FC-treated and control samples and LPS-treated and control samples, within each group (e.g., SCZ and healthy controls). Differences in biomarkers identified in each group were then compared between the SCZ and control groups. In certain embodiments, gene expression may also be directly compared between Poly I:C and LPS conditions. However, without wishing to limit the present invention to any theory or mechanism, it is believed to be advantageous to first compare each stimulated sample to its corresponding control within the SCZ group and the healthy control group, and then compare the resulting sets of differentially expressed genes between the groups.

[0064] The identified biomarker genes were those induced by the immunogenic stimulus (e.g., Poly I:C or LPS) when comparing stimulated versus unstimulated (control) samples within each group (e.g., SCZ and healthy controls) in a manner that differed between SCZ samples and healthy control samples. For example, the biomarker genes included ARHGAP44 (Rho GTPase activating protein 44), SLC9B1 (solute carrier family 9 member Bl), and CATSPER2P1 (transcribed_unprocessed_pseudogene). These genes exhibited significantly different expression levels between SCZ and control subjects, with expression either increased or decreased in SCZ relative to controls. In some embodiments, the identified biomarker genes may be further validated.

[0065] In certain embodiments, the methodology may be applied to a larger cohort, such as 15UA24-176, ARIZ 24.33 PCT Gallitano-Mendel and Jakobi September 25, 2025 subjects with schizophrenia and 15 matched healthy controls, with RNA sequencing conducted for comprehensive analysis. Additionally, a microarray chip may be designed to quantify the expression levels of validated genes that define gene pathways, distinguishing schizophrenia from control subjects.

[0066] EXAMPLE 2

[0067] The following is a non-limiting example of how the methods of the present invention may be used to identify one or more biomarkers for diagnosing Parkinson’s Disease (PD). It is to be understood that said example is not intended to limit the present invention in any way. Equivalents or substitutes are within the scope of the present invention.

[0068] Blood samples are collected from subjects diagnosed with Parkinson’s disease and healthy controls. Peripheral blood mononuclear cells (PBMCs) are isolated and cryopreserved until use. At the time of the experiment, PBMCs are thawed, live cells are counted microscopically, and equal numbers of cells are suspended in cell culture medium containing (1) a pesticide (stimulus) or (2) culture medium alone (control). The cells are plated and incubated at 37°C for approximately 24 hours, after which they are collected, lysed, and subjected to RNA extraction for sequencing.

[0069] RNA sequencing data are analyzed by comparing gene expression between pesticide-treated and control samples within each group (Parkinson’s disease and healthy controls). Differentially expressed genes identified within each group are then compared between the Parkinson’s disease and control groups to identify candidate biomarkers. Candidate biomarkers may include genes whose expression levels differ significantly between PD and control subjects, with expression either increased or decreased in PD compared to controls.

[0070] EMBODIMENTS

[0071] The following embodiments are intended to be illustrative only and not to be limiting in any way.

[0072] Embodiment 1: A method comprising: a) obtaining or having obtained a biological sample from a subject; b) incubating a first portion of the biological sample obtained with a solution comprising a stimulus; and c) measuring biomarker levels in the first portion of the biological sample and a second portion of the biological sample. In some embodiments, the method further comprises comparing biomarker levels in the first portion of the biologicalUA24-176, ARIZ 24.33 PCTGallitano-Mendel and JakobiSeptember 25, 2025 sample to a second portion of the biological sample. Embodiment 2: The method of embodiment 1, wherein the subject is diagnosed with a psychiatric disorder. Embodiment 3: The method of embodiment 1 or embodiment 2, wherein the second portion of the biological sample is incubated with a control solution. Embodiment 4: The method of any one of embodiments 1-3, further comprising: obtaining or having obtained a biological sample from a healthy subject; incubating a first portion of the biological sample obtained from the healthy subject with a solution comprising a stimulus; and measuring biomarker levels in the first portion of the biological sample from the healthy subject and a second portion of the biological sample from the healthy subject. In some embodiments, the method further comprises comparing biomarker levels in the first portion of the biological sample to a second portion of the biological sample. Embodiment 5: The method of embodiment 4, wherein the second portion of the biological sample is incubated with a control solution. Embodiment 6: The method of embodiment 4 or embodiment 5 further comprising comparing biomarker levels in the first portion of the subject diagnosed with the psychiatric disorder to biomarker levels in the first portion of a healthy control subject. Embodiment 7: The method of embodiment 4 or embodiment 5 further comprising identifying biomarkers that differ between the first and second portions of the subject diagnosed with the psychiatric disorder, and comparing those biomarkers to corresponding biomarkers identified between the first and second portions of the healthy control subject.

[0073] Embodiment 8: The method of any one of embodiments 1-7 wherein the biological sample comprises a blood sample. Embodiment 9: The method of embodiment 8, wherein the blood sample comprises peripheral blood mononuclear cells (white blood cells). Embodiment 10: The method of any one of embodiments 1-9, wherein the second portion of the biological sample is incubated with a control solution. Embodiment 11: The method of any one of embodiments 1-10 further comprising isolating or having isolated white blood cells from each sample obtained. Embodiment 12: The method of any one of embodiments 1-11 further comprising freezing each of the samples obtained and thawing each sample obtained before use. Embodiment 13: The method of embodiments 1-12 further comprising extracting RNA from the samples subjected to either the solution that comprises the immunogenic stimulus or the control solution. Embodiment 14: The method of any one of embodiments 1-13, wherein the stimulus is an immunogenic stimulus. Embodiment 15: The method of embodiment 14, wherein the immunogenic stimulus comprises lipopolysaccharide (LPS), polyinosinic-polycytidylic acid (Poly I:C), or a combination thereof. Embodiment 16: The method of embodiment 14, wherein the immunogenic stimuli comprises a virus or bacteria. Embodiment 17: The method of any oneUA24-176, ARIZ 24.33 PCTGallitano-Mendel and JakobiSeptember 25, 2025 of embodiments 1-13, wherein the stimulus is an environmental stimulus (e.g., environmental factors). Embodiment 18: The method of embodiment 17, wherein the environmental stimulus comprises pesticides or pollutants. Embodiment 19: The method of any one of embodiments 1-13, wherein the stimulus is a drug or chemical. Embodiment 20: The method of any one of embodiments 1-13, wherein the stimulus is a drug or chemical.

[0074] Embodiment 21: A method of identifying one or more biomarkers for diagnosing a psychiatric disorder, the method comprises: a) obtaining or having obtained a biological sample from a subject diagnosed with psychiatric disorders; b) incubating or having incubated a first portion of the biological sample obtained from a) to a solution comprising a stimulus; c) analyzing gene expression from the first stimulated portion of the biological sample and a second non-stimulated portion of the biological sample; wherein genes differentially expressed between the first stimulated portion of the biological sample and the second non-stimulated portion of the biological sample are identified as biomarkers. Embodiment 22: The method of embodiment 21, wherein the second portion of the biological sample is incubated with a control solution. Embodiment 23: The method of any one of embodiments 20 or embodiment 21 further comprising obtaining or having obtained a biological sample from a healthy control subject. Embodiment 24: The method of embodiment 23 further comprising: incubating or having incubated a first portion of the biological sample obtained from the healthy control subject to a solution comprising a stimulus; and analyzing gene expression from the first stimulated portion of the biological sample from the healthy control and a second non-stimulated portion of the biological sample from the healthy control. Embodiment 25: The method of embodiment 24, wherein the second portion of the biological sample is incubated with a control solution.

[0075] Embodiment 26: A method of identifying one or more biomarkers for diagnosing psychiatric disorder, the method comprises: a) obtaining or having obtained a biological sample from a subject diagnosed with a psychiatric disorder; b) obtaining or having obtained a biological sample from a healthy control subject; c) individually incubating or having incubated a first portion of the biological samples obtained from a) and b) to a solution comprising a stimulus; and d) analyzing gene expression from each biological sample from c). In some embodiments, biomarkers are identified by comparing genes differentially expressed between the first stimulated portion and a second non-stimulated portion of a biological sample from a subject diagnosed with the psychiatric disorder and comparing the resulting genes to those differentially expressed between the first stimulated portion and a second non-stimulated portion of aUA24-176, ARIZ 24.33 PCTGallitano-Mendel and JakobiSeptember 25, 2025 biological sample from a healthy control subject. Embodiment 27: The method of embodiment 26, further comprising comparing biomarker levels in the first portion of the biological sample from the subject diagnosed with the psychiatric disorder to biomarker levels in the first portion of the biological sample from the healthy control subject. Embodiment 28: The method of embodiment 26 or embodiment 27, further comprising incubating or having incubated a second portion of the biological samples obtained from a) and b) to a control solution. Embodiment 29: The method of embodiment 28, further comprising identifying biomarkers that differ between the first and second portions of the subject diagnosed with the psychiatric disorder, and comparing those biomarkers to corresponding biomarkers identified between the first and second portions of the healthy control subject.

[0076] Embodiment 30: A method of identifying one or more biomarkers for diagnosing a psychiatric disorder, the method comprises: a) obtaining or having obtained a biological sample from a subject diagnosed with a psychiatric disorder; b) obtaining or having obtained a biological sample from a healthy control subject; c) individually, subjecting or having subjected a first portion of the blood samples obtained from a) and b) to a solution comprising an immunogenic stimulus; d) individually, subjecting or having subjected a second portion of the biological samples obtained from a) and b) to a control solution; and e) analyzing gene expression from each biological sample from c) and d). In some embodiments, biomarkers are identified by comparing genes differentially expressed between the first stimulated portion and the second non-stimulated portion of a biological sample from a subject diagnosed with the psychiatric disorder and comparing the resulting genes to those differentially expressed between the first stimulated portion and the second non-stimulated portions of a biological sample from a healthy control subject. Embodiment 31: The method of embodiment 30, further comprising comparing biomarker levels in the first portion of the biological sample from the subject diagnosed with the psychiatric disorder to biomarker levels in the first portion of the biological sample from the healthy control subject. Embodiment 32: The method of embodiment 30, further comprising identifying biomarkers that differ between the first and second portions of the subject diagnosed with the psychiatric disorder, and comparing those biomarkers to corresponding biomarkers identified between the first and second portions of the healthy control subject.

[0077] Embodiment 33:The method of any one of embodiments 21-27, wherein the biological sample comprises a blood sample. Embodiment 34: The method of embodiment 33, wherein the blood sample comprises peripheral blood mononuclear cells (white blood cells). EmbodimentUA24-176, ARIZ 24.33 PCTGallitano-Mendel and JakobiSeptember 25, 202535: The method of any one of embodiments 21-34 further comprising isolating or having isolated white blood cells from each sample obtained. Embodiment 36: The method of any one of embodiments 21-34 further comprising freezing each of the samples obtained and thawing each sample obtained before use. Embodiment 37: The method of embodiments 21-36 further comprising extracting RNA from the samples subjected to either the solution comprises the immunogenic stimulus or the control solution. Embodiment 38: The method of any one of embodiments 21-37, wherein the stimulus is an immunogenic stimulus. Embodiment 39: The method of embodiment 38, wherein the immunogenic stimulus comprises lipopolysaccharide (LPS), polyinosinic-polycytidylic acid (Poly I:C), or a combination thereof. Embodiment 40: The method of embodiment 39, wherein the immunogenic stimuli comprises a virus or bacteria. Embodiment 41: The method of any one of embodiments 21-37, wherein the stimulus is an environmental stimulus. Embodiment 42: The method of embodiment 41, wherein the environmental stimulus comprises pesticides or pollutants. Embodiment 43: The method of any one of embodiments 21-37, wherein the stimulus is a drug or chemical. Embodiment 44: The method of any one of embodiments 21-37, wherein the stimulus is heat. Embodiment 45: The method of any one of embodiments 1-44, wherein the sample is incubated for about 12 hours. Embodiment 46: The method of any one of embodiments 1-44, wherein the sample is incubated for about 24 hours. Embodiment 47: The method of any one of embodiments 1-44, wherein the sample is incubated for about 48 hours.

[0078] As used herein, the term “about” refers to plus or minus 10% of the referenced number.

[0079] Although there has been shown and described the preferred embodiment of the present invention, it will be readily apparent to those skilled in the art that modifications may be made thereto which do not exceed the scope of the appended claims. Therefore, the scope of the invention is only to be limited by the following claims. In some embodiments, the figures presented in this patent application are drawn to scale, including the angles, ratios of dimensions, etc. In some embodiments, the figures are representative only and the claims are not limited by the dimensions of the figures. In some embodiments, descriptions of the inventions described herein using the phrase “comprising” includes embodiments that could be described as “consisting essentially of’ or “consisting of’, and as such the written description requirement for claiming one or more embodiments of the present invention using the phrase “consisting essentially of’ or “consisting of’ is met.

Claims

UA24-176, ARIZ 24.33 PCT Gallitano-Mendel and Jakobi September 25, 2025WHAT IS CLAIMED IS:

1. A method comprising: a) obtaining or having obtained a biological sample from a subject; b) incubating a first portion of the biological sample obtained with a solution comprising a stimulus; and c) measuring biomarker levels in the first portion of the biological sample to biomarker levels in a second portion of the biological sample.

2. The method of claim 1, wherein the subject is diagnosed with a psychiatric disorder.

3. The method of claim 1 or claim 2, wherein the second portion of the biological sample is incubated with a control solution.

4. The method of any one of claims 1-3, further comprising: obtaining or having obtained a biological sample from a healthy subject; incubating a first portion of the biological sample obtained from the healthy subject with a solution comprising a stimulus; and measuring biomarker levels in the first portion of the biological sample from the healthy subject to biomarker levels in a second portion of the biological sample from the healthy subject.

5. The method of claim 4, wherein the second portion of the biological sample is incubated with a control solution.

6. The method of claim 4 or claim 5 further comprising comparing biomarker levels in the first portion of the subject diagnosed with the psychiatric disorder to biomarker levels in the first portion of a healthy control subject.

7. The method of claim 4 or claim 5 further comprising identifying biomarkers that differ between the first and second portions of the subject diagnosed with the psychiatric disorder, and comparing those biomarkers to corresponding biomarkers identified between the first and second portions of the healthy control subject.UA24-176, ARIZ 24.33 PCT Gallitano-Mendel and Jakobi September 25, 20258. The method of any one of claims 1-7 wherein the biological sample comprises a blood sample.

9. The method of claim 8, wherein the blood sample comprises peripheral blood mononuclear cells.

10. The method of any one of claims 1-9, wherein the second portion of the biological sample is incubated with a control solution.

11. The method of any one of claims 1-10 further comprising isolating or having isolated white blood cells from each sample obtained.

12. The method of any one of claims 1-11 further comprising freezing each of the samples obtained and thawing each sample obtained before use.

13. The method of claims 1-12 further comprising extracting RNA from the samples subjected to either the solution that comprises the immunogenic stimulus or the control solution.

14. The method of any one of claims 1-13, wherein the stimulus is an immunogenic stimulus.

15. The method of claim 14, wherein the immunogenic stimulus comprises lipopolysaccharide (LPS), polyinosinic-polycytidylic acid (Poly I:C), or a combination thereof.

16. The method of claim 14, wherein the immunogenic stimulus comprises a virus or bacteria.

17. The method of any one of claims 1-13, wherein the stimulus is an environmental stimulus.

18. The method of claim 17, wherein the environmental stimulus comprises pesticides or pollutants.

19. The method of any one of claims 1-13, wherein the stimulus is a drug or chemical, wherein the chemical acts as a hormone mimic or a hormone disruptor.

20. The method of any one of claims 1-13, wherein the stimulus comprises heat.UA24-176, ARIZ 24.33 PCT Gallitano-Mendel and Jakobi September 25, 202521. The method of any one of claims 1-13, wherein the stimulus comprises one or more hormones; wherein the one or more hormones are stress-related hormones, such as cortisol, released in response to stress or starvation.

22. The method of any one of claims 1-13, wherein the immunogenic stimulus comprises a nutritional state; wherein the nutritional state is a state of starvation or famine.

23. A method of identifying one or more biomarkers for diagnosing a psychiatric disorder, the method comprises: a) obtaining or having obtained a biological sample from a subject diagnosed with psychiatric disorders; b) incubating or having incubated a first portion of the biological sample obtained from a) to a solution comprising a stimulus; c) analyzing gene expression from the first stimulated portion of the biological sample and a second non-stimulated portion of the biological sample; wherein genes differentially expressed between the first stimulated portion of the biological sample and the second non-stimulated portion of the biological sample are identified as biomarkers.

24. The method of claim 23, wherein the second portion of the biological sample is incubated with a control solution.

25. The method of any one of claims 23 or claim 24 further comprising obtaining or having obtained a biological sample from a healthy control subject.

26. The method of claim 25 further comprising: incubating or having incubated a first portion of the biological sample obtained from the healthy control subject to a solution comprising a stimulus; and analyzing gene expression from the first stimulated portion of the biological sample from the healthy control and a second non-stimulated portion of the biological sample from the healthy control.UA24-176, ARIZ 24.33 PCT Gallitano-Mendel and Jakobi September 25, 202527. The method of claim 26, wherein the second portion of the biological sample is incubated with a control solution.

28. A method of identifying one or more biomarkers for diagnosing psychiatric disorder, the method comprises: a) obtaining or having obtained a biological sample from a subject diagnosed with a psychiatric disorder; b) obtaining or having obtained a biological sample from a healthy control subject; c) individually incubating or having incubated a first portion of the biological samples obtained from a) and b) to a solution comprising a stimulus; and d) analyzing gene expression from each biological sample from c).

29. The method of claim 28, further comprising comparing biomarker levels in the first portion of the biological sample from the subject diagnosed with the psychiatric disorder to biomarker levels in the first portion of the biological sample from the healthy control subject.

30. The method of claim 28 or claim 29, further comprising incubating or having incubated a second portion of the biological samples obtained from a) and b) to a control solution.

31. The method any one of claims 28-30, further comprising identifying biomarkers that differ between the first and second portions of the subject diagnosed with the psychiatric disorder, and comparing those biomarkers to corresponding biomarkers identified between the first and second portions of the healthy control subject.

32. A method of identifying one or more biomarkers for diagnosing a psychiatric disorder, the method comprises: a) obtaining or having obtained a biological sample from a subject diagnosed with a psychiatric disorder; b) obtaining or having obtained a biological sample from a healthy control subject; c) individually, subjecting or having subjected a first portion of the blood samplesUA24-176, ARIZ 24.33 PCTGallitano-Mendel and JakobiSeptember 25, 2025 obtained from a) and b) to a solution comprising an immunogenic stimulus; d) individually, subjecting or having subjected a second portion of the biological samples obtained from a) and b) to a control solution; and e) analyzing gene expression from each biological sample from c) and d).

33. The method of claim 32, further comprising comparing biomarker levels in the first portion of the biological sample from the subject diagnosed with the psychiatric disorder to biomarker levels in the first portion of the biological sample from the healthy control subject.

34. The method of claim 32 or 33, further comprising identifying biomarkers that differ between the first and second portions of the subject diagnosed with the psychiatric disorder, and comparing those biomarkers to corresponding biomarkers identified between the first and second portions of the healthy control subject.

35. The method of any one of claims 23-34, wherein the biological sample comprises a blood sample.

36. The method of claim 35, wherein the blood sample comprises peripheral blood mononuclear cells.

37. The method of any one of claims 23-36 further comprising isolating or having isolated white blood cells from each sample obtained.

38. The method of any one of claims 23-36 further comprising freezing each of the samples obtained and thawing each sample obtained before use.

39. The method of any one of claims 23-38, wherein the immunogenic stimulus comprises lipopolysaccharide (LPS), polyinosinic-polycytidylic acid (Poly I:C), or a combination thereof.

40. The method of claims 23-39 further comprising extracting RNA from the samples subjected to either the solution comprises the immunogenic stimulus or the control solution.

41. The method of any one of claims 23-40, wherein the stimulus is an immunogenicUA24-176, ARIZ 24.33 PCT Gallitano-Mendel and Jakobi September 25, 2025 stimulus.

42. The method of claim 41, wherein the immunogenic stimulus comprises lipopolysaccharide (LPS), polyinosinic-polycytidylic acid (Poly I:C), or a combination thereof.

43. The method of claim 41, wherein the immunogenic stimulus comprises a virus or bacteria.

44. The method of any one of claims 23-40, wherein the stimulus is an environmental stimulus.

45. The method of claim 44, wherein the environmental stimulus comprises pesticides or pollutants.

46. The method of any one of claims 23-40, wherein the stimulus is a drug or chemical, wherein the chemical acts as a hormone mimic or a hormone disruptor.

47. The method of any one of claims 23-40, wherein the stimulus is heat.

48. The method of any one of claims 23-40, wherein the stimulus comprises one or more hormones; wherein the one or more hormones are stress-related hormones, such as cortisol, released in response to stress or starvation.

49. The method of any one of claims 23-40, wherein the immunogenic stimulus comprises a nutritional state; wherein the nutritional state is a state of starvation or famine.

50. The method of any one of claims 1-45, wherein the sample is incubated for about 12, 24, or 48 hours

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